A preparation method, product and application of stem cell-derived extracellular vesicles

Abstract

The present invention relates to a method for preparing extracellular vesicles derived from stem cells, specifically, constructing a nucleotide fragment of a gene sequence such as SEQ ID No.1 into an adenovirus plasmid to obtain a BMP9 overexpression recombinant adenovirus; then infecting immortalized mouse adipose-derived stem cells with the BMP9 overexpression recombinant adenovirus; and finally collecting extracellular vesicles in the cell supernatant to obtain stem cell-derived extracellular vesicles. The extracellular vesicle has low immunogenicity, exerts an osteogenic effect through a completely independent molecular mechanism, and its osteogenic ability is comparable to that of AdR‑B9, providing a highly efficient cell-free and virus-free treatment strategy for bone defect repair, which is of great significance in biomedical research and clinical application, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to a preparation method, product and application of stem cell-derived extracellular vesicles based on BMP9 engineering. Background Art

[0002] Bones are key components of the musculoskeletal system, facilitating movement, protecting internal organs, and storing essential nutrients such as calcium, phosphorus, and bone marrow. Studies have shown that about 5% to 10% of fractures caused by trauma or osteoporosis cannot heal completely. Currently, the treatments for fractures and large bone defects mainly include autologous bone, allogeneic bone, and synthetic materials, but each material has its inherent limitations and still cannot fully meet clinical needs.

[0003] Bone morphogenetic protein (BMP) belongs to the transforming growth factor-β (TGF-β) superfamily and plays an important role in inducing osteogenic differentiation of stem cells. Members such as BMP-2, BMP-7 and BMP-9 have significant effects in bone healing, among which BMP-9 mediated by recombinant adenovirus vector has the strongest osteogenic effect among the 14 known BMPs. The adenovirus gene vector system enters the cell through receptor-mediated endocytosis, and the adenovirus genome is transferred to the host cell nucleus, but it is not integrated into the host cell genome. It can transiently express foreign genes and has high safety. It has great potential in gene therapy. However, wild-type AAV is widely present in nature. 40%-80% of adults have been infected with AAV. When using recombinant adenovirus vectors for gene therapy, it may cause immune rejection; and the use of recombinant adenovirus is based on virus titer and infection rate, and there is no absolute dose-effect relationship.

[0004] Mesenchymal stem cells (MSCs) have become one of the most commonly used stem cells in cell therapy due to their immunomodulatory, tissue regeneration and protective abilities. However, there are still some problems with mesenchymal stem cell therapy, such as the difficulty in obtaining a stable phenotype cell source and the retention of cells in the pulmonary microvasculature leading to infusion-related toxicity. Preclinical studies and clinical trials in recent years have shown that the therapeutic effects of mesenchymal stem cells are mainly exerted through the growth factors, chemokines and cytokines they secrete. MSCs can secrete a large number of extracellular vesicles (EVs), which are considered to be paracrine signal mediators between cells and target cells, demonstrating the biological potential of MSCs and may become an alternative to cell therapy, promoting the development of cell-free therapy. In addition, extracellular vesicles derived from immortalized mesenchymal stem cells can be used in cell therapy, while immortalized cells themselves are not suitable. Due to the nanoscale size of extracellular vesicles, they can effectively migrate to specific tissues after administration and avoid accumulation in the pulmonary microvasculature, thereby reducing the risk of pulmonary embolism that may be caused when using cells. At present, mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have been identified to contain 304 bioactive molecules including proteins, lipids, DNA, mRNA and 150 miRNAs. Although MSC-EVs from different sources have some commonalities in function and their specific effects may vary, the osteogenesis effect of MSC-EVs is poor. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a method for preparing stem cell-derived extracellular vesicles based on BMP9 engineering, and also to provide B9-EVs obtained by the preparation method, and the use of the extracellular vesicles as a drug in the treatment of bone injuries.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] 1. A method for preparing extracellular vesicles derived from stem cells, the specific steps of the preparation method are:

[0008] 1) constructing a nucleotide fragment of the gene sequence as shown in SEQ ID No. 1 into an adenovirus plasmid to obtain a BMP9 overexpression recombinant adenovirus;

[0009] 2) Immortalized mouse adipose-derived stem cells were then infected with the BMP9 overexpressing recombinant adenovirus and cultured for 24-48 hours;

[0010] 3) collecting the cell supernatant, and then collecting the extracellular vesicles in the cell supernatant to obtain stem cell-derived extracellular vesicles.

[0011] Further, in step 1), the restriction sites EcoRⅠ and EcoRⅤ are designed at both ends of the sequence fragment shown in SEQ ID No.1, and the sequence shown in SEQ ID No.2 is synthesized; the synthetic sequence and the adenovirus vector plasmid pAdTrace-TO4 are cut with EcoRⅠ and EcoRⅤ; the BMP9 coding region is inserted into pAdTrace-TO4 with a ligase to construct a shuttle plasmid; the shuttle plasmid is cut with PmeI, and the recombinant adenovirus plasmid is constructed by electroporation of BJ5183 competent cells containing the backbone plasmid pAdEasy1; and the BMP9 overexpression recombinant adenovirus is obtained by transfection and packaging.

