Definition and Signatures of Lung Fibroblast Populations in Development and Fibrosis in Mice and Men

RATIONALE: Lung fibroblasts have shown high heterogeneity in both mouse and human during lung development and disease states, but no common consensus has been determined. METHODS: In the present study, we performed single cell RNA-sequencing (scRNA-seq) on mouse lung of different stages. We extracted scRNA-seq data of both mouse lung of different stages and normal and IPF lung from published studies. RESULTS: We profiled 7,477 cells from three E17.5 embryonic mouse lungs, and 2,002 mesenchymal cells were readily clustered into six individual fibroblast subpopulations. By checking known fibroblast subpopulation markers, we defined them as lipofibroblasts, myofibroblasts, proliferative fibroblasts, mesothelial cells, Ebf1 positive fibroblasts, and intermediate fibroblasts. Each fibroblast subpopulation showed clear gene expression signature by differential gene expression analysis. As lipofibroblasts emerged in canalicular stage of the developing lung, we defined a large pre-lipofibroblast cluster in both E12.5 and E14.5 lung fibroblasts from scRNA-seq data extracted from two published studies by checking known lipofibroblast specific markers. In addition, we identified a small cluster of myofibroblasts and a small cluster of mesothelial cells in both E12.5 and E14.5 lung fibroblasts and a small Ebf1+ fibroblast cluster in E14.5 lung fibroblasts. Most of these signature genes from each cluster can also be identified from lung scRNA-seq datasets from postnatal 1 day (P1), 7 days (P7) and 15 days (P15). We previously showed a cluster of lipofibroblasts in both normal and bleomycin-induced fibrotic lungs. However, the analysis included a number of immune cells. To correct that, we re-analyzed and combined the scRNA-seq data with two recently published datasets, and we clarified the lipofibroblast cluster in both normal mouse lung fibroblasts and bleomycin day 21 mouse lung fibroblasts. In healthy and IPF lungs, lipofibroblasts, myofibroblasts, and Ebf1 fibroblasts were easily identified, although some signature genes for each cluster were less consistent with the signatures recognized in mice. CONCLUSIONS AND FUTURE DIRECTIONS: By scRNA-seq analysis of lung fibroblasts of different developmental and fibrotic stages, we classified the lipofibroblast, myofibroblast, Ebf1+ and intermediate fibroblast subpopulations and the specific signature genes of each cluster were identified. Prospective isolation of these fibroblast subpopulations, localization of signature gene markers, and lineage-tracing each cluster are under way in the laboratory.