Characterization of MSCs-derived apoVs
Initially, we employed conventional methods to isolate, culture, and characterize mouse BMMSCs. The cells displayed the characteristic morphology (Fig. S1A) and demonstrated colony-forming ability, as well as the capacity for osteogenic and adipogenic differentiation (Fig. S1B-D). Additionally, the cells exhibited positive expression of markers associated with mouse MSCs (CD73, CD90, CD105, CD126, CD166, and Scal-1) and negative expression of markers related to hematopoietic stem cells (CD34, CD45) (Fig. S1E).
Subsequently, we isolated and characterized apoVs derived from mouse MSCs following the methodology outlined in our previous study [36]. To initiate apoptosis in MSCs, we utilized staurosporine (STS) and isolated apoVs through a carefully optimized gradient centrifugation procedure (Fig. S2). The cells displayed typical morphological changes and apoptotic responses after 16 hours of STS treatment, as observed through microscopy, immunofluorescent staining of cleaved-caspase 3, and flow cytometry analysis of annexin V and 7AAD expression (Fig. 1A-D). Representative transmission electron microscopy (TEM) images provided visual confirmation of the characteristic morphology of apoVs (Fig. 1E). To validate the size distribution of apoVs, we conducted a nanoparticle track analysis (NTA) assay (Fig. 1F and G). Moreover, nano flow cytometry and immunofluorescent staining analysis revealed significant expression of integrin alpha-5 (87.7%), calreticulin (75.6%), and calnexin (76.5%) in apoVs (Fig. 1H and I).
WT apoVs rather than OVX apoVs rescued the impaired therapeutic effect of OVX MSCs on osteoporosis
Analysis of X-ray and micro-CT scans revealed a significant decrease in local bone density and cancellous bone mass in the distal femurs of the OVX group compared to the sham group (Fig. 2A and B), indicating successful modeling. Following 4 weeks of WT MSCs treatment, there was a remarkable recovery in bone density, cancellous bone mass, bone volume/total volume (BV/TV), and bone mineral density (BMD) like that of the sham group. However, the BMD only exhibited a slight rebound in the OVX MSCs group, which was significantly lower than that of the WT MSCs group (Fig. 2A-C). These findings suggest that WT MSCs treatment effectively alleviates osteopenia symptoms in OVX mice, whereas treatment with OVX MSCs is ineffective. Importantly, it should be noted that when OVX MSCs were preconditioned in vitro with WT apoVs for 3 days and then systemically infused, there was a substantial increase in bone density, trabecular bone mass, BMD, and BV/TV comparable to the WT MSCs group. In contrast, the OVX apoVs group failed to show a similar effect (Fig. 2A-C). This indicates that in vitro stimulation with WT apoVs restores the impaired capability of OVX MSCs to effectively treat osteoporosis, rather than OVX apoVs.
The HE staining of distal femurs in mice revealed distinct findings. In comparison to the sham group, the OVX group exhibited a reduction in trabecular bone density, with most of the remaining trabecular bone being discontinuous. Additionally, there was a prominent presence of immune cell infiltration in the bone marrow cavity. Conversely, the WT MSCs group demonstrated mostly continuous and interwoven trabecular bone, with a significant increase in both total amount and density. Furthermore, only a small amount of infiltrating immune cells w observed. However, these positive effects were not observed in the OVX MSCs group. Interestingly, the beneficial outcomes of OVX MSCs were nearly equivalent to those of the WT MSCs group when subjected to preconditioning with WT apoVs. Conversely, treatment with OVX apoVs did not yield similar results (Fig. 2D and E).
Given that osteoporosis is a chronic inflammatory disease associated with a prolonged hyperimmune state, we proceeded to investigate the changes in CD3+ T cells, Treg cells (regulatory T cells), and Th cells (helper T cells) subsets following systemic infusion of MSCs. Through ELISA and flow cytometry analysis, we demonstrated that treatment with WT apoVs restored the ability of OVX MSCs to mitigate the overexpressed proinflammatory factors TNF-α, IFN-γ, and IL-17, associating with Th1 and Th17 cells, in the serum of OVX mice. Additionally, WT apoVs treatment reduced the excessive activation of CD3+ T cells and the Th1/Th17 subsets in splenic lymphocytes, while simultaneously elevating the depleted Treg subsets. A similar pattern was observed in the WT MSCs group, but no such effects were evident in the OVX MSCs and OVX MSCs + OVX apoVs group (Fig. 2F and G).
WT apoVs rather than OVX apoVs rescued the therapeutic effect of impaired OVX MSCs on the recovery of recipient MSCs in osteoporotic mice
It is widely acknowledged that stem cells play an indispensable role in maintaining tissue homeostasis [40–42]. Therefore, our next objective was to determine whether MSCs treatment could rescue the impaired recipient MSCs in osteoporotic mice. We isolated BMMSCs from the OVX mice who received systemic MSCs infusion and assessed their key biological functions. The BrdU labeling and continuous passage assays revealed an increase in the proliferation and population doubling rate of MSCs derived from the OVX mice. However, the infusion of MSCs failed to change the proliferation and population doubling rate of MSCs originating from the OVX mice (Fig. 3A and B).
