Accumulating evidence has demonstrated that apoptotic vesicles (apoVs) derived from mesenchymal stem cells (MSCs) play a vital role in bone regeneration, and possess superior capabilities compared to MSCs and other extracellular vesicles derived from MSCs (like exosomes). The osteoinductive effect of MSC-apoVs is attributed to their diverse contents, especially enriched proteins or microRNAs (miRNAs). To optimize their osteoinductive capacity, it is indispensable to determine the unique cargo profiles of MSC-apoVs. In our previous study, we established the protein landscape and identified the specific proteins of MSC-apoVs. However, the features and functions of miRNAs enriched in MSC-apoVs remain elusive. In this study, we compared MSCs, MSC-apoVs, and MSC-exosomes from two different types of MSCs. We established a unique miRNA map of MSC-apoVs and identified 7 miRNAs specifically enriched in apoVs compared to MSCs and MSC-exosomes, which can be used as apoV-specific miRNAs. Among these 7 specific miRNAs, hsa-miR-4485-3p is the most abundant and stable miRNA. Then, we explored whether it is the main motive force responsible for apoV osteoinductive function. Unexpectedly, hsa-miR-4485-3p enriched in apoVs is proven to inhibit osteogenesis but promote adipogenesis by targeting the AKT pathway. Tailored apoVs by downregulating hsa-miR-4485-3p exhibited a more powerful effect in bone regeneration than normal apoVs. Like releasing the brake, we acquired more powerful osteoinductive apoVs. In summary, we determined miRNA cargo, identified the specific miRNAs of MSC-apoVs, and constructed an optimized tailored apoVs with excellent osteo-inductivity which is promising in apoV-based therapy for bone regeneration.
Article
Tailored apoptotic vesicles promote bone regeneration by releasing the osteoinductive brake
https://doi.org/10.21203/rs.3.rs-3643782/v1
This work is licensed under a CC BY 4.0 License
Journal Publication
published 16 Apr, 2024
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apoptotic vesicle
mesenchymal stem cell
microRNAs
hsa-miR-4485-3p
bone regeneration
Apoptosis is a class of programmed cell death that commonly occurs in biological processes1–3, and approximately 200 billion cells undergo apoptosis in the human body per day4,5. During apoptosis, cells secret a large number of apoptotic vesicles (apoVs)6 containing plentiful contents, like proteins, RNAs, DNAs, lipids, etc7,8. Mounting evidence has revealed that apoVs play crucial roles in various physiological and pathophysiological events9–11. As a promising cell source for clinical therapies, mesenchymal stem cells (MSCs) are widely used in the treatment of multiple diseases12–14. In recent years, emerging studies have demonstrated that apoVs derived from MSCs (MSC-apoVs) contribute to MSC-mediated therapeutic efficacy15–17. Moreover, the direct application of MSC-apoVs shows therapeutic potential in a variety of diseases18–25. In line with these reports, our previous studies have determined the effective functions of MSC-apoVs in the treatment of bone loss26, wound healing27, hemophilia A28, type 2 diabetes29, multiple myeloma30, traumatic hemorrhage31, arthritis32, etc. These findings suggest the potent potential of MSC-apoV therapy in clinical settings.
Among multiple studies on apoVs, the utilization of apoVs in bone regeneration has attracted numerous attentions25,26,33. Compared to MSCs or other extracellular vesicles (EVs) derived from MSCs (like exosomes), MSC-apoVs exhibit higher yield, lower cost, and superior osteoinductivity34, and thus they represent a more promising approach for bone regeneration. Similar to other EVs, the osteoinductive effect of apoVs relies on their bioactive molecules, especially proteins and microRNAs (miRNAs)35. Since the constituent of apoVs is very heterogenous, one fundamental question is to identify the unique cargo profiles of apoVs, which is indispensable to figure out the underlying molecular mechanism and construct tailored apoVs that possess more powerful efficacy. In our previous study, we established the protein profile and identified the specific protein biomarkers of MSC-apoVs28. However, the features and functions of miRNAs enriched in MSC-apoVs remain largely unknown. Previous studies have shown that MSC-apoVs transfer diverse miRNAs to recipient MSCs and then promote the osteogenesis of recipient cells in vitro and in vivo16,26. However, specific miRNAs of MSC-apoVs and whether all these enriched miRNAs are responsible for their osteoinductive effect are unclear.
In this study, we compared MSCs, MSC-apoVs, and MSC-exosomes from two different types of MSCs (including human bone marrow mesenchymal stem cells, hBMMSCs and human adipose mesenchymal stem cells, hASCs). Through RNA sequencing and bioinformatics analyses, we determined the miRNA cargo of apoVs, and identified 7 miRNAs specifically enriched in apoVs compared to MSCs and MSC-exosomes, which can be used as apoV-specific miRNAs. Among these 7 specific miRNAs, hsa-miR-4485-3p showed much more abundant and stable expression compared to other 6 miRNAs. More interestingly, beyond our expectations, hsa-miR-4485-3p enriched in apoVs is proven to inhibit osteogenesis but promote adipogenesis by targeting the AKT pathway. The most specific miRNA is a brake, instead of an accelerator for osteoinductivity of apoVs, which may play a pivotal role in balancing bone metabolism. Furthermore, tailored apoVs by downregulating hsa-miR-4485-3p indeed exhibited more powerful osteoinductive effect than normal apoVs. Through releasing the osteoinductive brake, we acquired one kind of optimized apoVs for bone regeneration. Taken together, our findings reveal the whole miRNA cargo, determine the specific miRNAs of MSC-apoVs, and construct an optimized tailored apoVs with excellent osteoinductivity, which provide a potent rationale for novel tailored apoVs and promote establishment of optimized apoV-based therapy for bone regeneration.
