ABSTRACT While much progress has been made in understanding early cardiac development, the precise mechanisms that specify the different cardiomyocyte subtypes remain poorly understood. Recent data from our lab have shown that transient Foxa2 expression identifies a progenitor population with exclusive ventricular differentiation potential in the mouse heart. Here we have translated this concept to the human pluripotent stem cell (hPSC) system. Using a FOXA2-GFP reporter cell line we characterized expression of FOXA2 during hPSC cardiac differentiation and found that a subset of cardiac mesoderm precursors transiently expresses FOXA2 . Gene expression analysis of FOXA2+ and FOXA2- cardiac mesoderm revealed that both populations similarly express early cardiac specification markers such as PDGFRA, TBX5 , and ISL1 , while other key candidates including TBX20 and GATA4 are significantly upregulated in the FOXA2+ population. Isolation and subsequent differentiation of FOXA2+ and FOXA2- populations demonstrates their comparable differentiation potential to both cardiomyocytes and epicardial cells. However, cardiomyocytes derived from FOXA2+ precursors showed enhanced differentiation efficiency toward ventricular cardiomyocytes compared to cardiomyocytes derived from FOXA2- precursors. To identify new mechanisms that regulate ventricular specification, we performed small molecule screening and found that inhibition of the EGFR pathway strongly increased the cardiac mesoderm population in general, and the FOXA2+ precursors in particular. Finally, we have identified a combination of cell surface markers to specifically isolate FOXA2+ cardiac precursors. In summary, our results suggest that FOXA2+ cardiac mesoderm harbors ventricular-specific differentiation potential and isolation of these cells permits the generation of cultures enriched for ventricular cardiomyocytes. Generating such enriched cardiac populations will be relevant for regenerative medicine approaches, as well as for disease modeling from induced pluripotent stem cells.

Identification and characterization of hPSC-derived FOXA2+ progenitor cells with ventricular cardiac differentiation potential