Signature marker combinations of pluripotent stem cell-derived small extracellular vesicles and uses thereof

Abstract

The present application belongs to the field of biotechnology, and particularly relates to a characteristic marker combination of pluripotent stem cell-derived small extracellular vesicles and application thereof.The characteristic marker combination of pluripotent stem cell-derived small extracellular vesicles comprises transmembrane protein PODXL and glycosylation epitopes Tra-1-60 and Tra-1-81 thereof, and membrane surface antigen SSEA-4, and markers CD9, CD63 and CD81.Compared with the prior art, the present application uses these reliable characteristic markers of pluripotent stem cell-derived small extracellular vesicles and combinations thereof to perform nano-flow analysis at a single-vesicle level, so that the pluripotent stem cell-derived small extracellular vesicles can be identified simply and quickly.The present application has practical significance for product quality control and clinical transformation of pluripotent stem cell-derived small extracellular vesicles.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a combination of characteristic markers of pluripotent stem cell derived small extracellular vesicles and application thereof. BACKGROUND

[0002] Small extracellular vesicles (sEVs) are microvesicles with a phospholipid bilayer membrane structure secreted by living cells with a diameter less than 200 nm. The surface and interior of sEVs contain various bioactive substances such as proteins and nucleic acids. By delivering these bioactive ingredients, 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 similar biological functions as their parent stem cells, while avoiding potential risks such as tumorigenesis and vascular embolism of direct stem cell transplantation. In addition, stem cell derived sEVs also have the characteristics of low immunogenicity, and can pass through the skin, mucous membrane, blood-brain barrier, etc., and have great potential 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. Pluripotent stem cell derived small extracellular vesicles 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 show therapeutic effects in various aging-related degenerative diseases, including: cardiovascular and cerebrovascular diseases, bone, joint and tendon related diseases, skin aging related diseases, premature ovarian failure, etc. In addition, pluripotent stem cell derived sEVs also have the characteristics of easy separation and purification, easy mass production and storage, etc., and show unique advantages in clinical translation applications.

[0003] Currently, the scientific research and clinical translation of PSC-sEVs are rapidly developing. However, due to different production and separation and purification methods of sEVs, the characterization methods and detection indexes of sEVs are also complex and diverse. At present, there is still a lack of complete quality control standards in the field of sEVs. For PSC-sEVs, the main quality control indexes at present include: direct morphological observation under transmission electron microscope, size distribution of particle population, Western Blot identification of sEVs surface and internal markers (CD9, CD63, CD81, TSG101, etc.), and sEVs particle protein ratio. Through the above detection, it can be determined whether the particles in the sample are sEVs, and the quantity and purity of sEVs contained therein, but the cell source of sEVs cannot be distinguished. sEVs from different cell sources have great differences in biological function, and in practical application, the cell source of sEVs needs to be strictly identified, and the specific biological function is matched according to the cell source. For the clinical translation application of PSC-sEVs, its unique anti-aging function is crucial, therefore, for the identification of PSC-sEVs, not only the general property analysis of sEVs is needed, but also the cell source of sEVs must be confirmed. Unfortunately, at present, the specific detection index of PSC-sEVs is still lacking, and it is difficult to confirm that the product is PSC-sEVs through the direct identification of sEVs product.

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

[0005] 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 application provides a characteristic marker combination of pluripotent stem cell-derived small extracellular vesicles and an application thereof.

[0006] The object of the present application can be achieved by the following technical solutions:

[0007] The present application provides a characteristic marker combination of pluripotent stem cell-derived small extracellular vesicles, which comprises transmembrane protein Podocalyxin-like protein-1 (PODXL) and its glycosylation epitopes Tra-1-60 and Tra-1-81, and membrane surface antigen Stage-Specific Embryonic Antigen-4 (SSEA-4), as well as markers CD9, CD63 and CD81.

[0008] The application also provides a method for identifying pluripotent stem cell-derived small extracellular vesicles, which is based on a characteristic marker combination of the pluripotent stem cell-derived small extracellular vesicles to identify whether the small extracellular vesicles are pluripotent stem cell-derived small extracellular vesicles;

[0009] The method for detecting the characteristic marker combination of the pluripotent stem cell-derived small extracellular vesicles comprises the following steps:

[0010] 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 pluripotent stem cell-derived small extracellular vesicles.

