The effects of cell type and culture condition on the procoagulant activity of human mesenchymal stromal cell-derived extracellular vesicles

Abstract

BACKGROUND Mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) have great potential as a cell-free therapy in wound healing applications. Because EV populations are not equivalent, rigorous characterization is needed before clinical use. Although there has been much focus on their RNA composition and regenerative capabilities, relatively less is known regarding the effects of MSC cell type (adipose tissue [Ad-MSCs] or bone marrow [BM-MSCs]) and culture condition (monolayer or spheroid) on MSC-EV performance, including characteristics related to their ability to promote coagulation, which could determine EV safety if administered intravenously. METHODS The successful isolation of EVs derived from Ad-MSCs or BM-MSCs cultured in either monolayer or spheroid cultures was confirmed by NanoSight (particle size distribution) and Western blot (surface marker expression). Extracellular vesicle surface expression of procoagulant molecules (tissue factor and phosphatidylserine) was evaluated by flow cytometry. Extracellular vesicle thrombogenicity was tested using calibrated thrombogram, and clotting parameters were assessed using thromboelastography and a flow-based adhesion model simulating blood flow over a collagen-expressing surface. RESULTS The MSC cell type and culture condition did not impact EV size distribution. Extracellular vesicles from all groups expressed phosphatidylserine and tissue factor on their surfaces were functionally thrombogenic and tended to increase clotting rates compared to the negative control of serum-free media without EVs. On average, EVs did not form significantly larger or stronger clots than the negative control, regardless of cell source or culture condition. Additionally, EVs interfered with platelet adhesion in an in vitro flow-based assay. CONCLUSION Adipose-derived EVs were more thrombogenic and expressed higher amounts of phosphatidylserine. Our findings suggest that, like intact MSCs, source variability among EVs is an important factor when considering EVs for potential therapeutic purposes. LEVEL OF EVIDENCE Therapeutic care management, level II.

Comprehensive Proteomic Analysis of Exosomes Derived from Human Bone Marrow-, Adipose Tissue-, and Umbilical Cord- Mesenchymal Stem Cells

“…Our data further con rm that exosomes play a role in immunity primarily by the regulation of leukocyte-mediated immunity. In addition, based on GO analysis, AT-MSC exo may facilitate clotting, which is consistent with the ndings of Chance et al [43]. However, the clinical application for this aspect is still rare.…”

Extracellular Vesicles Derived From Platelets, Red Blood Cells, and Monocyte-Like Cells Differ Regarding Their Ability to Induce Factor XII-Dependent Thrombin Generation

“…This different ability of EVs derived from platelets and unstimulated monocyte-like cells to support coagulation under conditions allowing for contact activation led us to hypothesize that EVs from different cell types or from different environments, such as blood vs. cell culture settings, differ regarding their procoagulant characteristics. In support of this hypothesis, recent evidence from studies on human mesenchymal stromal cellderived extracellular vesicles suggests that the ability of EVs to trigger coagulation depends on the type and state of their parent cells (Christy et al, 2017;Chance et al, 2019).…”

Mesenchymal Stromal Cell Bioreactor for Ex Vivo Reprogramming of Human Immune Cells

“…Hours after conventional IV infusion, MSCs cannot be found in circulation [13][14][15][16] . In addition, different infused MSC products have been shown to display varying levels of procoagulant activity (via the expression of tissue factor) resulting in an instant innate immune attack that can compromise the safety and efficacy of the MSCs [17][18][19] . With these issues in mind, we have been developing an engineered approach to deliver MSC therapy, where the MSCs are inoculated in an ex vivo system, allowing the cells and the blood to communicate bi-directionally through distinct compartments 20 .…”