Specific marker combination of mesenchymal stem cell-derived small extracellular vesicles and use thereof

Abstract

Disclosed are a specific marker combination of mesenchymal stem cell-derived small extracellular vesicles and the use thereof. The characteristic marker combination comprises aminopeptidase N (CD13), integrin β-1 (CD29), membrane glycoprotein THY-1 (CD90), and markers CD9, CD63 and CD81. By using the characteristic marker combination of mesenchymal stem cell-derived small extracellular vesicles for nano-flow cytometry analysis at the single-vesicle level, mesenchymal stem cell-derived small extracellular vesicles can be identified. On the basis of the provided characteristic marker combination, specific modification and engineering on the basis of small extracellular vesicle membrane proteins can further be realized.

Description

Combination of specific biomarkers for small extracellular vesicles derived from mesenchymal stem cells and their applications Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a specific biomarker combination of small extracellular vesicles derived from mesenchymal stem cells and its application. Background Technology

[0002] Small extracellular vesicles (sEVs) are tiny vesicles with a diameter of less than 200 nm, composed of a phospholipid bilayer membrane. Almost all living cells can secrete sEVs, whose membrane surface and lumen typically contain bioactive components such as proteins, lipids, and nucleic acids. By delivering their own bioactive components to target cells, sEVs can mediate intercellular communication, influence the behavior of surrounding or distant cells, and perform specific biological functions. Among them, mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) have been increasingly discovered to play a role in disease treatment, particularly in promoting the repair and regeneration of damaged tissues and organs, attracting widespread attention from researchers.

[0003] MSC-sEVs contain phospholipids, proteins, RNA, microRNA, and DNA fragments from maternal stem cells. They can deliver these active substances from maternal stem cells to recipient cells, thereby exerting stem cell-like biological functions, such as promoting cell proliferation and migration, inhibiting apoptosis, regulating inflammatory responses, and promoting angiogenesis. Studies have shown that MSC-sEVs have a significant promoting effect on the repair and regeneration of damaged tissues in the heart, liver, kidneys, musculoskeletal system, and central nervous system. Furthermore, using MSC-sEVs for tissue repair and disease treatment can avoid the risks of tumorigenesis and vascular embolism associated with direct stem cell transplantation. MSC-sEVs also exhibit low immunogenicity, efficient crossing of various human barriers (skin, mucous membranes, blood-brain barrier, etc.), and ease of industrial production and transportation, demonstrating promising prospects for clinical translational applications.

[0004] Currently, the research and clinical translation of MSC-sEVs for various tissue repair and disease treatment applications are rapidly developing. However, there are significant differences between MSC-sEVs prepared by different research teams or manufacturers: First, MSCs have multiple tissue origins, and sEVs produced from MSCs of different tissue origins have potential differences; second, as adult stem cells, MSCs usually come from different donors, and there are significant individual differences between different donors; third, there are differences in the collection methods and purification methods of MSC-sEVs; fourth, the characterization techniques and indicators included in the detection of MSC-sEVs are also different. Existing indicators for detecting MSC-sEVs include: observing whether MSC-sEVs have the classic saucer-like vesicle structure under transmission electron microscopy, whether the particle size distribution is in the range of 30-200 nm, and Western blotting identification of sEVs positive markers (CD9, CD63, CD81, TSG101, etc.) and negative markers (GM130, Calnexin, etc.). Analyzing the above detection indicators can determine whether particles in a sample are sEVs, and the quantity and purity of sEVs contained therein. However, it cannot distinguish the cellular origin of the sEVs in the sample, nor can it achieve specific identification of sEVs from different cellular sources. Current research shows that sEVs from different cells can perform vastly different biological functions. In tissue repair and disease treatment applications, it is necessary to strictly identify the cellular origin of sEVs and apply them to different scenarios based on the biological functions matched to the cellular origin. For the clinical translational application of MSC-sEVs, their unique proliferative and inflammatory regulatory functions are crucial. Therefore, the identification of MSC-sEVs cannot only include general sEV property analysis indicators; it is essential to confirm the cellular origin of the sEVs. Unfortunately, there is still a lack of specific detection indicators for MSC-sEVs, making it difficult to confirm that a sample is MSC-sEVs through direct identification of sEV samples. This is a major obstacle limiting the clinical translational application of MSC-sEVs.

[0005] Therefore, finding specific biomarkers for MSC-sEVs and using these biomarkers to establish rapid and simple methods for identifying MSC-sEVs is of practical significance for promoting the clinical translation and application of MSC-sEVs.

[0006] Chinese patent CN117147817A discloses a characteristic biomarker combination for small extracellular vesicles derived from pluripotent stem cells and its application. The characteristic biomarker combination for small extracellular vesicles derived from pluripotent stem cells includes: the transmembrane protein PODXL and its glycosylated epitopes Tra-1-60 and Tra-1-81, the membrane surface antigen SSEA-4, and biomarkers CD9, CD63, and CD81. This application utilizes these reliable characteristic biomarkers for small extracellular vesicles derived from pluripotent stem cells and their combination for nanoflow cytometry analysis at the single vesicle level, enabling simple and rapid identification of small extracellular vesicles derived from mesenchymal stem cells. However, since the characteristic biomarkers for small extracellular vesicles from different cell sources are completely different, those skilled in the art cannot obtain a specific biomarker combination for small extracellular vesicles derived from mesenchymal stem cells based on this method. Summary of the Invention

[0007] In view of the technical deficiencies in the identification of mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) in the prior art, the present invention provides a combination of characteristic biomarkers for mesenchymal stem cell-derived small extracellular vesicles and their applications.

[0008] The objective of this invention can be achieved through the following technical solutions:

[0009] This invention first provides a characteristic biomarker combination for small extracellular vesicles derived from mesenchymal stem cells. This biomarker combination includes: aminopeptidase N (AMPN, CD antigen name CD13), recombinant integrin Beta 1 (ITGb1, CD antigen name CD29), membrane glycoprotein THY-1 (THY1, CD antigen name CD90), and biomarkers CD9, CD63, and CD81. For ease of description, the CD antigen names of the biomarkers will be used consistently throughout this text.

