Human Pluripotent Stem Cell-Derived Alveolar Organoids for Gene-editing and Lung Adenocarcinomas Modeling.

Human pluripotent stem cell (hPSC)-derived alveolar organoids have emerged as valuable tools for studying lung development, modeling pulmonary diseases, and drug discovery, though their application has been hindered by laborious differentiation protocols and technical complexity. Here, we present an hPSC-derived alveolar organoid (hALO) system with exceptional long-term expandability (>30 passages), efficient cryopreservation resilience, and streamlined production achieved through earlier 3D culture initiation and elimination of cell sorting requirements. Transcriptomic analysis across passages confirmed hALOs contain alveolar progenitors and AT2 lineages, recapitulating pseudoglandular-to-canalicular development while partially maintaining adult AT2 immune-related functions. The system permits alveolar epithelial differentiation via pharmacological modulation of WNT/YAP signaling or through orthotopic transplantation, while multiplex genetic engineering enables programmable disease modeling and adenocarcinoma pathogenesis studies. These versatile capabilities establish hALOs as a robust dual-phase platform for mechanistic investigation of lung epithelial biology and disease modeling across in vitro and in vivo environments.