CD133 expression in osteosarcoma and derivation of CD133⁺ cells.

PubMed

Li, Ji; Zhong, Xiao-Yan; Li, Zong-Yu; Cai, Jin-Fang; Zou, Lin; Li, Jian-Min; Yang, Tao; Liu, Wei

2013-02-01

Cluster of differentiation 133 (CD133) is recognized as a stem cell marker for normal and cancerous tissues. Using cell culture and real‑time fluorescent polymerase chain reaction, CD133 expression was analyzed in osteosarcoma tissue and Saos‑2 cell lines. In addition, cancer stem cell‑related gene expression in the Saos‑2 cell line was determined to explore the mechanisms underlying tumorigenesis and high drug resistance in osteosarcoma. CD133+ cells were found to be widely distributed in various types of osteosarcoma tissue. Following cell culture, cells entered the G2/M and S cell cycle stages from G0/G1. Levels of CD133+ cells decreased to normal levels rapidly over the course of cell culture. Colony forming efficiency was higher in the CD133+ compared with the CD133‑ subpopulation of Saos‑2 cells. Expression levels of stem cell‑related genes, including multidrug resistance protein 1 (MDR1) and sex determining region Y‑box 2 (Sox2) in the CD133+ subpopulation of cells were found to be significantly higher compared with the CD133‑ subpopulation. These observations indicate that CD133+ Saos‑2 cells exhibit stem cell characteristics, including low abundance, quiescence and a high potential to undergo differentiation, as well as expression of key stem cell regulatory and drug resistance genes, which may cause osteosarcoma and high drug resistance.

CD133 Is Not Suitable Marker for Isolating Melanoma Stem Cells from D10 Cell Line.

PubMed

Rajabi Fomeshi, Motahareh; Ebrahimi, Marzieh; Mowla, Seyed Javad; Firouzi, Javad; Khosravani, Pardis

2016-01-01

Cutaneous melanoma is the most hazardous malignancy of skin cancer with a high mortality rate. It has been reported that cancer stem cells (CSCs) are responsible for malignancy in most of cancers including melanoma. The aim of this study is to compare two common methods for melanoma stem cell enriching; isolating based on the CD133 cell surface marker and spheroid cell culture. In this experimental study, melanoma stem cells were enriched by fluorescence activated cell sorting (FACS) based on the CD133 protein expression and spheroid culture of D10 melanoma cell line,. To determine stemness features, the mRNA expression analysis of ABCG2, c-MYC, NESTIN, OCT4-A and -B genes as well as colony and spheroid formation assays were utilized in unsorted CD133(+), CD133(-) and spheroid cells. Significant differences of the two experimental groups were compared using student's t tests and a two-tailed value of P<0.05 was statistically considered as a significant threshold. Our results demonstrated that spheroid cells had more colony and spheroid forming ability, rather than CD133(+) cells and the other groups. Moreover, melanospheres expressed higher mRNA expression level of ABCG2, c-MYC, NESTIN and OCT4-A com- pared to other groups (P<0.05). Although CD133(+) derived melanoma cells represented stemness fea- tures, our findings demonstrated that spheroid culture could be more effective meth- od to enrich melanoma stem cells.

CD133 Is Not Suitable Marker for Isolating Melanoma Stem Cells from D10 Cell Line

PubMed Central

Rajabi Fomeshi, Motahareh; Ebrahimi, Marzieh; Mowla, Seyed Javad; Firouzi, Javad; Khosravani, Pardis

2016-01-01

Objective Cutaneous melanoma is the most hazardous malignancy of skin cancer with a high mortality rate. It has been reported that cancer stem cells (CSCs) are responsible for malignancy in most of cancers including melanoma. The aim of this study is to compare two common methods for melanoma stem cell enriching; isolating based on the CD133 cell surface marker and spheroid cell culture. Materials and Methods In this experimental study, melanoma stem cells were enriched by fluorescence activated cell sorting (FACS) based on the CD133 protein expression and spheroid culture of D10 melanoma cell line,. To determine stemness features, the mRNA expression analysis of ABCG2, c-MYC, NESTIN, OCT4-A and -B genes as well as colony and spheroid formation assays were utilized in unsorted CD133+, CD133- and spheroid cells. Significant differences of the two experimental groups were compared using student’s t tests and a two-tailed value of P<0.05 was statistically considered as a significant threshold. Results Our results demonstrated that spheroid cells had more colony and spheroid forming ability, rather than CD133+ cells and the other groups. Moreover, melanospheres expressed higher mRNA expression level of ABCG2, c-MYC, NESTIN and OCT4-A com- pared to other groups (P<0.05). Conclusion Although CD133+ derived melanoma cells represented stemness fea- tures, our findings demonstrated that spheroid culture could be more effective meth- od to enrich melanoma stem cells. PMID:27054115

Omega-3 Eicosapentaenoic Acid Decreases CD133 Colon Cancer Stem-Like Cell Marker Expression While Increasing Sensitivity to Chemotherapy

PubMed Central

De Carlo, Flavia; Witte, Theodore R.; Hardman, W. Elaine; Claudio, Pier Paolo

2013-01-01

Colorectal cancer is the third leading cause of cancer-related death in the western world. In vitro and in vivo experiments showed that omega-3 polyunsaturated fatty acids (n-3 PUFAs) can attenuate the proliferation of cancer cells, including colon cancer, and increase the efficacy of various anticancer drugs. However, these studies address the effects of n-3 PUFAs on the bulk of the tumor cells and not on the undifferentiated colon cancer stem-like cells (CSLCs) that are responsible for tumor formation and maintenance. CSLCs have also been linked to the acquisition of chemotherapy resistance and to tumor relapse. Colon CSLCs have been immunophenotyped using several antibodies against cellular markers including CD133, CD44, EpCAM, and ALDH. Anti-CD133 has been used to isolate a population of colon cancer cells that retains stem cells properties (CSLCs) from both established cell lines and primary cell cultures. We demonstrated that the n-3 PUFA, eicosapentaenoic acid (EPA), was actively incorporated into the membrane lipids of COLO 320 DM cells. 25 uM EPA decreased the cell number of the overall population of cancer cells, but not of the CD133 (+) CSLCs. Also, we observed that EPA induced down-regulation of CD133 expression and up-regulation of colonic epithelium differentiation markers, Cytokeratin 20 (CK20) and Mucin 2 (MUC2). Finally, we demonstrated that EPA increased the sensitivity of COLO 320 DM cells (total population) to both standard-of-care chemotherapies (5-Fluorouracil and oxaliplatin), whereas EPA increased the sensitivity of the CD133 (+) CSLCs to only 5-Fluorouracil. PMID:23874993

Identification of progenitor cancer stem cell in lentigo maligna melanoma.

PubMed

Bongiorno, M R; Doukaki, S; Malleo, F; Aricò, M

2008-07-01

The potential role of stem cells in neoplasia has aroused considerable interest over the past few years. A number of known biologic characteristics of melanomas support the theory that they may originate in a mutated stem cell. Melanocytic stem cell markers have been described recently. Moreover, the CD133 cells that show surface markers for CD34 are stem cells primitive. These stem cells are capable of differentiating into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. The identification of cancer stem/initiating cells with a crucial role in tumor formation may open up new pharmacologic perspectives. The purpose of this study is to detect the expression of CD133 and CD34, two putative markers of cancer stem cells in the lentigo maligna melanoma. Thirty cases of lentigo maligna melanoma were analyzed using indirect immunohistochemical staining. The vast majority of the samples analyzed showed the presence of rare cells, which were clearly positive for CD133 and CD34. Strong CD133 and CD34 staining was found in the outer root sheath of the mid-lower hair follicles, intermixed with atypical melanocytes extending along layers of the hair follicles. A number of these staminal cells were adjacent and intermixed with melanoma cells. This study supports the stem cell origin of this tumor and suggests that the precursor of the melanoma in question is a stem-like cell rather than the primitive melanoblast committed to be exclusively involved in melanocytic differentiation.

CD133 expression is not restricted to stem cells, and both CD133+ and CD133– metastatic colon cancer cells initiate tumors

PubMed Central

Shmelkov, Sergey V.; Butler, Jason M.; Hooper, Andrea T.; Hormigo, Adilia; Kushner, Jared; Milde, Till; St. Clair, Ryan; Baljevic, Muhamed; White, Ian; Jin, David K.; Chadburn, Amy; Murphy, Andrew J.; Valenzuela, David M.; Gale, Nicholas W.; Thurston, Gavin; Yancopoulos, George D.; D’Angelica, Michael; Kemeny, Nancy; Lyden, David; Rafii, Shahin

2008-01-01

Colon cancer stem cells are believed to originate from a rare population of putative CD133+ intestinal stem cells. Recent publications suggest that a small subset of colon cancer cells expresses CD133, and that only these CD133+ cancer cells are capable of tumor initiation. However, the precise contribution of CD133+ tumor-initiating cells in mediating colon cancer metastasis remains unknown. Therefore, to temporally and spatially track the expression of CD133 in adult mice and during tumorigenesis, we generated a knockin lacZ reporter mouse (CD133lacZ/+), in which the expression of lacZ is driven by the endogenous CD133 promoters. Using this model and immunostaining, we discovered that CD133 expression in colon is not restricted to stem cells; on the contrary, CD133 is ubiquitously expressed on differentiated colonic epithelium in both adult mice and humans. Using Il10–/–CD133lacZ mice, in which chronic inflammation in colon leads to adenocarcinomas, we demonstrated that CD133 is expressed on a full gamut of colonic tumor cells, which express epithelial cell adhesion molecule (EpCAM). Similarly, CD133 is widely expressed by human primary colon cancer epithelial cells, whereas the CD133– population is composed mostly of stromal and inflammatory cells. Conversely, CD133 expression does not identify the entire population of epithelial and tumor-initiating cells in human metastatic colon cancer. Indeed, both CD133+ and CD133– metastatic tumor subpopulations formed colonospheres in in vitro cultures and were capable of long-term tumorigenesis in a NOD/SCID serial xenotransplantation model. Moreover, metastatic CD133– cells form more aggressive tumors and express typical phenotypic markers of cancer-initiating cells, including CD44 (CD44+CD24–), whereas the CD133+ fraction is composed of CD44lowCD24+ cells. Collectively, our data suggest that CD133 expression is not restricted to intestinal stem or cancer-initiating cells, and during the metastatic transition, CD133+ tumor cells might give rise to the more aggressive CD133– subset, which is also capable of tumor initiation in NOD/SCID mice. PMID:18497886

CD133+ tumor initiating cells in a syngenic murine model of pancreatic cancer respond to Minnelide.

PubMed

Banerjee, Sulagna; Nomura, Alice; Sangwan, Veena; Chugh, Rohit; Dudeja, Vikas; Vickers, Selwyn M; Saluja, Ashok

2014-05-01

Pancreatic adenocarcinoma is the fourth leading cause for cancer-related mortality with a survival rate of less than 5%. Late diagnosis and lack of effective chemotherapeutic regimen contribute to these grim survival statistics. Relapse of any tumor is largely attributed to the presence of tumor-initiating cells (TIC) or cancer stem cells (CSC). These cells are considered as hurdles to cancer therapy as no known chemotherapeutic compound is reported to target them. Thus, there is an urgent need to develop a TIC-targeted therapy for pancreatic cancer. We isolated CD133(+) cells from a spontaneous pancreatic ductal adenocarcinoma mouse model and studied both surface expression, molecular markers of pancreatic TICs. We also studied tumor initiation properties by implanting low numbers of CD133(+) cells in immune competent mice. Effect of Minnelide, a drug currently under phase I clinical trial, was studied on the tumors derived from the CD133(+) cells. Our study showed for the first time that CD133(+) population demonstrated all the molecular markers for pancreatic TIC. These cells initiated tumors in immunocompetent mouse models and showed increased expression of prosurvival and proinvasive proteins compared to the CD133(-) non-TIC population. Our study further showed that Minnelide was very efficient in downregulating both CD133(-) and CD133(+) population in the tumors, resulting in a 60% decrease in tumor volume compared with the untreated ones. As Minnelide is currently under phase I clinical trial, its evaluation in reducing tumor burden by decreasing TIC as well as non-TIC population suggests its potential as an effective therapy. ©2014 AACR.

Predictive and prognostic effect of CD133 and cancer-testis antigens in stage Ib-IIIA non-small cell lung cancer.

PubMed

Su, Chunxia; Xu, Ying; Li, Xuefei; Ren, Shengxiang; Zhao, Chao; Hou, Likun; Ye, Zhiwei; Zhou, Caicun

2015-01-01

CD133 and cancer-testis antigens (CTAs) may be potential predicted markers of adjuvant chemotherapy or immune therapy, and they may be the independent prognostic factor of NSCLC. Nowadays, there is still no predictive biomarker identified for the use of adjuvant chemotherapy in non-small cell lung cancer (NSCLC) patients. To clarify the role of CD133 and CTAs as a predictive marker for adjuvant chemotherapy or prognostic factors of overall survival, we performed a retrospective study in 159 stage Ib-IIIA NSCLC patients receiving adjuvant chemotherapy or observe from April 2003 to March 2004 in our institute. Clinical data and gene anaylisis results were collected, while CD133 and three CTAs (MAGE-A4, NY-ESO-1, MAGE-A10) were determined according to their monoclonal antibodies such as CD133, 57B, D8.38 and 3GA11 by immunohistochemistry. All CTAs were more frequently expressed in squamous cell carcinoma (SCC) (50.0%, 26.9%, 34.6%) than in adenocarcinoma (16.2%, 16.2%, 16.2%). CD133 was more frequently found in patients with adenocarcinoma (P=0.044). Negative expression of CD133 was associated with a significantly longer overall survival compared to positive expression of CD133 (62.5 vs. 48.5 months, P=0.035). When combined with MAGEA4, NY-ESO-1or MAGE-A10, patients' OS showed significantly difference among different combination. (CD133-MAGEA4-/CD133-MAGEA4+/CD133+MAGEA4-/CD133+MAGEA4+: 65.6 months vs.51.5 months vs.32.2 months vs.19.8 months, P=0.000, CD133-NY-ESO-1-/ CD133+NY-ESO-1-/CD133-NY-ESO-1+/ CD133+NY-ESO-1+: 57.8 months vs. 55.7 months vs. 44.6 months vs. 28.5 months, P=0.000, CD133-MAGEA10-/CD133+ MAGEA10-/CD133-MAGEA10-/CD133+MAGEA10+: 66.2 months vs. 57.2 months vs. 48.8 months vs. 41.4 months, P=0.001). There is no difference between patients received adjuvant chemotherapy or not, but subgroup analysis showed that the patients with CD133+NY-ESO-1+ expression who received chemotherapy will survive longer than not receive adjuvant chemotherapy (received vs. not received, 52.1 vs. 27.1 months, P=0.020). In the subgroup with EGFR mutation/ALK translocation/Ros1 translocation/Ret fusion, the trend remained but without a statistically significant difference. Multivariate COX regression analysis showed that stage, CD133, CD133-MAGEA4- and CD133-NY-ESO-1- are independent prognostic factors. In conclusion, CTAs (MAGE-A4, NY-ESO-1, MAGE-A10) were more likely expressed in patients with squamous cell carcinoma and when CTAs combined with CD133, they can be better prognostic factors. Patients with CD133+NY-ESO-1+ expression may survive longer when treated with adjuvant chemotherapy, which indicates that the CD133 and CTAs might be a potential marker to guide adjuvant chemotherapy in this population.

Enhancing the Oncolytic Activity of CD133-Targeted Measles Virus: Receptor Extension or Chimerism with Vesicular Stomatitis Virus Are Most Effective

PubMed Central

Kleinlützum, Dina; Hanauer, Julia D. S.; Muik, Alexander; Hanschmann, Kay-Martin; Kays, Sarah-Katharina; Ayala-Breton, Camilo; Peng, Kah-Whye; Mühlebach, Michael D.; Abel, Tobias; Buchholz, Christian J.

2017-01-01

Therapy resistance and tumor recurrence are often linked to a small refractory and highly tumorigenic subpopulation of neoplastic cells, known as cancer stem cells (CSCs). A putative marker of CSCs is CD133 (prominin-1). We have previously described a CD133-targeted oncolytic measles virus (MV-CD133) as a promising approach to specifically eliminate CD133-positive tumor cells. Selectivity was introduced at the level of cell entry by an engineered MV hemagglutinin (H). The H protein was blinded for its native receptors and displayed a CD133-specific single-chain antibody fragment (scFv) as targeting domain. Interestingly, MV-CD133 was more active in killing CD133-positive tumors than the unmodified MV-NSe despite being highly selective for its target cells. To further enhance the antitumoral activity of MV-CD133, we here pursued arming technologies, receptor extension, and chimeras between MV-CD133 and vesicular stomatitis virus (VSV). All newly generated viruses including VSV-CD133 were highly selective in eliminating CD133-positive cells. MV-CD46/CD133 killed in addition CD133-negative cells being positive for the MV receptors. In an orthotopic glioma model, MV-CD46/CD133 and MVSCD-CD133, which encodes the super cytosine deaminase, were most effective. Notably, VSV-CD133 caused fatal neurotoxicity in this tumor model. Use of CD133 as receptor could be excluded as being causative. In a subcutaneous tumor model of hepatocellular cancer, VSV-CD133 revealed the most potent oncolytic activity and also significantly prolonged survival of the mice when injected intravenously. Compared to MV-CD133, VSV-CD133 infected a more than 104-fold larger area of the tumor within the same time period. Our data not only suggest new concepts and approaches toward enhancing the oncolytic activity of CD133-targeted oncolytic viruses but also raise awareness about careful toxicity testing of novel virus types. PMID:28695108

Is sphere assay useful for the identification of cancer initiating cells of the ovary?

PubMed

Martínez-Serrano, María José; Caballero-Baños, Miguel; Vilella, Ramon; Vidal, Laura; Pahisa, Jaume; Martínez-Roman, Sergio

2015-01-01

Current evidence suggests that the presence of tumor-initiating cells (TICs) in epithelial ovarian cancer (EOC) has a role in chemoresistance and relapse. Surface markers such as CD44(+)/CD24(-), CD117(+), and CD133(+) expression have been reported as potential markers for TICs related to ovarian cancer and tumorigenic cell lines. In this study, we have investigated if spheroid forms are TIC specific or whether they can also be produced by somatic stem cells from healthy tissue in vitro. In addition, we also investigated the specificity of surface markers to identify TICs from papillary serous EOC patients. Cells were obtained from fresh tumors from 10 chemotherapy-naive patients with EOC, and cells from ovarian and tubal epithelium were obtained from 5 healthy menopausal women undergoing surgery for benign pathology and cultured in standard and in selective medium. Cells forming nonadherent spheroids were considered TICs, and the adherent cells were considered as non-TIC-like. Percentages of CD24(+), CD44(+), CD117(+), CD133(+), and vascular endothelial growth factor receptor (VEGF-R)(+) cell surface markers were analyzed by flow cytometry. Four of 10 EOC cell tissues were excluded from the study. Tumor cells cultured in selective medium developed spheroid forms after 1 to 7 weeks in 5 of 6 EOC patients. No spheroid forms were observed in cultures of cells from healthy women. Unlike previously published data, low levels of CD24(+), CD44(+), CD117(+), and VEGF-R(+) expression were observed in spheroid cells, whereas expression of CD133(+) was moderate but higher in adherent cells from papillary serous EOC cells in comparison with adherent cells from controls. Papillary serous EOC contains TICs that form spheroids with low expression of CD44(+), CD24(+), CD117(+) and VEGF-R(+). Further research is required to find specific surface markers to identify papillary serous TICs.

Expression of CD133 confers malignant potential by regulating metalloproteinases in human hepatocellular carcinoma.

PubMed

Kohga, Keisuke; Tatsumi, Tomohide; Takehara, Tetsuo; Tsunematsu, Hinako; Shimizu, Satoshi; Yamamoto, Masashi; Sasakawa, Akira; Miyagi, Takuya; Hayashi, Norio

2010-06-01

Although CD133 expression is identified as a cancer stem cell marker of hepatocellular carcinoma (HCC), the detailed characteristics of HCC cells expressing CD133 remain unclear. We examined the malignant characteristics of CD133-expressing HCC cells. CD133-expressing cells could be detected with low frequency in 5 HCC tissues. We derived two different HCC cell lines by (1) transfection of CD133 siRNA in PLC/PRF/5 cells in (CD133si-PLC/PRF/5), and (2) by a magnetic cell sorting method that allowed to divide Huh7 cells into two CD133 positive (+) and negative (-) groups. CD133 knockdown in PLC/PRF/5 cells resulted in a decrease of the mRNA and protein expressions of matrix metalloproteinase (MMP)-2 and a disintegrin and metalloproteinase (ADAM)9. We next examined the malignant characteristics related to decreasing MMP-2 and ADAM9 in HCC cells. In CD133si-PLC/PRF/5 cells and CD133- Huh7 cells, invasiveness and vascular endothelial growth factor (VEGF) production, which are both related to the activity of MMP-2, were inhibited compared CD133-expressing HCC cells. We previously demonstrated that ADAM9 protease plays critical roles in the shedding of MHC class I-related chain A (MICA) which regulates the sensitivity of tumor cells to natural killer cells (NK). Decreasing ADAM9 expression in CD133si-PLC/PRF/5 cells and CD133- Huh7 cells resulted in an increase in membrane-bound MICA and a decrease in soluble MICA production. Both CD133si-PLC/PRF/5 cells and CD133- Huh7 cells were susceptible to NK activity, depending on the expression levels of membrane-bound MICA, but CD133-expressing HCC cells were not. These results demonstrate that CD133 expression in HCC cells confers malignant potential which may contribute to the survival of HCC cells. Copyright 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Isolation and identification of tumor-initiating cell properties in human gallbladder cancer cell lines using the marker cluster of differentiation 133.

PubMed

Yu, Jiwei; Tang, Zhaohui; Gong, Wei; Zhang, Mingdi; Quan, Zhiwei

2017-12-01

The present study aimed to isolate and identify the properties of the cluster of differentiation (CD)133 + subset in human gallbladder cancer cells. The CD133 + and CD133 - subpopulations of the GBC-SD cell line were separated using immunomagnetic separation, and the biological features of the two subpopulations were analyzed in vitro and in vivo . In particular, the present study aimed to determine whether the two subpopulations were resistant to anti-tumor reagents and to identify the underlying molecular mechanisms involved. Following cell sorting of GBC-SD cells using immunomagnetic beads, 90.2±2% of cells were identified as CD133 + . Immunofluorescence confirmed that CD133 was expressed at higher levels in the Cd133 + group compared with the CD133 - group. The proliferation of the CD133 + group was significantly increased compared with the CD133 - group in vitro and in vivo . Following treatment with fluorouracil or gemcitabine, cells in the CD133 + group exhibited a decreased sensitivity to these drugs. The number of transmembrane cells was significantly increased in the CD133 + group compared with the CD133 - group. In addition, the expression levels of ATP binding cassette subfamily G member 2, CD44, C-X-C motif chemokine receptor 4 (CXCR4), phosphorylated-protein kinase B (Akt) and CD133 in the CD133 + group were significantly increased, compared with those in the CD133 - group. In CD133 + GBC-SD cells, stromal cell-derived factor 1α (SDF-1α) or treatment with AMD3100, an inhibitor of CXCR4, promotes or suppresses the SDF-1α/CXCR4 axis, respectively, resulting in increased or decreased CD133 expression through the Akt signaling pathway. Inhibition of the Akt signaling pathway resulted in decreased CD133 expression in GBC-SD cells. Immunomagnetic beads were successfully used for isolation of the CD133 + subset from GBC-SD cells. Furthermore, the CD133 + subset revealed an increased potential for tumor formation, cell proliferation, invasion and resistance to chemotherapeutic agents with expression of stem cell-associated genes. Therefore, in GBC-SD cells, the CXCR4/Akt/CD133 signaling pathways may be activated.

Functional significance of CD105-positive cells in papillary renal cell carcinoma.

PubMed

Matak, Damian; Brodaczewska, Klaudia K; Szczylik, Cezary; Koch, Irena; Myszczyszyn, Adam; Lipiec, Monika; Lewicki, Slawomir; Szymanski, Lukasz; Zdanowski, Robert; Czarnecka, Anna M

2017-01-05

CD105 was postulated as a renal cell carcinoma (RCC) stem cell marker, and CD133 as a putative RCC progenitor. Hypoxia, a natural microenvironment that prevails in tumors, was also incorporated into the study, especially in terms of the promotion of hypothetical stem-like cell properties. Within this study, we verify the existence of CD105+ and CD133+ populations in selected papillary subtype RCC (pRCC) cell lines. Both populations were analyzed for correlation with stem-like cell properties, such as stemness gene expression, and sphere and colony formation. For the preliminary analysis, several RCC cell lines were chosen (786-O, SMKT-R2, Caki-2, 796-P, ACHN, RCC6) and the control was human kidney cancer stem cells (HKCSC) and renal cells of embryonic origin (ASE-5063). Four cell lines were chosen for further investigation: Caki-2 (one of the highest numbers of CD105+ cells; primary origin), ACHN (a low number of CD105+ cells; metastatic origin), HKCSC (putative positive control), and ASE-5063 (additional control). In 769-P and RCC6, we could not detect a CD105+ population. Hypoxia variously affects pRCC cell growth, and mainly diminishes the stem-like properties of cells. Furthermore, we could not observe the correlation of CD105 and/or CD133 expression with the enhancement of stem-like properties. Based on this analysis, CD105/CD133 cannot be validated as cancer stem cell markers of pRCC cell lines.

CD133-targeted gene transfer into long-term repopulating hematopoietic stem cells.

PubMed

Brendel, Christian; Goebel, Benjamin; Daniela, Abriss; Brugman, Martijn; Kneissl, Sabrina; Schwäble, Joachim; Kaufmann, Kerstin B; Müller-Kuller, Uta; Kunkel, Hana; Chen-Wichmann, Linping; Abel, Tobias; Serve, Hubert; Bystrykh, Leonid; Buchholz, Christian J; Grez, Manuel

2015-01-01

Gene therapy for hematological disorders relies on the genetic modification of CD34(+) cells, a heterogeneous cell population containing about 0.01% long-term repopulating cells. Here, we show that the lentiviral vector CD133-LV, which uses a surface marker on human primitive hematopoietic stem cells (HSCs) as entry receptor, transfers genes preferentially into cells with high engraftment capability. Transduction of unstimulated CD34(+) cells with CD133-LV resulted in gene marking of cells with competitive proliferative advantage in vitro and in immunodeficient mice. The CD133-LV-transduced population contained significantly more cells with repopulating capacity than cells transduced with vesicular stomatitis virus (VSV)-LV, a lentiviral vector pseudotyped with the vesicular stomatitis virus G protein. Upon transfer of a barcode library, CD133-LV-transduced cells sustained gene marking in vivo for a prolonged period of time with a 6.7-fold higher recovery of barcodes compared to transduced control cells. Moreover, CD133-LV-transduced cells were capable of repopulating secondary recipients. Lastly, we show that this targeting strategy can be used for transfer of a therapeutic gene into CD34(+) cells obtained from patients suffering of X-linked chronic granulomatous disease. In conclusion, direct gene transfer into CD133(+) cells allows for sustained long-term engraftment of gene corrected cells.

Photochemical internalization (PCI) of immunotoxins targeting CD133 is specific and highly potent at femtomolar levels in cells with cancer stem cell properties.

PubMed

Bostad, Monica; Berg, Kristian; Høgset, Anders; Skarpen, Ellen; Stenmark, Harald; Selbo, Pål K

2013-06-28

CD133 is a putative cancer stem cell (CSC) marker for a number of different cancers and is suggested to be a therapeutic target. Since also normal stem cells express CD133 it is of paramount importance that targeting strategies provide a specific and efficient delivery of cytotoxic drugs in only CD133-positive CSCs. In this study, we have employed photochemical internalization (PCI), a minimally invasive method for light-controlled, specific delivery of membrane-impermeable macromolecules from endocytic vesicles to the cytosol, to specifically target CD133-positive cancer cells. We demonstrate that PCI increases the cytotoxic effect of an immunotoxin (IT) targeting CD133-expressing cancer cells of colon (WiDr and HCT116) and pancreas (BxPC-3) origin. The IT consisted of the mAb CD133/1 (AC133) bound to the ribosome inactivating plant toxin saporin (anti-CD133/1-sap). We show that TPCS2a-PCI of anti-CD133/1-sap is specific, and highly cytotoxic at femto-molar concentrations. Specific binding and uptake of CD133/1, was shown by fluorescence microscopy and co-localization with TPCS2a in endosomes/lysosomes was determined by confocal microscopy. CD133(high) WiDr cells, isolated by fluorescence activated cell sorting, had a 7-fold higher capacity to initiate spheroids than CD133(low) cells (P<0.001) and were resistant to photodynamic therapy (PDT). However, PDT-resistance was bypassed by the PCI strategy. Tumor initiation and aggressive growth in athymic nude mice was obtained with only 10 CD133(high) cells in contrast to CD133(low) cells where substantially higher cell numbers were needed. The excellent high efficacy and selectivity of eliminating CD133-expressing cells by PCI warrant further pre-clinical evaluations of this novel therapeutic approach. Copyright © 2013 Elsevier B.V. All rights reserved.

Specific elimination of CD133+ tumor cells with targeted oncolytic measles virus.

PubMed

Bach, Patricia; Abel, Tobias; Hoffmann, Christopher; Gal, Zoltan; Braun, Gundula; Voelker, Iris; Ball, Claudia R; Johnston, Ian C D; Lauer, Ulrich M; Herold-Mende, Christel; Mühlebach, Michael D; Glimm, Hanno; Buchholz, Christian J

2013-01-15

Tumor-initiating cells (TIC) are critical yet evasive targets for the development of more effective antitumoral strategies. The cell surface marker CD133 is frequently used to identify TICs of various tumor entities, including hepatocellular cancer and glioblastoma. Here, we describe oncolytic measles viruses (MV) retargeted to CD133. The viruses, termed MV-141.7 and MV-AC133, infected and selectively lysed CD133(+) tumor cells. Both viruses exerted strong antitumoral effects on human hepatocellular carcinoma growing subcutaneously or multifocally in the peritoneal cavity of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Notably, the CD133-targeted viruses were more effective in prolonging survival than the parental MV-NSe, which is currently assessed as oncolytic agent in clinical trials. Interestingly, target receptor overexpression or increased spreading kinetics through tumor cells were excluded as being causative for the enhanced oncolytic activity of CD133-targeted viruses. MV-141.7 was also effective in mouse models of orthotopic glioma tumor spheres and primary colon cancer. Our results indicate that CD133-targeted measles viruses selectively eliminate CD133(+) cells from tumor tissue, offering a key tool for research in tumor biology and cancer therapy.

Human CD34(lo)CD133(lo) fetal liver cells support the expansion of human CD34(hi)CD133(hi) hematopoietic stem cells.

PubMed

Yong, Kylie Su Mei; Keng, Choong Tat; Tan, Shu Qi; Loh, Eva; Chang, Kenneth Te; Tan, Thiam Chye; Hong, Wanjin; Chen, Qingfeng

2016-09-01

We have recently discovered a unique CD34(lo)CD133(lo) cell population in the human fetal liver (FL) that gives rise to cells in the hepatic lineage. In this study, we further characterized the biological functions of FL CD34(lo)CD133(lo) cells. Our findings show that these CD34(lo)CD133(lo) cells express markers of both endodermal and mesodermal lineages and have the capability to differentiate into hepatocyte and mesenchymal lineage cells by ex vivo differentiation assays. Furthermore, we show that CD34(lo)CD133(lo) cells express growth factors that are important for human hematopoietic stem cell (HSC) expansion: stem cell factor (SCF), insulin-like growth factor 2 (IGF2), C-X-C motif chemokine 12 (CXCL12), and factors in the angiopoietin-like protein family. Co-culture of autologous FL HSCs and allogenic HSCs derived from cord blood with CD34(lo)CD133(lo) cells supports and expands both types of HSCs.These findings are not only essential for extending our understanding of the HSC niche during the development of embryonic and fetal hematopoiesis but will also potentially benefit adult stem cell transplantations in clinics because expanded HSCs demonstrate the same capacity as primary cells to reconstitute the human immune system and mediate long-term hematopoiesis in vivo. Together, CD34(lo)CD133(lo) cells not only serve as stem/progenitor cells for liver development but are also an essential component of the HSC niche in the human FL.

Cancer stem cell-like cells from a single cell of oral squamous carcinoma cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Felthaus, O.; Department of Oral and Maxillofacial Surgery, University of Regensburg; Ettl, T.

2011-04-01

Research highlights: {yields} Four oral squamous cancer cell lines (OSCCL) were analyzed for cancer stem cells (CSCs). {yields} Single cell derived colonies of OSCCL express CSC-marker CD133 differentially. {yields} Monoclonal cell lines showed reduced sensitivity for Paclitaxel. {yields} In situ CD133{sup +} cells are slow cycling (Ki67-) indicating a reduced drug sensitivity. {yields} CD133{sup +} and CSC-like cells can be obtained from single colony forming cells of OSCCL. -- Abstract: Resistance of oral squamous cell carcinomas (OSCC) to conventional chemotherapy or radiation therapy might be due to cancer stem cells (CSCs). The development of novel anticancer drugs requires a simplemore » method for the enrichment of CSCs. CSCs can be enriched from OSCC cell lines, for example, after cultivation in serum-free cell culture medium (SFM). In our study, we analyzed four OSCC cell lines for the presence of CSCs. CSC-like cells could not be enriched with SFM. However, cell lines obtained from holoclone colonies showed CSC-like properties such as a reduced rate of cell proliferation and a reduced sensitivity to Paclitaxel in comparison to cells from the parental lineage. Moreover, these cell lines differentially expressed the CSC-marker CD133, which is also upregulated in OSCC tissues. Interestingly, CD133{sup +} cells in OSCC tissues expressed little to no Ki67, the cell proliferation marker that also indicates reduced drug sensitivity. Our study shows a method for the isolation of CSC-like cell lines from OSCC cell lines. These CSC-like cell lines could be new targets for the development of anticancer drugs under in vitro conditions.« less

Blocking the NOTCH pathway can inhibit the growth of CD133-positive A549 cells and sensitize to chemotherapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Juntao; Mao, Zhangfan; Huang, Jie

2014-02-21

Highlights: • Notch signaling pathway members are expressed lower levels in CD133+ cells. • CD133+ cells are not as sensitive as CD133− cells to chemotherapy. • GSI could inhibit the growth of both CD133+ and CD133− cells. • Blockade of Notch signaling pathway enhanced the effect of chemotherapy with CDDP. • DAPT/CDDP co-therapy caused G2/M arrest and elimination in CD133+ cells. - Abstract: Cancer stem cells (CSCs) are believed to play an important role in tumor growth and recurrence. These cells exhibit self-renewal and proliferation properties. CSCs also exhibit significant drug resistance compared with normal tumor cells. Finding new treatmentsmore » that target CSCs could significantly enhance the effect of chemotherapy and improve patient survival. Notch signaling is known to regulate the development of the lungs by controlling the cell-fate determination of normal stem cells. In this study, we isolated CSCs from the human lung adenocarcinoma cell line A549. CD133 was used as a stem cell marker for fluorescence-activated cell sorting (FACS). We compared the expression of Notch signaling in both CD133+ and CD133− cells and blocked Notch signaling using the γ-secretase inhibitor DAPT (GSI-IX). The effect of combining GSI and cisplatin (CDDP) was also examined in these two types of cells. We observed that both CD133+ and CD133− cells proliferated at similar rates, but the cells exhibited distinctive differences in cell cycle progression. Few CD133+ cells were observed in the G{sub 2}/M phase, and there were half as many cells in S phase compared with the CD133− cells. Furthermore, CD133+ cells exhibited significant resistance to chemotherapy when treated with CDDP. The expression of Notch signaling pathway members, such as Notch1, Notch2 and Hes1, was lower in CD133+ cells. GSI slightly inhibited the proliferation of both cell types and exhibited little effect on the cell cycle. The inhibitory effects of DPP on these two types of cells were enhanced when combined with GSI. Interestingly, this effect was especially significant in CD133+ cells, suggesting that Notch pathway blockade may be a useful CSC-targeted therapy in lung cancer.« less

Chemoresistance of CD133{sup +} colon cancer may be related with increased survivin expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Mi-Ra; Ji, Sun-Young; Mia-Jan, Khalilullah

2015-07-31

CD133, putative cancer stem cell marker, deemed to aid chemoresistance. However, this claim has been challenged recently and we previously reported that patients with CD133{sup +} colon cancer have benefit from 5-fluorouracil (5-FU) chemotherapy incontrast to no benefit in patients with CD133{sup −} cancer. To elucidate the role of CD133 expression in chemoresistance, we silenced the CD133 expression in a colon cancer cell line and determined its effect on the biological characteristics downstream. We comparatively analyzed the sequential changes of MDR1, ABCG2, AKT1 and survivin expression and the result of proliferation assay (WST-1 assay) with 5-FU treatment in CD133{sup +}more » and siRNA-induced CD133{sup −} cells, derived from Caco-2 colon cancer cell line. 5-FU treatment induced significantly increase of the mRNA expression of MDR1, ABCG2 and AKT1genes, but not protein level. CD133 had little to no effect on the mRNA and protein expression of these genes. However, survivin expression at mRNA and protein level were significantly increased in CD133{sup +} cells compared with siRNA-induced CD133-cells and Mock (not sorted CD133{sup +} cells) at 96 h after siRNA transfection. The cytotoxicity assay demonstrated notable increase of chemoresistance to 5-FU treatment (10 μM) in CD133{sup +} cells at 96 h after siRNA transfection. From this study, we conclude that CD133{sup +} cells may have chemoresistance to 5-FU through the mechanism which is related with survivin expression, instead of MDR1, ABCG2 and AKT1 expression. Therefore a survivin inhibitor can be a new target for effective treatment of CD133{sup +} colon cancer. - Highlights: • We evaluate the role of CD133 in chemoresistance of colon cancer. • We compared the chemoresistance of CD133{sup +} cells and siRNA-induced CD133{sup −} cells. • CD133 had little to no effect on MDR1, ABCG2 and AKT1 expression. • Survivin expression and chemoresistance were increased in CD133{sup +} colon cancer cells.« less

Activating β-catenin signaling in CD133-positive dermal papilla cells increases hair inductivity

PubMed Central

Zhou, Linli; Yang, Kun; Xu, Mingang; Andl, Thomas; Millar, Sarah; Boyce, Steven; Zhang, Yuhang

2016-01-01

Bioengineering hair follicles using cells isolated from human tissue remains as a difficult task. Dermal papilla (DP) cells are known to guide the growth and cycling activities of hair follicles by interacting with keratinocytes. However, DP cells quickly lose their inductivity during in vitro passaging. Rodent DP cell cultures need external addition of chemical factors, including WNT and BMP molecules, to maintain the hair inductive property. CD133 is expressed by a small subpopulation of DP cells that are capable of inducing hair follicle formation in vivo. We report here that expression of a stabilized form of β-catenin promoted clonal growth of CD133-positive (CD133+) DP cells in in vitro three-dimensional hydrogel culture while maintaining expression of DP markers, including alkaline phosphatase (AP), CD133, and Integrin α8. After a two-week in vitro culture, cultured CD133+ DP cells with up-regulated β-catenin activity led to an accelerated in vivo hair growth in reconstituted skin than control cells. Further analysis showed that matrix cell proliferation and differentiation were significantly promoted in hair follicles when β-catenin signaling was upregulated in CD133+ DP cells. Our data highlight an important role for β-catenin signaling in promoting the inductive capability of CD133+ DP cells for in vitro expansion and in vivo hair follicle regeneration, which could potentially be applied to cultured human DP cells. PMID:27312243

Expression profiles of cancer stem cell markers: CD133, CD44, Musashi-1 and EpCAM in the cardiac mucosa-Barrett's esophagus-early esophageal adenocarcinoma-advanced esophageal adenocarcinoma sequence.

PubMed

Mokrowiecka, Anna; Veits, Lothar; Falkeis, Christina; Musial, Jacek; Kordek, Radzislaw; Lochowski, Mariusz; Kozak, Jozef; Wierzchniewska-Lawska, Agnieszka; Vieth, Michael; Malecka-Panas, Ewa

2017-03-01

Barrett's esophagus (BE), which develops as a result of gastroesophageal reflux disease, is a preneoplastic condition for esophageal adenocarcinoma (EAC). A new hypothesis suggests that cancer is a disease of stem cells, however, their expression and pathways in BE - EAC sequence are not fully elucidated yet. We used a panel of putative cancer stem cells markers to identify stem cells in consecutive steps of BE-related cancer progression. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded blocks from 58 patients with normal cardiac mucosa (n=5), BE (n=14), early EAC (pT1) from mucosal resection (n=17) and advanced EAC (pT1-T4) from postoperative specimens (n=22). Expression of the CD133, CD44, Musashi-1 and EpCAM was analyzed using respective monoclonal antibodies. All markers showed a heterogeneous expression pattern, mainly at the base of the crypts of Barrett's epithelium and EAC, with positive stromal cells in metaplastic and dysplastic lesions. Immuno-expression of EpCAM, CD44 and CD133 in cardiac mucosa was significantly lower (mean immunoreactivity score (IRS)=1.2; 0.0; 0.4; respectively) compared to their expression in Barrett's metaplasia (mean IRS=4.3; 0.14; 0.7; respectively), in early adenocarcinoma (mean IRS=4.4; 0.29; 1.3; respectively) and in advanced adenocarcinoma (mean IRS=6.6; 0.7; 2.7; respectively) (p<0.05). On the contrary, Musashi-1 expression was higher in BE and early ADC compared to GM and advanced ADC (NS). Our results suggest that the stem cells could be present in premalignant lesions. EpCAM, CD44 and CD133 expression could be candidate markers for BE progression, whereas Musashi-1 may be a marker of the small intestinal features of Barrett's mucosa. Copyright © 2016 Elsevier GmbH. All rights reserved.

Colon cancer stem cells dictate tumor growth and resist cell death by production of interleukin-4.

PubMed

Todaro, Matilde; Alea, Mileidys Perez; Di Stefano, Anna B; Cammareri, Patrizia; Vermeulen, Louis; Iovino, Flora; Tripodo, Claudio; Russo, Antonio; Gulotta, Gaspare; Medema, Jan Paul; Stassi, Giorgio

2007-10-11

A novel paradigm in tumor biology suggests that cancer growth is driven by stem-like cells within a tumor. Here, we describe the identification and characterization of such cells from colon carcinomas using the stem cell marker CD133 that accounts around 2% of the cells in human colon cancer. The CD133(+) cells grow in vitro as undifferentiated tumor spheroids, and they are both necessary and sufficient to initiate tumor growth in immunodeficient mice. Xenografts resemble the original human tumor maintaining the rare subpopulation of tumorigenic CD133(+) cells. Further analysis revealed that the CD133(+) cells produce and utilize IL-4 to protect themselves from apoptosis. Consistently, treatment with IL-4Ralpha antagonist or anti-IL-4 neutralizing antibody strongly enhances the antitumor efficacy of standard chemotherapeutic drugs through selective sensitization of CD133(+) cells. Our data suggest that colon tumor growth is dictated by stem-like cells that are treatment resistant due to the autocrine production of IL-4.

A CD133-expressing murine liver oval cell population with bilineage potential.

PubMed

Rountree, C Bart; Barsky, Lora; Ge, Shundi; Zhu, Judy; Senadheera, Shantha; Crooks, Gay M

2007-10-01

Although oval cells are postulated to be adult liver stem cells, a well-defined phenotype of a bipotent liver stem cell remains elusive. The heterogeneity of cells within the oval cell fraction has hindered lineage potential studies. Our goal was to identify an enriched population of bipotent oval cells using a combination of flow cytometry and single cell gene expression in conjunction with lineage-specific liver injury models. Expression of cell surface markers on nonparenchymal, nonhematopoietic (CD45-) cells were characterized. Cell populations were isolated by flow cytometry for gene expression studies. 3,5-Diethoxycarbonyl-1,4-dihydrocollidine toxic injury induced cell cycling and expansion specifically in the subpopulation of oval cells in the periportal zone that express CD133. CD133+CD45- cells expressed hepatoblast and stem cell-associated genes, and single cells coexpressed both hepatocyte and cholangiocyte-associated genes, indicating bilineage potential. CD133+CD45- cells proliferated in response to liver injury. Following toxic hepatocyte damage, CD133+CD45- cells demonstrated upregulated expression of the hepatocyte gene Albumin. In contrast, toxic cholangiocyte injury resulted in upregulation of the cholangiocyte gene Ck19. After 21-28 days in culture, CD133+CD45- cells continued to generate cells of both hepatocyte and cholangiocyte lineages. Thus, CD133 expression identifies a population of oval cells in adult murine liver with the gene expression profile and function of primitive, bipotent liver stem cells. In response to lineage-specific injury, these cells demonstrate a lineage-appropriate genetic response. Disclosure of potential conflicts of interest is found at the end of this article.

Visualization of CD44 and CD133 in Normal Pancreas and Pancreatic Ductal Adenocarcinomas

PubMed Central

Immervoll, Heike; Hoem, Dag; Steffensen, Ole Johnny; Miletic, Hrvoje; Molven, Anders

2011-01-01

Tumor-initiating cells of pancreatic ductal adenocarcinoma (PDAC) have been isolated based on expression of either CD133 or CD44. The authors aimed to visualize pancreatic cells simultaneously expressing both these cell surface markers by employing the same antibodies commonly used in cell-sorting studies. Normal and diseased pancreatic tissue, including 51 PDAC cases, were analyzed. CD44 and CD133 expression was determined by immunohistochemical double staining on formalin-fixed material and subcellular protein distribution evaluated by immunofluorescence/confocal microscopy. In the normal pancreas, CD44 and CD133 were coexpressed in the centroacinar regions but in non-overlapping subcellular compartments. As expected, CD44 was found mainly basolaterally, whereas CD133 was present on the apical/endoluminal membrane. This was also the case in chronically inflamed/atrophic pancreatic tissue and in PDAC. In some malignant ducts, CD44 was found at the apical cell membrane adjacent to but never overlapping with CD133 expression. CD44 level was significantly associated with the patient’s lymph node status. In conclusion, a CD44+/CD133+ cell population does exist in the normal and neoplastic pancreas. The preferentially centroacinar localization of the doubly positive cells in the normal parenchyma suggests that this population could be of particular interest in attempts to identify tumor-initiating cells in PDAC. This article contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. PMID:21411814

Physiologic oxygen concentration enhances the stem-like properties of CD133+ human glioblastoma cells in vitro.

PubMed

McCord, Amy M; Jamal, Muhammad; Shankavaram, Uma T; Shankavarum, Uma T; Lang, Frederick F; Camphausen, Kevin; Tofilon, Philip J

2009-04-01

In vitro investigations of tumor stem-like cells (TSC) isolated from human glioblastoma (GB) surgical specimens have been done primarily at an atmospheric oxygen level of 20%. To determine whether an oxygen level more consistent with in situ conditions affects their stem cell-like characteristics, we compared GB TSCs grown under conditions of 20% and 7% oxygen. Growing CD133(+) cells sorted from three GB neurosphere cultures at 7% O(2) reduced their doubling time and increased the self-renewal potential as reflected by clonogenicity. Furthermore, at 7% oxygen, the cultures exhibited an enhanced capacity to differentiate along both the glial and neuronal pathways. As compared with 20%, growth at 7% oxygen resulted in an increase in the expression levels of the neural stem cell markers CD133 and nestin as well as the stem cell markers Oct4 and Sox2. In addition, whereas hypoxia inducible factor 1alpha was not affected in CD133(+) TSCs grown at 7% O(2), hypoxia-inducible factor 2alpha was expressed at higher levels as compared with 20% oxygen. Gene expression profiles generated by microarray analysis revealed that reducing oxygen level to 7% resulted in the up-regulation and down-regulation of a significant number of genes, with more than 140 being commonly affected among the three CD133(+) cultures. Furthermore, Gene Ontology categories up-regulated at 7% oxygen included those associated with stem cells or GB TSCs. Thus, the data presented indicate that growth at the more physiologically relevant oxygen level of 7% enhances the stem cell-like phenotype of CD133(+) GB cells.

Significance of CD133 positive cells in four novel HPV-16 positive cervical cancer-derived cell lines and biopsies of invasive cervical cancer.

PubMed

Javed, Shifa; Sharma, Bal Krishan; Sood, Swati; Sharma, Sanjeev; Bagga, Rashmi; Bhattacharyya, Shalmoli; Rayat, Charan Singh; Dhaliwal, Lakhbir; Srinivasan, Radhika

2018-04-02

Cervical cancer is a major cause of cancer-related mortality in women in the developing world. Cancer Stem cells (CSC) have been implicated in treatment resistance and metastases development; hence understanding their significance is important. Primary culture from tissue biopsies of invasive cervical cancer and serial passaging was performed for establishing cell lines. Variable Number Tandem Repeat (VNTR) assay was performed for comparison of cell lines with their parental tissue. Tumorsphere and Aldefluor assays enabled isolation of cancer stem cells (CSC); immunofluorescence and flow cytometry were performed for their surface phenotypic expression in cell lines and in 28 tissue samples. Quantitative real-time PCR for stemness and epithelial-mesenchymal transition (EMT) markers, MTT cytotoxicity assay, cell cycle analysis and cell kinetic studies were performed. Four low-passage novel cell lines designated RSBS-9, - 14 and - 23 from squamous cell carcinoma and RSBS-43 from adenocarcinoma of the uterine cervix were established. All were HPV16+. VNTR assay confirmed their uniqueness and derivation from respective parental tissue. CSC isolated from these cell lines showed CD133 + phenotype. In tissue samples of untreated invasive cervical cancer, CD133 + CSCs ranged from 1.3-23% of the total population which increased 2.8-fold in radiation-resistant cases. Comparison of CD133 + with CD133 - bulk population cells revealed increased tumorsphere formation and upregulation of stemness and epithelial-mesenchymal transition (EMT) markers with no significant difference in cisplatin sensitivity. Low-passage cell lines developed would serve as models for studying tumor biology. Cancer Stem Cells in cervical cancer display CD133 + phenotype and are increased in relapsed cases and hence should be targeted for achieving remission.

Differentiation potential of human CD133 positive hematopoietic stem cells into motor neuron- like cells, in vitro.

PubMed

Moghaddam, Sepideh Alavi; Yousefi, Behnam; Sanooghi, Davood; Faghihi, Faezeh; Hayati Roodbari, Nasim; Bana, Nikoo; Joghataei, Mohammad Taghi; Pooyan, Paria; Arjmand, Babak

2017-12-01

Spinal cord injuries and motor neuron-related disorders impact on life of many patients around the world. Since pharmacotherapy and surgical approaches were not efficient to regenerate these types of defects; stem cell therapy as a good strategy to restore the lost cells has become the focus of interest among the scientists. Umbilical cord blood CD133 + hematopoietic stem cells (UCB- CD133 + HSCs) with self- renewal property and neural lineage differentiation capacity are ethically approved cell candidate for use in regenerative medicine. In this regard the aim of this study was to quantitatively evaluate the capability of these cells to differentiate into motor neuron-like cells (MNL), in vitro. CD133 + HSCs were isolated from human UCB using MACS system. After cell characterization using flow cytometry, the cells were treated with a combination of Retinoic acid, Sonic hedgehog, Brain derived neurotrophic factor, and B27 through a 2- step procedure for two weeks. The expression of MN-specific markers was examined using qRT- PCR, flow cytometry and immunocytochemistry. By the end of the two-week differentiation protocol, CD133 + cells acquired unipolar MNL morphology with thin and long neurites. The expression of Isl-1(62.15%), AChE (41.83%), SMI-32 (21.55%) and Nestin (17.46%) was detected using flow cytometry and immunocytochemistry. The analysis of the expression of PAX6, ISL-1, ACHE, CHAT and SMI-32 revealed that MNLs present these neural markers at levels comparable with undifferentiated cells. In Conclusion Human UCB- CD133 + HSCs are remarkably potent cell candidates to transdifferentiate into motor neuron-like cells, in vitro. Copyright © 2017. Published by Elsevier B.V.

Clonal Growth of Dermal Papilla Cells in Hydrogels Reveals Intrinsic Differences between Sox2-Positive and -Negative Cells In Vitro and In Vivo

PubMed Central

Driskell, Ryan R; Juneja, Vikram R; Connelly, John T; Kretzschmar, Kai; Tan, David W -M; Watt, Fiona M

2012-01-01

In neonatal mouse skin, two types of dermal papilla (DP) are distinguished by Sox2 expression: CD133+Sox2+ DP are associated with guard/awl/auchene hairs, whereas CD133+Sox2− DP are associated with zigzag (ZZ) hairs. We describe a three-dimensional hydrogel culture system that supports clonal growth of CD133+Sox2+, CD133+Sox2−, and CD133−Sox2− (non-DP) neonatal dermal cells. All three cell populations formed spheres that expressed the DP markers alkaline phosphatase, α8 integrin, and CD133. Nevertheless, spheres formed by CD133− cells did not efficiently support hair follicle formation in skin reconstitution assays. In the presence of freshly isolated P2 dermal cells, CD133+Sox2+ and CD133+Sox2− spheres contributed to the DP of both AA and ZZ hairs. Hair type did not correlate with sphere size. Sox2 expression was maintained in culture, but not induced significantly in Sox2− cells in vitro or in vivo, suggesting that Sox2+ cells are a distinct cellular lineage. Although Sox2+ cells were least efficient at forming spheres, they had the greatest ability to contribute to DP and non-DP dermis in reconstituted skin. As the culture system supports clonal growth of DP cells and maintenance of distinct DP cell types, it will be useful for further analysis of intrinsic and extrinsic signals controlling DP function. PMID:22189784

The Pluripotent Stem-Cell Marker Alkaline Phosphatase is Highly Expressed in Refractory Glioblastoma with DNA Hypomethylation.

PubMed

Iwadate, Yasuo; Suganami, Akiko; Tamura, Yutaka; Matsutani, Tomoo; Hirono, Seiichiro; Shinozaki, Natsuki; Hiwasa, Takaki; Takiguchi, Masaki; Saeki, Naokatsu

2017-02-01

Hypomethylation of genomic DNA induces stem-cell properties in cancer cells and contributes to the treatment resistance of various malignancies. To examine the correlation between the methylation status of stem-cell-related genes and the treatment outcomes in patients with glioblastoma (GBM). The genome-wide DNA methylation status was determined using HumanMethylation450 BeadChips, and the methylation status was compared between a group of patients with good prognosis (survival > 4 yr) and a group with poor prognosis (survival < 1 yr). Immunohistochemistry for proteins translated from hypomethylated genes, including alkaline phosphatase (ALPL), CD133, and CD44, was performed in 70 GBMs and 60 oligodendroglial tumors. The genomic DNA in refractory GBM was more hypomethylated than in GBM from patients with relatively long survival (P = .0111). Stem-cell-related genes including ALPL, CD133, and CD44 were also significantly hypomethylated. A validation study using immunohistochemistry showed that DNA hypomethylation was strongly correlated with high protein expression of ALPL, CD133, and CD44. GBM patients with short survival showed high expression of these stem-cell markers. Multivariate analysis confirmed that co-expression of ALPL + CD133 or ALPL + CD44 was a strong predictor of short survival. Anaplastic oligodendroglial tumors without isocitrate dehydrogenase 1 mutation were significantly correlated with high ALPL expression and poor survival. Accumulation of stem-cell properties due to aberrant DNA hypomethylation is associated with the refractory nature of GBM. Copyright © 2017 by the Congress of Neurological Surgeons

Heterodimeric bispecific single chain variable fragments (scFv) killer engagers (BiKEs) enhance NK-cell activity against CD133+ colorectal cancer cells

PubMed Central

JU, Schmohl; MK, Gleason; PR, Dougherty; JS, Miller; DA, Vallera

2015-01-01

Background Natural killer (NK) cells are potent cytotoxic lymphocytes that play a critical role in tumor immunosurveillance and control. Cancer stem cells (CSC) initiate and sustain tumor cell growth, mediate drug refractory cancer relapse and express the well-known surface marker CD133. Methods DNA fragments from two fully humanized single chain fragment variable (scFv) antibody recognizing CD16 on NK-cells and CD133 on CSC were genetically spliced forming a novel drug, 16 × 133 BiKE that simultaneously recognizes these antigen to facilitate an immunologic synapse. The anti-CD133 was created using a fusion protein prepared by fusing DNA fragments encoding the two extracellular domains of CD133. Immunization of mice with the resulting fusion protein generated an unique antibody that recognized the molecular framework and was species cross-reactive. Results In vitro 51chromium release cytotoxicity assays at both high and low effector:target ratios demonstrated the ability of the heterodimeric biological drug to greatly enhance NK-cell killing of human Caco-2 colorectal carcinoma cells known to overexpress CD133. The tumor associated antigen specificity of the drug for CD133 even enhanced NK-cell cytotoxicity against the NK-resistant human Burkitt's lymphoma Daudi cell line, which has less than 5% CD133 surface expression. Flow cytometry analysis revealed increases in NK-cell degranulation and Interferon-γ production upon co-culture with Caco-2 targets in the presence of the drug. Conclusion These studies demonstrate that the innate immune system can be effectively recruited to kill CSC using bispecific antibodies targeting CD133, and that this anti-CD133 scFv may be useful in this bispecific platform or, perhaps, in the design of more complex trispecific molecules for carcinoma therapy. PMID:26566946

CD133: Enhancement of Bone Healing by Local Transplantation of Peripheral Blood Cells in a Biologically Delayed Rat Osteotomy Model

PubMed Central

Preininger, Bernd; Duda, Georg; Gerigk, Hinnerk; Bruckner, Jonas; Ellinghaus, Agnes; Sass, F. Andrea; Perka, Carsten; Schmidt-Bleek, Katharina; Dienelt, Anke

2013-01-01

Sufficient angiogenesis is crucial during tissue regeneration and therefore also pivotal in bone defect healing. Recently, peripheral blood derived progenitor cells have been identified to have in addition to their angiogenic potential also osteogenic characteristics, leading to the hypothesis that bone regeneration could be stimulated by local administration of these cells. The aim of this study was to evaluate the angiogenic potential of locally administered progenitor cells to improve bone defect healing. Cells were separated from the peripheral blood of donor animals using the markers CD34 and CD133. Results of the in vitro experiments confirmed high angiogenic potential in the CD133(+) cell group. CD34(+) and CD133(+) cells were tested in an in vivo rat femoral defect model of delayed healing for their positive effect on the healing outcome. An increased callus formation and higher bone mineral density of callus tissue was found after the CD133(+) cell treatment compared to the group treated with CD34(+) cells and the control group without cells. Histological findings confirmed an increase in vessel formation and mineralization at day 42 in the osteotomy gap after CD133(+) cell transplantation. The higher angiogenic potential of CD133(+) cells from the in vitro experients therefore correlates with the in vivo data. This study demonstrates the suitability of angiogenic precursors to further bone healing and gives an indication that peripheral blood is a promising source for progenitor cells circumventing the problems associated with bone marrow extraction. PMID:23457441

Residential Proximity to Major Roadways Is Associated With Increased Levels of AC133+ Circulating Angiogenic Cells.

PubMed

DeJarnett, Natasha; Yeager, Ray; Conklin, Daniel J; Lee, Jongmin; O'Toole, Timothy E; McCracken, James; Abplanalp, Wes; Srivastava, Sanjay; Riggs, Daniel W; Hamzeh, Ihab; Wagner, Stephen; Chugh, Atul; DeFilippis, Andrew; Ciszewski, Tiffany; Wyatt, Brad; Becher, Carrie; Higdon, Deirdre; Ramos, Kenneth S; Tollerud, David J; Myers, John A; Rai, Shesh N; Shah, Jasmit; Zafar, Nagma; Krishnasamy, Sathya S; Prabhu, Sumanth D; Bhatnagar, Aruni

2015-11-01

Previous studies have shown that residential proximity to a roadway is associated with increased cardiovascular disease risk. Yet, the nature of this association remains unclear, and its effect on individual cardiovascular disease risk factors has not been assessed. The objective of this study was to determine whether residential proximity to roadways influences systemic inflammation and the levels of circulating angiogenic cells. In a cross-sectional study, cardiovascular disease risk factors, blood levels of C-reactive protein, and 15 antigenically defined circulating angiogenic cell populations were measured in participants (n=316) with moderate-to-high cardiovascular disease risk. Attributes of roadways surrounding residential locations were assessed using geographic information systems. Associations between road proximity and cardiovascular indices were analyzed using generalized linear models. Close proximity (<50 m) to a major roadway was associated with lower income and higher rates of smoking but not C-reactive protein levels. After adjustment for potential confounders, the levels of circulating angiogenic cells in peripheral blood were significantly elevated in people living in close proximity to a major roadway (CD31(+)/AC133(+), AC133(+), CD34(+)/AC133(+), and CD34(+)/45(dim)/AC133(+) cells) and positively associated with road segment distance (CD31(+)/AC133(+), AC133(+), and CD34(+)/AC133(+) cells), traffic intensity (CD31(+)/AC133(+) and AC133(+) cells), and distance-weighted traffic intensity (CD31(+)/34(+)/45(+)/AC133(+) cells). Living close to a major roadway is associated with elevated levels of circulating cells positive for the early stem marker AC133(+). This may reflect an increased need for vascular repair. Levels of these cells in peripheral blood may be a sensitive index of cardiovascular injury because of residential proximity to roadways. © 2015 American Heart Association, Inc.

Peripheral blood CD34+KDR+ endothelial progenitor cells are determinants of subclinical atherosclerosis in a middle-aged general population.

PubMed

Fadini, Gian Paolo; Coracina, Anna; Baesso, Ilenia; Agostini, Carlo; Tiengo, Antonio; Avogaro, Angelo; de Kreutzenberg, Saula Vigili

2006-09-01

Disruption of the endothelial layer is the first step in the atherogenic process. Experimental studies have shown that endothelial progenitor cells (EPCs) are involved in endothelial homeostasis and repair. Conversely, EPC depletion has been demonstrated in the setting of established atherosclerotic diseases. With this background, we evaluated whether variations in the number of EPCs are associated with subclinical atherosclerosis in healthy subjects. Carotid intima-media thickness (IMT), high-sensitive C-reactive protein (hsCRP), levels of circulating EPCs, and cardiovascular risk were compared in 137 healthy subjects. Six subpopulations of progenitor cells were determined by flow cytometry on the basis of the surface expression of CD34, CD133, and KDR antigens: CD34(+), CD133(+), CD34(+)CD133(+), CD34(+)KDR(+), CD133(+)KDR(+), and CD34(+)CD133(+)KDR(+). Among different antigenic profiles of EPCs, only CD34(+)KDR(+) cells were significantly reduced in subjects with increased IMT. Specifically, CD34(+)KDR(+) cells were inversely correlated with IMT, even after adjustment for hsCRP and 10-year Framingham risk and independently of other cardiovascular parameters. Depletion of CD34(+)KDR(+) EPCs is an independent predictor of early subclinical atherosclerosis in healthy subjects and may provide additional information beyond classic risk factors and inflammatory markers.

CD133-positive dermal papilla-derived Wnt ligands regulate postnatal hair growth

PubMed Central

Zhou, Linli; Yang, Kun; Carpenter, April; Lang, Richard A.; Andl, Thomas; Zhang, Yuhang

2016-01-01

Active Wnt/β-catenin signaling in the dermal papilla (DP) is required for postnatal hair cycling. In addition, maintenance of the hair-inducing ability of DP cells in vitro requires external addition of Wnt molecules. However, whether DP cells are a critical source of Wnt ligands and induce both autocrine and paracrine signaling cascades to promote adult hair follicle growth and regeneration remains elusive. To address this question, we generated an animal model that allows inducible ablation of Wntless (Wls), a transmembrane Wnt exporter protein, in CD133-positive (CD133+) DP cells. CD133+ cells have been shown to be a specific subpopulation of cells in the DP, which possesses the hair-inducing capability. Here, we show that ablation of Wls expression in CD133+ DP cells results in a shortened period of postnatal hair growth. Mutant hair follicles were unable to enter full anagen (hair growth stage) and progressed toward a rapid regression. Notably, reduced size of the DP and decreased expression of anagen DP marker, versican, were observed in hair follicles when CD133+ DP cells lost Wls expression. Further analysis showed that Wls-deficient CD133+ DP cells led to reduced proliferation and differentiation in matrix keratinocytes and melanocytes that are needed for the generation of the hair follicle structure and a pigmented hair shaft. These findings clearly demonstrate that Wnt ligands produced by CD133+ DP cells play an important role in postnatal hair growth by maintaining the inductivity of DP cells and mediating the signaling cross-talk between the mesenchyme and the epithelial compartment. PMID:27462123

Walnut Phenolic Extract and Its Bioactive Compounds Suppress Colon Cancer Cell Growth by Regulating Colon Cancer Stemness.

PubMed

Lee, Jisoo; Kim, Yoo-Sun; Lee, JaeHwan; Heo, Seung Chul; Lee, Kook Lae; Choi, Sang-Woon; Kim, Yuri

2016-07-21

Walnut has been known for its health benefits, including anti-cardiovascular disease and anti-oxidative properties. However, there is limited evidence elucidating its effects on cancer stem cells (CSCs) which represent a small subset of cancer cells that provide resistance against chemotherapy. This study aimed to evaluate the anti-CSCs potential of walnut phenolic extract (WPE) and its bioactive compounds, including (+)-catechin, chlorogenic acid, ellagic acid, and gallic acid. In the present study, CD133⁺CD44⁺ cells were isolated from HCT116 cells using fluorescence-activated cell sorting (FACS) and then treated with WPE. As a result, survival of the CD133⁺CD44⁺ HCT116 cells was inhibited and cell differentiation was induced by WPE. In addition, WPE down-regulated the CSC markers, CD133, CD44, DLK1, and Notch1, as well as the β-catenin/p-GSK3β signaling pathway. WPE suppressed the self-renewal capacity of CSCs. Furthermore, the WPE exhibited stronger anti-CSC effects than its individual bioactive compounds. Finally, the WPE inhibited specific CSC markers in primary colon cancer cells isolated from primary colon tumor. These results suggest that WPE can suppress colon cancer by regulating the characteristics of colon CSCs.

Walnut Phenolic Extract and Its Bioactive Compounds Suppress Colon Cancer Cell Growth by Regulating Colon Cancer Stemness

PubMed Central

Lee, Jisoo; Kim, Yoo-Sun; Lee, JaeHwan; Heo, Seung Chul; Lee, Kook Lae; Choi, Sang-Woon; Kim, Yuri

2016-01-01

Walnut has been known for its health benefits, including anti-cardiovascular disease and anti-oxidative properties. However, there is limited evidence elucidating its effects on cancer stem cells (CSCs) which represent a small subset of cancer cells that provide resistance against chemotherapy. This study aimed to evaluate the anti-CSCs potential of walnut phenolic extract (WPE) and its bioactive compounds, including (+)-catechin, chlorogenic acid, ellagic acid, and gallic acid. In the present study, CD133+CD44+ cells were isolated from HCT116 cells using fluorescence-activated cell sorting (FACS) and then treated with WPE. As a result, survival of the CD133+CD44+ HCT116 cells was inhibited and cell differentiation was induced by WPE. In addition, WPE down-regulated the CSC markers, CD133, CD44, DLK1, and Notch1, as well as the β-catenin/p-GSK3β signaling pathway. WPE suppressed the self-renewal capacity of CSCs. Furthermore, the WPE exhibited stronger anti-CSC effects than its individual bioactive compounds. Finally, the WPE inhibited specific CSC markers in primary colon cancer cells isolated from primary colon tumor. These results suggest that WPE can suppress colon cancer by regulating the characteristics of colon CSCs. PMID:27455311

A Paracrine Mechanism Accelerating Expansion of Human Induced Pluripotent Stem Cell-Derived Hepatic Progenitor-Like Cells

PubMed Central

Tsuruya, Kota; Chikada, Hiromi; Ida, Kinuyo; Anzai, Kazuya; Kagawa, Tatehiro; Inagaki, Yutaka; Mine, Tetsuya

2015-01-01

Hepatic stem/progenitor cells in liver development have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In this study, we focused on the cell surface molecules of human induced pluripotent stem (iPS) cell-derived hepatic progenitor-like cells (HPCs) and analyzed how these molecules modulate expansion of these cells. Human iPS cells were differentiated into immature hepatic lineage cells by cytokines. In addition to hepatic progenitor markers (CD13 and CD133), the cells were coimmunostained for various cell surface markers (116 types). The cells were analyzed by flow cytometry and in vitro colony formation culture with feeder cells. Twenty types of cell surface molecules were highly expressed in CD13+CD133+ cells derived from human iPS cells. Of these molecules, CD221 (insulin-like growth factor receptor), which was expressed in CD13+CD133+ cells, was quickly downregulated after in vitro expansion. The proliferative ability was suppressed by a neutralizing antibody and specific inhibitor of CD221. Overexpression of CD221 increased colony-forming ability. We also found that inhibition of CD340 (erbB2) and CD266 (fibroblast growth factor-inducible 14) signals suppressed proliferation. In addition, both insulin-like growth factor (a ligand of CD221) and tumor necrosis factor-like weak inducer of apoptosis (a ligand of CD266) were provided by feeder cells in our culture system. This study revealed the expression profiles of cell surface molecules in human iPS cell-derived HPCs and that the paracrine interactions between HPCs and other cells through specific receptors are important for proliferation. PMID:25808356

A Paracrine Mechanism Accelerating Expansion of Human Induced Pluripotent Stem Cell-Derived Hepatic Progenitor-Like Cells.

PubMed

Tsuruya, Kota; Chikada, Hiromi; Ida, Kinuyo; Anzai, Kazuya; Kagawa, Tatehiro; Inagaki, Yutaka; Mine, Tetsuya; Kamiya, Akihide

2015-07-15

Hepatic stem/progenitor cells in liver development have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In this study, we focused on the cell surface molecules of human induced pluripotent stem (iPS) cell-derived hepatic progenitor-like cells (HPCs) and analyzed how these molecules modulate expansion of these cells. Human iPS cells were differentiated into immature hepatic lineage cells by cytokines. In addition to hepatic progenitor markers (CD13 and CD133), the cells were coimmunostained for various cell surface markers (116 types). The cells were analyzed by flow cytometry and in vitro colony formation culture with feeder cells. Twenty types of cell surface molecules were highly expressed in CD13(+)CD133(+) cells derived from human iPS cells. Of these molecules, CD221 (insulin-like growth factor receptor), which was expressed in CD13(+)CD133(+) cells, was quickly downregulated after in vitro expansion. The proliferative ability was suppressed by a neutralizing antibody and specific inhibitor of CD221. Overexpression of CD221 increased colony-forming ability. We also found that inhibition of CD340 (erbB2) and CD266 (fibroblast growth factor-inducible 14) signals suppressed proliferation. In addition, both insulin-like growth factor (a ligand of CD221) and tumor necrosis factor-like weak inducer of apoptosis (a ligand of CD266) were provided by feeder cells in our culture system. This study revealed the expression profiles of cell surface molecules in human iPS cell-derived HPCs and that the paracrine interactions between HPCs and other cells through specific receptors are important for proliferation.

Enrichment of prostate cancer stem-like cells from human prostate cancer cell lines by culture in serum-free medium and chemoradiotherapy.

PubMed

Wang, Lei; Huang, Xing; Zheng, Xinmin; Wang, Xinghuan; Li, Shiwen; Zhang, Lin; Yang, Zhonghua; Xia, Zhiping

2013-01-01

The discovery of rare subpopulations of cancer stem cells (CSCs) has created a new focus in cancer research. As CSCs demonstrate resistance to chemoradiation therapy relative to other cancer cells, this allows the enrichment of CSC populations by killing apoptosis-susceptible cancer cells. In this study, three commonly used human prostate cancer (PCa) cell lines (DU145, PC-3 and LNCaP) were examined for their expression of the putative stem cell markers CD133 and CD44 via flow cytometric analysis. Under normal culture conditions, CD133(+)/CD44(+) cells were only present in the DU145 cell line, and comprised only a minor percentage (0.1% ± 0.01%) of the total population. However, the proportion of these CD133(+)/CD44(+) prostate CSCs could be increased in these cell lines via culture in serum-free medium (SFM), or through chemotherapy or radiotherapy. Indeed, after culture in SFM, the proportion of CD133(+)/CD44(+) cells in DU145 and PC-3 had increased to 10.3% and 3.0%, respectively. Moreover, the proportion had increased to 9.8% enriched by chemotherapy and 3.5% by radiotherapy in DU145. Colony-formation tests, cell invasion assays, and tumor xenografts in BALB/c nude mice were used to evaluate the stem cell properties of CD133(+)/CD44(+) PCa cells that were isolated via fluorescence-activated cell sorting (FACS). CD133(+)/CD44(+) cells had an enhanced colony-formation capability and invasive ability in vitro, and displayed greater tumorigenic properties in vivo. These results demonstrate the presence of CD133(+)/CD44(+) prostate CSCs in established PCa cell lines and that populations of these cells can be enriched by culture in SFM or chemoradiotherapy. Finding novel therapies to override chemoradiation resistance in the prostate CSCs is the key to improve long-term results in PCa management.

Endothelial progenitor cells in chronic obstructive pulmonary disease and emphysema

PubMed Central

Tracy, Russell P.; Parikh, Megha A.; Hoffman, Eric A.; Shimbo, Daichi; Austin, John H. M.; Smith, Benjamin M.; Hueper, Katja; Vogel-Claussen, Jens; Lima, Joao; Gomes, Antoinette; Watson, Karol; Kawut, Steven; Barr, R. Graham

2017-01-01

Endothelial injury is implicated in the pathogenesis of COPD and emphysema; however the role of endothelial progenitor cells (EPCs), a marker of endothelial cell repair, and circulating endothelial cells (CECs), a marker of endothelial cell injury, in COPD and its subphenotypes is unresolved. We hypothesized that endothelial progenitor cell populations would be decreased in COPD and emphysema and that circulating endothelial cells would be increased. Associations with other subphenotypes were examined. The Multi-Ethnic Study of Atherosclerosis COPD Study recruited smokers with COPD and controls age 50–79 years without clinical cardiovascular disease. Endothelial progenitor cell populations (CD34+KDR+ and CD34+KDR+CD133+ cells) and circulating endothelial cells (CD45dimCD31+CD146+CD133-) were measured by flow cytometry. COPD was defined by standard spirometric criteria. Emphysema was assessed qualitatively and quantitatively on CT. Full pulmonary function testing and expiratory CTs were measured in a subset. Among 257 participants, both endothelial progenitor cell populations, and particularly CD34+KDR+ endothelial progenitor cells, were reduced in COPD. The CD34+KDR+CD133+ endothelial progenitor cells were associated inversely with emphysema extent. Both endothelial progenitor cell populations were associated inversely with extent of panlobular emphysema and positively with diffusing capacity. Circulating endothelial cells were not significantly altered in COPD but were inversely associated with pulmonary microvascular blood flow on MRI. There was no consistent association of endothelial progenitor cells or circulating endothelial cells with measures of gas trapping. These data provide evidence that endothelial repair is impaired in COPD and suggest that this pathological process is specific to emphysema. PMID:28291826

Expression of CD44 and CD29 by PEComa cells suggests their possible origin of mesenchymal stem cells.

PubMed

Liu, Ruixue; Jia, Wei; Zou, Hong; Wang, Xinhua; Ren, Yan; Zhao, Jin; Wang, Lianghai; Li, Man; Qi, Yan; Shen, Yaoyuan; Liang, Weihua; Jiang, Jinfang; Sun, Zhenzhu; Pang, Lijuan; Li, Feng

2015-01-01

Perivascular epithelioid cell tumor (PEComa) is a rare mesenchymal tumor composed of histologically and immunohistochemically distinctive perivascular epithelioid cells. The perivascular epithelioid cell (PEC) co-expresses melanocytic and muscle markers. Since no normal counterpart to the PEC has ever been identified in any normal tissue, the cell origin of these tumors is still uncertain. Although, several hypotheses have recently been advanced to explain the histogenesis of PEComa, it remains unclear. The aim of this study was to discuss whether differential expression of stem cell-associated proteins could be used to aid in determining the histogenesis of PEComa. For this purpose, we detected the immunoexpression of 5 kinds of stem cell markers on PEComas, including CD29, CD44, CD133, ALDH1, and nestin. In addition to observed histopathologic morphology, we also performed PEComa relevant clinical diagnostic markers (HMB-45, SMA, melan-A, Desmin, Ki-67, S-100 and TFE3) to identify whether they belonged to PEComas. Our study included 13 PEComa samples, and we obtained positive immunoexpression results as follows: CD29 (13/13), CD44 (8/13), ALDH1 (10/13), nestin (1/13), and CD133 (0/13). Since CD44 and CD29 are surface proteins associated with MSCs, these results suggest that PEComa might arise from MSCs. However, whether MSCs are the origin of PEComa needs to be further explored in the future.

MUC4 stabilizes HER2 expression and maintains the cancer stem cell population in ovarian cancer cells.

PubMed

Ponnusamy, Moorthy P; Seshacharyulu, Parthasarathy; Vaz, Arokiapriyanka; Dey, Parama; Batra, Surinder K

2011-04-26

Recent evidence has suggested that the capability of cancer to grow, propagate and relapse after therapy is dependent on a small subset of the cell population within the tumor, called cancer stem cells. Therefore, this subpopulation of cells needs to be targeted with different approaches by identification of unique stem-cell specific target antigens. One of the well known tumor antigens is the epithelial cell mucin MUC4, which is aberrantly expressed in ovarian cancer as compared to the normal ovary and plays a pivotal role in the aggressiveness and metastasis of ovarian cancer cells. In the present study, we aimed to analyze the cancer stem cell population in MUC4 overexpressed ovarian cancer cells. MUC4 was ectopically overexpressed in SKOV3 ovarian cancer cells. Western blot analysis was performed for MUC4, HER2, CD133, ALDH1 and Shh expression in MUC4 overexpressed cells. Confocal analysis of MUC4, HER2 and CD133 was also done in the MUC4 overexpressed cells. CD133 and Hoechst33342 dye staining was used to analyze the cancer stem cell population via FACS method in SKOV3-MUC4 cells. MUC4 overexpressed SKOV3 cells showed an increased expression of HER2 compared to control cells. MUC4 overexpression leads to increased (0.1%) side population (SP) and CD133-positive cancer stem cells compared to the control cells. Interestingly, the tumor sphere type circular colony formation was observed only in the MUC4 overexpressed ovarian cancer cells. Furthermore, the cancer stem cell marker CD133 was expressed along with MUC4 in the isolated circular colonies as analyzed by both confocal and western blot analysis. HER2 and cancer stem cell specific marker ALDH1 along with Shh, a self-renewal marker, showed increased expression in the isolated circular colonies compared to MUC4-transfected cells. These studies demonstrate that MUC4 overexpression leads to an enriched ovarian cancer stem cell population either directly or indirectly through HER2. In future, this study would be helpful for MUC4-directed therapy for the ovarian cancer stem cell population.

MUC4 stabilizes HER2 expression and maintains the cancer stem cell population in ovarian cancer cells

PubMed Central

2011-01-01

Background Recent evidence has suggested that the capability of cancer to grow, propagate and relapse after therapy is dependent on a small subset of the cell population within the tumor, called cancer stem cells. Therefore, this subpopulation of cells needs to be targeted with different approaches by identification of unique stem-cell specific target antigens. One of the well known tumor antigens is the epithelial cell mucin MUC4, which is aberrantly expressed in ovarian cancer as compared to the normal ovary and plays a pivotal role in the aggressiveness and metastasis of ovarian cancer cells. In the present study, we aimed to analyze the cancer stem cell population in MUC4 overexpressed ovarian cancer cells. Methods MUC4 was ectopically overexpressed in SKOV3 ovarian cancer cells. Western blot analysis was performed for MUC4, HER2, CD133, ALDH1 and Shh expression in MUC4 overexpressed cells. Confocal analysis of MUC4, HER2 and CD133 was also done in the MUC4 overexpressed cells. CD133 and Hoechst33342 dye staining was used to analyze the cancer stem cell population via FACS method in SKOV3-MUC4 cells. Results MUC4 overexpressed SKOV3 cells showed an increased expression of HER2 compared to control cells. MUC4 overexpression leads to increased (0.1%) side population (SP) and CD133-positive cancer stem cells compared to the control cells. Interestingly, the tumor sphere type circular colony formation was observed only in the MUC4 overexpressed ovarian cancer cells. Furthermore, the cancer stem cell marker CD133 was expressed along with MUC4 in the isolated circular colonies as analyzed by both confocal and western blot analysis. HER2 and cancer stem cell specific marker ALDH1 along with Shh, a self-renewal marker, showed increased expression in the isolated circular colonies compared to MUC4-transfected cells. Conclusion These studies demonstrate that MUC4 overexpression leads to an enriched ovarian cancer stem cell population either directly or indirectly through HER2. In future, this study would be helpful for MUC4-directed therapy for the ovarian cancer stem cell population. PMID:21521521

Light-controlled endosomal escape of the novel CD133-targeting immunotoxin AC133-saporin by photochemical internalization - A minimally invasive cancer stem cell-targeting strategy.

PubMed

Bostad, Monica; Olsen, Cathrine Elisabeth; Peng, Qian; Berg, Kristian; Høgset, Anders; Selbo, Pål Kristian

2015-05-28

The cancer stem cell (CSC) marker CD133 is an attractive target to improve antitumor therapy. We have used photochemical internalization (PCI) for the endosomal escape of the novel CD133-targeting immunotoxin AC133-saporin (PCIAC133-saporin). PCI employs an endocytic vesicle-localizing photosensitizer, which generates reactive oxygen species upon light-activation causing a rupture of the vesicle membranes and endosomal escape of entrapped drugs. Here we show that AC133-saporin co-localizes with the PCI-photosensitizer TPCS2a, which upon light exposure induces cytosolic release of AC133-saporin. PCI of picomolar levels of AC133-saporin in colorectal adenocarcinoma WiDr cells blocked cell proliferation and induced 100% inhibition of cell viability and colony forming ability at the highest light doses, whereas no cytotoxicity was obtained in the absence of light. Efficient PCI-based CD133-targeting was in addition demonstrated in the stem-cell-like, triple negative breast cancer cell line MDA-MB-231 and in the aggressive malignant melanoma cell line FEMX-1, whereas no enhanced targeting was obtained in the CD133-negative breast cancer cell line MCF-7. PCIAC133-saporin induced mainly necrosis and a minimal apoptotic response based on assessing cleavage of caspase-3 and PARP, and the TUNEL assay. PCIAC133-saporin resulted in S phase arrest and reduced LC3-II conversion compared to control treatments. Notably, co-treatment with Bafilomycin A1 and PCIAC133-saporin blocked LC3-II conversion, indicating a termination of the autophagic flux in WiDr cells. For the first time, we demonstrate laser-controlled targeting of CD133 in vivo. After only one systemic injection of AC133-saporin and TPCS2a, a strong anti-tumor response was observed after PCIAC133-saporin. The present PCI-based endosomal escape technology represents a minimally invasive strategy for spatio-temporal, light-controlled targeting of CD133+ cells in localized primary tumors or metastasis. Copyright © 2015 Elsevier B.V. All rights reserved.

CD133+CD54+CD44+ circulating tumor cells as a biomarker of treatment selection and liver metastasis in patients with colorectal cancer

PubMed Central

Wang, Cun; Huang, Qiaorong; Meng, Wentong; Yu, Yongyang; Yang, Lie; Peng, Zhihai; Hu, Jiankun; Li, Yuan; Mo, Xianming; Zhou, Zongguang

2016-01-01

Introduction Liver is the most common site of distant metastasis in colorectal cancer (CRC). Early diagnosis and appropriate treatment selection decides overall prognosis of patients. However, current diagnostic measures were basically imaging but not functional. Circulating tumor cells (CTCs) known as hold the key to understand the biology of metastatic mechanism provide a novel and auxiliary diagnostic strategy for CRC with liver metastasis (CRC-LM). Results The expression of CD133+ and CD133+CD54+CD44+ cellular subpopulations were higher in the peripheral blood of CRC-LM patients when compared with those without metastasis (P<0.001). Multivariate analysis proved the association between the expression of CD133+CD44+CD54+ cellular subpopulation and the existence of CRC-LM (P<0.001). The combination of abdominal CT/MRI, CEA and the CD133+CD44+CD54+ cellular subpopulation showed increased detection and discrimination rate for liver metastasis, with a sensitivity of 88.2% and a specificity of 92.4%. Meanwhile, it also show accurate predictive value for liver metastasis (OR=2.898, 95% C.I.1.374–6.110). Materials and Method Flow cytometry and multivariate analysis was performed to detect the expression of cancer initiating cells the correlation between cellular subpopulations and liver metastasis in patients with CRC. The receiver operating characteristic curves combined with the area under the curve were generated to compare the predictive ability of the cellular subpopulation for liver metastasis with current CT and MRI images. Conclusions The identification, expression and application of CTC subpopulations will provide an ideal cellular predictive marker for CRC liver metastasis and a potential marker for further investigation. PMID:27764803

CD133-positive dermal papilla-derived Wnt ligands regulate postnatal hair growth.

PubMed

Zhou, Linli; Yang, Kun; Carpenter, April; Lang, Richard A; Andl, Thomas; Zhang, Yuhang

2016-10-01

Active Wnt/β-catenin signaling in the dermal papilla (DP) is required for postnatal hair cycling. In addition, maintenance of the hair-inducing ability of DP cells in vitro requires external addition of Wnt molecules. However, whether DP cells are a critical source of Wnt ligands and induce both autocrine and paracrine signaling cascades to promote adult hair follicle growth and regeneration remains elusive. To address this question, we generated an animal model that allows inducible ablation of Wntless (Wls), a transmembrane Wnt exporter protein, in CD133-positive (CD133+) DP cells. CD133+ cells have been shown to be a specific subpopulation of cells in the DP, which possesses the hair-inducing capability. Here, we show that ablation of Wls expression in CD133+ DP cells results in a shortened period of postnatal hair growth. Mutant hair follicles were unable to enter full anagen (hair growth stage) and progressed toward a rapid regression. Notably, reduced size of the DP and decreased expression of anagen DP marker, versican, were observed in hair follicles when CD133+ DP cells lost Wls expression. Further analysis showed that Wls-deficient CD133+ DP cells led to reduced proliferation and differentiation in matrix keratinocytes and melanocytes that are needed for the generation of the hair follicle structure and a pigmented hair shaft. These findings clearly demonstrate that Wnt ligands produced by CD133+ DP cells play an important role in postnatal hair growth by maintaining the inductivity of DP cells and mediating the signaling cross-talk between the mesenchyme and the epithelial compartment. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

Poly(lactic-co-glycolic acid) nanoparticles conjugated with CD133 aptamers for targeted salinomycin delivery to CD133+ osteosarcoma cancer stem cells

PubMed Central

Ni, Miaozhong; Xiong, Min; Zhang, Xinchao; Cai, Guoping; Chen, Huaiwen; Zeng, Qingmin; Yu, Zuochong

2015-01-01

Background Cancer stem cells (CSCs) possess the characteristics associated with normal stem cells and are responsible for cancer initiation, recurrence, and metastasis. CD133 is regarded as a CSCs marker of osteosarcoma, which is the most common primary bone malignancy in childhood and adolescence. Salinomycin, a polyether ionophore antibiotic, has been shown to kill various CSCs, including osteosarcoma CSCs. However, salinomycin displayed poor aqueous solubility that hinders its clinical application. The objective of this study was to develop salinomycin-loaded nanoparticles to eliminate CD133+ osteosarcoma CSCs. Methods The salinomycin-loaded PEGylated poly(lactic-co-glycolic acid) nanoparticles (SAL-NP) conjugated with CD133 aptamers (Ap-SAL-NP) were developed by an emulsion/solvent evaporation method, and the targeting and cytotoxicity of Ap-SAL-NP to CD133+ osteosarcoma CSCs were evaluated. Results The nanoparticles are of desired particle size (~150 nm), drug encapsulation efficiency (~50%), and drug release profile. After 48 hours treatment of the Saos-2 CD133+ osteosarcoma cells with drugs formulated in Ap-SAL-NP, SAL-NP, and salinomycin, the concentrations needed to kill 50% of the incubated cells were found to be 2.18, 10.72, and 5.07 μg/mL, respectively, suggesting that Ap-SAL-NP could be 4.92 or 2.33 fold more effective than SAL-NP or salinomycin, respectively. In contrast, Ap-SAL-NP was as effective as SAL-NP, and less effective than salinomycin in Saos-2 CD133− cells, suggesting that Ap-SAL-NP possess specific cytotoxicity toward Saos-2 CD133+ cells. Ap-SAL-NP showed the best therapeutic effect in Saos-2 osteosarcoma xenograft mice, compared with SAL-NP or salinomycin. Significantly, Ap-SAL-NP could selectively kill CD133+ osteosarcoma CSCs both in vitro and in vivo, as reflected by the tumorsphere formation and proportion of Saos-2 CD133+ cells. Conclusion Our results suggest that CD133 is a potential target for drug delivery to osteosarcoma CSCs and that it is possible to significantly inhibit the osteosarcoma growth by killing CD133+ osteosarcoma CSCs. We demonstrated that Ap-SAL-NP have the potential to target and kill CD133+ osteosarcoma CSCs. PMID:25848270

Osteocalcin expression by circulating endothelial progenitor cells in patients with coronary atherosclerosis.

PubMed

Gössl, Mario; Mödder, Ulrike I; Atkinson, Elizabeth J; Lerman, Amir; Khosla, Sundeep

2008-10-14

This study was designed to test whether patients with coronary atherosclerosis have increases in circulating endothelial progenitor cells (EPCs) expressing an osteogenic phenotype. Increasing evidence indicates a link between bone and the vasculature, and bone marrow and circulating osteogenic cells have been identified by staining for the osteoblastic marker, osteocalcin (OCN). Endothelial progenitor cells contribute to vascular repair, but repair of vascular injury may result in calcification. Using cell surface markers (CD34, CD133, kinase insert domain receptor [KDR]) to identify EPCs, we examined whether patients with coronary atherosclerosis had increases in the percentage of EPCs expressing OCN. We studied 72 patients undergoing invasive coronary assessment: control patients (normal coronary arteries and no endothelial dysfunction, n = 21) versus 2 groups with coronary atherosclerosis-early coronary atherosclerosis (normal coronary arteries but with endothelial dysfunction, n = 22) and late coronary atherosclerosis (severe, multivessel coronary artery disease, n = 29). Peripheral blood mononuclear cells were analyzed using flow cytometry. Compared with control patients, patients with early or late coronary atherosclerosis had significant increases (approximately 2-fold) in the percentage of CD34+/KDR+ and CD34+/CD133+/KDR+ cells costaining for OCN. Even larger increases were noted in the early and late coronary atherosclerosis patients in the percentage of CD34+/CD133-/KDR+ cells costaining for OCN (5- and 2-fold, p < 0.001 and 0.05, respectively). A higher percentage of EPCs express OCN in patients with coronary atherosclerosis compared with subjects with normal endothelial function and no structural coronary artery disease. These findings have potential implications for the mechanisms of vascular calcification and for the development of novel markers for coronary atherosclerosis.

Prognostic value of cancer stem cell marker CD133 expression in pancreatic ductal adenocarcinoma (PDAC): a systematic review and meta-analysis.

PubMed

Li, Xiaoping; Zhao, Haojie; Gu, Jianchun; Zheng, Leizhen

2015-01-01

CD133 is one of the most commonly used markers of pancreatic cancer stem cells (CSCs), which are characterized by their ability for self-renewal and tumorigenicity. Although the expression of CD133 has been reported to correlate with poor prognosis of PDAC in most literatures, some controversies still exist. In this study, we aimed to investigate the correlation between CD133 expression and prognosis and clinicopathological features in PDAC. A search in the Medline, EMBASE and Chinese CNKI (China National Knowledge Infrastructure) database (up to 1 March 2015) was performed using the following keywords pancreatic cancer, CD133, AC133, prominin-1 etc. Data from eligible studies were extracted and included into meta-analysis using a random effects model. Outcomes included overall survival and various clinicopathological features. We performed a final analysis of 723 patients from 11 evaluable studies for prognostic value and 687 patients from 12 evaluable studies for clinicopathological features. Our study shows that the pooled hazard ratio (HR) of overexpression CD133 for overall survival in PDAC was 0.58 (95% confidence interval (CI): 0.49-0.67) by univariate analysis and 0.73 (95% CI: 0.52-1.03) by multivariate analysis. With respect to clinicopathological features, CD133 overexpression by immunohistochemistry (IHC) method was closely correlated with clinical TNM stage (TNM stage III+IV, OR=0.32, 95% CI: 0.19-0.54), tumor differentiation (poor differentiation, OR=0.56, 95% CI: 0.37-0.83), and lymph node metastasis (N1, 3.15, 95% CI: 1.56-6.36) in patients with PDAC. Our meta-analysis results suggest that CD133 is an efficient prognostic factor in PDAC. Overexpression of CD133 was significantly associated with clinical TNM stage, tumor differentiation and lymph node metastasis.

Expression of CD44 and CD29 by PEComa cells suggests their possible origin of mesenchymal stem cells

PubMed Central

Liu, Ruixue; Jia, Wei; Zou, Hong; Wang, Xinhua; Ren, Yan; Zhao, Jin; Wang, Lianghai; Li, Man; Qi, Yan; Shen, Yaoyuan; Liang, Weihua; Jiang, Jinfang; Sun, Zhenzhu; Pang, Lijuan; Li, Feng

2015-01-01

Background: Perivascular epithelioid cell tumor (PEComa) is a rare mesenchymal tumor composed of histologically and immunohistochemically distinctive perivascular epithelioid cells. The perivascular epithelioid cell (PEC) co-expresses melanocytic and muscle markers. Since no normal counterpart to the PEC has ever been identified in any normal tissue, the cell origin of these tumors is still uncertain. Although, several hypotheses have recently been advanced to explain the histogenesis of PEComa, it remains unclear. Methods: The aim of this study was to discuss whether differential expression of stem cell-associated proteins could be used to aid in determining the histogenesis of PEComa. For this purpose, we detected the immunoexpression of 5 kinds of stem cell markers on PEComas, including CD29, CD44, CD133, ALDH1, and nestin. In addition to observed histopathologic morphology, we also performed PEComa relevant clinical diagnostic markers (HMB-45, SMA, melan-A, Desmin, Ki-67, S-100 and TFE3) to identify whether they belonged to PEComas. Results: Our study included 13 PEComa samples, and we obtained positive immunoexpression results as follows: CD29 (13/13), CD44 (8/13), ALDH1 (10/13), nestin (1/13), and CD133 (0/13). Conclusions: Since CD44 and CD29 are surface proteins associated with MSCs, these results suggest that PEComa might arise from MSCs. However, whether MSCs are the origin of PEComa needs to be further explored in the future. PMID:26722497

CD133 expression in oral lichen planus correlated with the risk for progression to oral squamous cell carcinoma.

PubMed

Sun, Lili; Feng, Jinqiu; Ma, Lihua; Liu, Wei; Zhou, Zengtong

2013-12-01

Oral lichen planus (OLP) is a potentially malignant disorder associated with an increased risk for progression to oral squamous cell carcinoma (OSCC). The objective of this study to determine protein expression of cancer stem cell marker CD133 in tissue samples of patients with OLP and evaluate the correlation between CD133 expression and the risk of progression to OSCC. In this longitudinal case-control study, a total of 110 patients with OLP who received a mean follow-up of 56 months were enrolled, including 100 patients who did not progress to OSCC and 10 patients who had progressed to OSCC. CD133 expression was determined using immunohistochemistry in samples from these patients. Analysis of 10 cases of normal oral mucosa and 6 cases of postmalignant OSCC form previously diagnosed OLP was also performed. The results showed that CD133 expression was observed in 29% cases of nonprogressing OLP and in 80% cases of progressing OLP (P = .002). CD133 was not expressed in normal oral mucosa, but it positively expressed in the 100% cases of OSCC. Logistic regression analysis revealed that the risk of malignant progression in the patients with CD133-positive expression was significantly higher than those with CD133 negativity (odds ratio, 9.79; 95% confidence interval, 1.96-48.92; P = .005). Collectively, CD133 expression was significantly associated with malignant progression in a longitudinal series of patients with OLP. Our findings suggested that CD133 may serve as a novel candidate biomarker for risk assessment of malignant potential of OLP. © 2013.

Characterization of a Distinct Population of Circulating Human Non-Adherent Endothelial Forming Cells and Their Recruitment via Intercellular Adhesion Molecule-3

PubMed Central

Thompson, Emma J.; Barrett, Jeffrey M.; Tooley, Katie; Sen, Shaundeep; Sun, Wai Yan; Grose, Randall; Nicholson, Ian; Levina, Vitalina; Cooke, Ira; Talbo, Gert; Lopez, Angel F.; Bonder, Claudine S.

2012-01-01

Circulating vascular progenitor cells contribute to the pathological vasculogenesis of cancer whilst on the other hand offer much promise in therapeutic revascularization in post-occlusion intervention in cardiovascular disease. However, their characterization has been hampered by the many variables to produce them as well as their described phenotypic and functional heterogeneity. Herein we have isolated, enriched for and then characterized a human umbilical cord blood derived CD133+ population of non-adherent endothelial forming cells (naEFCs) which expressed the hematopoietic progenitor cell markers (CD133, CD34, CD117, CD90 and CD38) together with mature endothelial cell markers (VEGFR2, CD144 and CD31). These cells also expressed low levels of CD45 but did not express the lymphoid markers (CD3, CD4, CD8) or myeloid markers (CD11b and CD14) which distinguishes them from ‘early’ endothelial progenitor cells (EPCs). Functional studies demonstrated that these naEFCs (i) bound Ulex europaeus lectin, (ii) demonstrated acetylated-low density lipoprotein uptake, (iii) increased vascular cell adhesion molecule (VCAM-1) surface expression in response to tumor necrosis factor and (iv) in co-culture with mature endothelial cells increased the number of tubes, tubule branching and loops in a 3-dimensional in vitro matrix. More importantly, naEFCs placed in vivo generated new lumen containing vasculature lined by CD144 expressing human endothelial cells (ECs). Extensive genomic and proteomic analyses of the naEFCs showed that intercellular adhesion molecule (ICAM)-3 is expressed on their cell surface but not on mature endothelial cells. Furthermore, functional analysis demonstrated that ICAM-3 mediated the rolling and adhesive events of the naEFCs under shear stress. We suggest that the distinct population of naEFCs identified and characterized here represents a new valuable therapeutic target to control aberrant vasculogenesis. PMID:23144795

Donor-derived stem-cells and epithelial mesenchymal transition in squamous cell carcinoma in transplant recipients.

PubMed

Verneuil, Laurence; Leboeuf, Christophe; Bousquet, Guilhem; Brugiere, Charlotte; Elbouchtaoui, Morad; Plassa, Louis-François; Peraldi, Marie-Noelle; Lebbé, Celeste; Ratajczak, Philippe; Janin, Anne

2015-12-08

Skin squamous-cell-carcinoma (SCC), is the main complication in long-term kidney-transplant recipients, and it can include donor-derived cells. Preclinical models demonstrated the involvement of epithelial mesenchymal transition (EMT) in the progression of skin SCC, and the role of Snail, an EMT transcription factor, in cancer stem-cell survival and expansion.Here, we studied stem-cells and EMT expression in SCCs and concomitant actinic keratoses (AK) in kidney-transplant recipients. In SCC and AK in 3 female recipients of male kidney-transplants, donor-derived Y chromosome in epidermal stem cells was assessed using combined XY-FISH/CD133 immunostaining, and digital-droplet-PCR on laser-microdissected CD133 expressing epidermal cells.For EMT study, double immunostainings of CD133 with vimentin or snail and slug, electron microscopy and immunostainings of keratinocytes junctions were performed. Digital droplet PCR was used to check CDH1 (E-cadherin) expression level in laser-microdissected cells co-expressing CD133 and vimentin or snail and slug.The numbers of Y-chromosome were assessed using digital droplet PCR in laser-microdissected cells co-expressing CD133 and vimentin, or snail and slug, and in CD133 positive cells not expressing any EMT maker. We identified donor-derived stem-cells in basal layers and invasive areas in all skin SCCs and in concomitant AKs, but not in surrounding normal skin.The donor-derived stem-cells expressed the EMT markers, vimentin, snail and slug in SCCs but not in AKs. The expression of the EMT transcription factor, SNAI1, was higher in stem-cells when they expressed vimentin. They were located in invasive areas of SCCs. In these areas, the expressions of claudin-1 and desmoglein 1 were reduced or absent, and within the basal layer there were features of basal membrane disappearance.Donor-derived stem cells were in larger numbers in stem cells co-expressing vimentin or snail and slug than in stem cells not expressing any EMT marker. We identified here donor-derived stem cells within skin SCC in kidney-transplant recipients. They were located in invasive areas of SCC and had EMT characteristics.

Accumulation of Multipotent Progenitor Cells on Polymethylpentene Membranes During Extracorporeal Membrane Oxygenation.

PubMed

Lehle, Karla; Friedl, Lucas; Wilm, Julius; Philipp, Alois; Müller, Thomas; Lubnow, Matthias; Schmid, Christof

2016-06-01

Multipotent progenitor cells were mobilized during pediatric extracorporeal membrane oxygenation (ECMO). We hypothesize that these cells also adhered onto polymethylpentene (PMP) fibers within the membrane oxygenator (MO) during adult ECMO support. Mononuclear cells were removed from the surface of explanted PMP-MOs (n = 16). Endothelial-like outgrowth and mesenchymal-like cells were characterized by flow cytometric analysis using different surface markers. Spindle-shaped attaching cells were identified early, but without proliferative activity. After long-term cultivation palisading type or cobblestone-type outgrowth cells with high proliferative activity appeared and were characterized as (i) leukocytoid CD45+/CD31+ (CD133+/VEGFR-II+/CD90+/CD14+/CD146dim/CD105dim); (ii) endothelial-like CD45-/CD31+ (VEGF-RII+/CD146+/CD105+/CD133-/CD14-/CD90-); and (iii) mesenchymal-like cells CD45-/CD31- (CD105+/CD90+/CD133dim/VEGFR-II-/CD146-/CD14-). The distribution of the cell populations depended on the MO and cultivation time. Endothelial-like cells formed capillary-like structures and did uptake Dil-acetylated low-density lipoprotein. Endothelial- and mesenchymal-like cells adhered on the surface of PMP-MOs. Further research is needed to identify the clinical relevance of these cells. Copyright © 2015 The Authors. Artificial Organs published by Wiley Periodicals, Inc. on behalf of International Center for Artificial Organs and Transplantation (ICAOT).

Prominin‐1/CD133: Lipid Raft Association, Detergent Resistance, and Immunodetection

PubMed Central

Karbanová, Jana; Lorico, Aurelio; Bornhäuser, Martin; Fargeas, Christine A.

2017-01-01

Summary The cell surface antigen prominin‐1 (alias CD133) has gained enormous interest in the past 2 decades and given rise to debates as to its utility as a biological stem and cancer stem cell marker. Important and yet often overlooked knowledge that is pertinent to its physiological function has been generated in other systems given its more general expression beyond primitive cells. This article briefly discusses the importance of particular biochemical features of CD133 with relation to its association with membrane microdomains (lipid rafts) and proper immunodetection. It also draws attention toward the adequate use of detergents and caveats that may apply to the interpretation of the results generated. Stem Cells Translational Medicine 2018;7:155–160 PMID:29271118

Mammary stem cell and macrophage markers are enriched in normal tissue adjacent to inflammatory breast cancer.

PubMed

Reddy, Jay P; Atkinson, Rachel L; Larson, Richard; Burks, Jared K; Smith, Daniel; Debeb, Bisrat G; Ruffell, Brian; Creighton, Chad J; Bambhroliya, Arvind; Reuben, James M; Van Laere, Steven J; Krishnamurthy, Savitri; Symmans, William F; Brewster, Abenaa M; Woodward, Wendy A

2018-06-01

We hypothesized that breast tissue not involved by tumor in inflammatory breast cancer (IBC) patients contains intrinsic differences, including increased mammary stem cells and macrophage infiltration, which may promote the IBC phenotype. Normal breast parenchyma ≥ 5 cm away from primary tumors was obtained from mastectomy specimens. This included an initial cohort of 8 IBC patients and 60 non-IBC patients followed by a validation cohort of 19 IBC patients and 25 non-IBC patients. Samples were immunostained for either CD44 + CD49f + CD133/2 + mammary stem cell markers or the CD68 macrophage marker and correlated with IBC status. Quantitation of positive cells was determined using inForm software from PerkinElmer. We also examined the association between IBC status and previously published tumorigenic stem cell and IBC tumor signatures in the validation cohort samples. 8 of 8 IBC samples expressed isolated CD44 + CD49f + CD133/2 + stem cell marked cells in the initial cohort as opposed to 0/60 non-IBC samples (p = 0.001). Similarly, the median number of CD44 + CD49f + CD133/2 + cells was significantly higher in the IBC validation cohort as opposed to the non-IBC validation cohort (25.7 vs. 14.2, p = 0.007). 7 of 8 IBC samples expressed CD68 + histologically confirmed macrophages in initial cohort as opposed to 12/48 non-IBC samples (p = 0.001). In the validation cohort, the median number of CD68 + cells in IBC was 3.7 versus 1.0 in the non-IBC cohort (p = 0.06). IBC normal tissue was positively associated with a tumorigenic stem cell signature (p = 0.02) and with a 79-gene IBC signature (p < 0.001). Normal tissue from IBC patients is enriched for both mammary stem cells and macrophages and has higher association with both a tumorigenic stem cell signature and IBC-specific tumor signature. Collectively, these data suggest that IBC normal tissue differs from non-IBC tissue. Whether these changes occur before the tumor develops or is induced by tumor warrants further investigation.

[Analysis of expression of cancer stem cell-related markers in orbital adenoid cystic carcinoma].

PubMed

Lin, Ting-ting; Zhu, Li-min; He, Yan-jin; Zhang, Hong

2011-08-01

To observe the expression and distribution of CD44, CD133, and ABCG2 in orbital adenoid cystic carcinoma (ACC) and investigate their correlations with pathological type and prognosis. Two steps method of immunohistochemical staining was employed in 33 cases of paraffin embedded surgical specimens of human orbital ACC, 5 cases of recurrence samples, 3 cases of an excised lacrimal gland caused by neither inflammation nor tumor diseases, and 6 cases of xenograft tumors in nude mice. A retrospective analysis was performed on the clinical material of these patients, which were collected from Jan. 1991 to Mar. 2009. The positive rate of CD44 was 54.5% (18/33), with 76.9% (10/13) in solid type and 40.0% (8/20) in adeno-tubiform type. There was no statistically significant difference between them (P = 0.072). In solid type the positive expression cells were often located at the marginal part of the cancer nest. In the adeno-tubiform type, positive cells were often located at the outer layer of the tubiform structure (myoepithelial cells). CD44 was also expressed in normal tissues. The positive rate of CD133 was 57.6% (19/33), with 76.9% (10/13) in solid type and 45.0% (9/20) in adeno-tubiform type. There was no significant difference between them (P = 0.087). CD133 antigen was expressed in either the cytoplasm or nucleus, or expressed in both the cytoplasm and nucleus. The positive rate of ABCG2 was 21.2% (7/33), with 30.77% (4/13) in solid type and 15.0% (3/20) in adeno-tubiform type. There was no significant difference between them (P = 0.393). Many positive cells surrounded the vessels in tumor tissues. There were no significant differences between different prognosis groups of these surface phenotypes. The correlative analysis results of three surface phenotypes showed that CD44(+) cells have positive correlation with CD133(+) cells (Spearman, r(s) = 0.416, P = 0.016). In six transplanted tumors of nude mice, the number of positive cases for CD44(+), CD133(+) and ABCG2(+) was 1, 1 and 4 cases, respectively. The expression of CD44, CD133 and ABCG2 in ACC may influence the progress of ACC. However, they cannot be used as the markers for the evaluation of the prognosis of this tumor.

