Vesicles and their uses

Abstract

Vesicles and uses thereof are provided. The vesicles are inducible vesicles, originating from stem cells or somatic cells, and possess a marker including syntaxin 4. These vesicles can specifically express syntaxin 4 at higher levels than exosomes in mesenchymal stem cells, making them distinguishable from characteristic markers of vesicles and exosomes derived from MSCs. The vesicles can exert a blood coagulation promoting effect in vitro and, after injection into the body, can improve the bleeding tendency of hemophilic mice, making them suitable for the treatment of bleeding tendency in hemophilia. The vesicles can be excreted via the skin and hair.

Description

[Technical Field]

[0001] The present invention is in the field of biomedicine and relates to vesicles and uses thereof. [Background technology]

[0002] Extracellular vesicles (EVs) are nanoscale carriers secreted by cells and contain proteins, nucleic acids, and various cytokines. EVs can interact with target cells via endocrine or paracrine mechanisms and play an important role in intercellular transport and communication. Research has shown that EV-mediated communication plays an important regulatory role in physiological and pathological processes, including immune regulation, tumor growth, angiogenesis, and wound repair. Currently, research in this field focuses primarily on exosomes. Exosomes are extracellular vesicles with diameters of approximately 30–150 nm that contain components such as RNA, lipids, and proteins. Exosomes are widely involved in various physiological and pathological processes in the body and can be used for the diagnosis, treatment, and prognosis of many diseases. To date, mesenchymal stem cells (MSCs) are considered to be the cells with the highest exosome-producing capacity. Extensive research has demonstrated that MSC-derived exosomes can mimic the biological functions of MSCs and play important regulatory roles in promoting cell growth and differentiation, repairing tissue defects, and more. Therefore, in recent years, MSC-derived exosome-based cellular vesicle therapy has made remarkable progress. However, exosome-based cellular vesicle therapy currently faces many challenges, including the complex and time-consuming exosome extraction and purification process, the high equipment and reagent requirements, and the relatively low physiological exosome yield. These shortcomings limit the clinical translation and application of exosome therapy.

[0003] Hemophilia is a group of bleeding disorders resulting from inherited blood coagulation disorders. Common characteristics include impaired production of activated thromboplastin, prolonged clotting time, a lifetime tendency to bleed even with minor trauma, and "idiopathic" bleeding even in the absence of significant trauma in severely ill patients. On May 11, 2018, five departments, including the National Health Commission, jointly compiled the "First Rare Disease List" listing hemophilia. Hemophilia is primarily divided into three types: hemophilia A, hemophilia B, and hemophilia C. Hemophilia A, i.e., factor VIII deficiency (VIII:C), is an X-linked recessive genetic disorder that is transmitted in females and affects males. Hemophilia B, i.e., factor IX (FIX) deficiency, is also an X-linked recessive genetic disorder and is less common than hemophilia A. Hemophilia C, or factor XI (FXI) deficiency, is an autosomal recessive genetic disorder and a rare form of hemophilia. Hemophilia A accounts for the highest incidence rate at 80%-85%, followed by hemophilia B at 15%-20%, and hemophilia C is rare. For a long time, exogenous blood coagulation factor injections have been used clinically as the primary intervention for treating hemophilia. However, this method has various problems, such as high treatment costs, a short duration of therapeutic effect, and a tendency to produce autoantibodies, making it an inappropriate and effective treatment option. Summary of the Invention

[0004] In some embodiments, the present invention provides vesicles derived from mesenchymal stem cells.

[0005] In some embodiments, the present invention provides a vesicle composition.

[0006] In some embodiments, the present invention provides a vesicle-containing pharmaceutical composition for hemophilia.

[0007] In some embodiments, the present invention provides a vesicle screening, identification, or extraction kit.

[0008] In some embodiments, the present invention provides a marker for a vesicle.

[0009] In some embodiments, the present invention provides methods for identifying or screening vesicles by markers.

[0010] In some embodiments, the present invention provides a method for preparing a vesicle.

[0011] In some embodiments, the present invention provides vesicles derived from somatic cells or stem cells, wherein the vesicles are inducible vesicles and the marker carried by the vesicles includes syntaxin 4.

[0012] In some embodiments, the present invention provides methods for treating, preventing, or ameliorating a disease or a complication of the disease in a subject. The methods include administering to the subject an effective amount of the vesicles, vesicle composition, or composition. The disease is a bleeding disorder. In some embodiments, the bleeding disorder includes bleeding due to a deficiency, low platelet count, and / or dysfunction of a blood clotting factor. In some embodiments, the bleeding disorder includes hemophilia, lupus hemorrhage, or Chediak-Higashi syndrome. In some embodiments, the hemophilia includes hemophilia A, hemophilia B, or hemophilia C. In some embodiments, the disease is hemophilia A.

[0013] In some embodiments, the stem cells include totipotent stem cells and pluripotent stem cells, hi some embodiments, the stem cells include mesenchymal stem cells and induced pluripotent stem cells (IPS).

[0014] In some embodiments, the somatic cells comprise an osteoblastic cell lineage.

[0015] In some embodiments, the cells may be primary culture cells or may be a pre-existing or established cell line.

[0016] In some embodiments, the cell line refers to an immortalized cell culture that can grow indefinitely under appropriate fresh medium and space.

[0017] In some embodiments, the cells may be an established cell line.

[0018] In some embodiments, the induced vesicles are vesicles produced by inducing apoptosis of the stem cells or somatic cells during their normal life span with an external force.

[0019] In some embodiments, the induced vesicles are produced by inducing apoptosis of stem cells or stem cells by a combination of one or more of the following methods: addition of Staurospora, ultraviolet irradiation, starvation, and heat stress.

[0020] In some embodiments, the markers carried by the vesicles further include one or more of Annexin V, Flotillin-1, Cadherin 11, and Integrin α5.

[0021] In some embodiments, the vesicles have a combination of the markers syntaxin 4, annexin V, flotillin-1, cadherin 11, and integrin alpha 5.

[0022] In some embodiments, the vesicles highly express the markers annexin V, flotillin-1, cadherin 11, integrin alpha 5, and syntaxin 4.

[0023] In some embodiments, the vesicles express higher levels of the markers annexin V, flotillin-1, cadherin 11, integrin alpha 5, and syntaxin 4 than MSCs or exosomes.

[0024] In some embodiments, the expression levels of the markers annexin V, flotillin-1, cadherin 11, integrin α5, and syntaxin 4 in the vesicles are about 1-2 times, 2-3 times, 1-3 times, 3-4 times, and 3-6 times, respectively, relative to the expression levels of the markers in exosomes derived from mesenchymal stem cells.

[0025] In some embodiments, the expression levels of the markers annexin V, flotillin-1, cadherin 11, integrin α5, and syntaxin 4 in the vesicles are about 1.5-2 times, 2.5-3 times, 1.5-2.5 times, 3.5-4 times, and 3.5-5 times higher, respectively, than the expression levels of the markers in exosomes derived from mesenchymal stem cells.

