Application of EPC Subgroups in Ischemic Diseases

Abstract

The present invention belongs to the field of biomedicine and discloses the application of EPC subsets in ischemic diseases. The present invention discovers for the first time the indicators for distinguishing EPC subsets: CD144, vWF, CD133 or phagocytic ability. Based on the above indicators, the present invention discloses methods for identifying, sorting, separating or enriching EPC subsets.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to the application of EPC subsets in ischemic diseases. Background Art

[0002] With the accelerating aging of the population, the incidence of peripheral arterial disease (PAD) has been increasing year by year. Approximately 10% of PAD patients further develop into critical limb ischemia (CLI), and the clinical manifestations include rest pain (lasting for more than 2 weeks), ulcers, or gangrene. The quality of life index of CLI patients is similar to that of patients with advanced cancer, and within 5 years after the initial diagnosis, the mortality rate exceeds 50% (Kitrou P, et al. Gene-based therapies in patients with critical limb ischemia. Expert Opin Biol Ther 2017, 17(4): 449-456.). Traditional surgical operations or endovascular treatments are currently the preferred clinical treatment methods for CLI patients, but there are still about 30% of patients who 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 treatments, and reducing the amputation rate and mortality rate are currently urgent clinical problems to be solved. Summary of the Invention

[0003] In order to solve the problems existing in the prior art, the inventors of the present invention, through extensive and in-depth research, for the first time discovered that late EPC rather than early EPC has the effect of treating ischemic diseases, especially limb ischemia, providing a new and effective treatment means for the treatment of ischemic diseases, especially limb ischemia.

[0004] The present invention also discovered biomarkers for distinguishing early EPC (early EPC) and late EPC (late EPC). The use of related biomarkers can be used for the identification, sorting, separation, and enrichment of different EPC subsets. It can also be used to determine the purity of differentiation products, quantify EPC subsets in samples, and screen candidate drugs for regulating EPC subsets.

[0005] Based on this, the present invention provides the following technical solutions:

[0006] The first aspect of the present invention provides the application of late EPC in the preparation of drugs for treating ischemic diseases.

[0007] The second aspect of the present invention provides a pharmaceutical composition for treating ischemic diseases, and the pharmaceutical composition includes late EPC.

[0008] The third aspect of the present invention provides a method for identifying EPC subsets, the method comprising determining the expression of at least one biomarker in cells, the biomarker comprising CD144, vWF or CD133; and

[0009] when it is determined that the cells have at least one of the following expression profiles, the EPC is a late EPC: CD144 高 / + , vWF 高 / + , CD 133 低 / - ; when it is determined that the cells have at least one of the following expression profiles, the EPC cells are early EPCs: CD133 高 / + , CD144 低 / - , vWF 低 / - .

[0010] The fourth aspect of the present invention provides a method for identifying EPC subsets, the method comprising detecting the phagocytic ability of cells.

[0011] The fifth aspect of the present invention provides a method for sorting, separating or enriching late EPCs from a cell population, selecting cells having at least one of the following expression profiles: CD144 高 / + , vWF 高 / + and / or CD 133 低 / - .

[0012] The sixth aspect of the present invention provides a method for sorting, separating or enriching early EPCs from a cell population, selecting cells having at least one of the following expression profiles CD133 高 / + , CD144 低 / - and / or vWF 低 / - .

[0013] The seventh aspect of the present invention provides a product, the product comprising a binding agent against the biomarker, the biomarker comprising CD144, vWF and / or CD133.

[0014] The eighth aspect of the present invention provides a method for determining the purity of a differentiation product, the method comprising:

[0015] detecting the expression level of at least one biomarker of cells in a 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.

[0016] The ninth aspect of the present invention provides a method for quantifying EPC subsets in a sample, the method comprising:

[0017] Detect the expression level of at least one biomarker of cells in a sample for detection purposes, where the biomarker is selected from CD144, vWF, and / or CD133; determine the content of EPC subsets according to the expression level of the biomarker.

[0018] The tenth aspect of the present invention provides a method for screening candidate drugs that regulate EPC subsets, the method comprising adding a drug to be screened to a cell population and detecting the expression level of a biomarker of the cells, where the biomarker is selected from CD144, vWF, and / or CD133; when the drug to be screened changes the expression level of the biomarker of the cells, the drug to be screened is a candidate drug that regulates EPC subsets.

[0019] The eleventh aspect of the present invention provides a method for treating ischemic diseases, the method comprising administering an effective amount of late EPC or the pharmaceutical composition described in the tenth aspect of the present invention to a subject.

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

[0021] The present invention has for the first time discovered that late EPC rather than early EPC can be used for the treatment of ischemic diseases, especially lower limb ischemia. Based on this, the therapeutic effect of endothelial progenitor cell treatment for ischemic diseases can be improved by increasing the content of the active ingredient late EPC in ischemic diseases.

[0022] The present invention has for the first time discovered biomarkers for distinguishing early and late EPC. Based on the biomarkers, they can be used for the identification, sorting, separation, and enrichment of different EPC subsets.

[0023] The present invention also provides a method for determining the purity of differentiation products, a method for quantifying EPC subsets in a sample, and a method for screening candidate drugs that regulate EPC subsets.

[0024] The present invention has for the first time discovered that phagocytic ability can be used to distinguish early EPC from late EPC. Description of the Drawings

[0025] Figure 1 It is a detection result diagram of early EPC cell surface marker genes;

[0026] Figure 2 It is a detection result diagram of late EPC cell surface marker genes;

[0027] Figure 3 It is a detection result diagram of early EPC surface marker genes by flow cytometry;

[0028] Figure 4 It is a detection result diagram of late EPC surface marker genes by flow cytometry;

[0029] Figure 5 It is a graph of the phagocytosis ability detection result;

[0030] Figure 6 It is a laser speckle blood flow detection graph;

[0031] Figure 7 It is a statistical graph of the lower limb ischemia rate of mice. Specific implementation manners

[0032] It should be understood that the terms used in the present invention are only for the purpose of describing specific implementation manners and are not intended to be limiting. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. Although other methods and materials similar to or equivalent to the methods and materials described in the present invention are available in the present invention, the present invention describes preferred materials and methods.

[0033] As used in the present invention, "subject", "individual" or "patient" are used interchangeably in the present invention and refer to any member of the phylum Chordata, including but not limited to humans and other primates, including non-human primates, such as rhesus monkeys, chimpanzees, and other monkeys and apes; farm animals, such as cows, sheep, pigs, goats, and horses; domestic mammals, such as dogs and cats; laboratory animals, including rabbits, mice, rats, and guinea pigs; birds, including poultry, wild birds, and game birds, such as chickens, turkeys, and other gallinaceous birds, ducks, and geese; and the like. The term does not denote a specific age or gender. Thus, the term includes adult, young, and neonatal individuals as well as males and females. In some embodiments, cells (such as stem cells, including pluripotent stem cells, progenitor cells, or specific tissue cells) are derived from a subject.