Investigating the Link between Molecular Subtypes of Glioblastoma, Epithelial-Mesenchymal Transition, and CD133 Cell Surface Protein

PubMed Central

Zarkoob, Hadi; Taube, Joseph H.; Singh, Sheila K.; Mani, Sendurai A.; Kohandel, Mohammad

2013-01-01

In this manuscript, we use genetic data to provide a three-faceted analysis on the links between molecular subclasses of glioblastoma, epithelial-to-mesenchymal transition (EMT) and CD133 cell surface protein. The contribution of this paper is three-fold: First, we use a newly identified signature for epithelial-to-mesenchymal transition in human mammary epithelial cells, and demonstrate that genes in this signature have significant overlap with genes differentially expressed in all known GBM subtypes. However, the overlap between genes up regulated in the mesenchymal subtype of GBM and in the EMT signature was more significant than other GBM subtypes. Second, we provide evidence that there is a negative correlation between the genetic signature of EMT and that of CD133 cell surface protein, a putative marker for neural stem cells. Third, we study the correlation between GBM molecular subtypes and the genetic signature of CD133 cell surface protein. We demonstrate that the mesenchymal and neural subtypes of GBM have the strongest correlations with the CD133 genetic signature. While the mesenchymal subtype of GBM displays similarity with the signatures of both EMT and CD133, it also exhibits some differences with each of these signatures that are partly due to the fact that the signatures of EMT and CD133 are inversely related to each other. Taken together these data shed light on the role of the mesenchymal transition and neural stem cells, and their mutual interaction, in molecular subtypes of glioblastoma multiforme. PMID:23734191

Aggressive Phenotype of Cells Disseminated via Hematogenous and Lymphatic Route in Breast Cancer Patients.

PubMed

Markiewicz, Aleksandra; Nagel, Anna; Szade, Jolanta; Majewska, Hanna; Skokowski, Jaroslaw; Seroczynska, Barbara; Stokowy, Tomasz; Welnicka-Jaskiewicz, Marzena; Zaczek, Anna J

2018-06-01

Intratumoral heterogeneity of breast cancer remains a major challenge in successful treatment. Failure of cancer therapies can also be accredited to inability to systemically eradicate cancer stem cells (CSCs). Recent evidence points to the role of epithelial-mesenchymal transition (EMT) in expanding the pool of tumor cells with CSCs features. Thus, we assessed expression level as well as heterogeneity of CSCs markers in primary tumors (PT), lymph node metastasis (LNM), and circulating tumor cells (CTCs)-enriched blood fractions in order to correlate them with signs of EMT activation as well as clinicopathological data of breast cancer patients. Level of CSCs markers (ALDH1, CD44, CD133, OCT-4, NANOG) and EMT markers was quantified in PT (N=107), LNM (N=56), and CTCs-enriched blood fractions (N=85). Heterogeneity of CSCs markers expression within each PT and LNM was assessed by calculating Gini Index. Percentage of ALDH1-positive cells was elevated in PT in comparison to LNM (P = .005). However, heterogeneity of the four CSCs markers: ALDH1 (P = .019), CD133 (P = .009), OCT-4 (P = .027), and CD44 (P < .001) was decreased in LNM. Samples classified as mesenchymal (post-EMT) showed elevated expression of CSCs markers (OCT-4 and CD44 in PT; OCT-4 in LNM; ALDH1, OCT-4, NANOG, CD44 in CTCs). Patients with mesenchymal-like CTCs had worse prognosis than patients with epithelial-like or no CTCs (P = .0025). CSCs markers are enriched in PT, LNM, and CTCs with mesenchymal features, but their heterogeneity is decreased in metastatic lymph nodes. Mesenchymal CTCs phenotype correlates with poor prognosis of the patients. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Reviewing and Updating the Major Molecular Markers for Stem Cells

PubMed Central

Calloni, Raquel; Cordero, Elvira Alicia Aparicio; Henriques, João Antonio Pêgas

2013-01-01

Stem cells (SC) are able to self-renew and to differentiate into many types of committed cells, making SCs interesting for cellular therapy. However, the pool of SCs in vivo and in vitro consists of a mix of cells at several stages of differentiation, making it difficult to obtain a homogeneous population of SCs for research. Therefore, it is important to isolate and characterize unambiguous molecular markers that can be applied to SCs. Here, we review classical and new candidate molecular markers that have been established to show a molecular profile for human embryonic stem cells (hESCs), mesenchymal stem cells (MSCs), and hematopoietic stem cells (HSCs). The commonly cited markers for embryonic ESCs are Nanog, Oct-4, Sox-2, Rex-1, Dnmt3b, Lin-28, Tdgf1, FoxD3, Tert, Utf-1, Gal, Cx43, Gdf3, Gtcm1, Terf1, Terf2, Lefty A, and Lefty B. MSCs are primarily identified by the expression of CD13, CD29, CD44, CD49e, CD54, CD71, CD73, CD90, CD105, CD106, CD166, and HLA-ABC and lack CD14, CD31, CD34, CD45, CD62E, CD62L, CD62P, and HLA-DR expression. HSCs are mainly isolated based on the expression of CD34, but the combination of this marker with CD133 and CD90, together with a lack of CD38 and other lineage markers, provides the most homogeneous pool of SCs. Here, we present new and alternative markers for SCs, along with microRNA profiles, for these cells. PMID:23336433

Glioma Stem Cells but Not Bulk Glioma Cells Upregulate IL-6 Secretion in Microglia/Brain Macrophages via Toll-like Receptor 4 Signaling.

PubMed

a Dzaye, Omar Dildar; Hu, Feng; Derkow, Katja; Haage, Verena; Euskirchen, Philipp; Harms, Christoph; Lehnardt, Seija; Synowitz, Michael; Wolf, Susanne A; Kettenmann, Helmut

2016-05-01

Peripheral macrophages and resident microglia constitute the dominant glioma-infiltrating cells. The tumor induces an immunosuppressive and tumor-supportive phenotype in these glioma-associated microglia/brain macrophages (GAMs). A subpopulation of glioma cells acts as glioma stem cells (GSCs). We explored the interaction between GSCs and GAMs. Using CD133 as a marker of stemness, we enriched for or deprived the mouse glioma cell line GL261 of GSCs by fluorescence-activated cell sorting (FACS). Over the same period of time, 100 CD133(+ )GSCs had the capacity to form a tumor of comparable size to the ones formed by 10,000 CD133(-) GL261 cells. In IL-6(-/-) mice, only tumors formed by CD133(+ )cells were smaller compared with wild type. After stimulation of primary cultured microglia with medium from CD133-enriched GL261 glioma cells, we observed an selective upregulation in microglial IL-6 secretion dependent on Toll-like receptor (TLR) 4. Our results show that GSCs, but not the bulk glioma cells, initiate microglial IL-6 secretion via TLR4 signaling and that IL-6 regulates glioma growth by supporting GSCs. Using human glioma tissue, we could confirm the finding that GAMs are the major source of IL-6 in the tumor context. © 2016 American Association of Neuropathologists, Inc. All rights reserved.

miR-1915 and miR-1225-5p Regulate the Expression of CD133, PAX2 and TLR2 in Adult Renal Progenitor Cells

PubMed Central

Costantino, Vincenzo; Curci, Claudia; Cox, Sharon N.; De Palma, Giuseppe; Schena, Francesco P.

2013-01-01

Adult renal progenitor cells (ARPCs) were recently identified in the cortex of the renal parenchyma and it was demonstrated that they were positive for PAX2, CD133, CD24 and exhibited multipotent differentiation ability. Recent studies on stem cells indicated that microRNAs (miRNAs), a class of noncoding small RNAs that participate in the regulation of gene expression, may play a key role in stem cell self-renewal and differentiation. Distinct sets of miRNAs are specifically expressed in pluripotent stem cells but not in adult tissues, suggesting a role for miRNAs in stem cell self-renewal. We compared miRNA expression profiles of ARPCs with that of mesenchymal stem cells (MSCs) and renal proximal tubular cells (RPTECs) finding distinct sets of miRNAs that were specifically expressed in ARPCs. In particular, miR-1915 and miR-1225-5p regulated the expression of important markers of renal progenitors, such as CD133 and PAX2, and important genes involved in the repair mechanisms of ARPCs, such as TLR2. We demonstrated that the expression of both the renal stem cell markers CD133 and PAX2 depends on lower miR-1915 levels and that the increase of miR-1915 levels improved capacity of ARPCs to differentiate into adipocyte-like and epithelial-like cells. Finally, we found that the low levels of miR-1225-5p were responsible for high TLR2 expression in ARPCs. Therefore, together, miR-1915 and miR-1225-5p seem to regulate important traits of renal progenitors: the stemness and the repair capacity. PMID:23861881

The requirement for freshly isolated human colorectal cancer (CRC) cells in isolating CRC stem cells.

PubMed

Fan, F; Bellister, S; Lu, J; Ye, X; Boulbes, D R; Tozzi, F; Sceusi, E; Kopetz, S; Tian, F; Xia, L; Zhou, Y; Bhattacharya, R; Ellis, L M

2015-02-03

Isolation of colorectal cancer (CRC) cell populations enriched for cancer stem cells (CSCs) may facilitate target identification. There is no consensus regarding the best methods for isolating CRC stem cells (CRC-SCs). We determined the suitability of various cellular models and various stem cell markers for the isolation of CRC-SCs. Established human CRC cell lines, established CRC cell lines passaged through mice, patient-derived xenograft (PDX)-derived cells, early passage/newly established cell lines, and cells directly from clinical specimens were studied. Cells were FAC-sorted for the CRC-SC markers CD44, CD133, and aldehyde dehydrogenase (ALDH). Sphere formation and in vivo tumorigenicity studies were used to validate CRC-SC enrichment. None of the markers studied in established cell lines, grown either in vitro or in vivo, consistently enriched for CRC-SCs. In the three other cellular models, CD44 and CD133 did not reliably enrich for stemness. In contrast, freshly isolated PDX-derived cells or early passage/newly established CRC cell lines with high ALDH activity formed spheres in vitro and enhanced tumorigenicity in vivo, whereas cells with low ALDH activity did not. PDX-derived cells, early passages/newly established CRC cell lines and cells from clinical specimen with high ALDH activity can be used to identify CRC-SC-enriched populations. Established CRC cell lines should not be used to isolate CSCs.

Glucose metabolism-targeted therapy and withaferin A are effective for epidermal growth factor receptor tyrosine kinase inhibitor-induced drug-tolerant persisters.

PubMed

Kunimasa, Kei; Nagano, Tatsuya; Shimono, Yohei; Dokuni, Ryota; Kiriu, Tatsunori; Tokunaga, Shuntaro; Tamura, Daisuke; Yamamoto, Masatsugu; Tachihara, Motoko; Kobayashi, Kazuyuki; Satouchi, Miyako; Nishimura, Yoshihiro

2017-07-01

In pathway-targeted cancer drug therapies, the relatively rapid emergence of drug-tolerant persisters (DTPs) substantially limits the overall therapeutic benefit. However, little is known about the roles of DTPs in drug resistance. In this study, we investigated the features of epidermal growth factor receptor-tyrosine kinase inhibitor-induced DTPs and explored a new treatment strategy to overcome the emergence of these DTPs. We used two EGFR-mutated lung adenocarcinoma cell lines, PC9 and II-18. They were treated with 2 μM gefitinib for 6, 12, or 24 days or 6 months. We analyzed the mRNA expression of the stem cell-related markers by quantitative RT-PCR and the expression of the cellular senescence-associated proteins. Then we sorted DTPs according to the expression pattern of CD133 and analyzed the features of sorted cells. Finally, we tried to ablate DTPs by glucose metabolism targeting therapies and a stem-like cell targeting drug, withaferin A. Drug-tolerant persisters were composed of at least two types of cells, one with the properties of cancer stem-like cells (CSCs) and the other with the properties of therapy-induced senescent (TIS) cells. The CD133 high cell population had CSC properties and the CD133 low cell population had TIS properties. The CD133 low cell population containing TIS cells showed a senescence-associated secretory phenotype that supported the emergence of the CD133 high cell population containing CSCs. Glucose metabolism inhibitors effectively eliminated the CD133 low cell population. Withaferin A effectively eliminated the CD133 high cell population. The combination of phloretin and withaferin A effectively suppressed gefitinib-resistant tumor growth. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Unravelling the mystery of stem/progenitor cells in human breast milk.

PubMed

Fan, Yiping; Chong, Yap Seng; Choolani, Mahesh A; Cregan, Mark D; Chan, Jerry K Y

2010-12-28

Mammary stem cells have been extensively studied as a system to delineate the pathogenesis and treatment of breast cancer. However, research on mammary stem cells requires tissue biopsies which limit the quantity of samples available. We have previously identified putative mammary stem cells in human breast milk, and here, we further characterised the cellular component of human breast milk. We identified markers associated with haemopoietic, mesenchymal and neuro-epithelial lineages in the cellular component of human breast milk. We found 2.6 ± 0.8% (mean ± SEM) and 0.7 ± 0.2% of the whole cell population (WCP) were found to be CD133+ and CD34+ respectively, 27.8 ± 9.1% of the WCP to be positive for Stro-1 through flow-cytometry. Expressions of neuro-ectodermal stem cell markers such as nestin and cytokeratin 5 were found through reverse-transcription polymerase chain reaction (RT-PCR), and in 4.17 ± 0.2% and 0.9 ± 0.2% of the WCP on flow-cytometry. We also established the presence of a side-population (SP) (1.8 ± 0.4% of WCP) as well as CD133+ cells (1.7 ± 0.5% of the WCP). Characterisation of the sorted SP and non-SP, CD133+ and CD133- cells carried out showed enrichment of CD326 (EPCAM) in the SP cells (50.6 ± 8.6 vs 18.1 ± 6.0, P-value  = 0.02). However, culture in a wide range of in vitro conditions revealed the atypical behaviour of stem/progenitor cells in human breast milk; in that if they are present, they do not respond to established culture protocols of stem/progenitor cells. The identification of primitive cell types within human breast milk may provide a non-invasive source of relevant mammary cells for a wide-range of applications; even the possibility of banking one's own stem cell for every breastfeeding woman.

Unravelling the Mystery of Stem/Progenitor Cells in Human Breast Milk

PubMed Central

Fan, Yiping; Chong, Yap Seng; Choolani, Mahesh A.; Cregan, Mark D.; Chan, Jerry K. Y.

2010-01-01

Background Mammary stem cells have been extensively studied as a system to delineate the pathogenesis and treatment of breast cancer. However, research on mammary stem cells requires tissue biopsies which limit the quantity of samples available. We have previously identified putative mammary stem cells in human breast milk, and here, we further characterised the cellular component of human breast milk. Methodology/Principal Findings We identified markers associated with haemopoietic, mesenchymal and neuro-epithelial lineages in the cellular component of human breast milk. We found 2.6±0.8% (mean±SEM) and 0.7±0.2% of the whole cell population (WCP) were found to be CD133+ and CD34+ respectively, 27.8±9.1% of the WCP to be positive for Stro-1 through flow-cytometry. Expressions of neuro-ectodermal stem cell markers such as nestin and cytokeratin 5 were found through reverse-transcription polymerase chain reaction (RT-PCR), and in 4.17±0.2% and 0.9±0.2% of the WCP on flow-cytometry. We also established the presence of a side-population (SP) (1.8±0.4% of WCP) as well as CD133+ cells (1.7±0.5% of the WCP). Characterisation of the sorted SP and non-SP, CD133+ and CD133- cells carried out showed enrichment of CD326 (EPCAM) in the SP cells (50.6±8.6 vs 18.1±6.0, P-value  = 0.02). However, culture in a wide range of in vitro conditions revealed the atypical behaviour of stem/progenitor cells in human breast milk; in that if they are present, they do not respond to established culture protocols of stem/progenitor cells. Conclusions/Significance The identification of primitive cell types within human breast milk may provide a non-invasive source of relevant mammary cells for a wide-range of applications; even the possibility of banking one's own stem cell for every breastfeeding woman. PMID:21203434

[Expression of AC133 vs. CD34 in acute childhood leukemias].

PubMed

Ebener, U; Brinkmann, A; Zotova, V; Niegemann, E; Wehner, S

2000-01-01

AC133, a newly discovered antigen on human progenitor cells, demonstrating 5-transmembranous domains is expressed by 30-60% out of all CD34+ cells. Our aim therefore was to investigate the extent of human stem-/progenitor cells expressing AC133 antigen in umbilical cord blood, peripheral blood without or following an application of granulocyte-colony stimulating factor (rhG-CSF). The main task was the investigation of bone marrow aspirates derived from children suffering from newly diagnosed acute leukemias, as well as from patients with a relapse or during a complete remission. The determination of antigen expression was done by application of flow cytometry (FACScan analysis) and the usage of newly developed monoclonal antibodies (AC133/1 and AC133/2; Miltenyi Biotec GmbH) in combination with monoclonal antibody directed against CD34-antigens (HPCA-2; BD). Our studies till now show average percentages in umbilical cord blood derived from 43 newborns about 0.294 +/- 0.165% AC133+ vs. 0.327 +/- 0.156% CD34+ hematopoietic stem-/progenitor cells (HSPC). In peripheral blood from 11 healthy donors we verified up to 0.15% CD34+ as well as AC133+ HSPC's. The concentration of progenitor cells was found to be obviously higher in peripheral blood from children with various diseases (neuroblastoma, rhabdomyosarcoma, ALL/AML) and undergoing application with rhG-CSF in order to be prepared for PBSC-transplantation. In those cases we found up to 3.51% AC133+ cells as well as slightly higher values (3.94%) for CD34 antigens. Additionally we quantified 128 bone marrow (BM) samples for AC133+ and CD34+ cells. In 10 BM samples, derived from patients without any neoplasia, the CD34+ cells were about 0.03% and 1.49%, whereas AC133 values were up to 0.64%. Bone marrow aspirates from 53 children with acute leukemias at time of diagnosis (ALL: n = 41/AML: n = 12) have been immunophenotyped and leukemic blast cells have been proved for AC133- and CD34 antigen expression. 32/41 (78%) of lymphoblastic leukemic cells showed to be positive for CD34 antigen and 24/41 (58%) demonstrated AC133 antigens. Interestingly there were 2 ALL-patients with pathological blast cells positive for AC133 but lacking of any CD34 antigens. 42% (5/12) of investigated AML patients showed CD34+ phenotype, on the other hand there were only 25% (3/12) with AC133+ phenotype. Similar values were found in relapsed patients (n = 18). In BM samples from patients during complete remission (n = 47) we could detect percentages up to 5.55% for CD34 and up to 1.25% for AC133 positive stem-/progenitor cells. Such quite high data may be explained by occasionally application of rhG-CSF therapy. Our results till now lead to the conclusion, that it seems to be useful, to recruit quantification of CD34+ HPSC by additionally detecting AC133 antigens. This new stem cell marker (AC133) may be of great value in case of autologous peripheral blood stem cell transplantation (PBSCT) because it could be an alternative to the usual CD34+ MACS selection system.

Evaluation of CD44 and CD133 as markers of liver cancer stem cells in Egyptian patients with HCV-induced chronic liver diseases versus hepatocellular carcinoma

PubMed Central

Rozeik, Mohammed Saeed; Hammam, Olfat Ali; Ali, Ali Ibrahim; Magdy, Mona; Khalil, Heba; Anas, Amgad; Abo el Hassan, Ahmed Abdelaleem; Rahim, Ali Abdel; El-Shabasy, Ahmed Ibrahim

2017-01-01

Background Cancer stem cells (CSCs) play a critical role in tumor development, progression, metastasis and recurrence. Aim To evaluate hepatic expression of CD44 and CD133 in Egyptian patients with HCV-induced chronic liver diseases and hepatocellular carcinomas (HCCs), and to assess its correlation with inflammatory activity scores, stages of fibrosis (in chronic hepatitis with or without cirrhosis) and grades of HCC. Methods This prospective case-control study was conducted on eighty subjects who attended the Tropical Diseases Department, Al-Azhar University Hospital, and in collaboration with Theodor Bilharz Research Institute (2014–2016). They were divided as follows: A) Control healthy group: Ten individuals with serologically negative HCV-Ab and HBsAg, and histopathologically normal liver, B) Seventy patients subdivided into 3 groups; Twenty subjects each, as: HCV-Ab+ non-cirrhotic, HCV-Ab+ cirrhotic and HCC. Necroinflammatory activity and fibrosis in non-neoplastic liver biopsies were scored according to the METAVIR scoring system. CD44 and CD133 immunostaining was evaluated in all groups semi-quantitatively using H score. Statistical analysis was performed by SPSS version 22, using independent-samples t-test. Results Our study showed a significant increase of mean CD44 & CD133 expression values with disease progression among the groups (p<0.05). Their expressions increased significantly with the inflammatory activity scores and stages of fibrosis, reaching the highest values in A3F4 score compared to A1F1 (p<0.05). Moreover, there was a significant increase of their expressions across HCC grades (p<0.05), however with no significant correlation with focal lesions size. Conclusion CSCs clusters exhibiting CD133+ and/or CD44+ profiles were identified in chronic hepatitis, liver cirrhosis and HCC. CD133 and CD44 expressions significantly corresponded to the increased inflammatory activity, fibrosis stages and higher tumor grades. Therefore, evaluation of CD44 and CD133 expression profiles as CSCs markers in non-neoplastic liver and HCCs can help in development of novel therapeutic agents for HCC targeting and prevention. PMID:28894525

Widespread Non-Hematopoietic Tissue Distribution by Transplanted Human Progenitor Cells with High Aldehyde Dehydrogenase Activity

PubMed Central

Hess, David A.; Craft, Timothy P.; Wirthlin, Louisa; Hohm, Sarah; Zhou, Ping; Eades, William C.; Creer, Michael H.; Sands, Mark S.; Nolta, Jan A.

2011-01-01

Transplanted adult progenitor cells distribute to peripheral organs and can promote endogenous cellular repair in damaged tissues. However, development of cell-based regenerative therapies has been hindered by the lack of pre-clinical models to efficiently assess multiple organ distribution and difficulty defining human cells with regenerative function. After transplantation into beta-glucuronidase (GUSB)-deficient NOD/SCID/MPSVII mice, we characterized the distribution of lineage depleted human umbilical cord blood-derived cells purified by selection using high aldehyde dehydrogenase activity (ALDH) with CD133 co-expression. ALDHhi or ALDHhiCD133+ cells produced robust hematopoietic reconstitution, and variable levels of tissue distribution in multiple organs. GUSB+ donor cells that co-expressed human (HLA-A,B,C) and hematopoietic (CD45+) cell surface markers were the primary cell phenotype found adjacent to the vascular beds of several tissues, including islet and ductal regions of mouse pancreata. In contrast, variable phenotypes were detected in the chimeric liver, with HLA+/CD45+ cells demonstrating robust GUSB expression adjacent to blood vessels, and CD45−/HLA− cells with diluted GUSB expression predominant in the liver parenchyma. However, true non-hematopoietic human (HLA+/CD45−) cells were rarely detected in other peripheral tissues, suggesting that these GUSB+/HLA−/CD45− cells in the liver were a result of downregulated human surface marker expression in vivo, not widespread seeding of non-hematopoietic cells. However, relying solely on continued expression of cell surface markers, as employed in traditional xenotransplantation models, may underestimate true tissue distribution. ALDH-expressing progenitor cells demonstrated widespread and tissue-specific distribution of variable cellular phenotypes, indicating that these adult progenitor cells should be explored in transplantation models of tissue damage. PMID:18055447

Evidence of In Vitro Preservation of Human Nephrogenesis at the Single-Cell Level.

PubMed

Pode-Shakked, Naomi; Gershon, Rotem; Tam, Gal; Omer, Dorit; Gnatek, Yehudit; Kanter, Itamar; Oriel, Sarit; Katz, Guy; Harari-Steinberg, Orit; Kalisky, Tomer; Dekel, Benjamin

2017-07-11

During nephrogenesis, stem/progenitor cells differentiate and give rise to early nephron structures that segment to proximal and distal nephron cell types. Previously, we prospectively isolated progenitors from human fetal kidney (hFK) utilizing a combination of surface markers. However, upon culture nephron progenitors differentiated and could not be robustly maintained in vitro. Here, by culturing hFK in a modified medium used for in vitro growth of mouse nephron progenitors, and by dissection of NCAM + /CD133 - progenitor cells according to EpCAM expression (NCAM + /CD133 - /EpCAM - , NCAM + /CD133 - /EpCAM dim , NCAM + /CD133 - /EpCAM bright ), we show at single-cell resolution a preservation of uninduced and induced cap mesenchyme as well as a transitioning mesenchymal-epithelial state. Concomitantly, differentiating and differentiated epithelial lineages are also maintained. In vitro expansion of discrete stages of early human nephrogenesis in nephron stem cell cultures may be used for drug screening on a full repertoire of developing kidney cells and for prospective isolation of mesenchymal or epithelial renal lineages for regenerative medicine. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Organotypic distribution of stem cell markers in formalin-fixed brain harboring glioblastoma multiforme.

PubMed

Schrot, Rudolph J; Ma, Joyce H; Greco, Claudia M; Arias, Angelo D; Angelastro, James M

2007-11-01

The role of stem cells in the origin, growth patterns, and infiltration of glioblastoma multiforme is a subject of intense investigation. One possibility is that glioblastoma may arise from transformed stem cells in the ventricular zone. To explore this hypothesis, we examined the distribution of two stem cell markers, activating transcription factor 5 (ATF5) and CD133, in an autopsy brain specimen from an individual with glioblastoma multiforme. A 41-year-old male with a right posterior temporal glioblastoma had undergone surgery, radiation, and chemotherapy. The brain was harvested within several hours after death. After formalin fixation, sectioning, and mapping of tumor location in the gross specimen, histologic specimens were prepared from tumor-bearing and grossly normal hemispheres. Fluorescence immunohistochemistry and colorimetric staining were performed for ATF5 and CD133. Both markers co-localized to the ependymal and subependymal zones on the side of the tumor, but not in the normal hemisphere or more rostrally in the affected hemisphere. ATF5 staining was especially robust within the diseased hemisphere in histologically normal ependyma. To our knowledge, this is the first in situ demonstration of stem cell markers in whole human brain. These preliminary results support the hypothesis that some glioblastomas may arise from the neurogenic zone of the lateral ventricle. The robust staining for ATF5 and CD133 in histologically normal ventricular zone suggests that an increase in periventricular stem cell activity occurred in this patient on the side of the tumor, either as a localized response to brain injury or as an integral component of oncogenesis and tumor recurrence.

Mobilization of Peripheral Blood Stem Cells and Changes in the Concentration of Plasma Factors Influencing their Movement in Patients with Panic Disorder.

PubMed

Jabłoński, Marcin; Mazur, Jolanta Kucharska; Tarnowski, Maciej; Dołęgowska, Barbara; Pędziwiatr, Daniel; Kubiś, Ewa; Budkowska, Marta; Sałata, Daria; Wysiecka, Justyna Pełka; Kazimierczak, Arkadiusz; Reginia, Artur; Ratajczak, Mariusz Z; Samochowiec, Jerzy

2017-04-01

In this paper we examined whether stem cells and factors responsible for their movement may serve as new biological markers of anxiety disorders. The study was carried out on a group of 30 patients diagnosed with panic disorder (examined before and after treatment), compared to 30 healthy individuals forming the control group. We examined the number of circulating HSCs (hematopoetic stem cells) (Lin-/CD45 +/CD34 +) and HSCs (Lin-/CD45 +/AC133 +), the number of circulating VSELs (very small embryonic-like stem cells) (Lin-/CD45-/CD34 +) and VSELs (Lin-/CD45-/AC133 +), as well as the concentration of complement components: C3a, C5a and C5b-9, SDF-1 (stromal derived factor) and S1P (sphingosine-1-phosphate). Significantly lower levels of HSCs (Lin-/CD45 +/AC133 +) have been demonstrated in the patient group compared to the control group both before and after treatment. The level of VSELs (Lin-/CD45-/CD133 +) was significantly lower in the patient group before treatment as compared to the patient group after treatment.The levels of factors responsible for stem cell movement were significantly lower in the patient group compared to the control group before and after treatment. It was concluded that the study of stem cells and factors associated with their movement can be useful in the diagnostics of panic disorder, as well as differentiating between psychotic and anxiety disorders.

[Lentivirus-mediated RNA interference of CD133 inhibits the proliferation of CD133(+) liver cancer stem cells and increases their cisplatin chemosensitivity].

PubMed

Lan, Xi; Wang, Yong; Cao, Shu; Zou, Dongling; Li, Fang; Li, Shaolin

2012-12-01

To study the effects of CD133 suppression by lentivirus-mediated RNA interference (RNAi) on the proliferation and chemosensitivity of CD133(+) cancer stem cells (CSCs) sorted from HepG2 cell line. CD133(+) and CD133- cells were sorted from HepG2 cell line by flow cytometry, and the expression of CD133 before and after cell sorting were detected. The stem cell property of sorted CD133(+) cells were validated by sphere-forming assay in vitro and xenograft experiments in vivo. Lentivirus-mediated short hairpin RNA (shRNA) targeting CD133 were transfected into CD133(+) cells, and CD133 mRNA and protein expressions of the transfected cells were detected by RT-PCR and Western blotting, respectively. Before and after the transfection, the proliferative ability of CD133(+) cells was evaluated by colony formation assay, and the cell growth inhibition rate and apoptosis following cisplatin exposure were detected using CCK-8 assay and flow cytometry. The sorted CD133(+) cells showed a high purity of (88.74∓3.19)%, as compared with the purity of (3.36∓1.80)% before cell sorting. CD133(+) cells showed a high tumor sphere formation ability and tumorigenesis capacity compared with CD133- cells. CD133 shRNA transfection significantly inhibited CD133 mRNA and protein expressions in CD133(+) cells (P<0.01), resulting also in a significantly lowered cell proliferative ability (P<0.01) and an increased growth inhibition rate (P<0.01) and obviously increased cell apoptosis (P<0.05) after cisplatin exposure. Lentivirus-mediated RNAi for CD133 suppression inhibits the proliferation of CD133(+) liver cancer stem cells and increases their chemosensitivity to cisplatin.

PKI-587 and sorafenib alone and in combination on inhibition of liver cancer stem cell proliferation

PubMed Central

Gedaly, Roberto; Galuppo, Roberto; Musgrave, Yolanda; Angulo, Paul; Hundley, Jonathan; Shah, Malay; Daily, Michael F.; Chen, Changguo; Cohen, Donald A.; Spear, Brett T.; Evers, B. Mark

2015-01-01

Background Deregulated Ras/Raf/mitogen-activated protein kinase and PI3 K/AKT/mTOR signaling pathways are significant in hepatocellular carcinoma proliferation (HCC). In this study we evaluated differences in the antiproliferative effect of dual PI3 K/Akt/mTOR and Ras/Raf/mitogen-activated protein kinase inhibition of non liver cancer stem cell lines (PLC and HuH7) and liver cancer stem cell (LCSC) lines (CD133, CD44, CD24, and aldehyde dehydrogenase 1-positive cells). Materials and methods Flow cytometry was performed on the resulting tumors to identify the LCSC markers CD133, CD44, CD24, and aldehyde dehydrogenase 1. Methylthiazol tetrazolium assay was used to assess cellular proliferation. Finally, a Western blot assay was used to evaluate for inhibition of specific enzymes in these two signaling pathways. Results Using flow cytometry, we found that LCSC contain 64.4% CD133 + cells, 83.2% CD44 + cells, and 96.4% CD24 + cells. PKI-587 and sorafenib caused inhibiton of LCSC and HCC cell proliferation. PLC cells were more sensitive to PKI-587 than LCSC or Huh7 (P < 0.001). Interestingly, HuH7 cells were more sensitive to sorafenib than LCSC or PLC cells. Additionally, combination therapy with PKI-587 and sorafenib caused significantly more inhibition than monotherapy in HuH7, PLC, and LCSC. Using the methylthiazol tetrazolium assay, we found that the LCSC proliferation was inhibited with sorafenib monotherapy 39% at 5 μM (P < 0.001; n = 12) and 67% by PKI-587 at 0.1 μM (P = 0.002, n = 12) compared with control. The combination of PKI-587 and sorafenib, however, synergistically inhibited LCSC proliferation by 86% (P = 0.002; n = 12). Conclusions LCSC (CD133+, CD44+, CD24+) were able to develop very aggressive tumors with low cell concentrations at 4 to 6 wk. Cells CD133+, CD44+, CD24+ demonstrated at least moderate resistance to therapy in vitro. The combination of PKI-587 and sorafenib was better than either drug alone at inhibiting of LCSC and on HCC cell proliferation. PMID:23769634

PKI-587 and sorafenib alone and in combination on inhibition of liver cancer stem cell proliferation.

PubMed

Gedaly, Roberto; Galuppo, Roberto; Musgrave, Yolanda; Angulo, Paul; Hundley, Jonathan; Shah, Malay; Daily, Michael F; Chen, Changguo; Cohen, Donald A; Spear, Brett T; Evers, B Mark

2013-11-01

Deregulated Ras/Raf/mitogen-activated protein kinase and PI3 K/AKT/mTOR signaling pathways are significant in hepatocellular carcinoma proliferation (HCC). In this study we evaluated differences in the antiproliferative effect of dual PI3 K/Akt/mTOR and Ras/Raf/mitogen-activated protein kinase inhibition of non liver cancer stem cell lines (PLC and HuH7) and liver cancer stem cell (LCSC) lines (CD133, CD44, CD24, and aldehyde dehydrogenase 1-positive cells). Flow cytometry was performed on the resulting tumors to identify the LCSC markers CD133, CD44, CD24, and aldehyde dehydrogenase 1. Methylthiazol tetrazolium assay was used to assess cellular proliferation. Finally, a Western blot assay was used to evaluate for inhibition of specific enzymes in these two signaling pathways. Using flow cytometry, we found that LCSC contain 64.4% CD133 + cells, 83.2% CD44 + cells, and 96.4% CD24 + cells. PKI-587 and sorafenib caused inhibiton of LCSC and HCC cell proliferation. PLC cells were more sensitive to PKI-587 than LCSC or Huh7 (P < 0.001). Interestingly, HuH7 cells were more sensitive to sorafenib than LCSC or PLC cells. Additionally, combination therapy with PKI-587 and sorafenib caused significantly more inhibition than monotherapy in HuH7, PLC, and LCSC. Using the methylthiazol tetrazolium assay, we found that the LCSC proliferation was inhibited with sorafenib monotherapy 39% at 5 μM (P < 0.001; n = 12) and 67% by PKI-587 at 0.1 μM (P = 0.002, n = 12) compared with control. The combination of PKI-587 and sorafenib, however, synergistically inhibited LCSC proliferation by 86% (P = 0.002; n = 12). LCSC (CD133+, CD44+, CD24+) were able to develop very aggressive tumors with low cell concentrations at 4 to 6 wk. Cells CD133+, CD44+, CD24+, which demonstrated at least moderate resistance to therapy in vitro. The combination of PKI-587 and sorafenib was better than either drug alone at inhibiting of LCSC and on HCC cell proliferation. Copyright © 2013 Elsevier Inc. All rights reserved.

Identification of Three Molecular and Functional Subtypes in Canine Hemangiosarcoma through Gene Expression Profiling and Progenitor Cell Characterization

PubMed Central

Gorden, Brandi H.; Kim, Jong-Hyuk; Sarver, Aaron L.; Frantz, Aric M.; Breen, Matthew; Lindblad-Toh, Kerstin; O'Brien, Timothy D.; Sharkey, Leslie C.; Modiano, Jaime F.; Dickerson, Erin B.

2015-01-01

Canine hemangiosarcomas have been ascribed to an endothelial origin based on histologic appearance; however, recent findings suggest that these tumors may arise instead from hematopoietic progenitor cells. To clarify this ontogenetic dilemma, we used genome-wide expression profiling of primary hemangiosarcomas and identified three distinct tumor subtypes associated with angiogenesis (group 1), inflammation (group 2), and adipogenesis (group 3). Based on these findings, we hypothesized that a common progenitor may differentiate into the three tumor subtypes observed in our gene profiling experiment. To investigate this possibility, we cultured hemangiosarcoma cell lines under normal and sphere-forming culture conditions to enrich for tumor cell progenitors. Cells from sphere-forming cultures displayed a robust self-renewal capacity and exhibited genotypic, phenotypic, and functional properties consistent with each of the three molecular subtypes seen in primary tumors, including expression of endothelial progenitor cell (CD133 and CD34) and endothelial cell (CD105, CD146, and αvβ3 integrin) markers, expression of early hematopoietic (CD133, CD117, and CD34) and myeloid (CD115 and CD14) differentiation markers in parallel with increased phagocytic capacity, and acquisition of adipogenic potential. Collectively, these results suggest that canine hemangiosarcomas arise from multipotent progenitors that differentiate into distinct subtypes. Improved understanding of the mechanisms that determine the molecular and phenotypic differentiation of tumor cells in vivo could change paradigms regarding the origin and progression of endothelial sarcomas. PMID:24525151

The role of CD133 in the identification and characterisation of tumour-initiating cells in non-small-cell lung cancer.

PubMed

Tirino, Virginia; Camerlingo, Rosa; Franco, Renato; Malanga, Donatella; La Rocca, Antonello; Viglietto, Giuseppe; Rocco, Gaetano; Pirozzi, Giuseppe

2009-09-01

Emerging evidence suggests that specific sub-populations of cancer cells with stem cell characteristics within the bulk of tumours are implicated in the pathogenesis of heterogeneous malignant tumours. The cells that drive tumour growth have been denoted cancer-initiating cells or cancer stem cells (hereafter CSCs). CSCs have been isolated initially from leukaemias and subsequently from several solid tumours including brain, breast, prostate, colon and lung cancer. This study aimed at isolating and characterising the population of tumour-initiating cells in non-small-cell lung cancer (NSCLC). Specimens of NSCLC obtained from 89 patients undergoing tumour resection at the Cancer National Institute of Naples were analysed. Three methods to isolate the tumour-initiating cells were used: (1) flow cytometry analysis for identification of positive cells for surface markers such as CD24, CD29, CD31, CD34, CD44, CD133 and CD326; (2) Hoechst 33342 dye exclusion test for the identification of a side-population characteristic for the presence of stem cells; (3) non-adherent culture condition able to form spheres with stem cell-like characteristics. Definition of the tumourigenic potential of the cells through soft agar assay and injection into NOD/SCID mice were used to functionally define (in vitro and in vivo) putative CSCs isolated from NSCLC samples. Upon flow cytometry analysis of NSCLC samples, CD133-positive cells were found in 72% of 89 fresh specimens analysed and, on average, represented 6% of the total cells. Moreover, the number of CD133-positive cells increased markedly when the cells, isolated from NSCLC specimens, were grown as spheres in non-adherent culture conditions. Cells from NSCLC, grown as spheres, when assayed in soft agar, give rise to a 3.8-fold larger number of colonies in culture and are more tumourigenic in non-obese diabetic (NOD)/severe combined immunodeficiency (SCID) mice compared with the corresponding adherent cells. We have isolated and characterised a population of CD133-positive cells from NSCLC that is able to give rise to spheres and can act as tumour-initiating cells.

Transmembrane protein CD9 is glioblastoma biomarker, relevant for maintenance of glioblastoma stem cells

PubMed Central

Podergajs, Neža; Motaln, Helena; Rajčević, Uroš; Verbovšek, Urška; Koršič, Marjan; Obad, Nina; Espedal, Heidi; Vittori, Miloš; Herold-Mende, Christel; Miletic, Hrvoje; Bjerkvig, Rolf; Turnšek, Tamara Lah

2016-01-01

The cancer stem cell model suggests that glioblastomas contain a subpopulation of stem-like tumor cells that reproduce themselves to sustain tumor growth. Targeting these cells thus represents a novel treatment strategy and therefore more specific markers that characterize glioblastoma stem cells need to be identified. In the present study, we performed transcriptomic analysis of glioblastoma tissues compared to normal brain tissues revealing sensible up-regulation of CD9 gene. CD9 encodes the transmembrane protein tetraspanin which is involved in tumor cell invasion, apoptosis and resistance to chemotherapy. Using the public REMBRANDT database for brain tumors, we confirmed the prognostic value of CD9, whereby a more than two fold up-regulation correlates with shorter patient survival. We validated CD9 gene and protein expression showing selective up-regulation in glioblastoma stem cells isolated from primary biopsies and in primary organotypic glioblastoma spheroids as well as in U87-MG and U373 glioblastoma cell lines. In contrast, no or low CD9 gene expression was observed in normal human astrocytes, normal brain tissue and neural stem cells. CD9 silencing in three CD133+ glioblastoma cell lines (NCH644, NCH421k and NCH660h) led to decreased cell proliferation, survival, invasion, and self-renewal ability, and altered expression of the stem-cell markers CD133, nestin and SOX2. Moreover, CD9-silenced glioblastoma stem cells showed altered activation patterns of the Akt, MapK and Stat3 signaling transducers. Orthotopic xenotransplantation of CD9-silenced glioblastoma stem cells into nude rats promoted prolonged survival. Therefore, CD9 should be further evaluated as a target for glioblastoma treatment. PMID:26573230

Wharton's jelly mesenchymal stromal cells have contrasting effects on proliferation and phenotype of cancer stem cells from different subtypes of lung cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vulcano, Francesca, E-mail: francesca.vulcano@iss.it; Milazzo, Luisa, E-mail: luisa.milazzo@iss.it; Ciccarelli, Carmela, E-mail: carmela.ciccarelli@univaq.it

Studies on the role of multipotent mesenchymal stromal cells (MSC) on tumor growth have reported both a tumor promoting and a suppressive effect. The aim of the present study was to determine the effect of MSC isolated from Wharton's jelly of umbilical cord (WJMSC) on lung cancer stem cells (LCSC) derived from human lung tumors: two adenocarcinomas (AC) and two squamous cell carcinomas (SCC). LCSC derived from SCC and AC expressed, to varying extents, the more relevant stem cell markers. The effect of WJMSC on LCSC was investigated in vitro using conditioned medium (WJ-CM): a proliferation increase in AC-LCSC wasmore » observed, with an increase in the ALDH+ and in the CD133+ cell population. By contrast, WJ-CM hampered the growth of SCC-LCSC, with an increase in the pre-G1 phase indicating the induction of apoptosis. Furthermore, the ALDH+ and CD133+ population was also reduced. In vivo, subcutaneous co-transplantation of AC-LCSC/WJMSC generated larger tumors than AC-LCSC alone, characterized by an increased percentage of CD133+ and CD166+ cells. By contrast, co-transplantation of WJMSC and SCC-LCSC did not affect the tumor size. Our results strongly suggest that WJMSC exert, both in vitro and in vivo, contrasting effects on LCSC derived from different lung tumor subtypes. - Highlights: • CM from WJMSC induces apoptosis of SCC-LCSC and reduction of ALDH+ and CD133+ cells. • Specificity of SCC-LCSC inhibition by WJ-CM is proved by the use of a CM from NHDF. • WJ-CM enhance AC-LCSC proliferation and increase CD133+ and ALDH+ cell fractions. • Coinjection of WJMSC with AC-LCSC increase tumor growth with SCC-LCSC has no effect.« less

Various types of stem cells, including a population of very small embryonic-like stem cells, are mobilized into peripheral blood in patients with Crohn's disease.

PubMed

Marlicz, Wojciech; Zuba-Surma, Ewa; Kucia, Magda; Blogowski, Wojciech; Starzynska, Teresa; Ratajczak, Mariusz Z

2012-09-01

Developmentally early cells, including hematopoietic stem progenitor cells (HSPCs), mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs), and very small embryonic-like stem cells (VSELs), are mobilized into peripheral blood (PB) in response to tissue/organ injury. We sought to determine whether these cells are mobilized into PB in patients with Crohn's disease (CD). Twenty-five patients with active CD, 20 patients in clinical remission, and 25 age-matched controls were recruited and PB samples harvested. The circulating CD133+/Lin-/CD45+ and CD34+/Lin-/CD45+ cells enriched for HSPCs, CD105+/STRO-1+/CD45- cells enriched for MSCs, CD34+/KDR+/CD31+/CD45-cells enriched for EPCs, and small CXCR4+CD34+CD133+ subsets of Lin-CD45- cells that correspond to the population of VSELs were counted by fluorescence-activated cell sorting (FACS) and evaluated by direct immunofluorescence staining for pluripotency embryonic markers and by reverse-transcription polymerase chain reaction (RT-PCR) for expression of messenger (m)RNAs for a panel of genes expressed in intestine epithelial stem cells. The serum concentration of factors involved in stem cell trafficking, such as stromal derived factor-1 (SDF-1), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) were measured by enzyme-linked immunosorbent assay (ELISA). Our data indicate that cells expressing markers for MSCs, EPCs, and small Oct-4+Nanog+SSEA-4+CXCR4+lin-CD45- VSELs are mobilized into PB in CD. The mobilized cells also expressed at the mRNA level genes playing a role in development and regeneration of gastrointestinal epithelium. All these changes were accompanied by increased serum concentrations of VEGF and HGF. CD triggers the mobilization of MSCs, EPCs, and VSELs, while the significance and precise role of these mobilized cells in repair of damaged intestine requires further study. Copyright © 2012 Crohn's & Colitis Foundation of America, Inc.

Prognostic role of CD133 expression in colorectal cancer: a meta-analysis.

PubMed

Wang, Ke; Xu, Jianjun; Zhang, Junshu; Huang, Jian

2012-12-05

CD133 has been identified as a putative cancer stem cell marker in colorectal cancer (CRC). However, the clinical and prognostic significance of CD133 in CRC remains controversial. Publications were identified which assessed the clinical or prognostic significance of CD133 in CRC up to October 2012. A meta-analysis was performed to clarify the association between CD133 expression and clinical outcomes. A total of 12 studies met the inclusion criteria, and comprised 3652 cases. Analysis of these data showed that CD133 was not significantly associated with the depth of CRC invasion (odds ratio [OR] = 1.44, 95% confidence interval [CI]: 0.77-2.68, Z = 1.15, P = 0.252) or tumor differentiation (OR = 0.63, 95% CI: 0.28-1.46, Z = -1.06, P = 0.286). Also, there was no statistically significant association of CD133 with lymph node metastasis (OR = 1.16, 95% CI: 0.87-1.54, Z = 1.05, P = 0.315) or lymphatic invasion (OR = 1.08, 95% CI: 0.81-1.43, Z = 0.53, P = 0.594). However, in identified studies, overexpression of CD133 was highly correlated with reduced overall survival (relative risk [RR] = 2.14, 95% CI: 1.45-3.17, Z = 3.81, P = 0.0001). CD133 may play an important role in the progression of CRC, and overexpression of CD133 is closely related with poorer patient survival. If these findings are confirmed by well-designed prospective studies, CD133 may be a useful maker for clinical applications.

Overexpression of microRNA-194 suppresses the epithelial-mesenchymal transition in targeting stem cell transcription factor Sox3 in endometrial carcinoma stem cells.

PubMed

Gong, Baolan; Yue, Yan; Wang, Renxiao; Zhang, Yi; Jin, Quanfang; Zhou, Xi

2017-06-01

The epithelial-mesenchymal transition is the key process driving cancer metastasis. MicroRNA-194 inhibits epithelial-mesenchymal transition in several cancers and its downregulation indicates a poor prognosis in human endometrial carcinoma. Self-renewal factor Sox3 induces epithelial-mesenchymal transition at gastrulation and is also involved epithelial-mesenchymal transition in several cancers. We intended to determine the roles of Sox3 in inducing epithelial-mesenchymal transition in endometrial cancer stem cells and the possible role of microRNA-194 in controlling Sox3 expression. Firstly, we found that Sox3 and microRNA-194 expressions were associated with the status of endometrial cancer stem cells in a panel of endometrial carcinoma tissue, the CD133+ cell was higher in tumorsphere than in differentiated cells, and overexpression of microRNA-194 would decrease CD133+ cell expression. Silencing of Sox3 in endometrial cancer stem cell upregulated the epithelial marker E-cadherin, downregulated the mesenchymal marker vimentin, and significantly reduced cell invasion in vitro; overexpression of Sox3 reversed these phenotypes. Furthermore, we discovered that the expression of Sox3 was suppressed by microRNA-194 through direct binding to the Sox3 3'-untranslated region. Ectopic expression of microRNA-194 in endometrial cancer stem cells induced a mesenchymal-epithelial transition by restoring E-cadherin expression, decreasing vimentin expression, and inhibiting cell invasion in vitro. Moreover, overexpression of microRNA-194 inhibited endometrial cancer stem cell invasion or metastasis in vivo by injection of adenovirus microRNA-194. These findings demonstrate the novel mechanism by which Sox3 contributes to endometrial cancer stem cell invasion and suggest that repression of Sox3 by microRNA-194 may have therapeutic potential to suppress endometrial carcinoma metastasis. The cancer stem cell marker, CD133, might be the surface marker of endometrial cancer stem cell.

Hepatitis B virus PreS1 facilitates hepatocellular carcinoma development by promoting appearance and self-renewal of liver cancer stem cells.

PubMed

Liu, Zhixin; Dai, Xuechen; Wang, Tianci; Zhang, Chengcheng; Zhang, Wenjun; Zhang, Wei; Zhang, Qi; Wu, Kailang; Liu, Fang; Liu, Yingle; Wu, Jianguo

2017-08-01

Hepatitis B virus (HBV) is a major etiologic agent of hepatocellular carcinoma (HCC). However, the molecular mechanism by which HBV infection contributes to HCC development is not fully understood. Here, we initially showed that HBV stimulates the production of cancer stem cells (CSCs)-related markers (CD133, CD117 and CD90) and CSCs-related genes (Klf4, Sox2, Nanog, c-Myc and Oct4) and facilitates the self-renewal of CSCs in human hepatoma cells. Cellular and clinical studies revealed that HBV facilitates hepatoma cell growth and migration, enhances white blood cell (WBC) production in the sera of patients, stimulates CD133 and CD117 expression in HCC tissues, and promotes the CSCs generation of human hepatoma cells and clinical cancer tissues. Detailed studies revealed that PreS1 protein of HBV is required for HBV-mediated CSCs generation. PreS1 activates CD133, CD117 and CD90 expression in normal hepatocyte derived cell line (L02) and human hepatoma cell line (HepG2 and Huh-7); facilitates L02 cells migration, growth and sphere formation; and finally enhances the abilities of L02 cells and HepG2 cells to induce tumorigeneses in nude mice. Thus, PreS1 acts as a new oncoprotein to play a key role in the appearance and self-renewal of CSCs during HCC development. Copyright © 2017 Elsevier B.V. All rights reserved.

Stem cell marker prominin-1/AC133 is expressed in duct cells of the adult human pancreas.

PubMed

Lardon, Jessy; Corbeil, Denis; Huttner, Wieland B; Ling, Zhidong; Bouwens, Luc

2008-01-01

Many efforts are spent in identifying stem cells in adult pancreas because these could provide a source of beta cells for cell-based therapy of type 1 diabetes. Prominin-1, particularly its specific glycosylation-dependent AC133 epitope, is expressed on stem/progenitor cells of various human tissues and can be used to isolate them. We, therefore, examined its expression in adult human pancreas. To detect prominin-1 protein, monoclonal antibody CD133/1 (AC133 clone), which recognizes the AC133 epitope, and the alphahE2 antiserum, which is directed against the human prominin-1 polypeptide, were used. Prominin-1 RNA expression was analyzed by real-time polymerase chain reaction. We report that all duct-lining cells of the pancreas express prominin-1. Most notably, the cells that react with the alphahE2 antiserum also react with the AC133 antibody. After isolation and culture of human exocrine cells, we found a relative increase in prominin-1 expression both at protein and RNA expression level, which can be explained by an enrichment of cells with ductal phenotype in these cultures. Our data show that pancreatic duct cells express prominin-1 and surprisingly reveal that its particular AC133 epitope is not an exclusive stem and progenitor cell marker.

Platelet released growth factors boost expansion of bone marrow derived CD34(+) and CD133(+) endothelial progenitor cells for autologous grafting.

PubMed

Lippross, Sebastian; Loibl, Markus; Hoppe, Sven; Meury, Thomas; Benneker, Lorin; Alini, Mauro; Verrier, Sophie

2011-01-01

Stem cell based autologous grafting has recently gained mayor interest in various surgical fields for the treatment of extensive tissue defects. CD34(+) and CD133(+) cells that can be isolated from the pool of bone marrow mononuclear cells (BMC) are capable of differentiating into mature endothelial cells in vivo. These endothelial progenitor cells (EPC) are believed to represent a major portion of the angiogenic regenerative cells that are released from bone marrow when tissue injury has occurred. In recent years tissue engineers increasingly looked at the process of vessel neoformation because of its major importance for successful cell grafting to replace damaged tissue. Up to now one of the greatest problems preventing a clinical application is the large scale of expansion that is required for such purpose. We established a method to effectively enhance the expansion of CD34(+) and CD133(+) cells by the use of platelet-released growth factors (PRGF) as a media supplement. PRGF were prepared from thrombocyte concentrates and used as a media supplement to iscove's modified dulbecco's media (IMDM). EPC were immunomagnetically separated from human bone morrow monocyte cells and cultured in IMDM + 10% fetal calf serum (FCS), IMDM + 5%, FCS + 5% PRGF and IMDM + 10% PRGF. We clearly demonstrate a statistically significant higher and faster cell proliferation rate at 7, 14, 21, and 28 days of culture when both PRGF and FCS were added to the medium as opposed to 10% FCS or 10% PRGF alone. The addition of 10% PRGF to IMDM in the absence of FCS leads to a growth arrest from day 14 on. In histochemical, immunocytochemical, and gene-expression analysis we showed that angiogenic and precursor markers of CD34(+) and CD133(+) cells are maintained during long-term culture. In summary, we established a protocol to boost the expansion of CD34(+) and CD133(+) cells. Thereby we provide a technical step towards the clinical application of autologous stem cell transplantation.

Skeletal myogenic differentiation of human urine-derived cells as a potential source for skeletal muscle regeneration.

PubMed

Chen, Wei; Xie, Minkai; Yang, Bin; Bharadwaj, Shantaram; Song, Lujie; Liu, Guihua; Yi, Shanhong; Ye, Gang; Atala, Anthony; Zhang, Yuanyuan

2017-02-01

Stem cells are regarded as possible cell therapy candidates for skeletal muscle regeneration. However, invasive harvesting of those cells can cause potential harvest-site morbidity. The goal of this study was to assess whether human urine-derived stem cells (USCs), obtained through non-invasive procedures, can differentiate into skeletal muscle linage cells (Sk-MCs) and potentially be used for skeletal muscle regeneration. In this study, USCs were harvested from six healthy individuals aged 25-55. Expression profiles of cell-surface markers were assessed by flow cytometry. To optimize the myogenic differentiation medium, we selected two from four different types of myogenic differentiation media to induce the USCs. Differentiated USCs were identified with myogenic markers by gene and protein expression. USCs were implanted into the tibialis anterior muscles of nude mice for 1 month. The results showed that USCs displayed surface markers with positive staining for CD24, CD29, CD44, CD73, CD90, CD105, CD117, CD133, CD146, SSEA-4 and STRO-1, and negative staining for CD14, CD31, CD34 and CD45. After myogenic differentiation, a change in morphology was observed from 'rice-grain'-like cells to spindle-shaped cells. The USCs expressed specific Sk-MC transcripts and protein markers (myf5, myoD, myosin, and desmin) after being induced with different myogenic culture media. Implanted cells expressed Sk-MC markers stably in vivo. Our findings suggest that USCs are able to differentiate into the Sk-MC lineage in vitro and after being implanted in vivo. Thus, they might be a potential source for cell injection therapy in the use of skeletal muscle regeneration. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

Identification of three molecular and functional subtypes in canine hemangiosarcoma through gene expression profiling and progenitor cell characterization.

PubMed

Gorden, Brandi H; Kim, Jong-Hyuk; Sarver, Aaron L; Frantz, Aric M; Breen, Matthew; Lindblad-Toh, Kerstin; O'Brien, Timothy D; Sharkey, Leslie C; Modiano, Jaime F; Dickerson, Erin B

2014-04-01

Canine hemangiosarcomas have been ascribed to an endothelial origin based on histologic appearance; however, recent findings suggest that these tumors may arise instead from hematopoietic progenitor cells. To clarify this ontogenetic dilemma, we used genome-wide expression profiling of primary hemangiosarcomas and identified three distinct tumor subtypes associated with angiogenesis (group 1), inflammation (group 2), and adipogenesis (group 3). Based on these findings, we hypothesized that a common progenitor may differentiate into the three tumor subtypes observed in our gene profiling experiment. To investigate this possibility, we cultured hemangiosarcoma cell lines under normal and sphere-forming culture conditions to enrich for tumor cell progenitors. Cells from sphere-forming cultures displayed a robust self-renewal capacity and exhibited genotypic, phenotypic, and functional properties consistent with each of the three molecular subtypes seen in primary tumors, including expression of endothelial progenitor cell (CD133 and CD34) and endothelial cell (CD105, CD146, and αvβ3 integrin) markers, expression of early hematopoietic (CD133, CD117, and CD34) and myeloid (CD115 and CD14) differentiation markers in parallel with increased phagocytic capacity, and acquisition of adipogenic potential. Collectively, these results suggest that canine hemangiosarcomas arise from multipotent progenitors that differentiate into distinct subtypes. Improved understanding of the mechanisms that determine the molecular and phenotypic differentiation of tumor cells in vivo could change paradigms regarding the origin and progression of endothelial sarcomas. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Non-Small Cell Lung Cancer Cells Expressing CD44 Are Enriched for Stem Cell-Like Properties

PubMed Central

Leung, Elaine Lai-Han; Fiscus, Ronald R.; Tung, James W.; Tin, Vicky Pui-Chi; Cheng, Lik Cheung; Sihoe, Alan Dart-Loon; Fink, Louis M.; Ma, Yupo; Wong, Maria Pik

2010-01-01

Background The cancer stem cell theory hypothesizes that cancers are perpetuated by cancer stem cells (CSC) or tumor initiating cells (TIC) possessing self-renewal and other stem cell-like properties while differentiated non-stem/initiating cells have a finite life span. To investigate whether the hypothesis is applicable to lung cancer, identification of lung CSC and demonstration of these capacities is essential. Methodology/Principal Finding The expression profiles of five stem cell markers (CD34, CD44, CD133, BMI1 and OCT4) were screened by flow cytometry in 10 lung cancer cell lines. CD44 was further investigated by testing for in vitro and in vivo tumorigenecity. Formation of spheroid bodies and in vivo tumor initiation ability were demonstrated in CD44+ cells of 4 cell lines. Serial in vivo tumor transplantability in nude mice was demonstrated using H1299 cell line. The primary xenografts initiated from CD44+ cells consisted of mixed CD44+ and CD44− cells in similar ratio as the parental H1299 cell line, supporting in vivo differentiation. Semi-quantitative Real-Time PCR (RT-PCR) showed that both freshly sorted CD44+ and CD44+ cells derived from CD44+-initiated tumors expressed the pluripotency genes OCT4/POU5F1, NANOG, SOX2. These stemness markers were not expressed by CD44− cells. Furthermore, freshly sorted CD44+ cells were more resistant to cisplatin treatment with lower apoptosis levels than CD44− cells. Immunohistochemical analysis of 141 resected non-small cell lung cancers showed tumor cell expression of CD44 in 50.4% of tumors while no CD34, and CD133 expression was observed in tumor cells. CD44 expression was associated with squamous cell carcinoma but unexpectedly, a longer survival was observed in CD44-expressing adenocarcinomas. Conclusion/Significance Overall, our results demonstrated that stem cell-like properties are enriched in CD44-expressing subpopulations of some lung cancer cell lines. Further investigation is required to clarify the role of CD44 in tumor cell renewal and cancer propagation in the in vivo environment. PMID:21124918

Characterization of Amniotic Stem Cells

PubMed Central

Koike, Chika; Zhou, Kaixuan; Takeda, Yuji; Fathy, Moustafa; Okabe, Motonori; Yoshida, Toshiko; Nakamura, Yukio; Kato, Yukio

2014-01-01

Abstract The amnion membrane is developed from embryo-derived cells, and amniotic cells have been shown to exhibit multidifferentiation potential. These cells represent a desirable source for stem cells for a variety of reasons. However, to date very few molecular analyses of amnion-derived cells have been reported, and efficient markers for isolating the stem cells remain unclear. This paper assesses the characterization of amnion-derived cells as stem cells by examining stemness marker expressions for amnion-derived epithelial cells and mesenchymal cells by flow cytometry, immunocytochemistry, and quantitative PCR. Flow cytometry revealed that amnion epithelial cells expressed CD133, CD 271, and TRA-1-60, whereas mecenchymal cells expressed CD44, CD73, CD90, and CD105. Immunohistochemistry showed that both cells expressed the stemness markers Oct3/4, Sox2, Klf4, and SSEA4. Stemness genes' expression in amnion epithelial cells, mesenchymal cells, fibroblast, bone marrow–derived mesenchymal stem cells (MSCs), and induced pluripotent stem cells (iPSCs) was compared by quantitative reverse-transcription polymerase chain reaction (RT-PCR). Amnion-derived epithelial cells and mesenchymal cells expressed Oct3/4, Nanog, and Klf4 more than bone marrow–derived MSCs. The sorted TRA1-60–positive cells expressed Oct3/4, Nanog, and Klf4 more than unsorted cells or TRA1-60–negative cells. TRA1-60 can be a marker for isolating amnion epithelial stem cells. PMID:25068631

CD24-Positive Cells from Normal Adult Mouse Liver Are Hepatocyte Progenitor Cells

PubMed Central

Qiu, Qiong; Hernandez, Julio Cesar; Dean, Adam M.; Rao, Pulivarthi H.

2011-01-01

The identification of specific cell surface markers that can be used to isolate liver progenitor cells will greatly facilitate experimentation to determine the role of these cells in liver regeneration and their potential for therapeutic transplantation. Previously, the cell surface marker, CD24, was observed to be expressed on undifferentiated bipotential mouse embryonic liver stem cells and 3,5-diethoxycarbonyl-1,4-dihydrocollidine-induced oval cells. Here, we describe the isolation and characterization of a rare, primary, nonhematopoietic, CD24+ progenitor cell population from normal, untreated mouse liver. By immunohistochemistry, CD24-expressing cells in normal adult mouse liver were colocalized with CK19-positive cholangiocytes. This nonhematopoietic (CD45−, Ter119−) CD24+ cell population isolated by flow cytometry represented 0.04% of liver cells and expressed several markers of liver progenitor/oval cells. The immunophenotype of nonhematopoietic CD24+ cells was CD133, Dlk, and Sca-1 high, but c-Kit, Thy-1, and CD34 low. The CD24+ cells had increased expression of CK19, epithelial cell adhesion molecule, Sox 9, and FN14 compared with the unsorted cells. Upon transplantation of nonhematopoietic CD24+ cells under the sub-capsule of the livers of Fah knockout mice, cells differentiated into mature functional hepatocytes. Analysis of X and Y chromosome complements were used to determine whether or not fusion of the engrafted cells with the recipient hepatocytes occurred. No cells were found that contained XXXY or any other combination of donor and host sex chromosomes as would be expected if cell fusion had occurred. These results suggested that CD24 can be used as a cell surface marker for isolation of hepatocyte progenitor cells from normal adult liver that are able to differentiate into hepatocytes. PMID:21361791

CD24-positive cells from normal adult mouse liver are hepatocyte progenitor cells.

PubMed

Qiu, Qiong; Hernandez, Julio Cesar; Dean, Adam M; Rao, Pulivarthi H; Darlington, Gretchen J

2011-12-01

The identification of specific cell surface markers that can be used to isolate liver progenitor cells will greatly facilitate experimentation to determine the role of these cells in liver regeneration and their potential for therapeutic transplantation. Previously, the cell surface marker, CD24, was observed to be expressed on undifferentiated bipotential mouse embryonic liver stem cells and 3,5-diethoxycarbonyl-1,4-dihydrocollidine-induced oval cells. Here, we describe the isolation and characterization of a rare, primary, nonhematopoietic, CD24+ progenitor cell population from normal, untreated mouse liver. By immunohistochemistry, CD24-expressing cells in normal adult mouse liver were colocalized with CK19-positive cholangiocytes. This nonhematopoietic (CD45-, Ter119-) CD24+ cell population isolated by flow cytometry represented 0.04% of liver cells and expressed several markers of liver progenitor/oval cells. The immunophenotype of nonhematopoietic CD24+ cells was CD133, Dlk, and Sca-1 high, but c-Kit, Thy-1, and CD34 low. The CD24+ cells had increased expression of CK19, epithelial cell adhesion molecule, Sox 9, and FN14 compared with the unsorted cells. Upon transplantation of nonhematopoietic CD24+ cells under the sub-capsule of the livers of Fah knockout mice, cells differentiated into mature functional hepatocytes. Analysis of X and Y chromosome complements were used to determine whether or not fusion of the engrafted cells with the recipient hepatocytes occurred. No cells were found that contained XXXY or any other combination of donor and host sex chromosomes as would be expected if cell fusion had occurred. These results suggested that CD24 can be used as a cell surface marker for isolation of hepatocyte progenitor cells from normal adult liver that are able to differentiate into hepatocytes.

Topical pimecrolimus versus betamethasone for oral lichen planus: a randomized clinical trial.

PubMed

Ezzatt, Ola M; Helmy, Iman M

2018-06-16

Oral lichen plans (OLP) is a potentially malignant inflammatory mucocutaneous disease. CD133 is an investigated surface marker for cancer stem-like cells (CSCs) that may be involved in tumor initiation in head and neck carcinomas. We compared short-term clinical effectiveness of topical pimecrolimus as selective inflammatory cytokine release inhibitor with betamethasone cream for erosive/atrophic OLP and investigated the influence of this therapy on CD133 expression. Thirty patients were randomly assigned into two equal groups to receive topical pimecrolimus (group I) or betamethasone (group II) four times daily for 4 weeks. A marker lesion in each patient were assessed at baseline using clinical score (CS) and visual analog scale (VAS) then at 1, 2, and 4 weeks and after 4 weeks of treatment-free period. CD133 expression was detected in pre- and post-treatment immunostained sections. Both drugs showed a reduction in CS, VAS, and CD133 expressions after treatment termination (p < 0.001). Pimecrolimus-treated lesions showed significant higher 1st week reduction in severity (33.1% (22.2)), pain score (57.53% (14.27)), less recurrence in follow-up period and less CD133 expression by the end of the 1st 4 weeks compared with betamethasone. Pimecrolimus showed earlier clinical response and less recurrence rate compared with standard topical corticosteroid in symptomatic OLP lesions, and both treatment reduced CD133-positive CSC population. The study proved the benefits of topical pimecrolimus in early management of painful lesions of OLP and its ability to inhibit CSCs, suggesting a possible role in reducing risk of malignant transformation.

Selection based on CD133 and high aldehyde dehydrogenase activity isolates long-term reconstituting human hematopoietic stem cells

PubMed Central

Hess, David A.; Wirthlin, Louisa; Craft, Timothy P.; Herrbrich, Phillip E.; Hohm, Sarah A.; Lahey, Ryan; Eades, William C.; Creer, Michael H.; Nolta, Jan A.

2006-01-01

The development of novel cell-based therapies requires understanding of distinct human hematopoietic stem and progenitor cell populations. We recently isolated reconstituting hematopoietic stem cells (HSCs) by lineage depletion and purification based on high aldehyde dehydrogenase activity (ALDHhiLin- cells). Here, we further dissected the ALDHhi-Lin- population by selection for CD133, a surface molecule expressed on progenitors from hematopoietic, endothelial, and neural lineages. ALDHhiCD133+Lin- cells were primarily CD34+, but also included CD34-CD38-CD133+ cells, a phenotype previously associated with repopulating function. Both ALDHhiCD133-Lin- and ALDHhiCD133+Lin- cells demonstrated distinct clonogenic progenitor function in vitro, whereas only the ALDHhiCD133+Lin- population seeded the murine bone marrow 48 hours after transplantation. Significant human cell repopulation was observed only in NOD/SCID and NOD/SCID β2M-null mice that received transplants of ALDHhiCD133+Lin- cells. Limiting dilution analysis demonstrated a 10-fold increase in the frequency of NOD/SCID repopulating cells compared with CD133+Lin- cells, suggesting that high ALDH activity further purified cells with repopulating function. Transplanted ALDHhiCD133+Lin- cells also maintained primitive hematopoietic phenotypes (CD34+CD38-) and demonstrated enhanced repopulating function in recipients of serial, secondary transplants. Cell selection based on ALDH activity and CD133 expression provides a novel purification of HSCs with long-term repopulating function and may be considered an alternative to CD34 cell selection for stem cell therapies. PMID:16269619

2-((Benzimidazol-2-yl)thio)-1-arylethan-1-ones: Synthesis, crystal study and cancer stem cells CD133 targeting potential.

PubMed

Abdel-Aziz, Hatem A; Ghabbour, Hazem A; Eldehna, Wagdy M; Al-Rashood, Sara T A; Al-Rashood, Khalid A; Fun, Hoong-Kun; Al-Tahhan, Mays; Al-Dhfyan, Abdullah

2015-11-02

In order to develop a potent anti-tumor agent that can target both cancer stem cells and the bulk of tumor cells, a series of 2-((benzimidazol-2-yl)thio)-1-arylethan-1-ones 5a-o was synthesized. All compounds were evaluated for their anti-proliferative activity towards colon HT-29 cancer cell line. In addition, their inhibitory effect against cell surface expression of CD133, a potent cancer stem cells (CSCs) marker, in the same cells was evaluated by flow cytometry at 10 μM. Compound 5l emerged as the most active anti-proliferative analog against HT-29 (IC50 = 18.83 ± 1.37 μM), that almost equipotent as 5-fluorouracil (IC50 = 15.83 ± 1.63 μM) with 50.11 ± 4.05% inhibition effect on CD133 expression, suggested dual targeted effect. Also, compounds 5h, 5j, 5k and 5m-o inhibited the expression of CD133 with more than 50%. The SAR study pointed out the significance of substitution of the pendent phenyl group with lipophilic electron-donating groups or replacing it by 2-thienyl or 2-furyl groups. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Identification of a distinct population of CD133+CXCR4+ cancer stem cells in ovarian cancer

PubMed Central

Cioffi, Michele; D’Alterio, Crescenzo; Camerlingo, Rosalba; Tirino, Virginia; Consales, Claudia; Riccio, Anna; Ieranò, Caterina; Cecere, Sabrina Chiara; Losito, Nunzia Simona; Greggi, Stefano; Pignata, Sandro; Pirozzi, Giuseppe; Scala, Stefania

2015-01-01

CD133 and CXCR4 were evaluated in the NCI-60 cell lines to identify cancer stem cell rich populations. Screening revealed that, ovarian OVCAR-3, -4 and -5 and colon cancer HT-29, HCT-116 and SW620 over expressed both proteins. We aimed to isolate cells with stem cell features sorting the cells expressing CXCR4+CD133+ within ovarian cancer cell lines. The sorted population CD133+CXCR4+ demonstrated the highest efficiency in sphere formation in OVCAR-3, OVCAR-4 and OVCAR-5 cells. Moreover OCT4, SOX2, KLF4 and NANOG were highly expressed in CD133+CXCR4+ sorted OVCAR-5 cells. Most strikingly CXCR4+CD133+ sorted OVCAR-5 and -4 cells formed the highest number of tumors when inoculated in nude mice compared to CD133−CXCR4−, CD133+CXCR4−, CD133−CXCR4+ cells. CXCR4+CD133+ OVCAR-5 cells were resistant to cisplatin, overexpressed the ABCG2 surface drug transporter and migrated toward the CXCR4 ligand, CXCL12. Moreover, when human ovarian cancer cells were isolated from 37 primary ovarian cancer, an extremely variable level of CXCR4 and CD133 expression was detected. Thus, in human ovarian cancer cells CXCR4 and CD133 expression identified a discrete population with stem cell properties that regulated tumor development and chemo resistance. This cell population represents a potential therapeutic target. PMID:26020117

Lower levels of circulating progenitor cells are associated with low physical function and performance in elderly men with impaired glucose tolerance: a pilot substudy from the VA Enhanced Fitness trial.

PubMed

Povsic, Thomas J; Sloane, Richard; Zhou, Jiying; Pieper, Carl F; Pearson, Megan P; Peterson, Eric D; Green, Jennifer B; Cohen, Harvey J; Morey, Miriam C

2013-12-01

Aging is marked by a decline in physical function. Although the biological underpinnings for this remain unclear, loss of regenerative capacity has been proposed as one cause of the loss of physical function that occurs over time. The quantity of circulating progenitor cells (CPCs) may be one reflection of regenerative capability. We sought to determine whether certain specific CPC subpopulations were associated with physical function. Baseline CPCs were measured in 129 randomized participants in the Enhanced Fitness clinical trial based on the cell surface markers CD34, CD133, CD146, and CD14 and aldehyde dehydrogenase (ALDH) activity. Physical function was assessed using usual and rapid gait speed, 6-minute walk distance, chair stand time, and balance time. Low counts of early angiogenic CPCs identified as CD34(+), CD34(+)CD133(+), and ALDH-bright (ALDH(br)) cells were associated with low usual gait speed (p < .005, p < .001, and p < .007), rapid gait speed (p < .001, p < .003, and p < .001), and 6-minute walking distance (all comparisons p < .001), and longer time required to complete five chair stands (p < .006, p < .002, and p < .004). CPC counts of mature endothelial or monocytic markers were not associated with physical function. The numbers of CD34(+) and ALDH(br) CPCs are significantly lower in patients with impaired physical function. Further studies are needed to determine the underlying causes for this association.

CD133+ cells derived from skeletal muscles of Duchenne muscular dystrophy patients have a compromised myogenic and muscle regenerative capability.

PubMed

Meng, Jinhong; Muntoni, Francesco; Morgan, Jennifer

2018-05-12

Cell-mediated gene therapy is a possible means to treat muscular dystrophies like Duchenne muscular dystrophy. Autologous patient stem cells can be genetically-corrected and transplanted back into the patient, without causing immunorejection problems. Regenerated muscle fibres derived from these cells will express the missing dystrophin protein, thus improving muscle function. CD133+ cells derived from normal human skeletal muscle contribute to regenerated muscle fibres and form muscle stem cells after their intra-muscular transplantation into an immunodeficient mouse model. But it is not known whether CD133+ cells derived from DMD patient muscles have compromised muscle regenerative function. To test this, we compared CD133+ cells derived from DMD and normal human muscles. DMD CD133+ cells had a reduced capacity to undergo myogenic differentiation in vitro compared with CD133+ cells derived from normal muscle. In contrast to CD133+ cells derived from normal human muscle, those derived from DMD muscle formed no satellite cells and gave rise to significantly fewer muscle fibres of donor origin, after their intra-muscular transplantation into an immunodeficient, non-dystrophic, mouse muscle. DMD CD133+ cells gave rise to more clones of smaller size and more clones that were less myogenic than did CD133+ cells derived from normal muscle. The heterogeneity of the progeny of CD133+ cells, combined with the reduced proliferation and myogenicity of DMD compared to normal CD133+ cells, may explain the reduced regenerative capacity of DMD CD133+ cells. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Imaging and Selective Elimination of Glioblastoma Stem Cells with Theranostic Near-Infrared-Labeled CD133-Specific Antibodies.

PubMed

Jing, Hua; Weidensteiner, Claudia; Reichardt, Wilfried; Gaedicke, Simone; Zhu, Xuekai; Grosu, Anca-Ligia; Kobayashi, Hisataka; Niedermann, Gabriele

2016-01-01

Near-infrared photoimmunotherapy (NIR-PIT), which employs monoclonal antibody (mAb)-phototoxic phthalocyanine dye IR700 conjugates, permits the specific, image-guided and spatiotemporally controlled elimination of tumor cells. Here, we report the highly efficient NIR-PIT of human tumor xenografts initiated from patient-derived cancer stem cells (CSCs). Using glioblastoma stem cells (GBM-SCs) expressing the prototypic CSC marker AC133/CD133, we also demonstrate here for the first time that NIR-PIT is highly effective against brain tumors. The intravenously injected theranostic AC133 mAb conjugate enabled the non-invasive detection of orthotopic gliomas by NIR fluorescence imaging, and reached AC133+ GBM-SCs at the invasive tumor front. AC133-targeted NIR-PIT induced the rapid cell death of AC133+ GBM-SCs and thereby strong shrinkage of both subcutaneous and invasively growing brain tumors. A single round of NIR-PIT extended the overall survival of mice with established orthotopic gliomas by more than a factor of two, even though the harmless NIR light was applied through the intact skull. Humanised versions of this theranostic agent may facilitate intraoperative imaging and histopathological evaluation of tumor borders and enable the highly specific and efficient eradication of CSCs.

Isolation and characteristics of CD133‑/A2B5+ and CD133‑/A2B5‑ cells from the SHG139s cell line.

PubMed

Han, Yong; Wang, Hangzhou; Huang, Yulun; Cheng, Zhe; Sun, Ting; Chen, Guilin; Xie, Xueshun; Zhou, Youxin; Du, Ziwei

2015-12-01

In glioma tissues, there are small cell populations with the capability of sustaining tumor formation. These cells are referred to as glioma stem cells (GSCs). However, the presence of subpopulations of GSCs, and the differences between each subpopulation remain to be fully elucidated. In the present study, CD133‑/A2B5‑ and CD133‑/A2B5+ cells from the SHG139 GSC cell line (SHG139s) were isolated using magnetic‑activated cell sorting. Following xenografting into nude mice, the two isolated subpopulations generated tumors. The characteristics of the two subpopulations were investigated extensively, and it was found that the two exhibited cancer stem cell characteristics. These cells expressed stem cell markers, exhibited a neurosphere‑like appearance, and were found to exhibit self‑renewal and multipotency capabilities. Subsequently, the self‑renewal and proliferation abilities of the two subpopulations were compared. It was found that the A2B5‑ cells had a higher proliferative index and a higher self‑renewal ability, compared with the A2B5+ cells. In addition, the A2B5‑ cells exhibited increased angiogenic ability. However, the invasion ability of the A2B5+ cells was higher than that of the A2B5‑ cells. Taken together, the results of the present study suggested that there are different cell subpopulations in GSCs, and each subpopulation has its own properties.

Overexpression of syndecan-1, MUC-1, and putative stem cell markers in breast cancer leptomeningeal metastasis: a cerebrospinal fluid flow cytometry study.

PubMed

Cordone, Iole; Masi, Serena; Summa, Valentina; Carosi, Mariantonia; Vidiri, Antonello; Fabi, Alessandra; Pasquale, Alessia; Conti, Laura; Rosito, Immacolata; Carapella, Carmine Maria; Villani, Veronica; Pace, Andrea

2017-04-11

Cancer is a mosaic of tumor cell subpopulations, where only a minority is responsible for disease recurrence and cancer invasiveness. We focused on one of the most aggressive circulating tumor cells (CTCs) which, from the primitive tumor, spreads to the central nervous system (CNS), evaluating the expression of prognostic and putative cancer stem cell markers in breast cancer (BC) leptomeningeal metastasis (LM). Flow cytometry immunophenotypic analysis of cerebrospinal fluid (CSF) samples (4.5 ml) was performed in 13 consecutive cases of BCLM. Syndecan-1 (CD138), MUC-1 (CD227) CD45, CD34, and the putative cancer stem cell markers CD15, CD24, CD44, and CD133 surface expression were evaluated on CSF floating tumor cells. The tumor-associated leukocyte population was also characterized. Despite a low absolute cell number (8 cell/μl, range 1-86), the flow cytometry characterization was successfully conducted in all the samples. Syndecan-1 and MUC-1 overexpression was documented on BC cells in all the samples analyzed; CD44, CD24, CD15, and CD133 in 77%, 75%, 70%, and 45% of cases, respectively. A strong syndecan-1 and MUC-1 expression was also documented by immunohistochemistry on primary breast cancer tissues, performed in four patients. The CSF tumor population was flanked by T lymphocytes, with a different immunophenotype between the CSF and peripheral blood samples (P ≤ 0.02). Flow cytometry can be successfully employed for solid tumor LM characterization even in CSF samples with low cell count. This in vivo study documents that CSF floating BC cells overexpress prognostic and putative cancer stem cell biomarkers related to tumor invasiveness, potentially representing a molecular target for circulating tumor cell detection and LM treatment monitoring, as well as a primary target for innovative treatment strategies. The T lymphocyte infiltration, documented in all CSF samples, suggests a possible involvement of the CNS lymphatic system in both lymphoid and cancer cell migration into and out of the meninges, supporting the extension of a new form of cellular immunotherapy to LM. Due to the small number of cases, validation on large cohorts of patients are warranted to confirm these findings and to evaluate the impact and value of these results for diagnosis and management of LM.

Targeting cancer stem cell-specific markers and/or associated signaling pathways for overcoming cancer drug resistance.

PubMed

Ranji, Peyman; Salmani Kesejini, Tayyebali; Saeedikhoo, Sara; Alizadeh, Ali Mohammad

2016-10-01

Cancer stem cells (CSCs) are a small subpopulation of tumor cells with capabilities of self-renewal, dedifferentiation, tumorigenicity, and inherent chemo-and-radio therapy resistance. Tumor resistance is believed to be caused by CSCs that are intrinsically challenging to common treatments. A number of CSC markers including CD44, CD133, receptor tyrosine kinase, aldehyde dehydrogenases, epithelial cell adhesion molecule/epithelial specific antigen, and ATP-binding cassette subfamily G member 2 have been proved as the useful targets for defining CSC population in solid tumors. Furthermore, targeting CSC markers through new therapeutic strategies will ultimately improve treatments and overcome cancer drug resistance. Therefore, the identification of novel strategies to increase sensitivity of CSC markers has major clinical implications. This review will focus on the innovative treatment methods such as nano-, immuno-, gene-, and chemotherapy approaches for targeting CSC-specific markers and/or their associated signaling pathways.

Downregulation of DNA-PKcs suppresses P-gp expression via inhibition of the Akt/NF-κB pathway in CD133-positive osteosarcoma MG-63 cells.

PubMed

Li, Ka; Li, Xin; Tian, Jiguang; Wang, Hongliang; Pan, Jingbo; Li, Jianmin

2016-10-01

The development of chemoresistance is closely linked to the plateau of the survival rate in osteosarcoma (OS) patients. CD133-positive (CD133+) OS cells are known as cancer stem cells (CSCs) in OS and exhibit the characteristic of chemoresistance. In this study, CD133+ and CD133‑negative (CD133‑) MG‑63 cells were isolated by magnetic activated cell sorting (MACS). We verified that CD133+ MG‑63 cells were more resistant to cisplatin (CDDP) than CD133‑ MG‑63 cells. DNA‑dependent protein kinase catalytic subunit (DNA‑PKcs) and P‑glycoprotein (P‑gp) were expressed at higher levels in the CD133+ MG‑63 cells compared with those levels in the CD133‑ MG‑63 cells, whereas downregulation of DNA‑PKcs by small interfering RNA (siRNA) decreased chemoresistance to CDDP and P‑gp expression at the mRNA and protein levels in these cells. This indicated that DNA‑PKcs was correlated with P‑gp expression in the CD133+ MG‑63 cells. The Akt/NF‑κB pathway was hyperactivated in the CD133+ MG‑63 cells, whereas inhibition of the Akt/NF‑κB pathway downregulated P‑gp expression. In addition, downregulation of DNA‑PKcs suppressed the activity of the Akt/NF‑κB pathway. These results revealed that downregulation of DNA‑PKcs could decrease P‑gp expression via suppression of the Akt/NF‑κB pathway in CD133+ MG‑63 cells. Therefore, inhibition of DNA‑PKcs decreases P‑gp expression and sensitizes OS CSCs to chemotherapeutic agents in vitro, which needs to be further validated in vivo.

SC-12CD133 SURFACE EXPRESSION INDICATES ASYMMETRIC INHERITANCE OF SIGNALING RECEPTORS DURING GLIOBLASTOMA CANCER STEM CELL MITOSIS

PubMed Central

Hitomi, Masahiro; Jarvis, Stephanie; Yogeswaran, Vid; Pfaff, Kayla; Lathia, Justin

2014-01-01

Asymmetric cell division, the mechanism by which stem cells generate progeny undergoing tissue specific differentiation and a self-renewing stem cell population, enables organogenesis, maintenance of tissue homeostasis, and tissue regeneration without depleting stem cell pools. Cancer stem cells (CSCs) have been identified in malignant cancers including glioblastoma (GBM) by virtue of their enhanced self-renewal capacity and ability to reconstitute an entire tumor with all types of cells found in the original tumor. CSCs also play pivotal roles in therapeutic resistance and are the focus of recent therapeutic development efforts. CSC maintenance is regulated by intrinsic stem cell transcription factors, as well as by multiple extrinsic factors in the tumor microenvironment. In addition to these factors, the mode of cell division plays a critical role in CSC maintenance as exemplified by normal stem cells. Previously, we demonstrated that asymmetric segregation of a CSC marker, CD133, at the time of mitosis correlated with fate determination of CSCs derived from clinical GBM patient samples. Utilizing quantitative immunofluorecsence, we detected that receptors for key signaling molecules critical for CSC maintenance were co-segregated with CD133. Inhibition of downstream signaling induced asymmetric cell death in one of the daughter cells. These data indicate that CD133 marks daughter cells with higher inheritance of molecules that facilitate self-renewal and that asymmetric cell division may benefit CSC survival by concentrating essential receptors to one daughter cell in addition to its potential role in increasing cellular heterogeneity of the tumor.

The number and function of circulating CD34+CD133+ progenitor cells decreased in stable coronary artery disease but not in acute myocardial infarction

PubMed Central

Kondo, Takahisa; Shintani, Satoshi; Maeda, Kengo; Hayashi, Mutsuharu; Inden, Yasuya; Numaguchi, Yasushi; Sugiura, Kaichiro; Morita, Yasuhiro; Kitamura, Tomoya; Kamiya, Haruo; Sone, Takahito; Ohno, Miyoshi; Murohara, Toyoaki

2010-01-01

Objective Circulating CD34+CD133+ cells are one of the main sources of circulating endothelial progenitor cells (EPCs). Age is inversely related to the number and function of CD34+CD133+ progenitor cells in stable coronary artery disease (CAD), but the relationship remains unclear in acute myocardial infarction (AMI). The authors aimed to clarify how ageing affects the number and function of mobilised CD34+CD133+ progenitor cells in AMI. Design and results Circulating CD34+CD133+ progenitor cells were measured by flow cytometry. Measurements were made at admission for CAD, or on day 7 after the onset of AMI. In stable CAD (n=131), circulating CD34+CD133+ cells decreased with age (r=−0.344, p<0.0001). In AMI, circulating CD34+CD133+ cells did not correlate with age (n=50), and multivariate analysis revealed that the decreased number of circulating CD34+CD133+ cells was associated with male sex and higher peak creatinine kinase. The ability to give rise to functional EPCs, which show good migratory and tube-forming capabilities, deteriorated among stable CAD subjects (n=10) compared with AMI subjects (N=6). Conclusions In stable CAD, the number and function of circulating CD34+CD133+ progenitor cells decreased with age, whereas those mobilised and circulating in AMI did not. PMID:27325937

[Inhibitory effect of ¹³¹I-CD133mAb combined with cisplatin on liver cancer cells in vitro and in a tumor-bearing mouse model].

PubMed

Chen, Xingyue; Hou, Yanli; Duan, Liqun; Tang, Min; Kang, Qiangqiang; Shu, Jin; Peng, Zhiping; Li, Shaolin

2014-06-01

To study the inhibitory effect of CD133 monoclonal antibody labeled with ¹³¹I (¹³¹I-CD133mAb) on Huh-7 human liver cancer cell line overexpressing CD133 antigen in vitro and in mouse models bearing the tumor cell xenograft. ¹³¹I-CD133mAb was prepared by chloramines-T method and evaluated for its stability. Flow cytometry and immunohistochemistry were used to detect the expression of CD133 in Huh-7 cells and in Huh-7 cell-derived tumors, respectively. Huh-7 cells treated with ¹³¹I-CD133mAb plus cisplatin (DDP), ¹³¹I -CD133mAb, DDP, or no treatment (blank control) were examined for cell proliferation suppression by MTT assay with the IC₅₀ calculated. BALB/c mice bearing subcutaneous Huh-7 cell xenograft in the right forelegs were treated with ¹³¹I -CD133mAb, DDP, or both every two days for two weeks. The tumor size and volume were measured twice a week, and pathological examination of the tumor was carried out after the treatments. The tumor inhibition rate was calculated and tumor cell apoptosis observed with HE staining. The labeling ratio of ¹³¹I-CD133mAb was 90.25% and the radiochemical purity was 97.78%. Huh-7 cells showed obviously higher CD133 expression than HepG2 cells. ¹³¹I-CD133mAb combined with DDP group resulted in a significantly higher tumor inhibition rate than other treatments in the tumor-bearing mice. ¹³¹I-CD133mAb can inhibit the growth of liver cancer cells with a high CD133 expression both in vivo and in vitro.

Adenovirus-mediated truncated Bid overexpression induced by the Cre/LoxP system promotes the cell apoptosis of CD133+ ovarian cancer stem cells.

PubMed

Long, Qifang; Yang, Ru; Lu, Weixian; Zhu, Weipei; Zhou, Jundong; Zheng, Cui; Zhou, Dongmei; Yu, Ling; Wu, Jinchang

2017-01-01

Cancer stem cells are a small subset of cancer cells that contribute to cancer progression, metastasis, chemoresistance and recurrence. CD133-positive (CD133+) ovarian cancer cells have been identified as ovarian cancer stem cells. Adenovirus-mediated gene therapy is an innovative therapeutic method for cancer treatment. In the present study, we aimed to develop a new gene therapy to specifically eliminate CD133+ ovarian cancer stem cells by targeting CD133. We used the Cre/LoxP system to augment the selective expression of the truncated Bid (tBid) gene as suicide gene therapy in CD133+ ovarian cancer stem cells. The adenovirus (Ad)-CD133-Cre expressing Cre recombinase under the control of the CD133 promoter and Ad-CMV-LoxP-Neo-LoxP-tBid expressing tBid under the control of the CMV promoter were successfully constructed using the Cre/LoxP switching system. The co-infection of Ad-CMV-LoxP-Neo-LoxP-tBid and Ad-CD133-Cre selectively induced tBid overexpression, which inhibited cell growth and triggered the cell apoptosis of CD133+ ovarian cancer stem cells. The Cre/LoxP system-mediated tBid overexpression activated the pro-apoptotic signaling pathway and augmented the cytotoxic effect of cisplatin in CD133+ ovarian cancer stem cells. Furthermore, in xenograft experiments, co-infection with the two recombinant adenoviruses markedly suppressed tumor growth in vivo and promoted cell apoptosis in tumor tissues. Taken together, the present study provides evidence that the adenovirus-mediated tBid overexpression induced by the Cre/LoxP system can effectively eliminate CD133+ ovarian cancer stem cells, representing a novel therapeutic strategy for the treatment of ovarian cancer.

3-Bromopyruvate inhibits cell proliferation and induces apoptosis in CD133+ population in human glioma.

PubMed

Xu, Dong-Qiang; Tan, Xiao-Yu; Zhang, Bao-Wei; Wu, Tao; Liu, Ping; Sun, Shao-Jun; Cao, Yin-Guang

2016-03-01

The study was aimed to investigate the role of 3-bromopyruvate in inhibition of CD133+ U87 human glioma cell population growth. The results demonstrated that 3-bromopyruvate inhibited the viability of both CD133+ and parental cells derived from U87 human glioma cell line. However, the 3-bromopyruvate-induced inhibition in viability was more prominent in CD133+ cells at 10 μM concentration after 48 h. Treatment of CD133+ cells with 3-bromopyruvate caused reduction in cell population and cell size, membrane bubbling, and degradation of cell membranes. Hoechst 33258 staining showed condensation of chromatin material and fragmentation of DNA in treated CD133+ cells after 48 h. 3-Bromopyruvate inhibited the migration rate of CD133+ cells significantly compared to the parental cells. Flow cytometry revealed that exposure of CD133+ cells to 3-bromopyruvate increased the cell population in S phase from 24.5 to 37.9 % with increase in time from 12 to 48 h. In addition, 3-bromopyruvate significantly enhanced the expression of Bax and cleaved caspase 3 in CD133+ cells compared to the parental cells. Therefore, 3-bromopyruvate is a potent chemotherapeutic agent for the treatment of glioma by targeting stem cells selectively.

Effect of colorectal cancer on the number of normal stem cells circulating in peripheral blood.

PubMed

Marlicz, Wojciech; Sielatycka, Katarzyna; Serwin, Karol; Kubis, Ewa; Tkacz, Marta; Głuszko, Rafał; Białek, Andrzej; Starzyńska, Teresa; Ratajczak, Mariusz Z

2016-12-01

Bone marrow (BM) residing stem cells are mobilized from their BM niches into peripheral blood (PB) in several pathological situations including tissue organ injury and systemic inflammation. We recently reported that the number of BM-derived stem cells (SCs) increases in patients with pancreatic and stomach cancer. Accordingly, we observed higher numbers of circulating very small embryonic/epiblast‑like stem cells (VSELs) and mesenchymal stem cells (MSCs) that were associated with the activation of pro-mobilizing complement cascade and an elevated level of sphingosine-1 phosphate (S1P) in PB plasma. We wondered if a similar correlation occurs in patients with colorectal cancer (CRC). A total of 46 patients were enrolled in this study: 17 with CRC, 18 with benign colonic adenomas (BCA) and 11 healthy individuals. By employing fluorescence-activated cell sorting (FACS) we evaluated the number of BM-derived SCs circulating in PB: i) CD34+/Lin-/CD45- and CD133-/Lin-/CD45- VSELs; ii) CD45-/CD105+/CD90+/CD29+ MSCs; iii) CD45-/CD34+/CD133+/KDR+ endothelial progenitor cells (EPCs); and iv) CD133+/Lin-/CD45+ or CD34+/Lin-/CD45+ cells enriched for hematopoietic stem/progenitor cells (HSPCs). In parallel, we measured in the PB parameters regulating the egress of SCs from BM into PB. In contrast to pancreatic and gastric cancer patients, CRC subjects presented neither an increase in the number of circulating SCs nor the activation of pro-mobilizing factors such as complement, coagulation and fibrinolytic cascade, circulating stromal derived factor 1 (SDF‑1), vascular endothelial growth factor (VEGF) and intestinal permeability marker (zonulin). In conclusion, mobilization of SCs in cancer patients depends on the type of malignancy and its ability to activate pro-mobilization cascades.

Bufalin inhibits the differentiation and proliferation of human osteosarcoma cell line hMG63-derived cancer stem cells.

PubMed

Chang, Yuewen; Zhao, Yongfang; Zhan, Hongsheng; Wei, Xiaoen; Liu, Tianjin; Zheng, Bo

2014-02-01

Cancer stem cells (CSCs) play an important role in drug resistance of tumor and are responsible for high recurrence rates. Agents that can suppress the proliferation and differentiation of CSCs would provide new opportunity to fight against tumor recurrence. In this study, we developed a new strategy to enrich CSCs in human osteosarcoma cell line hMG63. Using these CSCs as model, we tested the effect of bufalin, a traditional Chinese medicine, on the proliferation and differentiation of CSCs. hMG63 cells were cultured in poly-HEMA-treated dish and cancer stem cell-specific medium. In this nonadhesive culture system, hMG63 formed spheres, which were then collected and injected into the immunodeficient mice. Cisplatin was administered every 3 days for five times. The enriched xenograft tumors were cultured in cancer stem cell-specific medium again to form tumor spheres. Expression of cancer stem cell markers of these cells was measured by flow cytometry. These cells were then treated with bufalin, and the proliferation and differentiation ability were indicated by the expression level of molecular markers and the formation of sphere again in vitro. We obtained a low CD133+/CD44 cell population with high-level stem cell marker. When treated with bufalin, the sphere could not get attached to the flask and failed to differentiate, which was indicated by the stable expression of stem cell marker CD133 and OCT-4 in the condition permissive to differentiation. Treatment of bufalin also suppressed the single cells isolated from the sphere to form sphere again in the nonadhesive culture system, and a decreased expression of proliferation marker Ki67 was also detected in these cells. Sphere-formed and chemoresistant colon xenograft tumors in immunodeficient mice could enrich cancer stem cell population. Bufalin could inhibit proliferation and differentiation of CSCs.

Isolation and characterization of human CXCR4-positive pancreatic cells.

PubMed

Koblas, T; Zacharovová, K; Berková, Z; Mindlová, M; Girman, P; Dovolilová, E; Karasová, L; Saudek, F

2007-01-01

The existence of an adult PSC that may be used in the treatment of diabetes is still a matter of scientific debate as conclusive evidence of such a stem cell in the adult pancreas has not yet been presented. The main reason why putative PSC has not yet been identified is the lack of specific markers that may be used to isolate and purify them. In order to increase the list of potential PSC markers we have focused on the human pancreatic cells that express cell surface receptor CXCR4, a marker of stem cells derived from different adult tissues. Here we report that CXCR4-positive pancreatic cells express markers of pancreatic endocrine progenitors (neurogenin-3, nestin) and markers of pluripotent stem cells (Oct-4, Nanog, ABCG2, CD133, CD117). Upon in vitro differentiation, these cells form ILCC and produce key islet hormones including insulin. Based on our results, we assume that CXCR4 marks pancreatic endocrine progenitors and in combination with other cell surface markers may be used in the attempt to identify and isolate PSC.

Endothelial Progenitor Cell Levels Predict Future Physical Function: An Exploratory Analysis From the VA Enhanced Fitness Study.

PubMed

Povsic, Thomas J; Sloane, Richard; Pieper, Carl F; Pearson, Megan P; Peterson, Eric D; Cohen, Harvey J; Morey, Miriam C

2016-03-01

Levels of circulating progenitor cells (CPCs) are depleted with aging and chronic injury and are associated with level of physical functioning; however, little is known about the correlation of CPCs with longer-term measures of physical capabilities. We sought to determine the association of CPCs with future levels of physical function and with changes in physical function over time. CPCs were measured in 117 participants with impaired glucose tolerance in the Enhanced Fitness clinical trial based on the cell surface markers CD34 and CD133 and aldehyde dehydrogenase (ALDH) activity at baseline, 3 months, and 12 months. Physical function was assessed using usual and rapid gait speed, 6-minute walk distance, chair stand time, and SF-36 physical functioning score and reassessed at 3 and 12 months after clinical intervention. Higher baseline levels of CD133(+), CD34(+), CD133(+)CD34(+), and ALDH(br) were each highly predictive of faster gait speed and longer distance walked in 6 minutes at both 3 and 12 months. These associations remained robust after adjustment for age, body mass index, baseline covariates, and inflammation and were independent of interventions to improve physical fitness. Further, higher CPC levels predicted greater improvements in usual and rapid gait speed over 1 year. Baseline CPC levels are associated not only with baseline mobility but also with future physical function, including changes in gait speed. These findings suggest that CPC measurement may be useful as a marker of both current and future physiologic aging and functional decline. © The Author 2015. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

CD133: Beyond a Cancer Stem Cell Biomarker.

PubMed

Barzegar Behrooz, Amir; Syahir, Amir; Ahmad, Syahida

2018-06-18

CD133 (prominin-1), a pentaspan membrane glycoprotein, is one of the most well-characterized biomarkers used for the isolation of cancer stem cells (CSCs). The presence of CSCs is one of the main causes of tumor reversal and resilience. Accumulating evidence has shown that CD133 might be responsible for CSCs tumorigenesis, metastasis, and chemoresistance. It is now understood that CD133 interacts with the Wnt/β-catenin and PI3K-Akt signaling pathways. Moreover, CD133 can upregulate the expression of FLICE-like inhibitory protein (FLIP) in CD133-positive cells, inhibiting apoptosis. Additionally, CD133 can increase angiogenesis by activating the Wnt signaling pathway and increasing the expression of vascular endothelial growth factor-A (VEGF-A) and interleukin-8. Therefore, CD133 could be considered to be an "Achilles' heel" for CSCs, because by inhibiting this protein, the signaling pathways that are involved in cell proliferation will also be inhibited. By understanding the molecular biology of CD133, we can not only isolate stem cells, but can also utilize it as a therapeutic strategy. In this review, we summarize new insights into the fundamental cell biology of CD133 and discuss the involvement of CD133 in metastasis, metabolism, tumorigenesis, drug-resistance, apoptosis, and autophagy.

Transient receptor potential vanilloid-type 2 targeting on stemness in liver cancer.

PubMed

Hu, Zecheng; Cao, Xiaocheng; Fang, Yu; Liu, Guoxing; Xie, Chengzhi; Qian, Ke; Lei, Xiaohua; Cao, Zhenyu; Du, Huihui; Cheng, Xiangding; Xu, Xundi

2018-06-12

The malignant phenotype of the cells resulting from human liver cancer is driven by liver cancer stem-like cells (LCSLCs). Transient Receptor Potential Vanilloid-type 2 channel (TRPV2) contributes to the progression of different tumor types, including liver cancer. In the current study, the TRPV2 expression levels give rise to the effect on stemness in liver cancer cell lines. TRPV2 knockdown in HepG2 cells enhanced spheroid and colony formation, and expression levels of CD133, CD44 and ALDH1 whereas the opposite effects were observed in TRPV2 enforced expression in SMMC-7721 cells. Furthermore, TRPV2 overexpression restored inhibition of spheroid and colony formation, and stem cell markers expression in HepG2 cells with TRPV2 silencing. The addition of the TRPV2 agonist probenecid and the TRPV2 antagonist tranilast suppressed and/or increased in vitro spheroid and colony formation, and stem cell marker expression of LCSLCs and/or liver cancer cell lines, respectively. Notably, probenecid and tranilast significantly inhibited or promoted tumor growth of HepG2 xenografts in the severe combined immunodeficiency (SCID) mouse model, respectively. TRPV2 expression at protein levels revealed converse correlation with those of CD133 and CD44 in human hepatocellular carcinoma (HCC) tissue. Collectively, the data demonstrate that TRPV2 exert effects on stemness of liver cancer and is a potential target in the treatment of human liver cancer patients. Copyright © 2018. Published by Elsevier Masson SAS.

HBx drives alpha fetoprotein expression to promote initiation of liver cancer stem cells through activating PI3K/AKT signal pathway.

PubMed

Zhu, Mingyue; Li, Wei; Lu, Yan; Dong, Xu; Lin, Bo; Chen, Yi; Zhang, Xueer; Guo, Junli; Li, Mengsen

2017-03-15

Hepatitis B virus (HBV)-X protein (HBx) plays critical role in inducing the malignant transformation of liver cells. Alpha fetoprotein (AFP) expression is closely related to hepatocarcinogenesis. We report that Oct4, Klf4, Sox2 and c-myc expression positively associated with AFP(+)/HBV(+) hepatocellular carcinoma(HCC) tissues, and the expression of the stemness markers CD44, CD133 and EpCAM was significantly higher in AFP(+)/HBV(+) HCC tissues compared to normal liver tissues or AFP (-)/HBV(-) HCC tissues. AFP expression turned on prior to expression of Oct4, Klf4, Sox2 and c-myc, and the stemness markers CD44, CD133 and EpCAM in the normal human liver L-02 cell line or CHL cell lines upon transfection with MCV-HBx vectors. Stem-like cells generated more tumour colonies compared to primary cells, and xenografts induced tumourigenesis in nude mice. Expression of reprogramming-related proteins was significantly enhanced in HLE cells while transfected with pcDNA3.1-afp vectors. The specific PI3K inhibitor Ly294002 inhibited the effects of pcDNA3.1-afp vectors. AFP-siRNA vectors were able to inhibit tumour colony formation and reprogramming-related gene expression. Altogether, HBx stimulates AFP expression to induce natural reprogramming of liver cells, and AFP plays a critical role in promoting the initiation of HCC progenitor/stem cells. AFP may be a potential novel biotarget for combating HBV-induced hepatocarcinogenesis. © 2016 UICC.

Immunohistochemical Markers of Neural Progenitor Cells in the Early Embryonic Human Cerebral Cortex

PubMed Central

Vinci, L.; Ravarino, A.; Fanos, V.; Naccarato, A.G.; Senes, G.; Gerosa, C.; Bevilacqua, G.; Faa, G.; Ambu, R.

2016-01-01

The development of the human central nervous system represents a delicate moment of embryogenesis. The purpose of this study was to analyze the expression of multiple immunohistochemical markers in the stem/progenitor cells in the human cerebral cortex during the early phases of development. To this end, samples from cerebral cortex were obtained from 4 human embryos of 11 weeks of gestation. Each sample was formalin-fixed, paraffin embedded and immunostained with several markers including GFAP, WT1, Nestin, Vimentin, CD117, S100B, Sox2, PAX2, PAX5, Tβ4, Neurofilament, CD44, CD133, Synaptophysin and Cyclin D1. Our study shows the ability of the different immunohistochemical markers to evidence different zones of the developing human cerebral cortex, allowing the identification of the multiple stages of differentiation of neuronal and glial precursors. Three important markers of radial glial cells are evidenced in this early gestational age: Vimentin, Nestin and WT1. Sox2 was expressed by the stem/progenitor cells of the ventricular zone, whereas the postmitotic neurons of the cortical plate were immunostained by PAX2 and NSE. Future studies are needed to test other important stem/progenitor cells markers and to better analyze differences in the immunohistochemical expression of these markers during gestation. PMID:26972711

Self-renewal and circulating capacities of metastatic hepatocarcinoma cells required for collaboration between TM4SF5 and CD44

PubMed Central

Lee, Doohyung; Lee, Jung Weon

2015-01-01

Tumor metastasis involves circulating and tumor-initiating capacities of metastatic cancer cells. Hepatic TM4SF5 promotes EMT for malignant growth and migration. Hepatocellular carcinoma (HCC) biomarkers remain unexplored for metastatic potential throughout metastasis. Here, novel TM4SF5/CD44 interaction-mediated self-renewal and circulating tumor cell (CTC) capacities were mechanistically explored. TM4SF5-dependent sphere growth was correlated with CD133+, CD24-, ALDH activity, and a physical association between CD44 and TM4SF5. The TM4SF5/CD44 interaction activated c-Src/STAT3/ Twist1/ B mi1 signaling for spheroid formation, while disturbing the interaction, expression, or activity of any component in this signaling pathway inhibited spheroid formation. In serial xenografts of less than 5,000 cells/injection, TM4SF5-positive tumors exhibited locally-increased CD44 expression, suggesting tumor cell differentiation. TM4SF5-positive cells were identified circulating in blood 4 to 6 weeks after orthotopic liver-injection. Anti-TM4SF reagents blocked their metastasis to distal intestinal organs. Altogether, our results provide evidence that TM4SF5 promotes self-renewal and CTC properties supported by CD133+/TM4SF5+/CD44+(TM4SF5-bound)/ALDH+/ CD24- markers during HCC metastasis. [BMB Reports 2015; 48(3): 127-128] PMID:25772760

Improved Killing of Ovarian Cancer Stem Cells by Combining a Novel Chimeric Antigen Receptor-Based Immunotherapy and Chemotherapy.

PubMed

Klapdor, Rüdiger; Wang, Shuo; Hacker, Ulrich; Büning, Hildegard; Morgan, Michael; Dörk, Thilo; Hillemanns, Peter; Schambach, Axel

2017-10-01

Ovarian cancer represents the most lethal gynecological cancer. Although cytoreductive chemotherapy and surgery lead to complete macroscopic tumor removal, most of the patients in advanced stages suffer from recurrent disease and subsequently die. This may be explained by the activity of cancer stem cells (CSC), which are a subpopulation of cells with an elevated chemoresistance and an increased capacity for self-renewal and metastatic spread. Specifically targeting these cells by adoptive immunotherapy represents a promising strategy to reduce the risk for recurrent disease. This study selected the widely accepted CSC marker CD133 as a target for a chimeric antigen receptor (CAR)-based immunotherapeutic approach to treat ovarian cancer. A lentiviral vector was generated encoding a third-generation anti-CD133-CAR, and clinically used NK92 cells were transduced. These engineered natural killer (NK) cells showed specific killing against CD133-positive ovarian cancer cell lines and primary ovarian cancer cells cultured from sequential ascites harvests. Additionally, specific activation of these engineered NK cells was demonstrated via interferon-gamma secretion assays. To improve clinical efficacy of ovarian cancer treatment, the effect of the chemotherapeutic agent cisplatin was evaluated together with CAR-transduced NK cell treatment. It was demonstrated that NK cells remain cytotoxic and active under cisplatin treatment and, importantly, that sequential treatment with cisplatin followed by CAR-NK cells led to the strongest killing effect. The specific eradication of ovarian CSCs by anti-CD133-CAR expressing NK92 cells represents a promising strategy and, when confirmed in vivo, shall be the basis of future clinical studies with the aim to prevent recurrent disease.

