Rapid selection of mesenchymal stem and progenitor cells in primary prostate stromal cultures.

PubMed

Brennen, W Nathaniel; Kisteman, L Nelleke; Isaacs, John T

2016-05-01

Carcinoma-associated fibroblasts (CAFs) are a dominant component of the tumor microenvironment with pro-tumorigenic properties. Despite this knowledge, their physiologic origins remain poorly understood. Mesenchymal stem cells (MSCs) can be recruited from the bone marrow to areas of tissue damage and inflammation, including prostate cancer. MSCs can generate and have many overlapping properties with CAFs in preclinical models. Multiparameter flow cytometry and multipotent differentiation assays used to define MSCs in primary prostate stromal cultures derived from young (<25 yrs) organ donors and prostate cancer patients compared with bone marrow-derived stromal cultures. Population doubling times, population doublings, cell size, and differentiation potential determined under multiple culture conditions, including normoxia, hypoxia, and a variety of media. TGF-β measured by ELISA. MSCs and stromal progenitors are not only present in normal and malignant prostate tissue, but are quickly selected for in primary stromal cultures derived from these tissues; becoming the dominant population within just a few passages. Growth potential inversely associated with TGF-β concentrations. All conditions generated populations with an average cell diameter >15 µm. All cultures tested had the ability to undergo osteogenic and chondrogenic differentiation, but unlike bone marrow-derived MSCs, primary stromal cultures derived from normal prostate tissue lack adipogenic differentiation potential. In contrast, a subset of stromal cultures derived from prostate cancer patients retain the ability to differentiate into adipocytes; a property that is significantly suppressed under hypoxic conditions in both bone marrow- and prostate-derived MSCs. Primary prostate stromal cultures are highly enriched in cells with an MSC or stromal progenitor phenotype. The use of primary cultures such as these to study CAFs raises interesting implications when considering their overlapping properties. The lack of adipogenesis in stromal cultures derived from normal prostates suggests they have a lineage-restricted progenitor phenotype. The retention of adipogenic differentiation in cultures from a subset of prostate cancer patients suggests the active recruitment of less committed progenitors or MSCs from the bone marrow as a function of disease progression. This recruitment can potentially be exploited for prognostic purposes or a cell-based platform for the systemic delivery of cytotoxic agents to sites of prostate cancer. © 2016 Wiley Periodicals, Inc.

Presence of S100A8/Gr1-Positive Myeloid-Derived Suppressor Cells in Primary Tumors and Visceral Organs Invaded by Breast Carcinoma Cells.

PubMed

Tanriover, Gamze; Eyinc, Mehmet Berk; Aliyev, Elnur; Dilmac, Sayra; Erin, Nuray

2018-04-26

Increased S100A8/A9 expression in Gr1-positive cells has been shown in myeloid-derived suppressor cells and may play a role in the formation of a metastatic milieu. We aimed to determine S100A8/A9 expression alone and with coexpression of Gr1 (a myeloid marker) in primary tumor and visceral tissues invaded by metastatic breast carcinoma. Female BALB/c mice were injected with 4TLM, 4THM, and 67NR orthotopically. Confluent cells (75%-80%) were used. Primary tumor, lung, liver, and spleen tissue samples were removed 26 days after injection. Peripheral blood smears and metastasis assay were performed, as was immunohistochemistry and staining. S100A8/A9 immunoreactivity alone or coexpressed with Gr1 was found in primary tumors formed by 4TLM and 4THM cells, which was markedly higher than in primary tumors formed by nonmetastatic 67NR cells. Similarly, liver and lung tissues obtained from mice injected with 4TLM or 4THM cells were invaded by S100A8/A9-positive and Gr1-positive cells. Double-positive cells were markedly fewer in liver and lung tissues of animals injected with 67NR cells. S100A8/A9-positive cells were mostly localized in red pulp of spleens. We observed an increased number of neutrophils in the peripheral blood of mice injected with metastatic breast carcinoma cells. Tumor-derived factors may increase S100A8/A9-positive cells locally and systemically, and S100A8/A9-positive cells may provide an appropriate milieu for the formation of metastasis. Copyright © 2018 Elsevier Inc. All rights reserved.

DNER, an epigenetically modulated gene, regulates glioblastoma-derived neurosphere cell differentiation and tumor propagation.

PubMed

Sun, Peng; Xia, Shuli; Lal, Bachchu; Eberhart, Charles G; Quinones-Hinojosa, Alfredo; Maciaczyk, Jarek; Matsui, William; Dimeco, Francesco; Piccirillo, Sara M; Vescovi, Angelo L; Laterra, John

2009-07-01

Neurospheres derived from glioblastoma (GBM) and other solid malignancies contain neoplastic stem-like cells that efficiently propagate tumor growth and resist cytotoxic therapeutics. The primary objective of this study was to use histone-modifying agents to elucidate mechanisms by which the phenotype and tumor-promoting capacity of GBM-derived neoplastic stem-like cells are regulated. Using established GBM-derived neurosphere lines and low passage primary GBM-derived neurospheres, we show that histone deacetylase (HDAC) inhibitors inhibit growth, induce differentiation, and induce apoptosis of neoplastic neurosphere cells. A specific gene product induced by HDAC inhibition, Delta/Notch-like epidermal growth factor-related receptor (DNER), inhibited the growth of GBM-derived neurospheres, induced their differentiation in vivo and in vitro, and inhibited their engraftment and growth as tumor xenografts. The differentiating and tumor suppressive effects of DNER, a noncanonical Notch ligand, contrast with the previously established tumor-promoting effects of canonical Notch signaling in brain cancer stem-like cells. Our findings are the first to implicate noncanonical Notch signaling in the regulation of neoplastic stem-like cells and suggest novel neoplastic stem cell targeting treatment strategies for GBM and potentially other solid malignancies.

Pancreatic cancer cell lines as patient-derived avatars: genetic characterisation and functional utility.

PubMed

Knudsen, Erik S; Balaji, Uthra; Mannakee, Brian; Vail, Paris; Eslinger, Cody; Moxom, Christopher; Mansour, John; Witkiewicz, Agnieszka K

2018-03-01

Pancreatic ductal adenocarcinoma (PDAC) is a therapy recalcitrant disease with the worst survival rate of common solid tumours. Preclinical models that accurately reflect the genetic and biological diversity of PDAC will be important for delineating features of tumour biology and therapeutic vulnerabilities. 27 primary PDAC tumours were employed for genetic analysis and development of tumour models. Tumour tissue was used for derivation of xenografts and cell lines. Exome sequencing was performed on the originating tumour and developed models. RNA sequencing, histological and functional analyses were employed to determine the relationship of the patient-derived models to clinical presentation of PDAC. The cohort employed captured the genetic diversity of PDAC. From most cases, both cell lines and xenograft models were developed. Exome sequencing confirmed preservation of the primary tumour mutations in developed cell lines, which remained stable with extended passaging. The level of genetic conservation in the cell lines was comparable to that observed with patient-derived xenograft (PDX) models. Unlike historically established PDAC cancer cell lines, patient-derived models recapitulated the histological architecture of the primary tumour and exhibited metastatic spread similar to that observed clinically. Detailed genetic analyses of tumours and derived models revealed features of ex vivo evolution and the clonal architecture of PDAC. Functional analysis was used to elucidate therapeutic vulnerabilities of relevance to treatment of PDAC. These data illustrate that with the appropriate methods it is possible to develop cell lines that maintain genetic features of PDAC. Such models serve as important substrates for analysing the significance of genetic variants and create a unique biorepository of annotated cell lines and xenografts that were established simultaneously from same primary tumour. These models can be used to infer genetic and empirically determined therapeutic sensitivities that would be germane to the patient. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Establishment of a patient-derived orthotopic osteosarcoma mouse model.

PubMed

Blattmann, Claudia; Thiemann, Markus; Stenzinger, Albrecht; Roth, Eva K; Dittmar, Anne; Witt, Hendrik; Lehner, Burkhard; Renker, Eva; Jugold, Manfred; Eichwald, Viktoria; Weichert, Wilko; Huber, Peter E; Kulozik, Andreas E

2015-04-30

Osteosarcoma (OS) is the most common pediatric primary malignant bone tumor. As the prognosis for patients following standard treatment did not improve for almost three decades, functional preclinical models that closely reflect important clinical cancer characteristics are urgently needed to develop and evaluate new treatment strategies. The objective of this study was to establish an orthotopic xenotransplanted mouse model using patient-derived tumor tissue. Fresh tumor tissue from an adolescent female patient with osteosarcoma after relapse was surgically xenografted into the right tibia of 6 immunodeficient BALB/c Nu/Nu mice as well as cultured into medium. Tumor growth was serially assessed by palpation and with magnetic resonance imaging (MRI). In parallel, a primary cell line of the same tumor was established. Histology and high-resolution array-based comparative genomic hybridization (aCGH) were used to investigate both phenotypic and genotypic characteristics of different passages of human xenografts and the cell line compared to the tissue of origin. A primary OS cell line and a primary patient-derived orthotopic xenotranplanted mouse model were established. MRI analyses and histopathology demonstrated an identical architecture in the primary tumor and in the xenografts. Array-CGH analyses of the cell line and all xenografts showed highly comparable patterns of genomic progression. So far, three further primary patient-derived orthotopic xenotranplanted mouse models could be established. We report the first orthotopic OS mouse model generated by transplantation of tumor fragments directly harvested from the patient. This model represents the morphologic and genomic identity of the primary tumor and provides a preclinical platform to evaluate new treatment strategies in OS.

Compartmental culture of embryonic stem cell-derived neurons in microfluidic devices for use in axonal biology.

PubMed

Shin, Hwa Sung; Kim, Hyung Joon; Min, Seul Ki; Kim, Sung Hoon; Lee, Byung Man; Jeon, Noo Li

2010-08-01

Axonal pathology has been clearly implicated in neurodegenerative diseases making the compartmental culture of neurons a useful research tool. Primary neurons have already been cultured in compartmental microfluidic devices but their derivation from an animal is a time-consuming and difficult work and has a limit in their sources. Embryonic stem cell (ESC)-derived neurons (ESC_Ns) overcome this limit, since ESCs can be renewed without limit and can be differentiated into ESC_Ns by robust and reproducible protocols. In this research, ESC_Ns were derived from mouse ESCs in compartmental microfluidic devices, and their axons were isolated from the somal cell bodies. Once embryoid bodies (EBs) were localized in the microfluidic culture chamber, ESC_Ns spread out from the EBs and occupied the cell culture chamber. Their axons traversed the microchannels and finally were isolated from the somata, providing an arrangement comparable to dissociated primary neurons. This ESC_N compartmental microfluidic culture system not only offers a substitute for the primary neuron counterpart system but also makes it possible to make comparisons between the two systems.

Monocytic and granulocytic myeloid derived suppressor cells differentially regulate spatiotemporal tumour plasticity during metastatic cascade.

PubMed

Ouzounova, Maria; Lee, Eunmi; Piranlioglu, Raziye; El Andaloussi, Abdeljabar; Kolhe, Ravindra; Demirci, Mehmet F; Marasco, Daniela; Asm, Iskander; Chadli, Ahmed; Hassan, Khaled A; Thangaraju, Muthusamy; Zhou, Gang; Arbab, Ali S; Cowell, John K; Korkaya, Hasan

2017-04-06

It is widely accepted that dynamic and reversible tumour cell plasticity is required for metastasis, however, in vivo steps and molecular mechanisms are poorly elucidated. We demonstrate here that monocytic (mMDSC) and granulocytic (gMDSC) subsets of myeloid-derived suppressor cells infiltrate in the primary tumour and distant organs with different time kinetics and regulate spatiotemporal tumour plasticity. Using co-culture experiments and mouse transcriptome analyses in syngeneic mouse models, we provide evidence that tumour-infiltrated mMDSCs facilitate tumour cell dissemination from the primary site by inducing EMT/CSC phenotype. In contrast, pulmonary gMDSC infiltrates support the metastatic growth by reverting EMT/CSC phenotype and promoting tumour cell proliferation. Furthermore, lung-derived gMDSCs isolated from tumour-bearing animals enhance metastatic growth of already disseminated tumour cells. MDSC-induced 'metastatic gene signature' derived from murine syngeneic model predicts poor patient survival in the majority of human solid tumours. Thus spatiotemporal MDSC infiltration may have clinical implications in tumour progression.

Monocytic and granulocytic myeloid derived suppressor cells differentially regulate spatiotemporal tumour plasticity during metastatic cascade

PubMed Central

Ouzounova, Maria; Lee, Eunmi; Piranlioglu, Raziye; El Andaloussi, Abdeljabar; Kolhe, Ravindra; Demirci, Mehmet F.; Marasco, Daniela; Asm, Iskander; Chadli, Ahmed; Hassan, Khaled A.; Thangaraju, Muthusamy; Zhou, Gang; Arbab, Ali S.; Cowell, John K.; Korkaya, Hasan

2017-01-01

It is widely accepted that dynamic and reversible tumour cell plasticity is required for metastasis, however, in vivo steps and molecular mechanisms are poorly elucidated. We demonstrate here that monocytic (mMDSC) and granulocytic (gMDSC) subsets of myeloid-derived suppressor cells infiltrate in the primary tumour and distant organs with different time kinetics and regulate spatiotemporal tumour plasticity. Using co-culture experiments and mouse transcriptome analyses in syngeneic mouse models, we provide evidence that tumour-infiltrated mMDSCs facilitate tumour cell dissemination from the primary site by inducing EMT/CSC phenotype. In contrast, pulmonary gMDSC infiltrates support the metastatic growth by reverting EMT/CSC phenotype and promoting tumour cell proliferation. Furthermore, lung-derived gMDSCs isolated from tumour-bearing animals enhance metastatic growth of already disseminated tumour cells. MDSC-induced ‘metastatic gene signature' derived from murine syngeneic model predicts poor patient survival in the majority of human solid tumours. Thus spatiotemporal MDSC infiltration may have clinical implications in tumour progression. PMID:28382931

A Rapid Filter Insert-based 3D Culture System for Primary Prostate Cell Differentiation

PubMed Central

Tricoli, Lucas; Berry, Deborah L.; Albanese, Chris

2018-01-01

Conditionally reprogrammed cells (CRCs) provide a sustainable method for primary cell culture and the ability to develop extensive “living biobanks” of patient derived cell lines. For many types of epithelial cells, various three dimensional (3D) culture approaches have been described that support an improved differentiated state. While CRCs retain their lineage commitment to the tissue from which they are isolated, they fail to express many of the differentiation markers associated with the tissue of origin when grown under normal two dimensional (2D) culture conditions. To enhance the application of patient-derived CRCs for prostate cancer research, a 3D culture format has been defined that enables a rapid (2 weeks total) luminal cell differentiation in both normal and tumor-derived prostate epithelial cells. Herein, a filter insert-based format is described for the culturing and differentiation of both normal and malignant prostate CRCs. A detailed description of the procedures required for cell collection and processing for immunohistochemical and immunofluorescent staining are provided. Collectively the 3D culture format described, combined with the primary CRC lines, provides an important medium- to high- throughput model system for biospecimen-based prostate research. PMID:28287583

Analysis of soluble factors in conditioned media derived from primary cultures of cirrhotic liver of biliary atresia.

PubMed

Yamazaki, Taisuke; Wakai, Mariko; Enosawa, Shin; Tokiwa, Takayoshi

2017-06-01

Biliary atresia (BA) is a rare and serious liver disease in newborn infants. Previously, we reported that non-parenchymal cell (NPC) fractions from cirrhotic liver of BA may contain hepatic stem/progenitor cells in primary culture of NPC fractions. In this study, NPC fractions were subjected to primary or passage culture and found that clusters of hepatocyte-like cells appear even without adding hepatocyte growth factor (HGF) to the culture medium, but not in their passage culture used as a control. Based on these findings, conditioned media (CMs) were collected and soluble factors in the CMs were analyzed in order to elucidate the mechanism of the appearance of hepatocyte-like cells or their clusters. A large amount of active HGF consisting of α and β chains was detected in CMs derived from primary culture, but not in CMs from passage culture, as determined by western blot analysis, bone morphogenetic protein (BMP)-4, oncostatin M (OSM), and transforming growth factor (TGF)-β1 were not detected in any of the CMs. The number of hepatocyte-like cells in primary culture tended to decrease following treatment with the HGF receptor c-Met inhibitor, SU11274 in a dose-dependent manner. Furthermore, the clusters of hepatocyte-like cells tended to increase in size and number when freshly isolated NPC fractions were cultured in the presence of 10% of CMs collected after 3-4 wk of primary culture. In conclusion, these findings indicate that CMs derived from primary culture of NPC fractions of BA liver contain a large amount of active HGF, which may activate hepatic stem/progenitor cells and promote the appearance of hepatocyte-like cells or their clusters through HGF/c-Met signaling. The present study would lead to cell therapy using the patient's own cells for the treatment of BA.

Uniformity under in vitro conditions: Changes in the phenotype of cancer cell lines derived from different medulloblastoma subgroups.

PubMed

Chlapek, Petr; Zitterbart, Karel; Kren, Leos; Filipova, Lenka; Sterba, Jaroslav; Veselska, Renata

2017-01-01

Medulloblastoma comprises four main subgroups (WNT, SHH, Group 3 and Group 4) originally defined by transcriptional profiling. In primary medulloblastoma tissues, these groups are thought to be distinguishable using the immunohistochemical detection of β-catenin, filamin A, GAB1 and YAP1 protein markers. To investigate the utility of these markers for in vitro studies using medulloblastoma cell lines, immunoblotting and indirect immunofluorescence were employed for the detection of β-catenin, filamin A, GAB1 and YAP1 in both DAOY and D283 Med reference cell lines and the panel of six medulloblastoma cell lines derived in our laboratory from the primary tumor tissues of known molecular subgroups. Immunohistochemical detection of these markers was performed on formalin-fixed paraffin-embedded tissue of the matching primary tumors. The results revealed substantial divergences between the primary tumor tissues and matching cell lines in the immunoreactivity pattern of medulloblastoma-subgroup-specific protein markers. Regardless of the molecular subgroup of the primary tumor, all six patient-derived medulloblastoma cell lines exhibited a uniform phenotype: immunofluorescence showed the nuclear localization of YAP1, accompanied by strong cytoplasmic positivity for β-catenin and filamin A, as well as weak positivity for GAB1. The same immunoreactivity pattern was also found in both DAOY and D283 Med reference medulloblastoma cell lines. Therefore, we can conclude that various medulloblastoma cell lines tend to exhibit the same characteristics of protein marker expression under standard in vitro conditions. Such a finding emphasizes the importance of the analyses of primary tumors in clinically oriented medulloblastoma research and the urgent need to develop in vitro models of improved clinical relevance, such as 3D cultures and organotypic slice cultures.

Human brain metastatic stroma attracts breast cancer cells via chemokines CXCL16 and CXCL12.

PubMed

Chung, Brile; Esmaeili, Ali A; Gopalakrishna-Pillai, Sailesh; Murad, John P; Andersen, Emily S; Kumar Reddy, Naveen; Srinivasan, Gayathri; Armstrong, Brian; Chu, Caleb; Kim, Young; Tong, Tommy; Waisman, James; Yim, John H; Badie, Behnam; Lee, Peter P

2017-01-01

The tumor microenvironment is composed of heterogeneous populations of cells, including cancer, immune, and stromal cells. Progression of tumor growth and initiation of metastasis is critically dependent on the reciprocal interactions between cancer cells and stroma. Through RNA-Seq and protein analyses, we found that cancer-associated fibroblasts derived from human breast cancer brain metastasis express significantly higher levels of chemokines CXCL12 and CXCL16 than fibroblasts from primary breast tumors or normal breast. To further understand the interplay between cancer cells and cancer-associated fibroblasts from each site, we developed three-dimensional organoids composed of patient-derived primary or brain metastasis cancer cells with matching cancer-associated fibroblasts. Three-dimensional CAF aggregates generated from brain metastasis promote migration of cancer cells more effectively than cancer-associated fibroblast aggregates derived from primary tumor or normal breast stromal cells. Treatment with a CXCR4 antagonist and/or CXCL16 neutralizing antibody, alone or in combination, significantly inhibited migration of cancer cells to brain metastatic cancer-associated fibroblast aggregates. These results demonstrate that human brain metastasis cancer-associated fibroblasts potently attract breast cancer cells via chemokines CXCL12 and CXCL16, and blocking CXCR6-CXCL16/CXCR4-CXCL12 receptor-ligand interactions may be an effective therapy for preventing breast cancer brain metastasis.

Isolation and characterization of porcine adipose tissue-derived adult stem cells.

PubMed

Williams, Kellie J; Picou, Alicia A; Kish, Sharon L; Giraldo, Angelica M; Godke, Robert A; Bondioli, Kenneth R

2008-01-01

Stem cell characteristics such as self-renewal, differentiation and expression of CD34 and CD44 stem cell markers have not been identified in porcine adipose tissue-derived adult stem (ADAS) cells. The objective of this study was to develop a protocol for the isolation and culture of porcine adipose tissue-derived cells and to determine stem cell-like characteristics. Primary cultures were established and cell cultures were maintained. Cloning capacity was determined using a ring cloning procedure. Primary cultures and clones were differentiated and stained for multiple differentiated phenotypes. CD34 and CD44 messenger ribonucleic acid (mRNA) was isolated and reverse transcriptase polymerase chain reaction was used to compare expression profiles. An average of 2,700,000 nucleated cells/ml was isolated; 26% were adherent, and cells completed a cell cycle approximately every 3.3 days. Ring cloning identified 19 colonies. Primary cultures and clones were determined to differentiate along osteogenic, adipogenic and chondrogenic tissue lineages. The mRNA expression profiles showed CD34 expression was higher for undifferentiated ADAS cells versus differentiated cell types and the CD34 expression level was lower than that of CD44 among differentiated cells. Improved culture conditions and defined cellular characteristics of these porcine ADAS cells have been identified. Porcine ADAS can self-renew, can differentiate into multiple tissue lineages and they express CD34. Copyright 2008 S. Karger AG, Basel.

Promotion of haematopoietic activity in embryonic stem cells by the aorta-gonad-mesonephros microenvironment

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

Krassowska, Anna; Gordon-Keylock, Sabrina; Samuel, Kay

We investigated whether the in vitro differentiation of ES cells into haematopoietic progenitors could be enhanced by exposure to the aorta-gonadal-mesonephros (AGM) microenvironment that is involved in the generation of haematopoietic stem cells (HSC) during embryonic development. We established a co-culture system that combines the requirements for primary organ culture and differentiating ES cells and showed that exposure of differentiating ES cells to the primary AGM region results in a significant increase in the number of ES-derived haematopoietic progenitors. Co-culture of ES cells on the AM20-1B4 stromal cell line derived from the AGM region also increases haematopoietic activity. We concludemore » that factors promoting the haematopoietic activity of differentiating ES cells present in primary AGM explants are partially retained in the AM20.1B4 stromal cell line and that these factors are likely to be different to those required for adult HSC maintenance.« less

Multiple roles for elastic fibers in the skin.

PubMed

Starcher, Barry; Aycock, Ronnie L; Hill, Charles H

2005-04-01

Dermal elastic fibers are believed to have a primary role in providing elastic stretch and recoil to the skin. Here we compare the structural arrangement of dermal elastic fibers of chick skin and different animal species. Most elastic fibers in chick skin are derived from cells that line the feather follicle and/or smooth muscle that connects the pterial and apterial muscle bundles to feather follicles. Elastic fibers in the dermis of animals with single, primary hair follicles are derived from cells lining the hair follicle or from the ends of the pili muscle, which anchors the muscle to the matrix or to the hair follicle. Each follicle is interconnected with elastic fibers. Follicles of animals with primary and secondary (wool) hair follicles are also interconnected by elastic fibers, yet only the elastic fibers derived from the primary follicle are connected to each primary follicle. Only the primary hair follicles are connected to the pili muscle. Human skin, but not the skin of other primates, is significantly different from other animals with respect to elastic fiber organization and probably cell of origin. The data suggest that the primary role for elastic fibers in animals, with the possible exception of humans, is movement and/or placement of feathers or hair.

Thermo-responsive cell culture carrier: Effects on macrophage functionality and detachment efficiency.

PubMed

Rennert, Knut; Nitschke, Mirko; Wallert, Maria; Keune, Natalie; Raasch, Martin; Lorkowski, Stefan; Mosig, Alexander S

2017-01-01

Harvesting cultivated macrophages for tissue engineering purposes by enzymatic digestion of cell adhesion molecules can potentially result in unintended activation, altered function, or behavior of these cells. Thermo-responsive polymer is a promising tool that allows for gentle macrophage detachment without artificial activation prior to subculture within engineered tissue constructs. We therefore characterized different species of thermo-responsive polymers for their suitability as cell substrate and to mediate gentle macrophage detachment by temperature shift. Primary human monocyte- and THP-1-derived macrophages were cultured on thermo-responsive polymers and characterized for phagocytosis and cytokine secretion in response to lipopolysaccharide stimulation. We found that both cell types differentially respond in dependence of culture and stimulation on thermo-responsive polymers. In contrast to THP-1 macrophages, primary monocyte-derived macrophages showed no signs of impaired viability, artificial activation, or altered functionality due to culture on thermo-responsive polymers compared to conventional cell culture. Our study demonstrates that along with commercially available UpCell carriers, two other thermo-responsive polymers based on poly(vinyl methyl ether) blends are attractive candidates for differentiation and gentle detachment of primary monocyte-derived macrophages. In summary, we observed similar functionality and viability of primary monocyte-derived macrophages cultured on thermo-responsive polymers compared to standard cell culture surfaces. While this first generation of custom-made thermo-responsive polymers does not yet outperform standard culture approaches, our results are very promising and provide the basis for exploiting the unique advantages offered by custom-made thermo-responsive polymers to further improve macrophage culture and recovery in the future, including the covalent binding of signaling molecules and the reduction of centrifugation and washing steps. Optimizing these and other benefits of thermo-responsive polymers could greatly improve the culture of macrophages for tissue engineering applications.

Intravital multiphoton imaging reveals multicellular streaming as a crucial component of in vivo cell migration in human breast tumors

PubMed Central

Patsialou, Antonia; Bravo-Cordero, Jose Javier; Wang, Yarong; Entenberg, David; Liu, Huiping; Clarke, Michael; Condeelis, John S.

2014-01-01

Metastasis is the main cause of death in breast cancer patients. Cell migration is an essential component of almost every step of the metastatic cascade, especially the early step of invasion inside the primary tumor. In this report, we have used intravital multiphoton microscopy to visualize the different migration patterns of human breast tumor cells in live primary tumors. We used xenograft tumors of MDA-MB-231 cells as well as a low passage xenograft tumor from orthotopically injected patient-derived breast tumor cells. Direct visualization of human tumor cells in vivo shows two patterns of high-speed migration inside primary tumors: a. single cells and b. multicellular streams (i.e., cells following each other in a single file but without cohesive cell junctions). Critically, we found that only streaming and not random migration of single cells was significantly correlated with proximity to vessels, with intravasation and with numbers of elevated circulating tumor cells in the bloodstream. Finally, although the two human tumors were derived from diverse genetic backgrounds, we found that their migratory tumor cells exhibited coordinated gene expression changes that led to the same end-phenotype of enhanced migration involving activating actin polymerization and myosin contraction. Our data are the first direct visualization and assessment of in vivo migration within a live patient-derived breast xenograft tumor. PMID:25013744

Microbial-derived lithocholic acid and vitamin K2 drive the metabolic maturation of pluripotent stem cells-derived and fetal hepatocytes.

PubMed

Avior, Yishai; Levy, Gahl; Zimerman, Michal; Kitsberg, Daniel; Schwartz, Robert; Sadeh, Ronen; Moussaieff, Arieh; Cohen, Merav; Itskovitz-Eldor, Joseph; Nahmias, Yaakov

2015-07-01

The liver is the main organ responsible for the modification, clearance, and transformational toxicity of most xenobiotics owing to its abundance in cytochrome P450 (CYP450) enzymes. However, the scarcity and variability of primary hepatocytes currently limits their utility. Human pluripotent stem cells (hPSCs) represent an excellent source of differentiated hepatocytes; however, current protocols still produce fetal-like hepatocytes with limited mature function. Interestingly, fetal hepatocytes acquire mature CYP450 expression only postpartum, suggesting that nutritional cues may drive hepatic maturation. We show that vitamin K2 and lithocholic acid, a by-product of intestinal flora, activate pregnane X receptor (PXR) and subsequent CYP3A4 and CYP2C9 expression in hPSC-derived and isolated fetal hepatocytes. Differentiated cells produce albumin and apolipoprotein B100 at levels equivalent to primary human hepatocytes, while demonstrating an 8-fold induction of CYP450 activity in response to aryl hydrocarbon receptor (AhR) agonist omeprazole and a 10-fold induction in response to PXR agonist rifampicin. Flow cytometry showed that over 83% of cells were albumin and hepatocyte nuclear factor 4 alpha (HNF4α) positive, permitting high-content screening in a 96-well plate format. Analysis of 12 compounds showed an R(2) correlation of 0.94 between TC50 values obtained in stem cell-derived hepatocytes and primary cells, compared to 0.62 for HepG2 cells. Finally, stem cell-derived hepatocytes demonstrate all toxicological endpoints examined, including steatosis, apoptosis, and cholestasis, when exposed to nine known hepatotoxins. Our work provides fresh insights into liver development, suggesting that microbial-derived cues may drive the maturation of CYP450 enzymes postpartum. Addition of these cues results in the first functional, inducible, hPSC-derived hepatocyte for predictive toxicology. © 2015 by the American Association for the Study of Liver Diseases.

Proteomic profiling identifies the inorganic pyrophosphatase (PPA1) protein as a potential biomarker of metastasis in laryngeal squamous cell carcinoma.

PubMed

Bodnar, Magdalena; Luczak, Magdalena; Bednarek, Kinga; Szylberg, Lukasz; Marszalek, Andrzej; Grenman, Reidar; Szyfter, Krzysztof; Jarmuz-Szymczak, Malgorzata; Giefing, Maciej

2016-06-01

Relapse and metastasis are the main causes of unfavorable outcome in head and neck cancers. Whereas, understanding of the molecular background of these processes is far from being complete. Therefore, in this study we aimed to identify potential biomarker candidates of relapse and metastasis in laryngeal squamous cell carcinoma (LSCC) by combining the 2D electrophoresis based protein screen and immunohistochemical analysis of candidate proteins. We screened three groups of LSCC cell lines derived from primary tumors, recurrent tumors and metastases and identified seven proteins that differed significantly in relative abundance between the analyzed groups. Among the identified proteins were the heat shock proteins HSP60 and HSP70 that were significantly downregulated both in recurrences- and metastases-derived cell lines but not in primary tumor-derived cell lines. Moreover, we identified significant upregulation of the annexin V, calreticulin and the inorganic pyrophosphatase (PPA1) exclusively in the metastases-derived cell lines. As these upregulated proteins could potentially become novel biomarkers of metastasis, we have compared their abundance in primary tumor LSCC N(0) cases, primary tumor LSCC N(+) cases as well as in LSCC metastases N(+). Our results show an intense increase of cytoplasmic PPA1 abundance in the N(+) (p = 0.000042) compared to the N(0) group. In summary, we show a group of proteins deregulated in recurrences and metastases of LSCC. Moreover, we suggest the PPA1 protein as a potential new biomarker for metastasis in this cancer.

T cell-derived Lymphotoxin is Essential for anti-HSV-1 Humoral Immune Response.

PubMed

Yang, Kaiting; Liang, Yong; Sun, Zhichen; Xue, Diyuan; Xu, Hairong; Zhu, Mingzhao; Fu, Yang-Xin; Peng, Hua

2018-05-09

B cell-derived lymphotoxin (LT) is required for the development of follicular dendritic cell clusters for the formation of primary and secondary lymphoid follicles, but the role of T cell-derived LT in antibody response has not been well demonstrated. We observed that lymphotoxin-β-receptor (LTβR) signaling is essential for optimal humoral immune response and protection against an acute HSV-1 infection. Blocking the LTβR pathway caused poor maintenance of germinal center B (GC-B) cells and follicular helper T (Tfh) cells. Using bone marrow chimeric mice and adoptive transplantation, we determined that T cell-derived LT played an indispensable role in the humoral immune response to HSV-1. Up-regulation of IFNγ by the LTβR-Ig blockade impairs the sustainability of Tfh-like cells, thus leading to an impaired humoral immune response. Our findings have identified a novel role of T cell-derived LT in the humoral immune response against HSV-1 infection. IMPORTANCE Immunocompromised people are susceptible for HSV-1 infection and lethal recurrence, which could be inhibited by anti-HSV-1 humoral immune response in the host. This study sought to explore the role of T cell-derived LT in the anti-HSV-1 humoral immune response using LT-LTβR signaling deficient mice and the LTβR-Ig blockade. The data indicate that the T cell-derived LT may play an essential role in sustaining Tfh-like cells and ensure Tfh-like cells' migration into primary or secondary follicles for further maturation. This study provides insights for vaccine development against infectious diseases. Copyright © 2018 American Society for Microbiology.

Genetically engineered oncolytic Newcastle disease virus mediates cytolysis of prostate cancer stem like cells.

PubMed

Raghunath, Shobana; Pudupakam, Raghavendra Sumanth; Allen, Adria; Biswas, Moanaro; Sriranganathan, Nammalwar

2017-10-20

Oncolytic virotherapy is a promising novel approach that overcomes the limitations posed by radiation and chemotherapy. In this study, the oncolytic efficacy of a recombinant Newcastle disease virus (rNDV) BC-KLQL-GFP, against prostate cancer stem-like/tumor initiating cells was evaluated. Xenograft derived prostaspheres (XPS) induced tumor more efficiently than monolayer cell derived prostaspheres (MCPS) in nude mice. Primary and secondary XPS show enhanced self-renewal and clonogenic potential compared to MCPS. XPS also expressed embryonic stem cell markers, such as Nanog, CD44 and Nestin. Further, prostate specific antigen (PSA) activated recombinant Newcastle Disease Virus (rNDV) was selectively cytotoxic to tumor derived DU145 prostaspheres. An effective concentration (EC 50 ) of 0.11-0.14 multiplicity of infection was sufficient to cause prostasphere cell death in serum free culture. DU145 tumor xenograft derived prostaspheres were used as tumor surrogates as they were enriched for a putative tumor initiating cell population. PSA activated rNDV was efficient in inducing cell death of cells and prostaspheres derived from primary xenografts ex-vivo, thus signifying a potential in vivo efficacy. The EC 50 (∼0.1 MOI) for cytolysis of tumor initiating cells was slightly higher than that was required for the parental cell line, but within the therapeutic margin for safety and efficacy. Copyright © 2017 Elsevier B.V. All rights reserved.

Genetic response and morphologic characterization of chicken bone-marrow derived dendritic cells during infection with high and low pathogenic avian influenza viruses

USDA-ARS?s Scientific Manuscript database

Dendritic cells (DC) are professional antigen-presenting cells of the immune system that function to initiate primary immune responses. Progenitors of DCs are derived from haematopoietic stem cells in the bone marrow (BM) that migrate in non-lymphoid tissues to develop into immature DCs. Here, they ...

ESCDL-1, a new cell line derived from chicken embryonic stem cells, supports efficient replication of Mardiviruses

PubMed Central

Jean, Christian; Fragnet-Trapp, Laetitia; Rémy, Sylvie; Chabanne-Vautherot, Danièle; Montillet, Guillaume; Fuet, Aurélie; Denesvre, Caroline; Pain, Bertrand

2017-01-01

Marek’s disease virus is the etiological agent of a major lymphoproliferative disorder in poultry and the prototype of the Mardivirus genus. Primary avian somatic cells are currently used for virus replication and vaccine production, but they are largely refractory to any genetic modification compatible with the preservation of intact viral susceptibility. We explored the concept of induction of viral replication permissiveness in an established pluripotent chicken embryonic stem cell-line (cES) in order to derive a new fully susceptible cell-line. Chicken ES cells were not permissive for Mardivirus infection, but as soon as differentiation was triggered, replication of Marek’s disease virus was detected. From a panel of cyto-differentiating agents, hexamethylene bis (acetamide) (HMBA) was found to be the most efficient regarding the induction of permissiveness. These initial findings prompted us to analyse the effect of HMBA on gene expression, to derive a new mesenchymal cell line, the so-called ESCDL-1, and monitor its susceptibility for Mardivirus replication. All Mardiviruses tested so far replicated equally well on primary embryonic skin cells and on ESCDL-1, and the latter showed no variation related to its passage number in its permissiveness for virus infection. Viral morphogenesis studies confirmed efficient multiplication with, as in other in vitro models, no extra-cellular virus production. We could show that ESCDL-1 can be transfected to express a transgene and subsequently cloned without any loss in permissiveness. Consequently, ESCDL-1 was genetically modified to complement viral gene deletions thus yielding stable trans-complementing cell lines. We herein claim that derivation of stable differentiated cell-lines from cES cell lines might be an alternative solution to the cultivation of primary cells for virology studies. PMID:28406989

Isolation and culture of primary human pancreatic stellate cells that reflect the context of their tissue of origin.

PubMed

Strobel, Oliver; Dadabaeva, Nigora; Felix, Klaus; Hackert, Thilo; Giese, Nathalia A; Jesenofsky, Ralf; Werner, Jens

2016-02-01

Pancreatic stellate cells (PSCs) play a critical role in pancreatic ductal adenocarcinoma (PDAC). Activated PSCs are the main source of fibrosis in chronic pancreatitis and of desmoplasia in PDAC. The majority of studies on PSC are based on in vitro experiments relying on immortalized cell lines derived from diseased human pancreas or from animal models. These PSCs are usually activated and may not represent the biological context of their tissue of origin. (1) To isolate and culture primary human PSC from different disease contexts with minimal impact on their state of activation. (2) To perform a comparative analysis of phenotypes of PSC derived from different contexts. PSCs were isolated from normal pancreas, chronic pancreatitis, and PDAC using a hybrid method of digestion and outgrowth. To minimize activation by serum compounds, cells were cultured in a low-serum environment (2.5 % fetal bovine serum (FBS)). Expression patterns of commonly used markers for PSC phenotype and activity were compared between primary PSC lines derived from different contexts and correlated to expression in their original tissues. Isolation was successful from 14 of 17 tissues (82 %). Isolated PSC displayed stable viability and phenotype in low-serum environment. Expression profiles of isolated PSC and matched original tissues were closely correlated. PDAC-derived PSC tended to have a higher status of activation if compared to PSC derived from non-cancerous tissues. Primary human PSCs isolated from different contexts and cultured in a low-serum environment maintain a phenotype that reflects the stromal activity present in their tissue of origin.

Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells

PubMed Central

Ulm, Ashley; Mayhew, Christopher N.; Debley, Jason; Khurana Hershey, Gurjit K.; Ji, Hong

2016-01-01

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They are also involved in many airway diseases through their innate immune response and interaction with immune and airway stromal cells. NECs are of particular interest for studies in children due to their accessibility during clinical visits. Human induced pluripotent stem cells (iPSCs) have been generated from multiple cell types and are a powerful tool for modeling human development and disease, as well as for their potential applications in regenerative medicine. This is the first protocol to lay out methods for successful generation of iPSCs from NECs derived from pediatric participants for research purposes. It describes how to obtain nasal epithelial cells from children, how to generate primary NEC cultures from these samples, and how to reprogram primary NECs into well-characterized iPSCs. Nasal mucosa samples are useful in epidemiological studies related to the effects of air pollution in children, and provide an important tool for studying airway disease. Primary nasal cells and iPSCs derived from them can be a tool for providing unlimited material for patient-specific research in diverse areas of airway epithelial biology, including asthma and COPD research. PMID:27022951

Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells.

PubMed

Ulm, Ashley; Mayhew, Christopher N; Debley, Jason; Khurana Hershey, Gurjit K; Ji, Hong

2016-03-10

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They are also involved in many airway diseases through their innate immune response and interaction with immune and airway stromal cells. NECs are of particular interest for studies in children due to their accessibility during clinical visits. Human induced pluripotent stem cells (iPSCs) have been generated from multiple cell types and are a powerful tool for modeling human development and disease, as well as for their potential applications in regenerative medicine. This is the first protocol to lay out methods for successful generation of iPSCs from NECs derived from pediatric participants for research purposes. It describes how to obtain nasal epithelial cells from children, how to generate primary NEC cultures from these samples, and how to reprogram primary NECs into well-characterized iPSCs. Nasal mucosa samples are useful in epidemiological studies related to the effects of air pollution in children, and provide an important tool for studying airway disease. Primary nasal cells and iPSCs derived from them can be a tool for providing unlimited material for patient-specific research in diverse areas of airway epithelial biology, including asthma and COPD research.

Epidermal growth factor receptor expression in primary cultured human colorectal carcinoma cells.

PubMed Central

Tong, W. M.; Ellinger, A.; Sheinin, Y.; Cross, H. S.

1998-01-01

In situ hybridization on human colon tissue demonstrates that epidermal growth factor receptor (EGFR) mRNA expression is strongly increased during tumour progression. To obtain test systems to evaluate the relevance of growth factor action during carcinogenesis, primary cultures from human colorectal carcinomas were established. EGFR distribution was determined in 2 of the 27 primary cultures and was compared with that in well-defined subclones derived from the Caco-2 cell line, which has the unique property to differentiate spontaneously in vitro in a manner similar to normal enterocytes. The primary carcinoma-derived cells had up to three-fold higher total EGFR levels than the Caco-2 subclones and a basal mitotic rate at least fourfold higher. The EGFR affinity constant is 0.26 nmol l(-1), which is similar to that reported in Caco-2 cells. The proliferation rate of Caco-2 cells is mainly induced by EGF from the basolateral cell surface where the majority of receptors are located, whereas primary cultures are strongly stimulated from the apical side also. This corresponds to a three- to fivefold higher level of EGFR at the apical cell surface. This redistribution of EGFR to apical plasma membranes in advanced colon carcinoma cells suggests that autocrine growth factors in the colon lumen may play a significant role during tumour progression. Images Figure 1 Figure 2 PMID:9667648

Combined platelet and plasma derivatives enhance proliferation of stem/progenitor cells maintaining their differentiation potential.

PubMed

Muraglia, Anita; Todeschi, Maria Rosa; Papait, Andrea; Poggi, Alessandro; Spanò, Raffaele; Strada, Paolo; Cancedda, Ranieri; Mastrogiacomo, Maddalena

2015-12-01

Platelet derivatives have been proposed as alternatives to animal sera given that for cell therapy applications, the use of fetal bovine/calf serum (FBS/FCS) is subjected to severe limitations for safety and ethical concerns. We developed a cell culture medium additive obtained by the combination of two blood-derived standardized components. A platelet lysate (PL) and a platelet-poor plasma (PPP) were produced in a lyophilized form. Each component was characterized for its growth factor content (platelet-derived growth factor-BB/vascular endothelial growth factor). PL and PPP were used as single components or in combination in different ratio at cumulative 5% final concentration in the culture medium. The single components were less effective than the component combination. In primary cell cultures (bone marrow stromal cells, adipose derived adult stem cells, osteoblasts, chondrocytes, umbilical cord-derived mesenchymal stromal cells, lymphocytes), the PL/PPP supplement promoted an increased cell proliferation in respect to the standard FCS culture in a dose-dependent manner, maintaining the cell functionality, clonogenicity, phenotype and differentiative properties throughout the culture. At a different component ratio, the supplement was also used to support proliferation of a cell line (U-937). The PL/PPP supplement is an efficient cell culture medium additive that can replace FCS to promote cell proliferation. It can outdo FCS, especially when adopted in primary cultures from tissue biopsies. Moreover, the dual component nature of the supplement allows the researcher to determine the more appropriate ratio of the two components for the nutritional and functional requirements of the cell type of interest. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Targeting PRMT5 as a Novel Radiosensitization Approach for Primary and Recurrent Prostate Cancer Treatment

DTIC Science & Technology

2014-08-01

acquisition of tumorigenicity in nude mice by lymphoblastoid cell line derived from patients with xeroderma pigmentosum group A. (2). Subtractive...isolation of genes contributing to the acquisition of tumorigenicity by lymphoblastoid cell line derived from xeroderma pigmentosum group A patient

Interferon beta 2/B-cell stimulatory factor type 2 shares identity with monocyte-derived hepatocyte-stimulating factor and regulates the major acute phase protein response in liver cells.

PubMed Central

Gauldie, J; Richards, C; Harnish, D; Lansdorp, P; Baumann, H

1987-01-01

One of the oldest and most preserved of the homeostatic responses of the body to injury is the acute phase protein response associated with inflammation. The liver responds to hormone-like mediators by the increased synthesis of a series of plasma proteins called acute phase reactants. In these studies, we examined the relationship of hepatocyte-stimulating factor derived from peripheral blood monocytes to interferon beta 2 (IFN-beta 2), which has been cloned. Antibodies raised against fibroblast-derived IFN-beta having neutralizing activity against both IFN-beta 1 and -beta 2 inhibited the major hepatocyte-stimulating activity derived from monocytes. Fibroblast-derived mediator elicited the identical stimulated response in human HepG2 cells and primary rat hepatocytes as the monocyte cytokine. Finally, recombinant-derived human B-cell stimulatory factor type 2 (IFN-beta 2) from Escherichia coli induced the synthesis of all major acute phase proteins studied in human hepatoma HepG2 and primary rat hepatocyte cultures. These data demonstrate that monocyte-derived hepatocyte-stimulating factor and IFN-beta 2 share immunological and functional identity and that IFN-beta 2, also known as B-cell stimulatory factor and hybridoma plasmacytoma growth factor, has the hepatocyte as a major physiologic target and thereby is essential in controlling the hepatic acute phase response. Images PMID:2444978

Potential Use of Autologous Renal Cells from Diseased Kidneys for the Treatment of Renal Failure.

PubMed

George, Sunil K; Abolbashari, Mehran; Jackson, John D; Aboushwareb, Tamer; Atala, Anthony; Yoo, James J

2016-01-01

Chronic kidney disease (CKD) occurs when certain conditions cause the kidneys to gradually lose function. For patients with CKD, renal transplantation is the only treatment option that restores kidney function. In this study, we evaluated primary renal cells obtained from diseased kidneys to determine whether their normal phenotypic and functional characteristics are retained, and could be used for cell therapy. Primary renal cells isolated from both normal kidneys (NK) and diseased kidneys (CKD) showed similar phenotypic characteristics and growth kinetics. The expression levels of renal tubular cell markers, Aquaporin-1 and E-Cadherin, and podocyte-specific markers, WT-1 and Nephrin, were similar in both NK and CKD kidney derived cells. Using fluorescence- activated cell sorting (FACS), specific renal cell populations were identified and included proximal tubular cells (83.1% from NK and 80.3% from CKD kidneys); distal tubular cells (11.03% from NK and 10.9% from CKD kidneys); and podocytes (1.91% from NK and 1.78% from CKD kidneys). Ultra-structural analysis using scanning electron microscopy (SEM) revealed microvilli on the apical surface of cultured cells from NK and CKD samples. Moreover, transmission electron microscopy (TEM) analysis showed a similar organization of tight junctions, desmosomes, and other intracellular structures. The Na+ uptake characteristics of NK and CKD derived renal cells were also similar (24.4 mmol/L and 25 mmol/L, respectively) and no significant differences were observed in the protein uptake and transport characteristics of these two cell isolates. These results show that primary renal cells derived from diseased kidneys such as CKD have similar structural and functional characteristics to their counterparts from a normal healthy kidney (NK) when grown in vitro. This study suggests that cells derived from diseased kidney may be used as an autologous cell source for renal cell therapy, particularly in patients with CKD or end-stage renal disease (ESRD).

Translating stem cell research: challenges at the research frontier.

PubMed

Magnus, David

2010-01-01

This paper will address the translation of basic stem cell research into clinical research. While "stem cell" trials are sometimes used to describe established practices of bone marrow transplantation or transplantation of primary cells derived from bone marrow, for the purposes of this paper, I am primarily focusing on stem cell trials which are far less established, including use of hESC derived stem cells. The central ethical challenges in stem cell clinical trials arise in frontier research, not in standard, well-established areas of research.

Exosomes isolated from cancer patients' sera transfer malignant traits and confer the same phenotype of primary tumors to oncosuppressor-mutated cells.

PubMed

Abdouh, Mohamed; Hamam, Dana; Gao, Zu-Hua; Arena, Vincenzo; Arena, Manuel; Arena, Goffredo Orazio

2017-08-30

Horizontal transfer of malignant traits from the primary tumor to distant organs, through blood circulating factors, has recently become a thoroughly studied metastatic pathway to explain cancer dissemination. Recently, we reported that oncosuppressor gene-mutated human cells undergo malignant transformation when exposed to cancer patients' sera. We also observed that oncosuppressor mutated cells would show an increased uptake of cancer-derived exosomes and we suggested that oncosuppressor genes might protect the integrity of the cell genome by blocking integration of cancer-derived exosomes. In the present study, we tested the hypothesis that cancer patients' sera-derived exosomes might be responsible for the malignant transformation of target cells and that oncosuppressor mutation would promote their increased uptake. We also sought to unveil the mechanisms behind the hypothesized phenomena. We used human BRCA1 knockout (BRCA1-KO) fibroblasts as target cells. Cells were treated in vitro with cancer patients' sera or cancer patients' sera-derived exosomes. Treated cells were injected into NOD-SCID mice. Immunohistochemical analyses were performed to determine the differentiation state of the xenotransplants. Mass spectrometry analyses of proteins from cancer exosomes and the BRCA1-KO fibroblasts' membrane were performed to investigate possible de novo expression of molecules involved in vesicles uptake. Blocking of the identified molecules in vitro was performed and in vivo experiments were conducted to confirm the role of these molecules in the malignant transformation carried out by cancer-derived exosomes. Cells treated with exosomes isolated from cancer patients' sera underwent malignant transformation and formed tumors when transplanted into immunodeficient mice. Histological analyses showed that the tumors were carcinomas that differentiated into the same lineage of the primary tumors of blood donors. Oncosuppressor mutation promoted the de novo expression, on the plasma membrane of target cells, of receptors, responsible for the increased uptake of cancer-derived exosomes. The selective blocking of these receptors inhibited the horizontal transfer of malignant traits. These findings strengthen the hypothesis that oncogenic factors transferred via circulating cancer exosomes, induce malignant transformation of target cells even at distance. Oncosuppressor genes might protect the integrity of the cell genome by inhibiting the uptake of cancer-derived exosomes.

The retinoblastoma gene is frequently altered leading to loss of expression in primary breast tumours.

PubMed

Varley, J M; Armour, J; Swallow, J E; Jeffreys, A J; Ponder, B A; T'Ang, A; Fung, Y K; Brammar, W J; Walker, R A

1989-06-01

We have analysed the organisation of the retinoblastoma (RB1) gene in 77 primary breast carcinomas, in metastatic tissue derived from 16 of those primary tumours, and in a variety of benign breast lesions. Expression of RB1 was also assessed in most samples by immunohistochemical detection of the RB1 protein in tissue sections. Structural abnormalities to RB1 were detected in DNA from 15/77 (19%) of primary breast carcinomas examined. Where DNA was available from metastatic tissue derived from such primary tumours, the same aberration could be detected. No alterations were seen in benign breast lesions. 16/56 (29%) of tumours examined for expression by immunohistochemical methods showed a proportion of tumour cells to be completely negative for the RB1 protein. All tumours in which a structural alteration to RB1 was detected had a proportion of negative cells, except for one case where all cells were positive. Several primary tumour samples were identified where there was no detectable structural change to the gene, but there was loss of expression in some tumour cells. The data presented here demonstrate that changes to the RB1 gene leading to loss of expression of both alleles are frequent in primary human breast tumours.

Induced pluripotent stem cell-derived limbal epithelial cells (LiPSC) as a cellular alternative for in vitro ocular toxicity testing.

PubMed

Aberdam, Edith; Petit, Isabelle; Sangari, Linda; Aberdam, Daniel

2017-01-01

Induced pluripotent stem cells hold great potential to produce unlimited amount of differentiated cells as cellular source for regenerative medicine but also for in vitro drug screening and cytotoxicity tests. Ocular toxicity testing is mandatory to evaluate the risks of drugs and cosmetic products before their application to human patients by preventing eye irritation or insult. Since the global ban to use animals, many human-derived alternatives have been proposed, from ex-vivo enucleated postmortem cornea, primary corneal cell culture and immortalized corneal epithelial cell lines. All of them share limitations for their routine use. Using an improved protocol, we derived limbal epithelial cells from human induced pluripotent stem cells, named LiPSC, that are able to be passaged and differentiate further into corneal epithelial cells. Comparative RT-qPCR, immunofluorescence staining, flow cytometry analysis and zymography assays demonstrate that LiPSC are morphologically and molecularly similar to the adult stem cells. Moreover, contrary to HCE, LiPSC and primary limbal cells display similarly sensitive to cytotoxicity treatment among passages. Our data strongly suggest that LiPSC could become a powerful alternative cellular model for cosmetic and drug tests.

Induced pluripotent stem cell-derived limbal epithelial cells (LiPSC) as a cellular alternative for in vitro ocular toxicity testing

PubMed Central

Aberdam, Edith; Petit, Isabelle; Sangari, Linda

2017-01-01

Induced pluripotent stem cells hold great potential to produce unlimited amount of differentiated cells as cellular source for regenerative medicine but also for in vitro drug screening and cytotoxicity tests. Ocular toxicity testing is mandatory to evaluate the risks of drugs and cosmetic products before their application to human patients by preventing eye irritation or insult. Since the global ban to use animals, many human-derived alternatives have been proposed, from ex-vivo enucleated postmortem cornea, primary corneal cell culture and immortalized corneal epithelial cell lines. All of them share limitations for their routine use. Using an improved protocol, we derived limbal epithelial cells from human induced pluripotent stem cells, named LiPSC, that are able to be passaged and differentiate further into corneal epithelial cells. Comparative RT-qPCR, immunofluorescence staining, flow cytometry analysis and zymography assays demonstrate that LiPSC are morphologically and molecularly similar to the adult stem cells. Moreover, contrary to HCE, LiPSC and primary limbal cells display similarly sensitive to cytotoxicity treatment among passages. Our data strongly suggest that LiPSC could become a powerful alternative cellular model for cosmetic and drug tests. PMID:28640863

Virus-host interactions in persistently FMDV-infected cells derived from bovine pharynx

USDA-ARS?s Scientific Manuscript database

Foot-and-mouth disease virus (FMDV) produces a disease in cattle characterized by vesicular lesions and a persistent infection with asymptomatic low-level production of virus. Here we describe the establishment of a persistently infected primary cell culture derived from bovine pharynx tissue (PBPT)...

Induced Pluripotent Stem Cells: A novel frontier in the study of human primary immunodeficiencies

PubMed Central

Pessach, Itai M.; Ordovas-Montanes, Jose; Zhang, Shen-Ying; Casanova, Jean-Laurent; Giliani, Silvia; Gennery, Andrew R.; Al-Herz, Waleed; Manos, Philip D.; Schlaeger, Thorsten M.; Park, In-Hyun; Rucci, Francesca; Agarwal, Suneet; Mostoslavsky, Gustavo; Daley, George Q.; Notarangelo, Luigi D.

2010-01-01

Background The novel ability to epigenetically reprogram somatic cells into induced pluripotent stem cells through the exogenous expression of transcription promises to revolutionize the study of human diseases. Objective Here we report on the generation of 25 induced pluripotent stem cell lines from 6 patients with various forms of Primary Immunodeficiencies, affecting adaptive and/or innate immunity. Methods Patients’ dermal fibroblasts were reprogrammed by expression of four transcription factors, OCT4, SOX2, KLF4, and c-MYC using a single excisable polycistronic lentiviral vector. Results Induced pluripotent stem cells derived from patients with primary immunodeficiencies show a stemness profile that is comparable to that observed in human embryonic stem cells. Following in vitro differentiation into embryoid bodies, pluripotency of the patient-derived indiced pluripotent stem cells lines was demonstrated by expression of genes characteristic of each of the three embryonic layers. We have confirmed the patient-specific origin of the induced pluripotent stem cell lines, and ascertained maintenance of karyotypic integrity. Conclusion By providing a limitless source of diseased stem cells that can be differentiated into various cell types in vitro, the repository of induced pluripotent stem cell lines from patients with primary immunodeficiencies represents a unique resource to investigate the pathophysiology of hematopoietic and extra-hematopoietic manifestations of these diseases, and may assist in the development of novel therapeutic approaches based on gene correction. PMID:21185069

Newly-derived neuroblastoma cell lines propagated in serum-free media recapitulate the genotype and phenotype of primary neuroblastoma tumours.

PubMed

Bate-Eya, Laurel T; Ebus, Marli E; Koster, Jan; den Hartog, Ilona J M; Zwijnenburg, Danny A; Schild, Linda; van der Ploeg, Ida; Dolman, M Emmy M; Caron, Huib N; Versteeg, Rogier; Molenaar, Jan J

2014-02-01

Recently protocols have been devised for the culturing of cell lines from fresh tumours under serum-free conditions in defined neural stem cell medium. These cells, frequently called tumour initiating cells (TICs) closely retained characteristics of the tumours of origin. We report the isolation of eight newly-derived neuroblastoma TICs from six primary neuroblastoma tumours and two bone marrow metastases. The primary tumours from which these TICs were generated have previously been fully typed by whole genome sequencing (WGS). Array comparative genomic hybridisation (aCGH) analysis showed that TIC lines retained essential characteristics of the primary tumours and exhibited typical neuroblastoma chromosomal aberrations such as MYCN amplification, gain of chromosome 17q and deletion of 1p36. Protein analysis showed expression for neuroblastoma markers MYCN, NCAM, CHGA, DBH and TH while haematopoietic markers CD19 and CD11b were absent. We analysed the growth characteristics and confirmed tumour-forming potential using sphere-forming assays, subcutaneous and orthotopic injection of these cells into immune-compromised mice. Affymetrix mRNA expression profiling of TIC line xenografts showed an expression pattern more closely mimicking primary tumours compared to xenografts from classical cell lines. This establishes that these neuroblastoma TICs cultured under serum-free conditions are relevant and useful neuroblastoma tumour models. Copyright © 2013 Elsevier Ltd. All rights reserved.

A 3D Human Renal Cell Carcinoma-on-a-Chip for the Study of Tumor Angiogenesis.

PubMed

Miller, Chris P; Tsuchida, Connor; Zheng, Ying; Himmelfarb, Jonathan; Akilesh, Shreeram

2018-06-01

Tractable human tissue-engineered 3D models of cancer that enable fine control of tumor growth, metabolism, and reciprocal interactions between different cell types in the tumor microenvironment promise to accelerate cancer research and pharmacologic testing. Progress to date mostly reflects the use of immortalized cancer cell lines, and progression to primary patient-derived tumor cells is needed to realize the full potential of these platforms. For the first time, we report endothelial sprouting induced by primary patient tumor cells in a 3D microfluidic system. Specifically, we have combined primary human clear cell renal cell carcinoma (ccRCC) cells from six independent donors with human endothelial cells in a vascularized, flow-directed, 3D culture system ("ccRCC-on-a-chip"). The upregulation of key angiogenic factors in primary human ccRCC cells, which exhibited unique patterns of donor variation, was further enhanced when they were cultured in 3D clusters. When embedded in the matrix surrounding engineered human vessels, these ccRCC tumor clusters drove potent endothelial cell sprouting under continuous flow, thus recapitulating the critical angiogenic signaling axis between human ccRCC cells and endothelial cells. Importantly, this phenotype was driven by a primary tumor cell-derived biochemical gradient of angiogenic growth factor accumulation that was subject to pharmacological blockade. Our novel 3D system represents a vascularized tumor model that is easy to image and quantify and is fully tunable in terms of input cells, perfusate, and matrices. We envision that this ccRCC-on-a-chip will be valuable for mechanistic studies, for studying tumor-vascular cell interactions, and for developing novel and personalized antitumor therapies. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Chromosome microduplication in somatic cells decreases the genetic stability of human reprogrammed somatic cells and results in pluripotent stem cells.

PubMed

Yu, Yang; Chang, Liang; Zhao, Hongcui; Li, Rong; Fan, Yong; Qiao, Jie

2015-05-12

Human pluripotent stem cells, including cloned embryonic and induced pluripotent stem cells, offer a limitless cellular source for regenerative medicine. However, their derivation efficiency is limited, and a large proportion of cells are arrested during reprogramming. In the current study, we explored chromosome microdeletion/duplication in arrested and established reprogrammed cells. Our results show that aneuploidy induced by somatic cell nuclear transfer technology is a key factor in the developmental failure of cloned human embryos and primary colonies from implanted cloned blastocysts and that expression patterns of apoptosis-related genes are dynamically altered. Overall, ~20%-53% of arrested primary colonies in induced plurpotent stem cells displayed aneuploidy, and upregulation of P53 and Bax occurred in all arrested primary colonies. Interestingly, when somatic cells with pre-existing chromosomal mutations were used as donor cells, no cloned blastocysts were obtained, and additional chromosomal mutations were detected in the resulting iPS cells following long-term culture, which was not observed in the two iPS cell lines with normal karyotypes. In conclusion, aneuploidy induced by the reprogramming process restricts the derivation of pluripotent stem cells, and, more importantly, pre-existing chromosomal mutations enhance the risk of genome instability, which limits the clinical utility of these cells.

Patient-derived osteosarcoma cells are resistant to methotrexate.

PubMed

Dos Santos Cavalcanti, Amanda; Meohas, Walter; Ribeiro, Gabriele de Oliveira; de Sá Lopes, Ana Cristina; Gholamin, Sharareh; Razavi, Mostafa; Hanae Kasai Brunswick, Taís; Avan, Amir; Matheus Guimarães, João Antonio; Leite Duarte, Maria Eugenia; Kahn, Suzana Assad

2017-01-01

Osteosarcoma is the most common primary bone tumor in children and young adults. The median survival of osteosarcoma patients has not significantly improved since 1990, despite administration of different classes of chemotherapy agents, such as methotrexate, cisplatin and doxorubicin. Cancer stem cells (CSCs) are responsible for the resistance of osteosarcoma to chemotherapy and OCT4, SOX2 and SSEA4 have been used to identify CSCs in osteosarcoma. Here, we used low-passage patient-derived osteosarcoma cells and osteosarcoma cells directly isolated from patients before and after chemotherapy treatments to evaluate the effects of chemotherapy on stem cell markers expression. We demonstrate that primary osteosarcoma cells are resistant to methotrexate treatment and sensitive to cisplatin and doxorubicin in vitro. We also verified that cisplatin and doxorubicin reduce the expression of SOX2 and OCT4 in primary osteosarcoma cells whereas methotrexate does not alter SOX2 and OCT4 expression, however it increases SSEA4 expression in primary osteosarcoma cells. Finally, we found that, although the combination treatment cisplatin plus doxorubicin inhibited the in vivo growth of osteosarcoma cells in NOD-SCID gamma mice subcutaneously injected with SaOs2, the combination treatment cisplatin plus doxorubicin plus methotrexate did not inhibit the in vivo growth of these cells. These observations may provide an explanation for the poor response of osteosarcomas to chemotherapy and point to the need of reevaluating the therapeutic strategies for human osteosarcomas.

Patient-derived osteosarcoma cells are resistant to methotrexate

PubMed Central

dos Santos Cavalcanti, Amanda; Meohas, Walter; Ribeiro, Gabriele de Oliveira; de Sá Lopes, Ana Cristina; Gholamin, Sharareh; Razavi, Mostafa; Hanae Kasai Brunswick, Taís; Avan, Amir; Matheus Guimarães, João Antonio; Leite Duarte, Maria Eugenia

2017-01-01

Osteosarcoma is the most common primary bone tumor in children and young adults. The median survival of osteosarcoma patients has not significantly improved since 1990, despite administration of different classes of chemotherapy agents, such as methotrexate, cisplatin and doxorubicin. Cancer stem cells (CSCs) are responsible for the resistance of osteosarcoma to chemotherapy and OCT4, SOX2 and SSEA4 have been used to identify CSCs in osteosarcoma. Here, we used low-passage patient-derived osteosarcoma cells and osteosarcoma cells directly isolated from patients before and after chemotherapy treatments to evaluate the effects of chemotherapy on stem cell markers expression. We demonstrate that primary osteosarcoma cells are resistant to methotrexate treatment and sensitive to cisplatin and doxorubicin in vitro. We also verified that cisplatin and doxorubicin reduce the expression of SOX2 and OCT4 in primary osteosarcoma cells whereas methotrexate does not alter SOX2 and OCT4 expression, however it increases SSEA4 expression in primary osteosarcoma cells. Finally, we found that, although the combination treatment cisplatin plus doxorubicin inhibited the in vivo growth of osteosarcoma cells in NOD-SCID gamma mice subcutaneously injected with SaOs2, the combination treatment cisplatin plus doxorubicin plus methotrexate did not inhibit the in vivo growth of these cells. These observations may provide an explanation for the poor response of osteosarcomas to chemotherapy and point to the need of reevaluating the therapeutic strategies for human osteosarcomas. PMID:28934267

A Primary Xenograft Model of Small Cell Lung Cancer Reveals Irreversible Changes in Gene Expression Imposed by Culture In-Vitro

PubMed Central

Daniel, Vincent C.; Marchionni, Luigi; Hierman, Jared S.; Rhodes, Jonathan T.; Devereux, Wendy L.; Rudin, Charles M.; Yung, Rex; Parmigani, Giovanni; Dorsch, Marion; Peacock, Craig D.; Watkins, D. Neil

2009-01-01

Traditional approaches to the preclinical investigation of cancer therapies rely on the use of established cell lines maintained in serum-based growth media. This is particularly true of small cell lung cancer (SCLC), where surgically resected tissue is rarely available. Recent attention has focused on the need for better models that preserve the integrity of cancer stem cell populations, as well as three-dimensional tumor-stromal interactions. Here we describe a primary xenograft model of SCLC in which endobronchial tumor specimens obtained from chemo-naive patients are serially propagated in vivo in immunodeficient mice. In parallel, cell lines grown in conventional tissue culture conditions were derived from each xenograft line, passaged for 6 months, and then re-implanted to generate secondary xenografts. Using the Affymetrix platform, we analyzed gene expression in primary xenograft, xenograft-derived cell line, and secondary xenograft, and compared these data to similar analyses of unrelated primary SCLC samples and laboratory models. When compared to normal lung, primary tumors, xenografts and cell lines displayed a gene expression signature specific for SCLC. Comparison of gene expression within the xenograft model identified a group of tumor-specific genes expressed in primary SCLC and xenografts that was lost during the transition to tissue culture, and that was not regained when the tumors were re-established as secondary xenografts. Such changes in gene expression may be a common feature of many cancer cell culture systems, with functional implications for the use of such models for preclinical drug development. PMID:19351829

Discovery of a drug targeting microenvironmental support for lymphoma cells by screening using patient-derived xenograft cells

PubMed Central

Sugimoto, Keiki; Hayakawa, Fumihiko; Shimada, Satoko; Morishita, Takanobu; Shimada, Kazuyuki; Katakai, Tomoya; Tomita, Akihiro; Kiyoi, Hitoshi; Naoe, Tomoki

2015-01-01

Cell lines have been used for drug discovery as useful models of cancers; however, they do not recapitulate cancers faithfully, especially in the points of rapid growth rate and microenvironment independency. Consequently, the majority of conventional anti-cancer drugs are less sensitive to slow growing cells and do not target microenvironmental support, although most primary cancer cells grow slower than cell lines and depend on microenvironmental support. Here, we developed a novel high throughput drug screening system using patient-derived xenograft (PDX) cells of lymphoma that maintained primary cancer cell phenotype more than cell lines. The library containing 2613 known pharmacologically active substance and off-patent drugs were screened by this system. We could find many compounds showing higher cytotoxicity than conventional anti-tumor drugs. Especially, pyruvinium pamoate showed the highest activity and its strong anti-tumor effect was confirmed also in vivo. We extensively investigated its mechanism of action and found that it inhibited glutathione supply from stromal cells to lymphoma cells, implying the importance of the stromal protection from oxidative stress for lymphoma cell survival and a new therapeutic strategy for lymphoma. Our system introduces a primary cancer cell phenotype into cell-based phenotype screening and sheds new light on anti-cancer drug development. PMID:26278963

Immortalized prairie vole-derived fibroblasts (VMF-K4DTs) can be transformed into pluripotent stem cells and provide a useful tool with which to determine optimal reprogramming conditions

PubMed Central

KATAYAMA, Masafumi; HIRAYAMA, Takashi; KIYONO, Tohru; ONUMA, Manabu; TANI, Tetsuya; TAKEDA, Satoru; NISHIMORI, Katsuhiko; FUKUDA, Tomokazu

2017-01-01

The cellular conditions required to establish induced pluripotent stem cells (iPSCs), such as the number of reprogramming factors and/or promoter selection, differ among species. The establishment of iPSCs derived from cells of previously unstudied species therefore requires the extensive optimization of programming conditions, including promoter selection and the optimal number of reprogramming factors, through a trial-and-error approach. While the four Yamanaka factors Oct3/4, Sox2, Klf4, and c-Myc are sufficient for iPSC establishment in mice, we reported previously that six reprogramming factors were necessary for the creation of iPSCs from primary prairie vole-derived cells. Further to this study, we now show detailed data describing the optimization protocol we developed in order to obtain iPSCs from immortalized prairie vole-derived fibroblasts. Immortalized cells can be very useful tools in the optimization of cellular reprogramming conditions, as cellular senescence is known to dramatically decrease the efficiency of iPSC establishment. The immortalized prairie vole cells used in this optimization were designated K4DT cells as they contained mutant forms of CDK4, cyclin D, and telomerase reverse transcriptase (TERT). We show that iPSCs derived from these immortalized cells exhibit the transcriptional silencing of exogenous reprogramming factors while maintaining pluripotent cell morphology. There were no observed differences between the iPSCs derived from primary and immortalized prairie vole fibroblasts. Our data suggest that cells that are immortalized with mutant CDK4, cyclin D, and TERT provide a useful tool for the determination of the optimal conditions for iPSC establishment. PMID:28331164

Immortalized prairie vole-derived fibroblasts (VMF-K4DTs) can be transformed into pluripotent stem cells and provide a useful tool with which to determine optimal reprogramming conditions.

PubMed

Katayama, Masafumi; Hirayama, Takashi; Kiyono, Tohru; Onuma, Manabu; Tani, Tetsuya; Takeda, Satoru; Nishimori, Katsuhiko; Fukuda, Tomokazu

2017-06-21

The cellular conditions required to establish induced pluripotent stem cells (iPSCs), such as the number of reprogramming factors and/or promoter selection, differ among species. The establishment of iPSCs derived from cells of previously unstudied species therefore requires the extensive optimization of programming conditions, including promoter selection and the optimal number of reprogramming factors, through a trial-and-error approach. While the four Yamanaka factors Oct3/4, Sox2, Klf4, and c-Myc are sufficient for iPSC establishment in mice, we reported previously that six reprogramming factors were necessary for the creation of iPSCs from primary prairie vole-derived cells. Further to this study, we now show detailed data describing the optimization protocol we developed in order to obtain iPSCs from immortalized prairie vole-derived fibroblasts. Immortalized cells can be very useful tools in the optimization of cellular reprogramming conditions, as cellular senescence is known to dramatically decrease the efficiency of iPSC establishment. The immortalized prairie vole cells used in this optimization were designated K4DT cells as they contained mutant forms of CDK4, cyclin D, and telomerase reverse transcriptase (TERT). We show that iPSCs derived from these immortalized cells exhibit the transcriptional silencing of exogenous reprogramming factors while maintaining pluripotent cell morphology. There were no observed differences between the iPSCs derived from primary and immortalized prairie vole fibroblasts. Our data suggest that cells that are immortalized with mutant CDK4, cyclin D, and TERT provide a useful tool for the determination of the optimal conditions for iPSC establishment.

The terminator mouse: salvation for primary cell culture.

PubMed

Kabgani, Nazanin; Moeller, Marcus J

2013-11-01

The Terminator had to come back from the future already several times in an effort to bring salvation to mankind. In the present issue of Kidney International, Guo et al. brought us a novel transgenic mouse model: the terminator mouse. This highly elegant mouse may facilitate significantly the derivation of primary cultures of a specific cell type from a tissue containing multiple cell populations.

Derivation of Human Skin Fibroblast Lines for Feeder Cells of Human Embryonic Stem Cells.

PubMed

Unger, Christian; Felldin, Ulrika; Rodin, Sergey; Nordenskjöld, Agneta; Dilber, Sirac; Hovatta, Outi

2016-02-03

After the first derivations of human embryonic stem cell (hESC) lines on fetal mouse feeder cell layers, the idea of using human cells instead of mouse cells as feeder cells soon arose. Mouse cells bear a risk of microbial contamination, and nonhuman immunogenic proteins are absorbed from the feeders to hESCs. Human skin fibroblasts can be effectively used as feeder cells for hESCs. The same primary cell line, which can be safely used for up to 15 passages after stock preparations, can be expanded and used for large numbers of hESC derivations and cultures. These cells are relatively easy to handle and maintain. No animal facilities or animal work is needed. Here, we describe the derivation, culture, and cryopreservation procedures for research-grade human skin fibroblast lines. We also describe how to make feeder layers for hESCs using these fibroblasts. Copyright © 2016 John Wiley & Sons, Inc.

Structural phenotyping of stem cell-derived cardiomyocytes.

PubMed

Pasqualini, Francesco Silvio; Sheehy, Sean Paul; Agarwal, Ashutosh; Aratyn-Schaus, Yvonne; Parker, Kevin Kit

2015-03-10

Structural phenotyping based on classical image feature detection has been adopted to elucidate the molecular mechanisms behind genetically or pharmacologically induced changes in cell morphology. Here, we developed a set of 11 metrics to capture the increasing sarcomere organization that occurs intracellularly during striated muscle cell development. To test our metrics, we analyzed the localization of the contractile protein α-actinin in a variety of primary and stem-cell derived cardiomyocytes. Further, we combined these metrics with data mining algorithms to unbiasedly score the phenotypic maturity of human-induced pluripotent stem cell-derived cardiomyocytes. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Primary human polarized small intestinal epithelial barriers respond differently to a hazardous and an innocuous protein.

PubMed

Eaton, A D; Zimmermann, C; Delaney, B; Hurley, B P

2017-08-01

An experimental platform employing human derived intestinal epithelial cell (IEC) line monolayers grown on permeable Transwell ® filters was previously investigated to differentiate between hazardous and innocuous proteins. This approach was effective at distinguishing these types of proteins and perturbation of monolayer integrity, particularly transepithelial electrical resistance (TEER), was the most sensitive indicator. In the current report, in vitro indicators of monolayer integrity, cytotoxicity, and inflammation were evaluated using primary (non-transformed) human polarized small intestinal epithelial barriers cultured on Transwell ® filters to compare effects of a hazardous protein (Clostridium difficile Toxin A [ToxA]) and an innocuous protein (bovine serum albumin [BSA]). ToxA exerted a reproducible decrease on barrier integrity at doses comparable to those producing effects observed from cell line-derived IEC monolayers, with TEER being the most sensitive indicator. In contrast, BSA, tested at concentrations substantially higher than ToxA, did not cause changes in any of the tested variables. These results demonstrate a similarity in response to certain proteins between cell line-derived polarized IEC models and a primary human polarized small intestinal epithelial barrier model, thereby reinforcing the potential usefulness of cell line-derived polarized IECs as a valid experimental platform to differentiate between hazardous and non-hazardous proteins. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Differences in lymphocyte developmental potential between human embryonic stem cell and umbilical cord blood–derived hematopoietic progenitor cells

PubMed Central

Martin, Colin H.; Woll, Petter S.; Ni, Zhenya; Zúñiga-Pflücker, Juan Carlos

2008-01-01

Hematopoietic progenitor cells derived from human embryonic stem cells (hESCs) develop into diverse mature hematopoietic lineages, including lymphocytes. Whereas functional natural killer (NK) cells can be efficiently generated in vitro from hESC-derived CD34+ cells, studies of T- and B-cell development from hESCs have been much more limited. Here, we demonstrate that despite expressing functional Notch-1, CD34+ cells from hESCs did not derive T cells when cocultured with OP9 cells expressing Delta-like 1, or in fetal thymus organ culture. hESC-derived CD34+ cells also did not produce B cells in vitro. In contrast, CD34+ cells isolated from UCB routinely generated T and B cells when cultured in the same conditions. Notably, both undifferentiated hESCs, and sorted hESC-derived populations with hematopoietic developmental potential exhibited constitutive expression of ID family genes and of transcriptional targets of stem cell factor–induced signaling. These pathways both inhibit T-cell development and promote NK-cell development. Together, these results demonstrate fundamental differences between hESC-derived hematopoietic progenitors and analogous primary human cells. Therefore, hESCs can be more readily supported to differentiate into certain cell types than others, findings that have important implications for derivation of defined lineage-committed populations from hESCs. PMID:18621931

Differences in lymphocyte developmental potential between human embryonic stem cell and umbilical cord blood-derived hematopoietic progenitor cells.

PubMed

Martin, Colin H; Woll, Petter S; Ni, Zhenya; Zúñiga-Pflücker, Juan Carlos; Kaufman, Dan S

2008-10-01

Hematopoietic progenitor cells derived from human embryonic stem cells (hESCs) develop into diverse mature hematopoietic lineages, including lymphocytes. Whereas functional natural killer (NK) cells can be efficiently generated in vitro from hESC-derived CD34(+) cells, studies of T- and B-cell development from hESCs have been much more limited. Here, we demonstrate that despite expressing functional Notch-1, CD34(+) cells from hESCs did not derive T cells when cocultured with OP9 cells expressing Delta-like 1, or in fetal thymus organ culture. hESC-derived CD34(+) cells also did not produce B cells in vitro. In contrast, CD34(+) cells isolated from UCB routinely generated T and B cells when cultured in the same conditions. Notably, both undifferentiated hESCs, and sorted hESC-derived populations with hematopoietic developmental potential exhibited constitutive expression of ID family genes and of transcriptional targets of stem cell factor-induced signaling. These pathways both inhibit T-cell development and promote NK-cell development. Together, these results demonstrate fundamental differences between hESC-derived hematopoietic progenitors and analogous primary human cells. Therefore, hESCs can be more readily supported to differentiate into certain cell types than others, findings that have important implications for derivation of defined lineage-committed populations from hESCs.

Establishment of primary cell cultures from fish calcified tissues

PubMed Central

Marques, Cátia L.; Rafael, Marta S.; Cancela, M. Leonor

2007-01-01

Fishes have been recently recognized as a suitable model organism to study vertebrate physiological processes, in particular skeletal development and tissue mineralization. However, there is a lack of well characterized in vitro cell systems derived from fish calcified tissues. We describe here a protocol that was successfully used to develop the first calcified tissue-derived cell cultures of fish origin. Vertebra and branchial arches collected from young gilthead seabreams were fragmented then submitted to the combined action of collagenase and trypsin to efficiently release cells embedded in the collagenous extracellular matrix. Primary cultures were maintained under standard conditions and spontaneously transformed to form continuous cell lines suitable for studying mechanisms of tissue mineralization in seabream. This simple and inexpensive protocol is also applicable to other calcified tissues and species by adjusting parameters to each particular case. PMID:19002990

Cloning of transgenic tobacco BY-2 cells; an efficient method to analyse and reduce high natural heterogeneity of transgene expression.

PubMed

Nocarova, Eva; Fischer, Lukas

2009-04-22

Phenotypic characterization of transgenic cell lines, frequently used in plant biology studies, is complicated because transgene expression in individual cells is often heterogeneous and unstable. To identify the sources and to reduce this heterogeneity, we transformed tobacco (Nicotiana tabacum L.) BY-2 cells with a gene encoding green fluorescent protein (GFP) using Agrobacterium tumefaciens, and then introduced a simple cloning procedure to generate cell lines derived from the individual transformed cells. Expression of the transgene was monitored by analysing GFP fluorescence in the cloned lines and also in lines obtained directly after transformation. The majority ( approximately 90%) of suspension culture lines derived from calli that were obtained directly from transformation consisted of cells with various levels of GFP fluorescence. In contrast, nearly 50% of lines generated by cloning cells from the primary heterogeneous suspensions consisted of cells with homogenous GFP fluorescence. The rest of the lines exhibited "permanent heterogeneity" that could not be resolved by cloning. The extent of fluorescence heterogeneity often varied, even among genetically identical clones derived from the primary transformed lines. In contrast, the offspring of subsequent cloning of the cloned lines was uniform, showing GFP fluorescence intensity and heterogeneity that corresponded to the original clone. The results demonstrate that, besides genetic heterogeneity detected in some lines, the primary lines often contained a mixture of epigenetically different cells that could be separated by cloning. This indicates that a single integration event frequently results in various heritable expression patterns, which are probably accidental and become stabilized in the offspring of the primary transformed cells early after the integration event. Because heterogeneity in transgene expression has proven to be a serious problem, it is highly advisable to use transgenes tagged with a visual marker for BY-2 transformation. The cloning procedure can be used not only for efficient reduction of expression heterogeneity of such transgenes, but also as a useful tool for studies of transgene expression and other purposes.

Human bone marrow-derived MSCs can home to orthotopic breast cancer tumors and promote bone metastasis

PubMed Central

Goldstein, Robert H; Reagan, Michaela R; Anderson, Kristen; Kaplan, David L; Rosenblatt, Michael

2010-01-01

American women have a nearly 25% lifetime risk of developing breast cancer, with 20–40% of these patients developing life-threatening metastases. Over 70% of patients presenting with metastases have skeletal involvement, which signals progression to an incurable stage. Tumor-stroma cell interactions are only superficially understood, specifically regarding the ability of stromal cells to affect metastasis. In vivo models show that exogenously supplied hBMSCs (human bone-marrow derived stem cells) migrate to breast cancer tumors, but no reports have shown endogenous hBMSC migration from the bone to primary tumors. Here we present a model of in vivo hBMSC migration from a physiologic human bone environment to human breast tumors. Further, hBMSCs alter tumor growth and bone metastasis frequency. hBMSCs may home to certain breast tumors based on tumor-derived TGF-β1. Moreover, at the primary tumor IL-17B/IL-17BR signaling may mediate interactions between hBMSCs and breast cancer cells (BCCs). PMID:21159629

Myeloid-derived suppressor cells

PubMed Central

Chandra, Dinesh; Gravekamp, Claudia

2013-01-01

While conventional anticancer therapies, including surgical resection, radiotherapy, and/or chemotherapy, are relatively efficient at eliminating primary tumors, these treatment modalities are largely ineffective against metastases. At least in part, this reflects the rather inefficient delivery of conventional anticancer agents to metastatic lesions. We have recently demonstrated that myeloid-derived suppressor cells (MDSCs) can be used as cellular missiles to selectively deliver a radioisotope-coupled attenuated variant of Listeria monocytogenes to both primary and metastatic neoplastic lesions in mice with pancreatic cancer. This novel immunotherapeutic intervention robustly inhibited tumor growth while promoting a dramatic decrease in the number of metastases. PMID:24427545

Myeloid-derived suppressor cells: Cellular missiles to target tumors.

PubMed

Chandra, Dinesh; Gravekamp, Claudia

2013-11-01

While conventional anticancer therapies, including surgical resection, radiotherapy, and/or chemotherapy, are relatively efficient at eliminating primary tumors, these treatment modalities are largely ineffective against metastases. At least in part, this reflects the rather inefficient delivery of conventional anticancer agents to metastatic lesions. We have recently demonstrated that myeloid-derived suppressor cells (MDSCs) can be used as cellular missiles to selectively deliver a radioisotope-coupled attenuated variant of Listeria monocytogenes to both primary and metastatic neoplastic lesions in mice with pancreatic cancer. This novel immunotherapeutic intervention robustly inhibited tumor growth while promoting a dramatic decrease in the number of metastases.

Uveal Melanoma Cell Lines: Where do they come from? (An American Ophthalmological Society Thesis).

PubMed

Jager, Martine J; Magner, J Antonio Bermudez; Ksander, Bruce R; Dubovy, Sander R

2016-08-01

To determine whether some of the most often used uveal melanoma cell lines resemble their original tumor. Analysis of the literature, patient charts, histopathology, mutations, chromosome status, HLA type, and expression of melanocyte markers on cell lines and their primary tumors. We examined five cell lines and the primary tumors from which they were derived. Four of the five examined primary tumors were unusual: one occupied the orbit, two were recurrences after prior irradiation, and one developed in an eye with a nevus of Ota. One cell line did not contain the GNA11 mutation, but it was present in the primary tumor. Three of the primary tumors had monosomy 3 (two of these lacked BAP1 expression); however, all five cell lines showed disomy 3 and BAP1 expression. All of the cell lines had gain of 8q. Two cell lines lacked expression of melanocyte markers, although these were present in the corresponding primary tumor. All cell lines could be traced back to their original uveal melanoma. Four of the five primary tumors were unusual. Cell lines often differed from their primary tumor in chromosome status and melanocyte markers. However, their specific chromosome aberrations and capacity to continue proliferation characterize them as uveal melanoma cell lines.

Uveal Melanoma Cell Lines: Where do they come from? (An American Ophthalmological Society Thesis)

PubMed Central

Jager, Martine J.; Magner, J. Antonio Bermudez; Ksander, Bruce R.; Dubovy, Sander R.

2016-01-01

Purpose To determine whether some of the most often used uveal melanoma cell lines resemble their original tumor. Methods Analysis of the literature, patient charts, histopathology, mutations, chromosome status, HLA type, and expression of melanocyte markers on cell lines and their primary tumors. We examined five cell lines and the primary tumors from which they were derived. Results Four of the five examined primary tumors were unusual: one occupied the orbit, two were recurrences after prior irradiation, and one developed in an eye with a nevus of Ota. One cell line did not contain the GNA11 mutation, but it was present in the primary tumor. Three of the primary tumors had monosomy 3 (two of these lacked BAP1 expression); however, all five cell lines showed disomy 3 and BAP1 expression. All of the cell lines had gain of 8q. Two cell lines lacked expression of melanocyte markers, although these were present in the corresponding primary tumor. Conclusions All cell lines could be traced back to their original uveal melanoma. Four of the five primary tumors were unusual. Cell lines often differed from their primary tumor in chromosome status and melanocyte markers. However, their specific chromosome aberrations and capacity to continue proliferation characterize them as uveal melanoma cell lines. PMID:28018010

Lens epithelial cells derived from alphaB-crystallin knockout mice demonstrate hyperproliferation and genomic instability.

PubMed

Andley, U P; Song, Z; Wawrousek, E F; Brady, J P; Bassnett, S; Fleming, T P

2001-01-01

alphaB-crystallin is a member of the small heat shock protein family and can act as a molecular chaperone preventing the in vitro aggregation of other proteins denatured by heat or other stress conditions. Expression of alphaB-crystallin increases in cells exposed to stress and enhanced in tumors of neuroectodermal origin and in many neurodegenerative diseases. In the present study, we examined the properties of lens epithelial cells derived from mice in which the alphaB-crystallin gene had been knocked out. Primary rodent cells immortalize spontaneously in tissue culture with a frequency of 10(-5) to 10(-6). Primary lens epithelial cells derived from alphaB-crystallin-/- mice produced hyperproliferative clones at a frequency of 7.6 x 10(-2), four orders of magnitude greater than predicted by spontaneous immortalization (1). Hyperproliferative alphaB-crystallin-/- cells were shown to be truly immortal since they have been passaged for more than 100 population doublings without any diminution in growth potential. In striking contrast to the wild-type cells, which were diploid, the alphaB-crystallin-/- cultures had a high proportion of tetraploid and higher ploidy cells, indicating that the loss of alphaB-crystallin is associated with an increase in genomic instability. Further evidence of genomic instability of alphaB-crystallin-/- cells was observed when primary cultures were infected with Ad12-SV40 hybrid virus. In striking contrast to wild-type cells, alphaB-crystallin-/- cells expressing SV40 T antigen exhibited a widespread cytocidal response 2 to 3 days after attaining confluence, indicating that SV40 T antigen enhanced the intrinsic genomic instability of alphaB-crystallin-/- lens epithelial cells. These observations suggest that the widely distributed molecular chaperone alphaB-crystallin may play an important nuclear role in maintaining genomic integrity.

Productive vs non-productive infection by cell-free Varicella zoster virus of human neurons derived from embryonic stem cells is dependent upon infectious viral dose

PubMed Central

Sloutskin, Anna; Kinchington, Paul R.; Goldstein, Ronald S.

2013-01-01

Varicella Zoster virus (VZV) productively infects humans causing varicella upon primary infection and herpes zoster upon reactivation from latency in neurons. In-vitro studies using cell-associated VZV infection have demonstrated productive VZV-infection, while a few recent studies of human neurons derived from stem cells incubated with cell-free, vaccine-derived VZV did not result in generation of infectious virus. In the present study, 90%-pure human embryonic stem cell-derived neurons were incubated with recombinant cell-free pOka-derived made with an improved method or with VZV vaccine. We found that cell-free pOka and vOka at higher multiplicities of infection elicited productive infection in neurons followed by spread of infection, cytopathic effect and release of infectious virus into the medium. These results further validate the use of this unlimited source of human neurons for studying unexplored aspects of VZV interaction with neurons such as entry, latency and reactivation. PMID:23769240

Mixed microencapsulation of rat primary hepatocytes and Sertoli cells improves the metabolic function in a D-galactosamine and lipopolysaccharide-induced rat model of acute liver failure.

PubMed

Zheng, Ming-Hua; Lin, Hai-Long; Qiu, Li-Xin; Cui, Yao-Li; Sun, Qing-Feng; Chen, Yong-Ping

2009-01-01

Hepatocyte transplantation is an alternative to transplantation of the whole liver. Compared with xenogeneic hepatocytes, primary hepatocytes have some advantages, such as a more powerful function and a smaller frequency of rejection caused by the host. Cell microencapsulation prevents direct access of host cells to the graft but cannot impede transfer of transplant-derived peptides, which can cross the physical barrier. Sertoli cells are central to the immune privilege demonstrated in the testis, and their actions have been utilized to protect cell transplants. Co-microencapsulating Sertoli cells with HepG2 cells has proved to be a valuable strategy in hepatocyte transplantation. Thus mixed microcapsules of primary rat hepatocytes and primary Sertoli cells may improve metabolic function in a d-galactosamine and lipopolysaccharide-induced rat model of acute liver failure.

Functional characterization of cell hybrids generated by induced fusion of primary porcine mesenchymal stem cells with an immortal murine cell line.

PubMed

Islam, M Q; Ringe, J; Reichmann, E; Migotti, R; Sittinger, M; da S Meirelles, L; Nardi, N B; Magnusson, P; Islam, K

2006-10-01

Bone marrow mesenchymal stem cells (MSC) integrate into various organs and contribute to the regeneration of diverse tissues. However, the mechanistic basis of the plasticity of MSC is not fully understood. The change of cell fate has been suggested to occur through cell fusion. We have generated hybrid cell lines by polyethylene-glycol-mediated cell fusion of primary porcine MSC with the immortal murine fibroblast cell line F7, a derivative of the GM05267 cell line. The hybrid cell lines display fibroblastic morphology and proliferate like immortal cells. They contain tetraploid to hexaploid porcine chromosomes accompanied by hypo-diploid murine chromosomes. Interestingly, many hybrid cell lines also express high levels of tissue-nonspecific alkaline phosphatase, which is considered to be a marker of undifferentiated embryonic stem cells. All tested hybrid cell lines retain osteogenic differentiation, a few of them also retain adipogenic potential, but none retain chondrogenic differentiation. Conditioned media from hybrid cells enhance the proliferation of both early-passage and late-passage porcine MSC, indicating that the hybrid cells secrete diffusible growth stimulatory factors. Murine F7 cells thus have the unique property of generating immortal cell hybrids containing unusually high numbers of chromosomes derived from normal cells. These hybrid cells can be employed in various studies to improve our understanding of regenerative biology. This is the first report, to our knowledge, describing the generation of experimentally induced cell hybrids by using normal primary MSC.

Generation of an iPS cell line from bone marrow derived mesenchymal stromal cells from an elderly patient.

PubMed

Megges, Matthias; Geissler, Sven; Duda, Georg N; Adjaye, James

2015-11-01

An induced pluripotent stem cell line was generated from primary human bone marrow derived mesenchymal stromal cells of a 74 year old donor using retroviruses harboring OCT4, SOX2, KLF4 and c-MYC in combination with the following inhibitors TGFβ receptor-SB 431542, MEK-PD325901, and p53-Pifithrin α. Pluripotency was confirmed both in vitro and in vivo. Copyright © 2015 Elsevier B.V. All rights reserved.

Co-culture of Gastric Organoids and Immortalized Stomach Mesenchymal Cells.