Characterization of MSC-apoVs
In the current study, staurosporine (STS) was used to induce apoptosis of MSCs. TUNEL staining result (Fig. 1a) showed that the majority of MSCs were observed as TUNEL-positive stained cells after treatment of STS for 12h. Then, we carried out differential centrifugation procedures presented in Fig. 1b to extract apoVs. For further identification, nanoparticle tracking analysis (NTA) was carried out and the results showed that the size distribution of apoVs was 100 to 1000 nm, and the average diameter of MSC-apoVs was about 192.6 nm (Fig. 1c). Transmission electron microscopic observation exhibited that apoVs are cup-shaped extracellular vesicles and have an average diameter of approximately 200 nm (Fig. 1d). To determine whether apoVs were uptake by MSCs, confocal microscopic images were scanned after co-incubation of PKH-26-labeled apoVs (red) and MSCs. The F-actin of MSCs was stained with phalloidin (green) while the nuclei of MSCs were stained with DAPI (blue). The views presented the internalization of apoVs by MSCs after 12 h and more distribution of apoVs in MSCs after 24 h, enabling apoVs to further exert effect on MSCs (Fig. 1e). In addition, western blotting revealed that apoVs highly expressed CD9, CD81, CD63, Fas, Calreticulin, CD44 and Integrin α-5(Fig. 1f), which are generally acknowledged as apoV-specific markers28. These results showed that MSC-apoVs possess properties consistent with apoVs commonly reported in previous studies in terms of morphology, size, biomarkers, and efferocytosis by MSCs26.
Specific miRNA profile of MSC-apoVs compared to MSCs, and MSC-exosomes
To figure out the specific miRNA profile of MSC-apoVs, a small RNA sequencing was conducted. Human bone marrow mesenchymal stem cells (hBMMSCs) and human adipose mesenchymal stem cells (hASCs) were used to perform RNA sequencing and subsequent verified experiments. ApoVs derived from hBMMSCs and hASCs were used for miRNA analysis, whereas parental MSCs and exosomes were used for control groups. A total of 3280 miRNAs was identified during small RNA sequencing analysis and miRNA expression levels of hBMMSCs, hBMMSC-apoVs, hBMMSC-exos, hASCs, hASC-apoVs and hASC-exos were recorded. The top 20 highly expressed miRNAs in hBMMSCs-derived apoVs and hASCs-derived apoVs compared to exosomes were shown in Table S1. It was found that up to 85% top 20 highly expressed miRNAs overlapped between hBMMSC-apoVs and hASC-apoVs, showing considerable consistency between miRNA patterns of MSC-apoVs. Adjusted p-value༜0.05 in combination with fold change ≥ 2 was set to identify the differential expression. Cluster heatmaps of differentially expressed genes (DEGs) among hBMMSCs, hBMMSC-apoVs, hBMMSC-exos, and hASCs, hASC-apoVs, hASC-exos were shown in Fig. 2a. 71 miRNAs were upregulated and 66 miRNAs were downregulated in hBMMSC-apoVs compared to hBMMSCs. 240 miRNAs were upregulated and 123 miRNAs were downregulated in hBMMSC-apoVs compared to hBMMSC-exos. In the meanwhile, 53 miRNAs were upregulated and 67 miRNAs were downregulated in hASC-apoVs compared to hASCs. 71 miRNAs were upregulated and 67 miRNAs were downregulated in hASC-apoVs compared to hASC-exos (Fig. 2b). The differentially expressed miRNAs were then intersected, and 7 miRNAs, namely hsa-miR-12136, hsa-miR-1973, hsa-miR-4454, hsa-miR-4485-3p, hsa-miR-6821-5p, novel-hsa-miR-115-3p, and novel-hsa-miR-264-3p, were screened out as MSC-apoV specific miRNAs and it is anticipated to be the candidate of unique miRNAs in MSC-apoVs compared to MSCs and MSC-exos (Fig. 2c, d). Then we shed light on the enrichment analysis of these 7 novel unique miRNAs in MSC-apoVs. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (Fig. S1a) showed that “Transport and catabolism” within the “Cellular Processes” domain; “Signal transduction” within the “Environmental Information Processing” domain; “Folding, sorting and degradation” and “Translation” within the “Genetic Information Processing” domain; “Infectious diseases: Viral” and “Cancers: Overview” within the “Human Diseases” domain; “Global and overview maps” within the “Metabolism” domain; and “Immune system”, “Endocrine system” within the “Organismal Systems” domain are predicted to be in relationship with these 7 miRNAs. Gene ontology (GO) analysis (Fig. S1b) revealed that “cellular process” in the field of “biological process”, “cell” and “cell part” in the field of “cellular component”, and “binding”, “catalytic activity” in the field of “molecular function” is associated with these 7 miRNAs. These results indicated that miRNAs enriched in MSC-apoVs play important roles in a wide variety of physiological and pathological processes. We then verified the expression level of these 7 candidate miRNAs through qRT-PCR (Fig. 2e), results showed that all these 7 candidates are specifically enriched in MSC-apoVs compared to MSCs and MSC-exos (including hBMMSCs and hASCs). Among these 7 miRNAs, hsa-miR-4485-3p is a lead candidate which show superior high expression specifically in MSC-apoVs. Therefore, we chose hsa-miR-4485-3p for further functional analysis. To sum up, MSCs, MSC-apoVs and MSC-exos possess distinct miRNA profiles, and 7 novel unique miRNAs that are specifically enriched in MSC-apoVs compared to MSCs and MSC-exos were screened out and were focused for further investigation.
