技术资料

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have the potential to provide an infinite source of tissues for regenerative medicine. Although defined xeno-free media have been developed,culture conditions for reliable propagation of hESCs still require considerable improvement. Here we show that recombinant E8 fragments of laminin isoforms (LM-E8s),which are the minimum fragments conferring integrin-binding activity,promote greater adhesion of hESCs and hiPSCs than do Matrigel and intact laminin isoforms. Furthermore,LM-E8s sustain long-term self-renewal of hESCs and hiPSCs in defined xeno-free media with dissociated cell passaging. We successfully maintained three hESC and two hiPSC lines on LM-E8s in three defined media for 10 passages. hESCs maintained high level expression of pluripotency markers,had a normal karyotype after 30 passages and could differentiate into all three germ layers. This culture system allows robust proliferation of hESCs and hiPSCs for therapeutic applications. View Publication

Human neural stem cells hold great promise for research and therapy in neural disease. We describe the generation of integration-free and expandable human neural progenitor cells (NPCs). We combined an episomal system to deliver reprogramming factors with a chemically defined culture medium to reprogram epithelial-like cells from human urine into NPCs (hUiNPCs). These transgene-free hUiNPCs can self-renew and can differentiate into multiple functional neuronal subtypes and glial cells in vitro. Although functional in vivo analysis is still needed,we report that the cells survive and differentiate upon transplant into newborn rat brain. View Publication

The use of human pluripotent stem cells (hPSCs) in cell therapy is hindered by the tumorigenic risk from residual undifferentiated cells. Here we performed a high-throughput screen of over 52,000 small molecules and identified 15 pluripotent cell-specific inhibitors (PluriSIns),nine of which share a common structural moiety. The PluriSIns selectively eliminated hPSCs while sparing a large array of progenitor and differentiated cells. Cellular and molecular analyses demonstrated that the most selective compound,PluriSIn 1,induces ER stress,protein synthesis attenuation,and apoptosis in hPSCs. Close examination identified this molecule as an inhibitor of stearoyl-coA desaturase (SCD1),the key enzyme in oleic acid biosynthesis,revealing a unique role for lipid metabolism in hPSCs. PluriSIn 1 was also cytotoxic to mouse blastocysts,indicating that the dependence on oleate is inherent to the pluripotent state. Finally,application of PluriSIn 1 prevented teratoma formation from tumorigenic undifferentiated cells. These findings should increase the safety of hPSC-based treatments. ?? 2013 Elsevier Inc. View Publication

Perhaps one of the most significant achievements in modern science is the discovery of human induced pluripotent stem cells (hiPSCs),which have paved the way for regeneration therapy using patients' own cells. Cardiomyocytes differentiated from hiPSCs (hiPSC-CMs) could be used for modelling patients with heart failure,for testing new drugs,and for cellular therapy in the future. However,the present cardiomyocyte differentiation protocols exhibit variable differentiation efficiency across different hiPSC lines,which inhibit the application of this technology significantly. Here,we demonstrate a novel myocyte differentiation protocol that can yield a significant,high percentage of cardiac myocyte differentiation (backslashtextgreater85%) in 2 hiPSC lines,which makes the fabrication of a human cardiac muscle patch possible. The established hiPSCs cell lines being examined include the transgene integrated UCBiPS7 derived from cord blood cells and non-integrated PCBC16iPS from skin fibroblasts. The results indicate that hiPSC-CMs derived from established hiPSC lines respond to adrenergic or acetylcholine stimulation and beat regularly for greater than 60 days. This data also demonstrates that this novel differentiation protocol can efficiently generate hiPSC-CMs from iPSC lines that are derived not only from fibroblasts,but also from blood mononuclear cells. View Publication