An In Vitro Expansion System for Generation of Human iPS Cell-Derived Hepatic Progenitor-Like Cells Exhibiting a Bipotent Differentiation Potential

PubMed Central

Yanagida, Ayaka; Ito, Keiichi; Chikada, Hiromi; Nakauchi, Hiromitsu; Kamiya, Akihide

2013-01-01

Hepatoblasts, hepatic stem/progenitor cells in liver development, have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In regenerative medicine and drug screening for the treatment of severe liver diseases, human induced pluripotent stem (iPS) cell-derived mature functional hepatocytes are considered to be a potentially good cell source. However, induction of proliferation of these cells is difficult ex vivo. To circumvent this problem, we generated hepatic progenitor-like cells from human iPS cells using serial cytokine treatments in vitro. Highly proliferative hepatic progenitor-like cells were purified by fluorescence-activated cell sorting using antibodies against CD13 and CD133 that are known cell surface markers of hepatic stem/progenitor cells in fetal and adult mouse livers. When the purified CD13highCD133+ cells were cultured at a low density with feeder cells in the presence of suitable growth factors and signaling inhibitors (ALK inhibitor A-83-01 and ROCK inhibitor Y-27632), individual cells gave rise to relatively large colonies. These colonies consisted of two types of cells expressing hepatocytic marker genes (hepatocyte nuclear factor 4α and α-fetoprotein) and a cholangiocytic marker gene (cytokeratin 7), and continued to proliferate over long periods of time. In a spheroid formation assay, these cells were found to express genes required for mature liver function, such as cytochrome P450 enzymes, and secrete albumin. When these cells were cultured in a suitable extracellular matrix gel, they eventually formed a cholangiocytic cyst-like structure with epithelial polarity, suggesting that human iPS cell-derived hepatic progenitor-like cells have a bipotent differentiation ability. Collectively these data indicate that this novel procedure using an in vitro expansion system is useful for not only liver regeneration but also for the determination of molecular mechanisms that regulate liver development. PMID:23935837

An in vitro expansion system for generation of human iPS cell-derived hepatic progenitor-like cells exhibiting a bipotent differentiation potential.

PubMed

Yanagida, Ayaka; Ito, Keiichi; Chikada, Hiromi; Nakauchi, Hiromitsu; Kamiya, Akihide

2013-01-01

Hepatoblasts, hepatic stem/progenitor cells in liver development, have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In regenerative medicine and drug screening for the treatment of severe liver diseases, human induced pluripotent stem (iPS) cell-derived mature functional hepatocytes are considered to be a potentially good cell source. However, induction of proliferation of these cells is difficult ex vivo. To circumvent this problem, we generated hepatic progenitor-like cells from human iPS cells using serial cytokine treatments in vitro. Highly proliferative hepatic progenitor-like cells were purified by fluorescence-activated cell sorting using antibodies against CD13 and CD133 that are known cell surface markers of hepatic stem/progenitor cells in fetal and adult mouse livers. When the purified CD13(high)CD133(+) cells were cultured at a low density with feeder cells in the presence of suitable growth factors and signaling inhibitors (ALK inhibitor A-83-01 and ROCK inhibitor Y-27632), individual cells gave rise to relatively large colonies. These colonies consisted of two types of cells expressing hepatocytic marker genes (hepatocyte nuclear factor 4α and α-fetoprotein) and a cholangiocytic marker gene (cytokeratin 7), and continued to proliferate over long periods of time. In a spheroid formation assay, these cells were found to express genes required for mature liver function, such as cytochrome P450 enzymes, and secrete albumin. When these cells were cultured in a suitable extracellular matrix gel, they eventually formed a cholangiocytic cyst-like structure with epithelial polarity, suggesting that human iPS cell-derived hepatic progenitor-like cells have a bipotent differentiation ability. Collectively these data indicate that this novel procedure using an in vitro expansion system is useful for not only liver regeneration but also for the determination of molecular mechanisms that regulate liver development.

The small molecule survivin inhibitor YM155 may be an effective treatment modality for colon cancer through increasing apoptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Wan Lu, E-mail: lvvlchina@msn.cn; Lee, Mi-Ra, E-mail: mira1125@yonsei.ac.kr; Cho, Mee-Yon, E-mail: meeyon@yonsei.ac.kr

Survivin has a known beneficial role in the survival of both cancer cells and normal cells. Therapies targeting survivin have been proposed as an alternative treatment modality for various tumors; however, finding the proper indication for this toxic therapy is critical for reducing unavoidable side effects. We recently observed that high survivin expression in CD133{sup +} cells is related to chemoresistance in Caco-2 colon cancer cells. However, the effect of survivin-targeted therapy on CD133{sup +} colon cancer is unknown. In this study, we investigated the roles of CD133 and survivin expression in colon cancer biology in vitro and comparatively analyzed themore » anticancer effects of survivin inhibitor on CD133{sup +} cells (ctrl-siRNA group) and small interfering RNA (siRNA)-induced CD133{sup −} cells (CD133-siRNA group) obtained from a single colon cancer cell line. CD133 knockdown via siRNA transfection did not change the tumorigenicity of cells, although in vitro survivin expression levels in CD133{sup +} cells were higher than those in siRNA-induced CD133{sup −} cells. The transfection procedure seemed to induce survivin expression. Notably, a significant number of CD133{sup −} cells (33.8%) was found in the cell colonies of the CD133-siRNA group. In the cell proliferation assay after treatment, YM155 and a combination of YM155 and 5-fluorouracil (5-FU) proved to be far more effective than 5-FU alone. A significantly increased level of apoptosis was observed with increasing doses of YM155 in all groups. However, significant differences in therapeutic effect and apoptosis among the mock, ctrl-siRNA, and CD133-siRNA groups were not detected. Survivin inhibitor is an effective treatment modality for colon cancer; however, the role of CD133 and the use of survivin expression as a biomarker for this targeted therapy must be verified.« less

CD133 antibody conjugation to decellularized human heart valves intended for circulating cell capture.

PubMed

Vossler, John D; Min Ju, Young; Williams, J Koudy; Goldstein, Steven; Hamlin, James; Lee, Sang Jin; Yoo, James J; Atala, Anthony

2015-09-03

The long term efficacy of tissue based heart valve grafts may be limited by progressive degeneration characterized by immune mediated inflammation and calcification. To avoid this degeneration, decellularized heart valves with functionalized surfaces capable of rapid in vivo endothelialization have been developed. The aim of this study is to examine the capacity of CD133 antibody-conjugated valve tissue to capture circulating endothelial progenitor cells (EPCs). Decellularized human pulmonary valve tissue was conjugated with CD133 antibody at varying concentrations and exposed to CD133 expressing NTERA-2 cl.D1 (NT2) cells in a microflow chamber. The amount of CD133 antibody conjugated on the valve tissue surface and the number of NT2 cells captured in the presence of shear stress was measured. Both the amount of CD133 antibody conjugated to the valve leaflet surface and the number of adherent NT2 cells increased as the concentration of CD133 antibody present in the surface immobilization procedure increased. The data presented in this study support the hypothesis that the rate of CD133(+) cell adhesion in the presence of shear stress to decellularized heart valve tissue functionalized by CD133 antibody conjugation increases as the quantity of CD133 antibody conjugated to the tissue surface increases.

Enhanced radiosensitivity and radiation-induced apoptosis in glioma CD133-positive cells by knockdown of SirT1 expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, C.-J.; Hsu, C.-C.; Department of Surgery, Chi-Mei Medical Center, Taipei, Taiwan

2009-03-06

CD133-expressing glioma cells play a critical role in tumor recovery after treatment and are resistant to radiotherapy. Herein, we demonstrated that glioblastoma-derived CD133-positive cells (GBM-CD133{sup +}) are capable of self-renewal and express high levels of embryonic stem cell genes and SirT1 compared to GBM-CD133{sup -} cells. To evaluate the role of SirT1 in GBM-CD133{sup +}, we used a lentiviral vector expressing shRNA to knock-down SirT1 expression (sh-SirT1) in GBM-CD133{sup +}. Silencing of SirT1 significantly enhanced the sensitivity of GBM-CD133{sup +} to radiation and increased the level of radiation-mediated apoptosis. Importantly, knock-down of SirT1 increased the effectiveness of radiotherapy in themore » inhibition of tumor growth in nude mice transplanted with GBM-CD133{sup +}. Kaplan-Meier survival analysis indicated that the mean survival rate of GBM-CD133{sup +} mice treated with radiotherapy was significantly improved by Sh-SirT1 as well. In sum, these results suggest that SirT1 is a potential target for increasing the sensitivity of GBM and glioblastoma-associated cancer stem cells to radiotherapy.« less

Hyperglycemic Stress Impairs the Stemness Capacity of Kidney Stem Cells in Rats.

PubMed

Yang, Guang; Jia, Yali; Li, Chunlin; Cheng, Qingli; Yue, Wen; Pei, Xuetao

2015-01-01

The incidence of acute kidney injury in patients with diabetes is significantly higher than that of patients without diabetes, and may be associated with the poor stemness capacity of kidney stem cells (KSCs) and limited recovery of injured renal tubules. To investigate the effects of hyperglycemic stress on KSC stemness, KSCs were isolated from the rat renal papilla and analyzed for their self-renewal and differentiation abilities. Our results showed that isolated KSCs expressed the mesenchymal stem cell markers N-cadherin, Nestin, CD133, CD29, CD90, and CD73. Moreover, KSCs co-cultured with hypoxia-injured renal tubular epithelial cell (RTECs) induced the expression of the mature epithelial cell marker CK18, suggesting that the KSCs could differentiate into RTECs in vitro. However, KSC proliferation, differentiation ability and tolerance to hypoxia were decreased in high-glucose cultures. Taken together, these results suggest the high-glucose microenvironment can damage the reparative ability of KSCs. It may result in a decreased of recovery capability of renal tubules from injury.

Cancer Stem Cell Hierarchy in Glioblastoma Multiforme

PubMed Central

Bradshaw, Amy; Wickremsekera, Agadha; Tan, Swee T.; Peng, Lifeng; Davis, Paul F.; Itinteang, Tinte

2016-01-01

Glioblastoma multiforme (GBM), an aggressive tumor that typically exhibits treatment failure with high mortality rates, is associated with the presence of cancer stem cells (CSCs) within the tumor. CSCs possess the ability for perpetual self-renewal and proliferation, producing downstream progenitor cells that drive tumor growth. Studies of many cancer types have identified CSCs using specific markers, but it is still unclear as to where in the stem cell hierarchy these markers fall. This is compounded further by the presence of multiple GBM and glioblastoma cancer stem cell subtypes, making investigation and establishment of a universal treatment difficult. This review examines the current knowledge on the CSC markers SALL4, OCT-4, SOX2, STAT3, NANOG, c-Myc, KLF4, CD133, CD44, nestin, and glial fibrillary acidic protein, specifically focusing on their use and validity in GBM research and how they may be utilized for investigations into GBM’s cancer biology. PMID:27148537

Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth

DOE Office of Scientific and Technical Information (OSTI.GOV)

Varga, Nora; Vereb, Zoltan; Rajnavoelgyi, Eva

2011-10-28

Highlights: Black-Right-Pointing-Pointer MSC like cells were derived from hESC by a simple and reproducible method. Black-Right-Pointing-Pointer Differentiation and immunosuppressive features of MSCl cells were similar to bmMSC. Black-Right-Pointing-Pointer MSCl cells as feeder cells support the undifferentiated growth of hESC. -- Abstract: Mesenchymal stem cell like (MSCl) cells were generated from human embryonic stem cells (hESC) through embryoid body formation, and isolated by adherence to plastic surface. MSCl cell lines could be propagated without changes in morphological or functional characteristics for more than 15 passages. These cells, as well as their fluorescent protein expressing stable derivatives, efficiently supported the growth ofmore » undifferentiated human embryonic stem cells as feeder cells. The MSCl cells did not express the embryonic (Oct4, Nanog, ABCG2, PODXL, or SSEA4), or hematopoietic (CD34, CD45, CD14, CD133, HLA-DR) stem cell markers, while were positive for the characteristic cell surface markers of MSCs (CD44, CD73, CD90, CD105). MSCl cells could be differentiated toward osteogenic, chondrogenic or adipogenic directions and exhibited significant inhibition of mitogen-activated lymphocyte proliferation, and thus presented immunosuppressive features. We suggest that cultured MSCl cells can properly model human MSCs and be applied as efficient feeders in hESC cultures.« less

Effect of Bmi-1-mediated NF-κB signaling pathway on the stem-like properties of CD133+ human liver cancer cells.

PubMed

Ma, De-Qiang; Zhang, Yin-Hua; Ding, De-Ping; Li, Juan; Chen, Lin-Li; Tian, You-You; Ao, Kang-Jian

2018-05-11

To investigate the impact of Bmi-1-mediated NF-κB pathway on the biological characteristics of CD133+ liver cancer stem cells (LCSCs). Flow cytometry was used to isolate CD133+ LCSC cells from Huh7, Hep3B, SK-hep1, and PLC/PRF-5 cells. CD133+ Huh7 cells were divided into Control, Blank, Bmi-1 siRNA, JSH-23 (NF-κB pathway inhibitor), and Bmi-1 + JSH-23 groups. The properties of CD133+ Huh7 cells were detected by the colony-formation and sphere-forming assays. Besides, Transwell assay was applied for the measurement of cell invasion and migration, immunofluorescence staining for the detection of NF-κB p65 nuclear translocation, and qRT-PCR and Western blotting for the determination of SOX2, NANOG, OCT4, Bmi-1, and NF-κB p65 expression. CD133+ Huh-7 cells were chosen as the experiment subjects after flow cytometry. Compared with CD133- Huh-7 cells, the expression of CD133, OCT4, SOX2, NANOG, Bmi-1, and NF-κB p65, the nuclear translocation of NF-κB p65, the number of cell colonies and Sphere formation, as well as the abilities of invasion and migration were observed to be increased in CD133+ Huh-7 cells, which was inhibited after treated with Bmi-1 siRNA or JSH-23, meanwhile, the cell cycle was arrested at the G0/G1 and S phases with apparently enhanced cell apoptosis. Importantly, no significant differences in the biological characteristics of CD133 + Huh-7 cells were found between the Blank group and Bmi-1 + JSH-23 group. Down-regulating Bmi-1 may inhibit the biological properties of CD133+ LCSC by blocking NF-κB signaling pathway, which lays a scientific foundation for the clinical treatment of liver cancer.

Flow cytometric techniques for detection of candidate cancer stem cell subpopulations in canine tumour models.

PubMed

Blacking, T M; Waterfall, M; Samuel, K; Argyle, D J

2012-12-01

The cancer stem cell (CSC) hypothesis proposes that tumour growth is maintained by a distinct subpopulation of 'CSC'. This study applied flow cytometric methods, reported to detect CSC in both primary and cultured cancer cells of other species, to identify candidate canine subpopulations. Cell lines representing diverse canine malignancies, and cells derived from spontaneous canine tumours, were evaluated for expression of stem cell-associated surface markers (CD34, CD44, CD117 and CD133) and functional properties [Hoecsht 33342 efflux, aldehyde dehydrogenase (ALDH) activity]. No discrete marker-defined subsets were identified within established cell lines; cells derived directly from spontaneous tumours demonstrated more heterogeneity, although this diminished upon in vitro culture. Functional assays produced variable results, suggesting context-dependency. Flow cytometric methods may be adopted to identify putative canine CSC. Whilst cell lines are valuable in assay development, primary cells may provide a more rewarding model for studying tumour heterogeneity in the context of CSC. However, it will be essential to fully characterize any candidate subpopulations to ensure that they meet CSC criteria. © 2011 Blackwell Publishing Ltd.

CD133 antisense suppresses cancer cell growth and increases sensitivity to cisplatin in vitro.

PubMed

Blancas-Mosqueda, Marisol; Zapata-Benavides, Pablo; Zamora-Ávila, Diana; Saavedra-Alonso, Santiago; Manilla-Muñoz, Edgar; Franco-Molina, Moisés; DE LA Peña, Carmen Mondragón; Rodríguez-Padilla, Cristina

2012-11-01

The increased incidence of cancer in recent years is associated with a high rate of mortality. Numerous types of cancer have a low percentage of CD133(+) cells, which have similar features to stem cells. The CD133 molecule is involved in apoptosis and cell proliferation. The aim of this study was to determine the biological effect of CD133 suppression and its role in the chemosensitization of cancer cell lines. RT-PCR and immunocytochemical analyses indicated that CD133 was expressed in the cancer cell lines B16F10, MCF7 and INER51. Downregulation of CD133 by transfection with an antisense sequence (As-CD133) resulted in a decrease in cancer cell viability of up to 52, 47 and 22% in B16F10, MCF-7 and INER51 cancer cell lines, respectively. This decreased viability appeared to be due to the induction of apoptosis. In addition, treatment with As-CD133 in combination with cisplatin had a synergic effect in all of the cancer cell lines analyzed, and in particular, significantly decreased the viability of B16F10 cancer cells compared with each treatment separately (3.1% viability for the combined treatment compared with 48% for 0.4 μg As-CD133 and 25% for 5 ng/μl cisplatin; P<0.05). The results indicate that the downregulation of CD133 by antisense is a potential therapeutic target for cancer and has a synergistic effect when administered with minimal doses of the chemotherapeutic drug cisplatin, suggesting that this combination strategy may be applied in cancer treatment.

Phenanthrene exposure induces cardiac hypertrophy via reducing miR-133a expression by DNA methylation

PubMed Central

Huang, Lixing; Xi, Zhihui; Wang, Chonggang; Zhang, Youyu; Yang, Zhibing; Zhang, Shiqi; Chen, Yixin; Zuo, Zhenghong

2016-01-01

Growing evidence indicates that there is an emerging link between environmental pollution and cardiac hypertrophy, while the mechanism is unclear. The objective of this study was to examine whether phenanthrene (Phe) could cause cardiac hypertrophy, and elucidate the molecular mechanisms involved. We found that: 1) Phe exposure increased the heart weight and cardiomyocyte size of rats; 2) Phe exposure led to enlarged cell size, and increased protein synthesis in H9C2 cells; 3) Phe exposure induced important markers of cardiac hypertrophy, such as atrial natriuretic peptide, B-type natriuretic peptide, and c-Myc in H9C2 cells and rat hearts; 4) Phe exposure perturbed miR-133a, CdC42 and RhoA, which were key regulators of cardiac hypertrophy, in H9C2 cells and rat hearts; 5) Phe exposure induced DNA methyltransferases (DNMTs) in H9C2 cells and rat hearts; 6) Phe exposure led to methylation of CpG sites within the miR-133a locus and reduced miR-133a expression in H9C2 cells; 7) DNMT inhibition and miR-133a overexpression could both alleviate the enlargement of cell size and perturbation of CdC42 and RhoA caused by Phe exposure. These results indicated that Phe could induce cardiomyocyte hypertrophy in the rat and H9C2 cells. The mechanism might involve reducing miR-133a expression by DNA methylation. PMID:26830171

MPEG-CS/Bmi-1RNAi Nanoparticles Synthesis and Its Targeted Inhibition Effect on CD133+ Laryngeal Stem Cells.

PubMed

Wei, Xudong; He, Jian; Wang, Jingyu; Wang, Wei

2018-03-01

Previous studies have confirmed that CD133+ cells in laryngeal tumor tissue have the characteristics of cancer stem cells. Bmi-1 gene expression is central to the tumorigenicity of CD133+ cells. In this study, we tried to develop a new siRNA carrier system using chitosan-methoxypolyethylene nanoparticles (CS-mPEG-NPs) that exhibit higher tumor-targeting ability and enhanced gene silencing efficacy in CD133+ tumor stem cells. It is hoped to block the self-renewal and kill the stem cells of laryngeal carcinoma. The mPEG-CS-Bmi-1RNAi-NPs were synthesized and their characters were checked. The changes in invasion ability and sensitivity to radiotherapy and chemotherapy of CD133+Hep-2 tumor cells were observed after Bmi-1 gene silencing. The mPEG-CS-Bmi-1RNAi-NPs synthesized in this experiment have a regular spherical form, a mean size of 139.70 ±6.40 nm, an encapsulation efficiency of 85.21 ± 1.94%, with drug loading capacity of 18.47 ± 1.83%, as well as low cytotoxicity, providing good protection to the loaded gene, strong resistance to nuclease degradation and high gene transfection efficiency. After Bmi-1 gene silencing, the invasion ability of CD133+ cells was weakened. Co-cultured with paclitaxel, the survival rates of CD133+Bmi-1RNAi cells were lower. After radiotherapy, the mean growth inhibition rate of CD133+/Bmi-1RNAi cells was significantly lower than CD133+ cells. In conclusion, the mPEG-CS nano-carrier is an ideal vector in gene therapy, while silencing the Bmi-1 gene can enhance the sensitivity of CD133+ tumor stem cells to chemoradiotherapy and abate their invasion ability.

Circulating Endothelial Progenitor Cells Present an Inflammatory Phenotype and Function in Patients With Alcoholic Liver Cirrhosis

PubMed Central

Kaur, Savneet; Sehgal, Rashi; Shastry, Saggere M.; McCaughan, Geoffrey; McGuire, Helen M.; Fazekas St de Groth, Barbara; Sarin, Shiv; Trehanpati, Nirupma; Seth, Devanshi

2018-01-01

Background and Aim: Endothelial progenitor cells (EPCs) have been implicated in liver injury and repair. However, the phenotype and potential of these heterogenous EPCs remain elusive. In particular, their involvement in the pathogenesis of alcoholic liver cirrhosis (ALC) remains unclear. The current study extensively characterized the phenotype and functions of EPCs to understand their role in ALC pathogenesis. Methods: Circulating EPCs were identified as CD34+CD133+CD31+ cells by flow cytometer in ALC patients (n = 7) and healthy controls (HC, n = 7). A comprehensive characterization of circulating EPCs using more than 30 phenotype markers was performed by mass cytometer time of flight (CyTOF) in an independent cohort of age and gender matched ALC patients (n = 4) and controls (n = 5). Ex vivo cultures of circulating EPCs from ALC patients (n = 20) and controls (n = 18) were also tested for their functions, including colony formation, LDL uptake, lectin binding and cytokine secretion (ELISA). Results: Three distinct populations of circulating EPCs (CD34+CD133+CD31+) were identified, classified on their CD45 expression (negative: CD45−; intermediate: CD45int; high: CD45hi). CD45int and CD45hi EPCs significantly increased in ALC patients compared to controls (p-val = 0.006). CyTOF data showed that CD45hi EPCs were distinct from CD45− and CD45int EPCs, with higher expression of T cell and myeloid markers, including CD3, CD4, HLA-DR, and chemokine receptors, CCR2, CCR5, CCR7, and CX3CR1. Similar to circulating EPCs, percentage of CD45hiCD34+CD31+ EPCs in ex-vivo cultures from patients, were significantly higher compared to controls (p < 0.05). Cultured EPCs from patients also showed increased LDL uptake, lectin binding and release of TNF-alpha, RANTES, FGF-2, and VEGF. Conclusions: We report the first extensive characterization of circulating human EPCs with distinct EPC subtypes. Increase in CD45hi EPC subtype in ALC patients with enhanced functions, inflammatory cytokines and angiogenic mediators in patients suggests an inflammatory role for these cells in ALC. PMID:29872403

CD133 antisense suppresses cancer cell growth and increases sensitivity to cisplatin in vitro

PubMed Central

BLANCAS-MOSQUEDA, MARISOL; ZAPATA-BENAVIDES, PABLO; ZAMORA-ÁVILA, DIANA; SAAVEDRA-ALONSO, SANTIAGO; MANILLA-MUÑOZ, EDGAR; FRANCO-MOLINA, MOISÉS; DE LA PEÑA, CARMEN MONDRAGÓN; RODRÍGUEZ-PADILLA, CRISTINA

2012-01-01

The increased incidence of cancer in recent years is associated with a high rate of mortality. Numerous types of cancer have a low percentage of CD133+ cells, which have similar features to stem cells. The CD133 molecule is involved in apoptosis and cell proliferation. The aim of this study was to determine the biological effect of CD133 suppression and its role in the chemosensitization of cancer cell lines. RT-PCR and immunocytochemical analyses indicated that CD133 was expressed in the cancer cell lines B16F10, MCF7 and INER51. Downregulation of CD133 by transfection with an antisense sequence (As-CD133) resulted in a decrease in cancer cell viability of up to 52, 47 and 22% in B16F10, MCF-7 and INER51 cancer cell lines, respectively. This decreased viability appeared to be due to the induction of apoptosis. In addition, treatment with As-CD133 in combination with cisplatin had a synergic effect in all of the cancer cell lines analyzed, and in particular, significantly decreased the viability of B16F10 cancer cells compared with each treatment separately (3.1% viability for the combined treatment compared with 48% for 0.4 μg As-CD133 and 25% for 5 ng/μl cisplatin; P<0.05). The results indicate that the downregulation of CD133 by antisense is a potential therapeutic target for cancer and has a synergistic effect when administered with minimal doses of the chemotherapeutic drug cisplatin, suggesting that this combination strategy may be applied in cancer treatment. PMID:23226746

MIA PaCa-2 and PANC-1 - pancreas ductal adenocarcinoma cell lines with neuroendocrine differentiation and somatostatin receptors.

PubMed

Gradiz, Rui; Silva, Henriqueta C; Carvalho, Lina; Botelho, Maria Filomena; Mota-Pinto, Anabela

2016-02-17

Studies using cell lines should always characterize these cells to ensure that the results are not distorted by unexpected morphological or genetic changes possibly due to culture time or passage number. Thus, the aim of this study was to describe those MIA PaCa-2 and PANC-1 cell line phenotype and genotype characteristics that may play a crucial role in pancreatic cancer therapeutic assays, namely neuroendocrine chemotherapy and peptide receptor radionuclide therapy. Epithelial, mesenchymal, endocrine and stem cell marker characterization was performed by immunohistochemistry and flow cytometry, and genotyping by PCR, gene sequencing and capillary electrophoresis. MIA PaCa-2 (polymorphism) expresses CK5.6, AE1/AE3, E-cadherin, vimentin, chromogranin A, synaptophysin, SSTR2 and NTR1 but not CD56. PANC-1 (pleomorphism) expresses CK5.6, MNF-116, vimentin, chromogranin A, CD56 and SSTR2 but not E-cadherin, synaptophysin or NTR1. MIA PaCA-1 is CD24(-), CD44(+/++), CD326(-/+) and CD133/1(-), while PANC-1 is CD24(-/+), CD44(+), CD326(-/+) and CD133/1(-). Both cell lines have KRAS and TP53 mutations and homozygous deletions including the first 3 exons of CDKN2A/p16(INK4A), but no SMAD4/DPC4 mutations or microsatellite instability. Both have neuroendocrine differentiation and SSTR2 receptors, precisely the features making them suitable for the therapies we propose to assay in future studies.

VCAM-1 expression is upregulated by CD34+/CD133+-stem cells derived from septic patients

PubMed Central

Remmé, Christoph; Betzen, Christian; Tönshoff, Burkhard; Yard, Benito A.; Beck, Grietje; Rafat, Neysan

2018-01-01

CD34+/CD133+- cells are a bone marrow derived stem cell population, which presumably contain vascular progenitor cells and are associated with improved vascular repair. In this study, we investigated whether the adhesion molecules ICAM-1 (intercellular adhesion molecule-1), VCAM-1 (vascular adhesion molecule-1), E-selectin und L-selectin, which are involved in homing of vascular stem cells, are upregulated by CD34+/CD133+-stem cells from septic patients and would be associated with improved clinical outcome. Peripheral blood mononuclear cells from intensive care unit (ICU) patients with (n = 30) and without sepsis (n = 10), and healthy volunteers (n = 15) were isolated using Ficoll density gradient centrifugation. The expression of VCAM-1, ICAM-1, E-selectin and L-selectin was detected on CD34+/CD133+-stem cells by flow cytometry. The severity of disease was assessed by the Simplified Acute Physiology Score (SAPS) II. Serum concentrations of vascular endothelial growth factor (VEGF) and angiopoietin (Ang)-2 were determined by Enzyme-linked immunosorbent assay. The expression of VCAM-1, ICAM-1, E-selectin and L-selectin by CD34+/CD133+-stem cells was significantly upregulated in septic patients, and correlated with sepsis severity. Furthermore, high expression of VCAM-1 by CD34+/CD133+-stem cells revealed a positive association with mortalitiy (p<0.05). Furthermore, significantly higher serum concentrations of VEGF and Ang-2 were found in septic patients, however none showed a strong association with survival. Our data suggest, that VCAM-1 upregulation on CD34+/CD133+-stem cells could play a crucial role in their homing in the course of sepsis. An increase in sepsis severity resulted in both and increase in CD34+/CD133+-stem cells and VCAM-1-expression by those cells, which might reflect an increase in need for vascular repair. PMID:29601599

Lgr5-positive cells are cancer stem cells in skin squamous cell carcinoma.

PubMed

Liu, Shunli; Gong, Zhenyu; Chen, Mingrui; Liu, Benli; Bian, Donghui; Wu, Kai

2014-11-01

Cancer stem cells (CSCs) in most human tumors are commonly identified and enriched using similar strategies for identifying normal stem cells, including flow cytometry assays for side population, high aldehyde dehydrogenase (ALDH) activity, and CD133 positivity. Thus, development of a method for isolating a specific cancer using cancer-specific characteristic appears to be potentially important. Here, we reported extremely high Lgr5 levels in the specimen from skin squamous cell carcinoma (SCC) in patients. Using SCC cell line A431, we detected high Lgr5 and CD133 levels in ALDH-high or side population from these cancer cells. To figure out whether Lgr5 is a marker of CSCs in SCC, we transfected A431 cells with a Lgr5-creERT-2A-DTR/Cag-Loxp-GFP-STOP-Loxp-RFP plasmid and purified transfected cells (tA431) based on GFP by flow cytometry. 4-Hydroxytamoxifen (4-OHT) was given to label Lgr5-positive cells with RFP, for comparison to GFP-positive Lgr5-negative cells. Lgr5-positive cells grew significantly faster than Lgr5-negative cells, and the fold increase in growth of Lgr5-positive vs Lgr5-negative cells is significantly higher than SP vs non-SP, or ALDH-high vs ALDH-low, or CD133-positive vs CD133-negative cells. Moreover, in Lgr5-negative population, Lgr5-positive re-appeared in culture with time, suggesting that Lgr5-positive cells can be regenerated from Lgr5-negative cells. Furthermore, the growth of tA431 cells significantly decreased upon a single dose of diphtheria toxin (DT)/4-OHT to eliminate Lgr5-positive cell lineage, while multiple doses of DT/4-OHT nearly completely inhibited tA431 cell growth. Taken together, our data provide compelling data to demonstrate that Lgr5-positive cells are CSCs in skin SCC.

CD24 negative lung cancer cells, possessing partial cancer stem cell properties, cannot be considered as cancer stem cells.

PubMed

Xu, Haineng; Mu, Jiasheng; Xiao, Jing; Wu, Xiangsong; Li, Maolan; Liu, Tianrun; Liu, Xinyuan

2016-01-01

Cancer stem cells (CSCs) play vital role in lung cancer progression, resistance, metastasis and relapse. Identifying lung CSCs makers for lung CSCs targeting researches are critical for lung cancer therapy. In this study, utilizing previous identified lung CSCs as model, we compared the expression of CD24, CD133 and CD44 between CSCs and non-stem cancer cells. Increased ratio of CD24- cells were found in CSCs. CD24- cells were then sorted by flow cytometry and their proliferative ability, chemo-resistance property and in vivo tumor formation abilities were detected. A549 CD24- cells formed smaller colonies, slower proliferated in comparison to A549 CD24+ cells. Besides, A549 CD24- exhibited stronger resistance to chemotherapy drug. However, A549 CD24- didn't exert any stronger tumor formation ability in vivo, which is the gold standard of CSCs. These results showed that CD24- A549 cells showed some properties of CSCs but not actually CSCs. This study provides evidence that CD24 cannot be considered as lung CSCs marker.

CD133+ cell content correlates with tumour growth in melanomas from skin with chronic sun-induced damage.

PubMed

González-Herrero, I; Romero-Camarero, I; Cañueto, J; Cardeñoso-Álvarez, E; Fernández-López, E; Pérez-Losada, J; Sánchez-García, I; Román-Curto, C

2013-10-01

Melanoma is responsible for almost 80% of the deaths attributed to skin cancer. Stem cells, defined by CD133 expression, have been implicated in melanoma tumour growth, but their specific role is still uncertain. We hypothesized that the phenotypic heterogeneity of human cutaneous melanomas is related to their content of CD133+ cells. We compared the percentages of CD133+ cells in 29 tumours from four classic types of melanoma: lentigo maligna melanoma (LMM), superficial spreading melanoma, nodular melanoma and acral lentiginous melanoma (ALM). Also, we compared the percentages of CD133+ cells in melanomas with different degrees of exposure to ultraviolet radiation: 16 melanomas from skin with chronic sun-induced damage and 13 melanomas from skin without such damage. We found a statistically significant increase of CD133+ cells in three different contexts: in melanomas arising on skin with signs of chronic sun-induced damage vs. nonexposed skin, in melanomas in situ vs. invasive melanomas, and in LMM vs. ALM. The proportions of CD133+ cells did not differ among samples of normal skin with different degrees of sun exposure. A distinct subpopulation of CD133+CXCR4+ cancer stem cells (CSCs) was identified and shown to be related to the invasive phenotype of the tumours. Here, we provide evidence showing, for the first time, that an increase in the CD133+ cell content is associated both with melanomas arising on skin with signs of chronic sun-induced damage and in melanomas in situ with better prognosis. Moreover, our study further confirms the existence of a subpopulation of CD133+CXCR4+ CSCs in cutaneous melanomas with invasive phenotype and poor prognosis. © 2013 British Association of Dermatologists.

Analyses of cell surface molecules on hepatic stem/progenitor cells in mouse fetal liver.

PubMed

Kakinuma, Sei; Ohta, Haruhiko; Kamiya, Akihide; Yamazaki, Yuji; Oikawa, Tsunekazu; Okada, Ken; Nakauchi, Hiromitsu

2009-07-01

Hepatic stem/progenitor cells possess active proliferative ability and the capacity for differentiation into hepatic and cholangiocytic lineages. Our group and others have shown that a prospectively defined population in mid-gestational fetal liver contains hepatic stem/progenitor cells. However, the phenotypes of such cells are incompletely elucidated. We analyzed the profile of cell-surface molecules on primary hepatic stem/progenitor cells. Expression of cell surface molecules on primary hepatic stem/progenitor cells in mouse mid-gestational fetal liver was analyzed using flow cytometric multicolor analyses and colony-formation assays. The potential of the cells for liver repopulation was examined by transplantation assay. We found that CD13 (aminopeptidase N) was detected on the cells of the previously reported (Dlk/Pref-1(+)) hepatic stem/progenitor fraction. Colony-formation assays revealed that the CD13(+) fraction, compared with the Dlk(+) fraction, of non-hematopoietic cells in fetal liver was enriched in hepatic stem/progenitor cells. Transplantation assay showed the former fraction exhibited repopulating potential in regenerating liver. Moreover, flow cytometric analysis for over 90 antigens demonstrated enrichment of hepatic stem/progenitor cells using several positive selection markers, including (hitherto unknown) CD13, CD73, CD106, and CD133. Our data indicated that CD13 is a positive selection marker for hepatic stem/progenitor cells in mid-gestational fetal liver.

Increase of endothelial progenitor cells in acute graft-versus-host disease after allogeneic haematopoietic stem cell transplantation for acute myeloid leukaemia.

PubMed

Medinger, Michael; Heim, Dominik; Gerull, Sabine; Halter, Jörg; Krenger, Werner; Buser, Andreas; Lengerke, Claudia; Bucher, Christoph; Passweg, Jakob

2016-08-01

Circulating endothelial progenitor cells (EPCs; CD31+ CD34(bright)CD133+ CD45(dim) cells) are novel markers of endothelial dysfunction and related to inflammatory processes such as acute graft-versus-host disease (aGvHD). 47 patients with acute myeloid leukaemia (AML) who were in complete remission as they underwent allogeneic hematopoietic stem cell transplantation with myeloablative conditioning with PBSC as stem cell source were enrolled in the study. Blood samples for the quantitative analysis of circulating EPC levels were drawn at different time points in patients with and without aGvHD. CD34+ VEGFR2/KDR+ CD133+ triple-positive cells identified among CD34+ cells by FACS. EPC were quantified and data are presented as cells/ml whole blood. Circulating EPC levels were not significantly different in patients with and without aGvHD prior to conditioning (baseline) and at the time of engraftment. However, at diagnosis of aGvHD≥grade 2, EPC levels increased whereas in patients without aGvHD the EPC levels remained significantly lower (3021±278 versus 2322±195 cells/ml; p<0.001). Patients with steroid-refractory aGvHD had high levels of EPC throughout. EPC levels fell in responding patients. Our results demonstrate that the number of circulating EPCs is increased in patients with aGvHD compared to patients without aGvHD. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cervical Cancer Stem Cells Selectively Overexpress HPV Oncoprotein E6 that Controls Stemness and Self-Renewal through Upregulation of HES1.

PubMed

Tyagi, Abhishek; Vishnoi, Kanchan; Mahata, Sutapa; Verma, Gaurav; Srivastava, Yogesh; Masaldan, Shashank; Roy, Bal Gangadhar; Bharti, Alok C; Das, Bhudev C

2016-08-15

Perturbation of keratinocyte differentiation by E6/E7 oncoproteins of high-risk human papillomaviruses that drive oncogenic transformation of cells in squamocolumnar junction of the uterine cervix may confer "stem-cell like" characteristics. However, the crosstalk between E6/E7 and stem cell signaling during cervical carcinogenesis is not well understood. We therefore examined the role of viral oncoproteins in stem cell signaling and maintenance of stemness in cervical cancer. Isolation and enrichment of cervical cancer stem-like cells (CaCxSLCs) was done from cervical primary tumors and cancer cell lines by novel sequential gating using a set of functional and phenotypic markers (ABCG2, CD49f, CD71, CD133) in defined conditioned media for assessing sphere formation and expression of self-renewal and stemness markers by FACS, confocal microscopy, and qRT-PCR. Differential expression level and DNA-binding activity of Notch1 and its downstream targets in CaCxSLCs as well as silencing of HPVE6/Hes1 by siRNA was evaluated by gel retardation assay, FACS, immunoblotting, and qRT-PCR followed by in silico and in vivo xenograft analysis. CaCxSLCs showed spheroid-forming ability, expressed self-renewal and stemness markers Oct4, Sox2, Nanog, Lrig1, and CD133, and selectively overexpressed E6 and HES1 transcripts in both cervical primary tumors and cancer cell lines. The enriched CaCxSLCs were highly tumorigenic and did recapitulate primary tumor histology in nude mice. siRNA silencing of HPVE6 or Hes1 abolished sphere formation, downregulated AP-1-STAT3 signaling, and induced redifferentiation. Our findings suggest the possible mechanism by which HPVE6 potentially regulate and maintain stem-like cancer cells through Hes1. Clin Cancer Res; 22(16); 4170-84. ©2016 AACR. ©2016 American Association for Cancer Research.

Glioblastoma stem cell differentiation into endothelial cells evidenced through live-cell imaging.

PubMed

Mei, Xin; Chen, Yin-Sheng; Chen, Fu-Rong; Xi, Shao-Yan; Chen, Zhong-Ping

2017-08-01

Glioblastoma cell-initiated vascularization is an alternative angiogenesis called vasculogenic mimicry. However, current knowledge on the mechanism of de novo vessel formation from glioblastoma stem cells (GSCs) is limited. Sixty-four glioblastoma samples from patients and 10 fluorescent glioma xenograft samples were examined by immunofluorescence staining for endothelial marker (CD34 and CD31) and glial cell marker (glial fibrillary acidic protein [GFAP]) expression. GSCs were then isolated from human glioblastoma tissue and CD133+/Sox2+ red fluorescent protein-containing (RFP)-GSC-1 cells were established. The ability of these cells to form vascular structures was examined by live-cell imaging of 3D cultures. CD34-GFAP or CD31-GFAP coexpressing glioblastoma-derived endothelial cells (GDEC) were found in 30 of 64 (46.9%) of clinical glioblastoma samples. In those 30 samples, GDEC were found to form vessel structures in 21 (70%) samples. Among 21 samples with GDEC vessels, the CD34+ GDEC vessels and CD31+ GDEC vessels accounted for about 14.16% and 18.08% of total vessels, respectively. In the xenograft samples, CD34+ GDEC were found in 7 out of 10 mice, and 4 out of 7 mice had CD34+ GDEC vessels. CD31+ GDEC were also found in 7 mice, and 4 mice had CD31+ GDEC vessels (10 mice in total). Through live-cell imaging, we observed gradual CD34 expression when cultured with vascular endothelial growth factor in some glioma cells, and a dynamic increase in endothelial marker expression in RFP-GSC-1 in vitro was recorded. Cells expressed CD34 (9.46%) after 6 hours in culture. The results demonstrated that GSCs may differentiate into endothelial cells and promote angiogenesis in glioblastomas. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Human dental pulp stem cells cultured in serum-free supplemented medium

PubMed Central

Bonnamain, Virginie; Thinard, Reynald; Sergent-Tanguy, Solène; Huet, Pascal; Bienvenu, Géraldine; Naveilhan, Philippe; Farges, Jean-Christophe; Alliot-Licht, Brigitte

2013-01-01

Growing evidence show that human dental pulp stem cells (DPSCs) could provide a source of adult stem cells for the treatment of neurodegenerative pathologies. In this study, DPSCs were expanded and cultured with a protocol generally used for the culture of neural stem/progenitor cells. Methodology: DPSC cultures were established from third molars. The pulp tissue was enzymatically digested and cultured in serum-supplemented basal medium for 12 h. Adherent (ADH) and non-adherent (non-ADH) cell populations were separated according to their differential adhesion to plastic and then cultured in serum-free defined N2 medium with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Both ADH and non-ADH populations were analyzed by FACS and/or PCR. Results: FACS analysis of ADH-DPSCs revealed the expression of the mesenchymal cell marker CD90, the neuronal marker CD56, the transferrin receptor CD71, and the chemokine receptor CXCR3, whereas hematopoietic stem cells markers CD45, CD133, and CD34 were not expressed. ADH-DPSCs expressed transcripts coding for the Nestin gene, whereas expression levels of genes coding for the neuronal markers β-III tubulin and NF-M, and the oligodendrocyte marker PLP-1 were donor dependent. ADH-DPSCs did not express the transcripts for GFAP, an astrocyte marker. Cells of the non-ADH population that grew as spheroids expressed Nestin, β-III tubulin, NF-M and PLP-1 transcripts. DPSCs that migrated out of the spheroids exhibited an odontoblast-like morphology and expressed a higher level of DSPP and osteocalcin transcripts than ADH-DPSCs. Conclusion: Collectively, these data indicate that human DPSCs can be expanded and cultured in serum-free supplemented medium with EGF and bFGF. ADH-DPSCs and non-ADH populations contained neuronal and/or oligodendrocyte progenitors at different stages of commitment and, interestingly, cells from spheroid structures seem to be more engaged into the odontoblastic lineage than the ADH-DPSCs. PMID:24376422

Hematopoietic Stem Cells in Neonates: Any Differences between Very Preterm and Term Neonates?

PubMed Central

Wisgrill, Lukas; Schüller, Simone; Bammer, Markus; Berger, Angelika; Pollak, Arnold; Radke, Teja Falk; Kögler, Gesine; Spittler, Andreas; Helmer, Hanns; Husslein, Peter; Gortner, Ludwig

2014-01-01

Background In the last decades, human full-term cord blood was extensively investigated as a potential source of hematopoietic stem and progenitor cells (HSPCs). Despite the growing interest of regenerative therapies in preterm neonates, only little is known about the biological function of HSPCs from early preterm neonates under different perinatal conditions. Therefore, we investigated the concentration, the clonogenic capacity and the influence of obstetric/perinatal complications and maternal history on HSPC subsets in preterm and term cord blood. Methods CD34+ HSPC subsets in UCB of 30 preterm and 30 term infants were evaluated by flow cytometry. Clonogenic assays suitable for detection of the proliferative potential of HSPCs were conducted. Furthermore, we analyzed the clonogenic potential of isolated HSPCs according to the stem cell marker CD133 and aldehyde dehydrogenase (ALDH) activity. Results Preterm cord blood contained a significantly higher concentration of circulating CD34+ HSPCs, especially primitive progenitors, than term cord blood. The clonogenic capacity of HSPCs was enhanced in preterm cord blood. Using univariate analysis, the number and clonogenic potential of circulating UCB HSPCs was influenced by gestational age, birth weight and maternal age. Multivariate analysis showed that main factors that significantly influenced the HSPC count were maternal age, gestational age and white blood cell count. Further, only gestational age significantly influenced the clonogenic potential of UCB HSPCs. Finally, isolated CD34+/CD133+, CD34+/CD133– and ALDHhigh HSPC obtained from preterm cord blood showed a significantly higher clonogenic potential compared to term cord blood. Conclusion We demonstrate that preterm cord blood exhibits a higher HSPC concentration and increased clonogenic capacity compared to term neonates. These data may imply an emerging use of HSPCs in autologous stem cell therapy in preterm neonates. PMID:25181353

CD133 overexpression correlates with clinicopathological features of gastric cancer patients and its impact on survival: a systematic review and meta-analysis.

PubMed

Yiming, Li; Yunshan, Guo; Bo, Ma; Yu, Zang; Tao, Wei; Gengfang, Liang; Dexian, Fan; Shiqian, Cui; Jianli, Jiang; Juan, Tang; Zhinan, Chen

2015-12-08

CD133 is one of the most commonly used markers of cancer stem cells (CSCs), which are characterized by their ability for self-renewal and tumorigenicity. However, the clinical and prognostic significance of CD133 in gastric cancer remains controversial. To clarify a precise determinant of the clinical significance of CD133, we conducted a systematic review and meta-analysis to elucidate the correlation of CD133 overexpression with prognosis and clinicopathological features of GC patients. A search in the Cochrane Library, PubMed, Medline, Web of Knowledge and Chinese CNKI, CBM (up to Jun 30, 2015) was performed using the following keywords gastric cancer, CD133, AC133, prominin-1, etc. Electronic searches were supplemented by hand searching reference lists, abstracts and proceedings from meetings. Outcomes included overall survival and various clinicopathological features. Two reviewers independently screened the literature according to the inclusion and exclusion criteria, extracted the data, and assessed the methodological quality of the included studies, and then RevMan 5.2.0 software was used for meta-analysis. A total of 603 gastric cancer patients from 8 studies were included. The results of the meta-analyses showed that, there were significant differences of CD133 expression in the following comparisons: gastric cancer tissues vs. normal esophageal tissue (OR = 3.49, 95% CI [2.48, 490], P < 0.00001), lymph node metastasis vs. non-lymph node metastasis (OR = 2.75, 95% CI [1.99, 3.81], P < 0.00001), distant metastasis vs. non-distant metastasis (OR = 2.38, 95%CI [1.47, 3.85], P < 0.0004), clinical stages III~IV vs. clinical stages I~II (OR = 2.83, 95% CI [2.13, 3.76], P < 0.00001), as well as the accumulative 5-year overall survival rates of CD133-positive vs. CD133-negative patients (OR = 0.23, 95% CI [0.16, 0.33], P < 0.00001). Overexpression of CD133 is associated with lymph node metastasis, distant metastasis, poor TNM stage. Additionally, CD133-positive gastric cancer patients had worse prognosis. Our results indicate that CD133 may be involved in the carcinogenesis of gastric cancer. Evaluation of cytoplasmic CD133 overexpression in gastric cancer tissue sections may be useful in the future as a novel prognostic factor. Nevertheless, due to the poor quality and small sample size of included trials, more well-designed multi-center randomized controlled trials should be performed.

ELK3 promotes the migration and invasion of liver cancer stem cells by targeting HIF-1α.

PubMed

Lee, Joon Ho; Hur, Wonhee; Hong, Sung Woo; Kim, Jung-Hee; Kim, Sung Min; Lee, Eun Byul; Yoon, Seung Kew

2017-02-01

Hepatocellular carcinoma (HCC) is the fifth most common solid cancer and the third most common cause of cancer-related mortality. HCC develops via a multistep process associated with genetic aberrations that facilitate HCC invasion and migration and promote metastasis. A growing body of evidence indicates that cancer stem cells (CSCs) are responsible for tumorigenesis, cancer cell invasion and metastasis. Despite the extremely small proportion of cancer cells represented by this subpopulation of HCC cells, CSCs play a key role in cancer metastasis and poor prognosis. ELK3 (Net/SAP-2/Erp) is a transcription factor that is activated by the Ras/extracellular signal-regulated kinase (ERK) signaling pathway. It plays several important roles in various physiological processes, including cell migration, invasion, wound healing, angiogenesis and tumorigenesis. In the present study, we investigated the role of ELK3 in cancer cell invasion and metastasis in CD133+/CD44+ liver cancer stem cells (LCSCs). We isolated LCSCs expressing CD133 and CD44 from Huh7 HCC cells and evaluated their metastatic potential using invasion and migration assays. We found that CD133+/CD44+ cells had increased metastatic potential compared with non-CD133+/CD44+ cells. We also demonstrated that ELK3 expression was upregulated in CD133+/CD44+ cells and that this aberration enhanced cell migration and invasion. In addition, we identified the molecular mechanism by which ELK3 promotes cancer cell migration and invasion. We found that silencing of ELK3 expression in CD133+/CD44+ LCSCs attenuated their metastatic potential by modulating the expression of heat shock-induced factor-1α (HIF-1α). Collectively, the results of the present study demonstrated that ELK3 overexpression promoted metastasis in CD133+/CD44+ cells by regulating HIF-1α expression and that silencing of ELK3 expression attenuated the metastatic potential of CD133+/CD44+ LCSCs. In conclusion, modulation of ELK3 expression may represent a novel therapeutic strategy for preventing HCC metastasis and invasion.

Prenatally fabricated autologous human living heart valves based on amniotic fluid derived progenitor cells as single cell source.

PubMed

Schmidt, Dörthe; Achermann, Josef; Odermatt, Bernhard; Breymann, Christian; Mol, Anita; Genoni, Michele; Zund, Gregor; Hoerstrup, Simon P

2007-09-11

A novel concept providing prenatally tissue engineered human autologous heart valves based on routinely obtained fetal amniotic fluid progenitors as single cell source is introduced. Fetal human amniotic progenitors were isolated from routinely sampled amniotic fluid and sorted using CD133 magnetic beads. After expansion and differentiation, cell phenotypes of CD133- and CD133+ cells were analyzed by immunohistochemistry and flowcytometry. After characterization, CD133- derived cells were seeded onto heart valve leaflet scaffolds (n=18) fabricated from rapidly biodegradable polymers, conditioned in a pulse duplicator system, and subsequently coated with CD133+ derived cells. After in vitro maturation, opening and closing behavior of leaflets was investigated. Neo-tissues were analyzed by histology, immunohistochemistry, and scanning electron microscopy (SEM). Extracellular matrix (ECM) elements and cell numbers were quantified biochemically. Mechanical properties were assessed by tensile testing. CD133- derived cells demonstrated characteristics of mesenchymal progenitors expressing CD44 and CD105. Differentiated CD133+ cells showed features of functional endothelial cells by eNOS and CD141 expression. Engineered heart valve leaflets demonstrated endothelialized tissue formation with production of ECM elements (GAG 80%, HYP 5%, cell number 100% of native values). SEM showed intact endothelial surfaces. Opening and closing behavior was sufficient under half of systemic conditions. The use of amniotic fluid as single cell source is a promising low-risk approach enabling the prenatal fabrication of heart valves ready to use at birth. These living replacements with the potential of growth, remodeling, and regeneration may realize the early repair of congenital malformations.

Phenotypical and Pharmacological Characterization of Stem-Like Cells in Human Pituitary Adenomas.

PubMed

Würth, Roberto; Barbieri, Federica; Pattarozzi, Alessandra; Gaudenzi, Germano; Gatto, Federico; Fiaschi, Pietro; Ravetti, Jean-Louis; Zona, Gianluigi; Daga, Antonio; Persani, Luca; Ferone, Diego; Vitale, Giovanni; Florio, Tullio

2017-09-01

The presence and functional role of tumor stem cells in benign tumors, and in human pituitary adenomas in particular, is a debated issue that still lacks a definitive formal demonstration. Fifty-six surgical specimens of human pituitary adenomas were processed to establish tumor stem-like cultures by selection and expansion in stem cell-permissive medium or isolating CD133-expressing cells. Phenotypic and functional characterization of these cells was performed (1) ex vivo, by immunohistochemistry analysis on paraffin-embedded tissues; (2) in vitro, attesting marker expression, proliferation, self-renewal, differentiation, and drug sensitivity; and (3) in vivo, using a zebrafish model. Within pituitary adenomas, we identified rare cell populations expressing stem cell markers but not pituitary hormones; we isolated and expanded in vitro these cells, obtaining fibroblast-free, stem-like cultures from 38 pituitary adenoma samples. These cells grow as spheroids, express stem cell markers (Oct4, Sox2, CD133, and nestin), show sustained in vitro proliferation as compared to primary cultures of differentiated pituitary adenoma cells, and are able to differentiate in hormone-expressing pituitary cells. Besides, pituisphere cells, apparently not tumorigenic in mice, engrafted in zebrafish embryos, inducing pro-angiogenic and invasive responses. Finally, pituitary adenoma stem-like cells express regulatory pituitary receptors (D2R, SSTR2, and SSTR5), whose activation by a dopamine/somatostatin chimeric agonist exerts antiproliferative effects. In conclusion, we provide evidence that human pituitary adenomas contain a subpopulation fulfilling biological and phenotypical signatures of tumor stem cells that may represent novel therapeutic targets for therapy-resistant tumors.

Endothelial progenitor cells proliferated via MEK-dependent p42 MAPK signaling pathway.

PubMed

Sandra, Ferry; Oktaviono, Yudi Her; Widodo, Mohammad Aris; Dirgantara, Yanni; Chouw, Angliana; Sargowo, Djanggan

2015-02-01

Endothelial progenitor cells (EPCs) clinical applications have been well reported. However, due to low number of EPCs that could be isolated, EPCs expansion study became one of the main focuses. Some optimized mediums to culture EPCs were currently available. However, the proliferation signaling pathway is not clearly disclosed yet. Peripheral blood was collected from eight healthy subjects, followed by mononuclear cells (MNCs) isolation. MNCs were then prepared and cultured for 2 days. After that, non-adherent cells were harvested and further cultured for 3 days. Resulted colony-forming unit (CFU)-Hill colonies were documented and enumerated under an inverted light microscope. To detect membrane markers, immunofluorescence was performed to detect CD34, VEGFR-2, and CD133. Cell documentation was conducted under a fluorescence microscope. To check cell proliferation, XTT Cell Proliferation Assay Kit was used according to kit insert. To detect possible activation of p44/42 MAPK, western blot was performed to detect p44/42 MAPK and phosphorylated p44/42 MAPK. All visualized bands were captured and quantified. Our results showed that EPCs markers (CD34, CD133 and VEGFR-2) were detected in 3 days culture. From XTT cell proliferation assay and CFU enumeration results, we found that EPCs proliferated significantly (p = 0.012) with addition of supplement. Phosphorylated-p42 MAPK expression of EPCs treated with supplement was significantly higher than the one of EPCs without treatment. Significant inhibition of p42 MAPK phosphorylation by U0126 was observed (p = 0.012). By pretreatment of U0126, number of viable cells and CFUs treated with supplement was significantly decreased (p = 0.012). Our results showed that MEK-dependent p42 MAPK pathway might play an important role in EPCs proliferation.

GMP-based CD133+ cells isolation maintains progenitor angiogenic properties and enhances standardization in cardiovascular cell therapy

PubMed Central

Gaipa, Giuseppe; Tilenni, Manuela; Straino, Stefania; Burba, Ilaria; Zaccagnini, Germana; Belotti, Daniela; Biagi, Ettore; Valentini, Marco; Perseghin, Paolo; Parma, Matteo; Campli, Cristiana Di; Biondi, Andrea; Capogrossi, Maurizio C; Pompilio, Giulio; Pesce, Maurizio

2010-01-01

Abstract The aim of the present study was to develop and validate a good manufacturing practice (GMP) compliant procedure for the preparation of bone marrow (BM) derived CD133+ cells for cardiovascular repair. Starting from available laboratory protocols to purify CD133+ cells from human cord blood, we implemented these procedures in a GMP facility and applied quality control conditions defining purity, microbiological safety and vitality of CD133+ cells. Validation of CD133+ cells isolation and release process were performed according to a two-step experimental program comprising release quality checking (step 1) as well as ‘proofs of principle’ of their phenotypic integrity and biological function (step 2). This testing program was accomplished using in vitro culture assays and in vivo testing in an immunosuppressed mouse model of hindlimb ischemia. These criteria and procedures were successfully applied to GMP production of CD133+ cells from the BM for an ongoing clinical trial of autologous stem cells administration into patients with ischemic cardiomyopathy. Our results show that GMP implementation of currently available protocols for CD133+ cells selection is feasible and reproducible, and enables the production of cells having a full biological potential according to the most recent quality requirements by European Regulatory Agencies. PMID:19627397

Human Myocardial Pericytes: Multipotent Mesodermal Precursors Exhibiting Cardiac Specificity

PubMed Central

Chen, William C.W.; Baily, James E.; Corselli, Mirko; Diaz, Mary; Sun, Bin; Xiang, Guosheng; Gray, Gillian A.; Huard, Johnny; Péault, Bruno

2015-01-01

Perivascular mesenchymal precursor cells (i.e. pericytes) reside in skeletal muscle where they contribute to myofiber regeneration; however, the existence of similar microvessel-associated regenerative precursor cells in cardiac muscle has not yet been documented. We tested whether microvascular pericytes within human myocardium exhibit phenotypes and multipotency similar to their anatomically and developmentally distinct counterparts. Fetal and adult human heart pericytes (hHPs) express canonical pericyte markers in situ, including CD146, NG2, PDGFRβ, PDGFRα, αSMA, and SM-MHC, but not CD117, CD133 and desmin, nor endothelial cell (EC) markers. hHPs were prospectively purified to homogeneity from ventricular myocardium by flow cytometry, based on a combination of positive- (CD146) and negative-selection (CD34, CD45, CD56, and CD117) cell lineage markers. Purified hHPs expanded in vitro were phenotypically similar to human skeletal muscle-derived pericytes (hSkMPs). hHPs express MSC markers in situ and exhibited osteo- chondro-, and adipogenic potentials but, importantly, no ability for skeletal myogenesis, diverging from pericytes of all other origins. hHPs supported network formation with/without ECs in Matrigel cultures; hHPs further stimulated angiogenic responses under hypoxia, markedly different from hSkMPs. The cardiomyogenic potential of hHPs was examined following 5-azacytidine treatment and neonatal cardiomyocyte co-culture in vitro, and intramyocardial transplantation in vivo. Results indicated cardiomyocytic differentiation in a small fraction of hHPs. In conclusion, human myocardial pericytes share certain phenotypic and developmental similarities with their skeletal muscle homologs, yet exhibit different antigenic, myogenic, and angiogenic properties. This is the first example of an anatomical restriction in the developmental potential of pericytes as native mesenchymal stem cells. PMID:25336400

Full GMP-Compliant Validation of Bone Marrow-Derived Human CD133+ Cells as Advanced Therapy Medicinal Product for Refractory Ischemic Cardiomyopathy

PubMed Central

Belotti, Daniela; Gaipa, Giuseppe; Bassetti, Beatrice; Cabiati, Benedetta; Spaltro, Gabriella; Biagi, Ettore; Parma, Matteo; Biondi, Andrea; Cavallotti, Laura; Gambini, Elisa; Pompilio, Giulio

2015-01-01

According to the European Medicine Agency (EMA) regulatory frameworks, Advanced Therapy Medicinal Products (ATMP) represent a new category of drugs in which the active ingredient consists of cells, genes, or tissues. ATMP-CD133 has been widely investigated in controlled clinical trials for cardiovascular diseases, making CD133+ cells one of the most well characterized cell-derived drugs in this field. To ensure high quality and safety standards for clinical use, the manufacturing process must be accomplished in certified facilities following standard operative procedures (SOPs). In the present work, we report the fully compliant GMP-grade production of ATMP-CD133 which aims to address the treatment of chronic refractory ischemic heart failure. Starting from bone marrow (BM), ATMP-CD133 manufacturing output yielded a median of 6.66 × 106 of CD133+ cells (range 2.85 × 106–30.84 × 106), with a viability ranged between 96,03% and 99,97% (median 99,87%) and a median purity of CD133+ cells of 90,60% (range 81,40%–96,20%). Based on these results we defined our final release criteria for ATMP-CD133: purity ≥ 70%, viability ≥ 80%, cellularity between 1 and 12 × 106 cells, sterile, and endotoxin-free. The abovementioned criteria are currently applied in our Phase I clinical trial (RECARDIO Trial). PMID:26495296

Full GMP-compliant validation of bone marrow-derived human CD133(+) cells as advanced therapy medicinal product for refractory ischemic cardiomyopathy.

PubMed

Belotti, Daniela; Gaipa, Giuseppe; Bassetti, Beatrice; Cabiati, Benedetta; Spaltro, Gabriella; Biagi, Ettore; Parma, Matteo; Biondi, Andrea; Cavallotti, Laura; Gambini, Elisa; Pompilio, Giulio

2015-01-01

According to the European Medicine Agency (EMA) regulatory frameworks, Advanced Therapy Medicinal Products (ATMP) represent a new category of drugs in which the active ingredient consists of cells, genes, or tissues. ATMP-CD133 has been widely investigated in controlled clinical trials for cardiovascular diseases, making CD133(+) cells one of the most well characterized cell-derived drugs in this field. To ensure high quality and safety standards for clinical use, the manufacturing process must be accomplished in certified facilities following standard operative procedures (SOPs). In the present work, we report the fully compliant GMP-grade production of ATMP-CD133 which aims to address the treatment of chronic refractory ischemic heart failure. Starting from bone marrow (BM), ATMP-CD133 manufacturing output yielded a median of 6.66 × 10(6) of CD133(+) cells (range 2.85 × 10(6)-30.84 × 10(6)), with a viability ranged between 96,03% and 99,97% (median 99,87%) and a median purity of CD133(+) cells of 90,60% (range 81,40%-96,20%). Based on these results we defined our final release criteria for ATMP-CD133: purity ≥ 70%, viability ≥ 80%, cellularity between 1 and 12 × 10(6) cells, sterile, and endotoxin-free. The abovementioned criteria are currently applied in our Phase I clinical trial (RECARDIO Trial).

Comparative characterization of stem cell marker expression, metabolic activity and resistance to doxorubicin in adherent and spheroid cells derived from the canine prostate adenocarcinoma cell line CT1258.

PubMed

Liu, Wen; Moulay, Mohammed; Willenbrock, Saskia; Roolf, Catrin; Junghanss, Christian; Ngenazahayo, Anaclet; Nolte, Ingo; Murua Escobar, Hugo

2015-04-01

Canine prostate cancer represents a spontaneous animal model for the human counterpart. Cells with stem cell-like character are considered to play a major role in therapeutic resistance and tumor relapse. Thus, the identification of markers allowing for recognition and characterization of these cells is essential. Expression of 12 stem cell marker genes in the canine prostate cancer cell line CT1258 and spheroid cells generated from these was analyzed by quantitative real-time PCR. In CT1258 and the generated spheroid cells, CD44 and CD133 expression was analyzed by flow cytometry, as well as proliferation and doxorubicin resistance. Integrin alpha-6 (ITGA6) expression and metabolic activity were significantly up-regulated in CT1258-derived spheroid cells, while doxorubicin resistance remained comparable. ITGA6 de-regulation and metabolic activity appear to be characteristic of the generated spheres, indicating potential intervention targets. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Colony-Forming Progenitor Cells in the Postnatal Mouse Liver and Pancreas Give Rise to Morphologically Distinct Insulin-Expressing Colonies in 3D Cultures

PubMed Central

Jin, Liang; Feng, Tao; Chai, Jing; Ghazalli, Nadiah; Gao, Dan; Zerda, Ricardo; Li, Zhuo; Hsu, Jasper; Mahdavi, Alborz; Tirrell, David A.; Riggs, Arthur D.; Ku, Hsun Teresa

2014-01-01

In our previous studies, colony-forming progenitor cells isolated from murine embryonic stem cell-derived cultures were differentiated into morphologically distinct insulin-expressing colonies. These colonies were small and not light-reflective when observed by phase-contrast microscopy (therefore termed “Dark” colonies). A single progenitor cell capable of giving rise to a Dark colony was termed a Dark colony-forming unit (CFU-Dark). The goal of the current study was to test whether endogenous pancreas, and its developmentally related liver, harbored CFU-Dark. Here we show that dissociated single cells from liver and pancreas of one-week-old mice give rise to Dark colonies in methylcellulose-based semisolid culture media containing either Matrigel or laminin hydrogel (an artificial extracellular matrix protein). CFU-Dark comprise approximately 0.1% and 0.03% of the postnatal hepatic and pancreatic cells, respectively. Adult liver also contains CFU-Dark, but at a much lower frequency (~0.003%). Microfluidic qRT-PCR, immunostaining, and electron microscopy analyses of individually handpicked colonies reveal the expression of insulin in many, but not all, Dark colonies. Most pancreatic insulin-positive Dark colonies also express glucagon, whereas liver colonies do not. Liver CFU-Dark require Matrigel, but not laminin hydrogel, to become insulin-positive. In contrast, laminin hydrogel is sufficient to support the development of pancreatic Dark colonies that express insulin. Postnatal liver CFU-Dark display a cell surface marker CD133+CD49flowCD107blow phenotype, while pancreatic CFU-Dark are CD133-. Together, these results demonstrate that specific progenitor cells in the postnatal liver and pancreas are capable of developing into insulin-expressing colonies, but they differ in frequency, marker expression, and matrix protein requirements for growth. PMID:25148366

Telomerase-immortalized non-malignant human prostate epithelial cells retain the properties of multipotent stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li Hongzhen; Zhou Jianjun; Miki, Jun

2008-01-01

Understanding prostate stem cells may provide insight into the origin of prostate cancer. Primary cells have been cultured from human prostate tissue but they usually survive only 15-20 population doublings before undergoing senescence. We report here that RC-170N/h/clone 7 cells, a clonal cell line from hTERT-immortalized primary non-malignant tissue-derived human prostate epithelial cell line (RC170N/h), retain multipotent stem cell properties. The RC-170N/h/clone 7 cells expressed a human embryonic stem cell marker, Oct-4, and potential prostate epithelial stem cell markers, CD133, integrin {alpha}2{beta}1{sup hi} and CD44. The RC-170N/h/clone 7 cells proliferated in KGM and Dulbecco's Modified Eagle Medium with 10% fetalmore » bovine serum and 5 {mu}g/ml insulin (DMEM + 10% FBS + Ins.) medium, and differentiated into epithelial stem cells that expressed epithelial cell markers, including CK5/14, CD44, p63 and cytokeratin 18 (CK18); as well as the mesenchymal cell markers, vimentin, desmin; the neuron and neuroendocrine cell marker, chromogranin A. Furthermore the RC170 N/h/clone 7 cells differentiated into multi tissues when transplanted into the sub-renal capsule and subcutaneously of NOD-SCID mice. The results indicate that RC170N/h/clone 7 cells retain the properties of multipotent stem cells and will be useful as a novel cell model for studying the mechanisms of human prostate stem cell differentiation and transformation.« less

Survivin Modulates Squamous Cell Carcinoma-Derived Stem-Like Cell Proliferation, Viability and Tumor Formation in Vivo

PubMed Central

Lotti, Roberta; Palazzo, Elisabetta; Petrachi, Tiziana; Dallaglio, Katiuscia; Saltari, Annalisa; Truzzi, Francesca; Quadri, Marika; Puviani, Mario; Maiorana, Antonino; Marconi, Alessandra; Pincelli, Carlo

2016-01-01

Squamous Cell Carcinoma-derived Stem-like Cells (SCC-SC) originate from alterations in keratinocyte stem cells (KSC) gene expression and sustain tumor development, invasion and recurrence. Since survivin, a KSC marker, is highly expressed in SCC-SC, we evaluate its role in SCC-SC cell growth and SCC models. Survivin silencing by siRNA decreases clonal growth of SCC keratinocytes and viability of total, rapidly adhering (RAD) and non-RAD (NRAD) cells from primary SCC. Similarly, survivin silencing reduces the expression of stem cell markers (OCT4, NOTCH1, CD133, β1-integrin), while it increases the level of differentiation markers (K10, involucrin). Moreover, survivin silencing improves the malignant phenotype of SCC 3D-reconstruct, as demonstrated by reduced epidermal thickness, lower Ki-67 positive cell number, and decreased expression of MMP9 and psoriasin. Furthermore, survivin depletion by siRNA in RasG12V-IκBα-derived tumors leads to smaller tumor formation characterized by lower mitotic index and reduced expression of the tumor-associated marker HIF1α, VEGF and CD51. Therefore, our results indicate survivin as a key gene in regulating SCC cancer stem cell formation and cSCC development. PMID:26771605

Sulfasalazine attenuates evading anticancer response of CD133-positive hepatocellular carcinoma cells.

PubMed

Song, Yeonhwa; Jang, Jaewoo; Shin, Tae-Hoon; Bae, Sang Mun; Kim, Jin-Sun; Kim, Kang Mo; Myung, Seung-Jae; Choi, Eun Kyung; Seo, Haeng Ran

2017-03-03

CD133-positive cells in hepatocellular carcinoma (HCC) exhibit cancer stem cell (CSC)-like properties as well as resistance to chemotherapeutic agents and ionizing radiation; however, their function remains unknown. In this paper, we identified a hitherto unknown mechanism to overcome CD133-induced resistance to anticancer therapy. We applied an alternative approach to enrich the CD133-positive HCC population by manipulating 3D culture conditions. Defense mechanisms against reactive oxygen species (ROS) in CSC spheroids were evaluated by fluorescence image-based phenotypic screening system. Further, we studied the effect of sulfasalazine on ROS defense system and synergistic therapeutic efficacy of anticancer therapies both in culture and in vivo HCC xenograft mouse model. Here, we found that oxidative stress increase CD133 expression in HCC and increased CD133 expression enhanced the capacity of the defense system against ROS, and thereby play a central role in resistance to liver cancer therapy. Moreover, ablation of CD133 attenuated not only the capacity for defense against ROS, but also chemoresistance, in HCC through decreasing glutathione (GSH) levels in vitro. Sulfasalazine, a potent xCT inhibitor that plays an important role in maintaining GSH levels, impaired the ROS defense system and increased the therapeutic efficacy of anticancer therapies in CD133-positive HCC but not CD133-negative HCC in vivo and in vitro. These results strongly indicate functional roles for CD133 in ROS defense and in evading anticancer therapies in HCC, and suggest that sulfasalazine, administered in combination with conventional chemotherapy, might be an effective strategy against CD133-positive HCC cells.

Circulating Bone Marrow-Derived CD45-/CD34+/CD133+/VEGF+ Endothelial Progenitor Cells in Adults with Crohn's Disease.

PubMed

Boltin, Doron; Kamenetsky, Zvi; Perets, Tsachi Tsadok; Snir, Yifat; Sapoznikov, Boris; Schmilovitz-Weiss, Hemda; Ablin, Jacob Nadav; Dickman, Ram; Niv, Yaron

2017-03-01

Circulating endothelial progenitor cells (EPCs) are bone marrow-derived stem cells able to migrate to sites of damaged endothelium and differentiate into endothelial cells. Altered EPC level and function have been described in various inflammatory diseases and have been shown to augment vasculogenesis in murine models. Previous studies of EPC in the context of Crohn's disease (CD) have yielded conflicting results. To determine whether the circulating levels of EPCs are changed in the context of CD. CD patients and healthy controls were recruited. Disease activity was assessed by CDAI. Peripheral blood mononuclear cells were isolated and EPC numbers evaluated by FACS analysis using anti-CD34, anti-VEGF receptor-2, anti-CD133, and anti-CD45 markers. Eighty-three subjects, including 32 CD patients and 51 controls were recruited, including 19 (59.4 %) and 23 (45 %) males (p = 0.26), aged 34.8 ± 14.9 and 43.3 ± 18.5 years (p = 0.64), in cases and controls, respectively. Mean CDAI was 147 ± 97, disease duration was 12.7 ± 11.1 years, and 28 (87.5 %) were receiving biologics for a mean duration of 21.7 ± 16.8 months. The mean level of peripheral EPCs in CD patients was 0.050 ± 0.086 percent and 0.007 ± 0.013 % in controls (p < 0.01). There was no significant correlation between EPC levels and age (r = -0.13, p = 0.47), CDAI (r = -0.26, p = 0.15), disease duration (r = -0.04, p = 0.84), or duration of treatment with biologics (r = 0.004, p = 0.99). EPCs are elevated in patients with CD. Further studies are needed to examine the function of EPCs and their possible role as a marker of disease severity or therapeutic response.

[The cultivation and identification of lacrimal gland adenoid cystic cancer stem cells].

PubMed

Lyu, Jianmei; He, Yanjin; Xie, Lianfeng; Liu, Xun; Zhu, Limin

2015-10-01

To isolate and cultivate the Lacrimal gland Adenoid Cystic Carcinoma cells line, study Cancer Stem Cells properties. Experimental study. Lacrimal gland adenoid cystic carcinoma cancer stem cells were cultivated in serum-free suspension culture and the morphological changes were observed. Cells were divided into two groups, the LACC-CSC experimental group and the LACC control group. The flow cytometry instrument was used to detect the expression of classical stem cell markers CD133 and ABCG2. Transwell chamber was used to detect the cancer stem cell aggressivity and differentiated into the vascular endothelial cells. The tumorigenic force in vitro xenotransplantation were applied. LACC cells can grow suspensively after vaccinated in serum free medium and form tumor microspheres after 10-12 days. Flow cytometry experiments showed that the expression ratio of stem cell markers CD133 in LACC-CSC was (35.67 ± 6.86)%, significantly different to LACC with (0.46 ± 0.48)%, (t = 8.867, P < 0.05). Similarly, the expression ratio of stem cell marker ABCG2 in LACC-CSC was (39.99 ± 4.54)%, significantly different to LACC with (6.75 ± 1.34)%, (t = -9.932, P < 0.05). In vitro experiment of Matrigel invasion, LACC-CSC went through the matrigel basement membrane averagely (32.60 ± 8.79)/HP contrary to LACC with average (10.20 ± 2.77)/HP after 24 hours, showing statistically significance (t = 5.433, P < 0.05) between the two groups. After training for 48 hours, the difference between two groups was still obvious (t = 5.779, P < 0.05) with LACC-CSC average (62.60 ± 4.83)/HP to LACC (44.00 ± 5.34)/HP. When induced by serum medium containing VEGF and bFGF, LACC-CSC grew adherent gradually and cell morphological changes occurred after continuous induction to long spindle cells. When cultured into three-dimensional matrix structure they formed vessel samples and expressed vascular endothelial marker CD31 and CD34. Transplanted tumor in vitro experiment, mice of LACC-CSC group grew tumors in 9 days with 100% tumorigenic rate, whereas LACC group 12 days with 100% tumorigenic rate. LACC-CSC can be obtained through serum-free culture method. LACC-CSC grew suspensively and expressed classical stem cell markers. LACC-CSC were identified as cancer stem cells with stronger migration and invasion. LACC-CSC have tumorigenic force and multi-directional differentiation potential with general characteristics of the stem cell.

Identification, expansion and characterization of cancer cells with stem cell properties from head and neck squamous cell carcinomas

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaseb, Hatem O.; Department of Clinical Pathology, National Cancer Institute; Fohrer-Ting, Helene

Head and neck squamous cell carcinoma (HNSCC) is a major public health concern. Recent data indicate the presence of cancer stem cells (CSC) in many solid tumors, including HNSCC. Here, we assessed the stem cell (SC) characteristics, including cell surface markers, radioresistance, chromosomal instability, and in vivo tumorigenic capacity of CSC isolated from HNSCC patient specimens. We show that spheroid enrichment of CSC from early and short-term HNSCC cell cultures was associated with increased expression of CD44, CD133, SOX2 and BMI1 compared with normal oral epithelial cells. On immunophenotyping, five of 12 SC/CSC markers were homogenously expressed in all tumormore » cultures, while one of 12 was negative, four of 12 showed variable expression, and two of the 12 were expressed heterogeneously. We showed that irradiated CSCs survived and retained their self-renewal capacity across different ionizing radiation (IR) regimens. Fluorescence in situ hybridization (FISH) analyses of parental and clonally-derived tumor cells revealed different chromosome copy numbers from cell to cell, suggesting the presence of chromosomal instability in HNSCC CSC. Further, our in vitro and in vivo mouse engraftment studies suggest that CD44+/CD66− is a promising, consistent biomarker combination for HNSCC CSC. Overall, our findings add further evidence to the proposed role of HNSCC CSCs in therapeutic resistance. - Highlights: • Spheroid enrichment selects cancer stem cells (CSC) from head & neck tumors (HNSCC). • Compared to normal epithelial cells, isolated CSC express increased SC/CSC markers. • Isolated CSC display enhanced radioresistance, clonogenicity and tumorigenicity. • HNSCC CSC express chromosomal instability. • CD44+/CD66− is a promising, consistent biomarker for HNSCC CSC.« less

Targeting glioma stem cells enhances anti-tumor effect of boron neutron capture therapy

PubMed Central

Sun, Ting; Li, Yanyan; Huang, Yulun; Zhang, Zizhu; Yang, Weilian; Du, Ziwei; Zhou, Youxin

2016-01-01

The uptake of (10)boron by tumor cells plays an important role for cell damage in boron neutron capture therapy (BNCT). CD133 is frequently expressed in the membrane of glioma stem cells (GSCs), resistant to radiotherapy and chemotherapy, and represents a potential therapeutic target. To increase (10)boron uptake in GSCs, we created a polyamido amine dendrimer, conjugated CD133 monoclonal antibodies, encapsulating mercaptoundecahydrododecaborate (BSH) in void spaces, and monitored the uptake of the bioconjugate nanoparticles by GSCs in vitro and in vivo. Fluorescence microscopy showed the specific uptake of the bioconjugate nanoparticles by CD133-positive GSCs. Treatment with the biconjugate nanoparticles resulted in a significant lethal effect after neutron radiation due to efficient and CD133-independent cellular targeting and uptake in CD133-expressing GSCs. A significantly longer survival occurred in combination with the biconjugate nanoparticles and BSH compared with BSH alone in human intracranial GBM models employing CD133-positive GSCs xenografts. Our data demonstrated that this bioconjugate nanoparticle targets human CD133-positive GSCs and is a potential boron agent in BNCT. PMID:27191269

Targeting glioma stem cells enhances anti-tumor effect of boron neutron capture therapy.

PubMed

Sun, Ting; Li, Yanyan; Huang, Yulun; Zhang, Zizhu; Yang, Weilian; Du, Ziwei; Zhou, Youxin

2016-07-12

The uptake of (10)boron by tumor cells plays an important role for cell damage in boron neutron capture therapy (BNCT). CD133 is frequently expressed in the membrane of glioma stem cells (GSCs), resistant to radiotherapy and chemotherapy, and represents a potential therapeutic target. To increase (10)boron uptake in GSCs, we created a polyamido amine dendrimer, conjugated CD133 monoclonal antibodies, encapsulating mercaptoundecahydrododecaborate (BSH) in void spaces, and monitored the uptake of the bioconjugate nanoparticles by GSCs in vitro and in vivo. Fluorescence microscopy showed the specific uptake of the bioconjugate nanoparticles by CD133-positive GSCs. Treatment with the biconjugate nanoparticles resulted in a significant lethal effect after neutron radiation due to efficient and CD133-independent cellular targeting and uptake in CD133-expressing GSCs. A significantly longer survival occurred in combination with the biconjugate nanoparticles and BSH compared with BSH alone in human intracranial GBM models employing CD133-positive GSCs xenografts. Our data demonstrated that this bioconjugate nanoparticle targets human CD133-positive GSCs and is a potential boron agent in BNCT.

Critical assessment of the efficiency of CD34 and CD133 antibodies for enrichment of rabbit hematopoietic stem cells.

PubMed

Vašíček, Jaromír; Shehata, Medhat; Schnabl, Susanne; Hilgarth, Martin; Hubmann, Rainer; Jäger, Ulrich; Bauer, Miroslav; Chrenek, Peter

2018-06-08

Rabbits have many hereditary diseases common to humans and are therefore a valuable model for regenerative disease and hematopoietic stem cell (HSC) therapies. Currently, there is no substantial data on the isolation and/or enrichment of rabbit HSCs. This study was initiated to evaluate the efficiency of the commercially available anti-CD34 and anti-CD133 antibodies for the detection and potential enrichment of rabbit HSCs from peripheral blood. PBMCs from rabbit and human blood were labelled with different clones of anti-human CD34 monoclonal antibodies (AC136, 581 and 8G12) and rabbit polyclonal CD34 antibody (pCD34) and anti-human CD133 monoclonal antibodies (AC133 and 293C3). Flow cytometry showed a higher percentage of rabbit CD34 + cells labelled by AC136 in comparison to the clone 581 and pCD34 (P<0.01). A higher percentage of rabbit CD133 + cells were also detected by 293C3 compared to the AC133 clone (P<0.01). Therefore, AC136 clone was used for the indirect immunomagnetic enrichment of rabbit CD34 + cells using magnetic-activated cell sorting (MACS). The enrichment of the rabbit CD34 + cells after sorting was low in comparison to human samples (2.4% vs. 39.6%). PCR analyses confirmed the efficient enrichment of human CD34 + cells and the low expression of CD34 mRNA in rabbit positive fraction. In conclusion, the tested antibodies might be suitable for detection, but not for sorting the rabbit CD34 + HSCs and new specific anti-rabbit CD34 antibodies are needed for efficient enrichment of rabbit HSCs. This article is protected by copyright. All rights reserved. © 2018 American Institute of Chemical Engineers.

GMP-conformant on-site manufacturing of a CD133+ stem cell product for cardiovascular regeneration.

PubMed

Skorska, Anna; Müller, Paula; Gaebel, Ralf; Große, Jana; Lemcke, Heiko; Lux, Cornelia A; Bastian, Manuela; Hausburg, Frauke; Zarniko, Nicole; Bubritzki, Sandra; Ruch, Ulrike; Tiedemann, Gudrun; David, Robert; Steinhoff, Gustav

2017-02-10

CD133 + stem cells represent a promising subpopulation for innovative cell-based therapies in cardiovascular regeneration. Several clinical trials have shown remarkable beneficial effects following their intramyocardial transplantation. Yet, the purification of CD133 + stem cells is typically performed in centralized clean room facilities using semi-automatic manufacturing processes based on magnetic cell sorting (MACS®). However, this requires time-consuming and cost-intensive logistics. CD133 + stem cells were purified from patient-derived sternal bone marrow using the recently developed automatic CliniMACS Prodigy® BM-133 System (Prodigy). The entire manufacturing process, as well as the subsequent quality control of the final cell product (CP), were realized on-site and in compliance with EU guidelines for Good Manufacturing Practice. The biological activity of automatically isolated CD133 + cells was evaluated and compared to manually isolated CD133 + cells via functional assays as well as immunofluorescence microscopy. In addition, the regenerative potential of purified stem cells was assessed 3 weeks after transplantation in immunodeficient mice which had been subjected to experimental myocardial infarction. We established for the first time an on-site manufacturing procedure for stem CPs intended for the treatment of ischemic heart diseases using an automatized system. On average, 0.88 × 10 6 viable CD133 + cells with a mean log 10 depletion of 3.23 ± 0.19 of non-target cells were isolated. Furthermore, we demonstrated that these automatically isolated cells bear proliferation and differentiation capacities comparable to manually isolated cells in vitro. Moreover, the automatically generated CP shows equal cardiac regeneration potential in vivo. Our results indicate that the Prodigy is a powerful system for automatic manufacturing of a CD133 + CP within few hours. Compared to conventional manufacturing processes, future clinical application of this system offers multiple benefits including stable CP quality and on-site purification under reduced clean room requirements. This will allow saving of time, reduced logistics and diminished costs.

Established breast cancer stem cell markers do not correlate with in vivo tumorigenicity of tumor-initiating cells

PubMed Central

LEHMANN, CHRISTIAN; JOBS, GABRIELE; THOMAS, MARKUS; BURTSCHER, HELMUT; KUBBIES, MANFRED

2012-01-01

The tumor-initiating capacity of primary human breast cancer cells is maintained in vitro by culturing these cells as spheres/aggregates. Inoculation of small cell numbers derived from these non-adherent cultures leads to rapid xenograft tumor formation in mice. Accordingly, injection of more differentiated monolayer cells derived from spheres results in significantly decelerated tumor growth. For our study, two breast cancer cell lines were generated from primary tumors and cultured as mammospheres or as their adherent counterparts. We examined the in vivo tumorigenicity of these cells by injecting serial dilutions into immunodeficient mice. Inoculation of 106 cells per mouse led to rapid tumor formation, irrespective of cell line or culture conditions. However, after injection of only 103 cells, solely sphere cells were highly tumorigenic. In vitro, we investigated differentiation markers, established breast CSC markers and conducted mRNA profiling. Cytokeratin 5 and 18 were increased in both monolayer cell types, indicating a more differentiated phenotype. All cell lines were CD24−/CD44+ and did not express CD133, CD326 or E-cadherin. ALDH1 activity was not detectable in any cell line. A verapamil-sensitive Hoechst side population was present in sphere cells, but there was no correlation with tumorigenicity in vivo. mRNA profiling did not reveal upregulation of relevant transcription factors. In vitro cell cycle kinetics and in vivo tumor doubling times displayed no difference between sphere and monolayer cultures. Our data indicate that intrinsic genetic and functional markers investigated are not indicative of the in vivo tumori-genicity of putative breast tumor-initiating cells. PMID:23042145

Possible association between stem-like hallmark and radioresistance in human cervical carcinoma cells.

PubMed

Kumazawa, Shoko; Kajiyama, Hiroaki; Umezu, Tomokazu; Mizuno, Mika; Suzuki, Shiro; Yamamoto, Eiko; Mitsui, Hiroko; Sekiya, Ryuichiro; Shibata, Kiyosumi; Kikkawa, Fumitaka

2014-05-01

We aimed to investigate the possibility of an association between a stem-like hallmark and radiotherapeutic sensitivity in human cervical carcinoma cells. Side-population (SP) cells and non-SP (NSP) cells in HeLa cells were isolated using flow cytometry and Hoechst 33342 efflux. We performed Western blot analysis to evaluate the expression of stem cell markers (CXCR4, Oct3/4, CD133, and SOX2) and apoptosis markers after irradiation. In addition, SP and NSP cells were injected into nude mice and we assessed subcutaneous tumor formation. To examine tolerance of irradiation, colony formation and apoptosis change were confirmed in the SP and NSP cells. SP cells showed a higher expression of CXCR4, Oct3/4, CD133, and SOX2 than NSP cells. The colony size of SP cells cultured on non-coated dishes was larger than that of NSP cells, and NSP cells were easily induced to undergo apoptosis. SP cells tended to form spheroids and showed a higher level of tumorigenicity compared with NSP cells. In addition, nude mice inoculated with SP cells showed greater tumor growth compared with NSP cells. SP cells showed a higher tumorigenicity and lower apoptotic potential, leading to enhanced radiotolerance. Tumor SP cells showed higher-level stem-cell-like characters and radioresistance than NSP cells. SP cells may be useful for new therapeutic approaches for radiation-resistant cervical cancer. © 2014 The Authors. Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology.

Circulating progenitor and angiogenic cell frequencies are abnormally static over pregnancy in women with preconception diabetes: A pilot study.

PubMed

Lima, Patricia D A; Chen, Zhilin; Tayab, Aysha; Murphy, Malia S Q; Pudwell, Jessica; Smith, Graeme N; Croy, B Anne

2017-01-01

Type 1 and 2 diabetes decrease the frequencies and functional capacities of circulating angiogenic cells (CAC). Diabetes also elevates gestational complications. These observations may be interrelated. We undertook pilot studies to address the hypothesis that preconception diabetes deviates known gestational increases in CACs. Cross-sectional study of type 1 diabetic, type 2 diabetic and normoglycemic pregnant women was conducted at 1st, 2nd, and 3rd trimester and compared to a 6mo postpartum surrogate baseline. Circulating progenitor cells (CPC; CD34+CD45dimSSlow) and CACs (CD34+CD45dimSSlow expressing CD133 without or with KDR) were quantified by flow cytometry and by colony assay (CFU-Hill). In pregnant normoglycemic women, CD34+CD45dimSSlow cell frequency was greater in 1st and 3rd trimester than postpartum but frequency of these cells was static over type 1 or 2 diabetic pregnancies. Type 1 and type 2 diabetic women showed CACs variance versus normal controls. Type 1 diabetic women had more total CD34+KDR+ CACs in 1st trimester and a higher ratio of CD133+KDR+ to total CD133+ cells in 1st and 2nd trimesters than control women, demonstrating an unbalance in CD133+KDR+ CACs. Type 2 diabetic women had more CD133+KDR+ CACs in 1st trimester and fewer CD133+KDR- CACs at mid-late pregnancy than normal pregnant women. Thus, pregnancy stage-specific physiological fluctuation in CPCs (CD34+) and CACs (CD133+KDR+ and CD133+KDR-) did not occur in type 1 and type 2 diabetic women. Early outgrowth colonies were stable across normal and diabetic pregnancies. Therefore, preconception diabetes blocks the normal dynamic pattern of CAC frequencies across gestation but does not alter colony growth. The differences between diabetic and typical women were seen at specific gestational stages that may be critical for initiation of the uterine vascular pathologies characterizing diabetic gestations.

Expression of the lysosomal-associated membrane protein-1 (LAMP-1) in astrocytomas

PubMed Central

Jensen, Stine S; Aaberg-Jessen, Charlotte; Christensen, Karina G; Kristensen, Bjarne

2013-01-01

Targeting of lysosomes is a novel therapeutic anti-cancer strategy for killing the otherwise apoptosis-resistant cancer cells. Such strategies are urgently needed for treatment of brain tumors, especially the glioblastoma, which is the most frequent and most malignant type. The aim of the present study was to investigate the presence of lysosomes in astrocytic brain tumors focussing also on the therapy resistant tumor stem cells. Expression of the lysosomal marker LAMP-1 (lysosomal-associated membrane protein-1) was investigated by immunohistochemistry in 112 formalin fixed paraffin embedded astrocytomas and compared with tumor grade and overall patient survival. Moreover, double immunofluorescence stainings were performed with LAMP-1 and the astrocytic marker GFAP and the putative stem cell marker CD133 on ten glioblastomas. Most tumors expressed the LAMP-1 protein in the cytoplasm of the tumor cells, while the blood vessels were positive in all tumors. The percentage of LAMP-1 positive tumor cells and staining intensities increased with tumor grade but variations in tumors of the same grade were also found. No association was found between LAMP-1 expression and patient overall survival in the individual tumor grades. LAMP-1/GFAP showed pronounced co-expression and LAMP-1/CD133 was co-expressed as well suggesting that tumor cells including the proposed tumor stem cells contain lysosomes. The results suggest that high amounts of lysosomes are present in glioblastomas and in the proposed tumor stem cells. Targeting of lysosomes may be a promising novel therapeutic strategy against this highly malignant neoplasm. PMID:23826410

Nitric oxide synthase 2 is required for conversion of pro-fibrogenic inflammatory CD133(+) progenitors into F4/80(+) macrophages in experimental autoimmune myocarditis.

PubMed

Blyszczuk, Przemyslaw; Berthonneche, Corrine; Behnke, Silvia; Glönkler, Marcel; Moch, Holger; Pedrazzini, Thierry; Lüscher, Thomas F; Eriksson, Urs; Kania, Gabriela

2013-02-01

Experimental autoimmune myocarditis (EAM) model mirrors important mechanisms of inflammatory dilated cardiomyopathy (iDCM). In EAM, inflammatory CD133(+) progenitors are a major cellular source of cardiac myofibroblasts in the post-inflammatory myocardium. We hypothesized that exogenous delivery of macrophage-colony-stimulating factor (M-CSF) can stimulate macrophage lineage differentiation of inflammatory progenitors and, therefore, prevent their naturally occurring myofibroblast fate in EAM. EAM was induced in wild-type (BALB/c) and nitric oxide synthase 2-deficient (Nos2(-/-)) mice and CD133(+) progenitors were isolated from inflamed hearts. In vitro, M-CSF converted inflammatory CD133(+) progenitors into nitric oxide-producing F4/80(+) macrophages and prevented transforming growth factor-β-mediated myofibroblast differentiation. Importantly, only a subset of heart-infiltrating CD133(+) progenitors expresses macrophage-specific antigen F4/80 in EAM. These CD133(+)/F4/80(hi) cells show impaired myofibrogenic potential compared with CD133(+)/F4/80(-) cells. M-CSF treatment of wild-type mice with EAM at the peak of disease markedly increased CD133(+)/F4/80(hi) cells in the myocardium, and CD133(+) progenitors isolated from M-CSF-treated mice failed to differentiate into myofibroblasts. In contrast, M-CSF was not effective in converting CD133(+) progenitors from inflamed hearts of Nos2(-/-) mice into macrophages, and M-CSF treatment did not result in increased CD133(+)/F4/80(hi) cell population in hearts of Nos2(-/-) mice. Accordingly, M-CSF prevented post-inflammatory fibrosis and left ventricular dysfunction in wild-type but not in Nos2(-/-) mice. Active and NOS2-dependent induction of macrophage lineage differentiation abrogates the myofibrogenic potential of heart-infiltrating CD133(+) progenitors. Modulating the in vivo differentiation fate of specific progenitors might become a novel approach for the treatment of inflammatory heart diseases.

Clinical relevance of miR-mediated HLA-G regulation and the associated immune cell infiltration in renal cell carcinoma.

PubMed

Jasinski-Bergner, Simon; Stoehr, Christine; Bukur, Juergen; Massa, Chiara; Braun, Juliane; Hüttelmaier, Stefan; Spath, Verena; Wartenberg, Roland; Legal, Wolfgang; Taubert, Helge; Wach, Sven; Wullich, Bernd; Hartmann, Arndt; Seliger, Barbara

2015-06-01

In human tumors of distinct origin including renal cell carcinoma (RCC), the non-classical human leukocyte antigen G (HLA-G) is frequently expressed, thereby inhibiting the cytotoxic activity of T and natural killer (NK) cells. Recent studies demonstrated a strong post-transcriptional gene regulation of the HLA-G by miR-152, -148A, -148B and -133A. Standard methods were applied to characterize the expression and function of HLA-G, HLA-G-regulatory microRNAs (miRs) and the immune cell infiltration in 453 RCC lesions using a tissue microarray and five RCC cell lines linking these results to clinical parameters. Direct interactions with HLA-G regulatory miRs and the HLA-G 3' untranslated region (UTR) were detected and the affinities of these different miRs to the HLA-G 3'-UTR compared. qPCR analyses and immunohistochemical staining revealed an inverse expression of miR-148A and -133A with the HLA-G protein in situ and in vitro . Stable miR overexpression caused a downregulation of HLA-G protein enhancing the NK and LAK cell-mediated cytotoxicity in in vitro CD107a activation assays revealing a HLA-G-dependent cytotoxic activity of immune effector cells. A significant higher frequency of CD3 + /CD8 + T cell lymphocytes, but no differences in the activation markers CD69, CD25 or in the presence of CD56 + , FoxP3 + and CD4 + immune cells were detected in HLA-G + compared to HLA-G - RCC lesions. This could be associated with higher WHO grade, but not with a disease-specific survival. These data suggest a miR-mediated control of HLA-G expression in RCC, which is associated with a distinct pattern of immune cell infiltration.

uPAR-controlled oncolytic adenoviruses eliminate cancer stem cells in human pancreatic tumors.

PubMed

Sobrevals, Luciano; Mato-Berciano, Ana; Urtasun, Nerea; Mazo, Adela; Fillat, Cristina

2014-01-01

Pancreatic tumors contain cancer stem cells highly resistant to chemotherapy. The identification of therapies that can eliminate this population of cells might provide with more effective treatments. In the current work we evaluated the potential of oncolytic adenoviruses to act against pancreatic cancer stem cells (PCSC). PCSC from two patient-derived xenograft models were isolated from orthotopic pancreatic tumors treated with saline, or with the chemotherapeutic agent gemcitabine. An enrichment in the number of PCSC expressing the cell surface marker CD133 and a marked enhancement on tumorsphere formation was observed in gemcitabine treated tumors. No significant increase in the CD44, CD24, and epithelial-specific antigen (ESA) positive cells was observed. Neoplastic sphere-forming cells were susceptible to adenoviral infection and exposure to oncolytic adenoviruses resulted in elevated cytotoxicity with both Adwt and the tumor specific AduPARE1A adenovirus. In vivo, intravenous administration of a single dose of AduPARE1A in human-derived pancreatic xenografts led to a remarkable anti-tumor effect. In contrast to gemcitabine AduPARE1A treatment did not result in PCSC enrichment. No enrichment on tumorspheres neither on the CD133(+) population was detected. Therefore our data provide evidences of the relevance of uPAR-controlled oncolytic adenoviruses for the elimination of pancreatic cancer stem cells. © 2013.

Repopulating hematopoietic stem cells from steady-state blood before and after ex vivo culture are enriched in CD34+CD133+CXCR4low fraction.

PubMed

Lapostolle, Véronique; Chevaleyre, Jean; Duchez, Pascale; Rodriguez, Laura; Vlaski-Lafarge, Marija; Sandvig, Ioanna; Brunet de la Grange, Philippe; Ivanovic, Zoran

2018-06-01

Feasibility of ex vivo expansion allows us to consider the steady-state peripheral blood as an alternative source of hematopoietic stem progenitor cells for transplantation when growth factor-induced cell mobilization is contraindicated or inapplicable. Ex vivo expansion dramatically enhances the in vivo reconstituting cell population from steady-state blood. In order to investigate phenotype and the expression of homing molecules, CD34, CD133, CD90, CD45RA, CD26 and CD9 expression was determined on sorted CD34+ cells according to CXCR4 (neg, low, bright) and CD133 expression before and after ex vivo expansion. Hematopoietic stem cell activity was determined in vivo on the basis of hematopoietic repopulation of primary and secondary recipients - NSG immuno-deficient mice. In vivo reconstituting cells in steady-state blood CD34+ cell fraction before expansion belong to the CD133+ population and are CXCR4low or, to a lesser extent, CXCR4neg, while after ex vivo expansion they are contained in only the CD133+CXCR4low cells. The failure of CXCR4bright population to engraft is probably due to the exclusive expression of CD26 by these cells. The limiting-dilution analysis showed that both repopulating cell number and individual proliferative capacity were enhanced by ex vivo expansion. Thus, steady-state peripheral blood cells exhibit a different phenotype compared to mobilized and cord blood ones, as well as to those issued from the bone marrow. This data represent the first phenotypic characterization of steady-state blood cells exhibiting short and long term hematopoietic reconstituting potential, which can be expanded ex vivo, a sine qua non for their subsequent use for transplantation. Copyright © 2018, Ferrata Storti Foundation.

Self-renewal of CD133(hi) cells by IL6/Notch3 signalling regulates endocrine resistance in metastatic breast cancer.

PubMed

Sansone, Pasquale; Ceccarelli, Claudio; Berishaj, Marjan; Chang, Qing; Rajasekhar, Vinagolu K; Perna, Fabiana; Bowman, Robert L; Vidone, Michele; Daly, Laura; Nnoli, Jennifer; Santini, Donatella; Taffurelli, Mario; Shih, Natalie N C; Feldman, Michael; Mao, Jun J; Colameco, Christopher; Chen, Jinbo; DeMichele, Angela; Fabbri, Nicola; Healey, John H; Cricca, Monica; Gasparre, Giuseppe; Lyden, David; Bonafé, Massimiliano; Bromberg, Jacqueline

2016-02-09

The mechanisms of metastatic progression from hormonal therapy (HT) are largely unknown in luminal breast cancer. Here we demonstrate the enrichment of CD133(hi)/ER(lo) cancer cells in clinical specimens following neoadjuvant endocrine therapy and in HT refractory metastatic disease. We develop experimental models of metastatic luminal breast cancer and demonstrate that HT can promote the generation of HT-resistant, self-renewing CD133(hi)/ER(lo)/IL6(hi) cancer stem cells (CSCs). HT initially abrogates oxidative phosphorylation (OXPHOS) generating self-renewal-deficient cancer cells, CD133(hi)/ER(lo)/OXPHOS(lo). These cells exit metabolic dormancy via an IL6-driven feed-forward ER(lo)-IL6(hi)-Notch(hi) loop, activating OXPHOS, in the absence of ER activity. The inhibition of IL6R/IL6-Notch pathways switches the self-renewal of CD133(hi) CSCs, from an IL6/Notch-dependent one to an ER-dependent one, through the re-expression of ER. Thus, HT induces an OXPHOS metabolic editing of luminal breast cancers, paradoxically establishing HT-driven self-renewal of dormant CD133(hi)/ER(lo) cells mediating metastatic progression, which is sensitive to dual targeted therapy.

Circulating progenitor and angiogenic cell frequencies are abnormally static over pregnancy in women with preconception diabetes: A pilot study

PubMed Central

Chen, Zhilin; Tayab, Aysha; Murphy, Malia S. Q.; Pudwell, Jessica; Smith, Graeme N.; Croy, B. Anne

2017-01-01

Type 1 and 2 diabetes decrease the frequencies and functional capacities of circulating angiogenic cells (CAC). Diabetes also elevates gestational complications. These observations may be interrelated. We undertook pilot studies to address the hypothesis that preconception diabetes deviates known gestational increases in CACs. Cross-sectional study of type 1 diabetic, type 2 diabetic and normoglycemic pregnant women was conducted at 1st, 2nd, and 3rd trimester and compared to a 6mo postpartum surrogate baseline. Circulating progenitor cells (CPC; CD34+CD45dimSSlow) and CACs (CD34+CD45dimSSlow expressing CD133 without or with KDR) were quantified by flow cytometry and by colony assay (CFU-Hill). In pregnant normoglycemic women, CD34+CD45dimSSlow cell frequency was greater in 1st and 3rd trimester than postpartum but frequency of these cells was static over type 1 or 2 diabetic pregnancies. Type 1 and type 2 diabetic women showed CACs variance versus normal controls. Type 1 diabetic women had more total CD34+KDR+ CACs in 1st trimester and a higher ratio of CD133+KDR+ to total CD133+ cells in 1st and 2nd trimesters than control women, demonstrating an unbalance in CD133+KDR+ CACs. Type 2 diabetic women had more CD133+KDR+ CACs in 1st trimester and fewer CD133+KDR- CACs at mid-late pregnancy than normal pregnant women. Thus, pregnancy stage-specific physiological fluctuation in CPCs (CD34+) and CACs (CD133+KDR+ and CD133+KDR-) did not occur in type 1 and type 2 diabetic women. Early outgrowth colonies were stable across normal and diabetic pregnancies. Therefore, preconception diabetes blocks the normal dynamic pattern of CAC frequencies across gestation but does not alter colony growth. The differences between diabetic and typical women were seen at specific gestational stages that may be critical for initiation of the uterine vascular pathologies characterizing diabetic gestations. PMID:28278173

Treatment of ovarian cancer by targeting the tumor stem cell-associated carbohydrate antigen, Sialyl-Thomsen-nouveau.

PubMed

Starbuck, Kristen; Al-Alem, Linah; Eavarone, David A; Hernandez, Silvia Fatima; Bellio, Chiara; Prendergast, Jillian M; Stein, Jenna; Dransfield, Daniel T; Zarrella, Bianca; Growdon, Whitfield B; Behrens, Jeff; Foster, Rosemary; Rueda, Bo R

2018-05-01

Recurrent ovarian cancer (OvCa) is thought to result in part from the inability to eliminate rare quiescent cancer stem cells (CSCs) that survive cytotoxic chemotherapy and drive tumor resurgence. The Sialyl-Thomsen-nouveau antigen (STn) is a carbohydrate moiety present on protein markers of CSCs in pancreatic, colon, and gastric malignancies. We have demonstrated that human OvCa cell lines contain varying levels of cells that independently express either STn or the ovarian CSC marker CD133. Here we determine co-expression of STn and CD133 in a subset of human OvCa cell lines. Analyses of colony and sphere forming capacity and of response to standard-of-care cytotoxic therapy suggest a subset of OvCa STn + cells display some CSC features. The effect of the anti-STn antibody-drug conjugates (ADCs) S3F-CL-MMAE and 2G12-2B2-CL-MMAE on OvCa cell viability in vitro and in vivo was also assessed. Treatment with S3F-CL-MMAE reduced the viability of two of three OvCa cell lines in vitro and exposure to either S3F-CL-MMAE or 2G12-2B2-CL-MMAE reduced OVCAR3-derived xenograft volume in vivo , depleting STn + tumor cells. In summary, STn + cells demonstrate some stem-like properties and specific therapeutic targeting of STn in ovarian tumors may be an effective clinical strategy to eliminate both STn + CSC and STn + non-CSC populations.

R-spondin1/Wnt-enhanced Ascl2 autoregulation controls the self-renewal of colorectal cancer progenitor cells.

PubMed

Ye, Jun; Liu, Shanxi; Shang, Yangyang; Chen, Haoyuan; Wang, Rongquan

2018-06-25

The Wnt signaling pathway controls stem cell identity in the intestinal epithelium and cancer stem cells (CSCs). The transcription factor Ascl2 (Wnt target gene) is fate decider of intestinal cryptic stem cells and colon cancer stem cells. It is unclear how Wnt signaling is translated into Ascl2 expression and keeping the self-renewal of CRC progenitor cells. We showed that the exogenous Ascl2 in colorectal cancer (CRC) cells activated the endogenous Ascl2 expression via a direct autoactivatory loop, including Ascl2 binding to its own promoter and further transcriptional activation. Higher Ascl2 expression in human CRC cancerous tissues led to greater enrichment in Ascl2 immunoprecipitated DNA within the Ascl2 promoter in the CRC cancerous sample than the peri-cancerous mucosa. Ascl2 binding to its own promoter and inducing further transcriptional activation of the Ascl2 gene was predominant in the CD133 + CD44 + CRC population. R-spondin1/Wnt activated Ascl2 expression dose-dependently in the CD133 + CD44 + CRC population, but not in the CD133 - CD44 - CRC population, which was caused by differences in Ascl2 autoregulation under R-spondin1/Wnt activation. R-spondin1/Wnt treatment in the CD133 + CD44 + or CRC CD133 - CD44 - populations exerted a different pattern of stemness maintenance, which was defined by alterations of the mRNA levels of stemness-associated genes, the protein expression levels (Bmi1, C-myc, Oct-4 and Nanog) and tumorsphere formation. The results indicated that Ascl2 autoregulation formed a transcriptional switch that was enhanced by Wnt signaling in the CD133 + CD44 + CRC population, thus conferring their self-renewal.

Establishment of primary cell culture and an intracranial xenograft model of pediatric ependymoma: a prospect for therapy development and understanding of tumor biology.

