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.