[0034] As used in the present invention, "induced pluripotent stem cell (iPSC)" refers to a class of pluripotent stem cells artificially derived from non-pluripotent cells (usually somatic cells). In some embodiments, the somatic cells are human cells. 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, thymocytes, and hematopoietic stem cells.

[0035] As used herein, "binding" refers to non-covalent interactions 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 "association" or "interaction" (e.g., if a first macromolecule interacts with a second macromolecule, the first macromolecule binds to the second macromolecule in a non-covalent manner). Some portions of the binding interaction can be sequence-specific.

[0036] As used herein, the term "regulation" refers to a change in the nature, activity, function, or quantity, degree, or number of physical molecules. Regulation can be determined by identifying any characteristic that is directly or indirectly affected by the expression of a target gene. These characteristics include, for example, changes at the RNA or protein level, protein activity, product level, gene expression, or reporter gene activity level.

[0037] Through extensive and in-depth research, by studying different EPC subsets, the present invention has discovered indicators that can be used to distinguish early and late EPCs, and further demonstrated that late EPCs, rather than early EPCs, have the effect of treating ischemic diseases, especially lower limb ischemia.

[0038] On the one hand, the present invention provides the use of late EPCs in the preparation of drugs for treating ischemic diseases.

[0039] In some embodiments, the late EPCs have at least one of the following expression profiles: CD144 高 / + , vWF 高 / + , CD133 低 / - .

[0040] In some embodiments, the late EPCs have the following expression profile: CD144 高 / + , vWF 高 / + and CD133 低 / - in combination.

[0041] In some embodiments, the late EPCs further have at least one of the following expression profiles: CD31 高 / + , CD34 高 / + and / or KDR 高 / + .

[0042] In some embodiments, the late EPCs have the following expression profile: CD144 高 / + , vWF 高 / + , CD133 低 / - , CD31 高 / + , CD34 高 / + and KDR 高 / + .

[0043] In some embodiments, the EPCs are derived from iPSCs.

[0044] In some embodiments, the ischemic disease is selected from lower limb ischemia.

[0045] On the other hand, the present invention provides a pharmaceutical composition for treating an ischemic disease, the pharmaceutical composition comprising late EPCs.

[0046] In some embodiments, the late EPCs have at least one of the following expression profiles: CD144 高 / + , vWF 高 / + , CD 133 低 / - .

[0047] In some embodiments, the late EPCs have the following expression profile: CD144 高 / + , vWF 高 / + and CD 133 低 / - in combination.

[0048] In some embodiments, the late EPCs further have at least one of the following expression profiles: CD31 高 / + , CD34 高 / + and / or KDR 高 / + .

[0049] In some embodiments, the late EPCs have the following expression profile: CD144 高 / + , vWF 高 / + , CD 133 低 / - , CD31 高 / + , CD34 高 / + and KDR 高 / + .

[0050] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0051] "Pharmaceutically acceptable" means a composition that is physiologically acceptable and generally does not cause gastrointestinal disorders, dizziness, allergic reactions such as those mentioned above or similar reactions when administered to humans. As a pharmaceutically acceptable carrier, there are, for example, non-parenteral administration carriers such as water, appropriate oils, physiological saline, aqueous glucose, and ethylene glycol, and stabilizers and preservatives may also be included. Appropriate stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Appropriate preservatives include benzalkonium chloride, methyl paraben, propyl paraben, and chlorobutanol.

[0052] Moreover, the above-mentioned pharmaceutical composition can also be administered by any device capable of moving the pharmaceutical composition towards the target cells.

[0053] The pharmaceutical composition of the present invention can be administered by any conventional route capable of reaching the target tissue. It can be administered parenterally, such as intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, but is not limited thereto.

[0054] In some embodiments, the pharmaceutical composition of the present invention can have any dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, infusions, internal solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, suspending agents, oils, lyophilized agents, and suppositories.

[0055] In some embodiments, the dosage form of the pharmaceutical composition is an infusion.

[0056] In some embodiments, the infusion of the present invention contains an infusion solution, such as normal saline, Ringer's lactate solution, compound electrolyte injection, 5% glucose injection, 20% HSA injection, succinylated gelatin injection, succinylated gelatin MIX injection, MZJ injection 1, MZJ injection 2, MZJ injection 3, human serum albumin injection, Plasmalyte A, potassium chloride injection, magnesium sulfate injection, sodium bicarbonate injection, glucose and 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 triphosphate disodium, lysine hydrochloride for injection, vitamin C injection, citicoline sodium chloride, fat-soluble vitamins for injection II, reduced glutathione for injection, cerebroprotein hydrolysate for injection, sodium deoxynucleotidate injection, multiple trace element injection II, mannitol injection, arginine hydrochloride injection, potassium chloride injection, cytidine triphosphate disodium for injection, ornithine aspartate for injection, etc.

[0057] The infusion of the present invention can also contain one or more additional agents, such as selected from solubilizers, wetting agents, emulsifiers, buffers, suspending aids, chelating agents, antioxidants, bacteriostatic agents, local anesthetics, isotonicity regulators, fillers, protective agents, and any combination thereof.

[0058] In some embodiments, the ischemic disease is selected from lower limb ischemia.

[0059] In yet another aspect, the present invention provides a method for identifying a subpopulation of EPCs, the method comprising determining the expression of at least one biomarker in the cells, the biomarker including CD144, vWF, or CD133; and

[0060] When it is determined that the 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 / - .

[0061] The biomarkers of the present invention can be detected individually, in combination, sequentially, simultaneously, synchronously, and / or in combination with other biomarkers, and the other biomarkers can be specific for endothelial progenitor cells.

[0062] In some embodiments, the expression of one or more of the biomarkers listed above is determined to determine the presence of EPC subsets in, for example, a composition, solution, cell aggregate, cell suspension, or tissue.

[0063] In some embodiments, the method further includes the step of identifying the cell as an EPC.

[0064] In some embodiments, determining the expression of at least one biomarker in a cell includes contacting the cell with a binding agent specific for the biomarker.

[0065] In the present invention, a binding agent refers to any compound, molecule, and / or system that functions to detect the presence / absence and / or expression or level of a biomarker in the present invention. Such a binding agent is capable of directly or indirectly detecting the biomarker and / or binding to the biomarker. In the present invention, additional moieties may be required to enhance the detection of the biomarker, for example, by / through amplified optical diffraction. Examples of binding agents and additional moieties include, but are not limited to, proteins (e.g., antigen-binding proteins such as antibodies or fragments thereof, enzymes such as horseradish peroxidase and alkaline phosphatase, etc.), polynucleotides (e.g., aptamers), and small molecules (e.g., metal nanoparticles).

