技术资料

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

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

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

Erasure of epigenetic memory is required to convert somatic cells towards pluripotency. Reactivation of the inactive X chromosome (Xi) has been used to model epigenetic reprogramming in mouse,but human studies are hampered by Xi epigenetic instability and difficulties in tracking partially reprogrammed iPSCs. Here we use cell fusion to examine the earliest events in the reprogramming-induced Xi reactivation of human female fibroblasts. We show that a rapid and widespread loss of Xi-associated H3K27me3 and XIST occurs in fused cells and precedes the bi-allelic expression of selected Xi-genes by many heterokaryons (30-50%). After cell division,RNA-FISH and RNA-seq analyses confirm that Xi reactivation remains partial and that induction of human pluripotency-specific XACT transcripts is rare (1%). These data effectively separate pre- and post-mitotic events in reprogramming-induced Xi reactivation and reveal a complex hierarchy of epigenetic changes that are required to reactivate the genes on the human Xi chromosome. View Publication

Williams syndrome is a genetic neurodevelopmental disorder characterized by an uncommon hypersociability and a mosaic of retained and compromised linguistic and cognitive abilities. Nearly all clinically diagnosed individuals with Williams syndrome lack precisely the same set of genes,with breakpoints in chromosome band 7q11.23 (refs 1-5). The contribution of specific genes to the neuroanatomical and functional alterations,leading to behavioural pathologies in humans,remains largely unexplored. Here we investigate neural progenitor cells and cortical neurons derived from Williams syndrome and typically developing induced pluripotent stem cells. Neural progenitor cells in Williams syndrome have an increased doubling time and apoptosis compared with typically developing neural progenitor cells. Using an individual with atypical Williams syndrome,we narrowed this cellular phenotype to a single gene candidate,frizzled 9 (FZD9). At the neuronal stage,layer V/VI cortical neurons derived from Williams syndrome were characterized by longer total dendrites,increased numbers of spines and synapses,aberrant calcium oscillation and altered network connectivity. Morphometric alterations observed in neurons from Williams syndrome were validated after Golgi staining of post-mortem layer V/VI cortical neurons. This model of human induced pluripotent stem cells fills the current knowledge gap in the cellular biology of Williams syndrome and could lead to further insights into the molecular mechanism underlying the disorder and the human social brain. View Publication

BACKGROUND The Activin A and bone morphogenetic protein (BMP) pathways are critical regulators of the immune system and of bone formation. Inappropriate activation of these pathways,as in conditions of congenital heterotopic ossification,are thought to activate an osteogenic program in endothelial cells. However,if and how this occurs in human endothelial cells remains unclear. METHODS We used a new directed differentiation protocol to create human induced pluripotent stem cell (hiPSC)-derived endothelial cells (iECs) from patients with fibrodysplasia ossificans progressiva (FOP),a congenital disease of heterotopic ossification caused by an activating R206H mutation in the Activin A type I receptor (ACVR1). This strategy allowed the direct assay of the cell-autonomous effects of ACVR1 R206H in the endogenous locus without the use of transgenic expression. These cells were challenged with BMP or Activin A ligand,and tested for their ability to activate osteogenesis,extracellular matrix production,and differential downstream signaling in the BMP/Activin A pathways. RESULTS We found that FOP iECs could form in conditions with low or absent BMP4. These conditions are not normally permissive in control cells. FOP iECs cultured in mineralization media showed increased alkaline phosphatase staining,suggesting formation of immature osteoblasts,but failed to show mature osteoblastic features. However,FOP iECs expressed more fibroblastic genes and Collagen 1/2 compared to control iECs,suggesting a mechanism for the tissue fibrosis seen in early heterotopic lesions. Finally,FOP iECs showed increased SMAD1/5/8 signaling upon BMP4 stimulation. Contrary to FOP hiPSCs,FOP iECs did not show a significant increase in SMAD1/5/8 phosphorylation upon Activin A stimulation,suggesting that the ACVR1 R206H mutation has a cell type-specific effect. In addition,we found that the expression of ACVR1 and type II receptors were different in hiPSCs and iECs,which could explain the cell type-specific SMAD signaling. CONCLUSIONS Our results suggest that the ACVR1 R206H mutation may not directly increase the formation of mature chondrogenic or osteogenic cells by FOP iECs. Our results also show that BMP can induce endothelial cell dysfunction,increase expression of fibrogenic matrix proteins,and cause differential downstream signaling of the ACVR1 R206H mutation. This iPSC model provides new insight into how human endothelial cells may contribute to the pathogenesis of heterotopic ossification. View Publication

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

Astrocytes are instrumental to major brain functions,including metabolic support,extracellular ion regulation,the shaping of excitatory signaling events and maintenance of synaptic glutamate homeostasis. Astrocyte dysfunction contributes to numerous developmental,psychiatric and neurodegenerative disorders. The generation of adult human fibroblast-derived induced pluripotent stem cells (iPSCs) has provided novel opportunities to study mechanisms of astrocyte dysfunction in human-derived cells. To overcome the difficulties of cell type heterogeneity during the differentiation process from iPSCs to astroglial cells (iPS astrocytes),we generated homogenous populations of iPS astrocytes using zinc-finger nuclease (ZFN) technology. Enhanced green fluorescent protein (eGFP) driven by the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter was inserted into the safe harbor adeno-associated virus integration site 1 (AAVS1) locus in disease and control-derived iPSCs. Astrocyte populations were enriched using Fluorescence Activated Cell Sorting (FACS) and after enrichment more than 99% of iPS astrocytes expressed mature astrocyte markers including GFAP,S100$\$,NFIA and ALDH1L1. In addition,mature pure GFP-iPS astrocytes exhibited a well-described functional astrocytic activity in vitro characterized by neuron-dependent regulation of glutamate transporters to regulate extracellular glutamate concentrations. Engraftment of GFP-iPS astrocytes into rat spinal cord grey matter confirmed in vivo cell survival and continued astrocytic maturation. In conclusion,the generation of GFAP::GFP-iPS astrocytes provides a powerful in vitro and in vivo tool for studying astrocyte biology and astrocyte-driven disease pathogenesis and therapy. 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

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

BACKGROUND: Constitutive promoters that ensure sustained and high level gene expression are basic research tools that have a wide range of applications,including studies of human embryology and drug discovery in human embryonic stem cells (hESCs). Numerous cellular/viral promoters that ensure sustained gene expression in various cell types have been identified but systematic comparison of their activities in hESCs is still lacking. METHODOLOGY/PRINCIPAL FINDINGS: We have quantitatively compared promoter activities of five commonly used constitutive promoters,including the human β-actin promoter (ACTB),cytomegalovirus (CMV),elongation factor-1α,(EF1α),phosphoglycerate kinase (PGK) and ubiquitinC (UbC) in hESCs. Lentiviral gene transfer was used to ensure stable integration of promoter-eGFP constructs into the hESCs genome. Promoter activities were quantitatively compared in long term culture of undifferentiated hESCs and in their differentiated progenies. CONCLUSION/SIGNIFICANCE: The ACTB,EF1α and PGK promoters showed stable activities during long term culture of undifferentiated hESCs. The ACTB promoter was superior by maintaining expression in 75-80% of the cells after 50 days in culture. During embryoid body (EB) differentiation,promoter activities of all five promoters decreased. Although the EF1α promoter was downregulated in approximately 50% of the cells,it was the most stable promoter during differentiation. Gene expression analysis of differentiated eGFP+ and eGFP- cells indicate that promoter activities might be restricted to specific cell lineages,suggesting the need to carefully select optimal promoters for constitutive gene expression in differentiated hESCs. View Publication