Markers for characterizing EPC subpopulations, EPC subpopulations and applications thereof

Abstract

The present invention belongs to the field of biomedicine, and discloses markers for characterizing EPC subpopulations, EPC subpopulations and applications thereof, and also discloses that phagocytic ability can be used as an indicator for distinguishing different EPC subpopulations. The present invention also discloses that late-stage EPC can be used for the treatment of erectile dysfunction.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to markers for characterizing EPC subpopulations, EPC subpopulations and applications thereof. Background Art

[0002] Erectile dysfunction (ED) refers to the inability of a man's penis to maintain or achieve an erection sufficient to complete a satisfactory sexual life, and the course of the disease lasts for more than 3 months. The incidence of ED is very high worldwide.

[0003] The occurrence of ED is extremely harmful. In the past, in most cases, erectile dysfunction was considered to be a purely psychogenic disorder. But current evidence shows that more than 80% of cases have organic causes (YAFI FA, JENKINS L, ALBERSEN M, et al. Erectile dysfunction [J]. Nat Rev Dis Primers, 2016, 2: 16003.). ED is closely related to heart disease, stroke, diabetes, etc. There is evidence that the presence of ED increases the risk of future cardiovascular events, including myocardial infarction, cerebrovascular events and all-cause mortality, and has a trend to increase the risk of cardiovascular death. Therefore, ED is an early manifestation of coronary artery and peripheral vascular disease. In addition, the severity of ED is positively correlated with the prevalence and severity of anxiety and depression. ED seriously affects the physical and mental health of patients, the relationship between husband and wife, and family stability, thus causing important social public health problems.

[0004] Currently, the EAU guidelines introduce methods for treating ED, such as oral medication, topical / intraurethral use of alprostadil, shock wave therapy, psychosocial intervention and treatment, hormone therapy, vacuum erection device, intracavernous injection therapy, and prosthesis implantation. In addition, stem cell therapy is currently a hot topic in the treatment of ED.

[0005] Currently, the first-line drug for the treatment of ED is phosphodiesterase type 5 (PDE5) inhibitors, but there are still many patients who do not respond to PDE5 inhibitors. Therefore, new intervention methods are urgently needed in the clinic to treat ischemic erectile dysfunction. Summary of the invention

[0006] In order to solve the problems existing in the prior art, the inventors of the present invention have conducted extensive and in-depth research and discovered for the first time that late EPC rather than early EPC has the effect of treating erectile dysfunction, providing a new and effective treatment for erectile dysfunction.

[0007] The present invention also discovered biomarkers for distinguishing early EPC (early EPC) from late EPC (late EPC), and the relevant markers can be used for the identification, sorting, separation and enrichment of different EPC subpopulations.

[0008] Based on this, the present invention provides the following technical solutions:

[0009] A first aspect of the present invention provides a method for identifying early / late EPCs, determining the expression of at least one biomarker in a cell, wherein the biomarker comprises CD144, vWF or CD133; and

[0010] When it is determined that the cell has at least one of the following expression profiles, the EPC is a late EPC: 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] A second aspect of the present invention provides a method for identifying early / late EPCs, the method comprising detecting the phagocytic ability of cells.

[0012] The third aspect of the present invention provides a method for sorting, isolating or enriching late EPCs from a cell population, selecting cells having at least one of the following expression profiles: CD144 高 / + 、vWF 高 / + and / or CD 133 低 / - .

[0013] A fourth aspect of the present invention provides a method for sorting, isolating or enriching early EPCs from a cell population, wherein cells having at least one of the following expression profiles are selected: CD133 高 / + 、CD144 低 / - and / or vWF 低 / - .

[0014] The fifth aspect of the present invention provides a product for identifying, enriching or isolating early / late EPCs, wherein the product comprises a binding agent capable of binding to biomarkers on cells, wherein the biomarkers include CD144, vWF and / or CD133.

[0015] The sixth aspect of the present invention provides a device or equipment for identifying, sorting, separating or enriching early / late EPCs, wherein the device or equipment is provided with a cell binding unit; the cell binding unit contains a reagent that binds to a biomarker, wherein the biomarker is CD144, vWF and / or CD133.

[0016] The seventh aspect of the present invention provides the use of biomarkers in identifying, sorting, isolating or enriching early / late EPCs, wherein the biomarkers include CD144, vWF and / or CD133.

[0017] The eighth aspect of the present invention provides the use of late EPC in the preparation of a drug for treating erectile dysfunction.

[0018] A ninth aspect of the present invention provides a pharmaceutical composition for treating erectile dysfunction, wherein the pharmaceutical composition comprises late EPC.

[0019] The tenth aspect of the present invention provides a method for treating erectile dysfunction, comprising administering an effective amount of advanced EPC or the pharmaceutical composition according to the ninth aspect of the present invention.

[0020] Advantages and beneficial effects of the present invention:

[0021] 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.

