技术资料

Wnt signaling is a key regulator of vertebrate heart development; however,specific roles for human cardiomyocyte development remain uncertain. Here we use human embryonic stem cells (hESCs) to analyze systematically in human cardiomyocyte development the expression of endogenous Wnt signaling components,monitor pathway activity,and dissect stage-specific requirements for canonical and noncanonical Wnt signaling mechanisms using small-molecule inhibitors. Our analysis suggests that WNT3 and WNT8A,via FZD7 and canonical signaling,regulate BRACHYURY expression and mesoderm induction; that WNT5A/5B,via ROR2 and noncanonical signaling,regulate MESP1 expression and cardiovascular development; and that later in development WNT2,WNT5A/5B,and WNT11,via FZD4 and FZD6,regulate functional cardiomyocyte differentiation via noncanonical Wnt signaling. Our findings confirm in human development previously proposed roles for canonical Wnt signaling in sequential stages of vertebrate cardiomyogenesis,and identify more precise roles for noncanonical signaling and for individual Wnt signal and Wnt receptor genes in human cardiomyocyte development. View Publication

Mitochondria are critical to neurogenesis,but the mechanisms of mitochondria in neurogenesis have not been well explored. We fully characterized mitochondrial alterations and function in relation to the development of human induced pluripotent stem cell (hiPSC)-derived dopaminergic (DA) neurons. Following directed differentiation of hiPSCs to DA neurons,mitochondria in these neurons exhibit pronounced changes during differentiation,including mature neurophysiology characterization and functional synaptic network formation. Inhibition of mitochondrial respiratory chains via application of complex IV inhibitor KCN (potassium cyanide) or complex I inhibitor rotenone restricted neurogenesis of DA neurons. These results demonstrated the direct importance of mitochondrial development and bioenergetics in DA neuronal differentiation. Our study also provides a neurophysiologic model of mitochondrial involvement in neurogenesis,which will enhance our understanding of the role of mitochondrial dysfunctions in neurodegenerative diseases. View Publication

Human embryonic stem cells (hESCs) is a potential unlimited ex vivo source of ventricular (V) cardiomyocytes (CMs),but hESC-VCMs and their engineered tissues display immature traits. In adult VCMs,sarcolemmal (sarc) and mitochondrial (mito) ATP-sensitive potassium (KATP) channels play crucial roles in excitability and cardioprotection. In this study,we aim to investigate the biological roles and use of sarcKATP and mitoKATP in hESC-VCM. We showed that SarcIK,ATP in single hESC-VCMs was dormant under baseline conditions,but became markedly activated by cyanide (CN) or the known opener P1075 with a current density that was ˜8-fold smaller than adult; These effects were reversible upon washout or the addition of GLI or HMR1098. Interestingly,sarcIK,ATP displayed a ˜3-fold increase after treatment with hypoxia (5% O2). MitoIK,ATP was absent in hESC-VCMs. However,the thyroid hormone T3 up-regulated mitoIK,ATP,conferring diazoxide protective effect on T3-treated hESC-VCMs. When assessed using a multi-cellular engineered 3D ventricular cardiac micro-tissue (hvCMT) system,T3 substantially enhanced the developed tension by 3-folds. Diazoxide also attenuated the decrease in contractility induced by simulated ischemia (1% O2). We conclude that hypoxia and T3 enhance the functionality of hESC-VCMs and their engineered tissues by selectively acting on sarc and mitoIK,ATP. View Publication

Wnt signaling plays essential roles in both embryonic pattern formation and postembryonic tissue homoestasis. High levels of Wnt activity repress foregut identity and facilitate hindgut fate through forming a gradient of Wnt signaling activity along the anterior-posterior axis. Here,we examined the mechanisms of Wnt signaling in hindgut development by differentiating human embryonic stem cells (hESCs) into the hindgut progenitors. We observed severe morphological changes when Wnt signaling was blocked by using Wnt antagonist Dkk1. We performed deep-transcriptome sequencing (RNA-seq) and identified 240 Wnt-activated genes and 2023 Wnt-repressed genes,respectively. Clusters of Wnt targets showed enrichment in specific biological functions,such as gastrointestinal or skeletal development" in the Wnt-activated targets and "neural or immune system development" in the Wnt-repressed targets. Moreover� View Publication

