Human mesenchymal stromal cell source and culture conditions influence extracellular vesicle angiogenic and metabolic effects on human endothelial cells in vitro

Abstract

BACKGROUND Mesenchymal stem/stromal cell (MSC)-derived extracellular vesicles (EVs) are a possible cell-free alternative to MSCs because they retain the regenerative potential of MSCs, while still mitigating some of their limitations (such as the possible elicitation of host immune responses). The promotion and restoration of angiogenesis, however, is an important component in treating trauma-related injuries, and has not been fully explored with EVs. Herein, we describe the effects of monolayer adipose-derived EVs, spheroid adipose-derived EVs (SAd-EVs), monolayer bone marrow-derived EVs (MBM-EVs), and spheroid bone marrow-derived EVs (SBM-EVs) on human umbilical vein endothelial cell (HUVEC) tube formation and mitochondrial respiration. METHODS The successful isolation of EVs derived from adipose MSCs or bone marrow MSCs in monolayer or spheroid cultures was confirmed by NanoSight (particle size distribution) and Western blot (surface marker expression). The EV angiogenic potential was measured using a 24-hour HUVEC tube formation assay. The EV effects on HUVEC mitochondrial function were evaluated using the Seahorse respirometer machine. RESULTS The number of junctions, branches, and the average length of branches formed at 24 hours of tube formation were significantly affected by cell and culture type; overall adipose-derived EVs outperformed bone marrow-derived EVs, and spheroid-derived EVs outperformed monolayer-derived EVs. Additionally, adipose-derived EVs resulted in significantly increased HUVEC mitochondrial maximal respiration and adenosine triphosphate (ATP) production, while only MBM-EVs negatively impacted HUVEC proton leak. CONCLUSION Adipose-derived EVs promoted HUVEC tube formation significantly more than bone marrow-derived EVs, while also maximizing HUVEC mitochondria function. Results demonstrate that, as with MSC therapies, it is possible to tailor EV culture and production to optimize therapeutic potential. LEVEL OF EVIDENCE Basic or Foundational Research.

A paradigm shift in cell-free approach: the emerging role of MSCs-derived exosomes in regenerative medicine

“…In addition, human embryonic mesenchymal stem/stromal cells (ES-MSCs) could elicit better immunomodulatory effect than BM- and AT-MSCs [ 70 ]. Moreover, EVs derived from AMSCs could promote angiogenesis significantly more than EVs obtained from BMMSCs, as shown by study of their effects on human umbilical vein endothelial cell (HUVEC) tube formation and mitochondrial respiration [ 71 ]. In the context of tumor therapy, BM- and UC-MSC-EVs reduced cell growth, whereas an opposite impacts was detected with AMSC-EVs on U87MG glioblastoma cells line.…”

Mesenchymal Stem Cell-Derived Extracellular Vesicles: Opportunities and Challenges for Clinical Translation

“…However, only few studies compared EVs from MSCs isolated from different sources. It has been recently reported that EVs from adipose tissue-derived MSCs (AD-MSCs) and from cardiac MSCs exhibit more potent angiogenic capacities than bone marrow-derived MSCs (BM-MSCs) (Chance et al, 2020;Kang et al, 2020). In addition, the capacity of EV production and secretory profile are higher in BM-MSCs than AD-MSCs, supporting a differential activity of EVs from different MSCs (Villatoro et al, 2019).…”

Tissue Regeneration Capacity of Extracellular Vesicles Isolated From Bone Marrow-Derived and Adipose-Derived Mesenchymal Stromal/Stem Cells

“…However, in vitro studies do provide insight into a few possible similarities and differences in ADMSCderived vs. BMMSC-derived EVs. For example, although EVs from both MSC sources generally expressed CD63, CD9, and CD81, and were negative for expression of either CD45, CD34, or calnexin (Katsuda et al, 2013;Del Fattore et al, 2015;Gouveia et al, 2015;Gualerzi et al, 2017;Bari et al, 2019;Chance et al, 2019;Villatoro et al, 2019;Chance et al, 2020;Hoang et al, 2020), ADMSC-derived EVs expressed higher levels of CD63, phosphatidylserine (Chance et al, 2019), and ceramides (Gualerzi et al, 2017), while BMMSC-derived EVs displayed more protein types and a higher protein content per cell (Villatoro et al, 2019). EV cargos and resulting in vitro effects were also observed to vary significantly depending on MSC-type.…”

“…For example, ADMSC-EVS expressed higher levels of HGF, whereas BMMSC-derived EVs expressed higher levels of VEGFA, FGF-2, and PDGF-BB and thus induced greater proliferation in dermal fibroblasts (Hoang et al, 2020). In direct comparison studies, ADMSC-derived EVs were shown to promote more HUVEC tube formation (Chance et al, 2020) and display higher thrombogenic activity (Chance et al, 2019) than BMMSC-derived EVs. Meanwhile, BMMSC-derived EVs increased IL-10 secretion by a factor of 1.8 in phytohemagglutinin -activated peripheral blood mononuclear cells compared to ADMSC-derived EVs (Bari et al, 2019).…”