[0011] When the four conditions of the proportion of PODXL / Tra-1-60 / Tra-1-81 positive particles being greater than 50%, the proportion of SSEA4 positive particles being greater than 50%, the proportion of CD9 positive particles being greater than 50%, and the proportion of CD9 / CD63 / CD81 positive particles being greater than 70% are not met at the same time, the small extracellular vesicles are identified as non-pluripotent stem cell-derived small extracellular vesicles.

[0012] In an embodiment of the application, the method for detecting the proportion of PODXL / Tra-1-60 / Tra-1-81 positive particles is as follows:

[0013] In the immunofluorescence staining, the three markers PODXL / Tra-1-60 / Tra-1-81 are jointly stained, that is, the fluorescently labeled aptamer molecules corresponding to the three markers PODXL, Tra-1-60 and Tra-1-81 are simultaneously added during the staining, and then the proportion of particles positive for PODXL / Tra-1-60 / Tra-1-81 is counted after incubation.

[0014] The method for detecting the proportion of SSEA4 positive particles is as follows:

[0015] In the immunofluorescence staining, the fluorescently labeled aptamer molecules corresponding to SSEA4 are added, and then the proportion of SSEA4 positive particles is counted after incubation.

[0016] The method for detecting the proportion of CD9 positive particles is as follows:

[0017] In the immunofluorescence staining, the fluorescently labeled aptamer molecules corresponding to CD9 are added, and then the proportion of CD9 positive particles is counted after incubation.

[0018] The method for detecting the proportion of CD9 / CD63 / CD81 positive particles is as follows:

[0019] The combined staining of three markers CD9 / CD63 / CD81 is performed in immunofluorescence staining, that is, the fluorescently labeled aptamer molecules corresponding to the three markers CD9, CD63 and CD81 are simultaneously added during staining, and then the proportion of particles positive for CD9 / CD63 / CD81 is counted after incubation.

[0020] In an embodiment of the present application, the sample is subjected to immunofluorescence staining using fluorescently labeled aptamer molecules capable of specifically recognizing 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.

[0021] In an embodiment of the present application, the fluorescently labeled aptamer molecules capable of specifically recognizing PODXL, Tra-1-60, Tra-1-80, SSEA-4, CD9, CD63 and CD81 are respectively: 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.

[0022] In an embodiment of the present application, before immunofluorescence staining is performed, the particle protein ratio is calculated, and the sample with a particle protein ratio (small extracellular vesicle purity index) 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.

[0023] If the particle concentration of the sample to be tested is too low before immunofluorescence staining is performed, the sample needs to be concentrated so that the particle protein ratio is greater than 1*10^8 particles / μg protein and the particle concentration is greater than 5*10^9 particles / mL, and then subsequent immunofluorescence staining and identification are performed.

[0024] In an embodiment of the present application, when the sample is subjected to immunofluorescence staining using fluorescently labeled aptamer molecules capable of specifically recognizing PODXL, Tra-1-60, Tra-1-80, SSEA-4, CD9, CD63 and CD81, the fluorescently labeled aptamer molecules and the sample are respectively incubated at 37°C for 30 min.

[0025] In one embodiment of the present application, after immunofluorescence staining, the free fluorescently labeled aptamer molecules that are not bound after immunofluorescence staining are removed to reduce the interference of free fluorescence, and the removal method is selected from ultracentrifugation, ultrafiltration or size exclusion chromatography.

[0026] In one embodiment of the present application, after immunofluorescence staining, the free fluorescently labeled aptamer molecules that are not bound after immunofluorescence staining are removed to reduce the interference of free fluorescence, and the removal method is selected from ultracentrifugation, ultrafiltration or size exclusion chromatography.

[0027] In one embodiment of the present application, the method for identifying pluripotent stem cell-derived small extracellular vesicles comprises the following steps:

[0028] (1) Sample quality control:

[0029] Before immunofluorescence staining, the particle protein ratio is calculated, and 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 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*10^8 particles / μg protein and the particle concentration is above 5*10^9 particles / mL, and then subjected to subsequent immunofluorescence staining and identification.

