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BACKGROUND Strategies on the basis of doxycycline-inducible lentiviruses in mouse cells allowed the examination of mechanisms governing somatic cell reprogramming. RESULTS Using a doxycycline-inducible human reprogramming system,we identified unreported miRs enhancing reprogramming efficiency. CONCLUSION We generated a drug-inducible human reprogramming reporter system as an invaluable tool for genetic or chemical screenings. SIGNIFICANCE These cellular systems provide a tool to enable the advancement of reprogramming technologies in human cells. Reprogramming of somatic cells into induced pluripotent stem cells is achieved by the expression of defined transcription factors. In the last few years,reprogramming strategies on the basis of doxycycline-inducible lentiviruses in mouse cells became highly powerful for screening purposes when the expression of a GFP gene,driven by the reactivation of endogenous stem cell specific promoters,was used as a reprogramming reporter signal. However,similar reporter systems in human cells have not been generated. Here,we describe the derivation of drug-inducible human fibroblast-like cell lines that express different subsets of reprogramming factors containing a GFP gene under the expression of the endogenous OCT4 promoter. These cell lines can be used to screen functional substitutes for reprogramming factors or modifiers of reprogramming efficiency. As a proof of principle of this system,we performed a screening of a library of pluripotent-enriched microRNAs and identified hsa-miR-519a as a novel inducer of reprogramming efficiency. View Publication

Hematopoiesis depends on the association of hematopoietic stem cells with stromal cells that constitute the hematopoietic microenvironment. The in vitro development of the endothelial cell from umbilical cord blood (UCB) is not well established and has met very limited success. In this study,UCB CD34(+) cells were cultured for 5 weeks in a stroma-free liquid culture system using thrombopoietin,flt3 ligand,and granulocyte-colony stimulating factor. By week 4-5,we found that firmly adherent fibroblast-like cells were established. These cells showed characteristics of endothelial cells expressing von Willebrand factor,human vascular cell adhesion molecule-1,human intracellular adhesion molecule-1,human CD31,E-selectin,and human macrophage. Furthermore,when comparing an ex vivo system without an established endothelial monolayer to an ex vivo system with an established endothelial monolayer,better expansion of total nucleated cells,CD34(+) cells,and colony-forming units (CFUs)-granulocyte-macrophage and CFUs-granulocyte-erythroid-megakaryocyte-macrophage were found during culture. This phenomenon was in part due to the fact that a significant reduction of apoptotic fractions was found in the CD34(+) cells,which were cultured on the adherent monolayer for up to 5 weeks. To gather quantitative data on the number of endothelial cells derived from a given number of CD34 cells,we performed limiting dilution assay by using Poisson distribution: the number of tested cells (linear scale) producing a 37% negative culture (logarithmic scale) is the number of cells containing one endothelial cell. By this method,one endothelial cell may be found from 314 CD34(+) cells after 5 weeks of culture. These results suggest that the UCB CD34(+) cell fraction contains endothelial cell precursors,establishing the hematopoietic microenvironment and providing the beneficial effects through downregulating apoptosis on UCB expansion protocols. These observations may provide insight for future cellular therapy or graft engineering. View Publication

Induced pluripotent stem (iPS) cells are being used increasingly to complement their embryonic counterparts to understand and develop the therapeutic potential of pluripotent cells. Our objectives were to identify an efficient cardiac differentiation protocol for human iPS cells as monolayers,and demonstrate that the resulting cardiac progenitors could provide a therapeutic benefit in a rodent model of myocardial infarction. Herein,we describe a 14-day protocol for efficient cardiac differentiation of human iPS cells as a monolayer,which routinely yielded a mixed population in which over 50% were cardiomyocytes,endothelium,or smooth muscle cells. When differentiating,cardiac progenitors from day 6 of this protocol were injected into the peri-infarct region of the rat heart; after coronary artery ligation and reperfusion,we were able to show that human iPS cell-derived cardiac progenitor cells engrafted,differentiated into cardiomyocytes and smooth muscle,and persisted for at least 10 weeks postinfarct. Hearts injected with iPS-derived cells showed a nonsignificant trend toward protection from decline in function after myocardial infarction,as assessed by magnetic resonance imaging at 10 weeks,such that the ejection fraction at 10 weeks in iPS treated hearts was 62%±4%,compared to that of control infarcted hearts at 45%±9% (Ptextless0.2). In conclusion,we demonstrated efficient cardiac differentiation of human iPS cells that gave rise to progenitors that were retained within the infarcted rat heart,and reduced remodeling of the heart after ischemic damage. View Publication

