Plating efficiency of CD34⁺ cells to form endothelial cell colonies.

A highly pure population of CD34⁺ cells was isolated from human FL cells and maintained in VEGF (10 ng/mL) and FGF-2 (2 ng/mL). VEGFR-2⁺ cells were isolated using Biotin-labeled anti-VEGFR-2 MoAb and Streptavidin Dynal magnetic beads. Isolated cells were incubated in EC-specific media (containing FGF-2⁺ and VEGF) in limiting dilutions approximating 1 single cell per well, and after 10-14 days endothelial colonies were quantified using Dil-Ac-LDL labeling. Typical Dil-Ac-LDL^± endothelial cells (A) within an adherent endothelial colony (A,B; phase contrast microscopy 350 × ) are shown. Maintaining of the endothelial colonies in the FGF-2 and VEGF resulted in the proliferation of characteristic endothelial cobblestone colonies (D), where most of cells were Dil-Ac-LDL⁺ (C,D; 200 × ).