技术资料

Human cytomegalovirus (HCMV) infection is one of the leading prenatal causes of congenital mental retardation and deformities world-wide. Access to cultured human neuronal lineages,necessary to understand the species specific pathogenic effects of HCMV,has been limited by difficulties in sustaining primary human neuronal cultures. Human induced pluripotent stem (iPS) cells now provide an opportunity for such research. We derived iPS cells from human adult fibroblasts and induced neural lineages to investigate their susceptibility to infection with HCMV strain Ad169. Analysis of iPS cells,iPS-derived neural stem cells (NSCs),neural progenitor cells (NPCs) and neurons suggests that (i) iPS cells are not permissive to HCMV infection,i.e.,they do not permit a full viral replication cycle; (ii) Neural stem cells have impaired differentiation when infected by HCMV; (iii) NPCs are fully permissive for HCMV infection; altered expression of genes related to neural metabolism or neuronal differentiation is also observed; (iv) most iPS-derived neurons are not permissive to HCMV infection; and (v) infected neurons have impaired calcium influx in response to glutamate. View Publication

The translational potential of mesenchymal stem/stromal cells (MSCs) is limited by their rarity in somatic organs,heterogeneity,and need for harvest by invasive procedures. Induced pluripotent stem cells (iPSCs) could be an advantageous source of MSCs,but attempts to derive MSCs from pluripotent cells have required cumbersome or untranslatable techniques,such as coculture,physical manipulation,sorting,or viral transduction. We devised a single-step method to direct mesengenic differentiation of human embryonic stem cells (ESCs) and iPSCs using a small molecule inhibitor. First,epithelial-like monolayer cells were generated by culturing ESCs/iPSCs in serum-free medium containing the transforming growth factor-β pathway inhibitor SB431542. After 10 days,iPSCs showed upregulation of mesodermal genes (MSX2,NCAM,HOXA2) and downregulation of pluripotency genes (OCT4,LEFTY1/2). Differentiation was then completed by transferring cells into conventional MSC medium. The resultant development of MSC-like morphology was associated with increased expression of genes,reflecting epithelial-to-mesenchymal transition. Both ESC- and iPSC-derived MSCs exhibited a typical MSC immunophenotype,expressed high levels of vimentin and N-cadherin,and lacked expression of pluripotency markers at the protein level. Robust osteogenic and chondrogenic differentiation was induced in vitro in ES-MSCs and iPS-MSCs,whereas adipogenic differentiation was limited,as reported for primitive fetal MSCs and ES-MSCs derived by other methods. We conclude that treatment with SB431542 in two-dimensional cultures followed by culture-induced epithelial-to-mesenchymal transition leads to rapid and uniform MSC conversion of human pluripotent cells without the need for embryoid body formation or feeder cell coculture,providing a robust,clinically applicable,and efficient system for generating MSCs from human iPSCs. View Publication

The study of the regulatory signaling hierarchies of human heart development is limited by a lack of model systems that can reproduce the precise developmental events that occur during human embryogenesis. The advent of human pluripotent stem cell (hPSC) technology and robust cardiac differentiation methods affords a unique opportunity to monitor the full course of cardiac induction in vitro. Here,we show that stage-specific activation of insulin signaling strongly inhibited cardiac differentiation during a monolayer-based differentiation protocol that used transforming growth factor β superfamily ligands to generate cardiomyocytes. However,insulin did not repress cardiomyocyte differentiation in a defined protocol that used small molecule regulators of canonical Wnt signaling. By examining the context of insulin inhibition of cardiomyocyte differentiation,we determined that the inhibitory effects by insulin required Wnt/β-catenin signaling and that the cardiomyocyte differentiation defect resulting from insulin exposure was rescued by inhibition of Wnt/β-catenin during the cardiac mesoderm (Nkx2.5+) stage. Thus,insulin and Wnt/β-catenin signaling pathways,as a network,coordinate to influence hPSC differentiation to cardiomyocytes,with the Wnt/β-catenin pathway dominant to the insulin pathway. Our study contributes to the understanding of the regulatory hierarchies of human cardiomyocyte differentiation and has implications for modeling human heart development. View Publication

