Characteristic marker combination of pluripotent stem cell-derived small extracellular vesicles and use thereof

Abstract

The present invention relates to the field of biotechnology, and in particular, relates to a characteristic marker combination of pluripotent stem cell-derived small extracellular vesicles and a use thereof. The characteristic marker combination of the pluripotent stem cell-derived small extracellular vesicles comprises: the transmembrane protein PODXL and the glycosylated epitopes Tra-1-60 and Tra-1-81 thereof, and the membrane surface antigen SSEA-4, as well as the markers CD9, CD63, and CD81. Compared with the prior art, the present invention utilizes the foregoing reliable characteristic markers of the pluripotent stem cell-derived small extracellular vesicles and the combination thereof to perform nanoflow analysis at the single vesicle level, which can achieve simple and rapid identification of the pluripotent stem cell-derived small extracellular vesicles. The present invention has practical significance for product quality control and clinical transformation of pluripotent stem cell-derived small extracellular vesicles.

Description

Characteristic marker combination of small extracellular vesicles derived from pluripotent stem cells and its application Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a characteristic marker combination of small extracellular vesicles derived from pluripotent stem cells and an application thereof. Background Art

[0002] Small extracellular vesicles (sEVs) are tiny vesicles with a diameter of less than 200 nm and a phospholipid bilayer membrane structure secreted by living cells. The surface and interior of sEVs contain a variety of bioactive substances such as proteins and nucleic acids. By delivering these bioactive components, sEVs can act as a medium for intercellular communication, affecting the biological behavior of adjacent or distant cells and exerting relevant biological functions. Among them, stem cell-derived sEVs can exert biological functions similar to their parent stem cells, while avoiding potential risks such as tumor formation and vascular embolism of direct stem cell transplantation. In addition, stem cell-derived sEVs also have low immunogenicity and can pass through the skin, mucous membranes, and blood-brain barriers, making them extremely promising in tissue repair and disease treatment. Among different types of stem cell-derived sEVs, pluripotent stem cell-derived small extracellular vesicles (PSC-sEVs) have unique anti-aging functions. Small extracellular vesicles derived from pluripotent stem cells are divided into embryonic stem cell-derived small extracellular vesicles (ESC-sEVs) and induced pluripotent stem cell-derived small extracellular vesicles (iPSC-sEVs). PSC-sEVs have shown therapeutic effects in a variety of age-related degenerative diseases, including cardiovascular and cerebrovascular diseases, bone and joint tendon diseases, skin aging-related diseases, and premature ovarian failure. In addition, pluripotent stem cell-derived sEVs are easy to isolate and purify, and are convenient for large-scale production and storage, showing unique advantages in clinical translational applications.

[0003] Currently, the scientific research and clinical translational application of PSC-sEVs are rapidly advancing. However, due to the diverse production, isolation, and purification methods of sEVs, the characterization methods and detection parameters of sEVs are equally complex and diverse, and the current sEV field lacks comprehensive quality control standards. For PSC-sEVs, the main quality control indicators currently include direct morphological observation under transmission electron microscopy, particle size distribution, Western blot analysis of sEV surface and internal markers (CD9, CD63, CD81, TSG101, etc.), and the particle-to-protein ratio of sEVs. These tests can determine whether the particles in the sample are sEVs, as well as the quantity and purity of the sEVs contained within them, but cannot distinguish the cellular origin of the sEVs. sEVs from different cell sources have significant differences in their biological functions. In practical applications, it is necessary to rigorously identify the cellular origin of sEVs and match them to specific biological functions. For the clinical translational application of PSC-sEVs, their unique anti-aging properties are crucial. Therefore, the identification of PSC-sEVs requires more than just general sEV property analysis; the cellular origin of the sEVs must also be confirmed. Unfortunately, specific detection indicators for PSC-sEVs are still lacking, making it difficult to directly identify sEV products as PSC-sEVs.

[0004] Therefore, analyzing the components of PSC-sEVs, finding specific markers, and establishing a rapid and simple identification method for PSC-sEVs are of practical significance for the clinical translation and application of PSC-sEVs.

[0005] Summary of the Invention

[0006] In view of the technical defects in the identification of pluripotent stem cell-derived small extracellular vesicles (PSC-sEVs) in the prior art, the present invention provides a characteristic marker combination of pluripotent stem cell-derived small extracellular vesicles and its application.

[0007] The purpose of the present invention can be achieved by the following technical solutions:

[0008] The present invention provides a characteristic marker combination of small extracellular vesicles derived from pluripotent stem cells, which includes the transmembrane protein Podocalyxin-like protein-1 (PODXL) and its glycosylated epitopes Tra-1-60 and Tra-1-81, the membrane surface antigen Stage-Specific Embryonic Antigen-4 (SSEA-4), and the markers CD9, CD63 and CD81.

[0009] The present invention also provides a method for identifying small extracellular vesicles derived from pluripotent stem cells, which identifies whether the small extracellular vesicles are derived from pluripotent stem cells based on a combination of characteristic markers of the small extracellular vesicles derived from pluripotent stem cells;

[0010] Based on the characteristic marker combination of pluripotent stem cell-derived small extracellular vesicles, immunofluorescence staining of the small extracellular vesicles to be detected was performed.

