Abstract
Recent reports have studied the use of cell populations from bone marrow, peripheral blood and umbilical cord blood (UCB) in mediating therapeutic angiogenesis in patients with coronary artery disease. We investigated the surface phenotype of UCB derived-mononuclear cells (MNC), and endothelial generating cells (EGC) by flow cytometry and in vitro functional migration studies. We used a murine hind-limb injury ischemia model to assess in vivo efficacy of the cell populations.
METHODS: MNC were isolated by density centrifugation and CD133+ cells were isolated by magnetic separation (Miltenyi). EGC were derived by adherence of CD133− cells cultured 16h on fibronectin-coated tissue culture plates in EGM2 media (Clonetics). Cell characterization included surface phenotype determined by flow cytometry for monocyte markers CD14 and CD11b, stromal markers CD73 and CD105, KDR (VEGFR2), and the receptor for SDF-1, CXCR4. Transwell plates with 5μm collagen coated filters (Costar) were used to observe chemotactic migration of MNC or EGC towards SDF-1 (100ng/mL). Following a 3 hour incubation, the cells migrated to the bottom wells was counted by flow cytometry with TruCOUNT™ tubes (BD Biosciences. NOD/SCID mice underwent right femoral artery ligation and were injected with cytokines (EGM2 media, n=10), MNC (n=7) or EGC (n=8), 1X 106 cells/mouse. Laser Doppler blood flow measurements were recorded weekly for four weeks.
RESULTS: Enhanced expression of CXCR4, CD105, KDR, CD14 and CD11b was found in the EGC cells generated after 16h culture on fibronectin.
Surface Phenotype of UCB MNC and EGC CD14 CD11b CD73 CD105 KDR CXCR4 MNC (n≥5) 8.1 ± 2.7 22.7 ± 6.6 5.1 ± 2.2 6.7 ± 2.1 7.1 ± 2.1 28.5 ± 5.8 EGC (n=5) 67.7 ± 9.5 80.0 ± 4.8 7.1 ± 4.0 33.8 ± 5.8 37.6 ± 6.4 64.6 ± 7.8
Functional assays showed increased migration of both MNC and EGC to SDF-1 compared to control media (4.9 ± 2.9, n=2 and 3.2 ± 0.6, n=3 fold increases respectively). With VEGF as a chemoattractant MNC exhibited a 1.5 fold increase over the negative control (n=1) and EGC showed a 1.4 ± 0.3 fold increase (n=2). In the murine hind-limb ischemia model the ratio of ischemic/non-ischemic limb blood flow was used to compare vasculogenic potential. There was improvement of blood flow 14 days after injection of the EGC cells (p=0.019). On days 21 and 28, blood flow ratio was higher than control but was not statistically significant (p=0.06). The difference between MNC and EGC was not significant at any time point (p> 0.05). Histological studies are ongoing.
CONCLUSION: In summary, UCB derived EGC exhibited monocyte and stromal surface antigen expression, migrated to an SDF-1 gradient, and mediated improved vascular blood flow. Ongoing studies are focused on direct cell vs. paracrine effects underlying observed neovasculogenesis mediated by EGC.
Activation antigen expression on human T cells. I. Analysis by two-colour flow cytometry of umbilical cord blood, adult blood and lymphoid tissue