Phenotypic characterization of large-scale expanded ECFCs. (A) Representative flow cytometry histograms of ECFCs after humanized large-scale expansion showing reactivity with EC-expressed marker molecules (right-shifted filled gray curves compared with black lined open curves of the appropriate isotype controls) and lack of reactivity with hematopoietic (CD14 and CD45) and stem cell-associated (CD90 and CD133) or activation (human leukocyte antigen class II type DR, HLA-DR) markers. (B-G) Representative cellular localization of (B) mesodermal cytoskeleton component vimentin, (C) CD146/melanoma and endothelial cell adhesion molecule detected by prototype antibody P1H12, (D) CD144/vascular endothelial-cadherin, (E) endothelial von Willebrand factor (VWF), (F) hematopoietic stem cell and vascular EC-specific CD31/platelet-endothelial cell adhesion molecule-1, compared with (G) one representative isotype control example. ECFCs were cryopreserved after large-scale expansion and thawed before seeding in chamber slides to obtain adherent cells for cytochemistry. Staining was done as specified in supplemental data. The brown color results from precipitation of the chromogen diaminobenzidine mediated by antibody binding to the target molecule. Images were captured with an Olympus (Hamburg, Germany) DP71 camera on an Olympus BX51 microscope (original magnification 100×/1.35 NA oil objective) with the Olympus cell D image acquisition software.