技术资料

We report that embryonic stem cells efficiently undergo differentiation in vitro to mesoderm and hematopoietic cells and that this in vitro system recapitulates days 6.5 to 7.5 of mouse hematopoietic development. Embryonic stem cells differentiated as embryoid bodies (EBs) develop erythroid precursors by day 4 of differentiation,and by day 6,more than 85% of EBs contain such cells. A comparative reverse transcriptase-mediated polymerase chain reaction profile of marker genes for primitive endoderm (collagen alpha IV) and mesoderm (Brachyury) indicates that both cell types are present in the developing EBs as well in normal embryos prior to the onset of hematopoiesis. GATA-1,GATA-3,and vav are expressed in both the EBs and embryos just prior to and/or during the early onset of hematopoiesis,indicating that they could play a role in the early stages of hematopoietic development both in vivo and in vitro. The initial stages of hematopoietic development within the EBs occur in the absence of added growth factors and are not significantly influenced by the addition of a broad spectrum of factors,including interleukin-3 (IL-3),IL-1,IL-6,IL-11,erythropoietin,and Kit ligand. At days 10 and 14 of differentiation,EB hematopoiesis is significantly enhanced by the addition of both Kit ligand and IL-11 to the cultures. Kinetic analysis indicates that hematopoietic precursors develop within the EBs in an ordered pattern. Precursors of the primitive erythroid lineage appear first,approximately 24 h before precursors of the macrophage and definitive erythroid lineages. Bipotential neutrophil/macrophage and multilineage precursors appear next,and precursors of the mast cell lineage develop last. The kinetics of precursor development,as well as the growth factor responsiveness of these early cells,is similar to that found in the yolk sac and early fetal liver,indicating that the onset of hematopoiesis within the EBs parallels that found in the embryo. View Publication

Mutations in WD40-repeat protein 62 (WDR62) are commonly associated with primary microcephaly and other developmental cortical malformations. We used human pluripotent stem cells (hPSC) to examine WDR62 function during human neural differentiation and model early stages of human corticogenesis. Neurospheres lacking WDR62 expression showed decreased expression of intermediate progenitor marker,TBR2,and also glial marker,S100β. In contrast,inhibition of c-Jun N-terminal kinase (JNK) signalling during hPSC neural differentiation induced upregulation of WDR62 with a corresponding increase in neural and glial progenitor markers,PAX6 and EAAT1,respectively. These findings may signify a role of WDR62 in specifying intermediate neural and glial progenitors during human pluripotent stem cell differentiation. View Publication

To better understand transcriptional regulation during human oogenesis and preimplantation development,we defined stage-specific transcription,which highlighted the cleavage stage as being highly distinctive. Here,we present multiple lines of evidence that a eutherian-specific multicopy retrogene,DUX4,encodes a transcription factor that activates hundreds of endogenous genes (for example,ZSCAN4,KDM4E and PRAMEF-family genes) and retroviral elements (MERVL/HERVL family) that define the cleavage-specific transcriptional programs in humans and mice. Remarkably,mouse Dux expression is both necessary and sufficient to convert mouse embryonic stem cells (mESCs) into 2-cell-embryo-like ('2C-like') cells,measured here by the reactivation of '2C' genes and repeat elements,the loss of POU5F1 (also known as OCT4) protein and chromocenters,and the conversion of the chromatin landscape (as assessed by transposase-accessible chromatin using sequencing (ATAC-seq)) to a state strongly resembling that of mouse 2C embryos. Thus,we propose mouse DUX and human DUX4 as major drivers of the cleavage or 2C state. View Publication

Human pluripotent stem cells (PSCs) exist in naive and primed states and provide important models to investigate the earliest stages of human development. Naive cells can be obtained through primed-to-naive resetting,but there are no reliable methods to prospectively isolate unmodified naive cells during this process. Here we report comprehensive profiling of cell surface proteins by flow cytometry in naive and primed human PSCs. Several naive-specific,but not primed-specific,proteins were also expressed by pluripotent cells in the human preimplantation embryo. The upregulation of naive-specific cell surface proteins during primed-to-naive resetting enabled the isolation and characterization of live naive cells and intermediate cell populations. This analysis revealed distinct transcriptional and X chromosome inactivation changes associated with the early and late stages of naive cell formation. Thus,identification of state-specific proteins provides a robust set of molecular markers to define the human PSC state and allows new insights into the molecular events leading to naive cell resetting. View Publication

