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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

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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

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

OBJECTIVE: In murine hematopoietic tissue,direct repopulation experiments have demonstrated that the side population (SP) represents a remarkable enrichment of hematopoietic stem cells. Human SP has been phenotyped as negative for lineage antigens as well as CD34. However,in the 9 years since the original publication,no long-term hematopoietic reconstitution has been reported for the adult human SP/CD34(-) subset. Elevated levels of aldehyde dehydrogenase (ALDH) have been demonstrated in murine and human progenitor cells when compared to other hematopoietic cells. METHODS: Here,we report the phenotype of human cord blood SP cells. We established the technique of simultaneous phenotyping,Hoechst exclusion,and ALDH labeling on murine tissues. We then performed the simultaneous analysis of phenotype,SP,and ALDH activity on human cord blood and bone marrow cells. Finally,we analyzed the phenotype and functional potential of human cord blood ALDH(+) cells to determine whether Lin(-)/CD34(-) cells are identified via this technique. RESULTS: We demonstrate that human Lin(-)/CD34(-)/ALDH(+) cells are capable of long-term repopulation. Although the SP technique identifies cells that overlap with the ALDH(+) cell population,this is restricted to the CD34(+) cell subset. CONCLUSION: Hoechst exclusion ability does not seem to be the method of choice for the isolation of human hematopoietic stem cells. View Publication

BACKGROUND Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) are in the foreground as a preferable application for treating diseases. However,the safety of hUC-MSCs after long-term culturing in vitro in serum-free medium remains unclear. METHODS hUC-MSCs were separated by adherent tissue culture. hUC-MSCs were cultured in serum-free MesenCult-XF medium and FBS-bases DMEM complete medium. At the 1st,3rd,5th,8th,10th,and 15th passage,the differentiation of MSCs into osteogenic,chondrogenic,and adipogenic cells was detected,and MTT,surface antigens were measured. Tumorigenicity was analyzed at the 15th passage. Conventional karyotyping was performed at passage 0,8,and 15. The telomerase activity of hUC-MSCs at passage 1-15 was analyzed. RESULTS Flow cytometry analysis showed that very high expression was detected for CD105,CD73,and CD90 and very low expression for CD45,CD34,CD14,CD79a,and HLA-DR. MSCs could differentiate into osteocytes,chondrocytes,and adipocytes in vitro. There was no obvious chromosome elimination,displacement,or chromosomal imbalance as determined from the guidelines of the International System for Human Cytogenetic Nomenclature. Telomerase activity was down-regulated significantly when the culture time was prolonged. Further,no tumors formed in rats injected with hUC-MSCs (P15) cultured in serum-free and in serum-containing conditions. CONCLUSION Our data showed that hUC-MSCs met the International Society for Cellular Therapy standards for conditions of long-term in vitro culturing at P15. Since hUC-MSCs can be safely expanded in vitro and are not susceptible to malignant transformation in serum-free medium,these cells are suitable for cell therapy. 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

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