技术资料

Amniogenesis-the development of amnion-is a critical developmental milestone for early human embryogenesis and successful pregnancy. However,human amniogenesis is poorly understood due to limited accessibility to peri-implantation embryos and a lack of in vitro models. Here we report an efficient biomaterial system to generate human amnion-like tissue in vitro through self-organized development of human pluripotent stem cells (hPSCs) in a bioengineered niche mimicking the in vivo implantation environment. We show that biophysical niche factors act as a switch to toggle hPSC self-renewal versus amniogenesis under self-renewal-permissive biochemical conditions. We identify a unique molecular signature of hPSC-derived amnion-like cells and show that endogenously activated BMP-SMAD signalling is required for the amnion-like tissue development by hPSCs. This study unveils the self-organizing and mechanosensitive nature of human amniogenesis and establishes the first hPSC-based model for investigating peri-implantation human amnion development,thereby helping advance human embryology and reproductive medicine. View Publication

The chromatin landscape and cellular metabolism both contribute to cell fate determination,but their interplay remains poorly understood. Using genome-wide siRNA screening,we have identified prohibitin (PHB) as an essential factor in self-renewal of human embryonic stem cells (hESCs). Mechanistically,PHB forms protein complexes with HIRA,a histone H3.3 chaperone,and stabilizes the protein levels of HIRA complex components. Like PHB,HIRA is required for hESC self-renewal. PHB and HIRA act together to control global deposition of histone H3.3 and gene expression in hESCs. Of particular note,PHB and HIRA regulate the chromatin architecture at the promoters of isocitrate dehydrogenase genes to promote transcription and,thus,production of α-ketoglutarate,a key metabolite in the regulation of ESC fate. Our study shows that PHB has an unexpected nuclear role in hESCs that is required for self-renewal and that it acts with HIRA in chromatin organization to link epigenetic organization to a metabolic circuit. View Publication

In this study,because excessive polycythemia is a predominant trait in some high-altitude dwellers (chronic mountain sickness [CMS] or Monge's disease) but not others living at the same altitude in the Andes,we took advantage of this human experiment of nature and used a combination of induced pluripotent stem cell technology,genomics,and molecular biology in this unique population to understand the molecular basis for hypoxia-induced excessive polycythemia. As compared with sea-level controls and non-CMS subjects who responded to hypoxia by increasing their RBCs modestly or not at all,respectively,CMS cells increased theirs remarkably (up to 60-fold). Although there was a switch from fetal to adult HgbA0 in all populations and a concomitant shift in oxygen binding,we found that CMS cells matured faster and had a higher efficiency and proliferative potential than non-CMS cells. We also established that SENP1 plays a critical role in the differential erythropoietic response of CMS and non-CMS subjects: we can convert the CMS phenotype into that of non-CMS and vice versa by altering SENP1 levels. We also demonstrated that GATA1 is an essential downstream target of SENP1 and that the differential expression and response of GATA1 and Bcl-xL are a key mechanism underlying CMS pathology. View Publication

Human pluripotent stem cells hold great promise for applications in drug discovery and regenerative medicine. Microfluidic technology is a promising approach for creating artificial microenvironments; however,although a proper 3D microenvironment is required to achieve robust control of cellular phenotypes,most current microfluidic devices provide only 2D cell culture and do not allow tuning of physical and chemical environmental cues simultaneously. Here,the authors report a 3D cellular microenvironment plate (3D-CEP),which consists of a microfluidic device filled with thermoresponsive poly(N-isopropylacrylamide)-β-poly(ethylene glycol) hydrogel (HG),which enables systematic tuning of both chemical and physical environmental cues as well as in situ cell monitoring. The authors show that H9 human embryonic stem cells (hESCs) and 253G1 human induced pluripotent stem cells in the HG/3D-CEP system maintain their pluripotent marker expression under HG/3D-CEP self-renewing conditions. Additionally,global gene expression analyses are used to elucidate small variations among different test environments. Interestingly,the authors find that treatment of H9 hESCs under HG/3D-CEP self-renewing conditions results in initiation of entry into the neural differentiation process by induction of PAX3 and OTX1 expression. The authors believe that this HG/3D-CEP system will serve as a versatile platform for developing targeted functional cell lines and facilitate advances in drug screening and regenerative medicine. View Publication

MicroRNAs (miRNA) are central regulators of diverse biological processes and are important in the regulation of stem cell self-renewal. One of the widely studied miRNA-protein regulators is the Lin28-Let-7 pair. In this study,we demonstrate that contrary to the well-established models of mouse ES cells (mESC) and transformed human cancer cells,the pluripotent state of human ES cells (hESC) involves expression of mature Let-7 family miRNAs with concurrent expression of all LIN28 proteins. We show that mature Let-7 miRNAs are regulated during hESC differentiation and have opposite expression profile with LIN28B. Moreover,mature Let-7 miRNAs fine tune the expression levels of LIN28B protein in pluripotent hESCs,whereas silencing of LIN28 proteins have no effect on mature Let-7 levels. These results bring novel information to the highly complex network of human pluripotency and suggest that maintenance of hESC pluripotency differs greatly from the mESCs in regard to LIN28-Let-7 regulation. View Publication