[0010] The present invention also provides a method for identifying small extracellular vesicles derived from mesenchymal stem cells, which identifies whether the small extracellular vesicles are derived from mesenchymal stem cells based on a combination of characteristic markers of the small extracellular vesicles derived from mesenchymal stem cells.

[0011] Based on a combination of characteristic biomarkers for small extracellular vesicles derived from mesenchymal stem cells, immunofluorescence staining was performed on the small extracellular vesicles to be detected.

[0012] When the proportion of CD13 positive granules is greater than 50%, the proportion of CD29 positive granules is greater than 50%, the proportion of CD90 positive granules is greater than 50%, the proportion of CD81 positive granules is greater than 50%, and the proportion of CD9 / CD63 / CD81 positive granules is greater than 70%, the small extracellular vesicles are identified as small extracellular vesicles derived from mesenchymal stem cells.

[0013] When none of the following five conditions are met simultaneously, small extracellular vesicles can be identified as small extracellular vesicles not derived from mesenchymal stem cells.

[0014] In one embodiment of the present invention, the method for detecting the proportion of CD13 positive particles is as follows:

[0015] During immunofluorescence staining, fluorescently labeled aptamer molecules corresponding to CD13 were added, and after incubation, the proportion of CD13 positive particles was counted.

[0016] The method for detecting the proportion of CD29 positive particles is as follows:

[0017] During immunofluorescence staining, fluorescently labeled aptamer molecules corresponding to CD29 were added, and after incubation, the proportion of CD29 positive particles was counted.

[0018] The method for detecting the proportion of CD90 positive particles is as follows:

[0019] During immunofluorescence staining, fluorescently labeled aptamer molecules corresponding to CD90 were added, and after incubation, the proportion of CD90 positive particles was counted.

[0020] The method for detecting the proportion of CD81 positive particles is as follows:

[0021] During immunofluorescence staining, fluorescently labeled aptamer molecules corresponding to CD81 were added, and after incubation, the proportion of CD81 positive particles was counted.

[0022] The method for detecting the ratio of CD9 / CD63 / CD81 positive particles is as follows:

[0023] During immunofluorescence staining, three markers CD9 / CD63 / CD81 were combined. That is, fluorescently labeled aptamer molecules corresponding to the three markers CD9, CD63 and CD81 were added at the same time during staining. After incubation, the proportion of particles that were simultaneously positive for CD9 / CD63 / CD81 was counted.

[0024] In one embodiment of the present invention, the sample is immunofluorescently stained using fluorescently labeled aptamer molecules that specifically recognize CD13, CD29, CD90, CD9, CD63, and CD81, wherein the aptamer molecules include, but are not limited to, antibodies and nucleic acid aptamers.

[0025] In one embodiment of the present invention, the fluorescently labeled aptamers that specifically recognize CD13, CD29, CD90, CD9, CD63, and CD81 are BB515 Anti-human CD13 Antibody, AF488 Anti-human CD29 Antibody, FITC Anti-human CD90 Antibody, AF488 Anti-Human CD9, AF488 Anti-Human CD63, and FITC Anti-Human CD81, respectively.

[0026] In one embodiment of the present invention, before performing immunofluorescence staining, the particle-to-protein ratio is calculated. Samples with a particle-to-protein ratio greater than 1*10^8 particles / μg protein and a particle concentration greater than 5*10^9 particles / mL can be subjected to subsequent immunofluorescence staining and identification.

[0027] If the particle concentration of the sample is too low before immunofluorescence staining, it needs to be concentrated so that the particle-to-protein ratio is greater than 1*10^8 particles / μg protein and the particle concentration is greater than 5*10^9 particles / mL before subsequent immunofluorescence staining and identification can be performed.

[0028] In one embodiment of the present invention, when performing immunofluorescence staining on a sample using fluorescently labeled aptamer molecules that specifically recognize CD13, CD29, CD90, CD9, CD63, and CD81, the fluorescently labeled aptamer molecules and the sample are incubated at 37°C for 30 min.

[0029] In one embodiment of the present invention, after immunofluorescence staining, unbound free fluorescently labeled aptamer molecules are removed to reduce free fluorescence interference. The removal method is selected from ultracentrifugation, ultrafiltration or size exclusion chromatography.

[0030] In one embodiment of the present invention, immunofluorescence staining is performed, and after removing unbound free fluorescently labeled aptamer molecules after immunofluorescence staining, nanoflow cytometry analysis at the single vesicle level is performed to obtain the proportions of CD13 positive particles, CD29 positive particles, CD90 positive particles, CD81 positive particles, and CD9 / CD63 / CD81 positive particles.

[0031] In one embodiment of the present invention, a method for identifying small extracellular vesicles derived from mesenchymal stem cells includes the following steps:

[0032] (1) Sample quality control:

[0033] Before immunofluorescence staining, the particle-to-protein ratio is calculated. Samples with a particle-to-protein ratio greater than 1*10^8 particles / μg protein and a particle concentration greater than 5*10^9 particles / mL can be subjected to subsequent immunofluorescence staining and identification. If the particle concentration of the sample is too low before immunofluorescence staining, it needs to be concentrated so that the particle-to-protein ratio is greater than 1*10^8 particles / μg protein and the particle concentration is greater than 5*10^9 particles / mL before subsequent immunofluorescence staining and identification can be performed.

[0034] (2) Immunofluorescence staining:

[0035] After aliquoting the samples, fluorescently labeled aptamer molecules corresponding to CD13, CD29, CD90, CD81 and CD9 / CD63 / CD81 were added in proportion. After vortexing, the samples were incubated at 37°C for 30 min.

