Figure 6.

MIS416-mediated increase of cytokines upregulate immunomodulative function of human adult stem cells, including umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs). (A) The levels of tumor necrosis factor (TNF), interferon (IFN)-γ, IL-6, and IL-12 in serum of mice were evaluated a day after MIS416 administration (day 2) by CBA analysis. (B–F) Combination of cytokines, IL-6 (25 ng/ml), IL-12 (20 ng/ml), and IFN-γ (20 ng/ml), were used to treat hUCB-MSCs for 24 h. (B) Representative of bright-field microscopy images of hUCB-MSCs, bar = 500 µm. Proliferation and cell viability of hUCB-MSCs were determined by (C) CCK-8 assay and (D) MTT assay. (E) The expression levels of COX2, iNOS, and IDO-1 in hUCB-MSCs were analyzed using western blot analysis. (F) hUCB-MNCs were co-cultured with hUCB-MSCs stimulated with the cytokines directly and their proliferation was determined by bromodeoxyuridine ELISA assay. n = 7–8 mice per group, two independent animal experiments were performed. In vitro experiments were performed in triplicate. Gel electrophoresis was conducted under the same experimental conditions, and images of blots were cropped. Uncropped blot images are shown in Figure S7 in Supplementary Material. (−): negative control group, (+): DSS administered group in (A) and ConA activated group in (F), M: MIS416-treated group, U: hUCB-MSCs, stimulated/U stimulated: cytokine cocktail-treated hUCB-MSCs. *P < 0.05, **P < 0.01, ****P < 0.0001. Results are shown as the mean ± SEM.