The enteric nervous system (ENS) is recognized as a second brain because of its complexity and its largely autonomic control of bowel function. Recent progress in studying the interactions between the ENS and the central nervous system (CNS) has implicated alterations of the gut/brain axis as a possible mechanism in the pathophysiology of autism spectrum disorders (ASDs),Parkinson's disease (PD) and other human CNS disorders,whereas the underlying mechanisms are largely unknown because of the lack of good model systems. Human induced pluripotent stem cells (hiPSCs) have the ability to proliferate indefinitely and differentiate into cells of all three germ layers,thus making iPSCs an ideal source of cells for disease modelling and cell therapy. Here,hiPSCs were induced to differentiate into neural crest stem cells (NCSCs) efficiently. When co-cultured with smooth muscle layers of ganglionic gut tissue,the NCSCs differentiated into different subtypes of mature enteric-like neurons expressing nitric oxide synthase (nNOS),vasoactive intestinal polypeptide (VIP),choline acetyltransferase (ChAT) or calretinin with typical electrophysiological characteristics of functional neurons. Furthermore,when they were transplanted into aneural or aganglionic chick,mouse or human gut tissues in ovo,in vitro or in vivo,hiPSC-derived NCSCs showed extensive migration and neural differentiation capacity,generating neurons and glial cells that expressed phenotypic markers characteristic of the enteric nervous system. Our results indicate that enteric NCSCs derived from hiPSCs supply a powerful tool for studying the pathogenesis of gastrointestinal disorders and brain/gut dysfunction and represent a potentially ideal cell source for enteric neural transplantation treatments.Molecular Psychiatry advance online publication,25 October 2016; doi:10.1038/mp.2016.191. View Publication

BACKGROUND Several compounds have been reported to induce translational readthrough of premature stop codons resulting in the production of full-length protein by interfering with ribosomal proofreading. Here we examined the effect of 2 of these compounds,gentamicin and PTC124,in human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes bearing nonsense mutations in the sodium channel gene SCN5A,which are associated with conduction disease and potential lethal arrhythmias. METHODS AND RESULTS We generated hiPSC from 2 patients carrying the mutations R1638X and W156X. hiPSC-derived cardiomyocytes from both patients recapitulated the expected electrophysiological phenotype,as evidenced by reduced Na(+) currents and action potential upstroke velocities compared with hiPSC-derived cardiomyocytes from 2 unrelated control individuals. While we were able to confirm the readthrough efficacy of the 2 drugs in Human Embryonic Kidney 293 cells,we did not observe rescue of the electrophysiological phenotype in hiPSC-derived cardiomyocytes from the patients. CONCLUSIONS We conclude that these drugs are unlikely to present an effective treatment for patients carrying the loss-of-function SCN5A gene mutations examined in this study. View Publication

Hypoplastic left heart syndrome (HLHS) is a clinically and anatomically severe form of congenital heart disease (CHD). Although prior studies suggest that HLHS has a complex genetic inheritance,its etiology remains largely unknown. The goal of this study was to characterize a risk gene in HLHS and its effect on HLHS etiology and outcome. We performed next-generation sequencing on a multigenerational family with a high prevalence of CHD/HLHS,identifying a rare variant in the α-myosin heavy chain (MYH6) gene. A case-control study of 190 unrelated HLHS subjects was then performed and compared with the 1000 Genomes Project. Damaging MYH6 variants,including novel,missense,in-frame deletion,premature stop,de novo,and compound heterozygous variants,were significantly enriched in HLHS cases (P textless 1 × 10(-5)). Clinical outcomes analysis showed reduced transplant-free survival in HLHS subjects with damaging MYH6 variants (P textless 1 × 10(-2)). Transcriptome and protein expression analyses with cardiac tissue revealed differential expression of cardiac contractility genes,notably upregulation of the β-myosin heavy chain (MYH7) gene in subjects with MYH6 variants (P textless 1 × 10(-3)). We subsequently used patient-specific induced pluripotent stem cells (iPSCs) to model HLHS in vitro. Early stages of in vitro cardiomyogenesis in iPSCs derived from two unrelated HLHS families mimicked the increased expression of MYH7 observed in vivo (P textless 1 × 10(-2)),while revealing defective cardiomyogenic differentiation. Rare,damaging variants in MYH6 are enriched in HLHS,affect molecular expression of contractility genes,and are predictive of poor outcome. These findings indicate that the etiology of MYH6-associated HLHS can be informed using iPSCs and suggest utility in future clinical applications. View Publication