[0036] (3) Removal of unbound free fluorescently labeled aptamer molecules after immunofluorescence staining:

[0037] After incubation, aptamer molecules that were not bound by immunofluorescence staining were removed by ultracentrifugation, ultrafiltration or size exclusion chromatography.

[0038] (4) Nanoflow cytometry detection: For samples after removing free fluorescently labeled aptamer molecules, after diluting with PBS to the optimal detection concentration (particle concentration of about 10^8 particles / mL), nanoflow cytometry is used to perform single vesicle level nanoparticle fluorescence analysis to obtain the proportion of CD13 positive particles, CD29 positive particles, CD90 positive particles, CD81 positive particles, and CD9 / CD63 / CD81 positive particles.

[0039] (5) Identification:

[0040] When the proportion of CD13 positive granules is greater than 50%, the proportion of CD29 positive granules is greater than 50%, the proportion of CD90 positive granules is greater than 50%, the proportion of CD81 positive granules is greater than 50%, and the proportion of CD9 / CD63 / CD81 positive granules is greater than 70%, the small extracellular vesicles are identified as small extracellular vesicles derived from mesenchymal stem cells.

[0041] When none of the following five conditions are met simultaneously, small extracellular vesicles can be identified as small extracellular vesicles not derived from mesenchymal stem cells.

[0042] In one embodiment of the present invention, when the sample is analyzed by a nanoflow cytometer, the fluorescence background signal of the sample is not significantly increased compared with the blank control, which verifies that the unbound free fluorescently labeled aptamer molecules have been removed.

[0043] The characteristic biomarker combination of CD13, CD29, and CD90, as well as the biomarkers CD9, CD63, and CD81 provided by this invention, have other applications, including but not limited to: serving as purity identification indicators and product quality control indicators for mesenchymal stem cell-derived small extracellular vesicles; serving as membrane protein modification sites for the engineering modification of mesenchymal stem cell-derived small extracellular vesicles; and serving as fusion gene sites for fluorescent proteins or other fluorescent staining targets for imaging, tracing, and pharmacokinetic analysis of mesenchymal stem cell-derived small extracellular vesicles.

[0044] This application overcomes the problem of the lack of specific identification indicators for MSC-sEVs in the prior art, and provides a combination of specific biomarkers for MSC-sEVs: aminopeptidase N (AMPN, CD antigen name: CD13), integrin β-1 (ITB1, CD antigen name: CD29), membrane glycoprotein THY-1 (THY1, CD antigen name: CD90), and biomarkers CD9, CD63, and CD81. The MSC-sEVs identification method provided by this invention involves immunofluorescence staining and nanoflow cytometry analysis at the single vesicle level using the specific biomarkers CD13, CD29, and CD90 of MSC-sEVs. If the proportion of CD13 positive particles in the sample is greater than 50%, and the proportion of CD29 positive particles is greater than 50%, and the proportion of CD90 positive particles is greater than 50%, and the proportion of CD81 positive particles is greater than 50%, and the proportion of CD9 / CD63 / CD81 positive particles is greater than 70%, then the sample can be identified as MSC-sEVs.

[0045] Compared with existing technologies, this invention utilizes a combination of characteristic biomarkers for mesenchymal stem cell-derived small extracellular vesicles (SMEVs) to perform nanoflow cytometry analysis at the single vesicle level, enabling simple and rapid identification of SMEVs. Furthermore, the combination of characteristic biomarkers for SMEVs based on these inventions also allows for specific modification and engineering of SMEV membrane proteins. This invention has practical significance for the distribution detection, product quality control, engineering modification, and clinical translation of SMEVs derived from mesenchymal stem cells. Attached Figure Description

[0046] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below.

[0047] Figure 1 is a flowchart of the operation for identifying MSC-sEVs according to the present invention.

[0048] Figure 2 shows the property characterization results of MSC-sEVs in this invention.

[0049] Figure 3 shows the nanoflow cytometry results of CD13, CD29 and CD90, the specific biomarkers for identifying MSC-sEVs, and CD9, CD63 and CD81, the general biomarkers for sEVs, as well as the single vesicle level of CD9 / CD63 / CD81.

[0050] Figure 4 shows the nanoflow cytometry results of the detection of CD13, CD29 and CD90; CD9, CD63 and CD81, and CD9 / CD63 / CD81 at the single vesicle level in sEVs samples from various cell sources according to the present invention (Figure 4 includes Figure 4-1, Figure 4-2, Figure 4-3 and Figure 4-4).

[0051] Figure 5 shows the nanoflow cytometry results of the detection of CD13, CD29 and CD90; CD9, CD63 and CD81, and CD9 / CD63 / CD81 at the single vesicle level in MSC-sEVs samples from other sources (Figure 5 includes Figure 5-1 and Figure 5-2).

[0052] Figure 6 shows the nanoflow cytometry results of the detection of CD13, CD29 and CD90; CD9, CD63 and CD81, and CD9 / CD63 / CD81 at the single vesicle level in MSC-sEVs samples from other donors (Figure 6 includes Figure 6-1 and Figure 6-2).

[0053] Figure 7 shows the nanoflow cytometry results of detecting other commonly used MSC cell identification markers on MSC-sEVs from different sources according to the present invention at the single vesicle level.

[0054] Figure 8 illustrates the application of this invention in labeling and tracing MSC-sEVs using specific biomarkers of MSC-sEVs. Detailed Implementation

[0055] To make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to specific examples.

[0056] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and any person skilled in the art may make similar extensions without departing from the spirit of the invention. The invention is not limited to the specific embodiments described below.

[0057] Example 1: Identification method of small extracellular vesicles derived from mesenchymal stem cells

[0058] The procedure for identifying MSC-sEVs is shown in Figure 1.

[0059] 1. Sample preparation: Prepare small extracellular vesicles (U-MSC-sEVs) derived from human umbilical cord mesenchymal stem cells.

[0060] 2. Characterization of sample properties

[0061] Morphology: The morphology of nanoparticles in the sample was observed using transmission electron microscopy.

