INDUCED PLURIPOTENT STEM CELL DIFFERENTIATION

诱导多能干细胞分化

• 批准号: 8173162
• 负责人: THADDEUS G GOLOS
• 金额: $ 3.1万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8173162
• 关键词: 3-Dimensional; Activins; Adherent Culture; Agreement; Animals; Cell Line; Cells; Computer Retrieval of Information on Scientific Projects Database; Culture Media; Cytokeratin; Dose; Embryo; Environment; Evaluation; Event; Exhibits; Extracellular Matrix; FGF2 gene; Funding; Grant; Human; Human Chorionic Gonadotropin; Immunohistochemistry; In Vitro; Institution; Lead; Light; Monitor; Pluripotent Stem Cells; Research; Research Personnel; Resources; Source; Staining method; Stains; Stem cells; Surface; Transforming Growth Factor beta; United States National Institutes of Health; Zebrafish; blastomere structure; bone morphogenetic protein 4; embryonic stem cell; human embryonic stem cell; induced pluripotent stem cell; matrigel; penis foreskin; response; stem cell differentiation; treatment duration; trophoblast; two-dimensional

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Objective: To define the differentiation potential of human induced pluripotent stem cells. 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 6 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. This research used WNPRC Stem Cell Resources.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 目的：明确人诱导多能干细胞的分化潜能。 滋养外胚层谱系的形成代表哺乳动物胚胎分化中最早的事件之一。源自人和动物胚胎的多能干细胞在自发或定向分化时形成滋养层谱系的能力方面表现出一系列分化潜力。这些观察结果可能反映了导致胚胎细胞永生化为胚胎干细胞的异质条件和事件。诱导多能干细胞（iPSC）为进一步阐明人类胚胎中滋养层形成的要求提供了机会。将人 iPSC 系 IMR90-1、-2 和 Foreskin-1、-2 和 -3 置于体外培养条件下，显示可诱导人 ESC 中的滋养层分化：用 BMP-4 处理，形成拟胚体，然后2 维或 3 维基质胶环境上的贴壁培养。通过人绒毛膜促性腺激素 (hCG) 的分泌以及 iPSC 形成的胚状体的组织学和免疫组织化学分析来监测滋养层分化。在所有细胞系中，用 BMP-4 而不是激活素、TGF-β、FGF4 或斑马鱼 FGF2 处理，会导致处理后 6 天内 hCG 分泌到培养基中的剂量相关性增加。同样，在基质胶包被的二维表面上贴壁培养拟胚体，在长达 4 周的培养过程中支持 hCG 表达的一致起始。有点出乎意料的是，当将 iPSC 衍生的胚状体放入 3 维基质胶外植体中时，hCG 的分泌上调不太一致。这与在这些 3-D 胚状体培养物中通过免疫组织化学检测 hCG 分泌的困难一致，尽管通过表面和生长细胞的细胞角蛋白染色表明滋养层分化。 我们得出的结论是，在滋养层分化方面，iPSC 对 BMP-4 处理的反应与人胚胎来源的多能干细胞相似，并且需要进一步评估 3 维细胞外基质环境中的分化。 这项研究使用了 WNPRC 干细胞资源。