[0022] The present invention first discovered that phagocytic ability can be used to distinguish early EPCs from late EPCs.

[0023] The present invention discovers for the first time that late EPCs rather than early EPCs can be used to treat erectile dysfunction. Based on this, the therapeutic effect of endothelial progenitor cells in treating erectile dysfunction can be improved by increasing the content of late EPCs, an effective ingredient for treating erectile dysfunction. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the result of early EPC cell surface marker gene detection;

[0025] Figure 2 This is the result of late EPC cell surface marker gene detection;

[0026] Figure 3 This is a graph showing the results of early EPC surface marker gene detection by flow cytometry;

[0027] Figure 4 This is the result of flow cytometry detection of late EPC surface marker gene;

[0028] Figure 5 This is a graph showing the results of the phagocytic ability test;

[0029] Figure 6 It is a graph showing the changes in animal body weight after drug administration;

[0030] Figure 7 This is a statistical chart of the number of times the penis was licked after subcutaneous injection of APO;

[0031] Figure 8 is the expression map of eNOS and CD 31 in the corpus cavernosum;

[0032] Fig. 9 This is a multiple immunofluorescence staining image of the corpus cavernosum.

[0033] Specific implementation method

[0034] The present invention has been extensively and deeply studied, and by studying different EPC subgroups, it has discovered indicators that can be used to distinguish between early and late EPCs, and further proved that late EPCs, rather than early EPCs, have the effect of treating erectile dysfunction.

[0035] In one aspect, the present invention provides a method for identifying early / late EPCs, comprising determining the expression of at least one biomarker in a cell, wherein the biomarker comprises CD144, vWF or CD133; and

[0036] When it is determined that the cell has at least one of the following expression profiles, the EPC is a late EPC: 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 / - .

[0037] In some embodiments, the late EPCs have the following expression profile: CD144 高 / + 、vWF 高 / + and CD 133 低 / - .

[0038] In some embodiments, the early EPCs have the following expression profile: CD133 高 / + 、CD144 低 / - 、vWF 低 / - .

[0039] In some embodiments, the EPCs are derived from iPSCs.

[0040] In some embodiments, the early EPC or late EPC further has at least one of the following expression profiles: CD31 高 / + 、CD34 高 / + and / or KDR 高 / + .

[0041] In a specific embodiment, the late EPC has the following expression profile: CD144 高 / + 、vWF 高 / + 、CD 133 低 / - 、CD31 高 / + 、CD34 高 / + and KDR 高 / + .

[0042] In a specific embodiment, the early EPC has the following expression profile: CD133 高 / + 、CD144 低 / - 、vWF 低 / - 、CD31 高 / + 、CD34 高 / + and KDR 高 / + .

[0043] In the present invention, any of a number of standard techniques well known to those skilled in the art can be used to implement actual monitoring or analysis of biomarkers. For example, the expression of biomarkers in cells can be detected by sequencing, such as single-cell sequencing; 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, laser capture microdissection, massively multiparametric mass cytometry, flow cytometry, and FACS analysis.

[0044] In some embodiments, determining the expression of at least one biomarker in a cell comprises contacting the cell with a binding agent for the biomarker.

[0045] In the present invention, a binding agent refers to any compound, molecule and / or system that functions to detect the presence / absence and / or expression or level of a biomarker in the present invention. Such binding agents are capable of directly or indirectly detecting and / or binding to biomarkers. In the present invention, additional parts may be required to enhance the detection of biomarkers, for example, by / through amplifying optical diffraction. Examples of binding agents and additional parts include, but are not limited to, proteins (e.g., antigen binding proteins such as antibodies or fragments thereof, enzymes such as horseradish peroxidase and alkaline phosphatase, etc.), polynucleotides (e.g., aptamers) and small molecules (e.g., metal nanoparticles).

[0046] Another aspect of the present invention provides a method for identifying early / late EPCs, the method comprising detecting the phagocytic ability of cells.

[0047] In some embodiments, late EPCs have greater phagocytic ability than early EPCs.

[0048] In some embodiments, the method detects the phagocytic ability of cells by detecting the uptake ability of acetylated LDL.

[0049] One aspect of the present invention provides a method for sorting, isolating or enriching late EPCs from a cell population, comprising selecting cells having at least one of the following expression profiles: CD144 高 / + 、vWF 高 / + and / or CD 133 低 / - .

[0050] In some embodiments, a method for sorting, isolating or enriching late EPCs from a cell population comprises selecting cells having the following expression profile: CD144 高 / + 、vWF 高 / + and CD 133 低 / - .

[0051] In some embodiments, the method of sorting, isolating or enriching late EPCs from a cell population further comprises further selecting cells having at least one of the following expression profiles: CD31 高 / + 、CD34 高 / + and / or KDR 高 / + .

