Marker for characterizing EPC subpopulation, EPC subpopulation, and use thereof

Abstract

A marker for characterizing an EPC subpopulation, an EPC subpopulation, and a use thereof. Indicators for distinguishing EPC subpopulations comprise CD144, vWF, CD133, or phagocytic capacity. On the basis of these indicators, identification, sorting, separation, or enrichment of EPC subpopulations can be achieved. Also provided are a use of late EPC in the preparation of a drug for treating stroke, erectile dysfunction, and ischemic diseases, and a method for treating stroke, erectile dysfunction, and ischemic diseases.

Description

Markers for characterizing EPC subpopulations, EPC subpopulations and applications thereof TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and relates to markers for characterizing EPC subpopulations, EPC subpopulations and applications thereof. BACKGROUND

[0002] Among today's medical challenges, blood vessel-related diseases such as stroke, erectile dysfunction (ED) and critical limb ischemia (CLI) pose a major public health problem due to their high incidence, limited treatment options and heavy disease burden.

[0003] Stroke, also known as apoplexy, is a disease of dysfunction of the cerebrovascular system caused by different blood vessel diseases, and is often accompanied by varying degrees of motor dysfunction and sensory dysfunction. Stroke is a major chronic non-communicable disease that seriously endangers the health of the Chinese people, and has five characteristics: high incidence, high disability rate, high mortality, high recurrence rate and heavy economic burden. Currently, the treatment options for ischemic stroke are very limited. The most effective treatment method in the acute phase is thrombolytic therapy, but due to the time window limitation, only a small number of patients can benefit from it, and most of the surviving patients have varying degrees of dysfunction. Other conventional methods include drug therapy, such as anti-platelet aggregation, statin lipid-lowering, blood pressure and blood sugar control, and surgical treatment, such as intracranial and extracranial vascular stent implantation and carotid endarterectomy, which can lead to various complications and side effects, such as increased risk of bleeding with anticoagulant therapy and infection with surgical treatment. Therefore, there is an urgent need for new intervention methods to treat ischemic stroke in clinical practice.

[0004] Erectile dysfunction (ED) is defined as the persistent inability to achieve or maintain a penile erection sufficient to permit satisfactory sexual performance, lasting for 3 months or more. The incidence of ED is high worldwide. ED is a huge hazard. In the past, erectile dysfunction was considered a purely psychogenic disorder in most cases. However, current evidence suggests that more than 80% of cases have organic causes. ED is closely related to heart disease, stroke, diabetes, and other diseases. Diseases such as diabetes can damage the endothelium of blood vessels, affect vascular elasticity, and increase the risk of ED. 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 of increasing cardiovascular mortality. 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 feelings of both spouses and family stability, thus causing important social and public health problems.

[0005] The EAU guidelines currently recommend oral medication, external / urethral use of alprostadil, shock wave therapy, psychosocial intervention and treatment, hormone therapy, vacuum erection devices, intracavernous injection therapy, and prosthesis implantation for the treatment of ED. In addition, stem cell therapy is currently a research hotspot for the treatment of ED. The first-line drug for the treatment of ED is phosphodiesterase type 5 (PDE5) inhibitors, but many patients do not respond to PDE5 inhibitors. Therefore, there is an urgent need for new interventions to treat ischemic erectile dysfunction in clinical practice.

[0006] With the acceleration of population aging, the incidence of peripheral arterial disease (PAD) is increasing year by year, and about 10% of PAD patients further develop into critical limb ischemia (CLI), which is clinically manifested as rest pain (lasting more than 2 weeks), ulcer, or gangrene. The quality of life index of CLI patients is similar to that of patients with advanced cancer, and the mortality rate is more than 50% within 5 years after the initial diagnosis. Traditional surgery or endovascular treatment is currently the preferred treatment for CLI patients, but about 30% of patients are not suitable for surgery or endovascular treatment due to severe complications. Therefore, improving limb blood perfusion and related clinical symptoms in CLI patients through non-surgical treatment, and reducing the amputation rate and mortality rate, are the current clinical problems that need to be solved.

[0007] In recent years, induced pluripotent stem cell-derived endothelial progenitor cells (iPSCs-EPCs) have shown potential in the treatment of cardiovascular and vascular-related diseases, but further optimization is needed to improve efficacy and provide new cell therapy directions for vascular-related diseases such as stroke, erectile dysfunction, and lower extremity ischemia. SUMMARY

[0008] To solve the problems existing in the prior art, the inventors of the present application first discovered biomarkers for distinguishing early EPCs (early EPCs) and late EPCs (late EPCs) through extensive and in-depth research, and further discovered that late EPCs, but not early EPCs, have the effect of treating stroke, erectile dysfunction, ischemic diseases, and especially lower extremity ischemia. Based on this, the present application provides the following technical solutions:

[0009] The first aspect of the present application provides a biomarker for identifying or detecting early / late EPCs, the biomarker comprising CD144, vWF and / or CD133.

[0010] The second aspect of the present application provides a product comprising a binding agent capable of binding to the biomarker described in the first aspect of the present application.

[0011] The third aspect of the present application provides a device or apparatus for identifying, enriching, isolating or sorting early / late EPCs, the device or apparatus comprising a cell binding unit configured to bind the biomarker described in the first aspect of the present application.

[0012] The fourth aspect of the present application provides a late EPC, the late EPC having at least one of the following expression profiles: CD144 高 / + , vWF 高 / + , CD133 低 / - .

[0013] The fifth aspect of the present application provides an early EPC, the early EPC having at least one of the following expression profiles: CD133 高 / + , CD144 低 / - , vWF 低 / - .

[0014] The sixth aspect of the present application provides a method for identifying, enriching, isolating or sorting early / late EPCs, determining the expression of the biomarker described in the first aspect of the present application in EPC cells, and when it is determined that the EPC cells have at least one of the following expression profiles, the EPC is a late EPC: CD144 高 / + , vWF 高 / + , CD133 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 / - .

[0015] The seventh aspect of the present application provides a method for identifying an EPC subpopulation, the method comprising detecting phagocytic capacity of a cell.

[0016] The eighth aspect of the present application provides use of the biomarker of the first aspect of the present application, the product of the second aspect of the present application, the device or apparatus of the third aspect of the present application in identifying, enriching, isolating or sorting early / late EPCs.

[0017] The ninth aspect of the present application provides use of the biomarker of the first aspect of the present application or the product of the second aspect of the present application in the preparation of a product for diagnosing a disease associated with early EPCs or late EPCs.

[0018] The tenth aspect of the present application provides use of the late EPC of the fourth aspect of the present application in the preparation of a medicament for treating a disease selected from one or more of stroke, erectile dysfunction, and ischemic diseases.

[0019] The eleventh aspect of the present application provides a pharmaceutical composition for treating a disease, the pharmaceutical composition comprising the late EPC of the fourth aspect of the present application, the disease being selected from one or more of stroke, erectile dysfunction, and ischemic diseases.

[0020] The twelfth aspect of the present application provides a method for treating a disease, the method comprising administering an effective amount of the late EPC of the fourth aspect of the present application and / or the pharmaceutical composition for treating a disease of the eleventh aspect of the present application, the disease being selected from one or more of stroke, erectile dysfunction, and ischemic diseases.

[0021] The thirteenth aspect of the present application provides a method for determining purity of a differentiated product, the method comprising:

[0022] detecting expression level of at least one biomarker of a cell in a sample of the differentiated product, the biomarker being selected from CD144, vWF and / or CD133; determining purity of the differentiated product according to the expression level of the biomarker.

[0023] The fourteenth aspect of the present application provides a method for quantifying an EPC subpopulation in a sample, the method comprising:

[0024] detecting expression level of at least one biomarker of cells in a sample of interest, the biomarker being selected from the group consisting of CD144, vWF and / or CD133; determining content of EPC subpopulation according to expression level of the biomarker.

[0025] The fifteenth aspect of the present application provides a method for screening candidate drugs for regulating EPC subpopulation, the method comprising adding a drug to be screened into a cell population, detecting expression level of a biomarker of the cells, the biomarker being selected from the group consisting of CD144, vWF and / or CD133; when the drug to be screened changes expression level of the biomarker of the cells, the drug to be screened is a candidate drug for regulating EPC subpopulation.

[0026] The present application has the advantages and beneficial effects of:

[0027] The present application first discovers biomarkers for distinguishing early EPCs and late EPCs, and based on the biomarkers, different EPC subpopulations can be identified, enriched, separated or sorted.

[0028] The present application also provides a method for determining purity of a differentiation product, a method for quantifying EPC subpopulation in a sample, and a method for screening candidate drugs for regulating EPC subpopulation.

[0029] The present application first discovers that phagocytic capacity can be used to distinguish early EPCs and late EPCs.

[0030] The present application first discovers that late EPCs but not early EPCs can be used for treating stroke, erectile dysfunction, ischemic diseases, especially lower limb ischemia, and based on this, the therapeutic effect of endothelial progenitor cells on stroke, erectile dysfunction, ischemic diseases, especially lower limb ischemia can be improved by increasing the content of late EPCs, which are effective components for treating stroke, erectile dysfunction, ischemic diseases, especially lower limb ischemia. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 is a diagram of detection results of early EPC cell surface marker genes;

[0032] Figure 2 is a diagram of detection results of late EPC cell surface marker genes;

[0033] Figure 3 is a diagram of detection results of early EPC surface marker genes by flow cytometry;

[0034] Figure 4 is a diagram of detection results of late EPC surface marker genes by flow cytometry;

[0035] Figure 5 is a diagram of detection results of phagocytic capacity;

[0036] Figure 6 is a diagram of detection results of TTC staining for determining the range of cerebral infarction;

[0037] Figure 7 is a graph of cerebral infarction area detection results;

[0038] Figure 8 is a graph of animal body weight change after administration;

[0039] Figure 9 is a graph of the number of times of licking the penis by subcutaneous injection of APO;

[0040] Figure 10 is a graph of the expression of eNOS and CD31 in the corpus cavernosum of the penis;

[0041] Figure 11 is a graph of multiple immunofluorescence staining of the corpus cavernosum of the penis;

[0042] Figure 12 is a graph of laser speckle blood flow detection;

[0043] Figure 13 is a graph of the rate of lower limb ischemia in mice. DETAILED DESCRIPTION

[0044] The present application is the result of extensive and in-depth research, through the study of different EPC subpopulations, a marker capable of distinguishing early and late EPCs is found, and it is further proved that late EPCs, but not early EPCs, have the effect of treating stroke, erectile dysfunction, ischemic diseases, especially lower limb ischemia.