[0030] (2) Immunofluorescence staining:

[0031] The sample is aliquoted into low adsorption tubes, and the corresponding fluorescently labeled aptamer molecules of PODXL / Tra-1-60 / Tra-1-81, SSEA4, CD9 and CD9 / CD63 / CD81 are added in proportion, vortexed and incubated at 37°C for 30 min.

[0032] The fluorescently labeled aptamer molecules capable of specifically recognizing PODXL, Tra-1-60, Tra-1-80, SSEA-4, CD9, CD63, and CD81 are respectively: 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;

[0033] (3) removing the free fluorescently labeled aptamer molecules not bound after immunofluorescence staining:

[0034] After the incubation, the free fluorescently labeled aptamer molecules not bound after immunofluorescence staining are removed by ultracentrifugation, ultrafiltration, or size exclusion chromatography;

[0035] (4) nano-flow cytometer detection: for the sample after removing the free fluorescently labeled aptamer molecules, the sample is diluted to an optimal detection concentration using PBS, and then a nano-flow cytometer is used to perform single-vesicle-level nanoparticle fluorescence analysis to obtain the PODXL / Tra-1-60 / Tra-1-81 positive particle ratio, the SSEA4 positive particle ratio, the CD9 positive particle ratio, and the CD9 / CD63 / CD81 positive particle ratio;

[0036] (5) identification:

[0037] When the PODXL / Tra-1-60 / Tra-1-81 positive particle ratio is greater than 50%, the SSEA4 positive particle ratio is greater than 50%, the CD9 positive particle ratio is greater than 50%, and the CD9 / CD63 / CD81 positive particle ratio is greater than 70%, the small extracellular vesicles are identified as pluripotent stem cell-derived small extracellular vesicles.

[0038] When the four conditions of the PODXL / Tra-1-60 / Tra-1-81 positive particle ratio being greater than 50%, the SSEA4 positive particle ratio being greater than 50%, the CD9 positive particle ratio being greater than 50%, and the CD9 / CD63 / CD81 positive particle ratio being greater than 70% are not simultaneously met, the small extracellular vesicles are identified as non-pluripotent stem cell-derived small extracellular vesicles.

[0039] In one embodiment of the present application, when the sample is subjected to fluorescence analysis using a nano-flow detector, the fluorescence background signal of the sample is not significantly increased compared with the blank control, verifying that the unbound free fluorescently labeled aptamer molecules have been removed.

[0040] In one embodiment of the present application, 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: as drug loading sites for PSC-sEVs, and as fusion gene sites for PSC-sEVs such as fluorescent proteins.

[0041] The present application overcomes the problem of lack of specific identification indicators for PSC-sEVs in the prior art, and provides specific markers for PSC-sEVs: transmembrane protein Podocalyxin-like protein-1 (PODXL) and its glycosylated epitopes Tra-1-60 and Tra-1-81, membrane surface antigen Stage-Specific Embryonic Antigen-4 (SSEA-4), and markers CD9, CD63, and CD81. The PSC-sEV identification method provided by the present application: immunofluorescence staining and single vesicle level nano-flow analysis of the specific markers of PSC-sEVs, PODXL and SSEA4, verify that the proportion of PODXL / Tra-1-60 / Tra-1-81 positive particles in the sample is greater than 50%, and the proportion of SSEA4 positive particles is greater than 50%, and 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 can be identified as PSC-sEVs.

[0042] Compared with the prior art, the present application uses these reliable characteristic markers of pluripotent stem cell-derived small extracellular vesicles and their combinations for single vesicle level nano-flow analysis, which can achieve simple and rapid identification of pluripotent stem cell-derived small extracellular vesicles. The present application has practical significance for product quality control and clinical transformation of pluripotent stem cell-derived small extracellular vesicles. BRIEF DESCRIPTION OF DRAWINGS

[0043] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced as follows.

[0044] Figure 1 The operation flow chart for identifying PSC-sEVs of the present application.

[0045] Figure 2 The property characterization result chart of PSC-sEVs in the present application.

[0046] Figure 3 Nano-flow analysis results of the specific markers PODXL / Tra-1-60 / Tra-1-81 and SSEA4 of PSC-sEVs of the present application, as well as the sEVs general markers CD9, CD63 and CD81, and the single vesicle level of CD9 / CD63 / CD81.

