Role of the environmental cues for beta-cell differentiation from ES cells

环境因素对 ES 细胞分化为 β 细胞的作用

• 批准号: 7631589
• 负责人: MANAMI HARA
• 金额: $ 23.1万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7631589
• 关键词: Basement membrane; Beta Cell; Cell Differentiation process; Cell Lineage; Cell Proliferation; Cells; Classification; Collagen Type IV; Color; Condition; Cues; D Cells; Development; Dimensions; ES Cell Line; Embryo; Endocrine; Endoderm; Environment; Epithelial; Extracellular Matrix; Genes; Goals; Green Fluorescent Proteins; In Vitro; Insulin; Integrins; Kidney Transplantation; Label; Laminin; Lead; Membrane Proteins; Mesenchyme; Methods; Monitor; Mus; NID gene; Nidogen; Organ; Organ Culture Techniques; Pancreas; Process; Proteins; Protocols documentation; Reporter; Reporter Genes; Reporting; Role; Signal Pathway; Signal Transduction; Staging; Structure; Structure of beta Cell of islet; Suspension substance; Suspensions; System; Testing; Time; Tubular formation; embryonic stem cell; fetal; in vivo; novel; progenitor; promoter; receptor; reconstitution; stem

The overall goal of this project is to determine culture conditions that promote differentiation of embryonic stem (ES) cells to pancreatic beta-cells. Various protocols reported to date are largely inefficient and not reproducible. Recent progress in establishing culture conditions to direct differentiation of ES cells to definitive endoderm as a first critical step has advanced the field. In this application, we propose to conduct stepwise optimization of culture conditions to direct differentiation of endoderm to pancreatic beta-cells to mimic the pancreatic development in vivo. To this end, we will utilize a unique mouse ES cell line in which pancreatic beta-cells are endogenously marked with green fluorescent protein (GFP) under the control of mouse insulin I promoter (MIP), in combination with sequential expression of stage-specific reporter genes. This novel system will enable us to study the process of beta-cell formation in real-time thereby allowing us to readily quantify beta-cells at each stage without destroying the culture, which will accelerate the identification of culture conditions that promote beta-cell differentiation. Furthermore, as our in vivo assessment of differentiation of MIP-GFP ES cells by renal transplantation in mice demonstrates, endocrine cell differentiation occurs forming the epithelial tubular structure that recapitulates fetal endocrine cell neogenesis, suggesting that the differentiation takes place in three-dimensions and is controlled by the environmental cues. Here we propose to define the environmental cues and signaling pathway components during fetal pancreatic development in Specific Aim 1. In parallel in Specific Aim 2, we will test the hypothesis that ES cell differentiation to pancreatic beta-cells mimics the fetal beta-cell differentiation. Followed by Specific Aim 3, we will aim at establishing the minimal artificial in vitro environment to direct differentiation of ES cells/definitive endoderm to beta-cells. This application has three specific aims.

该项目的总体目标是确定促进的文化条件 胚胎茎（ES）细胞与胰腺β细胞的分化。各种协议 迄今为止报告的效率很大，不可再现。最近的进展 建立培养条件以将ES细胞分化为确定的内胚层 作为第一个关键步骤，该领域已推进了领域。在此应用中，我们建议进行 逐步优化培养条件以将内胚层分化为 胰腺β细胞模仿体内胰腺发育。为此，我们将 利用独特的小鼠ES细胞系，其中胰腺β细胞是内源性的 在小鼠胰岛素I的控制下用绿色荧光蛋白（GFP）标记 启动子（MIP），结合阶段特异性报告基因的顺序表达 基因。这个新颖的系统将使我们能够研究β细胞形成的过程 实时，从而使我们在每个阶段都可以轻松地量化beta细胞 破坏文化，这将加速对文化条件的识别 促进β细胞分化。此外，作为我们的体内评估 通过肾移植在小鼠中分化MIP-GFP ES细胞，表明 内分泌细胞分化发生形成上皮管状结构 概括胎儿内分泌细胞的新作用，表明分化需要 放置三维，并由环境线索控制。我们在这里提出 定义胎儿期间的环境提示和信号通路组件 特定目的1中的胰腺发展。在特定目标2中并行，我们将测试 假设细胞与胰腺β细胞分化模仿胎儿β细胞 分化。其次是特定目标3，我们将旨在建立最小的 人工体外环境将ES细胞/确定性内胚层的分化转化为 Beta细胞。该应用程序具有三个具体目标。