[0012] Furthermore, the extracellular vesicles are collected by ultracentrifugation, polyethylene glycol precipitation or gradient centrifugation.

[0013] Furthermore, the ultracentrifugation method specifically involves centrifuging the collected supernatant at 1000g to 2000g to remove cell debris and large-size vesicles, then centrifuging the low-speed centrifugation supernatant at 50,000 to 100,000g to enrich the extracellular vesicles in the form of a precipitate, washing the vesicle precipitate with a buffer solution to remove impurities, and finally suspending the vesicles with a buffer solution to obtain extracellular vesicles.

[0014] Furthermore, the polyethylene glycol precipitation method specifically involves mixing the cell supernatant with a polyethylene glycol solution to form an extracellular vesicle-polyethylene glycol complex, then centrifuging to precipitate the complex, washing the vesicle precipitate with a buffer solution to remove impurities, and finally suspending the vesicles with a buffer solution to obtain extracellular vesicles.

[0015] Furthermore, the gradient centrifugation method specifically comprises centrifuging the cell supernatant at 300g-500g for 5-10 minutes, 2000g-3000g for 20-25 minutes, and 10000g-15000g for 30 minutes, collecting the supernatant in each of the previous centrifugation steps, and finally centrifuging at 80000-100000g for 70-90 minutes, collecting the precipitate, and resuspending it with buffer.

[0016] Furthermore, the same gradient centrifugation conditions can be used to prepare extracellular vesicles with higher purity.

[0017] 2. A stem cell-derived extracellular vesicle engineered based on BMP9 is also provided, and the preparation method is the same as above.

[0018] The extracellular vesicles have superior osteogenic ability than recombinant adenovirus overexpressing BMP9 and have low immunogenicity.

[0019] 3. The third objective of the present invention is to provide the use of the preparation method of the above-mentioned stem cell-derived extracellular vesicles in the preparation of drugs for the treatment of bone injuries.

[0020] 4. A fourth objective of the present invention is to provide the use of the above-mentioned stem cell-derived extracellular vesicles with excellent osteogenic ability in the preparation of drugs for treating bone injuries.

[0021] The beneficial effect of the present invention is that immortalized mouse adipose-derived stem cells are modified by using a recombinant adenovirus (AdR-B9) overexpressing BMP9, and stem cell-derived extracellular vesicles B9-EVs are extracted by gradient centrifugation and identified. B9-EVs not only have low immunogenicity, but also exert osteogenic effects through completely independent molecular mechanisms. When the dose reaches 60 μg / mL, the osteogenic ability is stronger than AdR-B9. In vitro and in vivo osteogenic experiments and subcutaneous immunization experiments all show that the B9-EVs prepared by the present invention effectively promote osteogenesis in a safe and effective manner without inducing an immune response. Further, the miRNA-seq of B9-EVs and the mRNA-seq of IMAD treated with a recombinant adenovirus (Ad-B9) overexpressing BMP9 jointly show that B9-EVs induce osteogenesis by changing the expression of osteogenesis-related mRNA regulated by different miRNAs carried by them, which is completely different from the osteogenesis mechanism of Ad-B9. In general, our research results show that the B9-EVs prepared by the present invention have great potential in bone tissue engineering. The extracellular vesicles B9-EVs are expected to replace recombinant adenovirus vectors to mediate BMP9-induced osteogenesis, providing a cell-free and virus-free treatment strategy for bone defect repair with natural biocompatibility, extremely low immunotoxicity, a clear dose-effect relationship, and high efficiency, which has important application value in both biology and medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0023] Figure 1 For the identification of B9-EVs after preparation, A: Fluorescence images were taken, and the overexpression efficiency of Bmp9 was verified by quantitative PCR (TqPCR). The results after 48 hours of infection are shown in the figure. The data analysis results show that "**" indicates p<0.01, AdR-B9 group vs. Ad-RFP group; B: Particle size (Nanoparticle tracking analysis, NTA) analysis of B9-EVs and MV-EVs; C: Representative transmission electron microscopy images of B9-EVs and MV-EVs, scale bars: 1000nm, 600nm, 200nm; D: Total protein was extracted and the protein expression levels of ALIX, TSG101, HSP70 and histone H3 were detected by Western blot. The blank control group was uninfected iMAD cells.

