Late endothelial progenitor cells and their application in the treatment of stroke

Abstract

The present invention belongs to the field of biomedicine, discloses late endothelial progenitor cells and their application in the treatment of stroke, and also discloses biomarkers or methods for identifying or detecting early / late endothelial cells.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to late endothelial progenitor cells and their application in the treatment of cerebral stroke. Background Art

[0002] Stroke, also known as apoplexy, is a cerebrovascular dysfunction disease caused by different vascular diseases, which is often accompanied by varying degrees of motor dysfunction and sensory dysfunction in clinical practice. Stroke is a major chronic non-communicable disease that seriously endangers the health of Chinese people. It has five major characteristics: high morbidity, high disability rate, high mortality rate, high recurrence rate, and heavy economic burden.

[0003] Currently, the treatment options for ischemic stroke are very limited. The most effective treatment in the acute phase is thrombolytic therapy, but due to the time window, only a few patients can benefit from it, and most surviving patients have residual functional impairments to varying degrees. Other conventional methods include drug therapy, such as antiplatelet aggregation, statins to lower blood lipids, control blood pressure and blood sugar, and surgical treatment, such as intracranial and extracranial endovascular stent implantation, carotid endarterectomy, etc., which may lead to various complications and side effects, such as anticoagulant therapy may increase the risk of bleeding, and surgical treatment may cause infection. Therefore, new intervention methods are urgently needed in the clinic to treat ischemic stroke.

[0004] Endothelial progenitor cells (EPCs) derived from iPSCs have shown great application potential in regenerative medicine and cell therapy, and have become an important tool for the treatment of cardiovascular diseases and other vascular-related diseases. However, the current therapeutic effect of EPCs for cardiovascular diseases is still limited and requires further research and optimization. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the inventors of the present invention have discovered for the first time, through extensive and in-depth research, a biomarker for distinguishing early EPC from late EPC, and further discovered that late EPC, rather than early EPC, has the effect of treating stroke. Based on this, the present application provides the following technical solutions:

[0006] A first aspect of the present invention provides a biomarker for identifying or detecting early / late EPCs, wherein the biomarker comprises CD144, vWF or CD133.

[0007] The second aspect of the present invention provides a reagent or kit for identifying, enriching or isolating early / late EPCs, wherein the reagent or kit comprises a binding agent capable of binding to the biomarker described in the first aspect of the present invention.

[0008] The third aspect of the present invention provides a late EPC, wherein the late EPC has at least one of the following expression profiles: CD144 高 / + 、vWF 高 / + 、CD 133 低 / - .

[0009] A fourth aspect of the present invention provides an early EPC, wherein the early EPC has at least one of the following expression profiles: CD133 高 / + 、CD144 低 / - 、vWF 低 / - .

[0010] The fifth aspect of the present invention provides a method for identifying, enriching or isolating early / late EPCs, wherein the expression of the biomarkers described in the first aspect of the present invention in EPC cells is determined, and when it is determined that the EPC cells have at least one of the following expression profiles, the EPCs are late EPCs: CD144 高 / + 、vWF 高 / + 、CD 133 低 / - When it is determined that the cell has at least one of the following expression profiles, the EPC cell is an early EPC: CD133 高 / + 、CD144 低 / - 、vWF 低 / - .

[0011] The sixth aspect of the present invention provides a device or apparatus for identifying, enriching or separating early / late EPCs, the device or apparatus comprising a cell binding unit, wherein the cell binding unit is configured to have a structure capable of binding to the biomarker described in the first aspect of the present invention.

[0012] The seventh aspect of the present invention provides the use of the biomarker described in the first aspect of the present invention, the reagent or kit described in the second aspect of the present invention, and the device or equipment described in the sixth aspect of the present invention in identifying, enriching or separating early / late EPCs.

[0013] The eighth aspect of the present invention provides the use of the biomarker described in the first aspect of the present invention or the reagent or kit described in the second aspect of the present invention in the preparation of a product for diagnosing a disease associated with early EPC or late EPC.

[0014] The ninth aspect of the present invention provides use of the late EPC described in the third aspect of the present invention in the preparation of a drug for treating stroke.

[0015] The tenth aspect of the present invention provides a pharmaceutical composition for treating stroke, wherein the pharmaceutical composition comprises the advanced EPC described in the third aspect of the present invention.

[0016] The eleventh aspect of the present invention provides a method for treating stroke, comprising administering an effective amount of advanced EPC or the pharmaceutical composition according to the tenth aspect of the present invention.

[0017] Advantages and beneficial effects of the present invention:

[0018] The present invention is the first to discover biomarkers that distinguish early and late EPCs, and based on the biomarkers, different EPC subpopulations can be identified, isolated and enriched.

[0019] The present invention discovers for the first time that late EPCs, rather than early EPCs, can be used to treat stroke. Based on this, the therapeutic effect of endothelial progenitor cells in treating stroke can be improved by increasing the content of late EPCs, an effective component for treating stroke. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the result of early EPC cell surface marker gene detection;

[0021] Figure 2 This is the result of late EPC cell surface marker gene detection;

[0022] Figure 3 This is a graph showing the results of early EPC surface marker gene detection by flow cytometry;

[0023] Figure 4 This is the result of flow cytometry detection of late EPC surface marker gene;

[0024] Figure 5 This is a graph showing the results of the phagocytic ability test;

[0025] Figure 6 This is the result of TTC staining to determine the scope of cerebral infarction;

[0026] Figure 7 This is a graph showing the results of cerebral infarction area detection.

[0027] Specific implementation method

[0028] After extensive and in-depth research, the present invention discovered markers that can be used to distinguish early and late EPCs by studying different EPC subgroups, and further proved that late EPCs, rather than early EPCs, have the effect of treating stroke.

[0029] In the present invention, the use of alternatives (such as "or") should be understood to refer to any one, all or any combination of the alternatives. The terms "include", "have" and "include" are used synonymously in the present invention, and these terms and their variations are intended to be interpreted as non-restrictive.

[0030] For purposes of the present application, the term "selection", "sorting", "division" or "separation" of selected cells, cell mass or cell subpopulation can be used interchangeably, and unless context indicates otherwise, represents that selected cells or defined cell subsets are removed from tissue samples and separated from other cells and pollutants that are not within the parameters defining this cell or cell mass. The EPC subpopulation separated will usually not be contaminated by other cell types and have the ability to allow the self-renewal of differentiation progeny. However, when the process produces cell mass, it should be understood that it is impractical to provide a composition with absolute purity. In such a case, by cell mass for the cell "enrichment" selected, wherein the cells of these selections then exist in the presence of various pollutants (comprising other cell types) of the function or characteristic of the selected cell subpopulation without substantial interference.

[0031] The terms "marker", "label" or "cell marker" have the same meaning and represent any property or characteristic in the form of a chemical or biological entity. Labels can be morphological, functional or biochemical in nature. In an embodiment of the present application, a label is a cytokine or surface antigen or membrane protein or cytoplasmic protein expressed by a cell under certain conditions (e.g., at a specific time point in the cell cycle or under a specific extracellular matrix) or expressed by a cell under certain conditions. More specifically, in the present application, it refers to labels that can indicate cells or cell subpopulations by means of their expression levels.

