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

 U19的整体申请说明(由申请人提供)：人体胃肠道在消化、吸收、分泌和免疫等广泛的过程中表现出各种和基本的作用。然而，这些同样重要的功能代表着一个严重的脆弱性，通过这种脆弱性，细菌和病毒胃肠道病原体在世界各地的人类中导致大量的发病率和死亡率。虽然动物模型对于研究传染病的基本机制是非常宝贵的，但一些最主要的胃肠道病原体与宿主组织之间的相互作用具有物种特异性和宿主限制性，特别是对于与人类共同进化的感染。因此，到目前为止，许多病原体的研究都需要动物适应的菌株，这些菌株不能准确地概括人类疾病的病理生理学和小鼠的寄主范围限制，或者是不能模拟正常组织特征的致癌转化的人类细胞系。近年来，原代组织培养为三维“类器官”，再现了多谱系分化和器官构筑，为体外模拟人类肠道病原体与人类胃肠上皮的相互作用提供了一种有前途的技术。这项应用的总体目标是通过(1)原代胃肠有机物与(2)细菌/病毒病原体和/或(3)免疫效应细胞的共培养，使用优化的3D人类胃肠有机物培养，创建各种人类肠道疾病和病原体相关特异性免疫反应的替代模型系统。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，成员包括Calvin Kuo(胃肠道有机物培养)、Manuel Amieva(幽门螺杆菌)、Harry Greenberg(轮状病毒)、Sarah海尔肖恩(微环境生物工程)、Sean Bendall(细胞TOF)、Elizabeth Mellins(免疫学)和Denise Monack(伤寒沙门氏菌)。这些PI共同构成了斯坦福大学肠道疾病合作研究中心(Stanford NAMSED CRC)计划。斯坦福NAMSED CRC由核心A-C(管理、有机物质生产、高级共培养工程和肠道免疫学单细胞统计“Access-GI”)和项目1-2组成，分别研究细菌和病毒病原体与人类GI有机物质的相互作用。为了实现这些目标，我们使用结合了仅上皮性或上皮性/间充质成分的人类有机体技术来创建细菌(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质量细胞仪、生物工程细胞外基质、微流体和基于CRISPR的基因编辑。总体而言，斯坦福NAMSED代表了一个协同小组，致力于将肠道病原体作为一个上皮免疫器官单位进行集成建模。

 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.