技术资料

Transcription factor-mediated reprograming is a powerful method to study cell fate changes. In this study,we demonstrate that the transcription factor Gata6 can initiate reprograming of multiple cell types to induced extraembryonic endoderm stem (iXEN) cells. Intriguingly,Gata6 is sufficient to drive iXEN cells from mouse pluripotent cells and differentiated neural cells. Furthermore,GATA6 induction in human embryonic stem (hES) cells also down-regulates pluripotency gene expression and up-regulates extraembryonic endoderm (ExEn) genes,revealing a conserved function in mediating this cell fate switch. Profiling transcriptional changes following Gata6 induction in mES cells reveals step-wise pluripotency factor disengagement,with initial repression of Nanog and Esrrb,then Sox2,and finally Oct4,alongside step-wise activation of ExEn genes. Chromatin immunoprecipitation and subsequent high-throughput sequencing analysis shows Gata6 enrichment near pluripotency and endoderm genes,suggesting that Gata6 functions as both a direct repressor and activator. Together,this demonstrates that Gata6 is a versatile and potent reprograming factor that can act alone to drive a cell fate switch from diverse cell types. View Publication

The hereditary spastic paraplegias (HSPs) are a heterogeneous group of motorneuron diseases characterized by progressive spasticity and paresis of the lower limbs. Mutations in Spastic Gait 4 (SPG4),encoding spastin,are the most frequent cause of HSP. To understand how mutations in SPG4 affect human neurons,we generated human induced pluripotent stem cells (hiPSCs) from fibroblasts of two patients carrying a c.1684CtextgreaterT nonsense mutation and from two controls. These SPG4 and control hiPSCs were able to differentiate into neurons and glia at comparable efficiency. All known spastin isoforms were reduced in SPG4 neuronal cells. The complexity of SPG4 neurites was decreased,which was paralleled by an imbalance of axonal transport with less retrograde movement. Prominent neurite swellings with disrupted microtubules were present in SPG4 neurons at an ultrastructural level. While some of these swellings contain acetylated and detyrosinated tubulin,these tubulin modifications were unchanged in total cell lysates of SPG4 neurons. Upregulation of another microtubule-severing protein,p60 katanin,may partially compensate for microtubuli dynamics in SPG4 neurons. Overexpression of the M1 or M87 spastin isoforms restored neurite length,branching,numbers of primary neurites and reduced swellings in SPG4 neuronal cells. We conclude that neurite complexity and maintenance in HSP patient-derived neurons are critically sensitive to spastin gene dosage. Our data show that elevation of single spastin isoform levels is sufficient to restore neurite complexity and reduce neurite swellings in patient cells. Furthermore,our human model offers an ideal platform for pharmacological screenings with the goal to restore physiological spastin levels in SPG4 patients. View Publication

Hoechst-effluxing cells (side population cells) are a rare subset of cells found in adult tissues that are highly enriched for stem and progenitor cell activity. To identify potential stem and progenitor cells during lung development,we generated gene expression profiles for CD45- and CD45+ side population cells in the embryonic day 17.5 lung. We found that side population cells comprise 1% of total embryonic day 17.5 lung cells (55% CD45+,45% CD45-). Gene profiling data demonstrated an overrepresentation of endothelial genes within the CD45- side population. We used expression of several distinct genes to identify two types of CD45- side population cells: 1) von Willebrand factor+/smooth muscle actin+ cells that reside in the muscular layer of select large vessels and 2) von Willebrand factor+/intercellular adhesion molecule+ cells that reside within the endothelial layer of select small vessels. Gene profiling of the CD45+ side population indicated an overrepresentation of genes associated with myeloid cell differentiation. Consistent with this,culturing CD45+ side population cells was associated with induction of mature dendritic markers (CD86). The microarray results suggested that expression of myeloperoxidase and proteinase-3 might be used to identify CD45+ side population cells. By immunohistochemistry,we found that myeloperoxidase+/proteinase-3+ cells represent a small subset of total CD45+ cells in the embryonic day 17.5 lung and that they reside in the mesenchyme and perivascular regions. This is the first detailed information regarding the phenotype and localization of side population cells in a developing organ. View Publication

Stem cells,especially human embryonic stem cells (hESCs),are useful models to study molecular mechanisms of human disorders that originate during gestation. Alcohol (ethanol,EtOH) consumption during pregnancy causes a variety of prenatal and postnatal disorders collectively referred to as fetal alcohol spectrum disorders (FASDs). To better understand the molecular events leading to FASDs,we performed a genome-wide analysis of EtOH's effects on the maintenance and differentiation of hESCs in culture. Gene Co-expression Network Analysis showed significant alterations in gene profiles of EtOH-treated differentiated or undifferentiated hESCs,particularly those associated with molecular pathways for metabolic processes,oxidative stress,and neuronal properties of stem cells. A genome-wide DNA methylome analysis revealed widespread EtOH-induced alterations with significant hypermethylation of many regions of chromosomes. Undifferentiated hESCs were more vulnerable to EtOH's effect than their differentiated counterparts,with methylation on the promoter regions of chromosomes 2,16 and 18 in undifferentiated hESCs most affected by EtOH exposure. Combined transcriptomic and DNA methylomic analysis produced a list of differentiation-related genes dysregulated by EtOH-induced DNA methylation changes,which likely play a role in EtOH-induced decreases in hESC pluripotency. DNA sequence motif analysis of genes epigenetically altered by EtOH identified major motifs representing potential binding sites for transcription factors. These findings should help in deciphering the precise mechanisms of alcohol-induced teratogenesis. ?? 2014 Published by Elsevier B.V. View Publication

