Study On The Effect And Molecular Mechanism Of Small Extracellular Vesicles On The Homeostasis Of Aging Mesenchymal Stem Cell

Research backgroundMesenchymal stem cells(MSCs)are essential players in multiple physiological processes in vivo and is a promising cell-based therapy for various diseases.Nonetheless,MSCs suffer from senescence with expansion culture.Senescent MSCs(Sen MSCs)exhibit irreversible cell cycle arrest and activation of the senescence-associated secretory phenotype(SASP)program,leading to a limitation for their clinical application.Small extracellular vesicles(sEV)are lipid bilayer membrane vesicles less than 200 nm in diameter secreted by cells.Currently,MSCs-derived sEV(MSC-sEV)have been studied mainly for their clinical therapeutic functions due to their outstanding advantages of low immunogenicity and high biosafety.However,the biological functions and related mechanisms of sEV in senescent exosome-secreting cells have not been well understood,especially in Sen MSCs.Research objectivesMSCs senescence severely limits their clinical application prospects.sEV has been reported to be involved in the regulation of cellular senescence.However,the biological role of sEV in Sen MSCs are poorly understood.Therefore,we investigated the biological functions and related mechanisms of sEV in the regulation of senescence in a replicative senescence model of MSCs.Research methodsFirst,we established a replicative senescence MSC model in vitro by serial passage.To identify the senescence of MSCs,senescence-associated β-galactosidase staining(SAβ-gal),qRT-PCR and Western blotting were used to examine the expression of senescence biomarkers.Then,we compared the MSC immunophenotype,adipogenic and osteogenic differentiation potential and SASP levels between Pre-Senescence MSCs(Pre-Sen MSCs)and Sen MSCs by flow cytometry,induction of adipogenic and osteogenic differentiation,Oil-red O staining,ALP staining,Alizarin red staining and ELISA.Next,sEV was isolated from conditioned medium of Pre-Sen MSCs or Sen MSCs using ultrafiltration,characterized by transmission electron microscopy(TEM),western blotting and nanoparticle tracking analysis(NTA),and quantified by NTA and BCA assay.Previous studies shown that sEV may act as cellular garbage bags to expel cellular metabolic wastes.Therefore,we speculated that the increased secretion of sEV from Sen MSCs may be aimed at maintaining cellular homeostasis.To prove this speculation,sEV secretion was inhibited by GW4869 and Sporoepoxide,and Annexin V-FITC/PI flow cytometry,yH2Ax immunofluorescence staining and CCK-8 assay were used to detected apoptosis,DNA damage and cell viability,respectively.Then,metabolomics profiling was performed on Pre-Sen MSCs derived sEV(Pre-Sen-sEV)and Sen MSCs derived sEV(Sen-sEV)to further investigate the molecular mechanisms by which sEV maintain Sen MSCs homeostasis.ResultsWhen MSCs were cultured to passage 16,the percentage of S A β-gal staining positive cells significantly increased to more than 80%,and expressed senescence-related biomarkers,indicating the successful establishment of the replicative senescence MSC model in vitro.We compared the phenotypic changes between Pre-Sen MSCs and Sen MSCs,and found that Sen MSCs exhibited a diminished adipogenic and osteogenic differentiation potential,and elevated SASP levels.Quantitative analysis of sEV revealed that sEV secretion was increased in Sen MSCs.Inhibition of sEV secretion led to increased apoptosis and DNA damage and decreased cell viability,suggesting that increased sEV secretion plays an important role in maintaining Sen MSCs homeostasis.Mechanistically,metabolomics profiling of Pre-Sen-sEV and Sen-sEV showed that lipid metabolites were significantly increased in Sen sEV,and these significantly up-regulated lipid metabolites have been shown to be toxic for inducing cellular senescence and apoptosis.Furthermore,KEGG analysis revealed that differential metabolites between Pre-Sen-sEV and Sen-sEV were mainly enriched in 25 signaling pathways,of which 21 metabolic pathways have been shown to be closely associated with senescence.ConclusionTaken together,our date indicated that serial passage of MSCs to P16 successfully established the replicative senescence MSC model in vitro,and that Sen MSCs showed a diminished adipogenic and osteogenic differentiation potential,elevated SASP levels.We focused on the sEV secretion of Sen MSCs and found that sEV secretion was increased in Sen MSCs.Inhibition of sEV secretion led to increased apoptosis and DNA damage and decreased cell viability,suggesting that increased sEV secretion plays an important role in maintaining Sen MSCs homeostasis.Metabolomics profiling suggesting that increased exosome secretion may be involved in maintaining Sen MSCs homeostasis at least in part by excreting deleterious lipids and regulating senescence-related metabolic pathways.