Evaluation of the therapeutic potential of metformin-pretreated tonsil-derived mesenchymal stem cells in a chronic colitis model

Background: Tonsil-derived mesenchymal stem cells (TMSCs), prepared after tonsillectomy in human, have many advantages including a short doubling time, high differentiation capacity, and mixed chimerism property. Metformin has an anti-inflammatory function involving inhibition of signal transducer and activator of transcription (STAT)-3. We aimed to evaluate the anti-inflammatory effect of metformin-pretreated TMSCs in a mouse model of chronic colitis. Methods: The optimal condition under which maximum decrease in STAT-3 level occurs was evaluated in vitro by treating 1×105 TMSCs with 0.2, 0.5, 1.0, and 2.0mM metformin and extracting protein at 4, 7, and 14 days. Eight-week-old C57BL/6 mice (n=60) were randomly assigned into 4 groups: normal, colitis, TMSC, and metformin-pretreated TMSC groups. Colitis was induced by oral administration of 1.5% dextran sulfate sodium (DSS) for 5 days followed by 5 days of drinking water continuously for 3 cycles (30 days). TMSCs and metformin-pretreated TMSCs were administered via intraperitoneal injection 4 times, on days 6, 9, 12, and 16. Control mice were injected with phosphate-buffered saline at the same time. The severity of the colitis was assessed by determining the disease activity index (DAI), body weight change, colon length, histologic grading, and cytokine levels. Results: The STAT-3 level was the lowest in TMSCs treated with 1mM metformin for 7 days, and this concentration was selected for the pretreatment. At 30 days after treatment, DAI (5.8±1.3 vs 2.6±1.0 vs 2.3±0.4, mean±standard error mean values for colitis, TMSC, and metformin-pretreated TMSC groups, respectively, p=0.025, ANOVA) and weight loss (-10.3±4.9% vs 6.8±4.3% vs 5.9±2.7%, p=0.007, ANOVA) showed significant improvement in TMSC and metformin-pretreated TMSC group than colitis group. However, colon length (p=0.524) and histologic improvement (p=0.054) were not significantly different between the groups. The survival rate for each group was 62.5%, 86.7%, and 100%, with the lowest rate recorded in the colitis group and the highest in the metformin-pretreated TMSC group (p=0.028, Kaplan-Meier analysis). IL-1β, a pro-inflammatory cytokine, showed higher levels in colitis group than TMSC and metformin-pretreated TMSC groups (p=0.196), whereas IL-10, an anti-inflammatory cytokine, showed opposite results (p=0.337), without statistical significance. Conclusions: To our knowledge, this was the first study to evaluate the effect of metformin-pretreated TMSCs in a chronic murine colitis model. Based on the several advantages of TMSCs over other mesenchymal stem cells in clinical use and the anti-inflammatory effect of metformin, TMSCs pretreated with metformin have clinical feasibility and favourable therapeutic potential for colitis treatment.