Figure 5.

Treatment with Nes⁺cMSCs, but Not Nes⁺bmMSCs, Largely Increases the Proportion of M2 Macrophages in the Infarcted Myocardium In Vivo and Regulates the Polarization of M2 Macrophages In Vitro

(A and B) CD68⁺MHC II⁺ M1 macrophages and CD68⁺CD206⁺ M2 macrophages were detected in the infarcted areas at 7 days post-AMI under fluorescence microscopy (A), and the percentages of CD68⁺MHC II⁺ M1 macrophages and CD68⁺CD206⁺ M2 macrophages in CD68⁺ total macrophages were calculated using a double-blind method (B). Scale bar, 20 μm; n = 5. (C) The percentage of CD68⁺CD206⁺ M2 macrophages was analyzed by flow cytometry after macrophages were co-cultured for 1, 2, and 3 days with or without Nes⁺cMSCs; n = 4. (D and E) CD68⁺CD206⁺ M2 macrophages were detected by immunofluorescence (IF) staining after 3 days of co-culture with or without Nes⁺cMSCs. Green indicates CD68⁺ macrophages, red indicates CD206⁺ M2 macrophages, and blue indicates nuclei (D). Scale bar, 20 μm. The percentage of CD68⁺CD206⁺ M2 macrophages was analyzed (E); n = 5. (F) The mRNA expressions of key macrophage differentiation-related enzymes (iNOS and Arg-1) were analyzed after 3 days of co-culture with or without Nes⁺cMSCs; n = 3. (G) The secretion levels of M1 macrophage-produced pro-inflammatory cytokines (TNF-α and IFN-γ) and M2 macrophage-secreted anti-inflammatory cytokines (IL-4 and IL-10) in supernatants after 3 days of co-culture with or without Nes⁺cMSCs, as examined by ELISA. Data are shown as mean ± SEM; n = 5. *p < 0.05, **p < 0.01, ***p < 0.001. n.s., not significant. LPS was used to activate macrophages toward a pro-inflammatory phenotype.