Human Endocrine Cell Development

描述(申请人提供)：在小鼠中，基本螺旋-环-螺旋转录因子NeUROG3(NeUROG3)对于胰腺和肠道的内分泌谱系的发育是必不可少的。带有NEUROG3突变的人类患有先天性肠道内分泌细胞丧失(肠源性无内分泌)1，但令人惊讶的是，C肽水平正常，并能耐受静脉输注葡萄糖，这表明NEUROG3功能不是人类胰腺β细胞发育所必需的。我们新的初步数据支持这一结论。这项应用探索了人类明显缺乏胰腺内分泌表型的原因。我们将确定是否由于NEUROG3活性的部分丧失而发生内分泌发育，或者NEUROG3功能不是人类内分泌发育所必需的。确定NEUROG3的作用是至关重要的，因为它被认为是从人类ES细胞产生胰腺内分泌细胞或通过从成人细胞类型新生产生胰腺内分泌细胞的治疗关键。考虑到携带NEUROG3突变的患者的罕见组织，从机械上研究肠道和胰腺内分泌细胞发育差异的基础是不可能的。为了能够进行这样的发育研究，我们建立了一种培养系统，使人类胚胎和诱导的多能干细胞(统称为PSCs)在体外有效地分化为胰腺或肠道组织。我们的新方法用于PSC来源的肠道组织，生成具有肠道所有主要功能细胞类型的上皮，包括肠内分泌细胞。此外，我们已经能够在肠道上皮细胞中敲除NEUROG3，导致人类肠道内分泌细胞几乎完全丧失。我们将使用人PSCs的胰腺和肠道内分泌细胞的发育作为模型，以确定NEUROG3依赖和独立的调节人类胰腺内分泌发育的途径。我们将首先确定NEUROG3水平对体外培养的人PSCs胰腺和肠道内分泌发育的影响。我们将通过观察NEUROG3水平对已知靶点和新途径的影响，确定肠道和胰腺对NEUROG3的差异需求的分子基础。最后，我们将在体外和体内研究NEUROG3水平对胰腺和肠道细胞功能的影响。总之，我们提出的目标构成了一项前所未有的NEUROG3在人类胰腺和肠道发育过程中的功能研究。我们的研究将毫不含糊地确定NEUROG3是否是胰腺和肠道内分泌细胞发育所必需的，如果不是必需的，则确定弥补NEUROG3缺失的分子途径。 与公众健康相关：葡萄糖稳态涉及由胰腺和肠道的内分泌细胞产生的多种激素的复杂活动。肠道和胰腺内分泌细胞之间的通讯是维持血糖稳态的关键。这一系统的紊乱会导致β细胞质量和/或功能的丧失，从而导致糖尿病。由于胰腺和肠道内分泌细胞在血糖稳态中的核心作用，了解这些细胞的起源和功能一直是当前和未来糖尿病治疗的重点。例如，对胰岛素反应的药理学处理一直是改善2型糖尿病患者胰岛β细胞功能的有效方法。几十年来，胰腺激素胰岛素一直被用来维持1型糖尿病患者的血糖稳态，而从干细胞生产β细胞仍然是许多研究人员未来治疗的重点。对小鼠的研究表明，NEUROG3是一种治疗关键，可以从ES细胞产生胰腺内分泌细胞，或者通过从成体细胞类型新生产生胰腺内分泌细胞。这项建议将明确确定NEUROG3是否对人类的β细胞和肠道内分泌发育都是必需的。无论在哪种情况下，这些研究都将通过确定NEUROG3的相关性和确定涉及人类β细胞发育的代偿替代途径，在糖尿病研究界产生广泛影响。

DESCRIPTION (provided by applicant): In mice, the basic Helix-loop-helix transcription factor Neurogenin 3 (NEUROG3) is essential for development of the endocrine lineage in both the pancreas and the intestines. Humans born with NEUROG3 mutations suffer from congenital loss of enteroendocrine cells (enteric anendocrinosis) 1 but surprisingly had normal C- peptide levels and tolerate intravenous infusion of glucose, suggesting that NEUROG3 function is not required for pancreatic beta-cell development in humans. Our new preliminary data supports this conclusion. This application with explore the cause for the apparent lack of a pancreatic endocrine phenotype in humans. We will determine if endocrine pancreas development occurred due to a partial loss of NEUROG3 activity, or alternatively NEUROG3 function is not required development of human endocrine pancreas. Identifying the role of NEUROG3 is critical since it is considered a therapeutic linchpin for generating pancreatic endocrine cells from human ES cells or via neogenesis from adult cell types. Given the rarity of tissues from patients harboring NEUROG3 mutations, it has been impossible to mechanistically investigate the basis for this difference in intestinal versus pancreatic endocrine cell development. To enable such a developmental study, we have established a culture system whereby human embryonic and induced pluripotent stem cells (collectively called PSCs) are efficiently differentiated into eithe pancreatic or intestinal tissue in vitro. Our novel method for PSC-derived intestinal tissue generates an epithelium with all of the major functioning cell types of the gut, including enteroendocrine cells. Moreover we have been able to knock down NEUROG3 in the intestinal epithelium resulting in nearly complete loss of human enteroendocrine cells. We will use development of pancreatic and intestinal endocrine cells from human PSCs as a model to identify NEUROG3-dependent and independent pathways that regulate human endocrine pancreas development. We will first determine the impact of NEUROG3 levels on pancreatic and intestinal endocrine development from human PSCs in vitro. We will identify the molecular basis for the differential requirement for NEUROG3 between the gut and pancreas by looking at the effects of NEUROG3 levels on known targets and novel pathways. Lastly we will investigate the impact of NEUROG3 levels on pancreatic and intestinal cell function in vitro and in vivo. Together, our proposed aims constitute an unprecedented functional study of NEUROG3 during human pancreas and intestinal development. Our studies will unambiguously determine if NEUROG3 is required for the development of pancreatic versus intestinal endocrine cells and if it is not required, identify the molecular pathways that compensate for absence of NEUROG3. PUBLIC HEALTH RELEVANCE: Glucose homeostasis involves the complex activity of a number of hormones produced by the endocrine cells of the pancreas and intestine. Communication between the intestinal and pancreatic endocrine cells is central to glucose homeostasis. Derangement of this system can lead to loss of beta-cell mass and/or function and result in Diabetes Mellitus. Due to the central role of pancreatic and intestinal endocrine cells i glucose homeostasis, understanding the origin and function of these cells has been the focus for current and future diabetic therapies. For example, pharmacologic manipulation of the incretin response has been an effective approach in improving the function of pancreatic beta-cells in patients with Type 2 diabetes. The pancreatic hormone insulin has been used for decades to maintain glucose homeostasis in patients with Type 1 diabetes, and production of beta-cells from stem cells continues to be a focus for many researchers as a future therapy. Studies in mice suggest that NEUROG3 is a therapeutic linchpin to generate pancreatic endocrine cells from ES cells or via neogenesis from adult cell types. This proposal will unambiguously determine if NEUROG3 is, or is not, required for both beta-cell and intestinal enteroendocrine development in HUMANS. In either case, these studies will have broad impact in the diabetes research community by determining the relevance of NEUROG3 and by identifying compensatory, alternative pathways involved in human beta-cell development.