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Human embryonic stem cells (hESCs) require specific niches for adhesion,expansion,and lineage-specific differentiation. In this study,we showed that a membrane substrate offers better tissue niches for hESC attachment,spreading,proliferation,and differentiation. The cell doubling time was shortened from 46.3±5.7 h for hESCs grown on solid substrates to 25.6±2.6 h for those on polyester (PE) membrane substrates with pore size of 0.4 μm. In addition,we observed an increase of approximately five- to ninefold of definitive endoderm marker gene expression in hESCs differentiated on PE or polyethylene terephthalate membrane substrates. Global gene expression analysis revealed upregulated expressions of a number of extracellular matrix and cell adhesion molecules in hESCs grown on membrane substrates. Further,an enhanced nuclear translocation of β-catenin was detected in these cells. These observations suggested the augmentation of Wnt signaling in hESCs grown on membrane substrates. These results also demonstrated that a membrane substrate can offer better physicochemical cues for enhancing in vitro hESC attachment,proliferation,and differentiation. View Publication

The mechanisms by which human embryonic stem cells (hESC) differentiate to endodermal lineage have not been extensively studied. Mathematical models can aid in the identification of mechanistic information. In this work we use a population-based modeling approach to understand the mechanism of endoderm induction in hESC,performed experimentally with exposure to Activin A and Activin A supplemented with growth factors (basic fibroblast growth factor (FGF2) and bone morphogenetic protein 4 (BMP4)). The differentiating cell population is analyzed daily for cellular growth,cell death,and expression of the endoderm proteins Sox17 and CXCR4. The stochastic model starts with a population of undifferentiated cells,wherefrom it evolves in time by assigning each cell a propensity to proliferate,die and differentiate using certain user defined rules. Twelve alternate mechanisms which might describe the observed dynamics were simulated,and an ensemble parameter estimation was performed on each mechanism. A comparison of the quality of agreement of experimental data with simulations for several competing mechanisms led to the identification of one which adequately describes the observed dynamics under both induction conditions. The results indicate that hESC commitment to endoderm occurs through an intermediate mesendoderm germ layer which further differentiates into mesoderm and endoderm,and that during induction proliferation of the endoderm germ layer is promoted. Furthermore,our model suggests that CXCR4 is expressed in mesendoderm and endoderm,but is not expressed in mesoderm. Comparison between the two induction conditions indicates that supplementing FGF2 and BMP4 to Activin A enhances the kinetics of differentiation than Activin A alone. This mechanistic information can aid in the derivation of functional,mature cells from their progenitors. While applied to initial endoderm commitment of hESC,the model is general enough to be applicable either to a system of adult stem cells or later stages of ESC differentiation. View Publication

The potential for human disease treatment using human pluripotent stem cells,including embryonic stem cells and induced pluripotent stem cells (iPSCs),also carries the risk of added genomic instability. Genomic instability is most often linked to DNA repair deficiencies,which indicates that screening/characterization of possible repair deficiencies in pluripotent human stem cells should be a necessary step prior to their clinical and research use. In this study,a comparison of DNA repair pathways in pluripotent cells,as compared to those in non-pluripotent cells,demonstrated that DNA repair capacities of pluripotent cell lines were more heterogeneous than those of differentiated lines examined and were generally greater. Although pluripotent cells had high DNA repair capacities for nucleotide excision repair,we show that ultraviolet radiation at low fluxes induced an apoptotic response in these cells,while differentiated cells lacked response to this stimulus,and note that pluripotent cells had a similar apoptotic response to alkylating agent damage. This sensitivity of pluripotent cells to damage is notable since viable pluripotent cells exhibit less ultraviolet light-induced DNA damage than do differentiated cells that receive the same flux. In addition,the importance of screening pluripotent cells for DNA repair defects was highlighted by an iPSC line that demonstrated a normal spectral karyotype,but showed both microsatellite instability and reduced DNA repair capacities in three out of four DNA repair pathways examined. Together,these results demonstrate a need to evaluate DNA repair capacities in pluripotent cell lines,in order to characterize their genomic stability,prior to their pre-clinical and clinical use. View Publication

Human embryonic stem cell-derived dendritic cells (hESC-DCs) may potentially provide a platform to generate off-the-shelf" therapeutic cancer vaccines. To apply hESC-DCs for cancer immunotherapy in a semiallogeneic setting� View Publication

