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.

Identification and Single-Cell Functional Characterization of an Endodermally Biased Pluripotent Substate in Human Embryonic Stem Cells.

PubMed

Allison, Thomas F; Smith, Andrew J H; Anastassiadis, Konstantinos; Sloane-Stanley, Jackie; Biga, Veronica; Stavish, Dylan; Hackland, James; Sabri, Shan; Langerman, Justin; Jones, Mark; Plath, Kathrin; Coca, Daniel; Barbaric, Ivana; Gokhale, Paul; Andrews, Peter W

2018-05-09

Human embryonic stem cells (hESCs) display substantial heterogeneity in gene expression, implying the existence of discrete substates within the stem cell compartment. To determine whether these substates impact fate decisions of hESCs we used a GFP reporter line to investigate the properties of fractions of putative undifferentiated cells defined by their differential expression of the endoderm transcription factor, GATA6, together with the hESC surface marker, SSEA3. By single-cell cloning, we confirmed that substates characterized by expression of GATA6 and SSEA3 include pluripotent stem cells capable of long-term self-renewal. When clonal stem cell colonies were formed from GATA6-positive and GATA6-negative cells, more of those derived from GATA6-positive cells contained spontaneously differentiated endoderm cells than similar colonies derived from the GATA6-negative cells. We characterized these discrete cellular states using single-cell transcriptomic analysis, identifying a potential role for SOX17 in the establishment of the endoderm-biased stem cell state. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Amnion-derived stem cells: in quest of clinical applications

PubMed Central

2011-01-01

In the promising field of regenerative medicine, human perinatal stem cells are of great interest as potential stem cells with clinical applications. Perinatal stem cells could be isolated from normally discarded human placentae, which are an ideal cell source in terms of availability, the fewer number of ethical concerns, less DNA damage, and so on. Numerous studies have demonstrated that some of the placenta-derived cells possess stem cell characteristics like pluripotent differentiation ability, particularly in amniotic epithelial (AE) cells. Term human amniotic epithelium contains a relatively large number of stem cell marker-positive cells as an adult stem cell source. In this review, we introduce a model theory of why so many AE cells possess stem cell characteristics. We also describe previous work concerning the therapeutic applications and discuss the pluripotency of the AE cells and potential pitfalls for amnion-derived stem cell research. PMID:21596003

Representations of stem cell clinics on Twitter.

PubMed

Kamenova, Kalina; Reshef, Amir; Caulfield, Timothy

2014-12-01

The practice of travelling abroad to receive unproven and unregulated stem cell treatments has become an increasingly problematic global phenomenon known as 'stem cell tourism'. In this paper, we examine representations of nine major clinics and providers of such treatments on the microblogging network Twitter. We collected and conducted a content analysis of Twitter posts (n = 363) by these establishments and by other users mentioning them, focusing specifically on marketing claims about treatment procedures and outcomes, discussions of safety and efficacy of stem cell transplants, and specific representations of patients' experiences. Our analysis has shown that there were explicit claims or suggestions of benefits associated with unproven stem cell treatments in approximately one third of the tweets and that patients' experiences, whenever referenced, were presented as invariably positive and as testimonials about the efficacy of stem cell transplants. Furthermore, the results indicated that the tone of most tweets (60.2 %) was overwhelmingly positive and there were rarely critical discussions about significant health risks associated with unproven stem cell therapies. When placed in the context of past research on the problems associated with the marketing of unproven stem cell therapies, this analysis of representations on Twitter suggests that discussions in social media have also remained largely uncritical of the stem cell tourism phenomenon, with inaccurate representations of risks and benefits for patients.

[Research progress of Lgr5-positive stem cells in the formation of organoid in 3D culture].

PubMed

He, Q Q; Li, A; Wang, M H; Gao, X

2018-06-07

Stem cell is critical to regeneration of tissue or organ of human. How to promote repair or regeneration in the tissues/organ using its pluripotency is always an important issue. Lgr5-possitive cell is one type of the stem cell-like cells capable of pluripotent differentiation in various tissues/organs of both humans and mice. Current study showed that single or small amount Lgr5-possitive stem cells can grow and form a plurality of organs in 3D culture system, and some organs can present similar biological and physiological properties with the progenitor they were derived. These studies provided new insight into future orientation, for example, Lgr5-possitive inner ear cells were confirmed as inner ear pluripotent cells population, the experiences obtained from organoid studies of Lgr5-possitive cells have certainly showed potential in the future study of inner ear stem cells. This review will focus on the recent progress associated with Lgr 5-positive stem cells forming organoids in the 3D culture.

Cell fate regulation in the shoot meristem.

PubMed

Laux, T; Mayer, K F

1998-04-01

The shoot meristem is a proliferative centre containing pluripotent stem cells that are the ultimate source of all cells and organs continuously added to the growing shoot. The progeny of the stem cells have two developmental options, either to renew the stem cell population or to leave the meristem and to differentiate, possibly according to signals from more mature tissue. The destiny of each cell depends on its position within the dynamic shoot meristem. Genetic data suggest a simple model in which graded positional information is provided by antagonistic gene functions and is interpreted by genes which regulate cell fate.

Fake news portrayals of stem cells and stem cell research.

PubMed

Marcon, Alessandro R; Murdoch, Blake; Caulfield, Timothy

2017-10-01

This study examines how stem cells and stem cell research are portrayed on websites deemed to be purveyors of distorted and dubious information. Content analysis was conducted on 224 articles from 2015 to 2016, compiled by searching with the keywords 'stem cell(s)' on a list of websites flagged for containing either 'fake' or 'junk science' news. Articles contained various exaggerated positive and negative claims about stem cells and stem cell science, health and science related conspiracy theories, and statements promoting fear and mistrust of conventional medicine. Findings demonstrate the existence of organized misinformation networks, which may lead the public away from accurate information and facilitate a polarization of public discourse.

FGF-2 signal promotes proliferation of cerebellar progenitor cells and their oligodendrocytic differentiation at early postnatal stage

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

Naruse, Masae; Shibasaki, Koji; Ishizaki, Yasuki, E-mail: yasukiishizaki@gunma-u.ac.jp

The origins and developmental regulation of cerebellar oligodendrocytes are largely unknown, although some hypotheses of embryonic origins have been suggested. Neural stem cells exist in the white matter of postnatal cerebellum, but it is unclear whether these neural stem cells generate oligodendrocytes at postnatal stages. We previously showed that cerebellar progenitor cells, including neural stem cells, widely express CD44 at around postnatal day 3. In the present study, we showed that CD44-positive cells prepared from the postnatal day 3 cerebellum gave rise to neurospheres, while CD44-negative cells prepared from the same cerebellum did not. These neurospheres differentiated mainly into oligodendrocytesmore » and astrocytes, suggesting that CD44-positive neural stem/progenitor cells might generate oligodendrocytes in postnatal cerebellum. We cultured CD44-positive cells from the postnatal day 3 cerebellum in the presence of signaling molecules known as mitogens or inductive differentiation factors for oligodendrocyte progenitor cells. Of these, only FGF-2 promoted survival and proliferation of CD44-positive cells, and these cells differentiated into O4+ oligodendrocytes. Furthermore, we examined the effect of FGF-2 on cerebellar oligodendrocyte development ex vivo. FGF-2 enhanced proliferation of oligodendrocyte progenitor cells and increased the number of O4+ and CC1+ oligodendrocytes in slice cultures. These results suggest that CD44-positive cells might be a source of cerebellar oligodendrocytes and that FGF-2 plays important roles in their development at an early postnatal stage. - Highlights: • CD44 is expressed in cerebellar neural stem/progenitor cells at postnatal day 3 (P3). • FGF-2 promoted proliferation of CD44-positive progenitor cells from P3 cerebellum. • FGF-2 promoted oligodendrocytic differentiation of CD44-positive progenitor cells. • FGF-2 increased the number of oligodendrocytes in P3 cerebellar slice culture.« less

Bulge Hair Follicle Stem Cells Accelerate Cutaneous Wound Healing in Rats.

PubMed

Heidari, Fatemeh; Yari, Abazar; Rasoolijazi, Homa; Soleimani, Mansoureh; Dehpoor, Ahmadreza; Sajedi, Nayereh; Joulai Veijouye, Sanaz; Nobakht, Maliheh

2016-04-01

Skin wound healing is a serious clinical problem especially after surgery and severe injury of the skin. Cell therapy is an innovative technique that can be applied to wound healing. One appropriate source of stem cells for therapeutic use is stem cells from the adult bulge of hair follicles. This study examined the effects of adult bulge hair follicle stem cells (HFSC) in wound healing. Hair follicle stem cells were obtained from rat vibrissa and labeled with DiI (Invitrogen, Carlsbad, CA), then special markers were detected using flow cytometry. A full-thickness excisional wound model was created and DiI-labeled HFSC were injected around the wound bed. Wound healing was recorded with digital photographs. Animals were sacrificed at 3, 7, or 14 days after surgery, and were used for the following histological analyses. Flow cytometry analysis showed that HFSC were CD34 positive and nestin positive, but K15 negative. Morphological analysis of HFSC-treated wounds exhibited accelerated wound closure. Histological analysis of hematoxylin and eosin stained and Masson's trichrome-stained photomicrographs showed significantly more re-epithelialization and dermal structural regeneration in HFSC-treated wounds than in the control group. Immunohistochemical analysis of CD31 protein-positive cells showed angiogenesis was also more significant in HFSC-treated wounds than in the control group. Hair follicle stem cells accelerate skin wound healing. Isolating HFSC from a small skin biopsy could repair less-extensive full-thickness skin wounds by autologous stem cells and overcome major challenges regarding the use of stem cells in clinical application, while avoiding immune rejection and ethical concerns.

Two sides of the same coin? Unraveling subtle differences between human embryonic and induced pluripotent stem cells by Raman spectroscopy.

PubMed

Parrotta, Elvira; De Angelis, Maria Teresa; Scalise, Stefania; Candeloro, Patrizio; Santamaria, Gianluca; Paonessa, Mariagrazia; Coluccio, Maria Laura; Perozziello, Gerardo; De Vitis, Stefania; Sgura, Antonella; Coluzzi, Elisa; Mollace, Vincenzo; Di Fabrizio, Enzo Mario; Cuda, Giovanni

2017-11-28

Human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells, hold enormous promise for many biomedical applications, such as regenerative medicine, drug testing, and disease modeling. Although induced pluripotent stem cells resemble embryonic stem cells both morphologically and functionally, the extent to which these cell lines are truly equivalent, from a molecular point of view, remains controversial. Principal component analysis and K-means cluster analysis of collected Raman spectroscopy data were used for a comparative study of the biochemical fingerprint of human induced pluripotent stem cells and human embryonic stem cells. The Raman spectra analysis results were further validated by conventional biological assays. Raman spectra analysis revealed that the major difference between human embryonic stem cells and induced pluripotent stem cells is due to the nucleic acid content, as shown by the strong positive peaks at 785, 1098, 1334, 1371, 1484, and 1575 cm -1 , which is enriched in human induced pluripotent stem cells. Here, we report a nonbiological approach to discriminate human induced pluripotent stem cells from their native embryonic stem cell counterparts.

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.

Nestin- and Doublecortin-Positive Cells Reside in Adult Spinal Cord Meninges and Participate in Injury-Induced Parenchymal Reaction

PubMed Central

Decimo, Ilaria; Bifari, Francesco; Rodriguez, Francisco Javier; Malpeli, Giorgio; Dolci, Sissi; Lavarini, Valentina; Pretto, Silvia; Vasquez, Sandra; Sciancalepore, Marina; Montalbano, Alberto; Berton, Valeria; Krampera, Mauro; Fumagalli, Guido

2011-01-01

Adult spinal cord has little regenerative potential, thus limiting patient recovery following injury. In this study, we describe a new population of cells resident in the adult rat spinal cord meninges that express the neural stem/precursor markers nestin and doublecortin. Furthermore, from dissociated meningeal tissue a neural stem cell population was cultured in vitro and subsequently shown to differentiate into functional neurons or mature oligodendrocytes. Proliferation rate and number of nestin- and doublecortin-positive cells increased in vivo in meninges following spinal cord injury. By using a lentivirus-labeling approach, we show that meningeal cells, including nestin- and doublecortin-positive cells, migrate in the spinal cord parenchyma and contribute to the glial scar formation. Our data emphasize the multiple roles of meninges in the reaction of the parenchyma to trauma and indicate for the first time that spinal cord meninges are potential niches harboring stem/precursor cells that can be activated by injury. Meninges may be considered as a new source of adult stem/precursor cells to be further tested for use in regenerative medicine applied to neurological disorders, including repair from spinal cord injury. Stem Cells 2011;29:2062–2076. PMID:22038821

The longest telomeres: a general signature of adult stem cell compartments

PubMed Central

Flores, Ignacio; Canela, Andres; Vera, Elsa; Tejera, Agueda; Cotsarelis, George; Blasco, María A.

2008-01-01

Identification of adult stem cells and their location (niches) is of great relevance for regenerative medicine. However, stem cell niches are still poorly defined in most adult tissues. Here, we show that the longest telomeres are a general feature of adult stem cell compartments. Using confocal telomere quantitative fluorescence in situ hybridization (telomapping), we find gradients of telomere length within tissues, with the longest telomeres mapping to the known stem cell compartments. In mouse hair follicles, we show that cells with the longest telomeres map to the known stem cell compartments, colocalize with stem cell markers, and behave as stem cells upon treatment with mitogenic stimuli. Using K15-EGFP reporter mice, which mark hair follicle stem cells, we show that GFP-positive cells have the longest telomeres. The stem cell compartments in small intestine, testis, cornea, and brain of the mouse are also enriched in cells with the longest telomeres. This constitutes the description of a novel general property of adult stem cell compartments. Finally, we make the novel finding that telomeres shorten with age in different mouse stem cell compartments, which parallels a decline in stem cell functionality, suggesting that telomere loss may contribute to stem cell dysfunction with age. PMID:18283121

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.

Isolation and Propagation of Mesenchymal Stem Cells from the Lacrimal Gland

PubMed Central

You, Samantha; Kublin, Claire L.; Avidan, Orna; Miyasaki, David

2011-01-01

Purpose. Previously, it was reported that the murine lacrimal gland is capable of repair after experimentally induced injury and that the number of stem/progenitor cells was increased during the repair phase (2–3 days after injury). The aim of the present study was to determine whether these cells can be isolated from the lacrimal gland and propagated in vitro. Methods. Lacrimal gland injury was induced by injection of interleukin (IL)-1, and injection of saline vehicle served as control. Two and half days after injection, the lacrimal glands were removed and used to prepare explants or acinar cells for tissue culture. Cells derived from the explants and the acinar cells were grown in DMEM supplemented with 10% fetal bovine serum. Cells were stained for the stem cells markers, nestin, vimentin, ABCG2, and Sca-1. Cell proliferation was measured using an antibody against Ki67 or a cell-counting kit. The adipogenic capability of these cells was also tested in vitro. Results. Results show that nestin-positive cells can be isolated from IL-1–injected, but not saline-injected, lacrimal glands. A population of nestin-positive cells was also positive for vimentin, an intermediate filament protein expressed by mesenchymal cells. In addition, cultured cells expressed two other markers of stem cells, ABCG2 and Sca-1. These cells proliferated in vitro and can be induced to form adipocytes, attesting to their mesenchymal stem cell property. Conclusions. Murine lacrimal glands contain mesenchymal stem cells that seem to play a pivotal role in tissue repair. PMID:21178145

STAT3 as a potential therapeutic target in ALDH+ and CD44+/CD24+ stem cell-like pancreatic cancer cells.

PubMed

Lin, Li; Jou, David; Wang, Yina; Ma, Haiyan; Liu, Tianshu; Fuchs, James; Li, Pui-Kai; Lü, Jiagao; Li, Chenglong; Lin, Jiayuh

2016-12-01

Persistent activation of signal transducers and activators of transcription 3 (STAT3) is commonly detected in many types of cancer including pancreatic cancer. Whether STAT3 is activated in stem cell-like pancreatic cancer cells and the effect of STAT3 inhibition, is still unknown. Flow cytometry was used to isolate pancreatic cancer stem-like cells which are identified by both aldehyde dehydrogenase (ALDH)-positive (ALDH+) as well as cluster of differentiation (CD) 44-positive/CD24-positive subpopulations (CD44+/CD24+). STAT3 activation and the effects of STAT3 inhibition by STAT3 inhibitors, LLL12, FLLL32, and Stattic in ALDH+ and CD44+/CD24+ cells were examined. Our results showed that ALDH+ and CD44+/CD24+ pancreatic cancer stem-like cells expressed higher levels of phosphorylated STAT3, an active form of STAT3, compared to ALDH-negative (ALDH-) and CD44-negative/CD24-negative (CD44-/CD24-) pancreatic cancer cells, suggesting that STAT3 is activated in pancreatic cancer stem-like cells. Small molecular STAT3 inhibitors inhibited STAT3 phosphorylation, STAT3 downstream target gene expression, cell viability, and tumorsphere formation in ALDH+ and CD44+/CD24+ cells. Our results indicate that STAT3 is a novel therapeutic target in pancreatic cancer stem-like cells and inhibition of activated STAT3 in these cells by STAT3 inhibitors may offer an effective treatment for pancreatic cancer.

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.

The specific linker phosphorylation of Smad2/3 indicates epithelial stem cells in stomach; particularly increasing in mucosae of Helicobacter-associated gastritis.

PubMed

Fukui, Toshiro; Kishimoto, Masanobu; Nakajima, Atsushi; Yamashina, Masao; Nakayama, Shinji; Kusuda, Takeo; Sakaguchi, Yutaku; Yoshida, Katsunori; Uchida, Kazushige; Nishio, Akiyoshi; Matsuzaki, Koichi; Okazaki, Kazuichi

2011-04-01

The gastric corpus and antrum are believed to contain epithelial stem cells in the isthmus. However, the lack of useful markers has hindered studies of their origin. We explored whether Smad2/3, phosphorylated at specific linker threonine residues (pSmad2/3L-Thr), could serve as a marker for stem cells. Stomachs, small intestines, and colons from Helicobacter felis-infected and noninfected C57BL/6 mice were examined. Double immunofluorescent staining of pSmad2/3L-Thr with Ki67, cytokeratin 8, or doublecortin and calcium/calmodulin-dependent protein kinase-like-1 (DCAMKL1) was performed, and pSmad2/3L-Thr immunostaining-positive cells were counted. After immunofluorescent staining, we stained the same sections with hematoxylin-eosin and observed these cells under a light microscope. In infected mice, pSmad2/3L-Thr immunostaining-positive cells were significantly increased in the corpus and antrum compared with those of noninfected mice (p < 0.0001). The number of Ki67 immunostaining-positive cells in the corpus and antrum of infected mice was also much greater than in the noninfected mice. Although pSmad2/3L-Thr immunostaining-positive cells were detected among the Ki67 cells, immunohistochemical co-localization of pSmad2/3L-Thr with Ki67 was never observed. pSmad2/3L-Thr immunostaining-positive cells showed immunohistochemical co-localization with cytokeratin 8, but some of them showed co-localization or adjacent localization with DCAMKL1 immunostaining-positive cells. Under a light microscope, pSmad2/3L-Thr immunostaining-positive cells indicated undifferentiated morphological features and were confirmed in the isthmus. In small intestines and colons, pSmad2/3L-Thr immunostaining-positive cells were detected in specific epithelial cells around crypt bases, where the respective putative stem cells are thought to exist. We have identified the significant expression of pSmad2/3L-Thr in specific epithelial cells of the murine stomach and have suggested these cells to be epithelial stem cells.

Cisplatin selects for stem-like cells in osteosarcoma by activating Notch signaling

PubMed Central

Yang, Jian; Gao, Tian; Simões, Bruno M.; Eyre, Rachel; Guo, Weichun; Clarke, Robert B.

2016-01-01

Notch signaling regulates normal stem cells and is also thought to regulate cancer stem cells (CSCs). Recent data indicate that Notch signaling plays a role in the development and progression of osteosarcoma, however the regulation of Notch in chemo-resistant stem-like cells has not yet been fully elucidated. In this study we generated cisplatin-resistant osteosarcoma cells by treating them with sub-lethal dose of cisplatin, sufficient to induce DNA damage responses. Cisplatin-resistant osteosarcoma cells exhibited lower proliferation, enhanced spheroid formation and more mesenchymal characteristics than cisplatin-sensitive cells, were enriched for Stro-1+/CD117+ cells and showed increased expression of stem cell-related genes. A similar effect was observed in vivo, and in addition in vivo tumorigenicity was enhanced during serial transplantation. Using several publicly available datasets, we identified that Notch expression was closely associated with osteosarcoma stem cells and chemotherapy resistance. We confirmed that cisplatin-induced enrichment of osteosarcoma stem cells was mediated through Notch signaling in vitro, and immunohistochemistry showed that cleaved Notch1 (NICD1) positive cells were significantly increased in a relapsed xenograft which had received cisplatin treatment. Furthermore, pretreatment with a γ-secretase inhibitor (GSI) to prevent Notch signalling inhibited cisplatin-enriched osteosarcoma stem cell activity in vitro, including Stro-1+/CD117+ double positive cells and spheroid formation capacity. The Notch inhibitor DAPT also prevented tumor recurrence in resistant xenograft tumors. Overall, our results show that cisplatin induces the enrichment of osteosarcoma stem-like cells through Notch signaling, and targeted inactivation of Notch may be useful for the elimination of CSCs and overcoming drug resistance. PMID:27102300

Nitrative DNA damage and Oct3/4 expression in urinary bladder cancer with Schistosomahaematobium infection

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

Ma, Ning; Thanan, Raynoo; Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Mie

Highlights: {yields} Oct3/4-positive cells increase in Schistosoma haematobium (SH)-associated bladder cancer. {yields} iNOS-dependent DNA lesion, 8-nitroguanine, was formed in Oct3/4-positive cells. {yields} 8-Nitroguanine formed in stem-like cells plays a role in SH-induced carcinogenesis. {yields} Mutant stem cells may participate in inflammation-related carcinogenesis. -- Abstract: To investigate whether mutant stem cells participate in inflammation-related carcinogenesis, we performed immunohistochemical analysis to examine nitrative and oxidative DNA lesions (8-nitroguanine and 8-oxodG) and a stem cell marker Oct3/4 in bladder tissues obtained from cystitis and bladder cancer patients infected with Schistosomahaematobium (S. haematobium). We also detected the expression of nuclear factor-{kappa}B (NF-{kappa}B) and induciblemore » nitric oxide synthase (iNOS), which lead to 8-nitroguanine formation. The staining intensity of 8-nitroguanine and 8-oxodG was significantly higher in bladder cancer and cystitis tissues than in normal tissues. iNOS expression was colocalized with NF-{kappa}B in 8-nitroguanine-positive tumor cells from bladder cancer patients. Oct3/4 expression was significantly increased in cells from S. haematobium-associated bladder cancer tissues in comparison to normal bladder and cancer tissues without infection. Oct3/4 was also expressed in epithelial cells of cystitis patients. Moreover, 8-nitroguanine was formed in Oct3/4-positive stem cells in S. haematobium-associated cystitis and cancer tissues. In conclusion, inflammation by S.haematobium infection may increase the number of mutant stem cells, in which iNOS-dependent DNA damage occurs via NF-{kappa}B activation, leading to tumor development.« less

β-Cell Replacement in Mice Using Human Type 1 Diabetes Nuclear Transfer Embryonic Stem Cells.

PubMed

Sui, Lina; Danzl, Nichole; Campbell, Sean R; Viola, Ryan; Williams, Damian; Xing, Yuan; Wang, Yong; Phillips, Neil; Poffenberger, Greg; Johannesson, Bjarki; Oberholzer, Jose; Powers, Alvin C; Leibel, Rudolph L; Chen, Xiaojuan; Sykes, Megan; Egli, Dieter

2018-01-01

β-Cells derived from stem cells hold great promise for cell replacement therapy for diabetes. Here we examine the ability of nuclear transfer embryonic stem cells (NT-ESs) derived from a patient with type 1 diabetes to differentiate into β-cells and provide a source of autologous islets for cell replacement. NT-ESs differentiate in vitro with an average efficiency of 55% into C-peptide-positive cells, expressing markers of mature β-cells, including MAFA and NKX6.1. Upon transplantation in immunodeficient mice, grafted cells form vascularized islet-like structures containing MAFA/C-peptide-positive cells. These β-cells adapt insulin secretion to ambient metabolite status and show normal insulin processing. Importantly, NT-ES-β-cells maintain normal blood glucose levels after ablation of the mouse endogenous β-cells. Cystic structures, but no teratomas, were observed in NT-ES-β-cell grafts. Isogenic induced pluripotent stem cell lines showed greater variability in β-cell differentiation. Even though different methods of somatic cell reprogramming result in stem cell lines that are molecularly indistinguishable, full differentiation competence is more common in ES cell lines than in induced pluripotent stem cell lines. These results demonstrate the suitability of NT-ES-β-cells for cell replacement for type 1 diabetes and provide proof of principle for therapeutic cloning combined with cell therapy. © 2017 by the American Diabetes Association.

Detection of cytomegalovirus DNA in fecal samples as a method for CMV enterocolitis diagnosis after allogeneic stem cell transplantation.

PubMed

Zavrelova, Alzbeta; Radocha, Jakub; Pliskova, Lenka; Paterova, Pavla; Vejrazkova, Eva; Cyrany, Jiri; Gabalec, Filip; Podhola, Miroslav; Zak, Pavel

2018-05-16

Cytomegalovirus enterocolitis is a rare but potentially life threatening complication after allogeneic stem cell transplantation. Its early diagnosis and treatment are essential for a successful outcome. To determine the potential benefit of fecal CMV DNA detection in the diagnosis of CMV colitis among stem cell transplant recipients. Biopsies from the lower gastrointestinal tract, taken during 69 episodes of diarrhea, were compared with fecal samples previously examined for CMV DNA in 45 patients after allogeneic stem cell transplantation. Six confirmed cases of CMV colitis were observed, with 16 out of 69 (23%) fecal samples proving positive for CMV DNA. Only one positive sample correlated with histologically confirmed CMV colitis, and 15 samples were evaluated as false positive. These results provide a 16.7% sensitivity and 76.2% specificity in the diagnosis of CMV enterocolitis. The examination of fecal samples for the presence of CMV DNA has very low potential in the diagnosis of CMV enterocolitis after allogeneic stem cell transplantation; therefore, a biopsy of the gastrointestinal mucosa is still warranted for correct diagnosis.

Evaluation of the maintenance of stemness, viability, and differentiation potential of gingiva-derived stem-cell spheroids.

PubMed

Lee, Sung-Il; Ko, Youngkyung; Park, Jun-Beom

2017-05-01

Gingiva-derived stem cells have been applied for tissue-engineering purposes and may be considered a favorable source of mesenchymal stem cells as harvesting stem cells from the mandible or maxilla may be performed with ease under local anesthesia. The present study was performed to fabricate stem-cell spheroids using concave microwells and to evaluate the maintenance of stemness, viability, and differentiation potential. Gingiva-derived stem cells were isolated, and the stem cells of 4×10 5 (group A) or 8×10 5 (group B) cells were seeded into polydimethylsiloxane-based, concave micromolds with 600 µm diameters. The morphology of the microspheres and the change of the diameters of the spheroids were evaluated. The viability of spheroids was qualitatively analyzed via Live/Dead kit assay. A cell viability analysis was performed on days 1, 3, 6, and 12 with Cell Counting Kit-8. The maintenance of stemness was evaluated with immunocytochemical staining using SSEA-4, TRA-1-60(R) (positive markers), and SSEA-1 (negative marker). Osteogenic, adipogenic, and chondrogenic differentiation potential was evaluated by incubating spheroids in osteogenic, adipogenic and chondrogenic induction medium, respectively. The gingiva-derived stem cells formed spheroids in the concave microwells. The diameters of the spheroids were larger in group A than in group B. The majority of cells in the spheroids emitted green fluorescence, indicating the presence of live cells at day 6. At day 12, the majority of cells in the spheroids emitted green fluorescence, and a small portion of red fluorescence was also noted, which indicated the presence of dead cells. The spheroids were positive for the stem-cell markers SSEA-4 and TRA-1-60(R) and were negative for SSEA-1, suggesting that these spheroids primarily contained undifferentiated human stem cells. Osteogenic, adipogenic, and chondrogenic differentiation was more evident with an increase of incubation time: Mineralized extracellular deposits were observed following Alizarin Red S staining at days 14 and 21; oil globules were increased at day 18 when compared with day 6; and Alcian blue staining was more evident at day 18 when compared with day 6. Within the limits of this study, stem-cell spheroids from gingival cells maintained the stemness, viability, and differentiation potential during the experimental periods. This method may be applied for a promising strategy for stem-cell therapy.

Hematopoietic stem cell loss and hematopoietic failure in severe aplastic anemia is driven by macrophages and aberrant podoplanin expression.

PubMed

McCabe, Amanda; Smith, Julianne N P; Costello, Angelica; Maloney, Jackson; Katikaneni, Divya; MacNamara, Katherine C

2018-05-17

Severe aplastic anemia results from profound hematopoietic stem cell loss. T cells and interferon gamma have long been associated with severe aplastic anemia, yet the underlying mechanisms driving hematopoietic stem cell loss remain unknown. Using a mouse model of severe aplastic anemia, we demonstrate that interferon gamma-dependent hematopoietic stem cell loss required macrophages. Interferon gamma was necessary for bone marrow macrophage persistence, despite loss of other myeloid cells and hematopoietic stem cells. Depleting macrophages or abrogating interferon gamma signaling specifically in macrophages did not impair T cell activation or interferon gamma production in the bone marrow but rescued hematopoietic stem cells and reduced mortality. Thus, macrophages are not required for induction of interferon gamma in severe aplastic anemia and rather act as sensors of interferon gamma. Macrophage depletion rescued thrombocytopenia, increased bone marrow megakaryocytes, preserved platelet-primed stem cells, and increased the platelet-repopulating capacity of transplanted hematopoietic stem cells. In addition to the hematopoietic effects, severe aplastic anemia induced loss of non-hematopoietic stromal populations, including podoplanin-positive stromal cells. However, a subset of podoplanin-positive macrophages was increased during disease, and blockade of podoplanin in mice was sufficient to rescue disease. Our data further our understanding of disease pathogenesis demonstrating a novel role for macrophages as sensors of interferon gamma, thus illustrating an important role for the microenvironment in pathogenesis of severe aplastic anemia. Copyright © 2018, Ferrata Storti Foundation.

The Effect of Laser Irradiation on Adipose Derived Stem Cell Proliferation and Differentiation

NASA Astrophysics Data System (ADS)

Abrahamse, H.; de Villiers, J.; Mvula, B.

2009-06-01

There are two fundamental types of stem cells: Embryonic Stem cells and Adult Stem cells. Adult Stem cells have a more restricted potential and can usually differentiate into a few different cell types. In the body these cells facilitate the replacement or repair of damaged or diseased cells in organs. Low intensity laser irradiation was shown to increase stem cell migration and stimulate proliferation and it is thought that treatment of these cells with laser irradiation may increase the stem cell harvest and have a positive effect on the viability and proliferation. Our research is aimed at determining the effect of laser irradiation on differentiation of Adipose Derived Stem Cells (ADSCs) into different cell types using a diode laser with a wavelength of 636 nm and at 5 J/cm2. Confirmation of stem cell characteristics and well as subsequent differentiation were assessed using Western blot analysis and cellular morphology supported by fluorescent live cell imaging. Functionality of subsequent differentiated cells was confirmed by measuring adenosine triphosphate (ATP) production and cell viability.

Phosphorylation of Smad2/3 at specific linker threonine indicates slow-cycling intestinal stem-like cells before reentry to cell cycle.

PubMed

Kishimoto, Masanobu; Fukui, Toshiro; Suzuki, Ryo; Takahashi, Yu; Sumimoto, Kimi; Okazaki, Takashi; Sakao, Masayuki; Sakaguchi, Yutaku; Yoshida, Katsunori; Uchida, Kazushige; Nishio, Akiyoshi; Matsuzaki, Koichi; Okazaki, Kazuichi

2015-02-01

Quiescent (slow-cycling) and active (rapid-cycling) stem cells are demonstrated in small intestines. We have identified significant expression of Smad2/3, phosphorylated at specific linker threonine residues (pSmad2/3L-Thr), in murine stomach, and suggested these cells are epithelial stem cells. Here, we explore whether pSmad2/3L-Thr could serve as a biomarker for small intestine and colon stem cells. We examined small intestines and colons from C57BL/6 mice and colons with dextran sulfate sodium (DSS)-induced colitis. We performed double-immunofluorescent staining of pSmad2/3L-Thr with Ki67, cytokeratin 8, chromogranin A, CDK4, DCAMKL1, and Musashi-1. Small intestines and colons from Lgr5-EGFP knock-in mice were examined by pSmad2/3L-Thr immunofluorescent staining. To examine BrdU label retention of pSmad2/3L-Thr immunostaining-positive cells, we collected specimens after BrdU administration and observed double-immunofluorescent staining of pSmad2/3L-Thr with BrdU. In small intestines and colons, pSmad2/3L-Thr immunostaining-strongly positive cells were detected around crypt bases. Immunohistochemical co-localization of pSmad2/3L-Thr with Ki67 was not observed. pSmad2/3L-Thr immunostaining-strongly positive cells showed co-localization with cytokeratin 8, CDK4, and Musashi-1 and different localization from chromogranin A and DCAMKL1 immunostaining-positive cells. Under a light microscope, pSmad2/3L-Thr immunostaining-strongly positive cells were morphologically undifferentiated. In Lgr5-EGFP knock-in mice, some but not all pSmad2/3L-Thr immunostaining-strongly positive cells showed co-localization with Lgr5. pSmad2/3L-Thr immunostaining-strongly positive cells showed co-localization with BrdU at 5, 10, and 15 days after administration. In DSS-induced colitis, pSmad2/3L-Thr and Ki67 immunostaining-positive cells increased in the regeneration phase and decreased in the injury phase. In murine small intestines and colons, we suggest pSmad2/3L-Thr immunostaining-strongly positive cells are epithelial stem-like cells just before reentry to the cell cycle.

Microvesicles released from human renal cancer stem cells stimulate angiogenesis and formation of lung premetastatic niche.

PubMed

Grange, Cristina; Tapparo, Marta; Collino, Federica; Vitillo, Loriana; Damasco, Christian; Deregibus, Maria Chiara; Tetta, Ciro; Bussolati, Benedetta; Camussi, Giovanni

2011-08-01

Recent studies suggest that tumor-derived microvesicles (MV) act as a vehicle for exchange of genetic information between tumor and stromal cells, engendering a favorable microenvironment for cancer development. Within the tumor mass, all cell types may contribute to MV shedding, but specific contributions to tumor progression have yet to be established. Here we report that a subset of tumor-initiating cells expressing the mesenchymal stem cell marker CD105 in human renal cell carcinoma releases MVs that trigger angiogenesis and promote the formation of a premetastatic niche. MVs derived only from CD105-positive cancer stem cells conferred an activated angiogenic phenotype to normal human endothelial cells, stimulating their growth and vessel formation after in vivo implantation in immunocompromised severe combined immunodeficient (SCID) mice. Furthermore, treating SCID mice with MVs shed from CD105-positive cells greatly enhanced lung metastases induced by i.v. injection of renal carcinoma cells. Molecular characterization of CD105-positive MVs defines a set of proangiogenic mRNAs and microRNAs implicated in tumor progression and metastases. Our results define a specific source of cancer stem cell-derived MVs that contribute to triggering the angiogenic switch and coordinating metastatic diffusion during tumor progression.

STAT3 signaling pathway is necessary for cell survival and tumorsphere forming capacity in ALDH{sup +}/CD133{sup +} stem cell-like human colon cancer cells

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

Lin, Li, E-mail: lin.796@osu.edu; Division of Cardiology, Department of Internal Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030; Fuchs, James

2011-12-16

Highlights: Black-Right-Pointing-Pointer The phosphorylated or activated form of STAT3 was expressed in colon cancer stem-like cells. Black-Right-Pointing-Pointer STAT3 inhibitor, FLLL32 inhibits P-STAT3 and STAT3 target genes in colon cancer stem-like cells. Black-Right-Pointing-Pointer Inhibition of STAT3 resulted in decreased cell viability and reduced numbers of tumorspheres. Black-Right-Pointing-Pointer STAT3 is required for survival and tumorsphere forming capacity in colon cancer stem-like cells. Black-Right-Pointing-Pointer Targeting STAT3 in cancer stem-like cells may offer a novel treatment approach for colon cancer. -- Abstract: Persistent activation of Signal Transducers and Activators of Transcription 3 (STAT3) is frequently detected in colon cancer. Increasing evidence suggests the existencemore » of a small population of colon cancer stem or cancer-initiating cells may be responsible for tumor initiation, metastasis, and resistance to chemotherapy and radiation. Whether STAT3 plays a role in colon cancer-initiating cells and the effect of STAT3 inhibition is still unknown. Flow cytometry was used to isolate colon cancer stem-like cells from three independent human colon cancer cell lines characterized by both aldehyde dehydrogenase (ALDH)-positive and CD133-positive subpopulation (ALDH{sup +}/CD133{sup +}). The effects of STAT3 inhibition in colon cancer stem-like cells were examined. The phosphorylated or activated form of STAT3 was expressed in colon cancer stem-like cells and was reduced by a STAT3-selective small molecular inhibitor, FLLL32. FLLL32 also inhibited the expression of potential STAT3 downstream target genes in colon cancer stem-like cells including survivin, Bcl-XL, as well as Notch-1, -3, and -4, which may be involved in stem cell function. Furthermore, FLLL32 inhibited cell viability and tumorsphere formation as well as induced cleaved caspase-3 in colon cancer stem-like cells. FLLL32 is more potent than curcumin as evidenced with lower IC50 in colon cancer stem-like cells. In summary, our results indicate that STAT3 is a novel therapeutic target in colon cancer stem-like cells and inhibition of STAT3 in cancer stem-like cells may offer a potential treatment for colorectal cancer.« less

Primitive Sca-1 Positive Bone Marrow HSC in Mouse Model of Aplastic Anemia: A Comparative Study through Flowcytometric Analysis and Scanning Electron Microscopy

PubMed Central

Chatterjee, Sumanta; Basak, Pratima; Das, Prosun; Das, Madhurima; Pereira, Jacintha Archana; Dutta, Ranjan Kumar; Chaklader, Malay; Chaudhuri, Samaresh; Law, Sujata

2010-01-01

Self-renewing Hematopoietic Stem Cells (HSCs) are responsible for reconstitution of all blood cell lineages. Sca-1 is the “stem cell antigen” marker used to identify the primitive murine HSC population, the expression of which decreases upon differentiation to other mature cell types. Sca-1+ HSCs maintain the bone marrow stem cell pool throughout the life. Aplastic anemia is a disease considered to involve primary stem cell deficiency and is characterized by severe pancytopenia and a decline in healthy blood cell generation system. Studies conducted in our laboratory revealed that the primitive Sca-1+ BM-HSCs (bone marrow hematopoietic stem cell) are significantly affected in experimental Aplastic animals pretreated with chemotherapeutic drugs (Busulfan and Cyclophosphamide) and there is increased Caspase-3 activity with consecutive high Annexin-V positivity leading to premature apoptosis in the bone marrow hematopoietic stem cell population in Aplastic condition. The Sca-1bright, that is, “more primitive” BM-HSC population was more affected than the “less primitive” BM-HSC Sca-1dim  population. The decreased cell population and the receptor expression were directly associated with an empty and deranged marrow microenvironment, which is evident from scanning electron microscopy (SEM). The above experimental evidences hint toward the manipulation of receptor expression for the benefit of cytotherapy by primitive stem cell population in Aplastic anemia cases. PMID:21048851

Recent Progress in Stem Cell Modification for Cardiac Regeneration

PubMed Central

Voronina, Natalia; Steinhoff, Gustav

2018-01-01

During the past decades, stem cell-based therapy has acquired a promising role in regenerative medicine. The application of novel cell therapeutics for the treatment of cardiovascular diseases could potentially achieve the ambitious aim of effective cardiac regeneration. Despite the highly positive results from preclinical studies, data from phase I/II clinical trials are inconsistent and the improvement of cardiac remodeling and heart performance was found to be quite limited. The major issues which cardiac stem cell therapy is facing include inefficient cell delivery to the site of injury, accompanied by low cell retention and weak effectiveness of remaining stem cells in tissue regeneration. According to preclinical and clinical studies, various stem cells (adult stem cells, embryonic stem cells, and induced pluripotent stem cells) represent the most promising cell types so far. Beside the selection of the appropriate cell type, researchers have developed several strategies to produce “second-generation” stem cell products with improved regenerative capacity. Genetic and nongenetic modifications, chemical and physical preconditioning, and the application of biomaterials were found to significantly enhance the regenerative capacity of transplanted stem cells. In this review, we will give an overview of the recent developments in stem cell engineering with the goal to facilitate stem cell delivery and to promote their cardiac regenerative activity. PMID:29535769

Presence of stem/progenitor cells in the rat penis.

PubMed

Lin, Guiting; Alwaal, Amjad; Zhang, Xiaoyu; Wang, Jianwen; Wang, Lin; Li, Huixi; Wang, Guifang; Ning, Hongxiu; Lin, Ching-Shwun; Xin, Zhongcheng; Lue, Tom F

2015-01-15

Tissue resident stem cells are believed to exist in every organ, and their identification is commonly done using a combination of immunostaining for putative stem cell markers and label-retaining cell (LRC) strategy. In this study, we employed these approaches to identify potential stem cells in the penis. Newborn rats were intraperitoneally injected with thymidine analog, 5-ethynyl-2-deoxyuridine (EdU), and their penis was harvested at 7 h, 3 days, 1 week, and 4 weeks. It was processed for EdU stains and immunofluorescence staining for stem cell markers A2B5, PCNA, and c-kit. EdU-positive cells were counted for each time point and co-localized with each stem cell marker, then isolated and cultured in vitro followed by their characterization using flowcytometry and immunofluorescence. At 7 h post-EdU injection, 410 ± 105.3 penile corporal cells were labeled in each cross-section (∼28%). The number of EdU-positive cells at 3 days increased to 536 ± 115.6, while their percentage dropped to 25%. Progressively fewer EdU-positive cells were present in the sacrificed rat penis at longer time points (1 and 4 weeks). They were mainly distributed in the subtunic and perisinusoidal spaces, and defined as subtunic penile progenitor cells (STPCs) and perisinusoidal penile progenitor cells (PPCs). These cells expressed c-kit, A2B5, and PCNA. After culturing in vitro, only ∼0.324% corporal cells were EdU-labeled LRCs and expressed A2B5/PCNA. Therefore, labeling of penis cells by EdU occurred randomly, and label retaining was not associated with expression of c-kit, A2B5, or PCNA. The penile LRCs are mainly distributed within the subtunic and perisinusoidal space.

[Morphofunctional organization of reserve stem cells providing for asexual and sexual reproduction of invertebrates].

PubMed

Isaeva, V V; Akhmadieva, A V; Aleksandriova, Ia N; Shukaliuk, A I

2009-01-01

Published and original data indicating evolutionary conservation of the morphofunctional organization of reserve stem cells providing for asexual and sexual reproduction of invertebrates are reviewed. Stem cells were studied in representatives of five animal types: archeocytes in sponge Oscarella malakhovi (Porifera), large interstitial cells in colonial hydroid Obelia longissima (Cnidaria), neoblasts in an asexual race of planarian Girardia tigrina (Platyhelmintes), stem cells in colonial rhizocephalans Peltogasterella gracilis, Polyascus polygenea, and Thylacoplethus isaevae (Arthropoda), and colonial ascidian Botryllus tuberatus (Chordata). Stem cells in animals of such diverse taxa feature the presence of germinal granules, are positive for proliferating cell nuclear antigen, demonstrate alkaline phosphatase activity (at marker of embryonic stem cells and primary germ cells in vertebrates), and rhizocephalan stem cells express the vasa-like gene (such genes are expressed in germline cells of different metazoans). The self-renewing pool of stem cells is the cellular basis of the reproductive strategy including sexual and asexual reproduction.

Un(MaSC)ing Stem Cell Dynamics in Mammary Branching Morphogenesis.

PubMed

Greenwood, Erin; Wrenn, Emma D; Cheung, Kevin J

2017-02-27

The properties of stem cells that participate in mammary gland branching morphogenesis remain contested. Reporting in Nature, Scheele et al. (2017) establish a model for post-pubertal mammary branching morphogenesis in which position-dependent, lineage-restricted stem cells undergo cell mixing in order to contribute to long-term growth. Copyright © 2017 Elsevier Inc. All rights reserved.

Purinergic Receptors in Quiescence and Localization of Leukemic Stem Cells

DTIC Science & Technology

2011-05-01

2011. Ewing’s Sarcoma Gene EWS regulates Hematopoietic Stem Cell Senescence. Blood, 117:1156-66. Conclusion How leukemia stem cells gained...findings contained in this report are those of the author( s ) and should not be construed as an official Department of the Army position, policy or...Receptors in Quiescence and Localization of Leukemic Stem Cells 5b. GRANT NUMBER W81XWH-09-1-0364 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR( S ) 5d

A rational approach for cancer stem-like cell isolation and characterization using CD44 and prominin-1(CD133) as selection markers

PubMed Central

Lee, Yi-Jen; Wu, Chang-Cheng; Li, Jhy-Wei; Ou, Chien-Chih; Hsu, Shih-Chung; Tseng, Hsiu-Hsueh; Kao, Ming-Ching; Liu, Jah-Yao

2016-01-01

The availability of adequate cancer stem cells or cancer stem-like cell (CSC) is important in cancer study. From ovarian cancer cell lines, SKOV3 and OVCAR3, we induced peritoneal ascites tumors in immunodeficient mice. Among the cells (SKOV3.PX1 and OVCAR3.PX1) from those tumors, we sorted both CD44 and CD133 positive cells (SKOV3.PX1_133+44+, OVCAR3.PX1_133+44+), which manifest the characteristics of self-renewal, multi-lineage differentiation, chemoresistance and tumorigenicity, those of cancer stem-like cells (CSLC). Intraperitoneal transplantation of these CD44 and CD133 positive cells resulted in poorer survival in the engrafted animals. Clinically, increased CD133 expression was found in moderately and poorly differentiated (grade II and III) ovarian serous cystadenocarcinomas. The ascites tumor cells from human ovarian cancers demonstrated more CD133 and CD44 expressions than those from primary ovarian or metastatic tumors and confer tumorigenicity in immunodeficient mice. Compared to their parental cells, the SKOV3.PX1_133+44+ and OVCAR3.PX1_133+44+ cells uniquely expressed 5 CD markers (CD97, CD104, CD107a, CD121a, and CD125). Among these markers, CD97, CD104, CD107a, and CD121a are significantly more expressed in the CD133+ and CD44+ double positive cells of human ovarian ascites tumor cells (Ascites_133+44+) than those from primary ovarian or metastatic tumors. The cancer stem-like cells were enriched from 3% to more than 70% after this manipulation. This intraperitoneal enrichment of cancer stem-like cells, from ovarian cancer cell lines or primary ovarian tumor, potentially provides an adequate amount of ovarian cancer stem-like cells for the ovarian cancer study and possibly benefits cancer therapy. PMID:27655682

An avian model for the reversal of neurobehavioral teratogenicity with neural stem cells

PubMed Central

Dotan, Sharon; Pinkas, Adi; Slotkin, Theodore A.; Yanai, Joseph

2010-01-01

A fast and simple model which uses lower animals on the evolutionary scale is beneficial for developing procedures for the reversal of neurobehavioral teratogenicity with neural stem cells. Here, we established a procedure for the derivation of chick neural stem cells, establishing embryonic day (E) 10 as optimal for progression to neuronal phenotypes. Cells were obtained from the embryonic cerebral hemispheres and incubated for 5–7 days in enriched medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (FGF2) according to a procedure originally developed for mice. A small percentage of the cells survived, proliferated and formed nestin-positive neurospheres. After removal of the growth factors to allow differentiation (5 days), 74% of the cells differentiated into all major lineages of the nervous system, including neurons (Beta III tubulin-positive, 54% of the total number of differentiated cells), astrocytes (GFAP-positive, 26%), and oligodendrocytes (O4-positive, 20%). These findings demonstrate that the cells were indeed neural stem cells. Next, the cells were transplanted in two allograft chick models; (1) direct cerebral transplantation to 24-hours-old chicks, followed by post-transplantation cell tracking at 24 hours, 6 days and 14 days, and (2) intravenous transplantation to chick embryos on E13, followed by cell tracking on E19. With both methods, transplanted cells were found in the brain. The chick embryo provides a convenient, precisely-timed and unlimited supply of neural progenitors for therapy by transplantation, as well as constituting a fast and simple model in which to evaluate the ability of neural stem cell transplantation to repair neural damage, steps that are critical for progress toward therapeutic applications. PMID:20211723

SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells.

PubMed

Dubois, Nicole C; Craft, April M; Sharma, Parveen; Elliott, David A; Stanley, Edouard G; Elefanty, Andrew G; Gramolini, Anthony; Keller, Gordon

2011-10-23

To identify cell-surface markers specific to human cardiomyocytes, we screened cardiovascular cell populations derived from human embryonic stem cells (hESCs) against a panel of 370 known CD antibodies. This screen identified the signal-regulatory protein alpha (SIRPA) as a marker expressed specifically on cardiomyocytes derived from hESCs and human induced pluripotent stem cells (hiPSCs), and PECAM, THY1, PDGFRB and ITGA1 as markers of the nonmyocyte population. Cell sorting with an antibody against SIRPA allowed for the enrichment of cardiac precursors and cardiomyocytes from hESC/hiPSC differentiation cultures, yielding populations of up to 98% cardiac troponin T-positive cells. When plated in culture, SIRPA-positive cells were contracting and could be maintained over extended periods of time. These findings provide a simple method for isolating populations of cardiomyocytes from human pluripotent stem cell cultures, and thereby establish a readily adaptable technology for generating large numbers of enriched cardiomyocytes for therapeutic applications.

Integrated Immunotherapy for Breast Cancer

DTIC Science & Technology

2015-09-01

patterns in these reconstructed co-cultured cancer cell /stromal cell 3D organoids (Figure 2). The role of mesenchymal stem cells in cancer Bone...marrow-derived mesenchymal stem cells (MSC) have been the subject of interest in solid tumor. Because of their ability to migrate to sites of inflammation...10 Figure 3. Characterization of ex-vivo expanded C57 B6 derived bone marrow mesenchymal stem cells . The cells are positive for CD44, CD140β

SOX10-positive cells emerge in the rat pituitary gland during late embryogenesis and start to express S100β.

PubMed

Ueharu, Hiroki; Yoshida, Saishu; Kanno, Naoko; Horiguchi, Kotaro; Nishimura, Naoto; Kato, Takako; Kato, Yukio

2018-04-01

In the pituitary gland, S100β-positive cells localize in the neurohypophysis and adenohypophysis but the lineage of the two groups remains obscure. S100β is often observed in many neural crest-derived cell types. Therefore, in this study, we investigate the origin of pituitary S100β-positive cells by immunohistochemistry for SOX10, a potent neural crest cell marker, using S100β-green fluorescence protein-transgenic rats. On embryonic day 21.5, a SOX10-positive cell population, which was also positive for the stem/progenitor cell marker SOX2, emerged in the pituitary stalk and posterior lobe and subsequently expanded to create a rostral-caudal gradient on postnatal day 3 (P3). Thereafter, SOX10-positive cells appeared in the intermediate lobe by P15, localizing to the boundary facing the posterior lobe, the gap between the lobule structures and the marginal cell layer, a pituitary stem/progenitor cell niche. Subsequently, there was an increase in SOX10/S100β double-positive cells; some of these cells in the gap between the lobule structures showed extended cytoplasm containing F-actin, indicating a feature of migration activity. The proportion of SOX10-positive cells in the postnatal anterior lobe was lower than 0.025% but about half of them co-localized with the pituitary-specific progenitor cell marker PROP1. Collectively, the present study identified that one of the lineages of S100β-positive cells is a SOX10-positive one and that SOX10-positive cells express pituitary stem/progenitor cell marker genes.

Epithelial-mesenchymal transition transcription factors control pluripotent adult stem cell migration in vivo in planarians

PubMed Central

Abnave, Prasad; Aboukhatwa, Ellen; Kosaka, Nobuyoshi; Thompson, James; Hill, Mark A.

2017-01-01

Migration of stem cells underpins the physiology of metazoan animals. For tissues to be maintained, stem cells and their progeny must migrate and differentiate in the correct positions. This need is even more acute after tissue damage by wounding or pathogenic infection. Inappropriate migration also underpins metastasis. Despite this, few mechanistic studies address stem cell migration during repair or homeostasis in adult tissues. Here, we present a shielded X-ray irradiation assay that allows us to follow stem cell migration in planarians. We demonstrate the use of this system to study the molecular control of stem cell migration and show that snail-1, snail-2 and zeb-1 EMT transcription factor homologs are necessary for cell migration to wound sites and for the establishment of migratory cell morphology. We also observed that stem cells undergo homeostatic migration to anterior regions that lack local stem cells, in the absence of injury, maintaining tissue homeostasis. This requires the polarity determinant notum. Our work establishes planarians as a suitable model for further in-depth study of the processes controlling stem cell migration in vivo. PMID:28893948

Phosphorylation of Smad2/3 at the specific linker threonine residue indicates slow-cycling esophageal stem-like cells before re-entry to the cell cycle.

PubMed

Takahashi, Y; Fukui, T; Kishimoto, M; Suzuki, R; Mitsuyama, T; Sumimoto, K; Okazaki, T; Sakao, M; Sakaguchi, Y; Yoshida, K; Uchida, K; Nishio, A; Matsuzaki, K; Okazaki, K

2016-01-01

The stem cell compartment in the esophageal epithelium is possibly located in the basal layer. We have identified significant expression of Smad2/3, phosphorylated at specific linker threonine residues (pSmad2/3L-Thr), in the epithelial cells of murine stomach and intestine, and have suggested that these cells are epithelial stem cells. In this study, we explore whether pSmad2/3L-Thr could serve as a biomarker for esophageal stem cells. We examined esophageal tissues from normal C57BL/6 mice and those with esophagitis. Double immunofluorescent staining of pSmad2/3L-Thr with Ki67, CDK4, p63, or CK14 was performed. After immunofluorescent staining, we stained the same sections with hematoxylin-eosin and observed these cells under a light microscope. We used the 5-bromo-2-deoxyuridine (BrdU) labeling assay to examine label retention of pSmad2/3L-Thr immunostaining-positive cells. We collected specimens 5, 10, 15 and 20 days after repeated BrdU administrations and observed double immunofluorescent staining of pSmad2/3L-Thr with BrdU. In the esophagus, pSmad2/3L-Thr immunostaining-positive cells were detected in the basal layer. These cells were detected between Ki67 immunostaining-positive cells, but they were not co-localized with Ki67. pSmad2/3L-Thr immunostaining-positive cells showed co-localization with CDK4, p63, and CK14. Under a light microscope, pSmad2/3L-Thr immunostaining-positive cells indicated undifferentiated morphological features. Until 20 days follow-up period, pSmad2/3L-Thr immunostaining-positive cells were co-localized with BrdU. pSmad2/3L-Thr immunostaining-positive cells significantly increased in the regeneration phase of esophagitis mucosae, as compared with control mice (esophagitis vs. 6.889 ± 0.676/cm vs. 4.293 ± 0.659/cm; P < 0.001). We have identified significant expression of pSmad2/3L-Thr in the specific epithelial cells of murine esophagi. We suggest that these cells are slow-cycling epithelial stem-like cells before re-entry to the cell cycle. © 2016 International Society for Diseases of the Esophagus.

Reduced hematopoietic stem cell frequency predicts outcome in acute myeloid leukemia.

PubMed

Wang, Wenwen; Stiehl, Thomas; Raffel, Simon; Hoang, Van T; Hoffmann, Isabel; Poisa-Beiro, Laura; Saeed, Borhan R; Blume, Rachel; Manta, Linda; Eckstein, Volker; Bochtler, Tilmann; Wuchter, Patrick; Essers, Marieke; Jauch, Anna; Trumpp, Andreas; Marciniak-Czochra, Anna; Ho, Anthony D; Lutz, Christoph

2017-09-01

In patients with acute myeloid leukemia and low percentages of aldehyde-dehydrogenase-positive cells, non-leukemic hematopoietic stem cells can be separated from leukemic cells. By relating hematopoietic stem cell frequencies to outcome we detected poor overall- and disease-free survival of patients with low hematopoietic stem cell frequencies. Serial analysis of matched diagnostic and follow-up samples further demonstrated that hematopoietic stem cells increased after chemotherapy in patients who achieved durable remissions. However, in patients who eventually relapsed, hematopoietic stem cell numbers decreased dramatically at the time of molecular relapse demonstrating that hematopoietic stem cell levels represent an indirect marker of minimal residual disease, which heralds leukemic relapse. Upon transplantation in immune-deficient mice cases with low percentages of hematopoietic stem cells of our cohort gave rise to leukemic or no engraftment, whereas cases with normal hematopoietic stem cell levels mostly resulted in multi-lineage engraftment. Based on our experimental data, we propose that leukemic stem cells have increased niche affinity in cases with low percentages of hematopoietic stem cells. To validate this hypothesis, we developed new mathematical models describing the dynamics of healthy and leukemic cells under different regulatory scenarios. These models suggest that the mechanism leading to decreases in hematopoietic stem cell frequencies before leukemic relapse must be based on expansion of leukemic stem cells with high niche affinity and the ability to dislodge hematopoietic stem cells. Thus, our data suggest that decreasing numbers of hematopoietic stem cells indicate leukemic stem cell persistence and the emergence of leukemic relapse. Copyright© 2017 Ferrata Storti Foundation.

Characterization of cell types during rat liver development.

PubMed

Fiegel, Henning C; Park, Jonas J h; Lioznov, Michael V; Martin, Andreas; Jaeschke-Melli, Stefan; Kaufmann, Peter M; Fehse, Boris; Zander, Axel R; Kluth, Dietrich

2003-01-01

Hepatic stem cells have been identified in adult liver. Recently, the origin of hepatic progenitors and hepatocytes from bone marrow was demonstrated. Hematopoietic and hepatic stem cells share the markers CD 34, c-kit, and Thy1. Little is known about liver stem cells during liver development. In this study, we investigated the potential stem cell marker Thy1 and hepatocytic marker CK-18 during liver development to identify putative fetal liver stem cell candidates. Livers were harvested from embryonic and fetal day (ED) 16, ED 18, ED 20, and neonatal ED 22 stage rat fetuses from Sprague-Dawley rats. Fetal livers were digested by collagenase-DNAse solution and purified by percoll centrifugation. Magnetic cell sorting (MACS) depletion of fetal liver cells was performed using OX43 and OX44 antibodies. Cells were characterized by immunocytochemistry for Thy1, CK-18, and proliferating cell antigen Ki-67 and double labeling for Thy1 and CK-18. Thy1 expression was found at all stages of liver development before and after MACS in immunocytochemistry. Thy1 positive cells were enriched after MACS only in early developmental stages. An enrichment of CK-18 positive cells was found after MACS at all developmental stages. Cells coexpressing Thy1 and CK-18 were identified by double labeling of fetal liver cell isolates. In conclusion, hepatic progenitor cells (CK-18 positive) in fetal rat liver express Thy1. Other progenitors express only CK-18. This indicates the coexistence of different hepatic cell compartments. Isolation and further characterization of such cells is needed to demonstrate their biologic properties.

The influence of the donor-recipient relationship on related donor reactions to stem cell donation.

PubMed

Labott, S; Pfammatter, A

2014-06-01

Previous research has begun to delineate the complicated reactions experienced by bone marrow and stem cell donors. The purpose of this study was to examine the influence of the donor-recipient relationship on the related donor's emotional reactions. Twenty-eight adult stem cell donors completed questionnaires before donation, 30 days post stem cell infusion, and 1 year after infusion. Questionnaires addressed the donor-recipient relationship, depression, mood, guilt and responsibility, self-esteem, ambivalence about donation and reactions to the donation itself. Results indicated that most donors reported little ambivalence about donation, and their reactions to the donation itself were generally positive. Closer and more positive donor-recipient relationships were associated with less anticipated guilt and responsibility if the transplant did not work. The relationships between the donor and the recipient did not change over time. Mood disturbance and depression were low overall, not related to the donor-recipient relationship, and did not significantly change over time. These results indicate that related stem cell donors are generally without significant emotional distress, and are comfortable with the donation process. Further, a more positive relationship with the recipient may help donors to avoid feeling guilty and responsible if the transplant does not work.

The Implications of the Cancer Stem Cell Hypothesis for Neuro-Oncology and Neurology.

PubMed

Rich, Jeremy N

2008-05-01

The cancer stem cell hypothesis posits that cancers contain a subset of neoplastic cells that propagate and maintain tumors through sustained self-renewal and potent tumorigenecity. Recent excitement has been generated by a number of reports that have demonstrated the existence of cancer stem cells in several types of brain tumors. Brain cancer stem cells - also called tumor initiating cells or tumor propagating cells - share features with normal neural stem cells but do not necessarily originate from stem cells. Although most cancers have only a small fraction of cancer stem cells, these tumor cells have been shown in laboratory studies to contribute to therapeutic resistance, formation of new blood vessels to supply the tumor, and tumor spread. As malignant brain tumors rank among the deadliest of all neurologic diseases, the identification of new cellular targets may have profound implications in neuro-oncology. Novel drugs that target stem cell pathways active in brain tumors have been efficacious against cancer stem cells suggesting that anti-cancer stem cell therapies may advance brain tumor therapy. The cancer stem cell hypothesis may have several implications for other neurologic diseases as caution must be exercised in activating stem cell maintenance pathways in cellular therapies for neurodegenerative diseases. The ability for a small fraction of cells to determine the overall course of a disease may also inform new paradigms of disease that may translate into improved patient outcomes.

S100β-Positive Cells of Mesenchymal Origin Reside in the Anterior Lobe of the Embryonic Pituitary Gland.

PubMed

Horiguchi, Kotaro; Yako, Hideji; Yoshida, Saishu; Fujiwara, Ken; Tsukada, Takehiro; Kanno, Naoko; Ueharu, Hiroki; Nishihara, Hiroto; Kato, Takako; Yashiro, Takashi; Kato, Yukio

2016-01-01

The anterior and intermediate lobes of the pituitary gland develop through invagination of the oral ectoderm and as they are endocrine tissues, they participate in the maintenance of vital functions via the synthesis and secretion of numerous hormones. We recently observed that several extrapituitary cells invade the anterior lobe of the developing pituitary gland. This raised the question of the origin(s) of these S100β-positive cells, which are not classic endocrine cells but instead comprise a heterogeneous cell population with plural roles, especially as stem/progenitor cells. To better understand the roles of these S100β-positive cells, we performed immunohistochemical analysis using several markers in S100β/GFP-TG rats, which express GFP in S100β-expressing cells under control of the S100β promoter. GFP-positive cells were present as mesenchymal cells surrounding the developing pituitary gland and at Atwell's recess but were not present in the anterior lobe on embryonic day 15.5. These cells were negative for SOX2, a pituitary stem/progenitor marker, and PRRX1, a mesenchyme and pituitary stem/progenitor marker. However, three days later, GFP-positive and PRRX1-positive (but SOX2-negative) cells were observed in the parenchyma of the anterior lobe. Furthermore, some GFP-positive cells were positive for vimentin, p75, isolectin B4, DESMIN, and Ki67. These data suggest that S100β-positive cells of extrapituitary origin invade the anterior lobe, undergoing proliferation and diverse transformation during pituitary organogenesis.

S100β-Positive Cells of Mesenchymal Origin Reside in the Anterior Lobe of the Embryonic Pituitary Gland

PubMed Central

Yoshida, Saishu; Fujiwara, Ken; Tsukada, Takehiro; Kanno, Naoko; Ueharu, Hiroki; Nishihara, Hiroto; Kato, Takako; Yashiro, Takashi; Kato, Yukio

2016-01-01

The anterior and intermediate lobes of the pituitary gland develop through invagination of the oral ectoderm and as they are endocrine tissues, they participate in the maintenance of vital functions via the synthesis and secretion of numerous hormones. We recently observed that several extrapituitary cells invade the anterior lobe of the developing pituitary gland. This raised the question of the origin(s) of these S100β-positive cells, which are not classic endocrine cells but instead comprise a heterogeneous cell population with plural roles, especially as stem/progenitor cells. To better understand the roles of these S100β-positive cells, we performed immunohistochemical analysis using several markers in S100β/GFP-TG rats, which express GFP in S100β-expressing cells under control of the S100β promoter. GFP-positive cells were present as mesenchymal cells surrounding the developing pituitary gland and at Atwell's recess but were not present in the anterior lobe on embryonic day 15.5. These cells were negative for SOX2, a pituitary stem/progenitor marker, and PRRX1, a mesenchyme and pituitary stem/progenitor marker. However, three days later, GFP-positive and PRRX1-positive (but SOX2-negative) cells were observed in the parenchyma of the anterior lobe. Furthermore, some GFP-positive cells were positive for vimentin, p75, isolectin B4, DESMIN, and Ki67. These data suggest that S100β-positive cells of extrapituitary origin invade the anterior lobe, undergoing proliferation and diverse transformation during pituitary organogenesis. PMID:27695124

Nestin- and doublecortin-positive cells reside in adult spinal cord meninges and participate in injury-induced parenchymal reaction.

PubMed

Decimo, Ilaria; Bifari, Francesco; Rodriguez, Francisco Javier; Malpeli, Giorgio; Dolci, Sissi; Lavarini, Valentina; Pretto, Silvia; Vasquez, Sandra; Sciancalepore, Marina; Montalbano, Alberto; Berton, Valeria; Krampera, Mauro; Fumagalli, Guido

2011-12-01

Adult spinal cord has little regenerative potential, thus limiting patient recovery following injury. In this study, we describe a new population of cells resident in the adult rat spinal cord meninges that express the neural stem/precursor markers nestin and doublecortin. Furthermore, from dissociated meningeal tissue a neural stem cell population was cultured in vitro and subsequently shown to differentiate into functional neurons or mature oligodendrocytes. Proliferation rate and number of nestin- and doublecortin-positive cells increased in vivo in meninges following spinal cord injury. By using a lentivirus-labeling approach, we show that meningeal cells, including nestin- and doublecortin-positive cells, migrate in the spinal cord parenchyma and contribute to the glial scar formation. Our data emphasize the multiple roles of meninges in the reaction of the parenchyma to trauma and indicate for the first time that spinal cord meninges are potential niches harboring stem/precursor cells that can be activated by injury. Meninges may be considered as a new source of adult stem/precursor cells to be further tested for use in regenerative medicine applied to neurological disorders, including repair from spinal cord injury. Copyright © 2011 AlphaMed Press.

Clinical application of adipose stem cells in plastic surgery.

PubMed

Kim, Yong-Jin; Jeong, Jae-Ho

2014-04-01

Adipose stem cells (ASCs) are a type of adult stem cells that share common characteristics with typical mesenchymal stem cells. In the last decade, ASCs have been shown to be a useful cell resource for tissue regeneration. The major role of regenerative medicine in this century is based on cell therapy in which ASCs hold a key position. Active research on this new type of adult stem cell has been ongoing and these cells now have several clinical applications, including fat grafting, overcoming wound healing difficulties, recovery from local tissue ischemia, and scar remodeling. The application of cultured cells will increase the efficiency of cell therapy. However, the use of cultured stem cells is strictly controlled by government regulation to ensure patient safety. Government regulation is a factor that can limit more versatile clinical application of ASCs. In this review, current clinical applications of ASCs in plastic surgery are introduced. Future stem cell applications in clinical field including culturing and banking of ASCs are also discussed in this review.

DMRT1 Is Required for Mouse Spermatogonial Stem Cell Maintenance and Replenishment.

PubMed

Zhang, Teng; Oatley, Jon; Bardwell, Vivian J; Zarkower, David

2016-09-01

Male mammals produce sperm for most of postnatal life and therefore require a robust germ line stem cell system, with precise balance between self-renewal and differentiation. Prior work established doublesex- and mab-3-related transcription factor 1 (Dmrt1) as a conserved transcriptional regulator of male sexual differentiation. Here we investigate the role of Dmrt1 in mouse spermatogonial stem cell (SSC) homeostasis. We find that Dmrt1 maintains SSCs during steady state spermatogenesis, where it regulates expression of Plzf, another transcription factor required for SSC maintenance. We also find that Dmrt1 is required for recovery of spermatogenesis after germ cell depletion. Committed progenitor cells expressing Ngn3 normally do not contribute to SSCs marked by the Id4-Gfp transgene, but do so when spermatogonia are chemically depleted using busulfan. Removal of Dmrt1 from Ngn3-positive germ cells blocks the replenishment of Id4-GFP-positive SSCs and recovery of spermatogenesis after busulfan treatment. Our data therefore reveal that Dmrt1 supports SSC maintenance in two ways: allowing SSCs to remain in the stem cell pool under normal conditions; and enabling progenitor cells to help restore the stem cell pool after germ cell depletion.

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

Whittaker, Peter A.

A brief outline of stem cells, stem cell therapy and therapeutic cloning is given. The position of therapeutic cloning with regard to other embryonic manipulations - IVF-based reproduction, embryonic stem formation from IVF embryos and reproductive cloning - is indicated. The main ethically challenging stages in therapeutic cloning are considered to be the nuclear transfer process including the source of eggs for this and the destruction of an embryo to provide stem cells for therapeutic use. The extremely polarised nature of the debate regarding the status of an early human embryo is noted, and some potential alternative strategies for preparingmore » immunocompatible pluripotent stem cells are indicated.« less

Effects of neurotrophin-3 on the differentiation of neural stem cells into neurons and oligodendrocytes

PubMed Central

Zhu, Guowei; Sun, Chongran; Liu, Weiguo

2012-01-01

In this study, cells from the cerebral cortex of fetal rats at pregnant 16 days were harvested and cultured with 20 μg/L neurotrophin-3. After 7 days of culture, immunocytochemical staining showed that, 22.4% of cells were positive for nestin, 10.5% were positive for β-III tubulin (neuronal marker), and 60.6% were positive for glial fibrillary acidic protein, but no cells were positive for O4 (oligodendrocytic marker). At 14 days, there were 5.6% nestin-, 9.6% β-III tubulin-, 81.1% glial fibrillary acidic protein-, and 2.2% O4-positive cells. In cells not treated with neurotrophin-3, some were nestin-positive, while the majority showed positive staining for glial fibrillary acidic protein. Our experimental findings indicate that neurotrophin-3 is a crucial factor for inducing neural stem cells differentiation into neurons and oligodendrocytes. PMID:25657683

Gastroesophageal reflux disease and its association with bronchiolitis obliterans syndrome in allogeneic hematopoietic stem cell transplant recipients.

PubMed

Khalid, Mohammed; Aljurf, Mahmoud; Saleemi, Sarfraz; Khan, Mohammed Qaseem; Khan, Basha; Ahmed, Shad; Ibrahim, Khalid El Tayeb; Mobeireek, Abdullah; Al Mohareb, Fahad; Chaudhri, Naeem

2013-06-01

Bronchiolitis obliterans syndrome is a significant postallogeneic hematopoietic stem cell transplant problem. Recent data in lung transplant patients suggest an association with gastroesophageal reflux disease and bronchiolitis obliterans syndrome. We studied posthematopoietic stem cell transplant patients with bronchiolitis obliterans syndrome for gastroesophageal reflux disease and its response to a proton pump inhibitor. Seven postallogeneic hematopoietic stem cell transplant patients with bronchiolitis obliterans syndrome were studied. Gastroesophageal reflux disease was assessed by 24-hour pH monitoring with a Bravo catheter-free radio pH capsule. Patients with positive gastroesophageal reflux disease were started on omeprazole. Pretreatment and posttreatment pulmonary function tests were done at 3-month intervals. Of 7 patients, 5 had positive results for gastroesophageal reflux disease (71%). Omeprazole had a disease-stabilizing effect on the patients' pulmonary function tests. Our study shows a significant association between bronchiolitis obliterans syndrome and gastroesophageal reflux disease in postallogeneic hematopoietic stem cell transplant patients. Use of omeprazole may have a disease-stabilizing effect in short-term follow-up.

Social media & stem cell science: examining the discourse.

PubMed

Adams, Amy; Lomax, Geoffrey; Santarini, Anthony

2011-11-01

Research suggests that the representation of scientific and medical issues in the traditional media such as newspapers, TV and radio is an important determinant of public opinion and related public policy outcomes, particularly with regard to attitudes toward stem cell research. With the emergence of social media, the discursive space around public policy issues has expanded to include a new demographic of media consumer who is directly involved in political action. However, little is known about the influence of social media on scientific public policy conversations. We analyzed Twitter posts on two topics relating to stem cell science and policy according to the originator and tone of the tweet, and whether the tweet was intended to be neutral or to further a stated policy position. This analysis provides a means for clarifying the role of social media in influencing public opinion of policy issues such as stem cell research and offers organizations a better understanding of how to more effectively apply social media to advancing their stem cell policy positions.

Isolation of Small SSEA-4-Positive Putative Stem Cells from the Ovarian Surface Epithelium of Adult Human Ovaries by Two Different Methods

PubMed Central

Virant-Klun, Irma; Skutella, Thomas; Hren, Matjaz; Gruden, Kristina; Cvjeticanin, Branko; Vogler, Andrej; Sinkovec, Jasna

2013-01-01

The adult ovarian surface epithelium has already been proposed as a source of stem cells and germinal cells in the literature, therefore it has been termed the “germinal epithelium”. At present more studies have confirmed the presence of stem cells expressing markers of pluripotency in adult mammalian ovaries, including humans. The aim of this study was to isolate a population of stem cells, based on the expression of pluripotency-related stage-specific embryonic antigen-4 (SSEA-4) from adult human ovarian surface epithelium by two different methods: magnetic-activated cell sorting and fluorescence-activated cell sorting. Both methods made it possible to isolate a similar, relatively homogenous population of small, SSEA-4-positive cells with diameters of up to 4 μm from the suspension of cells retrieved by brushing of the ovarian cortex biopsies in reproductive-age and postmenopausal women and in women with premature ovarian failure. The immunocytochemistry and genetic analyses revealed that these small cells—putative stem cells—expressed some primordial germ cell and pluripotency-related markers and might be related to the in vitro development of oocyte-like cells expressing some oocyte-specific transcription factors in the presence of donated follicular fluid with substances important for oocyte growth and development. The stemness of these cells needs to be further researched. PMID:23509763

CD44 staining of cancer stem-like cells is influenced by down-regulation of CD44 variant isoforms and up-regulation of the standard CD44 isoform in the population of cells that have undergone epithelial-to-mesenchymal transition.

PubMed

Biddle, Adrian; Gammon, Luke; Fazil, Bilal; Mackenzie, Ian C

2013-01-01

CD44 is commonly used as a cell surface marker of cancer stem-like cells in epithelial tumours, and we have previously demonstrated the existence of two different CD44(high) cancer stem-like cell populations in squamous cell carcinoma, one having undergone epithelial-to-mesenchymal transition and the other maintaining an epithelial phenotype. Alternative splicing of CD44 variant exons generates a great many isoforms, and it is not known which isoforms are expressed on the surface of the two different cancer stem-like cell phenotypes. Here, we demonstrate that cancer stem-like cells with an epithelial phenotype predominantly express isoforms containing the variant exons, whereas the cancer stem-like cells that have undergone an epithelial-to-mesenchymal transition down-regulate these variant isoforms and up-regulate expression of the standard CD44 isoform that contains no variant exons. In addition, we find that enzymatic treatments used to dissociate cells from tissue culture or fresh tumour specimens cause destruction of variant CD44 isoforms at the cell surface whereas expression of the standard CD44 isoform is preserved. This results in enrichment within the CD44(high) population of cancer stem-like cells that have undergone an epithelial-to-mesenchymal transition and depletion from the CD44(high) population of cancer stem-like cells that maintain an epithelial phenotype, and therefore greatly effects the characteristics of any cancer stem-like cell population isolated based on expression of CD44. As well as effecting the CD44(high) population, enzymatic treatment also reduces the percentage of the total epithelial cancer cell population staining CD44-positive, with potential implications for studies that aim to use CD44-positive staining as a prognostic indicator. Analyses of the properties of cancer stem-like cells are largely dependent on the ability to accurately identify and assay these populations. It is therefore critical that consideration be given to use of multiple cancer stem-like cell markers and suitable procedures for cell isolation in order that the correct populations are assayed.

Evaluation of the secretion and release of vascular endothelial growth factor from two-dimensional culture and three-dimensional cell spheroids formed with stem cells and osteoprecursor cells.

PubMed

Lee, Hyunjin; Lee, Sung-Il; Ko, Youngkyung; Park, Jun-Beom

2018-05-18

Co-culture has been applied in cell therapy, including stem cells, and has been reported to give enhanced functionality. In this study, stem-cell spheroids were formed in concave micromolds at different ratios of stem cells to osteoprecursor cells, and the amount of secretion of vascular endothelial growth factor (VEGF) was evaluated. Gingiva-derived stem cells and osteoprecursor cells in the amount of 6 × 105 were seeded on a 24-well culture plate or concave micromolds. The ratios of stem cells to osteoprecursor cells included: 0:4 (group 1), 1:3 (group 2), 2:2 (group 3), 3:1 (group 4), and 4:0 (group 5). The morphology of cells in a 2-dimensional culture (groups 1-5) showed a fibroblast-like appearance. The secretion of VEGF increased with the increase in stem cells, and a statistically significant increase was noted in groups 3, 4 and 5 when compared with the media-only group (p < 0.05). Osteoprecursor cells formed spheroids in concave microwells, and no noticeable change in the morphology was noted with the increase in stem cells. Spheroids containing stem cells were positive for the stem-cell markers SSEA-4. The secretion of VEGF from cell spheroids increased with the increase in stem cells. This study showed that cell spheroids formed with stem cells and osteoprecursor cells with different ratios, using microwells, had paracrine effects on the stem cells. The secretion of VEGF increased with the increase in stem cells. This stem-cell spheroid may be applied for tissue-engineering purposes.

Chemotherapy-Induced Depletion of OCT4-Positive Cancer Stem Cells in a Mouse Model of Malignant Testicular Cancer.

PubMed

Pierpont, Timothy M; Lyndaker, Amy M; Anderson, Claire M; Jin, Qiming; Moore, Elizabeth S; Roden, Jamie L; Braxton, Alicia; Bagepalli, Lina; Kataria, Nandita; Hu, Hilary Zhaoxu; Garness, Jason; Cook, Matthew S; Capel, Blanche; Schlafer, Donald H; Southard, Teresa; Weiss, Robert S

2017-11-14

Testicular germ cell tumors (TGCTs) are among the most responsive solid cancers to conventional chemotherapy. To elucidate the underlying mechanisms, we developed a mouse TGCT model featuring germ cell-specific Kras activation and Pten inactivation. The resulting mice developed malignant, metastatic TGCTs composed of teratoma and embryonal carcinoma, the latter of which exhibited stem cell characteristics, including expression of the pluripotency factor OCT4. Consistent with epidemiological data linking human testicular cancer risk to in utero exposures, embryonic germ cells were susceptible to malignant transformation, whereas adult germ cells underwent apoptosis in response to the same oncogenic events. Treatment of tumor-bearing mice with genotoxic chemotherapy not only prolonged survival and reduced tumor size but also selectively eliminated the OCT4-positive cancer stem cells. We conclude that the chemosensitivity of TGCTs derives from the sensitivity of their cancer stem cells to DNA-damaging chemotherapy. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

In delicate balance: stem cells and spinal cord injury advocacy.

PubMed

Parke, Sara; Illes, Judy

2011-09-01

Spinal cord injury (SCI) is a major focus for stem cell therapy (SCT). However, the science of SCT has not been well matched with an understanding of perspectives of persons with SCI. The online advocacy community is a key source of health information for primary stakeholders and their caregivers. In this study, we sought to characterize the content of SCI advocacy websites with respect to their discussion of SCT and stem cell tourism. We performed a comprehensive analysis of SCI advocacy websites identified through a web search and verified by expert opinion. Two independent researchers coded the information for major themes (e.g., scientific & clinical facts, research & funding, policy, ethics) and valence (positive, negative, balanced, neutral). Of the 40 SCI advocacy websites that met inclusion criteria, 50% (N=20) contained information about SCT. Less than 18% (N=7) contained information on stem cell tourism. There were more than ten times as many statements about SCT with a positive valence (N=67) as with a negative valence (N=6). Ethics-related SCT information comprised 20% (N=37) of the total content; the largest proportion of ethics-related content was devoted to stem cell tourism (80%, N=30 statements). Of those, the majority focused on the risks of stem cell tourism (N=16). Given the still-developing science behind SCT, the presence of cautionary information about stem cell tourism at advocacy sites is ethically appropriate. The absence of stem cell tourism information at the majority of advocacy sites represents a lost educational opportunity.

Isolation and evaluation of dental pulp stem cells from teeth with advanced periodontal disease.

PubMed

Derakhshani, Ali; Raoof, Maryam; Dabiri, Shahriar; Farsinejad, Ali Reza; Gorjestani, Hedayat; Yaghoobi, Mohammad Mehdi; Shokouhinejad, Noushin; Ehsani, Maryam

2015-04-01

Successful isolation of mesenchymal stem cells from waste tissues might be extremely promising for developing stem cell-based therapies. This study aimed to explore whether cells retrieved from teeth extracted due to advanced periodontal disease present mesenchymal stem cell-like properties. Pulp cells were isolated from 15 intact molars and 15 teeth with advanced periodontal disease. Cell proliferation and markers of mesenchymal stem cells were evaluated. Based on the RT-PCR and agarose gel electrophoresis, nucleostemin, Oct-4 and jmj2c, but not Nanog, were expressed in undifferentiated mesenchymal stem cells of both groups. Interestingly, diseased pulp exhibited higher gene expressions although it was not statistically significant. The average percentage of BrdU positive cells in the diseased group (84.4%, n = 5) was significantly higher than that of the control group (65.4%, n = 5) (t-test, P = 0.001). Our results indicate the successful isolation of mesenchymal stem cells from the pulp tissue of hopeless periodontally involved teeth.

Our Fat Future: Translating Adipose Stem Cell Therapy.

PubMed

Nordberg, Rachel C; Loboa, Elizabeth G

2015-09-01

Human adipose stem cells (hASCs) have the potential to treat patients with a variety of clinical conditions. Recent advancements in translational research, regulatory policy, and industry have positioned hASCs on the threshold of clinical translation. We discuss the progress and challenges of bringing adipose stem cell therapy into mainstream clinical use. This article details the advances made in recent years that have helped move human adipose stem cell therapy toward mainstream clinical use from a translational research, regulatory policy, and industrial standpoint. Four recurrent themes in translational technology as they pertain to human adipose stem cells are discussed: automated closed-system operations, biosensors and real-time monitoring, biomimetics, and rapid manufacturing. In light of recent FDA guidance documents, regulatory concerns about adipose stem cell therapy are discussed. Finally, an update is provided on the current state of clinical trials and the emerging industry that uses human adipose stem cells. This article is expected to stimulate future studies in translational adipose stem cell research. ©AlphaMed Press.

Neutral competition of stem cells is skewed by proliferative changes downstream of Hh and Hpo.

PubMed

Amoyel, Marc; Simons, Benjamin D; Bach, Erika A

2014-10-16

Neutral competition, an emerging feature of stem cell homeostasis, posits that individual stem cells can be lost and replaced by their neighbors stochastically, resulting in chance dominance of a clone at the niche. A single stem cell with an oncogenic mutation could bias this process and clonally spread the mutation throughout the stem cell pool. The Drosophila testis provides an ideal system for testing this model. The niche supports two stem cell populations that compete for niche occupancy. Here, we show that cyst stem cells (CySCs) conform to the paradigm of neutral competition and that clonal deregulation of either the Hedgehog (Hh) or Hippo (Hpo) pathway allows a single CySC to colonize the niche. We find that the driving force behind such behavior is accelerated proliferation. Our results demonstrate that a single stem cell colonizes its niche through oncogenic mutation by co-opting an underlying homeostatic process. © 2014 The Authors.

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

[The use of embryonic stem cells for medical-therapeutical purposes: a study of attitudes among Icelandic physicians, lawyers and clergymen.].

