Extracellular vesicles derived from clonal mesenchymal stromal cells preconditioned by indirect hypoxia modulate immune responses in diabetic mice more effectively than directly preconditioned vesicles.

BACKGROUND: Mesenchymal stem/stromal cells (MSCs) or MSC-derived extracellular vesicles (MSC-EVs) are considered potential modulators of the immune system in type 1 diabetes (T1D) therapy. On the other hand, preconditioning MSCs with inflammatory agents is a promising strategy to improve the therapeutic effects of these cells. Therefore, we performed various direct preconditioning regimens (IFN-γ, poly(I: C), LPS, and hypoxia) on clonal MSCs (cMSCs), and for indirect preconditioning regimens, the same regimens were applied to peripheral blood mononuclear cells (PBMCs). PBMC-derived concentrated conditioned media (CCM) was transferred into cMSC cultures to mimic in vivo conditions to obtain cMSC-EVs with better immunomodulatory effects and then selected hypoxia-induced cMSC-EVs and their source cells to determine their influence on immune responses in multiple low-dose/Streptozotocin (MLD/STZ)-induced mouse model. METHODS: Direct preconditioning regimens include four groups: IFN-γ (50 ng/ml), poly(I: C) (42.22 µg/ml), LPS (1 µg/ml), and hypoxia (1% oxygen). For indirect preconditioning, these regimens were applied to PBMCs, and PBMC-derived CCM was added to cMSC cultures. The resulting cMSC-CCM was assessed for the expression of anti-inflammatory, pro-inflammatory, and regenerative factors and its ability to inhibit lymphocyte proliferation. The isolated EVs from the most effective direct and indirect preconditioned cMSC-CCM groups and their source cells were transplanted in MLD/STZ-induced mouse models of T1D and followed for 73 days. RESULTS: In vitro characterization revealed that direct and indirect hypoxia preconditioning were the most effective groups among the different preconditioning regimens. Therefore, four treatment groups, including direct hypoxia-induced cMSC-EVs (dH-MSC-EVs), indirect hypoxia-induced MSC-derived EVs (iH-MSC-EVs), and their cell sources (dH-MSCs and iH-MSCs), were selected for EV isolation and animal studies. The in vivo study revealed that the fasting blood glucose (FBG) trend decreased in the four treatment groups compared with the sham group. However, the effects of direct/indirect hypoxia on cMSC-EVs were associated with an increase in the levels of anti-inflammatory cytokines (IL-4 and IL-10) in the serum of the MLD/STZ-induced mouse models compared with their parental cell counterparts. Indeed, iH-MSC-EVs attenuate immune responses in MLD/STZ-induced mouse models. Furthermore, compared with their source cells and dH-MSC-EVs, iH-MSC-EVs significantly reduced the levels of proinflammatory cytokines (TNF-α and IL-6) in the serum of the MLD/STZ-induced mouse models. CONCLUSION: Our results showed the effectiveness of direct/indirect hypoxia preconditioning for cMSCs to modulate immune responses in the MLD/STZ-induced mice model. This study demonstrated that the immunomodulatory potential of iH-MSC-EVs is significantly greater than that of dH-MSC-EVs and their source cells. Our results suggest iH-MSC-EVs, could be a potential option for modulating the immune response in T1D.