Abstract 9617: Real Angiogenic Potential of Early or Late Outgrowth Endothelial Progenitor Cells is Rigorously Dependent on the Time of Emergence

Abstract

Background: Recent studies have suggested that late outgrowth endothelial progenitor cells (EPCs) emerging from cultured peripheral blood mononuclear cells (PBMNCs) might have higher angiogenic potential than that of classically defined early outgrowth EPCs (EOCs). However, it still remains undetermined which of EPC subpopulations rigorously defined by the time of emergence has the highest therapeutic angiogenic potency. Methods and Results: Human PBMNCs freshly isolated were cultured under the endothelial cell conditions. EOCs were defined as the cells attached on culture plates at days 3 to 7, and late outgrowth EPCs were defined as the cells forming clusters with cobble stone appearance at days 10 to 30. In addition, late outgrowth EPCs were newly defined according to the dates of colony emergence as follows: MOC at days 10-16, LOC at days 17-23 and VOC at days 24-30. Flow cytometry analyses revealed that the expression patterns of cell surface antigens relating to hematopoietic/endothelial lineages varied among EPC subpopulations, e.g., EOC: CD31 + CD34 + CD14 + CD105 low and LOC: CD31 + CD34 + CD14 - CD105 high . Using in vitro assays, we found that LOCs had the highest proliferation and tube formation potentials along with the highest expression of eNOS. Then, each EPC subpopulation was intravenously injected into immunocompromised mice at days 1, 3, 5 and 7 after unilateral hindlimb ischemia surgery (each 1×10 5 cells per time). Four weeks after surgery, the mice in LOC-injected group showed significantly enhanced blood flow recovery (blood flow ratios of ischemic/non ischemic leg: 1.02±0.02 [LOC group] versus 0.60±0.16 to 0.85±0.14 [other groups], P <0.05) and augmented capillary collateral formation in ischemic leg. To evaluate how much percentage of CD31 + endothelial cells (ECs) of generated capillaries were derived from the injected EPCs, the GFP-labeled EPCs were injected into the mice with hindlimb ischemia. The proportions of human EPC-derived ECs among a total of CD31 + ECs in ischemic muscle were 14.3±2.8% in LOC group and 4.0±1.8% to 9.2±2.3% in other groups ( P <0.05). Conclusions: Late outgrowth EPCs emerging at days 17-23 in ex vivo culture of human PBMNCs are superior to other EPC subpopulations as to the therapeutic angiogenic potency.