Alveolar Basal Cells Differentiate towards Secretory Epithelial- and Aberrant Basaloid-like Cells In Vitro

In idiopathic pulmonary fibrosis (IPF), keratin (KRT)17+/KRT5+ basal and KRT17+/KRT5- aberrant basaloid cells are atypically present within the alveolar space. We previously described the fibrosis-enriched outgrowth of alveolar basal cells from peripheral fibrotic lung tissue. Using single cell RNA sequencing (scRNA-seq), we here characterize the transcriptome of these cultured alveolar basal cells under different culture conditions. Methods: Fibrotic peripheral lung tissue pieces were placed in DMEM growth medium. Outgrown cells were analysed by scRNA-seq, TaqMan-PCR or immunofluorescence (IF) either directly or after medium change to an epithelial cell specific medium (Cnt-PR-A). Results: A fraction of alveolar basal cells cultured in DMEM growth medium showed close transcriptomic similarities to IPF basal cells. However, although they expressed KRT5, the transcriptome of the majority of cells matched best to the transcriptome of recently described KRT17+/KRT5- aberrant basaloid cells, co-expressing the canonical basal cell marker KRT17 and mesenchymal cell marker (VIM, FN1). A smaller fraction of cells matched best to secretory epithelial cells. Two differentiation gradients from basal to aberrant basaloid-like cells and basal to secretory epithelial-like cells were apparent. Interestingly, these differentiation paths seemed reversed when the cell culture medium was changed to Cnt-PR-A. Conclusions: Our results suggest that cultured alveolar basal cells have the capacity to differentiate towards secretory epithelial-like cells and to aberrant basaloid-like cells. However, due to the persistent expression of KRT5, a complete differentiation towards aberrant basaloid cells did not seem to be achieved in our culture conditions. Importantly, differentiation seemed reversible by changing the cells microenvironment. Determining specific factors influencing these differentiation paths may help to define novel drug targets for IPF therapy.