[0026] In some embodiments, the expression levels of the markers annexin V, flotillin-1, cadherin 11, integrin α5, and syntaxin 4 in the vesicles are approximately 1.5-1.9-fold, 2.5-2.9-fold, 1.8-2.5-fold, 3.5-3.9-fold, and 4-5-fold higher, respectively, than the expression levels of the markers in exosomes derived from mesenchymal stem cells.

[0027] In some embodiments, the expression levels of the markers annexin V, flotillin-1, cadherin 11, integrin α5, and syntaxin 4 in the vesicles are approximately 1.76-fold, 2.81-fold, 2.41-fold, 3.68-fold, and 4.45-fold, respectively, relative to the expression levels of the markers in exosomes derived from mesenchymal stem cells.

[0028] In some embodiments, the exosomes do not express syntaxin 4, whereas the vesicles of the invention express syntaxin 4.

[0029] In some embodiments, the exosomes do not simultaneously express annexin V, flotillin-1, cadherin 11, integrin α5, and syntaxin 4, whereas the vesicles of the invention simultaneously express annexin V, flotillin-1, cadherin 11, integrin α5, and syntaxin 4.

[0030] In some embodiments, the vesicles and exosomes are derived from allogeneic MSCs.

[0031] In some embodiments, analysis of IEV surface membrane proteins by flow cytometry revealed that IEVs derived from MSCs can express surface proteins similar to those of MSCs, such as CD29, CD44, CD73, CD166 positive, CD34, and CD45 negative, and can also express ubiquitous surface proteins of extracellular vesicles, such as CD9, CD63, CD81, and C1q.

[0032] In some embodiments, the induced vesicles are produced by inducing apoptosis in mesenchymal stem cells by adding staurosporine, ultraviolet irradiation, starvation, heat stress, or a combination thereof.

[0033] In some embodiments, the vesicles are produced by inducing mesenchymal stem cells with staurosporine.

[0034] In some embodiments, the number of generations of the mesenchymal stem cells may be, but is not limited to, 2 to 5 generations.

[0035] In some embodiments, the concentration of staurosporine is about 1 nM-10,000 nM. In some embodiments, the concentration of staurosporine is about 100 nM-10,000 nM. In some embodiments, the concentration of staurosporine is about 500 nM-10,000 nM. In some embodiments, the concentration of staurosporine is about 500-1,000 nM. In some embodiments, the concentration of staurosporine is about 500-900 nM. In some embodiments, the concentration of staurosporine is about 500-800 nM.

[0036] In some embodiments, the vesicles have a diameter of about 0.03-6 μM. In some embodiments, the vesicles have a diameter of about 0.03-4.5 μM. In some embodiments, the vesicles have a diameter of about 0.03-1 μM. In some embodiments, the vesicles have a diameter of about 0.04-1 μM. In some embodiments, the vesicles have a diameter of about 0.05-1 μM. In some embodiments, the vesicles have a diameter of about 0.1-1 μM. In some embodiments, the vesicles have a diameter of about 0.15-1 μM.

[0037] In some embodiments, the present invention further provides a vesicle composition comprising the vesicle.

[0038] In some embodiments, the vesicle composition further comprises other vesicles described in the art, including, but not limited to, exosomes, migratory bodies, microvesicles, and ectosomes.

[0039] In some embodiments, the number of vesicles is about 65-100% of the vesicle composition.

[0040] In some embodiments, the vesicles comprise about 75-98% of the vesicle composition.

[0041] In some embodiments, the vesicles comprise about 80-96% of the vesicle composition.

[0042] In some embodiments, the present invention further provides a composition comprising the vesicle or the vesicle composition.

[0043] In some embodiments, the composition comprises a pharmaceutical, a food, a health function product, a cosmetic, an additive, or an intermediate.

[0044] In some embodiments, the composition is a pharmaceutical agent.

[0045] In some embodiments, the composition further comprises a pharmaceutically or immunologically acceptable carrier.

[0046] In some embodiments, the dosage form of the composition is selected from a lyophilized powder for injection, an injection, a tablet, a capsule, a kit, or a patch.

[0047] In some embodiments, the vesicles are used as drug carriers.

[0048] In some embodiments, the invention further provides a reagent or kit for screening, identifying, or extracting said vesicles, comprising a detection reagent for one or more of the markers Annexin V, Flotillin-1, Cadherin 11, Integrin α5, and Syntaxin 4.

[0049] In some embodiments, the detection reagent for the marker detects the expression level of the gene for the marker.

[0050] In some embodiments, the detection reagent for the marker detects the expression level of mRNA of the marker.

[0051] In some embodiments, the detection reagent for the marker detects the expression level of the protein of the marker.

[0052] In some embodiments, the detection reagent for the marker is one or more of a fluorescent qPCR dye, a fluorescent qPCR primer, a fluorescent qPCR probe, an antibody, an antibody functional fragment, and a conjugated antibody.

[0053] In some embodiments, the kit is one or more selected from a qPCR kit, a Western blotting detection kit, a flow cytometry analysis kit, an immunohistochemistry detection kit, and an ELISA kit.

[0054] In some embodiments, the kit is selected from a flow cytometry analysis kit.

[0055] In some embodiments, the present invention further provides use of said vesicles, said vesicle composition, or said pharmaceutical composition in the preparation of a product for treating, preventing, or ameliorating a disease or a complication of said disease, including liver disease and hemophilia.

[0056] In some embodiments, the disease is hemophilia. The vesicles can exert a significant blood coagulation promoting effect in vitro and significantly improve the bleeding tendency of hemophilic mice after intravenous injection, making them suitable for the treatment of bleeding tendency in hemophilia, and have good prospects for application.

[0057] In some embodiments, the disease is hemophilia A.

[0058] In some embodiments, the product comprises a pharmaceutical, a food, a health function product, a cosmetic, an additive, or an intermediate product.

[0059] When the vesicles are used to treat a disease, the vesicles can be administered by a route selected from the group consisting of intravenous injection, intramuscular injection, subcutaneous injection, intrathecal injection or infusion, and intraorgan injection. For example, intravenous injection can be via the tail vein. Inorgan injection includes injection into an anatomical space, such as the gallbladder, gastrointestinal cavity, esophagus, pulmonary system (by inhalation), and / or bladder.

[0060] For example, intraperitoneal injection, which is a gastrointestinal injection, can achieve the same therapeutic effect as tail vein injection, and intraperitoneal injection is superior to tail vein injection in terms of safety and operability.

[0061] In some embodiments, the invention further provides a method of screening or identifying the vesicles, the method comprising detecting one or more of the markers Annexin V, Flotillin-1, Cadherin 11, Integrin α5, and Syntaxin 4.

[0062] If the detection result of the marker is positive, it is determined to be the vesicle.

[0063] In some embodiments, the expression result of the marker is compared with a control, and if the expression level is significantly higher than that of the control, it can be determined as a positive result. The control may be other conventional vesicles or exosomes (which may include one or more of exosomes, migratory bodies, microvesicles, and ectosomes), or other vesicles or exosomes derived from mesenchymal stem cells.

[0064] Among the markers, syntaxin 4 is preferred. In some embodiments, when the expression level of syntaxin 4 in the detected vesicles is 2-6 times (more preferably 4-5 times) or more that of exosomes (e.g., exosomes derived from allogeneic cells), the vesicles are determined to be induced vesicles.