[0011] When the proportion of PODXL / Tra-1-60 / Tra-1-81-positive particles is greater than 50%, the proportion of SSEA4-positive particles is greater than 50%, the proportion of CD9-positive particles is greater than 50%, and the proportion of CD9 / CD63 / CD81-positive particles is greater than 70%, the small extracellular vesicles are identified as small extracellular vesicles derived from pluripotent stem cells;

[0012] When the following four conditions are not met simultaneously: the proportion of PODXL / Tra-1-60 / Tra-1-81 positive particles is greater than 50%, the proportion of SSEA4 positive particles is greater than 50%, the proportion of CD9 positive particles is greater than 50%, and the proportion of CD9 / CD63 / CD81 positive particles is greater than 70%, the small extracellular vesicles are identified as small extracellular vesicles derived from non-pluripotent stem cells.

[0013] In one embodiment of the present invention, the method for detecting the ratio of PODXL / Tra-1-60 / Tra-1-81 positive particles is:

[0014] During immunofluorescence staining, the three markers PODXL / Tra-1-60 / Tra-1-81 were jointly stained. That is, fluorescently labeled aptamer molecules corresponding to the three markers PODXL, Tra-1-60, and Tra-1-81 were added simultaneously during the staining. After incubation, the proportion of particles that were simultaneously positive for PODXL / Tra-1-60 / Tra-1-81 was counted.

[0015] The method for detecting the proportion of SSEA4-positive particles is:

[0016] During immunofluorescence staining, fluorescently labeled aptamer molecules corresponding to SSEA4 were added, and after incubation, the proportion of SSEA4-positive particles was counted;

[0017] The method for detecting the proportion of CD9 positive particles is:

[0018] During immunofluorescence staining, fluorescently labeled aptamer molecules corresponding to CD9 were added, and after incubation, the proportion of CD9-positive particles was counted;

[0019] The method for detecting the ratio of CD9 / CD63 / CD81 positive particles is:

[0020] During immunofluorescence staining, the three markers CD9 / CD63 / CD81 were jointly stained. That is, fluorescently labeled aptamer molecules corresponding to the three markers CD9, CD63, and CD81 were added simultaneously during staining. After incubation, the proportion of particles that were simultaneously positive for CD9 / CD63 / CD81 was counted.

[0021] In one embodiment of the present invention, immunofluorescence staining of the sample is performed using fluorescently labeled aptamer molecules that can specifically recognize PODXL, Tra-1-60, Tra-1-80, SSEA-4, CD9, CD63, and CD81. The aptamer molecules include but are not limited to specific antibodies and nucleic acid aptamers.

[0022] In one embodiment of the present invention, the fluorescently labeled aptamer molecules that can specifically recognize PODXL, Tra-1-60, Tra-1-80, SSEA-4, CD9, CD63, and CD81 are: AF488 anti-human PODXL Antibody, AF488 anti-human TRA-1-60-R Antibody, AF488 anti-human TRA-1-81 Antibody, AF488 anti-human SSEA-4 Antibody, AF488 Mouse Anti-Human CD9, AF488 Mouse Anti-Human CD63, and FITC Mouse Anti-Human CD81.

[0023] In one embodiment of the present invention, before immunofluorescence staining, the granule-protein ratio is calculated. Samples with a granule-protein ratio (an indicator of small extracellular vesicle purity) greater than 1*10^8 particles / μg protein and a particle concentration above 5*10^9 particles / mL can be subjected to subsequent immunofluorescence staining and identification.

[0024] If the particle concentration of the sample to be tested is too low before immunofluorescence staining, it needs to be concentrated so that the particle-protein ratio of the sample is greater than 1*10^8 particles / μg protein and the particle concentration is above 5*10^9 particles / mL before subsequent immunofluorescence staining and identification.

[0025] In one embodiment of the present invention, when immunofluorescence staining of a sample is performed using fluorescently labeled aptamer molecules that can specifically recognize PODXL, Tra-1-60, Tra-1-80, SSEA-4, CD9, CD63, and CD81, the fluorescently labeled aptamer molecules and the sample are incubated at 37° C. for 30 minutes.

[0026] In one embodiment of the present invention, after immunofluorescence staining, unbound free fluorescently labeled aptamer molecules are removed to reduce interference from free fluorescence, and the removal method is selected from ultracentrifugation, ultrafiltration or size exclusion chromatography.

[0027] 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 is performed at the single vesicle level to obtain the PODXL / Tra-1-60 / Tra-1-81 positive particle ratio, SSEA4 positive particle ratio, CD9 positive particle ratio, and CD9 / CD63 / CD81 positive particle ratio.

