Airway basal cell culture

Human and mouse basal cells were isolated and cultured using the established protocols (Mou et al., 2016; Levardon et al., 2018). Briefly, basal cells were maintained in Small Airway Epithelial Cell Medium (Lonza, CC-3118 or Promocell medium, C-21170) with 5–10 μM ROCKi (Tocris, 1254), 1 μM A-83–01 (Tocris, 2939), 0.1 μM DMH-1 (Tocris, 4126) and 0.5 μM CHIR99021 (Tocris, 4423). Human cells were cultured on plates pre-coated with laminin-enriched 804G-conditioned medium, while mouse cells were cultured on plain plates without pre-coating. To expand, cells were dissociated with trypsin and re-seeded at 1:10 ratio. Mucociliary differentiation of airway basal cells in ALI was performed as previously described (Mou et al., 2016). Briefly, airway basal cells were seeded onto 0.4 μm transwell membranes pre-coated with 804G-conditioned medium at a cell density of > 6000 cells/mm² and were allowed to attach for a minimum of 12 hours. After the removal of unattached cells, the medium was replaced with complete Pneumacult-ALI medium (StemCell Technology, Cat. 05001) filling both the upper and lower chambers. The next day, the ALI medium was added only to the lower chamber to initiate differentiation at the airway-liquid interface. The ALI medium was changed daily until differentiation was well established after 14 days as a standard protocol. To lineage label PNECs in ALI culture derived from Ascl1-CreERT2;Rosa(tmRed) mice, 4-OH-tamoxifen (1 μM, Sigma-Aldrich, H7904) was added to day 15 ALI culture. After 24 hours, the culture was rinsed with saline three times to remove 4-OH-tamoxifen. Cultures were fixed for staining on day 17–19 in ALI. For the measurement of serotonin secretion, ALI culture was replaced with fresh media and after 2 days, media were collected for ELISA for 5-HT using a commercialized kit.