Development of Novel Human Alveolar Epithelial Cell Models to Study Distal Lung Biology and Disease

Abstract

Many acute and chronic lung diseases affect the distal lung alveoli. Although airway-derived human cell lines exist, alveolar epithelial cell (AEC)-derived lines are needed to better model these diseases. By first expanding human primary AECs in medium containing ROCK inhibitor, Y-27632, then transducing with lentivirus carrying Simian virus 40 Large T antigen (SV40 LgT), we have generated and characterized three novel immortalized cell lines from separate lung donors. These cell lines grow as epithelial monolayers expressing lung progenitor markers SOX9 and SOX2, with little to no expression of mature AEC markers. Co-cultured in three-dimensions (3D) with lung fibroblasts, the cells form NKX2-1+ organoids expressing mature AEC markers AQP5 and GPRC5A. Single cell RNA-sequencing of an AEC line in 2D versus 3D revealed increased cellular heterogeneity and induction of cytokine and lipoprotein signaling, consistent with organoid formation. Activating WNT and FGF pathways resulted in larger organoids. Our approach appears to yield lung progenitor lines that retain a genetic and structural memory of their alveolar cell lineage despite long-term expansion and whose differentiation may be modulated under various 3D conditions. These cell lines provide a valuable new system to model the distal lung in vitro.