Mononuclear cells (MNCs) isolated from peripheral blood of patients contained a heterogeneous population of progenitor cells. More than 90% of the cells exhibit endothelial progenitor cell (EPC) characteristics, manifested by dil-acetylated low-density lipoprotein uptake and lectin (UEA-1) binding. They were further documented by showing the expression of vascular epithelium-cadherin, kinase-insert domain-containing receptor, CD34 and AC133 by flow cytometry.
In patients receiving cell infusion, 250 ml of venous blood was collected immediately after random assignment. Mononuclear cells (MNCs) were isolated from peripheral blood of patients, by Ficoll density gradient centrifugation and cultured on fibronectin-coated dishes in Medium 199 supplemented with human serum (20%) drawn from each individual patient, and VEGF (50 ng/ml). After 5 days in culture, cells were detached with ethylenediaminetetraacetic acid (0.5 mmol/l), washed 3 times with heparinized saline before final resuspension in 10 ml heparinized saline. Filtration was carried out to prevent cell clotting and microembolization.