[0028] In one embodiment of the present invention, a method for identifying small extracellular vesicles derived from pluripotent stem cells comprises the following steps:

[0029] (1) Sample quality control:

[0030] Before immunofluorescence staining, calculate the particle-protein ratio. Samples with a particle-protein ratio greater than 1*10^8 particles / μg protein and a particle concentration above 5*10^9 particles / mL can undergo subsequent immunofluorescence staining and identification. If the particle concentration of the sample to be tested is too low before immunofluorescence staining, the sample needs to be concentrated to a particle-protein ratio greater than 1*10^8 particles / μg protein and a particle concentration above 5*10^9 particles / mL before subsequent immunofluorescence staining and identification.

[0031] (2) Immunofluorescence staining:

[0032] The samples were dispensed into low-adsorption tubes, and fluorescently labeled aptamer molecules corresponding to PODXL / Tra-1-60 / Tra-1-81, SSEA4, CD9, and CD9 / CD63 / CD81 were added in proportion. After vortexing, the tubes were incubated at 37°C for 30 min.

[0033] The fluorescently labeled aptamer molecules that can specifically recognize PODXL, Tra-1-60, Tra-1-80, SSEA-4, CD9, CD63, and CD81 are: AF488 anti-human PODXL Antibody, AF488 anti-human TRA-1-60-R Antibody, AF488 anti-human TRA-1-81 Antibody, AF488 anti-human SSEA-4 Antibody, AF488 Mouse Anti-Human CD9, AF488 Mouse Anti-Human CD63, FITC Mouse Anti-Human CD81;

[0034] (3) Removal of unbound free fluorescently labeled aptamer molecules after immunofluorescence staining:

[0035] After the incubation, the free fluorescently labeled aptamer molecules that were not bound after immunofluorescence staining are removed by ultracentrifugation, ultrafiltration or size exclusion chromatography;

[0036] (4) Nanoflow cytometry detection: After removing the free fluorescently labeled aptamer molecules, the sample was diluted with PBS to the optimal detection concentration, and then a nanoflow cytometry was used to perform single vesicle-level nanoparticle fluorescence analysis to obtain the ratio of PODXL / Tra-1-60 / Tra-1-81 positive particles, the ratio of SSEA4 positive particles, the ratio of CD9 positive particles, and the ratio of CD9 / CD63 / CD81 positive particles;

[0037] (5) Identification:

[0038] When the proportion of PODXL / Tra-1-60 / Tra-1-81-positive particles is greater than 50%, the proportion of SSEA4-positive particles is greater than 50%, the proportion of CD9-positive particles is greater than 50%, and the proportion of CD9 / CD63 / CD81-positive particles is greater than 70%, the small extracellular vesicles are identified as small extracellular vesicles derived from pluripotent stem cells;

[0039] When the following four conditions are not met simultaneously: the proportion of PODXL / Tra-1-60 / Tra-1-81 positive particles is greater than 50%, the proportion of SSEA4 positive particles is greater than 50%, the proportion of CD9 positive particles is greater than 50%, and the proportion of CD9 / CD63 / CD81 positive particles is greater than 70%, the small extracellular vesicles are identified as small extracellular vesicles derived from non-pluripotent stem cells.

[0040] In one embodiment of the present invention, when a sample is subjected to fluorescence analysis using a nanoflow cytometer, the fluorescence background signal of the sample is not significantly increased compared to the blank control, verifying that the unbound free fluorescently labeled aptamer molecules have been removed.

[0041] In one embodiment of the present invention, the characteristic markers of pluripotent stem cell-derived small extracellular vesicles PODXL / Tra-1-60 / Tra-1-81 and SSEA-4 also have other applications, including but not limited to: serving as drug-mounting sites for drug delivery in PSC-sEVs, and serving as gene sites fused with fluorescent proteins for tracing of PSC-sEVs.

[0042] The present invention overcomes the problem of the lack of specific identification indicators for PSC-sEVs in the prior art and provides specific markers for PSC-sEVs: the transmembrane protein Podocalyxin-like protein-1 (PODXL) and its glycosylated epitopes Tra-1-60 and Tra-1-81, the membrane surface antigen Stage-Specific Embryonic Antigen-4 (SSEA-4), and the markers CD9, CD63, and CD81. The present invention provides a method for identifying PSC-sEVs: immunofluorescence staining and single-vesicle nanoflow cytometry analysis of the specific markers PODXL and SSEA4 for PSC-sEVs are performed to verify that the proportion of PODXL / Tra-1-60 / Tra-1-81-positive particles in the sample is greater than 50%, the proportion of SSEA4-positive particles is greater than 50%, the proportion of CD9-positive particles is greater than 50%, and the proportion of CD9 / CD63 / CD81-positive particles is greater than 70%. The sample can be identified as PSC-sEVs.

[0043] Compared to existing technologies, the present invention utilizes these reliable characteristic markers of pluripotent stem cell-derived small extracellular vesicles and their combination for single-vesicle nanoflow cytometry analysis, enabling simple and rapid identification of pluripotent stem cell-derived small extracellular vesicles. This invention has practical significance for product quality control and clinical translation of pluripotent stem cell-derived small extracellular vesicles. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments.

[0045] FIG1 is a flowchart of the operation of identifying PSC-sEVs according to the present invention.

[0046] FIG2 is a diagram showing the property characterization results of PSC-sEVs in the present invention.