[0062] Concentration & Particle Size: The particle concentration and particle size distribution of the sample were detected using a nanoflow cytometer.

[0063] sEVs marker proteins: Western blot analysis was used to analyze sEVs surface markers.

[0064] Total protein content: The total protein concentration in the sample was determined using the BCA method.

[0065] The results are shown in Figure 2. It can be seen that U-MSC-sEVs exhibit a typical saucer-like vesicle structure; nanoflow cytometry analysis showed that the particle size distribution was between 30 and 200 nm. Furthermore, Western blot analysis confirmed the expression of sEVs positive markers CD9, CD63, TSG101, and Alix, while the absence of sEVs negative markers Calnexin and GM130. Therefore, it can be concluded that the prepared U-MSC-sEVs samples are all typical small extracellular vesicles.

[0066] 3. Sample Quality Control: Calculate the particle-to-protein ratio. Samples with a particle-to-protein ratio greater than 1*10^8 particles / μg protein can be used for subsequent identification. Before identification, ensure that the particle concentration of the sample is above 5*10^9 particles / mL. If the particle concentration of the sample is too low, it needs to be concentrated.

[0067] 4. Immunofluorescence staining: Samples were aliquoted into 100 μL tubes, and fluorescently labeled antibodies corresponding to CD13, CD29, CD90, CD9, CD63, and CD81 were added to each tube in the specified proportions. One tube of sample also contained fluorescent antibodies for CD9, CD63, and CD81. After vortexing, in this example, the samples were incubated at 37°C for 30 min.

[0068] Antibody information and usage ratios are as follows: BB515 Anti-human CD13 Antibody (BD, 566034, 1:20), AF488 Anti-human CD29 Antibody (Biolegend, 303015, 1:20), FITC Anti-human CD90 (Biolegend, 328107, 1:20), AF488 Mouse Anti-Human CD9 (NanoFCM, NHA009-A488-50T, 1:10), AF488 Mouse Anti-Human CD63 (NanoFCM, NHA063-A488-50T, 1:10), FITC Mouse Anti-Human CD81 (NanoFCM, NHA-FITC-50T, 1:10).

[0069] 5. Removal of free antibodies (This section uses ultracentrifugation as an example; other methods such as ultrafiltration or size exclusion can be used to remove free antibodies): After incubation, transfer the liquid to 1 mL ultracentrifuge tubes, add 1 mL of PBS to each tube for dilution, and ultracentrifuge at 100,000 g for 17 min at 4°C (Beckman Coulter MAX-XP centrifuge, MLA-150 rotor). After ultracentrifugation, discard the supernatant, resuspend in 1 mL of PBS, and ultracentrifuge again at 100,000 g for 17 min at 4°C. Discard the supernatant and resuspend in 200 μL of PBS.

[0070] 6. Nanoflow cytometry detection: For samples after removing free antibodies, dilute to the optimal detection concentration with PBS (10-fold dilution in this example; however, due to slight differences in experimental conditions, 5-50-fold dilution can be performed in other examples of this invention). Perform single-vesicle level nanoparticle fluorescence analysis using a nanoflow cytometer (nFCM, Xiamen Fuliu Biotechnology). If the proportion of CD13-positive particles in the sample is greater than 50%, and the proportion of CD29-positive particles is greater than 50%, and the proportion of CD90-positive particles is greater than 50%, and the proportion of CD81-positive particles is greater than 50%, and the proportion of CD9 / CD63 / CD81-positive particles is greater than 70%, then the sample can be identified as MSC-sEVs (as shown in Figure 3).

[0071] In Figure 3, "Isotype" represents the isotype control, which uses immunoglobulins of the same species, subtype, dose, and subtype as the primary antibody to eliminate background staining caused by nonspecific antibody binding to the cell surface. The content of CD9 positive particles in U-MSC-sEVs was 38.5%, CD63 positive particles in U-MSC-sEVs was 43.3%, CD81 positive particles in U-MSC-sEVs was 63.8%, CD9 / CD63 / CD81 positive particles in U-MSC-sEVs was 84.1%, CD13 positive particles in U-MSC-sEVs was 81.7%, CD29 positive particles in U-MSC-sEVs was 73.5%, and CD90 positive particles in U-MSC-sEVs was 76.1%.

[0072] In Figure 3, the detection of CD9 / CD63 / CD81 refers to the combined staining of the three markers CD9 / CD63 / CD81 during immunofluorescence staining, i.e., the antibodies corresponding to the three markers are added simultaneously during staining. The proportion of positive particles of CD9 / CD63 / CD81 Combination refers to the proportion of positive particles after simultaneous staining with CD9 / CD63 / CD81. Since CD9, CD63, and CD81 are all universal sEVs markers, this embodiment detects the positive rate of each marker individually, as well as the positive rate of the CD9 / CD63 / CD81 combination.

[0073] The results shown in Figure 3 confirm that the sample is identified as MSC-sEVs if the proportion of CD13 positive particles is greater than 50% (specifically, the content of CD13 positive particles in U-MSC-sEVs is 81.7%), the proportion of CD29 positive particles is greater than 50% (specifically, the content of CD29 positive particles in U-MSC-sEVs is 73.5%), the proportion of CD90 positive particles is greater than 50% (specifically, the content of CD90 positive particles in U-MSC-sEVs is 76.1%), the proportion of CD81 positive particles is greater than 50% (specifically, the content of CD81 positive particles in U-MSC-sEVs is 63.8%), and the proportion of Combination of CD9 / CD63 / CD81 positive particles is greater than 70% (specifically, the content of Combination of CD9 / CD63 / CD81 positive particles in U-MSC-sEVs is 84.1%).