Recent genomic approaches have revealed that the repertoire of RNA Pol III-transcribed genes varies in different human cell types,and that this variation is likely determined by a combination of the chromatin landscape,cell-specific DNA-binding transcription factors,and collaboration with RNA Pol II. Although much is known about this regulation in differentiated human cells,there is presently little understanding of this aspect of the Pol III system in human ES cells. Here,we determine the occupancy profiles of Pol III components in human H1 ES cells,and also induced pluripotent cells,and compare to known profiles of chromatin,transcription factors,and RNA expression. We find a relatively large fraction of the Pol III repertoire occupied in human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). In ES cells we find clear correlations between Pol III occupancy and active chromatin. Interestingly,we find a highly significant fraction of Pol III-occupied genes with adjacent binding events by pluripotency factors in ES cells,especially NANOG. Notably,in human ES cells we find H3K27me3 adjacent to but not overlapping many active Pol III loci. We observe in all such cases,a peak of H3K4me3 and/or RNA Pol II,between the H3K27me3 and Pol III binding peaks,suggesting that H3K4me3 and Pol II activity may “insulate�? Pol III from neighboring repressive H3K27me3. Further,we find iPSCs have a larger Pol III repertoire than their precursors. Finally,the active Pol III genome in iPSCs is not completely reprogrammed to a hESC like state and partially retains the transcriptional repertoire of the precursor. Together,our correlative results are consistent with Pol III binding and activity in human ES cells being enabled by active/permissive chromatin that is shaped in part by the pluripotency network of transcription factors and RNA Pol II activity. View Publication

Realization of clinical potential of human pluripotent stem cells (hPSCs) in bone regenerative medicine requires development of simple and safe biomaterials for expansion of hPSCs followed by directing their lineage commitment to osteoblasts. In the present study,a chemically defined peptide-decorated polycaprolactone (PCL) nanofibrous microenvironment was prepared through electrospinning technology and subsequent conjugation with vitronectin peptide to promote the culture and osteogenic potential of hPSCs in vitro. The results indicated that hPSCs successfully proliferated and maintained their pluripotency on the biointerface of peptide-conjugated nanofibers without Matrigel under defined conditions. Moreover,the prepared niche exhibited an appealing ability in promoting directed differentiation of hPSCs to osteoblastic phenotype without embryoid body formation step,determined from the cell morphological alteration,alkaline phosphate activity,and osteogenesis-related gene expression,as well as protein production. Such well-defined,xeno-free,and safe nanofiber scaffolds that allow the survival and facilitate osteo-differentiation of hPSCs provide a novel platform for hPSCs differentiation via cell-nanofiber interplay,and possess great value in accelerating the translational perspectives of hPSCs in bone tissue engineering. View Publication

Predictive toxicology using stem cells or their derived tissues has gained increasing importance in biomedical and pharmaceutical research. Here,we show that toxicity category prediction by support vector machines (SVMs),which uses qRT-PCR data from 20 categorized chemicals based on a human embryonic stem cell (hESC) system,is improved by the adoption of gene networks,in which network edge weights are added as feature vectors when noisy qRT-PCR data fail to make accurate predictions. The accuracies of our system were 97.5-100% for three toxicity categories: neurotoxins (NTs),genotoxic carcinogens (GCs) and non-genotoxic carcinogens (NGCs). For two uncategorized chemicals,bisphenol-A and permethrin,our system yielded reasonable results: bisphenol-A was categorized as an NGC,and permethrin was categorized as an NT; both predictions were supported by recently published papers. Our study has two important features: (i) as the first study to employ gene networks without using conventional quantitative structure-activity relationships (QSARs) as input data for SVMs to analyze toxicogenomics data in an hESC validation system,it uses additional information of gene-to-gene interactions to significantly increase prediction accuracies for noisy gene expression data; and (ii) using only undifferentiated hESCs,our study has considerable potential to predict late-onset chemical toxicities,including abnormalities that occur during embryonic development. View Publication

Human induced pluripotent stem cells (iPSCs) can be derived from various types of somatic cells by transient overexpression of 4 Yamanaka factors (OCT4,SOX2,C-MYC,and KLF4). Patient-specific iPSC derivatives (e.g.,neuronal,cardiac,hepatic,muscular,and endothelial cells [ECs]) hold great promise in drug discovery and regenerative medicine. In this study,we aimed to evaluate whether the cellular origin can affect the differentiation,in vivo behavior,and single-cell gene expression signatures of human iPSC-derived ECs. We derived human iPSCs from 3 types of somatic cells of the same individuals: fibroblasts (FB-iPSCs),ECs (EC-iPSCs),and cardiac progenitor cells (CPC-iPSCs). We then differentiated them into ECs by sequential administration of Activin,BMP4,bFGF,and VEGF. EC-iPSCs at early passage (10 textless P textless 20) showed higher EC differentiation propensity and gene expression of EC-specific markers (PECAM1 and NOS3) than FB-iPSCs and CPC-iPSCs. In vivo transplanted EC-iPSC-ECs were recovered with a higher percentage of CD31(+) population and expressed higher EC-specific gene expression markers (PECAM1,KDR,and ICAM) as revealed by microfluidic single-cell quantitative PCR (qPCR). In vitro EC-iPSC-ECs maintained a higher CD31(+) population than FB-iPSC-ECs and CPC-iPSC-ECs with long-term culturing and passaging. These results indicate that cellular origin may influence lineage differentiation propensity of human iPSCs; hence,the somatic memory carried by early passage iPSCs should be carefully considered before clinical translation. View Publication