Regarding the differentiation property, we demonstrated that WT apoVs effectively restored the therapeutic impact of OVX MSCs on the restoration of osteogenic and adipogenic differentiation capacity to a similar degree as WT MSCs. However, OVX apoVs did not exhibit such an effect. This was evident from the formation of calcium nodules and lipid droplets, as well as the expression of osteogenic markers including runt-related transcription factor 2 (Runx2) and alkaline phosphatase (ALP), and adipogenic markers, peroxisome proliferator-activated receptor γ (PPARγ) and lipoprotein lipase (LPL) (Fig. 3C-F).
Given the crucial role of their MSCs in regulating the host immune system and maintaining immunological homeostasis [43, 44], we investigated the immunoregulatory capacity of recipient MSCs following the administration of exogenous MSCs. It was observed that preconditioning with WT apoVs, in contrast to OVX apoVs, restored the reparative effect of exogenous OVX MSCs on the recovery of recipient MSCs' immunoregulatory capacity. This was evident in the induction of T cell apoptosis comparable to that showed in the WT MSCs group (Fig. 3G and H).
WT apoVs rather than OVX apoVs recovered the impaired biological functions of OVX MSCs in vitro
Based on the observation that WT apoVs effectively restored the therapeutic effect of OVX MSCs on osteoporosis, we were intrigued to investigate whether apoVs could have a similar effect on damaged cells in vitro. Therefore, we assessed the proliferation, osteogenic and adipogenic differentiation, and immunoregulatory properties of OVX MSCs after 3 days of apoVs treatment. Interestingly, it was found that apoVs did not impact the proliferation and population doubling rate of OVX MSCs (Fig. 4A and B). However, in line with our expectations, WT apoVs, but not OVX apoVs, promoted osteogenic differentiation and inhibited adipogenic differentiation of OVX MSCs. This was evident from the increased mineralization nodules formation, elevated ALP and Runx2 expression, decreased lipid droplet formation, and reduced LPL and PPARγ expression (Fig. 4C-F), resembling the characteristics of the WT MSCs group. Furthermore, flow cytometry analysis demonstrated that WT apoVs restored the immunoregulatory capacity of OVX MSCs to a level similar to that of WT MSCs, while OVX apoVs did not produce the same effect (Fig. 4G and H). These results indicated that WT apoVs possessed the ability to transform damaged cells into a relatively normal state similar to WT cells.
The crosstalk of TGF-β/Smad 2/3 and Wnt/β-catenin signaling pathway plays a central role in the reparative process of WT apoVs on the OVX MSCs
After observing the beneficial reparative effects of WT ApoVs on diseased MSCs in both in vitro and in vivo settings, further exploration into the underlying mechanisms is warranted. To explore this, we conducted a western blot analysis to assess the activation status of several key signaling pathways associated with osteogenic differentiation and inflammation, namely TGF-β/Smad 2/3, mTOR, ERK, Wnt/β-catenin, PI3K/AKT, and NFkB pathways. Our findings revealed that the TGF-β/Smad 2/3 signaling was activated, while the Wnt/β-catenin signaling was inhibited in OVX MSCs compared to WT MSCs. Interestingly, treatment with WT ApoVs restored the abnormal activation state of the aforementioned signaling pathways, while OVX ApoVs failed to produce a similar effect. Since the changes in other signaling pathways did not align with the observed phenotypic alterations in cells, we deemed the TGF-β/Smad 2/3 and Wnt/β-catenin pathways as the main focus of our study for further investigation (Fig. 5A).
We proceeded to investigate the reciprocal relationship between the two signaling pathways. To achieve this, we utilized CHIR-99021, a GSK-3β inhibitor, to activate Wnt/β-catenin signaling, and SB-431542, a Src family kinase inhibitor, to inhibit TGF-β/Smad 2/3 signaling. Through this experimental approach, we demonstrated that downregulating TGF-β/Smad 2/3 signaling in OVX MSCs subsequently led to upregulation of Wnt/β-catenin signaling. However, the opposite scenario was not feasible. These findings indicated that, in OVX MSCs, TGF-β/Smad 2/3 signaling acted upstream of Wnt/β-catenin signaling (Fig. 5B).
As the TGF-β/Smad 2/3 signaling pathway follows a typical receptor-ligand binding activation mode, our next objective was to investigate the activation process of this pathway in OVX MSCs. Initially, we examined the expression level of TGF-β1 in the supernatant of culture medium, and our findings revealed that apoVs did not have any impact on TGF-β1 secretion in MSCs (Fig. 5C). Therefore, we directed our focus towards the effect of apoVs on the expression of TGF-β receptors and Dickkopf 1 (Dkk1), a significant inhibitor of the Wnt/β-catenin signaling pathway. Our results showed that while the expression of TGF-β receptor 1 (TGF-βR1) remained constant in the four groups, TGF-β receptor 2 (TGF-βR2) was increased in OVX MSCs, but this increase was mitigated by treatment with WT apoVs. Similar findings were observed regarding the expression level of Dkk1 (Fig. 5D). Thus, WT apoVs exhibited inhibitory effects on the TGF-β/Smad 2/3 signaling pathway while simultaneously activating the Wnt/β-catenin signaling pathway through the downregulation of TGF-βR2 and DKK-1.