PubMed

Oskarsson, Trausti; Guðmundsson, Flóki; Sigurðsson, Jóhann Agúst; Getz, Linn; Arnason, Vilhjálmur

2003-06-01

To study the bioethical standpoints among three groups of Icelandic professionals in relation to the use of embryonic stem cells for medical-therapeutical purposes. In June 2002, a questionnaire was sent by mail to a random sample of 284 doctors and 293 lawyers, as well as all 168 practicing clergymen in Iceland. The participants' position in relation to the use of embryonic stem cells for therapeutical purposes was elicited through general questions as well as case examples. 290 questionnaires (39%) were returned. 62% of participants believed the embryo to have an ethical status superior to that of biologically comparable life forms. 20% of respondents considered its status as equal to that of a grown human being, whilst 18% considered it equal to biologically comparable primitive life forms. There was a difference between the respondent groups (p<0,05). A vast majority believed the use of embryonic stem cells for therapeutical purposes to be justifiable, although the origin of the stem cells appeared to make a difference to many respondents. 8% of participants took an unconditional position against the use of embryonic stem cells. Among those who considered the use of embryonic stem cells with a therapeutic aim to be justifiable, 71% believed that embryonic stem cells should only be utilized to treat diseases of a severe nature. 64% of participants defended the idea of therapeutic cloning with the intention to treat a patient with Parkinson's disease, but the case history elicited considerable difference between professional groups. Clergymen and lawyers tended to hold firmer attitudes, clergymen against and lawyers for the use of stem cells, whilst medical doctors as a group positioned themselves more towards the middle. Female respondents generally took a more modest stand whilst males were more likely to take a firmer stand in both directions. A vast majority (87%) of the participants believed there to be a need for public debate in relation to the use of embryonic stem cells for therapeutical purposes. Overall, participants views in relation to the use of embryonic stem cells for medical purposes were rather liberal. There were however significant differences between professional groups. The relatively high tolerance in regard to therapeutic cloning is interesting in view of the considerable controversy over this topic in many countries. There appears to be fertile ground for a public debate about the use of embryonic stem cells for medical purposes in Iceland.

Recruitment of bone marrow-derived cells to the periodontal ligament via the stromal cell-derived factor-1/C-X-C chemokine receptor type 4 axis.

PubMed

Kaku, M; Kitami, M; Rosales Rocabado, J M; Ida, T; Akiba, Y; Uoshima, K

2017-08-01

The periodontal ligament (PDL) is a non-mineralized connective tissue that exists between the alveolar bone and root surface cementum and plays important roles in tooth function. The PDL harbors a remarkable reserve of multipotent stem cells, which maintain various types of cells. However, the sources of these stem cells, other than their developmental origin, are not well understood. To elucidate the recruitment of bone marrow (BM)-derived stem cells in the PDL, green fluorescent protein (GFP)-expressing BM-derived cells were transplanted into the femoral BM of immunodeficient rats, and the distribution and expression of stem cell markers in the PDL were analyzed in vivo. To evaluate the functional significance of BM-derived cells to the PDL, tooth replantation was performed and the expression of stromal cell-derived factor (SDF)-1, a critical chemotactic signal for mesenchymal stem cell recruitment, was analyzed. To confirm the SDF-1-dependency of BM-derived cell migration to the PDL, PDL-conditioned medium (CM) was prepared, and BM-derived cell migration was analyzed using a transwell culture system. Four weeks after cell transplantation, GFP-positive cells were detected in the PDL, and some of them were also positive for stem cell markers (i.e., CD29, SSEA4, and αSMA). Seven days after tooth replantation, the number of GFP- and SDF-1-positive cells significantly increased in PDL. Concurrently, the concentration of SDF-1 and the number of colony-forming units of fibroblasts in peripheral blood were increased. BM-derived cell migration increased in PDL-CM and was inhibited by an inhibitor of C-X-C chemokine receptor type 4 (CXCR4), an SDF-1 receptor. These results indicate that stem cells and their progeny in PDL are not only derived from their developmental origin but are also supplied from the BM via the blood as the need arises. Moreover, this BM-derived cell recruitment appears to be regulated, at least partially, by the SDF-1/CXCR4 axis. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

P63 EXPRESSION LEVELS IN SIDE POPULATION AND LOW LIGHT SCATTERING OCULAR SURFACE EPITHELIAL CELLS

PubMed Central

Epstein, Seth P; Wolosin, J. Mario; Asbell, Penny A

2005-01-01

Purpose Because stem cells exhibit high self-renewal capacity, slow cycling, and high proliferative potential, and one of many markers postulated for epithelial stem cells, p63, is challenged by widespread expression within stem cell–free regions, we examined p63 expression in these stem cell–associated cohorts compared with their controls. Methods Rabbit limbocorneal cryosections, cytospun cell-sorted (by fluorescence-activated cell sorter) side population (SP) and low side scatter (LSSC) cells, and limbal epithelial cells over feeders were stained for p63 by indirect immunofluorescence. Clones were fixed and stained daily for 7 days. Image analysis measured p63 intensity, plotting it against colony size. Results All basal limbal cells were positive for p63, yet only 5% to 7% expressed high p63 intensities, 40% intermediate, and the majority low. Side population cells were less than 1% of total cells. The average intensity of SP staining was three times that of controls. Subpopulations displaying stemlike features exhibited highest p63 expression. Replication rates of isolated cells differed. Day 5 colonies contained 256 (16 hours/cycle) to two (96 hours/cycle) cells. Whereas all cells were positive for p63, intensity in slow-cycling cells was three to four times that in rapidly proliferating congeners. Increased cell doublings did not decrease fluorescence. Conclusions Results suggest that p63 concentration is maximal in stem cells and decreases with differentiation. High p63 levels seem to correlate with cells of the SP and LSSC phenotypes, indicating high cell stemness. With identification of stem cells, further studies can elucidate their use in supporting ocular surface health. PMID:17057802

HPV Infection of the Head and Neck Region and Its Stem Cells.

PubMed

Pullos, A N; Castilho, R M; Squarize, C H

2015-11-01

The human papillomavirus (HPV) is an etiologic agent associated with the development of head and neck squamous carcinoma (HNSCC)-in particular, oropharyngeal squamous cell carcinoma. The HPV-positive HNSCC is characterized by genetic alterations, clinical progression, and therapeutic response, which are distinct from HPV-negative head and neck cancers, suggesting that virus-associated tumors constitute a unique entity among head and neck cancers. Malignant stem cells, or cancer stem cells, are a subpopulation of tumor cells that self-renew, initiate new tumors upon transplantation, and are resistant to therapy, and their discovery has revealed novel effects of oncovirus infection in cancer. In this review, we provide a virus-centric view and novel insights into HPV-positive head and neck pathogenesis. We discuss the influence of cancer stem cells, HPV oncoproteins, altered molecular pathways, and mutations in cancer initiation and cancer progression. We compiled a catalogue of the mutations associated with HPV-positive HNSCC, which may be a useful resource for genomic-based studies aiming to develop personalized therapies. We also explain recent changes in mass vaccination campaigns against HPV and the potential long-term impact of vaccinations on the prevention and treatment of HPV-positive head and neck cancers. © International & American Associations for Dental Research 2015.

Large-scale production of embryonic red blood cells from human embryonic stem cells.

PubMed

Olivier, Emmanuel N; Qiu, Caihong; Velho, Michelle; Hirsch, Rhoda Elison; Bouhassira, Eric E

2006-12-01

To develop a method to produce in culture large number of erythroid cells from human embryonic stem cells. Human H1 embryonic stem cells were differentiated into hematopoietic cells by coculture with a human fetal liver cell line, and the resulting CD34-positive cells were expanded in vitro in liquid culture using a three-step method. The erythroid cells produced were then analyzed by light microscopy and flow cytometry. Globin expression was characterized by quantitative reverse-transcriptase polymerase chain reaction and by high-performance liquid chromatography. CD34-positive cells produced from human embryonic stem cells could be efficiently differentiated into erythroid cells in liquid culture leading to a more than 5000-fold increase in cell number. The erythroid cells produced are similar to primitive erythroid cells present in the yolk sac of early human embryos and did not enucleate. They are fully hemoglobinized and express a mixture of embryonic and fetal globins but no beta-globin. We have developed an experimental protocol to produce large numbers of primitive erythroid cells starting from undifferentiated human embryonic stem cells. As the earliest human erythroid cells, the nucleated primitive erythroblasts, are not very well characterized because experimental material at this stage of development is very difficult to obtain, this system should prove useful to answer a number of experimental questions regarding the biology of these cells. In addition, production of mature red blood cells from human embryonic stem cells is of great potential practical importance because it could eventually become an alternate source of cell for transfusion.

Responsible Translation of Stem Cell Research: An Assessment of Clinical Trial Registration and Publications.

PubMed

Fung, Moses; Yuan, Yan; Atkins, Harold; Shi, Qian; Bubela, Tania

2017-05-09

We assessed the extent to which the publication of clinical trial results of innovative cell-based interventions reflects International Society for Stem Cell Research best practice guidelines. We assessed: (1) characteristics and time to publication of completed trials; (2) quality of reported trials; and (3) results of published trials. We identified and analyzed publications from 1,052 novel stem cell clinical trials: 179 (45.4%) of 393 completed trials had published results; 48 trials were registered by known stem cell tourism clinics, none of which reported results. Completed non-industry-sponsored trials initially published more rapidly, but differences with industry-sponsored trials decreased over time. Most publications reported safety, and 67.3% (mainly early-stage trials) reported positive outcomes. A higher proportion of industry trials reported positive efficacy. Heightened patient expectations for stem cell therapies give rise to ethical obligations for the transparent conduct of clinical trials. Reporting guidelines need to be developed that are specific to early-phase clinical trials. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Temperature variation and distribution of living cells within tree stems: implications for stem respiration modeling and scale-up.

PubMed

Stockfors, J

2000-09-01

Few studies have examined variation in respiration rates within trees, and even fewer studies have focused on variation caused by within-stem temperature differences. In this study, stem temperatures at 40 positions in the stem of one 30-year-old Norway spruce (Picea abies (L.) Karst.) were measured during 40 days between July 1994 and June 1995. The temperature data were used to simulate variations in respiration rate within the stem. The simulations assumed that the temperature-respiration relationship was constant (Q10 = 2) for all days and all stem positions. Total respiration for the whole stem was calculated by interpolating the temperature between the thermocouples and integrating the respiration rates in three dimensions. Total respiration rate of the stem was then compared to respiration rate scaled up from horizontal planes at the thermocouple heights (40, 140, 240 and 340 cm) on a surface area and on a sapwood volume basis. Simulations were made for three distributions of living cells in the stems: one with a constant 5% fraction of living cells, disregarding depth into the stem; one with a living cell fraction decreasing linearly with depth into the stem; and one with an exponentially decreasing fraction of living cells. Mean temperature variation within the stem was 3.7 degrees C, and was more than 10 degrees C for 8% of the time. The maximum measured temperature difference was 21.5 degrees C. The corresponding mean variation in respiration was 35% and was more than 50% for 24% of the time. Scaling up respiration rates from different heights between 40 and 240 cm to the whole stem produced an error of 2 to 58% for the whole year. For a single sunny day, the error was between 2 and 72%. Thus, within-stem variations in temperature may significantly affect the accuracy of scaling respiration data obtained from small samples to whole trees. A careful choice of chamber position and basis for scaling is necessary to minimize errors from variation in temperature.

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

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.

Congenital anomalies

PubMed Central

Kunisaki, Shaun M.

2012-01-01

Over the past decade, amniotic fluid-derived stem cells have emerged as a novel, experimental approach for the treatment of a wide variety of congenital anomalies diagnosed either in utero or postnatally. There are a number of unique properties of amniotic fluid stem cells that have allowed it to become a major research focus. These include the relative ease of accessing amniotic fluid cells in a minimally invasive fashion by amniocentesis as well as the relatively rich population of progenitor cells obtained from a small aliquot of fluid. Mesenchymal stem cells, c-kit positive stem cells, as well as induced pluripotent stem cells have all been derived from human amniotic fluid in recent years. This article gives a pediatric surgeon’s perspective on amniotic fluid stem cell therapy for the management of congenital anomalies. The current status in the use of amniotic fluid-derived stem cells, particularly as they relate as substrates in tissue engineering-based applications, is described in various animal models. A roadmap for further study and eventual clinical application is also proposed. PMID:22986340

Improved Isolation, Proliferation, and Differentiation Capacity of Mouse Ovarian Putative Stem Cells.

PubMed

Yazdekhasti, Hossein; Hosseini, Marzieh Agha; Rajabi, Zahra; Parvari, Soraya; Salehnia, Mojdeh; Koruji, Morteza; Izadyar, Fariborz; Aliakbari, Fereshte; Abbasi, Mehdi

2017-04-01

The recent discovery of ovarian stem cells in postnatal mammalian ovaries, also referred to as putative stem cells (PSCs), and their roles in mammalian fertility has challenged the long-existing theory that women are endowed with a certain number of germ cells. The rare amount of PSCs is the major limitation for utilizing them through different applications. Therefore, this study was conducted in six phases to find a way to increase the number of Fragilis- and mouse vasa homolog (MVH)-positive sorted cells from 14-day-old NMRI strain mice. Results showed that there is a population of Fragilis- and MVH-positive cells with pluripotent stem cell characteristics, which can be isolated and expanded for months in vitro. PSCs increase their proliferation capacity under the influence of some mitogenic agents, and our results showed that different doses of stem cell factor (SCF) induce PSC proliferation with the maximum increase observed at 50 ng/mL. SCF was also able to increase the number of Fragilis- and MVH-positive cells after sorting by magnetic-activated cell sorting and enhance colony formation efficiency in sorted cells. Differentiation capacity assay indicated that there is a basic level of spontaneous differentiation toward oocyte-like cells during 3 days of culture. However, relative gene expression was significantly higher in the follicle-stimulating hormone-treated groups, especially in the Fragilis- sorted PSCs. We suggest that higher number of PSCs provides us either a greater source of energy that can be injected into energy-impaired oocytes in women with a history of repeat IVF failure or a good source for research.

Epithelial-mesenchymal transition transcription factors control pluripotent adult stem cell migration in vivo in planarians.

PubMed

Abnave, Prasad; Aboukhatwa, Ellen; Kosaka, Nobuyoshi; Thompson, James; Hill, Mark A; Aboobaker, A Aziz

2017-10-01

Migration of stem cells underpins the physiology of metazoan animals. For tissues to be maintained, stem cells and their progeny must migrate and differentiate in the correct positions. This need is even more acute after tissue damage by wounding or pathogenic infection. Inappropriate migration also underpins metastasis. Despite this, few mechanistic studies address stem cell migration during repair or homeostasis in adult tissues. Here, we present a shielded X-ray irradiation assay that allows us to follow stem cell migration in planarians. We demonstrate the use of this system to study the molecular control of stem cell migration and show that snail-1 , snail-2 and zeb-1 EMT transcription factor homologs are necessary for cell migration to wound sites and for the establishment of migratory cell morphology. We also observed that stem cells undergo homeostatic migration to anterior regions that lack local stem cells, in the absence of injury, maintaining tissue homeostasis. This requires the polarity determinant notum Our work establishes planarians as a suitable model for further in-depth study of the processes controlling stem cell migration in vivo . © 2017. Published by The Company of Biologists Ltd.

In vitro cementoblast-like differentiation of postmigratory neural crest-derived p75{sup +} stem cells with dental follicle cell conditioned medium

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

Wen, Xiujie; Liu, Luchuan; Deng, Manjing

Cranial neural crest-derived cells (CNCCs) play important role in epithelial–mesenchymal interactions during tooth morphogenesis. However, the heterogeneity of CNCCs and their tendency to spontaneously differentiate along smooth muscle or osteoblast lineages in vitro limit further understanding of their biological properties. We studied the differentiation properties of isolated rat embryonic postmigratory CNCCs, expressing p75 neurotrophin receptor (p75NTR). These p75NTR positive (p75{sup +}) CNCCs, isolated using fluorescence activated cell sorter, exhibited fibroblast-like morphology and characteristics of mesenchymal stem cells. Incubation of p75{sup +} CNCCs in dental follicle cell conditioned medium (DFCCM) combined with dentin non-collagenous proteins (dNCPs), altered their morphological features tomore » cementoblast-like appearance. These cells also showed low proliferative activity, high ALP activity and significantly increased calcified nodule formation. Markers related to mineralization or specific to cementoblast lineage were highly expressed in dNCPs/DFCCM-treated p75{sup +} cells, suggesting their differentiation along cementoblast-like lineage. p75{sup +} stem cells selected from postmigratory CNCCs represent a pure stem cell population and could be used as a stem cell model for in vitro studies due to their intrinsic ability to differentiate to neuronal cells and transform from neuroectoderm to ectomesenchyme. They can provide a potential stem cell resource for tooth engineering studies and help to further investigate mechanisms of epithelial–mesenchymal interactions in tooth morphogenesis. - Highlights: • Cranial neural crest-derived cells (CNCCs) take part in tooth morphogenesis. • positive (p75{sup +}) CNCCs are fibroblast-like and resemble mesenchymal stem cells. • p75{sup +} CNCCs in dental follicle cell medium (DFCCM/dNCP) appear like cementoblasts. • DFCCM/dNCP-treated p75{sup +} cells express cementoblast specific mineralization markers. • p75{sup +} cells are pure stem cells and able to differentiate to neuronal cells.« less

Differentiation of isolated human umbilical cord mesenchymal stem cells into neural stem cells

PubMed Central

Chen, Song; Zhang, Wei; Wang, Ji-Ming; Duan, Hong-Tao; Kong, Jia-Hui; Wang, Yue-Xin; Dong, Meng; Bi, Xue; Song, Jian

2016-01-01

AIM To investigate whether umbilical cord human mesenchymal stem cell (UC-MSC) was able to differentiate into neural stem cell and neuron in vitro. METHODS The umbilical cords were obtained from pregnant women with their written consent and the approval of the Clinic Ethnics Committee. UC-MSC were isolated by adherent culture in the medium contains 20% fetal bovine serum (FBS), then they were maintained in the medium contain 10% FBS and induced to neural cells in neural differentiation medium. We investigated whether UC-MSC was able to differentiate into neural stem cell and neuron in vitro by using flow cytometry, reverse transcriptase-polymerase chain reaction (RT-PCR) and immunofluorescence (IF) analyzes. RESULTS A substantial number of UC-MSC was harvested using the tissue explants adherent method at about 2wk. Flow cytometric study revealed that these cells expressed common markers of MSCs, such as CD105 (SH2), CD73 (SH3) and CD90. After induction of differentiation of neural stem cells, the cells began to form clusters; RT-PCR and IF showed that the neuron specific enolase (NSE) and neurogenic differentiation 1-positive cells reached 87.3%±14.7% and 72.6%±11.8%, respectively. Cells showed neuronal cell differentiation after induced, including neuron-like protrusions, plump cell body, obviously and stronger refraction. RT-PCR and IF analysis showed that microtubule-associated protein 2 (MAP2) and nuclear factor-M-positive cells reached 43.1%±10.3% and 69.4%±19.5%, respectively. CONCLUSION Human umbilical cord derived MSCs can be cultured and proliferated in vitro and differentiate into neural stem cells, which may be a valuable source for cell therapy of neurodegenerative eye diseases. PMID:26949608

Replacement of Lost Lgr5-Positive Stem Cells through Plasticity of Their Enterocyte-Lineage Daughters.

PubMed

Tetteh, Paul W; Basak, Onur; Farin, Henner F; Wiebrands, Kay; Kretzschmar, Kai; Begthel, Harry; van den Born, Maaike; Korving, Jeroen; de Sauvage, Frederic; van Es, Johan H; van Oudenaarden, Alexander; Clevers, Hans

2016-02-04

Intestinal crypts display robust regeneration upon injury. The relatively rare secretory precursors can replace lost stem cells, but it is unknown if the abundant enterocyte progenitors that express the Alkaline phosphate intestinal (Alpi) gene also have this capacity. We created an Alpi-IRES-CreERT2 (Alpi(CreER)) knockin allele for lineage tracing. Marked clones consist entirely of enterocytes and are all lost from villus tips within days. Genetic fate-mapping of Alpi(+) cells before or during targeted ablation of Lgr5-expressing stem cells generated numerous long-lived crypt-villus "ribbons," indicative of dedifferentiation of enterocyte precursors into Lgr5(+) stems. By single-cell analysis of dedifferentiating enterocytes, we observed the generation of Paneth-like cells and proliferative stem cells. We conclude that the highly proliferative, short-lived enterocyte precursors serve as a large reservoir of potential stem cells during crypt regeneration. Copyright © 2016 Elsevier Inc. All rights reserved.

Transfer and detection of freshly isolated or cultured chicken (Gallus gallus) and exotic species' embryonic gonadal germ stem cells in host embryos.

PubMed

Imus, Nastassja; Roe, Mandi; Charter, Suellen; Durrant, Barbara; Jensen, Thomas

2014-06-01

The management of captive avian breeding programs increasingly utilizes various artificial reproductive technologies, including in ovo sexing of embryos to adjust population sex ratios. Currently, however, no attention has been given to the loss of genetic diversity following sex-selective incubation, even with respect to individuals from critically endangered species. This project evaluated the possibility of using xenotransfer of embryonic gonadal germline stem cells (GGCs) for future reintroduction of their germplasm into the gene pool. We examined and compared the host gonad colonization of freshly isolated and 3 day (3d) cultured donor GGCs from chicken and 13 species of exotic embryos. Following 3d-culture of GGCs, there was a significant increase in the percentage of stem cell marker (SSEA-1, -3, -4) positive cells. However, the percentage of positive host gonads with chicken donor-derived cells decreased from 68% (fresh) to 22% (3d), while the percentage of exotic species donor-cells positive host gonads decreased from 61% (fresh) to 49% (3d-cultured). Donor GGCs from both chicken and exotic species were localized within the caudal endoderm, including the region encompassing the gonadal ridge by 16 hours post-injection. Furthermore, donor-derived cells isolated from stage 36 host embryos were antigenic for anti SSEA-1, VASA/DDX4 and EMA-1 antibodies, presumably indicating maintenance of stem cell identity. This study demonstrates that GGCs from multiple species can migrate to the gonadal region and maintain presumed stemness following xenotransfer into a chicken host embryo, suggesting that germline stem cell migration is highly conserved in birds.

EGFR-mediated interleukin enhancer-binding factor 3 contributes to formation and survival of cancer stem-like tumorspheres as a therapeutic target against EGFR-positive non-small cell lung cancer.

PubMed

Cheng, Chun-Chia; Chou, Kuei-Fang; Wu, Cheng-Wen; Su, Nai-Wen; Peng, Cheng-Liang; Su, Ying-Wen; Chang, Jungshan; Ho, Ai-Sheng; Lin, Huan-Chau; Chen, Caleb Gon-Shen; Yang, Bi-Ling; Chang, Yu-Cheng; Chiang, Ya-Wen; Lim, Ken-Hong; Chang, Yi-Fang

2018-02-01

YM155, an inhibitor of interleukin enhancer-binding factor 3 (ILF3), significantly suppresses cancer stemness property, implying that ILF3 contributes to cell survival of cancer stem cells. However, the molecular function of ILF3 inhibiting cancer stemness remains unclear. This study aimed to uncover the potential function of ILF3 involving in cell survival of epidermal growth factor receptor (EGFR)-positive lung stem-like cancer, and to investigate the potential role to improve the efficacy of anti-EGFR therapeutics. The association of EGFR and ILF3 in expression and regulations was first investigated in this study. Lung cancer A549 cells with deprivation of ILF3 were created by the gene-knockdown method and then RNAseq was applied to identify the putative genes regulated by ILF3. Meanwhile, HCC827- and A549-derived cancer stem-like cells were used to investigate the role of ILF3 in the formation of cancer stem-like tumorspheres. We found that EGFR induced ILF3 expression, and YM155 reduced EGFR expression. The knockdown of ILF3 reduced not only EGFR expression in mRNA and protein levels, but also cell proliferation in vitro and in vivo, demonstrating that ILF3 may play an important role in contributing to cancer cell survival. Moreover, the knockdown and inhibition of ILF3 by shRNA and YM155, respectively, reduced the formation and survival of HCC827- and A549-derived tumorspheres through inhibiting ErbB3 (HER3) expression, and synergized the therapeutic efficacy of afatinib, a tyrosine kinase inhibitor, against EGFR-positive A549 lung cells. This study demonstrated that ILF3 plays an oncogenic like role in maintaining the EGFR-mediated cellular pathway, and can be a therapeutic target to improve the therapeutic efficacy of afatinib. Our results suggested that YM155, an ILF3 inhibitor, has the potential for utilization in cancer therapy against EGFR-positive lung cancers. Copyright © 2017 Elsevier B.V. All rights reserved.

Mesenchymal and embryonic characteristics of stem cells obtained from mouse dental pulp.

PubMed

Guimarães, Elisalva Teixeira; Cruz, Gabriela Silva; de Jesus, Alan Araújo; Lacerda de Carvalho, Acácia Fernandes; Rogatto, Silvia Regina; Pereira, Lygia da Veiga; Ribeiro-dos-Santos, Ricardo; Soares, Milena Botelho Pereira

2011-11-01

Several studies have demonstrated that human dental pulp is a source of mesenchymal stem cells. To better understand the biological properties of these cells we isolated and characterized stem cells from the dental pulp of EGFP transgenic mice. The pulp tissue was gently separated from the roots of teeth extracted from C57BL/6 mice, and cultured under appropriate conditions. Flow cytometry, RT-PCR, light microscopy (staining for alkaline phosphatase) and immunofluorescence were used to investigate the expression of stem cell markers. The presence of chromosomal abnormalities was evaluated by G banding. The mouse dental pulp stem cells (mDPSC) were highly proliferative, plastic-adherent, and exhibited a polymorphic morphology predominantly with stellate or fusiform shapes. The presence of cell clusters was observed in cultures of mDPSC. Some cells were positive for alkaline phosphatase. The karyotype was normal until the 5th passage. The Pou5f1/Oct-4 and ZFP42/Rex-1, but not Nanog transcripts were detected in mDPSC. Flow cytometry and fluorescence analyses revealed the presence of a heterogeneous population positive for embryonic and mesenchymal cell markers. Adipogenic, chondrogenic and osteogenic differentiation was achieved after two weeks of cell culture under chemically defined in vitro conditions. In addition, some elongated cells spontaneously acquired a contraction capacity. Our results reinforce that the dental pulp is an important source of adult stem cells and encourage studies on therapeutic potential of mDPSC in experimental disease models. Copyright © 2011 Elsevier Ltd. All rights reserved.

Establishment of a Novel Lingual Organoid Culture System: Generation of Organoids Having Mature Keratinized Epithelium from Adult Epithelial Stem Cells

NASA Astrophysics Data System (ADS)

Hisha, Hiroko; Tanaka, Toshihiro; Kanno, Shohei; Tokuyama, Yoko; Komai, Yoshihiro; Ohe, Shuichi; Yanai, Hirotsugu; Omachi, Taichi; Ueno, Hiroo

2013-11-01

Despite the strong need for the establishment of a lingual epithelial cell culture system, a simple and convenient culture method has not yet been established. Here, we report the establishment of a novel lingual epithelium organoid culture system using a three-dimensional matrix and growth factors. Histological analyses showed that the generated organoids had both a stratified squamous epithelial cell layer and a stratum corneum. Very recently, we showed via a multicolor lineage tracing method that Bmi1-positive stem cells exist at the base of the epithelial basal layer in the interpapillary pit. Using our new culture system, we found that organoids could be generated by single Bmi1-positive stem cells and that in the established organoids, multiple Bmi1-positive stem cells were generated at the outermost layer. Moreover, we observed that organoids harvested at an early point in culture could be engrafted and maturate in the tongue of recipient mice and that the organoids generated from carcinogen-treated mice had an abnormal morphology. Thus, this culture system presents valuable settings for studying not only the regulatory mechanisms of lingual epithelium but also lingual regeneration and carcinogenesis.

Asymmetric segregation and self-renewal of hematopoietic stem and progenitor cells with endocytic Ap2a2.

PubMed

Ting, Stephen B; Deneault, Eric; Hope, Kristin; Cellot, Sonia; Chagraoui, Jalila; Mayotte, Nadine; Dorn, Jonas F; Laverdure, Jean-Philippe; Harvey, Michael; Hawkins, Edwin D; Russell, Sarah M; Maddox, Paul S; Iscove, Norman N; Sauvageau, Guy

2012-03-15

The stem cell-intrinsic model of self-renewal via asymmetric cell division (ACD) posits that fate determinants be partitioned unequally between daughter cells to either activate or suppress the stemness state. ACD is a purported mechanism by which hematopoietic stem cells (HSCs) self-renew, but definitive evidence for this cellular process remains open to conjecture. To address this issue, we chose 73 candidate genes that function within the cell polarity network to identify potential determinants that may concomitantly alter HSC fate while also exhibiting asymmetric segregation at cell division. Initial gene-expression profiles of polarity candidates showed high and differential expression in both HSCs and leukemia stem cells. Altered HSC fate was assessed by our established in vitro to in vivo screen on a subcohort of candidate polarity genes, which revealed 6 novel positive regulators of HSC function: Ap2a2, Gpsm2, Tmod1, Kif3a, Racgap1, and Ccnb1. Interestingly, live-cell videomicroscopy of the endocytic protein AP2A2 shows instances of asymmetric segregation during HSC/progenitor cell cytokinesis. These results contribute further evidence that ACD is functional in HSC self-renewal, suggest a role for Ap2a2 in HSC activity, and provide a unique opportunity to prospectively analyze progeny from HSC asymmetric divisions.

Are nestin-positive mesenchymal stromal cells a better source of cells for CNS repair?

PubMed

Lindsay, Susan L; Barnett, Susan C

2017-06-01

In recent years there has been a great deal of research within the stem cell field which has led to the definition and classification of a range of stem cells from a plethora of tissues and organs. Stem cells, by classification, are considered to be pluri- or multipotent and have both self-renewal and multi-differentiation capabilities. Presently there is a great deal of interest in stem cells isolated from both embryonic and adult tissues in the hope they hold the therapeutic key to restoring or treating damaged cells in a number of central nervous system (CNS) disorders. In this review we will discuss the role of mesenchymal stromal cells (MSCs) isolated from human olfactory mucosa, with particular emphasis on their potential role as a candidate for transplant mediated repair in the CNS. Since nestin expression defines the entire population of olfactory mucosal derived MSCs, we will compare these cells to a population of neural crest derived nestin positive population of bone marrow-MSCs. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Generation of diverse neuronal subtypes in cloned populations of stem-like cells

PubMed Central

Varga, Balázs V; Hádinger, Nóra; Gócza, Elen; Dulberg, Vered; Demeter, Kornél; Madarász, Emília; Herberth, Balázs

2008-01-01

Background The central nervous tissue contains diverse subtypes of neurons with characteristic morphological and physiological features and different neurotransmitter phenotypes. The generation of neurons with defined neurotransmitter phenotypes seems to be governed by factors differently expressed along the anterior-posterior and dorsal-ventral body axes. The mechanisms of the cell-type determination, however, are poorly understood. Selected neuronal phenotypes had been generated from embryonic stem (ES) cells, but similar results were not obtained on more restricted neural stem cells, presumably due to the lack of homogeneous neural stem cell populations as a starting material. Results In the presented work, the establishment of different neurotransmitter phenotypes was investigated in the course of in vitro induced neural differentiation of a one-cell derived neuroectodermal cell line, in conjunction with the activation of various region-specific genes. For comparison, similar studies were carried out on the R1 embryonic stem (ES) and P19 multipotent embryonic carcinoma (EC) cells. In response to a short treatment with all-trans retinoic acid, all cell lines gave rise to neurons and astrocytes. Non-induced neural stem cells and self-renewing cells persisting in differentiated cultures, expressed "stemness genes" along with early embryonic anterior-dorsal positional genes, but did not express the investigated CNS region-specific genes. In differentiating stem-like cell populations, on the other hand, different region-specific genes, those expressed in non-overlapping regions along the body axes were activated. The potential for diverse regional specifications was induced in parallel with the initiation of neural tissue-type differentiation. In accordance with the wide regional specification potential, neurons with different neurotransmitter phenotypes developed. Mechanisms inherent to one-cell derived neural stem cell populations were sufficient to establish glutamatergic and GABAergic neuronal phenotypes but failed to manifest cathecolaminergic neurons. Conclusion The data indicate that genes involved in positional determination are activated along with pro-neuronal genes in conditions excluding any outside influences. Interactions among progenies of one cell derived neural stem cells are sufficient for the activation of diverse region specific genes and initiate different routes of neuronal specification. PMID:18808670

Effect of Dedifferentiation on Time to Mutation Acquisition in Stem Cell-Driven Cancers

PubMed Central

Jilkine, Alexandra; Gutenkunst, Ryan N.

2014-01-01

Accumulating evidence suggests that many tumors have a hierarchical organization, with the bulk of the tumor composed of relatively differentiated short-lived progenitor cells that are maintained by a small population of undifferentiated long-lived cancer stem cells. It is unclear, however, whether cancer stem cells originate from normal stem cells or from dedifferentiated progenitor cells. To address this, we mathematically modeled the effect of dedifferentiation on carcinogenesis. We considered a hybrid stochastic-deterministic model of mutation accumulation in both stem cells and progenitors, including dedifferentiation of progenitor cells to a stem cell-like state. We performed exact computer simulations of the emergence of tumor subpopulations with two mutations, and we derived semi-analytical estimates for the waiting time distribution to fixation. Our results suggest that dedifferentiation may play an important role in carcinogenesis, depending on how stem cell homeostasis is maintained. If the stem cell population size is held strictly constant (due to all divisions being asymmetric), we found that dedifferentiation acts like a positive selective force in the stem cell population and thus speeds carcinogenesis. If the stem cell population size is allowed to vary stochastically with density-dependent reproduction rates (allowing both symmetric and asymmetric divisions), we found that dedifferentiation beyond a critical threshold leads to exponential growth of the stem cell population. Thus, dedifferentiation may play a crucial role, the common modeling assumption of constant stem cell population size may not be adequate, and further progress in understanding carcinogenesis demands a more detailed mechanistic understanding of stem cell homeostasis. PMID:24603301

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.

Differentiation of Human Dental Pulp Stem Cells into Dopaminergic Neuron-like Cells in Vitro.

PubMed

Chun, So Young; Soker, Shay; Jang, Yu-Jin; Kwon, Tae Gyun; Yoo, Eun Sang

2016-02-01

We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.

CBX7 regulates stem cell-like properties of gastric cancer cells via p16 and AKT-NF-κB-miR-21 pathways.

PubMed

Ni, Su-Jie; Zhao, Li-Qin; Wang, Xiao-Feng; Wu, Zhen-Hua; Hua, Rui-Xi; Wan, Chun-Hua; Zhang, Jie-Yun; Zhang, Xiao-Wei; Huang, Ming-Zhu; Gan, Lu; Sun, Hua-Lin; Dimri, Goberdhan P; Guo, Wei-Jian

2018-02-08

Chromobox protein homolog 7 (CBX7), a member of the polycomb group (PcG) family of proteins, is involved in the regulation of cell proliferation and cancer progression. PcG family members, such as BMI, Mel-18, and EZH2, are integral constituents of the polycomb repressive complexes (PRCs) and have been known to regulate cancer stem cell (CSC) phenotype. However, the role of other PRCs' constituents such as CBX7 in the regulation of CSC phenotype remains largely elusive. This study was to investigate the role of CBX7 in regulating stem cell-like properties of gastric cancer and the underlying mechanisms. Firstly, the role of CBX7 in regulating stem cell-like properties of gastric cancer was investigated using sphere formation, Western blot, and xenograft tumor assays. Next, RNA interference and ectopic CBX7 expression were employed to determine the impact of CBX7 on the expression of CSC marker proteins and CSC characteristics. The expression of CBX7, its downstream targets, and stem cell markers were analyzed in gastric stem cell spheres, common cancer cells, and gastric cancer tissues. Finally, the pathways by which CBX7 regulates stem cell-like properties of gastric cancer were explored. We found that CBX7, a constituent of the polycomb repressive complex 1 (PRC1), plays an important role in maintaining stem cell-like characteristics of gastric cancer cells via the activation of AKT pathway and the downregulation of p16. Spearman rank correlation analysis showed positive correlations among the expression of CBX7 and phospho-AKT (pAKT), stem cell markers OCT-4, and CD133 in gastric cancer tissues. In addition, CBX7 was found to upregulate microRNA-21 (miR-21) via the activation of AKT-NF-κB pathway, and miR-21 contributes to CBX7-mediated CSC characteristics. CBX7 positively regulates stem cell-like characteristics of gastric cancer cells by inhibiting p16 and activating AKT-NF-κB-miR-21 pathway.

Acceleration of astrocytic differentiation in neural stem cells surviving X-irradiation.

PubMed

Ozeki, Ayumi; Suzuki, Keiji; Suzuki, Masatoshi; Ozawa, Hiroki; Yamashita, Shunichi

2012-03-28

Neural stem cells (NSCs) are highly susceptible to DNA double-strand breaks; however, little is known about the effects of radiation in cells surviving radiation. Although the nestin-positive NSCs predominantly became glial fibrillary acidic protein (GFAP)-positive in differentiation-permissive medium, little or no cells were GFAP positive in proliferation-permissive medium. We found that more than half of the cells surviving X-rays became GFAP positive in proliferation-permissive medium. Moreover, localized irradiation stimulated differentiation of cells outside the irradiated area. These results indicate for the first time that ionizing radiation is able to stimulate astrocyte-specific differentiation of surviving NSCs, whose process is mediated both by the direct activation of nuclear factor-κB and by the indirect bystander effect induced by X-irradiation.

Key action items for the stem cell field: looking ahead to 2014.

PubMed

Knoepfler, Paul S

2013-12-01

The stem cell field is at a critical juncture in late 2013. We find ourselves buoyed by building momentum for both transformative basic science discoveries and clinical translation of stem cells. Cellular reprogramming has given the field exciting new avenues as well. The overall prospect of novel stem cell-based therapies becoming a reality for patients in the coming years has never seemed higher. At the same time, we face serious challenges. Some of these challenges, such as stem cell tourism, are familiar to us, although even those are evolving in ways that require adaptability and action by the stem cell field. Other new challenges are also emerging, including an urgent need for formal physician training in stem cells, regulatory compliance balanced with innovation and U.S. Food and Drug Administration reform, and savvy educational outreach. Looking ahead to 2014, both the challenges and opportunities for the stem cell field require a proactive, thoughtful approach to maximize the potential for a positive impact from stem cell advances. In this study, I discuss the key action items for the field as we look ahead to the coming year and beyond.

Differentiation of female Oct4-GFP embryonic stem cells into germ lineage cells.

PubMed

Ma, Xin; Li, Peng; Sun, Xiang; Sun, Yifeng; Hu, Rong; Yuan, Ping

2018-04-01

Due to high infertility ratio nowadays, it is essential to explore efficient ways of enhancing mammalian reproductivity, in particular female reproductivity. Using female Oct4-GFP embryonic stem cells, we mimic the in vivo development procedure to induce ES cells into epiblast cell-like cells (EpiLCs) and then primordial germ cell-like cells (PGCLCs). GFP positive PGCLCs that showed typical PGC markers and epigenetic modification were efficiently obtained. Further transplantation of the GFP positive PGCLC and native ovary cell mixture into ovary of infertile mice revealed that both MVH and GFP positive cells could be developed in ovary, but no later developmental stage germ cells were observed. This study suggested that Oct4-GFP ES cells may be only suitable for tracing early germ cell development. © 2018 International Federation for Cell Biology.

CD44 Staining of Cancer Stem-Like Cells Is Influenced by Down-Regulation of CD44 Variant Isoforms and Up-Regulation of the Standard CD44 Isoform in the Population of Cells That Have Undergone Epithelial-to-Mesenchymal Transition

PubMed Central

Biddle, Adrian; Gammon, Luke; Fazil, Bilal; Mackenzie, Ian C.

2013-01-01

CD44 is commonly used as a cell surface marker of cancer stem-like cells in epithelial tumours, and we have previously demonstrated the existence of two different CD44high cancer stem-like cell populations in squamous cell carcinoma, one having undergone epithelial-to-mesenchymal transition and the other maintaining an epithelial phenotype. Alternative splicing of CD44 variant exons generates a great many isoforms, and it is not known which isoforms are expressed on the surface of the two different cancer stem-like cell phenotypes. Here, we demonstrate that cancer stem-like cells with an epithelial phenotype predominantly express isoforms containing the variant exons, whereas the cancer stem-like cells that have undergone an epithelial-to-mesenchymal transition down-regulate these variant isoforms and up-regulate expression of the standard CD44 isoform that contains no variant exons. In addition, we find that enzymatic treatments used to dissociate cells from tissue culture or fresh tumour specimens cause destruction of variant CD44 isoforms at the cell surface whereas expression of the standard CD44 isoform is preserved. This results in enrichment within the CD44high population of cancer stem-like cells that have undergone an epithelial-to-mesenchymal transition and depletion from the CD44high population of cancer stem-like cells that maintain an epithelial phenotype, and therefore greatly effects the characteristics of any cancer stem-like cell population isolated based on expression of CD44. As well as effecting the CD44high population, enzymatic treatment also reduces the percentage of the total epithelial cancer cell population staining CD44-positive, with potential implications for studies that aim to use CD44-positive staining as a prognostic indicator. Analyses of the properties of cancer stem-like cells are largely dependent on the ability to accurately identify and assay these populations. It is therefore critical that consideration be given to use of multiple cancer stem-like cell markers and suitable procedures for cell isolation in order that the correct populations are assayed. PMID:23437366

The potential for stem cells in cerebral palsy--piecing together the puzzle.

PubMed

Faulkner, Stuart D; Ruff, Crystal A; Fehlings, Michael G

2013-06-01

The substantial socioeconomic burden of a diagnosis of cerebral palsy, coupled with a positive anecdotal and media spin on stem cell treatments, drives many affected families to seek information and treatment outside of the current clinical and scientific realm. Preclinical studies using several types of stem and adult cells--including mesenchymal stem cells, neural precursor cells, olfactory ensheathing glia and Schwann cells--have demonstrated some regenerative and functional efficacy in neurologic paradigms. This paper describes the most common cell types investigated for transplant in vivo and summarizes the current state of early-phase clinical trials. It investigates the most relevant and promising coadministered therapies, including rehabilitation, drug targeting, magnetic stimulation, and bioengineering approaches. We highlight the need for adjunctive combinatorial strategies to successfully transfer stem cell treatments from bench to bedside. Copyright © 2013 Elsevier Inc. All rights reserved.

Helping Yourself by Offering Help: Mediators of Expressive Helping in Survivors of Hematopoietic Stem Cell Transplant.

PubMed

Williamson, Timothy J; Stanton, Annette L; Austin, Jane E; Valdimarsdottir, Heiddis B; Wu, Lisa M; Krull, Jennifer L; Rini, Christine M

2017-10-01

A randomized experiment by Rini et al. (Health Psychol. 33(12):1541-1551, 2014) demonstrated that expressive helping, which involves three expressive writing sessions regarding hematopoietic stem cell transplant, followed by one writing session directed toward helping other stem cell transplant recipients, reduced psychological distress and bothersome physical symptoms among stem cell transplant recipients with elevated survivorship problems, relative to a neutral writing control condition. The current study evaluated whether word use reflective of emotional expression, cognitive processing, and change in perspective mediates the effects of expressive helping. The essays of 67 stem cell transplant recipients with high survivorship problems were analyzed with Linguistic Inquiry and Word Count. Multiple mediation modeling was used to test the hypothesized mechanisms of expressive helping on distress and bothersome physical symptoms. Relative to the control condition, expressive helping produced significant reductions in psychological distress and marginal reductions in physical symptom bother in the analyzed subset of participants from the parent study. Results indicated that positive emotion word use significantly mediated effects of expressive helping on reduced distress, but only for participants who used average (compared to above or below average) rates of negative emotion words. Cognitive processing and change in perspective did not significantly mediate benefits of expressive helping. Expressive helping carried its positive effects on distress through participants' higher expression of positive emotions when coupled with moderate rates of negative emotions. Findings highlight the benefit of expressing both positive and negative emotions in stressful situations.

A natural stem cell therapy? How novel findings and biotechnology clarify the ethics of stem cell research

PubMed Central

Patel, P

2006-01-01

The natural replacement of damaged cells by stem cells occurs actively and often in adult tissues, especially rapidly dividing cells such as blood cells. An exciting case in Boston, however, posits a kind of natural stem cell therapy provided to a mother by her fetus—long after the fetus is born. Because there is a profound lack of medical intervention, this therapy seems natural enough and is unlikely to be morally suspect. Nevertheless, we feel morally uncertain when we consider giving this type of therapy to patients who would not naturally receive it. Much has been written about the ethics of stem cell research and therapy; this paper will focus on how recent advances in biotechnology and biological understandings of development narrow the debate. Here, the author briefly reviews current stem cell research practices, revisits the natural stem cell therapy case for moral evaluation, and ultimately demonstrates the importance of permissible stem cell research and therapy, even absent an agreement about the definition of when embryonic life begins. Although one promising technology, blighted ovum utilisation, uses fertilised but developmentally bankrupt eggs, it is argued that utilisation of unfertilised eggs to derive totipotent stem cells obviates the moral debate over when life begins. There are two existing technologies that fulfil this criterion: somatic cell nuclear transfer and parthenogenic stem cell derivation. Although these technologies are far from therapeutic, concerns over the morality of embryonic stem cell derivation should not hinder their advancement. PMID:16574879

Abortion, embryonic stem cell research, and waste.

PubMed

Jensen, David A

2008-01-01

Can one consistently deny the permissibility of abortion while endorsing the killing of human embryos for the sake of stem cell research? The question is not trivial; for even if one accepts that abortion is prima facie wrong in all cases, there are significant differences with many of the embryos used for stem cell research from those involved in abortion--most prominently, many have been abandoned in vitro, and appear to have no reasonably likely meaningful future. On these grounds one might think to maintain a strong position against abortion but endorse killing human embryos for the sake of stem cell research and its promising benefits. I will argue, however, that these differences are not decisive. Thus, one who accepts a strong view against abortion is committed to the moral impermissibility of killing human embryos for the sake of stem cell research. I do not argue for the moral standing of either abortion or the killing of embryos for stem cell research; I only argue for the relation between the two. Thus the conclusion is relevant to those with a strong view in favor of the permissibility of killing embryos for the sake of research as much as for those who may strongly oppose abortion; neither can consider their position in isolation from the other.

A role for adult TLX-positive neural stem cells in learning and behaviour.

PubMed

Zhang, Chun-Li; Zou, Yuhua; He, Weimin; Gage, Fred H; Evans, Ronald M

2008-02-21

Neurogenesis persists in the adult brain and can be regulated by a plethora of external stimuli, such as learning, memory, exercise, environment and stress. Although newly generated neurons are able to migrate and preferentially incorporate into the neural network, how these cells are molecularly regulated and whether they are required for any normal brain function are unresolved questions. The adult neural stem cell pool is composed of orphan nuclear receptor TLX-positive cells. Here, using genetic approaches in mice, we demonstrate that TLX (also called NR2E1) regulates adult neural stem cell proliferation in a cell-autonomous manner by controlling a defined genetic network implicated in cell proliferation and growth. Consequently, specific removal of TLX from the adult mouse brain through inducible recombination results in a significant reduction of stem cell proliferation and a marked decrement in spatial learning. In contrast, the resulting suppression of adult neurogenesis does not affect contextual fear conditioning, locomotion or diurnal rhythmic activities, indicating a more selective contribution of newly generated neurons to specific cognitive functions.

Chinese newspaper coverage of (unproven) stem cell therapies and their providers.

PubMed

Ogbogu, Ubaka; Du, Li; Rachul, Christen; Bélanger, Lisa; Caulfield, Timothy

2013-04-01

China is a primary destination for stem cell tourism, the phenomenon whereby patients travel abroad to receive unproven stem cell-based treatments that have not been approved in their home countries. Yet, much remains unknown about the state of the stem cell treatment industry in China and about how the Chinese view treatments and providers. Given the media's crucial role in science/health communication and in framing public dialogue, this study sought to examine Chinese newspaper portrayal and perceptions of stem cell treatments and their providers. Based on a content analysis of over 300 newspaper articles, the study revealed that while Chinese newspaper reporting is generally neutral in tone, it is also inaccurate, overly positive, heavily influenced by "interested" treatment providers and focused on the therapeutic uses of stem cells to address the health needs of the local population. The study findings suggest a need to counterbalance providers' influence on media reporting through strategies that encourage media uptake of accurate information about stem cell research and treatments.

DDX4 (DEAD box polypeptide 4) colocalizes with cancer stem cell marker CD133 in ovarian cancers

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

Kim, Ki Hyung; Biomedical Research Institute and Pusan Cancer Center, Pusan National University Hospital, Busan; Kang, Yun-Jeong

Highlights: • Germ cell marker DDX4 was significantly increased in ovarian cancer. • Ovarian cancer stem cell marker CD133 was significantly increased in ovarian cancer. • DDX4 and CD133 were mostly colocalized in various types of ovarian cancer tissues. • CD133 positive ovarian cancer cells also express DDX4 whereas CD133-negative cells did not possess DDX4. • Germ cell marker DDX4 has the potential of ovarian cancer stem cell marker. - Abstract: DDX4 (DEAD box polypeptide 4), characterized by the conserved motif Asp-Glu-Ala-Asp (DEAD), is an RNA helicase which is implicated in various cellular processes involving the alteration of RNA secondarymore » structure, such as translation initiation, nuclear and mitochondrial splicing, and ribosome and spliceosome assembly. DDX4 is known to be a germ cell-specific protein and is used as a sorting marker of germline stem cells for the production of oocytes. A recent report about DDX4 in ovarian cancer showed that DDX4 is overexpressed in epithelial ovarian cancer and disrupts a DNA damage-induced G2 checkpoint. We investigated the relationship between DDX4 and ovarian cancer stem cells by analyzing the expression patterns of DDX4 and the cancer stem cell marker CD133 in ovarian cancers via tissue microarray. Both DDX4 and CD133 were significantly increased in ovarian cancer compared to benign tumors, and showed similar patterns of expression. In addition, DDX4 and CD133 were mostly colocalized in various types of ovarian cancer tissues. Furthermore, almost all CD133 positive ovarian cancer cells also express DDX4 whereas CD133-negative cells did not possess DDX4, suggesting a strong possibility that DDX4 plays an important role in cancer stem cells, and/or can be used as an ovarian cancer stem cell marker.« less

Investigation of functional activity human dental pulp stem cells at acute and chronic pulpitis.

PubMed

Ustiashvili, M; Kordzaia, D; Mamaladze, M; Jangavadze, M; Sanodze, L

2014-09-01

It is already recognized that together with the other connective tissues organ-specific progenic stem cells are also found in postnatal dental pulp. This group of undifferentiated cells is only 1% of total cell population of the pulp. The aim of the study was the identification of stem cells in human dental pulp, detection of their localization and assessment of functional activity during inflammation process and/or at norm. The obtained results showed that at acute pulpitis the pulp stroma is hypocellular in comparison with the norm but cells proliferative activity is low. CD 133 and NCAM (CD 56) positive stem cells were found in perivascularl space of the pulp stroma and in Hohle layer. At process prolongation and transition to the chronic phase pulp stroma is hypercellular, the cells with large, rounded or oval-shaped nuclei with clear chromatin appear together with fibroblasts. They are distributed as about entire thickness of the stroma as especially Hohle layer. In such cells higher proliferative activity (Ki67 expression) was observed. The cells in the mentioned proliferation phase are intensively marked by CD133, the rate of which is high in Hohle layer and along it. A large number of NCAM (CD 56) positive cells appear in pulp stroma. During pulpitis an involvement of stem cells into the process of reparative dentinogenesis should be conducted stepwise. In acute cases of the disease, stem cell perivascularl mobilization and proliferation and its migration to Hohle layer occur in response to irritation /stimulation. Chronification of the process leads not only to the migration of stem cells to the periphery of the pulp but also s their В«maturationВ» (increase of NCAM expression in the stem cells), which causes an increase the number of dentin producing active odontoblasts and initiation of reparative dentinogenesis.

Generation of mature T cells from human hematopoietic stem and progenitor cells in artificial thymic organoids.

PubMed

Seet, Christopher S; He, Chongbin; Bethune, Michael T; Li, Suwen; Chick, Brent; Gschweng, Eric H; Zhu, Yuhua; Kim, Kenneth; Kohn, Donald B; Baltimore, David; Crooks, Gay M; Montel-Hagen, Amélie

2017-05-01

Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3 + TCR-αβ + single-positive CD8 + or CD4 + cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naive phenotypes, a diverse T cell receptor (TCR) repertoire and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen-specific cytotoxicity and near-complete lack of endogenous TCR Vβ expression, consistent with allelic exclusion of Vβ-encoding loci. ATOs provide a robust tool for studying human T cell differentiation and for the future development of stem-cell-based engineered T cell therapies.

Testing stem cell therapy in a rat model of inflammatory bowel disease: role of bone marrow stem cells and stem cell factor in mucosal regeneration.

PubMed

Qu, Bo; Xin, Guo-Rong; Zhao, Li-Xia; Xing, Hui; Lian, Li-Ying; Jiang, Hai-Yan; Tong, Jia-Zhao; Wang, Bei-Bei; Jin, Shi-Zhu

2014-01-01

The gastrointestinal (GI) mucosal cells turnover regularly under physiological conditions, which may be stimulated in various pathological situations including inflammation. Local epithelial stem cells appear to play a major role in such mucosal renewal or pathological regeneration. Less is clear about the involvement of multipotent stem cells from blood in GI repair. We attempted to explore a role of bone marrow mesenchymal stromal cells (BMMSCs) and soluble stem cell factor (SCF) in GI mucosa regeneration in a rat model of inflammatory bowel diseases (IBD). BMMSCs labelled with the fluorescent dye PKH26 from donor rats were transfused into rats suffering indomethacin-induced GI injury. Experimental effects by BMMSCs transplant and SCF were determined by morphometry of intestinal mucosa, double labeling of PKH26 positive BMMSCs with endogenous proliferative and intestinal cell markers, and western blot and PCR analyses of the above molecular markers in the recipient rats relative to controls. PKH26 positive BMMSCs were found in the recipient mucosa, partially colocalizing with the proliferating cell nuclear antigen (PCNA), Lgr5, Musashi-1 and ephrin-B3. mRNA and protein levels of PCNA, Lgr5, Musashi-1 and ephrin-B3 were elevated in the intestine in BMMSCs-treated rats, most prominent in the BMMSCs-SCF co-treatment group. The mucosal layer and the crypt layer of the small intestine were thicker in BMMSCs-treated rats, more evident in the BMMSCs-SCF co-treatment group. BMMSCs and SCF participate in but may play a synergistic role in mucosal cell regeneration following experimentally induced intestinal injury. Bone marrow stem cell therapy and SCF administration may be of therapeutic value in IBD.

Hepatic differentiation capability of rat bone marrow-derived mesenchymal stem cells and hematopoietic stem cells.

PubMed

Shu, Sai-Nan; Wei, Lai; Wang, Jiang-Hua; Zhan, Yu-Tao; Chen, Hong-Song; Wang, Yu

2004-10-01

To investigate the different effects of mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) on hepatic differentiation. MSCs from rat bone marrow were isolated and cultured by standard methods. HSCs from rat bone marrow were isolated and purified by magnetic activated cell sorting. Both cell subsets were induced. Morphology, RT-PCR and immunocytochemistry were used to identify the hepatic differentiation grade. MSCs exhibited round in shape after differentiation, instead of fibroblast-like morphology before differentiation. Albumin mRNA and protein were expressed positively in MSCs, without detection of alpha-fetoprotein (AFP). HSCs were polygonal in shape after differentiation. The expression of albumin signal decreased and AFP signal increased. The expression of CK18 was continuous in MSCs and HSCs both before and after induction. Both MSCs and HSCs have hepatic differentiation capabilities. However, their capabilities are not the same. MSCs can differentiate into mature hepatocyte-like cells, never expressing early hepatic specific genes, while Thy-1.1(+) cells are inclined to differentiate into hepatic stem cell-like cells, with an increasing AFP expression and a decreasing albumin signal. CK18 mRNA is positive in Thy-1.1(+) cells and MSCs, negative in Thy-1.1(-) cells. It seems that CK18 has some relationship with Thy-1.1 antigen, and CK18 may be a predictive marker of hepatic differentiation capability.

JNK signaling mediates EPHA2-dependent tumor cell proliferation, motility, and cancer stem cell-like properties in non-small cell lung cancer

PubMed Central

Song, Wenqiang; Ma, Yufang; Wang, Jialiang; Brantley-Sieders, Dana; Chen, Jin

2014-01-01

Recent genome-wide analyses in human lung cancer revealed that EPHA2 receptor tyrosine kinase is overexpressed in non-small cell lung cancer (NSCLC), and high levels of EPHA2 correlate with poor clinical outcome. However, the mechanistic basis for EPHA2-mediated tumor promotion in lung cancer remains poorly understood. Here we show that the JNK/c-JUN signaling mediates EPHA2-dependent tumor cell proliferation and motility. A screen of phospho-kinase arrays revealed a decrease in phospho-c-JUN levels in EPHA2 knockdown cells. Knockdown of EPHA2 inhibited p-JNK and p-c-JUN levels in approximately 50% of NSCLC lines tested. Treatment of parental cells with SP600125, a JNK inhibitor, recapitulated defects in EPHA2-deficient tumor cells; whereas constitutively activated JNK mutants were sufficient to rescue phenotypes. Knockdown of EPHA2 also inhibited tumor formation and progression in xenograft animal models in vivo. Furthermore, we investigated the role of EPHA2 in cancer stem-like cells. RNAi-mediated depletion of EPHA2 in multiple NSCLC lines decreased the ALDH positive cancer stem-like population and tumor spheroid formation in suspension. Depletion of EPHA2 in sorted ALDH positive populations markedly inhibited tumorigenicity in nude mice. Furthermore, analysis of a human lung cancer tissue microarray revealed a significant, positive association between EPHA2 and ALDH expression, indicating an important role for EPHA2 in human lung cancer stem-like cells. Collectively, these studies revealed a critical role of JNK signaling in EPHA2-dependent lung cancer cell proliferation and motility and a role for EPHA2 in cancer stem-like cell function, providing evidence for EPHA2 as a potential therapeutic target in NSCLC. PMID:24607842

The Use of Blood Vessel–Derived Stem Cells for Meniscal Regeneration and Repair

PubMed Central

OSAWA, AKI; HARNER, CHRISTOPHER D.; GHARAIBEH, BURHAN; MATSUMOTO, TOMOYUKI; MIFUNE, YUTAKA; KOPF, SEBASTIAN; INGHAM, SHEILA J. M.; SCHREIBER, VERENA; USAS, ARVYDAS; HUARD, JOHNNY

2015-01-01

Purpose Surgical repairs of tears in the vascular region of the meniscus usually heal better than repairs performed in the avascular region; thus, we hypothesized that this region might possess a richer supply of vascular-derived stem cells than the avascular region. Methods In this study, we analyzed 6 menisci extracted from aborted human fetuses and 12 human lateral menisci extracted from adult human subjects undergoing total knee arthroplasty. Menisci were immunostained for CD34 (a stem cell marker) and CD146 (a pericyte marker) in situ, whereas other menisci were dissected into two regions (peripheral and inner) and used to isolate meniscus-derived cells by flow cytometry. Cell populations expressing CD34 and CD146 were tested for their multi-lineage differentiation potentials, including chondrogenic, osteogenic, and adipogenic lineages. Fetal peripheral meniscus cells were transplanted by intracapsular injection into the knee joints of an athymic rat meniscal tear model. Rat menisci were extracted and histologically evaluated after 4 wk posttransplantation. Results Immunohistochemistry and flow cytometric analyses demonstrated that a higher number of CD34- and CD146-positive cells were found in the peripheral region compared with the inner region. The CD34- and CD146-positive cells isolated from the vascular region of both fetal and adult menisci demonstrated multilineage differentiation capacities and were more potent than cells isolated from the inner (avascular) region. Fetal CD34- and CD146-positive cells transplanted into the athymic rat knee joint were recruited into the meniscal tear sites and contributed to meniscus repair. Conclusions The vascularized region of the meniscus contains more stem cells than the avascular region. These meniscal-derived stem cells were multi-potent and contributed to meniscal regeneration. PMID:23247715

Isolation, identification and multipotential differentiation of mouse adipose tissue-derived stem cells.

PubMed

Taha, Masoumeh Fakhr; Hedayati, Vahideh

2010-08-01

Bone marrow and adipose tissue have provided two suitable sources of mesenchymal stem cells. Although previous studies have confirmed close similarities between bone marrow-derived stem cells (BM-MSCs) and adipose tissue-derived stem cells (ADSCs), the molecular phenotype of ADSCs is still poorly identified. In the present study, mouse ADSCs were isolated from the inguinal fat pad of 12-14 weeks old mice. Freshly isolated and three passaged ADSCs were analyzed for the expression of OCT4, Sca-1, c-kit and CD34 by RT-PCR. Three passaged ADSCs were analyzed by flow cytometry for the presence of CD11b, CD45, CD31, CD29 and CD44. Moreover, cardiogenic, adipogenic and neurogenic differentiation of ADSCs were induced in vitro. Freshly isolated ADSCs showed the expression of OCT4, Sca-1, c-kit and CD34, and two days cultured ADSCs were positively immunostained with anti-OCT4 monoclonal antibody. After three passages, the expression of OCT4, c-kit and CD34 eliminated, while the expression of Sca-1 showed a striking enhancement. These cells were identified positive for CD29 and CD44 markers, and they showed the lack of CD45 and CD31 expression. Three passaged ADSCs were differentiated to adipocyte-, cardiomyocyte- and neuron-like cells that were identified based on the positive staining with Sudan black, anti-cardiac troponin I antibody and anti-map-2 antibody, respectively. In conclusion, adipose tissue contains a stem cell population that seems to be a good multipotential cell candidate for the future cell replacement therapy. Copyright 2010 Elsevier Ltd. All rights reserved.

The SHH/Gli axis regulates CD90-mediated liver cancer stem cell function by activating the IL6/JAK2 pathway.

PubMed

Zhang, Ketao; Che, Siyao; Pan, Chuzhi; Su, Zheng; Zheng, Shangyou; Yang, Shanglin; Zhang, Huayao; Li, Wenda; Wang, Weidong; Liu, Jianping

2018-05-02

The cell surface antigen CD90 has recently been established as a promising marker for liver cancer stem cells. This study aimed to investigate potential implications of SHH/Gli signalling in CD90+ liver cancer stem cells. Correlation of the expression of SHH signalling components and CD90 in liver cancer cells and clinical tissues, as well as in enriched CD90+ liver cancer stem cells and the TCGA database, were analysed by quantitative RT-PCR, Western blotting and flow cytometry. Functional analysis was conducted by siRNA-mediated CD90, Gli1 and Gli3 gene knockdown, SHH treatment and application of the JAK2 inhibitor AZD1480 and IL6 neutralizing antibody in CD90+ liver cancer stem cells, followed by cell proliferation, migration, sphere formation and tumorigenicity assays. CD90 expression exhibited a high positive correlation with Gli1 and Gli3 in multiple liver cancer cell lines and human cancerous liver tissues, both of which showed a significant increase in liver cancer. Analysis of TCGA data revealed an association of CD90, Gli1 and Gli3 with a short overall survival and positive correlation between CD90 expression and Gli3 expression level. The stem cell potentials of CD90+ 97L liver cancer cells were greatly impaired by Gli1/3 knockdown with siRNA but enhanced by SHH treatment. Application of the JAK2 inhibitor AZD1480 and IL6 neutralizing antibody showed the CD90 and SHH/Gli-regulated liver cancer stem cell functions were mediated by the IL6/JAK2/STAT3 pathway. The stem cell properties of CD90+ liver cancer cells are regulated by the downstream SHH/Gli and IL6/JAK2/STAT3 signalling pathways. © 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Hedgehog signaling regulates the generation of ameloblast progenitors in the continuously growing mouse incisor

PubMed Central

Seidel, Kerstin; Ahn, Christina P.; Lyons, David; Nee, Alexander; Ting, Kevin; Brownell, Isaac; Cao, Tim; Carano, Richard A. D.; Curran, Tom; Schober, Markus; Fuchs, Elaine; Joyner, Alexandra; Martin, Gail R.; de Sauvage, Frederic J.; Klein, Ophir D.

2010-01-01

In many organ systems such as the skin, gastrointestinal tract and hematopoietic system, homeostasis is dependent on the continuous generation of differentiated progeny from stem cells. The rodent incisor, unlike human teeth, grows throughout the life of the animal and provides a prime example of an organ that rapidly deteriorates if newly differentiated cells cease to form from adult stem cells. Hedgehog (Hh) signaling has been proposed to regulate self-renewal, survival, proliferation and/or differentiation of stem cells in several systems, but to date there is little evidence supporting a role for Hh signaling in adult stem cells. We used in vivo genetic lineage tracing to identify Hh-responsive stem cells in the mouse incisor and we show that sonic hedgehog (SHH), which is produced by the differentiating progeny of the stem cells, signals to several regions of the incisor. Using a hedgehog pathway inhibitor (HPI), we demonstrate that Hh signaling is not required for stem cell survival but is essential for the generation of ameloblasts, one of the major differentiated cell types in the tooth, from the stem cells. These results therefore reveal the existence of a positive-feedback loop in which differentiating progeny produce the signal that in turn allows them to be generated from stem cells. PMID:20978073

Generation of mature T cells from human hematopoietic stem/progenitor cells in artificial thymic organoids

PubMed Central

Seet, Christopher S.; He, Chongbin; Bethune, Michael T.; Li, Suwen; Chick, Brent; Gschweng, Eric H.; Zhu, Yuhua; Kim, Kenneth; Kohn, Donald B.; Baltimore, David; Crooks, Gay M.; Montel-Hagen, Amélie

2017-01-01

Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3+TCRab+ single positive (SP) CD8+ or CD4+ cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports highly efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naïve phenotypes, a diverse TCR repertoire, and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen specific cytotoxicity and near complete lack of endogenous TCR Vβ expression, consistent with allelic exclusion of Vβ loci. ATOs provide a robust tool for studying human T cell development and stem cell based approaches to engineered T cell therapies. PMID:28369043

Key Action Items for the Stem Cell Field: Looking Ahead to 2014

PubMed Central

Knoepfler, Paul S.

2013-01-01

Abstract The stem cell field is at a critical juncture in late 2013. We find ourselves buoyed by building momentum for both transformative basic science discoveries and clinical translation of stem cells. Cellular reprogramming has given the field exciting new avenues as well. The overall prospect of novel stem cell–based therapies becoming a reality for patients in the coming years has never seemed higher. At the same time, we face serious challenges. Some of these challenges, such as stem cell tourism, are familiar to us, although even those are evolving in ways that require adaptability and action by the stem cell field. Other new challenges are also emerging, including an urgent need for formal physician training in stem cells, regulatory compliance balanced with innovation and U.S. Food and Drug Administration reform, and savvy educational outreach. Looking ahead to 2014, both the challenges and opportunities for the stem cell field require a proactive, thoughtful approach to maximize the potential for a positive impact from stem cell advances. In this study, I discuss the key action items for the field as we look ahead to the coming year and beyond. PMID:24147571

Stem cell tourism and doctors' duties to minors--a view from Canada.

PubMed

Zarzeczny, Amy; Caulfield, Timothy

2010-05-01

While the clinical promise of much stem cell research remains largely theoretical, patients are nonetheless pursuing unproven stem cell therapies in jurisdictions around the world--a phenomenon referred to as "stem cell tourism." These treatments are generally advertised on a direct-to-consumer basis via the Internet. Research shows portrayals of stem cell medicine on such websites are overly optimistic and the claims made are unsubstantiated by published evidence. However, anecdotal evidence suggests that parents are pursuing these "treatments" for their children, despite potential physical and financial risk. Physicians are in a unique position as they can be expected to be involved in, or privy to, such decisions. In this paper, we consider what duties physicians may have toward minor patients whose parents/guardians wish to engage in stem cell tourism on their behalf. We use the Canadian perspective to address the broadly relevant issues raised by this trend.

Biophysical regulation of stem cell differentiation.

PubMed

Govey, Peter M; Loiselle, Alayna E; Donahue, Henry J

2013-06-01

Bone adaptation to its mechanical environment, from embryonic through adult life, is thought to be the product of increased osteoblastic differentiation from mesenchymal stem cells. In parallel with tissue-scale loading, these heterogeneous populations of multipotent stem cells are subject to a variety of biophysical cues within their native microenvironments. Bone marrow-derived mesenchymal stem cells-the most broadly studied source of osteoblastic progenitors-undergo osteoblastic differentiation in vitro in response to biophysical signals, including hydrostatic pressure, fluid flow and accompanying shear stress, substrate strain and stiffness, substrate topography, and electromagnetic fields. Furthermore, stem cells may be subject to indirect regulation by mechano-sensing osteocytes positioned to more readily detect these same loading-induced signals within the bone matrix. Such paracrine and juxtacrine regulation of differentiation by osteocytes occurs in vitro. Further studies are needed to confirm both direct and indirect mechanisms of biophysical regulation within the in vivo stem cell niche.

Isolating LacZ-expressing cells from mouse inner ear tissues using flow cytometry.

PubMed

Jan, Taha A; Chai, Renjie; Sayyid, Zahra N; Cheng, Alan G

2011-12-23

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae. The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown. To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Generation of H1 PAX6WT/EGFP reporter cells to purify PAX6 positive neural stem/progenitor cells.

PubMed

Wu, Wei; Liu, Juli; Su, Zhenghui; Li, Zhonghao; Ma, Ning; Huang, Ke; Zhou, Tiancheng; Wang, Linli

2018-08-25

Neural conversion from human pluripotent cells (hPSCs) is a potential therapy to neurological disease in the future. However, this is still limited by efficiency and stability of existed protocols used for neural induction from hPSCs. To overcome this obstacle, we developed a reporter system to screen PAX6 + neural progenitor/stem cells using transcription activator like effector nuclease (TALEN). We found that knock-in 2 A-EGFP cassette into PAX6 exon of human embryonic stem cells H1 with TALEN-based homology recombination could establish PAX6 WT/EGFP H1 reporter cell line fast and efficiently. This reporter cell line could differentiate into PAX6 and EGFP double positive neural progenitor/stem cells (NPCs/NSCs) after neural induction. Those PAX6 WT/EGFP NPCs could be purified, expanded and specified to post-mitotic neurons in vitro efficiently. With this reporter cell line, we also screened out 1 NPC-specific microRNA, hsa-miR-99a-5p, and 3 ESCs-enriched miRNAs, hsa-miR-302c-5p, hsa-miR-512-3p and hsa-miR-518 b. In conclusion, the TALEN-based neural stem cell screening system is safe and efficient and could help researcher to acquire adequate and pure neural progenitor cells for further application. Copyright © 2018 Elsevier Inc. All rights reserved.

Endodermal differentiation of human pluripotent stem cells to insulin-producing cells in 3D culture

PubMed Central

Takeuchi, Hiroki; Nakatsuji, Norio; Suemori, Hirofumi

2014-01-01

Insulin-producing cells (IPCs) derived from human pluripotent stem cells (hPSCs) may be useful in cell therapy and drug discovery for diabetes. Here, we examined various growth factors and small molecules including those previously reported to develop a robust differentiation method for induction of mature IPCs from hPSCs. We established a protocol that induced PDX1-positive pancreatic progenitor cells at high efficiency, and further induced mature IPCs by treatment with forskolin, dexamethasone, Alk5 inhibitor II and nicotinamide in 3D culture. The cells that differentiated into INSULIN-positive and C-PEPTIDE-positive cells secreted insulin in response to glucose stimulation, indicating a functional IPC phenotype. We also found that this method was applicable to different types of hPSCs. PMID:24671046

The number of stem cells in the subependymal zone of the adult rodent brain is correlated with the number of ependymal cells and not with the volume of the niche.

PubMed

Kazanis, Ilias; Ffrench-Constant, Charles

2012-05-01

The mammalian subependymal zone (SEZ; often called subventricular) situated at the lateral walls of the lateral ventricles of the brain contains a pool of relatively quiescent adult neural stem cells whose neurogenic activity persists throughout life. These stem cells are positioned in close proximity both to the ependymal cells that provide the cerebrospinal fluid interface and to the blood vessel endothelial cells, but the relative contribution of these 2 cell types to stem cell regulation remains undetermined. Here, we address this question by analyzing a naturally occurring example of volumetric scaling of the SEZ in a comparison of the mouse SEZ with the larger rat SEZ. Our analysis reveals that the number of stem cells in the SEZ niche is correlated with the number of ependymal cells rather than with the volume, thereby indicating the importance of ependymal-derived factors in the formation and function of the SEZ. The elucidation of the factors generated by ependymal cells that regulate stem cell numbers within the SEZ is, therefore, of importance for stem cell biology and regenerative neuroscience.

Molecular assessment, characterization, and differentiation of theca stem cells imply the presence of mesenchymal and pluripotent stem cells in sheep ovarian theca layer.

PubMed

Adib, Samane; Valojerdi, Mojtaba Rezazadeh

2017-10-01

The ability of ovarian theca stem cells to differentiate into oocyte and theca cells may lead to a major advancement in reproductive biology and infertility treatments. However, there is little information about function, growth and differentiation potential of these immature cells. In this study adult sheep theca stem cells (TSCs) characteristics, and differentiation potential into osteocyte-like cells (OSLCs), adipocyte-like cells (ALCs), theca progenitor-like cells (TPCs), and oocyte-like cells (OLCs) were investigated. TSCs were isolated, cultured, and compared with mesenchymal stem cells (MSCs), fibroblast cells (FCs), and pluripotent embryonic ovarian cells (EO). Adherent TSCs were morphologically similar to FCs. Cell cycle analysis showed high proliferation capacity of TSCs. TSCs were positive for the mesenchymal cells surface markers, and also expressed POU5F1. Differentiation potential of TSCs into OSLCs and ALCs were confirmed by alizarin red and oil red staining respectively. OSTEOCALCIN and COL1 were expressed in OSLCs. ALCs were positive for PPARα and LPL. TPCs expressed theca specific genes (GLI2, GLI3, PTCH1, CYP17A1, 3β-HSD and LHR) and secreted testosterone, dehydroepiandrostenedione (DHEA), androstenedione, progesterone and estradiol. Lipid droplets in these steroid cells were viewed by oil red staining. OLCs expressed oocyte-specific marker genes including, ZP3, ZP2, GDF9, SYCP3, PRDM1, STELLA, FRAGILIS, DAZL, as well as POU5F1, and showed separated sphere structure. Our results indicated that TSCs derived from ovarian follicles contain MSCs and pluripotent stem cells (PSCs) that can be differentiated into lineages of mesenchymal origin and are capable of differentiation into TPCs and OLCs under in vitro conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Paracrine effects and heterogeneity of marrow-derived stem/progenitor cells: relevance for the treatment of respiratory diseases.

PubMed

Conese, Massimo; Carbone, Annalucia; Castellani, Stefano; Di Gioia, Sante

2013-01-01

Stem cell-based treatment may represent a hope for the treatment of acute lung injury and pulmonary fibrosis, and other chronic lung diseases, such as cystic fibrosis, asthma and chronic obstructive pulmonary disease (COPD). It is well established in preclinical models that bone marrow-derived stem and progenitor cells exert beneficial effects on inflammation, immune responses and repairing of damage in virtually all lung-borne diseases. While it was initially thought that the positive outcome was due to a direct engraftment of these cells into the lung as endothelial and epithelial cells, paracrine factors are now considered the main mechanism through which stem and progenitor cells exert their therapeutic effect. This knowledge has led to the clinical use of marrow cells in pulmonary hypertension with endothelial progenitor cells (EPCs) and in COPD with mesenchymal stromal (stem) cells (MSCs). Bone marrow-derived stem cells, including hematopoietic stem/progenitor cells, MSCs, EPCs and fibrocytes, encompass a wide array of cell subsets with different capacities of engraftment and injured tissue-regenerating potential. The characterization/isolation of the stem cell subpopulations represents a major challenge to improve the efficacy of transplantation protocols used in regenerative medicine and applied to lung disorders. Copyright © 2013 S. Karger AG, Basel.

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.

CD34+ Testicular Stromal Cells Support Long-Term Expansion of Embryonic and Adult Stem and Progenitor Cells

PubMed Central

Kim, Jiyeon; Seandel, Marco; Falciatori, Ilaria; Wen, Duancheng; Rafii, Shahin

2010-01-01

Stem cells reside in specialized microenvironments created by supporting stromal cells that orchestrate self-renewal and lineage-specific differentiation. However, the precise identity of the cellular and molecular pathways that support self-renewal of stem cells is not known. For example, long-term culture of prototypical stem cells, such as adult spermatogonial stem and progenitor cells (SPCs), in vitro has been impeded by the lack of an optimal stromal cell line that initiates and sustains proliferation of these cells. Indeed, current methods, including the use of mouse embryonic fibroblasts (MEFs), have not been efficient and have generally led to inconsistent results. Here, we report the establishment of a novel CD34-positive cell line, referred to as JK1, derived from mouse testicular stromal cells that not only facilitated long-term SPC culture but also allowed faithful generation of SPCs and multipotent stem cells. SPCs generated on JK1 maintained key features of germ line stem cells, including expression of PLZF, DAZL, and GCNA. Furthermore, these feeders also promoted the long-term cultivation of other types of primitive cells including multi-potent adult spermatogonial-derived stem cells, pluripotent murine embryonic stem cells, and embryonic germ cells derived from primordial germ cells. Stem cells could be passaged serially and still maintained expression of characteristic markers such as OCT4 and NANOG in vitro, as well as the ability to generate all three germ layers in vivo. These results indicate that the JK1 cell line is capable of promoting long-term culture of primitive cells. As such, this cell line allows for identification of stromal-derived factors that support long-term proliferation of various types of stem cells and constitutes a convenient alternative to other types of feeder layers. PMID:18669907

Sox2+ Stem Cells Contribute to All Epithelial Lineages of the Tooth via Sfrp5+ Progenitors

PubMed Central

Juuri, Emma; Saito, Kan; Ahtiainen, Laura; Seidel, Kerstin; Tummers, Mark; Hochedlinger, Konrad; Klein, Ophir D.; Thesleff, Irma; Michon, Frederic

2012-01-01

SUMMARY The continuously growing mouse incisor serves as a valuable model to study stem cell regulation during organ renewal. Epithelial stem cells are localized in the proximal end of the incisor in the labial cervical loop. Here, we show that the transcription factor Sox2 is a specific marker for these stem cells. Sox2+ cells became restricted to the labial cervical loop during tooth morphogenesis, and they contributed to the renewal of enamel-producing ameloblasts as well as all other epithelial cell lineages of the tooth. The early progeny of Sox2-positive stem cells transiently expressed the Wnt inhibitor Sfrp5. Sox2 expression was regulated by the tooth initiation marker FGF8 and specific miRNAs, suggesting a fine-tuning to maintain homeostasis of the dental epithelium. The identification of Sox2 as a marker for the dental epithelial stem cells will facilitate further studies on their lineage segregation and differentiation during tooth renewal. PMID:22819339

Imaging transplanted stem cells in real time using an MRI dual-contrast method

PubMed Central

Ngen, Ethel J.; Wang, Lee; Kato, Yoshinori; Krishnamachary, Balaji; Zhu, Wenlian; Gandhi, Nishant; Smith, Barbara; Armour, Michael; Wong, John; Gabrielson, Kathleen; Artemov, Dmitri

2015-01-01

Stem cell therapies are currently being investigated for the repair of brain injuries. Although exogenous stem cell labelling with superparamagnetic iron oxide nanoparticles (SPIONs) prior to transplantation provides a means to noninvasively monitor stem cell transplantation by magnetic resonance imaging (MRI), monitoring cell death is still a challenge. Here, we investigate the feasibility of using an MRI dual-contrast technique to detect cell delivery, cell migration and cell death after stem cell transplantation. Human mesenchymal stem cells were dual labelled with SPIONs and gadolinium-based chelates (GdDTPA). The viability, proliferation rate, and differentiation potential of the labelled cells were then evaluated. The feasibility of this MRI technique to distinguish between live and dead cells was next evaluated using MRI phantoms, and in vivo using both immune-competent and immune-deficient mice, following the induction of brain injury in the mice. All results were validated with bioluminescence imaging. In live cells, a negative (T2/T2*) MRI contrast predominates, and is used to track cell delivery and cell migration. Upon cell death, a diffused positive (T1) MRI contrast is generated in the vicinity of the dead cells, and serves as an imaging marker for cell death. Ultimately, this technique could be used to manage stem cell therapies. PMID:26330231

Imaging transplanted stem cells in real time using an MRI dual-contrast method.

PubMed

Ngen, Ethel J; Wang, Lee; Kato, Yoshinori; Krishnamachary, Balaji; Zhu, Wenlian; Gandhi, Nishant; Smith, Barbara; Armour, Michael; Wong, John; Gabrielson, Kathleen; Artemov, Dmitri

2015-09-02

Stem cell therapies are currently being investigated for the repair of brain injuries. Although exogenous stem cell labelling with superparamagnetic iron oxide nanoparticles (SPIONs) prior to transplantation provides a means to noninvasively monitor stem cell transplantation by magnetic resonance imaging (MRI), monitoring cell death is still a challenge. Here, we investigate the feasibility of using an MRI dual-contrast technique to detect cell delivery, cell migration and cell death after stem cell transplantation. Human mesenchymal stem cells were dual labelled with SPIONs and gadolinium-based chelates (GdDTPA). The viability, proliferation rate, and differentiation potential of the labelled cells were then evaluated. The feasibility of this MRI technique to distinguish between live and dead cells was next evaluated using MRI phantoms, and in vivo using both immune-competent and immune-deficient mice, following the induction of brain injury in the mice. All results were validated with bioluminescence imaging. In live cells, a negative (T2/T2*) MRI contrast predominates, and is used to track cell delivery and cell migration. Upon cell death, a diffused positive (T1) MRI contrast is generated in the vicinity of the dead cells, and serves as an imaging marker for cell death. Ultimately, this technique could be used to manage stem cell therapies.

Role of CD146 Enrichment in Purification of Stem Cells Derived from Dental Pulp Polyp.

PubMed

Tavangar, Maryam Sadat; Hosseini, Seyed-Mojtaba; Dehghani-Nazhvani, Ali; Monabati, Ahmad

2017-01-01

Hyperplastic pulpitis (pulp polyp) tissues contains cells with stem cell properties similar to that of the dental pulp stem cells (DPSCs). It has also been shown that CD146 enrichment can homogenize the cultures of DPSCs and enhance the colony forming potentials of their cultures. This study determines whether CD146 enrichment can help purifying the stem cells from heterogeneous cultures of the pulp polyp derived stem cells (PPSCs). Healthy dental pulps and pulp polyp tissues were enzymatically digested and the harvested single cells were sorted according to the presence of CD146 marker. The sorted cells were seeded directly for colony forming unit (CFU) assays of the negative and positive portions. Flowcytometric antigen panel and differentiation assays were used to see if these cells conform with mesenchymal stems cells (MSCs) definition. Differences between the between groups was assessed using independent t-test. The level of significance was set at 0.05. Normal pulp tissue derived cells formed higher colonies (42.5±16.8 per 10 4 cells) than the pulp polyp (17.75±8.9 per 10 4 cells) ( P =0.015). The CD146 positive portion of the polyp derived cells formed an average of 91.5±29.7 per 10 4 cells per CFU. On the other hand, CD146 negative portion did not show any colonies ( P <0.001). Both resources showed cells with flowcytometric antigen panel and differentiation potentials conforming to MSC definition. The entire CFU of PPSCs were formed within CD146 enriched portion. It seems that CD146 enrichment may reduce the number of possible fibroblasts of the pulp polyps and may further homogenize the culture of the PPSCs.

Detection of Catheter-Related Bloodstream Infections by the Differential-Time-to-Positivity Method and Gram Stain-Acridine Orange Leukocyte Cytospin Test in Neutropenic Patients after Hematopoietic Stem Cell Transplantation

PubMed Central

Krause, R.; Auner, H. W.; Gorkiewicz, G.; Wölfler, A.; Daxboeck, F.; Linkesch, W.; Krejs, G. J.; Wenisch, C.; Reisinger, E. C.

2004-01-01

For febrile neutropenic patients who received hematopoietic stem cell transplantation, the Gram stain-acridine orange leukocyte cytospin (AOLC) test and the differential-time-to-positivity method (DTP) were performed. As a diagnostic tool for catheter-related bloodstream infections in these patients, the Gram stain-AOLC test has a lower sensitivity than does the DTP method but acceptable positive and negative predictive values. PMID:15472355

NSAID-activated gene 1 mediates pro-inflammatory signaling activation and paclitaxel chemoresistance in type I human epithelial ovarian cancer stem-like cells.

