技术资料

The naïve pluripotent state has been shown in mice to lead to broad and more robust developmental potential relative to primed mouse epiblast cells. The human naïve ES cell state has eluded derivation without the use of transgenes,and forced expression of OCT4,KLF4,and KLF2 allows maintenance of human cells in a naïve state [Hanna J,et al. (2010) Proc Natl Acad Sci USA 107(20):9222-9227]. We describe two routes to generate nontransgenic naïve human ES cells (hESCs). The first is by reverse toggling of preexisting primed hESC lines by preculture in the histone deacetylase inhibitors butyrate and suberoylanilide hydroxamic acid,followed by culture in MEK/ERK and GSK3 inhibitors (2i) with FGF2. The second route is by direct derivation from a human embryo in 2i with FGF2. We show that human naïve cells meet mouse criteria for the naïve state by growth characteristics,antibody labeling profile,gene expression,X-inactivation profile,mitochondrial morphology,microRNA profile and development in the context of teratomas. hESCs can exist in a naïve state without the need for transgenes. Direct derivation is an elusive,but attainable,process,leading to cells at the earliest stage of in vitro pluripotency described for humans. Reverse toggling of primed cells to naïve is efficient and reproducible. View Publication

AIM To enumerate and characterize mesenchymal stem cells (MSC) derived from human embryonic stem cells (hESC) for clinical application. MATERIALS & METHODS hESC were differentiated into hESC-MSC and characterized by the expression of surface markers using flow cytometry. hESC-MSC were evaluated with respect to growth kinetics,colony-forming potential,as well as osteogenic and adipogenic differentiation capacity. Immunosuppressive effects were assessed using peripheral blood mononuclear cell (PBMC) proliferation and cytotoxicity assays. RESULTS hESC-MSC showed similar morphology,and cell surface markers as adipose (AMSC) and bone marrow-derived MSC (BMSC). hESC-MSC exhibited a higher growth rate during early in vitro expansion and equivalent adipogenic and osteogenic differentiation and colony-forming potential as AMSC and BMSC. hESC-MSC demonstrated similar immunosuppressive effects as AMSC and BMSC. CONCLUSION hESC-MSC were comparable to BMSC and AMSC and hence can be used as an alternative source of MSC for clinical applications. View Publication

Differentiation of human stem cells to hepatocytes is crucial for industrial applications as well as to develop new therapeutic strategies for liver disease. The protocol described here,using sequentially growth factors known to play a role in liver embryonic development,efficiently differentiates human embryonic stem cells (hESC) as well as human-induced pluripotent stem cells (hiPSC) to hepatocytes by directing them through defined embryonic intermediates,namely,mesendoderm/definitive endoderm and hepatoblast and hepatocyte phenotype. After 28 days,the final differentiated progeny is a mixture of cells,comprising cells with characteristics of hepatoblasts and a smaller cell fraction with morphological and phenotypical features of mature hepatocytes. An extensive functional characterization of the stem cell progeny should be used to confirm that differentiated cells display functional characteristics of mature hepatocytes including albumin secretion,glycogen storage,and several detoxifying functions such as urea production,bilirubin conjugation,glutathione S-transferase activity,cytochrome activity and drug transporter activity. View Publication

Alcohol consumption has long been associated with a majority of liver diseases and has been found to influence both fetal and adult liver functions. In spite of being one of the major causes of morbidity and mortality in the world,currently,there are no effective strategies that can prevent or treat alcoholic liver disease (ALD),due to a lack of human-relevant research models. Recent success in generation of functionally active mature hepatocyte-like cells from human-induced pluripotent cells (iPSCs) enables us to better understand the effects of alcohol on liver functions. Here,we describe the method and effect of alcohol exposure on multistage hepatic cell types derived from human iPSCs,in an attempt to recapitulate the early stages of liver tissue injury associated with ALD. We exposed different stages of iPSC-induced hepatic cells to ethanol at a pathophysiological concentration. In addition to stage-specific molecular markers,we measured several key cellular parameters of hepatocyte injury,including apoptosis,proliferation,and lipid accumulation. View Publication

