Gastroids and Colonoids as 4D Models of Gastrointestinal Infection

摘要 哺乳动物胃肠道组织培养模型为阐明胃肠道感染提供了重要工具 发病机制；然而，目前这样的模型缺乏方法来询问人类的作用 胃肠道对感染的动态平衡反应中的衰老和生物节律。在项目3中，我们提出了小说《4-- 推进胃肠道三大病原体幽门螺杆菌研究的“维度”实验平台 (Hp)、艰难梭菌(CD)和产志贺毒素的大肠杆菌(STEC)。使用可诱导的多能干细胞 从人血清中分离的细胞(IPSC)，我们将产生两种类型的类器官：人胃类器官 (HGOS)和人类结肠有机化合物(HCOs)。使用患者活检的胃肠道上皮组织，我们将 产生两种类型的肠样体：来自胃上皮的人类“胃样体”和来自胃上皮的人类“结肠型”。 结肠上皮。器官类和肠类将被设计和转换为荧光和 细胞周期和昼夜节律的生物发光记者。我们将使用这些记者进行单细胞和 人群水平实时分析上皮细胞周转和昼夜节律：A)hGO和胃类 感染Hp和b)HCOs和类结肠菌感染Cd毒素A和B以及志贺毒素。 GI器官平台和肠道平台将来自婴儿、青少年、年轻人和老年人群体 成年人。在具体目标1中，我们将确定：a)哪种平台(器官平台和肠道平台)是最好的模型 每种病原体，以及b)病源年龄对感染反应的影响程度。我们 假设hGO和HCOs在病原体的细胞受体表达方面是“新生”的 和毒素，而胃样和结肠样体将反映捐赠者的组织成熟度。我们进一步 假设来自老年人的肠状突将非常容易受到 由于年龄相关的胃肠道干细胞再生障碍而引起的感染。在目标2中，我们将确定：a) 一天中的时间影响胃肠道上皮对感染的反应，b)胃肠道病原体扰乱上皮的计时， 以及c)昼夜节律中与年龄相关的扰动会调节胃肠道感染的结果。总而言之，这些 特定的目标将把我们的NAMSED项目和核心聚集在一起，以实现发展稳健的共同目标 胃肠道感染的体外模型，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.