技术资料

Human U1 small nuclear (sn)RNA,required for splicing of pre-mRNA,is encoded by genes on chromosome 1 (1p36). Imperfect copies of these U1 snRNA genes,also located on chromosome 1 (1q12-21),were thought to be pseudogenes. However,many of these variant" (v)U1 snRNA genes produce fully processed transcripts. Using antisense oligonucleotides to block the activity of a specific vU1 snRNA in HeLa cells� View Publication

BackgroundX-chromosome inactivation silences one X chromosome in females to achieve dosage compensation with the single X chromosome in males. While most genes are silenced on the inactive X chromosome,the gene for the long non-coding RNA XIST is silenced on the active X chromosome and expressed from the inactive X chromosome with which the XIST RNA associates,triggering silencing of the chromosome. In mouse,an alternative Xist promoter,P2 is also the site of YY1 binding,which has been shown to serve as a tether between the Xist RNA and the DNA of the chromosome. In humans there are many differences from the initial events of mouse Xist activation,including absence of a functional antisense regulator Tsix,and absence of strictly paternal inactivation in extraembryonic tissues,prompting us to examine regulatory regions for the human XIST gene.ResultsWe demonstrate that the female-specific DNase hypersensitivity site within XIST is specific to the inactive X chromosome and correlates with transcription from an internal P2 promoter. P2 is located within a CpG island that is differentially methylated between males and females and overlaps conserved YY1 binding sites that are only bound on the inactive X chromosome where the sites are unmethylated. However,YY1 binding is insufficient to drive P2 expression or establish the DHS,which may require a development-specific factor. Furthermore,reduction of YY1 reduces XIST transcription in addition to causing delocalization of XIST.ConclusionsThe differentially methylated DNase hypersensitive site within XIST marks the location of an alternative promoter,P2,that generates a transcript of unknown function as it lacks the A repeats that are critical for silencing. In addition,this region binds YY1 on the unmethylated inactive X chromosome,and depletion of YY1 untethers the XIST RNA as well as decreasing transcription of XIST. View Publication

The blood-brain barrier (BBB) is a critical component of the central nervous system (CNS) that regulates the flux of material between the blood and the brain. Because of its barrier properties,the BBB creates a bottleneck to CNS drug delivery. Human in vitro BBB models offer a potential tool to screen pharmaceutical libraries for CNS penetration as well as for BBB modulators in development and disease,yet primary and immortalized models respectively lack scalability and robust phenotypes. Recently,in vitro BBB models derived from human pluripotent stem cells (hPSCs) have helped overcome these challenges by providing a scalable and renewable source of human brain microvascular endothelial cells (BMECs). We have demonstrated that hPSC-derived BMECs exhibit robust structural and functional characteristics reminiscent of the in vivo BBB. Here,we provide a detailed description of the methods required to differentiate and functionally characterize hPSC-derived BMECs to facilitate their widespread use in downstream applications. View Publication

It has been suggested that the isolation of scalable populations of limbal stem cells may lead to radical changes in ocular therapy. In particular,the derivation and transplantation of corneal stem cells from these populations may result in therapies providing clinical normality of the diseased or damaged cornea. Although feasible in theory,the lack of donor material in sufficient quantity and quality currently limits such a strategy. A potential scalable source of corneal cells could be derived from pluripotent stem cells (PSCs). We developed an in vitro and serum-free corneal differentiation model which displays significant promise. Our stepwise differentiation model was designed with reference to development and gave rise to cells which displayed similarities to epithelial progenitor cells which can be specified to cells displaying a corneal epithelial phenotype. We believe our approach is novel,provides a robust model of human development and in the future,may facilitate the generation of corneal epithelial cells that are suitable for clinical use. Additionally,we demonstrate that following continued cell culture,stem cell-derived corneal epithelial cells undergo transdifferentiation and exhibit squamous metaplasia and therefore,also offer an in vitro model of disease. View Publication