[0065] In some embodiments, the present invention provides use of a detection reagent for the marker in the preparation of a reagent or kit for detecting or identifying vesicles, wherein the marker comprises one or more of annexin V, flotillin-1, cadherin 11, integrin α5, and syntaxin 4, and the reagent or kit further comprises a control reagent, wherein the control reagent comprises one or more of exosomes, mobile bodies, microvesicles, and ectosomes, and the test sample is determined to be positive when the expression level of the marker in the test sample is higher than that of the control reagent.

[0066] In some embodiments, the control reagent is an exosome.

[0067] In some embodiments, when the expression level of syntaxin 4 in the test sample is 2-6 times or more that of exosomes, the test sample is determined to be a vesicle.

[0068] In some embodiments, when the expression level of syntaxin 4 in the test sample is 4-5 times or more that of exosomes, the vesicles are determined to be induced vesicles (e.g., induced vesicles).

[0069] In some embodiments, the present invention provides a method for preparing the vesicles, which comprises inducing stem cells or somatic cells to produce the vesicles by adding an apoptosis-inducing agent.

[0070] In some embodiments, the method includes the steps of (1) culturing mesenchymal stem cells, (2) collecting a culture supernatant of the mesenchymal stem cells, and (3) isolating vesicles from the culture supernatant of step (2).

[0071] In some embodiments, the step (1) of culturing mesenchymal stem cells comprises the step (4) of isolating mesenchymal stem cells from tissue, and the step (5) of adding a medium to culture the mesenchymal stem cells and contacting the mesenchymal stem cells in the medium with an apoptosis-inducing agent.

[0072] In some embodiments, the apoptosis-inducing agent comprises a combination of one or more of staurosporine, ultraviolet irradiation, starvation, and heat stress.

[0073] In some embodiments, the apoptosis-inducing agent is staurosporine.

[0074] In some embodiments, the concentration of staurosporine is about 500-1000 nM. In some embodiments, the concentration of staurosporine is about 500-900 nM. In some embodiments, the concentration of staurosporine is about 500-800 nM.

[0075] In some embodiments, the time period for treating the cells with the apoptosis-inducing agent in step (5) is about 16-24 hours.

[0076] In some embodiments, in step (3), the method for separating vesicles includes separating the vesicles by ultracentrifugation.

[0077] In some embodiments, in the present invention, a single MSC can produce 300-2000 follicles.

[0078] In some embodiments, the step of separating the vesicles by ultracentrifugation includes step (a) of centrifuging the collected culture supernatant a first time and collecting the supernatant; step (b) of centrifuging the supernatant collected in step (a) a second time and collecting the supernatant; step (c) of centrifuging the supernatant collected in step (b) a third time and collecting the precipitate; step (d) of centrifuging the precipitate collected in step (c) a fourth time and collecting the precipitate; and step (e) of centrifuging the precipitate collected in step (d) a fifth time and collecting the precipitate.

[0079] In some embodiments, the first centrifugation involves centrifugation at about 500-1500 g for 5-30 minutes. In some embodiments, the first centrifugation involves centrifugation at about 500-1000 g for 5-20 minutes. In some embodiments, the first centrifugation involves centrifugation at about 500-900 g for 5-15 minutes. In some embodiments, the second centrifugation involves centrifugation at about 1000-3000 g for 5-30 minutes. In some embodiments, the second centrifugation involves centrifugation at about 1500-2500 g for 5-20 minutes. In some embodiments, the second centrifugation involves centrifugation at about 1500-2200 g for 5-15 minutes. In some embodiments, the third centrifugation involves centrifugation at about 10,000-30,000 g for 15-60 minutes. In some embodiments, the third centrifugation involves centrifugation at about 12,000-25,000 g for 20-60 minutes. In some embodiments, the third centrifugation involves centrifugation at about 12,000-20,000 g for 20-40 minutes. In some embodiments, the fourth centrifugation involves centrifugation at about 10,000-30,000 g for 15-60 minutes. In some embodiments, the fourth centrifugation involves centrifugation at about 12,000-25,000 g for 20-60 minutes. In some embodiments, the fourth centrifugation involves centrifugation at about 12,000-20,000 g for 20-40 minutes.

[0080] In some embodiments, vesicles bearing a specific marker are obtained by enrichment using an enrichment method for that specific marker. After obtaining sufficient vesicles, the medium is collected, and the specific vesicles are purified and isolated from the medium. This can be achieved by any suitable method known in the art. Such methods include, for example, the primitive method of isolating exosomes by differential ultracentrifugation; and newer methods such as polymer precipitation (ExoQuick™ from SBI, Palo Alto, CA), immunoaffinity capture (Greening et al., 2015, Methods in Molecular Biology), and immunomagnetic capture (Exo-FLOW™, SBI).

[0081] Immunoaffinity purification is a method for selectively capturing specific vesicles based on surface markers. High-affinity binding between streptavidin-coated magnetic beads and biotinylated capture antibodies allows for efficient vesicle capture. After elution, the captured vesicles retain their intact structure and biological activity. Based on the principles of the present invention, the vesicles can specifically and highly express annexin V, flotillin-1, cadherin 11, integrin α5, and syntaxin 4. Therefore, the present invention allows for the separation, purification, or enrichment of vesicles using this method.

[0082] In some embodiments, the vesicles can be enriched using immunomagnetic bead technology. The immunomagnetic beads can be obtained by conjugating monoclonal antibodies to magnetic beads. The monoclonal antibodies can include one or more of anti-syntaxin 4 antibody, anti-annexin V antibody, anti-flotillin-1 antibody, anti-cadherin 11 antibody, and anti-integrin α5 antibody.

[0083] In some embodiments, the present invention further provides inducible vesicles, which are derived from IPS cells and are subcellular products produced by intervening or inducing apoptosis of IPS cells while they are in their normal viability.

[0084] As used herein, the term "enriched" includes the separation of one or more vesicles from any other vesicles present in a sample, or the presence of said vesicles at a higher total percentage content in a composition comprising the vesicles than it would be found to be present in a biological tissue.

[0085] In one embodiment, the enriched vesicles are not separated from the sample, and any diagnostics are performed on the vesicles while they are still in the sample. The sample can be placed on a glass slide and examined under a microscope; in this embodiment, the vesicles are detected but not separated.

[0086] In another embodiment, the concentrated vesicles are separated from the sample.

[0087] Immunomagnetic bead-based separation (IMS) is a new immunological technology that has been developed in recent years. Immunomagnetic beads (IMBs) can be bound to activated protein antibodies and can also be attracted to a magnet. After processing, the antibodies are bound to the magnetic beads, which then serve as carriers for the antibodies. When the antibodies bind to specific antigenic substances on the magnetic beads, antigen-antibody-magnetic bead immune complexes are formed. These complexes are separated from other substances by mechanical movement under the influence of magnetic force, achieving the goal of isolating specific antigens. Immunomagnetic beads (IMBs) are a platform that can be used in any field that uses the antigen-antibody binding principle. They have achieved remarkable results in medical and biological applications, such as bone marrow transplantation, or the separation of stem cells, organelles, cancer cells, hormones, pathogens, and toxins. In recent years, IMB has been widely used in the separation and detection of mycotoxins in samples such as food, water, biological samples, and the environment due to its high sensitivity and specificity, and shows good prospects for development and application.