[0032] In this application, "marker" refers to any part that can be used to identify the desired EPC. For example, the marker can be a polypeptide molecule expressed on the cell of interest, such as a "surface marker". A specific marker can be present only in the cell of interest, or encompass the cell of interest, or the detectable level of the marker in the cell of interest is high enough compared to other cells, so that the cell of interest can be identified using any of a variety of methods known in the art. Those skilled in the art will understand that expression is a relative term, and other cell types have different expressions.

[0033] One aspect of the present invention provides a biomarker for identifying or detecting early / late EPCs, wherein the biomarker comprises CD144, vWF or CD133.

[0034] In some embodiments, the CD144 and vWF are highly expressed in late EPCs, and CD133 is not expressed or is lowly expressed in late endothelial progenitor cells.

[0035] In some embodiments, the CD144 and vWF are not expressed or are lowly expressed in early EPCs.

[0036] In some embodiments, the biomarkers further include CD31, CD34 and / or KDR.

[0037] In some embodiments, the EPCs are derived from iPSCs.

[0038] Another aspect of the present invention provides a reagent or kit for identifying, enriching or isolating early / late EPCs, wherein the reagent or kit comprises a binding agent capable of binding to the biomarker described in the first aspect of the present invention.

[0039] The terms "binding agent", "binding molecule" and "binding entity" are synonymous and can be used interchangeably. In the context of the present application, a binding agent binds to, recognizes, interacts with, reacts with or otherwise associates with a selectable marker on the cell subpopulation. Exemplary binding agents may include, but are not limited to, antibodies or fragments thereof, antigens, aptamers, nucleic acids (e.g., DNA and RNA), proteins (e.g., receptors, enzymes, enzyme inhibitors, enzyme substrates, ligands), peptides, lectins, fatty acids or lipids, and polysaccharides.

[0040] In some embodiments, the binding agent is selected from a nucleic acid, a ligand, an enzyme, a substrate, and / or an antibody.

[0041] In some embodiments, the binding agent is selected from probes that hybridize to the aforementioned biomarkers.

[0042] In some embodiments, the binding agents are selected from primers that bind to the aforementioned biomarkers.

[0043] In some embodiments, the binding agent is selected from an antibody that binds to a protein or protein fragment encoded by the biomarker.

[0044] In the present application, "antibody" is used in the broadest sense, and specifically covers, for example, monoclonal antibodies, polyclonal antibodies, antibodies with multi-epitope specificity, single-chain antibodies, multispecific antibodies and antibody fragments. Such antibodies can be chimeric, humanized, human and synthetic. In some embodiments, antibodies include single domain antibodies, further engineered molecules (such as, but not limited to double antibodies, three antibodies, four antibodies, mini antibodies, etc.), Fab fragments, Fab' fragments, F(ab')2 fragments, Fd fragments, Fv fragments, single-chain Fv (scFv) molecules, scFv dimers, BsFv molecules, dsFv molecules, (dsFv)2 molecules, dsFv-dsFv' molecules, Fv fragments, dAb fragments, bispecific antibodies, ds double antibodies, nanobodies, domain antibodies, bivalent domain antibodies, and the minimum recognition unit composed of amino acid residues that simulate the hypervariable region of antibodies (e.g., isolated complementary determining regions (CDRs)).

[0045] In some embodiments, the binding agent is linked to a reporter molecule.

[0046] In some embodiments, the reporter molecule is selected from a fluorescent substance, a radioactive substance and / or an enzyme.

[0047] In the present application, actual monitoring or analysis of biomarkers can be performed using any of a number of standard techniques well known to those skilled in the art. For example, cell surface marker expression can be analyzed by immunoassays, including but not limited to Western blotting, immunohistochemistry, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, immunofluorescence, protein A immunoassays, laser capture microdissection, massively multiparametric mass cytometry, flow cytometry, and FACS analysis.

[0048] In the present application, the kit can be used for patient diagnostic purposes in a clinical setting or for the characterization and / or enrichment of EPC subpopulations in research. The kit according to the present invention will include one or more containers, the container comprising a binding agent and a label or package insert on or in combination with the container. Suitable containers include, for example, bottles, vials, syringes, 96-well plates, and the like. The container can be formed from a variety of materials such as glass or plastic. This container holds one or more compositions, which contain binding agents that are effective for analyzing EPCs and optionally provide cells or cell subpopulations enriched or separated as described herein. Such a kit will generally contain a preparation of one or more binding agents in a suitable container, and in the case of a variety of binding agents, the binding agent can be in the same or different containers. The kit may also contain other pharmaceutically acceptable preparations for diagnosis or for labeling or modifying a sealed binding agent.

[0049] More specifically, these kits can have a single container containing one or more binding agents, with or without additional components, or they can have different containers for each component. In the case of providing a combination reporter for binding, separate solutions can be premixed in molar equivalents or in the case where one component exceeds the remaining components. Alternatively, the binding agent of the kit and any optional reporter molecules can be maintained separately in different containers before being applied to a subject or used in vitro. The kit can also include a second / third container device for containing sterile, pharmaceutically acceptable buffer or other diluents (such as antibacterial water for injection (BWFI), phosphate buffered saline (PBS), Ringer solution and glucose solution).

[0050] When the components of the test kit are provided in one or more liquid solutions, the liquid solution is an aqueous solution in some embodiments, particularly preferably a sterile aqueous solution. However, the components of the test kit can be provided as dry powders. When reagents or components are provided as dry powders, the powder can be reconstituted by adding a suitable solvent. It is contemplated that the solvent can also be provided in another container.

[0051] In another aspect, the present invention provides late EPCs, wherein the late EPCs have at least one of the following expression profiles: CD144 高 / + 、vWF 高 / + 、CD 133 低 / - .

[0052] In some embodiments, the late EPCs have the following expression profile: CD144 高 / + 、vWF 高 / + and CD 133 低 / - .

[0053] In some embodiments, the late EPCs have at least one of the following expression profiles: CD31 高 / + 、CD34 高 / + and / or KDR 高 / + .

[0054] In some embodiments, the late EPCs have the following expression profile: CD144 高 / + 、vWF 高 / + 、CD 133 低 / - 、CD31 高 / + 、CD34 高 / + and KDR 高 / + .

[0055] In some embodiments, the EPCs are derived from iPSCs.

[0056] Another aspect of the present invention provides an early EPC having at least one of the following expression profiles: CD133 高 / + 、CD144 低 / - 、vWF 低 / - .

[0057] In some embodiments, the early EPCs have the following expression profile: CD133 高 / + 、CD144 低 / - and vWF 低 / - .

[0058] In some embodiments, the early EPCs have at least one of the following expression profiles: CD31 高 / + 、CD34 高 / + and / or KDR高 / + .

[0059] In some embodiments, the early EPCs have the following expression profile: CD133 高 / + 、CD144 低 / - 、vWF 低 / - 、CD31 高 / + 、CD34 高 / + and KDR 高 / + .

