Unraveling the Therapeutic Potential of Small Extracellular Vesicles: A Proteomic Perspective on Heterogeneity, Functionality, and Stability

Abstract

Small extracellular vesicles (sEVs), secreted by all cells, stand at the forefront of cell-free therapy, offering therapeutic and regenerative benefits that can surpass their parent cells. Rich in bioactive cargo with anti-inflammatory and immunomodulatory properties, sEVs offer considerable potential for accelerating wound healing, anti-aging, and dermatological applications. Their promising bioactivity has thus led to increasing commercialization in the cosmeceutical industry. Although characterization studies have confirmed the presence of therapeutic proteins within sEVs, their cargo is highly influenced by multiple factors such as cell type and manufacturing conditions, contributing to the substantial heterogeneity reported in literature. The lack of comparative quantitative analysis on the impact of these factors makes it challenging to optimize them, hindering the manufacture of high-quality sEV-based formulations for commercial use. The rise of proteomic characterization studies and advancements in bioinformatics enable a systematic cross-study analysis, presenting opportunities to link process conditions with sEV composition and potential biological function, enabling broader bioprocess optimization through careful control of process variables. This work presents a meta-analysis of current sEV proteomics literature, screening over 400 articles published in the past 15 years. Among the 52 relevant studies that published their datasets, 40% of all proteins were only identified in a single study, highlighting the high variability of sEVs. To account for variations in cell source, culture medium, and analytical techniques, a mixed effects model was employed. The model, however, was only able to explain 25-60% of the total variance, suggesting that underreported factors, such as induction methods, may have a greater influence on sEV composition than previously believed. In general, mesenchymal stem cell-derived sEVs were found to contain, on average, a greater proportion of proteins involved in wound healing and skin therapeutic pathways, making them promising candidates for therapeutic applications. While current sEV literature was found to be highly variable, a majority of studies used a data dependent acquisition (DDA) approach, which lacks reproducibility and comprehensiveness. Thus, a data independent acquisition (DIA) approach was chosen to characterize the proteome of a commercial sEV product derived from Wharton’s jelly mesenchymal stem cells (WJ-MSCs), manufactured using a specialized protocol to enhance wound healing and skin therapeutic properties. This approach resulted in the identification of substantially more proteins, with 90% overlapping with those identified across multiple studies in literature. Through the development of bioinformatics workflows, sEVs derived from WJ-MSCs were found to have the greatest number of proteins involved in wound healing and anti-aging pathways. Evaluation of the two mass spectrometry acquisition approaches further confirmed the greater reproducibility offered by DIA, underscoring the need for its widespread adoption in sEV research. Analysis of multiple lots and storage at different temperatures revealed the general consistency and stability of sEVs, making them suitable for commercial applications. Time-related degradation had the greatest impact on protein content, indicating the need for future efforts focused on preservation strategies. This work reaffirms the therapeutic potential of MSC-derived sEVs and emphasizes the critical need for standardization in methodologies and reporting practices to enable further optimization of sEV-based formulations.