Brain-derived microvascular endothelial cells (BMECs),which play a central role in blood brain barrier (BBB),can be used for the evaluation of drug transport into the brain. Although human BMEC cell lines have already been reported,they lack original properties such as barrier integrity. Pluripotent stem cells (PSCs) can be used for various applications such as regenerative therapy,drug screening,and pathological study. In the recent study,an induction method of BMECs from PSCs has been established,making it possible to more precisely study the in vitro human BBB function. Here,using induced pluripotent stem (iPS) cell-derived BMECs,we examined the effects of oxygen-glucose deprivation (OGD) and OGD/reoxygenation (OGD/R) on BBB permeability. OGD disrupted the barrier function,and the dysfunction was rapidly restored by re-supply of the oxygen and glucose. Interestingly,TNF-α,which is known to be secreted from astrocytes and microglia in the cerebral ischemia,prevented the restoration of OGD-induced barrier dysfunction in an apoptosis-independent manner. Thus,we could establish the in vitro BBB disease model that mimics the cerebral ischemia by using iPS cell-derived BMECs. View Publication

BACKGROUND CHD8 (chromodomain helicase DNA-binding protein 8),which codes for a member of the CHD family of ATP-dependent chromatin-remodeling factors,is one of the most commonly mutated genes in autism spectrum disorders (ASD) identified in exome-sequencing studies. Loss of function mutations in the gene have also been found in schizophrenia (SZ) and intellectual disabilities and influence cancer cell proliferation. We previously reported an RNA-seq analysis carried out on neural progenitor cells (NPCs) and monolayer neurons derived from induced pluripotent stem (iPS) cells that were heterozygous for CHD8 knockout (KO) alleles generated using CRISPR-Cas9 gene editing. A significant number of ASD and SZ candidate genes were among those that were differentially expressed in a comparison of heterozygous KO lines (CHD8(+/-)) vs isogenic controls (CHD8(+/-)),including the SZ and bipolar disorder (BD) candidate gene TCF4,which was markedly upregulated in CHD8(+/-) neuronal cells. METHODS In the current study,RNA-seq was carried out on CHD8(+/-) and isogenic control (CHD8(+/+)) cerebral organoids,which are 3-dimensional structures derived from iPS cells that model the developing human telencephalon. RESULTS TCF4 expression was,again,significantly upregulated. Pathway analysis carried out on differentially expressed genes (DEGs) revealed an enrichment of genes involved in neurogenesis,neuronal differentiation,forebrain development,Wnt/β-catenin signaling,and axonal guidance,similar to our previous study on NPCs and monolayer neurons. There was also significant overlap in our CHD8(+/-) DEGs with those found in a transcriptome analysis carried out by another group using cerebral organoids derived from a family with idiopathic ASD. Remarkably,the top DEG in our respective studies was the non-coding RNA DLX6-AS1,which was markedly upregulated in both studies; DLX6-AS1 regulates the expression of members of the DLX (distal-less homeobox) gene family. DLX1 was also upregulated in both studies. DLX genes code for transcription factors that play a key role in GABAergic interneuron differentiation. Significant overlap was also found in a transcriptome study carried out by another group using iPS cell-derived neurons from patients with BD,a condition characterized by dysregulated WNT/β-catenin signaling in a subgroup of affected individuals. CONCLUSIONS Overall,the findings show that distinct ASD,SZ,and BD candidate genes converge on common molecular targets-an important consideration for developing novel therapeutics in genetically heterogeneous complex traits. 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

BACKGROUND & AIMS Developmental morphogens play an important role in coordinating the ductular reaction and portal fibrosis occurring in the setting of cholangiopathies. However,little is known about how membrane signaling events in ductular reactive cells (DRCs) are transduced into nuclear transcriptional changes to drive cholangiocyte maturation and matrix deposition. Therefore,the aim of this study was to investigate potential mechanistic links between cell signaling events and epigenetic regulators in DRCs. METHODS Using directed differentiation of induced pluripotent stem cells (iPSC),isolated DRCs,and in vivo models,we examine the mechanisms whereby sonic hedgehog (Shh) overcomes an epigenetic barrier in biliary precursors and promotes both cholangiocyte maturation and deposition of fibronectin (FN). RESULTS We demonstrate,for the first time,that Gli1 influences the differentiation state and fibrogenic capacity of iPSC-derived hepatic progenitors and isolated DRCs. We outline a novel pathway wherein Shh-mediated Gli1 binding in key cholangiocyte gene promoters overcomes an epigenetic barrier conferred by the polycomb protein,enhancer of zeste homolog 2 (EZH2) and initiates the transcriptional program of cholangiocyte maturation. We also define previously unknown functional Gli1 binding sites in the promoters of cytokeratin (CK)7,CK19,and FN. Our in vivo results show that EZH2 KO mice fed the choline-deficient,ethanolamine supplemented (CDE) diet have an exaggerated cholangiocyte expansion associated with more robust ductular reaction and increased peri-portal fibrosis. CONCLUSION We conclude that Shh/Gli1 signaling plays an integral role in cholangiocyte maturation in vitro by overcoming an EZH2-dependent epigenetic barrier and this mechanism also promotes biliary expansion in vivo. View Publication