beta 1 Integrins are known to regulate terminal differentiation and morphogenesis in the adult epidermis. We have investigated their role in the embryonic development of keratinocytes by comparing the differentiation of wild-type and beta 1-null mouse embryonal stem (ES) cells. By 12-15 days in culture,differentiation of embryonic or simple epithelial cells occurred in both ES cell populations,as detected by expression of keratins 8,18,and 19. From 21 days,expression of keratins 10 and 14 and of the cornified envelope precursor involucrin indicated that some of the wild-type cells had differentiated into keratinocytes. In contrast,keratinocyte markers were not expressed in beta 1-null cultures. The beta 1-null cells failed to express the alpha 2 and alpha 3 integrin subunits on the cell surface,consistent with the association of these a subunits with beta 1. Furthermore,alpha 6 and beta 4 expression was reduced in the beta 1-null cultures. Although beta 1-null ES cells failed to undergo differentiation into keratinocytes in vitro,they did form keratinocyte cysts expressing alpha 6 beta 4,keratins 1 and 14,and involucrin when allowed to form teratomas by subcutaneous injection in mice; furthermore,beta 1-null keratinocytes were found in the epidermis of a wild-type/beta 1-null chimeric mouse. As judged by immunofluorescence microscopy,extracellular matrix assembly was severely impaired in beta 1-null ES cell cultures,but not in the teratomas or chimeric mouse skin. We therefore speculate that the failure of beta 1-null cells to differentiate into keratinocytes in vitro may reflect an inability to assemble a basement membrane. View Publication

Embryonic stem cells,derived from the inner cell mass of murine blastocysts,can be maintained in a totipotent state in vitro. In appropriate conditions embryonic stem cells have been shown to differentiate in vitro into various derivatives of all three primary germ layers. We describe in this paper conditions to induce differentiation of embryonic stem cells reliably and at high efficiency into adipocytes. A prerequisite is to treat early developing embryonic stem cell-derived embryoid bodies with retinoic acid for a precise period of time. Retinoic acid could not be substituted by adipogenic hormones nor by potent activators of peroxisome proliferator-activated receptors. Treatment with retinoic acid resulted in the subsequent appearance of large clusters of mature adipocytes in embryoid body outgrowths. Lipogenic and lipolytic activities as well as high level expression of adipocyte specific genes could be detected in these cultures. Analysis of expression of potential adipogenic genes,such as peroxisome proliferator-activated receptors gamma and delta and CCAAT/enhancer binding protein beta,during differentiation of retinoic acid-treated embryoid bodies has been performed. The temporal pattern of expression of genes encoding these nuclear factors resembled that found during mouse embryogenesis. The differentiation of embryonic stem cells into adipocytes will provide an invaluable model for the characterisation of the role of genes expressed during the adipocyte development programme and for the identification of new adipogenic regulatory genes. View Publication

Although the source of embryonic stem (ES) cells presents ethical concerns,their use may lead to many clinical benefits if differentiated cell types can be derived from them and used to assemble functional organs. In pancreas,insulin is produced and secreted by specialized structures,islets of Langerhans. Diabetes,which affects 16 million people in the United States,results from abnormal function of pancreatic islets. We have generated cells expressing insulin and other pancreatic endocrine hormones from mouse ES cells. The cells self-assemble to form three-dimensional clusters similar in topology to normal pancreatic islets where pancreatic cell types are in close association with neurons. Glucose triggers insulin release from these cell clusters by mechanisms similar to those employed in vivo. When injected into diabetic mice,the insulin-producing cells undergo rapid vascularization and maintain a clustered,islet-like organization. View Publication

Differentiation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells into hepatocyte-like cells provides a platform to study the molecular basis of human hepatocyte differentiation,to develop cell culture models of liver disease,and to potentially provide hepatocytes for treatment of end-stage liver disease. Additionally,hepatocyte-like cells generated from human pluripotent stem cells could serve as platforms for drug discovery,determination of pharmaceutical-induced hepatotoxicity,and evaluation of idiosyncratic drug-drug interactions. Here,we describe a step-wise protocol previously developed in our laboratory that facilitates the highly efficient and reproducible differentiation of human pluripotent stem cells into hepatocyte-like cells. Our protocol uses defined culture conditions and closely recapitulates key developmental events that are found to occur during hepatogenesis. View Publication