PubMed

Pavon, Lorena Favaro; Sibov, Tatiana Tais; Caminada de Toledo, Silvia Regina; Mara de Oliveira, Daniela; Cabral, Francisco Romero; Gabriel de Souza, Jean; Boufleur, Pamela; Marti, Luciana C; Malheiros, Jackeline Moraes; Ferreira da Cruz, Edgar; Paiva, Fernando F; Malheiros, Suzana M F; de Paiva Neto, Manoel A; Tannús, Alberto; Mascarenhas de Oliveira, Sérgio; Silva, Nasjla Saba; Cappellano, Andrea Maria; Petrilli, Antonio Sérgio; Chudzinski-Tavassi, Ana Marisa; Cavalheiro, Sérgio

2018-04-24

Ependymoma (EPN), the third most common pediatric brain tumor, is a central nervous system (CNS) malignancy originating from the walls of the ventricular system. Surgical resection followed by radiation therapy has been the primary treatment for most pediatric intracranial EPNs. Despite numerous studies into the prognostic value of histological classification, the extent of surgical resection and adjuvant radiotherapy, there have been relatively few studies into the molecular and cellular biology of EPNs. We elucidated the ultrastructure of the cultured EPN cells and characterized their profile of immunophenotypic pluripotency markers (CD133, CD90, SSEA-3, CXCR4). We established an experimental EPN model by the intracerebroventricular infusion of EPN cells labeled with multimodal iron oxide nanoparticles (MION), thereby generating a tumor and providing a clinically relevant animal model. MRI analysis was shown to be a valuable tool when combined with effective MION labeling techniques to accompany EPN growth. We demonstrated that GFAP/CD133+CD90+/CD44+ EPN cells maintained key histopathological and growth characteristics of the original patient tumor. The characterization of EPN cells and the experimental model could facilitate biological studies and preclinical drug screening for pediatric EPNs. In this work, we established notoriously challenging primary cell culture of anaplastic EPNs (WHO grade III) localized in the posterior fossa (PF), using EPNs obtained from 1 to 10-year-old patients ( n = 07), and then characterized their immunophenotype and ultrastructure to finally develop a xenograft model.

Molecular and functional characterization of CD133+ stem/progenitor cells infused in patients with end-stage liver disease reveals their interplay with stromal liver cells.

PubMed

Catani, Lucia; Sollazzo, Daria; Bianchi, Elisa; Ciciarello, Marilena; Antoniani, Chiara; Foscoli, Licia; Caraceni, Paolo; Giannone, Ferdinando Antonino; Baldassarre, Maurizio; Giordano, Rosaria; Montemurro, Tiziana; Montelatici, Elisa; D'Errico, Antonia; Andreone, Pietro; Giudice, Valeria; Curti, Antonio; Manfredini, Rossella; Lemoli, Roberto Massimo

2017-12-01

Growing evidence supports the therapeutic potential of bone marrow (BM)-derived stem/progenitor cells for end-stage liver disease (ESLD). We recently demonstrated that CD133 + stem/progenitor cell (SPC) reinfusion in patients with ESLD is feasible and safe and improve, albeit transiently, liver function. However, the mechanism(s) through which BM-derived SPCs may improve liver function are not fully elucidated. Here, we characterized the circulating SPCs compartment of patients with ESLD undergoing CD133 + cell therapy. Next, we set up an in vitro model mimicking SPCs/liver microenvironment interaction by culturing granulocyte colony-stimulating factor (G-CSF)-mobilized CD133 + and LX-2 hepatic stellate cells. We found that patients with ESLD show normal basal levels of circulating hematopoietic and endothelial progenitors with impaired clonogenic ability. After G-CSF treatment, patients with ESLD were capable to mobilize significant numbers of functional multipotent SPCs, and interestingly, this was associated with increased levels of selected cytokines potentially facilitating SPC function. Co-culture experiments showed, at the molecular and functional levels, the bi-directional cross-talk between CD133 + SPCs and human hepatic stellate cells LX-2. Human hepatic stellate cells LX-2 showed reduced activation and fibrotic potential. In turn, hepatic stellate cells enhanced the proliferation and survival of CD133 + SPCs as well as their endothelial and hematopoietic function while promoting an anti-inflammatory profile. We demonstrated that the interaction between CD133 + SPCs from patients with ESLD and hepatic stellate cells induces significant functional changes in both cellular types that may be instrumental for the improvement of liver function in cirrhotic patients undergoing cell therapy. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Clinical and Pathological Significance of ER Stress Marker (BiP/GRP78 and PERK) Expression in Malignant Melanoma.

PubMed

Shimizu, Akira; Kaira, Kyoichi; Yasuda, Masahito; Asao, Takayuki; Ishikawa, Osamu

2017-01-01

Glucose-regulated protein of 78 kD (GRP78) also referred to as immunoglobulin heavy chain binding protein (BiP/GRP78) plays an important role in the endoplasmic reticulum (ER) stress. The level of BiP/GRP78 is highly elevated in various human cancers. The purpose of this study is to examine the prognostic significance of BiP/GRP78 expression in patients with malignant melanoma. A total of 133 malignant melanoma patients were analyzed, and tumor specimens were stained by immunohistochemistry for BiP/GRP78, PKR-like endoplasmic reticulum kinase (PERK), Ki-67, p53 and microvessel density (MVD) determined by CD34. BiP/GRP78 and PERK were highly expressed in 40 % (53/133) and 78 % (104/133), respectively. BiP/GRP78 disclosed a significant relationship with PERK expression, thickness, T factor, N factor, disease staging, cell proliferation (Ki-67) and MVD (CD34). By multivariate analysis, the high expression of BiP/GRP78 was identified as an independent prognostic factor for predicting poor survival against malignant melanoma. The increased BiP/GRP78 expression was clarified as an independent prognostic marker for predicting worse outcome. ER stress marker, BiP/GRP78 could be a powerful molecular target for the treatment of malignant melanoma.

Distinctive effects of CD34- and CD133-specific antibody-coated stents on re-endothelialization and in-stent restenosis at the early phase of vascular injury

PubMed Central

Wu, Xue; Yin, Tieying; Tian, Jie; Tang, Chaojun; Huang, Junli; Zhao, Yinping; Zhang, Xiaojuan; Deng, Xiaoyan; Fan, Yubo; Yu, Donghong; Wang, Guixue

2015-01-01

It is not clear what effects of CD34- and CD133-specific antibody-coated stents have on re-endothelialization and in-stent restenosis (ISR) at the early phase of vascular injury. This study aims at determining the capabilities of different coatings on stents (e.g. gelatin, anti-CD133 and anti-CD34 antibodies) to promote adhesion and proliferation of endothelial progenitor cells (EPCs). The in vitro study revealed that the adhesion force enabled the EPCs coated on glass slides to withstand flow-induced shear stress, so that allowing for the growth of the cells on the slides for 48 h. The in vivo experiment using a rabbit model in which the coated stents with different substrates were implanted showed that anti-CD34 and anti-CD133 antibody-coated stents markedly reduced the intima area and restenosis than bare mental stents (BMS) and gelatin-coated stents. Compared with the anti-CD34 antibody-coated stents, the time of cells adhesion was longer and earlier present in the anti-CD133 antibody-coated stents and anti-CD133 antibody-coated stents have superiority in re-endothelialization and inhibition of ISR. In conclusion, this study demonstrated that anti-CD133 antibody as a stent coating for capturing EPCs is better than anti-CD34 antibody in promoting endothelialization and reducing ISR. PMID:26813006

[LincRNA-ROR functions as a ceRNA to regulate Oct4, Sox2, and Nanog expression by sponging miR-145 and its effect on biologic characteristics of colonic cancer stem cells].

PubMed

Yan, Z Y; Sun, X C

2018-04-08

Objective: To investigate the impact of lincRNA-ROR, a ceRNA by binding miR-145 on the expression of the downstream genes Oct4, Sox2 and Nanog, and related biological characteristics of colon cancer stem cells, and to elucidate the clinical significance of this molecular regulatory network. Methods: Fifty-two cases of colorectal cancer tissue and adjacent tissue were collected at Nanyang City Central Hospital and Nanyang Second Hospital, Henan Province, from 2014 to 2016. Real-time quantitative polymerase chain reaction (qPCR) was used to detect the expression of lincRNA-ROR and miR-145 in colorectal cancer tissue and isolated colon cancer cells. The correlation between the expression of lincRNA-ROR, miR-145 and the clinicopathologic features of colon cancer was performed. CD44(-)CD133(-) and CD44(+) CD133(+) cells were isolated from SW1116 by using flow cytometry. The expression of CD44, CD133, Oct4, Sox2, Nanog, lincRNA-ROR and miR-145 in cells were detected by qPCR. The relationship between lincRNA-ROR, miR-145, Oct4, Sox2 and Nanog was analyzed by bioinformatics, dual luciferase reporter assay, qPCR and Western blot. The effects of silencing lincRNA-ROR on the proliferation and chemosensitivity of colon cancer stem cells were detected by MTT, colony formation. Results: LincRNA-ROR was frequently up-regulated and inversely correlated with miR-145 down-regulation in the colon cancer specimens( P <0.05). LincRNA-ROR was related to tumor size, lymph node involvement and distant metastasis( P <0.05), and miR-145 was found related to tumor size and tumor location( P <0.05). CD44(+) CD133(+) cells were successfully isolated from SW1116 by flow cytometry. The levels of CD44, CD133, Oct4, Sox2, Nanog, lincRNA-ROR in CD44(+) CD133(+) cells were significantly increased, while miR-145 was decreased compared with CD44(-)CD133(-)cells( P <0.05). The levels of CD44, CD133, lnc-ROR in CD44(+) CD133(+) cells were significantly reduced upon cell adherence, while miR-145 was significantly increased( P <0.05). Bioinformatics analysis revealed that lincRNA-ROR shared miRNA response elements with core transcription factors Oct4, Sox2 and Nanog. MiR-145 significantly inhibited the expression of lincRNA-ROR, Oct4, Sox2 and Nanog. Silencing lincRNA-ROR significantly inhibited colon cancer stem cells proliferation and increased the sensitivity to chemotherapy. Conclusions: Linc-ROR functions as a key ceRNA to prevent core TFs, e. g., Oct4, Sox2, Nanog, from miR-145-mediated suppression in colon cancer stem cells and regulates cell proliferation and chemosensitivity.The data may provide insights into the pathophysiological interactions of the components of genetic networks in the development of colon cancer and may lead to new therapies in the future.

miR-375 induces human decidua basalis-derived stromal cells to become insulin-producing cells.

PubMed

Shaer, Anahita; Azarpira, Negar; Vahdati, Akbar; Karimi, Mohammad Hosein; Shariati, Mehrdad

2014-09-01

This paper focuses on the development of renewable sources of isletreplacement tissue for the treatment of type I diabetes mellitus. Placental tissue-derived mesenchymal stem cells (MSCs) are a promising source for regenerative medicine due to their plasticity and easy availability. They have the potential to differentiate into insulin-producing cells. miR-375 is a micro RNA that is expressed in the pancreas and involved in islet development. Human placental decidua basalis MSCs (PDB-MSCs) were cultured from full-term human placenta. The immunophenotype of the isolated cells was checked for CD90, CD105, CD44, CD133 and CD34 markers. The MSCs (P3) were chemically transfected with hsa-miR-375. Total RNA was extracted 4 and 6 days after transfection. The expressions of insulin, NGN3, GLUT2, PAX4, PAX6, KIR6.2, NKX6.1, PDX1, and glucagon genes were evaluated using real-time qPCR. On day 6, we tested the potency of the clusters in response to the high glucose challenge and assessed the presence of insulin and NGN3 proteins via immunocytochemistry. Flow cytometry analysis confirmed that more than 90% of the cells were positive for CD90, CD105 and CD44 and negative for CD133 and CD34. Morphological changes were followed from day 2. Cell clusters formed during day 6. Insulin-producing clusters showed a deep red color with DTZ. The expression of pancreatic-specific transcription factors increased remarkably during the four days after transfection and significantly increased on day 7. The clusters were positive for insulin and NGN3 proteins, and C-peptide and insulin secretion increased in response to changes in the glucose concentration (2.8 mM and 16.7 mM). In conclusion, the MSCs could be programmed into functional insulin-producing cells by transfection of miR-375.

Autocrine interleukin-23 promotes self-renewal of CD133+ ovarian cancer stem-like cells.

PubMed

Wang, Dan; Xiang, Tong; Zhao, Zhongquan; Lin, Kailong; Yin, Pin; Jiang, Lupin; Liang, Zhiqing; Zhu, Bo

2016-11-15

Cancer stem cells (CSCs) are a group of cells which possess the ability of self-renewing and unlimited proliferation. And these CSCs are thought to be the cause of metastasis, recurrence and resistance. Recent study has found that pro-inflammatory cytokine and chemotactic factor mediate the self-renewing and differentiation of most of CSCs. Thus we speculate that ovarian cancer stem cells (OCSCs) can also maintain the ability of self-renewing and differentiation by releasing inflammatory factor. This report we discuss the biological characteristics and the specific molecular mechanism mediated by interleukin-23 (IL-23) and its receptor on the self-renewing of OCSCs. We found that OCSCs had high expression of IL-23 and IL-23R. IL-23 could promote the self-renewal ability of OCSCs and played a very important role to maintain the stable expression of stem cell markers in vitro. Moreover, we verified that IL-23 could maintain the potential tumorigenic of OCSCs in vivo and mediate the self-renewal ability and the formation of tumor in OCSCs by activating the signal pathways of STAT3 and NF-κB. In addition, human low differentiation tissues showed overexpression of IL-23. And IL-23 positively correlated to the expression level of CD133, Nanog and Oct4. In conclusion, Our discoveries demonstrate that autocrine IL-23 contribute to ovarian cancer malignancy through promoting the self-renewal of CD133+ ovarian cancer stem-like cells, and this suggests that IL-23 and its signaling pathway might serve as therapeutic targets for the treatment of ovarian cancer.

[Coronary disease extension determines mobilization of endothelial progenitor cells and cytokines after a first myocardial infarction with ST elevation].

PubMed

Jiménez-Navarro, Manuel F; González, Francisco Jesús; Caballero-Borrego, Juan; Marchal, Juan Antonio; Rodríguez-Losada, Noela; Carrillo, Esmeralda; García-Pinilla, José Manuel; Hernández-García, José M; Pérez-González, Rita; Ramírez, Gemma; Aránega, Antonia; de Teresa Galván, Eduardo

2011-12-01

Multivessel coronary disease is still a postinfarction prognostic marker despite new forms of reperfusion, such as primary angioplasty. The aim of this study was to determine the time sequence of various sets of endothelial progenitor cells and angiogenic cytokines (vascular endothelial growth factor, hepatocyte growth factor) according to the degree of extension of the postinfarction coronary disease. We studied the release kinetics in 32 patients admitted for a first myocardial infarction with ST elevation, grouped according to whether they had single or multivessel disease, and 26 controls. The patients had a higher number of endothelial progenitor cells and angiogenic cytokines than the controls at all 3 measurements (admission, day 3, and day 7) of the following subsets: CD34, CD34+CD133+, CD34+KDR+, and CD34+CD133+KDR+CD45+(weak); this latter was higher on day 7. The levels of these cell subsets were all higher in the patients with single-vessel disease and at all 3 measurements. The vascular endothelial growth factor levels were raised during the first week and the hepatocyte growth factor showed an early peak on admission for infarction. No significant differences were seen in the cytokines according to coronary disease extension. Although the release kinetics of different subsets of endothelial progenitor cells in patients with a first acute myocardial infarction with ST elevation was similar in those with single vessel disease to those with multivessel disease, the number of circulating endothelial progenitor cells was greater in the patients with single vessel disease. The vascular endothelial growth factor levels were raised during the first postinfarction week and the hepatocyte growth factor were higher on admission. Copyright © 2011 Sociedad Española de Cardiología. Published by Elsevier Espana. All rights reserved.

Identification and isolation from either adult human bone marrow or G-CSF-mobilized peripheral blood of CD34(+)/CD133(+)/CXCR4(+)/ Lin(-)CD45(-) cells, featuring morphological, molecular, and phenotypic characteristics of very small embryonic-like (VSEL) stem cells.

PubMed

Sovalat, Hanna; Scrofani, Maurice; Eidenschenk, Antoinette; Pasquet, Stéphanie; Rimelen, Valérie; Hénon, Philippe

2011-04-01

Recently, we demonstrated that normal human bone marrow (hBM)-derived CD34(+) cells, released into the peripheral blood after granulocyte colony-stimulating factor mobilization, contain cell subpopulations committed along endothelial and cardiac differentiation pathways. These subpopulations could play a key role in the regeneration of post-ischemic myocardial lesion after their direct intracardiac delivery. We hypothesized that these relevant cells might be issued from very small embryonic-like stem cells deposited in the BM during ontogenesis and reside lifelong in the adult BM, and that they could be mobilized into peripheral blood by granulocyte colony-stimulating factor. Samples of normal hBM and leukapheresis products harvested from cancer patients after granulocyte colony-stimulating factor mobilization were analyzed and sorted by multiparameter flow cytometry strategy. Immunofluorescence and reverse transcription quantitative polymerase chain reaction assays were performed to analyze the expression of typical pluripotent stem cells markers. A population of CD34(+)/CD133(+)/CXCR4(+)/Lin(-) CD45(-) immature cells was first isolated from the hBM or from leukapheresis products. Among this population, very small (2-5 μm) cells expressing Oct-4, Nanog, and stage-specific embryonic antigen-4 at protein and messenger RNA levels were identified. Our study supports the hypothesis that very small embryonic-like stem cells constitute a "mobile" pool of primitive/pluripotent stem cells that could be released from the BM into the peripheral blood under the influence of various physiological or pathological stimuli. In order to fully support that hBM- and leukapheresis product-derived very small embryonic-like stem cells are actually pluripotent, we are currently testing their ability to differentiate in vitro into cells from all three germ layers. Copyright © 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

Transforming growth factor-β decreases side population cells in hepatocellular carcinoma in vitro.

PubMed

Kim, Jong Bin; Lee, Seulki; Kim, Hye Ri; Park, Seo-Young; Lee, Minjong; Yoon, Jung-Hwan; Kim, Yoon Jun

2018-06-01

Hepatocellular carcinoma (HCC) can result from hepatitis B or C infection, fibrosis or cirrhosis. Transforming growth factor-β (TGF-β) is one of the main growth factors associated with fibrosis or cirrhosis progression in the liver, but its role is controversial in hepatocarcinogenesis. In the present study, the effect of TGF-β on the HCC Huh-7 and Huh-Bat cell lines was evaluated. To study the effect of TGF-β, Huh-7 and Huh-Bat cells were treated with TGF-β and a TGF-β receptor inhibitor (SB431542). Cell survival, cell cycle, numbers of side population (SP) cells and expression of the cancer stem cell marker cluster of differentiation (CD)133, epithelial-mesenchymal transition markers (E-cadherin, α-smooth muscle actin and vimentin) and TGF-β-regulated proteins [phospho-c-Jun N-terminal kinase (p-JNK), p-c-Jun and p-smad2] were investigated. TGF-β treatment resulted in decreased cell survival with a targeted effect on SP cells. Expression of CD133 and vimentin was upregulated by treatment with the TGF-β receptor antagonist SB431542, but not with TGF-β. By contrast, TGF-β induced accumulation of cells at G0/G1, and upregulated expression of p-JNK, p-c-Jun and p-smad2. However, these effects were blocked when cells were treated with TGF-β plus SB431542, indicating the specificity of the TGF-β effect. The present results indicated that TGF-β has anticancer effects mediated by survival inhibition of cancer stem cells, which may be developed as a novel therapy for HCC.

Treatment of ovarian cancer by targeting the tumor stem cell-associated carbohydrate antigen, Sialyl-Thomsen-nouveau

PubMed Central

Starbuck, Kristen; Al-Alem, Linah; Eavarone, David A.; Hernandez, Silvia Fatima; Bellio, Chiara; Prendergast, Jillian M.; Stein, Jenna; Dransfield, Daniel T.; Zarrella, Bianca; Growdon, Whitfield B.; Behrens, Jeff; Foster, Rosemary; Rueda, Bo R.

2018-01-01

Recurrent ovarian cancer (OvCa) is thought to result in part from the inability to eliminate rare quiescent cancer stem cells (CSCs) that survive cytotoxic chemotherapy and drive tumor resurgence. The Sialyl-Thomsen-nouveau antigen (STn) is a carbohydrate moiety present on protein markers of CSCs in pancreatic, colon, and gastric malignancies. We have demonstrated that human OvCa cell lines contain varying levels of cells that independently express either STn or the ovarian CSC marker CD133. Here we determine co-expression of STn and CD133 in a subset of human OvCa cell lines. Analyses of colony and sphere forming capacity and of response to standard-of-care cytotoxic therapy suggest a subset of OvCa STn+ cells display some CSC features. The effect of the anti-STn antibody-drug conjugates (ADCs) S3F-CL-MMAE and 2G12-2B2-CL-MMAE on OvCa cell viability in vitro and in vivo was also assessed. Treatment with S3F-CL-MMAE reduced the viability of two of three OvCa cell lines in vitro and exposure to either S3F-CL-MMAE or 2G12-2B2-CL-MMAE reduced OVCAR3-derived xenograft volume in vivo, depleting STn+ tumor cells. In summary, STn+ cells demonstrate some stem-like properties and specific therapeutic targeting of STn in ovarian tumors may be an effective clinical strategy to eliminate both STn+ CSC and STn+ non-CSC populations. PMID:29796189

Establishment of primary cell culture and an intracranial xenograft model of pediatric ependymoma: a prospect for therapy development and understanding of tumor biology

PubMed Central

Pavon, Lorena Favaro; Sibov, Tatiana Tais; Caminada de Toledo, Silvia Regina; Mara de Oliveira, Daniela; Cabral, Francisco Romero; Gabriel de Souza, Jean; Boufleur, Pamela; Marti, Luciana C.; Malheiros, Jackeline Moraes; Ferreira da Cruz, Edgar; Paiva, Fernando F.; Malheiros, Suzana M.F.; de Paiva Neto, Manoel A.; Tannús, Alberto; Mascarenhas de Oliveira, Sérgio; Silva, Nasjla Saba; Cappellano, Andrea Maria; Petrilli, Antonio Sérgio; Chudzinski-Tavassi, Ana Marisa; Cavalheiro, Sérgio

2018-01-01

Background Ependymoma (EPN), the third most common pediatric brain tumor, is a central nervous system (CNS) malignancy originating from the walls of the ventricular system. Surgical resection followed by radiation therapy has been the primary treatment for most pediatric intracranial EPNs. Despite numerous studies into the prognostic value of histological classification, the extent of surgical resection and adjuvant radiotherapy, there have been relatively few studies into the molecular and cellular biology of EPNs. Results We elucidated the ultrastructure of the cultured EPN cells and characterized their profile of immunophenotypic pluripotency markers (CD133, CD90, SSEA-3, CXCR4). We established an experimental EPN model by the intracerebroventricular infusion of EPN cells labeled with multimodal iron oxide nanoparticles (MION), thereby generating a tumor and providing a clinically relevant animal model. MRI analysis was shown to be a valuable tool when combined with effective MION labeling techniques to accompany EPN growth. Conclusions We demonstrated that GFAP/CD133+CD90+/CD44+ EPN cells maintained key histopathological and growth characteristics of the original patient tumor. The characterization of EPN cells and the experimental model could facilitate biological studies and preclinical drug screening for pediatric EPNs. Methods In this work, we established notoriously challenging primary cell culture of anaplastic EPNs (WHO grade III) localized in the posterior fossa (PF), using EPNs obtained from 1 to 10-year-old patients (n = 07), and then characterized their immunophenotype and ultrastructure to finally develop a xenograft model. PMID:29774098

Endothelial precursor cells promote angiogenesis in hepatocellular carcinoma.

PubMed

Sun, Xi-Tai; Yuan, Xian-Wen; Zhu, Hai-Tao; Deng, Zheng-Ming; Yu, De-Cai; Zhou, Xiang; Ding, Yi-Tao

2012-09-21

To investigate the role of bone marrow-derived endothelial progenitor cells (EPCs) in the angiogenesis of hepatocellular carcinoma (HCC). The bone marrow of HCC mice was reconstructed by transplanting green fluorescent protein (GFP) + bone marrow cells. The concentration of circulating EPCs was determined by colony-forming assays and fluorescence-activated cell sorting. Serum and tissue levels of vascular endothelial growth factor (VEGF) and colony-stimulating factor (CSF) were quantified by enzyme-linked immunosorbent assay. The distribution of EPCs in tumor and tumor-free tissues was detected by immunohistochemistry and real-time polymerase chain reaction. The incorporation of EPCs into hepatic vessels was examined by immunofluorescence and immunohistochemistry. The proportion of EPCs in vessels was then calculated. The HCC model was successful established. The flow cytometry analysis showed the mean percentage of CD133CD34 and CD133VEGFR2 double positive cells in HCC mice was 0.45% ± 0.16% and 0.20% ± 0.09% respectively. These values are much higher than in the sham-operation group (0.11% ± 0.13%, 0.05% ± 0.11%, n = 9) at 14 d after modeling. At 21 d, the mean percentage of circulating CD133CD34 and CD133VEGFR2 cells is 0.23% ± 0.19%, 0.25% ± 0.15% in HCC model vs 0.05% ± 0.04%, 0.12% ± 0.11% in control. Compared to the transient increase observed in controls, the higher level of circulating EPCs were induced by HCC. In addition, the level of serum VEGF and CSF increased gradually in HCC, reaching its peak 14 d after modeling, then slowly decreased. Consecutive sections stained for the CD133 and CD34 antigens showed that the CD133+ and CD34+ VEGFR2 cells were mostly recruited to HCC tissue and concentrated in tumor microvessels. Under fluorescence microscopy, the bone-marrow (BM)-derived cells labeled with GFP were concentrated in the same area. The relative levels of CD133 and CD34 gene expression were elevated in tumors, around 5.0 and 3.8 times that of the tumor free area. In frozen liver sections from HCC mice, cells co-expressing CD133 and VEGFR2 were identified by immunohistochemical staining using anti-CD133 and VEGFR2 antibodies. In tumor tissue, the double-positive cells were incorporated into vessel walls. In immunofluorescent staining. These CD31 and GFP double positive cells are direct evidence that tumor vascular endothelial cells (VECs) come partly from BM-derived EPCs. The proportion of GFP CD31 double positive VECs (out of all VECs) on day 21 was around 35.3% ± 21.2%. This is much higher than the value recorded on day 7 group (17.1% ± 8.9%). The expression of intercellular adhesion molecule 1, vascular adhesion molecule 1, and VEGF was higher in tumor areas than in tumor-free tissues. Mobilized EPCs were found to participate in tumor vasculogenesis of HCC. Inhibiting EPC mobilization or recruitment to tumor tissue may be an efficient strategy for treating HCC.

Clinical relevance of miR-mediated HLA-G regulation and the associated immune cell infiltration in renal cell carcinoma

PubMed Central

Jasinski-Bergner, Simon; Stoehr, Christine; Bukur, Juergen; Massa, Chiara; Braun, Juliane; Hüttelmaier, Stefan; Spath, Verena; Wartenberg, Roland; Legal, Wolfgang; Taubert, Helge; Wach, Sven; Wullich, Bernd; Hartmann, Arndt; Seliger, Barbara

2015-01-01

In human tumors of distinct origin including renal cell carcinoma (RCC), the non-classical human leukocyte antigen G (HLA-G) is frequently expressed, thereby inhibiting the cytotoxic activity of T and natural killer (NK) cells. Recent studies demonstrated a strong post-transcriptional gene regulation of the HLA-G by miR-152, −148A, −148B and −133A. Standard methods were applied to characterize the expression and function of HLA-G, HLA-G-regulatory microRNAs (miRs) and the immune cell infiltration in 453 RCC lesions using a tissue microarray and five RCC cell lines linking these results to clinical parameters. Direct interactions with HLA-G regulatory miRs and the HLA-G 3′ untranslated region (UTR) were detected and the affinities of these different miRs to the HLA-G 3′-UTR compared. qPCR analyses and immunohistochemical staining revealed an inverse expression of miR-148A and −133A with the HLA-G protein in situ and in vitro. Stable miR overexpression caused a downregulation of HLA-G protein enhancing the NK and LAK cell-mediated cytotoxicity in in vitro CD107a activation assays revealing a HLA-G-dependent cytotoxic activity of immune effector cells. A significant higher frequency of CD3+/CD8+ T cell lymphocytes, but no differences in the activation markers CD69, CD25 or in the presence of CD56+, FoxP3+ and CD4+ immune cells were detected in HLA-G+ compared to HLA-G− RCC lesions. This could be associated with higher WHO grade, but not with a disease-specific survival. These data suggest a miR-mediated control of HLA-G expression in RCC, which is associated with a distinct pattern of immune cell infiltration. PMID:26155421

Hematopoietic stem cell capture and directional differentiation into vascular endothelial cells for metal stent-coated chitosan/hyaluronic acid loading CD133 antibody.

PubMed

Zhang, Shixuan; Zhang, Fan; Feng, Bo; Fan, Qingyu; Yang, Feng; Shang, Debin; Sui, Jinghan; Zhao, Hong

2015-03-01

A series of metal stents coated with chitosan/hyaluronic acid (CS/HA) loading antibodies by electrostatic self-assembled method were prepared, and the types of cells captured by antibodies and their differentiation in vascular endothelial cells (ECs) evaluated by molecular biology and scanning electron microscope. The results showed that CD133 stent can selectively capture hematopoietic stem cells (HSC),which directionally differentiate into vascular ECs in peripheral blood by (CS/HA) induction, and simultaneously inhibit migration and proliferation of immune cells and vascular smooth muscle cells (MCs). CD34 stent can capture HSC, hematopoietic progenitor cells that differentiate into vascular ECs and immune cells, promoting smooth MCs growth, leading to thrombosis, inflammation, and rejection. CD133 stent can be implanted into miniature pig heart coronary and can repair vascular damage by capturing own HSC, thus contributing to the rapid natural vascular repair, avoiding inflammation and rejection, thrombosis and restenosis. These studies demonstrated that CD133 stent of HSC capture will be an ideal coated metal stent providing a new therapeutic approach for cardiovascular and cerebrovascular disease.

Nestin suppression attenuates invasive potential of endometrial cancer cells by downregulating TGF-β signaling pathway.

PubMed

Bokhari, Amber A; Baker, Tabari M; Dorjbal, Batsukh; Waheed, Sana; Zahn, Christopher M; Hamilton, Chad A; Maxwell, G Larry; Syed, Viqar

2016-10-25

Nestin, an intermediate filament protein and a stem cell marker is expressed in several tumors. Until recently, little was known about the expression levels and the role of Nestin in endometrial cancer. Compared to the immortalized endometrial epithelial cell line EM-E6/E7-TERT, endometrial cancer cell lines express high to moderate levels of Nestin. Furthermore, endometrial tumors and tumor cell lines have a cancer stem-like cell subpopulation expressing CD133. Among the cancer lines, AN3CA and KLE cells exhibited both a significantly higher number of CD133+ cells and expressed Nestin at higher levels than Ishikawa cells. Knockdown of Nestin in AN3CA and KLE increased cells in G0/G1 phase of the cell cycle, whereas overexpression in Ishikawa decreased cells in G0/G1 phase and increased cells in S-phase. Nestin knockdown cells showed increased p21, p27, and PNCA levels and decreased expression of cyclin-D1 and D3. In contrast, Nestin overexpression revealed an inverse expression pattern of cell cycle regulatory proteins. Nestin knockdown inhibited cancer cell growth and invasive potential by downregulating TGF-β signaling components, MMP-2, MMP-9, vimentin, SNAIL, SLUG, Twist, N-cadherin, and upregulating the epithelial cell marker E-cadherin whereas the opposite was observed with Nestin overexpressing Ishikawa cells. Nestin knockdown also inhibited, while overexpression promoted invadopodia formation and pFAK expression. Knockdown of Nestin significantly reduced tumor volume in vivo. Finally, progesterone inhibited Nestin expression in endometrial cancer cells. These results suggest that Nestin can be a therapeutic target for cancer treatment.

Nestin suppression attenuates invasive potential of endometrial cancer cells by downregulating TGF-β signaling pathway

PubMed Central

Bokhari, Amber A.; Baker, Tabari M.; Dorjbal, Batsukh; Waheed, Sana; Zahn, Christopher M.; Hamilton, Chad A.; Maxwell, G. Larry; Syed, Viqar

2016-01-01

Nestin, an intermediate filament protein and a stem cell marker is expressed in several tumors. Until recently, little was known about the expression levels and the role of Nestin in endometrial cancer. Compared to the immortalized endometrial epithelial cell line EM-E6/E7-TERT, endometrial cancer cell lines express high to moderate levels of Nestin. Furthermore, endometrial tumors and tumor cell lines have a cancer stem-like cell subpopulation expressing CD133. Among the cancer lines, AN3CA and KLE cells exhibited both a significantly higher number of CD133+ cells and expressed Nestin at higher levels than Ishikawa cells. Knockdown of Nestin in AN3CA and KLE increased cells in G0/G1 phase of the cell cycle, whereas overexpression in Ishikawa decreased cells in G0/G1 phase and increased cells in S-phase. Nestin knockdown cells showed increased p21, p27, and PNCA levels and decreased expression of cyclin-D1 and D3. In contrast, Nestin overexpression revealed an inverse expression pattern of cell cycle regulatory proteins. Nestin knockdown inhibited cancer cell growth and invasive potential by downregulating TGF-β signaling components, MMP-2, MMP-9, vimentin, SNAIL, SLUG, Twist, N-cadherin, and upregulating the epithelial cell marker E-cadherin whereas the opposite was observed with Nestin overexpressing Ishikawa cells. Nestin knockdown also inhibited, while overexpression promoted invadopodia formation and pFAK expression. Knockdown of Nestin significantly reduced tumor volume in vivo. Finally, progesterone inhibited Nestin expression in endometrial cancer cells. These results suggest that Nestin can be a therapeutic target for cancer treatment. PMID:27626172

Abnormal neural precursor cell regulation in the early postnatal Fragile X mouse hippocampus.

PubMed

Sourial, Mary; Doering, Laurie C

2017-07-01

The regulation of neural precursor cells (NPCs) is indispensable for a properly functioning brain. Abnormalities in NPC proliferation, differentiation, survival, or integration have been linked to various neurological diseases including Fragile X syndrome. Yet, no studies have examined NPCs from the early postnatal Fragile X mouse hippocampus despite the importance of this developmental time point, which marks the highest expression level of FMRP, the protein missing in Fragile X, in the rodent hippocampus and is when hippocampal NPCs have migrated to the dentate gyrus (DG) to give rise to lifelong neurogenesis. In this study, we examined NPCs from the early postnatal hippocampus and DG of Fragile X mice (Fmr1-KO). Immunocytochemistry on neurospheres showed increased Nestin expression and decreased Ki67 expression, which collectively indicated aberrant NPC biology. Intriguingly, flow cytometric analysis of the expression of the antigens CD15, CD24, CD133, GLAST, and PSA-NCAM showed a decreased proportion of neural stem cells (GLAST + CD15 + CD133 + ) and an increased proportion of neuroblasts (PSA-NCAM + CD15 + ) in the DG of P7 Fmr1-KO mice. This was mirrored by lower expression levels of Nestin and the mitotic marker phospho-histone H3 in vivo in the P9 hippocampus, as well as a decreased proportion of cells in the G 2 /M phases of the P7 DG. Thus, the absence of FMRP leads to fewer actively cycling NPCs, coinciding with a decrease in neural stem cells and an increase in neuroblasts. Together, these results show the importance of FMRP in the developing hippocampal formation and suggest abnormalities in cell cycle regulation in Fragile X. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Chitosan promotes cancer progression and stem cell properties in association with Wnt signaling in colon and hepatocellular carcinoma cells

PubMed Central

Chang, Po-Hsiang; Sekine, Keisuke; Chao, Hsiao-Mei; Hsu, Shan-hui; Chern, Edward

2017-01-01

Cancer stem cells (CSCs), a small population of cancer cells, have been considered to be the origin of cancer initiation, recurrence, and metastasis. Tumor microenvironment provides crucial signals for CSCs to maintain stem cell properties and promotes tumorigenesis. Therefore, establishment of an appropriate cell culture system to mimic the microenvironment for CSC studies is an important issue. In this study, we grew colon and hepatocellular carcinoma (HCC) cells on chitosan membranes and evaluated the tumor progression and the CSC properties. Experimental results showed that culturing cancer cells on chitosan increased cell motility, drug resistance, quiescent population, self-renewal capacity, and the expression levels of stemness and CSC marker genes, such as OCT4, NANOG, CD133, CD44, and EpCAM. Furthermore, we demonstrated that chitosan might activate canonical Wnt/β-catenin-CD44 axis signaling in CD44positive colon cancer cells and noncanonical Wnt-STAT3 signaling in CD44negative HCC cells. In conclusion, chitosan as culture substrates activated the essential signaling of CSCs and promoted CSC properties. The chitosan culture system provides a convenient platform for the research of CSC biology and screening of anticancer drugs. PMID:28367998

Current molecular markers for gastric progenitor cells and gastric cancer stem cells.

PubMed

Qiao, Xiaotan T; Gumucio, Deborah L

2011-07-01

Gastric stem and progenitor cells (GPC) play key roles in the homeostatic renewal of gastric glands and are instrumental in epithelial repair after injury. Until very recently, the existence of GPC could only be inferred by indirect labeling strategies. The last few years have seen significant progress in the identification of biomarkers that allow prospective identification of GPC. The analysis of these unique cell populations is providing new insights into the molecular underpinnings of gastric epithelial homeostasis and repair. Of closely related interest is the potential to identify so-called cancer stem cells, a rare subpopulation of tumor-initiating cells. Here, we review the current useful biomarkers for GPC, including: (a) those that have been demonstrated by lineage tracing to give rise to all gastric cell lineages (e.g., the villin-transgene marker as well as Lgr5); (b) those that give rise to a subset of gastric lineages (e.g., TFF2); (c) markers that recognize cryptic progenitors for metaplasia (e.g., MIST1), and (d) markers that have not yet been analyzed by lineage tracing (e.g., DCKL1/DCAMKL1, CD133/PROM1, and CD44). The study of these markers has been mostly limited to the mouse model, but the hope is that the rapid pace of recent breakthroughs in this animal model will soon lead to a greater understanding of human gastric stem cell biology and to new insights into gastric cancer, the second leading cause of cancer-related death worldwide.

Acoustic and hybrid 3D-printed electrochemical biosensors for the real-time immunodetection of liver cancer cells (HepG2).

PubMed

Damiati, Samar; Küpcü, Seta; Peacock, Martin; Eilenberger, Christoph; Zamzami, Mazin; Qadri, Ishtiaq; Choudhry, Hani; Sleytr, Uwe B; Schuster, Bernhard

2017-08-15

This study presents an efficient acoustic and hybrid three-dimensional (3D)-printed electrochemical biosensors for the detection of liver cancer cells. The biosensors function by recognizing the highly expressed tumor marker CD133, which is located on the surface of liver cancer cells. Detection was achieved by recrystallizing a recombinant S-layer fusion protein (rSbpA/ZZ) on the surface of the sensors. The fused ZZ-domain enables immobilization of the anti-CD133 antibody in a defined manner. These highly accessible anti-CD133 antibodies were employed as a sensing layer, thereby enabling the efficient detection of liver cancer cells (HepG2). The recognition of HepG2 cells was investigated in situ using a quartz crystal microbalance with dissipation monitoring (QCM-D), which enabled the label-free, real-time detection of living cells on the modified sensor surface under controlled conditions. Furthermore, the hybrid 3D additive printing strategy for biosensors facilitates both rapid development and small-scale manufacturing. The hybrid strategy of combining 3D-printed parts and more traditionally fabricated parts enables the use of optimal materials: a ceramic substrate with noble metals for the sensing element and 3D-printed capillary channels to guide and constrain the clinical sample. Cyclic voltammetry (CV) measurements confirmed the efficiency of the fabricated sensors. Most importantly, these sensors offer low-cost and disposable detection platforms for real-world applications. Thus, as demonstrated in this study, both fabricated acoustic and electrochemical sensing platforms can detect cancer cells and therefore may have further potential in other clinical applications and drug-screening studies. Copyright © 2017 Elsevier B.V. All rights reserved.

Granulocyte colony-stimulating factor mobilizes functional endothelial progenitor cells in patients with coronary artery disease.

PubMed

Powell, Tiffany M; Paul, Jonathan D; Hill, Jonathan M; Thompson, Michael; Benjamin, Moshe; Rodrigo, Maria; McCoy, J Philip; Read, Elizabeth J; Khuu, Hanh M; Leitman, Susan F; Finkel, Toren; Cannon, Richard O

2005-02-01

Endothelial progenitor cells (EPCs) that may repair vascular injury are reduced in patients with coronary artery disease (CAD). We reasoned that EPC number and function may be increased by granulocyte colony-stimulating factor (G-CSF) used to mobilize hematopoietic progenitor cells in healthy donors. Sixteen CAD patients had reduced CD34(+)/CD133(+) (0.0224+/-0.0063% versus 0.121+/-0.038% mononuclear cells [MNCs], P<0.01) and CD133(+)/VEGFR-2(+) cells, consistent with EPC phenotype (0.00033+/-0.00015% versus 0.0017+/-0.0006% MNCs, P<0.01), compared with 7 healthy controls. Patients also had fewer clusters of cells in culture, with out-growth consistent with mature endothelial phenotype (2+/-1/well) compared with 16 healthy subjects at high risk (13+/-4/well, P<0.05) or 14 at low risk (22+/-3/well, P<0.001) for CAD. G-CSF 10 microg/kg per day for 5 days increased CD34(+)/CD133(+) cells from 0.5+/-0.2/microL to 59.5+/-10.6/microL and CD133(+)/ VEGFR-2(+) cells from 0.007+/-0.004/microL to 1.9+/-0.6/microL (both P<0.001). Also increased were CD133(+) cells that coexpressed the homing receptor CXCR4 (30.4+/-8.3/microL, P<0.05). Endothelial cell-forming clusters in 10 patients increased to 27+/-9/well after treatment (P<0.05), with a decline to 9+/-4/well at 2 weeks (P=0.06). Despite reduced EPCs compared with healthy controls, patients with CAD respond to G-CSF with increases in EPC number and homing receptor expression in the circulation and endothelial out-growth in culture. Endothelial progenitor cells (EPCs) are reduced in coronary artery disease. Granulocyte colony-stimulating factor (CSF) administered to patients increased: (1) CD133+/VEGFR-2+ cells consistent with EPC phenotype; (2) CD133+ cells coexpressing the chemokine receptor CXCR4, important for homing of EPCs to ischemic tissue; and (3) endothelial cell-forming clusters in culture. Whether EPCs mobilized into the circulation will be useful for the purpose of initiating vascular growth and myocyte repair in coronary artery disease patients must be tested in clinical trials.

Targeted salinomycin delivery with EGFR and CD133 aptamers based dual-ligand lipid-polymer nanoparticles to both osteosarcoma cells and cancer stem cells.

PubMed

Chen, Fangyi; Zeng, Yibin; Qi, Xiaoxia; Chen, Yanchao; Ge, Zhe; Jiang, Zengxin; Zhang, Xinchao; Dong, Yinmei; Chen, Huaiwen; Yu, Zuochong

2018-06-10

We previously developed salinomycin (sali)-entrapped nanoparticles labeled with CD133 aptamers which could efficiently eliminate CD133 + osteosarcoma cancer stem cells (CSCs). However, sufficient evidences suggest that the simultaneous targeting both CSCs and cancer cells is pivotal in achieving preferable cancer therapeutic efficacy, due to the spontaneous conversion between cancer cells and CSCs. We hereby constructed sali-entrapped lipid-polymer nanoparticles labeled with CD133 and EGFR aptamers (CESP) to target both osteosarcoma cells and CSCs. The cytotoxicity of CESP in osteosarcoma cells and CSCs was superior to that of single targeting or nontargeted sali-loaded nanoparticles. Administration of CESP in vivo showed the best efficacy in inhibiting tumor growth than other controls in osteosarcoma-bearing mice. Thus, CESP was demonstrated to be capable of efficiently targeting both osteosarcoma CSCs and cancer cells, and it represents an effective potential approach to treat osteosarcoma. Copyright © 2018 Elsevier Inc. All rights reserved.

Intraportal Infusion of Bone Marrow Mononuclear or CD133+ Cells in Patients With Decompensated Cirrhosis: A Double-Blind Randomized Controlled Trial

PubMed Central

Mohamadnejad, Mehdi; Vosough, Massoud; Moossavi, Shirin; Nikfam, Sepideh; Mardpour, Soura; Akhlaghpoor, Shahram; Ashrafi, Mandana; Azimian, Vajiheh; Jarughi, Neda; Hosseini, Seyedeh-Esmat; Moeininia, Fatemeh; Bagheri, Mohamad; Sharafkhah, Maryam; Aghdami, Nasser

2016-01-01

The present study assessed the effects of intraportal infusions of autologous bone marrow-derived mononuclear cells (MNCs) and/or CD133+ cells on liver function in patients with decompensated cirrhosis. We randomly assigned 27 eligible patients to a placebo, MNCs, and/or CD133+ cells. Cell infusions were performed at baseline and month 3. We considered the absolute changes in the Model for End-Stage Liver Disease (MELD) scores at months 3 and 6 after infusion as the primary outcome. The participants and those who assessed the outcomes were unaware of the treatment intervention assignments. After 6 months, 9 patients were excluded because of liver transplantation (n = 3), hepatocellular carcinoma (n = 1), loss to follow-up (n = 3), and death (n = 2). The final analysis included 4 patients from the CD133+ group, 8 from the MNC group, and 6 from the placebo group. No improvement was seen in the MELD score at month 6 using either CD133+ cells or MNC infusions compared with placebo. However, at month 3 after infusion, a trend was seen toward a higher mean absolute change in the MELD score in patients who had received CD133+ cells compared with placebo (−2.00 ± 1.87 vs. −0.13 ± 1.46; p = .08). No significant adverse events occurred in the present study. A transient improvement in the MELD score was observed in subjects treated with CD133+ cells but not in the MNC or placebo group. Although the study was not powered to make definitive conclusions, the data justify further study of CD133+ therapy in cirrhotic patients. Significance Cell therapy is a new approach in liver disease. Several clinical experiments have been reported on the safety of bone marrow-derived stem cells to treat liver disorders. However, the effectiveness of these approaches in the long-term follow-ups of patients initiated controversial discussions among the scientific community. A double-blind randomized controlled trial was designed to address this concern scientifically. A transient improvement in the patients’ signs occurred; however, for a sustainable result, more work is needed. The results of multiple administrations of cells reported in the present study can be compared with the results from other single-injection studies. PMID:26659833

GFP Labeling and Hepatic Differentiation Potential of Human Placenta-Derived Mesenchymal Stem Cells.

PubMed

Yu, Jiong; Su, Xiaoru; Zhu, Chengxing; Pan, Qiaoling; Yang, Jinfeng; Ma, Jing; Shen, Leyao; Cao, Hongcui; Li, Lanjuan

2015-01-01

Stem cell-based therapy in liver diseases has received increasing interest over the past decade, but direct evidence of the homing and implantation of transplanted cells is conflicting. Reliable labeling and tracking techniques are essential but lacking. The purpose of this study was to establish human placenta-derived mesenchymal stem cells (hPMSCs) expressing green fluorescent protein (GFP) and to assay their hepatic functional differentiation in vitro. The GFP gene was transduced into hPMSCs using a lentivirus to establish GFP(+) hPMSCs. GFP(+) hPMSCs were analyzed for their phenotypic profile, viability and adipogenic, osteogenic and hepatic differentiation. The derived GFP(+) hepatocyte-like cells were evaluated for their metabolic, synthetic and secretory functions, respectively. GFP(+) hPMSCs expressed high levels of HLA I, CD13, CD105, CD73, CD90, CD44 and CD29, but were negative for HLA II, CD45, CD31, CD34, CD133, CD271 and CD79. They possessed adipogenic, osteogenic and hepatic differentiation potential. Hepatocyte-like cells derived from GFP(+) hPMSCs showed typical hepatic phenotypes. GFP gene transduction has no adverse influences on the cellular or biochemical properties of hPMSCs or markers. GFP gene transduction using lentiviral vectors is a reliable labeling and tracking method. GFP(+) hPMSCs can therefore serve as a tool to investigate the mechanisms of MSC-based therapy, including hepatic disease therapy. © 2015 S. Karger AG, Basel.

Inhibition of oxygen-glucose deprivation-induced apoptosis of human adipose-derived stem cells by genetic modification with antiapoptotic protein bcl-2.

PubMed

Cui, Ziwei; Shen, Liangyun; Lin, Yue; Wang, Shuqin; Zheng, Dongfeng; Tan, Qian

2014-08-01

Adipose-derived stem cells (ADSCs) have become a promising tool for a wide range of cell-based therapies. However, transplanted ADSCs do not survive well under ischemic conditions. In this study we aimed to inhibit oxygen-glucose deprivation (OGD)-induced apoptosis of human ADSCs by genetic modification with antiapoptotic protein Bcl-2. After isolation and culture, the phenotypes of human ADSCs at passage 3 were analyzed by flow cytometry. Then, genetic modification of ADSCs with Bcl-2 was carried out. Bcl-2 gene transfection was verified by Western blot analysis and multipotent differentiation properties were evaluated in Bcl-2-modified ADSCs (Bcl-2-ADSCs). Apoptosis was evaluated by a TUNEL assay under ischemic conditions induced by OGD. Apoptotic nuclei were also assessed and quantified by Hoechst staining. The cultured ADSCs expressed stem cell-associated markers CD29, CD34, CD44, and CD90, but not fibroblast marker HLA-DR or hematopoietic stem cell marker CD133. The Bcl-2 gene was transferred into ADSCs efficiently, and Bcl-2-ADSCs differentiated into adipocytes, chondrocytes, and osteoblasts. In addition, Bcl-2 overexpression reduced the percentage of apoptotic Bcl-2-ADSCs by 38 % under OGD. Our results indicate that Bcl-2 overexpression through gene transfection inhibits apoptosis of ADSCs under ischemic conditions. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Crosstalk-eliminated quantitative determination of aflatoxin B1-induced hepatocellular cancer stem cells based on concurrent monitoring of CD133, CD44, and aldehyde dehydrogenase1.

PubMed

Ju, Hee; Shim, Yumi; Arumugam, Parthasarathy; Song, Joon Myong

2016-01-22

Cancer stem cells (CSCs), known as tumor initiating cells, have become a critically important issue for cancer therapy. Although much research has demonstrated the induction of hepato cellular carcinoma by aflatoxin B1, the formation of hepatocellular CSCs and their quantitative determination is hardly reported. In this work, it was found that hepatocellular CSCs were produced from HepG2 cells by aflatoxin B1-induced mutation, and their amount was quantitatively determined using crosstalk-eliminated multicolor cellular imaging based on quantum dot (Qdot) nanoprobes and an acousto-optical tunable filter (AOTF). Hepatocellular CSCs were acquired via magnetic bead-based sorting and observed using concurrent detection of three different markers: CD133, CD44, and aldehyde dehydrogenase1 (ALDH1). The DNA mutation of HepG2 cells caused by aflatoxin B1 was quantitatively observed via absorbance spectra of aflatoxin B1-8, 9-epoxide-DNA adducts. The percentages of hepatocellular CSCs formed in the entire HepG2 cells were determined to be 9.77±0.65%, 10.9±1.39%, 11.4±1.32%, and 12.8±0.7%, respectively, at 0 μM, 5 μM, 10 μM, and 20 μM of aflatoxin B1. The results matched well with those obtained utilizing flow cytometry. This study demonstrates that aflatoxin mediated mutation induced the conversion of hepatic cancer cell to hepatic CSCs by using a Qdot based constructed multicolor cellular imaging system. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Neighbor of Punc E 11: expression pattern of the new hepatic stem/progenitor cell marker during murine liver development.

PubMed

Schievenbusch, Stephanie; Sauer, Elisabeth; Curth, Harald-Morten; Schulte, Sigrid; Demir, Münevver; Toex, Ulrich; Goeser, Tobias; Nierhoff, Dirk

2012-09-20

We have previously identified Neighbor of Punc E 11 (Nope) as a specific cell surface marker of stem/progenitor cells in the murine fetal liver that is also expressed in hepatocellular carcinoma. Here, we focus on the differential expression pattern of Nope during murine fetal and postnatal liver development as well as in a normal and regenerating adult liver including oval cell activation. In the fetal liver, Nope shows a constantly high expression level and is a useful surface marker for the identification of Dlk, E-cadherin, and CD133-positive hepatoblasts by flow cytometry. Postnatally, Nope expression declines rapidly and remains barely detectable in the adult liver as shown by quantitative real-time reverse-transcriptase polymerase chain reaction and western blot analyses. Immunohistochemically, costainings for Nope- and epithelial-specific markers (E-cadherin), markers of early hepatoblasts (alpha-fetoprotein), and biliary marker proteins (CK19) demonstrate that Nope is initially expressed on bipotent hepatoblasts and persists thereafter on commited hepatocytic as well as cholangiocytic progenitor cells during late fetal liver development. Postnatally, Nope loses its circular expression pattern and is specifically directed to the sinusoidal membrane of early hepatocytes. While Nope is only weakly expressed on cholangiocytes in the normal adult liver, activated stem/progenitor (oval) cells clearly coexpress Nope together with the common markers A6, EpCAM, and CD24 in the 3,5-diethoxycarbonyl-1,4-dihydrocollidine mouse model. In conclusion, Nope should be most useful in future research to define the differentiation stage of hepatic-specified cells of various sources and is a promising candidate to identify and isolate hepatic stem cells from the adult liver.

Hematopoietic Stem Cell Capture and Directional Differentiation into Vascular Endothelial Cells for Metal Stent-Coated Chitosan/Hyaluronic Acid Loading CD133 Antibody

PubMed Central

Zhang, Fan; Feng, Bo; Fan, Qingyu; Yang, Feng; Shang, Debin; Sui, Jinghan; Zhao, Hong

2015-01-01

A series of metal stents coated with chitosan/hyaluronic acid (CS/HA) loading antibodies by electrostatic self-assembled method were prepared, and the types of cells captured by antibodies and their differentiation in vascular endothelial cells (ECs) evaluated by molecular biology and scanning electron microscope. The results showed that CD133 stent can selectively capture hematopoietic stem cells (HSC),which directionally differentiate into vascular ECs in peripheral blood by (CS/HA) induction, and simultaneously inhibit migration and proliferation of immune cells and vascular smooth muscle cells (MCs). CD34 stent can capture HSC, hematopoietic progenitor cells that differentiate into vascular ECs and immune cells, promoting smooth MCs growth, leading to thrombosis, inflammation, and rejection. CD133 stent can be implanted into miniature pig heart coronary and can repair vascular damage by capturing own HSC, thus contributing to the rapid natural vascular repair, avoiding inflammation and rejection, thrombosis and restenosis. These studies demonstrated that CD133 stent of HSC capture will be an ideal coated metal stent providing a new therapeutic approach for cardiovascular and cerebrovascular disease. PMID:25404533

COMPARE CPM-RMI Trial: Intramyocardial Transplantation of Autologous Bone Marrow-Derived CD133+ Cells and MNCs during CABG in Patients with Recent MI: A Phase II/III, Multicenter, Placebo-Controlled, Randomized, Double-Blind Clinical Trial.

PubMed

Naseri, Mohammad Hassan; Madani, Hoda; Ahmadi Tafti, Seyed Hossein; Moshkani Farahani, Maryam; Kazemi Saleh, Davood; Hosseinnejad, Hossein; Hosseini, Saeid; Hekmat, Sepideh; Hossein Ahmadi, Zargham; Dehghani, Majid; Saadat, Alireza; Mardpour, Soura; Hosseini, Seyedeh Esmat; Esmaeilzadeh, Maryam; Sadeghian, Hakimeh; Bahoush, Gholamreza; Bassi, Ali; Amin, Ahmad; Fazeli, Roghayeh; Sharafi, Yaser; Arab, Leila; Movahhed, Mansour; Davaran, Saeid; Ramezanzadeh, Narges; Kouhkan, Azam; Hezavehei, Ali; Namiri, Mehrnaz; Kashfi, Fahimeh; Akhlaghi, Ali; Sotoodehnejadnematalahi, Fattah; Vosough Dizaji, Ahmad; Gourabi, Hamid; Syedi, Naeema; Shahverdi, Abdol Hosein; Baharvand, Hossein; Aghdami, Nasser

2018-07-01

The regenerative potential of bone marrow-derived mononuclear cells (MNCs) and CD133+ stem cells in the heart varies in terms of their pro-angiogenic effects. This phase II/III, multicenter and double-blind trial is designed to compare the functional effects of intramyocardial autologous transplantation of both cell types and placebo in patients with recent myocardial infarction (RMI) post-coronary artery bypass graft. This was a phase II/III, randomized, double-blind, placebo-controlled trial COMPARE CPM-RMI (CD133, Placebo, MNCs - recent myocardial infarction) conducted in accordance with the Declaration of Helsinki that assessed the safety and efficacy of CD133 and MNCs compared to placebo in patients with RMI. We randomly assigned 77 eligible RMI patients selected from 5 hospitals to receive CD133+ cells, MNC, or a placebo. Patients underwent gated single photon emission computed tomography assessments at 6 and 18 months post-intramyocardial transplantation. We tested the normally distributed efficacy outcomes with a mixed analysis of variance model that used the entire data set of baseline and between-group comparisons as well as within subject (time) and group×time interaction terms. There were no related serious adverse events reported. The intramyocardial transplantation of both cell types increased left ventricular ejection fraction by 9% [95% confidence intervals (CI): 2.14% to 15.78%, P=0.01] and improved decreased systolic wall thickening by -3.7 (95% CI: -7.07 to -0.42, P=0.03). The CD133 group showed significantly decreased non-viable segments by 75% (P=0.001) compared to the placebo and 60% (P=0.01) compared to the MNC group. We observed this improvement at both the 6- and 18-month time points. Intramyocardial injections of CD133+ cells or MNCs appeared to be safe and efficient with superiority of CD133+ cells for patients with RMI. Although the sample size precluded a definitive statement about clinical outcomes, these results have provided the basis for larger studies to confirm definitive evidence about the efficacy of these cell types (Registration Number: NCT01167751). Copyright© by Royan Institute. All rights reserved.

Pediatric medulloblastoma xenografts including molecular subgroup 3 and CD133+ and CD15+ cells are sensitive to killing by oncolytic herpes simplex viruses.

PubMed

Friedman, Gregory K; Moore, Blake P; Nan, Li; Kelly, Virginia M; Etminan, Tina; Langford, Catherine P; Xu, Hui; Han, Xiaosi; Markert, James M; Beierle, Elizabeth A; Gillespie, G Yancey

2016-02-01

Childhood medulloblastoma is associated with significant morbidity and mortality that is compounded by neurotoxicity for the developing brain caused by current therapies, including surgery, craniospinal radiation, and chemotherapy. Innate therapeutic resistance of some aggressive pediatric medulloblastoma has been attributed to a subpopulation of cells, termed cancer-initiating cells or cancer stemlike cells (CSCs), marked by the surface protein CD133 or CD15. Brain tumors characteristically contain areas of pathophysiologic hypoxia, which has been shown to drive the CSC phenotype leading to heightened invasiveness, angiogenesis, and metastasis. Novel therapies that target medulloblastoma CSCs are needed to improve outcomes and decrease toxicity. We hypothesized that oncolytic engineered herpes simplex virus (oHSV) therapy could effectively infect and kill pediatric medulloblastoma cells, including CSCs marked by CD133 or CD15. Using 4 human pediatric medulloblastoma xenografts, including 3 molecular subgroup 3 tumors, which portend worse patient outcomes, we determined the expression of CD133, CD15, and the primary HSV-1 entry molecule nectin-1 (CD111) by fluorescence activated cell sorting (FACS) analysis. Infectability and cytotoxicity of clinically relevant oHSVs (G207 and M002) were determined in vitro and in vivo by FACS, immunofluorescent staining, cytotoxicity assays, and murine survival studies. We demonstrate that hypoxia increased the CD133+ cell fraction, while having the opposite effect on CD15 expression. We established that all 4 xenografts, including the CSCs, expressed CD111 and were highly sensitive to killing by G207 or M002. Pediatric medulloblastoma, including Group 3 tumors, may be an excellent target for oHSV virotherapy, and a clinical trial in medulloblastoma is warranted. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Human endothelial precursor cells express tumor endothelial marker 1/endosialin/CD248.

PubMed

Bagley, Rebecca G; Rouleau, Cecile; St Martin, Thia; Boutin, Paula; Weber, William; Ruzek, Melanie; Honma, Nakayuki; Nacht, Mariana; Shankara, Srinivas; Kataoka, Shiro; Ishida, Isao; Roberts, Bruce L; Teicher, Beverly A

2008-08-01

Angiogenesis occurs during normal physiologic processes as well as under pathologic conditions such as tumor growth. Serial analysis of gene expression profiling revealed genes [tumor endothelial markers (TEM)] that are overexpressed in tumor endothelial cells compared with normal adult endothelial cells. Because blood vessel development of malignant tumors under certain conditions may include endothelial precursor cells (EPC) recruited from bone marrow, we investigated TEM expression in EPC. The expression of TEM1 or endosialin (CD248) and other TEM has been discovered in a population of vascular endothelial growth factor receptor 2+/CD31+/CD45-/VE-cadherin+ EPC derived from human CD133+/CD34+ cells. EPC share some properties with fully differentiated endothelial cells from normal tissue, yet reverse transcription-PCR and flow cytometry reveal that EPC express higher levels of endosialin at the molecular and protein levels. The elevated expression of endosialin in EPC versus mature endothelial cells suggests that endosialin is involved in the earlier stages of tumor angiogenesis. Anti-endosialin antibodies inhibited EPC migration and tube formation in vitro. In vivo, immunohistochemistry indicated that human EPC continued to express endosialin protein in a Matrigel plug angiogenesis assay established in nude mice. Anti-endosialin antibodies delivered systemically at 25 mg/kg were also able to inhibit circulating murine EPC in nude mice bearing s.c. SKNAS tumors. EPC and bone marrow-derived cells have been shown previously to incorporate into malignant blood vessels in some instances, yet they remain controversial in the field. The data presented here on endothelial genes that are up-regulated in tumor vasculature and in EPC support the hypothesis that the angiogenesis process in cancer can involve EPC.

The usefulness of three-dimensional cell culture in induction of cancer stem cells from esophageal squamous cell carcinoma cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fujiwara, Daisuke; Kato, Kazunori, E-mail: kzkatou@juntendo.ac.jp; Department of Atopy Research Center, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421

2013-05-17

Highlights: •Spheroids were created from esophageal carcinoma cells using NanoCulture® Plates. •The proportion of strongly ALDH-positive cells increased in 3-D culture. •Expression of cancer stem cell-related genes was enhanced in 3-D culture. •CA-9 expression was enhanced, suggesting hypoxia had been induced in 3-D culture. •Drug resistance was increased. 3-D culture is useful for inducing cancer stem cells. -- Abstract: In recent years, research on resistance to chemotherapy and radiotherapy in cancer treatment has come under the spotlight, and researchers have also begun investigating the relationship between resistance and cancer stem cells. Cancer stem cells are assumed to be present inmore » esophageal cancer, but experimental methods for identification and culture of these cells have not yet been established. To solve this problem, we created spheroids using a NanoCulture® Plate (NCP) for 3-dimensional (3-D) cell culture, which was designed as a means for experimentally reproducing the 3-D structures found in the body. We investigated the potential for induction of cancer stem cells from esophageal cancer cells. Using flow cytometry we analyzed the expression of surface antigen markers CD44, CD133, CD338 (ABCG2), CD318 (CDCP1), and CD326 (EpCAM), which are known cancer stem cell markers. None of these surface antigen markers showed enhanced expression in 3-D cultured cells. We then analyzed aldehyde dehydrogenase (ALDH) enzymatic activity using the ALDEFLUOR reagent, which can identify immature cells such as stem cells and precursor cells. 3-D-cultured cells were strongly positive for ALDH enzyme activity. We also analyzed the expression of the stem cell-related genes Sox-2, Nanog, Oct3/4, and Lin28 using RT-PCR. Expression of Sox-2, Nanog, and Lin28 was enhanced. Analysis of expression of the hypoxic surface antigen marker carbonic anhydrase-9 (CA-9), which is an indicator of cancer stem cell induction and maintenance, revealed that CA-9 expression was enhanced, suggesting that hypoxia had been induced. Comparison of cancer drug resistance using cisplatin and doxorubicin in 3-D-cultured esophageal cancer cells showed that cancer drug resistance had increased. These results indicate that 3-D culture of esophageal squamous cell carcinoma lines is a useful method for inducing cancer stem cells.« less

Isolation, molecular cloning and in vitro expression of rhesus monkey (Macaca mulatta) prominin-1.s1 complementary DNA encoding a potential hematopoietic stem cell antigen.

PubMed

Husain, S M; Shou, Y; Sorrentino, B P; Handgretinger, R

2006-10-01

Human prominin-1 (CD133 or AC133) is an important cell surface marker used to isolate primitive hematopoietic stem cells. The commercially available antibody to human prominin-1 does not recognize rhesus prominin-1. Therefore, we isolated, cloned and characterized the complementary DNA (cDNA) of rhesus prominin-1 gene and determined its coding potential. Following the nomenclature of prominin family of genes, we named this cDNA as rhesus prominin-1.s1. The amino acid sequence data of the putative rhesus prominin-1.s1 could be used in designing antigenic peptides to raise antibodies for use in isolation of pure populations of rhesus prominin-1(+) hematopoietic cells. To the best of our knowledge, there has been no previously published report about the isolation of a prominin-1 cDNA from rhesus monkey (Macaca mulatta).

Bmi-1 plays a critical role in the protection from acute tubular necrosis by mobilizing renal stem/progenitor cells.

PubMed

Lv, Xianhui; Yu, Zhenzhen; Xie, Chunfeng; Dai, Xiuliang; Li, Qing; Miao, Dengshun; Jin, Jianliang

2017-01-22

The regeneration of injured tubular cell occurs primarily from intrinsic renal stem/progenitor cells (RSCs) labeled with CD24 and CD133 after acute tubular necrosis (ATN). Bmi-1 plays a crucial role in regulating self-renewal, differentiation and aging of multiple adult stem cells and progenitor cells. Bmi-1 was rapidly elevated in the induction of adult kidney regeneration by renal injury. To determine whether Bmi-1 maintained mobilization of RSCs in the protection from ATN, glycerol-rhabdomyolysis-induced ATN were performed in wild type (WT) and Bmi-1-deficient (Bmi-1 -/- ) mice. Their ATN phenotypes were analyzed; CD24 and CD133 double positive (CD24 + CD133 + ) cells were measured; and the levels of serum urea nitrogen (SUN) and serum creatinine (SCr) were detected. We found that CD24 + CD133 + RSCs were mobilized in WT ATN mice with the increased expression of Bmi-1; Bmi-1 deficiency led to increased tubular cast formation and necrosis, elevated levels of SUN and SCr, decreased tubular proliferation, and immobilized ratio of RSCs in ATN. These findings indicated that Bmi-1 played a critical role in the protection from ATN by maintaining mobilization of RSCs and would be a novel therapeutic target for preventing the progression of ATN. Copyright © 2016 Elsevier Inc. All rights reserved.

Bmi-1 plays a critical role in the protection from acute tubular necrosis by mobilizing renal stem/progenitor cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lv, Xianhui; Yu, Zhenzhen; Xie, Chunfeng

The regeneration of injured tubular cell occurs primarily from intrinsic renal stem/progenitor cells (RSCs) labeled with CD24 and CD133 after acute tubular necrosis (ATN). Bmi-1 plays a crucial role in regulating self-renewal, differentiation and aging of multiple adult stem cells and progenitor cells. Bmi-1 was rapidly elevated in the induction of adult kidney regeneration by renal injury. To determine whether Bmi-1 maintained mobilization of RSCs in the protection from ATN, glycerol-rhabdomyolysis-induced ATN were performed in wild type (WT) and Bmi-1-deficient (Bmi-1{sup −/−}) mice. Their ATN phenotypes were analyzed; CD24 and CD133 double positive (CD24{sup +}CD133{sup +}) cells were measured; andmore » the levels of serum urea nitrogen (SUN) and serum creatinine (SCr) were detected. We found that CD24{sup +}CD133{sup +} RSCs were mobilized in WT ATN mice with the increased expression of Bmi-1; Bmi-1 deficiency led to increased tubular cast formation and necrosis, elevated levels of SUN and SCr, decreased tubular proliferation, and immobilized ratio of RSCs in ATN. These findings indicated that Bmi-1 played a critical role in the protection from ATN by maintaining mobilization of RSCs and would be a novel therapeutic target for preventing the progression of ATN.« less

Human primary CD34+ cells transplantation for critical limb ischemia.

PubMed

Lian, Weishuai; Hu, Xiaoxiao; Pan, Long; Han, Shilong; Cao, Chuanwu; Jia, Zhongzhi; Li, Maoquan

2018-06-11

The goal of this study was to characterize the properties of human CD34 + cells in culture and investigate the feasibility and efficacy of CD34 + transplantation in a mouse model of limb ischemia and in patients with no-option critical limb ischemia. Human CD34 + cells isolated from peripheral blood and grown in culture for up to four passages stained positively for the surface markers CD34 and CD133 and showed high viability after cryopreservation and recovery. Seven days after surgery to induce limb ischemia, ischemic muscles of nude mice were injected with CD34 + cells. Two weeks later, mice were scored for extent of ischemic injury, and muscle tissue was collected for immunohistochemical analysis of vascular endothelial cells and RT-PCR analysis of cytokine expression. Injury scores of CD34 + -treated, but not control, mice were significantly different before and after transplantation. Vascular density and expression of VEGF and bFGF mRNAs were also significantly increased in the treated mice. Patients with severe lower extremity arterial ischemia were injected with their own CD34 + cells in the affected calf, foot, or toe. Significant improvements were observed in peak pain-free walking time, ankle-brachial index, and transcutaneous partial oxygen pressure. These findings demonstrate that growth of human CD34 + cells in vitro and cryopreservations are feasible. Such cells may provide a renewable source of stem cells for transplantation, which appears to be a feasible, safe, and effective treatment for patients with critical limb ischemia. © 2018 Wiley Periodicals, Inc.

Circulating CD34+ progenitor cells and risk of mortality in a population with coronary artery disease.

PubMed

Patel, Riyaz S; Li, Qunna; Ghasemzadeh, Nima; Eapen, Danny J; Moss, Lauren D; Janjua, A Umair; Manocha, Pankaj; Kassem, Hatem Al; Veledar, Emir; Samady, Habib; Taylor, W Robert; Zafari, A Maziar; Sperling, Laurence; Vaccarino, Viola; Waller, Edmund K; Quyyumi, Arshed A

2015-01-16

Low circulating progenitor cell numbers and activity may reflect impaired intrinsic regenerative/reparative potential, but it remains uncertain whether this translates into a worse prognosis. To investigate whether low numbers of progenitor cells associate with a greater risk of mortality in a population at high cardiovascular risk. Patients undergoing coronary angiography were recruited into 2 cohorts (1, n=502 and 2, n=403) over separate time periods. Progenitor cells were enumerated by flow cytometry as CD45(med+) blood mononuclear cells expressing CD34, with additional quantification of subsets coexpressing CD133, vascular endothelial growth factor receptor 2, and chemokine (C-X-C motif) receptor 4. Coefficient of variation for CD34 cells was 2.9% and 4.8%, 21.6% and 6.5% for the respective subsets. Each cohort was followed for a mean of 2.7 and 1.2 years, respectively, for the primary end point of all-cause death. There was an inverse association between CD34(+) and CD34(+)/CD133(+) cell counts and risk of death in cohort 1 (β=-0.92, P=0.043 and β=-1.64, P=0.019, respectively) that was confirmed in cohort 2 (β=-1.25, P=0.020 and β=-1.81, P=0.015, respectively). Covariate-adjusted hazard ratios in the pooled cohort (n=905) were 3.54 (1.67-7.50) and 2.46 (1.18-5.13), respectively. CD34(+)/CD133(+) cell counts improved risk prediction metrics beyond standard risk factors. Reduced circulating progenitor cell counts, identified primarily as CD34(+) mononuclear cells or its subset expressing CD133, are associated with risk of death in individuals with coronary artery disease, suggesting that impaired endogenous regenerative capacity is associated with increased mortality. These findings have implications for biological understanding, risk prediction, and cell selection for cell-based therapies. © 2014 American Heart Association, Inc.

Multiplication free neural network for cancer stem cell detection in H-and-E stained liver images

NASA Astrophysics Data System (ADS)

Badawi, Diaa; Akhan, Ece; Mallah, Ma'en; Üner, Ayşegül; ćetin-Atalay, Rengül; ćetin, A. Enis

2017-05-01

Markers such as CD13 and CD133 have been used to identify Cancer Stem Cells (CSC) in various tissue images. It is highly likely that CSC nuclei appear as brown in CD13 stained liver tissue images. We observe that there is a high correlation between the ratio of brown to blue colored nuclei in CD13 images and the ratio between the dark blue to blue colored nuclei in H&E stained liver images. Therefore, we recommend that a pathologist observing many dark blue nuclei in an H&E stained tissue image may also order CD13 staining to estimate the CSC ratio. In this paper, we describe a computer vision method based on a neural network estimating the ratio of dark blue to blue colored nuclei in an H&E stained liver tissue image. The neural network structure is based on a multiplication free operator using only additions and sign operations. Experimental results are presented.

Pancreatic cancer stem cell markers and exosomes - the incentive push

PubMed Central

Heiler, Sarah; Wang, Zhe; Zöller, Margot

2016-01-01

Pancreatic cancer (PaCa) has the highest death rate and incidence is increasing. Poor prognosis is due to late diagnosis and early metastatic spread, which is ascribed to a minor population of so called cancer stem cells (CSC) within the mass of the primary tumor. CSC are defined by biological features, which they share with adult stem cells like longevity, rare cell division, the capacity for self renewal, differentiation, drug resistance and the requirement for a niche. CSC can also be identified by sets of markers, which for pancreatic CSC (Pa-CSC) include CD44v6, c-Met, Tspan8, alpha6beta4, CXCR4, CD133, EpCAM and claudin7. The functional relevance of CSC markers is still disputed. We hypothesize that Pa-CSC markers play a decisive role in tumor progression. This is fostered by the location in glycolipid-enriched membrane domains, which function as signaling platform and support connectivity of the individual Pa-CSC markers. Outside-in signaling supports apoptosis resistance, stem cell gene expression and tumor suppressor gene repression as well as miRNA transcription and silencing. Pa-CSC markers also contribute to motility and invasiveness. By ligand binding host cells are triggered towards creating a milieu supporting Pa-CSC maintenance. Furthermore, CSC markers contribute to the generation, loading and delivery of exosomes, whereby CSC gain the capacity for a cell-cell contact independent crosstalk with the host and neighboring non-CSC. This allows Pa-CSC exosomes (TEX) to reprogram neighboring non-CSC towards epithelial mesenchymal transition and to stimulate host cells towards preparing a niche for metastasizing tumor cells. Finally, TEX communicate with the matrix to support tumor cell motility, invasion and homing. We will discuss the possibility that CSC markers are the initial trigger for these processes and what is the special contribution of CSC-TEX. PMID:27468191

Lactobacillus delbrueckii subsp. lactis (strain CIDCA 133) stimulates murine macrophages infected with Citrobacter rodentium.

PubMed

Hugo, Ayelén A; Rolny, Ivanna S; Romanin, David; Pérez, Pablo F

2017-03-01

Citrobacter rodentium is a specific murine enteropathogen which causes diarrheal disease characterized by colonic hyperplasia and intestinal inflammation. Recruitment of neutrophils and macrophages constitute a key step to control the infection. Since modulation of the activity of professional phagocytic cells could contribute to improve host´s defences against C. rodentium, we investigated the effect of Lactobacillus delbrueckii subsp. lactis (strain CIDCA 133) on the interaction between murine macrophages (RAW 264.7) and C. rodentium. Phagocytosis, surface molecules and inducible nitric oxide synthase (iNOs) expression were determined by flow cytometry. Reactive oxygen species (ROS) were assessed by fluorescence microscopy. The presence of lactobacilli increased phagocytosis of C. rodentium whereas C. rodentium had no effect on lactobacilli internalization. Survival of internalized C. rodentium diminished when strain CIDCA 133 was present. CD-86, MHCII, iNOs expression and nitrite production were increased when C. rodentium and lactobacilli were present even though strain CIDCA 133 alone had no effect. Strain CIDCA 133 led to a strong induction of ROS activity which was not modified by C. rodentium. Lactobacillus delbrueckii subsp. lactis (strain CIDCA 133) is able to increase the activation of murine macrophages infected with C. rodentium. The sole presence of lactobacilli is enough to modify some stimulation markers (e.g. ROS induction) whereas other markers require the presence of both bacteria; thus, indicating a synergistic effect.

Crucial role of interleukin-4 in the survival of colon cancer stem cells.

PubMed

Francipane, Maria Giovanna; Alea, Mileidys Perez; Lombardo, Ylenia; Todaro, Matilde; Medema, J P; Stassi, Giorgio

2008-06-01

Colon tumors may be maintained by a rare fraction of cancer stem-like cells (CSC) that express the cell surface marker CD133. Self-renewing CSCs exhibit relatively greater resistance to clinical cytotoxic therapies and recent work suggests that this resistance may be mediated in part by an autocrine response to the immune cytokine interleukin 4 (IL-4). Blocking IL-4 signaling can sensitize CSCs to apoptotic stimuli and increase the in vivo efficacy of cytotoxic therapy. These findings suggest that inhibitors of IL-4 signaling may offer a new therapeutic tool in colon carcinoma.

IGF-1 contributes to the expansion of melanoma-initiating cells through an epithelial-mesenchymal transition process.

PubMed

Le Coz, Vincent; Zhu, Chaobin; Devocelle, Aurore; Vazquez, Aimé; Boucheix, Claude; Azzi, Sandy; Gallerne, Cindy; Eid, Pierre; Lecourt, Séverine; Giron-Michel, Julien

2016-12-13

Melanoma is a particularly virulent human cancer, due to its resistance to conventional treatments and high frequency of metastasis. Melanomas contain a fraction of cells, the melanoma-initiating cells (MICs), responsible for tumor propagation and relapse. Identification of the molecular pathways supporting MICs is, therefore, vital for the development of targeted treatments. One factor produced by melanoma cells and their microenvironment, insulin-like growth factor-1 (IGF- 1), is linked to epithelial-mesenchymal transition (EMT) and stemness features in several cancers.We evaluated the effect of IGF-1 on the phenotype and chemoresistance of B16-F10 cells. IGF-1 inhibition in these cells prevented malignant cell proliferation, migration and invasion, and lung colony formation in immunodeficient mice. IGF-1 downregulation also markedly inhibited EMT, with low levels of ZEB1 and mesenchymal markers (N-cadherin, CD44, CD29, CD105) associated with high levels of E-cadherin and MITF, the major regulator of melanocyte differentiation. IGF-1 inhibition greatly reduced stemness features, including the expression of key stem markers (SOX2, Oct-3/4, CD24 and CD133), and the functional characteristics of MICs (melanosphere formation, aldehyde dehydrogenase activity, side population). These features were associated with a high degree of sensitivity to mitoxantrone treatment.In this study, we deciphered new connections between IGF-1 and stemness features and identified IGF-1 as instrumental for maintaining the MIC phenotype. The IGF1/IGF1-R nexus could be targeted for the development of more efficient anti-melanoma treatments. Blocking the IGF-1 pathway would improve the immune response, decrease the metastatic potential of tumor cells and sensitize melanoma cells to conventional treatments.

Synergistic Integration of Mesenchymal Stem Cells and Hydrostatic Pressure in the Expansion and Maintenance of Human Hematopoietic/Progenitor Cells

PubMed Central

2018-01-01

Ex vivo expansion of hematopoietic stem/progenitor cell (HSPC) has been investigated to improve the clinical outcome of HSPC transplantation. However, ex vivo expansion of HSPCs still faces a major obstacle in that HPSCs tend to differentiate when proliferating. Here, we cocultured HSPCs with mesenchymal stem cells (MSCs) and divided the HSPCs into two fractions according to whether they came into adherent to MSCs or not. Additionally, we used hydrostatic pressure (HP) to mimic the physical conditions in vivo. Even nonadherent cells expanded to yield a significantly larger number of total nucleated cells (TNCs), adherent cells maintained the HSPC phenotype (CD34+, CD34+CD38−, and CD133+CD38−) to a greater extent than nonadherent cells and had superior clonogenic potential. Moreover, applying HP significantly increased the number of TNCs, the frequency of the immature HSPC phenotype, and the clonogenic potential. Furthermore, the genetic markers for the HSPC niche were significantly increased under HP. Our data suggest that the nonadherent fraction is the predominant site of HSPC expansion, whereas the adherent fraction seems to mimic the HSPC niche for immature cells. Moreover, HP has a synergistic effect on expansion and functional maintenance. This first study utilizing HP has a potential of designing clinically applicable expansion systems. PMID:29681947

Cancer-initiating cells derived from established cervical cell lines exhibit stem-cell markers and increased radioresistance

PubMed Central

2012-01-01

Background Cancer-initiating cells (CICs) are proposed to be responsible for the generation of metastasis and resistance to therapy. Accumulating evidences indicates CICs are found among different human cancers and cell lines derived from them. Few studies address the characteristics of CICs in cervical cancer. We identify biological features of CICs from four of the best-know human cell lines from uterine cervix tumors. (HeLa, SiHa, Ca Ski, C-4 I). Methods Cells were cultured as spheres under stem-cell conditions. Flow cytometry was used to detect expression of CD34, CD49f and CD133 antigens and Hoechst 33342 staining to identify side population (SP). Magnetic and fluorescence-activated cell sorting was applied to enrich and purify populations used to evaluate tumorigenicity in nude mice. cDNA microarray analysis and in vitro radioresistance assay were carried out under standard conditions. Results CICs, enriched as spheroids, were capable to generate reproducible tumor phenotypes in nu-nu mice and serial propagation. Injection of 1 × 103 dissociated spheroid cells induced tumors in the majority of animals, whereas injection of 1 × 105 monolayer cells remained nontumorigenic. Sphere-derived CICs expressed CD49f surface marker. Gene profiling analysis of HeLa and SiHa spheroid cells showed up-regulation of CICs markers characteristic of the female reproductive system. Importantly, epithelial to mesenchymal (EMT) transition-associated markers were found highly expressed in spheroid cells. More importantly, gene expression analysis indicated that genes required for radioresistance were also up-regulated, including components of the double-strand break (DSB) DNA repair machinery and the metabolism of reactive oxygen species (ROS). Dose-dependent radiation assay indicated indeed that CICs-enriched populations exhibit an increased resistance to ionizing radiation (IR). Conclusions We characterized a self-renewing subpopulation of CICs found among four well known human cancer-derived cell lines (HeLa, SiHa, Ca Ski and C-4 I) and found that they express characteristic markers of stem cell, EMT and radioresistance. The fact that CICs demonstrated a higher degree of resistance to radiation than differentiated cells suggests that specific detection and targeting of CICs could be highly valuable for the therapy of tumors from the uterine cervix. PMID:22284662

Esophageal cancer stem cells and implications for future therapeutics.

PubMed

Qian, Xia; Tan, Cheng; Wang, Feng; Yang, Baixia; Ge, Yangyang; Guan, Zhifeng; Cai, Jing

2016-01-01

Esophageal carcinoma (EC) is a lethal disease with high morbidity and mortality worldwide, and the incidence has been increasing in recent years. Although the diagnosis and treatment of EC have improved considerably, EC has rapidly progressed in the clinical setting and has a poor prognosis for its metastasis and recurrence. The general idea of cancer stem cells (CSCs) is primarily based on clinical and experimental observations, indicating the existence of a subpopulation of cells that can self-renew and differentiate. The EC stem cells, which can be isolated from normal pluripotent stem cells by applying similar biomarkers, may participate in promoting esophageal tumorigenesis through renewal and repair. In this review, major emphasis is given to CSC markers, altered CSC-specific pathways, and molecular targeting agents currently available to target CSCs of esophageal cancer. The roles of numerous markers (CD44, aldehyde dehydrogenase, CD133, and ATP-binding cassette subfamily G member 2) and developmental signaling pathways (Wnt/β-catenin, Notch, hedgehog, and Hippo) in isolating esophageal CSCs are discussed in detail. Targeting CSCs can be a logical strategy to treat EC, as these cells are responsible for carcinoma recurrence and chemoradiation resistance.

Update on the pathogenesis of Scleroderma: focus on circulating progenitor cells.

PubMed

Brunasso, Alexandra Maria Giovanna; Massone, Cesare

2016-01-01

In systemic sclerosis (SSc), the development of fibrosis seems to be a consequence of the initial ischemic process related to an endothelial injury. The initial trigger event in SSc is still unknown, but circulating progenitor cells (CPCs) might play a key role. Such cells have the ability to traffic into injury sites, exhibiting inflammatory features of macrophages, tissue remodeling properties of fibroblasts, and vasculogenesis functions of endothelial cells. The different subsets of CPCs described thus far in SSc arise from a pool of circulating monocyte precursors (CD14 (+) cells) and probably correspond to a different degree of differentiation of a single cell of origin. Several subsets of CPCs have been described in patients with SSc, all have a monocytic origin but may or may not express CD14, and all of these cells have the ability to give origin to endothelial cells, or collagen (Col)-producing cells, or both. We were able to identify six subsets of CPCs: pluripotent stem cells (CD14 (+), CD45 (+), and CD34 (+)), monocyte-derived multipotential cells (MOMCs) or monocyte-derived mesenchymal progenitors (CD14 (+), CD45 (+), CD34 (+), Col I (+), CD11b (+), CD68 (+), CD105 (+), and VEGFR1 (+)), early endothelial progenitor cells (EPCs) or monocytic pro-angiogenic hematopoietic cells or circulating hematopoietic cells (CD14 (+), CD45 (+), CD34 (low/-), VEGFR2 (+/-), CXCR4 (+), c-kit (+), and DC117 (+)), late EPCs (CD14 (-), CD133 (+), VEGFR2 (+), CD144 (+) [VE-cadherin (+)], and CD146 (+)), fibroblast-like cells (FLCs)/circulating Col-producing monocytes (CD14 (+), CD45 (+), CD34 (+/-), and Col I (+)), and fibrocytes (CD14 (-), CD45 (+), CD34 (+), Col I (+), and CXCR4 (+)). It has been demonstrated that circulating CD14 (+) monocytes with an activated phenotype are increased in patients with SSc when compared with normal subjects. CD14 (+), CD34 (+), and Col I (+) spindle-shaped cells have been found in increased numbers in lungs of SSc patients with interstitial lung disease. Elevated blood amounts of early EPCs have been found in patients with SSc by different groups of researchers and such levels correlate directly with the interstitial lung involvement. The prevalence of hematopoietic markers expressed by CPCs that migrate from blood into injury sites in SSc differs and changes according to the degree of differentiation. CXCR4 is the most commonly expressed marker, followed by CD34 and CD45 at an end stage of differentiation. Such difference also indicates a continuous process of cell differentiation that might relate to the SSc clinical phenotype (degree of fibrosis and vascular involvement). A deeper understanding of the role of each subtype of CPCs in the development of the disease will help us to better classify patients in order to offer them targeted approaches in the future.

Cancer stem cell marker Musashi-1 rs2522137 genotype is associated with an increased risk of lung cancer.

PubMed

Wang, Xu; Hu, Ji-Fan; Tan, Yehui; Cui, Jiuwei; Wang, Guanjun; Mrsny, Randall J; Li, Wei

2014-01-01

Gene single nucleotide polymorphisms (SNPs) have been extensively studied in association with development and prognosis of various malignancies. However, the potential role of genetic polymorphisms of cancer stem cell (CSC) marker genes with respect to cancer risk has not been examined. We conducted a case-control study involving a total of 1000 subjects (500 lung cancer patients and 500 age-matched cancer-free controls) from northeastern China. Lung cancer risk was analyzed in a logistic regression model in association with genotypes of four lung CSC marker genes (CD133, ALDH1, Musashi-1, and EpCAM). Using univariate analysis, the Musashi-1 rs2522137 GG genotype was found to be associated with a higher incidence of lung cancer compared with the TT genotype. No significant associations were observed for gene variants of CD133, ALDH1, or EpCAM. In multivariate analysis, Musashi-1 rs2522137 was still significantly associated with lung cancer when environmental and lifestyle factors were incorporated in the model, including lower BMI; family history of cancer; prior diagnosis of chronic obstructive pulmonary disease, pneumonia, or pulmonary tuberculosis; occupational exposure to pesticide; occupational exposure to gasoline or diesel fuel; heavier smoking; and exposure to heavy cooking emissions. The value of the area under the receiver-operating characteristic (ROC) curve (AUC) was 0.7686. To our knowledge, this is the first report to show an association between a Musashi-1 genotype and lung cancer risk. Further, the prediction model in this study may be useful in determining individuals with high risk of lung cancer.

Effect of The Receptor Activator of Nuclear Factor кB and RANK Ligand on In Vitro Differentiation of Cord Blood CD133(+) Hematopoietic Stem Cells to Osteoclasts.

PubMed

Kalantari, Nasim; Abroun, Saeid; Soleimani, Masoud; Kaviani, Saeid; Azad, Mehdi; Eskandari, Fatemeh; Habibi, Hossein

2016-01-01

Receptor activator of nuclear factor-kappa B ligand (RANKL) appears to be an osteoclast-activating factor, bearing an important role in the pathogenesis of multiple myeloma. Some studies demonstrated that U-266 myeloma cell line and primary myeloma cells expressed RANK and RANKL. It had been reported that the expression of myeloid and monocytoid markers was increased by co-culturing myeloma cells with hematopoietic stem cells (HSCs). This study also attempted to show the molecular mechanism of RANK and RANKL on differentiation capability of human cord blood HSC to osteoclast, as well as expression of calcitonin receptor (CTR) on cord blood HSC surface. In this experimental study, CD133(+) hematopoietic stem cells were isolated from umbilical cord blood and cultured in the presence of macrophage colony-stimulating factor (M-CSF) and RANKL. Osteoclast differentiation was characterized by using tartrate-resistant acid phosphatase (TRAP) staining, giemsa staining, immunophenotyping, and reverse transcription-polymerase chain reaction (RT-PCR) assay for specific genes. Hematopoietic stem cells expressed RANK before and after differentiation into osteoclast. Compared to control group, flow cytometric results showed an increased expression of RANK after differentiation. Expression of CTR mRNA showed TRAP reaction was positive in some differentiated cells, including osteoclast cells. Presence of RANKL and M-CSF in bone marrow could induce HSCs differentiation into osteoclast.

Radiosensitivity of Patient-Derived Glioma Stem Cell 3-Dimensional Cultures to Photon, Proton, and Carbon Irradiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chiblak, Sara; Tang, Zili; Molecular and Translational Radiation Oncology, Heidelberg Ion Therapy Center, Heidelberg Institute of Radiation Oncology, University of Heidelberg Medical School and National Center for Tumor Diseases, German Cancer Research Center, Heidelberg

Purpose: To investigate the radiosensitivity of primary glioma stem cell (GSC) cultures with different CD133 status in a 3-dimensional (3D) model after photon versus proton versus carbon irradiation. Methods and Materials: Human primary GSC spheroid cultures were established from tumor specimens of six consented glioblastoma patients. Human U87MG was used as a classical glioblastoma radioresistant cell line. Cell suspensions were generated by mechanical dissociation of GSC spheroids and embedded in a semi-solid 3D matrix before irradiation. Spheroid-like colonies were manually counted by microscopy. Cells were also recovered and quantified by fluorescence. CD133 expression and DNA damage were evaluated by flow cytometry.more » Results: The fraction of CD133{sup +} cells varied between 0.014% and 96% in the six GSC cultures and showed a nonsignificant correlation with plating efficiency and survival fractions. The 4 most photon-radioresistant GSC cultures were NCH644, NCH421k, NCH441, and NCH636. Clonogenic survival for proton irradiation revealed relative biologic effectiveness (RBE) in the range of 0.7-1.20. However, carbon irradiation rendered the photon-resistant GSC cultures sensitive, with average RBE of 1.87-3.44. This effect was partly attributed to impaired capability of GSC to repair carbon ion–induced DNA double-strand breaks as determined by residual DNA repair foci. Interestingly, radiosensitivity of U87 cells was comparable to GSC cultures using clonogenic survival as the standard readout. Conclusions: Carbon irradiation is effective in GSC eradication with similar RBE ranges approximately 2-3 as compared with non-stem GSC cultures (U87). Our data strongly suggest further exploration of GSC using classic radiobiology endpoints such as the here-used 3D clonogenic survival assay and integration of additional GSC-specific markers.« less

[Overexpressed miRNA-134b inhibits proliferation and invasion of CD133+ U87 glioma stem cells].

PubMed

Liu, Yifeng; Zhang, Baochao; Wen, Changming; Wen, Gongling; Zhou, Guoping; Zhang, Jingwei; He, Haifa; Wang, Ning; Li, Wei

2017-05-01

Objective To investigate the role of microRNA-134b (miR-134b) in the tumorigenesis of glioma stem cells (GSCs) and the possible molecular mechanism. Methods Real-time quantitative PCR (qRT-PCR) was used to evalate the expression of miR-134b in CD133 + and CD133 - U87 GSCs. A lentiviral vector overexpressing miR-134b in U87 GSCs was constructed, and the effect of miR-134b overexpression on matrix metalloproteinase-2 (MMP-2), MMP-9 and MMP-12 expressions at both mRNA and protein levels were detected by qRT-PCR and Western blotting, respectively. Transwell TM assay was performed to determine the effect of miR-134b overexpression on GSCs invasion ability. Tumor xenograft models in nude mice were established to evaluate the effect of miR-134b overexpression on tumorgenesis in vivo. Results The qRT-PCR showed that, compared with CD133 - cells, miR-134b was significantly down-regulated in CD133 + cells. Cell line over-expressing miR-134b was successfully established, and miR-134b was up-regulated significantly compared with empty vector control. Overexpression of miR-134b remarkably inhibited the invasion of U87 GSCs and the expression of MMP-12. However, overexpression of miR-134b did not affect MMP-2 and MMP-9 expressions. miR-134b also suppressed U87 GSCs xenograft growth in vivo. Tumor volume in tumor xenograft model group was significantly lower than that in control group, and tumor weight decreased by 42% in the former group. Conclusion Overexpression of miR-134b inhibits the growth and invasion of CD133 + GSCs.

Black TiO2-based nanoprobes for T1-weighted MRI-guided photothermal therapy in CD133 high expressed pancreatic cancer stem-like cells.

PubMed

Wang, Siqi; Ren, Wenzhi; Wang, Jianhua; Jiang, Zhenqi; Saeed, Madiha; Zhang, Lili; Li, Aiguo; Wu, Aiguo

2018-06-27

At present, transmembrane glycoprotein CD133 highly expressed pancreatic cancer stem cells (PCSCs), with the features of chemotherapeutic/radiotherapeutic resistance and exclusive tumorigenic potential, are considered as the primary cause of metastasis and recurrence in pancreatic cancer, and therefore are an effective target in the disease treatment. Furthermore, with the launch of precision medicine, multifunctional nanoprobes have been applied as an efficient strategy for the magnetic resonance imaging (MRI)-guided photothermal therapy (PTT) of pancreatic cancer. In this research, with the aim of achieving precise MRI-guided PTT in CD133 highly expressed PCSCs, novel bTiO2-Gd-CD133mAb nanoprobes were designed and successfully prepared by loading Gd-DOTA and CD133 monoclonal antibodies on black TiO2 nanoparticles. It was very interesting to find that the r1 relaxivity value of the nanoprobes was 34.394 mM-1 s-1, about 7.5 times that of commercial Magnevist (4.5624 mM-1 s-1), which indicates that the nanoprobes have good potential as MRI T1 contrast agents with excellent performance. Herein, CD133 highly expressed PANC-1 cells were selected and verified as PCSCs model. In vitro experiments demonstrated that the nanoprobes exhibited active-targeting ability in PANC-1 cells, and consequently could specially enhance T1-weighted MR imaging and 808 nm near-infrared (NIR)-triggered PTT efficiency in the PCSCs model. Our study not only provides a new strategy for the effective treatment of pancreatic cancer and its' stem cells, but also further broadens the application of black TiO2 in the field of cancer theranostics.

Exploring the clonal evolution of CD133/aldehyde-dehydrogenase-1 (ALDH1)-positive cancer stem-like cells from primary to recurrent high-grade serous ovarian cancer (HGSOC). A study of the Ovarian Cancer Therapy-Innovative Models Prolong Survival (OCTIPS) Consortium.

PubMed

Ruscito, Ilary; Cacsire Castillo-Tong, Dan; Vergote, Ignace; Ignat, Iulia; Stanske, Mandy; Vanderstichele, Adriaan; Ganapathi, Ram N; Glajzer, Jacek; Kulbe, Hagen; Trillsch, Fabian; Mustea, Alexander; Kreuzinger, Caroline; Benedetti Panici, Pierluigi; Gourley, Charlie; Gabra, Hani; Kessler, Mirjana; Sehouli, Jalid; Darb-Esfahani, Silvia; Braicu, Elena Ioana

2017-07-01

High-grade serous ovarian cancer (HGSOC) causes 80% of all ovarian cancer (OC) deaths. In this setting, the role of cancer stem-like cells (CSCs) is still unclear. In particular, the evolution of CSC biomarkers from primary (pOC) to recurrent (rOC) HGSOCs is unknown. Aim of this study was to investigate changes in CD133 and aldehyde dehydrogenase-1 (ALDH1) CSC biomarker expression in pOC and rOC HGSOCs. Two-hundred and twenty-four pOC and rOC intrapatient paired tissue samples derived from 112 HGSOC patients were evaluated for CD133 and ALDH1 expression using immunohistochemistry (IHC); pOCs and rOCs were compared for CD133 and/or ALDH1 levels. Expression profiles were also correlated with patients' clinicopathological and survival data. Some 49.1% of the patient population (55/112) and 37.5% (42/112) pOCs were CD133+ and ALDH1+ respectively. CD133+ and ALDH1+ samples were detected in 33.9% (38/112) and 36.6% (41/112) rOCs. CD133/ALDH1 coexpression was observed in 23.2% (26/112) and 15.2% (17/112) of pOCs and rOCs respectively. Pairwise analysis showed a significant shift of CD133 staining from higher (pOCs) to lower expression levels (rOCs) (p < 0.0001). Furthermore, all CD133 + pOC patients were International Federation of Gynaecology and Obstetrics (FIGO)-stage III/IV (p < 0.0001) and had significantly worse progression-free interval (PFI) (p = 0.04) and overall survival (OS) (p = 0.02). On multivariate analysis, CD133/ALDH1 coexpression in pOCs was identified as independent prognostic factor for PFI (HR: 1.64; 95% CI: 1.03-2.60; p = 0.036) and OS (HR: 1.71; 95% CI: 1.01-2.88; p = 0.045). Analysis on 52 pts patients with known somatic BRCA status revealed that BRCA mutations did not influence CSC biomarker expression. The study showed that CD133/ALDH1 expression impacts HGSOC patients' survival and first suggests that CSCs might undergo phenotypic change during the disease course similarly to non stem-like cancer cells, providing also a first evidence that there is no correlation between CSCs and BRCA status. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cytokeratin 19 (KRT19) has a Role in the Reprogramming of Cancer Stem Cell-Like Cells to Less Aggressive and More Drug-Sensitive Cells.

PubMed

Saha, Subbroto Kumar; Kim, Kyeongseok; Yang, Gwang-Mo; Choi, Hye Yeon; Cho, Ssang-Goo

2018-05-09

Cytokeratin 19 ( KRT19 ) is a cytoplasmic intermediate filament protein, which is responsible for structural rigidity and multipurpose scaffolds. In several cancers, KRT19 is overexpressed and may play a crucial role in tumorigenic transformation. In our previous study, we revealed the role of KRT19 as signaling component which mediated Wnt/NOTCH crosstalk through NUMB transcription in breast cancer. Here, we investigated the function of KRT19 in cancer reprogramming and drug resistance in breast cancer cells. We found that expression of KRT19 was attenuated in several patients-derived breast cancer tissues and patients with a low expression of KRT19 were significantly correlated with poor prognosis in breast cancer patients. Consistently, highly aggressive and drug-resistant breast cancer patient-derived cancer stem cell-like cells (konkuk university-cancer stem cell-like cell (KU-CSLCs)) displayed higher expression of cancer stem cell (CSC) markers, including ALDH1 , CXCR4 , and CD133 , but a much lower expression of KRT19 than that is seen in highly aggressive triple negative breast cancer MDA-MB231 cells. Moreover, we revealed that the knockdown of KRT19 in MDA-MB231 cells led to an enhancement of cancer properties, such as cell proliferation, sphere formation, migration, and drug resistance, while the overexpression of KRT19 in KU-CSLCs resulted in the significant attenuation of cancer properties. KRT19 regulated cancer stem cell reprogramming by modulating the expression of cancer stem cell markers ( ALDH1 , CXCR4 , and CD133 ), as well as the phosphorylation of Src and GSK3β (Tyr216). Therefore, our data may imply that the modulation of KRT19 expression could be involved in cancer stem cell reprogramming and drug sensitivity, which might have clinical implications for cancer or cancer stem cell treatment.

Cytokeratin 19 (KRT19) has a Role in the Reprogramming of Cancer Stem Cell-Like Cells to Less Aggressive and More Drug-Sensitive Cells

PubMed Central

Kim, Kyeongseok; Yang, Gwang-Mo; Choi, Hye Yeon

2018-01-01

Cytokeratin 19 (KRT19) is a cytoplasmic intermediate filament protein, which is responsible for structural rigidity and multipurpose scaffolds. In several cancers, KRT19 is overexpressed and may play a crucial role in tumorigenic transformation. In our previous study, we revealed the role of KRT19 as signaling component which mediated Wnt/NOTCH crosstalk through NUMB transcription in breast cancer. Here, we investigated the function of KRT19 in cancer reprogramming and drug resistance in breast cancer cells. We found that expression of KRT19 was attenuated in several patients-derived breast cancer tissues and patients with a low expression of KRT19 were significantly correlated with poor prognosis in breast cancer patients. Consistently, highly aggressive and drug-resistant breast cancer patient-derived cancer stem cell-like cells (konkuk university-cancer stem cell-like cell (KU-CSLCs)) displayed higher expression of cancer stem cell (CSC) markers, including ALDH1, CXCR4, and CD133, but a much lower expression of KRT19 than that is seen in highly aggressive triple negative breast cancer MDA-MB231 cells. Moreover, we revealed that the knockdown of KRT19 in MDA-MB231 cells led to an enhancement of cancer properties, such as cell proliferation, sphere formation, migration, and drug resistance, while the overexpression of KRT19 in KU-CSLCs resulted in the significant attenuation of cancer properties. KRT19 regulated cancer stem cell reprogramming by modulating the expression of cancer stem cell markers (ALDH1, CXCR4, and CD133), as well as the phosphorylation of Src and GSK3β (Tyr216). Therefore, our data may imply that the modulation of KRT19 expression could be involved in cancer stem cell reprogramming and drug sensitivity, which might have clinical implications for cancer or cancer stem cell treatment. PMID:29747452

Expression of Pluripotency Markers in Nonpluripotent Human Neural Stem and Progenitor Cells.

PubMed

Vincent, Per Henrik; Benedikz, Eirikur; Uhlén, Per; Hovatta, Outi; Sundström, Erik

2017-06-15

Nonpluripotent neural progenitor cells (NPCs) derived from the human fetal central nervous system were found to express a number of messenger RNA (mRNA) species associated with pluripotency, such as NANOG, REX1, and OCT4. The expression was restricted to small subpopulations of NPCs. In contrast to pluripotent stem cells, there was no coexpression of the pluripotency-associated genes studied. Although the expression of these genes rapidly declined during the in vitro differentiation of NPCs, we found no evidence that the discrete expression was associated with the markers of multipotent neural stem cells (CD133 + /CD24 lo ), the capacity of sphere formation, or high cell proliferation rates. The rate of cell death among NPCs expressing pluripotency-associated genes was also similar to that of other NPCs. Live cell imaging showed that NANOG- and REX1-expressing NPCs continuously changed morphology, as did the nonexpressing cells. Depletion experiments showed that after the complete removal of the subpopulations of NANOG- and REX1-expressing NPCs, the expression of these genes appeared in other NPCs within a few days. The percentage of NANOG- and REX1-expressing cells returned to that observed before depletion. Our results are best explained by a model in which there is stochastic transient expression of pluripotency-associated genes in proliferating NPCs.

Nanovehicles as a novel target strategy for hyperthermic intraperitoneal chemotherapy: a multidisciplinary study of peritoneal carcinomatosis

PubMed Central

Nowacki, Maciej; Wisniewski, Marek; Werengowska-Ciecwierz, Karolina; Roszek, Katarzyna; Czarnecka, Joanna; Łakomska, I.; Kloskowski, Tomasz; Tyloch, Dominik; Debski, Robert; Pietkun, Katarzyna; Pokrywczynska, Marta; Grzanka, Dariusz; Czajkowski, Rafał; Drewa, Gerard; Jundziłł, A.; Agyin, Joseph K.; Habib, Samy L.; Terzyk, Artur P.; Drewa, Tomasz

2015-01-01

In general, detection of peritoneal carcinomatosis (PC) occurs at the late stage when there is no treatment option. In the present study, we designed novel drug delivery systems that are functionalized with anti-CD133 antibodies. The C1, C2 and C3 complexes with cisplatin were introduced into nanotubes, either physically or chemically. The complexes were reacted with anti-CD133 antibody to form the labeled product of A0-o-CX-chem-CD133. Cytotoxicity screening of all the complexes was performed on CHO cells. Data showed that both C2 and C3 Pt-complexes are more cytotoxic than C1. Flow-cytometry analysis showed that nanotubes conjugated to CD133 antibody have the ability to target cells expressing the CD133 antigen which is responsible for the emergence of resistance to chemotherapy and disease recurrence. The shortest survival rate was observed in the control mice group (K3) where no hyperthermic intraperitoneal chemotherapy procedures were used. On the other hand, the longest median survival rate was observed in the group treated with A0-o-C1-chem-CD133. In summary, we designed a novel drug delivery system based on carbon nanotubes loaded with Pt-prodrugs and functionalized with anti-CD133 antibodies. Our data demonstrates the effectiveness of the new drug delivery system and provides a novel therapeutic modality in the treatment of melanoma. PMID:26254295

The Selective Centrifugation Ensures a Better In Vitro Isolation of ASCs and Restores a Soft Tissue Regeneration In Vivo.

PubMed

De Francesco, Francesco; Guastafierro, Antonio; Nicoletti, Gianfranco; Razzano, Sergio; Riccio, Michele; Ferraro, Giuseppe A

2017-05-12

Autologous fat grafting procedures in plastic surgery have been extensively used to reinforce soft tissue in congenital or acquired tissue impairments. With this background, the aim of this study is firstly to examine the impact of a selective centrifugation on existing adipose stem cells (ASCs) in terms of stemness profile maintenance and, secondly, to investigate the effect of restoring volume in reconstruction on patients affected by soft tissue damage. After centrifugation, the fat graft products were separated into two layers and subsequently examined in vitro for the expression of CD34, CD90, CD117, CD105, CD29, CD31, CD44, CD73, CD133, CD14 and CD45 markers by flow cytometry and gene expression analyses were performed for Sox2, WNT3A, END, CD44, FUT4, COLL1, CTNNB1, hbEGF, KRTLG, MMP2 and VIM genes. The results showed that in the middle-high density (MHD) layer there was a peak concentration of ASCs, compared to another layer obtained after centrifugation. Research carried out on patients under treatment for soft tissue regeneration using cells obtained from MHD layer selection will be fundamental in comparative analysis. These studies will lead to an adequate standardization of outcomes, provided that treatment is performed through cell selection. Therefore, a unique procedure in tissue reconstruction and regeneration through fat grafting is presented here.