[0024] Figure 2The results of the experiment on B9-EVs promoting osteogenic differentiation of stem cells in vitro, where A: Confocal microscopy images show the uptake of B9-EVs and MV-EVs by iMAD cells. Extracellular vesicles were stained with Dil (red), cytoskeleton was stained with phalloidin (green), and nucleus was stained with DAPI (blue); B: iMAD cells were treated with different concentrations of B9-EVs (a) and MV-EVs (b), and alkaline phosphatase (ALP) activity was detected on the third day. AdR- The B9 treatment group was the positive control. The data analysis results showed that "*" indicates p<0.05, "**" indicates p<0.01, B9-EVs group vs. MV-EVs group; C: iMAD cells were treated with 60μg / mL of B9-EVs and MV-EVs, and ALP staining was performed on the 5th day (left) and Alizarin red staining was performed on the 14th day (right); D: RT-qPCR analysis of the expression of osteogenic markers in iMAD cells treated with B9-EVs and MV-EVs at 36 hours. Data analysis showed that "*" indicates p<0.05, "**" indicates p<0.01, B9-EVs group vs. MV-EVs group.

[0025] Figure 3 Figure 1 is the result of the B9-EVs biosafety and immunogenicity experiment, where A is a schematic diagram of the operation process; B: 72 hours after injection, the changes in the total white blood cell (WBC), neutrophil, lymphocyte and eosinophil counts in the peripheral blood were evaluated; C: the levels of IgM, IgE and IgG were measured; D: the changes in the skin appearance of the injection site were observed 24 hours, 48 ​​hours and 72 hours after the last subcutaneous injection; E: H&E staining of the injection site and spleen, and histological examination was performed 72 hours after the last subcutaneous injection; F: Immunohistochemistry (IHC) staining was used to detect the expression of CD68, CD206, CD19 and CD3 at the injection site, 72 hours after the last injection.

[0026] Figure 4 The results of the experiment of B9-EVs promoting subcutaneous ectopic osteogenesis in nude mice are shown in Figure 1. A: After about 50% density of iMAD cells were treated with MV-EVs and B9-EVs for 36 hours, the cells were collected and injected into the subcutaneous tissue of nude mice. After 6 weeks of implantation, the subcutaneous mass was removed and μCT imaging was performed; B: Quantitative analysis of ectopic bone blocks was performed to evaluate the relevant parameters of osteogenesis. The data analysis results showed that "*" indicates p<0.05, "**" indicates p<0.01, B9-EVs group vs. MV-EVs group; C: H&E staining, Masson trichrome staining, safranin fast green staining and Alcian blue staining were performed on the subcutaneous mass.

[0027] Figure 5The results of the transcriptome analysis experiment of B9-EVs-induced osteogenesis, where A: Western blot detection diagram; B: Heat map of differentially expressed genes between B9-EVs and MV-EVs; C: Volcano map, in the B9-EVs treatment group, compared with the MV-EVs treatment group, 447 mRNAs were significantly upregulated (FDR<0.05, fold change>2), and 321 mRNAs were significantly downregulated (FDR<0.05, fold change>2); D: KEGG analysis of the top 30 signaling pathways ranked by P value in the B9-EVs treatment group; E: Volcano map, in the AdR-B9 treatment group, compared with the Ad-RFP treatment group, 398 mRNAs were significantly upregulated (FDR<0.05, fold change>2), and 337 mRNAs were significantly downregulated (FDR<0.05, fold change>2). change>2); F: KEGG analysis of the top 30 signaling pathways ranked by P value in the AdR-B9-treated group compared with the Ad-RFP-treated group; G: Figure 5 Cluster analysis of the FPKM values ​​of the 356 intersection mRNAs in H; H: Venn diagram showing the 356 intersection mRNAs co-expressed in the B9-EVs treatment group and the AdR-B9 treatment group; I: Based on Figure 5 The 356 mRNAs in H were analyzed by KEGG for 19 signaling pathways.

[0028] Figure 6 Figure 1 shows the results of the osteogenesis-related miRNA analysis experiment in B9-EVs, where A: heat map showing the expression of differentially expressed miRNAs in B9-EVs and MV-EVs; B: volcano map of differential miRNAs in MV-EVs and B9-EVs; C: the top 30 related KEGG signaling pathways enriched by differentially expressed miRNAs; D: Venn diagram of mRNAs in different groups; E: Figure 5 D: Cluster analysis of the FPKM values ​​of 98 mRNAs; F: KEGG enrichment analysis of 98 mRNAs; G: GO analysis of 98 mRNAs.

[0029] Figure 7 The results of the experiment to identify the unique miRNA-mRNA interaction osteogenesis mechanism of B9-EVs, where A: the regulatory network diagram of 98 mRNAs and miRNAs; B: the heat map showing the expression of 45 miRNAs in B9-EVs and MV-EVs.

