技术资料

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

Human pluripotent stem cell (hPSC) density is an important factor in self-renewal and differentiation fates; however,the mechanisms through which hPSCs sense cell density and process this information in making cell fate decisions remain to be fully understood. One particular pathway that may prove important in density-dependent signaling in hPSCs is the Hippo pathway,which is regulated by cell-cell contact and mechanosensing through the cytoskeleton and has been linked to the maintenance of stem cell pluripotency. To probe regulation of Hippo pathway activity in hPSCs,we assessed whether Hippo pathway transcriptional activator YAP was differentially modulated by cell density. At higher cell densities,YAP phosphorylation and localization to the cytoplasm increased,which led to decreased YAP-mediated transcriptional activity. Furthermore,total YAP protein levels diminished at high cell density due to the phosphorylation-targeted degradation of YAP. Inducible shRNA knockdown of YAP reduced expression of YAP target genes and pluripotency genes. Finally,the density-dependent increase of neuroepithelial cell differentiation was mitigated by shRNA knockdown of YAP. Our results suggest a pivotal role of YAP in cell density-mediated fate decisions in hPSCs. View Publication

Embryonic stem cells (ESCs) exhibit unrestricted and indefinite,but stringently controlled,proliferation,and can differentiate into any lineage in the body. In the current study,we test the hypothesis that expression of ribosomal RNA (rRNA) and ribosomal protein genes (RPGs) contribute to the ability of hESCs to proliferate indefinitely. Consistent with the accelerated growth rate of hESCs,we find that hESC lines H1 and H9 both exhibit significantly higher levels of rRNA when compared to a panel of normal and cancer human cell lines. Although many RPGs are expressed at levels that comparable to other human cell lines,a few RPGs also exhibit higher expression levels. In situ nuclear run-on assays reveal that both nucleoli in hESCs actively transcribe nascent rRNA. Employing genome-wide chromatin immunoprecipitation-deep sequencing and bioinformatics approaches,we discovered that,RPGs are dominantly marked by the activating H3K4me3 histone mark in the G1,M,and G2 phases of the cell cycle. Interestingly,the rDNA repeats are marked by the activating H3K4me3 only in the M phase,and repressive H3K27me3 histone mark in all three cell cycle phases. Bioinformatics analyses also reveal that Myc,a known regulator of cell growth and proliferation,occupies both the rRNA genes and RPGs. Functionally,down-regulation of Myc expression by siRNA results in a concomitant decrease in rRNA levels. Together,our results show that expression of rRNA,which is regulated by the Myc pluripotency transcription factor,and of RPGs in hESCs is associated with the activating H3K4me3 modification. J. Cell. Physiol. 231: 2007-2013,2016. textcopyright 2016 Wiley Periodicals,Inc. View Publication

Since the discovery of induced pluripotent stem cells (iPSCs),numerous approaches have been explored to improve the original protocol,which is based on a two-dimensional (2D) cell-culture system. Surprisingly,nothing is known about the effect of a more biologically faithful 3D environment on somatic-cell reprogramming. Here,we report a systematic analysis of how reprogramming of somatic cells occurs within engineered 3D extracellular matrices. By modulating microenvironmental stiffness,degradability and biochemical composition,we have identified a previously unknown role for biophysical effectors in the promotion of iPSC generation. We find that the physical cell confinement imposed by the 3D microenvironment boosts reprogramming through an accelerated mesenchymal-to-epithelial transition and increased epigenetic remodelling. We conclude that 3D microenvironmental signals act synergistically with reprogramming transcription factors to increase somatic plasticity. View Publication

Human induced pluripotent stem cells (hiPSCs) are capable of unlimited self-renewal and can generate nearly all cells in the body. Changes induced by different LSD1 activities on the regulation of hiPSC self-renewal and differentiation and the mechanism underlying such changes were determined. We used two different LSD1 inhibitors (phenelzine sulfate and tranylcypromine) and RNAi technique to inhibit LSD1 activity,and we obtained hiPSCs showing 71.3%,53.28%,and 31.33% of the LSD1 activity in normal hiPSCs. The cells still maintained satisfactory self-renewal capacity when LSD1 activity was at 71.3%. The growth rate of hiPSCs decreased and cells differentiated when LSD1 activity was at approximately 53.28%. The hiPSCs were mainly arrested in the G0/G1 phase and simultaneously differentiated into endodermal tissue when LSD1 activity was at 31.33%. Teratoma experiments showed that the downregulation of LSD1 resulted in low teratoma volume. When LSD1 activity was below 50%,pluripotency of hiPSCs was impaired,and the teratomas mainly comprised endodermal and mesodermal tissues. This phenomenon was achieved by regulating the critical balance between histone methylation and demethylation at regulatory regions of several key pluripotent and developmental genes. View Publication