[0045] In the present application, the use of alternatives (e.g. "or") should be understood as meaning any one of the alternatives, all or any combination thereof. In the present application, the terms "comprising", "having" and "including" are used synonymously, and these terms and variants thereof are intended to be interpreted non-limitingly.

[0046] For the purposes of the present application, the terms "selected", "sorted", "fractionated" or "isolated" cells, cell populations or cell subpopulations can be used interchangeably, and unless the context indicates otherwise, mean removal of the selected cells or defined subset of cells from a tissue sample and separation from other cells and contaminants that are not within the parameters defining such cells or cell population. Isolated EPC subpopulations will typically not be contaminated with other cell types by and have the capacity for self-renewal allowing the generation of differentiated progeny. However, when the process results in a cell population, it will be understood that it is not practical to provide compositions having absolute purity. In such cases, the cell population is "enriched" for the selected cells, wherein these selected cells then exist in the presence of various contaminants, including other cell types, that do not substantially interfere with the function or characteristics of the selected cell subpopulation.

[0047] The terms "marker", "markers" or "cell markers" are synonymous and mean any trait or characteristic in the form of a chemical or biological entity. The markers can be morphological, functional or biochemical in nature. In embodiments of the application, the markers are differentially or preferentially expressed by a particular cell type, or are expressed by a cell under certain conditions, e.g., a cytokine or surface antigen or membrane protein or cytoplasmic protein, etc., at a particular point in the cell cycle or under a particular extracellular matrix. More specifically in the present application are those markers by which a cell or subpopulation of cells can be indicated by the level of expression thereof.

[0048] In the present application "marker" means any moiety that can be used to identify a desired EPC. For example, a 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 sufficiently high compared to other cells 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 appreciate that expression is a relative term and that other cell types have different expression.

[0049] The terms "subject" or "patient" are used interchangeably. The term includes, but is not limited to, humans, non-human animals. The humans, non-human animals and includes all vertebrates, e.g., non-human primates, such as Rhesus monkeys, chimpanzees and other ape and monkey species; farm animals such as cows, sheep, pigs, goats and horses; domesticated subjects such as dogs and cats; laboratory animals including rabbits, rodents such as mice, rats and guinea pigs, and non-mammals such as birds, including poultry, game and sporting birds, such as chickens, turkeys and other galliformes, ducks and geese, amphibians, reptiles, and the like. The term does not indicate a specific age or sex. Thus, both adult and newborn subjects, as well as fetuses, whether male or female, are contemplated. The term "subject" also includes living organisms susceptible to a condition or disease state as generally disclosed throughout this specification, but not limited thereto. In one embodiment, examples of subjects include humans, dogs, cats, cows, goats, and mice, including transgenic species. In another embodiment, the subject is an experimental animal or animal surrogate as a model of disease, e.g., a dog.

[0050] As used herein, "induced pluripotent stem cell (iPSC)" refers to a type of pluripotent stem cell artificially derived from a non-pluripotent cell, typically a somatic cell. The somatic cell can be derived from a human or a non-human animal, as explained above. In some embodiments, the somatic cell is a human somatic cell. Examples of somatic cells include, but are not limited to, dermal fibroblasts, bone marrow-derived mesenchymal cells, cardiomyocytes, keratinocytes, hepatocytes, gastric cells, neural stem cells, lung cells, kidney cells, spleen cells, and pancreatic cells. Other examples of somatic cells include cells of the immune system, including but not limited to B cells, dendritic cells, granulocytes, natural lymphocytes, megakaryocytes, monocytes / macrophages, bone marrow-derived suppressor cells, natural killer (NK) cells, T cells, thymic cells, and hematopoietic stem cells.

[0051] As used herein, "bind" refers to a non-covalent interaction between macromolecules (e.g., between a protein and a polynucleotide, between polynucleotides, or between proteins, etc.). Such non-covalent interactions are also referred to as "associating" or "interacting" (e.g., if a first macromolecule interacts with a second macromolecule, the first macromolecule is bound to the second macromolecule in a non-covalent manner). Certain portions of the binding interaction can be sequence-specific.

[0052] As used herein, the term "modulate" refers to a change in the quantity, degree, or number of a property, activity, function, or physical molecule. Modulation can be determined by any feature that determines a direct or indirect effect on the expression of a target gene. These features include, for example, changes at the RNA or protein level, protein activity, product level, expression of a gene, or level of activity of a reporter gene.

[0053] A first aspect of the present application provides a biomarker for identifying or detecting early / late EPCs, the biomarker comprising CD144, vWF, and / or CD133.

[0054] In some embodiments, the CD144, vWF is highly expressed in late EPCs, and CD133 is not expressed or lowly expressed in late endothelial progenitor cells.

[0055] In some embodiments, the CD144, vWF is not expressed or lowly expressed in early EPCs.

[0056] In some embodiments, the biomarker further comprises CD31, CD34, and / or KDR.

[0057] In some embodiments, the EPCs are derived from iPSCs.

[0058] In another aspect of the application there is provided a product comprising a binding agent capable of binding to a biomarker as defined in the first aspect of the application.

[0059] In the present application, the terms "binding agent", "binding molecule" and "binding entity" are synonymous and used interchangeably. In the context of the present application, a binding agent binds to, recognises, interacts with, reacts with or otherwise associates with a selection marker on the subpopulation of cells. Exemplary binding agents can 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.

[0060] In some embodiments, the binding agent is selected from a nucleic acid, a ligand, an enzyme, a substrate and / or an antibody.

[0061] In some embodiments, the nucleic acid comprises DNA or RNA. Exemplary nucleic acids include probes, primers.

[0062] In some embodiments, the binding agent is selected from a probe that hybridises to a biomarker as previously described.

[0063] In some embodiments, the binding agent is selected from a primer that binds to a biomarker as previously described.

[0064] In some embodiments, the binding agent is selected from an antibody that binds to a protein or protein fragment encoded by the biomarker.

[0065] In the present application, "antibody" is used in the broadest sense and specifically covers, for example, monoclonal antibodies, polyclonal antibodies, antibodies with polyepitopic specificity, single-chain antibodies, multispecific antibodies and antibody fragments. Such antibodies can be chimeric, humanised, human and synthetic. In some embodiments, antibodies include single domain antibodies, further engineered molecules such as, but not limited to, diabodies, triabodies, tetrabodies, minibodies and the like, 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 diabodies, nanobodies, domain antibodies, bivalent domain antibodies and minimal recognition units consisting of the amino acid residues that mimic the hypervariable regions of antibodies (e.g., an isolated complementarity determining region (CDR)).

[0066] In some embodiments, the binding agent is linked to a detectable molecule.

[0067] In some embodiments, the detectable molecule can be selected from the group consisting of: a fluorescent molecule, a luminescent molecule, an absorbent molecule, a chemiluminescent molecule, a radioactive molecule, an enzymatic molecule, and a secondary antibody. By "detectable molecule" is meant to include any molecule that can be detected and the relative amount and / or relative position of the moiety is determined.

[0068] 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 a particular condition. For example, a fluorescent molecule can require exposure to radiation (i.e. light) at a particular wavelength and intensity to cause excitation of the fluorescent molecule, thereby enabling it to emit detectable fluorescence at a particular wavelength.

[0069] In some embodiments, the detectable molecule can be an enzyme that is capable of converting a (preferably non-detectable) substrate into a detectable product that can be visualized and / or detected. Examples of suitable enzymes for use can be those known for use in assays such as ELISA. These include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase or acetylcholinesterase.

[0070] In some embodiments, the detection moiety can be a radioactive molecule. Suitable radioactive molecules include those used in scintigraphic studies such as 99 mTc and 123 I. Other readily detectable moieties include spin labels such as 123 I, 131 I, 111 In, 19 F, 13 C, 15 N, 17 O, gadolinium, manganese or iron.

[0071] Radiolabelling or other labelling can be incorporated into the agents of the application (i.e. the proteins present in the sample of the method of the application and / or the binding agents of the application) in known ways. For example, if the binding moiety is a polypeptide, it can be biosynthesised or it can be synthesised by chemical amino acid synthesis using suitable amino acid precursors (including, for example, fluorine-19 in place of hydrogen). For example, 99m Tc, 123 I, 186 Rh, 188 Rh and 111 In can be attached, for example, via a cysteine residue in the binding moiety. Yttrium-90 can be attached via a lysine residue. 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.

[0072] In some embodiments, the detectable molecule can include a molecule that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means (e.g., fluorescent, chemiluminescent, or chemiluminescent), or any other appropriate means. In some embodiments, the detectable molecule can be a primary molecule (where the molecule contains a group that can be directly detected or generates a directly detectable group); or a secondary molecule (where the detectable molecule binds to another group to generate a detectable signal, e.g., common in immuno-labeling using secondary and tertiary antibodies). The detectable molecule can be linked to the agent by covalent or non-covalent means. Alternatively, the detectable label can be linked, e.g., by direct labeling, by ligand-receptor binding pairs or other such specific recognition molecules to effect binding of the molecule to the agent.

[0073] In some embodiments, the expression levels of the plurality of markers of the present application can be determined simultaneously (e.g., multiplexed) or in parallel.

[0074] In other embodiments, the gene expression products (proteins) associated with the biomarkers of the present application can be detected to determine the presence of the EPC subpopulation. These detection techniques are known to those of skill in the art and include, for example, ELISA (enzyme-linked immunosorbent assay), western blot, FACS, radioimmunoassay; (RIA); sandwich assays; fluorescence in situ hybridization (FISH); immunohistological staining; immunoelectrophoresis; immunoprecipitation; and immunofluorescence using detection reagents such as antibodies or protein binding agents.

[0075] In the present application, actual monitoring or analysis of the biomarkers can be performed using any of a number of standard techniques well known to those of skill 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, agglutuation assays, complement-fixation assays, immunoradioassay, fluorescent immunoassay, immunofluorescence, protein A immunoassay, laser capture microdissection, massively multiparametric mass cytometry, flow cytometry, and FACS analysis.

