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.