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. Our Wt1GFP/+;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, which are 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 utility of the model. 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.