Generating Human Intestinal Organoids with an ENS.

描述（由申请方提供）：胃肠道（GI）动力/功能障碍影响高达25%的美国人群。常见的肠动力障碍包括肠易激综合征和粪便失禁，而更罕见的形式，如先天性巨结肠症有遗传基础，并与缺乏或缺乏肠神经有关。目前GI动力/功能障碍的治疗计划范围从饮食改变到肠切除术，但是很少有药物可用于治疗控制性腹泻的主要缺陷。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，这本身就是低通量的。最近，我们已经建立了一种培养系统，通过人胚胎干细胞和诱导多能干细胞（统称为PSC）的定向分化来产生人肠组织“类器官”（HIO）。HIO是三维结构，包含在肠中发现的大多数上皮细胞和间充质细胞类型。然而，由于HIO缺乏肠神经系统，该系统不是研究GI动力障碍的有用平台。我们假设肠神经系统可以通过将神经嵴干细胞（NCSC）引入到分化过程中来构建成HIO。有几种成熟的方法在体外从PSC产生神经嵴细胞，我们建议使用PSC衍生的NCSC来构建含有肠神经元和神经胶质细胞的人肠类器官。在目标1中，我们提出通过修改已用于产生更多前部NCSC的现有方案来体外产生人PSC衍生的迷走神经NCSC。我们将测试PSC衍生的NCSC在体外的分化潜力，并将其植入鸡胚胎中。在目标2中，我们将使用NCSC来产生含有肠神经的人肠类器官。我们将使用几种方法，通过在类器官发育期间结合两种组织，将NCSC并入发育中的肠类器官中。我们还将操纵在胚胎ENS发育过程中发挥作用的信号传导途径，以促进NCSC在类器官中掺入、增殖和分化为ENS细胞类型。将通过标记物和功能分析ENS形成。含有ENS的体外肠器官系统的开发将是高通量研究的理想平台，该研究旨在确定改善GI动力障碍患者ENS功能的新疗法。!

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. !