METHODS AND RESULTS: We performed i

METHODS AND RESULTS: We performed immunohistochemical characterization of EC-CFUs and their mononuclear precursors. Using fluorescent-activated cell sorting, we evaluated the capacity of mononuclear subpopulations to generate EC-CFUs, and monitored their migratory behaviour when co-incubated with EC-CFUs. Time-lapse microscopy was used to observe colony maturation. Cellular proliferation within EC-CFUs was assessed using bromodeoxyuridine (BrdU) and anti-proliferative agents. EC-CFUs exhibited typical endothelial characteristics; however, several endothelial markers were weakly expressed or absent. Macrophage and lymphocyte antigens were intensely expressed. EC-CFUs readily incorporated BrdU, and failed to develop in the presence of anti-proliferative agents (P < 0.01; n = 12). Time-lapse microscopy demonstrated that the characteristic EC-CFU 'spindle cells' are not EC-CFU progeny, but are mononuclear cells migrating towards, and incorporating into colonies. Only CD14(+) monocytes were necessary for EC-CFU formation. CD14 expression was progressively down-regulated during colony maturation (P < 0.001; n = 6). Although unable to generate EC-CFUs directly, CD34(+) cells could differentiate into CD14(+) cells and potentiate EC-CFU formation.