PubMed

Bertaux-Skeirik, Nina; Centeno, Jomaris; Feng, Rui; Schumacher, Michael A; Shivdasani, Ramesh A; Zavros, Yana

2016-01-01

Three-dimensional primary epithelial-derived gastric organoids have recently been established as an important tool to study gastric development, physiology, and disease. Specifically, mouse-derived fundic gastric organoids (mFGOs) co-cultured with Immortalized Stomach Mesenchymal Cells (ISMCs) reflect expression patterns of mature fundic cell types seen in vivo, thus allowing for long-term in vitro studies of gastric epithelial cell physiology, regeneration, and bacterial-host interactions. Here, we describe the development and culture of mFGOs, co-cultured with ISMCs.

Transcriptional and Cell Cycle Alterations Mark Aging of Primary Human Adipose-Derived Stem Cells.

PubMed

Shan, Xiaoyin; Roberts, Cleresa; Kim, Eun Ji; Brenner, Ariana; Grant, Gregory; Percec, Ivona

2017-05-01

Adult stem cells play a critical role in the maintenance of tissue homeostasis and prevention of aging. While the regenerative potential of stem cells with low cellular turnover, such as adipose-derived stem cells (ASCs), is increasingly recognized, the study of chronological aging in ASCs is technically difficult and remains poorly understood. Here, we use our model of chronological aging in primary human ASCs to examine genome-wide transcriptional networks. We demonstrate first that the transcriptome of aging ASCs is distinctly more stable than that of age-matched fibroblasts, and further, that age-dependent modifications in cell cycle progression and translation initiation specifically characterize aging ASCs in conjunction with increased nascent protein synthesis and a distinctly shortened G1 phase. Our results reveal novel chronological aging mechanisms in ASCs that are inherently different from differentiated cells and that may reflect an organismal attempt to meet the increased demands of tissue and organ homeostasis during aging. Stem Cells 2017;35:1392-1401. © 2017 AlphaMed Press.

Comparison of Tumor- and Bone Marrow-Derived Mesenchymal Stromal/Stem Cells from Patients with High-Grade Osteosarcoma

PubMed Central

Le Nail, Louis-Romée; Brennan, Meadhbh; Rosset, Philippe; Piloquet, Philippe; Pichon, Olivier; Le Caignec, Cédric; Crenn, Vincent; Layrolle, Pierre; Hérault, Olivier; De Pinieux, Gonzague

2018-01-01

Osteosarcoma (OS) is suspected to originate from dysfunctional mesenchymal stromal/stem cells (MSC). We sought to identify OS-derived cells (OSDC) with potential cancer stem cell (CSC) properties by comparing OSDC to MSC derived from bone marrow of patients. This study included in vitro characterization with sphere forming assays, differentiation assays, cytogenetic analysis, and in vivo investigations of their tumorigenicity and tumor supportive capacities. Primary cell lines were isolated from nine high-grade OS samples. All primary cell lines demonstrated stromal cell characteristics. Compared to MSC, OSDC presented a higher ability to form sphere clones, indicating a potential CSC phenotype, and were more efficient at differentiation towards osteoblasts. None of the OSDC displayed the complex chromosome rearrangements typical of high grade OS and none of them induced tumors in immunodeficient mice. However, two OSDC demonstrated focused genomic abnormalities. Three out of seven, and six out of seven OSDC showed a supportive role on local tumor development, and on metastatic progression to the lungs, respectively, when co-injected with OS cells in nude mice. The observation of OS-associated stromal cells with rare genetic abnormalities and with the capacity to sustain tumor progression may have implications for future tumor treatments. PMID:29494553

Comparison of the reaction of bone-derived cells to enhanced MgCl2-salt concentrations.

PubMed

Burmester, Anna; Luthringer, Bérengère; Willumeit, Regine; Feyerabend, Frank

2014-01-01

Magnesium-based implants exhibit various advantages such as biodegradability and potential for enhanced in vivo bone formation. However, the cellular mechanisms behind this possible osteoconductivity remain unclear. To determine whether high local magnesium concentrations can be osteoconductive and exclude other environmental factors that occur during the degradation of magnesium implants, magnesium salt (MgCl2) was used as a model system. Because cell lines are preferred targets in studies of non-degradable implant materials, we performed a comparative study of 3 osteosarcoma-derived cell lines (MG63, SaoS2 and U2OS) with primary human osteoblasts. The correlation among cell count, viability, cell size and several MgCl2 concentrations was used to examine the influence of magnesium on proliferation in vitro. Moreover, bone metabolism alterations during proliferation were investigated by analyzing the expression of genes involved in osteogenesis. It was observed that for all cell types, the cell count decreases at concentrations above 10 mM MgCl2. However, detailed analysis showed that MgCl2 has a relevant but very diverse influence on proliferation and bone metabolism, depending on the cell type. Only for primary cells was a clear stimulating effect observed. Therefore, reliable results demonstrating the osteoconductivity of magnesium implants can only be achieved with primary osteoblasts.

Comparison of the reaction of bone-derived cells to enhanced MgCl2-salt concentrations

PubMed Central

Burmester, Anna; Luthringer, Bérengère; Willumeit, Regine; Feyerabend, Frank

2014-01-01

Magnesium-based implants exhibit various advantages such as biodegradability and potential for enhanced in vivo bone formation. However, the cellular mechanisms behind this possible osteoconductivity remain unclear. To determine whether high local magnesium concentrations can be osteoconductive and exclude other environmental factors that occur during the degradation of magnesium implants, magnesium salt (MgCl2) was used as a model system. Because cell lines are preferred targets in studies of non-degradable implant materials, we performed a comparative study of 3 osteosarcoma-derived cell lines (MG63, SaoS2 and U2OS) with primary human osteoblasts. The correlation among cell count, viability, cell size and several MgCl2 concentrations was used to examine the influence of magnesium on proliferation in vitro. Moreover, bone metabolism alterations during proliferation were investigated by analyzing the expression of genes involved in osteogenesis. It was observed that for all cell types, the cell count decreases at concentrations above 10 mM MgCl2. However, detailed analysis showed that MgCl2 has a relevant but very diverse influence on proliferation and bone metabolism, depending on the cell type. Only for primary cells was a clear stimulating effect observed. Therefore, reliable results demonstrating the osteoconductivity of magnesium implants can only be achieved with primary osteoblasts. PMID:25482335

Primary Human Uterine Leiomyoma Cell Culture Quality Control: Some Properties of Myometrial Cells Cultured under Serum Deprivation Conditions in the Presence of Ovarian Steroids.

PubMed

Bonazza, Camila; Andrade, Sheila Siqueira; Sumikawa, Joana Tomomi; Batista, Fabrício Pereira; Paredes-Gamero, Edgar J; Girão, Manoel J B C; Oliva, Maria Luiza V; Castro, Rodrigo Aquino

2016-01-01

Cell culture is considered the standard media used in research to emulate the in vivo cell environment. Crucial in vivo experiments cannot be conducted in humans and depend on in vitro methodologies such as cell culture systems. However, some procedures involving the quality control of cells in culture have been gradually neglected by failing to acknowledge that primary cells and cell lines change over time in culture. Thus, we report methods based on our experience for monitoring primary cell culture of human myometrial cells derived from uterine leiomyoma. We standardized the best procedure of tissue dissociation required for the study of multiple genetic marker systems that include species-specific antigens, expression of myofibroblast or myoblast markers, growth curve, serum deprivation, starvation by cell cycle synchronization, culture on collagen coated plates, and 17 β-estradiol (E2) and progesterone (P4) effects. The results showed that primary myometrial cells from patients with uterine leiomyoma displayed myoblast phenotypes before and after in vitro cultivation, and leiomyoma cells differentiated into mature myocyte cells under the appropriate differentiation-inducing conditions (serum deprivation). These cells grew well on collagen coated plates and responded to E2 and P4, which may drive myometrial and leiomyoma cells to proliferate and adhere into a focal adhesion complex involvement in a paracrine manner. The establishment of these techniques as routine procedures will improve the understanding of the myometrial physiology and pathogenesis of myometrium-derived diseases such as leiomyoma. Mimicking the in vivo environment of fibrotic conditions can prevent false results and enhance results that are based on cell culture integrity.

Primary Human Uterine Leiomyoma Cell Culture Quality Control: Some Properties of Myometrial Cells Cultured under Serum Deprivation Conditions in the Presence of Ovarian Steroids

PubMed Central

Sumikawa, Joana Tomomi; Batista, Fabrício Pereira; Paredes-Gamero, Edgar J.; Girão, Manoel J. B. C.; Oliva, Maria Luiza V.

2016-01-01

Cell culture is considered the standard media used in research to emulate the in vivo cell environment. Crucial in vivo experiments cannot be conducted in humans and depend on in vitro methodologies such as cell culture systems. However, some procedures involving the quality control of cells in culture have been gradually neglected by failing to acknowledge that primary cells and cell lines change over time in culture. Thus, we report methods based on our experience for monitoring primary cell culture of human myometrial cells derived from uterine leiomyoma. We standardized the best procedure of tissue dissociation required for the study of multiple genetic marker systems that include species-specific antigens, expression of myofibroblast or myoblast markers, growth curve, serum deprivation, starvation by cell cycle synchronization, culture on collagen coated plates, and 17 β-estradiol (E2) and progesterone (P4) effects. The results showed that primary myometrial cells from patients with uterine leiomyoma displayed myoblast phenotypes before and after in vitro cultivation, and leiomyoma cells differentiated into mature myocyte cells under the appropriate differentiation-inducing conditions (serum deprivation). These cells grew well on collagen coated plates and responded to E2 and P4, which may drive myometrial and leiomyoma cells to proliferate and adhere into a focal adhesion complex involvement in a paracrine manner. The establishment of these techniques as routine procedures will improve the understanding of the myometrial physiology and pathogenesis of myometrium-derived diseases such as leiomyoma. Mimicking the in vivo environment of fibrotic conditions can prevent false results and enhance results that are based on cell culture integrity. PMID:27391384

Putative porcine embryonic stem cell lines derived from aggregated four-celled cloned embryos produced by oocyte bisection cloning.

PubMed

Siriboon, Chawalit; Lin, Yu-Hsuan; Kere, Michel; Chen, Chun-Da; Chen, Lih-Ren; Chen, Chien-Hong; Tu, Ching-Fu; Lo, Neng-Wen; Ju, Jyh-Cherng

2015-01-01

We attempted to isolate ES cell lines using inner cell masses from high-quality cloned porcine blastocysts. After being seeded onto feeders, embryos had better (P < 0.05) attachment, outgrowth formation and primary colonization in both 2× and 3× aggregated cloned embryos (62.8, 42.6 and 12.8% vs. 76.2, 55.2 and 26.2%, respectively) compared to the non-aggregated group (41.6, 23.4 and 3.9%). Effects of feeder types (STO vs. MEF) and serum sources (FBS vs. KSR) on extraction of cloned embryo-derived porcine ES cells were examined. More (17.1%) ntES cell lines over Passage 3 were generated in the MEF/KSR group. However, ntES cells cultured in KSR-supplemented medium had a low proliferation rate with defective morphology, and eventually underwent differentiation or apoptosis subsequently. Approximately 26.1, 22.7 and 35.7% of primary colonies were formed after plating embryos in DMEM, DMEM/F12 and α-MEM media, respectively. Survival rates of ntES cells cultured in α-MEM, DMEM and DMEM/F12 were 16.7, 4.3 and 6.8%, respectively (P > 0.05). We further examined the beneficial effect of TSA treatment of 3× aggregated cloned embryos on establishment of ntES cell lines. Primary colony numbers and survival rates of ntES cells beyond passage 3 were higher (P < 0.05) in those derived from TSA-treated 3× blastocysts (36.7 and 26.7%) than from the non-treated aggregated group (23.1 and 11.5%). These cells, remaining undifferentiated over 25 passages, had alkaline phosphatase activity and expressed ES specific markers Oct4, Nanog, Sox2, and Rex01. Moreover, these ntES cells successfully differentiated into embryoid bodies (EBs) that expressed specific genes of all three germ layers after being cultured in LIF-free medium. In conclusion, we have successfully derived putative porcine ntES cells with high efficiency from quality cloned embryos produced by embryo aggregation, and optimized the ES cell culture system suitable for establishing and maintaining ntES cell lines in undifferentiated state.

Putative Porcine Embryonic Stem Cell Lines Derived from Aggregated Four-Celled Cloned Embryos Produced by Oocyte Bisection Cloning

PubMed Central

Siriboon, Chawalit; Lin, Yu-Hsuan; Kere, Michel; Chen, Chun-Da; Chen, Lih-Ren; Chen, Chien-Hong; Tu, Ching-Fu; Lo, Neng-Wen; Ju, Jyh-Cherng

2015-01-01

We attempted to isolate ES cell lines using inner cell masses from high-quality cloned porcine blastocysts. After being seeded onto feeders, embryos had better (P < 0.05) attachment, outgrowth formation and primary colonization in both 2× and 3× aggregated cloned embryos (62.8, 42.6 and12.8% vs. 76.2, 55.2 and 26.2%, respectively) compared to the non-aggregated group (41.6, 23.4 and 3.9%). Effects of feeder types (STO vs. MEF) and serum sources (FBS vs. KSR) on extraction of cloned embryo-derived porcine ES cells were examined. More (17.1%) ntES cell lines over Passage 3 were generated in the MEF/KSR group. However, ntES cells cultured in KSR-supplemented medium had a low proliferation rate with defective morphology, and eventually underwent differentiation or apoptosis subsequently. Approximately 26.1, 22.7 and 35.7% of primary colonies were formed after plating embryos in DMEM, DMEM/F12 and α-MEM media, respectively. Survival rates of ntES cells cultured in α-MEM, DMEM and DMEM/F12 were 16.7, 4.3 and 6.8%, respectively (P > 0.05). We further examined the beneficial effect of TSA treatment of 3× aggregated cloned embryos on establishment of ntES cell lines. Primary colony numbers and survival rates of ntES cells beyond passage 3 were higher (P < 0.05) in those derived from TSA-treated 3× blastocysts (36.7 and 26.7%) than from the non-treated aggregated group (23.1 and 11.5%). These cells, remaining undifferentiated over 25 passages, had alkaline phosphatase activity and expressed ES specific markers Oct4, Nanog, Sox2, and Rex01. Moreover, these ntES cells successfully differentiated into embryoid bodies (EBs) that expressed specific genes of all three germ layers after being cultured in LIF-free medium. In conclusion, we have successfully derived putative porcine ntES cells with high efficiency from quality cloned embryos produced by embryo aggregation, and optimized the ES cell culture system suitable for establishing and maintaining ntES cell lines in undifferentiated state. PMID:25680105

Immortalized Human Schwann Cell Lines Derived From Tumors of Schwannomatosis Patients.

PubMed

Ostrow, Kimberly Laskie; Donaldson, Katelyn; Blakeley, Jaishri; Belzberg, Allan; Hoke, Ahmet

2015-01-01

Schwannomatosis, a rare form of neurofibromatosis, is characterized predominantly by multiple, often painful, schwannomas throughout the peripheral nervous system. The current standard of care for schwannomatosis is surgical resection. A major obstacle to schwannomatosis research is the lack of robust tumor cell lines. There is a great need for mechanistic and drug discovery studies of schwannomatosis, yet appropriate tools are not currently available. Schwannomatosis tumors are difficult to grow in culture as they survive only a few passages before senescence. Our lab has extensive experience in establishing primary and immortalized human Schwann cell cultures from normal tissue that retain their phenotypes after immortalization. Therefore we took on the challenge of creating immortalized human Schwann cell lines derived from tumors from schwannomatosis patients. We have established and fully characterized 2 schwannomatosis cell lines from 2 separate patients using SV40 virus large T antigen. One patient reported pain and the other did not. The schwannomatosis cell lines were stained with S100B antibodies to confirm Schwann cell identity. The schwannomatosis cells also expressed the Schwann cell markers, p75NTR, S100B, and NGF after multiple passages. Cell morphology was retained following multiple passaging and freeze/ thaw cycles. Gene expression microarray analysis was used to compare the cell lines with their respective parent tumors. No differences in key genes were detected, with the exception that several cell cycle regulators were upregulated in the schwannomatosis cell lines when compared to their parent tumors. This upregulation was apparently a product of cell culturing, as the schwannomatosis cells exhibited the same expression pattern of cell cycle regulatory genes as normal primary human Schwann cells. Cell growth was also similar between normal primary and immortalized tumor cells in culture. Accurate cell lines derived directly from human tumors will serve as invaluable tools for advancing schwannomatosis research, including drug screening.

Immortalized Human Schwann Cell Lines Derived From Tumors of Schwannomatosis Patients

PubMed Central

Ostrow, Kimberly Laskie; Donaldson, Katelyn; Blakeley, Jaishri; Belzberg, Allan; Hoke, Ahmet

2015-01-01

Schwannomatosis, a rare form of neurofibromatosis, is characterized predominantly by multiple, often painful, schwannomas throughout the peripheral nervous system. The current standard of care for schwannomatosis is surgical resection. A major obstacle to schwannomatosis research is the lack of robust tumor cell lines. There is a great need for mechanistic and drug discovery studies of schwannomatosis, yet appropriate tools are not currently available. Schwannomatosis tumors are difficult to grow in culture as they survive only a few passages before senescence. Our lab has extensive experience in establishing primary and immortalized human Schwann cell cultures from normal tissue that retain their phenotypes after immortalization. Therefore we took on the challenge of creating immortalized human Schwann cell lines derived from tumors from schwannomatosis patients. We have established and fully characterized 2 schwannomatosis cell lines from 2 separate patients using SV40 virus large T antigen. One patient reported pain and the other did not. The schwannomatosis cell lines were stained with S100B antibodies to confirm Schwann cell identity. The schwannomatosis cells also expressed the Schwann cell markers, p75NTR, S100B, and NGF after multiple passages. Cell morphology was retained following multiple passaging and freeze/ thaw cycles. Gene expression microarray analysis was used to compare the cell lines with their respective parent tumors. No differences in key genes were detected, with the exception that several cell cycle regulators were upregulated in the schwannomatosis cell lines when compared to their parent tumors. This upregulation was apparently a product of cell culturing, as the schwannomatosis cells exhibited the same expression pattern of cell cycle regulatory genes as normal primary human Schwann cells. Cell growth was also similar between normal primary and immortalized tumor cells in culture. Accurate cell lines derived directly from human tumors will serve as invaluable tools for advancing schwannomatosis research, including drug screening. PMID:26657314

A comparison of the functionality and in vivo phenotypic stability of cartilaginous tissues engineered from different stem cell sources.

PubMed

Vinardell, Tatiana; Sheehy, Eamon J; Buckley, Conor T; Kelly, Daniel J

2012-06-01

Joint-derived stem cells are a promising alternative cell source for cartilage repair therapies that may overcome many of the problems associated with the use of primary chondrocytes (CCs). The objective of this study was to compare the in vitro functionality and in vivo phenotypic stability of cartilaginous tissues engineered using bone marrow-derived stem cells (BMSCs) and joint tissue-derived stem cells following encapsulation in agarose hydrogels. Culture-expanded BMSCs, fat pad-derived stem cells (FPSCs), and synovial membrane-derived stem cells (SDSCs) were encapsulated in agarose and maintained in a chondrogenic medium supplemented with transforming growth factor-β3. After 21 days of culture, constructs were either implanted subcutaneously into the back of nude mice for an additional 28 days or maintained for a similar period in vitro in either chondrogenic or hypertrophic media formulations. After 49 days of in vitro culture in chondrogenic media, SDSC constructs accumulated the highest levels of sulfated glycosaminoglycan (sGAG) (∼2.8% w/w) and collagen (∼1.8% w/w) and were mechanically stiffer than constructs engineered using other cell types. After subcutaneous implantation in nude mice, sGAG content significantly decreased for all stem cell-seeded constructs, while no significant change was observed in the control constructs engineered using primary CCs, indicating that the in vitro chondrocyte-like phenotype generated in all stem cell-seeded agarose constructs was transient. FPSCs and SDSCs appeared to undergo fibrous dedifferentiation or resorption, as evident from increased collagen type I staining and a dramatic loss in sGAG content. BMSCs followed a more endochondral pathway with increased type X collagen expression and mineralization of the engineered tissue. In conclusion, while joint tissue-derived stem cells possess a strong intrinsic chondrogenic capacity, further studies are needed to identify the factors that will lead to the generation of a more stable chondrogenic phenotype.

Insights in spatio-temporal characterization of human fetal neural stem cells.

PubMed

Martín-Ibáñez, Raquel; Guardia, Inés; Pardo, Mónica; Herranz, Cristina; Zietlow, Rike; Vinh, Ngoc-Nga; Rosser, Anne; Canals, Josep M

2017-05-01

Primary human fetal cells have been used in clinical trials of cell replacement therapy for the treatment of neurodegenerative disorders such as Huntington's disease (HD). However, human fetal primary cells are scarce and difficult to work with and so a renewable source of cells is sought. Human fetal neural stem cells (hfNSCs) can be generated from human fetal tissue, but little is known about the differences between hfNSCs obtained from different developmental stages and brain areas. In the present work we characterized hfNSCs, grown as neurospheres, obtained from three developmental stages: 4-5, 6-7 and 8-9weeks post conception (wpc) and four brain areas: forebrain, cortex, whole ganglionic eminence (WGE) and cerebellum. We observed that, as fetal brain development proceeds, the number of neural precursors is diminished and post-mitotic cells are increased. In turn, primary cells obtained from older embryos are more sensitive to the dissociation process, their viability is diminished and they present lower proliferation ratios compared to younger embryos. However, independently of the developmental stage of derivation proliferation ratios were very low in all cases. Improvements in the expansion rates were achieved by mechanical, instead of enzymatic, dissociation of neurospheres but not by changes in the seeding densities. Regardless of the developmental stage, neurosphere cultures presented large variability in the viability and proliferation rates during the initial 3-4 passages, but stabilized achieving significant expansion rates at passage 5 to 6. This was true also for all brain regions except cerebellar derived cultures that did not expand. Interestingly, the brain region of hfNSC derivation influences the expansion potential, being forebrain, cortex and WGE derived cells the most expandable compared to cerebellar. Short term expansion partially compromised the regional identity of cortical but not WGE cultures. Nevertheless, both expanded cultures were multipotent and kept the ability to differentiate to region specific mature neuronal phenotypes. Copyright © 2017 Elsevier Inc. All rights reserved.

Establishment of two new scirrhous gastric cancer cell lines: analysis of factors associated with disseminated metastasis.

PubMed Central

Yashiro, M.; Chung, Y. S.; Nishimura, S.; Inoue, T.; Sowa, M.

1995-01-01

Determination of the differences between cell lines which are derived from a primary tumour and a disseminated metastatic lesion from the same patient may aid in elucidating the factors associated with disseminated metastases. We report on the establishment and characterisation of two new scirrhous gastric cancer cell lines, designated OCUM-2M and OCUM-2D, derived from a 49-year-old female. OCUM-2M was derived from a primary gastric tumour, and OCUM-2D was derived from a sample of disseminated metastasis. The two cell lines were derived from the same patient. We investigated biological differences between the two cell lines to study mechanisms involved in disseminated metastasis. The growth activity of OCUM-2D cells as determined by doubling time and tumorigenicity was greater than that of OCUM-2M cells. The level of epidermal growth factor receptor (EGFR) expression in OCUM-2D cells was about twice that of OCUM-2M cells and the growth of OCUM-2D cells was stimulated more by epidermal growth factor (EGF) than that of OCUM-2M cells. The invasive activity of OCUM-2D cells was higher than that of OCUM-2M cells and was increased after addition of transforming growth factor-beta 1 (TGF-beta 1). An increase in the number of attached and spreading cells was found following the addition of 10 ng ml-1 TGF-beta 1. These findings suggest that high growth and invasive activity may play an important role in disseminated metastasis and that EGF and TGF-beta 1, which affect the growth and invasive activity of OCUM-2D cells, might be factors associated with metastasis in scirrhous gastric carcinoma. The two cell lines OCUM-2M and OCUM-2D should be beneficial for analysing mechanisms of tumour progression. Images Figure 1 Figure 5 Figure 6 Figure 7 Figure 10 PMID:7577468

Melanoma cell-derived exosomes promote epithelial-mesenchymal transition in primary melanocytes through paracrine/autocrine signaling in the tumor microenvironment

PubMed Central

Xiao, Deyi; Barry, Samantha; Kmetz, Daniel; Egger, Michael; Pan, Jianmin; Rai, Shesh N; Qu, Jifu; McMasters, Kelly M.; Hao, Hongying

2016-01-01

The tumor microenvironment is abundant with exosomes that are secreted by the cancer cells themselves. Exosomes are nanosized, organelle-like membranous structures that are increasingly being recognized as major contributors in the progression of malignant neoplasms. A critical element in melanoma progression is its propensity to metastasize, but little is known about how melanoma cell-derived exosomes modulate the microenvironment to optimize conditions for tumor progression and metastasis. Here, we provide evidence that melanoma cell-derived exosomes promote phenotype switching in primary melanocytes through paracrine/autocrine signaling. We found that the mitogen-activated protein kinase (MAPK) signaling pathway was activated during the exosome-mediated epithelial-to-mesenchymal transition (EMT)-resembling process, which promotes metastasis. Let-7i, an miRNA modulator of EMT, was also involved in this process. We further defined two other miRNA modulators of EMT (miR-191 and let-7a) in serum exosomes for differentiating stage I melanoma patients from non-melanoma subjects. These results provide the first strong molecular evidence that melanoma cell-derived exosomes promote the EMT-resembling process in the tumor microenvironment. Thus, novel strategies targeting EMT and modulating the tumor microenvironment may emerge as important approaches for the treatment of metastatic melanoma. PMID:27063098

Mechanisms of the epithelial-to-mesenchymal transition in sea urchin embryos

PubMed Central

Katow, Hideki

2015-01-01

Sea urchin mesenchyme is composed of the large micromere-derived spiculogenetic primary mesenchyme cells (PMC), veg2-tier macromere-derived non-spiculogenetic mesenchyme cells, the small micromere-derived germ cells, and the macro- and mesomere-derived neuronal mesenchyme cells. They are formed through the epithelial-to-mesenchymal transition (EMT) and possess multipotency, except PMCs that solely differentiate larval spicules. The process of EMT is associated with modification of epithelial cell surface property that includes loss of affinity to the apical and basal extracellular matrices, inter-epithelial cell adherens junctions and epithelial cell surface-specific proteins. These cell surface structures and molecules are endocytosed during EMT and utilized as initiators of cytoplasmic signaling pathways that often initiate protein phosphorylation to activate the gene regulatory networks. Acquisition of cell motility after EMT in these mesenchyme cells is associated with the expression of proteins such as Lefty, Snail and Seawi. Structural simplicity and genomic database of this model will further promote detailed EMT research. PMID:26716069

Periodontal-ligament-derived stem cells exhibit the capacity for long-term survival, self-renewal, and regeneration of multiple tissue types in vivo.

PubMed

Menicanin, Danijela; Mrozik, Krzysztof Marek; Wada, Naohisa; Marino, Victor; Shi, Songtao; Bartold, P Mark; Gronthos, Stan

2014-05-01

Primary periodontal ligament stem cells (PDLSCs) are known to possess multidifferentiation potential and exhibit an immunophenotype similar to that described for bone-marrow-derived mesenchymal stem cells. In the present study, bromo-deoxyuridine (BrdU)-labeled ovine PDLSCs implanted into immunodeficient mice survived after 8 weeks post-transplantation and exhibited the capacity to form bone/cementum-like mineralized tissue, ligament structures similar to Sharpey's fibers with an associated vasculature. To evaluate self-renewal potential, PDLSCs were recovered from harvested primary transplants 8 weeks post-transplantation that exhibit an immunophenotype and multipotential capacity comparable to primary PDLSCs. The re-derived PDLSCs isolated from primary transplants were implanted into secondary ectopic xenogeneic transplants. Histomorphological analysis demonstrated that four out of six donor re-derived PDLSC populations displayed a capacity to survive and form fibrous ligament structures and mineralized tissues associated with vasculature in vivo, although at diminished levels in comparison to primary PDLSCs. Further, the capacity for long-term survival and the potential role of PDLSCs in dental tissue regeneration were determined using an ovine preclinical periodontal defect model. Autologous ex vivo-expanded PDLSCs that were prelabeled with BrdU were seeded onto Gelfoam(®) scaffolds and then transplanted into fenestration defects surgically created in the periodontium of the second premolars. Histological assessment at 8 weeks post-implantation revealed surviving BrdU-positive PDLSCs associated with regenerated periodontium-related tissues, including cementum and bone-like structures. This is the first report to demonstrate the self-renewal capacity of PDLSCs using serial xenogeneic transplants and provides evidence of the long-term survival and tissue contribution of autologous PDLSCs in a preclinical periodontal defect model.

Periodontal-Ligament-Derived Stem Cells Exhibit the Capacity for Long-Term Survival, Self-Renewal, and Regeneration of Multiple Tissue Types in Vivo

PubMed Central

Menicanin, Danijela; Mrozik, Krzysztof Marek; Wada, Naohisa; Marino, Victor; Shi, Songtao; Bartold, P. Mark

2014-01-01

Primary periodontal ligament stem cells (PDLSCs) are known to possess multidifferentiation potential and exhibit an immunophenotype similar to that described for bone-marrow-derived mesenchymal stem cells. In the present study, bromo-deoxyuridine (BrdU)–labeled ovine PDLSCs implanted into immunodeficient mice survived after 8 weeks post-transplantation and exhibited the capacity to form bone/cementum-like mineralized tissue, ligament structures similar to Sharpey's fibers with an associated vasculature. To evaluate self-renewal potential, PDLSCs were recovered from harvested primary transplants 8 weeks post-transplantation that exhibit an immunophenotype and multipotential capacity comparable to primary PDLSCs. The re-derived PDLSCs isolated from primary transplants were implanted into secondary ectopic xenogeneic transplants. Histomorphological analysis demonstrated that four out of six donor re-derived PDLSC populations displayed a capacity to survive and form fibrous ligament structures and mineralized tissues associated with vasculature in vivo, although at diminished levels in comparison to primary PDLSCs. Further, the capacity for long-term survival and the potential role of PDLSCs in dental tissue regeneration were determined using an ovine preclinical periodontal defect model. Autologous ex vivo–expanded PDLSCs that were prelabeled with BrdU were seeded onto Gelfoam® scaffolds and then transplanted into fenestration defects surgically created in the periodontium of the second premolars. Histological assessment at 8 weeks post-implantation revealed surviving BrdU-positive PDLSCs associated with regenerated periodontium-related tissues, including cementum and bone-like structures. This is the first report to demonstrate the self-renewal capacity of PDLSCs using serial xenogeneic transplants and provides evidence of the long-term survival and tissue contribution of autologous PDLSCs in a preclinical periodontal defect model. PMID:24351050

Change in the activity of Cl-,HCO3(-)-ATPase in microsome fraction during early development of the sea urchin, Hemicentrotus pulcherrimus.

PubMed

Mitsunaga, K; Fujino, Y; Yasumasu, I

1986-12-01

In sea urchin embryos, primary mesenchyme cells, descendants from micromeres produced at the 16-cell stage, form spicules or CaCO3 deposits in their skeletal vacuoles, at the post-gastrula stage. Micromeres isolated at the 16-cell stage also differentiate into spicule-forming cells during their culture at the same time schedule as in the embryos. The present study was planned to observe change in the activity of Cl-,HCO3(-)-ATPase, which was expected to contribute to the carbonate supply for CaCO3 deposition, during development. ATP-hydrolysis in the microsome fraction, obtained from embryos of the sea urchin, Hemicentrotus pulcherrimus, and from micromere-derived cells in culture was stimulated by Cl- and HCO3- in the presence of ouabain and EGTA. The ATP-hydrolysis was inhibited by ethacrynic acid, an inhibitor of Cl-,HCO3(-)-ATPase. The activity of Cl-,HCO3(-)-ATPase in embryos and in micromere-derived cells increased during development, keeping pace with the rate of calcium deposition in spicules. Formation of calcified spicules in the cultured micromere-derived cells was inhibited by ethacrynic acid. These results indicate that Cl-,HCO3(-)-ATPase plays an important role in the mechanism of CaCO3 deposition in the primary mesenchyme cells.

Tissue Engineering Platforms to Replicate the Tumor Microenvironment of Multiple Myeloma.

PubMed

Zhang, Wenting; Lee, Woo Y; Zilberberg, Jenny

2017-01-01

We described here the manufacturing and implementation of two prototype perfusion culture devices designed primarily for the cultivation of difficult-to-preserve primary patient-derived multiple myeloma cells (MMC). The first device consists of an osteoblast (OSB)-derived 3D tissue scaffold constructed in a perfused microfluidic environment. The second platform is a 96-well plate-modified perfusion culture device that can be utilized to reconstruct several tissue and tumor microenvironments utilizing both primary human and murine cells. This culture device was designed and fabricated specifically to: (1) enable the preservation of primary MMC for downstream use in biological studies and chemosensitivity analyses and, (2) provide a high-throughput format that is compatible with plate readers specifically seeing that this system is built on an industry standard 96-well tissue culture plate.

Differentiation and Transplantation of Human Embryonic Stem Cell-Derived Hepatocytes

PubMed Central

Basma, Hesham; Soto-Gutiérrez, Alejandro; Yannam, Govardhana Rao; Liu, Liping; Ito, Ryotaro; Yamamoto, Toshiyuki; Ellis, Ewa; Carson, Steven D.; Sato, Shintaro; Chen, Yong; Muirhead, David; Navarro-Álvarez, Nalu; Wong, Ron; Roy-Chowdhury, Jayanta; Platt, Jeffrey L.; Mercer, David F.; Miller, John D.; Strom, Stephen C.; Kobayashi, Noaya; Fox, Ira J.

2009-01-01

Background & Aims The ability to obtain unlimited numbers of human hepatocytes would improve development of cell-based therapies for liver diseases, facilitate the study of liver biology and improve the early stages of drug discovery. Embryonic stem cells are pluripotent, can potentially differentiate into any cell type and could therefore be developed as a source of human hepatocytes. Methods To generate human hepatocytes, human embryonic stem cells were differentiated by sequential culture in fibroblast growth factor 2 and human Activin-A, hepatocyte growth factor, and dexamethasone. Functional hepatocytes were isolated by sorting for surface asialoglycoprotein receptor expression. Characterization was performed by real-time PCR, imunohistochemistry, immunoblot, functional assays and transplantation. Results Embryonic stem cell-derived hepatocytes expressed liver-specific genes but not genes representing other lineages, secreted functional human liver-specific proteins similar to those of primary human hepatocytes and demonstrated human hepatocyte cytochrome P450 metabolic activity. Serum from rodents given injections of embryonic stem cell-derived hepatocytes contained significant amounts of human albumin and alpha-1-antitrypsin. Colonies of cytokeratin-18 and human albumin-expressing cells were present in the livers of recipient animals. Conclusion Human embryonic stem cells can be differentiated into cells with many characteristics of primary human hepatocytes. Hepatocyte-like cells can be enriched and recovered based on asialoglycoprotein receptor expression and could potentially be used in drug discovery research and developed as therapeutics. PMID:19026649

Pan-Genotype Hepatitis E Virus Replication in Stem Cell-Derived Hepatocellular Systems.

PubMed

Wu, Xianfang; Dao Thi, Viet Loan; Liu, Peng; Takacs, Constantin N; Xiang, Kuanhui; Andrus, Linda; Gouttenoire, Jérôme; Moradpour, Darius; Rice, Charles M

2018-02-01

The 4 genotypes of hepatitis E virus (HEV) that infect humans (genotypes 1-4) vary in geographical distribution, transmission, and pathogenesis. Little is known about the properties of HEV or its hosts that contribute to these variations. Primary isolates grow poorly in cell culture; most studies have relied on variants adapted to cancer cell lines, which likely alter virus biology. We investigated the infection and replication of primary isolates of HEV in hepatocyte-like cells (HLCs) derived from human embryonic and induced pluripotent stem cells. Using a cell culture-adapted genotype 3 strain and primary isolates of genotypes 1 to 4, we compared viral replication kinetics, sensitivity to drugs, and ability of HEV to activate the innate immune response. We studied HLCs using quantitative reverse-transcriptase polymerase chain reaction and immunofluorescence assay and enzyme-linked immunosorbent assays. We used an embryonic stem cell line that can be induced to express the CRISPR-Cas9 machinery to disrupt the peptidylprolyl isomerase A gene, encoding cyclophilin A (CYPA), a protein reported to inhibit replication of cell culture-adapted HEV. We further modified this line to rescue expression of CYPA before terminal differentiation to HLCs and performed HEV infection studies. HLCs were permissive for infection by nonadapted, primary isolates of HEV genotypes 1 to 4. HEV infection of HLCs induced a replication-dependent type III interferon response. Replication of primary HEV isolates, unlike the cell culture-adapted strain, was not affected by disruption of the peptidylprolyl isomerase A gene or exposure to the CYPA inhibitor cyclosporine A. Cell culture adaptations alter the replicative capacities of HEV. HLCs offer an improved, physiologically relevant, and genetically tractable system for studying the replication of primary HEV isolates. HLCs could provide a model to aid development of HEV drugs and a system to guide personalized regimens, especially for patients with chronic hepatitis E who have developed resistance to ribavirin. Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.

Cell-to-Cell Contact and Nectin-4 Govern Spread of Measles Virus from Primary Human Myeloid Cells to Primary Human Airway Epithelial Cells.

PubMed

Singh, Brajesh K; Li, Ni; Mark, Anna C; Mateo, Mathieu; Cattaneo, Roberto; Sinn, Patrick L

2016-08-01

Measles is a highly contagious, acute viral illness. Immune cells within the airways are likely first targets of infection, and these cells traffic measles virus (MeV) to lymph nodes for amplification and subsequent systemic dissemination. Infected immune cells are thought to return MeV to the airways; however, the mechanisms responsible for virus transfer to pulmonary epithelial cells are poorly understood. To investigate this process, we collected blood from human donors and generated primary myeloid cells, specifically, monocyte-derived macrophages (MDMs) and dendritic cells (DCs). MDMs and DCs were infected with MeV and then applied to primary cultures of well-differentiated airway epithelial cells from human donors (HAE). Consistent with previous results obtained with free virus, infected MDMs or DCs were incapable of transferring MeV to HAE when applied to the apical surface. Likewise, infected MDMs or DCs applied to the basolateral surface of HAE grown on small-pore (0.4-μm) support membranes did not transfer virus. In contrast, infected MDMs and DCs applied to the basolateral surface of HAE grown on large-pore (3.0-μm) membranes successfully transferred MeV. Confocal microscopy demonstrated that MDMs and DCs are capable of penetrating large-pore membranes but not small-pore membranes. Further, by using a nectin-4 blocking antibody or recombinant MeV unable to enter cells through nectin-4, we demonstrated formally that transfer from immune cells to HAE occurs in a nectin-4-dependent manner. Thus, both infected MDMs and DCs rely on cell-to-cell contacts and nectin-4 to efficiently deliver MeV to the basolateral surface of HAE. Measles virus spreads rapidly and efficiently in human airway epithelial cells. This rapid spread is based on cell-to-cell contact rather than on particle release and reentry. Here we posit that MeV transfer from infected immune cells to epithelial cells also occurs by cell-to-cell contact rather than through cell-free particles. In addition, we sought to determine which immune cells transfer MeV infectivity to the human airway epithelium. Our studies are based on two types of human primary cells: (i) myeloid cells generated from donated blood and (ii) well-differentiated airway epithelial cells derived from donor lungs. We show that different types of myeloid cells, i.e., monocyte-derived macrophages and dendritic cells, transfer infection to airway epithelial cells. Furthermore, cell-to-cell contact is an important component of successful MeV transfer. Our studies elucidate a mechanism by which the most contagious human respiratory virus is delivered to the airway epithelium. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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

PubMed

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

2012-12-01

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

Directing adult human periodontal ligament-derived stem cells to retinal fate.

PubMed

Huang, Li; Liang, Jiajian; Geng, Yiqun; Tsang, Wai-Ming; Yao, Xiaowu; Jhanji, Vishal; Zhang, Mingzhi; Cheung, Herman S; Pang, Chi Pui; Yam, Gary Hin-Fai

2013-06-06

To investigate the retinal fate competence of human postnatal periodontal ligament (PDL)-derived stem cells (PDLSC) through a directed differentiation mimicking mammalian retinogenesis. Human teeth were collected from healthy subjects younger than 35 years old. Primary PDLSC were isolated by collagenase digestion and cultivated. PDLSC at passage 3 were cultured in the induction media containing Noggin (antagonist of bone morphogenic protein) and Dkk-1 (antagonist of Wnt/β-catenin signaling). Gene expression of neural crest cells, retinal progenitors, and retinal neurons, including photoreceptors, was revealed by RNA analyses, immunofluorescence, and flow cytometry. The neuronal-like property of differentiated cells in response to excitatory glutamate was examined by fluo-4-acetoxymethyl calcium imaging assay. Primary human PDLSC stably expressed marker genes for neural crest (Notch1, BMP2, Slug, Snail, nestin, and Tuj1), mesenchymal stem cell (CD44, CD90, and vimentin), and embryonic stem cell (c-Myc, Klf4, Nanog, and SSEA4). Under low attachment culture, PDLSC generated neurospheres expressing nestin, p75/NGFR, Pax6, and Tuj1 (markers of neural progenitors). When neurospheres were plated on Matrigel-coated surface, they exhibited rosette-like outgrowth. They expressed eye field transcription factors (Pax6, Rx, Lhx, Otx2). By flow cytometry, 94% of cells were Pax6(nuclear)Rx(+), indicative of retinal progenitors. At prolonged induction, they expressed photoreceptor markers (Nrl, rhodopsin and its kinase) and showed significant responsiveness to excitatory glutamate. Primary human PDLSC could be directed to retinal progenitors with competence for photoreceptor differentiation. Human neural crest-derived PDL is readily accessible and can be an ample autologous source of undifferentiated cells for retinal cell regeneration.

Differentiation "in vitro" of primary and immortalized porcine mesenchymal stem cells into cardiomyocytes for cell transplantation.

PubMed

Moscoso, I; Centeno, A; López, E; Rodriguez-Barbosa, J I; Santamarina, I; Filgueira, P; Sánchez, M J; Domínguez-Perles, R; Peñuelas-Rivas, G; Domenech, N

2005-01-01

Cell transplantation to regenerate injured tissues is a promising new treatment for patients suffering several diseases. Bone marrow contains a population of progenitor cells known as mesenchymal stem cells (MSCs), which have the capability to colonize different tissues, replicate, and differentiate into multilineage cells. Our goal was the isolation, characterization, and immortalization of porcine MSCs (pMSCs) to study their potential differentiation "in vitro" into cardiomyocytes. pMSCs were obtained from the aspirated bone marrow of Large-White pigs. After 4 weeks in culture, adherent cells were phenotypically characterized by flow cytometry and immunochemistry by using monoclonal antibodies. Primary pMSCs were transfected with the plasmid pRNS-1 to obtain continuous growing cloned cell lines. Fresh pMSCs and immortalized cells were treated with 5-azacytidine to differentiate them into cardiomyocytes. Flow cytometry analysis of isolated pMSCs demonstrated the following phenotype, CD90(pos), CD29(pos), CD44(pos), SLA-I(pos), CD106(pos), CD46(pos) and CD45(neg), CD14(neg), CD31(neg), and CD11b(neg), similar to that described for human MSC. We derived several stable immortalized MSC cell lines. One of these, called pBMC-2, was chosen for further characterization. After "in vitro" stimulation of both primary or immortalized cells with 5-azacytidine, we obtained different percentages (30%-50%) of cells with cardiomyocyte characteristics, namely, positive for alpha-Actin and T-Troponin. Thus, primary or immortalized pMSCs derived from bone marrow and cultured were able to differentiate "ex vivo" into cardiac-like muscle cells. These elements may be potentials tools to improve cardiac function in a swine myocardial infarct model.

Differential Activation of CD8+ Tumor-Specific Tc1 and Tc2 Cells by an IL-10-Producing Murine Plasmacytoma

PubMed Central

Pauels, Hans-Gerd; Becker, Christian; Kölsch, Eckehart

1998-01-01

The involvement of counteractive CD8+ T-cell subsets during tumor-specific immune responses was analyzed in a syngeneic murine plasmacytoma model. CD8+ Tc cells against the immunogenic IL-10-producing BALB/c plasmacytoma ADJ-PC-5 can be easily induced by immunization of BALB/c mice with X-irradiated ADJ-PC-5 tumor cells in vivo and in vitro. However, the failure of recipient mice to mount a protective Tc response against the tumor during early stages of a real or simulated tumor growth is not due to immunological ignorance, but depends on the induction of tumor-specific tolerance, involving a population of tumorinduced CD8+ T cells that are able to inhibit the generation of tumor-specific Tc cells in a primary ADJ-PC-5-specific MLTC, using IFN-γ as a suppressive factor. Whereas most longterm cultivated CD8+ ADJ-PC-5-specific Tc lines produce type-1 cytokines on stimulation, at least two of them, which were derived from a primary MLTC, display a type-2 cytokine spectrum. Furthermore, the primary in vitro Tc response against ADJ-PC-5 cells shows characteristics of a Tc2 response. The Tc response is strictly depending on tumor-derived IL-10. CD8+ Tc cells that are induced in a primary MLTC do not produce IFN-γ, and the tumor-specific Tc response is enhanced by IL-4 but suppressed by IFN-γ or IL-12. In contrast, ADJ-PC- 5-specific CD8+ Tc cells from immunized mice are IFN-γ producing Tc1 cells. Since the primary in vitro Tc response against the tumor is suppressed even by the smallest numbers of irradiated ADJ-PC-5-specific Tc1 cells via IFN-γ these Tc1 cells behave similar to the suppressive CD8+ T cells that are induced during early stages of ADJ-PC-5 tumorigenesis. PMID:9814607

Neurite outgrowth in human iPSC-derived neurons

EPA Pesticide Factsheets

Data on morphology of rat and human neurons in cell cultureThis dataset is associated with the following publication:Druwe, I., T. Freudenrich , K. Wallace , T. Shafer , and W. Mundy. Comparison of Human Induced PluripotentStem Cell-Derived Neurons and Rat Primary CorticalNeurons as In Vitro Models of Neurite Outgrowth. Applied In vitro Toxicology. Mary Ann Liebert, Inc., Larchmont, NY, USA, 2(1): 26-36, (2016).

Involvement of multiple myeloma cell-derived exosomes in osteoclast differentiation

PubMed Central

Raimondi, Lavinia; De Luca, Angela; Amodio, Nicola; Manno, Mauro; Raccosta, Samuele; Taverna, Simona; Bellavia, Daniele; Naselli, Flores; Fontana, Simona; Schillaci, Odessa; Giardino, Roberto; Fini, Milena; Tassone, Pierfrancesco; Santoro, Alessandra; De Leo, Giacomo; Giavaresi, Gianluca; Alessandro, Riccardo

2015-01-01

Bone disease is the most frequent complication in multiple myeloma (MM) resulting in osteolytic lesions, bone pain, hypercalcemia and renal failure. In MM bone disease the perfect balance between bone-resorbing osteoclasts (OCs) and bone-forming osteoblasts (OBs) activity is lost in favour of OCs, thus resulting in skeletal disorders. Since exosomes have been described for their functional role in cancer progression, we here investigate whether MM cell-derived exosomes may be involved in OCs differentiation. We show that MM cells produce exosomes which are actively internalized by Raw264.7 cell line, a cellular model of osteoclast formation. MM cell-derived exosomes positively modulate pre-osteoclast migration, through the increasing of CXCR4 expression and trigger a survival pathway. MM cell-derived exosomes play a significant pro-differentiative role in murine Raw264.7 cells and human primary osteoclasts, inducing the expression of osteoclast markers such as Cathepsin K (CTSK), Matrix Metalloproteinases 9 (MMP9) and Tartrate-resistant Acid Phosphatase (TRAP). Pre-osteoclast treated with MM cell-derived exosomes differentiate in multinuclear OCs able to excavate authentic resorption lacunae. Similar results were obtained with exosomes derived from MM patient's sera. Our data indicate that MM-exosomes modulate OCs function and differentiation. Further studies are needed to identify the OCs activating factors transported by MM cell-derived exosomes. PMID:25944696

Liver cell-derived microparticles activate hedgehog signaling and alter gene expression in hepatic endothelial cells.

PubMed

Witek, Rafal P; Yang, Liu; Liu, Renshui; Jung, Youngmi; Omenetti, Alessia; Syn, Wing-Kin; Choi, Steve S; Cheong, Yeiwon; Fearing, Caitlin M; Agboola, Kolade M; Chen, Wei; Diehl, Anna Mae

2009-01-01

Angiogenesis contributes to vascular remodeling during cirrhosis. In cirrhotic livers, cholangiocytes, and myofibroblastic hepatic stellate cells (MF-HSC) produce Hedgehog (Hh) ligands. During embryogenesis Hh ligands are released from ligand-producing cells in microparticles and activate Hh signaling in endothelial cells. We studied whether adult liver cell-derived microparticles contain Hh ligands that alter hepatic sinusoidal endothelial cells (SEC). MF-HSC and cholangiocytes were exposed to platelet-derived growth factor to induce Hh ligands; microparticles were isolated from medium, analyzed by transmission electron microscopy and immunoblots, and applied to Hh-reporter-containing cells. Microparticles were obtained from serum and bile of rats after bile duct ligation (BDL) or sham surgery and applied to normal primary liver SEC with or without cyclopamine, an Hh signaling inhibitor. Effects on SEC gene expression were evaluated by quantitative reverse-transcription polymerase chain reaction and immunoblotting. Hh target gene expression and SEC activation markers were compared in primary SEC and in liver sections from healthy and BDL rats. Platelet-derived growth factor-treated MF-HSC and cholangiocytes released exosome-enriched microparticles containing biologically-active Hh ligands. BDL increased release of Hh-containing exosome-enriched microparticles into plasma and bile. Transmission electron microscopy and immunoblots revealed similarities among microparticles from all sources; all microparticles induced similar Hh-dependent changes in SEC gene expression. SEC from healthy livers did not express Hh target genes or activation markers, but both were up-regulated in SEC after BDL. Hh-containing exosome-enriched microparticles released from liver cells alter hepatic SEC gene expression, suggesting a novel mechanism for cirrhotic vasculopathy.

Critical role for ERK1/2 in bone marrow and fetal liver–derived primary megakaryocyte differentiation, motility, and proplatelet formation

PubMed Central

Mazharian, Alexandra; Watson, Steve P.; Séverin, Sonia

2009-01-01

Objective Megakaryopoiesis and platelet formation is a multistep process through which hematopoietic progenitor cells develop into mature megakaryocytes (MKs) and form proplatelets. The present study investigates the regulation of different steps of megakaryopoiesis (i.e., differentiation, migration, and proplatelet formation) by extracellar signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) in two models of primary murine MKs derived from bone marrow (BM) cells and fetal liver (FL) cells. Materials and Methods A preparation of MKs was generated from BM obtained from femora and tibiae of C57BL6 mice. FL-derived MKs were obtained from the liver of mouse fetuses aged 13 to 15 days. Results For both cell populations, activation of MEK-ERK1/2 pathway by thrombopoietin was found to have a critical role in MK differentiation, regulating polyploidy and surface expression of CD34, GPIIb, and GPIb. The MEK-ERK1/2 pathway plays a major role in migration of BM-derived MKs toward a stromal-cell−derived factor 1α (SDF1α) gradient, whereas unexpectedly, FL-derived cells fail to migrate in response to the chemokine due to negligible expression of its receptor, CXCR4. The MEK-ERK1/2 pathway also plays a critical role in the generation of proplatelets. In contrast, p38MAPK pathway was not involved in any of these processes. Conclusion This report demonstrates a critical role of MEK-ERK1/2 pathway in MK differentiation, motility, and proplatelet formation. This study highlights several differences between BM- and FL-derived MKs, which are discussed. PMID:19619605

High CD49f expression is associated with osteosarcoma tumor progression: a study using patient-derived primary cell cultures.

PubMed

Penfornis, Patrice; Cai, David Z; Harris, Michael R; Walker, Ryan; Licini, David; Fernandes, Joseph D A; Orr, Griffin; Koganti, Tejaswi; Hicks, Chindo; Induru, Spandana; Meyer, Mark S; Khokha, Rama; Barr, Jennifer; Pochampally, Radhika R

2014-08-01

Overall prognosis for osteosarcoma (OS) is poor despite aggressive treatment options. Limited access to primary tumors, technical challenges in processing OS tissues, and the lack of well-characterized primary cell cultures has hindered our ability to fully understand the properties of OS tumor initiation and progression. In this study, we have isolated and characterized cell cultures derived from four central high-grade human OS samples. Furthermore, we used the cell cultures to study the role of CD49f in OS progression. Recent studies have implicated CD49f in stemness and multipotency of both cancer stem cells and mesenchymal stem cells. Therefore, we investigated the role of CD49f in osteosarcomagenesis. First, single cell suspensions of tumor biopsies were subcultured and characterized for cell surface marker expression. Next, we characterized the growth and differentiation properties, sensitivity to chemotherapy drugs, and anchorage-independent growth. Xenograft assays showed that cell populations expressing CD49f(hi) /CD90(lo) cell phenotype produced an aggressive tumor. Multiple lines of evidence demonstrated that inhibiting CD49f decreased the tumor-forming ability. Furthermore, the CD49f(hi) /CD90(lo) cell population is generating more aggressive OS tumor growth and indicating this cell surface marker could be a potential candidate for the isolation of an aggressive cell type in OSs. © 2014 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

EphA2 is a biomarker of hMSCs derived from human placenta and umbilical cord.

PubMed

Shen, Shih-Pei; Liu, Wei-Ting; Lin, Yun; Li, Yuan-Tsung; Chang, Chih-Hao; Chang, Fung-Wei; Wang, Le-Ming; Teng, Sen-Wen; Hsuan, Yogi

2015-12-01

The heterogeneous nature of mesenchymal stem cells (MSCs) and the absence of known MSC-specific biomarkers make it challenging to define MSC phenotypes and characteristics. In this study, we compared the phenotypic and functional features of human placenta-derived MSCs with those of human dermal fibroblasts in vitro in order to identify a biomarker that can be used to increase the purity of MSCs in a primary culture of placenta-derived cells. Liquid chromatography-tandem mass spectrometry analysis was used to analyze and compare the proteome of human placenta-derived MSCs with that of fibroblasts. Quantitative real-time polymerase chain reaction, immunofluorescence, and flow cytometry were used to determine expression levels of EphA2 in placenta-derived MSCs. EphA2-positive cells were enriched by magnetic-activated cell sorting or with a cell sorter. An shRNA-mediated EphA2 knockdown was used to assess the role of EphA2 in MSC response to Tumor necrosis factor (TNF)-α stimulation. Analysis of proteomics data from MSCs and fibroblasts resulted in the identification of the EphA2 surface protein biomarker, which could reliably distinguish MSCs from fibroblasts. EphA2 was significantly upregulated in placenta-derived MSCs when compared to fibroblasts. EphA2 played an important role in MSC migration in response to inflammatory stimuli, such as TNF-α. EphA2-enriched MSCs were also more responsive to inflammatory stimuli in vitro when compared to unsorted MSCs, indicating a role for EphA2 in the immunomodulatory functionality of MSCs. EphA2 can be used to distinguish and isolate MSCs from a primary culture of placenta-derived cells. EphA2-sorted MSCs exhibited superior responsiveness to TNF-α signaling in an inflammatory environment compared with unsorted MSCs or MSC-like cells. Copyright © 2015. Published by Elsevier B.V.

HtrA3 Is Downregulated in Cancer Cell Lines and Significantly Reduced in Primary Serous and Granulosa Cell Ovarian Tumors.

PubMed

Singh, Harmeet; Li, Ying; Fuller, Peter J; Harrison, Craig; Rao, Jyothsna; Stephens, Andrew N; Nie, Guiying

2013-01-01

Objective. The high temperature requirement factor A3 (HtrA3) is a serine protease homologous to bacterial HtrA. Four human HtrAs have been identified. HtrA1 and HtrA3 share a high degree of domain organization and are downregulated in a number of cancers, suggesting a widespread loss of these proteases in cancer. This study examined how extensively the HtrA (HtrA1-3) proteins are downregulated in commonly used cancer cell lines and primary ovarian tumors.Methods. RT-PCR was applied to various cancer cell lines (n=17) derived from the ovary, endometrium, testes, breast, prostate, and colon, and different subtypes of primary ovarian tumors [granulosa cell tumors (n=19), mucinous cystadenocarcinomas (n=6), serous cystadenocarcinomas (n=8)] and normal ovary (n = 9). HtrA3 protein was localized by immunohistochemistry.Results. HtrA3 was extensively downregulated in the cancer cell lines examined including the granulosa cell tumor-derived cell lines. In primary ovarian tumors, the HtrA3 was significantly lower in serous cystadenocarcinoma and granulosa cell tumors. In contrast, HtrA1 and HtrA2 were expressed in all samples with no significant differences between the control and tumors. In normal postmenopausal ovary, HtrA3 protein was localized to lutenizing stromal cells and corpus albicans. In serous cystadenocarcinoma, HtrA3 protein was absent in the papillae but detected in the mesenchymal cyst wall.Conclusion. HtrA3 is more extensively downregulated than HtrA1-2 in cancer cell lines. HtrA3, but not HtrA1 or HtrA2, was decreased in primary ovarian serous cystadenocarcinoma and granulosa cell tumors. This study provides evidence that HtrA3 may be the most relevant HtrA associated with ovarian malignancy.

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.

Generation and customization of biosynthetic excitable tissues for electrophysiological studies and cell-based therapies.

PubMed

Nguyen, Hung X; Kirkton, Robert D; Bursac, Nenad

2018-05-01

We describe a two-stage protocol to generate electrically excitable and actively conducting cell networks with stable and customizable electrophysiological phenotypes. Using this method, we have engineered monoclonally derived excitable tissues as a robust and reproducible platform to investigate how specific ion channels and mutations affect action potential (AP) shape and conduction. In the first stage of the protocol, we combine computational modeling, site-directed mutagenesis, and electrophysiological techniques to derive optimal sets of mammalian and/or prokaryotic ion channels that produce specific AP shape and conduction characteristics. In the second stage of the protocol, selected ion channels are stably expressed in unexcitable human cells by means of viral or nonviral delivery, followed by flow cytometry or antibiotic selection to purify the desired phenotype. This protocol can be used with traditional heterologous expression systems or primary excitable cells, and application of this method to primary fibroblasts may enable an alternative approach to cardiac cell therapy. Compared with existing methods, this protocol generates a well-defined, relatively homogeneous electrophysiological phenotype of excitable cells that facilitates experimental and computational studies of AP conduction and can decrease arrhythmogenic risk upon cell transplantation. Although basic cell culture and molecular biology techniques are sufficient to generate excitable tissues using the described protocol, experience with patch-clamp techniques is required to characterize and optimize derived cell populations.

Metastases to bones.

PubMed

Wick, Mark R

2014-01-01

Metastatic tumors involving the bones may derive from a number of visceral primary sites, and they can assume several histological appearances. In selected instances, diagnostic confusion with some primary bone tumors may eventuate, necessitating the use of adjunctive pathologic studies to reach a final interpretation. This review considers metastatic osseous neoplasms in the small-cell, large-polygonal-cell, and spindle-cell-pleomorphic microscopic categories. The use of immunohistology and molecular analysis to study such tumors is discussed. Copyright © 2014 Elsevier Inc. All rights reserved.

cGAS Senses Human Cytomegalovirus and Induces Type I Interferon Responses in Human Monocyte-Derived Cells

PubMed Central

Paijo, Jennifer; Döring, Marius; Spanier, Julia; Grabski, Elena; Nooruzzaman, Mohammed; Schmidt, Tobias; Witte, Gregor; Messerle, Martin; Hornung, Veit; Kaever, Volkhard; Kalinke, Ulrich

2016-01-01

Human cytomegalovirus (HCMV) infections of healthy individuals are mostly unnoticed and result in viral latency. However, HCMV can also cause devastating disease, e.g., upon reactivation in immunocompromised patients. Yet, little is known about human immune cell sensing of DNA-encoded HCMV. Recent studies indicated that during viral infection the cyclic GMP/AMP synthase (cGAS) senses cytosolic DNA and catalyzes formation of the cyclic di-nucleotide cGAMP, which triggers stimulator of interferon genes (STING) and thus induces antiviral type I interferon (IFN-I) responses. We found that plasmacytoid dendritic cells (pDC) as well as monocyte-derived DC and macrophages constitutively expressed cGAS and STING. HCMV infection further induced cGAS, whereas STING expression was only moderately affected. Although pDC expressed particularly high levels of cGAS, and the cGAS/STING axis was functional down-stream of STING, as indicated by IFN-I induction upon synthetic cGAMP treatment, pDC were not susceptible to HCMV infection and mounted IFN-I responses in a TLR9-dependent manner. Conversely, HCMV infected monocyte-derived cells synthesized abundant cGAMP levels that preceded IFN-I production and that correlated with the extent of infection. CRISPR/Cas9- or siRNA-mediated cGAS ablation in monocytic THP-1 cells and primary monocyte-derived cells, respectively, impeded induction of IFN-I responses following HCMV infection. Thus, cGAS is a key sensor of HCMV for IFN-I induction in primary human monocyte-derived DC and macrophages. PMID:27058035

GM-CSF production by glioblastoma cells has a functional role in eosinophil survival, activation and growth factor production for enhanced tumor cell proliferation

PubMed Central

Curran, Colleen S.; Evans, Michael D.; Bertics, Paul J.

2011-01-01

Medicinal interventions of limited efficacy are currently available for the treatment of glioblastoma multiforme (GBM), the most common and lethal primary brain tumor in adults. The eosinophil is a pivotal immune cell in the pathobiology of atopic disease that is also found to accumulate in certain tumor tissues. Inverse associations between atopy and GBM risk suggest that the eosinophil may play a functional role in certain tumor immune responses. To assess the potential interactions between eosinophils and GBM, human primary blood eosinophils were cultured with two separate human GBM-derived cell lines (A172, U87-MG) or conditioned media generated in the presence or absence of TNF-α. Results revealed differential eosinophil adhesion and increased survival in response to co-culture with GBM cell lines. Eosinophil responses to GBM cell line-conditioned media included increased survival, activation, CD11b expression and S100A9 release. Addition of GM-CSF neutralizing antibodies to GBM cell cultures or conditioned media reduced eosinophil adhesion, survival and activation, linking tumor cell-derived GM-CSF to the functions of eosinophils in the tumor microenvironment. Dexamethasone, which has been reported to inhibit eosinophil recruitment and shrink GBM lesions on contrast enhanced scans, reduced the production of tumor cell-derived GM-CSF. Furthermore, culture of GBM cells in eosinophil-conditioned media increased tumor cell viability, and generation of eosinophil-conditioned media in the presence of GM-CSF enhanced the effect. These data support the idea of a paracrine loop between GM-CSF producing tumors and eosinophil-derived growth factors in tumor promotion/progression. PMID:21705618

Magentic Cell labeling of primary and stem cell-derived pig hepatocytes for MRI-based cell tracking of heptocytes transplantation

USDA-ARS?s Scientific Manuscript database

Pig hepatocytes are an important investigational tool for optimizing hepatocyte transplantation schemes in both allogeneic and xenogeneic transplant scenarios. MRI can be used to serially monitor the transplanted cells, but only if the hepatocytes can be labeled with a magnetic particle. In this wo...

An open-label dose escalation study to evaluate the safety of administration of nonviral stromal cell-derived factor-1 plasmid to treat symptomatic ischemic heart failure.

PubMed

Penn, Marc S; Mendelsohn, Farrell O; Schaer, Gary L; Sherman, Warren; Farr, Maryjane; Pastore, Joseph; Rouy, Didier; Clemens, Ruth; Aras, Rahul; Losordo, Douglas W

2013-03-01

Preclinical studies indicate that adult stem cells induce tissue repair by activating endogenous stem cells through the stromal cell-derived factor-1:chemokine receptor type 4 axis. JVS-100 is a DNA plasmid encoding human stromal cell-derived factor-1. We tested in a phase 1, open-label, dose-escalation study with 12 months of follow-up in subjects with ischemic cardiomyopathy to see if JVS-100 improves clinical parameters. Seventeen subjects with ischemic cardiomyopathy, New York Heart Association class III heart failure, with an ejection fraction ≤40% on stable medical therapy, were enrolled to receive 5, 15, or 30 mg of JVS-100 via endomyocardial injection. The primary end points for safety and efficacy were at 1 and 4 months, respectively. The primary safety end point was a major adverse cardiac event. Efficacy end points were change in quality of life, New York Heart Association class, 6-minute walk distance, single photon emission computed tomography, N-terminal pro-brain natruretic peptide, and echocardiography at 4 and 12 months. The primary safety end point was met. At 4 months, all of the cohorts demonstrated improvements in 6-minute walk distance, quality of life, and New York Heart Association class. Subjects in the 15- and 30-mg dose groups exhibited improvements in 6-minute walk distance (15 mg: median [range]: 41 minutes [3-61 minutes]; 30 mg: 31 minutes [22-74 minutes]) and quality of life (15 mg: -16 points [+1 to -32 points]; 30 mg: -24 points [+17 to -38 points]) over baseline. At 12 months, improvements in symptoms were maintained. These data highlight the importance of defining the molecular mechanisms of stem cell-based tissue repair and suggest that overexpression of stromal cell-derived factor-1 via gene therapy is a strategy for improving heart failure symptoms in patients with ischemic cardiomyopathy.

Induced pluripotent stem cell models of Zellweger spectrum disorder show impaired peroxisome assembly and cell type-specific lipid abnormalities.

PubMed

Wang, Xiao-Ming; Yik, Wing Yan; Zhang, Peilin; Lu, Wange; Huang, Ning; Kim, Bo Ram; Shibata, Darryl; Zitting, Madison; Chow, Robert H; Moser, Ann B; Steinberg, Steven J; Hacia, Joseph G

2015-08-29

Zellweger spectrum disorder (PBD-ZSD) is a disease continuum caused by mutations in a subset of PEX genes required for normal peroxisome assembly and function. They highlight the importance of peroxisomes in the development and functions of the central nervous system, liver, and other organs. To date, the underlying bases for the cell-type specificity of disease are not fully elucidated. Primary skin fibroblasts from seven PBD-ZSD patients with biallelic PEX1, PEX10, PEX12, or PEX26 mutations and three healthy donors were transduced with retroviral vectors expressing Yamanaka reprogramming factors. Candidate induced pluripotent stem cells (iPSCs) were subject to global gene expression, DNA methylation, copy number variation, genotyping, in vitro differentiation and teratoma formation assays. Confirmed iPSCs were differentiated into neural progenitor cells (NPCs), neurons, oligodendrocyte precursor cells (OPCs), and hepatocyte-like cell cultures with peroxisome assembly evaluated by microscopy. Saturated very long chain fatty acid (sVLCFA) and plasmalogen levels were determined in primary fibroblasts and their derivatives. iPSCs were derived from seven PBD-ZSD patient-derived fibroblasts with mild to severe peroxisome assembly defects. Although patient and control skin fibroblasts had similar gene expression profiles, genes related to mitochondrial functions and organelle cross-talk were differentially expressed among corresponding iPSCs. Mitochondrial DNA levels were consistent among patient and control fibroblasts, but varied among all iPSCs. Relative to matching controls, sVLCFA levels were elevated in patient-derived fibroblasts, reduced in patient-derived iPSCs, and not significantly different in patient-derived NPCs. All cell types derived from donors with biallelic null mutations in a PEX gene showed plasmalogen deficiencies. Reporter gene assays compatible with high content screening (HCS) indicated patient-derived OPC and hepatocyte-like cell cultures had impaired peroxisome assembly. Normal peroxisome activity levels are not required for cellular reprogramming of skin fibroblasts. Patient iPSC gene expression profiles were consistent with hypotheses highlighting the role of altered mitochondrial activities and organelle cross-talk in PBD-ZSD pathogenesis. sVLCFA abnormalities dramatically differed among patient cell types, similar to observations made in iPSC models of X-linked adrenoleukodystrophy. We propose that iPSCs could assist investigations into the cell type-specificity of peroxisomal activities, toxicology studies, and in HCS for targeted therapies for peroxisome-related disorders.

Bone marrow-derived stromal cells are more beneficial cell sources for tooth regeneration compared with adipose-derived stromal cells.

PubMed

Ye, Lanfeng; Chen, Lin; Feng, Fan; Cui, Junhui; Li, Kaide; Li, Zhiyong; Liu, Lei

2015-10-01

Tooth loss is presently a global epidemic and tooth regeneration is thought to be a feasible and ideal treatment approach. Choice of cell source is a primary concern in tooth regeneration. In this study, the odontogenic differentiation potential of two non-dental-derived stem cells, adipose-derived stromal cells (ADSCs) and bone marrow-derived stromal cells (BMSCs), were evaluated both in vitro and in vivo. ADSCs and BMSCs were induced in vitro in the presence of tooth germ cell-conditioned medium (TGC-CM) prior to implantation into the omentum majus of rats, in combination with inactivated dentin matrix (IDM). Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the mRNA expression levels of odontogenic-related genes. Immunofluorescence and immunohistochemical assays were used to detect the protein levels of odontogenic-specific genes, such as DSP and DMP-1 both in vitro and in vivo. The results suggest that both ADSCs and BMSCs have odontogenic differentiation potential. However, the odontogenic potential of BMSCs was greater compared with ADSCs, showing that BMSCs are a more appropriate cell source for tooth regeneration. © 2015 International Federation for Cell Biology.

Morphology, cell viability, karyotype, expression of surface markers and plasticity of three human primary cell line cultures before and after the cryostorage in LN2 and GN2.

PubMed

Del Pino, Alberto; Ligero, Gertrudis; López, María B; Navarro, Héctor; Carrillo, Jose A; Pantoll, Siobhan C; Díaz de la Guardia, Rafael

2015-02-01

Primary cell line cultures from human skin biopsies, adipose tissue and tumor tissue are valuable samples for research and therapy. In this regard, their derivation, culture, storage, transport and thawing are important steps to be studied. Towards this end, we wanted to establish the derivation, and identify the culture characteristics and the loss of viability of three human primary cell line cultures (human adult dermal fibroblasts (hADFs), human adult mesenchymal stem cells (hMSCs), and primary culture of tumor cells from lung adenocarcinoma (PCTCLA)). Compared to fresh hADFs, hMSCs and PCTCLA, thawed cells stored in a cryogenic Dewar tanks with liquid nitrogen (LN2), displayed 98.20% ± 0.99, 95.40% ± 1.41 and 93.31% ± 3.83 of cell viability, respectively. Thawed cells stored in a Dry Vapor Shipper container with gas phase (GN2), for 20 days, in addition displayed 4.61% ± 2.78, 3.70% ± 4.09 and 9.13% ± 3.51 of average loss of cells viability, respectively, showing strong correlation between the loss of viability in hADFs and the number of post-freezing days in the Dry Vapor Shipper. No significant changes in morphological characteristics or in the expression of surface markers (being hADFs, hMSCs and PCTCLA characterized by positive markers CD73+; CD90+; CD105+; and negative markers CD14-; CD20-; CD34-; and CD45-; n=2) were found. Chromosome abnormalities in the karyotype were not found. In addition, under the right conditions hMSCs were differentiated into adipogenic, osteogenic and chondrogenic lineages in vitro. In this paper, we have shown the characteristics of three human primary cell line cultures when they are stored in LN2 and GN2. Copyright © 2014 Elsevier Inc. All rights reserved.

Culture Medium Supplements Derived from Human Platelet and Plasma: Cell Commitment and Proliferation Support

PubMed Central

Muraglia, Anita; Nguyen, Van Thi; Nardini, Marta; Mogni, Massimo; Coviello, Domenico; Dozin, Beatrice; Strada, Paolo; Baldelli, Ilaria; Formica, Matteo; Cancedda, Ranieri; Mastrogiacomo, Maddalena

2017-01-01

Present cell culture medium supplements, in most cases based on animal sera, are not fully satisfactory especially for the in vitro expansion of cells intended for human cell therapy. This paper refers to (i) an heparin-free human platelet lysate (PL) devoid of serum or plasma components (v-PL) and (ii) an heparin-free human serum derived from plasma devoid of PL components (Pl-s) and to their use as single components or in combination in primary or cell line cultures. Human mesenchymal stem cells (MSC) primary cultures were obtained from adipose tissue, bone marrow, and umbilical cord. Human chondrocytes were obtained from articular cartilage biopsies. In general, MSC expanded in the presence of Pl-s alone showed a low or no proliferation in comparison to cells grown with the combination of Pl-s and v-PL. Confluent, growth-arrested cells, either human MSC or human articular chondrocytes, treated with v-PL resumed proliferation, whereas control cultures, not supplemented with v-PL, remained quiescent and did not proliferate. Interestingly, signal transduction pathways distinctive of proliferation were activated also in cells treated with v-PL in the absence of serum, when cell proliferation did not occur, indicating that v-PL could induce the cell re-entry in the cell cycle (cell commitment), but the presence of serum proteins was an absolute requirement for cell proliferation to happen. Indeed, Pl-s alone supported cell growth in constitutively activated cell lines (U-937, HeLa, HaCaT, and V-79) regardless of the co-presence of v-PL. Plasma- and plasma-derived serum were equally able to sustain cell proliferation although, for cells cultured in adhesion, the Pl-s was more efficient than the plasma from which it was derived. In conclusion, the cells expanded in the presence of the new additives maintained their differentiation potential and did not show alterations in their karyotype. PMID:29209609

Inhibition of Super-Enhancer Activity in Autoinflammatory Site-Derived T Cells Reduces Disease-Associated Gene Expression.

PubMed

Peeters, Janneke G C; Vervoort, Stephin J; Tan, Sander C; Mijnheer, Gerdien; de Roock, Sytze; Vastert, Sebastiaan J; Nieuwenhuis, Edward E S; van Wijk, Femke; Prakken, Berent J; Creyghton, Menno P; Coffer, Paul J; Mokry, Michal; van Loosdregt, Jorg

2015-09-29

The underlying molecular mechanisms for many autoimmune diseases are poorly understood. Juvenile idiopathic arthritis (JIA) is an exceptionally well-suited model for studying autoimmune diseases due to its early onset and the possibility to analyze cells derived from the site of inflammation. Epigenetic profiling, utilizing primary JIA patient-derived cells, can contribute to the understanding of autoimmune diseases. With H3K27ac chromatin immunoprecipitation, we identified a disease-specific, inflammation-associated, typical enhancer and super-enhancer signature in JIA patient synovial-fluid-derived CD4(+) memory/effector T cells. RNA sequencing of autoinflammatory site-derived patient T cells revealed that BET inhibition, utilizing JQ1, inhibited immune-related super-enhancers and preferentially reduced disease-associated gene expression, including cytokine-related processes. Altogether, these results demonstrate the potential use of enhancer profiling to identify disease mediators and provide evidence for BET inhibition as a possible therapeutic approach for the treatment of autoimmune diseases. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Evaluation and identification of hepatitis B virus entry inhibitors using HepG2 cells overexpressing a membrane transporter NTCP.

PubMed

Iwamoto, Masashi; Watashi, Koichi; Tsukuda, Senko; Aly, Hussein Hassan; Fukasawa, Masayoshi; Fujimoto, Akira; Suzuki, Ryosuke; Aizaki, Hideki; Ito, Takayoshi; Koiwai, Osamu; Kusuhara, Hiroyuki; Wakita, Takaji

2014-01-17

Hepatitis B virus (HBV) entry has been analyzed using infection-susceptible cells, including primary human hepatocytes, primary tupaia hepatocytes, and HepaRG cells. Recently, the sodium taurocholate cotransporting polypeptide (NTCP) membrane transporter was reported as an HBV entry receptor. In this study, we established a strain of HepG2 cells engineered to overexpress the human NTCP gene (HepG2-hNTCP-C4 cells). HepG2-hNTCP-C4 cells were shown to be susceptible to infection by blood-borne and cell culture-derived HBV. HBV infection was facilitated by pretreating cells with 3% dimethyl sulfoxide permitting nearly 50% of the cells to be infected with HBV. Knockdown analysis suggested that HBV infection of HepG2-hNTCP-C4 cells was mediated by NTCP. HBV infection was blocked by an anti-HBV surface protein neutralizing antibody, by compounds known to inhibit NTCP transporter activity, and by cyclosporin A and its derivatives. The infection assay suggested that cyclosporin B was a more potent inhibitor of HBV entry than was cyclosporin A. Further chemical screening identified oxysterols, oxidized derivatives of cholesterol, as inhibitors of HBV infection. Thus, the HepG2-hNTCP-C4 cell line established in this study is a useful tool for the identification of inhibitors of HBV infection as well as for the analysis of the molecular mechanisms of HBV infection. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Naïve CD8 T cell activation by liver bone marrow-derived cells leads to a "neglected" IL-2low Bimhigh phenotype, poor CTL function and cell death.

PubMed

Holz, Lauren E; Benseler, Volker; Vo, Michelle; McGuffog, Claire; Van Rooijen, Nico; McCaughan, Geoffrey W; Bowen, David G; Bertolino, Patrick

2012-10-01

The occurrence of primary CD8 T cell activation within the liver, unique among the non-lymphoid organs, is now well accepted. However, the outcome of intrahepatic T cell activation remains controversial. We have previously reported that activation initiated by hepatocytes results in a tolerogenic phenotype characterized by low expression of CD25 and IL-2, poor cytotoxic T lymphocyte (CTL) function, and excessive expression of the pro-apoptotic protein Bim. To investigate whether this phenotype was due to activation in the absence of co-stimulation, we generated bone marrow (bm) radiation chimeras in which adoptively transferred naïve transgenic CD8 T cells were activated in the presence of co-stimulation by liver bm-derived cells. Despite expressing pro-inflammatory cytokines, high levels of CD25 and CD54, donor T cells activated by liver bm-derived cells did not produce detectable IL-2 and displayed poor CTL function, suggesting incomplete acquisition of effector function. Simultaneously, these cells expressed high levels of Bim and died by neglect. Transfer of Bim-deficient T cells resulted in increased T cell numbers. These results imply that expression of CD25 and CD54 is co-stimulation dependent and distinguishes T cell activated by hepatocytes and liver bm-derived cells. In contrast, low expression of IL-2, poor CTL function and excess Bim production represent a more universal phenotype defining T cells undergoing primary activation by both types of hepatic antigen presenting cells (APC). These results have important implications for transplantation, in which all liver antigen presenting cells contribute to activation of T cells specific for the allograft. Copyright © 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Requirement of the Listeria monocytogenes broad-range phospholipase PC-PLC during infection of human epithelial cells.

PubMed

Gründling, Angelika; Gonzalez, Mark D; Higgins, Darren E

2003-11-01

In this study, we investigated the requirement of the Listeria monocytogenes broad-range phospholipase C (PC-PLC) during infection of human epithelial cells. L. monocytogenes is a facultative intracellular bacterial pathogen of humans and a variety of animal species. After entering a host cell, L. monocytogenes is initially surrounded by a membrane-bound vacuole. Bacteria promote their escape from this vacuole, grow within the host cell cytosol, and spread from cell to cell via actin-based motility. Most infection studies with L. monocytogenes have been performed with mouse cells or an in vivo mouse model of infection. In all mouse-derived cells tested, the pore-forming cytolysin listeriolysin O (LLO) is absolutely required for lysis of primary vacuoles formed during host cell entry. However, L. monocytogenes can escape from primary vacuoles in the absence of LLO during infection of human epithelial cell lines Henle 407, HEp-2, and HeLa. Previous studies have shown that the broad-range phospholipase C, PC-PLC, promotes lysis of Henle 407 cell primary vacuoles in the absence of LLO. Here, we have shown that PC-PLC is also required for lysis of HEp-2 and HeLa cell primary vacuoles in the absence of LLO expression. Furthermore, our results indicated that the amount of PC-PLC activity is critical for the efficiency of vacuolar lysis. In an LLO-negative derivative of L. monocytogenes strain 10403S, expression of PC-PLC has to increase before or upon entry into human epithelial cells, compared to expression in broth culture, to allow bacterial escape from primary vacuoles. Using a system for inducible PC-PLC expression in L. monocytogenes, we provide evidence that phospholipase activity can be increased by elevated expression of PC-PLC or Mpl, the enzyme required for proteolytic activation of PC-PLC. Lastly, by using the inducible PC-PLC expression system, we demonstrate that, in the absence of LLO, PC-PLC activity is not only required for lysis of primary vacuoles in human epithelial cells but is also necessary for efficient cell-to-cell spread. We speculate that the additional requirement for PC-PLC activity is for lysis of secondary double-membrane vacuoles formed during cell-to-cell spread.

Quantitative assessment of neurite outgrowth in human embryonic stem cell derived hN2 cells using automated high-content image analysis

EPA Science Inventory

Throughout development neurons undergo a number of morphological changes including neurite outgrowth from the cell body. Exposure to neurotoxic chemicals that interfere with this process may result in permanent deficits in nervous system function. Traditionally, rodent primary ne...

Platelets secrete stromal cell-derived factor 1alpha and recruit bone marrow-derived progenitor cells to arterial thrombi in vivo.

PubMed

Massberg, Steffen; Konrad, Ildiko; Schürzinger, Katrin; Lorenz, Michael; Schneider, Simon; Zohlnhoefer, Dietlind; Hoppe, Katharina; Schiemann, Matthias; Kennerknecht, Elisabeth; Sauer, Susanne; Schulz, Christian; Kerstan, Sandra; Rudelius, Martina; Seidl, Stefan; Sorge, Falko; Langer, Harald; Peluso, Mario; Goyal, Pankaj; Vestweber, Dietmar; Emambokus, Nikla R; Busch, Dirk H; Frampton, Jon; Gawaz, Meinrad

2006-05-15

The accumulation of smooth muscle and endothelial cells is essential for remodeling and repair of injured blood vessel walls. Bone marrow-derived progenitor cells have been implicated in vascular repair and remodeling; however, the mechanisms underlying their recruitment to the site of injury remain elusive. Here, using real-time in vivo fluorescence microscopy, we show that platelets provide the critical signal that recruits CD34+ bone marrow cells and c-Kit+ Sca-1+ Lin- bone marrow-derived progenitor cells to sites of vascular injury. Correspondingly, specific inhibition of platelet adhesion virtually abrogated the accumulation of both CD34+ and c-Kit+ Sca-1+ Lin- bone marrow-derived progenitor cells at sites of endothelial disruption. Binding of bone marrow cells to platelets involves both P-selectin and GPIIb integrin on platelets. Unexpectedly, we found that activated platelets secrete the chemokine SDF-1alpha, thereby supporting further primary adhesion and migration of progenitor cells. These findings establish the platelet as a major player in the initiation of vascular remodeling, a process of fundamental importance for vascular repair and pathological remodeling after vascular injury.

Purification and differentiation of human adipose-derived stem cells by membrane filtration and membrane migration methods

PubMed Central

Lin, Hong Reng; Heish, Chao-Wen; Liu, Cheng-Hui; Muduli, Saradaprasan; Li, Hsing-Fen; Higuchi, Akon; Kumar, S. Suresh; Alarfaj, Abdullah A.; Munusamy, Murugan A.; Hsu, Shih-Tien; Chen, Da-Chung; Benelli, Giovanni; Murugan, Kadarkarai; Cheng, Nai-Chen; Wang, Han-Chow; Wu, Gwo-Jang

2017-01-01

Human adipose-derived stem cells (hADSCs) are easily isolated from fat tissue without ethical concerns, but differ in purity, pluripotency, differentiation ability, and stem cell marker expression, depending on the isolation method. We isolated hADSCs from a primary fat tissue solution using: (1) conventional culture, (2) a membrane filtration method, (3) a membrane migration method where the primary cell solution was permeated through membranes, adhered hADSCs were cultured, and hADSCs migrated out from the membranes. Expression of mesenchymal stem cell markers and pluripotency genes, and osteogenic differentiation were compared for hADSCs isolated by different methods using nylon mesh filter membranes with pore sizes ranging from 11 to 80 μm. hADSCs isolated by the membrane migration method had the highest MSC surface marker expression and efficient differentiation into osteoblasts. Osteogenic differentiation ability of hADSCs and MSC surface marker expression were correlated, but osteogenic differentiation ability and pluripotent gene expression were not. PMID:28071738

Conditionally reprogrammed primary airway epithelial cells maintain morphology, lineage and disease specific functional characteristics.

PubMed

Martinovich, Kelly M; Iosifidis, Thomas; Buckley, Alysia G; Looi, Kevin; Ling, Kak-Ming; Sutanto, Erika N; Kicic-Starcevich, Elizabeth; Garratt, Luke W; Shaw, Nicole C; Montgomery, Samuel; Lannigan, Francis J; Knight, Darryl A; Kicic, Anthony; Stick, Stephen M

2017-12-21

Current limitations to primary cell expansion led us to test whether airway epithelial cells derived from healthy children and those with asthma and cystic fibrosis (CF), co-cultured with an irradiated fibroblast feeder cell in F-medium containing 10 µM ROCK inhibitor could maintain their lineage during expansion and whether this is influenced by underlying disease status. Here, we show that conditionally reprogrammed airway epithelial cells (CRAECs) can be established from both healthy and diseased phenotypes. CRAECs can be expanded, cryopreserved and maintain phenotypes over at least 5 passages. Population doublings of CRAEC cultures were significantly greater than standard cultures, but maintained their lineage characteristics. CRAECs from all phenotypes were also capable of fully differentiating at air-liquid interface (ALI) and maintained disease specific characteristics including; defective CFTR channel function cultures and the inability to repair wounds. Our findings indicate that CRAECs derived from children maintain lineage, phenotypic and importantly disease-specific functional characteristics over a specified passage range.

In Vitro Derivation and Propagation of Spermatogonial Stem Cell Activity from Mouse Pluripotent Stem Cells.

PubMed

Ishikura, Yukiko; Yabuta, Yukihiro; Ohta, Hiroshi; Hayashi, Katsuhiko; Nakamura, Tomonori; Okamoto, Ikuhiro; Yamamoto, Takuya; Kurimoto, Kazuki; Shirane, Kenjiro; Sasaki, Hiroyuki; Saitou, Mitinori

2016-12-06

The in vitro derivation and propagation of spermatogonial stem cells (SSCs) from pluripotent stem cells (PSCs) is a key goal in reproductive science. We show here that when aggregated with embryonic testicular somatic cells (reconstituted testes), primordial germ cell-like cells (PGCLCs) induced from mouse embryonic stem cells differentiate into spermatogonia-like cells in vitro and are expandable as cells that resemble germline stem cells (GSCs), a primary cell line with SSC activity. Remarkably, GSC-like cells (GSCLCs), but not PGCLCs, colonize adult testes and, albeit less effectively than GSCs, contribute to spermatogenesis and fertile offspring. Whole-genome analyses reveal that GSCLCs exhibit aberrant methylation at vulnerable regulatory elements, including those critical for spermatogenesis, which may restrain their spermatogenic potential. Our study establishes a strategy for the in vitro derivation of SSC activity from PSCs, which, we propose, relies on faithful epigenomic regulation. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

An avian cell line designed for production of highly attenuated viruses.

PubMed

Jordan, Ingo; Vos, Ad; Beilfuss, Stefanie; Neubert, Andreas; Breul, Sabine; Sandig, Volker

2009-01-29

Several viral vaccines, including highly promising vectors such as modified vaccinia Ankara (MVA), are produced on chicken embryo fibroblasts. Dependence on primary cells complicates production especially in large vaccination programs. With primary cells it is also not possible to create packaging lines for replication-deficient vectors that are adapted to proliferation in an avian host. To obviate requirement for primary cells permanent lines from specific tissues of muscovy duck were derived (AGE1.CR, CS, and CA) and further modified: we demonstrate that stable expression of the structural gene pIX from human adenovirus increases titers for unrelated poxvirus in the avian cells. This augmentation appears to be mediated via induction of heat shock and thus provides a novel cellular substrate that may allow further attenuation of vaccine strains.

Establishment of primary mixed cell cultures from spontaneous canine mammary tumors: Characterization of classic and new cancer-associated molecules

PubMed Central

Gentile, Luciana B.; Nagamine, Marcia K.; Biondi, Luiz R.; Sanches, Daniel S.; Toyota, Fábio; Giovani, Tatiane M.; de Jesus, Isis P.; da Fonseca, Ivone I. M.; Queiroz-Hazarbassanov, Nicolle; Diaz, Bruno L.; Salles Gomes, Cristina de O. Massoco

2017-01-01

There are many factors which make canine cancer like cancer in humans. The occurrence of spontaneous mammary tumors in pet dogs, tumor genetics, molecular targets and exposure to the same environmental risk factors are among these factors. Therefore, the study of canine cancer can provide useful information to the oncology field. This study aimed to establish and characterize a panel of primary mixed cell cultures obtained from spontaneous canine mammary tumors. Eight established cell cultures obtained from one normal mammary gland, one complex adenoma, one mixed adenoma, two complex carcinomas and two mixed carcinomas were analyzed. The gene expression levels of classic molecular cancer players such as fibroblast growth factor receptor (FGFR) 2, breast cancer (BRCA) 1, BRCA2 and estrogen receptor (ESR) 1 were evaluated. For the first time, three orphan nuclear receptors, estrogen-related receptors (ERRs) α, β and γ were studied in canine mammary cancer. The highest expression level of ERRα was observed in complex carcinoma-derived cell culture, while the highest levels of ERRβ and γ were observed in cells derived from a mixed carcinoma. Meanwhile, complex carcinomas presented the highest levels of expression of ESR1, BRCA1 and FGFR2 among all samples. BRCA2 was found exclusively in complex adenoma. The transcription factor GATA3 had its highest levels in mixed carcinoma samples and its lowest levels in complex adenoma. Proliferation assays were also performed to evaluate the mixed cell cultures response to ER ligands, genistein and DES, both in normoxia and hypoxic conditions. Our results demonstrate that morphological and functional studies of primary mixed cell cultures derived from spontaneous canine mammary tumors are possible and provide valuable tool for the study of various stages of mammary cancer development. PMID:28945747

Profiling post-translational modifications of histones in human monocyte-derived macrophages.

PubMed

Olszowy, Pawel; Donnelly, Maire Rose; Lee, Chanho; Ciborowski, Pawel

2015-01-01

Histones and their post-translational modifications impact cellular function by acting as key regulators in the maintenance and remodeling of chromatin, thus affecting transcription regulation either positively (activation) or negatively (repression). In this study we describe a comprehensive, bottom-up proteomics approach to profiling post-translational modifications (acetylation, mono-, di- and tri-methylation, phosphorylation, biotinylation, ubiquitination, citrullination and ADP-ribosylation) in human macrophages, which are primary cells of the innate immune system. As our knowledge expands, it becomes more evident that macrophages are a heterogeneous population with potentially subtle differences in their responses to various stimuli driven by highly complex epigenetic regulatory mechanisms. To profile post-translational modifications (PTMs) of histones in macrophages we used two platforms of liquid chromatography and mass spectrometry. One platform was based on Sciex5600 TripleTof and the second one was based on VelosPro Orbitrap Elite ETD mass spectrometers. We provide side-by-side comparison of profiling using two mass spectrometric platforms, ion trap and qTOF, coupled with the application of collisional induced and electron transfer dissociation. We show for the first time methylation of a His residue in macrophages and demonstrate differences in histone PTMs between those currently reported for macrophage cell lines and what we identified in primary cells. We have found a relatively low level of histone PTMs in differentiated but resting human primary monocyte derived macrophages. This study is the first comprehensive profiling of histone PTMs in primary human MDM. Our study implies that epigenetic regulatory mechanisms operative in transformed cell lines and primary cells are overlapping to a limited extent. Our mass spectrometric approach provides groundwork for the investigation of how histone PTMs contribute to epigenetic regulation in primary human macrophages.