[0076] In some embodiments, the product comprises a reagent or a kit.

[0077] In the present application, the kits can be used in a clinical setting for patient diagnostic purposes or in research for the characterization and / or enrichment of EPC subpopulations. A kit according to the present application will include one or more containers containing the binding agents and a label or package insert on or associated with the container(s). Suitable containers include, for example, bottles, vials, syringes, 96-well plates, etc. The containers can be formed from a variety of materials such as glass or plastic. Such containers can hold one or more compositions, which include binding agents effective for analyzing EPCs and, optionally, providing enriched or isolated cells or cell subpopulations as described herein. Typically, such kits will contain one or more preparations of binding agents in suitable containers, and where multiple binding agents are involved, the binding agents can be in the same or different containers. The kits can also contain other pharmaceutically acceptable formulations for use in diagnosis or for labeling or modifying the bound agents.

[0078] More specifically, the kits can have a single container with one or more binding agents, with or without additional components, or they can have different containers for each component. Where a combination reporter molecule is provided for binding, the individual solutions can be pre-mixed in molar equivalents or where one component is in excess of the remaining components. Alternatively, the binding agents of the kit and any optional reporter molecules can be maintained separately within different containers prior to administration to a subject or use in vitro. The kits can also include second / third container means for containing sterile, pharmaceutically-acceptable buffers or other diluents, e.g., bacteriostatic water for injection (BWFI), phosphate-buffered saline (PBS), Ringer's solution, and dextrose solution.

[0079] When the components of the kit are provided in one or more liquid solutions, the liquid solution is in some embodiments an aqueous solution, particularly preferably a sterile aqueous solution. However, the components of the kit can be provided as dry powders. When the reagents or components are provided as dry powders, the powders can be reconstituted by the addition of a suitable solvent. It is contemplated that the solvent can also be provided in another container.

[0080] The kits can also include instructions for using the kit components to perform a method. The instructions for performing the method are typically recorded on a suitable recording medium. For example, the instructions can be recorded in a tabular format on paper or plastic, etc. The instructions can be present in the kit in a variety of forms. For example, the instructions can be printed on a paper label, in the packaging material, etc. Electronic storage media, optical storage media, and various other forms of storage media can also be used.

[0081] In some embodiments, the product is a product for identifying, enriching, isolating or sorting early / late EPCs.

[0082] In one aspect the present application provides a device or apparatus for identifying, enriching, isolating or sorting early / late EPCs, said device or apparatus comprising a cell binding unit configured to bind a biomarker according to the first aspect of the present application.

[0083] In another aspect the present application provides a late EPC having at least one of the following expression profiles: CD144 高 / + , vWF 高 / + , CD 133 低 / - .

[0084] In some embodiments the late EPC has the following expression profile: CD144 高 / + , vWF 高 / + and CD 133 低 / - .

[0085] In some embodiments the late EPC has at least one of the following expression profiles: CD31 高 / + , CD34 高 / + and / or KDR 高 / + .

[0086] In some embodiments the late EPC has the following expression profile: CD144 高 / + , vWF 高 / + , CD 133 低 / - , CD31 高 / + , CD34 高 / + and KDR 高 / + .

[0087] In some embodiments the EPC is derived from an iPSC.

[0088] In another aspect the present application provides an early EPC having at least one of the following expression profiles: CD133 高 / + , CD144 低 / - , vWF 低 / - .

[0089] In some embodiments the early EPC has the following expression profile: CD133 高 / + , CD144 低 / - and vWF 低 / - .

[0090] In some embodiments the early EPC has at least one of the following expression profiles: CD31 高 / + , CD34 高 / + and / or KDR 高 / + .

[0091] In some embodiments, the early EPCs have the following expression profile: CD133 高 / + , CD144 低 / - , vWF 低 / - , CD31 高 / + , CD34 高 / + , and KDR 高 / + .

[0092] In some embodiments, the EPCs are derived from iPSCs.

[0093] Another aspect of the application provides a method of identifying, enriching, isolating or sorting early / late EPCs, determining the expression of the biomarkers described supra in the EPC cells, the EPCs being late EPCs when it is determined that the EPC cells have at least one of the following expression profile: CD144 高 / + , vWF 高 / + , CD133 低 / - ; the EPC cells being early EPCs when it is determined that the cells have at least one of the following expression profile: CD133 高 / + , CD144 低 / - , vWF 低 / - .

[0094] In some embodiments, the EPCs are derived from iPSCs.

[0095] In some embodiments, the early EPCs or late EPCs further have at least one of the following expression profile: CD31 高 / + , CD34 高 / + , and / or KDR 高 / + .

[0096] In some embodiments, the late EPCs have the following expression profile: CD144 高 / + , vWF 高 / + , CD133 低 / - , CD31 高 / + , CD34 高 / + , and KDR 高 / + .

[0097] In some embodiments, the early EPCs have the following expression profile: CD133 高 / + , CD144 低 / - , vWF 低 / - , CD31 高 / + , CD34 高 / + , and KDR 高 / + .

[0098] The present invention uses the term "enriched" or variants thereof to describe a population of cells 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 when compared to a population of untreated cells (e.g., cells in their native environment).

[0099] In one example, the term "enriched" refers to a proportion or percentage of a subpopulation of EPC cells that is greater than the proportion or percentage of EPCs in the population of cells from which it was originally contained.

[0100] In some embodiments, the method of identifying, enriching, isolating or sorting comprises contacting the cells with a binding agent for the biomarker and directly or indirectly detecting the presence of the complex formed.

[0101] In some embodiments, the method further comprises sorting the EPCs bound to the binding agent. Methods for isolating and selecting cells based on expression of cell surface markers are familiar to those of skill in the art. These include, but are not limited to, affinity-based interactions, affinity panning, magnetic beads (e.g., Dynabeads) or flow cytometry.

[0102] In some embodiments, the sorting or isolation technique comprises fluorescence activated cell sorting, magnetic assisted cell sorting, substrate assisted cell sorting, laser mediated cleavage, fluorometry, flow cytometry or microscopy.

[0103] In some embodiments, the cells can be isolated by binding to an immobilized support and then harvested by removing the cells from the support.

[0104] Harvesting of the cells can be achieved by collecting the isolated cells into a suitable container or collection dish, test tube, etc.

[0105] In some embodiments, the method is a method of identifying, enriching, isolating or sorting early / late EPCs from a population of cells.

[0106] The biomarkers of the present invention can be detected individually, in combination, sequentially, simultaneously, synchronously and / or in conjunction with other biomarkers, which can be specific for endothelial progenitor cells.

[0107] In some embodiments, the expression of one or more of the biomarkers listed above is determined to determine the presence of a subpopulation of EPCs in, for example, a composition, solution, cell aggregate, cell suspension or tissue.

[0108] In some embodiments, the expression of at least one biomarker in a cell is determined, comprising contacting the cell with a binding agent for the biomarker.

[0109] In some embodiments, biomarker expression can be assessed by any of a variety of well-known methods for detecting transcriptional molecules (e.g., mRNA) or protein expression. Non-limiting examples of such methods include immunological methods for detection, protein purification methods, protein function or activity assays, nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods.

[0110] Various amplification and detection methods can be used to aid in the detection of expression of biomarkers. For example, reverse transcription of mRNA into cDNA prior to further analysis (e.g., polymerase chain reaction, RT-PCR) is within the scope of the present application. Other known amplification methods that can be utilized in the present application include, but are not limited to, "NASBA" or "3SR" technology; Q-beta amplification; strand displacement amplification; target-mediated amplification; ligase chain reaction (LCR); self-sustained sequence replication (SSR); and transcription amplification.

[0111] Methods of detecting, characterizing, and / or quantifying nucleic acid sequences; and methods for detecting, characterizing, and / or quantifying mRNA expression are known to those of skill in the art and include, but are not limited to, for example, PCR procedures, RT-PCR, quantitative PCR or RT-PCR, Northern blot analysis, differential gene expression, RNA protection assay, microarray analysis, hybridization assay, serial analysis of gene expression (SAGE), hybridization-based quantitative assay using digital barcodes, multiplex RT-PCR, digital droplet PCR (ddPCR), qRT-PCR, qPCR, ultraviolet spectroscopy, DNA sequencing, RNA sequencing, next generation sequencing, including RNAseq, lysing-based hybridization assay utilizing branched DNA signal amplification, such as QuantiGene 2.0 Single Plex, and branched DNA analysis methods.

[0112] Non-limiting examples of nucleic acid sequencing technologies, for example for DNA sequencing and RNA sequencing, include Maxam-Gilbert sequencing, Sanger sequencing (i.e., chain termination), sequencing by synthesis (SBS), ligation sequencing, pyrophosphorolysis sequencing, single molecule real-time sequencing, MiSeq sequencing, massively parallel signature sequencing (MPSS), polymerase colony sequencing, 454 sequencing, nanopore sequencing. The present application also includes, but is not limited to, next generation sequencing technologies.

[0113] Non-limiting examples of next generation sequencing technologies include, for example, Ion Torrent, Illumina, SOLiD, 454; massively parallel signature sequencing solid phase, reversible dye terminator sequencing; and DNA nanoball sequencing. Digital barcoding quantification analysis can include BeadArray (Illumina), xMAP system (Luminex), nCounter (Nanostring), high throughput genomics (HTG) molecular, BioMark (Fluidigm), or Wafergen microarray. Detection methods can include DASL (Illumina), RNA-Seq (Illumina), Truseq (Illumina), SureSelect (Agilent), Bioanalyzer (Agilent), and TaqMan (ThermoFisher).

[0114] Another aspect of the present application provides a method of identifying a subpopulation of EPCs, the method comprising detecting phagocytic capacity of the cells.

[0115] In some embodiments of the method of identifying a subpopulation of EPCs, or the method of identifying early / late EPCs, the late EPCs have stronger phagocytic capacity than the early EPCs.

[0116] In some embodiments, the method detects phagocytic capacity of the cells by detecting uptake capacity of acetylated LDL.

[0117] An aspect of the present application provides use of the biomarker, product, device or apparatus as described above in identifying, enriching, isolating or sorting early / late EPCs.

[0118] Another aspect of the present application provides use of the biomarker or product as described above in the manufacture of a product for diagnosing a disease associated with early EPCs or late EPCs.