[0060] In some embodiments, the EPCs are derived from iPSCs.

[0061] Another aspect of the present invention provides a method for identifying, enriching or isolating early / late EPCs, wherein the expression of the biomarkers described above in the present invention in EPC cells is determined, and when it is determined that the EPC cells have at least one of the following expression profiles, the EPCs are late EPCs: CD144 高 / + 、vWF 高 / + 、CD 133 低 / - When it is determined that the cell has at least one of the following expression profiles, the EPC cell is an early EPC: CD133 高 / + 、CD144 低 / - 、vWF 低 / - .

[0062] The term "enriched" or variations thereof are used herein to describe a cell population in which the proportion or percentage of cells of a particular cell type or the proportion or percentage of multiple particular cell types is increased in the cell population when compared to a population of untreated cells (e.g., cells in their natural environment).

[0063] In one example, the term "enriched" refers to a proportion or percentage of EPCs in a subpopulation of cells that is greater than the proportion or percentage of EPCs in the cell population that originally comprised it.

[0064] In some embodiments, the identification, enrichment or isolation methods comprise contacting a cell with a binding agent for a biomarker and directly or indirectly detecting the presence of the formed complex.

[0065] In some embodiments, the method further comprises sorting the EPCs bound to the binding agent. Methods for separating and selecting cells based on the expression of cell surface markers are familiar to those skilled in the art. Including but not limited to affinity-based interactions, affinity panning, magnetic beads (e.g., Dynabeads), or flow cytometry.

[0066] In some embodiments, the sorting or separation technique comprises fluorescence activated cell sorting, magnetic assisted cell sorting, substrate assisted cell sorting, laser mediated cutting, fluorimetry, flow cytometry, or microscopy.

[0067] In some embodiments, cells can be isolated by binding to an immobilized support and then harvested by removing the cells from the support.

[0068] Harvesting of cells can be achieved by collecting the separated cells into a suitable container or collection dish, test tube, or the like.

[0069] In one aspect, the present invention provides a device or apparatus for identifying, enriching or separating early / late EPCs, the device or apparatus comprising a cell binding unit, wherein the cell binding unit is configured to be able to bind to the biomarker described in the first aspect of the present invention.

[0070] In one aspect, the present invention provides the use of the aforementioned biomarkers in identifying, enriching or isolating early / late EPCs.

[0071] Another aspect of the present invention provides the use of the aforementioned reagent or kit for identifying, enriching or separating early / late EPCs in identifying, enriching or separating early / late EPCs.

[0072] In another aspect, the present invention provides the use of the aforementioned reagent or kit for identifying, enriching or separating early / late EPCs in identifying, enriching or separating early / late EPCs.

[0073] Another aspect of the present invention provides the use of the aforementioned biomarkers in the preparation of products for diagnosing diseases associated with early EPC or late EPC.

[0074] In one aspect, the present invention also provides the use of the above-mentioned reagent or kit for enriching or separating early / late EPCs in the preparation of products for diagnosing diseases associated with early EPCs or late EPCs.

[0075] One aspect of the present invention provides the use of the aforementioned late EPC in the preparation of a drug for treating stroke.

[0076] In some embodiments, stroke includes ischemic stroke and hemorrhagic stroke. Ischemic stroke includes transient ischemic attack, atherosclerotic thrombotic cerebral infarction, lacunar cerebral infarction, and cerebral embolism. Hemorrhagic stroke includes cerebral hemorrhage and subarachnoid hemorrhage.

[0077] In one aspect, the present invention provides a pharmaceutical composition for treating cerebral stroke, wherein the pharmaceutical composition comprises the advanced EPC described above in the present invention.

[0078] In some embodiments, late EPCs are obtained by the methods of the examples of the present application.

[0079] In some embodiments, late EPCs are obtained by the isolation or enrichment methods described above.

[0080] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0081] "Pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not usually cause gastrointestinal disorders, allergic reactions such as dizziness or similar reactions when administered to a subject. In addition, the composition comprising a pharmaceutically acceptable carrier can be a variety of dosage forms for oral or parenteral administration. Commonly used carriers such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and other diluents or excipients can be used to prepare during formulation. The above-mentioned carrier can be selected from one or more of the group consisting of lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, saline, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, dextrin, calcium carbonate, propylene glycol and liquid paraffin, but is not limited thereto, and all common carriers can be used. The above-mentioned components can be added alone or in combination to the mixture of late EPC as the above-mentioned active ingredient.

[0082] In some embodiments, solid dosage forms for oral administration may include tablets, pills, powders, granules, capsules, etc. Such solid preparations can be prepared by mixing one or more excipients into one or more mixtures, for example, mixed starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition, lubricants such as magnesium stearate and talcum powder can also be used in addition to simple excipients. Liquid preparations for oral administration include suspensions, internal solutions, emulsions, syrups, etc., and in addition to water and liquid paraffin, which are widely used as simple diluents, a variety of excipients such as wetting agents, sweeteners, aromatics, preservatives, etc. can also be included.

[0083] In some embodiments, preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, oils, freeze-dried preparations, suppositories, etc. Non-aqueous solvents and suspending agents can use propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable lipids such as ethyl oleate, etc. The base of the suppository can use witepsol, polyethylene glycol, Tween 61, cocoa butter, lauric acid ester, glycerin, gelatin, etc.

[0084] In some embodiments, the pharmaceutical composition of the present invention can have any dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, suspensions, oils, lyophilized agents and suppositories. The base of the suppository can use witepsol, polyethylene glycol, Tween 61, cocoa butter, lauric acid esters, glycerin, gelatin, etc.

[0085] In some embodiments, the pharmaceutical composition further comprises other drugs for treating stroke. Other drugs for treating stroke include, but are not limited to, thrombolytic drugs such as alteplase, urokinase, streptokinase, recombinant streptokinase, lumbrokinase, thrombinase, etc., neuroprotective drugs such as citicoline, brain protein extract, calf serum deproteinized extract, ganglioside, edaravone, etc., calcium channel blockers such as nimodipine, cinnarizine, flunarizine, etc., and brain metabolism activators such as piracetam, oxiracetam, aniracetam, etc.

[0086] In one aspect, the present invention provides a method for treating cerebral stroke, comprising administering to a subject an effective amount of the advanced EPC as described above or the pharmaceutical composition as described above.

[0087] A "therapeutically effective amount" will be the amount of an agent that can prevent or at least slow down (mitigate) a medical condition such as a stroke. The dosage and administration of the disclosed cell or pharmaceutical composition can be determined by a person of ordinary skill in the field of clinical pharmacology or pharmacokinetics. The effective amount of the present application's active agent used in treatment will depend on, for example, the treatment goal, the route of administration, and the patient's condition. Accordingly, the therapist may need to adjust the dosage and change the route of administration as needed to obtain the best therapeutic effect.

