Fig. 7: Lineage tracing of cell fate for endogenous cardiac fibroblasts following induction via IL-β administration.

Lineage tracing of Pdgfrα+ cardiac fibroblasts was performed to confirm that these explicit cells become CD4-expressing cells in association with IL β1 induction. a Generation of the lineage tracing mouse model in which tamoxifen-dependent Cre recombinase is expressed under the influence of a Pdgfra promoter. Combined with a Cre-dependent tdTomato (red fluorescence) expressing reporter, the allele permanently labels all resident cardiac fibroblasts. b Experimental scheme in which PdgfraCreERT2/+; Rosa26tdT/tdT mice are given tamoxifen chow for two weeks prior to harvesting to tag cardiac fibroblasts in vivo. c Flow cytometric sorting of tdTomato labeled cardiac fibroblasts. Bracket shows the tdTomato positive population that is sorted and cultured. d Live red fluorescence imaging of cultured fibroblasts. Throughout all of the steps of the culturing protocol, tdTomato signal has been maintained due to the permanent recombination of the reporter allele. (Scale bar is 100 mm). e Flow cytometry analysis showing CD45⁺ and CD4⁺ phenotype acquisition of tdTomato+ fibroblasts following IL1β treatment. f Flow cytometry analysis demonstrating that originally Pdgfra+ cardiac fibroblasts do not maintain their fibroblast markers upon transitioning to immune phenotype. g Normalized histogram analysis of Pdgfra positivity among tdTomato⁺CD45⁺ cells. h Normalized histogram analysis of CD4 positivity from flow cytometry among tdTomato⁺ cells. i Quantification derived from flow cytometry of both mouse and human fibroblasts that acquired CD4 phenotype, shown as a percentage of CD4 cells/total cells sampled. Each experiment has a biological n = 3 and technical replicates of n = 3.