Airway Basal Stem Cell Differentiation

Tracheal cells were harvested and cultured as previously described (Mou et al., 2016 (link)). Briefly, cells were cultured and expanded in complete SAGM (small airway epithelial cell growth medium; Lonza, CC-3118) containing a TGF-β/BMP4/WNT antagonist cocktail and 5 μM ROCK inhibitor Y-27632 (Selleckbio, S1049). To generate ALI cultures, airway basal stem cells were seeded onto transwell membranes and differentiated using PneumaCult-ALI Medium (StemCell, 05001). Air-liquid cultures were fully differentiated after 14 days as demonstrated by the formation of air-liquid interface and the beating of cilia.