Multilineage murine lung stem cells generate 3D bronchioalveolar organoids 2 for analysis of developmental processes and disease modeling

Organoid models derived from mouse and human stem cells have recently emergedas a powerful tool to study organ development and disease. We have established athreedimensional (3D) murine bronchioalveolar lung organoid (BALO) model thatallows clonal expansion and self-organization of FACS-sorted bronchioalveolar stemcells upon co-culture with lung mesenchymal cells. BALO yield a branched 3Dstructure within 21 days of culture mimicking the cellular composition of thebronchioalveolar compartment, as defined by single cell RNA sequencing andmicroscopic phenotyping. In addition, BALO support engraftment and maintenance ofthe cellular phenotype of injected tissue-resident macrophages. We also demonstratethat BALO recapitulate lung developmental defects after knock-down of criticalregulatory genes and permit viral infection and replication. We conclude that theBALO model enables 3D reconstruction of the bronchioalveolar lung compartmentwith its unique cellular composition, thereby opening numerous new avenues tostudy lung development, infection, and regenerative processes in vitro.