Abstract 3423: Cultured Human Bone Marrow CD31+ Cells Have Higher Angio-vasculogenic Properties Than Uncultured Cells And Showed Endothelial Cell Differentiation Capacity In Hindlimb Ischemia
We previously reported that mouse bone marrow (BM) and human peripheral blood derived CD31+ cells have hemangiogenic activities. The aims of the study were first to determine whether culture of human BM CD31+ cells hold higher angio-vasculogenic activities than uncultured CD31+ cells (CD31-U) and second to investigate whether these cells have transdifferentiation potential into endothelial cells in vivo for a long-term period. CD31+ cells were isolated and cultured in 3 different conditions using various cytokines and mediums mimicking EPC (E), MSC (M) and HSC (H) (E: VEGF-A, HGF, bFGF, IGF; M: 20% serum; H: SCF, Tpo, Flt-3L) for 10 days. FACS analysis showed that CD31+ cells in E (CD31-E) maintained vascular gene expression such as CD31 (92.3%), KDR (42.3%), and Tie-2 (45.6%). In the Matrigel tube formation assay, CD31-E has higher tube length than other conditions. Real-time PCR showed that CD31-E express higher levels of angiogenic genes (fold increase: angiopoietin-2, 7.8±2.9; HGF, 4.4±1.6; PDGF, 17.2±4.3; PlGF, 2.8±0.5; MMP-9, 46.4±5.7) than CD31-U. However, CD31-E express lower level of the inflammatory genes (fold decrease: Interferonγ, 6.7±1.4; IL-1; 5.1±1.3; IL-6, 2.7±0.6) than CD31-U. To investigate therapeutic potential, we surgically induced hindlimb ischemia in athymic nude mice, and injected CD31-E, M, H, U and PBS into the ischemic limbs (n=8, each). The CD31-E group showed a higher limb salvage rate than other groups [total salvage/tip necrosis/amputation; E 5/3/0, H 3/4/1, M 2/5/1, U 3/3/2, PBS 0/3/5]. Laser Doppler imaging revealed markedly increased blood perfusion at 14 days in CD31-E injected limbs, compared to CD31-U (0.55±0.08 vs 0.42±0.05, P<0.05). Capillary densities were increased in CD31-E (2.1 fold, P<0.05) compared to CD31-U. Histologic analysis demonstrated that a fraction of injected CD31-E exhibit endothelial phenotypes during the follow-up period of 6~12 months. The present study demonstrated that CD31+ cells cultured in defined media containing angiogenic cytokines have higher angio-vasculogenic potential and exerted better post-ischemia recovery. These data suggested that cultured CD31+ cell are a promising source for treating ischemic cardiovascular diseases.