[0088] The terms "subject" or "patient" can be used interchangeably. The term includes, but is not limited to, humans, non-human animals, for example, non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic subjects such as dogs and cats; laboratory animals, including rodents such as mice, rats and guinea pigs, etc. The term does not indicate a specific age or gender. Therefore, it is expected to cover adult and newborn subjects and fetuses, whether male or female. The term "subject" also includes living organisms susceptible to disorders or disease states as generally disclosed throughout this specification (but not limited thereto). Examples of subjects include humans, dogs, cats, cattle, goats and mice, including transgenic species.

[0089] The terms "non-human animals" and "non-human mammals" are used interchangeably herein and include all vertebrates, e.g., mammals, such as non-human primates (especially higher primates), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, cows, and non-mammals such as chickens, amphibians, reptiles, etc. In one embodiment, the subject is a human. In another embodiment, the subject is an experimental animal or animal surrogate as a disease model, such as a dog.

[0090] The present invention will be further described in detail below in conjunction with the accompanying drawings and examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the examples where specific conditions are not specified are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0091] Example 1 Directed differentiation and identification of early EPC

[0092] Preparation of culture medium

[0093] Preparation of E8 complete medium: Prepare E8 complete medium according to Table 1 and store at 4°C for up to 2 weeks.

[0094] Table 1E8 Complete medium preparation table

[0095] Reagents Manufacturer Part Number Final concentration / volume E8 Basal Medium Stemcell 05991 450mL E8 25×Supplement Stemcell 05992 20mL Penicillin-Streptomycin Invitrogen 15140122 1％

[0096] Preparation of differentiation basal medium: Prepare differentiation basal medium according to Table 2 and store at 4°C for up to 1 month.

[0097] Table 2 Preparation of differentiation basal medium

[0098]

[0099] 1. iPSCs recovery and passaging

[0100] 1.1 Matrigel coating

[0101] (1) In an ice box, open the 1.5 mL EP tube containing matrigel with the help of hemostats.

[0102] (2) Pipette 100 μL of pre-cooled coating solution into the EP tube containing matrigel, pipette and vortex repeatedly, and then pipette the supernatant back into the centrifuge tube. This process needs to be repeated several times, and the matrigel should be dissolved as quickly as possible. Do not rewarm the tube. The dilution of matrigel is 1:400.

[0103] (3) Take out the pre-cooled objects to be coated and place them on an ice box. Mix the coating solution with an electric pipette and use a suitable measuring vessel to coat the following volumes: T75: 12 mL per bottle; T25: 4 mL per bottle; 6-well plate: 2 mL per well; 12-well plate: 1 mL per well; 24-well plate: 0.5 mL per well; 48-well plate: 0.25 mL per well.

[0104] (4) Place the coated culture flask / plate in a 37°C, 5% CO2 incubator and incubate overnight.

[0105] 1.2 iPSCs recovery

[0106] (1) Prepare E8 complete medium containing 10 μM Y-27632 according to the amount of medium required for recovery. Add 1 μL Y-27632 (10 mM) stock solution per mL of medium and preheat at room temperature.

[0107] (2) Prepare 10-15 mL of E8 medium and preheat it in a 37°C water bath.

[0108] (3) Take out a cryovial from the liquid nitrogen tank and immediately place it in a 37°C water bath. Shake it back and forth for 60-90 seconds to completely melt the frozen cell suspension.

[0109] (4) Once the cells in the cryotube are completely thawed (liquid), immediately remove them from the hot water bucket, thoroughly disinfect the surface of the cryotube with 75% alcohol, and place them in the clean bench. When the cryotube is about to thaw, place the centrifuge tube containing E8 culture medium preheated to 37°C in the clean bench after disinfection with alcohol.

[0110] (5) Under strict aseptic operation conditions, remove the cell suspension from the cryopreservation tube and inject it into a 15 mL centrifuge tube with preheated E8 medium. Gently pipette 2 to 3 times and centrifuge at 200 g for 5 min. After centrifugation, discard the supernatant.

[0111] (6) Add E8 medium containing 10 μM Y-27632, gently blow 2 to 3 times, and remove the Matrigel-coated plate / culture bottle from the incubator. Aspirate the coating solution and gently add the cell suspension along the uncoated surface (do not add directly onto the coating layer). Place the culture dish in the incubator for culture.

[0112] Note: When thawing, choose the inoculation container according to the number of cells. Generally, 500,000 to 800,000 cells are inoculated in a 6-well plate, 800,000 to 1,000,000 cells are inoculated in a T25 flask, and 1,000,000 to 2,000,000 cells are inoculated in a T75 flask.

[0113] 1.3 iPSCs digestion and passaging;

[0114] (1) Prepare TrypLE working solution: pipette 5 mL of DPBS into a new 15 mL centrifuge tube, then add 5 mL of TrypLE stock solution and mix well to obtain the working solution.

[0115] (2) Prepare E8 complete medium containing 10 μM Y-27632 according to the amount of culture medium required for subculturing. Add 1 μL of Y-27632 (10 mM) stock solution per mL of culture medium.

[0116] (3) Take out the well plate / culture bottle to be subcultured from the incubator, discard the supernatant, and wash twice with DPBS (the amount of DPBS used each time should not be less than the amount of the original culture medium), each time for 1 min (when washing, leave the DPBS in the well / bottle for 30-45 seconds before aspirating it).

[0117] (4) After adding TrypLE working solution (add about 1 mL of TrypLE working solution to the six-well plate and about 2 mL of TrypLE working solution to the T25 bottle), place the plate in an incubator and incubate for 2-5 min. During this time, the cells can be observed under a microscope and shrink, become round, and disperse.

[0118] (5) Gently tap the culture flask / plate to detach the cells from the bottom of the plate, then gently pipette up and down several times, and finally add DMEM / F12 to terminate digestion.

[0119] (6) After balancing, centrifuge at 200 g for 5 min. After centrifugation, discard the supernatant, gently shake the bottom of the centrifuge tube, add 5 mL of E8 complete medium containing 10 μM Y-27632 to resuspend the cells, inoculate the cell suspension into a T25 flask coated with matrigel, and place the culture flask in a 37°C, 5% CO2 incubator for static culture.

[0120] (7) After 24 h of culture, the medium was completely replaced with fresh E8 complete medium.

[0121] (8) Thereafter, the medium was completely replaced every day, and cells were subcultured when the confluence reached about 70% to 80%.

[0122] Note: The number of iPSCs cells seeded is about 8000 cells / cm 2 According to different iPSCs lines, the subculture density can be adjusted so that the interval between subcultures of iPSCs cells is 5 to 7 days.

[0123] 2. iPSCs induce early EPC differentiation process:

[0124] 2.1hiPSCs monolayer adherent cell formation (Day-1);

[0125] (1) Prepare TrypLE working solution: pipette 5 mL of DPBS into a new 15 mL centrifuge tube, then add 5 mL of TrypLE stock solution and mix well to obtain the TrypLE working solution.

[0126] (2) According to the amount of culture medium required for subculturing, prepare E8 complete culture medium containing 1% PS and 10 μM Y-27632 (ROCKi), and add 1 μL Y-27632 (10 mM) stock solution per mL of culture medium.