The continued success of pluripotent stem cell research is ultimately dependent on access to reliable and defined reagents for the consistent culture and cryopreservation of undifferentiated,pluripotent cells. The development of defined and feeder-independent culture media has provided a platform for greater reproducibility and standardization in this field. Here we provide detailed protocols for the use of mTeSR™1 and TeSR™2 with various cell culture matrices as well as defined cryopreservation protocols for human embryonic and human induced pluripotent stem cells. View Publication

To search for genes that promote hematopoietic development from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs),we overexpressed several known hematopoietic regulator genes in hESC/iPSC-derived CD34(+)CD43(-) endothelial cells (ECs) enriched in hemogenic endothelium (HE). Among the genes tested,only Sox17,a gene encoding a transcription factor of the SOX family,promoted cell growth and supported expansion of CD34(+)CD43(+)CD45(-/low) cells expressing the HE marker VE-cadherin. SOX17 was expressed at high levels in CD34(+)CD43(-) ECs compared with low levels in CD34(+)CD43(+)CD45(-) pre-hematopoietic progenitor cells (pre-HPCs) and CD34(+)CD43(+)CD45(+) HPCs. Sox17-overexpressing cells formed semiadherent cell aggregates and generated few hematopoietic progenies. However,they retained hemogenic potential and gave rise to hematopoietic progenies on inactivation of Sox17. Global gene-expression analyses revealed that the CD34(+)CD43(+)CD45(-/low) cells expanded on overexpression of Sox17 are HE-like cells developmentally placed between ECs and pre-HPCs. Sox17 overexpression also reprogrammed both pre-HPCs and HPCs into HE-like cells. Genome-wide mapping of Sox17-binding sites revealed that Sox17 activates the transcription of key regulator genes for vasculogenesis,hematopoiesis,and erythrocyte differentiation directly. Depletion of SOX17 in CD34(+)CD43(-) ECs severely compromised their hemogenic activity. These findings suggest that SOX17 plays a key role in priming hemogenic potential in ECs,thereby regulating hematopoietic development from hESCs/iPSCs. View Publication

Pluripotent cells represent a powerful tool for tissue regeneration,but their clinical utility is limited by their propensity to form teratomas. Little is known about their interaction with the surrounding niche following implantation and how this may be applied to promote survival and functional engraftment. In this study,we evaluated the ability of an osteogenic microniche consisting of a hydroxyapatite-coated,bone morphogenetic protein-2-releasing poly-L-lactic acid scaffold placed within the context of a macroenvironmental skeletal defect to guide in vivo differentiation of both embryonic and induced pluripotent stem cells. In this setting,we found de novo bone formation and participation by implanted cells in skeletal regeneration without the formation of a teratoma. This finding suggests that local cues from both the implanted scaffold/cell micro- and surrounding macroniche may act in concert to promote cellular survival and the in vivo acquisition of a terminal cell fate,thereby allowing for functional engraftment of pluripotent cells into regenerating tissue. View Publication

