Venous samples were minced and digested with Liberase 2 (Roche, Basel, Switzerland). For culture selection, 2×105 cells/mL were plated in the presence of Human Medium (HM; complete NeuroCult medium, StemCell Technologies Inc, Vancouver, British Columbia, Canada) that contained basic fibroblast growth factor (10 ng/mL) and epidermal growth factor (20 ng/mL; both from R&D Systems, Minneapolis, Minn) on uncoated wells (n=5).
CD34pos/CD31-negative (CD31neg) cells were isolated from saphenous vein digests either by magnetic bead–assisted cell sorting (MACS, Miltenyi Biotec, Bergisch Gladbach, Germany; n=6) or by fluorescence-activated cell sorting (n 2). Fluorescence-activated cell sorting was performed with a high-speed cell sorter (MoFlo, Beckman Coulter, Fullerton, Colo). Purity of the preparations was assessed by flow cytometry analysis. Differentiation/expansion was performed by seeding CD34posCD31neg cells obtained with each method on fibronectin-coated plates in endothelial growth medium (EGM2, Lonza, Basel, Switzerland) that contained 2% fetal bovine serum (Lonza).
CD34pos/CD31-negative (CD31neg) cells were isolated from saphenous vein digests either by magnetic bead–assisted cell sorting (MACS, Miltenyi Biotec, Bergisch Gladbach, Germany; n=6) or by fluorescence-activated cell sorting (n 2). Fluorescence-activated cell sorting was performed with a high-speed cell sorter (MoFlo, Beckman Coulter, Fullerton, Colo). Purity of the preparations was assessed by flow cytometry analysis. Differentiation/expansion was performed by seeding CD34posCD31neg cells obtained with each method on fibronectin-coated plates in endothelial growth medium (EGM2, Lonza, Basel, Switzerland) that contained 2% fetal bovine serum (Lonza).
