Abstract
<title>Abstract</title> <p> Two-dimensional gastric organoid-derived mucosoids provide a physiologically relevant model for studying epithelial differentiation and function. We demonstrated that withdrawal of WNT3a (WNT) and TGFβ inhibitor promotes differentiation and compartmentalization in human gastric mucosoids without impairing functionality. This results in a phenotype resembling the <italic>in vivo</italic> antral glands. Compared with WNT/TGFβi <sup>+</sup> conditions, WNT/TGFβi <sup>-</sup> cultures exhibited increased cell height, enhanced barrier function, and reduced cell density, accompanied by reduced stemness markers and increased tight junction and secreted factors. Phenotypically, WNT/TGFβi⁻ cultures resembled the antral gland tip, showing increased MUC5AC expression, whereas WNT/TGFβi⁺ condition maintained gland base–like features with higher nuclear density. We applied the adenylyl cyclase activator Forskolin (FSK), which enhanced expression of mucus and hormone-related marker without additional WNT supplementation. Differential proteomic confirmed that withdrawal of WNT and TGFβ inhibitor shift cells from stemness toward differentiated epithelial states with enhanced tight junctions and secretory features. FSK promoted a more parietal cell-like state, dependent on the absence of WNT and TGFβ inhibition. Proteomic analysis revealed that the mucus layer has a protein composition distinct from matched cellular lysates, enriched in extracellular matrix, immune, antimicrobial, coagulation, and GPCR-related proteins. This mucus signature was conserved across organoid lines despite marked donor-to-donor variability. WNT, TGFβ, and cAMP signalling further shaped the composition of the mucus proteome while preserving patient-specific protein signatures. We show that the withdrawal of WNT and TGFβ inhibition alone is sufficient to drive broad epithelial differentiation of cells and the enrichment of multiple differentiated subpopulations, without the need for supplementation of additional growth factors. At the same time, standard culture protocols can be used to preserve the proliferative zone, enabling study of both compartments of the gland. Together, these findings establish gastric mucosoids as a versatile model to investigate epithelial differentiation, functional compartmentalization, and secretion with enhanced <italic>in vitro</italic> differentiation. </p>