[0030] Figure 8Figure 2: The results of differential analysis of miRNA and mRNA. In iMAD, after treatment with B9-EVs and MV-EVs for 36 hours, RT-qPCR analysis was performed on down-regulated miRNA (A) and up-regulated miRNA (B). “*” indicates p<0.05, “**” indicates p<0.01, B9-EVs group vs. MV-EVs group; C: RT-qPCR analysis of target mRNA after treatment of iMAD with B9-EVs and MV-EVs for 36 hours. “*” indicates p<0.05, “**” indicates p<0.01, B9-EVs group vs. MV-EVs group; D: RT-qPCR analysis of target mRNA after treatment of iMAD with Ad-RFP and AdR-B9 for 36 hours; E: Regulatory network diagram of significant target mRNA in B9-EVs; F: Regulatory network diagram of significant related miRNA in B9-EVs. DETAILED DESCRIPTION

[0031] The preferred embodiment of the present invention will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. The experimental methods for which specific conditions are not specified in the embodiments are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0032] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0033] Immortalized mouse adipose-derived mesenchymal stem cells (iMAD) are extracted from mouse embryos and immortalized.

[0034] The animal experiments in this invention were approved by the Ethics Committee for the Use of Research and Experimental Animals of Chongqing Medical University, and all experimental procedures were performed in accordance with the guidelines.

[0035] Other reagents were from Thermo Fisher Scientific (Waltham, MA) or Millipore Sigma (St. Louis, MO).

[0036] Example 1

[0037] Preparation and identification of BMP9 engineered vesicles (B9-EVs):

[0038] 1. The nucleic acid sequence of mouse BMP9 coding region (NM_019506.4) is SEQ ID No. 1:

[0039]

[0040] 2. Chemically synthesize the nucleic acid fragment of mouse BMP9 coding region (NM_019506.4), and design restriction sites EcoRⅠ and EcoRⅤ at both ends of the fragment, respectively. The synthetic sequence is SEQ ID No.2:

[0041] GAATTC GATATC

[0042] 3. Use EcoRⅠ and EcoRⅤ to treat the synthetic sequence and adenovirus vector plasmid pAdTrace-TO4 respectively, use T4 ligase to insert the BMP9 coding region into pAdTrace-TO4, and the constructed shuttle plasmid is named pAdTrace-BMP9;

[0043] 4. The shuttle plasmid named pAdTrace-BMP9 was cut at the PmeI restriction site to expose the left and right arms. The shuttle plasmid and the left and right arms on the backbone plasmid were homologously recombined by electroporation into BJ5183 competent cells containing the backbone plasmid pAdEasy1 to construct the recombinant adenovirus plasmid pAd-BMP9;

[0044] 5. Construction of mouse BMP9 overexpression recombinant adenovirus AdR-B9: transfect the recombinant adenovirus plasmid pAd-BMP9 into the tool cell 293pTP for adenovirus packaging, amplify the adenovirus in HEK-293 cells, observe the fluorescence under a fluorescence microscope, and prepare 10 10 PFU / mL recombinant adenovirus AdR-B9, the optimal infection titer was measured in the target cells before use;

[0045] 6. Preparation of B9-EVs using gradient centrifugation: Immortalized mouse adipose-derived mesenchymal stem cells (iMAD) with a density of about 50% were inoculated in T75 culture dishes (x5) and routinely cultured in DMEM containing 10% FBS and double antibodies at 37°C and 5% CO2. The cells were infected with recombinant adenovirus AdR-B9. After 48 hours, the cells were observed under a fluorescence microscope. The virus infection rate was about 50-70%; fluorescent images were taken, and RNA was extracted. qPCR was used to verify the overexpression of Bmp9 in the AdR-B9 group ( Figure 1 A). After culturing in DMEM medium without fetal bovine serum for 48 hours, the cell supernatant was collected and subjected to gradient centrifugation, i.e., centrifugation at 300g for 5 minutes, 2,000g for 25 minutes, and 10,000g for 30 minutes. The supernatant was collected at each of the previous centrifugations, and finally centrifuged at 100,000g for 70 minutes, the supernatant was discarded, and the precipitate was collected to obtain the extracellular vesicles, which were resuspended in 50-100 μL PBS. If you need to collect extracellular vesicles with higher purity, add 30 mL of PBS buffer to the precipitate, and then collect the precipitate under the same gradient centrifugation conditions to obtain extracellular vesicles with higher purity. The obtained extracellular vesicles are named B9-EVs, and the centrifuged B9-EVs are resuspended in 50-100 μL PBS.

[0046] 7. Western Blot: EVs and cells were lysed using a protein extraction kit (Cat# BC3710, Solarbio, China) to determine relative protein expression levels. Proteins were electrophoresed on polyacrylamide gels and transferred to polyvinylidene difluoride membranes. After blocking with skim milk powder, the proteins adsorbed on the membrane were detected with the following primary antibodies: anti-Alix antibody (Bimake; Cat#A5508), anti-Tsg101 antibody (Selleck; Cat#A5378), anti-Hsp70 antibody (Bimake; Cat#A5502), anti-histone H3 antibody (Bimake; Cat#A5885), and anti-β-actin antibody (Proteintech; Cat#66009-1-Ig), anti-Bmp9 antibody (Proteintech; Cat#17769-1-AP), anti-Smad5 (Proteintech; Cat#12167-1-AP), and anti-phospho-Smad5 (Abcam, Cat#ab92698). Horseradish peroxidase-conjugated secondary antibodies (1:5000 dilution; ZSGB-BIG; peroxidase-labeled rabbit anti-goat IgG or peroxidase-labeled goat anti-mouse IgG, Cat#ZB-2301 or 2305) were used. Immunoreactive signals were visualized using an enhanced chemiluminescence (ECL) kit (Cat#BL523A, Biosharp, China) and recorded using a Bio-Rad ChemiDoc imager (Hercules, USA).

