Effects of MSCs Preconditioning on an Experimental In Vitro Model of Alzheimer’s Disease

Oxidative neuronal damage and long-term microglial inflammation are key events in the pathogenesis of Alzheimer's disease. Conditioned media (CM) from mesenchymal stromal/stem cells (MSCs) are known to have neuroprotective effects due to a wide range of dissolved neurotrophic, antioxidant and anti-inflammatory factors. The therapeutic effect of CM from MSCs can be enhanced by pretreatment of MSCs. Thus, the aim of this study was to investigate the neuroprotective properties of the secretome of MSCs with activated TLR3 (prMSCs) and pretreated with hypoxia mimetic (hMSCs) in an experimental in vitro model of Alzheimer's disease. In this study, the immunophenotype of MSCs was proved by flow cytometry, assessed through their differentiation potential using cytochemical reactions, and immunosuppressive properties after preconditioning were evaluated (qRT-PCR, ELISA). The effect of CM from prMSCs and hMSCs on neuroinflammation and H2O2-induced neurotoxicity was investigated in an in vitro model by qRT-PCR, ELISA, MTT assay and CM-H2DCFDA assay. This research demonstrated for the first time that TLR3 activation in MSCs enhances the immunosuppressive properties of CM in relation to proinflammatory M1-macrophages, and prevents the development of neuroinflammation by maintaining the balance between M1/M2 types of microglia. This process is achieved by decreasing the secretion of proinflammatory cytokines (IL-1β, IL-6, TNF-α) and increasing IL-4, IL-10 and TGF-β. At the same time, the neurotrophic properties of hMSCs were demonstrated for the first time. Consequently, the application of CM from hMSCs resulted in a reduction in apoptosis induced by oxidative stress in PC12 neuron-like cells. These results were accompanied by an increase in the production of growth factors, including VEGF. Furthermore, this study was the first to demonstrate that preconditioning MSCs with poly(I:C) or the hypoxia mimetic CoCl2 resulted in enhanced neuroprotective properties of CMs in an in vitro model of Alzheimer's disease, by increasing BCL2 expression, activating the Nrf2/ARE pathway and IL-6 production. To conclude, the obtained results of this study are of interest for further development of Alzheimer's disease therapy using conditioned media from MSCs.