ISOLATION, CHARACTERIZATION AND DIFFERENTIATION POTENTIAL OF CARDIAC PROGENITOR CELLS IN ADULT PIGS

Cardiac progenitor cells have been recently identified in the post-natal heart. However, the experiments carried out so far were predominantly performed on mice and humans. Considering the potential future application of these cells for human cell therapy, we propose the pig as an intermediate preclinical model, due to the morphological and functional affinity of the porcine species with the human. Pig adult cardiac progenitors were isolated from explants of adult pig hearts and cultured in complete DMEM medium. Cells were analyzed for their expansion ability and for the expression of surface markers. All lines were positive for CD44 and CD34. Only a small percentage of cells expressed CD31, while screening for CD45 gave a negative result in all the lines obtained, regardless to their origin. Molecular characterization was performed using primers designed for pluripotency, mesenchymal and cardiac related genes. These cells were shown to actively transcribe for c-kit, Oct4, Gata6, CD31, CD34, CD44, Mesp1, Mesp2, Mef2a, Nkx2.5, ANP, Cx43, Cardiac Actinin, Tbx5 and Tbx18. Staining with Desmin, Connexin43, MF20, Rhodamine Phalloidin, α-Tropomyosin and Smooth Muscle Actin antibodies demonstrated that adult pig cardiac progenitors could differentiate into cardiomyocytes and smooth muscle cells, when exposed to the appropriate inducing media. Fusion experiments revealed that pig cardiac progenitor cells have the ability to fuse with fetal rat cardiomyocytes, reflecting a distinct property common to many types of cardiac progenitors. The data obtained indicate that adult porcine cardiac progenitor cells can be isolated and cultured in vitro. These cells display a high molecular affinity and share many differentiation properties with a cardiac progenitor cell population recently described in weanling pigs. This suggests that cardiac progenitors, although more abundant and functional in the neonatal and post weaning period, are present and active in adult porcine hearts and may represent an interesting tool for preclinical and translational studies of stem cell-based cardiovascular therapy.