Proteomic Analysis of Mesenchymal Stromal Cell-Derived Extracellular Vesicles and Reconstructed Membrane Particles.

Hector Tejeda-Mora,Jeroen Demmers,Carla C Baan,Eleuterio Lombardo,Martin J Hoogduijn,Marlies E J Reinders,Leticia G Leon,Bertram Bleck,Ana Merino

doi:10.3390/ijms222312935

Abstract

Extracellular vesicles (EV) derived from mesenchymal stromal cells (MSC) are a potential therapy for immunological and degenerative diseases. However, large-scale production of EV free from contamination by soluble proteins is a major challenge. The generation of particles from isolated membranes of MSC, membrane particles (MP), may be an alternative to EV. In the present study we generated MP from the membranes of lysed MSC after removal of the nuclei. The yield of MP per MSC was 1 × 105 times higher than EV derived from the same number of MSC. To compare the proteome of MP and EV, proteomic analysis of MP and EV was performed. MP contained over 20 times more proteins than EV. The proteins present in MP evidenced a multi-organelle origin of MP. The projected function of the proteins in EV and MP was very different. Whilst proteins in EV mainly play a role in extracellular matrix organization, proteins in MP were interconnected in diverse molecular pathways, including protein synthesis and degradation pathways and demonstrated enzymatic activity. Treatment of MSC with IFNγ led to a profound effect on the protein make up of EV and MP, demonstrating the possibility to modify the phenotype of EV and MP through modification of parent MSC. These results demonstrate that MP are an attractive alternative to EV for the development of potential therapies. Functional studies will have to demonstrate therapeutic efficacy of MP in preclinical disease models.

Highlights

Mesenchymal stromal cells (MSC) play an important role in immunomodulatory and regenerative processes by interacting with a variety of immune and progenitor cell types
extracellular vesicles (EV) were obtained from 24 h MSC conditioned medium, and membrane particles (MP) were generated from trypsinised MSC (Figure 1)
An important observation was that compared to EV, there was little contamination of MP by soluble proteins

Summary

Introduction

Mesenchymal stromal cells (MSC) play an important role in immunomodulatory and regenerative processes by interacting with a variety of immune and progenitor cell types. A major part of these interactions are mediated via the MSC secretome, which includes a range of soluble immune and trophic mediators [1] and extracellular vesicles (EV) When they were first identified, EV were shown to play a role in controlled shedding of factors to the extracellular space [2], but it has become clear that EV contain functional proteins, microRNAs and even depolarized mitochondria, and carry signals to target cells [3,4,5]. MSC-derived EV have for instance been shown to polarize inflammatory macrophages to regulatory macrophages [8], induce apoptosis of subsets of T lymphocytes [9] and stimulate neuron branching and outgrowth [10]. In addition to their biological effects, MSC-derived EV are reported to modulate disease onset and progression in, amongst others, models of acute kidney injury [11,12], myocardial ischemia [13] and lung injury [14] by MSC-derived EV

Methods

Results

Conclusion