In Vitro Characteristics of Umbilical Cord Blood Derived Mononuclear Cells, CD133+ Cells and Endothelial Generating Cells and Their Effectiveness In Vivo in Mediating Neovascularization in a Murine Model of Hind-Limb Ischemia.

Recent reports have studied the use of various cell populations from bone marrow, peripheral blood and umbilical cord blood (UCB) in mediating therapeutic angiogenesis. We sought to investigate the surface phenotype of UCB derived-mononuclear cells (MNC), CD133+ cells and endothelial generating cells (EGC) using flow cytometry and in vitro functional migration studies. A murine hind-limb injury ischemia model was used to assess in vivo efficacy of the different cell populations. METHODS: Mononuclear cells were isolated by density centrifugation. CD133+ cells were isolated from UCB MNC by magnetic separation (Miltenyi). EGC were derived by adherence of the CD133- cells overnight on fibronectin-coated tissue culture plates in EGM2 media (Clonetics). Surface phenotype was determined by flow cytometry for stem cell markers, CD133 and CD34, stromal markers CD73 and CD105, KDR (VEGFR2), and the receptor for SDF-1, CXCR4. Modified Boyden chambers (Neuroprobe) were used to observe chemotactic migration of MNC, CD133, EGC or the combination of CD133 and EGC towards SDF-1 (100ng/mL) compared to control wells containing media alone. In the in vivo studies, 40 NOD.SCID mice underwent right femoral artery ligation. Mice were randomized into five study groups: Cytokines (EGM2 media, n=5), MNC (n=5), CD133 (n=5), EGC (n=7), or CD133 and EGC (1:2 ratio, n=4). Laser Doppler blood flow measurements were recorded weekly for four weeks and the ratio of ischemic/non-ischemic leg was calculated. At day 28, tissue samples were harvested for histological studies. RESULTS: Surface phenotype by flow cytometry showed an average purity of 78.67% +/− 2.41% for the selection of CD133. In addition, an enhancement of the expression of CXCR4 was seen in the EGC population after overnight exposure to fibronectin and EGM2 media. Surface Phenotype of UCB MNC, CD133 and EGC | | MNC | CD133 | EGC | |:----- | ----- | ----- | ----- | | CD34 | 4.03 | 87.42 | 3.40 | | CD133 | 3.19 | 78.67 | 5.06 | | CD73 | 5.07 | N/A | 7.15 | | CD105 | 6.74 | 22.74 | 33.78 | | KDR | 7.14 | 3.59 | 37.65 | | CXCR4 | 28.54 | 8.66 | 64.57 | The in vitro functional migration assays showed increased migration of MNC, EGC, and CD133 with EGC to SDF-1, where the CD133 cells alone showed no increased migration compared to control media. In the in vivo murine model of hind-limb ischemia, the blood flow ratio of ischemic/non-ischemic limb was used to estimate the rate of blood flow recovery. The rates of blood flow recovery were 0.0198 (CD133), 0.02 (CD133 and EGC), 0.0163 (MNC), 0.016 (EGC) and 0.0123 (cytokines). The rates were significantly different between CD133 and cytokines (p=0.011) and between CD133 and EGC compared to cytokines (p=0.011). The difference between MNC and cytokines (p=0.156) and between EGC and cytokines (p=0.176) was not significant. Histological studies are ongoing. CONCLUSION: Surface phenotype of UCB-derived MNC, CD133, and EGC were compared by flow cytometry. The in vitro functional chemotactic capacity toward SDF-1 of these cell types was determined by migration assays. The infusion of CD133+ or the combination of CD133 and EGC cells augmented the rate of blood flow recovery in the in vivo murine hind-limb model of ischemia compared to the crude MNC prep or the ECG alone.