In Vitro Characterization of Umbilical Cord Blood CD133+ and In Vivo Functionality in Response to Ischemia in a Murine Hind-Limb Injury Model.

Previous reports have demonstrated efficacy of cellular therapy in mediating therapeutic angiogenesis in response to ischemia. We sought to determine the potential efficacy of adult umbilical cord blood (UCB) derived selected CD133⁺ cells in the murine hind limb ischemia model and to characterize these cells by surface phenotype and functionality prior to injection.

Methods and Results: Mononuclear cells (MNC) from UCB were labeled with CD133⁺ conjugated magnetic beads, followed by automated sorting through magnetic columns (Miltenyi). Routine yield of CD133⁺ cells was 0.51 ± 0.2% of MNC, with a purity of 78.7 ± 2.4% (n=30). Surface expression in the UCB CD133⁺ population was 3.59 ± 1.49% KDR(VEGFR2), 8.66 ± 3.79% CXCR4 and 22.74 ± 2.84% CD105 compared to 7.14 ± 2.15% KDR, 28.54 ± 5.81% CXCR4 and 6.74 ± 2.07% CD105 in the UCB MNC population. Transwell plates with 5μm collagen coated filters (Costar) were used to observe chemotactic migration of MNC or CD133⁺ cells towards SDF-1 (100ng/mL) compared to control wells containing media alone. Following a 3 hour incubation, the cells migrating to the bottom wells were counted by flow cytometry with TruCOUNT™ tubes (BD Biosciences). MNC and CD133⁺ cells migration to SDF showed a 4.9 ± 2.9 and 1.8 ± 0.7 fold increase over the negative control respectively. To test vasculogenic functionality of these selected cell populations, NOD/SCID mice underwent ligation of the right femoral artery and were randomized into 3 study groups: control (endothelial media with cytokines), non-selected MNC (1 x 10⁶ cells/mouse) or CD133⁺ (0.5 x 10⁶ cells/mouse) given via intracardiac injection immediately after injury. Doppler flow measurements were taken on both limbs each week for 4 weeks and the ratio of perfusion in the ischemic/healthy limb was calculated. At 28 days, perfusion ratios were statistically higher in study groups receiving CD133⁺ cells from UCB, 0.55 ± 0.07 (n=8) compared to cytokine controls 0.39 ± 0.02 (n=10, p=0.019). Mice receiving MNC did not show statistically significant improvement over control animals 0.42 ± 0.06 (n=7, p=0.27).

Conclusion: Surface phenotyping was notable for increased expression of the receptor for SDF-1, CXCR4 on MNC when compared to CD133⁺ cells. In vitro functional assays showed that CD133+ and MNC exhibited increased chemotactic migration to SDF-1. In vivo studies showed that injection of UCB CD133⁺ cells improved blood flow compared with cytokines alone in the murine hind limb injury model, highlighting the vasculogenic potential of CD133⁺ cells from UCB.