FACS analysis of undifferentiated MAPCs and MAPCs cultured with VEGF. MAPCs (after 40 population doublings; donor age, 28 years) were replated at 2 × 10⁴ cells/cm² in fibronectin-coated wells in serum-free defined medium without EGF or PDGF-BB, but with 10 ng/ml VEGF. Undifferentiated MAPCs at day 0, or VEGF-induced cells recovered after short trypsinization after 3, 9, or 14 days of culture, were stained with Ab’s against β2-microglobulin, HLA class I, MUC18, Flk1, Flt1, VCAM, CD62P, CD62E, vWF, CD31, CD34, CD36, AC133, VE-cadherin, or control IgG. Cells were analyzed by FACS. Plots show isotype control IgG staining profile (thin line) versus specific Ab staining profile (thick line). Each analysis shown is one representative example from a total of three donors. Values on x axes indicate intensity log. (a) Phenotype of undifferentiated MAPCs. MAPCs express low levels of β2-microglobulin, Flk1, Flt1, and AC133, but do not stain with any of the other anti-endothelial markers. (b) Phenotype of MAPCs cultured for 14 days with 10 ng/ml VEGF. MAPCs express high levels of most endothelial markers associated with endothelial cells, but lose expression of AC133. (c) Phenotype of MAPCs cultured for 3–9 days with 10 ng/ml VEGF. MAPCs lose expression of AC133 by day 3 of culture with VEGF, and acquire expression of Tek by day 3, and vWF, CD34, and MUC18 by day 9. β2-mic, β2-microglobulin; VE-cad, VE-cadherin.