[0127] (3) Take out the well plate / culture bottle to be subcultured from the incubator, discard the supernatant, and wash twice with DPBS (the amount of DPBS used each time should not be less than the amount of the original culture medium), each time for 1 min (when washing, leave the DPBS in the well / bottle for 30-45 seconds before aspirating it).

[0128] (4) After adding TrypLE working solution (add about 1 mL of TrypLE working solution to the six-well plate and about 2 mL of TrypLE working solution to the T25 bottle), place the plate in an incubator and incubate for 2-5 min. During this time, the cells can be observed under a microscope and shrink, become round, and disperse.

[0129] (5) Gently tap the culture flask / plate to detach the cells from the bottom of the plate, then gently blow and swirl several times with a pipette, and finally add DMEM / F12 to terminate digestion. Pipette an appropriate amount of cell suspension for counting.

[0130] (6) After balancing, centrifuge at 200g for 5 min. After centrifugation, discard the supernatant, gently shake the bottom of the centrifuge tube, and resuspend in E8 complete medium containing 10 μM Y-27632 according to different cell densities. After the cells are fully mixed, drop the cell suspension into the wells of the culture plate, and the inoculation cell density is 8000 cells / cm 2 , placed in a 37°C, 5% CO2 incubator for static culture.

[0131] (7) After culturing for 24 h, the cells were washed twice with DPBS and then induced to differentiate.

[0132] 2.2 Mesendoderm differentiation induction process (Day 0-1)

[0133] (1) Prepare an appropriate amount of mesendoderm induction medium and preheat it in a 37°C water bath.

[0134] (2) Aspirate the original culture medium and add an appropriate amount of DPBS to wash the cells.

[0135] (3) Add mesendoderm induction medium and then place in a 37°C, 5% CO2 incubator for 24 hours.

[0136] Table 3 Preparation of mesendoderm differentiation medium

[0137] Reagents Manufacturer Part Number Final concentration Differentiation-based culture —— —— 1× CHIR 99021 Selleck S2924 10μM

[0138] 2.3 Mesoderm differentiation induction process (Day 1-3)

[0139] (1) Prepare an appropriate amount of mesoderm-specific culture medium and preheat it in a 37°C water bath.

[0140] (2) Aspirate the original culture medium and add an appropriate amount of DPBS to wash the cells.

[0141] (3) Add mesoderm-specific culture medium and then place in a 37°C, 5% CO2 incubator for 48 h.

[0142] Table 4 Preparation of mesoderm differentiation medium

[0143] Reagents Manufacturer Part Number Final concentration Differentiation-based culture —— —— 1× VEGF165 Peprotech AF-100-20 20 ng / mL FGF Peprotech AF-100-18B 20 ng / mL

[0144] 2.4 Operational procedures for early EPC differentiation induction (Day 3-9)

[0145] (1) Prepare an appropriate amount of early EPC differentiation medium and preheat it in a 37°C water bath.

[0146] (2) After 48 hours of mesoderm specialization, the original culture medium was discarded; an appropriate amount of DPBS was added to wash the cells, and the cells were digested to single cells with TrypLE working solution to terminate cell digestion. After centrifugation at 200g for 5 minutes, the cells were resuspended in early EPC differentiation medium and cultured at 10,000 cells / cm 2 The cells were inoculated and then placed in a 37°C, 5% CO2 incubator for static culture.

[0147] (3) Thereafter, the medium for early EPC differentiation was replaced with fresh medium on days 4 and 5 of differentiation.

[0148] (4) On the 6th day of differentiation, the original culture medium was discarded; an appropriate amount of DPBS was added to wash the cells, and the cells were digested to single cells with TrypLE working solution to terminate the cell digestion. After centrifugation at 200g for 5 min, the cells were resuspended in early EPC maintenance medium and cultured at 1000 cells / cm 2 The cells were inoculated and then placed in a 37°C, 5% CO2 incubator for static culture.

[0149] (5) Thereafter, fresh early EPC maintenance medium was replaced on days 7 and 8 of differentiation.

[0150] (6) On the 9th day of differentiation, the original culture medium was discarded; an appropriate amount of DPBS was added to wash the cells, and the cells were digested into single cells with TrypLE working solution to terminate the cell digestion. After centrifugation at 200g for 5 min, the early EPC cells were harvested for freezing and quality inspection.

[0151] Table 5 Preparation of early EPC differentiation medium

[0152] Reagents Manufacturer Part Number Final concentration Differentiation-based culture —— —— 1× VEGF165 Peprotech AF-100-20 20 ng / mL FGF Peprotech AF-100-18B 20 ng / mL SCF Peprotech 300-07 50ng / mL Flt-3L Peprotech AF-300-19 10 ng / mL TPO Peprotech AF-300-18 30 ng / mL IL-3 Peprotech AF-200-03 10 ng / mL BMP-4 Peprotech 120-05ET 10 ng / mL IL-6 Peprotech 200-06 10 ng / mL IGF1 Peprotech 100-11 25 ng / mL

[0153] Table 6 Preparation of early EPC maintenance medium

[0154] Reagents Manufacturer Part Number Final concentration Differentiation-based culture —— —— 1× VEGF165 Peprotech AF-100-20 20 ng / mL

[0155] Example 2 Directed differentiation and identification of late EPC

[0156] Preparation of basal culture medium

[0157] Preparation of mesoderm induction basal medium: Prepare it according to the specifications and proportions shown in Table 7 and store it at 4°C for up to 1 month. B-27minus insulin should be packaged in single doses and stored at -20°C. Avoid repeated freezing and thawing. Take out one tube of each medium one day before use and thaw it overnight at 4°C, or thaw it at room temperature on the same day.

[0158] Table 7 Preparation of mesoderm induction basal medium

[0159] Total volume 1000mL 500mL 100mL 50mL DMEM / F12 medium 500mL 250mL 50mL 25mL Neurobasal medium 500mL 250mL 50mL 25mL N-2(100X) 10 mL 5mL 1mL 0.5mL B-27minus insulin(50X) 20mL 10mL 2mL 1mL β-Mercaptoethanol 1mL 0.5mL 0.1mL 0.05mL

[0160] Preparation of EPCs induction basal medium: Prepare it immediately before use according to the specifications and proportions shown in Table 8, and store it at 4°C for up to 1 month. Among them, StemPro-34 Supplement needs to be divided into single-use amounts and stored at -20°C. Avoid repeated freezing and thawing. Take out one tube and thaw it at 4°C overnight one day before use, or thaw it at room temperature on the same day.

[0161] Table 8 Preparation of EPCs induction basal medium

[0162] Total volume 500mL 100mL 50mL StemPro-34SFM medium 500mL 100mL 50mL StemPro-34Supplement 13mL 2.6mL 1.3mL GlutaMAX(100X) 5 mL 1 mL 0.5mL

[0163] 1. Recovery and Passaging of iPSCs

[0164] (1) Take out a tube of frozen iPSC cells from the iPSCs working bank and quickly transfer it to the laboratory. Place it in a hot water bath at 38℃-40℃ and shake it gently. When only tiny (about the size of a millet grain) crystals remain in the cryopreservation tube, thoroughly disinfect the surface of the cryopreservation tube with 75% alcohol and place it in an ultra-clean workbench.