[0052] In some embodiments, the method of sorting, isolating or enriching late EPCs from a cell population comprises selecting cells having the following expression profile: CD144 高 / + 、vWF 高 / + 、CD 133 低 / - 、CD31 高 / + 、CD34 高 / + and KDR 高 / + .

[0053] In some embodiments, the method further comprises culturing the late EPCs.

[0054] One aspect of the present invention provides a method for sorting, isolating or enriching early EPCs from a cell population, comprising selecting cells having at least one of the following expression profiles: CD133 高 / + 、CD144 低 / - and / or vWF 低 / - .

[0055] In some embodiments, a method for sorting, isolating or enriching early EPCs from a cell population comprises selecting cells having the following expression profiles: CD133 高 / + 、CD144 低 / - and vWF 低 / - .

[0056] In some embodiments, the method of sorting, isolating or enriching early EPCs from a cell population further comprises selecting cells having at least one of the following expression profiles: CD31 高 / + 、CD34 高 / + and / or KDR 高 / + .

[0057] In a specific embodiment, the method of sorting, isolating or enriching early EPCs from a cell population comprises selecting cells having the following expression profile: CD133 高 / + 、CD144 低 / - 、vWF 低 / - 、CD31 高 / + 、CD34 高 / + and KDR 高 / + .

[0058] 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.

[0059] In some embodiments, cells can be isolated by binding to an immobilized support and then harvested by removing the cells from the support.

[0060] Harvesting of cells can be achieved by collecting the separated cells into a suitable container or collection dish, test tube, or the like.

[0061] One aspect of the present invention provides a product for identifying, enriching or isolating early / late EPCs, the product comprising a binding agent capable of binding to a biomarker on a cell, wherein the biomarker comprises CD144, vWF and / or CD133.

[0062] In some embodiments, the biomarkers include a combination of CD144, vWF, and CD133.

[0063] In some embodiments, the biomarkers further include CD31, CD34 and / or KDR.

[0064] The terms "binding agent", "binding molecule" and "binding entity" are synonymous and can be used interchangeably. In the context of the present invention, 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.

[0065] In some embodiments, the binding agent is selected from a nucleic acid, a ligand, an enzyme, a substrate, and / or an antibody.

[0066] In the present invention, "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 mimic the hypervariable region of antibodies (e.g., isolated complementary determining regions (CDRs)).

[0067] In some embodiments, the nucleic acid comprises DNA or RNA.Exemplary nucleic acids include probes and primers.

[0068] In some embodiments, the binding agent has a detectable molecule attached thereto.

[0069] In some embodiments, the detectable molecule can be selected from: fluorescent molecules, luminescent molecules, chemiluminescent molecules, radioactive molecules, enzymatic molecules and secondary antibodies."Detectable molecule" is meant to include any molecule that can be detected and the relative amount and / or relative position of the moiety determined.

[0070] The detectable molecule can be a fluorescent molecule and / or a luminescent molecule and / or a chemiluminescent molecule that can be detected when exposed to specific conditions. For example, a fluorescent molecule may need to be exposed to radiation (i.e., light) at a specific wavelength and intensity to cause excitation of the fluorescent molecule, thereby enabling it to emit detectable fluorescence at a specific wavelength that can be detected.

[0071] In some embodiments, the detectable molecule can be an enzyme that can convert a (preferably undetectable) substrate into a detectable product that can be visualized and / or detected. Examples of suitable enzymes used can be those enzymes known for use in tests such as ELISA. Examples include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase.

[0072] In some embodiments, the detection moiety can be a radioactive molecule. Suitable radioactive molecules include those used in scintigraphic studies. 99 mTc and 123 I. Other easily detectable moieties include, for example, spin labels for use in magnetic resonance imaging (MRI), such as 123 I. 131 I. 111 In, 19 F. 13 C. 15 N. 17 O, gadolinium, manganese, or iron.

[0073] Radiolabels or other labels may be incorporated into the reagents of the invention (i.e., proteins present in a sample of the methods of the invention and / or binding agents of the invention) in a known manner. For example, if the binding moiety is a polypeptide, it may be biosynthesized or may be synthesized by chemical amino acid synthesis using appropriate amino acid precursors (containing, for example, fluorine-19 in place of hydrogen). 99m Tc, 123 I. 186 Rh, 188 Rh and 111 Labels such as In can be attached, for example, via cysteine ​​residues in the binding moiety. Yttrium-90 can be attached via lysine residues. Methods for conjugating other detectable molecules (such as enzymatic molecules, fluorescent molecules, luminescent molecules, chemiluminescent molecules, or radioactive molecules) to proteins are well known in the art.

[0074] In some embodiments, the product can be provided in any form, an exemplary form being selected from a kit.

[0075] In the present invention, 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 enriched or separated cells or cell subpopulations 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 multiple 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 marking or modifying a sealed binding agent.

[0076] 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).

[0077] 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.

