技术资料

Human embryonic stem cells (hESCs) and their differentiated progeny allow for investigation of important changes/events during normal embryonic development. Currently most of the research is focused on proteinacous changes occurring as a result of differentiation of stem cells and little is known about changes in cell surface glycosylation patterns. Identification of cell lineage specific glycans can help in understanding their role in maintenance,proliferation and differentiation. Furthermore,these glycans can serve as markers for isolation of homogenous populations of cells. Using a panel of eight biotinylated lectins,the glycan expression of hESCs,hESCs-derived human neural progenitors (hNP) cells,and hESCs-derived mesenchymal progenitor (hMP) cells was investigated. Our goal was to identify glycans that are unique for hNP cells and use the corresponding lectins for cell isolation. Flow cytometry and immunocytochemistry were used to determine expression and localization of glycans,respectively,in each cell type. These results show that the glycan expression changes upon differentiation of hESCs and is different for neural and mesenchymal lineage. For example,binding of PHA-L lectin is low in hESCs (14±4.4%) but significantly higher in differentiated hNP cells (99±0.4%) and hMP cells (90±3%). Three lectins: VVA,DBA and LTL have low binding in hESCs and hMP cells,but significantly higher binding in hNP cells. Finally,VVA lectin binding was used to isolate hNP cells from a mixed population of hESCs,hNP cells and hMP cells. This is the first report that compares glycan expression across these human stem cell lineages and identifies significant differences. Also,this is the first study that uses VVA lectin for isolation for human neural progenitor cells. View Publication

The midbody is a singular organelle formed between daughter cells during cytokinesis and required for their final separation. Midbodies persist in cells long after division as midbody derivatives (MB(d)s),but their fate is unclear. Here we show that MB(d)s are inherited asymmetrically by the daughter cell with the older centrosome. They selectively accumulate in stem cells,induced pluripotent stem cells and potential cancer 'stem cells' in vivo and in vitro. MB(d) loss accompanies stem-cell differentiation,and involves autophagic degradation mediated by binding of the autophagic receptor NBR1 to the midbody protein CEP55. Differentiating cells and normal dividing cells do not accumulate MB(d)s and possess high autophagic activity. Stem cells and cancer cells accumulate MB(d)s by evading autophagosome encapsulation and exhibit low autophagic activity. MB(d) enrichment enhances reprogramming to induced pluripotent stem cells and increases the in vitro tumorigenicity of cancer cells. These results indicate unexpected roles for MB(d)s in stem cells and cancer 'stem cells'. View Publication

Human pluripotent stem cells hold promising potential in many therapeutics applications including regenerative medicine and drug discovery. Over the past three decades,embryonic stem cell research has illustrated that embryonic stem cells possess two important and distinct properties: the ability to continuously self-renew and the ability to differentiate into all specialized cell types. In this article,we will discuss the continuing evolution of human pluripotent stem cell culture by examining requirements needed for the maintenance of self-renewal in vitro. We will also elaborate on the future direction of the field toward generating a robust and completely defined culture system,which has brought forth collaborations amongst biologists and engineers. As human pluripotent stem cell research progresses towards identifying solutions for debilitating diseases,it will be critical to establish a defined,reproducible and scalable culture system to meet the requirements of these clinical applications. View Publication

Measuring spatial and temporal patterns of cytochemical variation in human embryonic stem cell (hESC) colonies is necessary for understanding the role of cellular communication in spontaneous differentiation,the mechanisms of biological niche creation,and structure-generating developmental processes. Such insights will ultimately facilitate directed differentiation and therewith promote advances in tissue engineering and regenerative medicine. However,the patterns of cytochemical inhomogeneities of hESC colonies are not well studied and their causes are not fully understood. We used Raman spectroscopic mapping to contrast supracellular variations in cytochemical composition across pluripotent and partly differentiated hESC colonies to gain a better understanding of the early-stage (i.e.,5 days) effects of the differentiation process on the nature and evolution of these patterns. Higher protein-to-nucleic acid ratios,a differentiation status indicator observed previously using Raman spectroscopy,confirmed reported results that spontaneous differentiation is more pronounced on the edges of a colony than elsewhere. In addition,pluripotent and partly differentiated colonies also showed higher lipid concentrations relative to nucleic acids at colony edges in contrast to relative glycogen concentrations,which were up to 400% more pronounced in the colony centers compared to their edges. Pluripotent and partly differentiated colonies differed,with the latter having higher average protein-to-nucleic acid and lipid-to-nucleic acid ratios but a lower glycogen-to-nucleic acid ratio. In both cases,cell density,pluripotency,and high glycogen appeared to vary in tandem. Spatial variations in glycogen- and protein-to-nucleic acid ratios have features on the order of 100 μm and larger. These dimensions are consistent with those reported for stem cell niches and suggest that cytochemical inhomogeneities may provide colony-level information about niches and niche formation. These results demonstrate Raman mapping to be a potentially useful technique for revealing the complexities in the spatial organization of hESC cultures and thus for monitoring the evolution of engineered hESC niches. View Publication

View Publication

View Publication

To gain insight into the molecular regulation of human heart development,a detailed comparison of the mRNA and miRNA transcriptomes across differentiating human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and biopsies from fetal,adult,and hypertensive human hearts was performed. Gene ontology analysis of the mRNA expression levels of the hiPSCs differentiating into cardiomyocytes revealed 3 distinct groups of genes: pluripotent specific,transitional cardiac specification,and mature cardiomyocyte specific. Hierarchical clustering of the mRNA data revealed that the transcriptome of hiPSC cardiomyocytes largely stabilizes 20 days after initiation of differentiation. Nevertheless,analysis of cells continuously cultured for 120 days indicated that the cardiomyocytes continued to mature toward a more adult-like gene expression pattern. Analysis of cardiomyocyte-specific miRNAs (miR-1,miR-133a/b,and miR-208a/b) revealed an miRNA pattern indicative of stem cell to cardiomyocyte specification. A biostatistitical approach integrated the miRNA and mRNA expression profiles revealing a cardiomyocyte differentiation miRNA network and identified putative mRNAs targeted by multiple miRNAs. Together,these data reveal the miRNA network in human heart development and support the notion that overlapping miRNA networks re-enforce transcriptional control during developmental specification. 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