[0088] In the immunomagnetic bead method described in the present invention, magnetic beads bound to specific antibodies are used to bind to target vesicles having specific surface antigens, and then the target vesicles are adsorbed by a magnetic field and extracted.

[0089] In some specific embodiments of the present invention, the specific method for enriching vesicles is as follows: immunomagnetic beads coated with one or more antibodies selected from the group consisting of anti-syntaxin 4 antibody, anti-annexin V antibody, anti-flotillin-1 antibody, anti-cadherin 11 antibody, and anti-integrin α5 antibody are added to a cell culture supernatant containing vesicles, thereby isolating vesicles capable of specifically binding to one or more antibodies selected from the group consisting of anti-syntaxin 4 antibody, anti-annexin V antibody, anti-flotillin-1 antibody, anti-cadherin 11 antibody, and anti-integrin α5 antibody, thereby achieving the goal of enriching specific vesicles. In some preferred embodiments, vesicles isolated using immunomagnetic beads simultaneously coated with anti-syntaxin 4 antibody, anti-annexin V antibody, anti-flotillin-1 antibody, anti-cadherin 11 antibody, and anti-integrin α5 antibody have the highest purity and the highest therapeutic efficacy for diseases such as hemophilia A.

[0090] In some preferred embodiments, combinatorial optimization is performed based on the centrifugation method to remove impurities such as cells and cell debris to obtain a cell culture supernatant, and immunomagnetic beads coated with one or more antibodies selected from the group consisting of anti-syntaxin 4 antibody, anti-annexin V antibody, anti-flotillin-1 antibody, anti-cadherin 11 antibody, and anti-integrin α5 antibody are then added to the cell culture supernatant to separate vesicles that can specifically bind to the antibodies, thereby achieving the goal of concentrating specific vesicles.

[0091] In some embodiments, the mesenchymal stem cells are derived from, but not limited to, a human or mouse.

[0092] In some embodiments, the mesenchymal stem cells include, but are not limited to, bone marrow-derived mesenchymal stem cells, allantoic fluid-derived mesenchymal stem cells, oral cavity-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, periosteum-derived stem cells, or a combination thereof.

[0093] In some embodiments, the mesenchymal stem cells are selected from bone marrow-derived mesenchymal cells, adipose-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and oral cavity-derived mesenchymal stem cells. [Brief explanation of the drawings]

[0094] [Figure 1] Figures 1A to 1E show the results of flow cytometry detection of surface markers of isolated BMMSCs. [Figure 2] 10 is an operation flowchart of the second embodiment. [Figure 3] Statistical results of the number of IEVs produced by MSCs (106 MSCs) analyzed by flow cytometry. [Figure 4] Figures 4A to 4F show the diameter detection of IEV particles. Figure 4A shows the particle size distribution of IEVs detected by flow cytometry. Figure 4B shows the analysis of scattered light intensity of IEVs using side scatter (SSC), showing the particle size distribution of IEVs. Figure 4C shows the analysis of scattered light intensity of IEVs using standardized small particle microspheres manufactured by Bangs Laboratories, showing the particle size distribution of IEVs. Figure 4D shows IEVs observed by transmission electron microscopy (TEM), showing the particle size distribution of IEVs. Figure 4E shows nanoparticle tracking analysis (NTA), showing the particle size distribution of IEVs. Figure 4F shows the particle size detection of IEVs at the single vesicle level using nanoflow cytometry detection technology, showing the particle size distribution of IEVs. [Figure 5] Figures 5A to 5K show the results of flow cytometric analysis of IEV surface membrane proteins. [Figure 6]Figures 6A to 6D show the content analysis of IEVs. Figure 6A shows the results of quantitative proteomics analysis of MSCs, MSC-exosomes, and MSC-IEVs using DIA quantification technology. Figure 6B shows a heat map generated by screening for proteins specifically expressed by IEVs. Figure 6C shows the results of GO enrichment analysis of differential proteins for IEV-expressed annexin V, flotillin-1, cadherin 11, integrin α5, and syntaxin 4. Figure 6D shows the results of Western blot analysis of MSCs, MSC-exosomes, and MSC-IEV-expressed annexin V, flotillin-1, cadherin 11, integrin α5, and syntaxin 4. [Figure 7] Figure 1 shows the procoagulant effect of IEVs in vivo in hemophilia A mice. [Figure 8] Figures 8A to 8D show the changes in the levels of each blood coagulation factor after IEV injection into hemophilia A mice. Figure 8A shows the changes in blood coagulation factor VIII, Figure 8B shows the changes in vWF, Figure 8C shows the changes in tissue factor (TF), and Figure 8D shows the changes in prothrombin. [Figure 9] Figures 9A and 9B show the effects of PS and TF blockade on the in vivo therapeutic efficacy of IEV in a hemophilia A mouse model. Figure 9C shows that IEV injection treatment in lpr mice significantly improved their bleeding tendency. Figure 9D shows that IEV injection treatment in CHS mice significantly improved their bleeding tendency. [Figure 10] The figure shows a comparison of the therapeutic effects of IEVs and exosomes derived from allogeneic MSCs on hemophilia A mice. Here, WT refers to wild-type mice, the HA group refers to a hemophilia A mouse model, HA+IEV refers to IEV treatment in a hemophilia A mouse model, HA+PS-IEV refers to PS-negative IEVs in a hemophilia A mouse model, HA+TF-IEV refers to TF-negative IEVs in a hemophilia A mouse model, and HA+exosomes refers to exosome treatment in a hemophilia A mouse model. [Figure 11]Morphology of IEVs derived from MC3T3-E1 and hBMMSCs under a light microscope. [Figure 12] Flow cytometry detection results of the diameter distribution of IEV particles derived from MC3T3-E1 and hBMMSC. [Figure 13] Figures 13A to 13C show that IEVs can be excreted via the skin and hair. Figure 13A is a schematic diagram of the dynamic metabolism of IEVs at the skin surface. Figure 13B shows that IEVs gradually migrate from the subcutaneous tissue to the dermis and epidermis over time. Figure 13C shows that PKH26-IEVs were found to be present in hair follicles plucked from the surface of mice on day 7. [Figure 14] This is a diagram showing the death process of hiPSCs and hUCMSCs photographed using a High Content Analysis (HCA) system. [Figure 15] The apoptosis rate of hiPSCs and hUCMSCs induced to apoptosis was analyzed by flow cytometry, demonstrating that most cells underwent apoptosis. [Figure 16] Analysis of the positive rate of Annexin 5 expression by flow cytometry showed that the expression rate was over 80% for both hiPSCs and hUCMSCs. [Figure 17] The particle sizes of two types of IEVs detected by nanoparticle tracking analysis (NTA) are shown. [Figure 18] The numbers of IEVs produced by two types of IEV detected by nanoparticle tracking analysis (NTA) are shown. [Figure 19] The potentials of two types of IEVs detected by nanoparticle tracking analysis (NTA) are shown. DETAILED DESCRIPTION OF THE INVENTION

[0095] The technical solutions of the present invention will be further described below through specific examples. The specific examples do not limit the scope of protection of the present invention. Any non-essential modifications and adjustments made by those skilled in the art based on the concept of the present invention will still fall within the scope of protection of the present invention.

