Cells are collected and cultured from peripheral venous blood of patients. Using FACS analysis, endothelial progenitor cells (EPCs) were characterized as CD34+/KDR+/CD45+(low). EPCs were additionally characterized by DiI-Ac-LDL uptake, a colony-forming units (CFU) assay, and an in-vitro tube formation assay.
Mononuclear cells (MNCs) were separated from the blood sample using Ficoll density gradient centrifugation. After isolation, MNCs were washed twice with Medium 199 (M199) and re-suspended in growth medium supplemented with M199 and fetal calf serum (FBS, 20%), VEGF (10 ng/ml), FGF-2(2 ng/ml), penicillin (100 U/milliliter), and streptomycin (100 μg/milliliter). MNCs were then plated on human fibronectin-coated plates. In order to avoid contamination with mature endothelial cells, the nonadherent cells were collected 48 h thereafter and replated onto fibronectin-coated 24-well plates for a final assessment of the number of colonies. Growth medium was changed every 3 days.