Establishment of a human in vitro model for airway epithelial repair and regeneration

Abstract

The human airway epithelium is constantly exposed to airborne noxious substances and thus susceptible to airway injury. Our knowledge on mechanisms of airway regeneration largely derives from mouse models where exposition to e.g. naphthalene (NP) or polidocanol (PL) causes depletion of bronchial epithelial cells followed by epithelial regeneration. Given the major differences in cellular composition in the upper respiratory epithelium between humans and mice, we set out to develop a human in vitro model for the study of airway injury and regeneration. Primary human bronchial epithelial cells (phBECs, N=3) were fully differentiated at the air-liquid interface, followed by treatment with NP or PL and quantification of cell loss. After PL treatment, regeneration of a fully differentiated bronchial epithelium was monitored by immunofluorescent stainings, qRT-PCR and transepithelial electrical resistance over 4 weeks. For proof-of-concept, regeneration was studied in absence and presence of DAPT, an inhibitor of Notch signaling. In contrast to the mouse model, NP treatment of phBECs did not induce cell death, let alone specifically deplete club cells. PL treatment led to a dose-dependent loss of epithelial cells with an IC50 of 0.047% PL. Treatment of phBECs with 0.04% PL resulted in a loss of differentiated cell types followed by subsequent regeneration within 2-4 weeks, characterized by emergence of goblet, ciliated and club cells at an expense of basal cells. Presence of DAPT, an inhibitor of Notch signaling, inhibited the formation of club and goblet cells during regeneration. Treatment of PhBECs with PL allows for the elucidation of mechanisms involved in human airway repair and regeneration