A comprehensive proteomics profiling identifies NRP1 as a novel identity marker of human bone marrow mesenchymal stromal cell-derived small extracellular vesicles

Abstract

BackgroundClinical applications have shown extracellular vesicles (EVs) to be a major paracrine effector in therapeutic responses produced by human mesenchymal stromal/stem cells (hMSCs). As the regenerative capacity of EVs is mainly ascribed to the transfer of proteins and RNA composing its cargo, and to the activity attributed by the protein surface markers, we sought to profile the protein composition of small EVs released from hMSCs to identify hMSC-EV biomarkers with potential clinical relevance.MethodsSmall EVs were produced and qualified from five human bone marrow MSC donors at low passage following a 48-h culture in exosome-depleted medium further processed by steps of centrifugation, filtration, and precipitation. Quantitative proteomic analysis comparing the protein profile of the EVs released from hMSCs and their parental cell was conducted using tandem mass tag labeling combined to mass spectrometry (LC-MS/MS) to identify enriched EV protein markers.ResultsNanoparticle tracking analysis showed no differences in the EV concentration and size among the five hMSC donors (1.83 × 1010 ± 3.23 × 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 commonly found exosomal (CD63/CD81) and hMSC (CD105/CD44/CD146) markers from released EVs in addition to surface mediators of migration (CD29 and MCSP). Quantitative proteomic identified 270 proteins significantly enriched by at least twofold in EVs released from hMSCs as compared to parental hMSCs, where neuropilin 1 (NRP1) was identified among 21 membrane-bound proteins regulating the migration and invasion of cells, as well as chemotaxis and vasculogenesis. Validation by western blot of multiple batches of EVs confirmed consistent enrichment of NRP1 in the nanovesicles released from all five hMSC donors.ConclusionThe identification and verification of NRP1 as a novel enriched surface marker from multiple batches of EVs derived from multiple hMSC donors may serve as a biomarker for the assessment and measurement of EVs for therapeutic uses.

Hollow-fiber bioreactor production of extracellular vesicles from human bone marrow mesenchymal stromal cells yields nanovesicles that mirrors the immuno-modulatory antigenic signature of the producer cell

“…Furthermore, CD40 antigen was detected on EVs, further supporting the immuno-modulatory role of MSC-EVs as the CD40 ligand/CD40 pathway is widely recognized for its prominent role in immune regulation and homeostasis. Our previous work identified MSC-known antigens (CD73, CD90, CD44, CD146, and MSCP) on EVs, as well as integrins (CD29e and CD49), which were also confirmed herein [6] with a different cohort of four MSC donors grown under different culture conditions. These results not only highlight consistency for the expression of these markers among multiple MSC donors and culture conditions, but also further confirm that EV profile mirrors the one of the cell origin, which further reinforces the EV-to-cell mirroring notion previously described [6,53,54].…”

“…EVs are cell-released nano-sized membrane vesicles that can distribute systematically and cross the blood-brain barrier to act as mediators of cell-cell communication [3][4][5]. Through the transfer of their bioactive payload (cytokines, growth factors, signaling lipids, mRNAs, and regulatory miRNAs) and/or through the binding via their membrane-bound molecules (receptors, lipids, integrins, and glycans), EVs can regulate cell/tissue responses, and more broadly, have immune-modulating effects [6][7][8][9][10]. Their native membrane constituents and intrinsic abilities to be transferred from one cell to another may play a role in their enhanced bioavailability and lower immunogenicity and prospects as a new biological nanoplatforms for drug delivery or diagnostic purpose [11,12].…”

Paracrine Mechanisms of Mesenchymal Stromal Cells in Angiogenesis

“…More recently, a proteomic analysis identified significant enrichment of hundreds of proteins in BM-MSC-derived EVs compared to parental BM-MSCs. Among these proteins, neuropilin 1 (NRP1) is interestingly known to regulate vasculogenesis, chemotaxis, migration, and invasion [59]. Such comprehensive proteomic techniques may be used to ultimately unravel the MSCderived EV content and assess their angiogenic potential.…”

Mesenchymal stem cells secretome: The cornerstone of cell-free regenerative medicine

“…Proteomic analyses have provided further information on the protein contents of EVs in the last few years. In 2019, Munshi et al [ 119 ] carried out a quantitative proteomic analysis comparing the protein profile of MSC-EVs and their parental cells. This study established a list of 270 proteins enriched in the EVs by at least two-fold.…”

“…Although the main function of NRP1 is related to neural development, vascular development involvement has been proved as well, since its interaction with several members of the VEGF family induce cell migration resulting in pro-angiogenic activity. NRP1 also regulates platelet-derived growth factor receptors (PDGFR) and NRP-1/PDGFR cross-talk is crucial for vascular remodelling[ 119 ]. A summary of the main proteins in the EVS cargo with a role in bone homeostasis is shown in Table 1 .…”