Fig. 6

The beneficial effects of hMuSCs on acute liver injury require IDO. (A) PBMCs stained with CFSE were co-cultured with hMuSCs (2.5 × 10⁴ cells per well in 48-well plates) at a ratio of 1 : 20 (hMuSC : PBMC) for 72 h in the presence of anti-human CD3 antibody (1 µg/ml) with or without the IDO inhibitor 1-MT (0.5 mM). CFSE fluorescence intensity reduction of PBMCs was detected by flow cytometry to measure the proliferation of PBMCs (n = 4). (B) Efficiency of shRNA-mediated IDO knockdown in hMuSCs (n = 3). The original images of blots are in the Supplementary Information. (C) PBMCs stained with CFSE were co-cultured with Scrambed-shRNA hMuSCs or IDO-shRNA hMuSCs (2.5 × 10⁴ cells per well in 48-well plates) at graded ratios (hMuSC : PBMC) for 72 h in the presence of anti-human CD3 antibody (1 µg/ml). CFSE fluorescence intensity reduction of PBMCs was detected by flow cytometry to measure the proliferation of PBMCs (n = 4). (D) Mice were intravenously injected with ConA (15 mg/kg) to induce acute liver injury; 0.5 h later, Scrambled-shRNA hMuSCs or IDO-shRNA hMuSCs (5 × 10⁵) were transfused to suffering mice in the same way. After 12 h, serum and liver tissues were sampled. (E) Serum levels of AST and ALT were measured. (F) IFN-γ and IL-12 levels in the serum were assayed by ELISA. (G) Representative H&E-stained liver sections and percentages of tissue necrosis. Scale bar, 250 μm. (H) Absolute numbers of MNCs in liver tissues were calculated. Absolute numbers of CD3⁺ T cells were determined by flow cytometry (Control: n = 5, ConA + PBS: n = 5, ConA + Scrambled hMuSC: n = 5, ConA + IDO-shRNA hMuSC: n = 5). Values are presented as mean ± SEM. Statistical analysis was performed by one-way analysis of variance test (A), two-tailed unpaired t test (B) or Mann-Whitney test (E-H).