hsa-miR-4485-3p overexpressed apoVs inhibited MSC osteogenic differentiation and promoted MSC adipogenic differentiation in vitro
In the current study, we explored whether hsa-miR-4485-3p, as a lead candidate miRNA in apoVs shown in qRT-PCR, contributes to MSC fate choices. We performed qRT-PCR to examine the involvement of hsa-miR-4485-3p in MSC osteogenic and adipogenic differentiation. Its expression level was relatively low and subsequently increased during osteogenic induction, while it was initially low and upregulated afterward during adipogenic induction (Fig. S2a, b). Afterward, we transfected lentiviruses to overexpress hsa-miR-4485-3p (mimics) and RNA oligo to knockdown hsa-miR-4485-3p (inhibitor) to MSCs and proved via qRT-PCR that the expression level of hsa-miR-4485-3p in MSC-apoVs could be upregulated or downregulated in MSC-apoVs by hsa-miR-4485-3p overexpression or knockdown in the parent cells (Fig. S2c-f). To investigate the optimal concentration of apoVs in vitro, we set up concentration gradients of 62.5 ng/ml, 125 ng/ml, 250 ng/ml, 500 ng/ml, and 1000 ng/ml. The CCK-8 assay revealed that 0-250 ng/ml mi-NC, inhi-NC, mimics, and inhibitor apoVs have no distinctive effect on the viability of MSCs. With the increase in concentration (500 ng/ml, 1000 ng/ml), apoVs impaired the viability of MSCs (Fig. S2g, h). The relative mRNA expression level of the osteogenic differentiation marker RUNX2 and the adipogenic differentiation marker PPARγ revealed that 250 ng/ml mimics apoVs and inhibitor apoVs changed osteogenic and adipogenic capability of MSCs to the maximum extent compared with respective NC apoVs after 7 days of inductive culture (Fig. S2i-l). Therefore, the optimal concentration of apoVs is 250 ng/ml and this concentration is used in the subsequent in vitro experiments.
ALP staining (Fig. 3a) and ARS staining (Fig. 3b) of MSCs indicated that the increase of hsa-miR-4485-3p in apoVs inhibited the promoting effect of NC apoVs on osteogenic differentiation. ALP activity quantification (Fig. 3c) and ARS level quantification (Fig. 3d) displayed similar results. Moreover, the protein expression of RUNX2 declined during 7 days of osteogenesis in the group treated with mimics apoVs compared with NC apoVs (Fig. 3e, f). We carried out qRT-PCR, showing that ALP (Fig. 3g) and RUNX2 (Fig. 3h) mRNA expression levels on day 7, OCN (Fig. 3i) and BMP2 (Fig. 3j) mRNA expression levels on day 14 were relatively low on the presence of mimics apoVs compared with NC apoVs when MSCs were treated with OM.
To further evaluate the effect of hsa-miR-4485-3p in apoVs on adipogenesis, MSCs treated by PM, AM, AM + NC apoVs, AM + mimics apoVs were subjected to Oil red O staining (Fig. 3k) and Oil red quantity (Fig. 3l), which indicated that the up-regulation of hsa-miR-4485-3p in apoVs reversed the suppression effect of apoVs on MSC adipogenesis. Determined by qRT-PCR, the relative PPARγ and CEBPα mRNA expression levels were up-regulated in the group with the presence of mimics apoVs (Fig. 3m, n). Furthermore, protein expression of PPARγ detected by western blot and its quantification (Fig. 3o, p) exhibited the results likewise. In brief, hsa-miR-4485-3p overexpression in apoVs exerted a positive impact on adipogenic differentiation and inhibited osteogenic differentiation of MSCs in vitro.
hsa-miR-4485-3p knockdown apoVs enhanced MSC osteogenic differentiation and reduced MSC adipogenic differentiation in vitro
For repeated validation, ALP staining (Fig. 4a), ARS staining (Fig. 4b) and ALP, ARS quantification (Fig. 4c, d), western blot (Fig. 4e, f) and qRT-PCR (Fig. 4g, h, i, j) results of hsa-miR-4485-3p silencing apoVs showed that hsa-miR-4485-3p downregulation in apoVs strengthened the promoting effect of apoVs on the osteogenic potential of MSCs. In addition, the loss of hsa-miR-4485-3p in apoVs further inhibited MSC adipogenic differentiation potential, which is proved by Oil red O staining (Fig. 4k), Oil red quantity (Fig. 4l), qRT-PCR (Fig. 4m, n) and western blot experiments (Fig. 4o, p). These above results showed that hsa-miR-4485-3p in apoVs inhibited osteogenesis and promoted adipogenesis in MSCs in vitro.