Changes in the frequencies of human hematopoietic stem and progenitor cells with age and site

PubMed Central

Farrell, TL; McGuire, TR; Bilek, L; Brusnahan, SK; Jackson, JD; Lane, JT; Garvin, KL; O'Kane, BJ; Berger, AM; Tuljapurkar, SR; Kessinger, MA; Sharp, JG

2013-01-01

This study enumerated CD45hi/CD34+ and CD45hi/CD133+ human hematopoietic stem cells (HSC) and granulocyte-monocyte colony forming (GM-CFC) progenitor cells in blood and trochanteric and femoral bone marrow in 233 individuals. Stem cell frequencies were determined by multi-parameter flow cytometry employing an internal control to determine the intrinsic variance of the assays. Progenitor cell frequency was determined using a standard colony assay technique. The frequency of outliers from undetermined methodological causes was highest for blood but less than 5% for all values. The frequency of CD45hi/CD133+ cells correlated highly with the frequency of CD45hi/CD34+ cells in trochanteric and femoral bone marrow. The frequency of these HSC populations in trochanteric and femoral bone marrow rose significantly with age. In contrast, there was no significant trend of either of these cell populations with age in the blood. Trochanteric marrow GM-CFC progenitor cells showed no significant trends with age, but femoral marrow GM-CFC trended downward with age, potentially because of the reported conversion of red marrow at this site to fat with age. Hematopoietic stem and progenitor cells exhibited changes in frequencies with age that differed between blood and bone marrow. We previously reported that side population (SP) multipotential HSC, that include the precursors of CD45hi/CD133+ and CD45hi/CD34+, decline with age. Potentially the increases in stem cell frequencies in the intermediate compartment between SP and GM progenitor cells observed in this study represent a compensatory increase for the loss of more potent members of the HSC hierarchy. PMID:24246745

Grating coupled SPR microarray analysis of proteins and cells in blood from mice with breast cancer.

PubMed

Mendoza, A; Torrisi, D M; Sell, S; Cady, N C; Lawrence, D A

2016-01-21

Biomarker discovery for early disease diagnosis is highly important. Of late, much effort has been made to analyze complex biological fluids in an effort to develop new markers specific for different cancer types. Recent advancements in label-free technologies such as surface plasmon resonance (SPR)-based biosensors have shown promise as a diagnostic tool since there is no need for labeling or separation of cells. Furthermore, SPR can provide rapid, real-time detection of antigens from biological samples since SPR is highly sensitive to changes in surface-associated molecular and cellular interactions. Herein, we report a lab-on-a-chip microarray biosensor that utilizes grating-coupled surface plasmon resonance (GCSPR) and grating-coupled surface plasmon coupled fluorescence (GCSPCF) imaging to detect circulating tumor cells (CTCs) from a mouse model (FVB-MMTV-PyVT). GCSPR and GCSPCF analysis was accomplished by spotting antibodies to surface cell markers, cytokines and stress proteins on a nanofabricated GCSPR microchip and screening blood samples from FVB control mice or FVB-MMTV-PyVT mice with developing mammary carcinomas. A transgenic MMTV-PyVT mouse derived cancer cell line was also analyzed. The analyses indicated that CD24, CD44, CD326, CD133 and CD49b were expressed in both cell lines and in blood from MMTV-PyVT mice. Furthermore, cytokines such as IL-6, IL-10 and TNF-α, along with heat shock proteins HSP60, HSP27, HSc70(HSP73), HSP90 total, HSP70/HSc70, HSP90, HSP70, HSP90 alpha, phosphotyrosine and HSF-1 were overexpressed in MMTV-PyVT mice.

Overexpression of CYP3A4 in a COLO 205 Colon Cancer Stem Cell Model in vitro

PubMed Central

Olszewski, Ulrike; Liedauer, Richard; Ausch, Christoph; Thalhammer, Theresia; Hamilton, Gerhard

2011-01-01

Cancer stem cells (CSCs) seem to constitute a subpopulation of tumor cells that escape from chemotherapy and cause recurrent disease. Low proliferation rates, protection in a stem cell niche and overexpression of drug resistance proteins are considered to confer chemoresistance. We established an in vitro colon CSC-like model using the COLO 205 cell line, which revealed transiently increased expression of CD133 when transferred to serum-free stem cell culture medium. Assessment of global gene expression of COLO 205 cells under these conditions identified a set of upregulated genes including cytochrome P450 3A4 (CYP3A4) and aldehyde dehydrogenase 1A1 (ALDH1A1), as confirmed by real-time qPCR. ALDH1A1 is a CSC marker for certain tumor entities and confers resistance to cyclophosphamide. CYP3A4 is expressed in liver and colon and its overexpression seems particularly relevant in colon cancer, since it inactivates irinotecan and other xenobiotics, such as taxols and vinca alkaloids. In conclusion, this COLO 205 model provides evidence for CD133 induction concomitant with overexpression of CYP3A4, which, together with ATP-binding cassette, subfamily G, member 2 (ABCG2) and others, may have a role in chemoresistant colon CSCs and a negative impact on disease-free survival in colon cancer patients. PMID:24212669

Coexistence of Eph receptor B1 and ephrin B2 in port-wine stain endothelial progenitor cells contributes to clinicopathological vasculature dilatation.

PubMed

Tan, W; Wang, J; Zhou, F; Gao, L; Yin, R; Liu, H; Sukanthanag, A; Wang, G; Mihm, M C; Chen, D-B; Nelson, J S

2017-12-01

Port-wine stain (PWS) is a vascular malformation characterized by progressive dilatation of postcapillary venules, but the molecular pathogenesis remains obscure. To illustrate that PWS endothelial cells (ECs) present a unique molecular phenotype that leads to pathoanatomical PWS vasculatures. Immunohistochemistry and transmission electron microscopy were used to characterize the ultrastructure and molecular phenotypes of PWS blood vessels. Primary culture of human dermal microvascular endothelial cells and in vitro tube formation assay were used for confirmative functional studies. Multiple clinicopathological features of PWS blood vessels during the development and progression of the disease were shown. There were no normal arterioles and venules observed phenotypically and morphologically in PWS skin; arterioles and venules both showed differentiation impairments, resulting in a reduction of arteriole-like vasculatures and defects in capillary loop formation in PWS lesions. PWS ECs showed stemness properties with expression of endothelial progenitor cell markers CD133 and CD166 in non-nodular lesions. They also expressed dual venous/arterial identities, Eph receptor B1 (EphB1) and ephrin B2 (EfnB2). Co-expression of EphB1 and EfnB2 in normal human dermal microvascular ECs led to the formation of PWS-like vasculatures in vitro, for example larger-diameter and thick-walled capillaries. PWS ECs are differentiation-impaired, late-stage endothelial progenitor cells with a specific phenotype of CD133 + /CD166 + /EphB1 + /EfnB2 + , which form immature venule-like pathoanatomical vasculatures. The disruption of normal EC-EC interactions by coexistence of EphB1 and EfnB2 contributes to progressive dilatation of PWS vasculatures. © 2017 British Association of Dermatologists.

Novel strategies targeting cancer stem cells through phytochemicals and their analogs

PubMed Central

Dandawate, Prasad; Padhye, Subhash; Ahmad, Aamir

2013-01-01

Cancer stem cells (CSCs) are cells that exist within a tumor with a capacity of self-renewal and an ability to differentiate, giving rise to heterogeneous populations of cancer cells. These cells are increasingly being implicated in resistance to conventional therapeutics and have also been implicated in tumor recurrence. Several cellular signaling pathways including Notch, Wnt, phosphoinositide-3-kinase–Akt–mammalian target of rapamycin pathways, and known markers such as CD44, CD133, CD166, ALDH, etc. have been associated with CSCs. Here, we have reviewed our current understanding of self-renewal pathways and factors that help in the survival of CSCs with special emphasis on those that have been documented to be modulated by well characterized natural agents such as curcumin, sulforaphane, resveratrol, genistein, and epigallocatechin gallate. With the inclusion of a novel derivative of curcumin, CDF, we showcase how natural agents can be effectively modified to increase their efficacy, particularly against CSCs. We hope that this article will generate interest among researchers for further mechanistic and clinical studies exploiting the cancer preventive and therapeutic role of nutraceuticals by targeted elimination of CSCs. PMID:24076568

MUC4 down-regulation reverses chemoresistance of pancreatic cancer stem/progenitor cells and their progenies.

PubMed

Mimeault, Murielle; Johansson, Sonny L; Senapati, Shantibhusan; Momi, Navneet; Chakraborty, Subhankar; Batra, Surinder K

2010-09-01

The present study was undertaken to estimate the therapeutic benefit to down-regulate the MUC4 mucin for reversing chemoresistance of pancreatic cancer (PC) stem/progenitor cells and their progenies. The results have revealed that MUC4 mucin is overexpressed in CD133(+) and CD133(-) pancreatic cells (PCs) detected in patient's adenocarcinoma tissues while no significant expression was seen in normal pancreatic tissues. The gain- and loss-of-function analyses have indicated that the overexpression of MUC4 in PC lines is associated with a higher resistance to the anti-proliferative, anti-invasive and apoptotic effects induced by gemcitabine. Importantly, the treatment of the MUC4-overexpressing CD18/HPAF-Src cells with gemcitabine resulted in an enrichment of the side population (SP) cells expressing CD133 while the total PC cells including non-SP cells detected in MUC4 knockdown CD18/HPAF-shMUC4 cells were responsive to the cytotoxic effects induced by gemcitabine. These data suggest that the MUC4 down-regulation may constitute a potential therapeutic strategy for improving the efficacy of gemcitabine to eradicate the total PC cell mass, and thereby preventing disease relapse. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

Curcumin inhibits bladder cancer stem cells by suppressing Sonic Hedgehog pathway.

PubMed

Wang, Dengdian; Kong, Xiaochuan; Li, Yuan; Qian, Weiwei; Ma, Jiaxing; Wang, Daming; Yu, Dexin; Zhong, Caiyun

2017-11-04

Cancer stem cells (CSCs) is responsible for the recurrence of human cancers. Thus, targeting CSCs is considered to be a valid way for human cancer treatment. Curcumin is a major component of phytochemicals that exerts potent anticancer activities. However, the effect of curcumin on bladder cancer stem cells (BCSCs) remains to be elucidated. In this study, we investigated the mechanism of curcumin suppressing bladder cancer stem cells. In this study, UM-UC-3 and EJ cells were cultured in serum-free medium (SFM) to form cell spheres that was characterized as BCSCs. Then cell spheres were separately treated with different concentrations of curcumin and purmorphamine. Cell cycle analysis were used to determine the percentage of cells in different phases. Western blot and quantitative real-time PCR analysis were used to detect the expression of relative molecules. Immunofluorescence staining analysis were also utilized to measure the protein level of CD44. We found that CSC markers, including CD44, CD133, ALDH1-A1, OCT-4 and Nanog, were obviously highly expressed in cell spheres. Moreover, we observed that curcumin reduced the cell spheres formation, decreased the expression of CSC markers, suppressed cell proliferation and induced cell apoptosis. We also found that curcumin inhibited the activation of Shh pathway, while the inhibitory effects of curcumin on BCSCs could be weakened by upregulation of Sonic Hedgehog (Shh) pathway. Altogether, these data suggested that curcumin inhibited the activities of BCSCs through suppressing Shh pathway, which might be an effective chemopreventive agent for bladder cancer intervention. Copyright © 2017. Published by Elsevier Inc.

Relevance of mortalin to cancer cell stemness and cancer therapy

PubMed Central

Yun, Chae-Ok; Bhargava, Priyanshu; Na, Youjin; Lee, Jung-Sun; Ryu, Jihoon; Kaul, Sunil C.; Wadhwa, Renu

2017-01-01

Mortalin/mtHsp70 is a member of Hsp70 family of proteins. Enriched in a large variety of cancers, it has been shown to contribute to the process of carcinogenesis by multiple ways including inactivation of tumor suppressor p53 protein, deregulation of apoptosis and activation of EMT signaling. In this study, we report that upregulation of mortalin contributes to cancer cell stemness. Several cancer cell stemness markers, such as ABCG2, OCT-4, CD133, ALDH1, CD9, MRP1 and connexin were upregulated in mortalin-overexpressing cells that showed higher ability to form spheroids. These cells also showed higher migration, and were less responsive to a variety of cancer chemotherapeutic drugs. Of note, knockdown of mortalin by specific shRNA sensitized these cells to all the drugs used in this study. We report that low doses of anti-mortalin molecules, MKT-077 and CAPE, also caused similar sensitization of cancer cells to chemotherapeutic drugs and hence are potential candidates for effective cancer chemotherapy. PMID:28165047

Peripheral blood "endothelial progenitor cells" are derived from monocyte/macrophages and secrete angiogenic growth factors.

PubMed

Rehman, Jalees; Li, Jingling; Orschell, Christie M; March, Keith L

2003-03-04

Endothelial progenitor cells (EPCs) have been isolated from peripheral blood and can enhance angiogenesis after infusion into host animals. It is not known whether the proangiogenic effects are a result of such events as endothelial differentiation and subsequent proliferation of EPCs or secondary to secretion of angiogenic growth factors. Human EPCs were isolated as previously described, and their phenotypes were confirmed by uptake of acetylated LDL and binding of ulex-lectin. EPC proliferation and surface marker expression were analyzed by flow cytometry, and conditioned medium was assayed for growth factors. The majority of EPCs expressed monocyte/macrophage markers such as CD14 (95.7+/-0.3%), Mac-1 (57.6+/-13.5%), and CD11c (90.8+/-4.9%). A much lower percentage of cells expressed the specific endothelial marker VE-cadherin (5.2+/-0.7%) or stem/progenitor-cell markers AC133 (0.16+/-0.05%) and c-kit (1.3+/-0.7%). Compared with circulating monocytes, cultured EPCs showed upregulation of monocyte activation and macrophage differentiation markers. EPCs did not demonstrate any significant proliferation but did secrete the angiogenic growth factors vascular endothelial growth factor, hepatocyte growth factor, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor. Our findings suggest that acetylated LDL(+)ulex-lectin(+) cells, commonly referred to as EPCs, do not proliferate but release potent proangiogenic growth factors. The majority of acetylated LDL(+)ulex-lectin(+) cells are derived from monocyte/macrophages. The findings of low proliferation and endothelial differentiation suggest that their angiogenic effects are most likely mediated by growth factor secretion. These findings may allow for development of novel angiogenic therapies relying on secreted growth factors or on recruitment of endogenous monocytes/macrophages to sites of ischemia.

A mesenchymal-like phenotype and expression of CD44 predict lack of apoptotic response to sorafenib in liver tumor cells.

PubMed

Fernando, Joan; Malfettone, Andrea; Cepeda, Edgar B; Vilarrasa-Blasi, Roser; Bertran, Esther; Raimondi, Giulia; Fabra, Àngels; Alvarez-Barrientos, Alberto; Fernández-Salguero, Pedro; Fernández-Rodríguez, Conrado M; Giannelli, Gianluigi; Sancho, Patricia; Fabregat, Isabel

2015-02-15

The multikinase inhibitor sorafenib is the only effective drug in advanced cases of hepatocellular carcinoma (HCC). However, response differs among patients and effectiveness only implies a delay. We have recently described that sorafenib sensitizes HCC cells to apoptosis. In this work, we have explored the response to this drug of six different liver tumor cell lines to define a phenotypic signature that may predict lack of response in HCC patients. Results have indicated that liver tumor cells that show a mesenchymal-like phenotype, resistance to the suppressor effects of transforming growth factor beta (TGF-β) and high expression of the stem cell marker CD44 were refractory to sorafenib-induced cell death in in vitro studies, which correlated with lack of response to sorafenib in nude mice xenograft models of human HCC. In contrast, epithelial-like cells expressing the stem-related proteins EpCAM or CD133 were sensitive to sorafenib-induced apoptosis both in vitro and in vivo. A cross-talk between the TGF-β pathway and the acquisition of a mesenchymal-like phenotype with up-regulation of CD44 expression was found in the HCC cell lines. Targeted CD44 knock-down in the mesenchymal-like cells indicated that CD44 plays an active role in protecting HCC cells from sorafenib-induced apoptosis. However, CD44 effect requires a TGF-β-induced mesenchymal background, since the only overexpression of CD44 in epithelial-like HCC cells is not sufficient to impair sorafenib-induced cell death. In conclusion, a mesenchymal profile and expression of CD44, linked to activation of the TGF-β pathway, may predict lack of response to sorafenib in HCC patients. © 2014 UICC.

Update on the pathogenesis of Scleroderma: focus on circulating progenitor cells

PubMed Central

Brunasso, Alexandra Maria Giovanna; Massone, Cesare

2016-01-01

In systemic sclerosis (SSc), the development of fibrosis seems to be a consequence of the initial ischemic process related to an endothelial injury. The initial trigger event in SSc is still unknown, but circulating progenitor cells (CPCs) might play a key role. Such cells have the ability to traffic into injury sites, exhibiting inflammatory features of macrophages, tissue remodeling properties of fibroblasts, and vasculogenesis functions of endothelial cells. The different subsets of CPCs described thus far in SSc arise from a pool of circulating monocyte precursors (CD14 + cells) and probably correspond to a different degree of differentiation of a single cell of origin. Several subsets of CPCs have been described in patients with SSc, all have a monocytic origin but may or may not express CD14, and all of these cells have the ability to give origin to endothelial cells, or collagen (Col)-producing cells, or both. We were able to identify six subsets of CPCs: pluripotent stem cells (CD14 +, CD45 +, and CD34 +), monocyte-derived multipotential cells (MOMCs) or monocyte-derived mesenchymal progenitors (CD14 +, CD45 +, CD34 +, Col I +, CD11b +, CD68 +, CD105 +, and VEGFR1 +), early endothelial progenitor cells (EPCs) or monocytic pro-angiogenic hematopoietic cells or circulating hematopoietic cells (CD14 +, CD45 +, CD34 low/−, VEGFR2 +/−, CXCR4 +, c-kit +, and DC117 +), late EPCs (CD14 −, CD133 +, VEGFR2 +, CD144 + [VE-cadherin +], and CD146 +), fibroblast-like cells (FLCs)/circulating Col-producing monocytes (CD14 +, CD45 +, CD34 +/−, and Col I +), and fibrocytes (CD14 −, CD45 +, CD34 +, Col I +, and CXCR4 +). It has been demonstrated that circulating CD14 + monocytes with an activated phenotype are increased in patients with SSc when compared with normal subjects. CD14 +, CD34 +, and Col I + spindle-shaped cells have been found in increased numbers in lungs of SSc patients with interstitial lung disease. Elevated blood amounts of early EPCs have been found in patients with SSc by different groups of researchers and such levels correlate directly with the interstitial lung involvement. The prevalence of hematopoietic markers expressed by CPCs that migrate from blood into injury sites in SSc differs and changes according to the degree of differentiation. CXCR4 is the most commonly expressed marker, followed by CD34 and CD45 at an end stage of differentiation. Such difference also indicates a continuous process of cell differentiation that might relate to the SSc clinical phenotype (degree of fibrosis and vascular involvement). A deeper understanding of the role of each subtype of CPCs in the development of the disease will help us to better classify patients in order to offer them targeted approaches in the future. PMID:27158466

FoxO1-negative cells are cancer stem-like cells in pancreatic ductal adenocarcinoma.

PubMed

Song, Weifeng; Li, Qi; Wang, Lei; Huang, Weiyi; Wang, Liwei

2015-06-11

Flow cytometry assays using aldehyde dehydrogenase (ALDH) activity or CD133 positivity to isolate cancer stem cells (CSCs) are widely applied but have limitations. Thus, characterization of CSC makers for a specific cancer is potentially important. We have previously shown that miR-21 regulates cancer cell growth via FoxO1 in pancreatic ductal adenocarcinoma (PDAC). Here, we areported evidence of FoxO1-negative PDAC cells as CSCs in PDAC. Both ALDH-high and CD133-high cell fractions isolated from PDAC of the patients expressed high levels of miR-21 and null FoxO1. Cultured PDAC cells were virally transduced with GFP under FoxO1 promoter. GFP (FoxO1)-null PDAC cells expressed high levels of miR-21, and grew more quickly than FoxO1-positive PDAC cells. Moreover, the fold increases in growth of FoxO1-negative vs FoxO1-positive cells were greater than CD133-high vs CD133-low cells, or ALDH-high vs ALDH-low cells. Further, FoxO1-negative cells formed tumor spheres in culture and developed tumors after serial adoptive transplantation into NOD/SCID mice, while the FoxO1-positive cells did not. Finally, selective elimination of FoxO1-negative cells completely inhibited the growth of PDAC cells. Together, these data suggest that FoxO1-negative cells as CSCs in PDAC, and targeting FoxO1-negative cells in PDAC may provide better therapeutic outcome.

The low chamber pancreatic cancer cells had stem-like characteristics in modified transwell system: is it a novel method to identify and enrich cancer stem-like cells?

PubMed

Wang, Dongqing; Zhu, Haitao; Liu, Yanfang; Liu, Qing; Xie, Xiaodong; Zhou, Yuepeng; Zhang, Lirong; Zhu, Yan; Zhang, Zhijian; Su, Zhaoliang

2014-01-01

Cancer stem cells (CSCs) or cancer-initiating cells (CICs) play an important role in tumor initiation, progression, metastasis, chemoresistance, and recurrence. It is important to construct an effective method to identify and isolate CSCs for biotherapy of cancer. During the past years, many researchers had paid more attention to it; however, this method was still on seeking. Therefore, compared to the former methods that were used to isolate the cancer stem cell, in the present study, we tried to use modified transwell system to isolate and enrich CSCs from human pancreatic cancer cell lines (Panc-1). Our results clearly showed that the lower chamber cells in modified transwell system were easily forming spheres; furthermore, these spheres expressed high levels of stem cell markers (CD133/CD44/CD24/Oct-4/ESA) and exhibited chemoresistance, underwent epithelial-to-mesenchymal transition (EMT), and possessed the properties of self-renewal in vitro and tumorigenicity in vivo. Therefore, we speculated that modified transwell assay system, as a rapid and effective method, can be used to isolate and enrich CSCs.

Feline mammary carcinoma stem cells are tumorigenic, radioresistant, chemoresistant and defective in activation of the ATM/p53 DNA damage pathway

PubMed Central

Pang, L.Y.; Blacking, T.M.; Else, R.W.; Sherman, A.; Sang, H.M.; Whitelaw, B.A.; Hupp, T.R.; Argyle, D.J.

2013-01-01

Cancer stem cells were identified in a feline mammary carcinoma cell line by demonstrating expression of CD133 and utilising the tumour sphere assay. A population of cells was identified that had an invasive, mesenchymal phenotype, expressed markers of pluripotency and enhanced tumour formation in the NOD-SCID mouse and chick embryo models. This population of feline mammary carcinoma stem cells was resistant to chemotherapy and radiation, possibly due to aberrant activation of the ATM/p53 DNA damage pathway. Epithelial–mesenchymal transition was a feature of the invasive phenotype. These data demonstrate that cancer stem cells are a feature of mammary cancer in cats. PMID:23219486

Concurrent Isolation of 3 Distinct Cardiac Stem Cell Populations From a Single Human Heart Biopsy.

PubMed

Monsanto, Megan M; White, Kevin S; Kim, Taeyong; Wang, Bingyan J; Fisher, Kristina; Ilves, Kelli; Khalafalla, Farid G; Casillas, Alexandria; Broughton, Kathleen; Mohsin, Sadia; Dembitsky, Walter P; Sussman, Mark A

2017-07-07

The relative actions and synergism between distinct myocardial-derived stem cell populations remain obscure. Ongoing debates on optimal cell population(s) for treatment of heart failure prompted implementation of a protocol for isolation of multiple stem cell populations from a single myocardial tissue sample to develop new insights for achieving myocardial regeneration. Establish a robust cardiac stem cell isolation and culture protocol to consistently generate 3 distinct stem cell populations from a single human heart biopsy. Isolation of 3 endogenous cardiac stem cell populations was performed from human heart samples routinely discarded during implantation of a left ventricular assist device. Tissue explants were mechanically minced into 1 mm 3 pieces to minimize time exposure to collagenase digestion and preserve cell viability. Centrifugation removes large cardiomyocytes and tissue debris producing a single cell suspension that is sorted using magnetic-activated cell sorting technology. Initial sorting is based on tyrosine-protein kinase Kit (c-Kit) expression that enriches for 2 c-Kit + cell populations yielding a mixture of cardiac progenitor cells and endothelial progenitor cells. Flowthrough c-Kit - mesenchymal stem cells are positively selected by surface expression of markers CD90 and CD105. After 1 week of culture, the c-Kit + population is further enriched by selection for a CD133 + endothelial progenitor cell population. Persistence of respective cell surface markers in vitro is confirmed both by flow cytometry and immunocytochemistry. Three distinct cardiac cell populations with individualized phenotypic properties consistent with cardiac progenitor cells, endothelial progenitor cells, and mesenchymal stem cells can be successfully concurrently isolated and expanded from a single tissue sample derived from human heart failure patients. © 2017 American Heart Association, Inc.

Curcumin inhibits cancer stem cell phenotypes in ex vivo models of colorectal liver metastases, and is clinically safe and tolerable in combination with FOLFOX chemotherapy

PubMed Central

James, Mark I.; Iwuji, Chinenye; Irving, Glen; Karmokar, Ankur; Higgins, Jennifer A.; Griffin-Teal, Nicola; Thomas, Anne; Greaves, Peter; Cai, Hong; Patel, Samita R.; Morgan, Bruno; Dennison, Ashley; Metcalfe, Matthew; Garcea, Giuseppe; Lloyd, David M.; Berry, David P.; Steward, William P.; Howells, Lynne M.; Brown, Karen

2015-01-01

In vitro and pre-clinical studies have suggested that addition of the diet-derived agent curcumin may provide a suitable adjunct to enhance efficacy of chemotherapy in models of colorectal cancer. However, the majority of evidence for this currently derives from established cell lines. Here, we utilised patient-derived colorectal liver metastases (CRLM) to assess whether curcumin may provide added benefit over 5-fluorouracil (5-FU) and oxaliplatin (FOLFOX) in cancer stem cell (CSC) models. Combination of curcumin with FOLFOX chemotherapy was then assessed clinically in a phase I dose escalation study. Curcumin alone and in combination significantly reduced spheroid number in CRLM CSC models, and decreased the number of cells with high aldehyde dehydrogenase activity (ALDHhigh/CD133−). Addition of curcumin to oxaliplatin/5-FU enhanced anti-proliferative and pro-apoptotic effects in a proportion of patient-derived explants, whilst reducing expression of stem cell-associated markers ALDH and CD133. The phase I dose escalation study revealed curcumin to be a safe and tolerable adjunct to FOLFOX chemotherapy in patients with CRLM (n = 12) at doses up to 2 grams daily. Curcumin may provide added benefit in subsets of patients when administered with FOLFOX, and is a well-tolerated chemotherapy adjunct. PMID:25979230

Molecular markers in glioma.

PubMed

Ludwig, Kirsten; Kornblum, Harley I

2017-09-01

Gliomas are the most malignant and aggressive form of brain tumors, and account for the majority of brain cancer related deaths. Malignant gliomas, including glioblastoma are treated with radiation and temozolomide, with only a minor benefit in survival time. A number of advances have been made in understanding glioma biology, including the discovery of cancer stem cells, termed glioma stem cells (GSC). Some of these advances include the delineation of molecular heterogeneity both between tumors from different patients as well as within tumors from the same patient. Such research highlights the importance of identifying and validating molecular markers in glioma. This review, intended as a practical resource for both clinical and basic investigators, summarizes some of the more well-known molecular markers (MGMT, 1p/19q, IDH, EGFR, p53, PI3K, Rb, and RAF), discusses how they are identified, and what, if any, clinical relevance they may have, in addition to discussing some of the specific biology for these markers. Additionally, we discuss identification methods for studying putative GSC's (CD133, CD15, A2B5, nestin, ALDH1, proteasome activity, ABC transporters, and label-retention). While much research has been done on these markers, there is still a significant amount that we do not yet understand, which may account for some conflicting reports in the literature. Furthermore, it is unlikely that the investigator will be able to utilize one single marker to prospectively identify and isolate GSC from all, or possibly, any gliomas.

Whole-Transcriptome Analysis of CD133+CD144+ Cancer Stem Cells Derived from Human Laryngeal Squamous Cell Carcinoma Cells.

PubMed

Wu, Yongyan; Zhang, Yuliang; Niu, Min; Shi, Yong; Liu, Hongliang; Yang, Dongli; Li, Fei; Lu, Yan; Bo, Yunfeng; Zhang, Ruiping; Li, Zhenyu; Luo, Hongjie; Cui, Jiajia; Sang, Jiangwei; Xiang, Caixia; Gao, Wei; Wen, Shuxin

2018-06-27

CD133+CD44+ cancer stem cells previously isolated from laryngeal squamous cell carcinoma (LSCC) cell lines showed strong malignancy and tumorigenicity. However, the molecular mechanism underlying the enhanced malignancy remained unclear. Cell proliferation assay, spheroid-formation experiment, RNA sequencing (RNA-seq), miRNA-seq, bioinformatic analysis, quantitative real-time PCR, migration assay, invasion assay, and luciferase reporter assay were used to identify differentially expressed mRNAs, lncRNAs, circRNAs and miRNAs, construct transcription regulatory network, and investigate functional roles and mechanism of circRNA in CD133+CD44+ laryngeal cancer stem cells. Differentially expressed genes in TDP cells were mainly enriched in the biological processes of cell differentiation, regulation of autophagy, negative regulation of cell death, regulation of cell growth, response to hypoxia, telomere maintenance, cellular response to gamma radiation, and regulation of apoptotic signaling, which are closely related to the malignant features of tumor cells. We constructed the regulatory network of differentially expressed circRNAs, miRNAs and mRNAs. qPCR findings for the expression of key genes in the network were consistent with the sequencing data. Moreover, our data revealed that circRNA hg19_circ_0005033 promotes proliferation, migration, invasion, and chemotherapy resistance of laryngeal cancer stem cells. This study provides potential biomarkers and targets for LSCC diagnosis and therapy, and provide important evidences for the heterogeneity of LSCC cells at the transcription level. © 2018 The Author(s). Published by S. Karger AG, Basel.

Comparative Gene Expression Profiling of Primary and Metastatic Renal Cell Carcinoma Stem Cell-Like Cancer Cells

PubMed Central

Czarnecka, Anna M.; Lewicki, Sławomir; Helbrecht, Igor; Brodaczewska, Klaudia; Koch, Irena; Zdanowski, Robert; Król, Magdalena; Szczylik, Cezary

2016-01-01

Background Recent advancement in cancer research has shown that tumors are highly heterogeneous, and multiple phenotypically different cell populations are found in a single tumor. Cancer development and tumor growth are driven by specific types of cells—stem cell-like cancer cells (SCLCCs)—which are also responsible for metastatic spread and drug resistance. This research was designed to verify the presence of SCLCCs in renal cell cancer cell lines. Subsequently, we aimed to characterize phenotype and cell biology of CD105+ cells, defined previously as renal cell carcinoma tumor-initiating cells. The main goal of the project was to describe the gene-expression profile of stem cell-like cancer cells of primary tumor and metastatic origin. Materials and Methods Real-time PCR analysis of stemness genes (Oct-4, Nanog and Ncam) and soft agar colony formation assay were conducted to check the stemness properties of renal cell carcinoma (RCC) cell lines. FACS analysis of CD105+ and CD133+ cells was performed on RCC cells. Isolated CD105+ cells were verified for expression of mesenchymal markers—CD24, CD146, CD90, CD73, CD44, CD11b, CD19, CD34, CD45, HLA-DR and alkaline phosphatase. Hanging drop assay was used to investigate CD105+ cell-cell cohesion. Analysis of free-floating 3D spheres formed by isolated CD105+ was verified, as spheres have been hypothesized to contain undifferentiated multipotent progenitor cells. Finally, CD105+ cells were sorted from primary (Caki-2) and metastatic (ACHN) renal cell cancer cell lines. Gene-expression profiling of sorted CD105+ cells was performed with Agilent’s human GE 4x44K v2 microarrays. Differentially expressed genes were further categorized into canonical pathways. Network analysis and downstream analysis were performed with Ingenuity Pathway Analysis. Results Metastatic RCC cell lines (ACHN and Caki-1) demonstrated higher colony-forming ability in comparison to primary RCC cell lines. Metastatic RCC cell lines harbor numerous CD105+ cell subpopulations and have higher expression of stemness genes (Oct-4 and Nanog). CD105+ cells adopt 3D grape-like floating structures under handing drop conditions. Sorted CD105+ cells are positive for human mesenchymal stem cell (MSC) markers CD90, CD73, CD44, CD146, and alkaline phosphatase activity, but not for CD24 and hematopoietic lineage markers CD34, CD11b, CD19, CD45, and HLA-DR. 1411 genes are commonly differentially expressed in CD105+ cells (both from primary [Caki-2] and metastatic RCC [ACHN] cells) in comparison to a healthy kidney epithelial cell line (ASE-5063). TGF-β, Wnt/β-catenine, epithelial-mesenchymal transition (EMT), Rap1 signaling, PI3K-Akt signaling, and Hippo signaling pathway are deregulated in CD105+ cells. TGFB1, ERBB2, and TNF are the most significant transcriptional regulators activated in these cells. Conclusions All together, RCC-CD105+ cells present stemlike properties. These stem cell-like cancer cells may represent a novel target for therapy. A unique gene-expression profile of CD105+ cells could be used as initial data for subsequent functional studies and drug design. PMID:27812180

Profiling of normal and malignant breast tissue show CD44high/CD24low phenotype as a predominant stem/progenitor marker when used in combination with Ep-CAM/CD49f markers

PubMed Central

2013-01-01

Background Accumulating evidence supports cancer to initiate and develop from a small population of stem-like cells termed as cancer stem cells (CSC). The exact phenotype of CSC and their counterparts in normal mammary gland is not well characterized. In this study our aim was to evaluate the phenotype and function of stem/progenitor cells in normal mammary epithelial cell populations and their malignant counterparts. Methods Freshly isolated cells from both normal and malignant human breasts were sorted using 13 widely used stem/progenitor cell markers individually or in combination by multi-parametric (up to 9 colors) cell sorting. The sorted populations were functionally evaluated by their ability to form colonies and mammospheres, in vitro. Results We have compared, for the first time, the stem/progenitor markers of normal and malignant breasts side-by-side. Amongst all markers tested, we found CD44high/CD24low cell surface marker combination to be the most efficient at selecting normal epithelial progenitors. Further fractionation of CD44high/CD24low positive cells showed that this phenotype selects for luminal progenitors within Ep-CAMhigh/CD49f + cells, and enriches for basal progenitors within Ep-CAM-/low/CD49f + cells. On the other hand, primary breast cancer samples, which were mainly luminal Ep-CAMhigh, had CD44high/CD24low cells among both CD49fneg and CD49f + cancer cell fractions. However, functionally, CSC were predominantly CD49f + proposing the use of CD44high/CD24low in combination with Ep-CAM/CD49f cell surface markers to further enrich for CSC. Conclusion Our study clearly demonstrates that both normal and malignant breast cells with the CD44high/CD24low phenotype have the highest stem/progenitor cell ability when used in combination with Ep-CAM/CD49f reference markers. We believe that this extensive characterization study will help in understanding breast cancer carcinogenesis, heterogeneity and drug resistance. PMID:23768049

The Selective Centrifugation Ensures a Better In Vitro Isolation of ASCs and Restores a Soft Tissue Regeneration In Vivo

PubMed Central

De Francesco, Francesco; Guastafierro, Antonio; Nicoletti, Gianfranco; Razzano, Sergio; Riccio, Michele; Ferraro, Giuseppe A.

2017-01-01

Autologous fat grafting procedures in plastic surgery have been extensively used to reinforce soft tissue in congenital or acquired tissue impairments. With this background, the aim of this study is firstly to examine the impact of a selective centrifugation on existing adipose stem cells (ASCs) in terms of stemness profile maintenance and, secondly, to investigate the effect of restoring volume in reconstruction on patients affected by soft tissue damage. After centrifugation, the fat graft products were separated into two layers and subsequently examined in vitro for the expression of CD34, CD90, CD117, CD105, CD29, CD31, CD44, CD73, CD133, CD14 and CD45 markers by flow cytometry and gene expression analyses were performed for Sox2, WNT3A, END, CD44, FUT4, COLL1, CTNNB1, hbEGF, KRTLG, MMP2 and VIM genes. The results showed that in the middle-high density (MHD) layer there was a peak concentration of ASCs, compared to another layer obtained after centrifugation. Research carried out on patients under treatment for soft tissue regeneration using cells obtained from MHD layer selection will be fundamental in comparative analysis. These studies will lead to an adequate standardization of outcomes, provided that treatment is performed through cell selection. Therefore, a unique procedure in tissue reconstruction and regeneration through fat grafting is presented here. PMID:28498335

Mammalian-enabled (MENA) protein enhances oncogenic potential and cancer stem cell-like phenotype in hepatocellular carcinoma cells.

PubMed

Hu, Kunpeng; Huang, Pinzhu; Luo, Hui; Yao, Zhicheng; Wang, Qingliang; Xiong, Zhiyong; Lin, Jizong; Huang, He; Xu, Shilei; Zhang, Peng; Liu, Bo

2017-08-01

Mammalian-enabled (MENA) protein is an actin-regulatory protein that influences cell motility and adhesion. It is known to play a role in tumorigenicity of hepatocellular carcinoma (HCC) but the underlying molecular mechanism remains unknown. This study aimed to investigate the oncogenic potential of MENA and its capacity to regulate cancer stem cell (CSC)-like phenotypes in HCC cells. Real-time-PCR and western blot were used to assess mRNA and protein levels of target genes in human HCC tissue specimens and HCC cell lines, respectively. Stable MENA-overexpressing HCC cells were generated from HCC cell lines. Transwell cell migration and colony formation assays were employed to evaluate tumorigenicity. Ectopic expression of MENA significantly enhanced cell migration and colony-forming ability in HCC cells. Overexpression of MENA upregulated several hepatic progenitor/stem cell markers in HCC cells. A high MENA protein level was associated with high mRNA levels of MENA, CD133, cytokeratin 19 (CK19), and epithelial cell adhesion molecule (EpCAM) in human HCC tissues. Overexpression of MENA enhanced epithelial-to-mesenchymal transition (EMT) markers, extracellular signal-regulated kinases (ERK) phosphorylation, and the level of β-catenin in HCC cells. This study demonstrated that overexpression of MENA in HCC cells promoted stem cell markers, EMT markers, and tumorigenicity. These effects may involve, at least partially, the ERK and β-catenin signaling pathways.

Humoral Activity of Cord Blood-Derived Stem/Progenitor Cells: Implications for Stem Cell-Based Adjuvant Therapy of Neurodegenerative Disorders

PubMed Central

Paczkowska, Edyta; Kaczyńska, Katarzyna; Pius-Sadowska, Ewa; Rogińska, Dorota; Kawa, Miłosz; Ustianowski, Przemysław; Safranow, Krzysztof; Celewicz, Zbigniew; Machaliński, Bogusław

2013-01-01

Background Stem/progenitor cells (SPCs) demonstrate neuro-regenerative potential that is dependent upon their humoral activity by producing various trophic factors regulating cell migration, growth, and differentiation. Herein, we compared the expression of neurotrophins (NTs) and their receptors in specific umbilical cord blood (UCB) SPC populations, including lineage-negative, CD34+, and CD133+ cells, with that in unsorted, nucleated cells (NCs). Methods and Results The expression of NTs and their receptors was detected by QRT-PCR, western blotting, and immunofluorescent staining in UCB-derived SPC populations (i.e., NCs vs. lineage-negative, CD34+, and CD133+ cells). To better characterize, global gene expression profiles of SPCs were determined using genome-wide RNA microarray technology. Furthermore, the intracellular production of crucial neuro-regenerative NTs (i.e., BDNF and NT-3) was assessed in NCs and lineage-negative cells after incubation for 24, 48, and 72 h in both serum and serum-free conditions. We discovered significantly higher expression of NTs and NT receptors at both the mRNA and protein level in lineage-negative, CD34+, and CD133+ cells than in NCs. Global gene expression analysis revealed considerably higher expression of genes associated with the production and secretion of proteins, migration, proliferation, and differentiation in lineage-negative cells than in CD34+ or CD133+ cell populations. Notably, after short-term incubation under serum-free conditions, lineage-negative cells and NCs produced significantly higher amounts of BDNF and NT-3 than under steady-state conditions. Finally, conditioned medium (CM) from lineage-negative SPCs exerted a beneficial impact on neural cell survival and proliferation. Conclusions Collectively, our findings demonstrate that UCB-derived SPCs highly express NTs and their relevant receptors under steady-state conditions, NT expression is greater under stress-related conditions and that CM from SPCs favorable influence neural cell proliferation and survival. Understanding the mechanisms governing the characterization and humoral activity of subsets of SPCs may yield new therapeutic strategies that might be more effective in treating neurodegenerative disorders. PMID:24391835

Humoral activity of cord blood-derived stem/progenitor cells: implications for stem cell-based adjuvant therapy of neurodegenerative disorders.

PubMed

Paczkowska, Edyta; Kaczyńska, Katarzyna; Pius-Sadowska, Ewa; Rogińska, Dorota; Kawa, Miłosz; Ustianowski, Przemysław; Safranow, Krzysztof; Celewicz, Zbigniew; Machaliński, Bogusław

2013-01-01

Stem/progenitor cells (SPCs) demonstrate neuro-regenerative potential that is dependent upon their humoral activity by producing various trophic factors regulating cell migration, growth, and differentiation. Herein, we compared the expression of neurotrophins (NTs) and their receptors in specific umbilical cord blood (UCB) SPC populations, including lineage-negative, CD34(+), and CD133(+) cells, with that in unsorted, nucleated cells (NCs). The expression of NTs and their receptors was detected by QRT-PCR, western blotting, and immunofluorescent staining in UCB-derived SPC populations (i.e., NCs vs. lineage-negative, CD34(+), and CD133(+) cells). To better characterize, global gene expression profiles of SPCs were determined using genome-wide RNA microarray technology. Furthermore, the intracellular production of crucial neuro-regenerative NTs (i.e., BDNF and NT-3) was assessed in NCs and lineage-negative cells after incubation for 24, 48, and 72 h in both serum and serum-free conditions. We discovered significantly higher expression of NTs and NT receptors at both the mRNA and protein level in lineage-negative, CD34(+), and CD133(+) cells than in NCs. Global gene expression analysis revealed considerably higher expression of genes associated with the production and secretion of proteins, migration, proliferation, and differentiation in lineage-negative cells than in CD34(+) or CD133(+) cell populations. Notably, after short-term incubation under serum-free conditions, lineage-negative cells and NCs produced significantly higher amounts of BDNF and NT-3 than under steady-state conditions. Finally, conditioned medium (CM) from lineage-negative SPCs exerted a beneficial impact on neural cell survival and proliferation. Collectively, our findings demonstrate that UCB-derived SPCs highly express NTs and their relevant receptors under steady-state conditions, NT expression is greater under stress-related conditions and that CM from SPCs favorable influence neural cell proliferation and survival. Understanding the mechanisms governing the characterization and humoral activity of subsets of SPCs may yield new therapeutic strategies that might be more effective in treating neurodegenerative disorders.

Molecular cloning and characterization of markers and cytokines for equid myeloid cells.

PubMed

Steinbach, Falko; Stark, Robert; Ibrahim, Sherif; Gawad, Eman Abd-El; Ludwig, Hanns; Walter, Jakob; Commandeur, Ulrich; Mauel, Susanne

2005-10-18

The myeloid cell system comprises of monocytes, macrophages (MPhi), dendritic cells (DC), Kupffer cells, osteoclasts or microglia and is also known as the mononuclear phagocytic system (MPS). Essential cytokines to differentiate or activate these cells include GM-CSF or IL-4. Important markers for characterization include CD1, CD14, CD68, CD163 and CD206. All these markers, however, were not cloned or further characterized in equids by use of monoclonal antibodies earlier. To overcome this problem with the present study, two approaches were used. First, we cloned equine cytokines and markers, and second we analyzed cross-reactivity of human homologues or anti-human monoclonal antibodies. For cloning of equine cytokines and markers, we used degenerate primers delineated from other species, or equine-specific primers based on previous information in Genbank. Flow cytometry was used to determine the expression of markers on myeloid cells. Cross-reactivity could be shown for anti-human CD14, CD163 and mannose receptor (CD206) mAbs. Surface markers such as CD1 and CD68 that distinguish MPhi and DC were cloned and sequenced. According to blast homology, equine CD1a and CD1b could be identified and distinguished. With the resulting information, dendritic cells and macrophages of horses may be characterized.

Construction of an Aptamer-SiRNA Chimera-Modified Tissue-Engineered Blood Vessel for Cell-Type-Specific Capture and Delivery.

PubMed

Chen, Wen; Zeng, Wen; Sun, Jiansen; Yang, Mingcan; Li, Li; Zhou, Jingting; Wu, Yangxiao; Sun, Jun; Liu, Ge; Tang, Rui; Tan, Ju; Zhu, Chuhong

2015-06-23

The application of tissue-engineered blood vessels (TEBVs) is the main developmental direction of vascular replacement therapy. Due to few and/or dysfunctional endothelial progenitor cells (EPCs), it is difficult to successfully construct EPC capture TEBVs in diabetes. RNA has a potential application in cell protection and diabetes treatment, but poor specificity and low efficiency of RNA transfection in vivo limit the application of RNA. On the basis of an acellular vascular matrix, we propose an aptamer-siRNA chimera-modified TEBV that can maintain a satisfactory patency in diabetes. This TEBV consists of two parts, CD133-adenosine kinase (ADK) chimeras and a TEBV scaffold. Our results showed that CD133-ADK chimeras could selectively capture the CD133-positive cells in vivo, and then captured cells can internalize the bound chimeras to achieve RNA self-transfection. Subsequently, CD133-ADK chimeras were cut into ADK siRNA by a dicer, resulting in depletion of ADK. An ADK-deficient cell may act as a bioreactor that sustainably releases adenosine. To reduce nonspecific RNA transfection, we increased the proportion of HAuCl4 during the material preparation, through which the transfection capacity of polyethylenimine (PEI)/polyethylene glycol (PEG)-capped gold nanoparticles (PEI/PEG-AuNPs) was significantly decreased and the ability of TEBV to resist tensile and liquid shear stress was greatly enhanced. PEG and 2'-O-methyl modification was used to enhance the in vivo stability of RNA chimeras. At day 30 postgrafting, the patency rate of CD133-ADK chimera-modified TEBVs reached 90% in diabetic rats and good endothelialization was observed.

Sustainability of CD24 expression, cell proliferation and migration, cisplatin-resistance, and caspase-3 expression during mesenchymal-epithelial transition induced by the removal of TGF-β1 in A549 lung cancer cells.

PubMed

Kim, Seong-Kwan; Park, Jin-A; Zhang, Dan; Cho, Sang-Hyun; Yi, Hee; Cho, Soo-Min; Chang, Byung-Joon; Kim, Jin-Suk; Shim, Jae-Han; Abd El-Aty, A M; Shin, Ho-Chul

2017-08-01

Epithelial-mesenchymal transition (EMT) is a notable mechanism underlying cancer cell metastasis. Transforming growth factor β1 (TGF-β1) has been used to induce EMT; however, there is a lack of information regarding the role of TGF-β1 in mesenchymal-epithelial transition (MET). In the present study, EMT was induced in A549 lung cancer cells using TGF-β1 (TGF-β1-treated group) and MET was induced sequentially from the TGF-β1-treated group by removing the TGF-β1 (MET/return group). Untreated A549 lung cancer cells were used as a control. Characteristic features, including cancer stem cell markers [cluster of differentiation (CD)24, CD44 and CD133], cell proliferation and migration and diverse intracellular mechanisms, were observed in all groups. Using western blot analysis, the TGF-β1-treated group demonstrated increased vimentin and reduced E-cadherin expression, whereas the MET/return group demonstrated the opposite trend. Among cancer stem cell markers, the population of CD24 low cells was reduced in the TGF-β1-treated group. Furthermore, the G2/M phase cell cycle population, cisplatin-sensitivity, and cell proliferation and migration ability were increased in the TGF-β1-treated group. These features were unaltered in the MET/return group when compared to the TGF-β1-treated group. Immunoblotting revealed an increase in the levels of SMAD3, phosphorylated SMAD3, phosphorylated extracellular signal-regulated kinase and caspase-3, and a decrease in active caspase-3 levels in the TGF-β1-treated group. Increased caspase-3 and reduced active caspase-3 levels were observed in the MET/return group, similar to those in the TGF-β1-treated group; however, levels of other signalling proteins were unchanged compared with the control group. EMT induced by TGF-β1 was not preserved; however, stemness-associated properties (CD24 expression, caspase-3 expression, cell proliferation and cisplatin-resistance) were sustained following removal of TGF-β1.

Cancer stem-like cells of ovarian clear cell carcinoma are enriched in the ALDH-high population associated with an accelerated scavenging system in reactive oxygen species.

PubMed

Mizuno, T; Suzuki, N; Makino, H; Furui, T; Morii, E; Aoki, H; Kunisada, T; Yano, M; Kuji, S; Hirashima, Y; Arakawa, A; Nishio, S; Ushijima, K; Ito, K; Itani, Y; Morishige, K

2015-05-01

In ovarian cancer cases, recurrence after chemotherapy is frequently observed, suggesting the involvement of ovarian cancer stem-like cells (CSCs). The chemoresistance of ovarian clear cell carcinomas is particularly strong in comparison to other epithelial ovarian cancer subtypes. We investigated the relationship between a CSC marker, aldehyde dehydrogenase 1 (ALDH1), and clinical prognosis using ovarian clear cell carcinoma tissue samples. Furthermore, we investigated the antioxidant mechanism by which CSCs maintain a lower reactive oxygen species (ROS) level, which provides protection from chemotherapeutic agents. Immunohistochemical staining was performed to examine the CSC markers (CD133, CD44, ALDH1) using ovarian clear cell carcinoma tissue samples (n=81). Clear cell carcinoma cell lines (KOC-7C, OVTOKO) are separated into the ALDH-high and ALDH-low populations by ALDEFLUOR assay and fluorescence-activated cell sorting (FACS). We compared the intracellular ROS level, mRNA level of the antioxidant enzymes and Nrf2 expression of the two populations. High ALDH1 expression levels are related to advanced stage in clear cell carcinoma cases. ALDH1 expression significantly reduced progression free survival. Other markers are not related to clinical stage and prognosis. ALDH-high cells contained a lower ROS level than ALDH-low cells. Antioxidant enzymes were upregulated in ALDH-high cells. ALDH-high cells showed increased expression of Nrf2, a key transcriptional factor of the antioxidant system. ALDH-positive CSCs might have increased Nrf2-induced antioxidant scavengers, which lower ROS level relevant to chemoresistance in ovarian clear cell carcinoma. Copyright © 2014 Elsevier Inc. All rights reserved.

Characterization of cancer stem cell properties of CD24 and CD26-positive human malignant mesothelioma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamazaki, Hiroto; Naito, Motohiko; Ghani, Farhana Ishrat

2012-03-16

Highlights: Black-Right-Pointing-Pointer We focused on CD24 and CD26 for further analysis of CSC properties in MM. Black-Right-Pointing-Pointer Their expressions were correlated with chemoresistance, cell growth, and invasion. Black-Right-Pointing-Pointer Their expressions were also correlated with several cancer related genes. Black-Right-Pointing-Pointer The expression of each marker was correlated with different CSC property in Meso1. Black-Right-Pointing-Pointer Phosphorylation of ERK by EGF was regulated by expression of CD26, but not CD24. -- Abstract: Malignant mesothelioma (MM) is an asbestos-related malignancy characterized by rapid growth and poor prognosis. In our previous study, we have demonstrated that several cancer stem cell (CSC) markers correlated with CSCmore » properties in MM cells. Among these markers, we focused on two: CD24, the common CSC marker, and CD26, the additional CSC marker. We further analyzed the CSC properties of CD24 and CD26-positve MM cells. We established RNAi-knockdown cells and found that these markers were significantly correlated with chemoresistance, proliferation, and invasion potentials in vitro. Interestingly, while Meso-1 cells expressed both CD24 and CD26, the presence of each of these two markers was correlated with different CSC property. In addition, downstream signaling of these markers was explored by microarray analysis, which revealed that their expressions were correlated with several cancer-related genes. Furthermore, phosphorylation of ERK by EGF stimulation was significantly affected by the expression of CD26, but not CD24. These results suggest that CD24 and CD26 differentially regulate the CSC potentials of MM and could be promising targets for CSC-oriented therapy.« less

Efficacy of T Regulatory Cells, Th17 Cells and the Associated Markers in Monitoring Tuberculosis Treatment Response

PubMed Central

Agrawal, Sonali; Parkash, Om; Palaniappan, Alangudi Natarajan; Bhatia, Ashok Kumar; Kumar, Santosh; Chauhan, Devendra Singh; Madhan Kumar, M.

2018-01-01

Treatment monitoring is an essential aspect for tuberculosis (TB) disease management. Sputum smear microscopy is the only available tool for monitoring, but it suffers from demerits. Therefore, we sought to evaluate markers and cellular subsets of T regulatory (Treg) cells and T helper (Th) 17 cells in pulmonary TB patients (PTB) for TB treatment monitoring. Peripheral blood mononuclear cells (PBMCs) were stimulated in vitro (with purified protein derivative (PPD)) overnight which was followed by a polychromatic flow cytometry approach to study Treg and Th17 markers and cellular subsets in PTB (n = 12) undergoing antituberculous treatment (ATT). The baseline levels of these markers and cellular subsets were evaluated in normal healthy subjects (NHS). We observed a significant decrease in the expression of CD25 (p<0.01) marker and percentage of T-cell subsets like CD4+CD25+ (p<0.001) and CD4+CD25+CD39+ (p<0.05) at the end of intensive phase (IP) as well as in the continuation phase (CP) of ATT. A decrease in CD25 marker expression and percentage of CD4+CD25+ T cell subset showed a positive correlation to sputum conversion both in high and low sputum positive PTB. In eight PTB with cavitary lesions, only CD4+CD25+FoxP3 Treg subset manifested a significant decrease at the end of CP. Thus, results of this study show that CD25 marker and CD4+CD25+ T cells can serve as better markers for monitoring TB treatment efficacy. The Treg subset CD4+CD25+FoxP3 may be useful for prediction of favorable response in PTB with extensive lung lesions. However, these findings have to be evaluated in a larger patient cohort. PMID:29472922

USP1 targeting impedes GBM growth by inhibiting stem cell maintenance and radioresistance

PubMed Central

Lee, Jin-Ku; Chang, Nakho; Yoon, Yeup; Yang, Heekyoung; Cho, Heejin; Kim, Eunhee; Shin, Yongjae; Kang, Wonyoung; Oh, Young Taek; Mun, Gyeong In; Joo, Kyeung Min; Nam, Do-Hyun; Lee, Jeongwu

2016-01-01

Background Clinical benefits from standard therapies against glioblastoma (GBM) are limited in part due to intrinsic radio- and chemoresistance of GBM and inefficient targeting of GBM stem-like cells (GSCs). Novel therapeutic approaches that overcome treatment resistance and diminish stem-like properties of GBM are needed. Methods We determined the expression levels of ubiquitination-specific proteases (USPs) by transcriptome analysis and found that USP1 is highly expressed in GBM. Using the patient GBM-derived primary tumor cells, we inhibited USP1 by shRNA-mediated knockdown or its specific inhibitor pimozide and evaluated the effects on stem cell marker expression, proliferation, and clonogenic growth of tumor cells. Results USP1 was highly expressed in gliomas relative to normal brain tissues and more preferentially in GSC enrichment marker (CD133 or CD15) positive cells. USP1 positively regulated the protein stability of the ID1 and CHEK1, critical regulators of DNA damage response and stem cell maintenance. Targeting USP1 by RNA interference or treatment with a chemical USP1 inhibitor attenuated clonogenic growth and survival of GSCs and enhanced radiosensitivity of GBM cells. Finally, USP1 inhibition alone or in combination with radiation significantly prolonged the survival of tumor-bearing mice. Conclusion USP1-mediated protein stabilization promotes GSC maintenance and treatment resistance, thereby providing a rationale for USP1 inhibition as a potential therapeutic approach against GBM. PMID:26032834

Identification of cancer stem cell markers in human malignant mesothelioma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ghani, Farhana Ishrat; Yamazaki, Hiroto; Iwata, Satoshi

2011-01-14

Research highlights: {yields} We performed serial transplantation of surgical samples and established new cell lines of malignant mesothelioma. {yields} SP cell and expressions of CD9/CD24/CD26 were often observed in mesothelioma cell lines. {yields} SP and CD24{sup +} cells proliferated by asymmetric cell division-like manner. CD9{sup +} and CD24{sup +} cells have higher potential to generate spheroid colony. {yields} The marker-positive cells have clear tendency to generate larger tumors in mice. -- Abstract: Malignant mesothelioma (MM) is an aggressive and therapy-resistant neoplasm arising from the pleural mesothelial cells and usually associated with long-term asbestos exposure. Recent studies suggest that tumors containmore » cancer stem cells (CSCs) and their stem cell characteristics are thought to confer therapy-resistance. However, whether MM cell has any stem cell characteristics is not known. To understand the molecular basis of MM, we first performed serial transplantation of surgical samples into NOD/SCID mice and established new cell lines. Next, we performed marker analysis of the MM cell lines and found that many of them contain SP cells and expressed several putative CSC markers such as CD9, CD24, and CD26. Interestingly, expression of CD26 closely correlated with that of CD24 in some cases. Sorting and culture assay revealed that SP and CD24{sup +} cells proliferated by asymmetric cell division-like manner. In addition, CD9{sup +} and CD24{sup +} cells have higher potential to generate spheroid colony than negative cells in the stem cell medium. Moreover, these marker-positive cells have clear tendency to generate larger tumors in mouse transplantation assay. Taken together, our data suggest that SP, CD9, CD24, and CD26 are CSC markers of MM and could be used as novel therapeutic targets.« less

Evolution of Cancer Stem-like Cells in Endocrine-Resistant Metastatic Breast Cancers Is Mediated by Stromal Microvesicles.

PubMed

Sansone, Pasquale; Berishaj, Marjan; Rajasekhar, Vinagolu K; Ceccarelli, Claudio; Chang, Qing; Strillacci, Antonio; Savini, Claudia; Shapiro, Lauren; Bowman, Robert L; Mastroleo, Chiara; De Carolis, Sabrina; Daly, Laura; Benito-Martin, Alberto; Perna, Fabiana; Fabbri, Nicola; Healey, John H; Spisni, Enzo; Cricca, Monica; Lyden, David; Bonafé, Massimiliano; Bromberg, Jacqueline

2017-04-15

The hypothesis that microvesicle-mediated miRNA transfer converts noncancer stem cells into cancer stem cells (CSC) leading to therapy resistance remains poorly investigated. Here we provide direct evidence supporting this hypothesis, by demonstrating how microvesicles derived from cancer-associated fibroblasts (CAF) transfer miR-221 to promote hormonal therapy resistance (HTR) in models of luminal breast cancer. We determined that CAF-derived microvesicles horizontally transferred miR-221 to tumor cells and, in combination with hormone therapy, activated an ER lo /Notch hi feed-forward loop responsible for the generation of CD133 hi CSCs. Importantly, microvesicles from patients with HTR metastatic disease expressed high levels of miR-221. We further determined that the IL6-pStat3 pathway promoted the biogenesis of onco-miR-221 hi CAF microvesicles and established stromal CSC niches in experimental and patient-derived breast cancer models. Coinjection of patient-derived CAFs from bone metastases led to de novo HTR tumors, which was reversed with IL6R blockade. Finally, we generated patient-derived xenograft (PDX) models from patient-derived HTR bone metastases and analyzed tumor cells, stroma, and microvesicles. Murine and human CAFs were enriched in HTR tumors expressing high levels of CD133 hi cells. Depletion of murine CAFs from PDX restored sensitivity to HT, with a concurrent reduction of CD133 hi CSCs. Conversely, in models of CD133 neg , HT-sensitive cancer cells, both murine and human CAFs promoted de novo HT resistance via the generation of CD133 hi CSCs that expressed low levels of estrogen receptor alpha. Overall, our results illuminate how microvesicle-mediated horizontal transfer of genetic material from host stromal cells to cancer cells triggers the evolution of therapy-resistant metastases, with potentially broad implications for their control. Cancer Res; 77(8); 1927-41. ©2017 AACR . ©2017 American Association for Cancer Research.

Automatic cell cloning assay for determining the clonogenic capacity of cancer and cancer stem-like cells.

PubMed

Fedr, Radek; Pernicová, Zuzana; Slabáková, Eva; Straková, Nicol; Bouchal, Jan; Grepl, Michal; Kozubík, Alois; Souček, Karel

2013-05-01

The clonogenic assay is a well-established in vitro method for testing the survival and proliferative capability of cells. It can be used to determine the cytotoxic effects of various treatments including chemotherapeutics and ionizing radiation. However, this approach can also characterize cells with different phenotypes and biological properties, such as stem cells or cancer stem cells. In this study, we implemented a faster and more precise method for assessing the cloning efficiency of cancer stem-like cells that were characterized and separated using a high-speed cell sorter. Cell plating onto a microplate using an automatic cell deposition unit was performed in a single-cell or dilution rank mode by the fluorescence-activated cell sorting method. We tested the new automatic cell-cloning assay (ACCA) on selected cancer cell lines and compared it with the manual approach. The obtained results were also compared with the results of the limiting dilution assay for different cell lines. We applied the ACCA to analyze the cloning capacity of different subpopulations of prostate and colon cancer cells based on the expression of the characteristic markers of stem (CD44 and CD133) and cancer stem cells (TROP-2, CD49f, and CD44). Our results revealed that the novel ACCA is a straightforward approach for determining the clonogenic capacity of cancer stem-like cells identified in both cell lines and patient samples. Copyright © 2013 International Society for Advancement of Cytometry.

A comparative study of the structural organization of spheres derived from the adult human subventricular zone and glioblastoma biopsies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vik-Mo, Einar Osland, E-mail: e.o.vik-mo@medisin.uio.no; Department of Neurosurgery, Oslo University Hospital, Oslo; Sandberg, Cecilie

2011-04-15

Sphere forming assays have been useful to enrich for stem like cells in a range of tumors. The robustness of this system contrasts the difficulties in defining a stem cell population based on cell surface markers. We have undertaken a study to describe the cellular and organizational composition of tumorspheres, directly comparing these to neurospheres derived from the adult human subventricular zone (SVZ). Primary cell cultures from brain tumors were found to contain variable fractions of cells positive for tumor stem cell markers (CD133 (2-93%)/SSEA1 (3-15%)/CXCR4 (1-72%)). All cultures produced tumors upon xenografting. Tumorspheres contained a heterogeneous population of cells,more » but were structurally organized with stem cell markers present at the core of spheres, with markers of more mature glial progenitors and astrocytes at more peripheral location. Ultrastructural studies showed that tumorspheres contained a higher fraction of electron dense cells in the core than the periphery (36% and 19%, respectively). Neurospheres also contained a heterogeneous cell population, but did not have an organization similar to tumorspheres. Although tumorspheres clearly display irregular and neoplastic cells, they establish an organized structure with an outward gradient of differentiation. We suggest that this organization is central in maintaining the tumor stem cell pool.« less

Autologous CD133+ bone marrow cells and bypass grafting for regeneration of ischaemic myocardium: the Cardio133 trial.

PubMed

Nasseri, Boris A; Ebell, Wolfram; Dandel, Michael; Kukucka, Marian; Gebker, Rolf; Doltra, Adelina; Knosalla, Christoph; Choi, Yeong-Hoon; Hetzer, Roland; Stamm, Christof

2014-05-14

Intra-myocardial transplantation of CD133(+) bone marrow stem cells (BMC) yielded promising results in clinical pilot trials. We now performed the double-blinded, randomized, placebo-controlled CARDIO133 trial to determine its impact on left ventricular (LV) function and clinical symptoms. Sixty patients with chronic ischaemic heart disease and impaired LV function (left ventricular ejection fraction, LVEF <35%) were randomized to undergo either coronary artery bypass grafting (CABG) and injection of CD133(+) BMC in the non-transmural, hypokinetic infarct border zone (CD133), or CABG and placebo injection (placebo). Pre-operative LVEF was 27 ± 6% in CD133 patients and 26 ± 6% in placebo patients. Outcome was assessed after 6 months, and the primary endpoint was LVEF measured by cardiac magnetic resonance imaging (MRI) at rest. The incidence of adverse events was similar in both groups. There was no difference in 6-min walking distance, Minnesota Living with Heart Failure score, or Canadian Cardiovascular Society (CCS) class between groups at follow-up, and New York Heart Association class improved more in the placebo group (P = 0.004). By cardiac MRI, LVEF at 6 months was 33 ± 8% in the placebo group and 31 ± 7% in verum patients (P = 0.3), with an average inter-group difference of -2.1% (95% CI -6.3 to 2.1). Systolic or diastolic LV dimensions at 6 months were not different, either. In the CD133 group, myocardial perfusion at rest recovered in more LV segments than in the placebo group (9 vs. 2%, P < 0.001). Scar mass decreased by 2.2 ± 5 g in CD133(+) patients (P = 0.05), but was unchanged in the placebo group (0.3 ± 4 g, P = 0.7; inter-group difference in change = 2 g (95% CI -1.1 to 5)). By speckle-tracking echocardiography, cell-treated patients showed a better recovery of regional wall motion when the target area was posterior. Although there may be some improvements in scar size and regional perfusion, intra-myocardial injection of CD133(+) BMC has no effect on global LV function and clinical symptoms. Improvements in regional myocardial function are only detectable in patients with posterior infarction, probably because the interventricular septum after anterior infarction is not accessible by trans-epicardial injection. This trial was registered at http://www.clinicaltrials.gov under NCT00462774. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.

Diversity of trends of viremia and T-cell markers in experimental acute feline immunodeficiency virus infection.

PubMed

Roche, Sylvain; El Garch, Hanane; Brunet, Sylvie; Poulet, Hervé; Iwaz, Jean; Ecochard, René; Vanhems, Philippe

2013-01-01

The early events of human immunodeficiency virus infection seem critical for progression toward disease and antiretroviral therapy initiation. We wanted to clarify some still unknown prognostic relationships between inoculum size and changes in various immunological and virological markers. Feline immunodeficiency virus infection could be a helpful model. Viremia and T-cell markers (number of CD4, CD8, CD8β(low)CD62L(neg) T-cells, CD4/CD8 ratio, and percentage of CD8β(low)CD62L(neg) cells among CD8 T-cells) were measured over 12 weeks in 102 cats infected with different feline immunodeficiency virus strains and doses. Viremia and T-cell markers trajectory groups were determined and the dose-response relationships between inoculum titres and trajectory groups investigated. Cats given the same inoculum showed different patterns of changes in viremia and T-cell markers. A statistically significant positive dose-response relationship was observed between inoculum titre and i) viremia trajectory-groups (r = 0.80, p<0.01), ii) CD8β(low)CD62L(neg) cell-fraction trajectory-groups (r = 0.56, p<0.01). Significant correlations were also found between viremia and the CD4/CD8 ratio and between seven out of ten T-cell markers. In cats, the infectious dose determines early kinetics of viremia and initial CD8+ T-cell activation. An expansion of the CD8β(low)CD62L(neg) T-cells might be an early predictor of progression toward disease. The same might be expected in humans but needs confirmation.

Expression of the myeloid-associated marker CD33 is not an exclusive factor for leukemic plasmacytoid dendritic cells.

PubMed

Garnache-Ottou, Francine; Chaperot, Laurence; Biichle, Sabeha; Ferrand, Christophe; Remy-Martin, Jean-Paul; Deconinck, Eric; de Tailly, Patrick Darodes; Bulabois, Bénédicte; Poulet, Jacqueline; Kuhlein, Emilienne; Jacob, Marie-Christine; Salaun, Véronique; Arock, Michel; Drenou, Bernard; Schillinger, Françoise; Seilles, Estelle; Tiberghien, Pierre; Bensa, Jean-Claude; Plumas, Joel; Saas, Philippe

2005-02-01

A new entity of acute leukemia coexpressing CD4(+)CD56(+) markers without any other lineage-specific markers has been identified recently as arising from lymphoid-related plasmacytoid dendritic cells (pDCs). In our laboratory, cells from a patient with such CD4(+)CD56(+) lineage-negative leukemia were unexpectedly found to also express the myeloid marker CD33. To confirm the diagnosis of pDC leukemia despite the CD33 expression, we demonstrated that the leukemic cells indeed exhibited pDC phenotypic and functional properties. In 7 of 8 other patients with CD4(+)CD56(+) pDC malignancies, we were able to confirm that the tumor cells expressed CD33 although with variable expression levels. CD33 expression was shown by flow cytometry, reverse transcriptase-polymerase chain reaction, and immunoblot analysis. Furthermore, CD33 monoclonal antibody stimulation of purified CD4(+)CD56(+) leukemic cells led to cytokine secretion, thus confirming the presence of a functional CD33 on these leukemic cells. Moreover, we found that circulating pDCs in healthy individuals also weakly express CD33. Overall, our results demonstrate that the expression of CD33 on CD4(+)CD56(+) lineage-negative cells should not exclude the diagnosis of pDC leukemia and underline that pDC-specific markers should be used at diagnosis for CD4(+)CD56(+) malignancies.

Targeting colon cancer stem cells using a new curcumin analogue, GO-Y030

PubMed Central

Lin, L; Liu, Y; Li, H; Li, P-K; Fuchs, J; Shibata, H; Iwabuchi, Y; Lin, J

2011-01-01

Background: Persistent activation of signal transducers and activators of transcription 3 (STAT3) is commonly detected in many types of cancer, including colon cancer. To date, whether STAT3 is activated and the effects of STAT3 inhibition by a newly developed curcumin analogue, GO-Y030, in colon cancer stem cells are still unknown. Methods: Flow cytometry was used to isolate colon cancer stem cells, which are characterised by both aldehyde dehydrogenase (ALDH)-positive and CD133-positive subpopulations (ALDH+/CD133+). The levels of STAT3 phosphorylation and the effects of STAT3 inhibition by a newly developed curcumin analogue, GO-Y030, that targets STAT3 in colon cancer stem cells were examined. Results: Our results observed that ALDH+/CD133+ colon cancer cells expressed higher levels of phosphorylated STAT3 than ALDH-negative/CD133-negative colon cancer cells, suggesting that STAT3 is activated in colon cancer stem cells. GO-Y030 and curcumin inhibited STAT3 phosphorylation, cell viability, tumoursphere formation in colon cancer stem cells. GO-Y030 also reduced STAT3 downstream target gene expression and induced apoptosis in colon cancer stem cells. Furthermore, GO-Y030 suppressed tumour growth of cancer stem cells from both SW480 and HCT-116 colon cancer cell lines in the mouse model. Conclusion: Our results indicate that STAT3 is a novel therapeutic target in colon cancer stem cells, and inhibition of activated STAT3 in cancer stem cells by GO-Y030 may offer an effective treatment for colorectal cancer. PMID:21694723

Targeting colon cancer stem cells using a new curcumin analogue, GO-Y030.

PubMed

Lin, L; Liu, Y; Li, H; Li, P-K; Fuchs, J; Shibata, H; Iwabuchi, Y; Lin, J

2011-07-12

Persistent activation of signal transducers and activators of transcription 3 (STAT3) is commonly detected in many types of cancer, including colon cancer. To date, whether STAT3 is activated and the effects of STAT3 inhibition by a newly developed curcumin analogue, GO-Y030, in colon cancer stem cells are still unknown. Flow cytometry was used to isolate colon cancer stem cells, which are characterised by both aldehyde dehydrogenase (ALDH)-positive and CD133-positive subpopulations (ALDH(+)/CD133(+)). The levels of STAT3 phosphorylation and the effects of STAT3 inhibition by a newly developed curcumin analogue, GO-Y030, that targets STAT3 in colon cancer stem cells were examined. Our results observed that ALDH(+)/CD133(+) colon cancer cells expressed higher levels of phosphorylated STAT3 than ALDH-negative/CD133-negative colon cancer cells, suggesting that STAT3 is activated in colon cancer stem cells. GO-Y030 and curcumin inhibited STAT3 phosphorylation, cell viability, tumoursphere formation in colon cancer stem cells. GO-Y030 also reduced STAT3 downstream target gene expression and induced apoptosis in colon cancer stem cells. Furthermore, GO-Y030 suppressed tumour growth of cancer stem cells from both SW480 and HCT-116 colon cancer cell lines in the mouse model. Our results indicate that STAT3 is a novel therapeutic target in colon cancer stem cells, and inhibition of activated STAT3 in cancer stem cells by GO-Y030 may offer an effective treatment for colorectal cancer.

Cancer stem cells CD133 inhibition and cytotoxicity of certain 3-phenylthiazolo[3,2-a]benzimidazoles: design, direct synthesis, crystal study and in vitro biological evaluation.

PubMed

Al-Ansary, Ghada H; Eldehna, Wagdy M; Ghabbour, Hazem A; Al-Rashood, Sara T A; Al-Rashood, Khalid A; Eladwy, Radwa A; Al-Dhfyan, Abdullah; Kabil, Maha M; Abdel-Aziz, Hatem A

2017-12-01

Cancer stem cells (CSCs) have been objects of intensive study since their identification in 1994. Adopting a structural rigidification approach, a novel series of 3-phenylthiazolo[3,2-a]benzimidazoles 4a-d was designed and synthesised, in an attempt to develop potent anticancer agent that can target the bulk of tumour cells and CSCs. The anti-proliferative activity of the synthesised compounds was evaluated against two cell lines, namely; colon cancer HT-29 and triple negative breast cancer MDA-MB-468 cell lines. Also, their inhibitory activity against the cell surface expression of CD133 was examined. In particular, compound 4b emerged as a promising hit molecule as it manifested good antineoplastic potency against both tested cell lines (IC 50  = 9 and 12 μM, respectively), beside its ability to inhibit the cell surface expression of CD133 by 50% suggesting a promising potential of effectively controlling the tumour by eradicating the tumour bulk and inhibiting the proliferation of the CSCs. Moreover, compounds 4a and 4c showed moderate activity against HT-29 (IC 50  = 21 and 29 μM, respectively) and MDA-MB-468 (IC 50  = 23 and 24 μM, respectively) cell lines, while they inhibited the CD133 expression by 14% and 48%, respectively. Finally, a single crystal X-ray diffraction was recorded for compound 4d.

Small G protein Rac GTPases regulate the maintenance of glioblastoma stem-like cells in vitro and in vivo.

PubMed

Lai, Yun-Ju; Tsai, Jui-Cheng; Tseng, Ying-Ting; Wu, Meng-Shih; Liu, Wen-Shan; Lam, Hoi-Ian; Yu, Jei-Hwa; Nozell, Susan E; Benveniste, Etty N

2017-03-14

Glioblastoma is the most common and aggressive malignant brain tumor in adults. The existence of glioblastoma stem cells (GSCs) or stem-like cells (stemloids) may account for its invasiveness and high recurrence. Rac proteins belong to the Rho small GTPase subfamily which regulates cell movement, proliferation, and survival. To investigate whether Rac proteins can serve as therapeutic targets for glioblastoma, especially for GSCs or stemloids, we examined the potential roles of Rac1, Rac2 and Rac3 on the properties of tumorspheres derived from glioblastoma cell lines. Tumorspheres are thought to be glioblastoma stem-like cells. We showed that Rac proteins promote the STAT3 and ERK activation and enhance cell proliferation and colony formation of glioblastoma stem-like cells. Knockdown of Rac proteins reduces the expression of GSC markers, such as CD133 and Sox2. The in vivo effects of Rac proteins in glioblastoma were further studied in zebrafish and in the mouse xenotransplantation model. Knocking-down Rac proteins abolished the angiogenesis effect induced by the injected tumorspheres in zebrafish model. In the CD133+-U373-tumorsphere xenotransplanted mouse model, suppression of Rac proteins decreased the incidence of tumor formation and inhibited the tumor growth. Moreover, knockdown of Rac proteins reduced the sphere forming efficiency of cells derived from these tumors. In conclusion, not only Rac1 but also Rac2 and 3 are important for glioblastoma tumorigenesis and can serve as the potential therapeutic targets against glioblastoma and its stem-like cells.

Small G protein Rac GTPases regulate the maintenance of glioblastoma stem-like cells in vitro and in vivo

PubMed Central

Lai, Yun-Ju; Tsai, Jui-Cheng; Tseng, Ying-Ting; Wu, Meng-Shih; Liu, Wen-Shan; Lam, Hoi-Ian; Yu, Jei-Hwa; Nozell, Susan E.; Benveniste, Etty N.

2017-01-01

Glioblastoma is the most common and aggressive malignant brain tumor in adults. The existence of glioblastoma stem cells (GSCs) or stem–like cells (stemloids) may account for its invasiveness and high recurrence. Rac proteins belong to the Rho small GTPase subfamily which regulates cell movement, proliferation, and survival. To investigate whether Rac proteins can serve as therapeutic targets for glioblastoma, especially for GSCs or stemloids, we examined the potential roles of Rac1, Rac2 and Rac3 on the properties of tumorspheres derived from glioblastoma cell lines. Tumorspheres are thought to be glioblastoma stem-like cells. We showed that Rac proteins promote the STAT3 and ERK activation and enhance cell proliferation and colony formation of glioblastoma stem-like cells. Knockdown of Rac proteins reduces the expression of GSC markers, such as CD133 and Sox2. The in vivo effects of Rac proteins in glioblastoma were further studied in zebrafish and in the mouse xenotransplantation model. Knocking-down Rac proteins abolished the angiogenesis effect induced by the injected tumorspheres in zebrafish model. In the CD133+-U373-tumorsphere xenotransplanted mouse model, suppression of Rac proteins decreased the incidence of tumor formation and inhibited the tumor growth. Moreover, knockdown of Rac proteins reduced the sphere forming efficiency of cells derived from these tumors. In conclusion, not only Rac1 but also Rac2 and 3 are important for glioblastoma tumorigenesis and can serve as the potential therapeutic targets against glioblastoma and its stem-like cells. PMID:28160553

Emphysema and soluble CD14 are associated with pulmonary nodules in HIV-infected patients: implications for lung cancer screening.

PubMed

Triplette, Matthew; Sigel, Keith M; Morris, Alison; Shahrir, Shahida; Wisnivesky, Juan P; Kong, Chung Y; Diaz, Phillip T; Petraglia, Alycia; Crothers, Kristina

2017-07-31

Lung cancer screening may benefit HIV-infected (HIV) smokers because of an elevated risk of lung cancer, but may have unique harms because of HIV-specific risk factors for false-positive screens. This study seeks to understand whether inflammatory biomarkers and markers of chronic lung disease are associated with noncalcified nodules at least 4 mm (NCN) in HIV compared with uninfected patients. This is a cohort study of Examinations of HIV-Associated Lung Emphysema (EXHALE), including 158 HIV and 133 HIV-uninfected participants. Participants underwent a laboratory assessment [including measurement of D-dimer, interleukin 6, and soluble CD14 (sCD14)], chest computed tomography (CT), and pulmonary function testing. We created multivariable logistic regression models to determine predictors of NCN in the participants stratified by HIV status, with attention to semiqualitative scoring of radiographic emphysema, markers of pulmonary function, and inflammatory biomarkers. Of the 291 participants, 69 had NCN on chest CT. As previously reported, there was no difference in prevalence of these nodules by HIV status. Emphysema and elevated sCD14 demonstrated an association with NCN in HIV participants independent of smoking status, CD4 cell count, HIV viral load, and pulmonary function. Emphysema and sCD14, a marker of immune activation, was associated with a higher prevalence of NCN on chest CT in HIV participants. Patients with chronic immune activation and emphysema may be at higher risk for both false-positive findings and incident lung cancer, thus screening in this group requires further study to understand the balance of benefits and harms.

HER2-specific T cells target primary glioblastoma stem cells and induce regression of autologous experimental tumors.

PubMed

Ahmed, Nabil; Salsman, Vita S; Kew, Yvonne; Shaffer, Donald; Powell, Suzanne; Zhang, Yi J; Grossman, Robert G; Heslop, Helen E; Gottschalk, Stephen

2010-01-15

Glioblastoma multiforme (GBM) is the most aggressive human primary brain tumor and is currently incurable. Immunotherapies have the potential to target GBM stem cells, which are resistant to conventional therapies. Human epidermal growth factor receptor 2 (HER2) is a validated immunotherapy target, and we determined if HER2-specific T cells can be generated from GBM patients that will target autologous HER2-positive GBMs and their CD133-positive stem cell compartment. HER2-specific T cells from 10 consecutive GBM patients were generated by transduction with a retroviral vector encoding a HER2-specific chimeric antigen receptor. The effector function of HER2-specific T cells against autologous GBM cells, including CD133-positive stem cells, was evaluated in vitro and in an orthotopic murine xenograft model. Stimulation of HER2-specific T cells with HER2-positive autologous GBM cells resulted in T-cell proliferation and secretion of IFN-gamma and interleukin-2 in a HER2-dependent manner. Patients' HER2-specific T cells killed CD133-positive and CD133-negative cells derived from primary HER2-positive GBMs, whereas HER2-negative tumor cells were not killed. Injection of HER2-specific T cells induced sustained regression of autologous GBM xenografts established in the brain of severe combined immunodeficient mice. Gene transfer allows the reliable generation of HER2-specific T cells from GBM patients, which have potent antitumor activity against autologous HER2-positive tumors including their putative stem cells. Hence, the adoptive transfer of HER2-redirected T cells may be a promising immunotherapeutic approach for GBM.

Verification of TREX1 as a promising indicator of judging the prognosis of osteosarcoma.

PubMed

Feng, Jinyi; Lan, Ruilong; Cai, Guanxiong; Lin, Jinluan; Wang, Xinwen; Lin, Jianhua; Han, Deping

2016-11-24

The study aimed to explore the correlation between the expression of TREX1 and the metastasis and the survival time of patients with osteosarcoma as well as biological characteristics of osteosarcoma cells for the prognosis judgment of osteosarcoma. The correlation between the expression of TREX1 protein and the occurrence of pulmonary metastasis in 45 cases of osteosarcoma was analyzed. The CD133 + and CD133 - cell subsets of osteosarcoma stem cells were sorted by the flow cytometry. The tumorsphere culture, clone formation, growth curve, osteogenic and adipogenic differentiation, tumor-formation ability in nude mice, sensitivity of chemotherapeutic drugs, and other cytobiology behaviors were compared between the cell subsets in two groups; the expressions of stem cell-related genes Nanog and Oct4 were compared; The expressions of TREX1 protein and mRNA were compared between the cell subsets in two groups. The data was statistically analyzed. The measurement data between the two groups were compared using t test. The count data between the two groups were compared using χ 2 test and Kaplan-Meier survival analysis. A P value <0.05 indicated that the difference was statistically significant. The expression of TREX1 protein in patients with osteosarcoma in the metastasis group was significantly lower than that in the non-metastasis group. The difference was statistically significant (P < 0.05). Up to the last follow-up visit, the former average survival time was significantly lower than that of the latter, and the difference was statistically significant (P < 0.05). The expression of TREX1 in human osteosarcoma CD133 + cell subsets was significantly lower than that in CD133 - cell subsets. Stemness-related genes Nanog and Oct4 were highly expressed in human osteosarcoma CD133 + cell subsets with lower expression of TREX1; the biological characteristics identification experiment showed that human CD133 + cell subsets with low TREX1 expression could form tumorspheres, the number of colony forming was more, the cell proliferation ability was strong, the osteogenic and adipogenic differentiation potential was big, the tumor-forming ability in nude mice was strong, and the sensibility of chemotherapeutics drugs on cisplatin was low. The expression of TREX1 may be related to metastasis in patients with osteosarcoma. The expression of TREX1 was closely related to the cytobiology characteristics of osteosarcoma stem cell. TREX1 can play an important role in the occurrence and development processes. And, TREX1 is expected to become an effective new index for the evaluation of the prognosis.

A1E reduces stemness and self-renewal in HPV 16-positive cervical cancer stem cells.

PubMed

Kwon, Taeho; Bak, Yesol; Ham, Sun-Young; Yu, Dae-Yeul; Yoon, Do-Young

2016-02-02

Cervical cancer is the second most common cancer in females. Recent reports have revealed the critical role of cervical cancer stem cells (CSCs) in tumorigenicity and metastasis. Previously we demonstrated that A1E exerts an anti-proliferative action, which inhibits the growth of cervical cancer cells. A1E is composed of 11 oriental medicinal herbs. Cervical cancer cell culture, wund healing and invasion assay, flow cytometry, sheroid formation assay, and wstern blot assays were performed in HPV 16-positive SiHa cell and HPV 16-negative C33A cells. A1E targets the E6 and E7 oncogenes; thus, A1E significantly inhibited proliferation of human papilloma virus (HPV) 16-positive SiHa cells, it did not inhibit the proliferation of HPV-negative C33A cells. Accordingly, we investigated whether A1E can regulate epithelial-to-mesenchymal transition (EMT), CSC self-renewal, and stemness-related gene expression in cervical cancer cells. Down rgulation of cell migration, cell invasion, and EMT was observed in A1E-treated SiHa cells. Specifically, A1E-treated SiHa cells showed significant decreases in OCT-3/4 and Sox2 expression levels and in sphere formation. Moreover, CSCs makers ALDH+ and ALDH, CD133 double positive cell were significantly decreased in A1E-treated SiHa cells. However, A1E treatment did not down regulate ALDH+ expression and the number of ALDH/CD133 double positive cells in C33A cells. Taken together, A1E can inhibit CSCs and reduce the expression of stemness markers. Treating CSCs with A1E may be a potential therapy for cervical cancer.

Prostate Cancer Stem Cell-Targeted Efficacy of a New-Generation Taxoid, SBT-1214 and Novel Polyenolic Zinc-Binding Curcuminoid, CMC2.24

PubMed Central

Botchkina, Galina I.; Zuniga, Edison S.; Rowehl, Rebecca H.; Park, Rosa; Bhalla, Rahuldev; Bialkowska, Agnieszka B.; Johnson, Francis; Golub, Lorne M.; Zhang, Yu; Ojima, Iwao; Shroyer, Kenneth R.

2013-01-01

Background Prostate cancer is the second leading cause of cancer death among men. Multiple evidence suggests that a population of tumor-initiating, or cancer stem cells (CSCs) is responsible for cancer development and exceptional drug resistance, representing a highly important therapeutic target. The present study evaluated CSC-specific alterations induced by new-generation taxoid SBT-1214 and a novel polyenolic zinc-binding curcuminoid, CMC2.24, in prostate CSCs. Principal Findings The CD133high/CD44high phenotype was isolated from spontaneously immortalized patient-derived PPT2 cells and highly metastatic PC3MM2 cells. Weekly treatment of the NOD/SCID mice bearing PPT2- and PC3MM3-induced tumors with the SBT-1214 led to dramatic suppression of tumor growth. Four of six PPT2 and 3 of 6 PC3MM2 tumors have shown the absence of viable cells in residual tumors. In vitro, SBT-1214 (100nM-1µM; for 72 hr) induced about 60% cell death in CD133high/CD44+/high cells cultured on collagen I in stem cell medium (in contrast, the same doses of paclitaxel increased proliferation of these cells). The cytotoxic effects were increased when SBT-1214 was combined with the CMC2.24. A stem cell-specific PCR array assay revealed that this drug combination mediated massive inhibition of multiple constitutively up-regulated stem cell-related genes, including key pluripotency transcription factors. Importantly, this drug combination induced expression of p21 and p53, which were absent in CD133high/CD44high cells. Viable cells that survived this treatment regimen were no longer able to induce secondary spheroids, exhibited significant morphological abnormalities and died in 2-5 days. Conclusions We report here that the SBT-1214 alone, or in combination with CMC2.24, possesses significant activity against prostate CD133high/CD44+/high tumor-initiating cells. This drug combination efficiently inhibits expression of the majority of stem cell-related genes and pluripotency transcription factors. In addition, it induces a previously absent expression of p21 and p53 (“gene wake-up”), which can potentially reverse drug resistance by increasing sensitivity to anti-cancer drugs. PMID:24086245

Ciprofloxacin mediates cancer stem cell phenotypes in lung cancer cells through caveolin-1-dependent mechanism.

PubMed

Phiboonchaiyanan, Preeyaporn Plaimee; Kiratipaiboon, Chayanin; Chanvorachote, Pithi

2016-04-25

Cancer stem cells (CSCs), a subpopulation of cancer cells with high aggressive behaviors, have been identified in many types of cancer including lung cancer as one of the key mediators driving cancer progression and metastasis. Here, we have reported for the first time that ciprofloxacin (CIP), a widely used anti-microbial drug, has a potentiating effect on CSC-like features in human non-small cell lung cancer (NSCLC) cells. CIP treatment promoted CSC-like phenotypes, including enhanced anchorage-independent growth and spheroid formation. The known lung CSC markers: CD133, CD44, ABCG2 and ALDH1A1 were found to be significantly increased, while the factors involving in epithelial to mesenchymal transition (EMT): Slug and Snail, were depleted. Also, self-renewal transcription factors Oct-4 and Nanog were found to be up-regulated in CIP-treated cells. The treatment of CIP on CSC-rich populations obtained from secondary spheroids resulted in the further increase of CSC markers. In addition, we have proven that the mechanistic insight of the CIP induced stemness is through Caveolin-1 (Cav-1)-dependent mechanism. The specific suppression of Cav-1 by stably transfected Cav-1 shRNA plasmid dramatically reduced the effect of CIP on CSC markers as well as the CIP-induced spheroid formation ability. Cav-1 was shown to activate protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) pathways in CSC-rich population; however, such an effect was rarely found in the main lung cancer cells population. These findings reveal a novel effect of CIP in positively regulating CSCs in lung cancer cells via the activation of Cav-1, Akt and ERK, and may provoke the awareness of appropriate therapeutic strategy in cancer patients. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

СD44+/CD24- markers of cancer stem cells in patients with breast cancer of different molecular subtypes.

PubMed

Chekhun, S V; Zadvorny, T V; Tymovska, Yu O; Anikusko, M F; Novak, O E; Polishchuk, L Z

2015-03-01

To determine frequency of tumors with immunohistochemical markers of cancer stem cells (CSC) CD44+/CD24- in patients with breast cancer (BC) of different molecular subtype and to evaluate their prognostic value. Surgical material of 132 patients with BC stage I-II, age from 23 to 75 years, mean age - 50.2 ± 3.1 years was studied. Clinical, immunohistochemical (expression CD44+/CD24-), morphological, statistical. BC is characterized by heterogeneity of molecular subtypes and expression of markers (CD44+/CD24-). Immunohistochemical study of expression of CSC markers in surgical material has detected their expression in 34 (25.4%) patients with BC of different molecular subtypes. The highest frequency of cells with expression of CSC marker was observed in patients with basal molecular subtype (44.8% patients). Most of BC patients with phenotype CD44+/CD24 had stage I of tumor process (34.3%). Statistical processing of data has showen that Yule colligation coefficient equaled 0.28 (р > 0.05) that argues poor correlation between stage of tumor process and number of tumors with positive expression of CSC markers. Statistical processing of data has showen high correlation between presence of cells with expression of CSC markers and metastases of BC in regional lymph nodes (Yule colligation coefficient equals 0.943; р < 0.5). Difference in overall survival of patients with BC of basal molecular subtype depending on expression of CSC CD44+/CD24- markers was detected. Survival of patients with basal BC was reliably higher at lack in tumors of cells with CSC markers CD44+/CD24- and, correspondingly, lower at presence of such cells (р < 0.05). In patients with BC of luminal (A and B), HER-2-positive subtypes, significant change in survival of patients depending on expression of CSC markers was not determined (р > 0.05). Significance of tumor cells with markers CD44+/CD24- within the limits of molecular subtype of BC may be additional criterion for advanced biological characteristic of BC, and in patients with BC of basal molecular subtype - for predictive evaluation of individual potential of tumor to aggressive clinical course.

Universal monitoring of minimal residual disease in acute myeloid leukemia.

PubMed

Coustan-Smith, Elaine; Song, Guangchun; Shurtleff, Sheila; Yeoh, Allen Eng-Juh; Chng, Wee Joo; Chen, Siew Peng; Rubnitz, Jeffrey E; Pui, Ching-Hon; Downing, James R; Campana, Dario

2018-05-03

Optimal management of acute myeloid leukemia (AML) requires monitoring of treatment response, but minimal residual disease (MRD) may escape detection. We sought to identify distinctive features of AML cells for universal MRD monitoring. We compared genome-wide gene expression of AML cells from 157 patients with that of normal myeloblasts. Markers encoded by aberrantly expressed genes, including some previously associated with leukemia stem cells, were studied by flow cytometry in 240 patients with AML and in nonleukemic myeloblasts from 63 bone marrow samples. Twenty-two (CD9, CD18, CD25, CD32, CD44, CD47, CD52, CD54, CD59, CD64, CD68, CD86, CD93, CD96, CD97, CD99, CD123, CD200, CD300a/c, CD366, CD371, and CX3CR1) markers were aberrantly expressed in AML. Leukemia-associated profiles defined by these markers extended to immature CD34+CD38- AML cells; expression remained stable during treatment. The markers yielded MRD measurements matching those of standard methods in 208 samples from 52 patients undergoing chemotherapy and revealed otherwise undetectable MRD. They allowed MRD monitoring in 129 consecutive patients, yielding prognostically significant results. Using a machine-learning algorithm to reduce high-dimensional data sets to 2-dimensional data, the markers allowed a clear visualization of MRD and could detect 1 leukemic cell among more than 100,000 normal cells. The markers uncovered in this study allow universal and sensitive monitoring of MRD in AML. In combination with contemporary analytical tools, the markers improve the discrimination between leukemic and normal cells, thus facilitating data interpretation and, hence, the reliability of MRD results. National Cancer Institute (CA60419 and CA21765); American Lebanese Syrian Associated Charities; National Medical Research Council of Singapore (1299/2011); Viva Foundation for Children with Cancer, Children's Cancer Foundation, Tote Board & Turf Club, and Lee Foundation of Singapore.

Interplay of macrophages and T cells in the lung vasculature.

PubMed

Gerasimovskaya, Evgenia; Kratzer, Adelheid; Sidiakova, Asya; Salys, Jonas; Zamora, Martin; Taraseviciene-Stewart, Laimute

2012-05-15

In severe pulmonary arterial hypertension (PAH), vascular lesions are composed of phenotypically altered vascular and inflammatory cells that form clusters or tumorlets. Because macrophages are found in increased numbers in intravascular and perivascular space in human PAH, here we address the question whether macrophages play a role in pulmonary vascular remodeling and whether accumulation of macrophages in the lung vasculature could be compromised by the immune system. We used the mouse macrophage cell line RAW 264.7 because these cells are resistant to apoptosis, have high proliferative capacity, and resemble cells in the plexiform lesions that tend to pile up instead of maintaining a monolayer. Cells were characterized by immunocytochemistry with cell surface markers (Lycopersicon Esculentum Lectin, CD117, CD133, FVIII, CD31, VEGFR-2, and S100). Activated, but not quiescent, T cells were able to suppress RAW 264.7 cell proliferative and migration activity in vitro. The carboxyfluorescein diacetate-labeled RAW 264.7 cells were injected into the naïve Sprague Dawley (SD) rat and athymic nude rat. Twelve days later, cells were found in the lung vasculature of athymic nude rats that lack functional T cells, contributing to vascular remodeling. No labeled RAW 264.7 cells were detected in the lungs of immune-competent SD rats. Our data demonstrate that T cells can inhibit in vitro migration and in vivo accumulation of macrophage-like cells.

Benzene exposure is associated with cardiovascular disease risk.

PubMed

Abplanalp, Wesley; DeJarnett, Natasha; Riggs, Daniel W; Conklin, Daniel J; McCracken, James P; Srivastava, Sanjay; Xie, Zhengzhi; Rai, Shesh; Bhatnagar, Aruni; O'Toole, Timothy E

2017-01-01

Benzene is a ubiquitous, volatile pollutant present at high concentrations in toxins (e.g. tobacco smoke) known to increase cardiovascular disease (CVD) risk. Despite its prevalence, the cardiovascular effects of benzene have rarely been studied. Hence, we examined whether exposure to benzene is associated with increased CVD risk. The effects of benzene exposure in mice were assessed by direct inhalation, while the effects of benzene exposure in humans was assessed in 210 individuals with mild to high CVD risk by measuring urinary levels of the benzene metabolite trans,trans-muconic acid (t,t-MA). Generalized linear models were used to assess the association between benzene exposure and CVD risk. Mice inhaling volatile benzene had significantly reduced levels of circulating angiogenic cells (Flk-1+/Sca-1+) as well as an increased levels of plasma low-density lipoprotein (LDL) compared with control mice breathing filtered air. In the human cohort, urinary levels of t,t-MA were inversely associated several populations of circulating angiogenic cells (CD31+/34+/45+, CD31+/34+/45+/AC133-, CD34+/45+/AC133+). Although t,t-MA was not associated with plasma markers of inflammation or thrombosis, t,t-MA levels were higher in smokers and in individuals with dyslipidemia. In smokers, t,t-MA levels were positively associated with urinary metabolites of nicotine (cotinine) and acrolein (3-hydroxymercapturic acid). Levels of t,t-MA were also associated with CVD risk as assessed using the Framingham Risk Score and this association was independent of smoking. Thus, benzene exposure is associated with increased CVD risk and deficits in circulating angiogenic cells in both smokers and non-smokers.

Identification and validation of multiple cell surface markers of clinical-grade adipose-derived mesenchymal stromal cells as novel release criteria for good manufacturing practice-compliant production.

PubMed

Camilleri, Emily T; Gustafson, Michael P; Dudakovic, Amel; Riester, Scott M; Garces, Catalina Galeano; Paradise, Christopher R; Takai, Hideki; Karperien, Marcel; Cool, Simon; Sampen, Hee-Jeong Im; Larson, A Noelle; Qu, Wenchun; Smith, Jay; Dietz, Allan B; van Wijnen, Andre J

2016-08-11

Clinical translation of mesenchymal stromal cells (MSCs) necessitates basic characterization of the cell product since variability in biological source and processing of MSCs may impact therapeutic outcomes. Although expression of classical cell surface markers (e.g., CD90, CD73, CD105, and CD44) is used to define MSCs, identification of functionally relevant cell surface markers would provide more robust release criteria and options for quality control. In addition, cell surface expression may distinguish between MSCs from different sources, including bone marrow-derived MSCs and clinical-grade adipose-derived MSCs (AMSCs) grown in human platelet lysate (hPL). In this work we utilized quantitative PCR, flow cytometry, and RNA-sequencing to characterize AMSCs grown in hPL and validated non-classical markers in 15 clinical-grade donors. We characterized the surface marker transcriptome of AMSCs, validated the expression of classical markers, and identified nine non-classical markers (i.e., CD36, CD163, CD271, CD200, CD273, CD274, CD146, CD248, and CD140B) that may potentially discriminate AMSCs from other cell types. More importantly, these markers exhibit variability in cell surface expression among different cell isolates from a diverse cohort of donors, including freshly prepared, previously frozen, or proliferative state AMSCs and may be informative when manufacturing cells. Our study establishes that clinical-grade AMSCs expanded in hPL represent a homogeneous cell culture population according to classical markers,. Additionally, we validated new biomarkers for further AMSC characterization that may provide novel information guiding the development of new release criteria. Use of Autologous Bone Marrow Aspirate Concentrate in Painful Knee Osteoarthritis (BMAC): Clinicaltrials.gov NCT01931007 . Registered August 26, 2013. MSC for Occlusive Disease of the Kidney: Clinicaltrials.gov NCT01840540 . Registered April 23, 2013. Mesenchymal Stem Cell Therapy in Multiple System Atrophy: Clinicaltrials.gov NCT02315027 . Registered October 31, 2014. Efficacy and Safety of Adult Human Mesenchymal Stem Cells to Treat Steroid Refractory Acute Graft Versus Host Disease. Clinicaltrials.gov NCT00366145 . Registered August 17, 2006. A Dose-escalation Safety Trial for Intrathecal Autologous Mesenchymal Stem Cell Therapy in Amyotrophic Lateral Sclerosis. Clinicaltrials.gov NCT01609283 . Registered May 18, 2012.

CD133(+)/CD44(+)/Oct4(+)/Nestin(+) stem-like cells isolated from Panc-1 cell line may contribute to multi-resistance and metastasis of pancreatic cancer.

PubMed

Wang, Dongqing; Zhu, Haitao; Zhu, Ying; Liu, Yanfang; Shen, Huiling; Yin, Ruigen; Zhang, Zhijian; Su, Zhaoliang

2013-05-01

Pancreatic cancer is an aggressive malignant disease. Owing to the lack of early symptoms, accompanied by extensive metastasis and high resistance to chemotherapy, pancreatic adenocarcinoma becomes the fourth leading cause of cancer-related deaths. In this study, we identified a subpopulation of cells isolated from the Panc-1 cell line and named pancreatic cancer stem-like cells. These Panc-1 stem-like cells expressed high levels of CD133/CD44/Oct4/Nestin. Compared to Panc-1 cells, Panc-1 stem-like cells were resistant to gemcitabine and expressed high levels of MDR1; furthermore, Panc-1 stem-like cells have high anti-apoptotic, but weak proliferative potential. These results indicated that Panc-1 stem-like cells, as a novel group, may be a potential major cause of pancreatic cancer multidrug resistance and extensive metastasis. Copyright © 2012 Elsevier GmbH. All rights reserved.

A combination of valproic acid sodium salt, CHIR99021, E-616452, tranylcypromine, and 3-Deazaneplanocin A causes stem cell-like characteristics in cancer cells.

PubMed

Sha, Shuang; Zhai, Yuanfen; Lin, Chengzhao; Wang, Heyong; Chang, Qing; Song, Shuang; Ren, Mingqiang; Liu, Gentao

2017-08-08

Many studies are based on the hypothesis that recurrence and drug resistance in lung carcinoma are due to a subpopulation of cancer stem-like cells (CSLCs) in solid tumors. Therefore it is crucial to screen for and recognize lung CSLCs. In this study, we stimulated non-small cell lung cancer (NSCLC) A549 cells to display stem cell-like characteristics using a combination of five small molecule compounds. The putative A549 stem cells activated an important CSLC marker, CD133 protein, as well multiple CSLC-related genes including ATP-binding cassette transporter G2 (ABCG2), C-X-C chemokine receptor type 4 (CXCR4), NESTIN, and BMI1. The A549 stem-like cells displayed resistance to the chemotherapeutic drugs etoposide and cisplatin, epithelial-to-mesenchymal transition properties, and increased protein expression levels of NOTCH1 and Hes Family bHLH Transcription Factor 1 (HES1). When A549 cells were pretreated with a NOTCH signaling pathway inhibitor before compound induction, expression of the NOTCH1 target gene HES1 was reduced. This demonstrated that the NOTCH signaling pathway in the putative A549 stem-like cells had been activated. Together, the results of our study showed that a combination of five small molecule agents could transform A549 cells into putative stem-like cells, and that these compounds could also elevate CD133 and ABCG2 protein expression levels in H460 cells. This study provides a convenient method for obtaining lung CSLCs, which may be an effective strategy for developing lung carcinoma treatments.

Gliosarcomas arising from the pineal gland region: uncommon localization and rare tumors.

PubMed

Sugita, Yasuo; Terasaki, Mizuhiko; Tanigawa, Ken; Ohshima, Koichi; Morioka, Motohiro; Higaki, Koichi; Nakagawa, Setsuko; Shimokawa, Shoko; Nakashima, Susumu

2016-02-01

Gliosarcomas are a variant of glioblastomas and present a biphasic pattern, with coexisting glial and mesenchymal components. In this study, two unusual cases are presented. Case 1 is a 52-year-old woman with a headache and memory disturbance for a month. Case 2 is an 18-year-old man with a headache lasting two weeks. In both cases, an MRI revealed enhancing T1-low to iso, T2-iso to high intensity lesions in the pineal gland region. Histologically, in case 1, the tumor showed spindle cell proliferation with disorganized fascicles and cellular pleomorphism. Tumor cells variously exhibited oncocytic transformation. Immunohistochemically, most of the spindle tumor cells were positive for myoglobin and desmin. Some of the tumor cells were positive for GFAP and S-100 protein. On the other hand, all tumor cells were positive for CD133, Musashi1, and SOX-2 which are the markers of neural stem cells. In case 2, the tumor showed monotonous proliferation of short spindle cells with disorganized fascicles and cellular atypism. The morphological distinction between glial and mesenchymal components was not apparent. Immunohistochemically, most of the spindle tumor cells were positive for desmin. Glial tumor cells that were dispersed within the sarcoma as single cells were positive for GFAP. In addition, all tumor cells were positive for CD133, Musashi1 and SOX-2. Based on these microscopic appearances, and immunohistochemical findings, these cases were diagnosed as gliosarcomas arising from the pineal gland region. These results also indicated that pluripotential cancer stem cells differentiated into glial and muscle cell lines at the time of tumor growth. In a survey of previous publications on gliosarcoma arising from the pineal gland, these cases are the second and third reports found in English scientific writings. © 2015 Japanese Society of Neuropathology.

Human cancer stem cells are a target for cancer prevention using (-)-epigallocatechin gallate.

PubMed

Fujiki, Hirota; Sueoka, Eisaburo; Rawangkan, Anchalee; Suganuma, Masami

2017-12-01

Our previous experiments show that the main constituent of green-tea catechins, (-)-epigallocatechin gallate (EGCG), completely prevents tumor promotion on mouse skin initiated with 7,12-dimethylbenz(a)anthracene followed by okadaic acid and that EGCG and green tea extract prevent cancer development in a wide range of target organs in rodents. Therefore, we focused our attention on human cancer stem cells (CSCs) as targets of cancer prevention and treatment with EGCG. The numerous reports concerning anticancer activity of EGCG against human CSCs enriched from cancer cell lines were gathered from a search of PubMed, and we hope our review of the literatures will provide a broad selection for the effects of EGCG on various human CSCs. Based on our theoretical study, we discuss the findings as follows: (1) Compared with the parental cells, human CSCs express increased levels of the stemness markers Nanog, Oct4, Sox2, CD44, CD133, as well as the EMT markers, Twist, Snail, vimentin, and also aldehyde dehydrogenase. They showed decreased levels of E-cadherin and cyclin D1. (2) EGCG inhibits the transcription and translation of genes encoding stemness markers, indicating that EGCG generally inhibits the self-renewal of CSCs. (3) EGCG inhibits the expression of the epithelial-mesenchymal transition phenotypes of human CSCs. (4) The inhibition of EGCG of the stemness of CSCs was weaker compared with parental cells. (5) The weak inhibitory activity of EGCG increased synergistically in combination with anticancer drugs. Green tea prevents human cancer, and the combination of EGCG and anticancer drugs confers cancer treatment with tissue-agnostic efficacy.

Enrichment and characterization of cancer stem cells from a human non-small cell lung cancer cell line.

PubMed

Zhao, Changhong; Setrerrahmane, Sarra; Xu, Hanmei

2015-10-01

Tumor cells from the same origin comprise different cell populations. Among them, cancer stem cells (CSCs) have higher tumorigenicity. It is necessary to enrich CSCs to determine an effective way to suppress and eliminate them. In the present study, using the non-adhesive culture system, tumor spheres were successfully generated from human A549 non-small cell lung cancer (NSCLC) cell line within 2 weeks. Compared to A549 adherent cells, sphere cells had a higher self-renewal ability and increased resistance to cytotoxic drugs. Sphere cells were more invasive and expressed stem cell markers including octamer‑binding transcription factor 4 (Oct4) and sex-determining region Y-box 2 (Sox2) at high levels. CD133, a disputed marker of lung CSCs, was also upregulated. Tumor sphere cells showed higher tumorigenic ability in vivo, indicating that more CSCs were enriched in the sphere cells. More blood vessels were formed in the tumor generated by sphere cells suggesting the interaction between CSCs and blood vessel. A reliable model of enriching CSCs from the human A549 NSCLC cell line was established that was simple and cost-effective compared to other methods.

CD45RA, a specific marker for leukaemia stem cell sub-populations in acute myeloid leukaemia.

