Extracellular matrix stiffness directs region-specific lung epithelial differentiation revealed by hPSC-derived lung organoids

Regional epithelial lineages of the human respiratory system reside within an extracellular matrix (ECM) whose mechanics vary along the airway–alveolar axis, yet how ECM stiffness directs epithelial fates remains unclear. Here, utilizing human pluripotent stem cell-derived lung organoids embedded in stiffness-tunable hydrogels as an in vitro model, we show ECM stiffness governs region-specific epithelial differentiation. Stepwise softening of ECM stiffness yields airway organoids with proximal-to-distal airway epithelial compositions and biomimetic physiological functions. During alveolar differentiation, increased stiffness promotes alveolar type 2 (AT2) and type 1 (AT1) maturation and drives AT2-to-AT1 transition. Furthermore, RNA sequencing reveals ECM stiffness regulates epithelial fates primarily through mechanotransduction pathways. Finally, these organoids reproduce the infection tropisms of SARS-CoV-2 variants. Together, this research elucidates ECM stiffness as a critical determinant of epithelial cell fate specification and region-specific lung organoid generation, which offers a valuable in vitro model for studying region-specific lung development, diseases pathogenesis, and drug screening.