[0066] In some embodiments, biomarker expression can be evaluated by any of a variety of well-known methods for detecting transcriptional molecules (e.g., mRNA) or protein expression. Non-limiting examples of these 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.

[0067] A variety of amplification and detection methods can be used to assist in the detection of biomarker expression. For example, reverse transcription of mRNA into cDNA (e.g., polymerase chain reaction, RT-PCR) prior to further analysis is within the scope of the present invention. Other known amplification methods that can be utilized in the present invention include, but are not limited to, the "NASBA" or "3SR" techniques; Q-beta amplification; strand displacement amplification; target-mediated amplification; ligase chain reaction (LCR); self-sustained sequence replication (SSR); and transcription amplification.

[0068] Methods for detecting, characterizing, and / or quantifying nucleic acid sequences; and methods for detecting, characterizing, and / or quantifying mRNA expression are known to those skilled 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 assays, microarray analysis, hybridization assays, serial analysis of gene expression (SAGE), hybridization based on digital barcode quantification, multiplex RT-PCR, digital droplet PCR (ddPCR), qRT-PCR, qPCR, ultraviolet spectroscopy, DNA sequencing, RNA sequencing, next-generation sequencing, including RNAseq, lysate-based hybridization assays utilizing branched DNA signal amplification, such as QuantiGene 2.0 Single Plex, and branched DNA analysis methods.

[0069] Non-limiting examples of nucleic acid sequencing techniques, such as for DNA sequencing and RNA sequencing, include Maxam-Gilbert sequencing, Sanger sequencing (i.e., chain termination), sequencing by synthesis (SBS), ligation sequencing, pyrosequencing, single molecule real-time sequencing, MiSeq sequencing, massively parallel signature sequencing (MPSS), polymerase cloning sequencing, 454 sequencing, nanopore sequencing. The present invention also includes, but is not limited to, next-generation sequencing technologies.

[0070] 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 barcode quantification can include BeadArray (Illumina), xMAP system (Luminex), nCounter (Nanostring), high-throughput genomics (HTG) molecules, BioMark (Fluidigm), or Wafergen microarrays. Detection methods can include DASL (Illumina), RNA-Seq (Illumina), TruSeq (Illumina), SureSelect (Agilent), Bioanalyzer (Agilent), and TaqMan (ThermoFisher).

[0071] Another aspect of the present invention provides a method for identifying a subpopulation of EPCs, the method comprising detecting the phagocytic ability of the cells.

[0072] In some embodiments, the late EPC subpopulation has a stronger phagocytic ability compared to the early EPC subpopulation.

[0073] In some embodiments, the method detects the phagocytic ability of the cells by detecting the uptake ability of acetylated LDL.

[0074] Another aspect of the present invention provides a method for sorting, isolating or enriching late EPCs from a cell population, selecting cells having at least one of the following expression profiles: CD144 高 / + 、vWF 高 / + and / or CD133 低 / - .

[0075] In some embodiments, the method comprises cells selected from the following expression profiles: CD144 高 / + 、vWF 高 / + and CD133 低 / - .

[0076] In some embodiments, the method further comprises further selecting cells having at least one of the following expression profiles: CD31 高 / + 、CD34 高 / + and / or KDR 高 / + .

[0077] In some embodiments, the method comprises selecting cells having the following expression profiles: CD144 高 / + 、vWF 高 / + and / or CD133 低 / - 、CD31 高 / +, CD34 高 / + and KDR 高 / + .

[0078] In some embodiments, the method further comprises culturing the late EPCs.

[0079] Another aspect of the present invention provides a method for sorting, isolating or enriching early EPCs from a cell population, selecting cells having at least one of the following expression profiles: CD133 高 / + , CD144 低 / - and / or vWF 低 / - .

[0080] In some embodiments, the method comprises selecting cells having the following expression profiles: CD133 高 / + , CD144 低 / - and vWF 低 / - .

[0081] In some embodiments, the method further comprises further selecting cells having at least one of the following expression profiles: CD31 高 / + , CD34 高 / + and KDR 高 / + .

[0082] In some embodiments, the method comprises selecting cells having the following expression profiles: CD133 高 / + , CD144 低 / - and / or vWF 低 / - , CD31 高 / + , CD34 高 / + and KDR 高 / + .

[0083] In some embodiments, the sorting or separation technique includes fluorescence-activated cell sorting, magnetic-activated cell sorting, substrate-assisted cell sorting, laser-mediated dissection, fluorometry, flow cytometry or microscopy.

[0084] In some embodiments, cells can be separated by binding to a fixed support and then harvested by removing the cells from the support.

[0085] Harvesting of the cells can be achieved by collecting the separated cells into a suitable container or collection dish, test tube, etc.

[0086] Another aspect of the present invention provides a product comprising a binding agent against the biomarker, the biomarker comprising CD144, vWF and / or CD133.

[0087] In some embodiments, the biomarker comprises a combination of CD144, vWF and CD133.

[0088] In some embodiments, the biomarker further comprises CD31, CD34, and / or KDR.

[0089] In some embodiments, the biomarker comprises a combination of CD144, vWF, CD133, CD31, CD34, and KDR.

[0090] In some embodiments, the binding agent is selected from nucleic acids, ligands, enzymes, substrates, and / or antibodies.

[0091] In the present invention, "antibody" is used in the broadest sense and specifically encompasses, for example, monoclonal antibodies, polyclonal antibodies, antibodies with multi-epitope specificity, single-chain antibodies, multispecific antibodies, and antibody fragments. Such antibodies can be chimeric, humanized, human, and synthetic.

[0092] In some embodiments, a detectable label is attached to the binding agent. Detectable labels can include, for example, absorbent dyes, fluorescent dyes, or radioactive labels. Detectable labels, methods for detecting them, and methods for incorporating them into reagents (such as antibodies and nucleic acid probes) are well known in the art.

[0093] In some embodiments, detectable labels can include labels detectable by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means (such as fluorescence, chemifluorescence, or chemiluminescence), or any other suitable means. Detectable labels can be primary labels (where the label contains a group that can be directly detected or generates a group that can be directly detected); or secondary labels (where the detectable label binds to another group to generate a detectable signal, for example, the use of secondary and tertiary antibodies commonly used in immunoassays). Detectable labels can be attached to the reagent by covalent or non-covalent means. Alternatively, the detectable label can be attached, for example, by directly labeling a molecule that binds to the reagent through a ligand-receptor binding pair arrangement or other such specific recognition molecules. Detectable labels can include, but are not limited to, radioisotopes, bioluminescent compounds, chromophores, antibodies, chemiluminescent compounds, fluorescent compounds, metal chelates, and enzymes.

[0094] In some embodiments, the expression levels of multiple markers of the present invention can be determined simultaneously (such as multiplex detection) or in parallel.