PubMed

Kersten, Bas; Valkering, Matthijs; Wouters, Rolf; van Amerongen, Rosa; Hanekamp, Diana; Kwidama, Zinia; Valk, Peter; Ossenkoppele, Gert; Zeijlemaker, Wendelien; Kaspers, Gertjan; Cloos, Jacqueline; Schuurhuis, Gerrit J

2016-04-01

Chemotherapy resistant leukaemic stem cells (LSC) are thought to be responsible for relapses after therapy in acute myeloid leukaemia (AML). Flow cytometry can discriminate CD34(+) CD38(-) LSC and normal haematopoietic stem cells (HSC) by using aberrant expression of markers and scatter properties. However, not all LSC can be identified using currently available markers, so new markers are needed. CD45RA is expressed on leukaemic cells in the majority of AML patients. We investigated the potency of CD45RA to specifically identify LSC and HSC and improve LSC quantification. Compared to our best other markers (CLL-1, also termed CLEC12A, CD33 and CD123), CD45RA was the most reliable marker. Patients with high percentages (>90%) of CD45RA on CD34(+) CD38(-) LSC have 1·69-fold higher scatter values compared to HSC (P < 0·001), indicating a more mature CD34(+) CD38(-) phenotype. Patients with low (<10%) or intermediate (10-90%) CD45RA expression on LSC showed no significant differences to HSC (1·12- and 1·15-fold higher, P = 0·31 and P = 0·44, respectively). CD45RA-positive LSC tended to represent more favourable cytogenetic/molecular markers. In conclusion, CD45RA contributes to more accurate LSC detection and is recommended for inclusion in stem cell tracking panels. CD45RA may contribute to define new LSC-specific therapies and to monitor effects of anti-LSC treatment. © 2016 John Wiley & Sons Ltd.

Does the liposuction method influence the phenotypic characteristic of human adipose-derived stem cells?

PubMed

Bajek, Anna; Gurtowska, Natalia; Gackowska, Lidia; Kubiszewska, Izabela; Bodnar, Magdalena; Marszałek, Andrzej; Januszewski, Rafał; Michalkiewicz, Jacek; Drewa, Tomasz

2015-05-14

Adipose-derived stem cells (ASCs) possess a high differentiation and proliferation potential. However, the phenotypic characterization of ASCs is still difficult. Until now, there is no extensive analysis of ASCs markers depending on different liposuction methods. Therefore, the aim of the present study was to analyse 242 surface markers and determine the differences in the phenotypic pattern between ASCs obtained during mechanical and ultrasound-assisted liposuction. ASCs were isolated from healthy donors, due to mechanical and ultrasound-assisted liposuction and cultured in standard medium to the second passage. Differentiation potential and markers expression was evaluated to confirm the mesenchymal nature of cells. Then, the BD LyoplateTM Human Cell Surface Marker Screening Panel was used. Results shown that both population of ASCs are characterized by high expression of markers specific for ASCs: cluster of differentiation (CD)9, CD10, CD34, CD44, CD49d, CD54, CD55, CD59, CD71 and low expression of CD11a, CD11c and CD144. Moreover, we have noticed significant differences in antigen expression in 58 markers from the 242 studied. Presented study shows for the first time that different liposuction methods are not a significant factor which can influence the expression of human ASCs surface markers. © 2015 The Authors.

Identification of a unique hepatocellular carcinoma line, Li-7, with CD13(+) cancer stem cells hierarchy and population change upon its differentiation during culture and effects of sorafenib.

PubMed

Yamada, Takeshi; Abei, Masato; Danjoh, Inaho; Shirota, Ryoko; Yamashita, Taro; Hyodo, Ichinosuke; Nakamura, Yukio

2015-04-11

Cancer stem cell (CSC) research has highlighted the necessity of developing drugs targeting CSCs. We investigated a hepatocellular carcinoma (HCC) cell line that not only has CSC hierarchy but also shows phenotypic changes (population changes) upon differentiation of CSC during culture and can be used for screening drugs targeting CSC. Based on a hypothesis that the CSC proportion should decrease upon its differentiation into progenitors (population change), we tested HCC cell lines (HuH-7, Li-7, PLC/PRF/5, HLF, HLE) before and after 2 months culture for several markers (CD13, EpCAM, CD133, CD44, CD90, CD24, CD166). Tumorigenicity was tested using nude mice. To evaluate the CSC hierarchy, we investigated reconstructivity, proliferation, ALDH activity, spheroid formation, chemosensitivity and microarray analysis of the cell populations sorted by FACS. Only Li-7 cells showed a population change during culture: the proportion of CD13 positive cells decreased, while that of CD166 positive cells increased. The high tumorigenicity of the Li-7 was lost after the population change. CD13(+)/CD166(-) cells showed slow growth and reconstructed the bulk Li-7 populations composed of CD13(+)/CD166(-), CD13(-)/CD166(-) and CD13(-)/CD166(+) fractions, whereas CD13(-)/CD166(+) cells showed rapid growth but could not reproduce any other population. CD13(+)/CD166(-) cells showed high ALDH activity, spheroid forming ability and resistance to 5-fluorouracil. Microarray analysis demonstrated higher expression of stemness-related genes in CD166(-) than CD166(+) fraction. These results indicated a hierarchy in Li-7 cells, in which CD13(+)/CD166(-) and CD13(-)/CD166(+) cells serve as slow growing CSCs and rapid growing progenitors, respectively. Sorafenib selectively targeted the CD166(-) fraction, including CD13(+) CSCs, which exhibited higher mRNA expression for FGF3 and FGF4, candidate biomarkers for sorafenib. 5-fluorouracil followed by sorafenib inhibited the growth of bulk Li-7 cells more effectively than the reverse sequence or either alone. We identified a unique HCC line, Li-7, which not only shows heterogeneity for a CD13(+) CSC hierarchy, but also undergoes a "population change" upon CSC differentiation. Sorafenib targeted the CSC in vitro, supporting the use of this model for screening drugs targeting the CSC. This type of "heterogeneous, unstable" cell line may prove more useful in the CSC era than conventional "homogeneous, stable" cell lines.

Dietary intake of phytoestrogen is associated with increased circulating endothelial progenitor cells in patients with cardiovascular disease.

PubMed

Chan, Yap-Hang; Lam, Tai-Hing; Lau, Kui-Kai; Yiu, Kai-Hang; Siu, Chung-Wah; Li, Sheung-Wai; Chan, Hiu-Ting; Tam, Sidney; Lau, Chu-Pak; Tse, Hung-Fat

2011-06-01

Endogenous estrogen is known to positively influence the level and functionality of endothelial progenitor cells (EPC). However, the effect of phytoestrogen on EPC is unknown. Isoflavone is a major component of phytoestrogen. This study aims to investigate if the intake of isoflavone has any impact on the circulating level of EPC. We studied 102 consecutive patients (mean age: 66.5 ± 9.5 years, 78% male, all female post-menopausal) with cardiovascular disease (atherothrombotic stroke 62%, coronary artery disease 38%). Circulating levels of CD133(+) EPC were determined by flow cytometry. Non-invasive pulse wave velocity (PWV) was measured. Long-term intake of isoflavone was determined by a validated food frequency questionnaire. Isoflavone intake was positively associated with circulating CD133(+) EPC (r = 0.31, p = 0.001). Patients with circulating CD133(+) EPC <10th percentile had significantly lower isoflavone intake than patients with CD133(+)EPC ≥10th percentile (4.6 ± 3.7 mg/day versus 19.3 ± 30.2 mg/day, p < 0.001). A significant overall linear trend of circulating EPC across increasing tertiles of isoflavone intake was observed (p = 0.004). Adjusted for potential confounders, increased isoflavone intake from the 1st to the 3rd tertile independently predicted increased circulating CD133(+) EPC level by 221 cells/µl (95%CI: 71.4 to 369.8, relative increase 160%, p = 0.004). Gender was not a significant factor (p > 0.05). Furthermore, circulating CD133(+) EPC <10th percentile was independently predictive of increased PWV by 261.7 cm/s (95% CI: 37.1 to 486.2, p = 0.024). The study demonstrated that circulating EPC increased by more than one fold in patients with cardiovascular disease who had higher intake of isoflavone, suggesting that isoflavone may confer vascular protection through enhanced endothelial repair.

Phenotypic profile of expanded NK cells in chronic lymphoproliferative disorders: a surrogate marker for NK-cell clonality

PubMed Central

Bárcena, Paloma; Jara-Acevedo, María; Tabernero, María Dolores; López, Antonio; Sánchez, María Luz; García-Montero, Andrés C.; Muñoz-García, Noemí; Vidriales, María Belén; Paiva, Artur; Lecrevisse, Quentin; Lima, Margarida; Langerak, Anton W.; Böttcher, Sebastian; van Dongen, Jacques J.M.

2015-01-01

Currently, the lack of a universal and specific marker of clonality hampers the diagnosis and classification of chronic expansions of natural killer (NK) cells. Here we investigated the utility of flow cytometric detection of aberrant/altered NK-cell phenotypes as a surrogate marker for clonality, in the diagnostic work-up of chronic lymphoproliferative disorders of NK cells (CLPD-NK). For this purpose, a large panel of markers was evaluated by multiparametric flow cytometry on peripheral blood (PB) CD56low NK cells from 60 patients, including 23 subjects with predefined clonal (n = 9) and polyclonal (n = 14) CD56low NK-cell expansions, and 37 with CLPD-NK of undetermined clonality; also, PB samples from 10 healthy adults were included. Clonality was established using the human androgen receptor (HUMARA) assay. Clonal NK cells were found to show decreased expression of CD7, CD11b and CD38, and higher CD2, CD94 and HLADR levels vs. normal NK cells, together with a restricted repertoire of expression of the CD158a, CD158b and CD161 killer-associated receptors. In turn, NK cells from both clonal and polyclonal CLPD-NK showed similar/overlapping phenotypic profiles, except for high and more homogeneous expression of CD94 and HLADR, which was restricted to clonal CLPD-NK. We conclude that the CD94hi/HLADR+ phenotypic profile proved to be a useful surrogate marker for NK-cell clonality. PMID:26556869

Gastric cancer stem cells in gastric carcinogenesis, progression, prevention and treatment

PubMed Central

Li, Kang; Dan, Zeng; Nie, Yu-Qiang

2014-01-01

In recent decades, the study of the mechanism of tumorigenesis has brought much progress to cancer treatment. However, cancer stem cell (CSC) theory has changed previous views of tumors, and has provided a new method for treatment of cancer. The discovery of CSCs and their characteristics have contributed to understanding the molecular mechanism of tumor genesis and development, resulting in a new effective strategy for cancer treatment. Gastric CSCs (GCSCs) are the basis for the onset of gastric cancer. They may be derived from gastric stem cells in gastric tissues, or bone marrow mesenchymal stem cells. As with other stem cells, GCSCs highly express drug-resistance genes such as aldehyde dehydrogenase and multidrug resistance, which are resistant to chemotherapy and thus form the basis of drug resistance. Many specific molecular markers such as CD44 and CD133 have been used for identification and isolation of GCSCs, diagnosis and grading of gastric cancer, and research on GCSC-targeted therapy for gastric cancer. Therefore, discussion of the recent development and advancements in GCSCs will be helpful for providing novel insight into gastric cancer treatment. PMID:24833872

Increased expression of CD44 is associated with more aggressive behavior in clear cell renal cell carcinoma.

PubMed

Zanjani, Leili Saeednejad; Madjd, Zahra; Abolhasani, Maryam; Rasti, Arezoo; Fodstad, Oystein; Andersson, Yvonne; Asgari, Mojgan

2018-01-01

Although CD44 has been suggested as a prognostic marker in renal cell carcinoma (RCC), the prognostic significance of this marker in three main subtypes of RCC is still unclear. Thus, the present study was conducted to evaluate the expression and prognostic significance of CD44 as a cancer stem cell marker in different histological subtypes of RCC. Methodology & results: CD44 expression was evaluated in 206 well-defined renal tumor samples using immunohistochemistry on tissue microarrays. Higher CD44 expression was associated with more aggressive behavior, tumor progression and worse prognosis in clear cell RCC (ccRCC) but not in papillary and chromophobe RCC subtypes. Cancer stem cell marker CD44 may be a promising target for cancer treatment only in ccRCC.

Ovarian Cancers: Genetic Abnormalities, Tumor Heterogeneity and Progression, Clonal Evolution and Cancer Stem Cells.

PubMed

Testa, Ugo; Petrucci, Eleonora; Pasquini, Luca; Castelli, Germana; Pelosi, Elvira

2018-02-01

Four main histological subtypes of ovarian cancer exist: serous (the most frequent), endometrioid, mucinous and clear cell; in each subtype, low and high grade. The large majority of ovarian cancers are diagnosed as high-grade serous ovarian cancers (HGS-OvCas). TP53 is the most frequently mutated gene in HGS-OvCas; about 50% of these tumors displayed defective homologous recombination due to germline and somatic BRCA mutations, epigenetic inactivation of BRCA and abnormalities of DNA repair genes; somatic copy number alterations are frequent in these tumors and some of them are associated with prognosis; defective NOTCH, RAS/MEK, PI3K and FOXM1 pathway signaling is frequent. Other histological subtypes were characterized by a different mutational spectrum: LGS-OvCas have increased frequency of BRAF and RAS mutations; mucinous cancers have mutation in ARID1A , PIK3CA , PTEN , CTNNB1 and RAS . Intensive research was focused to characterize ovarian cancer stem cells, based on positivity for some markers, including CD133, CD44, CD117, CD24, EpCAM, LY6A, ALDH1. Ovarian cancer cells have an intrinsic plasticity, thus explaining that in a single tumor more than one cell subpopulation, may exhibit tumor-initiating capacity. The improvements in our understanding of the molecular and cellular basis of ovarian cancers should lead to more efficacious treatments.

Ovarian Cancers: Genetic Abnormalities, Tumor Heterogeneity and Progression, Clonal Evolution and Cancer Stem Cells

PubMed Central

Castelli, Germana; Pelosi, Elvira

2018-01-01

Four main histological subtypes of ovarian cancer exist: serous (the most frequent), endometrioid, mucinous and clear cell; in each subtype, low and high grade. The large majority of ovarian cancers are diagnosed as high-grade serous ovarian cancers (HGS-OvCas). TP53 is the most frequently mutated gene in HGS-OvCas; about 50% of these tumors displayed defective homologous recombination due to germline and somatic BRCA mutations, epigenetic inactivation of BRCA and abnormalities of DNA repair genes; somatic copy number alterations are frequent in these tumors and some of them are associated with prognosis; defective NOTCH, RAS/MEK, PI3K and FOXM1 pathway signaling is frequent. Other histological subtypes were characterized by a different mutational spectrum: LGS-OvCas have increased frequency of BRAF and RAS mutations; mucinous cancers have mutation in ARID1A, PIK3CA, PTEN, CTNNB1 and RAS. Intensive research was focused to characterize ovarian cancer stem cells, based on positivity for some markers, including CD133, CD44, CD117, CD24, EpCAM, LY6A, ALDH1. Ovarian cancer cells have an intrinsic plasticity, thus explaining that in a single tumor more than one cell subpopulation, may exhibit tumor-initiating capacity. The improvements in our understanding of the molecular and cellular basis of ovarian cancers should lead to more efficacious treatments. PMID:29389895

Characterization of hepatic progenitors from human fetal liver during second trimester.

PubMed

Rao, Mekala-Subba; Khan, Aleem-Ahmed; Parveen, Nyamath; Habeeb, Mohammed-Aejaz; Habibullah, Chittoor-Mohammed; Pande, Gopal

2008-10-07

To enrich hepatic progenitors using epithelial cell adhesion molecule (EpCAM) as a marker from human fetal liver and investigate the expression of human leukocyte antigen (HLA) and their markers associated with hepatic progenitor cells. EpCAM +ve cells were isolated using magnetic cell sorting (MACS) from human fetuses (n = 10) at 15-25 wk gestation. Expression of markers for hepatic progenitors such as albumin, alpha-fetoprotein (AFP), CD29 (integrin beta1), CD49f (integrin alpha6) and CD90 (Thy 1) was studied by using flow cytometry, immunocytochemistry and RT-PCR; HLA class I (A, B, C) and class II (DR) expression was studied by flow cytometry only. FACS analysis indicated that EpCAM +ve cells were positive for CD29, CD49f, CD90, CD34, HLA class I, albumin and AFP but negative for HLA class II (DR) and CD45. RT PCR showed that EpCAM +ve cells expressed liver epithelial markers (CK18), biliary specific marker (CK19) and hepatic markers (albumin, AFP). On immunocytochemical staining, EpCAM +ve cells were shown positive signals for CK18 and albumin. Our study suggests that these EpCAM +ve cells can be used as hepatic progenitors for cell transplantation with a minimum risk of alloreactivity and these cells may serve as a potential source for enrichment of hepatic progenitor.

CXCR6 identifies a putative population of retained human lung T cells characterised by co-expression of activation markers.

PubMed

Morgan, Angela J; Guillen, Cristina; Symon, Fiona A; Birring, Surinder S; Campbell, James J; Wardlaw, Andrew J

2008-01-01

Expressions of activation markers have been described on the surface of T cells in the blood and the lung in both health and disease. We have studied the distribution of activation markers on human lung T cells and have found that only certain populations exist. Importantly, the presence or absence of some markers appears to predict those of others, in particular cells which express CD103 also express CD49a and CD69, whereas cells which do not express CD69 also do not express CD49a or CD103. In view of the paucity of activation marker expression in the peripheral blood, we have hypothesised that these CD69+, CD49a+, and CD103+ (triple positive) cells are retained in the lung, possess effector function (IFNgamma secretion) and express particular chemokine receptors which allow them to be maintained in this environment. We have found that the ability of the triple negative cells to secrete IFNgamma is significantly less than the triple positive cells, suggesting that the expression of activation markers can highlight a highly specialised effector cell. We have studied the expression of 14 chemokine receptors and have found that the most striking difference between the triple negative cells and the triple positive cells is the expression of CXCR6 with 12.8+/-9.8% of triple negative cells expressing CXCR6 compared to 89.5+/-5.5% of triple positive cells. We propose therefore that CXCR6 may play an important role in the retention of T cells within the lung.

Granulysin-Expressing CD4+ T Cells as Candidate Immune Marker for Tuberculosis during Childhood and Adolescence

PubMed Central

Mueller, Henrik; Faé, Kellen C.; Magdorf, Klaus; Ganoza, Christian A.; Wahn, Ulrich; Guhlich, Ute; Feiterna-Sperling, Cornelia; Kaufmann, Stefan H. E.

2011-01-01

Background Granulysin produced by cytolytic T cells directly contributes to immune defense against tuberculosis (TB). We investigated granulysin as a candidate immune marker for childhood and adolescent TB. Methods Peripheral blood mononuclear cells (PBMC) from children and adolescents (1–17 years) with active TB, latent TB infection (LTBI), nontuberculous mycobacteria (NTM) infection and from uninfected controls were isolated and restimulated in a 7-day restimulation assay. Intracellular staining was then performed to analyze antigen-specific induction of activation markers and cytotoxic proteins, notably, granulysin in CD4+ CD45RO+ memory T cells. Results CD4+ CD45RO+ T cells co-expressing granulysin with specificity for Mycobacterium tuberculosis (Mtb) were present in high frequency in TB-experienced children and adolescents. Proliferating memory T cells (CFSElowCD4+CD45RO+) were identified as main source of granulysin and these cells expressed both central and effector memory phenotype. PBMC from study participants after TB drug therapy revealed that granulysin-expressing CD4+ T cells are long-lived, and express several activation and cytotoxicity markers with a proportion of cells being interferon-gamma-positive. In addition, granulysin-expressing T cell lines showed cytolytic activity against Mtb-infected target cells. Conclusions Our data suggest granulysin expression by CD4+ memory T cells as candidate immune marker for TB infection, notably, in childhood and adolescence. PMID:22216262

Enrichment of the Cancer Stem Phenotype in Sphere Cultures of Prostate Cancer Cell Lines Occurs through Activation of Developmental Pathways Mediated by the Transcriptional Regulator ΔNp63α

PubMed Central

Portillo-Lara, Roberto; Alvarez, Mario Moisés

2015-01-01

Background Cancer stem cells (CSC) drive prostate cancer tumor survival and metastasis. Nevertheless, the development of specific therapies against CSCs is hindered by the scarcity of these cells in prostate tissues. Suspension culture systems have been reported to enrich CSCs in primary cultures and cell lines. However, the molecular mechanisms underlying this phenomenon have not been fully explored. Methodology/Principal Findings We describe a prostasphere assay for the enrichment of CD133+ CSCs in four commercial PCa cell lines: 22Rv1, DU145, LNCaP, and PC3. Overexpression of CD133, as determined by flow cytometric analysis, correlated with an increased clonogenic, chemoresistant, and invasive potential in vitro. This phenotype is concordant to that of CSCs in vivo. Gene expression profiling was then carried out using the Cancer Reference panel and the nCounter system from NanoString Technologies. This analysis revealed several upregulated transcripts that can be further explored as potential diagnostic markers or therapeutic targets. Furthermore, functional annotation analysis suggests that ΔNp63α modulates the activation of developmental pathways responsible for the increased stem identity of cells growing in suspension cultures. Conclusions/Significance We conclude that profiling the genetic mechanisms involved in CSC enrichment will help us to better understand the molecular pathways that underlie CSC pathophysiology. This platform can be readily adapted to enrich and assay actual patient samples, in order to design patient-specific therapies that are aimed particularly against CSCs. PMID:26110651

Icariin protects rats against 5/6 nephrectomy-induced chronic kidney failure by increasing the number of renal stem cells.

PubMed

Huang, Zhongdi; He, Liqun; Huang, Di; Lei, Shi; Gao, Jiandong

2015-10-21

Chronic kidney disease poses a serious health problem worldwide with increasing prevalence and lack of effective treatment. This study aimed to investigate the mechanism of icariin in alleviating chronic renal failure induced by 5/6 nephrectomy in rats. The chronic renal failure model was established by a two-phased 5/6 nephrectomy procedure. The model rats were given daily doses of water or icariin for 8 weeks. The kidney morphology was checked by HE staining. The levels of blood urea nitrogen, serum creatinine, and serum uric acid were measured by colometric methods. The expression of specified genes was analyzed by quantitative real-time PCR and immunohistochemical staining. The number of renal stem/progenitor cells was analyzed by CD133 and CD24 immunohistochemical staining. Icariin protected against CDK-caused damages to kidney histology and improved renal function, significantly reduced levels of BUN, creatinine, and uric acid. Icariin inhibited the expression level of TGF-β1 whereas upregulated HGF, BMP-7, WT-1, and Pax2 expression. Moreover, ccariin significantly increased the expression of CD24, CD133, Osr1, and Nanog in remnant kidney and the numbers of CD133(+)/CD24(+) renal stem/progenitor cells. These data demonstrated that icariin effectively alleviated 5/6 nephrectomy induced chronic renal failure through increasing renal stem/progenitor cells.

A novel long non-coding RNA, hypoxia-inducible factor-2α promoter upstream transcript, functions as an inhibitor of osteosarcoma stem cells in vitro.

PubMed

Wang, Yongcheng; Yao, Jie; Meng, Haoye; Yu, Zhiguo; Wang, Zhigang; Yuan, Xueling; Chen, Hong; Wang, Aiyuan

2015-04-01

Long non‑coding RNAs (lncRNAs) have recently been identified as novel modulators of malignant tumors. However, the function of lncRNAs in cancer stem cells (CSCs) remains to be elucidated. The present study aimed to investigate the regulating role of a novel lncRNA, hypoxia‑inducible factor‑2α (HIF‑2α) promoter upstream transcript (HIF2PUT), in osteosarcoma stem cells. The expression levels of HIF2PUT were assessed by quantitative polymerase chain reaction in 17 osteosarcoma tissue specimens, and the correlation between the expression of HIF2PUT and its host transcript‑HIF‑2α was determined. In functional experiments, HIF2PUT expression was knocked down by small interfering RNAs, or overexpressed by transfection with pcDNA‑HIF2PUT, in order to evaluate the effects of HIF2PUT on cell proliferation, migration, expression rate of osteosarcoma stem cell marker CD133, and stem sphere‑forming ability in MG63 cells. HIF2PUT expression levels were positively correlated with HIF‑2α in osteosarcoma tissues. Overexpression of HIF2PUT markedly inhibited cell proliferation and migration, decreased the percentage of CD133 expressing cells, and impaired the osteosarcoma stem sphere‑forming ability of the MG63 cells. Whereas, knockdown of HIF2PUT expression had the opposite effect. Furthermore, altering the expression of HIF2PUT resulted in a concomitant change to HIF‑2α mRNA expression. These results indicate that the lncRNA HIF2PUT may be a novel regulatory factor of osteosarcoma stem cells, which may exert its function partly by controlling HIF‑2α expression. Further studies regarding HIF2PUT may provide a novel therapeutic target of osteosarcoma in the future.

TLX activates MMP-2, promotes self-renewal of tumor spheres in neuroblastoma and correlates with poor patient survival.

PubMed

Chavali, P L; Saini, R K R; Zhai, Q; Vizlin-Hodzic, D; Venkatabalasubramanian, S; Hayashi, A; Johansson, E; Zeng, Z-j; Mohlin, S; Påhlman, S; Hansford, L; Kaplan, D R; Funa, K

2014-10-30

Nuclear orphan receptor TLX (Drosophila tailless homolog) is essential for the maintenance of neural stem/progenitor cell self-renewal, but its role in neuroblastoma (NB) is not well understood. Here, we show that TLX is essential for the formation of tumor spheres in three different NB cell lines, when grown in neural stem cell media. We demonstrate that the knock down of TLX in IMR-32 cells diminishes its tumor sphere-forming capacity. In tumor spheres, TLX is coexpressed with the neural progenitor markers Nestin, CD133 and Oct-4. In addition, TLX is coexpressed with the migratory neural progenitor markers CD15 and matrix metalloproteinase-2 (MMP-2) in xenografts of primary NB cells from patients. Subsequently, we show the effect of TLX on the proliferative, invasive and migratory properties of IMR-32 cells. We attribute this to the recruitment of TLX to both MMP-2 and Oct-4 gene promoters, which resulted in the respective gene activation. In support of our findings, we found that TLX expression was high in NB patient tissues when compared with normal peripheral nervous system tissues. Further, the Kaplan-Meier estimator indicated a negative correlation between TLX expression and survival in 88 NB patients. Therefore, our results point at TLX being a crucial player in progression of NB, by promoting self-renewal of NB tumor-initiating cells and altering their migratory and invasive properties.

TLX activates MMP-2, promotes self-renewal of tumor spheres in neuroblastoma and correlates with poor patient survival

PubMed Central

Chavali, P L; Saini, R K R; Zhai, Q; Vizlin-Hodzic, D; Venkatabalasubramanian, S; Hayashi, A; Johansson, E; Zeng, Z-j; Mohlin, S; Påhlman, S; Hansford, L; Kaplan, D R; Funa, K

2014-01-01

Nuclear orphan receptor TLX (Drosophila tailless homolog) is essential for the maintenance of neural stem/progenitor cell self-renewal, but its role in neuroblastoma (NB) is not well understood. Here, we show that TLX is essential for the formation of tumor spheres in three different NB cell lines, when grown in neural stem cell media. We demonstrate that the knock down of TLX in IMR-32 cells diminishes its tumor sphere-forming capacity. In tumor spheres, TLX is coexpressed with the neural progenitor markers Nestin, CD133 and Oct-4. In addition, TLX is coexpressed with the migratory neural progenitor markers CD15 and matrix metalloproteinase-2 (MMP-2) in xenografts of primary NB cells from patients. Subsequently, we show the effect of TLX on the proliferative, invasive and migratory properties of IMR-32 cells. We attribute this to the recruitment of TLX to both MMP-2 and Oct-4 gene promoters, which resulted in the respective gene activation. In support of our findings, we found that TLX expression was high in NB patient tissues when compared with normal peripheral nervous system tissues. Further, the Kaplan–Meier estimator indicated a negative correlation between TLX expression and survival in 88 NB patients. Therefore, our results point at TLX being a crucial player in progression of NB, by promoting self-renewal of NB tumor-initiating cells and altering their migratory and invasive properties. PMID:25356871

Investigating Cell Surface Markers on Normal Hematopoietic Stem Cells in Three Different Niche Conditions

PubMed Central

Garg, Swati; Madkaikar, Manisha

2013-01-01

Hematopoietic stem cells are of therapeutic interest to the clinicians and researchers due to their promising assistance in management of malignant and inherited hematological conditions. Evaluation of cell surface markers using multiparametric flow cytometry is a well adapted qualitative measure of cells in question for many years. An artillery of these markers has been studied in hematological malignancies and related disorders. However, their role and differential expression on normal hematopoietic stem cells from clinically available sources is not always described carefully. In the present study, we attempted to evaluate expression of CD44, CD90, CD96 and CD123 in three clinically available sources of normal HSCs (Hematopoietic stem cells). Sources of HSCs in the present study involved umbilical cord blood (UCB), normal bone marrow (NBM) and bone marrow from idiopathic thrombocytopenic purpura (ITP) patients (IBM). CD44 is an important homing receptor while CD90 is involved in maintaining stem cell quiescent. CD96 is known to be leukemia specific marker and CD123 is involved in stem cell differentiation and survival. We observed a significant difference in expression CD44, CD90 and CD123 on normal HSCs derived from umbilical cord and ITP marrow. CD96 was highly expressed on HSCs obtained from ITP marrow. Investigating expression of these markers on normal HSCs in different niches will be helpful in correlating their function with niche condition and delineating their ‘abnormal’ expression from the normal. PMID:24386557

Investigating cell surface markers on normal hematopoietic stem cells in three different niche conditions.

PubMed

Garg, Swati; Madkaikar, Manisha; Ghosh, Kanjaksha

2013-11-01

Hematopoietic stem cells are of therapeutic interest to the clinicians and researchers due to their promising assistance in management of malignant and inherited hematological conditions. Evaluation of cell surface markers using multiparametric flow cytometry is a well adapted qualitative measure of cells in question for many years. An artillery of these markers has been studied in hematological malignancies and related disorders. However, their role and differential expression on normal hematopoietic stem cells from clinically available sources is not always described carefully. In the present study, we attempted to evaluate expression of CD44, CD90, CD96 and CD123 in three clinically available sources of normal HSCs (Hematopoietic stem cells). Sources of HSCs in the present study involved umbilical cord blood (UCB), normal bone marrow (NBM) and bone marrow from idiopathic thrombocytopenic purpura (ITP) patients (IBM). CD44 is an important homing receptor while CD90 is involved in maintaining stem cell quiescent. CD96 is known to be leukemia specific marker and CD123 is involved in stem cell differentiation and survival. We observed a significant difference in expression CD44, CD90 and CD123 on normal HSCs derived from umbilical cord and ITP marrow. CD96 was highly expressed on HSCs obtained from ITP marrow. Investigating expression of these markers on normal HSCs in different niches will be helpful in correlating their function with niche condition and delineating their 'abnormal' expression from the normal.

Exhaustion of Activated CD8 T Cells Predicts Disease Progression in Primary HIV-1 Infection

PubMed Central

Hickling, Stephen; Hurst, Jacob; Meyerowitz, Jodi; Willberg, Christian B.; Robinson, Nicola; Brown, Helen; Kinloch, Sabine; Babiker, Abdel; Nwokolo, Nneka; Fox, Julie; Fidler, Sarah; Phillips, Rodney; Frater, John

2016-01-01

The rate at which HIV-1 infected individuals progress to AIDS is highly variable and impacted by T cell immunity. CD8 T cell inhibitory molecules are up-regulated in HIV-1 infection and associate with immune dysfunction. We evaluated participants (n = 122) recruited to the SPARTAC randomised clinical trial to determine whether CD8 T cell exhaustion markers PD-1, Lag-3 and Tim-3 were associated with immune activation and disease progression. Expression of PD-1, Tim-3, Lag-3 and CD38 on CD8 T cells from the closest pre-therapy time-point to seroconversion was measured by flow cytometry, and correlated with surrogate markers of HIV-1 disease (HIV-1 plasma viral load (pVL) and CD4 T cell count) and the trial endpoint (time to CD4 count <350 cells/μl or initiation of antiretroviral therapy). To explore the functional significance of these markers, co-expression of Eomes, T-bet and CD39 was assessed. Expression of PD-1 on CD8 and CD38 CD8 T cells correlated with pVL and CD4 count at baseline, and predicted time to the trial endpoint. Lag-3 expression was associated with pVL but not CD4 count. For all exhaustion markers, expression of CD38 on CD8 T cells increased the strength of associations. In Cox models, progression to the trial endpoint was most marked for PD-1/CD38 co-expressing cells, with evidence for a stronger effect within 12 weeks from confirmed diagnosis of PHI. The effect of PD-1 and Lag-3 expression on CD8 T cells retained statistical significance in Cox proportional hazards models including antiretroviral therapy and CD4 count, but not pVL as co-variants. Expression of ‘exhaustion’ or ‘immune checkpoint’ markers in early HIV-1 infection is associated with clinical progression and is impacted by immune activation and the duration of infection. New markers to identify exhausted T cells and novel interventions to reverse exhaustion may inform the development of novel immunotherapeutic approaches. PMID:27415828

Monoamine Oxidase Deficiency Causes Prostate Atrophy and Reduces Prostate Progenitor Cell Activity.

PubMed

Yin, Lijuan; Li, Jingjing; Liao, Chun-Peng; Jason Wu, Boyang

2018-04-10

Monoamine oxidases (MAOs) degrade a number of biogenic and dietary amines, including monoamine neurotransmitters, and play an essential role in many biological processes. Neurotransmitters and related neural events have been shown to participate in the development, differentiation, and maintenance of diverse tissues and organs by regulating the specialized cellular function and morphological structures of innervated organs such as the prostate. Here we show that mice lacking both MAO isoforms, MAOA and MAOB, exhibit smaller prostate mass and develop epithelial atrophy in the ventral and dorsolateral prostates. The cellular composition of prostate epithelium showed reduced CK5 + or p63 + basal cells, accompanied by lower Sca-1 expression in p63 + basal cells, but intact differentiated CK8 + luminal cells in MAOA/B-deficient mouse prostates. MAOA/B ablation also decreased epithelial cell proliferation without affecting cell apoptosis in mouse prostates. Using a human prostate epithelial cell line, we found that stable knockdown of MAOA and MAOB impaired the capacity of prostate stem cells to form spheres, coinciding with a reduced CD133 + /CD44 + /CD24 - stem cell population and less expression of CK5 and select stem cell markers, including ALDH1A1, TROP2, and CD166. Alternative pharmacological inhibition of MAOs also repressed prostate cell stemness. In addition, we found elevated expression of MAOA and MAOB in epithelial and/or stromal components of human prostate hyperplasia samples compared with normal prostate tissues. Taken together, our findings reveal critical roles for MAOs in the regulation of prostate basal progenitor cells and prostate maintenance. Stem Cells 2018. © AlphaMed Press 2018.

Epithelial-mesenchymal transition in breast epithelial cells treated with cadmium and the role of Snail.

PubMed

Wei, Zhengxi; Shan, Zhongguo; Shaikh, Zahir A

2018-04-01

Epidemiological and experimental studies have implicated cadmium (Cd) with breast cancer. In breast epithelial MCF10A and MDA-MB-231 cells, Cd has been shown to promote cell growth. The present study examined whether Cd also promotes epithelial-mesenchymal transition (EMT), a hallmark of cancer progression. Human breast epithelial cells consisting of non-cancerous MCF10A, non-metastatic HCC 1937 and HCC 38, and metastatic MDA-MB-231 were treated with 1 or 3 μM Cd for 4 weeks. The MCF10A epithelial cells switched to a more mesenchymal-like morphology, which was accompanied by a decrease in the epithelial marker E-cadherin and an increase in the mesenchymal markers N-cadherin and vimentin. In both non-metastatic HCC 1937 and HCC 38 cells, treatment with Cd decreased the epithelial marker claudin-1. In addition, E-cadherin also decreased in the HCC 1937 cells. Even the mesenchymal-like MDA-MB-231 cells exhibited an increase in the mesenchymal marker vimentin. These changes indicated that prolonged treatment with Cd resulted in EMT in both normal and cancer-derived breast epithelial cells. Furthermore, both the MCF10A and MDA-MB-231 cells labeled with Zcad, a dual sensor for tracking EMT, demonstrated a decrease in the epithelial marker E-cadherin and an increase in the mesenchymal marker ZEB-1. Treatment of cells with Cd significantly increased the level of Snail, a transcription factor involved in the regulation of EMT. However, the Cd-induced Snail expression was completely abolished by actinomycin D. Luciferase reporter assay indicated that the expression of Snail was regulated by Cd at the promotor level. Snail was essential for Cd-induced promotion of EMT in the MDA-MB-231 cells, as knockdown of Snail expression blocked Cd-induced cell migration. Together, these results indicate that Cd promotes EMT in breast epithelial cells and does so by modulating the transcription of Snail. Copyright © 2018 Elsevier Inc. All rights reserved.

Influence of different types of pulp treatment during isolation in the obtention of human dental pulp stem cells

PubMed Central

Viña-Almunia, Jose; Borras, Consuelo; Gambini, Juan; El Alamy, Marya; Viña, Jose

2016-01-01

Background Different methods have been used in order to isolate dental pulp stem cells. The aim of this study was to study the effect of different types of pulp treatment during isolation, under 3% O2 conditions, in the time needed and the efficacy for obtaining dental pulp stem cells. Material and Methods One hundred and twenty dental pulps were used to isolate dental pulp stem cells treating the pulp tissue during isolation using 9 different methods, using digestive, disgregation, or mechanical agents, or combining them. The cells were positive for CD133, Oct4, Nestin, Stro-1, CD34 markers, and negative for the hematopoietic cell marker CD-45, thus confirming the presence of mesenchymal stem cells. The efficacy of dental pulp stem cells obtention and the minimum time needed to obtain such cells comparing the 9 different methods was analyzed. Results Dental pulp stem cells were obtained from 97 of the 120 pulps used in the study, i.e. 80.8% of the cases. They were obtained with all the methods used except with mechanical fragmentation of the pulp, where no enzymatic digestion was performed. The minimum time needed to isolate dental pulp stem cells was 8 hours, digesting with 2mg/ml EDTA for 10 minutes, 4mg/ml of type I collagenase, 4mg/ml of type II dispase for 40 minutes, 13ng/ml of thermolysine for 40 minutes and sonicating the culture for one minute. Conclusions Dental pulp stem cells were obtained in 97 cases from a series of 120 pulps. The time for obtaining dental pulp stem cells was reduced maximally, without compromising the obtention of the cells, by combining digestive, disgregation, and mechanical agents. Key words:Dental pulp stem cells, mesenchymal stem cells, isolation method. PMID:26946201

Effects of shiga toxin 2 on cellular regeneration mechanisms in primary and three-dimensional cultures of human renal tubular epithelial cells.

PubMed

Márquez, Laura B; Araoz, Alicia; Repetto, Horacio A; Ibarra, Fernando R; Silberstein, Claudia

2016-10-01

Shiga toxin (Stx)-producing Escherichia coli (STEC) causes post-diarrheal Hemolytic Uremic Syndrome (HUS), which is one of the most common causes of acute renal failure in children in Argentine. The aim of the present work was to study the effects of Shiga toxin type 2 (Stx2) on regenerative mechanisms of primary cultures of human cortical renal tubular epithelial cells (HRTEC) and three-dimensional (3D) cultures of HRTEC. Primary cultures of HRTEC were able to develop tubular structures when grown in matrigel, which showed epithelial cells surrounding a central lumen resembling the original renal tubules. Exposure to Stx2 inhibited tubulogenesis in 3D-HRTEC cultures. Moreover, a significant increase in apoptosis, and decrease in cell proliferation was observed in tubular structures of 3D-HRTEC exposed to Stx2. A significant reduction in cell migration and vimentin expression levels was observed in HRTEC primary cultures exposed to Stx2, demonstrating that the holotoxin affected HRTEC dedifferentiation. Furthermore, a decreased number of cells expressing CD133 progenitor marker was found in HRTEC cultures treated with Stx2. The CD133 positive cells also expressed the Stx receptor globotriaosylceramide, which may explain their sensitivity to Stx2. In conclusion, Stx2 affects the regenerative processes of human renal tubular epithelial cells in vitro, by inhibiting cell dedifferentiation mechanisms, as well as tubules restoration. The development of 3D-HRTEC cultures that resemble original human renal proximal tubules is a novel in vitro model to study renal epithelial repair mechanisms after injury. Copyright © 2016 Elsevier Ltd. All rights reserved.

Interleukin-6 controls uterine Th9 cells and CD8(+) T regulatory cells to accelerate parturition in mice.

PubMed

Gomez-Lopez, Nardhy; Olson, David M; Robertson, Sarah A

2016-01-01

Interleukin-6 (IL6) is a determinant of the timing of parturition and birth in mice. We previously demonstrated that genetic IL6 deficiency delays parturition by ~24 h, and this is restored by administration of exogenous IL6. In this study, we have investigated whether IL6 influences the number or phenotypes of T cells or other leukocytes in uterine decidual tissue at the maternal-fetal interface. In late gestation, decidual leukocytes in Il6 null mutant (Il6(-/-)) mice exhibit an altered profile, characterized by reduced numbers of cells expressing the monocyte/macrophage marker F4/80 or the T-cell marker CD4, increased cells expressing the natural killer (NK) cell marker CD49b or the dendritic cell marker CD11c, but no change in cells expressing the neutrophil marker Ly6G. These changes are specific to late pregnancy, as similar differences in decidual leukocytes were not evident in mid-gestation Il6(-/-) mice. The IL6-regulated changes in decidual NK and dendritic cells appear secondary to local recruitment, as no comparable changes occurred in peripheral blood of Il6(-/-) mice. When exogenous IL6 was administered to restore normal timing of parturition, a partial reversal of the altered leukocyte profile was observed, with a 10% increase in the proportion of decidual CD4(+) T cells, a notable 60% increase in CD8(+) T cells including CD8(+)CD25(+)Foxp3(+) regulatory T cells and a 60% reduction in CD4(+)IL9(+) Th9 cells. Together these findings suggest that IL6-controlled accumulation of decidual CD4(+) T cells and CD8(+) regulatory T cells, with an associated decline in decidual Th9 cells, is instrumental for progressing parturition in mice.

Physiological β-catenin signaling controls self-renewal networks and generation of stem-like cells from nasopharyngeal carcinoma.

PubMed

Cheng, Yue; Cheung, Arthur Kwok Leung; Ko, Josephine Mun Yee; Phoon, Yee Peng; Chiu, Pui Man; Lo, Paulisally Hau Yi; Waterman, Marian L; Lung, Maria Li

2013-09-27

A few reports suggested that low levels of Wnt signaling might drive cell reprogramming, but these studies could not establish a clear relationship between Wnt signaling and self-renewal networks. There are ongoing debates as to whether and how the Wnt/β-catenin signaling is involved in the control of pluripotency gene networks. Additionally, whether physiological β-catenin signaling generates stem-like cells through interactions with other pathways is as yet unclear. The nasopharyngeal carcinoma HONE1 cells have low expression of β-catenin and wild-type expression of p53, which provided a possibility to study regulatory mechanism of stemness networks induced by physiological levels of Wnt signaling in these cells. Introduction of increased β-catenin signaling, haploid expression of β-catenin under control by its natural regulators in transferred chromosome 3, resulted in activation of Wnt/β-catenin networks and dedifferentiation in HONE1 hybrid cell lines, but not in esophageal carcinoma SLMT1 hybrid cells that had high levels of endogenous β-catenin expression. HONE1 hybrid cells displayed stem cell-like properties, including enhancement of CD24(+) and CD44(+) populations and generation of spheres that were not observed in parental HONE1 cells. Signaling cascades were detected in HONE1 hybrid cells, including activation of p53- and RB1-mediated tumor suppressor pathways, up-regulation of Nanog-, Oct4-, Sox2-, and Klf4-mediated pluripotency networks, and altered E-cadherin expression in both in vitro and in vivo assays. qPCR array analyses further revealed interactions of physiological Wnt/β-catenin signaling with other pathways such as epithelial-mesenchymal transition, TGF-β, Activin, BMPR, FGFR2, and LIFR- and IL6ST-mediated cell self-renewal networks. Using β-catenin shRNA inhibitory assays, a dominant role for β-catenin in these cellular network activities was observed. The expression of cell surface markers such as CD9, CD24, CD44, CD90, and CD133 in generated spheres was progressively up-regulated compared to HONE1 hybrid cells. Thirty-four up-regulated components of the Wnt pathway were identified in these spheres. Wnt/β-catenin signaling regulates self-renewal networks and plays a central role in the control of pluripotency genes, tumor suppressive pathways and expression of cancer stem cell markers. This current study provides a novel platform to investigate the interaction of physiological Wnt/β-catenin signaling with stemness transition networks.

DHFR and MDR1 upregulation is associated with chemoresistance in osteosarcoma stem-like cells

PubMed Central

Lee, Yu-Hsien; Yang, Hui-Wen; Yang, Li-Chiu; Lu, Ming-Yi; Tsai, Lo-Lin; Yang, Shun-Fa; Huang, Yu-Feng; Chou, Ming-Yung; Yu, Cheng-Chia; Hu, Fang-Wei

2017-01-01

Tumor-initiating cells (TICs) are defined as a specialized subset of cells with tumor-initiating capacity that can initiate tumor growth, tumor relapse and metastasis. In the present study, osteosarcoma TICs (OS-TICs) were isolated and enriched from the osteosarcoma U2OS and MG-63 cell lines using sphere formation assays and serum-depleted media. These enriched OS-TICs showed the expression of several typical cancer stemness markers, including octamer-binding transcription factor 4, Nanog homeobox, cluster of differentiation (CD)117, Nestin and CD133, and the expression of ATP binding cassette subfamily G member 2, multidrug resistance protein 1 (MDR1) and dihydrofolate reductase (DHFR). Notably, in vitro and in vivo tumorigenic properties were enhanced in these OS-TICs. Additionally, methotrexate and doxorubicin are the most widely used anticancer agents against osteosarcoma, and the observed enhanced chemoresistance of OS-TICs to these two agents could be associated with the upregulation of DHFR and MDR1. These findings suggest that the upregulation of DHFR and MDR1 is associated with the development of chemoresistance of OS-TICs. PMID:28693150

Molecular Biomarkers of Cancer Stem/Progenitor Cells Associated with Progression, Metastases, and Treatment Resistance of Aggressive Cancers

PubMed Central

Mimeault, Murielle; Batra, Surinder K.

2014-01-01

The validation of novel diagnostic, prognostic, and predictive biomarkers and therapeutic targets in tumor cells is of critical importance for optimizing the choice and efficacy of personalized therapies. Importantly, recent advances have led to the identification of gene-expression signatures in cancer cells, including cancer stem/progenitor cells, in the primary tumors, exosomes, circulating tumor cells (CTC), and disseminated cancer cells at distant metastatic sites. The gene-expression signatures may help to improve the accuracy of diagnosis and predict the therapeutic responses and overall survival of patients with cancer. Potential biomarkers in cancer cells include stem cell–like markers [CD133, aldehyde dehydrogenase (ALDH), CD44, and CD24], growth factors, and their cognate receptors [epidermal growth factor receptor (EGFR), EGFRvIII, and HER2], molecules associated with epithelial–mesenchymal transition (EMT; vimentin, N-cadherin, snail, twist, and Zeb1), regulators of altered metabolism (phosphatidylinositol-3′ kinase/Akt/mTOR), and drug resistance (multidrug transporters and macrophage inhibitory cytokine-1). Moreover, different pluripotency-associated transcription factors (Oct3/4, Nanog, Sox2, and Myc) and microRNAs that are involved in the epigenetic reprogramming and acquisition of stem cell–like properties by cancer cells during cancer progression may also be exploited as molecular biomarkers to predict the risk of metastases, systemic treatment resistance, and disease relapse of patients with cancer. PMID:24273063

Removing the cells from adult bone marrow derived stem cell therapy does not eliminate cardioprotection.

PubMed

Yasin, Mohammed

2013-04-01

The debate as to whether adult stem cell therapy is regenerative or not continues. The non-regenerative benefits of adult bone marrow-derived stem cell therapy were investigated by testing whether the supernatant derived from unfractionated bone marrow mononuclear cells might be cardioprotective in an animal model of myocardial ischaemia-reperfusion injury. Regional myocardial reperfusion injury was acquired by 25 min reversible left anterior descending coronary artery (LAD) occlusion followed by 2 h reperfusion, in anaesthetized Wistar male rats. Unfractionated bone marrow mononuclear cells (BMMNC) isolated from sibling Wistar male rat whole bone marrow were phenotyped by fluorescence activated cell sorting flowcytometry for the haematopoietic stem cell surface markers c-kit, CD34, CD45 and CD133. Animals subjected to regional myocardial reperfusion injury received either 10 million BMMNC or BMMNC supernatant (BMS); both were collected in 0.5 ml phosphate-buffered saline and delivered by intravenous bolus at the onset of reperfusion. The left ventricular region distal to the LAD occlusion point was excised for measurement of myocardial infarct size and proteomic analysis, which was used to identify whether there were any differences in myocardial proteins associated with intravenous injection of either BMMNC or BMS. BMMNC were phenotyped to be c-kit(+) (7 ± 1%), CD34(+) (7 ± 1%), CD45(+) (54 ± 6%), CD133(+) (15 ± 1%). The supernatant reduced myocardial infarct size (BMS 34 ± 2%, n = 15 vs control 57 ± 2%, n = 7, P < 0.0001), which was comparable to the reduction in infarct size afforded by the injection of cells (BMMNC 33 ± 3% vs control 57 ± 2%, n = 10, P < 0.0001). Proteomics of hearts treated with either BMS or BMMNC demonstrated higher expression of (i) anti-apoptotic signal transduction protein: 14-3-3-epsilon (1.5-fold); (ii) anti-oxidants: peroxiredoxin-6 (2.1-fold); (iii) heat shock proteins: alpha B-crystallin (1.7-fold), heat shock protein 72 (2.8-fold), tumour necrosis factor receptor-1 associated protein (2.3-fold), ischaemia responsive protein-94 (1.6-fold); (iv) glycolytic protein: glyceraldehyde-3-phosphate dehydrogenase (2.3-fold); (v) mitochondrial respiratory proteins: mitochondrial aconitase (4.7-fold), voltage-dependent anion-selective channel protein-1 (VDAC-1) (2.7-fold). Regional myocardial reperfusion injury can be attenuated by intravenous administration of either BMMNC or BMS at the onset of reperfusion, which suggests adult stem cells mediate non-regenerative cardioprotection.

Equine Mesenchymal Stromal Cells Retain a Pericyte-Like Phenotype

PubMed Central

Sheldrake, Tara A.; Dawson, Lucy; Menghini, Timothy; Rink, Burgunde Elisabeth; Amilon, Karin; Khan, Nusrat; Péault, Bruno; Donadeu, Francesc Xavier

2017-01-01

Mesenchymal stem/stromal cells (MSCs) have been used in human and equine regenerative medicine, and interest in exploiting their potential has increased dramatically over the years. Despite significant effort to characterize equine MSCs, the actual origin of these cells and how much of their native phenotype is maintained in culture have not been determined. In this study, we investigated the relationship between MSCs, derived from adipose tissue (AT) and bone marrow (BM), and pericytes in the horse. Both pericyte (CD146, NG2, and αSMA) and MSC (CD29, CD90, and CD73) markers were detected in equine AT and colocalized around blood vessels. Importantly, as assessed by flow cytometry, both pericyte (CD146, NG2, and αSMA) and MSC (CD29, CD44, CD90, and CD105) markers were present in a majority (≥90%) of cells in cultures of AT-MSCs and BM-MSCs; however, levels of pericyte markers were variable within each of those populations. Moreover, the expression of pericyte markers was maintained for at least eight passages in both AT-MSCs and BM-MSCs. Hematopoietic (CD45) and endothelial (CD144) markers were also detected at low levels in MSCs by quantitative polymerase chain reaction (qPCR). Finally, in coculture experiments, AT-MSCs closely associated with networks produced by endothelial cells, resembling the natural perivascular location of pericytes in vivo. Our results indicate that equine MSCs originate from perivascular cells and moreover maintain a pericyte-like phenotype in culture. Therefore, we suggest that, in addition to classical MSC markers, pericyte markers such as CD146 could be used when assessing and characterizing equine MSCs. PMID:28376684

Equine Mesenchymal Stromal Cells Retain a Pericyte-Like Phenotype.

PubMed

Esteves, Cristina L; Sheldrake, Tara A; Dawson, Lucy; Menghini, Timothy; Rink, Burgunde Elisabeth; Amilon, Karin; Khan, Nusrat; Péault, Bruno; Donadeu, Francesc Xavier

2017-07-01

Mesenchymal stem/stromal cells (MSCs) have been used in human and equine regenerative medicine, and interest in exploiting their potential has increased dramatically over the years. Despite significant effort to characterize equine MSCs, the actual origin of these cells and how much of their native phenotype is maintained in culture have not been determined. In this study, we investigated the relationship between MSCs, derived from adipose tissue (AT) and bone marrow (BM), and pericytes in the horse. Both pericyte (CD146, NG2, and αSMA) and MSC (CD29, CD90, and CD73) markers were detected in equine AT and colocalized around blood vessels. Importantly, as assessed by flow cytometry, both pericyte (CD146, NG2, and αSMA) and MSC (CD29, CD44, CD90, and CD105) markers were present in a majority (≥90%) of cells in cultures of AT-MSCs and BM-MSCs; however, levels of pericyte markers were variable within each of those populations. Moreover, the expression of pericyte markers was maintained for at least eight passages in both AT-MSCs and BM-MSCs. Hematopoietic (CD45) and endothelial (CD144) markers were also detected at low levels in MSCs by quantitative polymerase chain reaction (qPCR). Finally, in coculture experiments, AT-MSCs closely associated with networks produced by endothelial cells, resembling the natural perivascular location of pericytes in vivo. Our results indicate that equine MSCs originate from perivascular cells and moreover maintain a pericyte-like phenotype in culture. Therefore, we suggest that, in addition to classical MSC markers, pericyte markers such as CD146 could be used when assessing and characterizing equine MSCs.

Apigenin inhibited hypoxia induced stem cell marker expression in a head and neck squamous cell carcinoma cell line.

PubMed

Ketkaew, Yuwaporn; Osathanon, Thanaphum; Pavasant, Prasit; Sooampon, Sireerat

2017-02-01

Cancer stem cells contribute to tumor recurrence, and a hypoxic environment is critical for maintaining cancer stem cells. Apigenin is a natural product with anticancer activity. However, the effect of apigenin on cancer stem cells remains unclear. Our aim was to investigate the effect of apigenin on cancer stem cell marker expression in head and neck squamous cell carcinoma cells under hypoxia. We used three head and neck squamous cell carcinoma cell lines; HN-8, HN-30, and HSC-3. The mRNA expression of cancer stem cell markers was determined by semiquantitative RT-PCR and Real-time PCR. The cytotoxic effect of apigenin was determined by MTT colorimetric assay. Flow cytometry was used to reveal the number of cells expressing cancer stem cell surface markers. HN-30 cells, a cancer cell line from the pharynx, showed the greatest response to hypoxia by increasing their expression of CD44, CD105, NANOG, OCT-4, REX-1, and VEGF. Apigenin significantly decreased HN-30 cell viability in dose- and time-dependent manners. In addition, 40μM apigenin significantly down-regulated the mRNA expression of CD44, NANOG, and CD105. Consistent with these results, the hypoxia-induced increase in CD44 + cells, CD105 + cells, and STRO-1 + cells was significantly abolished by apigenin. Apigenin suppresses cancer stem cell marker expression and the number of cells expressing cell surface markers under hypoxia. Copyright © 2016 Elsevier Ltd. All rights reserved.

Temporal and spatial changes of cells positive for stem-like markers in different compartments and stages of human colorectal adenoma-carcinoma sequence

PubMed Central

Cui, Guanglin; Xu, Gang; Zhu, Li; Pang, Zhigang; Zheng, Wei; Li, Zhenfeng; Yuan, Aping

2017-01-01

Considerable evidence supports the idea that stem-like cells may play an essential role during the development of colorectal cancer (CRC). To accomplish this aim, we use immunohistochemistry (IHC) and double IHC with different potential stem-like markers, anti-musashi (Msi), anti-CD133, anti- LGR5 and anti-ALDH1 to examine the presentation of stem-like cells in different compartments including adenoma/CRC epithelium, transitional crypts and tumor stroma in colorectal adenoma and CRC. The results showed that cells positive for stem-like markers were remarkably increased in number and frequently observed in the adenoma/CRC epithelium, transitional crypts and tumor stroma. Notably, the population of cells positive for stem-liker markers was expanded from the base to the middle part of the transitional crypt in both adenoma and CRC tissues, reflecting that stem-like cells are likely involved in the process of colorectal tumorigenesis. Counting results showed that the grading scores of cells positive for LGR5 and ALDH1 in the adenoma/CRC epithelium were significantly increased relative with the control epithelium, and associated with the degree of dysplasia in the adenoma and node involvement in the CRC (all P < 0.05). In addition, the density of cells positive for stem–like markers in the adenomatous/cancerous stroma was also increased and paralleled an increase in the density of proliferative stromal cells labeled by PCNA, which were primarily identified as vimentin positive fibroblasts. Our results have revealed a changed temporal and spatial presentation of stem-like markers in different stages of human colorectal adenoma-carcinoma sequence, which might be a hallmark of the adenoma-carcinoma transition. PMID:28484082

Fetal bovine bone marrow is a rich source of CD34+ hematopoietic progenitors with myelo-monocytic colony-forming activity.

PubMed

Pessa-Morikawa, Tiina; Niku, Mikael; Iivanainen, Antti

2012-03-01

The CD34 glycoprotein is an important marker of hematopoietic stem cells. We used a polyclonal rabbit anti-bovine CD34 antibody to stain fetal and adult bovine bone marrow cells. Flow cytometry revealed a low side scatter (SSC(low)) population of cells that were CD34(+) but negative for leukocyte lineage markers CD11b, CD14 or CD2. Hematopoietic colony assays with CD34(+) and CD34(-) bone marrow cells suggested that the colony-forming potential in SSC(low) bone marrow cells was confined to the CD34(+) fraction. In contrast, this population was not enriched for cells expressing high aldehyde dehydrogenase activity, a metabolic marker that has been used to characterize hematopoietic stem cells. Thus, the CD34 antigen can be used to identify and isolate bovine bone marrow cells exhibiting clonogenic potential in vitro. Moreover, the proportion of CD34(+) cells is very high in fetal bovine bone marrow, indicating it as a rich source of hematopoietic progenitors. Copyright © 2011 Elsevier Ltd. All rights reserved.

Porcine cluster of differentiation (CD) markers 2018 update.

PubMed

Dawson, Harry D; Lunney, Joan K

2018-06-01

Pigs are a major source of food worldwide; preventing and treating their infectious diseases is essential, requiring a thorough understanding of porcine immunity. The use of pigs as models for human physiology is a growing area; progress in this area has been limited because the immune toolkit is not robust. The international community has established cluster of differentiation (CD) markers for assessing cells involved in immunity as well as characterizing numerous other cells like stem cells. Overall, for humans 419 proteins have been designated as CD markers, each reacting with a defined set of antibodies (Abs). This paper summarizes current knowledge of swine CD markers and identifies 359 corresponding CD proteins in pigs. A broad-based literature and vendor search was conducted to identify defined sets of monoclonal (mAbs) and polyclonal Abs (pAbs) reacting with porcine CD markers along with other reagents (fusion proteins, ELISAs, PCR assays, and gene edited cell and pig models). This process identified over 800 reagents that are reportedly reactive with 266 pig CD markers. Despite this number, there is a great need to develop and characterize additional CD marker reagents, particularly mAbs, for pig research. There are numerous high priority targets: reagents for the characterization of porcine innate lymphoid cells, polarized macrophages and T regulatory cells and for the detection of porcine CD45 isoforms. Overall, improved technologies and genomics have contributed to dramatic increases in our knowledge of the pig, its immune system, disease and vaccine responses, and utility as a biomedical model. The development of more CD reagents will clearly advance these initiatives. Published by Elsevier Ltd.

Benzene exposure is associated with cardiovascular disease risk

PubMed Central

Riggs, Daniel W.; Conklin, Daniel J.; McCracken, James P.; Srivastava, Sanjay; Xie, Zhengzhi; Rai, Shesh; Bhatnagar, Aruni; O’Toole, Timothy E.

2017-01-01

Benzene is a ubiquitous, volatile pollutant present at high concentrations in toxins (e.g. tobacco smoke) known to increase cardiovascular disease (CVD) risk. Despite its prevalence, the cardiovascular effects of benzene have rarely been studied. Hence, we examined whether exposure to benzene is associated with increased CVD risk. The effects of benzene exposure in mice were assessed by direct inhalation, while the effects of benzene exposure in humans was assessed in 210 individuals with mild to high CVD risk by measuring urinary levels of the benzene metabolite trans,trans-muconic acid (t,t-MA). Generalized linear models were used to assess the association between benzene exposure and CVD risk. Mice inhaling volatile benzene had significantly reduced levels of circulating angiogenic cells (Flk-1+/Sca-1+) as well as an increased levels of plasma low-density lipoprotein (LDL) compared with control mice breathing filtered air. In the human cohort, urinary levels of t,t-MA were inversely associated several populations of circulating angiogenic cells (CD31+/34+/45+, CD31+/34+/45+/AC133–, CD34+/45+/AC133+). Although t,t-MA was not associated with plasma markers of inflammation or thrombosis, t,t-MA levels were higher in smokers and in individuals with dyslipidemia. In smokers, t,t-MA levels were positively associated with urinary metabolites of nicotine (cotinine) and acrolein (3-hydroxymercapturic acid). Levels of t,t-MA were also associated with CVD risk as assessed using the Framingham Risk Score and this association was independent of smoking. Thus, benzene exposure is associated with increased CVD risk and deficits in circulating angiogenic cells in both smokers and non-smokers. PMID:28886060

Optimizing of the basophil activation test: Comparison of different basophil identification markers.

PubMed

Eberlein, Bernadette; Hann, Rebekka; Eyerich, Stefanie; Pennino, Davide; Ring, Johannes; Schmidt-Weber, Carsten B; Buters, Jeroen

2015-01-01

Flowcytometric identification of basophils is a prerequisite for measuring activation of basophils with IgE-dependent or IgE-independent stimuli. Aim of this study was to compare different marker combinations in a simultaneous multicolor flowcytometric measurement. Ten patients with a grass pollen allergy and three controls were included in the study. Basophilic cells were gated by using anti-CCR3, anti-IgE, anti-CRTH2, anti-CD203c, and anti-CD3. Cells were activated by a monoclonal anti-FcεRI antibody, N-formyl-methionyl-leucyl-phenylalanine (fMLP), and the allergen extract Phleum pratense. The activation marker anti-CD63 was used. The highest relative number of basophils was found with anti-CCR3+ cells, anti-IgE+ and anti-IgE+ /anti-CD203c+ cells, the lowest with CRTH2+/CD203c+/CD3- cells. A very good and good concordance of CCR3+ cells was seen with CCR3+/CD3- cells and CRTH2+/CD203c+/CD3- cells in all experiments. The contamination of the CCR3+ population with CD3+ cells and the contamination of the IgE+-population with CCR3- cells and CD203- cells were the lowest compared to all other marker combinations. As the highest relative number of basophils was identified by anti-CCR3 followed by the anti-IgE and anti-IgE/antiCD203c positive population in most cases, these markers can generally be recommended for identification of basophils. If a basophil population with very high purity is needed, anti-IgE should be chosen. © 2014 International Clinical Cytometry Society.

Extended flow cytometry characterization of normal bone marrow progenitor cells by simultaneous detection of aldehyde dehydrogenase and early hematopoietic antigens: implication for erythroid differentiation studies

PubMed Central

Mirabelli, Peppino; Di Noto, Rosa; Lo Pardo, Catia; Morabito, Paolo; Abate, Giovanna; Gorrese, Marisa; Raia, Maddalena; Pascariello, Caterina; Scalia, Giulia; Gemei, Marica; Mariotti, Elisabetta; Del Vecchio, Luigi

2008-01-01

Background Aldehyde dehydrogenase (ALDH) is a cytosolic enzyme highly expressed in hematopoietic precursors from cord blood and granulocyte-colony stimulating factor mobilized peripheral blood, as well as in bone marrow from patients with acute myeloblastic leukemia. As regards human normal bone marrow, detailed characterization of ALDH+ cells has been addressed by one single study (Gentry et al, 2007). The goal of our work was to provide new information about the dissection of normal bone marrow progenitor cells based upon the simultaneous detection by flow cytometry of ALDH and early hematopoietic antigens, with particular attention to the expression of ALDH on erythroid precursors. To this aim, we used three kinds of approach: i) multidimensional analytical flow cytometry, detecting ALDH and early hematopoietic antigens in normal bone marrow; ii) fluorescence activated cell sorting of distinct subpopulations of progenitor cells, followed by in vitro induction of erythroid differentiation; iii) detection of ALDH+ cellular subsets in bone marrow from pure red cell aplasia patients. Results In normal bone marrow, we identified three populations of cells, namely ALDH+CD34+, ALDH-CD34+ and ALDH+CD34- (median percentages were 0.52, 0.53 and 0.57, respectively). As compared to ALDH-CD34+ cells, ALDH+CD34+ cells expressed the phenotypic profile of primitive hematopoietic progenitor cells, with brighter expression of CD117 and CD133, accompanied by lower display of CD38 and CD45RA. Of interest, ALDH+CD34- population disclosed a straightforward erythroid commitment, on the basis of three orders of evidences. First of all, ALDH+CD34- cells showed a CD71bright, CD105+, CD45- phenotype. Secondly, induction of differentiation experiments evidenced a clear-cut expression of glycophorin A (CD235a). Finally, ALDH+CD34- precursors were not detectable in patients with pure red cell aplasia (PRCA). Conclusion Our study, comparing surface antigen expression of ALDH+/CD34+, ALDH-/CD34+ and ALDH+/CD34- progenitor cell subsets in human bone marrow, clearly indicated that ALDH+CD34- cells are mainly committed towards erythropoiesis. To the best of our knowledge this finding is new and could be useful for basic studies about normal erythropoietic differentiation as well as for enabling the employment of ALDH as a red cell marker in polychromatic flow cytometry characterization of bone marrow from patients with aplastic anemia and myelodysplasia. PMID:18510759

Progenitor Cells from Cartilage: Grade Specific Differences in Stem Cell Marker Expression

PubMed Central

Mazor, Marija; Cesaro, Annabelle; Ali, Mazen; Best, Thomas M.; Lespessaille, Eric; Toumi, Hechmi

2017-01-01

Recent research has confirmed the presence of Mesenchymal stem cell (MSC)-like progenitors (MPC) in both normal and osteoarthritic cartilage. However, there is only limited information concerning how MPC markers are expressed with osteoarthritis (OA) progression. The purpose of this study was to compare the prevalence of various MPC markers in different OA grades. Human osteoarthritic tibial plateaus were obtained from ten patients undergoing total knee replacement. Each sample had been classified into a mild or severe group according to OARSI scoring. Tissue was taken from each specimen and mRNA expression levels of CD105, CD166, Notch 1, Sox9, Acan and Col II A1 were measured at day 0 and day 14 (2 weeks in vitro). Furthermore, MSC markers: Nucleostemin, CD90, CD73, CD166, CD105 and Notch 1 were studied by immunofluorescence. mRNA levels of MSC markers did not differ between mild and severe OA at day 0. At day 14, protein analysis showed that proliferated cells from both sources expressed all 6 MSC markers. Only cells from the mild OA subjects resulted in a significant increase of mRNA CD105 and CD166 after in vitro expansion. Moreover, cells from the mild OA subjects showed significantly higher levels of CD105, Sox9 and Acan compared with those from severe OA specimens. Results confirmed the presence of MSC markers in mild and severe OA tissue at both mRNA and protein levels. We found significant differences between cells obtained from mild compared to severe OA specimens suggests that mild OA derived cells may have a greater MSC potential. PMID:28805694

Establishment of a pancreatic cancer stem cell model using the SW1990 human pancreatic cancer cell line in nude mice.

PubMed

Pan, Yan; Gao, Song; Hua, Yong-Qiang; Liu, Lu-Ming

2015-01-01

To establish a pancreatic cancer stem cell model using human pancreatic cancer cells in nude mice to provide a platform for pancreatic cancer stem cell research. To establish pancreatic cancer xenografts using human pancreatic cancer cell line SW1990, nude mice were randomly divided into control and gemcitabine groups. When the tumor grew to a volume of 125 mm3, they treated with gemcitabine at a dose of 50 mg/kg by intraperitoneal injection of 0.2 ml in the gemcitabine group, while the mice in control group were treated with the same volume of normal saline. Gemcitabine was given 2 times a week for 3 times. When the model was established, the proliferation of pancreatic cancer stem cells was observed by clone formation assay, and the protein and/or mRNA expression of pancreatic stem cell surface markers including CD24, CD44, CD133, ALDH, transcription factors containing Oct-4, Sox-2, Nanog and Gli, the key nuclear transcription factor in Sonic Hedgehog signaling pathway was detected by Western blot and/or RT-PCR to verify the reliability of this model. This model is feasible and safe. During the establishment, no mice died and the weight of nude mice maintained above 16.5 g. The clone forming ability in gemcitabine group was stronger than that of the control group (p<0.01). In gemcitabine group, the protein expression of pancreatic cancer stem cell surface markers including CD44, and ALDH was up-regulated, the protein and mRNA expression of nuclear transcription factor including Oct-4, Sox-2 and Nanog was also significantly increased (P<0.01). In addition, the protein expression of key nuclear transcription factor in Sonic Hedgehog signaling pathway, Gli-1, was significantly enhanced (p<0.01). The pancreatic cancer stem cell model was successfully established using human pancreatic cancer cell line SW1990 in nude mice. Gemcitabine could enrich pancreatic cancer stem cells, simultaneously accompanied by the activation of Sonic Hedgehog signaling pathway.

Isolation and characterisation of mesenchymal stem/stromal cells in the ovine endometrium.

PubMed

Letouzey, Vincent; Tan, Ker Sin; Deane, James A; Ulrich, Daniela; Gurung, Shanti; Ong, Y Rue; Gargett, Caroline E

2015-01-01

Mesenchymal stem/stromal cells (MSC) were recently discovered in the human endometrium. These cells possess key stem cell properties and show promising results in small animal models when used for preclinical tissue engineering studies. A small number of surface markers have been identified that enrich for MSC from bone marrow and human endometrium, including the Sushi Domain-containing 2 (SUSD2; W5C5) and CD271 markers. In preparation for developing a large animal preclinical model for urological and gynecological tissue engineering applications we aimed to identify and characterise MSC in ovine endometrium and determine surface markers to enable their prospective isolation. Ovine endometrium was obtained from hysterectomised ewes following progesterone synchronisation, dissociated into single cell suspensions and tested for MSC surface markers and key stem cell properties. Purified stromal cells were obtained by flow cytometry sorting with CD49f and CD45 to remove epithelial cells and leukocytes respectively, and MSC properties investigated. There was a small population CD271+ stromal cells (4.5 ± 2.3%) in the ovine endometrium. Double labelling with CD271 and CD49f showed that the sorted CD271+CD49f- stromal cell population possessed significantly higher cloning efficiency, serial cloning capacity and a qualitative increased ability to differentiate into 4 mesodermal lineages (adipocytic, smooth muscle, chondrocytic and osteoblastic) than CD271-CD49f- cells. Immunolabelling studies identified an adventitial perivascular location for ovine endometrial CD271+ cells. This is the first study to characterise MSC in the ovine endometrium and identify a surface marker profile identifying their location and enabling their prospective isolation. This knowledge will allow future preclinical studies with a large animal model that is well established for pelvic organ prolapse research.

Curcumin and metformin-mediated chemoprevention of oral cancer is associated with inhibition of cancer stem cells.

PubMed

Siddappa, Gangotri; Kulsum, Safeena; Ravindra, Doddathimmasandra Ramanjanappa; Kumar, Vinay V; Raju, Nalini; Raghavan, Nisheena; Sudheendra, Holalugunda Vittalamurthy; Sharma, Anupam; Sunny, Sumsum P; Jacob, Tina; Kuruvilla, Binu T; Benny, Merina; Antony, Benny; Seshadri, Mukund; Lakshminarayan, Padma; Hicks, Wesley; Suresh, Amritha; Kuriakose, Moni A

2017-11-01

Effective chemoprevention is critical for improving outcomes of oral cancer. As single agents, curcumin and metformin are reported to exhibit chemopreventive properties, in vitro as well as in patients with oral cancer. In this study, the chemopreventive efficacy of this drug combination was tested in a 4-nitro quinoline-1-oxide (4NQO) induced mice oral carcinogenesis model. Molecular analysis revealed a cancer stem cell (CSC)-driven oral carcinogenic progression in this model, wherein a progressive increase in the expression of CSC-specific markers (CD44 and CD133) was observed from 8th to 25th week, at transcript (40-100-fold) and protein levels (P ≤ 0.0001). Chemopreventive treatment of the animals at 17th week with curcumin and metformin indicated that the combination regimen decreased tumor volume when compared to the control arm (0.69+0.03 vs 6.66+2.4 mm 3 ; P = 0.04) and improved overall survival of the animals (P = 0.03). Assessment of the molecular status showed an overall downregulation of CSC markers in the treatment arms as compared to the untreated control. Further, in vitro assessment of the treatment on the primary cells generated from progressive stages of 4NQO-induced mice tissue showed a concordant and consistent downregulation of the CSC markers following combination treatment (P < 0.05). The treatment also inhibited the migratory and self-renewal properties of these cells; the effect of which was prominent in the cultures of early dysplastic tissue (P < 0.002). Collectively, our observations suggest that the combination of curcumin and metformin may improve chemopreventive efficacy against oral squamous cell carcinoma through a CSC-associated mechanism. © 2017 Wiley Periodicals, Inc.

Expression of microRNA-133 inhibits epithelial-mesenchymal transition in lung cancer cells by directly targeting FOXQ1.

PubMed

Xiao, Bo; Liu, Huazhen; Gu, Zeyun; Ji, Cheng

2016-10-01

MicroRNA (miR) was implicated in the tumorigenesis of many types of cancer, but no study was conducted on the exact role of miR-133 in lung cancer. We have identified miR-133 as a putative regulator of FOXQ1 expression, and investigated the potential involvement of miR-133 in the migration and invasion of lung cancer cells, as well as the underlying molecular mechanism. MiR-133 directly targeted and down-regulated FOXQ1 expression, which in turn reduced TGF-β level. MiR-133 was down-regulated in lung cancer cell lines A549 and HCC827, and its re-expression significantly inhibited the migration and invasion of the lung cancer cells. Further investigation revealed that this inhibition was caused by reversing the epithelial-mesenchymal transition, evidenced by miR-133 induced elevation of epithelial marker E-cadherin, and reduction of mesenchymal marker Vimentin. Our study is the first to identify miR-133 as a biomarker for lung cancer. It functions to down-regulate FOXQ1, and inhibit epithelial-mesenchymal transition, which antagonizes lung cancer tumorigenesis. Therefore our data support the role of miR-133 as a potential molecular therapeutic tool in treating lung cancer. Copyright © 2015 SEPAR. Publicado por Elsevier España, S.L.U. All rights reserved.

CD271 Defines a Stem Cell-Like Population in Hypopharyngeal Cancer

PubMed Central

Imai, Takayuki; Tamai, Keiichi; Oizumi, Sayuri; Oyama, Kyoko; Yamaguchi, Kazunori; Sato, Ikuro; Satoh, Kennichi; Matsuura, Kazuto; Saijo, Shigeru; Sugamura, Kazuo; Tanaka, Nobuyuki

2013-01-01

Cancer stem cells contribute to the malignant phenotypes of a variety of cancers, but markers to identify human hypopharyngeal cancer (HPC) stem cells remain poorly understood. Here, we report that the CD271+ population sorted from xenotransplanted HPCs possesses an enhanced tumor-initiating capability in immunodeficient mice. Tumors generated from the CD271+ cells contained both CD271+ and CD271− cells, indicating that the population could undergo differentiation. Immunohistological analyses of the tumors revealed that the CD271+ cells localized to a perivascular niche near CD34+ vasculature, to invasive fronts, and to the basal layer. In accordance with these characteristics, a stemness marker, Nanog, and matrix metalloproteinases (MMPs), which are implicated in cancer invasion, were significantly up-regulated in the CD271+ compared to the CD271− cell population. Furthermore, using primary HPC specimens, we demonstrated that high CD271 expression was correlated with a poor prognosis for patients. Taken together, our findings indicate that CD271 is a novel marker for HPC stem-like cells and for HPC prognosis. PMID:23626764

Advanced glycation end products, carotid atherosclerosis, and circulating endothelial progenitor cells in patients with end-stage renal disease.

PubMed

Ueno, Hiroki; Koyama, Hidenori; Fukumoto, Shinya; Tanaka, Shinji; Shoji, Takuhito; Shoji, Tetsuo; Emoto, Masanori; Tahara, Hideki; Inaba, Masaaki; Kakiya, Ryusuke; Tabata, Tsutomu; Miyata, Toshio; Nishizawa, Yoshiki

2011-04-01

Numbers of endothelial progenitor cells (EPCs) have been shown to be decreased in subjects with end-stage renal disease (ESRD), the mechanism of which remained poorly understood. In this study, mutual association among circulating EPC levels, carotid atherosclerosis, serum pentosidine, and skin autofluorescence, a recently established noninvasive measure of advanced glycation end products accumulation, was examined in 212 ESRD subjects undergoing hemodialysis. Numbers of circulating EPCs were measured as CD34+ CD133+ CD45(low) VEGFR2+ cells and progenitor cells as CD34+ CD133+ CD45(low) fraction by flow cytometry. Skin autofluorescence was assessed by the autofluorescence reader; and serum pentosidine, by enzyme-linked immunosorbent assay. Carotid atherosclerosis was determined as intimal-medial thickness (IMT) measured by ultrasound. Circulating EPCs were significantly and inversely correlated with skin autofluorescence in ESRD subjects (R = -0.216, P = .002), but not with serum pentosidine (R = -0.079, P = .25). Circulating EPCs tended to be inversely associated with IMT (R = -0.125, P = .069). Intimal-medial thickness was also tended to be correlated positively with skin autofluorescence (R = 0.133, P = .054) and significantly with serum pentosidine (R = 0.159, P = .019). Stepwise multiple regression analyses reveal that skin autofluorescence, but not serum pentosidine and IMT, was independently associated with low circulating EPCs. Of note, skin autofluorescence was also inversely and independently associated with circulating progenitor cells. Thus, tissue accumulated, but not circulating, advanced glycation end products may be a determinant of a decrease in circulating EPCs in ESRD subjects. Copyright © 2011 Elsevier Inc. All rights reserved.

USP1 targeting impedes GBM growth by inhibiting stem cell maintenance and radioresistance.

PubMed

Lee, Jin-Ku; Chang, Nakho; Yoon, Yeup; Yang, Heekyoung; Cho, Heejin; Kim, Eunhee; Shin, Yongjae; Kang, Wonyoung; Oh, Young Taek; Mun, Gyeong In; Joo, Kyeung Min; Nam, Do-Hyun; Lee, Jeongwu

2016-01-01

Clinical benefits from standard therapies against glioblastoma (GBM) are limited in part due to intrinsic radio- and chemoresistance of GBM and inefficient targeting of GBM stem-like cells (GSCs). Novel therapeutic approaches that overcome treatment resistance and diminish stem-like properties of GBM are needed. We determined the expression levels of ubiquitination-specific proteases (USPs) by transcriptome analysis and found that USP1 is highly expressed in GBM. Using the patient GBM-derived primary tumor cells, we inhibited USP1 by shRNA-mediated knockdown or its specific inhibitor pimozide and evaluated the effects on stem cell marker expression, proliferation, and clonogenic growth of tumor cells. USP1 was highly expressed in gliomas relative to normal brain tissues and more preferentially in GSC enrichment marker (CD133 or CD15) positive cells. USP1 positively regulated the protein stability of the ID1 and CHEK1, critical regulators of DNA damage response and stem cell maintenance. Targeting USP1 by RNA interference or treatment with a chemical USP1 inhibitor attenuated clonogenic growth and survival of GSCs and enhanced radiosensitivity of GBM cells. Finally, USP1 inhibition alone or in combination with radiation significantly prolonged the survival of tumor-bearing mice. USP1-mediated protein stabilization promotes GSC maintenance and treatment resistance, thereby providing a rationale for USP1 inhibition as a potential therapeutic approach against GBM. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Identification of active and quiescent adipose vascular stromal cells.

PubMed

Lin, Guiting; Xin, Zhongcheng; Zhang, Haiyang; Banie, Lia; Wang, Guifang; Qiu, Xuefeng; Ning, Hongxiu; Lue, Tom F; Lin, Ching-Shwun

2012-02-01

Recent studies have demonstrated the existence of both active and quiescent stem cells in bone marrow, hair follicle and intestine. We attempted to identify active and quiescent vascular stromal cells (VSC) in adipose tissue. For identification of active VSC, adult rats were injected intraperitoneally with thymidine analog 5-ethynyl-2-deoxyuridine (EdU) and their subcutaneous tissue harvested 3 days later. For identification of quiescent VSC, newborn rats were injected intraperitoneally with EdU and their subcutaneous tissue harvested 9 weeks later. The harvested adipose tissues were examined for the co-localization of EdU with VSC marker CD34, smooth muscle marker SMA, endothelial marker RECA and pericyte marker CD140b. In adult rat adipose tissues harvested 3 days after EdU injection, there were 28.80 ± 8.70 (mean ± SD) EdU+ cells/100 × microscopic field, and approximately 6.2% of cell nuclei were labeled with EdU. The percentages of EdU+ cells expressing the following markers were approximately: 84 for CD34, 5.6 for RECA (rat endothelial marker), 3.7 for SMA and 14.8 for CD140b. In the adipose tissues of newborn rats that were harvested 9 weeks after EdU injection, the percentages of EdU+ cells expressing the following markers were approximately: 76 for CD34, 1.8 for RECA, 0 for SMA and 12.9 for CD140b. In both the short-term (active) and long-term (quiescent) EdU-labeled adipose tissues, the EdU label was consistently co-localized with CD34 and in the proximity of CD140b stain or in the adventitia. Both active and quiescent VSC expressed CD34 and localized to capillaries and the adventitia of larger blood vessels.

Tetraspecific scFv construct provides NK cell mediated ADCC and self-sustaining stimuli via insertion of IL-15 as a cross-linker

PubMed Central

Schmohl, Joerg U.; Felices, Martin; Todhunter, Deborah; Taras, Elizabeth; Miller, Jeffrey S.; Vallera, Daniel A.

2016-01-01

Background The design of a highly effective anti-cancer immune-engager would include targeting of highly drug refractory cancer stem cells (CSC). The design would promote effective antibody-dependent cell-mediated cytotoxicity (ADCC) and simultaneously promote costimulation to expand and self-sustain the effector NK cell population. Based on our bispecific NK cell engager platform we constructed a tetraspecific killer engager (TetraKE) comprising single-chain variable fragments (scFvs) binding FcγRIII (CD16) on NK cells, EpCAM on carcinoma cells and CD133 on cancer stem cells in order to promote ADCC. Furthermore, an Interleukin (IL)-15-crosslinker enhanced NK cell related proliferation resulting in a highly active drug termed 1615EpCAM133. Results Proliferation assays showed TetraKE promoted proliferation and enhanced NK cell survival. Drug-target binding, NK cell related degranulation, and IFN-γ production was specific for both tumor related antigens in EpCAM and CD133 bearing cancer cell lines. The TetraKE showed higher killing activity and superior dose dependent degranulation. Cytokine profiling showed a moderately enhanced IFN-γ production, enhanced GM-CSF production, but no evidence of induction of excessive cytokine release. Methods Assembly and synthesis of hybrid genes encoding the TetraKE were performed using DNA shuffling and ligation. The TetraKE was tested for efficacy, specificity, proliferation, survival, and cytokine production using carcinoma cell lines and functional assays measuring NK cell activity. Conclusion 1615EpCAM133 combines improved induction of ADCC with enhanced proliferation, limited cytokine response, and prolonged survival and proliferation of NK cells. By linking scFv-related targeting of carcinoma and CSCs with a sustaining IL-15 signal, our new construct shows great promise to target cancer and CSCs. PMID:27650544

Characterization of aldehyde dehydrogenase 1 high ovarian cancer cells: Towards targeted stem cell therapy.

PubMed

Sharrow, Allison C; Perkins, Brandy; Collector, Michael I; Yu, Wayne; Simons, Brian W; Jones, Richard J

2016-08-01

The cancer stem cell (CSC) paradigm hypothesizes that successful clinical eradication of CSCs may lead to durable remission for patients with ovarian cancer. Despite mounting evidence in support of ovarian CSCs, their phenotype and clinical relevance remain unclear. We and others have found high aldehyde dehydrogenase 1 (ALDH(high)) expression in a variety of normal and malignant stem cells, and sought to better characterize ALDH(high) cells in ovarian cancer. We compared ALDH(high) to ALDH(low) cells in two ovarian cancer models representing distinct subtypes: FNAR-C1 cells, derived from a spontaneous rat endometrioid carcinoma, and the human SKOV3 cell line (described as both serous and clear cell subtypes). We assessed these populations for stem cell features then analyzed expression by microarray and qPCR. ALDH(high) cells displayed CSC properties, including: smaller size, quiescence, regenerating the phenotypic diversity of the cell lines in vitro, lack of contact inhibition, nonadherent growth, multi-drug resistance, and in vivo tumorigenicity. Microarray and qPCR analysis of the expression of markers reported by others to enrich for ovarian CSCs revealed that ALDH(high) cells of both models showed downregulation of CD24, but inconsistent expression of CD44, KIT and CD133. However, the following druggable targets were consistently expressed in the ALDH(high) cells from both models: mTOR signaling, her-2/neu, CD47 and FGF18/FGFR3. Based on functional characterization, ALDH(high) ovarian cancer cells represent an ovarian CSC population. Differential gene expression identified druggable targets that have the potential for therapeutic efficacy against ovarian CSCs from multiple subtypes. Copyright © 2016 Elsevier Inc. All rights reserved.

γ/δ T cell subsets in human aging using the classical α/β T cell model.

PubMed

Vasudev, Anusha; Ying, Crystal Tan Tze; Ayyadhury, Shamini; Puan, Kia Joo; Andiappan, Anand Kumar; Nyunt, Ma Shwe Zin; Shadan, Nurhidaya Binte; Mustafa, Seri; Low, Ivy; Rotzschke, Olaf; Fulop, Tamas; Ng, Tze Pin; Larbi, Anis

2014-10-01

Aging is associated with an increased susceptibility to infections and diseases. It has also been associated with reduced functionality and altered distribution of immune cells, especially T cells. Whereas classical α/β T cells, especially CD8(+) T cells, were shown to be highly susceptible to aging, the effects of viral persistent stimulations on the fate of γ/δ T cells are much less documented. Healthy, elderly individuals of Chinese ethnical background were recruited under the aegis of SLAS-II. In this observational study, γ/δ T cell populations were characterized by flow cytometry and compared with the α/β CD4(+) and CD8(+) T cells in elderly and young controls. In our study, we identified a reduced frequency of γ/δ T cells but not α/β T cells with aging. The classical markers of α/β T cell aging, including CD28, CD27, and CD57, did not prove significant for γ/δ T cells. The extreme range of expression of these markers in γ/δ T cells was responsible for the lack of relationship between γ/δ T cell subsets, CD4/CD8 ratio, and anti-CMV titers that was significant for α/β T cells and, especially, CD8(+) T cells. Although markers of aging for γ/δ T cells are not clearly identified, our data collectively suggest that the presence of CD27 γ/δ T cells is associated with markers of α/β T cell aging. © 2014 Society for Leukocyte Biology.

Transcriptional Networks in Single Perivascular Cells Sorted from Human Adipose Tissue Reveal a Hierarchy of Mesenchymal Stem Cells.

PubMed

Hardy, W Reef; Moldovan, Nicanor I; Moldovan, Leni; Livak, Kenneth J; Datta, Krishna; Goswami, Chirayu; Corselli, Mirko; Traktuev, Dmitry O; Murray, Iain R; Péault, Bruno; March, Keith

2017-05-01

Adipose tissue is a rich source of multipotent mesenchymal stem-like cells, located in the perivascular niche. Based on their surface markers, these have been assigned to two main categories: CD31 - /CD45 - /CD34 + /CD146 - cells (adventitial stromal/stem cells [ASCs]) and CD31 - /CD45 - /CD34 - /CD146 + cells (pericytes [PCs]). These populations display heterogeneity of unknown significance. We hypothesized that aldehyde dehydrogenase (ALDH) activity, a functional marker of primitivity, could help to better define ASC and PC subclasses. To this end, the stromal vascular fraction from a human lipoaspirate was simultaneously stained with fluorescent antibodies to CD31, CD45, CD34, and CD146 antigens and the ALDH substrate Aldefluor, then sorted by fluorescence-activated cell sorting. Individual ASCs (n = 67) and PCs (n = 73) selected from the extremities of the ALDH-staining spectrum were transcriptionally profiled by Fluidigm single-cell quantitative polymerase chain reaction for a predefined set (n = 429) of marker genes. To these single-cell data, we applied differential expression and principal component and clustering analysis, as well as an original gene coexpression network reconstruction algorithm. Despite the stochasticity at the single-cell level, covariation of gene expression analysis yielded multiple network connectivity parameters suggesting that these perivascular progenitor cell subclasses possess the following order of maturity: (a) ALDH br ASC (most primitive); (b) ALDH dim ASC; (c) ALDH br PC; (d) ALDH dim PC (least primitive). This order was independently supported by specific combinations of class-specific expressed genes and further confirmed by the analysis of associated signaling pathways. In conclusion, single-cell transcriptional analysis of four populations isolated from fat by surface markers and enzyme activity suggests a developmental hierarchy among perivascular mesenchymal stem cells supported by markers and coexpression networks. Stem Cells 2017;35:1273-1289. © 2017 AlphaMed Press.

Comparison of differentiation potential of male mouse adipose tissue and bone marrow derived-mesenchymal stem cells into germ cells

PubMed Central

Hosseinzadeh Shirzeily, Maryam; Pasbakhsh, Parichehr; Amidi, Fardin; Mehrannia, Kobra; Sobhani, Aligholi

2013-01-01

Background: Recent publications about differentiation of stem cells to germ cells have motivated researchers to make new approaches to infertility. In vitro production of germ cells improves understanding differentiation process of male and female germ cells. Due to the problem of using embryonic stem cells (ESC), it’s necessary the mentioned cells be replaced with some adult multi-potent stem cells in laboratories. Objective: The aim of this study was to obtain germ cells from appropriate source beyond ESC and compare differential potentials of adipocytes derived stem cells (ADMSCs) with bone marrow derived stem cells (BMMSCs). Materials and Methods: To find multi-potential entity, after providing purified ADMSCs and BMMSCs, differentiation to osteoblast and adipocyte was confirmed by using appropriate culture medium. To confirm mesenchymal lineage production superficial markers (expression of CD90 and CD44 and non-expression of CD45 and CD31) were investigated by flowcytometry. Then the cells were differentiated to germ cells in inductive medium containing retinoic acid for 7days. To evaluate germ cells characteristic markers [Dazl (Deleted in azoospermia-like), Mvh (Mouse vasa homolog gene), Stra8 (Stimulated by retinoic acid) and Scp3 (Synaptonemal complex protein 3)] flowcytometry, imunoflorescence and real time PCR were used. Results: Both types of cells were able to differentiate into osteoblast and adipocyte cells and presentation of stem cell superficial markers (CD90, CD44) and absence of endothelial and blood cell markers (CD31, CD45) were confirmative The flowcytometry, imunoflorescence and real time PCR results showed remarkable expression of germ cells characteristic markers (Mvh, Dazl, Stra8, and Scp3). Conclusion: It was found that although ADMSCs were attained easier and also cultured and differentiated rapidly, germ cell markers were expressed in BMMSCs significantly more than ADMSCs. This article extracted from M.Sc. thesis. (Maryam Hosseinzadeh Shirzeily) PMID:24639722

Mesenchymal stem cells in the Wharton's jelly of the human umbilical cord.

PubMed

Wang, Hwai-Shi; Hung, Shih-Chieh; Peng, Shu-Tine; Huang, Chun-Chieh; Wei, Hung-Mu; Guo, Yi-Jhih; Fu, Yu-Show; Lai, Mei-Chun; Chen, Chin-Chang

2004-01-01

The Wharton's jelly of the umbilical cord contains mucoid connective tissue and fibroblast-like cells. Using flow cytometric analysis, we found that mesenchymal cells isolated from the umbilical cord express matrix receptors (CD44, CD105) and integrin markers (CD29, CD51) but not hematopoietic lineage markers (CD34, CD45). Interestingly, these cells also express significant amounts of mesenchymal stem cell markers (SH2, SH3). We therefore investigated the potential of these cells to differentiate into cardiomyocytes by treating them with 5-azacytidine or by culturing them in cardiomyocyte-conditioned medium and found that both sets of conditions resulted in the expression of cardiomyocyte markers, namely N-cadherin and cardiac troponin I. We also showed that these cells have multilineage potential and that, under suitable culture conditions, are able to differentiate into cells of the adipogenic and osteogenic lineages. These findings may have a significant impact on studies of early human cardiac differentiation, functional genomics, pharmacological testing, cell therapy, and tissue engineering by helping to eliminate worrying ethical and technical issues.

The Impact of Sex Work Interruption on Blood-Derived T Cells in Sex Workers from Nairobi, Kenya.

PubMed

Omollo, Kenneth; Boily-Larouche, Geneviève; Lajoie, Julie; Kimani, Makobu; Cheruiyot, Julianna; Kimani, Joshua; Oyugi, Julius; Fowke, Keith Raymond

Unprotected sexual intercourse exposes the female genital tract (FGT) to semen-derived antigens, which leads to a proinflammatory response. Studies have shown that this postcoital inflammatory response can lead to recruitment of activated T cells to the FGT, thereby increasing risk of HIV infection. The purpose of this study was to evaluate the impact of sex work on activation and memory phenotypes of peripheral T cells among female sex workers (FSW) from Nairobi, Kenya. Thirty FSW were recruited from the Pumwani Sex Workers Cohort, 10 in each of the following groups: HIV-exposed seronegative (at least 7 years in active sex work), HIV positive, and New Negative (HIV negative, less than 3 years in active sex work). Blood was obtained at three different phases (active sex work, abstinence from sex work-sex break, and following resumption of sex work). Peripheral blood mononuclear cells were isolated and stained for phenotypic markers (CD3, CD4, CD8, and CD161), memory phenotype markers (CD45RA and CCR7), activation markers (CD69, HLA-DR, and CD95), and the HIV coreceptor (CCR5). T-cell populations were compared between groups. In HIV-positive women, CD8+CCR5+ T cells declined at the sex break period, while CD4+CD161+ T cells increased when returning to sex work. All groups showed no significant changes in systemic T-cell activation markers following the interruption of sex work, however, significant reductions in naive CD8+ T cells were noted. For each of the study points, HIV positives had higher effector memory and CD8+CD95+ T cells and lower naive CD8+ T cells than the HIV-uninfected groups. Interruption of sex work had subtle effects on systemic T-cell memory phenotypes.

A double blinded, placebo-controlled pilot study to examine reduction of CD34 +/CD117 +/CD133 + lymphoma progenitor cells and duration of remission induced by neoadjuvant valspodar in dogs with large B-cell lymphoma

PubMed Central

Ito, Daisuke; Childress, Michael; Mason, Nicola; Winter, Amber; O’Brien, Timothy; Henson, Michael; Borgatti, Antonella; Lewellen, Mitzi; Krick, Erika; Stewart, Jane; Lahrman, Sarah; Rajwa, Bartek; Scott, Milcah C; Seelig, Davis; Koopmeiners, Joseph; Ruetz, Stephan; Modiano, Jaime

2017-01-01

We previously described a population of lymphoid progenitor cells (LPCs) in canine B-cell lymphoma defined by retention of the early progenitor markers CD34 and CD117 and “slow proliferation” molecular signatures that persist in the xenotransplantation setting. We examined whether valspodar, a selective inhibitor of the ATP binding cassette B1 transporter (ABCB1, a.k.a., p-glycoprotein/multidrug resistance protein-1) used in the neoadjuvant setting would sensitize LPCs to doxorubicin and extend the length of remission in dogs with therapy naïve large B-cell lymphoma. Twenty dogs were enrolled into a double-blinded, placebo controlled study where experimental and control groups received oral valspodar (7.5 mg/kg) or placebo, respectively, twice daily for five days followed by five treatments with doxorubicin 21 days apart with a reduction in the first dose to mitigate the potential side effects of ABCB1 inhibition. Lymph node and blood LPCs were quantified at diagnosis, on the fourth day of neoadjuvant period, and 1-week after the first chemotherapy dose. Valspodar therapy was well tolerated. There were no differences between groups in total LPCs in lymph nodes or peripheral blood, nor in event-free survival or overall survival. Overall, we conclude that valspodar can be administered safely in the neoadjuvant setting for canine B-cell lymphoma; however, its use to attenuate ABCB1 + cells does not alter the composition of lymph node or blood LPCs, and it does not appear to be sufficient to prolong doxorubicin-dependent remissions in this setting. PMID:28357033

Dendritic Cell-Based Genetic Immunotherapy for Ovarian Cancer

DTIC Science & Technology

2007-12-01

CAR. CD40 is a surface marker expressed by DCs that plays a crucial role in their maturation and subsequent stimulation of T cells. DC infection with... surface . CD40 is a cell surface marker expressed by DCs, is crucial for their maturation and the subsequent activation of the immune system by the DCs...cell surface . CD40 is a cell surface marker expressed by DCs, is crucial for their maturation and the subsequent activation of the immune system by the

Burn-injury affects gut-associated lymphoid tissues derived CD4+ T cells.

PubMed

Fazal, Nadeem; Shelip, Alla; Alzahrani, Alhusain J

2013-01-01

After scald burn-injury, the intestinal immune system responds to maintain immune balance. In this regard CD4+T cells in Gut-Associated Lymphoid Tissues (GALT), like mesenteric lymph nodes (MLN) and Peyer's patches (PP) respond to avoid immune suppression following major injury such as burn. Therefore, we hypothesized that the gut CD4+T cells become dysfunctional and turn the immune homeostasis towards depression of CD4+ T cell-mediated adaptive immune responses. In the current study we show down regulation of mucosal CD4+ T cell proliferation, IL-2 production and cell surface marker expression of mucosal CD4+ T cells moving towards suppressive-type. Acute burn-injury lead to up-regulation of regulatory marker (CD25+), down regulation of adhesion (CD62L, CD11a) and homing receptor (CD49d) expression, and up-regulation of negative co-stimulatory (CTLA-4) molecule. Moreover, CD4+CD25+ T cells of intestinal origin showed resistance to spontaneous as well as induced apoptosis that may contribute to suppression of effector CD4+ T cells. Furthermore, gut CD4+CD25+ T cells obtained from burn-injured animals were able to down-regulate naïve CD4+ T cell proliferation following adoptive transfer of burn-injured CD4+CD25+ T cells into sham control animals, without any significant effect on cell surface activation markers. Together, these data demonstrate that the intestinal CD4+ T cells evolve a strategy to promote suppressive CD4+ T cell effector responses, as evidenced by enhanced CD4+CD25+ T cells, up-regulated CTLA-4 expression, reduced IL-2 production, tendency towards diminished apoptosis of suppressive CD4+ T cells, and thus lose their natural ability to regulate immune homeostasis following acute burn-injury and prevent immune paralysis.

Prognostic value of CD44 expression in penile squamous cell carcinoma: a pilot study.

PubMed

Minardi, Daniele; Lucarini, Guendalina; Filosa, Alessandra; Zizzi, Antonio; Simonetti, Oriana; Offidani, Anna Maria; d'Anzeo, Gianluca; Di Primio, Roberto; Montironi, Rodolfo; Muzzonigro, Giovanni

2012-10-01

Several studies have reported on the prognostic value of molecular markers for metastasis risk and survival in penile squamous cell carcinoma (SCC) patients. The usefulness of CD44 expression as such a marker has been studied in different tumors, but not in penile SCC. Our aim was to determine whether CD44 expression may serve as a prognostic marker for lymph node metastasis and survival in penile SCC patients. CD44 immunoistochemical expression was investigated in tissue specimens from 39 patients with penile SCC. CD44 cell positivity, staining intensity and distribution were analyzed and correlated with tumor stage, grade, lymph node status and disease-specific survival. CD44 expression was detected in epithelial cells of both intratumoral and normal tissues with different intensities and staining distributions. In normal tissues CD44 protein was mainly detected in cell membranes, whereas in the tumor compartments it was found in both the cell membranes and the cytoplasm. The intensities and percentages of CD44 expressing cells did not correlate with tumor stage and/or grade. Seventy-three percent of the patients with lymph node metastasis showed high intensities of CD44 staining, as compared to 44% of the patients without lymph node metastasis (P = 0.03). Lymph node-positive patients showed both cytoplasmic and membranous CD44 expression. High CD44 expression was found to be significantly correlated with a decreased 5 year overall survival (P = 0.01). CD44 levels and patterns of expression can be considered as markers for penile SCC aggressiveness and, in addition, may serve as predictive markers for lymph node metastasis, also in patients with clinically negative lymph nodes. CD44 expression may provide prognostic information for penile SCC patients, next to classical clinical-pathological factors.

The CD11a and Endothelial Protein C Receptor Marker Combination Simplifies and Improves the Purification of Mouse Hematopoietic Stem Cells

PubMed Central

Karimzadeh, Alborz; Scarfone, Vanessa M.; Varady, Erika; Chao, Connie; Grathwohl, Karin; Fathman, John W.; Fruman, David A.; Serwold, Thomas

2018-01-01

Abstract Hematopoietic stem cells (HSCs) are the self‐renewing multipotent progenitors to all blood cell types. Identification and isolation of HSCs for study has depended on the expression of combinations of surface markers on HSCs that reliably distinguish them from other cell types. However, the increasing number of markers required to isolate HSCs has made it tedious, expensive, and difficult for newcomers, suggesting the need for a simpler panel of HSC markers. We previously showed that phenotypic HSCs could be separated based on expression of CD11a and that only the CD11a negative fraction contained true HSCs. Here, we show that CD11a and another HSC marker, endothelial protein C receptor (EPCR), can be used to effectively identify and purify HSCs. We introduce a new two‐color HSC sorting method that can highly enrich for HSCs with efficiencies comparable to the gold standard combination of CD150 and CD48. Our results demonstrate that adding CD11a and EPCR to the HSC biologist's toolkit improves the purity of and simplifies isolation of HSCs. stem cells translational medicine 2018;7:468–476 PMID:29543389

[The expression and significance of CD(276) and CD(133) in colorectal cancer and precancerous lesions].

PubMed

Lu, G F; Huang, L N; Ren, J L; Hu, G M; Zheng, Z H; Wu, J X; Zhu, Y P; Tang, F A

2018-06-01

In order to study the significance of CD(276) and CD(133) in the development and progression of colorectal cancer (CRC), the expression of CD(276) and CD(133) was detected by immunohistochemistry in CRC and precancerous lesions. The results showed that the intensity of CD(276) and CD(133) in CRC samples was higher than that in adenoma group and non-adenoma group. CD(276) and CD(133) single and double positive expression were significantly correlated with CRC lymph node metastasis, distant metastasis and survival. CD(276) and CD(133) are significantly correlated to the development and progression of CRC and associated with poor prognosis.

Normal T-cell activation in elite controllers with preserved CD4+ T-cell counts.

PubMed

Bansal, Anju; Sterrett, Sarah; Erdmann, Nathan; Westfall, Andrew O; Dionne-Odom, Jodie; Overton, Edgar T; Goepfert, Paul A

2015-11-01

HIV elite controllers suppress HIV viremia without antiretroviral therapy (ART), yet previous studies demonstrated that elite controllers maintain an activated T-cell phenotype. Chronic immune activation has detrimental consequences and thus ART has been advocated for all elite controllers. However, elite controllers are not a clinically homogenous group. Since CD4% is among the best predictors of AIDS-related events, in the current study, we assessed whether this marker can be used to stratify elite controllers needing ART. Sixteen elite controllers were divided into two groups based on CD4% (EC > 40% and EC ≤40%), and T-cell subsets were analyzed for markers of memory/differentiation (CD45RA, CCR7, CD28), activation (CD38/HLA-DR), immunosenescence (CD57), costimulation (CD73, CD28) and exhaustion (PD-1, CD160, Tim-3). Monocyte subsets (CD14, CD16) were also analyzed and sCD14 levels were quantified using ELISA. In the EC group, expression of activation, exhaustion, and immunosensescence markers on T cells were significantly reduced compared with the EC group and similar to the seronegative controls. The EC group expressed higher levels of costimulatory molecules CD28 and CD73 and had lower levels of monocyte activation (HLA-DR expression) with a reduced frequency of inflammatory monocyte (CD14 CD16) subset. Furthermore, the EC group maintained a stable CD4% during a median follow-up of 6 years. Elite controllers with preserved CD4T cells (EC) have normal T-cell and monocyte phenotypes and therefore may have limited benefit from ART. CD4% can be an important marker for evaluating future studies aimed at determining the need for ART in this group of individuals.

Isolation and characterisation of human gingival margin-derived STRO-1/MACS+ and MACS− cell populations

PubMed Central

El-Sayed, Karim M Fawzy; Paris, Sebastian; Graetz, Christian; Kassem, Neemat; Mekhemar, Mohamed; Ungefroren, Hendrick; Fändrich, Fred; Dörfer, Christof

2015-01-01

Recently, gingival margin-derived stem/progenitor cells isolated via STRO-1/magnetic activated cell sorting (MACS) showed remarkable periodontal regenerative potential in vivo. As a second-stage investigation, the present study's aim was to perform in vitro characterisation and comparison of the stem/progenitor cell characteristics of sorted STRO-1-positive (MACS+) and STRO-1-negative (MACS−) cell populations from the human free gingival margin. Cells were isolated from the free gingiva using a minimally invasive technique and were magnetically sorted using anti-STRO-1 antibodies. Subsequently, the MACS+ and MACS− cell fractions were characterized by flow cytometry for expression of CD14, CD34, CD45, CD73, CD90, CD105, CD146/MUC18 and STRO-1. Colony-forming unit (CFU) and multilineage differentiation potential were assayed for both cell fractions. Mineralisation marker expression was examined using real-time polymerase chain reaction (PCR). MACS+ and MACS− cell fractions showed plastic adherence. MACS+ cells, in contrast to MACS− cells, showed all of the predefined mesenchymal stem/progenitor cell characteristics and a significantly higher number of CFUs (P<0.01). More than 95% of MACS+ cells expressed CD105, CD90 and CD73; lacked the haematopoietic markers CD45, CD34 and CD14, and expressed STRO-1 and CD146/MUC18. MACS− cells showed a different surface marker expression profile, with almost no expression of CD14 or STRO-1, and more than 95% of these cells expressed CD73, CD90 and CD146/MUC18, as well as the haematopoietic markers CD34 and CD45 and CD105. MACS+ cells could be differentiated along osteoblastic, adipocytic and chondroblastic lineages. In contrast, MACS− cells demonstrated slight osteogenic potential. Unstimulated MACS+ cells showed significantly higher expression of collagen I (P<0.05) and collagen III (P<0.01), whereas MACS− cells demonstrated higher expression of osteonectin (P<0.05; Mann–Whitney). The present study is the first to compare gingival MACS+ and MACS− cell populations demonstrating that MACS+ cells, in contrast to MACS− cells, harbour stem/progenitor cell characteristics. This study also validates the effectiveness of the STRO-1/MACS+ technique for the isolation of gingival stem/progenitor cells. Human free gingival margin-derived STRO-1/MACS+ cells are a unique renewable source of multipotent stem/progenitor cells. PMID:25257881

Prom1 Function in Development, Intestinal Inflammation, and Intestinal Tumorigenesis

PubMed Central

Karim, Baktiar O.; Rhee, Ki-Jong; Liu, Guosheng; Yun, Kyuson; Brant, Steven R.

