fig3

Figure 3. miRNA profiling of ASC-derived EVs and sASC-EVs. (A) Venn diagram summarizing miRNAs detected by NGS across five donors. A total of 749 distinct miRNAs were identified in ASC-EVs and 285 in sASC-EVs, with 271 miRNAs shared between the two groups, 478 detected exclusively in ASC-EVs, and 14 uniquely identified in sASC-EVs; (B) Heatmap of the Top-30 most abundant miRNAs ranked according to expression levels in sASC-EVs. Colors represent average miRNA expression after Rlog transformation. The majority of these highly expressed miRNAs were also among the most abundant in ASC-EVs, indicating strong concordance between pre- and post-sorted EV preparations; (C) PCA based on Rlog-transformed expression values of the 271 shared miRNAs, showing clear discrimination between ASC-EVs and sASC-EVs despite the overall high correlation in miRNA expression profiles; (D) Volcano plot depicting differential miRNA expression between sASC-EVs and ASC-EVs. The significance threshold was set at -log10 adjusted P-value > 1.3 (adjusted P < 0.05), n = 5 donors, identifying 38 differentially expressed miRNAs, with upregulation in sASC-EVs as the predominant effect of sorting; (E) Count distribution analysis of the 478 miRNAs detected exclusively in ASC-EVs. Bars indicate the number of miRNAs showing zero counts in 3, 4, or all 5 post-sorting sASC-EV samples, supporting their classification as absent in sASC-Evs; (F) Heatmap of ASC-EV-specific miRNAs, displaying average Rlog-transformed expression levels across donors. Color intensity reflects relative abundance, with red and blue indicating higher and lower expression, respectively, and highlighting the generally low abundance of these miRNAs (Rlog < 6). ASC: Adipose-derived stromal cell; EVs: extracellular vesicles; sASC: sorted adipose-derived stromal cell; miRNA: microRNA; NGS: next-generation sequencing; Rlog: regularized log transformation; PCA: principal component analysis.