[0047] Figure 3 shows the nanoflow cytometry analysis results of the present invention for identifying the specific markers PODXL / Tra-1-60 / Tra-1-81 and SSEA4 for PSC-sEVs, as well as the universal markers CD9, CD63 and CD81 for sEVs, and the single vesicle level of CD9 / CD63 / CD81.

[0048] Figure 4 shows the nanoflow cytometry analysis results of the present invention for detecting PODXL / Tra-1-60 / Tra-1-81, SSEA4, as well as CD9, CD63 and CD81 at the single vesicle level in sEVs samples from various cell sources (Figure 4 includes Figure 4-1 and Figure 4-2).

[0049] FIG5 shows the nanoflow cytometry analysis results of the present invention for detecting other commonly used PSC cell identification markers at the single vesicle level on PSC-sEV.

[0050] FIG6 shows the test results of the present invention when the sample quality control fails before testing.

[0051] FIG7 shows the detection results when the unbound free fluorescent antibody is not completely removed (the background fluorescence level is too high) after immunofluorescence staining in the present invention. DETAILED DESCRIPTION

[0052] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0053] In the following description, many specific details are set forth to fully understand the present invention, but the present invention can also be implemented in other ways different from those described herein. The technology in this field can be similarly extended without violating the connotation of the present invention. The present invention is not limited to the specific embodiments below.

[0054] Example 1: Method for Identifying Small Extracellular Vesicles Derived from Pluripotent Stem Cells

[0055] The operational process for identifying PSC-sEVs is shown in Figure 1.

[0056] 1. Sample preparation: Prepare embryonic stem cell-derived small extracellular vesicles (ESC-sEVs) and induced pluripotent stem cell-derived small extracellular vesicles (iPSC-sEVs).

[0057] 2. Sample property characterization

[0058] Morphology: Transmission electron microscopy was used to observe the morphology of nanoparticles in the samples.

[0059] Concentration & Particle Size: Nanoflow cytometry is used to measure the particle concentration and particle size distribution of the sample.

[0060] sEVs marker proteins: sEVs surface markers were analyzed by western blot.

[0061] Total protein content: The total protein concentration in the samples was determined using the BCA assay.

[0062] The results, shown in Figure 2, show that both ESC-sEVs and iPSC-sEVs exhibit typical saucer-shaped vesicle structures, with a particle size distribution ranging from 30 to 200 nm. Western blot analysis revealed the expression of positive sEV markers CD9, CD63, TSG101, and Alix, while negative sEV markers Calnexin and GM130 were absent. This confirms that both the prepared ESC-sEV and iPSC-sEV samples are typical small extracellular vesicles.

[0063] 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 proceed to subsequent identification. Before identification, ensure that the particle concentration of the sample is above 5*10^9 particles / mL. If the particle concentration is too low, the sample needs to be concentrated.

[0064] 4. Immunofluorescence staining: Aliquot 100 μL of sample into low-adhesion tubes and add fluorescently labeled antibodies corresponding to CD9, CD63, CD81, and SSEA4 in the appropriate proportions. To one tube, add fluorescent antibodies against PODXL, Tra-1-60, and Tra-1-81. To another tube, add fluorescent antibodies against CD9, CD63, and CD81. Vortex and incubate at 37°C for 30 min.

[0065] Antibody information and usage ratios are as follows: AF488 Mouse Anti-Human CD9 (NanoFCM, NHA009-A488-50T, 1:10-1:40), AF488 Mouse Anti-Human CD63 (NanoFCM, NHA063-A488-50T, 1:10-1:40), FITC Mouse Anti-Human CD81 (NanoFCM, NHA-FITC-50T, 1:10-1:40), AF488 anti-human PODXL Antibody (Abcam, ab208254, 1:50-1:200), AF488 anti-human SSEA-4 Antibody (BioLegend, 330411, 1:20-1:100), AF488 anti-human TRA-1-60-R Antibody(BioLegend,330613,1:50-1:200), AF488anti-human TRA-1-81 Antibody(BioLegend,330709,1:50-1:200).

[0066] 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 solution to 1 mL ultracentrifuge tubes. Add 1 mL of PBS to each tube for dilution. Ultracentrifuge at 100,000 g for 17 minutes 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 minutes at 4°C. Discard the supernatant and resuspend in 200 μL of PBS.

[0067] 6. Nanoflow cytometry: After removing free antibodies, the sample was diluted with PBS to the optimal detection concentration (10-fold dilution in this example, but based on slight differences in experimental conditions, a 5-20-fold dilution can be performed in other examples of the present invention). Single-vesicle nanoparticle fluorescence analysis was performed using a nanoflow cytometer (nFCM, Xiamen Fuliu Bio). If the proportion of PODXL / Tra-1-60 / Tra-1-81-positive particles in the sample was greater than 50%, the proportion of SSEA4-positive particles was greater than 50%, the proportion of CD9-positive particles was greater than 50%, and the proportion of CD9 / CD63 / CD81-positive particles was greater than 70%, the sample was identified as PSC-sEVs (as shown in Figure 3).