[0095] In other embodiments, gene expression products (proteins) associated with the biomarkers of the present invention can be detected to determine the presence of EPC subsets. These detection techniques are known to those skilled in the art and include, for example, ELISA (enzyme-linked immunosorbent assay), western blot, FACS, radioimmunoassay; (RIA); sandwich assay; fluorescence in situ hybridization (FISH); immunohistological staining; immunoelectrophoresis; immunoprecipitation; and immunofluorescence using detection reagents (such as antibodies or protein binders).

[0096] In some embodiments, the product is selected from the group consisting of kits.

[0097] The kit may further include instructions for using the kit components to perform the method. Instructions for performing the method are typically recorded on a suitable recording medium. For example, the instructions may be printed on a substrate such as paper or plastic. The instructions may be present in the kit in the form of a package insert or on a label of the container of the kit or its components (e.g., associated with the package or subpackage).

[0098] Another aspect of the present invention provides a method for determining the purity of a differentiated product, the method comprising:

[0099] detecting the expression level of at least one biomarker of cells in a sample of the differentiated product, the biomarker being selected from CD144, vWF, and / or CD133; and determining the purity of the differentiated product based on the expression level of the biomarker.

[0100] In some embodiments, the cells in the sample are present in the form of a cell mixture, cell aggregate, or tissue.

[0101] Another aspect of the present invention provides a method for quantifying an EPC subset in a sample, the method comprising:

[0102] detecting the expression level of at least one biomarker of cells in a target sample, the biomarker being selected from CD144, vWF, and / or CD133; and determining the content of the EPC subset based on the expression level of the biomarker.

[0103] In some embodiments, at least one cell having the following expression profile is a late EPC: CD144 高 / + , vWF 高 / + , CD133 低 / - .

[0104] In some embodiments, at least one cell having the following expression profile is an early EPC: CD133 高 / + , CD144 低 / - and / or vWF 低 / - .

[0105] In some embodiments, the target sample comprises a cell mixture of EPCs.

[0106] In some embodiments, the target sample comprises a mixture of various types of cells. When the target sample comprises a mixture of various types of cells, the method for quantifying the EPC subpopulation in the quantitative sample may include, in addition to detecting the expression level of the aforementioned biomarker, detecting the expression level of a biomarker for confirming EPC.

[0107] On the other hand, the present invention provides a method for screening a candidate drug for regulating the EPC subpopulation, the method comprising adding a substance to be screened to a cell population and detecting the expression level of a biomarker of the cell, the biomarker being selected from CD144, vWF, and / or CD133; when the substance to be screened changes the expression level of the biomarker of the cell, the substance to be screened is a candidate drug for regulating the EPC subpopulation.

[0108] In some embodiments, if a substance to be screened is added and the biomarker has an increased following expression profile: CD144 高 / + , vWF 高 / + , CD133 低 / - , then the substance to be screened is a candidate drug for promoting late EPCs.

[0109] In some embodiments, if a substance to be screened is added and the biomarker has an increased following expression profile: CD133 高 / + , CD144 低 / - and / or vWF 低 / - , then the substance to be screened is a candidate drug for promoting early EPCs.

[0110] On the one hand, the present invention provides a method for treating an ischemic disease, the method comprising administering to a subject an effective amount of late EPCs or the pharmaceutical composition described above in the present invention.

[0111] In some embodiments, the late EPCs for treatment can be obtained by the method in the examples of the present application.

[0112] In some embodiments, the late EPCs for treatment can be obtained by the method for separating or enriching late EPCs described above.

[0113] "A therapeutically effective amount" will be the amount of an active agent that is capable of preventing or at least slowing (mitigating) a medical condition such as an ischemic disease, particularly lower limb ischemia. The dosage and administration of the cell 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 on, for example, the treatment objective, the route of administration, and the condition of the patient. Accordingly, it may be necessary for the therapist to adjust the dosage and change the route of administration as needed to achieve the best therapeutic effect.

[0114] The present invention will be further described in detail below with reference to the accompanying drawings and examples. The following examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. For experimental methods not specified in the examples, they are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer.

[0115] Example 1 Directed Differentiation and Identification of Early EPCs (Early Endothelial Progenitor Cells)

[0116] Preparation of Culture Medium

[0117] Preparation of E8 Complete Medium: Prepare E8 complete medium according to Table 1, and it can be stored at 4°C for up to 2 weeks.

[0118] Table 1 Preparation Table of E8 Complete Medium

[0119] Reagent Manufacturer Catalog Number Final Concentration / Volume E8 Basal Medium Stemcell 05991 450 mL E8 25× Supplement Stemcell 05992 20 mL Penicillin-Streptomycin Invitrogen 15140122 1％

[0120] Preparation of Differentiation Basal Medium: Prepare differentiation basal medium according to Table 2, and it can be stored at 4°C for up to 1 month.

[0121] Table 2 Preparation Table of Differentiation Basal Medium

[0122]

[0123] 1. Resuscitation and Passage of iPSCs

[0124] 1.1 Coating with Matrigel

[0125] (1) In an ice box, use hemostats to open a 1.5 mL EP tube containing matrigel.

[0126] (2) Pipette 100 μL of pre-cooled coating solution into the EP tube containing matrigel. After repeated pipetting, aspirate the supernatant back into the centrifuge tube. This process needs to be repeated multiple times to dissolve matrigel as quickly as possible without rewarming. The dilution of matrigel is 1:400.

[0127] (3) Take out the pre-cooled material to be coated and place it on an ice box. After mixing the coating solution with an electric pipette, perform coating according to the following volumes using a suitable measuring device: 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.

[0128] (4) Place the coated culture bottle / plate in an incubator at 37 °C and 5% CO₂ overnight.

[0129] 1.2 iPSCs Resuscitation

[0130] (1) Prepare E8 complete medium containing 10 μM Y-27632 according to the amount of medium required for resuscitation. Add 1 μL of Y-27632 (10 mM) stock solution to each milliliter of medium and preheat it at room temperature.

[0131] (2) Prepare 10 - 15 mL of E8 medium and preheat it in a 37 °C water bath.

[0132] (3) Take out 1 cryotube from the liquid nitrogen tank and immediately put it into a 37 °C water bath, shaking it back and forth continuously for 60 - 90 sec to completely thaw the frozen cell suspension.

[0133] (4) Once the cells in the cryotube are completely thawed (liquid), immediately take it out of the hot water bath, thoroughly disinfect the surface of the cryotube with 75% alcohol, and put it into the laminar flow hood. When the cryotube is about to thaw, place a centrifuge tube containing preheated E8 medium at 37 °C in the laminar flow hood after alcohol disinfection.

