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

描述(申请人提供)：人类胚胎干细胞(HESCs)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，产生的胰岛素产生细胞很少，在体外对葡萄糖没有反应。只有在体内移植后，细胞才能获得葡萄糖反应性。开启葡萄糖响应性的分子开关仍然是个谜。最近的研究强调了被称为microRNA(MiRNA)的小非编码RNA在发育过程中调节基因表达的核心作用。这项建议的首要目标是开发一种对葡萄糖反应的干细胞来源的胰岛素阳性细胞的快速筛查，并表征对获得这一重要表型重要的遗传和表观遗传机制。作为这一建议的起点，我们已经对阶段5分化的hESCs与人类胰岛细胞进行了miRNA微阵列分析。虽然已知这两个群体都由不同的细胞类型组成，但我们的初步分析显示，几个miRNAs的水平存在显著差异，包括miR-375-a miRNA，此前其他组织表明miR-375-a对维持胰腺β细胞质量很重要，因此支持我们可能找到调节β细胞特异性基因的miRNAs，包括那些调节葡萄糖反应性的miRNAs。我们还开始开发一种新型的荧光报告器，它可以识别胰岛素产生细胞，并报告胰岛素的分泌。在这里，我们概述了一种系统的方法来测试miRNAs在调节葡萄糖反应性中的作用。我们的具体目标是：1.开发一种既能识别胰岛素阳性细胞又能报告胰岛素分泌的荧光报告程序，作为一种快速筛选激活葡萄糖反应基因的新方案的工具。2.确定参与葡萄糖反应基因调控的miRNA候选基因。我们将在阶段4或-5的hESCs中表达荧光报告，并使用荧光激活细胞分选(FACS)分离胰岛素阳性但葡萄糖无反应的阶段-5 hESC来源的细胞。我们将使用差异miRNA微阵列方法将它们的miRNA图谱与人类β细胞进行比较。3.利用群体和单细胞生理和遗传分析测试候选调节miRNAs的功能活性。 与公共卫生相关：人类胚胎干细胞治疗1型糖尿病迄今被阻止，因为尽管科学家可以诱使干细胞转变为制造胰岛素的细胞，但这些细胞在暴露在糖中时不会释放胰岛素。这项工作的目的是用一种荧光标记来标记胰岛素制造细胞，这种标记可以快速告诉我们，当细胞暴露在糖中时，细胞是否正在分泌。这个标记物将帮助我们找出激活细胞的方法&S对糖的反应，它将帮助我们证明胰岛素正在适当地分泌，以响应糖的刺激。

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.