hsa-miR-4485-3p in apoVs attenuated MSC osteogenic differentiation and enhanced MSC adipogenic differentiation in vivo
To determine the role of hsa-miR-4485-3p in apoVs in vivo, nude mice were implanted with β-TCP carrier scaffold loaded with MSCs cultured in PM, PM + 250ng/ml inhi-NC apoVs, PM + 250ng/ml inhibitor apoVs, PM + 250ng/ml mi-NC apoVs, and PM + 250ng/ml mimics apoVs in the dorsal subcutaneous area. H&E staining of ectopic bone formation tissues showed that neo-generated bone was reduced significantly (Fig. 5a), and Masson staining (Fig. 5b) revealed that less collagen tissue was formed in PM + mimics apoVs group compared to PM + mi-NC apoVs group. In the meanwhile, the area of newly formed bone-like tissue and collagen structure was remarkably increased in the PM + inhibitor apoVs group than in the PM + inhi-NC group (Fig. 5c, d). The above results showed that hsa-miR-4485-3p in apoVs attenuated MSC osteogenic differentiation ability in vivo.
To further investigate the effect on adipogenesis of hsa-miR-4485-3p in apoVs in vivo, we cultured MSCs in PM, AM, AM + 250ng/ml inhibitor apoVs, AM + 250ng/ml inhi-NC apoVs, AM + 250ng/ml mimics apoVs, and AM + mi-NC apoVs separately for 7 days, combined them with collagen sponge scaffolds, and then implanted the mixture into nude mice. Both H&E and Oil Red O staining illustrated that group supplement with mimics apoVs had much more lipid droplets compared to mi-NC apoVs (Fig. 5e, f). Otherwise, fewer adipose-like tissue was shown in the histological sections of H&E and Oil Red O staining in AM + inhibitor apoVs group than AM + inhi-NC apoVs group (Fig. 5g, h). These results suggest that hsa-miR-4485-3p in apoVs enhanced the MSC adipogenic differentiation ability in vivo. In conclusion, our current study revealed that hsa-miR-4485-3p in apoVs acts as a switch point in MSC fate commitment towards adipogenesis and against osteogenesis in vitro and in vivo.
hsa-miR-4485-3p in apoVs control MSC fate commitment through regulating AKT pathway
To provide insight into the mechanism by which hsa-miR-4485-3p in apoVs regulate MSC fate commitment, we carried out RNA sequencing and the DEGs between OM + inhibitor apoVs and OM + inhi-NC apoVs groups were shown in the form of volcano plot and heatmap in Fig. 6a, b. p-value༜0.05 in combination with fold change༞1 was set to identify the differential expression. The results demonstrated that 614 genes were upregulated and 497 genes were downregulated in OM + inhibitor apoVs group compared to OM + inhi-NC apoVs group. The KEGG enrichment analysis of DEGs further provided some hints that the AKT pathway may be upregulated in the OM + inhibitor apoVs group compared to OM + inhi-NC apoVs group (Fig. 6c). Then we conducted western blot of several crucial signaling pathways, such as NF-κB, Wnt, Smad and Erk signaling pathways (Fig. S3), which were reported previously to be related to the MSC fate commitment and confirmed that AKT signaling pathway was more significantly influenced by hsa-miR-4485-3p in apoVs compared to other pathways (Fig. 6d). Then, we used MK-2206, which is a powerful inhibiting agent for AKT pathway. Western blot and ORS staining results (Fig. 6e, f) demonstrated that MK-2206 rescued the suppression in adipogenesis of inhibitor apoVs, whereas western blot, ALP and ARS staining results (Fig. 6g, h) showed that in osteogenic induction, MK-2206 reversed the promoting effect in osteogenesis of inhibitor apoVs. In summary, these in-depth mechanism investigations proved that hsa-miR-4485-3p in apoVs negatively regulates osteogenic differentiation and positively regulates adipogenic differentiation by regulating the AKT signaling pathway.
Tailored apoVs are more effective in bone restoration of osteoporosis
Integrating the existing results, we found that MSC-derived hsa-miR-4485-3p knockdown apoVs appear to promote MSC osteogenic differentiation to a greater extent in vitro and in vivo compared to NC apoVs, so we named apoVs reducing expression of has-miR-4485-3p as tailored apoVs for bone regeneration. We first injected DiR-labeled apoVs to detect the biodistribution pattern of apoVs after systemic injection through the tail vein. Whole-body fluorescence imaging (Fig. S4) revealed that NC apoVs and inhibitor apoVs went through hepatic metabolism, in the meanwhile, NC apoVs and inhibitor apoVs both enriched in the femurs and the targeting level increased during 48h. To demonstrate the clinical significance of tailored apoVs, we further verified its effect on the prevention of osteoporosis in estrogen deficiency mice and on the treatment of osteoporosis in aged mice. We established an estrogen deficiency-induced osteoporosis mouse model through an ovariectomy (OVX) operation and purchased a senescence-induced osteoporosis mouse model. Then we injected phosphate-buffered saline (PBS), NC apoVs and inhibitor apoVs solution into mice via the tail vein at the dose of 20 µg per 30 g weight according to the previous study since the confirmed biocompatibility26. 8 weeks later, the femurs were collected and scanned by micro-CT. The longitudinal section, cross-section, and three-dimensional reconstruction views demonstrated that massive bone deterioration occurred in the femur of OVX and aged mice, and a distinct improvement in trabecular bone restoration was observed in the inhibitor apoVs group compared to NC apoVs group in both types of osteoporosis models. H&E staining also exhibited that bone impairment in both osteoporosis models was partially reversed by inhibitor apoVs and the restorative effect of inhibitor apoVs was more obvious (Fig. 7a-d, i-k). The bone histomorphometry quantitative analysis of bone mineral density (BMD), bone volume/total volume (BV/TV), and trabecular number (Tb. N) parameters were higher, and trabecular spacing (Tb. Sp) parameter were lower in experimental groups (Fig. 7e-h, l-o). These results suggested that tailored apoVs have excellent delivery capacity to long bones and are more effective in suppressing bone mass impairment in osteoporosis caused by ovariotomy and aging.