[0068] In Figure 3, "Isotype" indicates an isotype control, which uses an immunoglobulin 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 content of CD9 in ESC-sEVs was 67.2%, and the content of CD9 in iPSC-sEVs was 71.4%. The content of CD63 in ESC-sEVs was 49.0%, and the content of CD63 in iPSC-sEVs was 43.3%. The content of CD81 in ESC-sEVs was 41.9%, and the content of CD81 in iPSC-sEVs was 44.6%. The content of CD9 / CD63 / CD81-positive particles in ESC-sEVs was 74.1%, and the content of CD9 / CD63 / CD81-positive particles in iPSC-sEVs was 76.1%. The content of PODXL-positive particles in ESC-sEVs was 52.0%, and the content of PODXL-positive particles in iPSC-sEVs was 53.8%. The content of SSEA4-positive particles in ESC-sEVs was 76.0%, and the content of SSEA4-positive particles in iPSC-sEVs was 76.9%.

[0069] In Figure 3, the detection of the specific markers PODXL / Tra-1-60 / Tra-1-81 refers to the combined staining of these three markers during immunofluorescence staining, i.e., antibodies corresponding to these three markers are added simultaneously during the staining. The detection of CD9 / CD63 / CD81 refers to the combined staining of these three markers during immunofluorescence staining, i.e., antibodies corresponding to these three markers are added simultaneously during the staining. The PODXL / Tra-1-60 / Tra-1-81 positive particle ratio refers to the proportion of positive particles after simultaneous staining for PODXL / Tra-1-60 / Tra-1-81.

[0070] Since CD9, CD63, and CD81 are all universal sEVs markers, in this example, the positive rate of each marker was detected separately, as well as the positive rate of the CD9 / CD63 / CD81 combination.

[0071] The results shown in Figure 3 verify that the proportion of PODXL / Tra-1-60 / Tra-1-81 positive particles in the sample is greater than 50% (specifically, the content of PODXL / Tra-1-60 / Tra-1-81 positive particles in ESC-sEVs is 51.4%, and the content of PODXL / Tra-1-60 / Tra-1-81 positive particles in iPSC-sEVs is 59.7%), the proportion of SSEA4 positive particles is greater than 50%, the proportion of CD9 positive particles is greater than 50%, and the proportion of CD9 / CD63 / CD81 positive particles is greater than 70%, then the sample is identified as PSC-sEVs.

[0072] Example 2: Identification of small extracellular vesicles from other cell sources (non-PSC-sEVs)

[0073] 1. Sample preparation: Prepare small extracellular vesicles derived from human umbilical cord mesenchymal stem cells (MSC-sEVs), small extracellular vesicles derived from human fibroblasts (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), and small extracellular vesicles derived from human breast cancer cells (MDA-MB-231-sEVs).

[0074] 2. Following steps 2-6 of Example 1, the samples were subjected to quality control, immunofluorescence staining, and free fluorescent antibody removal. The proportions of positive particles for the universal markers CD9, CD63, and CD81, as well as the proportions of positive particles for the PSC-sEVs-specific markers PODXL / Tra-1-60 / Tra-1-81 and SSEA4, were detected using a nanoflow cytometer.

[0075] In Figure 4 (including Figure 4-1 and Figure 4-2), Isotype represents isotype control (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 contents of CD9 in 293T-sEVs, HFF1-sEVs, MSC-sEVs, A549-sEVs, H460-sEVs, MCF7-sEVs, and MDA-MB-231-sEVs were 31.4, 37.5, 35.4, 30.3, 35.9, 43.4, and 32.0%, respectively. The contents of CD63 in 293T-sEVs, HFF1-sEVs, MSC-sEVs, A549-sEVs, H460-sEVs, MCF7-sEVs, and MDA-MB-231-sEVs were 31.4, 37.5, 35.4, 30.3, 35.9, 43.4, and 32.0%, respectively. The contents of CD81 in 293T-sEVs, HFF1-sEVs, MSC-sEVs, A549-sEVs, H460-sEVs, MCF7-sEVs and MDA-MB-231-sEVs were 43.9, 43.5, 49.3, 42.5, 34.6, 46.2 and 51.9%, respectively; the contents of CD81 in 293T-sEVs, HFF1-sEVs, MSC-sEVs, A549-sEVs, H460-sEVs, MCF7-sEVs and MDA-MB-231-sEVs were 37.6, 42.2, 58.2, 26.7, 43.1, 27.7 and 33.4%, respectively; The contents of D63 / CD81 positive particles in 293T-sEVs, HFF1-sEVs, MSC-sEVs, A549-sEVs, H460-sEVs, MCF7-sEVs, and MDA-MB-231-sEVs were 62.0, 63.8, 75.5, 52.0, 57.0, 65.9, and 65.0%, respectively, and the contents of PODXL positive particles in 293T-sEVs, HFF1-sEVs, MSC-sEVs, A549-sEVs, H460-sEVs, MCF7-sEVs, and MDA-MB-231-sEVs were 62.0, 63.8, 75.5, 52.0, 57.0, 65.9, and 65.0%, respectively. The contents of SSEA4-positive particles in 293T-sEVs, HFF1-sEVs, MSC-sEVs, A549-sEVs, H460-sEVs, MCF7-sEVs, and MDA-MB-231-sEVs were 1.1, 1.4, 3.0, 9.5, 7.6, 8.7, and 20%, respectively. The contents of SSEA4-positive particles in 293T-sEVs, HFF1-sEVs, MSC-sEVs, A549-sEVs, H460-sEVs, MCF7-sEVs, and MDA-MB-231-sEVs were 4.8, 5.3, 2.5, 4.9, 7.1, 7.6, and 14.7%, respectively.