PubMed

Kim, Ki-Hyung; Park, Seong-Hwan; Do, Kee Hun; Kim, Juil; Choi, Kyung Un; Moon, Yuseok

2016-11-01

Epithelial ovarian cancer (EOC) remains the most lethal gynecologic malignancy in developed countries. Chronic endogenous sterile pro-inflammatory responses are strongly linked to EOC progression and chemoresistance to anti-cancer therapeutics. In the present study, the activity of epithelial NF-κB, a key pro-inflammatory transcription factor, was enhanced with the progress of EOC. This result was mechanistically linked with an increased expression of NSAID-Activated Gene 1 (NAG-1) in MyD88-positive type I EOC stem-like cells, compared with that in MyD88-negative type II EOC cells. Elevated NAG-1 as a potent biomarker of poor prognosis in the ovarian cancer was positively associated with the levels of NF-κB activation, chemokines and stemness markers in type I EOC cells. In terms of signal transduction, NAG-1-activated SMAD-linked and non-canonical TGFβ-activated kinase 1 (TAK-1)-activated pathways contributed to NF-κB activation and the subsequent induction of some chemokines and cancer stemness markers. In addition to effects on NF-κB-dependent gene regulation, NAG-1 was involved in expression of EGF receptor and subsequent activation of EGF receptor-linked signaling. The present study also provided evidences for links between NAG-1-linked signaling and chemoresistance in ovarian cancer cells. NAG-1 and pro-inflammatory NF-κB were positively associated with resistance to paclitaxel in MyD88-positive type I EOC cells. Mechanistically, this chemoresistance occurred due to enhanced activation of the SMAD-4- and non-SMAD-TAK-1-linked pathways. All of the present data suggested NAG-1 protein as a crucial mediator of EOC progression and resistance to the standard first-line chemotherapy against EOC, particularly in MyD88-positive ovarian cancer stem-like cells.

Characterization of TLX expression in neural stem cells and progenitor cells in adult brains.

PubMed

Li, Shengxiu; Sun, Guoqiang; Murai, Kiyohito; Ye, Peng; Shi, Yanhong

2012-01-01

TLX has been shown to play an important role in regulating the self-renewal and proliferation of neural stem cells in adult brains. However, the cellular distribution of endogenous TLX protein in adult brains remains to be elucidated. In this study, we used immunostaining with a TLX-specific antibody to show that TLX is expressed in both neural stem cells and transit-amplifying neural progenitor cells in the subventricular zone (SVZ) of adult mouse brains. Then, using a double thymidine analog labeling approach, we showed that almost all of the self-renewing neural stem cells expressed TLX. Interestingly, most of the TLX-positive cells in the SVZ represented the thymidine analog-negative, relatively quiescent neural stem cell population. Using cell type markers and short-term BrdU labeling, we demonstrated that TLX was also expressed in the Mash1+ rapidly dividing type C cells. Furthermore, loss of TLX expression dramatically reduced BrdU label-retaining neural stem cells and the actively dividing neural progenitor cells in the SVZ, but substantially increased GFAP staining and extended GFAP processes. These results suggest that TLX is essential to maintain the self-renewing neural stem cells in the SVZ and that the GFAP+ cells in the SVZ lose neural stem cell property upon loss of TLX expression. Understanding the cellular distribution of TLX and its function in specific cell types may provide insights into the development of therapeutic tools for neurodegenerative diseases by targeting TLX in neural stem/progenitors cells.

Characterization of TLX Expression in Neural Stem Cells and Progenitor Cells in Adult Brains

PubMed Central

Li, Shengxiu; Sun, Guoqiang; Murai, Kiyohito; Ye, Peng; Shi, Yanhong

2012-01-01

TLX has been shown to play an important role in regulating the self-renewal and proliferation of neural stem cells in adult brains. However, the cellular distribution of endogenous TLX protein in adult brains remains to be elucidated. In this study, we used immunostaining with a TLX-specific antibody to show that TLX is expressed in both neural stem cells and transit-amplifying neural progenitor cells in the subventricular zone (SVZ) of adult mouse brains. Then, using a double thymidine analog labeling approach, we showed that almost all of the self-renewing neural stem cells expressed TLX. Interestingly, most of the TLX-positive cells in the SVZ represented the thymidine analog-negative, relatively quiescent neural stem cell population. Using cell type markers and short-term BrdU labeling, we demonstrated that TLX was also expressed in the Mash1+ rapidly dividing type C cells. Furthermore, loss of TLX expression dramatically reduced BrdU label-retaining neural stem cells and the actively dividing neural progenitor cells in the SVZ, but substantially increased GFAP staining and extended GFAP processes. These results suggest that TLX is essential to maintain the self-renewing neural stem cells in the SVZ and that the GFAP+ cells in the SVZ lose neural stem cell property upon loss of TLX expression.Understanding the cellular distribution of TLX and its function in specific cell types may provide insights into the development of therapeutic tools for neurodegenerative diseases by targeting TLX in neural stem/progenitors cells. PMID:22952666

Pituitary stem cells drop their mask.

PubMed

Vankelecom, Hugo

2012-01-01

The pituitary gland represents the organism's endocrine hub, integrating central and peripheral inputs to generate the appropriate hormonal signals that govern key physiological processes. To meet the changing endocrine demands, the gland has to flexibly remodel its hormone-producing cell compartment. Mechanisms underlying pituitary cellular plasticity, as well as homeostatic turnover, are poorly understood. Similar to other tissues, resident stem cells may participate in the generation of newborn cells. Although in the past recurrently postulated to exist, pituitary stem cells remained obscure until the quest recently regained momentum, resulting in a surge of studies that designated very strong candidates for the stem/progenitor cell position. The cells identified express stem cell-associated markers and signaling factors, as well as transcriptional regulators that play essential roles during pituitary embryogenesis. They exhibit the stem cell properties of multilineage differentiation and prominent efflux capacity ("side population" phenotype), and display a topographical pattern reminiscent of niche-like configurations. Yet, the stem cell tenet of long-term self-renewal remains to be unequivocally demonstrated. Taken together, pituitary stem cells commence to drop their mask. While their "face gradually becomes visible, the "character" they play in the pituitary awaits further disclosure. The aim of this review is to highlight the recent progress in pituitary stem/progenitor cell identification by sketching the historical context, describing the new findings with inclusion of critical and cautionary reflections, proposing a tentative stem/progenitor cell model, and pointing out remaining gaps and challenges. The recent acceleration in pituitary stem cell research may announce an exciting era in this endocrine field.

High oxygen condition facilitates the differentiation of mouse and human pluripotent stem cells into pancreatic progenitors and insulin-producing cells.

PubMed

Hakim, Farzana; Kaitsuka, Taku; Raeed, Jamiruddin Mohd; Wei, Fan-Yan; Shiraki, Nobuaki; Akagi, Tadayuki; Yokota, Takashi; Kume, Shoen; Tomizawa, Kazuhito

2014-04-04

Pluripotent stem cells have potential applications in regenerative medicine for diabetes. Differentiation of stem cells into insulin-producing cells has been achieved using various protocols. However, both the efficiency of the method and potency of differentiated cells are insufficient. Oxygen tension, the partial pressure of oxygen, has been shown to regulate the embryonic development of several organs, including pancreatic β-cells. In this study, we tried to establish an effective method for the differentiation of induced pluripotent stem cells (iPSCs) into insulin-producing cells by culturing under high oxygen (O2) conditions. Treatment with a high O2 condition in the early stage of differentiation increased insulin-positive cells at the terminus of differentiation. We found that a high O2 condition repressed Notch-dependent gene Hes1 expression and increased Ngn3 expression at the stage of pancreatic progenitors. This effect was caused by inhibition of hypoxia-inducible factor-1α protein level. Moreover, a high O2 condition activated Wnt signaling. Optimal stage-specific treatment with a high O2 condition resulted in a significant increase in insulin production in both mouse embryonic stem cells and human iPSCs and yielded populations containing up to 10% C-peptide-positive cells in human iPSCs. These results suggest that culturing in a high O2 condition at a specific stage is useful for the efficient generation of insulin-producing cells.

Generation and characterization of rat liver stem cell lines and their engraftment in a rat model of liver failure

PubMed Central

Kuijk, Ewart W.; Rasmussen, Shauna; Blokzijl, Francis; Huch, Meritxell; Gehart, Helmuth; Toonen, Pim; Begthel, Harry; Clevers, Hans; Geurts, Aron M.; Cuppen, Edwin

2016-01-01

The rat is an important model for liver regeneration. However, there is no in vitro culture system that can capture the massive proliferation that can be observed after partial hepatectomy in rats. We here describe the generation of rat liver stem cell lines. Rat liver stem cells, which grow as cystic organoids, were characterized by high expression of the stem cell marker Lgr5, by the expression of liver progenitor and duct markers, and by low expression of hepatocyte markers, oval cell markers, and stellate cell markers. Prolonged cultures of rat liver organoids depended on high levels of WNT-signalling and the inhibition of BMP-signaling. Upon transplantation of clonal lines to a Fah−/− Il2rg−/− rat model of liver failure, the rat liver stem cells engrafted into the host liver where they differentiated into areas with FAH and Albumin positive hepatocytes. Rat liver stem cell lines hold potential as consistent reliable cell sources for pharmacological, toxicological or metabolic studies. In addition, rat liver stem cell lines may contribute to the development of regenerative medicine in liver disease. To our knowledge, the here described liver stem cell lines represent the first organoid culture system in the rat. PMID:26915950

How electromagnetic fields can influence adult stem cells: positive and negative impacts.

PubMed

Maziarz, Aleksandra; Kocan, Beata; Bester, Mariusz; Budzik, Sylwia; Cholewa, Marian; Ochiya, Takahiro; Banas, Agnieszka

2016-04-18

The electromagnetic field (EMF) has a great impact on our body. It has been successfully used in physiotherapy for the treatment of bone disorders and osteoarthritis, as well as for cartilage regeneration or pain reduction. Recently, EMFs have also been applied in in vitro experiments on cell/stem cell cultures. Stem cells reside in almost all tissues within the human body, where they exhibit various potential. These cells are of great importance because they control homeostasis, regeneration, and healing. Nevertheless, stem cells when become cancer stem cells, may influence the pathological condition. In this article we review the current knowledge on the effects of EMFs on human adult stem cell biology, such as proliferation, the cell cycle, or differentiation. We present the characteristics of the EMFs used in miscellaneous assays. Most research has so far been performed during osteogenic and chondrogenic differentiation of mesenchymal stem cells. It has been demonstrated that the effects of EMF stimulation depend on the intensity and frequency of the EMF and the time of exposure to it. However, other factors may affect these processes, such as growth factors, reactive oxygen species, and so forth. Exploration of this research area may enhance the development of EMF-based technologies used in medical applications and thereby improve stem cell-based therapy and tissue engineering.

Stem cell research as innovation: expanding the ethical and policy conversation.

PubMed

Dresser, Rebecca

2010-01-01

Research using human embryonic stem cells raises an array of complex ethical issues, including, but by no means limited to, the moral status of developing human life. Unfortunately much of the public discussion fails to take into account this complexity. Advocacy for liberal and conservative positions on human embryonic stem cell research can be simplistic and misleading. Ethical concepts such as truth-telling, scientific integrity, and social justice should be part of the debate over federal support for human embryonic stem cell research. Moreover, the debate should be conducted in accord with principles of deliberative democracy, including respect for people holding competing views.

Push-pull strategy in the regulation of postembryonic root development.

PubMed

Choe, Goh; Lee, Ji-Young

2017-02-01

Unlike animals, plants continue to grow throughout their lives. The stem cell niche, protected in meristems of shoots and roots, enables this process. In the root, stem cells produce precursors for highly organized cell types via asymmetric cell divisions. These precursors, which are "transit-amplifying cells," actively divide for several rounds before entering into differentiation programs. In this review, we highlight positive feedback regulation between shoot- and root-ward signals during the postembryonic root growth, which is reminiscent of a "push-pull strategy" in business parlance. This property of molecular networks underlies the regulation of stem cells and their organizer, the "quiescent center," as well as of the signaling between stem cell niche, transit-amplifying cells, and beyond. Copyright © 2016 Elsevier Ltd. All rights reserved.

Curcumin Promotes Autophagic Survival of a Sub-Set of Colon Cancer Stem Cells, which are Ablated by DCLK1-siRNA

PubMed Central

Kantara, Carla; O’Connell, Malaney; Sarkar, Shubhashish; Moya, Stephanie; Ullrich, Robert; Singh, Pomila

2014-01-01

Curcumin is known to induce apoptosis of cancer cells by different mechanisms, but its effects on cancer stem-like cells have been less investigated. Here we report that curcumin promotes the survival of DCLK1-positive colon cancer stem-like cells (CSC), potentially confounding application of its anticancer properties. At optimal concentrations, curcumin greatly reduced expression levels of stem cell markers (DCLK1/CD44/ALDHA1/Lgr5/Nanog) in 3D spheroid cultures and tumor xenografts derived from colon cancer cells. However, curcumin unexpectedly induced proliferation and autophagic survival of a subset of DCLK1-positive CSCs. Spheroid cultures were disintegrated by curcumin in vitro but re-grew within 30–40 days of treatment, suggesting a survival benefit from autophagy, permitting long-term persistence of CRC. Notably, RNAi-mediated silencing of DCLK1 triggered apoptotic cell death of colon cancer cells in vitro and in vivo, and abolished CRC survival in response to curcumin; combination of DCLK1-siRNA and curcumin dramatically reversed CSC phenotype, contributing to attenuation of the growth of spheroid cultures and tumor xenografts. Taken together, our findings confirm a role of DCLK1 in colon cancer stem cells and highlight DCLK1 as a target to enhance antitumor properties of curcumin. PMID:24626093

Trophic Effects and Regenerative Potential of Mobilized Mesenchymal Stem Cells From Bone Marrow and Adipose Tissue as Alternative Cell Sources for Pulp/Dentin Regeneration.

PubMed

Murakami, Masashi; Hayashi, Yuki; Iohara, Koichiro; Osako, Yohei; Hirose, Yujiro; Nakashima, Misako

2015-01-01

Dental pulp stem cell (DPSC) subsets mobilized by granulocyte-colony-stimulating factor (G-CSF) are safe and efficacious for complete pulp regeneration. The supply of autologous pulp tissue, however, is very limited in the aged. Therefore, alternative sources of mesenchymal stem/progenitor cells (MSCs) are needed for the cell therapy. In this study, DPSCs, bone marrow (BM), and adipose tissue (AD)-derived stem cells of the same individual dog were isolated using G-CSF-induced mobilization (MDPSCs, MBMSCs, and MADSCs). The positive rates of CXCR4 and G-CSFR in MDPSCs were similar to MADSCs and were significantly higher than those in MBMSCs. Trophic effects of MDPSCs on angiogenesis, neurite extension, migration, and antiapoptosis were higher than those of MBMSCs and MADSCs. Pulp-like loose connective tissues were regenerated in all three MSC transplantations. Significantly higher volume of regenerated pulp and higher density of vascularization and innervation were observed in response to MDPSCs compared to MBMSC and MADSC transplantation. Collagenous matrix containing dentin sialophosphoprotein (DSPP)-positive odontoblast-like cells was the highest in MBMSCs and significantly higher in MADSCs compared to MDPSCs. MBMSCs and MADSCs, therefore, have potential for pulp regeneration, although the volume of regenerated pulp tissue, angiogenesis, and reinnervation, were less. Thus, in conclusion, an alternative cell source for dental pulp/dentin regeneration are stem cells from BM and AD tissue.

Implantation of Allogenic Synovial Stem Cells Promotes Meniscal Regeneration in a Rabbit Meniscal Defect Model

PubMed Central

Horie, Masafumi; Driscoll, Matthew D.; Sampson, H. Wayne; Sekiya, Ichiro; Caroom, Cyrus T.; Prockop, Darwin J.; Thomas, Darryl B.

2012-01-01

Update This article was updated on May 16, 2012, because of a previous error. The legend for Figures 7-A and 7-B that had previously read “Representative macroscopic appearance (Fig. 7-A) and histological sections (Fig. 7-B) of the meniscal defect one day to twelve weeks after the implantation of GFP-positive green fluorescent protein under fluorescence” now reads “Representative macroscopic appearance (Fig. 7-A) and histological sections (Fig. 7-B) of the meniscal defect one day to twelve weeks after the implantation of GFP-positive synovial mesenchymal stem cells under fluorescence.” Background: Indications for surgical meniscal repair are limited, and failure rates remain high. Thus, new ways to augment repair and stimulate meniscal regeneration are needed. Mesenchymal stem cells are multipotent cells present in mature individuals and accessible from peripheral connective tissue sites, including synovium. The purpose of this study was to quantitatively evaluate the effect of implantation of synovial tissue-derived mesenchymal stem cells on meniscal regeneration in a rabbit model of partial meniscectomy. Methods: Synovial mesenchymal stem cells were harvested from the knee of one New Zealand White rabbit, expanded in culture, and labeled with a fluorescent marker. A reproducible 1.5-mm cylindrical defect was created in the avascular portion of the anterior horn of the medial meniscus bilaterally in fifteen additional rabbits. Allogenic synovial mesenchymal stem cells suspended in phosphate-buffered saline solution were implanted into the right knees, and phosphate-buffered saline solution alone was placed in the left knees. Meniscal regeneration was evaluated histologically at four, twelve, and twenty-four weeks for (1) quantity and (2) quality (with use of an established three-component scoring system). A similar procedure was performed in four additional rabbits with use of green fluorescent protein-positive synovial mesenchymal stem cells for the purpose of tracking progeny following implantation. Results: The quantity of regenerated tissue in the group that had implantation of synovial mesenchymal stem cells was greater at all end points, reaching significance at four and twelve weeks (p < 0.05). Tissue quality scores were also superior in knees treated with mesenchymal stem cells compared with controls at all end points, achieving significance at twelve and twenty-four weeks (3.8 versus 2.8 at four weeks [p = 0.29], 5.7 versus 1.7 at twelve weeks [p = 0.008], and 6.0 versus 3.9 at twenty-four weeks [p = 0.021]). Implanted cells adhered to meniscal defects and were observed in the regenerated tissue, where they differentiated into type-I and II collagen-expressing cells, at up to twenty-four weeks. Conclusions: Synovial mesenchymal stem cells adhere to sites of meniscal injury, differentiate into cells resembling meniscal fibrochondrocytes, and enhance both quality and quantity of meniscal regeneration. Clinical Relevance: These results may stimulate further exploration into the utility of synovial mesenchymal stem cells in the treatment of meniscal injury in large animals and humans. PMID:22517386

PERSPECTIVES ON CANCER STEM CELLS IN OSTEOSARCOMA

PubMed Central

Basu-Roy, Upal; Basilico, Claudio; Mansukhani, Alka

2012-01-01

Osteosarcoma is an aggressive pediatric tumor of growing bones that, despite surgery and chemotherapy, is prone to relapse. These mesenchymal tumors are derived from progenitor cells in the osteoblast lineage that have accumulated mutations to escape cell cycle checkpoints leading to excessive proliferation and defects in their ability to differentiate appropriately into mature bone-forming osteoblasts. Like other malignant tumors, osteosarcoma is often heterogeneous, consisting of phenotypically distinct cells with features of different stages of differentiation. The cancer stem cell hypothesis posits that tumors are maintained by stem cells and it is the incomplete eradication of a refractory population of tumor-initiating stem cells that accounts for drug resistance and tumor relapse. In this review we present our current knowledge about the biology of osteosarcoma stem cells from mouse and human tumors, highlighting new insights and unresolved issues in the identification of this elusive population. We focus on factors and pathways that are implicated in maintaining such cells, and differences from paradigms of epithelial cancers. Targeting of the cancer stem cells in osteosarcoma is a promising avenue to explore to develop new therapies for this devastating childhood cancer. PMID:22659734

Huperzine A protects neural stem cells against Aβ-induced apoptosis in a neural stem cells and microglia co-culture system

PubMed Central

Zhu, Ning; Lin, Jizong; Wang, Kewan; Wei, Meidan; Chen, Qingzhuang; Wang, Yong

2015-01-01

Objectives: This study aims to explore whether Huperzine A (HupA) could protect neural stem cells against amyloid beta-peptide Aβ induced apoptosis in a neural stem cells (NSCs) and microglia co-culture system. Methods: Rat NSCs and microglial cells were isolated, cultured and identified with immunofluorescence Assays (IFA). Co-culture systems of NSCs and microglial cells were employed using Transwell Permeable Supports. The effects of Aβ1-42 on NSCs were studied in 4 groups using co-culture systems: NSCs, Aβ+NSCs, co-culture and Aβ+co-culture groups. Bromodeoxyuridine (BrdU) incorporation and flow cytometry were utilized to assess the differences of proliferation, differentiation and apoptosis of NSCs between the groups. LQ test was performed to assess the amounts of IL-6, TNF-α and MIP-α secreted, and flow cytometry and Western blotting were used to assess apoptosis of NSCs and the expressions of Bcl-2 and Bax in each group. Results: IFA results showed that isolated rat NSCs were nestin-positive and microglial cells were CD11b/c-positive. Among all the groups, the Aβ+co-culture group has the lowest BrdU expression level, the lowest MAP2-positive, ChAT-positive cell counts and the highest NSC apoptosis rate. Smaller amounts of IL-6, TNF-α and MIP-α were being secreted by microglial cells in the HupA+Aβ+co-culture group compared with those in the Aβ+ co-culture group. Also the Bcl-2: Bax ratio was much higher in the HupA+Aβ+co-culture group than in the Aβ+co-culture group. Conclusions: HupA inhibits cell apoptosis through restraining microglia’s inflammatory response induced by Aβ1-42. PMID:26261518

Embryonic Stem Cell-Based Cardiopatches Improve Cardiac Function in Infarcted Rats

PubMed Central

Vallée, Jean-Paul; Hauwel, Mathieu; Lepetit-Coiffé, Matthieu; Bei, Wang; Montet-Abou, Karin; Meda, Paolo; Gardier, Stephany; Zammaretti, Prisca; Kraehenbuehl, Thomas P.; Herrmann, Francois; Hubbell, Jeffrey A.

2012-01-01

Pluripotent stem cell-seeded cardiopatches hold promise for in situ regeneration of infarcted hearts. Here, we describe a novel cardiopatch based on bone morphogenetic protein 2-primed cardiac-committed mouse embryonic stem cells, embedded into biodegradable fibrin matrices and engrafted onto infarcted rat hearts. For in vivo tracking of the engrafted cardiac-committed cells, superparamagnetic iron oxide nanoparticles were magnetofected into the cells, thus enabling detection and functional evaluation by high-resolution magnetic resonance imaging. Six weeks after transplantation into infarcted rat hearts, both local (p < .04) and global (p < .015) heart function, as well as the left ventricular dilation (p < .0011), were significantly improved (p < .001) as compared with hearts receiving cardiopatches loaded with iron nanoparticles alone. Histological analysis revealed that the fibrin scaffolds had degraded over time and clusters of myocyte enhancer factor 2-positive cardiac-committed cells had colonized most of the infarcted myocardium, including the fibrotic area. De novo CD31-positive blood vessels were formed in the vicinity of the transplanted cardiopatch. Altogether, our data provide evidence that stem cell-based cardiopatches represent a promising therapeutic strategy to achieve efficient cell implantation and improved global and regional cardiac function after myocardial infarction. PMID:23197784

Bone marrow cells stained by azide-conjugated Alexa fluors in the absence of an alkyne label.

PubMed

Lin, Guiting; Ning, Hongxiu; Banie, Lia; Qiu, Xuefeng; Zhang, Haiyang; Lue, Tom F; Lin, Ching-Shwun

2012-09-01

Thymidine analog 5-ethynyl-2'-deoxyuridine (EdU) has recently been introduced as an alternative to 5-bromo-2-deoxyuridine (BrdU) for cell labeling and tracking. Incorporation of EdU into replicating DNA can be detected by azide-conjugated fluors (eg, Alexa-azide) through a Cu(i)-catalyzed click reaction between EdU's alkyne moiety and azide. While this cell labeling method has proven to be valuable for tracking transplanted stem cells in various tissues, we have found that some bone marrow cells could be stained by Alexa-azide in the absence of EdU label. In intact rat femoral bone marrow, ~3% of nucleated cells were false-positively stained, and in isolated bone marrow cells, ~13%. In contrast to true-positive stains, which localize in the nucleus, the false-positive stains were cytoplasmic. Furthermore, while true-positive staining requires Cu(i), false-positive staining does not. Reducing the click reaction time or reducing the Alexa-azide concentration failed to improve the distinction between true- and false-positive staining. Hematopoietic and mesenchymal stem cell markers CD34 and Stro-1 did not co-localize with the false-positively stained cells, and these cells' identity remains unknown.

Expression of the stem cell factor in fibroblasts, endothelial cells, and macrophages in periapical tissues in human chronic periapical diseases.

PubMed

Shen, S Q; Wang, R; Huang, S G

2017-03-08

Stem cell factor (SCF), an important stem cell cytokine, has multiple functions. Fibroblasts (FBs), mature mast cells, endothelial cells (ECs), and eosinophil granulocytes can produce SCF in the inflammatory process. Therefore, we aimed to observe SCF expression in FBs, ECs, and macrophages (MPs) in periapical tissues in human chronic periapical disease and investigate the effects of cells expressing SCF in pathogenesis of the disease. Healthy (N = 20), periapical cyst (N = 15), and periapical granuloma (N = 15) tissues were fixed in 10% formalin for 48 h, embedded in paraffin, and stained with hematoxylin and eosin to observe histological changes. SCF expression was observed in FBs, ECs, and MPs in periapical tissues by double immunofluorescence. CD334, CD31, and CD14 are specific markers of FBs, ECs, and MPs, respectively. Results showed that densities of CD334-SCF double-positive FBs, CD31-SCF double-positive ECs, and CD14-SCF double-positive MPs were significantly increased in periapical tissue groups (P < 0.01). There were no significant differences in CD334-SCF double-positive FB and CD31-SCF double-positive EC levels between the two periapical tissue groups (P > 0.05). CD14-SCF double-positive MP density was considerably higher in periapical granulomas than in cysts (P < 0.01). FB, EC, and MP levels were significantly high and densities of CD334-SCF double-positive FBs, CD31-SCF double-positive ECs, and CD14-SCF double-positive MPs improved considerably in chronic periapical tissues, suggesting that the cells might be related to occurrence, development, and pathogenesis of chronic periapical disease.

Radiation-Induced Dedifferentiation of Head and Neck Cancer Cells Into Cancer Stem Cells Depends on Human Papillomavirus Status.

PubMed

Vlashi, Erina; Chen, Allen M; Boyrie, Sabrina; Yu, Garrett; Nguyen, Andrea; Brower, Philip A; Hess, Clayton B; Pajonk, Frank

2016-04-01

To test the hypothesis that the radiation response of cancer stem cells (CSCs) in human papillomavirus (HPV)-positive and HPV-negative head and neck squamous cell carcinoma (HNSCC) differs and is not reflected in the radiation response of the bulk tumor populations, that radiation therapy (RT) can dedifferentiate non-stem HNSCC cells into CSCs, and that radiation-induced dedifferentiation depends on the HPV status. Records of a cohort of 162 HNSCC patients were reviewed, and their outcomes were correlated with their HPV status. Using a panel of HPV-positive and HPV-negative HNSCC cell lines expressing a reporter for CSCs, we characterized HPV-positive and HPV-negative lines via flow cytometry, sphere-forming capacity assays in vitro, and limiting dilution assays in vivo. Non-CSCs were treated with different doses of radiation, and the dedifferentiation of non-CSCs into CSCs was investigated via flow cytometry and quantitative reverse transcription-polymerase chain reaction for re-expression of reprogramming factors. Patients with HPV-positive tumors have superior overall survival and local-regional control. Human papillomavirus-positive HNSCC cell lines have lower numbers of CSCs, which inversely correlates with radiosensitivity. Human papillomavirus-negative HNSCC cell lines lack hierarchy owing to enhanced spontaneous dedifferentiation. Non-CSCs from HPV-negative lines show enhanced radiation-induced dedifferentiation compared with HPV-positive lines, and RT induced re-expression of Yamanaka reprogramming factors. Supporting the favorable prognosis of HPV-positive HNSCCs, we show that (1) HPV-positive HNSCCs have a lower frequency of CSCs; (2) RT can dedifferentiate HNSCC cells into CSCs; and (3) radiation-induced dedifferentiation depends on the HPV status of the tumor. Copyright © 2016 Elsevier Inc. All rights reserved.

Colon Stem Cell and Crypt Dynamics Exposed by Cell Lineage Reconstruction

PubMed Central

Itzkovitz, Shalev; Elbaz, Judith; Maruvka, Yosef E.; Segev, Elad; Shlush, Liran I.; Dekel, Nava; Shapiro, Ehud

2011-01-01

Stem cell dynamics in vivo are often being studied by lineage tracing methods. Our laboratory has previously developed a retrospective method for reconstructing cell lineage trees from somatic mutations accumulated in microsatellites. This method was applied here to explore different aspects of stem cell dynamics in the mouse colon without the use of stem cell markers. We first demonstrated the reliability of our method for the study of stem cells by confirming previously established facts, and then we addressed open questions. Our findings confirmed that colon crypts are monoclonal and that, throughout adulthood, the process of monoclonal conversion plays a major role in the maintenance of crypts. The absence of immortal strand mechanism in crypts stem cells was validated by the age-dependent accumulation of microsatellite mutations. In addition, we confirmed the positive correlation between physical and lineage proximity of crypts, by showing that the colon is separated into small domains that share a common ancestor. We gained new data demonstrating that colon epithelium is clustered separately from hematopoietic and other cell types, indicating that the colon is constituted of few progenitors and ruling out significant renewal of colonic epithelium from hematopoietic cells during adulthood. Overall, our study demonstrates the reliability of cell lineage reconstruction for the study of stem cell dynamics, and it further addresses open questions in colon stem cells. In addition, this method can be applied to study stem cell dynamics in other systems. PMID:21829376

Detection of the relatively slow-growing Propionibacterium acnes in seven matrices of blood components and advanced therapeutical medicinal products.

PubMed

Arlt, Nicole; Rothe, Remo; Juretzek, Thomas; Peltroche, Heidrun; Tonn, Torsten; Moog, Rainer

2017-06-01

Relatively slow-growing bacteria like Propionibacterium acnes represent a challenge for quality control investigations in sterility release testing of blood components and advanced therapeutic medicinal products (ATMPs). A convenient validation with 7 matrices was performed using buffy coat, stem cells, islet cells, natural killer cells, red blood cells, platelets and plasma in the microbial detection system Bact/Alert ® 3D incubator. All matrix samples were spiked twofold with Propionibacterium acnes with approximately 50 colony forming units (CFUs) per bottle in iAST and iNST culture bottles for 14days using a multishot bioball. Additionally, the stem cell preparations were also incubated in iFAplus and iFNplus culture bottles, which include neutralizing polymers. The Bact/Alert ® 3D-System detected Propionibacterium acnes in anaerobic culture bottles in buffy coat [3.3 d (=positive signal day to detection as mean value)], red blood cells [3.2 d], platelets [3.3], plasma [3.7 d], natural killer cells [3.3 d] and islet cells [4.9 d], resp. No growth of Propionibacterium was found in autologous stem cells using iAST and iNST culture bottles. However, Propionibacterium was safely detected in the iFNplus culture bottle with polymers in the stem cell matrix. A successful validation of media was performed. Our study shows that Bact/Alert ® 3D-System safely detects the relatively slow-growing bacterium Propionibacterium acnes in different matrices in a practical way except stem cells. Using the iFNplus culture bottle for stem cell products positive signals were observed. Copyright © 2017 Elsevier Ltd. All rights reserved.

Transcriptional control of stem cell fate by E2Fs and pocket proteins

PubMed Central

Julian, Lisa M.; Blais, Alexandre

2015-01-01

E2F transcription factors and their regulatory partners, the pocket proteins (PPs), have emerged as essential regulators of stem cell fate control in a number of lineages. In mammals, this role extends from both pluripotent stem cells to those encompassing all embryonic germ layers, as well as extra-embryonic lineages. E2F/PP-mediated regulation of stem cell decisions is highly evolutionarily conserved, and is likely a pivotal biological mechanism underlying stem cell homeostasis. This has immense implications for organismal development, tissue maintenance, and regeneration. In this article, we discuss the roles of E2F factors and PPs in stem cell populations, focusing on mammalian systems. We discuss emerging findings that position the E2F and PP families as widespread and dynamic epigenetic regulators of cell fate decisions. Additionally, we focus on the ever expanding landscape of E2F/PP target genes, and explore the possibility that E2Fs are not simply regulators of general ‘multi-purpose’ cell fate genes but can execute tissue- and cell type-specific gene regulatory programs. PMID:25972892

Analysis for apoptosis and necrosis on adipocytes, stromal vascular fraction, and adipose-derived stem cells in human lipoaspirates after liposuction.

PubMed

Wang, Wei Z; Fang, Xin-Hua; Williams, Shelley J; Stephenson, Linda L; Baynosa, Richard C; Wong, Nancy; Khiabani, Kayvan T; Zamboni, William A

2013-01-01

Adipose-derived stem cells have become the most studied adult stem cells. The authors examined the apoptosis and necrosis rates for adipocyte, stromal vascular fraction, and adipose-derived stem cells in fresh human lipoaspirates. Human lipoaspirate (n = 8) was harvested using a standard liposuction technique. Stromal vascular fraction cells were separated from adipocytes and cultured to obtain purified adipose-derived stem cells. A panel of stem cell markers was used to identify the surface phenotypes of cultured adipose-derived stem cells. Three distinct stem cell subpopulations (CD90/CD45, CD105/CD45, and CD34/CD31) were selected from the stromal vascular fraction. Apoptosis and necrosis were determined by annexin V/propidium iodide assay and analyzed by flow cytometry. The cultured adipose-derived stem cells demonstrated long-term proliferation and differentiation evidenced by cell doubling time and positive staining with oil red O and alkaline phosphatase. Isolated from lipoaspirates, adipocytes exhibited 19.7 ± 3.7 percent apoptosis and 1.1 ± 0.3 percent necrosis; stromal vascular fraction cells revealed 22.0 ± 6.3 percent of apoptosis and 11.2 ± 1.9 percent of necrosis; stromal vascular fraction cells had a higher rate of necrosis than adipocytes (p < 0.05). Among the stromal vascular fraction cells, 51.1 ± 3.7 percent expressed CD90/CD45, 7.5 ± 1.0 percent expressed CD105/CD45, and 26.4 ± 3.8 percent expressed CD34/CD31. CD34/CD31 adipose-derived stem cells had lower rates of apoptosis and necrosis compared with CD105/CD45 adipose-derived stem cells (p < 0.05). Adipose-derived stem cells had a higher rate of apoptosis and necrosis than adipocytes. However, the extent of apoptosis and necrosis was significantly different among adipose-derived stem cell subpopulations.

HOX and TALE signatures specify human stromal stem cell populations from different sources.

PubMed

Picchi, Jacopo; Trombi, Luisa; Spugnesi, Laura; Barachini, Serena; Maroni, Giorgia; Brodano, Giovanni Barbanti; Boriani, Stefano; Valtieri, Mauro; Petrini, Mario; Magli, Maria Cristina

2013-04-01

Human stromal stem cell populations reside in different tissues and anatomical sites, however a critical question related to their efficient use in regenerative medicine is whether they exhibit equivalent biological properties. Here, we compared cellular and molecular characteristics of stromal stem cells derived from the bone marrow, at different body sites (iliac crest, sternum, and vertebrae) and other tissues (dental pulp and colon). In particular, we investigated whether homeobox genes of the HOX and TALE subfamilies might provide suitable markers to identify distinct stromal cell populations, as HOX proteins control cell positional identity and, together with their co-factors TALE, are involved in orchestrating differentiation of adult tissues. Our results show that stromal populations from different sources, although immunophenotypically similar, display distinct HOX and TALE signatures, as well as different growth and differentiation abilities. Stromal stem cells from different tissues are characterized by specific HOX profiles, differing in the number and type of active genes, as well as in their level of expression. Conversely, bone marrow-derived cell populations can be essentially distinguished for the expression levels of specific HOX members, strongly suggesting that quantitative differences in HOX activity may be crucial. Taken together, our data indicate that the HOX and TALE profiles provide positional, embryological and hierarchical identity of human stromal stem cells. Furthermore, our data suggest that cell populations derived from different body sites may not represent equivalent cell sources for cell-based therapeutical strategies for regeneration and repair of specific tissues. Copyright © 2012 Wiley Periodicals, Inc.

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

Role of neural stem cell activity in postweaning development of the sexually dimorphic nucleus of the preoptic area in rats.

PubMed

He, Zhen; Ferguson, Sherry A; Cui, Li; Greenfield, L John; Paule, Merle G

2013-01-01

The sexually dimorphic nucleus of the preoptic area (SDN-POA) has received increased attention due to its apparent sensitivity to estrogen-like compounds found in food and food containers. The mechanisms that regulate SDN-POA volume remain unclear as is the extent of postweaning development of the SDN-POA. Here we demonstrate that the female Sprague-Dawley SDN-POA volume increased from weaning to adulthood, although this increase was not statistically significant as it was in males. The number of cells positive for Ki67, a marker of cell proliferation, in both the SDN-POA and the hypothalamus was significantly higher at weaning than at adulthood in male rats. In contrast, the number of Ki67-positive cells was significantly higher in the hypothalamus but not in the SDN-POA (p>0.05) at weaning than at adulthood in female rats. A subset of the Ki67-positive cells in the SDN-POA displayed the morphology of dividing cells. Nestin-immunoreactivity delineated a potential macroscopic neural stem cell niche in the rostral end of the 3rd ventricle. In conclusion, stem cells may partially account for the sexually dimorphic postweaning development of the SDN-POA.

[Isolation and identification of human periodontal ligament stem cells in vitro].

PubMed

Shen, Tao; Chang, Hui-jun; Jian, Cong-xiang; Yang, Yan-chun; Zhou, Ji-xiang

2011-02-01

To isolate and identify human periodontal ligament stem cells (PDLSC) by improved methods and assess the characteristics of PDLSC ex vivo. The periodontal ligament cells were obtained from the healthy impacted third molars and teeth extracted for orthodontic purposes and used to isolate PDLSC by limiting dilution assay. PDLSC were cultured and expanded in alpha-MEM supplemented with 10% FBS. Colony-forming assay, immunohistochemistry, flow cytometry, osteogenic and adipogenic induction were used to identify PDLSC. The obtained cells had high colony-forming efficiency and were positive staining for vimentin and negative for pancytokeratin. Flow cytometry revealed that the isolated cells were positive for STRO-1 and CD146 antibodies and most were in the G0/G1 phase of cell cycle. Under specific conditions, they could differentiate to the osteoblast and adipocyte lineages in vitro. Limiting dilution assay is an effective method to isolate PDLSC and the single-cell-derived colonies demonstrate the properties of stem cells in vitro.

Stem Cell-Like Differentiation Potentials of Endometrial Side Population Cells as Revealed by a Newly Developed In Vivo Endometrial Stem Cell Assay

PubMed Central

Miyazaki, Kaoru; Maruyama, Tetsuo; Masuda, Hirotaka; Yamasaki, Akiko; Uchida, Sayaka; Oda, Hideyuki; Uchida, Hiroshi; Yoshimura, Yasunori

2012-01-01

Background Endometrial stem/progenitor cells contribute to the cyclical regeneration of human endometrium throughout a woman's reproductive life. Although the candidate cell populations have been extensively studied, no consensus exists regarding which endometrial population represents the stem/progenitor cell fraction in terms of in vivo stem cell activity. We have previously reported that human endometrial side population cells (ESP), but not endometrial main population cells (EMP), exhibit stem cell-like properties, including in vivo reconstitution of endometrium-like tissues when xenotransplanted into immunodeficient mice. The reconstitution efficiency, however, was low presumably because ESP cells alone could not provide a sufficient microenvironment (niche) to support their stem cell activity. The objective of this study was to establish a novel in vivo endometrial stem cell assay employing cell tracking and tissue reconstitution systems and to examine the stem cell properties of ESP through use of this assay. Methodology/Principal Findings ESP and EMP cells isolated from whole endometrial cells were infected with lentivirus to express tandem Tomato (TdTom), a red fluorescent protein. They were mixed with unlabeled whole endometrial cells and then transplanted under the kidney capsule of ovariectomized immunodeficient mice. These mice were treated with estradiol and progesterone for eight weeks and nephrectomized. All of the grafts reconstituted endometrium-like tissues under the kidney capsules. Immunofluorescence revealed that TdTom-positive cells were significantly more abundant in the glandular, stromal, and endothelial cells of the reconstituted endometrium in mice transplanted with TdTom-labeled ESP cells than those with TdTom-labeled EMP cells. Conclusions/Significance We have established a novel in vivo endometrial stem cell assay in which multi-potential differentiation can be identified through cell tracking during in vivo endometrial tissue reconstitution. Using this assay, we demonstrated that ESP cells differentiated into multiple endometrial lineages in the niche provided by whole endometrial cells, indicating that ESP cells are genuine endometrial stem/progenitor cells. PMID:23226538

Canonical Wnt Signaling as a Specific Marker for Normal and Tumorigenic Mammary Stem Cells

DTIC Science & Technology

2012-02-01

These cells were identified by  flow   cytometry  to detect cells that were positive for CD24 and CD49f.   2. We have established that activation of Wnt...09-1-0072 TITLE: Canonical Wnt Signaling as a Specific marker for Normal and Tumorigenic Mammary Stem Cells PRINCIPAL...activation of canonical Wnt signaling may be a very specific marker for mammary stem cells and be a target for transformation that results in the

Histone Deacetylase Inhibitors Stimulate Dedifferentiation of Human Breast Cancer Cells through WNT/β-catenin Signaling

PubMed Central

Debeb, Bisrat G; Lacerda, Lara; Xu, Wei; Larson, Richard; Solley, Travis; Atkinson, Rachel; Sulman, Erik P.; Ueno, Naoto T; Krishnamurthy, Savitri; Reuben, James M; Buchholz, Thomas A; Woodward, Wendy A

2015-01-01

Recent studies have shown that differentiated cancer cells can de-differentiate into cancer stem cells (CSCs) although to date no studies have reported whether this transition is influenced by systemic anti-cancer agents. Valproic acid (VA) is a histone deacetylase (HDAC) inhibitor that promotes self renewal and expansion of hematopietic stem cells and facilitates the generation of induced pluripotent stem cells from somatic cells and is currently being investigated in breast cancer clinical trials. We hypothesized that HDAC inhibitors reprogram differentiated cancer cells towards the more resistant stem cell-like state. Two highly aggressive breast cancer cell lines, SUM159 and MDA-231, were FACS-sorted based on ALDH activity and subsequently ALDH-negative and ALDH-positive cells were treated with one of two known HDAC inhibitors, VA or SAHA (suberoylanilide hydroxamic acid). In addition, primary tumor cells from patients with metastatic breast cancer were evaluated for ALDH activity following treatment with HDAC inhibitors. We demonstrate that single cell sorted ALDH- negative cells spontaneously generated ALDH-positive cells in vitro. Treatment of ALDH-negative cells with HDAC inhibitors promoted the expansion of ALDH-positive cells and increased mammosphere forming efficiency. Most importantly, it significantly increased the tumor-initiating capacity of ALDH- negative cells in limiting dilution outgrowth assays. Moreover, while HDAC inhibitors upregulated β-catenin expression and significantly increased WNT reporter activity, a TCF4 dominant negative construct abolished HDAC-inhibitor induced expansion of CSCs. These results demonstrate that HDAC inhibitors promote the expansion of breast CSCs through dedifferentiation and have important clinical implications for the use of HDAC inhibitors in the treatment of cancer. PMID:22961641

Latent progenitor cells as potential regulators for tympanic membrane regeneration

NASA Astrophysics Data System (ADS)

Kim, Seung Won; Kim, Jangho; Seonwoo, Hoon; Jang, Kyung-Jin; Kim, Yeon Ju; Lim, Hye Jin; Lim, Ki-Taek; Tian, Chunjie; Chung, Jong Hoon; Choung, Yun-Hoon

2015-06-01

Tympanic membrane (TM) perforation, in particular chronic otitis media, is one of the most common clinical problems in the world and can present with sensorineural healing loss. Here, we explored an approach for TM regeneration where the latent progenitor or stem cells within TM epithelial layers may play an important regulatory role. We showed that potential TM stem cells present highly positive staining for epithelial stem cell markers in all areas of normal TM tissue. Additionally, they are present at high levels in perforated TMs, especially in proximity to the holes, regardless of acute or chronic status, suggesting that TM stem cells may be a potential factor for TM regeneration. Our study suggests that latent TM stem cells could be potential regulators of regeneration, which provides a new insight into this clinically important process and a potential target for new therapies for chronic otitis media and other eardrum injuries.

Successful isolation of viable adipose-derived stem cells from human adipose tissue subject to long-term cryopreservation: positive implications for adult stem cell-based therapeutics in patients of advanced age.

PubMed

Devitt, Sean M; Carter, Cynthia M; Dierov, Raia; Weiss, Scott; Gersch, Robert P; Percec, Ivona

2015-01-01

We examined cell isolation, viability, and growth in adipose-derived stem cells harvested from whole adipose tissue subject to different cryopreservation lengths (2-1159 days) from patients of varying ages (26-62 years). Subcutaneous abdominal adipose tissue was excised during abdominoplasties and was cryopreserved. The viability and number of adipose-derived stem cells isolated were measured after initial isolation and after 9, 18, and 28 days of growth. Data were analyzed with respect to cryopreservation duration and patient age. Significantly more viable cells were initially isolated from tissue cryopreserved <1 year than from tissue cryopreserved >2 years, irrespective of patient age. However, this difference did not persist with continued growth and there were no significant differences in cell viability or growth at subsequent time points with respect to cryopreservation duration or patient age. Mesenchymal stem cell markers were maintained in all cohorts tested throughout the duration of the study. Consequently, longer cryopreservation negatively impacts initial live adipose-derived stem cell isolation; however, this effect is neutralized with continued cell growth. Patient age does not significantly impact stem cell isolation, viability, or growth. Cryopreservation of adipose tissue is an effective long-term banking method for isolation of adipose-derived stem cells in patients of varying ages.

Honeybee product therapeutic as stem cells homing for ovary failure.

PubMed

Safitri, Erma; Widiyatno, Thomas V; Prasetyo, R Heru

2016-11-01

Complexity of the method of isolation, cultivation in vitro and the expensive cost of transplantation process of stem cells, it would require an innovation to homing and differentiation of stem cells and increase folliculogenesis. The stem cells homing was achieved through the provision of food or beverages derived from natural materials like honeybee product. Through honeybee product, there will be homing of stem cells and accompany with the sources from the body itself will take place in regeneration of the ovary. Female rats model of degenerative ovary was obtained through food fasting but still have drinking water for 5 days. It caused malnutrition and damage of the ovarian tissue. The administration of 50% honeybee product (T1) was performed for 10 consecutive days, while the positive control group (T0+) was fasted and not given honeybee product and the negative control (T0-) not fasted and without honeybee product. Observations were taken for homing of stem cells, raised of folliculogenesis, differentiation of stem cells, and regeneration of the ovarian tissue using routine H&E staining. Homing of stem cells shown the vascular endothelial growth factor and granulocyte colony-stimulating factor expression; enhancement of folliculogenesis was indicated by an increase of follicle dee Graaf count; enhancement of differentiation of stem cells was indicated by growth differentiation factor-9 expression; and regeneration of ovarian tissue indicated by intact ovarian tissue with growing follicles. Honeybee product can be induced endogenous stem cells in regeneration of ovary failure due to malnutrition.

Stearidonic and eicosapentaenoic acids inhibit interleukin-6 expression in ob/ob mouse adipose stem cells via toll-like receptor-2-mediated pathways

USDA-ARS?s Scientific Manuscript database

Increases in adipose tissue weight positively correlates with increased circulating inflammatory cytokines such as interleukin-6 (IL-6). We previously have shown that adipose stem cell produce significantly higher levels of IL-6 when compared to other cell types in the adipose tissue in genetically ...

The role of CD133 in normal human prostate stem cells and malignant cancer-initiating cells.

PubMed

Vander Griend, Donald J; Karthaus, Wouter L; Dalrymple, Susan; Meeker, Alan; DeMarzo, Angelo M; Isaacs, John T

2008-12-01

Resolving the specific cell of origin for prostate cancer is critical to define rational targets for therapeutic intervention and requires the isolation and characterization of both normal human prostate stem cells and prostate cancer-initiating cells (CIC). Single epithelial cells from fresh normal human prostate tissue and prostate epithelial cell (PrEC) cultures derived from them were evaluated for the presence of subpopulations expressing stem cell markers and exhibiting stem-like growth characteristics. When epithelial cell suspensions containing cells expressing the stem cell marker CD133+ are inoculated in vivo, regeneration of stratified human prostate glands requires inductive prostate stromal cells. PrEC cultures contain a small subpopulation of CD133+ cells, and fluorescence-activated cell sorting-purified CD133+ PrECs self-renew and regenerate cell populations expressing markers of transit-amplifying cells (DeltaNp63), intermediate cells (prostate stem cell antigen), and neuroendocrine cells (CD56). Using a series of CD133 monoclonal antibodies, attachment and growth of CD133+ PrECs requires surface expression of full-length glycosylated CD133 protein. Within a series of androgen receptor-positive (AR+) human prostate cancer cell lines, CD133+ cells are present at a low frequency, self-renew, express AR, generate phenotypically heterogeneous progeny negative for CD133, and possess an unlimited proliferative capacity, consistent with CD133+ cells being CICs. Unlike normal adult prostate stem cells, prostate CICs are AR+ and do not require functional CD133. This suggests that (a) AR-expressing prostate CICs are derived from a malignantly transformed intermediate cell that acquires "stem-like activity" and not from a malignantly transformed normal stem cell and (b) AR signaling pathways are a therapeutic target for prostate CICs.

Are neural crest stem cells the missing link between hematopoietic and neurogenic niches?

PubMed

Coste, Cécile; Neirinckx, Virginie; Gothot, André; Wislet, Sabine; Rogister, Bernard

2015-01-01

Hematopoietic niches are defined as cellular and molecular microenvironments that regulate hematopoietic stem cell (HSC) function together with stem cell autonomous mechanisms. Many different cell types have been characterized as contributors to the formation of HSC niches, such as osteoblasts, endothelial cells, Schwann cells, and mesenchymal progenitors. These mesenchymal progenitors have themselves been classified as CXC chemokine ligand (CXCL) 12-abundant reticular (CAR) cells, stem cell factor expressing cells, or nestin-positive mesenchymal stem cells (MSCs), which have been recently identified as neural crest-derived cells (NCSCs). Together, these cells are spatially associated with HSCs and believed to provide appropriate microenvironments for HSC self-renewal, differentiation, mobilization and hibernation both by cell-cell contact and soluble factors. Interestingly, it appears that regulatory pathways governing the hematopoietic niche homeostasis are operating in the neurogenic niche as well. Therefore, this review paper aims to compare both the regulation of hematopoietic and neurogenic niches, in order to highlight the role of NCSCs and nervous system components in the development and the regulation of the hematopoietic system.

Embryonic origin and Hox status determine progenitor cell fate during adult bone regeneration.

PubMed

Leucht, Philipp; Kim, Jae-Beom; Amasha, Raimy; James, Aaron W; Girod, Sabine; Helms, Jill A

2008-09-01

The fetal skeleton arises from neural crest and from mesoderm. Here, we provide evidence that each lineage contributes a unique stem cell population to the regeneration of injured adult bones. Using Wnt1Cre::Z/EG mice we found that the neural crest-derived mandible heals with neural crest-derived skeletal stem cells, whereas the mesoderm-derived tibia heals with mesoderm-derived stem cells. We tested whether skeletal stem cells from each lineage were functionally interchangeable by grafting mesoderm-derived cells into mandibular defects, and vice versa. All of the grafting scenarios, except one, healed through the direct differentiation of skeletal stem cells into osteoblasts; when mesoderm-derived cells were transplanted into tibial defects they differentiated into osteoblasts but when transplanted into mandibular defects they differentiated into chondrocytes. A mismatch between the Hox gene expression status of the host and donor cells might be responsible for this aberration in bone repair. We found that initially, mandibular skeletal progenitor cells are Hox-negative but that they adopt a Hoxa11-positive profile when transplanted into a tibial defect. Conversely, tibial skeletal progenitor cells are Hox-positive and maintain this Hox status even when transplanted into a Hox-negative mandibular defect. Skeletal progenitor cells from the two lineages also show differences in osteogenic potential and proliferation, which translate into more robust in vivo bone regeneration by neural crest-derived cells. Thus, embryonic origin and Hox gene expression status distinguish neural crest-derived from mesoderm-derived skeletal progenitor cells, and both characteristics influence the process of adult bone regeneration.

Establishment of autologous embryonic stem cells derived from preantral follicle culture and oocyte parthenogenesis.

PubMed

Lee, Seung Tae; Choi, Mun Hwan; Lee, Eun Ju; Gong, Seung Pyo; Jang, Mi; Park, Sang Hyun; Jee, Hyang; Kim, Dae Yong; Han, Jae Yong; Lim, Jeong Mook

2008-11-01

To evaluate whether autologous embryonic stem cells can be established without generating clone embryos. Prospective model study. Gamete and stem cell biotechnology laboratory in Seoul National University, Seoul, Korea. F1 hybrid B6D2F1 mice. Preantral follicles were cultured, and oocytes matured in the follicles were parthenogenetically activated. Preimplantation development and stem cell characterization. More intrafollicular oocytes that were retrieved from secondary follicles matured and developed into blastocysts after parthenogenesis than those that were retrieved from primary follicles. Of those 35 blastocysts derived from 193 parthenotes, one line of colony-forming cells was established from the culturing of early secondary follicles. The established cells were positive for embryonic stem cell-specific markers and had normal diploid karyotype and telomerase activity. They differentiated into embryoid bodies in vitro and teratomas in vivo. Inducible differentiation of the established cells into neuronal lineage cells also was possible. Autologous embryonic stem cells can be established by preantral follicle culture and oocyte parthenogenesis. A combined technique of follicle culture and oocyte parthenogenesis that does not use developmentally competent oocytes has the potential to replace somatic cell nuclear transfer for autologous cell therapy.

Investigation of the expression of RIF1 gene on head and neck, pancreatic and brain cancer and cancer stem cells.

PubMed

GursesCila, Hacer E; Acar, Muradiye; Barut, Furkan B; Gunduz, Mehmet; Grenman, Reidar; Gunduz, Esra

2016-12-01

Recent studies have shown that cancer stem cells are resistant to chemotherapy. The aim of this study was to compare RIF1 gene expression in head and neck, pancreatic cancer and glioma cell lines and the cancer stem cells isolated from these cell lines. UT-SCC-74 from Turku University and UT-SCC-74B primary tumor metastasis and neck cancer cell lines, YKG-1 glioma cancer cell line from RIKEN, pancreatic cancer cell lines and ASPC-1 cells from ATCC were grown in cell culture. To isolate cancer stem cells, ALDH-1 for UT-SCC-74 and UT-SCC-74B cell line, CD-133 for YKG-1 cell line and CD-24 for ASPC-1 cell line, were used as markers of cancer stem cells. RNA isolation was performed for both cancer lines and cancer stem cells. RNAs were converted to cDNA. RIF1 gene expression was performed by qRT-PCR analysis. RIF1 gene expression was compared with cancer cell lines and cancer stem cells isolated from these cell lines. The possible effect of RIF1 gene was evaluated. In the pancreatic cells, RIF1 gene expression in the stem cell-positive cell line was 256 time that seen in the stem cell-negative cell line. Considering the importance of RIF1 in NHEJ and of NHEJ in pancreatic cancer, RIF1 may be one of the genes that plays an important role in the diagnoses and therapeutic treatment of pancreatic cancer. The results of head and neck and brain cancers are inconclusive and further studies are required to elucidate the connection between RIF1 gene and these other types of cancers.

Impact of electromagnetic fields on stem cells: common mechanisms at the crossroad between adult neurogenesis and osteogenesis

PubMed Central

Leone, Lucia; Podda, Maria Vittoria; Grassi, Claudio

2015-01-01

In the recent years adult neural and mesenchymal stem cells have been intensively investigated as effective resources for repair therapies. In vivo and in vitro studies have provided insights on the molecular mechanisms underlying the neurogenic and osteogenic processes in adulthood. This knowledge appears fundamental for the development of targeted strategies to manipulate stem cells. Here we review recent literature dealing with the effects of electromagnetic fields on stem cell biology that lends support to their use as a promising tool to positively influence the different steps of neurogenic and osteogenic processes. We will focus on recent studies revealing that extremely-low frequency electromagnetic fields enhance adult hippocampal neurogenesis by inducing epigenetic modifications on the regulatory sequences of genes responsible for neural stem cell proliferation and neuronal differentiation. In light of the emerging critical role played by chromatin modifications in maintaining the stemness as well as in regulating stem cell differentiation, we will also attempt to exploit epigenetic changes that can represent common targets for electromagnetic field effects on neurogenic and osteogenic processes. PMID:26124705

Engineered stem cell mimics to enhance stroke recovery.

PubMed

George, Paul M; Oh, Byeongtaek; Dewi, Ruby; Hua, Thuy; Cai, Lei; Levinson, Alexa; Liang, Xibin; Krajina, Brad A; Bliss, Tonya M; Heilshorn, Sarah C; Steinberg, Gary K

2018-06-13

Currently, no medical therapies exist to augment stroke recovery. Stem cells are an intriguing treatment option being evaluated, but cell-based therapies have several challenges including developing a stable cell product with long term reproducibility. Since much of the improvement observed from cellular therapeutics is believed to result from trophic factors the stem cells release over time, biomaterials are well-positioned to deliver these important molecules in a similar fashion. Here we show that essential trophic factors secreted from stem cells can be effectively released from a multi-component hydrogel system into the post-stroke environment. Using our polymeric system to deliver VEGF-A and MMP-9, we improved recovery after stroke to an equivalent degree as observed with traditional stem cell treatment in a rodent model. While VEGF-A and MMP-9 have many unique mechanisms of action, connective tissue growth factor (CTGF) interacts with both VEGF-A and MMP-9. With our hydrogel system as well as with stem cell delivery, the CTGF pathway is shown to be downregulated with improved stroke recovery. Copyright © 2018 Elsevier Ltd. All rights reserved.

Hydrogen sulphide increases hepatic differentiation of human tooth pulp stem cells compared with human bone marrow stem cells.

PubMed

Okada, M; Ishkitiev, N; Yaegaki, K; Imai, T; Tanaka, T; Fukuda, M; Ono, S; Haapasalo, M

2014-12-01

To determine the differences in stem cell properties, in hepatic differentiation and in the effects of hydrogen sulphide (H2 S) on hepatic differentiation between human bone marrow stem cells (hBMC) and stem cells from human exfoliated primary tooth pulp (SHED). CD117(+) cells were magnetically separated and subjected to hepatic differentiation. CD117(+) cell lineages were characterized for transcription factors indicative of stem cells by qRT-PCR. For the last 9 days of the differentiation, the test cells were exposed to 0.1 ng mL(-1) H2 S. Immunocytochemistry and flow cytometry of albumin, alpha-fetoprotein and carbamoyl phosphate synthetase were carried out after differentiation. Urea concentration and glycogen synthesis were also determined. Genes expressed in SHED were also expressed in BMC. No difference in expression level of hepatic markers was shown by immunofluorescence. SHED showed more positive cells than hBMC (P < 0.01). H2 S increased the number of positive cells in both cultures (P < 0.01). Urea concentration and glycogen synthesis increased significantly after H2 S exposure (P < 0.001 and P < 0.05, respectively). Real-time PCR data were analysed by RT(2) profiler RT-PCR Array Data Analysis version 3.5 (Qiagen), and ELISA data were analysed by Bonferroni's multiple comparison using Windows spss version 16 (SPSS Inc, Chicago, IL, USA). Bonferroni's multiple comparison test was also carried out after angle transformation for the percentage data of flow cytometer using Windows spss(®) version 16 (SPSS Inc). Statistical significance was accepted at P < 0.05. Stem cells from human exfoliated primary tooth pulp and BMC have similar properties. The level of hepatic differentiation in SHED compared with BMC was the same or higher. H2 S increased the level of hepatic differentiation. © 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd.

Chondrogenesis of infrapatellar fat pad derived adipose stem cells in 3D printed chitosan scaffold.

PubMed

Ye, Ken; Felimban, Raed; Traianedes, Kathy; Moulton, Simon E; Wallace, Gordon G; Chung, Johnson; Quigley, Anita; Choong, Peter F M; Myers, Damian E

2014-01-01

Infrapatellar fat pad adipose stem cells (IPFP-ASCs) have been shown to harbor chondrogenic potential. When combined with 3D polymeric structures, the stem cells provide a source of stem cells to engineer 3D tissues for cartilage repair. In this study, we have shown human IPFP-ASCs seeded onto 3D printed chitosan scaffolds can undergo chondrogenesis using TGFβ3 and BMP6. By week 4, a pearlescent, cartilage-like matrix had formed that penetrated the top layers of the chitosan scaffold forming a 'cap' on the scaffold. Chondrocytic morphology showed typical cells encased in extracellular matrix which stained positively with toluidine blue. Immunohistochemistry demonstrated positive staining for collagen type II and cartilage proteoglycans, as well as collagen type I. Real time PCR analysis showed up-regulation of collagen type II, aggrecan and SOX9 genes when IPFP-ASCs were stimulated by TGFβ3 and BMP6. Thus, IPFP-ASCs can successfully undergo chondrogenesis using TGFβ3 and BMP6 and the cartilage-like tissue that forms on the surface of 3D-printed chitosan scaffold may prove useful as an osteochondral graft.

p75 neurotrophin receptor positive dental pulp stem cells: new hope for patients with neurodegenerative disease and neural injury.

PubMed

Dai, Jie-wen; Yuan, Hao; Shen, Shun-yao; Lu, Jing-ting; Zhu, Xiao-fang; Yang, Tong; Zhang, Jiang-fei; Shen, Guo-fang

2013-08-01

Neurodegenerative diseases and neural injury are 2 of the most feared disorders that afflict humankind by leading to permanent paralysis and loss of sensation. Cell based treatment for these diseases had gained special interest in recent years. Previous studies showed that dental pulp stem cells (DPSCs) could differentiate toward functionally active neurons both in vitro and in vivo, and could promote neuranagenesis through both cell-autonomous and paracrine neuroregenerative activities. Some of these neuroregenerative activities were unique to tooth-derived stem cells and superior to bone marrow stromal cells. However, DPSCs used in most of these studies were mixed and unfractionated dental pulp cells that contain several types of cells, and most were fibroblast cells while just contain a small portion of DPSCs. Thus, there might be weaker ability of neuranagenesis and more side effects from the fibroblast cells that cannot differentiate into neural cells. p75 neurotrophin receptor (p75NTR) positive DPSCs subpopulation was derived from migrating cranial neural crest cells and had been isolated from DPSCs, which had capacity of differentiation into neurons and repairing neural system. In this article, we hypothesize that p75NTR positive DPSCs simultaneously have greater propensity for neuronal differentiation and fewer side effects from fibroblast, and in vivo transptantation of autologous p75NTR positive DPSCs is a novel method for neuranagenesis. This will bring great hope to patients with neurodegenerative disease and neural injury.

Single Cell-Based Vector Tracing in Patients with ADA-SCID Treated with Stem Cell Gene Therapy.

PubMed

Igarashi, Yuka; Uchiyama, Toru; Minegishi, Tomoko; Takahashi, Sirirat; Watanabe, Nobuyuki; Kawai, Toshinao; Yamada, Masafumi; Ariga, Tadashi; Onodera, Masafumi

2017-09-15

Clinical improvement in stem cell gene therapy (SCGT) for primary immunodeficiencies depends on the engraftment levels of genetically corrected cells, and tracing the transgene in each hematopoietic lineage is therefore extremely important in evaluating the efficacy of SCGT. We established a single cell-based droplet digital PCR (sc-ddPCR) method consisting of the encapsulation of a single cell into each droplet, followed by emulsion PCR with primers and probes specific for the transgene. A fluorescent signal in a droplet indicates the presence of a single cell carrying the target gene in its genome, and this system can clearly determine the ratio of transgene-positive cells in the entire population at the genomic level. Using sc-ddPCR, we analyzed the engraftment of vector-transduced cells in two patients with severe combined immunodeficiency (SCID) who were treated with SCGT. Sufficient engraftment of the transduced cells was limited to the T cell lineage in peripheral blood (PB), and a small percentage of CD34 + cells exhibited vector integration in bone marrow, indicating that the transgene-positive cells in PB might have differentiated from a small population of stem cells or lineage-restricted precursor cells. sc-ddPCR is a simplified and powerful tool for the detailed assessment of transgene-positive cell distribution in patients treated with SCGT.

Prolonged Growth Hormone/Insulin/Insulin-like Growth Factor Nutrient Response Signaling Pathway as a Silent Killer of Stem Cells and a Culprit in Aging.

PubMed

Ratajczak, Mariusz Z; Bartke, Andrzej; Darzynkiewicz, Zbigniew

2017-08-01

The dream of slowing down the aging process has always inspired mankind. Since stem cells are responsible for tissue and organ rejuvenation, it is logical that we should search for encoded mechanisms affecting life span in these cells. However, in adult life the hierarchy within the stem cell compartment is still not very well defined, and evidence has accumulated that adult tissues contain rare stem cells that possess a broad trans-germ layer differentiation potential. These most-primitive stem cells-those endowed with pluripotent or multipotent differentiation ability and that give rise to other cells more restricted in differentiation, known as tissue-committed stem cells (TCSCs) - are of particular interest. In this review we present the concept supported by accumulating evidence that a population of so-called very small embryonic-like stem cells (VSELs) residing in adult tissues positively impacts the overall survival of mammals, including humans. These unique cells are prevented in vertebrates from premature depletion by decreased sensitivity to growth hormone (GH), insulin (INS), and insulin-like growth factor (IGF) signaling, due to epigenetic changes in paternally imprinted genes that regulate their resistance to these factors. In this context, we can envision nutrient response GH/INS/IGF signaling pathway as a lethal factor for these most primitive stem cells and an important culprit in aging.

Radiation-Induced Dedifferentiation of Head and Neck Cancer Cells Into Cancer Stem Cells Depends on Human Papillomavirus Status

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

Vlashi, Erina, E-mail: evlashi@mednet.ucla.edu; Jonsson Comprehensive Cancer Center at UCLA, Los Angeles, California; Chen, Allen M.

Purpose: To test the hypothesis that the radiation response of cancer stem cells (CSCs) in human papillomavirus (HPV)-positive and HPV-negative head and neck squamous cell carcinoma (HNSCC) differs and is not reflected in the radiation response of the bulk tumor populations, that radiation therapy (RT) can dedifferentiate non-stem HNSCC cells into CSCs, and that radiation-induced dedifferentiation depends on the HPV status. Methods and Materials: Records of a cohort of 162 HNSCC patients were reviewed, and their outcomes were correlated with their HPV status. Using a panel of HPV-positive and HPV-negative HNSCC cell lines expressing a reporter for CSCs, we characterized HPV-positivemore » and HPV-negative lines via flow cytometry, sphere-forming capacity assays in vitro, and limiting dilution assays in vivo. Non-CSCs were treated with different doses of radiation, and the dedifferentiation of non-CSCs into CSCs was investigated via flow cytometry and quantitative reverse transcription–polymerase chain reaction for re-expression of reprogramming factors. Results: Patients with HPV-positive tumors have superior overall survival and local–regional control. Human papillomavirus–positive HNSCC cell lines have lower numbers of CSCs, which inversely correlates with radiosensitivity. Human papillomavirus–negative HNSCC cell lines lack hierarchy owing to enhanced spontaneous dedifferentiation. Non-CSCs from HPV-negative lines show enhanced radiation-induced dedifferentiation compared with HPV-positive lines, and RT induced re-expression of Yamanaka reprogramming factors. Conclusions: Supporting the favorable prognosis of HPV-positive HNSCCs, we show that (1) HPV-positive HNSCCs have a lower frequency of CSCs; (2) RT can dedifferentiate HNSCC cells into CSCs; and (3) radiation-induced dedifferentiation depends on the HPV status of the tumor.« less

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

The Effect of In Vivo Mobilization of Bone Marrow Stem Cells on the Pancreas of Diabetic Albino Rats (A Histological & Immunohistochemical Study)

PubMed Central

Ismail, Zeinab Mohamed Kamel; Kamel, Ashraf Mahmoud Fawzy; Yacoub, Mira Farouk Youssef; Aboulkhair, Alshaymaa Gamal

2013-01-01

Background and Objectives The rapidly increasing number of diabetic patients across the world drew the attention to develop more effective therapeutic approaches. Recent investigations on newly differentiated insulin producing cells (IPCs) revealed that they could be derived from embryonic, adult mesenchymal and hematopoietic stem cells. This work was planned to evaluate the role of StemEnhance (Aphanizomenon flos-aquae [AFA] plant extract) in mobilizing naturally occurring bone marrow stem cells as well as in improving streptozotocin-induced diabetic rats. Methods and Results Twenty adult male albino rats were divided into four groups namely the control, the diabetic, the positive control-StemEnhance and the diabetic-StemEnhance groups. After diabetes induction by streptozotocin (STZ), rats received StemEnhance for four weeks. The mean number of blood CD34 immunopositive cells was measured by flowcytometry and random blood sugar was measured weekly. The pancreas was removed from the sacrificed rats and processed for staining with H&E and immunohistochemical staining for CD34+ve and insulin +ve cells. CD34+ve cells increased in the blood after introduction of StemEnhance. CD34+ve cells were observed in the pancreas and the insulin producing cells in the islets of Langerhans were increased from the second to the fourth week of treatment. Blood glucose level improved but it was still higher than the control level after four weeks of StemEnhance treatment. Conclusions This work points to the significant role of StemEnhance in stem cell mobilization and the improvement of diabetes mellitus. PMID:24298369

From the basics to application of cell therapy, a steppingstone to the conquest of neurodegeneration: a meeting report.

PubMed

Park, Dong-Hyuk; Eve, David J; Borlongan, Cesario V; Klasko, Stephen K; Cruz, L Eduardo; Sanberg, Paul R

2009-02-01

The annual meeting of the American Society for Neural Therapy and Repair (ASNTR) showcases the latest research trends in neurodegenerative disease and the related medical regenerative science. The 2008 ASNTR meeting covered a variety of different topics ranging from basic research to exploration of currently unknown pathogenesis and mechanisms for specific neurodegenerative disease such as Parkinson's disease, Alzheimer's disease, or stroke. This included studies to characterize stem cells, such as neural stem cells, embryonic stem cells, bone marrow mesenchymal stem cells, and human umbilical cord blood cells, for transplantation and the conditions necessary to maximize the efficacy of endogenous and exogenous stem cells, such as isolation, purification, differentiation, and migration. Moreover, a number of studies looked at methods for more advanced application of transplantation of cells or specific factors, through tissue engineering or manipulation beyond simple injection. Finally, well-known or previously un-known dietary supplementation or pharmacological materials that can affect the nervous system positively or negatively, were also important topics.

Characterization of mesenchymal stem cells from human dental pulp, preapical follicle and periodontal ligament.

PubMed

Navabazam, Ali Reza; Sadeghian Nodoshan, Fatemeh; Sheikhha, Mohammad Hasan; Miresmaeili, Sayyed Mohsen; Soleimani, Mehrdad; Fesahat, Farzaneh

2013-03-01

Human dental stem cells have high proliferative potential for self-renewal that is important to the regenerative capacity of the tissue. Objective : The aim was to isolate human dental pulp stem cells (DPSC), periodontal ligament stem cells (PDLSC) and periapical follicle stem cells (PAFSC) for their potential role in tissue regeneration. In this experimental study, the postnatal stem cells were isolated from dental pulp, preapical follicle and periodontal ligament .The cells were stained for different stem cell markers by immunocytochemistry. To investigate the mesenchymal nature of cells, differentiation potential along osteoblastic and adipogenic lineages and gene expression profile were performed. For proliferation potential assay, Brdu staining and growth curve tests were performed. Finally, all three cell types were compared together regarding their proliferation, differentiation and displaying phenotype. The isolated cell populations have similar fibroblastic like morphology and expressed all examined cell surface molecule markers. These cells were capable of differentiating into osteocyte with different capability and adipocyte with the same rate. PAFSCs showed more significant proliferation rate than others. Reverse transcriptase PCR (RT-PCR) for nanog, oct4, Alkaline phosphatase (ALP) and glyceraldehydes-3-phosphate dehydrogenease (GADPH) as control gene showed strong positive expression of these genes in all three isolated cell types. PDLSCs, DPSCs and PAFSCs exist in various tissues of the teeth and can use as a source of mesenchymal stem cells for developing bioengineered organs and also in craniomaxillofacial reconstruction with varying efficiency in differentiation and proliferation.

Centrosome misorientation mediates slowing of the cell cycle under limited nutrient conditions in Drosophila male germline stem cells

PubMed Central

Roth, Therese M.; Chiang, C.-Y. Ason; Inaba, Mayu; Yuan, Hebao; Salzmann, Viktoria; Roth, Caitlin E.; Yamashita, Yukiko M.

2012-01-01

Drosophila male germline stem cells (GSCs) divide asymmetrically, balancing self-renewal and differentiation. Although asymmetric stem cell division balances between self-renewal and differentiation, it does not dictate how frequently differentiating cells must be produced. In male GSCs, asymmetric GSC division is achieved by stereotyped positioning of the centrosome with respect to the stem cell niche. Recently we showed that the centrosome orientation checkpoint monitors the correct centrosome orientation to ensure an asymmetric outcome of the GSC division. When GSC centrosomes are not correctly oriented with respect to the niche, GSC cell cycle is arrested/delayed until the correct centrosome orientation is reacquired. Here we show that induction of centrosome misorientation upon culture in poor nutrient conditions mediates slowing of GSC cell proliferation via activation of the centrosome orientation checkpoint. Consistently, inactivation of the centrosome orientation checkpoint leads to lack of cell cycle slowdown even under poor nutrient conditions. We propose that centrosome misorientation serves as a mediator that transduces nutrient information into stem cell proliferation, providing a previously unappreciated mechanism of stem cell regulation in response to nutrient conditions. PMID:22357619

Stem cell senescence. Effects of REAC technology on telomerase-independent and telomerase-dependent pathways.

PubMed

Rinaldi, S; Maioli, M; Pigliaru, G; Castagna, A; Santaniello, S; Basoli, V; Fontani, V; Ventura, C

2014-09-16

Decline in the gene expression of senescence repressor Bmi1, and telomerase, together with telomere shortening, underlay senescence of stem cells cultured for multiple passages. Here, we investigated whether the impairment of senescence preventing mechanisms can be efficiently counteracted by exposure of human adipose-derived stem cells to radio electric asymmetrically conveyed fields by an innovative technology, named Radio Electric Asymmetric Conveyer (REAC). Due to REAC exposure, the number of stem cells positively stained for senescence associated β-galactosidase was significantly reduced along multiple culturing passages. After a 90-day culture, REAC-treated cells exhibited significantly higher transcription of Bmi1 and enhanced expression of other stem cell pluripotency genes and related proteins, compared to unexposed cells. Transcription of the catalytic telomerase subunit (TERT) was also increased in REAC-treated cells at all passages. Moreover, while telomere shortening occurred at early passages in both REAC-treated and untreated cells, a significant rescue of telomere length could be observed at late passages only in REAC-exposed cells. Thus, REAC-asymmetrically conveyed radio electric fields acted on a gene and protein expression program of both telomerase-independent and telomerase-dependent patterning to optimize stem cell ability to cope with senescence progression.

Isolation, in vitro culture and identification of a new type of mesenchymal stem cell derived from fetal bovine lung tissues.

PubMed

Hu, Pengfei; Pu, Yabin; Li, Xiayun; Zhu, Zhiqiang; Zhao, Yuhua; Guan, Weijun; Ma, Yuehui

2015-09-01

Lung‑derived mesenchymal stem cells (LMSCs) are considered to be important in lung tissue repair and regenerative processes. However, the biological characteristics and differentiation potential of LMSCs remain to be elucidated. In the present study, fetal lung‑derived mesenchymal stem cells (FLMSCs) were isolated from fetal bovine lung tissues by collagenase digestion. The in vitro culture conditions were optimized and stabilized and the self‑renewal ability and differentiation potential were evaluated. The results demonstrated that the FLMSCs were morphologically consistent with fibroblasts, were able to be cultured and passaged for at least 33 passages and the cell morphology and proliferative ability were stable during the first 10 passages. In addition, FLMSCs were found to express CD29, CD44, CD73 and CD166, however, they did not express hematopoietic cell specific markers, including CD34, CD45 and BOLA‑DRα. The growth kinetics of FLMSCs consisted of a lag phase, a logarithmic phase and a plateau phase, and as the passages increased, the proliferative ability of cells gradually decreased. The majority of FLMSCs were in G0/G1 phase. Following osteogenic induction, FLMSCs were positive for the expression of osteopontin and collagen type I α2. Following neurogenic differentiation, the cells were morphologically consistent with neuronal cells and positive for microtubule‑associated protein 2 and nestin expression. It was concluded that the isolated FLMSCs exhibited typical characteristics of mesenchymal stem cells and that the culture conditions were suitable for their proliferation and the maintenance of stemness. The present study illustrated the potential application of lung tissue as an adult stem cell source for regenerative therapies.

Stem cell isolation by a morphology-based selection method in postnatal mouse ovary.

PubMed

Parvari, Soraya; Abbasi, Mehdi; Abbasi, Niloufar; Malek, Valliollah Gerayeli; Amidi, Fardin; Aval, Fereydoon Sargolzaei; Roudkenar, Mehryar Habibi; Izadyar, Fariburz

2015-06-19

An increasing body of evidence has emerged regarding the existence and function of spermatogonial stem cells (SSCs); however, their female counterparts are the subject of extensive debate. Theoretically, ovarian germ stem cells (GSCs) have to reside in the murine ovary to support and replenish the follicle pool during the reproductive life span. Recently, various methods have been recruited to isolate and describe aspects of ovarian GSCs, but newer and more convenient strategies in isolation are still growing. Herein, a morphology-based method was used to isolate GSCs. A cell suspension of mouse neonatal ovaries was cultured. Colonies of GSCs were harvested mechanically and cultivated on mouse embryonic fibroblasts (MEF). Alkaline phosphatase activity was assessed to verify stemness features of cells in colonies. Expression of germ and stem cell specific genes (Oct-4, Nanog, Fragilis, C-kit, Dazl, and Mvh) was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Immunofluorescence of Oct4, Dazl, Mvh, and SSEA-1 was also performed. Small colonies without a clear border appeared during the first 4 days of culture, and the size of colonies increased rapidly. Cells in colonies were positive for alkaline phosphatase activity. Reverse transcription-polymerase chain reaction showed that Oct-4, Fragilis, C-kit, Nanog, Mvh, and Dazl were expressed in colony-forming cells. Immunofluorescence revealed a positive signal for Oct4, Dazl, Mvh, and SSEA-1 in colonies as well. The applicability of morphological selection for isolation of GSCs was verified. This method is easier and more economical than other techniques. The availability of ovarian stem cells can motivate further studies in development of oocyte and cell-based therapies.

Stem cell isolation by a morphology-based selection method in postnatal mouse ovary

PubMed Central

Parvari, Soraya; Abbasi, Niloufar; Malek, Valliollah Gerayeli; Amidi, Fardin; Aval, Fereydoon Sargolzaei; Roudkenar, Mehryar Habibi; Izadyar, Fariburz

2015-01-01

Introduction An increasing body of evidence has emerged regarding the existence and function of spermatogonial stem cells (SSCs); however, their female counterparts are the subject of extensive debate. Theoretically, ovarian germ stem cells (GSCs) have to reside in the murine ovary to support and replenish the follicle pool during the reproductive life span. Recently, various methods have been recruited to isolate and describe aspects of ovarian GSCs, but newer and more convenient strategies in isolation are still growing. Herein, a morphology-based method was used to isolate GSCs. Material and methods A cell suspension of mouse neonatal ovaries was cultured. Colonies of GSCs were harvested mechanically and cultivated on mouse embryonic fibroblasts (MEF). Alkaline phosphatase activity was assessed to verify stemness features of cells in colonies. Expression of germ and stem cell specific genes (Oct-4, Nanog, Fragilis, C-kit, Dazl, and Mvh) was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Immunofluorescence of Oct4, Dazl, Mvh, and SSEA-1 was also performed. Results Small colonies without a clear border appeared during the first 4 days of culture, and the size of colonies increased rapidly. Cells in colonies were positive for alkaline phosphatase activity. Reverse transcription-polymerase chain reaction showed that Oct-4, Fragilis, C-kit, Nanog, Mvh, and Dazl were expressed in colony-forming cells. Immunofluorescence revealed a positive signal for Oct4, Dazl, Mvh, and SSEA-1 in colonies as well. Conclusions The applicability of morphological selection for isolation of GSCs was verified. This method is easier and more economical than other techniques. The availability of ovarian stem cells can motivate further studies in development of oocyte and cell-based therapies. PMID:26170863

Side population in human glioblastoma is non-tumorigenic and characterizes brain endothelial cells

PubMed Central

Golebiewska, Anna; Bougnaud, Sébastien; Stieber, Daniel; Brons, Nicolaas H. C.; Vallar, Laurent; Hertel, Frank; Klink, Barbara; Schröck, Evelin; Bjerkvig, Rolf

2013-01-01

The identification and significance of cancer stem-like cells in malignant gliomas remains controversial. It has been proposed that cancer stem-like cells display increased drug resistance, through the expression of ATP-binding cassette transporters that detoxify cells by effluxing exogenous compounds. Here, we investigated the ‘side population’ phenotype based on efflux properties of ATP-binding cassette transporters in freshly isolated human glioblastoma samples and intracranial xenografts derived thereof. Using fluorescence in situ hybridization analysis on sorted cells obtained from glioblastoma biopsies, as well as human tumour xenografts developed in immunodeficient enhanced green fluorescence protein-expressing mice that allow an unequivocal tumour-stroma discrimination, we show that side population cells in human glioblastoma are non-neoplastic and exclusively stroma-derived. Tumour cells were consistently devoid of efflux properties regardless of their genetic background, tumour ploidy or stem cell associated marker expression. Using multi-parameter flow cytometry we identified the stromal side population in human glioblastoma to be brain-derived endothelial cells with a minor contribution of astrocytes. In contrast with their foetal counterpart, neural stem/progenitor cells in the adult brain did not display the side population phenotype. Of note, we show that CD133-positive cells often associated with cancer stem-like cells in glioblastoma biopsies, do not represent a homogenous cell population and include CD31-positive endothelial cells. Interestingly, treatment of brain tumours with the anti-angiogenic agent bevacizumab reduced total vessel density, but did not affect the efflux properties of endothelial cells. In conclusion our findings contribute to an unbiased identification of cancer stem-like cells and stromal cells in brain neoplasms, and provide novel insight into the complex issue of drug delivery to the brain. Since efflux properties of endothelial cells are likely to compromise drug availability, transiently targeting ATP-binding cassette transporters may be a valuable therapeutic strategy to improve treatment effects in brain tumours. PMID:23460667

Cancer stem cells and cell size: A causal link?

PubMed

Li, Qiuhui; Rycaj, Kiera; Chen, Xin; Tang, Dean G

2015-12-01

The majority of normal animal cells are 10-20 μm in diameter. Many signaling mechanisms, notably PI3K/Akt/mTOR, Myc, and Hippo pathways, tightly control and coordinate cell growth, cell size, cell division, and cell number during homeostasis. These regulatory mechanisms are frequently deregulated during tumorigenesis resulting in wide variations in cell sizes and increased proliferation in cancer cells. Here, we first review the evidence that primitive stem cells in adult tissues are quiescent and generally smaller than their differentiated progeny, suggesting a correlation between small cell sizes with the stemness. Conversely, increased cell size positively correlates with differentiation phenotypes. We then discuss cancer stem cells (CSCs) and present some evidence that correlates cell sizes with CSC activity. Overall, a causal link between CSCs and cell size is relatively weak and remains to be rigorously assessed. In the future, optimizing methods for isolating cells based on size should help elucidate the connection between cancer cell size and CSC characteristics. Copyright © 2015 Elsevier Ltd. All rights reserved.

FSH-FSHR3-stem cells in ovary surface epithelium: basis for adult ovarian biology, failure, aging, and cancer.

