Abstract
IntroductionThis study assessed whether mesenchymal stem cell (MSC)-derived extracellular vesicles influenced ageing and pluripotency markers in cell cultures where they are added.MethodsMSC-derived extracellular vesicles from old and young rat bone marrows were isolated by ultracentrifugation and were characterised by western blotting, nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). They were added to young and old MSC cultures. Real-time quantitative reverse transcription polymerase chain reactions and western blot analysis were performed to check the markers of ageing (vinculin and lamin A), pluripotency markers (Nanog and Oct4) and components of the mTOR signalling pathway (Rictor, Raptor, AKT and mTOR) in these cell populations. Subsequently, microRNA (miR)-188-3p expression was transiently inhibited in young MSCs to demonstrate the influence of mTOR2 on MSC ageing.ResultsIncubation with young MSC-derived extracellular vesicles decreased the levels of ageing markers and components of the mTOR pathway and increased the pluripotency markers from old MSC populations. By contrast, incubation of young MSCs with old MSC-derived extracellular vesicles generated the reverse effects. Inhibition of miR-188-3p expression in young MSCs produced extracellular vesicles that when incubated with old MSCs produced an increase in the levels of Rictor, as well as a decrease of phosphor-AKT, as indicated by a significant decrease in beta-galactosidase staining.ConclusionsMSC-derived extracellular vesicles affected the behaviour of MSC cultures, based on their composition, which could be modified in vitro. These experiments represented the basis for the development of new therapies against ageing-associated diseases using MSC-derived extracellular vesicles.
Highlights
This study assessed whether mesenchymal stem cell (MSC)-derived extracellular vesicles influenced ageing and pluripotency markers in cell cultures where they are added
There are two signalling complexes, mammalian target of rapamycin (mTOR) complex 1 and mTOR complex 2. mTORC2 plays an important role in endothelial senescence, evident by an increase in binding of Rictor, which is an essential component of mTORC2directed phosphorylation of mTOR at Ser2481 and AKT, producing an increase of β-galactosidase staining in senescence [7]
MicroRNA-188-3p has been shown to be a key regulator of age-associated bone loss in the bone marrow through its target on Rictor [8], and insights into the mechanism involved in MSC ageing suggest that interventions with miRNAs could modify the function of MSCs and their derived extracellular vesicles [4]
Summary
This study assessed whether mesenchymal stem cell (MSC)-derived extracellular vesicles influenced ageing and pluripotency markers in cell cultures where they are added. We demonstrated that immune profiles of MSC-derived EVs have age-dependent differences and they can be modified using miRNA [4]. MicroRNA (miR)-188-3p has been shown to be a key regulator of age-associated bone loss in the bone marrow through its target on Rictor [8], and insights into the mechanism involved in MSC ageing suggest that interventions with miRNAs could modify the function of MSCs and their derived extracellular vesicles [4]. This study assessed whether EVs could modify MSC markers of ageing in vitro and whether Rictor has a key role through the mTORC2 pathway involving the ageing process
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