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The discovery of induced pluripotent stem cells(iPSCs) is a promising advancement in the field of regenerative medicine. Previous studies have indicated that the teratoma-forming propensity of iPSCs is variable; however,the relationship between tumorigenic potential and genomic instability in human iPSCs (HiPSCs) remains to be fully elucidated. Here,we evaluated the malignant potential of HiPSCs by using both colony formation assays and tumorigenicity tests. We demonstrated that HiPSCs formed tumorigenic colonies when grown in cancer cell culture medium and produced malignancies in immunodeficient mice. Furthermore,we analyzed genomic instability in HiPSCs using whole-genome copy number variation analysis and determined that the extent of genomic instability was related with both the cells' propensity to form colonies and their potential for tumorigenesis. These findings indicate a risk for potential malignancy of HiPSCs derived from genomic instability and suggest that quality control tests,including comprehensive tumorigenicity assays and genomic integrity validation,should be rigorously executed before the clinical application of HiPSCs. In addition,HiPSCs should be generated through the use of combined factors or other approaches that decrease the likelihood of genomic instability. View Publication

BACKGROUND: Neural induction of human pluripotent stem cells often yields heterogeneous cell populations that can hamper quantitative and comparative analyses. There is a need for improved differentiation and enrichment procedures that generate highly pure populations of neural stem cells (NSC),glia and neurons. One way to address this problem is to identify cell-surface signatures that enable the isolation of these cell types from heterogeneous cell populations by fluorescence activated cell sorting (FACS). METHODOLOGY/PRINCIPAL FINDINGS: We performed an unbiased FACS- and image-based immunophenotyping analysis using 190 antibodies to cell surface markers on naïve human embryonic stem cells (hESC) and cell derivatives from neural differentiation cultures. From this analysis we identified prospective cell surface signatures for the isolation of NSC,glia and neurons. We isolated a population of NSC that was CD184(+)/CD271(-)/CD44(-)/CD24(+) from neural induction cultures of hESC and human induced pluripotent stem cells (hiPSC). Sorted NSC could be propagated for many passages and could differentiate to mixed cultures of neurons and glia in vitro and in vivo. A population of neurons that was CD184(-)/CD44(-)/CD15(LOW)/CD24(+) and a population of glia that was CD184(+)/CD44(+) were subsequently purified from cultures of differentiating NSC. Purified neurons were viable,expressed mature and subtype-specific neuronal markers,and could fire action potentials. Purified glia were mitotic and could mature to GFAP-expressing astrocytes in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: These findings illustrate the utility of immunophenotyping screens for the identification of cell surface signatures of neural cells derived from human pluripotent stem cells. These signatures can be used for isolating highly pure populations of viable NSC,glia and neurons by FACS. The methods described here will enable downstream studies that require consistent and defined neural cell populations. View Publication

We present an integrated platform comprised of a biomimetic substrate and physiologically aligned human pluripotent stem cell-derived cardiomyocytes (CMs) with optical detection and algorithms to monitor subtle changes in cardiac properties under various conditions. In the native heart,anisotropic tissue structures facilitate important concerted mechanical contraction and electrical propagation. To recapitulate the architecture necessary for a physiologically accurate heart response,we have developed a simple way to create large areas of aligned CMs with improved functional properties using shrink-wrap film. Combined with simple bright field imaging,obviating the need for fluorescent labels or beads,we quantify and analyze key cardiac contractile parameters. To evaluate the performance capabilities of this platform,the effects of two drugs,E-4031 and isoprenaline,were examined. Cardiac cells supplemented with E-4031 exhibited an increase in contractile duration exclusively due to prolonged relaxation peak. Notably,cells aligned on the biomimetic platform responded detectably down to a dosage of 3nm E-4031,which is lower than the IC50 in the hERG channel assay. Cells supplemented with isoprenaline exhibited increased contractile frequency and acceleration. Interestingly,cells grown on the biomimetic substrate were more responsive to isoprenaline than those grown on the two control surfaces,suggesting topography may help induce more mature ion channel development. This simple and low-cost platform could thus be a powerful tool for longitudinal assays as well as an effective tool for drug screening and basic cardiac research. ?? 2013 Elsevier Ltd. View Publication

