Stanford Cooperative Research Center for Novel, Alternative Model Systems for Enteric Diseases

 U19总体申请描述（由申请人提供）：人体胃肠道在消化、吸收、分泌和免疫等广泛过程中发挥着多种重要作用。然而，这些相同的关键功能代表了细菌和病毒胃肠道病原体在全世界人类中诱导大量发病率和死亡率的显著脆弱性。虽然动物模型对于研究传染病的基本机制是非常宝贵的，但一些最突出的胃肠道病原体与宿主组织之间的相互作用是物种特异性的，并且受宿主限制，特别是对于与人类共同进化的感染。因此，迄今为止，许多病原体的研究需要动物适应的菌株，其不能准确地概括小鼠中的人类疾病病理生理学和宿主范围限制，或者可替代地，不能模拟正常组织特征的致癌转化的人类细胞系。 最近的进展，在培养的原代组织作为三维“类器官”，再现多谱系分化和器官结构代表了一个有前途的技术，在体外模拟人类肠道病原体与人类胃肠道上皮细胞的相互作用。本申请的总体目标是使用优化的3D人胃肠类器官培养物，通过（1）原代胃和肠类器官与（2）细菌/病毒病原体和/或（3）免疫效应细胞的共培养，为各种人肠道疾病和病原体相关的特异性免疫应答创建替代模型系统。因此，我们在斯坦福大学组建了一个多学科、协作和协同的团队，包括卡尔文·郭（胃肠类器官培养）、曼努埃尔·阿米耶娃（幽门螺杆菌）、哈里·格林伯格（轮状病毒）、莎拉·海尔斯霍恩（微环境生物工程）、肖恩·本道尔（细胞飞行时间）、伊丽莎白·梅林斯（免疫学）和丹尼斯·莫纳克（伤寒沙门氏菌）。这些PI共同组成了斯坦福大学肠道疾病新型替代模型合作研究中心（斯坦福大学NAMSED CRC）计划。 斯坦福大学NAMSED CRC由核心A-C（肠道免疫学“ACCESS-GI”的管理、类器官生产和高级共培养工程和单细胞统计学）和项目1-2组成，分别研究细菌和病毒病原体与人类GI类器官的相互作用。为了实现这些目标，我们采用人类类器官技术，结合上皮细胞或上皮/间充质成分，以创建细菌（幽门螺杆菌，伤寒沙门氏菌，鼠伤寒沙门氏菌）或病毒（轮状病毒）感染的组织特异性模型，有或没有免疫效应细胞或刺激。我们部署了创新的支持技术，包括CyTOF质谱仪、生物工程细胞外基质、微流体和基于CRISPR的基因编辑。总的来说，斯坦福大学NAMSED代表了一个致力于肠道病原体作为上皮免疫类器官单位的综合建模的协同小组。

 DESCRIPTION OF THE OVERALL U19 APPLICATION (provided by applicant): The human gastrointestinal tract exhibits diverse and essential roles in processes as widespread as digestion, absorption, secretion and immunity. However, these same crucial functions represent a significant vulnerability by which bacterial and viral gastrointestinal pathogens induce substantial morbidity and mortality in humans worldwide. While animal models are invaluable to the study of basic mechanisms of infectious disease, the interactions between some of the most prominent gastrointestinal pathogens with host tissues are species specific and host restricted, particularly for infections that have co-evolved with humans. Thus, the study of many pathogens has heretofore required animal-adapted strains that do not accurately recapitulate the human disease pathophysiology and host range restriction in mice, or alternatively oncogenically transformed human cell lines that do not model normal tissue characteristics. Recent advances in culture of primary tissues as 3-dimensional "organoids" that reproduce multilineage differentiation and organ architecture represent a promising technology to modeling the interaction of human enteric pathogens with human gastrointestinal epithelium in vitro. The overall goal of this application is to create alternative model systems for a variety of human enteric diseases and pathogen-associated specific immune responses using optimized 3D human gastrointestinal organoid cultures via the co-culture of (1) primary gastric and intestinal organoids with (2) bacterial/viral pathogens and/or (3) immune effector cells. We have thus assembled a multidisciplinary, collaborative, and synergistic team at Stanford University including Calvin Kuo (gastrointestinal organoid culture), Manuel Amieva (Helicobacter pylori), Harry Greenberg (Rotavirus), Sarah Heilshorn (microenvironment bioengineering), Sean Bendall (CyTOF), Elizabeth Mellins (immunology), and Denise Monack (Salmonella Typhi). Together, these PIs form the Stanford Novel, Alternative Models for Enteric Diseases Cooperative Research Center (Stanford NAMSED CRC) Program. The Stanford NAMSED CRC is composed of Cores A-C (Administration, Organoid Production, and Advanced Co-Culture Engineering and Single Cell Statistics of Gut Immunology "ACCESS-GI"), and Projects 1-2, which study bacterial and viral pathogen interactions with human GI organoids, respectively. Towards these goals, we employ human organoid technologies incorporating epithelial-only or epithelial/mesenchymal components to create tissue-specific models of bacterial (Helicobacter pylori, Salmonella Typhi, Salmonella Typhimurium) or viral (rotavirus) infections, with or without immune effector cells or stimuli. We deploy innovative supporting technologies including CyTOF mass cytometry, bioengineered extracellular matrices, microfluidics and CRISPR-based gene editing. Overall, the Stanford NAMSED represents a synergistic group devoted to the integrated modeling of enteric pathogens as an epithelial-immune organoid unit.