[0078] One aspect of the present invention provides a device or apparatus for identifying, sorting, separating or enriching early / late EPCs, wherein the device or apparatus is provided with a cell binding unit; the cell binding unit contains a reagent that binds to a biomarker, wherein the biomarker is CD144, vWF and / or CD133.

[0079] In some embodiments, the biomarkers further include CD31, CD34 and / or KDR.

[0080] One aspect of the present invention provides the use of biomarkers in identifying, sorting, isolating or enriching early / late EPCs, wherein the biomarkers include CD144, vWF and / or CD133.

[0081] One aspect of the present invention provides the use of late EPC in the preparation of a medicament for treating erectile dysfunction.

[0082] In the present invention, the main disease-related causes of erectile dysfunction include aging, atherosclerosis, chronic kidney disease, diabetes, hypertension and antihypertensive drugs, pelvic surgery and radiation therapy, and psychological anxiety. Erectile dysfunction can be caused by many disorders including neurogenic, arterial and veno-occlusive dysfunction and other conditions that lead to incomplete relaxation of smooth muscle. Therefore, the method of the present invention can treat, prevent or improve the symptoms of a disease or condition, such as selected from the group consisting of: such as aging, atherosclerosis, chronic kidney disease, diabetes, hypertension, side effects from drugs (such as antihypertensive drugs), pelvic surgery, radiation therapy and psychological anxiety, wherein the symptom is erectile dysfunction.

[0083] In some embodiments, late EPCs promote the expression levels of eNOS and CD31 in the corpus cavernosum.

[0084] In some embodiments, late EPCs promote the expression levels of PODXL and eNOS in the corpus cavernosum.

[0085] In some embodiments, the late EPCs have at least one of the following expression profiles: CD144 高 / + 、vWF 高 / + 、CD 133 低 / - .

[0086] In some embodiments, the late EPCs also have at least one of the following expression profiles: CD31 高 / + 、CD34 高 / + and / or KDR 高 / + .

[0087] In some embodiments, the EPCs are derived from iPSCs.

[0088] In some embodiments, iPSCs are derived from somatic cells. Somatic cells can be derived from humans or non-human animals, including, for example, humans and other primates, including non-human primates, such as rhesus macaques, chimpanzees, and other monkeys and apes; farm animals, such as cattle, sheep, pigs, goats, and horses; domestic mammals, such as dogs and cats; laboratory animals, including rabbits, mice, rats, and guinea pigs; birds, including poultry, wild birds, and game birds, such as chickens, turkeys, and other gallinaceous birds, ducks, and geese, and the like.

[0089] One aspect of the present invention provides a pharmaceutical composition for treating erectile dysfunction, comprising late EPC.

[0090] In some embodiments, the late EPC is obtained by the method in the examples of the present application.

[0091] In some embodiments, the late EPC is obtained by the aforementioned method of sorting, isolating or enriching late EPC from a cell population. By the method, the content or proportion of late EPC in the cell population can be increased.

[0092] In some embodiments, the pharmaceutical composition further comprises other drugs for treating erectile dysfunction, including but not limited to hormones, PDE5 inhibitors such as sildenafil, tadalafil, vardenafil, and avanafil.

[0093] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0094] "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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] One aspect of the present invention provides a method for treating erectile dysfunction, comprising administering to a subject an effective amount of advanced EPC or the pharmaceutical composition described above in the present invention.

[0099] In some embodiments, the late EPC has a biomarker as described above. As an alternative embodiment, the late EPC is obtained by the method in the examples of the present application. As another alternative embodiment, the late EPC is obtained by the method of sorting, isolating or enriching the late EPC from the cell population as described above. By the method, the content or proportion of the late EPC in the cell population can be increased.

[0100] A "therapeutically effective amount" would be an amount of an active agent that is capable of preventing or at least slowing down (mitigating) a medical condition such as erectile dysfunction. The dosage and administration of the disclosed cells or pharmaceutical compositions can be determined by one of ordinary skill in the art of clinical pharmacology or pharmacokinetics. The effective amount of an active agent of the invention used therapeutically will depend, for example, on the therapeutic goal, the route of administration, and the patient's condition. Accordingly, it may be necessary for the therapist to adjust the dosage and change the route of administration as needed to obtain the best therapeutic effect.

[0101] 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; and the like. The term does not indicate a specific age or gender. Therefore, it is intended 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.

[0102] When used for treatment, the "dosage regimen" may vary according to a variety of factors, including the activity of the particular composition used, the patient's age, weight, general health, sex and diet, administration time, route of administration, excretion rate, drug combination, and the severity of the particular disease to be prevented or treated. Although the dosage of the pharmaceutical composition can vary according to the patient's condition and weight, the severity of the disease, the form of the drug, and the route and duration of administration, it can be appropriately selected by those skilled in the art, and administration can be performed once a day or several times a day. This dosage is not meant to limit the scope of the present invention in any way. The pharmaceutical composition of the present invention can be formulated in the form of pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, or suspensions.