[0119] An aspect of the present application provides use of the late EPCs as described above in the manufacture of a medicament for a disease selected from one or more of stroke, erectile dysfunction, and ischemic diseases.

[0120] In some embodiments, the stroke comprises ischemic stroke and hemorrhagic stroke. The ischemic stroke comprises transient ischemic attack, atherosclerotic thrombotic cerebral infarction, lacunar cerebral infarction, cerebral embolism. The hemorrhagic stroke comprises intracerebral hemorrhage, subarachnoid hemorrhage.

[0121] In the present invention, the main disease-related causes of erectile dysfunction include aging, atherosclerosis, chronic kidney disease, diabetes, hypertension and anti-hypertensive drugs, pelvic surgery and radiation therapy, and psychological anxiety. Erectile dysfunction can be caused by a number of disorders including neurogenic, arterial, and veno-occlusive dysfunction, as well as other conditions that result in incomplete relaxation of smooth muscle. Thus, the methods of the present invention can treat, prevent, or ameliorate symptoms of a disease or condition, e.g., selected from the group consisting of: aging, atherosclerosis, chronic kidney disease, diabetes, hypertension, side effects from drugs (e.g., anti-hypertensive drugs), pelvic surgery, radiation therapy, and psychological anxiety, wherein the symptoms are erectile dysfunction.

[0122] In some embodiments, the late EPCs promote the expression level of eNOS, CD31 in the corpus cavernosum of the penis.

[0123] In some embodiments, the late EPCs promote the expression level of eNOS, CD31 in the corpus cavernosum of the penis.

[0124] In some embodiments, the ischemic disease is selected from lower extremity ischemia.

[0125] In some embodiments, the late EPCs are obtained by the methods of the embodiments of the present invention.

[0126] In some embodiments, the late EPCs are obtained by the isolation or enrichment methods described above.

[0127] In some embodiments, the pharmaceutical composition further comprises other drugs for treating stroke. The other drugs for treating stroke include, but are not limited to, thrombolytic drugs such as alteplase, urokinase, streptokinase, recombinant streptokinase, lumbrukinase, streptokinase, etc., neuroprotective drugs such as citicoline, brain protein extract, deproteinized calf serum extract, ganglioside, edaravone, etc., calcium channel blockers such as nimodipine, cinnarizine, flunarizine, etc., and brain metabolism activators such as bilaracitan, olracitan, anracitan, etc.

[0128] In some embodiments, the pharmaceutical composition further comprises other drugs for treating erectile dysfunction. The drugs include, but are not limited to, hormones, PDE5 inhibitors such as sildenafil, tadalafil, vardenafil, avanafil.

[0129] In some embodiments, the pharmaceutical composition described above further comprises a pharmaceutically acceptable carrier.

[0130] In the present application, "pharmaceutically acceptable" means a composition that is physiologically acceptable and does not usually cause allergic reactions such as gastrointestinal disorders, dizziness, or the like, or reactions similar thereto, upon administration to a subject. Also, the composition including a pharmaceutically acceptable carrier can be in various dosage forms for oral or parenteral administration. At the time of formulation, a diluent or an excipient such as a filler, a bulking agent, a binding agent, a wetting agent, a disintegrating agent, a surfactant, and the like, which are generally used, can be used to prepare. The above-mentioned carrier can be one or more selected from the group consisting of lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, physiological saline, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, dextrin, calcium carbonate, propylene glycol, and liquid paraffin, but is not limited thereto, and all of the usual carriers can be used. For example, a non-oral administration carrier such as water, an appropriate oil, aqueous glucose, and ethylene glycol, and the like, can also include a stabilizer and a preservative. An appropriate stabilizer is sodium bisulfite, sodium sulfite, or an antioxidant such as ascorbic acid. An appropriate preservative is benzalkonium chloride, methylparaben, or propylparaben, and chlorobutanol. The above-mentioned ingredients can be added to the mixture of the late EPCs as the above-mentioned effective ingredient, alone or in combination.

[0131] Also, the above-mentioned pharmaceutical composition can be administered by any device capable of moving the pharmaceutical composition to the target cell.

[0132] The pharmaceutical composition of the present application can be administered by any usual route capable of reaching the target tissue. It can be administered non-orally, for example, intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, but is not limited thereto. It can also be administered parenterally.

[0133] In some embodiments, the pharmaceutical composition of the present application can have any dosage form selected from the group consisting of a tablet, a pill, a powder, a granule, a capsule, a suspension, an infusion, an intraliquid, an emulsion, a syrup, a sterilized aqueous solution, a non-aqueous solvent, a suspension, an oil, a freeze-dried agent, and a suppository.

[0134] In some embodiments, the solid dosage form for oral administration can include a tablet, a pill, a powder, a granule, a capsule, and the like, and such a solid preparation can be mixed with one or more excipients to one or more mixtures, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like, to prepare. Also, in addition to the simple excipient, a lubricant such as magnesium stearate, talc, and the like, can be used.

[0135] Liquid preparations for oral administration can be suspensions, solutions, emulsions, syrups, etc., and in addition to water, which is most frequently used as the simple diluent, various other excipients, such as wetting agents, sweetening agents, aromatizing agents, preserving agents, etc., can be included.

[0136] In some embodiments, the formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, oil solutions, lyophilized formulations, suppositories, etc. Non-aqueous solvents, suspending agents can use propylene glycol, polyethylene glycol, vegetable oils such as olive oil, ethyl oleate, etc. injectable lipids, etc. The base of the suppository can use witepsol, polyethylene glycol, tween 61, cocoa butter, lauric acid ester, glycerol, gelatin, etc.

[0137] In some embodiments, the dosage form of the pharmaceutical composition is an infusion.

[0138] In some embodiments, the infusion of the present application contains infusion solutions such as normal saline, Ringer's lactated solution, compound electrolyte injection, 5% glucose injection, 20% HSA injection, succinyl gelatin injection, succinyl gelatin MIX injection, MZJ injection 1, MZJ injection 2, MZJ injection 3, human serum albumin injection, bruce force A, potassium chloride injection, magnesium sulfate injection, sodium bicarbonate injection, glucose sodium chloride injection, compound sodium chloride injection (Ringer's solution), dextran 20 glucose injection (small molecule), amino acid injection, hydroxyethyl starch 40 sodium chloride injection, hydroxyethyl starch 40 sodium chloride injection, hydroxyethyl starch 40 sodium chloride injection, low molecular weight heparin calcium for injection, heparin sodium injection, coenzyme A for injection, cytidine disodium triphosphate, lysine hydrochloride for injection, vitamin C injection, cytidine disodium triphosphate, fat-soluble vitamin II for injection, reduced glutathione for injection, brain protein hydrolysate for injection, deoxyribonucleotide sodium injection, multiple trace element injection II, mannitol injection, arginine hydrochloride injection, potassium chloride injection, cytidine disodium triphosphate for injection, ornithine aspartate for injection, etc.

[0139] The infusion of the present application can also contain one or more additional agents, such as selected from the group consisting of solubilizing agents, humectants, emulsifiers, buffers, suspending agents, chelating agents, antioxidants, bacteriostatic agents, local anesthetics, isotonicity adjusting agents, fillers, protective agents, and any combination thereof.

[0140] In one aspect, the present application provides a method for treating a disease, the method comprising administering an effective amount of the late EPCs according to the fourth aspect of the present application and / or the pharmaceutical composition for treating a disease according to the eleventh aspect of the present application, the disease being selected from one or more of the group consisting of stroke, erectile dysfunction, and ischemic disease.

[0141] In some embodiments, the ischemic disease is selected from lower extremity ischemia.

[0142] A "therapeutically effective amount" will be that amount of an active agent that is capable of preventing or at least diminishing (reducing) a medical condition such as stroke, erectile dysfunction, ischemic disease, particularly lower extremity ischemia. The dosage and administration of the cells or pharmaceutical compositions of the present disclosure can be determined by one of ordinary skill in the art of clinical pharmacology or pharmacokinetics. The effective amount of the active agent of the present invention used therapeutically will depend, e.g., on the therapeutic objectives, the route of administration, and the condition of the patient. Accordingly, it can be necessary to adjust the dosage and vary the route of administration to obtain the optimal therapeutic effect.

[0143] In some embodiments, the late EPCs have the biomarkers as described above. As an alternative embodiment, the late EPCs are obtained by the methods in the examples of the present invention. As another alternative embodiment, the late EPCs are obtained by the methods of identifying, enriching, isolating or sorting late EPCs as described above. As another alternative embodiment, the late EPCs are obtained by the methods of sorting, isolating or enriching late EPCs from a cell population as described above. By the methods, the content or proportion of late EPCs in a cell population can be increased.

[0144] For therapeutic use, "dosage regimen" can vary according to many factors, including the activity of the particular composition used, the age, body weight, general health, sex, and diet of the patient, the time of administration, the route of administration, the rate of excretion, the combination with other drugs, and the severity of the particular disease being treated. Although the dosage of the pharmaceutical composition can vary according to the patient's condition and body weight, the severity of the disease, the form of the drug, and the route and duration of administration, it can be appropriately selected by one skilled in the art, and the administration can be performed once a day or several times a day. The dosage does not mean 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 a pill, a sugar-coated tablet, a capsule, a liquid, a gel, a syrup, a slurry, or a suspension.

[0145] Another aspect of the present invention provides a method of determining the purity of a differentiated product, the method comprising:

[0146] detecting the expression level of at least one biomarker of cells in a sample of the differentiated product, the biomarker selected from the group consisting of CD144, vWF and / or CD133; determining the purity of the differentiated product according to the expression level of the biomarker.

[0147] In some embodiments, the cells in the sample are present in the form of a cell mixture, a cell aggregate, or a tissue.

[0148] Another aspect of the present invention provides a method of quantifying a subpopulation of EPCs in a sample, the method comprising:

[0149] detecting the expression level of at least one biomarker of the cells in the sample of interest, the biomarker being selected from the group consisting of CD144, vWF and / or CD133; determining the content of the EPC subpopulation according to the expression level of the biomarker.

[0150] In some embodiments, the at least one cell having the following expression profile is a late EPC: CD144 高 / + , vWF 高 / + , CD 133 低 / - .

[0151] In some embodiments, the at least one cell having the following expression profile is an early EPC: CD133 高 / + , CD144 低 / - and / or vWF 低 / - .

