搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

Increasing adipocyte size as well as numbers is important in the development of obesity and type 2 diabetes,with adipocytes being generated from mesenchymal precursor cells. This process includes the determination of mesenchymal stem cells (MSC) into preadipocytes (PA) and the differentiation of PA into mature fat cells. Although the process of differentiation has been highly investigated,the determination in humans is poorly understood. In this study,we compared human MSC and human committed PA on a cellular and molecular level to gain further insights into the regulatory mechanisms in the determination process. Both cell types showed similar morphology and expression patterns of common mesenchymal and hematopoietic surface markers. However,although MSC were able to differentiate into adipocytes and osteocytes,PA were only able to undergo adipogenesis,indicating that PA lost their multipotency during determination. WNT-5a expression showed significantly higher levels in MSC compared with PA suggesting that WNT-5a down-regulation might be important in the determination process. Indeed,incubation of human MSC in medium containing neutralizing WNT-5a antibodies abolished their ability to undergo osteogenesis,although adipogenesis was still possible. An opposite effect was achieved using recombinant WNT-5a protein. On a molecular level,WNT-5a was found to promote c-Jun N-terminal kinase-dependent intracellular signaling in MSC. Activation of this noncanonical pathway resulted in the induction of osteopontin expression further indicating pro-osteogenic effects of WNT-5a. Our data suggest that WNT-5a is necessary to maintain osteogenic potential of MSC and that inhibition of WNT-5a signaling therefore plays a role in their determination into PA in humans. View Publication

Reprogramming of a limited number of human cell types has been achieved through ectopic expression of four transcription factors to yield induced pluripotent stem (iPS) cells that closely resemble human embryonic stem cells (ESCs). Here,we determined functional and epigenetic properties of iPS cells generated from human umbilical vein endothelial cells (HUVEC) by conventional method of direct reprogramming. Retroviral overexpression of four transcription factors resets HUVEC to the pluripotency. Human endothelial cell-derived iPS (endo-iPS) cells were similar to human ESCs in morphology,gene expression,in vitro and in vivo differentiation capacity. Endo-iPS cells were efficiently differentiated in vitro into endothelial cells. Using genome-wide methylation profiling we show that promoter elements of endothelial specific genes were methylated following reprogramming whereas pluripotency-related gene promoters were hypomethylated similar to levels observed in ESCs. Genome-wide methylation analysis of CpG sites located in the functional regions of over than 14,000 genes indicated that human endo-iPS cells were highly similar to human ES cells,although differences in methylation levels of 46 genes were found. Overall CpG methylation of promoter regions in the pluripotent cells was higher than in somatic. We also show that during reprogramming female human endo-iPS cells exhibited reactivation of the somatically silenced X chromosome. Our findings demonstrate that iPS cells can be generated from human endothelial cells and reprogramming resets epigenetic status of endothelial cells to pluripotency. View Publication

A hallmark feature of glioblastoma is its strong self-renewal potential and immature differentiation state,which contributes to its plasticity and therapeutic resistance. Here,integrated genomic and biological analyses identified PLAGL2 as a potent protooncogene targeted for amplification/gain in malignant gliomas. Enhanced PLAGL2 expression strongly suppresses neural stem cell (NSC) and glioma-initiating cell differentiation while promoting their self-renewal capacity upon differentiation induction. Transcriptome analysis revealed that these differentiation-suppressive activities are attributable in part to PLAGL2 modulation of Wnt/beta-catenin signaling. Inhibition of Wnt signaling partially restores PLAGL2-expressing NSC differentiation capacity. The identification of PLAGL2 as a glioma oncogene highlights the importance of a growing class of cancer genes functioning to impart stem cell-like characteristics in malignant cells. View Publication

Teratogens,substances that may cause fetal abnormalities during development,are responsible for a significant number of birth defects. Animal models used to predict teratogenicity often do not faithfully correlate to human response. Here,we seek to develop a more predictive developmental toxicity model based on an in vitro method that utilizes both human embryonic stem (hES) cells and metabolomics to discover biomarkers of developmental toxicity. We developed a method where hES cells were dosed with several drugs of known teratogenicity then LC-MS analysis was performed to measure changes in abundance levels of small molecules in response to drug dosing. Statistical analysis was employed to select for specific mass features that can provide a prediction of the developmental toxicity of a substance. These molecules can serve as biomarkers of developmental toxicity,leading to better prediction of teratogenicity. In particular,our work shows a correlation between teratogenicity and changes of greater than 10% in the ratio of arginine to asymmetric dimethylarginine levels. In addition,this study resulted in the establishment of a predictive model based on the most informative mass features. This model was subsequently tested for its predictive accuracy in two blinded studies using eight drugs of known teratogenicity,where it correctly predicted the teratogenicity for seven of the eight drugs. Thus,our initial data shows that this platform is a robust alternative to animal and other in vitro models for the prediction of the developmental toxicity of chemicals that may also provide invaluable information about the underlying biochemical pathways. ?? 2010 Elsevier Inc. View Publication

