Isolation and Characterization of Endothelial Colony-Forming Cells

Primary human ECFC were isolated, characterized, and cultivated as previously described by others and our own studies [12 (link),46 (link),47 (link)]. Briefly, maternal peripheral (PBMC) or venous cord blood mononuclear cells (CBMC) were isolated by density gradient centrifugation and PBMC or CBMC were plated (5 × 10⁷ cells/well) onto collagen-coated 6-well plates (BD Bioscience, Heidelberg, Germany) containing endothelial growth medium 2 (EGM-2, Lonza, Basel, Switzerland), supplemented with the supplier’s recommended concentrations of growth factors (10% FBS and 1% penicillin–streptomycin). After 10–21 days of cultivation, ECFC appeared as adherent single layers of cobblestone-shaped, late-outgrowth cells that formed colonies (>50 cells). Flow cytometric analyses to confirm the ECFC phenotype were performed using surface markers CD31, CD133, and CD45, as well as appropriate isotype controls (Supplemental Figure S1).