Functional Pdgfra fibroblast heterogeneity in normal and fibrotic mouse lung

Using, scRNA Seq transcriptomic analysis, we report that Pdgra+ fibroblasts in mouse lung have significant heterogenity in normal and fibrotic lung, and key pathways involved in lipogenic and fibrogenic pathways govern the transition of normal lipofibroblasts from a normal to a myofibroblast phenotype following lung injury Pdgfra-GFP reporter mice were dosed with either 2 U/Kg bleomycin or PBS. On day 14 post-exposure, lungs (3 mice/treatment) were harvested, cells enzymatically digested, and GFP+ cells sorted using FACS to prepare live cells for scRNA Seq. Sequencing was done using the 10X Genomics Chromium Platform, and following processing with Cell Ranger v3.0.1 was done to generate an initial cell by gene count matrix. Samples were combined in Seurat v3 and clustered into 11 unique clusters based on top gene marker expression. Data was analyzed using Ingenuity Pathway Analysis, Pseudotime trajectories prepared with SlingShot, and IF staining conducted for verification. For repetive bleomyin exposure, Pdgfra-GFP mice were dosed with bleomycin or PBS once weekly for 3 weeks (bleomycin at 1, 1, and 0.5 U/Kg). Two weeks after the last exposure, lungs (3 mice/treatment) were harvested, cells enzymatically digested, and GFP+ cells sorted using FACS. Sequencing was done using the 10X Genomics Chromium Platform, and following processing with Cell Ranger v3.0.1 was done to generate an initial cell by gene count matrix. Samples were combined in Seurat v3 and clustered into 11 unique clusters based on top gene marker expression. Data was analyzed using Ingenuity Pathway Analysis, Pseudotime trajectories prepared with SlingShot, and IF staining conducted for verification.