技术资料

Induced pluripotent stem cells (iPSCs) hold enormous potential for the development of personalized in vitro disease models,genomic health analyses,and autologous cell therapy. Here we describe the generation of T lymphocyte-derived iPSCs from small,clinically advantageous volumes of non-mobilized peripheral blood. These T-cell derived iPSCs (TiPS") retain a normal karyotype and genetic identity to the donor. They share common characteristics with human embryonic stem cells (hESCs) with respect to morphology� View Publication

Induced pluripotent stem cells (iPSCs) provide an unprecedented opportunity for modeling of human diseases in vitro,as well as for developing novel approaches for regenerative therapy based on immunologically compatible cells. In this study,we employed an OP9 differentiation system to characterize the hematopoietic and endothelial differentiation potential of seven human iPSC lines obtained from human fetal,neonatal,and adult fibroblasts through reprogramming with POU5F1,SOX2,NANOG,and LIN28 and compared it with the differentiation potential of five human embryonic stem cell lines (hESC,H1,H7,H9,H13,and H14). Similar to hESCs,all iPSCs generated CD34(+)CD43(+) hematopoietic progenitors and CD31(+)CD43(-) endothelial cells in coculture with OP9. When cultured in semisolid media in the presence of hematopoietic growth factors,iPSC-derived primitive blood cells formed all types of hematopoietic colonies,including GEMM colony-forming cells. Human induced pluripotent cells (hiPSCs)-derived CD43(+) cells could be separated into the following phenotypically defined subsets of primitive hematopoietic cells: CD43(+)CD235a(+)CD41a(+/-) (erythro-megakaryopoietic),lin(-)CD34(+)CD43(+)CD45(-) (multipotent),and lin(-)CD34(+)CD43(+)CD45(+) (myeloid-skewed) cells. Although we observed some variations in the efficiency of hematopoietic differentiation between different hiPSCs,the pattern of differentiation was very similar in all seven tested lines obtained through reprogramming of human fetal,neonatal,or adult fibroblasts with three or four genes. Although several issues remain to be resolved before iPSC-derived blood cells can be administered to humans for therapeutic purposes,patient-specific iPSCs can already be used for characterization of mechanisms of blood diseases and for identification of molecules that can correct affected genetic networks. View Publication

MicroRNAs (miRNAs) are a newly discovered endogenous class of small noncoding RNAs that play important posttranscriptional regulatory roles by targeting mRNAs for cleavage or translational repression. Accumulating evidence now supports the importance of miRNAs for human embryonic stem cell (hESC) self-renewal,pluripotency,and differentiation. However,with respect to induced pluripotent stem cells (iPSC),in which embryonic-like cells are reprogrammed from adult cells using defined factors,the role of miRNAs during reprogramming has not been well-characterized. Determining the miRNAs that are associated with reprogramming should yield significant insight into the specific miRNA expression patterns that are required for pluripotency. To address this lack of knowledge,we use miRNA microarrays to compare the microRNA-omes" of human iPSCs� View Publication

The human body is composed of diverse cell types with distinct functions. Although it is known that lineage specification depends on cell-specific gene expression,which in turn is driven by promoters,enhancers,insulators and other cis-regulatory DNA sequences for each gene,the relative roles of these regulatory elements in this process are not clear. We have previously developed a chromatin-immunoprecipitation-based microarray method (ChIP-chip) to locate promoters,enhancers and insulators in the human genome. Here we use the same approach to identify these elements in multiple cell types and investigate their roles in cell-type-specific gene expression. We observed that the chromatin state at promoters and CTCF-binding at insulators is largely invariant across diverse cell types. In contrast,enhancers are marked with highly cell-type-specific histone modification patterns,strongly correlate to cell-type-specific gene expression programs on a global scale,and are functionally active in a cell-type-specific manner. Our results define over 55,000 potential transcriptional enhancers in the human genome,significantly expanding the current catalogue of human enhancers and highlighting the role of these elements in cell-type-specific gene expression. View Publication

Despite the recent identification of the transcriptional regulatory circuitry involving SOX2,NANOG,and OCT-4,the intracellular signaling networks that control pluripotency of human embryonic stem cells (hESCs) remain largely undefined. Here,we demonstrate an essential role for the serine/threonine protein kinase mammalian target of rapamycin (mTOR) in regulating hESC long-term undifferentiated growth. Inhibition of mTOR impairs pluripotency,prevents cell proliferation,and enhances mesoderm and endoderm activities in hESCs. At the molecular level,mTOR integrates signals from extrinsic pluripotency-supporting factors and represses the transcriptional activities of a subset of developmental and growth-inhibitory genes,as revealed by genome-wide microarray analyses. Repression of the developmental genes by mTOR is necessary for the maintenance of hESC pluripotency. These results uncover a novel signaling mechanism by which mTOR controls fate decisions in hESCs. Our findings may contribute to effective strategies for tissue repair and regeneration. View Publication

Cripto is a developmental oncoprotein that signals via mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK),phosphatidylinositol 3-kinase (PI3K)/Akt and Smad2/3 pathways. However,the molecular basis for Cripto coupling to these pathways during embryogenesis and tumorigenesis is not fully understood. In this regard,we recently demonstrated that Cripto forms a cell surface complex with the HSP70 family member glucose-regulated protein-78 (GRP78). Here,we provide novel functional evidence demonstrating that cell surface GRP78 is a necessary mediator of Cripto signaling in human tumor,mammary epithelial and embryonic stem cells. We show that targeted disruption of the cell surface Cripto/GRP78 complex using shRNAs or GRP78 immunoneutralization precludes Cripto activation of MAPK/PI3K pathways and modulation of activin-A,activin-B,Nodal and transforming growth factor-beta1 signaling. We further demonstrate that blockade of Cripto binding to cell surface GRP78 prevents Cripto from increasing cellular proliferation,downregulating E-Cadherin,decreasing cell adhesion and promoting pro-proliferative responses to activin-A and Nodal. Thus,disrupting the Cripto/GRP78 binding interface blocks oncogenic Cripto signaling and may have important therapeutic value in the treatment of cancer. View Publication