The sodium bicarbonate co-transporter (NBC) is the major bicarbonate-dependent acid-base transporter in mammalian astrocytes and has been implicated in ischemic brain injury. A malfunction of astrocytes could have great impact on the outcome of stroke due to their participation in the formation of blood-brain barrier,synaptic transmission,and electrolyte balance in the human brain. Nevertheless,the role of NBC in the ischemic astrocyte death has not been well understood. In this work,we obtained skin biopsies from healthy human subjects and had their fibroblasts grown in culture and reprogrammed into human-induced pluripotent stem cells (hiPSCs). These hiPSCs were further differentiated into neuroprogenitor cells (NPCs) and then into human astrocytes. These astrocytes express GFAP and S100β and readily propagate calcium waves upon mechanical stimulation. Using pH-sensitive dye BCECF [2',7'-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein] and qPCR technique,we have confirmed that these astrocytes express functional NBC including electrogenic NBC (NBCe). In addition,astrocytes exposed to an ischemic solution (IS) that mimics the ischemic penumbral environment enhanced both mRNA and protein expression level of NBCe1 in astrocytes. Using IS and a generic NBC blocker S0859,we have studied the involvement of NBC in IS-induced human astrocytes death. Our results show that a 30μM S0859 induced a 97.5±1.6% (n=10) cell death in IS-treated astrocytes,which is significantly higher than 43.6±4.5%,(n=10) in the control group treated with IS alone. In summary,a NBC blocker exaggerates IS-induced cell death,suggesting that NBC activity is essential for astrocyte survival when exposed to ischemic penumbral environment. View Publication

Fate allocation in the gastrulating embryo is spatially organized as cells differentiate into specialized cell types depending on their positions with respect to the body axes. There is a need for in vitro protocols that allow the study of spatial organization associated with this developmental transition. Although embryoid bodies and organoids can exhibit some spatial organization of differentiated cells,methods that generate embryoid bodies or organoids do not yield consistent and fully reproducible results. Here,we describe a micropatterning approach in which human embryonic stem cells are confined to disk-shaped,submillimeter colonies. After 42 h of BMP4 stimulation,cells form self-organized differentiation patterns in concentric radial domains,which express specific markers associated with the embryonic germ layers,reminiscent of gastrulating embryos. Our protocol takes 3 d; it uses commercial microfabricated slides (from CYTOO),human laminin-521 (LN-521) as extracellular matrix coating,and either conditioned or chemically defined medium (mTeSR). Differentiation patterns within individual colonies can be determined by immunofluorescence and analyzed with cellular resolution. Both the size of the micropattern and the type of medium affect the patterning outcome. The protocol is appropriate for personnel with basic stem cell culture training. This protocol describes a robust platform for quantitative analysis of the mechanisms associated with pattern formation at the onset of gastrulation. View Publication

Patient-derived induced pluripotent stem cells (iPSCs) provide a novel tool to investigate the pathophysiology of poorly known diseases,in particular those affecting the nervous system,which has been difficult to study for its lack of accessibility. In this emerging and promising field,recent iPSCs studies are mostly used as proof-of-principle" experiments that are confirmatory of previous findings obtained from animal models and postmortem human studies; its promise as a discovery tool is just beginning to be realized. A recent number of studies point to the functional similarities between in vitro neurogenesis and in vivo neuronal development� View Publication

The mechanisms underlying human germ cell development are largely unknown,partly due to the scarcity of primordial germ cells and the inaccessibility of the human germline to genetic analysis. Human embryonic stem cells can differentiate to germ cells in vitro and can be genetically modified to study the genetic requirements for germ cell development. Here,we studied NANOS3 and DAZL,which have critical roles in germ cell development in several species,via their over expression in human embryonic stem cells using global transcriptional analysis,in vitro germ cell differentiation,and in vivo germ cell formation assay by xenotransplantation. We found that NANOS3 over expression prolonged pluripotency and delayed differentiation. In addition,we observed a possible connection of NANOS3 with inhibition of apoptosis. For DAZL,our results suggest a post-transcriptional regulation mechanism in hES cells. In addition,we found that DAZL suppressed the translation of OCT4,and affected the transcription of several genes associated with germ cells,cell cycle arrest,and cell migration. Furthermore,DAZL over expressed cells formed spermatogonia-like colonies in a rare instance upon xenotransplantation. These data can be used to further elucidate the role of NANOS3 and DAZL in germ cell development both in vitro and in vivo. View Publication

