Development of novel in vitro human alveolar epithelial cell models to study distal lung biology and disease [bulk RNA-seq]

To address the need for human alveolar epithelial cell (AEC)-derived lines to more suitably model distal lung diseases, we have generated and characterized novel immortalized cell lines derived from human AECs. We used a combination of the ROCK inhibitor, Y-27632, and lentiviral transduction of SV40 Large T antigen of previously cryopreserved isolated human alveolar epithelial type 2 (AT2) cells to generate immortalized AECs. These AEC lines proliferate well on standard tissue culture dishes forming an epithelial monolayer and express lung progenitor markers SOX9 and SOX2. When grown in 3D culture with lung fibroblasts, the cells form NKX2-1+ organoids expressing more mature alveolar lung markers, AQP5 and GPRC5A. Single cell RNA-sequencing of one AEC line comparing cells in 2D versus 3D revealed increased cellular heterogeneity and an induction of cytokine and lipoprotein signaling in 3D culture, reflecting interactions with the microenvironment during organoid formation. Taken together, these data show our novel progenitor-like AEC lines retain a genetic and structural memory of their alveolar cell lineage despite long-term expansion, providing a valuable new system to model the distal lung in vitro. We compared our novel AEC lines transcriptomically to 12 primary AECs (AT2 and AT1-like cells), 3 lung fibroblasts, fetal lung tissue, and 18 lung adenocarcinoma (LUAD) cell lines. The LUAD cell data were downloaded from the publicly available resource DBTSS. Two of the 3 lung fibroblast samples and the fetal lung tissue were downloaded from ENCODE. Four of the primary AEC data were previously published. We profiled 7 human AEC lines generated in the study (one cell line sequenced with a replicate), 1 human lung fibroblast cell line, purified human AT2 cells from two lung donors, purified human AT2 cells from the same two donors transdifferentiated on Transwell filters for 2, 4, and 6 days into alveolar type 1 (AT1)-like cells. The remaining samples used in the entire analyses were taken from previously published work and publicly available datasets, as discussed in the paper.