[0047] Figure 4 is the nano-flow analysis results of the present application in detecting PODXL / Tra-1-60 / Tra-1-81, SSEA4, as well as the single vesicle level of CD9, CD63 and CD81 in sEVs samples of various different cell sources (Figure 4 includes Figure 4-1 and Figure 4-2 ).

[0048] Figure 5 Nano-flow analysis results of the present application in detecting the single vesicle level of other commonly used PSC cell markers on PSC-sEVs.

[0049] Figure 6 Detection results when the sample quality control before detection in the present application is unqualified.

[0050] Figure 7 Detection results when the removal of unbound free fluorescent antibody after immunofluorescence staining in the present application is incomplete (background fluorescence level is too high). DETAILED DESCRIPTION

[0051] In order to make the above objectives, features and advantages of the present application more obvious and easy to understand, the specific embodiments of the present application will be described in detail below.

[0052] In the following description, many specific details are set forth in order to provide a thorough understanding of the present application, but the present application can also be implemented in other ways different from those described herein, and any person skilled in the art can make similar generalizations without departing from the connotation of the present application, and the present application is not limited by the following specific embodiments.

[0053] Example 1: Identification method of small extracellular vesicles from pluripotent stem cells

[0054] Reference Figure 1 .

[0055] 1. Sample preparation: Prepare embryonic stem cell-derived small extracellular vesicles (ESC-sEVs) and induced pluripotent stem cell-derived small extracellular vesicles (iPSC-sEVs).

[0056] 2. Characterization of sample properties

[0057] Morphology: The morphology of nanoparticles in the sample was observed using transmission electron microscopy.

[0058] Concentration & Particle Size: The particle concentration and particle size distribution of the sample were detected using a nanoflow cytometer.

[0059] sEVs marker proteins: Western blot analysis was used to analyze sEVs surface markers.

[0060] Total protein content: The total protein concentration in the sample was determined using the BCA method.

[0061] The results are as follows Figure 2 As shown, both ESC-sEVs and iPSC-sEVs exhibit typical saucer-like vesicle structures; their particle size distribution is between 30 and 200 nm; Western blot analysis revealed that sEVs expressed positive markers CD9, CD63, TSG101, and Alix, while sEVs did not express negative markers Calnexin and GM130. Therefore, it can be concluded that the prepared ESC-sEVs and iPSC-sEVs samples are typical small extracellular vesicles.

[0062] 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.

[0063] 4. Immunofluorescence staining: Samples were aliquoted into 100 μL tubes and fluorescently labeled antibodies corresponding to CD9, CD63, CD81, and SSEA4 were added in the specified proportions. Another tube contained PODXL, Tra-1-60, and Tra-1-81 fluorescent antibodies, and a third tube contained CD9, CD63, and CD81 fluorescent antibodies. After vortexing, the samples were incubated at 37°C for 30 min in this example.

[0064] The antibody information and use examples are as follows: AF488 Mouse Anti-Human CD9 (Nano FCM, NHA009-A488-50T, 1:10-1:40), AF488 Mouse Anti-Human CD63 (Nano FCM, NHA063-A488-50T, 1:10-1:40), FITC Mouse Anti-Human CD81 (Nano FCM, NHA-FITC-50T, 1:10-1:40), AF488 anti-human PODXL Antibody (Abeam, 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), AF488 anti-human TRA-1-81 Antibody (BioLegend, 330709, 1:50-1:200).

[0065] 5. Remove free antibodies (this part takes ultracentrifugation as an example, and other methods such as ultrafiltration or size exclusion can be used to remove free antibodies): After incubation, transfer the liquid to a 1 mL ultracentrifuge tube, add 1 mL of PBS dilution to each ultracentrifuge tube, and centrifuge at 100 000 g at 4°C for 17 min (Beckman Coulter MAX-XP centrifuge, MLA-150 rotor). After ultracentrifugation, aspirate the supernatant, resuspend with 1 mL of PBS, and centrifuge again at 100 000 g at 4°C for 17 min. After aspirating the supernatant, resuspend with 200 μL of PBS.