PLGA/pDA-tailored apoVs scaffold promoted more bone formation in rat calvarial defects
We produced PLGA/pDA-apoVs composite scaffolds in our previous study and demonstrated that apoVs are well integrated with PLGA/pDA scaffold, allowing its effective and biocompatible promotion of bone formation in rat calvarial defects 26. Therefore, we used this complex in the current study, and results showed that these scaffolds slowly released apoVs to the surrounding environment (Fig. 8a, b). Then we established a rat skull defect model to figure out the role of tailored apoVs in localized bone loss. Micro-CT scanning image (Fig. 8c) displayed that in the blank group, there were no signs of healing; with the aid of PLGA/pDA scaffolds, there was a little new bone tissue along the edges of the skull defects. In addition, rats implanted with PLGA/pDA-NC apoVs scaffolds formed more new bone tissue, while the PLGA/pDA-tailored apoVs scaffolds caused a significantly greater amount of new bone formation. Quantitative detection of BMD (Fig. 8d) and BV/TV (Fig. 8e) also revealed that more new bone tissue was generated in the rats treated with PLGA/pDA-tailored apoVs scaffolds than in the rats treated with PLGA/pDA-NC apoVs scaffolds. H&E (Fig. 8f) and Masson (Fig. 8g) staining further confirmed the superiority of the PLGA/pDA-tailored apoVs scaffolds in rat calvarial defects.
Apoptosis is an indispensable physiological activity that maintains homeostasis in organisms and plays a pivotal role in tissue regeneration. MSC is one of the most appealing cell sources used in bone tissue engineering, therefore, recent advances in exploring the functional products of MSC apoptosis attracted widespread attention to MSC-apoVs. MSC-apoVs have striking advantages, which exhibit the potential to break through the limitations in the large-scale clinical translation of cell-based therapy and exosome-based therapy36,37.
The heterogeneity in bioactive constituents of MSC-apoVs results in the exhibition of variant functional properties35, and may impede the precisive treatment of bone-related diseases based on MSC-apoVs. Among diverse molecular contents loading in apoVs, miRNA is of great importance, and it is estimated that 60% of mammalian gene expression is regulated by miRNAs38. Besides, several miRNAs are emerging as key regulators in MSC osteogenesis39,40. Therefore, studies concerning specific enriched miRNAs in MSC-apoVs and detailed functions of these miRNAs in MSC-apoV osteoinductive ability are urgently needed. We used small RNA sequencing in this study to compare two types of MSCs (including hBMMSCs and hASCs), MSC-apoVs and MSC-exos comprehensively. A total of 3280 miRNAs were identified and all miRNA expression levels were recorded. The differentially expressed miRNAs were intersected and 7 miRNAs that are specifically enriched in MSC-apoVs compared to MSCs and MSC-exosomes were screened out and verified through qRT-PCR. We found that miRNAs share great similarity between hBMMSCs-apoVs and hASCs-apoVs. However, the miRNA expression of MSCs, MSC-apoVs and MSC-exos demonstrated obvious differences. It is noteworthy that, the amount of distinct miRNA is generally between 100–400. While in our previous study28, more than 5600 proteins in total were identified, and thousands of distinct proteins were detected between MSC-apoVs and MSC-exos. Taken together, the results showed that the distinction is more obvious in proteomics between MSC-apoVs and MSC-exos, and indicated the difference in the cargo sorting mechanism of miRNA and protein from its mother cells in apoVs biogenesis41. Recent literature elucidated that miRNA secretion into exosomes depends on EXOmotifs in miRNA, and insertion of EXOmotifs in miRNA leads to a reduction of target gene expression level in distant cells42. This work unveiled the miRNA selection mechanism in exosomes, and drove an urgent need to explore the sorting mechanism of apoV cargo. Zheng et al. identified proteins with conserved EV-sorting abilities and demonstrated that the genetic fusion of specific proteins to scaffold proteins can achieve the stable and effective generation of EV delivery vehicles43. This research offered a new method to improve EVs based on their cargo-loading mechanism. ApoV is also known as a next-generation drug delivery modality, it is vital to figure out the underlying mechanisms of specific cargo loading processes into apoVs, which may provide clues to improve apoV-mediated therapies.