Human ES cells (hESC) exposed to bone morphogenic protein 4 (BMP4) in the absence of FGF2 have become widely used for studying trophoblast development,but the soundness of this model has been challenged by others,who concluded that differentiation was primarily toward mesoderm rather than trophoblast. Here we confirm that hESC grown under the standard conditions on a medium conditioned by mouse embryonic fibroblasts in the presence of BMP4 and absence of FGF2 on a Matrigel substratum rapidly convert to an epithelium that is largely KRT7+ within 48 h,with minimal expression of mesoderm markers,including T (Brachyury). Instead,they begin to express a series of trophoblast markers,including HLA-G,demonstrate invasive properties that are independent of the continued presence of BMP4 in the medium,and,over time,produce extensive amounts of human chorionic gonadotropin,progesterone,placental growth factor,and placental lactogen. This process of differentiation is not dependent on conditioning of the medium by mouse embryonic fibroblasts and is accelerated in the presence of inhibitors of Activin and FGF2 signaling,which at day 2 provide colonies that are entirely KRT7+ and in which the majority of cells are transiently CDX2+. Colonies grown on two chemically defined media,including the one in which BMP4 was reported to drive mesoderm formation,also differentiate at least partially to trophoblast in response to BMP4. The experiments demonstrate that the in vitro BMP4/hESC model is valid for studying the emergence and differentiation of trophoblasts. View Publication

We report that a single growth factor,NM23-H1,enables serial passaging of both human ES and iPS cells in the absence of feeder cells,their conditioned media or bFGF in a fully defined xeno-free media on a novel defined,xeno-free surface. Stem cells cultured in this system show a gene expression pattern indicative of a more naïve" state than stem cells grown in bFGF-based media. NM23-H1 and MUC1* growth factor receptor cooperate to control stem cell self-replication. By manipulating the multimerization state of NM23-H1� View Publication

In this study,we sought to establish a novel method to prospectively and dynamically identify live human oligodendrocyte precursor cells (OPCs) and oligodendrocyte lineage cells from brain dissociates and pluripotent stem cell culture. We selected a highly conserved enhancer element of the Sox10 gene,known as MCS5,which directs reporter expression to oligodendrocyte lineage cells in mouse and zebrafish. We demonstrate that lentiviral Sox10-MCS5 induced expression of GFP at high levels in a subpopulation of human CD140a/PDGF??R-sorted OPCs as well as their immature oligodendrocyte progeny. Furthermore,we show that almost all Sox10-MCS5:GFPhigh cells expressed OPC antigen CD140a and human OPCs expressing SOX10,OLIG2,and PDGFRA mRNAs could be prospectively identified using GFP based fluorescence activated cells sorting alone. Additionally,we established a human induced pluripotent cell (iPSC) line transduced with the Sox10-MCS5:GFP reporter using a Rex-Neo cassette. Similar to human primary cells,GFP expression was restricted to embryoid bodies containing both oligodendrocyte progenitor and oligodendrocyte cells and co-localized with NG2 and O4-positive cells respectively. As such,we have developed a novel reporter system that can track oligodendrocyte commitment in human cells,establishing a valuable tool to improve our understanding and efficiency of human oligodendrocyte derivation. ?? 2013 Elsevier Inc. View Publication

Huntington's disease is caused by expanded CAG repeats in HTT,conferring toxic gain of function on mutant HTT (mHTT) protein. Reducing mHTT amounts is postulated as a strategy for therapeutic intervention. We conducted genome-wide RNA interference screens for genes modifying mHTT abundance and identified 13 hits. We tested 10 in vivo in a Drosophila melanogaster Huntington's disease model,and 6 exhibited activity consistent with the in vitro screening results. Among these,negative regulator of ubiquitin-like protein 1 (NUB1) overexpression lowered mHTT in neuronal models and rescued mHTT-induced death. NUB1 reduces mHTT amounts by enhancing polyubiquitination and proteasomal degradation of mHTT protein. The process requires CUL3 and the ubiquitin-like protein NEDD8 necessary for CUL3 activation. As a potential approach to modulating NUB1 for treatment,interferon-β lowered mHTT and rescued neuronal toxicity through induction of NUB1. Thus,we have identified genes modifying endogenous mHTT using high-throughput screening and demonstrate NUB1 as an exemplar entry point for therapeutic intervention of Huntington's disease. View Publication