Subsequently, we proceeded to evaluate the distinct effects of these two signaling pathways on the osteogenic differentiation and immunoregulatory capacity of OVX MSCs. Remarkably, it was observed that either inhibiting the TGF-β/Smad 2/3 signaling pathway or promoting the Wnt/β-catenin signaling pathway effectively facilitated the osteogenic differentiation in OVX MSCs (Fig. 5E and F). Furthermore, the immunoregulatory capacity was restored when the Wnt/β-catenin signaling pathway was promoted (Fig. 5G and H). These findings suggested that the aforementioned signaling pathways may play a crucial role in shaping the phenotype of osteoporosis by regulating the osteogenic differentiation and immunoregulatory capacity of the host MSCs.
miR-145a-5p was responsible for the WT apoVs-mediated rescue of impaired OVX MSCs in vitro
A multitude of studies have provided evidence that apoVs and other extracellular vesicles exert their regulatory functions by transmitting internally encapsulated miRNAs, thus influencing the expression of specific target genes in recipient cells [45–47]. With this understanding, we employed three widely utilized miRNA target gene prediction databases, namely Targetscan, miRDB, and miRWalk, to identify potential miRNAs that target TGF-βR2. Through an intersection of the search results, three miRNAs (miR-93-5p, miR-145a-5p, and miR-294-3p) emerged as promising candidates (Fig. S3). Subsequently, we validated the expression of these three miRNAs in different cell groups and apoVs using real-time PCR. Our findings indicated a significant decrease in miR-145a-5p levels in OVX MSCs and OVX apoVs compared to WT MSCs and WT apoVs, respectively. Conversely, when treated with WT apoVs instead of OVX apoVs, the expression of miR-145a-5p increased, which contrasted with the observed variation in TGF-βR2 expression across the groups. Nevertheless, there is no statistically significant differences in the expression of miR-93-5p and miR-294-3p among the various cell groups and apoVs (Fig. 6A and B).
To confirm the mechanism by which apoVs influence miR-145a-5p expression, we initially evaluated the expression of the primary transcript of miR-145a-5p (pri-miR-145a-5p) in the different cell groups. Our findings demonstrated that OVX MSCs exhibited lower levels of pri-miR-145a-5p, which kept stable following apoVs treatment (Fig. 6C). In addition, we utilized actinomycin D to inhibit RNA synthesis and observed no discernible changes in miR-145a-5p expression (Fig. 6D). the results suggest that the decrease in pri-miR-145a-5p synthesis in OVX MSCs leads to reduced expression of mature miR-145a-5p after cleavage. Importantly, apoVs treatment did not significantly influence pri-miR-145a-5p synthesis, indicating that WT apoVs primarily enhance the expression of mature miR-145a-5p in OVX MSCs through direct cargo delivery and reuse.
Subsequently, we sought to dissect the prospective effect of miR-145a-5p on the TGF-β/Smad 2/3 and Wnt/β-catenin signaling pathways, also on the osteogenic differentiation and immune regulation in OVX MSCs. To assess the functionality of miR-145a-5p, we utilized micro-RNA mimics and inhibitors to manipulate its expression. As demonstrated in Fig. 6E-L, when miR-145a-5p was upregulated using mimics, there was a reduction in the expression of TGF-βR2 and Dkk1. Consequently, the TGF-β/Smad 2/3 signaling pathway was inhibited, while the Wnt/β-catenin signaling pathway was promoted, mediating the restoration of osteogenic differentiation and immune regulation. Conversely, the observed trends were reversed when miR-145a-5p was suppressed using an inhibitor.
miR-145a-5p was critical in the apoVs-mediated rescue of the therapeutic effect of OVX MSCs on osteoporosis in vivo
Based on our data, which demonstrated the involvement of miR-145a-5p in the restoration of impaired OVX MSCs mediated by WT apoVs in vitro, we formulated the hypothesis that the apoVs-mediated rescue of the therapeutic effects of OVX MSCs on osteoporosis might be partially dependent on miR-145a-5p. To investigate this, we manipulated the expression of miR-145a-5p using mimics and an inhibitor, revealing that the overexpressed miR-145a-5p in OVX MSCs significantly enhanced their therapeutic effect on the osteopenic phenotype. This was evident from microCT scans (Fig. 7A and B) and H&E staining (Fig. 7C and D) of distal femurs in OVX mice, as well as from ELISA (Fig. 7E) and flow cytometry analysis (Fig. 7F) indicating improvements in the hyperimmune state of the host. Conversely, the inhibition of miR-145a-5p in WT MSCs yielded opposite results, as the beneficial effects associated with the infusion of WT MSCs were significantly diminished, reaching levels comparable to OVX MSCs treatment (Fig. 7A-F). All evaluation indicators were consistent with those presented in Fig. 2. These findings strongly suggested that miR-145a-5p is indispensable in the apoVs-mediated rescue of the therapeutic effects of OVX MSCs in the context of osteoporosis.