Proteomic Profiling of Mesenchymal Stem Cell-Derived Extracellular Vesicles: Impact of Isolation Methods on Protein Cargo

Abstract

Extracellular vesicles (EVs) are nanosized vesicles with a lipid bilayer that are secreted by cells and play a critical role in cell-to-cell communication. Despite the promising reports regarding their diagnostic and therapeutic potential, the utilization of EVs in the clinical setting is limited due to insufficient information about their cargo and a lack of standardization in isolation and analysis methods. Considering protein cargos in EVs as key contributors to their therapeutic potency, we conducted a tandem mass tag (TMT) quantitative proteomics analysis of three subpopulations of mesenchymal stem cell (MSC)-derived EVs obtained through three different isolation techniques: ultracentrifugation (UC), high-speed centrifugation (HS), and ultracentrifugation on sucrose cushion (SU). Subsequently, we checked EV marker expression, size distribution, and morphological characterization, followed by bioinformatic analysis. The bioinformatic analysis of the proteome results revealed that these subpopulations exhibit distinct molecular and functional characteristics. The choice of isolation method impacts the proteome of isolated EVs by isolating different subpopulations of EVs. Specifically, EVs isolated through the high-speed centrifugation (HS) method exhibited a higher abundance of ribosomal and mitochondrial proteins. Functional apoptosis assays comparing isolated mitochondria with different EV isolation methods revealed that HS-EVs, but not other EVs, induced early apoptosis in cancer cells. On the other hand, EVs isolated using the sucrose cushion (SU) and ultracentrifugation (UC) methods demonstrated a higher abundance of proteins primarily involved in the immune response, cellLJcell interactions, and extracellular matrix interactions. Our analyses unveil notable disparities in proteins and associated biological functions among EV subpopulations, underscoring the importance of meticulously selecting isolation methods and resultant EV subpopulations based on the intended application.

Application of Multiomics Approach to Investigate the Therapeutic Potentials of Stem Cell-derived Extracellular Vesicle Subpopulations for Alzheimer’s Disease

“…Daps for each EV subpopulation were identified using one-way ANOVA followed by a multiple comparison analysis with Tukey's multiple comparison test in SPSS (26.0), and P-values <0.05 were considered statistically significant herein instead of doing multiple test correction analysis; we used log2 fold change cutoff > |0.263| as the additional criteria of defining DAPs 16 . EVs were classified into three different subpopulations with different sizes and size distributions that were isolated through different isolation methods, including high-speed centrifuge (HS-EVs) with higher size distribution ranging from 50-1500 nm, ultracentrifuge (UC-EVs) with size distribution ranging from 100-300 nm and ultracentrifugation on sucrose cushion (SU-EVs) with size distribution of 300-1500 nm 16 . The EV subpopulation was compared in binary mode, and we included two-sided comparisons to facilitate comparison with AD data.…”