Dengue Virus Infection of Mast Cells Triggers Endothelial Cell Activation ▿

PubMed Central

Brown, Michael G.; Hermann, Laura L.; Issekutz, Andrew C.; Marshall, Jean S.; Rowter, Derek; Al-Afif, Ayham; Anderson, Robert

2011-01-01

Vascular perturbation is a hallmark of severe forms of dengue disease. We show here that antibody-enhanced dengue virus infection of primary human cord blood-derived mast cells (CBMCs) and the human mast cell-like line HMC-1 results in the release of factor(s) which activate human endothelial cells, as evidenced by increased expression of the adhesion molecules ICAM-1 and VCAM-1. Endothelial cell activation was prevented by pretreatment of mast cell-derived supernatants with a tumor necrosis factor (TNF)-specific blocking antibody, thus identifying TNF as the endothelial cell-activating factor. Our findings suggest that mast cells may represent an important source of TNF, promoting vascular endothelial perturbation following antibody-enhanced dengue virus infection. PMID:21068256

Mammary Cancer and Activation of Transposable Elements

DTIC Science & Technology

2015-03-01

regularly hold meetings. • Completed Y1 4-6 6. • Preliminary Methyl-MAPS analysis of pilot virgin samples • This material was never received. Based...construct the libraries for sequencing. A strategic decision was made to hold the material for validation, rather than attempt library construction. Y2 10...derived adipo- cytes and ADS-derived induced pluripotent stem cells (ADS-iPSCs) (19) and primary mouse ES cells to isolated sperm and oocytes (20). We

Primary intraosseous squamous cell carcinoma: a devil in disguise

PubMed Central

Pardhe, Nilesh; Bhagalia, Sanjay; Nayak, Prathibha Anand; Sireesha, Sundaragiri Krishna

2013-01-01

Primary intraosseous squamous cell carcinoma (PIOSCC) is a rare central jaw carcinoma derived from odontogenic epithelial remnants. When the tumour arises in an existing cyst, it may be difficult to recognise early PIOSCC and carry out necessary treatment. We report a case diagnosed in a 50-year-old man where timely intervention was delayed and prognosis was affected and the case diagnosed eventually by comprehensive clinical, radiological and histopathological examination. PMID:23749827

Platelets secrete stromal cell–derived factor 1α and recruit bone marrow–derived progenitor cells to arterial thrombi in vivo

PubMed Central

Massberg, Steffen; Konrad, Ildiko; Schürzinger, Katrin; Lorenz, Michael; Schneider, Simon; Zohlnhoefer, Dietlind; Hoppe, Katharina; Schiemann, Matthias; Kennerknecht, Elisabeth; Sauer, Susanne; Schulz, Christian; Kerstan, Sandra; Rudelius, Martina; Seidl, Stefan; Sorge, Falko; Langer, Harald; Peluso, Mario; Goyal, Pankaj; Vestweber, Dietmar; Emambokus, Nikla R.; Busch, Dirk H.; Frampton, Jon; Gawaz, Meinrad

2006-01-01

The accumulation of smooth muscle and endothelial cells is essential for remodeling and repair of injured blood vessel walls. Bone marrow–derived progenitor cells have been implicated in vascular repair and remodeling; however, the mechanisms underlying their recruitment to the site of injury remain elusive. Here, using real-time in vivo fluorescence microscopy, we show that platelets provide the critical signal that recruits CD34+ bone marrow cells and c-Kit+ Sca-1+ Lin− bone marrow–derived progenitor cells to sites of vascular injury. Correspondingly, specific inhibition of platelet adhesion virtually abrogated the accumulation of both CD34+ and c-Kit+ Sca-1+ Lin− bone marrow–derived progenitor cells at sites of endothelial disruption. Binding of bone marrow cells to platelets involves both P-selectin and GPIIb integrin on platelets. Unexpectedly, we found that activated platelets secrete the chemokine SDF-1α, thereby supporting further primary adhesion and migration of progenitor cells. These findings establish the platelet as a major player in the initiation of vascular remodeling, a process of fundamental importance for vascular repair and pathological remodeling after vascular injury. PMID:16618794

Lysophosphatidylcholine acyltransferase1 overexpression promotes oral squamous cell carcinoma progression via enhanced biosynthesis of platelet-activating factor.

PubMed

Shida-Sakazume, Tomomi; Endo-Sakamoto, Yosuke; Unozawa, Motoharu; Fukumoto, Chonji; Shimada, Ken; Kasamatsu, Atsushi; Ogawara, Katsunori; Yokoe, Hidetaka; Shiiba, Masashi; Tanzawa, Hideki; Uzawa, Katsuhiro

2015-01-01

The relevance of lysophosphatidylcholine acyltransferase1 (LPCAT1), a cytosolic enzyme in the remodeling pathway of phosphatidylcholine metabolism, in oral squamous cell carcinoma (OSCC) is unknown. We investigated LPCAT1 expression and its functional mechanism in OSCCs. We analyzed LPCAT1 mRNA and protein expression levels in OSCC-derived cell lines. Immunohistochemistry was performed to identify correlations between LPCAT1 expression levels and primary OSCCs clinicopathological status. We established LPCAT1 knockdown models of the OSCC-derived cell lines (SAS, Ca9-22) for functional analysis and examined the association between LPCAT1 expression and the platelet-activating factor (PAF) concentration and PAF-receptor (PAFR) expression. LPCAT1 mRNA and protein were up-regulated significantly (p<0.05) in OSCC-derived cell lines compared with human normal oral keratinocytes. Immunohistochemistry showed significantly (p<0.05) elevated LPCAT1 expression in primary OSCCs compared with normal counterparts and a strong correlation between LPCAT1-positive OSCCs and tumoral size and regional lymph node metastasis. In LPCAT1 knockdown cells, cellular proliferation and invasiveness decreased significantly (p<0.05); cellular migration was inhibited compared with control cells. Down-regulation of LPCAT1 resulted in a decreased intercellular PAF concentration and PAFR expression. LPCAT1 was overexpressed in OSCCs and correlated with cellular invasiveness and migration. LPCAT1 may contribute to tumoral growth and metastasis in oral cancer.

Selection of appropriate isolation method based on morphology of blastocyst for efficient derivation of buffalo embryonic stem cells.

PubMed

Kumar, R; Ahlawat, S P S; Sharma, M; Verma, O P; Sai Kumar, G; Taru Sharma, G

2014-03-01

The efficiency of embryonic stem cell (ESC) derivation from all species except for rodents and primates is very low. There are however, multiple interests in obtaining pluripotent cells from these animals with main expectations in the fields of transgenesis, cloning, regenerative medicine and tissue engineering. Researches are being carried out in laboratories throughout the world to increase the efficiency of ESC isolation for their downstream applications. Thus, the present study was undertaken to study the effect of different isolation methods based on the morphology of blastocyst for efficient derivation of buffalo ESCs. Embryos were produced in vitro through the procedures of maturation, fertilization and culture. Hatched blastocysts or isolated inner cell masses (ICMs) were seeded on mitomycin-C inactivated buffalo fetal fibroblast monolayer for the development of ESC colonies. The ESCs were analyzed for alkaline phosphatase activity, expression of pluripotency markers and karyotypic stability. Primary ESC colonies were obtained after 2-5 days of seeding hatched blastocysts or isolated ICMs on mitomycin-C inactivated feeder layer. Mechanically isolated ICMs attached and formed primary cell colonies more efficiently than ICMs isolated enzymatically. For derivation of ESCs from poorly defined ICMs intact hatched blastocyst culture was the most successful method. Results of this study implied that although ESCs can be obtained using all three methods used in this study, efficiency varies depending upon the morphology of blastocyst and isolation method used. So, appropriate isolation method must be selected depending on the quality of blastocyst for efficient derivation of ESCs.

Erythroid Kruppel-like factor (EKLF) is recruited to the γ-globin gene promoter as a co-activator and is required for γ-globin gene induction by short-chain fatty acid derivatives

PubMed Central

Perrine, Susan P.; Mankidy, Rishikesh; Boosalis, Michael S.; Bieker, James J.; Faller, Douglas V.

2011-01-01

Objectives The erythroid Kruppel-like factor (EKLF) is an essential transcription factor for β-type globin gene switching, and specifically activates transcription of the adult β-globin gene promoter. We sought to determine if EKLF is also required for activation of the γ-globin gene by short-chain fatty acid (SCFA) derivatives, which are now entering clinical trials. Methods The functional and physical interaction of EKLF and co-regulatory molecules with the endogenous human globin gene promoters was studied in primary human erythroid progenitors and cell lines, using chromatin immunoprecipitation (ChIP) assays and genetic manipulation of the levels of EKLF and co-regulators. Results and conclusions Knockdown of EKLF prevents SCFA-induced expression of the γ-globin promoter in a stably expressed μLCRβprRlucAγprFluc cassette, and prevents induction of the endogenous γ-globin gene in primary human erythroid progenitors. EKLF is actively recruited to endogenous γ-globin gene promoters after exposure of primary human erythroid progenitors, and murine hematopoietic cell lines, to SCFA derivatives. The core ATPase BRG1 subunit of the human SWI/WNF complex, a ubiquitous multimeric complex that regulates gene expression by remodeling nucleosomal structure, is also required for γ-globin gene induction by SCFA derivatives. BRG1 is actively recruited to the endogenous γ-globin promoter of primary human erythroid progenitors by exposure to SCFA derivatives, and this recruitment is dependent upon the presence of EKLF. These findings demonstrate that EKLF, and the co-activator BRG1, previously demonstrated to be required for definitive or adult erythropoietic patterns of globin gene expression, are co-opted by SCFA derivatives to activate the fetal globin genes. PMID:19220418

Primary Cilium-Regulated EG-VEGF Signaling Facilitates Trophoblast Invasion.

PubMed

Wang, Chia-Yih; Tsai, Hui-Ling; Syu, Jhih-Siang; Chen, Ting-Yu; Su, Mei-Tsz

2017-06-01

Trophoblast invasion is an important event in embryo implantation and placental development. During these processes, endocrine gland-derived vascular endothelial growth factor (EG-VEGF) is the key regulator mediating the crosstalk at the feto-maternal interface. The primary cilium is a cellular antenna receiving environmental signals and is crucial for proper development. However, little is known regarding the role of the primary cilium in early human pregnancy. Here, we demonstrate that EG-VEGF regulates trophoblast cell invasion via primary cilia. We found that EG-VEGF activated ERK1/2 signaling and subsequent upregulation of MMP2 and MMP9, thereby facilitating cell invasion in human trophoblast HTR-8/SVneo cells. Inhibition of ERK1/2 alleviated the expression of MMPs and trophoblast cell invasion after EG-VEGF treatment. In addition, primary cilia were observed in all the trophoblast cell lines tested and, more importantly, in human first-trimester placental tissue. The receptor of EG-VEGF, PROKR1, was detected in primary cilia. Depletion of IFT88, the intraflagellar transporter required for ciliogenesis, inhibited primary cilium growth, thereby ameliorating ERK1/2 activation, MMP upregulation, and trophoblast cell invasion promoted by EG-VEGF. These findings demonstrate a novel function of primary cilia in controlling EG-VEGF-regulated trophoblast invasion and reveal the underlying molecular mechanism. J. Cell. Physiol. 232: 1467-1477, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Pharmacological Inhibition of the Protein Kinase MRK/ZAK Radiosensitizes Medulloblastoma.

PubMed

Markowitz, Daniel; Powell, Caitlin; Tran, Nhan L; Berens, Michael E; Ryken, Timothy C; Vanan, Magimairajan; Rosen, Lisa; He, Mingzu; Sun, Shan; Symons, Marc; Al-Abed, Yousef; Ruggieri, Rosamaria

2016-08-01

Medulloblastoma is a cerebellar tumor and the most common pediatric brain malignancy. Radiotherapy is part of the standard care for this tumor, but its effectiveness is accompanied by significant neurocognitive sequelae due to the deleterious effects of radiation on the developing brain. We have previously shown that the protein kinase MRK/ZAK protects tumor cells from radiation-induced cell death by regulating cell-cycle arrest after ionizing radiation. Here, we show that siRNA-mediated MRK depletion sensitizes medulloblastoma primary cells to radiation. We have, therefore, designed and tested a specific small molecule inhibitor of MRK, M443, which binds to MRK in an irreversible fashion and inhibits its activity. We found that M443 strongly radiosensitizes UW228 medulloblastoma cells as well as UI226 patient-derived primary cells, whereas it does not affect the response to radiation of normal brain cells. M443 also inhibits radiation-induced activation of both p38 and Chk2, two proteins that act downstream of MRK and are involved in DNA damage-induced cell-cycle arrest. Importantly, in an animal model of medulloblastoma that employs orthotopic implantation of primary patient-derived UI226 cells in nude mice, M443 in combination with radiation achieved a synergistic increase in survival. We hypothesize that combining radiotherapy with M443 will allow us to lower the radiation dose while maintaining therapeutic efficacy, thereby minimizing radiation-induced side effects. Mol Cancer Ther; 15(8); 1799-808. ©2016 AACR. ©2016 American Association for Cancer Research.

CMV seronegative donors: Effect on clinical severity of CMV infection and reconstitution of CMV-specific immunity.

PubMed

van der Heiden, P L J; van Egmond, H M; Veld, S A J; van de Meent, M; Eefting, M; de Wreede, L C; Halkes, C J M; Falkenburg, J H F; Marijt, W A F; Jedema, I

2018-04-18

Cytomegalovirus (CMV)-specific T-cells are crucial to prevent CMV disease. CMV seropositive recipients transplanted with stem cells from a CMV seronegative allogeneic donor (R + D - ) may be at risk for CMV disease due to absence of donor CMV-specific memory T-cells in the graft. We analyzed the duration of CMV reactivations and the incidence of CMV disease in R + D - and R + D + patients after alemtuzumab-based T-cell depleted allogeneic stem cell transplantation (TCD alloSCT). To determine the presence of donor-derived primary CMV-specific T-cell responses we analyzed the origin of CMV-specific T-cells in R + D - patients. The duration of CMV reactivations (54 versus 38 days, respectively, p = 0.048) and the incidence of CMV disease (0.14 versus 0.02, p = 0.003 at 1 year after alloSCT) were higher in R + D - patients compared to R + D + patients. In R + D - patients, CMV-specific CD4 + and CD8 + T-cells were mainly of recipient origin. However, in 53% of R + D - patients donor-derived CMV-specific T-cells were detected within the first year. In R + D - patients, immunity against CMV was predominantly mediated by recipient T-cells. Nevertheless, donor CMV serostatus significantly influenced the clinical severity of CMV reactivations indicating the role of CMV-specific memory T-cells transferred with the graft, despite the ultimate formation of primary donor-derived CMV-specific T-cell responses in R + D - patients. Copyright © 2018 Elsevier B.V. All rights reserved.

The neuroprotective effect of rat adipose tissue-derived mesenchymal stem cell-conditioned medium on cortical neurons using an in vitro model of SCI inflammation.

PubMed

Szekiova, Eva; Slovinska, Lucia; Blasko, Juraj; Plsikova, Jana; Cizkova, Dasa

2018-04-01

Objectives In this study, a new approach was used with an in vitro model in which neural cells were exposed to conditioned media from the injured spinal cord (SCI-CM) mimicking a local inflammatory microenvironment . Subsequently, the neuroprotective effect of rat adipose tissue-derived msesenchymal stem cell-conditioned media (ATMSC-CM) was investigated through a cell-free based therapy, which was used to treat cortical neurons and astrocytes under inflammation. Methods Primary cell cultures isolated from postnatal day (P6) Wistar rat brain cortex were exposed to SCI-CM derived from the central lesion, rostral and caudal segments of injured spinal cord. After 48 h incubation, the SCI-CM was replaced and primary cultures were cultivated either in DMEM media alone or in ATMSC-CM for 72 h. The impact of ATMSC-CM on the viability of neurons and astrocytes was assessed using a CyQUANT® Direct Cell Proliferation Assay Kit as well as immunocytochemistry analysis. Results Immunocytochemical analysis revealed significant decrease in the number of MAP2 positive neurons exposed to SCI-CM compared to Control. Protection by ATMSC-CM was associated with increased survival of neurons compared to primary culture cultivated in DMEM media alone. The ATMSC-CM effect on astrocytes was more variable and without any significant impact. Conclusion The results demonstrate that SCI-CM mimicking inflammation can reduce cortical neuron survival, and subsequent exposure to ATMSC-CM can stabilize the neuronal population most likely via released neuroprotective and trophic factors. In addition, astrogliosis was not affected by ATMSC-CM.

Patient-derived Mammosphere and Xenograft Tumour Initiation Correlates with Progression to Metastasis.

PubMed

Eyre, Rachel; Alférez, Denis G; Spence, Kath; Kamal, Mohamed; Shaw, Frances L; Simões, Bruno M; Santiago-Gómez, Angélica; Sarmiento-Castro, Aida; Bramley, Maria; Absar, Mohammed; Saad, Zahida; Chatterjee, Sumohan; Kirwan, Cliona; Gandhi, Ashu; Armstrong, Anne C; Wardley, Andrew M; O'Brien, Ciara S; Farnie, Gillian; Howell, Sacha J; Clarke, Robert B

2016-12-01

Breast cancer specific mortality results from tumour cell dissemination and metastatic colonisation. Identification of the cells and processes responsible for metastasis will enable better prevention and control of metastatic disease, thus reducing relapse and mortality. To better understand these processes, we prospectively collected 307 patient-derived breast cancer samples (n = 195 early breast cancers (EBC) and n = 112 metastatic samples (MBC)). We assessed colony-forming activity in vitro by growing isolated cells in both primary (formation) and secondary (self-renewal) mammosphere culture, and tumour initiating activity in vivo through subcutaneous transplantation of fragments or cells into mice. Metastatic samples formed primary mammosphere colonies significantly more frequently than early breast cancers and had significantly higher primary mammosphere colony formation efficiency (0.9 % vs. 0.6 %; p < 0.0001). Tumour initiation in vivo was significantly higher in metastatic than early breast cancer samples (63 % vs. 38 %, p = 0.04). Of 144 breast cancer samples implanted in vivo, we established 20 stable patient-derived xenograft (PDX) models at passage 2 or greater. Lung metastases were detected in mice from 14 PDX models. Mammosphere colony formation in vitro significantly correlated with the ability of a tumour to metastasise to the lungs in vivo (p = 0.05), but not with subcutaneous tumour initiation. In summary, the breast cancer stem cell activities of colony formation and tumour initiation are increased in metastatic compared to early samples, and predict metastasis in vivo. These results suggest that breast stem cell activity will predict for poor outcome tumours, and therapy targeting this activity will improve outcomes for patients with metastatic disease.

Acrylamide-induced apoptosis in rat primary astrocytes and human astrocytoma cell lines.

PubMed

Lee, Jiann-Gwu; Wang, Yan-Shiu; Chou, Chin-Cheng

2014-06-01

This study aimed to evaluate the acrylamide (ACR)-induced apoptotic effects on rat primary astrocytes and three human astrocytoma-derived cell lines (U-1240 MG, U-87 MG, and U-251 MG). As determined through the MTT assay, treatment with 1 and 2 mM ACR for 24-72 h resulted in decreased cell viability in all cells. Decreases in cell viability could be blocked in all cells with the exception of U-251 MG cells by Z-DEVD FMK. ACR-induced dose-dependent apoptotic effects were also demonstrated by increases in the sub-G1 phase cell population in all cells. The decreased expressions of pro-caspase 3, 8, and 9 and the interruption of the mitochondrial membrane potential were observed in all cells. Exposure to 2 mM ACR for 48 h resulted in increased Bax/Bcl-2 ratios in primary astrocytes and U-87 MG cells, whereas the overexpression of Bcl-2 was observed in U-1240 MG and U-251 MG cells. The ACR-induced increases in the levels of p53 and pp53 in primary astrocytes could be attenuated by caffeine. These results suggest the existence of a common apoptotic pathway among all cell types and that U-87 MG cells may be a suitable substitute in vitro model for primary astrocytes in future studies on ACR-induced neurotoxicity. Copyright © 2014 Elsevier Ltd. All rights reserved.

Functional Comparison of Induced Pluripotent Stem Cell- and Blood-Derived GPIIbIIIa Deficient Platelets

PubMed Central

Haas, Jessica; Sandrock-Lang, Kirstin; Gärtner, Florian; Jung, Christian Billy; Zieger, Barbara; Parrotta, Elvira; Kurnik, Karin; Sinnecker, Daniel; Wanner, Gerhard; Laugwitz, Karl-Ludwig; Massberg, Steffen; Moretti, Alessandra

2015-01-01

Human induced pluripotent stem cells (hiPSCs) represent a versatile tool to model genetic diseases and are a potential source for cell transfusion therapies. However, it remains elusive to which extent patient-specific hiPSC-derived cells functionally resemble their native counterparts. Here, we generated a hiPSC model of the primary platelet disease Glanzmann thrombasthenia (GT), characterized by dysfunction of the integrin receptor GPIIbIIIa, and compared side-by-side healthy and diseased hiPSC-derived platelets with peripheral blood platelets. Both GT-hiPSC-derived platelets and their peripheral blood equivalents showed absence of membrane expression of GPIIbIIIa, a reduction of PAC-1 binding, surface spreading and adherence to fibrinogen. We demonstrated that GT-hiPSC-derived platelets recapitulate molecular and functional aspects of the disease and show comparable behavior to their native counterparts encouraging the further use of hiPSC-based disease models as well as the transition towards a clinical application. PMID:25607928

Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET

PubMed Central

Peinado, Héctor; Alečković, Maša; Lavotshkin, Simon; Matei, Irina; Costa-Silva, Bruno; Moreno-Bueno, Gema; Hergueta-Redondo, Marta; Williams, Caitlin; García-Santos, Guillermo; Nitadori-Hoshino, Ayuko; Hoffman, Caitlin; Badal, Karen; Garcia, Benjamin A.; Callahan, Margaret K.; Yuan, Jianda; Martins, Vilma R.; Skog, Johan; Kaplan, Rosandra N.; Brady, Mary S.; Wolchok, Jedd D.; Chapman, Paul B.; Kang, Yibin; Bromberg, Jacqueline; Lyden, David

2013-01-01

Tumor-derived exosomes are emerging mediators of tumorigenesis with tissue-specific addresses and messages. We explored the function of melanoma-derived exosomes in the formation of primary tumor and metastases in mouse and human subjects. Exosomes from highly metastatic melanoma increased the metastatic behavior of primary tumors by permanently “educating” bone marrow (BM) progenitors via the MET receptor. Melanoma-derived exosomes also induced vascular leakiness at pre-metastatic sites, and reprogrammed BM progenitors towards a c-Kit+Tie2+Met+ pro-vasculogenic phenotype. Reducing Met expression in exosomes diminished the pro-metastatic behavior of BM cells. Importantly, MET expression was elevated in circulating CD45−C-KITlow/+TIE2+ BM progenitors from metastatic melanoma subjects. RAB1a, RAB5b, RAB7, and RAB27a were highly expressed in melanoma cells and Rab27a RNA interference decreased exosome production, preventing BM education, tumor growth and metastasis. Finally, we identified an exosome-specific “melanoma signature” with prognostic and therapeutic potential, comprised of TYRP2, VLA-4, HSP70, an HSP90 isoform and the MET oncoprotein. PMID:22635005

Human primary mixed brain cultures: preparation, differentiation, characterization and application to neuroscience research.

PubMed

Ray, Balmiki; Chopra, Nipun; Long, Justin M; Lahiri, Debomoy K

2014-09-16

Culturing primary cortical neurons is an essential neuroscience technique. However, most cultures are derived from rodent brains and standard protocols for human brain cultures are sparse. Herein, we describe preparation, maintenance and major characteristics of a primary human mixed brain culture, including neurons, obtained from legally aborted fetal brain tissue. This approach employs standard materials and techniques used in the preparation of rodent neuron cultures, with critical modifications. This culture has distinct differences from rodent cultures. Specifically, a significant numbers of cells in the human culture are derived from progenitor cells, and the yield and survival of the cells grossly depend on the presence of bFGF. In the presence of bFGF, this culture can be maintained for an extended period. Abundant productions of amyloid-β, tau and proteins make this a powerful model for Alzheimer's research. The culture also produces glia and different sub-types of neurons. We provide a well-characterized methodology for human mixed brain cultures useful to test therapeutic agents under various conditions, and to carry forward mechanistic and translational studies for several brain disorders.

Characterization of Reemerging Chikungunya Virus

PubMed Central

Sourisseau, Marion; Schilte, Clémentine; Casartelli, Nicoletta; Trouillet, Céline; Guivel-Benhassine, Florence; Rudnicka, Dominika; Sol-Foulon, Nathalie; Roux, Karin Le; Prevost, Marie-Christine; Fsihi, Hafida; Frenkiel, Marie-Pascale; Blanchet, Fabien; Afonso, Philippe V; Ceccaldi, Pierre-Emmanuel; Ozden, Simona; Gessain, Antoine; Schuffenecker, Isabelle; Verhasselt, Bruno; Zamborlini, Alessia; Saïb, Ali; Rey, Felix A; Arenzana-Seisdedos, Fernando; Desprès, Philippe; Michault, Alain; Albert, Matthew L; Schwartz, Olivier

2007-01-01

An unprecedented epidemic of chikungunya virus (CHIKV) infection recently started in countries of the Indian Ocean area, causing an acute and painful syndrome with strong fever, asthenia, skin rash, polyarthritis, and lethal cases of encephalitis. The basis for chikungunya disease and the tropism of CHIKV remain unknown. Here, we describe the replication characteristics of recent clinical CHIKV strains. Human epithelial and endothelial cells, primary fibroblasts and, to a lesser extent, monocyte-derived macrophages, were susceptible to infection and allowed viral production. In contrast, CHIKV did not replicate in lymphoid and monocytoid cell lines, primary lymphocytes and monocytes, or monocyte-derived dendritic cells. CHIKV replication was cytopathic and associated with an induction of apoptosis in infected cells. Chloroquine, bafilomycin-A1, and short hairpin RNAs against dynamin-2 inhibited viral production, indicating that viral entry occurs through pH-dependent endocytosis. CHIKV was highly sensitive to the antiviral activity of type I and II interferons. These results provide a general insight into the interaction between CHIKV and its mammalian host. PMID:17604450

Critical role of dendritic cell-derived IL-27 in antitumor immunity through regulating the recruitment and activation of NK and NKT cells.

PubMed

Wei, Jun; Xia, Siyuan; Sun, Huayan; Zhang, Song; Wang, Jingya; Zhao, Huiyuan; Wu, Xiaoli; Chen, Xi; Hao, Jianlei; Zhou, Xinglong; Zhu, Zhengmao; Gao, Xiang; Gao, Jian-xin; Wang, Puyue; Wu, Zhenzhou; Zhao, Liqing; Yin, Zhinan

2013-07-01

Critical roles of IL-27 in autoimmune diseases and infections have been reported; however, the contribution of endogenous IL-27 to tumor progression remains elusive. In this study, by using IL-27p28 conditional knockout mice, we demonstrate that IL-27 is critical in protective immune response against methyl-cholanthrene-induced fibrosarcoma and transplanted B16 melanoma, and dendritic cells (DCs) are the primary source. DC-derived IL-27 is required for shaping tumor microenvironment by inducing CXCL-10 expression in myeloid-derived suppressor cells and regulating IL-12 production from DCs, which lead to the recruitment and activation of NK and NKT cells resulting in immunological control of tumors. Indeed, reconstitution of IL-27 or CXCL-10 in tumor site significantly inhibits tumor growth and restores the number and activation of NK and NKT cells. In summary, our study identifies a previous unknown critical role of DC-derived IL-27 in NK and NKT cell-dependent antitumor immunity through shaping tumor microenvironment, and sheds light on developing novel therapeutic approaches based on IL-27.

Isolation and expansion of human pluripotent stem cell-derived hepatic progenitor cells by growth factor defined serum-free culture conditions.

PubMed

Fukuda, Takayuki; Takayama, Kazuo; Hirata, Mitsuhi; Liu, Yu-Jung; Yanagihara, Kana; Suga, Mika; Mizuguchi, Hiroyuki; Furue, Miho K

2017-03-15

Limited growth potential, narrow ranges of sources, and difference in variability and functions from batch to batch of primary hepatocytes cause a problem for predicting drug-induced hepatotoxicity during drug development. Human pluripotent stem cell (hPSC)-derived hepatocyte-like cells in vitro are expected as a tool for predicting drug-induced hepatotoxicity. Several studies have already reported efficient methods for differentiating hPSCs into hepatocyte-like cells, however its differentiation process is time-consuming, labor-intensive, cost-intensive, and unstable. In order to solve this problem, expansion culture for hPSC-derived hepatic progenitor cells, including hepatic stem cells and hepatoblasts which can self-renewal and differentiate into hepatocytes should be valuable as a source of hepatocytes. However, the mechanisms of the expansion of hPSC-derived hepatic progenitor cells are not yet fully understood. In this study, to isolate hPSC-derived hepatic progenitor cells, we tried to develop serum-free growth factor defined culture conditions using defined components. Our culture conditions were able to isolate and grow hPSC-derived hepatic progenitor cells which could differentiate into hepatocyte-like cells through hepatoblast-like cells. We have confirmed that the hepatocyte-like cells prepared by our methods were able to increase gene expression of cytochrome P450 enzymes upon encountering rifampicin, phenobarbital, or omeprazole. The isolation and expansion of hPSC-derived hepatic progenitor cells in defined culture conditions should have advantages in terms of detecting accurate effects of exogenous factors on hepatic lineage differentiation, understanding mechanisms underlying self-renewal ability of hepatic progenitor cells, and stably supplying functional hepatic cells. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

PubMed Central

Dong, Yifei; Arif, Arif A.; Poon, Grace F. T.; Hardman, Blair; Dosanjh, Manisha; Johnson, Pauline

2016-01-01

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function. PMID:27404290

Distinct Mechanisms of Ferritin Delivery to Lysosomes in Iron-Depleted and Iron-Replete Cells ▿

PubMed Central

Asano, Takeshi; Komatsu, Masaaki; Yamaguchi-Iwai, Yuko; Ishikawa, Fuyuki; Mizushima, Noboru; Iwai, Kazuhiro

2011-01-01

Ferritin is a cytosolic protein that stores excess iron, thereby protecting cells from iron toxicity. Ferritin-stored iron is believed to be utilized when cells become iron deficient; however, the mechanisms underlying the extraction of iron from ferritin have yet to be fully elucidated. Here, we demonstrate that ferritin is degraded in the lysosome under iron-depleted conditions and that the acidic environment of the lysosome is crucial for iron extraction from ferritin and utilization by cells. Ferritin was targeted for degradation in the lysosome even under iron-replete conditions in primary cells; however, the mechanisms underlying lysosomal targeting of ferritin were distinct under depleted and replete conditions. In iron-depleted cells, ferritin was targeted to the lysosome via a mechanism that involved autophagy. In contrast, lysosomal targeting of ferritin in iron-replete cells did not involve autophagy. The autophagy-independent pathway of ferritin delivery to lysosomes was deficient in several cancer-derived cells, and cancer-derived cell lines are more resistant to iron toxicity than primary cells. Collectively, these results suggest that ferritin trafficking may be differentially regulated by cell type and that loss of ferritin delivery to the lysosome under iron-replete conditions may be related to oncogenic cellular transformation. PMID:21444722

Glycolysis is the primary bioenergetic pathway for cell motility and cytoskeletal remodeling in human prostate and breast cancer cells.

PubMed

Shiraishi, Takumi; Verdone, James E; Huang, Jessie; Kahlert, Ulf D; Hernandez, James R; Torga, Gonzalo; Zarif, Jelani C; Epstein, Tamir; Gatenby, Robert; McCartney, Annemarie; Elisseeff, Jennifer H; Mooney, Steven M; An, Steven S; Pienta, Kenneth J

2015-01-01

The ability of a cancer cell to detach from the primary tumor and move to distant sites is fundamental to a lethal cancer phenotype. Metabolic transformations are associated with highly motile aggressive cellular phenotypes in tumor progression. Here, we report that cancer cell motility requires increased utilization of the glycolytic pathway. Mesenchymal cancer cells exhibited higher aerobic glycolysis compared to epithelial cancer cells while no significant change was observed in mitochondrial ATP production rate. Higher glycolysis was associated with increased rates of cytoskeletal remodeling, greater cell traction forces and faster cell migration, all of which were blocked by inhibition of glycolysis, but not by inhibition of mitochondrial ATP synthesis. Thus, our results demonstrate that cancer cell motility and cytoskeleton rearrangement is energetically dependent on aerobic glycolysis and not oxidative phosphorylation. Mitochondrial derived ATP is insufficient to compensate for inhibition of the glycolytic pathway with regard to cellular motility and CSK rearrangement, implying that localization of ATP derived from glycolytic enzymes near sites of active CSK rearrangement is more important for cell motility than total cellular ATP production rate. These results extend our understanding of cancer cell metabolism, potentially providing a target metabolic pathway associated with aggressive disease.

Glycolysis is the primary bioenergetic pathway for cell motility and cytoskeletal remodeling in human prostate and breast cancer cells

PubMed Central

Shiraishi, Takumi; Verdone, James E.; Huang, Jessie; Kahlert, Ulf D.; Hernandez, James R.; Torga, Gonzalo; Zarif, Jelani C.; Epstein, Tamir; Gatenby, Robert; McCartney, Annemarie; Elisseeff, Jennifer H.; Mooney, Steven M.; An, Steven S.; Pienta, Kenneth J.

2015-01-01

The ability of a cancer cell to detach from the primary tumor and move to distant sites is fundamental to a lethal cancer phenotype. Metabolic transformations are associated with highly motile aggressive cellular phenotypes in tumor progression. Here, we report that cancer cell motility requires increased utilization of the glycolytic pathway. Mesenchymal cancer cells exhibited higher aerobic glycolysis compared to epithelial cancer cells while no significant change was observed in mitochondrial ATP production rate. Higher glycolysis was associated with increased rates of cytoskeletal remodeling, greater cell traction forces and faster cell migration, all of which were blocked by inhibition of glycolysis, but not by inhibition of mitochondrial ATP synthesis. Thus, our results demonstrate that cancer cell motility and cytoskeleton rearrangement is energetically dependent on aerobic glycolysis and not oxidative phosphorylation. Mitochondrial derived ATP is insufficient to compensate for inhibition of the glycolytic pathway with regard to cellular motility and CSK rearrangement, implying that localization of ATP derived from glycolytic enzymes near sites of active CSK rearrangement is more important for cell motility than total cellular ATP production rate. These results extend our understanding of cancer cell metabolism, potentially providing a target metabolic pathway associated with aggressive disease. PMID:25426557

Isolation, Characterization, and Functional Analysis of Ferret Lymphatic Endothelial Cells

PubMed Central

Berendam, Stella J.; Fallert-Junecko, Beth A.; Murphy-Corb, Michael A.; Fuller, Deborah H.; Reinhart, Todd A.

2014-01-01

The lymphatic endothelium (LE) serves as a conduit for transport of immune cells and soluble antigens from peripheral tissues to draining lymph nodes (LNs), contributing to development of host immune responses and possibly dissemination of microbes. Lymphatic endothelial cells (LECs) are major constituents of the lymphatic endothelium. These specialized cells could play important roles in initiation of host innate immune responses through sensing of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs), including toll-like receptors (TLRs). LECs secrete pro-inflammatory cytokines and chemokines to create local inflammatory conditions for recruitment of naïve antigen presenting cells (APCs) such as dendritic cells (DCs) to sites of infection and/or vaccine administration. In this study, we examined the innate immune potential of primary LEC populations derived from multiple tissues of an animal model for human infectious diseases -- the ferret. We generated a total of six primary LEC populations from lung, tracheal, and mesenteric LN tissues from three different ferrets. Standard RT-PCR characterization of these primary LECs showed that they varied in their expression of LEC markers. The ferret LECs were examined for their ability to respond to poly I:C (TLR3 and RIG-1 ligand) and other known TLR ligands as measured by production of proinflammatory cytokine (IFNα, IL6, IL10, Mx1, and TNFα) and chemokine (CCL5, CCL20, and CXCL10) mRNAs using real time RT-PCR. Poly I:C exposure induced robust proinflammatory responses by all of the primary ferret LECs. Chemotaxis was performed to determine the functional activity of CCL20 produced by the primary lung LECs and showed that the LEC-derived CCL20 was abundant and functional. Taken together, our results continue to reveal the innate immune potential of primary LECs during pathogen-host interactions and expand our understanding of the roles of LECs might play in health and disease in animal models. PMID:25540877

Isolation, characterization, and functional analysis of ferret lymphatic endothelial cells.

PubMed

Berendam, Stella J; Fallert Junecko, Beth A; Murphey-Corb, Michael A; Fuller, Deborah H; Reinhart, Todd A

2015-02-15

The lymphatic endothelium (LE) serves as a conduit for transport of immune cells and soluble antigens from peripheral tissues to draining lymph nodes (LNs), contributing to development of host immune responses and possibly dissemination of microbes. Lymphatic endothelial cells (LECs) are major constituents of the lymphatic endothelium. These specialized cells could play important roles in initiation of host innate immune responses through sensing of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs), including toll-like receptors (TLRs). LECs secrete pro-inflammatory cytokines and chemokines to create local inflammatory conditions for recruitment of naïve antigen presenting cells (APCs) such as dendritic cells (DCs) to sites of infection and/or vaccine administration. In this study, we examined the innate immune potential of primary LEC populations derived from multiple tissues of an animal model for human infectious diseases - the ferret. We generated a total of six primary LEC populations from lung, tracheal, and mesenteric LN tissues from three different ferrets. Standard RT-PCR characterization of these primary LECs showed that they varied in their expression of LEC markers. The ferret LECs were examined for their ability to respond to poly I:C (TLR3 and RIG-I ligand) and other known TLR ligands as measured by production of proinflammatory cytokine (IFNα, IL6, IL10, Mx1, and TNFα) and chemokine (CCL5, CCL20, and CXCL10) mRNAs using real time RT-PCR. Poly I:C exposure induced robust proinflammatory responses by all of the primary ferret LECs. Chemotaxis was performed to determine the functional activity of CCL20 produced by the primary lung LECs and showed that the LEC-derived CCL20 was abundant and functional. Taken together, our results continue to reveal the innate immune potential of primary LECs during pathogen-host interactions and expand our understanding of the roles LECs might play in health and disease in animal models. Copyright © 2014 Elsevier B.V. All rights reserved.

P-selectin- and heparanase-dependent antimetastatic activity of non-anticoagulant heparins.

PubMed

Hostettler, Nina; Naggi, Annamaria; Torri, Giangiacomo; Ishai-Michaeli, Riva; Casu, Benito; Vlodavsky, Israel; Borsig, Lubor

2007-11-01

Vascular cell adhesion molecules, P- and L-selectins, facilitate metastasis of cancer cells in mice by mediating interactions with platelets, endothelium, and leukocytes. Heparanase is an endoglycosidase that degrades heparan sulfate of extracellular matrix, thereby promoting tumor invasion and metastasis. Heparin is known to efficiently attenuate metastasis in different tumor models. Here we identified modified, nonanticoagulant species of heparin that specifically inhibit selectin-mediated cell-cell interactions, heparanase enzymatic activity, or both. We show that selective inhibition of selectin interactions or heparanase with specific heparin derivatives in mouse models of MC-38 colon carcinoma and B16-BL6 melanoma attenuates metastasis. Selectin-specific heparin derivatives attenuated metastasis of MC-38 carcinoma, but heparanase-specific derivatives had no effect, in accordance with the virtual absence of heparanase activity in these cells. Heparin derivatives had no further effect on metastasis in mice deficient in P- and L-selectin, indicating that selectins are the primary targets of heparin antimetastatic activity. Selectin-specific and heparanase-specific derivatives attenuated metastasis of B16-BL6 melanomas to a similar extent. When mice were injected with a derivative containing both heparanase and selectin inhibitory activity, no additional attenuation of metastasis could be observed. Thus, selectin-specific heparin derivatives efficiently attenuated metastasis of both tumor cell types whereas inhibition of heparanase led to reduction of metastasis only in tumor cells producing heparanase.

Efficient establishment of primary fibroblast cultures from the hawksbill sea turtle (Eretmochelys imbricata).

PubMed

Fukuda, Tomokazu; Kurita, Jun; Saito, Tomomi; Yuasa, Kei; Kurita, Masanobu; Donai, Kenichiro; Nitto, Hiroshi; Soichi, Makoto; Nishimori, Katsuhiko; Uchida, Takafumi; Isogai, Emiko; Onuma, Manabu; Sone, Hideko; Oseko, Norihisa; Inoue-Murayama, Miho

2012-12-01

The hawksbill sea turtle (Eretmochelys imbricata) is a critically endangered species at a risk of extinction. Preservation of the genomic and cellular information of endangered animals is important for future genetic and biological studies. Here, we report the efficient establishment of primary fibroblast cultures from skin tissue of the hawksbill sea turtle. We succeeded in establishing 19 primary cultures from 20 hawksbill sea turtle individuals (a success rate of 95%). These cells exhibited a fibroblast-like morphology and grew optimally at a temperature of 26°C, but experienced a loss of viability when cultured at 37°C. Chromosomal analysis using the primary cells derived here revealed that hawksbill sea turtles have a 2n = 56 karyotype. Furthermore, we showed that our primary cell cultures are free of several fish-related viruses, and this finding is important for preservation purposes. To our knowledge, this report is the first to describe primary cell cultures established from normal tissues of the hawksbill sea turtle. The results will contribute to the preservation of biodiversity, especially for the sea turtles that are critically endangered owing to human activities.

Phosphoproteomics of Primary Cells Reveals Druggable Kinase Signatures in Ovarian Cancer.

PubMed

Francavilla, Chiara; Lupia, Michela; Tsafou, Kalliopi; Villa, Alessandra; Kowalczyk, Katarzyna; Rakownikow Jersie-Christensen, Rosa; Bertalot, Giovanni; Confalonieri, Stefano; Brunak, Søren; Jensen, Lars J; Cavallaro, Ugo; Olsen, Jesper V

2017-03-28

Our understanding of the molecular determinants of cancer is still inadequate because of cancer heterogeneity. Here, using epithelial ovarian cancer (EOC) as a model system, we analyzed a minute amount of patient-derived epithelial cells from either healthy or cancerous tissues by single-shot mass-spectrometry-based phosphoproteomics. Using a multi-disciplinary approach, we demonstrated that primary cells recapitulate tissue complexity and represent a valuable source of differentially expressed proteins and phosphorylation sites that discriminate cancer from healthy cells. Furthermore, we uncovered kinase signatures associated with EOC. In particular, CDK7 targets were characterized in both EOC primary cells and ovarian cancer cell lines. We showed that CDK7 controls cell proliferation and that pharmacological inhibition of CDK7 selectively represses EOC cell proliferation. Our approach defines the molecular landscape of EOC, paving the way for efficient therapeutic approaches for patients. Finally, we highlight the potential of phosphoproteomics to identify clinically relevant and druggable pathways in cancer. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Improved bone marrow stromal cell adhesion on micropatterned titanium surfaces.

PubMed

Iskandar, Maria E; Cipriano, Aaron F; Lock, Jaclyn; Gott, Shannon C; Rao, Masaru P; Liu, Huinan

2012-01-01

Implant longevity is desired for all bone replacements and fixatives. Titanium (Ti) implants fail due to lack of juxtaposed bone formation, resulting in implant loosening. Implant surface modifications have shown to affect the interactions between the implant and bone. In clinical applications, it is crucial to improve osseointegration and implant fixation at the implant and bone interface. Moreover, bone marrow derived cells play a significant role for implant and tissue integration. Therefore, the objective of this study is to investigate how surface micropatterning on Ti influences its interactions with bone marrow derived cells containing mesenchymal and hematopoietic stem cells. Bone marrow derived mesenchymal stem cells (BMSC) have the capability of differentiating into osteoblasts that contribute to bone growth, and therefore implant/bone integration. Hematopoietic stem cell derivatives are precursor cells that contribute to inflammatory response. By using all three cells naturally contained within bone marrow, we mimic the physiological environment to which an implant is exposed. Primary rat bone marrow derived cells were seeded onto Ti with surfaces composed of arrays of grooves of equal width and spacing ranging from 0.5 to 50 µm, fabricated using a novel plasma-based dry etching technique. Results demonstrated enhanced total cell adhesion on smaller micrometer-scale Ti patterns compared with larger micrometer-scale Ti patterns, after 24-hr culture. Further studies are needed to determine bone marrow derived cell proliferation and osteogenic differentiation potential on micropatterned Ti, and eventually nanopatterned Ti.

Tie2 Expressing Monocytes in the Spleen of Patients with Primary Myelofibrosis

PubMed Central

Campanelli, Rita; Fois, Gabriela; Catarsi, Paolo; Poletto, Valentina; Villani, Laura; Erba, Benedetta Gaia; Maddaluno, Luigi; Jemos, Basilio; Salmoiraghi, Silvia; Guglielmelli, Paola; Abbonante, Vittorio; Di Buduo, Christian Andrea; Balduini, Alessandra; Iurlo, Alessandra; Barosi, Giovanni; Rosti, Vittorio; Massa, Margherita

2016-01-01

Primary myelofibrosis (PMF) is a Philadelphia-negative (Ph−) myeloproliferative disorder, showing abnormal CD34+ progenitor cell trafficking, splenomegaly, marrow fibrosis leading to extensive extramedullary haematopoiesis, and abnormal neoangiogenesis in either the bone marrow or the spleen. Monocytes expressing the angiopoietin-2 receptor (Tie2) have been shown to support abnormal angiogenic processes in solid tumors through a paracrine action that takes place in proximity to the vessels. In this study we investigated the frequency of Tie2 expressing monocytes in the spleen tissue samples of patients with PMF, and healthy subjects (CTRLs), and evaluated their possible role in favouring spleen angiogenesis. We show by confocal microscopy that in the spleen tissue of patients with PMF, but not of CTRLs, the most of the CD14+ cells are Tie2+ and are close to vessels; by flow cytometry, we found that Tie2 expressing monocytes were Tie2+CD14lowCD16brightCDL62−CCR2− (TEMs) and their frequency was higher (p = 0.008) in spleen tissue-derived mononuclear cells (MNCs) of patients with PMF than in spleen tissue-derived MNCs from CTRLs undergoing splenectomy for abdominal trauma. By in vitro angiogenesis assay we evidenced that conditioned medium of immunomagnetically selected spleen tissue derived CD14+ cells of patients with PMF induced a denser tube like net than that of CTRLs; in addition, CD14+Tie2+ cells sorted from spleen tissue derived single cell suspension of patients with PMF show a higher expression of genes involved in angiogenesis than that found in CTRLs. Our results document the enrichment of Tie2+ monocytes expressing angiogenic genes in the spleen of patients with PMF, suggesting a role for these cells in starting/maintaining the pathological angiogenesis in this organ. PMID:27281335

Tumor associated CD70 expression is involved in promoting tumor migration and macrophage infiltration in GBM.

PubMed

Ge, Haitao; Mu, Luyan; Jin, Linchun; Yang, Changlin; Chang, Yifan Emily; Long, Yu; DeLeon, Gabriel; Deleyrolle, Loic; Mitchell, Duane A; Kubilis, Paul S; Lu, Dunyue; Qi, Jiping; Gu, Yunhe; Lin, Zhiguo; Huang, Jianping

2017-10-01

Tumor migration/metastasis and immunosuppression are major obstacles in effective cancer therapy. Incidentally, these 2 hurdles usually coexist inside tumors, therefore making therapy significantly more complicated, as both oncogenic mechanisms must be addressed for successful therapeutic intervention. Our recent report highlights that the tumor expression of a TNF family member, CD70, is correlated with poor survival for primary gliomas. In this study, we investigated how CD70 expression by GBM affects the characteristics of tumor cells and the tumor microenvironment. We found that the ablation of CD70 in primary GBM decreased CD44 and SOX2 gene expression, and inhibited tumor migration, growth and the ability to attract monocyte-derived M2 macrophages in vitro. In the tumor microenvironment, CD70 was associated with immune cell infiltrates, such as T cells; myeloid-derived suppressor cells; and monocytes/macrophages based on the RNA-sequencing profile. The CD163+ macrophages were far more abundant than T cells were. This overwhelming level of macrophages was identified only in GBM and not in low-grade gliomas and normal brain specimens, implying their tumor association. CD70 was detected only on tumor cells, not on macrophages, and was highly correlated with CD163 gene expression in primary GBM. Additionally, the co-expression of the CD70 and CD163 genes was found to correlate with decreased survival for patients with primary GBM. Together, these data suggest that CD70 expression is involved in promoting tumor aggressiveness and immunosuppression via tumor-associated macrophage recruitment/activation. Our current efforts to target this molecule using chimeric antigen receptor T cells hold great potential for treating patients with GBM. © 2017 UICC.

Generation of a human airway epithelium derived basal cell line with multipotent differentiation capacity

PubMed Central

2013-01-01

Background As the multipotent progenitor population of the airway epithelium, human airway basal cells (BC) replenish the specialized differentiated cell populations of the mucociliated airway epithelium during physiological turnover and repair. Cultured primary BC divide a limited number of times before entering a state of replicative senescence, preventing the establishment of long-term replicating cultures of airway BC that maintain their original phenotype. Methods To generate an immortalized human airway BC cell line, primary human airway BC obtained by brushing the airway epithelium of healthy nonsmokers were infected with a retrovirus expressing human telomerase (hTERT). The resulting immortalized cell line was then characterized under non-differentiating and differentiating air-liquid interface (ALI) culture conditions using ELISA, TaqMan quantitative PCR, Western analysis, and immunofluorescent and immunohistochemical staining analysis for cell type specific markers. In addition, the ability of the cell line to respond to environmental stimuli under differentiating ALI culture was assessed. Results We successfully generated an immortalized human airway BC cell line termed BCi-NS1 via expression of hTERT. A single cell derived clone from the parental BCi-NS1 cells, BCi-NS1.1, retains characteristics of the original primary cells for over 40 passages and demonstrates a multipotent differentiation capacity into secretory (MUC5AC, MUC5B), goblet (TFF3), Clara (CC10) and ciliated (DNAI1, FOXJ1) cells on ALI culture. The cells can respond to external stimuli such as IL-13, resulting in alteration of the normal differentiation process. Conclusion Development of immortalized human airway BC that retain multipotent differentiation capacity over long-term culture should be useful in understanding the biology of BC, the response of BC to environmental stress, and as a target for assessment of pharmacologic agents. PMID:24298994

Primary brain tumors, neural stem cell, and brain tumor cancer cells: where is the link?

PubMed Central

Germano, Isabelle; Swiss, Victoria; Casaccia, Patrizia

2010-01-01

The discovery of brain tumor-derived cells (BTSC) with the properties of stem cells has led to the formulation of the hypothesis that neural stem cells could be the cell of origin of primary brain tumors (PBT). In this review we present the most common molecular changes in PBT, define the criteria of identification of BTSC and discuss the similarities between the characteristics of these cells and those of the endogenous population of neural stem cells (NPCs) residing in germinal areas of the adult brain. Finally, we propose possible mechanisms of cancer initiation and progression and suggest a model of tumor initiation that includes intrinsic changes of resident NSC and potential changes in the microenvironment defining the niche where the NSC reside. PMID:20045420

Defined, serum/feeder-free conditions for expansion and drug screening of primary B-acute lymphoblastic leukemia.

PubMed

Jiang, Zhiwu; Wu, Di; Ye, Wei; Weng, Jianyu; Lai, Peilong; Shi, Pengcheng; Guo, Xutao; Huang, Guohua; Deng, Qiuhua; Tang, Yanlai; Zhao, Hongyu; Cui, Shuzhong; Lin, Simiao; Wang, Suna; Li, Baiheng; Wu, Qiting; Li, Yangqiu; Liu, Pentao; Pei, Duanqing; Du, Xin; Yao, Yao; Li, Peng

2017-12-05

Functional screening for compounds represents a major hurdle in the development of rational therapeutics for B-acute lymphoblastic leukemia (B-ALL). In addition, using cell lines as valid models for evaluating responses to novel drug therapies raises serious concerns, as cell lines are prone to genotypic/phenotypic drift and loss of heterogeneity in vitro . Here, we reported that OP9 cells, not OP9-derived adipocytes (OP9TA), support the growth of primary B-ALL cells in vitro . To identify the factors from OP9 cells that support the growth of primary B-ALL cells, we performed RNA-Seq to analyze the gene expression profiles of OP9 and OP9TA cells. We thus developed a defined, serum/feeder-free condition (FI76V) that can support the expansion of a range of clinically distinct primary B-ALL cells that still maintain their leukemia-initiating ability. We demonstrated the suitability of high-throughput drug screening based on our B-ALL cultured conditions. Upon screening 378 kinase inhibitors, we identified a cluster of 17 kinase inhibitors that can efficiently kill B-ALL cells in vitro . Importantly, we demonstrated the synergistic cytotoxicity of dinaciclib/BTG226 to B-ALL cells. Taken together, we developed a defined condition for the ex vivo expansion of primary B-ALL cells that is suitable for high-throughput screening of novel compounds.

Evidence for Altered Glutamine Metabolism in Human Immunodeficiency Virus Type 1 Infected Primary Human CD4+ T Cells

PubMed Central

Hegedus, Andrea; Kavanagh Williamson, Maia; Khan, Mariam B.; Dias Zeidler, Julianna; Da Poian, Andrea T.; El-Bacha, Tatiana; Struys, Eduard A.

2017-01-01

Abstract Glutamine is a conditionally essential amino acid that is an important metabolic resource for proliferating tissues by acting as a proteinogenic amino acid, a nitrogen donor for biosynthetic reactions and as a substrate for the citric acid or tricarboxylic acid cycle. The human immunodeficiency virus type 1 (HIV-1) productively infects activated CD4+ T cells that are known to require glutamine for proliferation and for carrying out effector functions. As a virus, HIV-1 is furthermore entirely dependent on host metabolism to support its replication. In this study, we compared HIV-1 infected with uninfected activated primary human CD4+ T cells with regard to glutamine metabolism. We report that glutamine concentrations are elevated in HIV-1-infected cells and that glutamine is important to support HIV-1 replication, although the latter is closely linked to the glutamine dependency of cell survival. Metabolic tracer experiments showed that entry of glutamine-derived carbon into the citric acid cycle is unaffected by HIV-1 infection, but that there is an increase in the secretion of glutamine-derived glutamic acid from HIV-1-infected cells. Western blotting of key enzymes that metabolize glutamine revealed marked differences in the expression of glutaminase isoforms, KGA and CAG, as well as the PPAT enzyme that targets glutamine-derived nitrogen toward nucleotide synthesis. Altogether, this demonstrates that infection of CD4+ T cells with HIV-1 leads to considerable changes in the cellular glutamine metabolism. PMID:28844150

Evidence for Altered Glutamine Metabolism in Human Immunodeficiency Virus Type 1 Infected Primary Human CD4+ T Cells.

PubMed

Hegedus, Andrea; Kavanagh Williamson, Maia; Khan, Mariam B; Dias Zeidler, Julianna; Da Poian, Andrea T; El-Bacha, Tatiana; Struys, Eduard A; Huthoff, Hendrik

2017-12-01

Glutamine is a conditionally essential amino acid that is an important metabolic resource for proliferating tissues by acting as a proteinogenic amino acid, a nitrogen donor for biosynthetic reactions and as a substrate for the citric acid or tricarboxylic acid cycle. The human immunodeficiency virus type 1 (HIV-1) productively infects activated CD4 + T cells that are known to require glutamine for proliferation and for carrying out effector functions. As a virus, HIV-1 is furthermore entirely dependent on host metabolism to support its replication. In this study, we compared HIV-1 infected with uninfected activated primary human CD4 + T cells with regard to glutamine metabolism. We report that glutamine concentrations are elevated in HIV-1-infected cells and that glutamine is important to support HIV-1 replication, although the latter is closely linked to the glutamine dependency of cell survival. Metabolic tracer experiments showed that entry of glutamine-derived carbon into the citric acid cycle is unaffected by HIV-1 infection, but that there is an increase in the secretion of glutamine-derived glutamic acid from HIV-1-infected cells. Western blotting of key enzymes that metabolize glutamine revealed marked differences in the expression of glutaminase isoforms, KGA and CAG, as well as the PPAT enzyme that targets glutamine-derived nitrogen toward nucleotide synthesis. Altogether, this demonstrates that infection of CD4 + T cells with HIV-1 leads to considerable changes in the cellular glutamine metabolism.

A mutual activation loop between breast cancer cells and myeloid-derived suppressor cells facilitates spontaneous metastasis through IL-6 trans-signaling in a murine model.

PubMed

Oh, Keunhee; Lee, Ok-Young; Shon, Suh Youn; Nam, Onyou; Ryu, Po Mee; Seo, Myung Won; Lee, Dong-Sup

2013-01-01

Tumor cell interactions with the microenvironment, especially those of bone-marrow-derived myeloid cells, are important in various aspects of tumor metastasis. Myeloid-derived suppressor cells (MDSCs) have been suggested to constitute tumor-favoring microenvironments. In this study, we elucidated a novel mechanism by which the MDSCs can mediate spontaneous distant metastasis of breast cancer cells. Murine breast cancer cells, 4T1 and EMT6, were orthotopically grafted into the mammary fat pads of syngeneic BALB/c mice. CD11b(+)Gr-1(+) MDSCs in the spleen, liver, lung and primary tumor mass were analyzed. To evaluate the role of MDSCs in the distant metastasis, MDSCs were depleted or reconstituted in tumor-bearing mice. To evaluate whether MDSCs in the metastasizing tumor microenvironment affect breast cancer cell behavior, MDSCs and cancer cells were co-cultivated. To investigate the role of MDSCs in in vivo metastasis, we blocked the interactions between MDSCs and cancer cells. Using a murine breast cancer cell model, we showed that murine breast cancer cells with high IL-6 expression recruited more MDSCs and that the metastasizing capacity of cancer cells paralleled MDSC recruitment in tumor-bearing mice. Metastasizing, but not non-metastasizing, tumor-derived factors induced MDSCs to increase IL-6 production and full activation of recruited MDSCs occurred in the primary tumor site and metastatic organ in the vicinity of metastasizing cancer cells, but not in lymphoid organs. In addition, tumor-expanded MDSCs expressed Adam-family proteases, which facilitated shedding of IL-6 receptor, thereby contributing to breast cancer cell invasiveness and distant metastasis through IL-6 trans-signaling. The critical role of IL-6 trans-signaling was confirmed in both the afferent and efferent pathways of metastasis. In this study, we showed that metastasizing cancer cells induced higher MDSCs infiltration and prompted them to secret exaggerated IL-6 as well as soluble IL-6Ra, which, in turn, triggered a persistent increase of pSTAT3 in tumor cells. This potential tumor-MDSC axis involving IL-6 trans-signaling directly affected breast cancer cell aggressiveness, leading to spontaneous metastasis.

Discovery of Benzofuran Derivatives that Collaborate with Insulin-Like Growth Factor 1 (IGF-1) to Promote Neuroprotection.

PubMed

Wakabayashi, Takeshi; Tokunaga, Norihito; Tokumaru, Kazuyuki; Ohra, Taiichi; Koyama, Nobuyuki; Hayashi, Satoru; Yamada, Ryuji; Shirasaki, Mikio; Inui, Yoshitaka; Tsukamoto, Tetsuya

2016-05-26

A series of benzofuran derivatives with neuroprotective activity in collaboration with IGF-1 was discovered using a newly developed cell-based assay involving primary neural cells prepared from rat hippocampal and cerebral cortical tissues. A structure-activity relationship study identified compound 8 as exhibiting potent activity and brain penetrability. An in vitro pharmacological study demonstrated that although IGF-1 and 8 individually exhibited the neuroprotective effect, the latter acted in collaboration with IGF-1 to enhance neuroprotective activity.

Blood dendritic cells interact with splenic marginal zone B cells to initiate T-independent immune responses.

PubMed

Balázs, Mercedesz; Martin, Flavius; Zhou, Tong; Kearney, John

2002-09-01

Marginal zone (MZ) and B1 B lymphocytes participate jointly in the early immune response against T-independent (TI) particulate antigens. Here we show that blood-derived neutrophil granulocytes and CD11c(lo) immature dendritic cells (DC) are the primary cells that efficiently capture and transport particulate bacteria to the spleen. In a systemic infection, CD11c(lo) DC, but not neutrophils, provide critical survival signals, which can be inhibited by TACI-Fc, to antigen-specific MZ B cells and promote their differentiation into IgM-secreting plasmablasts. In a local TI response, peritoneal cavity macrophages provide similar support to B1 B-derived Ag-specific blasts. In the absence of soluble TACI ligands, Ag-activated MZ- and B1-derived blasts lack survival signals and undergo apoptosis, resulting in severely impaired antibody responses.

Liver Cell-Derived Microparticles Activate Hedgehog Signaling and Alter Gene Expression in Hepatic Endothelial Cells

PubMed Central

Witek, Rafal P.; Yang, Liu; Liu, Renshui; Jung, Youngmi; Omenetti, Alessia; Syn, Wing-Kin; Choi, Steve S.; Cheong, Yeiwon; Fearing, Caitlin M.; Agboola, Kolade M.; Chen, Wei; Diehl, Anna Mae

2013-01-01

Background & Aims Angiogenesis contributes to vascular remodeling during cirrhosis. In cirrhotic livers, cholangiocytes and myofibroblastic hepatic stellate cells (MF-HSC) produce Hedgehog (Hh) ligands. During embryogenesis Hh ligands are released from ligand-producing cells in microparticles and activate Hh signaling in endothelial cells. We studied whether adult liver cell-derived microparticles contain Hh ligands that alter hepatic sinusoidal endothelial cells (SEC). Methods MF-HSCs and cholangiocytes were exposed to platelet-derived growth factor (PDGF) to induce Hh ligands; microparticles were isolated from medium, analyzed by transmission electron microscopy (TEM) and immunoblots, and applied to Hh-reporter containing cells. Microparticles were also obtained from serum and bile of rats after bile duct ligation (BDL) or sham surgery and applied to normal primary liver SEC with or without cyclopamine, a Hh signaling inhibitor. Effects on SEC gene expression were evaluated by QRT-PCR and immunoblotting. Finally, Hh target gene expression and SEC activation markers were compared in primary SEC and in liver sections from healthy and BDL rats. Results PDGF-treated MF-HSC and cholangiocytes released exosome-enriched microparticles containing biologically active Hh ligands. BDL also increased release of Hh-containing exosome-enriched microparticles into plasma and bile. TEM and immunoblots revealed similarities among microparticles from all sources; all microparticles induced similar Hh-dependent changes in SEC gene expression. SEC from healthy livers did not express Hh target genes or activation markers, but both were up-regulated in SEC after BDL. Conclusions Hh-containing exosome-enriched microparticles released from liver cells alter hepatic SEC gene expression, suggesting a novel mechanism for cirrhotic vasculopathy. PMID:19013163

Sex Reporting in Preclinical Microbiological and Immunological Research.

PubMed

Potluri, Tanvi; Engle, Kyrra; Fink, Ashley L; Vom Steeg, Landon G; Klein, Sabra L

2017-11-14

Both sex (i.e., biological construct of male and female) and gender (i.e., social construct of masculine and feminine) impact the pathogenesis of diseases, including those caused by microbial infections. Following the 2015 NIH policy for consideration of sex as a biological variable in preclinical research, in 2018, authors of papers published in primary-research American Society for Microbiology (ASM) journals will be asked to report the sex of the research subjects and animals and of materials derived directly from them. To address the need for sex reporting in ASM journals, we systematically reviewed 2,928 primary-research articles published in six primary-research ASM journals ( Antimicrobial Agents and Chemotherapy , Clinical and Vaccine Immunology , Infection and Immunity , Journal of Bacteriology , Journal of Virology , and mBio ) in 2016. Approximately 37% of animal studies and 9% of primary cell culture papers published in 2016 would have been affected by the new sex-reporting policy. For animal studies (i.e., studies with any nonhuman vertebrate hosts), most published papers either did not report the sex of the animals or used only female animals, and a minority used only males or both sexes. For published studies using primary cells from diverse animal species (i.e., humans and nonhuman vertebrates), almost all studies failed to report the sex of donors from which the cells were isolated. We believe that reporting the sex of animals and even of the donors of derived cells could improve the rigor and reproducibility of research conducted in microbiology and immunology and published in ASM journals. Copyright © 2017 Potluri et al.

Damage of photoreceptor-derived cells in culture induced by light emitting diode-derived blue light

PubMed Central

Kuse, Yoshiki; Ogawa, Kenjiro; Tsuruma, Kazuhiro; Shimazawa, Masamitsu; Hara, Hideaki

2014-01-01

Our eyes are increasingly exposed to light from the emitting diode (LED) light of video display terminals (VDT) which contain much blue light. VDTs are equipped with televisions, personal computers, and smart phones. The present study aims to clarify the mechanism underlying blue LED light-induced photoreceptor cell damage. Murine cone photoreceptor-derived cells (661 W) were exposed to blue, white, or green LED light (0.38 mW/cm2). In the present study, blue LED light increased reactive oxygen species (ROS) production, altered the protein expression level, induced the aggregation of short-wavelength opsins (S-opsin), resulting in severe cell damage. While, blue LED light damaged the primary retinal cells and the damage was photoreceptor specific. N-Acetylcysteine (NAC), an antioxidant, protected against the cellular damage induced by blue LED light. Overall, the LED light induced cell damage was wavelength-, but not energy-dependent and may cause more severe retinal photoreceptor cell damage than the other LED light. PMID:24909301

Metastatic Melanoma Secreted IL-10 Down-Regulates CD1 Molecules on Dendritic Cells in Metastatic Tumor Lesions

PubMed Central

Gerlini, Gianni; Tun-Kyi, Adrian; Dudli, Christa; Burg, Günter; Pimpinelli, Nicola; Nestle, Frank O.

2004-01-01

CD1 molecules are expressed by antigen-presenting cells such as dendritic cells and mediate primary immune responses to lipids and glycolipids which have been shown to be expressed by various tumors. Glycolipids are expressed by melanoma cells but, despite their immunogenicity, no efficient spontaneous immune responses are elicited. As IL-10 has previously been shown to down-regulate CD1a on dendritic cells and is known to be expressed by various melanoma cell lines, we investigated if melanoma-derived IL-10 could down-regulate CD1 molecule expression on dendritic cells as a possible way to circumvent immune recognition. We found that CD1a, CD1b, CD1c, and CD1d were significantly down-regulated on dendritic cells in metastatic (n = 10) but not in primary melanoma lesions (n = 10). We further detected significantly higher IL-10 protein levels in metastatic than in primary melanomas. Moreover, supernatants from metastatic melanomas were significantly more effective in down-regulating CD1 molecules on dendritic cells than supernatants from primary melanoma cultures. This effect was blocked using a neutralizing IL-10 antibody in a dose dependent manner. Our findings suggest that metastatic but not primary melanomas can down-regulate CD1 molecules on infiltrating dendritic cells by secreting IL-10 which may represent a novel way to escape the immune response directed against the tumor. PMID:15579430

Quantitative assessment of neurite outgrowth in human embryonic stem-cell derived neurons using automated high-content image analysis

EPA Science Inventory

During development neurons undergo a number of morphological changes including neurite outgrowth from the cell body. Exposure to neurotoxicants that interfere with this process may cause in permanent deficits in nervous system function. While many studies have used rodent primary...

Reactivating p53 and Inducing Tumor Apoptosis (RITA) Enhances the Response of RITA-Sensitive Colorectal Cancer Cells to Chemotherapeutic Agents 5-Fluorouracil and Oxaliplatin.

PubMed

Wiegering, Armin; Matthes, Niels; Mühling, Bettina; Koospal, Monika; Quenzer, Anne; Peter, Stephanie; Germer, Christoph-Thomas; Linnebacher, Michael; Otto, Christoph

2017-04-01

Colorectal carcinoma (CRC) is the most common cancer of the gastrointestinal tract with frequently dysregulated intracellular signaling pathways, including p53 signaling. The mainstay of chemotherapy treatment of CRC is 5-fluorouracil (5FU) and oxaliplatin. The two anticancer drugs mediate their therapeutic effect via DNA damage-triggered signaling. The small molecule reactivating p53 and inducing tumor apoptosis (RITA) is described as an activator of wild-type and reactivator of mutant p53 function, resulting in elevated levels of p53 protein, cell growth arrest, and cell death. Additionally, it has been shown that RITA can induce DNA damage signaling. It is expected that the therapeutic benefits of 5FU and oxaliplatin can be increased by enhancing DNA damage signaling pathways. Therefore, we highlighted the antiproliferative response of RITA alone and in combination with 5FU or oxaliplatin in human CRC cells. A panel of long-term established CRC cell lines (n=9) including p53 wild-type, p53 mutant, and p53 null and primary patient-derived, low-passage cell lines (n=5) with different p53 protein status were used for this study. A substantial number of CRC cells with pronounced sensitivity to RITA (IC 50 <3.0 μmol/l) were identified within established (4/9) and primary patient-derived (2/5) CRC cell lines harboring wild-type or mutant p53 protein. Sensitivity to RITA appeared independent of p53 status and was associated with an increase in antiproliferative response to 5FU and oxaliplatin, a transcriptional increase of p53 targets p21 and NOXA, and a decrease in MYC mRNA. The effect of RITA as an inducer of DNA damage was shown by a strong elevation of phosphorylated histone variant H2A.X, which was restricted to RITA-sensitive cells. Our data underline the primary effect of RITA, inducing DNA damage, and demonstrate the differential antiproliferative effect of RITA to CRC cells independent of p53 protein status. We found a substantial number of RITA-sensitive CRC cells within both panels of established CRC cell lines and primary patient-derived CRC cell lines (6/14) that provide a rationale for combining RITA with 5FU or oxaliplatin to enhance the antiproliferative response to both chemotherapeutic agents. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Acetate supplementation induces growth arrest of NG2/PDGFRα-positive oligodendroglioma-derived tumor-initiating cells.

PubMed

Long, Patrick M; Tighe, Scott W; Driscoll, Heather E; Moffett, John R; Namboodiri, Aryan M A; Viapiano, Mariano S; Lawler, Sean E; Jaworski, Diane M

2013-01-01

Cancer is associated with globally hypoacetylated chromatin and considerable attention has recently been focused on epigenetic therapies. N-acetyl-L-aspartate (NAA), the primary storage form of acetate in the brain, and aspartoacylase (ASPA), the enzyme responsible for NAA catalysis to generate acetate and ultimately acetyl-Coenzyme A for histone acetylation, are reduced in oligodendroglioma. The short chain triglyceride glyceryl triacetate (GTA), which increases histone acetylation and inhibits histone deacetylase expression, has been safely used for acetate supplementation in Canavan disease, a leukodystrophy due to ASPA mutation. We demonstrate that GTA induces cytostatic G0 growth arrest of oligodendroglioma-derived cells in vitro, without affecting normal cells. Sodium acetate, at doses comparable to that generated by complete GTA catalysis, but not glycerol also promoted growth arrest, whereas long chain triglycerides promoted cell growth. To begin to elucidate its mechanism of action, the effects of GTA on ASPA and acetyl-CoA synthetase protein levels and differentiation of established human oligodendroglioma cells (HOG and Hs683) and primary tumor-derived oligodendroglioma cells that exhibit some features of cancer stem cells (grade II OG33 and grade III OG35) relative to an oligodendrocyte progenitor line (Oli-Neu) were examined. The nuclear localization of ASPA and acetyl-CoA synthetase-1 in untreated cells was regulated during the cell cycle. GTA-mediated growth arrest was not associated with apoptosis or differentiation, but increased expression of acetylated proteins. Thus, GTA-mediated acetate supplementation may provide a safe, novel epigenetic therapy to reduce the growth of oligodendroglioma cells without affecting normal neural stem or oligodendrocyte progenitor cell proliferation or differentiation.

Acetate Supplementation Induces Growth Arrest of NG2/PDGFRα-Positive Oligodendroglioma-Derived Tumor-Initiating Cells

PubMed Central

Long, Patrick M.; Tighe, Scott W.; Driscoll, Heather E.; Moffett, John R.; Namboodiri, Aryan M. A.; Viapiano, Mariano S.; Lawler, Sean E.; Jaworski, Diane M.

2013-01-01

Cancer is associated with globally hypoacetylated chromatin and considerable attention has recently been focused on epigenetic therapies. N-acetyl-L-aspartate (NAA), the primary storage form of acetate in the brain, and aspartoacylase (ASPA), the enzyme responsible for NAA catalysis to generate acetate and ultimately acetyl-Coenzyme A for histone acetylation, are reduced in oligodendroglioma. The short chain triglyceride glyceryl triacetate (GTA), which increases histone acetylation and inhibits histone deacetylase expression, has been safely used for acetate supplementation in Canavan disease, a leukodystrophy due to ASPA mutation. We demonstrate that GTA induces cytostatic G0 growth arrest of oligodendroglioma-derived cells in vitro, without affecting normal cells. Sodium acetate, at doses comparable to that generated by complete GTA catalysis, but not glycerol also promoted growth arrest, whereas long chain triglycerides promoted cell growth. To begin to elucidate its mechanism of action, the effects of GTA on ASPA and acetyl-CoA synthetase protein levels and differentiation of established human oligodendroglioma cells (HOG and Hs683) and primary tumor-derived oligodendroglioma cells that exhibit some features of cancer stem cells (grade II OG33 and grade III OG35) relative to an oligodendrocyte progenitor line (Oli-Neu) were examined. The nuclear localization of ASPA and acetyl-CoA synthetase-1 in untreated cells was regulated during the cell cycle. GTA-mediated growth arrest was not associated with apoptosis or differentiation, but increased expression of acetylated proteins. Thus, GTA-mediated acetate supplementation may provide a safe, novel epigenetic therapy to reduce the growth of oligodendroglioma cells without affecting normal neural stem or oligodendrocyte progenitor cell proliferation or differentiation. PMID:24278309

Claudin-2 Promotes Breast Cancer Liver Metastasis by Facilitating Tumor Cell Interactions with Hepatocytes

PubMed Central

Tabariès, Sébastien; Dupuy, Fanny; Dong, Zhifeng; Monast, Anie; Annis, Matthew G.; Spicer, Jonathan; Ferri, Lorenzo E.; Omeroglu, Atilla; Basik, Mark; Amir, Eitan; Clemons, Mark

2012-01-01

We previously identified claudin-2 as a functional mediator of breast cancer liver metastasis. We now confirm that claudin-2 levels are elevated in liver metastases, but not in skin metastases, compared to levels in their matched primary tumors in patients with breast cancer. Moreover, claudin-2 is specifically expressed in liver-metastatic breast cancer cells compared to populations derived from bone or lung metastases. The increased liver tropism exhibited by claudin-2-expressing breast cancer cells requires claudin-2-mediated interactions between breast cancer cells and primary hepatocytes. Furthermore, the reduction of the claudin-2 expression level, either in cancer cells or in primary hepatocytes, diminishes these heterotypic cell-cell interactions. Finally, we demonstrate that the first claudin-2 extracellular loop is essential for mediating tumor cell-hepatocyte interactions and the ability of breast cancer cells to form liver metastases in vivo. Thus, during breast cancer liver metastasis, claudin-2 shifts from acting within tight-junctional complexes to functioning as an adhesion molecule between breast cancer cells and hepatocytes. PMID:22645303

Exosomes derived from pancreatic cancer cells induce activation and profibrogenic activities in pancreatic stellate cells.

PubMed

Masamune, Atsushi; Yoshida, Naoki; Hamada, Shin; Takikawa, Tetsuya; Nabeshima, Tatsuhide; Shimosegawa, Tooru

2018-01-01

Pancreatic cancer cells (PCCs) interact with pancreatic stellate cells (PSCs), which play a pivotal role in pancreatic fibrogenesis, to develop the cancer-conditioned tumor microenvironment. Exosomes are membrane-enclosed nanovesicles, and have been increasingly recognized as important mediators of cell-to-cell communications. The aim of this study was to clarify the effects of PCC-derived exosomes on cell functions in PSCs. Exosomes were isolated from the conditioned medium of Panc-1 and SUIT-2 PCCs. Human primary PSCs were treated with PCC-derived exosomes. PCC-derived exosomes stimulated the proliferation, migration, activation of ERK and Akt, the mRNA expression of α-smooth muscle actin (ACTA2) and fibrosis-related genes, and procollagen type I C-peptide production in PSCs. Ingenuity pathway analysis of the microarray data identified transforming growth factor β1 and tumor necrosis factor as top upstream regulators. PCCs increased the expression of miR-1246 and miR-1290, abundantly contained in PCC-derived exosomes, in PSCs. Overexpression of miR-1290 induced the expression of ACTA2 and fibrosis-related genes in PSCs. In conclusion, PCC-derived exosomes stimulate activation and profibrogenic activities in PSCs. Exosome-mediated interactions between PSCs and PCCs might play a role in the development of the tumor microenvironment. Copyright © 2017 Elsevier Inc. All rights reserved.

Artificial Extracellular Matrices with Oversulfated Glycosaminoglycan Derivatives Promote the Differentiation of Osteoblast-Precursor Cells and Premature Osteoblasts

PubMed Central

Hempel, Ute; Preissler, Carolin; Möller, Stephanie; Becher, Jana; Rauner, Martina; Hofbauer, Lorenz C.; Dieter, Peter

2014-01-01

Sulfated glycosaminoglycans (GAG) are components of the bone marrow stem cell niche and to a minor extent of mature bone tissue with important functions in regulating stem cell lineage commitment and differentiation. We anticipated that artificial extracellular matrices (aECM) composed of collagen I and synthetically oversulfated GAG derivatives affect preferentially the differentiation of osteoblast-precursor cells and early osteoblasts. A set of gradually sulfated chondroitin sulfate and hyaluronan derivatives was used for the preparation of aECM. All these matrices were analysed with human bone marrow stromal cells to identify the most potent aECM and to determine the influence of the degree and position of sulfate groups and the kind of disaccharide units on the osteogenic differentiation. Oversulfated GAG derivatives with a sulfate group at the C-6 position of the N-acetylglycosamine revealed the most pronounced proosteogenic effect as determined by tissue nonspecific alkaline phosphatase activity and calcium deposition. A subset of the aECM was further analysed with different primary osteoblasts and cell lines reflecting different maturation stages to test whether the effect of sulfated GAG derivatives depends on the maturation status of the cells. It was shown that the proosteogenic effect of aECM was most prominent in early osteoblasts. PMID:24864267

Comparative Chondrogenesis of Human Cell Sources in 3D Scaffolds

PubMed Central

Tıg̑lı, R. Seda; Ghosh, Sourabh; Laha, Michael M.; Shevde, Nirupama K.; Daheron, Laurence; Gimble, Jeffrey; Gümüşdereliog̑lu, Menemşe; Kaplan, David L.

2009-01-01

Cartilage tissue can be engineered by starting from a diversity of cell sources, including stem-cell based and primary cell-based platforms. Selecting an appropriate cell source for the process of cartilage tissue engineering or repair is critical and challenging due to the variety of cell options available. In this study, cellular responses of isolated human chondrocytes, human embryonic stem cells and mesenchymal stem cells (MSCs) derived from three sources, human embryonic stem cells, bone marrow and adipose tissue, were assessed for chondrogenic potential in 3D culture. All cell sources were characterized by FACS analysis to compare expression of some surface markers. The cells were differentiated in two different biomaterial matrices, silk and chitosan scaffolds, in the presence and absence of bone morphogenetic protein 6 (BMP-6) along with the standard chondrogenic differentiating factors. Embryonic stem cells derived MSCs showed unique characteristics with preserved chondrogenic phenotype in both scaffolds with regard to chondrogenesis, as determined by real time RT-PCR, histological and microscopic analyses. After 4 weeks of cultivation, embryonic stem cells derived MSCs were promising for chondrogenesis, particularly in the silk scaffolds with BMP-6. The results suggest that cell source differences are important to consider with regard to chondrogenic outcomes and with the variables addressed here, the human embryonic stem cells derived MSCs were the preferred cell source. PMID:19382119

Synthetic Lethality as a Targeted Approach to Advanced Prostate Cancer

DTIC Science & Technology

2013-03-01

cell line was derived from primary human prostate epithelial cells by transformation with human papilloma virus. While not tumorigenic, they do...normal cells and tissues has no significant adverse effects. Inhibition of PKCδ in human and murine cells containing an activated Ras protein, however...initiates rapid and profound apoptosis. In this work, we are testing the hypothesis that inhibition or down-regulation of PKCδ in human and murine

Compounds with species and cell type specific toxicity identified in a 2000 compound drug screen of neural stem cells and rat mixed cortical neurons.

PubMed

Malik, Nasir; Efthymiou, Anastasia G; Mather, Karly; Chester, Nathaniel; Wang, Xiantao; Nath, Avindra; Rao, Mahendra S; Steiner, Joseph P

2014-12-01

Human primary neural tissue is a vital component for the quick and simple determination of chemical compound neurotoxicity in vitro. In particular, such tissue would be ideal for high-throughput screens that can be used to identify novel neurotoxic or neurotherapeutic compounds. We have previously established a high-throughput screening platform using human induced pluripotent stem cell (iPSC)-derived neural stem cells (NSCs) and neurons. In this study, we conducted a 2000 compound screen with human NSCs and rat cortical cells to identify compounds that are selectively toxic to each group. Approximately 100 of the tested compounds showed specific toxicity to human NSCs. A secondary screen of a small subset of compounds from the primary screen on human iPSCs, NSC-derived neurons, and fetal astrocytes validated the results from >80% of these compounds with some showing cell specific toxicity. Amongst those compounds were several cardiac glycosides, all of which were selectively toxic to the human cells. As the screen was able to reliably identify neurotoxicants, many with species and cell-type specificity, this study demonstrates the feasibility of this NSC-driven platform for higher-throughput neurotoxicity screens. Published by Elsevier B.V.

Genome-wide analysis of AR binding and comparison with transcript expression in primary human fetal prostate fibroblasts and cancer associated fibroblasts.

PubMed

Nash, Claire; Boufaied, Nadia; Mills, Ian G; Franco, Omar E; Hayward, Simon W; Thomson, Axel A

2017-05-05

The androgen receptor (AR) is a transcription factor, and key regulator of prostate development and cancer, which has discrete functions in stromal versus epithelial cells. AR expressed in mesenchyme is necessary and sufficient for prostate development while loss of stromal AR is predictive of prostate cancer progression. Many studies have characterized genome-wide binding of AR in prostate tumour cells but none have used primary mesenchyme or stroma. We applied ChIPseq to identify genomic AR binding sites in primary human fetal prostate fibroblasts and patient derived cancer associated fibroblasts, as well as the WPMY1 cell line overexpressing AR. We identified AR binding sites that were specific to fetal prostate fibroblasts (7534), cancer fibroblasts (629), WPMY1-AR (2561) as well as those common among all (783). Primary fibroblasts had a distinct AR binding profile versus prostate cancer cell lines and tissue, and showed a localisation to gene promoter binding sites 1 kb upstream of the transcriptional start site, as well as non-classical AR binding sequence motifs. We used RNAseq to define transcribed genes associated with AR binding sites and derived cistromes for embryonic and cancer fibroblasts as well as a cistrome common to both. These were compared to several in vivo ChIPseq and transcript expression datasets; which identified subsets of AR targets that were expressed in vivo and regulated by androgens. This analysis enabled us to deconvolute stromal AR targets active in stroma within tumour samples. Taken together, our data suggest that the AR shows significantly different genomic binding site locations in primary prostate fibroblasts compared to that observed in tumour cells. Validation of our AR binding site data with transcript expression in vitro and in vivo suggests that the AR target genes we have identified in primary fibroblasts may contribute to clinically significant and biologically important AR-regulated changes in prostate tissue. Copyright © 2017. Published by Elsevier B.V.

Differentiation and characterization of human pluripotent stem cell-derived brain microvascular endothelial cells.

PubMed

Stebbins, Matthew J; Wilson, Hannah K; Canfield, Scott G; Qian, Tongcheng; Palecek, Sean P; Shusta, Eric V

2016-05-15

The blood-brain barrier (BBB) is a critical component of the central nervous system (CNS) that regulates the flux of material between the blood and the brain. Because of its barrier properties, the BBB creates a bottleneck to CNS drug delivery. Human in vitro BBB models offer a potential tool to screen pharmaceutical libraries for CNS penetration as well as for BBB modulators in development and disease, yet primary and immortalized models respectively lack scalability and robust phenotypes. Recently, in vitro BBB models derived from human pluripotent stem cells (hPSCs) have helped overcome these challenges by providing a scalable and renewable source of human brain microvascular endothelial cells (BMECs). We have demonstrated that hPSC-derived BMECs exhibit robust structural and functional characteristics reminiscent of the in vivo BBB. Here, we provide a detailed description of the methods required to differentiate and functionally characterize hPSC-derived BMECs to facilitate their widespread use in downstream applications. Copyright © 2015 Elsevier Inc. All rights reserved.

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.

In vitro cytotoxicity and differential cellular sensitivity of derivatives of diamino acids. II. N1-methyl, N1-allyl, N1-(2-chloroethyl) and N1-propargyl nitrosoureas.

PubMed

Dulude, H; Salvador, R; Gallant, G

1995-01-01

The in vitro cytotoxicity and differential cellular sensitivity of a series of new N1-methyl, N1-allyl, N1-2-chloroethyl and N1-propargyl nitrosourea derivatives of diamino acids were determined in the National Cancer Institute's primary antitumor drug screen. The compounds tested showed an in vitro anticancer activity similar to commercialized nitrosoureas such as CCNU, BCNU, MeCCNU, chlorozotocin, streptozotocin and PCNU. The alkylating moiety of the nitrosoureas seems to play a role in the general selectivity of our compounds. The N1-methyl and N1-2-chloroethyl nitrosourea derivatives are more selective for central nervous system cell lines, the N1-allyl nitrosourea derivatives are more selective for lung cancer cell lines and the N1-propargyl nitrosoureas are more selective for leukemia cell lines.

The Terminator mouse is a diphtheria toxin-receptor knock-in mouse strain for rapid and efficient enrichment of desired cell lineages.

PubMed

Guo, Jian-Kan; Shi, Hongmei; Koraishy, Farrukh; Marlier, Arnaud; Ding, Zhaowei; Shan, Alan; Cantley, Lloyd G

2013-11-01

Biomedical research often requires primary cultures of specific cell types, which are challenging to obtain at high purity in a reproducible manner. Here we engineered the murine Rosa26 locus by introducing the diphtheria toxin receptor flanked by loxP sites. The resultant strain was nicknamed the Terminator mouse. This approach results in diphtheria toxin-receptor expression in all non-Cre expressing cell types, making these cells susceptible to diphtheria toxin exposure. In primary cultures of kidney cells derived from the Terminator mouse, over 99.99% of cells were dead within 72 h of diphtheria toxin treatment. After crossing the Terminator with the podocin-Cre (podocyte specific) mouse or the Ggt-Cre (proximal tubule specific) mouse, diphtheria toxin treatment killed non-Cre expressing cells but spared podocytes and proximal tubule cells, respectively, enriching the primary cultures to over 99% purity, based on both western blotting and immunostaining of marker proteins. Thus, the Terminator mouse can be a useful tool to selectively and reproducibly obtain even low-abundant cell types at high quantity and purity.

Photodynamic treatment (PDT) of endometrium primary cultures serving as an in-vitro-model for endometriosis

NASA Astrophysics Data System (ADS)

Werter, Wiebke; Viereck, Volker; Keckstein, J.; Steiner, Rudolf W.; Rueck, Angelika C.

1994-05-01

As a new treatment model for endometriosis, photodynamic therapy (PDT) was applied to endometrium cultures. Endometriosis is a benign disease. Therefore primary cultures were used instead of cell lines. Endometrium is composed of epithelial and stromal cells which can also be found in primary culture. While stromal cells take a polygonal shape in culture, epithelial cells form cell colonies. PSIII (Photasan III), which is similar to hematorporphyrin derivate (HpD), meso-tetra (4-sulfonatophenyl) porphyrin (TPPS4), which posses a high fluorescence quantum yield and may be useful in fluorescence diagnosis of subtle endometriotic spots, and methylene blue (MB), a vital dye with phototoxic properties, were used as photosensitizers. Different sensitizer concentrations and incubation times were applied. The highest phototoxicity was observed for PSIII; TPPS4 and MB were less phototoxic. We compared our results with the sensitivity of cell lines described in the literature. The necessary irradiation to destroy stromal cells was relatively high but still in the same dimension as for cell lines. However they were even more sensitive than epithelial cells. This was true for all sensitizers used.

Photodynamic therapy (PDT) of endometrium primary cultures serving as an in-vitro model for endometriosis

NASA Astrophysics Data System (ADS)

Herter, Wiebke; Viereck, Volker; Keckstein, J.; Steiner, Rudolf W.; Rueck, Angelika C.

1994-05-01

As a new treatment model for endometriosis, photodynamic therapy (PDT) was applied to endometrium cultures. Endometriosis is a benign disease. Therefore primary cultures were used instead of cell lines. Endometrium is composed of epithelial and stromal cells which can also be found in primary culture. While stromal cells take a polygonal shape in culture, epithelial cells form cell colonies. PSIII (Photasan III), which is similar to hematorporphyrin derivate (HpD), meso-tetra (4-sulfonatophenyl) porphyrin (TPPS4), which posses a high fluorescence quantum yield and may be useful in fluorescence diagnosis of subtle endometriotic spots, and methylene blue (MB), a vital dye with phototoxic properties, were used as photosensitizers. Different sensitizer concentrations and incubation times were applied. The highest phototoxicity was observed for PSIII; TPPS4 and MB were less phototoxic. We compared our results with the sensitivity of cell lines described in the literature. The necessary irradiation to destroy stromal cells was relatively high but still in the same dimension as for cell lines. However they were even more sensitive than epithelial cells. This was true for all sensitizers used.

In Vitro Analysis of Breast Cancer Cell Line Tumourspheres and Primary Human Breast Epithelia Mammospheres Demonstrates Inter- and Intrasphere Heterogeneity

PubMed Central

Vargas, Ana Cristina; Keith, Patricia; Reid, Lynne; Wockner, Leesa; Amiri, Marjan Askarian; Sarkar, Debina; Simpson, Peter T.; Clarke, Catherine; Schmidt, Chris W.; Reynolds, Brent A.

2013-01-01

Mammosphere and breast tumoursphere culture have gained popularity as in vitro assays for propagating and analysing normal and cancer stem cells. Whether the spheres derived from different sources or parent cultures themselves are indeed single entities enriched in stem/progenitor cells compared to other culture formats has not been fully determined. We surveyed sphere-forming capacity across 26 breast cell lines, immunophenotyped spheres from six luminal- and basal-like lines by immunohistochemistry and flow cytometry and compared clonogenicity between sphere, adherent and matrigel culture formats using in vitro functional assays. Analyses revealed morphological and molecular intra- and inter-sphere heterogeneity, consistent with adherent parental cell line phenotypes. Flow cytometry showed sphere culture does not universally enrich for markers previously associated with stem cell phenotypes, although we found some cell-line specific changes between sphere and adherent formats. Sphere-forming efficiency was significantly lower than adherent or matrigel clonogenicity and constant over serial passage. Surprisingly, self-renewal capacity of sphere-derived cells was similar/lower than other culture formats. We observed significant correlation between long-term-proliferating-cell symmetric division rates in sphere and adherent cultures, suggesting functional overlap between the compartments sustaining them. Experiments with normal primary human mammary epithelia, including sorted luminal (MUC1+) and basal/myoepithelial (CD10+) cells revealed distinct luminal-like, basal-like and mesenchymal entities amongst primary mammospheres. Morphological and colony-forming-cell assay data suggested mammosphere culture may enrich for a luminal progenitor phenotype, or induce reversion/relaxation of the basal/mesenchymal in vitro selection occurring with adherent culture. Overall, cell line tumourspheres and primary mammospheres are not homogenous entities enriched for stem cells, suggesting a more cautious approach to interpreting data from these assays and careful consideration of its limitations. Sphere culture may represent an alternative 3-dimensional culture system which rather than universally ‘enriching’ for stem cells, has utility as one of a suite of functional assays that provide a read-out of progenitor activity. PMID:23750209

Direct cytotoxicity evaluation of 63S bioactive glass and bone-derived hydroxyapatite particles using yeast model and human chondrocyte cells by microcalorimetry.

PubMed

Doostmohammadi, A; Monshi, A; Fathi, M H; Karbasi, S; Braissant, O; Daniels, A U

2011-10-01

In this study, the cytotoxicity evaluation of prepared 63S bioactive glass and bone-derived hydroxyapatite particles with yeast and human chondrocyte cells was carried out using isothermal micro-nano calorimetry (IMNC), which is a new method for studying cell/biomaterial interactions. Bioactive glass particles were made via sol-gel method and hydroxyapatite was obtained from bovine bone. Elemental analysis was carried out by XRF and EDXRF. Amorphous structure of the glass and completely crystalline structure of HA were detected by XRD analysis. Finally, the cytotoxicity of bioactive glass and bone-derived HA particles with yeast and cultured human chondrocyte cells was evaluated using IMNC. The results confirmed the viability, growth and proliferation of human chondrocyte cells in contact with 63S bioactive glass, and bone-derived HA particles. Also the results indicated that yeast model which is much easier to handle, can be considered as a good proxy and can provide a rapid primary estimate of the ranges to be used in assays involving human cells. All of these results confirmed that IMNC is a convenient method which caters to measuring the cell-biomaterial interactions alongside the current methods.

Establishment and cryopreservation of a giant panda skeletal muscle-derived cell line.

PubMed

Yu, Fang-Jian; Zeng, Chang-Jun; Zhang, Yan; Wang, Cheng-Dong; Xiong, Tie-Yi; Fang, Sheng-Guo; Zhang, He-Min

2015-06-01

The giant panda Ailuropoda melanoleuca is an endangered species and is a symbol for wildlife conservation. Although efforts have been made to protect this rare and endangered species through breeding and conservative biology, the long-term preservation of giant panda genome resources (gametes, tissues, organs, genomic libraries, etc.) is still a practical option. In this study, the giant panda skeletal muscle-derived cell line was successfully established via primary explants culture and cryopreservation techniques. The population doubling time of giant panda skeletal cells was approximately 33.8 h, and this population maintained a high cell viability before and after cryopreservation (95.6% and 90.7%, respectively). The two skeletal muscle-specific genes SMYD1 and MYF6 were expressed and detected by RT-PCR in the giant panda skeletal muscle-derived cell line. Karyotyping analysis revealed that the frequencies of giant panda skeletal muscle cells showing a chromosome number of 2n=42 ranged from 90.6∼94.2%. Thus, the giant panda skeletal muscle-derived cell line provides a vital resource and material platform for further studies and is likely to be useful for the protection of this rare and endangered species.

