Porcine cardiac mesangioblast isolation, expansion, characterization and differentiation ability

Different cardiac stem/progenitor cells have been recently identified in the post-natal heart. In particular cardiac mesoangioblasts are self-renewing progenitors with high spontaneous cardiac differentiation that can be expanded in vitro for systemic delivery, and, upon transplantation, they regenerate the infarcted myocardium. The information available are confined to the mouse and heavily limited by the enormous diversities between the mouse and the human. Here we propose the use of the pig as a model and describe experiments aiming for isolation, clonal expansion and characterization of pig mesoangioblasts. Adult cardiac stem cells were derived from aorta, ventricle and atrium of porcine hearts. Explants were cut into small pieces and plated on 0.1% gelatin. Within 7 days, putative mesoangioblasts started coming out from the explants. These cells were collected, purified and grown in DMEM 10% FBS. They were then analyzed for their expansion ability, monitoring their growth for 21 days. They were sorted, based on mesoangioblast related surface markers by flow cytometry and scored for stem cell and cardiac specific gene expression by PCR analysis. Their differentiation ability in response to adequate culture conditions, was assessed by immunocytochemistry/ confocal analysis, using antibodies raised against connexin-43, alfa-actin and alfa-tropomiosin. The results obtained indicate that pig cardiac mesoangioblasts can be isolated, these cells express many markers, common to the mouse and human. They can efficiently differentiate into cardiomyocites, skeletal and smooth muscle, although with different efficiency. Altogether the studies with these cells will allow us to propose the use of cardiac mesoangioblasts for a human stem cell-based theraphy.