[0047] 8. In the above step 3, the BMP9 sequence is not inserted into the shuttle plasmid, but only the red fluorescent protein (RFP) is inserted. The remaining steps 4 and 5 are the same to prepare the recombinant adenovirus Ad-RFP overexpressing RFP. Ad-RFP is also used as a control to infect iMAD cells with a density of about 50%. The remaining operation steps are the same as step 6 to prepare MV-EVs as a control.

[0048] Figure 1 Figure B is the particle size analysis (Nanoparticle tracking analysis, NTA) of B9-EVs and RFP-EVs. NTA shows that the average diameters of B9-EVs and MV-EVs are mainly between 100 and 300 nm. Figure 1Figure C shows representative transmission electron microscopy images of B9-EVs and MV-EVs, where scale bars are 1000 nm, 600 nm, and 200 nm. TEM images show that both types of EVs have a typical cup-shaped structure. Total extracellular vesicle protein was extracted and the protein expression levels of ALIX, TSG101, HSP70, and histone H3 were detected by Western blot. The blank control group was composed of intracellular proteins of uninfected iMAD cells. The results are shown in Figure 2. Figure 1 As shown in D, Western blot analysis further confirmed the presence of characteristic markers of extracellular vesicles in both EVs, including ALIX, TSG101, and HSP70, and the absence of Histone H3 (a protein present in the cell nucleus). The above results collectively indicate that BMP9-engineered extracellular vesicles B9-EVs and blank MV-EVs were successfully prepared.

[0049] Example 2

[0050] Example of analysis of B9-EVs-induced osteogenic differentiation potential of stem cells in vitro:

[0051] iMAD cells were infected with AdR-B9 and Ad-RFP (negative control), and the extracted extracellular vesicles were named B9-EVs and MV-EVs (negative control), respectively. B9-EVs and MV-EVs were stained with Dil (red) dye, the cytoskeleton was stained with phalloidin (green), and the cell nucleus was stained with DAPI (blue).

[0052] Confocal microscopy images showed that both B9-EVs and MV-EVs could be successfully taken up by iMAD cells ( Figure 2 A), ensuring that EVs can smoothly enter cells through endocytosis. In order to verify the osteogenic ability of B9-EVs in vitro, iMAD cells were treated with different doses of B9-EVs and MV-EVs, and AdR-B9 was used as a positive control. The results of alkaline phosphatase (ALP) activity detection showed that with the increase of B9-EVs concentration, the cell ALP activity gradually increased; when the concentration was about 60μg / mL, the ALP activity was higher than that of the AdR-B9 group, while in the Ad-RFP group, no significant changes in ALP were observed ( Figure 2 ALP staining of iMAD cells treated with B9-EVs and AdR-B9 was positive on day 5 ( Figure 2 By day 14, matrix mineralization deposition was significantly increased in the B9-EVs and AdR-B9-treated groups, whereas almost no mineralization was observed in the MV-EVs-treated group ( Figure 2RT-qPCR results further showed that the expression of osteogenic gene markers Opn, Osx, Ocn, Runx2, Alpl, Acan, and Sox9 in iMAD cells treated with B9-EVs was significantly upregulated compared with the MV-EVs group. These results indicate that B9-EVs can significantly induce osteogenic differentiation of stem cells in vitro, and the osteogenic capacity can increase with increasing doses. The osteogenic capacity of 60μg / mL dose of B9-EVs is comparable to that of AdR-B9.

[0053] Experimental Example 3.

[0054] B9-EVs biosafety and immunogenicity analysis example:

[0055] To explore whether B9-EVs could trigger an immune response in the host, we performed a subcutaneous immune characterization experiment ( Figure 3 The specific operation process is as follows: 5 μg B9-EVs were dissolved in 20 μL saline diluent and injected subcutaneously into C57BL / 6J mice once a week for 3 consecutive weeks. An equal volume of saline was used as a control. The mice were killed 24 hours, 48 ​​hours, and 72 hours after the last injection for subsequent operations. In the B9-EVs-treated group, there was no significant change in the counts of white blood cells (WBC), lymphocytes, neutrophils, and eosinophils in the peripheral blood 72 hours after the last injection ( Figure 3 B), and the levels of IgE, IgG, and IgM in serum did not fluctuate significantly ( Figure 3 In addition, no visible lumps or papules were observed at the injection site at any time point ( Figure 3 Middle D). H&E staining results showed that no accumulation of inflammatory cells or abnormal tissue structure was observed in the skin layer around the injection site, and no abnormal changes were observed in the spleen morphology ( Figure 3 Immunohistochemical staining further showed that the number of macrophages (marked by CD68 and CD206) and T lymphocytes (marked by CD3 and CD19) around the injection site did not increase after 72 hours ( Figure 3 (F) Taken together, these results suggest that topical application of B9-EVs does not induce an immunogenic response.

