Directed differentiation with mechanical simulation enhances alveolar-like features in human pluripotent stem cell-derived lung epithelium

Human pluripotent stem cell (hPSC)-derived lung models remain limited by inefficient progenitor specification and incomplete alveolar maturation. Here, we present a robust and scalable differentiation strategy that reproducibly generates high-purity ( > 85%) fetal lung progenitors without cell sorting. These cells form distal bud tip organoids that recapitulate human fetal late tip and late stalk epithelial populations with multilineage potential. We show that direct co-culture with human lung fibroblasts, combined with Notch inhibition, promotes efficient differentiation toward fetal AT1- and AT2-like epithelial states, supported by transcriptional, protein, and ultrastructural analyses. Importantly, paracrine signaling alone is insufficient, highlighting a requirement for direct epithelial–mesenchymal interaction. We further integrated a microfluidic air-liquid interface culture system incorporating cyclic stretch. Under these conditions, we observed activation of mechanotransduction-associated and developmental signaling programs together with structural and transcriptional changes consistent with more mature alveolar-like epithelial states. This platform establishes a physiologically relevant model for human alveologenesis and provides a foundation for disease modeling and regenerative applications. hPSC-derived distal bud tip organoids that resemble fetal alveolar progenitors are developed and used to show that air-liquid interface culture and cyclic stretch enhance alveolar epithelial maturation and increased segregation of AT1- and AT2-associated cell states.