Abstract 15911: Reduced Senescence and Enhanced Proliferation of Human Cardiac Progenitor Cells Isolated and Expanded Under Reduced Oxygen Tension

Abstract

Background: Oxygen availability at the cellular level in vivo is significantly lower (~1-7%) than that used for typical ambient cell culture conditions (21%). Here we investigated whether prolonged culture at reduced O2 concentrations affected proliferation, senescence and oxidative stress of human cardiac progenitor cells (hCPCs). Methods: hCPCs positive for c-kit and negative for lineage markers (ckit+/Lin-) were isolated from right atrial tissues obtained from five infants during repair of congenital heart defects and expanded for a maximum of 10 passages in varying O2 concentrations (1, 5, and 21%); all manipulations were performed at the target O2 in a hypoxic chamber. Cellular phenotype was confirmed by ICC staining and flow cytometry. Doubling time, oxidative stress (8-OH-deoxyguanosine [8OHdG], protein carbonyl formation) and senescence markers (telomere length, telomerase activity, P16ink4a staining) were measured. Results: Reducing ambient O2 from 21% to 1% did not alter cell surface marker expression. Culture and expansion at 21% O2 markedly accelerated hCPC senescence compared to 1% or 5% O2, as indicated by increased P16ink4a positive hCPCs and greater loss of telomere length and telomerase activity; much of this damage appeared to occur during early passage and expansion. Both protein carbonyl and 8OHdG formation progressively increased in 21% O2, whereas these oxidative injury markers showed little change at 1 and 5% O2 concentrations. hCPCs that were cultured at either 5% or 1% O2 demonstrated shorter doubling times with resultant higher cell yields during in vitro expansion. Conclusion: Culturing ckit+/Lin- hCPCs at lower oxygen tension minimizes oxidative damage, reduces senescence, and enhances proliferative potential during long-term culture; expansion at 1% ambient O2 appeared to be most effective. This relatively straightforward modification may further understanding of the biology of CPCs and their regenerative potential.