Small Extracellular Vesicles Maintain Homeostasis of Senescent Mesenchymal Stem Cells at Least Through Excreting Harmful Lipids

Mesenchymal stem cells (MSCs) play an essential role in multiple physiological processes in vivo and a promising cell-based therapy for various diseases. Nonetheless, MSCs suffer from senescence with expansion culture, leading to a limitation for their clinical application. Recently, it was reported that small extracellular vesicles (sEVs) are involved in regulation of senescence in tumor cells and fibroblasts. However, the biological roles of sEVs in senescent MSCs (Sen MSCs) are poorly understood. In this study, we established a replicative senescence model of MSCs by successive passages and compared the phenotypic changes between presenescent MSCs (Pre-Sen MSCs) and Sen MSCs and found that Sen MSCs exhibited a diminished adipogenic and osteogenic differentiation potential and elevated senescence-associated secretory phenotype levels. In addition, we found that sEV secretion was increased in Sen MSCs, and inhibition of sEV secretion led to apoptosis, DNA damage, and decreased cell viability, suggesting that increased sEV secretion plays an important role in maintaining Sen MSC homeostasis. To further investigate the molecular mechanisms, metabolomic profiling of Pre-Sen MSC-derived sEVs (Pre-Sen-sEVs) and Sen MSC-derived sEVs (Sen-sEVs) was performed. The results showed that lipid metabolites were significantly increased in Sen-sEVs and these significantly upregulated lipid metabolites were shown to be toxic for inducing cellular senescence and apoptosis in previous studies. Kyoto Encyclopedia of Genes and Genomes analysis revealed enrichment of differential metabolites between Pre-Sen-sEVs and Sen-sEVs mainly in 25 signaling pathways, of which 21 metabolic pathways have been shown to be closely associated with senescence. Taken together, our findings suggested that increased sEV secretion maintains Sen MSC homeostasis, at least in part, by excreting harmful lipids, thus providing new insights into the regulation of senescence by sEVs.