Multiple studies show that tumor suppressor p53 is a barrier to dedifferentiation; whether this is strictly due to repression of proliferation remains a subject of debate. Here,we show that p53 plays an active role in promoting differentiation of human embryonic stem cells (hESCs) and opposing self-renewal by regulation of specific target genes and microRNAs. In contrast to mouse embryonic stem cells,p53 in hESCs is maintained at low levels in the nucleus,albeit in a deacetylated,inactive state. In response to retinoic acid,CBP/p300 acetylates p53 at lysine 373,which leads to dissociation from E3-ubiquitin ligases HDM2 and TRIM24. Stabilized p53 binds CDKN1A to establish a G(1) phase of cell cycle without activation of cell death pathways. In parallel,p53 activates expression of miR-34a and miR-145,which in turn repress stem cell factors OCT4,KLF4,LIN28A,and SOX2 and prevent backsliding to pluripotency. Induction of p53 levels is a key step: RNA-interference-mediated knockdown of p53 delays differentiation,whereas depletion of negative regulators of p53 or ectopic expression of p53 yields spontaneous differentiation of hESCs,independently of retinoic acid. Ectopic expression of p53R175H,a mutated form of p53 that does not bind DNA or regulate transcription,failed to induce differentiation. These studies underscore the importance of a p53-regulated network in determining the human stem cell state. View Publication

Precise,robust and scalable directed differentiation of pluripotent stem cells is an important goal with respect to disease modeling or future therapies. Using the AggreWell™400 system we have standardized the differentiation of human embryonic and induced pluripotent stem cells to a neuronal fate using defined conditions. This allows reproducibility in replicate experiments and facilitates the direct comparison of cell lines. Since the starting point for EB formation is a single cell suspension,this protocol is suitable for standard and novel methods of pluripotent stem cell culture. Moreover,an intermediate population of neural precursor cells,which are routinely textgreater95% NCAM(pos) and Tra-1-60(neg) by FACS analysis,may be expanded and frozen prior to differentiation allowing a convenient starting point for downstream experiments. View Publication

Enteroendocrine cells of the gastrointestinal (GI) tract play a central role in metabolism,digestion,satiety and lipid absorption,yet their development remains poorly understood. Here we show that Arx,a homeodomain-containing transcription factor,is required for the normal development of mouse and human enteroendocrine cells. Arx expression is detected in a subset of Neurogenin3 (Ngn3)-positive endocrine progenitors and is also found in a subset of hormone-producing cells. In mice,removal of Arx from the developing endoderm results in a decrease of enteroendocrine cell types including gastrin-,glucagon/GLP-1-,CCK-,secretin-producing cell populations and an increase of somatostatin-expressing cells. This phenotype is also observed in mice with endocrine-progenitor-specific Arx ablation suggesting that Arx is required in the progenitor for enteroendocrine cell development. In addition,depletion of human ARX in developing human intestinal tissue results in a profound deficit in expression of the enteroendocrine cell markers CCK,secretin and glucagon while expression of a pan-intestinal epithelial marker,CDX2,and other non-endocrine markers remained unchanged. Taken together,our findings uncover a novel and conserved role of Arx in mammalian endocrine cell development and provide a potential cause for the chronic diarrhea seen in both humans and mice carrying Arx mutations. View Publication

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4,Sox2,Klf4 and c-myc (OSKM) in the presence of sodium butyrate (1-3). We used this method to reprogram late passage (textgreaterp10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665,Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81,a surface marker of pluripotent cells,separated from non-reprogrammed fibroblasts and manually passaged (4,5). These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions,directly from the reprogramming plate. Starting from the first passage,hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4,as well as nuclear markers Oct3/4,Sox2 and Nanog. The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals( 6),human newborn fibroblasts,as well as human keratinocytes. View Publication

Embryonic stem cell (ESC) identity and self-renewal is maintained by extrinsic signaling pathways and intrinsic gene regulatory networks. Here,we show that three members of the Ccr4-Not complex,Cnot1,Cnot2,and Cnot3,play critical roles in maintaining mouse and human ESC identity as a protein complex and inhibit differentiation into the extraembryonic lineages. Enriched in the inner cell mass of blastocysts,these Cnot genes are highly expressed in ESC and downregulated during differentiation. In mouse ESCs,Cnot1,Cnot2,and Cnot3 are important for maintenance in both normal conditions and the 2i/LIF medium that supports the ground state pluripotency. Genetic analysis indicated that they do not act through known self-renewal pathways or core transcription factors. Instead,they repress the expression of early trophectoderm (TE) transcription factors such as Cdx2. Importantly,these Cnot genes are also necessary for the maintenance of human ESCs,and silencing them mainly lead to TE and primitive endoderm differentiation. Together,our results indicate that Cnot1,Cnot2,and Cnot3 represent a novel component of the core self-renewal and pluripotency circuitry conserved in mouse and human ESCs. View Publication