[0074] Example 2: Identification of small extracellular vesicles (non-MSC-sEVs) from other cell sources

[0075] 1. Sample preparation: Prepare small extracellular vesicles derived from human fibroblasts (HDF-sEVs and HFF1-sEVs), small extracellular vesicles derived from human renal epithelial cells (293T-sEVs), small extracellular vesicles derived from human non-small cell lung cancer cells (A549-sEVs), small extracellular vesicles derived from human large cell lung cancer cells (H460-sEVs), small extracellular vesicles derived from human breast cancer cells (MCF7-sEVs), small extracellular vesicles derived from human breast cancer cells (MDA-MB-231-sEVs), and small extracellular vesicles derived from human osteosarcoma cells (143B-sEVs).

[0076] 2. Following steps 2-6 in Example 1, after quality control, immunofluorescence staining, and removal of free fluorescent antibodies, the samples were analyzed using a nanoflow cytometer to detect the combination of mesenchymal stem cell specific markers: CD13, CD29, CD90, and the proportion of positive particles of CD9, CD63, CD81, and Combination of CD9 / CD63 / CD81.

[0077] In Figure 4 (including Figures 4-1 and 4-2), Isotype represents the isotype control, which uses immunoglobulins of the same species, subtype, dose, and subtype as the primary antibody to eliminate background staining caused by nonspecific binding of the antibody to the cell surface. The concentrations of CD9 positive particles in HDF-sEVs, HFF1-sEVs, 293T-sEVs, A549-sEVs, H460-sEVs, MCF7-sEVs, MDA-MB-231-sEVs, and 143B-sEVs were 37.3%, 30.4%, 34.6%, 36.0%, 33.0%, 49.0%, 46.7%, and 42.9%, respectively. The concentrations of CD63 positive particles in HDF-sEVs, HFF1-sEVs, 293T-sEVs, A549-sEVs, H460-sEVs, MCF7-sEVs, MDA-MB-231-sEVs, and MDA-MB-231-sEVs were 42.9%, respectively. The contents of 31-sEVs and 143B-sEVs were 47.3%, 43.2%, 41.5%, 42.4%, 42.4%, 45.2%, 43.7%, and 40.6%, respectively. The contents of CD81 positive particles in HDF-sEVs, HFF1-sEVs, 293T-sEVs, A549-sEVs, H460-sEVs, MCF7-sEVs, MDA-MB-231-sEVs, and 143B-sEVs were 20.8%, 44.8%, 40.5%, 38.3%, 40.8%, 36.6%, 39.2%, and 41.4%, respectively. The concentrations of CD9 / CD63 / CD81 positive particles in HDF-sEVs, HFF1-sEVs, 293T-sEVs, A549-sEVs, H460-sEVs, MCF7-sEVs, MDA-MB-231-sEVs, and 143B-sEVs were 57.7%, 62.8%, 53.3%, 59.0%, 56.3%, 62.1%, 64.3%, and 54.9%, respectively. The concentrations of CD13 positive particles in HDF-sEVs, HFF1-sEVs, 293T-sEVs, A549-sEVs, H460-sEVs, MCF7-sEVs, and MDA-MB-231-sEVs were 54.9%, 62.8%, 53.3%, 59.0%, 56.3%, 62.1%, 64.3%, and 54.9%, respectively. The contents of Vs, MDA-MB-231-sEVs, and 143B-sEVs were 74.3%, 56.2%, 28.2%, 42.3%, 18.6%, 25.2%, 24.9%, and 57.5%, respectively. The contents of CD29 positive particles in HDF-sEVs, HFF1-sEVs, 293T-sEVs, A549-sEVs, H460-sEVs, MCF7-sEVs, MDA-MB-231-sEVs, and 143B-sEVs were 25.7%, 36.9%, 26.0%, 24.6%, 14.5%, 39.0%, 78.8%, and 42.1%, respectively.The contents of CD90 positive particles in HDF-sEVs, HFF1-sEVs, 293T-sEVs, A549-sEVs, H460-sEVs, MCF7-sEVs, MDA-MB-231-sEVs, and 143B-sEVs were 57.4%, 32.8%, 0.9%, 2.4%, 0.8%, 1.6%, 0.9%, and 2.8%, respectively.

[0078] In Figure 4, the detection of CD9 / CD63 / CD81 refers to the combined staining of the three markers CD9 / CD63 / CD81 during immunofluorescence staining, i.e., the antibodies corresponding to the three markers are added simultaneously during staining. The proportion of positive particles of CD9 / CD63 / CD81 Combination refers to the proportion of positive particles after simultaneous staining with CD9 / CD63 / CD81. Since CD9, CD63, and CD81 are all universal sEVs markers, this embodiment detects the positive rate of each marker individually, as well as the positive rate of the CD9 / CD63 / CD81 combination.

[0079] As shown in Figure 4, small extracellular vesicles (non-MSC-sEVs) from other cell sources cannot simultaneously possess the following five characteristics: CD13 positive granule ratio greater than 50%, CD29 positive granule ratio greater than 50%, CD90 positive granule ratio greater than 50%, CD81 positive granule ratio greater than 50%, and CD9 / CD63 / CD81 positive granule ratio greater than 70%. When these five conditions are not met simultaneously, the small extracellular vesicles can be identified as small extracellular vesicles from non-mesenchymal stem cells.

[0080] As can be seen from Figure 4, the identification of MSC-sEVs requires that the proportion of CD13 positive particles be greater than 50%, the proportion of CD29 positive particles be greater than 50%, the proportion of CD90 positive particles be greater than 50%, the proportion of CD81 positive particles be greater than 50%, and the proportion of CD9 / CD63 / CD81 positive particles be greater than 70%. All five conditions are indispensable, and other non-MSC-sEVs cannot meet all five conditions at the same time.

[0081] Example 3: Identification of sEVs from MSCs of other sources

[0082] 1. Sample preparation: Prepare small extracellular vesicle samples derived from human-induced pluripotent stem cells (i-MSC-sEVs) and human adipose-derived mesenchymal stem cells (A-MSC-sEVs).

