3.3 MicroRNA profiles of EVs during expansion and differentiation
Among the variety of molecules that are contained in EVs, miRNAs have attracted the most attention because of their regulatory roles in gene expression [45]. For this reason, we determined the content of miRNAs within our EVs. We then moved onto next-generation sequencing to determine the miRNA profiles contained within our EVs. A total of 306 miRNAs were identified in all samples (Table S1) . Clustering analysis of the miRNA expression in the six groups of EVs revealed marked differences in miRNA expression, and the software system was able to determine biological pathways that were significantly impacted by the identified miRNAs (Figure 4A and Table S2) . Among the differentially expressed miRNAs in D0 versus D21 (Table S3),miR-186, miR-210, miR-181c-5p, and miR-146a-5p were increased two-fold and miR-133 and miR-485 were decreased two-fold in the last stage. Meanwhile, miR-10, miR-27a, miR-192 increased which have been reported as late markers of osteogenesis, and miR-31 decreased. In addition, miR-10 and miR-138 were expressed six-fold higher in BMSCs versus PSCs(Figure 4B, C) . The top 34 KEGG pathways were also reported which indicated the potential upregulated target genes of the 301 miRNAs that were identified in both BMSC and PSC EVs (Figure S1) . Of the potential KEGG pathways analyzed Wnt signaling was of particular interest due to its role in osteogenic differentiation of MSCs [46].