A fully defined protocol and PEG-based platform for probing transcriptional responses to matrix cues in human iPSC-derived intestinal organoids

Human intestinal organoids (hIOs) are powerful models of development and disease, yet reliance on ill-defined murine basement membrane extracts limits reproducibility, mechanistic interrogation of extracellular matrix (ECM) cues, and translational relevance. Here, we establish a fully defined, animal product-free workflow for generating human induced pluripotent stem cell (iPSC)-derived hIOs, combining recombinantly produced human protein substrates for early 2D differentiation with synthetic polyethylene glycol (PEG) hydrogels for 3D culture. This platform decouples biochemical and mechanical matrix cues, enabling independent control of ligand presentation and stiffness while robustly supporting hIO growth and intestinal epithelial patterning. Transcriptomic analyses showed that epithelial cells from PEG-cultured hIOs align closely with human duodenal epithelium and are comparable to Matrigel-grown controls, while exhibiting subtle shifts in markers associated with epithelial maturation. Systemic modulation of PEG stiffness revealed that epithelial cells retain sensitivity to mechanical cues in 3D culture, whereas mesenchymal transcriptional programs were more strongly influenced by matrix composition and integrin-mediated adhesion than by stiffness alone. PEG-cultured hIOs also remained responsive to inflammatory stimulation. Together, this work establishes a defined and mechanically tunable organoid culture platform that decouples biochemical and mechanical matrix cues, providing a versatile framework for organoid mechanobiology across tissue engineering and disease modeling applications.