[0083] 2. Following steps 2-6 in Example 1, after quality control, immunofluorescence staining, and removal of free fluorescent antibodies, the samples were analyzed using a nanoflow cytometer to detect the combination of mesenchymal stem cell specific markers: CD13, CD29, CD90, and the proportion of positive particles of CD9, CD63, CD81, and Combination of CD9 / CD63 / CD81.

[0084] Figure 5 shows that the contents of CD9 positive particles in i-MSC-sEVs and A-MSC-sEVs were 58.5% and 40.1%, respectively; the contents of CD63 positive particles in i-MSC-sEVs and A-MSC-sEVs were 47.4% and 53.0%, respectively; and the contents of CD81 positive particles in i-MSC-sEVs and A-MSC-sEVs were 66.2% and 75.3%, respectively. The proportions of CD9 / CD63 / CD81 positive particles in i-MSC-sEVs were... The contents of Vs and A-MSC-sEVs were 80.1% and 80.4%, respectively; the contents of CD13 positive particles in i-MSC-sEVs and A-MSC-sEVs were 81.5% and 92.1%, respectively; the contents of CD29 positive particles in i-MSC-sEVs and A-MSC-sEVs were 82.0% and 76.8%, respectively; and the contents of CD90 positive particles in i-MSC-sEVs and A-MSC-sEVs were 77.8% and 66.7%, respectively.

[0085] Figure 5 shows the results verifying that the proportion of CD13 positive particles in the samples was greater than 50% (specifically, the content of CD13 positive particles in i-MSC-sEVs and A-MSC-sEVs was 81.5% and 92.1%, respectively); the proportion of CD29 positive particles was greater than 50% (specifically, the content of CD29 positive particles in i-MSC-sEVs and A-MSC-sEVs was 82.0% and 76.8%, respectively); the proportion of CD90 positive particles was greater than 50% (specifically, the content of CD90 positive particles in i-MSC-sEVs and A-MSC-sEVs was 77.8% and 66.7%, respectively); the proportion of CD81 positive particles was greater than 50% (specifically, the content of CD81 positive particles in i-MSC-sEVs and A-MSC-sEVs was 66.2% and 75.3%, respectively); and the proportion of CD9 / CD63 / CD81 positive particles was greater than 70% (specifically, the proportion of CD9 / CD63 / CD81 positive particles was greater than 70%). If the contents of CD9 / CD63 / CD81 positive particles in i-MSC-sEVs and A-MSC-sEVs are 80.1% and 80.4%, respectively, then the small extracellular vesicle samples derived from mesenchymal stem cells that are induced pluripotent stem cells are identified as MSC-sEVs, and the small extracellular vesicles derived from human adipose mesenchymal stem cells are identified as MSC-sEVs.

[0086] Example 4: Identification of sEVs from MSCs of different donors

[0087] 1. Sample preparation: Prepare small extracellular vesicles (U-MSC2-sEVs and U-MSC3-sEVs) derived from umbilical cord mesenchymal stem cells from two other donors, as well as small extracellular vesicles (A-MSC2-sEVs and A-MSC3-sEVs) derived from adipose mesenchymal stem cells from two other donors.

[0088] 2. Following steps 2-6 in Example 1, after quality control, immunofluorescence staining, and removal of free fluorescent antibodies, the samples were analyzed using a nanoflow cytometer to detect the combination of mesenchymal stem cell specific markers: CD13, CD29, CD90, and the proportion of positive particles of CD9, CD63, CD81, and Combination of CD9 / CD63 / CD81.

[0089] As shown in Figure 6, as in Figure 5, the contents of CD9 positive particles in U-MSC2-sEVs, U-MSC3-sEVs, A-MSC2-sEVs, and A-MSC3-sEVs were 60.7%, 37.9%, 48.2%, and 58.2%, respectively; the contents of CD63 positive particles in U-MSC2-sEVs, U-MSC3-sEVs, A-MSC2-sEVs, and A-MSC3-sEVs were 46.7%, 50.8%, 49.1%, and 50.3%, respectively; and the contents of CD81 positive particles in U-MSC2-sEVs, U-MSC3-sEVs, A-MSC2-sEVs, and A-MSC3-sEVs were 64.5%, 66.6%, 65.2%, and 71.9%, respectively. The contents of CD9 / CD63 / CD81 positive particles in U-MSC2-sEVs and U-MSC3-sEVs were... The contents of CD13-positive particles in U-MSC2-sEVs, U-MSC3-sEVs, A-MSC2-sEVs, and A-MSC3-sEVs were 82.1%, 76.0%, 80.4%, and 80.3%, respectively. The contents of CD13-positive particles in U-MSC2-sEVs, U-MSC3-sEVs, A-MSC2-sEVs, and A-MSC3-sEVs were 83.2%, 83.5%, 85.1%, and 91.0%, respectively. The contents of CD29-positive particles in U-MSC2-sEVs, U-MSC3-sEVs, A-MSC2-sEVs, and A-MSC3-sEVs were 74.1%, 77.3%, 64.2%, and 80.1%, respectively. The contents of CD90-positive particles in U-MSC2-sEVs, U-MSC3-sEVs, A-MSC2-sEVs, and A-MSC3-sEVs were 73.5%, 70.2%, 66.8%, and 70.2%, respectively.

