Abstract 903: Human Cardiac Progenitor Cells
Samples of human atrial and ventricular myocardium were collected at surgery and cultured by the primary explant technique. Outgrowth of spindle-shaped cells from the myocardial samples was apparent at ~4 days after seeding and, at 3 weeks, clusters of ~5,000–7,000 cells were obtained. At P0, cells positive for the stem cell antigens c-kit, MDR1, and Sca-1-like were identified. These cells constituted 2%, 0.5%, and 1 % of the entire myocardial cell population, respectively. Subsequently, c-kitPOS cells were sorted and following their expansion by serial passages from P1 to P8 were characterized by FACS analysis. C-kitPOS cells were negative for markers of hematopoietic progenitor cells including CD34, CD45 and CD133, and a panel of lineage-specific hematopoietic cell epitopes. Additionally, only a small fraction of the c-kitPOS cells expressed cytoplasmic or membrane proteins of myocytes, smooth muscle cells, and endothelial cells. Single cell clones were obtained and c-kitPOS clonogenic cells in differentiating medium acquired the myocyte, smooth muscle cells and endothelial cell lineages. The expression of human mRNA for c-kit, Nkx2.5, myosin light chain 2v, connexin 43, smooth muscle myosin heavy chain, and von Willebrand factor was detected by real time RT-PCR. Differentiating human myocytes alone or infected with EGFP and then co-cultured with neonatal rat myocytes contracted in response to electrical stimulation and showed calcium transient. C-kitPOS cells in culture continued to grow up to P8 undergoing ~25 population doublings. From P1 to P8, a 70% average of the cells was c-kitPOS and a 50% average of the cells was cycling. Telomeric length decreased slightly from P4 to P9 without reaching a critical shortening as documented by the lack of increase in the fraction of p16INK4a-positive cells at the late passages. These observations indicate that the vast majority of the cells retained a young phenotype during in vitro expansion. At P12, however, the percentage of p16INK4a-positive cells increased markedly together with shortening of telomeres. In conclusion, clonogenic derived c-kitPOS cardiac stem cells can be well characterized and expanded in vitro before their autologous administration to patients with chronic heart failure.