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 胚胎干（ES）细胞向胰腺β细胞的分化。各种协议 迄今为止报告的大多是低效的，不可复制的。研究进展 建立培养条件以指导ES细胞分化为定形内胚层 作为第一个关键步骤，已经推动了该领域的发展。在本申请中，我们建议进行 逐步优化培养条件以指导内胚层分化为 胰腺β细胞以模拟体内胰腺发育。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.这种新的系统将使我们能够研究β细胞形成的过程， 实时，从而使我们能够容易地量化β细胞在每个阶段， 破坏文化，这将加速文化条件的识别， 促进β细胞分化。此外，作为我们对 通过肾移植在小鼠中分化MIP-GFP ES细胞证明， 内分泌细胞分化形成上皮管状结构， 概括了胎儿内分泌细胞的新生，这表明分化需要 在三维空间中放置，并由环境线索控制。在这里我们建议 以确定胎儿期的环境线索和信号通路成分， 具体目标1中的胰腺发育。在具体目标2中，我们将测试 胚胎干细胞向胰腺β细胞分化模拟胎儿β细胞的假说 分化其次是具体目标3，我们将致力于建立最低限度的 人工体外环境，以指导ES细胞/定形内胚层分化为 β细胞这项申请有三个具体目标。