Fraction of MHCII and EpCAM expression characterizes distal lung epithelial cells for alveolar type 2 cell isolation

Koichi Hasegawa, Atsuyasu Sato, Kazuya Tanimura, Kiyoshi Uemasu, Yoko Hamakawa, Yoshinori Fuseya, Susumu Sato, Shigeo Muro, Toyohiro Hirai

Alveolar type 2 (AT2) cells play important roles in maintaining adult lung homeostasis. AT2 cells isolated from the lung have revealed the cell-specific functions of AT2 cells. Comprehensive molecular and transcriptional profiling of purified AT2 cells would be helpful for elucidating the underlying mechanisms of their cell-specific functions. To enable the further purification of AT2 cells, we aimed to discriminate AT2 cells from non-AT2 lung epithelial cells based on surface antigen expression via fluorescence activated cell sorting (FACS). Single-cell suspensions obtained from enzymatically digested murine lungs were labeled for surface antigens (CD45/CD31/epithelial cell adhesion molecule (EpCAM)/ major histocompatibility complex class II (MHCII)) and for pro-surfactant protein C (proSP-C), followed by FACS analysis for surface antigen expression on AT2 cells. AT2 cells were sorted, and purity was evaluated by immunofluorescence and FACS. This newly developed strategy for AT2 cell isolation was validated in different strains and ages of mice, as well as in a lung injury model. FACS analysis revealed that EpCAM(+) epithelial cells existed in 3 subpopulations based on EpCAM and MHCII expression: EpCAM(med)MHCII(+) cells (Population1:P1), EpCAM(hi)MHCII(-) cells (P2), and EpCAM(low)MHCII(-) cells (P3). proSP-C(+) cells were enriched in P1 cells, and the purity values of the sorted AT2 cells in P1 were 99.0% by immunofluorescence analysis and 98.0% by FACS analysis. P2 cells were mainly composed of ciliated cells and P3 cells were composed of AT1 cells, respectively, based on the gene expression analysis and immunofluorescence. EpCAM and MHCII expression levels were not significantly altered in different strains or ages of mice or following lipopolysaccharide (LPS)-induced lung injury. We successfully classified murine distal lung epithelial cells based on EpCAM and MHCII expression. The discrimination of AT2 cells from non-AT2 epithelial cells resulted in the isolation of pure AT2 cells. Highly pure AT2 cells will provide accurate and deeper insights into the cell-specific mechanisms of alveolar homeostasis.