[0056] Experimental Example 4.

[0057] Example of analysis of the ectopic osteogenic potential of B9-EVs-induced stem cells in nude mice:

[0058] iMAD cells were treated with MV-EVs, B9-EVs, and AdR-B9, respectively, and the cells were collected and injected subcutaneously into nude mice. After 6 weeks of observation, μCT images showed that obvious osteogenesis occurred in the AdR-B9 and B9-EVs treatment groups, while no bone formation was observed in the MV-EVs group ( Figure 4 Quantitative analysis by μCT further showed that the mean bone density, tissue volume (TV), bone volume (BV), BV / TV ratio, trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular spacing (Tb.Sp) in the AdR-B9 and B9-EVs treatment groups were significantly increased compared with the MV-EVs treatment group ( Figure 4 The results of H&E staining, Masson's trichrome staining, safranin fast green staining, and Alcian blue staining showed that both the AdR-B9 and B9-EVs treatment groups showed obvious mature bone and cartilage formation, while the MV-EVs group showed almost no mature bone and cartilage ( Figure 4 These results suggest that B9-EVs exhibit similar ectopic osteogenic capacity as AdR-B9 in vivo.

[0059] Experimental Example 5.

[0060] Example of mRNA transcriptomic analysis after B9-EVs induced stem cells:

[0061] In the molecular mechanism of BMP9-induced osteogenesis, BMP9 activates the intracellular R-SMAD signaling pathway by binding to the cell surface receptors BMPR1 and BMPR2. This pathway further regulates the transcription of osteogenesis-related genes after the complex enters the cell nucleus. After the sub-confluent iMAD cells were treated with B9-EVs or MV-EVs for 36 hours, total RNA was collected for RNA sequencing analysis. The total protein of B9-EVs was directly extracted for Western blot detection of BMP9 expression. Sub-confluent iMAD cells were treated with B9-EVs, MV-EVs, AdR-B9 and Ad-RFP, respectively. The total cell protein after 72 hours was taken for Western blot analysis to evaluate the expression levels of BMP9, SMAD5 and p-SMAD5. The SMAD5 and p-SMAD5 samples were intracellular proteins extracted 72 hours after the cells were treated with adenovirus and B9-EVs, respectively. In this study, the Western blot results are shown in the following table. Figure 5 As shown in A, Western blot showed that BMP9 expression was significantly upregulated in iMAD cells treated with AdR-B9. Compared with AdR-B9, B9-EVs contained only a very small amount of BMP9 protein, which indicates that the osteogenic ability exhibited by B9-EVs in Examples 2 and 4 is not achieved through BMP9.

[0062] Further, B9-EVs and MV-EVs were treated in iMAD cells, and RNA-seq analysis was performed. FPKM value cluster analysis was performed on 768 differentially expressed mRNAs in the B9-EVs-treated group and the MV-EVs-treated group. The differential gene heat map showed that the mRNA expression profiles of 768 genes in iMAD cells treated with B9-EVs changed significantly ( Figure 5 B), of which 447 genes were down-regulated and 321 genes were up-regulated ( Figure 5 C). KEGG analysis showed that these 768 genes were enriched in a variety of osteogenesis-related signaling pathways, including the PI3K-AKT pathway, calcium ion signaling pathway, MAPK pathway, TGF-β pathway, and pathways related to regulating stem cell pluripotency ( Figure 5 In addition, GO enrichment analysis showed that B9-EVs play a role in biological processes such as cell differentiation, extracellular matrix formation, and angiogenesis.

[0063] In addition, iMAD cells were infected with AdR-B9 and Ad-RFP, respectively, and mRNA-seq analysis was performed. The results showed that the expression profiles of 735 mRNAs changed significantly in iMAD cells induced by AdR-B9, of which 398 genes were downregulated and 337 genes were upregulated ( Figure 5 Middle E). KEGG enrichment analysis showed that these 735 mRNAs were enriched in calcium signaling pathway, TGF-β signaling pathway and related pathways regulating stem cell pluripotency ( Figure 5 F). GO enrichment analysis further found that AdR-B9-treated iMAD cells also showed significant enrichment in biological processes such as cartilage development, vascular development, and BMP signaling pathways. The Venn diagram showed that 356 differentially expressed mRNAs were significantly changed in B9-EVs-treated and AdR-B9-treated iMAD cells ( Figure 5 The heat map of these 356 mRNAs showed significant differences in the expression profiles induced by B9-EVs and AdR-B9 treatment ( Figure 5 KEGG analysis of these 356 mRNAs showed that the TGF-β signaling pathway directly regulated by BMP9 had the highest enrichment level ( Figure 5 These results indicate that B9-EVs and AdR-B9 share some of the same osteogenic mechanisms, as well as unique osteogenic mechanisms (a total of 412 genes), which deserve further exploration.

