Corneal Nerve Regeneration via MSC-Derived EVs: Tissue Source and Culture Dimensionality Dictate miRNA Cargo and Therapeutic Efficacy.

Extracellular vesicles (EVs) are emerging as critical mediators of intercellular communication and tissue repair, offering a promising cell-free platform for regenerative therapies. In the cornea, sensory nerves are critical for maintaining epithelial integrity and ocular homeostasis. Nerve injury resulting from trauma, surgery, or disease leads to persistent epithelial defects and impaired vision, with limited treatment options. Here, the neuro-regenerative potential of mesenchymal stem cell-derived EVs (MSC-EVs) isolated from human cornea (Co-MSC) and bone marrow (BM-MSC) cultured under 2D and 3D conditions is investigated. EVs are characterized by nanoparticle tracking analysis, ExoView profiling, and Western blot, and their effects on nerve regeneration are evaluated using primary trigeminal ganglion neurons in vitro and a murine corneal injury model in vivo. EVs from both tissue sources promoted neurite outgrowth; however, 3D-derived EVs demonstrate superior efficacy compared to 2D-derived EVs in vitro and in vivo. Co-MSC-EVs show a consistent trend toward enhanced regenerative effects over BM-MSC-EVs. Small RNA sequencing reveals that EV cargo is influenced by both tissue origin and culture dimensionality, with Co-MSC-EVs enriched in miRNAs regulating the extracellular matrix and immune pathways, while BM-MSC-EVs are enriched in neurotrophic signaling miRNAs. These findings support the rational design of MSC-EV-based therapies for neuro-ophthalmic repair.