[0076] In Figure 4, the detection of the specific markers PODXL / Tra-1-60 / Tra-1-81 refers to the combined staining of these three markers during immunofluorescence staining, i.e., antibodies corresponding to these three markers are added simultaneously during the staining. The detection of CD9 / CD63 / CD81 refers to the combined staining of these three markers during immunofluorescence staining, i.e., antibodies corresponding to these three markers are added simultaneously during the staining. The PODXL / Tra-1-60 / Tra-1-81 positive particle ratio refers to the proportion of positive particles after simultaneous staining for PODXL / Tra-1-60 / Tra-1-81.

[0077] As shown in Figure 4 , small extracellular vesicles from other cell sources (non-PSC-sEVs) cannot simultaneously possess the following four characteristics: the proportion of PODXL / Tra-1-60 / Tra-1-81-positive particles is greater than 50%, the proportion of SSEA4-positive particles is greater than 50%, the proportion of CD9-positive particles is greater than 50%, and the proportion of CD9 / CD63 / CD81-positive particles is greater than 70%. Therefore, it can be identified that the sample is not PSC-sEVs.

[0078] It can be concluded from Figure 4 that the identification of PSC-sEVs requires that the proportion of PODXL / Tra-1-60 / Tra-1-81 positive particles is greater than 50%, the proportion of SSEA4 positive particles is greater than 50%, the proportion of CD9 positive particles is greater than 50%, and the proportion of CD9 / CD63 / CD81 positive particles is greater than 70%. These four conditions are indispensable. Other non-PSC-sEVs cannot meet all four conditions at the same time.

[0079] Example 3: Identification of Common PSC Markers on the Surface of PSC-sEVs

[0080] 1. Among the commonly used PSC markers, SSEA4, Tra-1-60, and Tra-1-81, which are subcellularly localized on the membrane, were selected as detection indicators.

[0081] 2. Perform quality control on the samples according to steps 2-5 of Example 1, perform immunofluorescence staining for SSEA4, Tra-1-60, and Tra-1-81, respectively. Then, remove the free fluorescent antibodies and use a nanoflow cytometer to detect the proportion of SSEA4, Tra-1-60, and Tra-1-81 positive particles in the PSC-sEV samples.

[0082] As shown in Figure 5, the proportion of SSEA4-positive particles in the PSC-sEV sample is greater than 50% (76.0), while the proportion of Tra-1-60-positive particles is less than 20% (6.4%), and the proportion of Tra-1-81-positive particles is less than 20% (9.8%). This result shows that although Tra-1-60 and Tra-1-81 are markers of PSC cells, they cannot be used alone as markers of PSC-sEVs. Not all PSC markers are markers of PSC-sEVs, and they must be used in combination with PODXL / Tra-1-60 / Tra-1-81.

[0083] Example 4: Identification Method of Small Extracellular Vesicles Derived from Pluripotent Stem Cells (Sample Quality Control Failed)

[0084] 1. Sample preparation: Prepare PSC-sEV samples with high purity (particle-to-protein ratio of 3*10^8 particles / μg protein) and normal particle concentration (2*10^10 particles / mL); PSC-sEV samples with high purity (particle-to-protein ratio of 3*10^8 particles / μg protein) and low particle concentration (1*10^9 particles / mL); and PSC-sEV samples with poor purity (particle-to-protein ratio of 6*10^7 particles / μg protein).

[0085] 2. After immunofluorescence staining and free fluorescent antibody removal according to steps 4-5 of Example 1, the samples were detected using a nanoflow cytometer for universal marker CD9 and the ratio of CD9 / CD63 / CD81 positive particles. The results are shown in FIG6 .

[0086] The detection of specific markers CD9 / CD63 / CD81 in Figure 6 refers to the combined staining of these three markers during immunofluorescence staining, i.e., the use of antibodies corresponding to these three markers. The PODXL / Tra-1-60 / Tra-1-81 positive particle ratio refers to the proportion of positive particles after simultaneous PODXL / Tra-1-60 / Tra-1-81 staining.

[0087] The results in Figure 6 show that for qualified PSC-sEV 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, the proportion of CD9-positive particles was greater than 50% (70.3%), and the proportion of CD9 / CD63 / CD81-positive particles was greater than 70% (72.0%). For samples with a particle-to-protein ratio less than 1*10^8 particles / μg protein or a particle concentration less than 5*10^9 particles / mL, the proportion of CD9-positive particles was less than 50% (28.8 or 45.4%), and the proportion of CD9 / CD63 / CD81-positive particles was less than 70% (66.2 or 54.7%). In general, samples that fail quality control (insufficient concentration and purity) do not meet the identification requirements for PSC-sEVs and cannot be identified as PSC-sEVs.