Three-dimensional wet-electrospun poly(lactic acid)/multi-wall carbon nanotubes scaffold induces differentiation of human menstrual blood-derived stem cells into germ-like cells.

PubMed

Eyni, Hossein; Ghorbani, Sadegh; Shirazi, Reza; Salari Asl, Leila; P Beiranvand, Shahram; Soleimani, Masoud

2017-09-01

Infertility caused by the disruption or absence of germ cells is a major and largely incurable medical problem. Germ cells (i.e., sperm or egg) play a key role in the transmission of genetic and epigenetic information across generations. Generation of gametes derived in vitro from stem cells hold promising prospects which could potentially help infertile men and women. Menstrual blood-derived stem cells are a unique stem cell source. Evidence suggests that menstrual blood-derived stem cells exhibit a multi-lineage potential and have attracted extensive attention in regenerative medicine. To maintain the three-dimensional structure of natural extra cellular matrices in vitro, scaffolds can do this favor and mimic a microenvironment for cell proliferation and differentiation. According to previous studies, poly(lactic acid) and multi-wall carbon nanotubes have been introduced as novel and promising biomaterials for the proliferation and differentiation of stem cells. Some cell types have been successfully grown on a matrix containing carbon nanotubes in tissue engineering but there is no report for this material to support stem cells differentiation into germ cells lineage. This study designed a 3D wet-electrospun poly(lactic acid) and poly(lactic acid)/multi-wall carbon nanotubes composite scaffold to compare infiltration, proliferation, and differentiation potential of menstrual blood-derived stem cells toward germ cell lineage with 2D culture. Our primary data revealed that the fabricated scaffold has mechanical and biological suitable qualities for supporting and attachments of stem cells. The differentiated menstrual blood-derived stem cells tracking in scaffolds using scanning electron microscopy confirmed cell attachment, aggregation, and distribution on the porous scaffold. Based on the differentiation assay by RT-PCR analysis, stem cells and germ-like cells markers were expressed in 3D groups as well as 2D one. It seems that poly(lactic acid)/multi-wall carbon nanotubes scaffold-seeded menstrual blood-derived stem cells could be viewed as a novel, safe, and accessible construct for these cells, as they enhance germ-like generation from menstrual blood-derived stem cells.

Generation of B-cell chronic lymphocytic leukemia (B-CLL)-reactive T-cell lines and clones from HLA class I-matched donors using modified B-CLL cells as stimulators: implications for adoptive immunotherapy.

PubMed

Hoogendoorn, M; Wolbers, J Olde; Smit, W M; Schaafsma, M R; Barge, R M Y; Willemze, R; Falkenburg, J H F

2004-07-01

Allogeneic stem cell transplantation following reduced-intensity conditioning is being evaluated in patients with advanced B-cell chronic lymphocytic leukemia (B-CLL). The curative potential of this procedure is mediated by donor-derived alloreactive T cells, resulting in a graft-versus-leukemia effect. However, B-CLL may escape T-cell-mediated immune reactivity since these cells lack expression of costimulatory molecules. We examined the most optimal method to transform B-CLL cells into efficient antigen-presenting cells (APC) using activating cytokines, by triggering toll-like receptors (TLRs) using microbial pathogens and by CD40 stimulation with CD40L-transfected fibroblasts. CD40 activation in the presence of IL-4 induced strongest upregulation of costimulatory and adhesion molecules on B-CLL cells and induced the production of high amounts of IL-12 by the leukemic cells. In contrast to primary B-CLL cells as stimulator cells, these malignant APCs were capable of inducing the generation of B-CLL-reactive CD8(+) CTL lines and clones from HLA class I-matched donors. These CTL lines and clones recognized and killed primary B-CLL as well as patient-derived lymphoblasts, but not donor cells. These results show the feasibility of ex vivo generation of B-CLL-reactive CD8(+) CTLs. This opens new perspectives for adoptive immunotherapy, following allogeneic stem cell transplantation in patients with advanced B-CLL.

Mineralization and Expression of Col1a1-3.6GFP Transgene in Primary Dental Pulp Culture

PubMed Central

Balic, Anamaria; Rodgers, Barbara; Mina, Mina

2008-01-01

We have examined and compared the effects of various differentiation-inducing media on mineralization, cell morphology and expression of pOBCol3.6GFP (3.6-GFP) in primary dental pulp cultures derived from 3.6-GFP transgenic mice. Our results show that media containing ascorbic acid only could not induce mineralization in primary dental pulp cultures. On the other hand, media containing ascorbic acid and β-glycerophosphate induced formation of mineralized matrix-containing dentin. The amount of mineralized matrix was increased by addition of dexamethasone. Cells treated with ascorbic acid and β-glycerophosphate were fibroblast like and cells treated with dexamethasone were cuboidal. In all culture conditions, high levels of 3.6-GFP were expressed in areas of mineralization PMID:18781059

Differentiation of Inflammation-Responsive Astrocytes from Glial Progenitors Generated from Human Induced Pluripotent Stem Cells.

PubMed

Santos, Renata; Vadodaria, Krishna C; Jaeger, Baptiste N; Mei, Arianna; Lefcochilos-Fogelquist, Sabrina; Mendes, Ana P D; Erikson, Galina; Shokhirev, Maxim; Randolph-Moore, Lynne; Fredlender, Callie; Dave, Sonia; Oefner, Ruth; Fitzpatrick, Conor; Pena, Monique; Barron, Jerika J; Ku, Manching; Denli, Ahmet M; Kerman, Bilal E; Charnay, Patrick; Kelsoe, John R; Marchetto, Maria C; Gage, Fred H

2017-06-06

Astrocyte dysfunction and neuroinflammation are detrimental features in multiple pathologies of the CNS. Therefore, the development of methods that produce functional human astrocytes represents an advance in the study of neurological diseases. Here we report an efficient method for inflammation-responsive astrocyte generation from induced pluripotent stem cells (iPSCs) and embryonic stem cells. This protocol uses an intermediate glial progenitor stage and generates functional astrocytes that show levels of glutamate uptake and calcium activation comparable with those observed in human primary astrocytes. Stimulation of stem cell-derived astrocytes with interleukin-1β or tumor necrosis factor α elicits a strong and rapid pro-inflammatory response. RNA-sequencing transcriptome profiling confirmed that similar gene expression changes occurred in iPSC-derived and primary astrocytes upon stimulation with interleukin-1β. This protocol represents an important tool for modeling in-a-dish neurological diseases with an inflammatory component, allowing for the investigation of the role of diseased astrocytes in neuronal degeneration. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Development, Characterization, and Pluripotency Analysis of Buffalo (Bubalus bubalis) Embryonic Stem Cell Lines Derived from In Vitro–Fertilized, Hand-Guided Cloned, and Parthenogenetic Embryos

PubMed Central

Shah, Syed Mohmad; Saini, Neha; Ashraf, Syma; Zandi, Mohammad; Manik, Radhey Sham; Singla, Suresh Kumar; Palta, Prabhat

2015-01-01

Abstract We present the derivation, characterization, and pluripotency analysis of three buffalo embryonic stem cell (buESC) lines, from in vitro–fertilized, somatic cell nuclear–transferred, and parthenogenetic blastocysts. These cell lines were developed for later differentiation into germ lineage cells and elucidation of the signaling pathways involved. The cell lines were established from inner cell masses (ICMs) that were isolated manually from the in vitro–produced blastocysts. Most of the ICMs (45–55%) resulted in formation of primary colonies that were subcultured after 8–10 days, leading subsequently to the formation of three buESC lines, one from each blastocyst type. All the cell lines expressed stem cell markers, such as Alkaline Phosphatase, OCT4, NANOG, SSEA1, SSEA4, TRA-1-60, TRA-1-81, SOX2, REX1, CD-90, STAT3, and TELOMERASE. They differentiated into all three germ layers as determined by ectodermal, mesodermal, and endodermal RNA and protein markers. All of the cell lines showed equal expression of pluripotency markers as well as equivalent differentiation potential into all the three germ layers. The static suspension culture–derived embryoid bodies (EBs) showed greater expression of all the three germ layer markers as compared to hanging drop culture–derived EBs. When analyzed for germ layer marker expression, EBs derived from 15% fetal bovine serum (FBS)-based spontaneous differentiation medium showed greater differentiation across all the three germ layers as compared to those derived from Knock-Out Serum Replacement (KoSR)-based differentiation medium. PMID:26168169

Self-targeting of TNF-releasing cancer cells in preclinical models of primary and metastatic tumors.

PubMed

Dondossola, Eleonora; Dobroff, Andrey S; Marchiò, Serena; Cardó-Vila, Marina; Hosoya, Hitomi; Libutti, Steven K; Corti, Angelo; Sidman, Richard L; Arap, Wadih; Pasqualini, Renata

2016-02-23

Circulating cancer cells can putatively colonize distant organs to form metastases or to reinfiltrate primary tumors themselves through a process termed "tumor self-seeding." Here we exploit this biological attribute to deliver tumor necrosis factor alpha (TNF), a potent antitumor cytokine, directly to primary and metastatic tumors in a mechanism that we have defined as "tumor self-targeting." For this purpose, we genetically engineered mouse mammary adenocarcinoma (TSA), melanoma (B16-F10), and Lewis lung carcinoma cells to produce and release murine TNF. In a series of intervention trials, systemic administration of TNF-expressing tumor cells was associated with reduced growth of both primary tumors and metastatic colonies in immunocompetent mice. We show that these malignant cells home to tumors, locally release TNF, damage neovascular endothelium, and induce massive cancer cell apoptosis. We also demonstrate that such tumor-cell-mediated delivery avoids or minimizes common side effects often associated with TNF-based therapy, such as acute inflammation and weight loss. Our study provides proof of concept that genetically modified circulating tumor cells may serve as targeted vectors to deliver anticancer agents. In a clinical context, this unique paradigm represents a personalized approach to be translated into applications potentially using patient-derived circulating tumor cells as self-targeted vectors for drug delivery.

Pluripotent stem cell-derived organoids: using principles of developmental biology to grow human tissues in a dish.

PubMed

McCauley, Heather A; Wells, James M

2017-03-15

Pluripotent stem cell (PSC)-derived organoids are miniature, three-dimensional human tissues generated by the application of developmental biological principles to PSCs in vitro The approach to generate organoids uses a combination of directed differentiation, morphogenetic processes, and the intrinsically driven self-assembly of cells that mimics organogenesis in the developing embryo. The resulting organoids have remarkable cell type complexity, architecture and function similar to their in vivo counterparts. In the past five years, human PSC-derived organoids with components of all three germ layers have been generated, resulting in the establishment of a new human model system. Here, and in the accompanying poster, we provide an overview of how principles of developmental biology have been essential for generating human organoids in vitro , and how organoids are now being used as a primary research tool to investigate human developmental biology. © 2017. Published by The Company of Biologists Ltd.

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

Differentiation-Dependent Energy Production and Metabolite Utilization: A Comparative Study on Neural Stem Cells, Neurons, and Astrocytes.

PubMed

Jády, Attila Gy; Nagy, Ádám M; Kőhidi, Tímea; Ferenczi, Szilamér; Tretter, László; Madarász, Emília

2016-07-01

While it is evident that the metabolic machinery of stem cells should be fairly different from that of differentiated neurons, the basic energy production pathways in neural stem cells (NSCs) or in neurons are far from clear. Using the model of in vitro neuron production by NE-4C NSCs, this study focused on the metabolic changes taking place during the in vitro neuronal differentiation. O2 consumption, H(+) production, and metabolic responses to single metabolites were measured in cultures of NSCs and in their neuronal derivatives, as well as in primary neuronal and astroglial cultures. In metabolite-free solutions, NSCs consumed little O2 and displayed a higher level of mitochondrial proton leak than neurons. In stem cells, glycolysis was the main source of energy for the survival of a 2.5-h period of metabolite deprivation. In contrast, stem cell-derived or primary neurons sustained a high-level oxidative phosphorylation during metabolite deprivation, indicating the consumption of own cellular material for energy production. The stem cells increased O2 consumption and mitochondrial ATP production in response to single metabolites (with the exception of glucose), showing rapid adaptation of the metabolic machinery to the available resources. In contrast, single metabolites did not increase the O2 consumption of neurons or astrocytes. In "starving" neurons, neither lactate nor pyruvate was utilized for mitochondrial ATP production. Gene expression studies also suggested that aerobic glycolysis and rapid metabolic adaptation characterize the NE-4C NSCs, while autophagy and alternative glucose utilization play important roles in the metabolism of stem cell-derived neurons.

Establishment and genetic characterization of an immortal tumor cell line derived from intestinal-type sinonasal adenocarcinoma.

PubMed

Pérez-Escuredo, Jhudit; García Martínez, Jorge; García-Inclán, Cristina; Vivanco, Blanca; Costales, María; Álvarez Marcos, César; Llorente, José Luis; Hermsen, Mario A

2011-02-01

Intestinal-type sinonasal adenocarcinoma (ITAC) is a rare tumor etiologically related to professional exposure to wood dust. The overall prognosis is poor, mainly due to the difficulty to resect the tumor completely in this anatomically complex region. Therefore, there is great need for alternative treatments. However, the lack of a good tumor model system for ITAC has hampered the development and testing of new therapeutic agents. Here, we report the establishment and characterization of the first human ITAC cell line named ITAC-3. The cell line was initiated from small explants of a T4bN0M0 colonic type ITAC from the ethmoid sinus. Growth and invasion parameters as well as genetic characteristics were analyzed. The population doubling time was 18 h and the cell line was capable of invasion in matrigel. Chromosomal analysis showed a tetraploid karyotype with both numerical and structural aberrations. High resolution microarray CGH analysis identified many copy number alterations, including homozygous deletions. TP53 carried a mutation c.818G>T in exon eight concurring with a strong nuclear protein overexpression. Immunohistochemical analysis showed protein overexpression of EGFR and normal expression of β-catenin and p16. This is the first report of the establishment of a cell line derived from a primary ITAC. The genomic profile of the cell line was the same as the primary tumor from which it was derived. This new cell line will be a useful tool for the development and testing of new therapeutic agents for this tumor type.

ETS-1 Expression Is Hypoxia-independent in Glioblastoma-derived Endothelial and Mesenchymal Stem-like Cells.

PubMed

Koessinger, Dominik; Albrecht, Valerie; Faber, Florian; Jaehnert, Irene; Schichor, Christian

2018-06-01

Tumor cells infiltrating the brain are a typical hallmark of glioblastoma. Invasiveness of glioma cells has been associated with ETS proto-oncogene 1 (ETS-1). In non-glial tumors, ETS-1 expression has been linked to hypoxia. However, it is not known whether hypoxia regulates ETS-1 expression in glioblastoma. The spatial distribution of ETS-1 expression in primary glioblastoma was assessed using immunohistochemistry. ETS-1 expression in glioblastoma-derived mesenchymal stem-like cells (gbMSLCs) was determined using immunocytochemistry. The effect of hypoxia on ETS-1 expression of gbMSLCs, glioma cell lines and glioblastoma-derived endothelial cells was assessed using polymerase chain reaction and immunoblotting. Our immunohistochemical studies revealed ETS-1 expression in stromal and endothelial glioblastoma cells. Stromal ETS-1 expression in glioblastoma correlated with microvessel density. gbMSLCs were found to express ETS-1. In all examined cell lines, ETS-1 transcription and expression were independent of hypoxia. In glioblastoma, ETS-1-expression is not dependent on hypoxia, but correlates with tumor vascularization. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Pseudoislets as primary islet replacements for research: report on a symposium at King's College London, London UK.

PubMed

Persaud, Shanta J; Arden, Catherine; Bergsten, Peter; Bone, Adrian J; Brown, James; Dunmore, Simon; Harrison, Moira; Hauge-Evans, Astrid; Kelly, Catriona; King, Aileen; Maffucci, Tania; Marriott, Claire E; McClenaghan, Neville; Morgan, Noel G; Reers, Christina; Russell, Mark A; Turner, Mark D; Willoughby, Emma; Younis, Mustafa Y G; Zhi, Z L; Jones, Peter M

2010-01-01

Laboratory-based research aimed at understanding processes regulating insulin secretion and mechanisms underlying β-cell dysfunction and loss in diabetes often makes use of rodents, as these processes are in many respects similar between rats/mice and humans. Indeed, a rough calculation suggests that islets have been isolated from as many as 150,000 rodents to generate the data contained within papers published in 2009 and the first four months of 2010. Rodent use for islet isolation has been mitigated, to a certain extent, by the availability of a variety of insulin-secreting cell lines that are used by researchers world-wide. However, when maintained as monolayers the cell lines do not replicate the robust, sustained secretory responses of primary islets which limits their usefulness as islet surrogates. On the other hand, there have been several reports that configuration of MIN6 β-cells, derived from a mouse insulinoma, as three-dimensional cell clusters termed ‘pseudoislets’ largely recapitulates the function of primary islet β-cells. The Diabetes Research Group at King’s College London has been using the MIN6 pseudoislet model for over a decade and they hosted a symposium on “Pseudoislets as primary islet replacements for research”, which was funded by the UK National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs), in London on 15th and 16th April 2010. This small, focused meeting was conceived as an opportunity to consolidate information on experiences of working with pseudoislets between different UK labs, and to introduce the theory and practice of pseudoislet culture to laboratories working with islets and/or β-cell lines but who do not currently use pseudoislets. This short review summarizes the background to the development of the cell line-derived pseudoislet model, the key messages arising from the symposium and emerging themes for future pseudoislet research.

Acute myeloid leukemia originates from a hierarchy of leukemic stem cell classes that differ in self-renewal capacity.

PubMed

Hope, Kristin J; Jin, Liqing; Dick, John E

2004-07-01

Emerging evidence suggests cancer stem cells sustain neoplasms; however, little is understood of the normal cell initially targeted and the resultant cancer stem cells. We show here, by tracking individual human leukemia stem cells (LSCs) in nonobese diabetic-severe combined immunodeficiency mice serially transplanted with acute myeloid leukemia cells, that LSCs are not functionally homogeneous but, like the normal hematopoietic stem cell (HSC) compartment, comprise distinct hierarchically arranged LSC classes. Distinct LSC fates derived from heterogeneous self-renewal potential. Some LSCs emerged only in recipients of serial transplantation, indicating they divided rarely and underwent self-renewal rather than commitment after cell division within primary recipients. Heterogeneity in LSC self-renewal potential supports the hypothesis that they derive from normal HSCs. Furthermore, normal developmental processes are not completely abolished during leukemogenesis. The existence of multiple stem cell classes shows the need for LSC-targeted therapies.

Innovative approaches to establish and characterize primary cultures: an ex vivo 3D system and the zebrafish model.

PubMed

Liverani, Chiara; La Manna, Federico; Groenewoud, Arwin; Mercatali, Laura; Van Der Pluijm, Gabri; Pieri, Federica; Cavaliere, Davide; De Vita, Alessandro; Spadazzi, Chiara; Miserocchi, Giacomo; Bongiovanni, Alberto; Recine, Federica; Riva, Nada; Amadori, Dino; Tasciotti, Ennio; Snaar-Jagalska, Ewa; Ibrahim, Toni

2017-02-15

Patient-derived specimens are an invaluable resource to investigate tumor biology. However, in vivo studies on primary cultures are often limited by the small amount of material available, while conventional in vitro systems might alter the features and behavior that characterize cancer cells. We present our data obtained on primary dedifferentiated liposarcoma cells cultured in a 3D scaffold-based system and injected into a zebrafish model. Primary cells were characterized in vitro for their morphological features, sensitivity to drugs and biomarker expression, and in vivo for their engraftment and invasiveness abilities. The 3D culture showed a higher enrichment in cancer cells than the standard monolayer culture and a better preservation of liposarcoma-associated markers. We also successfully grafted primary cells into zebrafish, showing their local migratory and invasive abilities. Our work provides proof of concept of the ability of 3D cultures to maintain the original phenotype of ex vivo cells, and highlights the potential of the zebrafish model to provide a versatile in vivo system for studies with limited biological material. Such models could be used in translational research studies for biomolecular analyses, drug screenings and tumor aggressiveness assays. © 2016. Published by The Company of Biologists Ltd.

CAR-Engineered NK Cells Targeting Wild-Type EGFR and EGFRvIII Enhance Killing of Glioblastoma and Patient-Derived Glioblastoma Stem Cells.

PubMed

Han, Jianfeng; Chu, Jianhong; Keung Chan, Wing; Zhang, Jianying; Wang, Youwei; Cohen, Justus B; Victor, Aaron; Meisen, Walter H; Kim, Sung-hak; Grandi, Paola; Wang, Qi-En; He, Xiaoming; Nakano, Ichiro; Chiocca, E Antonio; Glorioso, Joseph C; Kaur, Balveen; Caligiuri, Michael A; Yu, Jianhua

2015-07-09

Glioblastoma (GB) remains the most aggressive primary brain malignancy. Adoptive transfer of chimeric antigen receptor (CAR)-modified immune cells has emerged as a promising anti-cancer approach, yet the potential utility of CAR-engineered natural killer (NK) cells to treat GB has not been explored. Tumors from approximately 50% of GB patients express wild-type EGFR (wtEGFR) and in fewer cases express both wtEGFR and the mutant form EGFRvIII; however, previously reported CAR T cell studies only focus on targeting EGFRvIII. Here we explore whether both wtEGFR and EGFRvIII can be effectively targeted by CAR-redirected NK cells to treat GB. We transduced human NK cell lines NK-92 and NKL, and primary NK cells with a lentiviral construct harboring a second generation CAR targeting both wtEGFR and EGFRvIII and evaluated the anti-GB efficacy of EGFR-CAR-modified NK cells. EGFR-CAR-engineered NK cells displayed enhanced cytolytic capability and IFN-γ production when co-cultured with GB cells or patient-derived GB stem cells in an EGFR-dependent manner. In two orthotopic GB xenograft mouse models, intracranial administration of NK-92-EGFR-CAR cells resulted in efficient suppression of tumor growth and significantly prolonged the tumor-bearing mice survival. These findings support intracranial administration of NK-92-EGFR-CAR cells represents a promising clinical strategy to treat GB.

Comparative photodynamic therapy cytotoxicity of mannose-conjugated chlorin and talaporfin sodium in cultured human and rat cells.

PubMed

Shinoda, Yo; Takahashi, Tsutomu; Akimoto, Jiro; Ichikawa, Megumi; Yamazaki, Hiromi; Narumi, Atsushi; Yano, Shigenobu; Fujiwara, Yasuyuki

2017-01-01

Photodynamic therapy (PDT) is a Food and Drug Administration authorized method for cancer treatment, which uses photosensitizer and laser photo-irradiation to generate reactive oxygen species to induce cell death in tumors. Photosensitizers have been progressively developed, from first to third generation, with improvements in cell specificity, reduced side effects and toxicity, increased sensitivity for irradiation and reduced persistence of photosensitizer in healthy cells. These improvements have been achieved by basic comparative experiments between current and novel photosensitizers using cell lines; however, photosensitizers should be carefully evaluated because they may have cell type specificity. In the present study, we compared a third-generation photosensitizer, β-mannose-conjugated chlorin (β-M-chlorin), with the second generation, talaporfin sodium (NPe6), using seven different rat and human cell lines and a neuronal/glial primary culture prepared from rat embryos. NPe6 was more effective than β-M-chlorin in human-derived cell lines, and β-M-chlorin was more effective than NPe6 in rat primary cultures and rat-derived cell lines, except for the rat pheochromocytoma cell line, PC12. These differences of phototoxicity in different cell types are not because of differences in photosensitivity between the photosensitizers, but rather are associated with different distribution and accumulation rates in the different cell types. These data suggest that evaluation of photosensitizers for PDT should be carried out using as large a variety of cell types as possible because each photosensitizer may have cell type specificity.

Generation of Mesenchymal-Like Stem Cells From Urine in Pediatric Patients.

PubMed

He, W; Zhu, W; Cao, Q; Shen, Y; Zhou, Q; Yu, P; Liu, X; Ma, J; Li, Y; Hong, K

2016-01-01

Mesenchymal stem cells (MSCs) have been widely used for regenerative medicine. Traditionally, the procedures of MSC isolation are usually invasive and time-consuming. Urine is merely a body waste, and recent studies have suggested that urine represents an alternative source of stem cells. We, therefore, determined whether the possibility of isolating mesenchymal-like stem cells was practical from human urine. A total of 16 urine samples were collected from pediatric patients. Urine-derived cells were isolated, expanded, and identified for specific cell surface markers using flow cytometry. Cell morphology was observed by microscopy. Osteogenic and adipogenic differentiation potential were determinded by culturing cells in specific induction medium, and assessed by alkaline phosphatase and oil red O stainings, respectively. Clones were established and passaged successfully from primary cultures of urine cells. Cultured urine-derived cells at passage 3 were fusiform and arranged with certain directionality. Urine-derived cells at passage 5 displayed expressions of cell surface markers (CD29, CD105, CD166, CD90, and CD13). There was no expression of the general hematopoietic cell markers (CD45, CD34, and HLA-DR). Under in vitro induction conditions, urine-derived cells at passage 5 were able to differentiate into osteoblasts, but not adipocytes. Urine may be a noninvasive source for mesenchymal-like stem cells. These cells could potentially provide a new source of autologous stem cells for regenerative medicine and cell therapy. Copyright © 2016 Elsevier Inc. All rights reserved.

Selective impact of CDK4/6 suppression on patient-derived models of pancreatic cancer.

PubMed

Witkiewicz, Agnieszka K; Borja, Nicholas A; Franco, Jorge; Brody, Jonathan R; Yeo, Charles J; Mansour, John; Choti, Michael A; McCue, Peter; Knudsen, Erik S

2015-06-30

Pancreatic ductal adenocarcinoma (PDA) harbors an exceedingly poor prognosis, and is generally considered a therapy-recalcitrant disease due to poor response to conventional chemotherapy coupled with non-actionable genetic drivers (e.g. KRAS mutations). However, PDA frequently loses p16ink4a, thereby leading to deregulation of CDK4/6. Surprisingly, in established cell models and xenografts, CDK4/6 inhibition has a modest effect on proliferation and resistance develops rapidly. To determine if such weak response was an intrinsic feature of PDA, we developed primary tumor explants that maintain the tumor environment and recapitulate feuture of primary PDA. The CDK4/6 inhibitor PD-0332991 was highly efficient at suppressing proliferation in 14 of the 15 explants. In the single resistant explant, we identified the rare loss of the RB tumor suppressor as the basis for resistance. Patient-derived xenografts (PDXs) were developed in parallel, and unlike the xenografts emerging from established cell lines, the PDXs maintained the histoarchitecture of the primary tumor. These PDXs were highly sensitive to CDK4/6 inhibition, yielding a complete suppression of PDA proliferation. Together, these data indicate that primary PDA is sensitive to CDK4/6 inhibition, that specific biomarkers can delineate intrinsic resistance, and that established cell line models may not represent an adequate means for evaluating therapeutic sensitivities.

Drug Transporter Protein Quantification of Immortalized Human Lung Cell Lines Derived from Tracheobronchial Epithelial Cells (Calu-3 and BEAS2-B), Bronchiolar-Alveolar Cells (NCI-H292 and NCI-H441), and Alveolar Type II-like Cells (A549) by Liquid Chromatography-Tandem Mass Spectrometry.

PubMed

Sakamoto, Atsushi; Matsumaru, Takehisa; Yamamura, Norio; Suzuki, Shinobu; Uchida, Yasuo; Tachikawa, Masanori; Terasaki, Tetsuya

2015-09-01

Understanding the mechanisms of drug transport in the human lung is an important issue in pulmonary drug discovery and development. For this purpose, there is an increasing interest in immortalized lung cell lines as alternatives to primary cultured lung cells. We recently reported the protein expression in human lung tissues and pulmonary epithelial cells in primary culture, (Sakamoto A, Matsumaru T, Yamamura N, Uchida Y, Tachikawa M, Ohtsuki S, Terasaki T. 2013. J Pharm Sci 102(9):3395-3406) whereas comprehensive quantification of protein expressions in immortalized lung cell lines is sparse. Therefore, the aim of the present study was to clarify the drug transporter protein expression of five commercially available immortalized lung cell lines derived from tracheobronchial cells (Calu-3 and BEAS2-B), bronchiolar-alveolar cells (NCI-H292 and NCI-H441), and alveolar type II cells (A549), by liquid chromatography-tandem mass spectrometry-based approaches. Among transporters detected, breast cancer-resistance protein in Calu-3, NCI-H292, NCI-H441, and A549 and OCTN2 in BEAS2-B showed the highest protein expression. Compared with data from our previous study,(Sakamoto A, Matsumaru T, Yamamura N, Uchida Y, Tachikawa M, Ohtsuki S, Terasaki T. 2013. J Pharm Sci 102(9):3395-3406) NCI-H441 was the most similar with primary lung cells from all regions in terms of protein expression of organic cation/carnitine transporter 1 (OCTN1). In conclusion, the protein expression profiles of transporters in five immortalized lung cell lines were determined, and these findings may contribute to a better understanding of drug transport in immortalized lung cell lines. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

Nuclear DNA Methylation and Chromatin Condensation Phenotypes Are Distinct Between Normally Proliferating/Aging, Rapidly Growing/Immortal, and Senescent Cells

PubMed Central

Gertych, Arkadiusz; Tajbakhsh, Jian

2013-01-01

This study reports on probing the utility of in situ chromatin texture features such as nuclear DNA methylation and chromatin condensation patterns — visualized by fluorescent staining and evaluated by dedicated three-dimensional (3D) quantitative and high-throughput cell-by-cell image analysis — in assessing the proliferative capacity, i.e. growth behavior of cells: to provide a more dynamic picture of a cell population with potential implications in basic science, cancer diagnostics/prognostics and therapeutic drug development. Two types of primary cells and four different cancer cell lines were propagated and subjected to cell-counting, flow cytometry, confocal imaging, and 3D image analysis at various points in culture. Additionally a subset of primary and cancer cells was accelerated into senescence by oxidative stress. DNA methylation and chromatin condensation levels decreased with declining doubling times when primary cells aged in culture with the lowest levels reached at the stage of proliferative senescence. In comparison, immortal cancer cells with constant but higher doubling times mostly displayed lower and constant levels of the two in situ-derived features. However, stress-induced senescent primary and cancer cells showed similar levels of these features compared with primary cells that had reached natural growth arrest. With regards to global DNA methylation and chromatin condensation levels, aggressively growing cancer cells seem to take an intermediate level between normally proliferating and senescent cells. Thus, normal cells apparently reach cancer-cell equivalent stages of the two parameters at some point in aging, which might challenge phenotypic distinction between these two types of cells. Companion high-resolution molecular profiling could provide information on possible underlying differences that would explain benign versus malign cell growth behaviors. PMID:23562889

Nuclear DNA methylation and chromatin condensation phenotypes are distinct between normally proliferating/aging, rapidly growing/immortal, and senescent cells.

PubMed

Oh, Jin Ho; Gertych, Arkadiusz; Tajbakhsh, Jian

2013-03-01

This study reports on probing the utility of in situ chromatin texture features such as nuclear DNA methylation and chromatin condensation patterns - visualized by fluorescent staining and evaluated by dedicated three-dimensional (3D) quantitative and high-throughput cell-by-cell image analysis - in assessing the proliferative capacity, i.e. growth behavior of cells: to provide a more dynamic picture of a cell population with potential implications in basic science, cancer diagnostics/prognostics and therapeutic drug development. Two types of primary cells and four different cancer cell lines were propagated and subjected to cell-counting, flow cytometry, confocal imaging, and 3D image analysis at various points in culture. Additionally a subset of primary and cancer cells was accelerated into senescence by oxidative stress. DNA methylation and chromatin condensation levels decreased with declining doubling times when primary cells aged in culture with the lowest levels reached at the stage of proliferative senescence. In comparison, immortal cancer cells with constant but higher doubling times mostly displayed lower and constant levels of the two in situ-derived features. However, stress-induced senescent primary and cancer cells showed similar levels of these features compared with primary cells that had reached natural growth arrest. With regards to global DNA methylation and chromatin condensation levels, aggressively growing cancer cells seem to take an intermediate level between normally proliferating and senescent cells. Thus, normal cells apparently reach cancer-cell equivalent stages of the two parameters at some point in aging, which might challenge phenotypic distinction between these two types of cells. Companion high-resolution molecular profiling could provide information on possible underlying differences that would explain benign versus malign cell growth behaviors.

Tumor tropism of intravenously injected human-induced pluripotent stem cell-derived neural stem cells and their gene therapy application in a metastatic breast cancer model.

PubMed

Yang, Jing; Lam, Dang Hoang; Goh, Sally Sallee; Lee, Esther Xingwei; Zhao, Ying; Tay, Felix Chang; Chen, Can; Du, Shouhui; Balasundaram, Ghayathri; Shahbazi, Mohammad; Tham, Chee Kian; Ng, Wai Hoe; Toh, Han Chong; Wang, Shu

2012-05-01

Human pluripotent stem cells can serve as an accessible and reliable source for the generation of functional human cells for medical therapies. In this study, we used a conventional lentiviral transduction method to derive human-induced pluripotent stem (iPS) cells from primary human fibroblasts and then generated neural stem cells (NSCs) from the iPS cells. Using a dual-color whole-body imaging technology, we demonstrated that after tail vein injection, these human NSCs displayed a robust migratory capacity outside the central nervous system in both immunodeficient and immunocompetent mice and homed in on established orthotopic 4T1 mouse mammary tumors. To investigate whether the iPS cell-derived NSCs can be used as a cellular delivery vehicle for cancer gene therapy, the cells were transduced with a baculoviral vector containing the herpes simplex virus thymidine kinase suicide gene and injected through tail vein into 4T1 tumor-bearing mice. The transduced NSCs were effective in inhibiting the growth of the orthotopic 4T1 breast tumor and the metastatic spread of the cancer cells in the presence of ganciclovir, leading to prolonged survival of the tumor-bearing mice. The use of iPS cell-derived NSCs for cancer gene therapy bypasses the sensitive ethical issue surrounding the use of cells derived from human fetal tissues or human embryonic stem cells. This approach may also help to overcome problems associated with allogeneic transplantation of other types of human NSCs. Copyright © 2012 AlphaMed Press.

Establishment and proteomic characterization of a novel cell line, NCC-UPS2-C1, derived from a patient with undifferentiated pleomorphic sarcoma.

PubMed

Oyama, Rieko; Kito, Fusako; Sakumoto, Marimu; Shiozawa, Kumiko; Toki, Shunichi; Yoshida, Akihiko; Kawai, Akira; Kondo, Tadashi

2018-03-01

Undifferentiated pleomorphic sarcoma (UPS) is an aggressive mesenchymal malignancy requiring novel therapeutic approaches to improve clinical outcome. Patient-derived cancer cell lines are an essential tool for investigating molecular mechanisms underlying cancer initiation and development; however, there is a lack of patient-derived cell lines of UPS available for research. The objective of this study was to develop a patient-derived cell model of UPS. A cell line designated NCC-UPS2-C1 was established from the primary tumor tissue of an 84-yr-old female patient with UPS. The short tandem repeat pattern of NCC-UPS2-C1 cells was identical to that of the original tumor and distinct from that of any other cell lines deposited in public cell banks. NCC-UPS2-C1 cells were maintained as a monolayer culture for over 80 passages during 30 mo and exhibited spindle-like morphology, continuous growth, and ability for spheroid formation and invasion. Proteomic profiling using mass spectrometry and functional treemap analysis revealed that the original tumor and the derived NCC-UPS2-C1 cells had similar but distinct protein expression patterns. Our results indicate that a novel UPS cell line was successfully established and could be used to study UPS development and effects of anti-cancer drugs. However, the revealed difference between proteomes of the original tumor and NCC-UPS2-C1 cells should be further investigated to determine the appropriate applications of this cell line in UPS research.

The avian cell line AGE1.CR.pIX characterized by metabolic flux analysis

PubMed Central

2014-01-01

Background In human vaccine manufacturing some pathogens such as Modified Vaccinia Virus Ankara, measles, mumps virus as well as influenza viruses are still produced on primary material derived from embryonated chicken eggs. Processes depending on primary cell culture, however, are difficult to adapt to modern vaccine production. Therefore, we derived previously a continuous suspension cell line, AGE1.CR.pIX, from muscovy duck and established chemically-defined media for virus propagation. Results To better understand vaccine production processes, we developed a stoichiometric model of the central metabolism of AGE1.CR.pIX cells and applied flux variability and metabolic flux analysis. Results were compared to literature dealing with mammalian and insect cell culture metabolism focusing on the question whether cultured avian cells differ in metabolism. Qualitatively, the observed flux distribution of this avian cell line was similar to distributions found for mammalian cell lines (e.g. CHO, MDCK cells). In particular, glucose was catabolized inefficiently and glycolysis and TCA cycle seem to be only weakly connected. Conclusions A distinguishing feature of the avian cell line is that glutaminolysis plays only a minor role in energy generation and production of precursors, resulting in low extracellular ammonia concentrations. This metabolic flux study is the first for a continuous avian cell line. It provides a basis for further metabolic analyses to exploit the biotechnological potential of avian and vertebrate cell lines and to develop specific optimized cell culture processes, e.g. vaccine production processes. PMID:25077436

The avian cell line AGE1.CR.pIX characterized by metabolic flux analysis.

PubMed

Lohr, Verena; Hädicke, Oliver; Genzel, Yvonne; Jordan, Ingo; Büntemeyer, Heino; Klamt, Steffen; Reichl, Udo

2014-07-30

In human vaccine manufacturing some pathogens such as Modified Vaccinia Virus Ankara, measles, mumps virus as well as influenza viruses are still produced on primary material derived from embryonated chicken eggs. Processes depending on primary cell culture, however, are difficult to adapt to modern vaccine production. Therefore, we derived previously a continuous suspension cell line, AGE1.CR.pIX, from muscovy duck and established chemically-defined media for virus propagation. To better understand vaccine production processes, we developed a stoichiometric model of the central metabolism of AGE1.CR.pIX cells and applied flux variability and metabolic flux analysis. Results were compared to literature dealing with mammalian and insect cell culture metabolism focusing on the question whether cultured avian cells differ in metabolism. Qualitatively, the observed flux distribution of this avian cell line was similar to distributions found for mammalian cell lines (e.g. CHO, MDCK cells). In particular, glucose was catabolized inefficiently and glycolysis and TCA cycle seem to be only weakly connected. A distinguishing feature of the avian cell line is that glutaminolysis plays only a minor role in energy generation and production of precursors, resulting in low extracellular ammonia concentrations. This metabolic flux study is the first for a continuous avian cell line. It provides a basis for further metabolic analyses to exploit the biotechnological potential of avian and vertebrate cell lines and to develop specific optimized cell culture processes, e.g. vaccine production processes.

Use of Social Support during Communication about Sickle Cell Carrier Status

PubMed Central

Bradford, Lisa; Roedl, Sara J.; Christopher, Stephanie A.; Farrell, Michael H.

2012-01-01

Objective To examine the use of social support behaviors by primary care providers during delivery of positive newborn screening results for Sickle Cell Anemia carrier status. Methods Transcripts from 125 primary care providers who conveyed Sickle Cell Anemia carrier status to standardized parents were content analyzed using categories derived from Cutrona and Suhr’s social support taxonomy. Frequencies and cross-tabulation matrices were calculated to study providers’ social support utilization. Results Results showed most primary care providers (80%) incorporate social support behaviors into delivery of Sickle Cell Anemia carrier results and most frequently employed social network (61.6%) and informational support (38.4%) behaviors. Providers used tangible aid (8%), esteem (1.6%), and emotional support (9.6%) behaviors less frequently. Conclusion Cutrona and Suhr’s taxonomy may be a useful tool for assessing supportive communication during the delivery of Sickle Cell Anemia carrier status and could be incorporated into population scale assessments of communication quality assurance. Practice Implications Primary care providers may need training in how to adapt supportive behaviors to parents’ needs during communication of Sickle Cell Anemia carrier status. They also may benefit from specific training about how to use esteem and emotional support. PMID:22658247

Automated patch clamp on mESC-derived cardiomyocytes for cardiotoxicity prediction.

PubMed

Stoelzle, Sonja; Haythornthwaite, Alison; Kettenhofen, Ralf; Kolossov, Eugen; Bohlen, Heribert; George, Michael; Brüggemann, Andrea; Fertig, Niels

2011-09-01

Cardiovascular side effects are critical in drug development and have frequently led to late-stage project terminations or even drug withdrawal from the market. Physiologically relevant and predictive assays for cardiotoxicity are hence strongly demanded by the pharmaceutical industry. To identify a potential impact of test compounds on ventricular repolarization, typically a variety of ion channels in diverse heterologously expressing cells have to be investigated. Similar to primary cells, in vitro-generated stem cell-derived cardiomyocytes simultaneously express cardiac ion channels. Thus, they more accurately represent the native situation compared with cell lines overexpressing only a single type of ion channel. The aim of this study was to determine if stem cell-derived cardiomyocytes are suited for use in an automated patch clamp system. The authors show recordings of cardiac ion currents as well as action potential recordings in readily available stem cell-derived cardiomyocytes. Besides monitoring inhibitory effects of reference compounds on typical cardiac ion currents, the authors revealed for the first time drug-induced modulation of cardiac action potentials in an automated patch clamp system. The combination of an in vitro cardiac cell model with higher throughput patch clamp screening technology allows for a cost-effective cardiotoxicity prediction in a physiologically relevant cell system.

Long-term Culture of Human iPS Cell-derived Telencephalic Neuron Aggregates on Collagen Gel.

PubMed

Oyama, Hiroshi; Takahashi, Koji; Tanaka, Yoshikazu; Takemoto, Hiroshi; Haga, Hisashi

2018-01-01

It takes several months to form the 3-dimensional morphology of the human embryonic brain. Therefore, establishing a long-term culture method for neuronal tissues derived from human induced pluripotent stem (iPS) cells is very important for studying human brain development. However, it is difficult to keep primary neurons alive for more than 3 weeks in culture. Moreover, long-term adherent culture to maintain the morphology of telencephalic neuron aggregates induced from human iPS cells is also difficult. Although collagen gel has been widely used to support long-term culture of cells, it is not clear whether human iPS cell-derived neuron aggregates can be cultured for long periods on this substrate. In the present study, we differentiated human iPS cells to telencephalic neuron aggregates and examined long-term culture of these aggregates on collagen gel. The results indicated that these aggregates could be cultured for over 3 months by adhering tightly onto collagen gel. Furthermore, telencephalic neuronal precursors within these aggregates matured over time and formed layered structures. Thus, long-term culture of telencephalic neuron aggregates derived from human iPS cells on collagen gel would be useful for studying human cerebral cortex development.Key words: Induced pluripotent stem cell, forebrain neuron, collagen gel, long-term culture.

Infection rate and tissue localization of murine IL-12p40-producing monocyte-derived CD103(+) lung dendritic cells during pulmonary tuberculosis.

PubMed

Leepiyasakulchai, Chaniya; Taher, Chato; Chuquimia, Olga D; Mazurek, Jolanta; Söderberg-Naucler, Cecilia; Fernández, Carmen; Sköld, Markus

2013-01-01

Non-hematopoietic cells, including lung epithelial cells, influence host immune responses. By co-culturing primary alveolar epithelial cells and monocytes from naïve donor mice, we show that alveolar epithelial cells support monocyte survival and differentiation in vitro, suggesting a role for non-hematopoietic cells in monocyte differentiation during the steady state in vivo. CD103(+) dendritic cells (αE-DC) are present at mucosal surfaces. Using a murine primary monocyte adoptive transfer model, we demonstrate that αE-DC in the lungs and pulmonary lymph nodes are monocyte-derived during pulmonary tuberculosis. The tissue localization may influence the functional potential of αE-DC that accumulate in Mycobacterium tuberculosis-infected lungs. Here, we confirm the localization of αE-DC in uninfected mice beneath the bronchial epithelial cell layer and near the vascular wall, and show that αE-DC have a similar distribution in the lungs during pulmonary tuberculosis and are detected in the bronchoalveolar lavage fluid from infected mice. Lung DC can be targeted by M. tuberculosis in vivo and play a role in bacterial dissemination to the draining lymph node. In contrast to other DC subsets, only a fraction of lung αE-DC are infected with the bacterium. We also show that virulent M. tuberculosis does not significantly alter cell surface expression levels of MHC class II on infected cells in vivo and that αE-DC contain the highest frequency of IL-12p40(+) cells among the myeloid cell subsets in infected lungs. Our results support a model in which inflammatory monocytes are recruited into the M. tuberculosis-infected lung tissue and, depending on which non-hematopoietic cells they interact with, differentiate along different paths to give rise to multiple monocyte-derived cells, including DC with a distinctive αE-DC phenotype.

Infection Rate and Tissue Localization of Murine IL-12p40-Producing Monocyte-Derived CD103+ Lung Dendritic Cells during Pulmonary Tuberculosis

PubMed Central

Leepiyasakulchai, Chaniya; Taher, Chato; Chuquimia, Olga D.; Mazurek, Jolanta; Söderberg-Naucler, Cecilia; Fernández, Carmen; Sköld, Markus

2013-01-01

Non-hematopoietic cells, including lung epithelial cells, influence host immune responses. By co-culturing primary alveolar epithelial cells and monocytes from naïve donor mice, we show that alveolar epithelial cells support monocyte survival and differentiation in vitro, suggesting a role for non-hematopoietic cells in monocyte differentiation during the steady state in vivo. CD103+ dendritic cells (αE-DC) are present at mucosal surfaces. Using a murine primary monocyte adoptive transfer model, we demonstrate that αE-DC in the lungs and pulmonary lymph nodes are monocyte-derived during pulmonary tuberculosis. The tissue localization may influence the functional potential of αE-DC that accumulate in Mycobacterium tuberculosis-infected lungs. Here, we confirm the localization of αE-DC in uninfected mice beneath the bronchial epithelial cell layer and near the vascular wall, and show that αE-DC have a similar distribution in the lungs during pulmonary tuberculosis and are detected in the bronchoalveolar lavage fluid from infected mice. Lung DC can be targeted by M. tuberculosis in vivo and play a role in bacterial dissemination to the draining lymph node. In contrast to other DC subsets, only a fraction of lung αE-DC are infected with the bacterium. We also show that virulent M. tuberculosis does not significantly alter cell surface expression levels of MHC class II on infected cells in vivo and that αE-DC contain the highest frequency of IL-12p40+ cells among the myeloid cell subsets in infected lungs. Our results support a model in which inflammatory monocytes are recruited into the M. tuberculosis-infected lung tissue and, depending on which non-hematopoietic cells they interact with, differentiate along different paths to give rise to multiple monocyte-derived cells, including DC with a distinctive αE-DC phenotype. PMID:23861965

Evaluation of Stem Cell-Derived Red Blood Cells as a Transfusion Product Using a Novel Animal Model.

PubMed

Shah, Sandeep N; Gelderman, Monique P; Lewis, Emily M A; Farrel, John; Wood, Francine; Strader, Michael Brad; Alayash, Abdu I; Vostal, Jaroslav G

2016-01-01

Reliance on volunteer blood donors can lead to transfusion product shortages, and current liquid storage of red blood cells (RBCs) is associated with biochemical changes over time, known as 'the storage lesion'. Thus, there is a need for alternative sources of transfusable RBCs to supplement conventional blood donations. Extracorporeal production of stem cell-derived RBCs (stemRBCs) is a potential and yet untapped source of fresh, transfusable RBCs. A number of groups have attempted RBC differentiation from CD34+ cells. However, it is still unclear whether these stemRBCs could eventually be effective substitutes for traditional RBCs due to potential differences in oxygen carrying capacity, viability, deformability, and other critical parameters. We have generated ex vivo stemRBCs from primary human cord blood CD34+ cells and compared them to donor-derived RBCs based on a number of in vitro parameters. In vivo, we assessed stemRBC circulation kinetics in an animal model of transfusion and oxygen delivery in a mouse model of exercise performance. Our novel, chronically anemic, SCID mouse model can evaluate the potential of stemRBCs to deliver oxygen to tissues (muscle) under resting and exercise-induced hypoxic conditions. Based on our data, stem cell-derived RBCs have a similar biochemical profile compared to donor-derived RBCs. While certain key differences remain between donor-derived RBCs and stemRBCs, the ability of stemRBCs to deliver oxygen in a living organism provides support for further development as a transfusion product.

Nephrotoxicity testing in vitro--what we know and what we need to know.

PubMed Central

Pfaller, W; Gstraunthaler, G

1998-01-01

The kidney is affected by many chemicals. Some of the chemicals may even contribute to end-stage renal disease and thus contribute considerably to health care costs. Because of the large functional reserve of the kidney, which masks signs of dysfunction, early diagnosis of renal disease is often difficult. Although numerous studies aimed at understanding the mechanisms underlying chemicals and drugs that target various renal cell types have delivered enough understanding for a reasonable risk assessment, there is still an urgent need to better understand the mechanisms leading to renal cell injury and organ dysfunction. The increasing use of in vitro techniques using isolated renal cells, nephron fragments, or cell cultures derived from specific renal cell types has improved our insight into the molecular mechanisms involved in nephrotoxicity. A short overview is given on the various in vitro systems currently used to clarify mechanistic aspects leading to sublethal or lethal injury of the functionally most important nephron epithelial cells derived from various species. Whereas freshly isolated cells and nephron fragments appear to represent a sufficient basis to study acute effects (hours) of nephrotoxins, e.g., on cell metabolism, primary cultures of these cells are more appropriate to study long-term effects. In contrast to isolated cells and fragments, however, primary cultures tend to first lose several of their in vivo metabolic properties during culture, and second to have only a limited life span (days to weeks). Moreover, establishing such primary cultures is a time-consuming and laborious procedure. For that reason many studies have been carried out on renal cell lines, which are easy to cultivate in large quantities and which have an unlimited life span. Unfortunately, none of the lines display a state of differentiation comparable to that of freshly isolated cells or their primary cultures. Most often they lack expression of key functions (e.g., gluconeogenesis or organic anion transport) of their in vivo correspondents. Therefore, the use of cell lines for assessment of nephrotoxic mechanisms will be limited to those functions the lines express. Upcoming molecular biology approaches such as the transduction of immortalizing genes into primary cultures and the utilization of cells from transgenic animals may in the near future result in the availability of highly differentiated renal cells with markedly extended life spans and near in vivo characteristics that may facilitate the use of renal cell culture for routine screening of nephrotoxins. Images Figure 1 Figure 2 PMID:9599703

Id1 suppresses anti-tumour immune responses and promotes tumour progression by impairing myeloid cell maturation.

PubMed

Papaspyridonos, Marianna; Matei, Irina; Huang, Yujie; do Rosario Andre, Maria; Brazier-Mitouart, Helene; Waite, Janelle C; Chan, April S; Kalter, Julie; Ramos, Ilyssa; Wu, Qi; Williams, Caitlin; Wolchok, Jedd D; Chapman, Paul B; Peinado, Hector; Anandasabapathy, Niroshana; Ocean, Allyson J; Kaplan, Rosandra N; Greenfield, Jeffrey P; Bromberg, Jacqueline; Skokos, Dimitris; Lyden, David

2015-04-29

A central mechanism of tumour progression and metastasis involves the generation of an immunosuppressive 'macroenvironment' mediated in part through tumour-secreted factors. Here we demonstrate that upregulation of the Inhibitor of Differentiation 1 (Id1), in response to tumour-derived factors, such as TGFβ, is responsible for the switch from dendritic cell (DC) differentiation to myeloid-derived suppressor cell expansion during tumour progression. Genetic inactivation of Id1 largely corrects the myeloid imbalance, whereas Id1 overexpression in the absence of tumour-derived factors re-creates it. Id1 overexpression leads to systemic immunosuppression by downregulation of key molecules involved in DC differentiation and suppression of CD8 T-cell proliferation, thus promoting primary tumour growth and metastatic progression. Furthermore, advanced melanoma patients have increased plasma TGFβ levels and express higher levels of ID1 in myeloid peripheral blood cells. This study reveals a critical role for Id1 in suppressing the anti-tumour immune response during tumour progression and metastasis.

Quantification of Mesenchymal Stem Cells (MSCs) at sites of human prostate cancer.

PubMed

Brennen, W Nathaniel; Chen, Shuangling; Denmeade, Samuel R; Isaacs, John T

2013-01-01

Circulating bone marrow-derived Mesenchymal Stem Cells (BM-MSCs) have an innate tropism for tumor tissue in response to the inflammatory microenvironment present in malignant lesions. The prostate is bombarded by numerous infectious and inflammatory insults over a lifetime. Chronic inflammation is associated with CXCL12, CCL5, and CCL2, which are highly overexpressed in prostate cancer. Among other cell types, these chemoattractant stimuli recruit BM-MSCs to the tumor. MSCs are minimally defined as plastic-adhering cells characterized by the expression of CD90, CD73, and CD105 in the absence of hematopoietic markers, which can differentiate into osteoblasts, chondrocytes, and adipocytes. MSCs are immunoprivileged and have been implicated in tumorigenesis through multiple mechanisms, including promoting proliferation, angiogenesis, and metastasis, in addition to the generation of an immunosuppressive microenvironment. We have demonstrated that MSCs represent 0.01-1.1% of the total cells present in core biopsies from primary human prostatectomies. Importantly, these analyses were performed on samples prior to expansion in tissue culture. MSCs in these prostatectomy samples are FAP-, CD90-, CD73-, and CD105-positive, and CD14-, CD20-, CD34-, CD45-, and HLA-DR-negative. Additionally, like BM-MSCs, these prostate cancer-derived stromal cells (PrCSCs) were shown to differentiate into osteoblasts, adipocytes and chondrocytes. In contrast to primary prostate cancer-derived epithelial cells, fluorescently-labeled PrCSCs and BM-MSCs were both shown to home to CWR22RH prostate cancer xenografts following IV injection. These studies demonstrate that not only are MSCs present in sites of prostate cancer where they may contribute to carcinogenesis, but these cells may also potentially be used to deliver cytotoxic or imaging agents for therapeutic and/or diagnostic purposes.

Saquinavir inhibits early events associated with establishment of HIV-1 infection: potential role for protease inhibitors in prevention.

PubMed

Stefanidou, Martha; Herrera, Carolina; Armanasco, Naomi; Shattock, Robin J

2012-08-01

The maturation of newly formed human immunodeficiency virus type 1 (HIV-1) virions is a critical step for the establishment of productive infection. We investigated the potential of saquinavir (SQV), a protease inhibitor (PI) used in highly active antiretroviral therapy (HAART), as a candidate microbicide. SQV inhibited replication of clade B and clade C isolates in a dose-dependent manner in all cellular models tested: PM-1 CD4 T cells, peripheral blood mononuclear cells (PBMCs), monocyte-derived macrophages (MDMs), and immature monocyte-derived dendritic cells (iMDDCs). SQV also inhibited production of infectious virus in cervical, penile, and colorectal explants cocultured with T cells. Moreover, SQV demonstrated inhibitory potency against trans infection of T cells by in vitro-derived dendritic cells and by primary dendritic cells that emigrate from penile and cervical tissue explants. No cellular or tissue toxicity was detected in the presence of SQV, suggesting that this drug could be considered for development as a component of an effective microbicide, capable of blocking viral maturation and transmission of HIV-1 at mucosal surfaces.

Donor‐Dependent and Other Nondefined Factors Have Greater Influence on the Hepatic Phenotype Than the Starting Cell Type in Induced Pluripotent Stem Cell Derived Hepatocyte‐Like Cells

PubMed Central

Heslop, James A.; Kia, Richard; Pridgeon, Christopher S.; Sison‐Young, Rowena L.; Liloglou, Triantafillos; Elmasry, Mohamed; Fenwick, Stephen W.; Mills, John S.; Kitteringham, Neil R.; Park, Bong K.

2017-01-01

Abstract Drug‐induced liver injury is the greatest cause of post‐marketing drug withdrawal; therefore, substantial resources are directed toward triaging potentially dangerous new compounds at all stages of drug development. One of the major factors preventing effective screening of new compounds is the lack of a predictive in vitro model of hepatotoxicity. Primary human hepatocytes offer a metabolically relevant model for which the molecular initiating events of hepatotoxicity can be examined; however, these cells vary greatly between donors and dedifferentiate rapidly in culture. Induced pluripotent stem cell (iPSC)‐derived hepatocyte‐like cells (HLCs) offer a reproducible, physiologically relevant and genotypically normal model cell; however, current differentiation protocols produce HLCs with a relatively immature phenotype. During the reprogramming of somatic cells, the epigenome undergoes dramatic changes; however, this “resetting” is a gradual process, resulting in an altered differentiation propensity, skewed toward the lineage of origin, particularly in early passage cultures. We, therefore, performed a comparison of human hepatocyte‐ and dermal fibroblast‐derived iPSCs, assessing the impact of epigenetic memory at all stages of HLC differentiation. These results provide the first isogenic assessment of the starting cell type in human iPSC‐derived HLCs. Despite a trend toward improvement in hepatic phenotype in albumin secretion and gene expression, few significant differences in hepatic differentiation capacity were found between hepatocyte and fibroblast‐derived iPSCs. We conclude that the donor and inter‐clonal differences have a greater influence on the hepatocyte phenotypic maturity than the starting cell type. Therefore, it is not necessary to use human hepatocytes for generating iPSC‐derived HLCs. Stem Cells Translational Medicine 2017;6:1321–1331 PMID:28456008

Enhancer connectome in primary human cells identifies target genes of disease-associated DNA elements

PubMed Central

Mumbach, Maxwell R; Satpathy, Ansuman T; Boyle, Evan A; Dai, Chao; Gowen, Benjamin G; Cho, Seung Woo; Nguyen, Michelle L; Rubin, Adam J; Granja, Jeffrey M; Kazane, Katelynn R; Wei, Yuning; Nguyen, Trieu; Greenside, Peyton G; Corces, M Ryan; Tycko, Josh; Simeonov, Dimitre R; Suliman, Nabeela; Li, Rui; Xu, Jin; Flynn, Ryan A; Kundaje, Anshul; Khavari, Paul A; Marson, Alexander; Corn, Jacob E; Quertermous, Thomas; Greenleaf, William J; Chang, Howard Y

2018-01-01

The challenge of linking intergenic mutations to target genes has limited molecular understanding of human diseases. Here we show that H3K27ac HiChIP generates high-resolution contact maps of active enhancers and target genes in rare primary human T cell subtypes and coronary artery smooth muscle cells. Differentiation of naive T cells into T helper 17 cells or regulatory T cells creates subtype-specific enhancer–promoter interactions, specifically at regions of shared DNA accessibility. These data provide a principled means of assigning molecular functions to autoimmune and cardiovascular disease risk variants, linking hundreds of noncoding variants to putative gene targets. Target genes identified with HiChIP are further supported by CRISPR interference and activation at linked enhancers, by the presence of expression quantitative trait loci, and by allele-specific enhancer loops in patient-derived primary cells. The majority of disease-associated enhancers contact genes beyond the nearest gene in the linear genome, leading to a fourfold increase in the number of potential target genes for autoimmune and cardiovascular diseases. PMID:28945252

Understanding pathogenetic aspects and clinical presentation of primary effusion lymphoma (PEL) through its derived cell lines

PubMed Central

Carbone, Antonino; Cesarman, Ethel; Gloghini, Annunziata; Drexler, Hans G.

2013-01-01

Primary effusion lymphoma (PEL) is a very rare subgroup of B-cell lymphomas presenting as pleural, peritoneal and pericardial neoplastic effusions in the absence of a solid tumor mass or recognizable nodal involvement. There is strong evidence that Kaposi’s sarcoma associated herpesvirus (KSHV) is a causal agent of PEL. PEL tumor cells are latently infected by KSHV with consistent expression of several viral proteins and microRNAs that can affect cellular proliferation, differentiation and survival. The most relevant data on pathogenesis and biology of KSHV have been provided by studies on PEL derived cell lines. Fourteen continuous cell lines have been established from the malignant effusions of patients with AIDS-and non-AIDS-associated PEL. These KSHV+ EBV+/− cell lines are wellcharacterized, authenticated and mostly available from public biological ressource centers. The PEL cell lines display unique features and are clearly distinct from other lymphoma cell lines. PEL cell lines represent an indispensable tool for the understanding of KSHV biology and its impact on the clinical manifestation of PEL. Studies on PEL cell lines have shown that a number of viral genes, expressed during latency or lytic life cycle, have effects on cell binding, proliferation, angiogenesis and inflammation. Also PEL cell lines are important model systems for the study of the pathology of PEL including the lack of invasive or destructive growth patterns and the peculiar propensity of PEL to involve body cavity surfaces. PMID:20051807

Primary EBV infection of human umbilical cord lymphocytes and EBV genome-negative lymphoblastoid cell lines (BJAB and Ramos).

PubMed

Takimoto, T; Sato, H; Ogura, H

1986-01-01

The appearance of Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA) and induction of EBV-induced early antigen (EA) in human umbilical cord blood lymphocytes (HUCLs) and two EBV genome-negative Burkitt's lymphoma (BL) lines (BJAB and Ramos) were studied by infection with EBVs prepared from three different cell lines: marmoset cell line (B95-8) derived from infections mononucleosis, BL-derived cell line (P3HR-1) and human epithelial hybrid cell line (NPC-KT) derived from nasopharyngeal carcinoma. B95-8 virus can transform HUCLs but cannot superinfect Raji cells. P3HR-1 virus can transform HUCLs cells but cannot transform HUCLs. NPC-KT virus can transform HUCLs and can superinfect Raji cells. We have examined the time sequence of EBNA appearance and EA induction in HUCLs, BJAB cells and Ramos cells, in order to determine if three different strains of EBV differ in their abilities to infect their cells. We found that all three strains of EBV can induce EBNA in HUCLs, BJAB cells and Ramos cells. On the other hand, we found that P3HR-1 virus and NPC-KT virus can induce EA in BJAB cells and Ramos cells, but B95-8 virus cannot induce EA in their cells.

Human GAPDH Is a Target of Aspirin’s Primary Metabolite Salicylic Acid and Its Derivatives

PubMed Central

Manohar, Murli; Harraz, Maged M.; Park, Sang-Wook; Schroeder, Frank C.; Snyder, Solomon H.; Klessig, Daniel F.

2015-01-01

The plant hormone salicylic acid (SA) controls several physiological processes and is a key regulator of multiple levels of plant immunity. To decipher the mechanisms through which SA’s multiple physiological effects are mediated, particularly in immunity, two high-throughput screens were developed to identify SA-binding proteins (SABPs). Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) from plants (Arabidopsis thaliana) was identified in these screens. Similar screens and subsequent analyses using SA analogs, in conjunction with either a photoaffinity labeling technique or surface plasmon resonance-based technology, established that human GAPDH (HsGAPDH) also binds SA. In addition to its central role in glycolysis, HsGAPDH participates in several pathological processes, including viral replication and neuronal cell death. The anti-Parkinson’s drug deprenyl has been shown to suppress nuclear translocation of HsGAPDH, an early step in cell death and the resulting cell death induced by the DNA alkylating agent N-methyl-N’-nitro-N-nitrosoguanidine. Here, we demonstrate that SA, which is the primary metabolite of aspirin (acetyl SA) and is likely responsible for many of its pharmacological effects, also suppresses nuclear translocation of HsGAPDH and cell death. Analysis of two synthetic SA derivatives and two classes of compounds from the Chinese medicinal herb Glycyrrhiza foetida (licorice), glycyrrhizin and the SA-derivatives amorfrutins, revealed that they not only appear to bind HsGAPDH more tightly than SA, but also exhibit a greater ability to suppress translocation of HsGAPDH to the nucleus and cell death. PMID:26606248

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

Blastic Plasmacytoid Dendritic Cell Neoplasm Is Dependent on BCL2 and Sensitive to Venetoclax.

PubMed

Montero, Joan; Stephansky, Jason; Cai, Tianyu; Griffin, Gabriel K; Cabal-Hierro, Lucia; Togami, Katsuhiro; Hogdal, Leah J; Galinsky, Ilene; Morgan, Elizabeth A; Aster, Jon C; Davids, Matthew S; LeBoeuf, Nicole R; Stone, Richard M; Konopleva, Marina; Pemmaraju, Naveen; Letai, Anthony; Lane, Andrew A

2017-02-01

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive hematologic malignancy with dismal outcomes for which no standard therapy exists. We found that primary BPDCN cells were dependent on the antiapoptotic protein BCL2 and were uniformly sensitive to the BCL2 inhibitor venetoclax, as measured by direct cytotoxicity, apoptosis assays, and dynamic BH3 profiling. Animals bearing BPDCN patient-derived xenografts had disease responses and improved survival after venetoclax treatment in vivo Finally, we report on 2 patients with relapsed/refractory BPDCN who received venetoclax off-label and experienced significant disease responses. We propose that venetoclax or other BCL2 inhibitors undergo expedited clinical evaluation in BPDCN, alone or in combination with other therapies. In addition, these data illustrate an example of precision medicine to predict treatment response using ex vivo functional assessment of primary tumor tissue, without requiring a genetic biomarker. Therapy for BPDCN is inadequate, and survival in patients with the disease is poor. We used primary tumor cell functional profiling to predict BCL2 antagonist sensitivity as a common feature of BPDCN, and demonstrated in vivo clinical activity of venetoclax in patient-derived xenografts and in 2 patients with relapsed chemotherapy-refractory disease. Cancer Discov; 7(2); 156-64. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 115. ©2016 American Association for Cancer Research.

Primary Cell Culture of Live Neurosurgically Resected Aged Adult Human Brain Cells and Single Cell Transcriptomics.

PubMed

Spaethling, Jennifer M; Na, Young-Ji; Lee, Jaehee; Ulyanova, Alexandra V; Baltuch, Gordon H; Bell, Thomas J; Brem, Steven; Chen, H Isaac; Dueck, Hannah; Fisher, Stephen A; Garcia, Marcela P; Khaladkar, Mugdha; Kung, David K; Lucas, Timothy H; O'Rourke, Donald M; Stefanik, Derek; Wang, Jinhui; Wolf, John A; Bartfai, Tamas; Grady, M Sean; Sul, Jai-Yoon; Kim, Junhyong; Eberwine, James H

2017-01-17

Investigation of human CNS disease and drug effects has been hampered by the lack of a system that enables single-cell analysis of live adult patient brain cells. We developed a culturing system, based on a papain-aided procedure, for resected adult human brain tissue removed during neurosurgery. We performed single-cell transcriptomics on over 300 cells, permitting identification of oligodendrocytes, microglia, neurons, endothelial cells, and astrocytes after 3 weeks in culture. Using deep sequencing, we detected over 12,000 expressed genes, including hundreds of cell-type-enriched mRNAs, lncRNAs and pri-miRNAs. We describe cell-type- and patient-specific transcriptional hierarchies. Single-cell transcriptomics on cultured live adult patient derived cells is a prime example of the promise of personalized precision medicine. Because these cells derive from subjects ranging in age into their sixties, this system permits human aging studies previously possible only in rodent systems. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Leukaemia cell of origin identified by chromatin landscape of bulk tumour cells

PubMed Central

George, Joshy; Uyar, Asli; Young, Kira; Kuffler, Lauren; Waldron-Francis, Kaiden; Marquez, Eladio; Ucar, Duygu; Trowbridge, Jennifer J.

2016-01-01

The precise identity of a tumour's cell of origin can influence disease prognosis and outcome. Methods to reliably define tumour cell of origin from primary, bulk tumour cell samples has been a challenge. Here we use a well-defined model of MLL-rearranged acute myeloid leukaemia (AML) to demonstrate that transforming haematopoietic stem cells (HSCs) and multipotent progenitors results in more aggressive AML than transforming committed progenitor cells. Transcriptome profiling reveals a gene expression signature broadly distinguishing stem cell-derived versus progenitor cell-derived AML, including genes involved in immune escape, extravasation and small GTPase signal transduction. However, whole-genome profiling of open chromatin reveals precise and robust biomarkers reflecting each cell of origin tested, from bulk AML tumour cell sampling. We find that bulk AML tumour cells exhibit distinct open chromatin loci that reflect the transformed cell of origin and suggest that open chromatin patterns may be leveraged as prognostic signatures in human AML. PMID:27397025

Establishment and characterization of a unique 1 microm diameter liver-derived progenitor cell line.

PubMed

Aravalli, Rajagopal N; Behnan Sahin, M; Cressman, Erik N K; Steer, Clifford J

2010-01-01

Liver-derived progenitor cells (LDPCs) are recently identified novel stem/progenitor cells from healthy, unmanipulated adult rat livers. They are distinct from other known liver stem/progenitor cells such as the oval cells. In this study, we have generated a LDPC cell line RA1 by overexpressing the simian virus 40 (SV40) large T antigen (TAg) in primary LDPCs. This cell line was propagated continuously for 55 passages in culture, after which it became senescent. Interestingly, following transformation with SV40 TAg, LDPCs decreased in size significantly and the propagating cells measured 1 microm in diameter. RA1 cells proliferated in vitro with a doubling time of 5-7 days, and expressed cell surface markers of LDPCs. In this report, we describe the characterization of this novel progenitor cell line that might serve as a valuable model to study liver cell functions and stem cell origin of liver cancers. Copyright 2009 Elsevier Inc. All rights reserved.

Comparison of the interferon-tau expression from primary trophectoderm outgrowths derived from IVP, NT, and parthenogenote bovine blastocysts.

PubMed

Talbot, Neil C; Powell, Anne M; Ocón, Olga M; Caperna, Thomas J; Camp, Mary; Garrett, Wesley M; Ealy, Alan D

2008-02-01

The expression of interferon-tau (IFN-tau) is essential for bovine embryo survival in the uterus. An evaluation of IFN-tau production from somatic cell nuclear transfer (NT)-embryo-derived primary trophectoderm cultures in comparison to trophectoderm cultured from parthenogenote (P) and in vitro matured, fertilized, and cultured (IVP) bovine embryos was performed. In Experiment 1, the success/failure ratio for primary trophectoderm colony formation was similar for IVP and NT blastocysts [IVP = 155/29 (84%); NT 104/25 (81%)], but was decreased (P = .05) for P blastocysts [54/43 (56%)]. Most trophectoderm colonies reached diameters of at least 1 cm within 3-4 weeks, and at this time, 72 hr conditioned cell culture medium was measured for IFN-tau concentration by antiviral activity assay. The amount of IFN-tau produced by IVP-outgrowths [4311 IU/mL (n = 155)] was greater (P < .05) than that from NT- [626 IU/mL (n = 104)] and P - [1595 IU/mL (n = 54)] derived trophectoderm. Differential expression of IFN-tau was confirmed by immunoblotting. In Experiment 2, colony formation was again similar for IVP and NT blastocysts [IVP = 70/5 (93%); NT 67/1 (99%)] and less (P < .05) for P blastocysts [65/27 (70%)]. Analysis of trophectoderm colony size after 23 days in culture showed a similar relationship with P-derived colonies being significantly smaller in comparison to IVP and NT colonies. A differential expression of IFN-tau was also observed again, but this time as measured over time in culture. Maximal IFN-tau production was found at day-14 of primary culture and diminished to a minimum by the 23rd day. 2007 Wiley-Liss, Inc

Kinin effects on ion transport in monolayers of HCA-7 cells, a line from a human colonic adenocarcinoma.

PubMed

Cuthbert, A W; Kirkland, S C; MacVinish, L J

1985-09-01

Using epithelial monolayers of HCA-7 cells, derived from a primary human colonic adenocarcinoma and grown on pervious supports, it is shown that responses to lysylbradykinin can be elicited from either side. It is proposed that kinin receptors are inserted into both apical and basolateral membrane domains.

ANGPTL2 increases bone metastasis of breast cancer cells through enhancing CXCR4 signaling

PubMed Central

Masuda, Tetsuro; Endo, Motoyoshi; Yamamoto, Yutaka; Odagiri, Haruki; Kadomatsu, Tsuyoshi; Nakamura, Takayuki; Tanoue, Hironori; Ito, Hitoshi; Yugami, Masaki; Miyata, Keishi; Morinaga, Jun; Horiguchi, Haruki; Motokawa, Ikuyo; Terada, Kazutoyo; Morioka, Masaki Suimye; Manabe, Ichiro; Iwase, Hirotaka; Mizuta, Hiroshi; Oike, Yuichi

2015-01-01

Bone metastasis of breast cancer cells is a major concern, as it causes increased morbidity and mortality in patients. Bone tissue-derived CXCL12 preferentially recruits breast cancer cells expressing CXCR4 to bone metastatic sites. Thus, understanding how CXCR4 expression is regulated in breast cancer cells could suggest approaches to decrease bone metastasis of breast tumor cells. Here, we show that tumor cell-derived angiopoietin-like protein 2 (ANGPTL2) increases responsiveness of breast cancer cells to CXCL12 by promoting up-regulation of CXCR4 in those cells. In addition, we used a xenograft mouse model established by intracardiac injection of tumor cells to show that ANGPTL2 knockdown in breast cancer cells attenuates tumor cell responsiveness to CXCL12 by decreasing CXCR4 expression in those cells, thereby decreasing bone metastasis. Finally, we found that ANGPTL2 and CXCR4 expression levels within primary tumor tissues from breast cancer patients are positively correlated. We conclude that tumor cell-derived ANGPTL2 may increase bone metastasis by enhancing breast tumor cell responsiveness to CXCL12 signaling through up-regulation of tumor cell CXCR4 expression. These findings may suggest novel therapeutic approaches to treat metastatic breast cancer. PMID:25773070

Mechanism of Hormonal Regulation of CAD Gene Expression in Human Breast Cancer Cells

DTIC Science & Technology

2006-05-01

showed that PGJ2, troglitazone, and ciglitazone inhibit E2-stimulated cell proliferation of a leiomyoma -derived cell line and human primary leiomyoma ...cultures and also inhibit ER-mediated gene expression and protein expression. These results suggested that in uterine leiomyomas PPARγ activation is...peroxisome proliferator-activated receptor gamma ligands in uterine leiomyoma . Cancer Res. 63, 1221-1227. Hulka, BS., Liu, ET., and Lininger, RA., 1994

Interaction of Synuclein and Inflammation in Dopaminergic Neurodegeneration

DTIC Science & Technology

2010-07-01

synuclein in cell culture. Specific Aim II In vivo studies: we are now optimizing immunostaining for human -synuclein in order to distinguish...responsible for this response. Recent cell culture experiments seem to point us in the right direction for the answer as it has been shown that human BE-M17...synuclein. Furthermore, Zhou et al (2002) showed that, in primary cell cultures derived from the embryonic human mesencephalon overexpressing either

Evaluation of the Adult Goldfish Brain as a Model for the Study of Progenitor Cells

DTIC Science & Technology

2007-08-27

embryo [34]. ESCs are able to differentiate into all derivatives of the three primary germ layers: ectoderm, endoderm, and mesoderm, and they are...postnatal brain is their functional and anatomical destiny . Based on many reports investigating neurogenesis, the majority of newly produced cells...Homeodomain-bearing transcriptional factor. Expression is specific to early embryos and pluripotential stem cells. Key molecule involved in the

Comparative human cellular radiosensitivity: I. The effect of SV40 transformation and immortalisation on the gamma-irradiation survival of skin derived fibroblasts from normal individuals and from ataxia-telangiectasia patients and heterozygotes.

PubMed

Arlett, C F; Green, M H; Priestley, A; Harcourt, S A; Mayne, L V

1988-12-01

We have compared cell killing following 60Co gamma irradiation in 22 primary human fibroblast strains, nine SV40-immortalized human fibroblast lines and seven SV40-transformed pre-crisis human fibroblast cultures. We have examined material from normal individuals, from ataxia-telangiectasia (A-T) patients and from A-T heterozygotes. We have confirmed the greater sensitivity of A-T derived cells to gamma radiation. The distinction between A-T and normal cells is maintained in cells immortalized by SV40 virus but the immortal cells are more gamma radiation resistant than the corresponding primary fibroblasts. Cells transformed by plasmids (pSV3gpt and pSV3neo) expressing SV40 T-antigen, both pre- and post-crisis, show this increased resistance, indicating that it is expression of SV40 T-antigen, rather than immortalization per se which is responsible for the change. We use D0, obtained from a straight line fit, and D, estimated from a multitarget curve, as parameters to compare radiosensitivity. We suggest that both have their advantages; D0 is perhaps more reproducible, but D is more realistic when comparing shouldered and non-shouldered data.

Bone morphogenetic protein 9 (BMP9) induces effective bone formation from reversibly immortalized multipotent adipose-derived (iMAD) mesenchymal stem cells.

PubMed

Lu, Shun; Wang, Jing; Ye, Jixing; Zou, Yulong; Zhu, Yunxiao; Wei, Qiang; Wang, Xin; Tang, Shengli; Liu, Hao; Fan, Jiaming; Zhang, Fugui; Farina, Evan M; Mohammed, Maryam M; Song, Dongzhe; Liao, Junyi; Huang, Jiayi; Guo, Dan; Lu, Minpeng; Liu, Feng; Liu, Jianxiang; Li, Li; Ma, Chao; Hu, Xue; Lee, Michael J; Reid, Russell R; Ameer, Guillermo A; Zhou, Dongsheng; He, Tongchuan

2016-01-01

Regenerative medicine and bone tissue engineering using mesenchymal stem cells (MSCs) hold great promise as an effective approach to bone and skeletal reconstruction. While adipose tissue harbors MSC-like progenitors, or multipotent adipose-derived cells (MADs), it is important to identify and characterize potential biological factors that can effectively induce osteogenic differentiation of MADs. To overcome the time-consuming and technically challenging process of isolating and culturing primary MADs, here we establish and characterize the reversibly immortalized mouse multipotent adipose-derived cells (iMADs). The isolated mouse primary inguinal MAD cells are reversibly immortalized via the retrovirus-mediated expression of SV40 T antigen flanked with FRT sites. The iMADs are shown to express most common MSC markers. FLP-mediated removal of SV40 T antigen effectively reduces the proliferative activity and cell survival of iMADs, indicating the immortalization is reversible. Using the highly osteogenic BMP9, we find that the iMADs are highly responsive to BMP9 stimulation, express multiple lineage regulators, and undergo osteogenic differentiation in vitro upon BMP9 stimulation. Furthermore, we demonstrate that BMP9-stimulated iMADs form robust ectopic bone with a thermoresponsive biodegradable scaffold material. Collectively, our results demonstrate that the reversibly immortalized iMADs exhibit the characteristics of multipotent MSCs and are highly responsive to BMP9-induced osteogenic differentiation. Thus, the iMADs should provide a valuable resource for the study of MAD biology, which would ultimately enable us to develop novel and efficacious strategies for MAD-based bone tissue engineering.

Effect of Microcystin-LR on Cultured Rat Endothelial Cells

DTIC Science & Technology

1990-02-26

protection, silymarin , and dithioerythritol 19. ABSTRACT (Continue on reverse if necessary and identify by block number) Primary cultureLs of adult rat...8217 22.g-Ldenine nucleotides, and a small reduction of cell density in endothelial cell monolayers._ Silymarin at 0.2 mM but not(dithioerythritol;at 2.5...monolayers. Silymarin at 0.2 mM but not dithioerythritol at 2.5 mM, partially protected changes in endothelial cells produced by supernatants derived

Decursinol and decursin protect primary cultured rat cortical cells from glutamate-induced neurotoxicity.

PubMed

Kang, So Young; Kim, Young Choong

2007-06-01

We previously reported six neuroprotective decursinol derivatives, coumarins from Angelica gigas (Umbelliferae) roots. To elucidate the action patterns of decursinol derivatives, we investigated the neuroprotective effects of decursinol and decursin, which showed highly significant activity and were major constituents of A. gigas, using primary cultures of rat cortical cells in-vitro. At concentrations of 0.1-10.0 microM, both decursinol and decursin exerted a significant neuroprotective activity pretreatment and throughout treatment. In addition, decursin had a neuroprotective impact in the post-treatment paradigm implying that decursin might possess different action mechanisms from that of decursinol in the protection of neurons against glutamate injury. Both decursinol and decursin effectively reduced the glutamate-induced increased intracellular calcium ([Ca(2+)](i)) in cortical cells, suggesting that these two coumarins may exert neuroprotection by reducing calcium influx by overactivation of glutamate receptors. This suggestion was supported by the result that decursinol and decursin protected neurons against kainic acid (KA)-induced neurotoxicity better than against that induced by N-methyl-D-aspartate (NMDA). Moreover, both decursinol and decursin significantly prevented glutamate-induced decreases in glutathione, a cellular antioxidant, and glutathione peroxidase activity. In addition, both compounds efficiently reduced the overproduction of cellular peroxide in glutamate-injured cortical cells. These results suggested that both decursinol and decursin protected primary cultured rat cortical cells against glutamate-induced oxidative stress by both reducing calcium influx and acting on the cellular antioxidative defence system. Moreover, decursin is considered to probably have a different action mechanism from that of decursinol in protecting cortical cells against glutamate injury.

Enamel Matrix Derivative Promote Primary Human Pulp Cell Differentiation and Mineralization

PubMed Central

Riksen, Elisabeth Aurstad; Landin, Maria A.; Reppe, Sjur; Nakamura, Yukio; Lyngstadaas, Ståle Petter; Reseland, Janne E.

2014-01-01

Enamel matrix derivative (EMD) has been found to induce reactive dentin formation; however the molecular mechanisms involved are unclear. The effect of EMD (5–50 μg/mL) on primary human pulp cells were compared to untreated cells and cells incubated with 10−8 M dexamethasone (DEX) for 1, 2, 3, 7, and 14 days in culture. Expression analysis using Affymetrix microchips demonstrated that 10 μg/mL EMD regulated several hundred genes and stimulated the gene expression of proteins involved in mesenchymal proliferation and differentiation. Both EMD and DEX enhanced the expression of amelogenin (amel), and the dentinogenic markers dentin sialophosphoprotein (DSSP) and dentin matrix acidic phosphoprotein 1 (DMP1), as well as the osteogenic markers osteocalcin (OC, BGLAP) and collagen type 1 (COL1A1). Whereas, only EMD had effect on alkaline phosphatase (ALP) mRNA expression, the stimulatory effect were verified by enhanced secretion of OC and COL1A from EMD treated cells, and increased ALP activity in cell culture medium after EMD treatment. Increased levels of interleukin-6 (IL-6), interleukin-8 (IL-8), and monocyte chemoattractant proteins (MCP-1) in the cell culture medium were also found. Consequently, the suggested effect of EMD is to promote differentiation of pulp cells and increases the potential for pulpal mineralization to favor reactive dentine formation. PMID:24857913

Interleukin (IL)-1 Receptor Signaling on Graft Parenchymal Cells Regulates Memory and De Novo Donor-Reactive CD8 T Cell Responses to Cardiac Allografts1

PubMed Central

Iida, Shoichi; Tsuda, Hidetoshi; Tanaka, Toshiaki; Kish, Danielle D.; Abe, Toyofumi; Su, Charles A.; Abe, Ryo; Tanabe, Kazunari; Valujskikh, Anna; Baldwin, William M.; Fairchild, Robert L.

2016-01-01

Reperfusion of organ allografts induces a potent inflammatory response that directs rapid memory T cell, neutrophil and macrophage graft infiltration and their activation to express functions mediating graft tissue injury. The role of cardiac allograft IL-1 receptor signaling in this early inflammation and the downstream primary alloimmune response was investigated. When compared to complete MHC-mismatched wild type cardiac allografts, IL-1R−/− allografts had marked decreases in endogenous memory CD8 T cell and neutrophil infiltration and expression of proinflammatory mediators at early times after transplant whereas endogenous memory CD4 T cell and macrophage infiltration was not decreased. IL-1R−/− allograft recipients also had marked decreases in de novo donor-reactive CD8, but not CD4, T cell development to IFN-γ-producing cells. CD8 T cell-mediated rejection of IL-1R−/− cardiac allografts took 3 weeks longer than wild type allografts. Cardiac allografts from reciprocal bone marrow reconstituted IL-1R−/−/wild type chimeric donors indicated that IL-1R signaling on graft non-hematopoietic-derived, but not bone marrow-derived, cells is required for the potent donor-reactive memory and primary CD8 T cell alloimmune responses observed in response to wild type allografts. These studies implicate IL-1R-mediated signals by allograft parenchymal cells in generating the stimuli provoking development and elicitation of optimal alloimmune responses to the grafts. PMID:26856697

Factors affecting the loss of MED12-mutated leiomyoma cells during in vitro growth.

PubMed

Bloch, Jeannine; Holzmann, Carsten; Koczan, Dirk; Helmke, Burkhard Maria; Bullerdiek, Jörn

2017-05-23

Uterine leiomyomas (UL) are the most prevalent symptomatic human tumors at all and somatic mutations of the gene encoding mediator subcomplex 12 (MED12) constitute the most frequent driver mutations in UL. Recently, a rapid loss of mutated cells during in vitro growth of UL-derived cell cultures was reported, resulting in doubts about the benefits of UL-derived cell cultures. To evaluate if the rapid loss of MED12-mutated cells in UL cell cultures depends on in vitro passaging, we set up cell cultures from nine UL from 40-50 year old Caucasian patients with at least one UL. Cultured UL cells were investigated for loss of MED12-mutated cells. Genetic characterization of native tumor samples and adjacent myometrium was done by array analysis. "Aged" primary cultures without passaging were compared to cells of three subsequent passages. Comparative analyses of the mutated/non-mutated ratios between native tissue, primary cells, and cultured tumor cells revealed a clear decrease of MED12-mutated cells. None of the tumors showed gross alterations of the array profiles, excluding the presence of gross genomic imbalances besides the MED12 mutations as a reason for the intertumoral variation in the loss of MED12-mutated cells. Albeit at a lesser rate, loss of MED12-mutated cells from cell cultures of UL occurs even without passaging thus indicating the requirement of soluble factors or matrix components lacking in vitro. Identification of these factors can help to understand the mechanisms of the growth of the most frequent type of uterine leiomyomas and to decipher novel drug targets.

High content screening of patient-derived cell lines highlights the potential of non-standard chemotherapeutic agents for the treatment of glioblastoma.

PubMed

Yu, Kenny Kwok-Hei; Taylor, Jessica T; Pathmanaban, Omar N; Youshani, Amir Saam; Beyit, Deniz; Dutko-Gwozdz, Joanna; Benson, Roderick; Griffiths, Gareth; Peers, Ian; Cueppens, Peter; Telfer, Brian A; Williams, Kaye J; McBain, Catherine; Kamaly-Asl, Ian D; Bigger, Brian W

2018-01-01

Glioblastoma (GBM) is the most common primary brain malignancy in adults, yet survival outcomes remain poor. First line treatment is well established, however disease invariably recurs and improving prognosis is challenging. With the aim of personalizing therapy at recurrence, we have established a high content screening (HCS) platform to analyze the sensitivity profile of seven patient-derived cancer stem cell lines to 83 FDA-approved chemotherapy drugs, with and without irradiation. Seven cancer stem cell lines were derived from patients with GBM and, along with the established cell line U87-MG, each patient-derived line was cultured in tandem in serum-free conditions as adherent monolayers and three-dimensional neurospheres. Chemotherapeutics were screened at multiple concentrations and cells double-stained to observe their effect on both cell death and proliferation. Sensitivity was classified using high-throughput algorithmic image analysis. Cell line specific drug responses were observed across the seven patient-derived cell lines. Few agents were seen to have radio-sensitizing effects, yet some drug classes showed a marked difference in efficacy between monolayers and neurospheres. In vivo validation of six drugs suggested that cell death readout in a three-dimensional culture scenario is a more physiologically relevant screening model and could be used effectively to assess the chemosensitivity of patient-derived GBM lines. The study puts forward a number of non-standard chemotherapeutics that could be useful in the treatment of recurrent GBM, namely mitoxantrone, bortezomib and actinomycin D, whilst demonstrating the potential of HCS to be used for personalized treatment based on the chemosensitivity profile of patient tumor cells.

Early dissemination seeds metastasis in breast cancer

PubMed Central

Hosseini, Hedayatollah; Obradović, Milan M.S.; Hoffmann, Martin; Harper, Kathryn; Sosa, Maria Soledad; Werner-Klein, Melanie; Nanduri, Lahiri Kanth; Werno, Christian; Ehrl, Carolin; Maneck, Matthias; Patwary, Nina; Haunschild, Gundula; Gužvić, Miodrag; Reimelt, Christian; Grauvogl, Michael; Eichner, Norbert; Weber, Florian; Hartkopf, Andreas; Taran, Florin-Andrei; Brucker, Sara Y.; Fehm, Tanja; Rack, Brigitte; Buchholz, Stefan; Spang, Rainer; Meister, Gunter; Aguirre-Ghiso, Julio A.; Klein, Christoph A.

2016-01-01

Accumulating data suggest that metastatic dissemination often occurs early during tumour formation but the mechanisms of early metastatic spread have not yet been addressed. Here, we studied metastasis in a HER2-driven mouse breast cancer model and found that progesterone-induced signalling triggered migration of cancer cells from early lesions shortly after HER2 activation, but promoted proliferation in advanced primary tumour cells. The switch from migration to proliferation was regulated by elevated HER2 expression and increased tumour cell density involving miRNA-mediated progesterone receptor (PGR) down-regulation and was reversible. Cells from early, low-density lesions displayed more stemness features than cells from dense, advanced tumours, migrated more and founded more metastases. Strikingly, we found that at least 80% of metastases were derived from early disseminated cancer cells (DCC). Karyotypic and phenotypic analysis of human disseminated cancer cells and primary tumours corroborated the relevance of these findings for human metastatic dissemination. PMID:27974799

The secreted factors responsible for pre-metastatic niche formation: old sayings and new thoughts.

PubMed

Peinado, Héctor; Lavotshkin, Simon; Lyden, David

2011-04-01

Metastasis is a multistep process that requires acquisition of malignant cell phenotypes which allow tumor cells to escape from the primary tumor site. Each of the steps during metastatic progression involves co-evolution of the tumor and its microenvironment. Although tumor cells are the driving force of metastasis, new findings suggest that the host cells within the tumor microenvironment play a key role in influencing metastatic behavior. Many of these contributing cells are derived from the bone marrow; in particular, recruited bone marrow progenitor cells generate the "pre-metastatic niche" to which the tumor cells metastasize. Analysis of the molecular mechanisms involved in pre-metastatic niche formation has revealed that secreted soluble factors are key players in bone marrow cell mobilization during metastasis. In addition, membrane vesicles derived from both tumor and host cells have recently been recognized as new candidates with important roles in the promotion of tumor growth and metastasis. This review describes old ideas and presents new insights into the role of tumor and bone marrow-derived microvesicles and exosomes in pre-metastatic niche formation and metastasis. Copyright © 2011 Elsevier Ltd. All rights reserved.

Optimizing Culture Medium Composition to Improve Oligodendrocyte Progenitor Cell Yields In Vitro from Subventricular Zone-Derived Neural Progenitor Cell Neurospheres

PubMed Central

Franco, Paula G.; Pasquini, Juana M.; Silvestroff, Lucas

2015-01-01

Neural Stem and Progenitor Cells (NSC/NPC) are gathering tangible recognition for their uses in cell therapy and cell replacement therapies for human disease, as well as a model system to continue research on overall neural developmental processes in vitro. The Subventricular Zone is one of the largest NSC/NPC niches in the developing mammalian Central Nervous System, and persists through to adulthood. Oligodendrocyte progenitor cell (OPC) enriched cultures are usefull tools for in vitro studies as well as for cell replacement therapies for treating demyelination diseases. We used Subventricular Zone-derived NSC/NPC primary cultures from newborn mice and compared the effects of different growth factor combinations on cell proliferation and OPC yield. The Platelet Derived Growth Factor-AA and BB homodimers had a positive and significant impact on OPC generation. Furthermore, heparin addition to the culture media contributed to further increase overall culture yields. The OPC generated by this protocol were able to mature into Myelin Basic Protein-expressing cells and to interact with neurons in an in vitro co-culture system. As a whole, we describe an optimized in vitro method for increasing OPC. PMID:25837625

Macrophages discriminate glycosylation patterns of apoptotic cell-derived microparticles.

