Adipose-derived Mesenchymal Stem Cells and Retinal Pigment Epithelial Cells Interactions in Stress Environment via Tunneling Nanotubes

Abstract This study aims to demonstrate the formation of TNTs between AdMSCs and RPE-1 and their alterations in response to experimental stress conditions. Serum starvation was employed as a stress condition to induce TNTs between the AdMSC and RPE-1. The presence of TNTs was demonstrated through immunofluorescence microscopy while scanning electron microscopy was utilized to determine the average thickness. Cell viabilities were assessed after stress by CTG, and H2DCFH-DA probes evaluated the cells’ reactive oxygen species (ROS) levels. Further, JC-1 labeled mitochondrial exchange between cells via TNTs was supported by videos. A transmembrane culture system was employed to inhibit TNT formation. In this study, we investigated the role of TNTs in facilitating intercellular communication and mitochondrial transfer between AdMSCs and RPE-1 under stress. We found that TNT-mediated mitochondrial transfer from AdMSCs to RPE-1 helps to reduce ROS levels and improve cell viability. We demonstrated that direct interaction between AdMSCs and RPE-1 was crucial for stress recovery. Co-culture enhanced viability and sustained retinal epithelial cell function after stress-induced damage. Mechanical inhibition of TNT formation decreased cell viability and increased ROS levels, indicating the importance of TNTs in cellular protection. The findings can provide a new perspective on the therapeutic potential of stem cell-based therapy in protecting RPE against stress-induced damage and promoting tissue regeneration. Competing Interest Statement The authors have declared no competing interest. Footnotes The "Isolation and culture of adipose-derived mesenchymal stem cells (AdMSCs)" 2.2 section was revised to ensure clarity, conciseness, and alignment with standardized reporting practices. Key updates include emphasizing the source and processing of AdMSCs, specifying the culture conditions and passage number used for experiments, and providing detailed descriptions of characterization methods, including flow cytometry markers and differentiation assays. The revision ensures the methodology is transparent and reproducible while maintaining a clear connection to the cited reference (Gozel et al., 2024). Abbreviations - AdMSCs - Adipose-derived mesenchymal stem cells - MSCs - Mesenchymal stem cells - RPE-1 - Retinal Pigment Epithelial Cell Line - TNTs - Tunneling nanotubes - ROS - Reactive oxygen species - DPBS - Dulbecco’s phosphate-buffered saline - DMEM-LG - Dulbecco’s Modified Eagle Medium - low glucose - P/S - penicillin/streptomycin - FBS - Fetal Bovine Serum - PBS - Phosphate-buffered saline - PFA - Paraformaldehyde - SF - Serum Free - H2DCFH-DA - 2,7-dichlorodihydrofluorescein diacetate - CTG - CellTiter-Glo - IF - Immunofluorescence - SEM - Scanning electron microscopy - DIC - Differential Interference - RGC - Retinal ganglion cells - CEC - Corneal epithelium cells