[0088] Example 5: Identification Method of Small Extracellular Vesicles Derived from Pluripotent Stem Cells (Incomplete Removal of Free Antibodies)

[0089] 1. Perform sample quality control and immunofluorescence staining according to steps 2-4 in Example 1.

[0090] 2. Free fluorescent antibodies were removed according to step 5 of Example 1. After immunofluorescence staining of another tube of sample was completed, the liquid was transferred to a 1 mL ultracentrifuge tube, 1 mL of PBS was added to the ultracentrifuge tube for dilution, and ultracentrifuged at 100,000 g for 17 min at 4°C. After the ultracentrifugation, the supernatant was discarded, and 200 uL of PBS was added for resuspending and used directly for detection (compared to the step of removing free fluorescent antibodies in Example 1, one ultracentrifugation process was omitted, and free antibodies were not completely removed).

[0091] 3. Nanoflow cytometry was used to measure the proportion of particles positive for the universal markers CD9, CD9 / CD63 / CD81, and the PSC-sEV-specific marker PODXL / Tra-1-60 / Tra-1-81. As shown in Figure 7, for PSC-sEV samples with good removal of unbound free fluorescent antibodies (low background fluorescence), the proportion of CD9-positive particles was greater than 50%, the proportion of CD9 / CD63 / CD81-positive particles was greater than 70%, and the proportion of PODXL / Tra-1-60 / Tra-1-81-positive particles was greater than 50%. For PSC-sEV samples with incomplete removal of unbound free fluorescent antibodies (excessively high background fluorescence), the proportion of CD9-positive particles was less than 20%, the proportion of CD9 / CD63 / CD81-positive particles was less than 30%, and the proportion of PODXL / Tra-1-60 / Tra-1-81-positive particles was less than 20%. In general, if free antibodies are not completely removed during the identification of PSC-sEVs samples, the results will not meet the identification requirements of PSC-sEVs and cannot be identified as PSC-sEVs.

[0092] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. 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 present invention, should be within the scope of protection of the present invention.

Claims

1. A characteristic marker combination of small extracellular vesicles derived from pluripotent stem cells, characterized in that: The characteristic marker combination includes: transmembrane protein PODXL and its glycosylated epitopes Tra-1-60 and Tra-1-81, membrane surface antigen SSEA-4, and markers CD9, CD63 and CD81.

2. A method for identifying small extracellular vesicles derived from pluripotent stem cells, characterized in that: Identifying whether the small extracellular vesicles are pluripotent stem cell-derived small extracellular vesicles based on the characteristic marker combination of the pluripotent stem cell-derived small extracellular vesicles according to claim 1; Based on the characteristic marker combination of pluripotent stem cell-derived small extracellular vesicles, immunofluorescence staining of the small extracellular vesicles to be detected was performed. When the proportion of PODXL / Tra-1-60 / Tra-1-81 positive particles is greater than 50%, the proportion of SSEA4 positive particles is greater than 50%, the proportion of CD9 positive particles is greater than 50%, and the proportion of CD9 / CD63 / CD81 positive particles is greater than 70%, the small extracellular vesicles are identified as small extracellular vesicles derived from pluripotent stem cells; When the four conditions that the proportion of PODXL / Tra-1-60 / Tra-1-81 positive particles is greater than 50%, the proportion of SSEA4 positive particles is greater than 50%, the proportion of CD9 positive particles is greater than 50%, and the proportion of CD9 / CD63 / CD81 positive particles is greater than 70% are not met at the same time, the small extracellular vesicles are identified as small extracellular vesicles derived from non-pluripotent stem cells.

3. A method for identifying small extracellular vesicles derived from pluripotent stem cells according to claim 2, characterized in that: The method for detecting the proportion of PODXL / Tra-1-60 / Tra-1-81 positive particles is: During immunofluorescence staining, the three markers PODXL / Tra-1-60 / Tra-1-81 were jointly stained, that is, the fluorescently labeled aptamer molecules corresponding to the three markers PODXL, Tra-1-60, and Tra-1-81 were added simultaneously during the staining, and then after incubation, the proportion of particles that were simultaneously positive for PODXL / Tra-1-60 / Tra-1-81 was counted; The method for detecting the proportion of SSEA4 positive particles is: During immunofluorescence staining, the fluorescently labeled aptamer molecules corresponding to SSEA4 were added, and then after incubation, the proportion of SSEA4-positive particles was counted; The method for detecting the proportion of CD9 positive particles is: During immunofluorescence staining, fluorescently labeled aptamer molecules corresponding to CD9 were added, and then after incubation, the proportion of CD9-positive particles was counted; The method for detecting the ratio of CD9 / CD63 / CD81 positive particles is: During immunofluorescence staining, the three markers CD9 / CD63 / CD81 were jointly stained, that is, the fluorescently labeled aptamer molecules corresponding to the three markers CD9, CD63, and CD81 were added simultaneously during the staining, and then 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 pluripotent stem cells according to claim 3, characterized in that: The samples were subjected to immunofluorescence staining using fluorescently labeled aptamer molecules that can specifically recognize PODXL, Tra-1-60, Tra-1-80, SSEA-4, CD9, CD63, and CD81.