2014-01-01

Prom1/CD133 has been identified in colorectal, hepatocellular, and pancreatic cancer as a cancer stem cell marker and has been used as such to predict colon cancer recurrence in humans. Its potential molecular function as well as its role as a marker of intestinal regeneration is still not fully known. We evaluated the role of Prom1 in intestinal regeneration in inflammatory bowel disease (IBD), determined the function of Prom1, and characterized the effect of a lack of Prom1 on intestinal tumor formation in animal models. Our results suggest that Apc mutations lead to an increase in Prom1 expressing cells in the intestinal crypt stem cell compartment and in early intestinal adenomas. Also, Prom1 knockout mice are more susceptible to intestinal tumor formation. We conclude that Prom1 likely plays a role in regulating intestinal homeostasis and that these results clearly illustrate the role of Prom1 in intestinal regeneration. We further conclude that Prom1 may provide a novel therapeutic target for patients with gastrointestinal conditions such as IBD, short bowel syndrome, and colorectal cancer. PMID:25452936

Adult Palatum as a Novel Source of Neural Crest-Related Stem Cells

PubMed Central

Widera, Darius; Zander, Christin; Heidbreder, Meike; Kasperek, Yvonne; Noll, Thomas; Seitz, Oliver; Saldamli, Belma; Sudhoff, Holger; Sader, Robert; Kaltschmidt, Christian; Kaltschmidt, Barbara

2009-01-01

Somatic neural and neural crest stem cells are promising sources for cellular therapy of several neurodegenerative diseases. However, because of practical considerations such as inadequate accessibility of the source material, the application of neural crest stem cells is strictly limited. The secondary palate is a highly regenerative and heavily innervated tissue, which develops embryonically under direct contribution of neural crest cells. Here, we describe for the first time the presence of nestin-positive neural crest-related stem cells within Meissner corpuscles and Merkel cell-neurite complexes located in the hard palate of adult Wistar rats. After isolation, palatal neural crest-related stem cells (pNC-SCs) were cultivated in the presence of epidermal growth factor and fibroblast growth factor under serum-free conditions, resulting in large amounts of neurospheres. We used immunocytochemical techniques and reverse transcriptase-polymerase chain reaction to assess the expression profile of pNC-SCs. In addition to the expression of neural crest stem cell markers such as Nestin, Sox2, and p75, we detected the expression of Klf4, Oct4, and c-Myc. pNC-SCs differentiated efficiently into neuronal and glial cells. Finally, we investigated the potential expression of stemness markers within the human palate. We identified expression of stem cell markers nestin and CD133 and the transcription factors needed for reprogramming of somatic cells into pluripotent cells: Sox2, Oct4, Klf4, and c-Myc. These data show that cells isolated from palatal rugae form neurospheres, are highly plastic, and express neural crest stem cell markers. In addition, pNC-SCs may have the ability to differentiate into functional neurons and glial cells, serving as a starting point for therapeutic studies. Stem Cells 2009;27:1899–1910 PMID:19544446

Monitoring α4β7 integrin expression on circulating CD4+ T cells as a surrogate marker for tracking intestinal CD4+ T cell loss in SIV infection

PubMed Central

Wang, Xiaolei; Xu, Huanbin; Gill, Amy F.; Pahar, Bapi; Kempf, Doty; Rasmussen, Terri; Lackner, Andrew A.; Veazey, Ronald S.

2013-01-01

Intestinal CD4+ T cells are rapidly and profoundly depleted in HIV-infected patients and SIV-infected macaques. However, monitoring intestinal cells in humans is difficult, and identifying surrogate markers in the blood, which correlate with loss or restoration of intestinal CD4+ T cells could be helpful in monitoring the success of therapeutic strategies and vaccine candidates. Recent studies indicate HIV utilizes the intestinal homing molecule α4β7 for attachment and signaling of CD4+ T cells, suggesting this molecule may play a central role in HIV pathogenesis. Here we compared β7HIGH integrin expression on CD4+ T cells in blood with loss of CD4+ T cells in the intestine of macaques throughout SIV infection. The loss of β7HIGH CD4+ T cells in blood closely paralleled the loss of intestinal CD4+ T cells, and proved to be a more reliable marker of intestinal CD4+ T cell loss than monitoring CCR5+ memory CD4+ T cells. These data are consistent with a recent hypothesis that α4β7 plays a role in the selective depletion of intestinal CD4+ T cells, and indicate that monitoring β7HIGH expression on CD4+ T cells in the blood may be a useful surrogate for estimating intestinal CD4+ T cell loss and restoration in HIV-infected patients. PMID:19710637

Blastic plasmacytoid dendritic cell neoplasm: report of two pediatric cases.

PubMed

Dharmani, Preeti Ashok; Mittal, Neha Manish; Subramanian, P G; Galani, Komal; Badrinath, Yajamanam; Amare, Pratibha; Gujral, Sumeet

2015-01-01

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare subtype of acute leukemia that typically follows a highly aggressive clinical course in adults, whereas experience in children with this disease is very limited. We report cases of two children in whom bone marrow showed infiltration by large atypical monocytoid 'blast-like' cells which on immunophenotyping expressed CD4, CD56, HLA-DR and CD33 while were negative for CD34 other T-cell, B-cell and myeloid markers. The differential diagnoses considered were AML, T/NK-cell leukemia and acute undifferentiated leukemia. Additional markers CD303/BDCA-2 and CD123 which are recently validated plasmacytoid dendritic cell markers were done which helped us clinch the diagnosis of this rare neoplasm. An accurate diagnosis of BPDCN is essential in order to provide prompt treatment. Due to its rarity and only recent recognition as a distinct clinicopathological entity, no standardized therapeutic approach has been established for BPDCN.

A putative mesenchymal stem cells population isolated from adult human testes.

PubMed

Gonzalez, R; Griparic, L; Vargas, V; Burgee, K; Santacruz, P; Anderson, R; Schiewe, M; Silva, F; Patel, A

2009-08-07

Mesenchymal stem cells (MSCs) isolated from several adult human tissues are reported to be a promising tool for regenerative medicine. In order to broaden the array of tools for therapeutic application, we isolated a new population of cells from adult human testis termed gonadal stem cells (GSCs). GSCs express CD105, CD166, CD73, CD90, STRO-1 and lack hematopoietic markers CD34, CD45, and HLA-DR which are characteristic identifiers of MSCs. In addition, GSCs express pluripotent markers Oct4, Nanog, and SSEA-4. GSCs propagated for at least 64 population doublings and exhibited clonogenic capability. GSCs have a broad plasticity and the potential to differentiate into adipogenic, osteogenic, and chondrogenic cells. These studies demonstrate that GSCs are easily obtainable stem cells, have growth kinetics and marker expression similar to MSCs, and differentiate into mesodermal lineage cells. Therefore, GSCs may be a valuable tool for therapeutic applications.

Cancer stem cell markers in patterning differentiation and in prognosis of oral squamous cell carcinoma.

PubMed

Mohanta, Simple; Siddappa, Gangotri; Valiyaveedan, Sindhu Govindan; Dodda Thimmasandra Ramanjanappa, Ravindra; Das, Debashish; Pandian, Ramanan; Khora, Samanta Sekhar; Kuriakose, Moni Abraham; Suresh, Amritha

2017-06-01

Differentiation is a major histological parameter determining tumor aggressiveness and prognosis of the patient; cancer stem cells with their slow dividing and undifferentiated nature might be one of the factors determining the same. This study aims to correlate cancer stem cell markers (CD44 and CD147) with tumor differentiation and evaluate their subsequent effect on prognosis. Immunohistochemical analysis in treatment naïve oral cancer patients (n = 53) indicated that the expression of CD147 was associated with poorly differentiated squamous cell carcinoma and moderately differentiated squamous cell carcinoma (p < 0.01). Furthermore, co-expression analysis showed that 45% each of moderately differentiated squamous cell carcinoma and poorly differentiated squamous cell carcinoma patients were CD44 high /CD147 high as compared to only 10% of patients with well-differentiated squamous cell carcinoma. A three-way analysis indicated that differentiation correlated with recurrence and survival (p < 0.05) in only the patients with CD44 high /CD147 high cohort. Subsequently, relevance of these cancer stem cell markers in patterning the differentiation characteristics was evaluated in oral squamous cell carcinoma cell lines originating from different grades of oral cancer. Flowcytometry-based analysis indicated an increase in CD44 + /CD147 + cells in cell lines of poorly differentiated squamous cell carcinoma (94.35 ± 1.14%, p < 0.001) and moderately differentiated squamous cell carcinoma origin (93.49 ± 0.47%, p < 0.001) as compared to cell line of well-differentiated squamous cell carcinoma origin (23.12% ± 0.49%). Expression profiling indicated higher expression of cancer stem cell and epithelial-mesenchymal transition markers in SCC029B (poorly differentiated squamous cell carcinoma originated; p ≤ 0.001), which was further translated into increased spheroid formation, migration, and invasion (p < 0.001) as compared to cell line of well-differentiated squamous cell carcinoma origin. This study suggests that CD44 and CD147 together improve the prognostic efficacy of tumor differentiation; in vitro results further point out that these markers might be determinant of differentiation characteristics, imparting properties of increased self-renewal, migration, and invasion.

The CD11a and Endothelial Protein C Receptor Marker Combination Simplifies and Improves the Purification of Mouse Hematopoietic Stem Cells.

PubMed

Karimzadeh, Alborz; Scarfone, Vanessa M; Varady, Erika; Chao, Connie; Grathwohl, Karin; Fathman, John W; Fruman, David A; Serwold, Thomas; Inlay, Matthew A

2018-06-01

Hematopoietic stem cells (HSCs) are the self-renewing multipotent progenitors to all blood cell types. Identification and isolation of HSCs for study has depended on the expression of combinations of surface markers on HSCs that reliably distinguish them from other cell types. However, the increasing number of markers required to isolate HSCs has made it tedious, expensive, and difficult for newcomers, suggesting the need for a simpler panel of HSC markers. We previously showed that phenotypic HSCs could be separated based on expression of CD11a and that only the CD11a negative fraction contained true HSCs. Here, we show that CD11a and another HSC marker, endothelial protein C receptor (EPCR), can be used to effectively identify and purify HSCs. We introduce a new two-color HSC sorting method that can highly enrich for HSCs with efficiencies comparable to the gold standard combination of CD150 and CD48. Our results demonstrate that adding CD11a and EPCR to the HSC biologist's toolkit improves the purity of and simplifies isolation of HSCs. Stem Cells Translational Medicine 2018;7:468-476. © 2018 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Plasmacytoid dendritic cell leukaemia/lymphoma: towards a well defined entity?

PubMed

Garnache-Ottou, Francine; Feuillard, Jean; Saas, Philippe

2007-02-01

CD4(+)/CD56(+) haematodermic neoplasm or 'early' plasmacytoid dendritic cell leukaemia/lymphoma (pDCL) was described as a disease entity in the last World Health Organisation/European Organisation for Research and Treatment of Cancer classification for cutaneous lymphomas. These leukaemia/lymphomas co-express CD4 and CD56 without any other lineage-specific markers and have been identified as arising from plasmacytoid dendritic cells. Despite a fairly homogeneous pattern of markers expressed by most pDCL, numerous distinctive features (e.g. cytological aspects and aberrant marker expression) have been reported. This may be related to the 'lineage-independent developmental' programme of dendritic cells, which may be able to develop from either immature or already committed haematopoietic progenitors. This highlights the need for specific validated markers to diagnose such aggressive leukaemia. Here, we propose--among others (e.g. T-cell leukaemia 1)--blood dendritic cell antigen-2 and high levels of CD123 expression as potential markers. In addition, we propose a multidisciplinary approach including several fields of haematology to improve pDCL diagnosis.

[Cordyceps sinensis enhances lymphocyte proliferation and CD markers expression in simulated microgravity environment].

PubMed

Hao, Tong; Li, Jun-Jie; Du, Zhi-Yan; Duan, Cui-Mi; Wang, Yan-Meng; Wang, Chang-Yong; Song, Jing-Ping; Wang, Lin-Jie; Li, Ying-Hui; Wang, Yan

2012-10-01

This study was aimed to explore the effect of cordyceps sinensis enhancing lymphocyte proliferation and surface CD marker expression in simulated microgravity environment. The splenic lymphocytes were separated from mice and cultured in the rotary cell culture system simulated microgravity environment. The cells were treated with different concentration of cordyceps sinensis solution (0, 6.25, 12.5, 25 and 50 µg/ml) for 24, 48 and 72 h respectively, then the cells were harvested, and analyzed for cell proliferation and the expression of cell surface markers (CD4 and CD8). The results showed that under simulated microgravity environment, the lymphocyte proliferation was inhibited. When the concentration of cordyceps sinensis was 25 or 50 µg/ml, the lymphocyte proliferation, CD4 and CD8 expressions all increased, but 50 µg/ml cordyceps sinensis could inhibit the proliferation ability with the time prolonging. It is concluded that the suitable concentration of cordyceps sinensis displayed the ability to enhance the lymphocyte proliferation and CD marker expression in simulated microgravity environment. These results may be valuable for screening drugs which can be potentially against immunosuppression under simulated microgravity.

Sequential CD34 cell fractionation by magnetophoresis in a magnetic dipole flow sorter.

PubMed

Schneider, Thomas; Karl, Stephan; Moore, Lee R; Chalmers, Jeffrey J; Williams, P Stephen; Zborowski, Maciej

2010-01-01

Cell separation and fractionation based on fluorescent and magnetic labeling procedures are common tools in contemporary research. These techniques rely on binding of fluorophores or magnetic particles conjugated to antibodies to target cells. Cell surface marker expression levels within cell populations vary with progression through the cell cycle. In an earlier work we showed the reproducible magnetic fractionation (single pass) of the Jurkat cell line based on the population distribution of CD45 surface marker expression. Here we present a study on magnetic fractionation of a stem and progenitor cell (SPC) population using the established acute myelogenous leukemia cell line KG-1a as a cell model. The cells express a CD34 cell surface marker associated with the hematopoietic progenitor cell activity and the progenitor cell lineage commitment. The CD34 expression level is approximately an order of magnitude lower than that of the CD45 marker, which required further improvements of the magnetic fractionation apparatus. The cells were immunomagnetically labeled using a sandwich of anti-CD34 antibody-phycoerythrin (PE) conjugate and anti-PE magnetic nanobead and fractionated into eight components using a continuous flow dipole magnetophoresis apparatus. The CD34 marker expression distribution between sorted fractions was measured by quantitative PE flow cytometry (using QuantiBRITE PE calibration beads), and it was shown to be correlated with the cell magnetophoretic mobility distribution. A flow outlet addressing scheme based on the concept of the transport lamina thickness was used to control cell distribution between the eight outlet ports. The fractional cell distributions showed good agreement with numerical simulations of the fractionation based on the cell magnetophoretic mobility distribution in the unsorted sample.

[Comparative immunophenotypic characterization of human and monkey permanent lymphoid culture cells].

PubMed

Agrba, V Z; Lapin, B A; Medvedeva, N M; Ignatova, I E; Karal-Ogly, D D

2007-01-01

The aim of the study was to define the comparative immunophenotypic characteristics ofwidely spread lymphoid cell cultures, derived from Burkett's lymphoma named as Raji and P3HR-1 in comparison with analogous monkey cultures. It has been shown that P3HR-1 culture consists of similar type cells - activated B-lymphocytes CD23 with k phenotype, which demonstrates its monoclonality. Raji culture includes cells with markers of immature B-lymphocytes CD10 and CD24, as well as elements expressing CD10 antigens. T-cell markers were found in none of the cultures. In contrast to human cells, monkey lymphoid culture expressed both B- and T-cell markers. Moreover, in one of them, obtained from a green monkey, T-cells of suppressor type (CD8) prevailed. The immunophenotypic characteristics of primate lymphoid cell cultures, revealed by the study, are of great importance for their proper application to medical and biological studies.

Characterization and Classification of Mesenchymal Stem Cells in Several Species Using Surface Markers for Cell Therapy Purposes.

PubMed

Ghaneialvar, Hori; Soltani, Leila; Rahmani, Hamid Reza; Lotfi, Abbas Sahebghadam; Soleimani, Masoud

2018-01-01

Mesenchymal stem cells are multipotent cells capable of replicating as undifferentiated cells, and have the potential of differentiating into mesenchymal tissue lineages such as osteocytes, adipocytes and chondrocytes. Such lineages can then be used in cell therapy. The aim of present study was to characterize bone marrow derived mesenchymal stem cells in four different species, including: sheep, goat, human and mouse. Human bone-marrow mesenchymal stem cells were purchased, those of sheep and goat were isolated from fetal bone marrow, and those of mouse were collected by washing bone cavity of femur and tibia with DMEM/F12. Using flow-cytometry, they were characterized by CD surface antigens. Furthermore, cells of third passage were examined for their osteogenic and adipogenic differentiation potential by oil red and alizarin red staining respectively. According to the results, CD markers studied in the four groups of mesenchymal stem cells showed a different expression. Goat and sheep expressed CD44 and CD166, and weakly expressed CD34, CD45, CD105 and CD90. Similarly, human and mouse mesenchymal cells expressed CD44, CD166, CD105 and CD90 whereas the expression of CD34 and CD45 was negative. In conclusion, although all mesenchymal stem cells display plastic adherence and tri-lineage differentiation, not all express the same panel of surface antigens described for human mesenchymal stem cells. Additional panel of CD markers are necessary to characterize regenerative potential and possible application of these stem cells in regenerative medicine and implantology.

CD68/macrosialin: not just a histochemical marker.

PubMed

Chistiakov, Dimitry A; Killingsworth, Murry C; Myasoedova, Veronika A; Orekhov, Alexander N; Bobryshev, Yuri V

2017-01-01

CD68 is a heavily glycosylated glycoprotein that is highly expressed in macrophages and other mononuclear phagocytes. Traditionally, CD68 is exploited as a valuable cytochemical marker to immunostain monocyte/macrophages in the histochemical analysis of inflamed tissues, tumor tissues, and other immunohistopathological applications. CD68 alone or in combination with other cell markers of tumor-associated macrophages showed a good predictive value as a prognostic marker of survival in cancer patients. Lowression of CD68 was found in the lymphoid cells, non-hematopoietic cells (fibroblasts, endothelial cells, etc), and tumor cells. Cell-specific CD68 expression and differentiated expression levels are determined by the complex interplay between transcription factors, regulatory transcriptional elements, and epigenetic factors. Human CD68 and its mouse ortholog macrosialin belong to the family of LAMP proteins located in the lysosomal membrane and share many structural similarities such as the presence of the LAMP-like domain. Except for a second LAMP-like domain present in LAMPs, CD68/microsialin has a highly glycosylated mucin-like domain involved in ligand binding. CD68 has been shown to bind oxLDL, phosphatidylserine, apoptotic cells and serve as a receptor for malaria sporozoite in liver infection. CD68 is mainly located in the endosomal/lysosomal compartment but can rapidly shuttle to the cell surface. However, the role of CD68 as a scavenger receptor remains to be confirmed. It seems that CD68 is not involved in binding bacterial/viral pathogens, innate, inflammatory or humoral immune responses, although it may potentially be involved in antigen processing/presentation. CD68 could be functionally important in osteoclasts since its deletion leads to reduced bone resorption capacity. The role of CD68 in atherosclerosis is contradictory.

Role of lymphocyte-specific protein tyrosine kinase (LCK) in the expansion of glioma-initiating cells by fractionated radiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Rae-Kwon; Yoon, Chang-Hwan; Hyun, Kyung-Hwan

2010-11-26

Research highlights: {yields} Activation of Lymphocyte-specific protein tyrosine kinase (LCK) is involved in the fractionated radiation-induced expansion of glioma stem-like cells. {yields} Inhibition of LCK prevents acquisition of fractionated radiation-induced resistance to chemotherapeutic treatment. {yields} LCK activity is critical for the maintenance of self-renewal in glioma stem-like cells. -- Abstract: Brain cancers frequently recur or progress as focal masses after treatment with ionizing radiation. Radiation used to target gliomas may expand the cancer stem cell population and enhance the aggressiveness of tumors; however, the mechanisms underlying the expansion of cancer stem cell population after radiation have remained unclear. In thismore » study, we show that LCK (lymphocyte-specific protein tyrosine kinase) is involved in the fractionated radiation-induced expansion of the glioma-initiating cell population and acquisition of resistance to anticancer treatments. Fractionated radiation caused a selective increase in the activity of LCK, a Src family non-receptor tyrosine kinase. The activities of other Src family kinases Src, Fyn, and Lyn were not significantly increased. Moreover, knockdown of LCK expression with a specific small interfering RNA (siRNA) effectively blocked fractionated radiation-induced expansion of the CD133{sup +} cell population. siRNA targeting of LCK also suppressed fractionated radiation-induced expression of the glioma stem cell marker proteins CD133, Nestin, and Musashi. Expression of the known self-renewal-related proteins Notch2 and Sox2 in glioma cells treated with fractionated radiation was also downregulated by LCK inhibition. Moreover, siRNA-mediated knockdown of LCK effectively restored the sensitivity of glioma cells to cisplatin and etoposide. These results indicate that the non-receptor tyrosine kinase LCK is critically involved in fractionated radiation-induced expansion of the glioma-initiating cell population and decreased cellular sensitivity to anticancer treatments. These findings may provide pivotal insights in the context of fractionated radiation-based therapeutic interventions in brain cancer.« less

Generation and Characterisation of Cisplatin-Resistant Non-Small Cell Lung Cancer Cell Lines Displaying a Stem-Like Signature

PubMed Central

Barr, Martin P.; Gray, Steven G.; Hoffmann, Andreas C.; Hilger, Ralf A.; Thomale, Juergen; O’Flaherty, John D.; Fennell, Dean A.; Richard, Derek; O’Leary, John J.; O’Byrne, Kenneth J.

2013-01-01

Introduction Inherent and acquired cisplatin resistance reduces the effectiveness of this agent in the management of non-small cell lung cancer (NSCLC). Understanding the molecular mechanisms underlying this process may result in the development of novel agents to enhance the sensitivity of cisplatin. Methods An isogenic model of cisplatin resistance was generated in a panel of NSCLC cell lines (A549, SKMES-1, MOR, H460). Over a period of twelve months, cisplatin resistant (CisR) cell lines were derived from original, age-matched parent cells (PT) and subsequently characterized. Proliferation (MTT) and clonogenic survival assays (crystal violet) were carried out between PT and CisR cells. Cellular response to cisplatin-induced apoptosis and cell cycle distribution were examined by FACS analysis. A panel of cancer stem cell and pluripotent markers was examined in addition to the EMT proteins, c-Met and β-catenin. Cisplatin-DNA adduct formation, DNA damage (γH2AX) and cellular platinum uptake (ICP-MS) was also assessed. Results Characterisation studies demonstrated a decreased proliferative capacity of lung tumour cells in response to cisplatin, increased resistance to cisplatin-induced cell death, accumulation of resistant cells in the G0/G1 phase of the cell cycle and enhanced clonogenic survival ability. Moreover, resistant cells displayed a putative stem-like signature with increased expression of CD133+/CD44+cells and increased ALDH activity relative to their corresponding parental cells. The stem cell markers, Nanog, Oct-4 and SOX-2, were significantly upregulated as were the EMT markers, c-Met and β-catenin. While resistant sublines demonstrated decreased uptake of cisplatin in response to treatment, reduced cisplatin-GpG DNA adduct formation and significantly decreased γH2AX foci were observed compared to parental cell lines. Conclusion Our results identified cisplatin resistant subpopulations of NSCLC cells with a putative stem-like signature, providing a further understanding of the cellular events associated with the cisplatin resistance phenotype in lung cancer. PMID:23349823

Characterization of novel biomarkers in selecting for subtype specific medulloblastoma phenotypes.

PubMed

Liang, Lisa; Aiken, Christopher; McClelland, Robyn; Morrison, Ludivine Coudière; Tatari, Nazanin; Remke, Marc; Ramaswamy, Vijay; Issaivanan, Magimairajan; Ryken, Timothy; Del Bigio, Marc R; Taylor, Michael D; Werbowetski-Ogilvie, Tamra E

2015-11-17

Major research efforts have focused on defining cell surface marker profiles for characterization and selection of brain tumor stem/progenitor cells. Medulloblastoma is the most common primary malignant pediatric brain cancer and consists of 4 molecular subgroups: WNT, SHH, Group 3 and Group 4. Given the heterogeneity within and between medulloblastoma variants, surface marker profiles may be subtype-specific. Here, we employed a high throughput flow cytometry screen to identify differentially expressed cell surface markers in self-renewing vs. non-self-renewing SHH medulloblastoma cells. The top 25 markers were reduced to 4, CD271/p75NTR/NGFR, CD106/VCAM1, EGFR and CD171/NCAM-L1, by evaluating transcript levels in SHH tumors relative to samples representing the other variants. However, only CD271/p75NTR/NGFR and CD171/NCAM-L1 maintain differential expression between variants at the protein level. Functional characterization of CD271, a low affinity neurotrophin receptor, in cell lines and primary cultures suggested that CD271 selects for lower self-renewing progenitors or stem cells. Moreover, CD271 levels were negatively correlated with expression of SHH pathway genes. Our study reveals a novel role for CD271 in SHH medulloblastoma and suggests that targeting CD271 pathways could lead to the design of more selective therapies that lessen the broad impact of current treatments on developing nervous systems.

Spatiotemporal analysis of putative notochordal cell markers reveals CD24 and keratins 8, 18, and 19 as notochord‐specific markers during early human intervertebral disc development

PubMed Central

Rodrigues‐Pinto, Ricardo; Berry, Andrew; Piper‐Hanley, Karen; Hanley, Neil; Richardson, Stephen M.

2016-01-01

ABSTRACT In humans, the nucleus pulposus (NP) is composed of large vacuolated notochordal cells in the fetus but, soon after birth, becomes populated by smaller, chondrocyte‐like cells. Although animal studies indicate that notochord‐derived cells persist in the adult NP, the ontogeny of the adult human NP cell population is still unclear. As such, identification of unique notochordal markers is required. This study was conducted to determine the spatiotemporal expression of putative human notochordal markers to aid in the elucidation of the ontogeny of adult human NP cells. Human embryos and fetuses (3.5–18 weeks post‐conception (WPC)) were microdissected to isolate the spine anlagens (notochord and somites/sclerotome). Morphology of the developing IVD was assessed using hematoxylin and eosin. Expression of keratin (KRT) 8, KRT18, KRT19, CD24, GAL3, CD55, BASP1, CTGF, T, CD90, Tie2, and E‐cadherin was assessed using immunohistochemistry. KRT8, KRT18, KRT19 were uniquely expressed by notochordal cells at all spine levels at all stages studied; CD24 was expressed at all stages except 3.5 WPC. While GAL3, CD55, BASP1, CTGF, and T were expressed by notochordal cells at specific stages, they were also co‐expressed by sclerotomal cells. CD90, Tie2, and E‐cadherin expression was not detectable in developing human spine cells at any stage. This study has identified, for the first time, the consistent expression of KRT8, KRT18, KRT19, and CD24 as human notochord‐specific markers during early IVD development. Thus, we propose that these markers can be used to help ascertain the ontogeny of adult human NP cells. © 2016 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. J Orthop Res 34:1327–1340, 2016. PMID:26910849

Spatiotemporal analysis of putative notochordal cell markers reveals CD24 and keratins 8, 18, and 19 as notochord-specific markers during early human intervertebral disc development.

PubMed

Rodrigues-Pinto, Ricardo; Berry, Andrew; Piper-Hanley, Karen; Hanley, Neil; Richardson, Stephen M; Hoyland, Judith A

2016-08-01

In humans, the nucleus pulposus (NP) is composed of large vacuolated notochordal cells in the fetus but, soon after birth, becomes populated by smaller, chondrocyte-like cells. Although animal studies indicate that notochord-derived cells persist in the adult NP, the ontogeny of the adult human NP cell population is still unclear. As such, identification of unique notochordal markers is required. This study was conducted to determine the spatiotemporal expression of putative human notochordal markers to aid in the elucidation of the ontogeny of adult human NP cells. Human embryos and fetuses (3.5-18 weeks post-conception (WPC)) were microdissected to isolate the spine anlagens (notochord and somites/sclerotome). Morphology of the developing IVD was assessed using hematoxylin and eosin. Expression of keratin (KRT) 8, KRT18, KRT19, CD24, GAL3, CD55, BASP1, CTGF, T, CD90, Tie2, and E-cadherin was assessed using immunohistochemistry. KRT8, KRT18, KRT19 were uniquely expressed by notochordal cells at all spine levels at all stages studied; CD24 was expressed at all stages except 3.5 WPC. While GAL3, CD55, BASP1, CTGF, and T were expressed by notochordal cells at specific stages, they were also co-expressed by sclerotomal cells. CD90, Tie2, and E-cadherin expression was not detectable in developing human spine cells at any stage. This study has identified, for the first time, the consistent expression of KRT8, KRT18, KRT19, and CD24 as human notochord-specific markers during early IVD development. Thus, we propose that these markers can be used to help ascertain the ontogeny of adult human NP cells. © 2016 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. J Orthop Res 34:1327-1340, 2016. © 2016 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc.

Intracoronary injection of CD34-cells in chronic ischemic heart failure: 7 years follow-up of the DanCell study.

PubMed

Hansen, Morten; Nyby, Sebastian; Eifer Møller, Jacob; Videbæk, Lars; Kassem, Moustapha; Barington, Torben; Thayssen, Per; Diederichsen, Axel Cosmus Pyndt

2014-01-01

Seven years ago, the DanCell study was carried out to test the hypothesis of improvement in left ventricular ejection fraction (LVEF) following repeated intracoronary injections of autologous bone marrow-derived stem cells (BMSCs) in patients suffering from chronic ischemic heart failure. In this post hoc analysis, the long-term effect of therapy is assessed. 32 patients [mean age 61 (SD ± 9), 81% males] with systolic dysfunction (LVEF 33 ± 9%) received two repeated intracoronary infusions (4 months apart) of autologous BMSCs (1,533 ± 765 × 10(6) BMSCs including 23 ± 11 × 10(6) CD34(+) cells and 14 ± 7 × 10(6) CD133(+) cells). Patients were followed for 7 years and deaths were recorded. During follow-up, 10 patients died (31%). In univariate regression analysis, the total number of BMSCs, CD34(+) cell count and CD133(+) cell count did not significantly correlate with survival (hazard ratio: 0.999, 95% CI: 0.998-1.000, p = 0.24; hazard ratio: 0.94, 95% CI: 0.88-1.01, p = 0.10, and hazard ratio: 0.96, 95% CI: 0.87-1.07, p = 0.47, respectively). After adjustment for baseline variables in multivariate regression analysis, the CD34(+) cell count was significantly associated with survival (hazard ratio: 0.90, 95% CI: 0.82-1.00, p = 0.04). Intracoronary injections of a high number of CD34(+) cells may have a beneficial effect on chronic ischemic heart failure in terms of long-term survival.

Manufacture of endothelial colony-forming progenitor cells from steady-state peripheral blood leukapheresis using pooled human platelet lysate.

PubMed

Siegel, Georg; Fleck, Erika; Elser, Stefanie; Hermanutz-Klein, Ursula; Waidmann, Marc; Northoff, Hinnak; Seifried, Erhard; Schäfer, Richard

2018-05-01

Endothelial colony-forming progenitor cells (ECFCs) are promising candidates for cell therapies. However, ECFC translation to the clinic requires optimized isolation and manufacture technologies according to good manufacturing practice (GMP). ECFCs were manufactured from steady-state peripheral blood (PB) leukapheresis (11 donors), using GMP-compliant technologies including pooled human platelet (PLT) lysate, and compared to human umbilical cord endothelial cells, human aortic endothelial cells, and human cerebral microvascular endothelial cells. Specific variables assessed were growth kinetics, phenotype, trophic factors production, stimulation of tube formation, and Dil-AcLDL uptake. ECFCs could be isolated from PB leukapheresis units with mean processed volume of 5411 mL and mean white blood cell (WBC) concentration factor of 8.74. The mean frequency was 1.44 × 10 -8 ECFCs per WBC, corresponding to a mean of 177.8 ECFCs per apheresis unit. Expandable for up to 12 cumulative population doublings, calculated projection showed that approximately 730 × 10 3 ECFCs could be manufactured from 1 apheresis unit. ECFCs produced epidermal growth factor, hepatocyte growth factor, vascular endothelial growth factor (VEGF)-A, PLT-derived growth factor-B, interleukin-8, and monocyte chemoattractant protein-1, featured high potential for capillary-like tubes formation, and showed no telomerase activity. They were characterized by CD29, CD31, CD44, CD105, CD117, CD133, CD144, CD146, and VEGF-R2 expression, with the most common subpopulation CD34+CD117-CD133-. Compared to controls, ECFCs featured greater Dil-AcLDL uptake and higher expression of CD29, CD31, CD34, CD44, CD144, and VEGF-R2. Here we show that isolation of ECFCs with proangiogenic profile from steady-state PB leukapheresis is feasible, marking a first step toward ECFC product manufacture according to GMP. © 2018 AABB.

HA117 endows HL60 cells with a stem-like signature by inhibiting the degradation of DNMT1 via its ability to down-regulate expression of the GGL domain of RGS6

PubMed Central

Li, Shuangshuang; Wu, Huan; Wang, Yi; Li, Xiaoqing; Guo, Yuxia; Liang, Shaoyan

2017-01-01

All-trans retinoic acid (ATRA) induces complete remission in almost all patients with acute promyelocytic leukemia (APL) via its ability to induce the in vivo differentiation of APL blasts. However, prolonged ATRA treatment can result in drug resistance. In previous studies, we generated a multi-drug-resistant HL60/ATRA cell line and found it to contain a new drug resistance-related gene segment, HA117. In this study, we demonstrate that ATRA induces multi-drug-resistant subpopulations of HL60 cells with a putative stem-like signature by up-regulating the expression of the new gene segment HA117. Western blot analysis and quantitative real-time PCR demonstrated that HA117 causes alternative splicing of regulator of G-protein signaling 6 (RGS6) and down-regulation of the expression of the GGL domain of RGS6, which plays an important role in DNA methyltransferase 1 (DNMT1) degradation. Moreover, DNMT1 expression was increased in multi-drug resistance HL60/ATRA cells. Knockdown of HA117 restored expression of the GGL domain and blocked DNMT1 expression. Moreover, resistant cells displayed a putative stem-like signature with increased expression of cancer steam cell markers CD133 and CD123. The stem cell marker, Nanog, was significantly up-regulated. In conclusion, our study shows that HA117 potentially promotes the stem-like signature of the HL60/ATRA cell line by inhibiting by the ubiquitination and degradation of DNMT1 and by down-regulating the expression of the GGL domain of RGS6. These results throw light on the cellular events associated with the ATRA-induced multi-drug resistance phenotype in acute leukemia. PMID:28665981

Human Uterine Leiomyoma Stem/Progenitor Cells Expressing CD34 and CD49b Initiate Tumors In Vivo

PubMed Central

Ono, Masanori; Moravek, Molly B.; Coon, John S.; Navarro, Antonia; Monsivais, Diana; Dyson, Matthew T.; Druschitz, Stacy A.; Malpani, Saurabh S.; Serna, Vanida A.; Qiang, Wenan; Chakravarti, Debabrata; Kim, J. Julie; Bulun, Serdar E.

2015-01-01

Context: Uterine leiomyoma is the most common benign tumor in reproductive-age women. Using a dye-exclusion technique, we previously identified a side population of leiomyoma cells exhibiting stem cell characteristics. However, unless mixed with mature myometrial cells, these leiomyoma side population cells did not survive or grow well in vitro or in vivo. Objective: The objective of this study was to identify cell surface markers to isolate leiomyoma stem/progenitor cells. Design: Real-time PCR screening was used to identify cell surface markers preferentially expressed in leiomyoma side population cells. In vitro colony-formation assay and in vivo tumor-regeneration assay were used to demonstrate functions of leiomyoma stem/progenitor cells. Results: We found significantly elevated CD49b and CD34 gene expression in side population cells compared with main population cells. Leiomyoma cells were sorted into three populations based on the expression of CD34 and CD49b: CD34+/CD49b+, CD34+/CD49b−, and CD34−/CD49b− cells, with the majority of the side population cells residing in the CD34+/CD49b+ fraction. Of these populations, CD34+/CD49b+ cells expressed the lowest levels of estrogen receptor-α, progesterone receptor, and α-smooth muscle actin, but the highest levels of KLF4, NANOG, SOX2, and OCT4, confirming their more undifferentiated status. The stemness of CD34+/CD49b+ cells was also demonstrated by their strongest in vitro colony-formation capacity and in vivo tumor-regeneration ability. Conclusions: CD34 and CD49b are cell surface markers that can be used to enrich a subpopulation of leiomyoma cells possessing stem/progenitor cell properties; this technique will accelerate efforts to develop new therapies for uterine leiomyoma. PMID:25658015

Cell Homogeneity Indispensable for Regenerative Medicine by Cultured Human Corneal Endothelial Cells.

PubMed

Hamuro, Junji; Toda, Munetoyo; Asada, Kazuko; Hiraga, Asako; Schlötzer-Schrehardt, Ursula; Montoya, Monty; Sotozono, Chie; Ueno, Morio; Kinoshita, Shigeru

2016-09-01

To identify the subpopulation (SP) among heterogeneous cultured human corneal endothelial cells (cHCECs) devoid of cell-state transition applicable for cell-based therapy. Subpopulation presence in cHCECs was confirmed via surface CD-marker expression level by flow cytometry. CD markers effective for distinguishing distinct SPs were selected by analyzing those on established cHCECs with a small cell area and high cell density. Contrasting features among three typical cHCEC SPs was confirmed by PCR array for extracellular matrix (ECM). Combined analysis of CD markers was performed to identify the SP (effector cells) applicable for therapy. ZO-1 and Na+/K+ ATPase, CD200, and HLA expression were compared among heterogeneous SPs. Flow cytometry analysis identified the effector cell expressing CD166+CD105-CD44-∼+/-CD26-CD24-, but CD200-, and the presence of other SPs with CD166+ CD105-CD44+++ (CD26 and CD24, either + or -) was confirmed. PCR array revealed three distinct ECM expression profiles. Some SPs expressed ZO-1 and Na+/K+ ATPase at comparable levels with effector cells, while only one SP expressed CD200, but not on effector cells. Human leukocyte antigen expression was most reduced in the effector SP. The proportion of effector cells (E-ratio) inversely paralleled donor age and decreased during prolonged culture passages. The presence of Rho-associated protein kinase (ROCK) inhibitor increased the E-ratio in cHCECs. The average area of effector cells was approximately 200∼220 μm2, and the density of cHCECs exceeded 2500 cells/mm2. A specified cultured effector cell population sharing the surface phenotypes with mature HCECs in corneal tissues may serve as an alternative to donor corneas for the treatment of corneal endothelial dysfunction.

CD32-Expressing CD4 T Cells Are Phenotypically Diverse and Can Contain Proviral HIV DNA.

PubMed

Martin, Genevieve E; Pace, Matthew; Thornhill, John P; Phetsouphanh, Chansavath; Meyerowitz, Jodi; Gossez, Morgane; Brown, Helen; Olejniczak, Natalia; Lwanga, Julianne; Ramjee, Gita; Kaleebu, Pontiano; Porter, Kholoud; Willberg, Christian B; Klenerman, Paul; Nwokolo, Nneka; Fox, Julie; Fidler, Sarah; Frater, John

2018-01-01

Efforts to both characterize and eradicate the HIV reservoir have been limited by the rarity of latently infected cells and the absence of a specific denoting biomarker. CD32a (FcγRIIa) has been proposed to be a marker for an enriched CD4 T cell HIV reservoir, but this finding remains controversial. Here, we explore the expression of CD32 on CD3 + CD4 + cells in participants from two primary HIV infection studies and identify at least three distinct phenotypes (CD32 low , CD32 + CD14 + , and CD32 high ). Of note, CD4 negative enrichment kits remove the majority of CD4 + CD32 + T cells, potentially skewing subsequent analyses if used. CD32 high CD4 T cells had higher levels of HLA-DR and HIV co-receptor expression than other subsets, compatible with their being more susceptible to infection. Surprisingly, they also expressed high levels of CD20, TCRαβ, IgD, and IgM (but not IgG), markers for both T cells and naïve B cells. Compared with other populations, CD32 low cells had a more differentiated memory phenotype and high levels of immune checkpoint receptors, programmed death receptor-1 (PD-1), Tim-3, and TIGIT. Within all three CD3 + CD4 + CD32 + phenotypes, cells could be identified in infected participants, which contained HIV DNA. CD32 expression on CD4 T cells did not correlate with HIV DNA or cell-associated HIV RNA (both surrogate measures of overall reservoir size) or predict time to rebound viremia following treatment interruption, suggesting that it is not a dominant biomarker for HIV persistence. Our data suggest that while CD32 + T cells can be infected with HIV, CD32 is not a specific marker of the reservoir although it might identify a population of HIV enriched cells in certain situations.

Differential expression of CD44 and CD24 markers discriminates the epitheliod from the fibroblastoid subset in a sarcomatoid renal carcinoma cell line: evidence suggesting the existence of cancer stem cells in both subsets as studied with sorted cells.

PubMed

Hsieh, Chin-Hsuan; Hsiung, Shih-Chieh; Yeh, Chi-Tai; Yen, Chih-Feng; Chou, Yah-Huei Wu; Lei, Wei-Yi; Pang, See-Tong; Chuang, Cheng-Keng; Liao, Shuen-Kuei

2017-02-28

Epithelioid and fibroblastoid subsets coexist in the human sarcomatoid renal cell carcinoma (sRCC) cell line, RCC52, according to previous clonal studies. Herein, using monoclonal antibodies to CD44 and CD24 markers, we identified and isolated these two populations, and showed that CD44bright/CD24dim and CD44bright/CD24bright phenotypes correspond to epithelioid and fibroblastoid subsets, respectively. Both sorted subsets displayed different levels of tumorigenicity in xenotransplantation, indicating that each harbored its own cancer stem cells (CSCs). The CD44bright/CD24bright subset, associated with higher expression of MMP-7, -8 and TIMP-1 transcripts, showed greater migratory/invasive potential than the CD44bright/CD24dim subset, which was associated with higher expression of MMP-2, -9 and TIMP-2 transcripts. Both subsets differentially expressed stemness gene products c-Myc, Oct4A, Notch1, Notch2 and Notch3, and the RCC stem cell marker, CD105 in 4-5% of RCC52 cells. These results suggest the presence of CSCs in both sRCC subsets for the first time and should therefore be considered potential therapeutic targets for this aggressive malignancy.

Defining adipose tissue-derived stem cells in tissue and in culture.

PubMed

Lin, Ching-Shwun; Xin, Zhong-Cheng; Deng, Chun-Hua; Ning, Hongxiu; Lin, Guiting; Lue, Tom F

2010-06-01

Adipose tissue-derived stem cells (ADSC) are routinely isolated from the stromal vascular fraction (SVF) of homogenized adipose tissue. Similar to other types of mesenchymal stem cells (MSC), ADSC remain difficult to define due to the lack of definitive cellular markers. Still, many types of MSC, including ADSC, have been shown to reside in a perivascular location, and increasing evidence shows that both MSC and ADSC may in fact be vascular stem cells (VSC). Locally, these cells differentiate into smooth muscle and endothelial cells that are assembled into newly formed blood vessels during angiogenesis and neovasculogenesis. Additionally, MSC or ADSC can also differentiate into tissue cells such as adipocytes in the adipose tissue. Systematically, MSC or ADSC are recruited to injury sites where they participate in the repair/regeneration of the injured tissue. Due to the vasculature's dynamic capacity for growth and multipotential nature for diversification, VSC in tissue are individually at various stages and on different paths of differentiation. Therefore, when isolated and put in culture, these cells are expected to be heterogeneous in marker expression, renewal capacity, and differentiation potential. Although this heterogeneity of VSC does impose difficulties and cause confusions in basic science studies, its impact on the development of VSC as a therapeutic cell source has not been as apparent, as many preclinical and clinical trials have reported favorable outcomes. With this understanding, ADSC are generally defined as CD34+CD31- although loss of CD34 expression in culture is well documented. In adipose tissue, CD34 is localized to the intima and adventitia of blood vessels but not the media where cells expressing alpha-smooth muscle actin (SMA) exist. By excluding the intima, which contains the CD34+CD31+ endothelial cells, and the media, which contains the CD34-CD31- smooth muscle cells, it leaves the adventitia as the only possible location for the CD34+ ADSC. In the capillary, CD34 and CD140b (a pericyte marker) are mutually exclusively expressed, thus suggesting that pericytes are not the CD34+ ADSC. Many other cellular markers for vascular cells, stem cells, and stem cell niche have also been investigated as possible ADSC markers. Particularly the best-known MSC marker STRO-1 has been found either expressed or not expressed in cultured ADSC. In the adipose tissue, STRO-1 appears to be expressed exclusively in the endothelium of certain but not all blood vessels, and thus not associated with the CD34+ ADSC. In conclusion, we believe that ADSC exist as CD34+CD31-CD104b-SMA- cells in the capillary and in the adventitia of larger vessels. In the capillary these cells coexist with pericytes and endothelial cells, both of which are possibly progenies of ADSC (or more precisely VSC). In the larger vessels, these ADSC or VSC exist as specialized fibroblasts (having stem cell properties) in the adventitia.

Methylisoindigo preferentially kills cancer stem cells by interfering cell metabolism via inhibition of LKB1 and activation of AMPK in PDACs.

PubMed

Cheng, Xinlai; Kim, Jee Young; Ghafoory, Shahrouz; Duvaci, Tijen; Rafiee, Roya; Theobald, Jannick; Alborzinia, Hamed; Holenya, Pavlo; Fredebohm, Johannes; Merz, Karl-Heinz; Mehrabi, Arianeb; Hafezi, Mohammadreza; Saffari, Arash; Eisenbrand, Gerhard; Hoheisel, Jörg D; Wölfl, Stefan

2016-06-01

Pancreatic ductal adenocarcinoma (PDAC) clinically has a very poor prognosis. No small molecule is available to reliably achieve cures. Meisoindigo is chemically related to the natural product indirubin and showed substantial efficiency in clinical chemotherapy for CML in China. However, its effect on PDAC is still unknown. Our results showed strong anti-proliferation effect of meisoindigo on gemcitabine-resistant PDACs. Using a recently established primary PDAC cell line, called Jopaca-1 with a larger CSCs population as model, we observed a reduction of CD133+ and ESA+/CD44+/CD24+ populations upon treatment and concomitantly a decreased expression of CSC-associated genes, and reduced cellular mobility and sphere formation. Investigating basic cellular metabolic responses, we detected lower oxygen consumption and glucose uptake, while intracellular ROS levels increased. This was effectively neutralized by the addition of antioxidants, indicating an essential role of the cellular redox balance. Further analysis on energy metabolism related signaling revealed that meisoindigo inhibited LKB1, but activated AMPK. Both of them were involved in cellular apoptosis. Additional in situ hybridization in tissue sections of PDAC patients reproducibly demonstrated co-expression and -localization of LKB1 and CD133 in malignant areas. Finally, we detected that CD133+/CD44+ were more vulnerable to meisoindigo, which could be mimicked by LKB1 siRNAs. Our results provide the first evidence, to our knowledge, that LKB1 sustains the CSC population in PDACs and demonstrate a clear benefit of meisoindigo in treatment of gemcitabine-resistant cells. This novel mechanism may provide a promising new treatment option for PDAC. Copyright © 2016 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Isolation of a circulating CD45−, CD34dim cell population and validation of their endothelial phenotype

PubMed Central

Tropea, Margaret M.; Harper, Bonnie J. A.; Graninger, Grace M.; Phillips, Terry M.; Ferreyra, Gabriela; Mostowski, Howard S.; Danner, Robert L.; Suffredini, Anthony F.; Solomon, Michael A.

2016-01-01

Summary Accurately detecting circulating endothelial cells (CECs) is important since their enumeration has been proposed as a biomarker to measure injury to the vascular endothelium. However, there is no single methodology for determining CECs in blood, making comparison across studies difficult. Many methods for detecting CECs rely on characteristic cell surface markers and cell viability indicators, but lack secondary validation. Here, a CEC population in healthy adult human subjects was identified by flow cytometry as CD45−, CD34dim that is comparable to a previously described CD45−, CD31bright population. In addition, nuclear staining with 7-aminoactinomycin D (7-AAD) was employed as a standard technique to exclude dead cells. Unexpectedly, the CD45−, CD34dim, 7-AAD− CECs lacked surface detectable CD146, a commonly used marker of CECs. Furthermore, light microscopy revealed this cell population to be composed primarily of large cells without a clearly defined nucleus. Nevertheless, immunostains still demonstrated the presence of the lectin Ulex europaeus and van Willebrand factor. Ultramicro analytical immunochemistry assays for the endothelial cell proteins CD31, CD34, CD62E, CD105, CD141, CD144 and vWF indicated these cells possess an endothelial phenotype. However, only a small amount of RNA, which was mostly degraded, could be isolated from these cells. Thus the majority of CECs in healthy individuals as defined by CD45−, CD34dim, and 7-AAD− have shed their CD146 surface marker and are senescent cells without an identifiable nucleus and lacking RNA of sufficient quantity and quality for transcriptomal analysis. This study highlights the importance of secondary validation of CEC identification. PMID:25057108

Influence of Bovine Serum Lipids and Fetal Bovine Serum on the Expression of Cell Surface Markers in Cultured Bovine Preadipocytes.

PubMed

Sandhu, Mansur A; Jurek, Sandra; Trappe, Susanne; Kolisek, Martin; Sponder, Gerhard; Aschenbach, Jörg R

2017-01-01

To establish the influence of fetal bovine serum (FBS) and bovine serum lipids (BSL) on cell differentiation marker expression, bovine adipose-derived stem cells from subcutaneous tissue were incubated for 14 days in 4 types of differentiation media containing 10% FBS and 10 µL/mL BSL (TRT-1), no FBS and 10 µL/mL of BSL (TRT-2), 10% FBS and no BSL (TRT-3), or no supplements (TRT-4). Cells were subjected to Nile red staining, immunocytochemistry (CD73, CD90, CD105, DLK1, FabP4), and quantitative real-time PCR (CD73, CD90, CD105, FabP4). The number of cells presenting FabP4 and the percentage of mature adipocytes with large lipid droplets were increased in TRT-2, accompanied by a robust increase in FabP4 mRNA abundance and a decrease in DLK1-positive cells. In preadipocytes, CD73 was present around the nucleus and translocated towards cell membranes during differentiation. Although the percentage of CD73-positive cells was not different among treatments, its mRNA abundance, immunocytochemical staining intensity, and translocation towards cell membranes were decreased when the medium contained no FBS (TRT-2 and TRT-4). All cells showed a diffuse distribution of CD90 and CD105 and remained positive for these markers irrespective of the treatment. However, the CD90 and CD105 mRNA abundance was decreased in TRT-2 and TRT-4; i.e., in media containing no FBS. The presence of FBS increased the absolute number of cell nuclei as assessed by DAPI fluorescence. Our results suggest that bovine subcutaneous preadipocytes display typical stem cell markers. The differentiation into mature adipocytes is promoted by BSL, whereas FBS endorses cell proliferation. © 2017 S. Karger AG, Basel.

Differentiation Affects the Release of Exosomes from Colon Cancer Cells and Their Ability to Modulate the Behavior of Recipient Cells.

PubMed

Lucchetti, Donatella; Calapà, Federica; Palmieri, Valentina; Fanali, Caterina; Carbone, Federica; Papa, Alfredo; De Maria, Ruggero; De Spirito, Marco; Sgambato, Alessandro

2017-07-01

Exosomes are involved in intercellular communication. We previously reported that sodium butyrate-induced differentiation of HT29 colon cancer cells is associated with a reduced CD133 expression. Herein, we analyzed the role of exosomes in the differentiation of HT29 cells. Exosomes were prepared using ultracentrifugation. Gene expression levels were evaluated by real-time PCR. The cell proliferation rate was assessed by MTT assay and with the electric cell-substrate impedance sensing system, whereas cell motility was assessed using the scratch test and confocal microscopy. Sodium butyrate-induced differentiation of HT29 and Caco-2 cells increased the levels of released exosomes and their expression of CD133. Cell differentiation and the decrease of cellular CD133 expression levels were prevented by blocking multivesicular body maturation. Exosomes released by HT29 differentiating cells carried increased levels of miRNAs, induced an increased proliferation and motility of both colon cancer cells and normal fibroblasts, increased the colony-forming efficiency of cancer cells, and reduced the sodium butyrate-induced differentiation of HT29 cells. Such effects were associated with an increased phosphorylation level of both Src and extracellular signal regulated kinase proteins and with an increased expression of epithelial-to-mesenchymal transition-related genes. Release of exosomes is affected by differentiation of colon cancer cells; exosomes might be used by differentiating cells to get rid of components that are no longer necessary but might continue to exert their effects on recipient cells. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Cancer stem cell mediated acquired chemoresistance in head and neck cancer can be abrogated by aldehyde dehydrogenase 1 A1 inhibition.

PubMed

Kulsum, Safeena; Sudheendra, Holalugunda Vittalamurthy; Pandian, Ramanan; Ravindra, Doddathimmasandra Ramanjanappa; Siddappa, Gangotri; R, Nisheena; Chevour, Priyanka; Ramachandran, Balaji; Sagar, Milind; Jayaprakash, Aravindakshan; Mehta, Alka; Kekatpure, Vikram; Hedne, Naveen; Kuriakose, Moni A; Suresh, Amritha

2017-02-01

Chemoresistance leading to disease relapse is one of the major challenges to improve outcome in head and neck cancers. Cancer Stem Cells (CSCs) are increasingly being implicated in chemotherapy resistance, this study investigates the correlation between CSC behavior and acquired drug resistance in in vitro cell line models. Cell lines resistant to Cisplatin (Cal-27 CisR, Hep-2 CisR) and 5FU (Cal-27 5FUR) with high Resistance Indices (RI) were generated (RI ≥ 3) by short-term treatment of head and neck squamous cell carcinoma (HNSCC) cell lines with chemotherapeutic drugs (Cisplatin, Docetaxel, 5FU), using a dose-incremental strategy. The cell lines (Cal-27 DoxR, Hep-2 DoxR, Hep-2 5FUR) that showed low RI, nevertheless had a high cross resistance to Cisplatin/5FU (P < 0.05). Cal-27 CisR and DoxR showed 12-14% enrichment of CD44+ cells, while CisR/5FUR showed 4-6% increase in ALDH1A1+ cells as compared to parental cells (P < 0.05). Increased expression of stem cell markers (CD44, CD133, NOTCH1, ALDH1A1, OCT4, SOX2) in these cell lines, correlated with enhanced spheroid/colony formation, migratory potential, and increased in vivo tumor burden (P < 0.05). Inhibition of ALDH1A1 in Cal-27 CisR led to down regulation of the CSC markers, reduction in migratory, self-renewal and tumorigenic potential (P < 0.05) accompanied by an induction of sensitivity to Cisplatin (P < 0.05). Further, ex vivo treatment of explants (n = 4) from HNSCC patients with the inhibitor (NCT-501) in combination with Cisplatin showed a significant decrease in proliferating cells as compared to individual treatment (P = 0.001). This study hence suggests an ALDH1A1-driven, CSC-mediated mechanism in acquired drug resistance of HNSCC, which may have therapeutic implications. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

mTOR plays critical roles in pancreatic cancer stem cells through specific and stemness-related functions

NASA Astrophysics Data System (ADS)

Matsubara, Shyuichiro; Ding, Qiang; Miyazaki, Yumi; Kuwahata, Taisaku; Tsukasa, Koichiro; Takao, Sonshin

2013-11-01

Pancreatic cancer is characterized by near-universal mutations in KRAS. The mammalian target of rapamycin (mTOR), which functions downstream of RAS, has divergent effects on stem cells. In the present study, we investigated the significance of the mTOR pathway in maintaining the properties of pancreatic cancer stem cells. The mTOR inhibitor, rapamycin, reduced the viability of CD133+ pancreatic cancer cells and sphere formation which is an index of self-renewal of stem-like cells, indicating that the mTOR pathway functions to maintain cancer stem-like cells. Further, rapamycin had different effects on CD133+ cells compared to cyclopamine which is an inhibitor of the Hedgehog pathway. Thus, the mTOR pathway has a distinct role although both pathways maintain pancreatic cancer stem cells. Therefore, mTOR might be a promising target to eliminate pancreatic cancer stem cells.

Effectiveness of Vascular Markers (Immunohistochemical Stains) in Soft Tissue Sarcomas.

PubMed

Naeem, Namra; Mushtaq, Sajid; Akhter, Noreen; Hussain, Mudassar; Hassan, Usman

2018-05-01

To ascertain the effectiveness of IHC markers of vascular origin like CD31, CD34, FLI1 and ERG in vascular soft tissue sarcomas including angiosarcomas, Kaposi sarcomas, epithelioid hemangioendothelioma and a non-vascular soft tissue sarcoma (Epithelioid sarcoma). Descriptive study. Shaukat Khanum Memorial Cancer Hospital and Research Centre, Lahore, from 2011 to 2017. Diagnosed cases of angiosarcomas (n=48), epithelioid hemangioendothelioma (n=9), Kaposi sarcoma (n=9) and epithelioid sarcoma (n=20) were selected. Immunohistochemical staining as performed on formalin fixed paraffin embedded sections. The sections were stained for the following markers: CD34 (VENTANA clone Q Bend 10), CD31 (Leica clone 1 A 10), FLI1 (CELL MARQUE clone MRQ-1) and ERG (CELL MARQUE clone EP111). A complete panel of CD34, CD31 and ERG was applied on 8/48 cases of angiosarcomas with triple positivity in 6 cases. Eight cases showed positivity for only CD31 and ERG and 2 cases showed positivity for only ERG. A complete panel of CD34, CD31 and ERG was applied on 3/9 cases of epithelioid hemangioendothelioma with positivity for all markers in 2 cases. Combined positivity for ERG and CD34 was seen in 2 cases and on 4 cases only CD31 immunohistochemical was solely applied with 100% positivity. FLI1 was not applied on any case. Among 9 cases of Kaposi sarcoma, ERG, CD34 and CD31 in combination were applied on only 1 case with triple positivity. Remaining cases show positivity for either CD34, CD31 or FLI1. Majority of cases of epithelioid sarcomas were diagnosed on the basis of cytokeratin and CD34 positivity with loss of INI1. The other vascular markers showed negativity in all cases. Among these four markers, ERG immunohistochemical stain is highly effective for endothelial differentiation due to its specific nuclear staining pattern in normal blood vessel endothelial cells (internal control) as well as neoplastic cells of vascular tumors and lack of background staining.

Zonal hierarchy of differentiation markers and nestin expression during oval cell mediated rat liver regeneration.

PubMed

Koenig, Sarah; Probst, Irmelin; Becker, Heinz; Krause, Petra

2006-12-01

Oval cells constitute a heterogeneous population of proliferating progenitors found in rat livers following carcinogenic treatment (2-acetylaminofluorene and 70% hepatectomy). The aim of this study was to investigate the cellular pattern of various differentiation and cell type markers in this model of liver regeneration. Immunophenotypic characterisation revealed at least two subtypes emerging from the portal field. First, a population of oval cells formed duct-like structures and expressed bile duct (CD49f) as well as hepatocytic markers (alpha-foetoprotein, CD26). Second, a population of non-ductular oval cells was detected between and distally from the ductules expressing the neural marker nestin and the haematopoietic marker Thy1. Following oval cell isolation, a subset of the nestin-positive cells was shown to co-express hepatocytic and epithelial markers (albumin, CD26, pancytokeratin) and could be clearly distinguished from anti-desmin reactive hepatic stellate cells. The gene expression profiles (RT-PCR) of isolated oval cells and oval cell liver tissue were found to be similar to foetal liver (ED14). The present results suggest that the two oval cell populations are organised in a zonal hierarchy with a marker gradient from the inner (displaying hepatocytic and biliary markers) to the outer zone (showing hepatocytic and extrahepatic progenitor markers) of the proliferating progeny clusters.

Antibody-immobilized column for quick cell separation based on cell rolling.

PubMed

Mahara, Atsushi; Yamaoka, Tetsuji

2010-01-01

Cell separation using methodological standards that ensure high purity is a very important step in cell transplantation for regenerative medicine and for stem cell research. A separation protocol using magnetic beads has been widely used for cell separation to isolate negative and positive cells. However, not only the surface marker pattern, e.g., negative or positive, but also the density of a cell depends on its developmental stage and differentiation ability. Rapid and label-free separation procedures based on surface marker density are the focus of our interest. In this study, we have successfully developed an antiCD34 antibody-immobilized cell-rolling column, that can separate cells depending on the CD34 density of the cell surfaces. Various conditions for the cell-rolling column were optimized including graft copolymerization, and adjustment of the column tilt angle, and medium flow rate. Using CD34-positive and -negative cell lines, the cell separation potential of the column was established. We observed a difference in the rolling velocities between CD34-positive and CD34-negative cells on antibody-immobilized microfluidic device. Cell separation was achieved by tilting the surface 20 degrees and the increasing medium flow. Surface marker characteristics of the isolated cells in each fraction were analyzed using a cell-sorting system, and it was found that populations containing high density of CD34 were eluted in the delayed fractions. These results demonstrate that cells with a given surface marker density can be continuously separated using the cell rolling column.

Primary adipose-derived stem cells enriched by growth factor treatment improves cell adaptability toward cardiovascular differentiation in a rodent model of acute myocardial infarction.

PubMed

Chang, Jui-Chih; Lee, Ping-Chun; Lin, Yu-Chun; Lee, Kung-Wei; Hsu, Shan-hui

2011-01-01

The heterogeneous cell population in primary adipose-derived adult stem cells (ADAS) and difficulty in keeping their primitive properties have posed certain limitations on using these cells for cell therapy. Therefore, our objective was to generate a population of cells enriched from the adipose stromal-vascular fraction (SVF) with greater differentiation potential than ADAS and to explore the mechanism behind the repair of the injured myocardium in vivo. The distinct population of adipose stromal cells was enriched by immediate treatment of the growth factor cocktail (EGF and PDGF-BB) to the freshly isolated SVF. These cells (ADAS-GFs) had distinct cell morphology from ADAS and in average had a smaller size. They presented co-expression of CD140a (pericytic markers) and CD34 (hematopoietic marker), more obvious mesenchymal (CD13, CD29, CD44, CD90 and CD117) markers, but rare KDR, and were negative for CD45 and CD31. ADAS-GFs not only spontaneously expressed endothelial cell markers and formed capillary-like tubes on Matrigel but also clearly expressed early cardiomyocyte marker genes when embedded in methylcellulose-based medium. In Sprague-Dawley (SD) rats with left anterior descending artery (LAD)-induced myocardial infarction (MI), the ADAS-GFs transplanted group had the left ventricular function significantly improved compared with the ADAS transplanted group or the control group at 12 weeks post transplantation. The immunofluorescence staining revealed that the transplanted ADAS-GFs expressed GATA4, betamyosin heavy chain and troponin T protein but not vWF. More capillaries were also observed around the infarcted zone in the ADAS-GFs transplanted group. These data suggested that ADAS-GFs with a higher proangiogenic potential may restore the cardiac function of infarcted myocardium via the direct cardiomyocyte differentiation as well as angiogenesis recruitment.

Acidic conditions induce the suppression of CD86 and CD54 expression in THP-1 cells.

PubMed

Mitachi, Takafumi; Mezaki, Minori; Yamashita, Kunihiko; Itagaki, Hiroshi

2018-01-01

To evaluate the sensitization potential of chemicals in cosmetics, using non-animal methods, a number of in vitro safety tests have been designed. Current assays are based on the expression of cell surface markers, such as CD86 and CD54, which are associated with the activation of dendritic cells, in skin sensitization tests. However, these markers are influenced by culture conditions through activating danger signals. In this study, we investigated the relationship between extracellular pH and the expression of the skin sensitization test human cell line activation test (h-CLAT) markers CD86 and CD54. We measured expression levels after THP-1 cells were exposed to representative contact allergens, i.e., 2,4-dinitrochlorobenzene and imidazolidinyl urea, under acidic conditions. These conditions were set by exposure to hydrochloric acid, lactic acid, and citric acid. An acidic extracellular pH (6-7) suppressed the augmentation of CD86 and CD54 levels by the sensitizer. Additionally, when the CD86/CD54 expression levels were suppressed, a reduction in the intracellular pH was confirmed. Furthermore, we observed that Na + /H + exchanger 1 (NHE-1), a protein that contributes to the regulation of extracellular/intracellular pH, is involved in CD86 and CD54 expression. These findings suggest that the extracellular/intracellular pH has substantial effects on in vitro skin sensitization markers and should be considered in evaluations of the safety of mixtures and commercial products in the future.

Characterization of the Murine Myeloid Precursor Cell Line MuMac-E8

PubMed Central

Fricke, Stephan; Riemschneider, Sina; Kohlschmidt, Janine; Hilger, Nadja; Fueldner, Christiane; Knauer, Jens; Sack, Ulrich; Emmrich, Frank; Lehmann, Jörg

2014-01-01

Starting point for the present work was the assumption that the cell line MuMac-E8 represents a murine cell population with stem cell properties. Preliminary studies already pointed to the expression of stem-cell associated markers and a self-regenerative potential of the cells. The cell line MuMac-E8 should be examined for their differential stage within stem cell hierarchy. MuMac-E8 cells were derived from a chimeric mouse model of arthritis. It could be shown that MuMac-E8 cells express mRNA of some genes associated with pluripotent stem cells (Nanog, Nucleostemin), of genes for hematopoietic markers (EPCR, Sca-1, CD11b, CD45), for the mesenchymal marker CD105 and of genes for the neural markers Pax-6 and Ezrin. In methylcellulose and May-Grünwald-Giemsa staining, hematopoietic colonies were obtained but the hematopoietic system of lethally irradiated mice could not be rescued. Osteogenic differentiation was not detectable. Thus, it became evident that MuMac-E8 represents not a stem cell line. However, MuMac-E8 cells expressed several myeloid surface markers (i.e. CD11b, F4/80, CD14, CD64), showed phagocytosis and is capable of producing nitric oxide. Thus, this cell line seems to be arrested an advanced stage of myeloid differentiation. Adherence data measured by impedance-based real-time cell analysis together with cell morphology data suggested that MuMac-E8 represents a new macrophage precursor cell line exhibiting weak adherence. This cell line is suitable as an in-vitro model for testing of macrophage functions. Moreover, it might be also useful for differentiation or reprogramming studies. PMID:25546418

Liver-specific gene expression in cultured human hematopoietic stem cells.

PubMed

Fiegel, Henning C; Lioznov, Michael V; Cortes-Dericks, Lourdes; Lange, Claudia; Kluth, Dietrich; Fehse, Boris; Zander, Axel R

2003-01-01

Hematopoietic and hepatic stem cells share characteristic markers such as CD34, c-kit, and Thy1. Based on the recent observations that hepatocytes may originate from bone marrow, we investigated the potential of CD34(+) bone marrow cells to differentiate into hepatocytic cells in vitro. CD34(+) and CD34(-) human bone marrow cells were separated by magnetic cell sorting. Cells were cultured on a collagen matrix in a defined medium containing hepatocyte growth factor. Cell count and size were measured by flow cytometry, and reverse transcription polymerase chain reaction was carried out for the liver-specific markers CK-19 and albumin. During cell culture, CD34(+) cells showed an increasing cell number and proliferative activity as assessed by Ki-67 staining. Under the specified culture conditions, CD34(+) cells expressed albumin RNA and CK-19 RNA after 28 days, whereas CD34(-) cells did not show liver-specific gene expression. The results indicate that CD34(+) adult human bone marrow stem cells can differentiate into hepatocytic cells in vitro.

Activated T cell subsets in human type 1 diabetes: evidence for expansion of the DR+ CD30+ subpopulation in new-onset disease

PubMed Central

Baker, C; Chang, L; Elsegood, K A; Bishop, A J; Gannon, D H; Narendran, P; Leech, N J; Dayan, C M

2007-01-01

An important limitation in T cell studies of human autoimmune (type 1) diabetes is lack of direct access to cells infiltrating the pancreas. We hypothesized that cells recently released from the pancreas into the blood might express a characteristic combination of markers of activation. We therefore examined the recently activated circulating T cell population [CD3+, human leucocyte antigen D-related (HLA-DR+)] using cytokine production and 10 additional subset markers [CD69, CD25, CD122, CD30, CD44v6, CD57, CD71, CCR3 (CD193), CCR5 (CD195) or CXCR3 (CD183)], comparing newly diagnosed adult (ND) (age 18–40 years) patients (n = 19) to patients with diabetes for > 10 years [long-standing (LS), n = 19] and HLA-matched controls (C, n = 16). CD3+ DR+ cells were enriched by two-step immunomagnetic separation. No differences in basal or stimulated production of interleukin (IL)-4, IL-10, IL-13 or interferon (IFN)-γ by CD3+ DR+ enriched cells were observed between the different groups of subjects. However, among the CD3+ DR+ population, significant expansions appeared to be present in the very small CD30+, CD69+ and CD122+ subpopulations. A confirmatory study was then performed using new subjects (ND = 26, LS = 15), three-colour flow cytometry, unseparated cells and three additional subset markers (CD38, CD134, CD4/CD25). This confirmed the expansion of the CD3+ DR+ CD30+ subpopulation in ND subjects. We conclude that a relative expansion in the T cell subpopulation with the activated phenotype CD3+ DR+ CD30+ is seen in the peripheral blood of subjects with newly diagnosed type 1 diabetes. This subpopulation represents less than 0·7% of circulating T cells and may provide a rich source of disease-specific T cells that can be isolated from blood. PMID:17302896

CD133 expression in well-differentiated pancreatic neuroendocrine tumors: a potential predictor of progressive clinical courses.

PubMed

Sakai, Yasuhiro; Hong, Seung-Mo; An, Soyeon; Kim, Joo Young; Corbeil, Denis; Karbanová, Jana; Otani, Kyoko; Fujikura, Kohei; Song, Ki-Byung; Kim, Song Cheol; Akita, Masayuki; Nanno, Yoshihide; Toyama, Hirochika; Fukumoto, Takumi; Ku, Yonson; Hirose, Takanori; Itoh, Tomoo; Zen, Yoh

2017-03-01

The present study aimed to elucidate whether the stemness molecule, CD133, is expressed in well-differentiated pancreatic neuroendocrine tumors (PanNETs; World Health Organization grades 1 and 2) and establish its clinical relevance using 2 separate cohorts. In the first series (n = 178) in which tissue microarrays were available, immunohistochemistry revealed that CD133 was expressed in 14 cases (8%). CD133+ PanNETs had higher TNM stages (P < .01), more frequent lymphovascular invasion (P = .01), and higher recurrence rates (P = .01). In the second cohort (n = 56), the expression of CD133 and CK19 was examined in whole tissue sections. CD133 and CK19 were positive in 10 (18%) and 36 (64%) cases, respectively. CD133 expression correlated with higher pT scores (P < .01), the presence of microscopic venous infiltration (P = .03), and shorter disease-free periods (P < .01). When cases were divided into grade 1 and 2 neoplasms, patients with CD133+ PanNET continued to have shorter disease-free periods than did those with CD133- tumors in both groups (P < .01 and P = .02, respectively). Although CK19+ cases had shorter disease-free periods than did CK19- cases in the whole cohort (P = .02), this difference was less apparent in subanalyses of grade 1 and 2 cases. CD133 expression also appeared to be an independent predictive factor for tumor recurrence in a multivariate analysis (P = .018). The CD133 phenotype was identical between primary and metastatic foci in 17 of 18 cases from which tissues of metastatic deposits were available. In conclusion, the combination of CD133 phenotyping and World Health Organization grading may assist in stratifying patients in terms of the risk of progressive clinical courses. Copyright © 2016 Elsevier Inc. All rights reserved.

Acute leukemia coexpressing myeloid, B- and T-lineage associated markers: multiparameter analysis of criteria defining lineage commitment and maturational stage in a case of undifferentiated leukemia.

PubMed

Meckenstock, G; Heyll, A; Schneider, E M; Hildebrandt, B; Runde, V; Aul, C; Bartram, C R; Ludwig, W D; Schneider, W

1995-02-01

Coexpression of myeloid, B-, and T-lineage associated markers was found in a patient with morphologically and cytochemically undifferentiated acute leukemia. Surface marker analysis using two-color immunofluorescence staining characterized blast cells to express CD34, CD38, CD117, and class II antigens, coexpressing TdT, CD4, CD7, CD13, CD19, and CD33. Cytoplasmic expression of myeloperoxidase, CD3, and CD22 could not be demonstrated. Monosomy for chromosome 7 was found by cytogenetic analysis. The absence of clonal rearrangements of immunoglobulin or T-cell receptor genes was shown by Southern blot analysis. Using a 3H-thymidine incorporation assay, DNA synthesis of leukemic blasts could be stimulated by IL-3, IL-6 and G-CSF in vitro. The present case did not offer specific criteria of lineage commitment. Corresponding to an equivalent counterpart in normal hematopoiesis, the involved cell population may reflect an early, most immature developmental stage within a multipotent progenitor cell compartment.

Downregulation of Melanoma Cell Adhesion Molecule (MCAM/CD146) Accelerates Cellular Senescence in Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells.

PubMed

Jin, Hye Jin; Kwon, Ji Hye; Kim, Miyeon; Bae, Yun Kyung; Choi, Soo Jin; Oh, Wonil; Yang, Yoon Sun; Jeon, Hong Bae

2016-04-01

Therapeutic applications of mesenchymal stem cells (MSCs) for treating various diseases have increased in recent years. To ensure that treatment is effective, an adequate MSC dosage should be determined before these cells are used for therapeutic purposes. To obtain a sufficient number of cells for therapeutic applications, MSCs must be expanded in long-term cell culture, which inevitably triggers cellular senescence. In this study, we investigated the surface markers of human umbilical cord blood-derived MSCs (hUCB-MSCs) associated with cellular senescence using fluorescence-activated cell sorting analysis and 242 cell surface-marker antibodies. Among these surface proteins, we selected the melanoma cell adhesion molecule (MCAM/CD146) for further study with the aim of validating observed expression differences and investigating the associated implications in hUCB-MSCs during cellular senescence. We observed that CD146 expression markedly decreased in hUCB-MSCs following prolonged in vitro expansion. Using preparative sorting, we found that hUCB-MSCs with high CD146 expression displayed high growth rates, multilineage differentiation, expression of stemness markers, and telomerase activity, as well as significantly lower expression of the senescence markers p16, p21, p53, and senescence-associated β-galactosidase, compared with that observed in hUCB-MSCs with low-level CD146 expression. In contrast, CD146 downregulation with small interfering RNAs enhanced the senescence phenotype. In addition, CD146 suppression in hUCB-MSCs caused downregulation of other cellular senescence regulators, including Bmi-1, Id1, and Twist1. Collectively, our results suggest that CD146 regulates cellular senescence; thus, it could be used as a therapeutic marker to identify senescent hUCB-MSCs. One of the fundamental requirements for mesenchymal stem cell (MSC)-based therapies is the expansion of MSCs during long-term culture because a sufficient number of functional cells is required. However, long-term growth inevitably induces cellular senescence, which potentially causes poor clinical outcomes by inducing growth arrest and the loss of stem cell properties. Thus, the identification of markers for evaluating the status of MSC senescence during long-term culture may enhance the success of MSC-based therapy. This study provides strong evidence that CD146 is a novel and useful marker for predicting senescence in human umbilical cord blood-derived MSCs (hUCB-MSCs), and CD146 can potentially be applied in quality-control assessments of hUCB-MSC-based therapy. ©AlphaMed Press.

High BAALC expression associates with other molecular prognostic markers, poor outcome, and a distinct gene-expression signature in cytogenetically normal patients younger than 60 years with acute myeloid leukemia: a Cancer and Leukemia Group B (CALGB) study

PubMed Central

Langer, Christian; Radmacher, Michael D.; Ruppert, Amy S.; Whitman, Susan P.; Paschka, Peter; Mrózek, Krzysztof; Baldus, Claudia D.; Vukosavljevic, Tamara; Liu, Chang-Gong; Ross, Mary E.; Powell, Bayard L.; de la Chapelle, Albert; Kolitz, Jonathan E.; Larson, Richard A.; Marcucci, Guido

2008-01-01

BAALC expression is considered an independent prognostic factor in cytogenetically normal acute myeloid leukemia (CN-AML), but has yet to be investigated together with multiple other established prognostic molecular markers in CN-AML. We analyzed BAALC expression in 172 primary CN-AML patients younger than 60 years of age, treated similarly on CALGB protocols. High BAALC expression was associated with FLT3-ITD (P = .04), wild-type NPM1 (P < .001), mutated CEBPA (P = .003), MLL-PTD (P = .009), absent FLT3-TKD (P = .005), and high ERG expression (P = .05). In multivariable analysis, high BAALC expression independently predicted lower complete remission rates (P = .04) when adjusting for ERG expression and age, and shorter survival (P = .04) when adjusting for FLT3-ITD, NPM1, CEBPA, and white blood cell count. A gene-expression signature of 312 probe sets differentiating high from low BAALC expressers was identified. High BAALC expression was associated with overexpression of genes involved in drug resistance (MDR1) and stem cell markers (CD133, CD34, KIT). Global microRNA-expression analysis did not reveal significant differences between BAALC expression groups. However, an analysis of microRNAs that putatively target BAALC revealed a potentially interesting inverse association between expression of miR-148a and BAALC. We conclude that high BAALC expression is an independent adverse prognostic factor and is associated with a specific gene-expression profile. PMID:18378853

Lung cells from neonates show a mesenchymal stem cell phenotype.

PubMed

Hennrick, Kenneth T; Keeton, Angela G; Nanua, Suparna; Kijek, Theresa G; Goldsmith, Adam M; Sajjan, Umadevi S; Bentley, J Kelley; Lama, Vibha N; Moore, Bethany B; Schumacher, Robert E; Thannickal, Victor J; Hershenson, Marc B

2007-06-01

Mesenchymal stem cells have been isolated from adult bone marrow, peripheral blood, adipose tissue, trabecular bone, articular synovium, and bronchial submucosa. We hypothesized that the lungs of premature infants undergoing mechanical ventilation contain fibroblast-like cells with features of mesenchymal stem cells. Tracheal aspirate fluid from mechanically ventilated, premature (< 30 wk gestation) infants 7 days old or younger was obtained from routine suctioning and plated on plastic culture dishes. A total of 11 of 20 patients studied demonstrated fibroblast-like cells, which were identified as early as 6 hours after plating. Cells were found to express the mesenchymal stem cell markers STRO-1, CD73, CD90, CD105, and CD166, as well as CCR2b, CD13, prolyl 4-hydroxylase, and alpha-smooth muscle actin. Cells were negative for the hematopoietic and endothelial cell markers CD11b, CD31, CD34, or CD45. Tracheal aspirate monocyte chemoattractant protein-1/CCL2 levels were ninefold higher in aspirates in which fibroblast-like cells were found, and cells demonstrated chemotaxis in response to monocyte chemoattractant protein. Placement of cells into appropriate media resulted in adipogenic, osteogenic, and myofibroblastic differentiation. Patients from whom mesenchymal stem cells were isolated tended to require more days of mechanical ventilation and supplemental oxygen. Together, these data demonstrate that tracheal aspirate fluid from premature, mechanically ventilated infants contains fibroblasts with cell markers and differentiation potential typically found in mesenchymal stem cells.

Number of Langerhans cells is decreased in premalignant keratosis and skin cancers.

PubMed

Shevchuk, Z; Filip, A; Shevchuk, V; Kashuba, E

2014-03-01

It was shown earlier that a number of CD207 positive Langerhans cells was lower in basal cell carcinomas than in the normal epidermis. Moreover, benign skin lesions presented a higher number of Langerhans cells when they were compared to malignant tumors. To count Langerhans cells, assessing expression levels of CD1A and CD207 markers in actinic keratosis, basal and squamous cell carcinomas, compared with the normal skin. Comparison of Langerhans cells might give a valuable prognostic marker for skin cancer. Immunohistochemistry and methods of statistics were used. Expression of CD1A and CD207 markers was assessed in tumor samples of actinic keratosis, cutaneous basal and squamous cell carcinomas, in comparison with the normal skin. In each cohort there were 40 patients (and 11 healthy individuals). We have shown that the number of Langerhans cells is considerably lower in cutaneous basal and squamous cell carcinomas, compared with their number in the normal skin (p < 0.0001). CD1A expression correlated with CD207 expression only in the control group. There was no correlation in actinic keratosis, basal and squamous cell carcinoma. This may suggest an alteration of Langerhans cells phenotype in skin neoplastic diseases, making the number of Langerhans cells a valuable prognostic factor for skin tumors.

Increase of CD69, CD161 and CD94 on NK cells in women with recurrent spontaneous abortion and in vitro fertilization failure.

PubMed

Ghafourian, Mehri; Karami, Najmeh; Khodadadi, Ali; Nikbakht, Roshan

2014-06-01

Recurrent spontaneous abortion (RSA) and in vitro fertilization (IVF) failure with unknown causes are the controversial issues that are probably related to the immune system. To compare circulating NK cells expressing activation and inhibition surface markers between patients with RSA and IVF failure with those of healthy multiparous and successful IVF control women, respectively. In this case-control study peripheral blood samples were collected from 43 patients who included 23 women with RSA and 20 with IVF failure, plus 43 healthy control women comprising of 36 normal multiparous women and seven women with successful IVF. The expression of CD69, CD94 and CD161 surface markers on CD56+NK cells were assessed using specific monoclonal antibodies by flowcytometry. The percentage of NK cells increased significantly in patients with RSA and in women with IVF failure in comparison to healthy multiparous and successful IVF control groups (p<0.001). The overall expression of CD69, CD94, CD161 were also increased significantly on NK cells in both patient groups compared to control groups (p<0.001). Elevated expression of CD69 and CD161 on NK cells can be considered as immunological risk markers in RSA and IVF failure. However, it is not clear if high expression of CD94 on peripheral blood NK cells is related to abnormal activity of endometrial NK cells.

Enrichment of herpes simplex virus type 2 (HSV-2) reactive mucosal T cells in the human female genital tract.

PubMed

Posavad, C M; Zhao, L; Dong, L; Jin, L; Stevens, C E; Magaret, A S; Johnston, C; Wald, A; Zhu, J; Corey, L; Koelle, D M

2017-09-01

Local mucosal cellular immunity is critical in providing protection from HSV-2. To characterize and quantify HSV-2-reactive mucosal T cells, lymphocytes were isolated from endocervical cytobrush and biopsy specimens from 17 HSV-2-infected women and examined ex vivo for the expression of markers associated with maturation and tissue residency and for functional T-cell responses to HSV-2. Compared with their circulating counterparts, cervix-derived CD4+ and CD8+ T cells were predominantly effector memory T cells (CCR7-/CD45RA-) and the majority expressed CD69, a marker of tissue residency. Co-expression of CD103, another marker of tissue residency, was highest on cervix-derived CD8+ T cells. Functional HSV-2 reactive CD4+ and CD8+ T-cell responses were detected in cervical samples and a median of 17% co-expressed CD103. HSV-2-reactive CD4+ T cells co-expressed IL-2 and were significantly enriched in the cervix compared with blood. This first direct ex vivo documentation of local enrichment of HSV-2-reactive T cells in the human female genital mucosa is consistent with the presence of antigen-specific tissue-resident memory T cells. Ex vivo analysis of these T cells may uncover tissue-specific mechanisms of local control of HSV-2 to assist the development of vaccine strategies that target protective T cells to sites of HSV-2 infection.

Lung cancer stem cells and implications for future therapeutics.

PubMed

Wang, Jing; Li, Ze-hong; White, James; Zhang, Lin-bo

2014-07-01

Lung cancer is the most dreaded of all cancers because of the higher mortality rates associated with it worldwide. The various subtypes of lung cancer respond differently to a particular treatment regime, which makes the therapeutic interventions all the more complicated. The concept of cancer stem cells (CSCs) is based primarily on the clinical and experimental observations that indicate the existence of a subpopulation of cells with the capacity to self-renew and differentiate as well as show increased resistance to radiation and chemotherapy. They are considered as the factors responsible for the cases of tumor relapse. The CSCs may have significant role in the development of lung tumorigenesis based on the identification of the CSCs which respond during injury. The properties of multi-potency and self-renewal are shared in common by the lung CSCs with the normal pluripotent stem cells which can be isolated using the similar markers. This review deals with the origin and characteristics of the lung cancer stem cells. The role of different markers used to isolate lung CSCs like CD44, ALDH (aldehyde dehydrogenase), CD133 and ABCG2 (ATP binding cassette sub family G member 2) have been discussed in detail. Analysis of the developmental signaling pathways such as Wnt/β-catenin, Notch, hedgehog in the regulation and maintenance of the lung CSCs have been done. Finally, before targeting the lung CSC biomarkers for potential therapeutics, challenges faced in lung cancer stem cell research need to be taken into account. With the accepted notion that the CSCs are to blame for cancer relapse and drug resistance, targeting them can be an important aspect of lung cancer therapy in the future.

Expression of surface markers on the human monocytic leukaemia cell line, THP-1, as indicators for the sensitizing potential of chemicals.

PubMed

An, Susun; Kim, Seoyoung; Huh, Yong; Lee, Tae Ryong; Kim, Han-Kon; Park, Kui-Lea; Eun, Hee Chul

2009-04-01

Evaluation of skin sensitization potential is an important part of the safety assessment of cosmetic ingredients and topical drugs. Recently, evaluation of changes in surface marker expression induced in dendritic cells (DC) or DC surrogate cell lines following exposure to chemicals represents one approach for in vitro test methods. The study aimed to test the change of expression patterns of surface markers on THP-1 cells by chemicals as a predictive in vitro method for contact sensitization. We investigated the expression of CD54, CD86, CD83, CD80, and CD40 after a 1-day exposure to sensitizers (1-chloro-2,4-dinitrobenzene; 2,4-dinitrofluorobenzene; benzocaine; 5-chloro-2-methyl-4-isothiazolin-3-one; hexyl cinnamic aldehyde; eugenol; nickel sulfate hexahydrate; potassium dichromate; cobalt sulfate; 2-mercaptobenzothiazole; and ammonium tetrachloroplatinate) and non-sensitizers (sodium lauryl sulfate, benzalkonium chloride, lactic acid, salicylic acid, isopropanol, and dimethyl sulphoxide). The test concentrations were 0.1x, 0.5x, and 1x of the 50% inhibitory concentration, and the relative fluorescence intensity was used as an expression indicator. By evaluating the expression patterns of CD54, CD86, and CD40, we could classify the chemicals as sensitizers or non-sensitizers, but CD80 and CD83 showed non-specific patterns of expression. These data suggest that the THP-1 cells are good model for screening contact sensitizers and CD40 could be a useful marker complementary to CD54 and CD86.

A small population of resident limb bud mesenchymal cells express few MSC-associated markers, but the expression of these markers is increased immediately after cell culture.

PubMed

Marín-Llera, Jessica Cristina; Chimal-Monroy, Jesús

2018-05-01

Skeletal progenitors are derived from resident limb bud mesenchymal cells of the vertebrate embryos. However, it remains poorly understood if they represent stem cells, progenitors, or multipotent mesenchymal stromal cells (MSC). Derived-MSC of different adult tissues under in vitro experimental conditions can differentiate into the same cellular lineages that are present in the limb. Here, comparing non-cultured versus cultured mesenchymal limb bud cells, we determined the expression of MSC-associated markers, the in vitro differentiation capacity and their gene expression profile. Results showed that in freshly isolated limb bud mesenchymal cells, the proportion of cells expressing Sca1, CD44, CD105, CD90, and CD73 is very low and a low expression of lineage-specific genes was observed. However, recently seeded limb bud mesenchymal cells acquired Sca1 and CD44 markers and the expression of the key differentiation genes Runx2 and Sox9, while Scx and Pparg genes decreased. Also, their chondrogenic differentiation capacity decreased through cellular passages while the osteogenic increased. Our findings suggest that the modification of the cell adhesion process through the in vitro method changed the limb mesenchymal cell immunophenotype leading to the expression and maintenance of common MSC-associated markers. These findings could have a significant impact on MSC study and isolation strategy because they could explain common variations observed in the MSC immunophenotype in different tissues. © 2018 International Federation for Cell Biology.

CD146 Expression Influences Periapical Cyst Mesenchymal Stem Cell Properties.

PubMed

Paduano, Francesco; Marrelli, Massimo; Palmieri, Francesca; Tatullo, Marco

2016-10-01

Recent studies have identified a new human dental derived progenitor cell population with multi-lineage differentiation potential referred to as human periapical cyst mesenchymal stem cells (hPCy-MSCs). In the present study, we compared two subpopulations of hPCy-MSCs characterised by the low or high expression of CD146 to establish whether this expression can regulate their stem cell properties. Using flow cytometry, we evaluated the stem cell marker profile of hPCy-MSCs during passaging. Furthermore, CD146 Low and CD146 High cells were sorted by magnetic beads and subsequently both cell populations were evaluated for differences in their proliferation, self-renewal, stem cell surface markers, stemness genes expression and osteogenic differentiation potential.We found that hPCy-MSCs possessed a stable expression of several mesenchymal stem cell surface markers, whereas CD146 expression declined during passaging.In addition, sorted CD146 Low cells proliferated significantly faster, displayed higher colony-forming unit-fibroblast capacity and showed higher expression of Klf4 when compared to the CD146 High subset. Significantly, the osteogenic potential of hPCy-MSCs was greater in the CD146 Low than in CD146 High population. These results demonstrate that CD146 is spontaneously downregulated with passaging at both mRNA and protein levels and that the high expression of CD146 reduces the proliferative, self-renewal and osteogenic differentiation potential of hPCy-MSCs. In conclusion, our study demonstrates that changes in the expression of CD146 can influence the stem cell properties of hPCy-MSCs.

CD4+ T Cells Expressing PD-1, TIGIT and LAG-3 Contribute to HIV Persistence during ART

PubMed Central

Fromentin, Rémi; Bakeman, Wendy; Lawani, Mariam B.; Khoury, Gabriela; Hartogensis, Wendy; DaFonseca, Sandrina; Killian, Marisela; Epling, Lorrie; Hoh, Rebecca; Sinclair, Elizabeth; Hecht, Frederick M.; Bacchetti, Peter; Deeks, Steven G.; Lewin, Sharon R.; Sékaly, Rafick-Pierre; Chomont, Nicolas

2016-01-01

HIV persists in a small pool of latently infected cells despite antiretroviral therapy (ART). Identifying cellular markers expressed at the surface of these cells may lead to novel therapeutic strategies to reduce the size of the HIV reservoir. We hypothesized that CD4+ T cells expressing immune checkpoint molecules would be enriched in HIV-infected cells in individuals receiving suppressive ART. Expression levels of 7 immune checkpoint molecules (PD-1, CTLA-4, LAG-3, TIGIT, TIM-3, CD160 and 2B4) as well as 4 markers of HIV persistence (integrated and total HIV DNA, 2-LTR circles and cell-associated unspliced HIV RNA) were measured in PBMCs from 48 virally suppressed individuals. Using negative binomial regression models, we identified PD-1, TIGIT and LAG-3 as immune checkpoint molecules positively associated with the frequency of CD4+ T cells harboring integrated HIV DNA. The frequency of CD4+ T cells co-expressing PD-1, TIGIT and LAG-3 independently predicted the frequency of cells harboring integrated HIV DNA. Quantification of HIV genomes in highly purified cell subsets from blood further revealed that expressions of PD-1, TIGIT and LAG-3 were associated with HIV-infected cells in distinct memory CD4+ T cell subsets. CD4+ T cells co-expressing the three markers were highly enriched for integrated viral genomes (median of 8.2 fold compared to total CD4+ T cells). Importantly, most cells carrying inducible HIV genomes expressed at least one of these markers (median contribution of cells expressing LAG-3, PD-1 or TIGIT to the inducible reservoir = 76%). Our data provide evidence that CD4+ T cells expressing PD-1, TIGIT and LAG-3 alone or in combination are enriched for persistent HIV during ART and suggest that immune checkpoint blockers directed against these receptors may represent valuable tools to target latently infected cells in virally suppressed individuals. PMID:27415008

Immunophenotypic characterization and tenogenic differentiation of mesenchymal stromal cells isolated from equine umbilical cord blood.

PubMed

Mohanty, Niharika; Gulati, Baldev R; Kumar, Rajesh; Gera, Sandeep; Kumar, Pawan; Somasundaram, Rajesh K; Kumar, Sandeep

2014-06-01

Mesenchymal stem cells (MSCs) isolated from umbilical cord blood (UCB) in equines have not been well characterized with respect to the expression of pluripotency and mesenchymal markers and for tenogenic differentiation potential in vitro. The plastic adherent fibroblast-like cells isolated from 13 out of 20 UCB samples could proliferate till passage 20. The cells expressed pluripotency markers (OCT4, NANOG, and SOX2) and MSC surface markers (CD90, CD73, and CD105) by RT-PCR, but did not express CD34, CD45, and CD14. On immunocytochemistry, the isolated cells showed expression of CD90 and CD73 proteins, but tested negative for CD34 and CD45. In flow cytometry, CD29, CD44, CD73, and CD90 were expressed by 96.36 ± 1.28%, 93.40 ± 0.70%, 73.23 ± 1.29% and 46.75 ± 3.95% cells, respectively. The UCB-MSCs could be differentiated to tenocytes by culturing in growth medium supplemented with 50 ng/ml of BMP-12 by day 10. The differentiated cells showed the expression of mohawk homeobox (Mkx), collagen type I alpha 1 (Col1α1), scleraxis (Scx), tenomodulin (Tnmd) and decorin (Dcn) by RT-PCR. In addition, flow cytometry detected tenomodulin and decorin protein in 95.65 ± 2.15% and 96.30 ± 1.00% of differentiated cells in comparison to 11.30 ± 0.10% and 19.45 ± 0.55% cells, respect vely in undifferentiated control cells. The findings support the observation that these cells may be suitable for therapeutic applications, including ruptured tendons in racehorses.

Role of immune activation in CD4+ T-cell depletion in HIV-1 infected Indian patients.

PubMed

Vajpayee, M; Kaushik, S; Sreenivas, V; Mojumdar, K; Mendiratta, S; Chauhan, N K

2009-01-01

The correlation of immune activation with CD4(+) depletion and HIV-1 disease progression has been evidenced by several studies involving mainly clade B virus. However, this needs to be investigated in developing countries such as India predominately infected with clade C virus. In a cross-sectional study of 68 antiretroviral treatment naïve, HIV-1 infected Indian patients, we studied the association between CD4(+) T cells, plasma HIV-1 RNA levels, and immune activation markers using unadjusted and adjusted correlative analyses. Significant negative correlations of higher magnitude were observed between the CD4(+) T cell percentages and plasma HIV-1 RNA levels in the study population when adjusted for the effects of immune activation markers. However, the negative association of CD4(+) T cells with immune activation markers remained unaffected when controlled for the effects of plasma HIV-1 RNA levels. Our results support the important role of immune activation in CD4(+) T cell depletion and disease progression during untreated HIV-1 infection.

Immunosenescence-Related Transcriptomic and Immunologic Changes in Older Individuals Following Influenza Vaccination

PubMed Central

Kennedy, Richard B.; Ovsyannikova, Inna G.; Haralambieva, Iana H.; Oberg, Ann L.; Zimmermann, Michael T.; Grill, Diane E.; Poland, Gregory A.

2016-01-01

The goal of annual influenza vaccination is to reduce mortality and morbidity associated with this disease through the generation of protective immune responses. The objective of the current study was to examine markers of immunosenescence and identify immunosenescence-related differences in gene expression, gene regulation, cytokine secretion, and immunologic changes in an older study population receiving seasonal influenza A/H1N1 vaccination. Surprisingly, prior studies in this cohort revealed weak correlations between immunosenescence markers and humoral immune response to vaccination. In this report, we further examined the relationship of each immunosenescence marker (age, T cell receptor excision circle frequency, telomerase expression, percentage of CD28− CD4+ T cells, percentage of CD28− CD8+ T cells, and the CD4/CD8 T cell ratio) with additional markers of immune response (serum cytokine and chemokine expression) and measures of gene expression and/or regulation. Many of the immunosenescence markers indeed correlated with distinct sets of individual DNA methylation sites, miRNA expression levels, mRNA expression levels, serum cytokines, and leukocyte subsets. However, when the individual immunosenescence markers were grouped by pathways or functional terms, several shared biological functions were identified: antigen processing and presentation pathways, MAPK, mTOR, TCR, BCR, and calcium signaling pathways, as well as key cellular metabolic, proliferation and survival activities. Furthermore, the percent of CD4+ and/or CD8+ T cells lacking CD28 expression also correlated with miRNAs regulating clusters of genes known to be involved in viral infection. Integrated (DNA methylation, mRNA, miRNA, and protein levels) network biology analysis of immunosenescence-related pathways and genesets identified both known pathways (e.g., chemokine signaling, CTL, and NK cell activity), as well as a gene expression module not previously annotated with a known function. These results may improve our ability to predict immune responses to influenza and aid in new vaccine development, and highlight the need for additional studies to better define and characterize immunosenescence. PMID:27853459

Cell expression patterns of CD147 in N-diethylnitrosamine/phenobarbital-induced mouse hepatocellular carcinoma.

PubMed

Lu, Meng; Wu, Jiao; He, Feng; Wang, Xi-Long; Li, Can; Chen, Zhi-Nan; Bian, Huijie

2015-02-01

Overexpression of CD147/basigin in hepatic cells promotes the progression of hepatocellular carcinoma (HCC). Whether CD147 also expressed in liver non-parenchymal cells and associated with HCC development was unknown. The aim of the study was to explore time-dependent cell expression patterns of CD147 in a widely accepted N-diethylnitrosamine/phenobarbital (DEN/PB)-induced HCC mouse model. Liver samples collected at month 1-12 of post-DEN/PB administration were assessed the localization of CD147 in hepatocytes, endothelial cells, hepatic stellate cells, and macrophages. Immunohistochemistry analysis showed that CD147 was upregulated in liver tumors during month 1-8 of DEN/PB induction. Expression of CD147 was positively correlated with cytokeratin 18, a hepatocyte marker (r = 0.7857, P = 0.0279), CD31 (r = 0.9048, P = 0.0046), an endothelial cell marker, and CD68, a macrophage marker (r = 0.7619, P = 0.0368). A significant correlation was also observed between CD147 and alpha-smooth muscle actin (r = 0.8857, P = 0.0333) at DEN/PB initiation and early stage of tumor formation. Immunofluorescence and fluorescence in situ hybridization showed that CD147 co-expressed with cytokeratin 18, CD31, alpha-smooth muscle actin, and CD68. Moreover, there existed positive correlations between CD147 and microvessel density (r = 0.7857, P = 0.0279), CD147 and Ki-67 (r = 0.9341, P = 0.0022) in the development of DEN/PB-induced HCC. In conclusion, our results demonstrated that CD147 was upregulated in the liver parenchymal and mesenchymal cells and involved in angiogenesis and tumor cell proliferation in the development of DEN/PB-induced HCC.

Impairment of Circulating CD4⁺CD25⁺GARP⁺ regulatory T cells in patients with acute coronary syndrome.

PubMed

Meng, Kai; Zhang, Wei; Zhong, Yucheng; Mao, Xiaobo; Lin, Yingzhong; Huang, Ying; Lang, Mingjian; Peng, Yudong; Zhu, Zhengfeng; Liu, Yuzhou; Zhao, Xiaoqi; Yu, Kunwu; Wu, Bangwei; Ji, Qingwei; Zeng, Qiutang

2014-01-01

Atherosclerosis (AS) is an inflammatory and immune disease. Regulatory T cells (Tregs) suppress the activation of T cells and have been shown to play a protective role during the pathogenesis of AS. However, specific markers for Tregs are lacking. Recently, glycoprotein A repetitions predominant (GARP) was discovered as a specific marker of activated Tregs, and we therefore utilized GARP as a specific surface marker for Tregs in the current study. To assess whether GARP(+) Tregs are downregulated in patients with acute coronary syndrome (ACS), we examined CD4(+)CD25(+)GARP(+) T cell frequencies as well as their associated cytokines and suppressive function. Additionally, we compared GARP expression to that of FOXP3, which may be more sensitive as a marker of activated Tregs in patients with ACS. Patients with ACS demonstrated a significant decrease in circulating CD4(+)CD25(+)GARP(+) Tregs. Moreover, the suppressive function of Tregs and levels of related cytokines were also impaired in ACS patients compared to those with stable angina (SA) or normal coronary artery (NCA). Additionally, after TCR stimulation, peripheral blood mononuclear cells (PBMCs) from patients with ACS exhibited a decrease in CD4(+)CD25(+)GARP(+) Tregs. These fnding indicate that circulating CD4(+)CD25(+)GARP(+) Tregs are impaired in patients withACS. Thus, targeting GARP may promote the protective function of Tregs in ACS. © 2014 S. Karger AG, Basel.

Infiltration of γ⁢δ T cells, IL-17+ T cells and FoxP3+ T cells in human breast cancer

PubMed Central

Allaoui, Roni; Hagerling, Catharina; Desmond, Eva; Warfvinge, Carl-Fredrik; Jirström, Karin; Leandersson, Karin

2017-01-01

BACKGROUND: Tumor-infiltrating lymphocytes (TILs) have a strong prognostic value in various forms of cancers. These data often refer to use of the pan-T cell marker CD3, or the cytotoxic T lymphocyte marker CD8α. However, T cells are a heterogeneous group of cells with a wide array of effector mechanisms ranging from immunosuppression to cytotoxicity. OBJECTIVE: In this study we have investigated the prognostic effects of some unconventional T cell subtypes in breast cancer; γ⁢δ T cells, IL-17+ T cells and FoxP3+ T cells (Tregs) in relation to the conventional CD3 and CD8α T cell markers. METHODS: This was done using immunohistochemistry on a human breast cancer tissue microarray consisting of 498 consecutive cases of primary breast cancer. RESULTS: Infiltration of γ⁢δ T cells and T cell infiltration in general (CD3), correlated with a good prognosis, while Treg infiltration with a worse. Infiltration of γ⁢δ T cells was associated with a significantly improved clinical outcome in all breast cancer subtypes except triple negative tumors. Only infiltration of either CD3+ or CD8α+ cells was independently associated with better prognosis for all breast cancer patients. CONCLUSIONS: This study sheds further light on the prognostic impact of various T cell subtypes in breast cancer. PMID:29060923

Simultaneous isolation of vascular endothelial cells and mesenchymal stem cells from the human umbilical cord.

PubMed

Kadam, Sachin S; Tiwari, Shubha; Bhonde, Ramesh R

2009-01-01

The umbilical cord represents the link between mother and fetus during pregnancy. This cord is usually discarded as a biological waste after the child's birth; however, its importance as a "store house" of stem cells has been explored recently. We developed a method of simultaneous isolation of endothelial cells (ECs) from the vein and mesenchymal stem cells from umbilical cord Wharton's jelly of the same cord. The isolation protocol has been simplified, modified, and improvised with respect to choice of enzyme and enzyme mixture, digestion time, cell yield, cell growth, and culture medium. Isolated human umbilical vascular ECs (hUVECs) were positive for von-Willibrand factor, a classical endothelial marker, and could form capillary-like structures when seeded on Matrigel, thus proving their functionality. The isolated human umbilical cord mesenchymal stem cells (hUCMSCs) were found positive for CD44, CD90, CD 73, and CD117 and were found negative for CD33, CD34, CD45, and CD105 surface markers; they were also positive for cytoskeleton markers of smooth muscle actin and vimentin. The hUCMSCs showed multilineage differentiation potential and differentiated into adipogenic, chondrogenic, osteogenic, and neuronal lineages under influence of lineage specific differentiation medium. Thus, isolating endothelial cells as well as mesenchymal cells from the same umbilical cord could lead to complete utilization of the available tissue for the tissue engineering and cell therapy.

Adoptive cell therapy for lymphoma with CD4 T cells depleted of CD137-expressing regulatory T cells.

PubMed

Goldstein, Matthew J; Kohrt, Holbrook E; Houot, Roch; Varghese, Bindu; Lin, Jack T; Swanson, Erica; Levy, Ronald

2012-03-01

Adoptive immunotherapy with antitumor T cells is a promising novel approach for the treatment of cancer. However, T-cell therapy may be limited by the cotransfer of regulatory T cells (T(reg)). Here, we explored this hypothesis by using 2 cell surface markers, CD44 and CD137, to isolate antitumor CD4 T cells while excluding T(regs). In a murine model of B-cell lymphoma, only CD137(neg)CD44(hi) CD4 T cells infiltrated tumor sites and provided protection. Conversely, the population of CD137(pos)CD44hi CD4 T cells consisted primarily of activated T(regs). Notably, this CD137(pos) T(reg) population persisted following adoptive transfer and maintained expression of FoxP3 as well as CD137. Moreover, in vitro these CD137(pos) cells suppressed the proliferation of effector cells in a contact-dependent manner, and in vivo adding the CD137(pos)CD44(hi) CD4 cells to CD137(neg)CD44(hi) CD4 cells suppressed the antitumor immune response. Thus, CD137 expression on CD4 T cells defined a population of activated T(regs) that greatly limited antitumor immune responses. Consistent with observations in the murine model, human lymphoma biopsies also contained a population of CD137(pos) CD4 T cells that were predominantly CD25(pos)FoxP3(pos) T(regs). In conclusion, our findings identify 2 surface markers that can be used to facilitate the enrichment of antitumor CD4 T cells while depleting an inhibitory T(reg) population.

CD44-positive cells are candidates for astrocyte precursor cells in developing mouse cerebellum.

PubMed

Cai, Na; Kurachi, Masashi; Shibasaki, Koji; Okano-Uchida, Takayuki; Ishizaki, Yasuki

2012-03-01

Neural stem cells are generally considered to be committed to becoming precursor cells before terminally differentiating into either neurons or glial cells during neural development. Neuronal and oligodendrocyte precursor cells have been identified in several areas in the murine central nervous system. The presence of astrocyte precursor cells (APCs) is not so well understood. The present study provides several lines of evidence that CD44-positive cells are APCs in the early postnatal mouse cerebellum. In developing mouse cerebellum, CD44-positive cells, mostly located in the white matter, were positive for the markers of the astrocyte lineage, but negative for the markers of mature astrocytes. CD44-positive cells were purified from postnatal cerebellum by fluorescence-activated cell sorting and characterized in vitro. In the absence of any signaling molecule, many cells died by apoptosis. The surviving cells gradually expressed glial fibrillary acidic protein, a marker for mature astrocytes, indicating that differentiation into mature astrocytes is the default program for these cells. The cells produced no neurospheres nor neurons nor oligodendrocytes under any condition examined, indicating these cells are not neural stem cells. Leukemia inhibitory factor greatly promoted astrocytic differentiation of CD44-positive cells, whereas bone morphogenetic protein 4 (BMP4) did not. Fibroblast growth factor-2 was a potent mitogen for these cells, but was insufficient for survival. BMP4 inhibited activation of caspase-3 and greatly promoted survival, suggesting a novel role for BMP4 in the control of development of astrocytes in cerebellum. We isolated and characterized only CD44 strongly positive large cells and discarded small and/or CD44 weakly positive cells in this study. Further studies are necessary to characterize these cells to help determine whether CD44 is a selective and specific marker for APCs in the developing mouse cerebellum. In conclusion, we succeeded in preparing APC candidates from developing mouse cerebellum, characterized them in vitro, and found that BMPs are survival factors for these cells.

Specific skin lesions in chronic myelomonocytic leukemia: a spectrum of myelomonocytic and dendritic cell proliferations: a study of 42 cases.