[0066] 6. Nano flow cytometer detection: After removing the free antibodies, dilute the sample to the optimal detection concentration (in this example, dilute 10 times, but based on slight differences in experimental conditions, dilution of 5-20 times can be performed in other embodiments of the application), and use the nano flow cytometer (nFCM, Xiamen Fu Flow Biotechnology) to perform single vesicle level nanoparticle fluorescence analysis. 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%, and identify the sample as PSC-sEVs (e.g. Figure 3 ).

[0067] Figure 3 Isotype indicates the isotype control (Isotype Control), which uses the same species source, same subtype, same dose and same immunoglobulin and subtype of the primary antibody, to eliminate the background staining due to non-specific binding of antibodies to the cell surface. The content of CD9 in ESC-sEVs is 67.2%, the content of CD9 in iPSC-sEVs is 71.4%, the content of CD63 in ESC-sEVs is 49.0%, the content of CD63 in iPSC-sEVs is 43.3%, the content of CD81 in ESC-sEVs is 41.9%, the content of CD81 in iPSC-sEVs is 44.6%, the content of CD9 / CD63 / CD81 positive particles in ESC-sEVs is 74.1%, the content of CD9 / CD63 / CD81 positive particles in iPSC-sEVs is 76.1%, the content of PODXL positive particles in ESC-sEVs is 52.0%, the content of PODXL positive particles in iPSC-sEVs is 53.8%, the content of SSEA4 positive particles in ESC-sEVs is 76.0%, and the content of SSEA4 positive particles in iPSC-sEVs is 76.9%.

[0068] Figure 3 The detection of specific markers PODXL / Tra-1-60 / Tra-1-81 refers to the combined staining of three markers PODXL / Tra-1-60 / Tra-1-81 during immunofluorescence staining, that is, the corresponding antibodies of the three markers are added at the same time during staining. The detection of CD9 / CD63 / CD81 refers to the combined staining of three markers CD9 / CD63 / CD81 during immunofluorescence staining, that is, the corresponding antibodies of the three markers are added at the same time during staining. The proportion of PODXL / Tra-1-60 / Tra-1-81 positive particles refers to the proportion of positive particles after simultaneous PODXL / Tra-1-60 / Tra-1-81 staining.

[0069] Since CD9, CD63 and CD81 are all general sEV markers, in this embodiment, the positive rate of each marker is detected separately, and the positive rate of CD9 / CD63 / CD81 combination is also detected.

[0070] Figure 3The results shown verify that the proportion of PODXL / Tra-1-60 / Tra-1-81 positive particles in the sample is greater than 50% (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%, and the sample is identified as PSC-sEVs.

[0071] Example Two: Identification of small extracellular vesicles (non-PSC-sEVs) of other cell sources

[0072] 1. Sample preparation: Prepare human umbilical cord mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs), human fibroblast-derived small extracellular vesicles (HFF1-sEVs), human kidney epithelial cell-derived small extracellular vesicles (293T-sEVs), human non-small cell lung cancer cell-derived small extracellular vesicles (A549-sEVs), human large cell lung cancer cell-derived small extracellular vesicles (H460-sEVs), human breast cancer cell-derived small extracellular vesicles (MCF7-sEVs), and human breast cancer cell-derived small extracellular vesicles (MDA-MB-231-sEVs).

[0073] 2. After quality control, immunofluorescence staining and free fluorescent antibody removal of the samples according to steps 2-6 in Example One, use a nano-flow cytometer to detect the proportion of positive particles of general markers CD9, CD63, and CD81, and the proportion of positive particles of PSC-sEVs specific markers PODXL / Tra-1-60 / Tra-1-81 and SSEA4.

