Fig. 4

Transplantation of IL10-MSCs alternatively activated M2 microglia/macrophages at the lesion site. A Heat map of the most differentially expressed genes involved in regulating immune and macrophage polarization between IL10-MSCs and MSCs. The alternatively activated M2 and M1 microglia/macrophages at lesion regions were identified by immunostaining against CD11b/Arg1 and CD11b/iNOS, respectively. B, D At W2 after SCI, IL10-MSC treatment significantly increased the ratio of Arg-1⁺ cells to CD11b⁺ cells compared with MSC graft and vehicle control. C, E The ratio of iNOS⁺ cells to CD11b⁺ cells in the IL10-MSC group were significantly decreased compared with the other two groups. F, G The mRNA expression of iNOS and Arg1 was assessed in injured sites. Consistent with immunostaining results, IL10-MSCs treatment significantly improved the mRNA expression of Arg-1 at lesion site compared with the other two groups, and decreased mRNA expression of iNOS, compared with control group. H–L Cytometry markers and gating strategy were performed to distinguish the resident microglia and infiltrated macrophage at lesion site. H Gates to exclude debris and cell aggregates in FSC-A/SSC-A and FSC-A/FSC-H plots. Representative flow cytometry gating strategy correspond to resident microglia (CD11b^hi+/CD45^lo/CCR2⁻) and infiltrated macrophages (CD11b^lo/CD45^hi/CCR2⁺). Activated microglia/macrophage gating was further stratified by incubation with activation markers Arg1 and iNOS. The area outside the purple line represents the resident microglia, and the area within the red line represents the infiltrated macrophages. I, J M1 and M2 macrophages were identified according to iNOS and Arg1 expression. Graph shows the ratio of M1 to M2 infiltrated macrophages at the injured spinal cord. K, L Ratio of M1 to M2 resident microglia cells at injured spinal cord. Data represented as mean ± SEM; ***p < 0.001, **p < 0.01, *p < 0.05; n = 6–8 per group (B, D), n = 4 per group (I–L); Scale bars, 50 um (B, D)