3.4. EV-stimulated osteogenic differentiation of BMSCs
EVs derived from PSCs and BMSCs during different stages of
differentiation were applied as stimuli to treat undifferentiated BMSCs.
After 7 and 14 days of treatment, the ALP activity was measured as an
early indicator of cell differentiation towards the osteogenic lineage(Figure 5). Both BMSC-D21 and PSC-D21 EVs significantly
increased ALP activity at 7 days of culture in comparison to the
positive control in OM. At 14 days, almost all EV groups showed a
significant increase in ALP activity, except for BMSC-D0.Alizarin Red S staining (Figure 6A) and quantification assay(Figure 6B) demonstrated that calcium deposition significantly
increased after 14-day induction by all EVs in comparison to the
positive control in OM.
4. Discussion
The therapeutic potential of BMSC-derived EVs has been well documented
[47-50]. Their role in bone remodeling has been investigated bothin vitro and in vivo, and it has been shown that many of
the key factors for regulating bone remodeling are targeted by miRNAs
contained within the EVs [51, 52]. Researchers have focused mainly
on utilizing these EVs and their biologically active content to address
pathological bone diseases such as osteoporosis, but these EVs also
exhibit the potential to improve stem cell differentiation within
tissue-engineered bone scaffolds due to their ability to accelerate bone
mineral deposition. Most studies utilize EVs derived from BMSCs or
mineralizing osteoblasts to achieve this effect, so there exists a lack
of knowledge on utilizing alternate cell sources to harvest EVs with the
potential to improve bone mineral formation [53].
In this study, we compared EVs derived from BMSCs and PSCs during the
process of osteogenic differentiation in an attempt to better understand
the role that cell source and culture conditions have on the therapeutic
effect of these EVs. It has been shown that the miRNA profile within EVs
changes depending on the stage of differentiation [54]; however,
this effect has not been studied while comparing different mesenchymal
stem cells. We isolated and characterized EVs derived from BMSCs and
PSCs, and investigated the differentiation capabilities of EVs secreted
under different stages of osteogenic differentiation. Within this study,
we aimed to determine the effects of both cell type and differentiation
stage, on EV content, and the downstream effect on the regulation of
osteogenic differentiation [54]. By doing so, we have proven the
feasibility of utilizing alternative cell sources of EVs for future
studies involving osteogenic differentiation.
The isolated EV groups did not exhibit any statistically significant
differences in size, regardless of cell source. This confirms the
consistency of the isolation process utilized for EV separation. When
comparing between differentiation time within the same cell source, the
isolated EVs exhibited larger size distributions at D21 compared to D0
with p = 0.004 for BMSC-derived and p =0.0369 for
PSC-derived EVs. This may be due to the larger cell membrane surface
area available for EV formation since the cell morphology of MSCs become
less spindle-like due to the cells differentiating into osteoblast-like
cells [55].
Characterizing the protein concentration between cell groups yielded
large differences between cell source with the PSC-derived EVs
consistently yielding higher protein concentrations. When analyzing the
lipid concentrations of each EV group, we found that the PSC-derived EVs
also had significantly higher lipid concentrations at all time points
with the exception of D21. The trend of isolating both higher protein
concentrations and lipid concentrations from PSCs suggests that there
was a larger total yield of EVs from these cells when compared to BMSCs.
When comparing the protein to lipid ratio, which is a common quality
assurance step to ensure that the isolation method is not co-isolating
unwanted proteins, the PSC-derived EVs had slightly higher protein to
lipid ratios than BMSCs. All groups fell within an acceptable range of a
protein to lipid ratio < 5, which was previously described by
Osteikoetxea et al [56]. Another interesting result is that as the
time of differentiation increased, the PSCs yielded lower lipid
concentrations while the lipid concentration increased for BMSCs. Taken
together with the in vitro data, which dosages were based off of
protein concentrations, this implies that the PSC-derived EVs required a
lower total number of EVs to achieve the same targeted differentiation
effect as the BMSC-derived EVs.
The ability of PSC-derived EVs to be internalized by BMSCs was an
important aspect of this study, suggesting that the EVs are not
internalized by a cell-specific pathway. It was beyond the focus of this
study to identify the individual surface proteins and glycoproteins
expressed on each EV group or to determine the efficiency at which these
EVs were internalized. But, by fluorescently labeling the EV membrane it
was observed that the EVs were internalized by cells in vitroregardless of cell source or differentiation time. Taken in combination
with the results of the differentiation experiments, it can be assumed
that EVs were internalized efficiently enough to achieve the targeted
effect.