[0074] Figure 4 (includes Figure 4-1 and Figure 4-2) in which Isotype indicates Isotype Control, using the same species origin, same subtype, same dose and same immunoglobulin and subtype of the immunoglobulin as the primary antibody, for eliminating background staining due to non-specific binding of the antibody to the cell surface. The content of CD9 in 293T-sEVs, HFF1-sEVs, MSC-sEVs, A549-sEVs, H460-sEVs, MCF7-sEVs, MDA-MB-231-sEVs was 31.4, 37.5, 35.4, 30.3, 35.9, 43.4, 32.0%, respectively, the content of CD63 in 293T-sEVs, HFF1-sEVs, MSC-sEVs, A549-sEVs, H460-sEVs, MCF7-sEVs, MDA-MB-231-sEVs was 43.9, 43.5, 49.3, 42.5, 34.6, 46.2, 51.9%, respectively, the content of CD81 in 293T-sEVs, HFF1-sEVs, MSC-sEVs, A549-sEVs, H460-sEVs, MCF7-sEVs, MDA-MB-231-sEVs was 37.6, 42.2, 58.2, 26.7, 43.1, 27.7, 33.4%, respectively, the content of CD9 / CD63 / CD81 positive particles in 293T-sEVs, HFF1-sEVs, MSC-sEVs, A549-sEVs, H460-sEVs, MCF7-sEVs, MDA-MB-231-sEVs was 62.0, 63.8, 75.5, 52.0, 57.0, 65.9, 65.0%, respectively, the content of PODXL positive particles in 293T-sEVs, HFF1-sEVs, MSC-sEVs, A549-sEVs, H460-sEVs, MCF7-sEVs, MDA-MB-231-sEVs was 1.1, 1.4, 3.0, 9.5, 7.6, 8.7, 20%, respectively, the content of SSEA4 positive particles in 293T-sEVs, HFF1-sEVs, MSC-sEVs, A549-sEVs, H460-sEVs, MCF7-sEVs, MDA-MB-231-sEVs was 4.8, 5.3, 2.5, 4.9, 7.1, 7.6, 14.7%, respectively.

[0075] The detection of the specific marker PODXL / Tra-1-60 / Tra-1-81 in FIG. 4 refers to the combined staining of the three markers PODXL / Tra-1-60 / Tra-1-81 when immunofluorescence staining is performed, that is, the corresponding antibodies of the three markers are added at the same time during staining. The detection of CD9 / CD63 / CD81 refers to the combined staining of the three markers CD9 / CD63 / CD81 when immunofluorescence staining is performed, that is, the corresponding antibodies of the three markers are added at the same time during staining. The proportion of PODXL / Tra-1-60 / Tra-1-81 positive particles refers to the proportion of positive particles after simultaneous PODXL / Tra-1-60 / Tra-1-81 staining.

[0076] As can be seen from FIG. 4, other cell-derived small extracellular vesicles (non-PSC-sEVs) cannot simultaneously have the following four characteristics: the proportion of PODXL / Tra-1-60 / Tra-1-81 positive particles is greater than 50%, and the proportion of SSEA4 positive particles is greater than 50%, and the proportion of CD9 positive particles is greater than 50%, and the proportion of CD9 / CD63 / CD81 positive particles is greater than 70%, so the sample can be identified as not PSC-sEVs.

[0077] As can be seen from FIG. 4, the conclusion can be seen that the identification of PSC-sEVs requires that the proportion of PODXL / Tra-1-60 / Tra-1-81 positive particles is greater than 50%, and the proportion of SSEA4 positive particles is greater than 50%, and the proportion of CD9 positive particles is greater than 50%, and the proportion of CD9 / CD63 / CD81 positive particles is greater than 70%, and all four conditions must be met. Other non-PSC-sEVs cannot simultaneously meet the four conditions.

[0078] Example Three, Identification of PSC Common Markers on the Surface of PSC-sEVs

[0079] 1. Among the commonly used markers of PSCs, SSEA4, Tra-1-60, and Tra-1-81, which are subcellularly localized on the membrane, are selected as detection indicators.

[0080] 2. According to steps 2-5 in Example One, the sample is subjected to quality control, and immunofluorescence staining of SSEA4, Tra-1-60, and Tra-1-81 is performed, respectively, and then the free fluorescent antibody is removed. The nano-flow cytometer is used to detect the proportion of SSEA4, Tra-1-60, and Tra-1-81 positive particles in the PSC-sEV sample, respectively.

[0081] As Figure 5It can be seen that the proportion of SSEA4 positive particles in 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 markers of PSCs are markers of PSC-sEVs, and the combination of PODXL / Tra-1-60 / Tra-1-81 must be used.