Deciding to exit pluripotency and undergo differentiation is of singular importance for pluripotent cells,including embryonic stem cells (ESCs). The molecular mechanisms for these decisions to differentiate,as well as reversing those decisions during induced pluripotency (iPS),have focused largely on transcriptomic controls. Here,we explore the role of translational control for the maintenance of pluripotency and the decisions to differentiate. Global protein translation is significantly reduced in hESCs compared to their differentiated progeny. Furthermore,p70 S6K activation is restricted in hESCs compared to differentiated fibroblast-like cells. Disruption of p70 S6K-mediated translation by rapamycin or siRNA knockdown in undifferentiated hESCs does not alter cell viability or expression of the pluripotency markers Oct4 and Nanog. However,expression of constitutively active p70 S6K,but not wild-type p70 S6K,induces differentiation. Additionally,hESCs exhibit high levels of the mTORC1/p70 S6K inhibitory complex TSC1/TSC2 and preferentially express more rapamycin insensitive mTORC2 compared to differentiated cells. siRNA-mediated knockdown of both TSC2 and Rictor elevates p70 S6K activation and induces differentiation of hESCs. These results suggest that hESCs tightly regulate mTORC1/p70 S6K-mediated protein translation to maintain a pluripotent state as well as implicate a novel role for protein synthesis as a driving force behind hESC differentiation. View Publication

Mesenchymal stromal cells (MSCs) may be derived from a variety of tissues,with human umbilical cord (UC) providing an abundant and noninvasive source. Human UC-MSCs share similar in vitro immunosuppressive properties as MSCs obtained from bone marrow and cord blood. However,the mechanisms and cellular interactions used by MSCs to control immune responses remain to be fully elucidated. In this paper,we report that suppression of mitogen-induced T cell proliferation by human UC-,bone marrow-,and cord blood-MSCs required monocytes. Removal of monocytes but not B cells from human adult PBMCs (PBMNCs) reduced the immunosuppressive effects of MSCs on T cell proliferation. There was rapid modulation of a number of cell surface molecules on monocytes when PBMCs or alloantigen-activated PBMNCs were cultured with UC-MSCs. Indomethacin treatment significantly inhibited the ability of UC-MSCs to suppress T cell proliferation,indicating an important role for PGE(2). Monocytes purified from UC-MSC coculture had significantly reduced accessory cell and allostimulatory function when tested in subsequent T cell proliferation assays,an effect mediated in part by UC-MSC PGE(2) production and enhanced by PBMNC alloactivation. Therefore,we identify monocytes as an essential intermediary through which UC-MSCs mediate their suppressive effects on T cell proliferation. View Publication

The differentiation of patient-derived induced pluripotent stem cells (iPSCs) to committed fates such as neurons,muscle and liver is a powerful approach for understanding key parameters of human development and disease. Whether undifferentiated iPSCs themselves can be used to probe disease mechanisms is uncertain. Dyskeratosis congenita is characterized by defective maintenance of blood,pulmonary tissue and epidermal tissues and is caused by mutations in genes controlling telomere homeostasis. Short telomeres,a hallmark of dyskeratosis congenita,impair tissue stem cell function in mouse models,indicating that a tissue stem cell defect may underlie the pathophysiology of dyskeratosis congenita. Here we show that even in the undifferentiated state,iPSCs from dyskeratosis congenita patients harbour the precise biochemical defects characteristic of each form of the disease and that the magnitude of the telomere maintenance defect in iPSCs correlates with clinical severity. In iPSCs from patients with heterozygous mutations in TERT,the telomerase reverse transcriptase,a 50% reduction in telomerase levels blunts the natural telomere elongation that accompanies reprogramming. In contrast,mutation of dyskerin (DKC1) in X-linked dyskeratosis congenita severely impairs telomerase activity by blocking telomerase assembly and disrupts telomere elongation during reprogramming. In iPSCs from a form of dyskeratosis congenita caused by mutations in TCAB1 (also known as WRAP53),telomerase catalytic activity is unperturbed,yet the ability of telomerase to lengthen telomeres is abrogated,because telomerase mislocalizes from Cajal bodies to nucleoli within the iPSCs. Extended culture of DKC1-mutant iPSCs leads to progressive telomere shortening and eventual loss of self-renewal,indicating that a similar process occurs in tissue stem cells in dyskeratosis congenita patients. These findings in iPSCs from dyskeratosis congenita patients reveal that undifferentiated iPSCs accurately recapitulate features of a human stem cell disease and may serve as a cell-culture-based system for the development of targeted therapeutics. View Publication

