Extracellular vesicle bioactivity and potential for clinical development are determined by mesenchymal stromal cell clonal subtype

Abstract

Background Mesenchymal stromal cells (MSCs) have been used in numerous clinical trials but very few reach phase 3 or market authorisation. Progress is often hampered by the use of non-clonal, heterogeneous and uncharacterised MSC cultures and lack of mechanistic understanding. There is limited evidence of MSC engraftment in vivo and disease resolution may be the result of the paracrine effects of the MSC secretome, rather than the cells per se. Extracellular vesicles (EVs) are key components of the MSC secretome and there is growing interest in the use of EVs as cell-free therapies. However, like MSCs, heterogeneity can exist within any therapeutic EV pool. Here we used immortalised clonal MSC lines, termed Y201 and Y202, to examine how MSC phenotype influences EV character and function. Methods EVs were isolated by ultracentrifugation and characterised by nano-sizing, ultrastructural morphometric analysis, western blotting, mass spectrometry and miRNA screening. Bioactivity was determined by phosphorylation of ERK1/2, proliferation and T cell polarisation assays and using two in vivo models of inflammatory disease. Results EVs from Y201 and Y202 MSCs were morphologically similar, however, Y201 EVs were more abundant in EV biomarkers versus Y202 EVs, with an enhanced miRNA and proteomic content. Computational analysis of the Y201 EV proteome identified significant enrichment in matrix-associated proteins, predicted to contribute to an elaborate EV corona particularly abundant in RGD-containing proteins fibronectin and MFG-E8, which was confirmed by western blotting. Y201 EVs, but not Y202 EVs, significantly increased the proliferation of articular chondrocytes in a dose-dependent manner, and the proliferative effect of Y201 EVs was mediated at least in part via an RGD (integrin)-FAK-ERK1/2 axis. Both Y201 and Y202 EV subsets significantly reduced proliferative index scores of activated T cells. However, only Y201 EVs, not Y202 EVs, suppressed disease activity compared to controls in different in vivo models of inflammatory peritonitis and arthritis.

1. Introduction

Mesenchymal stromal cells (MSCs, often referred to as mesenchymal stem cells) can exhibit trilineage differentiation capacity (osteogenic, chondrogenic and adipogenic) and immuno-suppressive properties [1–3]. Largely based on these biological functions, MSCs have been used in numerous clinical trials targeting musculoskeletal and inflammatory disorders but the range of clinical indications is very broad and success is variable [4–8]. Although phase 1 and 2 clinical trials continue to rise, very few reach phase 3 [4, 7]. Progress has been hampered largely by the use of non-clonal, heterogeneous and uncharacterised MSC cultures and lack of mechanistic understanding [5]. In addition, there is limited evidence of MSC engraftment in vivo [9, 10], and disease resolution may be the direct result of MSC apoptosis [11] and/or the paracrine effects of the MSC secretome, rather than the cells per se [12–17].

Key components of the MSC secretome are extracellular vesicles (EVs). The term EV describes a heterogeneous population of secreted membrane vesicles [18] that have the ability to exchange biological components between cells, thereby acting as signalling vehicles [19–21]. There are different types of EVs, frequently categorised as exosomes, microvesicles (MVs), and apoptotic bodies based on their secretory process and size [18, 22]. Exosomes have a typical diameter of 30–150 nm [23] and are formed during the maturation of multivesicular bodies (MVBs). Biogenesis of intraluminal vesicles occurs by the inward budding of the limiting membrane of the MVB followed by scission [24, 25]. Intraluminal vesicles are released as exosomes into extracellular space during the fusion of the MVB with the plasma membrane [26]. In contrast, MVs are formed by the outward budding of the plasma membrane [27] ranging in size from 100 nm to 1000 nm in diameter [23]. Apoptotic bodies are larger membrane-bound fragments (50–5000 nm in diameter) that are generated as cells undergo apoptosis [23]. Apoptotic bodies are no longer considered the inert debris of dying cells, indeed they have important functions in many aspects of tissue homeostasis [28].