[0082] Example Four: Identification method of pluripotent stem cell-derived small extracellular vesicles (sample quality control unqualified)

[0083] 1. Sample preparation: Prepare PSC-sEV samples with high purity (particle protein ratio of 3*10^8 particles / ug protein), normal particle concentration (2*10^10 particles / mL); PSC-sEV samples with high purity (particle protein ratio of 3*10^8 particles / ug protein), low particle concentration (1*10^9 particles / mL); PSC-sEV samples with poor purity (particle protein ratio of 6*10^7 particles / ug protein).

[0084] 2. After immunofluorescence staining and free fluorescent antibody removal of the samples according to steps 4-5 in Example One, use a nano-flow cytometer to detect the universal marker CD9 and the proportion of CD9 / CD63 / CD81 positive particles. The results are shown in Figure 6

[0085] Figure 6 The detection of CD9 / CD63 / CD81 specific markers refers to the combined staining of three markers CD9 / CD63 / CD81 during immunofluorescence staining, that is, three marker antibodies are added at the same time during staining. The proportion of PODXL / Tra-1-60 / Tra-1-81 positive particles refers to the proportion of positive particles after simultaneous staining of PODXL / Tra-1-60 / Tra-1-81.

[0086] Figure 6 ​The results show that for qualified PSC-sEV samples with a particle protein ratio greater than 1*10^8 particles / μg protein and a particle concentration higher than 5*10^9 particles / mL, the proportion of CD9 positive particles is greater than 50% (70.3%), and the proportion of CD9 / CD63 / CD81 positive particles is greater than 70% (72.0%). For samples with a particle protein ratio lower than 1*10^8 particles / μg protein or a particle concentration lower than 5*10^9 particles / mL, the proportion of CD9 positive particles is lower than 50% (28.8 or 45.4%), and the proportion of CD9 / CD63 / CD81 positive particles is lower than 70% (66.2 or 54.7%). Overall, samples that fail quality control (insufficient concentration and purity) cannot meet the identification requirements of PSC-sEVs and cannot be identified as PSC-sEVs.

[0087] Example Five: Identification method of pluripotent stem cell-derived small extracellular vesicles (incomplete removal of free antibodies)

[0088] 1. Perform sample quality control and immunofluorescence staining according to steps 2-4 in Example One.

[0089] 2. Remove free fluorescent antibodies according to step 5 in Example One. In addition, after immunofluorescence staining of the sample, transfer the liquid to a 1 mL ultracentrifuge tube, add 1 mL of PBS dilution to the ultracentrifuge tube, and ultracentrifuge at 4°C, 100 000g, for 17 min. After ultracentrifugation, discard the supernatant, resuspend with 200 uL of PBS, and directly use for detection (compared to the step of removing free fluorescent antibodies in Example One, one ultracentrifugation process is reduced, and the removal of free antibodies is incomplete).

[0090] 3. Use a nano-flow cytometer to detect the proportion of universal markers CD9, CD9 / CD63 / CD81, and PSC-sEV specific markers PODXL / Tra-1-60 / Tra-1-81 positive particles. The results are as follows Figure 7As shown, for PSC-sEV samples with better removal of unbound free fluorescent antibody (lower background fluorescence level), the proportion of CD9 positive particles is greater than 50%, the proportion of CD9 / CD63 / CD81 positive particles is greater than 70%, and the proportion of PODXL / Tra-1-60 / Tra-1-81 positive particles is greater than 50%. For PSC-sEV samples with incomplete removal of unbound free fluorescent antibody (excessive background fluorescence level), the proportion of CD9 positive particles is less than 20%, the proportion of CD9 / CD63 / CD81 positive particles is less than 30%, and the proportion of PODXL / Tra-1-60 / Tra-1-81 positive particles is less than 20%. In general, if the free antibody is not completely removed, the results will not meet the identification requirements of PSC-sEVs, and the PSC-sEVs cannot be identified.

[0091] The foregoing description of the examples has been presented for the purposes of conduction and enabling those of ordinary skill in the art to make and use the application. Modifications to, and variations of, the described examples are possible and are within the scope of the application. Accordingly, the application is not limited to the above described examples, but is instead defined by the appended claims in light of the foregoing disclosure.

