Characterization of EPCs expressing KDR, vascular endothelial cadherin (VE-cadherin), von Willebrand factor (VWF), and CD-133. Colonies and outgrowing cells were characterized by their high expression of KDR compared with other antigens. A representative panel of studies from 1 patient is shown. (A) A large colony manifests predominant and intense expression of membrane KDR (red) and less intense expression of VE-cadherin staining by a smaller percentage of cells (green). (B) Colonies grew smaller by day 21, expressed VWF (green), and showed increased VE-cadherin expression (red). (C) Outgrowing EPCs expressed CD133 (red) and KDR (green). (D) Compared with VWF (green), VE-cadherin (red) was also localized to the poles of outgrowing EPCs. (E) Outgrowing EPCs had consistently intense staining for KDR (red), and all cells also expressed VE-cadherin (green) and (F) VWF (green). Cultures were maintained on 96-well laminin-coated plates in M199 medium containing 50 μg/mL heparin, 50 μg/mL endothelial cell growth medium, and 20% fetal calf serum at standard culture conditions and were fixed for immunostaining at the time points indicated on the left. Images were generated at the projections of the z-stacks at 1024 × 1024 pixels.