[0165] (2) Use a pipette to transfer the cell suspension to a 15 mL centrifuge tube, add 5 mL of DMEM / F12 Medium preheated at 37°C, and centrifuge at 200 g for 5 min at room temperature.

[0166] (3) After centrifugation, discard the supernatant with a pipette. Then, add 1 mL of 37°C preheated E8 complete medium containing ROCKi (1 μL of ROCKi storage solution per mL of medium) using a 1 mL pipette tip. Gently pipette to resuspend the cell pellet and aspirate 10 μL for cell counting and viability detection.

[0167] (4) Take out the prepared Matrigel-coated cell culture plate from the 37°C / 5% CO2 cell culture incubator, take a T25 culture flask as an example, remove the liquid, and add 5 mL of E8 complete medium containing ROCKi preheated at 37°C.

[0168] (5) Then slowly add an appropriate amount of cell dilution solution (seeding density 1.0-1.5×10 4 Cell clusters / cm 2 ), put the cell bottle into a 37℃ / 5% CO2 cell culture incubator, shake it back and forth and left and right for about 10 times (short, flat, and fast) to ensure that the cells are evenly distributed on the bottom of the culture bottle, and then let it stand overnight. The iPSCs inoculated after recovery are at the P13 generation.

[0169] (6) After 24 hours, replace with fresh E8 complete medium preheated at 37°C. Repeat the medium change every day until the cell aggregation degree increases to 70-80%, or the diameter of any single colony is larger than 10× the microscopic field of view. Then, iPS cells can be passaged according to steps (3)-(7).

[0170] (7) The inoculation density of each generation is fixed at 1.0-1.5×10 4 Cell clusters / cm 2 The fixed amplification time is 4 days, and the cells are subcultured every 4 days. After the P14 generation has been amplified for 4 days, the next step is entered.

[0171] 2. Operational procedures for establishing iPSCs monolayer differentiation (Day-1)

[0172] (1) After formally starting the induction of iPSCs differentiation, the cells were centrifuged normally, the supernatant was removed, and an appropriate amount of E8 complete medium containing ROCK preheated at 37°C was added. The cell pellet was resuspended by gently pipetting, and then the resuspended cell fluid was counted and the viability was tested.

[0173] (2) Take out four Matrigel-coated T75 culture flasks from the 37°C / 5% CO2 cell culture incubator, remove the liquid, and add 13 mL of 37°C preheated E8 complete medium containing ROCK to each flask.

[0174] (3) The initial seeding density of iPSCs induced differentiation should be controlled at 3.0×10 4 ~4.0×10 4cells / cm 2 According to the density of the cell resuspension after counting, add an appropriate volume of the cell resuspension into the Matrigel-coated T75 culture flask prepared above.

[0175] (4) Place the cell plate in a 37°C / 5% CO2 cell culture incubator, shake it back and forth and left and right for about 10 times to ensure that the cells are evenly distributed on the culture plate, and then let it stand overnight.

[0176] (5) After 24 hours, observe the polymerization degree of iPSCs after inoculation. If the polymerization degree reaches 15-25%, you can directly proceed to the subsequent formal induction step. If the polymerization degree does not reach 15%, you can replace it with fresh E8 complete medium preheated at 37°C and appropriately extend the iPSCs culture time to 12-24 hours.

[0177] 3. Operational procedures for the induction of lateral plate mesoderm (Day 0-3)

[0178] (1) After the iPSC cells are seeded, when the degree of aggregation reaches about 15-25%, formal differentiation induction begins, which is defined as Day 0. Remove the old culture medium in the T75 flask, wash it with 10 mL DPBS, and then add 30 mL of 37°C preheated mesoderm induction complete medium-1 to each flask, followed by incubation in a 37°C / 5% CO2 cell culture incubator for 17-18 hours.

[0179] Table 9 Mesoderm Induction Complete Medium-1

[0180]

[0181] (2) After 17-18 hours of incubation (Day 1), iPS cells gradually spread out, the nuclear-cytoplasmic ratio decreased, and the cell area increased. Remove the old culture medium in the T75 flask, wash it with 10 mL DPBS, and then add 50 ml of 37°C preheated mesoderm induction complete medium-2 to each flask, and then incubate in a 37°C / 5% CO2 cell culture incubator for 2 days without changing the medium.

[0182] Table 10 Mesoderm induction complete medium-2

[0183]

[0184]

[0185] 4. Operational procedures for induction of late EPCs (Day 3-6)

[0186] (1) After the formation of the lateral plate mesoderm cells on Day 3, remove the old culture medium in the T75 flask, wash it with 10 mL of DPBS, and then add 30 mL of EPCs induction complete medium (prepared according to the table below) preheated at 37°C to each flask. Return the culture plate to the 37°C / 5% CO2 cell culture incubator and incubate for 24 hours.

[0187] Table 11 Preparation of complete medium for EPCs induction

[0188]

[0189] (2) One day later (Day 4), repeat the medium change operation of Day 3, and add 30 mL of 37°C preheated EPCs induction complete medium (prepared according to the above table) to each bottle. Incubate for 24 hours until Day 5, and EPCs with a higher EC tendency will be induced.

[0190] (3) At least 1 hour before enzymatic dissociation and replating of EPCs on Day 5, prepare 8 Fibronectin-coated T175 cell culture flasks.

[0191] (4) Remove all old culture medium from T75 flasks, wash twice with DPBS, then add 3 mL of TrypLE to each flask and place in a 37°C / 5% CO2 cell culture incubator for 3-5 min. Observe the degree of cell detachment under a microscope until most of the cells begin to float.

[0192] (5) Gently shake the bottom of the culture flask until most of the cells fall off in a quicksand-like state. Add 12 mL of DMEM / F12 Medium to neutralize the digestion effect of TrypLE. Use a pipette to gently blow the detached cells to resuspend them. Then transfer them to a centrifuge tube and take an appropriate amount of cells for counting.

[0193] (6) Centrifuge at 200 g for 5 min at room temperature. During this time, prepare complete EPC maintenance medium (prepare according to the table below).

[0194] Table 12 Preparation of complete culture medium for EPCs maintenance

[0195]

[0196] (7) Remove the Fibronectin working solution from the T175 bottle that has been standing at room temperature for 1 hour, and rinse twice with an appropriate amount of double distilled water. Remove the supernatant of the centrifuged cells, take an appropriate amount of prepared EPCs maintenance complete medium to resuspend the cells, and then add the cells (one T75 bottle to two T175 bottles) evenly to the T175 bottles (35 ml / T175 bottle).

[0197] (8) Place the culture flask in a 37°C / 5% CO2 cell culture incubator and let it stand overnight.

[0198] (9) 24 h later (Day 6), the cells were allowed to adhere and washed once with an appropriate amount of DPBS. Each T175 flask was then digested with 6 mL of TrypLE and terminated with 24 mL of DMEM / F12. The cells were centrifuged at 200 g for 5 min and the supernatant was removed.