[0096] In some embodiments of the present invention, IEV is an abbreviation for induced vesicles and may also be referred to as induced extracellular vesicles (IEVs). Induced extracellular vesicles refer to subcellular products produced by apoptosis induced or induced in progenitor cells (e.g., stem cells) during normal cell survival. Typically, such subcellular products have a membrane structure, express apoptosis markers, and partially contain genetic material, DNA. The inventors have discovered that induced extracellular vesicles are distinct from cells and conventional extracellular vesicles (e.g., exosomes). In some embodiments, the cells in a normal cell state are, for example, non-apoptotic cells, non-senescent cells, cells not arrested in growth due to senescence, cells not resuscitated after freezing, cells not undergoing abnormal growth due to malignant transformation, or undamaged cells. In some embodiments, the cells in a normal cell state are collected from cells that have reached 80-100% confluence during cell culture. In some embodiments, the normally viable cells are obtained from cells in logarithmic growth phase. In some embodiments, the normally viable cells are obtained from primary cells and their subcultures derived from human or mouse tissue. In some embodiments, the normally viable cells are obtained from an established cell line or cell strain. In some embodiments, the progenitor cells are obtained from early cells.

[0097] In the present invention, IEV is the same as IEVs. In the present invention, STS is staurosporine. In the present invention, Exosomes are exosomes.

[0098] "Comprise" or "contain" means that compositions (e.g., media) and methods include the recited elements, but do not exclude other elements. When used to define compositions and methods, "consisting essentially of" means excluding other elements of any significant significance to the intended combination. Thus, a composition consisting essentially of the elements defined herein does not exclude other materials or steps that do not materially affect the basic and novel characteristics of the invention for which protection is sought. "Consisting of" means excluding trace elements and substantial method steps from other compositions. All embodiments defined by each of these terms are within the scope of the present invention.

[0099] An "effective amount" refers to an amount sufficient to achieve a beneficial or desired result (e.g., enhancing an immune response, treating, preventing, or ameliorating a medical condition (disease, infection, etc.)). An effective amount can be administered in one or more applications, doses, or doses. The appropriate dose depends on the body weight, age, health, disease or condition being treated, and the route of administration.

[0100] As used herein, terms such as "high expression" are intended to include increasing the expression of a nucleic acid or protein to a level higher than that contained in prior art vesicles (e.g., exosomes).

[0101] As used herein, "pharmaceutically acceptable carrier" refers to any standard pharmaceutical carrier, such as an injectable lyophilized powder, injection, tablet, capsule, kit, or patch. Typically, such carriers contain excipients such as starch, emulsions, sugars, certain types of clay, gelatin, stearic acid or its salts, magnesium stearate or calcium stearate, talc, vegetable fats or oils, gums, glycols, or other known excipients. These carriers may further contain flavorings, coloring agents, or other ingredients. Examples of pharmaceutically acceptable carriers include, but are not limited to, water, saline, buffer solutions, and inert, non-toxic solids (e.g., mannitol, talc). Compositions containing such carriers are prepared by known, conventional methods. Depending on the desired mode of administration and intended use, the compositions may be in the form of solid, semi-solid, or liquid dosage forms, such as powders, particles, crystals, liquids, suspensions, liposomes, pastes, creams, ointments, etc., suitable for administration in relatively precise unit doses.

[0102] In the present invention, the components in a "composition" may be present as a mixture or packaged separately. Each of the separately packaged components may contain an adjuvant. The adjuvant refers to a means capable of pharmacologic support of the therapeutic effect of a drug. When the components in a composition are packaged separately, the separately packaged components may be administered simultaneously or in any order, in which case one drug is administered to a patient for treatment, followed by the administration of another drug. The patient is a mammalian subject, particularly a human.

[0103] In the present invention, the "composition" may exist in a form in which one component is encapsulated in another component. In some embodiments, in the composition, the inducible vesicle encapsulates a drug for treating or preventing a disease within the inducible vesicle as a drug carrier.

[0104] In the present invention, the corresponding reagents are from the following sources: penicillin / streptomycin solution (BIOSOURCE; P303-100), glutamine (BIOSOURCE; P300-100), dexamethasone sodium phosphate (Sigma; D-8893), α-MEM (Gibco; 12571-063), and 2-ME (GIBCO; 21985-023).

[0105] Example 1: Isolation and culture of MSCs Following the guidelines of the animal ethics committee, mice were euthanized with excessive CO2. Under sterile conditions, the tibia and femur were removed and the attached muscle and connective tissue were removed. The metaphysis was then separated to expose the marrow cavity. The marrow cavity was repeatedly flushed with PBS containing 10% fetal bovine serum (FBS) using a 10 mL sterile syringe. The cells were then filtered through a 70 μm pore size cell filter, centrifuged at 500 g for 5 min, and the supernatant was removed. The cell pellet at the bottom was collected, resuspended in PBS, and centrifuged at 500 g for another 5 min to obtain the final cell pellet. Next, the cells were sorted by flow cytometry to obtain BMMSCs using CD34- and CD90+ as the sorting standard. Finally, the cells were resuspended in Dex(-) medium, seeded into 10 cm cell culture dishes, and cultured at 37°C with 5% CO2. After 24 hours, non-adherent cells in the supernatant were removed by aspiration and washed with PBS. Dex(-) medium was added and the culture continued. After one week, the same amount of Dex(+) medium was added, and after another week, dense primary BMMSC colonies were observed. BMMSCs were digested with trypsin and incubated at 37°C for subcultivation. The Dex(+) medium was then replaced every three days, and the cultures were passaged once the culture dish filled. Subsequent experiments were performed using P2 BMMSCs.

[0106] The components of the Dex(-) culture medium are shown in Table 1, and the components of the Dex(+) culture medium are shown in Table 2.

[0107] [Table 1]

[0108] [Table 2]

[0109] The purity of the isolated BMMSCs was evaluated by analyzing surface markers using flow cytometry. For surface marker identification, P2 BMMSCs were digested with trypsin, collected, washed once with PBS, and then cultured at 5 × 10 5 The cells were resuspended in 3% FBS-containing PBS at a density of 1 / mL, and 1 μL of PE fluorescent dye-conjugated antibodies against CD29, CD44, CD90, CD45, and CD34 was added (1 μL was added to the blank group). The cells were incubated at 4°C in the dark for 30 min, washed twice with PBS, and then subjected to flow cytometry. The results of flow cytometry are shown in Figures 1A-1E. As can be seen from the results, the isolated cells were bone marrow mesenchymal stem cells (BMMSCs).

[0110] Example 2: Obtaining inducible vesicles MSCs (bone marrow-derived MSCs, BMMSCs) cultured up to the second passage in Example 1 were further cultured in the medium (Dex(+) culture medium) in Example 1 until they reached 80%-90% confluence. After that, they were rinsed twice with PBS, and apoptosis was induced by adding serum-free medium (α-MEM medium) containing 500 nM STS. The cells were then incubated at 37°C for 24 hours, and the cell supernatant was collected and used for isolation and extraction of IEVs.