Induced pluripotent stem cell (iPSC)-derived cortical neurons potentially present a powerful new model to understand corticogenesis and neurological disease. Previous work has established that differentiation protocols can produce cortical neurons,but little has been done to characterize these at cellular resolution. In particular,it is unclear to what extent in vitro two-dimensional,relatively disordered culture conditions recapitulate the development of in vivo cortical layer identity. Single-cell multiplex reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) was used to interrogate the expression of genes previously implicated in cortical layer or phenotypic identity in individual cells. Totally,93.6% of single cells derived from iPSCs expressed genes indicative of neuronal identity. High proportions of single neurons derived from iPSCs expressed glutamatergic receptors and synaptic genes. And,68.4% of iPSC-derived neurons expressing at least one layer marker could be assigned to a laminar identity using canonical cortical layer marker genes. We compared single-cell RNA-seq of our iPSC-derived neurons to available single-cell RNA-seq data from human fetal and adult brain and found that iPSC-derived cortical neurons closely resembled primary fetal brain cells. Unexpectedly,a subpopulation of iPSC-derived neurons co-expressed canonical fetal deep and upper cortical layer markers. However,this appeared to be concordant with data from primary cells. Our results therefore provide reassurance that iPSC-derived cortical neurons are highly similar to primary cortical neurons at the level of single cells but suggest that current layer markers,although effective,may not be able to disambiguate cortical layer identity in all cells. View Publication

BACKGROUND The incidence of neurological complications and fatalities associated with Hand,Foot & Mouth disease has increased over recent years,due to emergence of newly-evolved strains of Enterovirus 71 (EV71). In the search for new antiviral therapeutics against EV71,accurate and sensitive in vitro cellular models for preliminary studies of EV71 pathogenesis is an essential prerequisite,before progressing to expensive and time-consuming live animal studies and clinical trials. METHODS This study thus investigated whether neural lineages derived from pluripotent human embryonic stem cells (hESC) can fulfil this purpose. EV71 infection of hESC-derived neural stem cells (NSC) and mature neurons (MN) was carried out in vitro,in comparison with RD and SH-SY5Y cell lines. RESULTS Upon assessment of post-infection survivability and EV71 production by the various types,it was observed that NSC were significantly more susceptible to EV71 infection compared to MN,RD (rhabdomyosarcoma) and SH-SY5Y cells,which was consistent with previous studies on mice. The SP81 peptide had significantly greater inhibitory effect on EV71 production by NSC and MN compared to the cancer-derived RD and SH-SY5Y cell lines. CONCLUSIONS Hence,this study demonstrates that hESC-derived neural lineages can be utilized as in vitro models for studying EV71 pathogenesis and for screening of antiviral therapeutics. View Publication

BACKGROUND: Many retinal degenerative diseases are caused by the loss of retinal ganglion cells (RGCs). Autosomal dominant optic atrophy is the most common hereditary optic atrophy disease and is characterized by central vision loss and degeneration of RGCs. Currently,there is no effective treatment for this group of diseases. However,stem cell therapy holds great potential for replacing lost RGCs of patients. Compared with embryonic stem cells,induced pluripotent stem cells (iPSCs) can be derived from adult somatic cells,and they are associated with fewer ethical concerns and are less prone to immune rejection. In addition,patient-derived iPSCs may provide us with a cellular model for studying the pathogenesis and potential therapeutic agents for optic atrophy.backslashnbackslashnMETHODS: In this study,iPSCs were obtained from patients carrying an OPA1 mutation (OPA1 (+/-) -iPSC) that were diagnosed with optic atrophy. These iPSCs were differentiated into putative RGCs,which were subsequently characterized by using RGC-specific expression markers BRN3a and ISLET-1.backslashnbackslashnRESULTS: Mutant OPA1 (+/-) -iPSCs exhibited significantly more apoptosis and were unable to efficiently differentiate into RGCs. However,with the addition of neural induction medium,Noggin,or estrogen,OPA1 (+/-) -iPSC differentiation into RGCs was promoted.backslashnbackslashnCONCLUSIONS: Our results suggest that apoptosis mediated by OPA1 mutations plays an important role in the pathogenesis of optic atrophy,and both noggin and β-estrogen may represent potential therapeutic agents for OPA1-related optic atrophy. View Publication

