BM-derived CD133⁺CXCR4⁺ cells enrich for neovascularization potential. The retinal injury model that shows the most robust BM contribution was used to identify BM-derived cells with direct neovasculogenic potential. (A) BM isolated from wild-type C57Bl/6 mice was analyzed for CD133 and CXCR4 expression. (B) Flow cytometric analysis of gated CD133⁺CXCR4⁺ cells shows an expression pattern that includes markers known to be found on BM-derived cells that participate in neovasculogenesis. CD133⁺CXCR4⁺ cells express endothelial cell surface markers such as VEGFR2, CD31, VE-cadherin, and tie 2. They also express CD45, CD117 (c-kit), Sca-1, VLA-4, CD11b, CD44, CD150, and CD135 (flt-3). (C) Kinetic analysis of CD133⁺CXCR4⁺ cell mobilization into the peripheral blood of mice after retinal injury showed an increase in cell number that correlated with SDF-1α levels in blood serum (n = 6; *P < .05). (D) Retinal flat mounts of injured eyes after induction of retinal ischemic injury that were adoptively transplanted with 10⁶ BM-derived CD133⁺CXCR4⁺DsRed⁺ cells showed contribution to retinal neovascularization (n = 6). Untreated left eye (negative control) is also shown (scale bars represent 100 μm). Similar results were observed with GFP⁺ BM (data not shown; E) CD133⁺CXCR4⁺DsRed⁺ cell transplanted eyes that underwent induction of retinal ischemic injury with the added step of intravitreal injection with PBS containing an anti–SDF-1α– or anti–CXCR4-neutralizing antibody to a final concentration of 1 μg/mL are shown. Note the absence of newly formed vessels from CD133⁺CXCR4⁺DsRed⁺ BM cells under these conditions. As a control, treated eyes that underwent induction of retinal ischemic injury were treated with PBS containing an isotype-matched control antibody (n = 6; scale bars represent 100 μm). All animals were perfused with FITC-dextran to show functional vasculature.