Obtaining highly purified differentiated cells via directed differentiation from human pluripotent stem cells (hPSCs) is an essential step for their clinical application. Among the various conditions that should be optimized,the precise role and contribution of the extracellular matrix (ECM) during differentiation are relatively unclear. Here,using a short fragment of laminin 411 (LM411-E8),an ECM predominantly expressed in the vascular endothelial basement membrane,we demonstrate that the directed switching of defined ECMs robustly yields highly-purified (textgreater95%) endothelial progenitor cells (PSC-EPCs) without cell sorting from hPSCs in an integrin-laminin axis-dependent manner. Single-cell RNA-seq analysis revealed that LM411-E8 resolved intercellular transcriptional heterogeneity and escorted the progenitor cells to the appropriate differentiation pathway. The PSC-EPCs gave rise to functional endothelial cells both in vivo and in vitro. We therefore propose that sequential switching of defined matrices is an important concept for guiding cells towards desired fate. View Publication

The differentiation of human pluripotent stem cells toward the hepatocyte lineage can potentially provide an unlimited source of functional hepatocytes for transplantation and extracorporeal bioartificial liver applications. It is anticipated that the quantities of cells needed for these applications will be in the order of 10(9)-10(10) cells,because of the size of the liver. An ideal differentiation protocol would be to enable directed differentiation to the hepatocyte lineage with simultaneous cell expansion. We introduced a cell expansion stage after the commitment of human embryonic stem cells to the endodermal lineage,to allow for at least an eightfold increase in cell number,with continuation of cell maturation toward the hepatocyte lineage. The progressive changes in the transcriptome were measured by expression array,and the expression dynamics of certain lineage markers was measured by mass cytometry during the differentiation and expansion process. The findings revealed that while cells were expanding they were also capable of progressing in their differentiation toward the hepatocyte lineage. In addition,our transcriptome,protein and functional studies,including albumin secretion,drug-induced CYP450 expression and urea production,all indicated that the hepatocyte-like cells obtained with or without cell expansion are very similar. This method of simultaneous cell expansion and hepatocyte differentiation should facilitate obtaining large quantities of cells for liver cell applications. View Publication

PURPOSE Nonexudative (dry) age-related macular degeneration (AMD),a leading cause of blindness in the elderly,is associated with the loss of retinal pigmented epithelium (RPE) cells and the development of geographic atrophy,which are areas devoid of RPE cells and photoreceptors. One possible treatment option would be to stimulate RPE attachment and proliferation to replace dying/dysfunctional RPE and bring about wound repair. Clinical trials are underway testing injections of RPE cells derived from pluripotent stem cells to determine their safety and efficacy in treating AMD. However,the factors regulating RPE responses to AMD-associated lesions are not well understood. Here,we use cell culture to investigate the role of RhoA coiled coil kinases (ROCKs) in human embryonic stem cell-derived RPE (hESC-RPE) attachment,proliferation,and wound closure. METHODS H9 hESC were spontaneously differentiated into RPE cells. hESC-RPE cells were treated with a pan ROCK1/2 or a ROCK2 only inhibitor; attachment,and proliferation and cell size within an in vitro scratch assay were examined. RESULTS Pharmacological inhibition of ROCKs promoted hESC-RPE attachment and proliferation,and increased the rate of closure of in vitro wounds. ROCK inhibition decreased phosphorylation of cofilin and myosin light chain,suggesting that regulation of the cytoskeleton underlies the mechanism of action of ROCK inhibition. CONCLUSIONS ROCK inhibition promotes attachment,proliferation,and wound closure in H9 hESC-RPE cells. ROCK isoforms may have different roles in wound healing. TRANSLATIONAL RELEVANCE Modulation of the ROCK-cytoskeletal axis has potential in stimulating wound repair in transplanted RPE cells and attachment in cellular therapies. View Publication

Gap junction-mediated cell-cell interactions are highly conserved and play essential roles in cell survival,proliferation,differentiation and patterning. We report that Connexin 32 (Cx32)-mediated gap junctional intercellular communication (GJIC) is necessary for human embryonic stem cell-derived hepatocytes (hESC-Heps) during step-wise hepatic lineage restriction and maturation. Vitamin K2,previously shown to promote Cx32 expression in mature hepatocytes,up-regulated Cx32 expression and GJIC activation during hepatic differentiation and maturation,resulting in significant increases of hepatic markers expression and hepatocyte functions. In contrast,negative Cx32 regulator 2-aminoethoxydiphenyl borate blocked hESC-to-hepatocyte maturation and muted hepatocyte functions through disruption of GJIC activities. Dynamic gap junction organization and internalization are phosphorylation-dependent and the p38 mitogen-activated protein kinases pathway (MAPK) can negatively regulate Cxs through phosphorylation-dependent degradation of Cxs. We found that p38 MAPK inhibitor SB203580 improved maturation of hESC-Heps correlating with up-regulation of Cx32; by contrast,the p38 MAPK activator,anisomycin,blocked hESC-Heps maturation correlating with down-regulation of Cx32. These results suggested that Cx32 is essential for cell-cell interactions that facilitate driving hESCs through hepatic-lineage maturation. Regulators of both Cx32 and other members of its pathways maybe used as a promising approach on regulating hepatic lineage restriction of pluripotent stem cells and optimizing their functional maturation. View Publication