[0111] IEVs were isolated and extracted from the collected culture supernatant according to the process shown in Figure 2. The specific procedure involved centrifugation at 800 g for 10 minutes, followed by collection of the supernatant, centrifugation at 2000 g for 10 minutes, collection of the supernatant, centrifugation at 16000 g for 30 minutes, removal of the supernatant, resuspension of the IEVs in sterile PBS, and subsequent centrifugation at 16000 g for 30 minutes, removal of the supernatant, and resuspension of the IEVs in 300-500 μL of sterile PBS.

[0112] Comparative Example 1: Isolation and extraction of exosomes derived from allogeneic MSCs MSCs (bone marrow-derived MSCs, BMMSCs) cultured up to the second passage in Example 1 were further cultured in the medium in Example 1 until they became 80%-90% confluent, then rinsed twice with PBS, serum-free medium was added, and the cells were incubated at 37°C for 48 hours. The cell supernatant was collected and used for exosome isolation and extraction.

[0113] The extraction procedure involves centrifugation at 800g for 10 minutes, collecting the supernatant, centrifugation at 2000g for 10 minutes, collecting the supernatant, centrifugation at 16000g for 30 minutes, collecting the supernatant, centrifugation at 120000g for 90 minutes, removing the supernatant, resuspending the pellet in sterile PBS, centrifugation at 120000g for another 90 minutes, removing the supernatant, collecting the exosomes at the bottom, and resuspending them in sterile PBS.

[0114] Example 3: Analysis of IEV 1. Quantification of IEVs and analysis of membrane proteins The IEVs obtained in Example 2 were quantitatively analyzed by flow cytometry. Measurements were made at 1 hour, 4 hours, 8 hours, 16 hours, and 24 hours. 6 MSCs were induced for 1, 4, 8, 16, and 24 hours, and then 0.76 × 10 8 , 1.29×10 8 , 1.95×10 8 , 2.48×10 8 , 3.14 × 10 8 As can be seen, after 24 hours of induction, a single MSC was able to produce 300 IEVs (Figure 3).

[0115] Flow cytometry revealed that the IEV particle size distribution was concentrated within the 1 μm range, accounting for 94.97% (Figure 4A). Side scatter (SSC) analysis also showed that the IEV scattering intensity was concentrated within the 1 μm range (Figure 4B). Furthermore, analysis of the IEV scattering intensity using standardized small-particle microspheres (0.2 μm, 0.5 μm, and 1 μm) from Bangs Laboratories revealed that the IEV particle size was concentrated within the 0.2 μm range (Figure 4C). Transmission electron microscopy (TEM) observations were similar to the flow cytometry results, showing that most vesicles had diameters of 200 nm and below (Figure 4D). Nanoparticle tracking analysis (NTA) results, consistent with the TEM results, showed that the average IEV particle size was 169 nm (Figure 4E). The results of single-vesicle particle size detection using cutting-edge nanoflow cytometry detection technology also showed that the average particle size of IEVs was 100.63 nm (Figure 4F).

[0116] Flow cytometry analysis of the surface membrane proteins of IEVs extracted in Example 2 revealed that MSC-derived IEVs expressed surface proteins similar to those of MSCs, i.e., CD29, CD44, CD73, CD166 positive, CD34, CD45 negative, and the ubiquitous extracellular vesicle surface proteins CD9, CD63, CD81, and C1q (Figures 5A-5K).

[0117] 2. Content analysis of IEV Using protein DIA quantification technology, quantitative proteomic analysis of BMMSCs, MSC-exosomes (extracted in Comparative Example 1), and MSC-IEVs (obtained in Example 2) was completed. As a result, the protein content expression of MSC-exosomes and MSC-IEVs was relatively highly consistent with that of the parent cells, and 170 proteins were specifically and highly expressed in IEVs (Figure 6A).

[0118] Bioinformatics analysis screened for proteins specifically overexpressed in IEVs and generated a heatmap (Figure 6B). Furthermore, combined with GO enrichment analysis of differential proteins, we found that IEVs specifically overexpressed annexin V, flotillin-1, cadherin 11, integrin α5, and syntaxin 4. The expression levels of these five characteristic molecules were significantly upregulated in IEVs compared with exosomes derived from allogeneic MSCs. Specifically, the expression levels of the markers annexin V, flotillin-1, cadherin 11, integrin α5, and syntaxin 4 in IEVs were 1.76-fold, 2.81-fold, 2.41-fold, 3.68-fold, and 4.45-fold higher than those in exosomes, respectively (Figure 6C). Finally, Western blotting further validated the results, which were consistent with the DIA quantitative analysis (Figure 6D).

[0119] MSC-exosomes: exosomes derived from BMMSCs MSC-IEV: IEV derived from BMMSC The MSCs in the content analysis and the MSCs from which exosomes and IEVs were extracted were the same BMMSC cell line.

[0120] Example 4: Use of IEVs derived from MSCs in the treatment of mice with bleeding disorders and mechanistic studies (1) Use of IEVs in the treatment of hemophilic mice In vitro blood coagulation experiments were performed to examine the in vitro blood coagulation promoting effects of IEVs obtained in Example 2 and exosomes extracted in Comparative Example 1. The results are shown in Table 3. IEVs were able to shorten the in vitro clotting time of most plasma samples, demonstrating superior blood coagulation promoting effects compared to exosomes. However, in plasma lacking factors II, V, and X, IEVs were unable to exert their in vitro blood coagulation promoting effects, indicating that the in vitro blood coagulation promoting effects of IEVs are concentrated upstream of the common blood coagulation pathway.

[0121] [Table 3]

[0122] Hemophilia A mice (blood coagulation factor VIII deficiency) were used as a model, and 9 × 10 8 The in vivo blood coagulation promoting effect of IEVs was observed. The results are shown in Figure 7. After treatment with IEVs, the bleeding tendency of hemophilic mice was significantly improved, and the therapeutic effect was stably maintained for 14 days.

[0123] The experimental results show that IEV can exert a significant blood coagulation promoting effect in vitro and significantly improve bleeding tendency after in vivo injection, making it applicable to the improvement of bleeding tendency in hemophilia A.

[0124] The levels of various blood coagulation factors in mouse plasma were simultaneously detected, and none of factor VIII, vWF, tissue factor (TF), and prothrombin were significantly altered (Figures 8A, 8B, 8C, and 8D).

[0125] In a hemophilia A mouse model, normal IEVs, PS-negative IEVs, and TF-negative IEVs were injected, respectively, and tail-cut experiments were performed 7 days later. The results are shown in Figures 9A and 9B. Blockade of PS and TF did not affect the in vivo therapeutic effect of IEVs, preliminarily demonstrating that the mechanism by which IEVs treat hemophilia mice is independent of PS and TF. Previous literature has shown that the procoagulant effect of extracellular vesicles is highly dependent on PS and TF on their surface. However, the in vivo results of IEV experiments are inconsistent with previous studies, suggesting that IEVs may exert their procoagulant effect through a novel mechanism in vivo.