[0134] (5) Under strict aseptic conditions, take out the cell suspension in the cryotube and inject it into a 15 mL centrifuge tube containing preheated E8 medium. Gently pipette 2 - 3 times and centrifuge at 200 g for 5 min. After centrifugation, discard the supernatant.

[0135] (6) Add E8 medium containing 10 μM Y-27632, gently pipette 2 - 3 times, and take out the Matrigel-coated well plate / culture bottle from the incubator. Aspirate the coating solution and gently add the cell suspension along the uncoated surface (do not add directly onto the coated layer). Place the culture dish in the incubator for culture.

[0136] Note: When resuscitating, select the inoculation container according to the cell quantity. Generally, inoculate 500,000 - 800,000 cells into a 6-well plate, 800,000 - 1,000,000 cells into a T25 bottle, and 1,000,000 - 2,000,000 cells into a T75 bottle.

[0137] 1.3 iPSCs Digestion and Subculture;

[0138] (1) Prepare TrypLE working solution: Pipette 5 mL of DPBS into a new 15 mL centrifuge tube, then add 5 mL of TrypLE stock solution. Mix well to obtain the working solution.

[0139] (2) Prepare E8 complete medium containing 10 μM Y-27632 according to the amount of medium required for passage. Add 1 μL of Y-27632 (10 mM) stock solution to each milliliter of medium.

[0140] (3) Take out the well plate / culture flask to be passaged from the incubator, aspirate the supernatant, wash twice with DPBS (the amount of DPBS used each time should be no less than the amount of the original medium), 1 min each time (when washing, place the DPBS in the well / flask for 30 - 45 sec and then aspirate).

[0141] (4) After adding TrypLE working solution (add about 1 mL of TrypLE working solution to a six-well plate and about 2 mL of TrypLE working solution to a T25 flask), incubate in the incubator for 2 - 5 min. During this period, observe under the microscope. The cells shrink, become round and disperse.

[0142] (5) Gently tap the culture flask / plate to make the cells detach from the bottom of the plate, then gently pipette up and down several times, and finally add DMEM / F12 to terminate digestion.

[0143] (6) After balancing, centrifuge at 200 g for 5 min. After centrifugation, aspirate the supernatant, gently shake the bottom of the centrifuge tube, add 5 mL of E8 complete medium containing 10 μM Y-27632 to resuspend, and inoculate the cell suspension into a T25 flask coated with matrigel. Place the culture flask in an incubator at 37°C and 5% CO2 for static culture.

[0144] (7) After culturing for 24 h, perform a complete medium change with fresh E8 complete medium.

[0145] (8) Thereafter, perform a complete medium change operation every day. When the cell confluence is about 70% - 80%, passage the cells.

[0146] Note: The seeding density of iPSCs cells is about 8000 cells / cm 2 , according to different iPSCs lines, the passage density can be adjusted so that the interval passage time of iPSCs cells is 5 - 7 days.

[0147] 2. Procedure for inducing early differentiation of EPCs from iPSCS:

[0148] 2.1 Formation of hiPSCs monolayer adherent cells (Day - 1);

[0149] (1) Prepare TrypLE working solution: Pipette 5 mL of DPBS into a new 15 mL centrifuge tube, then add 5 mL of TrypLE stock solution. Mix well to obtain the TrypLE working solution.

[0150] (2) According to the amount of medium required for subculture, prepare E8 complete medium containing 1% PS and 10 μM Y-27632 (ROCKi). Add 1 μL of Y-27632 (10 mM) stock solution to each milliliter of medium.

[0151] (3) Take out the well plate / flask to be subcultured from the incubator, aspirate the supernatant, wash twice with DPBS (the amount of DPBS used each time should be no less than the amount of the original medium), 1 min each time (when washing, place the DBPS in the well / flask for 30 - 45 sec and then aspirate).

[0152] (4) After adding TrypLE working solution (add about 1 mL of TrypLE working solution to a six-well plate and about 2 mL to a T25 flask), incubate in the incubator for 2 - 5 min. During this period, observe under the microscope. The cells shrink, become round and disperse.

[0153] (5) Gently tap the culture flask / plate to detach the cells from the bottom of the plate, then gently pipette up and down several times, and finally add DMEM / F12 to terminate digestion. Aspirate an appropriate amount of cell suspension for counting.

[0154] (6) After balancing, centrifuge at 200 g for 5 min. After centrifugation, aspirate the supernatant, gently shake the bottom of the centrifuge tube, and resuspend according to different cell densities with E8 complete medium containing 10 μM Y-27632. After the cells are fully mixed, drop the cell suspension into the wells of the culture plate, and the seeding cell density is 8000 cells / cm 2 , and place it in a 37°C, 5% CO2 incubator for static culture.

[0155] (7) After culturing for 24 h, wash twice with DPBS and then proceed with subsequent induction and differentiation.

[0156] 2.2 Operation procedure for endoderm induction and differentiation (Day0 - 1)

[0157] (1) Prepare an appropriate amount of endoderm induction medium and preheat it in a 37°C water bath.

[0158] (2) Aspirate the original culture medium and add an appropriate amount of DPBS to wash the cells.

[0159] (3) Add the endoderm induction medium, and then place it in a 37°C, 5% CO2 incubator for static culture for 24 h.

[0160] Table 3 Preparation table of endoderm differentiation medium

[0161] Reagent Manufacturer Catalog Number Final Concentration Differentiation Basal Culture —— —— 1× CHIR 99021 Selleck S2924 10 μM

[0162] 2.3 Operation Process of Mesoderm Induction and Differentiation (Day1 - 3)

[0163] (1) Prepare an appropriate amount of mesoderm - specific medium and pre - heat it in a 37°C water bath.

[0164] (2) Aspirate the original culture medium and add an appropriate amount of DPBS to wash the cells.

[0165] (3) Add the mesoderm - specific medium and then incubate statically in a 37°C, 5% CO₂ incubator for 48 h.

[0166] Table 4 Preparation Table of Mesoderm Differentiation Medium

[0167] Reagent Manufacturer Catalog Number Final Concentration Differentiation Basal Culture —— —— 1× VEGF165 Peprotech AF-100-20 20 ng / mL bFGF Peprotech AF-100-18B 20 ng / mL

[0168] 2.4 Operation Process of Early EPC Induction and Differentiation (Day3 - 9)

[0169] (1) Prepare an appropriate amount of early EPC differentiation medium and pre - heat it in a 37°C water bath.

[0170] (2) After 48 h of mesoderm specialization, aspirate the original culture medium; add an appropriate amount of DPBS to wash the cells, digest the cells with TrypLE working solution until single - cell suspension, terminate cell digestion, centrifuge at 200g for 5 min, and then resuspend the cells with early EPC differentiation medium. Inoculate the cells at a density of 10000 cells / cm 2 ; then incubate statically in a 37°C, 5% CO₂ incubator.

