Cell culture differentiation and proliferation conditions influence the in vitro regeneration of the human airway epithelium

The human airway mucociliary epithelium can be recapitulated <italic>in vitro</italic> using primary cells cultured at an air–liquid interface (ALI), a reliable surrogate to perform pathophysiological studies. Given the significant variability among media used in ALI-cultured human airway epithelial cells, our study aimed to assess the effects of different media (BEGM, PneumaCult, Half & Half, and Clancy) on cell-type distribution through single-cell RNA sequencing and imaging. Our findings reveal that these media markedly influence cell composition, gene expression profiles, cell signaling pathways, and epithelial morphology. Notably, we identified that proliferation in PneumaCult-Ex Plus biases cells toward a secretory fate, highlighting the critical impact of proliferation media on epithelial differentiation characteristics. To further investigate genes potentially affected long-term by proliferation in this medium, we examined chromatin bound to histone marks associated with cellular memory in proliferative basal cells. This analysis revealed <italic>SPDEF</italic> priming, a key transcription factor for goblet cell differentiation. Collectively, our data provide a comprehensive framework for evaluating the effects of culture conditions on airway epithelial differentiation, guiding the selection of optimal media for studies focused on specific processes such as cilia formation, mucus biology, or viral infection. We also outline essential parameters for documenting airway epithelial cell fate and morphology.