Intestinal Organoids as a model system for studying enteric disease

 U19总体申请描述（由申请人提供）：本提案将使用人胃肠道类器官作为模型系统。我们将整合国家的最先进的方法，与协作和协同的研究方法来研究胃肠道疾病。我们将开发体外胃肠道模型，复制人体肠道的生物结构。这些组织将概括人类生理学和疾病病理学，并纳入疾病和人类宿主反应的关键成分。我们的重点将涵盖从基础研究（发病机制）到产品开发的整个过程。对于没有相关动物模型的胃肠道病原体，另一个长期目标是开发可用于IND启动研究的模型系统。我们将通过以下具体目标来推进这一领域：目标1。标题：H.使用人胃类器官的幽门宿主感染。研究项目1将使用新开发的胃类器官系统来研究幽门螺杆菌。这些研究有可能促进我们对H. pylori改变胃干细胞的命运，并推进我们对H.幽门螺杆菌是一种致癌物质。 目标2.标题：人类肠道类器官研究产毒素细菌病原体。研究项目II将使用人类肠道类器官作为一种新型模型系统，研究两种致炎性肠道病原体，艰难梭菌和产滋贺毒素（Stx）的大肠杆菌（STEC）。这些研究有可能促进我们对C. difficile和STEC，并创建用于评估潜在疗法的模型系统。 目标3：标题：作为胃肠道感染4D模型的人类胃和结肠。研究项目III将开发离体模型，以确定年龄依赖性和时间GI上皮反应：a）H。pylori感染，以及B）艰难梭菌（CD）和产滋贺毒素大肠杆菌（STEC）感染及其在人结肠类器官（HCO）和类结肠中的主要毒素。 目标4。技术传播。我们将推进和鼓励教育和培训活动，目前与本提案有关的研究人员，通过其他NAMSED奖资助的研究人员，以及一般科学界感兴趣的成员。 目标5。类器官工程核心人类类器官核心将开发报告细胞系，其中荧光蛋白在特定谱系中表达。shRNA敲除和CRISPR靶向细胞系将产生缺乏特定谱系的人类胃肠道类器官，四环素调节系统将用于扩增特定谱系。

 DESCRIPTION OF THE OVERALL U19 APPLICATION (provided by applicant): This proposal will use human gastrointestinal organoids as a model system. We will integrate state-of-the-art methodologies, with collaborative and synergistic research approaches to the study of gastrointestinal diseases. We will develop in vitro gastrointestinal models that replicate the biological structures of the human intestinal tract. These tissues will recapitulate human physiology and disease pathology, and incorporate components critical to disease and human host response. Our focus will span the pipeline from basic research (pathogenesis) to product development. For gastrointestinal pathogens that have no relevant animal models, another long-term goal is to develop model systems that can be used in IND-enabling studies. We will advance the field through the following Specific Aims: Aim 1. Title: Mechanisms of H. pylori-host infection using human gastric organoids. Research Project 1 will use the newly developed gastric organoid system to study Helicobacter pylori. The proposed studies have the potential to advance our understanding of the mechanisms by which H. pylori alters gastric stem cell fate, and advance our understanding of the role of H. pylori as a carcinogen. Aim 2. Title: Human Intestinal Organoids to Study Toxigenic Bacterial Pathogens. Research Project II will use human intestinal organoids as a novel model system to study two toxigenic enteric pathogens, Clostridium difficile and Shiga toxin (Stx) producing Escherichia coli (STEC). The proposed studies have the potential to advance our understanding of the pathogenic processes of C. difficile and STEC and create model systems for the evaluation of potential therapeutics. Aim 3. Title: Human gastroids and colonoids as 4D models of gastrointestinal infection. Research Project III will develop ex vivo models to determine age-dependent and temporal GI epithelial responses to: a) H. pylori infection in human gastric organoids (HGOs) and gastroids and b) Clostridium difficile (CD) and Shiga toxin-producing Escherichia coli (STEC) infection and their major toxins in human colonic organoids (HCOs) and colonoids. Aim 4. Technology dissemination. We will advance and encourage educational and training activities to investigators currently affiliated with this proposal, investigators funded through other NAMSED awards, and interested members of the scientific community in general. Aim 5. Organoid Engineering Core. The human organoid core will develop reporter lines in which fluorescent proteins are expressed in specific lineages. shRNA knockdown and CRISPR targeted lines will give rise to human gastro-intestinal organoids lacking specific lineages, and tetracycline-regulated systems will be used to expand specific lineages.