[0171] (3) Thereafter, replace the fresh early EPC differentiation medium on the 4th and 5th days of differentiation.

[0172] (4) On the 6th day of differentiation, aspirate the original culture medium; add an appropriate amount of DPBS to wash the cells, digest the cells with TrypLE working solution until single - cell suspension, terminate cell digestion, centrifuge at 200g for 5 min, and then resuspend the cells with early EPC maintenance medium. Inoculate the cells at a density of 1000 cells / cm 2 ; then incubate statically in a 37°C, 5% CO₂ incubator.

[0173] (5) Thereafter, replace the fresh early EPC maintenance medium on the 7th and 8th days of differentiation.

[0174] (6) On the 9th day of differentiation, aspirate the original culture medium; add an appropriate amount of DPBS to wash the cells, digest the cells with TrypLE working solution until single - cell suspension, terminate cell digestion, centrifuge at 200g for 5 min, and then harvest the early EPC cells for cryopreservation and quality inspection.

[0175] Table 5 Preparation Table of Early EPC Differentiation Medium

[0176]

[0177]

[0178] Table 6 Preparation Table of Early EPC Maintenance Medium

[0179] Reagent Manufacturer Catalog Number Final Concentration Differentiation Basal Culture —— —— 1× VEGF165 Peprotech AF-100-20 20 ng / mL

[0180] Example 2 Directional Differentiation and Identification of Late EPC (late EPC)

[0181] Preparation of Basal Medium

[0182] Preparation of Mesoderm Induction Basal Medium: Prepare it freshly according to the specifications and ratios shown in Table 7, and it can be stored at 4°C for up to 1 month at most. Among them, B-27 minus insulin needs to be aliquoted according to the single-use amount and stored at -20°C. Avoid repeated freezing and thawing. Take out 1 tube each 1 day before use and thaw overnight at 4°C for use, or let it thaw at room temperature and stand still on the same day.

[0183] Table 7 Preparation Table of Mesoderm Induction Basal Medium

[0184] Total Volume 1000 mL 500 mL 100 mL 50 mL DMEM / F12 medium 500 mL 250 mL 50 mL 25 mL Neurobasal medium 500 mL 250 mL 50 mL 25 mL N-2(100X) 10 mL 5 mL 1 mL 0.5 mL B-27 minus insulin(50X) 20 mL 10 mL 2 mL 1 mL β-Mercaptoethanol 1 mL 0.5 mL 0.1 mL 0.05 mL

[0185] Preparation of EPCs Induction Basal Medium: Prepare it freshly according to the specifications and ratios shown in Table 8, and it can be stored at 4°C for up to 1 month at most. Among them, StemPro-34 Supplement needs to be aliquoted according to the single-use amount and stored at -20°C. Avoid repeated freezing and thawing. Take out 1 tube 1 day before use and thaw overnight at 4°C for use, or let it thaw at room temperature and stand still on the same day.

[0186] Table 8 Preparation Table of EPCs Induction Basal Medium

[0187] Total Volume 500 mL 100 mL 50 mL StemPro-34SFM medium 500 mL 100 mL 50 mL StemPro-34 Supplement 13 mL 2.6 mL 1.3 mL GlutaMAX(100X) 5 mL 1 mL 0.5 mL

[0188] 1. Resuscitation and Passage of iPSCs

[0189] (1) Take out a tube of cryopreserved iPSC cells from the iPSCs working library and quickly transfer it to the laboratory. Place it in a hot water bath with a water temperature of 38°C - 40°C and gently shake. When there are only tiny (about the size of millet grains) crystals left in the cryopreservation tube, thoroughly disinfect the surface of the cryopreservation tube with 75% alcohol and put it into the laminar flow hood.

[0190] (2) Use a pipette to transfer the cell suspension to a 15 mL centrifuge tube, add 5 mL of pre-warmed DMEM / F12 Medium at 37°C, and centrifuge at 200 g for 5 min at room temperature.

[0191] (3) After centrifugation, gently aspirate and discard the supernatant with a pipette. Then, add 1 mL of pre-warmed (37 °C) E8 complete medium containing ROCKi (add 1 μL of ROCKi stock solution per milliliter of medium) using a 1 mL pipette tip. Gently pipette up and down to resuspend the cell pellet, and aspirate 10 μL for cell counting and viability detection.

[0192] (4) Take out the prepared Matrigel-coated cell culture plate from the 37 °C / 5% CO2 cell culture incubator. Taking a T25 culture flask as an example, remove the liquid and add 5 mL of pre-warmed (37 °C) E8 complete medium containing ROCKi.

[0193] (5) Then, slowly add an appropriate amount of cell dilution solution to the T25 flask (seeding density 1.0 - 1.5×10 4 cell clusters / cm 2 ). Place the cell flask in the 37 °C / 5% CO2 cell culture incubator and shake it about 10 times in each of the front, back, left, and right directions (short, flat, and fast) to ensure that the cells are evenly distributed on the bottom surface of the culture flask as much as possible. Then, let it stand overnight. The iPSCs inoculated after recovery are at passage P13.

[0194] (6) After 24 h, replace with fresh pre-warmed (37 °C) E8 complete medium. 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× the microscope field of view. Then, the iPS cell passage can be carried out according to steps (3) - (7).

[0195] (7) The inoculation density for each passage is fixed at 1.0 - 1.5×10 4 cell clusters / cm 2 . The fixed amplification time is 4 days, and passage is carried out every 4 days. After 4 days of amplification at passage P14, proceed to the subsequent steps.

[0196] 2. Operation procedure for establishing monolayer differentiation of iPSCs (Day - 1)

[0197] (1) After officially starting the induction and differentiation of iPSCs, after normal centrifugation of the cells, remove the supernatant, add an appropriate amount of pre-warmed (37 °C) E8 complete medium containing ROCK, gently pipette up and down to resuspend the cell pellet, and then perform cell counting and viability detection on the resuspended cell suspension.

[0198] (2) Take out 4 Matrigel-coated T75 culture flasks from the 37 °C / 5% CO2 cell culture incubator, remove the liquid, and add 13 mL of pre-warmed (37 °C) E8 complete medium containing ROCK to each flask.

[0199] (3) The initial seeding density for the induction and differentiation of iPSCs needs to be controlled at 3.0×10 4 ~4.0×104 cells / cm 2 , according to the density of the cell suspension after counting, add an appropriate volume of the cell suspension to the Matrigel-coated T75 culture flask prepared above.

[0200] (4) Place the cell plate in a 37°C / 5% CO2 cell culture incubator and shake it about 10 times in each of the front, back, left, and right directions to ensure that the cells are evenly distributed on the culture plate surface as much as possible, and then let it stand overnight.

