Abstract 1745: Highly Effective Restoration of Blood Flow in ApoE−/− Mice Ischemic Hindlimbs from CD34+/M-cadherin+ Adult Bone Marrow Cell Population
Injection of adult bone marrow cells in animal models of ischemia promotes angiogenesis, but the specific precursors mediating these potential benefits have not been well defined. Currently we focus on identifying and characterizing a subpopulation of cells that possess high efficacy and capacity in alleviating hindlimb ischemia. The proximal femoral artery of ApoE −/− mice was occluded by 2 adjacent ligatures followed by intrafemoral injection of 25 x 106 GFP+ unfractionated bone marrow mononuclear cells (BMMNC). 7 days after GFP+ BMMNC delivery, immunostaining was performed on leg cross-sections to detect myogenic and endothelial markers expressed on delivered cells. Subsequently, by multi-parameter flow cytometry, the selected panel of myo-endothelial markers was used to verify the presence and co-expression of freshly isolated BMMNC. Further characterization and isolation of BMMNC was carried out by flow analysis and cell sorting. 3 x 105 of each group of sorted cells was injected into ApoE −/− ischemic hindlimbs. Hindlimb blood flow of ischemic and normal limbs was monitored every 7 days post-surgery up to 60 days by Laser Doppler imager. No immunosuppressant manipulations were performed. The engraftment and distribution of CD34+ and M-cadherin+ GFP+ cells in ischemic hindlimbs was observed by immunohistochemistry. Through multi-parameter flow analyses of BMMNC, we illustrated a rare population of CD34+ /Mcad+ cells (0.02% ± 0.003%). Additionally this specific cell subset was enriched with CXCR4 (85.68% ± 12.65%), VEGFR2 (63.17% ± 8.13%), Sca-1 (70.06% ± 9.65%) and Tie-2 (59.32% ± 8.44%). Laser Doppler analysis revealed that 3 x 105 CD34+/Mcad+ cells had significant blood flow recovery on ischemic hindlimbs as early as 14 days compared to CD34+ (74.82% ± 11.62% vs. 55.4% ± 13.03, n = 5, p < 0.05), and to Mcad+ (74.82% ± 11.62% vs. 35.84% ± 7.16, n= 5, p < 0.001). We demonstrated that a distinctively rare cell population enriched with key myoendothelial homing properties is effective in restoration of blood flow in mouse hindlimb ischemia. Importantly, the specific cell markers used to isolate this promising cell population are present on adult human bone marrow cells and bear significant clinical potential.