Regulation of pancreatic islet cell fate

项目总结/摘要 目前正在进行重大的研究工作，以了解和预防型的发病机制 1型和2型糖尿病，两者都与功能性胰岛素产生B细胞的逐渐丧失有关。 使用人多能干细胞（hPSC）群体作为免疫调节的替代来源已经取得了进展。 胰岛细胞;然而，尚未有效生产功能齐全的纯B细胞群。 办妥了一批此外，目前的方案导致取决于干细胞的高度可变的分化。 来源、鉴别程序和用户。此外，内源性和干细胞衍生的胰岛细胞都显示出 固有的表型不稳定性，并经常经历自发的去分化和/或重编程， 病理生理条件，导致细胞特性丧失和表型功能障碍。这些 挑战表明，仍然有很大的需要，以确定精确的转录和表观遗传 促进胰岛细胞特性的特化和维持的机制。 我们和其他人已经证明，B细胞特性的发育和维持需要 几个非冗余转录程序的连续活性。尽管许多转录因子 对于维持a和B细胞表型至关重要的蛋白质已经被表征，我们缺乏一个完整的 了解细胞特异性基因程序是如何调节的，以指定和维持这些密切相关的基因。 细胞谱系在小鼠中，并且有不同的报告与它们各自在人类胰腺中的作用有关 发展这一建议将使我们能够更全面地了解基本分子 胰岛细胞分化和维持胰岛细胞身份/功能的潜在机制;这些知识 将为改进人类胰岛细胞分化方案和确定更好的治疗方法铺平道路 治疗糖尿病中的胰岛细胞功能障碍。这些研究是通过增加获得新的和改进的 分子技术此外，我们在啮齿动物模型中的研究提供了重要的生物学和 生理背景，其将被直接翻译到hPSC分化系统中。我们的首要目标是 表征调节小鼠a vs B细胞程序的差异分子机制。我们将专注于 NKX2.2、NKX6.1、KLF 4和CHD 4在调节内分泌细胞命运中的组合分子活性 决策我们还将采取一种无偏的方法，使用组合的单细胞ATAC-Seq和RNA-Seq， 表征与体内胰岛细胞命运决定相关的转录景观。第二个目标将 表征染色质修饰蛋白质CHD 4在调节B细胞发育和功能中的作用，和 调节NKX2.2和NKX6.1的调节活性。第三个目标将开始将这些知识应用于 从hPSC定向分化人胰岛细胞。我们建议确定必要和充分的 NKX2.2和NKX6.1在调节a和B细胞命运决定中的功能。我们还将利用这个平台来识别 在这些谱系选择期间，由NKX2.2和NKX6.1调节的染色质和基因表达景观。

Project Summary/Abstract Significant research efforts are currently underway to understand and prevent the pathogeneses of Type 1 and Type 2 diabetes, both of which are associated with the gradual loss of functional insulin-producing b cells. Progress has been made with using human pluripotent stem cell (hPSC) populations as alternative sources of islet cells; however, the efficient production of pure populations of fully functional b cells has not yet been achieved. Furthermore, the current protocols result in highly variable differentiations depending on the stem cell source, differentiation procedure and user. In addition, both endogenous and stem cell-derived islet cells display inherent phenotypic instability and often undergo spontaneous dedifferentiation and/or reprogramming in pathophysiological conditions, which results in the loss of cellular identity and phenotypic dysfunction. These challenges suggest that there is still a great need to identify the precise transcriptional and epigenetic mechanisms that promote the specification and maintenance of islet cell identities. We and others have demonstrated that development and maintenance of b cell identity requires the continuous activity of several non-redundant transcriptional programs. Although many of the transcription factors that are essential for maintaining a and b cell phenotypes have been characterized, we lack a complete understanding of how cell-specific gene programs are regulated to specify and maintain these closely related cell lineages in mice, and there are varying reports related to their respective roles in human pancreas development. This proposal will allow us to gain a more complete understanding of the basic molecular mechanisms underlying islet cell differentiation and the maintenance of islet cell identity/function; this knowledge will pave the way to improved human islet cell differentiation protocols and the identification of better treatments for islet cell dysfunctions in diabetes. These studies are facilitated by increased access to novel and improved molecular technologies. Furthermore, our studies in rodent models are providing important biological and physiological context that will be directly translated into the hPSC differentiation system. Our first aim will characterize the differential molecular mechanisms that regulate a vs b cell programs in mice. We will focus on the combined molecular activities of NKX2.2, NKX6.1, KLF4 and CHD4 in regulating endocrine cell fate decisions. We will also take an unbiased approach using combined single cell ATAC-Seq and RNA-Seq to characterize the transcriptional landscape associated with islet cell fate decisions in vivo. The second aim will characterize the role of the chromatin modifier protein CHD4 in regulating b cell development and function, and in modulating NKX2.2 and NKX6.1 regulatory activities. The third aim will begin to apply this knowledge to the directed differentiation of human islet cells from hPSCs. We propose to identify the necessary and sufficient functions of NKX2.2 and NKX6.1 in regulating a and b cell fate decisions. We will also use this platform to identify the chromatin and gene expression landscapes regulated by NKX2.2 and NKX6.1 during these lineage choices.