An innovative in vitro model for studying the biology of cardiac fibroblasts originating from the epicardium

Embryonic epicardium is a major source of cardiac fibroblasts (CFs), which play essential roles in heart development and response to heart injury. In this study, we developed a novel mouse model to identify distinct populations of epicardium-derived CFs based on WT1 expression. Our Wt1 ^GFP/+ ;Wt1Cre;ROSA26-tdRFP model enables lineage tracing of WT1Cre-labeled (RFP⁺) fibroblasts and the identification of cells actively expressing WT1 (GFP⁺). Flow cytometry at early postnatal stages showed that RFP⁺ cells form a heterogeneous stromal population, with 20.13% co-expressing GFP, indicating persistent WT1 expression in a subset. We successfully immortalized RFP⁺ cardiac stromal cells, highly enriched in fibroblasts, by excluding other Wt1Cre-active cell types. Through culture condition optimization, we could selectively expand or differentiate specific fibroblast subpopulations, increasing the model’s utility. These immortalized cells, carrying an integrated WT1 reporter system, provide a robust in vitro platform to study fibroblast activation, differentiation, and plasticity under defined conditions.