[0199] (10) Use a pipette to take up a small amount of DPBS and gently blow to resuspend each cell pellet. Then transfer the pellet to the same 50 mL centrifuge tube to ensure that the total volume is not less than 40 mL. Centrifuge at 200 g for 5 min and discard the supernatant.

[0200] (11) Resuspend and wash again with 40 mL of DPBS, and take 50 μL for counting and viability detection.

[0201] (12) Based on the cell density obtained from the test and the quantity required for quality control, take the corresponding volume of cell suspension and send it to the quality inspection department for various quality tests of the product.

[0202] (13) The remaining cell suspension was centrifuged again at 200 g for 5 min and cryopreserved as EPCs stock solution according to the subsequent operation procedures.

[0203] Example 3 Immunofluorescence detection of EPC molecular markers

[0204] Table 13 Reagents

[0205]

[0206]

[0207] Table 14 Working fluid

[0208]

[0209] Steps:

[0210] (1) Accurately count the cryopreserved EPC cells after passage or resuscitation, and calculate the total number of cells based on the counting results. Take about 1×10 EPC cells by volume ratio. 6 cells, divided into 8 wells, each well has about 100,000-150,000 cells. The final volume of EPC complete medium in each well is 0.5 mL. Mark the inoculation wells for the purpose of the experiment, the test person and the inoculation time. Place in a 37℃ 5% CO2 cell culture incubator and culture until the confluence reaches more than 60%.

[0211] (2) Aspirate and discard the liquid in the used wells and wash three times with 500 μL DPBS.

[0212] (3) Add 200 μL of fixative (4% paraformaldehyde) to each well and incubate at room temperature for 20 min.

[0213] (4) Aspirate the fixative and wash three times with 200 μL DPBS.

[0214] (5) Add 200 μL of 0.1% Triton-X 100 and incubate at room temperature for 15 min.

[0215] (6) Discard the supernatant and wash three times with 200 μL DPBS.

[0216] (7) Add 200 μL of 5% BSA blocking solution to each well and let stand at room temperature for 30 min.

[0217] (8) Aspirate the supernatant from the test wells and add 200 μL of primary antibody working solution (5% BSA 100-fold diluted primary antibody as primary antibody working solution) and incubate overnight at 4°C. Negative control wells were left untreated and incubated overnight at 4°C.

[0218] (9) Discard the supernatant and wash three times with 200 μL DPBS.

[0219] (10) Add 200 μL of secondary antibody working solution (dilute the secondary antibody 100-fold in DPBS to prepare the secondary antibody working solution) to each well and incubate at room temperature in the dark for 1 h.

[0220] (11) Discard the supernatant and wash three times with 200 μL DPBS.

[0221] (12) Add 200 μL of DAPI working solution (DAPI diluted 200 times in DPBS as DAPI working solution) to each well and incubate at room temperature for 2 min in the dark. Wait for observation and photography under a microscope in a dark room.

[0222] Experimental results and conclusions:

[0223] Immunofluorescence test results Figure 1 and Figure 2 As shown, both early EPC and late EPC highly expressed CD31, CD34 and KDR; late EPC also highly expressed CD144 and vWF, but early EPC lowly expressed or even did not express CD144 and vWF.

[0224] Conclusion: CD144 and vWF can be used as marker genes to distinguish early EPC from late EPC. Example 4 Detection of EPC molecular markers by flow cytometry

[0225] Table 15 Reagents

[0226]

[0227] Steps:

[0228] (1) Accurately count the EPC cells of the cryopreserved test sample after passage or thawing, and calculate the total number of cells based on the counting results. Take about 5×10 6 cells, centrifuged at 350g for 5 min, and transferred to a 1.5mL EP tube with 1mL DPBS. The cells collected in this step should be prepared into a single-cell suspension using an appropriate method to prevent cell clumps from blocking the flow system or affecting data accuracy.

[0229] (2) The test samples were grouped, packaged and labeled in 1.5 mL EP tubes according to the experimental settings. Seven groups were set up, with 100 μL of cell suspension in each tube, labeled Blank (blank control group), 647 and 488 (negative control group), KDR, CD34, CD31 and CD133 respectively.

[0230] (3) Prepare a mixture of DPBS and antibody at a ratio of 100:5 μL and incubate at room temperature in the dark for 30 min. Centrifuge for 120 s in a microcentrifuge, discard the supernatant and break up the cell pellet.

[0231] (4) Washing: Add 500 μL of DPBS to each tube for washing, centrifuge for 120 seconds, discard the supernatant and break up the cell pellet. Repeat the washing operation twice, for a total of 3 times.

[0232] (5) Flow cytometry: Add 200 μL of DPBS to each tube of sample to be tested and resuspend it. Then load the sample onto the flow cytometer. After the test is completed, save the document and analyze the flow cytometry results using analysis software.

[0233] Experimental results and conclusions:

[0234] Test results such as Figure 3 and 4 As shown, both early EPC and late EPC highly expressed CD31, CD34 and KDR. Early EPC also expressed CD133, while late EPC expressed low or even no CD133. Conclusion: CD133 can be used as one of the surface markers of early EPC and late EPC.

[0235] Example 5 In vitro tube formation experiment to detect the angiogenesis ability of EPC

[0236] (1) The EPCs prepared in Example 1 and Example 2 were cultured in T25 culture flasks for 3 days, the culture supernatant in the T25 cell culture flasks was discarded, the cells were washed twice with 3 mL DPBS, 1 mL TrypLE enzymatic solution was added, and the cells were placed in a 37° C. 5% CO2 cell culture incubator for digestion for 3 to 5 min. The degree of cell detachment was observed under a microscope until most of the cells began to float.

[0237] (2) Gently shake the bottom of the culture flask until most of the cells fall off in a quicksand-like state. Then add 2 mL of culture supernatant to neutralize the digestion effect of TrypLE. Use a pipette to gently blow the detached cells to resuspend them. Then transfer all the cell resuspension to a 15 mL centrifuge tube and centrifuge at 300 g for 5 min at room temperature.

[0238] (3) After centrifugation, discard the supernatant, add 0.5 mL of EPC maintenance complete medium to resuspend all cell pellets, and transfer all cell resuspension to a 15 mL centrifuge tube.

[0239] (4) Take 50 μL of cell resuspension and calculate the total number of cells.

[0240] (5) Take an appropriate amount of cell resuspension and place it in a new 15 mL centrifuge tube. Add EPC tube complete medium to a total volume of 350 μL and a total cell volume of 3.50 × 10 5 cells.

[0241] (6) After slowly pipetting twice with a 200 μL pipette tip, 100 μL of cell suspension per well was inoculated into a Matrigel-coated 48-well cell culture plate incubated at 37°C / 5% CO2 for 30 min, 0.1 mL / well, i.e., 1.0×10 5 cells / well, and place the 48-well cell culture plate in a 37°C / 5% CO2 cell culture incubator for static culture.

[0242] (7) After 6 hours, turn on the OLYMPUS optical inverted microscope, camera system, and computer and put them in standby mode.

[0243] Place the 48-well cell culture plate to be tested on the stage of the inverted microscope, adjust the light source and field of view of the inverted microscope, observe the tube formation results of the test sample, take photos and record them, and archive the photos.

