Production of MSC overexpressing TSG-6. (A) The mRNA level of TSG-6 in CSII-EF-MCS- and CSII-EF-TSG-6-expressing hMSC cells was determined via real-time PCR. (B) TSG-6 stable transfection to hMSCs was confirmed by immunoblotting for Wip1 (top panel). Equal protein loading was verified by α-tubulin immunoblotting (middle panel). Secretion of TSG-6 in hMSCs was confirmed in the conditioned media by immunoblotting for TSG-6 (bottom panel). (C) TSG-6 expression in TSG-6-hMSC was validated by immunostaining with anti-TSG-6 antibody (left panel, TSG-6). GM130 was counterstained as a Golgi complex marker (middle panel, GM130). DAPI was used for nuclear counterstaining (right panel, DAPI). (D) CD4+ T cells from mouse splenocytes were cultured in the presence of CD3 antibody (1 μg/ml of α-CD3). T cells proliferation was determined by FACS analysis after CFSE staining. CM either from hMSCs (B10) or TSG-6-hMSCs were incubated for three days in the presence of CD3 antibody. Culture medium for hMSCs was used as the control medium. CFSE, carboxyfluorescein succinimidyl ester; CM, conditioned medium; DAPI, 4′,6-diamidino-2-phenylindole; FACS, fluorescence activated cell sorting; hMSCs, human mesenchymal stem cells; MSCs, mesenchymal stem cells; TSG-6, tumor necrosis factor-inducible gene 6 protein.