Human embryonic stem cells (hESCs) offer tremendous potential for not only treating neurological disorders but also for their ability to serve as vital reagents to model and investigate human disease. To further our understanding of a key protein involved in Alzheimer disease pathogenesis,we stably overexpressed amyloid precursor protein (APP) in hESCs. Remarkably,we found that APP overexpression in hESCs caused a rapid and robust differentiation of pluripotent stem cells toward a neural fate. Despite maintenance in standard hESC media,up to 80% of cells expressed the neural stem cell marker nestin,and 65% exhibited the more mature neural marker β-3 tubulin within just 5 days of passaging. To elucidate the mechanism underlying the effects of APP on neural differentiation,we examined the proteolysis of APP and performed both gain of function and loss of function experiments. Taken together,our results demonstrate that the N-terminal secreted soluble forms of APP (in particular sAPPβ) robustly drive neural differentiation of hESCs. Our findings not only reveal a novel and intriguing role for APP in neural lineage commitment but also identify a straightforward and rapid approach to generate large numbers of neurons from human embryonic stem cells. These novel APP-hESC lines represent a valuable tool to investigate the potential role of APP in development and neurodegeneration and allow for insights into physiological functions of this protein. View Publication

Oncogenic transformation in Ewing sarcoma tumors is driven by the fusion oncogene EWS-FLI1. However,despite the well-established role of EWS-FLI1 in tumor initiation,the development of models of Ewing sarcoma in human cells with defined genetic elements has been challenging. Here,we report a novel approach to model the initiation of Ewing sarcoma tumorigenesis that exploits the developmental and pluripotent potential of human embryonic stem cells. The inducible expression of EWS-FLI1 in embryoid bodies,or collections of differentiating stem cells,generates cells with properties of Ewing sarcoma tumors,including characteristics of transformation. These cell lines exhibit anchorage-independent growth,a lack of contact inhibition and a strong Ewing sarcoma gene expression signature. Furthermore,these cells also demonstrate a requirement for the persistent expression of EWS-FLI1 for cell survival and growth,which is a hallmark of Ewing sarcoma tumors.Oncogene advance online publication,12 October 2015; doi:10.1038/onc.2015.368. View Publication

Replication stress causes DNA damage at fragile sites in the genome. DNA damage at telomeres can initiate breakage-fusion-bridge cycles and chromosome instability,which can result in replicative senescence or tumor formation. Little is known about the extent of replication stress or telomere dysfunction in human embryonic stem cells (hESCs). hESCs are grown in culture with the expectation of being used therapeutically in humans,making it important to minimize the levels of replication stress and telomere dysfunction. Here,the hESC line UCSF4 was cultured in a defined medium with growth factor Activin A,exogenous nucleosides,or DNA polymerase inhibitor aphidicolin. We used quantitative fluorescence in situ hybridization to analyze individual telomeres for dysfunction and observed that it can be increased by aphidicolin or Activin A. In contrast,adding exogenous nucleosides relieved dysfunction,suggesting that telomere dysfunction results from replication stress. Whether these findings can be applied to other hESC lines remains to be determined. However,because the loss of telomeres can lead to chromosome instability and cancer,we conclude that hESCs grown in culture for future therapeutic purposes should be routinely checked for replication stress and telomere dysfunction. View Publication

The tentative clinical application of human pluripotent stem cells (hPSCs),such as human embryonic stem cells and human induced pluripotent stem cells,is restricted by the possibility of xenogenic contamination resulting from the use of mouse embryonic fibroblasts (MEFs) as a feeder layer. Therefore,we investigated hPSC cultures on biomaterials with different elasticities that were grafted with different nanosegments. We prepared dishes coated with polyvinylalcohol-co-itaconic acid hydrogels grafted with an oligopeptide derived from vitronectin (KGGPQVTRGDVFTMP) with elasticities ranging from 10.3 to 30.4 kPa storage moduli by controlling the crosslinking time. The hPSCs cultured on the stiffest substrates (30.4 kPa) tended to differentiate after five days of culture,whereas the hPSCs cultured on the optimal elastic substrates (25 kPa) maintained their pluripotency for over 20 passages under xeno-free conditions. These results indicate that cell culture matrices with optimal elasticity can maintain the pluripotency of hPSCs in culture. View Publication

AIM To generate human embryonic stem cell derived corneal endothelial cells (hESC-CECs) for transplantation in patients with corneal endothelial dystrophies. MATERIALS AND METHODS Feeder-free hESC-CECs were generated by a directed differentiation protocol. hESC-CECs were characterized by morphology,expression of corneal endothelial markers,and microarray analysis of gene expression. RESULTS hESC-CECs were nearly identical morphologically to primary human corneal endothelial cells,expressed Zona Occludens 1 (ZO-1) and Na+/K+ATPase$\$1 (ATPA1) on the apical surface in monolayer culture,and produced the key proteins of Descemet's membrane,Collagen VIII$\$1 and VIII$\$2 (COL8A1 and 8A2). Quantitative PCR analysis revealed expression of all corneal endothelial pump transcripts. hESC-CECs were 96% similar to primary human adult CECs by microarray analysis. CONCLUSION hESC-CECs are morphologically similar,express corneal endothelial cell markers and express a nearly identical complement of genes compared to human adult corneal endothelial cells. hESC-CECs may be a suitable alternative to donor-derived corneal endothelium. View Publication