PubMed

Bhartiya, Deepa; Singh, Jarnail

2015-01-01

Despite extensive research, genetic basis of premature ovarian failure (POF) and ovarian cancer still remains elusive. It is indeed paradoxical that scientists searched for mutations in FSH receptor (FSHR) expressed on granulosa cells, whereas more than 90% of cancers arise in ovary surface epithelium (OSE). Two distinct populations of stem cells including very small embryonic-like stem cells (VSELs) and ovarian stem cells (OSCs) exist in OSE, are responsible for neo-oogenesis and primordial follicle assembly in adult life, and are modulated by FSH via its alternatively spliced receptor variant FSHR3 (growth factor type 1 receptor acting via calcium signaling and the ERK/MAPK pathway). Any defect in FSH-FSHR3-stem cell interaction in OSE may affect folliculogenesis and thus result in POF. Ovarian aging is associated with a compromised microenvironment that does not support stem cell differentiation into oocytes and further folliculogenesis. FSH exerts a mitogenic effect on OSE and elevated FSH levels associated with advanced age may provide a continuous trigger for stem cells to proliferate resulting in cancer, thus supporting gonadotropin theory for ovarian cancer. Present review is an attempt to put adult ovarian biology, POF, aging, and cancer in the perspective of FSH-FSHR3-stem cell network that functions in OSE. This hypothesis is further supported by the recent understanding that: i) cancer is a stem cell disease and OSE is the niche for ovarian cancer stem cells; ii) ovarian OCT4-positive stem cells are regulated by FSH; and iii) OCT4 along with LIN28 and BMP4 are highly expressed in ovarian cancers. © 2015 Society for Reproduction and Fertility.

Regeneration of hepatocyte 'buds' in cirrhosis from intrabiliary stem cells.

PubMed

Falkowski, Olga; An, Hee Jung; Ianus, I Andreea; Chiriboga, Luis; Yee, Herman; West, A Brian; Theise, Neil D

2003-09-01

In massive hepatic necrosis, hepatic stem cells constitute a canal of Hering derived, cytokeratin 19 (CK19) positive 'ductular reaction' (DR). Whether DRs in cirrhosis are activated stem cells (so called 'buds') or biliary metaplasia of cholestatic, injured hepatocytes is still debated. We investigate derivation of intraseptal hepatocytes (ISHs) from DRs and from the biliary tree in cirrhosis. Explants of hepatitis B and C, alcohol, primary biliary cirrhosis and primary sclerosing cholangitis-related cirrhosis were examined. ISHs were quantified and their associations with DRs and cholestasis recorded. 3D-reconstruction of ISHs and nearby bile ducts was performed in blocks from hepatitis C and primary sclerosing cholangitis cirrhosis. Seven hundred seventy five/830 (94%) ISHs were associated with CK19 positive DRs. ISHs without ductular reactions were more likely to show cholestatic features (P<0.0001). In 3D, ISHs were seen to bud directly from the biliary tree. In summary: ISHs: (1) are usually associated with stem cell-like DRs; (2) are rarely cholestatic, leaving the associated DRs unexplained; and (3) are linked to the biliary tree in 3D. Dynamic proliferation rates in hepatitis C over time suggest that hepatocyte replication diminishes in late stages, with an associated activation of the biliary stem cell compartment. We therefore suggest that the biliary tree, from at least its smaller branches up to the canals of Hering, are composed of or at least harbor facultative hepatic stem cells, and that ISH largely represent 'buds' of newly formed hepatocytes.

Differentiation of oligodendrocyte progenitor cells from dissociated monolayer and feeder-free cultured pluripotent stem cells.

PubMed

Yamashita, Tomoko; Miyamoto, Yuki; Bando, Yoshio; Ono, Takashi; Kobayashi, Sakurako; Doi, Ayano; Araki, Toshihiro; Kato, Yosuke; Shirakawa, Takayuki; Suzuki, Yutaka; Yamauchi, Junji; Yoshida, Shigetaka; Sato, Naoya

2017-01-01

Oligodendrocytes myelinate axons and form myelin sheaths in the central nervous system. The development of therapies for demyelinating diseases, including multiple sclerosis and leukodystrophies, is a challenge because the pathogenic mechanisms of disease remain poorly understood. Primate pluripotent stem cell-derived oligodendrocytes are expected to help elucidate the molecular pathogenesis of these diseases. Oligodendrocytes have been successfully differentiated from human pluripotent stem cells. However, it is challenging to prepare large amounts of oligodendrocytes over a short amount of time because of manipulation difficulties under conventional primate pluripotent stem cell culture methods. We developed a proprietary dissociated monolayer and feeder-free culture system to handle pluripotent stem cell cultures. Because the dissociated monolayer and feeder-free culture system improves the quality and growth of primate pluripotent stem cells, these cells could potentially be differentiated into any desired functional cells and consistently cultured in large-scale conditions. In the current study, oligodendrocyte progenitor cells and mature oligodendrocytes were generated within three months from monkey embryonic stem cells. The embryonic stem cell-derived oligodendrocytes exhibited in vitro myelinogenic potency with rat dorsal root ganglion neurons. Additionally, the transplanted oligodendrocyte progenitor cells differentiated into myelin basic protein-positive mature oligodendrocytes in the mouse corpus callosum. This preparative method was used for human induced pluripotent stem cells, which were also successfully differentiated into oligodendrocyte progenitor cells and mature oligodendrocytes that were capable of myelinating rat dorsal root ganglion neurons. Moreover, it was possible to freeze, thaw, and successfully re-culture the differentiating cells. These results showed that embryonic stem cells and human induced pluripotent stem cells maintained in a dissociated monolayer and feeder-free culture system have the potential to generate oligodendrocyte progenitor cells and mature oligodendrocytes in vitro and in vivo. This culture method could be applied to prepare large amounts of oligodendrocyte progenitor cells and mature oligodendrocytes in a relatively short amount of time.

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

Hopx expression defines a subset of multipotent hair follicle stem cells and a progenitor population primed to give rise to K6+ niche cells

PubMed Central

Takeda, Norifumi; Jain, Rajan; LeBoeuf, Matthew R.; Padmanabhan, Arun; Wang, Qiaohong; Li, Li; Lu, Min Min; Millar, Sarah E.; Epstein, Jonathan A.

2013-01-01

The mammalian hair follicle relies on adult resident stem cells and their progeny to fuel and maintain hair growth throughout the life of an organism. The cyclical and initially synchronous nature of hair growth makes the hair follicle an ideal system with which to define homeostatic mechanisms of an adult stem cell population. Recently, we demonstrated that Hopx is a specific marker of intestinal stem cells. Here, we show that Hopx specifically labels long-lived hair follicle stem cells residing in the telogen basal bulge. Hopx+ cells contribute to all lineages of the mature hair follicle and to the interfollicular epidermis upon epidermal wounding. Unexpectedly, our analysis identifies a previously unappreciated progenitor population that resides in the lower hair bulb of anagen-phase follicles and expresses Hopx. These cells co-express Lgr5, do not express Shh and escape catagen-induced apoptosis. They ultimately differentiate into the cytokeratin 6-positive (K6) inner bulge cells in telogen, which regulate the quiescence of adjacent hair follicle stem cells. Although previous studies have suggested that K6+ cells arise from Lgr5-expressing lower outer root sheath cells in anagen, our studies indicate an alternative origin, and a novel role for Hopx-expressing lower hair bulb progenitor cells in contributing to stem cell homeostasis. PMID:23487314

Stem cell bioprinting for applications in regenerative medicine.

PubMed

Tricomi, Brad J; Dias, Andrew D; Corr, David T

2016-11-01

Many regenerative medicine applications seek to harness the biologic power of stem cells in architecturally complex scaffolds or microenvironments. Traditional tissue engineering methods cannot create such intricate structures, nor can they precisely control cellular position or spatial distribution. These limitations have spurred advances in the field of bioprinting, aimed to satisfy these structural and compositional demands. Bioprinting can be defined as the programmed deposition of cells or other biologics, often with accompanying biomaterials. In this concise review, we focus on recent advances in stem cell bioprinting, including performance, utility, and applications in regenerative medicine. More specifically, this review explores the capability of bioprinting to direct stem cell fate, engineer tissue(s), and create functional vascular networks. Furthermore, the unique challenges and concerns related to bioprinting living stem cells, such as viability and maintaining multi- or pluripotency, are discussed. The regenerative capacity of stem cells, when combined with the structural/compositional control afforded by bioprinting, provides a unique and powerful tool to address the complex demands of tissue engineering and regenerative medicine applications. © 2016 New York Academy of Sciences.

Osteoblastic differentiation of human stem cells derived from bone marrow and periodontal ligament under the effect of enamel matrix derivative and transforming growth factor-beta.

PubMed

Houshmand, Behzad; Behnia, Hossein; Khoshzaban, Ahad; Morad, Golnaz; Behrouzi, Gholamreza; Dashti, Seyedeh Ghazaleh; Khojasteh, Arash

2013-01-01

To increase the understanding of the applicability of biomaterials and growth factors in enhancing stem cell-based bone regeneration modalities, this study evaluated the effects of enamel matrix derivative (EMD) and recombinant human transforming growth factor-beta (rhTGF-β) on osteoblastic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) as well as human periodontal ligament stem cells (hPDLSCs). hBMSCs and hPDLSCs were obtained, and identification of stem cell surface markers was performed according to the criteria of the International Society for Cellular Therapy. Each group of stem cells was separately treated with a serial dilution of EMD (10, 50, and 100 μg/mL) or rhTGF-β (10 ng/mL). Osteoblastic differentiation was examined through in vitro matrix mineralization by alizarin red staining, and mRNA expression of osteopontin and osteonectin was determined by quantitative reverse-transcriptase polymerase chain reaction. hPDLSCs were further assessed for osteocalcin mRNA expression. Stem cells cultured in osteogenic medium were employed as a standard positive control group. In none of the experimental groups were bone-related mRNAs detected subsequent to treatment with EMD for 5, 10, and 15 days. Alizarin red staining on day 21 was negative in EMD-treated BMSC and PDLSC cultures. In rhTGF-β-supplemented BMSC culture, expression of osteonectin mRNA was demonstrated on day 15, which was statistically comparable to the positive control group. Nevertheless, extracellular matrix mineralization was inhibited in both groups of stem cells. Within the limitations of this study, it could be concluded that EMD with a concentration of 10, 50, or 100 μg/mL has no appreciable effect on osteoblastic differentiation of BMSCs and PDLSCs. Application of rhTGF-β increased osteonectin mRNA expression in BMSCs. This finding corroborates the hypothesis that TGF-β might be involved in early osteoblastic maturation.

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.

Stem Cells in Aggregate Form to Enhance Chondrogenesis in Hydrogels

PubMed Central

Sridharan, BanuPriya; Lin, Staphany M.; Hwu, Alexander T.; Laflin, Amy D.; Detamore, Michael S.

2015-01-01

There are a variety of exciting hydrogel technologies being explored for cartilage regenerative medicine. Our overall goal is to explore whether using stem cells in an aggregate form may be advantageous in these applications. 3D stem cell aggregates hold great promise as they may recapitulate the in vivo skeletal tissue condensation, a property that is not typically observed in 2D culture. We considered two different stem cell sources, human umbilical cord Wharton’s jelly cells (hWJCs, currently being used in clinical trials) and rat bone marrow-derived mesenchymal stem cells (rBMSCs). The objective of the current study was to compare the influence of cell phenotype, aggregate size, and aggregate number on chondrogenic differentiation in a generic hydrogel (agarose) platform. Despite being differing cell sources, both rBMSC and hWJC aggregates were consistent in outperforming cell suspension control groups in biosynthesis and chondrogenesis. Higher cell density impacted biosynthesis favorably, and the number of aggregates positively influenced chondrogenesis. Therefore, we recommend that investigators employing hydrogels consider using cells in an aggregate form for enhanced chondrogenic performance. PMID:26719986

Magnetic resonance imaging with superparamagnetic iron oxide fails to track the long-term fate of mesenchymal stem cells transplanted into heart.

PubMed

Ma, Ning; Cheng, Huaibing; Lu, Minjie; Liu, Qiong; Chen, Xiuyu; Yin, Gang; Zhu, Hao; Zhang, Lianfeng; Meng, Xianmin; Tang, Yue; Zhao, Shihua

2015-03-12

MRI for in vivo stem cell tracking remains controversial. Here we tested the hypothesis that MRI can track the long-term fate of the superparamagnetic iron oxide (SPIO) nanoparticles labelled mesenchymal stem cells (MSCs) following intramyocardially injection in AMI rats. MSCs (1 × 10(6)) from male rats doubly labeled with SPIO and DAPI were injected 2 weeks after myocardial infarction. The control group received cell-free media injection. In vivo serial MRI was performed at 24 hours before cell delivery (baseline), 3 days, 1, 2, and 4 weeks after cell delivery, respectively. Serial follow-up MRI demonstrated large persistent intramyocardial signal-voids representing SPIO during the follow-up of 4 weeks, and MSCs did not moderate the left ventricular dysfunction. The TUNEL analysis confirmed that MSCs engrafted underwent apoptosis. The histopathological studies revealed that the site of cell injection was infiltrated by inflammatory cells progressively and the iron-positive cells were macrophages identified by CD68 staining, but very few or no DAPI-positive stem cells at 4 weeks after cells transplantation. The presence of engrafted cells was confirmed by real-time PCR, which showed that the amount of Y-chromosome-specific SRY gene was consistent with the results. MRI may not reliably track the long-term fate of SPIO-labeled MSCs engraftment in heart.

Environmental microbial contamination in a stem cell bank.

PubMed

Cobo, F; Concha, A

2007-04-01

The aim of this study was to evaluate the main environmental microbial contaminants of the clean rooms in our stem cell bank. We have measured the microbial air contamination by both passive and active air sampling and the microbial monitoring of surfaces by means of Rodac plates. The environmental monitoring tests were carried out in accordance with the guidelines of European Pharmacopeia and US Pharmacopeia. The micro-organisms were identified by means of an automated system (VITEK 2). During the monitoring, the clean rooms are continually under good manufacturing practices specifications. The most frequent contaminants were Gram-positive cocci. The main contaminants in our stem cell bank were coagulase-negative staphylococci and other opportunistic human pathogens. In order to assure the levels of potential contamination in both embryonic and adult stem cell lines, a continuous sampling of air particles and testing for viable microbiological contamination is necessary. This study is the first evaluation of the environmental contaminants in stem cell banks and can serve as initial evaluation for these establishments. The introduction of environmental monitoring programmes in the processing of stem cell lines could diminish the risk of contamination in stem cell cultures.

Optimal culture conditions are critical for efficient expansion of human testicular somatic and germ cells in vitro.

PubMed

Gat, Itai; Maghen, Leila; Filice, Melissa; Wyse, Brandon; Zohni, Khaled; Jarvi, Keith; Lo, Kirk C; Gauthier Fisher, Andrée; Librach, Clifford

2017-03-01

To optimize culture conditions for human testicular somatic cells (TSCs) and spermatogonial stem cells. Basic science study. Urology clinic and stem cell research laboratory. Eight human testicular samples. Testicular tissues were processed by mechanical and enzymatic digestion. Cell suspensions were subjected to differential plating (DP) after which floating cells (representing germ cells) were removed and attached cells (representing TSCs) were cultured for 2 passages (P0-P1) in StemPro-34- or DMEM-F12-based medium. Germ cell cultures were established in both media for 12 days. TSC cultures: proliferation doubling time (PDT), fluorescence-activated cell sorting for CD90, next-generation sequencing for 89 RNA transcripts, immunocytochemistry for TSC and germ cell markers, and conditioned media analysis; germ cell cultures: number of aggregates. TSCs had significantly prolonged PDT in DMEM-F12 versus StemPro-34 (319.6 ± 275.8 h and 110.5 ± 68.3 h, respectively). The proportion of CD90-positive cells increased after P1 in StemPro-34 and DMEM-F12 (90.1 ± 10.8% and 76.5 ± 17.4%, respectively) versus after DP (66.3 ± 7%). Samples from both media after P1 clustered closely in the principle components analysis map whereas those after DP did not. After P1 in either medium, CD90-positive cells expressed TSC markers only, and fibroblast growth factor 2 and bone morphogenetic protein 4 were detected in conditioned medium. A higher number of germ cell aggregates formed in DMEM-F12 (59 ± 39 vs. 28 ± 17, respectively). Use of DMEM-F12 reduces TSC proliferation while preserving their unique characteristics, leading to improved germ cell aggregates formation compared with StemPro-34, the standard basal medium used in the majority of previous reports. Copyright © 2017. Published by Elsevier Inc.

Epigenetic regulation of open chromatin in pluripotent stem cells

PubMed Central

Kobayashi, Hiroshi; Kikyo, Nobuaki

2014-01-01

The recent progress in pluripotent stem cell research has opened new avenues of disease modeling, drug screening, and transplantation of patient-specific tissues that had been unimaginable until a decade ago. The central mechanism underlying pluripotency is epigenetic gene regulation; the majority of cell signaling pathways, both extracellular and cytoplasmic, eventually alter the epigenetic status of their target genes during the process of activating or suppressing the genes to acquire or maintain pluripotency. It has long been thought that the chromatin of pluripotent stem cells is globally open to enable the timely activation of essentially all genes in the genome during differentiation into multiple lineages. The current article reviews descriptive observations and the epigenetic machinery relevant to what is supposed to be globally open chromatin in pluripotent stem cells. This includes microscopic appearance, permissive gene transcription, chromatin remodeling complexes, histone modifications, DNA methylation, noncoding RNAs, dynamic movement of chromatin proteins, nucleosome accessibility and positioning, and long-range chromosomal interactions. Detailed analyses of each element, however, have revealed that the globally open chromatin hypothesis is not necessarily supported by some of the critical experimental evidence, such as genome-wide nucleosome accessibility and nucleosome positioning. Further understanding of the epigenetic gene regulation is expected to determine the true nature of the so-called globally open chromatin in pluripotent stem. PMID:24695097

The CXCL16-CXCR6 chemokine axis in glial tumors.

PubMed

Hattermann, Kirsten; Held-Feindt, Janka; Ludwig, Andreas; Mentlein, Rolf

2013-07-15

Since chemokines and their receptors play a pivotal role in tumors, we investigated the CXCL16-CXCR6-axis in human astroglial tumors. The transmembrane chemokine CXCL16 is heavily expressed by tumor, microglial and endothelial cells in situ and in vitro. In contrast, the receptor CXCR6 is restricted in glioblastomas to a small subset of proliferating cells positive for the stem-cell markers Musashi, Nanog, Sox2 and Oct4. In particular, the vast majority (about 90%) of Musashi-positive cells stained also for CXCR6. Thus, CXCL16 is highly expressed by glial tumor and stroma cells whereas CXCR6 defines a subset of cells with stem cell character. Copyright © 2013 Elsevier B.V. All rights reserved.

Combinatorial Gata2 and Sca1 expression defines hematopoietic stem cells in the bone marrow niche

PubMed Central

Suzuki, Norio; Ohneda, Osamu; Minegishi, Naoko; Nishikawa, Mitsuo; Ohta, Takayuki; Takahashi, Satoru; Engel, James Douglas; Yamamoto, Masayuki

2006-01-01

The interaction between stem cells and their supportive microenvironment is critical for their maintenance, function, and survival. Whereas hematopoietic stem cells (HSCs) are among the best characterized of tissue stem cells, their precise site of residence (referred to as the niche) in the adult bone marrow has not been precisely defined. In this study, we found that a Gata2 promoter directs activity in all HSCs. We show that HSCs can be isolated efficiently from bone marrow cells by following Gata2-directed GFP fluorescence, and that they can also be monitored in vivo. Each individual GFP-positive cell lay in a G0/G1 cell cycle state, in intimate contact with osteoblasts beside the endosteum, at the edge of the bone marrow. We conclude that the HSC niche is composed of solitary cells and that adult bone marrow HSC are not clustered. PMID:16461905

Isolation and Characterization of Prostate Cancer Stem Cells

DTIC Science & Technology

2009-08-01

The Prostate Manuscript ID: PROS-09-224.R1 Wiley - Manuscript type: Original Article Date Submitted by the Author: 18-Sep-2009 Complete List of ...subpopulation of basal cells has stem cell characteristics raises some interesting questions about the cell of origin for prostate cancer. Can both basal...positively for AMACR and the retention of 7 a p63+ basal layer, the basal-derived lesions fulfill the histologic criteria used

Mesenchymal stem cells attenuate blood-brain barrier leakage after cerebral ischemia in mice.

PubMed

Cheng, Zhuo; Wang, Liping; Qu, Meijie; Liang, Huaibin; Li, Wanlu; Li, Yongfang; Deng, Lidong; Zhang, Zhijun; Yang, Guo-Yuan

2018-05-03

Ischemic stroke induced matrixmetallo-proteinase-9 (MMP-9) upregulation, which increased blood-brain barrier permeability. Studies demonstrated that mesenchymal stem cell therapy protected blood-brain barrier disruption from several cerebrovascular diseases. However, the underlying mechanism was largely unknown. We therefore hypothesized that mesenchymal stem cells reduced blood-brain barrier destruction by inhibiting matrixmetallo-proteinase-9 and it was related to intercellular adhesion molecule-1 (ICAM-1). Adult ICR male mice (n = 118) underwent 90-min middle cerebral artery occlusion and received 2 × 10 5 mesenchymal stem cell transplantation. Neurobehavioral outcome, infarct volume, and blood-brain barrier permeability were measured after ischemia. The relationship between myeloperoxidase (MPO) activity and ICAM-1 release was further determined. We found that intracranial injection of mesenchymal stem cells reduced infarct volume and improved behavioral function in experimental stroke models (p < 0.05). IgG leakage, tight junction protein loss, and inflammatory cytokines IL-1β, IL-6, and TNF-α reduced in mesenchymal stem cell-treated mice compared to the control group following ischemia (p < 0.05). After transplantation, MMP-9 was decreased in protein and activity levels as compared with controls (p < 0.05). Furthermore, myeloperoxidase-positive cells and myeloperoxidase activity were decreased in mesenchymal stem cell-treated mice (p < 0.05). The results showed that mesenchymal stem cell therapy attenuated blood-brain barrier disruption in mice after ischemia. Mesenchymal stem cells attenuated the upward trend of MMP-9 and potentially via downregulating ICAM-1 in endothelial cells. Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) pathway may influence MMP-9 expression of neutrophils and resident cells, and ICAM-1 acted as a key factor in the paracrine actions of mesenchymal stem cell.

Stem cells technology: a powerful tool behind new brain treatments.

PubMed

Duru, Lucienne N; Quan, Zhenzhen; Qazi, Talal Jamil; Qing, Hong

2018-06-18

Stem cell research has recently become a hot research topic in biomedical research due to the foreseen unlimited potential of stem cells in tissue engineering and regenerative medicine. For many years, medicine has been facing intense challenges, such as an insufficient number of organ donations that is preventing clinicians to fulfill the increasing needs. To try and overcome this regrettable matter, research has been aiming at developing strategies to facilitate the in vitro culture and study of stem cells as a tool for tissue regeneration. Meanwhile, new developments in the microfluidics technology brought forward emerging cell culture applications that are currently allowing for a better chemical and physical control of cellular microenvironment. This review presents the latest developments in stem cell research that brought new therapies to the clinics and how the convergence of the microfluidics technology with stem cell research can have positive outcomes on the fields of regenerative medicine and high-throughput screening. These advances will bring new translational solutions for drug discovery and will upgrade in vitro cell culture to a new level of accuracy and performance. We hope this review will provide new insights into the understanding of new brain treatments from the perspective of stem cell technology especially regarding regenerative medicine and tissue engineering.

Wilms' tumor blastemal stem cells dedifferentiate to propagate the tumor bulk.

PubMed

Shukrun, Rachel; Pode-Shakked, Naomi; Pleniceanu, Oren; Omer, Dorit; Vax, Einav; Peer, Eyal; Pri-Chen, Sara; Jacob, Jasmine; Hu, Qianghua; Harari-Steinberg, Orit; Huff, Vicki; Dekel, Benjamin

2014-07-08

An open question remains in cancer stem cell (CSC) biology whether CSCs are by definition at the top of the differentiation hierarchy of the tumor. Wilms' tumor (WT), composed of blastema and differentiated renal elements resembling the nephrogenic zone of the developing kidney, is a valuable model for studying this question because early kidney differentiation is well characterized. WT neural cell adhesion molecule 1-positive (NCAM1(+)) aldehyde dehydrogenase 1-positive (ALDH1(+)) CSCs have been recently isolated and shown to harbor early renal progenitor traits. Herein, by generating pure blastema WT xenografts, composed solely of cells expressing the renal developmental markers SIX2 and NCAM1, we surprisingly show that sorted ALDH1(+) WT CSCs do not correspond to earliest renal stem cells. Rather, gene expression and proteomic comparative analyses disclose a cell type skewed more toward epithelial differentiation than the bulk of the blastema. Thus, WT CSCs are likely to dedifferentiate to propagate WT blastema.

Wilms’ Tumor Blastemal Stem Cells Dedifferentiate to Propagate the Tumor Bulk

PubMed Central

Shukrun, Rachel; Pode-Shakked, Naomi; Pleniceanu, Oren; Omer, Dorit; Vax, Einav; Peer, Eyal; Pri-Chen, Sara; Jacob, Jasmine; Hu, Qianghua; Harari-Steinberg, Orit; Huff, Vicki; Dekel, Benjamin

2014-01-01

Summary An open question remains in cancer stem cell (CSC) biology whether CSCs are by definition at the top of the differentiation hierarchy of the tumor. Wilms’ tumor (WT), composed of blastema and differentiated renal elements resembling the nephrogenic zone of the developing kidney, is a valuable model for studying this question because early kidney differentiation is well characterized. WT neural cell adhesion molecule 1-positive (NCAM1+) aldehyde dehydrogenase 1-positive (ALDH1+) CSCs have been recently isolated and shown to harbor early renal progenitor traits. Herein, by generating pure blastema WT xenografts, composed solely of cells expressing the renal developmental markers SIX2 and NCAM1, we surprisingly show that sorted ALDH1+ WT CSCs do not correspond to earliest renal stem cells. Rather, gene expression and proteomic comparative analyses disclose a cell type skewed more toward epithelial differentiation than the bulk of the blastema. Thus, WT CSCs are likely to dedifferentiate to propagate WT blastema. PMID:25068119

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.

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.

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.

Dynamics associated with spontaneous differentiation of ovarian stem cells in vitro

PubMed Central

2014-01-01

Background Recent studies suggest that ovarian germ line stem cells replenish oocyte-pool in adult stage, and challenge the central doctrine of ‘fixed germ cell pool’ in mammalian reproductive biology. Two distinct populations of spherical stem cells with high nucleo-cytoplasmic ratio have been recently identified in the adult mammalian ovary surface epithelium (OSE) including nuclear OCT-4A positive very small embryonic-like (VSELs) and cytoplasmic OCT-4 expressing ovarian germ stem cells (OGSCs). Three weeks culture of scraped OSE cells results in spontaneous differentiation of the stem cells into oocyte-like, parthenote-like, embryoid body-like structures and also embryonic stem cell-like colonies whereas epithelial cells attach and transform into a bed of mesenchymal cells. Present study was undertaken, to further characterize ovarian stem cells and to comprehend better the process of spontaneous differentiation of ovarian stem cells into oocyte-like structures in vitro. Methods Ovarian stem cells were enriched by immunomagnetic sorting using SSEA-4 as a cell surface marker and were further characterized. Stem cells and clusters of OGSCs (reminiscent of germ cell nests in fetal ovaries), were characterized by immuno-localization for stem and germ cell specific markers and spontaneous differentiation in OSE cultures was studied by live cell imaging. Results Differential expression of markers specific for pluripotent VSELs (nuclear OCT-4A, SSEA-4, CD133), OGSCs (cytoplasmic OCT-4) primordial germ cells (FRAGILIS, STELLA, VASA) and germ cells (DAZL, GDF-9, SCP-3) were studied. Within one week of culture, stem cells became bigger in size, developed abundant cytoplasm, differentiated into germ cells, revealed presence of Balbiani body-like structure (mitochondrial cloud) and exhibited characteristic cytoplasmic streaming. Conclusions Presence of germ cell nests, Balbiani body-like structures and cytoplasmic streaming extensively described during fetal ovary development, are indeed well recapitulated during in vitro oogenesis in adult OSE cultures along with characteristic expression of stem/germ cell/oocyte markers. Further studies are required to assess the genetic integrity of in vitro derived oocytes before harnessing their clinical potential. Advance in our knowledge about germ cell differentiation from stem cells will enable researchers to design better in vitro strategies which in turn may have relevance to reproductive biology and regenerative medicine. PMID:24568237

Stem cell-mediated osteogenesis: therapeutic potential for bone tissue engineering

PubMed Central

Neman, Josh; Hambrecht, Amanda; Cadry, Cherie; Jandial, Rahul

2012-01-01

Intervertebral disc degeneration often requires bony spinal fusion for long-term relief. Current arthrodesis procedures use bone grafts from autogenous bone, allogenic backed bone, or synthetic materials. Autogenous bone grafts can result in donor site morbidity and pain at the donor site, while allogenic backed bone and synthetic materials have variable effectiveness. Given these limitations, researchers have focused on new treatments that will allow for safe and successful bone repair and regeneration. Mesenchymal stem cells have received attention for their ability to differentiate into osteoblasts, cells that synthesize new bone. With the recent advances in scaffold and biomaterial technology as well as stem cell manipulation and transplantation, stem cells and their scaffolds are uniquely positioned to bring about significant improvements in the treatment and outcomes of spinal fusion and other injuries. PMID:22500114

Stem cell-mediated osteogenesis: therapeutic potential for bone tissue engineering.

PubMed

Neman, Josh; Hambrecht, Amanda; Cadry, Cherie; Jandial, Rahul

2012-01-01

Intervertebral disc degeneration often requires bony spinal fusion for long-term relief. Current arthrodesis procedures use bone grafts from autogenous bone, allogenic backed bone, or synthetic materials. Autogenous bone grafts can result in donor site morbidity and pain at the donor site, while allogenic backed bone and synthetic materials have variable effectiveness. Given these limitations, researchers have focused on new treatments that will allow for safe and successful bone repair and regeneration. Mesenchymal stem cells have received attention for their ability to differentiate into osteoblasts, cells that synthesize new bone. With the recent advances in scaffold and biomaterial technology as well as stem cell manipulation and transplantation, stem cells and their scaffolds are uniquely positioned to bring about significant improvements in the treatment and outcomes of spinal fusion and other injuries.

Plant Growth Biophysics: the Basis for Growth Asymmetry Induced by Gravity

NASA Technical Reports Server (NTRS)

Cosgrove, D.

1985-01-01

The identification and quantification of the physical properties altered by gravity when plant stems grow upward was studied. Growth of the stem in vertical and horizontal positions was recorded by time lapse photography. A computer program that uses a cubic spline fitting algorithm was used to calculate the growth rate and curvature of the stem as a function of time. Plant stems were tested to ascertain whether cell osmotic pressure was altered by gravity. A technique for measuring the yielding properties of the cell wall was developed.

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

In vitro induction effect of 1,25(OH)2D3 on differentiation of hair follicle stem cell into keratinocyte.

PubMed

Joulai Veijouyeh, Sanaz; Mashayekhi, Farhad; Yari, Abazar; Heidari, Fatemeh; Sajedi, Nayereh; Moghani Ghoroghi, Fatemeh; Nobakht, Maliheh

2017-02-01

Stem cells are characterized by self-renewal and differentiation capabilities. The bulge hair follicle stem cells (HFSCs) are able to convert to epithelial components. The active metabolite of vitamin D, 1,25(OH) 2 D 3 , plays important roles in this differentiation process. In the present study has found that 1,25(OH) 2 D 3 induces the HFSCs differentiation into keratinocyte. HFSCs are isolated from rat whiskers and cultivated in DMEM medium. To isolate bulge stem cell population, flow cytometry and immunocytochemistry using K15, CD34 and nestin biomarkers were performed. In order to accelerate the HFSCs differentiation into eratinocyte, HFSCs were treated with 10 -12 M, 1,25(OH) 2 D 3 every 48 h for a week. Immunocytochemistry results showed that bulge stem cells are nestin and CD34 positive but K15 negative before differentiation. Subsequently flow cytometry results, showed that the expression of nestin, CD34 and K15 were 70.96%, 93.03% and 6.88% respectively. After differentiation, the immunocytochemical and flow cytometry results indicated that differentiated cells have positive reaction to K15 with 68.94% expression level. It was concluded that 10 -12 M, 1,25(OH) 2 D 3 could induce the HFSCs differentiation into keratinocytes. Copyright © 2017 Chang Gung University. Published by Elsevier B.V. All rights reserved.

Stem cell science in India: emerging economies and the politics of globalization.

PubMed

Salter, Brian; Cooper, Melinda; Dickins, Amanda; Cardo, Valentina

2007-01-01

The globalization of stem cell science is increasingly being shaped by the emerging economies of the Asia/Pacific region. Undaunted and unhampered by the more established views of the commercialization of science, countries such as India are constructing models of innovation, policies and patterns of investment that challenge such orthodoxies. This report examines the position of India within the globalization of stem cell science, its adjustments to the developing knowledge market in this field and its particular contribution to the likely future of this promising bioeconomy.

The "Growing" Reality of the Neurological Complications of Global "Stem Cell Tourism".

PubMed

Julian, Katie; Yuhasz, Nick; Hollingsworth, Ethan; Imitola, Jaime

2018-04-01

"Stem cell tourism" is defined as the unethical practice of offering unproven cellular preparations to patients suffering from various medical conditions. This phenomenon is rising in the field of neurology as patients are requesting information and opportunities for treatment with stem cells for incurable conditions such as multiple sclerosis and amyotrophic lateral sclerosis, despite their clinical research and experimental designation. Here, we review the recent trends in "stem cell tourism" in both the United States and abroad, and discuss the recent reports of neurological complications from these activities. Finally, we frame critical questions for the field of neurology regarding training in the ethical, legal, and societal issues of the global "stem cell tourism," as well as suggest strategies to alleviate this problem. Although there are ongoing legitimate clinical trials with stem cells for neurological diseases, procedures offered by "stem cell clinics" cannot be defined as clinical research. They lack the experimental and state-of-the-art framework defined by peers and the FDA that focus on human research that safeguard the protection of human subjects against economical exploitation, unwanted side effects, and futility of unproven procedures. "Stem cell tourism" ultimately exploits therapeutic hope of patients and families with incurable neurological diseases and can put in danger the legitimacy of stem cell research as a whole. We posit that an improvement in education, regulation, legislation, and involvement of authorities in global health in neurology and neurosurgery is required. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

Role of Neural Stem Cell Activity in Postweaning Development of the Sexually Dimorphic Nucleus of the Preoptic Area in Rats

PubMed Central

He, Zhen; Ferguson, Sherry A.; Cui, Li; Greenfield, L. John; Paule, Merle G.

2013-01-01

The sexually dimorphic nucleus of the preoptic area (SDN-POA) has received increased attention due to its apparent sensitivity to estrogen-like compounds found in food and food containers. The mechanisms that regulate SDN-POA volume remain unclear as is the extent of postweaning development of the SDN-POA. Here we demonstrate that the female Sprague-Dawley SDN-POA volume increased from weaning to adulthood, although this increase was not statistically significant as it was in males. The number of cells positive for Ki67, a marker of cell proliferation, in both the SDN-POA and the hypothalamus was significantly higher at weaning than at adulthood in male rats. In contrast, the number of Ki67-positive cells was significantly higher in the hypothalamus but not in the SDN-POA (p>0.05) at weaning than at adulthood in female rats. A subset of the Ki67-positive cells in the SDN-POA displayed the morphology of dividing cells. Nestin-immunoreactivity delineated a potential macroscopic neural stem cell niche in the rostral end of the 3rd ventricle. In conclusion, stem cells may partially account for the sexually dimorphic postweaning development of the SDN-POA. PMID:23383001

Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy

NASA Technical Reports Server (NTRS)

Askari, Arman T.; Unzek, Samuel; Popovic, Zoran B.; Goldman, Corey K.; Forudi, Farhad; Kiedrowski, Matthew; Rovner, Aleksandr; Ellis, Stephen G.; Thomas, James D.; DiCorleto, Paul E.;

Differentiation of Human Dental Stem Cells Reveal a Role for microRNA-218

PubMed Central

Gay, Isabel; Cavender, Adriana; Peto, David; Sun, Zhao; Speer, Aline; Cao, Huojun; Amendt, Brad A.

2013-01-01

Background Regeneration of the lost periodontium is the ultimate goal of periodontal therapy. Advances in tissue engineering have demonstrated the multilineage potential and plasticity of adult stem cells located in the periodontal apparatus. However, it remains unclear how epigenetic mechanisms controlling signals determine tissue specification and cell lineage decisions. To date, no data is available on micro-RNAs (miRNAs) activity behind human-derived dental stem cells. Methods In this study, we isolated periodontal ligament stem cells (PDLSCs), dental pulp stem cells (DPSCs), and gingival stem cells (GSCs) from extracted third molars; human bone marrow stem cells (BMSCs) were used as a positive control. The expression of OCT4A and NANOG was confirmed in these undifferentiated cells. All cells were cultured under osteogenic inductive conditions and RUNX2 expression was analyzed as a marker of mineralized tissue differentiation. A miRNA expression profile was obtained at baseline and after osteogenic induction in all cell types. Results RUNX2 expression demonstrated the successful osteogenic induction of all cell types, which was confirmed by alizarin red stain. The analysis of 765 miRNAs demonstrated a shift in miRNA expression occurred in all four stem cell types, including a decrease in hsa-mir-218 across all differentiated cell populations. Hsa-mir-218 targets RUNX2 and decreases RUNX2 expression in undifferentiated human dental stem cells (DSCs). DSC mineralized tissue type differentiation is associated with a decrease in hsa-mir-218 expression. Conclusions These data reveal a miRNA regulated pathway for the differentiation of human DSCs and a select network of human microRNAs that control DSC osteogenic differentiation. PMID:23662917

Pluripotent stem cell-derived radial glia-like cells as stable intermediate for efficient generation of human oligodendrocytes.

PubMed

Gorris, Raphaela; Fischer, Julia; Erwes, Kim Lina; Kesavan, Jaideep; Peterson, Daniel A; Alexander, Michael; Nöthen, Markus M; Peitz, Michael; Quandel, Tamara; Karus, Michael; Brüstle, Oliver

2015-12-01

Neural precursor cells (NPCs) derived from human pluripotent stem cells (hPSCs) represent an attractive tool for the in vitro generation of various neural cell types. However, the developmentally early NPCs emerging during hPSC differentiation typically show a strong propensity for neuronal differentiation, with more limited potential for generating astrocytes and, in particular, for generating oligodendrocytes. This phenomenon corresponds well to the consecutive and protracted generation of neurons and GLIA during normal human development. To obtain a more gliogenic NPC type, we combined growth factor-mediated expansion with pre-exposure to the differentiation-inducing agent retinoic acid and subsequent immunoisolation of CD133-positive cells. This protocol yields an adherent and self-renewing population of hindbrain/spinal cord radial glia (RG)-like neural precursor cells (RGL-NPCs) expressing typical neural stem cell markers such as nestin, ASCL1, SOX2, and PAX6 as well as RG markers BLBP, GLAST, vimentin, and GFAP. While RGL-NPCs maintain the ability for tripotential differentiation into neurons, astrocytes, and oligodendrocytes, they exhibit greatly enhanced propensity for oligodendrocyte generation. Under defined differentiation conditions promoting the expression of the major oligodendrocyte fate-determinants OLIG1/2, NKX6.2, NKX2.2, and SOX10, RGL-NPCs efficiently convert into NG2-positive oligodendroglial progenitor cells (OPCs) and are subsequently capable of in vivo myelination. Representing a stable intermediate between PSCs and OPCs, RGL-NPCs expedite the generation of PSC-derived oligodendrocytes with O4-, 4860-, and myelin basic protein (MBP)-positive cells that already appear within 7 weeks following growth factor withdrawal-induced differentiation. Thus, RGL-NPCs may serve as robust tool for time-efficient generation of human oligodendrocytes from embryonic and induced pluripotent stem cells. © 2015 Wiley Periodicals, Inc.

Combination therapy with carfilzomib, lenalidomide and dexamethasone (KRd) results in an unprecedented purity of the stem cell graft in newly diagnosed patients with myeloma.

PubMed

Tageja, Nishant; Korde, Neha; Kazandjian, Dickran; Panch, Sandhya; Manasanch, Elisabet; Bhutani, Manisha; Kwok, Mary; Mailankody, Sham; Yuan, Constance; Stetler-Stevenson, Maryalice; Leitman, Susan F; Sportes, Claude; Landgren, Ola

2018-05-04

Still, many physicians give 4 cycles of combination therapy to multiple myeloma patients prior to collection of stem cells for autologous bone marrow transplant. This tradition originates from older doxorubicin-containing regiments which limited the number of cycles due to cumulative cardiotoxicity. Using older regiments, most patients had residual myeloma cells in their autologous stem-cell grafts during collection. Emerging data show that newly diagnosed multiple myeloma patients treated with modern carfilzomib/lenalidomide/dexamethasone (KRd) therapy, on average, take 6 cycles until reaching minimal residual disease (MRD) negativity. We assessed newly diagnosed patients treated with KRd focusing MRD status both in the individual patient's bone marrow, and the corresponding autologous hematopoietic progenitor cell grafts during collection. Per protocol, stem-cell collection was allowed after 4 to 8 cycles of KRd. We found similar stem-cell yield independent of the number of cycles of KRd. At stem-cell collection, 11/30 patients (36.6%) were MRD negative in their bone marrow; all 11 patients had MRD negative hematopoietic progenitor cell grafts. Furthermore, 18/19 patients who were MRD positive in their bone marrows also had MRD negative hematopoietic progenitor cell grafts. These observations support 6 cycles of KRd as an efficacious and safe induction strategy prior to stem-cell collection.

A new method for cryopreserving adipose-derived stem cells: an attractive and suitable large-scale and long-term cell banking technology.

PubMed

De Rosa, Alfredo; De Francesco, Francesco; Tirino, Virginia; Ferraro, Giuseppe A; Desiderio, Vincenzo; Paino, Francesca; Pirozzi, Giuseppe; D'Andrea, Francesco; Papaccio, Gianpaolo

2009-12-01

Recent studies have shown potential ways for improving stem cell cryopreservation. The major need for autologous stem cell use is a long-term storage: this arises from the humans' hope of future use of their own cells. Therefore, it is important to evaluate the cell potential of vitality and differentiation before and after cryopreservation. Although several studies have shown a long-term preservation of adipose tissue, a few of them focused their attention to stem cells. The aim of this study was to evaluate the fate of cryopreserved stem cells collected from adipose tissue and stored at low a temperature in liquid nitrogen through an optimal cryopreservation solution (using slowly cooling in 6% threalose, 4% dimethyl sulfoxide, and 10% fetal bovine serum) and to develop a novel approach to efficiently preserve adipose-derived stem cells (ASCs) for future clinical applications. Results showed that stem cells, after being thawed, are still capable of differentiation and express all surface antigens detected before storage, confirming the integrity of their biology. In particular, ASCs differentiated into adipocytes, showed diffuse positivity for PPARgamma and adiponectin, and were also able to differentiate into endothelial cells without addition of angiogenic factors. Therefore, ASCs can be long-term cryopreserved, and this, due to their great numbers, is an attractive tool for clinical applications as well as of impact for the derived market.

Commentary: on bone marrow stem cells and openmindedness.

PubMed

Mezey, Eva

2004-02-01

Several lines of evidence support the concept that pluripotent stem cells reside in the hematopoietic system of adults, but each has been questioned for valid reasons. Thus, the results reported to date after infusion of bone marrow stem cells, may be due to cell fusion, non-physiological de-differentiation and subsequent differentiation to lineages directed by the culture environment, microchimerism, or transdifferentiation. Several authors have suggested complex ways of investigating each of these possibilities, but in no case are any of the suggested protocols complete, nor will they rule out other possible causes of the results observed to date. Determining the nature, origin, and characteristics of adult cells is important and interesting, but the important question at this time is not what happens physiologically, but what we can do with these cells therapeutically. Research addressing therapeutic endpoints now takes a pivotal position in studies of nonembryonic stem cells.

TOO MANY MOUTHS promotes cell fate progression in stomatal development of Arabidopsis stems.

PubMed

Bhave, Neela S; Veley, Kira M; Nadeau, Jeanette A; Lucas, Jessica R; Bhave, Sanjay L; Sack, Fred D

2009-01-01

Mutations in TOO MANY MOUTHS (TMM), which encodes a receptor-like protein, cause stomatal patterning defects in Arabidopsis leaves but eliminate stomatal formation in stems. Stomatal development in wild-type and tmm stems was analyzed to define TMM function. Epidermal cells in young tmm stems underwent many asymmetric divisions characteristic of entry into the stomatal pathway. The resulting precursor cells, meristemoids, appropriately expressed cell fate markers such as pTMM:GFP. However, instead of progressing developmentally by forming a guard mother cell, the meristemoids arrested, dedifferentiated, and enlarged. Thus asymmetric divisions are necessary but not sufficient for stomatal formation in stems, and TMM promotes the fate and developmental progression of early precursor cells. Comparable developmental and mature stomatal phenotypes were also found in tmm hypocotyls and in the proximal flower stalk. TMM is also a positive regulator of meristemoid division in leaves suggesting that TMM generally promotes meristemoid activity. Our results are consistent with a model in which TMM interacts with other proteins to modulate precursor cell fate and progression in an organ and domain-specific manner. Finally, the consistent presence of a small number of dedifferentiated meristemoids in mature wild-type stems suggests that precursor cell arrest is a normal feature of Arabidopsis stem development.

Running rescues defective adult neurogenesis by shortening the length of the cell cycle of neural stem and progenitor cells.

PubMed

Farioli-Vecchioli, Stefano; Mattera, Andrea; Micheli, Laura; Ceccarelli, Manuela; Leonardi, Luca; Saraulli, Daniele; Costanzi, Marco; Cestari, Vincenzo; Rouault, Jean-Pierre; Tirone, Felice

2014-07-01

Physical exercise increases the generation of new neurons in adult neurogenesis. However, only few studies have investigated the beneficial effects of physical exercise in paradigms of impaired neurogenesis. Here, we demonstrate that running fully reverses the deficient adult neurogenesis within the hippocampus and subventricular zone of the lateral ventricle, observed in mice lacking the antiproliferative gene Btg1. We also evaluated for the first time how running influences the cell cycle kinetics of stem and precursor subpopulations of wild-type and Btg1-null mice, using a new method to determine the cell cycle length. Our data show that in wild-type mice running leads to a cell cycle shortening only of NeuroD1-positive progenitor cells. In contrast, in Btg1-null mice, physical exercise fully reactivates the defective hippocampal neurogenesis, by shortening the S-phase length and the overall cell cycle duration of both neural stem (glial fibrillary acidic protein(+) and Sox2(+)) and progenitor (NeuroD1(+)) cells. These events are sufficient and necessary to reactivate the hyperproliferation observed in Btg1-null early-postnatal mice and to expand the pool of adult neural stem and progenitor cells. Such a sustained increase of cell proliferation in Btg1-null mice after running provides a long-lasting increment of proliferation, differentiation, and production of newborn neurons, which rescues the impaired pattern separation previously identified in Btg1-null mice. This study shows that running positively affects the cell cycle kinetics of specific subpopulations of newly generated neurons and suggests that the plasticity of neural stem cells without cell cycle inhibitory control is reactivated by running, with implications for the long-term modulation of neurogenesis. © 2014 AlphaMed Press.

Purification of cardiomyocytes from differentiating pluripotent stem cells using molecular beacons that target cardiomyocyte-specific mRNA.

PubMed

Ban, Kiwon; Wile, Brian; Kim, Sangsung; Park, Hun-Jun; Byun, Jaemin; Cho, Kyu-Won; Saafir, Talib; Song, Ming-Ke; Yu, Shan Ping; Wagner, Mary; Bao, Gang; Yoon, Young-Sup

2013-10-22

Although methods for generating cardiomyocytes from pluripotent stem cells have been reported, current methods produce heterogeneous mixtures of cardiomyocytes and noncardiomyocyte cells. Here, we report an entirely novel system in which pluripotent stem cell-derived cardiomyocytes are purified by cardiomyocyte-specific molecular beacons (MBs). MBs are nanoscale probes that emit a fluorescence signal when hybridized to target mRNAs. Five MBs targeting mRNAs of either cardiac troponin T or myosin heavy chain 6/7 were generated. Among 5 MBs, an MB that targeted myosin heavy chain 6/7 mRNA (MHC1-MB) identified up to 99% of HL-1 cardiomyocytes, a mouse cardiomyocyte cell line, but <3% of 4 noncardiomyocyte cell types in flow cytometry analysis, which indicates that MHC1-MB is specific for identifying cardiomyocytes. We delivered MHC1-MB into cardiomyogenically differentiated pluripotent stem cells through nucleofection. The detection rate of cardiomyocytes was similar to the percentages of cardiac troponin T- or cardiac troponin I-positive cardiomyocytes, which supports the specificity of MBs. Finally, MHC1-MB-positive cells were sorted by fluorescence-activated cell sorter from mouse and human pluripotent stem cell differentiating cultures, and ≈97% cells expressed cardiac troponin T or cardiac troponin I as determined by flow cytometry. These MB-based sorted cells maintained their cardiomyocyte characteristics, which was verified by spontaneous beating, electrophysiological studies, and expression of cardiac proteins. When transplanted in a myocardial infarction model, MB-based purified cardiomyocytes improved cardiac function and demonstrated significant engraftment for 4 weeks without forming tumors. We developed a novel cardiomyocyte selection system that allows production of highly purified cardiomyocytes. These purified cardiomyocytes and this system can be valuable for cell therapy and drug discovery.

Establishing a public umbilical cord blood stem cell bank for South Africa: an enquiry into public acceptability.

PubMed

Meissner-Roloff, Madelein; Pepper, Michael S

2013-12-01

South Africa (SA) faces a large unmet need for bone marrow (BM) transplantation, which could be alleviated in part by establishing a public umbilical cord blood stem cell bank (UCB SCB). Umbilical cord blood is an increasingly utilised source of hematopoietic stem cells for BM transplantation in addition to BM or mobilized peripheral blood stem cells. Establishing a public UCB SCB would therefore be a positive step towards improving the quality of health care in SA by providing for an important unmet need. This study takes the form of an enquiry into the acceptability of establishing a public bank through an interview with and questionnaire completed by mothers-to-be in the antenatal clinic of a large public hospital in SA. Initial results are positive, with 85 % of the participants in favour of establishing a public UCB SCB in SA. This initial probe will serve as a model for a more comprehensive national enquiry into public support and acceptability in different clinics, hospitals and provinces in SA.

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.

CD44 and ALDH1 immunoexpression as prognostic indicators of invasion and metastasis in oral squamous cell carcinoma.

PubMed

Ortiz, Rafael Carneiro; Lopes, Nathália Martins; Amôr, Nadia Ghinelli; Ponce, José Burgos; Schmerling, Cláudia Kliemann; Lara, Vanessa Soares; Moyses, Raquel Ajub; Rodini, Camila Oliveira

2018-05-23

Tumour metastasis has been associated with cancer stem cells, a small population with stem-like cells properties, higher rate of migration and metastatic potential compared to cells from the tumour bulk. Our aim was to evaluate the immunoexpression of the putative cancer stem cell biomarkers ALDH1 and CD44 in primary tumour and corresponding metastatic lymph nodes. Tumour tissue specimens (n=50) and corresponding metastatic lymph nodes (n=25) were surgically obtained from 50 patients with oral squamous cell carcinoma and submitted to immunohistochemistry. CD44 and ALDH1 were semi-quantitatively scored according to the proportion and intensity of positive cells within the invasive front and metastatic lymph nodes as a whole. A combined score was obtained by multiplying both parameters and later dichotomized into a final score classified as low (≤ 2) or high (> 2) immunoexpression. ALDH1 and CD44 immunoexpression was detected in both tumour sites, although the means of ALDH1 (P = 0.0985) and CD44 (P = 0.4220) cells were higher in metastasis compared to primary tumours. ALDH1 high was positively associated (P = 0.0184) with angiolymphatic invasion, while CD44 high was positively associated (P = 0.0181) with metastasis (N+). At multivariate analysis, CD44 significantly increased the odds of lymph node metastasis, regardless of T stage (OR=8,24; 1,64-65,64, p=0,0088). CD44 immunoexpression was a significant predictor of lymph node metastasis, while ALDH1 high immunostaining was associated with angiolymphatic invasion. Altogether, it suggests that immunoexpression of CD44 and ALDH1 links the cancer stem cell phenotype with oral squamous cell carcinoma invasion and metastasis. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

TCPs, WUSs, and WINDs: families of transcription factors that regulate shoot meristem formation, stem cell maintenance, and somatic cell differentiation.

PubMed

Ikeda, Miho; Ohme-Takagi, Masaru

2014-01-01

In contrast to somatic mammalian cells, which cannot alter their fate, plant cells can dedifferentiate to form totipotent callus cells and regenerate a whole plant, following treatment with specific phytohormones. However, the regulatory mechanisms and key factors that control differentiation-dedifferentiation and cell totipotency have not been completely clarified in plants. Recently, several plant transcription factors that regulate meristem formation and dedifferentiation have been identified and include members of the TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP), WUSCHEL (WUS), and WOUND INDUCED DEDIFFERENTIATION (WIND1) families. WUS and WIND positively control plant cell totipotency, while TCP negatively controls it. Interestingly, TCP is a transcriptional activator that acts as a negative regulator of shoot meristem formation, and WUS is a transcriptional repressor that positively maintains totipotency of the stem cells of the shoot meristem. We describe here the functions of TCP, WUS, and WIND transcription factors in the regulation of differentiation-dedifferentiation by positive and negative transcriptional regulators.

A Positive Regulatory Loop between a Wnt-Regulated Non-coding RNA and ASCL2 Controls Intestinal Stem Cell Fate.

PubMed

Giakountis, Antonis; Moulos, Panagiotis; Zarkou, Vasiliki; Oikonomou, Christina; Harokopos, Vaggelis; Hatzigeorgiou, Artemis G; Reczko, Martin; Hatzis, Pantelis

2016-06-21

The canonical Wnt pathway plays a central role in stem cell maintenance, differentiation, and proliferation in the intestinal epithelium. Constitutive, aberrant activity of the TCF4/β-catenin transcriptional complex is the primary transforming factor in colorectal cancer. We identify a nuclear long non-coding RNA, termed WiNTRLINC1, as a direct target of TCF4/β-catenin in colorectal cancer cells. WiNTRLINC1 positively regulates the expression of its genomic neighbor ASCL2, a transcription factor that controls intestinal stem cell fate. WiNTRLINC1 interacts with TCF4/β-catenin to mediate the juxtaposition of its promoter with the regulatory regions of ASCL2. ASCL2, in turn, regulates WiNTRLINC1 transcriptionally, closing a feedforward regulatory loop that controls stem cell-related gene expression. This regulatory circuitry is highly amplified in colorectal cancer and correlates with increased metastatic potential and decreased patient survival. Our results uncover the interplay between non-coding RNA-mediated regulation and Wnt signaling and point to the diagnostic and therapeutic potential of WiNTRLINC1. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Development of a novel method for amniotic fluid stem cell storage.

PubMed

Zavatti, Manuela; Beretti, Francesca; Casciaro, Francesca; Comitini, Giuseppina; Franchi, Fabrizia; Barbieri, Veronica; Bertoni, Laura; De Pol, Anto; La Sala, Giovanni B; Maraldi, Tullia

2017-08-01

Current procedures for collection of human amniotic fluid stem cells (hAFSCs) indicate that cells cultured in a flask for 2 weeks can then be used for research. However, hAFSCs can be retrieved directly from a small amount of amniotic fluid that can be obtained at the time of diagnostic amniocentesis. The aim of this study was to determine whether direct freezing of amniotic fluid cells is able to maintain or improve the potential of a sub-population of stem cells. We compared the potential of the hAFSCs regarding timing of freezing, cells obtained directly from amniotic fluid aspiration (D samples) and cells cultured in a flask before freezing (C samples). Colony-forming-unit ability, proliferation, morphology, stemness-related marker expression, senescence, apoptosis and differentiation potential of C and D samples were compared. hAFSCs isolated from D samples expressed mesenchymal stem cells markers until later passages, had a good proliferation rate and exhibited differentiation capacity similar to hAFSCs of C samples. Interestingly, direct freezing induced a higher concentration of cells positive for pluripotency stem cell markers, without teratoma formation in vivo. This study suggests that minimal processing may be adequate for the banking of amniotic fluid cells, avoiding in vitro passages before the storage and exposure to high oxygen concentration, which affect stem cell properties. This technique might be a cost-effective and reasonable approach to the process of Good Manufacturing Process accreditation for stem-cell banks. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Efficient and rapid derivation of primitive neural stem cells and generation of brain subtype neurons from human pluripotent stem cells.

PubMed

Yan, Yiping; Shin, Soojung; Jha, Balendu Shekhar; Liu, Qiuyue; Sheng, Jianting; Li, Fuhai; Zhan, Ming; Davis, Janine; Bharti, Kapil; Zeng, Xianmin; Rao, Mahendra; Malik, Nasir; Vemuri, Mohan C

2013-11-01

Human pluripotent stem cells (hPSCs), including human embryonic stem cells and human induced pluripotent stem cells, are unique cell sources for disease modeling, drug discovery screens, and cell therapy applications. The first step in producing neural lineages from hPSCs is the generation of neural stem cells (NSCs). Current methods of NSC derivation involve the time-consuming, labor-intensive steps of an embryoid body generation or coculture with stromal cell lines that result in low-efficiency derivation of NSCs. In this study, we report a highly efficient serum-free pluripotent stem cell neural induction medium that can induce hPSCs into primitive NSCs (pNSCs) in 7 days, obviating the need for time-consuming, laborious embryoid body generation or rosette picking. The pNSCs expressed the neural stem cell markers Pax6, Sox1, Sox2, and Nestin; were negative for Oct4; could be expanded for multiple passages; and could be differentiated into neurons, astrocytes, and oligodendrocytes, in addition to the brain region-specific neuronal subtypes GABAergic, dopaminergic, and motor neurons. Global gene expression of the transcripts of pNSCs was comparable to that of rosette-derived and human fetal-derived NSCs. This work demonstrates an efficient method to generate expandable pNSCs, which can be further differentiated into central nervous system neurons and glia with temporal, spatial, and positional cues of brain regional heterogeneity. This method of pNSC derivation sets the stage for the scalable production of clinically relevant neural cells for cell therapy applications in good manufacturing practice conditions.

Adipose Derived Stem Cells for Corneal Wound Healing after Laser Induced Corneal Lesions in Mice.

PubMed

Zeppieri, Marco; Salvetat, Maria Letizia; Beltrami, Antonio; Cesselli, Daniela; Russo, Rossella; Alcalde, Ignacio; Merayo-Lloves, Jesús; Brusini, Paolo; Parodi, Pier Camillo

2017-12-05

The aim of our study was to assess the clinical effectiveness of topical adipose derived stem cell (ADSC) treatment in laser induced corneal wounds in mice by comparing epithelial repair, inflammation, and histological analysis between treatment arms. Corneal lesions were performed on both eyes of 40 mice by laser induced photorefractive keratectomy. All eyes were treated with topical azythromycin bid for three days. Mice were divided in three treatment groups ( n = 20), which included: control, stem cells and basic serum; which received topical treatment three times daily for five consecutive days. Biomicroscope assessments and digital imaging were performed by two masked graders at 30, 54, 78, 100, and 172 h to analyze extent of fluorescein positive epithelial defect, corneal inflammation, etc. Immunohistochemical techniques were used in fixed eyes to assess corneal repair markers Ki67, α Smooth Muscle Actin (α-SMA) and E-Cadherin. The fluorescein positive corneal lesion areas were significantly smaller in the stem cells group on days 1 ( p < 0.05), 2 ( p < 0.02) and 3. The stem cell treated group had slightly better and faster re-epithelization than the serum treated group in the initial phases. Comparative histological data showed signs of earlier and better corneal repair in epithelium and stromal layers in stem cell treated eyes, which showed more epithelial layers and enhanced wound healing performance of Ki67, E-Cadherin, and α-SMA. Our study shows the potential clinical and histological advantages in the topical ADSC treatment for corneal lesions in mice.

Adipose Derived Stem Cells for Corneal Wound Healing after Laser Induced Corneal Lesions in Mice

PubMed Central

Salvetat, Maria Letizia; Beltrami, Antonio; Cesselli, Daniela; Russo, Rossella; Merayo-Lloves, Jesús; Brusini, Paolo; Parodi, Pier Camillo

2017-01-01

The aim of our study was to assess the clinical effectiveness of topical adipose derived stem cell (ADSC) treatment in laser induced corneal wounds in mice by comparing epithelial repair, inflammation, and histological analysis between treatment arms. Corneal lesions were performed on both eyes of 40 mice by laser induced photorefractive keratectomy. All eyes were treated with topical azythromycin bid for three days. Mice were divided in three treatment groups (n = 20), which included: control, stem cells and basic serum; which received topical treatment three times daily for five consecutive days. Biomicroscope assessments and digital imaging were performed by two masked graders at 30, 54, 78, 100, and 172 h to analyze extent of fluorescein positive epithelial defect, corneal inflammation, etc. Immunohistochemical techniques were used in fixed eyes to assess corneal repair markers Ki67, α Smooth Muscle Actin (α-SMA) and E-Cadherin. The fluorescein positive corneal lesion areas were significantly smaller in the stem cells group on days 1 (p < 0.05), 2 (p < 0.02) and 3. The stem cell treated group had slightly better and faster re-epithelization than the serum treated group in the initial phases. Comparative histological data showed signs of earlier and better corneal repair in epithelium and stromal layers in stem cell treated eyes, which showed more epithelial layers and enhanced wound healing performance of Ki67, E-Cadherin, and α-SMA. Our study shows the potential clinical and histological advantages in the topical ADSC treatment for corneal lesions in mice. PMID:29206194

Optimization of a serum-free culture medium for mouse embryonic stem cells using design of experiments (DoE) methodology.

PubMed

Knöspel, Fanny; Schindler, Rudolf K; Lübberstedt, Marc; Petzolt, Stephanie; Gerlach, Jörg C; Zeilinger, Katrin

2010-12-01

The in vitro culture behaviour of embryonic stem cells (ESC) is strongly influenced by the culture conditions. Current culture media for expansion of ESC contain some undefined substances. Considering potential clinical translation work with such cells, the use of defined media is desirable. We have used Design of Experiments (DoE) methods to investigate the composition of a serum-free chemically defined culture medium for expansion of mouse embryonic stem cells (mESC). Factor screening analysis according to Plackett-Burman revealed that insulin and leukaemia inhibitory factor (LIF) had a significant positive influence on the proliferation activity of the cells, while zinc and L: -cysteine reduced the cell growth. Further analysis using minimum run resolution IV (MinRes IV) design indicates that following factor adjustment LIF becomes the main factor for the survival and proliferation of mESC. In conclusion, DoE screening assays are applicable to develop and to refine culture media for stem cells and could also be employed to optimize culture media for human embryonic stem cells (hESC).

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

Isolation of stem-like cells from spontaneous feline mammary carcinomas: phenotypic characterization and tumorigenic potential.

PubMed

Barbieri, Federica; Wurth, Roberto; Ratto, Alessandra; Campanella, Chiara; Vito, Guendalina; Thellung, Stefano; Daga, Antonio; Cilli, Michele; Ferrari, Angelo; Florio, Tullio

2012-04-15

Current carcinogenesis theory states that only a small subset of tumor cells, the cancer stem cells or tumor initiating cells (TICs), are responsible for tumor formation and progression. Human breast cancer-initiating cells have been identified as CD44-expressing cells, which retain tumorigenic activity and display stem cell-like properties. Spontaneous feline mammary carcinoma (FMC) is an aggressive cancer, which shows biological similarities to the human tumor counterpart. We report the isolation and phenotypic characterization of FMC-derived stem/progenitor cells, showing in vitro self-renewal, long-lasting proliferation and in vivo tumorigenicity. Twenty-one FMC samples were collected, histologically classified and characterized for the expression of Ki67, EGFR, ER-α and CD44, by immunohistochemistry. By culture in stem cell permissive conditions, we isolated, from 13 FMCs, a CD44-positive subpopulation able to survive and proliferate in vitro as mammospheres of different sizes and morphologies. When injected in NOD/SCID mice, FMC stem-like cells initiate tumors, generating cell heterogeneity and recapitulating the original histotype. In serum-containing medium, spheroid cells showed differentiation properties as shown by morphological changes, the loss of CD44 expression and tumorigenic potential. These data show that stem-defined culture of FMC enriches for TICs and validate the use of these cells as a suitable model for comparative oncology studies of mammary biology and testing therapeutic strategies aimed at eradicating TICs. Copyright © 2012 Elsevier Inc. All rights reserved.

Tracing the destiny of mesenchymal stem cells from embryo to adult bone marrow and white adipose tissue via Pdgfrα expression.

PubMed

Miwa, Hiroyuki; Era, Takumi

2018-01-29

Mesenchymal stem cells (MSCs) are somatic stem cells that can be derived from adult bone marrow (BM) and white adipose tissue (WAT), and that display multipotency and self-renewal capacity. Although MSCs are essential for tissue formation and have already been used in clinical therapy, the origins and markers of these cells remain unknown. In this study, we first investigated the developmental process of MSCs in mouse embryos using the gene encoding platelet-derived growth factor receptor α ( Pdgfra ) as a marker. We then traced cells expressing Pdgfra and other genes (brachyury, Sox1 and Pmx1 ) in various mutant mouse embryos until the adult stage. This tracing of MSC origins and destinies indicates that embryonic MSCs emerge in waves and that almost all adult BM MSCs and WAT MSCs originate from mesoderm and embryonic Pdgfrα-positive cells. Furthermore, we demonstrate that adult Pdgfrα-positive cells are involved in some pathological conditions. © 2018. Published by The Company of Biologists Ltd.

Inhibition of FOXC2 restores epithelial phenotype and drug sensitivity in prostate cancer cells with stem-cell properties

PubMed Central

Paranjape, A N; Soundararajan, R; Werden, S J; Joseph, R; Taube, J H; Liu, H; Rodriguez-Canales, J; Sphyris, N; Wistuba, I; Miura, N; Dhillon, J; Mahajan, N; Mahajan, K; Chang, J T; Ittmann, M; Maity, S N; Logothetis, C; Tang, D G; Mani, S A

2016-01-01

Advanced prostate adenocarcinomas enriched in stem-cell features, as well as variant androgen receptor (AR)-negative neuroendocrine (NE)/small-cell prostate cancers are difficult to treat, and account for up to 30% of prostate cancer-related deaths every year. While existing therapies for prostate cancer such as androgen deprivation therapy (ADT), destroy the bulk of the AR-positive cells within the tumor, eradicating this population eventually leads to castration-resistance, owing to the continued survival of AR-/lo stem-like cells. In this study, we identified a critical nexus between p38MAPK signaling, and the transcription factor Forkhead Box Protein C2 (FOXC2) known to promote cancer stem-cells and metastasis. We demonstrate that prostate cancer cells that are insensitive to ADT, as well as high-grade/NE prostate tumors, are characterized by elevated FOXC2, and that targeting FOXC2 using a well-tolerated p38 inhibitor restores epithelial attributes and ADT-sensitivity, and reduces the shedding of circulating tumor cells in vivo with significant shrinkage in the tumor mass. This study thus specifies a tangible mechanism to target the AR-/lo population of prostate cancer cells with stem-cell properties. PMID:26804168

Application of stem cell/growth factor system, as a multimodal therapy approach in regenerative medicine to improve cell therapy yields.

PubMed

Pourrajab, Fatemeh; Babaei Zarch, Mojtaba; Baghi Yazdi, Mohammad; Rahimi Zarchi, Abolfazl; Vakili Zarch, Abbas

2014-04-15

Stem cells hold a great promise for regenerative medicine, especially for replacing cells in infarcted organ that hardly have any intrinsic renewal capacity, including heart and brain. Signaling pathways that regulate pluripotency or lineage-specific gene and protein expression have been the major focus of stem cell research. Between them, there are some well known signaling pathways such as GF/GFR systems, SDF-1α/CXC4 ligand receptor interaction and PI3K/Akt signaling, and cytokines may regulate cell fate decisions, and can be utilized to positively influence cell therapy outcomes or accentuate synergistic compliance. For example, contributing factors in the progression of heart failure are both the loss of cardiomyocytes after myocardial infarction, and the absence of an adequate endogenous repair signaling. Combining cell engraftment with therapeutic signaling factor delivery is more exciting in terms of host progenitor/donor stem cell survival and proliferation. Thus stem cell-based therapy, besides triggering signaling pathways through GF/GFR systems can become a realistic option in regenerative processes for replacing lost cells and reconstituting the damaged organ, as before. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Monitoring Notch Signaling-Associated Activation of Stem Cell Niches within Injured Dental Pulp

PubMed Central

Mitsiadis, Thimios A.; Catón, Javier; Pagella, Pierfrancesco; Orsini, Giovanna; Jimenez-Rojo, Lucia

2017-01-01

Dental pulp stem/progenitor cells guarantee tooth homeostasis, repair and regeneration throughout life. The decision between renewal and differentiation of these cells is influenced by physical and molecular interactions with stromal cells and extracellular matrix molecules forming the specialized microenvironment of dental pulp stem cell niches. Here we study the activation of putative pulp niches after tooth injury through the upregulation of Notch signaling pathway. Notch1, Notch2, and Notch3 molecules were used as markers of dental pulp stem/progenitor cells. Upon dental injury, Notch1 and Notch3 are detected in cells related to vascular structures suggesting a role of these proteins in the activation of specific pulpal perivascular niches. In contrast, a population of Notch2-positive cells that are actively proliferative is observed in the apical part of the pulp. Kinetics of these cells is followed up with a lipophilic DiI labeling, showing that apical pulp cells migrate toward the injury site where dynamic regenerative/repair events occur. The knowledge of the activation and regulation of dental pulp stem/progenitor cells within their niches in pathologic conditions may be helpful for the realization of innovative dental treatments in the near future. PMID:28611689

Stem cells and cancer of the stomach and intestine.

PubMed

Vries, Robert G J; Huch, Meritxell; Clevers, Hans

2010-10-01

Cancer in the 21st century has become the number one cause of death in developed countries. Although much progress has been made in improving patient survival, tumour relapse is one of the important causes of cancer treatment failure. An early observation in the study of cancer was the heterogeneity of tumours. Traditionally, this was explained by a combination of genomic instability of tumours and micro environmental factors leading to diverse phenotypical characteristics. It was assumed that cells in a tumour have an equal capacity to propagate the cancer. This model is currently known as the stochastic model. Recently, the Cancer stem cell model has been proposed to explain the heterogeneity of a tumour and its progression. According to this model, the heterogeneity of tumours is the result of aberrant differentiation of tumour cells into the cells of the tissue the tumour originated from. Tumours were suggested to contain stem cell-like cells, the cancer stem cells or tumour-initiating cells, which are uniquely capable of propagating a tumour much like normal stem cells fuel proliferation and differentiation in normal tissue. In this review we discuss the normal stem cell biology of the stomach and intestine followed by both the stochastic and cancer stem cell models in light of recent findings in the gastric and intestinal systems. The molecular pathways underlying normal and tumourigenic growth have been well studied, and recently the stem cells of the stomach and intestine have been identified. Furthermore, intestinal stem cells were identified as the cells-of-origin of colon cancer upon loss of the tumour suppressor APC. Lastly, several studies have proposed the positive identification of a cancer stem cell of human colon cancer. At the end we compare the cancer stem cell model and the stochastic model. We conclude that clonal evolution of tumour cells resulting from genetic mutations underlies tumour initiation and progression in both cancer models. This implies that at any point during tumour development any tumour cell can revert to a cancer stem cell after having gained a clonal advantage over the original cancer stem cell. Therefore, these models represent two sides of the same coin. Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Aberrant epithelial GREM1 expression initiates colonic tumorigenesis from cells outside the stem cell niche.

PubMed

Davis, Hayley; Irshad, Shazia; Bansal, Mukesh; Rafferty, Hannah; Boitsova, Tatjana; Bardella, Chiara; Jaeger, Emma; Lewis, Annabelle; Freeman-Mills, Luke; Giner, Francesc Castro; Rodenas-Cuadrado, Pedro; Mallappa, Sreelakshmi; Clark, Susan; Thomas, Huw; Jeffery, Rosemary; Poulsom, Richard; Rodriguez-Justo, Manuel; Novelli, Marco; Chetty, Runjan; Silver, Andrew; Sansom, Owen James; Greten, Florian R; Wang, Lai Mun; East, James Edward; Tomlinson, Ian; Leedham, Simon John

2015-01-01

Hereditary mixed polyposis syndrome (HMPS) is characterized by the development of mixed-morphology colorectal tumors and is caused by a 40-kb genetic duplication that results in aberrant epithelial expression of the gene encoding mesenchymal bone morphogenetic protein antagonist, GREM1. Here we use HMPS tissue and a mouse model of the disease to show that epithelial GREM1 disrupts homeostatic intestinal morphogen gradients, altering cell fate that is normally determined by position along the vertical epithelial axis. This promotes the persistence and/or reacquisition of stem cell properties in Lgr5-negative progenitor cells that have exited the stem cell niche. These cells form ectopic crypts, proliferate, accumulate somatic mutations and can initiate intestinal neoplasia, indicating that the crypt base stem cell is not the sole cell of origin of colorectal cancer. Furthermore, we show that epithelial expression of GREM1 also occurs in traditional serrated adenomas, sporadic premalignant lesions with a hitherto unknown pathogenesis, and these lesions can be considered the sporadic equivalents of HMPS polyps.

Mammary molecular portraits reveal lineage-specific features and progenitor cell vulnerabilities.

PubMed

Casey, Alison E; Sinha, Ankit; Singhania, Rajat; Livingstone, Julie; Waterhouse, Paul; Tharmapalan, Pirashaanthy; Cruickshank, Jennifer; Shehata, Mona; Drysdale, Erik; Fang, Hui; Kim, Hyeyeon; Isserlin, Ruth; Bailey, Swneke; Medina, Tiago; Deblois, Genevieve; Shiah, Yu-Jia; Barsyte-Lovejoy, Dalia; Hofer, Stefan; Bader, Gary; Lupien, Mathieu; Arrowsmith, Cheryl; Knapp, Stefan; De Carvalho, Daniel; Berman, Hal; Boutros, Paul C; Kislinger, Thomas; Khokha, Rama

2018-06-19

The mammary epithelium depends on specific lineages and their stem and progenitor function to accommodate hormone-triggered physiological demands in the adult female. Perturbations of these lineages underpin breast cancer risk, yet our understanding of normal mammary cell composition is incomplete. Here, we build a multimodal resource for the adult gland through comprehensive profiling of primary cell epigenomes, transcriptomes, and proteomes. We define systems-level relationships between chromatin-DNA-RNA-protein states, identify lineage-specific DNA methylation of transcription factor binding sites, and pinpoint proteins underlying progesterone responsiveness. Comparative proteomics of estrogen and progesterone receptor-positive and -negative cell populations, extensive target validation, and drug testing lead to discovery of stem and progenitor cell vulnerabilities. Top epigenetic drugs exert cytostatic effects; prevent adult mammary cell expansion, clonogenicity, and mammopoiesis; and deplete stem cell frequency. Select drugs also abrogate human breast progenitor cell activity in normal and high-risk patient samples. This integrative computational and functional study provides fundamental insight into mammary lineage and stem cell biology. © 2018 Casey et al.

Human adipose-derived stem cells (ADSC) and human periodontal ligament stem cells (PDLSC) as cellular substrates of a toxicity prediction assay.

PubMed

Corrêa, Natássia Caroline Resende; Kuligovski, Crisciele; Paschoal, Ariane Caroline Campos; Abud, Ana Paula Ressetti; Rebelatto, Carmen Lucia Kuniyoshi; Leite, Lidiane Maria Boldrini; Senegaglia, Alexandra Cristina; Dallagiovanna, Bruno; Aguiar, Alessandra Melo de

2018-02-01

With the increasing need to develop in vitro assays to replace animal use, human stem cell-derived methods are emerging and showing outstanding contributions to the toxicological screening of substances. Adult human stem cells such as adipose-derived stem cells (ADSC) and periodontal ligament stem cells (PDLSC) were used as cell substrates for a cytotoxicity assay and toxicity prediction using the neutral red uptake (NRU) assay. First, primary cell cultures from three independent donors, from each tissue source, were characterized as mesenchymal stem cells (MSC) by plastic adherence and appropriate immunophenotype for MSC markers (positive for CD90, CD73, and CD105 and negative for CD11b, CD34, CD45, HLADR, and CD19). Furthermore, ADSC and PDLSC were able to differentiate into adipocytes and osteoblasts when maintained under the same culture conditions previously established for the NRU assay. NRU assays for three reference test substances were performed. R 2 was higher than 0.85 for all conditions, showing the feasibility to calculate IC 50 values. The IC 50 values were then used to predict the LD 50 of the test substances, which were comparable to previous results and the ICCVAM standard test report. Primary ADSC and PDLSC showed the potential to be considered as additional models for use in cytotoxicity assays. Copyright © 2017 Elsevier Inc. All rights reserved.

Fascin Is Critical for the Maintenance of Breast Cancer Stem Cell Pool Predominantly via the Activation of the Notch Self-Renewal Pathway.

PubMed

Barnawi, Rayanah; Al-Khaldi, Samiyah; Majed Sleiman, Ghida; Sarkar, Abdullah; Al-Dhfyan, Abdullah; Al-Mohanna, Falah; Ghebeh, Hazem; Al-Alwan, Monther

2016-12-01

An emerging dogma shows that tumors are initiated and maintained by a subpopulation of cancer cells that hijack some stem cell features and thus referred to as "cancer stem cells" (CSCs). The exact mechanism that regulates the maintenance of CSC pool remains largely unknown. Fascin is an actin-bundling protein that we have previously demonstrated to be a major regulator of breast cancer chemoresistance and metastasis, two cardinal features of CSCs. Here, we manipulated fascin expression in breast cancer cell lines and used several in vitro and in vivo approaches to examine the relationship between fascin expression and breast CSCs. Fascin knockdown significantly reduced stem cell-like phenotype (CD44 hi /CD24 lo and ALDH + ) and reversal of epithelial to mesenchymal transition. Interestingly, expression of the embryonic stem cell transcriptional factors (Oct4, Nanog, Sox2, and Klf4) was significantly reduced when fascin expression was down-regulated. Functionally, fascin-knockdown cells were less competent in forming colonies and tumorspheres, consistent with lower basal self-renewal activity and higher susceptibility to chemotherapy. Fascin effect on CSC chemoresistance and self-renewability was associated with Notch signaling. Activation of Notch induced the relevant downstream targets predominantly in the fascin-positive cells. Limiting-dilution xenotransplantation assay showed higher frequency of tumor-initiating cells in the fascin-positive group. Collectively, our data demonstrated fascin as a critical regulator of breast CSC pool at least partially via activation of the Notch self-renewal signaling pathway and modification of the expression embryonic transcriptional factors. Targeting fascin may halt CSCs and thus presents a novel therapeutic approach for effective treatment of breast cancer. Stem Cells 2016;34:2799-2813 Video Highlight: https://youtu.be/GxS4fJ_Ow-o. © 2016 AlphaMed Press.

Cell type transcriptome atlas for the planarian Schmidtea mediterranea.

PubMed

Fincher, Christopher T; Wurtzel, Omri; de Hoog, Thom; Kravarik, Kellie M; Reddien, Peter W

2018-05-25

The transcriptome of a cell dictates its unique cell type biology. We used single-cell RNA sequencing to determine the transcriptomes for essentially every cell type of a complete animal: the regenerative planarian Schmidtea mediterranea. Planarians contain a diverse array of cell types, possess lineage progenitors for differentiated cells (including pluripotent stem cells), and constitutively express positional information, making them ideal for this undertaking. We generated data for 66,783 cells, defining transcriptomes for known and many previously unknown planarian cell types and for putative transition states between stem and differentiated cells. We also uncovered regionally expressed genes in muscle, which harbors positional information. Identifying the transcriptomes for potentially all cell types for many organisms should be readily attainable and represents a powerful approach to metazoan biology. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Ethical questions to ponder in the European stem cell patent debate.

PubMed

Curley, Duncan; Sharples, Andrew

2006-01-01

Patents may be refused in Europe on ethical grounds. Whereas in the past this issue has arisen only infrequently, recent developments in human embryonic stem cell research have given rise to conflicting opinions in Europe as to the approach that should be adopted in relation to patents. The United Kingdom Patent Office has adopted a positive policy towards inventions involving human embryonic stem cells, but the European Patent Office has to date refused to grant patent applications involving similar subject-matter. A series of legal questions on the role of ethics in granting European patents is now to be considered for clarification by the European Patent Office. The answers to these questions should eventually resolve the debate on the patenting of human embryonic stem cells throughout Europe.

Effect of calcium sprays on mechanical strength and cell wall fractions of herbaceous peony (Paeonia lactiflora pall.) inflorescence stems.

PubMed

Li, Chengzhong; Tao, Jun; Zhao, Daqiu; You, Chao; Ge, Jintao

2012-01-01

Calcium is an essential element and imparts significant structural rigidity to the plant cell walls, which provide the main mechanical support to the entire plant. In order to increase the mechanical strength of the inflorescence stems of herbaceous peony, the stems are treated with calcium chloride. The results shows that preharvest sprays with 4% (w/v) calcium chloride three times after bud emergence are the best at strengthening "Da Fugui" peonies' stems. Calcium sprays increased the concentrations of endogenous calcium, total pectin content as well as cell wall fractions in herbaceous peonies stems, and significantly increased the contents of them in the top segment. Correlation analysis showed that the breaking force of the top segment of peonies' stems was positively correlated with the ratio of water insoluble pectin to water soluble pectin (R = 0.673) as well as lignin contents (R = 0.926) after calcium applications.

Enhanced expression of PKM2 associates with the biological properties of cancer stem cells from A549 human lung cancer cells.

PubMed

Guo, Chang-Ying; Yan, Chen; Luo, Lan; Goto, Shinji; Urata, Yoshishige; Xu, Jian-Jun; Wen, Xiao-Ming; Kuang, Yu-Kang; Tou, Fang-Fang; Li, Tao-Sheng

2017-04-01

Cancer cells express the M2 isoform of glycolytic enzyme pyruvate kinase (PKM2) for favoring the survival under a hypoxic condition. Considering the relative low oxygen microenvironment in stem cell niche, we hypothesized that an enhanced PKM2 expression associates with the biological properties of cancer stem cells. We used A549 human lung cancer cell line and surgical resected lung cancer tissue samples from patients for experiments. We confirmed the co-localization of PKM2 and CD44, a popular marker for cancer stem cells in lung cancer tissue samples from patients. The expression of PKM2 was clearly observed in approximately 80% of the A549 human lung cancer cells. Remarkably, enhanced expression of PKM2 was specially observed in these cells that also positively expressed CD44. Downregulation of PKM2 in CD44+ cancer stem cells by siRNA significantly impaired the potency for spheroid formation, decreased the cell survival under fetal bovine serum deprivation and hypoxic conditions, but increased their sensitivity to anti-cancer drug of cisplatin and γ-ray. The enhanced expression of PKM2 seems to associate with the biological properties of cancer stem cells from A549 human lung cancer cells. Selective targeting of PKM2 may provide a new strategy for cancer therapy, especially for patients with therapeutic resistance.

The E3 Ligase Axotrophin/MARCH-7: Protein Expression Profiling of Human Tissues Reveals Links to Adult Stem Cells

PubMed Central

Szigyarto, Cristina A.; Sibbons, Paul; Williams, Gill; Uhlen, Mathias; Metcalfe, Su M.

2010-01-01

Axotrophin/MARCH-7 was first identified in mouse embryonic stem cells as a neural stem cell gene. Using the axotrophin/MARCH-7 null mouse, we discovered profound effects on T lymphocyte responses, including 8-fold hyperproliferation and 5-fold excess release of the stem cell cytokine leukemia inhibitory factor (LIF). Our further discovery that axotrophin/MARCH-7 is required for targeted degradation of the LIF receptor subunit gp190 implies a direct role in the regulation of LIF signaling. Bioinformatics studies revealed a highly conserved RING-CH domain in common with the MARCH family of E3-ubiquitin ligases, and accordingly, axotrophin was renamed “MARCH-7.” To probe protein expression of human axotrophin/MARCH-7, we prepared antibodies against different domains of the protein. Each antibody bound its specific target epitope with high affinity, and immunohistochemistry cross-validated target specificity. Forty-eight human tissue types were screened. Epithelial cells stained strongly, with trophoblasts having the greatest staining. In certain tissues, specific cell types were selectively positive, including neurons and neuronal progenitor cells in the hippocampus and cerebellum, endothelial sinusoids of the spleen, megakaryocytes in the bone marrow, crypt stem cells of the small intestine, and alveolar macrophages in the lung. Approximately 20% of central nervous system neuropils were positive. Notably, axotrophin/MARCH-7 has an expression profile that is distinct from that of other MARCH family members. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. (J Histochem Cytochem 58:301–308, 2010) PMID:19901269

Functionalizing Ascl1 with Novel Intracellular Protein Delivery Technology for Promoting Neuronal Differentiation of Human Induced Pluripotent Stem Cells.