[0152] In some embodiments, the sample of interest comprises a mixture of cells of different types.

[0153] In some embodiments, the sample of interest comprises a mixture of cells of different types, and when the sample of interest comprises a mixture of cells of different types, the method for quantifying the EPC subpopulation in the sample can comprise detecting the expression level of a marker confirming EPCs in addition to detecting the expression level of the aforementioned biomarkers.

[0154] Another aspect of the present application provides a method for screening a candidate drug for modulating the EPC subpopulation, the method comprising adding a substance to be screened to a cell population, detecting the expression level of a biomarker of the cells, the biomarker being selected from the group consisting of CD144, vWF and / or CD133; when the substance to be screened changes the expression level of the marker of the cells, the substance to be screened is a candidate drug for modulating the EPC subpopulation.

[0155] In some embodiments, if the addition of the substance to be screened results in an increased expression profile of the following markers: CD144 高 / + , vWF 高 / + , CD 133 低 / - , the substance to be screened is a candidate drug for promoting late EPCs.

[0156] In some embodiments, if the addition of the substance to be screened results in an increased expression profile of the following markers: CD133 高 / + , CD144 低 / - and / or vWF 低 / - , the substance to be screened is a candidate drug for promoting early EPCs.

[0157] The application will be further described in conjunction with the following drawings and examples. The following examples are only used to illustrate the present application and not intended to limit the scope of the present application. The experimental methods not specified in the examples are usually carried out according to the conventional conditions or the conditions suggested by the manufacturers.

[0158] Example 1 Early EPC (Early EPC) directed differentiation and identification

[0159] Preparation of culture medium

[0160] Preparation of E8 complete medium: E8 complete medium was prepared according to Table 1, and stored at 4°C for a maximum of 2 weeks.

[0161] Table 1 Preparation table of E8 complete medium

[0162]

[0163] Preparation of differentiation base medium: The differentiation base medium was prepared according to Table 2, and stored at 4°C for a maximum of 1 month.

[0164] Table 2 Preparation table of differentiation base medium

[0165]

[0166] 1. iPSCs resuscitation and subculture

[0167] 1.1 Matrigel coating

[0168] (1) In an ice box, open the 1.5 mL EP tube containing matrigel with a hemostat.

[0169] (2) Take 100 μL of pre-cooled coating solution into the EP tube containing matrigel, and after repeated blowing, suck the supernatant back into the centrifuge tube. This process needs to be repeated several times to make the matrigel dissolve quickly, and do not re-warm. The dilution of matrigel is 1:400.

[0170] (3) Take out the pre-cooled to-be-coated material and place it on the ice box. Mix the coating solution with an electric pipette, and coat according to the following volume: 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.

[0171] (4) Place the coated culture bottle / plate in a 37°C, 5% CO2 incubator overnight.

[0172] 1.2 iPSCs resuscitation

[0173] (1) According to the amount of medium required for recovery, prepare E8 complete medium containing 10 μM Y-27632, add 1 μL Y-27632 (10 mM) stock solution per milliliter of medium, and preheat at room temperature.

[0174] (2) Prepare 10-15 mL of E8 medium, and preheat in a 37°C water bath.

[0175] (3) Take one cryopreservation tube from the liquid nitrogen tank and immediately place it in a 37°C water bath. Shake back and forth constantly for 60-90 seconds to completely thaw the frozen cell suspension.

[0176] (4) Once the cells in the cryopreservation tube are completely thawed (liquid), remove it from the hot water tank and sterilize the surface of the cryopreservation tube with 75% alcohol. Place it in a clean bench. When the cryopreservation tube is about to melt, sterilize the centrifuge tube containing the preheated E8 medium at 37°C with alcohol and place it in the clean bench.

[0177] (5) Under strict aseptic conditions, take the cell suspension in the cryopreservation tube and inject it into the preheated E8 medium 15 mL centrifuge tube. Gently blow 2-3 times, centrifuge at 200 g for 5 min, and discard the supernatant after centrifugation.

[0178] (6) Add E8 medium containing 10 μM Y-27632, gently blow 2-3 times, and remove the Matrigel-coated well plate / culture bottle from the incubator. Suck the coating liquid and gently add the cell suspension along the uncoated side (do not directly add to the coated layer). Place the culture dish in the incubator for culture.

[0179] Note: When recovering, choose the container for inoculation according to the number of cells. Generally, 50-80 million cells are inoculated in a 6-well plate, 80-100 million cells are inoculated in a T25 bottle, and 100-200 million cells are inoculated in a T75 bottle.

[0180] 1.3 iPSCs digestion and subculture;

[0181] (1) Prepare TrypLE working solution: take 5 mL DPBS into a new 15 mL centrifuge tube, then add 5 mL TrypLE stock solution, mix well to obtain the working solution.

[0182] (2) According to the amount of medium required for subculture, prepare E8 complete medium containing 10 μM Y-27632, add 1 μL Y-27632 (10 mM) stock solution per milliliter of medium.

[0183] (3) Take out the well plate / culture bottle to be subcultured from the incubator, discard the supernatant, and wash twice with DPBS (each time the amount of DPBS is not less than the amount of the original culture medium), 1 min each time (when washing, place the DPBS in the well / bottle for 30-45 seconds before sucking it out).

[0184] (4) After adding TrypLE working solution (about 1 mL TrypLE working solution for a six-well plate, and about 2 mL TrypLE working solution for a T25 bottle), incubate in the incubator for 2-5 min, and observe under a microscope during the incubation. When the cells shrink and disperse, the incubation can be stopped.

[0185] (5) Gently tap the culture bottle / plate to make the cells detach from the bottom of the plate, then blow gently several times with a pipette, and finally add DMEM / F12 to terminate the digestion.

[0186] (6) After equilibration, 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, resuspend the cells, and inoculate the cell suspension into a matrigel-coated T25 bottle. Place the culture bottle in a 37°C, 5% CO2 incubator and incubate.

[0187] (7) After 24 h of culture, replace the medium with fresh E8 complete medium.

[0188] (8) Thereafter, perform full medium replacement operation every day. When the confluence of the cells is about 70%-80%, perform subculture.

[0189] Note: The inoculation amount of iPSCs cells is about 8000 cells / cm 2 , according to different iPSCs lines, the subculture density can be adjusted, and the interval time of subculture of iPSCs cells is 5-7 days.

[0190] 2. Early EPC differentiation process of iPSCS:

[0191] 2.1 hiPSCs monolayer adherent cell formation (Day-1);

[0192] (1) Prepare TrypLE working solution: take 5 mL DPBS into a new 15 mL centrifuge tube, then add 5 mL TrypLE stock solution, mix well, and the mixture is TrypLE working solution.

[0193] (2) According to the amount of culture medium required for subculture, prepare E8 complete medium containing 1% PS and 10 μM Y-27632 (ROCKi), and add 1 μL Y-27632 (10 mM) stock solution per milliliter of culture medium.

[0194] (3) Take out the well plate / culture bottle to be subcultured from the incubator, discard the supernatant, and wash twice with DPBS (each time the amount of DPBS is not less than the amount of the original culture medium), 1 min each time (when washing, place the DBPS in the hole / vial for 30-45 sec before sucking it out).

[0195] (4) After adding TrypLE working solution (about 1 mL TrypLE working solution for a six-well plate, and about 2 mL TrypLE working solution for a T25 bottle), incubate in the incubator for 2-5 min, and observe under a microscope during the incubation. When the cells shrink and disperse, stop the incubation.

[0196] (5) Gently tap the culture bottle / plate to make the cells detach from the plate bottom, then blow gently several times with a pipette, and finally add DMEM / F12 to terminate the digestion. Count the appropriate amount of cell suspension.

[0197] (6) After equilibration, centrifuge at 200 g for 5 min. After centrifugation, discard the supernatant, gently shake the bottom of the centrifuge tube, and resuspend with E8 complete medium containing 10 μM Y-27632 according to different cell densities. After thoroughly mixing the cells, drop the cell suspension into the culture plate wells, and inoculate the cells at a density of 8000 cells / cm 2 in a 37°C, 5% CO2 incubator.

[0198] (7) After 24 h of culture, wash twice with DPBS before subsequent induction and differentiation.

[0199] 2.2 Operation procedure for mesendoderm induction and differentiation (Day 0-1)

[0200] (1) Prepare an appropriate amount of mesendoderm induction medium and preheat it in a 37°C water bath.

[0201] (2) Discard the original culture medium and wash the cells with an appropriate amount of DPBS.

[0202] (3) Add the mesendoderm induction medium, and then incubate in a 37°C, 5% CO2 incubator for 24 h.

[0203] Table 3 Preparation table of mesendoderm differentiation medium

[0204]

[0205] 2.3 Operation procedure for mesoderm induction and differentiation (Day 1-3)

[0206] (1) Prepare an appropriate amount of mesoderm specification medium and preheat it in a 37°C water bath.

[0207] (2) Discard the original culture medium and wash the cells with an appropriate amount of DPBS.

[0208] (3) Add mesoderm specialization medium, and then place in a 37 °C, 5% CO2 incubator for static culture for 48 h.

[0209] Table 4 Preparation table of mesoderm differentiation medium

[0210]

[0211] 2.4 Operation procedure of early EPC induced differentiation (Day 3-9)

[0212] (1) Prepare an appropriate amount of early EPC differentiation medium, and preheat in a 37 °C water bath.

[0213] (2) After mesoderm specialization for 48 h, discard the original culture medium; wash the cells with an appropriate amount of DPBS, digest the cells to single cells with TrypLE working solution, centrifuge at 200 g for 5 min after stopping cell digestion, resuspend the cells in early EPC differentiation medium, inoculate cells at 10000 cells / cm 2 , and then place in a 37 °C, 5% CO2 incubator for static culture.

[0214] (3) Thereafter, replace the fresh early EPC differentiation medium on the 4th day and the 5th day of differentiation.

[0215] (4) On the 6th day of differentiation, discard the original culture medium; wash the cells with an appropriate amount of DPBS, digest the cells to single cells with TrypLE working solution, centrifuge at 200 g for 5 min after stopping cell digestion, resuspend the cells in early EPC maintenance medium, inoculate cells at 1000 cells / cm 2 , and then place in a 37 °C, 5% CO2 incubator for static culture.

