Small RNA sequencing of small extracellular vesicles secreted by umbilical cord mesenchymal stem cells following replicative senescence

Background: Umbilical cord mesenchymal stem cells (UCMSC) are subsets of multipotent stem cells involved in immune modulation, tissue regeneration, and antimicrobial defense. Cellular senescence is associated with the onset of aging-related diseases and small extracellular vesicles (sEVs) are important mediators of senescence and aging. Objective: However, little is known about the role and function of microRNAs (miRNAs) carried by UCMSC-derived sEVs. To analyze the expression profiles of miRNAs secreted by senescent UCMSC, small RNA sequencing of the miRNAs within the sEVs was performed in this study. Methods: UCMSC cultures underwent serial passaging beyond passage number 20 to achieve replicative senescence, which was confirmed by various methods, including increased senescence-associated β-gal staining and cytokine secretion levels. sEVs derived from non-senescent and senescent UCMSC were isolated and characterized by nanoparticle tracking analysis, transmission electron microscopy, and immunoblot analysis. Results: Small RNA sequencing of the miRNAs within the sEVs revealed senescence-associated differences in the miRNA composition, as shown by the upregulation of miR-122-5p and miR-146a-5p, and downregulation of miR-125b-5p and miR-29-3p. In addition, total RNA sequencing analysis showed that PENK, ITGA8, and TSIX were upregulated, whereas AKR1B10, UNC13D, and IL21R were downregulated by replicative senescence in UCMSC. In sEVs, upregulated genes were linked to downregulated miRNAs, and vice versa. In the gene-concept network analysis, five gynecologic terms were retrieved. Conclusions: The study provides an insight into the cellular characteristics of UCMSC following replicative senescence and emphasizes the importance of monitoring passage numbers of UCMSC for further therapeutic use.