Generating Human Intestinal Organoids with an ENS.

使用 ENS 生成人类肠道类器官。

• 批准号: 8415736
• 负责人: James M Wells
• 金额: $ 34.43万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-24 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8415736
• 关键词: 3-Dimensional; Activities of Daily Living; Affect; Animals; Anterior; Cell Proliferation; Cells; Cephalic; Chickens; Congenital Megacolon; Cues; Development; Diet; Embryo; Embryonic Development; Endoderm; Endothelin; Engraftment; Enteral; Enteric Nervous System; Enterocytes; Enteroendocrine Cell; Environment; Enzymes; Epithelial; Epithelium; Excision; Fecal Incontinence; Fibroblast Growth Factor; Fibroblasts; Food; Functional Imaging; Functional disorder; GDNF gene; Gastrointestinal Diseases; Gastrointestinal Motility; Gastrointestinal tract structure; Gene Expression; Genetic; Germ Layers; Goblet Cells; HOI; Human; In Vitro; Intestinal Diseases; Intestinal Motility; Intestines; Irritable Bowel Syndrome; Mesenchymal; Mesenchyme; Mesoderm; Methods; Molecular; Movement; Muscle Contraction; Myofibroblast; Nerve; Nervous System Physiology; Neural Crest; Neural Crest Cell; Neuroglia; Neurons; Organoids; Paneth Cells; Pathway interactions; Patients; Pattern; Peristalsis; Pharmaceutical Preparations; Plastics; Population; Process; Protocols documentation; Research; Screening procedure; Signal Pathway; Signal Transduction; Smooth Muscle; Smooth Muscle Actin Staining Method; Somites; Specific qualifier value; Staging; Stem cells; Structure; Study models; System; Testing; Tissues; Tretinoin; Villus; base; body system; cell motility; cell type; gastrointestinal epithelium; hindbrain; improved; induced pluripotent stem cell; migration; motility disorder; nervous system development; notch protein; progenitor; response; stem cell differentiation; three dimensional structure; tool; treatment planning

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. ! PUBLIC HEALTH RELEVANCE: 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. This proposal aims to generate human intestinal organoids containing functional epithelium, surrounded by smooth muscle that is innervated by enteric nerves. This research tool would allow for unprecedented studies of the cellular and molecular basis of gut motility in humans and be an ideal screening platform for drugs that regulate gut motility.

描述（由申请人提供）：胃肠 (GI) 动力/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症和大便失禁，而更罕见的形式如先天性巨结肠具有遗传基础，并且与肠神经缺失或缺乏有关。目前针对胃肠道运动/功能障碍的治疗计划范围从饮食改变到肠切除，但针对控制蠕动的主要缺陷的药物很少。胃肠道疾病研究的一大障碍是它在很大程度上依赖于体内动物研究，而这些研究本质上通量较低。最近，我们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道组织“类器官”（HIO）。 HIO 是三维结构，包含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 HIO 缺乏肠神经系统，该系统并不是研究胃肠道运动障碍的有用平台。我们假设，通过将神经嵴干细胞 (NCSC) 引入分化过程，可以将肠神经系统构建到 HIO 中。有几种成熟的方法可以在体外从 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道类器官。在目标 1 中，我们建议通过修改用于生成更多前 NCSC 的现有方案，在体外生成人类 PSC 衍生的迷走神经 NCSC。我们将测试 PSC 衍生的 NCSC 的体外分化潜力以及植入鸡胚胎后的分化潜力。在目标 2 中，我们将使用 NCSC 生成含有肠神经的人类肠道类器官。我们将使用多种方法，通过在类器官发育过程中结合两种组织，将 NCSC 纳入正在发育的肠道类器官中。我们还将操纵在胚胎 ENS 发育过程中发挥作用的信号通路，以促进 NCSC 掺入、增殖和分化为类器官中的 ENS 细胞类型。 ENS 的形成将通过标记和功能进行分析。开发含有 ENS 的体外肠道器官系统将是高通量研究的理想平台，旨在确定新疗法以改善胃肠道运动障碍患者的 ENS 功能。 ！ 公共卫生相关性：胃肠道 (GI) 动力/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症和大便失禁，而更罕见的形式如先天性巨结肠具有遗传基础，并且与肠神经缺失或缺乏有关。目前针对胃肠道运动/功能障碍的治疗计划范围从饮食改变到肠切除，但针对控制蠕动的主要缺陷的药物很少。胃肠道疾病研究的一大障碍是它在很大程度上依赖于体内动物研究，而这些研究本质上通量较低。该提案旨在产生含有功能性上皮的人类肠道类器官，其周围是由肠神经支配的平滑肌。该研究工具将允许对人类肠道运动的细胞和分子基础进行前所未有的研究，并成为调节肠道运动药物的理想筛选平台。