G-CSF–induced mobilization of BM cells in mice receiving transplants of single CD34^–c-kit⁺Sca-1⁺ lineage^– side population (CD34^–KSL-SP) cells or whole BM cells following MI. (A) Experimental protocol. Single EGFP⁺ CD34^–KSL-SP cells and radioprotective EGFP^– BM cells were transplanted into the recipient mice. (B) The nucleated cell counts (NCCs) for peripheral blood samples obtained 24 hours after the last injection of G-CSF (n = 10) or saline (n = 10) are shown. ▪ indicates EGFP⁺ cells; ▨, EGFP^– cells. NS indicates not significant; *P < .0001. Bars indicate the standard error. (C-F) Panels show representative results for immunofluorescent analysis using anti–GFP antibody and TOTO-3 dye in the hearts of mice receiving translplants of w-BM cells or single CD34^–c-kit⁺Sca-1⁺ lineage^– side population (CD34^–KSL-SP) cells separated from the BM of transgenic EGFP mice. The green and blue signals indicate EGFP and nuclei, respectively. Saline-treated mice (G-CSF[–] mice) (C) and G-CSF–treated mice (G-CSF (+) mice) (D) in the w-BM group. G-CSF(–) mice (E) and G-CSF (+) mice (F) in the CD34^–KSL-SP group. Bars indicate 200 μm. (G) The control experiment using C57BL/6J mice as donors revealed that no green fluorescence was detected in the whole heart. Bar indicates 50 μm. (H-J) Coimmunostaining with anti-GFP and anti–actinin antibodies in the infarcted myocardium of G-CSF(+) mice transplanted with w-BM (H, I) or single CD34^–KSL-SP cells (J). The green, red, and blue signals indicate EGFP, actinin, and nuclei, respectively. (H, I) In the w-BM group, some of the EGFP⁺ cells in the infarcted area were positive also for actinin and showed striation, indicating that they had differentiated into cardiomyocytes. (J) In contrast, very few EGFP⁺ actinin⁺ cells were seen in the infarcted area in the CD34^–KSL-SP group. Bars indicate 50 μm (H, I) and 20 μm (J).