技术资料

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

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

Human embryonic stem cell line BJNhem20-pCAG-tdTomato was generated using non-viral method. The construct pCAG-tdTomato was transfected using microporation procedure. This fluorescent hESC line can help to study heterogeneity within individual cells in hESC colonies by enabling live tracking of their growth,migration and differentiation properties. This cell line also serves as a resource for additional transgene introduction/knock-out/knock-in generation in a fluorescent background and allows ease of analysis in studies involving cell mixing. View Publication

Human iPSC line L749.1 was generated from fibroblasts of a patient with Leigh syndrome associated with a heteroplasmic mutation in the MT-ATP6 gene. Reprogramming factors OCT4,SOX2,CMYC and KLF4 were delivered using retroviruses. View Publication

Objective Friedreich's ataxia (FRDA) is an autosomal recessive trinucleotide repeat expansion disorder caused by epigenetic silencing of the frataxin gene (FXN). Current research suggests that damage and variation of mitochondrial DNA (mtDNA) contribute to the molecular pathogenesis of FRDA. We sought to establish the extent of the mutation burden across the mitochondrial genome in FRDA cells and investigate the molecular mechanisms connecting FXN downregulation and the acquisition of mtDNA damage. Methods Damage and mutation load in mtDNA of a panel of FRDA and control fibroblasts were determined using qPCR and next-generation MiSeq sequencing,respectively. The capacity of FRDA and control cells to repair oxidative lesions in their mtDNA was measured using a quantitative DNA damage assay. Comprehensive RNA sequencing gene expression analyses were conducted to assess the status of DNA repair and metabolism genes in FRDA cells. Results Acute or prolonged downregulation of FXN expression resulted in a significant increase in mtDNA damage that translated to a significant elevation of mutation load in mtDNA. The predominant mutations identified throughout the mtDNA were CtextgreaterT,GtextgreaterA transitions (P = 0.007). Low FXN expression reduced capacity to repair oxidative damage in mtDNA. Downregulation of FXN expression strongly correlated (r = 0.73) with decreased levels of base excision repair (BER) DNA glycosylase NTHL1. Interpretation Downregulation of FXN expression in FRDA cells elevates mtDNA damage,increases mutation load of the mitochondrial genome,and diminishes DNA repair capacity. Progressive accumulation of mtDNA mutations in vulnerable FRDA patient cells reduces mitochondrial fitness ultimately leading to cell death. View Publication

Pluripotent stem cell-derived cardiomyocytes (CMs) have great potential in the development of new therapies for cardiovascular disease. In particular,human induced pluripotent stem cells (iPSCs) may prove especially advantageous due to their pluripotency,their self-renewal potential,and their ability to create patient-specific cell lines. Unfortunately,pluripotent stem cell-derived CMs are immature,with characteristics more closely resembling fetal CMs than adult CMs,and this immaturity has limited their use in drug screening and cell-based therapies. Extracellular matrix (ECM) influences cellular behavior and maturation,as does the geometry of the environment-two-dimensional (2D) versus three-dimensional (3D). We therefore tested the hypothesis that native cardiac ECM and 3D cultures might enhance the maturation of iPSC-derived CMs in vitro. We demonstrate that maturation of iPSC-derived CMs was enhanced when cells were seeded into a 3D cardiac ECM scaffold,compared with 2D culture. 3D cardiac ECM promoted increased expression of calcium-handling genes,Junctin,CaV1.2,NCX1,HCN4,SERCA2a,Triadin,and CASQ2. Consistent with this,we find that iPSC-derived CMs in 3D adult cardiac ECM show increased calcium signaling (amplitude) and kinetics (maximum upstroke and downstroke) compared with cells in 2D. Cells in 3D culture were also more responsive to caffeine,likely reflecting an increased availability of calcium in the sarcoplasmic reticulum. Taken together,these studies provide novel strategies for maturing iPSC-derived CMs that may have applications in drug screening and transplantation therapies to treat heart disease. View Publication

PURPOSE To model keratoconus (KC) using induced pluripotent stem cells (iPSC) generated from fibroblasts of both KC and normal human corneal stroma by a viral method. METHODS Both normal and KC corneal fibroblasts from four human donors were reprogramed directly by delivering reprogramming factors in a single virus using 2A self-cleaving" peptides� View Publication

Insights into the expression of pacemaker-speci�?c markers in human induced pluripotent stemcell (hiPSC)-derived cardiomyocyte subtypes can facilitate the enrichment and track differentia-tion and maturation of hiPSC-derived pacemaker-like cardiomyocytes. To date,no study hasdirectly assessed gene expression in each pacemaker-,atria-,and ventricular-like cardiomyocytesubtype derived from hiPSCs since currently the subtypes of these immature cardiomyocytescan only be identi�?ed by action potential pro�?les. Traditional acquisition of action potentialsusing patch-clamp recordings renders the cells unviable for subsequent analysis. We circum-vented these issues by acquiring the action potential pro�?le of a single cell optically followedby assessment of protein expression through immunostaining in that same cell. Our same-single-cell analysis for the �?rst time revealed expression of proposed pacemaker-speci�?cmarkers—hyperpolarization-activated cyclic nucleotide-modulated (HCN)4 channel and Islet(Isl)1—at the protein level in all three hiPSC-derived cardiomyocyte subtypes. HCN4 expressionwas found to be higher in pacemaker-like hiPSC-derived cardiomyocytes than atrial- andventricular-like subtypes but its downregulation over time in all subtypes diminished the differ-ences. Isl1 expression in pacemaker-like hiPSC-derived cardiomyocytes was initially not statisti-cally different than the contractile subtypes but did become statistically higher than ventricular-like cells with time. Our observations suggest that although HCN4 and Isl1 are differentiallyexpressed in hiPSC-derived pacemaker-like relative to ventricular-like cardiomyocytes,thesemarkers alone are insuf�?cient in identifying hiPSC-derived pacemaker-like cardiomyocytes. 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

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

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

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