2.5. MicroRNA profiling of EVs
The total miRNA of each EV group was extracted using a miRCURY RNA Isolation Kit (Qiagen, Venlo, Netherlands), according to the manufacturer’s protocol. Total RNA including the miRNA fraction was used as the starting material to prepare cDNA libraries using CleanTagTM Library kit from TriLink Biotechnologies (cat # L-3206). In brief, adapters are ligated to the 5’ phosphate and 3’ hydroxyl groups, reverse transcribed, PCR amplified, and then purified using AMPure® XP Beads. Each cDNA Library was validated and checked for size distribution using a High Sensitivity DNA kit on the Agilent 2100 Bioanalyzer. The quantity of each library was measured using the Qubit 3.0 (Thermo Fisher) and loaded on an Illumina Mid Output 150 cycle kit and sequenced using the Illumina NextSeq 500.
Differentially expressed miRNAs, between PSC-EVs and BMSC-EVs, as well as from the early (D0) and late (D21) stages of osteogenic differentiation were further analyzed to predict their target genes and pathways. The target genes of candidate miRNAs were predicted based on two algorithms, DIANA-microT-CDS and DIANA-TarBase v7.0 [37, 38]. The microT threshold was set at a score of 0.8 when microT-CDS algorithms were utilized for target gene prediction. DIANA-mirPath v.3 (http://www.microrna.gr/miRPathv3) [38] was used to perform the hierarchical clustering of miRNAs, and all known KEGG pathways [39] based on their interaction levels using predicted miRNA targets provided by the DIANA-microT-CDS algorithm [40, 41] and/or experimentally validated miRNA interactions derived from DIANA-TarBase v7.0 [42]. The option gene union in the software was selected to merge the results. The graphical output of the program provides an overview of the pathways modulated by selected miRNAs, facilitating the interpretation and presentation of the analysis results. The statistical significance value associated with the identified biological pathways was calculated automatically by the mirPath software, in which Benjamini and Hochberg’s false discovery rate (FDR) was applied with the significant threshold set at 0.05. The FDR is a statistical method for controlling incorrect rejections of the null hypothesis when conducting multiple comparisons which is common during miRNA analysis. This is in accordance with Wang et al. [43] who reported miRNA pathways utilizing the same software systems.
2.6. EV-stimulated osteogenic differentiation of BMSCs
BMSCs (passage 3) were seeded at a density of 18,000 cells/cm2 in 24-well plates in EV-free OM. After overnight attachment, the culture medium was replaced by media containing EVs. In total, six different EV groups were tested. Both PSC and BMSC EVs isolated at different osteogenic differentiation time points (D0, D7, and D21) were used. The BMSCs were continuously treated with 10 μg/mL EVs in all in vitro experiments and media was changed every 3 days.
2.7. Alkaline phosphatase (ALP) assay
After 7 and 14 days of treatment, BMSCs were rinsed with DMEM and lysed using Triton X-100 (one-step kit, Thermo Fisher Scientific). The ALP activity was measured using p -nitrophenyl phosphate as the substrate. The quantity of p -nitrophenol produced was considered directly proportional to the ALP activity according to the protocol.