Isolation and proteomic profiling of mesenchymal stem cell-derived extracellular vesicles: Unlocking new frontiers in regenerative and translational medicine

Abstract

BACKGROUND

Extracellular vesicles (EVs) are small particles that range between 30-150 nanometers in size and are made of two phospholipid bilayers. The EVs play an important role in cell-to-cell communication to perform functional roles in regeneration. Despite the therapeutic prospects for their use in clinical settings, the use of EVs in standard daily clinical practice is limited, which is due to limited information about their cargo. Even though there are no universally accepted “standardized EV body isolations”, the described methods yield enough isolated cargoes to identify different functional proteins.

AIM

To perform a proteomic analysis of EVs from bone marrow-derived mesenchymal stem cells (BMSCs) and to identify core and unique protein signatures of BMSC-derived EVs, explore the enriched pathways and biological processes they influence, and highlight potential biomarkers or therapeutic targets relevant to regenerative medicine and disease modulation.

METHODS

EVs were isolated from mesenchymal stromal cells derived from the bone marrow of healthy male donors using standard techniques. EVs were identified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein was isolated from the EVs and subjected to liquid chromatography and mass spectrometry analysis using liquid chromatography and mass spectrometry detection. Metascape (https://metascape.org/) was used to perform Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of common proteins in all samples. A minimum overlap of ≥ 2 proteins and a P-value < 0.01 was considered statistically significant. The STRING database was utilized to predict protein-protein interactions for common proteins and intersecting proteins involved in diseases.

RESULTS

Proteomic analysis revealed that 180 proteins were recognized in sample one, 130 in sample two, 113 in sample three, and 170 in sample four. Fifty-three proteins were identified, and their sizes ranged from 11 kDa to 245 kDa. Among all four BMSC-derived EV samples, 53 proteins were consistently present across all samples. The unique protein signatures may reflect donor-specific or culture-specific differences and could represent potential biomarkers for patient stratification if validated in larger datasets. Fifteen of the identified proteins were found to be related to regenerative medicine, and five proteins very important in neurological disease/disorder relevance.

CONCLUSION

Our study reveals that different proteins can be identified and separated from BMSCs, providing a window of opportunity to test the functionality of EVs in trials without directly using BMSCs. It remains to be seen whether these individual EVs will exert the same physiological levels as BMSCs in influencing the behavior of recipient cells in modifying disease processes, affecting the immune system, and facilitating tissue regeneration.

Keywords: Extracellular vesicles; Extracellular vesicle analysis; Proteomics; Extracellular vesicle characterization; Isolation methods

Core Tip: In regenerative medicine, mesenchymal stem cell (MSC)-based therapy is used to repair diseased or damaged tissue. The technology of culturing MSCs is expensive and not available everywhere. MSC-derived extracellular vesicles have gained attention as a potential therapeutic alternative to MSCs themselves. Therefore, scientists should be able to isolate and identify individual proteins and their functionality, which can be useful for therapeutic purposes. We used proteomics to isolate and identify 53 proteins from MSC-derived extracellular vesicles, 15 of which can be targeted in the field of regenerative medicine.