技术资料

Surface ectoderm (SE) cells give rise to structures including the epidermis and ectodermal associated appendages such as hair,eye,and the mammary gland. In this study,we validate a protocol that utilizes BMP4 and the $$-secretase inhibitor DAPT to induce SE differentiation from human induced pluripotent stem cells (hiPSCs). hiPSC-differentiated SE cells expressed markers suggesting their commitment to the SE lineage. Computational analyses using integrated quantitative transcriptomic and proteomic profiling reveal that TGF$$ superfamily signaling pathways are preferentially activated in SE cells compared with hiPSCs. SE differentiation can be enhanced by selectively blocking TGF$$-RI signaling. We also show that SE cells and neural ectoderm cells possess distinct gene expression patterns and signaling networks as indicated by functional Ingenuity Pathway Analysis. Our findings advance current understanding of early human SE cell development and pave the way for modeling of SE-derived tissue development,studying disease pathogenesis,and development of regenerative medicine approaches. View Publication

AIMS/HYPOTHESIS Endothelial cells (ECs) play an essential role in pancreatic organogenesis. We hypothesise that effective in vitro interactions between human microvascular endothelial cells (HMECs) and human pluripotent stem cells (hPSCs) results in the generation of functional pancreatic beta cells. METHODS Embryoid bodies (EBs) derived from hPSCs were cultured alone (controls) or with ECs in collagen gels. Subsequently,cells were analysed for pancreatic beta cell markers,and then isolated and expanded. Insulin secretion in response to glucose was evaluated in vitro by static and dynamic (perifusion) assays,and in vivo by EB transplantation into immunodeficient mice. RESULTS Co-cultured EBs had a higher expression of mature beta cells markers and enhanced insulin secretion in vitro,compared with controls. In mice,transplanted EBs had higher levels of human C-peptide secretion with a significant reduction in hyperglycaemia after the selective destruction of native pancreatic beta cells. In addition,there was significant in vitro upregulation of bone morphogenetic proteins 2 and 4 (BMP-2,4) in co-cultured cells,compared with controls. CONCLUSIONS/INTERPRETATION ECs provide essential signalling in vitro,such as activation of the BMP pathway,for derivation of functional insulin-producing beta cells from hPSCs. View Publication

Progress toward finding a cure for muscle diseases has been slow because of the absence of relevant cellular models and the lack of a reliable source of muscle progenitors for biomedical investigation. Here we report an optimized serum-free differentiation protocol to efficiently produce striated,millimeter-long muscle fibers together with satellite-like cells from human pluripotent stem cells (hPSCs) in vitro. By mimicking key signaling events leading to muscle formation in the embryo,in particular the dual modulation of Wnt and bone morphogenetic protein (BMP) pathway signaling,this directed differentiation protocol avoids the requirement for genetic modifications or cell sorting. Robust myogenesis can be achieved in vitro within 1 month by personnel experienced in hPSC culture. The differentiating culture can be subcultured to produce large amounts of myogenic progenitors amenable to numerous downstream applications. Beyond the study of myogenesis,this differentiation method offers an attractive platform for the development of relevant in vitro models of muscle dystrophies and drug screening strategies,as well as providing a source of cells for tissue engineering and cell therapy approaches. View Publication

Efficient derivation of endothelial cells and their progenitors from human pluripotent stem cells (hPSCs) can facilitate studies of human vascular development,disease modeling,drug discovery,and cell-based therapy. Here we provide a detailed protocol for directing hPSCs to functional endothelial cells and their progenitors in a completely defined,growth factor- and serum-free system by temporal modulation of Wnt/$$-catenin signaling via small molecules. We demonstrate a 10-day,two-stage process that recapitulates endothelial cell development,in which hPSCs first differentiate to endothelial progenitors that then generate functional endothelial cells and smooth muscle cells. Methods to characterize endothelial cell identity and function are also described. View Publication