MSC-apoVs regulate bone metabolism and promote bone repair as a whole16,26,44. Hsa-miR-4485-3p, as a lead candidate which is the most abundant and stable miRNA among 7 apoV-specific miRNAs, was expected to be an accelerator in our preliminary assumption. Hsa-miR-4485-3p is 20 nucleotides long and it is a product of the hsa-mir-4485 precursor. Hsa-miR-4485 has been reported to be related to cancers45, infectious diseases46,47, and bone fracture healing48. Very interestingly and unexpectedly, we found hsa-miR-4485-3p in MSC-apoVs actually act as an inhibitor of osteogenesis and an enhancer of adipogenesis, that is, a brake for osteoindictivity of MSC-apoVs. Our results showed that in the early stage of MSC osteogenesis, the expression level of hsa-miR-4485-3p decreased, however, in the late stage of MSC osteogenesis, the expression level of hsa-miR-4485-3p remarkably increased. These results indicated that the underlying reason could be that hsa-miR-4485-3p in apoVs exerts a substantial effect on balancing bone metabolism.
ApoVs, as key communication mediators, are carriers of specific miRNAs and offer a viable solution to prevent miRNAs from rapid degradation and transport miRNA messengers for bone homeostasis mediation49. Based on the fact that hsa-miR-4485-3p knockdown MSC-apoVs enhanced the osteoinductive effect of innate MSC-apoVs, hsa-miR-4485-3p knockdown MSC-apoVs were exploited as optimized tailored apoVs in bone regeneration. Systemic infusion of tailored apoVs represents a more effective approach to treating osteoporosis caused by estrogen deficiency and senescence. What’s more, tailored apoVs combined with PLGA/pDA scaffold significantly enhanced the new bone formation of maxillofacial bone defects. In the present medical context, the importance of precision medicine has gained more and more attention. Notably, the majority of investigations in tailored extracellular vesicles have focused on the addition of the favorable elements 50,51, like step on the accelerator of osteogenesis. However, the above evidence in our study indicated that by performing subtraction in the particular component, hsa-miR-4485-3p, it is possible to achieve an additive effect on the osteoinductive ability of MSC-apoVs, like releasing the brake. This approach presents new avenues to improve the therapeutic efficiency and achieve precise treatment of apoVs.
Our study still has limitations. Firstly, the direct target gene of hsa-miR-4485-3p in MSC-apoVs and functional analysis of other apoV-specific miRNAs still remain elusive and will be carried out in our upcoming research. Secondly, we confirmed that hsa-miR-4485-3p overexpressed MSC-apoVs can promote MSC adipogenetic differentiation in vivo. Considering that cutaneous damage is also a thorny issue in regenerative medicine, and apoVs are expected to be a novel agent for the skin-wound healing process52,53, problems need to be addressed whether hsa-miR-4485-3p overexpressed apoVs are more effective in the soft tissue engineering. Besides the above limitations, to achieve optimal clinical application, the distribution and pharmacokinetics of tailored apoVs should be further discussed in our subsequent research. What’s more, the aforementioned research offers a novel perspective, suggesting that the production of more potent apoVs for osteoinduction might be feasible by adding the accelerator and releasing the brake simultaneously. This “working along both lines” approach merits subsequent investigation and has promising clinical prospects.
In conclusion, our investigation expanded our understanding of MSC-apoV specific miRNA profile, and paved the way for novel strategies to achieve precisive treatment in regenerative medicine.
Proliferation, osteogenic and adipogenic induction of human MSCs
hBMMSCs and hASCs were purchased from Science Cell Research Laboratories (San Diego, CA, USA). hBMMSCs were cultured in the proliferation medium (PM) which contained 10% FBS, α-MEM, and 1% streptomycin-penicillin. hASCs were cultured in the PM which contained 10% FBS, DMEM, and 1% streptomycin-penicillin. The osteogenic medium (OM) is a mixture of standard PM and 0.2 mM ascorbic acid, 10 mM β-glycerophosphate, and 10 nM dexamethasone. The adipogenic medium (AM) contained standard PM and 200 µM indomecin, 100 nM dexamethasone, 50 nM insulin and 500 µM 3-isobutyl-1-methylxanthin.
Induction of apoptosis, isolation, and identification of apoVs
The culture medium was substituted by α-MEM/DMEM with 500 nM staurosporine (STS) (Enzo Life Sciences) to induce MSC apoptosis. Cell apoptosis was observed using fluorescence microscopy (Olympus) after MSCs were labeled by the TUNEL apoptosis detection kit (Applygen, C0003-20) and rhodamine fluorescein (red) dUTP solution. After 12 hours, apoVs were extracted as previously described by differential centrifugation steps (Fig. 1b) 39. The Pierce BCA Assay Kit (Thermo Scientific) was used to quantify apoVs concentration.
The morphology of apoVs was scanned using an HT7700 transmission electron microscopy (Hitachi) after apoVs were deposited onto a carbon-coated copper net and stained twice with 1% uranyl acetate. Size distribution evaluation of apoVs was performed via a Nano Sight NS300 (Malvern) according to the manufacturer’s instructions. ApoV markers (CD9, CD81, CD63, Fas, Calreticulin, CD44, and Integrin α-5) detection was carried out by western blotting as mentioned below.
