Proteomic Characterization of Extracellular Vesicles from Human Neural Precursor Cells: A Promising Advanced Therapy for Neurodegenerative Diseases

Priscila Elias Ferreira Stricker,1,* Nathalia Barth de Oliveira,1,* Bassam Felipe Mogharbel,1 Ana Carolina Irioda,1 Nádia Nascimento da Rosa,1 Larissa Lührs,1 Claudia Sayuri Saçaki,1 Isadora Munhoz da Rocha,2 Lysangela Ronalte Alves,2 Saloe Bispo Poubel,1 Julia Cardoso da Silva,3 Paulo Costa Carvalho,3 Juliana Saldanha da Gama Fischer,3 Katherine Athayde Teixeira de Carvalho1 1Pelé Pequeno Príncipe Research Institute, Child and Adolescent Health Research & Pequeno Príncipe Faculties, Advanced Therapy and Cellular Biotechnology in Regenerative Medicine Department, Curitiba, PR, Brazil; 2Gene Expression Regulation Laboratory, Carlos Chagas Institute, FIOCRUZ, Curitiba, PR, Brazil; 3Computational Mass Spectrometry Group, Carlos Chagas Institute, FIOCRUZ, Curitiba, PR, Brazil*These authors contributed equally to this workCorrespondence: Katherine Athayde Teixeira de Carvalho, Pelé Pequeno Príncipe Research Institute, Child and Adolescent Health Research & Pequeno Príncipe Faculties, Av. Munhoz da Rocha, 490 - Juvevê, Curitiba, PR, 80035-000, Brazil, Tel +55-41-3310-1034, Email [email protected]: The therapeutic effect of stem cells is attributed to their direct maturation into somatic cells and their paracrine effects, which influence the extracellular environment. One such component released is extracellular vesicles containing proteins and genetic materials with immunomodulatory functions and facilitating cell-to-cell communication.Purpose: The study’s main objective was to characterize extracellular vesicles (EVs) from Human Neural Precursor Cells (hNPCs).Methods: Wharton’s Jelly mesenchymal stem cells (WJ-MSCs) were isolated by explant technique and characterized by flow cytometry and trilineage differentiation. The hNPCs obtained from neurospheres were produced by seeding WJ-MSCs on a natural functional biopolymer matrix. EVs derived from WJ-MSCs and hNPCs were isolated by precipitation methodology and characterized by flow cytometry, nanoparticle tracking analysis (NTA), scanning electron microscopy (TEM), and proteomic.Results: hNPCs expressed proteins and genes characteristic of neural precursor cells. The EVs were characterized by flow cytometry and showed varied expression for the markers CD63, CD9, and CD81, indicating different subpopulations based on their origin of formation. NTA and TEM of the EVs exhibited characteristic size, shape, and structural integrity consistent with the criteria established by the International Society for Extracellular Vesicles (ISEV). EV-hNPCs function enrichment analysis of the proteomic results showed that these vesicles presented abundant proteins directly involved in neuronal biological processes such as plasticity, transduction, postsynaptic density, and overall brain development.Discussion: The results indicate that EVs derived from hNPCs maintain key neural precursor characteristics and exhibit marker variability, suggesting distinct subpopulations. Their structural integrity aligns with ISEV standards, supporting their potential as reliable biological entities. The proteomic analysis highlights their role in neuronal functions, reinforcing their applicability in neurodegenerative research and therapeutic strategies.Conclusion: The EVs were successfully isolated from hNPCs with abundant proteins involved in neuronal processes, making them attractive for acellular therapies to treat neurodegenerative diseases.Keywords: mesenchymal stem cell, Wharton’s Jelly, neurospheres, neural precursor cells, extracellular vesicles