Generating Human Intestinal Organoids with an ENS.

描述(申请人提供)：胃肠道(GI)运动/功能障碍影响多达25%的美国人口。常见的肠道动力障碍包括肠易激综合征和大便失禁，而更罕见的形式，如先天性巨结肠，则有遗传基础，并与肠神经缺失或缺乏有关。目前针对胃肠道动力/功能障碍的治疗计划从改变饮食到肠切除，然而，针对受控蠕动的主要缺陷的药物很少。胃肠道疾病研究的一个障碍是，它在很大程度上依赖于体内动物研究，而这些研究本身就是低吞吐量的。最近，我们建立了一种培养系统，通过对人类胚胎和诱导的多能干细胞(统称为PSCs)进行定向分化来生成人类肠道组织类有机物(HIO)。HIO是一种三维结构，含有肠道中发现的大多数上皮细胞和间充质细胞类型。然而，由于HIOS缺乏肠道神经系统，该系统不是研究胃肠动力障碍的有用平台。我们假设，通过在分化过程中引入神经脊干细胞(NCSC)，可以将肠道神经系统构建成HIOS。有几种成熟的方法可以在体外从PSC中获得神经脊细胞，我们建议使用PSC来源的NCSCs来构建含有肠神经元和神经胶质细胞的人肠道器官。在目标1中，我们建议通过修改现有的用于产生更多前部NCSC的方案，在体外培养人PSCs来源的迷走神经NCSC。我们将检测PSC来源的神经干细胞在体外和植入鸡胚胎后的分化潜能。在目标2中，我们将使用神经干细胞产生含有肠神经的人类肠道器官。我们将使用几种方法将NCSCs整合到发育中的肠道器官中，在器官发育过程中结合这两个组织。我们还将操纵在胚胎ENS发育过程中发挥作用的信号通路，以促进NCSCs在类器官中并入、增殖和分化为ENS细胞类型。ENS的形成将通过标记物和功能进行分析。含有ENS的体外肠道器官系统的开发将是高通量研究的理想平台，旨在寻找新的治疗方法来改善胃肠道动力障碍患者的ENS功能。好了！

DESCRIPTION (provided by applicant): Gastrointestinal (GI) motility/functional disorders affect up to 25% of the US population. Common intestinal motility disorders include Irritable Bowel Syndrome and Fecal Incontinence, whereas more rare forms such as Hirschsprung's Disease have a genetic basis and are associated with absence or paucity of enteric nerves. Current treatment plans for GI motility/functional disorders range from changes in diet to bowel resection, however there are very few drugs available that target the primary deficiencies in controlled peristalsis. One barrier to research of GI disease is that it has largely relied on in vvo animal studies, which are intrinsically low throughput. Recently, we have established a culture system to generate human intestinal tissue "organoids" (HIOs) through directed differentiation of human embryonic and induced pluripotent stem cells (collectively called PSCs). HIOs are three-dimensional structures containing most epithelial and mesenchymal cell types found in the intestine. However, due to lack of an enteric nervous system in HIOs, the system is not a useful platform to study GI motility disorders. We hypothesize that the enteric nervous system can be built into HIOs by introducing neural crest stem cells (NCSC) into the differentiation process. There are several well-established methods to generate neural crest cells from PSCs in vitro and we propose to use PSC-derived NCSCs to construct human intestinal organoids containing enteric neurons and glial cells. In aim 1 we propose to generate human PSCs-derived vagal NCSC in vitro by modifying existing protocols that have been used to generate more anterior NCSCs. We will test the differentiation potential of PSC-derived NCSCs in vitro and following engraftment into chicken embryos. In aim 2, we will use NCSCs to generate human intestinal organoids containing enteric nerves. We will use several approaches to incorporate NCSCs into developing intestinal organoids by combining the two tissues during organoid development. We will also manipulate signaling pathways that function during embryonic ENS development to promote incorporation, proliferation and differentiation of NCSCs into ENS cell types in organoids. ENS formation will be analyzed by markers and by function. Development of an in vitro intestinal organ system containing an ENS would be an ideal platform for high throughput studies aimed at identifying new therapies to improve ENS function in patients with GI motility disorders. !