PubMed

Robinson, Meghan; Chapani, Parv; Styan, Tara; Vaidyanathan, Ranjani; Willerth, Stephanie Michelle

2016-08-01

Pluripotent stem cells can become any cell type found in the body. Accordingly, one of the major challenges when working with pluripotent stem cells is producing a highly homogenous population of differentiated cells, which can then be used for downstream applications such as cell therapies or drug screening. The transcription factor Ascl1 plays a key role in neural development and previous work has shown that Ascl1 overexpression using viral vectors can reprogram fibroblasts directly into neurons. Here we report on how a recombinant version of the Ascl1 protein functionalized with intracellular protein delivery technology (Ascl1-IPTD) can be used to rapidly differentiate human induced pluripotent stem cells (hiPSCs) into neurons. We first evaluated a range of Ascl1-IPTD concentrations to determine the most effective amount for generating neurons from hiPSCs cultured in serum free media. Next, we looked at the frequency of Ascl1-IPTD supplementation in the media on differentiation and found that one time supplementation is sufficient enough to trigger the neural differentiation process. Ascl1-IPTD was efficiently taken up by the hiPSCs and enabled rapid differentiation into TUJ1-positive and NeuN-positive populations with neuronal morphology after 8 days. After 12 days of culture, hiPSC-derived neurons produced by Ascl1-IPTD treatment exhibited greater neurite length and higher numbers of branch points compared to neurons derived using a standard neural progenitor differentiation protocol. This work validates Ascl1-IPTD as a powerful tool for engineering neural tissue from pluripotent stem cells.

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.

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

Barbieri, Federica; Wurth, Roberto; Ratto, Alessandra

Current carcinogenesis theory states that only a small subset of tumor cells, the cancer stem cells or tumor initiating cells (TICs), are responsible for tumor formation and progression. Human breast cancer-initiating cells have been identified as CD44-expressing cells, which retain tumorigenic activity and display stem cell-like properties. Spontaneous feline mammary carcinoma (FMC) is an aggressive cancer, which shows biological similarities to the human tumor counterpart. We report the isolation and phenotypic characterization of FMC-derived stem/progenitor cells, showing in vitro self-renewal, long-lasting proliferation and in vivo tumorigenicity. Twenty-one FMC samples were collected, histologically classified and characterized for the expression of Ki67,more » EGFR, ER-{alpha} and CD44, by immunohistochemistry. By culture in stem cell permissive conditions, we isolated, from 13 FMCs, a CD44-positive subpopulation able to survive and proliferate in vitro as mammospheres of different sizes and morphologies. When injected in NOD/SCID mice, FMC stem-like cells initiate tumors, generating cell heterogeneity and recapitulating the original histotype. In serum-containing medium, spheroid cells showed differentiation properties as shown by morphological changes, the loss of CD44 expression and tumorigenic potential. These data show that stem-defined culture of FMC enriches for TICs and validate the use of these cells as a suitable model for comparative oncology studies of mammary biology and testing therapeutic strategies aimed at eradicating TICs. -- Highlights: Black-Right-Pointing-Pointer Feline mammary carcinoma contain a sub-population of stem-like cells expressing CD44 Black-Right-Pointing-Pointer These grow as spheres in serum-free medium and self-renew Black-Right-Pointing-Pointer Isolated stem-like cancer cells initiate tumor in immunodeficient mice Black-Right-Pointing-Pointer Xenografted tumors are phenotypically similar to the original tumor Black-Right-Pointing-Pointer Upon differentiation, cells grow as monolayers, loosing the tumorigenic potential.« less

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

In vitro expansion of Lin+ and Lin- mononuclear cells from human peripheral blood

NASA Astrophysics Data System (ADS)

Norhaiza, H. Siti; Rohaya, M. A. W.; Zarina, Z. A. Intan; Hisham, Z. A. Shahrul

2013-11-01

Haematopoietic stem cells (HSCs) are used in the therapy of blood disorders due to the ability of these cells to reconstitute haematopoietic lineage cells when transplanted into myeloablative recipients. However, substantial number of cells is required in order for the reconstitution to take place. Since HSCs present in low frequency, larger number of donor is required to accommodate the demand of transplantable HSCs. Therefore, in vitro expansion of HSCs will have profound impact on clinical purposes. The aim of this study was to expand lineage negative (Lin-) stem cells from human peripheral blood. Total peripheral blood mononuclear cells (PBMNCs) were fractionated from human blood by density gradient centrifugation. Subsequently, PBMNCs were subjected to magnetic assisted cell sorter (MACS) which depletes lineage positive (Lin+) mononuclear cells expressing lineage positive markers such as CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, and CD235a to obtained Lin- cell population. The ability of Lin+ and Lin- to survive in vitro was explored by culturing both cell populations in complete medium consisting of Alpha-Minimal Essential Medium (AMEM) +10% (v/v) Newborn Calf Serum (NBCS)+ 2% (v/v) pen/strep. In another experiment, Lin+ and Lin- were cultured with complete medium supplemented with 10ng/mL of the following growth factors: stem cell factor (SCF), interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF), 2IU/mL of Erythropoietin (Epo) and 20ng/mL of IL-6. Three samples were monitored in static culture for 22 days. The expansion potential was assessed by the number of total viable cells, counted by trypan blue exclusion assay. It was found that Lin+ mononuclear cells were not able to survive either in normal proliferation medium or proliferation medium supplemented with cytokines. Similarly, Lin- stem cells were not able to survive in proliferation medium however, addition of cytokines into the proliferation medium support Lin- stem cells for at least 18 days. The Lin- stem cells started to response to the cytokines added as early as Day 2 of culture. It is concluded that Lin- stem cells can be expanded in vitro by culturing in proliferation medium supplemented with cytokines.

The transplantation of neural stem cells and predictive factors in hematopoietic recovery in irradiated mice.

PubMed

Filip, S; Mokrý, J; Karbanová, J; Vávrová, J; Vokurková, J; Bláha, M; English, D

2005-04-01

A number of surprising observations have shown that stem cells, in suitable conditions, have the ability to produce a whole spectrum of cell types, regardless, whether these tissues are derived from the same germ layer or not. This phenomenon is called stem cell plasticity, which means that tissue-specific stem cells are mutually interchangeable. In our experiments, as a model, we used neural stem cells (NSCs) harvested from fetal (E14-15) neocortex and beta-galactosidase positive. In the first experiment we found that on days 12 and 30 after sub-lethal irradiation (LD 8.5 Gy) and (beta-galactosidase(+)) NSCs transplantation all mice survived, just as the group with bone marrow transplantation. Moreover, the bone marrow of mice transplanted NSCs contained the number of CFU-GM colonies with beta-galactosidase(+) cells which was as much as 50% higher. These differences were statistically significant, p<0.001. In the second experiment, we studied kinetics of (beta-galactosidase(+)) NSCs after their transplantation to sub-lethally irradiated mice. Histochemistry of tissues was performed on days 12 and 30 post-transplantation, and beta-galactosidase(+) cells were detected with the help of histochemical examination of removed tissues (lung, liver, spleen, thymus, and skeletal muscle). In tissues removed on day 12 post-transplantation, we found a significantly higher number of beta-galactosidase(+) cells in the spleen and thymus on day 30. While we presumed the presence beta-galactosidase(+) cells in the spleen, as spleen and reticuloendothelial system represent an important retaining system for different cell types, the presence of beta-galactosidase(+) cells in the thymus was rather surprising but very interesting. This indicates a certain mutual and close interconnection of transplanted stem cells and immune system in an adult organism. In the third experiment, we verified the mutual interchange of Sca-1 surface antigen in the bone marrow cells and NSCs before transplantation. Analysis of this antigen showed 24.8% Sca-1 positive cells among the bone marrow cells, while NSCs were Sca-1 negative. Our experiments show that NSCs share hemopoietic identity and may significantly influence the recovery of damaged hematopoiesis but do not have typical superficial markers as HSCs. This result is important for the determination of predictive factors for hemopoiesis recovery, for stem cell plasticity and for their use in the cell therapy.

Human embryonic stem cell research: an intercultural perspective.

PubMed

Walters, LeRoy

2004-03-01

In 1998, researchers discovered that embryonic stem cells could be derived from early human embryos. This discovery has raised a series of ethical and public-policy questions that are now being confronted by multiple international organizations, nations, cultures, and religious traditions. This essay surveys policies for human embryonic stem cell research in four regions of the world, reports on the recent debate at the United Nations about one type of such research, and reviews the positions that various religious traditions have adopted regarding this novel type of research. In several instances the religious traditions seem to have influenced the public-policy debates.

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

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.

Therapeutic Potential of Human Adipose-Derived Stem/Stromal Cell Microspheroids Prepared by Three-Dimensional Culture in Non-Cross-Linked Hyaluronic Acid Gel.

PubMed

Mineda, Kazuhide; Feng, Jingwei; Ishimine, Hisako; Takada, Hitomi; Doi, Kentaro; Kuno, Shinichiro; Kinoshita, Kahori; Kanayama, Koji; Kato, Harunosuke; Mashiko, Takanobu; Hashimoto, Ichiro; Nakanishi, Hideki; Kurisaki, Akira; Yoshimura, Kotaro

2015-12-01

Three-dimensional culture of mesenchymal stem/stromal cells for spheroid formation is known to enhance their therapeutic potential for regenerative medicine. Spheroids were prepared by culturing human adipose-derived stem/stromal cells (hASCs) in a non-cross-linked hyaluronic acid (HA) gel and compared with dissociated hASCs and hASC spheroids prepared using a nonadherent dish. Preliminary experiments indicated that a 4% HA gel was the most appropriate for forming hASC spheroids with a relatively consistent size (20-50 µm) within 48 hours. Prepared spheroids were positive for pluripotency markers (NANOG, OCT3/4, and SOX-2), and 40% of the cells were SSEA-3-positive, a marker of the multilineage differentiating stress enduring or Muse cell. In contrast with dissociated ASCs, increased secretion of cytokines such as hepatocyte growth factor was detected in ASC spheroids cultured under hypoxia. On microarray ASC spheroids showed upregulation of some pluripotency markers and downregulation of genes related to the mitotic cell cycle. After ischemia-reperfusion injury to the fat pad in SCID mice, local injection of hASC spheroids promoted tissue repair and reduced the final atrophy (1.6%) compared with that of dissociated hASCs (14.3%) or phosphate-buffered saline (20.3%). Part of the administered hASCs differentiated into vascular endothelial cells. ASC spheroids prepared in a HA gel contain undifferentiated cells with therapeutic potential to promote angiogenesis and tissue regeneration after damage. This study shows the therapeutic value of human adipose-derived stem cell spheroids prepared in hyarulonic acid gel. The spheroids have various benefits as an injectable cellular product and show therapeutic potential to the stem cell-depleted conditions such as diabetic chronic skin ulcer. ©AlphaMed Press.

Epstein-Barr-Virus-Positive B-Cell Lymphoma of Recipient Origin Despite of the Elimination of Clonally EBV-Infected T Cells by Allogeneic Stem Cell Transplantation in a Patient with Chronic Active EBV Infection

PubMed Central

Nagasawa, Masayuki

2012-01-01

A 20-year-old patient with chronic active EBV infection (CAEBV) received peripheral blood stem cell transplantation (PBSCT) from HLA-one-locus-mismatched mother. Although EB-virus-infected T cells were eliminated after PBSCT, she developed EB-virus-positive B-cell lymphoma of recipient origin in the brain. By reducing the immunosuppressive therapy, the initial lesion disappeared. However, another lesion in the opposite lateral brain appeared later and was resistant to further reduction of immunosuppressive therapy. EBV-DNA was persistently negative after PBSCT in the peripheral blood. This case is suggestive in management of EBV reactivation after SCT and understanding alloimmune response to EBV. PMID:23213608

Molecular cloning and production of caprine recombinant Oct4 protein for generation induced pluripotent stem cells.

PubMed

Singhal, Dinesh K; Singhal, Raxita; Malik, Hruda N; Singh, Surender; Kumar, Sudarshan; Kaushik, Jai K; Mohanty, Ashok K; Malakar, Dhruba

2015-12-01

Oct4, pluripotency marker and transcription factor, expresses in embryonic stem cells. It plays a pivotal role in determination of stem cells fate. Up and down regulation of Oct4 causes differentiation of embryonic stem cells. It is one of the main transcription factors which remained concerned in every study related to induced pluripotent stem cell. Here, we report the production of goat Oct4 protein using plasmid and lentiviral based vectors. Firstly, Oct4 ORF was cloned in pAcGFP1-N1 plasmid vector and positive clones were screened with colony PCR. Oct4 was over-expressed in CHO-K1 cell line and expression was confirmed by observing green florescent protein expression in CHO-K1 cells. Secondly, Oct4 lentiviral expression construct has been prepared using pLenti-gw vector. Oct4 ORF was cloned into pLenti4/V5-DEST vector and viral particles were produced in 293FT cells. Oct4 viral particles were used to infect goat fibroblast cells. Oct4 expression was observed and confirmed in transfected goat fibroblast cells using RT-PCR. Detection of Oct4 protein in western blotting assay affirmed the capacity of over-expression of our Oct4 lentiviral vector. The lentiviral expression construct and recombinant Oct4 protein may be used for reprogramming of somatic cell into induced pluripotent stem cell.

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.

Hair Follicle-Associated Pluripotent (HAP) Stem Cells in Gelfoam® Histoculture for Use in Spinal Cord Repair.

PubMed

Liu, Fang; Hoffman, Robert M

2018-01-01

The stem cell marker, nestin, is expressed in the hair follicle, both in cells in the bulge area (BA) and the dermal papilla (DP). Nestin-expressing hair follicle-associated-pluripotent (HAP) stem cells of both the BA and DP have been previously shown to be able to form neurons, heart muscle cells, and other non-follicle cell types. The ability of the nestin-expressing HAP stem cells from the BA and DP to repair spinal cord injury was compared. Nestin-expressing HAP stem cells from both the BA and DP grew very well on Gelfoam ® . The HAP stem cells attached to the Gelfoam ® within 1 h. They grew along the grids of the Gelfoam ® during the first 2 or 3 days. Later they spread into the Gelfoam ® . After transplantation of Gelfoam ® cultures of nestin-expressing BA or DP HAP stem cells into the injured spinal cord (including the Gelfoam ® ) nestin-expressing BA and DP cells were observed to be viable over 100 days post-surgery. Hematoxylin and eosin (H&E) staining showed connections between the transplanted cells and the host spine tissue. Immunohistochemistry showed many Tuj1-, Isl 1/2, and EN1-positive cells and nerve fibers in the transplanted area of the spinal cord after BA Gelfoam ® or DP Gelfoam ® cultures were transplanted to the spine. The spinal cord of mice was injured to effect hind-limb paralysis. Twenty-eight days after transplantation with BA or DP HAP stem cells on Gelfoam ® to the injured area of the spine, the mice recovered normal locomotion.

A reserve stem cell population in small intestine renders Lgr5-positive cells dispensable

PubMed Central

Tian, Hua; Biehs, Brian; Warming, Soren; Leong, Kevin G.; Rangell, Linda; Klein, Ophir D.; de Sauvage, Frederic J.

2014-01-01

The small intestine epithelium renews every 2 to 5 days, making it one of the most regenerative mammalian tissues. Genetic inducible fate mapping studies have identified two principal epithelial stem cell pools in this tissue. One pool consists of columnar Lgr5-expressing cells that cycle rapidly and are present predominantly at the crypt base1. The other pool consists of Bmi1-expressing cells that largely reside above the crypt base2. However, the relative functions of these two pools and their interrelationship are not understood. Here, we specifically ablated Lgr5-expressing cells using a diphtheria toxin receptor (DTR) gene knocked into the Lgr5 locus. We found that complete loss of the Lgr5-expressing cells did not perturb homeostasis of the epithelium, indicating that other cell types can compensate for elimination of this population. After ablation of Lgr5-expressing cells, progeny production by Bmi1-expressing cells increased, suggesting that Bmi1-expressing stem cells compensate for the loss of Lgr5-expressing cells. Indeed, lineage tracing showed that Bmi1-expressing cells gave rise to Lgr5-expressing cells, pointing to a hierarchy of stem cells in the intestinal epithelium. Our results demonstrate that Lgr5-expressing cells are dispensable for normal intestinal homeostasis. In the absence of these cells, the Bmi1-expressing cells can serve as an alternative stem cell pool, providing the first experimental evidence for the interrelationship between these populations. The Bmi1-expressing stem cells may represent both a reserve stem cell pool in case of injury to the small intestine epithelium and a source for replenishment of the Lgr5-expressing cells under non-pathological conditions. PMID:21927002

Electrophysiological properties of prion-positive cardiac progenitors derived from murine embryonic stem cells.

PubMed

Fujii, Hiroshi; Ikeuchi, Yu; Kurata, Yasutaka; Ikeda, Nobuhito; Bahrudin, Udin; Li, Peili; Nakayama, Yuji; Endo, Ryo; Hasegawa, Akira; Morikawa, Kumi; Miake, Junichiro; Yoshida, Akio; Hidaka, Kyoko; Morisaki, Takayuki; Ninomiya, Haruaki; Shirayoshi, Yasuaki; Yamamoto, Kazuhiro; Hisatome, Ichiro

2012-01-01

The prion protein (PrP) has been reported to serve as a surface maker for isolation of cardiomyogenic progenitors from murine embryonic stem (ES) cells. Although PrP-positive cells exhibited automaticity, their electrophysiological characteristics remain unresolved. The aim of the present study was therefore to investigate the electrophysiological properties of PrP-positive cells in comparison with those of HCN4p-or Nkx2.5-positive cells. Differentiation of AB1, HCN5p-EGFP and hcgp7 ES cells into cardiac progenitors was induced by embryoid body (EB) formation. EBs were dissociated and cells expressing PrP, HCN4-EGFP and/or Nkx2.5-GFP were collected via flow cytometry. Sorted cells were subjected to reverse transcriptase-polymerase chain reaction, immunostaining and patch-clamp experiments. PrP-positive cells expressed mRNA of undifferentiation markers, first and second heart field markers, and cardiac-specific genes and ion channels, indicating their commitment to cardiomyogenic progenitors. PrP-positive cells with automaticity showed positive and negative chronotropic responses to isoproterenol and carbamylcholine, respectively. Hyperpolarization-activated cation current (I(f)) was barely detectable, whereas Na(+) and L-type Ca(2+) channel currents were frequently observed. Their spontaneous activity was slowed by inhibition of sarcoplasmic reticulum Ca(2+) uptake and release but not by blocking I(f). The maximum diastolic potential of their spontaneous firings was more depolarized than that of Nkx2.5-GFP-positive cells. PrP-positive cells contained cardiac progenitors that separated from the lineage of sinoatrial node cells. PrP can be used as a marker to enrich nascent cardiac progenitors.

Cell lineages of the embryo of the nematode Caenorhabditis elegans.

PubMed

Deppe, U; Schierenberg, E; Cole, T; Krieg, C; Schmitt, D; Yoder, B; von Ehrenstein, G

1978-01-01

Embryogenesis of the free-living soil nematode Caenorhabditis elegans produces a juvenile having about 550 cells at hatching. We have determined the lineages of 182 cells by tracing the divisions of individual cells in living embryos. An invariant pattern of cleavage divisions of the egg generates a set of stem cells. These stem cells are the founders of six stem cell lineages. Each lineage has its own clock--i.e., an autonomous rhythm of synchronous cell divisions. The rhythms are maintained in spite of extensive cellular rearrangement. The rate and the orientation of the cell divisions of the cell lineages are essentially invariant among individuals. Thus, the destiny of cells seems to depend primarily on their lineage history. The anterior position of the site of origin of the stem cells in the egg relates to the rate of the cell cycle clock, suggesting intracellular preprogramming of the uncleaved egg. We used a technique that allows normal embryogenesis, from the fertilized egg to hatching, outside the parent under a cover glass. Embryogenesis was followed microscopically with Nomarski interference optics and high-resolution video recording.

New Monoclonal Antibodies to Defined Cell Surface Proteins on Human Pluripotent Stem Cells.

PubMed

O'Brien, Carmel M; Chy, Hun S; Zhou, Qi; Blumenfeld, Shiri; Lambshead, Jack W; Liu, Xiaodong; Kie, Joshua; Capaldo, Bianca D; Chung, Tung-Liang; Adams, Timothy E; Phan, Tram; Bentley, John D; McKinstry, William J; Oliva, Karen; McMurrick, Paul J; Wang, Yu-Chieh; Rossello, Fernando J; Lindeman, Geoffrey J; Chen, Di; Jarde, Thierry; Clark, Amander T; Abud, Helen E; Visvader, Jane E; Nefzger, Christian M; Polo, Jose M; Loring, Jeanne F; Laslett, Andrew L

2017-03-01

The study and application of human pluripotent stem cells (hPSCs) will be enhanced by the availability of well-characterized monoclonal antibodies (mAbs) detecting cell-surface epitopes. Here, we report generation of seven new mAbs that detect cell surface proteins present on live and fixed human ES cells (hESCs) and human iPS cells (hiPSCs), confirming our previous prediction that these proteins were present on the cell surface of hPSCs. The mAbs all show a high correlation with POU5F1 (OCT4) expression and other hPSC surface markers (TRA-160 and SSEA-4) in hPSC cultures and detect rare OCT4 positive cells in differentiated cell cultures. These mAbs are immunoreactive to cell surface protein epitopes on both primed and naive state hPSCs, providing useful research tools to investigate the cellular mechanisms underlying human pluripotency and states of cellular reprogramming. In addition, we report that subsets of the seven new mAbs are also immunoreactive to human bone marrow-derived mesenchymal stem cells (MSCs), normal human breast subsets and both normal and tumorigenic colorectal cell populations. The mAbs reported here should accelerate the investigation of the nature of pluripotency, and enable development of robust cell separation and tracing technologies to enrich or deplete for hPSCs and other human stem and somatic cell types. Stem Cells 2017;35:626-640. © 2016 The Authors Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Aging induced loss of stemness with concomitant gain of myogenic properties of a pure population of CD34(+)/CD45(-) muscle derived stem cells.

PubMed

Bose, Bipasha; Shenoy, P Sudheer

2016-01-01

Aging is accompanied by the functional decline of cells, tissues, and organs, as well as, a striking increase in susceptibility to a wide range of diseases. Within a tissue, both differentiated cells and adult stem cells are susceptible to intrinsic and extrinsic changes while aging. Muscle derived stem cells (MDSCs) are tissue specific stem cells which have been studied well for their multipotential nature. Although there are reports relating to diminished function and regenerative capacity of aged MDSCs as compared to their young counterparts, not much has been reported relating to the concomitant gain in unipotent nature of aged MDSCs. In this study, we report an inverse correlation between aging and expression of adult/mesenchymal stem cell markers and a direct correlation between aging and myogenecity in MDSCs. Aged MDSCs were able to generate a greater number of dystrophin positive myofibres, as compared to, the young MDSCs when transplanted in muscle of dystrophic mice. Our data, therefore, suggests that aging stress adds to the decline in stem cell characteristics with a concomitant increase in unipotency, in terms of, myogenecity of MDSCs. This study, hence, also opens the possibilities of using unipotent aged MDSCs as potential candidates for transplantation in patients with muscular dystrophies. Copyright © 2015. Published by Elsevier Ltd.

Manganese-Enhanced Magnetic Resonance Imaging Enables In Vivo Confirmation of Peri-Infarct Restoration Following Stem Cell Therapy in a Porcine Ischemia-Reperfusion Model.

PubMed

Dash, Rajesh; Kim, Paul J; Matsuura, Yuka; Ikeno, Fumiaki; Metzler, Scott; Huang, Ngan F; Lyons, Jennifer K; Nguyen, Patricia K; Ge, Xiaohu; Foo, Cheryl Wong Po; McConnell, Michael V; Wu, Joseph C; Yeung, Alan C; Harnish, Phillip; Yang, Phillip C

2015-07-27

The exact mechanism of stem cell therapy in augmenting the function of ischemic cardiomyopathy is unclear. In this study, we hypothesized that increased viability of the peri-infarct region (PIR) produces restorative benefits after stem cell engraftment. A novel multimodality imaging approach simultaneously assessed myocardial viability (manganese-enhanced magnetic resonance imaging [MEMRI]), myocardial scar (delayed gadolinium enhancement MRI), and transplanted stem cell engraftment (positron emission tomography reporter gene) in the injured porcine hearts. Twelve adult swine underwent ischemia-reperfusion injury. Digital subtraction of MEMRI-negative myocardium (intrainfarct region) from delayed gadolinium enhancement MRI-positive myocardium (PIR and intrainfarct region) clearly delineated the PIR in which the MEMRI-positive signal reflected PIR viability. Human amniotic mesenchymal stem cells (hAMSCs) represent a unique population of immunomodulatory mesodermal stem cells that restored the murine PIR. Immediately following hAMSC delivery, MEMRI demonstrated an increased PIR viability signal compared with control. Direct PIR viability remained higher in hAMSC-treated hearts for >6 weeks. Increased PIR viability correlated with improved regional contractility, left ventricular ejection fraction, infarct size, and hAMSC engraftment, as confirmed by immunocytochemistry. Increased MEMRI and positron emission tomography reporter gene signal in the intrainfarct region and the PIR correlated with sustained functional augmentation (global and regional) within the hAMSC group (mean change, left ventricular ejection fraction: hAMSC 85±60%, control 8±10%; P<0.05) and reduced chamber dilatation (left ventricular end-diastole volume increase: hAMSC 24±8%, control 110±30%; P<0.05). The positron emission tomography reporter gene signal of hAMSC engraftment correlates with the improved MEMRI signal in the PIR. The increased MEMRI signal represents PIR viability and the restorative potential of the injured heart. This in vivo multimodality imaging platform represents a novel, real-time method of tracking PIR viability and stem cell engraftment while providing a mechanistic explanation of the therapeutic efficacy of cardiovascular stem cells. © 2015 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Fabrication of a co-culture micro-bioreactor device for efficient hepatic differentiation of human induced pluripotent stem cells (hiPSCs).

PubMed

Kehtari, Mousa; Zeynali, Bahman; Soleimani, Masoud; Kabiri, Mahboubeh; Seyedjafari, Ehsan

2018-04-27

Primary hepatocytes, as the gold standard cell type for in vitro models, lose their characteristic morphology and functions after few days. There is an urgent need to develop physiologically relevant models that recapitulate liver microenvironment to obtain mature hepatocyte from stem cells. We designed and fabricated a micro-bioreactor device mimicking the physiological shear stress and cell-cell interaction in liver sinusoid microenvironment. Induced pluripotent stem cells (iPSCs) were co-cultured with human umbilical vein endothelial cells (HUVECs) in the micro-bioreactor device with continuous perfusion of hepatic differentiation medium (100 μL/h). Simulation results showed that flow field inside our perfusion device was uniform and shear stress was adjusted to physiological condition (<2 dyne/cm 2 ). IPSCs-derived hepatocytes (iPSCs-Heps) that were cultured in micro-bioreactor device showed a higher level of hepatic markers compared to those in static condition. Flow cytometry and immunocytochemistry analysis revealed iPSCs cultured in the device sequentially acquired characteristics of definitive endodermal cells (SOX17 positive), hepatoblasts (AFP positive) and mature hepatocyte (ALB positive). Moreover, the albumin and urea secretion were significantly higher in micro-bioreactor device than those cultured in culture dishes during experiment. Thus, based on our results, we propose our micro-bioreactor as a beneficial device to generate mature hepatocytes for drug screening and basic research.

Cellular reprogramming in skin cancer.

PubMed

Song, Ihn Young; Balmain, Allan

2015-06-01

Early primitive stem cells have long been viewed as the cancer cells of origin (tumor initiating target cells) due to their intrinsic features of self-renewal and longevity. However, emerging evidence suggests a surprising capacity for normal committed cells to function as reserve stem cells upon reprogramming as a consequence of tissue damage resulting in inflammation and wound healing. This results in an alternative concept positing that tumors may originate from differentiated cells that can re-acquire stem cell properties due to genetic or epigenetic reprogramming. It is likely that both models are correct, and that a continuum of potential cells of origin exists, ranging from early primitive stem cells to committed progenitor or even terminally differentiated cells. A combination of the nature of the target cell and the specific types of gene mutations introduced determine tumor cell lineage, as well as potential for malignant conversion. Evidence from mouse skin models of carcinogenesis suggests that initiated cells at different stages within a stem cell hierarchy have varying degrees of requirement for reprogramming (e.g. inflammation stimuli), depending on their degree of differentiation. This article will present evidence in favor of these concepts that has been developed from studies of several mouse models of skin carcinogenesis. Copyright © 2014 Elsevier Ltd. All rights reserved.

Effect of sertraline on proliferation and neurogenic differentiation of human adipose-derived stem cells

PubMed Central

Razavi, Shahnaz; Jahromi, Maliheh; Amirpour, Nushin; Khosravizadeh, Zahra

2014-01-01

Background: Antidepressant drugs are commonly employed for anxiety and mood disorders. Sertraline is extensively used as antidepressant in clinic. In addition, adipose tissue represents an abundant and accessible source of adult stem cells with the ability to differentiate in to multiple lineages. Therefore, human adipose-derived stem cells (hADSCs) may be useful for autologous transplantation. Materials and Methods: In the present study, we assessed the effect of antidepressant drug Sertraline on the proliferation and neurogenic differentiation of hADSCs using MTT assay and immunofluorescence technique respectively. Results: MTT assay analysis showed that 0.5 μM Sertraline significantly increased the proliferation rate of hADSCs induced cells (P < 0.05), while immunofluorescent staining indicated that Sertraline treatment during neurogenic differentiation could be decreased the percentage of glial fibrillary acidic protein and Nestin-positive cells, but did not significantly effect on the percentage of MAP2 positive cells. Conclusion: Overall, our data show that Sertraline can be promoting proliferation rate during neurogenic differentiation of hADSCs after 6 days post-induction, while Sertraline inhibits gliogenesis of induced hADSCs. PMID:24800186

SPECIFICITIES OF ENDOMETRIAL PROLIFERATION/STEM CELL INDEX DISTRIBUTION IN ENDOMETRIOID CARCINOMA OF DIFFERENT GRADE OF MALIGNANCY.

PubMed

Kikalishvili, N; Beriashvili, R; Muzashvili, T; Burkadze, G

2018-03-01

Endometrial neoplasia is the most common malignant tumor of female genital system in developed countries. The incidence of endometrial cancer has increased in the last years and despite advances in diagnosis and treatment, the death rates have steadily been increasing over the past 20 years. Therefore aspects of endometrial cancer development, pathogenesis and effective treatment is especially urgent to this day, as much of the risk for endometrial cancer development is influenced by the environment and lifestyle. Endometrial stem cells take the special place among somatic stem cells of female reproductive system-the detection of them and identification of their location in the complex cellular hierarchy still remains challenging. Further study of endometrial stem cells will clarify their role in gynecologic pathologies associated with hyper-proliferative states of endometrium. The aim of our study was to explore the specificities of endometrial proliferative/stem cell index distribution under endometrioid carcinoma of different grade of malignancy. The study represents a retrospective research. The coded and depersonalized material data from Acad. N. Kipshidze Central University Clinic was used in the study. 3 study groups - 1st study group "Endometrioid Carcinoma Grade 1" (14 cases), 2nd study group "Endometrioid Carcinoma Grade 2" (23 cases) and 3rd study group "Endometrioid Carcinoma Grade 3" were selected from routine histopathology tissue specimens of uterus. Hematoxilyn-eosin technology and immunohistochemistry with proliferation marker ki67 and stem cell marker CD146 was performed. The proliferative/stem cell index was calculated by the ratio of Ki67-positive cell percentage value divided by CD146-positive cell percentage value. The study showed that in the 1st study group labeled as "Endometrioid Carcinoma Grade 1", the proliferative/stem cell index ranges between 21.7 and 25.5. Its mean average value in the age distribution subgroups accounts for: 1.1) reproductive age - 22.4; 1.2) menopause - 23.5; 1.3) post-menopause - 24.8. Proliferative/stem cell index reaches its maximum in the samples retrieved from post-menopause age, and decreases significantly in reproductive age individuals. In the 2nd study group labeled as "Endometrioid Carcinoma Grade 2", the proliferative/stem cell index increases and ranges within the interval 23.2-27.8. Its mean average value in the age distribution subgroups accounts for: 2.1) reproductive age -23.7; 2.2) menopause - 24.2; 2.3) post-menopause - 25.8. In the 3rd study group labeled as "Endometrioid Carcinoma Grade 3", the proliferative/stem cell index markedly increases and ranges within the interval 25.8-29.4. Its mean average value in the age distribution subgroups accounts for: 3.1) reproductive age - 28.4; 3.2) menopause - 28.5; 3.3) post-menopause - 28.5. It was found that average value of proliferative/stem cell index in the 1st and 2nd study groups (EC Grade 1/2) keeps the same tendencies of increase in age subgroups as well as at endometrial hyperplasia conditions - in particular in both study groups increase in value of the proliferative/stem cell index in age subgroups makes about 1% (1st study group-0,97%, 2nd study group-0,96%). What about 3rd study group (EC Grade 3) average value of proliferative/stem cell index in age subgroups is almost the same. It was found that average value of proliferative/stem cell index in endometrioid carcinoma most markedly differs from the norm in post-menopause period. The study showed that average value of proliferative/stem cell index in endometrioid carcinoma cases (EC Grade 1/2) tends to increase with age like endometrial hyperplasia conditions, in contrast with the norm, where it is observed to progressively decrease with aging. The attention should be given to the fact that the mean average value of proliferative/stem cell index in endometrioid carcinoma Grade 3 is almost constant.

Ex vivo preservation and expansion of human limbal epithelial stem cells on amniotic membrane cultures.

PubMed

Meller, D; Pires, R T F; Tseng, S C G

2002-04-01

Amniotic membrane (AM) transplantation effectively expands the remaining limbal epithelial stem cells in patients with partial limbal stem cell deficiency. The authors investigated whether this action could be produced ex vivo. The outgrowth rate on AM was compared among explants derived from human limbus, peripheral cornea, and central cornea. For outgrowth of human limbal epithelial cells (HLEC), cell cycle kinetics were measured by BrdU labelling for 1 or 7 days, of which the latter was also chased in primary cultures, secondary 3T3 fibroblast cultures, and in athymic Balb/c mice following a brief treatment with a phorbol ester. Epithelial morphology was studied by histology and transmission electron microscopy, and phenotype was defined by immunostaining with monoclonal antibodies to keratins and mucins. Outgrowth rate was 0/22 (0%) and 2/24 (8.3%) for central and peripheral corneal explants, respectively, but was 77/80 (96.2%) for limbal explants (p <0.0001). 24 hour BrdU labelling showed a uniformly low (that is, less than 5%) labelling index in 65% of the limbal explants, but a mixed pattern with areas showing a high (that is, more than 40%) labelling index in 35% of limbal explants, and in all (100%) peripheral corneal explants. Continuous BrdU labelling for 7 days detected a high labelling index in 61.5% of the limbal explants with the remainder still retaining a low labelling index. A number of label retaining cells were noted after 7 day labelling followed by 14 days of chase in primary culture or by 21 days of chase after transplantation to 3T3 fibroblast feeder layers. After exposure to phorbol 12-myristate 13-acetate for 24 hours and 7 day labelling, HLEC transplanted in athymic mice still showed a number of label retaining basal cells after 9 days of chase. HLEC cultured on AM were strongly positive for K14 keratin and MUC4 and slightly positive in suprabasal cells for K3 keratin but negative for K12 keratin, AMEM2, and MUC5AC. After subcutaneous implantation in athymic mice, the resultant epithelium was markedly stratified and the basal epithelial cells were strongly positive for K14 keratin, while the suprabasal epithelial cells were strongly positive for K3 keratin and MUC4, and the entire epithelium was negative for K12 keratin and MUC5A/C. These data support the notion that AM cultures preferentially preserve and expand limbal epithelial stem cells that retain their in vivo properties of slow cycling, label retaining, and undifferentiation. This finding supports the feasibility of ex vivo expansion of limbal epithelial stem cells for treating patients with total limbal stem cell deficiency using a small amount of donor limbal tissue.

Mechanisms of cellular therapy in respiratory diseases.

PubMed

Abreu, Soraia C; Antunes, Mariana A; Pelosi, Paolo; Morales, Marcelo M; Rocco, Patricia R M

2011-09-01

Stem cells present a variety of clinical implications in the lungs. According to their origin, these cells can be divided into embryonic and adult stem cells; however, due to the important ethical and safety limitations that are involved in the embryonic stem cell use, most studies have chosen to focus on adult stem cell therapy. This article aims to present and clarify the recent advances in the field of stem cell biology, as well as to highlight the effects of mesenchymal stem cell (MSC) therapy in the context of acute lung injury/acute respiratory distress syndrome and chronic disorders such as lung fibrosis and chronic obstructive pulmonary disease. For this purpose, we performed a critical review of adult stem cell therapies, covering the main clinical and experimental studies published in Pubmed databases in the past 11 years. Different characteristics were extracted from these articles, such as: the experimental model, strain, cellular type and administration route used as well as the positive or negative effects obtained. There is evidence for beneficial effects of MSC on lung development, repair, and remodeling. The engraftment in the injured lung does not occur easily, but several studies report that paracrine factors can be effective in reducing inflammation and promoting tissue repair. MSC releases several growth factors and anti-inflammatory cytokines that regulate endothelial and epithelial permeability and reduce the severity of inflammation. A better understanding of the mechanisms that control cell division and differentiation, as well as of their paracrine effects, is required to enable the optimal use of bone marrow-derived stem cell therapy to treat human respiratory diseases.

Nano Let‑7b sensitization of eliminating esophageal cancer stem‑like cells is dependent on blockade of Wnt activation of symmetric division.

PubMed

Pang, Yamei; Liu, Jian; Li, Xiang; Zhang, Yiwen; Zhang, Boxiang; Zhang, Jing; Du, Ning; Xu, Chongwen; Liang, Rui; Ren, Hong; Tang, Shou-Ching; Sun, Xin

2017-10-01

The poor therapy response and poor prognosis of esophageal cancer has made it one of the most malignant carcinoma, and the complicated multidisciplinary treatment failed to achieve a long-term disease-free survival. To diagnose esophageal cancer at an earlier stage, and to improve the effect of anticancer therapy would improve the therapeutic efficacy. After retrospective analysis of the cancer samples of patients who received esophagectomy, we found the relevance between ratio of either ALDH1 or CD133-positive cancer stem cells and 2-year recurrence. Higher ratios of cancer stem cells indicated later clinical stages, and Wnt signaling activation was more frequent in later esophageal carcinoma. Further in bench studies, we explored the suppressive roles and the mechanisms involved in Let‑7 on self-renewal in ECA‑109 and ECA‑9706 esophageal cancer stem cells. Isolated cancer stem cells naturally divide symmetrically and are therapy resistant. Therapy of fluorouracil and docetaxel both enriched the stem cells, proving the resistant characteristics of cancer stem cells. Wnt activation stimulated more symmetric division of stem cells, resulting in self-renewal promotion, which could be blocked by Let‑7 overexpression. Furthermore, enforced Let‑7 sensitized the stem cells to chemotherapies in a Wnt pathway inhibition-dependent manner, contributing to Let‑7 sensitization of chemotherapeutic response. Wnt activation weakened the suppressive Let‑7b through the sponge functions of CCAT-1, forming the negative feedback loop of Let‑7b/Wnt/CCAT1. These results identified the crucial participation of stem cells in esophageal cancer occurrence and progression as the potent indicator, and also indicate the potential powerful agent of Let‑7 nano-particles in treatment of cancer.

Effect of essential amino acids on enteroids: Methionine deprivation suppresses proliferation and affects differentiation in enteroid stem cells

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

Saito, Yuki; Iwatsuki, Ken; Hanyu, Hikaru

We investigated the effects of essential amino acids on intestinal stem cell proliferation and differentiation using murine small intestinal organoids (enteroids) from the jejunum. By selectively removing individual essential amino acids from culture medium, we found that 24 h of methionine (Met) deprivation markedly suppressed cell proliferation in enteroids. This effect was rescued when enteroids cultured in Met deprivation media for 12 h were transferred to complete medium, suggesting that Met plays an important role in enteroid cell proliferation. In addition, mRNA levels of the stem cell marker leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) decreased in enteroids grown in Met deprivationmore » conditions. Consistent with this observation, Met deprivation also attenuated Lgr5-EGFP fluorescence intensity in enteroids. In contrast, Met deprivation enhanced mRNA levels of the enteroendocrine cell marker chromogranin A (ChgA) and markers of K cells, enterochromaffin cells, goblet cells, and Paneth cells. Immunofluorescence experiments demonstrated that Met deprivation led to an increase in the number of ChgA-positive cells. These results suggest that Met deprivation suppresses stem cell proliferation, thereby promoting differentiation. In conclusion, Met is an important nutrient in the maintenance of intestinal stem cells and Met deprivation potentially affects cell differentiation. - Highlights: • Met influences the proliferation of enteroids. • Met plays a crucial role in the maintenance of stem cells. • Met deprivation potentially promotes differentiation into secretory cells.« less

Fusion-independent expression of functional ACh receptors in mouse mesoangioblast stem cells contacting muscle cells

PubMed Central

Grassi, Francesca; Pagani, Francesca; Spinelli, Gabriele; Angelis, Luciana De; Cossu, Giulio; Eusebi, Fabrizio

2004-01-01

Mesoangioblasts are vessel-associated fetal stem cells that can be induced to differentiate into skeletal muscle, both in vitro and in vivo. Whether this is due to fusion or to transdifferentiation into bona fide satellite cells is still an open question, for mesoangioblasts as well as for other types of stem cells. The early steps of satellite cell myogenic differentiation involve MyoD activation, membrane hyperpolarization and the appearance of ACh sensitivity and gap junctional communication. If mesoangioblasts differentiate into satellite cells, these characteristics should be observed in stem cells prior to fusion into multinucleated myotubes. We have investigated the functional properties acquired by mononucleated green fluorescent protein (GFP)-positive mesoangioblasts co-cultured with differentiating C2C12 myogenic cells, using the patch-clamp technique. Mesoangioblasts whose membrane contacted myogenic cells developed a hyperpolarized membrane resting potential and ACh-evoked current responses. Dye and electrical coupling was observed among mesoangioblasts but not between mesoangioblasts and myotubes. Mouse MyoD was detected by RT-PCR both in single, mononucleated mesoangioblasts co-cultured with C2C12 myotubes and in the total mRNA from mouse mesoangioblasts co-cultured with human myotubes, but not in human myotubes or stem cells cultured in isolation. In conclusion, when co-cultured with muscle cells, mesoangioblasts acquire many of the functional characteristics of differentiating satellite cells in the absence of cell fusion, strongly indicating that these stem cells undergo transdifferentiation into satellite cells, when exposed to a myogenic environment. PMID:15319417

Sox2, Tlx, Gli3, and Her9 converge on Rx2 to define retinal stem cells in vivo

PubMed Central

Reinhardt, Robert; Centanin, Lázaro; Tavhelidse, Tinatini; Inoue, Daigo; Wittbrodt, Beate; Concordet, Jean-Paul; Martinez-Morales, Juan Ramón; Wittbrodt, Joachim

2015-01-01

Transcriptional networks defining stemness in adult neural stem cells (NSCs) are largely unknown. We used the proximal cis-regulatory element (pCRE) of the retina-specific homeobox gene 2 (rx2) to address such a network. Lineage analysis in the fish retina identified rx2 as marker for multipotent NSCs. rx2-positive cells located in the peripheral ciliary marginal zone behave as stem cells for the neuroretina, or the retinal pigmented epithelium. We identified upstream regulators of rx2 interrogating the rx2 pCRE in a trans-regulation screen and focused on four TFs (Sox2, Tlx, Gli3, and Her9) activating or repressing rx2 expression. We demonstrated direct interaction of the rx2 pCRE with the four factors in vitro and in vivo. By conditional mosaic gain- and loss-of-function analyses, we validated the activity of those factors on regulating rx2 transcription and consequently modulating neuroretinal and RPE stem cell features. This becomes obvious by the rx2-mutant phenotypes that together with the data presented above identify rx2 as a transcriptional hub balancing stemness of neuroretinal and RPE stem cells in the adult fish retina. PMID:25908840

Sox2, Tlx, Gli3, and Her9 converge on Rx2 to define retinal stem cells in vivo.

PubMed

Reinhardt, Robert; Centanin, Lázaro; Tavhelidse, Tinatini; Inoue, Daigo; Wittbrodt, Beate; Concordet, Jean-Paul; Martinez-Morales, Juan Ramón; Wittbrodt, Joachim

2015-06-03

Transcriptional networks defining stemness in adult neural stem cells (NSCs) are largely unknown. We used the proximal cis-regulatory element (pCRE) of the retina-specific homeobox gene 2 (rx2) to address such a network. Lineage analysis in the fish retina identified rx2 as marker for multipotent NSCs. rx2-positive cells located in the peripheral ciliary marginal zone behave as stem cells for the neuroretina, or the retinal pigmented epithelium. We identified upstream regulators of rx2 interrogating the rx2 pCRE in a trans-regulation screen and focused on four TFs (Sox2, Tlx, Gli3, and Her9) activating or repressing rx2 expression. We demonstrated direct interaction of the rx2 pCRE with the four factors in vitro and in vivo. By conditional mosaic gain- and loss-of-function analyses, we validated the activity of those factors on regulating rx2 transcription and consequently modulating neuroretinal and RPE stem cell features. This becomes obvious by the rx2-mutant phenotypes that together with the data presented above identify rx2 as a transcriptional hub balancing stemness of neuroretinal and RPE stem cells in the adult fish retina. © 2015 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

The Role of Stem Cells in the Treatment of Cerebral Palsy: a Review.

PubMed

Kiasatdolatabadi, Anahita; Lotfibakhshaiesh, Nasrin; Yazdankhah, Meysam; Ebrahimi-Barough, Somayeh; Jafarabadi, Mina; Ai, Arman; Sadroddiny, Esmaeil; Ai, Jafar

2017-09-01

Cerebral palsy (CP) is a neuromuscular disease due to injury in the infant's brain. The CP disorder causes many neurologic dysfunctions in the patient. Various treatment methods have been used for the management of CP disorder. However, there has been no absolute cure for this condition. Furthermore, some of the procedures which are currently used for relief of symptoms in CP cause discomfort or side effects in the patient. Recently, stem cell therapy has attracted a huge interest as a new therapeutic method for treatment of CP. Several investigations in animal and human with CP have demonstrated positive potential of stem cell transplantation for the treatment of CP disorder. The ultimate goal of this therapeutic method is to harness the regenerative capacity of the stem cells causing a formation of new tissues to replace the damaged tissue. During the recent years, there have been many investigations on stem cell therapy. However, there are still many unclear issues regarding this method and high effort is needed to create a technology as a perfect treatment. This review will discuss the scientific background of stem cell therapy for cerebral palsy including evidences from current clinical trials.

Preclinical Assessment of the Proliferation Capacity of Gingival and Periodontal Ligament Stem Cells from Diabetic Patients.

PubMed

Assem, Mostafa; Kamal, Samia; Sabry, Dina; Soliman, Nadia; Aly, Riham M

2018-02-15

Stem cells have recently received great interest as potential therapeutics alternative for a variety of diseases. The oral and maxillofacial region, in particular, encompasses a variety of distinctive mesenchymal (MSC) populations and is characterized by a potent multilineage differentiation capacity. In this report, we aimed to investigate the effect of diabetes on the proliferation potential of stem cells isolated from controlled diabetic patients (type 2) and healthy individuals. The proliferation rate of gingival and periodontal derived stem cells isolated from diabetic & healthy individuals were compared using MTT Assay. Expression levels of Survivin in isolated stem cells from all groups were measured by qRt - PCR. There was a significantly positive correlation between proliferation rate and expression of Survivin in all groups which sheds light on the importance of Survivin as a reliable indicator of proliferation. The expression of Survivin further confirmed the proliferation results from MTT Assay where the expression of stem cells from non - diabetic individuals was higher than diabetic patients. Taking together all the results, it could be concluded that PDLSC and GSC are promising candidates for autologous regenerative therapy due to their ease of accessibility in addition to their high proliferative rates.

Religion and the public ethics of stem-cell research: Attitudes in Europe, Canada and the United States.

PubMed

Allum, Nick; Allansdottir, Agnes; Gaskell, George; Hampel, Jürgen; Jackson, Jonathan; Moldovan, Andreea; Priest, Susanna; Stares, Sally; Stoneman, Paul

2017-01-01

We examine international public opinion towards stem-cell research during the period when the issue was at its most contentious. We draw upon representative sample surveys in Europe and North America, fielded in 2005 and find that the majority of people in Europe, Canada and the United States supported stem-cell research, providing it was tightly regulated, but that there were key differences between the geographical regions in the relative importance of different types of ethical position. In the U.S., moral acceptability was more influential as a driver of support for stem-cell research; in Europe the perceived benefit to society carried more weight; and in Canada the two were almost equally important. We also find that public opinion on stem-cell research was more strongly associated with religious convictions in the U.S. than in Canada and Europe, although many strongly religious citizens in all regions approved of stem-cell research. We conclude that if anything public opinion or 'public ethics' are likely to play an increasingly important role in framing policy and regulatory regimes for sensitive technologies in the future.

Liver fibrosis alleviation after co-transplantation of hematopoietic stem cells with mesenchymal stem cells in patients with thalassemia major.

PubMed

Ghavamzadeh, Ardeshir; Sotoudeh, Masoud; Hashemi Taheri, Amir Pejman; Alimoghaddam, Kamran; Pashaiefar, Hossein; Jalili, Mahdi; Shahi, Farhad; Jahani, Mohammad; Yaghmaie, Marjan

2018-02-01

The aims of this study are to determine the replacement rate of damaged hepatocytes by donor-derived cells in sex-mismatched recipient patients with thalassemia major and to determine whether co-transplantation of mesenchymal stem cells and hematopoietic stem cells (HSCs) can alleviate liver fibrosis. Ten sex-mismatched donor-recipient pairs who received co-transplantation of HSCs with mesenchymal stem cells were included in our study. Liver biopsy was performed before transplantation. Two other liver biopsies were performed between 2 and 5 years after transplantation. The specimens were studied for the presence of donor-derived epithelial cells or hepatocytes using fluorescence in situ hybridization by X- and Y-centromeric probes and immunohistochemical staining for pancytokeratin, CD45, and a hepatocyte-specific antigen. All sex-mismatched tissue samples demonstrated donor-derived hepatocyte independent of donor gender. XY-positive epithelial cells or hepatocytes accounted for 11 to 25% of the cells in histologic sections of female recipients in the first follow-up. It rose to 47-95% in the second follow-up. Although not statistically significant, four out of ten patients showed signs of improvement in liver fibrosis. Our results showed that co-transplantation of HSC with mesenchymal stem cells increases the rate of replacement of recipient hepatocytes by donor-derived cells and may improve liver fibrosis.

Highly osteogenic PDL stem cell clones specifically express elevated levels of ICAM1, ITGB1 and TERT.

PubMed

Sununliganon, Laddawun; Singhatanadgit, Weerachai

2012-01-01

Cells derived from the periodontal ligament (PDL) have previously been reported to have stem cell-like characteristics (PDL stem cells; PDLSCs) and play an important part in bone engineering, including that of alveolar bone. However, these populations have been heterogeneous, and thus far no specific marker has yet been established from adult human stem cells derived from PDL tissue. We have previously isolated highly purified single cell-derived PDLSC clones and delineated their phenotypic and functional characteristics. In this report, we further obtained three homogeneous and distinct PDLSC clones demonstrating low, moderate and high mineralized matrix forming ability-namely PC12, PC4 and PC3, respectively, and the expression of mesenchymal stem cell pathway-specific genes in these clones was investigated. PCR array revealed that the expression of intercellular adhesion molecule 1 (ICAM1), integrin beta 1 (ITGB1) and telomerase reverse transcriptase (TERT) was associated with highly osteogenic PDLSC clones, as determined by the expression of key osteoblastic markers and their ability to form alizarin red S positive mineralized matrix in vitro. The present results suggest that these three mesenchymal stem cell-associated markers could potentially be used to isolate PDLSCs with high osteogenic capability for engineering new bone.

Niclosamide inhibits epithelial-mesenchymal transition and tumor growth in lapatinib-resistant human epidermal growth factor receptor 2-positive breast cancer.

PubMed

Liu, Junjun; Chen, Xiaosong; Ward, Toby; Mao, Yan; Bockhorn, Jessica; Liu, Xiaofei; Wang, Gen; Pegram, Mark; Shen, Kunwei

2016-02-01

Acquired resistance to lapatinib, a human epidermal growth factor receptor 2 kinase inhibitor, remains a clinical problem for women with human epidermal growth factor receptor 2-positive advanced breast cancer, as metastasis is commonly observed in these patients. Niclosamide, an anti-helminthic agent, has recently been shown to exhibit cytotoxicity to tumor cells with stem-like characteristics. This study was designed to identify the mechanisms underlying lapatinib resistance and to determine whether niclosamide inhibits lapatinib resistance by reversing epithelial-mesenchymal transition. Here, two human epidermal growth factor receptor 2-positive breast cancer cell lines, SKBR3 and BT474, were exposed to increasing concentrations of lapatinib to establish lapatinib-resistant cultures. Lapatinib-resistant SKBR3 and BT474 cells exhibited up-regulation of the phenotypic epithelial-mesenchymal transition markers Snail, vimentin and α-smooth muscle actin, accompanied by activation of nuclear factor-кB and Src and a concomitant increase in stem cell marker expression (CD44(high)/CD24(low)), compared to naive lapatinib-sensitive SKBR3 and BT474 cells, respectively. Interestingly, niclosamide reversed epithelial-mesenchymal transition, induced apoptosis and inhibited cell growth by perturbing aberrant signaling pathway activation in lapatinib-resistant human epidermal growth factor receptor 2-positive cells. The ability of niclosamide to alleviate stem-like phenotype development and invasion was confirmed. Collectively, our results demonstrate that lapatinib resistance correlates with epithelial-mesenchymal transition and that niclosamide inhibits lapatinib-resistant cell viability and epithelial-mesenchymal transition. These findings suggest a role of niclosamide or derivatives optimized for more favorable bioavailability not only in reversing lapatinib resistance but also in reducing metastatic potential during the treatment of human epidermal growth factor receptor 2-positive breast cancer. Copyright © 2015 Elsevier Ltd. All rights reserved.

Self-assembled dual-modality contrast agents for non-invasive stem cell tracking via near-infrared fluorescence and magnetic resonance imaging.

PubMed

Liu, Hong; Tan, Yan; Xie, Lisi; Yang, Lei; Zhao, Jing; Bai, Jingxuan; Huang, Ping; Zhan, Wugen; Wan, Qian; Zou, Chao; Han, Yali; Wang, Zhiyong

2016-09-15

Stem cells hold great promise for treating various diseases. However, one of the main drawbacks of stem cell therapy is the lack of non-invasive image-tracking technologies. Although magnetic resonance imaging (MRI) and near-infrared fluorescence (NIRF) imaging have been employed to analyse cellular and subcellular events via the assistance of contrast agents, the sensitivity and temporal resolution of MRI and the spatial resolution of NIRF are still shortcomings. In this study, superparamagnetic iron oxide nanocrystals and IR-780 dyes were co-encapsulated in stearic acid-modified polyethylenimine to form a dual-modality contrast agent with nano-size and positive charge. These resulting agents efficiently labelled stem cells and did not influence the cellular viability and differentiation. Moreover, the labelled cells showed the advantages of dual-modality imaging in vivo. Copyright © 2016 Elsevier Inc. All rights reserved.

Extracellular Matrix from Periodontal Ligament Cells Could Induce the Differentiation of Induced Pluripotent Stem Cells to Periodontal Ligament Stem Cell-Like Cells.

PubMed

Hamano, Sayuri; Tomokiyo, Atsushi; Hasegawa, Daigaku; Yoshida, Shinichiro; Sugii, Hideki; Mitarai, Hiromi; Fujino, Shoko; Wada, Naohisa; Maeda, Hidefumi

2018-01-15

The periodontal ligament (PDL) plays an important role in anchoring teeth in the bone socket. Damage to the PDL, such as after severe inflammation, can be treated with a therapeutic strategy that uses stem cells derived from PDL tissue (PDLSCs), a strategy that has received intense scrutiny over the past decade. However, there is an insufficient number of PDLSCs within the PDL for treating such damage. Therefore, we sought to induce the differentiation of induced pluripotent stem (iPS) cells into PDLSCs as an initial step toward PDL therapy. To this end, we first induced iPS cells into neural crest (NC)-like cells. We then captured the p75 neurotrophic receptor-positive cells (iPS-NC cells) and cultured them on an extracellular matrix (ECM) produced by human PDL cells (iPS-NC-PDL cells). These iPS-NC-PDL cells showed reduced expression of embryonic stem cell and NC cell markers as compared with iPS and iPS-NC cells, and enrichment of mesenchymal stem cell markers. The cells also had a higher proliferative capacity, multipotency, and elevated expression of PDL-related markers than iPS-NC cells cultured on fibronectin and laminin (iPS-NC-FL cells) or ECM produced by human skin fibroblast cells (iPS-NC-SF cells). Overall, we present a culture method to produce high number of PDLSC-like cells from iPS cells as a first step toward a strategy for PDL regeneration.

Inhibition of EGFR Induces a c-MET Driven Stem Cell Population in Glioblastoma

PubMed Central

Jun, Hyun Jung; Bronson, Roderick T.; Charest, Al

2015-01-01

Glioblastoma multiforme (GBM) is the most lethal form of primary brain tumors, characterized by highly invasive and aggressive tumors that are resistant to all current therapeutic options. GBMs are highly heterogeneous in nature and contain a small but highly tumorigenic and self-renewing population of stem or initiating cells (Glioblastoma stem cells or GSCs). GSCs have been shown to contribute to tumor propagation and resistance to current therapeutic modalities. Recent studies of human GBMs have elucidated the genetic alterations common in these tumors, but much remains unknown about specific signaling pathways that regulate GSCs. Here we identify a distinct fraction of cells in a genetically engineered mouse model of EGFR-driven GBM that respond to anti-EGFR therapy by inducing high levels of c-MET expression. The MET positive cells displayed clonogenic potential and long-term self-renewal ability in vitro and are capable of differentiating into multiple lineages. The MET positive GBM cells are resistant to radiation and highly tumorigenic in vivo. Activation of MET signaling led to an increase in expression of the stemness transcriptional regulators Oct4, Nanog and Klf4. Pharmacological inhibition of MET activity in GSCs prevented the activation of Oct4, Nanog and Klf4 and potently abrogated stemness. Finally, the MET expressing cells were preferentially localized in perivascular regions of mouse tumors consistent with their function as GSCs. Together, our findings indicate that EGFR inhibition in GBM induces MET activation in GSCs, which is a functional requisite for GSCs activity and thus represents a promising therapeutic target. PMID:24115218

Characteristics of Notch2(+) pancreatic cancer stem-like cells and the relationship with centroacinar cells.

PubMed

Zhou, Zhu-Chao; Dong, Qiang-Gang; Fu, De-Liang; Gong, Yi-Yi; Ni, Quan-Xing

2013-08-01

Notch2, a surface marker in cell lines, is used to isolate, identify and localise pancreatic cancer stem-like cells and is a target for therapy of these cells. Sphere formation was induced in Panc-1 and Bxpc-3 pancreatic cancer cell lines, and Notch2(+) cells were separated from Bxpc-3 and Panc-1 cell lines by magnetic activated cell sorting (MACS). Expression of stem cell-related markers, OCT4, Nanog and PDX1, were measured by immunofluorescent (IF) staining. Expression of Notch2 was also determined immunohistochemically in pancreatic tissues. Notch2(+) cells were transplanted in subcutaneous of mice. AQP1 and AQP5 were also measured by IF in Bxpc-3 cells. The Notch signal pathway inhibitor, Compound E (CE), was used to treat Notch2(+) Bxpc-3 cells, and their vitalities were subsequently measured by the CCK-8 method. Positive expression of OCT4, Nanog and PDX1 was observed in Notch2(+) cells. Notch2(+) cells at centroacinar cell (CAC) and terminal ductal locations expressed AQP1 and AQP5. They were strongly tumourigenic in mice, and CE inhibited proliferation of Notch2(+) Bxpc-3 cells to some degree. OCT4 and Nanog can be used as markers of self-renewal in pancreatic cancer stem cells. Notch2(+) cells in human pancreatic cancer Bxpc-3 and Panc-1 cell lines had the properties of cancer stem cells. The results suggest that Notch2(+) pancreatic cancer stem-like cells had a close relationship with CAC. © 2013 International Federation for Cell Biology.

Isolation and gene expression analysis of single potential human spermatogonial stem cells.

PubMed

von Kopylow, K; Schulze, W; Salzbrunn, A; Spiess, A-N

2016-04-01

It is possible to isolate pure populations of single potential human spermatogonial stem cells without somatic contamination for down-stream applications, for example cell culture and gene expression analysis. We isolated pure populations of single potential human spermatogonial stem cells (hSSC) without contaminating somatic cells and analyzed gene expression of these cells via single-cell real-time RT-PCR. The isolation of a pure hSSC fraction could enable clinical applications such as fertility preservation for prepubertal boys and in vitro-spermatogenesis. By utilizing largely nonspecific markers for the isolation of spermatogonia (SPG) and hSSC, previously published cell selection methods are not able to deliver pure target cell populations without contamination by testicular somatic cells. However, uniform cell populations free of somatic cells are necessary to guarantee defined growth conditions in cell culture experiments and to prevent unintended stem cell differentiation. Fibroblast growth factor receptor 3 (FGFR3) is a cell surface protein of human undifferentiated A-type SPG and a promising candidate marker for hSSC. It is exclusively expressed in small, non-proliferating subgroups of this spermatogonial cell type together with the pluripotency-associated protein and spermatogonial nuclear marker undifferentiated embryonic cell transcription factor 1 (UTF1). We specifically selected the FGFR3-positive spermatogonial subpopulation from two 30 mg biopsies per patient from a total of 37 patients with full spermatogenesis and three patients with meiotic arrest. We then employed cell selection with magnetic beads in combination with a fluorescence-activated cell sorter antibody directed against human FGFR3 to tag and visually identify human FGFR3-positive spermatogonia. Positively selected and bead-labeled cells were subsequently picked with a micromanipulator. Analysis of the isolated cells was carried out by single-cell real-time RT-PCR, real-time RT-PCR, immunocytochemistry and live/dead staining. Single-cell real-time RT-PCR and real-time RT-PCR of pooled cells indicate that bead-labeled single cells express FGFR3 with high heterogeneity at the mRNA level, while bead-unlabeled cells lack FGFR3 mRNA. Furthermore, isolated cells exhibit strong immunocytochemical staining for the stem cell factor UTF1 and are viable. The cell population isolated in this study has to be tested for their potential stem cell characteristics via xenotransplantation. Due to the small amount of the isolated cells, propagation by cell culture will be essential. Other potential hSSC without FGFR3 surface expression will not be captured with the provided experimental design. The technical approach as developed in this work could encourage the scientific community to test other established or novel hSSC markers on single SPG that present with potential stem cell-like features. The project was funded by the DFG Research Unit FOR1041 Germ cell potential (SCH 587/3-2) and DFG grants to K.v.K. (KO 4769/2-1) and A.-N.S. (SP 721/4-1). The authors declare no competing interests. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Differentiation of Mouse Ovarian Stem Cells Toward Oocyte-Like Structure by Coculture with Granulosa Cells.

PubMed

Parvari, Soraya; Yazdekhasti, Hossein; Rajabi, Zahra; Gerayeli Malek, Valliollah; Rastegar, Tayebeh; Abbasi, Mehdi

2016-11-01

An increasing body of evidence has confirmed existence and function of ovarian stem cells (OSCs). In this study, a novel approach on differentiation of OSCs into oocyte-like cells (OLCs) has been addressed. Recently, different methods have been recruited to isolate and describe aspects of OSCs, but newer and more convenient strategies in isolation are still growing. Herein, a morphology-based method was used to isolate OSCs. Cell suspension of mouse neonatal ovaries was cultured and formed colonies were harvested mechanically and cultivated on mouse embryonic fibroblasts. For differentiation induction, colonies transferred on inactive granulosa cells. Results showed that cells in colonies were positive for alkaline phosphatase activity and reverse transcription-polymerase chain reaction (RT-PCR) confirmed the pluripotency characteristics of cells. Immunofluorescence revealed a positive signal for OCT4, DAZL, MVH, and SSEA1 in colonies as well. Results of RT-PCR and immunofluorescence confirmed that some OLCs were generated within the germ stem cell (GSCs) colonies. The applicability of morphological selection for isolation of GSCs was verified. This method is easier and more economic than other techniques. Our results demonstrate that granulosa cells were effective in inducing the differentiation of OSCs into OLCs through direct cell-to-cell contacts.

Reciprocal activation between STAT3 and miR-181b regulates the proliferation of esophageal cancer stem-like cells via the CYLD pathway.

PubMed

Xu, Dan-Dan; Zhou, Peng-Jun; Wang, Ying; Zhang, Li; Fu, Wu-Yu; Ruan, Bi-Bo; Xu, Hai-Peng; Hu, Chao-Zhi; Tian, Lu; Qin, Jin-Hong; Wang, Sheng; Wang, Xiao; Li, Yi-Cheng; Liu, Qiu-Ying; Ren, Zhe; Zhang, Rong; Wang, Yi-Fei

2016-05-17

Recent studies have suggested that cancer cells contain subpopulations that can initiate tumor growth, self-renew, and maintain tumor cell growth. However, for esophageal cancer cells, the relationship between STAT3, microRNAs and cancer stem cells remains unclear. Serum-free culture was used to enrich esophageal cancer stem-like cells (ECSLC). Flow cytometry determined the proportion of ECSLC. qPCR were performed to examine expression level of stemness factors, mesenchymal markers, ATP-binding cassette (ABC) transporters, STAT3, miR-181b, CYLD. Western blot were performed to analyze the expression of STAT3, p-STAT3 and CYLD (cylindromatosis). BALB/c mice xenograft studies were conducted to evaluate the tumorigenicity of enriched ECSLC. Sphere formation assay and colony formation assays were employed to analyze the relationship between STAT3 and miR-181b. Luciferase assays were used to evaluate activity which CYLD is a target of miR-181b. Sphere formation cells (SFCs) with properties of ECSLC were enriched. Enriched SFCs in serum-free suspension culture exhibited cancer stem-like cell properties and increased single-positive CD44 + CD24-, stemness factor, mesenchymal marker expression ABC transporters and tumorigenicity in vivo compared with the parental cells. Additionally, we found that reciprocal activation between STAT3 and miR-181b regulated SFCs proliferation. Moreover, STAT3 directly activated miR-181b transcription in SFCs and miR-181b then potentiated p-STAT3 activity. Luciferase assays indicated that CYLD was a direct and functional target of miR-181b. The mutual regulation between STAT3 and miR-181b in SFCs was required for proliferation and apoptosis resistance. STAT3 and miR-181b control each other's expression in a positive feedback loop that regulates SFCs via CYLD pathway. These findings maybe is helpful for targeting ECSLC and providing approach for esophageal cancer treatments.

Human adipose tissue-derived tenomodulin positive subpopulation of stem cells: A promising source of tendon progenitor cells.

PubMed

Gonçalves, A I; Gershovich, P M; Rodrigues, M T; Reis, R L; Gomes, M E

2018-03-01

Cell-based therapies are of particular interest for tendon and ligament regeneration given the low regenerative potential of these tissues. Adipose tissue is an abundant source of stem cells, which may be employed for the healing of tendon lesions. However, human adult multipotent adipose-derived stem cells (hASCs) isolated from the stromal vascular fraction of adipose tissue originate highly heterogeneous cell populations that hinder their use in specific tissue-oriented applications. In this study, distinct subpopulations of hASCs were immunomagnetic separated and their tenogenic differentiation capacity evaluated in the presence of several growth factors (GFs), namely endothelial GF, basic-fibroblast GF, transforming GF-β1 and platelet-derived GF-BB, which are well-known regulators of tendon development, growth and healing. Among the screened hASCs subpopulations, tenomodulin-positive cells were shown to be more promising for tenogenic applications and therefore this subpopulation was further studied, assessing tendon-related markers (scleraxis, tenomodulin, tenascin C and decorin) both at gene and protein level. Additionally, the ability for depositing collagen type I and III forming extracellular matrix structures were weekly assessed up to 28 days. The results obtained indicated that tenomodulin-positive cells exhibit phenotypical features of tendon progenitor cells and can be biochemically induced towards tenogenic lineage, demonstrating that this subset of hASCs can provide a reliable source of progenitor cells for therapies targeting tendon regeneration. Copyright © 2017 John Wiley & Sons, Ltd.

Characterization of stem cells in Dupuytren's disease.

PubMed

Hindocha, S; Iqbal, S A; Farhatullah, S; Paus, R; Bayat, A

2011-02-01

Dupuytren's disease (DD) is a common fibroproliferative disease of unknown origin. The source of abnormal cells leading to DD formation remains underexplored. In addition to fascia, palmar skin and fat-derived cells may be a potential source of cells causing DD. This study aimed to profile haematopoietic and mesenchymal stem cells in different DD tissue components compared with tissue removed at carpal tunnel surgery (control). Biopsies were taken from the diseased cord, nodule, perinodular fat and skin overlying the nodule of ten patients with DD and compared with control tissue from seven patients having surgery for carpal tunnel syndrome. Fluorescence-activated cell sorting (FACS), immunohistochemistry and quantitative real-time polymerase chain reaction (QRT-PCR) were used to identify expression of selected stem cell markers. FACS and QRT-PCR analysis identified the highest RNA expression and number of cells positive for adipocyte stem cell markers (CD13 and CD29) in the DD nodule in comparison with carpal tunnel control tissue (P = 0·053). CD34 RNA was overexpressed, and a higher percentage of these cells was present in DD skin compared with carpal tunnel skin (P = 0·001). Each structural component of DD (cord, nodule, perinodular fat and skin) had distinct stem cell populations. These findings support the hypothesis that DD may result from mesenchymal progenitor cell expansion.

Targeting PTPRK-RSPO3 colon tumours promotes differentiation and loss of stem-cell function.

PubMed

Storm, Elaine E; Durinck, Steffen; de Sousa e Melo, Felipe; Tremayne, Jarrod; Kljavin, Noelyn; Tan, Christine; Ye, Xiaofen; Chiu, Cecilia; Pham, Thinh; Hongo, Jo-Anne; Bainbridge, Travis; Firestein, Ron; Blackwood, Elizabeth; Metcalfe, Ciara; Stawiski, Eric W; Yauch, Robert L; Wu, Yan; de Sauvage, Frederic J

2016-01-07

Colorectal cancer remains a major unmet medical need, prompting large-scale genomics efforts in the field to identify molecular drivers for which targeted therapies might be developed. We previously reported the identification of recurrent translocations in R-spondin genes present in a subset of colorectal tumours. Here we show that targeting RSPO3 in PTPRK-RSPO3-fusion-positive human tumour xenografts inhibits tumour growth and promotes differentiation. Notably, genes expressed in the stem-cell compartment of the intestine were among those most sensitive to anti-RSPO3 treatment. This observation, combined with functional assays, suggests that a stem-cell compartment drives PTPRK-RSPO3 colorectal tumour growth and indicates that the therapeutic targeting of stem-cell properties within tumours may be a clinically relevant approach for the treatment of colorectal tumours.

Transplanted hematopoietic stem cells demonstrate impaired sarcoglycan expression after engraftment into cardiac and skeletal muscle.

PubMed

Lapidos, Karen A; Chen, Yiyin E; Earley, Judy U; Heydemann, Ahlke; Huber, Jill M; Chien, Marcia; Ma, Averil; McNally, Elizabeth M

2004-12-01

Pluripotent bone marrow-derived side population (BM-SP) stem cells have been shown to repopulate the hematopoietic system and to contribute to skeletal and cardiac muscle regeneration after transplantation. We tested BM-SP cells for their ability to regenerate heart and skeletal muscle using a model of cardiomyopathy and muscular dystrophy that lacks delta-sarcoglycan. The absence of delta-sarcoglycan produces microinfarcts in heart and skeletal muscle that should recruit regenerative stem cells. Additionally, sarcoglycan expression after transplantation should mark successful stem cell maturation into cardiac and skeletal muscle lineages. BM-SP cells from normal male mice were transplanted into female delta-sarcoglycan-null mice. We detected engraftment of donor-derived stem cells into skeletal muscle, with the majority of donor-derived cells incorporated within myofibers. In the heart, donor-derived nuclei were detected inside cardiomyocytes. Skeletal muscle myofibers containing donor-derived nuclei generally failed to express sarcoglycan, with only 2 sarcoglycan-positive fibers detected in the quadriceps muscle from all 14 mice analyzed. Moreover, all cardiomyocytes with donor-derived nuclei were sarcoglycan-negative. The absence of sarcoglycan expression in cardiomyocytes and skeletal myofibers after transplantation indicates impaired differentiation and/or maturation of bone marrow-derived stem cells. The inability of BM-SP cells to express this protein severely limits their utility for cardiac and skeletal muscle regeneration.

Stromal cell-derived factor-1 significantly induces proliferation, migration, and collagen type I expression in a human periodontal ligament stem cell subpopulation.

PubMed

Du, Lingqian; Yang, Pishan; Ge, Shaohua

2012-03-01

The pivotal role of chemokine stromal cell-derived factor-1 (SDF-1) in bone marrow mesenchymal stem cells recruitment and tissue regeneration has already been reported. However, its roles in human periodontal ligament stem cells (PDLSCs) remain unknown. PDLSCs are regarded as candidates for periodontal tissue regeneration and are used in stem cell-based periodontal tissue engineering. The expression of chemokine receptors on PDLSCs and the migration of these cells induced by chemokines and their subsequent function in tissue repair may be a crucial procedure for periodontal tissue regeneration. PDL tissues were obtained from clinically healthy premolars extracted for orthodontic reasons and used to isolate single-cell colonies by the limited-dilution method. Immunocytochemical staining was used to detect the expression of the mesenchymal stem cell marker STRO-1. Differentiation potentials were assessed by alizarin-red staining and oil-red O staining. The expression of SDF-1 receptor CXCR4 was evaluated by real-time polymerase chain reaction (PCR) and immunocytochemical staining. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and bromodeoxyuridine incorporation assay were used to determine the viability and proliferation of the PDLSC subpopulation. Expression of collagen type I and alkaline phosphatase was detected by real-time PCR to determine the effect of SDF-1 on cells differentiation. Twenty percent of PDL single-cell colonies expressed STRO-1 positively, and this specific subpopulation was positive for CXCR4 and formed minerals and lipid vacuoles after 4 weeks induction. SDF-1 significantly increased proliferation and stimulated the migration of this PDLSC subpopulation at concentrations between 100 and 400 ng/mL. CXCR4 neutralizing antibody could block cell proliferation and migration, suggesting that SDF-1 exerted its effects on cells through CXCR4. SDF-1 promoted collagen type I level significantly but had little effect on alkaline phosphatase level. SDF-1 may have the potential of promoting periodontal tissue regeneration by the mechanism of guiding PDLSCs to destructive periodontal tissue, promoting their activation and proliferation and influencing the differentiation of these stem cells.

Stem cell collection in unmanipulated HLA-haploidentical/mismatched related transplantation with combined granulocyte-colony stimulating factor-mobilised blood and bone marrow for patients with haematologic malignancies: the impact of donor characteristics and procedural settings.

PubMed

Zhang, C; Chen, X-H; Zhang, X; Gao, L; Gao, L; Kong, P-Y; Peng, X-G; Sun, A-H; Gong, Y; Zeng, D-F; Wang, Q-Y

2010-06-01

Unmanipulated haploidentical/mismatched related transplantation with combined granulocyte-colony stimulating factor-mobilised peripheral blood stem cells (G-PBSCs) and granulocyte-colony stimulating factor-mobilised bone marrow (G-BM) has been developed as an alternative transplantation strategy for patients with haematologic malignancies. However, little information is available about the factors predicting the outcome of peripheral blood stem cell (PBSC) collection and bone marrow (BM) harvest in this transplantation. The effects of donor characteristics and procedure factors on CD34(+) cell yield were investigated. A total of 104 related healthy donors received granulocyte-colony stimulating factor (G-CSF) followed by PBSC collection and BM harvest. Male donors had significantly higher yields compared with female donors. In multiple regression analysis for peripheral blood collection, age and flow rate were negatively correlated with cell yield, whereas body mass index, pre-aphaeresis white blood cell (WBC) and circulating immature cell (CIC) counts were positively correlated with cell yields. For BM harvest, age was negatively correlated with cell yields, whereas pre-BM collection CIC counts were positively correlated with cell yield. All donors achieved the final product of >or=6 x10(6) kg(-1) recipient body weight. This transplantation strategy has been shown to be a feasible approach with acceptable outcomes in stem cell collection for patients who received HLA-haploidentical/mismatched transplantation with combined G-PBSCs and G-BM. In donors with multiple high-risk characteristics for poor aphaeresis CD34(+) cell yield, BM was an alternative source.

c-Kit-positive cardiac stem cells nested in hypoxic niches are activated by stem cell factor reversing the aging myopathy.

PubMed

Sanada, Fumihiro; Kim, Junghyun; Czarna, Anna; Chan, Noel Yan-Ki; Signore, Sergio; Ogórek, Barbara; Isobe, Kazuya; Wybieralska, Ewa; Borghetti, Giulia; Pesapane, Ada; Sorrentino, Andrea; Mangano, Emily; Cappetta, Donato; Mangiaracina, Chiara; Ricciardi, Mario; Cimini, Maria; Ifedigbo, Emeka; Perrella, Mark A; Goichberg, Polina; Choi, Augustine M; Kajstura, Jan; Hosoda, Toru; Rota, Marcello; Anversa, Piero; Leri, Annarosa

2014-01-03

Hypoxia favors stem cell quiescence, whereas normoxia is required for stem cell activation, but whether cardiac stem cell (CSC) function is regulated by the hypoxic/normoxic state of the cell is currently unknown. A balance between hypoxic and normoxic CSCs may be present in the young heart, although this homeostatic control may be disrupted with aging. Defects in tissue oxygenation occur in the old myocardium, and this phenomenon may expand the pool of hypoxic CSCs, which are no longer involved in myocyte renewal. Here, we show that the senescent heart is characterized by an increased number of quiescent CSCs with intact telomeres that cannot re-enter the cell cycle and form a differentiated progeny. Conversely, myocyte replacement is controlled only by frequently dividing CSCs with shortened telomeres; these CSCs generate a myocyte population that is chronologically young but phenotypically old. Telomere dysfunction dictates their actual age and mechanical behavior. However, the residual subset of quiescent young CSCs can be stimulated in situ by stem cell factor reversing the aging myopathy. Our findings support the notion that strategies targeting CSC activation and growth interfere with the manifestations of myocardial aging in an animal model. Although caution has to be exercised in the translation of animal studies to human beings, our data strongly suggest that a pool of functionally competent CSCs persists in the senescent heart and that this stem cell compartment can promote myocyte regeneration effectively, partly correcting the aging myopathy.

Isolation and characterization of the trophectoderm from the Arabian camel (Camelus dromedarius).

PubMed

Saadeldin, Islam M; Swelum, Ayman Abdel-Aziz; Elsafadi, Mona; Moumen, Abdullah F; Alzahrani, Faisal A; Mahmood, Amer; Alfayez, Musaad; Alowaimer, Abdullah N

2017-09-01

We isolated and characterized trophoblast from in vivo-derived camel embryos and compared with embryonic stem-like cells. Camel embryos were flushed on day 8 post-insemination and used to derive trophectoderm and embryonic stem-like cells under feeder-free culture conditions using a basement membrane matrix. Embryos were evaluated for the expression of POU5F1, MYC, KLF4, SOX2, CDX2, and KRT8 mRNA transcripts by relative quantitative polymerase chain reaction. Camel embryos grew and expanded to ∼4.5 mm and maintained their vesicular shape in vitro for 21 days post-insemination. Trophoblast and embryonic stem-like cell lines grew under feeder-free culture conditions and showed distinct morphological criteria and normal chromosomal counts. Embryonic stem-like cells showed positive staining in the alkaline phosphatase reaction. Trophoblast cells showed a significant increase in CDX2, KRT8, KLF4, and SOX2 expression compared with embryonic stem-like cells and whole embryos. Embryonic stem-like cells showed a significant decrease in CDX2 expression and increase in SOX2 and KRT8 expression compared to embryonic expression. POU5F1 and MYC expression showed no difference between embryos and both cell lines. We characterized embryo survival in vitro, particularly the derivation of trophectoderm and embryonic stem-like cells, providing a foundation for further analysis of early embryonic development and placentation in camels. Copyright © 2017 Elsevier Ltd. All rights reserved.

Can civil lawsuits stem the tide of direct-to-consumer marketing of unproven stem cell interventions.

PubMed

Horner, Claire; Tenenbaum, Evelyn; Sipp, Douglas; Master, Zubin

2018-01-01

The sale of unproven stem cell interventions (SCIs) by commercial entities has proliferated in highly developed countries, most notably in the USA. Yet, there have been few criminal prosecutions and regulatory enforcement actions against providers who have violated laws and best practice standards due to the lack of resources and legal ambiguity. While the stem cell research community has invested much in protecting patients and preventing the growth of this industry, some patients are seeking remedies under civil law to hold stem cell clinics responsible for fraudulent practices. Several patients have filed lawsuits against providers demanding compensation for physical injuries caused by unproven treatments and financial losses due to fraud and false advertising. Lawsuits can be used as a tool not only to compensate plaintiffs but also to achieve positive public health and policy outcomes. In this paper, we explore the value of a public health litigation strategy as a countermeasure against the exploitative practices of the unproven SCI industry by analyzing stem cell lawsuits and comparing them with other major public health litigation efforts. We argue that stem cell lawsuits complement other approaches to reining in unsafe practices. In particular, stem cell lawsuits could intensify publicity and raise awareness of the harms of unproven treatments, set legal precedent, reshape the media narrative from one focused on the right to try or practice to one highlighting the need for adequate safety and efficacy standards, and encourage authorities to turn their attention to policy reform and enforcement.

Asialoglycoprotein receptor 1 is a specific cell-surface marker for isolating hepatocytes derived from human pluripotent stem cells

PubMed Central

Peters, Derek T.; Henderson, Christopher A.; Warren, Curtis R.; Friesen, Max; Xia, Fang; Becker, Caroline E.; Musunuru, Kiran; Cowan, Chad A.

2016-01-01

ABSTRACT Hepatocyte-like cells (HLCs) are derived from human pluripotent stem cells (hPSCs) in vitro, but differentiation protocols commonly give rise to a heterogeneous mixture of cells. This variability confounds the evaluation of in vitro functional assays performed using HLCs. Increased differentiation efficiency and more accurate approximation of the in vivo hepatocyte gene expression profile would improve the utility of hPSCs. Towards this goal, we demonstrate the purification of a subpopulation of functional HLCs using the hepatocyte surface marker asialoglycoprotein receptor 1 (ASGR1). We analyzed the expression profile of ASGR1-positive cells by microarray, and tested their ability to perform mature hepatocyte functions (albumin and urea secretion, cytochrome activity). By these measures, ASGR1-positive HLCs are enriched for the gene expression profile and functional characteristics of primary hepatocytes compared with unsorted HLCs. We have demonstrated that ASGR1-positive sorting isolates a functional subpopulation of HLCs from among the heterogeneous cellular population produced by directed differentiation. PMID:27143754

Wound Healing and Angiogenesis through Combined Use of a Vascularized Tissue Flap and Adipose-Derived Stem Cells in a Rat Hindlimb Irradiated Ischemia Model.

PubMed

Yoshida, Shuhei; Yoshimoto, Hiroshi; Hirano, Akiyoshi; Akita, Sadanori

2016-05-01

Treatment of critical limb ischemia is sometimes difficult because of the patient's condition, and some novel approaches are needed. The hindlimbs of Sprague-Dawley rats, after 20-Gy x-ray irradiation and surgical occlusion, were divided into four groups: with a superficial fascial flap, 5.0 × 10 adipose-derived stromal/stem cells, and both combined. The rats were tested for laser tissue blood flow, immunohistologic blood vessel density, and foot paw punch hole wound healing. Green fluorescent protein-tagged Sprague-Dawley rats were used for further investigation by cell tracking for 2 weeks. Laser tissue blood flow demonstrated a significant increase in the combined treatment of flap and adipose-derived stem cells at both 1 and 2 weeks. There were no significant differences between the treatment groups treated with flaps alone and those treated with adipose-derived stem cells alone. Wound healing was significantly increased following combined treatment at 1 week, and there was no wound by 2 weeks except for the no-flap and no-adipose-derived stem cell group. The number of vessels depicted by von Willebrand factor showed a significant increase in the combined treatment group, at both 1 week and 2 weeks. In the cell tracking group, at 2 weeks, the green fluorescent protein-tagged adipose-derived stem cells were significantly more positive in the no-flap group than in the flap group. Adipose-derived stem cells may be a potent cell source in irradiated and occluded limbs by enhancing tissue blood flow and blood vessel density. Adipose-derived stem cells may play an important role in some difficult ischemic conditions in terms of wound healing.

