Influence of oligodendrocyte precursor cell-derived exosomes on cell therapy of experimental autoimmune encephalomyelitis

Author(s)

Sarah Ingrid Pinto Santos

• Document type: Doctoral Thesis
• Press: Pirassununga.
• Institution: Universidade de São Paulo (USP). Faculdade de Zootecnica e Engenharia de Alimentos (FZE/BT)
• Defense date: 2024-04-16

Examining board members

Carlos Eduardo Ambrósio; Luciana Politti Cartarozzi; Alexandre Leite Rodrigues de Oliveira; Kelly Cristine Santos Roballo; Juliano Coelho da Silveira

• Advisor: Carlos Eduardo Ambrósio

Abstract

Multiple sclerosis is a chronic inflammatory disease of the central nervous system characterized by autoimmune destruction of the myelin sheath, leading to irreversible and progressive functional deficits in patients. Pre-clinical studies involving the use of neural stem cells (NSCs) have already demonstrated their potential in neuronal regeneration and remyelination. However, the exclusive application of cellular therapy has not proven sufficient to achieve satisfactory therapeutic levels. Recognizing these limitations, there arises the need to combine cellular therapy with other adjunctive protocols. In this context, extracellular vesicles (EVs) may contribute to intercellular communication, stimulating the production of remyelination-associated proteins and lipids, and providing trophic support to axons. This study aimed to evaluate the therapeutic efficacy of the combination of NSCs and EVs derived from oligodendrocyte precursor cells (OPCs) in an animal model of multiple sclerosis. The project unfolded through three experimental phases. In Phase 1, NSCs were isolated and cultured from the subventricular zone, characterized, and differentiated into OPCs. Gene expression analysis and immunocytochemistry confirmed the identity of these cells both before and after differentiation. In Phase 2, exosomes were isolated through ultracentrifugation and characterized using Western Blotting for exosomal protein detection, transmission electron microscopy, and nanoparticle tracking analysis. NSCs were supplemented with OPC-derived EVs, with no observed differences at the transcriptional level. However, an increase in GFAP+ cells was noted. Phase 3 involved the experimental therapy of C57BL/6 mice induced with experimental autoimmune encephalomyelitis (EAE), grouped according to the established protocol. Groups included control, treated with NSCs, treated with OPC-derived EVs, and treated with the combination of both. Treatments were clinically evaluated through scores and daily weight, microscopically through immunohistochemistry, immunomodulation through flow cytometry and tracking of cells and stained exosomes. The animals showed significant clinical improvement and weight gain with the treatments. However, only treatments involving EVs led to immune modulation, shifting the Th1 to Th2 lymphocyte profile, generating an anti-inflammatory response. Cells were predominantly found in the spleen, while exosomes were primarily identified in the lymph nodes. Microscopic evaluation of the spinal cord 15 days after treatment revealed a reduction in reactive microgliosis and astrogliosis in groups treated with EVs. There was no reduction in demyelination, and minimal differences were observed in axonal damage assessed by synaptophysin. The results indicate the potential therapeutic use of OPC-derived EVs in attenuating inflammation and promoting recovery in EAE. (AU)

• FAPESP's process: 21/09869-6 - Influence of oligodendrocyte-derived exosomes on cell therapy of experimental autoimmune encephalomyelitis
• Grantee: Sarah Ingrid Pinto Santos
• Support Opportunities: Scholarships in Brazil - Doctorate