5. The method for identifying small extracellular vesicles derived from pluripotent stem cells according to claim 4, characterized in that: The fluorescently labeled aptamer molecules that can specifically recognize PODXL, Tra-1-60, Tra-1-80, SSEA-4, CD9, CD63, and CD81 are: AF488 anti-human PODXL Antibody, AF488 anti-human TRA-1-60-R Antibody, AF488 anti-human TRA-1-81Antibody, AF488 anti-human SSEA-4Antibody, AF488 Mouse Anti-Human CD9, AF488 Mouse Anti-Human CD63, and FITC Mouse Anti-Human CD81.

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

7. The method for identifying small extracellular vesicles derived from pluripotent stem cells according to claim 2, characterized in that: When the samples were immunofluorescently stained using fluorescently labeled aptamer molecules that could specifically recognize PODXL, Tra-1-60, Tra-1-80, SSEA-4, CD9, CD63, and CD81, the fluorescently labeled aptamer molecules and the samples were incubated at 37° C. for 30 min.

8. The method for identifying small extracellular vesicles derived from pluripotent stem cells according to claim 2, characterized in that: After immunofluorescence staining, the free fluorescently labeled aptamer molecules that are not bound after immunofluorescence staining are removed to reduce interference from free fluorescence, and the removal method is selected from ultracentrifugation, ultrafiltration or size exclusion chromatography.

9. The method for identifying small extracellular vesicles derived from pluripotent stem cells according to claim 2, characterized in that: After immunofluorescence staining was performed and the unbound free fluorescently labeled aptamer molecules were removed, nanoflow cytometry analysis at the single vesicle level was performed to obtain the PODXL / Tra-1-60 / Tra-1-81 positive particle ratio, SSEA4 positive particle ratio, CD9 positive particle ratio and CD9 / CD63 / CD81 positive particle ratio.

10. The method for identifying small extracellular vesicles derived from pluripotent stem cells according to claim 2, characterized in that: The following steps are involved: (1) Sample quality control: Before immunofluorescence staining, calculate the particle-protein ratio. Samples with a particle-protein ratio greater than 1*10^8particles / μg protein and a particle concentration of more than 5*10^9particles / mL can be subjected to subsequent immunofluorescence staining and identification. If the particle concentration of the sample to be tested is too low before immunofluorescence staining, it is necessary to concentrate the sample so that the particle-protein ratio is greater than 1*10^8particles / μg protein and the particle concentration is more than 5*10^9particles / mL before subsequent immunofluorescence staining and identification; (2) Immunofluorescence staining: The samples were dispensed into low-adsorption tubes, and fluorescently labeled aptamer molecules corresponding to PODXL / Tra-1-60 / Tra-1-81, SSEA4, CD9, and CD9 / CD63 / CD81 were added in proportion, vortexed, and incubated at 37°C for 30 min; The fluorescently labeled aptamer molecules that can specifically recognize PODXL, Tra-1-60, Tra-1-80, SSEA-4, CD9, CD63, and CD81 are: AF488 anti-human PODXL Antibody, AF488 anti-human TRA-1-60-R Antibody, AF488 anti-human TRA-1-81Antibody, AF488 anti-human SSEA-4Antibody, AF488 Mouse Anti-Human CD9, AF488 Mouse Anti-Human CD63, FITC Mouse Anti-Human CD81; (3) Removal of unbound free fluorescently labeled aptamer molecules after immunofluorescence staining: After the incubation, a method selected from ultracentrifugation, ultrafiltration or size exclusion chromatography is used to remove the free fluorescently labeled aptamer molecules that are not bound after immunofluorescence staining; (4) Nanoflow cytometry detection: After removing the free fluorescently labeled aptamer molecules, the samples were diluted with PBS to the optimal detection concentration, and then the nanoparticle fluorescence analysis at the single vesicle level was performed using a nanoflow cytometry detector to obtain the PODXL / Tra-1-60 / Tra-1-81 positive particle ratio, SSEA4 positive particle ratio, CD9 positive particle ratio, and CD9 / CD63 / CD81 positive particle ratio; (5) Identification: When the proportion of PODXL / Tra-1-60 / Tra-1-81 positive particles is greater than 50%, the proportion of SSEA4 positive particles is greater than 50%, the proportion of CD9 positive particles is greater than 50%, and the proportion of CD9 / CD63 / CD81 positive particles is greater than 70%, the small extracellular vesicles are identified as small extracellular vesicles derived from pluripotent stem cells; When the four conditions that the proportion of PODXL / Tra-1-60 / Tra-1-81 positive particles is greater than 50%, the proportion of SSEA4 positive particles is greater than 50%, the proportion of CD9 positive particles is greater than 50%, and the proportion of CD9 / CD63 / CD81 positive particles is greater than 70% are not met at the same time, the small extracellular vesicles are identified as small extracellular vesicles derived from non-pluripotent stem cells.

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