Hemorrhagic cystitis after allogeneic hematopoietic stem cell transplants is the complex result of BK virus infection, preparative regimen intensity and donor type

PubMed Central

de Padua Silva, Leandro; Patah, Poliana A.; Saliba, Rima M.; Szewczyk, Nicholas A.; Gilman, Lisa; Neumann, Joyce; Han, Xiang-Yang; Tarrand, Jeffrey; Ribeiro, Rachel; Gulbis, Alison; Shpall, Elizabeth J.; Jones, Roy; Popat, Uday; Walker, Julia A.; Petropoulos, Demetrios; Chiattone, Alexandre; Stewart, John; El-Zimaity, Maha; Anderlini, Paolo; Giralt, Sergio; Champlin, Richard E; de Lima, Marcos

2010-01-01

Background Hemorrhagic cystitis is a common cause of morbidity after allogeneic stem cell transplantation, frequently associated with BK virus infection. We hypothesized that patients with positive BK viruria before unrelated or mismatched related donor allogeneic hematopoietic stem cell transplantation have a higher incidence of hemorrhagic cystitis. Design and Methods To test this hypothesis, we prospectively studied 209 patients (median age 49 years, range 19–71) with hematologic malignancies who received bone marrow (n=78), peripheral blood (n=108) or umbilical cord blood (n=23) allogeneic hematopoietic stem cell transplantation after myeloablative (n=110) or reduced intensity conditioning (n=99). Donors were unrelated (n=201) or haploidentical related (n=8). Results Twenty-five patients developed hemorrhagic cystitis. Pre-transplant BK viruria detected by quantitative PCR was positive in 96 patients. The one-year cumulative incidence of hemorrhagic cystitis was 16% in the PCR-positive group versus 9% in the PCR-negative group (P=0.1). The use of umbilical cord blood or a haploidentical donor was the only significant predictor of the incidence of hemorrhagic cystitis on univariate analysis. There was also a trend for a higher incidence after myeloablative conditioning. Multivariate analysis showed that patients who had a positive PCR pre-transplant and received haploidentical or cord blood grafts with myeloablative conditioning had a significantly higher risk of developing hemorrhagic cystitis (58%) than all other recipients (7%, P<0.001). Conclusions Hemorrhagic cystitis is the result of a complex interaction of donor type, preparative regimen intensity, and BK viruria. PMID:20410183

SOX2 regulates self-renewal and tumorigenicity of stem-like cells of head and neck squamous cell carcinoma.

PubMed

Lee, S H; Oh, S-Y; Do, S I; Lee, H J; Kang, H J; Rho, Y S; Bae, W J; Lim, Y C

2014-11-25

Head and neck squamous cell carcinomas (HNSCCs) display cellular heterogeneity and contain cancer stem cells (CSCs). Sex-determining region Y [SRY]-box (SOX)2 is an important regulator of embryonic stem cell fate and is aberrantly expressed in several types of human tumours. Nonetheless, the role of SOX2 in HNSCC remains unclear. We created cells ectopically expressing SOX2 from previously established HNSCC cells and examined the cell proliferation, self-renewal capacity, and chemoresistance of these cells compared with control cells. In addition, we knocked down SOX2 in primary spheres obtained from HNSCC tumour tissue and assessed the attenuation of stemness-associated traits in these cells in vitro and in vivo. Furthermore, we examined the clinical relevance of SOX2 expression in HNSCC patients. SOX2 is aberrantly expressed in primary tissue of HNSCC patients but not in healthy tissue. SOX2 expression correlated with tumour recurrence and poor prognosis of HNSCC patients. Ectopic expression of SOX2 induced cell proliferation via cyclin B1 expression and stemness-associated features, such as self-renewal and chemoresistance. In addition, a knockdown of SOX2 in HNSCC CSCs attenuated their self-renewal capacity, chemoresistance (through ABCG2 suppression), invasion capacity (via snail downregulation), and in vivo tumorigenicity. These results suggest that SOX2 may have important roles in the 'stemness' and progression of HNSCC. Targeting SOX2-positive tumour cells (CSCs) could be a new therapeutic strategy in HNSCCs.

Effect of Photobiomodulation on Mesenchymal Stem Cells.

PubMed

Fekrazad, Reza; Asefi, Sohrab; Allahdadi, Mahdi; Kalhori, Katayoun A M

2016-11-01

The purpose of this study was to review available literature about the effect of photobiomodulation (PBM) on mesenchymal stem cells (MSCs). The effects of coherent and noncoherent light sources such as low-level lasers and light-emitting diodes (LEDs) on cells and tissues, known as PBM, form the basis of photomedicine. This treatment technique effects cell function, proliferation, and migration, and plays an important role in tissue regeneration. Stem cells have been found to be helpful elements in tissue regeneration, and the combination of stem cell therapy and laser therapy appears to positively affect treatment results. An electronic search in PubMed was conducted of publications from the previous 12 years. English language articles related to the subject were found using selected key words. The full texts of potentially suitable articles were assessed according to inclusion and exclusion criteria. After evaluation, 30 articles were deemed relevant according to the inclusion criteria. The energy density of the laser was 0.7-9 J/cm 2 . The power used for visible light was 30-110 mW and that used for infrared light was 50-800 mW. Nearly all studies showed that low-level laser therapy had a positive effect on cell proliferation. Similar outcomes were found for LED; however, some studies suggest that the laser alone is not effective, and should be used as an adjunct tool. PBM has positive effects on MSCs. This review concluded that doses of 0.7-4 J/cm 2 and wavelengths of 600-700 nm are appropriate for light therapy. The results were dependent upon different parameters; therefore, optimization of parameters used in light therapy to obtain favorable results is required to provide more accurate comparison.

Isolation, Characterization, and Differentiation of Dental Pulp Stem Cells in Ferrets.

PubMed

Homayounfar, Negar; Verma, Prashant; Nosrat, Ali; El Ayachi, Ikbale; Yu, Zongdong; Romberg, Elaine; Huang, George T-J; Fouad, Ashraf F

2016-03-01

The ferret canine tooth has been introduced as a suitable model for studying dental pulp regeneration. The aim of this study was to isolate and characterize ferret dental pulp stem cells (fDPSCs) and their differentiation potential. Dental pulp stem cells were isolated from freshly extracted ferret canine teeth. The cells were examined for the expression of stem cell markers STRO-1, CD90, CD105, and CD146. The osteo/odontogenic and adipogenic differentiation potential of fDPSCs was evaluated. Osteogenic and odontogenic marker genes were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR) on days 1, 4, and 8 after osteo/odontogenic induction of fDPSCs including dentin sialophosphoprotein (DSPP), dentin matrix protein-1, osteopontin, and alkaline phosphatase. Human dental pulp cells were used as the control. The results were analyzed using 3-way analysis of variance. fDPSCs were positive for STRO1, CD90, and CD105 and negative for CD146 markers with immunohistochemistry. fDPSCs showed strong osteogenic and weak adipogenic potential. The overall expression of DSPP was not significantly different between fDPSCs and human dental pulp cells. The expression of DSPP in osteo/odontogenic media was significantly higher in fDPSCs on day 4 (P < .01). The overall expression of dentin matrix protein-1, osteopontin, and alkaline phosphatase was significantly higher in fDPSCs (P = .0005). fDPSCs were positive for several markers of dental pulp stem cells resembling human DPSCs and appeared to show a stronger potential to differentiate to osteoblastic rather than odontoblastic lineage. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Lgr5-EGFP marks taste bud stem/progenitor cells in posterior tongue

PubMed Central

Yee, Karen K.; Li, Yan; Redding, Kevin M.; Iwatsuki, Ken; Margolskee, Robert F.; Jiang, Peihua

2013-01-01

Until recently, reliable markers for adult stem cells have been lacking for many regenerative mammalian tissues. Lgr5 (leucine-rich repeat-containing G-protein coupled receptor 5) has been identified as a marker for adult stem cells in intestine, stomach, and hair follicle; Lgr5-expressing cells give rise to all types of cells in these tissues. Taste epithelium also regenerates constantly, yet the identity of adult taste stem cells remains elusive. In this study, we found that Lgr5 is strongly expressed in cells at the bottom of trench areas at the base of circumvallate and foliate taste papillae and weakly expressed in the basal area of taste buds and that Lgr5-expressing cells in posterior tongue are a subset of K14-positive epithelial cells. Lineage-tracing experiments using an inducible Cre knock-in allele in combination with Rosa26-LacZ and Rosa26-tdTomato reporter strains showed that Lgr5-expressing cells gave rise to taste cells, perigemmal cells, along with self-renewing cells at the bottom of trench areas at the base of circumvallate and foliate papillae. Moreover, using subtype-specific taste markers, we found that Lgr5-expressing cell progeny include all three major types of adult taste cells. Our results indicate that Lgr5 may mark adult taste stem or progenitor cells in the posterior portion of the tongue. PMID:23377989

Lgr5-EGFP marks taste bud stem/progenitor cells in posterior tongue.

PubMed

Yee, Karen K; Li, Yan; Redding, Kevin M; Iwatsuki, Ken; Margolskee, Robert F; Jiang, Peihua

2013-05-01

Until recently, reliable markers for adult stem cells have been lacking for many regenerative mammalian tissues. Lgr5 (leucine-rich repeat-containing G-protein-coupled receptor 5) has been identified as a marker for adult stem cells in intestine, stomach, and hair follicle; Lgr5-expressing cells give rise to all types of cells in these tissues. Taste epithelium also regenerates constantly, yet the identity of adult taste stem cells remains elusive. In this study, we found that Lgr5 is strongly expressed in cells at the bottom of trench areas at the base of circumvallate (CV) and foliate taste papillae and weakly expressed in the basal area of taste buds and that Lgr5-expressing cells in posterior tongue are a subset of K14-positive epithelial cells. Lineage-tracing experiments using an inducible Cre knockin allele in combination with Rosa26-LacZ and Rosa26-tdTomato reporter strains showed that Lgr5-expressing cells gave rise to taste cells, perigemmal cells, along with self-renewing cells at the bottom of trench areas at the base of CV and foliate papillae. Moreover, using subtype-specific taste markers, we found that Lgr5-expressing cell progeny include all three major types of adult taste cells. Our results indicate that Lgr5 may mark adult taste stem or progenitor cells in the posterior portion of the tongue. Copyright © 2013 AlphaMed Press.

Anti-CD20 Radioimmunotherapy Before Chemotherapy and Stem Cell Transplant in Treating Patients With High-Risk B-Cell Malignancies

ClinicalTrials.gov

2018-03-13

Burkitt Lymphoma; CD20-Positive Neoplastic Cells Present; Diffuse Large B-Cell Lymphoma; Indolent Non-Hodgkin Lymphoma; Mantle Cell Lymphoma; Recurrent B-Cell Non-Hodgkin Lymphoma; Refractory Mature B-Cell Non-Hodgkin Lymphoma

Gravitropism in Higher Plant Shoots 1

PubMed Central

Sliwinski, Julianne E.; Salisbury, Frank B.

1984-01-01

Cross and longitudinal sections were prepared for light microscopy from vertical control plants (Xanthium strumarium L. Chicago strain), free-bending horizontal stems, plants restrained 48 hours in a horizontal position, and plants restrained 48 hours and then released, bending immediately about 130°. Top cells of free-bending stems shrink or elongate little; bottom cells continue to elongate. In restrained stems, bottom cells elongate some and increase in diameter; top cells elongate about as much but decrease in diameter. Upon release, bottom cells elongate more and decrease in diameter, while top cells shorten and increase in diameter, accounting for the bend. During restraint, bottom cells take up water while tissue pressures increase; top cells fail to take up water although tissue pressures are decreasing. Settling of amyloplasts was observed in cells of the starch sheath. Removal of different amounts of stem (Xanthium; Lycopersicon esculentum Miller, cv Bonny Best; Ricinus communis L. cv Yolo Wonder) showed that perception of gravity occurs in the bending (elongation) zone, although bending of fourth and fifth internodes from the top was less than in uncut controls. Uniform application of 1% indoleacetic acid in lanolin to cut stem surfaces partially restored bending. Reversing the gradient in tension/compression in horizontal stems (top under compression, bottom under tension) did not affect gravitropic bending. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 Fig. 10 PMID:16663939

Medical societies, patient education initiatives, public debate and marketing of unproven stem cell interventions.

PubMed

Weiss, Daniel J; Turner, Leigh; Levine, Aaron D; Ikonomou, Laertis

2018-02-01

Businesses marketing unproven stem cell interventions proliferate within the U.S. and in the larger global marketplace. There have been global efforts by scientists, patient advocacy groups, bioethicists, and public policy experts to counteract the uncontrolled and premature commercialization of stem cell interventions. In this commentary, we posit that medical societies and associations of health care professionals have a particular responsibility to be an active partner in such efforts. We review the role medical societies can and should play in this area through patient advocacy and awareness initiatives. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Dynamics of gene expression with positive feedback to histone modifications at bivalent domains

NASA Astrophysics Data System (ADS)

Huang, Rongsheng; Lei, Jinzhi

2018-03-01

Experiments have shown that in embryonic stem cells, the promoters of many lineage-control genes contain “bivalent domains”, within which the nucleosomes possess both active (H3K4me3) and repressive (H3K27me3) marks. Such bivalent modifications play important roles in maintaining pluripotency in embryonic stem cells. Here, to investigate gene expression dynamics when there are regulations in bivalent histone modifications and random partition in cell divisions, we study how positive feedback to histone methylation/demethylation controls the transition dynamics of the histone modification patterns along with cell cycles. We constructed a computational model that includes dynamics of histone marks, three-stage chromatin state transitions, transcription and translation, feedbacks from protein product to enzymes to regulate the addition and removal of histone marks, and the inheritance of nucleosome state between cell cycles. The model reveals how dynamics of both nucleosome state transition and gene expression are dependent on the enzyme activities and feedback regulations. Results show that the combination of stochastic histone modification at each cell division and the deterministic feedback regulation work together to adjust the dynamics of chromatin state transition in stem cell regenerations.

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

Fanconi anemia genes are highly expressed in primitive CD34+ hematopoietic cells

PubMed Central

Aubé, Michel; Lafrance, Matthieu; Brodeur, Isabelle; Delisle, Marie-Chantal; Carreau, Madeleine

2003-01-01

Background Fanconi anemia (FA) is a complex recessive genetic disease characterized by progressive bone marrow failure (BM) and a predisposition to cancer. We have previously shown using the Fancc mouse model that the progressive BM failure results from a hematopoietic stem cell defect suggesting that function of the FA genes may reside in primitive hematopoietic stem cells. Methods Since genes involved in stem cell differentiation and/or maintenance are usually regulated at the transcription level, we used a semiquantitative RT-PCR method to evaluate FA gene transcript levels in purified hematopoietic stem cells. Results We show that most FA genes are highly expressed in primitive CD34-positive and negative cells compared to lower levels in more differentiated cells. However, in CD34- stem cells the Fancc gene was found to be expressed at low levels while Fancg was undetectable in this population. Furthermore, Fancg expression is significantly decreased in Fancc -/- stem cells as compared to wild-type cells while the cancer susceptibility genes Brca1 and Fancd1/Brac2 are upregulated in Fancc-/- hematopoietic cells. Conclusions These results suggest that FA genes are regulated at the mRNA level, that increased Fancc expression in LTS-CD34+ cells correlates with a role at the CD34+ differentiation stage and that lack of Fancc affects the expression of other FA gene, more specifically Fancg and Fancd1/Brca2, through an unknown mechanism. PMID:12809565

Aberrant epithelial GREM1 expression initiates colonic tumorigenesis from cells outside of the crypt base stem cell niche

PubMed Central

Bansal, Mukesh; Rafferty, Hannah; Boitsova, Tatjana; Bardella, Chiara; Jaeger, Emma; Lewis, Annabelle; Freeman-Mills, Luke; Giner, Francesc Castro; Rodenas-Cuadrado, Pedro; Mallappa, Sreelakshmi; Clark, Susan; Thomas, Huw; Jeffery, Rosemary; Poulsom, Richard; Rodriguez-Justo, Manuel; Novelli, Marco; Chetty, Runjan; Silver, Andrew; Sansom, Owen James; Greten, Florian R; Wang, Lai Mun; East, James Edward; Tomlinson, Ian; Leedham, Simon John

2015-01-01

Hereditary mixed polyposis syndrome (HMPS) is characterised by the development of mixed morphology colorectal tumours and is caused by a 40 kb duplication that results in aberrant epithelial expression of the mesenchymal Bone Morphogenetic Protein antagonist, GREM1. Here we use HMPS tissue and a mouse model of the disease to show that epithelial GREM1 disrupts homeostatic intestinal morphogen gradients, altering cell-fate, that is normally determined by position along the vertical epithelial axis. This promotes the persistence and/or reacquisition of stem-cell properties in Lgr5 negative (non-expressing) progenitor cells that have exited the stem-cell niche. These cells form ectopic crypts, proliferate, accumulate somatic mutations and can initiate intestinal neoplasia, indicating that the crypt base stem-cell is not the sole cell-of-origin of colorectal cancer. Furthermore, we show that epithelial expression of GREM1 also occurs in traditional serrated adenomas, sporadic pre-malignant lesions with a hitherto unknown pathogenesis and these lesions can be considered the sporadic equivalents of HMPS polyps. PMID:25419707

Slow-Adhering Stem Cells Derived from Injured Skeletal Muscle Have Improved Regenerative Capacity

DTIC Science & Technology

2011-08-01

images. Flow Cytometry Assay of Stem Cell Markers SASCs (1 105) isolated from noninjured or injured muscle were collected and washed twice with...muscle. Results of flow cytometry further verified Sca-1 and CD34 expression in isolated SASCs, and a greater percentage of cells were positive for Sca-1...from both injured and control noninjured muscle were analyzed using flow cytometry for the immunofluorescent signal of Sca-1 and CD34. Results

Sulforaphane targets cancer stemness and tumor initiating properties in oral squamous cell carcinomas via miR-200c induction.

PubMed

Liu, Chia-Ming; Peng, Chih-Yu; Liao, Yi-Wen; Lu, Ming-Yi; Tsai, Meng-Lun; Yeh, Jung-Chun; Yu, Chuan-Hang; Yu, Cheng-Chia

2017-01-01

Cancer stem cells (CSCs) are deemed as the driving force of tumorigenesis in oral squamous cell carcinomas (OSCCs). In this study, we investigated the chemotherapeutic effect of sulforaphane, a dietary component from broccoli sprouts, on targeting OSCC-CSCs. The effect of sulforaphane on normal oral epithelial cells (SG) and sphere-forming OSCC-CSCs isolated from SAS and GNM cells was examined. ALDH1 activity and CD44 positivity of OSCC-CSCs with sulforaphane treatment was assessed by flow cytometry analysis. In vitro and in vivo tumorigenicity assays of OSCC-CSCs with sulforaphane treatment were presented. We observed that the sulforaphane dose-dependently eliminated the proliferation rate of OSCC-CSCs, whereas the inhibition on SG cells proliferation was limited. Cancer stemness properties including self-renewal, CD44 positivity, and ALDH1 activity were also decreased in OSCC-CSCs with different doses of sulforaphane treatment. Moreover, sulforaphane treatment of OSCC-CSCs decreased the migration, invasion, clonogenicity, and in vivo tumorigenicity of xenograghts. Sulforaphane treatment resulted in a dose-dependent increase in the levels of tumor suppressive miR200c. These lines of evidence suggest that sulforaphane can suppress the cancer stemness and tumor-initiating properties in OSCC-CSCs both in vitro and in vivo. Copyright © 2016. Published by Elsevier B.V.

Monoclonal antibodies targeting non-small cell lung cancer stem-like cells by multipotent cancer stem cell monoclonal antibody library.

PubMed

Cao, Kaiyue; Pan, Yunzhi; Yu, Long; Shu, Xiong; Yang, Jing; Sun, Linxin; Sun, Lichao; Yang, Zhihua; Ran, Yuliang

2017-02-01

Cancer stem cells (CSCs) are a rare subset of cancer cells that play a significant role in cancer initiation, spreading, and recurrence. In this study, a subpopulation of lung cancer stem-like cells (LCSLCs) was identified from non-small cell lung carcinoma cell lines, SPCA-1 and A549, using serum-free suspension sphere-forming culture method. A monoclonal antibody library was constructed using immunized BLAB/c mice with the multipotent CSC cell line T3A-A3. Flow cytometry analysis showed that 33 mAbs targeted antigens can be enriched in sphere cells compared with the parental cells of SPCA-1 and A549 cell lines. Then, we performed functional antibody screening including sphere-forming inhibiting and invasion inhibiting assay. The results showed that two antibodies, 12C7 and 9B8, notably suppressed the self-renewal and invasion of LCSLCs. Fluorescence-activated cell sorting (FACs) found that the positive cells recognized by mAbs, 12C7 or 9B8, displayed features of LCSLCs. Interestingly, we found that these two antibodies recognized different subsets of cells and their combination effect was superior to the individual effect both in vitro and in vivo. Tissue microarrays were applied to detect the expression of the antigens targeted by these two antibodies. The positive expression of 12C7 and 9B8 targeted antigen was 84.4 and 82.5%, respectively, which was significantly higher than that in the non-tumor lung tissues. In conclusion, we screened two potential therapeutic antibodies that target different subsets of LCSLCs.

Development of Gonadotropin-Releasing Hormone-Secreting Neurons from Human Pluripotent Stem Cells.

PubMed

Lund, Carina; Pulli, Kristiina; Yellapragada, Venkatram; Giacobini, Paolo; Lundin, Karolina; Vuoristo, Sanna; Tuuri, Timo; Noisa, Parinya; Raivio, Taneli

2016-08-09

Gonadotropin-releasing hormone (GnRH) neurons regulate human puberty and reproduction. Modeling their development and function in vitro would be of interest for both basic research and clinical translation. Here, we report a three-step protocol to differentiate human pluripotent stem cells (hPSCs) into GnRH-secreting neurons. Firstly, hPSCs were differentiated to FOXG1, EMX2, and PAX6 expressing anterior neural progenitor cells (NPCs) by dual SMAD inhibition. Secondly, NPCs were treated for 10 days with FGF8, which is a key ligand implicated in GnRH neuron ontogeny, and finally, the cells were matured with Notch inhibitor to bipolar TUJ1-positive neurons that robustly expressed GNRH1 and secreted GnRH decapeptide into the culture medium. The protocol was reproducible both in human embryonic stem cells and induced pluripotent stem cells, and thus provides a translational tool for investigating the mechanisms of human puberty and its disorders. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Leptin and Adiponectin Modulate the Self-renewal of Normal Human Breast Epithelial Stem Cells.

PubMed

Esper, Raymond M; Dame, Michael; McClintock, Shannon; Holt, Peter R; Dannenberg, Andrew J; Wicha, Max S; Brenner, Dean E

2015-12-01

Multiple mechanisms are likely to account for the link between obesity and increased risk of postmenopausal breast cancer. Two adipokines, leptin and adiponectin, are of particular interest due to their opposing biologic functions and associations with breast cancer risk. In the current study, we investigated the effects of leptin and adiponectin on normal breast epithelial stem cells. Levels of leptin in human adipose explant-derived conditioned media positively correlated with the size of the normal breast stem cell pool. In contrast, an inverse relationship was found for adiponectin. Moreover, a strong linear relationship was observed between the leptin/adiponectin ratio in adipose conditioned media and breast stem cell self-renewal. Consistent with these findings, exogenous leptin stimulated whereas adiponectin suppressed breast stem cell self-renewal. In addition to local in-breast effects, circulating factors, including leptin and adiponectin, may contribute to the link between obesity and breast cancer. Increased levels of leptin and reduced amounts of adiponectin were found in serum from obese compared with age-matched lean postmenopausal women. Interestingly, serum from obese women increased stem cell self-renewal by 30% compared with only 7% for lean control serum. Taken together, these data suggest a plausible explanation for the obesity-driven increase in postmenopausal breast cancer risk. Leptin and adiponectin may function as both endocrine and paracrine/juxtacrine factors to modulate the size of the normal stem cell pool. Interventions that disrupt this axis and thereby normalize breast stem cell self-renewal could reduce the risk of breast cancer. ©2015 American Association for Cancer Research.

MELK and EZH2 Cooperate to Regulate Medulloblastoma Cancer Stem-like Cell Proliferation and Differentiation.

PubMed

Liu, Hailong; Sun, Qianwen; Sun, Youliang; Zhang, Junping; Yuan, Hongyu; Pang, Shuhuan; Qi, Xueling; Wang, Haoran; Zhang, Mingshan; Zhang, Hongwei; Yu, Chunjiang; Gu, Chunyu

2017-09-01

Medulloblastoma is the most common malignant brain tumor in children. Although accumulated research has suggested that cancer stem-like cells play a key role in medulloblastoma tumorigenesis, the specific molecular mechanism regarding proliferation remains elusive. Here, we reported more abundant expression of maternal embryonic leucine-zipper kinase (MELK) and enhancer of zeste homolog 2 (EZH2) in medulloblastoma stem-like cells than in neural stem cells and the interaction between the two proteins could mediate the self-renewal of sonic hedgehog subtype medulloblastoma. In human medulloblastoma, extensive nodularity and large-cell/anaplastic subgroups differed according to the staining levels of MELK and EZH2 from the other two subgroups. The proportion of MELK- or EZH2-positive staining status could be considered as a potential indicator for survival. Mechanistically, MELK bound to and phosphorylated EZH2, and its methylation was induced by EZH2 in medulloblastoma, which could regulate the proliferation of cancer stem-like cells. In xenografts, loss of MELK or EZH2 attenuated medulloblastoma stem-like cell-derived tumor growth and promoted differentiation. These findings indicate that MELK-induced phosphorylation and EZH2-mediated methylation in MELK/EZH2 pathway are essential for medulloblastoma stem-like cell-derived tumor proliferation, thereby identifying a potential therapeutic strategy for these patients. Implications: This study demonstrates that the interaction occurring between MELK and EZH2 promotes self-proliferation and stemness, thus representing an attractive therapeutic target and potential candidate for diagnosis of medulloblastoma. Mol Cancer Res; 15(9); 1275-86. ©2017 AACR . ©2017 American Association for Cancer Research.

Reprogramming of non-genomic estrogen signaling by the stemness factor SOX2 enhances the tumor-initiating capacity of breast cancer cells.

PubMed

Vazquez-Martin, Alejandro; Cufí, Sílvia; López-Bonet, Eugeni; Corominas-Faja, Bruna; Cuyàs, Elisabet; Vellon, Luciano; Iglesias, Juan Manuel; Leis, Olatz; Martín, Angel G; Menendez, Javier A

2013-11-15

The restoration of pluripotency circuits by the reactivation of endogenous stemness factors, such as SOX2, may provide a new paradigm in cancer development. The tumoral stem cell reprogramming hypothesis, i.e., the ability of stemness factors to redirect normal and differentiated tumor cells toward a less-differentiated and stem-like state, adds new layers of complexity to cancer biology, because the effects of such reprogramming may remain dormant until engaged later in response to (epi)genetic and/or (micro)environmental events. To test this hypothesis, we utilized an in vitro model of a SOX2-overexpressing cancer stem cell (CSC)-like cellular state that was recently developed in our laboratory by employing Yamanaka's nuclear reprogramming technology in the estrogen receptor α (ERα)-positive MCF-7 breast cancer cell line. Despite the acquisition of distinct molecular features that were compatible with a breast CSC-like cellular state, such as strong aldehyde dehydrogenase activity, as detected by ALDEFLUOR, and overexpression of the SSEA-4 and CD44 breast CSC markers, the tumor growth-initiating ability of SOX2-overexpressing CSC-like MCF-7 cells solely occurred in female nude mice supplemented with estradiol when compared with MCF-7 parental cells. Ser118 phosphorylation of estrogen receptor α (ERα), which is a pivotal integrator of the genomic and nongenomic E 2/ERα signaling pathways, drastically accumulated in nuclear speckles in the interphase nuclei of SOX2-driven CSC-like cell populations. Moreover, SOX2-positive CSC-like cells accumulated significantly higher numbers of actively dividing cells, and the highest levels of phospho-Ser118-ERα occurred when chromosomes lined up on a metaphase plate. The previously unrecognized link between E 2/ERα signaling and SOX2-driven stem cell circuitry may significantly impact our current understanding of breast cancer initiation and progression, i.e., SOX2 can promote non-genomic E 2 signaling that leads to nuclear phospho-Ser118-ERα, which ultimately exacerbates genomic ER signaling in response to E 2. Because E 2 stimulation has been recently shown to enhance breast tumor-initiating cell survival by downregulating miR-140, which targets SOX2, the establishment of a bidirectional cross-talk interaction between the stem cell self-renewal regulator, SOX2, and the local and systemic ability of E 2 to increase breast CSC activity may have profound implications for the development of new CSC-directed strategies for breast cancer prevention and therapy.

The IGF-1 Receptor Identifies a Pool of Human Cardiac Stem Cells with Superior Therapeutic Potential for Myocardial Regeneration

PubMed Central

D’Amario, Domenico; Cabral-Da-Silva, Mauricio; Zheng, Hanqiao; Fiorini, Claudia; Goichberg, Polina; Steadman, Elisabeth; Ferreira-Martins, João; Sanada, Fumihiro; Piccoli, Marco; Cappetta, Donato; D’Alessandro, David A.; Michler, Robert E.; Hosoda, Toru; Anastasia, Luigi; Rota, Marcello; Leri, Annarosa; Anversa, Piero; Kajstura, Jan

2012-01-01

Rationale Age and coronary artery disease may negatively affect the function of human cardiac stem cells (hCSCs) and their potential therapeutic efficacy for autologous cell transplantation in the failing heart. Objective Insulin-like growth factor 1 (IGF-1) and 2 (IGF-2), and angiotensin II (Ang II) and their receptors, IGF-1R, IGF-2R and AT1R, were characterized in c-kit-positive-hCSCs to establish whether these systems would allow us to separate hCSC classes with different growth reserve in the aging and diseased myocardium. Methods and Results C-kit-positive-hCSCs were collected from myocardial samples obtained from 24 patients, 48 to 86 years of age, undergoing elective cardiac surgery for coronary artery disease. The expression of IGF-1R in hCSCs recognized a young cell phenotype defined by long telomeres, high telomerase activity, enhanced cell proliferation and attenuated apoptosis. In addition to IGF-1, IGF-1R-positive-hCSCs secreted IGF-2 that promoted myocyte differentiation. Conversely, the presence of IGF-2R and AT1R, in the absence of IGF-1R, identified senescent hCSCs with impaired growth reserve and increased susceptibility to apoptosis. The ability of IGF-1R-positive-hCSCs to regenerate infarcted myocardium was then compared with that of unselected c-kit-positive-hCSCs. IGF-1R-positive-hCSCs improved cardiomyogenesis and vasculogenesis. Pretreatment of IGF-1R-positive-hCSCs with IGF-2 resulted in the formation of more mature myocytes and superior recovery of ventricular structure. Conclusions hCSCs expressing only IGF-1R synthesize both IGF-1 and IGF-2, which are potent modulators of stem cell replication, commitment to the myocyte lineage and myocyte differentiation, pointing to this hCSC subset as the ideal candidate cell for the management of human heart failure. PMID:21546606

Identification of Multipotent Stem/Progenitor Cells in Murine Sclera

PubMed Central

Tsai, Chia-Ling; Wu, Pei-Chang; Fini, M. Elizabeth; Shi, Songtao

2011-01-01

Purpose. The sclera forms the fibrous outer coat of the eyeball and acts as a supportive framework. The purpose of this study was to examine whether the sclera contains mesenchymal stem/progenitor cells. Method. Scleral tissue from C57BL6/J mice was separated from the retina and choroid and subsequently enzyme digested to release single cells. Proliferation capacity, self-renewal capacity, and ability for multipotent differentiation were analyzed by BrdU labeling, flow cytometry, reverse transcriptase–polymerase chain reaction, immunocytochemistry, and in vivo transplantation. Results. The scleral stem/progenitor cells (SSPCs) possessed clonogenic and high doubling capacities. These cells were positive for the mesenchymal markers Sca-1, CD90.2, CD44, CD105, and CD73 and negative for the hematopoietic markers CD45, CD11b, Flk1, CD34, and CD117. In addition to expressing stem cell genes ABCG2, Six2, Notch1, and Pax6, SSPCs were able to differentiate to adipogenic, chondrogenic, and neurogenic lineages. Conclusions. This study indicates that the sclera contains multipotent mesenchymal stem cells. Further study of SSPCs may help elucidate the cellular and molecular mechanism of scleral diseases such as scleritis and myopia. PMID:21788434

3D Graphene Oxide-encapsulated Gold Nanoparticles to Detect Neural Stem Cell Differentiation

PubMed Central

Kim, Tae-Hyung; Lee, Ki-Bum; Choi, Jeong-Woo

2013-01-01

Monitoring of stem cell differentiation and pluripotency is an important step for the practical use of stem cells in the field of regenerative medicine. Hence, a new non-destructive detection tool capable of in situ monitoring of stem cell differentiation is highly needed. In this study, we report a 3D graphene oxide-encapsulated gold nanoparticle that is very effective for the detection of the differentiation potential of neural stem cells (NSCs) based on surface-enhanced Raman spectroscopy (SERS). A new material, 3D GO-encapsulated gold nanoparticle, is developed to induce the double enhancement effect of graphene oxide and gold nanoparticle on SERS signals which is only effective for undifferentiated NSCs. The Raman peaks achieved from undifferentiated NSCs on the graphene oxide (GO)-encapsulated gold nanoparticles were 3.5 times higher than peaks obtained from normal metal structures and were clearly distinguishable from those of differentiated cells. The number of C=C bonds and the raman instensity at 1656cm−1 was found to show a positive correlation, which matches the differentiation state of the NSCs. Moreover, the substrate composed of 3D GO-encapsulated gold nanoparticles was also effective at distinguishing the differentiation state of single NSC by using electrochemical and electrical techniques. Hence, the proposed technique can be used as a powerful non-destructive in situ monitoring tool for the identification of the differentiation potential of various kinds of stem cells (mesenchymal, hematopoietic, and neural stem cells). PMID:23937915

Growing Stem Cells: The Impact of Federal Funding Policy on the U.S. Scientific Frontier

ERIC Educational Resources Information Center

Furman, Jeffrey L.; Murray, Fiona; Stern, Scott

2012-01-01

This paper articulates a citation-based approach to science policy evaluation and employs that approach to investigate the impact of the United States' 2001 policy regarding the federal funding of human embryonic stem cell (hESC) research. We evaluate the impact of the policy on the level of U.S. hESC research, the U.S. position at the knowledge…

Translational Control in Bone Marrow Failure

DTIC Science & Technology

2015-05-01

HCLS1 associated protein X-1 (HAX1), cause hereditary forms of neutropenia . Previously, competing hypotheses have posited that mutant forms of...derived induced pluripotent stem cell (iPSC) model of ELANE-associated neutropenia . During the second year of this project, in order to facilitate...pathology. 3 2. KEY WORDS neutropenia bone marrow failure neutrophil elastase ELANE HAX1 alternate translation induced pluripotent stem cells (iPSC

Pericarditis mediated by respiratory syncytial virus in a hematopoietic stem cell transplant patient.

PubMed

Rubach, M P; Pavlisko, E N; Perfect, J R

2013-08-01

We describe a case of pericarditis and large pericardial effusion in a 63-year-old African-American man undergoing autologous hematopoietic stem cell transplant for multiple myeloma. Pericardial tissue biopsy demonstrated fibrinous pericarditis, and immunohistochemistry stains were positive for respiratory syncytial virus. The patient improved with oral ribavirin and intravenous immune globulin infusions. © 2013 John Wiley & Sons A/S.

[Expression of c-MPL in leukemic stem cells from acute myeloid leukemia patients].

PubMed

Yu, Pei; Qiu, Shao-Wei; Rao, Qing; Lin, Dong; Xing, Hai-Yan; Tang, Ke-Jing; Tian, Zheng; Wang, Min; Wang, Jian-Xiang

2012-10-01

This study was aimed to investigate the expression of c-MPL in acute myeloid leukemia (AML) and the correlation of the c-MPL expression with CD34 and CD38, so as to define the expression of c-MPL in leukemic stem cells. The expression levels of CD34, CD38 and c-MPL were detected by flow cytometry in bone marrow cells from 29 newly diagnosed AML patients. The relationship of c-MPL positive cell ratio with clinical parameters and correlation of c-MPL with CD34 and CD38 expression in AML patients were analyzed. The results showed that expression level of c-MPL in AML patients was significantly higher than that of normal controls (P < 0.05), and the expression level of c-MPL did not correlate with age, sex, white blood cell count, AML1-ETO fusion gene and remission after chemotherapy, but the expression of c-MPL in M2 and M5 patients was higher than that of normal control (P < 0.05). Expression of c-MPL in CD34 positive AML patients was obviously higher than that in CD34 negative AML patients (P < 0.01). c-MPL was significantly higher expressed in CD34(+) cells than that in CD34(-) cells (P < 0.001), while c-MPL expression was not significantly different between CD34(+)CD38(-) and CD34(+)CD38(-) cell groups. Positive correlation between c-MPL and CD34 expression was observed (r = 0.380, P = 0.042). It is concluded that expression of c-MPL is higher in AML patients, and positively correlates with the expression level of CD34. The c-MPL expresses in leukemic stem cells.

BONE MARROW MESENCHYMAL STEM CELLS ARE PROGENITORS IN VITRO FOR INNER EAR HAIR CELLS

PubMed Central

Jeon, Sang-Jun; Oshima, Kazuo; Heller, Stefan; Edge, Albert S.B.

2011-01-01

Stem cells have been demonstrated in the inner ear but they do not spontaneously divide to replace damaged sensory cells. Mesenchymal stem cells (MSC) from bone marrow have been reported to differentiate into multiple lineages including neurons, and we therefore asked whether MSCs could generate sensory cells. Overexpression of the prosensory transcription factor, Math1, in sensory epithelial precursor cells induced expression of myosin VIIa, espin, Brn3c, p27Kip, and jagged2, indicating differentiation to inner ear sensory cells. Some of the cells displayed F-actin positive protrusions in the morphology characteristic of hair cell stereociliary bundles. Hair cell markers were also induced by culture of mouse MSC-derived cells in contact with embryonic chick inner ear cells, and this induction was not due to a cell fusion event, because the chick hair cells could be identified with a chick-specific antibody and chick and mouse antigens were never found in the same cell. PMID:17113786

Adipose-derived stem cell: a better stem cell than BMSC.

PubMed

Zhu, Yanxia; Liu, Tianqing; Song, Kedong; Fan, Xiubo; Ma, Xuehu; Cui, Zhanfeng

2008-08-01

To further study the proliferation and multi-differentiation potentials of adipose-derived stem cells (ADSCs), the cells were isolated with improved methods and their growth curves were achieved with cck-8. Surface protein expression was analyzed by flow cytometry to characterize the cell phenotype. The multi-lineage potential of ADSCs was testified by differentiating cells with adipogenic, chondrogenic, osteogenic, and myogenic inducers. The results showed that about 5 x 10(5) stem cells could be obtained from 400 to 600 mg adipose tissue. The ADSCs can be continuously cultured in vitro for up to 1 month without passage and they have several logarithmic growth phases during the culture period. Also, the flow cytometry analysis showed that ADSCs expressed high levels of stem cell-related antigens (CD13, CD29, CD44, CD105, and CD166), while did not express hematopoiesis-related antigens CD34 and CD45, and human leukocyte antigen HLA-DR was also negative. Moreover, stem cell-related transcription factors, Nanog, Oct-4, Sox-2, and Rex-1 were positively expressed in ADSCs. The expression of alkaline phosphatase (ALP) was detected in the early osteogenic induction and the calcified nodules were observed by von Kossa staining. Intracellular lipid droplets could be observed by Oil Red staining. Differentiated cardiomyocytes were observed by connexin43 fluorescent staining. In order to obtain more stem cells, we can subculture ADSCs every 14 days instead of the normal 5 days. ADSCs still keep strong proliferation ability, maintain their phenotypes, and have stronger multi-differentiation potential after 25 passages. Copyright 2008 John Wiley & Sons, Ltd.

Translating Stem Cell Research to Cardiac Disease Therapies: Pitfalls and Prospects for Improvement

PubMed Central

Rosen, Michael R.; Myerburg, Robert J.; Francis, Darrel P.; Cole, Graham D.; Marbán, Eduardo

2014-01-01

Over the past 2 decades, there have been numerous stem cell studies focused on cardiac diseases, ranging from proof-of-concept to phase 2 trials. This series of articles focuses on the legacy of these studies and the outlook for future treatment of cardiac diseases with stem cell therapies. The first section by Rosen and Myerburg is an independent review that analyzes the basic science and translational strategies supporting the rapid advance of stem cell technology to the clinic, the philosophies behind them, trial designs, and means for going forward that may impact favorably on progress. The second and third sections were collected in response to the initial section of this review. The commentary by Francis and Cole discusses the Rosen and Myerburg review and details how trial outcomes can be affected by noise, poor trial design (particularly the absence of blinding), and normal human tendencies toward optimism and denial. The final, independent article by Marbán takes a different perspective concerning the potential for positive impact of stem cell research applied to heart disease and future prospects for its clinical application. PMID:25169179

[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.

In vitro expansion of Lin{sup +} and Lin{sup −} mononuclear cells from human peripheral blood

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

Norhaiza, H. Siti; Zarina, Z. A. Intan; Hisham, Z. A. Shahrul

2013-11-27

Haematopoietic stem cells (HSCs) are used in the therapy of blood disorders due to the ability of these cells to reconstitute haematopoietic lineage cells when transplanted into myeloablative recipients. However, substantial number of cells is required in order for the reconstitution to take place. Since HSCs present in low frequency, larger number of donor is required to accommodate the demand of transplantable HSCs. Therefore, in vitro expansion of HSCs will have profound impact on clinical purposes. The aim of this study was to expand lineage negative (Lin{sup −}) stem cells from human peripheral blood. Total peripheral blood mononuclear cells (PBMNCs)more » were fractionated from human blood by density gradient centrifugation. Subsequently, PBMNCs were subjected to magnetic assisted cell sorter (MACS) which depletes lineage positive (Lin{sup +}) mononuclear cells expressing lineage positive markers such as CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, and CD235a to obtained Lin{sup −} cell population. The ability of Lin{sup +} and Lin{sup −} to survive in vitro was explored by culturing both cell populations in complete medium consisting of Alpha-Minimal Essential Medium (AMEM) +10% (v/v) Newborn Calf Serum (NBCS)+ 2% (v/v) pen/strep. In another experiment, Lin{sup +} and Lin{sup −} were cultured with complete medium supplemented with 10ng/mL of the following growth factors: stem cell factor (SCF), interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF), 2IU/mL of Erythropoietin (Epo) and 20ng/mL of IL-6. Three samples were monitored in static culture for 22 days. The expansion potential was assessed by the number of total viable cells, counted by trypan blue exclusion assay. It was found that Lin{sup +} mononuclear cells were not able to survive either in normal proliferation medium or proliferation medium supplemented with cytokines. Similarly, Lin{sup −} stem cells were not able to survive in proliferation medium however, addition of cytokines into the proliferation medium support Lin{sup −} stem cells for at least 18 days. The Lin{sup −} stem cells started to response to the cytokines added as early as Day 2 of culture. It is concluded that Lin{sup −} stem cells can be expanded in vitro by culturing in proliferation medium supplemented with cytokines.« less

Images of cloning and stem cell research in editorial cartoons in the United States.

PubMed

Giarelli, Ellen

2006-01-01

Through semiotic analysis of manifest and latent meanings in editorial cartoons, the author uncovers how cloning and stem cell research are represented in a popular mass medium. She identified 86 editorial cartoons published in the United States between 2001 and 2004 that referred to cloning and 20 that referred to stem cell research. Cartoonists portrayed people individually 224 times and 4 times in groups of more than 10. Men were portrayed in 64% of cartoons. Stem cell research was depicted as having a potential positive value, and cloning was depicted negatively. Some major messages are that cloning will lead to the mass production of evil, cloning creates monsters, and politics will influence who or what will be cloned. Analyzing popular images can allow access to public understanding about genetic technology and evaluation of public beliefs, preconceptions, and expectations as the public is educated on the use and value of services.

A New Case of Syringocystadenocarcinoma Papilliferum: A Rare Pathology for a Wide-Ranging Comprehension

PubMed Central

Paradiso, Beatrice; Bianchini, Enzo; Cifelli, Pierangelo; Cavazzini, Luigi; Lanza, Giovanni

2014-01-01

We report a new case of p63/cytokeratin 7 (CK7) positive syringocystadenocarcinoma papilliferum (SCACP), on the shoulder of an 88-year-old man, with superficial dermal infiltration and squamoid differentiation. We describe the 24th case of SCACP, the malignant counterpart of syringocystadenoma papilliferum (SCAP). At the present, we do not know whether SCACP arises from eccrine or apocrine glands because of the contrasting opinions in the literature. Only few histochemical and ultrastructural studies have previously advised that SCACP could arise from pluripotent stem cells. Through our case, we wish to suggest the stem cell-like properties of the syringocystadenocarcinoma papilliferum. This rare neoplasm shows two different patterns of stem cell marker expression in the glandular and squamous components, respectively. For the double phenotype of SCACP, we propose it like an intriguing model to study histogenesis and stem cell properties for more wide-ranging epithelial tumors. PMID:24959179

The Role of Stem Cell Therapeutics in Wound Healing: Current Understanding and Future Directions.

PubMed

Sorice, Sarah; Rustad, Kristine C; Li, Alexander Y; Gurtner, Geoffrey C

2016-09-01

Chronic wounds present unique challenges for healthcare providers as they place patients at increased risk for various morbidities and mortality. Advances in wound care technology have expanded the treatment options available for wound management, but few products fully address the underlying core deficiencies responsible for the development of poorly healing wounds. In the future, addressing these derangements will undoubtedly play a key role in the treatment of these patients. Broad enthusiasm has surrounded the field of stem cell biology, which has shown great promise in repairing damaged tissues across numerous disease phenotypes. In this review, we provide a comprehensive review of the literature and evaluate the present landscape of wound therapeutics while discussing the rationales and allure behind stem cell-based products. We further propose 2 challenges that remain as new stem cell-based therapies are being developed and as this technology moves toward clinical translation. Given the relatively young age of this newer technology in wound healing, numerous challenges continue to surround its effective use including identifying the ideal population of stem cells to use and determining the optimal cell delivery method. However, significant forward progress has been made, with several clinical trials beginning to demonstrate reliable clinical benefit. The upward trajectory of stem cell technologies provides an exciting opportunity to positively impact patient outcomes through the controlled application of regenerative cell-based therapy.

The effect of age and medical comorbidities on in vitro myoblast expansion in women with and without pelvic organ prolapse.

PubMed

Price, Danielle Markle; Lane, Felicia L; Craig, Jocelyn B; Nistor, Gabriel; Motakef, Saba; Pham, Quynh-Ahn; Keirstead, Hans

2014-01-01

This is an observational study is designed to assess the influence of age, prolapse and medical co-morbidities on myogenic stem cells growth in-vitro. A biopsy of the rectus abdominus muscle was obtained during surgery in patients with and without pelvic organ prolapse (POP). Nuclei number and fiber count were correlated with patient's age, presence of POP, and medical comorbidities. Efficiency of expansion of myogenic stem cells in vitro was calculated. The percentage of Pax7-, MyoD-, and desmin-positive cells was correlated with age, POP status, and medical comorbidities. A total of 17 specimens were obtained; 13 specimens were available for histologic analysis. There was no correlation between patient's age, POP status or medical comorbidities and nuclei or fiber count, growth rate, or the percentage of Pax7- and MyoD-positive cells. Patients with 2 to 4 medical comorbidities were noted to have a significantly lower percentage of desmin-positive cells. Specimens with a higher nuclear count had significantly better cellular expansion. Data were analyzed using Kruskal-Wallis or Wilcoxon rank sum statistics. Multiple medical comorbidities but not patient's age or POP status influenced in vitro myogenic stem cell growth. These data suggest that patients with advancing age or POP may be acceptable autologous donors if treatment of urinary or anal incontinence requires myoblast transplantation.

In vitro differentiation of human tooth germ stem cells into endothelial- and epithelial-like cells.

PubMed

Doğan, Ayşegül; Demirci, Selami; Şahin, Fikrettin

2015-01-01

Current clinical techniques in dental practice include stem cell and tissue engineering applications. Dental stem cells are promising primary cell source for mainly tooth tissue engineering. Interaction of mesenchymal stem cell with epithelial and endothelial cells is strictly required for an intact tooth morphogenesis. Therefore, it is important to investigate whether human tooth germ stem cells (hTGSCs) derived from wisdom tooth are suitable for endothelial and epithelial cell transformation in dental tissue regeneration approaches. Differentiation into endothelial and epithelial cell lineages were mimicked under defined conditions, confirmed by real time PCR, western blotting and immunocytochemical analysis by qualitative and quantitative methods. HUVECs and HaCaT cells were used as positive controls for the endothelial and epithelial differentiation assays, respectively. Immunocytochemical and western blotting analysis revealed that terminally differentiated cells expressed cell-lineage markers including CD31, VEGFR2, VE-Cadherin, vWF (endothelial cell markers), and cytokeratin (CK)-17, CK-19, EpCaM, vimentin (epithelial cell markers) in significant levels with respect to undifferentiated control cells. Moreover, high expression levels of VEGFR1, VEGFR2, VEGF, CK-18, and CK-19 genes were detected in differentiated endothelial and epithelial-like cells. Endothelial-like cells derived from hTGSCs were cultured on Matrigel, tube-like structure formations were followed as an indication for functional endothelial differentiation. hTGSCs successfully differentiate into various cell types with a broad range of functional abilities using an in vitro approach. These findings suggest that hTGSCs may serve a potential stem cell source for tissue engineering and cell therapy of epithelial and endothelial tissue. © 2014 International Federation for Cell Biology.

Effective and persistent antitumor activity of HER2-directed CAR-T cells against gastric cancer cells in vitro and xenotransplanted tumors in vivo.

PubMed

Song, Yanjing; Tong, Chuan; Wang, Yao; Gao, Yunhe; Dai, Hanren; Guo, Yelei; Zhao, Xudong; Wang, Yi; Wang, Zizheng; Han, Weidong; Chen, Lin

2017-03-10

Human epidermal growth factor receptor 2 (HER2) proteins are overexpressed in a high proportion of gastric cancer (GC) cases and affect the maintenance of cancer stem cell (CSC) subpopulations, which are used as targets for the clinical treatment of patients with HER2-positive GC. Despite improvements in survival, numerous HER2-positive patients fail treatment with trastuzumab, highlighting the need for more effective therapies. In this study, we generated a novel type of genetically modified human T cells, expressing a chimeric antigen receptor (CAR), and targeting the GC cell antigen HER2, which harbors the CD137 and CD3ζ moieties. Our findings show that the expanded CAR-T cells, expressing an increased central memory phenotype, were activated by the specific recognition of HER2 antigens in an MHC-independent manner, and effectively killed patient-derived HER2-positive GC cells. In HER2-positive xenograft tumors, CAR-T cells exhibited considerably enhanced tumor inhibition ability, long-term survival, and homing to targets, compared with those of non-transduced T cells. The sphere-forming ability and in vivo tumorigenicity of patient-derived gastric cancer stem-like cells, expressing HER2 and the CD44 protein, were also inhibited. Our results support the future development and clinical application of this adoptive immunotherapy in patients with HER2-positive advanced GC.

Anti-cancer stem cell activity of a hedgehog inhibitor GANT61 in estrogen receptor-positive breast cancer cells.

PubMed

Kurebayashi, Junichi; Koike, Yoshikazu; Ohta, Yusuke; Saitoh, Wataru; Yamashita, Tetsumasa; Kanomata, Naoki; Moriya, Takuya

2017-05-01

Estradiol (E2) increases not only the cell growth but also the cancer stem cell (CSC) proportion in estrogen receptor (ER)-positive breast cancer cells. It has been suggested that the non-canonical hedgehog (Hh) pathway activated by E2 plays an important role in the regulation of CSC proportion in ER-positive breast cancer cells. We studied anti-CSC activity of a non-canonical Hh inhibitor GANT61 in ER-positive breast cancer cells. Effects of GANT61 on the cell growth, cell cycle progression, apoptosis and CSC proportion were investigated in four ER-positive breast cancer cell lines. CSC proportion was measured using either the mammosphere assay or CD44/CD24 assay. Expression levels of pivotal molecules in the Hh pathway were measured. Combined effects of GANT61 with antiestrogens on the anti-cell growth and anti-CSC activities were investigated. E2 significantly increased the cell growth and CSC proportion in all ER-positive cell lines. E2 increased the expression levels of glioma-associated oncogene (GLI) 1 and/or GLI2. GANT61 decreased the cell growth in association with a G1-S cell cycle retardation and increased apoptosis. GANT61 decreased the E2-induced CSC proportion measured by the mammosphere assay in all cell lines. Antiestrogens also decreased the E2-induced cell growth and CSC proportion. Combined treatments of GANT61 with antiestrogens additively enhanced anti-cell growth and/or anti-CSC activities in some ER-positive cell lines. In conclusion, the non-canonical Hh inhibitor GANT61 inhibited not only the cell growth but also the CSC proportion increased by E2 in ER-positive breast cancer cells. GANT61 enhanced anti-cell growth and/or anti-CSC activities of antiestrogens in ER-positive cell lines. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Zinc Promotes Adipose-Derived Mesenchymal Stem Cell Proliferation and Differentiation towards a Neuronal Fate.

PubMed

Moon, Mi-Young; Kim, Hyun Jung; Choi, Bo Young; Sohn, Min; Chung, Tae Nyoung; Suh, Sang Won

2018-01-01

Zinc is an essential element required for cell division, migration, and proliferation. Under zinc-deficient conditions, proliferation and differentiation of neural progenitors are significantly impaired. Adipose-derived mesenchymal stem cells (AD-MSCs) are multipotent stem cells that can differentiate into neurons. The aim of this study was to evaluate the effect of zinc on AD-MSC proliferation and differentiation. We initially examined the effect of zinc on stem cell proliferation at the undifferentiated stage. AD-MSCs showed high proliferation rates on day 6 in 30  μ M and 100  μ M of ZnCl 2 . Zinc chelation inhibited AD-MSC proliferation via downregulation of ERK1/2 activity. We then assessed whether zinc was involved in cell migration and neurite outgrowth during differentiation. After three days of neuronal differentiation, TUJ-1-positive cells were observed, implying that AD-MSCs had differentiated into early neuron or neuron-like cells. Neurite outgrowth was increased in the zinc-treated group, while the CaEDTA-treated group showed diminished, shrunken neurites. Furthermore, we showed that zinc promoted neurite outgrowth via the inactivation of RhoA and led to the induction of neuronal gene expression (MAP2 and nestin) in differentiated stem cells. Taken together, zinc promoted AD-MSC proliferation and affected neuronal differentiation, mainly by increasing neurite outgrowth.

Assessment of Safety and Functional Efficacy of Stem Cell-Based Therapeutic Approaches Using Retinal Degenerative Animal Models

PubMed Central

Lin, Tai-Chi; Zhu, Danhong; Hinton, David R.; Clegg, Dennis O.; Humayun, Mark S.

2017-01-01

Dysfunction and death of retinal pigment epithelium (RPE) and or photoreceptors can lead to irreversible vision loss. The eye represents an ideal microenvironment for stem cell-based therapy. It is considered an “immune privileged” site, and the number of cells needed for therapy is relatively low for the area of focused vision (macula). Further, surgical placement of stem cell-derived grafts (RPE, retinal progenitors, and photoreceptor precursors) into the vitreous cavity or subretinal space has been well established. For preclinical tests, assessments of stem cell-derived graft survival and functionality are conducted in animal models by various noninvasive approaches and imaging modalities. In vivo experiments conducted in animal models based on replacing photoreceptors and/or RPE cells have shown survival and functionality of the transplanted cells, rescue of the host retina, and improvement of visual function. Based on the positive results obtained from these animal experiments, human clinical trials are being initiated. Despite such progress in stem cell research, ethical, regulatory, safety, and technical difficulties still remain a challenge for the transformation of this technique into a standard clinical approach. In this review, the current status of preclinical safety and efficacy studies for retinal cell replacement therapies conducted in animal models will be discussed. PMID:28928775

In Vitro T-Cell Generation From Adult, Embryonic, and Induced Pluripotent Stem Cells: Many Roads to One Destination.

PubMed

Smith, Michelle J; Webber, Beau R; Mohtashami, Mahmood; Stefanski, Heather E; Zúñiga-Pflücker, Juan Carlos; Blazar, Bruce R

2015-11-01

T lymphocytes are critical mediators of the adaptive immune system and have the capacity to serve as therapeutic agents in the areas of transplant and cancer immunotherapy. While T cells can be isolated and expanded from patients, T cells derived in vitro from both hematopoietic stem/progenitor cells (HSPCs) and human pluripotent stem cells (hPSCs) offer great potential advantages in generating a self-renewing source of T cells that can be readily genetically modified. T-cell differentiation in vivo is a complex process requiring tightly regulated signals; providing the correct signals in vitro to induce T-cell lineage commitment followed by their development into mature, functional, single positive T cells, is similarly complex. In this review, we discuss current methods for the in vitro derivation of T cells from murine and human HSPCs and hPSCs that use feeder-cell and feeder-cell-free systems. Furthermore, we explore their potential for adoption for use in T-cell-based therapies. © 2015 AlphaMed Press.

Zic-Proteins Are Repressors of Dopaminergic Forebrain Fate in Mice and C. elegans.

PubMed

Tiveron, Marie-Catherine; Beclin, Christophe; Murgan, Sabrina; Wild, Stefan; Angelova, Alexandra; Marc, Julie; Coré, Nathalie; de Chevigny, Antoine; Herrera, Eloisa; Bosio, Andreas; Bertrand, Vincent; Cremer, Harold

2017-11-01

In the postnatal forebrain regionalized neural stem cells along the ventricular walls produce olfactory bulb (OB) interneurons with varying neurotransmitter phenotypes and positions. To understand the molecular basis of this region-specific variability we analyzed gene expression in the postnatal dorsal and lateral lineages in mice of both sexes from stem cells to neurons. We show that both lineages maintain transcription factor signatures of their embryonic site of origin, the pallium and subpallium. However, additional factors, including Zic1 and Zic2, are postnatally expressed in the dorsal stem cell compartment and maintained in the lineage that generates calretinin-positive GABAergic neurons for the OB. Functionally, we show that Zic1 and Zic2 induce the generation of calretinin-positive neurons while suppressing dopaminergic fate in the postnatal dorsal lineage. We investigated the evolutionary conservation of the dopaminergic repressor function of Zic proteins and show that it is already present in C. elegans SIGNIFICANCE STATEMENT The vertebrate brain generates thousands of different neuron types. In this work we investigate the molecular mechanisms underlying this variability. Using a genomics approach we identify the transcription factor signatures of defined neural stem cells and neuron populations. Based thereon we show that two related transcription factors, Zic1 and Zic2, are essential to control the balance between two defined neuron types in the postnatal brain. We show that this mechanism is conserved in evolutionary very distant species. Copyright © 2017 the authors 0270-6474/17/3710611-13$15.00/0.

Sonic hedgehog promotes stem-cell potential of Mueller glia in the mammalian retina

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

Wan Jin; Zheng Hua; Xiao Honglei

2007-11-16

Mueller glia have been demonstrated to display stem-cell properties after retinal damage. Here, we report this potential can be regulated by Sonic hedgehog (Shh) signaling. Shh can stimulate proliferation of Mueller glia through its receptor and target gene expressed on them, furthermore, Shh-treated Mueller glia are induced to dedifferentiate by expressing progenitor-specific markers, and then adopt cell fate of rod photoreceptor. Inhibition of signaling by cyclopamine inhibits proliferation and dedifferentiation. Intraocular injection of Shh promotes Mueller glia activation in the photoreceptor-damaged retina, Shh also enhances neurogenic potential by producing more rhodopsin-positive photoreceptors from Mueller glia-derived cells. Together, these results providemore » evidences that Mueller glia act as potential stem cells in mammalian retina, Shh may have therapeutic effects on these cells for promoting the regeneration of retinal neurons.« less

Store-Operated Calcium Entries Control Neural Stem Cell Self-Renewal in the Adult Brain Subventricular Zone.

PubMed

Domenichini, Florence; Terrié, Elodie; Arnault, Patricia; Harnois, Thomas; Magaud, Christophe; Bois, Patrick; Constantin, Bruno; Coronas, Valérie

2018-05-01

The subventricular zone (SVZ) is the major stem cell niche in the brain of adult mammals. Within this region, neural stem cells (NSC) proliferate, self-renew and give birth to neurons and glial cells. Previous studies underlined enrichment in calcium signaling-related transcripts in adult NSC. Because of their ability to mobilize sustained calcium influxes in response to a wide range of extracellular factors, store-operated channels (SOC) appear to be, among calcium channels, relevant candidates to induce calcium signaling in NSC whose cellular activities are continuously adapted to physiological signals from the microenvironment. By Reverse Transcription Polymerase Chain Reaction (RT-PCR), Western blotting and immunocytochemistry experiments, we demonstrate that SVZ cells express molecular actors known to build up SOC, namely transient receptor potential canonical 1 (TRPC1) and Orai1, as well as their activator stromal interaction molecule 1 (STIM1). Calcium imaging reveals that SVZ cells display store-operated calcium entries. Pharmacological blockade of SOC with SKF-96365 or YM-58483 (also called BTP2) decreases proliferation, impairs self-renewal by shifting the type of SVZ stem cell division from symmetric proliferative to asymmetric, thereby reducing the stem cell population. Brain section immunostainings show that TRPC1, Orai1, and STIM1 are expressed in vivo, in SOX2-positive SVZ NSC. Injection of SKF-96365 in brain lateral ventricle diminishes SVZ cell proliferation and reduces the ability of SVZ cells to form neurospheres in vitro. The present study combining in vitro and in vivo approaches uncovers a major role for SOC in the control of SVZ NSC population and opens new fields of investigation for stem cell biology in health and disease. Stem Cells 2018;36:761-774. © AlphaMed Press 2018.

Liver repopulation by c-Met-positive stem/progenitor cells isolated from the developing rat liver.

PubMed

Suzuki, Atsushi; Zheng, Yun-wen; Fukao, Katashi; Nakauchi, Hiromitsu; Taniguchi, Hideki

2004-01-01

Self-renewing stem cells responsible for tissue or organ development and regeneration have been recently described. To isolate such cells using flow cytometry, it should be required to find molecules expressing on their cell surfaces. We have previously reported that, on cells fulfilling the criteria for hepatic stem cells, the hepatocyte growth factor receptor c-Met is expressed specifically in the developing mouse liver. In this study, to determine whether c-Met is an essential marker for hepatic stem cells in other animal strains, we examined the potential for in vivo liver-repopulation in sorted fetal rat-derived c-Met+ cells using the retrorsine model. Using flow cytometry and monoclonal antibodies for c-Met and leukocyte common antigen CD45, fetal rat liver cells were fractionated according to the expression of these molecules. Then, cells in each cell subpopulation were sorted and transplanted into the retrorsine-treated adult rats with two-third hepatectomy. At 9 months post transplant, frequency of liver-repopulation was examined by qualitative and quantitative analyses. When we transplanted c-Met+ CD45- sorted cells, many donor-derived cells formed colonies that included mature hepatocytes expressing albumin and containing abundant glycogen in their cytoplasm. In contrast, c-Met- cells and CD45+ cells could not repopulate damaged recipient livers. High enrichment of liver-repopulating cells was conducted by sorting of c-Met+ cells from the developing rat liver. This result suggests that c-Met/HGF interaction plays a crucial role for stem cell growth, differentiation, and self-renewal in rat liver organogenesis. Since the c-Met is also expressed in the fetal mouse-derived hepatic stem cells, this molecule could be expected to be an essential marker for such cell population in the various animal strains, including human.

Transcriptional programming during cell wall maturation in the expanding Arabidopsis stem.

PubMed

Hall, Hardy; Ellis, Brian

2013-01-25

Plant cell walls are complex dynamic structures that play a vital role in coordinating the directional growth of plant tissues. The rapid elongation of the inflorescence stem in the model plant Arabidopsis thaliana is accompanied by radical changes in cell wall structure and chemistry, but analysis of the underlying mechanisms and identification of the genes that are involved has been hampered by difficulties in accurately sampling discrete developmental states along the developing stem. By creating stem growth kinematic profiles for individual expanding Arabidopsis stems we have been able to harvest and pool developmentally-matched tissue samples, and to use these for comparative analysis of global transcript profiles at four distinct phases of stem growth: the period of elongation rate increase, the point of maximum growth rate, the point of stem growth cessation and the fully matured stem. The resulting profiles identify numerous genes whose expression is affected as the stem tissues pass through these defined growth transitions, including both novel loci and genes identified in earlier studies. Of particular note is the preponderance of highly active genes associated with secondary cell wall deposition in the region of stem growth cessation, and of genes associated with defence and stress responses in the fully mature stem. The use of growth kinematic profiling to create tissue samples that are accurately positioned along the expansion growth continuum of Arabidopsis inflorescence stems establishes a new standard for transcript profiling analyses of such tissues. The resulting expression profiles identify a substantial number of genes whose expression is correlated for the first time with rapid cell wall extension and subsequent fortification, and thus provide an important new resource for plant biologists interested in gene discovery related to plant biomass accumulation.

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

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

Characterization of human skeletal stem and bone cell populations using dielectrophoresis.

PubMed

Ismail, A; Hughes, M P; Mulhall, H J; Oreffo, R O C; Labeed, F H

2015-02-01

Dielectrophoresis (DEP) is a non-invasive cell analysis method that uses differences in electrical properties between particles and surrounding medium to determine a unique set of cellular properties that can be used as a basis for cell separation. Cell-based therapies using skeletal stem cells are currently one of the most promising areas for treating a variety of skeletal and muscular disorders. However, identifying and sorting these cells remains a challenge in the absence of unique skeletal stem cell markers. DEP provides an ideal method for identifying subsets of cells without the need for markers by using their dielectric properties. This study used a 3D dielectrophoretic well chip device to determine the dielectric characteristics of two osteosarcoma cell lines (MG-63 and SAOS-2) and an immunoselected enriched skeletal stem cell fraction (STRO-1 positive cell) of human bone marrow. Skeletal cells were exposed to a series of different frequencies to induce dielectrophoretic cell movement, and a model was developed to generate the membrane and cytoplasmic properties of the cell populations. Differences were observed in the dielectric properties of MG-63, SAOS-2 and STRO-1 enriched skeletal populations, which could potentially be used to sort cells in mixed populations. This study provide evidence of the ability to characterize different human skeletal stem and mature cell populations, and acts as a proof-of-concept that dielectrophoresis can be exploited to detect, isolate and separate skeletal cell populations from heterogeneous bone marrow cell populations. Copyright © 2012 John Wiley & Sons, Ltd.

Silencing PRDM14 expression by an innovative RNAi therapy inhibits stemness, tumorigenicity, and metastasis of breast cancer

PubMed Central

Taniguchi, Hiroaki; Hoshino, Daisuke; Moriya, Chiharu; Zembutsu, Hitoshi; Nishiyama, Nobuhiro; Yamamoto, Hiroyuki; Kataoka, Kazunori; Imai, Kohzoh

2017-01-01

PR domain zinc finger protein 14 (PRDM14) maintains stemness in embryonic stem cells via epigenetic mechanisms. Although PRDM14 is elevated in several cancers, it is unclear if and how PRDM14 confers stem cell-like properties and epigenetic changes to cancer cells. Here, we examined the phenotypic characteristics and epigenetic and gene expression profiles of cancer cells that differentially express PRDM14, and assessed the potential of PRDM14-targeted cancer therapy. PRDM14 expression was markedly increased in many different cancer types and correlated with poor survival of breast cancer patients. PRDM14 conferred stem cell-like phenotypes to cancer cells and regulated the expression of genes involved in cancer stemness, metastasis, and chemoresistance. PRDM14 also reduced the methylation of proto-oncogene and stemness gene promoters and PRDM14-binding regions were primarily occupied by histone H3 Lys-4 trimethylation (H3K4me3), both of which are positively correlated with gene expression. Moreover, strong PRDM14 binding sites coincided with promoters containing both H3K4me3 and H3K27me3 histone marks. Using calcium phosphate hybrid micelles as an RNAi delivery system, silencing of PRDM14 expression by chimera RNAi reduced tumor size and metastasis in vivo without causing adverse effects. Conditional loss of PRDM14 function also improved survival of MMTV-Wnt-1 transgenic mice, a spontaneous model of murine breast cancer. Our findings suggest that PRDM14 inhibition may be an effective and novel therapy for cancer stem cells. PMID:28423353

Stem/Progenitor Cell–Mediated De Novo Regeneration of Dental Pulp with Newly Deposited Continuous Layer of Dentin in an In Vivo Model

PubMed Central

Yamaza, Takayoshi; Shea, Lonnie D.; Djouad, Farida; Kuhn, Nastaran Z.; Tuan, Rocky S.; Shi, Songtao

2010-01-01

The ultimate goal of this study is to regenerate lost dental pulp and dentin via stem/progenitor cell–based approaches and tissue engineering technologies. In this study, we tested the possibility of regenerating vascularized human dental pulp in emptied root canal space and producing new dentin on existing dentinal walls using a stem/progenitor cell–mediated approach with a human root fragment and an immunocompromised mouse model. Stem/progenitor cells from apical papilla and dental pulp stem cells were isolated, characterized, seeded onto synthetic scaffolds consisting of poly-D,L-lactide/glycolide, inserted into the tooth fragments, and transplanted into mice. Our results showed that the root canal space was filled entirely by a pulp-like tissue with well-established vascularity. In addition, a continuous layer of dentin-like tissue was deposited onto the canal dentinal wall. This dentin-like structure appeared to be produced by a layer of newly formed odontoblast-like cells expressing dentin sialophosphoprotein, bone sialoprotein, alkaline phosphatase, and CD105. The cells in regenerated pulp-like tissue reacted positively to anti-human mitochondria antibodies, indicating their human origin. This study provides the first evidence showing that pulp-like tissue can be regenerated de novo in emptied root canal space by stem cells from apical papilla and dental pulp stem cells that give rise to odontoblast-like cells producing dentin-like tissue on existing dentinal walls. PMID:19737072

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

Isolation and Characterization of Human Anterior Cruciate Ligament-Derived Vascular Stem Cells

PubMed Central

Matsumoto, Tomoyuki; Ingham, Sheila M.; Mifune, Yutaka; Osawa, Aki; Logar, Alison; Usas, Arvydas; Kuroda, Ryosuke; Kurosaka, Masahiro; Fu, Freddie H.

2012-01-01

The anterior cruciate ligament (ACL) usually fails to heal after rupture mainly due to the inability of the cells within the ACL tissue to establish an adequate healing process, making graft reconstruction surgery a necessity. However, some reports have shown that there is a healing potential of ACL with primary suture repair. Although some reports showed the existence of mesenchymal stem cell-like cells in human ACL tissues, their origin still remains unclear. Recently, blood vessels have been reported to represent a rich supply of stem/progenitor cells with a characteristic expression of CD34 and CD146. In this study, we attempted to validate the hypothesis that CD34- and CD146-expressing vascular cells exist in hACL tissues, have a potential for multi-lineage differentiation, and are recruited to the rupture site to participate in the intrinsic healing of injured ACL. Immunohistochemistry and flow cytometry analysis of hACL tissues demonstrated that it contains significantly more CD34 and CD146-positive cells in the ACL ruptured site compared with the noninjured midsubstance. CD34+CD45− cells isolated from ACL ruptured site showed higher expansionary potentials than CD146+CD45− and CD34−CD146−CD45− cells, and displayed higher differentiation potentials into osteogenic, adipogenic, and angiogenic lineages than the other cell populations. Immunohistochemistry of fetal and adult hACL tissues demonstrated a higher number of CD34 and CD146-positive cells in the ACL septum region compared with the midsubstance. In conclusion, our findings suggest that the ACL septum region contains a population of vascular-derived stem cells that may contribute to ligament regeneration and repair at the site of rupture. PMID:21732814

Loss of quiescence and self-renewal capacity of hematopoietic stem cell in an in vitro leukemic niche.

PubMed

Vanegas, Natalia-Del Pilar; Vernot, Jean-Paul

2017-01-01

Leukemic and mesenchymal stem cells interact in the leukemic microenvironment and affect each other differently. This interplay has also important implications for the hematopoietic stem cell (HSC) biology and function. This study evaluated human HSC self-renewal potential and quiescence in an in vitro leukemic niche without leukemic cells. A leukemic niche was established by co-culturing mesenchymal stem cells with a fresh conditioned medium obtained from a leukemic (REH) cell line. After 3 days, the REH-conditioned medium was removed and freshly isolated CD34+ at a density of up to 100,000 cells/ml were added to the leukemic niche. CD34+ cell evaluations (cell cycle, self-renewal gene expression and migration capacity) were performed after 3 further days of co-culture. Additionally, we preliminary investigated the soluble factors present in the leukemic niche and their effect on the mesenchymal stem cells. Statistical significance was assessed by Student's t test or the nonparametric test Kolmogorov-Smirnov. By co-culturing normal mesenchymal stem cells with the REH-conditioned medium we showed that hematopoietic stem cells, normally in a quiescent state, enter cell cycle and proliferate. This loss of quiescence was accompanied by an increased expression of Ki-67 and c-Myc, two well-known cell proliferation-associated markers. Two central regulators of quiescence GATA2 and p53 were also down regulated. Importantly, two genes involved in HSC self-renewal, Klf4 and the histone-lysine N -methyltransferase enzyme Ezh2, were severely affected. On the contrary, c-Kit expression, the stem cell factor receptor, was upregulated in hematopoietic stem cells when compared to the normal niche. Interestingly, mesenchymal stem cells incubated with the REH-conditioned medium stopped growing, showed a flattened morphology with the appearance of small vacuoles, and importantly, became positive for the senescence-associated beta-galactosidase activity. Evaluation of the leukemic-conditioned medium showed increased IL-6 and IL-8, suggesting that these cytokines could be responsible for the observed changes. Our results showed that quiescence and self-renewal are severely affected in this leukemic niche. This in vitro leukemic niche, established without leukemic cells, will facilitate HSC gene expression evaluation and the development of therapeutic agents aimed to neutralize soluble factors and the cell signaling pathways involved in HSC alterations.

The development of human mast cells. An historical reappraisal

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

Ribatti, Domenico, E-mail: domenico.ribatti@uniba.it

2016-03-15

The understanding of mast cell (MC) differentiation is derived mainly from in vitro studies of different stages of stem and progenitor cells. The hematopoietic lineage development of human MCs is unique compared to other myeloid-derived cells. Human MCs originate from CD34{sup +}/CD117{sup +}/CD13{sup +}multipotent hematopoietic progenitors, which undergo transendothelial recruitment into peripheral tissues, where they complete differentiation. Stem cell factor (SCF) is a major chemotactic factor for MCs and their progenitors. SCF also elicits cell-cell and cell-substratum adhesion, facilitates the proliferation, and sustains the survival, differentiation, and maturation, of MCs. Because MC maturation is influenced by local microenvironmental factors, differentmore » MC phenotypes can develop in different tissues and organs. - Highlights: • Human mast cells originate from CD34/CD117/CD13 positive multipotent hematopoietic progenitors. • Stem cell factor is a major chemotactic factor for mast cells and their progenitors. • Different mast cell phenotypes can develop in different tissues and organs.« less

Adult neural stem cell cycling in vivo requires thyroid hormone and its alpha receptor.

PubMed

Lemkine, G F; Raj, A; Alfama, G; Turque, N; Hassani, Z; Alegria-Prévot, O; Samarut, J; Levi, G; Demeneix, B A

2005-05-01

Thyroid hormones (TH) are essential for brain development. However, information on if and how this key endocrine factor affects adult neurogenesis is fragmentary. We thus investigated the effects of TH on proliferation and apoptosis of stem cells in the subventricular zone (SVZ), as well as on migration of transgene-tagged neuroblasts out of the stem cell niche. Hypothyroidism significantly reduced all three of these processes, inhibiting generation of new cells. To determine the mechanisms relaying TH action in the SVZ, we analyzed which receptor was implicated and whether the effects were played out directly at the level of the stem cell population. The alpha TH receptor (TRalpha), but not TRbeta, was found to be expressed in nestin positive progenitor cells of the SVZ. Further, use of TRalpha mutant mice showed TRalpha to be required to maintain full proliferative activity. Finally, a direct TH transcriptional effect, not mediated through other cell populations, was revealed by targeted gene transfer to stem cells in vivo. Indeed, TH directly modulated transcription from the c-myc promoter reporter construct containing a functional TH response element containing TRE but not from a mutated TRE sequence. We conclude that liganded-TRalpha is critical for neurogenesis in the adult mammalian brain.

Ovarian stem cells are always accompanied by very small embryonic-like stem cells in adult mammalian ovary.

PubMed

Bhartiya, Deepa

2015-11-05

Existing dogma that a female is born with fixed number of eggs was challenged by the detection of stem cells in adult mammalian ovary. Data has accumulated in support of ovarian stem cells (OSCs) proliferation, maintenance in culture, formation of germ cell nests and differentiation into oocytes and primordial follicle assembly using different strategies. Flow cytometry analysis identified >8 μm OSCs which are DDX1 positive and are considered equivalent to spermatogonial stem cells (SSCs) in testis. Analysis of both ovarian and testicular smears obtained after enzymatic digestion has led to the identification of an additional stem cell population termed very small embryonic-like stem cells (VSELs). VSELs and OSCs/SSCs differ from each other in their size and OCT-4 expression. VSELs express pluripotent markers including nuclear OCT-4 whereas OSCs/SSCs express cytoplasmic OCT-4 suggesting a differentiated state. VSELs can be studied by flow cytometry as small sized cells which are LIN-/CD45-/Sca-1+. We have reported 0.02 ± 0.008, 0.03 ± 0.017 and 0.08 ± 0.03 % of total cells as VSELs in normal, chemoablated and after FSH treatment to chemoablated mouse ovary. VSELs have remained poorly studied till now because of their very small size and rare occurrence. Spinning cells obtained after enzymatic digestion of ovarian tissue at a speed of 1000G (rather than 1200 rpm) throughout processing allows reliable detection of the VSELs by flow cytometry. VSELs exist in aged, chemoablated and non-functional ovary and providing a healthy niche to support their function offers an interesting strategy to manage infertility.

Vaccine Therapy in Reducing the Frequency of Cytomegalovirus Events in Patients With Hematologic Malignancies Undergoing Donor Stem Cell Transplant

ClinicalTrials.gov

2017-12-15

Accelerated Phase Chronic Myelogenous Leukemia, BCR-ABL1 Positive; Adult Acute Lymphoblastic Leukemia in Remission; Adult Acute Myeloid Leukemia in Remission; Adult Hodgkin Lymphoma; Adult Non-Hodgkin Lymphoma; Chronic Phase Chronic Myelogenous Leukemia, BCR-ABL1 Positive; Cytomegaloviral Infection; Hematopoietic and Lymphoid Cell Neoplasm; HLA-A*0201 Positive Cells Present; Myelodysplastic Syndrome; Adult Lymphoblastic Lymphoma; Chronic Lymphocytic Leukemia; Myelofibrosis; Myeloproliferative Neoplasm

Successful treatment of post-transplant relapsed acute myeloid leukemia with FLT3 internal tandem duplication using the combination of induction chemotherapy, donor lymphocyte infusion, sorafenib and azacitidine. Report of three cases

PubMed Central

Campregher, Paulo Vidal; de Mattos, Vinicius Renan Pinto; Salvino, Marco Aurélio; Santos, Fabio Pires de Souza; Hamerschlak, Nelson

2017-01-01

ABSTRACT Acute myeloid leukemia is a hematopoietic stem cell neoplastic disease associated with high morbidity and mortality. The presence of FLT3 internal tandem duplication mutations leads to high rates of relapse and decreased overall survival. Patients with FLT3 internal tandem duplication are normally treated with hematopoietic stem cell transplantation in first complete remission. Nevertheless, the incidence of post-transplant relapse is considerable in this group of patients, and the management of this clinical condition is challenging. The report describes the outcomes of patients with FLT3 internal tandem duplication positive acute myeloid leukemia who relapsed after allogeneic hematopoietic stem cell transplantation and were treated with the combination of re-induction chemotherapy, donor lymphocyte infusion, sorafenib and azacitidine. Three cases are described and all patients achieved prolonged complete remission with the combined therapy. The combination of induction chemotherapy followed by donor lymphocyte infusion, and the maintenance with azacitidine and sorafenib can be effective approaches in the treatment of post-hematopoietic stem cell transplant and relapsed FLT3 internal tandem duplication positive acute myeloid leukemia patients. This strategy should be further explored in the context of clinical trials. PMID:28746590

IDH1R132H in Neural Stem Cells: Differentiation Impaired by Increased Apoptosis

PubMed Central

Rosiak, Kamila; Smolarz, Maciej; Stec, Wojciech J.; Peciak, Joanna; Grzela, Dawid; Winiecka-Klimek, Marta; Stoczynska-Fidelus, Ewelina; Krynska, Barbara; Piaskowski, Sylwester; Rieske, Piotr

2016-01-01

Background The high frequency of mutations in the isocitrate dehydrogenase 1 (IDH1) gene in diffuse gliomas indicates its importance in the process of gliomagenesis. These mutations result in loss of the normal function and acquisition of the neomorphic activity converting α-ketoglutarate to 2-hydroxyglutarate. This potential oncometabolite may induce the epigenetic changes, resulting in the deregulated expression of numerous genes, including those related to the differentiation process or cell survivability. Methods Neural stem cells were derived from human induced pluripotent stem cells following embryoid body formation. Neural stem cells transduced with mutant IDH1R132H, empty vector, non-transduced and overexpressing IDH1WT controls were differentiated into astrocytes and neurons in culture. The neuronal and astrocytic differentiation was determined by morphology and expression of lineage specific markers (MAP2, Synapsin I and GFAP) as determined by real-time PCR and immunocytochemical staining. Apoptosis was evaluated by real-time observation of Caspase-3 activation and measurement of PARP cleavage by Western Blot. Results Compared with control groups, cells expressing IDH1R132H retained an undifferentiated state and lacked morphological changes following stimulated differentiation. The significant inhibitory effect of IDH1R132H on neuronal and astrocytic differentiation was confirmed by immunocytochemical staining for markers of neural stem cells. Additionally, real-time PCR indicated suppressed expression of lineage markers. High percentage of apoptotic cells was detected within IDH1R132H-positive neural stem cells population and their derivatives, if compared to normal neural stem cells and their derivatives. The analysis of PARP and Caspase-3 activity confirmed apoptosis sensitivity in mutant protein-expressing neural cells. Conclusions Our study demonstrates that expression of IDH1R132H increases apoptosis susceptibility of neural stem cells and their derivatives. Robust apoptosis causes differentiation deficiency of IDH1R132H-expressing cells. PMID:27145078

Clonal origin of Epstein-Barr virus (EBV)-infected T/NK-cell subpopulations in EBV-positive T/NK-cell lymphoproliferative disorders of childhood.

PubMed

Ohga, Shouichi; Ishimura, Masataka; Yoshimoto, Goichi; Miyamoto, Toshihiro; Takada, Hidetoshi; Tanaka, Tamami; Ohshima, Koichi; Ogawa, Yoshiyasu; Imadome, Ken-Ichi; Abe, Yasunobu; Akashi, Koichi; Hara, Toshiro

2011-05-01

In Japan, chronic active Epstein-Barr virus infection (CAEBV) may manifest with infection of T-cells or NK-cells, clonal lymphoid proliferations, and overt lymphoid malignancy. These EBV-positive lymphoproliferative disorders (EBV(+)LPD) of childhood are related to, but distinct from the infectious mononucleosis-like CAEBV seen in Western populations. The clonal nature of viral infection within lymphoid subsets of patients with EBV(+)LPD of childhood is not well described. Viral distribution and clonotype were assessed within T-cell subsets, NK-cells, and CD34(+)stem cells following high purity cell sorting. Six Japanese patients with EBV(+)LPD of childhood (3 T-cell LPD and 3 NK-cell LPD) were recruited. Prior to immunochemotherapy, viral loads and clonal analyses of T-cell subsets, NK-cells, and CD34(+)stem cells were studied by high-accuracy cell sorting (>99.5%), Southern blotting and real-time polymerase chain reaction. Patient 1 had a monoclonal proliferation of EBV-infected γδT-cells and carried a lower copy number of EBV in αβT-cells. Patients 2 and 3 had clonal expansions of EBV-infected CD4(+)T-cells, and lower EBV load in NK-cells. Patients 4, 5 and 6 had EBV(+)NK-cell expansions with higher EBV load than T-cells. EBV-terminal repeats were determined as clonal bands in the minor targeted populations of 5 patients. The size of terminal repeats indicated the same clonotype in minor subsets as in the major subsets of four patients. EBV was not, however, detected in the bone marrow-derived CD34(+)stem cells of patients. A single EBV clonotype may infect multiple NK-cell and T-cell subsets of patients with EBV(+)LPD of childhood. CD34(+)stem cells are spared, suggesting infection of more differentiated elements. Copyright © 2011 Elsevier B.V. All rights reserved.