Progenitor cells have been extensively studied and therapeutically applied in tissue reconstructive therapy. Stromal vascular fraction (SVF) cells,which are derived from adipose tissue,may represent a potential source of the cells which undergo phenotypical differentiation into many lineages both in vitro as well as in vivo. The goal of this study was to check whether human SVF cells may differentiate into cardiomyocyte-like entities. Human SVF cells were induced to differentiate by their incubation in Methocult medium in the presence of SCF,IL-3 and IL-6. Morphological transformation of the cells was monitored using optical light microscope,whereas changes in expression of the genes typical for cardiac phenotype were measured by qRT-PCR. Incubation of the human SVF cells in the medium that promotes cardiomyocyte differentiation in vitro resulted in formation of myotubule-like structures accompanied by up-regulation of the myocardium-characteristic genes,such as GATA,MEF2C,MYOD1,but not ANP. Human SVF cells differentiate into cardiomyocyte-like cells in the presence of the certain set of myogenesis promoting cytokines. View Publication

Corneal transparency depends on a unique extracellular matrix secreted by stromal keratocytes,mesenchymal cells of neural crest lineage. Derivation of keratocytes from human embryonic stem (hES) cells could elucidate the keratocyte developmental pathway and open a potential for cell-based therapy for corneal blindness. This study seeks to identify conditions inducing differentiation of pluripotent hES cells to the keratocyte lineage. Neural differentiation of hES cell line WA01(H1) was induced by co-culture with mouse PA6 fibroblasts. After 6 days of co-culture,hES cells expressing cell-surface NGFR protein (CD271,p75NTR) were isolated by immunoaffinity adsorption,and cultured as a monolayer for one week. Keratocyte phenotype was induced by substratum-independent pellet culture in serum-free medium containing ascorbate. Gene expression,examined by quantitative RT-PCR,found hES cells co-cultured with PA6 cells for 6 days to upregulate expression of neural crest genes including NGFR,SNAI1,NTRK3,SOX9,and MSX1. Isolated NGFR-expressing cells were free of PA6 feeder cells. After expansion as a monolayer,mRNAs typifying adult stromal stem cells were detected,including BMI1,KIT,NES,NOTCH1,and SIX2. When these cells were cultured as substratum-free pellets keratocyte markers AQP1,B3GNT7,PTDGS,and ALDH3A1 were upregulated. mRNA for keratocan (KERA),a cornea-specific proteoglycan,was upregulated more than 10,000 fold. Culture medium from pellets contained high molecular weight keratocan modified with keratan sulfate,a unique molecular component of corneal stroma. These results show hES cells can be induced to differentiate into keratocytes in vitro. Pluripotent stem cells,therefore,may provide a renewable source of material for development of treatment of corneal stromal opacities. View Publication

BACKGROUND: Human embryonic stem cells (hESC) provide a unique model to study early events in human development. The hESC-derived cells can potentially be used to replace or restore different tissues including neuronal that have been damaged by disease or injury.backslashnbackslashnMETHODOLOGY AND PRINCIPAL FINDINGS: The cells of two different hESC lines were converted to neural rosettes using adherent and chemically defined conditions. The progenitor cells were exposed to retinoic acid (RA) or to human recombinant basic fibroblast growth factor (bFGF) in the late phase of the rosette formation. Exposing the progenitor cells to RA suppressed differentiation to rostral forebrain dopamine neural lineage and promoted that of spinal neural tissue including motor neurons. The functional characteristics of these differentiated neuronal precursors under both,rostral (bFGF) and caudalizing (RA) signals were confirmed by patch clamp analysis.backslashnbackslashnCONCLUSIONS/SIGNIFICANCE: These findings suggest that our differentiation protocol has the capacity to generate region-specific and electrophysiologically active neurons under in vitro conditions without embryoid body formation,co-culture with stromal cells and without presence of cells of mesodermal or endodermal lineages. View Publication

Human induced pluripotent stem cells (iPSCs) can give rise to multiple cell types and hold great promise in regenerative medicine and disease-modeling applications. We have developed a reliable two-step protocol to generate human mammary-like organoids from iPSCs. Non-neural ectoderm-cell-containing spheres,referred to as mEBs,were first differentiated and enriched from iPSCs using MammoCult medium. Gene expression profile analysis suggested that mammary gland function-associated signaling pathways were hallmarks of 10-day differentiated mEBs. We then generated mammary-like organoids from 10-day mEBs using 3D floating mixed gel culture and a three-stage differentiation procedure. These organoids expressed common breast tissue,luminal,and basal markers,including estrogen receptor,and could be induced to produce milk protein. These results demonstrate that human iPSCs can be directed in vitro toward mammary lineage differentiation. Our findings provide an iPSC-based model for studying regulation of normal mammary cell fate and function as well as breast disease development. View Publication