ApoVs uptake assay in vitro
ApoVs were labeled with PKH-26 using a red fluorescent cell labeling kit (Umibio). After staining, the apoVs were washed and the supernatant was collected to be used as negative control (NC). 250 ng/ml labeled apoVs solution and NC solution were incubated with MSCs for 12 h or 24 h, then 5 µg/ml phalloidin (Sigma-Aldrich) and 6-diamidine-2-phenylindole (DAPI) was added. Fluorescence imaging was taken by a confocal LSM 5 EXCITER microscope (Carl Zeiss).
Isolation of exosomes
Fetal bovine serum (FBS) free of exosomes was obtained by ultracentrifugation at 120,000 g for 18h. After cells were cultured with a medium free of exosomes for 2 days, cell supernatants were collected and centrifuged at 300 g for 10 min, 3000 g for 10 min, and 20 000 g for 30 min. Then, the leftover supernatant was subjected to 0.22 µm filter (Millipore), following ultra-centrifugation at 120 000 g for 120 min to extract exosomes.
Lentivirus infection
Recombined lentiviruses overexpressing hsa-miR-4485-3p (mimics) and the negative control (mi-NC) were purchased from GenePhama (Suzhou, China). The sequences were as follows: mimics: 5’-TAACGGCCGCGGTACCCTAA-3’; mi-NC: 5’-TTCTCCGAACGTGTCACGT-3’. To carry out transfection, the cells at 30–40% confluence were exposed to the viral supernatant. After 2 days, 1 mg/ml puromycin (Sigma-Aldrich, USA) was added to select stably transfected cells.
microRNA and plasmids transient infection
RNA oligo with 2’ Ome modification to knockdown hsa-miR-4485-3p (inhibitor) and its negative control (inhi-NC) were obtained from GenePhama (Suzhou, China). The sequences were as follows: inhibitor: 5’-UUAGGGUACCGCGGCCGUUA-3’; inhi-NC: 5’-CAGUACUUUUGUGUAGUACAA-3’. The cells were transfected using Lipofectamine 3000 (Invitrogen).
Cell counting kit-8 assay
MSCs were cultured in PM or PM supplemented with 62.5, 125, 250, and 500 ng/ml MSC-apoVs, respectively. The viability of MSCs was evaluated with a Cell Counting Kit-8 (CCK8, Dojindo Laboratories, Kumamoto, Japan) at day 0, 1, 3, 5, and 7. The same treatment methods are repeated in three wells. Optical density values were measured at 450nm in an ELX808 reader (BioTek).
Alkaline phosphatase (ALP), Alizarin red S (ARS) staining and quantification
MSCs were soaking in 95% ethanol for fixation. For ALP staining, a BCIP/NBT staining kit (CoWin Biotech, Jiangsu, China) was used. ALP activity was measured using optical density values at 520 nm after treatment of the ALP assay kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China). For ARS staining, MSCs were incubated with 40mM filtered 2% Alizarin red buffer (Sigma-Aldrich, USA). 100 nM cetylpyridine solution was used for at least 1 hour to quantify calcium-bound ARS. The absorbance at 562 nm was detected spectrophotometrically.
Oil red O staining and quantification
MSCs were fixed in 10% neutral formalin and washed by 60% isopropanol. Then Oil red O working solution (Sigma-Aldrich) was added and MSCs were observed using a microscope. 100% isopropanol was used to dissolve the lipid droplets for quantitative evaluation, then the absorbance at 500 nm was measured.
RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA of MSCs was extracted through TRIzol (Invitrogen, Carlsbad, CA, USA). RNA of apoVs and exosomes were extracted using miRNeasy Mini Kit (QIAGEN). Purity and concentration of RNA were measured using a NanoDrop 8000 spectrophotometer (Pierce Thermo Scientific, Waltham, MA, USA). For mRNA detection, the PrimeScript RT Reagent Kit (#RR037A; Takara, Tokyo, Japan) was used to produce cDNA. For miRNA evaluation, miDETECT A Track™ qRT-PCR Starter Kit (Ribo Bio) was used to produce cDNA. Then qRT-PCR assays were conducted using SYBR Green Master Mix (Yeasen Biotechnology, Shanghai, China) and ABI Prism 7500 Real-Time PCR Detection System (Applied Biosystems, Foster City, CA, USA). The primer sequences involved are presented in Table 1. Expression levels were normalized to GAPDH and U6.
Western blot
MSCs were lysed and the Pierce BCA protein assay kit was used to detect protein concentration. Aliquots (40 µg) of protein were transferred to PVDF membranes after electrophoresis in 10% SES-PAGE (Millipore, Billerica, MA, USA). The membranes were incubated with diluted anti-RUNX2, anti-PPARγ, anti-GAPDH, anti-β-catenin (ProteinTech, Wuhan, China), anti-CD9, anti-CD81, anti-Fas, anti-Integrin alpha-5, anti-CD44, anti-p65 (Abcam, Cambridge, UK), anti-Calreticulin (Cell Signaling Technology), anti-AKT1 (Santa Cruz, Dallas, Texas, USA), anti-p-AKT1, anti-ERK and anti-p-ERK (Abclonal, Wuhan, China) and secondary antibodies (Cell Signaling). The bands were detected by the ECL Kit (NCM bio, Suzhou, China). For analysis, Image J software (National Institutes of Health, USA) was used.