[0201] (5) After 24 hours, observe the polymerization degree after the iPSCs are seeded. If the polymerization degree reaches 15-25%, the subsequent formal induction steps can be directly entered. If the polymerization degree does not reach 15%, replace it with fresh E8 complete medium preheated at 37°C and appropriately extend the iPSCs culture time to 12-24 hours.

[0202] 3. Operating procedure for the induction and formation of lateral plate mesoderm (Day0-3)

[0203] (1) When the polymerization degree reaches about 15-25% after the iPSC cells are seeded, start the formal induction and differentiation, which is defined as Day0. Remove the old medium in the T75 flask, wash it once with 10 mL of DPBS, and then add 30 mL of mesoderm induction complete medium-1 preheated at 37°C to each flask, and then incubate it in a 37°C / 5% CO2 cell culture incubator for 17-18 hours.

[0204] Table 9 Mesoderm induction complete medium-1

[0205]

[0206] (2) After incubating for 17-18 hours (Day1), the iPS cells gradually spread out, the nuclear-cytoplasmic ratio decreases, and the cell area increases. Remove the old medium in the T75 flask, wash it once with 10 mL of DPBS, and then add 50 ml of mesoderm induction complete medium-2 preheated at 37°C to each flask, and then incubate it in a 37°C / 5% CO2 cell culture incubator for 2 days without changing the medium.

[0207] Table 10 Mesoderm induction complete medium-2

[0208]

[0209] 4. Operating procedure for the induction and formation of late EPCs (Day3-6)

[0210] (1) After the formation of the Day3 lateral plate mesoderm cells, remove the old medium in the T75 flask, wash it once with 10 mL of DPBS, and then add 30 mL of pre-warmed (at 37 °C) complete EPCs induction medium (prepared according to the following table) to each flask. Place the culture plate back into the 37 °C / 5% CO2 cell culture incubator and incubate for 24 hours.

[0211] Table 11 Preparation table of complete EPCs induction medium

[0212]

[0213]

[0214] (2) One day later (Day4), repeat the medium change operation of Day3, and add 30 mL of pre-warmed (at 37 °C) complete EPCs induction medium (prepared according to the above table) to each flask. Incubate for 24 hours until Day5, and the induction of EPCs with a high EC tendency is formed.

[0215] (3) At least 1 hour before enzymatic digestion and reseeding of EPCs on Day5, prepare 8 Fibronectin-coated T175 cell culture flasks.

[0216] (4) Remove all the old medium in the T75 flasks, wash twice with DPBS, then add 3 mL of TrypLE to each flask, place it in the 37 °C / 5% CO2 cell culture incubator for 3 - 5 minutes, observe the degree of cell detachment under the microscope until most of the cells start to float.

[0217] (5) Gently shake the bottom of the culture flask. After most of the cells have detached in a flowing sand-like state, add 12 mL of DMEM / F12 Medium to neutralize the digestion of TrypLE, gently pipette the detached cells to resuspend them, then transfer them to a centrifuge tube, and take an appropriate amount of cells for counting.

[0218] (6) Centrifuge at 200 g for 5 minutes at room temperature. During this period, prepare the complete EPCs maintenance medium (prepared according to the following table).

[0219] Table 12 Preparation table of complete EPCs maintenance medium

[0220]

[0221] (7) Remove the Fibronectin working solution in the T175 flasks that have been standing at room temperature for 1 hour, and rinse twice with an appropriate amount of double-distilled water. After centrifugation, discard the supernatant of the cells, take an appropriate amount of the prepared complete EPCs maintenance medium to resuspend the cells, and then evenly add the cells (transferring from 1 T75 flask to 2 T175 flasks) to the T175 flasks (35 ml / T175 flask).

[0222] (8) Place the culture flask in a 37 °C / 5% CO2 cell culture incubator and let it stand overnight.

[0223] (9) After 24 h (Day 6), the cells adhered to the wall. Wash them 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. Remove the supernatant after centrifugation at 200 g for 5 min.

[0224] (10) Use a pipette to aspirate a small amount of DPBS and gently pipette to resuspend each cell pellet, then transfer them to the same 50 mL centrifuge tube, ensuring that the total volume is not less than 40 mL. Remove the supernatant after centrifugation at 200 g for 5 min.

[0225] (11) Resuspend and wash again with 40 mL of DPBS, and take 50 μL for cell counting and viability detection.

[0226] (12) According to the cell density obtained from the detection, take the corresponding volume of the cell suspension according to the specified quantity required for quality control and send it to the quality inspection department for various quality inspections of the product.

[0227] (13) Centrifuge the remaining cell suspension again at 200 g for 5 min and freeze it in the form of EPCs stock solution according to the subsequent operation procedure.

[0228] Example 3 Detection of EPC Molecular Markers by Immunofluorescence

[0229] Table 13 Reagents

[0230]

[0231] Table 14 Working Solutions

[0232]

[0233]

[0234] Operation Steps:

[0235] (1) Accurately count the frozen EPC cell test samples after subculture or resuscitation, and calculate the total cell count according to the counting results. Take about 1×10 6 cells of EPCs by volume ratio, divide them into 8 wells, about 100,000 - 150,000 per well. The final volume of the complete culture medium for EPCs in each well is 0.5 mL. Mark the well positions for the test purpose, the tester, and the inoculation time. Place them in a 37 °C 5% CO2 cell culture incubator and culture until the confluence reaches more than 60%.

[0236] (2) Aspirate the liquid in the used well positions and wash three times with 500 μL of DPBS.

[0237] (3) Add 200 μL of fixative (4% paraformaldehyde) to each well and incubate at room temperature for 20 min.

[0238] (4) Aspirate the fixative and wash three times with 200 μL of DPBS.

[0239] (5) Add 200 μL of 0.1% Triton-X 100 and incubate at room temperature for 15 min.

[0240] (6) Aspirate the supernatant and wash three times with 200 μL of DPBS.

[0241] (7) Add 200 μL of 5% BSA blocking solution to each well and let stand at room temperature for 30 min.

[0242] (8) For the test wells, aspirate the supernatant and add 200 μL of primary antibody working solution (dilute the primary antibody 100-fold with 5% BSA to prepare the primary antibody working solution), and incubate overnight at 4°C. Do not treat the negative control wells and incubate overnight at 4°C.

[0243] (9) Aspirate the supernatant and wash 3 times with 200 μL of DPBS.

[0244] (10) Add 200 μL of secondary antibody working solution (dilute the secondary antibody 100-fold with DPBS to prepare the secondary antibody working solution) to each well and incubate for 1 h at room temperature in the dark.

[0245] (11) Aspirate the supernatant and wash 3 times with 200 μL of DPBS.

[0246] (12) Add 200 μL of DAPI working solution (dilute DAPI 200-fold with DPBS to prepare the DAPI working solution) to each well and incubate for 2 min at room temperature in the dark. In a darkroom environment, wait to observe and photograph under the microscope.

