Human Endocrine Cell Development

描述（由申请人提供）：在小鼠中，碱性螺旋-环-螺旋转录因子Neurogenin 3（NEUROG 3）对于胰腺和肠中内分泌谱系的发育至关重要。出生时具有NEUROG 3突变的人患有先天性肠内分泌细胞缺失（肠内分泌缺乏症）1，但令人惊讶的是具有正常的C肽水平并耐受葡萄糖静脉输注，这表明NEUROG 3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。本申请旨在探索人类明显缺乏胰腺内分泌表型的原因。我们将确定内分泌胰腺的发育是否是由于NEUROG 3活性的部分丧失而发生的，或者NEUROG 3功能不是人类内分泌胰腺发育所必需的。确定NEUROG 3的作用至关重要，因为它被认为是从人ES细胞或通过成体细胞类型新生产生胰腺内分泌细胞的治疗关键。鉴于携带NEUROG 3突变的患者组织的罕见性，不可能从机制上研究肠内分泌细胞发育与胰腺内分泌细胞发育差异的基础。为了能够进行这样的发育研究，我们建立了一种培养系统，其中人胚胎干细胞和诱导多能干细胞（统称为PSC）在体外有效地分化成胰腺或肠组织。我们用于PSC衍生的肠组织的新方法产生具有肠道的所有主要功能细胞类型的上皮，包括肠内分泌细胞。此外，我们已经能够敲低肠上皮中的NEUROG 3，导致人类肠内分泌细胞几乎完全丧失。我们将使用人PSC的胰腺和肠内分泌细胞的发育作为模型来鉴定调节人内分泌胰腺发育的NEUROG 3依赖性和独立性途径。我们将首先确定NEUROG 3水平对体外人PSC胰腺和肠道内分泌发育的影响。我们将通过观察NEUROG 3水平对已知靶点和新途径的影响，确定肠道和胰腺对NEUROG 3差异需求的分子基础。最后，我们将在体外和体内研究NEUROG 3水平对胰腺和肠细胞功能的影响。总之，我们提出的目标构成了人类胰腺和肠道发育过程中NEUROG 3前所未有的功能研究。我们的研究将明确确定NEUROG 3是否是胰腺与肠内分泌细胞发育所必需的，如果不需要，则确定补偿NEUROG 3缺失的分子途径。 公共卫生相关性：葡萄糖稳态涉及胰腺和肠内分泌细胞产生的多种激素的复杂活性。肠道和胰腺内分泌细胞之间的沟通是葡萄糖稳态的核心。该系统的紊乱可导致β细胞质量和/或功能的丧失，并导致糖尿病。由于胰腺和肠内分泌细胞在葡萄糖稳态中的核心作用，了解这些细胞的起源和功能已成为当前和未来糖尿病治疗的焦点。例如，肠促胰岛素反应的药理学操作已经是改善2型糖尿病患者胰腺β细胞功能的有效方法。几十年来，胰腺激素胰岛素一直用于维持1型糖尿病患者的葡萄糖稳态，而从干细胞中产生β细胞仍然是许多研究人员关注的焦点，作为未来的治疗方法。在小鼠中的研究表明，NEUROG 3是从ES细胞或通过成体细胞类型的新生产生胰腺内分泌细胞的治疗关键。该建议将明确确定NEUROG 3是否是人类β细胞和肠道内分泌发育所需的。在任何一种情况下，这些研究将通过确定NEUROG 3的相关性和确定参与人类β细胞发育的补偿性替代途径，在糖尿病研究界产生广泛的影响。

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.