技术资料

Long non-coding RNAs (lncRNAs) are a numerous class of newly discovered genes in the human genome,which have been proposed to be key regulators of biological processes,including stem cell pluripotency and neurogenesis. However,at present very little functional characterization of lncRNAs in human differentiation has been carried out. In the present study,we address this using human embryonic stem cells (hESCs) as a paradigm for pluripotency and neuronal differentiation. With a newly developed method,hESCs were robustly and efficiently differentiated into neurons,and we profiled the expression of thousands of lncRNAs using a custom-designed microarray. Some hESC-specific lncRNAs involved in pluripotency maintenance were identified,and shown to physically interact with SOX2,and PRC2 complex component,SUZ12. Using a similar approach,we identified lncRNAs required for neurogenesis. Knockdown studies indicated that loss of any of these lncRNAs blocked neurogenesis,and immunoprecipitation studies revealed physical association with REST and SUZ12. This study indicates that lncRNAs are important regulators of pluripotency and neurogenesis,and represents important evidence for an indispensable role of lncRNAs in human brain development. View Publication

We demonstrate that dissociated human pluripotent stem cells (PSCs) are intrinsically programmed to form lumens. PSCs form two-cell cysts with a shared apical domain within 20 hr of plating; these cysts collapse to form monolayers after 5 days. Expression of pluripotency markers is maintained throughout this time. In two-cell cysts,an apical domain,marked by EZRIN and atypical PKC$\$,is surrounded by apically targeted organelles (early endosomes and Golgi). Molecularly,actin polymerization,regulated by ARP2/3 and mammalian diaphanous-related formin 1 (MDIA),promotes lumen formation,whereas actin contraction,mediated by MYOSIN-II,inhibits this process. Finally,we show that lumenal shape can be manipulated in bioengineered micro-wells. Since lumen formation is an indispensable step in early mammalian development,this system can provide a powerful model for investigation of this process in a controlled environment. Overall,our data establish that lumenogenesis is a fundamental cell biological property of human PSCs. View Publication

Human pluripotent stem cells (hPSCs),including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs),share the properties of unlimited self-renewal and the capacity to become any cell type in the body,making them well suited for regenerative medicine and cell therapy. So far,almost all hPSC lines have been directly or indirectly exposed to animal-derived products,which would hinder their use for clinical purposes. One of the biggest challenges in this area is to remove animal components from the derivation,propagation,and cryopreservation of hPSCs. Moreover,the presence of undefined components of animal or human origin in culture system may interfere with the interpretation of the effect of exogenous agents on the growth and differentiation of hPSCs and are prone to significant variability. To explore hPSC expansion in defined,xeno-free conditions,2 different groups of culture systems were used to culture different hESC and hiPSC lines. Our results suggested that (1) medium,matrix,and exogenous factors have synergistic effects on the adhesion and growth of hPSCs; (2) cooperation of exogenous factors including basic fibroblast growth factor,Rho-associated kinase inhibitor (ROCK),and other growth factors is critical for hPSC adhesion and proliferation; (3) basal media have different effects on hPSC attachment to the culture surface; and (4) a medium or matrix component can work synergistically in one culture system,and not at all in another. In this study,we found that Vitronectin/TeSR2 and PDL/HEScGRO (Y-27632) systems were optimal for maintaining the long-term culture of 3 hESC lines and 2 hiPSC lines under defined,xeno-free conditions. View Publication

We present a predictive bioprocess design strategy employing cell- and molecular-level analysis of rate-limiting steps in human pluripotent stem cell (hPSC) expansion and differentiation,and apply it to produce definitive endoderm (DE) progenitors using a scalable directed-differentiation technology. We define a bioprocess optimization parameter (L; targeted cell Loss) and,with quantitative cell division tracking and fate monitoring,identify and overcome key suspension bioprocess bottlenecks. Adapting process operating conditions to pivotal parameters (single cell survival and growth rate) in a cell-line-specific manner enabled adherent-equivalent expansion of hPSCs in feeder- and matrix-free defined-medium suspension culture. Predominantly instructive differentiation mechanisms were found to underlie a subsequent 18-fold expansion,during directed differentiation,to high-purity DE competent for further commitment along pancreatic and hepatic lineages. This study demonstrates that iPSC expansion and differentiation conditions can be prospectively specified to guide the enhanced production of target cells in a scale-free directed differentiation system. View Publication

Pluripotent stem cells are able to give rise to all cell types of the organism. There are two sources for human pluripotent stem cells: embryonic stem cells (ESCs) derived from surplus blastocysts created for in vitro fertilization and induced pluripotent stem cells (iPSCs) generated by reprogramming of somatic cells. ESCs have been an area of intense research during the past decade,and two clinical trials have been recently approved. iPSCs were created only recently,and most of the research has been focused on the iPSC generation protocols and investigation of mechanisms of direct reprogramming. The iPSC technology makes possible to derive pluripotent stem cells from any patient. However,there are a number of hurdles to be overcome before iPSCs will find a niche in practice. In this review,we discuss differences and similarities of the two pluripotent cell types and assess prospects for application of these cells in biomedicine. 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