[0244] Experimental results and conclusions:

[0245] The experimental results show that both early EPC and late EPC have the ability to form tubes (A), but there is no significant difference in the number of branches (B) and the average number of nodes (C) of tubes formed between early EPC and late EPC. There is a certain difference in the average tube length between early EPC and late EPC.

[0246] Conclusion: The ability to form tubes needs to be comprehensively judged from different dimensions. The above results show that both early EPC and late EPC have the ability to form tubes, but they cannot be used as a criterion for distinguishing early EPC from late EPC.

[0247] Example 6 Identification of late EPCs by acetylated LDL uptake and Ulex oleraceus lectin binding assay

[0248] Table 16 Reagents

[0249]

[0250] method:

[0251] (1) Accurately count the cryopreserved EPC cells after passage or resuscitation, and calculate the total number of cells based on the counting results. Take about 1×10 EPC cells by volume ratio. 6 cells, divided into 8 wells, each well has about 100,000-150,000 cells. The final volume of EPC complete medium in each well is 0.5 mL. Mark the well position for the purpose of the experiment, the test subject and the time of inoculation. Place in a 37℃ 5% CO2 cell culture incubator and culture until the confluence reaches more than 60% and then prepare for staining.

[0252] (2) Add fluorescent dye to the above detection wells at a ratio of 1 μL Dil-Ac-LDL per 100 μL culture medium and incubate at 37°C for 4 h.

[0253] (3) Aspirate the liquid in the wells and wash three times with DPBS, soaking for 5 min each time.

[0254] (4) Add 200 μL of 4% polymethanol to each well and fix for 10 min.

[0255] (5) Aspirate the liquid in the wells and wash three times with DPBS.

[0256] (6) Add 200 μL of FITC-UEA-Ⅰ working solution to each well and incubate at room temperature for 1 h.

[0257] (7) Aspirate the liquid in the wells, wash three times with DPBS, and add 200 μL DPBS.

[0258] (8) Observe and photograph under a fluorescence microscope in a dark room.

[0259] Experimental results and conclusions:

[0260] The experimental results are as follows Figure 5As shown, red fluorescence marks acetylated LDL and green fluorescence marks UEA-Ⅰ. Acetylated low-density lipoprotein (Ac-LDL) cannot bind to LDL receptors after being modified, and is then taken up by macrophages and endothelial cells containing scavenger receptors that specifically recognize modified LDL, producing foam cells. Ulex oleraceus agglutinin (UEA) is a protein extracted from urease. Lectins play an important role in cell recognition and adhesion reactions, mainly promoting cell-to-cell adhesion. Lectins have more than one sugar-binding site and can therefore participate in cell recognition and adhesion to link different cells. As can be seen from the figure, acetylated low-density lipoprotein (Ac-LDL) is rarely or even cannot be taken up by early EPC; late EPC can efficiently take up acetylated low-density lipoprotein (Ac-LDL).

[0261] Conclusion: There are significant differences in the phagocytic ability of acetylated low-density lipoprotein (Ac-LDL) between early EPC and late EPC, and late EPC has stronger phagocytic ability, which can be used as a functional test to distinguish early EPC from late EPC cells.

[0262] Example 7 Non-clinical study on the treatment of acute ischemic stroke with endothelial progenitor cells (EPCs)

[0263] Twenty-four male SD rats were selected and divided into four groups: sham operation group, model control group, early EPC and late EPC treatment groups, with 6 animals in each group. The animals were occluded by suture embolism to establish a permanent ischemic model of the middle cerebral artery. The test article EPCs (1×10 7 cells / kg) were injected into the tail vein once 24 hours after ischemia. Eight days after surgery, the animals were dissected and the brain tissue was stained with TTC to determine the scope of cerebral infarction. The protective effect of the test article on acute ischemic stroke in rats was evaluated by the scope of cerebral infarction.

[0264] The experimental results are as follows Figure 6 and Figure 7 As shown in the data, in the MCAO rat cerebral infarction model, the blood supply to the infarct area was significantly restored in the late EPC treatment group after administration, indicating that late EPC has a protective effect on acute rat ischemic stroke, while there was no significant difference in the blood supply to the infarct area between the early EPC treatment group and the model control after administration, indicating that early EPC has almost no protective effect on acute rat ischemic stroke.

[0265] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A method for identifying, enriching or isolating early / late EPCs, characterized in that: Determine the expression of biomarkers in EPC cells, and when it is determined that the EPC cells have the following expression profile, the EPC is a late EPC: CD144 高 / + 、vWF 高 / + and CD 133 低 / - ; When the cell is determined to have the following expression profile, the EPC cell is an early EPC: CD133 高 / + 、CD144 低 / - and vWF 低 / - ; The biomarkers include CD144, vWF and CD133; The EPCs are derived from iPSCs.

2. The method according to claim 1, characterized in that The method comprises contacting the cell with a binding agent for the biomarker.

3. The method according to claim 2, characterized in that The method also includes sorting the EPCs that bind to the binding agent.

4. The method according to claim 3, characterized in that The sorting techniques include fluorescence activated cell sorting, magnetic assisted cell sorting, substrate assisted cell sorting, laser mediated cutting, fluorimetry, flow cytometry or microscopy.

5. A device or apparatus for identifying, enriching or isolating early / late EPCs, characterized in that: The device or apparatus comprises a cell binding unit, wherein the cell binding unit is configured to have a structure capable of binding a biomarker; The biomarkers include CD144, vWF and CD133; The device or apparatus determines the expression of biomarkers in EPC cells, and when it is determined that the EPC cells have the following expression profile, the EPC is a late EPC: CD144 高 / + 、vWF 高 / + and CD 133 低 / - ; When the cell is determined to have the following expression profile, the EPC cell is an early EPC: CD133 高 / + 、CD144 低 / - and vWF 低 / - ; The EPCs are derived from iPSCs.

6. The device or apparatus according to claim 5, characterized in that The biomarkers also include CD31, CD34 and / or KDR.

7. Use of the biomarkers CD144, vWF and CD133, and the device or apparatus according to any one of claims 5 to 6 in identifying, enriching or isolating early / late EPCs; The method comprises determining the expression of biomarkers in EPC cells, wherein the EPC cells are late EPC cells when the EPC cells are determined to have the following expression profiles: CD144 高 / + 、vWF 高 / + and CD 133 低 / - When it is determined that the cell has the following expression profile, the EPC cell is an early EPC: CD133 高 / + 、CD144 低 / - and vWF 低 / - ; The EPCs are derived from iPSCs.

8. Use of late EPC in the preparation of a drug for treating stroke, wherein the late EPC has the following expression profile: CD144 高 / + 、vWF 高 / + and CD 133 低 / - ; The late EPCs are derived from iPSCs.

9. Use of late EPC in the preparation of a drug for treating stroke, wherein the late EPC has the following expression profile: CD144 高 / + 、vWF 高 / + 、CD 133 低 / - 、CD31 高 / + 、CD34 高 / + and KDR 高 / + ; The late EPCs are derived from iPSCs.

Images