It was outside this study’s scope to experimentally confirm the effect
of each miRNA identified within our EVs. We observed a trend within both
PSC and BMSC-derived EVs that resulted in the improved mineral
deposition in 2D in vitro culture when delivering EVs from cells
that were exposed to longer osteogenic differentiation times. This hints
that the trend for miRNA profiles observed in BMSCs between stages of
differentiation is consistent within PSCs as well [54]. It is
unclear whether these miRNAs are solely responsible for the osteogenic
differentiation effect identified in the D21 group of EVs. It is much
more likely that the effects observed are a result of a combination of
bioactive molecules contained within the EVs, including these miRNAs.
Regardless, we identified the relative expression levels of over 300
miRNAs and analyzed the differences in expression between cell source
and stage of differentiation in an attempt to gain insight on whether
these EVs are comparable in terms of miRNA profile.
Among the the miRNAs differentially expressed between PSCs and BMSCs,
miR-10b was dramatically increased in EVs from the D21 BMSC-derived EVs
and was about 123 folds higher than PSCs (Table S1 ). The
TargetScan 7.2 software, which predicts biological targets of miRNAs,
precited that miR-10b can affect ID4 and indirectly promote osteoblast
differentiation by enhancing RunX2 transcriptional activity [57].
Furthermore, it has been reported that miR-10b promotes the migration of
MSCs into the bone microenvironment [33, 58]. It was also determined
that the expression level of miR-21 increased 2 fold higher late-stage
PSC and BMSC EVs in comparison to D0 EVs (Table S1). The
upregulation of miR-21 promotes osteogenesis through the
PI3K/AKT/B-actin pathway and has effects on the mRNA expression RUNX2,
ALK, and OCN [59]. MiR-126-5p has also been found to regulate
osteoclast differentiation and plays a role in bone remodeling through
the inhibition of MMP-13 [60]. Pathway analysis revealed the
differentially expressed miRNAs targeted genes through multiple
signaling pathways such as Wnt signaling (Figure S1),MAPkinase, and TGFβ pathways (Table S2). The varied,
non-consistent miRNA expressions between cell type and differentiation
stage suggest a much more general trend towards osteogenesis, through
many different pathways, as opposed to the expression of specific
osteo-miRNAs that affect individual genes or pathways.
There were 3 miRNAs associated with increased osteogenesis that were
expressed significantly higher in late-stage PSCs when compared to
BMSCs. Most were associated with promoting osteoblast differentiation
such as miR-146, miR-515, and miR-520a (Table S3). The PSCs
also significantly expressed two miRNAs, miR-512 and miR516b that were
associated with inhibiting osteogenic differentiation and downregulating
osteoblast differentiation, respectively. There were also various other
osteo-related miRNAs that were downregulated at the later stages of
differentiation such as miR-133 which is associated with the inhibition
of osteoblast differentiation [61]. Although its expression
decreased over time, it was still expressed approximately 2-fold higher
at late-stage PSC EV in comparison to BMSC EV. Other negative regulators
of osteogenesis, such as let-7i, were found to be 6-fold higher in BMSC
EVs than PMSC EVs while still maintaining the trend of decreasing
expression at later time points of differentiation. The importance of
the expression levels of individual miRNAs remains unclear until further
studies can identify their specific functions. When analyzing the trends
of osteo-related miRNA, these miRNA expression levels are still
consistent with the general trend towards increased osteogenesis in the
late-stage EVs regardless of cell source. This was confirmed within vitro uptake and differentiation experiments.
Based on our in vitro experiments, EVs derived from late-stage
differentiation were able to achieve faster differentiation rates and
improved mineral deposition when compared to our control. This is the
result of delivering osteogenic miRNA as well as other potential
osteogenic factors contained within the vesicles. Although the cell
source had a large impact on the variety of miRNA contained within the
EVs, the overall osteogenic impact was similar between the groups when
comparing EVs derived at the same stage. All of the in vitroexperiments chosen have been previously used to confirm osteogenic
differentiation [62]. The improved bone mineral deposition resulting
from delivering late-stage PSC and BMSC-derived EVs could potentially
lead to the development of enhanced bone tissue engineering scaffolds or
even improve fracture or non-union healing.
All of this data taken together signifies a new candidate from which to
isolate osteogenic EVs. PSC’s ease of donor availability, fast
proliferation time, and high EV potency improve the likelihood that they
will make a suitable candidate for clinical translation. The ability to
easily scale up the isolation methods for these EVs, combined with their
enhanced osteogenic differentiation rates, demonstrate their viability
for future studies involving bone tissue engineering.