MicroRNAs (miRNA) comprise a group of short ribonucleic acid molecules implicated in regulation of key biological processes and functions at the post-transcriptional level. Ionizing radiation (IR) causes DNA damage and generally triggers cellular stress response. However,the role of miRNAs in IR-induced response in human embryonic stem cells (hESC) has not been defined yet. Here,by using system biology approaches,we show for the first time,that miRNAome undergoes global alterations in hESC (H1 and H9 lines) after IR. Interrogation of expression levels of 1,090 miRNA species in irradiated hESC showed statistically significant changes in 54 genes following 1 Gy of X-ray exposures; global miRNAome alterations were found to be highly temporally and cell line--dependent in hESC. Time-course studies showed that the 16 hr miRNAome radiation response of hESC is much more robust compared to 2 hr-response signature (only eight genes),and may be involved in regulating the cell cycle. Quantitative real-time PCR performed on some miRNA species confirms the robustness of our miRNA microarray platform. Positive regulation of differentiation-,cell cycle-,ion transport- and endomembrane system-related processes were predicted to be negatively affected by miRNAome changes in irradiated hESC. Our findings reveal a fundamental role of miRNAome in modulating the radiation response,and identify novel molecular targets of radiation in hESC. View Publication

HUCB (human umbilical cord blood) has been frequently used in clinical allogeneic HSC (haemopoietic stem cell) transplant. However,HUCB is poorly recognized as a rich source of MSC (mesenchymal stem cell). The aim of this study has been to establish a new method for isolating large number of MSC from HUCB to recognize it as a good source of MSC. HUCB samples were collected from women following their elective caesarean section. The new method (Clot Spot method) was carried out by explanting HUCB samples in mesencult complete medium and maintained in 37°C,in a 5% CO2 and air incubator. MSC presence was established by quantitative and qualitative immunophenotyping of cells and using FITC attached to MSC phenotypic markers (CD29,CD73,CD44 and CD105). Haematopoietic antibodies (CD34 and CD45) were used as negative control. MSC differentiation was examined in neurogenic and adipogenic media. Immunocytochemistry was carried out for the embryonic markers: SOX2 (sex determining region Y-box 2),OLIG-4 (oligodendrocyte-4) and FABP-4 (fatty acid binding protein-4). The new method was compared with the conventional Rosset Sep method. MSC cultures using the Clot Spot method showed 3-fold increase in proliferation rate compared with conventional method. Also,the cells showed high expression of MSC markers CD29,CD73,CD44 and CD105,but lacked the expression of specific HSC markers (CD34 and CD45). The isolated MSC showed some differentiation by expressing the neurogenic (SOX2 and Olig4) and adipogenic (FABP-4) markers respectively. In conclusion,HUCB is a good source of MSC using this new technique. View Publication

BACKGROUND: Fetal alcohol spectrum disorders (FASD) are a leading cause of neurodevelopmental disability. The mechanisms underlying FASD are incompletely understood,and biomarkers to identify those at risk are lacking. Here,we perform metabolomic analysis of embryoid bodies and neural lineages derived from human embryonic stem (hES) cells to identify the neural secretome produced in response to ethanol (EtOH) exposure. METHODS: WA01 and WA09 hES cells were differentiated into embryoid bodies,neural progenitors,or neurons. Cells along this progression were cultured for 4 days with 0,0.1,or 0.3% EtOH. Supernatants were subjected to C18 chromatography followed by ESI-QTOF-MS. Features were annotated using public databases,and the identities of 4 putative biomarkers were confirmed with purified standards and comparative MS/MS. RESULTS: EtOH treatment induced statistically significant changes to metabolite abundance in human embryoid bodies (180 features),neural progenitors (76 features),and neurons (42 features). There were no shared significant features between different cell types. Fifteen features showed a dose-response to EtOH. Four chemical identities were confirmed: L-thyroxine,5'-methylthioadenosine,and the tryptophan metabolites,L-kynurenine and indoleacetaldehyde. One feature with a putative annotation of succinyladenosine was significantly increased in both EtOH treatments. Additional features were selective to EtOH treatment but were not annotated in public databases. CONCLUSIONS: EtOH exposure induces statistically significant changes to the metabolome profile of human embryoid bodies,neural progenitors,and neurons. Several of these metabolites are normally present in human serum,suggesting their usefulness as potential serum FASD biomarkers. These findings suggest the biochemical pathways that are affected by EtOH in the developing nervous system and delineate mechanisms of alcohol injury during human development. View Publication