Pure isolation, culture, and postinjury lineage tracing of mouse visceral pleural mesothelial cells.

Pleural mesothelial cells (PMCs) are a major component of the pleura. The involvement of visceral PMCs has been suggested in several pleural or subpleural lung diseases, thus highlighting the importance of animal experiments using these cells. However, in wild-type rodent lungs, no direct and pure isolation method for visceral PMCs has been established so far. Using reanalyzed single-cell RNA sequencing data, we identified that mesothelin (Msln), a specifically expressed gene in visceral PMCs, can be useful for live cell sorting. After collecting cells by scraping the visceral pleura, MSLN+EpCAM-CD45-CD31-PDGFRα-CD146- cells with large size (≥1.4 times the median value of forward scatter in total cells) mostly exhibited WT1 protein (WT1 positivity in immunocytochemistry: 93.3% ± 0.8%). The sorted PMCs were culturable, and they responded generally predictably to several growth factors and cytokines, including genetic changes suggestive of a TGF-β-induced mesothelial-to-mesenchymal transition. In vivo experiments performed using PMC-specific reporter mice (Wt1-creERT2; tdTomato) demonstrated that intrapleural administration of bleomycin with carbon induced proliferation of PMCs in the visceral pleura. Importantly, some PMC-derived cells differentiated into α-smooth muscle actin-positive myofibroblasts with decreased MSLN expression, indicating that our method using MSLN is suitable only for uninjured lungs. In summary, we propose a novel and qualified method for visceral PMC isolation, which will aid in the elucidation of the mechanisms underlying pleura-related human lung diseases.