PubMed

Vitte, Franck; Fabiani, Bettina; Bénet, Claire; Dalac, Sophie; Balme, Brigitte; Delattre, Claire; Vergier, Béatrice; Beylot-Barry, Marie; Vignon-Pennamen, Dominique; Ortonne, Nicolas; Algros, Marie Paule; Carlotti, Agnès; Samaleire, Dimitri; Frouin, Eric; Levy, Anne; Laroche, Liliane; Theate, Ivan; Monnien, Franck; Mugneret, Francine; Petrella, Tony

2012-09-01

Chronic myelomonocytic leukemia (CMML) is a rare clonal hematopoietic disorder that can also involve the skin. The histopathology of these skin lesions is not clearly defined, and few data are available in the literature. To better understand tumoral skin involvements in CMML we carried out an extensive, retrospective clinicopathologic study of 42 cases selected from the database of the French Study Group of Cutaneous Lymphomas. On the basis of clinical data, morphology, and phenotype we identified 4 clinicopathologic profiles representing 4 distinct groups. The first group comprised myelomonocytic cell tumors (n=18), exhibiting a proliferation of granulocytic or monocytic blast cells, which were CD68 and/or MPO positive but negative for dendritic cell markers. The second group comprised mature plasmacytoid dendritic cell tumors (n=16), denoted by a proliferation of mature plasmacytoid dendritic cells, which were CD123, TCL1, and CD303 positive but CD56, CD1a, and S100 negative. The third group comprised blastic plasmacytoid dendritic cell tumors (n=4), characterized by a proliferation of monomorphous medium-sized blast cells, which were CD4, CD56, CD123, TCL1 positive but CD1a and S100 negative. The fourth group consisted of a putatively novel category of tumor that we named blastic indeterminate dendritic cell tumors (n=4), distinguished by a proliferation of large blast cells that not only exhibited monocytic markers but also the dendritic markers CD1a and S100. These 4 groups showed distinctive outcomes. Finally, we showed, by fluorescence in situ hybridization analysis, a clonal link between bone marrow disease and skin lesions in 4 patients. Herein, we have described a novel scheme for pathologists and physicians to handle specific lesions in CMML, which correspond to a spectrum of myelomonocytic and dendritic cell proliferations with different outcomes. A minimal panel of immunohistochemical markers including CD68, CD1a, S100, Langerin, and CD123 is necessary to make the correct classification in this spectrum of cutaneous CMML tumors, in which dendritic cell lineage plays an important role.

[Expression of embryonic markers in pterygium derived mesenchymal cells].

PubMed

Pascual, G; Montes, M A; Pérez-Rico, C; Pérez-Kohler, B; Bellón, J M; Buján, J

2010-12-01

Destruction of the limbal epithelium barrier is the most important mechanism of pterygium formation (conjunctiva proliferation, encroaching onto the cornea). It is thought to arise from activated and proliferating limbal epithelial stem cells. The objective of this study is to evaluate the presence of undifferentiated mesenchymal cells (stem cells) in cultured cells extracted from human pterygium. Cells from 6 human pterygium were isolated by explantation and placed in cultures with amniomax medium. Once the monolayer was reached the cells were seeded onto 24 well microplates. The cells were studied in the second sub-culture. The immunohistochemical expression of different embryonic stem cell markers, OCT3/4 and CD9, was analysed. The differentiated phenotypes were characterised with the monoclonal antibodies anti-CD31, α-actin and vimentin. All the cell populations obtained from pterygium showed vimentin expression. Less than 1% of the cells were positive for CD31 and α-actin markers. The majority of the cell population was positive for OCT3/4 and CD9. The cell population obtained from pterygium expressed mesenchymal cell phenotype and embryonic markers, such us OCT3/4 and CD9. This undifferentiated population could be involved in the large recurrence rate of this type of tissue after surgery. Copyright © 2010 Sociedad Española de Oftalmología. Published by Elsevier Espana. All rights reserved.

CD20-Positive nodal natural killer/T-cell lymphoma with cutaneous involvement.

PubMed

Tsai, Yi-Chiun; Chen, Chi-Kuan; Wu, Yu-Hung

2015-09-01

CD20-positive natural killer (NK)/T-cell lymphoma is extremely rare. We describe a case of a CD20-positive nodal NK/T-cell lymphoma with cutaneous involvement in a 32-year-old man. The patient presented with fever, night sweats, right inguinal lymphadenopathy and multiple violaceous to erythematous nodules and plaques on the back and bilateral legs. Immunohistochemical analysis showed diffusely and strongly positive staining for CD3, CD3 epsilon, CD43, CD56, TIA-1 and CD20 but negative staining for other B-cell markers, including CD79a and PAX-5 and T-cell markers CD5 and CD7. The tumor cell nuclei were diffusely positive for Epstein-Barr virus-encoded RNA in situ hybridization. A partial clinical response was observed after chemotherapy, indicated by the decreased size of the lymph nodes and skin lesions. It is a diagnostic challenge to deal with lymphoma cells that present with the surface proteins of both T- and B-cells. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Resistance to Cell Death and Its Modulation in Cancer Stem Cells

PubMed Central

Safa, Ahmad R.

2017-01-01

Accumulating evidence has demonstrated that human cancers arise from various tissues of origin that initiate from cancer stem cells (CSCs) or cancer-initiating cells. The extrinsic and intrinsic apoptotic pathways are dysregulated in CSCs, and these cells play crucial roles in tumor initiation, progression, cell death resistance, chemo- and radiotherapy resistance, and tumor recurrence. Understanding CSC-specific signaling proteins and pathways is necessary to identify specific therapeutic targets that may lead to the development of more efficient therapies selectively targeting CSCs. Several signaling pathways—including the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR), maternal embryonic leucine zipper kinase (MELK), NOTCH1, and Wnt/β-catenin—and expression of the CSC markers CD133, CD24, CD44, Oct4, Sox2, Nanog, and ALDH1A1 maintain CSC properties. Studying such pathways may help to understand CSC biology and lead to the development of potential therapeutic interventions to render CSCs more sensitive to cell death triggered by chemotherapy and radiation therapy. Moreover, recent demonstrations of dedifferentiation of differentiated cancer cells into CSC-like cells have created significant complexity in the CSCs hypothesis. Therefore, any successful therapeutic agent or combination of drugs for cancer therapy must eliminate not only CSCs but differentiated cancer cells and the entire bulk of tumor cells. This review article expands on the CSC hypothesis and paradigm with respect to major signaling pathways and effectors that regulate CSC apoptosis resistance. Moreover, selective CSC apoptotic modulators and their therapeutic potential for making tumors more responsive to therapy are discussed. The use of novel therapies, including small-molecule inhibitors of specific proteins in signaling pathways that regulate stemness, proliferation and migration of CSCs, immunotherapy, and noncoding microRNAs may provide better means of treating CSCs. PMID:27915972

Functional heterogeneity of side population cells in skeletal muscle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Uezumi, Akiyoshi; Ojima, Koichi; Fukada, So-ichiro

2006-03-17

Skeletal muscle regeneration has been exclusively attributed to myogenic precursors, satellite cells. A stem cell-rich fraction referred to as side population (SP) cells also resides in skeletal muscle, but its roles in muscle regeneration remain unclear. We found that muscle SP cells could be subdivided into three sub-fractions using CD31 and CD45 markers. The majority of SP cells in normal non-regenerating muscle expressed CD31 and had endothelial characteristics. However, CD31{sup -}CD45{sup -} SP cells, which are a minor subpopulation in normal muscle, actively proliferated upon muscle injury and expressed not only several regulatory genes for muscle regeneration but also somemore » mesenchymal lineage markers. CD31{sup -}CD45{sup -} SP cells showed the greatest myogenic potential among three SP sub-fractions, but indeed revealed mesenchymal potentials in vitro. These SP cells preferentially differentiated into myofibers after intramuscular transplantation in vivo. Our results revealed the heterogeneity of muscle SP cells and suggest that CD31{sup -}CD45{sup -} SP cells participate in muscle regeneration.« less

CD44 Is a Negative Cell Surface Marker for Pluripotent Stem Cell Identification during Human Fibroblast Reprogramming

PubMed Central

Vaz, Candida; Tanavde, Vivek; Lakshmipathy, Uma

2014-01-01

Induced pluripotent stem cells (iPSCs) are promising tools for disease research and cell therapy. One of the critical steps in establishing iPSC lines is the early identification of fully reprogrammed colonies among unreprogrammed fibroblasts and partially reprogrammed intermediates. Currently, colony morphology and pluripotent stem cell surface markers are used to identify iPSC colonies. Through additional clonal characterization, we show that these tools fail to distinguish partially reprogrammed intermediates from fully reprogrammed iPSCs. Thus, they can lead to the selection of suboptimal clones for expansion. A subsequent global transcriptome analysis revealed that the cell adhesion protein CD44 is a marker that differentiates between partially and fully reprogrammed cells. Immunohistochemistry and flow cytometry confirmed that CD44 is highly expressed in the human parental fibroblasts used for the reprogramming experiments. It is gradually lost throughout the reprogramming process and is absent in fully established iPSCs. When used in conjunction with pluripotent cell markers, CD44 staining results in the clear identification of fully reprogrammed cells. This combination of positive and negative surface markers allows for easier and more accurate iPSC detection and selection, thus reducing the effort spent on suboptimal iPSC clones. PMID:24416407

Expansion of PD-1-positive effector CD4 T cells in an experimental model of SLE: contribution to the self-organized criticality theory.

PubMed

Miyazaki, Yumi; Tsumiyama, Ken; Yamane, Takashi; Ito, Mitsuhiro; Shiozawa, Shunichi

2013-04-18

We have developed a systems biology concept to explain the origin of systemic autoimmunity. From our studies of systemic lupus erythematosus (SLE) we have concluded that this disease is the inevitable consequence of over-stimulating the host's immune system by repeated exposure to antigen to levels that surpass a critical threshold, which we term the system's "self-organized criticality". We observed that overstimulation of CD4 T cells in mice led to the development of autoantibody-inducing CD4 T cells (aiCD4 T) capable of generating various autoantibodies and pathological lesions identical to those observed in SLE. We show here that this is accompanied by the significant expansion of a novel population of effector T cells characterized by expression of programmed death-1 (PD-1)-positive, CD27(low), CD127(low), CCR7(low) and CD44(high)CD62L(low) markers, as well as increased production of IL-2 and IL-6. In addition, repeated immunization caused the expansion of CD8 T cells into fully-matured cytotoxic T lymphocytes (CTL) that express Ly6C(high)CD122(high) effector and memory markers. Thus, overstimulation with antigen leads to the expansion of a novel effector CD4 T cell population that expresses an unusual memory marker, PD-1, and that may contribute to the pathogenesis of SLE.

Medullospheres from DAOY, UW228 and ONS-76 cells: increased stem cell population and proteomic modifications.

PubMed

Zanini, Cristina; Ercole, Elisabetta; Mandili, Giorgia; Salaroli, Roberta; Poli, Alice; Renna, Cristiano; Papa, Valentina; Cenacchi, Giovanna; Forni, Marco

2013-01-01

Medulloblastoma (MB) is an aggressive pediatric tumor of the Central Nervous System (CNS) usually treated according to a refined risk stratification. The study of cancer stem cells (CSC) in MB is a promising approach aimed at finding new treatment strategies. The CSC compartment was studied in three characterized MB cell lines (DAOY, UW228 and ONS-76) grown in standard adhesion as well as being grown as spheres, which enables expansion of the CSC population. MB cell lines, grown in adherence and as spheres, were subjected to morphologic analysis at the light and electron microscopic level, as well as cytofluorimetric determinations. Medullospheres (MBS) were shown to express increasingly immature features, along with the stem cells markers: CD133, Nestin and β-catenin. Proteomic analysis highlighted the differences between MB cell lines, demonstrating a unique protein profile for each cell line, and minor differences when grown as spheres. In MBS, MALDI-TOF also identified some proteins, that have been linked to tumor progression and resistance, such as Nucleophosmin (NPM). In addition, immunocytochemistry detected Sox-2 as a stemness marker of MBS, as well as confirming high NPM expression. Culture conditioning based on low attachment flasks and specialized medium may provide new data on the staminal compartment of CNS tumors, although a proteomic profile of CSC is still elusive for MB.

Medullospheres from DAOY, UW228 and ONS-76 Cells: Increased Stem Cell Population and Proteomic Modifications

PubMed Central

Zanini, Cristina; Ercole, Elisabetta; Mandili, Giorgia; Salaroli, Roberta; Poli, Alice; Renna, Cristiano; Papa, Valentina; Cenacchi, Giovanna; Forni, Marco

2013-01-01

Background Medulloblastoma (MB) is an aggressive pediatric tumor of the Central Nervous System (CNS) usually treated according to a refined risk stratification. The study of cancer stem cells (CSC) in MB is a promising approach aimed at finding new treatment strategies. Methodology/Principal Findings The CSC compartment was studied in three characterized MB cell lines (DAOY, UW228 and ONS-76) grown in standard adhesion as well as being grown as spheres, which enables expansion of the CSC population. MB cell lines, grown in adherence and as spheres, were subjected to morphologic analysis at the light and electron microscopic level, as well as cytofluorimetric determinations. Medullospheres (MBS) were shown to express increasingly immature features, along with the stem cells markers: CD133, Nestin and β-catenin. Proteomic analysis highlighted the differences between MB cell lines, demonstrating a unique protein profile for each cell line, and minor differences when grown as spheres. In MBS, MALDI-TOF also identified some proteins, that have been linked to tumor progression and resistance, such as Nucleophosmin (NPM). In addition, immunocytochemistry detected Sox-2 as a stemness marker of MBS, as well as confirming high NPM expression. Conclusions/Significance Culture conditioning based on low attachment flasks and specialized medium may provide new data on the staminal compartment of CNS tumors, although a proteomic profile of CSC is still elusive for MB. PMID:23717474

EMMPRIN (CD147) is induced by C/EBPβ and is differentially expressed in ALK+ and ALK- anaplastic large-cell lymphoma.

PubMed

Schmidt, Janine; Bonzheim, Irina; Steinhilber, Julia; Montes-Mojarro, Ivonne A; Ortiz-Hidalgo, Carlos; Klapper, Wolfram; Fend, Falko; Quintanilla-Martínez, Leticia

2017-09-01

Anaplastic lymphoma kinase-positive (ALK+) anaplastic large-cell lymphoma (ALCL) is characterized by expression of oncogenic ALK fusion proteins due to the translocation t(2;5)(p23;q35) or variants. Although genotypically a T-cell lymphoma, ALK+ ALCL cells frequently show loss of T-cell-specific surface antigens and expression of monocytic markers. C/EBPβ, a transcription factor constitutively overexpressed in ALK+ ALCL cells, has been shown to play an important role in the activation and differentiation of macrophages and is furthermore capable of transdifferentiating B-cell and T-cell progenitors to macrophages in vitro. To analyze the role of C/EBPβ for the unusual phenotype of ALK+ ALCL cells, C/EBPβ was knocked down by RNA interference in two ALK+ ALCL cell lines, and surface antigen expression profiles of these cell lines were generated using a Human Cell Surface Marker Screening Panel (BD Biosciences). Interesting candidate antigens were further analyzed by immunohistochemistry in primary ALCL ALK+ and ALK- cases. Antigen expression profiling revealed marked changes in the expression of the activation markers CD25, CD30, CD98, CD147, and CD227 after C/EBPβ knockdown. Immunohistochemical analysis confirmed a strong, membranous CD147 (EMMPRIN) expression in ALK+ ALCL cases. In contrast, ALK- ALCL cases showed a weaker CD147 expression. CD274 or PD-L1, an immune inhibitory receptor ligand, was downregulated after C/EBPβ knockdown. PD-L1 also showed stronger expression in ALK+ ALCL compared with ALK- ALCL, suggesting an additional role of C/EBPβ in ALK+ ALCL in generating an immunosuppressive environment. Finally, no expression changes of T-cell or monocytic markers were detected. In conclusion, surface antigen expression profiling demonstrates that C/EBPβ plays a critical role in the activation state of ALK+ ALCL cells and reveals CD147 and PD-L1 as important downstream targets. The multiple roles of CD147 in migration, adhesion, and invasion, as well as T-cell activation and proliferation suggest its involvement in the pathogenesis of ALCL.

Upregulation of CD147 Promotes Metastasis of Cholangiocarcinoma by Modulating the Epithelial-to-Mesenchymal Transitional Process.

PubMed

Dana, Paweena; Kariya, Ryusho; Vaeteewoottacharn, Kulthida; Sawanyawisuth, Kanlayanee; Seubwai, Wunchana; Matsuda, Kouki; Okada, Seiji; Wongkham, Sopit

2017-08-07

CD147 is a transmembrane protein that can induce the expression and activity of matrix metalloproteinases (MMPs). Expression of CD147 has been shown to potentiate cell migration, invasion, and metastasis of cancer. In this study, the critical role of CD147 in metastasis was elucidated using CD147-overexpressing cholangiocarcinoma (CCA) cells in vitro and in vivo. The molecular mechanism, demonstrated herein, supported the hypothesis that metastasis increased in CD147-overexpressing cells. Five CD147-overexpressing clones (Ex-CD147) were established from a low CD147-expressing CCA cell line, KKU-055, using lentivirus containing pReceiver-Lenti-CD147. The metastatic capability was determined using the tail vein injection mouse model and an in vitro 3D invasion assay. Liver colonization was assessed using anti-HLA class I immunohistochemistry. Adhesion abilities, cytoskeletal arrangements, MMP activities, the expressions of adhesion molecules, and epithelial-mesenchymal transitional markers were analyzed. All Ex-CD147 clones exhibited a high CD147 expression and high liver colonization in the tail vein-injected mouse model, whereas parental cells lacked this ability. Ex-CD147 clones exhibited metastatic phenotypes (i.e., an increase in F-actin rearrangement) and cell invasion and a decrease in cell adhesion. The molecular mechanisms were shown to be via the induction of MMP-2 activity and enhancement of epithelial-mesenchymal transitions. An increase in mesenchymal markers Slug, vimentin, and N-cadherin, and a decrease in epithelial markers E-cadherin and claudin-1, together with suppression of the adhesion molecule ICAM-1, were observed in the Ex-CD147 clones. Moreover, suppression of CD147 expression using siCD147 in two CCA cell lines with high CD147 expression significantly decreased cell migration and invasion of these CCA cells. These findings emphasize the essential role of CD147 in CCA metastasis and suggest CD147 as a promising target for the effective treatment of CCA.

Chemo Resistance of Breast Cancer Stem Cells

DTIC Science & Technology

2006-05-01

for stem cell markers , including CD44+ CD24- lin- by flow cytometry following chemotherapy residual tumor is assayed. As shown in Figure 3 below...the tumor and thus may contribute to relapse following therapy. This was to be accomplished by utilizing mouse xenograft models as well as markers ...limitation of utilizing these markers is that they are not suitable for immunochemical detection of tumor stem cells since they require flow cytometric

[Expression of molecular markers detected by immunohistochemistry and risk of lymph node metastasis in stage T1 and T2 colorecrectal cancers].

PubMed

Wang, Fu-long; Wan, De-sen; Lu, Zhen-hai; Fang, Yu-jing; Li, Li-ren; Chen, Gong; Wu, Xiao-jun; Ding, Pei-rong; Kong, Ling-heng; Lin, Jun-zhong; Pan, Zhi-zhong

2013-04-01

To study the molecular risk factors of lymph node metastasis in stage T1 and T2 colorectal cancers by tissue microarray and immunohistochemistry techniques. Two hundred and three patients with stage T1 and T2 colorectal carcinoma who underwent radical surgery from 1999 to 2010 in our department were included in this study. Their clinicopathological data were retrospectively analyzed. Expression of the following 14 molecular markers were selected and assayed by tissue microarray and immunohistochemistry: VEGFR-3, HER2, CD44v6, CXCR4, TIMP-1, EGFR, IGF-1R, IGF-2, IGFBP-1, ECAD, MMP-9, RKIP, CD133, MSI. Chi-squared test and logistic regression were used to evaluate the variables as potential risk factors for lymph node metastasis. The positive expression rates of biomarkers were as following: VEGFR-3 (44.3%), EGFR (30.5%), HER-2 (28.1%), IGF-1R (63.5%), IGF-2 (44.8%), IGFBP-1 (70.9%), ECAD (45.8%), CD44v6 (51.2%), MMP-9 (44.3%), TIMP-1 (41.4%), RKIP (45.3%), CXCR4 (40.9%), and CD133 (49.8%). The positive rate of MSI expression was 22.2%. Both univariate and multivariate analyses showed that VEGFR-3, HER-2, and TIMP-1 were significant predictors of lymph node metastasis. Univariate analysis showed that CD44v6 and CXCR4 were significant significant predictors of lymph node metastasis. VEGFR-3, HER2 and TIMP-1 are independent factors for lymph node metastasis in stage T1 and T2 colorectal cancers.

Expression of CD105 cancer stem cell marker in three subtypes of renal cell carcinoma.

PubMed

Saeednejad Zanjani, Leili; Madjd, Zahra; Abolhasani, Maryam; Shariftabrizi, Ahmad; Rasti, Arezoo; Asgari, Mojgan

2018-01-01

CD105 is recently described as a cancer stem cell (CSC) marker. The present study was aimed to investigate the expression and prognostic significance of the CSC marker CD105 in different histological subtypes of renal cell carcinoma (RCC). Expression of CD105 was evaluated using immunohistochemistry in RCC samples on tissue microarrays including clear cell RCCs (ccRCCs), papillary, and chromophobe RCCs. The association between CD105 expression and clinicopathological features as well as survival outcomes was determined. In ccRCC, increased tumoral cytoplasmic and endothelial expression of CD105 were significantly associated with advanced stage, renal vein invasion, and microvascular invasion (MVI). In addition, MVI was associated with a worse overall survival (OS). Moreover, in multivariate analysis tumor stage and nuclear grade were independent prognostic factors for OS both in case of tumoral cytoplasmic and endothelial CD105 expression. Additionally, CD105 expression was found to be a predictor of worse OS in univariate analysis. However, in papillary and chromophobe RCC, no significant association was found between CD105 expression and clinicopathological parameters or prognosis. We showed that CD105 expression was associated with more aggressive tumor behavior, more advanced disease, and worse prognosis in ccRCC but not in the other RCC subtypes.

Cancer stem cells in head and neck squamous cell carcinoma: a review.

PubMed

Satpute, Pranali Shirish; Hazarey, Vinay; Ahmed, Riyaz; Yadav, Lalita

2013-01-01

Research indicates that a small population of cancer cells is highly tumorigenic, endowed with the capacity for self-renewal, and has the ability to differentiate into cells that constitute the bulk of tumors. These cells are considered the "drivers" of the tumorigenic process in some tumor types, and have been named cancer stem cells (CSC). Epithelial-mesenchymal transition (EMT) appears to be involved in the process leading to the acquisition of stemness by epithelial tumor cells. Through this process, cells acquire an invasive phenotype that may contribute to tumor recurrence and metastasis. CSC have been identified in human head and neck squamous cell carcinomas (HNSCC) using markers such as CD133 and CD44 expression, and aldehyde dehydrogenase (ALDH) activity. Head and neck cancer stem cells reside primarily in perivascular niches in the invasive fronts where endothelial-cell initiated events contribute to their survival and function. Clinically, CSC enrichment has been shown to be enhanced in recurrent disease, treatment failure and metastasis. CSC represent a novel target of study given their slow growth and innate mechanisms conferring treatment resistance. Further understanding of their unique phenotype may reveal potential molecular targets to improve therapeutic and survival outcomes in patients with HNSCC. Here, we discuss the state-of-the-knowledge on the pathobiology of cancer stem cells, with a focus on the impact of these cells on head and neck tumor progression, metastasis and recurrence due to treatment failure.

Markers for human brain pericytes and smooth muscle cells.

PubMed

Smyth, Leon C D; Rustenhoven, Justin; Scotter, Emma L; Schweder, Patrick; Faull, Richard L M; Park, Thomas I H; Dragunow, Mike

2018-06-07

Brain pericytes and vascular smooth muscle cells (vSMCs) are a critical component of the neurovascular unit and are important in regulating cerebral blood flow and blood-brain barrier integrity. Identification of subtypes of mural cells in tissue and in vitro is important to any study of their function, therefore we identified distinct mural cell morphologies in neurologically normal post-mortem human brain. Further, the distribution of mural cell markers platelet-derived growth factor receptor-β (PDGFRβ), α-smooth muscle actin (αSMA), CD13, neural/glial antigen-2 (NG2), CD146 and desmin was examined. We determined that PDGFRβ, NG2, CD13, and CD146 were expressed in capillary-associated pericytes. NG2, and CD13 were also present on vSMCs in large vessels, however abundant CD146 and desmin staining was also detected in vSMCs on large vessels, co-labelling with αSMA. To determine whether cultures recapitulated observations from tissue, primary human brain pericytes derived from neurologically normal autopsies were analysed for the presence of pericyte markers by immunocytochemistry, western blotting and qPCR. The proteins observed in brain pericytes in tissue (PDGFRβ, αSMA, desmin, CD146, CD13, and NG2) were present in vitro, validating a panel of proteins that can be used to label brain pericytes and vSMCs in tissue and in vitro. Finally, we showed that the proteins CD146 and desmin that are expressed on large vessels in situ, are also selective markers of a smooth muscle cell phenotype in vitro. Copyright © 2018 Elsevier B.V. All rights reserved.

Isolation and characterization of equine endometrial mesenchymal stromal cells.

PubMed

Rink, B Elisabeth; Amilon, Karin R; Esteves, Cristina L; French, Hilari M; Watson, Elaine; Aurich, Christine; Donadeu, F Xavier

2017-07-12

Equine mesenchymal stromal/stem cells (MSCs) are most commonly harvested from bone marrow (BM) or adipose tissue, requiring the use of surgical procedures. By contrast, the uterus can be accessed nonsurgically, and may provide a more readily available cell source. While human endometrium is known to harbor mesenchymal precursor cells, MSCs have not been identified in equine endometrium. This study reports the isolation, culture, and characterization of MSCs from equine endometrium. The presence of MSC and pericyte markers in endometrial sections was determined using immunohistochemistry. Stromal cells were harvested and cultured after separation of epithelial cells from endometrial fragments using Mucin-1-bound beads. For comparison, MSCs were also harvested from BM. The expression of surface markers in endometrial and BM-derived MSCs was characterized using flow cytometry and quantitative polymerase chain reaction. MSCs were differentiated in vitro into adipogenic, chondrogenic, osteogenic, and smooth muscle lineages. Typical markers of MSCs (CD29, CD44, CD90, and CD105) and pericytes (NG2 and CD146) were localized in the equine endometrium. Both endometrial and BM MSCs grew clonally and robustly expressed MSC and pericyte markers in culture while showing greatly reduced or negligible expression of hematopoietic markers (CD45, CD34) and MHC-II. Additionally, both endometrial and BM MSCs differentiated into adipogenic, osteogenic, and chondrogenic lineages in vitro, and endometrial MSCs had a distinct ability to undergo smooth muscle differentiation. We have demonstrated for the first time the presence of cells in equine endometrium that fulfill the definition of MSCs. The equine endometrium may provide an alternative, easily accessible source of MSCs, not only for therapeutic regeneration of the uterus, but also for other tissues where MSCs from other sources are currently being used therapeutically.

Articular cartilage-derived cells hold a strong osteogenic differentiation potential in comparison to mesenchymal stem cells in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Salamon, Achim, E-mail: achim.salamon@med.uni-rostock.de; Jonitz-Heincke, Anika, E-mail: anika.jonitz@med.uni-rostock.de; Adam, Stefanie, E-mail: stefanie.adam@med.uni-rostock.de

Cartilaginous matrix-degenerative diseases like osteoarthritis (OA) are characterized by gradual cartilage erosion, and also by increased presence of cells with mesenchymal stem cell (MSC) character within the affected tissues. Moreover, primary chondrocytes long since are known to de-differentiate in vitro and to be chondrogenically re-differentiable. Since both findings appear to conflict with each other, we quantitatively assessed the mesenchymal differentiation potential of OA patient cartilage-derived cells (CDC) towards the osteogenic and adipogenic lineage in vitro and compared it to that of MSC isolated from adipose tissue (adMSC) of healthy donors. We analyzed expression of MSC markers CD29, CD44, CD105, andmore » CD166, and, following osteogenic and adipogenic induction in vitro, quantified their expression of osteogenic and adipogenic differentiation markers. Furthermore, CDC phenotype and proliferation were monitored. We found that CDC exhibit an MSC CD marker expression pattern similar to adMSC and a similar increase in proliferation rate during osteogenic differentiation. In contrast, the marked reduction of proliferation observed during adipogenic differentiation of adMSC was absent in CDC. Quantification of differentiation markers revealed a strong osteogenic differentiation potential for CDC, however almost no capacity for adipogenic differentiation. Since in the pathogenesis of OA, cartilage degeneration coincides with high bone turnover rates, the high osteogenic differentiation potential of OA patient-derived CDC may affect clinical therapeutic regimens aiming at autologous cartilage regeneration in these patients. - Highlights: • We analyze the mesenchymal differentiation capacity of cartilage-derived cells (CDC). • CDC express mesenchymal stem cell (MSC) markers CD29, CD44, CD105, and CD166. • CDC and MSC proliferation is reduced in adipogenesis and increased in osteogenesis. • Adipogenic differentiation is virtually absent in CDC, but strong in MSC. • Osteogenic differentiation is significantly stronger for CDC than for MSC.« less

Generation of Mesenchymal-Like Stem Cells From Urine in Pediatric Patients.

PubMed

He, W; Zhu, W; Cao, Q; Shen, Y; Zhou, Q; Yu, P; Liu, X; Ma, J; Li, Y; Hong, K

2016-01-01

Mesenchymal stem cells (MSCs) have been widely used for regenerative medicine. Traditionally, the procedures of MSC isolation are usually invasive and time-consuming. Urine is merely a body waste, and recent studies have suggested that urine represents an alternative source of stem cells. We, therefore, determined whether the possibility of isolating mesenchymal-like stem cells was practical from human urine. A total of 16 urine samples were collected from pediatric patients. Urine-derived cells were isolated, expanded, and identified for specific cell surface markers using flow cytometry. Cell morphology was observed by microscopy. Osteogenic and adipogenic differentiation potential were determinded by culturing cells in specific induction medium, and assessed by alkaline phosphatase and oil red O stainings, respectively. Clones were established and passaged successfully from primary cultures of urine cells. Cultured urine-derived cells at passage 3 were fusiform and arranged with certain directionality. Urine-derived cells at passage 5 displayed expressions of cell surface markers (CD29, CD105, CD166, CD90, and CD13). There was no expression of the general hematopoietic cell markers (CD45, CD34, and HLA-DR). Under in vitro induction conditions, urine-derived cells at passage 5 were able to differentiate into osteoblasts, but not adipocytes. Urine may be a noninvasive source for mesenchymal-like stem cells. These cells could potentially provide a new source of autologous stem cells for regenerative medicine and cell therapy. Copyright © 2016 Elsevier Inc. All rights reserved.

Comprehensive Mass Cytometry Analysis of Cell Cycle, Activation, and Coinhibitory Receptors Expression in CD4 T Cells from Healthy and HIV-Infected Individuals.

PubMed

Corneau, Aurélien; Cosma, Antonio; Even, Sophie; Katlama, Christine; Le Grand, Roger; Frachet, Véronique; Blanc, Catherine; Autran, Brigitte

2017-01-01

Mass cytometry allows large multiplex analysis of cell cycle stages together with differentiation, activation, and exhaustion markers, allowing further assessment of the quiescence status of resting CD4 T cells. Peripheral blood CD4 T lymphocytes from 8 individuals, 4 healthy donors, and 4 HIV-infected on antiretroviral treatment (T) were stained with the same 26 monoclonal antibodies and dyes targeting surface and intracellular markers of differentiation, activation, exhaustion, and cell cycle stages. Samples were run on a CYTOF-2. Patterns of naïve [TN] CD4 T cells strongly differed from all other memory subsets central-memory (CM), transitional-memory (TM), effector-memory (EM), and terminally differentiated RA-expressing (TEMRA) subsets, while stem-cell memory (SCM) and T follicular-helper cells (TfH) were close to CM and TM cells with the highest percentages in cell cycle. EM and TEMRA were the most altered by HIV infection, with an increased frequency of activated and cycling cells. Activation markers and coinhibitory receptor expression differed among cell cycle stages, with HLA-DR fitting better than CD25 or CD38 with cycle, and opposite PD-1 gradients along differentiation and cell cycle. "Resting" DR-CD25- CD4+ T cells contained similar amounts of cells in G1 than the activated DR ± CD25± ones but three fold lower cells in S-G2-M. This broad multiplex mass cytometry analysis demonstrates some subsets of the so-called "resting" CD25-DR- CD4+ T cells contain noticeable amounts of cells into cycle or expressing coinhibitory receptors, opening new avenues for a redefinition of resting peripheral blood CD4 T cells harboring the HIV reservoirs. © 2016 International Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.

Differential marker expression by cultures rich in mesenchymal stem cells

PubMed Central

2013-01-01

Background Mesenchymal stem cells have properties that make them amenable to therapeutic use. However, the acceptance of mesenchymal stem cells in clinical practice requires standardized techniques for their specific isolation. To date, there are no conclusive marker (s) for the exclusive isolation of mesenchymal stem cells. Our aim was to identify markers differentially expressed between mesenchymal stem cell and non-stem cell mesenchymal cell cultures. We compared and contrasted the phenotype of tissue cultures in which mesenchymal stem cells are rich and rare. By initially assessing mesenchymal stem cell differentiation, we established that bone marrow and breast adipose cultures are rich in mesenchymal stem cells while, in our hands, foreskin fibroblast and olfactory tissue cultures contain rare mesenchymal stem cells. In particular, olfactory tissue cells represent non-stem cell mesenchymal cells. Subsequently, the phenotype of the tissue cultures were thoroughly assessed using immuno-fluorescence, flow-cytometry, proteomics, antibody arrays and qPCR. Results Our analysis revealed that all tissue cultures, regardless of differentiation potential, demonstrated remarkably similar phenotypes. Importantly, it was also observed that common mesenchymal stem cell markers, and fibroblast-associated markers, do not discriminate between mesenchymal stem cell and non-stem cell mesenchymal cell cultures. Examination and comparison of the phenotypes of mesenchymal stem cell and non-stem cell mesenchymal cell cultures revealed three differentially expressed markers – CD24, CD108 and CD40. Conclusion We indicate the importance of establishing differential marker expression between mesenchymal stem cells and non-stem cell mesenchymal cells in order to determine stem cell specific markers. PMID:24304471

Mycoplasma detection and elimination are necessary for the application of stem cell from human dental apical papilla to tissue engineering and regenerative medicine.

PubMed

Kim, Byung-Chul; Kim, So Yeon; Kwon, Yong-Dae; Choe, Sung Chul; Han, Dong-Wook; Hwang, Yu-Shik

2015-01-01

Recently, postnatal stem cells from dental papilla with neural crest origin have been considered as one of potent stem cell sources in regenerative medicine regarding their multi-differentiation capacity and relatively easy access. However, almost human oral tissues have been reported to be infected by mycoplasma which gives rise to oral cavity in teeth, and mycoplasma contamination of ex-vivo cultured stem cells from such dental tissues and its effect on stem cell culture has received little attention. In this study, mycoplama contamination was evaluated with stem cells from apical papilla which were isolated from human third molar and premolars from various aged patients undergoing orthodontic therapy. The ex-vivo expanded stem cells from apical papilla were found to express stem cell markers such as Stro-1, CD44, nestin and CD133, but mycoplama contamination was detected in almost all cell cultures of the tested 20 samples, which was confirmed by mycoplasma-specific gene expression and fluorescence staining. Such contaminated mycoplasma could be successfully eliminated using elimination kit, and proliferation test showed decreased proliferation activity in mycoplasma-contaminated cells. After elimination of contaminated mycoplasma, stem cells from apical papilla showed osteogenic and neural lineage differentiation under certain culture conditions. Our study proposes that the evaluation of mycoplasma contamination and elimination process might be required in the use of stem cells from apical papilla for their potent applications to tissue engineering and regenerative medicine.

Vascular Regeneration in a Basal Chordate Is Due to the Presence of Immobile, Bi-Functional Cells

PubMed Central

Braden, Brian P.; Taketa, Daryl A.; Pierce, James D.; Kassmer, Susannah; Lewis, Daniel D.; De Tomaso, Anthony W.

2014-01-01

The source of tissue turnover during homeostasis or following injury is usually due to proliferation of a small number of resident, lineage-restricted stem cells that have the ability to amplify and differentiate into mature cell types. We are studying vascular regeneration in a chordate model organism, Botryllus schlosseri, and have previously found that following surgical ablation of the extracorporeal vasculature, new tissue will regenerate in a VEGF-dependent process within 48 hrs. Here we use a novel vascular cell lineage tracing methodology to assess regeneration in parabiosed individuals and demonstrate that the source of regenerated vasculature is due to the proliferation of pre-existing vascular resident cells and not a mobile progenitor. We also show that these cells are bi-potential, and can reversibly adopt two fates, that of the newly forming vessels or the differentiated vascular tissue at the terminus of the vasculature, known as ampullae. In addition, we show that pre-existing vascular resident cells differentially express progenitor and differentiated cell markers including the Botryllus homologs of CD133, VEGFR-2, and Cadherin during the regenerative process. PMID:24736432

Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells: a joint statement of the International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy (ISCT).

PubMed

Bourin, Philippe; Bunnell, Bruce A; Casteilla, Louis; Dominici, Massimo; Katz, Adam J; March, Keith L; Redl, Heinz; Rubin, J Peter; Yoshimura, Kotaro; Gimble, Jeffrey M

2013-06-01

Adipose tissue is a rich and very convenient source of cells for regenerative medicine therapeutic approaches. However, a characterization of the population of adipose-derived stromal and stem cells (ASCs) with the greatest therapeutic potential remains unclear. Under the authority of International Federation of Adipose Therapeutics and International Society for Cellular Therapy, this paper sets out to establish minimal definitions of stromal cells both as uncultured stromal vascular fraction (SVF) and as an adherent stromal/stem cells population. Phenotypic and functional criteria for the identification of adipose-derived cells were drawn from the literature. In the SVF, cells are identified phenotypically by the following markers: CD45-CD235a-CD31-CD34+. Added value may be provided by both a viability marker and the following surface antigens: CD13, CD73, CD90 and CD105. The fibroblastoid colony-forming unit assay permits the evaluation of progenitor frequency in the SVF population. In culture, ASCs retain markers in common with other mesenchymal stromal/stem cells (MSCs), including CD90, CD73, CD105, and CD44 and remain negative for CD45 and CD31. They can be distinguished from bone-marrow-derived MSCs by their positivity for CD36 and negativity for CD106. The CFU-F assay is recommended to calculate population doublings capacity of ASCs. The adipocytic, chondroblastic and osteoblastic differentiation assays serve to complete the cell identification and potency assessment in conjunction with a quantitative evaluation of the differentiation either biochemically or by reverse transcription polymerase chain reaction. The goal of this paper is to provide initial guidance for the scientific community working with adipose-derived cells and to facilitate development of international standards based on reproducible parameters. Copyright © 2013 International Society for Cellular Therapy. All rights reserved.

Downregulation of mitochondrial UQCRB inhibits cancer stem cell-like properties in glioblastoma.

PubMed

Jung, Narae; Kwon, Ho Jeong; Jung, Hye Jin

2018-01-01

Glioblastoma stem cell targeted therapies have become a powerful strategy for the treatment of this deadliest brain tumor. We demonstrate for the first time that downregulation of mitochondrial ubiquinol-cytochrome c reductase binding protein (UQCRB) inhibits the cancer stem cell-like properties in human glioblastoma cells. The synthetic small molecules targeting UQCRB significantly suppressed not only the self-renewal capacity such as growth and neurosphere formation, but also the metastatic potential such as migration and invasion of glioblastoma stem‑like cells (GSCs) derived from U87MG and U373MG at subtoxic concentrations. Notably, the UQCRB inhibitors repressed c‑Met-mediated downstream signal transduction and hypoxia‑inducible factor‑1α (HIF‑1α) activation, thereby reducing the expression levels of GSC markers including CD133, Nanog, Oct4 and Sox2 in the GSCs. Furthermore, the UQCRB inhibitors decreased mitochondrial ROS generation and mitochondrial membrane potential in the GSCs, indicating that they regulate the mitochondrial function in GSCs. Indeed, the knockdown of UQCRB gene by UQCRB siRNA significantly inhibited the cancer stem cell-like phenotypes as well as the expression of stemness markers by blocking mitochondrial ROS/HIF‑1α/c‑Met pathway in U87MG GSCs. These findings suggest that UQCRB and its inhibitors could be a new therapeutic target and lead compounds for eliminating cancer stem cells in glioblastoma.

CD34 cells from patients with trisomy 8 myelodysplastic syndrome (MDS) express early apoptotic markers but avoid programmed cell death by up-regulation of antiapoptotic proteins

PubMed Central

Pfannes, Loretta; Chen, Gubin; Shah, Simant; Solomou, Elena E.; Barrett, John; Young, Neal S.

2007-01-01

CD34 cells from patients with trisomy 8 myelodysplastic syndrome (MDS) are distinguished from other MDS cells and from normal hematopoietic cells by their pronounced expression of apoptotic markers. Paradoxically, trisomy 8 clones can persist in patients with bone marrow failure and expand following immunosuppression. We previously demonstrated up-regulation of c-myc and CD1 by microarray analysis. Here, we confirmed these findings by real-time polymerase chain reaction (PCR), demonstrated up-regulation of survivin, c-myc, and CD1 protein expression, and documented comparable colony formation by annexin+ trisomy 8− CD34+ and annexin− CD34 cells. There were low levels of DNA degradation in annexin+ trisomy 8 CD34 cells, which were comparable with annexin− CD34 cells. Trisomy 8 cells were resistant to apoptosis induced by gamma irradiation. Knock-down of survivin by siRNA resulted in preferential loss of trisomy 8 cells. These results suggest that trisomy 8 cells undergo incomplete apoptosis and are nonetheless capable of colony formation and growth. PMID:17090657

CD25 is expressed by canine cutaneous mast cell tumors but not by cutaneous connective tissue mast cells.

PubMed

Meyer, A; Gruber, A D; Klopfleisch, R

2012-11-01

Canine cutaneous mast cell tumors (MCT) of different histological grades have distinct biological behaviors. However, little is known about underlying molecular mechanisms that lead to tumor development and increasing malignancy with higher tumor grade. Recent studies have identified the interleukin-2 receptor (IL-2R) subunits CD25 and CD2 as markers that distinguish nonneoplastic from neoplastic mast cells in human systemic mastocytosis. In this study, their potential as a marker for canine MCT and their possible impact on MCT carcinogenesis were evaluated. mRNA expression levels of both genes were compared between grade 1 (n = 12) and grade 3 (n = 8) MCT, and protein expression levels of CD25 were compared in 90 MCT of different tumor grades. mRNA expression levels of both CD25 and CD2 were upregulated in grade 3 MCT. In contrast, CD25 protein was expressed by fewer tumor cells and at decreased levels in grade 3 tumors, while most grade 1 MCT had strong CD25 protein expression. Moreover, CD25 was not expressed by nonneoplastic, resting cutaneous mast cells, while few presumably activated mast cells in tissue samples from dogs with allergic dermatitis had weak CD25 expression. Taken together, these findings suggest that CD25 may play a critical role in early MCT development and may be a stimulatory factor in grade 1 MCT, while grade 3 MCT seem to be less dependent on CD25. Because of the low number of CD25-positive tumor cells in high-grade tumors, the usefulness of CD25 as a tumor marker is, however, questionable.

Prognostic relevance of miR-137 in patients with hepatocellular carcinoma.

PubMed

Sakabe, Tomohiko; Azumi, Junya; Umekita, Yoshihisa; Toriguchi, Kan; Hatano, Etsuro; Hirooka, Yasuaki; Shiota, Goshi

2017-02-01

Cancer stem cells (CSCs) play a pivotal role in progression, metastasis and recurrence of cancer. Therefore, it is clinically useful to identify the relevant CSC marker that is associated with prognosis of hepatocellular carcinoma (HCC) and clarify its genetic and biological characteristics. Expression of four CSC markers, CD13, EpCAM, CD44 and CD44v9, was examined in 99 HCC patients. Biological and cDNA/miRNA microarray data were compared among CD44-positive/-negative HCC cells and normal hepatic cells. The significance of the representative miRNAs was examined with regard to prognosis of additional 110 HCC patients. CD44-positive HuH7 cells proliferated faster and showed a greater sphere forming ability than CD44-negative HuH7 cells. CD44-positive HuH7 cells exhibited higher expression of specific genes involved in resistance to reactive oxygen species, anticancer drugs and tumour invasion than CD44-negative HCC cells. Higher expression of six miRNAs was observed in CD44-positive HuH7 cells, CD44-negative HuH7 cells, and human normal hepatic cells in that order. Of the six miRNAs, miR-137 was closely associated with overall and cancer-specific survivals, as well as with invasion of hepatic vein, hepatic artery, portal vein and bile duct, and alpha-foetoprotein in additional 110 HCC patients. miR-137 may serve as a prognostic marker in patients with HCC and may be a potential target for the elimination of liver CSCs. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

CD34(+) Liver Cancer Stem Cells Were Formed by Fusion of Hepatobiliary Stem/Progenitor Cells with Hematopoietic Precursor-Derived Myeloid Intermediates.

PubMed

Zeng, Changjun; Zhang, Yanling; Park, Su Cheol; Eun, Jong Ryeol; Nguyen, Ngoc Tue; Tschudy-Seney, Benjamin; Jung, Yong Jin; Theise, Neil D; Zern, Mark A; Duan, Yuyou

2015-11-01

A large number of cancer stem cells (CSCs) were identified and characterized; however, the origins and formation of CSCs remain elusive. In this study, we examined the origination of the newly identified CD34(+) liver CSC (LCSC). We found that CD34(+) LCSC coexpressed liver stem cell and myelomonocytic cell markers, showing a mixed phenotype, a combination of hepatobiliary stem/progenitor cells (HSPCs) and myelomonocytic cells. Moreover, human xenografts produced by CD34(+) LCSCs and the parental cells, which CD34(+) LCSC was isolated from, coexpressed liver cancer and myelomonocytic markers, also demonstrating mixed phenotypes. The xenografts and the parental cells secreted albumin demonstrating their hepatocyte origin and also expressed cytokines [interleukin (IL)-1b, IL-6, IL-12A, IL-18, tumor necrosis factor-alpha (TNF-α), and CSF1] and chemokines (IL-8, CCL2, and CCL5). Expression of these cytokines and chemokines responded to the stimuli [interferon-γ (INF-γ), IL-4, and lipopolysaccharide (LPS)]. Furthermore, human xenografts and the parental cells phagocytized Escherichia coli. CD34(+) LCSC coexpressed CD45, demonstrating that its origin appears to be from a hematopoietic precursor. The percentage of cells positive for OV6, CD34, and CD31, presenting the markers of HSPC, hematopoietic, and myelomonocytic cells, increased under treatment of CD34(+) LCSC with a drug. Cytogenetic analysis showed that CD34(+) LCSC contained a greater number of chromosomes. HBV DNA integrations and mutations in CD34(+) LCSC and the parental cells were identical to those in the literature or the database. Thus, these results demonstrated that CD34(+) LCSCs were formed by fusion of HSPC with CD34(+) hematopoietic precursor-derived myeloid intermediates; it appears that this is the first report that human CSCs have been formed by the fusion. Therefore, it represents a significant step toward better understanding of the formation of human CSC and the diverse origins of liver cancers.

Expression of Master Regulators of T-cell, Helper T-cell and Follicular Helper T-cell Differentiation in Angioimmunoblastic T-cell Lymphoma.

PubMed

Matsumoto, Yosuke; Nagoshi, Hisao; Yoshida, Mihoko; Kato, Seiichi; Kuroda, Junya; Shimura, Kazuho; Kaneko, Hiroto; Horiike, Shigeo; Nakamura, Shigeo; Taniwaki, Masafumi

2017-11-01

Objective It has been postulated that the normal counterpart of angioimmunoblastic T-cell lymphoma (AITL) is the follicular helper T-cell (TFH). Recent immunological studies have identified several transcription factors responsible for T-cell differentiation. The master regulators associated with T-cell, helper T-cell (Th), and TFH differentiation are reportedly BCL11B, Th-POK, and BCL6, respectively. We explored the postulated normal counterpart of AITL with respect to the expression of the master regulators of T-cell differentiation. Methods We performed an immunohistochemical analysis in 15 AITL patients to determine the expression of the master regulators and several surface markers associated with T-cell differentiation. Results BCL11B was detected in 10 patients (67%), and the surface marker of T-cells (CD3) was detected in all patients. Only 2 patients (13%) expressed the marker of naïve T-cells (CD45RA), but all patients expressed the marker of effector T-cells (CD45RO). Nine patients expressed Th-POK (60%), and 7 (47%) expressed a set of surface antigens of Th (CD4-positive and CD8-negative). In addition, BCL6 and the surface markers of TFH (CXCL13, PD-1, and SAP) were detected in 11 (73%), 8 (53%), 14 (93%), and all patients, respectively. Th-POK-positive/BCL6-negative patients showed a significantly shorter overall survival (OS) than the other patients (median OS: 33.0 months vs. 74.0 months, p=0.020; log-rank test). Conclusion Many of the AITL patients analyzed in this study expressed the master regulators of T-cell differentiation. The clarification of the diagnostic significance and pathophysiology based on the expression of these master regulators in AITL is expected in the future.

Regulatory role of periodontal ligament fibroblasts for innate immune cell function and differentiation.

PubMed

Konermann, Anna; Stabenow, Dirk; Knolle, Percy A; Held, Stefanie A E; Deschner, James; Jäger, Andreas

2012-10-01

Innate immunity is crucial for an effective host defense against pathogenic microorganisms in periodontal tissues. As periodontal ligament (PDL) cells synthesize immunomodulatory cytokines, the aim of this in vitro study was to investigate whether these cells can interact with innate immune cells. Resting and inflammatory primed (IL-1β, TNF-α, HMGB1) human PDL cells were co-cultured with human monocyte-derived dendritic cells or macrophages. Migration, phenotypic maturation and modulation of phagocytosis of Porphyromonas gingivalis by immune cells were investigated upon co-culture with PDL cells and/or their released soluble factors. PDL cells interacted with immune cells under both non-inflammatory and inflammatory conditions. Immune cell migration was significantly enhanced by co-culture with PDL cells, which also affected their phenotypic maturation both through cell-cell contact and through released soluble mediators. The dendritic cell maturation markers CD83 and CD86 were upregulated as much as both 'alternatively activated' M2 macrophage maturation markers CD23 and CD163. In contrast, the 'classically activated' M1 macrophage maturation marker CD64 was downregulated. Finally, PDL cells significantly enhanced the phagocytosis of Porphyromonas gingivalis by immune cells. Our experiments revealed that PDL cells are not only structural elements of the periodontium, but actively influence immune responses by interaction with innate immune cells.

Variation in primary and culture-expanded cells derived from connective tissue progenitors in human bone marrow space, bone trabecular surface and adipose tissue.

PubMed

Qadan, Maha A; Piuzzi, Nicolas S; Boehm, Cynthia; Bova, Wesley; Moos, Malcolm; Midura, Ronald J; Hascall, Vincent C; Malcuit, Christopher; Muschler, George F

2018-03-01

Connective tissue progenitors (CTPs) embody the heterogeneous stem and progenitor cell populations present in native tissue. CTPs are essential to the formation and remodeling of connective tissue and represent key targets for tissue-engineering and cell-based therapies. To better understand and characterize CTPs, we aimed to compare the (i) concentration and prevalence, (ii) early in vitro biological behavior and (iii) expression of surface-markers and transcription factors among cells derived from marrow space (MS), trabecular surface (TS), and adipose tissues (AT). Cancellous-bone and subcutaneous-adipose tissues were collected from 8 patients. Cells were isolated and cultured. Colony formation was assayed using Colonyze software based on ASTM standards. Cell concentration ([Cell]), CTP concentration ([CTP]) and CTP prevalence (P CTP ) were determined. Attributes of culture-expanded cells were compared based on (i) effective proliferation rate and (ii) expression of surface-markers CD73, CD90, CD105, SSEA-4, SSEA-3, SSEA-1/CD15, Cripto-1, E-Cadherin/CD324, Ep-CAM/CD326, CD146, hyaluronan and transcription factors Oct3/4, Sox-2 and Nanog using flow cytometry. Mean [Cell], [CTP] and P CTP were significantly different between MS and TS samples (P = 0.03, P = 0.008 and P= 0.0003), respectively. AT-derived cells generated the highest mean total cell yield at day 6 of culture-4-fold greater than TS and more than 40-fold greater than MS per million cells plated. TS colonies grew with higher mean density than MS colonies (290 ± 11 versus 150 ± 11 cell per mm 2 ; P = 0.0002). Expression of classical-mesenchymal stromal cell (MSC) markers was consistently recorded (>95%) from all tissue sources, whereas all the other markers were highly variable. The prevalence and biological potential of CTPs are different between patients and tissue sources and lack variation in classical MSC markers. Other markers are more likely to discriminate differences between cell populations in biological performance. Understanding the underlying reasons for variation in the concentration, prevalence, marker expression and biological potential of CTPs between patients and source tissues and determining the means of managing this variation will contribute to the rational development of cell-based clinical diagnostics and targeted cell-based therapies. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Establishment and Characterization of Immortalized Human Amniotic Epithelial Cells

PubMed Central

Zhou, Kaixuan; Koike, Chika; Yoshida, Toshiko; Okabe, Motonori; Fathy, Moustafa; Kyo, Satoru; Kiyono, Tohru; Saito, Shigeru

2013-01-01

Abstract Human amniotic epithelial cells (HAEs) have a low immunogenic profile and possess potent immunosuppressive properties. HAEs also have several characteristics similar to stem cells, and they are discarded after parturition. Thus, they could potentially be used in cell therapy with fewer ethical problems. HAEs have a short life, so our aim is to establish and characterize immortalized human amniotic epithelial cells (iHAEs). HAEs were introduced with viral oncogenes E6/E7 and with human telomerase reverse transcriptase (hTERT) to create iHAEs. These iHAEs have proliferated around 200 population doublings (PDs) for at least 12 months. High expression of stem cell markers (Oct 3/4, Nanog, Sox2, Klf4) and epithelial markers (CK5, CK18) were detected by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). These iHAEs were expanded in ultra-low-attachment dishes to form spheroids similarly to epithelial stem/precursor cells. High expression of mesenchymal (CD44, CD73, CD90, CD105) and somatic (CD24, CD29, CD271, Nestin) stem cell markers was detected by flow cytometry. The iHAEs showed adipogenic, osteogenic, neuronal, and cardiac differentiation abilities. In conclusion, the immortalization of HAEs with the characteristics of stem cells has been established, allowing these iHAEs to become useful for cell therapy and regenerative medicine. PMID:23298399

Renal F4/80+CD11c+ Mononuclear Phagocytes Display Phenotypic and Functional Characteristics of Macrophages in Health and in Adriamycin Nephropathy

PubMed Central

Wang, Yiping; Wang, Xin Maggie; Lu, Junyu; Lee, Vincent W.S.; Ye, Qianling; Nguyen, Hanh; Zheng, Guoping; Zhao, Ye; Alexander, Stephen I.; Harris, David C.H.

2015-01-01

Conventional markers of macrophages (Mфs) and dendritic cells (DCs) lack specificity and often overlap, leading to confusion and controversy regarding the precise function of these cells in kidney and other diseases. This study aimed to identify the phenotype and function of renal mononuclear phagocytes (rMPs) expressing key markers of both Mфs and DCs. F4/80+CD11c+ cells accounted for 45% of total rMPs in normal kidneys and in those from mice with Adriamycin nephropathy (AN). Despite expression of the DC marker CD11c, these double-positive rMPs displayed the features of Mфs, including Mф-like morphology, high expression of CD68, CD204, and CD206, and high phagocytic ability but low antigen-presenting ability. F4/80+CD11c+ cells were found in the cortex but not in the medulla of the kidney. In AN, F4/80+CD11c+ cells displayed an M1 Mф phenotype with high expression of inflammatory mediators and costimulatory factors. Adoptive transfer of F4/80+CD11c+ cells separated from diseased kidney aggravated renal injury in AN mice. Furthermore, adoptive transfer of common progenitors revealed that kidney F4/80+CD11c+ cells were derived predominantly from monocytes, but not from pre-DCs. In conclusion, renal F4/80+CD11c+ cells are a major subset of rMPs and display Mф-like phenotypic and functional characteristics in health and in AN. PMID:25012165

Renal F4/80+ CD11c+ mononuclear phagocytes display phenotypic and functional characteristics of macrophages in health and in adriamycin nephropathy.

PubMed

Cao, Qi; Wang, Yiping; Wang, Xin Maggie; Lu, Junyu; Lee, Vincent W S; Ye, Qianling; Nguyen, Hanh; Zheng, Guoping; Zhao, Ye; Alexander, Stephen I; Harris, David C H

2015-02-01

Conventional markers of macrophages (Mфs) and dendritic cells (DCs) lack specificity and often overlap, leading to confusion and controversy regarding the precise function of these cells in kidney and other diseases. This study aimed to identify the phenotype and function of renal mononuclear phagocytes (rMPs) expressing key markers of both Mфs and DCs. F4/80(+)CD11c(+) cells accounted for 45% of total rMPs in normal kidneys and in those from mice with Adriamycin nephropathy (AN). Despite expression of the DC marker CD11c, these double-positive rMPs displayed the features of Mфs, including Mф-like morphology, high expression of CD68, CD204, and CD206, and high phagocytic ability but low antigen-presenting ability. F4/80(+)CD11c(+) cells were found in the cortex but not in the medulla of the kidney. In AN, F4/80(+)CD11c(+) cells displayed an M1 Mф phenotype with high expression of inflammatory mediators and costimulatory factors. Adoptive transfer of F4/80(+)CD11c(+) cells separated from diseased kidney aggravated renal injury in AN mice. Furthermore, adoptive transfer of common progenitors revealed that kidney F4/80(+)CD11c(+) cells were derived predominantly from monocytes, but not from pre-DCs. In conclusion, renal F4/80(+)CD11c(+) cells are a major subset of rMPs and display Mф-like phenotypic and functional characteristics in health and in AN. Copyright © 2015 by the American Society of Nephrology.

Blastic plasmacytoid dendritic cell neoplasms: clinico-immunohistochemical correlations in a series of 91 patients.

PubMed

Julia, Fanny; Dalle, Stephane; Duru, Gerard; Balme, Brigitte; Vergier, Béatrice; Ortonne, Nicolas; Vignon-Pennamen, Marie D; Costes-Martineau, Valérie; Lamant, Laurence; Dalac, Sophie; Delattre, Claire; Déchelotte, Pierre; Courville, Philippe; Carlotti, Agnès; De Muret, Anne; Fraitag, Sylvie; Levy, Annie; Mitchell, Andrew; Petrella, Tony

2014-05-01

Blastic plasmacytoid dendritic cell neoplasm is a rare clinicopathologic entity, characterized by strong skin tropism and a poor prognosis. The diagnosis is generally made by skin biopsy with appropriate immunohistochemical studies. To identify potential biological prognostic factors for blastic plasmacytoid dendritic cell neoplasm, we performed an extended clinico-immunohistochemical study on a series of 91 well-documented cases collected since 1995 by the French Study Group on Cutaneous Lymphomas. Skin biopsies were analyzed using a panel of 12 immunohistochemical markers (CD4, CD56, CD123, CD303, TCL1, CD68, CD2, CD7, TdT, Ki-67, S100, and MX-1). The results were correlated with survival. The 5 most characteristic markers of this entity (CD4, CD56, CD123, CD303, and TCL1) were expressed simultaneously in only 46% of patients. However, when 4 markers were expressed the diagnosis could still be reliably made without resorting to any additional stains. Expression of TdT and/or S100 correlated with varying degrees of maturation. Statistical survival analyses showed that CD303 expression and high proliferative index (Ki-67) were significantly associated with longer survival.

Different culture media affect growth characteristics, surface marker distribution and chondrogenic differentiation of human bone marrow-derived mesenchymal stromal cells.

PubMed

Hagmann, Sebastien; Moradi, Babak; Frank, Sebastian; Dreher, Thomas; Kämmerer, Peer Wolfgang; Richter, Wiltrud; Gotterbarm, Tobias

2013-07-30

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) play an important role in modern tissue engineering, while distinct variations of culture media compositions and supplements have been reported. Because MSCs are heterogeneous regarding their regenerative potential and their surface markers, these parameters were compared in four widely used culture media compositions. MSCs were isolated from bone marrow and expanded in four established cell culture media. MSC yield/1000 MNCs, passage time and growth index were observed. In P4, typical MSC surface markers were analysed by fluorescence cytometry. Additionally, chondrogenic, adipogenic and osteogenic differentiation potential were evaluated. Growth index and P0 cell yield varied importantly between the media. The different expansion media had a significant influence on the expression of CD10, CD90, CD105, CD140b CD146 and STRO-1. While no significant differences were observed regarding osteogenic and adipogenic differentiation, chondrogenic differentiation was superior in medium A as reflected by GAG/DNA content. The choice of expansion medium can have a significant influence on growth, differentiation potential and surface marker expression of mesenchymal stromal cells, which is of fundamental importance for tissue engineering procedures.

Photochemical internalisation, a minimally invasive strategy for light-controlled endosomal escape of cancer stem cell-targeting therapeutics.

PubMed

Selbo, Pål Kristian; Bostad, Monica; Olsen, Cathrine Elisabeth; Edwards, Victoria Tudor; Høgset, Anders; Weyergang, Anette; Berg, Kristian

2015-08-01

Despite progress in radio-, chemo- and photodynamic-therapy (PDT) of cancer, treatment resistance still remains a major problem for patients with aggressive tumours. Cancer stem cells (CSCs) or tumour-initiating cells are intrinsically and notoriously resistant to conventional cancer therapies and are proposed to be responsible for the recurrence of tumours after therapy. According to the CSC hypothesis, it is imperative to develop novel anticancer agents or therapeutic strategies that take into account the biology and role of CSCs. The present review outlines our recent study on photochemical internalisation (PCI) using the clinically relevant photosensitiser TPCS2a/Amphinex® as a rational, non-invasive strategy for the light-controlled endosomal escape of CSC-targeting drugs. PCI is an intracellular drug delivery method based on light-induced ROS-generation and a subsequent membrane-disruption of endocytic vesicles, leading to cytosolic release of the entrapped drugs of interest. In different proof-of-concept studies we have demonstrated that PCI of CSC-directed immunotoxins targeting CD133, CD44, CSPG4 and EpCAM is a highly specific and effective strategy for killing cancer cells and CSCs. CSCs overexpressing CD133 are PDT-resistant; however, this is circumvented by PCI of CD133-targeting immunotoxins. In view of the fact that TPCS2a is not a substrate of the efflux pumps ABCG2 and P-glycoprotein (ABCB1), the PCI-method is a promising anti-CSC therapeutic strategy. Due to a laser-controlled exposure, PCI of CSC-targeting drugs will be confined exclusively to the tumour tissue, suggesting that this drug delivery method has the potential to spare distant normal stem cells.

The XC chemokine receptor 1 is a conserved selective marker of mammalian cells homologous to mouse CD8α+ dendritic cells

PubMed Central

Crozat, Karine; Guiton, Rachel; Contreras, Vanessa; Feuillet, Vincent; Dutertre, Charles-Antoine; Ventre, Erwan; Vu Manh, Thien-Phong; Baranek, Thomas; Storset, Anne K.; Marvel, Jacqueline; Boudinot, Pierre; Hosmalin, Anne; Schwartz-Cornil, Isabelle

2010-01-01

Human BDCA3+ dendritic cells (DCs) were suggested to be homologous to mouse CD8α+ DCs. We demonstrate that human BDCA3+ DCs are more efficient than their BDCA1+ counterparts or plasmacytoid DCs (pDCs) in cross-presenting antigen and activating CD8+ T cells, which is similar to mouse CD8α+ DCs as compared with CD11b+ DCs or pDCs, although with more moderate differences between human DC subsets. Yet, no specific marker was known to be shared between homologous DC subsets across species. We found that XC chemokine receptor 1 (XCR1) is specifically expressed and active in mouse CD8α+, human BDCA3+, and sheep CD26+ DCs and is conserved across species. The mRNA encoding the XCR1 ligand chemokine (C motif) ligand 1 (XCL1) is selectively expressed in natural killer (NK) and CD8+ T lymphocytes at steady-state and is enhanced upon activation. Moreover, the Xcl1 mRNA is selectively expressed at high levels in central memory compared with naive CD8+ T lymphocytes. Finally, XCR1−/− mice have decreased early CD8+ T cell responses to Listeria monocytogenes infection, which is associated with higher bacterial loads early in infection. Therefore, XCR1 constitutes the first conserved specific marker for cell subsets homologous to mouse CD8α+ DCs in higher vertebrates and promotes their ability to activate early CD8+ T cell defenses against an intracellular pathogenic bacteria. PMID:20479118

Characterization of pancreatic stem cells derived from adult human pancreas ducts by fluorescence activated cell sorting.

PubMed

Lin, Han-Tso; Chiou, Shih-Hwa; Kao, Chung-Lan; Shyr, Yi-Ming; Hsu, Chien-Jen; Tarng, Yih-Wen; Ho, Larry L-T; Kwok, Ching-Fai; Ku, Hung-Hai

2006-07-28

To isolate putative pancreatic stem cells (PSCs) from human adult tissues of pancreas duct using serum-free, conditioned medium. The characterization of surface phenotype of these PSCs was analyzed by flow cytometry. The potential for pancreatic lineage and the capability of beta-cell differentiation in these PSCs were evaluated as well. By using serum-free medium supplemented with essential growth factors, we attempted to isolate the putative PSCs which has been reported to express nestin and pdx-1. The Matrigel(TM) was employed to evaluate the differential capacity of isolated cells. Dithizone staining, insulin content/secretion measurement, and immunohistochemistry staining were used to monitor the differentiation. Fluorescence activated cell sorting (FACS) was used to detect the phenotypic markers of putative PSCs. A monolayer of spindle-like cells was cultivated. The putative PSCs expressed pdx-1 and nestin. They were also able to differentiate into insulin-, glucagon-, and somatostatin-positive cells. The spectrum of phenotypic markers in PSCs was investigated; a similarity was revealed when using human bone marrow-derived stem cells as the comparative experiment, such as CD29, CD44, CD49, CD50, CD51, CD62E, PDGFR-alpha, CD73 (SH2), CD81, CD105(SH3). In this study, we successfully isolated PSCs from adult human pancreatic duct by using serum-free medium. These PSCs not only expressed nestin and pdx-1 but also exhibited markers attributable to mesenchymal stem cells. Although work is needed to elucidate the role of these cells, the application of these PSCs might be therapeutic strategies for diabetes mellitus.

Low CD1c + myeloid dendritic cell counts correlated with a high risk of rapid disease progression during early HIV-1 infection.

PubMed

Diao, Yingying; Geng, Wenqing; Fan, Xuejie; Cui, Hualu; Sun, Hong; Jiang, Yongjun; Wang, Yanan; Sun, Amy; Shang, Hong

2015-08-19

During early HIV-1 infection (EHI), the interaction between the immune response and the virus determines disease progression. Although CD1c + myeloid dendritic cells (mDCs) can trigger the immune response, the relationship between CD1c + mDC alteration and disease progression has not yet been defined. EHI changes in CD1c + mDC counts, surface marker (CD40, CD86, CD83) expression, and IL-12 secretion were assessed by flow cytometry in 29 patients. When compared with the normal controls, patients with EHI displayed significantly lower CD1c + mDC counts and IL-12 secretion and increased surface markers. CD1c + mDC counts were positively correlated with CD4+ T cell counts and inversely associated with viral loads. IL-12 secretion was only positively associated with CD4+ T cell counts. Rapid progressors had lower counts, CD86 expression, and IL-12 secretion of CD1c + mDCs comparing with typical progressors. Kaplan-Meier analysis and Cox regression models suggested patients with low CD1c + mDC counts (<10 cells/μL) had a 4-fold higher risk of rapid disease progression than those with high CD1c + mDC counts. However, no relationship was found between surface markers or IL-12 secretion and disease progression. During EHI, patients with low CD1c + mDC counts were more likely to experience rapid disease progression than those with high CD1c + mDC counts.

Comparative analysis of activation induced marker (AIM) assays for sensitive identification of antigen-specific CD4 T cells

PubMed Central

Cirelli, Kimberly M.; Dan, Jennifer M.; Morou, Antigoni; Daigneault, Audrey; Brassard, Nathalie; Silvestri, Guido; Routy, Jean-Pierre; Havenar-Daughton, Colin; Crotty, Shane

2017-01-01

The identification and study of antigen-specific CD4 T cells, both in peripheral blood and in tissues, is key for a broad range of immunological research, including vaccine responses and infectious diseases. Detection of these cells is hampered by both their rarity and their heterogeneity, in particular with regards to cytokine secretion profiles. These factors prevent the identification of the total pool of antigen-specific CD4 T cells by classical methods. We have developed assays for the highly sensitive detection of such cells by measuring the upregulation of surface activation induced markers (AIM). Here, we compare two such assays based on concurrent expression of CD69 plus CD40L (CD154) or expression of OX40 plus CD25, and we develop additional AIM assays based on OX40 plus PD-L1 or 4-1BB. We compare the relative sensitivity of these assays for detection of vaccine and natural infection-induced CD4 T cell responses and show that these assays identify distinct, but overlapping populations of antigen-specific CD4 T cells, a subpopulation of which can also be detected on the basis of cytokine synthesis. Bystander activation had minimal effect on AIM markers. However, some T regulatory cells upregulate CD25 upon antigen stimulation. We therefore validated AIM assays designed to exclude most T regulatory cells, for both human and non-human primate (NHP, Macaca mulatta) studies. Overall, through head-to-head comparisons and methodological improvements, we show that AIM assays represent a sensitive and valuable method for the detection of antigen-specific CD4 T cells. PMID:29065175

Comparative analysis of activation induced marker (AIM) assays for sensitive identification of antigen-specific CD4 T cells.

PubMed

Reiss, Samantha; Baxter, Amy E; Cirelli, Kimberly M; Dan, Jennifer M; Morou, Antigoni; Daigneault, Audrey; Brassard, Nathalie; Silvestri, Guido; Routy, Jean-Pierre; Havenar-Daughton, Colin; Crotty, Shane; Kaufmann, Daniel E

2017-01-01

The identification and study of antigen-specific CD4 T cells, both in peripheral blood and in tissues, is key for a broad range of immunological research, including vaccine responses and infectious diseases. Detection of these cells is hampered by both their rarity and their heterogeneity, in particular with regards to cytokine secretion profiles. These factors prevent the identification of the total pool of antigen-specific CD4 T cells by classical methods. We have developed assays for the highly sensitive detection of such cells by measuring the upregulation of surface activation induced markers (AIM). Here, we compare two such assays based on concurrent expression of CD69 plus CD40L (CD154) or expression of OX40 plus CD25, and we develop additional AIM assays based on OX40 plus PD-L1 or 4-1BB. We compare the relative sensitivity of these assays for detection of vaccine and natural infection-induced CD4 T cell responses and show that these assays identify distinct, but overlapping populations of antigen-specific CD4 T cells, a subpopulation of which can also be detected on the basis of cytokine synthesis. Bystander activation had minimal effect on AIM markers. However, some T regulatory cells upregulate CD25 upon antigen stimulation. We therefore validated AIM assays designed to exclude most T regulatory cells, for both human and non-human primate (NHP, Macaca mulatta) studies. Overall, through head-to-head comparisons and methodological improvements, we show that AIM assays represent a sensitive and valuable method for the detection of antigen-specific CD4 T cells.

Peripheral T-Cell Lymphoma with Aberrant Expression of CD19, CD20, and CD79a: Case Report and Literature Review

PubMed Central

Matnani, Rahul G.; Stewart, Rachel L.; Pulliam, Joseph; Jennings, Chester D.; Kesler, Melissa

2013-01-01

A case of lymphoma of T-cell derivation with aberrant expression of three B-cell lineage markers (CD19, CD20, and CD79a), which was diagnosed on a left axillary excision, is described. Immunohistochemical studies and flow cytometry analysis demonstrated neoplastic cells expressing CD3, CD19, CD20, and CD79a with absence of CD4, CD8, CD10, CD30, CD34, CD56, CD68, TDT, MPO, PAX-5, and surface immunoglobulin. Gene rearrangement studies performed on paraffin blocks demonstrated monoclonal T-cell receptor gamma chain rearrangement with no evidence of clonal heavy chain rearrangement. The neoplastic cells were negative for Epstein-Barr virus (EBV) or Human Herpes Virus 8 (HHV-8). At the time of diagnosis, the PET scan demonstrated hypermetabolic neoplastic cells involving the left axilla, bilateral internal jugular areas, mediastinum, right hilum, bilateral lungs, and spleen. However, bone marrow biopsy performed for hemolytic anemia revealed normocellular bone marrow with trilineage maturation. The patient had no evidence of immunodeficiency or infection with EBV or HHV-8. This is the first reported case of a mature T-cell lymphoma with aberrant expression of three B-cell lineage markers. The current report also highlights the need for molecular gene rearrangement studies to determine the precise lineage of ambiguous neoplastic clones. PMID:24066244

Ex vivo tetramer staining and cell surface phenotyping for early activation markers CD38 and HLA-DR to enumerate and characterize malaria antigen-specific CD8+ T-cells induced in human volunteers immunized with a Plasmodium falciparum adenovirus-vectored malaria vaccine expressing AMA1.

PubMed

Schwenk, Robert; Banania, Glenna; Epstein, Judy; Kim, Yohan; Peters, Bjoern; Belmonte, Maria; Ganeshan, Harini; Huang, Jun; Reyes, Sharina; Stryhn, Anette; Ockenhouse, Christian F; Buus, Soren; Richie, Thomas L; Sedegah, Martha

2013-10-29

Malaria is responsible for up to a 600,000 deaths per year; conveying an urgent need for the development of a malaria vaccine. Studies with whole sporozoite vaccines in mice and non-human primates have shown that sporozoite-induced CD8+ T cells targeting liver stage antigens can mediate sterile protection. There is a need for a direct method to identify and phenotype malaria vaccine-induced CD8+ T cells in humans. Fluorochrome-labelled tetramers consisting of appropriate MHC class I molecules in complex with predicted binding peptides derived from Plasmodium falciparum AMA-1 were used to label ex vivo AMA-1 epitope specific CD8+ T cells from research subjects responding strongly to immunization with the NMRC-M3V-Ad-PfCA (adenovirus-vectored) malaria vaccine. The identification of these CD8+ T cells on the basis of their expression of early activation markers was also investigated. Analyses by flow cytometry demonstrated that two of the six tetramers tested: TLDEMRHFY: HLA-A*01:01 and NEVVVKEEY: HLA-B*18:01, labelled tetramer-specific CD8+ T cells from two HLA-A*01:01 volunteers and one HLA-B*18:01 volunteer, respectively. By contrast, post-immune CD8+ T cells from all six of the immunized volunteers exhibited enhanced expression of the CD38 and HLA-DRhi early activation markers. For the three volunteers with positive tetramer staining, the early activation phenotype positive cells included essentially all of the tetramer positive, malaria epitope- specific CD8+ T cells suggesting that the early activation phenotype could identify all malaria vaccine-induced CD8+ T cells without prior knowledge of their exact epitope specificity. The results demonstrated that class I tetramers can identify ex vivo malaria vaccine antigen-specific CD8+ T cells and could therefore be used to determine their frequency, cell surface phenotype and transcription factor usage. The results also demonstrated that vaccine antigen-specific CD8+ T cells could be identified by activation markers without prior knowledge of their antigen-specificity, using a subunit vaccine for proof-of-concept. Whether, whole parasite or adjuvanted protein vaccines will also induce {CD38 and HLA-DRhi}+ CD8+ T cell populations reflective of the antigen-specific response will the subject of future investigations.

Cytokine Expression, Natural Killer Cell Activation, and Phenotypic Changes in Lymphoid Cells from Rhesus Macaques during Acute Infection with Pathogenic Simian Immunodeficiency Virus

PubMed Central

Giavedoni, Luis D.; Velasquillo, M. Cristina; Parodi, Laura M.; Hubbard, Gene B.; Hodara, Vida L.

2000-01-01

We studied the innate and adaptive immune system of rhesus macaques infected with the virulent simian immunodeficiency virus isolate SIVmac251 by evaluating natural killer (NK) cell activity, cytokine levels in plasma, humoral and virological parameters, and changes in the activation markers CD25 (interleukin 2R [IL-2R] α chain), CD69 (early activation marker), and CD154 (CD40 ligand) in lymphoid cells. We found that infection with SIVmac251 induced the sequential production of interferon-α/β (IFN-α/β), IL-18, and IL-12. IFN-γ, IL-4, and granulocyte-macrophage colony-stimulating factor were undetected in plasma by the assays used. NK cell activity peaked at 1 to 2 weeks postinfection and paralleled changes in viral loads. Maximum expression of CD69 on CD3−CD16+ lymphocytes correlated with NK cytotoxicity during this period. CD25 expression, which is associated with proliferation, was static or slightly down-regulated in CD4+ T cells from both peripheral blood (PB) and lymph nodes (LN). CD69, which is normally present in LN CD4+ T cells and absent in peripheral blood leukocyte (PBL) CD4+ T cells, was down-regulated in LN CD4+ T cells and up-regulated in PBL CD4+ T cells immediately after infection. CD8+ T cells increased CD69 but not CD25 expression, indicating the activation of this cellular subset in PB and LN. Finally, CD154 was transiently up-regulated in PBL CD4+ T cells but not in LN CD4+ T cells. Levels of antibodies to SIV Gag and Env did not correlate with the level of activation of CD154, a critical costimulatory molecule for T-cell-dependent immunity. In summary, we present the first documented evidence that the innate immune system of rhesus macaques recognizes SIV infection by sequential production of proinflammatory cytokines and transient activation of NK cytotoxic activity. Additionally, pathogenic SIV induces drastic changes in the level of activation markers on T cells from different anatomic compartments. These changes involve activation in the absence of proliferation, indicating that activation-induced cell death may cause some of the reported increase in lymphocyte turnover during SIV infection. PMID:10644334

Acid ceramidase and its inhibitors: a de novo drug target and a new class of drugs for killing glioblastoma cancer stem cells with high efficiency.

PubMed

Doan, Ninh B; Alhajala, Hisham; Al-Gizawiy, Mona M; Mueller, Wade M; Rand, Scott D; Connelly, Jennifer M; Cochran, Elizabeth J; Chitambar, Christopher R; Clark, Paul; Kuo, John; Schmainda, Kathleen M; Mirza, Shama P

2017-12-22

Glioblastoma remains the most common, malignant primary cancer of the central nervous system with a low life expectancy and an overall survival of less than 1.5 years. The treatment options are limited and there is no cure. Moreover, almost all patients develop recurrent tumors, which typically are more aggressive. Therapeutically resistant glioblastoma or glioblastoma stem-like cells (GSCs) are hypothesized to cause this inevitable recurrence. Identifying prognostic biomarkers of glioblastoma will potentially advance knowledge about glioblastoma tumorigenesis and enable discovery of more effective therapies. Proteomic analysis of more than 600 glioblastoma-specific proteins revealed, for the first time, that expression of acid ceramidase (ASAH1) is associated with poor glioblastoma survival. CD133+ GSCs express significantly higher ASAH1 compared to CD133- GSCs and serum-cultured glioblastoma cell lines, such as U87MG. These findings implicate ASAH1 as a plausible independent prognostic marker, providing a target for a therapy tailored toward GSCs. We further demonstrate that ASAH1 inhibition increases cellular ceramide level and induces apoptosis. Strikingly, U87MG cells, and three different patient-derived glioblastoma stem-like cancer cell lines were efficiently killed, through apoptosis, by three different known ASAH1 inhibitors with IC50's ranging from 11-104 μM. In comparison, the standard glioblastoma chemotherapy agent, temozolomide, had minimal GSC-targeted effects at comparable or even higher concentrations (IC50 > 750 μM against GSCs). ASAH1 is identified as a de novo glioblastoma drug target, and ASAH1 inhibitors, such as carmofur, are shown to be highly effective and to specifically target glioblastoma GSCs. Carmofur is an ASAH1 inhibitor that crosses the blood-brain barrier, a major bottleneck in glioblastoma treatment. It has been approved in Japan since 1981 for colorectal cancer therapy. Therefore, it is poised for repurposing and translation to glioblastoma clinical trials.

Novel Adult Stem Cells for Peripheral Nerve Regeneration

DTIC Science & Technology

2012-09-01

were also positive for MSC surface marker CD29 and CD44 (Fig. 1F-G). However, CD29 and CD44 are also expressed in SMCs, so we will not use these non...tubulin. In addition, MVSCs were negative for perivascular MSC marker CD146 (Fig. 1H) and SMC progenitor marker Sca-1 (Fig. 1I). MVSCs were also...University of California, Berkeley, California 94720, USA. 2 UC Berkeley-UCSF Graduate Program in Bioengineering, Berkeley, California 94720, USA. 3

Respiratory Syncytial Virus (RSV) Infects CD4+ T Cells: Frequency of Circulating CD4+ RSV+ T Cells as a Marker of Disease Severity in Young Children.

PubMed

Raiden, Silvina; Sananez, Inés; Remes-Lenicov, Federico; Pandolfi, Julieta; Romero, Cecilia; De Lillo, Leonardo; Ceballos, Ana; Geffner, Jorge; Arruvito, Lourdes

2017-04-01

Although human airway epithelial cells are the main target of respiratory syncytial virus (RSV), it also infects immune cells, such as macrophages and B cells. Whether T cells are permissive to RSV infection is unknown. We sought to analyze the permissiveness of CD4+ T cells to RSV infection. CD4+ and CD8+ T cells from cord blood, healthy young children, and adults were challenged by RSV or cocultured with infected HEp-2 cells. Infection, phenotype, and cytokine production by T cells were analyzed by flow cytometry or enzyme-linked immunosorbent assay. Expression of RSV antigens by circulating CD4+ T cells from infected children was analyzed by flow cytometry, and disease severity was defined by standard criteria. CD4+ and CD8+ T cells were productively infected by RSV. Infection decreased interleukin 2 and interferon γ production as well as the expression of CD25 and Ki-67 by activated CD4+ T cells. Respiratory syncytial virus antigens were detected in circulating CD4+ and CD8+ T cells during severe RSV infection of young children. Interestingly, the frequency of CD4+ RSV+ T cells positively correlated with disease severity. Respiratory syncytial virus infects CD4+ and CD8+ T cells and compromises T-cell function. The frequency of circulating CD4+ RSV+ T cells might represent a novel marker of severe infection. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

CD30 Receptor-Targeted Lentiviral Vectors for Human Induced Pluripotent Stem Cell-Specific Gene Modification.

PubMed

Friedel, Thorsten; Jung-Klawitter, Sabine; Sebe, Attila; Schenk, Franziska; Modlich, Ute; Ivics, Zoltán; Schumann, Gerald G; Buchholz, Christian J; Schneider, Irene C

2016-05-01

Cultures of induced pluripotent stem cells (iPSCs) often contain cells of varying grades of pluripotency. We present novel lentiviral vectors targeted to the surface receptor CD30 (CD30-LV) to transfer genes into iPSCs that are truly pluripotent as demonstrated by marker gene expression. We demonstrate that CD30 expression is restricted to SSEA4(high) cells of human iPSC cultures and a human embryonic stem cell line. When CD30-LV was added to iPSCs during routine cultivation, efficient and exclusive transduction of cells positive for the pluripotency marker Oct-4 was achieved, while retaining their pluripotency. When added during the reprogramming process, CD30-LV solely transduced cells that became fully reprogrammed iPSCs as confirmed by co-expression of endogenous Nanog and the reporter gene. Thus, CD30-LV may serve as novel tool for the selective gene transfer into PSCs with broad applications in basic and therapeutic research.

Metformin improves circulating endothelial cells and endothelial progenitor cells in type 1 diabetes: MERIT study.

PubMed

Ahmed, Fahad W; Rider, Rachel; Glanville, Michael; Narayanan, Kilimangalam; Razvi, Salman; Weaver, Jolanta U

2016-08-26

Type 1 diabetes is associated with increased cardiovascular disease (CVD). Decreased endothelial progenitor cells (EPCs) number plays a pivotal role in reduced endothelial repair and development of CVD. We aimed to determine if cardioprotective effect of metformin is mediated by increasing circulating endothelial progenitor cells (cEPCs), pro-angiogenic cells (PACs) and decreasing circulating endothelial cells (cECs) count whilst maintaining unchanged glycemic control. This study was an open label and parallel standard treatment study. Twenty-three type 1 diabetes patients without overt CVD were treated with metformin for 8 weeks (treatment group-TG). They were matched with nine type 1 diabetes patients on standard treatment (SG) and 23 age- and sex-matched healthy volunteers (HC). Insulin dose was adjusted to keep unchanged glycaemic control. cEPCs and cECs counts were determined by flow cytometry using surface markers CD45(dim)CD34(+)VEGFR-2(+) and CD45(dim)CD133(-)CD34(+)CD144(+) respectively. Peripheral blood mononuclear cells were cultured to assess changes in PACs number, function and colony forming units (CFU-Hill's colonies). At baseline TG had lower cEPCs, PACs, CFU-Hills' colonies and PACs adhesion versus HC (p < 0.001-all variables) and higher cECs versus HC (p = 0.03). Metformin improved cEPCs, PACs, CFU-Hill's colonies number, cECs and PACs adhesion (p < 0.05-all variables) to levels seen in HC whilst HbA1c (one-way ANOVA p = 0.78) and glucose variability (average glucose, blood glucose standard deviation, mean amplitude of glycaemic excursion, continuous overall net glycaemic action and area under curve) remained unchanged. No changes were seen in any variables in SG. There was an inverse correlation between CFU-Hill's colonies with cECs. Metformin has potential cardio-protective effect through improving cEPCs, CFU-Hill's colonies, cECs, PACs count and function independently of hypoglycaemic effect. This finding needs to be confirmed by long term cardiovascular outcome studies in type 1 diabetes. Trial registration ISRCTN26092132.

Breast implant-associated, ALK-negative, T-cell, anaplastic, large-cell lymphoma: establishment and characterization of a model cell line (TLBR-1) for this newly emerging clinical entity.

PubMed

Lechner, Melissa G; Lade, Stephen; Liebertz, Daniel J; Prince, H Miles; Brody, Garry S; Webster, Howard R; Epstein, Alan L

2011-04-01

Primary lymphomas of the breast are very rare (0.2-1.5% of breast malignancies) and the vast majority (95%) are of B-cell origin. Recently, 40 cases of clinically indolent anaplastic large-cell kinase (ALK)-negative, T-cell, anaplastic, non-Hodgkin lymphomas (T-ALCL) have been reported worldwide. A tumor biopsy specimen from a patient in this series was obtained for characterization. By using a human stromal feeder layer and IL-2, a novel cell line, TLBR-1, was established from this biopsy and investigated by using cytogenetics and various biomolecular methods. Immunoperoxidase staining of the tumor biopsy showed a CD30/CD8/CD4 coexpressing T-cell population that was epithelial membrane antigen (EMA)(+) and perforin(+) . Multiplex polymerase chain reaction (PCR) of TCRγ genes showed monoclonality that suggested a T-cell origin, yet pan-T markers CD2/5/7, anaplastic large-cell kinase (ALK)-1, pancytokeratins, CD20, CD56, and Epstein-Barr virus (EBV) by in situ hybridization (ISH) were negative. TLBR-1 is IL-2 dependent, has a relatively long doubling time (55 hours), and displays different cellular shapes in culture. Cytogenetic analysis of tumor and TLBR-1 cells confirmed a highly anaplastic cell population with a modal number of 47 chromosomes lacking t(2;5). PCR screens for EBV and human T-lymphotropic virus types 1 and 2 (HTLV-1/2) were negative. Fluorescence-activated cell-sorting (FACS) analysis showed strong positivity for CD4/8, CD30, CD71, and CD26 expression, and antigen presentation (HLA-DR(+) CD80(+) CD86(+) ), IL-2 signaling (CD25(+) CD122(+) ), and NK (CD56(+) ) markers, and Western blots demonstrated strong Notch1 expression. Severe combined immunodeficiency (SCID) mouse TLBR-1 heterotransplants recapitulated the histology and marker characteristics of the original tumor. TLBR-1, a novel ALK-negative, T-cell, anaplastic, large-cell lymphoma, closely resembles the original biopsy and represents an important tool for studying this newly recognized disease entity. Copyright © 2010 American Cancer Society.

Breast Implant-Associated, ALK-Negative, T-Cell, Anaplastic, Large-Cell Lymphoma: Establishment and Characterization of a Model Cell Line (TLBR-1) for This Newly Emerging Clinical Entity

PubMed Central

Lechner, Melissa G.; Lade, Stephen; Liebertz, Daniel J.; Prince, H. Miles; Brody, Garry S.; Webster, Howard R.; Epstein, Alan L.

2014-01-01

BACKGROUND Primary lymphomas of the breast are very rare (0.2–1.5% of breast malignancies) and the vast majority (95%) are of B-cell origin. Recently, 40 cases of clinically indolent anaplastic large-cell kinase (ALK)-negative, T-cell, anaplastic, non-Hodgkin lymphomas (T-ALCL) have been reported worldwide. METHODS A tumor biopsy specimen from a patient in this series was obtained for characterization. By using a human stromal feeder layer and IL-2, a novel cell line, TLBR-1, was established from this biopsy and investigated by using cytogenetics and various biomolecular methods. RESULTS Immunoperoxidase staining of the tumor biopsy showed a CD30/CD8/CD4 coexpressing T-cell population that was epithelial membrane antigen (EMA)+ and perforin+. Multiplex polymerase chain reaction (PCR) of TCRγ genes showed monoclonality that suggested a T-cell origin, yet pan-T markers CD2/5/7, anaplastic large-cell kinase (ALK)-1, pancytokeratins, CD20, CD56, and Epstein-Barr virus (EBV) by in situ hybridization (ISH) were negative. TLBR-1 is IL-2 dependent, has a relatively long doubling time (55 hours), and displays different cellular shapes in culture. Cytogenetic analysis of tumor and TLBR-1 cells confirmed a highly anaplastic cell population with a modal number of 47 chromosomes lacking t(2;5). PCR screens for EBV and human T-lymphotropic virus types 1 and 2 (HTLV-1/2) were negative. Fluorescence-activated cell-sorting (FACS) analysis showed strong positivity for CD4/8, CD30, CD71, and CD26 expression, and antigen presentation (HLA-DR+CD80+CD86+), IL-2 signaling (CD25+CD122+), and NK (CD56+) markers, and Western blots demonstrated strong Notch1 expression. Severe combined immunodeficiency (SCID) mouse TLBR-1 heterotransplants recapitulated the histology and marker characteristics of the original tumor. CONCLUSIONS TLBR-1, a novel ALK-negative, T-cell, anaplastic, large-cell lymphoma, closely resembles the original biopsy and represents an important tool for studying this newly recognized disease entity. PMID:21425149

Cell therapy: a therapeutic alternative to treat focal cartilage lesions.

PubMed

Gimeno, M J; Maneiro, E; Rendal, E; Ramallal, M; Sanjurjo, L; Blanco, F J

2005-11-01

Human mesenchymal stem cells (MSCs) are present in most of the tissue matrix, taking part in their regeneration when injury or damage occurs. The aim of this study was to investigate the presence of cells with pluripotential characteristics in synovial membranes from osteoarthritic (OA) patients and the capacity of these cells to differentiate to chondrocytes. Synovial membranes (n = 8) from OA patients were digested with collagenase. Isolated cells were cultured with DMEM, 20% FBS, and FGFb10 ng/mL. Cells from second subculture were used to carry out phenotypic characterization experiments (flow cytometry analysis with 11 monoclonal antibodies) and chondrogenic differentiation experiments(micropellet cultured in chondrogenic medium). Chondrogenic differentiation of cells was assessment by quantification of cartilage extracellular matrix components by following techniques: Safranin O, Toluidine Blue, and Alcian Blue stains to detect proteoglycans and immunohistochemistry to detect type I and II collagen. Flow cytometry analyses showed that in our population more than 90% of cells were positive for MSC markers: CD29 (95%), CD44 (90%), CD73 (95%), CD90 (98%). Cells were negative for hematopoietic markers (CD11b, CD34, and CD45). Furthermore, cells showed positive stain to multipotent markers such as CD117 (c-kit) (98%), CD166 (74%), and STRO-1 (88%) and to quiescent satellite cells like PAX-7 (35%). The micropellet analyses showed that the culture of these cells with TGFbeta-3 for 2 and 3 weeks stimulates proteoglycan and collagen type II synthesis. Both molecules are characteristic of hyaline articular cartilage. In this work, we demonstrate the presence of a cellular population with MSC characteristics in synovial tissue from OA patients. As MSC takes part in reparative processes of adult tissues, these cells could play an important role in OA pathogenesis and treatment.

Antiretroviral therapy in HIV-1-infected individuals with CD4 count below 100 cells/mm3 results in differential recovery of monocyte activation

PubMed Central

Patro, Sean C.; Azzoni, Livio; Joseph, Jocelin; Fair, Matthew G.; Sierra-Madero, Juan G.; Rassool, Mohammed S.; Sanne, Ian; Montaner, Luis J.

2016-01-01

Reversal of monocyte and macrophage activation and the relationship to viral suppression and T cell activation are unknown in patients with advanced HIV-1 infection, initiating antiretroviral therapy. This study aimed to determine whether reduction in biomarkers of monocyte and macrophage activation would be reduced in conjunction with viral suppression and resolution of T cell activation. Furthermore, we hypothesized that the addition of CCR5 antagonism (by maraviroc) would mediate greater reduction of monocyte/macrophage activation markers than suppressive antiretroviral therapy alone. In the CCR5 antagonism to decrease the incidence of immune reconstitution inflammatory syndrome study, antiretroviral therapy-naïve patients received maraviroc or placebo in addition to standard antiretroviral therapy. PBMCs and plasma from 65 patients were assessed during 24 wk of antiretroviral therapy for biomarkers of monocyte and macrophage activation. Markers of monocyte and macrophage activation were reduced significantly by 24 wk, including CD14++CD16+ intermediate monocytes (P < 0.0001), surface CD163 (P = 0.0004), CD169 (P < 0.0001), tetherin (P = 0.0153), and soluble CD163 (P < 0.0001). A change in CD38+, HLA-DR+ CD8 T cells was associated with changes in CD169 and tetherin expression. Maraviroc did not affect biomarkers of monocyte/macrophage activation but resulted in greater percentages of CCR5-positive monocytes in PBMC. HIV-1 suppression after 24 wk of antiretroviral therapy, with or without maraviroc, demonstrates robust recovery in monocyte subset activation markers, whereas soluble markers of activation demonstrate minimal decrease, qualitatively differentiating markers of monocyte/macrophage activation in advanced disease. PMID:26609048

Coexpression of CD14 and CD326 discriminate hepatic precursors in the human fetal liver.

PubMed

Fomin, Marina E; Tai, Lung-Kuo; Bárcena, Alicia; Muench, Marcus O

2011-07-01

The molecular and cellular profile of liver cells during early human development is incomplete, complicating the isolation and study of hepatocytes, cholangiocytes, and hepatic stem cells from the complex amalgam of hepatic and hematopoietic cells, that is, the fetal liver. Epithelial cell adhesion molecule, CD326, has emerged as a marker of hepatic stem cells, and lipopolysaccharide receptor CD14 is known to be expressed on adult hepatocytes. Using flow cytometry, we studied the breadth of CD326 and CD14 expression in midgestation liver. Both CD45(+) hematopoietic and CD45(-) nonhematopoietic cells expressed CD326. Moreover, diverse cell types expressing CD326 were revealed among CD45(-) cells by costaining for CD14. Fluorescence-activated cell sorting was used to isolate nonhematopoietic cells distinguished by expression of high levels of CD326 and low CD14 (CD326(++)CD14(lo)), which were characterized for gene expression associated with liver development. CD326(++)CD14(lo) cells expressed the genes albumin, α-fetoprotein, hepatic nuclear factor 3α, prospero-related homeobox 1, cytochrome P450 3A7, α(1)-antitrypsin, and transferrin. Proteins expressed included cell-surface CD24, CD26, CD29, CD34, CD49f, CD243, and CD324 and, in the cytoplasm, cytokeratins-7/8 (CAM 5.2 antigen) and some cytokeratin-19. Cultured CD326(++)CD14(lo) cells yielded albumin(+) hepatocytes, cytokeratin-19(+) cholangiocytes, and hepatoblasts expressing both markers. Using epifluorescence microscopy we observed CD326 and CD14 expression on fetal hepatocytes comprising the liver parenchyma, as well as on cells associated with ductal plates and surrounding large vessels. These findings indicate that expression of CD14 and CD326 can be used to identify functionally distinct subsets of fetal liver cells, including CD326(++)CD14(lo) cells, representing a mixture of parenchymal cells, cholangiocytes, and hepatoblasts.

T- and NK-cell populations with regulatory phenotype and markers of apoptosis in circulating lymphocytes of patients with CIN3 or microcarcinoma of the cervix: evidence for potential mechanisms of immune suppression.

PubMed

Kurmyshkina, Olga V; Kovchur, Pavel I; Schegoleva, Ludmila V; Volkova, Tatyana O

2017-01-01

Processes and mechanisms responsible for systemic immune suppression in early-stage cervical cancer remain substantially underinvestigated. In this work, we focused on studying the frequencies of circulating regulatory T (CD4 and CD8 Tregs) and NK (NKregs) cells in parallel with assessment of apoptotic markers expression in T cells from patients with preinvasive and microinvasive cervical cancer, with the aim to determine whether up-regulation of apoptosis-associated markers in Т lymphocytes accompanies cervical cancer development and correlates with the change in percentages of regulatory cell populations at systemic level during the initial stages of invasive cervical cancer progression. Fourty two women with histologically confirmed cervical intraepithelial neoplasia grade 3 (CIN3, including carcinoma in situ) or cervical cancer (stage IA) and 30 healthy women (control) were enrolled in the study. Peripheral blood samples were taken immediately before surgery or any treatment and immediately subjected to multicolor flow cytometry. Analysis of a combination of CD4/CD8, CD25, CD127, and FoxP3 markers revealed a statistically significant increase in the frequencies of Tregs within both the CD4 and CD8 subsets of circulating lymphocytes in patients with CIN3 and stage IA cancer. In contrast, lower numbers of NKregs (defined as CD16 dim/neg CD56 bright subpopulation) and increased CD56 dim /CD56 bright NK ratio were found in patients compared to controls, with the percentage of CD16 bright CD56 dim cells (major subtype of circulating NKs) showing no difference. Patients also exhibited an increased expression of CD95 in total peripheral blood T lymphocytes, along with increased level of Annexin V binding to CD95-positive cells, suggesting higher susceptibility of T cells to apoptosis and potential involvement of CD95-dependent pathway in early-stage cervical cancer. Differential analysis of CD4 and CD8 T cells revealed different trends in the change of CD95 expression, confirming that this change likely has different functional significance for these two subsets. A search for correlations between the phenotypic parameters analyzed in this study was performed to demonstrate that women with early neoplastic lesions of the cervix, such as carcinoma in situ and microinvasive carcinoma, displayed a coordinated increase in expression of Treg markers in circulating lymphocytes, along with more pronounced cross-relationships between Treg numbers, CD95 expression on T cells, and apoptosis, compared to the control group. The results of this study suggest that a diversity of immune regulatory mechanisms that provide support for initial stages of invasive growth in cervical cancer patients includes systemic changes in the ratios between the principal regulatory and effector lymphocyte populations both within adaptive and innate immunity.

Wnt interaction and extracellular release of prominin-1/CD133 in human malignant melanoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rappa, Germana; College of Pharmacy, Roseman University of Health Sciences, Henderson, NV 89104; Mercapide, Javier

2013-04-01

Prominin-1 (CD133) is the first identified gene of a novel class of pentaspan membrane glycoproteins. It is expressed by various epithelial and non-epithelial cells, and notably by stem and cancer stem cells. In non-cancerous cells such as neuro-epithelial and hematopoietic stem cells, prominin-1 is selectively concentrated in plasma membrane protrusions, and released into the extracellular milieu in association with small vesicles. Previously, we demonstrated that prominin-1 contributes to melanoma cells pro-metastatic properties and suggested that it may constitute a molecular target to prevent prominin-1-expressing melanomas from colonizing and growing in lymph nodes and distant organs. Here, we report that threemore » distinct pools of prominin-1 co-exist in cultures of human FEMX-I metastatic melanoma. Morphologically, in addition to the plasma membrane localization, prominin-1 is found within the intracellular compartments, (e.g., Golgi apparatus) and in association with extracellular membrane vesicles. The latter prominin-1–positive structures appeared in three sizes (small, ≤40 nm; intermediates ∼40–80 nm, and large, >80 nm). Functionally, the down-regulation of prominin-1 in FEMX-I cells resulted in a significant reduction of number of lipid droplets as observed by coherent anti-Stokes Raman scattering image analysis and Oil red O staining, and surprisingly in a decrease in the nuclear localization of beta-catenin, a surrogate marker of Wnt activation. Moreover, the T-cell factor/lymphoid enhancer factor (TCF/LEF) promoter activity was 2 to 4 times higher in parental than in prominin-1-knockdown cells. Collectively, our results point to Wnt signaling and/or release of prominin-1–containing membrane vesicles as mediators of the pro-metastatic activity of prominin-1 in FEMX-I melanoma. - Highlights: ► First report of release of prominin-1–containing microvesicles from cancer cells. ► Pro-metastatic role of prominin-1–containing microvesicles in FEMX-I melanoma. ► Down-regulation of prominin-1 results in decreased nuclear localization of β-catenin. ► Wnt signaling as mediator of the pro-metastatic activity of prominin-1.« less

A novel anticancer agent SNG1153 inhibits growth of lung cancer stem/progenitor cells

PubMed Central

Wang, Jing; Zhu, Hai; Han, Yuqing; Jin, Mingji; Wang, Jun; Zhou, Congya; Ma, Junfeng; Lin, Qingcong; Wang, Zhaoyi; Meng, Kun; Fu, Xueqi

2016-01-01

Lung cancer is the leading cause of cancer-related death in both men and women. Lung cancer contains a small population of cancer cells with stem-like features known as cancer stem cells (CSCs). CSCs are often more resistant to current therapeutic treatments. Thus, it is urgent to develop a novel agent that is able to inhibit CSCs growth. In this study, we examined the ability of SNG1153, a novel chemical agent to inhibit the growth of lung CSCs. We found that SNG1153 inhibited growth and induced apoptosis in established lung cancer cells. We also found that SNG1153 inhibited the tumorsphere formation and decreased CD133-positive (lung CSC marker) cancer cells. SNG1153 was able to attenuate tumor formation in NOD/SCID (non-obese diabetic/severe combined immunodeficient) mice injected with lung tumorsphere cells. We further demonstrated that SNG1153 induced β-catenin phosphorylation and down-regulated β-catenin. Our results thus demonstrate that SNG1153 effectively inhibits the growth of lung CSCs and suggest that SNG1153 may be a novel therapeutic agent to treat human lung cancer. PMID:27281614

CD44 Interacts with HIF-2α to Modulate the Hypoxic Phenotype of Perinecrotic and Perivascular Glioma Cells.

PubMed

Johansson, Elinn; Grassi, Elisa S; Pantazopoulou, Vasiliki; Tong, Bei; Lindgren, David; Berg, Tracy J; Pietras, Elin J; Axelson, Håkan; Pietras, Alexander

2017-08-15

Hypoxia-inducible factors enhance glioma stemness, and glioma stem cells have an amplified hypoxic response despite residing within a perivascular niche. Still, little is known about differential HIF regulation in stem versus bulk glioma cells. We show that the intracellular domain of stem cell marker CD44 (CD44ICD) is released at hypoxia, binds HIF-2α (but not HIF-1α), enhances HIF target gene activation, and is required for hypoxia-induced stemness in glioma. In a glioma mouse model, CD44 was restricted to hypoxic and perivascular tumor regions, and in human glioma, a hypoxia signature correlated with CD44. The CD44ICD was sufficient to induce hypoxic signaling at perivascular oxygen tensions, and blocking CD44 cleavage decreased HIF-2α stabilization in CD44-expressing cells. Our data indicate that the stem cell marker CD44 modulates the hypoxic response of glioma cells and that the pseudo-hypoxic phenotype of stem-like glioma cells is achieved by stabilization of HIF-2α through interaction with CD44, independently of oxygen. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

CMV induces expansion of highly polyfunctional CD4+ T cell subset coexpressing CD57 and CD154.

PubMed

Pera, Alejandra; Vasudev, Anusha; Tan, Crystal; Kared, Hassen; Solana, Rafael; Larbi, Anis

2017-02-01

CD4 + T cells are essential for human CMV infection control. CMV-specific CD4 + T cells possess antiviral functions and participate in anti-CMV humoral/cellular responses. In the elderly, CMV infection impairs immunity to other viruses and has been traditionally associated with T cell senescence; however, recent results suggest that, in younger people, CMV confers immune protection against other pathogens (heterologous immunity). To shed light on this controversy, we analyzed latent CMV infection effects on the quality of young individuals' immune response, specifically, the presence of polyfunctional T cells through an extensive phenotypic and functional characterization of the CD4 + T cell subset. CD154 expression, degranulation (CD107a), and cytokine production (IFN-γ, TNF-α, and IL-2) as well as T cell phenotype markers (CD57, CD28, and CD27) were analyzed. We demonstrate that CD4 + T cells that coexpress CD57 and CD154, which are exclusively present in CMV-positive individuals, are the most polyfunctional CD4 + subset, whereas CD4 + CD27 + CD28 - T cells associate with lower polyfunctionality. Conversely, the frequency of CD4 + CD28 + T cells correlates with higher polyfunctionality of CD4 + CD57 - T cells from CMV-seronegative individuals and CD4 + CD57 + CD154 + T cells from CMV-seropositive individuals. Thus, polyfunctionality is a property of central memory CD4 + T cells in CMV-seronegative individuals, whereas after CMV infection, polyfunctional T cells become highly differentiated, which allows efficient eradication of infections. We extend previous observations of the impact of CMV on CD8 + T cell functionality to the CD4 + T cell compartment, revealing CD57 as a polyfunctionality marker of T cells which expands after CMV infection. CD57 + T cells have been associated with inflammatory conditions, but their potential role in the response against infectious disease and vaccination should now be investigated. © Society for Leukocyte Biology.

Influence of Partial O₂ Pressure on the Adhesion, Proliferation, and Osteogenic Differentiation of Human Dental Pulp Stem Cells on β-Tricalcium Phosphate Scaffold.

PubMed

Viña-Almunia, Jose; Mas-Bargues, Cristina; Borras, Consuelo; Gambini, Juan; El Alami, Marya; Sanz-Ros, Jorge; Peñarrocha, Miguel; Vina, Jose

To analyze, in vitro, the influence of O₂ pressure on the adhesion, proliferation, and osteogenic differentiation of human dental pulp stem cells (DPSC) on β-tricalcium phosphate (β-TCP) scaffold. DPSC, positive for the molecular markers CD133, Oct4, Nestin, Stro-1, and CD34, and negative for CD45, were isolated from extracted third molars. Experiments were started by seeding 200,000 cells on β-TCP cultured under 3% or 21% O₂ pressure. No osteogenic medium was used. Eight different cultures were performed at each time point under each O₂ pressure condition. Cell adhesion, proliferation, and differentiation over the biomaterial were evaluated at 7, 13, 18, and 23 days of culture. Cell adhesion was determined by light microscopy, proliferation by DNA quantification, and osteogenic differentiation by alkaline phosphatase (ALP) activity analysis. DPSC adhered to β-TCP with both O₂ conditions. Cell proliferation was found from day 7 of culture. Higher values were recorded at 3% O₂ in each time point. Statistically significant differences were recorded at 23 days of culture (P = .033). ALP activity was not detectable at 7 days. There was, however, an increase in ALP activity over time in both groups. At 13, 18, and 23 days of culture, higher ALP activity was recorded under 3% O₂ pressure. Statistical differences were found at day 23 (P = .014). DPSC display capacity of adhering to β-TCP under 3% or 21% O₂ pressure conditions. Cell proliferation on β-TCP phosphate is significantly higher at 3% than at 21% O₂ pressure, the most frequently used O₂ tension. β-TCP can itself promote osteogenic differentiation of DPSC and is enhanced under 3% O₂ compared with 21%.