Intestinal Organoids as a model system for studying enteric disease

 U19整体申请说明(由申请人提供)：该提案将使用人类胃肠道有机化合物作为模型系统。我们将把最先进的方法与协作和协同的研究方法整合到胃肠道疾病的研究中。我们将开发复制人类肠道生物结构的体外胃肠道模型。这些组织将概括人类生理和疾病病理，并包含对疾病和人类宿主反应至关重要的成分。我们的重点将跨越从基础研究(发病机制)到产品开发的整个管道。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研究的模型系统。我们将通过以下具体目标推动这一领域的发展：目标1.标题：利用人胃类器官研究幽门螺杆菌宿主感染的机制。研究项目1将使用新开发的胃器官系统来研究幽门螺杆菌。这些研究有可能促进我们对幽门螺杆菌改变胃干细胞命运的机制的理解，并促进我们对幽门螺杆菌作为致癌物的作用的理解。目的2.标题：人体肠道有机物研究产毒细菌病原菌。研究计划二将使用人类肠道有机化合物作为一个新的模型系统来研究两种产毒的肠道病原体，艰难梭菌和志贺毒素(STX)产生的大肠杆菌(STEC)。拟议的研究有可能促进我们对艰难梭菌和STEC致病过程的理解，并为评估潜在的治疗方法创建模型系统。目的3.标题：人类胃液和结肠作为胃肠道感染的4D模型。研究项目III将开发体外模型，以确定胃上皮细胞对以下情况的年龄依赖性和时间性反应：a)幽门螺杆菌在人胃器官(HGO)和胃液中的感染；b)艰难梭菌(CD)和产志贺毒素的大肠杆菌(STEC)的感染及其在人结肠器官(HCOs)和结肠腺中的主要毒素。目标4.技术传播。我们将推进和鼓励目前与这项提议有关的研究人员、由NAMSED其他奖项资助的研究人员以及科学界感兴趣的成员开展的教育和培训活动。目标5.有机化合物工程核心。人类类器官核心将发展报告系，在其中荧光蛋白在特定的谱系中表达。ShRNA敲除和CRISPR靶向株将导致人类胃肠器官缺乏特定谱系，四环素调控系统将用于扩大特定谱系。

 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.