[0064] Experimental Example 6.

[0065] Example of high-throughput sequencing analysis of miRNA after B9-EVs induced stem cells:

[0066] To clarify the role of miRNAs in EVs in bone development and regeneration, we analyzed the changes in miRNA expression profiles in B9-EVs by high-throughput sequencing. Gene expression heat map analysis showed that 81 miRNAs were differentially expressed in B9-EVs compared with MV-EVs ( Figure 6 The volcano plot further showed that compared with MV-EVs, 64 miRNAs were significantly downregulated (P value<0.05, fold change>2) and 17 miRNAs were significantly upregulated (P value<0.05, fold change>2) in B9-EVs ( Figure 6 Middle B). KEGG analysis results showed that these differentially expressed miRNAs were significantly enriched in signaling pathways related to osteogenesis, such as PI3K-AKT, MAPK, calcium signaling pathway, and Wnt signaling pathway ( Figure 6 (C) GO enrichment analysis revealed that these miRNAs were highly correlated with biological processes related to tissue repair, including cell differentiation, extracellular matrix synthesis, and vascular remodeling.

[0067] By analyzing these 81 differentially expressed miRNAs, we predicted 15,222 potential target mRNAs and identified 464 mRNAs that appeared in the intersection of 15,222 targets and 768 mRNAs, indicating that these mRNAs may be directly regulated by miRNAs in B9-EVs ( Figure 6 In addition, the results of the intersection of mRNAs targeted by down-regulated miRNAs (14,641) and up-regulated differentially expressed mRNAs in B9-EVs (321) are also shown. Considering the regulatory mode of miRNA on mRNA, we screened out 196 mRNAs, some of which interacted with down-regulated miRNAs ( Figure 6 D, b), and the other part interacts with upregulated miRNAs ( Figure 6 These 247 mRNAs may be directly targeted by miRNAs in B9-EVs. Notably, the expression of 98 mRNAs was significantly changed in iMAD cells treated with B9-EVs ( Figure 6 D, d). The heat map shows the differential expression of these 98 mRNAs in iMAD cells treated with AdR-B9 and B9-EVs ( Figure 6 Further KEGG and GO enrichment analysis showed that these mRNAs play an important role in osteogenesis-related signaling pathways, especially in the PI3K and Notch signaling pathways, and are closely related to biological processes such as cell differentiation and vascular development ( Figure 6F and G). These results highlight the potential therapeutic value of EVs in regenerative medicine. At the same time, our study also showed that the changes of certain miRNAs in B9-EVs may regulate osteogenesis-related mRNAs in iMAD cells through the PI3K and Notch signaling pathways, and this mechanism may be different from the mechanism of action when directly using AdR-B9 treatment.

[0068] Experimental Example 7.

[0069] Analysis example of B9-EVs inducing stem cell osteogenesis through a specific miRNA-mRNA regulatory network:

[0070] We further Figure 6 45 miRNAs that may regulate 98 mRNAs were screened out from the miRNA-seq data of A ( Figure 7 The heat map shows the differential expression levels of these 45 miRNAs between the B9-EVs group and the MV-EVs group ( Figure 7 (B). The top 27 miRNAs were selected based on the P value ranking. Among these miRNAs, 21 were reported to be associated with osteogenesis, of which 16 were downregulated and 5 were upregulated. In iMAD, after 36 h of treatment with B9-EVs and MV-EVs, RT-qPCR analysis was performed on downregulated miRNAs (A) and upregulated miRNAs (B). TqPCR validation results showed that miR-200a-3p, miR-127-3p, miR-103-3p, miR-5106, miR-504-5p, miR-409-3p, miR-6240, miR-10b-5p, miR-182-5p, miR-183-5p, miR-205-5p, and miR-200c-3p were all downregulated in the B9-EVs group ( Figure 8 In contrast, miR-224-5p, miR-92a-3p, and miR-1198-5p were upregulated ( Figure 8 (B). Seven miRNAs that did not reach the expected expression were excluded. Among the 14 expected expressed miRNAs, we selected 18 mRNAs known to be involved in osteogenesis or tissue repair from the initial 98 mRNAs for further study. TqPCR results showed that mRNAs such as Fat4, Adamts12, Atp2b4, Ptx3, Bdnf, Efna3, Glis1, and Htr2a were upregulated after iMAD cells were treated with AdR-B9 EVs, while mRNAs such as Gbp4, Ankrd6, Camk2b, Dtx4, and Vcam1 were downregulated ( Figure 8(C). Five mRNAs that were not expressed as expected were excluded. We also verified the expression of 13 mRNAs in iMAD cells directly treated with AdR-B9. The results showed that only Ptx3 was upregulated in both AdR-B9EVs-treated and directly AdR-B9-treated iMAD cells, indicating that the expression of the other 12 mRNAs was significantly changed only in B9-EVs-treated cells. Figure 8 D is the RT-qPCR analysis of target mRNA after iMAD was treated with Ad-RFP and AdR-B9 for 36 hours.