PubMed

Bilyy, Rostyslav O; Shkandina, Tanya; Tomin, Andriy; Muñoz, Luis E; Franz, Sandra; Antonyuk, Volodymyr; Kit, Yuriy Ya; Zirngibl, Matthias; Fürnrohr, Barbara G; Janko, Christina; Lauber, Kirsten; Schiller, Martin; Schett, Georg; Stoika, Rostyslav S; Herrmann, Martin

2012-01-02

Inappropriate clearance of apoptotic remnants is considered to be the primary cause of systemic autoimmune diseases, like systemic lupus erythematosus. Here we demonstrate that apoptotic cells release distinct types of subcellular membranous particles (scMP) derived from the endoplasmic reticulum (ER) or the plasma membrane. Both types of scMP exhibit desialylated glycotopes resulting from surface exposure of immature ER-derived glycoproteins or from surface-borne sialidase activity, respectively. Sialidase activity is activated by caspase-dependent mechanisms during apoptosis. Cleavage of sialidase Neu1 by caspase 3 was shown to be directly involved in apoptosis-related increase of surface sialidase activity. ER-derived blebs possess immature mannosidic glycoepitopes and are prioritized by macrophages during clearance. Plasma membrane-derived blebs contain nuclear chromatin (DNA and histones) but not components of the nuclear envelope. Existence of two immunologically distinct types of apoptotic blebs may provide new insights into clearance-related diseases.

Functional screening assays with neurons generated from pluripotent stem cell-derived neural stem cells.

PubMed

Efthymiou, Anastasia; Shaltouki, Atossa; Steiner, Joseph P; Jha, Balendu; Heman-Ackah, Sabrina M; Swistowski, Andrzej; Zeng, Xianmin; Rao, Mahendra S; Malik, Nasir

2014-01-01

Rapid and effective drug discovery for neurodegenerative disease is currently impeded by an inability to source primary neural cells for high-throughput and phenotypic screens. This limitation can be addressed through the use of pluripotent stem cells (PSCs), which can be derived from patient-specific samples and differentiated to neural cells for use in identifying novel compounds for the treatment of neurodegenerative diseases. We have developed an efficient protocol to culture pure populations of neurons, as confirmed by gene expression analysis, in the 96-well format necessary for screens. These differentiated neurons were subjected to viability assays to illustrate their potential in future high-throughput screens. We have also shown that organelles such as nuclei and mitochondria could be live-labeled and visualized through fluorescence, suggesting that we should be able to monitor subcellular phenotypic changes. Neurons derived from a green fluorescent protein-expressing reporter line of PSCs were live-imaged to assess markers of neuronal maturation such as neurite length and co-cultured with astrocytes to demonstrate further maturation. These studies confirm that PSC-derived neurons can be used effectively in viability and functional assays and pave the way for high-throughput screens on neurons derived from patients with neurodegenerative disorders.

Hypoxia enhances the wound-healing potential of adipose-derived stem cells in a novel human primary keratinocyte-based scratch assay.

PubMed

Riis, Simone; Newman, Rhonda; Ipek, Hilal; Andersen, Jens I; Kuninger, David; Boucher, Shayne; Vemuri, Mohan C; Pennisi, Cristian P; Zachar, Vladimir; Fink, Trine

2017-03-01

Preclinical studies have suggested that paracrine factors from adipose-derived stem cells (ASCs) promote the healing of chronic wounds, and that the exposure of ASCs to hypoxia enhances their wound healing effect. To aid the translation of these findings into clinical use, robust wound models are necessary to explore each aspect of wound healing. The aspect of re-epithelization is often studied in a scratch assay based on transformed keratinocytes. However, there are concerns regarding the validity of this model, since these cell lines differ from normal keratinocytes, both in terms of proliferative capacity and differentiation, and sensitivity to environmental cues. In this study, the main challenge of using primary keratinocytes to examine the effects of ASCs was identified to be their different requirements for calcium in the culture media. We confirmed that a high calcium content led to morphological and cytoskeletal changes in primary keratinocytes, and demonstrated that a low calcium content compromised the growth of ASCs. We found that it is possible to perform the wound healing assay with primary keratinocytes, if the conditioned media from the ASCs is dialyzed to reduce the calcium concentration. Additionally, using this model of re-epithelization, conditioned media from normoxic ASCs was shown to markedly increase the rate of wound closure by primary keratinocytes, and this effect was significantly enhanced with media from the hypoxia-exposed ASCs. These findings, which are in line with the observations from previous in vivo studies, highlight the validity of this modified assay to investigate the wound healing properties of ASCs in vitro.

Construction of an in vitro primary lung co-culture platform derived from New Zealand white rabbits

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

Powell, Joshua D.; Hess, Becky M.; Hutchison, Janine R.

2015-05-01

We report the construction of an in vitro three dimensional (3D) co-culture platform consisting of differentiated lung epithelial cells and monocytes from New Zealand white rabbits. Rabbit lung epithelial cells were successfully grown at air-liquid interface, produced mucus, and expressed both sialic acid alpha-2,3 and alpha-2,6. Blood-derived CD14+ monocytes were deposited above the epithelial layer resulting in the differentiation of a subset of monocytes into CD11c+ cells within the co-culture. These proof-of-concept findings provide a convenient means to comparatively study in vitro versus in vivo rabbit lung responses as they relate to inhalation or lung-challenge studies.

Effect of Secreted Molecules of Human Embryonic Stem Cell-Derived Mesenchymal Stem Cells on Acute Hepatic Failure Model.

PubMed

Lotfinia, Majid; Kadivar, Mehdi; Piryaei, Abbas; Pournasr, Behshad; Sardari, Soroush; Sodeifi, Niloofar; Sayahpour, Forugh-Azam; Baharvand, Hossein

2016-12-15

Adult tissue-derived mesenchymal stem cells (MSCs) show tremendous promise for a wide array of therapeutic applications predominantly through paracrine activity. Recent reports showed that human embryonic stem cell (ESC)-derived MSCs are an alternative for regenerative cellular therapy due to manufacturing large quantities of MSCs from a single donor. However, no study has been reported to uncover the secretome of human ESC-MSCs as treatment of an acute liver failure (ALF) mouse model. We demonstrated that human ESC-MSCs showed similar morphology and cell surface markers compared with bone marrow-derived MSCs. ESC-MSCs exhibited a higher growth rate during early in vitro expansion, along with adipogenic and osteogenic differentiation potential. Treatment with ESC-MSC-conditioned medium (CM) led to statistically significant enhancement of primary hepatocyte viability and increased immunomodulatory interleukin-10 secretion from lipopolysaccharide-induced human blood mononuclear cells. Analysis of the MSCs secretome by a protein array screen showed an association between higher frequencies of secretory proteins such as vascular endothelial growth factor (VEGF) and regulation of cell proliferation, cell migration, the development process, immune system process, and apoptosis. In this thioacetamide-induced mouse model of acute liver injury, we observed that systemic infusion of VEGF led to significant survival. These data have provided the first experimental evidence of the therapeutic potential of human ESC-MSC-derived molecules. These molecules show trophic support to hepatocytes, which potentially creates new avenues for the treatment of ALF, as an inflammatory condition.

Versican-Derived Matrikines Regulate Batf3-Dendritic Cell Differentiation and Promote T Cell Infiltration in Colorectal Cancer.

PubMed

Hope, Chelsea; Emmerich, Philip B; Papadas, Athanasios; Pagenkopf, Adam; Matkowskyj, Kristina A; Van De Hey, Dana R; Payne, Susan N; Clipson, Linda; Callander, Natalie S; Hematti, Peiman; Miyamoto, Shigeki; Johnson, Michael G; Deming, Dustin A; Asimakopoulos, Fotis

2017-09-01

Colorectal cancer originates within immunologically complex microenvironments. To date, the benefits of immunotherapy have been modest, except in neoantigen-laden mismatch repair-deficient tumors. Approaches to enhance tumor-infiltrating lymphocytes in the tumor bed may substantially augment clinical immunotherapy responses. In this article, we report that proteolysis of the tolerogenic matrix proteoglycan versican (VCAN) strongly correlated with CD8 + T cell infiltration in colorectal cancer, regardless of mismatch repair status. Tumors displaying active VCAN proteolysis and low total VCAN were associated with robust (10-fold) CD8 + T cell infiltration. Tumor-intrinsic WNT pathway activation was associated with CD8 + T cell exclusion and VCAN accumulation. In addition to regulating VCAN levels at the tumor site, VCAN proteolysis results in the generation of bioactive fragments with novel functions (VCAN-derived matrikines). Versikine, a VCAN-derived matrikine, enhanced the generation of CD103 + CD11c hi MHCII hi conventional dendritic cells (cDCs) from Flt3L-mobilized primary bone marrow-derived progenitors, suggesting that VCAN proteolysis may promote differentiation of tumor-seeding DC precursors toward IRF8- and BATF3-expressing cDCs. Intratumoral BATF3-dependent DCs are critical determinants for T cell antitumor immunity, effector T cell trafficking to the tumor site, and response to immunotherapies. Our findings provide a rationale for testing VCAN proteolysis as a predictive and/or prognostic immune biomarker and VCAN-derived matrikines as novel immunotherapy agents. Copyright © 2017 by The American Association of Immunologists, Inc.

Cutting Edge: Inflammasome Activation in Primary Human Macrophages Is Dependent on Flagellin

PubMed Central

Kortmann, Jens; Brubaker, Sky W.

2015-01-01

Murine NLR family, apoptosis inhibitory protein (Naip)1, Naip2, and Naip5/6 are host sensors that detect the cytosolic presence of needle and rod proteins from bacterial type III secretion systems and flagellin, respectively. Previous studies using human-derived macrophage-like cell lines indicate that human macrophages sense the cytosolic needle protein, but not bacterial flagellin. In this study, we show that primary human macrophages readily sense cytosolic flagellin. Infection of primary human macrophages with Salmonella elicits robust cell death and IL-1β secretion that is dependent on flagellin. We show that flagellin detection requires a full-length isoform of human Naip. This full-length Naip isoform is robustly expressed in primary macrophages from healthy human donors, but it is drastically reduced in monocytic tumor cells, THP-1, and U937, rendering them insensitive to cytosolic flagellin. However, ectopic expression of full-length Naip rescues the ability of U937 cells to sense flagellin. In conclusion, human Naip functions to activate the inflammasome in response to flagellin, similar to murine Naip5/6. PMID:26109648

Novel phage display-derived neuroblastoma-targeting peptides potentiate the effect of drug nanocarriers in preclinical settings.

PubMed

Loi, Monica; Di Paolo, Daniela; Soster, Marco; Brignole, Chiara; Bartolini, Alice; Emionite, Laura; Sun, Jessica; Becherini, Pamela; Curnis, Flavio; Petretto, Andrea; Sani, Monica; Gori, Alessandro; Milanese, Marco; Gambini, Claudio; Longhi, Renato; Cilli, Michele; Allen, Theresa M; Bussolino, Federico; Arap, Wadih; Pasqualini, Renata; Corti, Angelo; Ponzoni, Mirco; Marchiò, Serena; Pastorino, Fabio

2013-09-10

Molecular targeting of drug delivery nanocarriers is expected to improve their therapeutic index while decreasing their toxicity. Here we report the identification and characterization of novel peptide ligands specific for cells present in high-risk neuroblastoma (NB), a childhood tumor mostly refractory to current therapies. To isolate such targeting moieties, we performed combined in vitro/ex-vivo phage display screenings on NB cell lines and on tumors derived from orthotopic mouse models of human NB. By designing proper subtractive protocols, we identified phage clones specific either for the primary tumor, its metastases, or for their respective stromal components. Globally, we isolated 121 phage-displayed NB-binding peptides: 26 bound the primary tumor, 15 the metastatic mass, 57 and 23 their respective microenvironments. Of these, five phage clones were further validated for their specific binding ex-vivo to biopsies from stage IV NB patients and to NB tumors derived from mice. All five clones also targeted tumor cells and vasculature in vivo when injected into NB-bearing mice. Coupling of the corresponding targeting peptides with doxorubicin-loaded liposomes led to a significant inhibition in tumor volume and enhanced survival in preclinical NB models, thereby paving the way to their clinical development. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

p19-targeted ABD-derived protein variants inhibit IL-23 binding and exert suppressive control over IL-23-stimulated expansion of primary human IL-17+ T-cells.

PubMed

Křížová, Lucie; Kuchař, Milan; Petroková, Hana; Osička, Radim; Hlavničková, Marie; Pelák, Ondřej; Černý, Jiří; Kalina, Tomáš; Malý, Petr

2017-03-01

Interleukin-23 (IL-23), a heterodimeric cytokine of covalently bound p19 and p40 proteins, has recently been closely associated with development of several chronic autoimmune diseases such as psoriasis, psoriatic arthritis or inflammatory bowel disease. Released by activated dendritic cells, IL-23 interacts with IL-23 receptor (IL-23R) on Th17 cells, thus promoting intracellular signaling, a pivotal step in Th17-driven pro-inflammatory axis. Here, we aimed to block the binding of IL-23 cytokine to its cell-surface receptor by novel inhibitory protein binders targeted to the p19 subunit of human IL-23. To this goal, we used a combinatorial library derived from a scaffold of albumin-binding domain (ABD) of streptococcal protein G, and ribosome display selection, to yield a collection of ABD-derived p19-targeted variants, called ILP binders. From 214 clones analyzed by ELISA, Western blot and DNA sequencing, 53 provided 35 different sequence variants that were further characterized. Using in silico docking in combination with cell-surface competition binding assay, we identified a group of inhibitory candidates that substantially diminished binding of recombinant p19 to the IL-23R on human monocytic THP-1 cells. Of these best p19-blockers, ILP030, ILP317 and ILP323 inhibited IL-23-driven expansion of IL-17-producing primary human CD4 +  T-cells. Thus, these novel binders represent unique IL-23-targeted probes useful for IL-23/IL-23R epitope mapping studies and could be used for designing novel p19/IL-23-targeted anti-inflammatory biologics.

Differentiation-Dependent Energy Production and Metabolite Utilization: A Comparative Study on Neural Stem Cells, Neurons, and Astrocytes

PubMed Central

Jády, Attila Gy.; Nagy, Ádám M.; Kőhidi, Tímea; Ferenczi, Szilamér; Tretter, László

2016-01-01

While it is evident that the metabolic machinery of stem cells should be fairly different from that of differentiated neurons, the basic energy production pathways in neural stem cells (NSCs) or in neurons are far from clear. Using the model of in vitro neuron production by NE-4C NSCs, this study focused on the metabolic changes taking place during the in vitro neuronal differentiation. O2 consumption, H+ production, and metabolic responses to single metabolites were measured in cultures of NSCs and in their neuronal derivatives, as well as in primary neuronal and astroglial cultures. In metabolite-free solutions, NSCs consumed little O2 and displayed a higher level of mitochondrial proton leak than neurons. In stem cells, glycolysis was the main source of energy for the survival of a 2.5-h period of metabolite deprivation. In contrast, stem cell-derived or primary neurons sustained a high-level oxidative phosphorylation during metabolite deprivation, indicating the consumption of own cellular material for energy production. The stem cells increased O2 consumption and mitochondrial ATP production in response to single metabolites (with the exception of glucose), showing rapid adaptation of the metabolic machinery to the available resources. In contrast, single metabolites did not increase the O2 consumption of neurons or astrocytes. In “starving” neurons, neither lactate nor pyruvate was utilized for mitochondrial ATP production. Gene expression studies also suggested that aerobic glycolysis and rapid metabolic adaptation characterize the NE-4C NSCs, while autophagy and alternative glucose utilization play important roles in the metabolism of stem cell-derived neurons. PMID:27116891

Antitumor activity of the investigational proteasome inhibitor MLN9708 in mouse models of B-cell and plasma cell malignancies.

PubMed

Lee, Edmund C; Fitzgerald, Michael; Bannerman, Bret; Donelan, Jill; Bano, Kristen; Terkelsen, Jennifer; Bradley, Daniel P; Subakan, Ozlem; Silva, Matthew D; Liu, Ray; Pickard, Michael; Li, Zhi; Tayber, Olga; Li, Ping; Hales, Paul; Carsillo, Mary; Neppalli, Vishala T; Berger, Allison J; Kupperman, Erik; Manfredi, Mark; Bolen, Joseph B; Van Ness, Brian; Janz, Siegfried

2011-12-01

The clinical success of the first-in-class proteasome inhibitor bortezomib (VELCADE) has validated the proteasome as a therapeutic target for treating human cancers. MLN9708 is an investigational proteasome inhibitor that, compared with bortezomib, has improved pharmacokinetics, pharmacodynamics, and antitumor activity in preclinical studies. Here, we focused on evaluating the in vivo activity of MLN2238 (the biologically active form of MLN9708) in a variety of mouse models of hematologic malignancies, including tumor xenograft models derived from a human lymphoma cell line and primary human lymphoma tissue, and genetically engineered mouse (GEM) models of plasma cell malignancies (PCM). Both cell line-derived OCI-Ly10 and primary human lymphoma-derived PHTX22L xenograft models of diffuse large B-cell lymphoma were used to evaluate the pharmacodynamics and antitumor effects of MLN2238 and bortezomib. The iMyc(Cα)/Bcl-X(L) GEM model was used to assess their effects on de novo PCM and overall survival. The newly developed DP54-Luc-disseminated model of iMyc(Cα)/Bcl-X(L) was used to determine antitumor activity and effects on osteolytic bone disease. MLN2238 has an improved pharmacodynamic profile and antitumor activity compared with bortezomib in both OCI-Ly10 and PHTX22L models. Although both MLN2238 and bortezomib prolonged overall survival, reduced splenomegaly, and attenuated IgG2a levels in the iMyc(Cα)/Bcl-X(L) GEM model, only MLN2238 alleviated osteolytic bone disease in the DP54-Luc model. Our results clearly showed the antitumor activity of MLN2238 in a variety of mouse models of B-cell lymphoma and PCM, supporting its clinical development. MLN9708 is being evaluated in multiple phase I and I/II trials. ©2011 AACR.

Elevated Mitochondrial Reactive Oxygen Species and Cellular Redox Imbalance in Human NADPH-Oxidase-Deficient Phagocytes

PubMed Central

Sundqvist, Martina; Christenson, Karin; Björnsdottir, Halla; Osla, Veronica; Karlsson, Anna; Dahlgren, Claes; Speert, David P.; Fasth, Anders; Brown, Kelly L.; Bylund, Johan

2017-01-01

Chronic granulomatous disease (CGD) is caused by mutations in genes that encode the NADPH-oxidase and result in a failure of phagocytic cells to produce reactive oxygen species (ROS) via this enzyme system. Patients with CGD are highly susceptible to infections and often suffer from inflammatory disorders; the latter occurs in the absence of infection and correlates with the spontaneous production of inflammatory cytokines. This clinical feature suggests that NADPH-oxidase-derived ROS are not required for, or may even suppress, inflammatory processes. Experimental evidence, however, implies that ROS are in fact required for inflammatory cytokine production. By using a myeloid cell line devoid of a functional NADPH-oxidase and primary CGD cells, we analyzed intracellular oxidants, signs of oxidative stress, and inflammatory cytokine production. Herein, we demonstrate that phagocytes lacking a functional NADPH-oxidase, namely primary CGD phagocytes and a gp91phox-deficient cell line, display elevated levels of ROS derived from mitochondria. Accordingly, these cells, despite lacking the major source of cellular ROS, display clear signs of oxidative stress, including an induced expression of antioxidants and altered oxidation of cell surface thiols. These observed changes in redox state were not due to abnormalities in mitochondrial mass or membrane integrity. Finally, we demonstrate that increased mitochondrial ROS enhanced phosphorylation of ERK1/2, and induced production of IL8, findings that correlate with previous observations of increased MAPK activation and inflammatory cytokine production in CGD cells. Our data show that elevated baseline levels of mitochondria-derived oxidants lead to the counter-intuitive observation that CGD phagocytes are under oxidative stress and have enhanced MAPK signaling, which may contribute to the elevated basal production of inflammatory cytokines and the sterile inflammatory manifestations in CGD. PMID:29375548

Evaluation of human embryonic stem cells and their differentiated fibroblastic progenies as cellular models for in vitro genotoxicity screening.

PubMed

Vinoth, Kumar Jayaseelan; Manikandan, Jayapal; Sethu, Swaminathan; Balakrishnan, Lakshmidevi; Heng, Alexis; Lu, Kai; Hande, Manoor Prakash; Cao, Tong

2014-08-20

This study evaluated human embryonic stem cells (hESC) and their differentiated fibroblastic progenies as cellular models for genotoxicity screening. The DNA damage response of hESCs and their differentiated fibroblastic progenies were compared to a fibroblastic cell line (HEPM, CRL1486) and primary cultures of peripheral blood lymphocytes (PBL), upon exposure to Mitomycin C, gamma irradiation and H2O2. It was demonstrated that hESC-derived fibroblastic progenies (H1F) displayed significantly higher chromosomal aberrations, micronuclei formation and double strand break (DSB) formation, as compared to undifferentiated hESC upon exposure to genotoxic stress. Nevertheless, H1F cell types displayed comparable sensitivities to genotoxic challenge as HEPM and PBL, both of which are representative of somatic cell types commonly used for genotoxicity screening. Subsequently, transcriptomic and pathways analysis identified differential expression of critical genes involved in cell death and DNA damage response upon exposure to gamma irradiation. The results thus demonstrate that hESC-derived fibroblastic progenies are as sensitive as commonly-used somatic cell types for genotoxicity screening. Moreover, hESCs have additional advantages, such as their genetic normality compared to immortalized cell lines, as well as their amenability to scale-up for producing large, standardized quantities of cells for genotoxicity screening on an industrial scale, something which can never be achieved with primary cell cultures. Copyright © 2014. Published by Elsevier B.V.

The lack of CD131 and the inhibition of Neuro-2a growth by carbamylated erythropoietin.

PubMed

Ding, Jing; Li, Qin-Ying; Yu, Jie-Zhong; Wang, Xin; Lu, Chuan-Zhen; Ma, Cun-Gen; Xiao, Bao-Guo

2015-02-01

Recombinant human erythropoietin (EPO), a glycohormone, is one of the leading biopharmaceutical products, while carbamylated erythropoietin (CEPO), an EPO derivative, is attracting widespread interest due to its neuroprotective effects without erythropoiesis in several cells and animal models. However, exogenous EPO promotes an angiogenic response from tumor cells and is associated with tumor growth, but knowledge of CEPO on tumor growth is lacking. Here we show that CEPO, but not EPO, inhibited Neuro-2a growth and viability. As expected, CEPO--unlike EPO--did not activate JAK-2 either in primary neurons or in Neuro-2a cells. Interestingly, CEPO did not induce GDNF expression and subsequent AKT activation in Neuro-2a cells. Before CEPO/EPO treatment, glial cell line-derived neurotrophic factor (GDNF) neutralization and GFR receptor blocking decreased the viability of EPO-treated Neuro-2a cells but did not influence CEPO-treated Neuro-2a cells. As compared to primary neurons, the expression of CD131, as a receptor complex binding to CEPO, is almost lacking in Neuro-2a cells. In BABL/C-nu mice, CEPO did not promote the growth of Neuro-2a cells nor extended the survival time compared to mice treated with EPO. The results indicate that CEPO did not promote tumor growth because of lower expression of CD131 and subsequent dysfunction of CD131/GDNF/AKT pathway in Neuro-2a cells, revealing its therapeutic potential in future clinical application.

Fibroblasts derived from long-lived insulin receptor substrate 1 null mice are not resistant to multiple forms of stress

PubMed Central

Page, Melissa M; Sinclair, Amy; Robb, Ellen L; Stuart, Jeffrey A; Withers, Dominic J; Selman, Colin

2014-01-01

Reduced signalling through the insulin/insulin-like growth factor-1 signalling (IIS) pathway is a highly conserved lifespan determinant in model organisms. The precise mechanism underlying the effects of the IIS on lifespan and health is currently unclear, although cellular stress resistance may be important. We have previously demonstrated that mice globally lacking insulin receptor substrate 1 (Irs1−/−) are long-lived and enjoy a greater period of their life free from age-related pathology compared with wild-type (WT) controls. In this study, we show that primary dermal fibroblasts and primary myoblasts derived from Irs1−/− mice are no more resistant to a range of oxidant and nonoxidant chemical stressors than cells derived from WT mice. PMID:25059507

Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation.

PubMed

Lanik, Wyatt E; Xu, Lily; Luke, Cliff J; Hu, Elise Z; Agrawal, Pranjal; Liu, Victoria S; Kumar, Rajesh; Bolock, Alexa M; Ma, Congrong; Good, Misty

2018-02-15

Human small intestinal enteroids are derived from the crypts and when grown in a stem cell niche contain all of the epithelial cell types. The ability to establish human enteroid ex vivo culture systems are important to model intestinal pathophysiology and to study the particular cellular responses involved. In recent years, enteroids from mice and humans are being cultured, passaged, and banked away for future use in several laboratories across the world. This enteroid platform can be used to test the effects of various treatments and drugs and what effects are exerted on different cell types in the intestine. Here, a protocol for establishing primary stem cell-derived small intestinal enteroids derived from neonatal mice and premature human intestine is provided. Moreover, this enteroid culture system was utilized to test the effects of species-specific breast milk. Mouse breast milk can be obtained efficiently using a modified human breast pump and expressed mouse milk can then be used for further research experiments. We now demonstrate the effects of expressed mouse, human, and donor breast milk on the growth and proliferation of enteroids derived from neonatal mice or premature human small intestine.

From skin biopsy to neurons through a pluripotent intermediate under Good Manufacturing Practice protocols.

PubMed

Karumbayaram, Saravanan; Lee, Peiyee; Azghadi, Soheila F; Cooper, Aaron R; Patterson, Michaela; Kohn, Donald B; Pyle, April; Clark, Amander; Byrne, James; Zack, Jerome A; Plath, Kathrin; Lowry, William E

2012-01-01

The clinical application of human-induced pluripotent stem cells (hiPSCs) requires not only the production of Good Manufacturing Practice-grade (GMP-grade) hiPSCs but also the derivation of specified cell types for transplantation under GMP conditions. Previous reports have suggested that hiPSCs can be produced in the absence of animal-derived reagents (xenobiotics) to ease the transition to production under GMP standards. However, to facilitate the use of hiPSCs in cell-based therapeutics, their progeny should be produced not only in the absence of xenobiotics but also under GMP conditions requiring extensive standardization of protocols, documentation, and reproducibility of methods and product. Here, we present a successful framework to produce GMP-grade derivatives of hiPSCs that are free of xenobiotic exposure from the collection of patient fibroblasts, through reprogramming, maintenance of hiPSCs, identification of reprogramming vector integration sites (nrLAM-PCR), and finally specification and terminal differentiation of clinically relevant cells. Furthermore, we developed a primary set of Standard Operating Procedures for the GMP-grade derivation and differentiation of these cells as a resource to facilitate widespread adoption of these practices.

A hanging drop culture method to study terminal erythroid differentiation.

PubMed

Gutiérrez, Laura; Lindeboom, Fokke; Ferreira, Rita; Drissen, Roy; Grosveld, Frank; Whyatt, David; Philipsen, Sjaak

2005-10-01

To design a culture method allowing the quantitative and qualitative analysis of terminal erythroid differentiation. Primary erythroid progenitors derived either from mouse tissues or from human umbilical cord blood were differentiated using hanging drop cultures and compared to methylcellulose cultures. Cultured cells were analyzed by FACS to assess differentiation. We describe a practical culture method by adapting the previously described hanging drop culture system to conditions allowing terminal differentiation of primary erythroid progenitors. Using minimal volumes of media and small numbers of cells, we obtained quantitative terminal erythroid differentiation within two days of culture in the case of murine cells and 4 days in the case of human cells. The established methods for ex vivo culture of primary erythroid progenitors, such as methylcellulose-based burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) assays, allow the detection of committed erythroid progenitors but are of limited value to study terminal erythroid differentiation. We show that the application of hanging drop cultures is a practical alternative that, in combination with clonogenic assays, enables a comprehensive assessment of the behavior of primary erythroid cells ex vivo in the context of genetic and drug-induced perturbations.

A correlative and quantitative imaging approach enabling characterization of primary cell-cell communication: Case of human CD4+ T cell-macrophage immunological synapses.

PubMed

Kasprowicz, Richard; Rand, Emma; O'Toole, Peter J; Signoret, Nathalie

2018-05-22

Cell-to-cell communication engages signaling and spatiotemporal reorganization events driven by highly context-dependent and dynamic intercellular interactions, which are difficult to capture within heterogeneous primary cell cultures. Here, we present a straightforward correlative imaging approach utilizing commonly available instrumentation to sample large numbers of cell-cell interaction events, allowing qualitative and quantitative characterization of rare functioning cell-conjugates based on calcium signals. We applied this approach to examine a previously uncharacterized immunological synapse, investigating autologous human blood CD4 + T cells and monocyte-derived macrophages (MDMs) forming functional conjugates in vitro. Populations of signaling conjugates were visualized, tracked and analyzed by combining live imaging, calcium recording and multivariate statistical analysis. Correlative immunofluorescence was added to quantify endogenous molecular recruitments at the cell-cell junction. By analyzing a large number of rare conjugates, we were able to define calcium signatures associated with different states of CD4 + T cell-MDM interactions. Quantitative image analysis of immunostained conjugates detected the propensity of endogenous T cell surface markers and intracellular organelles to polarize towards cell-cell junctions with high and sustained calcium signaling profiles, hence defining immunological synapses. Overall, we developed a broadly applicable approach enabling detailed single cell- and population-based investigations of rare cell-cell communication events with primary cells.

Novel Derivative of Benzofuran Induces Cell Death Mostly by G2/M Cell Cycle Arrest through p53-dependent Pathway but Partially by Inhibition of NF-κB*

PubMed Central

Manna, Sunil K.; Bose, Julie S.; Gangan, Vijay; Raviprakash, Nune; Navaneetha, Thota; Raghavendra, Pongali B.; Babajan, Banaganapalli; Kumar, Chitta S.; Jain, Swatantra K.

2010-01-01

The Dracaena resin is widely used in traditional medicine as an anticancer agent, and benzofuran lignan is the active component. In this report, we provide evidence that the synthetic derivative of benzofuran lignan (Benfur) showed antitumor activities. It induced apoptosis in p53-positive cells. Though it inhibited endotoxin-induced nuclear factor κB (NF-κB) activation in both p53-positive and -negative cells, the activation of caspase 3 was observed in p53-positive cells. It showed partial cell death effect in both p53-positive and -negative cells through inhibition of NF-κB. Cell cycle analysis using flow cytometry showed that treatment with this novel benozofuran lignan derivative to Jurkat T-cells, but not U-937 cells, resulted in a G2/M arrest in a dose- and time-dependent manner. It increased amounts of p21, p27, and cyclin B, but not phospho-Rb through p53 nuclear translocation in Jurkat T-cells, but not in U-937 cells. It inhibited amounts of MDM2 (murine double minute 2) by repressing the transcription factor Sp1, which was also proved in silico. It induced cell death in tumor cells, but not in primary T-cells. Overall, our data suggest that Benfur-mediated cell death is partially dependent upon NF-κB, but predominantly dependent on p53. Thus, this novel benzofuran lignan derivative can be effective chemopreventive or chemotherapeutic agent against malignant T-cells. PMID:20472557

A fat option for the pig: Hepatocytic differentiated mesenchymal stem cells for translational research

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

Brückner, Sandra, E-mail: sandra.brueckner@medizin.uni-leipzig.de; Tautenhahn, Hans-Michael, E-mail: hans-michael.tautenhahn@medizin.uni-leipzig.de; TRM, Translational Centre for Regenerative Medicine, Philipp-Rosenthal-Str. 55, Leipzig D-04103

Study background: Extended liver resection is the only curative treatment option of liver cancer. Yet, the residual liver may not accomplish the high metabolic and regenerative capacity needed, which frequently leads to acute liver failure. Because of their anti-inflammatory and -apoptotic as well as pro-proliferative features, mesenchymal stem cells differentiated into hepatocyte-like cells might provide functional and regenerative compensation. Clinical translation of basic research requires pre-clinical approval in large animals. Therefore, we characterized porcine mesenchymal stem cells (MSC) from adipose tissue and bone marrow and their hepatocyte differentiation potential for future assessment of functional liver support after surgical intervention inmore » the pig model. Methods: Mesenchymal surface antigens and multi-lineage differentiation potential of porcine MSC isolated by collagenase digestion either from bone marrow or adipose tissue (subcutaneous/visceral) were assessed by flow cytometry. Morphology and functional properties (urea-, glycogen synthesis and cytochrome P450 activity) were determined during culture under differentiation conditions and compared with primary porcine hepatocytes. Results: MSC from porcine adipose tissue and from bone marrow express the typical mesenchymal markers CD44, CD29, CD90 and CD105 but not haematopoietic markers. MSC from both sources displayed differentiation into the osteogenic as well as adipogenic lineage. After hepatocyte differentiation, expression of CD105 decreased significantly and cells adopted the typical polygonal morphology of hepatocytes. Glycogen storage was comparable in adipose tissue- and bone marrow-derived cells. Urea synthesis was about 35% lower in visceral than in subcutaneous adipose tissue-derived MSC. Cytochrome P450 activity increased significantly during differentiation and was twice as high in hepatocyte-like cells generated from bone marrow as from adipose tissue. Conclusion: The hepatocyte differentiation of porcine adipose tissue-derived MSC was shown for the first time yielding hepatocyte-like cells with specific functions similar in bone marrow and subcutaneous adipose tissue-derived MSC. That makes them good pre-clinical candidates for supportive approaches after liver resection in the pig. - Highlights: • First time to show hepatocytic differentiation of porcine adipose tissue-derived MSC. • Hepatocytic-differentiated MSC display metabolic qualities of primary hepatocytes. • Metabolic potency varies between differentiated MSC from different tissues. • MSC are good candidates for pre-clinical evaluation of stem cell-based therapies.« less

Pancreatic ductal cells acquire mesenchymal characteristics through cell fusion with bone marrow-derived mesenchymal stem cells and SIRT1 attenuates the apoptosis of hybrid cells.

PubMed

Gou, Shanmiao; Liu, Tao; Li, Xiangsheng; Cui, Jing; Wan, Chidan; Wang, Chunyou

2012-01-01

Bone marrow-derived mesenchymal stem cells (bMSCs) contribute to tissue repair and regeneration. Cell fusion between somatic cells and bMSCs to form hybrid cells may have an important role in tissue repair through the subsequent reprogramming of the somatic cell nucleus. Few studies have assessed the mesenchymal characteristics of fusion-induced hybrid cells and their survival mechanisms. In this study, we investigated the effect of cell fusion on the biological characteristics of pancreatic ductal cells (PDCs) and on the survival mechanism of hybrid cells. To this end, we generated mouse-mouse hybrid cells in vitro by polyethylene glycol-mediated fusion of primary mouse bMSCs with primary mouse PDCs. Hybrid cells showed an enhanced capacity for proliferation and self-renewal compared with PDCs. No PDC had the capacity for anchorage-independent growth or invasion into Matrigel, but some hybrid cells were able to form colonies in soft agar and invade Matrigel. Expression of the tumor suppressor protein p53, which initiates apoptosis, was detected in hybrid cells but not in PDCs or bMSCs. However, the p53 deacetylase, sirtuin 1 (SIRT1), was also detected in hybrid cells, and the level of acetylated p53, the active form, was low. The addition of nicotinamide (Nam) inhibited the deacetylation activity of SIRT1 on p53 and induced cell apoptosis in hybrid cells. This study demonstrated that PDCs could obtain high proliferation rates, self-renewal capabilities, and mesenchymal characteristics by fusion with bMSCs. SIRT1 expression in the hybrid cells attenuated their apoptosis. Copyright © 2012 S. Karger AG, Basel.

Transformation of primary chick embryo fibroblasts by Marek's disease virus.

PubMed

Buranathai, C; Rodriguez, J; Grose, C

1997-12-08

Marek's disease virus (MDV) is an alphaherpesvirus, which can mediate the malignant transformation of lymphocytes to form lymphomas in chickens. In this study, we demonstrate that MDV can transform primary chick embryo fibroblasts (CEF). The cell line derived from primary CEF infected with the GA strain of MDV was called CEM(MDV). The fibroblast nature of CEM(MDV) was verified by absence of cytokeratin type II. The CEM(MDV) phenotype differed from either primary CEF or MDV-infected CEF. CEM(MDV) were extensively vacuolated, with unusual multilamellar structures in the cytoplasm, The nuclei were considerably larger than those in primary CEF and were uniformly positive for proliferating cell nuclear antigen. The cell line was subcultured for more than 10 generations; however, CEM(MDV) did not support a fully productive MDV infection, because complete nucleocapsids were not detected and infectivity assays showed that cell line produced no infectious virus. PCR analyses demonstrated that this cell line carried both polypeptide 38 (pp38) and Meq DNA, MDV-specific genes associated with transformation. In addition, examination by laser scanning confocal microscopy revealed that CEM(MDV) constitutively produced MDV MEQ protein in nuclei and pp38 as well as glycoprotein B in the cytoplasm and on the plasma membrane. Growth in soft agar assay demonstrated that CEM(MDV) formed colonies, similar to HeLa and human melanoma cells. Retroviral insertion was not detected in DNA from the CEM(MDV) line.

Recombinant rabbit leukemia inhibitory factor and rabbit embryonic fibroblasts support the derivation and maintenance of rabbit embryonic stem cells.

PubMed

Xue, Fei; Ma, Yinghong; Chen, Y Eugene; Zhang, Jifeng; Lin, Tzu-An; Chen, Chien-Hong; Lin, Wei-Wen; Roach, Marsha; Ju, Jyh-Cherng; Yang, Lan; Du, Fuliang; Xu, Jie

2012-08-01

The rabbit is a classical experimental animal species. A major limitation in using rabbits for biomedical research is the lack of germ-line-competent rabbit embryonic stem cells (rbESCs). We hypothesized that the use of homologous feeder cells and recombinant rabbit leukemia inhibitory factor (rbLIF) might improve the chance in deriving germ-line-competent rbES cells. In the present study, we established rabbit embryonic fibroblast (REF) feeder layers and synthesized recombinant rbLIF. We derived a total of seven putative rbESC lines, of which two lines (M5 and M23) were from culture Condition I using mouse embryonic fibroblasts (MEFs) as feeders supplemented with human LIF (hLIF) (MEF+hLIF). Another five lines (R4, R9, R15, R21, and R31) were derived from Condition II using REFs as feeder cells supplemented with rbLIF (REF+rbLIF). Similar derivation efficiency was observed between these two conditions (8.7% vs. 10.2%). In a separate experiment with 2×3 factorial design, we examined the effects of feeder cells (MEF vs. REF) and LIFs (mLIF, hLIF vs. rbLIF) on rbESC culture. Both Conditions I and II supported satisfactory rbESC culture, with similar or better population doubling time and colony-forming efficiency than other combinations of feeder cells with LIFs. Rabbit ESCs derived and maintained on both conditions displayed typical ESC characteristics, including ESC pluripotency marker expression (AP, Oct4, Sox2, Nanog, and SSEA4) and gene expression (Oct4, Sox2, Nanog, c-Myc, Klf4, and Dppa5), and the capacity to differentiate into three primary germ layers in vitro. The present work is the first attempt to establish rbESC lines using homologous feeder cells and recombinant rbLIF, by which the rbESCs were derived and maintained normally. These cell lines are unique resources and may facilitate the derivation of germ-line-competent rbESCs.

Induced Pluripotent Stem (iPS) Cells in Dentistry: A Review

PubMed Central

Malhotra, Neeraj

2016-01-01

iPS cells are derived from somatic cells via transduction and expression of selective transcription factors. Both viral-integrating (like retroviral) and non-integrating (like, mRNA or protein-based) techniques are available for the production of iPS cells. In the field of dentistry, iPS cells have been derived from stem cells of apical papilla, dental pulp stem cells, and stem cells from exfoliated deciduous teeth, gingival and periodontal ligament fibroblasts, and buccal mucosa fibroblasts. iPS cells have the potential to differentiate into all derivatives of the 3 primary germ layers i.e. ectoderm, endoderm, and mesoderm. They are autogeneically accessible, and can produce patient-specific or disease-specific cell lines without the issue of ethical controversy. They have been successfully tested to produce mesenchymal stem cells-like cells, neural crest-like cells, ameloblasts-like cells, odontoblasts-like cells, and osteoprogenitor cells. These cells can aid in regeneration of periodontal ligament, alveolar bone, cementum, dentin-pulp complex, as well as possible Biotooth formation. However certain key issues like, epigenetic memory of iPS cells, viral-transduction, tumorgenesis and teratoma formation need to be overcome, before they can be successfully used in clinical practice. The article discusses the sources, pros and cons, and current applications of iPS cells in dentistry with an emphasis on encountered challenges and their solutions. PMID:27572712

MUTZ-3 derived Langerhans cells in human skin equivalents show differential migration and phenotypic plasticity after allergen or irritant exposure

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

Kosten, Ilona J.; Spiekstra, Sander W.; Gruijl, Tanja D. de

After allergen or irritant exposure, Langerhans cells (LC) undergo phenotypic changes and exit the epidermis. In this study we describe the unique ability of MUTZ-3 derived Langerhans cells (MUTZ-LC) to display similar phenotypic plasticity as their primary counterparts when incorporated into a physiologically relevant full-thickness skin equivalent model (SE-LC). We describe differences and similarities in the mechanisms regulating LC migration and plasticity upon allergen or irritant exposure. The skin equivalent consisted of a reconstructed epidermis containing primary differentiated keratinocytes and CD1a{sup +} MUTZ-LC on a primary fibroblast-populated dermis. Skin equivalents were exposed to a panel of allergens and irritants. Topicalmore » exposure to sub-toxic concentrations of allergens (nickel sulfate, resorcinol, cinnamaldehyde) and irritants (Triton X-100, SDS, Tween 80) resulted in LC migration out of the epidermis and into the dermis. Neutralizing antibody to CXCL12 blocked allergen-induced migration, whereas anti-CCL5 blocked irritant-induced migration. In contrast to allergen exposure, irritant exposure resulted in cells within the dermis becoming CD1a{sup −}/CD14{sup +}/CD68{sup +} which is characteristic of a phenotypic switch of MUTZ-LC to a macrophage-like cell in the dermis. This phenotypic switch was blocked with anti-IL-10. Mechanisms previously identified as being involved in LC activation and migration in native human skin could thus be reproduced in the in vitro constructed skin equivalent model containing functional LC. This model therefore provides a unique and relevant research tool to study human LC biology in situ under controlled in vitro conditions, and will provide a powerful tool for hazard identification, testing novel therapeutics and identifying new drug targets. - Highlights: • MUTZ-3 derived Langerhans cells integrated into skin equivalents are fully functional. • Anti-CXCL12 blocks allergen-induced MUTZ-LC migration. • Anti-CCL5 blocks irritant-induced MUTZ-LC migration. • Irritant mediated MUTZ-LC trans-differentiation to macrophage-like cell in dermis. • Trans-differentiation of MUTZ-LC is IL-10 dependent.« less

Which has more stem-cell characteristics: Müller cells or Müller cells derived from in vivo culture in neurospheres?

PubMed

Ji, Hong-Pei; Xiong, Yu; Zhang, En-Dong; Song, Wei-Tao; Gao, Zhao-Lin; Yao, Fei; Sun, Hong; Zhou, Rong-Rong; Xia, Xiao-Bo

2017-01-01

Müller cells can be acquired from in vitro culture or a neurosphere culture system. Both culture methods yield cells with progenitor-cell characteristics that can differentiate into mature nervous cells. We compared the progenitor-cell traits of Müller cells acquired from each method. Primary murine Müller cells were isolated in serum culture media and used to generate Müller cells derived from neurospheres in serum-free culture conditions. Gene expression of neural progenitor cell markers was examined by Q-PCR in the two groups. Expression of rhodopsin and the cone-rod homeobox protein CRX were assessed after induction with 1 μM all-trans retinoic acid (RA) for 7 days. After more than four passages, many cells were large, flattened, and difficult to passage. A spontaneously immortalized Müller cell line was not established. Three-passage neurospheres yielded few new spheres. Genes coding for Nestin, Sox2, Chx10, and Vimentin were downregulated in cells derived from neurospheres compared to the cells from standard culture, while Pax6 was upregulated. Müller cells from both culture methods were induced into rod photoreceptors, but expression of rhodopsin and CRX was greater in the Müller cells from the standard culture. Both culture methods yielded cells with stem-cell characteristics that can be induced into rod photoreceptor neurons by RA. Serum had no influence on the "stemness" of the cells. Cells from standard culture had greater "stemness" than cells derived from neurospheres. The standard Müller cells would seem to be the best choice for transplantation in cell replacement therapy for photoreceptor degeneration.

Static Mechanical Loading Influences the Expression of Extracellular Matrix and Cell Adhesion Proteins in Vaginal Cells Derived From Premenopausal Women With Severe Pelvic Organ Prolapse.

PubMed

Kufaishi, Hala; Alarab, May; Drutz, Harold; Lye, Stephen; Shynlova, Oksana

2016-08-01

Primary human vaginal cells derived from women with severe pelvic organ prolapse (POP-HVCs) demonstrate altered cellular characteristics as compared to cells derived from asymptomatic women (control-HVCs). Using computer-controllable Flexcell stretch unit, we examined whether POP-HVCs react differently to mechanical loading as compared to control-HVCs by the expression of extracellular matrix (ECM) components, cell-ECM adhesion proteins, and ECM degrading and maturating enzymes. Vaginal tissue biopsies from premenopausal patients with Pelvic Organ Prolapse Quantification System stage ≥3 (n = 8) and asymptomatic controls (n = 7) were collected during vaginal hysterectomy or repair. Human vaginal cells were isolated by enzymatic digestion, seeded on collagen (COLI)-coated plates, and stretched (24 hours, 25% elongation). Total RNA was extracted, and 84 genes were screened using Human ECM and Adhesion Molecules polymerase chain reaction array; selected genes were verified by quantitative reverse transcription-polymerase chain reaction. Stretch-conditioned media (SCM) were collected and analyzed by protein array, immunoblotting, and zymography. In mechanically stretched control-HVCs, transcript levels of integrins (ITGA1, ITGA4, ITGAV, and ITGB1) and matrix metalloproteinases (MMPs) 2, 8, and 13 were downregulated (P < .05); in POP-HVCs, MMP1, MMP3, and MMP10, ADAMTS8 and 13, tissue inhibitor of metalloproteinases (TIMPs) 1 to 3, ITGA2, ITGA4, ITGA6, ITGB1, contactin (CNTN1), catenins (A1 and B1), and laminins (A3 and C1) were significantly upregulated, whereas COLs (1, 4, 5, 6, 11, and 12) and LOXL1 were downregulated. Human vaginal cells massively secrete MMPs and TIMPs proteins; MMP1, MMP8, MMP9 protein expression and MMP2 gelatinase activity were increased, whereas TIMP2 decreased in SCM from POP-HVCs compared to control-HVCs. Primary human vaginal cells derived from women with severe pelvic organ prolapse and control-HVCs react differentially to in vitro mechanical stretch. Risk factors that induce stretch may alter ECM composition and cell-ECM interaction in pelvic floor tissue leading to the abatement of pelvic organ support and subsequent POP development. © The Author(s) 2016.

Mammary Cancer and Activation of Transposable Elements

DTIC Science & Technology

2013-09-01

formal monthly electronic lab meeting between Peaston lab and Edwards lab. And regularly hold meetings. • An informal schedule was set up with a plan...cytes and ADS-derived induced pluripotent stem cells (ADS-iPSCs) (19) and primary mouse ES cells to isolated sperm and oocytes (20). We selected an...051 59 5 92% H9-IMR90 5875 7 669 782 605 58 91% oocyte - ES cell (mouse) 4727 1 204 883 334 25 93% sperm - ES cell (mouse) 4580 4 364 748 1027 104 91

Unique and shared inflammatory profiles of human brain endothelia and pericytes.

PubMed

Smyth, Leon C D; Rustenhoven, Justin; Park, Thomas I-H; Schweder, Patrick; Jansson, Deidre; Heppner, Peter A; O'Carroll, Simon J; Mee, Edward W; Faull, Richard L M; Curtis, Maurice; Dragunow, Mike

2018-05-11

Pericytes and endothelial cells are critical cellular components of the blood-brain barrier (BBB) and play an important role in neuroinflammation. To date, the majority of inflammation-related studies in endothelia and pericytes have been carried out using immortalised cell lines or non-human-derived cells. Whether these are representative of primary human cells is unclear and systematic comparisons of the inflammatory responses of primary human brain-derived pericytes and endothelia has yet to be performed. To study the effects of neuroinflammation at the BBB, primary brain endothelial cells and pericytes were isolated from human biopsy tissue. Culture purity was examined using qPCR and immunocytochemistry. Electrical cell-substrate impedance sensing (ECIS) was used to determine the barrier properties of endothelial and pericyte cultures. Using immunocytochemistry, cytometric bead array, and ECIS, we compared the responses of endothelia and pericytes to a panel of inflammatory stimuli (IL-1β, TNFα, LPS, IFN-γ, TGF-β 1 , IL-6, and IL-4). Secretome analysis was performed to identify unique secretions of endothelia and pericytes in response to IL-1β. Endothelial cells were pure, moderately proliferative, retained the expression of BBB-related junctional proteins and transporters, and generated robust TEER. Both endothelia and pericytes have the same pattern of transcription factor activation in response to inflammatory stimuli but respond differently at the secretion level. Secretome analysis confirmed that endothelia and pericytes have overlapping but distinct secretome profiles in response to IL-1β. We identified several cell-type specific responses, including G-CSF and GM-CSF (endothelial-specific), and IGFBP2 and IGFBP3 (pericyte-specific). Finally, we demonstrated that direct addition of IL-1β, TNFα, LPS, and IL-4 contributed to the loss of endothelial barrier integrity in vitro. Here, we identify important cell-type differences in the inflammatory response of brain pericytes and endothelia and provide, for the first time, a comprehensive profile of the secretions of primary human brain endothelia and pericytes which has implications for understanding how inflammation affects the cerebrovasculature.

HER2 expression identifies dynamic functional states within circulating breast cancer cells.

PubMed

Jordan, Nicole Vincent; Bardia, Aditya; Wittner, Ben S; Benes, Cyril; Ligorio, Matteo; Zheng, Yu; Yu, Min; Sundaresan, Tilak K; Licausi, Joseph A; Desai, Rushil; O'Keefe, Ryan M; Ebright, Richard Y; Boukhali, Myriam; Sil, Srinjoy; Onozato, Maristela L; Iafrate, Anthony J; Kapur, Ravi; Sgroi, Dennis; Ting, David T; Toner, Mehmet; Ramaswamy, Sridhar; Haas, Wilhelm; Maheswaran, Shyamala; Haber, Daniel A

2016-09-01

Circulating tumour cells in women with advanced oestrogen-receptor (ER)-positive/human epidermal growth factor receptor 2 (HER2)-negative breast cancer acquire a HER2-positive subpopulation after multiple courses of therapy. In contrast to HER2-amplified primary breast cancer, which is highly sensitive to HER2-targeted therapy, the clinical significance of acquired HER2 heterogeneity during the evolution of metastatic breast cancer is unknown. Here we analyse circulating tumour cells from 19 women with ER + /HER2 - primary tumours, 84% of whom had acquired circulating tumour cells expressing HER2. Cultured circulating tumour cells maintain discrete HER2 + and HER2 - subpopulations: HER2 + circulating tumour cells are more proliferative but not addicted to HER2, consistent with activation of multiple signalling pathways; HER2 - circulating tumour cells show activation of Notch and DNA damage pathways, exhibiting resistance to cytotoxic chemotherapy, but sensitivity to Notch inhibition. HER2 + and HER2 - circulating tumour cells interconvert spontaneously, with cells of one phenotype producing daughters of the opposite within four cell doublings. Although HER2 + and HER2 - circulating tumour cells have comparable tumour initiating potential, differential proliferation favours the HER2 + state, while oxidative stress or cytotoxic chemotherapy enhances transition to the HER2 - phenotype. Simultaneous treatment with paclitaxel and Notch inhibitors achieves sustained suppression of tumorigenesis in orthotopic circulating tumour cell-derived tumour models. Together, these results point to distinct yet interconverting phenotypes within patient-derived circulating tumour cells, contributing to progression of breast cancer and acquisition of drug resistance.

Advanced Good Cell Culture Practice for human primary, stem cell-derived and organoid models as well as microphysiological systems.

PubMed

Pamies, David; Bal-Price, Anna; Chesné, Christophe; Coecke, Sandra; Dinnyes, Andras; Eskes, Chantra; Grillari, Regina; Gstraunthaler, Gerhard; Hartung, Thomas; Jennings, Paul; Leist, Marcel; Martin, Ulrich; Passier, Robert; Schwamborn, Jens C; Stacey, Glyn N; Ellinger-Ziegelbauer, Heidrun; Daneshian, Mardas

2018-04-13

A major reason for the current reproducibility crisis in the life sciences is the poor implementation of quality control measures and reporting standards. Improvement is needed, especially regarding increasingly complex in vitro methods. Good Cell Culture Practice (GCCP) was an effort from 1996 to 2005 to develop such minimum quality standards also applicable in academia. This paper summarizes recent key developments in in vitro cell culture and addresses the issues resulting for GCCP, e.g. the development of induced pluripotent stem cells (iPSCs) and gene-edited cells. It further deals with human stem-cell-derived models and bioengineering of organo-typic cell cultures, including organoids, organ-on-chip and human-on-chip approaches. Commercial vendors and cell banks have made human primary cells more widely available over the last decade, increasing their use, but also requiring specific guidance as to GCCP. The characterization of cell culture systems including high-content imaging and high-throughput measurement technologies increasingly combined with more complex cell and tissue cultures represent a further challenge for GCCP. The increasing use of gene editing techniques to generate and modify in vitro culture models also requires discussion of its impact on GCCP. International (often varying) legislations and market forces originating from the commercialization of cell and tissue products and technologies are further impacting on the need for the use of GCCP. This report summarizes the recommendations of the second of two workshops, held in Germany in December 2015, aiming map the challenge and organize the process or developing a revised GCCP 2.0.

Crystallographic Information Resources

ERIC Educational Resources Information Center

Glasser, Leslie

2016-01-01

Crystallographic information provides the fundamental basis for understanding the properties and behavior of materials. This data, such as chemical composition, unit cell dimensions, space group, and atomic positions, derives from the primary literature--that is, from published experimental measurement or theoretical calculation. Although the…

Human vascular tissue models formed from human induced pluripotent stem cell derived endothelial cells

PubMed Central

Belair, David G.; Whisler, Jordan A.; Valdez, Jorge; Velazquez, Jeremy; Molenda, James A.; Vickerman, Vernella; Lewis, Rachel; Daigh, Christine; Hansen, Tyler D.; Mann, David A.; Thomson, James A.; Griffith, Linda G.; Kamm, Roger D.; Schwartz, Michael P.; Murphy, William L.

2015-01-01

Here we describe a strategy to model blood vessel development using a well-defined iPSC-derived endothelial cell type (iPSC-EC) cultured within engineered platforms that mimic the 3D microenvironment. The iPSC-ECs used here were first characterized by expression of endothelial markers and functional properties that included VEGF responsiveness, TNF-α-induced upregulation of cell adhesion molecules (MCAM/CD146; ICAM1/CD54), thrombin-dependent barrier function, shear stress-induced alignment, and 2D and 3D capillary-like network formation in Matrigel. The iPSC-ECs also formed 3D vascular networks in a variety of engineering contexts, yielded perfusable, interconnected lumen when co-cultured with primary human fibroblasts, and aligned with flow in microfluidics devices. iPSC-EC function during tubule network formation, barrier formation, and sprouting was consistent with that of primary ECs, and the results suggest a VEGF-independent mechanism for sprouting, which is relevant to therapeutic anti-angiogenesis strategies. Our combined results demonstrate the feasibility of using a well-defined, stable source of iPSC-ECs to model blood vessel formation within a variety of contexts using standard in vitro formats. PMID:25190668

Engraftment of human induced pluripotent stem cell-derived hepatocytes in immunocompetent mice via 3D co-aggregation and encapsulation

PubMed Central

Song, Wei; Lu, Yen-Chun; Frankel, Angela S.; An, Duo; Schwartz, Robert E.; Ma, Minglin

2015-01-01

Cellular therapies for liver diseases and in vitro models for drug testing both require functional human hepatocytes (Hum-H), which have unfortunately been limited due to the paucity of donor liver tissues. Human pluripotent stem cells (hPSCs) represent a promising and potentially unlimited cell source to derive Hum-H. However, the hepatic functions of these hPSC-derived cells to date are not fully comparable to adult Hum-H and are more similar to fetal ones. In addition, it has been challenging to obtain functional hepatic engraftment of these cells with prior studies having been done in immunocompromised animals. In this report, we demonstrated successful engraftment of human induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (iPS-H) in immunocompetent mice by pre-engineering 3D cell co-aggregates with stromal cells (SCs) followed by encapsulation in recently developed biocompatible hydrogel capsules. Notably, upon transplantation, human albumin and α1-antitrypsin (A1AT) in mouse sera secreted by encapsulated iPS-H/SCs aggregates reached a level comparable to the primary Hum-H/SCs control. Further immunohistochemistry of human albumin in retrieved cell aggregates confirmed the survival and function of iPS-H. This proof-of-concept study provides a simple yet robust approach to improve the engraftment of iPS-H, and may be applicable to many stem cell-based therapies. PMID:26592180

Mechanistic insights into the antileukemic activity of hyperforin.

PubMed

Billard, C; Merhi, F; Bauvois, B

2013-01-01

Hyperforin is a prenylated phloroglucinol present in the medicinal plant St John's wort (Hypericum perforatum). The compound has many biological properties, including antidepressant, anti-inflammatory, antibacterial and antitumor activities. This review focuses on the in vitro antileukemic effects of purified hyperforin and related mechanisms in chronic lymphoid leukemia (CLL) and acute myeloid leukemia (AML) - conditions that are known for their resistance to chemotherapy. Hyperforin induces apoptosis in both CLL and AML cells. In AML cell lines and primary AML cells, hyperforin directly inhibits the kinase activity of the serine/threonine protein kinase B/AKT1, leading to activation of the pro-apoptotic Bcl-2 family protein Bad through its non-phosphorylation by AKT1. In primary CLL cells, hyperforin acts by stimulating the expression of the pro-apoptotic Bcl-2 family member Noxa (possibly through the inhibition of proteasome activity). Other hyperforin targets include matrix metalloproteinase-2 in AML cells and vascular endothelial growth factor and matrix metalloproteinase-9 in CLL cells - two mediators of cell migration and angiogenesis. In summary, hyperforin targets molecules involved in signaling pathways that control leukemic cell proliferation, survival, apoptosis, migration and angiogenesis. Hyperforin also downregulates the expression of P-glycoprotein, a protein that is involved in the resistance of leukemia cells to chemotherapeutic agents. Lastly, native hyperforin and its stable derivatives show interesting in vivo properties in animal models. In view of their low toxicity, hyperforin and its derivatives are promising antileukemic agents and deserve further investigation in vivo.

Surface coatings of ZnO nanoparticles mitigate differentially a host of transcriptional, protein and signalling responses in primary human olfactory cells

PubMed Central

2013-01-01

Background Inhaled nanoparticles have been reported in some instances to translocate from the nostril to the olfactory bulb in exposed rats. In close proximity to the olfactory bulb is the olfactory mucosa, within which resides a niche of multipotent cells. Cells isolated from this area may provide a relevant in vitro system to investigate potential effects of workplace exposure to inhaled zinc oxide nanoparticles. Methods Four types of commercially-available zinc oxide (ZnO) nanoparticles, two coated and two uncoated, were examined for their effects on primary human cells cultured from the olfactory mucosa. Human olfactory neurosphere-derived (hONS) cells from healthy adult donors were analyzed for modulation of cytokine levels, activation of intracellular signalling pathways, changes in gene-expression patterns across the whole genome, and compromised cellular function over a 24 h period following exposure to the nanoparticles suspended in cell culture medium. Results ZnO nanoparticle toxicity in hONS cells was mediated through a battery of mechanisms largely related to cell stress, inflammatory response and apoptosis, but not activation of mechanisms that repair damaged DNA. Surface coatings on the ZnO nanoparticles mitigated these cellular responses to varying degrees. Conclusions The results indicate that care should be taken in the workplace to minimize generation of, and exposure to, aerosols of uncoated ZnO nanoparticles, given the adverse responses reported here using multipotent cells derived from the olfactory mucosa. PMID:24144420

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

PubMed Central

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

2012-01-01

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

Spontaneous myogenic differentiation of Flk-1-positive cells from adult pancreas and other nonmuscle tissues.

PubMed

Di Rocco, Giuliana; Tritarelli, Alessandra; Toietta, Gabriele; Gatto, Ilaria; Iachininoto, Maria Grazia; Pagani, Francesca; Mangoni, Antonella; Straino, Stefania; Capogrossi, Maurizio C

2008-02-01

At the embryonic or fetal stages, autonomously myogenic cells (AMCs), i.e., cells able to spontaneously differentiate into skeletal myotubes, have been identified from several different sites other than skeletal muscle, including the vascular compartment. However, in the adult animal, AMCs from skeletal muscle-devoid tissues have been described in only two cases. One is represented by thymic myoid cells, a restricted population of committed myogenic progenitors of unknown derivation present in the thymic medulla; the other is represented by a small subset of adipose tissue-associated cells, which we recently identified. In the present study we report, for the first time, the presence of spontaneously differentiating myogenic precursors in the pancreas and in other skeletal muscle-devoid organs such as spleen and stomach, as well as in the periaortic tissue of adult mice. Immunomagnetic selection procedures indicate that AMCs derive from Flk-1(+) progenitors. Individual clones of myogenic cells from nonmuscle organs are morphologically and functionally indistinguishable from skeletal muscle-derived primary myoblasts. Moreover, they can be induced to proliferate in vitro and are able to participate in muscle regeneration in vivo. Thus, we provide evidence that fully competent myogenic progenitors can be derived from the Flk-1(+) compartment of several adult tissues that are embryologically unrelated to skeletal muscle.

Identification of Primary Transcriptional Regulation of Cell Cycle-Regulated Genes upon DNA Damage

PubMed Central

Zhou, Tong; Chou, Jeff; Mullen, Thomas E.; Elkon, Rani; Zhou, Yingchun; Simpson, Dennis A.; Bushel, Pierre R.; Paules, Richard S.; Lobenhofer, Edward K.; Hurban, Patrick; Kaufmann, William K.

2007-01-01

The changes in global gene expression in response to DNA damage may derive from either direct induction or repression by transcriptional regulation or indirectly by synchronization of cells to specific cell cycle phases, such as G1 or G2. We developed a model that successfully estimated the expression levels of >400 cell cycle-regulated genes in normal human fibroblasts based on the proportions of cells in each phase of the cell cycle. By isolating effects on the gene expression associated with the cell cycle phase redistribution after genotoxin treatment, the direct transcriptional target genes were distinguished from genes for which expression changed secondary to cell synchronization. Application of this model to ionizing radiation (IR)-treated normal human fibroblasts identified 150 of 406 cycle-regulated genes as putative direct transcriptional targets of IR-induced DNA damage. Changes in expression of these genes after IR treatment derived from both direct transcriptional regulation and cell cycle synchronization. PMID:17404513

Mechanical signals promote osteogenic fate through a primary cilia-mediated mechanism

PubMed Central

Chen, Julia C.; Hoey, David A.; Chua, Mardonn; Bellon, Raymond; Jacobs, Christopher R.

2016-01-01

It has long been suspected, but never directly shown, that bone formed to accommodate an increase in mechanical loading is related to the creation of osteoblasts from skeletal stem cells. Indeed, biophysical stimuli potently regulate osteogenic lineage commitment in vitro. In this study, we transplanted bone marrow cells expressing green fluorescent protein, to enable lineage tracing, and subjected mice to a biophysical stimulus, to elicit a bone-forming response. We detected cells derived from transplanted progenitors embedded within the bone matrix near active bone-forming surfaces in response to loading, demonstrating for the first time, that mechanical signals enhance the homing and attachment of bone marrow cells to bone surfaces and the commitment to an osteogenic lineage of these cells in vivo. Furthermore, we used an inducible Cre/Lox recombination system to delete kinesin family member 3A (Kif3a), a gene that is essential for primary cilia formation, at will in transplanted cells and their progeny, regardless of which tissue may have incorporated them. Disruption of the mechanosensing organelle, the primary cilium in a progenitor population, significantly decreased the amount of bone formed in response to mechanical stimulation. The collective results of our study directly demonstrate that, in a novel experimental stem cell mechanobiology model, mechanical signals enhance osteogenic lineage commitment in vivo and that the primary cilium contributes to this process.—Chen, J. C., Hoey, D. A., Chua, M., Bellon, R., Jacobs, C. R. Mechanical signals promote osteogenic fate through a primary cilia-mediated mechanism. PMID:26675708

PACAP receptor pharmacology and agonist bias: analysis in primary neurons and glia from the trigeminal ganglia and transfected cells

PubMed Central

Walker, C S; Sundrum, T; Hay, D L

2014-01-01

Background and Purpose A major challenge in the development of new medicines targeting GPCRs is the ability to quantify drug action in physiologically relevant models. Primary cell models that closely resemble the clinically relevant in vivo site of drug action are important translational tools in drug development. However, pharmacological studies in these models are generally very limited due to the methodology used. Experimental Approach We used a neuropeptide system to demonstrate the applicability of using highly sensitive signalling assays in primary cells. We quantified the action of pituitary adenylate cyclase-activating peptide (PACAP)-38, PACAP-27 and vasoactive intestinal polypeptide in primary cultures of neurons and glia derived from rat trigeminal ganglia (TG), comparing our observations to transfected cells. Key Results PACAP-responsive receptors in rat trigeminal neurons, glia and transfected PAC1n receptors were pharmacologically distinct. PACAP-38, but not PACAP-27, activated ERK in glia, while both forms stimulated cellular cAMP production. PACAP(6–38) also displayed cell-type-dependent, agonist-specific, antagonism. Conclusions and Implications The complexity of PACAP pharmacology in the TG may help to direct, more effectively, the development of disease treatments targeting the PACAP receptor. We suggest that these methodologies are broadly applicable to other primary cell types of human or animal origin, and that our approach may allow more thorough characterization of ligand properties in physiologically relevant cell types. PMID:24303997

Establishment and Characterization of Immortalized Minipig Neural Stem Cell Line

PubMed Central

Choi, Sung S.; Yoon, Seung-Bin; Lee, Sang-Rae; Kim, Sun-Uk; Cha, Young Joo; Lee, Daniel; Kim, Seung U.; Chang, Kyu-Tae; Lee, Hong J.

2017-01-01

Despite the increasing importance of minipigs in biomedical research, there has been relatively little research concerning minipig-derived adult stem cells as a promising research tool that could be used to develop stem cell-based therapies. We first generated immortalized neural stem cells (iNSCs) from primary minipig olfactory bulb cells (pmpOBCs) and defined the characteristics of the cell line. Primary neural cells were prepared from minipig neonate olfactory bulbs and immortalized by infection with retrovirus carrying the v-myc gene. The minipig iNSCs (mpiNSCs) had normal karyotypes and expressed NSC-specific markers, including nestin, vimentin, Musashi1, and SOX2, suggesting a similarity to human NSCs. On the basis of the global gene expression profiles from the microarray analysis, neurogenesis-associated transcript levels were predominantly altered in mpiNSCs compared with pmpOBCs. These findings increase our understanding of minipig stem cells and contribute to the utility of mpiNSCs as resources for immortalized stem cell experiments. PMID:27524466

Standardized cryopreservation of human primary cells.

PubMed

Ramos, Thomas V; Mathew, Aby J; Thompson, Maria L; Ehrhardt, Rolf O

2014-09-02

Cryopreservation is the use of low temperatures to preserve structurally intact living cells. The cells that survive the thermodynamic journey from the 37 °C incubator to the -196 °C liquid nitrogen storage tank are free from the influences of time. Thus, cryopreservation is a critical component of cell culture and cell manufacturing protocols. Successful cryopreservation of human cells requires that the cells be derived from patient samples that are collected in a standardized manner, and carefully handled from blood draw through cell isolation. Furthermore, proper equipment must be in place to ensure consistency, reproducibility, and sterility. In addition, the correct choice and amount of cryoprotectant agent must be added at the correct temperature, and a controlled rate of freezing (most commonly 1 °C/min) must be applied prior to a standardized method of cryogenic storage. This appendix describes how human primary cells can be frozen for long-term storage and thawed for growth in a tissue culture vessel. Copyright © 2014 John Wiley & Sons, Inc.

Establishment and Characterization of Immortalized Minipig Neural Stem Cell Line.

PubMed

Choi, Sung S; Yoon, Seung-Bin; Lee, Sang-Rae; Kim, Sun-Uk; Cha, Young Joo; Lee, Daniel; Kim, Seung U; Chang, Kyu-Tae; Lee, Hong J

2017-02-16

Despite the increasing importance of minipigs in biomedical research, there has been relatively little research concerning minipig-derived adult stem cells as a promising research tool that could be used to develop stem cell-based therapies. We first generated immortalized neural stem cells (iNSCs) from primary minipig olfactory bulb cells (pmpOBCs) and defined the characteristics of the cell line. Primary neural cells were prepared from minipig neonate olfactory bulbs and immortalized by infection with retrovirus carrying the v-myc gene. The minipig iNSCs (mpiNSCs) had normal karyotypes and expressed NSC-specific markers, including nestin, vimentin, Musashi1, and SOX2, suggesting a similarity to human NSCs. On the basis of the global gene expression profiles from the microarray analysis, neurogenesis-associated transcript levels were predominantly altered in mpiNSCs compared with pmpOBCs. These findings increase our understanding of minipig stem cells and contribute to the utility of mpiNSCs as resources for immortalized stem cell experiments.

HGF Secreted by Activated Kupffer Cells Induces Apoptosis of Plasmodium-Infected Hepatocytes

PubMed Central

Gonçalves, Lígia Antunes; Rodo, Joana; Rodrigues-Duarte, Lurdes; de Moraes, Luciana Vieira; Penha-Gonçalves, Carlos

2017-01-01

Malaria liver stage infection is an obligatory parasite development step and represents a population bottleneck in Plasmodium infections, providing an advantageous target for blocking parasite cycle progression. Parasite development inside hepatocytes implies a gross cellular insult evoking innate host responses to counteract intra-hepatocytic infection. Using primary hepatocyte cultures, we investigated the role of Kupffer cell-derived hepatocyte growth factor (HGF) in malaria liver stage infection. We found that Kupffer cells from Plasmodium-infected livers produced high levels of HGF, which trigger apoptosis of infected hepatocytes through a mitochondrial-independent apoptosis pathway. HGF action in infected hepatocyte primary cultures results in a potent reduction of parasite yield by specifically sensitizing hepatocytes carrying established parasite exo-erythrocytic forms to undergo apoptosis. This apoptosis mechanism is distinct from cell death that is spontaneously induced in infected cultures and is governed by Fas signaling modulation through a mitochondrial-dependent apoptosis pathway. This work indicates that HGF and Fas signaling pathways are part of an orchestrated host apoptosis response that occurs during malaria liver stage infection, decreasing the success of infection of individual hepatocytes. Our results raise the hypothesis that paracrine signals derived from Kupffer cell activation are implicated in directing death of hepatocytes infected with the malaria parasite. PMID:28220125

HGF Secreted by Activated Kupffer Cells Induces Apoptosis of Plasmodium-Infected Hepatocytes.

PubMed

Gonçalves, Lígia Antunes; Rodo, Joana; Rodrigues-Duarte, Lurdes; de Moraes, Luciana Vieira; Penha-Gonçalves, Carlos

2017-01-01

Malaria liver stage infection is an obligatory parasite development step and represents a population bottleneck in Plasmodium infections, providing an advantageous target for blocking parasite cycle progression. Parasite development inside hepatocytes implies a gross cellular insult evoking innate host responses to counteract intra-hepatocytic infection. Using primary hepatocyte cultures, we investigated the role of Kupffer cell-derived hepatocyte growth factor (HGF) in malaria liver stage infection. We found that Kupffer cells from Plasmodium -infected livers produced high levels of HGF, which trigger apoptosis of infected hepatocytes through a mitochondrial-independent apoptosis pathway. HGF action in infected hepatocyte primary cultures results in a potent reduction of parasite yield by specifically sensitizing hepatocytes carrying established parasite exo-erythrocytic forms to undergo apoptosis. This apoptosis mechanism is distinct from cell death that is spontaneously induced in infected cultures and is governed by Fas signaling modulation through a mitochondrial-dependent apoptosis pathway. This work indicates that HGF and Fas signaling pathways are part of an orchestrated host apoptosis response that occurs during malaria liver stage infection, decreasing the success of infection of individual hepatocytes. Our results raise the hypothesis that paracrine signals derived from Kupffer cell activation are implicated in directing death of hepatocytes infected with the malaria parasite.

Bioinformatic analysis of primary endothelial cell gene array data illustrated by the analysis of transcriptome changes in endothelial cells exposed to VEGF-A and PlGF.

PubMed

Schoenfeld, Jonathan; Lessan, Khashayar; Johnson, Nicola A; Charnock-Jones, D Stephen; Evans, Amanda; Vourvouhaki, Ekaterini; Scott, Laurie; Stephens, Richard; Freeman, Tom C; Saidi, Samir A; Tom, Brian; Weston, Gareth C; Rogers, Peter; Smith, Stephen K; Print, Cristin G

2004-01-01

We recently published a review in this journal describing the design, hybridisation and basic data processing required to use gene arrays to investigate vascular biology (Evans et al. Angiogenesis 2003; 6: 93-104). Here, we build on this review by describing a set of powerful and robust methods for the analysis and interpretation of gene array data derived from primary vascular cell cultures. First, we describe the evaluation of transcriptome heterogeneity between primary cultures derived from different individuals, and estimation of the false discovery rate introduced by this heterogeneity and by experimental noise. Then, we discuss the appropriate use of Bayesian t-tests, clustering and independent component analysis to mine the data. We illustrate these principles by analysis of a previously unpublished set of gene array data in which human umbilical vein endothelial cells (HUVEC) cultured in either rich or low-serum media were exposed to vascular endothelial growth factor (VEGF)-A165 or placental growth factor (PlGF)-1(131). We have used Affymetrix U95A gene arrays to map the effects of these factors on the HUVEC transcriptome. These experiments followed a paired design and were biologically replicated three times. In addition, one experiment was repeated using serial analysis of gene expression (SAGE). In contrast to some previous studies, we found that VEGF-A and PlGF consistently regulated only small, non-overlapping and culture media-dependant sets of HUVEC transcripts, despite causing significant cell biological changes.

Astrocytes mediated the nootropic and neurotrophic effects of Sarsasapogenin-AA13 via upregulating brain-derived neurotrophic factor.

PubMed

Dong, Dong; Mao, Yu; Huang, Cui; Jiao, Qian; Pan, Hui; Ma, Lei; Wang, Rui

2017-01-01

Rhizoma Anemarrhena , a widely used traditional Chinese medicine, has previously been shown to have neuroprotective effect. Sarsasapogenin-AA13 (AA13) is a novel synthetic derivative of Sarsasapogenin, which is extracted from Rhizoma Anemarrhena . The aim of this study is to investigate the nootropic and neurotrophic effects of AA13 and underlying mechanisms. In vitro , cell viability of rat primary astrocytes treated with AA13 and neurons cultured with conditioned medium of AA13-treated rat primary astrocytes was tested by MTT assays. In vivo , a pharmacological model of cognitive impairment induced by scopolamine was employed and spatial memory of the mice was assessed by Morris water maze. This study found that AA13 increased cell viability of primary astrocytes and AA13-treated astrocyte-conditioned medium enhanced the survival rate of primary neurons. Interestingly, AA13 markedly enhanced the level of BDNF in astrocytes. Furthermore, AA13 (6 mg/kg) improved the cognitive deficits in animal models (p<0.05) and BDNF and PSD95 levels were increased in brain. Therefore, we hypothesize that AA13 exerts nootropic and neurotrophic activities through astrocytes mediated upregulation of BDNF secretion. The results suggest that AA13 could be a potential compound for cognitive impairment after further research.

Cancer-associated fibroblasts affect breast cancer cell gene expression, invasion and angiogenesis.

PubMed

Eiro, Noemi; González, Lucía; Martínez-Ordoñez, Anxo; Fernandez-Garcia, Belen; González, Luis O; Cid, Sandra; Dominguez, Francisco; Perez-Fernandez, Román; Vizoso, Francisco J

2018-03-01

It has been reported that stromal cell features may affect the clinical outcome of breast cancer patients. Cancer associated fibroblasts (CAFs) represent one of the most abundant cell types within the breast cancer stroma. Here, we aimed to explore the influence of CAFs on breast cancer gene expression, as well as on invasion and angiogenesis. qRT-PCR was used to evaluate the expression of several cancer progression related genes (S100A4, TGFβ, FGF2, FGF7, PDGFA, PDGFB, VEGFA, IL-6, IL-8, uPA, MMP2, MMP9, MMP11 and TIMP1) in the human breast cancer-derived cell lines MCF-7 and MDA-MB-231, before and after co-culture with CAFs. Stromal mononuclear inflammatory cell (MIC) MMP11 expression was used to stratify primary tumors. In addition, we assessed the in vitro effects of CAFs on both MDA-MB-231 breast cancer cell invasion and endothelial cell (HUVEC) tube formation. We found that the expression levels of most of the genes tested were significantly increased in both breast cancer-derived cell lines after co-culture with CAFs from either MMP11+ or MMP11- MIC tumors. IL-6 and IL-8 showed an increased expression in both cancer-derived cell lines after co-culture with CAFs from MMP11+ MIC tumors. We also found that the invasive and angiogenic capacities of, respectively, MDA-MB-231 and HUVEC cells were increased after co-culture with CAFs, especially those from MMP11+ MIC tumors. Our data indicate that tumor-derived CAFs can induce up-regulation of genes involved in breast cancer progression. Our data additionally indicate that CAFs, especially those derived from MMP11+ MIC tumors, can promote breast cancer cell invasion and angiogenesis.

Establishment of rat embryonic stem-like cells from the morula using a combination of feeder layers.

PubMed

Sano, Chiaki; Matsumoto, Asako; Sato, Eimei; Fukui, Emiko; Yoshizawa, Midori; Matsumoto, Hiromichi

2009-08-01

Embryonic stem (ES) cells are characterized by pluripotency, in particular the ability to form a germline on injection into blastocysts. Despite numerous attempts, ES cell lines derived from rat embryos have not yet been established. The reason for this is unclear, although certain intrinsic biological differences among species and/or strains have been reported. Herein, using Wistar-Imamichi rats, specific characteristics of preimplantation embryos are described. At the blastocyst stage, Oct4 (also called Pou5f1) was expressed in both the inner cell mass (ICM) and the trophectoderm (TE), whereas expression of Cdx2 was localized to the TE. In contrast, at an earlier stage, expression of Oct4 was detected in all the nuclei in the morula. These stages were examined using a combination of feeder layers (rat embryonic fibroblast [REF] for primary outgrowth and SIM mouse embryo-derived thioguanine- and ouabain-resistant [STO] cells for passaging) to establish rat ES-like cell lines. The rat ES-like cell lines obtained from the morula maintained expression of Oct4 over long-term culture, whereas cell lines derived from blastocysts lost pluripotency during early passage. The morula-derived ES-like cell lines showed Oct4 expression in a long-term culture, even after cryogenic preservation, thawing and EGFP transfection. These results indicate that rat ES-like cell lines with long-term Oct4 expression can be established from the morula of Wistar-Imamichi rats using a combination of feeder layers.

Efficient nucleofection of primary human B cells and B-CLL cells induces apoptosis, which depends on the microenvironment and on the structure of transfected nucleic acids.

PubMed

Seiffert, M; Stilgenbauer, S; Döhner, H; Lichter, P

2007-09-01

Accumulation of neoplastic cells in B-cell chronic lymphocytic leukemia (B-CLL) is thought to be due to intrinsic defects in the apoptotic machinery of the leukemic cells or to an altered, survival-stimulating microenvironment in vivo. Despite their long survival in vivo, B-CLL cells undergo rapid spontaneous apoptosis ex vivo. To maintain survival in vitro, we established a coculture system using the human bone marrow-derived stromal cell line HS-5. The microenvironment in these cocultures lead to B-CLL cell survival for at least several months and therefore provided a tool for valid in vitro analysis, mimicking the in vivo situation. Although primary B lymphocytes are notoriously resistant to most gene transfer techniques, we achieved high transfection efficiency and cell viability in this coculture system by using a nucleofection-based strategy. Surprisingly, the introduction of circular plasmid DNA into B cells and B-CLL cells induced rapid apoptosis, which was independent of the type of transgene used, but dependent on the DNA concentration. However, transfection of these cells with mRNA was highly efficient and resulted in sustained cell viability and potent transgene expression. The described procedure represents a new approach to study gene function in primary B cells and B-CLL cells.

Vitamin D fails to prevent serum starvation- or staurosporine-induced apoptosis in human and rat osteosarcoma-derived cell lines

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

Witasp, Erika; Division of Biochemical Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm; Gustafsson, Ann-Catrin

2005-05-13

Previous studies have suggested that 1,25(OH){sub 2}D{sub 3}, the active form of vitamin D{sub 3}, may increase the survival of bone-forming osteoblasts through an inhibition of apoptosis. On the other hand, vitamin D{sub 3} has also been shown to trigger apoptosis in human cancer cells, including osteosarcoma-derived cell lines. In the present study, we show that 1,25(OH){sub 2}D{sub 3} induces a time- and dose-dependent loss of cell viability in the rat osteosarcoma cell line, UMR-106, and the human osteosarcoma cell line, TE-85. We were unable, however, to detect nuclear condensation, phosphatidylserine externalization, or other typical signs of apoptosis in thismore » model. Moreover, 1,25(OH){sub 2}D{sub 3} failed to protect against apoptosis induced by serum starvation or incubation with the protein kinase inhibitor, staurosporine. These in vitro findings are thus at variance with several previous reports in the literature and suggest that induction of or protection against apoptosis of bone-derived cells may not be a primary function of vitamin D{sub 3}.« less

Interpretation of field potentials measured on a multi electrode array in pharmacological toxicity screening on primary and human pluripotent stem cell-derived cardiomyocytes.

PubMed

Tertoolen, L G J; Braam, S R; van Meer, B J; Passier, R; Mummery, C L

2018-03-18

Multi electrode arrays (MEAs) are increasingly used to detect external field potentials in electrically active cells. Recently, in combination with cardiomyocytes derived from human (induced) pluripotent stem cells they have started to become a preferred tool to examine newly developed drugs for potential cardiac toxicity in pre-clinical safety pharmacology. The most important risk parameter is proarrhythmic activity in cardiomyocytes which can cause sudden cardiac death. Whilst MEAs can provide medium- to high- throughput noninvasive assay platform, the translation of a field potential to cardiac action potential (normally measured by low-throughput patch clamp) is complex so that accurate assessment of drug risk to the heart is in practice still challenging. To address this, we used computational simulation to study the theoretical relationship between aspects of the field potential and the underlying cardiac action potential. We then validated the model in both primary mouse- and human pluripotent (embryonic) stem cell-derived cardiomyocytes showing that field potentials measured in MEAs could be converted to action potentials that were essentially identical to those determined directly by electrophysiological patch clamp. The method significantly increased the amount of information that could be extracted from MEA measurements and thus combined the advantages of medium/high throughput with more informative readouts. We believe that this will benefit the analysis of drug toxicity screening of cardiomyocytes using in time and accuracy. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Exposure to Cell Phone Radiation Up-Regulates Apoptosis Genes in Primary Cultures of Neurons and Astrocytes

PubMed Central

Zhao, Tian-Yong; Zou, Shi-Ping; Knapp, Pamela E.

2007-01-01

The health effects of cell phone radiation exposure are a growing public concern. This study investigated whether expression of genes related to cell death pathways are dysregulated in primary cultured neurons and astrocytes by exposure to a working GSM (Global System for Mobile Communication) cell phone rated at a frequency of 1900 MHz. Primary cultures were exposed to cell phone emissions for 2 hrs. We used array analysis and real-time RT-PCR to show up-regulation of caspase-2, caspase-6 and Asc (apoptosis associated speck-like protein containing a card) gene expression in neurons and astrocytes. Upregulation occurred in both “on” and “stand-by” modes in neurons, but only in “on” mode in astrocytes. Additionally, astrocytes showed up-regulation of the Bax gene. The effects are specific since up-regulation was not seen for other genes associated with apoptosis, such as caspase-9 in either neurons and astrocytes, or Bax in neurons. The results show that even relatively short-term exposure to cell phone radiofrequency emissions can up-regulate elements of apoptotic pathways in cells derived from the brain, and that neurons appear to be more sensitive to this effect than astrocytes. PMID:17187929

Production of platelet-derived endothelial cell growth factor by normal and transformed human cells in culture.

PubMed Central

Usuki, K; Heldin, N E; Miyazono, K; Ishikawa, F; Takaku, F; Westermark, B; Heldin, C H

1989-01-01

Platelet-derived endothelial cell growth factor (PD-ECGF) is a 45-kDa endothelial cell mitogen which has angiogenic properties in vivo. We report here that human foreskin fibroblasts, a human squamous cell carcinoma cell line, and 2 out of the 3 human thyroid carcinoma cell lines investigated produce PD-ECGF, whereas 21 other cell lines examined do not. The positive cell lines contained a 1.8-kilobase PD-ECGF mRNA, and a 45-kDa protein could be demonstrated in lysates of the cell lines by immunoblotting and immunoprecipitation using a specific antiserum against PD-ECGF. Furthermore, the cell lysates contained mitogenic activity for endothelial cells that was neutralized by the PD-ECGF antiserum. PD-ECGF was found to be secreted only slowly from the producer cells, consistent with the previous finding that the primary translation product lacks a signal sequence. The restricted expression and intracellular sequestration of PD-ECGF imply a strictly controlled function in endothelial cell proliferation and angiogenesis. Aberrant production of PD-ECGF may play a role in tumor angiogenesis. Images PMID:2678104

Deficiency of Kruppel-like factor KLF4 in mammary tumor cells inhibits tumor growth and pulmonary metastasis and is accompanied by compromised recruitment of myeloid-derived suppressor cells

PubMed Central

Yu, Fang; Shi, Ying; Wang, Junfeng; Li, Juan; Fan, Daping; Ai, Walden

2013-01-01

Increasing evidence indicates that myeloid-derived suppressor cells (MDSCs) negatively regulate immune responses during tumor progression, inflammation and infection. However, the underlying molecular mechanisms of their development and mobilization remain to be fully delineated. Kruppel-like factor KLF4 is a transcription factor that has an oncogenic function in breast cancer development, but its function in tumor microenvironment, a critical component for tumorigenesis, has not been examined. By using a spontaneously metastatic 4T1 breast cancer mouse model and an immunodeficient NOD/SCID mouse model, we demonstrated that KLF4 knockdown delayed tumor development and inhibited pulmonary metastasis, which was accompanied by decreased accumulation of MDSCs in bone marrow, spleens and primary tumors. Mechanistically, we found that KLF4 knockdown resulted in a significant decrease of circulating GM-CSF, an important cytokine for MDSC biology. Consistently, recombinant GM-CSF restored the frequency of MDSCs in purified bone marrow cells incubated with conditioned medium from KLF4 deficient cells. In addition, we identified CXCL5 as a critical mediator to enhance the expression and function of GM-CSF. Reduced CXCL5 expression by KLF4 knockdown in primary tumors and breast cancer cells was correlated with a decreased GM-CSF expression in our mouse models. Finally, we found that CXCL5/CXCR2 axis facilitated MDSC migration and that anti-GM-CSF antibodies neutralized CXCL5-induced accumulation of MDSCs. Taken together, our data suggest that KLF4 modulates maintenance of MDSCs in bone marrow by inducing GM-CSF production via CXCL5 and regulates recruitment of MDSCs into the primary tumors through the CXCL5/CXCR2 axis, both of which contribute to KLF4-mediated mammary tumor development. PMID:23737434

Donor-Dependent and Other Nondefined Factors Have Greater Influence on the Hepatic Phenotype Than the Starting Cell Type in Induced Pluripotent Stem Cell Derived Hepatocyte-Like Cells.

PubMed

Heslop, James A; Kia, Richard; Pridgeon, Christopher S; Sison-Young, Rowena L; Liloglou, Triantafillos; Elmasry, Mohamed; Fenwick, Stephen W; Mills, John S; Kitteringham, Neil R; Goldring, Chris E; Park, Bong K

2017-05-01

Drug-induced liver injury is the greatest cause of post-marketing drug withdrawal; therefore, substantial resources are directed toward triaging potentially dangerous new compounds at all stages of drug development. One of the major factors preventing effective screening of new compounds is the lack of a predictive in vitro model of hepatotoxicity. Primary human hepatocytes offer a metabolically relevant model for which the molecular initiating events of hepatotoxicity can be examined; however, these cells vary greatly between donors and dedifferentiate rapidly in culture. Induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (HLCs) offer a reproducible, physiologically relevant and genotypically normal model cell; however, current differentiation protocols produce HLCs with a relatively immature phenotype. During the reprogramming of somatic cells, the epigenome undergoes dramatic changes; however, this "resetting" is a gradual process, resulting in an altered differentiation propensity, skewed toward the lineage of origin, particularly in early passage cultures. We, therefore, performed a comparison of human hepatocyte- and dermal fibroblast-derived iPSCs, assessing the impact of epigenetic memory at all stages of HLC differentiation. These results provide the first isogenic assessment of the starting cell type in human iPSC-derived HLCs. Despite a trend toward improvement in hepatic phenotype in albumin secretion and gene expression, few significant differences in hepatic differentiation capacity were found between hepatocyte and fibroblast-derived iPSCs. We conclude that the donor and inter-clonal differences have a greater influence on the hepatocyte phenotypic maturity than the starting cell type. Therefore, it is not necessary to use human hepatocytes for generating iPSC-derived HLCs. Stem Cells Translational Medicine 2017;6:1321-1331. © 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

IL-1 Receptor Signaling on Graft Parenchymal Cells Regulates Memory and De Novo Donor-Reactive CD8 T Cell Responses to Cardiac Allografts.

PubMed

Iida, Shoichi; Tsuda, Hidetoshi; Tanaka, Toshiaki; Kish, Danielle D; Abe, Toyofumi; Su, Charles A; Abe, Ryo; Tanabe, Kazunari; Valujskikh, Anna; Baldwin, William M; Fairchild, Robert L

2016-03-15

Reperfusion of organ allografts induces a potent inflammatory response that directs rapid memory T cell, neutrophil, and macrophage graft infiltration and their activation to express functions mediating graft tissue injury. The role of cardiac allograft IL-1 receptor (IL-1R) signaling in this early inflammation and the downstream primary alloimmune response was investigated. When compared with complete MHC-mismatched wild-type cardiac allografts, IL-1R(-/-) allografts had marked decreases in endogenous memory CD8 T cell and neutrophil infiltration and expression of proinflammatory mediators at early times after transplant, whereas endogenous memory CD4 T cell and macrophage infiltration was not decreased. IL-1R(-/-) allograft recipients also had marked decreases in de novo donor-reactive CD8, but not CD4, T cell development to IFN-γ-producing cells. CD8 T cell-mediated rejection of IL-1R(-/-) cardiac allografts took 3 wk longer than wild-type allografts. Cardiac allografts from reciprocal bone marrow reconstituted IL-1R(-/-)/wild-type chimeric donors indicated that IL-1R signaling on graft nonhematopoietic-derived, but not bone marrow-derived, cells is required for the potent donor-reactive memory and primary CD8 T cell alloimmune responses observed in response to wild-type allografts. These studies implicate IL-1R-mediated signals by allograft parenchymal cells in generating the stimuli-provoking development and elicitation of optimal alloimmune responses to the grafts. Copyright © 2016 by The American Association of Immunologists, Inc.

17β-Estradiol activates GPER- and ESR1-dependent pathways inducing apoptosis in GC-2 cells, a mouse spermatocyte-derived cell line.

PubMed

Chimento, Adele; Sirianni, Rosa; Casaburi, Ivan; Ruggiero, Carmen; Maggiolini, Marcello; Andò, Sebastiano; Pezzi, Vincenzo

2012-05-15

In mammals, spontaneous apoptosis is observed particularly in differentiating spermatogonia and in spermatocytes. 17β-Estradiol (E2) in primary rat pachytene spermatocytes (PS) binds estrogen receptor α (ESR1) and GPER to activate EGFR/ERK/c-Jun pathway leading to up regulation of proapoptotic factor bax. Aim of this study was to clarify the effector pathway(s) controlling spermatocytes apoptosis using as model GC-2 cells, an immortalized mouse pachytene spermatocyte-derived cell line, which reproduces primary cells responses to E2. In fact, in GC-2 cells we observed that ESR1 and GPER activation caused rapid ERK and c-Jun phosphorylation, bax up-regulation, events associated with apoptosis. We further investigated the apoptotic mechanism demonstrating that E2, as well as ESR1 and GPER specific agonists, induced sustained ERK, c-Jun and p38 phosphorylation, Cytochrome c release, caspase 3 and endogenous substrate Poly (ADP-ribose) polymerase (PARP) activation and increased expression of cell cycle inhibitor p21. When ESR1 or GPER expression was silenced, E2 was still able to decrease cell proliferation, only the concomitant silencing abolished E2 effect. These results indicate that GC-2 cells are a valid cell model to study E2-dependent apoptosis in spermatocytes and show that E2, activating both ESR1 and GPER, is able to induce an ERK1/2, c-Jun and p38-dependent mitochondrion apoptotic pathway in this cell type. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Production, characterisation, and cytocompatibility of porous titanium-based particulate scaffolds.

