Fig. 1

MSCs can convert conventional T cells into Foxp3-expressing Tregs with strong immunosuppressive capacity. CD4⁺ effector T cells and DCs isolated and cultured with allogeneic MSCs in Transwell system under four conditions as described in Methods. a CD4⁺CD25⁻ purity before culturing with MSCs. b CD4⁺ T cells harvested, and CD4⁺CD25⁺FOXP3⁺ regulatory T cells measured by flow cytometry after 72 h and 5 days of coculturing with MSCs. c Total mRNA extracted from MSC-induced Tregs after 6 h, 12 h, 24 h, 48 h, 72 h, and 5 days of coculture, and FOXP3 mRNA expression measured by real-time PCR. Allogeneic MLR performed; CD4⁺CD25⁻ effector T cells isolated 5 days after MLR used as negative control, and TGF-β-induced Treg cells used as positive control. mRNA samples normalized to expression of GAPDH and compared with negative control. d For suppression assay, MSC-cultured T cells isolated after 72 h and added to T cells that had been stimulated by allogeneic DCs. After 48 h, BrdU used to measure proliferation of CD4⁺ T cells; proliferation of these cells compared with that of MLR control group consisting of T cells cultured in presence of allogeneic DCs. e MSCs and T cells cocultured for 3 days. Total T cells (cells in suspension) collected and CD4⁺CD25⁺ population isolated via Treg isolation kit. These newly induced Tregs were further put in new MLR encountering freshly isolated CD4⁺CD25⁻ responder cells in a 1/10 ratio (1 Treg/10 Tconvs) in presence of anti-CD3 and anti-CD28 activation antibodies. Suppressive assay then investigated by analysis of CFSE expression. Data presented as mean ± SEM; n = 2 (a) and n = 4 (b, c) independent experiments. Significant results: *p < 0.05; ***p < 0.001. d day, DC dendritic cell, iTreg induced Treg, LPS lipopolysaccharide, MSC mesenchymal stem cell, MSC-DC MSC-treated DC, MLR mixed lymphocyte reaction, TC allogeneic CD4⁺CD25⁻ T cells