Fig. 1.

AdIL-10-transduced MSCs are immunomodulatory and not immunogenic. a To confirm the immunomodulatory potential of hMSCs on activated murine lymphocytes, untransduced, AdNull-transduced and AdIL-10-transduced hMSCs were co-cultured with ionomycin and PMA-stimulated C57BL/6 lymphocytes for 4 days at a ratio of 1:10. Murine lymphocyte proliferation was measured by CFSE dilution and flow cytometric detection of CFSE fluorescent peaks. Untransduced, AdNull-transduced and AdIL-10-transduced MSCs reduced the proliferation of stimulated lymphocytes, confirming their immunosuppressive capacity. Values represent the mean ± SD of three biological replicates. Statistical analysis was performed using one-way ANOVA, followed by Bonferroni’s multiple comparisons test. ***p < 0.001, indicating a significant difference compared with non-co-cultured stimulated lymphocytes. b To assess potential immunostimulatory activity of hMSCs, untransduced, AdNull-transduced and AdIL-10-transduced hMSCs were co-cultured with unstimulated C57BL/6 lymphocytes for 5 days, at a ratio of 1:5. Despite their xenogeneic origin, hMSCs did not induce proliferation of unstimulated C57BL/6 lymphocytes beyond basal levels. c To confirm immunosuppressive activity of vIL-10 overexpressed by AdIL-10-transduced MSCs, LPS-stimulated murine macrophages (RAW264.7) were treated with conditioned medium (CM) from untransduced, AdNull-transduced or AdIL-10-transduced hMSCs (100 ng/ml vIL-10), or 100 ng/ml recombinant vIL-10 (rvIL-10) alone. CM harvested from AdIL-10-transduced MSCs as well as rvIL-10 significantly reduced TNF-α production by LPS-activated macrophages. Values represent the mean ± SD of three biological replicates. Statistical significance was determined by a one-way ANOVA with Bonferroni’s multiple comparisons test. *p < 0.05 **p < 0.005, ***p < 0.001. Asterisks indicate significantly different from AdIL-10 MSC CM. LPS lipopolysaccharide, MSC mesenchymal stem cell, TNF-α tumour necrosis factor alpha