PubMed

Luthringer, B J C; Ali, F; Akaichi, H; Feyerabend, F; Ebel, T; Willumeit, R

2013-10-01

Despite its non-matching mechanical properties titanium remains the preferred metal implant material in orthopaedics. As a consequence in some cases stress shielding effect occurs, leading to implant loosening, osteopenia, and finally revision surgery. Porous metal scaffolds to allow easier specialised cells ingrowth with mechanical properties closer to the ones of bone can overcome this problem. This should improve healing processes, implant integration, and dynamic strength of implants retaining. Three Ti-6Al-4V materials were metal injection moulded and tailored porosities were effectively achieved. After microstructural and mechanical characterisation, two different primary cells of mesenchymal origin (human umbilical cord perivascular cells and human bone derived cells which revealed to be two pertinent models) as well as one cell line originated from primary osteogenic sarcoma, Saos-2, were bestowed to investigate cell-material interaction on genomic and proteome levels. Biological examinations disclosed that no material has negative impact on early adhesion, proliferation or cell viability. An efficient cell ingrowth into material with an average porosity of 25-50 μm was proved.

Comparative Study of Influenza Virus Replication in MDCK Cells and in Primary Cells Derived from Adenoids and Airway Epithelium

PubMed Central

Ikizler, Mine R.; Kawaoka, Yoshihiro; Rudenko, Larisa G.; Treanor, John J.; Subbarao, Kanta; Wright, Peter F.

2012-01-01

Although clinical trials with human subjects are essential for determination of safety, infectivity, and immunogenicity, it is desirable to know in advance the infectiousness of potential candidate live attenuated influenza vaccine strains for human use. We compared the replication kinetics of wild-type and live attenuated influenza viruses, including H1N1, H3N2, H9N2, and B strains, in Madin-Darby canine kidney (MDCK) cells, primary epithelial cells derived from human adenoids, and human bronchial epithelium (NHBE cells). Our data showed that despite the fact that all tissue culture models lack a functional adaptive immune system, differentiated cultures of human epithelium exhibited the greatest restriction for all H1N1, H3N2, and B vaccine viruses studied among three cell types tested and the best correlation with their levels of attenuation seen in clinical trials with humans. In contrast, the data obtained with MDCK cells were the least predictive of restricted viral replication of live attenuated vaccine viruses in humans. We were able to detect a statistically significant difference between the replication abilities of the U.S. (A/Ann Arbor/6/60) and Russian (A/Leningrad/134/17/57) cold-adapted vaccine donor strains in NHBE cultures. Since live attenuated pandemic influenza vaccines may potentially express a hemagglutinin and neuraminidase from a non-human influenza virus, we assessed which of the three cell cultures could be used to optimally evaluate the infectivity and cellular tropism of viruses derived from different hosts. Among the three cell types tested, NHBE cultures most adequately reflected the infectivity and cellular tropism of influenza virus strains with different receptor specificities. NHBE cultures could be considered for use as a screening step for evaluating the restricted replication of influenza vaccine candidates. PMID:22915797

R-ketorolac Targets Cdc42 and Rac1 and Alters Ovarian Cancer Cell Behaviors Critical for Invasion and Metastasis

PubMed Central

Guo, Yuna; Kenney, Shelby Ray; Muller, Carolyn Y.; Adams, Sarah; Rutledge, Teresa; Romero, Elsa; Murray-Krezan, Cristina; Prekeris, Rytis; Sklar, Larry A.; Hudson, Laurie G.; Wandinger-Ness, Angela

2015-01-01

Cdc42 (cell division control protein 42) and Rac1 (Ras-related C3 botulinum toxin substrate 1) are attractive therapeutic targets in ovarian cancer based on established importance in tumor cell migration, adhesion and invasion. Despite a predicted benefit, targeting GTPases has not yet been translated to clinical practice. We previously established that Cdc42 and constitutively active Rac1b are overexpressed in primary ovarian tumor tissues. Through high throughput screening and computational shape homology approaches we identified R-ketorolac as a Cdc42 and Rac1 inhibitor; distinct from the anti-inflammatory, cyclooxygenase inhibitory activity of S-ketorolac. In the present study, we establish R-ketorolac as an allosteric inhibitor of Cdc42 and Rac1. Cell-based assays validate R-ketorolac activity against Cdc42 and Rac1. Studies on immortalized human ovarian adenocarcinoma cells (SKOV3ip), and primary, patient-derived ovarian cancer cells show R-ketorolac is a robust inhibitor of growth factor or serum dependent Cdc42 and Rac1 activation with a potency and cellular efficacy similar to small molecule inhibitors of Cdc42 (CID2950007/ML141) and Rac1 (NSC23766). Furthermore, GTPase inhibition by R-ketorolac reduces downstream p21-activated kinases (PAK1/PAK2) effector activation by >80%. Multiple assays of cell behavior using SKOV3ip and primary patient-derived ovarian cancer cells show that R-ketorolac significantly inhibits cell adhesion, migration and invasion. In sum, we provide evidence for R-ketorolac as direct inhibitor of Cdc42 and Rac1 that is capable of modulating downstream GTPase-dependent, physiological responses, which are critical to tumor metastasis. Our findings demonstrate the selective inhibition of Cdc42 and Rac1 GTPases by an FDA approved drug-racemic ketorolac that can be used in humans. PMID:26206334

R-Ketorolac Targets Cdc42 and Rac1 and Alters Ovarian Cancer Cell Behaviors Critical for Invasion and Metastasis.

PubMed

Guo, Yuna; Kenney, S Ray; Muller, Carolyn Y; Adams, Sarah; Rutledge, Teresa; Romero, Elsa; Murray-Krezan, Cristina; Prekeris, Rytis; Sklar, Larry A; Hudson, Laurie G; Wandinger-Ness, Angela

2015-10-01

Cdc42 (cell division control protein 42) and Rac1 (Ras-related C3 botulinum toxin substrate 1) are attractive therapeutic targets in ovarian cancer based on established importance in tumor cell migration, adhesion, and invasion. Despite a predicted benefit, targeting GTPases has not yet been translated to clinical practice. We previously established that Cdc42 and constitutively active Rac1b are overexpressed in primary ovarian tumor tissues. Through high-throughput screening and computational shape homology approaches, we identified R-ketorolac as a Cdc42 and Rac1 inhibitor, distinct from the anti-inflammatory, cyclooxygenase inhibitory activity of S-ketorolac. In the present study, we establish R-ketorolac as an allosteric inhibitor of Cdc42 and Rac1. Cell-based assays validate R-ketorolac activity against Cdc42 and Rac1. Studies on immortalized human ovarian adenocarcinoma cells (SKOV3ip) and primary patient-derived ovarian cancer cells show that R-ketorolac is a robust inhibitor of growth factor or serum-dependent Cdc42 and Rac1 activation with a potency and cellular efficacy similar to small-molecule inhibitors of Cdc42 (CID2950007/ML141) and Rac1 (NSC23766). Furthermore, GTPase inhibition by R-ketorolac reduces downstream p21-activated kinases (PAK1/PAK2) effector activation by >80%. Multiple assays of cell behavior using SKOV3ip and primary patient-derived ovarian cancer cells show that R-ketorolac significantly inhibits cell adhesion, migration, and invasion. In summary, we provide evidence for R-ketorolac as a direct inhibitor of Cdc42 and Rac1 that is capable of modulating downstream GTPase-dependent, physiologic responses, which are critical to tumor metastasis. Our findings demonstrate the selective inhibition of Cdc42 and Rac1 GTPases by an FDA-approved drug, racemic ketorolac, that can be used in humans. ©2015 American Association for Cancer Research.

PEA and luteolin synergistically reduce mast cell-mediated toxicity and elicit neuroprotection in cell-based models of brain ischemia.

PubMed

Parrella, Edoardo; Porrini, Vanessa; Iorio, Rosa; Benarese, Marina; Lanzillotta, Annamaria; Mota, Mariana; Fusco, Mariella; Tonin, Paolo; Spano, PierFranco; Pizzi, Marina

2016-10-01

The combination of palmitoylethanolamide (PEA), an endogenous fatty acid amide belonging to the family of the N-acylethanolamines, and the flavonoid luteolin has been found to exert neuroprotective activities in a variety of mouse models of neurological disorders, including brain ischemia. Indirect findings suggest that the two molecules can reduce the activation of mastocytes in brain ischemia, thus modulating crucial cells that trigger the inflammatory cascade. Though, no evidence exists about a direct effect of PEA and luteolin on mast cells in experimental models of brain ischemia, either used separately or in combination. In order to fill this gap, we developed a novel cell-based model of severe brain ischemia consisting of primary mouse cortical neurons and cloned mast cells derived from mouse fetal liver (MC/9 cells) subjected to oxygen and glucose deprivation (OGD). OGD exposure promoted both mast cell degranulation and the release of lactate dehydrogenase (LDH) in a time-dependent fashion. MC/9 cells exacerbated neuronal damage in neuron-mast cells co-cultures exposed to OGD. Likewise, the conditioned medium derived from OGD-exposed MC/9 cells induced significant neurotoxicity in control primary neurons. PEA and luteolin pre-treatment synergistically prevented the OGD-induced degranulation of mast cells and reduced the neurotoxic potential of MC/9 cells conditioned medium. Finally, the association of the two drugs promoted a direct synergistic neuroprotection even in pure cortical neurons exposed to OGD. In summary, our results indicate that mast cells release neurotoxic factors upon OGD-induced activation. The association PEA-luteolin actively reduces mast cell-mediated neurotoxicity as well as pure neurons susceptibility to OGD. Copyright © 2016 Elsevier B.V. All rights reserved.

Marking cell lineages in living tissues.

PubMed

Kurup, Smita; Runions, John; Köhler, Uwe; Laplaze, Laurent; Hodge, Sarah; Haseloff, Jim

2005-05-01

We have generated a novel genetic system to visualize cell lineages in living tissues at high resolution. Heat shock was used to trigger the excision of a specific transposon and activation of a fluorescent marker gene. A histone-YFP marker was used to allow identification of cell lineages and easy counting of cells. Constitutive expression of a green fluorescent membrane protein was used to provide a precise outline of all surrounding cells. Marked lineages can be induced from specific cells within the organism by targeted laser irradiation, and the fate of the marked cells can be followed non-invasively. We have used the system to map cell lineages originating from the initials of primary and lateral roots in Arabidopsis. The lineage marking technique enabled us to measure the differential contribution of primary root pericycle cell files to developing lateral root primordia. The majority of cells in an emerging lateral root primordium derive from the central file of pericycle founder cells while off-centre founder cells contribute only a minor proliferation of tissue near the base of the root. The system shows great promise for the detailed study of cell division during morphogenesis.

Ex vivo expanded natural killer cells from breast cancer patients and healthy donors are highly cytotoxic against breast cancer cell lines and patient-derived tumours.

PubMed

Shenouda, Mira M; Gillgrass, Amy; Nham, Tina; Hogg, Richard; Lee, Amanda J; Chew, Marianne V; Shafaei, Mahsa; Aarts, Craig; Lee, Dean A; Hassell, John; Bane, Anita; Dhesy-Thind, Sukhbinder; Ashkar, Ali A

2017-07-01

Natural killer (NK) cells play a critical role in cancer immunosurveillance. Recent developments in NK cell ex-vivo expansion makes it possible to generate millions of activated NK cells from a small volume of peripheral blood. We tested the functionality of ex vivo expanded NK cells in vitro against breast cancer cell lines and in vivo using a xenograft mouse model. The study aim was to assess functionality and phenotype of expanded NK cells from breast cancer patients against breast cancer cell lines and autologous primary tumours. We used a well-established NK cell co-culture system to expand NK cells ex vivo from healthy donors and breast cancer patients and examined their surface marker expression. Moreover, we tested the ability of expanded NK cells to lyse the triple negative breast cancer and HER2-positive breast cancer cell lines MDA-MB-231 and MDA-MB-453, respectively. We also tested their ability to prevent tumour growth in vivo using a xenograft mouse model. Finally, we tested the cytotoxicity of expanded NK cells against autologous and allogeneic primary breast cancer tumours in vitro. After 3 weeks of culture we observed over 1000-fold expansion of NK cells isolated from either breast cancer patients or healthy donors. We also showed that the phenotype of expanded NK cells is comparable between those from healthy donors and cancer patients. Moreover, our results confirm the ability of ex vivo expanded NK cells to lyse tumour cell lines in vitro. While the cell lines examined had differential sensitivity to NK cell killing we found this was correlated with level of major histocompatibility complex (MHC) class I expression. In our in vivo model, NK cells prevented tumour establishment and growth in immunocompromised mice. Finally, we showed that NK cells expanded from the peripheral blood of breast cancer patients show high cytotoxicity against allogeneic and autologous patient-derived tumour cells in vitro. NK cells from breast cancer patients can be expanded similarly to those from healthy donors, have a high cytotoxic ability against breast cancer cell lines and patient-derived tumour cells, and can be compatible with current cancer treatments to restore NK cell function in cancer patients.

Inhibition of TGF-β Signaling in SHED Enhances Endothelial Differentiation.

PubMed

Xu, J G; Gong, T; Wang, Y Y; Zou, T; Heng, B C; Yang, Y Q; Zhang, C F

2018-02-01

Low efficiency of deriving endothelial cells (ECs) from adult stem cells hampers their utilization in tissue engineering studies. The purpose of this study was to investigate whether suppression of transforming growth factor beta (TGF-β) signaling could enhance the differentiation efficiency of dental pulp-derived stem cells into ECs. We initially used vascular endothelial growth factor A (VEGF-A) to stimulate 2 dental pulp-derived stem cells (dental pulp stem cells and stem cells from human exfoliated deciduous teeth [SHED]) and compared their differentiation capacity into ECs. We further evaluated whether the vascular endothelial growth factor receptor I (VEGF-RI)-specific ligand placental growth factor-1 (PlGF-1) could mediate endothelial differentiation. Finally, we investigated whether the TGF-β signaling inhibitor SB-431542 could enhance the inductive effect of VEGF-A on endothelial differentiation, as well as the underlying mechanisms involved. ECs differentiated from dental pulp-derived stem cells exhibited the typical phenotypes of primary ECs, with SHED possessing a higher endothelial differentiation potential than dental pulp stem cells. VEGFR1-specific ligand-PLGF exerted a negligible effect on SHED-ECs differentiation. Compared with VEGF-A alone, the combination of VEGF-A and SB-431542 significantly enhanced the endothelial differentiation of SHED. The presence of SB-431542 inhibited the phosphorylation of Suppressor of Mothers Against Decapentaplegic 2/3 (SMAD2/3), allowing for VEGF-A-dependent phosphorylation and upregulation of VEGFR2. Our results indicate that the combination of VEGF-A and SB-431542 could enhance the differentiation of dental pulp-derived stem cells into endothelial cells, and this process is mediated through enhancement of VEGF-A-VEGFR2 signaling and concomitant inhibition of TGF-β-SMAD2/3 signaling.

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.

Akt Suppression of TGFβ Signaling Contributes to the Maintenance of Vascular Identity in Embryonic Stem Cell-Derived Endothelial Cells

PubMed Central

Israely, Edo; Ginsberg, Michael; Nolan, Daniel; Ding, Bi-Sen; James, Daylon; Elemento, Olivier; Rafii, Shahin; Rabbany, Sina Y

2016-01-01

The ability to generate and maintain stable in vitro cultures of mouse endothelial cells (EC) has great potential for genetic dissection of the numerous pathologies involving vascular dysfunction as well as therapeutic applications. However, previous efforts at achieving sustained cultures of primary stable murine vascular cells have fallen short, and the cellular requirements for EC maintenance in vitro remain undefined. In this study, we have generated vascular ECs from mouse embryonic stem (ES) cells, and show that active Akt is essential to their survival and propagation as homogeneous monolayers in vitro. These cells harbor the phenotypical, biochemical, and functional characteristics of ECs, and expand throughout long-term cultures, while maintaining their angiogenic capacity. Moreover, Akt-transduced embryonic ECs form functional perfused vessels in vivo that anastomose with host blood vessels. We provide evidence for a novel function of Akt in stabilizing EC identity, whereby the activated form of the protein protects mouse ES cell-derived ECs from TGFβ-mediated transdifferentiation by downregulating SMAD3. These findings identify a role for Akt in regulating the developmental potential of ES cell-derived ECs, and demonstrate that active Akt maintains endothelial identity in embryonic ECs by interfering with active TGFβ-mediated processes that would ordinarily usher these cells to alternate fates. PMID:23963623

Akt suppression of TGFβ signaling contributes to the maintenance of vascular identity in embryonic stem cell-derived endothelial cells.

PubMed

Israely, Edo; Ginsberg, Michael; Nolan, Daniel; Ding, Bi-Sen; James, Daylon; Elemento, Olivier; Rafii, Shahin; Rabbany, Sina Y

2014-01-01

The ability to generate and maintain stable in vitro cultures of mouse endothelial cells (ECs) has great potential for genetic dissection of the numerous pathologies involving vascular dysfunction as well as therapeutic applications. However, previous efforts at achieving sustained cultures of primary stable murine vascular cells have fallen short, and the cellular requirements for EC maintenance in vitro remain undefined. In this study, we have generated vascular ECs from mouse embryonic stem (ES) cells and show that active Akt is essential to their survival and propagation as homogeneous monolayers in vitro. These cells harbor the phenotypical, biochemical, and functional characteristics of ECs and expand throughout long-term cultures, while maintaining their angiogenic capacity. Moreover, Akt-transduced embryonic ECs form functional perfused vessels in vivo that anastomose with host blood vessels. We provide evidence for a novel function of Akt in stabilizing EC identity, whereby the activated form of the protein protects mouse ES cell-derived ECs from TGFβ-mediated transdifferentiation by downregulating SMAD3. These findings identify a role for Akt in regulating the developmental potential of ES cell-derived ECs and demonstrate that active Akt maintains endothelial identity in embryonic ECs by interfering with active TGFβ-mediated processes that would ordinarily usher these cells to alternate fates. © AlphaMed Press.

The generation and functional characterization of induced pluripotent stem cells from human intervertebral disc nucleus pulposus cells.

PubMed

Zhu, Yanxia; Liang, Yuhong; Zhu, Hongxia; Lian, Cuihong; Wang, Liang; Wang, Yiwei; Gu, Hongsheng; Zhou, Guangqian; Yu, Xiaoping

2017-06-27

Disc degenerative disease (DDD) is believed to originate in the nucleus pulposus (NP) region therefore, it is important to obtain a greater number of active NP cells for the study and therapy of DDD. Human induced pluripotent stem cells (iPSCs) are a powerful tool for modeling the development of DDD in humans, and have the potential to be applied in regenerative medicine. NP cells were isolated from DDD patients following our improved method, and then the primary NP cells were reprogramed into iPSCs with Sendai virus vectors encoding 4 factors. Successful reprogramming of iPSCs was verified by the expression of surface markers and presence of teratoma. Differentiation of iPSCs into NP-like cells was performed in a culture plate or in hydrogel, whereby skin fibroblast derived-iPSCs were used as a control. Results demonstrated that iPSCs derived from NP cells displayed a normal karyotype, expressed pluripotency markers, and formed teratoma in nude mice. NP induction of iPSCs resulted in the expression of NP cell specific matrix proteins and related genes. Non-induced NP derived-iPSCs also showed some NP-like phenotype. Furthermore, NP-derived iPSCs differentiate much better in hydrogel than that in a culture plate. This is a novel method for the generation of iPSCs from NP cells of DDD patients, and we have successfully differentiated these iPSCs into NP-like cells in hydrogel. This method provides a novel treatment of DDD by using patient-specific NP cells in a relatively simple and straightforward manner.

Pediatric brain tumor cancer stem cells: cell cycle dynamics, DNA repair, and etoposide extrusion

PubMed Central

Hussein, Deema; Punjaruk, Wiyada; Storer, Lisa C.D.; Shaw, Lucy; Ottoman, Ramadan; Peet, Andrew; Miller, Suzanne; Bandopadhyay, Gagori; Heath, Rachel; Kumari, Rajendra; Bowman, Karen J.; Braker, Paul; Rahman, Ruman; Jones, George D.D.; Watson, Susan; Lowe, James; Kerr, Ian D.; Grundy, Richard G.; Coyle, Beth

2011-01-01

Reliable model systems are needed to elucidate the role cancer stem cells (CSCs) play in pediatric brain tumor drug resistance. The majority of studies to date have focused on clinically distinct adult tumors and restricted tumor types. Here, the CSC component of 7 newly established primary pediatric cell lines (2 ependymomas, 2 medulloblastomas, 2 gliomas, and a CNS primitive neuroectodermal tumor) was thoroughly characterized. Comparison of DNA copy number with the original corresponding tumor demonstrated that genomic changes present in the original tumor, typical of that particular tumor type, were retained in culture. In each case, the CSC component was approximately 3–4-fold enriched in neurosphere culture compared with monolayer culture, and a higher capacity for multilineage differentiation was observed for neurosphere-derived cells. DNA content profiles of neurosphere-derived cells expressing the CSC marker nestin demonstrated the presence of cells in all phases of the cell cycle, indicating that not all CSCs are quiescent. Furthermore, neurosphere-derived cells demonstrated an increased resistance to etoposide compared with monolayer-derived cells, having lower initial DNA damage, potentially due to a combination of increased drug extrusion by ATP-binding cassette multidrug transporters and enhanced rates of DNA repair. Finally, orthotopic xenograft models reflecting the tumor of origin were established from these cell lines. In summary, these cell lines and the approach taken provide a robust model system that can be used to develop our understanding of the biology of CSCs in pediatric brain tumors and other cancer types and to preclinically test therapeutic agents. PMID:20978004

Pediatric brain tumor cancer stem cells: cell cycle dynamics, DNA repair, and etoposide extrusion.

PubMed

Hussein, Deema; Punjaruk, Wiyada; Storer, Lisa C D; Shaw, Lucy; Othman, Ramadhan; Ottoman, Ramadan; Peet, Andrew; Miller, Suzanne; Bandopadhyay, Gagori; Heath, Rachel; Kumari, Rajendra; Bowman, Karen J; Braker, Paul; Rahman, Ruman; Jones, George D D; Watson, Susan; Lowe, James; Kerr, Ian D; Grundy, Richard G; Coyle, Beth

2011-01-01

Reliable model systems are needed to elucidate the role cancer stem cells (CSCs) play in pediatric brain tumor drug resistance. The majority of studies to date have focused on clinically distinct adult tumors and restricted tumor types. Here, the CSC component of 7 newly established primary pediatric cell lines (2 ependymomas, 2 medulloblastomas, 2 gliomas, and a CNS primitive neuroectodermal tumor) was thoroughly characterized. Comparison of DNA copy number with the original corresponding tumor demonstrated that genomic changes present in the original tumor, typical of that particular tumor type, were retained in culture. In each case, the CSC component was approximately 3-4-fold enriched in neurosphere culture compared with monolayer culture, and a higher capacity for multilineage differentiation was observed for neurosphere-derived cells. DNA content profiles of neurosphere-derived cells expressing the CSC marker nestin demonstrated the presence of cells in all phases of the cell cycle, indicating that not all CSCs are quiescent. Furthermore, neurosphere-derived cells demonstrated an increased resistance to etoposide compared with monolayer-derived cells, having lower initial DNA damage, potentially due to a combination of increased drug extrusion by ATP-binding cassette multidrug transporters and enhanced rates of DNA repair. Finally, orthotopic xenograft models reflecting the tumor of origin were established from these cell lines. In summary, these cell lines and the approach taken provide a robust model system that can be used to develop our understanding of the biology of CSCs in pediatric brain tumors and other cancer types and to preclinically test therapeutic agents.

Thymocyte emigration is mediated by active movement away from stroma-derived factors

PubMed Central

Poznansky, Mark C.; Olszak, Ivona T.; Evans, Richard H.; Wang, Zhengyu; Foxall, Russell B.; Olson, Douglas P.; Weibrecht, Kathryn; Luster, Andrew D.; Scadden, David T.

2002-01-01

T cells leave the thymus at a specific time during differentiation and do not return despite elaboration of known T cell chemoattractants by thymic stroma. We observed differentiation stage–restricted egress of thymocytes from an artificial thymus in which vascular structures or hemodynamics could not have been playing a role. Hypothesizing that active movement of cells away from a thymic product may be responsible, we demonstrated selective reduction in emigration from primary thymus by inhibitors of active movement down a concentration gradient (chemofugetaxis). Immature intrathymic precursors were insensitive to an emigration signal, whereas mature thymocytes and peripheral blood T cells were sensitive. Thymic stroma was noted to elaborate at least two proteins capable of inducing emigration, one of which was stromal cell–derived factor-1. Thymic emigration is mediated, at least in part, by specific fugetaxis-inducing factors to which only mature cells respond. PMID:11956248

Ajoene inhibits both primary tumor growth and metastasis of B16/BL6 melanoma cells in C57BL/6 mice.

PubMed

Taylor, Peter; Noriega, Raquel; Farah, Carla; Abad, María-Jesús; Arsenak, Miriam; Apitz, Rafael

2006-08-08

Ajoene is an organosulphur compound derived from garlic with important effects on several membrane-associated processes such as platelet aggregation, as well as being cytotoxic for tumor cell lines in vitro. In the present study, we investigated the effect of ajoene on different cell types in vitro, as well as its inhibitory effects on both primary tumors and metastasis in a mouse model. We found ajoene to inhibit tumor cell growth in vitro, but also to inhibit strongly metastasis to lung in the B16/BL6 melanoma tumor model in C57BL/6 mice. As far as we are aware, this is the first report of the anti-metastatic effect of ajoene. Ajoene also inhibited tumor-endothelial cell adhesion, as well as the in vivo TNF-alpha response to lipopolysaccharide. Possible mechanisms of its antitumoral activity are discussed in the light of these results.

In Vitro Generation of Functional Liver Organoid-Like Structures Using Adult Human Cells.

PubMed

Ramachandran, Sarada Devi; Schirmer, Katharina; Münst, Bernhard; Heinz, Stefan; Ghafoory, Shahrouz; Wölfl, Stefan; Simon-Keller, Katja; Marx, Alexander; Øie, Cristina Ionica; Ebert, Matthias P; Walles, Heike; Braspenning, Joris; Breitkopf-Heinlein, Katja

2015-01-01

In this study we used differentiated adult human upcyte® cells for the in vitro generation of liver organoids. Upcyte® cells are genetically engineered cell strains derived from primary human cells by lenti-viral transduction of genes or gene combinations inducing transient proliferation capacity (upcyte® process). Proliferating upcyte® cells undergo a finite number of cell divisions, i.e., 20 to 40 population doublings, but upon withdrawal of proliferation stimulating factors, they regain most of the cell specific characteristics of primary cells. When a defined mixture of differentiated human upcyte® cells (hepatocytes, liver sinusoidal endothelial cells (LSECs) and mesenchymal stem cells (MSCs)) was cultured in vitro on a thick layer of Matrigel™, they self-organized to form liver organoid-like structures within 24 hours. When further cultured for 10 days in a bioreactor, these liver organoids show typical functional characteristics of liver parenchyma including activity of cytochromes P450, CYP3A4, CYP2B6 and CYP2C9 as well as mRNA expression of several marker genes and other enzymes. In summary, we hereby describe that 3D functional hepatic structures composed of primary human cell strains can be generated in vitro. They can be cultured for a prolonged period of time and are potentially useful ex vivo models to study liver functions.

Dual mTORC1/2 inhibition induces anti-proliferative effect in NF1-associated plexiform neurofibroma and malignant peripheral nerve sheath tumor cells

PubMed Central

Hivelin, Mikael; Nusbaum, Patrick; Hubas, Arnaud; Laurendeau, Ingrid; Lantieri, Laurent; Wolkenstein, Pierre; Vidaud, Michel; Pasmant, Eric; Chapuis, Nicolas; Parfait, Béatrice

2016-01-01

Approximately 30-50% of individuals with Neurofibromatosis type 1 develop benign peripheral nerve sheath tumors, called plexiform neurofibromas (PNFs). PNFs can undergo malignant transformation to highly metastatic malignant peripheral nerve sheath tumors (MPNSTs) in 5-10% of NF1 patients, with poor prognosis. No effective systemic therapy is currently available for unresectable tumors. In tumors, the NF1 gene deficiency leads to Ras hyperactivation causing the subsequent activation of the AKT/mTOR and Raf/MEK/ERK pathways and inducing multiple cellular responses including cell proliferation. In this study, three NF1-null MPNST-derived cell lines (90-8, 88-14 and 96-2), STS26T sporadic MPNST cell line and PNF-derived primary Schwann cells were used to test responses to AZD8055, an ATP-competitive “active-site” mTOR inhibitor. In contrast to rapamycin treatment which only partially affected mTORC1 signaling, AZD8055 induced a strong inhibition of mTORC1 and mTORC2 signaling in MPNST-derived cell lines and PNF-derived Schwann cells. AZD8055 induced full blockade of mTORC1 leading to an efficient decrease of global protein synthesis. A higher cytotoxic effect was observed with AZD8055 compared to rapamycin in the NF1-null MPNST-derived cell lines with IC50 ranging from 70 to 140 nM and antiproliferative effect was confirmed in PNF-derived Schwann cells. Cell migration was impaired by AZD8055 treatment and cell cycle analysis showed a G0/G1 arrest. Combined effects of AZD8055 and PD0325901 MEK inhibitor as well as BRD4 (BromoDomain-containing protein 4) inhibitors showed a synergistic antiproliferative effect. These data suggest that NF1-associated peripheral nerve sheath tumors are an ideal target for AZD8055 as a single molecule or in combined therapies. PMID:26840085

Development of an efficient, non-viral transfection method for studying gene function and bone growth in human primary cranial suture mesenchymal cells reveals that the cells respond to BMP2 and BMP3.

PubMed

Dwivedi, Prem P; Anderson, Peter J; Powell, Barry C

2012-08-03

Achieving efficient introduction of plasmid DNA into primary cultures of mammalian cells is a common problem in biomedical research. Human primary cranial suture cells are derived from the connective mesenchymal tissue between the bone forming regions at the edges of the calvarial plates of the skull. Typically they are referred to as suture mesenchymal cells and are a heterogeneous population responsible for driving the rapid skull growth that occurs in utero and postnatally. To better understand the molecular mechanisms involved in skull growth, and in abnormal growth conditions, such as craniosynostosis, caused by premature bony fusion, it is essential to be able to easily introduce genes into primary bone forming cells to study their function. A comparison of several lipid-based techniques with two electroporation-based techniques demonstrated that the electroporation method known as nucleofection produced the best transfection efficiency. The parameters of nucleofection, including cell number, amount of DNA and nucleofection program, were optimized for transfection efficiency and cell survival. Two different genes and two promoter reporter vectors were used to validate the nucleofection method and the responses of human primary suture mesenchymal cells by fluorescence microscopy, RT-PCR and the dual luciferase assay. Quantification of bone morphogenetic protein (BMP) signalling using luciferase reporters demonstrated robust responses of the cells to both osteogenic BMP2 and to the anti-osteogenic BMP3. A nucleofection protocol has been developed that provides a simple and efficient, non-viral alternative method for in vitro studies of gene and protein function in human skull growth. Human primary suture mesenchymal cells exhibit robust responses to BMP2 and BMP3, and thus nucleofection can be a valuable method for studying the potential competing action of these two bone growth factors in a model system of cranial bone growth.

Fast isolation and expansion of multipotent cells from adipose tissue based on chitosan-selected primary culture.

PubMed

Huang, Guo-Shiang; Tseng, Ting-Chen; Dai, Niann-Tzyy; Fu, Keng-Yen; Dai, Lien-Guo; Hsu, Shan-Hui

2015-10-01

Adipose-derived adult stem cells (ASCs) have gained much attention because of their multipotency and easy access. Here we describe a novel chitosan-based selection (CS) system instead of the conventional plastic adherence (PA) to obtain the primary ASCs. The minimal amount of adipose tissue for consistent isolation of ASCs is reduced from 10 mL to 5 mL. The selection is based on the specific interaction between cells and chitosan materials, which separate ASCs by forming spheroids during primary culture. The primary culture period was reduced from 4 days to one day and more ASCs (ten-fold expansion) were achieved in a week. The average duration for obtaining 1 × 10(7) cells takes about seven days from 5 mL of adipose tissue, compared to 14 days using the conventional PA method from 10 mL of adipose tissue. The replicative senescence of CS-ASCs is not evident until the fifteenth passage (vs. eighth for the PA-ASCs). The obtained ASCs (CS-ASCs) have less doubling time for the same passage of cells and show greater stemness than those obtained from the conventional PA method (PA-ASCs). Moreover, CS-ASCs undergo trilineage differentiation more effectively than PA-ASCs. The greater differentiation potential of CS-ASCs may be associated with the enrichment and maintenance of CD271 positive cells by chitosan selection of primary culture. Copyright © 2015 Elsevier Ltd. All rights reserved.

Direct observation of CD4 T cell morphologies and their cross-sectional traction force derivation on quartz nanopillar substrates using focused ion beam technique

NASA Astrophysics Data System (ADS)

Kim, Dong-Joo; Kim, Gil-Sung; Hyung, Jung-Hwan; Lee, Won-Yong; Hong, Chang-Hee; Lee, Sang-Kwon

2013-07-01

Direct observations of the primary mouse CD4 T cell morphologies, e.g., cell adhesion and cell spreading by culturing CD4 T cells in a short period of incubation (e.g., 20 min) on streptavidin-functionalized quartz nanopillar arrays (QNPA) using a high-content scanning electron microscopy method were reported. Furthermore, we first demonstrated cross-sectional cell traction force distribution of surface-bound CD4 T cells on QNPA substrates by culturing the cells on top of the QNPA and further analysis in deflection of underlying QNPA via focused ion beam-assisted technique.

Adipose-Derived Stem Cells in Functional Bone Tissue Engineering: Lessons from Bone Mechanobiology

PubMed Central

Bodle, Josephine C.; Hanson, Ariel D.

2011-01-01

This review aims to highlight the current and significant work in the use of adipose-derived stem cells (ASC) in functional bone tissue engineering framed through the bone mechanobiology perspective. Over a century of work on the principles of bone mechanosensitivity is now being applied to our understanding of bone development. We are just beginning to harness that potential using stem cells in bone tissue engineering. ASC are the primary focus of this review due to their abundance and relative ease of accessibility for autologous procedures. This article outlines the current knowledge base in bone mechanobiology to investigate how the knowledge from this area has been applied to the various stem cell-based approaches to engineering bone tissue constructs. Specific emphasis is placed on the use of human ASC for this application. PMID:21338267

Effect of N-Acetylcysteine on Adipose-Derived Stem Cell and Autologous Fat Graft Survival in a Mouse Model.

PubMed

Gillis, Joshua; Gebremeskel, Simon; Phipps, Kyle D; MacNeil, Lori A; Sinal, Christopher J; Johnston, Brent; Hong, Paul; Bezuhly, Michael

2015-08-01

Autologous fat grafting is a popular reconstructive technique, but is limited by inconsistent graft retention. The authors examined whether a widely available, clinically safe antioxidant, N-acetylcysteine, could improve adipose-derived stem cell survival and graft take when added to tumescent solution during fat harvest. Inguinal fat pads were harvested from C57BL/6 mice using tumescent solution with or without N-acetylcysteine. Flow cytometric, proliferation, and differentiation assays were performed on isolated primary adipose-derived stem cells and 3T3-L1 preadipocytes treated with or without hydrogen peroxide and/or N-acetylcysteine. N-Acetylcysteine-treated or control grafts were injected under recipient mouse scalps and assessed by serial micro-computed tomographic volumetric analysis. Explanted grafts underwent immunohistochemical analysis. In culture, N-acetylcysteine protected adipose-derived stem cells from oxidative stress and improved cell survival following hydrogen peroxide treatment. Combined exposure to both N-acetylcysteine and hydrogen peroxide led to a 200-fold increase in adipose-derived stem cell proliferation, significantly higher than with either agent alone. N-Acetylcysteine decreased differentiation of adipose-derived stem cells into mature adipocytes, as evidenced by decreased transcription of adipocyte differentiation markers and reduced Oil Red-O staining. In vivo, N-acetylcysteine treatment resulted in improved graft retention at 3 months compared with control (46 versus 17 percent; p = 0.027). N-Acetylcysteine-treated grafts demonstrated less fibrosis and inflammation, and a 33 percent increase in adipocyte density compared with controls (p < 0.001) that was not associated with increased vascularity. These findings provide proof of principle for the addition of N-acetylcysteine to tumescent harvest solution in the clinical setting to optimize fat graft yields.

Nuclear fusion-independent smooth muscle differentiation of human adipose-derived stem cells induced by a smooth muscle environment.

PubMed

Zhang, Rong; Jack, Gregory S; Rao, Nagesh; Zuk, Patricia; Ignarro, Louis J; Wu, Benjamin; Rodríguez, Larissa V

2012-03-01

Human adipose-derived stem cells hASC have been isolated and were shown to have multilineage differentiation capacity. Although both plasticity and cell fusion have been suggested as mechanisms for cell differentiation in vivo, the effect of the local in vivo environment on the differentiation of adipose-derived stem cells has not been evaluated. We previously reported the in vitro capacity of smooth muscle differentiation of these cells. In this study, we evaluate the effect of an in vivo smooth muscle environment in the differentiation of hASC. We studied this by two experimental designs: (a) in vivo evaluation of smooth muscle differentiation of hASC injected into a smooth muscle environment and (b) in vitro evaluation of smooth muscle differentiation capacity of hASC exposed to bladder smooth muscle cells. Our results indicate a time-dependent differentiation of hASC into mature smooth muscle cells when these cells are injected into the smooth musculature of the urinary bladder. Similar findings were seen when the cells were cocultured in vitro with primary bladder smooth muscle cells. Chromosomal analysis demonstrated that microenvironment cues rather than nuclear fusion are responsible for this differentiation. We conclude that cell plasticity is present in hASCs, and their differentiation is accomplished in the absence of nuclear fusion. Copyright © 2011 AlphaMed Press.

Primary hepatic carcinoid tumor: case report and literature review

PubMed Central

Camargo, Éden Sartor; Viveiros, Marcelo de Melo; Corrêa, Isaac José Felippe; Robles, Laercio; Rezende, Marcelo Bruno

2014-01-01

Primary hepatic carcinoid tumors are extremely rare neoplasms derived from hormone-producing neuroendocrine cells. It is difficult to make their diagnosis before biopsy, surgical resection or necropsy. A recent publication described only 94 cases of these tumors. There is no sex predilection and apparently it has no association with cirrhosis or preexisting hepatic disease. The most effective treatment is hepatectomy, and resection is determined by size and location of the lesions. PMID:25628206

Aryl Hydrocarbon Receptor activation by diesel exhaust particles mediates epithelium-derived cytokines expression in severe allergic asthma.

PubMed

Weng, Chih-Ming; Wang, Chun-Hua; Lee, Meng-Jung; He, Jung-Re; Huang, Hsin-Yu; Chao, Ming-Wei; Chung, Kian Fan; Kuo, Han-Pin

2018-04-19

Exposure to environmental pollutants promotes Th2 cell responses. Aryl hydrocarbon receptor (AhR) activation aggravates allergic responses. Epithelium-derived thymic stromal lymphopoietin (TSLP), interleukin (IL)-25 and IL-33 are implicated in the dysregulation of Th2 immune responses in severe allergic asthma. Bronchial biopsies of 28 allergic severe asthma and 6 mild asthma subjects from highly polluted areas were analyzed for AhR nuclear translocation (NT), cytokine expression and gene activation. Cultured primary epithelial cells were stimulated with diesel exhausted particles (DEP) to determine AhR-mediated IL-33, Il-25 and TSLP synthesis and release. Primary bronchial epithelial cells exposed to DEP showed up-regulation of IL-33, IL-25 and TSLP. These effects were abolished by knock-down of AhR by siRNA. Increased AhR/ARNT binding to promoters of IL-33, IL-25, and TSLP was found using chromatin immunoprecipitation (ChIP) assay. Allergic severe asthma with high AhR NT had higher bronchial gene and protein expression of IL-33, IL-25 and TSLP. These patients derived clinical benefit from anti-IgE treatment. AhR activation by DEP mediates up-regulation of IL-33, IL-25 and TSLP with Th2 activation, potentially linking environmental pollution and allergic severe asthma. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Assessing commercial opportunities for autologous and allogeneic cell-based products.

PubMed

Smith, Devyn M

2012-09-01

The two primary cell sources used to produce cell-based therapies are autologous (self-derived) and allogeneic (derived from a donor). This analysis attempts to compare and contrast the two approaches in order to understand whether there is an emerging preference in the market. While the current clinical trials underway are slightly biased to autologous approaches, it is clear that both cell-based approaches are being aggressively pursued. This analysis also breaks down the commercial advantages of each cell-based approach, comparing both cost of goods and the ideal indication type for each. While allogeneic therapies have considerable advantages over autologous therapies, they do have a distinct disadvantage regarding potential immunogenicity. The introduction of the hybrid autologous business model provides the ability for autologous-based therapies to mitigate some of the advantages that allogeneic cell-based therapies enjoy, including cost of goods. Finally, two case studies are presented that demonstrate that there is sufficient space for both autologous and allogeneic cell-based therapies within a single disease area.

Isolation and Characterization of Ischemia-Derived Astrocytes (IDAs) with Ability to Transactivate Quiescent Astrocytes

PubMed Central

Villarreal, Alejandro; Rosciszewski, Gerardo; Murta, Veronica; Cadena, Vanesa; Usach, Vanina; Dodes-Traian, Martin M.; Setton-Avruj, Patricia; Barbeito, Luis H.; Ramos, Alberto J.

2016-01-01

Reactive gliosis involving activation and proliferation of astrocytes and microglia, is a widespread but largely complex and graded glial response to brain injury. Astroglial population has a previously underestimated high heterogeneity with cells differing in their morphology, gene expression profile, and response to injury. Here, we identified a subset of reactive astrocytes isolated from brain focal ischemic lesions that show several atypical characteristics. Ischemia-derived astrocytes (IDAs) were isolated from early ischemic penumbra and core. IDA did not originate from myeloid precursors, but rather from pre-existing local progenitors. Isolated IDA markedly differ from primary astrocytes, as they proliferate in vitro with high cell division rate, show increased migratory ability, have reduced replicative senescence and grow in the presence of macrophages within the limits imposed by the glial scar. Remarkably, IDA produce a conditioned medium that strongly induced activation on quiescent primary astrocytes and potentiated the neuronal death triggered by oxygen-glucose deprivation. When re-implanted into normal rat brains, eGFP-IDA migrated around the injection site and induced focal reactive gliosis. Inhibition of gamma secretases or culture on quiescent primary astrocytes monolayers facilitated IDA differentiation to astrocytes. We propose that IDA represent an undifferentiated, pro-inflammatory, highly replicative and migratory astroglial subtype emerging from the ischemic microenvironment that may contribute to the expansion of reactive gliosis. Main Points: Ischemia-derived astrocytes (IDA) were isolated from brain ischemic tissue IDA show reduced replicative senescence, increased cell division and spontaneous migration IDA potentiate death of oxygen-glucose deprived cortical neurons IDA propagate reactive gliosis on quiescent astrocytes in vitro and in vivo Inhibition of gamma secretases facilitates IDA differentiation to astrocytes PMID:27313509

Impaired clearance of early apoptotic cells mediated by inhibitory IgG antibodies in patients with primary Sjögren's syndrome.

PubMed

Manoussakis, Menelaos N; Fragoulis, George E; Vakrakou, Aigli G; Moutsopoulos, Haralampos M

2014-01-01

Deficient efferocytosis (i.e. phagocytic clearance of apoptotic cells) has been frequently reported in systemic lupus erythematosus (SLE). Todate, patients with primary Sjögren's syndrome (SS) have not been assessed for phagocytosis of apoptotic cells (ApoCell-phagocytosis) and of particulate targets (microbeads, MB-phagocytosis). ApoCell-phagocytosis and MB-phagocytosis were comparatively assessed by flow cytometry in peripheral blood specimens and monocyte-derived macrophage (MDM) preparations from healthy blood donors (HBD) and consecutive SS, SLE and rheumatoid arthritis (RA) patients. Cross-admixture ApoCell-phagocytosis experiments were also performed using phagocytes from HBD or patients, and apoptotic cells pretreated with whole sera or purified serum IgG derived from patients or HBD. Compared to HBD, approximately half of SS and SLE patients studied (but not RA) manifested significantly reduced ApoCell-phagocytosis (p<0.001) and MB-phagocytosis (p<0.003) by blood-borne phagocytes that correlated inversely with disease activity (p≤0.004). In cross-admixture assays, healthy monocytes showed significantly reduced ApoCell-phagocytosis when fed with apoptotic cells that were pretreated with sera or purified serum IgG preparations from SS and SLE patients (p<0.0001, compared to those from HBD or RA). Such aberrant effect of the SS and SLE sera and IgG preparations correlated linearly with their content of IgG antibodies against apoptotic cells (p≤0.0001). Phagocytic dysfunction maybe also present in certain SS and SLE patients, as supported by deficient capacity of MDM for ApoCell-phagocytosis and MB-phagocytosis under patients' serum-free conditions. Similarly to SLE, efferocytosis is frequently impaired in SS and is primarily due to the presence of inhibitory IgG anti-ApoCell antibodies and secondarily to phagocytes' dysfunction.

Generation of Osteosarcomas from a Combination of Rb Silencing and c‐Myc Overexpression in Human Mesenchymal Stem Cells

PubMed Central

Wang, Jir‐You; Wu, Po‐Kuei; Chen, Paul Chih‐Hsueh; Lee, Chia‐Wen

2016-01-01

Abstract Osteosarcoma (OS) was a malignant tumor occurring with unknown etiology that made prevention and early diagnosis difficult. Mesenchymal stem cells (MSCs), which were found in bone marrow, were claimed to be a possible origin of OS but with little direct evidence. We aimed to characterize OS cells transformed from human MSCs (hMSCs) and identify their association with human primary OS cells and patient survival. Genetic modification with p53 or retinoblastoma (Rb) knockdown and c‐Myc or Ras overexpression was applied for hMSC transformation. Transformed cells were assayed for proliferation, differentiation, tumorigenecity, and gene expression profile. Only the combination of Rb knockdown and c‐Myc overexpression successfully transformed hMSCs derived from four individual donors, with increasing cell proliferation, decreasing cell senescence rate, and increasing ability to form colonies and spheres in serum‐free medium. These transformed cells lost the expression of certain surface markers, increased in osteogenic potential, and decreased in adipogenic potential. After injection in immunodeficient mice, these cells formed OS‐like tumors, as evidenced by radiographic analyses and immunohistochemistry of various OS markers. Microarray with cluster analysis revealed that these transformed cells have gene profiles more similar to patient‐derived primary OS cells than their normal MSC counterparts. Most importantly, comparison of OS patient tumor samples revealed that a combination of Rb loss and c‐Myc overexpression correlated with a decrease in patient survival. This study successfully transformed human MSCs to OS‐like cells by Rb knockdown and c‐Myc overexpression that may be a useful platform for further investigation of preventive and target therapy for human OS. Stem Cells Translational Medicine 2017;6:512–526 PMID:28191765

18ß-glycyrrhetinic acid derivative promotes proliferation, migration and aquaporin-3 expression in human dermal fibroblasts.

PubMed

Hung, Chi-Feng; Hsiao, Chien-Yu; Hsieh, Wen-Hao; Li, Hsin-Ju; Tsai, Yi-Ju; Lin, Chun-Nan; Chang, Hsun-Hsien; Wu, Nan-Lin

2017-01-01

Licorice (Glycyrrhiza) species have been widely used as a traditional medicine and a natural sweetener in foods. The 18β-glycyrrhetinic acid (18β-GA) is a bioactive compound in licorice that exhibits potential anti-cancer, anti-inflammatory, and anti-microbial activities. Many synthesized derivatives of 18β-GA have been reported to be cytotoxic and suggested for the treatment of malignant diseases. In this study, we explored the possible pharmacological roles of an 18β-GA derivative in skin biology using primary human dermal fibroblasts and HaCaT keratinocytes as cell models. We found that this 18β-GA derivative did not cause cell death, but significantly enhanced the proliferation of dermal fibroblasts and HaCaT keratinocytes. A scratch wound healing assay revealed that the 18β-GA derivative promoted the migration of fibroblasts. Due to the important role of aquaporin-3 in cell migration and proliferation, we also investigated the expression of aquaporin-3 and found this compound up-regulated the expression of aquaporin-3 in dermal fibroblasts and HaCaT keratinocytes. In dermal fibroblasts, the 18β-GA derivative induced the phosphorylation of Akt, ERK, and p38. The inhibitor of Akt predominantly suppressed the 18β-GA derivative-induced expression of aquaporin-3. Collectively, this compound had a positive effect on the proliferation, migration, and aquaporin-3 expression of skin cells, implying its potential role in the treatment of skin diseases characterized by impaired wound healing or dermal defects.

RNF17 blocks promiscuous activity of PIWI proteins in mouse testes.

PubMed

Wasik, Kaja A; Tam, Oliver H; Knott, Simon R; Falciatori, Ilaria; Hammell, Molly; Vagin, Vasily V; Hannon, Gregory J

2015-07-01

PIWI proteins and their associated piRNAs protect germ cells from the activity of mobile genetic elements. Two classes of piRNAs—primary and secondary—are defined by their mechanisms of biogenesis. Primary piRNAs are processed directly from transcripts of piRNA cluster loci, whereas secondary piRNAs are generated in an adaptive amplification loop, termed the ping-pong cycle. In mammals, piRNA populations are dynamic, shifting as male germ cells develop. Embryonic piRNAs consist of both primary and secondary species and are mainly directed toward transposons. In meiotic cells, the piRNA population is transposon-poor and largely restricted to primary piRNAs derived from pachytene piRNA clusters. The transition from the embryonic to the adult piRNA pathway is not well understood. Here we show that RNF17 shapes adult meiotic piRNA content by suppressing the production of secondary piRNAs. In the absence of RNF17, ping-pong occurs inappropriately in meiotic cells. Ping-pong initiates piRNA responses against not only transposons but also protein-coding genes and long noncoding RNAs, including genes essential for germ cell development. Thus, the sterility of Rnf17 mutants may be a manifestation of a small RNA-based autoimmune reaction. © 2015 Wasik et al.; Published by Cold Spring Harbor Laboratory Press.

RNF17 blocks promiscuous activity of PIWI proteins in mouse testes

PubMed Central

Wasik, Kaja A.; Tam, Oliver H.; Knott, Simon R.; Falciatori, Ilaria; Hammell, Molly; Vagin, Vasily V.; Hannon, Gregory J.

2015-01-01

PIWI proteins and their associated piRNAs protect germ cells from the activity of mobile genetic elements. Two classes of piRNAs—primary and secondary—are defined by their mechanisms of biogenesis. Primary piRNAs are processed directly from transcripts of piRNA cluster loci, whereas secondary piRNAs are generated in an adaptive amplification loop, termed the ping-pong cycle. In mammals, piRNA populations are dynamic, shifting as male germ cells develop. Embryonic piRNAs consist of both primary and secondary species and are mainly directed toward transposons. In meiotic cells, the piRNA population is transposon-poor and largely restricted to primary piRNAs derived from pachytene piRNA clusters. The transition from the embryonic to the adult piRNA pathway is not well understood. Here we show that RNF17 shapes adult meiotic piRNA content by suppressing the production of secondary piRNAs. In the absence of RNF17, ping-pong occurs inappropriately in meiotic cells. Ping-pong initiates piRNA responses against not only transposons but also protein-coding genes and long noncoding RNAs, including genes essential for germ cell development. Thus, the sterility of Rnf17 mutants may be a manifestation of a small RNA-based autoimmune reaction. PMID:26115953

Cellular conservation of endangered midget buffalo (Lowland Anoa, Bubalus quarlesi) by establishment of primary cultured cell, and its immortalization with expression of cell cycle regulators.

PubMed

Fukuda, Tomokazu; Iino, Yuuka; Eitsuka, Takahiro; Onuma, Manabu; Katayama, Masafumi; Murata, Koichi; Inoue-Murayama, Miho; Hara, Kumiko; Isogai, Emiko; Kiyono, Tohru

2016-10-01

Lowland Anoa has become endangered due to hunting and human activity. Protection and breeding of endangered species in a controlled environment is the best way of conservation. However, it is not possible to adopt this approach for all endangered species because of the cost involved and the ever-increasing number of critically endangered species. In consideration of these limitations to the conventional conservation methods, we established a primary cell culture of endangered buffalo (Lowland Anoa, Bubalus quarlesi), for the preservation of this biological resource. In addition, we introduced human derived, mutant cyclin dependent kinase 4 (CDK4), Cyclin D, and telomerase reverse transcriptase (TERT) into the primary cells. The successful introduction of these three genes was confirmed by western blot with specific antibodies, and enzymatic activity. We also showed that the expression of mutant CDK4, Cyclin D, and TERT allows us to efficiently establish an immortalized cell line, with an intact chromosome pattern, from Lowland Anoa. To the best of our knowledge, this study is the first investigation that established an immortalized cell line of an endangered wild animal species.

In Vitro Evaluation of Nonnucleoside Reverse Transcriptase Inhibitors UC-781 and TMC120-R147681 as Human Immunodeficiency Virus Microbicides†

PubMed Central

Van Herrewege, Yven; Michiels, Jo; Van Roey, Jens; Fransen, Katrien; Kestens, Luc; Balzarini, Jan; Lewi, Paul; Vanham, Guido; Janssen, Paul

2004-01-01

The nonnucleoside reverse transcriptase inhibitors UC-781 and TMC120-R147681 (Dapivirine) effectively prevented human immunodeficiency virus (HIV) infection in cocultures of monocyte-derived dendritic cells and T cells, representing primary targets in sexual transmission. Both drugs had a favorable therapeutic index. A 24-h treatment with 1,000 nM UC-781 or 100 nM TMC120-R147681 prevented cell-free HIV infection, whereas 10-fold-higher concentrations blocked cell-associated HIV. PMID:14693562

Evaluation of selected biomarkers for the detection of chemical sensitization in human skin: a comparative study applying THP-1, MUTZ-3 and primary dendritic cells in culture.

PubMed

Hitzler, Manuel; Bergert, Antje; Luch, Andreas; Peiser, Matthias

2013-09-01

Dendritic cells (DCs) exhibit the unique capacity to induce T cell differentiation and proliferation, two processes that are crucially involved in allergic reactions. By combining the exclusive potential of DCs as the only professional antigen-presenting cells of the human body with the well known handling advantages of cell lines, cell-based alternative methods aimed at detecting chemical sensitization in vitro commonly apply DC-like cells derived from myeloid cell lines. Here, we present the new biomarkers programmed death-ligand 1 (PD-L1), DC immunoreceptor (DCIR), IL-16, and neutrophil-activating protein-2 (NAP-2), all of which have been detectable in primary human DCs upon exposure to chemical contact allergens. To evaluate the applicability of DC-like cells in the prediction of a chemical's sensitization potential, the expression of cell surface PD-L1 and DCIR was analyzed. In contrast to primary DCs, only minor subpopulations of MUTZ-3 and THP-1 cells presented PD-L1 or DCIR at their surface. After exposure to increasing concentrations of nickel and cinnamic aldehyde, the expression level of PD-L1 and DCIR revealed much stronger affected on monocyte-derived DCs (MoDCs) or Langerhans cells (MoLCs) when compared to THP-1 and MUTZ-3 cells. Applying protein profiler arrays we further identified the soluble factors NAP-2, IL-16, IL-8 and MIP-1α as sensitive biomarkers showing the capacity to discriminate sensitizing from non-sensitizing chemicals or irritants. An allergen-specific release of IL-8 and MIP-1α could be detected in the supernatants of MoDCs and MoLCs and also in MUTZ-3 and THP-1 cells, though at much lower levels. On the protein and transcriptional level, NAP-2 and IL-16 indicated sensitizers most sensitively and specifically in MoDCs. Altogether, we have proven the reciprocal regulated surface molecules PD-L1 and DCIR and the soluble factors MIP-1α, NAP-2 and IL-16 as reliable biomarkers for chemical sensitization. We further show that primary DCs are significantly different in their phenotype and function compared to DC-like cell lines. Since they demonstrated higher absolute values and a broader range in biomarker expression, we propose that MoDCs represent an optimal and robust sensor test system well suited to identify and classify chemicals with an allergic potential. Copyright © 2013 Elsevier Ltd. All rights reserved.

An isogenic blood-brain barrier model comprising brain endothelial cells, astrocytes, and neurons derived from human induced pluripotent stem cells.

PubMed

Canfield, Scott G; Stebbins, Matthew J; Morales, Bethsymarie Soto; Asai, Shusaku W; Vatine, Gad D; Svendsen, Clive N; Palecek, Sean P; Shusta, Eric V

2017-03-01

The blood-brain barrier (BBB) is critical in maintaining a physical and metabolic barrier between the blood and the brain. The BBB consists of brain microvascular endothelial cells (BMECs) that line the brain vasculature and combine with astrocytes, neurons and pericytes to form the neurovascular unit. We hypothesized that astrocytes and neurons generated from human-induced pluripotent stem cells (iPSCs) could induce BBB phenotypes in iPSC-derived BMECs, creating a robust multicellular human BBB model. To this end, iPSCs were used to form neural progenitor-like EZ-spheres, which were in turn differentiated to neurons and astrocytes, enabling facile neural cell generation. The iPSC-derived astrocytes and neurons induced barrier tightening in primary rat BMECs indicating their BBB inductive capacity. When co-cultured with human iPSC-derived BMECs, the iPSC-derived neurons and astrocytes significantly elevated trans-endothelial electrical resistance, reduced passive permeability, and improved tight junction continuity in the BMEC cell population, while p-glycoprotein efflux transporter activity was unchanged. A physiologically relevant neural cell mixture of one neuron: three astrocytes yielded optimal BMEC induction properties. Finally, an isogenic multicellular BBB model was successfully demonstrated employing BMECs, astrocytes, and neurons from the same donor iPSC source. It is anticipated that such an isogenic facsimile of the human BBB could have applications in furthering understanding the cellular interplay of the neurovascular unit in both healthy and diseased humans. Read the Editorial Highlight for this article on page 843. © 2016 International Society for Neurochemistry.

Adhesion Molecule Expression and Function of Primary Endothelial Cells in Benign and Malignant Tissues Correlates with Proliferation

PubMed Central

Sievert, Wolfgang; Tapio, Soile; Breuninger, Stephanie; Gaipl, Udo; Andratschke, Nicolaus; Trott, Klaus-Rüdiger; Multhoff, Gabriele

2014-01-01

Background Comparative analysis of the cellular biology of the microvasculature in different tissues requires the availability of viable primary endothelial cells (ECs). This study describes a novel method to isolate primary ECs from healthy organs, repair blastemas and tumors as examples of non-proliferating and proliferating benign and malignant tissues and their functional characterization. Methodology/Principal Findings Single cell suspensions from hearts, lungs, repair blastemas and tumors were incubated consecutively with an anti-CD31 antibody and magnetic micro-beads, coupled to a derivative of biotin and streptavidin, respectively. Following magnetic bead separation, CD31-positive ECs were released by biotin-streptavidin competition. In the absence of micro-beads, ECs became adherent to plastic surfaces. ECs from proliferating repair blastemas and tumors were larger and exhibited higher expression densities of CD31, CD105 and CD102 compared to those from non-proliferating normal tissues such as heart and lung. The expression density of CD34 was particularly high in tumor-derived ECs, and that of CD54 and CD144 in ECs of repair blastemas. Functionally, ECs of non-proliferating and proliferating tissues differed in their capacity to form tubes in matrigel and to align under flow conditions. Conclusions/Significance This method provides a powerful tool to generate high yields of viable, primary ECs of different origins. The results suggest that an altered expression of adhesion molecules on ECs in proliferating tissues contribute to loss of EC function that might cause a chaotic tumor vasculature. PMID:24632811

Calreticulin and Jak2 as Chaperones for MPL: Insights into MPN Pathogenesis

DTIC Science & Technology

2017-11-01

cell surface; and 2) CRISPR -Cas9 gene editing used to repair MPL T814C mutation in transfected cell lines and primary umbilical cord blood-derived...line has then been sub-cloned and individual clones were screened for robust Mpl expression using WB before being further modified (Fig. 1). CRISPR ...rescue its function and response to its ligand (Fig. 6). Genetic editing (using CRISPR /Cas9) performed on cells carrying the W272R mutation restored

Decreased proliferative, migrative and neuro-differentiative potential of postnatal rat enteric neural crest-derived cells during culture in vitro

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

Yu, Hui; Institute of Neurobiology, Environment and Genes Related to Diseases Key Laboratory of Chinese Ministry of Education, Xi’an Jiaotong University, No 96, Yan Ta Xi Road, Xi’an 710061, Shaanxi; Pan, Wei-Kang

A growing body of evidence supports the potential use of enteric neural crest-derived cells (ENCCs) as a cell replacement therapy for Hirschsprung's disease. Based on previous observations of robust propagation of primary ENCCs, as opposed to their progeny, it is suggested that their therapeutic potential after in vitro expansion may be restricted. We therefore examined the growth and differentiation activities and phenotypic characteristics of continuous ENCC cultures. ENCCs were isolated from the intestines of postnatal rats and were identified using an immunocytochemical approach. During continuous ENCC culture expansion, proliferation, migration, apoptosis, and differentiation potentials were monitored. The Cell Counting Kit-8more » was used for assessment of ENCC vitality, Transwell inserts for cell migration, immunocytochemistry for cell counts and identification, and flow cytometry for apoptosis. Over six continuous generations, ENCC proliferation potency was reduced and with prolonged culture, the ratio of migratory ENCCs was decreased. The percentage of apoptosis showed an upward trend with prolonged intragenerational culture, but showed a downward trend with prolonged culture of combined generations. Furthermore, the percentage of peripherin{sup +} cells decreased whilst the percentage of GFAP{sup +} cells increased with age. The results demonstrated that alterations in ENCC growth characteristics occur with increased culture time, which may partially account for the poor results of proposed cell therapies. - Highlights: • Differences were identified between primary and daughter ENCCs. • Daughter ENCCs had reduced proliferation, migration and differentiation. • Daughter ENCCs also had increased apoptosis. • These altered characteristics warrant further investigation.« less

Exosomes enriched in stemness/metastatic-related mRNAS promote oncogenic potential in breast cancer.

PubMed

Rodríguez, Marta; Silva, Javier; Herrera, Alberto; Herrera, Mercedes; Peña, Cristina; Martín, Paloma; Gil-Calderón, Beatriz; Larriba, María Jesús; Coronado, M Josés; Soldevilla, Beatriz; Turrión, Víctor S; Provencio, Mariano; Sánchez, Antonio; Bonilla, Félix; García-Barberán, Vanesa

2015-12-01

Cancer cells efficiently transfer exosome contents (essentially mRNAs and microRNAs) to other cell types, modifying immune responses, cell growth, angiogenesis and metastasis. Here we analyzed the exosomes release by breast tumor cells with different capacities of stemness/metastasis based on CXCR4 expression, and evaluated their capacity to generate oncogenic features in recipient cells. Breast cancer cells overexpressing CXCR4 showed an increase in stemness-related markers, and in proliferation, migration and invasion capacities. Furthermore, recipient cells treated with exosomes from CXCR4-cells showed increased in the same abilities. Moreover, inoculation of CXCR4-cell-derived exosomes in immunocompromised mice stimulated primary tumor growth and metastatic potential. Comparison of nucleic acids contained into exosomes isolated from patients revealed a "stemness and metastatic" signature in exosomes of patients with worse prognosis. Finally, our data supported the view that cancer cells with stem-like properties show concomitant metastatic behavior, and their exosomes stimulate tumor progression and metastasis. Exosomes-derived nucleic acids from plasma of breast cancer patients are suitable markers in the prognosis of such patients.

Modeling human endothelial cell transformation in vascular neoplasias

PubMed Central

Wen, Victoria W.; MacKenzie, Karen L.

2013-01-01

Endothelial cell (EC)-derived neoplasias range from benign hemangioma to aggressive metastatic angiosarcoma, which responds poorly to current treatments and has a very high mortality rate. The development of treatments that are more effective for these disorders will be expedited by insight into the processes that promote abnormal proliferation and malignant transformation of human ECs. The study of primary endothelial malignancy has been limited by the rarity of the disease; however, there is potential for carefully characterized EC lines and animal models to play a central role in the discovery, development and testing of molecular targeted therapies for vascular neoplasias. This review describes molecular alterations that have been identified in EC-derived neoplasias, as well as the processes that underpin the immortalization and tumorigenic conversion of ECs. Human EC lines, established through the introduction of defined genetic elements or by culture of primary tumor tissue, are catalogued and discussed in relation to their relevance as models of vascular neoplasia. PMID:24046386

Upregulation of CYP 450s expression of immortalized hepatocyte-like cells derived from mesenchymal stem cells by enzyme inducers

PubMed Central

2011-01-01

Background The strenuous procurement of cultured human hepatocytes and their short lives have constrained the cell culture model of cytochrome P450 (CYP450) induction, xenobiotic biotransformation, and hepatotoxicity. The development of continuous non-tumorous cell line steadily containing hepatocyte phenotypes would substitute the primary hepatocytes for these studies. Results The hepatocyte-like cells have been developed from hTERT plus Bmi-1-immortalized human mesenchymal stem cells to substitute the primary hepatocytes. The hepatocyte-like cells had polygonal morphology and steadily produced albumin, glycogen, urea and UGT1A1 beyond 6 months while maintaining proliferative capacity. Although these hepatocyte-like cells had low basal expression of CYP450 isotypes, their expressions could be extensively up regulated to 80 folds upon the exposure to enzyme inducers. Their inducibility outperformed the classical HepG2 cells. Conclusion The hepatocyte-like cells contained the markers of hepatocytes including CYP450 isotypes. The high inducibility of CYP450 transcripts could serve as a sensitive model for profiling xenobiotic-induced expression of CYP450. PMID:21961524

Dedifferentiation of Human Primary Thyrocytes into Multilineage Progenitor Cells without Gene Introduction

PubMed Central

Saenko, Vladimir; Suzuki, Masatoshi; Matsuse, Michiko; Ohtsuru, Akira; Kumagai, Atsushi; Uga, Tatsuya; Yano, Hiroshi; Nagayama, Yuji; Yamashita, Shunichi

2011-01-01

While identification and isolation of adult stem cells have potentially important implications, recent reports regarding dedifferentiation/reprogramming from differentiated cells have provided another clue to gain insight into source of tissue stem/progenitor cells. In this study, we developed a novel culture system to obtain dedifferentiated progenitor cells from normal human thyroid tissues. After enzymatic digestion, primary thyrocytes, expressing thyroglobulin, vimentin and cytokeratin-18, were cultured in a serum-free medium called SAGM. Although the vast majority of cells died, a small proportion (∼0.5%) survived and proliferated. During initial cell expansion, thyroglobulin/cytokeratin-18 expression was gradually declined in the proliferating cells. Moreover, sorted cells expressing thyroid peroxidase gave rise to proliferating clones in SAGM. These data suggest that those cells are derived from thyroid follicular cells or at least thyroid-committed cells. The SAGM-grown cells did not express any thyroid-specific genes. However, after four-week incubation with FBS and TSH, cytokeratin-18, thyroglobulin, TSH receptor, PAX8 and TTF1 expressions re-emerged. Moreover, surprisingly, the cells were capable of differentiating into neuronal or adipogenic lineage depending on differentiating conditions. In summary, we have developed a novel system to generate multilineage progenitor cells from normal human thyroid tissues. This seems to be achieved by dedifferentiation of thyroid follicular cells. The presently described culture system may be useful for regenerative medicine, but the primary importance will be as a tool to elucidate the mechanisms of thyroid diseases. PMID:21556376

Extensive and systematic rewiring of histone post-translational modifications in cancer model systems.

PubMed

Noberini, Roberta; Osti, Daniela; Miccolo, Claudia; Richichi, Cristina; Lupia, Michela; Corleone, Giacomo; Hong, Sung-Pil; Colombo, Piergiuseppe; Pollo, Bianca; Fornasari, Lorenzo; Pruneri, Giancarlo; Magnani, Luca; Cavallaro, Ugo; Chiocca, Susanna; Minucci, Saverio; Pelicci, Giuliana; Bonaldi, Tiziana

2018-05-04

Histone post-translational modifications (PTMs) generate a complex combinatorial code that regulates gene expression and nuclear functions, and whose deregulation has been documented in different types of cancers. Therefore, the availability of relevant culture models that can be manipulated and that retain the epigenetic features of the tissue of origin is absolutely crucial for studying the epigenetic mechanisms underlying cancer and testing epigenetic drugs. In this study, we took advantage of quantitative mass spectrometry to comprehensively profile histone PTMs in patient tumor tissues, primary cultures and cell lines from three representative tumor models, breast cancer, glioblastoma and ovarian cancer, revealing an extensive and systematic rewiring of histone marks in cell culture conditions, which includes a decrease of H3K27me2/me3, H3K79me1/me2 and H3K9ac/K14ac, and an increase of H3K36me1/me2. While some changes occur in short-term primary cultures, most of them are instead time-dependent and appear only in long-term cultures. Remarkably, such changes mostly revert in cell line- and primary cell-derived in vivo xenograft models. Taken together, these results support the use of xenografts as the most representative models of in vivo epigenetic processes, suggesting caution when using cultured cells, in particular cell lines and long-term primary cultures, for epigenetic investigations.

Extensive and systematic rewiring of histone post-translational modifications in cancer model systems

PubMed Central

Noberini, Roberta; Osti, Daniela; Miccolo, Claudia; Richichi, Cristina; Lupia, Michela; Corleone, Giacomo; Hong, Sung-Pil; Colombo, Piergiuseppe; Pollo, Bianca; Fornasari, Lorenzo; Pruneri, Giancarlo; Magnani, Luca; Cavallaro, Ugo; Chiocca, Susanna; Minucci, Saverio; Pelicci, Giuliana; Bonaldi, Tiziana

2018-01-01

Abstract Histone post-translational modifications (PTMs) generate a complex combinatorial code that regulates gene expression and nuclear functions, and whose deregulation has been documented in different types of cancers. Therefore, the availability of relevant culture models that can be manipulated and that retain the epigenetic features of the tissue of origin is absolutely crucial for studying the epigenetic mechanisms underlying cancer and testing epigenetic drugs. In this study, we took advantage of quantitative mass spectrometry to comprehensively profile histone PTMs in patient tumor tissues, primary cultures and cell lines from three representative tumor models, breast cancer, glioblastoma and ovarian cancer, revealing an extensive and systematic rewiring of histone marks in cell culture conditions, which includes a decrease of H3K27me2/me3, H3K79me1/me2 and H3K9ac/K14ac, and an increase of H3K36me1/me2. While some changes occur in short-term primary cultures, most of them are instead time-dependent and appear only in long-term cultures. Remarkably, such changes mostly revert in cell line- and primary cell-derived in vivo xenograft models. Taken together, these results support the use of xenografts as the most representative models of in vivo epigenetic processes, suggesting caution when using cultured cells, in particular cell lines and long-term primary cultures, for epigenetic investigations. PMID:29618087

Recombinant Rabbit Leukemia Inhibitory Factor and Rabbit Embryonic Fibroblasts Support the Derivation and Maintenance of Rabbit Embryonic Stem Cells

PubMed Central

Xue, Fei; Ma, Yinghong; Chen, Y. Eugene; Zhang, Jifeng; Lin, Tzu-An; Chen, Chien-Hong; Lin, Wei-Wen; Roach, Marsha; Ju, Jyh-Cherng; Yang, Lan; Du, Fuliang

2012-01-01

Abstract The rabbit is a classical experimental animal species. A major limitation in using rabbits for biomedical research is the lack of germ-line-competent rabbit embryonic stem cells (rbESCs). We hypothesized that the use of homologous feeder cells and recombinant rabbit leukemia inhibitory factor (rbLIF) might improve the chance in deriving germ-line-competent rbES cells. In the present study, we established rabbit embryonic fibroblast (REF) feeder layers and synthesized recombinant rbLIF. We derived a total of seven putative rbESC lines, of which two lines (M5 and M23) were from culture Condition I using mouse embryonic fibroblasts (MEFs) as feeders supplemented with human LIF (hLIF) (MEF+hLIF). Another five lines (R4, R9, R15, R21, and R31) were derived from Condition II using REFs as feeder cells supplemented with rbLIF (REF+rbLIF). Similar derivation efficiency was observed between these two conditions (8.7% vs. 10.2%). In a separate experiment with 2×3 factorial design, we examined the effects of feeder cells (MEF vs. REF) and LIFs (mLIF, hLIF vs. rbLIF) on rbESC culture. Both Conditions I and II supported satisfactory rbESC culture, with similar or better population doubling time and colony-forming efficiency than other combinations of feeder cells with LIFs. Rabbit ESCs derived and maintained on both conditions displayed typical ESC characteristics, including ESC pluripotency marker expression (AP, Oct4, Sox2, Nanog, and SSEA4) and gene expression (Oct4, Sox2, Nanog, c-Myc, Klf4, and Dppa5), and the capacity to differentiate into three primary germ layers in vitro. The present work is the first attempt to establish rbESC lines using homologous feeder cells and recombinant rbLIF, by which the rbESCs were derived and maintained normally. These cell lines are unique resources and may facilitate the derivation of germ-line-competent rbESCs. PMID:22775411

Human Induced Pluripotent Stem Cell-Derived Podocytes Mature into Vascularized Glomeruli upon Experimental Transplantation.

PubMed

Sharmin, Sazia; Taguchi, Atsuhiro; Kaku, Yusuke; Yoshimura, Yasuhiro; Ohmori, Tomoko; Sakuma, Tetsushi; Mukoyama, Masashi; Yamamoto, Takashi; Kurihara, Hidetake; Nishinakamura, Ryuichi

2016-06-01

Glomerular podocytes express proteins, such as nephrin, that constitute the slit diaphragm, thereby contributing to the filtration process in the kidney. Glomerular development has been analyzed mainly in mice, whereas analysis of human kidney development has been minimal because of limited access to embryonic kidneys. We previously reported the induction of three-dimensional primordial glomeruli from human induced pluripotent stem (iPS) cells. Here, using transcription activator-like effector nuclease-mediated homologous recombination, we generated human iPS cell lines that express green fluorescent protein (GFP) in the NPHS1 locus, which encodes nephrin, and we show that GFP expression facilitated accurate visualization of nephrin-positive podocyte formation in vitro These induced human podocytes exhibited apicobasal polarity, with nephrin proteins accumulated close to the basal domain, and possessed primary processes that were connected with slit diaphragm-like structures. Microarray analysis of sorted iPS cell-derived podocytes identified well conserved marker gene expression previously shown in mouse and human podocytes in vivo Furthermore, we developed a novel transplantation method using spacers that release the tension of host kidney capsules, thereby allowing the effective formation of glomeruli from human iPS cell-derived nephron progenitors. The human glomeruli were vascularized with the host mouse endothelial cells, and iPS cell-derived podocytes with numerous cell processes accumulated around the fenestrated endothelial cells. Therefore, the podocytes generated from iPS cells retain the podocyte-specific molecular and structural features, which will be useful for dissecting human glomerular development and diseases. Copyright © 2016 by the American Society of Nephrology.

Evaluation of hematopoietic potential generated by transplantation of muscle-derived stem cells in mice.

PubMed

Farace, Francoise; Prestoz, Laetitita; Badaoui, Sabrina; Guillier, Martine; Haond, Celine; Opolon, Paule; Thomas, Jean-Leon; Zalc, Bernard; Vainchenker, William; Turhan, Ali G

2004-02-01

Muscle tissue of adult mice has been shown to contain stem cells with hematopoietic repopulation ability in vivo. To determine the functional characteristics of stem cells giving rise to this hematopoietic activity, we have performed hematopoietic reconstitution experiments by the use of muscle versus marrow transplantation in lethally irradiated mice and followed the fate of transplanted cells by Y-chimerism using PCR and fluorescence in situ hybridization (FISH) analysis. We report here that transplantation of murine muscle generate a major hematopoietic chimerism at the level of CFU-C, CFU-S, and terminally-differentiated cells in three generations of lethally irradiated mice followed up to 1 year after transplantation. This potential is totally abolished when muscle grafts were performed by the use of muscle from previously irradiated mice. As compared to marrow transplantation, muscle transplants were able to generate similar potencies to give rise to myeloid, T, B, and natural killer (NK) cells. Interestingly, marrow stem cells that have been generated in primary and then in secondary recipients were able to contribute efficiently to myofibers in the muscle tissue of tertiary recipients. Altogether, our data demonstrate that muscle-derived stem cells present a major hematopoietic repopulating ability with evidence of self-replication in vivo. They are radiation-sensitive and similar to marrow-derived stem cells in terms of their ability to generate multilineage hematopoiesis. Finally, our data demonstrate that muscle-derived hematopoietic stem cells do not lose their ability to contribute to myofiber generation after at least two rounds of serial transplantation, suggesting a potential that is probably equivalent to that generated by marrow transplantation.

Stromal derived factor-1 regulates bone morphogenetic protein 2-induced osteogenic differentiation of primary mesenchymal stem cells

PubMed Central

Hosogane, Naobumi; Huang, Zhiping; Rawlins, Bernard A.; Liu, Xia; Boachie-Adjei, Oheneba; Boskey, Adele L.; Zhu, Wei

2010-01-01

Stromal derived factor-1 (SDF-1) is a chemokine signaling molecule that binds to its transmembrane receptor CXC chemokine receptor-4 (CXCR4). While we previously detected that SDF-1 was co-required with bone morphogenetic protein 2 (BMP2) for differentiating mesenchymal C2C12 cells into osteoblastic cells, it is unknown whether SDF-1 is similarly involved in the osteogenic differentiation of mesenchymal stem cells (MSCs). Therefore, here we examined the role of SDF-1 signaling during BMP2-induced osteogenic differentiation of primary MSCs that were derived from human and mouse bone marrow. Our data showed that blocking of the SDF-1/CXCR4 signal axis or adding SDF-1 protein to MSCs significantly affected BMP2-induced alkaline phosphatase (ALP) activity and osteocalcin (OCN) synthesis, markers of preosteoblasts and mature osteoblasts, respectively. Moreover, disrupting the SDF-1 signaling impaired bone nodule mineralization during terminal differentiation of MSCs. Furthermore, we detected that blocking of the SDF-1 signaling inhibited the BMP2-induced early expression of Runt-related factor-2 (Runx2) and osterix (Osx), two “master” regulators of osteogenesis, and the SDF-1 effect was mediated via intracellular Smad and Erk activation. In conclusion, our results demonstrated a regulatory role of SDF-1 in BMP2-induced osteogenic differentiation of MSCs, as perturbing the SDF-1 signaling affected the differentiation of MSCs towards osteoblastic cells in response to BMP2 stimulation. These data provide novel insights into molecular mechanisms underlying MSC osteogenesis, and will contribute to the development of MSC therapies for enhancing bone formation and regeneration in broad orthopaedic situations. PMID:20362069

Endothelial cell-derived nitric oxide enhances aerobic glycolysis in astrocytes via HIF-1α-mediated target gene activation.

PubMed

Brix, Britta; Mesters, Jeroen R; Pellerin, Luc; Jöhren, Olaf

2012-07-11

Astrocytes exhibit a prominent glycolytic activity, but whether such a metabolic profile is influenced by intercellular communication is unknown. Treatment of primary cultures of mouse cortical astrocytes with the nitric oxide (NO) donor DetaNONOate induced a time-dependent enhancement in the expression of genes encoding various glycolytic enzymes as well as transporters for glucose and lactate. Such an effect was shown to be dependent on the hypoxia-inducible factor HIF-1α, which is stabilized and translocated to the nucleus to exert its transcriptional regulation. NO action was dependent on both the PI3K/Akt/mTOR and MEK signaling pathways and required the activation of COX, but was independent of the soluble guanylate cyclase pathway. Furthermore, as a consequence of NO treatment, an enhanced lactate production and release by astrocytes was evidenced, which was prevented by downregulating HIF-1α. Several brain cell types represent possible sources of NO. It was found that endothelial cells, which express the endothelial NO synthase (eNOS) isoform, constitutively produced the largest amount of NO in culture. When astrocytes were cocultured with primary cultures of brain vascular endothelial cells, stabilization of HIF-1α and an enhancement in glucose transporter-1, hexokinase-2, and monocarboxylate transporter-4 expression as well as increased lactate production was found in astrocytes. This effect was inhibited by the NOS inhibitor l-NAME and was not seen when astrocytes were cocultured with primary cultures of cortical neurons. Our findings suggest that endothelial cell-derived NO participates to the maintenance of a high glycolytic activity in astrocytes mediated by astrocytic HIF-1α activation.

The differentiation and isolation of mouse embryonic stem cells toward hepatocytes using galactose-carrying substrata.

PubMed

Meng, Qingyuan; Haque, Amranul; Hexig, Bayar; Akaike, Toshihiro

2012-02-01

A simple culture system to achieve the differentiation of embryonic stem (ES) cells toward hepatocytes with high efficiency is crucial in providing a cell source for the medical application. In this study, we report the effect of a matrix-dependent enrichment of ES cell-derived hepatocytes using immobilized poly(N-p-vinylbenzyl-4-O-β-D-galactopyranosyl-D-gluconamide) (PVLA) with E-cadherin-IgG Fc (E-cad-Fc) as a galactose-carrying substratum. PVLA and E-cad-Fc were confirmed to be stably co-adsorbed onto polystyrene surface by quartz crystal microbalance (QCM). We showed that the E-cad-Fc/PVLA hybrid substratum was efficient in culturing primary hepatocytes and maintaining liver functions, on which the undifferentiated ES cells also maintained high proliferative capability. Furthermore, ES cell-derived hepatocytes on this hybrid matrix expressed elevated level of liver specific genes and functions together with early expression of definitive hepatocyte marker, asialoglycoprotein receptor (ASGPR). Finally, we isolated a high percentage of cells (about 60%) with ASGPR expression after re-seeding onto PVLA-coated surface, and observed the elimination of the poorly differentiated cells (Gata6(+) and Sox17(+)) and the ones toward another cell lineage (brachyury(+) and Pdx1(+)). The system uses a glycopolymer as an extracellular substratum for isolation and enrichment of ES cell-derived hepatocytes with adequate homogeneity and functionality. Copyright © 2011 Elsevier Ltd. All rights reserved.

An autologous dendritic cell canine mammary tumor hybrid-cell fusion vaccine.

PubMed

Bird, R Curtis; Deinnocentes, Patricia; Church Bird, Allison E; van Ginkel, Frederik W; Lindquist, Joni; Smith, Bruce F

2011-01-01

Mammary cancer is among the most prevalent canine tumors and frequently resulting in death due to metastatic disease that is highly homologous to human breast cancer. Most canine tumors fail to raise effective immune reactions yet, some spontaneous remissions do occur. Hybrid canine dendritic cell-tumor cell fusion vaccines were designed to enhance antigen presentation and tumor immune recognition. Peripheral blood-derived autologous dendritic cell enriched populations were isolated from dogs based on CD11c(+) expression and fused with canine mammary tumor (CMT) cells for vaccination of laboratory Beagles. These hybrid cells were injected into popliteal lymph nodes of normal dogs, guided by ultrasound, and included CpG-oligonucleotide adjuvants. Three rounds of vaccination were delivered. Significant IgG responses were observed in all vaccinated dogs compared to vehicle-injected controls. Canine IgG antibodies recognized shared CMT antigens as was demonstrated by IgG-recognition of three unrelated/independently derived CMT cell lines, and recognition of freshly isolated, unrelated, primary biopsy-derived CMT cells. A bias toward an IgG2 isotype response was observed after two vaccinations in most dogs. Neither significant cytotoxic T cell responses were detected, nor adverse or side-effects due to vaccination or due to the induced immune responses noted. These data provide proof-of-principle for this cancer vaccine strategy and demonstrate the presence of shared CMT antigens that promote immune recognition of mammary cancer.

Paragangliomas arise through an autonomous vasculo-angio-neurogenic program inhibited by imatinib.

PubMed

Verginelli, Fabio; Perconti, Silvia; Vespa, Simone; Schiavi, Francesca; Prasad, Sampath Chandra; Lanuti, Paola; Cama, Alessandro; Tramontana, Lorenzo; Esposito, Diana Liberata; Guarnieri, Simone; Sheu, Artenca; Pantalone, Mattia Russel; Florio, Rosalba; Morgano, Annalisa; Rossi, Cosmo; Bologna, Giuseppina; Marchisio, Marco; D'Argenio, Andrea; Taschin, Elisa; Visone, Rosa; Opocher, Giuseppe; Veronese, Angelo; Paties, Carlo T; Rajasekhar, Vinagolu K; Söderberg-Nauclér, Cecilia; Sanna, Mario; Lotti, Lavinia Vittoria; Mariani-Costantini, Renato

2018-05-01

Tumours can be viewed as aberrant tissues or organs sustained by tumorigenic stem-like cells that engage into dysregulated histo/organogenetic processes. Paragangliomas, prototypical organoid tumours constituted by dysmorphic variants of the vascular and neural tissues found in normal paraganglia, provide a model to test this hypothesis. To understand the origin of paragangliomas, we built a biobank comprising 77 cases, 18 primary cultures, 4 derived cell lines, 80 patient-derived xenografts and 11 cell-derived xenografts. We comparatively investigated these unique complementary materials using morphofunctional, ultrastructural and flow cytometric assays accompanied by microRNA studies. We found that paragangliomas contain stem-like cells with hybrid mesenchymal/vasculoneural phenotype, stabilized and expanded in the derived cultures. The viability and growth of such cultures depended on the downregulation of the miR-200 and miR-34 families, which allowed high PDGFRA and ZEB1 protein expression levels. Both tumour tissue- and cell culture-derived xenografts recapitulated the vasculoneural paraganglioma structure and arose from mesenchymal-like cells through a fixed developmental sequence. First, vasculoangiogenesis organized the microenvironment, building a perivascular niche which in turn supported neurogenesis. Neuroepithelial differentiation was associated with severe mitochondrial dysfunction, not present in cultured paraganglioma cells, but acquired in vivo during xenograft formation. Vasculogenesis was the Achilles' heel of xenograft development. In fact, imatinib, that targets endothelial-mural signalling, blocked paraganglioma xenograft formation (11 xenografts from 12 cell transplants in the control group versus 2 out of 10 in the treated group, P = 0.0015). Overall our key results were unaffected by the SDHx gene carrier status of the patient, characterized for 70 out of 77 cases. In conclusion, we explain the biphasic vasculoneural structure of paragangliomas and identify an early and pharmacologically actionable phase of paraganglioma organization.

Colorectal cancer cell lines are representative models of the main molecular subtypes of primary cancer.

PubMed

Mouradov, Dmitri; Sloggett, Clare; Jorissen, Robert N; Love, Christopher G; Li, Shan; Burgess, Antony W; Arango, Diego; Strausberg, Robert L; Buchanan, Daniel; Wormald, Samuel; O'Connor, Liam; Wilding, Jennifer L; Bicknell, David; Tomlinson, Ian P M; Bodmer, Walter F; Mariadason, John M; Sieber, Oliver M

2014-06-15

Human colorectal cancer cell lines are used widely to investigate tumor biology, experimental therapy, and biomarkers. However, to what extent these established cell lines represent and maintain the genetic diversity of primary cancers is uncertain. In this study, we profiled 70 colorectal cancer cell lines for mutations and DNA copy number by whole-exome sequencing and SNP microarray analyses, respectively. Gene expression was defined using RNA-Seq. Cell line data were compared with those published for primary colorectal cancers in The Cancer Genome Atlas. Notably, we found that exome mutation and DNA copy-number spectra in colorectal cancer cell lines closely resembled those seen in primary colorectal tumors. Similarities included the presence of two hypermutation phenotypes, as defined by signatures for defective DNA mismatch repair and DNA polymerase ε proofreading deficiency, along with concordant mutation profiles in the broadly altered WNT, MAPK, PI3K, TGFβ, and p53 pathways. Furthermore, we documented mutations enriched in genes involved in chromatin remodeling (ARID1A, CHD6, and SRCAP) and histone methylation or acetylation (ASH1L, EP300, EP400, MLL2, MLL3, PRDM2, and TRRAP). Chromosomal instability was prevalent in nonhypermutated cases, with similar patterns of chromosomal gains and losses. Although paired cell lines derived from the same tumor exhibited considerable mutation and DNA copy-number differences, in silico simulations suggest that these differences mainly reflected a preexisting heterogeneity in the tumor cells. In conclusion, our results establish that human colorectal cancer lines are representative of the main subtypes of primary tumors at the genomic level, further validating their utility as tools to investigate colorectal cancer biology and drug responses. ©2014 American Association for Cancer Research.

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

Organ-specific isogenic metastatic breast cancer cell lines exhibit distinct Raman spectral signatures and metabolomes

PubMed Central

Winnard, Paul T.; Zhang, Chi; Vesuna, Farhad; Kang, Jeon Woong; Garry, Jonah; Dasari, Ramachandra Rao; Barman, Ishan; Raman, Venu

2017-01-01

Molecular characterization of organ-specific metastatic lesions, which distinguish them from the primary tumor, will provide a better understanding of tissue specific adaptations that regulate metastatic progression. Using an orthotopic xenograft model, we have isolated isogenic metastatic human breast cancer cell lines directly from organ explants that are phenotypically distinct from the primary tumor cell line. Label-free Raman spectroscopy was used and informative spectral bands were ascertained as differentiators of organ-specific metastases as opposed to the presence of a single universal marker. Decision algorithms derived from the Raman spectra unambiguously identified these isogenic cell lines as unique biological entities – a finding reinforced through metabolomic analyses that indicated tissue of origin metabolite distinctions between the cell lines. Notably, complementarity of the metabolomics and Raman datasets was found. Our findings provide evidence that metastatic spread generates tissue-specific adaptations at the molecular level within cancer cells, which can be differentiated with Raman spectroscopy. PMID:28145887

Organ-specific isogenic metastatic breast cancer cell lines exhibit distinct Raman spectral signatures and metabolomes.

PubMed

Winnard, Paul T; Zhang, Chi; Vesuna, Farhad; Kang, Jeon Woong; Garry, Jonah; Dasari, Ramachandra Rao; Barman, Ishan; Raman, Venu

2017-03-21

Molecular characterization of organ-specific metastatic lesions, which distinguish them from the primary tumor, will provide a better understanding of tissue specific adaptations that regulate metastatic progression. Using an orthotopic xenograft model, we have isolated isogenic metastatic human breast cancer cell lines directly from organ explants that are phenotypically distinct from the primary tumor cell line. Label-free Raman spectroscopy was used and informative spectral bands were ascertained as differentiators of organ-specific metastases as opposed to the presence of a single universal marker. Decision algorithms derived from the Raman spectra unambiguously identified these isogenic cell lines as unique biological entities - a finding reinforced through metabolomic analyses that indicated tissue of origin metabolite distinctions between the cell lines. Notably, complementarity of the metabolomics and Raman datasets was found. Our findings provide evidence that metastatic spread generates tissue-specific adaptations at the molecular level within cancer cells, which can be differentiated with Raman spectroscopy.

The Epithelial Cell-derived Atopic Dermatitis Cytokine TSLP Activates Neurons to Induce Itch

PubMed Central

Wilson, Sarah R.; Thé, Lydia; Batia, Lyn M.; Beattie, Katherine; Katibah, George E.; McClain, Shannan P.; Pellegrino, Maurizio; Estandian, Daniel M.; Bautista, Diana M.

2014-01-01

Summary Atopic dermatitis (AD) is a chronic itch and inflammatory disorder of the skin that affects one in ten people. Patients suffering from severe AD eventually progress to develop asthma and allergic rhinitis, in a process known as the “atopic march.” Signaling between epithelial cells and innate immune cells via the cytokine Thymic Stromal Lymphopoietin (TSLP) is thought to drive AD and the atopic march. Here we report that epithelial cells directly communicate to cutaneous sensory neurons via TSLP to promote itch. We identify the ORAI1/NFAT calcium signaling pathway as an essential regulator of TSLP release from keratinocytes, the primary epithelial cells of the skin. TSLP then acts directly on a subset of TRPA1-positive sensory neurons to trigger robust itch behaviors. Our results support a new model whereby calcium-dependent TSLP release by keratinocytes activates both primary afferent neurons and immune cells to promote inflammatory responses in the skin and airways. PMID:24094650

A cyclized peptide derived from alpha fetoprotein inhibits the proliferation of ER-positive canine mammary cancer cells.

