MODELING ENTERIC NERVOUS SYSTEM DEVELOPMENT AND HIRSCHSPRUNG'S DISASE IN HUMAN PLURIPOTENT STEM CELLS

摘要 人类肠道神经系统(ENS)是一个由大约5亿个神经元组成的复杂网络，起源于 肠神经脊束(ENC)是胃肠(GI)功能所必需的。ENS缺陷是导致许多 常见的人类疾病包括胃瘫、肠易激综合征(IBS)、幽门肥大 狭窄。先天性巨结肠症(HD)是一种严重的先天性缺陷，其特征是ENS缺乏 肠道远端的先兆称为无神经节细胞增多症。无神经节细胞增多症导致缺乏 适当的蠕动导致功能性结肠梗阻(“巨结肠”) 由于缺乏可接近的组织，人们对人类胚胎的发育知之甚少。对.的使用 人类多能干细胞(HPSCs)代表了一种新的、独特的获取人类胚胎干细胞的策略 开发、建模和潜在地治疗HD。我们团队最近的工作证明了 从人的PSC生成ENC的可行性，为该技术用于开发奠定了基础 基于药物和细胞的治疗HD和潜在的其他ENS障碍的策略。当前应用程序 在这样令人振奋的前期工作的基础上，实现三个具体目标： 在目标1中，我们将建立条件来描述和操纵神经元亚型的广泛资料库。 并在与已知靶细胞的创新共培养试验中测试它们的功能。在……里面 目标2，将ENC分化技术应用于hPSCs中的HD建模，并验证电流和识别 新的候选HD药物。在目标3中，我们将在小鼠模型中优化基于hPSC的细胞治疗方法 并进行机制研究，确定体内拯救HD动物的关键参数。 我们的研究是独一无二的，因为它代表了第一次可靠地重建和研究早期人类谱系的努力 体外培养。为这项提议而聚集的合作者在hPSC、ENS生物学、 化学筛选和细胞移植。ENS是一种没有被其他人充分研究的结构 尽管它对人类健康做出了重要贡献。我们的研究有可能直接产生新药和 HD的基于细胞的治疗。这项工作奠定了基础，并开发了更广泛的必要技术 社区研究ENS的发展和ENS在人类健康和疾病中的作用。

SUMMARY The human enteric nervous system (ENS) is a complex network of ~500 million neurons derived from the enteric neural crest (ENC) and essential for gastro-intestinal (GI) function. ENS defects are the cause of many common human disorders including gastroparesis, irritable bowel syndrome (IBS), hypertrophic pyloric stenosis. Hirschsprung's disease (HD) is a severe congenital defect characterized by the lack of ENS precursors in the distal portions of the gut referred to as aganglionosis. Aganglionosis results in the lack of proper peristalsis causing functional colonic obstruction ("megacolon") Development of the human ENS is poorly understood given the lack of accessible tissue. The use of human pluripotent stem cells (hPSCs) represent an novel and unique strategy to access human ENS development and to model and potentially treat HD. Very recent work from our group demonstrates the feasibility of generating ENC from human PSCs and sets the stage for using the technology for developing drug and cell based strategies in treating HD and potentially other ENS disorders. The current application builds on such exciting preliminary work to pursue three specific aims: In Aim 1, we will establish conditions to characterize and manipulate the broad repertory of neuronal subtypes and enteric glia from hPSCs and to test their function in innovative co-culture assays with known target cells. In Aim 2, we will apply ENC differentiation technology to model HD in hPSCs and to validate current and identify novel candidate HD drugs. In Aim 3, we will optimize hPSC-based cell therapy approaches in a mouse model of HD and perform mechanistic studies that define the critical parameters for in vivo rescue of HD animals. Our study is unique as it represents the first effort to reliably recreate and study early human ENS lineages in vitro. The collaborators assembled for this proposal have complementary expertise in hPSCs, ENS biology, chemical screening and cell transplantation. The ENS is a structure that is not sufficiently studied by others despite its important contribution to human health. Our study has the potential to directly yield novel drug and cell based treatments for HD. The work sets the stage and develops the technology necessary for the broader community to study ENS development and ENS function in human health and disease.