MSC-derived extracellular vesicles rescue ARPE-19 cells from H2O2-induced oxidative damage via antioxidant and anti-inflammatory mechanisms

Oxidative stress (OS) is a key factor in the pathogenesis of several eye conditions. Extracellular vesicles (EVs) isolated from human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) have emerged as a promising therapeutic agent due to their antioxidant and cytoprotective properties. This study evaluated the regenerative potential of MSC-derived EVs in a H 2 O 2 -induced oxidative stress model in retinal pigment epithelial (RPE) cells. ARPE-19 cells were cultured and subjected to OS by treatment with 200 µM H 2 O 2 for 2 h. Cell viability was assessed using the XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium) assay and CMFDA (5-chloromethylfluorescein diacetate) staining. OS and morphological changes were evaluated using 2′,7′-dichlorodihydrofluorescein diacetate (H 2 DCFDA), and mitochondrial membrane potential was measured using the JC-1 assay. EVs, isolated from hUC-MSCs, were characterized by scanning electron microscopy (SEM), dynamic light scattering (DLS), and atomic force microscopy (AFM). Functional assays included DiI-labeled EV uptake, scratch wound healing, ELISA, and qPCR analysis. Treatment with EVs significantly reduced intracellular reactive oxygen species (ROS) levels in H 2 O 2 -stressed ARPE-19 cells, restored cell viability, and enhanced both migration and proliferation. Uptake assays confirmed efficient internalization of EVs by ARPE-19 cells. Scratch assay results demonstrated accelerated wound closure in EV-treated groups. ELISA revealed a significant decrease in the pro-inflammatory marker IL-6 ( p < 0.001) and an upregulation of the anti-inflammatory marker IL-10 ( p < 0.01). Furthermore, gene expression analysis showed that EVs treatment upregulated OS-responsive genes ( p < 0.001), downregulated IL-6 ( p < 0.01) and stress-associated genes ( p < 0.001), and upregulated IL-10 ( p < 0.001) and apoptosis- and cell cycle-related genes ( p < 0.001), collectively indicating a protective effect of EVs against oxidative damage. MSC-derived EVs protect ARPE-19 from H 2 O 2 -induced OS by reducing ROS production, promoting cell survival, and enhancing migratory and proliferative capacity. These findings highlight the therapeutic potential of EVs as a safe, cell-free approach for managing OS-induced eye disease.