Ex vivo expansion of endothelial cells is important when applying cell therapy to therapeutic angiogenesis in ischemic tissues. Endothelial precursor cells (EPCs) from the umbilical cord blood are one of adult stem cell. In order to establish the culture system for EPCs, we examined the effects of the media and matrix on the differentiation of a subset of mononuclear cells to endothelial cells, and analyzed their endothelial-lineage phenotype.

Mononuclear cells isolated from human umbilical cord blood were cultured in a chamber slide coated with fibronectin or gelatin in a M199 medium supplemented with 10% fetal bovine serum (FBS) (the normal medium) or with 20% FBS and ECGS (the rich medium). Changes in the morphology and the attainment of DiI-ac-LDL uptake ability were examined during a 7 day period. The attached cells were immunostained for CD31, KDR, and vWF.

The fibronectin matrix gave rise to more attached cells than the gelatin matrix (about 1.5 fold). The numbers of attached cells were no different between the normal medium and the rich medium at day 3 and 7, and were about 12% of the seeded mononuclear cells. However, the cell size and the numbers of longer spindle-shaped cells increased with the rich medium. Moreover, there was no increase in cellular population, but a 2-3 fold increase in the cellular size between day 3 and 7. About 20-40% of the attached cells acquired the DiI-ac-LDL uptake ability at day 3, whereas more than 85% of the attached cells could be stained with fluorescent DiI-ac-LDL at day 7 (p<0.001). The attached cells after being cultured for 7 days were stained moderately with the antibodies of CD31, or KDR. However, the cells at day 7 were only weakly immunostained with the vWF antibody, whereas more than 90% of cells were strongly stained at day 14.

These results suggest that a subset of mononuclear cells derived from cord blood cells can give rise to cells with an endothelial cell-like phenotype, in vitro, at high percentages, which could be applied to in vivo vasculogenesis.