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BACKGROUND: Human pluripotent stem cells have the ability to generate all cell types present in the adult organism,therefore harboring great potential for the in vitro study of differentiation and for the development of cell-based therapies. Nonetheless their use may prove challenging as incomplete differentiation of these cells might lead to tumoregenicity. Interestingly,many cancer types have been reported to display metabolic modifications with features that might be similar to stem cells. Understanding the metabolic properties of human pluripotent stem cells when compared to their differentiated counterparts can thus be of crucial importance. Furthermore recent data has stressed distinct features of different human pluripotent cells lines,namely when comparing embryo-derived human embryonic stem cells (hESCs) and induced pluripotent stem cells (IPSCs) reprogrammed from somatic cells.backslashnbackslashnMETHODOLOGY/PRINCIPAL FINDINGS: We compared the energy metabolism of hESCs,IPSCs,and their somatic counterparts. Focusing on mitochondria,we tracked organelle localization and morphology. Furthermore we performed gene expression analysis of several pathways related to the glucose metabolism,including glycolysis,the pentose phosphate pathway and the tricarboxylic acid (TCA) cycle. In addition we determined oxygen consumption rates (OCR) using a metabolic extracellular flux analyzer,as well as total intracellular ATP levels by high performance liquid chromatography (HPLC). Finally we explored the expression of key proteins involved in the regulation of glucose metabolism.backslashnbackslashnCONCLUSIONS/FINDINGS: Our results demonstrate that,although the metabolic signature of IPSCs is not identical to that of hESCs,nonetheless they cluster with hESCs rather than with their somatic counterparts. ATP levels,lactate production and OCR revealed that human pluripotent cells rely mostly on glycolysis to meet their energy demands. Furthermore,our work points to some of the strategies which human pluripotent stem cells may use to maintain high glycolytic rates,such as high levels of hexokinase II and inactive pyruvate dehydrogenase (PDH). View Publication

Rett syndrome (RTT) is a progressive neurologic disorder representing one of the most common causes of mental retardation in females. To date mutations in three genes have been associated with this condition. Classic RTT is caused by mutations in the MECP2 gene,whereas variants can be due to mutations in either MECP2 or FOXG1 or CDKL5. Mutations in CDKL5 have been identified both in females with the early onset seizure variant of RTT and in males with X-linked epileptic encephalopathy. CDKL5 is a kinase protein highly expressed in neurons,but its exact function inside the cell is unknown. To address this issue we established a human cellular model for CDKL5-related disease using the recently developed technology of induced pluripotent stem cells (iPSCs). iPSCs can be expanded indefinitely and differentiated in vitro into many different cell types,including neurons. These features make them the ideal tool to study disease mechanisms directly on the primarily affected neuronal cells. We derived iPSCs from fibroblasts of one female with p.Q347X and one male with p.T288I mutation,affected by early onset seizure variant and X-linked epileptic encephalopathy,respectively. We demonstrated that female CDKL5-mutated iPSCs maintain X-chromosome inactivation and clones express either the mutant CDKL5 allele or the wild-type allele that serve as an ideal experimental control. Array CGH indicates normal isogenic molecular karyotypes without detection of de novo CNVs in the CDKL5-mutated iPSCs. Furthermore,the iPS cells can be differentiated into neurons and are thus suitable to model disease pathogenesis in vitro. View Publication

Stem cells hold great promise for therapies aimed at regenerating damaged tissue,drug screening and studying in vitro models of human disease. However,many challenges remain before these applications can become a reality. One such challenge is developing chemically defined and scalable culture conditions for derivation and expansion of clinically viable human pluripotent stem cells,as well as controlling their differentiation with high specificity. Interaction of stem cells with their extracellular microenvironment plays an important role in determining their differentiation commitment and functions. Regenerative medicine approaches integrating cell-matrix and cell-cell interactions,and soluble factors could lead to development of robust microenvironments to control various cellular responses. Indeed,several of these recent developments have provided significant insight into the design of microenvironments that can elicit the targeted cellular response. In this article,we will focus on some of these developments with an emphasis on matrix-mediated expansion of human pluripotent stem cells while maintaining their pluripotency. We will also discuss the role of matrix-based cues and cell-cell interactions in the form of soluble signals in directing stem cell differentiation into musculoskeletal lineages. View Publication

We present a method to perform absolute quantification of glycogen in human embryonic stem cells (hESCs) in situ based on the use of Raman microspectroscopy. The proposed quantification method was validated by comparison to a commonly used commercial glycogen assay kit. With Raman microspectroscopy,we could obtain the glycogen content of hESCs faster and apparently more accurately than with the kit. In addition,glycogen distributions across a colony could be obtained. Raman spectroscopy can provide reliable estimates of the in situ glycogen content in hESCs,and this approach should also be extensible to their other biochemical constituents as well as to other cell types. View Publication

Since the derivation of the first human embryonic stem cell (hESC) lines by Thomson and coworkers in 1998,more than 1,200 different hESC lines have been established worldwide. Nevertheless,there is still a recognized interest in the establishment of new lines of hESC,particularly from HLA types and ethnic groups currently underrepresented among the available lines. The methodology of hESC derivation has evolved significantly since 1998,when human LIF (hLIF) was used for maintenance of pluripotency. However,there are a number of different strategies for the several steps involved in establishing a new line of hESC. Here we make a survey of the most relevant parameters used between 1998 and 2010 for the derivation of the 375 hESC lines deposited in two international stem cell registries,and able to form teratomas in immunocompromised mice. Although we identify some trends in the methodology for establishing hESC lines,our data reveal a much greater heterogeneity of strategies than what is used for derivation of murine ESC lines,indicating that optimum conditions have not been consolidated yet,and thus,hESC establishment is still an evolving field of research. View Publication