[0103] 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.

[0104] Example 1 Directed differentiation and identification of early EPC

[0105] Preparation of culture medium

[0106] Preparation of E8 complete medium: Prepare E8 complete medium according to Table 1 and store at 4°C for up to 2 weeks.

[0107] Table 1E8 Complete medium preparation table

[0108] Reagents Manufacturer Part Number Final concentration / volume E8 Basal Medium Stemcell 05991 450mL E8 25×Supplement Stemcell 05992 20mL Penicillin-Streptomycin Invitrogen 15140122 1％

[0109] Preparation of differentiation basal medium: Prepare differentiation basal medium according to Table 2 and store at 4°C for up to 1 month.

[0110] Table 2 Preparation of differentiation basal medium

[0111]

[0112] 1. iPSCs recovery and passaging

[0113] 1.1 Matrigel coating

[0114] (1) In an ice box, open the 1.5 mL EP tube containing matrigel with the help of hemostats.

[0115] (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.

[0116] (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.

[0117] (4) Place the coated culture flask / plate at 37°C and 5% CO 2 Incubate overnight in an incubator.

[0118] 1.2 iPSCs recovery

[0119] (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.

[0120] (2) Prepare 10-15 mL of E8 medium and preheat it in a 37°C water bath.

[0121] (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.

[0122] (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.

[0123] (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.

[0124] (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.

[0125] 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.

[0126] 1.3 iPSCs digestion and passaging;

[0127] (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.

[0128] (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.

[0129] (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).

[0130] (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.

[0131] (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.

[0132] (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 cell suspension, inoculate the cell suspension in a T25 flask coated with matrigel, and place the culture flask at 37°C and 5% CO. 2 Culture statically in an incubator.

[0133] (7) After 24 h of culture, the medium was completely replaced with fresh E8 complete medium.

[0134] (8) Thereafter, the medium was completely replaced every day, and cells were subcultured when the confluence reached about 70% to 80%.

[0135] 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 iPSCs cell subculture interval is 5 to 7 days.

[0136] 2. iPSCs induce early EPC differentiation process:

[0137] 2.1hiPSCs monolayer adherent cell formation (Day-1);

[0138] (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.

[0139] (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.

[0140] (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).

[0141] (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.

[0142] (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.

[0143] (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.

[0144] (7) After culturing for 24 h, the cells were washed twice with DPBS and then induced to differentiate.

[0145] 2.2 Mesendoderm differentiation induction process (Day 0-1)

[0146] (1) Prepare an appropriate amount of mesendoderm induction medium and preheat it in a 37°C water bath.

[0147] (2) Aspirate the original culture medium and add an appropriate amount of DPBS to wash the cells.

[0148] (3) Add mesendoderm induction medium and place at 37°C, 5% CO 2 Incubate in an incubator for 24 hours.

[0149] Table 3 Preparation of mesendoderm differentiation medium

[0150] Reagents Manufacturer Part Number Final concentration Differentiation-based culture —— —— 1× CHIR 99021 Selleck S2924 10μM

[0151] 2.3 Mesoderm differentiation induction process (Day 1-3)

[0152] (1) Prepare an appropriate amount of mesoderm-specific culture medium and preheat it in a 37°C water bath.

[0153] (2) Aspirate the original culture medium and add an appropriate amount of DPBS to wash the cells.

[0154] (3) Add mesoderm-specific medium and place at 37°C, 5% CO 2 Incubate in an incubator for 48 hours.

[0155] Table 4 Preparation of mesoderm differentiation medium

[0156] 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

[0157] 2.4 Operational procedures for early EPC differentiation induction (Day 3-9)

[0158] (1) Prepare an appropriate amount of early EPC differentiation medium and preheat it in a 37°C water bath.

[0159] (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 Inoculate cells and place at 37°C, 5% CO 2 Culture in an incubator.

[0160] (3) Thereafter, the medium for early EPC differentiation was replaced with fresh medium on days 4 and 5 of differentiation.

[0161] (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 Inoculate cells and place at 37°C, 5% CO 2 Culture in an incubator.

[0162] (5) Thereafter, fresh early EPC maintenance medium was replaced on days 7 and 8 of differentiation.

[0163] (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.

[0164] Table 5 Preparation of early EPC differentiation medium

[0165] 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

[0166] Table 6 Preparation of early EPC maintenance medium

[0167] Reagents Manufacturer Part Number Final concentration Differentiation-based culture —— —— 1× VEGF165 Peprotech AF-100-20 20 ng / mL

[0168] Example 2 Directed differentiation and identification of late EPC

[0169] Preparation of basal culture medium

[0170] 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.

[0171] Table 7 Preparation of mesoderm induction basal medium

[0172] 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

[0173] 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.