[0090] Figure 6 shows the results confirming that the proportion of CD13 positive particles in the samples was greater than 50% (specifically, the contents of CD13 positive particles in U-MSC2-sEVs, U-MSC3-sEVs, A-MSC2-sEVs, and A-MSC3-sEVs were 83.2%, 83.5%, 85.1%, and 91.0%, respectively), and the proportion of CD29 positive particles was greater than 50% (specifically, the contents of CD29 positive particles in U-MSC2-sEVs, U-MSC3-sEVs, A-MSC2-sEVs, and A-MSC3-sEVs were 74.1%, 77.3%, 64.2%, and 80.1%, respectively). The proportion of CD90 positive particles was greater than 50% (the contents of CD90 positive particles in U-MSC2-sEVs, U-MSC3-sEVs, A-MSC2-sEVs, and A-MSC3-sEVs were 73.5%, 70.2%, 66.8%, and 70.2%, respectively), and the proportion of CD81 positive particles was greater than 50% (the specific contents of CD81 positive particles in U-MSC2-sEVs, U-MSC3-sEVs, A-MSC2-sEVs, and A-MSC3-sEVs were 64.5%, 66.6%, 65.2%, and 71.9%, respectively). If the proportion of CD9 / CD63 / CD81 positive particles is greater than 70% (specifically, the content of CD9 / CD63 / CD81 positive particles in U-MSC2-sEVs, U-MSC3-sEVs, A-MSC2-sEVs, and A-MSC3-sEVs are 82.1%, 76.0%, 80.4%, and 80.3%, respectively), then the small extracellular vesicle samples derived from umbilical cord mesenchymal stem cells from the other two donors are identified as MSC-sEVs, and the small extracellular vesicle samples derived from adipose-derived mesenchymal stem cells from the other two donors are identified as MSC-sEVs.

[0091] Example 5: Detection of common MSC markers on the surface of MSC-sEVs

[0092] 1. Select CD44, CD73, and CD105, commonly used markers for identifying MSCs, as detection indicators, and prepare three types of small extracellular vesicles derived from mesenchymal stem cells (U-MSC-sEVs, A-MSC-sEVs, and i-MSC-sEVs).

[0093] 2. Perform quality control on the samples according to steps 2-5 in Example 1. Immunofluorescence staining was performed on CD44, CD73, and CD105 respectively. Then, the free fluorescent antibodies were removed. The proportion of CD44, CD73, and CD105 positive particles in the MSC-sEVs samples was detected by nanoflow cytometer.

[0094] As shown in Figure 7, the proportion of CD73 positive particles in U-MSC-sEVs samples was less than 40% (23.9%), and the proportion of CD105 positive particles was less than 40% (3.6%); the proportion of CD44 positive particles in A-MSC-sEVs samples was less than 40% (26.5%), the proportion of CD73 positive particles was less than 40% (32.8%), and the proportion of CD105 positive particles was less than 20% (10.6%); and the proportion of CD73 positive particles in i-MSC-sEVs samples was less than 40% (17.9%), and the proportion of CD105 positive particles was less than 40% (12.6%). This result indicates that although CD44, CD73, and CD105 are commonly used markers for identifying MSC cells, they are not highly expressed in MSC-sEVs and cannot be used as markers for MSC-sEVs. Not all markers of MSCs are markers of MSC-sEVs. Only CD29 and CD90, mentioned earlier, are markers that are highly expressed in MSC-sEVs.

[0095] Example 6: Other applications based on MSC-sEVs specific biomarkers

[0096] 1. Prepare MSC-sEVs samples. Perform immunofluorescence staining and free antibody removal on the samples according to steps 2-5 in Example 1. Use a nanoflow cytometer to detect the labeling rate of MSC-sEVs after immunofluorescence staining with specific biomarker antibodies.

[0097] 2. The same MSC-sEVs were stained with the classic lipophilic membrane dye DiD. DiD was added to the MSC-sEVs sample at a ratio of 1:100 and incubated at 37°C in the dark for 30 min. The labeling rate of MSC-sEVs after staining with lipophilic membrane dye DiD was detected using a nanoflow cytometer.

[0098] 3. Equal amounts of MSC-sEVs stained with specific marker antibodies via immunofluorescence and MSC-sEVs stained with lipophilic membrane dye DiD were injected into mice via the tail vein, and then the MSC-sEVs were traced in vivo using a small animal imaging system.

[0099] As shown in Figure 8, the fluorescence labeling rate of MSC-sEVs after immunofluorescence staining with specific marker antibodies can reach 90%, while the labeling rate of MSC-sEVs with the lipophilic membrane dye DiD is only 50-60%. This indicates that labeling MSC-sEVs with specific markers is far more efficient than the traditional membrane dye labeling method. After injecting MSC-sEVs labeled by both methods into mice via the tail vein, it was observed that the fluorescence signal of MSC-sEVs labeled with specific markers was significantly stronger than that of the traditional membrane dye labeling method, demonstrating better in vivo tracing performance.

[0100] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A characteristic biomarker combination of small extracellular vesicles derived from mesenchymal stem cells, characterized in that, The characteristic marker combination includes: CD13, CD29, CD90, CD9, CD63, and CD81.

2. A method for identifying small extracellular vesicles derived from mesenchymal stem cells, characterized in that, The characteristic marker combination of mesenchymal stem cell-derived small extracellular vesicles as described in claim 1 is used to identify whether small extracellular vesicles are mesenchymal stem cell-derived small extracellular vesicles. Based on a combination of characteristic biomarkers for small extracellular vesicles derived from mesenchymal stem cells, immunofluorescence staining was performed on the small extracellular vesicles to be detected. When the proportion of CD13 positive granules is greater than 50%, the proportion of CD29 positive granules is greater than 50%, the proportion of CD90 positive granules is greater than 50%, the proportion of CD81 positive granules is greater than 50%, and the proportion of CD9 / CD63 / CD81 positive granules is greater than 70%, the small extracellular vesicles are identified as small extracellular vesicles derived from mesenchymal stem cells. When none of the following five conditions are met simultaneously, small extracellular vesicles can be identified as small extracellular vesicles not derived from mesenchymal stem cells.