Human induced pluripotent stem cells (hiPSCs) have potential for personalized and regenerative medicine. While most of the methods using these cells have focused on deriving homogenous populations of specialized cells,there has been modest success in producing hiPSC-derived organotypic tissues or organoids. Here we present a novel approach for generating and then co-differentiating hiPSC-derived progenitors. With a genetically engineered pulse of GATA-binding protein 6 (GATA6) expression,we initiate rapid emergence of all three germ layers as a complex function of GATA6 expression levels and tissue context. Within 2 weeks we obtain a complex tissue that recapitulates early developmental processes and exhibits a liver bud-like phenotype,including haematopoietic and stromal cells as well as a neuronal niche. Collectively,our approach demonstrates derivation of complex tissues from hiPSCs using a single autologous hiPSCs as source and generates a range of stromal cells that co-develop with parenchymal cells to form tissues. View Publication

MicroRNA expression profiling in human liver progenitor cells following hepatocytic differentiation identified miR-122 and miR-194 as the microRNAs most strongly upregulated during hepatocytic differentiation of progenitor cells. MiR-194 was also highly upregulated following hepatocytic differentiation of human embryonic stem cells (hESCs). Overexpression of miR-194 in progenitor cells accelerated their differentiation into hepatocytes,as measured by morphological features such as canaliculi and expression of hepatocytic markers. Overexpression of miR-194 in hESCs induced their spontaneous differentiation,a phenotype accompanied with accelerated loss of the pluripotent factors OCT4 and NANOG and decrease in mesoderm marker HAND1 expression. We then identified YAP1 as a direct target of miR-194. Inhibition of YAP1 strongly induced hepatocytic differentiation of progenitor cells and YAP1 overexpression reversed the miR-194-induced hepatocytic differentiation of progenitor cells. In conclusion,we identified miR-194 as a potent inducer of hepatocytic differentiation of progenitor cells and further identified YAP1 as a mediator of miR-194's effects on hepatocytic differentiation and liver progenitor cell fate. Stem Cells 2016;34:1284-1296. View Publication

Mutations in SCN1A,the gene encoding the α subunit of Nav1.1 channel,can cause epilepsies with wide ranges of clinical phenotypes,which are associated with the contrasting effects of channel loss-of-function or gain-of-function. In this project,CRISPR/Cas9- and TALEN-mediated genome-editing techniques were applied to induced pluripotent stem cell (iPSC)-based-disease model to explore the mechanism of epilepsy caused by SCN1A loss-of-function mutation. By fluorescently labeling GABAergic subtype in iPSC-derived neurons using CRISPR/Cas9,we for the first time performed electrophysiological studies on SCN1A-expressing neural subtype and monitored the postsynaptic activity of both inhibitory and excitatory types. We found that the mutation c.A5768G,which led to no current of Nav1.1 in exogenously transfected system,influenced the properties of not only Nav current amount,but also Nav activation in Nav1.1-expressing GABAergic neurons. The two alterations in Nav further reduced the amplitudes and enhanced the thresholds of action potential in patient-derived GABAergic neurons,and led to weakened spontaneous inhibitory postsynaptic currents (sIPSCs) in the patient-derived neuronal network. Although the spontaneous excitatory postsynaptic currents (sEPSCs) did not change significantly,when the frequencies of both sIPSCs and sEPSCs were further analyzed,we found the whole postsynaptic activity transferred from the inhibition-dominated state to excitation in patient-derived neuronal networks,suggesting that changes in sIPSCs alone were sufficient to significantly reverse the excitatory level of spontaneous postsynaptic activity. In summary,our findings fill the gap of our knowledge regarding the relationship between SCN1A mutation effect recorded on exogenously transfected cells and on Nav1.1-expressing neurons,and reveal the physiological basis underlying epileptogenesis caused by SCN1A loss-of-function mutation. View Publication

Cystic fibrosis (CF) is a recessive inherited disease associated with multiorgan damage that compromises epithelial and inflammatory cell function. Induced pluripotent stem cells (iPSCs) have significantly advanced the potential of developing a personalized cell-based therapy for diseases like CF by generating patient-specific stem cells that can be differentiated into cells that repair tissues damaged by disease pathology. The F508del mutation in airway epithelial cell-derived CF-iPSCs was corrected with small/short DNA fragments (SDFs) and sequence-specific TALENs. An allele-specific PCR,cyclic enrichment strategy gave ˜100-fold enrichment of the corrected CF-iPSCs after six enrichment cycles that facilitated isolation of corrected clones. The seamless SDF-based gene modification strategy used to correct the CF-iPSCs resulted in pluripotent cells that,when differentiated into endoderm/airway-like epithelial cells showed wild-type (wt) airway epithelial cell cAMP-dependent Cl ion transport or showed the appropriate cell-type characteristics when differentiated along mesoderm/hematopoietic inflammatory cell lineage pathways. View Publication