BACKGROUND: Induced pluripotent stem cells (iPSCs) can be differentiated into potentially unlimited lineages of cell types for use in autologous cell therapy. However,the efficiency of the differentiation procedure and subsequent function of the iPSC-derived cells may be influenced by epigenetic factors that the iPSCs retain from their tissues of origin; thus,iPSC-derived cells may be more effective for treatment of myocardial injury if the iPSCs were engineered from cardiac-lineage cells,rather than dermal fibroblasts. METHODS AND RESULTS: We show that human cardiac iPSCs (hciPSCs) can be generated from cardiac fibroblasts and subsequently differentiated into exceptionally pure (textgreater92%) sheets of cardiomyocytes (CMs). The hciPSCs passed through all the normal stages of differentiation before assuming a CM identity. When using the fibrin gel-enhanced delivery of hciPSC-CM sheets at the site of injury in infarcted mouse hearts,the engraftment rate was 31.91%+/-5.75% at Day 28 post transplantation. The hciPSC-CM in the sheet also appeared to develop a more mature,structurally aligned phenotype 28 days after transplantation and was associated with significant improvements in cardiac function,vascularity,and reduction in apoptosis. CONCLUSIONS: These data strongly support the potential of hciPSC-CM sheet transplantation for the treatment of heart with acute myocardial infarction. View Publication

BACKGROUND: Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have been successfully used to knock out endogenous genes in stem cell research. However,the deficiencies of current gene-based delivery systems may hamper the clinical application of these nucleases. A new delivery method that can improve the utility of these nucleases is needed.backslashnbackslashnRESULTS: In this study,we utilized a cell-penetrating peptide-based system for ZFN and TALEN delivery. Functional TAT-ZFN and TAT-TALEN proteins were generated by fusing the cell-penetrating TAT peptide to ZFN and TALEN,respectively. However,TAT-ZFN was difficult to purify in quantities sufficient for analysis in cell culture. Purified TAT-TALEN was able to penetrate cells and disrupt the gene encoding endogenous human chemokine (C-C motif) receptor 5 (CCR5,a co-receptor for HIV-1 entry into cells). Hypothermic treatment greatly enhanced the TAT-TALEN-mediated gene disruption efficiency. A 5% modification rate was observed in human induced pluripotent stem cells (hiPSCs) treated with TAT-TALEN as measured by the Surveyor assay.backslashnbackslashnCONCLUSIONS: TAT-TALEN protein-mediated gene disruption was applicable in hiPSCs and represents a promising technique for gene knockout in stem cells. This new technique may advance the clinical application of TALEN technology. View Publication

Peripheral blood is the easy-to-access,minimally invasive,and the most abundant cell source to use for cell reprogramming. The episomal vector is among the best approaches for generating integration-free induced pluripotent stem (iPS) cells due to its simplicity and affordability. Here we describe the detailed protocol for the efficient generation of integration-free iPS cells from peripheral blood mononuclear cells. With this optimized protocol,one can readily generate hundreds of iPS cell colonies from 1 ml of peripheral blood. View Publication

The fate decisions of human pluripotent stem (hPS) cells are governed by soluble and insoluble signals from the microenvironment. Many hPS cell differentiation protocols use Matrigel,a complex and undefined substrate that engages multiple adhesion and signaling receptors. Using defined surfaces programmed to engage specific cell-surface ligands (i.e.,glycosaminoglycans and integrins),the contribution of specific matrix signals can be dissected. For ectoderm and motor neuron differentiation,peptide-modified surfaces that can engage both glycosaminoglycans and integrins are effective. In contrast,surfaces that interact selectively with glycosaminoglycans are superior to Matrigel in promoting hPS cell differentiation to definitive endoderm and mesoderm. The modular surfaces were used to elucidate the signaling pathways underlying these differences. Matrigel promotes integrin signaling,which in turn inhibits mesendoderm differentiation. The data indicate that integrin-activating surfaces stimulate Akt signaling via integrin-linked kinase (ILK),which is antagonistic to endoderm differentiation. The ability to attribute cellular responses to specific interactions between the cell and the substrate offers new opportunities for revealing and controlling the pathways governing cell fate. View Publication