Defined Matrices Reveal Epithelial‐ and Mesenchymal‐Specific Responses to Stiffness and Adhesion in Human iPSC‐Derived Intestinal Organoids

Human intestinal organoids (hIOs) are powerful models for studying development and disease, yet dissecting how physical and matrix cues shape epithelial and mesenchymal responses remains challenging due to reliance on ill-defined matrices. Here, a fully defined hIO workflow was developed in which Matrigel was replaced by recombinant human substrates during pluripotent stem cell differentiation and by polyethylene glycol (PEG) hydrogels during 3D culture, while maintaining organoid viability and epithelial cytodifferentiation. Transcriptomic analyses show that both PEG- and Matrigel-cultured hIO epithelial cells are most similar to human duodenal reference populations, and have greater transcriptional similarity to fetal than adult duodenal references. Despite this overall similarity, PEG culture is associated with subtle shifts in epithelial differentiation-associated gene expression, together with altered mesenchymal ECM-remodelling and integrin-associated programs. Increasing matrix stiffness produces modest effects in epithelial cells, including increased MUC2-positive cells, whereas mesenchymal transcriptional responses to stiffness are limited. Together, these findings establish a fully defined hIO platform that retains responsiveness to inflammatory stimulation while enabling controlled investigation of how matrix composition and stiffness shape epithelial and mesenchymal cell states in models of intestinal development and fibrosis.