PubMed

Torres, Cristian Gabriel; Pino, Ana María; Sierralta, Walter Daniel

2009-06-01

The effects of estradiol (E2) and of an AFP-derived cyclized peptide (cP) on the proliferation of primary cultures of cancer cells isolated from spontaneous canine mammary tumors were studied. The cellular response to E2 and cP was related to the expression of estradiol receptor (isoforms alpha and beta). In ER-positive cells, 2 nM estradiol increased cell proliferation and the phosphorylation of ERK1/2; 2 microg/ml cP inhibited all these effects. Estradiol also increased HER2 immunoreactivity in ER-positive cells, an effect that was reverted to its basal values by cP. Estradiol stimulated in these cells the release of MMP2 and MMP9 and the shedding of HB-EGF, effects that the cP did not affect. ER-negative cells were refractory to estradiol or cP. All canine mammary tumor cells in culture responded to treatments analogously to human mammary cancer cells. Our results support the proposal of cP as a new, potentially effective therapeutic agent for the management of mammary cancer.

In vitro culture of various typed meningiomas and characterization of a human malignant meningioma cell line (HKBMM).

PubMed

Ishiwata, Isamu; Ishiwata, Chieko; Ishiwata, Emiko; Sato, Yoshiro; Kiguchi, Kazushige; Tachibana, Toshiaki; Ishikawa, Hiroshi

2004-12-01

We placed on culture the 13 cases of meningiomas, succeeded in making a primary culture of 10 cases and maintained 5 cases in vitro over considerable period of time (over three month), and one cell line derived from a malignant meningioma were established. In the early period of the primary culture, meningioma cells were spindle- or round-shaped cells. In the case of psammomatous type, the cultured cells were characterized as forming psammoma bodies. A cell line designated "HKBMM" was established from a human malignant meningioma occurred from frontal lobe. This line grew well without interruption for 5 years and was subcultivated over 120 times. The cells were spindle and fibrous in shape, and neoplastic and pleomorphic features, and multilayering without contact inhibition. The cells proliferated rapidly, and the population doubling time was about 29 hours. The chromosome number showed a wide distribution of aneuploidy. The mode was in the diploid range. The culture cells were easily transplanted into the subcutis of nude mice and produced the tumor resembling the original tumor.

Immunomodulatory Properties of Induced Pluripotent Stem Cell-Derived Mesenchymal Cells.

PubMed

Ng, Jia; Hynes, Kim; White, Gregory; Sivanathan, Kisha Nandini; Vandyke, Kate; Bartold, Peter Mark; Gronthos, Stan

2016-12-01

MSC-like populations derived from induced pluripotent stem cells (iPSC-MSC) serve as an alternative stem cell source due to their high proliferative capacity. In this study, we assessed the immunomodulatory potential of iPSC-MSC generated from periodontal ligament (PDL) and gingival (GF) tissue. The iPSC-MSC lines exhibited a similar level of suppression of mitogen-stimulated peripheral blood mononuclear cells (PBMNC) proliferation compared to their respective parental fibroblast populations in vitro. Moreover, iPSC-MSC demonstrated the ability to suppress T-cells effector cells, Th1/Th2/Th17 populations, and increase levels of Treg cells. In order to investigate the mechanisms involved, expression of common MSC-derived soluble factors known to supress lymphocyte proliferation were assessed in iPSC-MSC cultured with PBMNC with direct cell-cell contact or separated in transwells. Real-time PCR analysis of factors known to be involved in MSC mediated immune regulation, found a general trend of elevated IDO1 and IL6 transcript levels in iPSC-MSC lines and their respective primary cells co-cultured with activated PBMNC, with a wide range of gene expression levels between the different mesenchymal cell types. The results suggest that different iPSC-MSC may be useful as a potential alternative source of cells for future clinical use in therapeutic applications because of their potent immunosuppressive properties. J. Cell. Biochem. 117: 2844-2853, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

vif-negative human immunodeficiency virus type 1 persistently replicates in primary macrophages, producing attenuated progeny virus.

PubMed Central

Chowdhury, I H; Chao, W; Potash, M J; Sova, P; Gendelman, H E; Volsky, D J

1996-01-01

The vif gene of human immunodeficiency virus type 1 (HIV-1) is required for efficient infection of primary T lymphocytes. In this study, we investigated in detail the role of vif in productive infection of primary monocyte-derived macrophages (MDM). Viruses carrying missense or deletion mutations in vif were constructed on the background of the monocytotropic recombinant NLHXADA-GP. Using MDM from multiple donors, we found that vif mutants produced in complementing or partially complementing cell lines were approximately 10% as infectious as wild-type virus when assayed for incomplete, complete, and circularized viral DNA molecules by quantitative PCR amplification or for viral core antigen p24 production by enzyme-linked immunosorbent assay. We then determined the structure and infectivity of vif mutant HIV-1 by using MDM exclusively both for virus production and as targets for infection. Biosynthetic labeling and immunoprecipitation analysis of sucrose cushion-purified vif-negative HIV-1 made in MDM revealed that the virus had reduced p24 content compared with wild-type HIV-1. Cell-free MDM-derived vif mutant HIV-1 was infectious in macrophages as determined by the synthesis and maintenance of full-length viral DNA and by the produc- tion of particle-associated viral RNA, but its infectivity was approximately 2,500-fold lower than that of wild-type virus whose titer was determined in parallel by measurement of the viral DNA burden. MDM infected with MDM-derived vif-negative HIV-1 were able to transmit the virus to uninfected MDM by cocultivation, confirming the infectiousness of this virus. We conclude that mutations in vif significantly reduce but do not eliminate the capacity of HIV-1 to replicate and produce infectious progeny virus in primary human macrophages. PMID:8764044

vif-negative human immunodeficiency virus type 1 persistently replicates in primary macrophages, producing attenuated progeny virus.

PubMed

Chowdhury, I H; Chao, W; Potash, M J; Sova, P; Gendelman, H E; Volsky, D J

1996-08-01

The vif gene of human immunodeficiency virus type 1 (HIV-1) is required for efficient infection of primary T lymphocytes. In this study, we investigated in detail the role of vif in productive infection of primary monocyte-derived macrophages (MDM). Viruses carrying missense or deletion mutations in vif were constructed on the background of the monocytotropic recombinant NLHXADA-GP. Using MDM from multiple donors, we found that vif mutants produced in complementing or partially complementing cell lines were approximately 10% as infectious as wild-type virus when assayed for incomplete, complete, and circularized viral DNA molecules by quantitative PCR amplification or for viral core antigen p24 production by enzyme-linked immunosorbent assay. We then determined the structure and infectivity of vif mutant HIV-1 by using MDM exclusively both for virus production and as targets for infection. Biosynthetic labeling and immunoprecipitation analysis of sucrose cushion-purified vif-negative HIV-1 made in MDM revealed that the virus had reduced p24 content compared with wild-type HIV-1. Cell-free MDM-derived vif mutant HIV-1 was infectious in macrophages as determined by the synthesis and maintenance of full-length viral DNA and by the produc- tion of particle-associated viral RNA, but its infectivity was approximately 2,500-fold lower than that of wild-type virus whose titer was determined in parallel by measurement of the viral DNA burden. MDM infected with MDM-derived vif-negative HIV-1 were able to transmit the virus to uninfected MDM by cocultivation, confirming the infectiousness of this virus. We conclude that mutations in vif significantly reduce but do not eliminate the capacity of HIV-1 to replicate and produce infectious progeny virus in primary human macrophages.

Ex Vivo Maintenance of Primary Human Multiple Myeloma Cells through the Optimization of the Osteoblastic Niche.

PubMed

Zhang, Wenting; Gu, Yexin; Sun, Qiaoling; Siegel, David S; Tolias, Peter; Yang, Zheng; Lee, Woo Y; Zilberberg, Jenny

2015-01-01

We previously reported a new approach for culturing difficult-to-preserve primary patient-derived multiple myeloma cells (MMC) using an osteoblast (OSB)-derived 3D tissue scaffold constructed in a perfused microfluidic environment and a culture medium supplemented with patient plasma. In the current study, we used this biomimetic model to show, for the first time, that the long-term survival of OSB is the most critical factor in maintaining the ex vivo viability and proliferative capacity of MMC. We found that the adhesion and retention of MMC to the tissue scaffold was meditated by osteoblastic N-cadherin, as one of potential mechanisms that regulate MMC-OSB interactions. However, in the presence of MMC and patient plasma, the viability and osteogenic activity of OSB became gradually compromised, and consequently MMC could not remain viable over 3 weeks. We demonstrated that the long-term survival of both OSB and MMC could be enhanced by: (1) optimizing perfusion flow rate and patient-derived plasma composition in the culture medium and (2) replenishing OSB during culture as a practical means of prolonging MMC's viability beyond several weeks. These findings were obtained using a high-throughput well plate-based perfusion device from the perspective of optimizing the ex vivo preservation of patient-derived MM biospecimens for downstream use in biological studies and chemosensitivity analyses.

Ex Vivo Maintenance of Primary Human Multiple Myeloma Cells through the Optimization of the Osteoblastic Niche

PubMed Central

Zhang, Wenting; Gu, Yexin; Sun, Qiaoling; Siegel, David S.; Tolias, Peter; Yang, Zheng

2015-01-01

We previously reported a new approach for culturing difficult-to-preserve primary patient-derived multiple myeloma cells (MMC) using an osteoblast (OSB)-derived 3D tissue scaffold constructed in a perfused microfluidic environment and a culture medium supplemented with patient plasma. In the current study, we used this biomimetic model to show, for the first time, that the long-term survival of OSB is the most critical factor in maintaining the ex vivo viability and proliferative capacity of MMC. We found that the adhesion and retention of MMC to the tissue scaffold was meditated by osteoblastic N-cadherin, as one of potential mechanisms that regulate MMC-OSB interactions. However, in the presence of MMC and patient plasma, the viability and osteogenic activity of OSB became gradually compromised, and consequently MMC could not remain viable over 3 weeks. We demonstrated that the long-term survival of both OSB and MMC could be enhanced by: (1) optimizing perfusion flow rate and patient-derived plasma composition in the culture medium and (2) replenishing OSB during culture as a practical means of prolonging MMC’s viability beyond several weeks. These findings were obtained using a high-throughput well plate-based perfusion device from the perspective of optimizing the ex vivo preservation of patient-derived MM biospecimens for downstream use in biological studies and chemosensitivity analyses. PMID:25973790

Pancreatic cancer ascites xenograft–an expeditious model mirroring advanced therapeutic resistant disease

PubMed Central

Schvimer, Michael; Atias, Dikla; Halperin, Sharon; Buzhor, Ella; Raitses-Gurevich, Maria; Cohen, Keren; Pri-Chen, Sara; Wilson, Julie; Denroche, Robert E.; Lungu, Ilinca; Bartlett, John M.S.; Mbabaali, Faridah; Yarden, Yosef; Nataraj, Nishanth Belugali; Gallinger, Steven; Berger, Raanan

2017-01-01

Pancreatic ductal adenocarcinoma has limited treatment options. There is an urgent need for developing appropriate pre-clinical models recapitulating metastatic disease, the most common clinical scenario at presentation. Ascites accumulation occurs in up to 20–30% of patients with pancreatic cancer; this milieu represents a highly cellular research resource of metastatic peritoneal spread. In this study, we utilized pancreatic ascites/pleural effusion cancer cells to establish patient derived xenografts. Ascites/pleural effusion-patient derived xenografts were established from twelve independent cases. Xenografts were serially passed in nude mice and tissue bio-specimen banking has been established. Histopathology of emergent tumors demonstrates poorly to moderately differentiated, glandular and mucin producing tumors, mirroring morphology of primary pancreatic cancer tumors. Whole genome sequencing of six patient derived xenografts samples demonstrates common mutations and structural variations similar to those reported in primary pancreatic cancer. Xenograft tumors were dissociated to single-cells and in-vitro drug sensitivity screen assays demonstrated chemo-resistance, correlating with patient clinical scenarios, thus serving as a platform for clinically relevant translational research. Therefore, establishment of this novel ascites/pleural effusion patient derived xenograft model, with extensive histopathology and genomic characterization, opens an opportunity for the study of advanced aggressive pancreatic cancer. Characterization of metastatic disease and mechanisms of resistance to therapeutics may lead to the development of novel drug combinations. PMID:28489577

Barriers to Infection of Human Cells by Feline Leukemia Virus: Insights into Resistance to Zoonosis.

PubMed

Terry, Anne; Kilbey, Anna; Naseer, Asif; Levy, Laura S; Ahmad, Shamim; Watts, Ciorsdaidh; Mackay, Nancy; Cameron, Ewan; Wilson, Sam; Neil, James C

2017-03-01

The human genome displays a rich fossil record of past gammaretrovirus infections, yet no current epidemic is evident, despite environmental exposure to viruses that infect human cells in vitro Feline leukemia viruses (FeLVs) rank high on this list, but neither domestic nor workplace exposure has been associated with detectable serological responses. Nonspecific inactivation of gammaretroviruses by serum factors appears insufficient to explain these observations. To investigate further, we explored the susceptibilities of primary and established human cell lines to FeLV-B, the most likely zoonotic variant. Fully permissive infection was common in cancer-derived cell lines but was also a feature of nontransformed keratinocytes and lung fibroblasts. Cells of hematopoietic origin were generally less permissive and formed discrete groups on the basis of high or low intracellular protein expression and virion release. Potent repression was observed in primary human blood mononuclear cells and a subset of leukemia cell lines. However, the early steps of reverse transcription and integration appear to be unimpaired in nonpermissive cells. FeLV-B was subject to G→A hypermutation with a predominant APOBEC3G signature in partially permissive cells but was not mutated in permissive cells or in nonpermissive cells that block secondary viral spread. Distinct cellular barriers that protect primary human blood cells are likely to be important in protection against zoonotic infection with FeLV. IMPORTANCE Domestic exposure to gammaretroviruses such as feline leukemia viruses (FeLVs) occurs worldwide, but the basis of human resistance to infection remains incompletely understood. The potential threat is evident from the human genome sequence, which reveals many past epidemics of gammaretrovirus infection, and from recent cross-species jumps of gammaretroviruses from rodents to primates and marsupials. This study examined resistance to infection at the cellular level with the most prevalent human cell-tropic FeLV variant, FeLV-B. We found that blood cells are uniquely resistant to infection with FeLV-B due to the activity of cellular enzymes that mutate the viral genome. A second block, which appears to suppress viral gene expression after the viral genome has integrated into the host cell genome, was identified. Since cells derived from other normal human cell types are fully supportive of FeLV replication, innate resistance of blood cells could be critical in protecting against cross-species infection. Copyright © 2017 Terry et al.

Grafting polyethylenimine with quinoline derivatives for targeted imaging of intracellular Zn(2+) and logic gate operations.

PubMed

Pan, Yi; Shi, Yupeng; Chen, Junying; Wong, Chap-Mo; Zhang, Heng; Li, Mei-Jin; Li, Cheuk-Wing; Yi, Changqing

2016-12-01

In this study, a highly sensitive and selective fluorescent Zn(2+) probe which exhibited excellent biocompatibility, water solubility, and cell-membrane permeability, was facilely synthesized in a single step by grafting polyethyleneimine (PEI) with quinoline derivatives. The primary amino groups in the branched PEI can increase water solubility and cell permeability of the probe PEIQ, while quinoline derivatives can specifically recognize Zn(2+) and reduce the potential cytotoxicity of PEI. Basing on fluorescence off-on mechanism, PEIQ demonstrated excellent sensing capability towards Zn(2+) in absolute aqueous solution, where a high sensitivity with a detection limit as low as 38.1nM, and a high selectivity over competing metal ions and potential interfering amino acids, were achieved. Inspired by these results, elementary logic operations (YES, NOT and INHIBIT) have been constructed by employing PEIQ as the gate while Zn(2+) and EDTA as chemical inputs. Together with the low cytotoxicity and good cell-permeability, the practical application of PEIQ in living cell imaging was satisfactorily demonstrated, emphasizing its wide application in fundamental biology research. Copyright © 2016. Published by Elsevier B.V.

The potential of induced pluripotent stem cell derived hepatocytes.

PubMed

Hannoun, Zara; Steichen, Clara; Dianat, Noushin; Weber, Anne; Dubart-Kupperschmitt, Anne

2016-07-01

Orthotopic liver transplantation remains the only curative treatment for liver disease. However, the number of patients who die while on the waiting list (15%) has increased in recent years as a result of severe organ shortages; furthermore the incidence of liver disease is increasing worldwide. Clinical trials involving hepatocyte transplantation have provided encouraging results. However, transplanted cell function appears to often decline after several months, necessitating liver transplantation. The precise aetiology of the loss of cell function is not clear, but poor engraftment and immune-mediated loss appear to be important factors. Also, primary human hepatocytes (PHH) are not readily available, de-differentiate, and die rapidly in culture. Hepatocytes are available from other sources, such as tumour-derived human hepatocyte cell lines and immortalised human hepatocyte cell lines or porcine hepatocytes. However, all these cells suffer from various limitations such as reduced or differences in functions or risk of zoonotic infections. Due to their significant potential, one possible inexhaustible source of hepatocytes is through the directed differentiation of human induced pluripotent stem cells (hiPSCs). This review will discuss the potential applications and existing limitations of hiPSC-derived hepatocytes in regenerative medicine, drug screening, in vitro disease modelling and bioartificial livers. Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Intracranial AAV-sTRAIL combined with lanatoside C prolongs survival in an orthotopic xenograft mouse model of invasive glioblastoma.

PubMed

Crommentuijn, Matheus H W; Maguire, Casey A; Niers, Johanna M; Vandertop, W Peter; Badr, Christian E; Würdinger, Thomas; Tannous, Bakhos A

2016-04-01

Glioblastoma (GBM) is the most common malignant brain tumor in adults. We designed an adeno-associated virus (AAV) vector for intracranial delivery of secreted, soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) to GBM tumors in mice and combined it with the TRAIL-sensitizing cardiac glycoside, lanatoside C (lan C). We applied this combined therapy to two different GBM models using human U87 glioma cells and primary patient-derived GBM neural spheres in culture and in orthotopic GBM xenograft models in mice. In U87 cells, conditioned medium from AAV2-sTRAIL expressing cells combined with lan C induced 80% cell death. Similarly, lan C sensitized primary GBM spheres to sTRAIL causing over 90% cell death. In mice bearing intracranial U87 tumors treated with AAVrh.8-sTRAIL, administration of lan C caused a decrease in tumor-associated Fluc signal, while tumor size increased within days of stopping the treatment. Another round of lan C treatment re-sensitized GBM tumor to sTRAIL-induced cell death. AAVrh.8-sTRAIL treatment alone and combined with lanatoside C resulted in a significant decrease in tumor growth and longer survival of mice bearing orthotopic invasive GBM brain tumors. In summary, AAV-sTRAIL combined with lanatoside C induced cell death in U87 glioma cells and patient-derived GBM neural spheres in culture and in vivo leading to an increased in overall mice survival. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

The human chondrosarcoma HCS-2/8 cell line is responsive to BMP-7, but not to IL-1beta.

PubMed

Saas, Joachim; Gebauer, Matthias; Jacobi, Carsten; Haag, Jochen; Takigawa, Masaharu; Aigner, Thomas

2005-05-01

Cultures of primary chondrocytes as in vitro model systems for studying the cellular behavior of chondrocytes are notoriously difficult to cultivate and propagate. One way to circumvent these problems appears to be the use of immortalized/immortal chondrocytic cell lines. In the present study, we were interested whether the chondrosarcoma derived HCS-2/8 cells are suitable for studying major cellular reaction pattern in response to key anabolic (BMP-7) and catabolic (IL-1beta) factors. Therefore, we used cDNA array and real-time PCR technology in order to evaluate gene expression triggered by stimulation with IL-1beta (0,1-100 ng/ml) and BMP-7 in confluent monolayer cultures. HCS-2/8 cells hardly responded to IL-1beta, but showed good responsiveness to BMP-7. We found 12 genes up- and 17 significantly down-regulated by BMP-7 (out of 340 investigated genes). Besides the expected activation of anabolic genes chondrocytic cells after BMP-stimulation try to neutralize activation of the BMP-signalling cascade by expressing intra- and extracellular BMP-antagonists. Chondrosarcoma derived cell lines are a potential substitute for primary articular chondrocytes promising consistent expression of a differentiated chondrocyte phenotype with sufficient proliferative capacity. However, as shown by this study one needs to carefully select the cell line depending on the effects which one intends to study. In this respect, HCS-2/8 cells are a validated tool for studying BMP-effects on chondrocytes, but not e.g. effects of interleukin-1.

Establishment and proteomic characterization of NCC-LMS1-C1, a novel cell line of primary leiomyosarcoma of the bone.

PubMed

Sakumoto, Marimu; Takahashi, Mami; Oyama, Rieko; Takai, Yoko; Kito, Fusako; Shiozawa, Kumiko; Qiao, Zhiwei; Yoshida, Akihiko; Endo, Makoto; Kawai, Akira; Kondo, Tadashi

2017-10-01

Leiomyosarcoma (LMS) is one of most aggressive mesenchymal malignancies that differentiate towards smooth muscle. The clinical outcome of LMS patients is poor; as such, there is an urgent need for novel therapeutic approaches. Experimental models such as patient-derived cell lines are invaluable tools for pre-clinical studies. In the present study, we established a stable cell line from the tumor tissue of a patient with a primary LMS of the bone. Despite the urgent need for novel therapeutic strategies in LMS, there are only a few LMS cell lines available in public cell banks, none of which are primary to the bone. Bone primary LMS tumor tissues were sampled to establish cell lines. Morphological and proteomic analyses were performed and sensitivity to pazopanib was evaluated. NCC-LMS1-C1 cells were maintained for over 100 passages. The cells exhibited a spindle shape and aggressive growth; they also expressed smooth muscle actin, reflecting the original LMS tissue (i.e. smooth muscle cells). The cells also showed tumor characteristics such as colony formation on soft agar and sensitivity to pazopanib, doxorubicin and cisplatin, with half-maximal inhibitory concentrations of 4.5, 0.11 and 20 μM, respectively. Proteomic analyses by mass spectrometry and antibody array revealed some differences in the protein expression profiles of these cells as compared to the original tumor tissue. Our results indicate that the NCC-LMS1-C1 cell lines will be useful for LMS research. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The expression of xenobiotic-metabolizing enzymes in human prostate and in prostate epithelial cells (PECs) derived from primary cultures.

PubMed

Al-Buheissi, S Z; Cole, K J; Hewer, A; Kumar, V; Bryan, R L; Hudson, D L; Patel, H R; Nathan, S; Miller, R A; Phillips, D H

2006-06-01

Dietary heterocyclic amines (HCAs) are carcinogenic in rodent prostate requiring activation by enzymes such as cytochrome P450 (CYP) and N-acetyltransferase (NAT). We investigated by Western blotting and immunohistochemistry the expression of CYP1A1, CYP1A2, and NAT1 in human prostate and in prostate epithelial cells (PECs) derived from primary cultures and tested their ability to activate the dietary carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and its N-hydroxy metabolite (N-OH-IQ) to DNA-damaging moieties. Western blotting identified CYP1A1, CYP1A2, and NAT1. Immunohistochemistry localized NAT1 to the cytoplasm of PECs. Inter-individual variation was observed in the expression levels of CYP1A1, 1A2, and NAT1 (11, 75, and 35-fold, respectively). PECs expressed CYP1A1 and NAT1 but not CYP1A2. When incubated with IQ or N-OH-IQ, PECs formed DNA adducts indicating their ability to metabolically activate these compounds. Prostate cells possess the capacity to activate dietary carcinogens. PECs may provide a useful model system to study their role in prostate carcinogenesis.

Zika virus propagation and release in human fetal astrocytes can be suppressed by neutral sphingomyelinase-2 inhibitor GW4869.

PubMed

Huang, Yunlong; Li, Yuju; Zhang, Hainan; Zhao, Runze; Jing, Ran; Xu, Yinghua; He, Miao; Peer, Justin; Kim, Yeong C; Luo, Jiangtao; Tong, Zenghan; Zheng, Jialin

2018-01-01

Zika virus (ZIKV) is a neurotrophic flavivirus that is capable of infecting humans, leading to brain abnormalities during fetal development. The ZIKV infectivity in neural target cells remains poorly understood. Here, we found that ZIKV specifically infected glial fibrillary acidic protein- and S100B-positive primary human astrocytes derived from fetal brains. In contrast, neuron-specific Class III β-tubulin (TuJ1)-positive neurons in the astrocyte cultures and SOX2-positive neural progenitor cells derived from the fetal brains were less susceptible to ZIKV infection compared with astrocytes. The infected astrocytes released competent viral particles and manifested programmed cell death with a progressive cytopathic effect. Interestingly, ZIKV infection in human fetal astrocytes induced a significant increase of extracellular vesicles (EVs). Treatment with GW4869, a specific inhibitor of neutral sphingomyelinase-2, decreased EV levels, suppressed ZIKV propagation, and reduced the release of infectious virions in astrocytes. Therefore, ZIKV infects primary human fetal astrocytes and the infection can be suppressed by neutral sphingomyelinase-2 inhibitor GW4869. Further investigation into sphingomyelin metabolism and EVs may provide insights to the therapeutic treatment of ZIKV infection.

Nrf2-dependent induction of innate host defense via heme oxygenase-1 inhibits Zika virus replication

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

Huang, Hanxia; Falgout, Barry; Takeda, Kazuyo

We identified primary human monocyte-derived macrophages (MDM) as vulnerable target cells for Zika virus (ZIKV) infection. We demonstrate dramatic effects of hemin, the natural inducer of the heme catabolic enzyme heme oxygenase-1 (HO-1), in the reduction of ZIKV replication in vitro. Both LLC-MK2 monkey kidney cells and primary MDM exhibited hemin-induced HO-1 expression with major reductions of >90% in ZIKV replication, with little toxicity to infected cells. Silencing expression of HO-1 or its upstream regulatory gene, nuclear factor erythroid-related factor 2 (Nrf2), attenuated hemin-induced suppression of ZIKV infection, suggesting an important role for induction of these intracellular mediators in retardingmore » ZIKV replication. The inverse correlation between hemin-induced HO-1 levels and ZIKV replication provides a potentially useful therapeutic modality based on stimulation of an innate cellular response against Zika virus infection. - Highlights: •Hemin treatment protected monocyte-derived macrophages against Zika virus (ZIKV) infection. •Innate cellular protection against ZIKV infection correlated with Nrf2-dependent HO-1 expression. •Stimulation of innate cellular responses may provide a therapeutic strategy against ZIKV infection.« less

CRISPR/Cas9-based genetic correction for recessive dystrophic epidermolysis bullosa

PubMed Central

Webber, Beau R; Osborn, Mark J; McElroy, Amber N; Twaroski, Kirk; Lonetree, Cara-lin; DeFeo, Anthony P; Xia, Lily; Eide, Cindy; Lees, Christopher J; McElmurry, Ron T; Riddle, Megan J; Kim, Chong Jai; Patel, Dharmeshkumar D; Blazar, Bruce R; Tolar, Jakub

2016-01-01

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe disorder caused by mutations to the COL7A1 gene that deactivate production of a structural protein essential for skin integrity. Haematopoietic cell transplantation can ameliorate some of the symptoms; however, significant side effects from the allogeneic transplant procedure can occur and unresponsive areas of blistering persist. Therefore, we employed genome editing in patient-derived cells to create an autologous platform for multilineage engineering of therapeutic cell types. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system facilitated correction of an RDEB-causing COL7A1 mutation in primary fibroblasts that were then used to derive induced pluripotent stem cells (iPSCs). The resulting iPSCs were subsequently re-differentiated into keratinocytes, mesenchymal stem cells (MSCs) and haematopoietic progenitor cells using defined differentiation strategies. Gene-corrected keratinocytes exhibited characteristic epithelial morphology and expressed keratinocyte-specific genes and transcription factors. iPSC-derived MSCs exhibited a spindle morphology and expression of CD73, CD90 and CD105 with the ability to undergo adipogenic, chondrogenic and osteogenic differentiation in vitro in a manner indistinguishable from bone marrow-derived MSCs. Finally, we used a vascular induction strategy to generate potent definitive haematopoietic progenitors capable of multilineage differentiation in methylcellulose-based assays. In totality, we have shown that CRISPR/Cas9 is an adaptable gene-editing strategy that can be coupled with iPSC technology to produce multiple gene-corrected autologous cell types with therapeutic potential for RDEB. PMID:28250968

Antitumor Activity of the Investigational Proteasome Inhibitor MLN9708 in Mouse Models of B-cell and Plasma Cell Malignancies

PubMed Central

Lee, Edmund C.; Fitzgerald, Michael; Bannerman, Bret; Donelan, Jill; Bano, Kristen; Terkelsen, Jennifer; Bradley, Daniel P.; Subakan, Ozlem; Silva, Matthew D.; Liu, Ray; Pickard, Michael; Li, Zhi; Tayber, Olga; Li, Ping; Hales, Paul; Carsillo, Mary; Neppalli, Vishala T.; Berger, Allison J.; Kupperman, Erik; Manfredi, Mark; Bolen, Joseph B.; Van Ness, Brian; Janz, Siegfried

2012-01-01

Purpose The clinical success of the first-in-class proteasome inhibitor bortezomib (VELCADE) has validated the proteasome as a therapeutic target for treating human cancers. MLN9708 is an investigational proteasome inhibitor that, compared with bortezomib, has improved pharmacokinetics, pharmacodynamics, and antitumor activity in preclinical studies. Here, we focused on evaluating the in vivo activity of MLN2238 (the biologically active form of MLN9708) in a variety of mouse models of hematologic malignancies, including tumor xenograft models derived from a human lymphoma cell line and primary human lymphoma tissue, and genetically engineered mouse (GEM) models of plasma cell malignancies (PCM). Experimental Design Both cell line–derived OCI-Ly10 and primary human lymphoma–derived PHTX22L xenograft models of diffuse large B-cell lymphoma were used to evaluate the pharmacodynamics and antitumor effects of MLN2238 and bortezomib. The iMycCα/Bcl-XL GEM model was used to assess their effects on de novo PCM and overall survival. The newly developed DP54-Luc–disseminated model of iMycCα/ Bcl-XL was used to determine antitumor activity and effects on osteolytic bone disease. Results MLN2238 has an improved pharmacodynamic profile and antitumor activity compared with bortezomib in both OCI-Ly10 and PHTX22L models. Although both MLN2238 and bortezomib prolonged overall survival, reduced splenomegaly, and attenuated IgG2a levels in the iMycCα/Bcl-XL GEM model, only MLN2238 alleviated osteolytic bone disease in the DP54-Luc model. Conclusions Our results clearly showed the antitumor activity of MLN2238 in a variety of mouse models of B-cell lymphoma and PCM, supporting its clinical development. MLN9708 is being evaluated in multiple phase I and I/II trials. PMID:21903769

Human mammary microenvironment better regulates the biology of human breast cancer in humanized mouse model.

PubMed

Zheng, Ming-Jie; Wang, Jue; Xu, Lu; Zha, Xiao-Ming; Zhao, Yi; Ling, Li-Jun; Wang, Shui

2015-02-01

During the past decades, many efforts have been made in mimicking the clinical progress of human cancer in mouse models. Previously, we developed a human breast tissue-derived (HB) mouse model. Theoretically, it may mimic the interactions between "species-specific" mammary microenvironment of human origin and human breast cancer cells. However, detailed evidences are absent. The present study (in vivo, cellular, and molecular experiments) was designed to explore the regulatory role of human mammary microenvironment in the progress of human breast cancer cells. Subcutaneous (SUB), mammary fat pad (MFP), and HB mouse models were developed for in vivo comparisons. Then, the orthotopic tumor masses from three different mouse models were collected for primary culture. Finally, the biology of primary cultured human breast cancer cells was compared by cellular and molecular experiments. Results of in vivo mouse models indicated that human breast cancer cells grew better in human mammary microenvironment. Cellular and molecular experiments confirmed that primary cultured human breast cancer cells from HB mouse model showed a better proliferative and anti-apoptotic biology than those from SUB to MFP mouse models. Meanwhile, primary cultured human breast cancer cells from HB mouse model also obtained the migratory and invasive biology for "species-specific" tissue metastasis to human tissues. Comprehensive analyses suggest that "species-specific" mammary microenvironment of human origin better regulates the biology of human breast cancer cells in our humanized mouse model of breast cancer, which is more consistent with the clinical progress of human breast cancer.

Bufalin Inhibits the Differentiation and Proliferation of Cancer Stem Cells Derived from Primary Osteosarcoma Cells through Mir-148a.

PubMed

Chang, Yuewen; Zhao, Yongfang; Gu, Wei; Cao, Yuelong; Wang, Shuqiang; Pang, Jian; Shi, Yinyu

2015-01-01

Osteosarcoma (OS) is the second leading cause of cancer-related death in children and young adults. Chemoresistance is the most important cause of treatment failure in OS, largely resulting from presence of cancer stem cells (CSCs). However, CSCs isolated from cancer cell lines do not necessarily represent those from primary human tumors due to accumulation of genetic aberrations that increase with passage number. Therefore, studies on CSCs from primary OS may be more important for understanding the mechanisms driving the chemoresistance of CSCs in OS. We established a primary culture of OS cells, known as C1OS, from freshly resected tumor tissue. We further isolated CSCs from C1OS cells (C1OS-CSCs). We analyzed the effects of bufalin, a traditional Chinese medicine, on the stemness of C1OS-CSCs. We also analyzed the microRNA (miR) targets of bufalin on the stemness of C1OS-CSCs. Moreover, we examined these findings in the OS specimen. Bufalin inhibited the stemness of C1OS-CSCs. Moreover, we found that miR-148a appeared to be a target of bufalin, and miR-148a further regulated DNMT1 and p27 to control the stemness of OS cells. This mechanism was further confirmed in OS specimen. Our data suggest that bufalin may be a promising treatment for OS, and its function may be conducted through regulation of miR-148a. © 2015 S. Karger AG, Basel.

Digital transcriptome profiling of normal and glioblastoma-derived neural stem cells identifies genes associated with patient survival

PubMed Central

2012-01-01

Background Glioblastoma multiforme, the most common type of primary brain tumor in adults, is driven by cells with neural stem (NS) cell characteristics. Using derivation methods developed for NS cells, it is possible to expand tumorigenic stem cells continuously in vitro. Although these glioblastoma-derived neural stem (GNS) cells are highly similar to normal NS cells, they harbor mutations typical of gliomas and initiate authentic tumors following orthotopic xenotransplantation. Here, we analyzed GNS and NS cell transcriptomes to identify gene expression alterations underlying the disease phenotype. Methods Sensitive measurements of gene expression were obtained by high-throughput sequencing of transcript tags (Tag-seq) on adherent GNS cell lines from three glioblastoma cases and two normal NS cell lines. Validation by quantitative real-time PCR was performed on 82 differentially expressed genes across a panel of 16 GNS and 6 NS cell lines. The molecular basis and prognostic relevance of expression differences were investigated by genetic characterization of GNS cells and comparison with public data for 867 glioma biopsies. Results Transcriptome analysis revealed major differences correlated with glioma histological grade, and identified misregulated genes of known significance in glioblastoma as well as novel candidates, including genes associated with other malignancies or glioma-related pathways. This analysis further detected several long non-coding RNAs with expression profiles similar to neighboring genes implicated in cancer. Quantitative PCR validation showed excellent agreement with Tag-seq data (median Pearson r = 0.91) and discerned a gene set robustly distinguishing GNS from NS cells across the 22 lines. These expression alterations include oncogene and tumor suppressor changes not detected by microarray profiling of tumor tissue samples, and facilitated the identification of a GNS expression signature strongly associated with patient survival (P = 1e-6, Cox model). Conclusions These results support the utility of GNS cell cultures as a model system for studying the molecular processes driving glioblastoma and the use of NS cells as reference controls. The association between a GNS expression signature and survival is consistent with the hypothesis that a cancer stem cell component drives tumor growth. We anticipate that analysis of normal and malignant stem cells will be an important complement to large-scale profiling of primary tumors. PMID:23046790

Cysteine Dioxygenase 1 Is a Tumor Suppressor Gene Silenced by Promoter Methylation in Multiple Human Cancers

PubMed Central

Brait, Mariana; Ling, Shizhang; Nagpal, Jatin K.; Chang, Xiaofei; Park, Hannah Lui; Lee, Juna; Okamura, Jun; Yamashita, Keishi; Sidransky, David; Kim, Myoung Sook

2012-01-01

The human cysteine dioxygenase 1 (CDO1) gene is a non-heme structured, iron-containing metalloenzyme involved in the conversion of cysteine to cysteine sulfinate, and plays a key role in taurine biosynthesis. In our search for novel methylated gene promoters, we have analyzed differential RNA expression profiles of colorectal cancer (CRC) cell lines with or without treatment of 5-aza-2′-deoxycytidine. Among the genes identified, the CDO1 promoter was found to be differentially methylated in primary CRC tissues with high frequency compared to normal colon tissues. In addition, a statistically significant difference in the frequency of CDO1 promoter methylation was observed between primary normal and tumor tissues derived from breast, esophagus, lung, bladder and stomach. Downregulation of CDO1 mRNA and protein levels were observed in cancer cell lines and tumors derived from these tissue types. Expression of CDO1 was tightly controlled by promoter methylation, suggesting that promoter methylation and silencing of CDO1 may be a common event in human carcinogenesis. Moreover, forced expression of full-length CDO1 in human cancer cells markedly decreased the tumor cell growth in an in vitro cell culture and/or an in vivo mouse model, whereas knockdown of CDO1 increased cell growth in culture. Our data implicate CDO1 as a novel tumor suppressor gene and a potentially valuable molecular marker for human cancer. PMID:23028699

BL-038, a Benzofuran Derivative, Induces Cell Apoptosis in Human Chondrosarcoma Cells through Reactive Oxygen Species/Mitochondrial Dysfunction and the Caspases Dependent Pathway.

PubMed

Liu, Ju-Fang; Chen, Chien-Yu; Chen, Hsien-Te; Chang, Chih-Shiang; Tang, Chih-Hsin

2016-09-07

Chondrosarcoma is a highly malignant cartilage-forming bone tumor that has the capacity to invade locally and cause distant metastasis. Moreover, chondrosarcoma is intrinsically resistant to conventional chemotherapy or radiotherapy. The novel benzofuran derivative, BL-038 (2-amino-3-(2,6-dichlorophenyl)-6-(4-methoxyphenyl)benzofuran-4-yl acetate), has been evaluated for its anticancer effects in human chondrosarcoma cells. BL-038 caused cell apoptosis in two human chondrosarcoma cell lines, JJ012 and SW1353, but not in primary chondrocytes. Treatment of chondrosarcoma with BL-038 also induced reactive oxygen species (ROS) production. Furthermore, BL-038 decreased mitochondrial membrane potential (MMP) and changed mitochondrial-related apoptosis, by downregulating the anti-apoptotic activity members (Bcl-2, Bcl-xL) and upregulating pro-apoptotic members (Bax, Bak) of the B-cell lymphoma 2 (Bcl-2) family of proteins, key regulators of the apoptotic machinery in cells. These results demonstrate that in human chondrosarcoma cells, the apoptotic and cytotoxic effects of BL-038 are mediated by the intrinsic mitochondria-mediated apoptotic pathway, which in turn causes the release of cytochrome c, the activation of caspase-9 and caspase-3, and the cleavage of poly (ADP-ribose) polymerase (PARP), to elicit apoptosis response. Our results show that the benzofuran derivative BL-038 induces apoptosis in chondrosarcoma cells.

BL-038, a Benzofuran Derivative, Induces Cell Apoptosis in Human Chondrosarcoma Cells through Reactive Oxygen Species/Mitochondrial Dysfunction and the Caspases Dependent Pathway

PubMed Central

Liu, Ju-Fang; Chen, Chien-Yu; Chen, Hsien-Te; Chang, Chih-Shiang; Tang, Chih-Hsin

2016-01-01

Chondrosarcoma is a highly malignant cartilage-forming bone tumor that has the capacity to invade locally and cause distant metastasis. Moreover, chondrosarcoma is intrinsically resistant to conventional chemotherapy or radiotherapy. The novel benzofuran derivative, BL-038 (2-amino-3-(2,6-dichlorophenyl)-6-(4-methoxyphenyl)benzofuran-4-yl acetate), has been evaluated for its anticancer effects in human chondrosarcoma cells. BL-038 caused cell apoptosis in two human chondrosarcoma cell lines, JJ012 and SW1353, but not in primary chondrocytes. Treatment of chondrosarcoma with BL-038 also induced reactive oxygen species (ROS) production. Furthermore, BL-038 decreased mitochondrial membrane potential (MMP) and changed mitochondrial-related apoptosis, by downregulating the anti-apoptotic activity members (Bcl-2, Bcl-xL) and upregulating pro-apoptotic members (Bax, Bak) of the B-cell lymphoma 2 (Bcl-2) family of proteins, key regulators of the apoptotic machinery in cells. These results demonstrate that in human chondrosarcoma cells, the apoptotic and cytotoxic effects of BL-038 are mediated by the intrinsic mitochondria-mediated apoptotic pathway, which in turn causes the release of cytochrome c, the activation of caspase-9 and caspase-3, and the cleavage of poly (ADP-ribose) polymerase (PARP), to elicit apoptosis response. Our results show that the benzofuran derivative BL-038 induces apoptosis in chondrosarcoma cells. PMID:27618007

CD34 Antigen and the MPL Receptor Expression Defines a Novel Class of Human Cord Blood-Derived Primitive Hematopoietic Stem Cells.

PubMed

Matsuoka, Yoshikazu; Takahashi, Masaya; Sumide, Keisuke; Kawamura, Hiroshi; Nakatsuka, Ryusuke; Fujioka, Tatsuya; Sonoda, Yoshiaki

2017-06-09

In the murine hematopoietic stem cell (HSC) compartment, thrombopoietin (THPO)/MPL (THPO receptor) signaling plays an important role in the maintenance of adult quiescent HSCs. However, the role of THPO/MPL signaling in the human primitive HSC compartment has not yet been elucidated. We have identified very primitive human cord blood (CB)-derived CD34- severe combined immunodeficiency (SCID)-repopulating cells (SRCs) using the intra-bone marrow injection method. In this study, we investigated the roles of the MPL expression in the human primitive HSC compartment. The SRC activities of the highly purified CB-derived 18Lin-CD34+/-MPL+/- cells were analyzed using NOG mice. In the primary recipient mice, nearly all mice that received CD34+/-MPL+/- cells were repopulated with human CD45+ cells. Nearly all of these mice that received CD34+MPL+/- and CD34-MPL- cells showed a secondary repopulation. Interestingly, the secondary recipient mice that received CD34+/-MPL- cells showed a distinct tertiary repopulation. These results clearly indicate that the CD34+/- SRCs not expressing MPL sustain a long-term (LT) (>1 year) human cell repopulation in NOG mice. Moreover, CD34- SRCs generate CD34+CD38-CD90+ SRCs in vitro and in vivo. These findings provide a new concept that CD34-MPL- SRCs reside at the apex of the human HSC hierarchy.

Adipogenic Differentiation of Mesenchymal Stem Cells Alters Their Immunomodulatory Properties in a Tissue-Specific Manner.

PubMed

Munir, Hafsa; Ward, Lewis S C; Sheriff, Lozan; Kemble, Samuel; Nayar, Saba; Barone, Francesca; Nash, Gerard B; McGettrick, Helen M

2017-06-01

Chronic inflammation is associated with formation of ectopic fat deposits that might represent damage-induced aberrant mesenchymal stem cell (MSC) differentiation. Such deposits are associated with increased levels of inflammatory infiltrate and poor prognosis. Here we tested the hypothesis that differentiation from MSC to adipocytes in inflamed tissue might contribute to chronicity through loss of immunomodulatory function. We assessed the effects of adipogenic differentiation of MSC isolated from bone marrow or adipose tissue on their capacity to regulate neutrophil recruitment by endothelial cells and compared the differentiated cells to primary adipocytes from adipose tissue. Bone marrow derived MSC were immunosuppressive, inhibiting neutrophil recruitment to TNFα-treated endothelial cells (EC), but MSC-derived adipocytes were no longer able to suppress neutrophil adhesion. Changes in IL-6 and TGFβ1 signalling appeared critical for the loss of the immunosuppressive phenotype. In contrast, native stromal cells, adipocytes derived from them, and mature adipocytes from adipose tissue were all immunoprotective. Thus disruption of normal tissue stroma homeostasis, as occurs in chronic inflammatory diseases, might drive "abnormal" adipogenesis which adversely influences the behavior of MSC and contributes to pathogenic recruitment of leukocytes. Interestingly, stromal cells programmed in native fat tissue retain an immunoprotective phenotype. Stem Cells 2017;35:1636-1646. © 2017 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

A Conserved HIV-1-Derived Peptide Presented by HLA-E Renders Infected T-cells Highly Susceptible to Attack by NKG2A/CD94-Bearing Natural Killer Cells.

PubMed

Davis, Zachary B; Cogswell, Andrew; Scott, Hamish; Mertsching, Amanda; Boucau, Julie; Wambua, Daniel; Le Gall, Sylvie; Planelles, Vicente; Campbell, Kerry S; Barker, Edward

2016-02-01

Major histocompatibility class I (MHC-I)-specific inhibitory receptors on natural killer (NK) cells (iNKRs) tolerize mature NK cell responses toward normal cells. NK cells generate cytolytic responses to virus-infected or malignant target cells with altered or decreased MHC-I surface expression due to the loss of tolerizing ligands. The NKG2A/CD94 iNKR suppresses NK cell responses through recognition of the non-classical MHC-I, HLA-E. We used HIV-infected primary T-cells as targets in an in vitro cytolytic assay with autologous NK cells from healthy donors. In these experiments, primary NKG2A/CD94(+) NK cells surprisingly generated the most efficient responses toward HIV-infected T-cells, despite high HLA-E expression on the infected targets. Since certain MHC-I-presented peptides can alter recognition by iNKRs, we hypothesized that HIV-1-derived peptides presented by HLA-E on infected cells may block engagement with NKG2A/CD94, thereby engendering susceptibility to NKG2A/CD94(+) NK cells. We demonstrate that HLA-E is capable of presenting a highly conserved peptide from HIV-1 capsid (AISPRTLNA) that is not recognized by NKG2A/CD94. We further confirmed that HLA-C expressed on HIV-infected cells restricts attack by KIR2DL(+) CD56(dim) NK cells, in contrast to the efficient responses by CD56(bright) NK cells, which express predominantly NKG2A/CD94 and lack KIR2DLs. These findings are important since the use of NK cells was recently proposed to treat latently HIV-1-infected patients in combination with latency reversing agents. Our results provide a mechanistic basis to guide these future clinical studies, suggesting that ex vivo-expanded NKG2A/CD94(+) KIR2DL(-) NK cells may be uniquely beneficial.

Expression and cytokine regulation of immune recognition elements by normal human biliary epithelial and established liver cell lines in vitro.

PubMed

Cruickshank, S M; Southgate, J; Selby, P J; Trejdosiewicz, L K

1998-10-01

Biliary epithelial cells are targets of immune-mediated attack in conditions such as primary biliary cirrhosis and allograft rejection. This has been attributed to the ability of biliary epithelial cells to express ligands for T cell receptors. We aimed to investigate the expression of immune recognition elements and the effects of pro-inflammatory and anti-inflammatory cytokines on cell surface phenotypes of normal human biliary epithelial cells and established human liver-derived (PLC/PRF/5, HepG2, Hep3B and CC-SW) lines. Cells were cultured in the presence or absence of cytokines for 72 h, and expression of cell surface molecules was assessed by flow cytometry and immunofluorescence. All cell lines expressed MHC class I, ICAM-1 (CD54), LFA-3 (CD58) and EGF receptor, and all but Hep3B expressed Fas/Apo-1 (CD95). Unlike hepatocyte-derived cell lines, biliary epithelial cells and CC-SW expressed CD40 and CD44. As expected, IFNgamma and TNFalpha upregulated expression of ICAM-1, MHC class I and MHC class II, particularly in biliary epithelial cells. TGFbeta downregulated these molecules and downregulated CD95 on biliary epithelial cells, but upregulated LFA-3. The Th2 cytokines had little effect, although IL-4 upregulated CD95 expression on biliary epithelial cells. IFNgamma upregulated CD40 expression on biliary epithelial cells, CC-SW and HepG2. These findings imply that biliary epithelial cells may be capable of interacting with activated T lymphocytes via CD40 and LFA-3, which are thought to be important T cell accessory ligands for T cell activation in a B7-independent manner. Sensitivity to pro-inflammatory cytokines and expression of CD95 may explain why biliary epithelial cells are primary targets for autoimmune attack.

A Conserved HIV-1-Derived Peptide Presented by HLA-E Renders Infected T-cells Highly Susceptible to Attack by NKG2A/CD94-Bearing Natural Killer Cells

PubMed Central

Davis, Zachary B.; Cogswell, Andrew; Scott, Hamish; Mertsching, Amanda; Boucau, Julie; Wambua, Daniel; Le Gall, Sylvie; Planelles, Vicente; Campbell, Kerry S.; Barker, Edward

2016-01-01

Major histocompatibility class I (MHC-I)-specific inhibitory receptors on natural killer (NK) cells (iNKRs) tolerize mature NK cell responses toward normal cells. NK cells generate cytolytic responses to virus-infected or malignant target cells with altered or decreased MHC-I surface expression due to the loss of tolerizing ligands. The NKG2A/CD94 iNKR suppresses NK cell responses through recognition of the non-classical MHC-I, HLA-E. We used HIV-infected primary T-cells as targets in an in vitro cytolytic assay with autologous NK cells from healthy donors. In these experiments, primary NKG2A/CD94+ NK cells surprisingly generated the most efficient responses toward HIV-infected T-cells, despite high HLA-E expression on the infected targets. Since certain MHC-I-presented peptides can alter recognition by iNKRs, we hypothesized that HIV-1-derived peptides presented by HLA-E on infected cells may block engagement with NKG2A/CD94, thereby engendering susceptibility to NKG2A/CD94+ NK cells. We demonstrate that HLA-E is capable of presenting a highly conserved peptide from HIV-1 capsid (AISPRTLNA) that is not recognized by NKG2A/CD94. We further confirmed that HLA-C expressed on HIV-infected cells restricts attack by KIR2DL+ CD56dim NK cells, in contrast to the efficient responses by CD56bright NK cells, which express predominantly NKG2A/CD94 and lack KIR2DLs. These findings are important since the use of NK cells was recently proposed to treat latently HIV-1-infected patients in combination with latency reversing agents. Our results provide a mechanistic basis to guide these future clinical studies, suggesting that ex vivo-expanded NKG2A/CD94+ KIR2DL- NK cells may be uniquely beneficial. PMID:26828202

The Silk-protein Sericin Induces Rapid Melanization of Cultured Primary Human Retinal Pigment Epithelial Cells by Activating the NF-κB Pathway

PubMed Central

Eidet, J. R.; Reppe, S.; Pasovic, L.; Olstad, O. K.; Lyberg, T.; Khan, A. Z.; Fostad, I. G.; Chen, D. F.; Utheim, T. P.

2016-01-01

Restoration of the retinal pigment epithelial (RPE) cells to prevent further loss of vision in patients with age-related macular degeneration represents a promising novel treatment modality. Development of RPE transplants, however, requires up to 3 months of cell differentiation. We explored whether the silk protein sericin can induce maturation of primary human retinal pigment epithelial (hRPE) cells. Microarray analysis demonstrated that sericin up-regulated RPE-associated transcripts (RPE65 and CRALBP). Upstream analysis identified the NF-κB pathway as one of the top sericin-induced regulators. ELISA confirmed that sericin stimulates the main NF-κB pathway. Increased levels of RPE-associated proteins (RPE65 and the pigment melanin) in the sericin-supplemented cultures were confirmed by western blot, spectrophotometry and transmission electron microscopy. Sericin also increased cell density and reduced cell death following serum starvation in culture. Inclusion of NF-κB agonists and antagonists in the culture medium showed that activation of the NF-κB pathway appears to be necessary, but not sufficient, for sericin-induced RPE pigmentation. We conclude that sericin promotes pigmentation of cultured primary hRPE cells by activating the main NF-κB pathway. Sericin’s potential role in culture protocols for rapid differentiation of hRPE cells derived from embryonic or induced pluripotent stem cells should be investigated. PMID:26940175

The Silk-protein Sericin Induces Rapid Melanization of Cultured Primary Human Retinal Pigment Epithelial Cells by Activating the NF-κB Pathway.

PubMed

Eidet, J R; Reppe, S; Pasovic, L; Olstad, O K; Lyberg, T; Khan, A Z; Fostad, I G; Chen, D F; Utheim, T P

2016-03-04

Restoration of the retinal pigment epithelial (RPE) cells to prevent further loss of vision in patients with age-related macular degeneration represents a promising novel treatment modality. Development of RPE transplants, however, requires up to 3 months of cell differentiation. We explored whether the silk protein sericin can induce maturation of primary human retinal pigment epithelial (hRPE) cells. Microarray analysis demonstrated that sericin up-regulated RPE-associated transcripts (RPE65 and CRALBP). Upstream analysis identified the NF-κB pathway as one of the top sericin-induced regulators. ELISA confirmed that sericin stimulates the main NF-κB pathway. Increased levels of RPE-associated proteins (RPE65 and the pigment melanin) in the sericin-supplemented cultures were confirmed by western blot, spectrophotometry and transmission electron microscopy. Sericin also increased cell density and reduced cell death following serum starvation in culture. Inclusion of NF-κB agonists and antagonists in the culture medium showed that activation of the NF-κB pathway appears to be necessary, but not sufficient, for sericin-induced RPE pigmentation. We conclude that sericin promotes pigmentation of cultured primary hRPE cells by activating the main NF-κB pathway. Sericin's potential role in culture protocols for rapid differentiation of hRPE cells derived from embryonic or induced pluripotent stem cells should be investigated.

Genetic Causes of Human NK Cell Deficiency and Their Effect on NK Cell Subsets

PubMed Central

Mace, Emily M.; Orange, Jordan S.

2016-01-01

Human NK cells play critical roles in human host defense, particularly the control of viral infection and malignancy, and patients with congenital immunodeficiency affecting NK cell function or number can suffer from severe illness. The importance of NK cell function is particularly underscored in patients with primary immunodeficiency in which NK cells are the primary or sole affected population (NK cell deficiency, NKD). While NKD may lead to the absence of NK cells, we are also gaining an increasing appreciation of the effect that NKD may have on the generation of specific NK cell subsets. In turn, this leads to improved insights into the requirements for human NK cell subset generation, as well as their importance in immune homeostasis. The presence of inherently abnormally developed or functionally impaired NK cells, in particular, appears to be problematic in the way of interfering with normal human host defense and may be more impactful than low numbers of NK cells alone. Here, we review the known genetic causes of NKD and the insight that is derived by these into the requirements for human subset generation and, by extension, for NK cell-mediated immunity. PMID:27994588

Hepatocyte transplantation and advancements in alternative cell sources for liver-based regenerative medicine.

PubMed

Lee, Charlotte A; Sinha, Siddharth; Fitzpatrick, Emer; Dhawan, Anil

2018-06-01

Human hepatocyte transplantation has been actively perused as an alternative to liver replacement for acute liver failure and liver-based metabolic defects. Current challenges in this field include a limited cell source, reduced cell viability following cryopreservation and poor engraftment of cells into the recipient liver with consequent limited life span. As a result, alternative stem cell sources such as pluripotent stem cells, fibroblasts, hepatic progenitor cells, amniotic epithelial cells and mesenchymal stem/stromal cells (MSCs) can be used to generate induced hepatocyte like cells (HLC) with each technique exhibiting advantages and disadvantages. HLCs may have comparable function to primary human hepatocytes and could offer patient-specific treatment. However, long-term functionality of transplanted HLCs and the potential oncogenic risks of using stem cells have yet to be established. The immunomodulatory effects of MSCs are promising, and multiple clinical trials are investigating their effect in cirrhosis and acute liver failure. Here, we review the current status of hepatocyte transplantation, alternative cell sources to primary human hepatocytes and their potential in liver regeneration. We also describe recent clinical trials using hepatocytes derived from stem cells and their role in improving the phenotype of several liver diseases.

Efficient Transformation of Primary Human Mesenchymal Stromal Cells by Adenovirus Early Region 1 Oncogenes.

PubMed

Speiseder, Thomas; Hofmann-Sieber, Helga; Rodríguez, Estefanía; Schellenberg, Anna; Akyüz, Nuray; Dierlamm, Judith; Spruss, Thilo; Lange, Claudia; Dobner, Thomas

2017-01-01

Previous observations that human amniotic fluid cells (AFC) can be transformed by human adenovirus type 5 (HAdV-5) E1A/E1B oncogenes prompted us to identify the target cells in the AFC population that are susceptible to transformation. Our results demonstrate that one cell type corresponding to mesenchymal stem/stroma cells (hMSCs) can be reproducibly transformed by HAdV-5 E1A/E1B oncogenes as efficiently as primary rodent cultures. HAdV-5 E1-transformed hMSCs exhibit all properties commonly associated with a high grade of oncogenic transformation, including enhanced cell proliferation, anchorage-independent growth, increased growth rate, and high telomerase activity as well as numerical and structural chromosomal aberrations. These data confirm previous work showing that HAdV preferentially transforms cells of mesenchymal origin in rodents. More importantly, they demonstrate for the first time that human cells with stem cell characteristics can be completely transformed by HAdV oncogenes in tissue culture with high efficiency. Our findings strongly support the hypothesis that undifferentiated progenitor cells or cells with stem cell-like properties are highly susceptible targets for HAdV-mediated cell transformation and suggest that virus-associated tumors in humans may originate, at least in part, from infections of these cell types. We expect that primary hMSCs will replace the primary rodent cultures in HAdV viral transformation studies and are confident that these investigations will continue to uncover general principles of viral oncogenesis that can be extended to human DNA tumor viruses as well. It is generally believed that transformation of primary human cells with HAdV-5 E1 oncogenes is very inefficient. However, a few cell lines have been successfully transformed with HAdV-5 E1A and E1B, indicating that there is a certain cell type which is susceptible to HAdV-mediated transformation. Interestingly, all those cell lines have been derived from human embryonic tissue, albeit the exact cell type is not known yet. We show for the first time the successful transformation of primary human mesenchymal stromal cells (hMSCs) by HAdV-5 E1A and E1B. Further, we show upon HAdV-5 E1A and E1B expression that these primary progenitor cells exhibit features of tumor cells and can no longer be differentiated into the adipogenic, chondrogenic, or osteogenic lineage. Hence, primary hMSCs represent a robust and novel model system to elucidate the underlying molecular mechanisms of adenovirus-mediated transformation of multipotent human progenitor cells. Copyright © 2016 American Society for Microbiology.

Protection against acetaminophen-induced acute liver failure by omentum adipose tissue derived stem cells through the mediation of Nrf2 and cytochrome P450 expression.

PubMed

Huang, Yu-Jen; Chen, Poda; Lee, Chih-Yuan; Yang, Sin-Yu; Lin, Ming-Tsan; Lee, Hsuan-Shu; Wu, Yao-Ming

2016-01-19

Acetaminophen (APAP) overdose causes acute liver failure (ALF) in animals and humans via the rapid depletion of intracellular glutathione (GSH) and the generation of excess reactive oxygen species (ROS) that damage hepatocytes. Stem cell therapy is a potential treatment strategy for ALF. We isolated mesenchymal stem cells (MSCs) from mice omentum adipose tissue-derived stem cells (ASCs) and transplanted them into a mouse model of APAP-induced ALF to explore their therapeutic potential. In addition, we performed in vitro co-culture studies with omentum-derived ASCs and primary isolated hepatocytes to demonstrate the hepatoprotective effect of omentum-derived ASCs on hepatocytes that were subjected to APAP-induced damage. ASC transplantation significantly improved the survival rate of mice with ALF and attenuated the severity of APAP-induced liver damage by suppressing cytochrome P450 activity to reduce the accumulation of toxic nitrotyrosine and the upregulation of NF-E2-related factor 2 (Nrf2) expression, resulting in an increase in the subsequent antioxidant activity. These effects protected the hepatocytes from APAP-induced damage through the suppression of downstream MAPK signal activation and inflammatory cytokine production. our results demonstrate that omentum-derived ASCs are an alternative source of ASCs that regulate the antioxidant response and may represent a beneficial therapeutic strategy for ALF.

The Role of Immune and Inflammatory Cells in Idiopathic Pulmonary Fibrosis

PubMed Central

Desai, Omkar; Winkler, Julia; Minasyan, Maksym; Herzog, Erica L.

2018-01-01

The contribution of the immune system to idiopathic pulmonary fibrosis (IPF) remains poorly understood. While most sources agree that IPF does not result from a primary immunopathogenic mechanism, evidence gleaned from animal modeling and human studies suggests that innate and adaptive immune processes can orchestrate existing fibrotic responses. This review will synthesize the available data regarding the complex role of professional immune cells in IPF. The role of innate immune populations such as monocytes, macrophages, myeloid suppressor cells, and innate lymphoid cells will be discussed, as will the activation of these cells via pathogen-associated molecular patterns derived from invading or commensural microbes, and danger-associated molecular patterns derived from injured cells and tissues. The contribution of adaptive immune responses driven by T-helper cells and B cells will be reviewed as well. Each form of immune activation will be discussed in the context of its relationship to environmental and genetic factors, disease outcomes, and potential therapies. We conclude with discussion of unanswered questions and opportunities for future study in this area. PMID:29616220

Conditionally reprogrammed normal and primary tumor prostate epithelial cells: a novel patient-derived cell model for studies of human prostate cancer

PubMed Central

Timofeeva, Olga A.; Palechor-Ceron, Nancy; Li, Guanglei; Yuan, Hang; Krawczyk, Ewa; Zhong, Xiaogang; Liu, Geng; Upadhyay, Geeta; Dakic, Aleksandra; Yu, Songtao; Fang, Shuang; Choudhury, Sujata; Zhang, Xueping; Ju, Andrew; Lee, Myeong-Seon; Dan, Han C.; Ji, Youngmi; Hou, Yong; Zheng, Yun-Ling; Albanese, Chris; Rhim, Johng; Schlegel, Richard; Dritschilo, Anatoly; Liu, Xuefeng

2017-01-01

Our previous study demonstrated that conditional reprogramming (CR) allows the establishment of patient-derived normal and tumor epithelial cell cultures from a variety of tissue types including breast, lung, colon and prostate. Using CR, we have established matched normal and tumor cultures, GUMC-29 and GUMC-30 respectively, from a patient's prostatectomy specimen. These CR cells proliferate indefinitely in vitro and retain stable karyotypes. Most importantly, only tumor-derived CR cells (GUMC-30) produced tumors in xenografted SCID mice, demonstrating maintenance of the critical tumor phenotype. Characterization of cells with DNA fingerprinting demonstrated identical patterns in normal and tumor CR cells as well as in xenografted tumors. By flow cytometry, both normal and tumor CR cells expressed basal, luminal, and stem cell markers, with the majority of the normal and tumor CR cells expressing prostate basal cell markers, CD44 and Trop2, as well as luminal marker, CD13, suggesting a transit-amplifying phenotype. Consistent with this phenotype, real time RT-PCR analyses demonstrated that CR cells predominantly expressed high levels of basal cell markers (KRT5, KRT14 and p63), and low levels of luminal markers. When the CR tumor cells were injected into SCID mice, the expression of luminal markers (AR, NKX3.1) increased significantly, while basal cell markers dramatically decreased. These data suggest that CR cells maintain high levels of proliferation and low levels of differentiation in the presence of feeder cells and ROCK inhibitor, but undergo differentiation once injected into SCID mice. Genomic analyses, including SNP and INDEL, identified genes mutated in tumor cells, including components of apoptosis, cell attachment, and hypoxia pathways. The use of matched patient-derived cells provides a unique in vitro model for studies of early prostate cancer. PMID:28009986

Relationships Among Microbial Communities, Maternal Cells, Oligosaccharides, and Macronutrients in Human Milk.

PubMed

Williams, Janet E; Price, William J; Shafii, Bahman; Yahvah, Katherine M; Bode, Lars; McGuire, Mark A; McGuire, Michelle K

2017-08-01

Human milk provides all essential nutrients necessary for early life and is rich in nonnutrients, maternally derived (host) cells, and bacteria, but almost nothing is known about the interplay among these components. Research aim: The primary objective of this research was to characterize relationships among macronutrients, maternal cells, and bacteria in milk. Milk samples were collected from 16 women and analyzed for protein, lipid, fatty acid, lactose, and human milk oligosaccharide concentrations. Concentrations of maternal cells were determined using microscopy, and somatic cell counts were enumerated. Microbial ecologies were characterized using culture-independent methods. Absolute and relative concentrations of maternal cells were mostly consistent within each woman as were relative abundances of bacterial genera, and there were many apparent relationships between these factors. For instance, relative abundance of Serratia was negatively associated with somatic cell counts ( r = -.47, p < .0001) and neutrophil concentration ( r = -.38, p < .0006). Concentrations of several oligosaccharides were correlated with maternally derived cell types as well as somatic cell counts; for example, lacto-N-tetraose and lacto-N-neotetraose were inversely correlated with somatic cell counts ( r = -.64, p = .0082; r = -.52, p = .0387, respectively), and relative abundance of Staphylococcus was positively associated with total oligosaccharide concentration ( r = .69, p = .0034). Complex relationships between milk nutrients and bacterial community profile, maternal cells, and milk oligosaccharides were also apparent. These data support the possibility that profiles of maternally derived cells, nutrient concentrations, and the microbiome of human milk might be interrelated.

18ß-glycyrrhetinic acid derivative promotes proliferation, migration and aquaporin-3 expression in human dermal fibroblasts

PubMed Central

Hung, Chi-Feng; Hsiao, Chien-Yu; Hsieh, Wen-Hao; Li, Hsin-Ju; Tsai, Yi-Ju; Lin, Chun-Nan; Chang, Hsun-Hsien; Wu, Nan-Lin

2017-01-01

Licorice (Glycyrrhiza) species have been widely used as a traditional medicine and a natural sweetener in foods. The 18β-glycyrrhetinic acid (18β-GA) is a bioactive compound in licorice that exhibits potential anti-cancer, anti-inflammatory, and anti-microbial activities. Many synthesized derivatives of 18β-GA have been reported to be cytotoxic and suggested for the treatment of malignant diseases. In this study, we explored the possible pharmacological roles of an 18β-GA derivative in skin biology using primary human dermal fibroblasts and HaCaT keratinocytes as cell models. We found that this 18β-GA derivative did not cause cell death, but significantly enhanced the proliferation of dermal fibroblasts and HaCaT keratinocytes. A scratch wound healing assay revealed that the 18β-GA derivative promoted the migration of fibroblasts. Due to the important role of aquaporin-3 in cell migration and proliferation, we also investigated the expression of aquaporin-3 and found this compound up-regulated the expression of aquaporin-3 in dermal fibroblasts and HaCaT keratinocytes. In dermal fibroblasts, the 18β-GA derivative induced the phosphorylation of Akt, ERK, and p38. The inhibitor of Akt predominantly suppressed the 18β-GA derivative-induced expression of aquaporin-3. Collectively, this compound had a positive effect on the proliferation, migration, and aquaporin-3 expression of skin cells, implying its potential role in the treatment of skin diseases characterized by impaired wound healing or dermal defects. PMID:28813533

Umbilical Cord Blood-Derived Mesenchymal Stem Cells Inhibit, But Adipose Tissue-Derived Mesenchymal Stem Cells Promote, Glioblastoma Multiforme Proliferation

PubMed Central

Akimoto, Keiko; Kimura, Kenichi; Nagano, Masumi; Takano, Shingo; To'a Salazar, Georgina; Yamashita, Toshiharu

2013-01-01

Mesenchymal stem cells (MSCs) possess self-renewal and multipotential differentiation abilities, and they are thought to be one of the most reliable stem cell sources for a variety of cell therapies. Recently, cell therapy using MSCs has been studied as a novel therapeutic approach for cancers that show refractory progress and poor prognosis. MSCs from different tissues have different properties. However, the effect of different MSC properties on their application in anticancer therapies has not been thoroughly investigated. In this study, to characterize the anticancer therapeutic application of MSCs from different sources, we established two different kinds of human MSCs: umbilical cord blood-derived MSCs (UCB-MSCs) and adipose-tissue-derived MSCs (AT-MSCs). We used these MSCs in a coculture assay with primary glioblastoma multiforme (GBM) cells to analyze how MSCs from different sources can inhibit GBM growth. We found that UCB-MSCs inhibited GBM growth and caused apoptosis, but AT-MSCs promoted GBM growth. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick-end labeling assay clearly demonstrated that UCB-MSCs promoted apoptosis of GBM via tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). TRAIL was expressed more highly by UCB-MSCs than by AT-MSCs. Higher mRNA expression levels of angiogenic factors (vascular endothelial growth factor, angiopoietin 1, platelet-derived growth factor, and insulin-like growth factor) and stromal-derived factor-1 (SDF-1/CXCL12) were observed in AT-MSCs, and highly vascularized tumors were developed when AT-MSCs and GBM were cotransplanted. Importantly, CXCL12 inhibited TRAIL activation of the apoptotic pathway in GBM, suggesting that AT-MSCs may support GBM development in vivo by at least two distinct mechanisms—promoting angiogenesis and inhibiting apoptosis. The opposite effects of AT-MSCs and UCB-MSCs on GBM clearly demonstrate that differences must be considered when choosing a stem cell source for safety in clinical application. PMID:23231075

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.

Development of an encapsulated stem cell-based therapy for diabetes.

PubMed

Tomei, Alice Anna; Villa, Chiara; Ricordi, Camillo

2015-01-01

Islet transplantation can treat the most severe cases of type 1 diabetes but it currently requires deceased donor pancreata as an islet source and chronic immunosuppression to prevent rejection and recurrence of autoimmunity. Stem cell-derived insulin-producing cells may address the shortage of organ donors, whereas cell encapsulation may reduce or eliminate the requirement for immunosuppression, minimizing the risks associated with the islet transplantation procedure, and potentially prolonging graft survival. This review focuses on the design principles for immunoisolation devices and on stem cell differentiation into insulin-producing cell products. The reader will gain understanding of the different types of immunoisolation devices and the key parameters that affect the outcome of the encapsulated graft. Progresses in stem cell differentiation towards mature endocrine islet cells, including the most recent clinical trials and the challenges associated with the application of immunoisolation devices designed for primary islets to stem-cell products, are also discussed. Recent advancements in the field of stem cell-derived islet cell products and immunoisolation strategies hold great promise for type 1 diabetes. However, a combination product including both cells and an immunoisolation strategy still needs to be optimized and tested for safety and efficacy.

17-AAG and Apoptosis, Autophagy, and Mitophagy in Canine Osteosarcoma Cell Lines.

PubMed

Massimini, M; Palmieri, C; De Maria, R; Romanucci, M; Malatesta, D; De Martinis, M; Maniscalco, L; Ciccarelli, A; Ginaldi, L; Buracco, P; Bongiovanni, L; Della Salda, L

2017-05-01

Canine osteosarcoma is highly resistant to current chemotherapy; thus, clarifying the mechanisms of tumor cell resistance to treatments is an urgent need. We tested the geldanamycin derivative 17-AAG (17-allylamino-17-demethoxygeldanamycin) prototype of Hsp90 (heat shock protein 90) inhibitors in 2 canine osteosarcoma cell lines, D22 and D17, derived from primary and metastatic tumors, respectively. With the aim to understand the interplay between cell death, autophagy, and mitophagy, in light of the dual effect of autophagy in regulating cancer cell viability and death, D22 and D17 cells were treated with different concentrations of 17-AAG (0.5 μM, 1 μM) for 24 and 48 hours. 17-AAG-induced apoptosis, necrosis, autophagy, and mitophagy were assessed by transmission electron microscopy, flow cytometry, and immunofluorescence. A simultaneous increase in apoptosis, autophagy, and mitophagy was observed only in the D22 cell line, while D17 cells showed low levels of apoptotic cell death. These results reveal differential cell response to drug-induced stress depending on tumor cell type. Therefore, pharmacological treatments based on proapoptotic chemotherapy in association with autophagy regulators would benefit from a predictive in vitro screening of the target cell type.

Characterization of Cancer Stem-Like Cells Derived from Mouse Induced Pluripotent Stem Cells Transformed by Tumor-Derived Extracellular Vesicles

PubMed Central

Yan, Ting; Mizutani, Akifumi; Chen, Ling; Takaki, Mai; Hiramoto, Yuki; Matsuda, Shuichi; Shigehiro, Tsukasa; Kasai, Tomonari; Kudoh, Takayuki; Murakami, Hiroshi; Masuda, Junko; Hendrix, Mary J. C.; Strizzi, Luigi; Salomon, David S.; Fu, Li; Seno, Masaharu

2014-01-01

Several studies have shown that cancer niche can perform an active role in the regulation of tumor cell maintenance and progression through extracellular vesicles-based intercellular communication. However, it has not been reported whether this vesicle-mediated communication affects the malignant transformation of normal stem cells/progenitors. We have previously reported that the conditioned medium derived from the mouse Lewis Lung Carcinoma (LLC) cell line can convert mouse induced pluripotent stem cells (miPSCs) into cancer stem cells (CSCs), indicating that normal stem cells when placed in an aberrant microenvironment can give rise to functionally active CSCs. Here, we focused on the contribution of tumor-derived extracellular vesicles (tEVs) that are secreted from LLC cells to induce the transformation of miPSCs into CSCs. We isolated tEVs from the conditioned medium of LLC cells, and then the differentiating miPSCs were exposed to tEVs for 4 weeks. The resultant tEV treated cells (miPS-LLCev) expressed Nanog and Oct3/4 proteins comparable to miPSCs. The frequency of sphere formation of the miPS-LLCev cells in suspension culture indicated that the self-renewal capacity of the miPS-LLCev cells was significant. When the miPS-LLCev cells were subcutaneously transplanted into Balb/c nude mice, malignant liposarcomas with extensive angiogenesis developed. miPS-LLCevPT and miPS-LLCevDT, the cells established from primary site and disseminated liposarcomas, respectively, showed their capacities to self-renew and differentiate into adipocytes and endothelial cells. Moreover, we confirmed the secondary liposarcoma development when these cells were transplanted. Taken together, these results indicate that miPS-LLCev cells possess CSC properties. Thus, our current study provides the first evidence that tEVs have the potential to induce CSC properties in normal tissue stem cells/progenitors. PMID:25057308

Stem/progenitor cell-like properties of desmoglein 3dim cells in primary and immortalized keratinocyte lines.

PubMed

Wan, Hong; Yuan, Ming; Simpson, Cathy; Allen, Kirsty; Gavins, Felicity N E; Ikram, Mohammed S; Basu, Subham; Baksh, Nuzhat; O'Toole, Edel A; Hart, Ian R

2007-05-01

We showed previously that primary keratinocytes selected for low desmoglein 3 (Dsg3) expression levels exhibited increased colony-forming efficiency and heightened proliferative potential relative to cells with higher Dsg3 expression levels, characteristics consistent with a more "stem/progenitor cell-like" phenotype. Here, we have confirmed that Dsg3(dim) cells derived from cultured primary human adult keratinocytes have comparability with alpha(6)(bri)/CD71(dim) stem cells in terms of colony-forming efficiency. Moreover, these Dsg3(dim) cells exhibit increased reconstituting ability in in vitro organotypic culture on de-epidermalized dermis (DED); they are small, actively cycling cells, and they express elevated levels of various p63 isoforms. In parallel, using the two immortalized keratinocyte cell lines HaCaT and NTERT, we obtained essentially similar though occasionally different findings. Thus, reduced colony-forming efficiency by Dsg3(bri) cells consistently was observed in both cell lines even though the cell cycle profile and levels of p63 isoforms in the bri and dim populations differed between these two cell lines. Dsg3(dim) cells from both immortalized lines produced thicker and better ordered hierarchical structural organization of reconstituted epidermis relative to Dsg3(bri) and sorted control cells. Dsg3(dim) HaCaT cells also show sebocyte-like differentiation in the basal compartment of skin reconstituted after a 4-week organotypic culture. No differences in percentages of side population cells (also a putative marker of stem cells) were detected between Dsg3(dim) and Dsg3(bri) populations. Taken together our data indicate that Dsg3(dim) populations from primary human adult keratinocytes and long-term established keratinocyte lines possess certain stem/progenitor cell-like properties, although the side population characteristic is not one of these features. Disclosure of potential conflicts of interest is found at the end of this article.

Silencing the Snail-dependent RNA splice regulator ESRP1 drives malignant transformation of human pulmonary epithelial cells. | Office of Cancer Genomics

Cancer.gov

Epithelial-to-mesenchymal transition (EMT) is organized in cancer cells by a set of key transcription factors, but the significance of this process is still debated including in non-small cell lung cancer (NSCLC). Here we report increased expression of the EMT-inducing transcription factor Snail in premalignant pulmonary lesions, relative to histologically normal pulmonary epithelium. In immortalized human pulmonary epithelial cells and isogenic derivatives, we documented Snail-dependent anchorage-independent growth in vitro and primary tumor growth and metastatic behavior in vivo.

Proliferation inhibitory effect of human alpha interferon on primary explants of Burkitt lymphoma: inverse relationship to patient survival.

PubMed

Ernberg, I; Einhorn, S; Strander, H; Klein, G

1981-11-01

Eleven biopsies from 9 patients with Burkitt's lymphoma were tested for their sensitivity to the cell multiplication inhibitory activity of interferon. Three were resistant to interferon while 8 were sensitive to various degrees. Different biopsies from the same patient did not differ in interferon sensitivity. These results indicate that Burkitt's lymphoma cells might be resistant to interferon already in vivo as previously shown for some derived cell lines tested in vitro. The results imply an inverse relationship between patient survival and interferon sensitivity of the tumor cells.

Idiopathic Mast Cell Activation Syndrome With Associated Salicylate Intolerance.

PubMed

Rechenauer, Tobias; Raithel, Martin; Götze, Thomas; Siebenlist, Gregor; Rückel, Aline; Baenkler, Hanns-Wolf; Hartmann, Arndt; Haller, Florian; Hoerning, André

2018-01-01

Idiopathic mast cell activation syndrome can be a rare cause for chronic abdominal pain in children. It remains a diagnosis by exclusion that can be particularly challenging due to the vast variety of possible clinical manifestations. We present a 13-year-old boy who suffered from a multitude of unspecific complaints over a long period of time. In this case, an assessment of mast cell-derived metabolites and immunohistochemical analysis of bioptic specimen was worthwhile. After ruling out, primary (oncologic) and secondary causes for mast cell activation, pharmacologic treatment adapted to the patient's salicylate intolerance resulted in a major relief of symptoms.

Derivation and characterization of human embryonic stem cell lines from poor quality embryos.

PubMed

Liu, Weiqiang; Yin, Yifei; Long, Xiaolin; Luo, Yumei; Jiang, Yonghua; Zhang, Wenhong; Du, Hongzi; Li, Shaoying; Zheng, Yuhong; Li, Qing; Chen, Xinjie; Liao, Baoping; Xiao, Guohong; Wang, Weihua; Sun, Xiaofang

2009-04-01

Poor quality embryos discarded from in vitro fertilization (IVF) laboratories are good sources for deriving human embryonic stem cell (hESC) lines. In this study, 166 poor quality embryos donated from IVF centers on day 3 were cultured in a blastocyst medium for 2 days, and 32 early blastocysts were further cultured in a blastocyst optimum culture medium for additional 2 days so that the inner cell masses (ICMs) could be identified and isolated easily. The ICMs of 17 blastocysts were isolated by a mechanical method, while those of the other 15 blastocysts were isolated by immunosurgery. All isolated ICMs were inoculated onto a feeder layer for subcultivation. The rates of ICM attachment, primary ICM colony formation and the efficiency of hESC derivation were similar between the ICMs isolated by the two methods (P>0.05). As a result, four new hESC lines were established. Three cell lines had normal karyotypes and one had an unbalanced Robertsonian translocation. All cell lines showed normal hESC characteristics and had the differentiation ability. In conclusion, we established a stable and effective method for hESC isolation and culture, and it was confirmed that the mechanical isolation was an effective method to isolate ICMs from poor embryos. These results further indicate that hESC lines can be derived from poor quality embryos discarded by IVF laboratories.

Sustained synchronized neuronal network activity in a human astrocyte co-culture system

PubMed Central

Kuijlaars, Jacobine; Oyelami, Tutu; Diels, Annick; Rohrbacher, Jutta; Versweyveld, Sofie; Meneghello, Giulia; Tuefferd, Marianne; Verstraelen, Peter; Detrez, Jan R.; Verschuuren, Marlies; De Vos, Winnok H.; Meert, Theo; Peeters, Pieter J.; Cik, Miroslav; Nuydens, Rony; Brône, Bert; Verheyen, An

2016-01-01

Impaired neuronal network function is a hallmark of neurodevelopmental and neurodegenerative disorders such as autism, schizophrenia, and Alzheimer’s disease and is typically studied using genetically modified cellular and animal models. Weak predictive capacity and poor translational value of these models urge for better human derived in vitro models. The implementation of human induced pluripotent stem cells (hiPSCs) allows studying pathologies in differentiated disease-relevant and patient-derived neuronal cells. However, the differentiation process and growth conditions of hiPSC-derived neurons are non-trivial. In order to study neuronal network formation and (mal)function in a fully humanized system, we have established an in vitro co-culture model of hiPSC-derived cortical neurons and human primary astrocytes that recapitulates neuronal network synchronization and connectivity within three to four weeks after final plating. Live cell calcium imaging, electrophysiology and high content image analyses revealed an increased maturation of network functionality and synchronicity over time for co-cultures compared to neuronal monocultures. The cells express GABAergic and glutamatergic markers and respond to inhibitors of both neurotransmitter pathways in a functional assay. The combination of this co-culture model with quantitative imaging of network morphofunction is amenable to high throughput screening for lead discovery and drug optimization for neurological diseases. PMID:27819315

Establishment and Maintenance of Primary Fibroblast Repositories for Rare Diseases-Friedreich's Ataxia Example.

PubMed

Li, Yanjie; Polak, Urszula; Clark, Amanda D; Bhalla, Angela D; Chen, Yu-Yun; Li, Jixue; Farmer, Jennifer; Seyer, Lauren; Lynch, David; Butler, Jill S; Napierala, Marek

2016-08-01

Friedreich's ataxia (FRDA) represents a rare neurodegenerative disease caused by expansion of GAA trinucleotide repeats in the first intron of the FXN gene. The number of GAA repeats in FRDA patients varies from approximately 60 to <1000 and is tightly correlated with age of onset and severity of the disease symptoms. The heterogeneity of Friedreich's ataxia stresses the need for a large cohort of patient samples to conduct studies addressing the mechanism of disease pathogenesis or evaluate novel therapeutic candidates. Herein, we report the establishment and characterization of an FRDA fibroblast repository, which currently includes 50 primary cell lines derived from FRDA patients and seven lines from mutation carriers. These cells are also a source for generating induced pluripotent stem cell (iPSC) lines by reprogramming, as well as disease-relevant neuronal, cardiac, and pancreatic cells that can then be differentiated from the iPSCs. All FRDA and carrier lines are derived using a standard operating procedure and characterized to confirm mutation status, as well as expression of FXN mRNA and protein. Consideration and significance of creating disease-focused cell line and tissue repositories, especially in the context of rare and heterogeneous disorders, are presented. Although the economic aspect of creating and maintaining such repositories is important, the benefits of easy access to a collection of well-characterized cell lines for the purpose of drug discovery or disease mechanism studies overshadow the associated costs. Importantly, all FRDA fibroblast cell lines collected in our repository are available to the scientific community.

Establishment and Maintenance of Primary Fibroblast Repositories for Rare Diseases—Friedreich's Ataxia Example

PubMed Central

Li, Yanjie; Polak, Urszula; Clark, Amanda D.; Bhalla, Angela D.; Chen, Yu-Yun; Li, Jixue; Farmer, Jennifer; Seyer, Lauren; Lynch, David

2016-01-01

Friedreich's ataxia (FRDA) represents a rare neurodegenerative disease caused by expansion of GAA trinucleotide repeats in the first intron of the FXN gene. The number of GAA repeats in FRDA patients varies from approximately 60 to <1000 and is tightly correlated with age of onset and severity of the disease symptoms. The heterogeneity of Friedreich's ataxia stresses the need for a large cohort of patient samples to conduct studies addressing the mechanism of disease pathogenesis or evaluate novel therapeutic candidates. Herein, we report the establishment and characterization of an FRDA fibroblast repository, which currently includes 50 primary cell lines derived from FRDA patients and seven lines from mutation carriers. These cells are also a source for generating induced pluripotent stem cell (iPSC) lines by reprogramming, as well as disease-relevant neuronal, cardiac, and pancreatic cells that can then be differentiated from the iPSCs. All FRDA and carrier lines are derived using a standard operating procedure and characterized to confirm mutation status, as well as expression of FXN mRNA and protein. Consideration and significance of creating disease-focused cell line and tissue repositories, especially in the context of rare and heterogeneous disorders, are presented. Although the economic aspect of creating and maintaining such repositories is important, the benefits of easy access to a collection of well-characterized cell lines for the purpose of drug discovery or disease mechanism studies overshadow the associated costs. Importantly, all FRDA fibroblast cell lines collected in our repository are available to the scientific community. PMID:27002638

Validation of the DRACO Particle-in-Cell Code using Busek 200W Hall Thruster Experimental Data

DTIC Science & Technology

2008-07-23

and where sputtered material will be deposited on a spacecraft. 15 1.4: Thesis Overview The primary objective of this thesis is to use the DRACO...the second derivative of a fuction is given in Equation 2.6. These equations are calculated at a node and use the value at the node, kjif ,, , as...determine how the results different and which field solving method produces the best results. 4.3.3: Collision Model There are two primary methods

HIV-2 infects resting CD4+ T cells but not monocyte-derived dendritic cells.

PubMed

Chauveau, Lise; Puigdomenech, Isabel; Ayinde, Diana; Roesch, Ferdinand; Porrot, Françoise; Bruni, Daniela; Visseaux, Benoit; Descamps, Diane; Schwartz, Olivier

2015-01-13

Human Immunodeficiency Virus-type 2 (HIV-2) encodes Vpx that degrades SAMHD1, a cellular restriction factor active in non-dividing cells. HIV-2 replicates in lymphocytes but the susceptibility of monocyte-derived dendritic cells (MDDCs) to in vitro infection remains partly characterized. Here, we investigated HIV-2 replication in primary CD4+ T lymphocytes, both activated and non-activated, as well as in MDDCs. We focused on the requirement of Vpx for productive HIV-2 infection, using the reference HIV-2 ROD strain, the proviral clone GL-AN, as well as two primary HIV-2 isolates. All HIV-2 strains tested replicated in activated CD4+ T cells. Unstimulated CD4+ T cells were not productively infected by HIV-2, but viral replication was triggered upon lymphocyte activation in a Vpx-dependent manner. In contrast, MDDCs were poorly infected when exposed to HIV-2. HIV-2 particles did not potently fuse with MDDCs and did not lead to efficient viral DNA synthesis, even in the presence of Vpx. Moreover, the HIV-2 strains tested were not efficiently sensed by MDDCs, as evidenced by a lack of MxA induction upon viral exposure. Virion pseudotyping with VSV-G rescued fusion, productive infection and HIV-2 sensing by MDDCs. Vpx allows the non-productive infection of resting CD4+ T cells, but does not confer HIV-2 with the ability to efficiently infect MDDCs. In these cells, an entry defect prevents viral fusion and reverse transcription independently of SAMHD1. We propose that HIV-2, like HIV-1, does not productively infect MDDCs, possibly to avoid triggering an immune response mediated by these cells.

Human trophoblasts recruited T lymphocytes and monocytes into decidua by secretion of chemokine CXCL16 and interaction with CXCR6 in the first-trimester pregnancy.

PubMed

Huang, Yu; Zhu, Xiao-Yong; Du, Mei-Rong; Li, Da-Jin

2008-02-15

During human early pregnancy, fetus-derived trophoblasts come into direct contact with maternal immune cells at the maternofetal interface. At sites of placental attachment, invasive extravillous trophoblasts encounter decidual leukocytes (DLC) that accumulate within the decidua. Because we first found chemokine CXCL16 was highly expressed in and secreted by the first-trimester human trophoblasts previously, in this study we tested the hypothesis of whether the fetal trophoblasts can direct migration of maternal T lymphocyte and monocytes into decidua by secreting CXCL16. We analyzed the transcription and translation of CXCL16 in the isolated first-trimester human trophoblast, and examined the kinetic secretion of CXCL16 in the supernatant of the primary-cultured trophoblasts. We demonstrated that the sole receptor of CXCL16, CXCR6, is preferentially expressed in T lymphocytes, NKT cells, and monocytes, hardly expressed in two subsets of NK cells from either the peripheral blood or decidua. We further demonstrated the chemotactic activity of CXCL16 in the supernatant of the primary trophoblast on the peripheral mononuclear cells and DLC. Moreover, the CXCL16/CXCR6 interaction is involved in the migration of the peripheral T lymphocytes, gammadelta T cells, and monocytes, but not NKT cells. In addition, the trophoblast-conditioned medium could enrich PBMC subsets selectively to constitute a leukocyte population with similar composition to that of DLC, which suggests that the fetus-derived trophoblasts can attract T cells, gammadelta T cells, and monocytes by producing CXCL16 and interaction with CXCR6 on these cells, leading to forming a specialized immune milieu at the maternofetal interface.

Characterization of primary human mammary epithelial cells isolated and propagated by conditional reprogrammed cell culture.

PubMed

Jin, Liting; Qu, Ying; Gomez, Liliana J; Chung, Stacey; Han, Bingchen; Gao, Bowen; Yue, Yong; Gong, Yiping; Liu, Xuefeng; Amersi, Farin; Dang, Catherine; Giuliano, Armando E; Cui, Xiaojiang

2018-02-20

Conditional reprogramming methods allow for the inexhaustible in vitro proliferation of primary epithelial cells from human tissue specimens. This methodology has the potential to enhance the utility of primary cell culture as a model for mammary gland research. However, few studies have systematically characterized this method in generating in vitro normal human mammary epithelial cell models. We show that cells derived from fresh normal breast tissues can be propagated and exhibit heterogeneous morphologic features. The cultures are composed of CK18, desmoglein 3, and CK19-positive luminal cells and vimentin, p63, and CK14-positive myoepithelial cells, suggesting the maintenance of in vivo heterogeneity. In addition, the cultures contain subpopulations with different CD49f and EpCAM expression profiles. When grown in 3D conditions, cells self-organize into distinct structures that express either luminal or basal cell markers. Among these structures, CK8-positive cells enclosing a lumen are capable of differentiation into milk-producing cells in the presence of lactogenic stimulus. Furthermore, our short-term cultures retain the expression of ERα, as well as its ability to respond to estrogen stimulation. We have investigated conditionally reprogrammed normal epithelial cells in terms of cell type heterogeneity, cellular marker expression, and structural arrangement in two-dimensional (2D) and three-dimensional (3D) systems. The conditional reprogramming methodology allows generation of a heterogeneous culture from normal human mammary tissue in vitro . We believe that this cell culture model will provide a valuable tool to study mammary cell function and malignant transformation.

Efficient activation of human T cells of both CD4 and CD8 subsets by urease-deficient recombinant Mycobacterium bovis BCG that produced a heat shock protein 70-M. tuberculosis-derived major membrane protein II fusion protein.

PubMed

Mukai, Tetsu; Tsukamoto, Yumiko; Maeda, Yumi; Tamura, Toshiki; Makino, Masahiko

2014-01-01

For the purpose of obtaining Mycobacterium bovis bacillus Calmette-Guérin (BCG) capable of activating human naive T cells, urease-deficient BCG expressing a fusion protein composed of Mycobacterium tuberculosis-derived major membrane protein II (MMP-II) and heat shock protein 70 (HSP70) of BCG (BCG-DHTM) was produced. BCG-DHTM secreted the HSP70-MMP-II fusion protein and effectively activated human monocyte-derived dendritic cells (DCs) by inducing phenotypic changes and enhanced cytokine production. BCG-DHTM-infected DCs activated naive T cells of both CD4 and naive CD8 subsets, in an antigen (Ag)-dependent manner. The T cell activation induced by BCG-DHTM was inhibited by the pretreatment of DCs with chloroquine. The naive CD8(+) T cell activation was mediated by the transporter associated with antigen presentation (TAP) and the proteosome-dependent cytosolic cross-priming pathway. Memory CD8(+) T cells and perforin-producing effector CD8(+) T cells were efficiently produced from the naive T cell population by BCG-DHTM stimulation. Single primary infection with BCG-DHTM in C57BL/6 mice efficiently produced T cells responsive to in vitro secondary stimulation with HSP70, MMP-II, and M. tuberculosis-derived cytosolic protein and inhibited the multiplication of subsequently aerosol-challenged M. tuberculosis more efficiently than did vector control BCG. These results indicate that the introduction of MMP-II and HSP70 into urease-deficient BCG may be useful for improving BCG for control of tuberculosis.

Cell behavior of human mesenchymal stromal cells in response to silica/collagen based xerogels and calcium deficient culture conditions.

