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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

Disturbances in neuronal differentiation and function are an underlying factor of many brain disorders. Zinc homeostasis and signaling are important mediators for a normal brain development and function,given that zinc deficiency was shown to result in cognitive and emotional deficits in animal models that might be associated with neurodevelopmental disorders. One underlying mechanism of the observed detrimental effects of zinc deficiency on the brain might be impaired proliferation and differentiation of stem cells participating in neurogenesis. Thus,to examine the molecular mechanisms regulating zinc metabolism and signaling in differentiating neurons,using a protocol for motor neuron differentiation,we characterized the expression of zinc homeostasis genes during neurogenesis using human induced pluripotent stem cells (hiPSCs) and evaluated the influence of altered zinc levels on the expression of zinc homeostasis genes,cell survival,cell fate,and neuronal function. Our results show that zinc transporters are highly regulated genes during neuronal differentiation and that low zinc levels are associated with decreased cell survival,altered neuronal differentiation,and,in particular,synaptic function. We conclude that zinc deficiency in a critical time window during brain development might influence brain function by modulating neuronal differentiation. View Publication

Mutations in WD40-repeat protein 62 (WDR62) are commonly associated with primary microcephaly and other developmental cortical malformations. We used human pluripotent stem cells (hPSC) to examine WDR62 function during human neural differentiation and model early stages of human corticogenesis. Neurospheres lacking WDR62 expression showed decreased expression of intermediate progenitor marker,TBR2,and also glial marker,S100β. In contrast,inhibition of c-Jun N-terminal kinase (JNK) signalling during hPSC neural differentiation induced upregulation of WDR62 with a corresponding increase in neural and glial progenitor markers,PAX6 and EAAT1,respectively. These findings may signify a role of WDR62 in specifying intermediate neural and glial progenitors during human pluripotent stem cell differentiation. View Publication

Neurogenin3+ (Ngn3+) progenitor cells in the developing pancreas give rise to five endocrine cell types secreting insulin,glucagon,somatostatin,pancreatic polypeptide and ghrelin. Gastrin is a hormone produced primarily by G-cells in the stomach,where it functions to stimulate acid secretion by gastric parietal cells. Gastrin is expressed in the embryonic pancreas and is common in islet cell tumors,but the lineage and regulators of pancreatic gastrin+ cells are not known. We report that gastrin is abundantly expressed in the embryonic pancreas and disappears soon after birth. Some gastrin+ cells in the developing pancreas co-express glucagon,ghrelin or pancreatic polypeptide,but many gastrin+ cells do not express any other islet hormone. Pancreatic gastrin+ cells express the transcription factors Nkx6.1,Nkx2.2 and low levels of Pdx1,and derive from Ngn3+ endocrine progenitor cells as shown by genetic lineage tracing. Using mice deficient for key transcription factors we show that gastrin expression depends on Ngn3,Nkx2.2,NeuroD1 and Arx,but not Pax4 or Pax6. Finally,gastrin expression is induced upon differentiation of human embryonic stem cells to pancreatic endocrine cells expressing insulin. Thus,gastrin+ cells are a distinct endocrine cell type in the pancreas and an alternative fate of Ngn3+ cells. View Publication

Tunneling nanotubes (TnTs) are long,non-adherent,actin-based cellular extensions that act as conduits for transport of cellular cargo between connected cells. The mechanisms of nanotube formation and the effects of the tumor microenvironment and cellular signals on TnT formation are unknown. In the present study,we explored exosomes as potential mediators of TnT formation in mesothelioma and the potential relationship of lipid rafts to TnT formation. Mesothelioma cells co-cultured with exogenous mesothelioma-derived exosomes formed more TnTs than cells cultured without exosomes within 24-48. h; and this effect was most prominent in media conditions (low-serum,hyperglycemic medium) that support TnT formation (1.3-1.9-fold difference). Fluorescence and electron microscopy confirmed the purity of isolated exosomes and revealed that they localized predominantly at the base of and within TnTs,in addition to the extracellular environment. Time-lapse microscopic imaging demonstrated uptake of tumor exosomes by TnTs,which facilitated intercellular transfer of these exosomes between connected cells. Mesothelioma cells connected via TnTs were also significantly enriched for lipid rafts at nearly a 2-fold higher number compared with cells not connected by TnTs. Our findings provide supportive evidence of exosomes as potential chemotactic stimuli for TnT formation,and also lipid raft formation as a potential biomarker for TnT-forming cells. textcopyright 2014 Elsevier Inc. View Publication

The generation of functional retinal pigment epithelium (RPE) is of great therapeutic interest to the field of regenerative medicine and may provide possible cures for retinal degenerative diseases,including age-related macular degeneration (AMD). Although RPE cells can be produced from either embryonic stem cells or induced pluripotent stem cells,direct cell reprogramming driven by lineagedetermining transcription factors provides an immediate route to their generation. By monitoring a human RPE specific Best1::GFP reporter,we report the conversion of human fibroblasts into RPE lineage using defined sets of transcription factors. We found that Best1::GFP positive cells formed colonies and exhibited morphological and molecular features of early stage RPE cells. Moreover,they were able to obtain pigmentation upon activation of Retinoic acid (RA) and Sonic Hedgehog (SHH) signaling pathways. Our study not only established an ideal platform to investigate the transcriptional network regulating the RPE cell fate determination,but also provided an alternative strategy to generate functional RPE cells that complement the use of pluripotent stem cells for disease modeling,drug screening,and cell therapy of retinal degeneration. View Publication