[0126] (2) Use of IEVs in the treatment of lupus hemorrhagic mice Clinically, patients with systemic lupus erythematosus (SLE) often have a tendency to bleed, but the specific mechanism is currently unclear, and previous literature suggests that this is related to factors such as thrombocytopenia associated with SLE. Blood or platelet transfusions are often used as a treatment, but the effect is insufficient.

[0127] In this study, lpr mice were injected with IEV (extracted in Example 2) and subjected to a tail-cut experiment 7 days later. As a result, the bleeding tendency of lpr mice was significantly improved after treatment with IEV, and the therapeutic effect was stably maintained for 7 days (Figure 9C). Here, lpr mice are a representative animal model of SLE. As can be seen from the experimental results, IEV is applicable to the improvement of bleeding tendency associated with lupus erythematosus.

[0128] (3) Use of IEVs in the treatment of mice with Chediak-Higashi syndrome Chediak-Higashi syndrome (CHS syndrome) is an autosomal recessive disorder commonly found in offspring of consanguineous couples. The causative gene is the lysosomal transport regulator (LYST) gene. Mutations in the LYST gene often result in the production of abnormal LYST protein, which in turn causes platelet dysfunction. Patients present with a clinically significant bleeding tendency, and to date, no effective preventive or therapeutic measures have been available.

[0129] In this study, CHS mice were injected with IEV (obtained in Example 2) and subjected to a tail-cut experiment 10 days later. As a result, the bleeding tendency of CHS mice was significantly improved after treatment with IEV, and the therapeutic effect was stably maintained for 10 days (Figure 9D).

[0130] As can be seen from the experimental results, IEV is applicable to ameliorating the bleeding tendency of Chediak-Higashi syndrome mice.

[0131] Comparative Example 2 A mouse model of hemophilia A was treated with injections of IEVs (obtained in Example 2) and exosomes (extracted in Comparative Example 1) derived from allogeneic MSCs (9 × 10 8 As a result, IEVs were able to improve the bleeding tendency of mice, whereas exosomes had no significant therapeutic effect (Figure 10).

[0132] Comparison of the in vitro blood coagulation promoting effects of IEVs obtained in Example 2 and exosomes prepared in Comparative Example 1 The IEVs obtained in Example 2 had diameters within the ranges of 0.03 μm to 0.2 μm and 0.2 μm to 1 μm, expressed the markers syntaxin 4, annexin V, flotillin-1, cadherin 11, and integrin α5, and had a high in vitro blood coagulation promoting effect, whereas the exosomes prepared in Comparative Example 1 had diameters within the ranges of 0.03 μm to 0.15 μm, expressed the markers complement C1q, complement C3, thrombospondin-1, and thrombospondin-2, and had a relatively low in vitro blood coagulation promoting effect.

[0133] [Table 4]

[0134] Example 5 1. Cell culture of induced pluripotent stem cells (iPS cells) (1) Preparation of lentivirus 1 mL of DMEM was transferred to an EP tube, 5 μg of gene expression plasmid and 5 μg of vsvg plasmid were added, and 25 μL of liposomes were added and gently mixed at room temperature for 20 minutes. The mixture was added dropwise to the cultured GP2-293 cells (95% homogeneous mixture) and swirled to ensure uniform distribution. After 12 hours, the medium was replaced with DMEM + 10% heat-inactivated FBS + glutamine. 24 hours after the medium change, the medium containing the virus was collected, and another medium was collected 48 hours later.

[0135] (2) Induction of cell reprogramming Add 5 x 10 GP2-293 cells cultured in step (1) to each well (12-well plate). 5 After reaching 80% confluence, 100 ng of virus was added to 500-1000 μL / well of medium (DMEM + 10% FBS (heat inactivated) + glutamine), 4 μg / mL of polybrene was added, and the medium was then replaced with fresh medium. The above steps were repeated. Within 7 days, 5 × 10 4 The induced cells were seeded onto a 10 cm culture dish containing feeder cells (mEF). The next day, the medium was replaced with Es medium containing bFGF (4 ng / ml) and was changed every other day. After 5 days, the cells initiated cloning. If no Es-like clones were found after 40 days, the culture was considered to have failed.

[0136] (3) Cell passage After reaching 60% confluence, 0.5 ml of accutase was added to each dish and allowed to stand for 1 minute at room temperature. The dissociated cell aggregates were transferred to a 15 ml centrifuge tube, and any remaining aggregates were collected using an additional 2 ml of mTeSR1. A wash solution was added to the 15 ml tube. The 15 ml tube containing the cell aggregates was centrifuged at 200 g for 5 minutes at room temperature. The supernatant was then aspirated. The cells were resuspended, ensuring that they remained in an aggregated state. Human iPS cells and mTeSR1 were aggregated onto a new Matrigel-coated plate. The culture dish was placed in a 37°C incubator and moved quickly from side to side to ensure uniform distribution of the cell aggregates. Culture conditions were 37°C, 5% carbon dioxide, and 95% humidity. The culture medium was changed daily.

[0137] 2. Culture of MC3T3-E1 subclone 14 osteoblastic cell line Purchased MC3T3-E1 subclone 14 was rapidly thawed and centrifuged at 500 g for 5 minutes. The supernatant was removed, and the cell pellet at the bottom was collected. The cells were resuspended in Dex(-) medium and seeded into a 10 cm cell culture dish. Cultures were then grown at 37°C and 5% CO2. After the dish was filled, the cells were digested with trypsin and incubated at 37°C for subcultivation. The Dex(-) medium was then replaced every 3 days. The cells can be passaged multiple times. The components of the Dex(-) medium are listed in Table 5.

[0138] [Table 5]

[0139] 3. Analysis of IEV This example compares IEVs derived from osteoblasts MC3T3-E1, iPS cells, and human bone marrow mesenchymal stem cells (hBMMSCs). The methods for obtaining IEVs derived from these three types of cells were the same as in Example 2.

[0140] (1) Morphological detection As a result of the experiment, as shown in Figure 11, under a 400x optical microscope, the IEVs derived from iPS cells (iPSCs) and osteoblastic cells MC3T3-E1 were morphologically similar to those derived from human BMMSCs and were relatively irregular.

[0141] (2) Diameter detection of IEV particles The results were detected by flow cytometry. As shown in Figure 12, the particle size distribution of IEVs derived from the osteoblast cell line MC3T3-E1 and human bone marrow mesenchymal stem cells (hBMMSC) was similar.

[0142] (3) Detection of IEV surface markers The surface markers of IEVs derived from iPS cells (iPSCs) and human bone marrow mesenchymal stem cells (hBMMSCs) were detected by Western blotting. As shown in Figure 13, IEVs derived from both iPSCs and hBMMSCs highly expressed the IEV marker annexin V. Compared to hBMMSCs, IEVs derived from iPS cells expressed syntaxin 4 at relatively high levels.

[0143] Example 6: IEV can be excreted via skin and hair. 4 x 10 IEVs prepared in Example 2 6 The IEVs were isolated, labeled with DIR, resuspended in 200 μL of PBS, and injected systemically into nude BALB / c-nu / nu mice via the tail vein. After 1, 3, and 7 days of observation, the distribution of IEVs on the skin surface was detected using a bioimaging system. The results are shown in Figures 13A-13C.

