ECFCs function in vitro and in vivo after large-scale humanized expansion. (A-C) Representative transformed images of vascular network formation from 3 typical independent oligoclonal cultures and (D) 1 monoclonal ECFC culture compared with (E,F) 2 independent oligoclonal UCB-derived EPC networks under the same conditions on Matrigel. Nontransformed original phase-contrast microphotographs are documented in higher magnification (Figure S5). Images were captured with an Olympus Color View III camera on an Olympus IX51 microscope (original magnification 20×/0.4 NA objective) with the Olympus analySIS B acquisition software. (G) Serial image reconstruction of one representative complete vascular network created in a 0.4-cm² well of a 16-well glass chamber slide is shown (Videos S2,3). For in vivo neovasculogenesis, ECFCs from 2 donors were mixed with MSCs in Matrigel before injecting 0.2 mL of the composite subcutaneously in 4 nude mice per group (n = 24; 4 mice per ECFC source analyzed at 3 time points; a macroscopic view is shown in Figure S6D,E). (H) Topography of the histology is symbolized and (J) shown as a low magnification overview of a vimentin-labeled vascularized plug part. (K) Vimentin reactivity in the border area showing murine tissue (left half) in the direct vicinity of the human cell-containing area of the Matrigel plug as indicated in panel J. (M) Antihuman CD31, (N) antihuman VWF, (O,P) antimouse glycophorin A reactivity detected with antibody mTer119 within (O) human and (P) mouse vessels and (Q) representative isotype control reactivity of mouse red blood cell containing vasculature inside the plug.