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; 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）：胃肠道（GI）运动/功能障碍影响多达25％的美国人群。常见的肠道运动障碍包括肠易激综合征和粪便失禁，而诸如Hirschsprung氏病等罕见形式具有遗传基础，并且与肠神经的缺乏或缺乏有关。胃肠道运动/功能障碍的当前治疗计划范围从饮食的变化到排便，但是很少有药物以受控蠕动的主要缺陷为目标。胃肠道疾病研究的一个障碍是，它在很大程度上依赖于本质上低吞吐量的VVO动物研究。最近，我们建立了一个培养系统，通过指示人类胚胎和诱导的多能干细胞（统称称为PSC）来产生人类肠道组织“器官”（HIO）。 HIO是三维结构，其中包含肠中发现的大多数上皮和间质细胞类型。但是，由于HIO中缺乏肠神经系统，该系统并不是研究胃肠道运动障碍的有用平台。我们假设可以通过将神经Crest干细胞（NCSC）引入分化过程中，可以将肠神经系统内置到HIO中。有几种完善的方法可以在体外从PSC产生神经rest细胞，我们建议使用PSC衍生的NCSC构建含有肠神经元和神经胶质细胞的人肠癌。在AIM 1中，我们建议通过修改已用于生成更多前NCSC的现有协议来在体外生成人类PSC衍生的迷走神经NCSC。我们将在体外测试PSC衍生的NCSC的分化潜力，然后植入鸡肉胚胎。在AIM 2中，我们将使用NCSC来产生含有肠神经的人类肠道器官。我们将使用几种方法将NCSC纳入在器官发育过程中的两种组织，将NCSC纳入开发肠癌。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在器官中的ENS细胞类型中的掺入，增殖和分化。 ENS组将通过标记和功能分析。开发包含ENS的体外肠道器官系统将是旨在识别新疗法以改善胃肠道运动障碍患者的ENS功能的理想平台。呢 公共卫生相关性：胃肠道（GI）运动/功能障碍影响多达25％的美国人口。常见的肠道运动障碍包括肠易激综合征和粪便失禁，而诸如Hirschsprung氏病等罕见形式具有遗传基础，并且与肠神经的缺乏或缺乏有关。胃肠道运动/功能障碍的当前治疗计划范围从饮食的变化到排便，但是很少有药物以受控蠕动的主要缺陷为目标。胃肠道疾病研究的一个障碍是，它在很大程度上依赖于本质上低吞吐量的VVO动物研究。该提议旨在产生含有功能性上皮的人类肠道类器官，并被肠神经支配的平滑肌包围。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，并成为调节肠运动性的药物的理想筛选平台。