Comprehensive Proteomic Analysis and Characterization of Human Bone Marrow Mesenchymal Stem/Stromal Derived Extracellular Vesicles

Abstract

Extracellular vesicles (EVs) are considered to be a major paracrine effector in therapeutic responses produced by human mesenchymal stromal/stem cells (hMSCs). As the hMSC-EV regenerative capacity is mainly ascribed to the transfer of proteins and RNA composing the EV cargo and to the activity attributed by the protein surface markers, we sought to profile the protein composition of hBM-MSC-EVs using a quantitative proteomics analysis. hBMMSC-EVs were produced from 5 hBM-MSC donors characterized by Nanoparticle Tracking Analysis showed no differences in the hBM-MSC-EV concentration and size among the 5 donors (1.83 x 10¹⁰ ± 3.23 x 10⁹ /mL), with the mode particle size measuring at 109.3 ± 5.7 nm. Transmission Electron Microscopy confirmed the presence of nanovesicles with bilayer membranes. Flow cytometric analysis identified EVs expressing exosomal tetraspanins (CD63/81/9) and western blot analysis confirmed an enriched expression of MMP-2 and HSP90B1 in hBM-MSC-EVs. Quantitative proteomic analysis performed using Tandem Mass Tag labeling combined to LC-MS/MS identified 5108 proteins in parental hMSCs versus 782 proteins in hBM-MSC-EVs, of which 270 proteins were enriched by at least 2- fold in hBM-MSC-EVs vs hBM-MSCs. Proteomic analysis also confirmed the presence of known exosomal tetraspanins (CD63/151), integrins (alpha 5/CD49e and beta 1/CD29), and adhesions molecules such as Cadherin 5 type 2, as well as novel surface proteins such as NRP1 involved in cellular movement pathways important in migration and invasion of cells, as well as chemotaxis and vasculogenesis. Our hBM-MSC-EV production workflow and proteomics profiling of the protein composition of EVs from multiple hBM-MSC donors has yielded not only commonly reported exosomal and hMSC markers, but also novel mediators of cell-cell interactions which may help to unravel hMSC’s mechanism of action.