The in vitro effect of mesenchymal stem cell-conditioned medium onoxidative stress-related retinal damage

This study aims to evaluate the effect of conditioned medium (CM) derived from adipose and limbal tissue-derived mesenchymal stem cells (MSC) on cell survival, cell cycle, inflammation, autophagy and angiogenesis in retinal pigment epithelial (RPE) cells stressed with hydrogen peroxide (H₂O₂). In addition, the effect of storage conditions on the anti-inflammatory properties of adipose-derived MSC-CM (ADMSC-CM) and the protective effects of lyophilized ADMSC-CM against age-related autophagy impairment were investigated. MSC-CM was applied to RPE cells stressed with H₂O₂. Cell viability was assessed by MTT assay and cell cycle distribution was analyzed by flow cytometry. Inflammatory responses were measured by RT-qPCR and Western blotting for key cytokines and signaling proteins. Angiogenic potential was analyzed by VEGF ELISA and HUVEC migration and survival assays. The effects of storage conditions of ADMSC-CM were examined by considering the effects of concentrated and lyophilized CM on markers of inflammation. Finally, the protective role of lyophilized ADMSC-CM against autophagy mechanisms was evaluated by pretreatment and subsequent exposure of RPE cells to H₂O₂; cell viability, LDH release, ATP levels and mitochondrial function were analyzed. Both ADMSC-CM and LMSC-CM significantly increased RPE cell survival after H₂O₂ treatment. MSC-CM from both sources attenuated the G2/M phase arrest induced by H₂O₂. MSC-CM reduced the expression of inflammatory cytokines (IL-1β, IL-6 and IL-10) and inhibited the p38 MAPK signaling pathway. Lyophilized ADMSC-CM showed a significant reduction in inflammation markers compared to concentrated CM. MSC-CM decreased the Bax/Bcl-2 ratio and modulated autophagy-related proteins such as LC3 and p62. Pretreatment with lyophilized ADMSC-CM increased cell viability, reduced LDH release and maintained ATP levels. MSC-CM showed a significant alteration on HUVEC migration and survival, although it did not affect released VEGF levels. Lyophilized ADMSC-CM played a protective role against oxidative stress-induced mitochondrial damage by preserving mitochondrial membrane potential through autophagy pathway. In particular, MSC-CM derived from adipose tissue improves the survival and function of H₂O₂-treated RPE cells by modulating cell cycle arrest, inflammation and autophagy mechanisms. Lyophilized ADMSC-CM retains its anti-inflammatory properties and shows protective effects against age-related autophagy impairments, making it a promising candidate for clinical applications in the treatment of retinal diseases associated with oxidative stress and inflammation.