[0216] (5) Thereafter, replace the fresh early EPC maintenance medium on the 7th day and the 8th day of differentiation.

[0217] (6) On the 9th day of differentiation, discard the original culture medium; wash the cells with an appropriate amount of DPBS, digest the cells to single cells with TrypLE working solution, centrifuge at 200 g for 5 min after stopping cell digestion, harvest the early EPC cells for freezing and quality inspection.

[0218] Table 5 Preparation table of early EPC differentiation medium

[0219]

[0220] Table 6 Preparation table of early EPC maintenance medium

[0221]

[0222] Example Two Late EPC (late EPC) directed differentiation and identification

[0223] Preparation of basal medium

[0224] Preparation of mesoderm induction basal medium: prepare according to the specifications, proportions shown in Table 7, and use immediately, store at 4°C for a maximum of 1 month. Among them, B-27 minus insulin needs to be packaged according to the single dose, and stored at -20°C, avoid repeated freeze-thaw, 1 day before use, take out 1 tube 4°C overnight thawing for use, or thaw at room temperature on the same day.

[0225] Table 7 Preparation of mesoderm induction basal medium

[0226]

[0227] Preparation of EPCs induction basal medium: prepare according to the specifications, proportions shown in Table 8, and use immediately, store at 4°C for a maximum of 1 month. Among them, StemPro-34 Supplement needs to be packaged according to the single dose, and stored at -20°C, avoid repeated freeze-thaw, 1 day before use, take out 1 tube 4°C overnight thawing for use, or thaw at room temperature on the same day.

[0228] Table 8 Preparation of EPCs induction basal medium

[0229]

[0230] 1. Resuscitation and subculture of iPSCs

[0231] (1) Take a tube of frozen iPSC cells from the iPSC cell bank and quickly transfer it to the laboratory. Place it in a hot water bath at a water temperature of 38-40°C, gently shake it, and when only a small (about the size of a millet) crystal remains in the frozen tube, sterilize the surface of the frozen tube with 75% alcohol, and place it in a clean bench.

[0232] (2) Transfer the cell suspension to a 15 mL centrifuge tube with a pipette, add 5 mL of 37°C preheated DMEM / F12 Medium, and centrifuge at 200g at room temperature for 5 min.

[0233] (3) After centrifugation, gently aspirate the supernatant with a pipette, then add 1 mL of 37°C preheated E8 complete medium containing ROCKi (1 μL of ROCKi stock solution per mL of medium) with a pipette, gently resuspend the cell pellet by blowing, and take 10 μL for cell counting and viability detection.

[0234] (4) Take out the prepared Matrigel-coated cell culture plate from the 37°C / 5% CO2 cell incubator, for example with a T25 culture flask, remove the liquid, and add 5 mL of 37°C preheated E8 complete medium containing ROCKi.

[0235] (5) Then slowly add an appropriate amount of cell diluent (seeding density 1.0-1.5 x 10 4 cell clusters / cm 2 ) to the T25 bottle, place the cell bottle in a 37°C / 5% CO2 cell incubator, and shake it 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. Then stand overnight. The iPSCs inoculated after recovery are P13 generation.

[0236] (6) Replace the fresh 37°C preheated E8 complete medium after 24 h, repeat the medium replacement every day until the cell aggregation degree increases to 70-80%, or the diameter of any single colony is greater than 10 x microscope field, then follow steps (3)-(7) to passage the iPS cells.

[0237] (7) The inoculation density of each generation is fixed at 1.0-1.5 x 10 4 cell clusters / cm 2 , and the fixed expansion time is 4 days, with a passage every 4 days. After 4 days of expansion to P14 generation, proceed to the subsequent steps.

[0238] 2. Operation procedure for establishing iPSCs monolayer differentiation (Day-1)

[0239] (1) After the formal start of iPSCs induction differentiation, the cells are normally centrifuged, the supernatant is removed, an appropriate amount of 37°C preheated E8 complete medium containing ROCK is added, the cell precipitate is resuspended by gentle blowing, and then the resuspended cell suspension is counted and the viability is detected.

[0240] (2) Take out 4 Matrigel-coated T75 culture flasks from the 37°C / 5% CO2 cell incubator, remove the liquid, and add 13 mL of 37°C preheated E8 complete medium containing ROCK to each flask.

[0241] (3) The initial seeding density of iPSCs induction differentiation needs to be controlled at 3.0 x 10 4 ~4.0 x 10 4 cells / cm 2 , according to the density of the counted cell resuspension, an appropriate volume of cell resuspension is added to the prepared Matrigel-coated T75 culture flask.

[0242] (4) Put the cell plate into 37°C / 5% CO2 cell incubator, shake it around 10 times, try to make sure the cells are evenly distributed on the plate, then let it stand overnight.

[0243] (5) After 24 hours, observe the iPSCs' aggregation degree. If the aggregation degree reaches 15-25%, you can directly proceed to the subsequent formal induction step. If the aggregation degree does not reach 15%, replace the old medium with fresh 37°C preheated E8 complete medium and extend the iPSCs culture time to 12-24 hours.

[0244] 3. Operation procedure for lateral plate mesoderm induction (Day 0-3)

[0245] (1) When the iPSCs' aggregation degree reaches 15-25%, start the formal induction and differentiation, which is defined as Day 0. Remove the old medium in the T75 bottle, wash it with 10 mL DPBS, then add 30 mL 37°C preheated mesoderm induction complete medium-1 to each bottle, and then incubate it in a 37°C / 5% CO2 cell incubator for 17-18 hours.

[0246] Table 9. Mesoderm induction complete medium-1

[0247]

[0248] (2) After 17-18 hours of incubation (Day 1), the iPS cells gradually spread out, the nucleus-cytoplasm ratio decreases, and the cell area increases. Remove the old medium in the T75 bottle, wash it with 10 mL DPBS, then add 50 mL 37°C preheated mesoderm induction complete medium-2 to each bottle, and then incubate it in a 37°C / 5% CO2 cell incubator for 2 days without changing the liquid.

[0249] Table 10. Mesoderm induction complete medium-2

[0250]

[0251] 4. Operation procedure for late EPCs induction (Day 3-6)

[0252] (1) After the lateral plate mesoderm cells are formed on Day 3, remove the old medium in the T75 bottle, wash it with 10 mL DPBS, then add 30 mL 37°C preheated EPCs induction complete medium (prepared according to the table below) to each bottle. Put the culture plate back into the 37°C / 5% CO2 cell incubator and incubate it for 24 hours.

[0253] Table 11. EPCs induction complete medium preparation table

[0254]

[0255] (2) One day later (Day 4), repeat the Day 3 medium change procedure, add 30 mL of 37°C pre-warmed EPCs Induction Complete Medium (prepared as above) to each flask. Incubate for 24 hours to Day 5, when EPCs induction with higher EC bias has occurred.

[0256] (3) At least 1 hour before enzymatic dissociation and re-plating of EPCs on Day 5, prepare 8 Fibronectin-coated T175 flasks.

[0257] (4) Remove old medium from all T75 flasks, wash twice with DPBS, then add 3 mL of TrypLE to each flask, and place in a 37°C / 5% CO2incubator for 3-5 min. Check the degree of cell detachment under a microscope until most cells start to float.

[0258] (5) Gently shake the bottom of the flask, and when most cells have detached as a slurry, add 12 mL of DMEM / F12 Medium to neutralize the TrypLE, and resuspend the detached cells by gently pipetting with a pipette. Then transfer the cells to a centrifuge tube, and count the cells.

[0259] (6) Centrifuge at 200g for 5 min at room temperature. During this time, prepare EPCs Maintenance Complete Medium (prepared as below).

[0260] Table 12. EPCs Maintenance Complete Medium Preparation

[0261]

[0262] (7) Remove the Fibronectin working solution from the T175 flasks that have been standing at room temperature for 1 hour, and rinse twice with an appropriate amount of double-distilled water. Remove the supernatant from the centrifuged cells, resuspend the cells with an appropriate amount of prepared EPCs Maintenance Complete Medium, and then add the cells (2 T175 flasks per 1 T75 flask) to the T175 flasks (35 mL per T175 flask).

[0263] (8) Place the flasks in a 37°C / 5% CO2incubator, and let them stand overnight.

[0264] (9) After 24 h (Day 6), the cells are attached, and wash once with an appropriate amount of DPBS. Then digest each T175 flask with 6 mL of TrypLE, and terminate the digestion with 24 mL of DMEM / F12. Centrifuge at 200g for 5 min, and remove the supernatant.

[0265] (10) Use a pipette to take a small amount of DPBS and gently resuspend each cell pellet, then transfer to the same 50 mL centrifuge tube, ensuring the total volume is not less than 40 mL, 200g centrifugation for 5 min, then remove the supernatant.

[0266] (11) Resuspend and wash again with 40 mL of DPBS, take 50 μL for counting and viability detection.

[0267] (12) According to the detected cell density, take the corresponding volume of cell suspension according to the required number of quality control, and send it to the quality control department for various quality tests of the product.

[0268] (13) The remaining cell suspension was centrifuged at 200g for 5 min, and the EPCs were stored in the form of stock solution according to the subsequent operation procedure.

[0269] Example Three Immunofluorescence Detection of EPC Molecular Markers

[0270] Table 13 Reagents

[0271]

[0272] Table 14 Working Solution

[0273]

[0274] Operation Steps:

[0275] (1) Accurately count the passage or recovered frozen EPC cell test sample, calculate the total amount of cells according to the counting results. Take about 1 x 10 6 cells by volume ratio, divide into 8 holes, about 100-150 million per hole. The final volume of EPC complete medium in each hole is 0.5 mL. Label the hole position for test purpose, test person and inoculation time. Place in a 37°C 5% CO2 cell incubator and culture until the confluence reaches more than 60%.

[0276] (2) Discard the liquid in the hole, wash three times with 500 μL of DPBS.

[0277] (3) Add 200 μL of fixing solution (4% paraformaldehyde) to each hole, incubate at room temperature for 20 min.

[0278] (4) Discard the fixing solution and wash three times with 200 μL of DPBS.

[0279] (5) Add 200 μL of 0.1% Triton-X 100 and incubate at room temperature for 15 min.

[0280] (6) Discard the supernatant and wash three times with 200 μL of DPBS.

