Analysis of fate and multimodal effects of scaffolded hMSCs in vivo. (A) Immunostaining for CD90 and human heat shock protein 27 (hHSP27) showed long-term survival (≥6 wk postinjury) only of scaffolded hMSCs (arrowed cells in Insets for the framed area). The engrafted hMSCs were mostly CD90+ and primarily concentrated in the lesion epicenter, with sharply reduced presence in the adjacent parenchyma. (B) Costaining for hHSP27 demonstrated cytoplasmic BDNF in the engrafted hMSCs (Left); relative to the controls, mean expression level increased nearly fourfold in the scaffolded hMSC-treated group (Right: ^, comparing with hMSC alone or *, lesion only; n = 5 per group; P < 0.05). (C) Costaining for CD90 showed IL-10 expression in the engrafted hMSC cytoplasm (Left); expression level of IL-10 was significantly higher in the scaffolded hMSC-treated spinal cords (^, comparing with hMSC-only group and *, to lesion-only group; +, hMSC only vs. lesion only; n = 5 per group; P < 0.05, one-way ANOVA with Tukey’s post hoc test). (D) Confocal analysis of the scaffolded hMSC-treated spinal cord tissue detected no discernible IR to collagen I, II, and IV or ALP (bone tissue marker), despite persistent presence of CD90+ signals, suggesting that no mesenchymal phenotypic differentiation of hMSCs occurred in the injured spinal cords ≥6 wk after transplantation. (E) Confocal z-stack imaging confirmed that IDO was expressed mainly in the cytoplasm of donor hMSCs (CD90+, green) in the subacutely injured spinal cord (i.e., 7–10 d postinjury). (F) The number of infiltrated CD3+ (red) T-cells was significantly decreased (G) in the white matter areas 3 mm rostral (Left) and caudal (Right) to the epicenter, and in the gray matter 3 mm caudal to the epicenter (G,
Center). (H) Scaffolded hMSCs (CD90+, green) discernibly increased the number of macrophages manifesting M2 phenotype polarization (arginase 1 IR: green), whereas the number of activated microglia and macrophage (CD68+) was greatly reduced near the epicenter of the subacutely injured spinal cord, compared with lesion-only or hMSC-alone controls (n = 4 per group).