[0174] Table 8 Preparation of EPCs induction basal medium

[0175] 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

[0176] 1. Recovery and Passaging of iPSCs

[0177] (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.

[0178] (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.

[0179] (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.

[0180] (4) From 37°C / 5% CO 2 Take out the prepared Matrigel-coated cell culture plate from the cell culture incubator. Take T25 culture flask as an example, remove the liquid, and add 5 mL of E8 complete medium containing ROCKi preheated at 37°C.

[0181] (5) Then slowly add an appropriate amount of cell dilution solution (seeding density 1.0-1.5×10 4 Cell clusters / cm 2 ), place the cell flask in 37°C / 5% CO 2 In the cell culture incubator, shake the flask 10 times (short, flat, and fast) to ensure that the cells are evenly distributed on the bottom of the culture flask, and then let it stand overnight. The iPSCs inoculated after recovery are at the P13 generation.

[0182] (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).

[0183] (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.

[0184] 2. Operational procedures for establishing iPSCs monolayer differentiation (Day-1)

[0185] (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.

[0186] (2) From 37°C / 5% CO 2 Take out 4 Matrigel-coated T75 culture flasks from the cell culture incubator, remove the liquid, and add 13 mL of E8 complete medium containing ROCK preheated at 37°C to each flask.

[0187] (3) The initial seeding density of iPSCs induced differentiation should be controlled at 3.0×10 4 ~4.0×10 4 cells / 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.

[0188] (4) Place the cell plate in 37°C / 5% CO 2 In the cell culture incubator, shake the plate 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.

[0189] (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.

[0190] 3. Operational procedures for the induction of lateral plate mesoderm (Day 0-3)

[0191] (1) After the iPSC cells are seeded, the degree of aggregation reaches about 15-25%, and the formal induction of differentiation is started, 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, and then incubate at 37°C / 5% CO 2 Incubate cells in a cell culture incubator for 17-18 hours.

[0192] Table 9 Mesoderm Induction Complete Medium-1

[0193]

[0194] (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 at 37°C / 5% CO. 2 Incubate the cells in a culture incubator for 2 days without changing the medium.

[0195] Table 10 Mesoderm induction complete medium-2

[0196]

[0197]

[0198] 4. Operational procedures for induction of late EPCs (Day 3-6)

[0199] (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 37°C preheated EPCs induction complete medium (prepared as shown in the table below) to each flask. Return the culture plate to 37°C / 5% CO. 2 Incubate cells in a cell culture incubator for 24 hours.

[0200] Table 11 Preparation of complete medium for EPCs induction

[0201]

[0202] (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.

[0203] (3) At least 1 hour before enzymatic dissociation and replating of EPCs on Day 5, prepare 8 Fibronectin-coated T175 cell culture flasks.

[0204] (4) Remove all old culture medium from the T75 flasks, wash twice with DPBS, then add 3 mL of TrypLE to each flask and place at 37°C / 5% CO. 2 Incubate the cells in a cell culture incubator for 3-5 minutes and observe the degree of cell detachment under a microscope until most of the cells begin to float.

[0205] (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.

[0206] (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).

[0207] Table 12 Preparation of complete culture medium for EPCs maintenance

[0208]

[0209] (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).

[0210] (8) Place the culture flask in a 37°C / 5% CO 2 Incubate the cells in a cell culture incubator overnight.

[0211] (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.

[0212] (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.

[0213] (11) Resuspend and wash again with 40 mL of DPBS, and take 50 μL for counting and viability detection.

[0214] (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.

[0215] (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.

[0216] Example 3 Immunofluorescence detection of EPC molecular markers

[0217] Table 13 Reagents

[0218]

[0219]

[0220] Table 14 Working fluid

[0221]

[0222] Steps:

[0223] (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 experimenter and the inoculation time. Place at 37℃ 5% CO 2 The cells were cultured in a cell culture incubator until the confluence reached more than 60%.

[0224] (2) Aspirate the liquid in the used wells and wash three times with 500 μL DPBS.

[0225] (3) Add 200 μL of fixative (4% paraformaldehyde) to each well and incubate at room temperature for 20 min.

[0226] (4) Aspirate the fixative and wash three times with 200 μL DPBS.

[0227] (5) Add 200 μL of 0.1% Triton-X 100 and incubate at room temperature for 15 min.

[0228] (6) Discard the supernatant and wash three times with 200 μL DPBS.

[0229] (7) Add 200 μL of 5% BSA blocking solution to each well and let stand at room temperature for 30 min.

[0230] (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.

[0231] (9) Discard the supernatant and wash three times with 200 μL DPBS.

[0232] (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.

[0233] (11) Discard the supernatant and wash three times with 200 μL DPBS.

[0234] (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.

[0235] Experimental results and conclusions:

[0236] 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.

