Abstract

Branching morphogenesis is a fundamental process governing pulmonary development, epithelial differentiation, and functional maturation. Here, we report a multilevel branching lung organoid (BLO) model generated from primary tip epithelial cells using a robust, chemically defined protocol. These BLOs exhibit characteristic dendritic architectures with proximodistal patterning and alveolar-sac-like structures. Time-resolved single-cell RNA sequencing reveals that BLOs comprise both proximal airway and distal alveolar epithelial populations and recapitulate the developmental trajectories of lung epithelial lineages in vitro . Live-cell imaging further demonstrates that the spatial organization and branching dynamics of BLOs closely resemble those of native murine pulmonary epithelium in vivo . Upon transplantation into bleomycin-injured murine models, BLO-derived cells further mature and ameliorate pulmonary fibrosis. Together, these findings establish BLOs as a powerful platform for investigating lung branching morphogenesis, epithelial lineage specification and regeneration.