gms | German Medical Science

Objective: Spinal cord injury (SCI) is a devastating event for the patient with often a disastrous outcome and no regenerative treatment approach currently available. Transplantation of neural precursor cells (NPCs) has shown beneficial effects in improving functional recovery after SCI, which is partially attributed to their paracrine function via exosomes. This study aims to characterize NPC-derived exosomes (NPC-exos) and assess their properties for potential use as a SCI-treatment.

Methods: NPCs were isolated from the subventricular zone of E13.5-14.5 rat embryos and seeded on poly-ornithine and laminin precoated plates. After the expansion of the NPC-culture, the exosomes were isolated and purified from the medium with a three-step differential centrifugation and filtration (0.22-µm) method. NPC-exos were identified and quantified by nanoparticle tracking analysis (NTA), bicinchoninic acid protein assay (BCA), transmission electron microscope (TEM), and western blot with multiple exosomal specific markers. In addition, proteomic analysis of NPC-exos was performed with mass spectrometry. To evaluate the protective function of the NPC-exos, 6.0E10+8 particles of NPC-exos in vitro.

Results: The primary cell culture of purified rat NPCs could be successfully characterized by nestin and beta-tubulin III-expression in absence of NeuN-, GFAP-, and Olig2-expression. The identification of NPC-exos was performed with the markers CD63, CD81, and TSG101 in western blot analysis. The average size of the NPC-exos was 141±7nm measured by NTA and their typical cup-shaped morphology was confirmed by TEM. The molecular analysis showed that the NPC-exos exhibit a significant enrichment of certain proteins such as Syntaxin 1B, Netrin-1, and EphrinB1. Examined by Ingenuity Pathway Analysis (IPA), we found that those enriched proteins are involved with the activation of the synaptogenesis signaling pathway, and axon guidance signaling pathway.

Conclusion: Our experiments confirm that exosomes with typical features can be isolated and purified from a primary NPC culture. Furthermore, NPC-exos seem to carry a cargo that includes enriched synaptogenesis and axon guidance-related proteins. Thus, NPC-exos might be a potential non-cell-based treatment option for SCI and should be assessed in future studies.