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The ability of human embryonic stem cells and induced pluripotent stem cells to differentiate into all adult cell types greatly facilitates the study of human development,disease pathogenesis,and the generation of screening systems to identify novel therapeutic agents. Autologous cell therapies based on patient-derived induced pluripotent stem cells also hold great promise for the treatment and correction of many inherited and acquired diseases. The full potential of human pluripotent stem cells can be unleashed by genetically modifying a chosen locus with minimal impact on the remaining genome,which can be achieved by targeting genes by homologous recombination. This chapter will describe a protocol for gene modification of pluripotent stem cells by homologous recombination and several methods for the screening and identification of successfully modified clones. View Publication

The advent of human induced pluripotent stem cell (hiPSC) technology has produced patient-specific hiPSC derived cardiomyocytes (hiPSC-CMs) that can be used as a platform to study cardiac diseases and to explore new therapies.The ability to genetically manipulate hiPSC-CMs not only is essential for identifying the structural and/or functional role of a protein but can also provide valuable information regarding therapeutic applications. In this chapter,we describe protocols for culture,maintenance,and cardiac differentiation of hiPSCs. Then,we provide a basic procedure to transduce hiPSC-CMs. View Publication

Recombinant lentiviral vectors are powerful tools to stably manipulate human pluripotent stem cells. They can be used to deliver ectopic genes,shRNAs,miRNAs,or any possible genetic DNA sequence into diving and nondividing cells. Here we describe a general protocol for the production of self-inactivating lentiviral vector particles and their purification to high titers by either ultracentrifugation or ultrafiltration. Next we provide a basic procedure to transduce human pluripotent stem cells and propagate clonal cell lines. View Publication

Induced pluripotent stem cell (iPS cell) holds great potential for applications in regenerative medicine,drug discovery,and disease modeling. We describe here a practical method to generate human iPS cells from urine-derived cells (UCs) under feeder-free,virus-free,serum-free condition and without oncogene c-MYC. We showed that this approach could be applied in a large population with different genetic backgrounds. UCs are easily accessible and exhibit high reprogramming efficiency,offering advantages over other cell types used for the purpose of iPS generation. Using the approach described in this study,we have generated 93 iPS cell lines from 20 donors with diverse genetic backgrounds. The non-viral iPS cell bank with these cell lines provides a valuable resource for iPS cells research,facilitating future applications of human iPS cells. View Publication

Human embryonic stem cells (hESCs) are thought to be a promising resource for cell therapy,while it has to face the major problem of graft immunological rejection. Major histocompatibility complex (MHC) class I expressed on the cell surface is the major cause of graft rejection. Transporter associated with antigen presentation 1 (TAP1) and TAP-associated glycoprotein (TAPBP) play important roles in regulating MHC class I expression. In this study,we generated TAP1- and TAPBP-deficient hESC lines,respectively,using transcription activator-like effector nucleases technique. These cells showed deficient expression of MHC class I on the cell surface and reduced immunogenicity compared with wild types,but maintained normal pluripotency,karyotypes,and differentiation ability. Thus,our findings are instrumental in developing a universal cell resource with both pluripotency and hypo-immunogenicity for transplantation therapy in the future. View Publication

Human pluripotent stem cells (hPSCs) provide a valuable model for the study of human development and a means to generate a scalable source of cells for therapeutic applications. This protocol specifies cell fate efficiently into cardiac and endothelial lineages from hPSCs. The protocol takes 2 weeks to complete and requires experience in hPSC culture and differentiation techniques. Building on lessons taken from early development,this monolayer-directed differentiation protocol uses different concentrations of activin A and bone morphogenetic protein 4 (BMP4) to polarize cells into mesodermal subtypes that reflect mid-primitive-streak cardiogenic mesoderm and posterior-primitive-streak hemogenic mesoderm. This differentiation platform provides a basis for generating distinct cardiovascular progenitor populations that enable the derivation of cardiomyocytes and functionally distinct endothelial cell (EC) subtypes from cardiogenic versus hemogenic mesoderm with high efficiency without cell sorting. ECs derived from cardiogenic and hemogenic mesoderm can be matured into textgreater90% CD31(+)/VE-cadherin(+) definitive ECs. To test the functionality of ECs at different stages of differentiation,we provide methods for assaying the blood-forming potential and de novo lumen-forming activity of ECs. To our knowledge,this is the first protocol that provides a common platform for directed differentiation of cardiomyocytes and endothelial subtypes from hPSCs. This protocol yields endothelial differentiation efficiencies exceeding those of previously published protocols. Derivation of these cell types is a critical step toward understanding the basis of disease and generating cells with therapeutic potential. View Publication

Here we describe a protocol for generating 3D human intestinal tissues (called organoids) in vitro from human pluripotent stem cells (hPSCs). To generate intestinal organoids,pluripotent stem cells are first differentiated into FOXA2(+)SOX17(+) endoderm by treating the cells with activin A for 3 d. After endoderm induction,the pluripotent stem cells are patterned into CDX2(+) mid- and hindgut tissue using FGF4 and WNT3a. During this patterning step,3D mid- or hindgut spheroids bud from the monolayer epithelium attached to the tissue culture dish. The 3D spheroids are further cultured in Matrigel along with prointestinal growth factors,and they proliferate and expand over 1-3 months to give rise to intestinal tissue,complete with intestinal mesenchyme and epithelium comprising all of the major intestinal cell types. To date,this is the only method for efficiently directing the differentiation of hPSCs into 3D human intestinal tissue in vitro. View Publication