[0144] As can be seen in Figure 13A, IEVs were able to reach the skin surface, reaching their highest numbers on day 3 and nearly disappearing by day 7, demonstrating the dynamic metabolic process of IEVs at the skin surface (Figure 13A). Immunofluorescence results showed that after systemic injection of PKH26-IEVs into C57 mice, they gradually migrated from the subcutaneous tissue to the dermis and epidermis over time. On day 7, a large amount of IEVs was observed in the stratum corneum of the skin surface, suggesting that systemically injected IEVs were excreted with the shedding of the stratum corneum (Figure 13B). Furthermore, on day 7, PKH26-IEVs were observed in hair follicles plucked from the mouse body surface, indicating that systemically injected IEVs were metabolized as hair shed (Figure 13C). This study demonstrates that IEVs are excreted by skin and hair, demonstrating the safety of IEV injection or in vivo content increase.

[0145] Example 7 The hiPSCs were cultured in the same manner as in Example 5, and the hUCMSCs were also cultured by a method commonly used in the art. The hiPSCs may be, but are not limited to, 26th to 29th passages, and the hiPSCs specifically used in this example were 26th passage.The hUCMSCs may be, but are not limited to, 7th passages, and the hiPSCs specifically used in this example were 7th passage.

[0146] 1. Experimental method (1) hiPSCs and hUCMSCs were induced to undergo apoptosis with staurosporine (500 nM) for approximately 9 h (other steps were the same as in Example 2), and then stained with Annexin V (15 min) and 7AAD (3 min). The cell apoptosis rate was detected by flow cytometry. (2) IEVs were isolated from the supernatant of apoptotic cells in step (1), and the expression rate of annexin V was detected by flow cytometry. IEVs were extracted by differential centrifugation. The procedure included centrifugation at 800 g for 10 min, centrifugation at 2000 g for 5 min (other extraction steps were the same as in Example 2), centrifugation at 16000 g for 30 min, and centrifugation at 16000 g for 30 min to obtain IEVs. The cells were stained with Annexin V for 15 minutes and then subjected to flow cytometry.

[0147] 2. Experimental results (1) We used high-content imaging to capture the death processes of hiPSCs and hUCMSCs and found differences in their death processes. hiPSCs exhibited multicentric contraction centered on the nucleus and cytoplasm, followed by dendritic branching accompanied by bubble formation. Meanwhile, hUCMSCs exhibited unicentric contraction centered on the nucleus, accompanied by branching and bubble formation. The results are shown in Figure 14. (2) The apoptosis rate of hiPSCs and hUCMSCs that induce apoptosis was analyzed by flow cytometry, demonstrating that most cells underwent apoptosis. The results are shown in Figure 15. (3) The positive rate of Annexin 5 expression was detected by flow cytometry, and the expression rate was found to be over 80% in both hiPSCs and hUCMSCs. The results are shown in Figure 16. (4) Two types of IEVs were characterized by nanoparticle tracking analysis (NTA). 1) As a result, as shown in Figure 17, the particle size of IEVs derived from hiPSCs was approximately 100 nm, and the particle size of IEVs derived from hUCMSCs was approximately 180 nm. 2) As a result, as shown in Figure 18, the yield of IEVs derived from hiPSCs was 21,971 particles / hiPSC, and the yield of IEVs derived from hUCMSCs was 886 particles / hUCMSC. 3) As a result, as shown in Figure 19, the potential of IEVs derived from hiPSCs was approximately -12 mV, and the potential of IEVs derived from hUCMSCs was approximately -45 mV.

Claims

1. 1. A method for collecting data to detect whether an induced vesicle derived from a cell that is a stem cell or a somatic cell is present in a sample, comprising: detecting the expression level of syntaxin 4 in the sample.

2. The method of claim 1 , wherein the cells are mesenchymal stem cells.

3. The method of claim 1 , wherein the cells are induced pluripotent stem cells, osteoblastic cells, or bone marrow-derived mesenchymal stem cells.

4. If the expression level of syntaxin 4 in the sample is higher than the expression level of syntaxin 4 in exosomes, mitochondria, microvesicles, or ectosomes derived from the cells, 4. The method of claim 1, further comprising collecting data indicating the presence of the induced vesicles in the sample.

5. If the expression level of syntaxin 4 in the sample is 3-6 times higher than the expression level of syntaxin 4 in the exosomes, the migratory bodies, the microvesicles, or the ectosomes derived from the cells, The method of claim 4, further comprising providing data indicating the presence of the induced vesicles in the sample.

6. The method according to any one of claims 1 to 3, further comprising detecting the expression level of annexin V, flotillin-1, cadherin 11, or integrin α5 in the sample.

7. detecting the expression level of annexin V in the sample, and determining whether the expression level of annexin V in the sample is higher than the expression level of annexin V in exosomes, migratory bodies, microvesicles, or ectosomes derived from the cells; The expression level of flotillin-1 in the sample is detected, and the expression level of flotillin-1 in the sample is higher than the expression level of flotillin-1 in the exosomes, the migratory bodies, the microvesicles, or the ectosomes derived from the cells; detecting the expression level of cadherin-11 in the sample, and the expression level of cadherin-11 in the sample is higher than the expression level of cadherin-11 in the exosomes, the migratory bodies, the microvesicles, or the ectosomes derived from the cells; or detecting the expression level of integrin α5 in the sample, and determining whether the expression level of integrin α5 in the sample is higher than the expression level of integrin α5 in the exosomes, the migratory bodies, the microvesicles, or the ectosomes derived from the cells; and, If the expression level of syntaxin 4 in the sample is higher than the expression level of syntaxin 4 in the exosomes, the migratory bodies, the microvesicles, or the ectosomes derived from the cells, The method of claim 6, further comprising providing data indicating the presence of the induced vesicles in the sample.

8. detecting the expression level of annexin V in the sample, and the expression level of annexin V in the sample is 1-2 times higher than the expression level of annexin V in the exosomes, the migratory bodies, the microvesicles, or the ectosomes derived from the cells; The expression level of flotillin-1 in the sample is detected, and the expression level of flotillin-1 in the sample is 2-3 times higher than the expression level of flotillin-1 in the exosomes, the migratory bodies, the microvesicles, or the ectosomes derived from the cells; detecting the expression level of cadherin-11 in the sample, and determining that the expression level of cadherin-11 in the sample is 1-3 times higher than the expression level of cadherin-11 in the exosomes, migratory bodies, microvesicles, or ectosomes derived from the cells; or detecting the expression level of integrin α5 in the sample, wherein the expression level of integrin α5 in the sample is 3-4 times higher than the expression level of integrin α5 in the exosomes, the migratory bodies, the microvesicles, or the ectosomes derived from the cells; and, If the expression level of syntaxin 4 in the sample is 3-6 times higher than the expression level of syntaxin 4 in the exosomes, the mobile bodies, the microvesicles, or the ectosomes, The method of claim 7, further comprising providing data indicating the presence of the induced vesicles in the sample.

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