Recent successes in deriving human-induced pluripotent stem cells (hiPSCs) allow for the possibility of studying human neurons derived from patients with neurological diseases. Concomitant inhibition of the BMP and TGF-β1 branches of the TGF-β signaling pathways by the endogenous antagonist,Noggin,and the small molecule SB431542,respectively,induces efficient neuralization of hiPSCs,a method known as dual-SMAD inhibition. The use of small molecule inhibitors instead of their endogenous counterparts has several advantages including lower cost,consistent activity,and the maintenance of xeno-free culture conditions. We tested the efficacy of DMH1,a highly selective small molecule BMP-inhibitor for its potential to replace Noggin in the neuralization of hiPSCs. We compare Noggin and DMH1-induced neuralization of hiPSCs by measuring protein and mRNA levels of pluripotency and neural precursor markers over a period of seven days. The regulation of five of the six markers assessed was indistinguishable in the presence of concentrations of Noggin or DMH1 that have been shown to effectively inhibit BMP signaling in other systems. We observed that by varying the DMH1 or Noggin concentration,we could selectively modulate the number of SOX1 expressing cells,whereas PAX6,another neural precursor marker,remained the same. The level and timing of SOX1 expression have been shown to affect neural induction as well as neural lineage. Our observations,therefore,suggest that BMP-inhibitor concentrations need to be carefully monitored to ensure appropriate expression levels of all transcription factors necessary for the induction of a particular neuronal lineage. We further demonstrate that DMH1-induced neural progenitors can be differentiated into β3-tubulin expressing neurons,a subset of which also express tyrosine hydroxylase. Thus,the combined use of DMH1,a highly specific BMP-pathway inhibitor,and SB431542,a TGF-β1-pathway specific inhibitor,provides us with the tools to independently regulate these two pathways through the exclusive use of small molecule inhibitors. View Publication

Stochastic processes and imprinting,along with genetic factors,lead to monoallelic or allele-biased gene expression. Stochastic monoallelic expression fine-tunes information processing in immune cells and the olfactory system,and imprinting plays an important role in development. Recent studies suggest that both stochastic events and imprinting may be more widespread than previously considered. We are interested in allele-biased gene expression occurring in the brain because parent-of-origin effects suggestive of imprinting appear to play a role in the transmission of schizophrenia (SZ) and autism spectrum disorders (ASD) in some families. In addition,allele-biased expression could help explain monozygotic (MZ) twin discordance and reduced penetrance. The ability to study allele-biased expression in human neurons has been transformed with the advent of induced pluripotent stem cell (iPSC) technology and next generation sequencing. Using transcriptome sequencing (RNA-Seq) we identified 801 genes in differentiating neurons that were expressed in an allele-biased manner. These included a number of putative SZ and ASD candidates,such as A2BP1 (RBFOX1),ERBB4,NLGN4X,NRG1,NRG3,NRXN1,and NLGN1. Overall,there was a modest enrichment for SZ and ASD candidate genes among those that showed evidence for allele-biased expression (chi-square,p = 0.02). In addition to helping explain MZ twin discordance and reduced penetrance,the capacity to group many candidate genes affecting a variety of molecular and cellular pathways under a common regulatory process - allele-biased expression - could have therapeutic implications. View Publication

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have been shown to differentiate into primordial germ cells (PGCs) but not into spermatogonia,haploid spermatocytes,or spermatids. Here,we show that hESCs and hiPSCs differentiate directly into advanced male germ cell lineages,including postmeiotic,spermatid-like cells,in vitro without genetic manipulation. Furthermore,our procedure mirrors spermatogenesis in vivo by differentiating PSCs into UTF1-,PLZF-,and CDH1-positive spermatogonia-like cells; HIWI- and HILI-positive spermatocyte-like cells; and haploid cells expressing acrosin,transition protein 1,and protamine 1 (proteins that are uniquely found in spermatids and/or sperm). These spermatids show uniparental genomic imprints similar to those of human sperm on two loci: H19 and IGF2. These results demonstrate that male PSCs have the ability to differentiate directly into advanced germ cell lineages and may represent a novel strategy for studying spermatogenesis in vitro View Publication