Human pluripotent stem cells (hPSCs) provide a valuable model for the study of human development and a means to generate a scalable source of cells for therapeutic applications. This protocol specifies cell fate efficiently into cardiac and endothelial lineages from hPSCs. The protocol takes 2 weeks to complete and requires experience in hPSC culture and differentiation techniques. Building on lessons taken from early development,this monolayer-directed differentiation protocol uses different concentrations of activin A and bone morphogenetic protein 4 (BMP4) to polarize cells into mesodermal subtypes that reflect mid-primitive-streak cardiogenic mesoderm and posterior-primitive-streak hemogenic mesoderm. This differentiation platform provides a basis for generating distinct cardiovascular progenitor populations that enable the derivation of cardiomyocytes and functionally distinct endothelial cell (EC) subtypes from cardiogenic versus hemogenic mesoderm with high efficiency without cell sorting. ECs derived from cardiogenic and hemogenic mesoderm can be matured into textgreater90% CD31(+)/VE-cadherin(+) definitive ECs. To test the functionality of ECs at different stages of differentiation,we provide methods for assaying the blood-forming potential and de novo lumen-forming activity of ECs. To our knowledge,this is the first protocol that provides a common platform for directed differentiation of cardiomyocytes and endothelial subtypes from hPSCs. This protocol yields endothelial differentiation efficiencies exceeding those of previously published protocols. Derivation of these cell types is a critical step toward understanding the basis of disease and generating cells with therapeutic potential. View Publication

Human induced pluripotent stem cells (iPSCs) are a potential source of hepatocytes for liver transplantation to treat end-stage liver disease. In vitro differentiation of human iPSCs into hepatic cells has been achieved using a multi- stage differentiation protocol,but whether these cells are functional and capable of engrafting and regenerating diseased liver tissue is not clear. We show that human iPSC-derived hepatic cells at various differentiation stages can engraft the liver in a mouse transplantation model. Using the same differentiation and transplantation protocols,we also assessed the ability of human iPSCs derived from each of the three developmental germ layer tissues (that is,ectoderm,mesoderm,and endoderm) to regenerate mouse liver. These iPSC lines,with similar but distinct global DNA methylation patterns,differentiated into multistage hepatic cells with an efficiency similar to that of human embryonic stem cells. Human hepatic cells at various differentiation stages derived from iPSC lines of different origins successfully repopulated the liver tissue of mice with liver cirrhosis. They also secreted human-specific liver proteins into mouse blood at concentrations comparable to that of proteins secreted by human primary hepato- cytes. Our results demonstrate the engraftment and liver regenerative capabilities of human iPSC-derived multi- stage hepatic cells in vivo and suggest that human iPSCs of distinct origins and regardless of their parental epigenetic memory can efficiently differentiate along the hepatic lineage. 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

Human pluripotent stem cell (hPSC-) derived cardiomyocytes have potential applications in drug discovery,toxicity testing,developmental studies,and regenerative medicine. Before these cells can be reliably utilized,characterization of their functionality is required to establish their similarity to native cardiomyocytes. We tracked fluorescent beads embedded in 4.4-99.7 kPa polyacrylamide hydrogels beneath contracting neonatal rat cardiomyocytes and cardiomyocytes generated from hPSCs via growth-factor-induced directed differentiation to measure contractile output in response to changes in substrate mechanics. Contraction stress was determined using traction force microscopy,and morphology was characterized by immunocytochemistry for α-actinin and subsequent image analysis. We found that contraction stress of all types of cardiomyocytes increased with substrate stiffness. This effect was not linked to beating rate or morphology. We demonstrated that hPSC-derived cardiomyocyte contractility responded appropriately to isoprenaline and remained stable in culture over a period of 2 months. This study demonstrates that hPSC-derived cardiomyocytes have appropriate functional responses to substrate stiffness and to a pharmaceutical agent,which motivates their use in further applications such as drug evaluation and cardiac therapies. View Publication

The expression and function of several multidrug transporters (including ABCB1 and ABCG2) have been studied in human cancer cells and in mouse and human adult stem cells. However,the expression of ABCG2 in human embryonic stem cells (hESCs) remains unclear. Limited and contradictory results in the literature from two research groups have raised questions regarding its expression and function. In this study,we used quantitative real-time PCR,Northern blots,whole genome RNA sequencing,Western blots,and immunofluorescence microscopy to study ABCG2 expression in hESCs. We found that full-length ABCG2 mRNA transcripts are expressed in undifferentiated hESC lines. However,ABCG2 protein was undetectable even under embryoid body differentiation or cytotoxic drug induction. Moreover,surface ABCG2 protein was coexpressed with the differentiation marker stage-specific embryonic antigen-1 of hESCs,following constant BMP-4 signaling at days 4 and 6. This expression was tightly correlated with the downregulation of two microRNAs (miRNAs) (i.e.,hsa-miR-519c and hsa-miR-520h). Transfection of miRNA mimics and inhibitors of these two miRNAs confirmed their direct involvement in the regulation ABCG2 translation. Our findings clarify the controversy regarding the expression of the ABCG2 gene and also provide new insights into translational control of the expression of membrane transporter mRNAs by miRNAs in hESCs. View Publication