Gastroids and Colonoids as 4D Models of Gastrointestinal Infection

摘要 胃肠道（GI）感染的哺乳动物组织培养模型为阐明GI提供了重要工具 发病机制;然而，这种模型目前缺乏方法来询问人类的作用， 衰老和生物节律在胃肠道稳态反应感染。在项目3中，我们提出了新的“4- 三维”实验平台，以推进三个主要胃肠道病原体的研究：幽门螺杆菌 (HP)、艰难梭菌（Clostridium difficile，CD）和产志贺毒素E. coli（STEC）。利用诱导性多能干细胞 从人血清分离的iPSC细胞（iPSC），我们将产生两种类型的类器官：人胃类器官 （HGO）和人类结肠类器官（HCO）。使用患者活检的胃肠道上皮组织，我们将 产生两种类型的类肠：来自胃上皮的人“类肠”和来自胃上皮的人“类结肠”。 结肠上皮类器官和类肠细胞将被工程化并用荧光和 细胞周期和昼夜节律的生物发光报告者。我们将使用这些报告单细胞和 上皮更新和昼夜节律的群体水平实时分析：a）阻塞性和胃病 感染HP和B）感染CD毒素A和B以及滋贺毒素的HCO和类结肠。 GI类器官和肠类平台将来自婴儿、青少年、年轻人和老年人群体 成年人了在具体目标1中，我们将确定：a）哪种平台（类器官与类肠）是用于以下的上级模型 每种病原体，和B）来源的年龄影响对感染的反应的程度。我们 假设HPLCs和HCO在它们对病原体的细胞受体的表达方面将是“新生儿”样的 和毒素，而胃和结肠将反映供体的组织成熟度。我们进一步 假设来自老年人的类肠上皮细胞非常容易受到 由于年龄相关的GI干细胞再生障碍引起的感染。在目标2中，我们将确定是否： 一天中的时间影响GI上皮对感染的反应，B）GI病原体破坏上皮的计时， 和c）昼夜节律中与年龄相关的扰动调节GI感染的结果。总的来说，这些 具体目标将汇集我们的NAMSED项目和核心，共同目标是开发强大的 胃肠道感染的离体模型，在正确的时间为正确的患者带来突破性的治疗。

Abstract Mammalian tissue culture models of gastrointestinal (GI) infection provide important tools for elucidating GI mechanisms of pathogenesis; however, such models currently lack methods to interrogate the role of human aging and biological rhythms in GI homeostatic responses to infection. In Project 3, we propose novel “4- dimensional” experimental platforms to advance the study of three major GI pathogens: Helicobacter pylori (HP), Clostridium difficile (CD), and Shiga-toxin producing E. coli (STEC). Using inducible pluripotent stem cells (iPSC) isolated from human serum, we will generate two types of organoids: human gastric organoids (HGOs) and human colonic organoids (HCOs). Using GI epithelial tissue from patient biopsies, we will generate two types of enteroids: human “gastroids” from stomach epithelium and human “colonoids” from colon epithelium. Organoids and enteroids will be engineered and transduced with fluorescent and bioluminescent reporters of cell cycle and circadian rhythms. We will use these reporters for single-cell and population-level real-time analyses of epithelial turnover and circadian rhythms in: a) HGOs and gastroids infected with HP and b) HCOs and colonoids infected with CD toxins A and B and Shiga toxin. GI organoid and enteroid platforms will be derived from groups of infants, adolescents, young adults, and older adults. In Specific Aim 1, we will determine: a) which platform (organoid vs. enteroid) is the superior model for each pathogen, and b) the extent to which the age of the source influences response to infection. We hypothesize that HGOs and HCO's will be “neonatal” like in their expression of cellular receptors to pathogens and toxins, whereas gastroids and colonoids will reflect the tissue maturity of the donor. We further hypothesize that enteroids derived from older adults will be highly susceptible to adverse outcomes from infection due to age-related impairments in GI stem cell regeneration. In Aim 2, we will determine whether: a) the time of day influences GI epithelial response to infection, b) GI pathogens disrupt epithelial timekeeping, and c) age-related perturbations in circadian rhythms modulate outcomes to GI infection. Collectively, these Specific Aims will bring together our NAMSED Projects and Cores for the common goal of developing robust ex vivo models of GI infection that lead to breakthrough therapies for the right patient at the right time.