Rapidly cycling Lgr5+ stem cells are exquisitely sensitive to extrinsic dietary factors that modulate colon cancer risk.

PubMed

Kim, Eunjoo; Davidson, Laurie A; Zoh, Roger S; Hensel, Martha E; Salinas, Michael L; Patil, Bhimanagouda S; Jayaprakasha, Guddadarangavvanahally K; Callaway, Evelyn S; Allred, Clinton D; Turner, Nancy D; Weeks, Brad R; Chapkin, Robert S

2016-11-10

The majority of colon tumors are driven by aberrant Wnt signaling in intestinal stem cells, which mediates an efficient route toward initiating intestinal cancer. Natural lipophilic polyphenols and long-chain polyunsaturated fatty acids (PUFAs) generally suppress Wnt- and NF-κB- (nuclear factor-κ light-chain enhancer of activated B-cell) related pathways. However, the effects of these extrinsic agents on colonic leucine-rich repeat-containing G-protein-coupled receptor 5-positive (Lgr5 + ) stem cells, the cells of origin of colon cancer, have not been documented to date. Therefore, we examined the effect of n-3 PUFA and polyphenol (curcumin) combination on Lgr5 + stem cells during tumor initiation and progression in the colon compared with an n-6 PUFA-enriched control diet. Lgr5-EGFP-IRES- creERT2 knock-in mice were fed diets containing n-6 PUFA (control), n-3 PUFA, n-6 PUFA+curcumin or n-3 PUFA+curcumin for 3 weeks, followed by 6 azoxymethane (AOM) injections, and terminated 17 weeks after the last injection. To further elucidate the effects of the dietary bioactives at the tumor initiation stage, Lgr5 + stem cells were also assessed at 12 and 24 h post AOM injection. Only n-3 PUFA+curcumin feeding reduced nuclear β-catenin in aberrant crypt foci (by threefold) compared with control at the progression time point. n-3 PUFA+curcumin synergistically increased targeted apoptosis in DNA-damaged Lgr5 + stem cells by 4.5-fold compared with control at 12 h and maximally reduced damaged Lgr5 + stem cells at 24 h, down to the level observed in saline-treated mice. Finally, RNAseq analysis indicated that p53 signaling in Lgr5 + stem cells from mice exposed to AOM was uniquely upregulated only following n-3 PUFA+curcumin cotreatment. These novel findings demonstrate that Lgr5 + stem cells are uniquely responsive to external dietary cues following the induction of DNA damage, providing a therapeutic strategy for eliminating damaged Lgr5 + stem cells to reduce colon cancer initiation.

Rapidly cycling Lgr5+ stem cells are exquisitely sensitive to extrinsic dietary factors that modulate colon cancer risk

PubMed Central

Kim, Eunjoo; Davidson, Laurie A; Zoh, Roger S; Hensel, Martha E; Salinas, Michael L; Patil, Bhimanagouda S; Jayaprakasha, Guddadarangavvanahally K; Callaway, Evelyn S; Allred, Clinton D; Turner, Nancy D; Weeks, Brad R; Chapkin, Robert S

2016-01-01

The majority of colon tumors are driven by aberrant Wnt signaling in intestinal stem cells, which mediates an efficient route toward initiating intestinal cancer. Natural lipophilic polyphenols and long-chain polyunsaturated fatty acids (PUFAs) generally suppress Wnt- and NF-κB- (nuclear factor-κ light-chain enhancer of activated B-cell) related pathways. However, the effects of these extrinsic agents on colonic leucine-rich repeat-containing G-protein-coupled receptor 5-positive (Lgr5+) stem cells, the cells of origin of colon cancer, have not been documented to date. Therefore, we examined the effect of n-3 PUFA and polyphenol (curcumin) combination on Lgr5+ stem cells during tumor initiation and progression in the colon compared with an n-6 PUFA-enriched control diet. Lgr5-EGFP-IRES-creERT2 knock-in mice were fed diets containing n-6 PUFA (control), n-3 PUFA, n-6 PUFA+curcumin or n-3 PUFA+curcumin for 3 weeks, followed by 6 azoxymethane (AOM) injections, and terminated 17 weeks after the last injection. To further elucidate the effects of the dietary bioactives at the tumor initiation stage, Lgr5+ stem cells were also assessed at 12 and 24 h post AOM injection. Only n-3 PUFA+curcumin feeding reduced nuclear β-catenin in aberrant crypt foci (by threefold) compared with control at the progression time point. n-3 PUFA+curcumin synergistically increased targeted apoptosis in DNA-damaged Lgr5+ stem cells by 4.5-fold compared with control at 12 h and maximally reduced damaged Lgr5+ stem cells at 24 h, down to the level observed in saline-treated mice. Finally, RNAseq analysis indicated that p53 signaling in Lgr5+ stem cells from mice exposed to AOM was uniquely upregulated only following n-3 PUFA+curcumin cotreatment. These novel findings demonstrate that Lgr5+ stem cells are uniquely responsive to external dietary cues following the induction of DNA damage, providing a therapeutic strategy for eliminating damaged Lgr5+ stem cells to reduce colon cancer initiation. PMID:27831561

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.

Responsibilities of Health Care Professionals in Counseling and Educating Patients With Incurable Neurological Diseases Regarding "Stem Cell Tourism": Caveat Emptor.

PubMed

Bowman, Michelle; Racke, Michael; Kissel, John; Imitola, Jaime

2015-11-01

"Stem cell tourism" is a rising Internet-based industry that aims to offer unproven procedures to patients with incurable diseases. This unregulated activity is reaching the neurologist's office as well as across the world, as patients request information or clearance for such procedures. Herein, we posit the need for medical societies and licensing boards to bring this issue to the forefront of neurology because it has the potential to affect patient care with risk of morbidity and mortality, as well as to undermine public confidence in legitimate stem cell research for incurable neurological diseases such as multiple sclerosis and amyotrophic lateral sclerosis.

Epithelial Cell Rests of Malassez Contain Unique Stem Cell Populations Capable of Undergoing Epithelial–Mesenchymal Transition

PubMed Central

Xiong, Jimin; Mrozik, Krzysztof; Gronthos, Stan

2012-01-01

The epithelial cell rests of Malassez (ERM) are odontogenic epithelial cells located within the periodontal ligament matrix. While their function is unknown, they may support tissue homeostasis and maintain periodontal ligament space or even contribute to periodontal regeneration. We investigated the notion that ERM contain a subpopulation of stem cells that could undergo epithelial–mesenchymal transition and differentiate into mesenchymal stem-like cells with multilineage potential. For this purpose, ERM collected from ovine incisors were subjected to different inductive conditions in vitro, previously developed for the characterization of bone marrow mesenchymal stromal/stem cells (BMSC). We found that ex vivo-expanded ERM expressed both epithelial (cytokeratin-8, E-cadherin, and epithelial membrane protein-1) and BMSC markers (CD44, CD29, and heat shock protein-90β). Integrin α6/CD49f could be used for the enrichment of clonogenic cell clusters [colony-forming units-epithelial cells (CFU-Epi)]. Integrin α6/CD49f-positive-selected epithelial cells demonstrated over 50- and 7-fold greater CFU-Epi than integrin α6/CD49f-negative cells and unfractionated cells, respectively. Importantly, ERM demonstrated stem cell-like properties in their differentiation capacity to form bone, fat, cartilage, and neural cells in vitro. When transplanted into immunocompromised mice, ERM generated bone, cementum-like and Sharpey's fiber-like structures. Additionally, gene expression studies showed that osteogenic induction of ERM triggered an epithelial–mesenchymal transition. In conclusion, ERM are unusual cells that display the morphological and phenotypic characteristics of ectoderm-derived epithelial cells; however, they also have the capacity to differentiate into a mesenchymal phenotype and thus represent a unique stem cell population within the periodontal ligament. PMID:22122577

Alginate/hyaluronic acid hydrogel delivery system characteristics regulate the differentiation of periodontal ligament stem cells toward chondrogenic lineage.

PubMed

Ansari, Sahar; Diniz, Ivana M; Chen, Chider; Aghaloo, Tara; Wu, Benjamin M; Shi, Songtao; Moshaverinia, Alireza

2017-09-15

Cartilage tissue regeneration often presents a challenging clinical situation. Recently, it has been shown that Periodontal Ligament Stem Cells (PDLSCs) possess high chondrogenic differentiation capacity. In this study, we developed a stem cell delivery system based on alginate/hyaluronic acid (HA) loaded with TGF-β1 ligand, encapsulating PDLSCs; and investigated the chondrogenic differentiation of encapsulated cells in alginate/HA hydrogel microspheres in vitro and in vivo. The results showed that PDLSCs, as well as human bone marrow mesenchymal stem cells (hBMMSCs), as the positive control, were stained positive for both toluidine blue and alcian blue staining, while exhibiting high levels of gene expression related to chondrogenesis (Col II, Aggrecan and Sox-9), as assessed via qPCR. The quantitative PCR analyses exhibited that the chondrogenic differentiation of encapsulated MSCs can be regulated by the modulus of elasticity of hydrogel delivery system, confirming the vital role of the microenvironment, and the presence of inductive signals for viability and differentiation of MSCs. In vivo, histological and immunofluorescence staining for chondrogenic specific protein markers confirmed ectopic cartilage-like tissue regeneration inside transplanted hydrogels. PDLSCs presented significantly greater capability for chondrogenic differentiation than hBMMSCs (P < 0.05). Altogether, our findings confirmed that alginate/HA hydrogels encapsulating PDLSCs are a promising candidate for cartilage regeneration.

SHBG Is an Important Factor in Stemness Induction of Cells by DHT In Vitro and Associated with Poor Clinical Features of Prostate Carcinomas

PubMed Central

Ma, Yuanyuan; Liang, Dongming; Liu, Jian; Wen, Jian-Guo; Servoll, Einar; Waaler, Gudmund; Sæter, Thorstein; Axcrona, Karol; Vlatkovic, Ljiljana; Axcrona, Ulrika; Paus, Elisabeth; Yang, Yue; Zhang, Zhiqian; Kvalheim, Gunnar; Nesland, Jahn M.; Suo, Zhenhe

2013-01-01

Androgen plays a vital role in prostate cancer development. However, it is not clear whether androgens influence stem-like properties of prostate cancer, a feature important for prostate cancer progression. In this study, we show that upon DHT treatment in vitro, prostate cancer cell lines LNCaP and PC-3 were revealed with higher clonogenic potential and higher expression levels of stemness related factors CD44, CD90, Oct3/4 and Nanog. Moreover, sex hormone binding globulin (SHBG) was also simultaneously upregulated in these cells. When the SHBG gene was blocked by SHBG siRNA knock-down, the induction of Oct3/4, Nanog, CD44 and CD90 by DHT was also correspondingly blocked in these cells. Immunohistochemical evaluation of clinical samples disclosed weakly positive, and areas negative for SHBG expression in the benign prostate tissues, while most of the prostate carcinomas were strongly positive for SHBG. In addition, higher levels of SHBG expression were significantly associated with higher Gleason score, more seminal vesicle invasions and lymph node metastases. Collectively, our results show a role of SHBG in upregulating stemness of prostate cancer cells upon DHT exposure in vitro, and SHBG expression in prostate cancer samples is significantly associated with poor clinicopathological features, indicating a role of SHBG in prostate cancer progression. PMID:23936228

Metabolic analysis of radioresistant medulloblastoma stem-like clones and potential therapeutic targets.

PubMed

Sun, Lue; Moritake, Takashi; Ito, Kazuya; Matsumoto, Yoshitaka; Yasui, Hironobu; Nakagawa, Hidehiko; Hirayama, Aki; Inanami, Osamu; Tsuboi, Koji

2017-01-01

Medulloblastoma is a fatal brain tumor in children, primarily due to the presence of treatment-resistant medulloblastoma stem cells. The energy metabolic pathway is a potential target of cancer therapy because it is often different between cancer cells and normal cells. However, the metabolic properties of medulloblastoma stem cells, and whether specific metabolic pathways are essential for sustaining their stem cell-like phenotype and radioresistance, remain unclear. We have established radioresistant medulloblastoma stem-like clones (rMSLCs) by irradiation of the human medulloblastoma cell line ONS-76. Here, we assessed reactive oxygen species (ROS) production, mitochondria function, oxygen consumption rate (OCR), energy state, and metabolites of glycolysis and tricarboxylic acid cycle in rMSLCs and parental cells. rMSLCs showed higher lactate production and lower oxygen consumption rate than parental cells. Additionally, rMSLCs had low mitochondria mass, low endogenous ROS production, and existed in a low-energy state. Treatment with the metabolic modifier dichloroacetate (DCA) resulted in mitochondria dysfunction, glycolysis inhibition, elongated mitochondria morphology, and increased ROS production. DCA also increased radiosensitivity by suppression of the DNA repair capacity through nuclear oxidization and accelerated the generation of acetyl CoA to compensate for the lack of ATP. Moreover, treatment with DCA decreased cancer stem cell-like characters (e.g., CD133 positivity and sphere-forming ability) in rMSLCs. Together, our findings provide insights into the specific metabolism of rMSLCs and illuminate potential metabolic targets that might be exploited for therapeutic benefit in medulloblastoma.

Human blood and marrow side population stem cell and Stro-1 positive bone marrow stromal cell numbers decline with age, with an increase in quality of surviving stem cells: Correlation with cytokines

PubMed Central

Brusnahan, S.K.; McGuire, T.R.; Jackson, J.D.; Lane, J.T.; Garvin, K.L.; O’Kane, B.J.; Berger, A.M.; Tuljapurkar, S.R.; Kessinger, M.A.; Sharp, J.G.

2010-01-01

Hematological deficiencies increase with aging leading to anemias, reduced hematopoietic stress responses and myelodysplasias. This study tested the hypothesis that side population hematopoietic stem cells (SP-HSC) would decrease with aging, correlating with IGF-1 and IL-6 levels and increases in bone marrow fat. Marrow was obtained from the femoral head and trochanteric region of the femur at surgery for total hip replacement (N = 100). Whole trabecular marrow samples were ground in a sterile mortar and pestle and cellularity and fat content determined. Marrow and blood mononuclear cells were stained with Hoechst dye and the SP-HSC profiles acquired. Marrow stromal cells (MSC) were enumerated flow cytometrically employing the Stro-1 antibody, and clonally in the colony forming unit fibroblast (CFU-F) assay. Plasma levels of IGF-1 (ng/ml) and IL-6 (pg/ml) were measured by ELISA. SP-HSC in blood and bone marrow decreased with age but the quality of the surviving stem cells increased. MSC decreased non-significantly. IGF-1 levels (mean = 30.7, SEM = 2) decreased and IL-6 levels (mean = 4.4, SEM = 1) increased with age as did marrow fat (mean = 1.2 mm fat/g, SEM = 0.04). There were no significant correlations between cytokine levels or fat and SP-HSC numbers. Stem cells appear to be progressively lost with aging and only the highest quality stem cells survive. PMID:21035480

Metabolic analysis of radioresistant medulloblastoma stem-like clones and potential therapeutic targets

PubMed Central

Sun, Lue; Moritake, Takashi; Ito, Kazuya; Matsumoto, Yoshitaka; Yasui, Hironobu; Nakagawa, Hidehiko; Hirayama, Aki; Inanami, Osamu; Tsuboi, Koji

2017-01-01

Medulloblastoma is a fatal brain tumor in children, primarily due to the presence of treatment-resistant medulloblastoma stem cells. The energy metabolic pathway is a potential target of cancer therapy because it is often different between cancer cells and normal cells. However, the metabolic properties of medulloblastoma stem cells, and whether specific metabolic pathways are essential for sustaining their stem cell-like phenotype and radioresistance, remain unclear. We have established radioresistant medulloblastoma stem-like clones (rMSLCs) by irradiation of the human medulloblastoma cell line ONS-76. Here, we assessed reactive oxygen species (ROS) production, mitochondria function, oxygen consumption rate (OCR), energy state, and metabolites of glycolysis and tricarboxylic acid cycle in rMSLCs and parental cells. rMSLCs showed higher lactate production and lower oxygen consumption rate than parental cells. Additionally, rMSLCs had low mitochondria mass, low endogenous ROS production, and existed in a low-energy state. Treatment with the metabolic modifier dichloroacetate (DCA) resulted in mitochondria dysfunction, glycolysis inhibition, elongated mitochondria morphology, and increased ROS production. DCA also increased radiosensitivity by suppression of the DNA repair capacity through nuclear oxidization and accelerated the generation of acetyl CoA to compensate for the lack of ATP. Moreover, treatment with DCA decreased cancer stem cell-like characters (e.g., CD133 positivity and sphere-forming ability) in rMSLCs. Together, our findings provide insights into the specific metabolism of rMSLCs and illuminate potential metabolic targets that might be exploited for therapeutic benefit in medulloblastoma. PMID:28426747

The role of exogenous neural stem cells transplantation in cerebral ischemic stroke.

PubMed

Chen, Lukui; Qiu, Rong; Li, Lushen; He, Dan; Lv, Haiqin; Wu, Xiaojing; Gu, Ning

2014-11-01

To observe the effects of neural stem cells (NSCs) transplantation in rats' striatum and subventricular zone (SVZ) in rat models of focal cerebral ischemia and reperfusion. Hippocampus was extracted from fetal rats with 14 days of gestation. Suspension culture was used to isolate and culture the rat's NSCs. A cerebral ischemia and reperfusion rat's model was made on the left side of the brain through occlusion of the left middle cerebral artery. Neurological signs were assessed by Zea Longa's five-grade scale, with scores 1, 2, and 3 used to determine the successful establishment of the rat's model. The NSCs were stereotaxically injected into the left striatum 24 hours after the successful rat's model was built. Rats were then randomly divided into 5 groups, namely, normal group, sham operation group, ischemia group, PBS transplantation group, and NSCs transplantation group, each of which was observed on day 3, day 7, and day 14. The ischemia-related neurological deficits were assessed by using a 7-point evaluation criterion. Forelimb injuries were evaluated in all rats using the foot-fault approach. Infarct size changes were observed through TTC staining and cell morphology and structure in the infarct region were investigated by Nissl staining. Apoptosis and apoptosis-positive cell counts were studied by Tunel assay. Expressions of double-labeling positive cells in the striatum and subventricular zone (SVZ) were observed by BrdU/NeuN and BrdU/GFAP fluorescent double-labeling method and the number of positive cells in the striatum and SVZ was counted. Results from the differently treated groups showed that right hemiplegia occurred in the ischemia group, PBS transplantation group, and NSCs transplantation group in varying degrees. Compared with the former two groups, there was least hemiplegia in the NSCs transplantation group. The TTC staining assay showed that rats in the NSCs transplantation group had smaller infarct volume than those from the PBS transplantation group. The Nissl dyeing showed that there was a large area of neuronal necrosis and apoptosis in the ischemia and PBS transplantation groups, and damage was mainly focused in the striatum. Degeneration and damage of nerve cells were significantly reduced in the NSCs transplantation group. The Tunel assay showed that the number of apoptosis-positive cells in the NSCs transplantation group was less than that in the PBS transplantation group at each time point. Double immunofluorescent labeling showed that the proliferation of endogenous neural stem cells began at the third day, reaching the peak at the 7th day, and was significantly reduced at the 14th day in the SVZ. The number of BrdU/NeuN increased significantly in the NSCs transplantation group compared to that in the PBS transplantation group (P < 0.05). The number of BrdU/GFAP decreased significantly in the NSCs transplantation group compared to that of PBS transplantation group (P < 0.05). The number of BrdU/GFAP-positive cells in the striatum was observed to be much more in the PBS transplantation group than in the NSCs transplantation group. Both neurological deficits and coordination capacity of rats with cerebral ischemia were significantly improved via transplantation of the neural stem cells. In conclusion, transplantation of neural stem cells can therefore possibly promote the differentiation of endogenous NSCs into neurons and reduce their differentiation towards glial cells. Transplantation of the neural stem cells may also change the ischemic microenvironment of striatum, possibly inhibiting the proliferation of glial cells.

Hematopoietic Stem Cell Transplant in Adolescent and Young Adults With Fanconi Anemia Is Feasible With Acceptable Toxicity, With Those Surviving 100 Days Posttransplant Having Excellent Outcomes.

PubMed

Alhuraiji, Ahmad; Alzahrani, Hazza; Al Mohareb, Fahad; Chaudhri, Naeem; Alsharif, Fahad; Mohamed, Said; Rasheed, Walid; Aldawsari, Ghuzayel; Ahmed, Syed Osman; Aljurf, Mahmoud

2016-12-01

Fanconi anemia is a congenital bone marrow failure syndrome that is associated with congenital anomalies and increased risk of cancer. Hematopoietic stem cell transplant is a potentially curative modality for bone marrow failure in Fanconi anemia patients. Here, we report our center's experience regarding adolescent and young adult patients with Fanconi anemia and hematopoietic stem cell transplant. We conducted a retrospective patient record analyses of patients who presented at our center from 1988 to 2014. We included patients greater than 14 years old with confirmed Fanconi anemia based on positive chromosome breakage study and who underwent hematopoietic stem cell transplant at our institution. Our study group comprised 12 patients with Fanconi anemia who underwent hematopoietic stem cell transplant at our institution. The median age was 20 years (range, 14-31 y) with a female predominance of 83%. Low-dose cyclophosphamide (20-80 mg/kg)-based conditioning regimens were used with different combinations that included fludarabine, antithymocyte globulin, or total body irradiation. All patients had HLA-matched sibling grafts. In all patients, stem cell source was the bone marrow. All patients showed engraftment. Four patients (33%) developed acute graft-versus-host disease. Three patients (25%) died early before day 100 after hematopoietic stem cell transplant due to infectious complications, with 1 patient having steroid refractory acute graft-versus-host disease. Overall survival was 75% at a median follow-up of 43 months. All patients who survived are well and remained transfusion independent without evidence of secondary malignancy. Our findings support the feasibility of reduced intensity conditioning allogeneic hematopoietic stem cell transplant in older and more heavily pretreated patients with Fanconi anemia, especially for those who are engrafted.

Cementogenic potential of multipotential mesenchymal stem cells purified from the human periodontal ligament.

PubMed

Torii, Daisuke; Konishi, Kiyoshi; Watanabe, Nobuyuki; Goto, Shinichi; Tsutsui, Takeki

2015-01-01

The periodontal ligament (PDL) consists of a group of specialized connective tissue fibers embedded in the alveolar bone and cementum that are believed to contain progenitors for mineralized tissue-forming cell lineages. These progenitors may contribute to regenerative cell therapy or tissue engineering methods aimed at recovery of tissue formation and functions lost in periodontal degenerative changes. Some reports using immortal clonal cell lines of cementoblasts, which are cells containing mineralized tissue-forming cell lineages, have shown that their phenotypic alteration and gene expression are associated with mineralization. Immortal, multipotential PDL-derived cell lines may be useful biological tools for evaluating differentiation-inducing agents. In this study, we confirmed the gene expression and mineralization potential of primary and immortal human PDL cells and characterized their immunophenotype. Following incubation with mineralization induction medium containing β-glycerophosphate, ascorbic acid, and dexamethasone, normal human PDL (Pel) cells and an immortal derivative line (Pelt) cells showed higher levels of mineralization compared with cells grown in normal growth medium. Both cell types were positive for putative surface antigens of mesenchymal cells (CD44, CD73, CD90, and CD105). They were also positive for stage-specific embryonic antigen-3, a marker of multipotential stem cells. Furthermore, PDL cells expressed cementum attachment protein and cementum protein 1 when cultured with recombinant human bone morphogenetic protein-2 or -7. The results suggest that normal and immortal human PDL cells contain multipotential mesenchymal stem cells with cementogenic potential.

The polarity protein Baz forms a platform for the centrosome orientation during asymmetric stem cell division in the Drosophila male germline.

PubMed

Inaba, Mayu; Venkei, Zsolt G; Yamashita, Yukiko M

2015-03-20

Many stem cells divide asymmetrically in order to balance self-renewal with differentiation. The essence of asymmetric cell division (ACD) is the polarization of cells and subsequent division, leading to unequal compartmentalization of cellular/extracellular components that confer distinct cell fates to daughter cells. Because precocious cell division before establishing cell polarity would lead to failure in ACD, these two processes must be tightly coupled; however, the underlying mechanism is poorly understood. In Drosophila male germline stem cells, ACD is prepared by stereotypical centrosome positioning. The centrosome orientation checkpoint (COC) further serves to ensure ACD by preventing mitosis upon centrosome misorientation. In this study, we show that Bazooka (Baz) provides a platform for the correct centrosome orientation and that Baz-centrosome association is the key event that is monitored by the COC. Our work provides a foundation for understanding how the correct cell polarity may be recognized by the cell to ensure productive ACD.

Smad2/3 Linker Phosphorylation Is a Possible Marker of Pancreatic Stem/Progenitor Cells in the Regenerative Phase of Acute Pancreatitis.

PubMed

Sakao, Masayuki; Sakaguchi, Yutaku; Suzuki, Ryo; Takahashi, Yu; Kishimoto, Masanobu; Fukui, Toshiro; Uchida, Kazushige; Nishio, Akiyoshi; Matsuzaki, Koichi; Okazaki, Kazuichi

The aims of this study are to characterize cell proliferation and differentiation during regeneration after pancreatitis and pancreatic buds during development to evaluate the role of Smad2/3, phosphorylated at the specific linker threonine residues (pSmad2/3L-Thr) in positive cells. Male C57BL/6 mice received hourly intraperitoneal injections of cerulein and were analyzed after induced pancreatitis. Pancreatitis-affected tissue sections and pancreatic buds were immunostained for pSmad2/3L-Thr, with other markers thought to be stem/progenitor markers of the pancreas. pSmad2/3L-Thr immunostaining-positive cells increased as the pancreatitis progressed. The expression of pSmad2/3L-Thr was seen in acinar cells and ductlike tubular complexes. These results suggest that pSmad2/3L-Thr is expressed during acinar-ductal metaplasia. Immunohistochemical colocalization of pSmad2/3L-Thr with Ki67 was never observed. pSmad2/3L-Thr-positive cells may remain in an undifferentiated state. During the pancreatic development process, pSmad2/3L-Thr was expressed as other markers. pSmad2/3L-Thr develops in duct structure of the undifferentiated cell population in the last part of viviparity that acinar structure is formed clearly. pSmad2/3L-Thr expression occurs during acinar-ductal metaplasia after pancreatitis and may represent the contribution of stem cells and/or progenitor cells to the differentiation of the pancreas.

High-temperature, high-pressure optical cell

NASA Technical Reports Server (NTRS)

Harris, R. P. (Inventor); Holland, L. R. (Inventor); Smith, R. E. (Inventor)

1986-01-01

The invention is an optical cell for containment of chemicals under conditions of high temperature and high pressure. The cell is formed of a vitreous silica tube, two optical windows comprising a vitreous silica rod inserted into the ends of a tube, and fused into position in the tube ends. Windows are spaced apart to form a cavity enclosed by the tube and the windows. A hole is drilled radially through the tube and into the cavity. Another vitreous silica tube is fused to the silica tube around the hole to form the stem, which is perpendicular to the long axis of the tube. The open end of the stem is used to load chemicals into the cavity. Then the stem may be sealed, and if desired, it may be shortened in order to reduce the volume of the cavity, which extends into the stem.

Dental mesenchymal stem cells encapsulated in an alginate hydrogel co-delivery microencapsulation system for cartilage regeneration.

PubMed

Moshaverinia, Alireza; Xu, Xingtian; Chen, Chider; Akiyama, Kentaro; Snead, Malcolm L; Shi, Songtao

2013-12-01

Dental-derived mesenchymal stem cells (MSCs) are promising candidates for cartilage regeneration, with a high capacity for chondrogenic differentiation. This property helps make dental MSCs an advantageous therapeutic option compared to current treatment modalities. The MSC delivery vehicle is the principal determinant for the success of MSC-mediated cartilage regeneration therapies. The objectives of this study were to: (1) develop a novel co-delivery system based on TGF-β1 loaded RGD-coupled alginate microspheres encapsulating periodontal ligament stem cells (PDLSCs) or gingival mesenchymal stem cells (GMSCs); and (2) investigate dental MSC viability and chondrogenic differentiation in alginate microspheres. The results revealed the sustained release of TGF-β1 from the alginate microspheres. After 4 weeks of chondrogenic differentiation in vitro, PDLSCs and GMSCs as well as human bone marrow mesenchymal stem cells (hBMMSCs) (as positive control) revealed chondrogenic gene expression markers (Col II and Sox-9) via qPCR, as well as matrix positively stained by Toluidine Blue and Safranin-O. In animal studies, ectopic cartilage tissue regeneration was observed inside and around the transplanted microspheres, confirmed by histochemical and immunofluorescent staining. Interestingly, PDLSCs showed more chondrogenesis than GMSCs and hBMMSCs (p<0.05). Taken together, these results suggest that RGD-modified alginate microencapsulating dental MSCs make a promising candidate for cartilage regeneration. Our results highlight the vital role played by the microenvironment, as well as value of presenting inductive signals for viability and differentiation of MSCs. Copyright © 2013 Acta Materialia Inc. All rights reserved.

Sphingosylphosphorylcholine promotes the differentiation of resident Sca-1 positive cardiac stem cells to cardiomyocytes through lipid raft/JNK/STAT3 and β-catenin signaling pathways.

PubMed

Li, Wenjing; Liu, Honghong; Liu, Pingping; Yin, Deling; Zhang, Shangli; Zhao, Jing

2016-07-01

Resident cardiac Sca-1-positive (+) stem cells may differentiate into cardiomyocytes to improve the function of damaged hearts. However, little is known about the inducers and molecular mechanisms underlying the myogenic conversion of Sca-1(+) stem cells. Here we report that sphingosylphosphorylcholine (SPC), a naturally occurring bioactive lipid, induces the myogenic conversion of Sca-1(+) stem cells, as evidenced by the increased expression of cardiac transcription factors (Nkx2.5 and GATA4), structural proteins (cardiac Troponin T), transcriptional enhancer (Mef2c) and GATA4 nucleus translocation. First, SPC activated JNK and STAT3, and the JNK inhibitor SP600125 or STAT3 inhibitor stattic impaired the SPC-induced expression of cardiac transcription factors and GATA4 nucleus translocation, which suggests that JNK and STAT3 participated in SPC-promoted cardiac differentiation. Moreover, STAT3 activation was inhibited by SP600125, whereas JNK was inhibited by β-cyclodextrin as a lipid raft breaker, which indicates a lipid raft/JNK/STAT3 pathway involved in SPC-induced myogenic transition. β-Catenin, degraded by activated GSK3β, was inhibited by SPC. Furthermore, GSK3β inhibitors weakened but the β-catenin inhibitor promoted SPC-induced differentiation. We found no crosstalk between the lipid raft/JNK/STAT3 and β-catenin pathway. Our study describes a lipid, SPC, as an endogenic inducer of myogenic conversion in Sca-1(+) stem cells with low toxicity and high efficiency for uptake. Copyright © 2016 Elsevier B.V. All rights reserved.

[Distribution of human enterovirus 71 in brainstem of infants with brain stem encephalitis and infection mechanism].

PubMed

Hao, Bo; Gao, Di; Tang, Da-Wei; Wang, Xiao-Guang; Liu, Shui-Ping; Kong, Xiao-Ping; Liu, Chao; Huang, Jing-Lu; Bi, Qi-Ming; Quan, Li; Luo, Bin

2012-04-01

To explore the mechanism that how human enterovirus 71 (EV71) invades the brainstem and how intercellular adhesion molecules-1 (ICAM-1) participates by analyzing the expression and distribution of human EV71, and ICAM-1 in brainstem of infants with brain stem encephalitis. Twenty-two brainstem of infants with brain stem encephalitis were collected as the experimental group and 10 brainstems of fatal congenital heart disease were selected as the control group. The sections with perivascular cuffings were selected to observe EV71-VP1 expression by immunohistochemistry method and ICAM-1 expression was detected for the sections with EV71-VP1 positive expression. The staining image analysis and statistics analysis were performed. The experiment and control groups were compared. (1) EV71-VP1 positive cells in the experimental group were mainly astrocytes in brainstem with nigger-brown particles, and the control group was negative. (2) ICAM-1 positive cells showed nigger-brown. The expression in inflammatory cells (around blood vessels of brain stem and in glial nodules) and gliocytes increased. The results showed statistical difference comparing with control group (P < 0.05). The brainstem encephalitis can be used to diagnose fatal EV71 infection in infants. EV71 can invade the brainstem via hematogenous route. ICAM-1 may play an important role in the pathogenic process.

Regional Differences in Stem and Transit Cell Proliferation and Apoptosis in the Terminal Ileum and Colon of Mice After 12 Gy

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

Gandara, Ricardo M.C.; Mahida, Yashwant R., E-mail: yash.mahida@nottingham.ac.uk; Potten, Christopher S.

2012-03-01

Purpose: The intestinal epithelium has a high rate of cell turnover, which is regulated by stem cells located near the base of crypts. We aimed to investigate stem cell-dependent characteristics of cell proliferation, apoptosis, and crypt size in terminal ileum and different regions of the colon. Methods and Materials: Mice were studied under steady-state conditions and after radiation-induced stem cell apoptosis. Percentage of proliferating or apoptotic cells at a particular cell position (cp) along the crypt axis was expressed as labeling or apoptotic index. Results: Under steady-state conditions: crypt size was smallest in the ascending colon. In contrast to othermore » regions of the colon, the distribution profile of proliferating cells in ascending colon showed some similarity to that in the terminal ileum. Postirradiation: apoptotic cells were prominent at the bottom of the crypt of mid- and descending colon but in the ascending colon, they were seen with similar frequency from cp 1 to 4. During regeneration, a constant proliferative capacity was seen above Paneth cells in the terminal ileum. In the ascending (but not mid- or descending) colon, the profile of proliferating cells over the first 4 days after irradiation showed a similarity to that in the terminal ileum. Conclusions: Profiles of proliferating epithelial cells (under steady-state conditions and postirradiation) and apoptotic cells (postirradiation) suggest similarities in the location of stem cells in the ascending colon and terminal ileum.« less

Regeneration of Articular Cartilage in Lizard Knee from Resident Stem/Progenitor Cells

PubMed Central

Alibardi, Lorenzo

2015-01-01

The epiphysis of femur and tibia in the lizard Podarcis muralis can extensively regenerate after injury. The process involves the articular cartilage and metaphyseal (growth) plate after damage. The secondary ossification center present between the articular cartilage and the growth plate is replaced by cartilaginous epiphyses after about one month of regeneration at high temperature. The present study analyzes the origin of the chondrogenic cells from putative stem cells located in the growing centers of the epiphyses. The study is carried out using immunocytochemistry for the detection of 5BrdU-labeled long retaining cells and for the localization of telomerase, an enzyme that indicates stemness. The observations show that putative stem cells retaining 5BrdU and positive for telomerase are present in the superficial articular cartilage and metaphyseal growth plate located in the epiphyses. This observation suggests that these areas represent stem cell niches lasting for most of the lifetime of lizards. In healthy long bones of adult lizards, the addition of new chondrocytes from the stem cells population in the articular cartilage and the metaphyseal growth plate likely allows for slow, continuous longitudinal growth. When the knee is injured in the adult lizard, new populations of chondrocytes actively producing chondroitin sulfate proteoglycan are derived from these stem cells to allow for the formation of completely new cartilaginous epiphyses, possibly anticipating the re-formation of secondary centers in later stages. The study suggests that in this lizard species, the regenerative ability of the epiphyses is a pre-adaptation to the regeneration of the articular cartilage. PMID:26340619

Photoactivation of Endogenous Latent Transforming Growth Factor–β1 Directs Dental Stem Cell Differentiation for Regeneration

PubMed Central

Arany, Praveen R.; Cho, Andrew; Hunt, Tristan D.; Sidhu, Gursimran; Shin, Kyungsup; Hahm, Eason; Huang, George X.; Weaver, James; Chen, Aaron Chih-Hao; Padwa, Bonnie L.; Hamblin, Michael R.; Barcellos-Hoff, Mary Helen; Kulkarni, Ashok B.; Mooney, David J.

2014-01-01

Rapid advancements in the field of stem cell biology have led to many current efforts to exploit stem cells as therapeutic agents in regenerative medicine. However, current ex vivo cell manipulations common to most regenerative approaches create a variety of technical and regulatory hurdles to their clinical translation, and even simpler approaches that use exogenous factors to differentiate tissue-resident stem cells carry significant off-target side effects. We show that non-ionizing, low-power laser (LPL) treatment can instead be used as a minimally invasive tool to activate an endogenous latent growth factor complex, transforming growth factor–β1 (TGF-β1), that subsequently differentiates host stem cells to promote tissue regeneration. LPL treatment induced reactive oxygen species (ROS) in a dose-dependent manner, which, in turn, activated latent TGF-β1 (LTGF-β1) via a specific methionine residue (at position 253 on LAP). Laser-activated TGF-β1 was capable of differentiating human dental stem cells in vitro. Further, an in vivo pulp capping model in rat teeth demonstrated significant increase in dentin regeneration after LPL treatment. These in vivo effects were abrogated in TGF-β receptor II (TGF-βRII) conditional knockout (DSPPCreTGF-βRIIfl/fl) mice or when wild-type mice were given a TGF-βRI inhibitor. These findings indicate a pivotal role for TGF-β in mediating LPL-induced dental tissue regeneration. More broadly, this work outlines a mechanistic basis for harnessing resident stem cells with a light-activated endogenous cue for clinical regenerative applications. PMID:24871130

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

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.

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

Early immunotherapy using autologous adult stem cells reversed the effect of anti-pancreatic islets in recently diagnosed type 1 diabetes mellitus: preliminary results.

PubMed

Mesples, Alejandro; Majeed, Nasir; Zhang, Yun; Hu, Xiang

2013-10-14

Bone marrow stem cell treatment has been proven a promising therapeutic strategy and showed significant results given the strong immune modulating properties. We have investigated the safety and efficacy of autologous bone marrow stem cell transplantation through liver puncture in two patients with recently diagnosed type 1 diabetes mellitus. The procedure was approved by the Institutional Ethics Committee. In 2011, in three young patients, type 1 diabetes mellitus diagnosis was confirmed, with the presence of positive antibodies and ketoacidosis. Two patients was treated with autologous bone marrow stem cell stimulated with filgrastim and transplantation, through liver puncture, as immune modulators. One patients was treated with conventional treatment and participate in this experiment as a control group. The families of the patients signed the informed consent. No specific statistical analysis was performed. The patients had less than 8 years old, diagnosis of type 1 diabetes for less than 60 days, body mass index less than 22 kg/m2, normal complete blood count, coagulation and renal function, no lesions in target organs, glycosylated hemoglobin (HbA1c) level less than 13.70%, c-peptide level less than 0.67 ng/ml, positive results of Islets Cells Antibody (ICA), Glutamic Acid Decarboxylase (GAD) and insulin antibody. In two patients treated, the follow up at 12 months showed negative value in ICA, GAD and anti insulin antibody levels, with an increased levels of c peptide and decreased levels of blood glucose and HbA1c. Treatment with autologous bone marrow stem cells is easy and effective as it reversed the production and effect of anti pancreatic islet antibody and significantly resulted in an increased c-peptide concentration.

Regulatory role of hexosamine biosynthetic pathway on hepatic cancer stem cell marker CD133 under low glucose conditions

NASA Astrophysics Data System (ADS)

Lin, Shu-Hai; Liu, Tengfei; Ming, Xiaoyan; Tang, Zhi; Fu, Li; Schmitt-Kopplin, Philippe; Kanawati, Basem; Guan, Xin-Yuan; Cai, Zongwei

2016-02-01

Cancer was hypothesized to be driven by cancer stem cells (CSCs), but the metabolic determinants of CSC-like phenotype still remain elusive. Here, we present that hexosamine biosynthetic pathway (HBP) at least in part rescues cancer cell fate with inactivation of glycolysis. Firstly, metabolomic analysis profiled cellular metabolome in CSCs of hepatocellular carcinoma using CD133 cell-surface marker. The metabolic signatures of CD133-positive subpopulation compared to CD133-negative cells highlighted HBP as one of the distinct metabolic pathways, prompting us to uncover the role of HBP in maintenance of CSC-like phenotype. To address this, CSC-like phenotypes and cell survival were investigated in cancer cells under low glucose conditions. As a result, HBP inhibitor azaserine reduced CD133-positive subpopulation and CD133 expression under high glucose condition. Furthermore, treatment of N-Acetylglucosamine in part restores CD133-positive subpopulation when either 2.5 mM glucose in culture media or glycolytic inhibitor 2-deoxy-D-glucose in HCC cell lines was applied, enhancing CD133 expression as well as promoting cancer cell survival. Together, HBP might be a key metabolic determinant in the functions of hepatic CSC marker CD133.

The effect of Bcr-Abl protein tyrosine kinase on maturation and proliferation of primitive haematopoietic cells.

PubMed Central

Buckle, A. M.; Mottram, R.; Pierce, A.; Lucas, G. S.; Russell, N.; Miyan, J. A.; Whetton, A. D.

2000-01-01

BACKGROUND: Chronic Myeloid Leukaemia (CML) is characterised by the chromosomal translocation resulting in expression of the Bcr-Abl protein tyrosine kinase (PTK) in early stem cells and their progeny. However the precise nature of Bcr-Abl effects in primitive CML stem cells remains a matter of active debate. MATERIALS AND METHODS: Extremely primitive Bcr-Abl fusion positive cells were purified from patients with CML using multiparameter flow cytometric analysis of CD34, Thy, and lineage marker (Lin) expression, plus rhodamine-123 (Rh-123) brightness. Progenitor cells of increasing maturity were examined for cycling status by flow cytometry and their proliferative status directly correlated with cell phenotype. The activation status of a key transcription factor, signal transducers and activators of transcription (STAT-5), was also analyzed by immunocytochemistry. RESULTS: The most primitive stem cells currently defined (CD34+Lin-Thy+ Rh-1231o) were present as a lower proportion of the stem cell compartment (CD34+Lin-) of CML patients at presentation than of normal individuals (2.3% +/- 0.4 compared with 5.1% +/- 0.6 respectively). Conversely there was a significantly higher proportion of the more mature cells (CD34+Lin-Thy-Rh-123 hi) in CML patients than in normal individuals (79.3 +/- 1.8 compared with 70.9 +/- 3.3). No primitive subpopulation of CML CD34+Lin- cells was cycling to a significantly greater degree than cells from normal donors, in fact, late progenitor cells (CD34+Lin+) were cycling significantly less in CML samples than normal samples. STAT5, however, was observed to be activated in CML cells. CONCLUSIONS: We conclude that no subpopulation of CML stem cells displays significantly increased cell cycling. Thus, increased cycling cannot be a direct consequence of Bcr-Abl PTK acquisition in highly enriched stem cells from patients with CML. In vivo CML need not be considered a disease of unbridled stem cell proliferation, but a subtle defect in the balance between self renewal and maturation. PMID:11126203

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.

Asialoglycoprotein receptor 1 is a specific cell-surface marker for isolating hepatocytes derived from human pluripotent stem cells.

PubMed

Peters, Derek T; Henderson, Christopher A; Warren, Curtis R; Friesen, Max; Xia, Fang; Becker, Caroline E; Musunuru, Kiran; Cowan, Chad A

2016-05-01

Hepatocyte-like cells (HLCs) are derived from human pluripotent stem cells (hPSCs) in vitro, but differentiation protocols commonly give rise to a heterogeneous mixture of cells. This variability confounds the evaluation of in vitro functional assays performed using HLCs. Increased differentiation efficiency and more accurate approximation of the in vivo hepatocyte gene expression profile would improve the utility of hPSCs. Towards this goal, we demonstrate the purification of a subpopulation of functional HLCs using the hepatocyte surface marker asialoglycoprotein receptor 1 (ASGR1). We analyzed the expression profile of ASGR1-positive cells by microarray, and tested their ability to perform mature hepatocyte functions (albumin and urea secretion, cytochrome activity). By these measures, ASGR1-positive HLCs are enriched for the gene expression profile and functional characteristics of primary hepatocytes compared with unsorted HLCs. We have demonstrated that ASGR1-positive sorting isolates a functional subpopulation of HLCs from among the heterogeneous cellular population produced by directed differentiation. © 2016. Published by The Company of Biologists Ltd.

Long Term Liver Engraftment of Functional Hepatocytes Obtained from Germline Cell-Derived Pluripotent Stem Cells

PubMed Central

Fagoonee, Sharmila; Famulari, Elvira Smeralda; Silengo, Lorenzo; Tolosano, Emanuela; Altruda, Fiorella

2015-01-01

One of the major hurdles in liver gene and cell therapy is availability of ex vivo-expanded hepatocytes. Pluripotent stem cells are an attractive alternative. Here, we show that hepatocyte precursors can be isolated from male germline cell-derived pluripotent stem cells (GPSCs) using the hepatoblast marker, Liv2, and induced to differentiate into hepatocytes in vitro. These cells expressed hepatic-specific genes and were functional as demonstrated by their ability to secrete albumin and produce urea. When transplanted in the liver parenchyma of partially hepatectomised mice, Liv2-sorted cells showed regional and heterogeneous engraftment in the injected lobe. Moreover, approximately 50% of Y chromosome-positive, GPSC-derived cells were found in the female livers, in the region of engraftment, even one month after cell injection. This is the first study showing that Liv2-sorted GPSCs-derived hepatocytes can undergo long lasting engraftment in the mouse liver. Thus, GPSCs might offer promise for regenerative medicine. PMID:26323094

Curcumin promotes autophagic survival of a subset of colon cancer stem cells, which are ablated by DCLK1-siRNA.

PubMed

Kantara, Carla; O'Connell, Malaney; Sarkar, Shubhashish; Moya, Stephanie; Ullrich, Robert; Singh, Pomila

2014-05-01

Curcumin is known to induce apoptosis of cancer cells by different mechanisms, but its effects on cancer stem cells (CSC) have been less investigated. Here, we report that curcumin promotes the survival of DCLK1-positive colon CSCs, potentially confounding application of its anticancer properties. At optimal concentrations, curcumin greatly reduced expression levels of stem cell markers (DCLK1/CD44/ALDHA1/Lgr5/Nanog) in three-dimensional spheroid cultures and tumor xenografts derived from colon cancer cells. However, curcumin unexpectedly induced proliferation and autophagic survival of a subset of DCLK1-positive CSCs. Spheroid cultures were disintegrated by curcumin in vitro but regrew within 30 to 40 days of treatment, suggesting a survival benefit from autophagy, permitting long-term persistence of colorectal cancer. Notably, RNA interference-mediated silencing of DCLK1 triggered apoptotic cell death of colon cancer cells in vitro and in vivo, and abolished colorectal cancer survival in response to curcumin; combination of DCLK1-siRNA and curcumin dramatically reversed CSC phenotype, contributing to attenuation of the growth of spheroid cultures and tumor xenografts. Taken together, our findings confirm a role of DCLK1 in colon CSCs and highlight DCLK1 as a target to enhance antitumor properties of curcumin. ©2014 AACR.

Characterization of p75{sup +} ectomesenchymal stem cells from rat embryonic facial process tissue

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

Wen, Xiujie; Liu, Luchuan; Deng, Manjing

2012-10-12

Highlights: Black-Right-Pointing-Pointer Ectomesenchymal stem cells (EMSCs) were found to migrate to rat facial processes at E11.5. Black-Right-Pointing-Pointer We successfully sorted p75NTR positive EMSCs (p75{sup +} EMSCs). Black-Right-Pointing-Pointer p75{sup +} EMSCs up to nine passages showed relative stable proliferative activity. Black-Right-Pointing-Pointer We examined the in vitro multilineage potential of p75{sup +} EMSCs. Black-Right-Pointing-Pointer p75{sup +}EMSCs provide an in vitro model for tooth morphogenesis. -- Abstract: Several populations of stem cells, including those from the dental pulp and periodontal ligament, have been isolated from different parts of the tooth and periodontium. The characteristics of such stem cells have been reported as well.more » However, as a common progenitor of these cells, ectomesenchymal stem cells (EMSCs), derived from the cranial neural crest have yet to be fully characterized. The aim of this study was to better understand the characteristics of EMSCs isolated from rat embryonic facial processes. Immunohistochemical staining showed that EMSCs had migrated to rat facial processes at E11.5, while the absence of epithelial invagination or tooth-like epithelium suggested that any epithelial-mesenchymal interactions were limited at this stage. The p75 neurotrophin receptor (p75NTR), a typical neural crest marker, was used to select p75NTR-positive EMSCs (p75{sup +} EMSCs), which were found to show a homogeneous fibroblast-like morphology and little change in the growth curve, proliferation capacity, and cell phenotype during cell passage. They also displayed the capacity to differentiate into diverse cell types under chemically defined conditions in vitro. p75{sup +} EMSCs proved to be homogeneous, stable in vitro and potentially capable of multiple lineages, suggesting their potential for application in dental or orofacial tissue engineering.« less

YKL-40 is differentially expressed in human embryonic stem cells and in cell progeny of the three germ layers.

PubMed

Brøchner, Christian B; Johansen, Julia S; Larsen, Lars A; Bak, Mads; Mikkelsen, Hanne B; Byskov, Anne Grete; Andersen, Claus Yding; Møllgård, Kjeld

2012-03-01

The secreted glycoprotein YKL-40 participates in cell differentiation, inflammation, and cancer progression. High YKL-40 expression is reported during early human development, but its functions are unknown. Six human embryonic stem cell (hESC) lines were cultured in an atmosphere of low or high oxygen tension, in culture medium with or without basic fibroblast growth factor, and on feeder layers comprising mouse embryonic fibroblasts or human foreskin fibroblasts to evaluate whether hESCs and their progeny produced YKL-40 and to characterize YKL-40 expression during differentiation. Secreted YKL-40 protein and YKL-40 mRNA expression were measured by enzyme-linked immunosorbent assay (ELISA) and quantitative RT-PCR. Serial-sectioned colonies were stained for YKL-40 protein and for pluripotent hESC (OCT4, NANOG) and germ layer (HNF-3β, PDX1, CD34, p63, nestin, PAX6) markers. Double-labeling showed YKL-40 expression in OCT4-positive hESCs, PAX6-positive neuroectodermal cells, and HNF-3β-positive endodermal cells. The differentiating progeny showed strong YKL-40 expression. Abrupt transition between YKL-40 and OCT4-positive hESCs and YKL-40-positive ecto- and neuroectodermal lineages was observed within the same epithelial-like layer. YKL-40-positive cells within deeper layers lacked contact with OCT4-positive cells. YKL-40 may be important in initial cell differentiation from hESCs toward ectoderm and neuroectoderm, with retained epithelial morphology, whereas later differentiation into endoderm and mesoderm involves a transition into the deeper layers of the colony.

NOS2 expression in glioma cell lines and glioma primary cell cultures: correlation with neurosphere generation and SOX-2 expression.

PubMed

Palumbo, Paola; Miconi, Gianfranca; Cinque, Benedetta; Lombardi, Francesca; La Torre, Cristina; Dehcordi, Soheila Raysi; Galzio, Renato; Cimini, Annamaria; Giordano, Antonio; Cifone, Maria Grazia

2017-04-11

Nitric oxide has been implicated in biology and progression of glioblastoma (GBM) being able to influence the cellular signal depending on the concentration and duration of cell exposure. NOS2 (inducible nitric oxide synthase) have been proposed as a component of molecular profile of several tumors, including glioma, one of the most aggressive primary brain tumor featuring local cancer stem cells responsible for enhanced resistance to therapies and for tumor recurrence. Here, we investigated the NOS2 mRNA expression by reverse transcription-PCR in human glioma primary cultures at several grade of malignancy and glioma stem cell (GSC) derived neurospheres. Glioma cell lines were used as positive controls both in terms of stemness marker expression that of capacity of generating neurospheres. NOS2 expression was detected at basal levels in cell lines and primary cultures and appeared significantly up-regulated in cultures kept in the specific medium for neurospheres. The immunofluorescence analysis of all cell cultures to evaluate the levels of SOX-2, a stemness marker aberrantly up-regulated in GBM, was also performed. The potential correlation between NOS2 expression and ability to generate neurospheres and between NOS2 and SOX-2 levels was also verified. The results show that the higher NOS2 expression is detected in all primary cultures able to arise neurosphere. A high and significant correlation between NOS2 expression and SOX-2 positive cells (%) in all cell cultures maintained in standard conditions has been observed. The results shed light on the potential relevance of NOS2 as a prognostic factor for glioma malignancy and recurrence.

Investigation of MACC1 Gene Expression in Head and Neck Cancer and Cancer Stem Cells.

PubMed

Evran, Ebru; Şahin, Hilal; Akbaş, Kübra; Çiğdem, Sadik; Gündüz, Esra

2016-12-01

By investigating the MACC1 gene (metastasis-associated in colon cancer 1) in cancer stem cells (CSC) resistant to chemotherapy and in cancer stem cells (CSC) resistant to chemotherapy and in cancer cells (CS) sensitive to chemotherapy we determineda steady expression in both types of cells in head and neck cancer. In conformity with the result we examined if this gene could be a competitor gene for chemotherapy. According to literature, the MACC1 gene shows a clear expression in head and neck cancer cells [1]. Here we examined MACC1 expression in CSC and investigated it as a possible biomarker. Our experiments were performed in the UT -SCC -74 in primary head and neck cancer cell line. We examined the MACC -1 gene expression by Real Time PCR from both isolated CSC and CS. Expression of MACC -1 gene of cancer stem cells showed an two-fold increase compared with cancer cells. Based on the positive expression of MACC1 in both CS and CSC, this gene may serve as a potential biomarker in head and neck cancer. By comparing the results of this study with the novel features of MACC1, two important hypotheses could be examined. The first hypothesis is that MACC1 is a possible transcripton factor in colon cancer, which influences a high expression of CSC in head and neck and affects the expression of three biomarkers of the CSC control group biomarkers. The second hypothesisis is that the positive expression of MACC1 in patients with a malignant prognosis of tongue cancer, which belongs to head and neck cancer types, operates a faster development of CSC to cancer cells.

Directing Induced Pluripotent Stem Cell Derived Neural Stem Cell Fate with a Three-Dimensional Biomimetic Hydrogel for Spinal Cord Injury Repair.

PubMed

Fan, Lei; Liu, Can; Chen, Xiuxing; Zou, Yan; Zhou, Zhengnan; Lin, Chenkai; Tan, Guoxin; Zhou, Lei; Ning, Chenyun; Wang, Qiyou

2018-05-30

Current treatment approaches for spinal cord injuries (SCIs) are mainly based on cellular transplantation. Induced pluripotent stem cells (iPSCs) without supply constraints and ethical concerns have emerged as a viable treatment option for repairing neurological disorders. However, the primarily limitations in the neuroregeneration field are uncontrolled cell differentiation, and low cell viability caused by the ischemic environment. The mechanical property of three-dimensional (3D) hydrogel can be easily controlled and shared similar characteristics with nerve tissue, thus promoting cell survival and controlled cell differentiation. We propose the combination of a 3D gelatin methacrylate (GelMA) hydrogel with iPSC-derived NSCs (iNSCs) to promote regeneration after SCI. In vitro, the iNSCs photoencapsulated in the 3D GelMA hydrogel survived and differentiated well, especially in lower-moduli hydrogels. More robust neurite outgrowth and more neuronal differentiation were detected in the soft hydrogel group. To further evaluate the in vivo neuronal regeneration effect of the GelMA hydrogels, a mouse spinal cord transection model was generated. We found that GelMA/iNSC implants significantly promoted functional recovery. Further histological analysis showed that the cavity areas were significantly reduced, and less collagen was deposited in the GelMA/iNSC group. Furthermore, the GelMA and iNSC combined transplantation decreased inflammation by reducing activated macrophages/microglia (CD68-positive cells). Additionally, GelMA/iNSC implantation showed striking therapeutic effects of inhibiting GFAP-positive cells and glial scar formation while simultaneously promoting axonal regeneration. Undoubtedly, use of this 3D hydrogel stem cell-loaded system is a promising therapeutic strategy for SCI repair.

Cnn dynamics drive centrosome size asymmetry to ensure daughter centriole retention in Drosophila neuroblasts.

PubMed

Conduit, Paul T; Raff, Jordan W

2010-12-21

Centrosomes comprise a pair of centrioles surrounded by an amorphous network of pericentriolar material (PCM). In certain stem cells, the two centrosomes differ in size, and this appears to be important for asymmetric cell division [1, 2]. In some cases, centrosome asymmetry is linked to centriole age because the older, mother centriole always organizes more PCM than the daughter centriole, thus ensuring that the mother centriole is always retained in the stem cell after cell division [3]. This has raised the possibility that an "immortal" mother centriole may help maintain stem cell fate [4, 5]. It is unclear, however, how centrosome size asymmetry is generated in stem cells. Here we provide compelling evidence that centrosome size asymmetry in Drosophila neuroblasts is generated by the differential regulation of Cnn incorporation into the PCM at mother and daughter centrioles. Shortly after centriole separation, mother and daughter centrioles organize similar amounts of PCM, but Cnn incorporation is then rapidly downregulated at the mother centriole, while it is maintained at the daughter centriole. This ensures that the daughter centriole maintains its PCM and so its position at the apical cortex. Thus, the daughter centriole, rather than an "immortal" mother centriole, is ultimately retained in these stem cells.

Airway epithelial stem cells and the pathophysiology of chronic obstructive pulmonary disease.

PubMed

Randell, Scott H

2006-11-01

Characteristic pathologic changes in chronic obstructive pulmonary disease (COPD) include an increased fractional volume of bronchiolar epithelial cells, fibrous thickening of the airway wall, and luminal inflammatory mucus exudates, which are positively correlated with airflow limitation and disease severity. The mechanisms driving general epithelial expansion, mucous secretory cell hyperplasia, and mucus accumulation must relate to the effects of initial toxic exposures on patterns of epithelial stem and progenitor cell proliferation and differentiation, eventually resulting in a self-perpetuating, and difficult to reverse, cycle of injury and repair. In this review, current concepts in stem cell biology and progenitor-progeny relationships related to COPD are discussed, focusing on the factors, pathways, and mechanisms leading to mucous secretory cell hyperplasia and mucus accumulation in the airways. A better understanding of alterations in airway epithelial phenotype in COPD will provide a logical basis for novel therapeutic approaches.

Dose-escalation study for the targeting of CD44v+ cancer stem cells by sulfasalazine in patients with advanced gastric cancer (EPOC1205).

PubMed

Shitara, Kohei; Doi, Toshihiko; Nagano, Osamu; Imamura, Chiyo K; Ozeki, Takeshi; Ishii, Yuya; Tsuchihashi, Kenji; Takahashi, Shunji; Nakajima, Takako E; Hironaka, Shuichi; Fukutani, Miki; Hasegawa, Hiromi; Nomura, Shogo; Sato, Akihiro; Einaga, Yasuaki; Kuwata, Takeshi; Saya, Hideyuki; Ohtsu, Atsushi

2017-03-01

Cancer stem cells (CSCs) have enhanced mechanisms of protection from oxidative stress. A variant form of CD44 (CD44v), a major CSC marker, was shown to interact with xCT, a subunit of cystine-glutamate transporter, which maintains high levels of intracellular reduced glutathione (GSH) which defend the cell against oxidative stress. Sulfasalazine (SSZ) is an inhibitor of xCT and was shown to suppress the survival of CD44v-positive stem-like cancer cells both in vitro and in vivo. To find the dose of SSZ which can safely reduce the population of CD44v-positive cells in tumors, a dose-escalation study in patients with advanced gastric cancer was conducted. SSZ was given four times daily by oral administration with 2 weeks as one cycle. Tumor biopsies were obtained before and after 14 days of administration of SSZ to evaluate expression of CD44v and the intratumoral level of GSH. Eleven patients were enrolled and received a dosage from 8 to 12 g/day. Safety was confirmed up to a dosage of 12 g/day, which was considered the maximum tolerated dose. Among the eight patients with CD44v-positive cells in their pretreatment biopsy samples, the CD44v-positive cancer cell population appeared to be reduced in the posttreatment biopsy tissues of four patients. Intratumoral GSH levels were also decreased in two patients, suggesting biological effectiveness of SSZ at 8 g/day or greater. This is the first study of SSZ as an xCT inhibitor for targeting CSCs. Reduction of the levels of CD44v-positive cells and GSH was observed in some patients, consistent with the mode of action of SSZ in CSCs.

Mule Regulates the Intestinal Stem Cell Niche via the Wnt Pathway and Targets EphB3 for Proteasomal and Lysosomal Degradation.

PubMed

Dominguez-Brauer, Carmen; Hao, Zhenyue; Elia, Andrew J; Fortin, Jérôme M; Nechanitzky, Robert; Brauer, Patrick M; Sheng, Yi; Mana, Miyeko D; Chio, Iok In Christine; Haight, Jillian; Pollett, Aaron; Cairns, Robert; Tworzyanski, Leanne; Inoue, Satoshi; Reardon, Colin; Marques, Ana; Silvester, Jennifer; Cox, Maureen A; Wakeham, Andrew; Yilmaz, Omer H; Sabatini, David M; van Es, Johan H; Clevers, Hans; Sato, Toshiro; Mak, Tak W

2016-08-04

The E3 ubiquitin ligase Mule is often overexpressed in human colorectal cancers, but its role in gut tumorigenesis is unknown. Here, we show in vivo that Mule controls murine intestinal stem and progenitor cell proliferation by modulating Wnt signaling via c-Myc. Mule also regulates protein levels of the receptor tyrosine kinase EphB3 by targeting it for proteasomal and lysosomal degradation. In the intestine, EphB/ephrinB interactions position cells along the crypt-villus axis and compartmentalize incipient colorectal tumors. Our study thus unveils an important new avenue by which Mule acts as an intestinal tumor suppressor by regulation of the intestinal stem cell niche. Copyright © 2016 Elsevier Inc. All rights reserved.

Study of muscle cell dedifferentiation after skeletal muscle injury of mice with a Cre-Lox system.

PubMed

Mu, Xiaodong; Peng, Hairong; Pan, Haiying; Huard, Johnny; Li, Yong

2011-02-03

Dedifferentiation of muscle cells in the tissue of mammals has yet to be observed. One of the challenges facing the study of skeletal muscle cell dedifferentiation is the availability of a reliable model that can confidentially distinguish differentiated cell populations of myotubes and non-fused mononuclear cells, including stem cells that can coexist within the population of cells being studied. In the current study, we created a Cre/Lox-β-galactosidase system, which can specifically tag differentiated multinuclear myotubes and myotube-generated mononuclear cells based on the activation of the marker gene, β-galactosidase. By using this system in an adult mouse model, we found that β-galactosidase positive mononuclear cells were generated from β-galactosidase positive multinuclear myofibers upon muscle injury. We also demonstrated that these mononuclear cells can develop into a variety of different muscle cell lineages, i.e., myoblasts, satellite cells, and muscle derived stem cells. These novel findings demonstrated, for the first time, that cellular dedifferentiation of skeletal muscle cells actually occurs in mammalian skeletal muscle following traumatic injury in vivo.

Effect of nitrogen on the seasonal course of growth and maintenance respiration in stems of Norway spruce trees.

PubMed

Stockfors, Jan; Linder, Sune

1998-03-01

To determine effects of stem nitrogen concentration ([N]) on the seasonal course of respiration, rates of stem respiration of ten control and ten irrigated-fertilized (IL), 30-year-old Norway spruce trees (Picea abies (L.) Karst.), growing in northern Sweden, were measured on seven occasions from June 1993 to April 1994. To explore sources of seasonal variation and mechanisms of fertilization effects on respiration, we separated total respiration into growth and maintenance respiration for both xylem and phloem bark. Stem respiration increased in response to the IL treatment and was positively correlated with growth rate, volume of living cells and stem nitrogen content. However, no significant effect of IL treatment or [N] in the living cells was found for respiration per unit volume of live cells. Total stem respiration during the growing season (June to September) was estimated to be 16.7 and 29.7 mol CO(2) m(-2) for control and IL-treated trees, respectively. Respiration during the growing season accounted for approximately 64% of total annual respiration. Depending on the method, estimated growth respiration varied between 40 and 60% of total respiration during the growing season. Between 75 and 80% of the live cell volume in the stems was in the phloem, and phloem maintenance accounted for about 70% of maintenance respiration. Because most of the living cells were found in the phloem, and the living xylem cells were concentrated in the outer growth rings, we concluded that the best base for expressing rates of stem growth and maintenance respiration in young Norway spruce trees is stem surface area.

Symmetry breaking in human neuroblastoma cells

PubMed Central

Izumi, Hideki; Kaneko, Yasuhiko

2014-01-01

Asymmetric cell division (ACD) is a characteristic of cancer stem cells, which exhibit high malignant potential. However, the cellular mechanisms that regulate symmetric (self-renewal) and asymmetric cell divisions are mostly unknown. Using human neuroblastoma cells, we found that the oncosuppressor protein tripartite motif containing 32 (TRIM32) positively regulates ACD. PMID:27308367

Head and neck cancer stem cells: the effect of HPV--an in vitro and mouse study.

PubMed

Tang, Alice L; Owen, John H; Hauff, Samantha J; Park, Jung Je; Papagerakis, Silvana; Bradford, Carol R; Carey, Thomas E; Prince, Mark E

2013-08-01

To determine if the behavior of cancer stem cells (CSCs) is affected by human papillomavirus (HPV) status. An in vitro and in vivo analysis of HPV and CSCs. University laboratory. We isolated CSCs from HPV-positive and HPV-negative cell lines. Two HPV-negative cell lines underwent lentiviral transduction of E6/E7. Chemoresistence was determined using colony formation assays. Native HPV-positive and HPV E6/E7-transduced cells were compared for lung colonization after tail vein injection in NOD/SCID mice. The proportion of CSC is not significantly different in HPV-positive or HPV-negative head and neck squamous cell carcinoma (HNSCC) cell lines. The HNSCC CSCs are more resistant to cisplatin than the non-CSCs, but there were no significant differences between HPV-positive and HPV-negative cells. The HPV-negative cancer cells yielded low colony formation after cell sorting. After transduction with HPV E6/E7, increased colony formation was observed in both CSCs and non-CSCs. Results from tail vein injections yielded no differences in development of lung colonies between HPV E6/E7-transduced cells and nontransduced cells. Human papillomavirus status does not correlate with the proportion of CSCs present in HNSCC. The HPV-positive cells and those transduced with HPV E6/E7 have a greater clonogenicity than HPV-negative cells. The HNSCC CSCs are more resistant to cisplatin than non-CSCs. This suggests that common chemotherapeutic agents may shrink tumor bulk by eliminating non-CSCs, whereas CSCs have mechanisms that facilitate evasion of cell death. Human papillomavirus status does not affect CSC response to cisplatin therapy, suggesting that other factors explain the better outcomes for patients with HPV-positive cancer.

Fgf10-positive cells represent a progenitor cell population during lung development and postnatally

PubMed Central

El Agha, Elie; Herold, Susanne; Alam, Denise Al; Quantius, Jennifer; MacKenzie, BreAnne; Carraro, Gianni; Moiseenko, Alena; Chao, Cho-Ming; Minoo, Parviz; Seeger, Werner; Bellusci, Saverio

2014-01-01

The lung mesenchyme consists of a widely heterogeneous population of cells that play crucial roles during development and homeostasis after birth. These cells belong to myogenic, adipogenic, chondrogenic, neuronal and other lineages. Yet, no clear hierarchy for these lineages has been established. We have previously generated a novel Fgf10iCre knock-in mouse line that allows lineage tracing of Fgf10-positive cells during development and postnatally. Using these mice, we hereby demonstrate the presence of two waves of Fgf10 expression during embryonic lung development: the first wave, comprising Fgf10-positive cells residing in the submesothelial mesenchyme at early pseudoglandular stage (as well as their descendants); and the second wave, comprising Fgf10-positive cells from late pseudoglandular stage (as well as their descendants). Our lineage-tracing data reveal that the first wave contributes to the formation of parabronchial and vascular smooth muscle cells as well as lipofibroblasts at later developmental stages, whereas the second wave does not give rise to smooth muscle cells but to lipofibroblasts as well as an Nkx2.1- E-Cad- Epcam+ Pro-Spc+ lineage that requires further in-depth analysis. During alveologenesis, Fgf10-positive cells give rise to lipofibroblasts rather than alveolar myofibroblasts, and during adult life, a subpopulation of Fgf10-expressing cells represents a pool of resident mesenchymal stromal (stem) cells (MSCs) (Cd45- Cd31- Sca-1+). Taken together, we show for the first time that Fgf10-expressing cells represent a pool of mesenchymal progenitors in the embryonic and postnatal lung. Our findings suggest that Fgf10-positive cells could be useful for developing stem cell-based therapies for treating interstitial lung diseases. PMID:24353064

Directed Differentiation of Human-Induced Pluripotent Stem Cells to Mesenchymal Stem Cells.

PubMed

Lian, Qizhou; Zhang, Yuelin; Liang, Xiaoting; Gao, Fei; Tse, Hung-Fat

2016-01-01

Multipotent stromal cells, also known as mesenchymal stem cells (MSCs), possess great potential to generate a wide range of cell types including endothelial cells, smooth muscle cells, bone, cartilage, and lipid cells. This protocol describes in detail how to perform highly efficient, lineage-specific differentiation of human-induced pluripotent stem cells (iPSCs) with an MSCs fate. The approach uses a clinically compliant protocol with chemically defined media, feeder-free conditions, and a CD105 positive and CD24 negative selection to achieve a single cell-based MSCs derivation from differentiating human pluripotent cells in approximately 20 days. Cells generated with this protocol express typical MSCs surface markers and undergo adipogenesis, osteogenesis, and chondrogenesis similar to adult bone marrow-derived MSCs (BM-MSCs). Nonetheless, compared with adult BM-MSCs, iPSC-MSCs display a higher proliferative capacity, up to 120 passages, without obvious loss of self-renewal potential and constitutively express MSCs surface antigens. MSCs generated with this protocol have numerous applications, including expansion to large scale cell numbers for tissue engineering and the development of cellular therapeutics. This approach has been used to rescue limb ischemia, allergic disorders, and cigarette smoke-induced lung damage and to model mesenchymal and vascular disorders of Hutchinson-Gilford progeria syndrome (HGPS).

Conversion of partially reprogrammed cells to fully pluripotent stem cells is associated with further activation of stem cell maintenance- and gamete generation-related genes.

PubMed

Kim, Jong Soo; Choi, Hyun Woo; Choi, Sol; Seo, Han Geuk; Moon, Sung-Hwan; Chung, Hyung-Min; Do, Jeong Tae

2014-11-01

Somatic cells are reprogrammed to induced pluripotent stem cells (iPSCs) by overexpression of a combination of defined transcription factors. We generated iPSCs from mouse embryonic fibroblasts (with Oct4-GFP reporter) by transfection of pCX-OSK-2A (Oct4, Sox2, and Klf4) and pCX-cMyc vectors. We could generate partially reprogrammed cells (XiPS-7), which maintained more than 20 passages in a partially reprogrammed state; the cells expressed Nanog but were Oct4-GFP negative. When the cells were transferred to serum-free medium (with serum replacement and basic fibroblast growth factor), the XiPS-7 cells converted to Oct4-GFP-positive iPSCs (XiPS-7c, fully reprogrammed cells) with ESC-like properties. During the conversion of XiPS-7 to XiPS-7c, we found several clusters of slowly reprogrammed genes, which were activated at later stages of reprogramming. Our results suggest that partial reprogrammed cells can be induced to full reprogramming status by serum-free medium, in which stem cell maintenance- and gamete generation-related genes were upregulated. These long-term expandable partially reprogrammed cells can be used to verify the mechanism of reprogramming.

Traffic jam functions in a branched pathway from Notch activation to niche cell fate.

PubMed

Wingert, Lindsey; DiNardo, Stephen

2015-07-01

The niche directs key behaviors of its resident stem cells, and is thus crucial for tissue maintenance, repair and longevity. However, little is known about the genetic pathways that guide niche specification and development. The male germline stem cell niche in Drosophila houses two stem cell populations and is specified within the embryonic gonad, thus making it an excellent model for studying niche development. The hub cells that form the niche are specified early by Notch activation. Over the next few hours, these individual cells then cluster together and take up a defined position before expressing markers of hub cell differentiation. This timing suggests that there are other factors for niche development yet to be defined. Here, we have identified a role for the large Maf transcription factor Traffic jam (Tj) in hub cell specification downstream of Notch. Tj downregulation is the first detectable effect of Notch activation in hub cells. Furthermore, Tj depletion is sufficient to generate ectopic hub cells that can recruit stem cells. Surprisingly, ectopic niche cells in tj mutants remain dispersed in the absence of Notch activation. This led us to uncover a branched pathway downstream of Notch in which Bowl functions to direct hub cell assembly in parallel to Tj downregulation. © 2015. Published by The Company of Biologists Ltd.

Zinc Promotes Adipose-Derived Mesenchymal Stem Cell Proliferation and Differentiation towards a Neuronal Fate

PubMed Central

Moon, Mi-Young; Kim, Hyun Jung; Choi, Bo Young; Sohn, Min

2018-01-01

Zinc is an essential element required for cell division, migration, and proliferation. Under zinc-deficient conditions, proliferation and differentiation of neural progenitors are significantly impaired. Adipose-derived mesenchymal stem cells (AD-MSCs) are multipotent stem cells that can differentiate into neurons. The aim of this study was to evaluate the effect of zinc on AD-MSC proliferation and differentiation. We initially examined the effect of zinc on stem cell proliferation at the undifferentiated stage. AD-MSCs showed high proliferation rates on day 6 in 30 μM and 100 μM of ZnCl2. Zinc chelation inhibited AD-MSC proliferation via downregulation of ERK1/2 activity. We then assessed whether zinc was involved in cell migration and neurite outgrowth during differentiation. After three days of neuronal differentiation, TUJ-1-positive cells were observed, implying that AD-MSCs had differentiated into early neuron or neuron-like cells. Neurite outgrowth was increased in the zinc-treated group, while the CaEDTA-treated group showed diminished, shrunken neurites. Furthermore, we showed that zinc promoted neurite outgrowth via the inactivation of RhoA and led to the induction of neuronal gene expression (MAP2 and nestin) in differentiated stem cells. Taken together, zinc promoted AD-MSC proliferation and affected neuronal differentiation, mainly by increasing neurite outgrowth. PMID:29765417

Establishment of stem cell lines from nuclear transferred and parthenogenetically activated mouse oocytes for therapeutic cloning.

PubMed

Ju, Jin Young; Park, Chun Young; Gupta, Mukesh Kumar; Uhm, Sang Jun; Paik, Eun Chan; Ryoo, Zae Young; Cho, Youl Hee; Chung, Kil Saeng; Lee, Hoon Taek

2008-05-01

To establish embryonic stem cell lines from nuclear transfer of somatic cell nuclei isolated from the same oocyte donor and from parthenogenetic activation. The study also evaluated the effect of the micromanipulation procedure on the outcome of somatic cell nuclear transfer in mice. Randomized, prospective study. Hospital-based assisted reproductive technology laboratory. F(1) (C57BL/6 x 129P3/J) mice. Metaphase II-stage oocytes were either parthenogenetically activated or nuclear transferred with cumulus cell nuclei or parthenogenetically activated after a sham-manipulation procedure. Embryogenesis and embryonic stem cell establishment. The development rate to morula/blastocyst of nuclear transferred oocytes (27.9% +/- 5.9%) was significantly lower than that of the sham-manipulated (84.1% +/- 5.6%) or parthenogenetic (98.6% +/- 1.4%) groups. A sharp decrease in cleavage potential was obvious in the two- to four-cell transition for the nuclear transferred embryos (79.0% +/- 4.6% and 43.3% +/- 5.0%), implying incomplete nuclear reprogramming in arrested oocytes. However, the cleavage, as well as the development rate, of parthenogenetic and sham-manipulated groups did not differ significantly. The embryonic stem cell line establishment rate was higher from parthenogenetically activated oocytes (15.7%) than nuclear transferred (4.3%) or sham-manipulated oocytes (12.5%). Cell colonies from all groups displayed typical morphology of mice embryonic stem cells and could be maintained successfully with undifferentiated morphology after continuous proliferation for more than 120 passages still maintaining normal karyotype. All these cells were positive for mice embryonic stem cell markers such as Oct-4 and SSEA-1 based on immunocytochemistry and reverse transcriptase-polymerase chain reaction. The clonal origin of the ntES cell line and the parthenogenetic embryonic stem cell lines were confirmed by polymerase chain reaction analysis of the polymorphic markers. Blastocyst injection experiments demonstrated that these lines contributed to resulting chimeras and are germ-line competent. We report the establishment of ntES cell lines from somatic cells isolated from same individual. Our data also suggest that embryo micromanipulation procedure during the nuclear transfer procedure influences the developmental ability and embryonic stem cell establishment rate of nuclear transferred embryos.

Alternative generation of CNS neural stem cells and PNS derivatives from neural crest-derived peripheral stem cells.

PubMed

Weber, Marlen; Apostolova, Galina; Widera, Darius; Mittelbronn, Michel; Dechant, Georg; Kaltschmidt, Barbara; Rohrer, Hermann

2015-02-01

Neural crest-derived stem cells (NCSCs) from the embryonic peripheral nervous system (PNS) can be reprogrammed in neurosphere (NS) culture to rNCSCs that produce central nervous system (CNS) progeny, including myelinating oligodendrocytes. Using global gene expression analysis we now demonstrate that rNCSCs completely lose their previous PNS characteristics and acquire the identity of neural stem cells derived from embryonic spinal cord. Reprogramming proceeds rapidly and results in a homogenous population of Olig2-, Sox3-, and Lex-positive CNS stem cells. Low-level expression of pluripotency inducing genes Oct4, Nanog, and Klf4 argues against a transient pluripotent state during reprogramming. The acquisition of CNS properties is prevented in the presence of BMP4 (BMP NCSCs) as shown by marker gene expression and the potential to produce PNS neurons and glia. In addition, genes characteristic for mesenchymal and perivascular progenitors are expressed, which suggests that BMP NCSCs are directed toward a pericyte progenitor/mesenchymal stem cell (MSC) fate. Adult NCSCs from mouse palate, an easily accessible source of adult NCSCs, display strikingly similar properties. They do not generate cells with CNS characteristics but lose the neural crest markers Sox10 and p75 and produce MSC-like cells. These findings show that embryonic NCSCs acquire a full CNS identity in NS culture. In contrast, MSC-like cells are generated from BMP NCSCs and pNCSCs, which reveals that postmigratory NCSCs are a source for MSC-like cells up to the adult stage. © 2014 AlphaMed Press.

Isolation and characterization of stem cells derived from human third molar tooth germs of young adults: implications in neo-vascularization, osteo-, adipo- and neurogenesis.

PubMed

Yalvac, M E; Ramazanoglu, M; Rizvanov, A A; Sahin, F; Bayrak, O F; Salli, U; Palotás, A; Kose, G T

2010-04-01

A number of studies have reported in the last decade that human tooth germs contain multipotent cells that give rise to dental and peri-odontal structures. The dental pulp, third molars in particular, have been shown to be a significant stem cell source. In this study, we isolated and characterized human tooth germ stem cells (hTGSCs) from third molars and assessed the expression of developmentally important transcription factors, such as oct4, sox2, klf4, nanog and c-myc, to determine their pluri-potency. Flow-cytometry analysis revealed that hTGSCs were positive for CD73, CD90, CD105 and CD166, but negative for CD34, CD45 and CD133, suggesting that these cells are mesenchymal-like stem cells. Under specific culture conditions, hTGSCs differentiated into osteogenic, adipogenic and neurogenic cells, as well as formed tube-like structures in Matrigel assay. hTGSCs showed significant levels of expression of sox2 and c-myc messenger RNA (mRNA), and a very high level of expression of klf4 mRNA when compared with human embryonic stem cells. This study reports for the first time that hTGSCs express developmentally important transcription factors that could render hTGSCs an attractive candidate for future somatic cell re-programming studies to differentiate germs into various tissue types, such as neurons and vascular structures. In addition, these multipotential hTGSCs could be important stem cell sources for autologous transplantation.

Pigmentation Is Associated with Stemness Hierarchy of Progenitor Cells Within Cultured Limbal Epithelial Cells.

PubMed

Liu, Lei; Nielsen, Frederik Mølgaard; Emmersen, Jeppe; Bath, Chris; Hjortdal, Jesper Østergaard; Riis, Simone; Fink, Trine; Pennisi, Cristian Pablo; Zachar, Vladimir

2018-05-20

Ex-vivo cultured human limbal epithelial stem/progenitor cells (hLESCs) are the main source for regenerative therapy of limbal stem cell deficiency (LSCD), which is worldwide one of the major causes of corneal blindness. Despite many stemness-associated markers have been identified within the limbal niche, the phenotype of the earliest hLESCs has not been hitherto identified. We sought to confirm or refute the use of tumor protein p63 (p63) and ATP binding cassette subfamily B member 5 (ABCB5) as surrogate markers for hLESCs early within the limbal differentiation hierarchy. Based on a robust fluorescence-activated cell sorting (FACS) and subsequent RNA isolation protocol, a comprehensive transcriptomic profile was obtained from four subpopulations of cultured hLESCs. The subpopulations were defined by co-expression of two putative stem/progenitor markers, the p63 and ABCB5, and the corneal differentiation marker cytokeratin 3 (CK3). A comparative transcriptomic analysis yielded novel data that indicated association between pigmentation and differentiation, with the p63 positive populations being the most pigmented and immature of the progenitors. In contrast, ABCB5, either alone or in co-expression patterns, identified more committed progenitor cells with less pigmentation. In conclusion, p63 is superior to ABCB5 as a marker for stemness. This article is protected by copyright. All rights reserved. © 2018 AlphaMed Press.

Magnetic Resonance Imaging of Ferumoxytol-Labeled Human Mesenchymal Stem Cells in the Mouse Brain.

PubMed

Lee, Na Kyung; Kim, Hyeong Seop; Yoo, Dongkyeom; Hwang, Jung Won; Choi, Soo Jin; Oh, Wonil; Chang, Jong Wook; Na, Duk L

2017-02-01

The success of stem cell therapy is highly dependent on accurate delivery of stem cells to the target site of interest. Possible ways to track the distribution of MSCs in vivo include the use of reporter genes or nanoparticles. The U.S. Food and Drug Administration (FDA) has approved ferumoxytol (Feraheme® [USA], Rienso® [UK]) as a treatment for iron deficiency anemia. Ferumoxytol is an ultrasmall superparamagnetic iron oxide nanoparticle (USPIO) that has recently been used to track the fate of transplanted cells using magnetic resonance imaging (MRI). The major objectives of this study were to demonstrate the feasibility of labeling hUCB-MSCs with ferumoxytol and to observe, through MRI, the engraftment of ferumoxytol-labeled human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) delivered via stereotactic injection into the hippocampi of a transgenic mouse model of familial Alzheimer's disease (5XFAD). Ferumoxytol had no toxic effects on the viability or stemness of hUCB-MSCs when assessed in vitro. Through MRI, hypointense signals were discernible at the site where ferumoxytol-labeled human MSCs were injected. Iron-positive areas were also observed in the engrafted hippocampi. The results from this study support the use of nanoparticle labeling to monitor transplanted MSCs in real time as a follow-up for AD stem cell therapy in the clinical field.

Nature vs. nurture: gold perpetuates "stemness".

PubMed

Paul, Willi; Sharma, Chandra P; Deb, Kaushik Dilip

2011-01-01

Adult tissues contain quiescent reservoirs of multipotent somatic stem cells and pluripotent embryonic-like stem cells (ELSCs). Credited with regenerative properties gold is used across both -contemporary and -ancient medicines. Here, we show that gold exerted these effects by enhancing the pool of pluripotent ELSC while improving their stemness. We used hESCs as an in-vitro model to understand if gold could enhance self-renewal and pluripotency. Swarna-bhasma (SB), an ancient Indian gold microparticulate (41.1 nm), preparation, reduced spontaneous-differentiation, improved self-renewal, pluripotency and proliferation of hESCs. Colloidal gold-nanoparticles (GNP) (15.59 nm) were tested to confirm that the observations were attributable to nanoparticulate-gold. SB and GNP exposure: maintained -stemness, -karyotypic stability, enhanced pluripotency till day-12, increased average colony-sizes, and reduced the number of autonomously-derived differentiated FGFR1 positive fibroblast-niche-cells/colony. Particulate-gold induced upregulation of FGFR1 and IGF2 expression, and decrease in IGF1 secretion indicates IGF1/2 mediated support for enhanced pluripotency and self-renewal in hESCs.

