Fig. 3
Establishment and evaluation of systemic hAMSC administration using DMD mice. A Cytokine and chemokines expression in 3-day co-culture media of hAMSCs with muscle cells from WT (B10) or mdx mice was analyzed using the Proteome ProfilerTM Array. Changes in the expression levels circled with blue squares 1–5 are monocyte chemotactic protein-1 (MCP-1), chemokine (C-X-C motif) ligand 1(CXCL1)/growth-regulated protein α (GROα), granulocyte colony stimulating factor (G-CSF), IL-6, and IL-8, compared with WT cells. PC; positive control signals, NC; negative control. B Data quantifying signal intensity for the dots circled with blue squares in the array image (A). C Schematic representation of the repeated hAMSC treatment of mdx mice. For the short-term evaluation, mice received four treatments (hAMSC-mdx-4), whereas, for the long-term evaluation, we examined two groups that had received a total of four, or six treatments (hAMSC-mdx-4 or hAMSC-mdx-6). D Serum creatine kinase (CK) levels in each group of mice; 12- and 18-week-old mice of control mdx (n = 4, and 6), and four-time hAMSC-mdx (n = 6, and 7), using ELISA. E Quantification of IL-6 levels in the TA muscle lysate (300 μg protein) of wild type (WT, n = 3), control mdx (n = 5), and four-time hAMSC-mdx (n = 4) mice using ELISA. F Hematoxylin and eosin (H, E) staining of the TA and diaphragm muscle (original magnification, × 100 and × 40, each) of control mdx and four-time hAMSC-mdx. Scale bars, 200 μm. G Grip strength (g) and H normalized grip strength (g/g body weight, BW) of 12-week-old WT (n = 7), control mdx (n = 14), and four-time hAMSC-treated mdx (n = 10) mice. All data are presented as the mean ± SD, statistical differences are expressed relative to WT (*P < 0.05, **P < 0.01, ***P < 0.0005 and ****P < 0.0001) and control mdx (#P < 0.05, ##P < 0.01, and ###P < 0.0005) mice, t-test (D), and one-way ANOVA (Tukey’s post hoc test; E, G, H)