3. The method for identifying small extracellular vesicles derived from mesenchymal stem cells according to claim 2, characterized in that, The method for detecting the proportion of CD13 positive particles is as follows: During immunofluorescence staining, fluorescently labeled aptamer molecules corresponding to CD13 were added, and after incubation, the proportion of CD13 positive particles was counted. The method for detecting the proportion of CD29 positive particles is as follows: During immunofluorescence staining, fluorescently labeled aptamer molecules corresponding to CD29 were added, and after incubation, the proportion of CD29 positive particles was counted. The method for detecting the proportion of CD90 positive particles is as follows: During immunofluorescence staining, fluorescently labeled aptamer molecules corresponding to CD90 are added, and after incubation, the proportion of CD90 positive particles is counted. The method for detecting the proportion of CD81 positive particles is as follows: During immunofluorescence staining, fluorescently labeled aptamer molecules corresponding to CD81 were added, and after incubation, the proportion of CD81 positive particles was counted. The method for detecting the ratio of CD9 / CD63 / CD81 positive particles is as follows: During immunofluorescence staining, three markers CD9 / CD63 / CD81 were combined. That is, fluorescently labeled aptamer molecules corresponding to the three markers CD9, CD63 and CD81 were added at the same time during staining. After incubation, the proportion of particles that were simultaneously positive for CD9 / CD63 / CD81 was counted.

4. The method for identifying small extracellular vesicles derived from mesenchymal stem cells according to claim 3, characterized in that, Immunofluorescence staining of samples was performed using fluorescently labeled aptamer molecules that specifically recognize CD13, CD29, CD90, CD9, CD63, and CD81, wherein the aptamer molecules were selected from antibodies or nucleic acid aptamers. The fluorescently labeled aptamers that specifically recognize CD13, CD29, CD90, CD9, CD63, and CD81 are: BB515 Anti-human CD13 Antibody, AF488 Anti-human CD29 Antibody, FITC Anti-human CD90 Antibody, AF488 Anti-Human CD9, AF488 Anti-Human CD63, and FITC Anti-Human CD81.

5. The method for identifying small extracellular vesicles derived from mesenchymal stem cells according to claim 2, characterized in that, Before immunofluorescence staining, the particle-to-protein ratio was calculated. Samples with a particle-to-protein ratio greater than 1*10^8 particles / μg protein and a particle concentration greater than 5*10^9 particles / mL were subjected to subsequent immunofluorescence staining and identification. If the particle concentration of the sample to be tested is too low before immunofluorescence staining, it should be concentrated so that the particle-to-protein ratio of the sample is greater than 1*10^8 particles / μg protein and the particle concentration is greater than 5*10^9 particles / mL before subsequent immunofluorescence staining and identification.

6. The method for identifying small extracellular vesicles derived from mesenchymal stem cells according to claim 2, characterized in that, When performing immunofluorescence staining on samples using fluorescently labeled aptamer molecules that specifically recognize CD13, CD29, CD90, CD9, CD63, and CD81, the fluorescently labeled aptamer molecules and the samples were incubated at 37°C for 30 min.

7. The method for identifying small extracellular vesicles derived from mesenchymal stem cells according to claim 2, characterized in that, After immunofluorescence staining, unbound free fluorescently labeled aptamer molecules are removed to reduce free fluorescence interference. The removal method is selected from ultracentrifugation, ultrafiltration or size exclusion chromatography.

8. The method for identifying small extracellular vesicles derived from mesenchymal stem cells according to claim 2, characterized in that, Immunofluorescence staining was performed, and after removing unbound free fluorescently labeled aptamer molecules after immunofluorescence staining, nanoflow cytometry analysis at the single vesicle level was performed to obtain the proportions of CD13 positive particles, CD29 positive particles, CD90 positive particles, CD81 positive particles, and CD9 / CD63 / CD81 positive particles.

9. The method for identifying small extracellular vesicles derived from mesenchymal stem cells according to claim 2, characterized in that, Includes the following steps: (1) Sample quality control: Before immunofluorescence staining, the particle-to-protein ratio is calculated. Samples with a particle-to-protein ratio greater than 1*10^8 particles / μg protein and a particle concentration greater than 5*10^9 particles / mL are then subjected to subsequent immunofluorescence staining and identification. If the particle concentration of the sample is too low before immunofluorescence staining, it is concentrated to ensure that the particle-to-protein ratio is greater than 1*10^8 particles / μg protein and the particle concentration is greater than 5*10^9 particles / mL before proceeding with subsequent immunofluorescence staining and identification. (2) Immunofluorescence staining: After aliquoting the samples, fluorescently labeled aptamer molecules corresponding to CD13, CD29, CD90, CD81 and CD9 / CD63 / CD81 were added in proportion. After vortexing, the samples were incubated at 37°C for 30 min. (3) Removal of unbound free fluorescently labeled aptamer molecules after immunofluorescence staining: After incubation, aptamer molecules that were not bound by immunofluorescence staining were removed by ultracentrifugation, ultrafiltration or size exclusion chromatography. (4) Nanoflow cytometry detection: For samples after removing free fluorescently labeled aptamer molecules, after diluting with PBS to a suitable detection concentration, nanoflow cytometry is used to perform single vesicle level nanoparticle fluorescence analysis to obtain the proportion of CD13 positive particles, CD29 positive particles, CD90 positive particles, CD81 positive particles, and CD9 / CD63 / CD81 positive particles. (5) Identification: When the proportion of CD13 positive granules is greater than 50%, the proportion of CD29 positive granules is greater than 50%, the proportion of CD90 positive granules is greater than 50%, the proportion of CD81 positive granules is greater than 50%, and the proportion of CD9 / CD63 / CD81 positive granules is greater than 70%, the small extracellular vesicles are identified as small extracellular vesicles derived from mesenchymal stem cells. When none of the following five conditions are met simultaneously, small extracellular vesicles can be identified as small extracellular vesicles not derived from mesenchymal stem cells.

10. The application of the characteristic marker combination of small extracellular vesicles derived from mesenchymal stem cells as described in claim 1, characterized in that, The application of the combination of characteristic markers is selected from one of the following: Labeling and in vivo tracing of small extracellular vesicles derived from mesenchymal stem cells; Product testing and quality control; Protein engineering modification; and Drug load.