Cells release diverse types of vesicles constitutively or in response to proliferation, injury, inflammation, or stress. Extracellular vesicles (EVs) are crucial in intercellular communication, and there is emerging evidence for their roles in inflammation, cancer, and thrombosis. We investigated the thrombogenicity of platelet-derived EVs, which constitute the majority of circulating EVs in human blood, and assessed the contributions of phosphatidylserine and tissue factor exposure on thrombin generation. Addition of platelet EVs to vesicle-free human plasma induced thrombin generation in a dose-dependent manner, which was efficiently inhibited by annexin V, but not by anti-tissue factor antibodies, indicating that it was primarily due to the exposure of phosphatidylserine on platelet EVs. Platelet EVs exhibited higher thrombogenicity than EVs from unstimulated monocytic THP-1 cells, but blockade of contact activation significantly reduced thrombin generation by platelet EVs. Stimulation of monocytic cells with lipopolysaccharide enhanced their thrombogenicity both in the presence and in the absence of contact activation, and thrombin generation was efficiently blocked by anti-tissue factor antibodies. Our study provides evidence that irrespective of their cellular origin, EVs support the propagation of coagulation via the exposure of phosphatidylserine, while the expression of functional tissue factor on EVs appears to be limited to pathological conditions.

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.

No abstract

Mesenchymal stem cells (MSCs) are considered as primary candidates for cell-based therapies due to their multiple effects in regenerative medicine. Pre-conditioning of MSCs under physiological conditions—such as hypoxia, three-dimensional environments, and dynamic cultivation—prior to transplantation proved to optimize their therapeutic efficiency. When cultivated as three-dimensional aggregates or spheroids, MSCs display increased angiogenic, anti-inflammatory, and immunomodulatory effects as well as improved stemness and survival rates after transplantation, and cultivation under dynamic conditions can increase their viability, proliferation, and paracrine effects, alike. Only few studies reported to date, however, have utilized dynamic conditions for three-dimensional aggregate cultivation of MSCs. Still, the integration of dynamic bioreactor systems, such as spinner flasks or stirred tank reactors might pave the way for a robust, scalable bulk expansion of MSC aggregates or MSC-derived extracellular vesicles. This review summarizes recent insights into the therapeutic potential of MSC aggregate cultivation and focuses on dynamic generation and cultivation techniques of MSC aggregates.

As transmitters of biological information, extracellular vesicles (EVs) are crucial for the maintenance of physiological homeostasis, but also contribute to pathological conditions, such as thrombotic disorders. The ability of EVs to support thrombin generation has been linked to their exposure of phosphatidylserine, an anionic phospholipid that is normally restricted to the inner leaflet of the plasma membrane but exposed on the outer leaflet during EV biogenesis. Here, we investigated whether EVs of different cellular origin and from different settings, namely platelets and red blood cells from blood bank units and a monocyte-like cell line (THP-1), differ regarding their potential to support factor XII-dependent thrombin generation. EVs were isolated from blood products or THP-1 cell culture supernatants using differential centrifugation and characterized by a combination of flow cytometry, nanoparticle tracking analysis, and Western blotting. Soluble factors co-enriched during the isolation of EVs were depleted from blood-cell derived EV fractions using size exclusion chromatography, while proteins bound to the surface of EVs were degraded by mild protease treatment. We found that platelet-derived and red blood cell-derived EVs supported factor XII-dependent thrombin generation to comparable extents, while monocytic EVs failed to support thrombin generation when added to EV-depleted human plasma. We excluded a major contribution of co-enriched soluble proteins or of proteins bound to the EV surface to the thrombogenicity of blood cell-derived EVs. Our data suggest that the enhanced potential of blood cell-derived EVs to support thrombin generation is rather due to enhanced exposure of phosphatidylserine on the surface of blood cell-derived EVs. Extending these investigations to EVs from other cell types, such as mesenchymal stromal cells, will be crucial for their future therapeutic applications.