In vivo implantation of hMSCs
This study was approved by the Institutional Animal Care and Use Committee of the Peking University Health Science Center (approval number: LA2021006).
For heterotopic adipose tissue formation, MSCs were cultured with different treatments for 7 days and were seeded on collagen membrane scaffolds (Wuxi Biot Bioengineering Institute, Wuxi, China). For ectopic bone tissue formation, MSCs were combined with β-TCP (RB-SK-005 G). After anaesthetization by pentobarbital, the hybrids were imbedded into subcutaneous areas of the backs of nude mice. The implants were harvested after 6 weeks and fixed.
Construction of osteoporosis mouse model and tail vein injection of apoVs
8-week-old female BALB/c mice went through bilateral OVX to establish a mouse model of estrogen deficiency-induced bone loss. Other 8-week-old female BALB/c mice underwent a sham operation. 8 weeks after operation, apoVs solution and PBS were administered to the mice via tail vein once a week and the in vivo treatment dose of apoVs was 20 µg per 30 g weight, while PBS was in an equivalent volume as the apoVs solution. The 12-month-old female mice went through tail vein injection similarly. All mice were humanely euthanized after 8 weeks. The femurs were dissected and fixed.
Distribution of apoVs in vivo
ApoVs were incubated with 3,3’-dioctadecyloxacarbocyanin perchlorate (DiR, Keygen Biotech, Jiangsu, China) solution. The labeled apoVs were then injected intravenously via the tail vein. 24 h and 48 h later, the livers, lungs, spleens, hearts, kidneys, spines, femurs, mandibular bones, and cranial bones were collected and scanned by IVIS Lumina Series III (PerkinElmer, MA, USA).
Rehabilitation of cranial defects in rats using apoVs- PLGA/pDA scaffold
To produce apoVs-PLGA/pDA scaffolds, the PLGA/pDA scaffolds were immersed in 1 µg/µL apoVs solution for 12h. The scaffolds were immersed in normal saline solution and the solution was replenished each day to detect apoVs release by BCA. Rat models of 5 mm diameter skull defect were subjected to different treatments accordingly. After 8 weeks, the rats’ cranial bones were collected and fixed.
Histological staining and assessment
After fixation for at least 2 days, the harvested implants for adipose tissue formation were cut in half. The specimens for H&E and Masson staining were dehydrated and embedded in paraffin, cut into 5-µm tissue sections and then stained. Besides, the specimens for oil red staining were embedded in a Tissue-Tek OCT freezing medium, cut into 7-mm-thick sections, and then stained.
The bone tissues were soaked in 0.5 M EDTA for decalcification and then sectioned and performed by H&E staining as described above. The histological sections were observed and images were captured in microscopic view.
Micro-computed tomography (micro-CT)
Images of femurs were obtained at a current of 220 µA, voltage of 60 kV, and exposure time of 1500 ms, while images of cranial bones were obtained at current of 500µA, voltage of 80 kV, and exposure time of 1500 ms by an Inveon MM system (Siemens, Munich, Germany). Inveon Research Workplace (Siemens) were used to calculate bone morphometric parameters including BV/TV, Tb. Sp, Tb. N, and BMD. Three independent samples were included in very calculation. The analysis executors were blinded to the treatment group.
RNA sequencing analysis
1 µg/sample total RNA was used to construct the miRNA library. After purification of small RNA from total RNA and reverse transcription, sequencing of the final products (100–120 bp) was carried out on the BGISEQ-500 platform (BGI-Shenzhen, China). Clean tags were generated using SOAPnuke 1.5.2 software (BGI-Shenzhen, China). These tags were then mapped to the reference genome and various sRNA databases using Bowtie2 54. For mapping to the Rfam database (version 13.0), cmsearch55 was utilized. The expression levels were quantified by counting the absolute numbers of molecules identified by the unique molecular identifiers56. Data processing and enrichment analysis were conducted using the R (R-3.1.1) software package. Differential expression analysis was performed using the DEGSeq2 57.
Statistical analyses
Data were shown as means ± standard deviations. For statistical analysis, GraphPad software (GraphPad Software, Inc., La Jolla, CA, USA) was applied. Comparisons of two groups were evaluated through independent two-tailed Student’s t-tests, and one-way ANOVA analysis of variance followed by Tukey’s post hoc tests were conducted for multiple comparisons. A two-tailed value of p < 0.05 was considered statistically significant (* p < 0.05; ** p < 0.01; *** p < 0.001).
Data availability
The data that support the conclusions of the current study are available from the corresponding authors upon reasonable request.
Acknowledgments
This study was supported by grants from the National Natural Science Foundation of China (No. 82170929 and 81970908 to YL) and the Beijing Natural Science Foundation (No. 7222224 to XZ).
Author contributions
X.Z. and Y.L.: conception and design, financial support, and manuscript writing. Y.Z., Y.L., X. L., Y.D. and D.L.: laboratory work, and manuscript writing. Y.C.: laboratory work, collection and analyses of data, manuscript writing. All authors read and approved the final manuscript.
Additional information
Supplementary information.
Supplementary information accompanies the manuscript on the International Journal of Oral Science website http://www.nature.com/ijos.
Competing interests.
The authors declare that they have no competing interests.
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Table 1. Sequences of PCR primers.
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