Claims

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

2. A method for identifying small extracellular vesicles derived from pluripotent stem cells, characterized in that, The characteristic marker combination of small extracellular vesicles derived from pluripotent stem cells described in claim 1 is used to identify whether the small extracellular vesicles are derived from pluripotent stem cells. Based on a combination of characteristic biomarkers of small extracellular vesicles derived from pluripotent stem cells, immunofluorescence staining was performed on the small extracellular vesicles to be tested. 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 of PODXL / Tra-1-60 / Tra-1-81 positive granule ratio are not simultaneously met, the small extracellular vesicles are identified as small extracellular vesicles of non-pluripotent stem cell origin. A positive particle percentage of PODXL / Tra-1-60 / Tra-1-81 greater than 50% means that the percentage of positive particles in at least one of PODXL, Tra-1-60, and Tra-1-81 is greater than 50%. The percentage of positive particles for CD9 / CD63 / CD81 is greater than 70% means that the percentage of positive particles for at least one of CD9, CD63, and CD81 is greater than 70%.

3. The 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 as follows: During immunofluorescence staining, three markers PODXL / Tra-1-60 / Tra-1-81 were combined. That is, fluorescently labeled aptamer molecules corresponding to the three markers PODXL, Tra-1-60 and Tra-1-81 were added at the same time during staining. After incubation, the proportion of particles with at least one positive PODXL, Tra-1-60 and Tra-1-81 was counted. The method for detecting the proportion of SSEA4 positive particles is as follows: During immunofluorescence staining, fluorescently labeled aptamer molecules corresponding to SSEA4 were added, and after incubation, the proportion of SSEA4 positive particles was counted. The method for detecting the proportion of CD9 positive particles is as follows: During immunofluorescence staining, fluorescently labeled aptamer molecules corresponding to CD9 were added, and after incubation, the proportion of CD9 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 simultaneously during staining. After incubation, the proportion of particles with at least one positive CD9, CD63, and CD81 was counted.

4. The method for identifying small extracellular vesicles derived from pluripotent stem cells according to claim 3, characterized in that, Immunofluorescence staining of samples was performed using fluorescently labeled aptamer molecules that 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 aptamers that 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-RAntibody, 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.

6. The method for identifying small extracellular vesicles derived from pluripotent stem cells according to claim 2, characterized in that, 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 of 5×10^9 particles / mL or higher 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-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 can be performed.

7. The method for identifying small extracellular vesicles derived from pluripotent stem cells according to claim 2, characterized in that, When performing immunofluorescence staining on samples using fluorescently labeled aptamer molecules that 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, 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.

9. The method for identifying small extracellular vesicles derived from pluripotent 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 PODXL / Tra-1-60 / Tra-1-81 positive particles, SSEA4 positive particles, CD9 positive particles, and CD9 / CD63 / CD81 positive particles. The PODXL / Tra-1-60 / Tra-1-81 positive particle ratio refers to the proportion of particles that are positive for at least one of PODXL, Tra-1-60, and Tra-1-81. The CD9 / CD63 / CD81 positive particle ratio refers to the proportion of particles that are positive for at least one of CD9, CD63, and CD81.

10. The method for identifying small extracellular vesicles derived from pluripotent 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 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. (2) Immunofluorescence staining: The samples were aliquoted 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. The fluorescently labeled aptamers that 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-RAntibody, 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. (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 the optimal detection concentration, nanoflow cytometry was used to perform single vesicle level nanoparticle fluorescence analysis to obtain the proportions of PODXL / Tra-1-60 / Tra-1-81 positive particles, SSEA4 positive particles, CD9 positive particles, and CD9 / CD63 / CD81 positive particles. (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 of PODXL / Tra-1-60 / Tra-1-81 positive granule ratio are not simultaneously met, the small extracellular vesicles are identified as small extracellular vesicles of non-pluripotent stem cell origin. A positive particle percentage of PODXL / Tra-1-60 / Tra-1-81 greater than 50% means that the percentage of positive particles in at least one of PODXL, Tra-1-60, and Tra-1-81 is greater than 50%. The percentage of positive particles for CD9 / CD63 / CD81 is greater than 70% means that the percentage of positive particles for at least one of CD9, CD63, and CD81 is greater than 70%.

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