Abstract 1145: Functional Heterogeneity of Angiopoietin-1, -2 on Endothelial Progenitor Cell Fate, Assessed by Single Cell culture of Cord Blood CD133+cells for Endothelial Lineage Commitment Assay
Background; Angiopoietin-1, -2 (Ang-1, Ang-2) has been demonstrated to play a pivotal role in angio-vasculogenesis, i.e. Ang-1 for vascular maturation or Ang-2 for vascular sprouting. However, the developmental mechanism of endothelial progenitor cell (EPC) fate oriented by angiopoietins for vasculogenesis remains to be elucidated. Here, we report the assessment of primitive EPC fate determined by Ang-1,-2, using in vitro endothelial lineage commitment assay of single cord blood (CB)-CD133+ cells. Methods; Single CB-CD133+cells isolated by MACS were cultured in a suspension manner for 7 days in liquid culture containing TPO, SCF, IL-6, Flt-3 ligand, and VEGF with either of Ang-1 or Ang-2 at 200 ng/ml each. The cultured cells from single CB-CD133+cells were applied to vasculogenic methylcellulose culture for EPC colony forming assay (EPC-CFA). Results; EPC-CFA disclosed the analytic potential on the degree of differentiation and commitment of single CB-CD133+cells by the assessment of two types of EPC colonies with the individual cellular sizes, i.e. primitive CFU-small and/or definitive CFU-large EPCs produced by single CB-CD133+cell. Whole EPC colony producing potential was upregulated by both Ang-1 and Ang-2 (ratio of total CFU-EPC number; 1.49 fold in Ang-1, 1.39 fold in Ang-2 vs control, p < 0.05). Both the commitment and differentiation frequencies of EPC from CB-CD133+ cell were promoted by Ang-1, inversely the latter inhibited by Ang-2 (commitment= frequency of single CB-CD133+cell producing any CFU-EPC; 1.5 fold in Ang-1: differentiation= frequency ratio of single CB-CD133+cell producing definitive CFU-EPC/single CB-CD133+cell producing any CFU-EPC; 2 fold in Ang-1, 0.27 fold in Ang-2 vs control, p < 0.01). On the other hand, the expansion potential of primitive CFU-EPC from single CB-CD133+ cell was augmented by Ang-2, although downregulated by Ang-1 (expansion= number of primitive CFU-EPC/ single CB-CD133+cell producing any CFU-EPC; 0.78 fold in Ang-1, 1.6 fold in Ang-2 vs control, p< 0.05). Conclusion; A novel in vitro colony assay system to survey EPC fate of single CB-CD133+cells precisely provides the evidences of angiopoietin-1 mechanism for differentiation into definitive EPC and angiopoietin-2 for procurance of primitive.