Clonal alveolar organoid assays were performed as described previously with some modifications from the original protocol17 (link),37 (link),81 (link). In brief, 5 × 103 AT2 cells were isolated as described above and mixed with 5 x104 lung PDGFRα+ isolated cells from adult wild type mice as previously described19 (link) for mouse or MRC5 cells (ATCC CCL-171, tested negative for mycobacterial contamination, at no greater than passage 7) for human cultures. For the first two days of culture, ROCK inhibitor Y27632 (Sigma-Aldrich catalog #Y0503) was added to the media (SAGM; Lonza catalog #CC-3118). After two days of culture, Y276632 was removed and ligand treatments of organoids were performed using the following reagents at the indicated concentrations: ANA-12 0.02μg/mL (Alomone Labs catalog #A-215), recombinant human BDNF 0.05μg/mL (Alomone Labs catalog #B-250) and recombinant murine FGF7 0.01μg/mL (R&D Systems catalog #5028-KG). DMSO was used as a control for the ANA-12 and 2% BSA was used as a control for the BDNF and FGF7. Media was changed every 48 hours, and fresh ligands were included at each media change. After 21 days (or 28 days where indicated) organoids were imaged using an Olympus MVX10 microscope or EVOS FL microscope (Life Technologies).