Development of a Polidocanol-based Human In Vitro Model to Explore Airway Epithelial Repair

The human airway epithelium is a primary site of toxicant exposure and crucial in the pathogenesis of acute and chronic lung disease. In chronic lung disease (CLD), the airway epithelium is frequently altered and distorted, and its restoration is desirable. The mechanisms underlying human aberrant epithelial regeneration, however, are poorly understood. Importantly, our knowledge about airway epithelial injury and regeneration stems largely from mouse models; yet, airways differ considerably between mice and humans. We hypothesized that treatment of differentiated primary human bronchial epithelial cells (phBECs) with polidocanol (PDOC) or naphthalene would allow study of mechanisms of human airway epithelial injury and regeneration. Injury of differentiated phBECs with 0.04% but not 0.1% PDOC resulted in full restoration of a functional epithelium and epithelial barrier integrity as monitored by qRT-PCR analysis, immunofluorescence staining, and transepithelial electrical resistance measurements. Regeneration was associated with a transient but not parallel increase of p21⁺ and KRT17⁺ cells. Providing proof of concept, DAPT, an inhibitor of Notch signaling, blunted the restoration of secretory cell types after 0.04% PDOC injury. Differentiation of phBECs in the presence of cigarette smoke extract or ethanol as a first hit significantly impaired the regeneration capacity of phBECs. Although naphthalene is known to specifically induce club cell depletion in mouse airways, it failed to do so in phBECs. In conclusion, using fully differentiated phBECs treated with PDOC, we successfully established and thoroughly characterized a human in vitro system that will facilitate studies of mechanisms involved in susceptibility to injury as well as human airway repair and regeneration.