技术资料

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

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

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

In most human somatic cells,the lack of telomerase activity results in progressive telomere shortening during each cell division. Eventually,DNA damage responses triggered by critically short telomeres induce an irreversible cell cycle arrest termed replicative senescence. However,the cellular responses of human pluripotent stem cells to telomere uncapping remain unknown. We generated telomerase knockout human embryonic stem (ES) cells through gene targeting. Telomerase inactivation in ES cells results in progressive telomere shortening. Telomere DNA damage in ES cells and neural progenitor cells induces rapid apoptosis when telomeres are uncapped,in contrast to fibroblast cells that enter a state of replicative senescence. Significantly,telomerase inactivation limits the proliferation capacity of human ES cells without affecting their pluripotency. By targeting telomerase activity,we can functionally separate the two unique properties of human pluripotent stem cells,namely unlimited self-renewal and pluripotency. We show that the potential of ES cells to form teratomas in vivo is dictated by their telomere length. By controlling telomere length of ES cells through telomerase inactivation,we can inhibit teratoma formation and potentially improve the safety of cell therapies involving terminally differentiated cells as well as specific progenitor cells that do not require sustained cellular proliferation in vivo,and thus sustained telomerase activity. This article is protected by copyright. All rights reserved. View Publication

It has become increasingly clear that proper cellular control of pluripotency and differentiation is related to the regulation of rRNA synthesis. To further our understanding of the role that the regulation of rRNA synthesis has in pluripotency we monitored rRNA synthesis during the directed differentiation of human embryonic stem cells (hESCs). We discovered that the rRNA synthesis rate is reduced ˜50% within 6 hours of ACTIVIN A treatment. This precedes reductions in expression of specific stem cell markers and increases in expression of specific germ layer markers. The reduction in rRNA synthesis is concomitant with dissociation of the Pol I transcription factor,UBTF,from the rRNA gene promoter and precedes any increase to heterochromatin throughout the rRNA gene. To directly investigate the role of rRNA synthesis in pluripotency,hESCs were treated with the Pol I inhibitor,CX-5461. The direct reduction of rRNA synthesis by CX-5461 induces the expression of markers for all three germ layers,reduces the expression of pluripotency markers,and is overall similar to the ACTIVIN A induced changes. This work indicates that the dissociation of UBTF from the rRNA gene,and corresponding reduction in transcription,represent early regulatory events during the directed differentiation of pluripotent stem cells. View Publication

Fibrodysplasia ossificans progressiva (FOP) syndrome is caused by mutation of the gene ACVR1,encoding a constitutive active bone morphogenetic protein type I receptor (also called ALK2) to induce heterotopic ossification in the patient. To genetically correct it,we attempted to generate the mutant ALK2-iPSCs (mALK2-iPSCs) from FOP-human dermal fibroblasts. However,the mALK2 leads to inhibitory pluripotency maintenance,or impaired clonogenic potential after single-cell dissociation as an inevitable step,which applies gene-correction tools to induced pluripotent stem cells (iPSCs). Thus,current iPSC-based gene therapy approach reveals a limitation that is not readily applicable to iPSCs with ALK2 mutation. Here we developed a simplified one-step procedure by simultaneously introducing reprogramming and gene-editing components into human fibroblasts derived from patient with FOP syndrome,and genetically treated it. The mixtures of reprogramming and gene-editing components are composed of reprogramming episomal vectors,CRISPR/Cas9-expressing vectors and single-stranded oligodeoxynucleotide harboring normal base to correct ALK2 c.617GtextgreaterA. The one-step-mediated ALK2 gene-corrected iPSCs restored global gene expression pattern,as well as mineralization to the extent of normal iPSCs. This procedure not only helps save time,labor and costs but also opens up a new paradigm that is beyond the current application of gene-editing methodologies,which is hampered by inhibitory pluripotency-maintenance requirements,or vulnerability of single-cell-dissociated iPSCs. View Publication

Induced pluripotent stem cells (iPSCs) are pluripotent cells derived from adult somatic cells. After the pioneering work by Yamanaka,who first generated iPSCs by retroviral transduction of four reprogramming factors,several alternative methods to obtain iPSCs have been developed in order to increase the yield and safety of the process. However,the question remains open on whether the different reprogramming methods can influence the pluripotency features of the derived lines. In this study,three different strategies,based on retroviral vectors,episomal vectors,and Sendai virus vectors,were applied to derive iPSCs from human fibroblasts. The reprogramming efficiency of the methods based on episomal and Sendai virus vectors was higher than that of the retroviral vector-based approach. All human iPSC clones derived with the different methods showed the typical features of pluripotent stem cells,including the expression of alkaline phosphatase and stemness maker genes,and could give rise to the three germ layer derivatives upon embryoid bodies assay. Microarray analysis confirmed the presence of typical stem cell gene expression profiles in all iPSC clones and did not identify any significant difference among reprogramming methods. In conclusion,the use of different reprogramming methods is equivalent and does not affect gene expression profile of the derived human iPSCs. 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