In vitro–cultured bone marrow cells with EPC phenotype home at sites of VEGF-induced angiogenesis but are not incorporated into the vessel wall.(A) Experimental design. Balb/c mice adherent bone marrow cells were cultured in vitro for 15 days in differentiating (D) and nondifferentiating medium (ND; see text) in order to enrich for endothelial progenitor cells. After 15 days, the EPCs were labeled with the PKH26 red fluorescent die and administered intravenously to syngenic recipient mice previously injected with AAV-VEGF in the right tibialis anterior muscle. (B) Flow cytometry phenotypic profile of the cell populations before injection into the animals (mean ± SD of 3 different experiments). (C) Number of PKH16-positive cells infiltrating the sites of VEGF-induced neovascularization at day 15 after cell injection. The cells expanded ex vivo using the ND medium were recruited approximately 4 times more efficiently than those cultivated in differentiating medium (mean ± SD of 3 different experiments). (D) Immunofluorescence analysis for the visualization of CD31 (green) and PKH26 (red). Colocalization of the 2 markers in the same cells was highly infrequent. (E) Immunofluorescence analysis for the visualization of CD11b (green) and PKH26 (red); arrows indicate 2 in vitro–labeled recruited cells. Most of the recruited cells were positive for the myelocytic/monocytic marker CD11b.