[0281] (7) Add 200 μL 5% BSA blocking solution to each well, and stand at room temperature for 30 min.

[0282] (8) Discard the supernatant in the test well, and add 200 μL primary antibody working solution (5% BSA 100-fold diluted primary antibody, prepared as primary antibody working solution) to the well, and incubate at 4°C overnight. The negative control well is not treated, and incubated at 4°C overnight.

[0283] (9) Discard the supernatant, and wash with 200 μL DPBS for 3 times.

[0284] (10) Add 200 μL secondary antibody working solution (DPBS 100-fold diluted secondary antibody, prepared as secondary antibody working solution) to each well, and incubate at room temperature for 1 h.

[0285] (11) Discard the supernatant, and wash with 200 μL DPBS for 3 times.

[0286] (12) Add 200 μL DAPI working solution (DPBS 200-fold diluted DAPI, prepared as DAPI working solution) to each well, and incubate at room temperature for 2 min. in the dark room environment, and wait for observation and photography under the microscope.

[0287] Experimental results and conclusions:

[0288] The immunofluorescence detection results are shown in FIG. 1 and FIG. 2. Both early EPC and late EPC highly express CD31, CD34 and KDR; late EPC also highly express CD144 and vWF, but early EPC lowly express or even do not express CD144 and vWF.

[0289] Conclusion: CD144 and vWF can be used as marker genes for distinguishing early EPC and late EPC.

[0290] Example Four: Flow cytometry detection of EPC molecular markers

[0291] Table 15: Reagents

[0292]

[0293] Operation steps:

[0294] (1) Accurately count the EPC cells of the test sample after passage or resuscitation from freezing, and calculate the total amount of cells according to the counting result. Take about 5 x 10 6Cells were collected by centrifugation at 350g for 5 min, and then transferred to 1.5 mL EP tubes with 1 mL DPBS. The collected cells were prepared into single cell suspension to prevent the blockage of the liquid flow system or affect the accuracy of the data.

[0295] (2) The test sample was grouped, sub-packed and labeled in 1.5 mL EP tubes according to the experimental setup. Seven groups were set up, and each tube was sub-packed with 100 μL of cell suspension, and labeled as Blank (blank control group), 647 and 488 (negative control group), KDR, CD34, CD31 and CD133.

[0296] (3) The antibody was prepared with DPBS at a ratio of 100:5 μL, and incubated at room temperature for 30 min in the dark. The cell pellet was dispersed after centrifugation for 120 s and the supernatant was discarded.

[0297] (4) Washing: 500 μL of DPBS was added to each tube for washing, and the cell pellet was dispersed after centrifugation for 120 s. The washing operation was repeated twice, and the total washing was three times.

[0298] (5) Flow detection: 200 μL of DPBS was added to each test sample for resuspension, and the flow machine was detected. After the detection was completed, the document was saved, and the flow results were analyzed by analysis software.

[0299] Experimental results and conclusions:

[0300] The detection results are shown in Figures 3 and 4. Early EPC and late EPC both highly express CD31, CD34 and KDR. Early EPC also expresses CD133, and late EPC has low expression or even no expression of CD133.

[0301] Conclusion: CD133 can be used as one of the surface markers of early EPC and late EPC.

[0302] Example Five: In vitro tube formation experiment for detecting the vascularization ability of EPC

[0303] (1) The EPC prepared in Example 1 and Example 2 were respectively cultured in T25 culture bottles for 3 days. The culture supernatant in the T25 cell culture bottle was aspirated, washed with 3 mL of DPBS for 2 times, 1 mL of TrypLE enzyme solution was added, and the cell culture bottle was placed in a 37°C 5% CO2 cell incubator for 3-5 min. The degree of cell detachment was observed under a microscope until most of the cells began to float.

[0304] (2) Lightly shake the bottom of the culture bottle, and after most of the cells are detached in a quicksand-like manner, add 2 mL of culture supernatant to neutralize the digestion of TrypLE. Resuspend the detached cells by gently blowing them with a pipette gun, and then transfer all the cell suspensions to one 15 mL centrifuge tube and centrifuge at 300 g for 5 min at room temperature.

[0305] (3) After centrifugation, discard the supernatant, add 0.5 mL of EPC maintenance complete medium to resuspend all the cell precipitates, and transfer all the cell suspensions to one 15 mL centrifuge tube.

[0306] (4) Take 50 μL of the cell suspension and calculate the total amount of cells.

[0307] (5) Take an appropriate amount of cell suspension 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 amount of cells of 3.50 x 10 5 cells.

[0308] (6) After gently blowing twice with a 200 μL pipette tip, inoculate 100 μL of the cell suspension per well into a Matrigel-coated 48-well cell culture plate that has been incubated at 37°C / 5% CO2 for 30 min. The volume per well is 0.1 mL, and the number of cells per well is 1.0 x 10 5 cells / well. Place the 48-well cell culture plate in a 37°C / 5% CO2 cell incubator for static culture.

[0309] (7) After 6 h, turn on the OLYMPUS optical inverted microscope, photographing system, and computer, and place them in standby mode.

[0310] Place the 48-well cell culture plate to be cultured in the sample well 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, and take photographs for record. The photographs are archived.

[0311] Experimental results and conclusions:

[0312] The experimental results show that both early EPC and late EPC have tube formation ability (A), but there is no significant difference in the number of branches (B) and the average number of nodes (C) formed by early EPC and late EPC. The average tube formation length of early EPC and late EPC has certain differences.

[0313] Conclusion: Tube formation ability needs to be comprehensively judged from different dimensions. The above results show that both early EPC and late EPC have tube formation ability, but they cannot be used as a standard to distinguish early EPC and late EPC.

[0314] Example Six Acetylated LDL Uptake and Wisteria Floribunda Agglutinin Binding Assay to Identify Late EPCs

[0315] Table 16 Reagents

[0316]

[0317] Method:

[0318] (1) Accurately count the passage or resuscitated cryopreserved EPC cell samples, and calculate the total amount of cells according to the counting results. Take about 1 x 10 6 cells, and divide them into 8 wells, about 10-15 million per well. The final volume of EPC complete medium in each well is 0.5 mL. Label the test purpose, test person, and inoculation time of the inoculation well. Incubate in a 37°C 5% CO2 cell incubator until the confluence reaches more than 60%, and then prepare for staining.

[0319] (2) Add fluorescent dye to the above test wells at a ratio of 1 μL Dil-Ac-LDL per 100 μL medium, and incubate at 37°C for 4 h.

[0320] (3) Discard the liquid in the well, and wash with DPBS 3 times, each time soaking for 5 min.

[0321] (4) Add 200 μL of 4% polyethylene glycol to each well and fix for 10 min.

[0322] (5) Discard the liquid in the well, and wash with DPBS 3 times.

[0323] (6) Add 200 μL of FITC-UEA-I working solution to each well, and incubate at room temperature for 1 h.

[0324] (7) Discard the liquid in the well, and wash with DPBS 3 times, and add 200 μL of DPBS.

[0325] (8) Observe and photograph under a fluorescence microscope in a dark room environment.

[0326] Results and Conclusion:

[0327] The experimental results are shown in Figure 5, in which 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, thereby producing foam cells. Ulex europaeus agglutinin (UEA) is a protein extracted from Ulex europaeus. Agglutinins play an important role in cell recognition and adhesion reactions, and mainly promote cell adhesion. Agglutinins have more than one site that binds to sugars, and thus can participate in cell recognition and adhesion to link different cells together. As can be seen in the figure, acetylated low-density lipoprotein (Ac-LDL) is rarely or even cannot be taken up by early EPCs; late EPCs can efficiently take up acetylated low-density lipoprotein (Ac-LDL).

[0328] Conclusion: There is a significant difference in the phagocytosis of acetylated low-density lipoprotein (Ac-LDL) between early EPCs and late EPCs, and late EPCs have stronger phagocytosis, which can be used as a functional test to distinguish early EPCs and late EPCs.

[0329] Example Seven: Non-clinical study of endothelial progenitor cells (EPCs) in the treatment of acute ischemic stroke

[0330] Twenty-four male SD rats were selected and divided into four groups, namely a sham operation group, a model control group, an early EPC treatment group, and a late EPC treatment group, with six animals in each group. The animals were subjected to permanent ischemia by plugging the middle cerebral artery with a thread. The test product, endothelial progenitor cells EPCs (1 x 10 7 cells / kg), was administered once by tail vein injection at 24 h after ischemia. Eight days after the operation, the animals were dissected, and the brain tissue was subjected to TTC staining to determine the infarction range, so as to evaluate the protective effect of the test product on acute ischemic stroke in rats.

[0331] The experimental results are shown in Figures 6 and 7. In the MCAO rat cerebral infarction model, the late EPC treatment group significantly restored blood supply to the infarction area after administration, indicating that late EPCs have a protective effect on acute ischemic stroke in rats, while the early EPC treatment group had no significant difference in blood supply to the infarction area after administration compared with the model control, indicating that early EPCs have almost no protective effect on acute ischemic stroke in rats.

[0332] Example Eight: Effectiveness of EPC cells in erectile dysfunction (ED) mice

[0333] 1. Test animals: db / db spontaneous diabetes mice, 12-16 weeks old, male, 24; db / + wild control mice, 12 weeks old, male, 5.

[0334] 2. Test drugs: early / late EPC concentration is 1 x 10 8 cells / mL.

[0335] 3. Test grouping: all animals are grouped according to age, weight, blood glucose and random number method, and the minimum difference of each group is taken as the final grouping.

[0336] Table 17 Grouping

[0337]

[0338] 4. Detection index

[0339] (1) On the 0th, 4th, 7th, 10th, 14th, and 17th days of administration, the body weight of each group of mice was weighed, and adverse reactions were observed.

[0340] (2) After administration of G2, G3, and G4 groups, subcutaneous injection of APO was performed, and the licking behavior of the penis within 30 min (usually the symbolic action of animal ejaculation) was recorded as the counting standard.

[0341] (3) After 17 days of administration, the mice were treated by cervical dislocation, the corpus cavernosum was separated, 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 observed for co-localization and expression.

[0342] 5. Experimental results

[0343] (1) As shown in Figure 8, after administration of G2, G3, and G4 groups, the body weight of the animals did not change significantly, indicating that the drug had no effect on the metabolism of the animals, and that single administration of EPC through the penis was safe.