Multiple signalling pathways maintain human embryonic stem cells (hESC) in an undifferentiated state. Here we sought to define the significance of G protein signal transduction in the preservation of this state distinct from other cellular processes. Continuous treatment with drugs targeting G(αs)-,G(α-i/o)- and G(α-q/11)-subunit signalling mediators were assessed in independent hESC lines after 7days to discern effects on normalised alkaline phosphatase positive colony frequency vs total cell content. This identified PLCβ,intracellular free calcium and CAMKII kinase activity downstream of G(α-q/11) as of particular importance to the former. To confirm the significance of this finding we generated an agonist-responsive hESC line transgenic for a G(α-q/11) subunit-coupled receptor and demonstrated that an undifferentiated state could be promoted in the presence of an agonist without exogenously supplied bFGF and that this correlated with elevated intracellular calcium. Similarly,treatment of unmodified hESCs with a range of intracellular free calcium-modulating drugs in biologically defined mTESR culture system lacking exogenous bFGF promoted an hESC phenotype after 1week of continuous culture as defined by co-expression of OCT4 and NANOG. At least one of these drugs,lysophosphatidic acid significantly elevates phosphorylation of calmodulin and STAT3 in this culture system (ptextless0.05). These findings substantiate a role for G-protein and calcium signalling in undifferentiated hESC culture. View Publication

BACKGROUND: Human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) are a promising source of cells for regenerating myocardium. However,several issues,especially the large-scale preparation of hiPS-CMs and elimination of undifferentiated iPS cells,must be resolved before hiPS cells can be used clinically. The cell-sheet technique is one of the useful methods for transplanting large numbers of cells. We hypothesized that hiPS-CM-sheet transplantation would be feasible,safe,and therapeutically effective for the treatment of ischemic cardiomyopathy.backslashnbackslashnMETHODS AND RESULTS: Human iPS cells were established by infecting human dermal fibroblasts with a retrovirus carrying Oct3/4,Sox2,Klf4,and c-Myc. Cardiomyogenic differentiation was induced by WNT signaling molecules,yielding hiPS-CMs that were almost 90% positive for α-actinin,Nkx2.5,and cardiac troponin T. hiPS-CM sheets were created using thermoresponsive dishes and transplanted over the myocardial infarcts in a porcine model of ischemic cardiomyopathy induced by ameroid constriction of the left anterior descending coronary artery (n=6 for the iPS group receiving sheet transplantation and the sham-operated group; both groups received tacrolimus daily). Transplantation significantly improved cardiac performance and attenuated left ventricular remodeling. hiPS-CMs were detectable 8 weeks after transplantation,but very few survived long term. No teratoma formation was observed in animals that received hiPS-CM sheets.backslashnbackslashnCONCLUSIONS: The culture system used yields a large number of highly pure hiPS-CMs,and hiPS-CM sheets could improve cardiac function after ischemic cardiomyopathy. This newly developed culture system and the hiPS-CM sheets may provide a basis for the clinical use of hiPS cells in cardiac regeneration therapy. View Publication

Introduction: Human embryonic stem cells (hESC) provide an invaluable model for assessing the effects of environmental chemicals and drugs on human prenatal development. However,hESC are difficult to adapt to 96-well plate screening assays,because they survive best when plated as colonies,which are difficult to count and plate accurately. The purpose of this study is to present an experimental method and analysis procedure to accomplish reliable screening of toxicants using hESC. Methods: We present a method developed to rapidly and easily determine the number of cells in small colonies of hESC spectrophotometerically and then accurately dispense equivalent numbers of cells in 96-well plates. The MTT assay was used to evaluate plating accuracy,and the method was tested using known toxicants. Results: The quality of the plate set-up and analysis procedure was evaluated with NIH plate validation and assessment software. All statistical parameters measured by the software were acceptable,and no drift or edge effects were observed. The 96-well plate MTT assay with hESC was tested by performing a dose-response screen of commercial products,which contain a variety of chemicals. The screen was done using single wells/dose,and the reliability of this method was demonstrated in a subsequent screen of the same products repeated three times. The single and triple screens were in good agreement,and NOAELs and IC50s could be determined from the single screen. The effects of vapor from volatile chemicals were studied,and methods to monitor and avoid vapor effects were incorporated into the assay. Discussion: Our method overcomes the difficulty of using hESC for reliable quantitative 96-well plate assays. It enables rapid dose-response screening using equipment that is commonly available in laboratories that culture hESC. This method could have a broad application in studies of environmental chemicals and drugs using hESC as models of prenatal development. ?? 2012 Elsevier Inc. View Publication