Abstract 730: Identification and Characterization of Cardiac Stem Cells in the Pig Heart
The mammalian heart possesses a population of cardiac stem cells (CSCs) that form myocytes and coronary vessels. This recent discovery raises the possibility of using CSCs for cardiac regeneration if they could be isolated and expanded in vitro for subsequent therapeutic use. To achieve this goal, CSCs need to be collected and propagated from fragments of myocardium for their subsequent implantation within the same heart. We addressed these issues in the pig because this model is commonly utilized in preclinical studies. Primitive cells expressing the stem cell antigens c-kit, MDR1 and Sca-1-like were identified within the myocardium. Approximately 50% of cells positive for c-kit, MDR1, or Sca-1-like exhibited markers of cell commitment such as the cardiac transcription factor GATA4 and contractile and non-contractile proteins typical of cardiomyocytes, endothelial cells (ECs), and smooth muscle cells (SMCs). These characteristics were consistent with the multipotentiality of CSCs. Next, c-kit, MDR1, and Sca-1-like positive cells were separated and collected by dissociation of atrial samples from pigs undergoing surgery. Lineage negative (Lin−) CSCs did not express transcription factors and cytoplasmic proteins specific of cardiac cells and were negative for makers of hematopoietic SCs and hematopoietic cell lineages. There was one Lin-CSC/30,000 cardiac cells. For clonal analysis, c-kitPOS-CSCs were sorted and individually placed in a 96-well Terasaki plate. After 2–3 weeks, c-kitPOS-clones were identified; clonal efficiency was 0.4%. Clonogenic c-kitPOS-CSCs in culture continued to grow up to P9 undergoing 22 population doublings. Nearly 4,00,000 cells were derived from a single c-kitPOS-CSC. Average population doubling time was 47 hours. Clonogenic cells in differentiating medium acquired the phenotype of myocyte, EC and SMC lineages. These data indicate that c-kitPOS-CSCs possess the fundamental properties of stem cells: they are self-renewing, clonogenic and multipotent. In conclusion, CSCs can be successfully obtained from atrial samples of the pig heart and expanded in vitro to an extent that may have therapeutic applicability for autologous administration in vivo.