PubMed

Wagner, Alena-Svenja; Glenske, Kristina; Henß, Anja; Kruppke, Benjamin; Rößler, Sina; Hanke, Thomas; Moritz, Andreas; Rohnke, Marcus; Kressin, Monika; Arnhold, Stefan; Schnettler, Reinhard; Wenisch, Sabine

2017-07-04

Herein, we aim to elucidate osteogenic effects of two silica-based xerogels with different degrees of bioactivity on human bone-derived mesenchymal stromal cells by means of scanning electron microscopy, quantitative PCR enhanced osteogenic effects and the formation of an extracellular matrix which could be ascribed to the sample with lower bioactivity. Given the high levels of bioactivity, the cells revealed remarkable sensitivity to extremely low calcium levels of the media. Therefore, additional experiments were performed to elucidate cell behavior under calcium deficient conditions. The results refer to capacity of the bone-derived stromal cells to overcome calcium deficiency even though proliferation, migration and osteogenic differentiation capabilities were diminished. One reason for the differences of the cellular response (on tissue culture plates versus xerogels) to calcium deficiency seems to be the positive effect of silica. The silica could be detected intracellularly as shown by time of flight-secondary ion mass spectrometry after cultivation of primary cells for 21 days on the surfaces of the xerogels. Thus, the present findings refer to different osteogenic differentiation potentials of the xerogels according to the different degrees of bioactivity, and to the role of silica as a stimulator of osteogenesis. Finally, the observed pattern of connexin-based hemichannel gating supports the assumption that connexin 43 is a key factor for calcium-mediated osteogenesis in bone-derived mesenchymal stromal cells.

A computational integrative approach based on alternative splicing analysis to compare immortalized and primary cancer cells.

PubMed

Tripathi, Kumar Parijat; Granata, Ilaria; Guarracino, Mario Rosario

2017-10-01

Immortalized cell lines are widely used to study the effectiveness and toxicity of anti cancer drugs as well as to assess the phenotypic characteristics of cancer cells, such as proliferation and migration ability. Unfortunately, cell lines often show extremely different properties than tumor tissues. Also the primary cells, that are deprived of the in vivo environment, might adapt to artificial conditions, and differ from the tissue they should represent. Despite these considerations, cell lines are still one of the most used cancer models due to their availability and capability to expand without limitation, but the clinical relevance of their use is still a big issue in cancer research. Many studies tried to overcome this task, comparing cell lines and tumor samples through the definition of the genomic and transcriptomic differences. To this aim, most of them used nucleotide variation or gene expression data. Here we introduce a different strategy based on alternative splicing detection and integration of DNA and RNA sequencing data, to explore the differences between immortalized and tissue-derived cells at isoforms level. Furthermore, in order to better investigate the heterogeneity of both cell populations, we took advantage of a public available dataset obtained with a new simultaneous omics single cell sequencing methodology. The proposed pipeline allowed us to identify, through a computational and prediction approach, putative mutated and alternative spliced transcripts responsible for the dissimilarity between immortalized and primary hepato carcinoma cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

Increased Cardiac Myocyte Progenitors in Failing Human Hearts

PubMed Central

Kubo, Hajime; Jaleel, Naser; Kumarapeli, Asangi; Berretta, Remus M.; Bratinov, George; Shan, Xiaoyin; Wang, Hongmei; Houser, Steven R.; Margulies, Kenneth B.

2009-01-01

Background Increasing evidence, derived mainly from animal models, supports the existence of endogenous cardiac renewal and repair mechanisms in adult mammalian hearts that could contribute to normal homeostasis and the responses to pathological insults. Methods and Results Translating these results, we isolated small c-kit+ cells from 36 of 37 human hearts using primary cell isolation techniques and magnetic cell sorting techniques. The abundance of these cardiac progenitor cells was increased nearly 4-fold in patients with heart failure requiring transplantation compared with nonfailing controls. Polychromatic flow cytometry of primary cell isolates (<30 μm) without antecedent c-kit enrichment confirmed the increased abundance of c-kit+ cells in failing hearts and demonstrated frequent coexpression of CD45 in these cells. Immunocytochemical characterization of freshly isolated, c-kit–enriched human cardiac progenitor cells confirmed frequent coexpression of c-kit and CD45. Primary cardiac progenitor cells formed new human cardiac myocytes at a relatively high frequency after coculture with neonatal rat ventricular myocytes. These contracting new cardiac myocytes exhibited an immature phenotype and frequent electric coupling with the rat myocytes that induced their myogenic differentiation. Conclusions Despite the increased abundance and cardiac myogenic capacity of cardiac progenitor cells in failing human hearts, the need to replace these organs via transplantation implies that adverse features of the local myocardial environment overwhelm endogenous cardiac repair capacity. Developing strategies to improve the success of endogenous cardiac regenerative processes may permit therapeutic myocardial repair without cell delivery per se. PMID:18645055

Health and reproductive profiles of malaria antigen-producing transgenic goats derived by somatic cell nuclear transfer.

PubMed

Behboodi, E; Ayres, S L; Memili, E; O'Coin, M; Chen, L H; Reggio, B C; Landry, A M; Gavin, W G; Meade, H M; Godke, R A; Echelard, Y

2005-01-01

Nuclear transfer (NT) using transfected primary cells is an efficient approach for the generation of transgenic goats. However, reprogramming abnormalities associated with this process might result in compromised animals. We examined the health, reproductive performance, and milk production of four transgenic does derived from somatic cell NT. Goats were derived from two fetal cell lines, each transfected with a transgene expressing a different version of the MSP-1(42) malaria antigen, either glycosylated or non-glycosylated. Two female kids were produced per cell line. Health and growth of these NT animals were monitored and compared with four age-matched control does. There were no differences in birth and weaning weights between NT and control animals. The NT does were bred and produced a total of nine kids. The control does delivered five kids. The NT does expressing the glycosylated antigen lactated only briefly, probably as a result of over-expression of the MSP-1(42) protein. However, NT does expressing the non-glycosylated antigen had normal milk yields and produced the recombinant protein. These data demonstrated that the production of healthy transgenic founder goats by somatic cell NT is readily achievable and that these animals can be used successfully for the production of a candidate Malaria vaccine.

POSTNATAL PHENOTYPE AND LOCALIZATION OF SPINAL CORD V1 DERIVED INTERNEURONS

PubMed Central

Alvarez, Francisco J.; Jonas, Philip C.; Sapir, Tamar; Hartley, Robert; Berrocal, Maria C.; Geiman, Eric J.; Todd, Andrew J.; Goulding, Martyn

2010-01-01

Developmental studies identified four classes (V0, V1, V2, V3) of embryonic interneurons in the ventral spinal cord. Very little however is known about their adult phenotypes. In order to further characterize interneuron cell types in the adult, the location, neurotransmitter phenotype, calcium-buffering protein expression and axon distributions of V1-derived neurons in the mouse spinal cord was determined. In the mature (P20 and older) spinal cord, most V1-derived neurons are located in lateral LVII and in LIX, few in medial LVII and none in LVIII. Approximately 40% express calbindin and/or parvalbumin, while few express calretinin. Of seven groups of ventral interneurons identified according to calcium-buffering protein expression, two groups (1 and 4) correspond with V1-derived neurons. Group 1 are Renshaw cells and intensely express calbindin and coexpress parvalbumin and calretinin. They represent 9% of the V1 population. Group 4 express only parvalbumin and represent 27% of V1-derived neurons. V1-derived group 4 neurons receive contacts from primary sensory afferents and are therefore proprioceptive interneurons and the most ventral neurons in this group receive convergent calbindin-IR Renshaw cell inputs. This subgroup resembles Ia inhibitory interneurons (IaINs) and represents 13% of V1-derived neurons. Adult V1-interneuron axons target LIX and LVII and some enter the deep dorsal horn. V1-axons do not cross the midline. V1 derived axonal varicosities were mostly (>80%) glycinergic and a third were GABAergic. None were glutamatergic or cholinergic. In summary, V1 interneurons develop into ipsilaterally projecting, inhibitory interneurons that include Renshaw cells, Ia inhibitory interneurons and other unidentified proprioceptive interneurons. PMID:16255029

CD30 targeting with brentuximab vedotin: a novel therapeutic approach to primary effusion lymphoma

PubMed Central

Bhatt, Shruti; Ashlock, Brittany M.; Natkunam, Yasodha; Sujoy, Victoria; Chapman, Jennifer Rose; Ramos, Juan Carlos; Mesri, Enrique A.; Lossos, Izidore S.

2013-01-01

Primary effusion lymphoma (PEL) is an aggressive subtype of non-Hodgkin lymphoma characterized by short survival with current therapies, emphasizing the urgent need to develop new therapeutic approaches. Brentuximab vedotin (SGN-35) is an anti-CD30 monoclonal antibody (cAC10) conjugated by a protease-cleavable linker to a microtubule-disrupting agent, monomethyl auristatin E. Brentuximab vedotin is an effective treatment of relapsed CD30-expressing Classical Hodgkin and systemic anaplastic large cell lymphomas. Herein, we demonstrated that PEL cell lines and primary tumors express CD30 and thus may serve as potential targets for brentuximab vedotin therapy. In vitro treatment with brentuximab vedotin decreased cell proliferation, induced cell cycle arrest, and triggered apoptosis of PEL cell lines. Furthermore, in vivo brentuximab vedotin promoted tumor regression and prolonged survival of mice bearing previously reported UM-PEL-1 tumors as well as UM-PEL-3 tumors derived from a newly established and characterized Kaposi’s sarcoma-associated herpesvirus- and Epstein-Barr virus-positive PEL cell line. Overall, our results demonstrate for the first time that brentuximab vedotin may serve as an effective therapy for PEL and provide strong preclinical indications for evaluation of brentuximab vedotin in clinical studies of PEL patients. PMID:23838350

Primary cilia in gastric Gastrointestinal Stromal Tumours (GISTs): an ultrastructural study

PubMed Central

Castiella, Tomás; Muñoz, Guillermo; Luesma, María José; Santander, Sonia; Soriano, Mario; Junquera, Concepción

2013-01-01

Gastrointestinal stromal tumours (GISTs) are the most common mesenchymal (non-epithelial) neoplasms of the human gastrointestinal (GI) tract. They are thought to derive from interstitial cells of Cajal (ICCs) or an ICC progenitor based on immunophenotypical and ultrastructural similarities. Because ICCs show primary cilium, our hypothesis is based on the possibility that some of these neoplastic cells could also present it. To determine this, an exhaustive ultrastructural study has been developed on four gastric GISTs. Previous studies had demonstrated considerable variability in tumour cells with two dominating phenotypes, spindly and epithelioid. In addition to these two types, we have found another cell type reminiscent of adult ICCs with a voluminous nucleus surrounded by narrow perinuclear cytoplasm with long slender cytoplasmic processes. We have also noted the presence of small undifferentiated cells. In this study, we report for the first time the presence of primary cilia (PCs) in spindle and epithelioid tumour cells, an ultrastructural feature we consider of special interest that has hitherto been ignored in the literature dealing with the ultrastructure of GISTs. We also point out the frequent occurrence of multivesicular bodies (MVBs). The ultrastructural findings described in gastric GISTs in this study appear to be relevant considering the critical roles played by PCs and MVBs recently demonstrated in tumourigenic processes. PMID:23672577

Evaluating the Potential of Adipose Tissue-Derived MSCs as Anticancer Gene Delivery Vehicles to Bone-Metastasized Prostate Cancers

DTIC Science & Technology

2011-04-01

JP, Floyd ZE, Zvonic S , Smith A , Gravois A , Reiners E, Wu X, Kilroy G, Lefevre M , Gimble JM. Proteomic analysis of primary cultures of human adipose...In brief, in a 6-week old nu/nu mice (n=5/experiment), C4-2B cells (2 x106) were mixed with Matrigel and injected subcutaneously ( s /c), alone or...cells to TRAIL. Cell Mol Life Sci. 2010;67(8):1307-14. PMID: 20063037. 4: Matuskova M , Hlubinova K, Pastorakova A , Hunakova L, Altanerova V

Initiation and Characterization of Small Cell Lung Cancer Patient-Derived Xenografts from Ultrasound-Guided Transbronchial Needle Aspirates

PubMed Central

Anderson, Wade C.; Boyd, Michael B.; Aguilar, Jorge; Pickell, Brett; Laysang, Amy; Pysz, Marybeth A.; Bheddah, Sheila; Ramoth, Johanna; Slingerland, Brian C.; Dylla, Scott J.; Rubio, Edmundo R.

2015-01-01

Small cell lung cancer (SCLC) is a devastating disease with limited treatment options. Due to its early metastatic nature and rapid growth, surgical resection is rare. Standard of care treatment regimens remain largely unchanged since the 1980’s, and five-year survival lingers near 5%. Patient-derived xenograft (PDX) models have been established for other tumor types, amplifying material for research and serving as models for preclinical experimentation; however, limited availability of primary tissue has curtailed development of these models for SCLC. The objective of this study was to establish PDX models from commonly collected fine needle aspirate biopsies of primary SCLC tumors, and to assess their utility as research models of primary SCLC tumors. These transbronchial needle aspirates efficiently engrafted as xenografts, and tumor histomorphology was similar to primary tumors. Resulting tumors were further characterized by H&E and immunohistochemistry, cryopreserved, and used to propagate tumor-bearing mice for the evaluation of standard of care chemotherapy regimens, to assess their utility as models for tumors in SCLC patients. When treated with Cisplatin and Etoposide, tumor-bearing mice responded similarly to patients from whom the tumors originated. Here, we demonstrate that PDX tumor models can be efficiently established from primary SCLC transbronchial needle aspirates, even after overnight shipping, and that resulting xenograft tumors are similar to matched primary tumors in cancer patients by both histology and chemo-sensitivity. This method enables physicians at non-research institutions to collaboratively contribute to the rapid establishment of extensive PDX collections of SCLC, enabling experimentation with clinically relevant tissues and development of improved therapies for SCLC patients. PMID:25955027

Preparing the “Soil”: The Premetastatic Niche

PubMed Central

Kaplan, Rosandra N.; Rafii, Shahin; Lyden, David

2010-01-01

Current focus on cancer metastasis has centered on the intrinsic factors regulating the cell autonomous homing of the tumor cells to the metastatic site. Specific up-regulation of fibronectin and clustering of bone marrow–derived cellular infiltrates coexpressing matrix metalloproteinases in distant tissue sites before tumor cell arrival are proving to be indispensable for the initial stages of metastasis. These bone marrow–derived hematopoietic progenitors that express vascular endothelial growth factor receptor 1 mobilize in response to the unique array of growth factors produced by the primary tumor. Their arrival in distant sites represents early changes in the local microenvironment, termed the “premetastatic niche,” which dictate the pattern of metastatic spread. Focus on the early cellular and molecular events in cancer dissemination and selectivity will likely lead to new approaches to detect and prevent metastasis at its earliest inception. PMID:17145848

Expression of receptors for atrial natriuretic peptide on the murine bone marrow-derived stromal cells.

PubMed

Agui, T; Yamada, T; Legros, G; Nakajima, T; Clark, M; Peschel, C; Matsumoto, K

1992-05-01

Atrial natriuretic peptide (ANP) receptors were identified on both murine bone marrow-derived stromal cell lines A-3 and ALC and primary cultured cells using [125I]ANP binding assays and Northern blot analyses. The binding of [125I] ANP to the stromal cells was rapid, saturable, and of high affinity. The dissociation constants between ANP and its receptors on these cells showed no difference among cell types, while maximal binding capacity values were different among cell types. Competitive inhibition of [125I]ANP binding with C-atrial natriuretic factor, specific for ANP clearance receptor (ANPR-C), revealed that most of [125I]ANP-binding sites corresponded to ANPR-C. Northern blotting data corroborated that bone marrow-derived stromal cells expressed ANPR-C. However, in ALC cells, ANP biological receptors (either ANPR-A or ANPR-B), the mol wt of which is approximately 130K, were detected, and cGMP was accumulated after stimulation with ANP. On the other hand, in another stromal cell clone, A-3 cells, the expression of biological receptor was not detected in the affinity cross-linking and competitive inhibition experiments using [125I]ANP. However, A-3 cells accumulated cGMP by responding to ANPR-B-specific ligand, C-type natriuretic peptide. These results suggest that ALC cells equally express ANPR-A and ANPR-B, while A-3 cells express ANPR-B dominantly. Although the physiological roles of these receptors in the bone marrow is still not resolved, ANP is expected to play a role in the regulation of stromal cell functions in bone marrow.

Importance of being Nernst: Synaptic activity and functional relevance in stem cell-derived neurons

PubMed Central

Bradford, Aaron B; McNutt, Patrick M

2015-01-01

Functional synaptogenesis and network emergence are signature endpoints of neurogenesis. These behaviors provide higher-order confirmation that biochemical and cellular processes necessary for neurotransmitter release, post-synaptic detection and network propagation of neuronal activity have been properly expressed and coordinated among cells. The development of synaptic neurotransmission can therefore be considered a defining property of neurons. Although dissociated primary neuron cultures readily form functioning synapses and network behaviors in vitro, continuously cultured neurogenic cell lines have historically failed to meet these criteria. Therefore, in vitro-derived neuron models that develop synaptic transmission are critically needed for a wide array of studies, including molecular neuroscience, developmental neurogenesis, disease research and neurotoxicology. Over the last decade, neurons derived from various stem cell lines have shown varying ability to develop into functionally mature neurons. In this review, we will discuss the neurogenic potential of various stem cells populations, addressing strengths and weaknesses of each, with particular attention to the emergence of functional behaviors. We will propose methods to functionally characterize new stem cell-derived neuron (SCN) platforms to improve their reliability as physiological relevant models. Finally, we will review how synaptically active SCNs can be applied to accelerate research in a variety of areas. Ultimately, emphasizing the critical importance of synaptic activity and network responses as a marker of neuronal maturation is anticipated to result in in vitro findings that better translate to efficacious clinical treatments. PMID:26240679

Extracellular cell wall β(1,3)glucan is required to couple septation to actomyosin ring contraction.

PubMed

Muñoz, Javier; Cortés, Juan Carlos G; Sipiczki, Matthias; Ramos, Mariona; Clemente-Ramos, José Angel; Moreno, M Belén; Martins, Ivone M; Pérez, Pilar; Ribas, Juan Carlos

2013-10-28

Cytokinesis has been extensively studied in different models, but the role of the extracellular cell wall is less understood. Here we studied this process in fission yeast. The essential protein Bgs4 synthesizes the main cell wall β(1,3)glucan. We show that Bgs4-derived β(1,3)glucan is required for correct and stable actomyosin ring positioning in the cell middle, before the start of septum formation and anchorage to the cell wall. Consequently, β(1,3)glucan loss generated ring sliding, oblique positioned rings and septa, misdirected septum synthesis indicative of relaxed rings, and uncoupling between a fast ring and membrane ingression and slow septum synthesis, suggesting that cytokinesis can progress with defective septum pushing and/or ring pulling forces. Moreover, Bgs4-derived β(1,3)glucan is essential for secondary septum formation and correct primary septum completion. Therefore, our results show that extracellular β(1,3)glucan is required for cytokinesis to connect the cell wall with the plasma membrane and for contractile ring function, as proposed for the equivalent extracellular matrix in animal cells.

Extracellular cell wall β(1,3)glucan is required to couple septation to actomyosin ring contraction

PubMed Central

Muñoz, Javier; Cortés, Juan Carlos G.; Sipiczki, Matthias; Ramos, Mariona; Clemente-Ramos, José Angel; Moreno, M. Belén; Martins, Ivone M.; Pérez, Pilar

2013-01-01

Cytokinesis has been extensively studied in different models, but the role of the extracellular cell wall is less understood. Here we studied this process in fission yeast. The essential protein Bgs4 synthesizes the main cell wall β(1,3)glucan. We show that Bgs4-derived β(1,3)glucan is required for correct and stable actomyosin ring positioning in the cell middle, before the start of septum formation and anchorage to the cell wall. Consequently, β(1,3)glucan loss generated ring sliding, oblique positioned rings and septa, misdirected septum synthesis indicative of relaxed rings, and uncoupling between a fast ring and membrane ingression and slow septum synthesis, suggesting that cytokinesis can progress with defective septum pushing and/or ring pulling forces. Moreover, Bgs4-derived β(1,3)glucan is essential for secondary septum formation and correct primary septum completion. Therefore, our results show that extracellular β(1,3)glucan is required for cytokinesis to connect the cell wall with the plasma membrane and for contractile ring function, as proposed for the equivalent extracellular matrix in animal cells. PMID:24165938

A method for high purity intestinal epithelial cell culture from adult human and murine tissues for the investigation of innate immune function.

PubMed

Graves, Christina L; Harden, Scott W; LaPato, Melissa; Nelson, Michael; Amador, Byron; Sorenson, Heather; Frazier, Charles J; Wallet, Shannon M

2014-12-01

Intestinal epithelial cells (IECs) serve as an important physiologic barrier between environmental antigens and the host intestinal immune system. Thus, IECs serve as a first line of defense and may act as sentinel cells during inflammatory insults. Despite recent renewed interest in IEC contributions to host immune function, the study of primary IEC has been hindered by lack of a robust culture technique, particularly for small intestinal and adult tissues. Here, a novel adaptation for culture of primary IEC is described for human duodenal organ donor tissue as well as duodenum and colon of adult mice. These epithelial cell cultures display characteristic phenotypes and are of high purity. In addition, the innate immune function of human primary IEC, specifically with regard to Toll-like receptor (TLR) expression and microbial ligand responsiveness, is contrasted with a commonly used intestinal epithelial cell line (HT-29). Specifically, TLR expression at the mRNA level and production of cytokine (IFNγ and TNFα) in response to TLR agonist stimulation is assessed. Differential expression of TLRs as well as innate immune responses to ligand stimulation is observed in human-derived cultures compared to that of HT-29. Thus, use of this adapted method to culture primary epithelial cells from adult human donors and from adult mice will allow for more appropriate studies of IECs as innate immune effectors. Published by Elsevier B.V.

Melanoma exosomes deliver a complex biological payload that upregulates PTPN11 to suppress T lymphocyte function

PubMed Central

Wu, Yueting; Deng, Wentao; McGinley, Emily Chambers; Klinke, David J.

2017-01-01

Summary As exosomes are emerging as a new mode of intercellular communication, we hypothesized that the payload contained within exosomes is shaped by somatic evolution. To test this, we assayed the impact on primary CD8+ T cell function, a key mechanism for anti-tumor immunity, of exosomes derived from three melanoma-related cell lines. While morphologically similar, exosomes from each cell line were functionally different, as B16F0 exosomes dose-dependently suppressed T cell proliferation. In contrast, Cloudman S91 exosomes promoted T cell proliferation and Melan-A exosomes had a negligible effect on primary CD8+ T cells. Mechanistically, transcript profiling suggested that exosomal mRNA is enriched for full-length mRNAs that target immune-related pathways. Interestingly, B16F0 exosomes were unique in that they contained both protein and mRNA for Ptpn11, which inhibited T cell proliferation. Collectively, the results suggest that upregulation of PTPN11 by B16F0 exosomes to tumor infiltrating lymphocytes would bypass the extracellular control of the immune checkpoints. PMID:27930879

Attenuated expression of interferon-β and interferon-λ1 by human alternatively activated macrophages.

PubMed

El Fiky, Ashraf; Perreault, Roger; McGinnis, Gwendolyn J; Rabin, Ronald L

2013-12-01

Macrophages can be polarized into classically (CAM) or alternatively (AAM) activated macrophages with IFN-γ or IL-4, respectively. CAM are associated with type 1 immune responses and are implicated in autoimmunity; AAM are associated with type 2 responses and are implicated in allergic diseases. An impediment in investigating macrophage biology using primary human monocyte derived macrophages is the wide inter-donor heterogeneity and the limited quantity of cells that survive in vitro polarization. To overcome this impediment, we established a protocol to generate CAM and AAM cultures derived from the THP-1 human promonocytic cell line. In this report, we demonstrate that THP-CAM and -AAM express gene and protein markers that define their primary human monocyte derived counterparts, such as IL-1β, CXCL10, and CXCL11 for CAM, and MRC1, IL-4 and CCL22 for AAM. In addition, we demonstrate that STAT6 is selectively activated in THP-AAM which, upon LPS stimulation, have an attenuated or delayed expression of IFN-β, IFN-λ1, and IFN α/β pathway genes compared to their CAM counterparts. Taken together, these findings may help further investigate human diseases associated with the alternatively activated macrophage phenotype using this reproducible in vitro macrophage model. Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.

Prostate derived Ets transcription factor and Carcinoembryonic antigen related cell adhesion molecule 6 constitute a highly active oncogenic axis in breast cancer

PubMed Central

Mukhopadhyay, Alka; Khoury, Thaer; Stein, Leighton; Shrikant, Protul; Sood, Ashwani K

2013-01-01

We previously reported overexpression of Prostate derived Ets transcriptionfactor (PDEF) in breast cancer and its role in breast cancer progression, supportingPDEF as an attractive target in this cancer. The goal of this research was to identifyspecific PDEF induced molecules that, like PDEF, show overexpression in breast tumorsand a role in breast tumor progression. PDEF expression was down regulated byshRNA in MCF-7 human breast tumor cell line, and probes from PDEF down-regulatedand control MCF-7 cells were used to screen the HG-U133A human gene chips. Theseanalyses identified 1318 genes that were induced two-fold or higher by PDEF in MCF-7 cells. Further analysis of three of these genes, namely CEACAM6, S100A7 and B7-H4, in relation to PDEF in primary breast tumors showed that in 82% of ER+, 67%of Her2 overexpressing and 24% of triple-negative breast tumors both PDEF andCEACAM6 expression was elevated 10-fold or higher in comparison to normal breasttissue. Overall, 72% (94 of 131) of the primary breast tumors showed 10-fold orhigher expression of both PDEF and CEACAM6. In contrast, S100A7 and B7-H4 failedto show concordant elevated expression with PDEF in primary tumors. To determinethe significance of elevated PDEF and CEACAM6 expression to tumor phenotype, theirexpression was down regulated by specific siRNAs in human breast tumor cell lines. This resulted in the loss of viability of tumor cells in vitro, supporting an oncogenicrole for both PDEF and CEACAM6 in breast cancer. Together, these findings show thatPDEF-CEACAM6 is a highly active oncogenic axis in breast cancer and suggest thattargeting of these molecules should provide novel treatments for most breast cancerpatients. PMID:23592399

Basic fibroblast growth factor (bFGF) facilitates differentiation of adult dorsal root ganglia-derived neural stem cells toward Schwann cells by binding to FGFR-1 through MAPK/ERK activation.

PubMed

Gu, Yun; Xue, Chenbin; Zhu, Jianbin; Sun, Hualin; Ding, Fei; Cao, Zheng; Gu, Xiaosong

2014-04-01

Considerable research has been devoted to unraveling the regulation of neural stem cell (NSC) differentiation. The responses of NSCs to various differentiation-inducing stimuli, however, are still difficult to estimate. In this study, we aimed to search for a potent growth factor that was able to effectively induce differentiation of NSCs toward Schwann cells. NSCs were isolated from dorsal root ganglia (DRGs) of adult rats and identified by immunostaining. Three different growth factors were used to stimulate the differentiation of DRG-derived NSCs (DRG-NSCs). We found that among these three growth factors, bFGF was the strongest inducer for the glial differentiation of DRG-NSCs, and bFGF induced the generation of an increased number of Schwann cell-like cells as compared to nerve growth factor (NGF) and neuregulin1-β (NRG). These Schwann cell-like cells demonstrated the same characteristics as those of primary Schwann cells. Furthermore, we noted that bFGF-induced differentiation of DRG-NSCs toward Schwann cells might be mediated by binding to fibroblast growth factor receptor-1 (FGFR-1) through activation of MAPK/ERK signal pathway.

Isolation and characterization of human mesenchymal stem cells derived from synovial fluid by magnetic-activated cell sorting (MACS).

PubMed

Jia, Zhaofeng; Liang, Yujie; Xu, Xiao; Li, Xingfu; Liu, Qisong; Ou, Yangkan; Duan, Li; Zhu, Weimin; Lu, Wei; Xiong, Jianyi; Wang, Daping

2018-03-01

Mesenchymal stem cells (MSCs) are the primary source of cells used for cell-based therapy in tissue engineering. MSCs are found in synovial fluid, a source that could be conveniently used for cartilage tissue engineering. However, the purification and characterization of SF-MSCs has been poorly documented in the literature. Here, we outline an easy-to-perform approach for the isolation and culture of MSCs derived from human synovial fluid (hSF-MSCs). We have successfully purified hSF-MSCs using magnetic-activated cell sorting (MACS) using the MSC surface marker, CD90. Purified SF-MSCs demonstrate significant renewal capacity following several passages in culture. Furthermore, we demonstrated that MACS-sorted CD90 + cells could differentiated into osteoblasts, adipocytes, and chondrocytes in vitro. In addition, we show that these cells can generate cartilage tissue in micromass culture as well. This study demonstrates that MACS is a useful tool that can be used for the purification of hSF-MSCs from synovial fluid. The proliferation properties and ability to differentiate into chondrocytes make these hSF-MSCs a promising source of stem cells for applications in cartilage repair. © 2017 International Federation for Cell Biology.

Vascular Smooth Muscle Cells From Hypertensive Patient-Derived Induced Pluripotent Stem Cells to Advance Hypertension Pharmacogenomics.

PubMed

Biel, Nikolett M; Santostefano, Katherine E; DiVita, Bayli B; El Rouby, Nihal; Carrasquilla, Santiago D; Simmons, Chelsey; Nakanishi, Mahito; Cooper-DeHoff, Rhonda M; Johnson, Julie A; Terada, Naohiro

2015-12-01

Studies in hypertension (HTN) pharmacogenomics seek to identify genetic sources of variable antihypertensive drug response. Genetic association studies have detected single-nucleotide polymorphisms (SNPs) that link to drug responses; however, to understand mechanisms underlying how genetic traits alter drug responses, a biological interface is needed. Patient-derived induced pluripotent stem cells (iPSCs) provide a potential source for studying otherwise inaccessible tissues that may be important to antihypertensive drug response. The present study established multiple iPSC lines from an HTN pharmacogenomics cohort. We demonstrated that established HTN iPSCs can robustly and reproducibly differentiate into functional vascular smooth muscle cells (VSMCs), a cell type most relevant to vasculature tone control. Moreover, a sensitive traction force microscopy assay demonstrated that iPSC-derived VSMCs show a quantitative contractile response on physiological stimulus of endothelin-1. Furthermore, the inflammatory chemokine tumor necrosis factor α induced a typical VSMC response in iPSC-derived VSMCs. These studies pave the way for a large research initiative to decode biological significance of identified SNPs in hypertension pharmacogenomics. Treatment of hypertension remains suboptimal, and a pharmacogenomics approach seeks to identify genetic biomarkers that could be used to guide treatment decisions; however, it is important to understand the biological underpinnings of genetic associations. Mouse models do not accurately recapitulate individual patient responses based on their genetics, and hypertension-relevant cells are difficult to obtain from patients. Induced pluripotent stem cell (iPSC) technology provides a great interface to bring patient cells with their genomic data into the laboratory and to study hypertensive responses. As an initial step, the present study established an iPSC bank from patients with primary hypertension and demonstrated an effective and reproducible method of generating functional vascular smooth muscle cells. ©AlphaMed Press.

Age of the Donor Reduces the Ability of Human Adipose-Derived Stem Cells to Alleviate Symptoms in the Experimental Autoimmune Encephalomyelitis Mouse Model

PubMed Central

Scruggs, Brittni A.; Semon, Julie A.; Zhang, Xiujuan; Zhang, Shijia; Bowles, Annie C.; Pandey, Amitabh C.; Imhof, Kathleen M.P.; Kalueff, Allan V.; Gimble, Jeffrey M.

2013-01-01

There is a significant clinical need for effective therapies for primary progressive multiple sclerosis, which presents later in life (i.e., older than 50 years) and has symptoms that increase in severity without remission. With autologous mesenchymal stem cell therapy now in the early phases of clinical trials for all forms of multiple sclerosis (MS), it is necessary to determine whether autologous stem cells from older donors have therapeutic effectiveness. In this study, the therapeutic efficacy of human adipose-derived mesenchymal stem cells (ASCs) from older donors was directly compared with that of cells from younger donors for disease prevention. Mice were induced with chronic experimental autoimmune encephalomyelitis (EAE) using the myelin oligodendrocyte glycoprotein35–55 peptide and treated before disease onset with ASCs derived from younger (<35 years) or older (>60 years) donors. ASCs from older donors failed to ameliorate the neurodegeneration associated with EAE, and mice treated with older donor cells had increased central nervous system inflammation, demyelination, and splenocyte proliferation in vitro compared with the mice receiving cells from younger donors. Therefore, the results of this study demonstrated that donor age significantly affects the ability of human ASCs to provide neuroprotection, immunomodulation, and/or remyelination in EAE mice. The age-related therapeutic differences corroborate recent findings that biologic aging occurs in stem cells, and the differences are supported by evidence in this study that older ASCs, compared with younger donor cells, secrete less hepatocyte growth factor and other bioactive molecules when stimulated in vitro. These results highlight the need for evaluation of autologous ASCs derived from older patients when used as therapy for MS. PMID:24018793

Very late antigen-5 facilitates stromal progenitor cell differentiation into myofibroblast.

PubMed

Sen, Namita; Weingarten, Mark; Peter, Yakov

2014-11-01

Fibrotic disease is associated with abrogated stromal cell proliferation and activity. The precise identity of the cells that drive fibrosis remains obscure, in part because of a lack of information on their lineage development. To investigate the role of an early stromal progenitor cell (SPC) on the fibrotic process, we selected for, and monitored the stages of, fibroblast development from a previously reported free-floating anchorage-independent cell (AIC) progenitor population. Our findings demonstrate that organotypic pulmonary, cardiac, and renal fibroblast commitment follows a two-step process of attachment and remodeling in culture. Cell differentiation was confirmed by the inability of SPCs to revert to the free-floating state and functional mesenchymal stem/stromal cell (MSC) differentiation into osteoblast, adipocyte, chondrocyte, and fibroblastic lineages. The myofibroblastic phenotype was reflected by actin stress-fiber formation, α-smooth muscle production, and a greater than threefold increase in proliferative activity compared with that of the progenitors. SPC-derived pulmonary myofibroblasts demonstrated a more than 300-fold increase in fibronectin-1 (Fn1), collagen, type 1, α1, integrin α-5 (Itga5), and integrin β-1 (Itgb1) transcript levels. Very late antigen-5 (ITGA5/ITGB1) protein cluster formations were also prevalent on the differentiated cells. Normalized SPC-derived myofibroblast expression patterns reflected those of primary cultured lung myofibroblasts. Intratracheal implantation of pulmonary AICs into recipient mouse lungs resulted in donor cell FN1 production and evidence of epithelial derivation. SPC derivation into stromal tissue in vitro and in vivo and the observation that MSC and fibroblast lineages share a common ancestor could potentially lead to personalized antifibrotic therapies. ©AlphaMed Press.

Importance of Being Nernst: Synaptic Activity and Functional Relevance in Stem Cell-derived Neurons

DTIC Science & Technology

2015-07-26

neurodevelopmental stages. In some cases these factors can be controlled very precisely, such as by the addition of small molecules to promote exit from...neurogenesis[43]. These include markers of the different stages of neurodevelopment , starting from a stem cell state and expressing characteristics of a...neuroligin-3 mutations associated with autism cause post-synaptic dysfunction in iNs when co-cultured with primary neurons[163]. The iN field is still

Endothelial trans-differentiation in glioblastoma recurring after radiotherapy.

PubMed

De Pascalis, Ivana; Morgante, Liliana; Pacioni, Simone; D'Alessandris, Quintino Giorgio; Giannetti, Stefano; Martini, Maurizio; Ricci-Vitiani, Lucia; Malinverno, Matteo; Dejana, Elisabetta; Larocca, Luigi M; Pallini, Roberto

2018-04-30

We hypothesized that in glioblastoma recurring after radiotherapy, a condition whereby the brain endothelium undergoes radiation-induced senescence, tumor cells with endothelial phenotype may be relevant for tumor neovascularization. Matched glioblastoma samples obtained at primary surgery and at surgery for tumor recurrence after radiotherapy, all expressing epidermal growth factor receptor variant III (EGFRvIII), were assessed by a technique that combines fluorescent in situ hybridization (FISH) for the EGFR/CEP7 chromosomal probe with immunostaining for endothelial cells (CD31) and activated pericytes (α Smooth Muscle Actin). Five EGFRvIII-expressing paired primary/recurrent glioblastoma samples, in which the tumor cells showed EGFR/CEP7 amplification, were then assessed by CD31 and α Smooth Muscle Actin immunofluorescence. In glomeruloid bodies, the ratio between CD31+ cells with amplified EGFR/CEP7 signal and the total CD31+ cells was 0.23 ± 0.09 (mean ± sem) and 0.63 ± 0.07 in primary tumors and in recurrent ones, respectively (p < 0.002, Student-t test). In capillaries, the ratio of CD31+ cells with amplified EGFR/CEP7 over the total CD31+ cells lining the capillary lumen was 0.21 ± 0.06 (mean ± sem) and 0.42 ± 0.07 at primary surgery and at recurrence, respectively (p < 0.005, Student-t test). Expression of α Smooth Muscle Actin by cells with EGFR/CEP7 amplification was not observed. Then, in glioblastoma recurring after radiotherapy, where the brain endothelium suffers from radiation-induced cell senescence, tumor-derived endothelium plays a role in neo-vascularization.

Strategies for immortalization of primary hepatocytes

PubMed Central

Eva, Ramboer; Bram, De Craene; Joery, De Kock; Tamara, Vanhaecke; Geert, Berx; Vera, Rogiers; Mathieu, Vinken

2014-01-01

The liver has the unique capacity to regenerate in response to a damaging event. Liver regeneration is hereby largely driven by hepatocyte proliferation, which in turn relies on cell cycling. The hepatocyte cell cycle is a complex process that is tightly regulated by several well-established mechanisms. In vitro, isolated hepatocytes do not longer retain this proliferative capacity. However, in vitro cell growth can be boosted by immortalization of hepatocytes. Well-defined immortalization genes can be artificially overexpressed in hepatocytes or the cells can be conditionally immortalized leading to controlled cell proliferation. This paper discusses the current immortalization techniques and provides a state-of-the-art overview of the actually available immortalized hepatocyte-derived cell lines and their applications. PMID:24911463

Dual HER2 targeting impedes growth of HER2 gene-amplified uterine serous carcinoma xenografts.

PubMed

Groeneweg, Jolijn W; Hernandez, Silvia F; Byron, Virginia F; DiGloria, Celeste M; Lopez, Hector; Scialabba, Vanessa; Kim, Minji; Zhang, Ling; Borger, Darrell R; Tambouret, Rosemary; Foster, Rosemary; Rueda, Bo R; Growdon, Whitfield B

2014-12-15

Uterine serous carcinoma (USC) is an aggressive subtype of endometrial cancer that commonly harbors HER2 gene amplification. We investigated the effectiveness of HER2 inhibition using lapatinib and trastuzumab in vitro and in xenografts derived from USC cell lines and USC patient-derived xenografts. Immunohistochemistry and FISH were performed to assess HER2 expression in 42 primary USC specimens. ARK1, ARK2, and SPEC2 cell lines were treated with trastuzumab or lapatinib. Cohorts of mice harboring xenografts derived from ARK2 and SPEC2 cell lines and EnCa1 and EnCa2 primary human USC samples were treated with either vehicle, trastuzumab, lapatinib, or the combination of trastuzumab and lapatinib. Acute and chronic posttreatment tumor samples were assessed for downstream signaling alterations and examined for apoptosis and proliferation. HER2 gene amplification (24%) correlated significantly with HER2 protein overexpression (55%). All models were impervious to single-agent trastuzumab treatment. Lapatinib decreased in vitro proliferation of all cell lines and in vivo growth of HER2-amplified xenografts (ARK2, EnCa1). In addition, dual therapy with trastuzumab and lapatinib resulted in significant antitumor activity only in ARK2 and EnCa1 tumors. Dual HER2 therapy induced on target alteration of downstream MAPK and PI3K pathway mediators only in HER2-amplified models, and was associated with increased apoptosis and decreased proliferation. Although trastuzumab alone did not impact USC growth, dual anti-HER2 therapy with lapatinib led to improved inhibition of tumor growth in HER2-amplified USC and may be a promising avenue for future investigation. ©2014 American Association for Cancer Research.

Kc167, a widely used Drosophila cell line, contains an active primary piRNA pathway.

PubMed

Vrettos, Nicholas; Maragkakis, Manolis; Alexiou, Panagiotis; Mourelatos, Zissimos

2017-01-01

PIWI family proteins bind to small RNAs known as PIWI-interacting RNAs (piRNAs) and play essential roles in the germline by silencing transposons and by promoting germ cell specification and function. Here we report that the widely used Kc167 cell line, derived from Drosophila melanogaster embryos, expresses piRNAs that are loaded to Aub and Piwi. Kc167 piRNAs are produced by a canonical, primary piRNA biogenesis pathway, from phased processing of precursor transcripts by the Zuc endonuclease, Armi helicase, and dGasz mitochondrial scaffold protein. Kc167 piRNAs derive from cytoplasmic transcripts, notably tRNAs and mRNAs, and their abundance correlates with that of parent transcripts. The expression of Aub is robust in Kc167, that of Piwi is modest, while Ago3 is undetectable, explaining the lack of transposon-related piRNA amplification by the Aub-Ago3, ping-pong mechanism. We propose that the default state of the primary piRNA biogenesis machinery is random transcript sampling to allow generation of piRNAs from any transcript, including newly acquired retrotransposons. This state is unmasked in Kc167, likely because they do not express piRNA cluster transcripts in sufficient amounts and do not amplify transposon piRNAs. We use Kc167 to characterize an inactive isoform of Aub protein. Since most Kc167 piRNAs are genic, they can be mapped uniquely to the genome, facilitating computational analyses. Furthermore, because Kc167 is a widely used and well-characterized cell line that is easily amenable to experimental manipulations, we expect that it will serve as an excellent system to study piRNA biogenesis and piRNA-related factors. © 2016 Vrettos et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Characterizing the efficacy of cancer therapeutics in patient-derived xenograft models of metastatic breast cancer.

PubMed

Turner, Tia H; Alzubi, Mohammad A; Sohal, Sahib S; Olex, Amy L; Dozmorov, Mikhail G; Harrell, J Chuck

2018-03-12

Basal-like breast cancers are aggressive and often metastasize to vital organs. Treatment is largely limited to chemotherapy. This study aims to characterize the efficacy of cancer therapeutics in vitro and in vivo within the primary tumor and metastatic setting, using patient-derived xenograft (PDX) models. We employed two basal-like, triple-negative PDX models, WHIM2 and WHIM30. PDX cells, obtained from mammary tumors grown in mice, were treated with twelve cancer therapeutics to evaluate their cytotoxicity in vitro. Four of the effective drugs-carboplatin, cyclophosphamide, bortezomib, and dacarbazine-were tested in vivo for their efficacy in treating mammary tumors, and metastases generated by intracardiac injection of tumor cells. RNA sequencing showed that global gene expression of PDX cells grown in the mammary gland was similar to those tested in culture. In vitro, carboplatin was cytotoxic to WHIM30 but not WHIM2, whereas bortezomib, dacarbazine, and cyclophosphamide were cytotoxic to both lines. Yet, these drugs were ineffective in treating both primary and metastatic WHIM2 tumors in vivo. Carboplatin and cyclophosphamide were effective in treating WHIM30 mammary tumors and reducing metastatic burden in the brain, liver, and lungs. WHIM2 and WHIM30 metastases showed distinct patterns of cytokeratin and vimentin expression, regardless of treatment, suggesting that different tumor cell subpopulations may preferentially seed in different organs. This study highlights the utility of PDX models for studying the efficacy of therapeutics in reducing metastatic burden in specific organs. The differential treatment responses between two PDX models of the same intrinsic subtype, in both the primary and metastatic setting, recapitulates the challenges faced in treating cancer patients and highlights the need for combination therapies and predictive biomarkers.

Cyclin-dependent kinase 9 is a novel specific molecular target in adult T-cell leukemia/lymphoma.

PubMed

Narita, Tomoko; Ishida, Takashi; Ito, Asahi; Masaki, Ayako; Kinoshita, Shiori; Suzuki, Susumu; Takino, Hisashi; Yoshida, Takashi; Ri, Masaki; Kusumoto, Shigeru; Komatsu, Hirokazu; Imada, Kazunori; Tanaka, Yuetsu; Takaori-Kondo, Akifumi; Inagaki, Hiroshi; Scholz, Arne; Lienau, Philip; Kuroda, Taruho; Ueda, Ryuzo; Iida, Shinsuke

2017-08-31

Cyclin-dependent kinase 9 (CDK9), a subunit of the positive transcription elongation factor b (P-TEFb) complex, regulates gene transcription elongation by phosphorylating the C-terminal domain (CTD) of RNA polymerase II (RNAPII). The deregulation of CDK9/P-TEFb has important implications for many cancer types. BAY 1143572 is a novel and highly selective CDK9/P-TEFb inhibitor currently being investigated in phase 1 studies. We evaluated the therapeutic potential of BAY 1143572 in adult T-cell leukemia/lymphoma (ATL). As a result of CDK9 inhibition and subsequent inhibition of phosphorylation at serine 2 of the RNAPII CTD, BAY 1143572 decreased c-Myc and Mcl-1 levels in ATL-derived or human T-cell lymphotropic virus type-1 (HTLV-1)-transformed lines and primary ATL cells tested, leading to their growth inhibition and apoptosis. Median inhibitory concentrations for BAY 1143572 in ATL-derived or HTLV-1-transformed lines (n = 8), primary ATL cells (n = 11), and CD4 + cells from healthy volunteers (n = 5) were 0.535, 0.30, and 0.36 μM, respectively. Next, NOG mice were used as recipients of tumor cells from an ATL patient. BAY 1143572-treated ATL-bearing mice (once daily 12.5 mg/kg oral application) demonstrated significantly decreased ATL cell infiltration of the liver and bone marrow, as well as decreased human soluble interleukin-2 receptor levels in serum (reflecting the ATL tumor burden), compared with untreated mice (n = 8 for both). BAY 1143572-treated ATL-bearing mice demonstrated significantly prolonged survival compared with untreated ATL-bearing mice (n = 7 for both). Collectively, this study indicates that BAY 1143572 showed strong potential as a novel treatment of ATL. © 2017 by The American Society of Hematology.

Aronia melanocarpa juice induces a redox-sensitive p73-related caspase 3-dependent apoptosis in human leukemia cells.

PubMed

Sharif, Tanveer; Alhosin, Mahmoud; Auger, Cyril; Minker, Carole; Kim, Jong-Hun; Etienne-Selloum, Nelly; Bories, Pierre; Gronemeyer, Hinrich; Lobstein, Annelise; Bronner, Christian; Fuhrmann, Guy; Schini-Kerth, Valérie B

2012-01-01

Polyphenols are natural compounds widely present in fruits and vegetables, which have antimutagenic and anticancer properties. The aim of the present study was to determine the anticancer effect of a polyphenol-rich Aronia melanocarpa juice (AMJ) containing 7.15 g/L of polyphenols in the acute lymphoblastic leukemia Jurkat cell line, and, if so, to clarify the underlying mechanism and to identify the active polyphenols involved. AMJ inhibited cell proliferation, which was associated with cell cycle arrest in G(2)/M phase, and caused the induction of apoptosis. These effects were associated with an upregulation of the expression of tumor suppressor p73 and active caspase 3, and a downregulation of the expression of cyclin B1 and the epigenetic integrator UHRF1. AMJ significantly increased the formation of reactive oxygen species (ROS), decreased the mitochondrial membrane potential and caused the release of cytochrome c into the cytoplasm. Treatment with intracellular ROS scavengers prevented the AMJ-induced apoptosis and upregulation of the expression of p73 and active caspase 3. The fractionation of the AMJ and the use of identified isolated compounds indicated that the anticancer activity was associated predominantly with chlorogenic acids, some cyanidin glycosides, and derivatives of quercetin. AMJ treatment also induced apoptosis of different human lymphoblastic leukemia cells (HSB-2, Molt-4 and CCRF-CEM). In addition, AMJ exerted a strong pro-apoptotic effect in human primary lymphoblastic leukemia cells but not in human normal primary T-lymphocytes. Thus, the present findings indicate that AMJ exhibits strong anticancer activity through a redox-sensitive mechanism in the p53-deficient Jurkat cells and that this effect involves several types of polyphenols. They further suggest that AMJ has chemotherapeutic properties against acute lymphoblastic leukemia by selectively targeting lymphoblast-derived tumor cells.

Recombinant TAT-BMI-1 fusion protein induces ex vivo expansion of human umbilical cord blood-derived hematopoietic stem cells.

PubMed

Codispoti, Bruna; Rinaldo, Nicola; Chiarella, Emanuela; Lupia, Michela; Spoleti, Cristina Barbara; Marafioti, Maria Grazia; Aloisio, Annamaria; Scicchitano, Stefania; Giordano, Marco; Nappo, Giovanna; Lucchino, Valeria; Moore, Malcolm A S; Zhou, Pengbo; Mesuraca, Maria; Bond, Heather Mandy; Morrone, Giovanni

2017-07-04

Transplantation of hematopoietic stem cells (HSCs) is a well-established therapeutic approach for numerous disorders. HSCs are typically derived from bone marrow or peripheral blood after cytokine-induced mobilization. Umbilical cord blood (CB) represents an appealing alternative HSC source, but the small amounts of the individual CB units have limited its applications. The availability of strategies for safe ex vivo expansion of CB-derived HSCs (CB-HSCs) may allow to extend the use of these cells in adult patients and to avoid the risk of insufficient engraftment or delayed hematopoietic recovery.Here we describe a system for the ex vivo expansion of CB-HSCs based on their transient exposure to a recombinant TAT-BMI-1 chimeric protein. BMI-1 belongs to the Polycomb family of epigenetic modifiers and is recognized as a central regulator of HSC self-renewal. Recombinant TAT-BMI-1 produced in bacteria was able to enter the target cells via the HIV TAT-derived protein transduction peptide covalently attached to BMI-1, and conserved its biological activity. Treatment of CB-CD34+ cells for 3 days with repeated addition of 10 nM purified TAT-BMI-1 significantly enhanced total cell expansion as well as that of primitive hematopoietic progenitors in culture. Importantly, TAT-BMI-1-treated CB-CD34+ cells displayed a consistently higher rate of multi-lineage long-term repopulating activity in primary and secondary xenotransplants in immunocompromised mice. Thus, recombinant TAT-BMI-1 may represent a novel, effective reagent for ex vivo expansion of CB-HSC for therapeutic purposes.

Modulating glioma-mediated myeloid-derived suppressor cell development with sulforaphane

PubMed Central

Kumar, Ravi; de Mooij, Tristan; Peterson, Timothy E.; Kaptzan, Tatiana; Johnson, Aaron J.; Daniels, David J.; Parney, Ian F.

2017-01-01

Glioblastoma is the most common primary tumor of the brain and has few long-term survivors. The local and systemic immunosuppressive environment created by glioblastoma allows it to evade immunosurveillance. Myeloid-derived suppressor cells (MDSCs) are a critical component of this immunosuppression. Understanding mechanisms of MDSC formation and function are key to developing effective immunotherapies. In this study, we developed a novel model to reliably generate human MDSCs from healthy-donor CD14+ monocytes by culture in human glioma-conditioned media. Monocytic MDSC frequency was assessed by flow cytometry and confocal microscopy. The resulting MDSCs robustly inhibited T cell proliferation. A cytokine array identified multiple components of the GCM potentially contributing to MDSC generation, including Monocyte Chemoattractive Protein-1, interleukin-6, interleukin-8, and Macrophage Migration Inhibitory Factor (MIF). Of these, Macrophage Migration Inhibitory Factor is a particularly attractive therapeutic target as sulforaphane, a naturally occurring MIF inhibitor derived from broccoli sprouts, has excellent oral bioavailability. Sulforaphane inhibits the transformation of normal monocytes to MDSCs by glioma-conditioned media in vitro at pharmacologically relevant concentrations that are non-toxic to normal leukocytes. This is associated with a corresponding increase in mature dendritic cells. Interestingly, sulforaphane treatment had similar pro-inflammatory effects on normal monocytes in fresh media but specifically increased immature dendritic cells. Thus, we have used a simple in vitro model system to identify a novel contributor to glioblastoma immunosuppression for which a natural inhibitor exists that increases mature dendritic cell development at the expense of myeloid-derived suppressor cells when normal monocytes are exposed to glioma conditioned media. PMID:28666020

Hypoxia-cultured human adipose-derived mesenchymal stem cells are non-oncogenic and have enhanced viability, motility, and tropism to brain cancer

PubMed Central

Feng, Y; Zhu, M; Dangelmajer, S; Lee, Y M; Wijesekera, O; Castellanos, C X; Denduluri, A; Chaichana, K L; Li, Q; Zhang, H; Levchenko, A; Guerrero-Cazares, H; Quiñones-Hinojosa, A

2014-01-01

Adult human adipose-derived mesenchymal stem cells (hAMSCs) are multipotent cells, which are abundant, easily collected, and bypass the ethical concerns that plague embryonic stem cells. Their utility and accessibility have led to the rapid development of clinical investigations to explore their autologous and allogeneic cellular-based regenerative potential, tissue preservation capabilities, anti-inflammatory properties, and anticancer properties, among others. hAMSCs are typically cultured under ambient conditions with 21% oxygen. However, physiologically, hAMSCs exist in an environment of much lower oxygen tension. Furthermore, hAMSCs cultured in standard conditions have shown limited proliferative and migratory capabilities, as well as limited viability. This study investigated the effects hypoxic culture conditions have on primary intraoperatively derived hAMSCs. hAMSCs cultured under hypoxia (hAMSCs-H) remained multipotent, capable of differentiation into osteogenic, chondrogenic, and adipogenic lineages. In addition, hAMSCs-H grew faster and exhibited less cell death. Furthermore, hAMSCs-H had greater motility than normoxia-cultured hAMSCs and exhibited greater homing ability to glioblastoma (GBM) derived from brain tumor-initiating cells from our patients in vitro and in vivo. Importantly, hAMSCs-H did not transform into tumor-associated fibroblasts in vitro and were not tumorigenic in vivo. Rather, hAMSCs-H promoted the differentiation of brain cancer cells in vitro and in vivo. These findings suggest an alternative culturing technique that can enhance the function of hAMSCs, which may be necessary for their use in the treatment of various pathologies including stroke, myocardial infarction, amyotrophic lateral sclerosis, and GBM. PMID:25501828

Recombinant TAT-BMI-1 fusion protein induces ex vivo expansion of human umbilical cord blood-derived hematopoietic stem cells

PubMed Central

Codispoti, Bruna; Rinaldo, Nicola; Chiarella, Emanuela; Lupia, Michela; Spoleti, Cristina Barbara; Marafioti, Maria Grazia; Aloisio, Annamaria; Scicchitano, Stefania; Giordano, Marco; Nappo, Giovanna; Lucchino, Valeria; Moore, Malcolm A.S.; Zhou, Pengbo; Mesuraca, Maria

2017-01-01

Transplantation of hematopoietic stem cells (HSCs) is a well-established therapeutic approach for numerous disorders. HSCs are typically derived from bone marrow or peripheral blood after cytokine-induced mobilization. Umbilical cord blood (CB) represents an appealing alternative HSC source, but the small amounts of the individual CB units have limited its applications. The availability of strategies for safe ex vivo expansion of CB-derived HSCs (CB-HSCs) may allow to extend the use of these cells in adult patients and to avoid the risk of insufficient engraftment or delayed hematopoietic recovery. Here we describe a system for the ex vivo expansion of CB-HSCs based on their transient exposure to a recombinant TAT-BMI-1 chimeric protein. BMI-1 belongs to the Polycomb family of epigenetic modifiers and is recognized as a central regulator of HSC self-renewal. Recombinant TAT-BMI-1 produced in bacteria was able to enter the target cells via the HIV TAT-derived protein transduction peptide covalently attached to BMI-1, and conserved its biological activity. Treatment of CB-CD34+ cells for 3 days with repeated addition of 10 nM purified TAT-BMI-1 significantly enhanced total cell expansion as well as that of primitive hematopoietic progenitors in culture. Importantly, TAT-BMI-1-treated CB-CD34+ cells displayed a consistently higher rate of multi-lineage long-term repopulating activity in primary and secondary xenotransplants in immunocompromised mice. Thus, recombinant TAT-BMI-1 may represent a novel, effective reagent for ex vivo expansion of CB-HSC for therapeutic purposes. PMID:28187462

Neutralizing Activity of Broadly Neutralizing Anti-HIV-1 Antibodies against Clade B Clinical Isolates Produced in Peripheral Blood Mononuclear Cells.

PubMed

Cohen, Yehuda Z; Lorenzi, Julio C C; Seaman, Michael S; Nogueira, Lilian; Schoofs, Till; Krassnig, Lisa; Butler, Allison; Millard, Katrina; Fitzsimons, Tomas; Daniell, Xiaoju; Dizon, Juan P; Shimeliovich, Irina; Montefiori, David C; Caskey, Marina; Nussenzweig, Michel C

2018-03-01

Recently discovered broadly neutralizing antibodies (bNAbs) against HIV-1 demonstrate extensive breadth and potency against diverse HIV-1 strains and represent a promising approach for the treatment and prevention of HIV-1 infection. The breadth and potency of these antibodies have primarily been evaluated by using panels of HIV-1 Env-pseudotyped viruses produced in 293T cells expressing molecularly cloned Env proteins. Here we report on the ability of five bNAbs currently in clinical development to neutralize circulating primary HIV-1 isolates derived from peripheral blood mononuclear cells (PBMCs) and compare the results to those obtained with the pseudovirus panels used to characterize the bNAbs. The five bNAbs demonstrated significantly less breadth and potency against clinical isolates produced in PBMCs than against Env-pseudotyped viruses. The magnitude of this difference in neutralizing activity varied, depending on the antibody epitope. Glycan-targeting antibodies showed differences of only 3- to 4-fold, while antibody 10E8, which targets the membrane-proximal external region, showed a nearly 100-fold decrease in activity between published Env-pseudotyped virus panels and PBMC-derived primary isolates. Utilizing clonal PBMC-derived primary isolates and molecular clones, we determined that the observed discrepancy in bNAb performance is due to the increased sensitivity to neutralization exhibited by 293T-produced Env-pseudotyped viruses. We also found that while full-length molecularly cloned viruses produced in 293T cells exhibit greater sensitivity to neutralization than PBMC-derived viruses do, Env-pseudotyped viruses produced in 293T cells generally exhibit even greater sensitivity to neutralization. As the clinical development of bNAbs progresses, it will be critical to determine the relevance of each of these in vitro neutralization assays to in vivo antibody performance. IMPORTANCE Novel therapeutic and preventive strategies are needed to contain the HIV-1 epidemic. Antibodies with exceptional neutralizing activity against HIV-1 may provide several advantages to traditional HIV drugs, including an improved side-effect profile, a reduced dosing frequency, and immune enhancement. The activity of these antibodies has been established in vitro by utilizing HIV-1 Env-pseudotyped viruses derived from circulating viruses but produced in 293T cells by pairing Env proteins with a backbone vector. We tested PBMC-produced circulating viruses against five anti-HIV-1 antibodies currently in clinical development. We found that the activity of these antibodies against PBMC isolates is significantly less than that against 293T Env-pseudotyped viruses. This decline varied among the antibodies tested, with some demonstrating moderate reductions in activity and others showing an almost 100-fold reduction. As the development of these antibodies progresses, it will be critical to determine how the results of different in vitro tests correspond to performance in the clinic. Copyright © 2018 Cohen et al.

Liver stem/progenitor cells in the canals of Hering: cellular origin of hepatocellular carcinoma with bile duct tumor thrombi?

PubMed

Peng, Ningfu; Li, Lequn; Cai, Xiang; Tan, Shaozao; Wu, Ting

2010-12-01

It is generally believed that the invasion of hepatocellular carcinoma (HCC) into the biliary tree ultimately leads to the formation of bile duct tumor thrombi (BDTT). However, recent studies revealed that primary tumor might be small, even undetectable, and there was no histopathologic evidence of direct tumor invasion into bile duct wall in some patients. During the last decade, efforts on stem cell biology may shed light on the pathogenesis of BDTT. Presently, accumulating evidence supports the following notions: (1) the canals of Hering (CoH) are the most likely origin of liver stem/progenitor cells (LSPCs) in adult livers; (2) similar signalling pathways may regulate self-renewal in LSPCs and liver cancer cells, and a substantial proportion of liver tumors may often originate from the transformation of LSPCs; and (3) liver cancer contains rare cells with stem cell-like properties, which could derive from malignant transformation of LSPCs. Herein, we propose that HCC with BDTT, especially with small or undetectable primary lesion and/or no histopathologic evidence for bile duct invasion, might arise from LSPCs residing in the CoH and, possibly, some primary lesions are formed firstly within the intrahepatic biliary tree. When "tumor thrombi" extends mainly along bile duct, there might be "BDTT" alone; when it invades into surrounding parenchyma, there might often be small "primary tumor" with "BDTT". If this holds true, the putative type may be a particular subset of HCC, and most importantly it would facilitate our understanding of stem-cell origin of HCC.

HER2-specific T cells target primary glioblastoma stem cells and induce regression of autologous experimental tumors.

PubMed

Ahmed, Nabil; Salsman, Vita S; Kew, Yvonne; Shaffer, Donald; Powell, Suzanne; Zhang, Yi J; Grossman, Robert G; Heslop, Helen E; Gottschalk, Stephen

2010-01-15

Glioblastoma multiforme (GBM) is the most aggressive human primary brain tumor and is currently incurable. Immunotherapies have the potential to target GBM stem cells, which are resistant to conventional therapies. Human epidermal growth factor receptor 2 (HER2) is a validated immunotherapy target, and we determined if HER2-specific T cells can be generated from GBM patients that will target autologous HER2-positive GBMs and their CD133-positive stem cell compartment. HER2-specific T cells from 10 consecutive GBM patients were generated by transduction with a retroviral vector encoding a HER2-specific chimeric antigen receptor. The effector function of HER2-specific T cells against autologous GBM cells, including CD133-positive stem cells, was evaluated in vitro and in an orthotopic murine xenograft model. Stimulation of HER2-specific T cells with HER2-positive autologous GBM cells resulted in T-cell proliferation and secretion of IFN-gamma and interleukin-2 in a HER2-dependent manner. Patients' HER2-specific T cells killed CD133-positive and CD133-negative cells derived from primary HER2-positive GBMs, whereas HER2-negative tumor cells were not killed. Injection of HER2-specific T cells induced sustained regression of autologous GBM xenografts established in the brain of severe combined immunodeficient mice. Gene transfer allows the reliable generation of HER2-specific T cells from GBM patients, which have potent antitumor activity against autologous HER2-positive tumors including their putative stem cells. Hence, the adoptive transfer of HER2-redirected T cells may be a promising immunotherapeutic approach for GBM.

Human fetal enterocytes in vitro: modulation of the phenotype by extracellular matrix.

PubMed Central

Sanderson, I R; Ezzell, R M; Kedinger, M; Erlanger, M; Xu, Z X; Pringault, E; Leon-Robine, S; Louvard, D; Walker, W A

1996-01-01

The differentiation of small intestinal epithelial cells may require stimulation by microenvironmental factors in vivo. In this study, the effects of mesenchymal and luminal elements in nonmalignant epithelia] cells isolated from the human fetus were studied in vitro. Enterocytes from the human fetus were cultured and microenvironmental factors were added in stages, each stage more closely approximating the microenvironment in vivo. Four stages were examined: epithelial cells derived on plastic from intestinal culture and grown as a cell clone, the same cells grown on connective tissue support, primary epithelial explants grown on fibroblasts with a laminin base, and primary epithelial explants grown on fibroblasts and laminin with n-butyrate added to the incubation medium. The epithelial cell clone dedifferentiated when grown on plastic; however, the cells expressed cytokeratins and villin as evidence of their epithelial cell origin. Human connective tissue matrix from Engelbreth-Holm-Swarm sarcoma cells (Matrigel) modulated their phenotype: alkaline phosphatase activity increased, microvilli developed on their apical surface, and the profile of insulin-like growth factor binding proteins resembled that secreted by differentiated enterocytes. Epithelial cells taken directly from the human fetus as primary cultures and grown as explants on fibroblasts and laminin expressed greater specific enzyme activities in brush border membrane fractions than the cell clone. These activities were enhanced by the luminal molecule sodium butyrate. Thus the sequential addition of connective tissue and luminal molecules to nonmalignant epithelia] cells in vitro induces a spectrum of changes in the epithelial cell phenotype toward full differentiation. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8755542

A cell line resource derived from honey bee (Apis mellifera) embryonic tissues.

PubMed

Goblirsch, Michael J; Spivak, Marla S; Kurtti, Timothy J

2013-01-01

A major hindrance to the study of honey bee pathogens or the effects of pesticides and nutritional deficiencies is the lack of controlled in vitro culture systems comprised of honey bee cells. Such systems are important to determine the impact of these stress factors on the developmental and cell biology of honey bees. We have developed a method incorporating established insect cell culture techniques that supports sustained growth of honey bee cells in vitro. We used honey bee eggs mid to late in their embryogenesis to establish primary cultures, as these eggs contain cells that are progressively dividing. Primary cultures were initiated in modified Leibovitz's L15 medium and incubated at 32(°)C. Serial transfer of material from several primary cultures was maintained and has led to the isolation of young cell lines. A cell line (AmE-711) has been established that is composed mainly of fibroblast-type cells that form an adherent monolayer. Most cells in the line are diploid (2n = 32) and have the Apis mellifera karyotype as revealed by Giemsa stain. The partial sequence for the mitochondrial-encoded cytochrome c oxidase subunit I (Cox 1) gene in the cell line is identical to those from honey bee tissues and a consensus sequence for A. mellifera. The population doubling time is approximately 4 days. Importantly, the cell line is continuously subcultured every 10-14 days when split at a 1:3 ratio and is cryopreserved in liquid nitrogen. The cell culture system we have developed has potential application for studies aimed at honey bee development, genetics, pathogenesis, transgenesis, and toxicology.

Target sequence accessibility limits activation-induced cytidine deaminase activity in primary mediastinal B-cell lymphoma.

PubMed

Popov, Sergey W; Moldenhauer, Gerhard; Wotschke, Beate; Brüderlein, Silke; Barth, Thomas F; Dorsch, Karola; Ritz, Olga; Möller, Peter; Leithäuser, Frank

2007-07-15

Activation-induced cytidine deaminase (AID) initiates somatic hypermutation (SHM) and class switch recombination (CSR) in activated B lymphocytes and is potentially implicated in genomic instability of B-cell malignancies. For unknown reasons, B-cell neoplasms often lack SHM and CSR in spite of high AID expression. Here, we show that primary mediastinal B-cell lymphoma (PMBL), an immunoglobulin (Ig)-negative lymphoma that possesses hypermutated, class-switched Ig genes, expresses high levels of AID with an intact primary structure but does not do CSR in 14 of 16 cases analyzed. Absence of CSR coincided with low Ig germ-line transcription, whereas high level germ-line transcription was observed only in those two cases with active CSR. Interleukin-4/CD40L costimulation induced CSR and a marked up-regulation of germ-line transcription in the PMBL-derived cell line MedB-1. In the PMBL cell line Karpas 1106P, CSR was not inducible and germ-line transcription remained low on stimulation. However, Karpas 1106P, but not MedB-1, had ongoing SHM of the Ig gene and BCL6. These genes were transcribed in Karpas 1106P, whereas transcription was undetectable or low in MedB-1 cells. Thus, accessibility of the target sequences seems to be a major limiting factor for AID-dependent somatic gene diversification in PMBL.

Comparison of Human Induced Pluripotent Stem Cell-Derived Neurons and Rat Primary CorticalNeurons as In Vitro Models of Neurite Outgrowth

EPA Science Inventory

High-throughput assays that can quantify chemical-induced changes at the cellular and molecular level have been recommended for use in chemical safety assessment. High-throughput, high content imaging assays for the key cellular events of neurodevelopment have been proposed to ra...

Efficacy and safety of a porcine collagen sponge for cranial neurosurgery: a prospective case-control study.

PubMed

Brandão, Rafael Augusto Castro Santiago; Costa, Bruno Silva; Dellaretti, Marcos Antonio; de Carvalho, Gervásio Teles C; Faria, Marcello Penholate; de Sousa, Atos Alves

2013-01-01

The use of dural grafts is very useful when primary dural closure cannot be achieved. Our primary objective was to study the incidence of postoperative cerebrospinal fluid leak, including fistula and pseudomeningocele, and postoperative infection by comparing autologous material and a new collagen graft. A prospective nonrandomized study with a new collagen-based product derived from porcine cells (Peridry) was performed. It was used for dural replacement in 50 patients who underwent a variety of neurosurgical procedures requiring the use of a dural graft. These results were compared with a control group of 50 patients who were treated with autologous duraplasty material. The follow-up period was 3 months. Postoperative overall cerebrospinal fluid fistula occurred in 6% of both groups. No patient in the collagen group developed any sort of infection. One patient in the control developed osteomyelitis in the bone flap. The new collagen-based product derived from porcine cells (Peridry), compared with an autologous tissue, is safe, effective, easy to use, as well as time saving in cranial neurosurgery. Copyright © 2013 Elsevier Inc. All rights reserved.

Identification of Regulatory Factors for Mesenchymal Stem Cell-Derived Salivary Epithelial Cells in a Co-Culture System

PubMed Central

Park, Yun-Jong; Koh, Jin; Gauna, Adrienne E.; Chen, Sixue; Cha, Seunghee

2014-01-01

Patients with Sjögren’s syndrome or head and neck cancer patients who have undergone radiation therapy suffer from severe dry mouth (xerostomia) due to salivary exocrine cell death. Regeneration of the salivary glands requires a better understanding of regulatory mechanisms by which stem cells differentiate into exocrine cells. In our study, bone marrow-derived mesenchymal stem cells were co-cultured with primary salivary epithelial cells from C57BL/6 mice. Co-cultured bone marrow-derived mesenchymal stem cells clearly resembled salivary epithelial cells, as confirmed by strong expression of salivary gland epithelial cell-specific markers, such as alpha-amylase, muscarinic type 3 receptor, aquaporin-5, and cytokeratin 19. To identify regulatory factors involved in this differentiation, transdifferentiated mesenchymal stem cells were analyzed temporarily by two-dimensional-gel-electrophoresis, which detected 58 protein spots (>1.5 fold change, p<0.05) that were further categorized into 12 temporal expression patterns. Of those proteins only induced in differentiated mesenchymal stem cells, ankryin-repeat-domain-containing-protein 56, high-mobility-group-protein 20B, and transcription factor E2a were selected as putative regulatory factors for mesenchymal stem cell transdifferentiation based on putative roles in salivary gland development. Induction of these molecules was confirmed by RT-PCR and western blotting on separate sets of co-cultured mesenchymal stem cells. In conclusion, our study is the first to identify differentially expressed proteins that are implicated in mesenchymal stem cell differentiation into salivary gland epithelial cells. Further investigation to elucidate regulatory roles of these three transcription factors in mesenchymal stem cell reprogramming will provide a critical foundation for a novel cell-based regenerative therapy for patients with xerostomia. PMID:25402494

Color-Coded Imaging of Syngeneic Orthotopic Malignant Lymphoma Interacting with Host Stromal Cells During Metastasis.

PubMed

Matsumoto, Takuro; Suetsugu, Atsushi; Hasegawa, Kosuke; Nakamura, Miki; Aoki, Hitomi; Kunisada, Takahiro; Tsurumi, Hisashi; Shimizu, Masahito; Hoffman, Robert M

2016-04-01

The EL4 cell line was previously derived from a lymphoma induced in a C57/BL6 mouse by 9,10-dimethyl-1,2-benzanthracene. In a previous study, EL4 lymphoma cells expressing red fluorescent protein (EL4-RFP) were established and injected into the tail vein of C57/BL6 green fluorescent protein (GFP) transgenic mice. Metastasis was observed at multiple sites which were also enriched with host GFP-expressing stromal cells. In the present study, our aim was to establish an orthotopic model of EL4-RFP. In the present study, EL4-RFP lymphoma cells were injected in the spleen of C57/BL6 GFP transgenic mice as an orthotopic model of lymphoma. Resultant primary tumor and metastases were imaged with the Olympus FV1000 scanning laser confocal microscope. EL4-RFP metastasis was observed 21 days later. EL4-RFP tumors in the spleen (primary injection site), liver, supra-mediastinum lymph nodes, abdominal lymph nodes, bone marrow, and lung were visualized by color-coded imaging. EL4-RFP metastases in the liver, lymph nodes, and bone marrow in C57/BL6 GFP mice were rich in GFP stromal cells such as macrophages, fibroblasts, dendritic cells, and normal lymphocytes derived from the host animal. Small tumors were observed in the spleen, which were rich in host stromal cells. In the lung, no mass formation of lymphoma cells occurred, but lymphoma cells circulated in lung peripheral blood vessels. Phagocytosis of EL4-RFP lymphoma cells by macrophages, as well as dendritic cells and fibroblasts, were observed in culture. Color-coded imaging of the lymphoma microenvironment suggests an important role of stromal cells in lymphoma progression and metastasis. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Development of patient-derived xenograft models from a spontaneously immortal low-grade meningioma cell line, KCI-MENG1.

PubMed

Michelhaugh, Sharon K; Guastella, Anthony R; Varadarajan, Kaushik; Klinger, Neil V; Parajuli, Prahlad; Ahmad, Aamir; Sethi, Seema; Aboukameel, Amro; Kiousis, Sam; Zitron, Ian M; Ebrahim, Salah A; Polin, Lisa A; Sarkar, Fazlul H; Bollig-Fischer, Aliccia; Mittal, Sandeep

2015-07-15

There is a paucity of effective therapies for recurrent/aggressive meningiomas. Establishment of improved in vitro and in vivo meningioma models will facilitate development and testing of novel therapeutic approaches. A primary meningioma cell line was generated from a patient with an olfactory groove meningioma. The cell line was extensively characterized by performing analysis of growth kinetics, immunocytochemistry, telomerase activity, karyotype, and comparative genomic hybridization. Xenograft models using immunocompromised SCID mice were also developed. Histopathology of the patient tumor was consistent with a WHO grade I typical meningioma composed of meningothelial cells, whorls, and occasional psammoma bodies. The original tumor and the early passage primary cells shared the standard immunohistochemical profile consistent with low-grade, good prognosis meningioma. Low passage KCI-MENG1 cells were composed of two cell types with spindle and round morphologies, showed linear growth curve, had very low telomerase activity, and were composed of two distinct unrelated clones on cytogenetic analysis. In contrast, high passage cells were homogeneously round, rapidly growing, had high telomerase activity, and were composed of a single clone with a near triploid karyotype containing 64-66 chromosomes with numerous aberrations. Following subcutaneous and orthotopic transplantation of low passage cells into SCID mice, firm tumors positive for vimentin and progesterone receptor (PR) formed, while subcutaneous implant of high passage cells yielded vimentin-positive, PR-negative tumors, concordant with a high-grade meningioma. Although derived from a benign meningioma specimen, the newly-established spontaneously immortal KCI-MENG1 meningioma cell line can be utilized to generate xenograft tumor models with either low- or high-grade features, dependent on the cell passage number (likely due to the relative abundance of the round, near-triploid cells). These human meningioma mouse xenograft models will provide biologically relevant platforms from which to investigate differences in low- vs. high-grade meningioma tumor biology and disease progression as well as to develop novel therapies to improve treatment options for poor prognosis or recurrent meningiomas.

Implantation of GL261 neurospheres into C57/BL6 mice: a more reliable syngeneic graft model for research on glioma-initiating cells.

PubMed

Yi, Liang; Zhou, Chun; Wang, Bing; Chen, Tunan; Xu, Minhui; Xu, Lunshan; Feng, Hua

2013-08-01

Recent studies have demonstrated that inflammatory cells and inflammatory mediators are indispensable components of the tumor-initiating cell (TIC) niche and regulate the malignant behavior of TICs. However, conventional animal models for glioma-initiating cell (GIC) studies are based on the implantation of GICs from human glioblastoma (GBM) into immunodeficient mice without the regulation of immune system. Whether animal models can mimic the cellular microenvironment of malignancy and evaluate the biological features of GICs accurately is unclear. Here, we detected the biological features of neurosphere-like tumor cells derived from the murine GBM cell line GL261 (GL261-NS) and from primary human GBM (PGBM-NS) in vitro, injected GL261-NS into syngeneic C57/BL6 mouse brain and injected PGBM-NS into NOD/SCID mouse brain, respectively. The tumorigenic characteristics of the two different orthotopic transplantation models were analyzed and the histological discrepancy between grafts and human primary GBM was compared. We found that GICs enriched in GL261-NS, GL261-NS and PGBM-NS exhibited increased GIC potential and enhanced chemoresistance in vitro. GL261-NS was significantly more aggressive compared to GL261 adhesive cells (GL261-AC) in vivo and the enhanced aggression was more significant in syngeneic mice compared to immunodeficient mice. The discrepancy of tumorigenicity between GL261-NS and GL261-AC in C57/BL6 mice was also larger compared to that between PGBM-NS and PGBM-AC in immunodeficient mice. Syngrafts derived from GL261-NS in C57/BL6 mice corresponded to the human GBM histologically better, compared with xenografts derived from PGBM-NS in NOD/SCID mice, which lack inflammatory cells and inflammatory mediators. We conclude that the inflammatory niche is involved in the tumorigenicity of GICs and implantation of GL261-NS into C57/BL6 mice is a more reliable syngeneic graft model for in vivo study on GICs relative to the immunodeficiency model.

Platelet-derived growth factor-receptor alpha strongly inhibits melanoma growth in vitro and in vivo.

PubMed

Faraone, Debora; Aguzzi, Maria Simona; Toietta, Gabriele; Facchiano, Angelo M; Facchiano, Francesco; Magenta, Alessandra; Martelli, Fabio; Truffa, Silvia; Cesareo, Eleonora; Ribatti, Domenico; Capogrossi, Maurizio C; Facchiano, Antonio

2009-08-01

Cutaneous melanoma is the most aggressive skin cancer; it is highly metastatic and responds poorly to current therapies. The expression of platelet-derived growth factor receptors (PDGF-Rs) is reported to be reduced in metastatic melanoma compared with benign nevi or normal skin; we then hypothesized that PDGF-Ralpha may control growth of melanoma cells. We show here that melanoma cells overexpressing PDGF-Ralpha respond to serum with a significantly lower proliferation compared with that of controls. Apoptosis, cell cycle arrest, pRb dephosphorylation, and DNA synthesis inhibition were also observed in cells overexpressing PDGF-Ralpha. Proliferation was rescued by PDGF-Ralpha inhibitors, allowing to exclude nonspecific toxic effects and indicating that PDGF-Ralpha mediates autocrine antiproliferation signals in melanoma cells. Accordingly, PDGF-Ralpha was found to mediate staurosporine cytotoxicity. A protein array-based analysis of the mitogen-activated protein kinase pathway revealed that melanoma cells overexpressing PDGF-Ralpha show a strong reduction of c-Jun phosphorylated in serine 63 and of protein phosphatase 2A/Balpha and a marked increase of p38gamma, mitogen-activated protein kinase kinase 3, and signal regulatory protein alpha1 protein expression. In a mouse model of primary melanoma growth, infection with the Ad-vector overexpressing PDGF-Ralpha reached a significant 70% inhibition of primary melanoma growth (P < .001) and a similar inhibition of tumor angiogenesis. All together, these data demonstrate that PDGF-Ralpha strongly impairs melanoma growth likely through autocrine mechanisms and indicate a novel endogenous mechanism involved in melanoma control.

Biology Based Lung Cancer Model for Chronic Low Radon Exposures

NASA Astrophysics Data System (ADS)

TruÅ£ǎ-Popa, Lucia-Adina; Hofmann, Werner; Fakir, Hatim; Cosma, Constantin

2008-08-01

Low dose effects of alpha particles at the tissue level are characterized by the interaction of single alpha particles, affecting only a small fraction of the cells within that tissue. Alpha particle intersections of bronchial target cells during a given exposure period were simulated by an initiation-promotion model, formulated in terms of cellular hits within the cycle time of the cell (dose-rate) and then integrated over the whole exposure period (dose). For a given average number of cellular hits during the lifetime of bronchial cells, the actual number of single and multiple hits was selected from a Poisson distribution. While oncogenic transformation is interpreted as the primary initiation step, stimulated mitosis by killing adjacent cells is assumed to be the primary radiological promotion event. Analytical initiation and promotion functions were derived from experimental in vitro data on oncogenic transformation and cellular survival. To investigate the shape of the lung cancer risk function at chronic, low level exposures in more detail, additional biological factors describing the tissue response and operating specifically at low doses were incorporated into the initiation-promotion model. These mechanisms modifying the initial response at the cellular level were: adaptive response, genomic instability, induction of apoptosis by surrounding cells, and detrimental as well as protective bystander mechanisms. To quantify the effects of these mechanisms as functions of dose, analytical functions were derived from the experimental evidence presently available. Predictions of lung cancer risk, including these mechanisms, exhibit a distinct sublinear dose-response relationship at low exposures, particularly for very low exposure rates.

Development of donor-derived thymic lymphomas after allogeneic bone marrow transplantation in AKR/J mice

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

Yasumizu, R.; Hiai, H.; Sugiura, K.

1988-09-15

The transplantation of bone marrow cells from BALB/c (but not C57BL/6 and C3H/HeN) mice was observed to lead to the development of thymic lymphomas (leukemias) in AKR/J mice. Two leukemic cell lines, CAK1.3 and CAK4.4, were established from the primary culture of two thymic lymphoma, and surface phenotypes of these cell lines found to be H-2d and Thy-1.2+, indicating that these lymphoma cells are derived from BALB/c donor bone marrow cells. Further analyses of surface markers revealed that CAK1.3 is L3T4+ Lyt2+ IL2R-, whereas CAK4.4 is L3T4- Lyt2- IL2R+. Both CAK1.3 and CAK4.4 were transplantable into BALB/c but not AKR/Jmore » mice, further indicating that these cells are of BALB/c bone marrow donor origin. The cells were found to produce XC+-ecotropic viruses, but xenotropic and mink cell focus-forming viruses were undetectable. Inasmuch as thymic lymphomas are derived from bone marrow cells of leukemia-resistant BALB/c strain of mice under the allogeneic environment of leukemia-prone AKR/J mice, this animal model may serve as a useful tool not only for the analysis of leukemic relapse after bone marrow transplantation but also for elucidation of the mechanism of leukemogenesis.« less

CD34 Antigen and the MPL Receptor Expression Defines a Novel Class of Human Cord Blood-Derived Primitive Hematopoietic Stem Cells

PubMed Central

Matsuoka, Yoshikazu; Takahashi, Masaya; Sumide, Keisuke; Kawamura, Hiroshi; Nakatsuka, Ryusuke; Fujioka, Tatsuya; Sonoda, Yoshiaki

2017-01-01

In the murine hematopoietic stem cell (HSC) compartment, thrombopoietin (THPO)/MPL (THPO receptor) signaling plays an important role in the maintenance of adult quiescent HSCs. However, the role of THPO/MPL signaling in the human primitive HSC compartment has not yet been elucidated. We have identified very primitive human cord blood (CB)-derived CD34– severe combined immunodeficiency (SCID)-repopulating cells (SRCs) using the intra-bone marrow injection method. In this study, we investigated the roles of the MPL expression in the human primitive HSC compartment. The SRC activities of the highly purified CB-derived 18Lin–CD34+/–MPL+/– cells were analyzed using NOG mice. In the primary recipient mice, nearly all mice that received CD34+/–MPL+/– cells were repopulated with human CD45+ cells. Nearly all of these mice that received CD34+MPL+/– and CD34–MPL– cells showed a secondary repopulation. Interestingly, the secondary recipient mice that received CD34+/–MPL– cells showed a distinct tertiary repopulation. These results clearly indicate that the CD34+/– SRCs not expressing MPL sustain a long-term (LT) (>1 year) human cell repopulation in NOG mice. Moreover, CD34– SRCs generate CD34+CD38–CD90+ SRCs in vitro and in vivo. These findings provide a new concept that CD34–MPL– SRCs reside at the apex of the human HSC hierarchy. PMID:27938494

High-throughput screening in niche-based assay identifies compounds to target preleukemic stem cells

PubMed Central

Gerby, Bastien; Veiga, Diogo F.T.; Krosl, Jana; Nourreddine, Sami; Ouellette, Julianne; Haman, André; Lavoie, Geneviève; Fares, Iman; Tremblay, Mathieu; Litalien, Véronique; Ottoni, Elizabeth; Geoffrion, Dominique; Maddox, Paul S.; Chagraoui, Jalila; Hébert, Josée; Sauvageau, Guy; Kwok, Benjamin H.; Roux, Philippe P.

2016-01-01

Current chemotherapies for T cell acute lymphoblastic leukemia (T-ALL) efficiently reduce tumor mass. Nonetheless, disease relapse attributed to survival of preleukemic stem cells (pre-LSCs) is associated with poor prognosis. Herein, we provide direct evidence that pre-LSCs are much less chemosensitive to existing chemotherapy drugs than leukemic blasts because of a distinctive lower proliferative state. Improving therapies for T-ALL requires the development of strategies to target pre-LSCs that are absolutely dependent on their microenvironment. Therefore, we designed a robust protocol for high-throughput screening of compounds that target primary pre-LSCs maintained in a niche-like environment, on stromal cells that were engineered for optimal NOTCH1 activation. The multiparametric readout takes into account the intrinsic complexity of primary cells in order to specifically monitor pre-LSCs, which were induced here by the SCL/TAL1 and LMO1 oncogenes. We screened a targeted library of compounds and determined that the estrogen derivative 2-methoxyestradiol (2-ME2) disrupted both cell-autonomous and non–cell-autonomous pathways. Specifically, 2-ME2 abrogated pre-LSC viability and self-renewal activity in vivo by inhibiting translation of MYC, a downstream effector of NOTCH1, and preventing SCL/TAL1 activity. In contrast, normal hematopoietic stem/progenitor cells remained functional. These results illustrate how recapitulating tissue-like properties of primary cells in high-throughput screening is a promising avenue for innovation in cancer chemotherapy. PMID:27797342

Computational Discovery of Niclosamide Ethanolamine, a Repurposed Drug Candidate That Reduces Growth of Hepatocellular Carcinoma Cells In Vitro and in Mice by Inhibiting Cell Division Cycle 37 Signaling.

PubMed

Chen, Bin; Wei, Wei; Ma, Li; Yang, Bin; Gill, Ryan M; Chua, Mei-Sze; Butte, Atul J; So, Samuel

2017-06-01

Drug repositioning offers a shorter approval process than new drug development. We therefore searched large public datasets of drug-induced gene expression signatures to identify agents that might be effective against hepatocellular carcinoma (HCC). We searched public databases of messenger RNA expression patterns reported from HCC specimens from patients, HCC cell lines, and cells exposed to various drugs. We identified drugs that might specifically increase expression of genes that are down-regulated in HCCs and reduce expression of genes up-regulated in HCCs using a nonparametric, rank-based pattern-matching strategy based on the Kolmogorov-Smirnov statistic. We evaluated the anti-tumor activity of niclosamide and its ethanolamine salt (NEN) in HCC cell lines (HepG2, Huh7, Hep3B, Hep40, and PLC/PRF/5), primary human hepatocytes, and 2 mouse models of HCC. In one model of HCC, liver tumor development was induced by hydrodynamic delivery of a sleeping beauty transposon expressing an activated form of Ras (v12) and truncated β-catenin (N90). In another mouse model, patient-derived xenografts were established by implanting HCC cells from patients into livers of immunocompromised mice. Tumor growth was monitored by bioluminescence imaging. Tumor-bearing mice were fed a regular chow diet or a chow diet containing niclosamide or NEN. In a separate experiment using patient-derived xenografts, tumor-bearing mice were given sorafenib (the standard of care for patients with advanced HCC), NEN, or niclosamide alone; a combination of sorafenib and NEN; or a combination sorafenib and niclosamide in their drinking water, or regular water (control), and tumor growth was monitored. Based on gene expression signatures, we identified 3 anthelmintics that significantly altered the expression of genes that are up- or down-regulated in HCCs. Niclosamide and NEN specifically reduced the viability of HCC cells: the agents were at least 7-fold more cytotoxic to HCCs than primary hepatocytes. Oral administration of NEN to mice significantly slowed growth of genetically induced liver tumors and patient-derived xenografts, whereas niclosamide did not, coinciding with the observed greater bioavailability of NEN compared with niclosamide. The combination of NEN and sorafenib was more effective at slowing growth of patient-derived xenografts than either agent alone. In HepG2 cells and in patient-derived xenografts, administration of niclosamide or NEN increased expression of 20 genes down-regulated in HCC and reduced expression of 29 genes up-regulated in the 274-gene HCC signature. Administration of NEN to mice with patient-derived xenografts reduced expression of proteins in the Wnt-β-catenin, signal transducer and activator of transcription 3, AKT-mechanistic target of rapamycin, epidermal growth factor receptor-Ras-Raf signaling pathways. Using immunoprecipitation assays, we found NEN to bind cell division cycle 37 protein and disrupt its interaction with heat shock protein 90. In a bioinformatics search for agents that alter the HCC-specific gene expression pattern, we identified the anthelmintic niclosamide as a potential anti-tumor agent. Its ethanolamine salt, with greater bioavailability, was more effective than niclosamide at slowing the growth of genetically induced liver tumors and patient-derived xenografts in mice. Both agents disrupted interaction between cell division cycle 37 and heat shock protein 90 in HCC cells, with concomitant inhibition of their downstream signaling pathways. NEN might be effective for treatment of patients with HCC. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

An efficient and scalable pipeline for epitope tagging in mammalian stem cells using Cas9 ribonucleoprotein

PubMed Central

Dewari, Pooran Singh; Southgate, Benjamin; Mccarten, Katrina; Monogarov, German; O'Duibhir, Eoghan; Quinn, Niall; Tyrer, Ashley; Leitner, Marie-Christin; Plumb, Colin; Kalantzaki, Maria; Blin, Carla; Finch, Rebecca; Bressan, Raul Bardini; Morrison, Gillian; Jacobi, Ashley M; Behlke, Mark A; von Kriegsheim, Alex; Tomlinson, Simon; Krijgsveld, Jeroen

2018-01-01

CRISPR/Cas9 can be used for precise genetic knock-in of epitope tags into endogenous genes, simplifying experimental analysis of protein function. However, Cas9-assisted epitope tagging in primary mammalian cell cultures is often inefficient and reliant on plasmid-based selection strategies. Here, we demonstrate improved knock-in efficiencies of diverse tags (V5, 3XFLAG, Myc, HA) using co-delivery of Cas9 protein pre-complexed with two-part synthetic modified RNAs (annealed crRNA:tracrRNA) and single-stranded oligodeoxynucleotide (ssODN) repair templates. Knock-in efficiencies of ~5–30%, were achieved without selection in embryonic stem (ES) cells, neural stem (NS) cells, and brain-tumor-derived stem cells. Biallelic-tagged clonal lines were readily derived and used to define Olig2 chromatin-bound interacting partners. Using our novel web-based design tool, we established a 96-well format pipeline that enabled V5-tagging of 60 different transcription factors. This efficient, selection-free and scalable epitope tagging pipeline enables systematic surveys of protein expression levels, subcellular localization, and interactors across diverse mammalian stem cells. PMID:29638216

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.

Human primary myoblast cell cultures from non-diabetic insulin resistant subjects retain defects in insulin action.

PubMed Central

Thompson, D B; Pratley, R; Ossowski, V

1996-01-01

Insulin resistance is a predictor of the development of noninsulin-dependent diabetes mellitus (NIDDM) in humans. It is unclear whether insulin resistance is a primary defect leading to NIDDM or the result of hyperinsulinemia and hyperglycemia. To determine if insulin resistance is the result of extrinsic factors such as hyperinsulinemia primary skeletal muscle cell cultures were established from muscle biopsies from Pima Indians with differing in vivo insulin sensitivities. These cell cultures expressed a variety of muscle-specific phenotypes including the proteins alpha-actinin and myosin, muscle-specific creatine kinase activity, and RNA encoding GLUT4, MYF5, MYOD1, and MYOGENIN. Labeled glucose was used to measure the insulin-stimulated conversion of glucose to glycogen in these cultures. The in vivo rates of insulin-stimulated glycogen production (insulin resistance) were correlated with in vitro measures of glycogen production (P = 0.007, r = 0.58). This defect in insulin action is stable in a uniform culture environment and is retained over time. The retention of insulin resistance in myoblast derived cell cultures is consistent with the expression of an underlying biochemical defect in insulin resistant skeletal muscle. PMID:8941652