Engineering of human intestinal organoids containing immune cells

项目摘要/摘要 肠道病原体通过胃肠道感染人类宿主，导致严重的发病率、死亡率、 世界范围内的经济困难。肠道是一个复杂的器官，由一层上皮细胞组成， 固有层含有免疫细胞，以及一层组织成绒毛和隐窝的平滑肌细胞。 目前可用的人体肠道体外模型具有重大局限性，例如缺乏免疫细胞或 微生物区系，因此，了解人类肠道病原体在其本土宿主中的行为 环境带来了重大挑战。更接近人类肠道的系统是 需要更好地了解肠道疾病、宿主与病原体的相互作用，并促进 开发更有效的治疗方法。最近的研究表明，人类原代多能干细胞 可在体外分化为具有假腔的三维人类肠道器官(HIO) 以及极化的肠上皮，类似于活体肠道中的组织。重要的是，HIO 含有肠道上皮的主要分泌和吸收细胞类型。然而，重债穷国缺乏微折叠 (M)细胞和免疫细胞，它们对于诱导粘膜免疫和平衡适当的 对食物抗原、共生菌和病原体的耐受性和/或炎症反应。不平衡 肠道内环境的稳定会导致严重的疾病，如炎症性肠病(IBD)和M细胞 是提高口服疫苗效力的重要目标。因此，项目2的目标是开发免疫 含有M细胞和免疫细胞的HIO。我们假设免疫HIO，即与HIO共培养 免疫细胞将比目前的模型更忠实地模拟人类的肠道，因此将是 在研究宿主-病原体相互作用、药物开发和疾病发病机制方面具有优势。至 开发具有免疫细胞的复杂的人体肠道器官，我们将：1)建立和基准一个复合体 通过免疫细胞与免疫细胞共培养建立类器官模型；2)确定免疫细胞对 鼠伤寒沙门氏菌、艰难梭菌和沙门氏菌感染复合有机物模型 人类诺如病毒；以及3)确定具有IBD相关突变的免疫性HIO模型的适用性 疾病发病机制方面。微生物区系衍生产物对免疫HIO功能的影响 也将接受评估。我们的研究将与项目1协同，该项目将阐明HIO的相互作用 在没有免疫细胞的情况下与共生或致病细菌合作，以及将工程 HIO的平台，允许腔内和腔外流动和采样。我们建议的研究是 意义重大，因为它有望提供一种新的系统来促进肠道功能障碍的研究。这个 免疫HIO模型有可能快速开发有效的疫苗、抗感染药物和 IBD等肠道疾病的治疗选择。

PROJECT SUMMARY/ABSTRACT Enteric pathogens infect their human host via the gastrointestinal tract causing significant morbidity, mortality, and economic hardship worldwide. The intestine is a complex organ comprised of a layer of epithelial cells, the lamina propria containing immune cells, and a layer of smooth muscle cells organized into villi and crypts. Currently available in vitro models of the human intestine have major limitations, e.g., lack of immune cells or microbiota, and therefore, understanding how human enteric pathogens behave in their native host environment has presented significant challenges. Systems more closely mimicking the human intestine are needed to gain a better understanding of intestinal diseases, host - pathogen interactions and to facilitate the development of more effective therapies. Recent studies revealed that primary human pluripotent stem cells can be differentiated in vitro into three-dimensional human intestinal organoids (HIOs) with a pseudo-lumen and polarized intestinal epithelium similar to the organization found in the intestine in vivo. Importantly, HIOs contain the major secretory and absorptive cell types of the intestinal epithelium. However, HIOs lack microfold (M) cells and immune cells, which are critical for induction of mucosal immunity and balancing of appropriate tolerogenic and/or inflammatory responses to food antigens, commensal bacteria and pathogens. Imbalances in intestinal homeostasis result in devastating diseases such as inflammatory bowel disease (IBD), and M cells are important targets for improving oral vaccine efficacy. Therefore, the goal of Project 2 is to develop immune HIOs containing M cells and immune cells. We hypothesize that immune HIOs, i.e., HIOs co-cultured with immune cells, will more faithfully mimic the human intestine than current models and thus will be superior for studying host-pathogen interactions, drug development and disease pathogenesis. To develop complex human intestinal organoids with immune cells, we will: 1) establish and benchmark a complex organoid model by co-culturing immune cells with HIOs (immune HIOs); 2) determine the response of the complex organoid model to infection with Salmonella enterica serovar Typhimurium, Clostridium difficile and human norovirus; and 3) determine the suitability of immune HIOs with IBD-associated mutations to model aspects of disease pathogenesis. The effects of microbiota-derived products on the function of immune HIOs will also be evaluated. Our studies will synergize with Project 1, which will elucidate the interactions of HIOs with commensal or pathogenic bacteria in the absence of immune cells, and with Project 3, which will engineer a platform for the HIOs to allow intra- and extra-luminal flow and sampling. Our proposed research is significant because it is expected to provide a novel system to facilitate studies of intestinal dysfunction. The immune HIO model has the potential to fast-track development of effective vaccines, anti-infectives and treatment options for intestinal diseases like IBD.