BACKGROUND This study aimed to evaluate the effect of exosomes produced by human-induced pluripotent stem cell-derived mesenchymal stromal cells (hiPSC-MSCs-Exo) on hepatic ischemia-reperfusion (I/R) injury. METHODS Exosomes were isolated and concentrated from conditioned medium using ultracentrifugation and ultrafiltration. hiPSC-MSCs-Exo were injected systemically via the inferior vena cava in a rat model of 70% warm hepatic I/R injury,and the therapeutic effect was evaluated. The serum levels of transaminases (aspartate aminotransferase [AST] and alanine aminotransferase [ALT]) were measured using an automatic analyzer. The expression of inflammatory factors was measured using enzyme-linked immunosorbent assay (ELISA). Histological changes indicated changes in pathology and inflammatory infiltration in liver tissue. Apoptosis of hepatic cells in liver tissue was measured using terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) staining along with apoptotic markers. RESULTS hiPSCs were efficiently induced into hiPSC-MSCs with typical MSC characteristics. hiPSC-MSCs-Exo had diameters ranging from 50 to 60 nm and expressed exosomal markers (CD9,CD63 and CD81). Hepatocyte necrosis and sinusoidal congestion were markedly suppressed with a lower Suzuki score after hiPSC-MSCs-Exo administration. The levels of the hepatocyte injury markers AST and ALT were significantly lower in the treated group than in the control group. Inflammatory markers,such as tumor necrosis factor (TNF)-α,interleukin (IL)-6 and high mobility group box 1 (HMGB1),were significantly reduced after administration of hiPSC-MSCs-Exo,which suggests that the exosomes have a role in suppressing the inflammatory response. Additionally,in liver tissues from the experimental group,the levels of apoptotic markers,such as caspase-3 and bax,were significantly lower and the levels of oxidative markers,such as glutathione (GSH),glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD),were significantly higher than in the control group. These data point to an anti-apoptotic,anti-oxidative stress response role for hiPSC-MSCs-Exo. CONCLUSIONS Our results demonstrated that hiPSC-MSCs-Exo alleviate hepatic I/R injury,possibly via suppression of inflammatory responses,attenuation of the oxidative stress response and inhibition of apoptosis. View Publication

G protein-coupled receptors (GPCRs) are involved in many fundamental cellular responses such as growth,death,movement,transcription and excitation. Their roles in human stem cell neural specialization are not well understood. In this study,we aimed to identify GPCRs that may play a role in the differentiation of human embryonic stem cells (hESCs) to neural stem cells (NSCs). Using a feeder-free hESC neural differentiation protocol,we found that the expression of several chemokine receptors changed dramatically during the hESC/NSC transition. Especially,the expression of CXCR4 increased approximately 50 folds in NSCs compared to the original hESCs. CXCR4 agonist SDF-1 promoted,whereas the antagonist AMD3100 delayed the neural induction process. In consistence with antagonizing CXCR4,knockdown of CXCR4 in hESCs also blocked the neural induction and cells with reduced CXCR4 were rarely positive for Nestin and Sox1-staining. Taken together,our results suggest that CXCR4 is involved in the neural induction process of hESC and it might be considered as a target to facilitate NSC production from hESCs in regenerative medicine. View Publication

The mechanisms of how signaling pathways are coordinated and integrated for the maintenance of the self-renewal of human embryonic stem cells (hESCs) and the acquisition of pluripotency in reprogramming are still only partly understood. CDK1 is a key regulator of mitosis. Recently,CDK1 has been shown to be involved in regulating self-renewal of stem cells,even though the mechanistic role of how CDK1 regulates pluripotency is unknown. In this report,we aim to understand how CDK1 can control pluripotency by reducing CDK1 activity to a level that has no effect on cell cycle progression. We demonstrated that high levels of CDK1 is associated with the pluripotency stage of hESCs; and decreased CDK1 activity to a level without perturbing the cell cycle is sufficient to induce differentiation. CDK1 specifically targets the phosphorylation of PDK1 and consequently the activity of PI3K/Akt and its effectors ERK and GSK3β. Evidence of the reversion of inactive CDK1-mediated differentiation by the inhibition of Akt signaling effectors suggests that the CDK1-PDK1-PI3K/Akt kinase cascade is a functional signaling pathway for the pluripotency of hESCs. Moreover,cyclin B1-CDK1 complexes promote somatic reprogramming efficiency,probably by regulating the maturation of induced pluripotent stem cells (iPSCs),as cyclin B1 stimulates a higher cellular level of LIN28A,suggesting that monitoring iPSC factors could be a new path for the enhancement of reprogramming efficiency. Together,we demonstrate an essential role for the CDK1-PDK1-PI3K/Akt kinase signaling pathway in the regulation of self-renewal,differentiation,and somatic reprogramming,which provides a novel kinase cascade mechanism for pluripotency control and acquisition.Cell Death and Differentiation advance online publication,16 September 2016; doi:10.1038/cdd.2016.84. View Publication

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