Directed differentiation of human embryonic stem cells into glucose-responsive be

描述（由申请人提供）：人胚胎干细胞（hESC）为1型糖尿病的替代治疗提供了潜在的无限组织来源。然而，目前的分化方案如5阶段Novocell方案产生很少的胰岛素产生细胞，其在体外对葡萄糖无反应。只有在体内移植后，细胞才获得葡萄糖反应性。开启葡萄糖反应的分子开关仍然是一个谜。最近的研究强调了称为microRNA（miRNA）的小非编码RNA在发育过程中调控基因表达的核心作用。该提案的总体目标是开发对葡萄糖应答的干细胞衍生的胰岛素阳性细胞的快速筛选，并表征对于获得该重要表型重要的遗传和表观遗传（基于miRNA）机制。作为该提议的起点，我们已经对5期分化的hESC与人胰岛细胞群体进行了miRNA微阵列分析。尽管已知这两个群体由不同的细胞类型组成，但我们的初步分析显示了几种miRNA水平的显着差异，包括先前由其他小组证明对维持胰腺β细胞质量很重要的miR-375 - a miRNA，因此，支持我们可能发现调节β细胞特异性基因的miRNA的想法，包括那些调节葡萄糖反应性的miRNA。我们还开始开发一种新的荧光报告，识别胰岛素产生细胞，以及报告胰岛素分泌。在这里，我们概述了一个系统的方法来测试的作用，miRNA在调节葡萄糖反应。我们的具体目标是：1.开发一种荧光报告，既可以识别胰岛素阳性细胞，也可以报告胰岛素分泌，作为快速筛选激活葡萄糖反应基因的新方案的工具。2.鉴定用于调节葡萄糖反应基因的miRNA候选物。我们将在4期或5期hESC中表达荧光报告基因，并使用荧光激活细胞分选（FACS）分离胰岛素阳性但葡萄糖非应答的5期hESC衍生细胞。我们将使用差异miRNA微阵列方法将其miRNA谱与人类β细胞进行比较。3.使用群体和单细胞生理学和遗传学分析来测试候选调控miRNAs的功能活性。 公共卫生相关性：到目前为止，人类胚胎干细胞治疗1型糖尿病的方法一直受到阻碍，因为虽然科学家可以诱导干细胞转化为制造胰岛素的细胞，但这些细胞在接触糖时不会释放胰岛素。这项工作的目的是用荧光标记物标记制造胰岛素的细胞，这种标记物可以快速告诉我们细胞在暴露于糖时是否分泌胰岛素。这个标志物将帮助我们找到开启细胞对糖的反应的方法，它将帮助我们证明胰岛素是在对糖的刺激做出反应时适当分泌的。

DESCRIPTION (provided by applicant): Human embryonic stem cells (hESCs) offer a potentially unlimited source of tissue for replacement therapy in type 1 diabetes. However, current differentiation protocols like the 5-stage Novocell protocol yield few insulin-producing cells, which in vitro are unresponsive to glucose. Only after in vivo transplantation do the cells acquire glucose responsiveness. The molecular switches that turn on glucose responsiveness remain a mystery. Recent studies highlight a central role of small noncoding RNAs called microRNA (miRNA) in the regulation of gene expression during development. The overarching goals of this proposal are to develop a rapid screen for stem cell- derived insulin-positive cells that respond to glucose and to characterize the genetic and epigenetic (miRNA-based) mechanisms important for acquisition of this important phenotype. As a starting point for this proposal, we have already conducted a miRNA microarray analysis on populations of stage-5-differentiated hESCs vs. human islet cells. Although both populations are known to be composed of diverse cell types, our preliminary analysis showed striking differences in the level of several miRNAs, including miR-375 - a miRNA previously shown by other groups to be important for maintaining pancreatic beta cell mass, and, thus, lending support to the idea that we may find miRNAs that regulate beta-cell-specific genes, including those regulating glucose responsiveness. We have also initiated development of a novel fluorescent reporter that identifies insulin-producing cells, as well as reports insulin secretion. Here we outline a systematic approach to test the role of miRNAs in regulating glucose responsiveness. Our specific aims are: 1. Develop a fluorescent reporter that both identifies insulin-positive cells and reports insulin secretion, as a tool to rapidly screen new protocols to activate glucose-response genes. 2. Identify miRNA candidates for regulation of glucose-response genes. We will express the fluorescent reporter in Stage-4 or -5 hESCs and use fluorescence-activated cell sorting (FACS) to isolate insulin-positive but glucose non-responsive Stage-5 hESC-derived cells. We will compare their miRNA profiles with human beta cells using a differential miRNA microarray approach. 3. Test the functional activity of candidate regulatory miRNAs using population and single-cell physiological and genetic analysis. PUBLIC HEALTH RELEVANCE: Type 1 diabetes treatment by human embryonic stem cells has been blocked so far, because, while scientists can coax the stem cells to turn into cells that make insulin, those cells don<t release insulin when exposed to sugar. The aim of this work is to mark the insulin-making cells with a fluorescent marker that can tell us quickly whether a cell is secreting when exposed to sugar. This marker will help us to work out ways to turn on the cell<s response to sugars, and it will help us to prove that insulin is being secreted appropriately in response to sugar stimulation.