Long-Term Culture of Distal Airway Epithelial Cells Allows Differentiation Towards Alveolar Epithelial Cells Suited for Influenza Virus Studies

As the target organ for numerous pathogens, the lung epithelium exerts critical functions in health and disease. However, research in this area has been hampered by the quiescence of the alveolar epithelium under standard culture conditions. Here, we used human distal airway epithelial cells (DAECs) to generate alveolar epithelial cells. Long-term, robust growth of human DAECs was achieved using co-culture with feeder cells and supplementation with epidermal growth factor (EGF), Rho-associated protein kinase inhibitor Y27632, and the Notch pathway inhibitor dibenzazepine (DBZ). Removal of feeders and priming with DBZ and a cocktail of lung maturation factors prevented the spontaneous differentiation into airway club cells and instead induced differentiation to alveolar epithelial cells. We successfully transferred this approach to chicken distal airway cells, thus generating a zoonotic infection model that enables studies on influenza A virus replication. These cells are also amenable for gene knockdown using RNAi technology, indicating the suitability of the model for mechanistic studies into lung function and disease.