X-linked dystonia-parkinsonism (XDP) is a hereditary neurodegenerative disorder involving a progressive loss of striatal medium spiny neurons. The mechanisms underlying neurodegeneration are not known,in part because there have been few cellular models available for studying the disease. The XDP haplotype consists of multiple sequence variations in a region of the X chromosome containingTAF1,a large gene with at least 38 exons,and a multiple transcript system (MTS) composed of five unconventional exons. A previous study identified an XDP-specific insertion of a SINE-VNTR-Alu (SVA)-type retrotransposon in intron 32 ofTAF1,as well as a neural-specific TAF1 isoform,N-TAF1,which showed decreased expression in post-mortem XDP brain compared with control tissue. Here,we generated XDP patient and control fibroblasts and induced pluripotent stem cells (iPSCs) in order to further probe cellular defects associated with this disease. As initial validation of the model,we compared expression ofTAF1and MTS transcripts in XDP versus control fibroblasts and iPSC-derived neural stem cells (NSCs). Compared with control cells,XDP fibroblasts exhibited decreased expression ofTAF1transcript fragments derived from exons 32-36,a region spanning the SVA insertion site. N-TAF1,which incorporates an alternative exon (exon 34'),was not expressed in fibroblasts,but was detectable in iPSC-differentiated NSCs at levels that were ∼threefold lower in XDP cells than in controls. These results support the previous findings that N-TAF1 expression is impaired in XDP,but additionally indicate that this aberrant transcription might occur in neural cells at relatively early stages of development that precede neurodegeneration. View Publication

Efficient cryopreservation of human pluripotent stem cells (hPSCs) in chemically defined,xeno-free conditions is highly desirable for medical research and clinical applications such as cell-based therapies. Here we present a simple and effective slow freezing-rapid thawing protocol for the cryopreservation of feeder-free,single hPSCs. This cryopreservation protocol involves the supplementation of 10 % dimethyl sulfoxide (DMSO) and 10 $$M Rho-associated kinase inhibitor Y-27632 into two types of xeno-free,defined media supplements (Knockout Serum Replacement and TeSR2). High post-thaw cell recovery (˜90 %) and cell expansion (˜70 %) can be achieved using this protocol. The cryopreserved single cells retain the morphological characteristics of hPSCs and differentiation capabilities of pluripotent stem cells. View Publication

Lineage specific human embryonic stem cell (hESC) reporter cell line is a versatile tool for biological studies on real time monitoring of differentiation,physiological and biochemical features of special cell types and pathological mechanism of disease. Here we report the generation of ChAT-zsGreen reporter hESC line that express zsGreen under the control of the choline acetyltransferase (ChAT) promoter using CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats)/Cas9 system. We show that the ChAT-zsGreen hESC reporter cell lines retain the features of undifferentiated hESC. After cholinergic neuronal differentiation,cholinergic neurons were clearly labeled with green fluorescence protein (zsGreen). The ChAT-zsGreen reporter hESC lines are invaluable not only for the monitoring cholinergic neuronal differentiation but also for study physiological and biochemical hallmarks of cholinergic neurons. View Publication

Organotin compounds,such as tributyltin (TBT),are well-known endocrine disruptors. TBT is also known to cause various forms of cytotoxicity,including neurotoxicity and immunotoxicity. However,TBT toxicity has not been identified in normal stem cells. In the present study,we examined the effects of TBT on cell growth in human induced pluripotent stem cells (iPSCs). We found that exposure to nanomolar concentrations of TBT decreased intracellular ATP levels and inhibited cell viability in iPSCs. Because TBT suppressed energy production,which is a critical function of the mitochondria,we further assessed the effects of TBT on mitochondrial dynamics. Staining with MitoTracker revealed that nanomolar concentrations of TBT induced mitochondrial fragmentation. TBT also reduced the expression of mitochondrial fusion protein mitofusin 1 (Mfn1),and this effect was abolished by knockdown of the E3 ubiquitin ligase membrane-associated RING-CH 5 (MARCH5),suggesting that nanomolar concentrations of TBT could induce mitochondrial dysfunction via MARCH5-mediated Mfn1 degradation in iPSCs. Thus,mitochondrial function in normal stem cells could be used to assess cytotoxicity associated with metal exposure. View Publication

