Human Multi-Regional Gastric Assembloids Favour Epithelial Cell Differentiation and Functional Parietal Maturation [paediatric patients]

Patient-derived human organoids have the remarkable capacity to self-organise into more complex structures. However, to which extend the gastric organoids can recapitulate the human stomach functions remains unexplored. Here, we report how gastric region-specific organoids can self-assemble into complex multi-regional assembloids showing levels of differentiation unattainable with simpler models. The assembloid shows preserved fundus, body and antrum regionality and gastric-specific crosstalk pathways arise. Remarkably, the increased complexity and cross communication between the different gastric regions, allows for the arise of the elusive parietal cell type, responsible for the production of gastric acid, the essential function for food digestion. We showed functional response to drugs targeting ATPase H+/K+ and further maturation of the assembloid when transplanted in vivo. This advanced in vitro model, using patient-derived tissue-specific progenitors, successfully recapitulates the structural and functional characteristics of the human stomach, offering a promising strategy for studying developmental processes, tissue interactions, and disease mechanisms that were previously challenging to attain. Overall design: To determine whether gastric stem cells have the ability to retain the identity of the region of origin, we isolated mucosal biopsies from the antrum, body and fundus regions of three paediatric patients. The organoids were derived according to previously established protocol (Giobbe et al., Nat. Comm., 2021) and cultured in gastric-specific medium. We designed a system to allow the self-aggregation of human gastric organoids in more complex multi-regional structures defined as assembloids. Briefly, full grown organoids were released from Matrigel, resuspended in collagen I gel, and cultured in floating conditions. We firstly defined the self-assembly conditions for each region (single-region assembloid, SRA) and then multi-regional, in the order fundus-body-antrum (multi-regional assembloid, MRA).

患者来源的人类类器官（Patient-derived human organoids）具备卓越的自组织能力，可组装为更为复杂的结构。然而，胃类器官能够在多大程度上重现人类胃部的生理功能，这一问题仍有待探索。本研究报道了胃区域特异性类器官如何自组装为复杂的多区域类器官组装体（assembloids），其分化水平是简单模型无法企及的。该类器官组装体保留了胃底（fundus）、胃体（body）与胃窦（antrum）的区域特性，并激活了胃特异性的信号互作通路。值得注意的是，不同胃区域间复杂度的提升与交叉信号通信，使得此前难以获取的壁细胞（parietal cell）得以产生——这类细胞负责合成胃酸，而胃酸是食物消化过程中的核心必需功能物质。我们证实，该组装体对靶向ATP酶H+/K+的药物产生功能性响应，且在体内移植后可进一步成熟。本研究采用患者来源的组织特异性祖细胞构建的先进体外模型，成功重现了人类胃部的结构与功能特征，为研究此前难以实现的发育过程、组织互作及疾病机制提供了极具潜力的策略。

实验整体设计：
为探究胃干细胞是否能够保留其来源区域的身份特性，我们从3名儿科患者的胃窦、胃体与胃底区域分离获取了黏膜活检组织。类器官的构建遵循已发表的实验方案（Giobbe等，《自然·通讯》，2021年），并在胃特异性培养基中培养。我们开发了一套体系，可促使人类胃类器官自聚集形成更为复杂的多区域结构，即类器官组装体（assembloids）。简言之，将完全成熟的类器官从基质胶（Matrigel）中解离，重悬于I型胶原凝胶中，并在悬浮培养条件下培育。我们首先确定了单区域类器官组装体（single-region assembloid, SRA）的自组装条件，随后以胃底-胃体-胃窦的顺序构建多区域类器官组装体（multi-regional assembloid, MRA）。