Small RNA-seq of small extracellular vesicles derived from adipose-derived stem cells of young and old donors at different in vitro expansion stages

Adipose-derived stem cells (ASCs) and their small extracellular vesicles (sEVs) hold significant potential for regenerative medicine due to their capabilities for tissue repair. However, the effects of ASC heterogeneity on sEV characteristics and microRNA (miRNA) profiles remain unclear. We investigated ASCs heterogeneity and its changes with aging and cell expansion using bulk RNA-seq from young and old donors across various passages. Non-negative matrix factorization (NMF) was used to identify distinct ASC clusters. Small RNA sequencing was performed on sEVs collected from ASC culture supernatants to analyze miRNA profiles. NMF analysis revealed ASC heterogeneity, identifying clusters characterized by ISLR and genes associated with collagen and smooth muscle cells. The miRNA profiles of sEVs matched these ASC clusters, showing variations based on donor age and cell expansion. Functional enrichment analysis suggested that these miRNAs may have distinct functional roles. Our findings show that ASC heterogeneity and sEV miRNA content are significantly influenced by donor age and cell expansion. These variations underscore the necessity of selecting specific ASC-derived sEVs. Overall design: To explore how donor age and cell expansion influence the miRNA profiles of sEVs derived from ASCs, we isolated sEVs from ASCs of three young and three old donors at different passages. Small RNA sequencing was performed to profile miRNA content in sEVs and assess the correlation with ASC cluster distributions identified from RNA-seq.