Combining proteomics and in vitro approaches to evaluate regenerative effect of extracellular vesicles from bone marrow and cornea mesenchymal stem cells cultured in 2D and 3D system on corneal nerve regeneration

Abstract

Purpose : Corneal nerve damage, resulting from trauma, surgery, infection, or diseases like diabetes, can lead to ocular discomfort, reduced tear production, vision issues, or even blindness. Current treatments like eye drops are often ineffective in severe cases, highlighting the need for therapies that foster rapid corneal nerve regeneration. Building on our previous work demonstrating the regenerative potential of extracellular vesicles (EVs) from bone marrow mesenchymal stem cells (BM-MSCs EVs), this study compares the effects of EVs derived from BM-MSCs and corneal MSCs (Co-MSCs), both cultured in 2D and 3D systems, on corneal trigeminal ganglion (TG) nerve regeneration. We also examine the protein expression profiles of these MSCs using proteomics.

Methods : Human BM-MSCs and Co-MSCs were cultured in 2D and 3D systems. EVs were isolated and characterized using ultra-centrifugation, Nanosight and ExoView. Mouse TGs were treated with EVs to assess neuronal growth over 48hrs, followed by β3 tubulin immunostaining and confocal microscopy. Proteomics were applied to EVs to further analyze and elucidate the molecular composition and potential functional differences between the EVs derived from different sources and culture conditions.

Results : EVs from 3D cultures showed a substantial increase in concentration and higher levels of exosomal markers (CD63, CD81, CD91) compared to 2D cultures. Both BM-MSCs and Co-MSCs promoted nerve regeneration, with 3D-derived EVs significantly enhancing this effect and this increase was comparatively more in BM-MSCs. EVs proteomic analysis revealed distinct protein profiles for BM-MSCs and Co-MSCs, with a higher abundance of proteins in both 3D-derived EVs and again more protein expression in BM vs. cornea derived EVs.

Conclusions : EVs from 3D cultures demonstrated enhanced potency in promoting corneal nerve regeneration compared to 2D cultures. Both BM and Co-MSCs contributed to this regenerative effect, with BM- MSCs exhibiting a more pronounced impact. Proteomic analysis further revealed distinct and more abundant protein profiles in EVs derived from 3D cultures, particularly those from BM-MSCs, underscoring the potential of 3D culture systems in advancing therapeutic strategies for corneal nerve regeneration.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.