Tunneling nanotubes (TNTs) are ultrafine,filamentous actin-based cytoplasmic extensions which form spontaneously to connect cells at short and long-range distances. We have previously described long-range intercellular communication via TNTs connecting mesothelioma cells in vitro and demonstrated TNTs in intact tumors from patients with mesothelioma. Here,we investigate the ability of TNTs to mediate a viral thymidine kinase based bystander effect after oncolytic viral infection and administration of the nucleoside analog ganciclovir. Using confocal microscopy we assessed the ability of TNTs to propagate enhanced green fluorescent protein (eGFP),which is encoded by the herpes simplex virus NV1066,from infected to uninfected recipient cells. Using time-lapse imaging,we observed eGFP expressed in infected cells being transferred via TNTs to noninfected cells; additionally,increasing fluorescent activity in recipient cells indicated cell-to-cell transmission of the eGFP-expressing NV1066 virus had also occurred. TNTs mediated cell death as a form of direct cell-to-cell transfer following viral thymidine kinase mediated activation of ganciclovir,inducing a unique long-range form of the bystander effect through transmission of activated ganciclovir to nonvirus-infected cells. Thus,we provide proof-of-principle demonstration of a previously unknown and alternative mechanism for inducing apoptosis in noninfected recipient cells. The conceptual advance of this work is that TNTs can be harnessed for delivery of oncolytic viruses and of viral thymidine kinase activated drugs to amplify the bystander effect between cancer cells over long distances in stroma-rich tumor microenvironments. 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

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

Biophysical properties of the scaffolds such as the elastic modulus,have been recently shown to impact stem cell lineage commitment. On the other hand,the contribution of the Poisson's ratio,another important biophysical property,to the stem cell fate decision,has not been studied. Scaffolds with tunable Poisson's ratio (ν) (termed as auxetic scaffolds when Poisson's ratio is zero or negative) are anticipated to provide a spectrum of unique biophysical 3-D microenvironments to influence stem cell fate. To test this hypothesis,in the present work we fabricated auxetic polyurethane scaffolds (ν=0 to -0.45) and evaluated their effects on neural differentiation of mouse embryonic stem cells (ESCs) and human induced pluripotent stem cells (hiPSCs). Compared to the regular scaffolds (ν=+0.30) before auxetic conversion,the auxetic scaffolds supported smaller aggregate formation and higher expression of β-tubulin III upon neural differentiation. The influences of pore structure,Poisson's ratio,and elastic modulus on neural lineage commitment were further evaluated using a series of auxetic scaffolds. The results indicate that Poisson's ratio may confound the effects of elastic modulus,and auxetic scaffolds with proper pore structure and Poisson's ratio enhance neural differentiation. This study demonstrates that tuning the Poisson's ratio of the scaffolds together with elastic modulus and microstructure would enhance the capability to generate broader,more diversified ranges of biophysical 3-D microenvironments for the modulation of cellular differentiation. STATEMENT OF SIGNIFICANCE Biophysical signaling from the substrates and scaffolds plays a critical role in neural lineage commitment of pluripotent stem cells. While the contribution of elastic modulus has been well studied,the influence of Poisson's ratio along with microstructure of the scaffolds remains unknown largely due to the lack of technology to produce materials with tailorable Poisson's ratio. This study fabricated auxetic polyurethane scaffolds with different elastic modulus,Poisson's ratio and microstructure and evaluated neural differentiation of pluripotent stem cells. The findings add a novel angle to understand the impact of biophysical microenvironment on stem cell fate decisions. View Publication