[0237] 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

[0238] Table 15 Reagents

[0239]

[0240] Steps:

[0241] (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.

[0242] (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.

[0243] (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.

[0244] (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.

[0245] (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.

[0246] Experimental results and conclusions:

[0247] Test results such as Figure 3and 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.

[0248] Example 5 In vitro tube formation experiment to detect the angiogenesis ability of EPC

[0249] (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 hydrolysis solution was added, and the cells were placed at 37°C with 5% CO 2 Digest in a cell culture incubator for 3 to 5 minutes and observe the degree of cell detachment under a microscope until most of the cells begin to float.

[0250] (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.

[0251] (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.

[0252] (4) Take 50 μL of cell resuspension and calculate the total number of cells.

[0253] (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.

[0254] (6) After slowly pipetting twice with a 200 μL pipette tip, 100 μL of cell suspension was inoculated into each well and placed in a 37°C / 5% CO 2 In a 48-well cell culture plate coated with Matrigel incubated in a cell culture incubator for 30 min, 0.1 mL / well, i.e. 1.0 x 10 5 The 48-well cell culture plate was placed in 37°C / 5% CO 2 The cells were cultured statically in a cell culture incubator.

[0255] (7) After 6 hours, turn on the OLYMPUS optical inverted microscope, camera system, and computer and put them in standby mode.

[0256] 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.

[0257] Experimental results and conclusions:

[0258] 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.

[0259] 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.

[0260] Example 6 Identification of late EPCs by acetylated LDL uptake and Ulex oleraceus lectin binding assay

[0261] Table 16 Reagents

[0262]

[0263] method:

[0264] (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 experimenter and the inoculation time. Place at 37℃ 5% CO 2 When the cells reach a confluence of more than 60%, they are ready for staining.

[0265] (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.

[0266] (3) Aspirate the liquid in the wells and wash three times with DPBS, soaking for 5 min each time.

[0267] (4) Add 200 μL of 4% polymethanol to each well and fix for 10 min.

[0268] (5) Aspirate the liquid in the wells and wash three times with DPBS.

[0269] (6) Add 200 μL of FITC-UEA-Ⅰ working solution to each well and incubate at room temperature for 1 h.

[0270] (7) Aspirate the liquid in the wells, wash three times with DPBS, and add 200 μL DPBS.

[0271] (8) Observe and photograph under a fluorescence microscope in a dark room.

[0272] Experimental results and conclusions:

[0273] The experimental results are as follows Figure 5 As 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).

[0274] 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.

[0275] Example 7 Study on the effectiveness of EPC cells in erectile dysfunction (ED) mice

[0276] 1. Experimental animals: db / db spontaneous diabetic mice, 12-16 weeks old, male, 24 mice; db / + wild-type control mice, 12 weeks old, male, 5 mice.

[0277] 2. Test drug: Early / late EPC concentration is 1×10 8 cells / mL.

[0278] 3. Experimental grouping: All animals were grouped by random number method according to age, body weight and blood sugar, and the one with the smallest difference among the groups was taken as the final grouping.

[0279] Table 17 Grouping

[0280]

[0281] 4. Detection indicators

[0282] (1) The mice in each group were weighed on day 0, day 4, day 7, day 10, day 14, and day 17 after administration to observe for any adverse reactions.

[0283] (2) After drug administration, APO was injected subcutaneously in groups G2, G3, and G4, and the behavior of licking the penis within 30 minutes (generally a sign of the animal's ejaculation) was recorded as a counting standard.

[0284] (3) After 17 days of administration, the mice were dislocated by cervical dislocation, and the corpus cavernosum was isolated, fixed, sectioned, and immunohistochemistry was performed to detect the expression of eNOS and CD31 in the corpus cavernosum. Multiple immunofluorescence staining of TAGLN, PODXL, SLC1A3, and eNOS in the corpus cavernosum was used to observe the co-localization and expression of the corpus cavernosum.

[0285] 5. Experimental results

[0286] (1) The results are as follows Figure 6 As shown, after administration to groups G2, G3, and G4, there was no significant change in the body weight of the animals, proving that the drug had no effect on the metabolism of the animals, indicating that a single administration of EPC through the penis is safe.

[0287] (2) The results are as follows Figure 7 As shown in the figure, the number of licking the penis of animals in the G2 model group was significantly lower than that in the normal group, indicating that long-term diabetes reduced the sexual impulse behavior of animals. There was no significant difference in the sexual impulse behavior of animals in G2 and G3, indicating that early EPC could not increase sexual impulse behavior and had no therapeutic effect on ED; in the comparison between G2 and G4, the sexual impulse behavior of animals in G4 increased significantly, indicating that late EPC increased sexual impulse behavior and had a therapeutic effect on ED.

