Hypoxia Conditioned Mesenchymal Stem Cell-Derived Extracellular Vesicles Induce Increased Vascular Tube Formation in vitro.

Ciarra Almeria,Viktoria Weber,René Weiss,Dominik Egger,Michelle Roy,Carla Tripisciano,Cornelia Kasper

doi:10.3389/fbioe.2019.00292

Abstract

Mesenchymal stem/stromal cells (MSCs) display a variety of therapeutically relevant effects, such as the induction of angiogenesis, particularly under hypoxic conditions. It is generally recognized that MSCs exert their effects by secretion of paracrine factors and by stimulation of host cells. Furthermore, there is increasing evidence that some therapeutically relevant effects of MSCs are mediated by MSC-derived extracellular vesicles (EVs). Since our current knowledge on MSC-derived EVs released under hypoxic conditions is very limited, we aimed to characterize MSC-derived EVs from normoxic vs. hypoxic conditions (5% O2). Adipose-derived MSCs were grown under normoxic and hypoxic conditions, and EVs were analyzed by flow cytometry using lactadherin as a marker for EVs exposing phosphatidylserine, CD63 and CD81 as EV markers, as well as CD73 and CD90 as MSC surface markers. Particle concentration and size distribution were measured by nanoparticle tracking analysis (NTA), and the EV surface antigen signature was characterized using bead-based multiplex flow cytometry. Furthermore, we evaluated the potential of MSC-derived EVs obtained under hypoxic conditions to support angiogenesis using an in vitro assay with an hTERT-immortalized human umbilical vein endothelial cell (HUVEC) line. Proliferation and viability of MSCs were increased under hypoxic conditions. EV concentration, size, and surface signature did not differ significantly between normoxic and hypoxic conditions, with the exception of CD44, which was significantly upregulated on normoxic EVs. EVs from hypoxic conditions exhibited increased tube formation as compared to normoxic EVs or to the corresponding supernatants from both groups, indicating that tube formation is facilitated by EVs rather than by soluble factors. In conclusion, hypoxia conditioned MSC-derived EVs appear to be functionally more potent than normoxic MSC-derived EVs regarding the induction of angiogenesis.

Highlights

The application potential of mesenchymal stem cells (MSCs) in regenerative cell-based therapies has been gaining substantial interest (Squillaro et al, 2016; Mastrolia et al, 2019)
The population doubling level (PDL) and the cell density were significantly higher under hypoxic conditions
100 and 75% of all extracellular vesicles (EVs) were positive for the MSC markers CD73 and CD90, respectively, while 35 and 40% were positive for the tetraspanins CD63 or CD81

Summary

Introduction

The application potential of mesenchymal stem cells (MSCs) in regenerative cell-based therapies has been gaining substantial interest (Squillaro et al, 2016; Mastrolia et al, 2019). Three EV subgroups have been discriminated according to their size, and biogenesis: (i) apoptotic bodies (>1,000 nm) released during early apoptosis; (ii) microvesicles (100 to 1,000 nm) formed via outward budding of the plasma membrane; and (iii) exosomes (40 to 100 nm) secreted after fusion of multivesicular bodies with the plasma membrane (Cocucci and Meldolesi, 2015) Since these subgroups can overlap in size, and as markers for their unambiguous discrimination are lacking, the generic term extracellular vesicles is used to describe both exosomes and MVs in the context of this study (Thery et al, 2018). Multiplex bead-based flow cytometry has recently been introduced to characterize the surface marker profile of EVs, which mediates the interaction of EVs with their target cells (Wiklander et al, 2018) and is relevant to understand the molecular content and related functions of subsets of EVs and to identifying potential EV subsets with a defined therapeutic activity

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