技术资料

The recent Zika virus (ZIKV) outbreak,which mainly affected Brazil and neighbouring states,demonstrated the paucity of information concerning the epidemiology of several flaviruses,but also highlighted the lack of available agents with which to treat such emerging diseases. Here,we show that heparin,a widely used anticoagulant,while exerting a modest inhibitory effect on Zika Virus replication,fully prevents virus-induced cell death of human neural progenitor cells (NPCs). View Publication

Acute monocytic leukemia (AML-M5),a subtype of acute myeloid leukemia (AML),affects mostly young children and has poor prognosis. The mechanisms of treatment failure of AML-M5 are still unclear. In this study,we generated iPSC from THP-1 cells from a patient with AML-M5,using retroviruses encoding the pluripotency-associated genes (OCT3/4,SOX2,KLF4 and c-MYC). These AML-M5-derived iPSC showed features similar with those of human embryonic stem cells in terms of the morphology,gene expression,protein/antigen expression and differentiation capability. Parental-specific markers were down-regulated in these AML-M5-derived iPSCs. Expression of MLL-AF9 fusion gene (previously identified to be associated with pathogenesis of AML-M5) was observed in all iPSC clones as well as parental cells. We conclude that AML-M5-specific iPSC clones have been successfully developed. This disease model may provide a novel approach for future study of pathogenesis and therapeutic intervention of AML-M5. View Publication

Directed differentiation of stem cells offers a scalable solution to the need for human cell models recapitulating islet biology and T2D pathogenesis. We profiled mRNA expression at 6 stages of an induced pluripotent stem cell (iPSC) model of endocrine pancreas development from 2 donors,and characterized the distinct transcriptomic profiles associated with each stage. Established regulators of endodermal lineage commitment,such as SOX17 (log2 fold change [FC] compared to iPSCs = 14.2,p-value = 4.9 × 10(-5)) and the pancreatic agenesis gene GATA6 (log2 FC = 12.1,p-value = 8.6 × 10(-5)),showed transcriptional variation consistent with their known developmental roles. However,these analyses highlighted many other genes with stage-specific expression patterns,some of which may be novel drivers or markers of islet development. For example,the leptin receptor gene,LEPR,was most highly expressed in published data from in vivo-matured cells compared to our endocrine pancreas-like cells (log2 FC = 5.5,p-value = 2.0 × 10(-12)),suggesting a role for the leptin pathway in the maturation process. Endocrine pancreas-like cells showed significant stage-selective expression of adult islet genes,including INS,ABCC8,and GLP1R,and enrichment of relevant GO-terms (e.g. insulin secretion"; odds ratio = 4.2� View Publication

Loss-of-function studies are fundamental for dissecting gene function. Yet,methods to rapidly and effectively perturb genes in mammalian cells,and particularly in stem cells,are scarce. Here we present a system for simultaneous conditional regulation of two different proteins in the same mammalian cell. This system harnesses the plant auxin and jasmonate hormone-induced degradation pathways,and is deliverable with only two lentiviral vectors. It combines RNAi-mediated silencing of two endogenous proteins with the expression of two exogenous proteins whose degradation is induced by external ligands in a rapid,reversible,titratable and independent manner. By engineering molecular tuners for NANOG,CHK1,p53 and NOTCH1 in mammalian stem cells,we have validated the applicability of the system and demonstrated its potential to unravel complex biological processes. View Publication

The unique mental abilities of humans are rooted in the immensely expanded and folded neocortex,which reflects the expansion of neural progenitors,especially basal progenitors including basal radial glia (bRGs) and intermediate progenitor cells (IPCs). We found that constitutively active Sonic hedgehog (Shh) signaling expanded bRGs and IPCs and induced folding in the otherwise smooth mouse neocortex,whereas the loss of Shh signaling decreased the number of bRGs and IPCs and the size of the neocortex. SHH signaling was strongly active in the human fetal neocortex but Shh signaling was not strongly active in the mouse embryonic neocortex,and blocking SHH signaling in human cerebral organoids decreased the number of bRGs. Mechanistically,Shh signaling increased the initial generation and self-renewal of bRGs and IPC proliferation in mice and the initial generation of bRGs in human cerebral organoids. Thus,robust SHH signaling in the human fetal neocortex may contribute to bRG and IPC expansion and neocortical growth and folding. View Publication

Frontotemporal dementia (FTD) and other tauopathies characterized by focal brain neurodegeneration and pathological accumulation of proteins are commonly associated with tau mutations. However,the mechanism of neuronal loss is not fully understood. To identify molecular events associated with tauopathy,we studied induced pluripotent stem cell (iPSC)-derived neurons from individuals carrying the tau-A152T variant. We highlight the potential of in-depth phenotyping of human neuronal cell models for pre-clinical studies and identification of modulators of endogenous tau toxicity. Through a panel of biochemical and cellular assays,A152T neurons showed accumulation,redistribution,and decreased solubility of tau. Upregulation of tau was coupled to enhanced stress-inducible markers and cell vulnerability to proteotoxic,excitotoxic,and mitochondrial stressors,which was rescued upon CRISPR/Cas9-mediated targeting of tau or by pharmacological activation of autophagy. Our findings unmask tau-mediated perturbations of specific pathways associated with neuronal vulnerability,revealing potential early disease biomarkers and therapeutic targets for FTD and other tauopathies. View Publication

Mesenchymal stem cells (MSCs) are defined as non-hematopoietic,plastic-adherent,self-renewing cells that are capable of tri-lineage differentiation into bone,cartilage or fat in vitro. Thus,MSCs are promising candidates for cell-based medicine. However,classifications of MSCs have been defined retrospectively; moreover,this conventional criterion may be inaccurate due to contamination with other hematopoietic lineage cells. Human MSCs can be enriched by selection for LNGFR and THY-1,and this population may be analogous to murine PDGFR??+Sca-1+ cells,which are developmentally derived from neural crest cells (NCCs). Murine NCCs were labeled by fluorescence,which provided definitive proof of neural crest lineage,however,technical considerations prevent the use of a similar approach to determine the origin of human LNGFR+THY-1+ MSCs. To further clarify the origin of human MSCs,human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs) were used in this study. Under culture conditions required for the induction of neural crest cells,human ESCs and iPSCs-derived cells highly expressed LNGFR and THY-1. These LNGFR+THY-1+ neural crest-like cells,designated as LT-NCLCs,showed a strong potential to differentiate into both mesenchymal and neural crest lineages. LT-NCLCs proliferated to form colonies and actively migrated in response to serum concentration. Furthermore,we transplanted LT-NCLCs into chick embryos,and traced their potential for survival,migration and differentiation in the host environment. These results suggest that LNGFR+THY-1+ cells identified following NCLC induction from ESCs/iPSCs shared similar potentials with multipotent MSCs. 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