INDUCED PLURIPOTENT STEM CELL DIFFERENTIATION

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 目的：明确人诱导多能干细胞的分化潜能。 细胞。 进度： 滋养外胚层谱系的形成代表了 哺乳动物胚胎分化。来源于人和人的多潜能干细胞 动物胚胎表现出一系列分化潜力，在它们形成 滋养层细胞自发或定向分化的谱系。这些 观测结果可能反映了导致 胚胎细胞永生化为胚胎干细胞。诱导多能干细胞 Cells(IPSC)提供了进一步阐明以下要求的机会 人类胚胎中滋养层细胞的形成。人IPSC株系IMR90-1、-2和 包皮-1、-2和-3在体外培养条件下被诱导 BMP-4和类胚体对人胚胎干细胞滋养层细胞分化的影响 在二维或三维基质上形成后的贴壁培养 环境。用人绒毛膜分泌物监测滋养层细胞分化 绒毛膜促性腺激素(HCG)及组织学和免疫组织化学分析 由IPSC形成的类胚体。在所有细胞系中，BMP-4处理而不是 激活素、转化生长因子-β、成纤维细胞生长因子4或斑马鱼成纤维细胞生长因子2可引起剂量相关的增加。 治疗后6天内将hCG分泌到培养液中。同样， 类胚体在Matrigel涂层二维表面上的贴壁培养 支持在长达4周的培养中持续启动hCG的表达。 有些出乎意料的是，IPSC衍生的类胚体不那么一致地上调 HCG在三维Matrigel外植体中的分泌。这是最流行的 免疫组织化学检测hCG分泌困难的符合性 这些3D类胚体培养，尽管滋养层细胞分化是 表面和突起细胞的细胞角蛋白染色提示。我们的结论是 IPSC对BMP-4的反应与人类胚胎来源的相似 多能干细胞与滋养层细胞分化的关系，以及进一步 三维细胞外基质环境中分化的评估是 需要的。 出版物： 没有。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Objective: To define the differentiation potential of human induced pluripotent stem cells. PROGRESS: The formation of the trophectoderm lineage represents one of the earliest events in mammalian embryonic differentiation. Pluripotent stem cells derived from human and animal embryos exhibit a range of differentiation potential in their ability to form the trophoblast lineage upon spontaneous or directed differentiation. These observations may reflect the heterogeneous conditions and events that lead to the immortalization of embryonic cells to embryonic stem cells. Induced pluripotent stem cells (iPSC) provide an opportunity to shed further light on the requirements for trophoblast formation in human embryos. The human iPSC lines IMR90-1, -2 and Foreskin-1, -2 and -3 were subjected to in vitro culture conditions shown to induce trophoblast differentiation in human ESC: treatment with BMP-4, and embryoid body formation followed by adherent culture on 2-dimensional or 3-dimensional Matrigel environments. Trophoblast differentiation was monitored by secretion of human chorionic gonadotropin (hCG), and by histological and immunohistochemical analysis of embryoid bodies formed from iPSC. In all cell lines, treatment with BMP-4 but not activin, TGF-beta, FGF4 or zebrafish FGF2 resulted in a dose-related increase in the secretion of hCG into the culture medium within six days of treatment. Likewise, adherent culture of embryoid bodies on Matrigel-coated 2-dimensional surfaces supported consistent initiation of hCG expression during up to 4 weeks of culture. Somewhat unexpectedly, iPSC-derived embryoid bodies less consistently upregulated the secretion of hCG when placed into 3-dimensional Matrigel explants. This was in agreement with difficulty in detecting hCG secretion by immunohistochemistry in these 3-D embryoid body cultures, although trophoblast differentiation was suggested by cytokeratin staining of surface and outgrowth cells. We conclude that iPSC have similar responses to BMP-4 treatment as human embryo-derived pluripotent stem cells with regard to trophoblast differentiation, and that further evaluation of differentiation in 3-dimensional extracellular matrix environments is needed. PUBLICATIONS: None.