Summary Leber congenital amaurosis (LCA) is an inherited retinal dystrophy that causes childhood blindness. Photoreceptors are especially sensitive to an intronic mutation in the cilia-related gene CEP290,which causes missplicing and premature termination,but the basis of this sensitivity is unclear. Here,we generated differentiated photoreceptors in three-dimensional optic cups and retinal pigment epithelium (RPE) from iPSCs with this common CEP290 mutation to investigate disease mechanisms and evaluate candidate therapies. iPSCs differentiated normally into RPE and optic cups,despite abnormal CEP290 splicing and cilia defects. The highest levels of aberrant splicing and cilia defects were observed in optic cups,explaining the retinal-specific manifestation of this CEP290 mutation. Treating optic cups with an antisense morpholino effectively blocked aberrant splicing and restored expression of full-length CEP290,restoring normal cilia-based protein trafficking. These results provide a mechanistic understanding of the retina-specific phenotypes in CEP290 LCA patients and potential strategies for therapeutic intervention. View Publication

Alternative models for more rapid compound safety testing are of increasing demand. With emerging techniques using human pluripotent stem cells,the possibility of generating human in vitro models has gained interest,as factors related to species differences could be potentially eliminated. When studying potential neurotoxic effects of a compound it is of crucial importance to have both neurons and glial cells. We have successfully developed a protocol for generating in vitro 3D human neural tissues,using neural progenitor cells derived from human embryonic stem cells. These 3D neural tissues can be maintained for two months and undergo progressive differentiation. We showed a gradual decreased expression of early neural lineage markers,paralleled by an increase in markers specific for mature neurons,astrocytes and oligodendrocytes. At the end of the two-month culture period the neural tissues not only displayed synapses and immature myelin sheaths around axons,but electrophysiological measurements also showed spontaneous activity. Neurotoxicity testing - comparing non-neurotoxic to known neurotoxic model compounds - showed an expected increase in the marker of astroglial reactivity after exposure to known neurotoxicants methylmercury and trimethyltin. Although further characterization and refinement of the model is required,these results indicate its potential usefulness for in vitro neurotoxicity testing. View Publication

Retinitis pigmentosa (RP) is the most frequent form of inherited retinal dystrophy. RP is genetically heterogeneous and the genes identified to date encode proteins involved in a wide range of functional pathways,including photoreceptor development,phototransduction,the retinoid cycle,cilia,and outer segment development. Here we report the identification of biallelic mutations in Receptor Expression Enhancer Protein 6 (REEP6) in seven individuals with autosomal-recessive RP from five unrelated families. REEP6 is a member of the REEP/Yop1 family of proteins that influence the structure of the endoplasmic reticulum but is relatively unstudied. The six variants identified include three frameshift variants,two missense variants,and a genomic rearrangement that disrupts exon 1. Human 3D organoid optic cups were used to investigate REEP6 expression and confirmed the expression of a retina-specific isoform REEP6.1,which is specifically affected by one of the frameshift mutations. Expression of the two missense variants (c.383CtextgreaterT [p.Pro128Leu] and c.404TtextgreaterC [p.Leu135Pro]) and the REEP6.1 frameshift mutant in cultured cells suggest that these changes destabilize the protein. Furthermore,CRISPR-Cas9-mediated gene editing was used to produce Reep6 knock-in mice with the p.Leu135Pro RP-associated variant identified in one RP-affected individual. The homozygous knock-in mice mimic the clinical phenotypes of RP,including progressive photoreceptor degeneration and dysfunction of the rod photoreceptors. Therefore,our study implicates REEP6 in retinal homeostasis and highlights a pathway previously uncharacterized in retinal dystrophy. View Publication

Sex chromosome dosage compensation is essential in most metazoans,but the developmental timing and underlying mechanisms vary significantly,even among placental mammals. Here we identify human-specific mechanisms regulating X chromosome activity in early embryonic development. Single-cell RNA sequencing and imaging revealed co-activation and accumulation of the long noncoding RNAs (lncRNAs) XACT and XIST on active X chromosomes in both early human pre-implantation embryos and naive human embryonic stem cells. In these contexts,the XIST RNA adopts an unusual,highly dispersed organization,which may explain why it does not trigger X chromosome inactivation at this stage. Functional studies in transgenic mouse cells show that XACT influences XIST accumulation in cis. Our findings therefore suggest a mechanism involving antagonistic activity of XIST and XACT in controlling X chromosome activity in early human embryos,and they highlight the contribution of rapidly evolving lncRNAs to species-specific developmental mechanisms. View Publication