Here we introduce the representative method to culture HESCs under the feeder and feeder-free conditions,the former of which is used to maintain or expand undifferentiated HESCs,and the latter can be used for the preparation of pure HESCs RNA samples,or for screening factors influential on self-renewal of HESCs. We also describe a protocol and tips for conducting gene chip analysis focusing on widely used Affymetrix Microarrays. These techniques will provide us unprecedented scale of biological information that would illuminate a key to decipher complex signaling networks controlling pluripotency. View Publication

There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support,but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study,five separate laboratories,each with experience in human embryonic stem cell culture,used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods,with propagation in the presence of Knockout Serum Replacer,FGF-2,and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment,death,and differentiated morphology by phase contrast microscopy,for growth by serial cell counts,and for maintenance of stem cell surface marker expression by flow cytometry. Of the eight culture systems,only the control and those based on two commercial media,mTeSR1 and STEMPRO,supported maintenance of most cell lines for ten passages. Cultures grown in the remaining media failed before this point due to lack of attachment,cell death,or overt cell differentiation. Possible explanations for relative success of the commercial formulations in this study,and the lack of success with other formulations from academic groups compared to previously published results,include: the complex combination of growth factors present in the commercial preparations; improved development,manufacture,and quality control in the commercial products; differences in epigenetic adaptation to culture in vitro between different ES cell lines grown in different laboratories. View Publication

Human embryonic stem (hES) cell production of heparan sulfate influences cell fate and pluripotency. Human ES cells remain pluripotent in vitro through the action of growth factors signaling,and the activity of these factors depends on interaction with specific receptors and also with heparan sulfate. Here,we tested the hypothesis that matrix-associated heparan sulfate is enough to maintain hES cells under low fibroblast growth factor-2 concentration in the absence of live feeder cells. To pursue this goal,we compared hES cells cultured either on coated plates containing live murine embryonic fibroblasts (MEFs) or on a matrix derived from ethanol-fixed MEFs. hES cells were analyzed for the expression of pluripotency markers and the ability to form embryoid bodies. hES cells cultured either on live mouse fibroblasts or onto a matrix derived from fixed fibroblasts expressed similar levels of Oct-4,SOX-2,Nanog,TRA-1-60 and SSEA-4,and they were also able to form cavitated embryoid bodies. Heparan sulfate-depleted matrix lost the ability to support the adherence and growth of hES cells,confirming that this glycosaminoglycan,bound to the extracellular matrix,is enough for the growth and attachment of hES cells. Finally,we observed that the ethanol-fixed matrix decreases by 30% the levels of Neu5Gc in hES cells,indicating that this procedure reduces xeno-contamination. Our data suggest that matrix-bound heparan sulfate is required for the growth and pluripotency of hES cells and that ethanol-fixed MEFs may be used as a live cell"-free substrate for stem cells." View Publication

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RATIONALE: Molecular imaging has proven to be a vital tool in the characterization of stem cell behavior in vivo. However,the integration of reporter genes has typically relied on random integration,a method that is associated with unwanted insertional mutagenesis and positional effects on transgene expression.backslashnbackslashnOBJECTIVE: To address this barrier,we used genome editing with zinc finger nuclease (ZFN) technology to integrate reporter genes into a safe harbor gene locus (PPP1R12C,also known as AAVS1) in the genome of human embryonic stem cells and human induced pluripotent stem cells for molecular imaging.backslashnbackslashnMETHODS AND RESULTS: We used ZFN technology to integrate a construct containing monomeric red fluorescent protein,firefly luciferase,and herpes simplex virus thymidine kinase reporter genes driven by a constitutive ubiquitin promoter into a safe harbor locus for fluorescence imaging,bioluminescence imaging,and positron emission tomography imaging,respectively. High efficiency of ZFN-mediated targeted integration was achieved in both human embryonic stem cells and induced pluripotent stem cells. ZFN-edited cells maintained both pluripotency and long-term reporter gene expression. Functionally,we successfully tracked the survival of ZFN-edited human embryonic stem cells and their differentiated cardiomyocytes and endothelial cells in murine models,demonstrating the use of ZFN-edited cells for preclinical studies in regenerative medicine.backslashnbackslashnCONCLUSION: Our study demonstrates a novel application of ZFN technology to the targeted genetic engineering of human pluripotent stem cells and their progeny for molecular imaging in vitro and in vivo. View Publication