[0071] We selected miRNAs and mRNAs that showed statistically significant changes and negatively correlated expression (e.g. Figure 8 E and F). Based on these findings, we propose that miRNAs in B9-EVs, such as miR-200a-3p, miR-205-5p, miR-103-3p, miR-182-5p, miR-6240, miR-127-3p, miR-5106, miR-504-5p, miR-224-5p, and miR-1198-5p, enter iMAD cells and regulate the expression of osteogenesis-related mRNAs, including Htr2a, Efna3, Glis1, Bdnf, Ptx3, Adamts12, Fat4, Atp2b4, Ankrd6, Dtx4, Gbp4, and Vcam1. This regulatory mechanism promotes osteogenesis by exerting a different pathway from AdR-B9.

[0072] The above results suggest that B9-EVs have the same excellent osteogenic ability as AdR-B9. From the perspective of the mechanism of action, the osteogenic mechanisms of the two are different. B9-EVs induce new bone formation by regulating the expression of multiple osteogenic-related mRNAs regulated by different miRNAs. Overall, our research results show that B9-EVs have very strong application potential in bone tissue engineering and are expected to become an emerging strategy for tissue engineering to repair bone defects, and can be used as a drug in the clinical treatment of bone loss.

[0073] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A method for preparing extracellular vesicles derived from stem cells, characterized in that: The specific steps of the preparation method are: 1) constructing a nucleotide fragment of the gene sequence as shown in SEQ ID No. 1 into an adenovirus plasmid to obtain a BMP9 overexpression recombinant adenovirus; 2) Immortalized mouse adipose-derived stem cells were then infected with the BMP9 overexpressing recombinant adenovirus and cultured for 24-48 hours; 3) collecting the cell supernatant, and then collecting the extracellular vesicles in the cell supernatant to obtain stem cell-derived extracellular vesicles.

2. The method for preparing stem cell-derived extracellular vesicles according to claim 1, characterized in that: The specific construction in step 1) is as follows: the restriction sites EcoRⅠ and EcoRⅤ are designed at both ends of the sequence fragment shown in SEQ ID No.1, and the sequence shown in SEQ ID No.2 is synthesized; the synthesized sequence and the adenovirus vector plasmid pAdTrace-TO4 are cut with EcoRⅠ and EcoRⅤ; the BMP9 coding region is inserted into pAdTrace-TO4 with a ligase to construct a shuttle plasmid; the shuttle plasmid is cut with PmeI, and the recombinant adenovirus plasmid is constructed by electroporation of BJ5183 competent cells containing the backbone plasmid pAdEasy1; and the BMP9 overexpression recombinant adenovirus is obtained by transfection and packaging.

3. The method for preparing stem cell-derived extracellular vesicles according to claim 1, characterized in that: Collect extracellular vesicles using ultracentrifugation, polyethylene glycol precipitation, or gradient centrifugation.

4. The method for preparing stem cell-derived extracellular vesicles according to claim 3, characterized in that: The ultracentrifugation method specifically involves centrifuging the collected supernatant at 1000g to 2000g to remove cell debris and large-size vesicles, then centrifuging the low-speed centrifugation supernatant at 50,000 to 100,000g to enrich the extracellular vesicles in the form of a precipitate, washing the vesicle precipitate with a buffer solution to remove impurities, and finally suspending the vesicles with a buffer solution to obtain extracellular vesicles.

5. The method for preparing stem cell-derived extracellular vesicles according to claim 3, characterized in that: The polyethylene glycol precipitation method specifically involves mixing the cell supernatant with a polyethylene glycol solution to form an extracellular vesicle-polyethylene glycol complex, centrifuging to precipitate the complex, washing the vesicle precipitate with a buffer solution to remove impurities, and finally suspending the vesicle with a buffer solution to obtain extracellular vesicles.

6. The method for preparing stem cell-derived extracellular vesicles according to claim 3, characterized in that: The gradient centrifugation method specifically involves centrifuging the cell supernatant at 300g-500g for 5-10 minutes, 2000g-3000g for 20-25 minutes, and 10000g-15000g for 30 minutes. The supernatant is collected in each of the previous centrifugation steps, and finally centrifuged at 80000-100000g for 70-90 minutes, the precipitate is collected, and resuspended with buffer.

7. The method for preparing stem cell-derived extracellular vesicles according to claim 6, characterized in that: The same gradient centrifugation conditions can also be used to prepare extracellular vesicles with higher purity.

8. The stem cell-derived extracellular vesicles engineered based on BMP9 obtained by the preparation method of claims 1-7.

9. Use of the preparation method according to claims 1 to 7 in preparing drugs for treating bone damage.

10. Use of the stem cell-derived extracellular vesicles according to claim 8 in the preparation of a drug for treating bone damage.

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