[0288] (3) The results are as follows Figure 8 As shown, by comparing the G1 group, G2 group and G3 group, it can be seen that the expression of eNOS and CD31 in the G2 group and G3 group was significantly downregulated, indicating that the model group may have erectile dysfunction. At the same time, the administration of the G3 group did not increase the expression of eNOS and CD31, indicating that the injection of early EPC has no therapeutic effect on ED; by comparing the G1 group, G2 group and G4 group, the administration of the G4 group significantly increased the expression of eNOS and CD31, and the expression level was higher than that of the G1 group, indicating that the injection of late EPC has a therapeutic effect on ED.

[0289] (4) The results are as follows Fig. 9As shown in the figure, TAGLN is a vascular smooth muscle cell marker gene, PODXL is an endothelial cell marker gene, SLC1A3 is a cavernous fibroblast marker gene, and eNOS is a NO production precursor. Compared with the G1 group, G2 group and G3 group, the expression of TAGLN, PODXL, SLC1A3 and eNOS in the G3 group did not increase significantly, indicating that the injection of early EPC had no therapeutic effect on the ED mouse model; compared with the G1 group, G2 group and G4 group, the expression of TAGLN and SLC1A3 did not increase significantly, and the expression of PODXL and eNOS increased significantly, indicating that the injection of late EPC had a therapeutic effect on the ED mouse model, and its mechanism of action was to increase the amount of endothelial cells and the content of eNOS in the cavernous body, and had no adverse effects on vascular smooth muscle and cavernous fibroblasts.

[0290] 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 early / late EPCs, characterized in that: determining the expression of biomarkers in the cells, the biomarkers comprising CD144, vWF, and CD133; and The EPC is a late EPC when the cell is determined to have the following expression profile: CD144 高 / + 、vWF 高 / + 、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 / - 、vWF 低 / - ; The early EPC or late EPC also has at least one of the following expression profiles: CD31 高 / + 、CD34 高 / + and / or KDR 高 / + ; The EPCs are derived from iPSCs.

2. The method according to claim 1, characterized in that The late EPCs have the following expression profile: CD144 高 / + 、vWF 高 / + 、CD 133 低 / - 、CD31 高 / + 、CD34 高 / + and KDR 高 / + .

3. The method according to claim 1, characterized in that The early EPCs have the following expression profile: CD133 高 / + 、CD144 低 / - 、vWF 低 / - 、CD31 高 / + 、CD34 高 / + and KDR 高 / + .

4. The method according to any one of claims 1 to 3, characterized in that: Determining the expression of at least one biomarker in a cell comprises contacting the cell with a binding agent for the biomarker.

5. A method for sorting, isolating or enriching late EPCs from a cell population, characterized in that: Includes selection of cells with the following expression profiles: CD144 高 / + 、vWF 高 / + and CD 133 低 / - , the EPCs are derived from iPSCs; The method further comprises selecting cells having at least one of the following expression profiles: CD31 高 / + 、CD34 高 / + and / or KDR 高 / + .

6. The method according to claim 5, characterized in that The method comprises selecting cells having the following expression profile: CD144 高 / + 、vWF 高 / + 、CD 133 低 / - 、CD31 高 / + 、CD34 高 / + and KDR 高 / + .

7. The method according to claim 5, characterized in that The method further comprises culturing the late EPCs.

8. A method for sorting, isolating or enriching early EPCs from a cell population, characterized in that: This includes selecting cells with the following expression profiles: CD133 高 / + 、CD144 低 / - and vWF 低 / - , the EPCs are derived from iPSCs; The method further comprises selecting cells having at least one of the following expression profiles: CD31 高 / + 、CD34 高 / + and KDR 高 / + .

9. The method according to claim 8, characterized in that The method comprises selecting cells having the following expression profile: CD133 高 / + 、CD144 低 / - 、vWF 低 / - 、CD31 高 / + 、CD34 高 / + and KDR 高 / + .

10. Use of a biomarker in identifying, sorting, isolating or enriching early / late EPCs, characterized in that: The biomarkers include CD144, vWF and CD133, and the EPCs are derived from iPSCs; The biomarkers also include CD31, CD34 and / or KDR.

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

12. The use according to claim 11, characterized in that: The late EPC also has at least one of the following expression profiles: CD31 高 / + 、CD34 高 / + and / or KDR 高 / + .

13. The use according to claim 12, characterized in that: The late EPCs have the following expression profile: CD144 高 / + 、vWF 高 / + 、CD 133 低 / - 、CD31 高 / + 、CD34 高 / + and KDR 高 / + .

14. A pharmaceutical composition for treating erectile dysfunction, characterized in that: The pharmaceutical composition includes late EPCs having the following expression profiles: CD144 高 / + 、vWF 高 / + and CD133 低 / - , the EPCs are derived from iPSCs.

15. The pharmaceutical composition according to claim 14, characterized in that The pharmaceutical composition also includes other drugs for treating erectile dysfunction.

16. The pharmaceutical composition according to claim 14, characterized in that The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

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