[0344] (2) As shown in Figure 9, the number of licking the penis in G2 model group was significantly reduced compared with the normal group, indicating that long-term diabetes reduced the sexual impulse behavior of animals. G2 and G3 were compared, and there was no significant difference in the sexual impulse behavior of animals, indicating that early EPC could not increase the sexual impulse behavior and had no therapeutic effect on ED; G2 and G4 were compared, and the sexual impulse behavior of G4 animals was significantly increased, indicating that late EPC increased the sexual impulse behavior and had a therapeutic effect on ED.

[0345] (3) The results are shown in Figure 10. Comparison of G1 group, G2 group and G3 group shows that the expression of eNOS and CD31 in G2 group and G3 group is significantly down-regulated, indicating that the model group may develop erectile dysfunction. Meanwhile, the expression of eNOS and CD31 in G3 group is not improved by administration, suggesting that injection of early EPCs has no therapeutic effect on ED. Comparison of G1 group, G2 group and G4 group shows that the expression of eNOS and CD31 in G4 group is significantly improved, and the expression is higher than that in G1 group, suggesting that injection of late EPCs has therapeutic effect on ED.

[0346] (4) The results are shown in Figure 11. TAGLN is a marker gene of vascular smooth muscle cells, PODXL is a marker gene of endothelial cells, SLC1A3 is a marker gene of corpus cavernosum fibroblasts, and eNOS is a precursor of NO production. Comparison of G1 group, G2 group and G3 group shows that the expression of TAGLN, PODXL, SLC1A3 and eNOS in G3 group is not significantly increased, indicating that injection of early EPCs has no therapeutic effect on ED mouse model. Comparison of G1 group, G2 group and G4 group shows that the expression of TAGLN and SLC1A3 is not significantly increased, and the expression of PODXL and eNOS is significantly improved, indicating that injection of late EPCs has therapeutic effect on ED mouse model. The mechanism is to increase the amount of endothelial cells and the content of eNOS in the corpus cavernosum, and has no adverse effect on vascular smooth muscle and corpus cavernosum fibroblasts.

[0347] Example Nine Pharmacodynamic Study of EPCs in Treatment of Lower Limb Ischemia in Mice

[0348] Eighteen male Balb / c nude mice were selected and divided into three groups, namely model control group, early EPC treatment group and late EPC treatment group, with 6 animals in each group. The lower limb ischemia model was established by double ligation and cutting of the femoral artery. After modeling, the treatment groups were given intravenous injection of the test product endothelial progenitor cells EPCs (1 x 10 7 cells / kg); the model control group was only modeled without administration. Fourteen days after administration, lower limb scoring and laser speckle blood flow detection were performed to determine the lower limb ischemia and function, and to evaluate the therapeutic effect of the test product on lower limb ischemia in animals.

[0349] The results are shown in Figures 12 and 13. After treatment with early EPCs, the lower limb ischemia rate of animals in each treatment group showed a downward trend compared with the model control group, but there was no statistical difference, indicating that the therapeutic effect of early EPCs was not significant. After treatment with late EPCs, the lower limb ischemia rate of animals in each treatment group showed a significant downward trend compared with the model control group, and there was a statistical difference, indicating that late EPCs had therapeutic effect on lower limb ischemia.

[0350] The above description of the embodiments is only for understanding the method of the present application and its core idea. It should be noted that for those skilled in the art, without departing from the principles of the present application, the present application can be improved and modified in several ways, and these improvements and modifications will also fall within the protection scope of the claims of the present application.

Claims

1. A biomarker for identifying or detecting early / late EPCs, characterized in that, The biomarker comprises CD144, vWF and / or CD133; Preferably, the biomarker comprises a combination of CD144, vWF and CD133; Preferably, the CD144 and vWF are highly expressed in late EPCs, and the CD133 is not expressed or lowly expressed in late EPCs; Preferably, the CD144 and vWF are not expressed or lowly expressed in early EPCs; Preferably, the biomarker further comprises CD31, CD34 and / or KDR; Preferably, the EPCs are derived from iPSCs.

2. A product characterized by, The product comprises a binding agent capable of binding to the biomarker of claim 1; Preferably, the binding agent is selected from the group consisting of nucleic acid, ligand, enzyme, substrate and / or antibody; Preferably, the binding agent is linked to a detectable molecule; Preferably, the nucleic acid comprises a probe capable of hybridizing to the biomarker; Preferably, the nucleic acid comprises a primer capable of binding to the biomarker; Preferably, the antibody is an antibody capable of binding to a protein or protein fragment encoded by the biomarker; Preferably, the product comprises a reagent or kit; Preferably, the product is a product for identifying, enriching, isolating or sorting early / late EPCs.

3. An apparatus or device for identifying, enriching, isolating or sorting early / late EPCs, comprising, The device or apparatus comprises a cell binding unit configured to bind the biomarker of claim 1.

4. Late EPCs characterized by, The late EPCs have at least one of the following expression profiles, CD144 高 / + , vWF 高 / + , CD 133 低 / - ; Preferably, said late EPCs have the following expression profile: CD144 高 / + , vWF 高 / + , and CD 133 低 / - ; Preferably, said late EPCs have at least one of the following expression profiles: CD31 高 / + , CD34 高 / + , and / or KDR 高 / + . Preferably, said late EPCs have the following expression profile: CD144 高 / + , vWF 高 / + , CD 133 低 / - , CD31 高 / + , CD34 高 / + , and KDR 高 / + ; Preferably, the EPCs are derived from iPSCs.

5. Early EPC, characterized in that, The early EPCs have at least one of the following expression profiles: CD133 高 / + , CD144 低 / - , vWF 低 / - ; Preferably, said early EPCs have the following expression profile: CD133 高 / + , CD144 低 / - , and vWF 低 / - ; Preferably, said early EPCs have at least one of the following expression profiles: CD31 高 / + , CD34 高 / + , and / or KDR 高 / + . Preferably, said early EPCs have the following expression profile: CD133 高 / + , CD144 低 / - , vWF 低 / - , CD31 高 / + , CD34 高 / + and KDR 高 / + ; Preferably, the EPCs are derived from iPSCs.

6. A method of identifying, enriching, isolating or sorting early / late EPCs, characterized in that, determining expression of the biomarker of claim 1 in EPC cells, said EPCs being late EPCs when it is determined that said EPC cells have at least one of the following expression profiles: CD 144 高 / + , vWF 高 / + , CD 133 低 / - ; The EPC cells are early EPCs when it is determined that the cells have at least one of the following expression profiles: CD133 高 / + , CD144 低 / - , vWF 低 / - ; Preferably, the EPCs are derived from iPSCs; Preferably, said early EPCs or late EPCs further have at least one of the following expression profiles: CD31 高 / + , CD34 高 / + , and / or KDR 高 / + . Preferably, said late EPCs have the following expression profile: CD144 高 / + , vWF 高 / + , CD 133 低 / - , CD31 高 / + , CD34 高 / + , and KDR 高 / + ; Preferably, said early EPCs have the following expression profile: CD133 高 / + , CD144 低 / - , vWF 低 / - , CD31 高 / + , CD34 高 / + and KDR 高 / + ; Preferably, the method comprises contacting the cells with a binding agent of the biomarker; Preferably, the method further comprises sorting the EPCs bound by the binding agent; Preferably, the sorting technique comprises fluorescence activated cell sorting, magnetic assisted cell sorting, substrate assisted cell sorting, laser mediated cleavage, fluorometry, flow cytometry or microscopy; Preferably, the method is a method for identifying, enriching, isolating or sorting early / late EPCs from a cell population.

7. A method of identifying a subpopulation of EPCs, comprising, The method comprises detecting the phagocytic ability of the cells; Preferably, the phagocytic ability of the late EPC subpopulation is stronger than that of the early EPC subpopulation; Preferably, the method detects the phagocytic ability of the cells by detecting the uptake ability of acetylated LDL.

8. Use of the biomarker of claim 1, the product of claim 2, or the device or apparatus of claim 3 in identifying, enriching, isolating or sorting early / late EPCs.

9. Use of the biomarker of claim 1 or the product of claim 2 in the manufacture of a product for diagnosing a disease associated with early EPCs or late EPCs.

10. Use of the late EPCs of claim 4 in the manufacture of a medicament for treating a disease selected from one or more of stroke, erectile dysfunction, ischemic disease; Preferably, the ischemic disease is selected from lower limb ischemia.

11. A pharmaceutical composition for treating a disease, characterized by, The pharmaceutical composition comprises the late EPCs of claim 4, and the disease is selected from one or more of stroke, erectile dysfunction, ischemic disease; Preferably, the ischemic disease is selected from lower limb ischemia. Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier; Preferably, the pharmaceutical composition further comprises other drugs for treating stroke, erectile dysfunction, ischemic diseases; Preferably, the pharmaceutical composition is in the form of an infusion.

12. A method for treating a disease, characterized in that, The method comprises administering an effective amount of the late EPCs of claim 4 and / or the pharmaceutical composition for treating diseases of claim 11, the diseases being selected from one or more of stroke, erectile dysfunction, and ischemic diseases; Preferably, the ischemic disease is selected from lower extremity ischemia.

13. A method of determining the purity of a differentiated product, characterized by, The method comprises: detecting the expression level of at least one biomarker of the cells in the sample of the differentiation product, the biomarker being selected from CD144, vWF and / or CD133; determining the purity of the differentiation product according to the expression level of the biomarker; Preferably, the cells in the sample are in the form of a cell mixture, a cell aggregate or a tissue.

14. A method for quantifying EPC subpopulations in a sample, characterized in that, The method comprises: detecting the expression level of at least one biomarker of the cells in the sample of the differentiation product, the biomarker being selected from CD144, vWF and / or CD133; determining the purity of the differentiation product according to the expression level of the biomarker; 15. A method of screening for a candidate drug that modulates a subpopulation of EPCs, comprising: contacting a candidate drug with a subpopulation of EPCs; and determining whether the candidate drug modulates the subpopulation of EPCs. The method comprises adding a substance to be screened to the cell population, detecting the expression level of a biomarker of the cells, the biomarker being selected from CD144, vWF and / or CD133; when the substance to be screened changes the expression level of the marker of the cells, the substance to be screened is a candidate drug for regulating the subpopulation of EPCs.

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