ABSTRACT: Peripheral nerve injury (PNI) is a major clinical and public health challenge that frequently results in significant functional impairment and permanent disability. However, most peripheral nerve injury-repairing strategies offer unsatisfactory clinical outcomes. Exosomes act as key mediators of the paracrine effects of induced pluripotent stem cells (iPSC-Exos), showing significant therapeutic promise for various conditions. Still, their efficacy and safety remain a major concern. Therefore, this study investigates the regeneration potential and safety of iPSC -Exos in a rat sciatic nerve crush injury model. Sendai virus vectors were used to reprogram peripheral blood mononuclear cells (PBMCs) from healthy donors into iPSCs. The cells were subsequently examined for pluripotency and karyotype integrity and validated using in vivo teratoma assays. iPSC-Exos were isolated and identified using transmission electron microscopy, nanoparticle tracking analysis, western blotting, and cell viability. In vivo, iPSC-Exos was injected directly at the injury site. Gait analysis, grip strength evaluation, and mechanical pain were measured preoperation and at 7, 14, 21, and 28 days postoperation. Animals' weight, hematology indexes, key organ weight, and histological changes were evaluated. Morphological analysis of regenerated nerve and muscle was examined. The immunofluorescence staining evaluated axon regeneration, remyelination, Schwann cells (SCs), and angiogenesis in the sciatic nerve at 2 and 4 weeks post-operation. Real-time PCR was used to assess gene-level expression associated with nerve regeneration. RNA sequencing (RNA-seq) was conducted to identify differentially expressed genes (DEGs) and pathways in experimental groups. PBMC-derived iPSCs exhibited Embryonic Stem cells-like morphology, showed pluripotency markers, preserved a normal karyotype, and induced teratomas, including tissues from all germ layers. After isolating and validating exosomes, PKH26-labeled exosomes were efficiently absorbed by SCs and enhanced SCs proliferation in a concentration-dependent manner in vitro. Safety evaluations revealed no adverse effect on the overall body weight, organ weight, hematological parameters, or histological changes of key organs. Exosome injection significantly improved gait analysis, grip strength, pain response, gastrocnemius muscle atrophy, axonal regrowth, myelin formation, and neovascularization. Furthermore, quantitative Real-time PCR (qPCR) results indicated an increase in regeneration-associated transcripts. RNA-seq analysis identified the upregulation of genes involved in nerve repair, with enrichment of pathways such as PI3K-AKT signaling, focal adhesion, and calcium signaling.Exosomes derived from iPSCs show a safe, effective, and non-cell-based therapeutic approach for PNIs. Their ability to promote axonal regrowth, myelin formation, and angiogenesis demonstrates their potential for clinical applications in regenerative medicine.