Allogeneic peripheral blood stem cell transplantation for the treatment of chronic active Epstein-Barr virus infection.

PubMed

Fujii, N; Takenaka, K; Hiraki, A; Maeda, Y; Ikeda, K; Shinagawa, K; Ashiba, A; Munemasa, M; Sunami, K; Hiramatsu, Y; Ishimaru, F; Niiya, K; Yoshino, T; Harada, M

2000-10-01

The prognosis of chronic active Epstein-Barr virus infection (CAEBV) is very poor. We describe a 24-year-old male with severe CAEBV who was treated with allogeneic peripheral blood stem cell transplantation (allo-PBSCT). On admission, EBER-1 in lymphocytes infiltrating the liver, EBV-DNA in peripheral blood mononuclear cells (PBMC) and monoclonal NK cell proliferation were confirmed. After unsuccessful chemotherapy, he received an allo-PBSCT from his HLA-identical sister. Although he died of pulmonary hemorrhage on day +19, EBV-DNA was undetectable by PCR in PBMC, and the post-mortem liver showed no EBER-1-positive lymphocytes. This experience suggests that EBV-positive lymphocytes in CAEBV may be eradicated by allo-PBSCT, thereby raising the possibility of a new treatment modality. Bone Marrow Transplantation (2000) 26, 805-808.

ErbB2 and bone sialoprotein as markers for metastatic osteosarcoma cells

PubMed Central

Valabrega, G; Fagioli, F; Corso, S; Madon, E; Brach del Prever, A; Biasin, E; Linari, A; Aglietta, M; Giordano, S

2003-01-01

Osteosarcoma is the most common malignant bone neoplasia occurring in young patients in the first two decades of life, and represents 20% of all primitive malignant bone tumours. At present, treatment of metastatic osteosarcoma is unsatisfactory. High-dose chemotherapy followed by CD34+ leukapheresis rescue may improve these poor results. Neoplastic cells contaminating the apheresis may, however, contribute to relapse. To identify markers suitable for detecting osteosarcoma cells in aphereses we analysed the expression of bone-specific genes (Bone Sialoprotein (BSP) and Osteocalcin) and oncogenes (Met and ErbB2) in 22 patients with metastatic osteosarcoma and six healthy stem cell donors. The expression of these genes in aphereses of patients affected by metastatic osteosarcoma was assessed by RT–PCR and Southern blot analysis. Met and Osteocalcin proved to be not useful markers since they are positive in aphereses of both patients with metastatic osteosarcoma and healthy stem cell donors. On the contrary, BSP was expressed at significant levels in 85% of patients. Moreover, 18% of patients showed a strong and significantly positive (seven to 16 times higher than healthy stem cell donors) ErbB2 expression. In all positive cases, neoplastic tissue also expressed ErbB2. Our data show that ErbB2 can be a useful marker for tumour contamination in aphereses of patients affected by ErbB2-expressing osteosarcomas and that analysis of Bone Sialoprotein expression can be an alternative useful marker. PMID:12569382

Periodontal Biology: Stem Cells, Bmp2 Gene, Transcriptional Enhancers, and Use of Sclerostin Antibody and Pth for Treatment of Periodontal Disease and Bone Loss

PubMed Central

Harris, Stephen E; Rediske, Michael; Neitzke, Rebecca; Rakian, Audrey

2017-01-01

The periodontium is a complex tissue with epithelial components and a complex set of mesodermal derived alveolar bone, cellular and a cellular cementum, and tendon like ligaments (PDL). The current evidence demonstrates that the major pool of periodontal stem cells is derived from a population of micro vascular associated aSMA-positive stem/progenitor (PSC) cells that by lineage tracing form all three major mesodermal derived components of the periodontium. With in vitro aSMA+ stem cells, transcriptome and chip- seq experiments, the gene network and enhancer maps were determined at several differentiation states of the PSC. Current work on the role of the Bmp2 gene in the periodontal stem cell differentiation demonstrated that this Wnt regulated gene, Bmp2, is necessary for differentiation to all three major mesodermal derived component of the periodontium. The mechanism and use of Sclerostin antibody as an activator of Wnt signaling and Bmp2 gene as a potential route to treat craniofacial bone loss is discussed. As well, the mechanism and use of Pth in the treatment of periodontal bone loss or other craniofacial bone loss is presented in this review. PMID:29457146

Hair follicle stem cell proliferation, Akt and Wnt signaling activation in TPA-induced hair regeneration.

PubMed

Qiu, Weiming; Lei, Mingxing; Zhou, Ling; Bai, Xiufeng; Lai, Xiangdong; Yu, Yu; Yang, Tian; Lian, Xiaohua

2017-06-01

Regeneration of hair follicles relies on activation of hair follicle stem cells during telogen to anagen transition process in hair cycle. This process is rigorously controlled by intrinsic and environmental factors. 12-o-tetradecanoylphorbol-13-acetate (TPA), a tumor promoter, accelerates reentry of hair follicles into anagen phase. However, it is unclear that how TPA promotes the hair regeneration. In the present study, we topically applied TPA onto the dorsal skin of 2-month-old C57BL/6 female mice to examine the activity of hair follicle stem cells and alteration of signaling pathways during hair regeneration. We found that refractory telogen hair follicles entered anagen prematurely after TPA treatment, with the enhanced proliferation of CD34-positive hair follicle stem cells. Meanwhile, we observed Akt signaling was activated in epidermis, hair infundibulum, bulge and hair bulb, and Wnt signaling was also activated after hair follicle stem cells proliferation. Importantly, after overexpression of DKK1, a specific Wnt signaling inhibitor, the accelerated reentry of hair follicles into anagen induced by TPA was abolished. Our data indicated that TPA-induced hair follicle regeneration is associated with activation of Akt and Wnt/β-catenin signaling.

[Effect of electroacupuncture on differentiation and proliferation of hippocampal nerve stem cells in splenic asthenia pedo-rats].

PubMed

Zhuo, Yuan-yuan; Yang, Zhuo-xin; Wu, Jia-man

2011-10-01

To observe the effect of electroacupuncture (EA) on the differentiation and proliferation of nerve stem cells in the hippocampal dentate gyrus (DG) in splenic asthenia pedo-rats so as to study its central mechanism. A total of 72 SD male rats were randomly assigned to normal control group (n=24), model group (n=24) and EA group (n=24) which were further divided into 7 d, 14 d, 28 d and 49 d time-points (n=6). Splenic asthenia model was established by intraperitoneal injection of reserpine and gavage of Dahuang (Radix et Rhizoma Rhei) fluid. EA was applied to bilateral "Zusanli" (ST 36) and "Sanyinjiao" (SP 6) for 20 min, once daily for 7, 14, 28 and 49 days respectively. Brdu, Nestin, glial fibrillary acidic protein (GFAP), and neuron-specific enolase (NSE) expression in the DG of hippocampus were detected by immunohistochemistry double staining. Compared with the normal control group, the numbers of Brdu, Brdu/GFAP, Brdu/NSE Immunoreactive (IR) positive cells in the DG of hippocampus on day 7 and 14, and that of Brdu/Nestin IR-positive cells on day 7 were decreased considerably in the model group (P < 0.05). Compared to the model group, the numbers of hippocampal Brdu IR-positive cells at the 4 time-points, Brdu/Nestin and Brdu/GFAP on day 7, 14 and 49, and Brdu/NSE on day 7, 14 and 28 were increased significantly in the EA group (P < 0.05). No significant differences were found between the model and control groups in the numbers of hippocampal Brdu and Brdu/NSE IR-positive cells on day 28 and 49, in the number of Brdu/Nestin IR-positive cells on day 14 and 49, in the number of Brdu/GFAP IR-positive cells on day 28; and between the EA and model groups in the numbers of Brdu/Nestin and Brdu/GFAP IR-positive cells on day 28, and in the number of Brdu/NSE IR-positive cells on day 49 (P > 0.05). EA of ST 36 and SP 6 can effectively suppress splenic asthenia syndrome-induced decrease of the numbers of Brdu, Brdu/GFAP, Brdu/Nestin and Brdu/NSE IR-positive cells in the DG of hippocampus at the early stage in the splenic asthenia rats, which may contribute to its effect in improving splenic asthenia symptoms in clinic by promoting the proliferation and differentiation of some nerve stem cells in the hippocampus.

In vitro generation of type-II pneumocytes can be initiated in human CD34(+) stem cells.

PubMed

Srikanth, Lokanathan; Venkatesh, Katari; Sunitha, Manne Mudhu; Kumar, Pasupuleti Santhosh; Chandrasekhar, Chodimella; Vengamma, Bhuma; Sarma, Potukuchi Venkata Gurunadha Krishna

2016-02-01

Human CD34(+) stem cells differentiated into type-II pneumocytes in Dulbecco's modified Eagle medium (DMEM) having hydrocortisone, insulin, fibroblast growth factor (FGF), epidermal growth factor (EGF) and bovine serum albumin (BSA), expressing surfactant proteins-B (SP-B) and C (SP-C), alkaline phosphatase (ALP) and lysozyme. FACS-enumerated pure CD34(+) cells, isolated from human peripheral blood, were cultured in DMEM and showed positive reaction with anti-human CD34 monoclonal antibodies in immunocytochemistry. These cells were cultured in DMEM having hydrocortisone, insulin, FGF, EGF and BSA (HIFEB-D) medium having an air-liquid interface. They differentiated into type-II pneumocytes with expression of SP-B and SP-C genes and disappearance of CD34 expression as assessed using real-time PCR. In reverse transcription-PCR amplicons showed 208 and 907 bp confirming SP-B and SP-C expressions. These cells expressed ALP with an activity of 1.05 ± 0.09 mM ml(-1) min(-1) and lysozyme that killed E. coli. The successful differentiation of human CD34(+) stem cells into type-II pneumocytes, and transplantation of such cells obtained from the patient's stem cell could be the futuristic approach to regenerate diseased lung alveoli.

Generation of urine-derived induced pluripotent stem cells from a patient with phenylketonuria

PubMed Central

Qi, Zijuan; Cui, Yazhou; Shi, Liang; Luan, Jing; Zhou, Xiaoyan; Han, Jinxiang

2018-01-01

Summary The aim of the study was to establish an induced pluripotent stem cell line from urine-derived cells (UiPSCs) from a patient with phenylketonuria (PKU) in order to provide a useful research tool with which to examine the pathology of this rare genetic metabolic disease. Urine-derived epithelial cells (UCs) from a 15-year-old male patient with PKU were isolated and reprogrammed with integration-free episomal vectors carrying an OCT4, SOX2, KLF4, and miR-302-367 cluster. PKU-UiPSCs were verified as correct using alkaline phosphatase staining. Pluripotency markers were detected with real-time PCR and flow cytometry. Promoter methylation in two pluripotent genes, NANOG and OCT4, was analyzed using bisulphite sequencing. An embryoid body (EB) formation assay was also performed. An induced pluripotent stem cell line (iPSC) was generated from epithelial cells in urine from a patient with PKU. This cell line had increased expression of stem cell biomarkers, it efficiently formed EBs, it stained positive for alkaline phosphatase (ALP), and it had a marked decrease in promoter methylation in the NANOG and OCT4 genes. The PKU-UiPSCs created here had typical characteristics and are suitable for further differentiation.

Integration-deficient lentivectors: an effective strategy to purify and differentiate human embryonic stem cell-derived hepatic progenitors.

PubMed

Yang, Guanghua; Si-Tayeb, Karim; Corbineau, Sébastien; Vernet, Rémi; Gayon, Régis; Dianat, Noushin; Martinet, Clémence; Clay, Denis; Goulinet-Mainot, Sylvie; Tachdjian, Gérard; Tachdjian, Gérard; Burks, Deborah; Vallier, Ludovic; Bouillé, Pascale; Dubart-Kupperschmitt, Anne; Weber, Anne

2013-07-19

Human pluripotent stem cells (hPSCs) hold great promise for applications in regenerative medicine. However, the safety of cell therapy using differentiated hPSC derivatives must be improved through methods that will permit the transplantation of homogenous populations of a specific cell type. To date, purification of progenitors and mature cells generated from either embryonic or induced pluripotent stem cells remains challenging with use of conventional methods. We used lentivectors encoding green fluorescent protein (GFP) driven by the liver-specific apoliprotein A-II (APOA-II) promoter to purify human hepatic progenitors. We evaluated both integrating and integration-defective lentivectors in combination with an HIV integrase inhibitor. A human embryonic stem cell line was differentiated into hepatic progenitors using a chemically defined protocol. Subsequently, cells were transduced and sorted at day 16 of differentiation to obtain a cell population enriched in hepatic progenitor cells. After sorting, more than 99% of these APOA-II-GFP-positive cells expressed hepatoblast markers such as α-fetoprotein and cytokeratin 19. When further cultured for 16 days, these cells underwent differentiation into more mature cells and exhibited hepatocyte properties such as albumin secretion. Moreover, they were devoid of vector DNA integration. We have developed an effective strategy to purify human hepatic cells from cultures of differentiating hPSCs, producing a novel tool that could be used not only for cell therapy but also for in vitro applications such as drug screening. The present strategy should also be suitable for the purification of a broad range of cell types derived from either pluripotent or adult stem cells.

Differentiation Generates Paracrine Cell Pairs That Maintain Basaloid Mouse Mammary Tumors: Proof of Concept

PubMed Central

Kim, Soyoung; Goel, Shruti; Alexander, Caroline M.

2011-01-01

There is a paradox offered up by the cancer stem cell hypothesis. How are the mixed populations that are characteristic of heterogeneous solid tumors maintained at constant proportion, given their high, and different, mitotic indices? In this study, we evaluate a well-characterized mouse model of human basaloid tumors (induced by the oncogene Wnt1), which comprise mixed populations of mammary epithelial cells resembling their normal basal and luminal counterparts. We show that these cell types are substantially inter-dependent, since the MMTV LTR drives expression of Wnt1 ligand in luminal cells, whereas the functional Wnt1-responsive receptor (Lrp5) is expressed by basal cells, and both molecules are necessary for tumor growth. There is a robust tumor initiating activity (tumor stem cell) in the basal cell population, which is associated with the ability to differentiate into luminal and basal cells, to regenerate the oncogenic paracrine signaling cell pair. However, we found an additional tumor stem cell activity in the luminal cell population. Knowing that tumors depend upon Wnt1-Lrp5, we hypothesized that this stem cell must express Lrp5, and found that indeed, all the stem cell activity could be retrieved from the Lrp5-positive cell population. Interestingly, this reflects post-transcriptional acquisition of Lrp5 protein expression in luminal cells. Furthermore, this plasticity of molecular expression is reflected in plasticity of cell fate determination. Thus, in vitro, Wnt1-expressing luminal cells retro-differentiate to basal cell types, and in vivo, tumors initiated with pure luminal cells reconstitute a robust basal cell subpopulation that is indistinguishable from the populations initiated by pure basal cells. We propose this is an important proof of concept, demonstrating that bipotential tumor stem cells are essential in tumors where oncogenic ligand-receptor pairs are separated into different cell types, and suggesting that Wnt-induced molecular and fate plasticity can close paracrine loops that are usually separated into distinct cell types. PMID:21541292

In vitro chronotropic effects of Erythrina senegalensis DC (Fabaceae) aqueous extract on mouse heart slice and pluripotent stem cell-derived cardiomyocytes.

PubMed

Nembo, Erastus Nembu; Atsamo, Albert Donatien; Nguelefack, Télesphore Benoît; Kamanyi, Albert; Hescheler, Jürgen; Nguemo, Filomain

2015-05-13

Erythrina senegalensis DC (Fabaceae) bark is commonly used in sub-Saharan traditional medicine for the treatment of many diseases including gastrointestinal disorders and cardiovascular diseases. In this study, we investigated the effect of the aqueous extract of the stem bark of Erythrina senegalensis on the contractile properties of mouse ventricular slices and human induced pluripotent stem (hiPS) cell-derived cardiomyocytes. We also investigated the cytotoxic effect of the extract on mouse embryonic stem (ES) cells differentiating into cardiomyocytes (CMs). We used well-established electrophysiological technologies to assess the effect of Erythrina senegalensis aqueous extract (ESAE) on the beating activity of mouse ventricular slices, mouse ES and hiPS cell-derived CMs. To study the cytotoxic effect of our extract, differentiating mouse ES cells were exposed to different concentrations of ESAE. EB morphology was assessed by microscopy at different stages of differentiation whereas cell viability was measured by flow cytometry, fluorometry and immunocytochemistry. The electrical activity of CMs and heart slices were respectively captured by the patch clamp technique and microelectrode array (MEA) method following ESAE acute exposure. Our findings revealed that ESAE exhibits a biphasic chronotropic activity on mouse ventricular slices with an initial low dose (0.001 and 0.01 µg/mL) decrease in beating activity followed by a corresponding significant increase in chronotropic activity at higher doses above 10 µg/mL. The muscarinic receptor blocker, atropine abolished the negative chronotropic activity of ESAE, while propranolol successfully blocked its positive chronotropic activity. ESAE showed a significant dose-dependent positive chronotropic activity on hiPS cell-derived CMs. Also, though not significantly, ESAE decreased cell viability and increased total caspase-3/7 activity of mouse ES cells in a concentration-dependent manner. Erythrina senegalensis aqueous extract exhibits a biphasic chronotropic effect on mouse heart and a positive chronotropic activity on hiPS cell-derived CMs, suggesting a possible mechanism through muscarinic and β-adrenergic receptor pathways. Also, ESAE is not cytotoxic on mouse ES cells at concentrations up to 100 µg/mL. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Identification and Characterization of Cells with Cancer Stem Cell Properties in Human Primary Lung Cancer Cell Lines

PubMed Central

Suo, Zhenhe; Munthe, Else; Solberg, Steinar; Ma, Liwei; Wang, Mengyu; Westerdaal, Nomdo Anton Christiaan; Kvalheim, Gunnar; Gaudernack, Gustav

2013-01-01

Lung cancer (LC) with its different subtypes is generally known as a therapy resistant cancer with the highest morbidity rate worldwide. Therapy resistance of a tumor is thought to be related to cancer stem cells (CSCs) within the tumors. There have been indications that the lung cancer is propagated and maintained by a small population of CSCs. To study this question we established a panel of 15 primary lung cancer cell lines (PLCCLs) from 20 fresh primary tumors using a robust serum-free culture system. We subsequently focused on identification of lung CSCs by studying these cell lines derived from 4 representative lung cancer subtypes such as small cell lung cancer (SCLC), large cell carcinoma (LCC), squamous cell carcinoma (SCC) and adenocarcinoma (AC). We identified a small population of cells strongly positive for CD44 (CD44high) and a main population which was either weakly positive or negative for CD44 (CD44low/−). Co-expression of CD90 further narrowed down the putative stem cell population in PLCCLs from SCLC and LCC as spheroid-forming cells were mainly found within the CD44highCD90+ sub-population. Moreover, these CD44highCD90+ cells revealed mesenchymal morphology, increased expression of mesenchymal markers N-Cadherin and Vimentin, increased mRNA levels of the embryonic stem cell related genes Nanog and Oct4 and increased resistance to irradiation compared to other sub-populations studied, suggesting the CD44highCD90+ population a good candidate for the lung CSCs. Both CD44highCD90+ and CD44highCD90− cells in the PLCCL derived from SCC formed spheroids, whereas the CD44low/− cells were lacking this potential. These results indicate that CD44highCD90+ sub-population may represent CSCs in SCLC and LCC, whereas in SCC lung cancer subtype, CSC potentials were found within the CD44high sub-population. PMID:23469181

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.

Stem cell transplantation and mesenchymal cells to treat autoimmune diseases.

PubMed

Tyndall, Alan; van Laar, Jacob M

2016-06-01

Since the start of the international stem cell transplantation project in 1997, over 2000 patients have received a haematopoietic stem cell transplant (HSCT), mostly autologous, as treatment for a severe autoimmune disease, the majority being multiple sclerosis (MS), systemic sclerosis (SSc) and Crohn's disease. There was an overall 85% 5-year survival and 43% progression-free survival. Around 30% of patients in all disease subgroups had a complete response, often durable despite full immune reconstitution. In many cases, e.g. systemic sclerosis, morphological improvement such as reduction of skin collagen and normalization of microvasculature was documented, beyond any predicted known effects of intense immunosuppression alone. It is hoped that the results of the three running large prospective randomized controlled trials will allow modification of the protocols to reduce the high transplant-related mortality which relates to regimen intensity, age of patient, and comorbidity. Mesenchymal stromal cells (MSC), often incorrectly called stem cells, have been the intense focus of in vitro studies and animal models of rheumatic and other diseases over more than a decade. Despite multiple plausible mechanisms of action and a plethora of positive in vivo animal studies, few randomised controlled clinical trials have demonstrated meaningful clinical benefit in any condition so far. This could be due to confusion in cell product terminology, complexity of clinical study design and execution or agreement on meaningful outcome measures. Within the rheumatic diseases, SLE and rheumatoid arthritis (RA) have received most attention. Uncontrolled multiple trial data from over 300 SLE patients have been published from one centre suggesting a positive outcome; one single centre comparative study in 172 RA was positive. In addition, small numbers of patients with Crohn's disease, multiple sclerosis, primary Sjögren's disease, polymyositis/dermatomyositis and type II diabetes mellitus have received MSC therapeutically. The possible reasons for this apparent mismatch between expectation and clinical reality will be discussed. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Mesenchymal stem cell characteristics of dental pulp and periodontal ligament stem cells after in vivo transplantation.

PubMed

Lei, Ming; Li, Kun; Li, Bei; Gao, Li-Na; Chen, Fa-Ming; Jin, Yan

2014-08-01

Mesenchymal stem cells (MSCs) isolated from human postnatal dental pulp and periodontal ligament (PDL) tissues can give rise to multilineage differentiation in vitro and generate related dental tissues in vivo. However, the cell properties of human dental pulp stem cells (DPSCs) and PDL stem cells (PDLSCs) after in vivo implantation remain largely unidentified. In this study, cells were re-isolated from in vivo-generated dental pulp-like and PDL-like tissues (termed re-DPCs and re-PDLCs, respectively) as a result of ectopic transplantation of human DPSC and PDLSC sheets. The cell characteristics in terms of colony-forming ability, cell surface antigens and multi-differentiation potentials were all evaluated before and after implantation. It was found that re-DPCs and re-PDLCs were of human and mesenchymal origin and positive for MSC markers such as STRO-1, CD146, CD29, CD90 and CD105; and, to some extent, re-DPCs could maintain their colony forming abilities. Moreover, both cell types were able to form mineral deposits and differentiate into adipocytes and chondrocytes; however, quantitative analysis and related gene expression determination showed that the osteo-/chondro-differentiation capabilities of re-DPCs and re-PDLCs were significantly reduced compared to those of DPSCs and PDLSCs, respectively (P < 0.05); re-PDLCs showed a greater reduction potential than re-DPCs. We conclude that DPSCs and PDLSCs may maintain their MSC characteristics after in vivo implantation and, compared to PDLSCs, DPSCs appear much more stable under in vivo conditions. These findings provide additional cellular and molecular evidence that supports expanding the use of dental tissue-derived stem cells in cell therapy and tissue engineering. Copyright © 2014 Elsevier Ltd. All rights reserved.

Six years' experience of tolerance induction in renal transplantation using stem cell therapy.

PubMed

Vanikar, Aruna V; Trivedi, Hargovind L; Thakkar, Umang G

2018-02-01

Tolerance induction (TI) has been attempted with chimerism/clonal deletion. We report results of TI protocol (TIP) using stem cell therapy (SCT) included adipose derived mesenchymal stem cells (AD-MSC) and hematopoietic stem cells (HSC) in 10 living-donor related renal transplantation (LDRT) patients under non-myeloablative conditioning with Bortezomib, Methylprednisone, rabbit-anti-thymoglobulin and Rituximab, without using conventional immunosuppression. Transplantation was performed following acceptable lymphocyte cross-match, flow cross-match, single antigen assay and negative mixed lymphocyte reaction (MLR). Monitoring included serum creatinine (SCr), donor specific antibodies (DSA) and MLR. Protocol biopsies were planned after 100days and yearly in willing patients. Rescue immunosuppression was planned for rejection/DSA/positive MLR. Over mean 6±0.37year follow-up patient survival was 80% and death-censored graft survival was 90%. Mean SCr was 1.44±0.41mg/dL. This is the first clinical report of sustained TI in LDRT for 6years using SCT. Copyright © 2017 Elsevier Inc. All rights reserved.

IQGAP1 Is Involved in Enhanced Aggressive Behavior of Epithelial Ovarian Cancer Stem Cell-Like Cells During Differentiation.

PubMed

Huang, Lu; Xu, Shanshan; Hu, Dongxiao; Lu, Weiguo; Xie, Xing; Cheng, Xiaodong

2015-05-01

Wide metastasis is one of characteristics of ovarian cancer. Cancer stem cells, as a source in cancer invasion and metastasis, possess powerful potential of differentiation. Scaffolding IQ domain GTPase-activating protein 1 (IQGAP1) plays a key role in the invasion and metastasis of cancer cells, but IQGAP1's role in cancer stem cells including ovarian cancer was unclear. Spheroid culture with serum-free medium was used for enriching ovarian cancer stem cell-like cells (CSC-LCs) from 3AO cell line, and a medium with 10% fetal bovine serum was used to induce the differentiation of CSC-LCs. Immunofluorescence was for detecting the stem markers OCT4 and SOX2. The quantitative real-time-polymerase chain reaction and Western blotting were performed to determine the messenger RNA and protein expression of IQGAP1, respectively. The capacity of cell invasion was evaluated by transwell chamber assay. Ovarian CSC-LCs obtained through spheroid culture showed irregularly elongated appearance, CD24 negative, and OCT4 and SOX2 positive. IQGAP1 expression was decreased in ovarian CSC-LCs compared with parental 3AO cells, but increased de novo during the differentiation of CSC-LCs. Knockdown of IQGAP1 by specific small interfering RNA remarkably weakened invasion capacity of 2-day differentiated ovarian CSC-LCs. Increased IQGAP1 expression during the differentiation of CSC-LCs is involved in an aggressive cell behavior, which may contribute to metastasis of ovarian cancer.

Human blood and marrow side population stem cell and Stro-1 positive bone marrow stromal cell numbers decline with age, with an increase in quality of surviving stem cells: correlation with cytokines.

PubMed

Brusnahan, S K; McGuire, T R; Jackson, J D; Lane, J T; Garvin, K L; O'Kane, B J; Berger, A M; Tuljapurkar, S R; Kessinger, M A; Sharp, J G

2010-01-01

Hematological deficiencies increase with aging leading to anemias, reduced hematopoietic stress responses and myelodysplasias. This study tested the hypothesis that side population hematopoietic stem cells (SP-HSC) would decrease with aging, correlating with IGF-1 and IL-6 levels and increases in bone marrow fat. Marrow was obtained from the femoral head and trochanteric region of the femur at surgery for total hip replacement (N=100). Whole trabecular marrow samples were ground in a sterile mortar and pestle and cellularity and fat content determined. Marrow and blood mononuclear cells were stained with Hoechst dye and the SP-HSC profiles acquired. Marrow stromal cells (MSC) were enumerated flow cytometrically employing the Stro-1 antibody, and clonally in the colony forming unit fibroblast (CFU-F) assay. Plasma levels of IGF-1 (ng/ml) and IL-6 (pg/ml) were measured by ELISA. SP-HSC in blood and bone marrow decreased with age but the quality of the surviving stem cells increased. MSC decreased non-significantly. IGF-1 levels (mean=30.7, SEM=2) decreased and IL-6 levels (mean=4.4, SEM=1) increased with age as did marrow fat (mean=1.2mmfat/g, SEM=0.04). There were no significant correlations between cytokine levels or fat and SP-HSC numbers. Stem cells appear to be progressively lost with aging and only the highest quality stem cells survive. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Histopathological Comparison between Bone Marrow- and Periodontium-derived Stem Cells for Bone Regeneration in Rabbit Calvaria.

PubMed

Kadkhoda, Z; Safarpour, A; Azmoodeh, F; Adibi, S; Khoshzaban, A; Bahrami, N

2016-01-01

Periodontitis is an important oral disease. Stem cell therapy has found its way in treatment of many diseases. To evaluate the regenerative potential of periodontal ligament-derived stem cells (PDLSCs) and osteoblast differentiated from PDLSC in comparison with bone marrow-derived mesenchymal stem cells (BM-MSCs) and pre-osteoblasts in calvarial defects. After proving the existence of surface markers by flow cytometry, BM-MSCs were differentiated into osteoblasts. 5 defects were made on rabbit calvaria. 3 of them were first covered with collagen membrane and then with BM-MSCs, PDLSCs, and pre-osteoblasts. The 4(th) defect was filled with collagen membrane and the 5(th) one was served as control. After 4 weeks, histological (quantitative) and histomorphological (qualitative) surveys were performed. Both cell lineages were positive for CD-90 cell marker, which was specifically related to stem cells. Alizarin red staining was done for showing mineral material. RT-PCR set up for the expression of Cbfa1 gene, BMP4 gene, and PGLAP gene, confirmed osteoblast differentiation. The findings indicated that although PDLSCs and pre-osteoblasts could be used for bone regeneration, the rate of regeneration in BM-MSCs-treated cavities was more significant (p<0.0001). The obtained results are probably attributable to the effective micro-environmental signals caused by different bone types and the rate of cell maturation.

Heterogeneity of circulating epithelial tumour cells from individual patients with respect to expression profiles and clonal growth (sphere formation) in breast cancer.

PubMed

Pizon, M; Zimon, D; Carl, S; Pachmann, U; Pachmann, K; Camara, O

2013-01-01

The detection of tumour cells circulating in the peripheral blood of patients with breast cancer is a sign that cells have been able to leave the primary tumour and survive in the circulation. However, in order to form metastases, they require additional properties such as the ability to adhere, self-renew, and grow. Here we present data that a variable fraction among the circulating tumour cells detected by the Maintrac(®) approach expresses mRNA of the stem cell gene NANOG and of the adhesion molecule vimentin and is capable of forming tumour spheres, a property ascribed to tumour-initiating cells (TICs). Between ten and 50 circulating epithelial antigen-positive cells detected by the Maintrac approach were selected randomly from each of 20 patients with breast cancer before and after surgery and were isolated using automated capillary aspiration and deposited individually onto slides for expression profiling. In addition, the circulating tumour cells were cultured without isolation among the white blood cells from 39 patients with breast cancer in different stages of disease using culture methods favouring growth of epithelial cells. Although no epithelial cell adhesion molecule (EpCAM)-positive cells expressing stem cell genes or the adhesion molecule vimentin was detected before surgery, 10%-20% of the cells were found to be positive for mRNA of these genes after surgery. Tumour spheres from circulating cells of 39 patients with different stages of breast cancer were grown without previous isolation in a fraction increasing with the aggressivity of the tumour. Here we show that among the peripherally circulating tumour cells, a variable fraction is able to express stem cell and adhesion properties and can be grown into tumour spheres, a property ascribed to cells capable of initiating tumours and metastases.

Periodontal ligament versus bone marrow mesenchymal stem cells in combination with Bio-Oss scaffolds for ectopic and in situ bone formation: A comparative study in the rat.

PubMed

Yu, Bo-Han; Zhou, Qian; Wang, Zuo-Lin

2014-08-01

The aim of this study was to compare the osteogenic effects of periodontal ligament stem cells (PDLSCs) versus bone marrow mesenchymal stem cells (BMMSCs) in combination with Bio-Oss scaffolds on subcutaneous and critical-size defects in the immunodeficient rat calvarium. PDLSCs and BMMSCs were obtained from the same canine donor. Twenty-four rats were randomly assigned to one of four experimental groups (n = 6 each): group A (no-graft negative control), group B (Bio-Oss positive control), group C (BMMSC/Bio-Oss test group), and group D (PDLSC/Bio-Oss test group). Eight weeks post-transplantation, ectopic and in situ bone regeneration was evaluated by micro-computed tomography (µ-CT), histology, histomorphometry, and immunohistochemistry. The stem cell/Bio-Oss constructs were significantly superior to the controls in terms of their ability to promote osteogenesis (p < 0.01), while the PDLSC/Bio-Oss construct tended to be superior to the BMMSC/Bio-Oss construct. Thus, engineered stem cell/Bio-Oss complexes can successfully reconstruct critical-size defects in rats, and PDLSCs and BMMSCs are both suitable as seed cells. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Isolation and Expansion of Hepatic Stem-like Cells from a Healthy Rat Liver and their Efficient Hepatic Differentiation of under Well-defined Vivo Hepatic like Microenvironment in a Multiwell Bioreactor

PubMed Central

Giri, Shibashish; Acikgöz, Ali; Bader, Augustinus

2015-01-01

Background Currently, undifferentiated cells are found in all tissue and term as local stem cells which are quiescent in nature and less in number under normal healthy conditions but activate upon injury and repair the tissue or organs via automated activating mechanism. Due to very scanty presence of local resident somatic local stem cells in healthy organs, isolation and expansion of these adult stems is an immense challenge for medical research and cell based therapy. Particularly organ like liver, there is an ongoing controversy about existence of liver stem cells. Methods Herein, Hepatic stem cells population was identified during culture of primary hepatocyte cells upon immediate isolation of primary hepatocyte cells. These liver stem cells has been expanded extensively and differentiated into primary hepatocytes under defined culture conditions in a nanostructured self assembling peptides modular bioreactor that mimic the state of art of liver microenvironment and compared with Matrigel as a positive control. Nanostructured self assembling peptides were used a defined extracellular matrix and Matrigel was used for undefined extracellular matrix. Proliferation of hepatic stem cells was investigated by two strategies. First strategy is to provide high concentration of hepatocyte growth factor (HGF) and second strategy is to evaluate the role of recombinant human erythropoietin (rHuEPO) in presence of trauma/ischemia cytokines (IL-6, TNF-α). Expansion to hepatic differentiation is observed by morphological analysis and was evaluated for the expression of hepatocyte-specific genes using RT-PCR and biochemical methods. Results Hepatocyte-specific genes are well expressed at final stage (day 21) of differentiation period. The differentiated hepatocytes exhibited functional hepatic characteristics such as albumin secretion, urea secretion and cytochrome P450 expression. Additionally, immunofluorescence analysis revealed that hepatic stem cells derived hepatocytes exhibited mature hepatocyte markers (albumin, CK-19, CPY3A1, alpha 1-antitrypsin). Expansion and hepatic differentiation was efficiently in nanostructured self assembling peptides without such batch to batch variation while there was much variation in Matrigel coated bioreactor. In conclusion, the results of the study suggest that the nanostructured self assembling peptides coated bioreactor supports expansion as well as hepatic differentiation of liver stem cells which is superior than Matrigel. Conclusion This defined microenvironment conditions in bioreactor module can be useful for research involving bioartificial liver system, stem cell research and engineered liver tissue which could contribute to regenerative cell therapies or drug discovery and development. PMID:26155038

Isolation, expansion, and differentiation of goat adipose-derived stem cells.

PubMed

Ren, Yu; Wu, Haiqing; Zhou, Xueyuan; Wen, Jianxun; Jin, Muzi; Cang, Ming; Guo, Xudong; Wang, Qinglian; Liu, Dongjun; Ma, Yuzhen

2012-08-01

A goat adipose-derived stem cell (ADSC) line was established and compared to a rat line. Goat ADSC cells had normal diploidy after subculture. Proliferation of goat ADSCs was faster than rat cells in the same conditions. Both rat and goat ADSCs stained positively for vimentin, CD49d, CD44 and CD13, but stained negatively for CD34 and CD106. Bone nodules were apparent, and alizarin staining was positive after osteogenic induction. Cells expressing osteocalcin were positive by alkaline phosphatase (ALP) staining. After osteogenic induction, ossification nodules of goat ADSCs were larger than in rats, with dense ALP staining. Adipogenic induction resulting in lipid droplets and peroxisome proliferator-activated receptor (PPARγ2) expression were observed. Cartilage lacunae were formed and COL2A1 was expressed. More cartilage lacunae with better morphology were seen following differentiation of goat ADSC's using the hang-drop method. For goat ADSCs, results with both adherent-induced and hanging-drop induced cultures were better than for three-dimensional cultures. Copyright © 2012. Published by Elsevier India Pvt Ltd.

Application of a Novel Population of Multipotent Stem Cells Derived from Skin Fibroblasts as Donor Cells in Bovine SCNT

PubMed Central

Pan, Shaohui; Chen, Wuju; Liu, Xu; Xiao, Jiajia; Wang, Yanqin; Liu, Jun; Du, Yue; Wang, Yongsheng; Zhang, Yong

2015-01-01

Undifferentiated stem cells are better donor cells for somatic cell nuclear transfer (SCNT), resulting in more offspring than more differentiated cells. While various stem cell populations have been confirmed to exist in the skin, progress has been restricted due to the lack of a suitable marker for their prospective isolation. To address this fundamental issue, a marker is required that could unambiguously prove the differentiation state of the donor cells. We therefore utilized magnetic activated cell sorting (MACS) to separate a homogeneous population of small SSEA-4+ cells from a heterogeneous population of bovine embryonic skin fibroblasts (BEF). SSEA-4+ cells were 8-10 μm in diameter and positive for alkaline phosphatase (AP). The percentage of SSEA-4+ cells within the cultured BEF population was low (2-3%). Immunocytochemistry and PCR analyses revealed that SSEA-4+ cells expressed pluripotency-related markers, and could differentiate into cells comprising all three germ layers in vitro. They remained undifferentiated over 20 passages in suspension culture. In addition, cloned embryos derived from SSEA-4 cells showed significant differences in cleavage rate and blastocyst development when compared with those from BEF and SSEA-4− cells. Moreover, blastocysts derived from SSEA-4+ cells showed a higher total cell number and lower apoptotic index as compared to BEF and SSEA-4– derived cells. It is well known that nuclei from pluripotent stem cells yield a higher cloning efficiency than those from adult somatic cells, however, pluripotent stem cells are relatively difficult to obtain from bovine. The SSEA-4+ cells described in the current study provide an attractive candidate for SCNT and a promising platform for the generation of transgenic cattle. PMID:25602959

Application of a novel population of multipotent stem cells derived from skin fibroblasts as donor cells in bovine SCNT.

PubMed

Pan, Shaohui; Chen, Wuju; Liu, Xu; Xiao, Jiajia; Wang, Yanqin; Liu, Jun; Du, Yue; Wang, Yongsheng; Zhang, Yong

2015-01-01

Undifferentiated stem cells are better donor cells for somatic cell nuclear transfer (SCNT), resulting in more offspring than more differentiated cells. While various stem cell populations have been confirmed to exist in the skin, progress has been restricted due to the lack of a suitable marker for their prospective isolation. To address this fundamental issue, a marker is required that could unambiguously prove the differentiation state of the donor cells. We therefore utilized magnetic activated cell sorting (MACS) to separate a homogeneous population of small SSEA-4(+) cells from a heterogeneous population of bovine embryonic skin fibroblasts (BEF). SSEA-4(+) cells were 8-10 μm in diameter and positive for alkaline phosphatase (AP). The percentage of SSEA-4(+) cells within the cultured BEF population was low (2-3%). Immunocytochemistry and PCR analyses revealed that SSEA-4(+) cells expressed pluripotency-related markers, and could differentiate into cells comprising all three germ layers in vitro. They remained undifferentiated over 20 passages in suspension culture. In addition, cloned embryos derived from SSEA-4 cells showed significant differences in cleavage rate and blastocyst development when compared with those from BEF and SSEA-4(-) cells. Moreover, blastocysts derived from SSEA-4(+) cells showed a higher total cell number and lower apoptotic index as compared to BEF and SSEA-4(-) derived cells. It is well known that nuclei from pluripotent stem cells yield a higher cloning efficiency than those from adult somatic cells, however, pluripotent stem cells are relatively difficult to obtain from bovine. The SSEA-4(+) cells described in the current study provide an attractive candidate for SCNT and a promising platform for the generation of transgenic cattle.

Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells

PubMed Central

Akopian, Veronika; Beil, Stephen; Benvenisty, Nissim; Brehm, Jennifer; Christie, Megan; Ford, Angela; Fox, Victoria; Gokhale, Paul J.; Healy, Lyn; Holm, Frida; Hovatta, Outi; Knowles, Barbara B.; Ludwig, Tenneille E.; McKay, Ronald D. G.; Miyazaki, Takamichi; Nakatsuji, Norio; Oh, Steve K. W.; Pera, Martin F.; Rossant, Janet; Stacey, Glyn N.; Suemori, Hirofumi

2010-01-01

There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support, but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study, five separate laboratories, each with experience in human embryonic stem cell culture, used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods, with propagation in the presence of Knockout Serum Replacer, FGF-2, and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment, death, and differentiated morphology by phase contrast microscopy, for growth by serial cell counts, and for maintenance of stem cell surface marker expression by flow cytometry. Of the eight culture systems, only the control and those based on two commercial media, mTeSR1 and STEMPRO, supported maintenance of most cell lines for ten passages. Cultures grown in the remaining media failed before this point due to lack of attachment, cell death, or overt cell differentiation. Possible explanations for relative success of the commercial formulations in this study, and the lack of success with other formulations from academic groups compared to previously published results, include: the complex combination of growth factors present in the commercial preparations; improved development, manufacture, and quality control in the commercial products; differences in epigenetic adaptation to culture in vitro between different ES cell lines grown in different laboratories. PMID:20186512

Sub-physiological oxygen levels optimal for growth and survival of human atrial cardiac stem cells.

PubMed

RajendranNair, Deepthi Sreerengam; Karunakaran, Jayakumar; Nair, Renuka R

2017-08-01

Cardiac stem cells reside in niches where the oxygen levels are close to 3%. For cytotherapy, cells are conventionally expanded in ambient oxygen (21% O 2 ) which represents hyperoxia compared to the oxygen tension of niches. Cardiosphere-derived cells (CDCs) are then transplanted to host tissue with lower-O 2 levels. The high-O 2 gradient can reduce the efficacy of cultured cells. Based on the assumption that minimizing injury due to O 2 gradients will enhance the yield of functionally efficient cells, CDCs were cultured in 3% O 2 and compared with cells maintained in ambient O 2 . CDCs were isolated from human right atrial explants and expanded in parallel in 21 and 3% oxygen and compared with regard to survival, proliferation, and retention of stemness. Increased cell viability even in the tenth passage and enhanced cardiosphere formation was observed in cells expanded in 3% O 2 . The cell yield from seven passages was fourfold higher for cells cultured in 3% O 2 . Preservation of stemness in hypoxic environment was evident from the proportion of c-kit-positive cells and reduced myogenic differentiation. Hypoxia promoted angiogenesis and reduced the tendency to differentiate to noncardiac lineages (adipocytes and osteocytes). Mimicking the microenvironment at transplantation, when shifted to 5% O 2 , viability and proliferation rate were significantly higher for CDCs expanded in 3% O 2 . Expansion of CDCs, from atria in sub-physiological oxygen, helps in obtaining a higher yield of healthy cells with better preservation of stem cell characteristics. The cells so cultured are expected to improve engraftment and facilitate myocardial regeneration.

Spectrum of bacteremia in posthematopoietic stem cell transplant patients from an Indian center.

PubMed

Ghafur, A; Devarajan, V; Raj, R; Easow, J; Raja, T

2016-01-01

Despite the relatively low prevalence of Gram-positive bacteremic infections in Indian oncology patients, glycopeptides are extensively used for empirical management of febrile neutropenia. Our aim was to analyze the spectrum of bacteremia in posthematopoietic stem cell transplant (HSCT) recipients in our center and make a recommendation on glycopeptide use in this patient population. Retrospective analysis of bacteremic data from HSCT recipients in a tertiary care oncology and transplant center from South India, between 2011 and 2013. In 217 patients, 52 bacteremic episodes were identified. The majority of the isolates were Gram-negatives (88.4%) with very few Gram-positives (7.69%). Glycopeptides need not be included in the empirical antibiotic regimen in post-HSCT settings with very low Gram-positive infection rates.

Chondrocyte Differentiation of Human Endometrial Gland-Derived MSCs in Layered Cell Sheets

PubMed Central

Shimizu, Tatsuya; Yamato, Masayuki; Umezawa, Akihiro; Okano, Teruo

2013-01-01

Recently, regenerative medicine using engineered three-dimensional (3D) tissues has been focused. In the fields of cell therapy and regenerative medicine, mesenchymal stem cells (MSCs) are attractive autologous cell sources. While, in bioengineered tissues, a 3D environment may affect the differentiation of the stem cells, little is known regarding the effect of 3D environment on cellular differentiation. In this study, MSC differentiation in in vitro 3D tissue models was assessed by human endometrial gland-derived MSCs (hEMSCs) and cell sheet technology. hEMSC sheets were layered into cell-dense 3D tissues and were cultured on porous membranes. The tissue sections revealed that chondrocyte-like cells were found within the multilayered cell sheets even at 24 h after layering. Immunostainings of chondrospecific markers were positive within those cell sheet constructs. In addition, sulfated glycosaminoglycan accumulation within the tissues increased in proportion to the numbers of layered cell sheets. The findings suggested that a high cell density and hypoxic environment in 3D tissues by layering cell sheets might accelerate a rapid differentiation of hEMSCs into chondrocytes without the help of chondro-differentiation reagents. These tissue models using cell sheets would give new insights to stem cell differentiation in 3D environment and contribute to the future application of stem cells to cartilage regenerative therapy. PMID:24348153

Serum replacement with a growth factor-free synthetic substance in culture medium contributes to effective establishment of mouse embryonic stem cells of various origins.

PubMed

Lee, Seung Tae; Oh, Se Woong; Kim, Dae Yong; Han, Jae Yong; Moon, Shin Yong; Lim, Jeong Mook

2006-10-01

To evaluate whether serum replacement with growth factor-free synthetic substances contributed to the effective establishment of embryonic stem (ES) cells. Randomized, prospective model study. Gamete and stem cell biotechnology laboratory at Seoul National University in Korea. F1 (C57BL6 x DBA2) mice. Blastocysts of different origins were cultured in serum-replaced media. Embryonic stem cell establishment. Eight batches of ES cells were established from colony-forming inner cell mass cells after the replacement of fetal bovine serum (FBS) with synthetic knockout serum replacement (KSR) in mkDMEM. The established cells were positive for ES cell markers and formed both embryoid bodies in vitro and teratomas in vivo, but the established cell batches and control (transformed) ES cells responded differently to the culture media. Higher levels of cell viability were detected after the replacement with the 75:25 FBS-KSR mixture than with any other mixtures, and a gradual decrease in viability was detected as the KSR volume ratio was increased. The 75:25 FBS-KSR mixture-containing medium supported ES cell establishment of outbred ICR, F1, and F2 of C57BL6/DBA2; F1 parthenogenetic and ES cell-complemented tetraploid blastocysts; and single ES-cell cultures. A serum-replaced medium could be used for effective ES-cell establishment of various origins.

Daughters of the Enamel Organ: Development, Fate, and Function of the Stratum Intermedium, Stellate Reticulum, and Outer Enamel Epithelium

PubMed Central

Liu, Hui; Yan, Xiulin; Pandya, Mirali; Luan, Xianghong

2016-01-01

The tooth enamel organ (EO) is a complex epithelial cell assembly involved in multiple aspects of tooth development, including amelogenesis. The present study focuses on the role of the nonameloblast layers of the EO, the stratum intermedium, the stellate reticulum, and the outer enamel epithelium (OEE). The secretory stage stratum intermedium was distinguished by p63-positive epithelial stem cell marks, highly specific alkaline phosphatase labeling, as well as multiple desmosomes and gap junctions. At the location of the presecretory stage stellate reticulum, the pre-eruption EO prominently featured the papillary layer (PL) as a keratin immunopositive network of epithelial strands between tooth crowns and oral epithelium. PL cell strands contained numerous p63-positive epithelial stem cells, while BrdU proliferative cells were detected at the outer boundaries of the PL, suggesting that the stellate reticulum/PL epithelial cell sheath proliferated to facilitate an epithelial seal during tooth eruption. Comparative histology studies demonstrated continuity between the OEE and the general lamina of continuous tooth replacement in reptiles, and the outer layer of Hertwig's epithelial root sheath in humans, implicating the OEE as the formative layer for continuous tooth replacement and tooth root extension. Cell fate studies in organ culture verified that the cervical portion of the mouse molar EO gave rise to Malassez rest-like cell islands. Together, these studies indicate that the nonameloblast layers of the EO play multiple roles during odontogenesis, including the maintenance of several p63-positive stem cell reservoirs, a role during tooth root morphogenesis and tooth succession, a stabilizing function for the ameloblast layer, the facilitation of ion transport from the EO capillaries to the enamel layer, as well as safe and seamless tooth eruption. PMID:27611344

Daughters of the Enamel Organ: Development, Fate, and Function of the Stratum Intermedium, Stellate Reticulum, and Outer Enamel Epithelium.

PubMed

Liu, Hui; Yan, Xiulin; Pandya, Mirali; Luan, Xianghong; Diekwisch, Thomas G H

2016-09-09

The tooth enamel organ (EO) is a complex epithelial cell assembly involved in multiple aspects of tooth development, including amelogenesis. The present study focuses on the role of the nonameloblast layers of the EO, the stratum intermedium, the stellate reticulum, and the outer enamel epithelium (OEE). The secretory stage stratum intermedium was distinguished by p63-positive epithelial stem cell marks, highly specific alkaline phosphatase labeling, as well as multiple desmosomes and gap junctions. At the location of the presecretory stage stellate reticulum, the pre-eruption EO prominently featured the papillary layer (PL) as a keratin immunopositive network of epithelial strands between tooth crowns and oral epithelium. PL cell strands contained numerous p63-positive epithelial stem cells, while BrdU proliferative cells were detected at the outer boundaries of the PL, suggesting that the stellate reticulum/PL epithelial cell sheath proliferated to facilitate an epithelial seal during tooth eruption. Comparative histology studies demonstrated continuity between the OEE and the general lamina of continuous tooth replacement in reptiles, and the outer layer of Hertwig's epithelial root sheath in humans, implicating the OEE as the formative layer for continuous tooth replacement and tooth root extension. Cell fate studies in organ culture verified that the cervical portion of the mouse molar EO gave rise to Malassez rest-like cell islands. Together, these studies indicate that the nonameloblast layers of the EO play multiple roles during odontogenesis, including the maintenance of several p63-positive stem cell reservoirs, a role during tooth root morphogenesis and tooth succession, a stabilizing function for the ameloblast layer, the facilitation of ion transport from the EO capillaries to the enamel layer, as well as safe and seamless tooth eruption.

Translational Significance of p53 Loss of Heterozygosity in Breast Cancer

DTIC Science & Technology

2017-09-01

LOH), radiation , chemotherapy, Her2 positive breast cancer. 3. ACCOMPLISHMENTS: The major goals of the project. Major Task 1. Determine the...Intestinal Stem Cell-Derived Lineage Following  Radiation -Induced Gut Injury in Mice. Stem Cell Reorts. 6(6):815-24. 5. Snider AJ, Bialkowska AB, Ghaleb...and Amr M. Ghaleb. 2015. Krüppel-like factor 4 is a radioprotective factor for the intestine following γ- radiation - induced gut injury in mice. Am. J

JNK Controls the Onset of Mitosis in Planarian Stem Cells and Triggers Apoptotic Cell Death Required for Regeneration and Remodeling

PubMed Central

Almuedo-Castillo, María; Crespo, Xenia; Seebeck, Florian; Bartscherer, Kerstin; Salò, Emili; Adell, Teresa

2014-01-01

Regeneration of lost tissues depends on the precise interpretation of molecular signals that control and coordinate the onset of proliferation, cellular differentiation and cell death. However, the nature of those molecular signals and the mechanisms that integrate the cellular responses remain largely unknown. The planarian flatworm is a unique model in which regeneration and tissue renewal can be comprehensively studied in vivo. The presence of a population of adult pluripotent stem cells combined with the ability to decode signaling after wounding enable planarians to regenerate a complete, correctly proportioned animal within a few days after any kind of amputation, and to adapt their size to nutritional changes without compromising functionality. Here, we demonstrate that the stress-activated c-jun–NH2–kinase (JNK) links wound-induced apoptosis to the stem cell response during planarian regeneration. We show that JNK modulates the expression of wound-related genes, triggers apoptosis and attenuates the onset of mitosis in stem cells specifically after tissue loss. Furthermore, in pre-existing body regions, JNK activity is required to establish a positive balance between cell death and stem cell proliferation to enable tissue renewal, remodeling and the maintenance of proportionality. During homeostatic degrowth, JNK RNAi blocks apoptosis, resulting in impaired organ remodeling and rescaling. Our findings indicate that JNK-dependent apoptotic cell death is crucial to coordinate tissue renewal and remodeling required to regenerate and to maintain a correctly proportioned animal. Hence, JNK might act as a hub, translating wound signals into apoptotic cell death, controlled stem cell proliferation and differentiation, all of which are required to coordinate regeneration and tissue renewal. PMID:24922054

A role for GPx3 in activity of normal and leukemia stem cells

PubMed Central

Herault, Olivier; Hope, Kristin J.; Deneault, Eric; Mayotte, Nadine; Chagraoui, Jalila; Wilhelm, Brian T.; Cellot, Sonia; Sauvageau, Martin; Andrade-Navarro, Miguel A.; Hébert, Josée

2012-01-01

The determinants of normal and leukemic stem cell self-renewal remain poorly characterized. We report that expression of the reactive oxygen species (ROS) scavenger glutathione peroxidase 3 (GPx3) positively correlates with the frequency of leukemia stem cells (LSCs) in Hoxa9+Meis1-induced leukemias. Compared with a leukemia with a low frequency of LSCs, a leukemia with a high frequency of LSCs showed hypomethylation of the Gpx3 promoter region, and expressed high levels of Gpx3 and low levels of ROS. LSCs and normal hematopoietic stem cells (HSCs) engineered to express Gpx3 short hairpin RNA (shRNA) were much less competitive in vivo than control cells. However, progenitor cell proliferation and differentiation was not affected by Gpx3 shRNA. Consistent with this, HSCs overexpressing Gpx3 were significantly more competitive than control cells in long-term repopulation experiments, and overexpression of the self-renewal genes Prdm16 or Hoxb4 boosted Gpx3 expression. In human primary acute myeloid leukemia samples, GPX3 expression level directly correlated with adverse prognostic outcome, revealing a potential novel target for the eradication of LSCs. PMID:22508837

Regeneration of cervical reserve cell-like cells from human induced pluripotent stem cells (iPSCs): A new approach to finding targets for cervical cancer stem cell treatment.

PubMed

Sato, Masakazu; Kawana, Kei; Adachi, Katsuyuki; Fujimoto, Asaha; Yoshida, Mitsuyo; Nakamura, Hiroe; Nishida, Haruka; Inoue, Tomoko; Taguchi, Ayumi; Ogishima, Juri; Eguchi, Satoko; Yamashita, Aki; Tomio, Kensuke; Wada-Hiraike, Osamu; Oda, Katsutoshi; Nagamatsu, Takeshi; Osuga, Yutaka; Fujii, Tomoyuki

2017-06-20

Cervical reserve cells are epithelial progenitor cells that are pathologically evident as the origin of cervical cancer. Thus, investigating the characteristics of cervical reserve cells could yield insight into the features of cervical cancer stem cells (CSCs). In this study, we established a method for the regeneration of cervical reserve cell-like properties from human induced pluripotent stem cells (iPSCs) and named these cells induced reserve cell-like cells (iRCs). Approximately 70% of iRCs were positive for the reserve cell markers p63, CK5 and CK8. iRCs also expressed the SC junction markers CK7, AGR2, CD63, MMP7 and GDA. While iRCs expressed neither ERα nor ERβ, they expressed CA125. These data indicated that iRCs possessed characteristics of cervical epithelial progenitor cells. iRCs secreted higher levels of several inflammatory cytokines such as macrophage migration inhibitory factor (MIF), soluble intercellular adhesion molecule 1 (sICAM-1) and C-X-C motif ligand 10 (CXCL-10) compared with normal cervical epithelial cells. iRCs also expressed human leukocyte antigen-G (HLA-G), which is an important cell-surface antigen for immune tolerance and carcinogenesis. Together with the fact that cervical CSCs can originate from reserve cells, our data suggested that iRCs were potent immune modulators that might favor cervical cancer cell survival. In conclusion, by generating reserve cell-like properties from iPSCs, we provide a new approach that may yield new insight into cervical cancer stem cells and help find new oncogenic targets.

Multipotent progenitor cells are present in human peripheral blood.

PubMed

Cesselli, Daniela; Beltrami, Antonio Paolo; Rigo, Silvia; Bergamin, Natascha; D'Aurizio, Federica; Verardo, Roberto; Piazza, Silvano; Klaric, Enio; Fanin, Renato; Toffoletto, Barbara; Marzinotto, Stefania; Mariuzzi, Laura; Finato, Nicoletta; Pandolfi, Maura; Leri, Annarosa; Schneider, Claudio; Beltrami, Carlo Alberto; Anversa, Piero

2009-05-22

To determine whether the peripheral blood in humans contains a population of multipotent progenitor cells (MPCs), products of leukapheresis were obtained from healthy donor volunteers following the administration of granulocyte colony-stimulating factor. Small clusters of adherent proliferating cells were collected, and these cells continued to divide up to 40 population doublings without reaching replicative senescence and growth arrest. MPCs were positive for the transcription factors Nanog, Oct3/4, Sox2, c-Myc, and Klf4 and expressed several antigens characteristic of mesenchymal stem cells. However, they were negative for markers of hematopoietic stem/progenitor cells and bone marrow cell lineages. MPCs had a cloning efficiency of approximately 3%, and following their expansion, retained a highly immature phenotype. Under permissive culture conditions, MPCs differentiated into neurons, glial cells, hepatocytes, cardiomyocytes, endothelial cells, and osteoblasts. Moreover, the gene expression profile of MPCs partially overlapped with that of neural and embryonic stem cells, further demonstrating their primitive, uncommitted phenotype. Following subcutaneous transplantation in nonimmunosuppressed mice, MPCs migrated to distant organs and integrated structurally and functionally within the new tissue, acquiring the identity of resident parenchymal cells. In conclusion, undifferentiated cells with properties of embryonic stem cells can be isolated and expanded from human peripheral blood after granulocyte colony-stimulating factor administration. This cell pool may constitute a unique source of autologous cells with critical clinical import.

Characterization of Nestin-positive stem Leydig cells as a potential source for the treatment of testicular Leydig cell dysfunction

PubMed Central

Jiang, Mei Hua; Cai, Bing; Tuo, Ying; Wang, Jiancheng; Zang, Zhi Jun; Tu, Xiang'an; Gao, Yong; Su, Zhijian; Li, Weiqiang; Li, Guilan; Zhang, Min; Jiao, Jianwei; Wan, Zi; Deng, Chunhua; Lahn, Bruce T; Xiang, Andy Peng

2014-01-01

The ability to identify and isolate lineage-specific stem cells from adult tissues could facilitate cell replacement therapy. Leydig cells (LCs) are the primary source of androgen in the mammalian testis, and the prospective identification of stem Leydig cells (SLCs) may offer new opportunities for treating testosterone deficiency. Here, in a transgenic mouse model expressing GFP driven by the Nestin (Nes) promoter, we observed Nes-GFP+ cells located in the testicular interstitial compartment where SLCs normally reside. We showed that these Nes-GFP+ cells expressed LIFR and PDGFR-α, but not LC lineage markers. We further observed that these cells were capable of clonogenic self-renewal and extensive proliferation in vitro and could differentiate into neural or mesenchymal cell lineages, as well as LCs, with the ability to produce testosterone, under defined conditions. Moreover, when transplanted into the testes of LC-disrupted or aging models, the Nes-GFP+ cells colonized the interstitium and partially increased testosterone production, and then accelerated meiotic and post-meiotic germ cell recovery. In addition, we further demonstrated that CD51 might be a putative cell surface marker for SLCs, similar with Nestin. Taken together, these results suggest that Nes-GFP+ cells from the testis have the characteristics of SLCs, and our study would shed new light on developing stem cell replacement therapy for testosterone deficiency. PMID:25418539

Modeling Aggressive Medulloblastoma Using Human Induced Pluripotent Stem Cells

DTIC Science & Technology

2017-09-01

and Myc in turn induces expression of AT1R creating a positive feedback loop and development of aggression tumor phenotype. The therapeutic...strengths are the relevant expertise of the applicant and his collaborating team, the novel paracrine positive feedback loop in EC-tumor cell...to as MYC-driven MB. The molecular mechanisms that drive MYC hyper -activation in MB remain incompletely understood. MB cells in actual tumors interact

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.

Fluorescent nanodiamonds enable quantitative tracking of human mesenchymal stem cells in miniature pigs

NASA Astrophysics Data System (ADS)

Su, Long-Jyun; Wu, Meng-Shiue; Hui, Yuen Yung; Chang, Be-Ming; Pan, Lei; Hsu, Pei-Chen; Chen, Yit-Tsong; Ho, Hong-Nerng; Huang, Yen-Hua; Ling, Thai-Yen; Hsu, Hsao-Hsun; Chang, Huan-Cheng

2017-03-01

Cell therapy is a promising strategy for the treatment of human diseases. While the first use of cells for therapeutic purposes can be traced to the 19th century, there has been a lack of general and reliable methods to study the biodistribution and associated pharmacokinetics of transplanted cells in various animal models for preclinical evaluation. Here, we present a new platform using albumin-conjugated fluorescent nanodiamonds (FNDs) as biocompatible and photostable labels for quantitative tracking of human placenta choriodecidual membrane-derived mesenchymal stem cells (pcMSCs) in miniature pigs by magnetic modulation. With this background-free detection technique and time-gated fluorescence imaging, we have been able to precisely determine the numbers as well as positions of the transplanted FND-labeled pcMSCs in organs and tissues of the miniature pigs after intravenous administration. The method is applicable to single-cell imaging and quantitative tracking of human stem/progenitor cells in rodents and other animal models as well.

Tacrolimus, Bortezomib, & Thymoglobulin in Preventing Low Toxicity GVHD in Donor Blood Stem Cell Transplant Patients

ClinicalTrials.gov

2018-03-30

Acute Leukemia; Chronic Lymphocytic Leukemia; Chronic Myelogenous Leukemia, BCR-ABL1 Positive; Diffuse Large B-Cell Lymphoma; Follicular Lymphoma; Graft Versus Host Disease; Mantle Cell Lymphoma; Marginal Zone Lymphoma; Myelodysplastic Syndrome; Myelofibrosis; Myeloproliferative Neoplasm; Small Lymphocytic Lymphoma

c-Kit-Mediated Functional Positioning of Stem Cells to Their Niches Is Essential for Maintenance and Regeneration of Adult Hematopoiesis

PubMed Central

Kimura, Yuki; Ding, Bisen; Imai, Norikazu; Nolan, Daniel J.; Butler, Jason M.; Rafii, Shahin

2011-01-01

The mechanism by which hematopoietic stem and progenitor cells (HSPCs) through interaction with their niches maintain and reconstitute adult hematopoietic cells is unknown. To functionally and genetically track localization of HSPCs with their niches, we employed novel mutant loxPs, lox66 and lox71 and Cre-recombinase technology to conditionally delete c-Kit in adult mice, while simultaneously enabling GFP expression in the c-Kit-deficient cells. Conditional deletion of c-Kit resulted in hematopoietic failure and splenic atrophy both at steady state and after marrow ablation leading to the demise of the treated adult mice. Within the marrow, the c-Kit-expressing GFP+ cells were positioned to Kit ligand (KL)-expressing niche cells. This c-Kit-mediated cellular adhesion was essential for long-term maintenance and expansion of HSPCs. These results lay the foundation for delivering KL within specific niches to maintain and restore hematopoiesis. PMID:22046410

Monolayer culturing and cloning of human pluripotent stem cells on laminin-521-based matrices under xeno-free and chemically defined conditions.

PubMed

Rodin, Sergey; Antonsson, Liselotte; Hovatta, Outi; Tryggvason, Karl

2014-10-01

A robust method for culturing human pluripotent stem (hPS) cells under chemically defined and xeno-free conditions is an important tool for stem cell research and for the development of regenerative medicine. Here, we describe a protocol for monolayer culturing of Oct-4-positive hPS cells on a specific laminin-521 (LN-521) isoform, under xeno-free and chemically defined conditions. The cells are dispersed into single-cell suspension and then plated on LN-521 isoform at densities higher than 5,000 cells per cm², where they attach, migrate and survive by forming small monolayer cell groups. The cells avidly divide and expand horizontally until the entire dish is covered by a confluent monolayer. LN-521, in combination with E-cadherin, allows cloning of individual hPS cells in separate wells of 96-well plates without the presence of rho-associated protein kinase (ROCK) inhibitors or any other inhibitors of anoikis. Characterization of cells maintained for several months in culture reveals pluripotency with a minimal degree of genetic abnormalities.

Rapid micropatterning of cell lines and human pluripotent stem cells on elastomeric membranes.

PubMed

Paik, Isha; Scurr, David J; Morris, Bryan; Hall, Graham; Denning, Chris; Alexander, Morgan R; Shakesheff, Kevin M; Dixon, James E

2012-10-01

Tissue function during development and in regenerative medicine completely relies on correct cell organization and patterning at micro and macro scales. We describe a rapid method for patterning mammalian cells including human embryonic stem cells (HESCs) and induced pluripotent stem cells (iPSCs) on elastomeric membranes such that micron-scale control of cell position can be achieved over centimeter-length scales. Our method employs surface engineering of hydrophobic polydimethylsiloxane (PDMS) membranes by plasma polymerization of allylamine. Deposition of plasma polymerized allylamine (ppAAm) using our methods may be spatially restricted using a micro-stencil leaving faithful hydrophilic ppAAm patterns. We employed airbrushing to create aerosols which deposit extracellular matrix (ECM) proteins (such as fibronectin and Matrigel™) onto the same patterned ppAAm rich regions. Cell patterns were created with a variety of well characterized cell lines (e.g., NIH-3T3, C2C12, HL1, BJ6, HESC line HUES7, and HiPSC line IPS2). Individual and multiple cell line patterning were also achieved. Patterning remains faithful for several days and cells are viable and proliferate. To demonstrate the utility of our technique we have patterned cells in a variety of configurations. The ability to rapidly pattern cells at high resolution over macro scales should aid future tissue engineering efforts for regenerative medicine applications and in creating in vitro stem cell niches. Copyright © 2012 Wiley Periodicals, Inc.

Successful vitrification of human amnion-derived mesenchymal stem cells.

PubMed

Moon, Jeong Hee; Lee, Jung Ryeol; Jee, Byung Chul; Suh, Chang Suk; Kim, Seok Hyun; Lim, Hyun Jung; Kim, Hae Kwon

2008-08-01

A cryopreservation protocol for human amnion-derived mesenchymal stem cells (HAMs) is required because these cells cannot survive for long periods in culture. The aim of this study was to determine whether vitrification is a useful freezing method for storage of HAMs. HAMs were cryopreserved using vitrification method. The morphology and viability of thawed HAMs was evaluated by Trypan Blue staining. The expression of several embryonic stem cell (ESC) markers was evaluated using flow cytometry, RT-PCR and immunocytochemistry. Von Kossa, Oil Red O and Alcian Blue staining were used to asses the differentiation potential of thawed HAMs. The post-thawing viability of HAMs was 84.3 +/- 3.2% (Mean +/- SD, n = 10). The thawed HAMs showed morphological characteristics indistinguishable from the non-vitrified fresh HAMs. The expression of surface antigens (strong positive for CD44, CD49d, CD59, CD90, CD105 and HLA-ABC; weak positive for HLA-G; negative for CD31, CD34, CD45, CD106, CD117 and HLA-DR) and the expression of ESC markers [CK18, fibroblast growth factor-5, GATA-4, neural cell adhesion molecule, Nestin, Oct-4, stem cell factor, HLA-ABC, Vimentin, bone morphogenetic protein (BMP) 4, hepatocyte nuclear factor 4 alpha (HNF-4 alpha), Pax-6, alpha-fetoprotein, Brachyury, BMP-2, TRA-1-60, stage-specific embryonic antigen (SSEA-3, SSEA-4)] were maintained in the vitrified-thawed HAMs. The thawed HAMs retained ability to differentiate into osteoblasts, adipocytes and chondrocytes under appropriate culture conditions. Our results suggest that vitrification is a reliable and effective method for cryopreservation of HAMs.

Correlation of cancer stem cell markers and immune cell markers in resected non-small cell lung cancer.

PubMed

Huang, Zhaoqin; Yu, Haining; Zhang, Jianbo; Jing, Haiyan; Zhu, Wanqi; Li, Xiaolin; Kong, Lingling; Xing, Ligang; Yu, Jinming; Meng, Xiangjiao

2017-01-01

Background: Recent studies confirmed that immunotherapy showed prominent efficacy in non-small cell lung cancer (NSCLC). Cancer stem cells/cancer initiating cells are resistant to anticancer treatment. The purpose of the study was to analyze the correlation of cancer stem cells/cancer initiating cells and tumor-infiltrating immune cells in NSCLC. Methods: CD133, octamer 4 (OCT-4), CD8, CD56, human leukocyte antigen (HLA) class I and programmed death ligand-1 (PD-L1) were assessed in 172 resected NSCLC samples. The staining was analyzed and scored by the pathologist who was blinded to the clinical pathological data of the patients. Results: High CD8+ T cell infiltration was correlated significantly with squamous cell carcinoma histology (p=0.008). High PD-L1 expression (≥10%) was associated with high tumor status (p=0.043). Pearson's correlation test showed that CD56+ cells were negatively correlated with CD133 expression (r=-0.361, p<0.001) and weakly correlated with negative OCT-4 expression (r=-0.180, p=0.018). There was a strong positive correlation between CD8 and HLA class I (r=0.573, p<0.001). In the survival analysis, high CD8+ T cell infiltration is an independent predictor of improved disease-free survival and overall survival. Patients with low CD133 expression and high CD56 expression had a longer overall survival than those with high CD133 expression and/or low CD56 expression (p=0.013). Conclusion: There is a negative correlation between CD56+ cells and cancer stem cell markers. This correlation may confirm the possibility that natural killer cells can target CD133+ cancer stem cells/cancer initiating cells in non-small cell lung cancer.

The Meaning of Disease and Spiritual Responses to Stressors in Adults With Acute Leukemia Undergoing Hematopoietic Stem Cell Transplantation.

PubMed

Farsi, Zahra

2015-12-01

Some studies have shown that patients with cancer may experience significant spiritual distress as well as spiritual growth, that there is a positive association between spirituality and coping, and that positive religious coping predicts enhanced health outcomes. This study was designed to help explain how the meaning of disease and spiritual responses to threatening stressors influence the final experiential outcomes of adults with leukemia undergoing hematopoietic stem cell transplantation in Iran. This grounded theory study conducted in-depth interviews between 2009 and 2011 on 10 adults in Iran with leukemia undergoing hematopoietic stem cell transplantation. Recorded audio interviews were transcribed verbatim in Persian and coded and analyzed using Corbin and Strauss (2008)'s approach. Main categories that emerged from data included "experiencing the meaning of cancer"; "changing perceptions of death, life and health"; and "moving toward perfection and sublimity." "Finding meaning" was the main concept that defined the final outcome of the experience of participants. Understanding the meaning to patients of disease and treatments may help healthcare providers better appreciate the patients' perspective and improve the physician-patient relationship. Nurses are well positioned to play a decisive role in helping patients cope effectively with their treatment process and in helping ensure positive outcomes for treatments through their helping patients find the unique meaning of their experience.

Hybrid clone cells derived from human breast epithelial cells and human breast cancer cells exhibit properties of cancer stem/initiating cells.

PubMed

Gauck, Daria; Keil, Silvia; Niggemann, Bernd; Zänker, Kurt S; Dittmar, Thomas

2017-08-02

The biological phenomenon of cell fusion has been associated with cancer progression since it was determined that normal cell × tumor cell fusion-derived hybrid cells could exhibit novel properties, such as enhanced metastatogenic capacity or increased drug resistance, and even as a mechanism that could give rise to cancer stem/initiating cells (CS/ICs). CS/ICs have been proposed as cancer cells that exhibit stem cell properties, including the ability to (re)initiate tumor growth. Five M13HS hybrid clone cells, which originated from spontaneous cell fusion events between M13SV1-EGFP-Neo human breast epithelial cells and HS578T-Hyg human breast cancer cells, and their parental cells were analyzed for expression of stemness and EMT-related marker proteins by Western blot analysis and confocal laser scanning microscopy. The frequency of ALDH1-positive cells was determined by flow cytometry using AldeRed fluorescent dye. Concurrently, the cells' colony forming capabilities as well as the cells' abilities to form mammospheres were investigated. The migratory activity of the cells was analyzed using a 3D collagen matrix migration assay. M13HS hybrid clone cells co-expressed SOX9, SLUG, CK8 and CK14, which were differently expressed in parental cells. A variation in the ALDH1-positive putative stem cell population was observed among the five hybrids ranging from 1.44% (M13HS-7) to 13.68% (M13HS-2). In comparison to the parental cells, all five hybrid clone cells possessed increased but also unique colony formation and mammosphere formation capabilities. M13HS-4 hybrid clone cells exhibited the highest colony formation capacity and second highest mammosphere formation capacity of all hybrids, whereby the mean diameter of the mammospheres was comparable to the parental cells. In contrast, the largest mammospheres originated from the M13HS-2 hybrid clone cells, whereas these cells' mammosphere formation capacity was comparable to the parental breast cancer cells. All M13HS hybrid clones exhibited a mesenchymal phenotype and, with the exception of one hybrid clone, responded to EGF with an increased migratory activity. Fusion of human breast epithelial cells and human breast cancer cells can give rise to hybrid clone cells that possess certain CS/IC properties, suggesting that cell fusion might be a mechanism underlying how tumor cells exhibiting a CS/IC phenotype could originate.

Differentiation of human umbilical cord mesenchymal stem cells into dermal fibroblasts in vitro

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

Han, Yanfu; Chai, Jiake, E-mail: cjk304@126.com; Sun, Tianjun

2011-10-07

Highlights: {yields} Mesenchymal stem cells (MSCs) are potential seed cells for tissue-engineered skin. {yields} Tissue-derived umbilical cord MSCs (UCMSCs) can readily be isolated in vitro. {yields} We induce UCMSCs to differentiate into dermal fibroblasts via conditioned medium. {yields} Collagen type I and collagen type III mRNA level was higher in differentiated cells. {yields} UCMSCs-derived fibroblast-like cells strongly express fibroblast-specific protein. -- Abstract: Tissue-derived umbilical cord mesenchymal stem cells (UCMSCs) can be readily obtained, avoid ethical or moral constraints, and show excellent pluripotency and proliferation potential. UCMSCs are considered to be a promising source of stem cells in regenerative medicine. Inmore » this study, we collected newborn umbilical cord tissue under sterile conditions and isolated UCMSCs through a tissue attachment method. UCMSC cell surface markers were examined using flow cytometry. On the third passage, UCMSCs were induced to differentiate into dermal fibroblasts in conditioned induction media. The induction results were detected using immunofluorescence with a fibroblast-specific monoclonal antibody and real time PCR for type I and type III collagen. UCMSCs exhibited a fibroblast-like morphology and reached 90% confluency 14 to 18 days after primary culture. Cultured UCMSCs showed strong positive staining for CD73, CD29, CD44, CD105, and HLA-I, but not CD34, CD45, CD31, or HLA-DR. After differentiation, immunostaining for collagen type I, type III, fibroblast-specific protein, vimentin, and desmin were all strongly positive in induced cells, and staining was weak or negative in non-induced cells; total transcript production of collagen type I and collagen type III mRNA was higher in induced cells than in non-induced cells. These results demonstrate that UCMSCs can be induced to differentiate into fibroblasts with conditioned induction media and, in turn, could be used as seed cells for tissue-engineered dermis.« less

[Proliferative capacity of mesenchymal stem cells from human fetal bone marrow and their ability to differentiate into the derivative cell types of three embryonic germ layers].

PubMed

Wang, Yue-Chun; Zhang, Yuan

2008-06-25

Strong proliferative capacity and the ability to differentiate into the derivative cell types of three embryonic germ layers are the two important characteristics of embryonic stem cells. To study whether the mesenchymal stem cells from human fetal bone marrow (hfBM-MSCs) possess these embryonic stem cell-like biological characteristics, hfBM-MSCs were isolated from bone barrows and further purified according to the different adherence of different kinds of cells to the wall of culture flask. The cell cycle of hfBM-MSCs and MSC-specific surface markers such as CD29, CD44, etc were identified using flow cytometry. The expressions of human telomerase reverse transcriptase (hTERT), the embryonic stem cell-specific antigens, such as Oct4 and SSEA-4 were detected with immunocytochemistry at the protein level and were also tested by RT-PCR at the mRNA level. Then, hfBM-MSCs were induced to differentiate toward neuron cells, adipose cells, and islet B cells under certain conditions. It was found that 92.3% passage-4 hfBM-MSCs and 96.1% passage-5 hfBM-MSCs were at G(0)/G(1) phase respectively. hfBM-MSCs expressed CD44, CD106 and adhesion molecule CD29, but not antigens of hematopoietic cells CD34 and CD45, and almost not antigens related to graft-versus-host disease (GVHD), such as HLA-DR, CD40 and CD80. hfBM-MSCs expressed the embryonic stem cell-specific antigens such as Oct4, SSEA-4, and also hTERT. Exposure of these cells to various inductive agents resulted in morphological changes towards neuron-like cells, adipose-like cells, and islet B-like cells and they were tested to be positive for related characteristic markers. These results suggest that there are plenty of MSCs in human fetal bone marrow, and hfBM-MSCs possess the embryonic stem cell-like biological characteristics, moreover, they have a lower immunogenic nature. Thus, hfBM-MSCs provide an ideal source for tissue engineering and cellular therapeutics.

Nestin in the epididymis is expressed in vascular wall cells and is regulated during postnatal development and in case of testosterone deficiency.

PubMed

Reckmann, Ansgar N; Tomczyk, Claudia U M; Davidoff, Michail S; Michurina, Tatyana V; Arnhold, Stefan; Müller, Dieter; Mietens, Andrea; Middendorff, Ralf

2018-01-01

Vascular smooth muscle cells (SMCs), distinguished by the expression of the neuronal stem cell marker nestin, may represent stem cell-like progenitor cells in various organs including the testis. We investigated epididymal tissues of adult nestin-GFP mice, rats after Leydig cell depletion via ethane dimethane sulfonate (EDS), rats and mice during postnatal development and human tissues. By use of Clarity, a histochemical method to illustrate a three-dimensional picture, we could demonstrate nestin-GFP positive cells within the vascular network. We localized nestin in the epididymis in proliferating vascular SMCs by colocalization with both smooth muscle actin and PCNA, and it was distinct from CD31-positive endothelial cells. The same nestin localization was found in the human epididymis. However, nestin was not found in SMCs of the epididymal duct. Nestin expression is high during postnatal development of mouse and rat and down-regulated towards adulthood when testosterone levels increase. Nestin increases dramatically in rats after Leydig cell ablation with EDS and subsequently low testosterone levels. Interestingly, during this period, the expression of androgen receptor in the epididymis is low and increases until nestin reaches normal levels of adulthood. Here we show that nestin, a common marker for neuronal stem cells, is also expressed in the vasculature of the epididymis. Our results give new insights into the yet underestimated role of proliferating nestin-expressing vascular SMCs during postnatal development and repair of the epididymis.

Paroxetine Can Enhance Neurogenesis during Neurogenic Differentiation of Human Adipose-derived Stem Cells

PubMed Central

Jahromi, Maliheh; Razavi, Shahnaz; Amirpour, Nushin; Khosravizadeh, Zahra

2016-01-01

Background: Some antidepressant drugs can promote neuronal cell proliferation in vitro as well as hippocampal neurogenesis in human and animal models. Furthermore, adipose tissue is an available source of adult stem cells with the ability to differentiate in to multiple lineages. Therefore, human Adipose-Derived Stem Cells (hAD-SCs) may be a suitable source for regenerative medical applications. Since there is no evidence for the effect of Paroxetine as the most commonly prescribed antidepressant drug for neurogenic potential of hADSCs, an attempt was made to determine the effect of Paroxetine on proliferation and neural differentiation of hADSCs. Methods: ADSCs were isolated from human abdominal fat. These cells differentiated to neuron-like cells and were treated with Paroxetine. 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) assay and immunofluorescence technique were used for assessment of cell proliferation and neurogenic differentiation potential of induced cells, respectively. Results: MTT assay analysis showed that Paroxetine significantly increased the proliferation rate of induced hADSCs (p<0.05), while immunofluorescent staining indicated that Paroxetine treatment during neurogenic differentiation could enhance the mean percentage of Nestin and MAP2 (Microtubule-associated protein-2) positive cells but the mean percentage of GFAP (Glial acidic fibrillary protein) positive cells significantly decreased relative to control group (p<0.05). Conclusion: Our results provide evidence that Paroxetine can promote proliferation and differentiation rate during neurogenic differentiation of hADSCs. Moreover, Paroxetine can reduce gliogenesis of induced hADSCs during neurogenic differentiation. PMID:27920882

To Be or Not to Be a Flatworm: The Acoel Controversy

PubMed Central

Arendt, Detlev; Borgonie, Gaëtan; Funayama, Noriko; Gschwentner, Robert; Hartenstein, Volker; Hobmayer, Bert; Hooge, Matthew; Hrouda, Martina; Ishida, Sachiko; Kobayashi, Chiyoko; Kuales, Georg; Nishimura, Osamu; Pfister, Daniela; Rieger, Reinhard; Salvenmoser, Willi; Smith, Julian; Technau, Ulrich; Tyler, Seth; Agata, Kiyokazu; Salzburger, Walter; Ladurner, Peter

2009-01-01

Since first described, acoels were considered members of the flatworms (Platyhelminthes). However, no clear synapomorphies among the three large flatworm taxa - the Catenulida, the Acoelomorpha and the Rhabditophora - have been characterized to date. Molecular phylogenies, on the other hand, commonly positioned acoels separate from other flatworms. Accordingly, our own multi-locus phylogenetic analysis using 43 genes and 23 animal species places the acoel flatworm Isodiametra pulchra at the base of all Bilateria, distant from other flatworms. By contrast, novel data on the distribution and proliferation of stem cells and the specific mode of epidermal replacement constitute a strong synapomorphy for the Acoela plus the major group of flatworms, the Rhabditophora. The expression of a piwi-like gene not only in gonadal, but also in adult somatic stem cells is another unique feature among bilaterians. These two independent stem-cell-related characters put the Acoela into the Platyhelminthes-Lophotrochozoa clade and account for the most parsimonious evolutionary explanation of epidermal cell renewal in the Bilateria. Most available multigene analyses produce conflicting results regarding the position of the acoels in the tree of life. Given these phylogenomic conflicts and the contradiction of developmental and morphological data with phylogenomic results, the monophyly of the phylum Platyhelminthes and the position of the Acoela remain unresolved. By these data, both the inclusion of Acoela within Platyhelminthes, and their separation from flatworms as basal bilaterians are well-supported alternatives. PMID:19430533

[Human stem cells from apical papilla can regenerate dentin-pulp complex].

PubMed

Xiong, Huacui; Chen, Ke; Huang, Yibin; Liu, Caiqi

2013-10-01

To regenerate dentin-pulp complex by tissue engineering with human stem cells from apical papilla cells (SCAP) as the seed cells. SCAP was separated from from normal human impacted third molars with immature roots by outgrowth culture. The cells were then cultured in the differentiation medium for 3 weeks or in normal medium for 60 days, and analyzed for mineralization potential by Alizarin red staining. The osteo/odontogenic markers including alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OC) and dentin sialoprotein (DSP) were investigated by immunofluorescence staining and reverse transcription-polymerase chain reaction. The co-cultured mixture of SCAP and HA/TCP, or HA/TCP alone was implanted subcutaneously on the back of nude mice for 8 weeks, and the implants were collected and examined by HE and immunohistochemical staining. Round alizarin red-positive nodules formed in the isolated cells after cell culture in the differentiation medium for 3 weeks or in normal medium for 60 days with positive staining for osteo/odontogenic markers. SCAP with HA/TCP could regenerate pulp-dentin complex-like tissue in nude mice. The cells near the dentin-like tissue were positive for DSP. No mineral tissue was found in mice receiving HA/TCP implantation. SCAP may serve as a promising seed cell for dentin-pulp complex tissue engineering.