Human gastric multi-regional assembloids for functional parietal maturation

Patient-derived human organoids have the remarkable capacity to self-organise into more complex structures. However, to what extent 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 four 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). Last, we implanted subcutaneously in immunodeficient NSG mice the MRA (implanted MRA). We also characterized native tissues (mucosal controls).