The identification of stem cells within a mixed population of cells is a major hurdle for stem cell biology--in particular,in the identification of induced pluripotent stem (iPS) cells during the reprogramming process. Based on the selective expression of stem cell surface markers,a method to specifically infect stem cells through antibody-conjugated lentiviral particles has been developed that can deliver both visual markers for live-cell imaging as well as selectable markers to enrich for iPS cells. Antibodies recognizing SSEA4 and CD24 mediated the selective infection of the iPS cells over the parental human fibroblasts,allowing for rapid expansion of these cells by puromycin selection. Adaptation of the vector allows for the selective marking of human embryonic stem (hES) cells for their removal from a population of differentiated cells. This method has the benefit that it not only identifies stem cells,but that specific genes,including positive and negative selection markers,regulatory genes or miRNA can be delivered to the targeted stem cells. The ability to specifically target gene delivery to human pluripotent stem cells has broad applications in tissue engineering and stem cell therapies. View Publication

The emerging models of human embryonic stem cell (hESC) self-organizing organoids provide a valuable in vitro platform for studying self-organizing processes that presumably mimic in vivo human developmental events. Here we report that through a chemical screen,we identified two novel and structurally similar small molecules BIR1 and BIR2 which robustly induced the self-organization of a balloon-shaped three-dimensional structure when applied to two-dimensional adherent hESC cultures in the absence of growth factors. Gene expression analyses and functional assays demonstrated an endothelial identity of this balloon-like structure,while cell surface marker analyses revealed a VE-cadherin+CD31+CD34+KDR+CD43???putative endothelial progenitor population. Furthermore,molecular marker labeling and morphological examinations characterized several other distinct DiI-Ac-LDL+multi-cellular modules and a VEGFR3+sprouting structure in the balloon cultures that likely represented intermediate structures of balloon-formation. View Publication

The ability to yield glucose-responsive pancreatic beta-cells from human pluripotent stem cells in vitro will facilitate the development of the cell replacement therapies for the treatment of Type 1 Diabetes. Here,through the sequential in vitro targeting of selected signaling pathways,we have developed an abbreviated five-stage protocol (25-30 days) to generate human Embryonic Stem Cell-Derived Beta-like Cells (ES-DBCs). We showed that Geltrex,as an extracellular matrix,could support the generation of ES-DBCs more efficiently than that of the previously described culture systems. The activation of FGF and Retinoic Acid along with the inhibition of BMP,SHH and TGF-beta led to the generation of 75% NKX6.1+/NGN3+ Endocrine Progenitors. The inhibition of Notch and tyrosine kinase receptor AXL,and the treatment with Exendin-4 and T3 in the final stage resulted in 35% mono-hormonal insulin positive cells,1% insulin and glucagon positive cells and 30% insulin and NKX6.1 co-expressing cells. Functionally,ES-DBCs were responsive to high glucose in static incubation and perifusion studies,and could secrete insulin in response to successive glucose stimulations. Mitochondrial metabolic flux analyses using Seahorse demonstrated that the ES-DBCs could efficiently metabolize glucose and generate intracellular signals to trigger insulin secretion. In conclusion,targeting selected signaling pathways for 25-30 days was sufficient to generate ES-DBCs in vitro. The ability of ES-DBCs to secrete insulin in response to glucose renders them a promising model for the in vitro screening of drugs,small molecules or genes that may have potential to influence beta-cell function. View Publication