Mesenchymal stem cell secretome induced the acquisition of anti-inflammatory phenotype in rat cortical microglia in vitro

Abstract

Introduction: Here, we aimed to address the impact of mesenchymal stem cells-conditioned medium (MSCs-CM) on M1/M2 phenotype shifting of rat microglia after 48 hours in vitro. Methods: Rat neonatal cortical microglia were randomly allocated into four different groups as follows: Control; MSC-CM; IL-4; and Anti-IL-4 groups. In the MSC-CM, microglia were treated with MSC condition media. In the interleukin-4 (IL-4), cells received 20 ng/mL IL-4; conceived as a positive M2 control group). In the Anti-IL-4, the combination of IL-4 peptide and anti-IL-4 antibody was used. After 48 hours, protein levels of Iba1, CD86, and CD206 were monitored using immunofluorescence imaging and flow cytometry analysis. Results: According to immunofluorescence imaging, 48-hour incubation of rat cortical microglia with stem cells condition media increased protein levels of CD206 and decreased CD86, showing the polarization of microglia toward M2 type lineage compared to the non-treated control group. We found a similar trend in the group that received IL-4. By contrast, the incubation of microglia with anti-IL-4 antibody blunted the stimulatory effect of IL-4 to promote M2-type microglia. Similar to the immunofluorescence data, flow cytometry analysis revealed a significant increase of CD206 positive microglia after exposure to MSC-CM and IL-4 (P<0.05). Treatment with anti-IL-4 antibody significantly reduced the percent of CD206 positive cells, showing the inhibition of M1-to-M2 phenotype acquisition. Conclusion: The current study highlighted a notable anti-inflammatory effect of MSC secretome on cortical microglia by promoting M1-to-M2 phenotype acquisition.