[0247] Experimental results and conclusions:

[0248] The results of immunofluorescence detection are as Figure 1 and Figure 2 shown. Both early EPC and late EPC highly express CD31, CD34, and KDR; late EPC also highly expresses CD144 and vWF, but early EPC weakly expresses or even does not express CD144 and vWF.

[0249] Conclusion: CD144 and vWF can be used as marker genes to distinguish early EPC from late EPC. Example 4 Detection of EPC molecular markers by flow cytometry

[0250] Table 15 Reagents

[0251]

[0252] Operation steps:

[0253] (1)Precisely count the cryopreserved test EPC cells after subculture or resuscitation, and calculate the total cell count according to the counting result. Take approximately 5×10 6 cells of the test sample by volume ratio. After centrifugation at 350 g for 5 min, transfer them to a 1.5 mL EP tube with 1 mL of DPBS. The cells collected in this step need to be prepared into a single-cell suspension by an appropriate method to prevent cell clumps from blocking the liquid flow system or affecting data accuracy.

[0254] (2)Group, aliquot, and label the test sample in 1.5 mL EP tubes according to the experimental settings. Set 7 groups, aliquot 100 μL of cell suspension into each tube, and label them as Blank (blank control group), 647 and 488 (negative control groups), KDR, CD34, CD31, and CD133 respectively.

[0255] (3)Prepare a mixture of DPBS and antibody at a ratio of 100:5 μL, incubate at room temperature in the dark for 30 min. Centrifuge with a microcentrifuge for 120 s, discard the supernatant, and disperse the cell pellet.

[0256] (4)Washing: Add 500 μL of DPBS to each tube for washing, centrifuge with a microcentrifuge for 120 s, discard the supernatant, and disperse the cell pellet. Repeat the washing operation 2 more times, for a total of 3 washes.

[0257] (5)Flow cytometry detection: Resuspend 200 μL of DPBS in each tube of the test sample to be detected, perform flow cytometry on the machine, save the document after the detection is completed, and analyze the flow cytometry results with analysis software.

[0258] Experimental results and conclusions:

[0259] The detection results are as Figure 3 and 4 shown. Both early EPC and late EPC highly express CD31, CD34, and KDR. Early EPC also expresses CD133, while late EPC has low expression or even no expression of CD133. Conclusion: CD133 can be used as one of the surface markers of early EPC and late EPC.

[0260] Example 5 Detection of the angiogenesis ability of EPC by in vitro tube formation assay

[0261] (1) Take the EPCs prepared in Example 1 and Example 2 respectively and culture them in a T25 cell culture flask for 3 days. Aspirate and discard the culture supernatant in the T25 cell culture flask, wash twice with 3 mL of DPBS, add 1 mL of TrypLE enzyme digestion solution, and place it in a 37°C 5% CO2 cell culture incubator for digestion for 3 - 5 minutes. Observe the degree of cell detachment under a microscope until most of the cells start to float.

[0262] (2) Gently shake the bottom of the culture flask. After most of the cells have detached in a flowing sand-like state, add 2 mL of culture supernatant to neutralize the digestion effect of TrypLE. Gently pipette the detached cells to resuspend them, and then transfer all the cell suspension to a 15 mL centrifuge tube. Centrifuge at 300 g for 5 minutes at room temperature.

[0263] (3) After centrifugation, discard the supernatant, add 0.5 mL of EPC maintenance complete medium to resuspend all the cell pellets, and transfer all the cell suspension to a 15 mL centrifuge tube.

[0264] (4) Take 50 μL of the cell suspension and calculate the total number of cells.

[0265] (5) Take an appropriate amount of the cell suspension and place it in a new 15 mL centrifuge tube. Supplement with EPC complete medium for the tube to make the total volume 350 μL, and the total number of cells is 3.50×10 5 cells.

[0266] (6) After slowly pipetting 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 incubated in a 37°C / 5% CO2 cell culture incubator for 30 minutes, 0.1 mL per well, that is, 1.0x10 5 cells / well. Place the 48-well cell culture plate in a 37°C / 5% CO2 cell culture incubator and let it stand for culture.

[0267] (7) After 6 hours, turn on the OLYMPUS inverted optical microscope, the camera system and the computer, and make them in the standby state.

[0268] Place the 48-well cell culture plate to be tested on the stage of the inverted microscope, adjust the light source and field of view of the inverted microscope, observe the results of the test article in the tube, take pictures and record, and archive the pictures.

[0269] Experimental results and conclusions:

[0270] The experimental results showed that both early EPCs and late EPCs had the ability to form tubes (A). However, when statistically analyzing the number of branches (B) and the average number of nodes (C) in the tube formation of early EPCs and late EPCs, there was no significant difference. There was a certain difference in the average tube formation length between early EPCs and late EPCs.

[0271] Conclusion: The ability to form tubes needs to be comprehensively judged from different dimensions. The above results indicate that both early EPCs and late EPCs have the ability to form tubes, but it cannot be used as a standard to distinguish between early EPCs and late EPCs.

[0272] Example 6 Identification of late EPCs by acetylated LDL uptake and Ulex europaeus agglutinin binding assay

[0273] Table 16 Reagents

[0274]

[0275] Method:

[0276] (1) Accurately count the frozen EPC cell test samples after subculture or resuscitation, and calculate the total cell count according to the counting results. Take about 1×10 6 cells of EPCs by volume ratio, divide them into 8 wells, about 100,000 - 150,000 per well. The final volume of the complete medium for EPCs in each well is 0.5 mL. Mark the inoculation well positions for test purposes, the tester, and the inoculation time. Place them in a 37°C, 5% CO2 cell culture incubator and culture until the confluence reaches more than 60% before preparing for staining.

[0277] (2) Add the fluorescent dye Dil-Ac-LDL to the above detection wells at a ratio of 1 μL per 100 μL of medium, and incubate at 37°C for 4 h.

[0278] (3) Aspirate the liquid in the wells and wash 3 times with DPBS, soak for 5 min each time.

[0279] (4) Add 200 μL of 4% paraformaldehyde to each well and fix for 10 min.

[0280] (5) Aspirate the liquid in the wells and wash 3 times with DPBS.

[0281] (6) Add 200 μL of FITC-UEA-I working solution to each well and incubate at room temperature for 1 h.

[0282] (7) Aspirate the liquid in the wells and wash 3 times with DPBS, then add 200 μL of DPBS.

[0283] (8) Observe and take pictures under a fluorescence microscope in a dark room environment.

[0284] Experimental Results and Conclusions:

[0285] The experimental results are as Figure 5 shown. Among them, acetylated LDL is labeled with red fluorescence, and UEA-I is labeled with green fluorescence. Acetylated low-density lipoprotein (Ac-LDL) cannot bind to the LDL receptor after being modified, and thus is taken up by macrophages and endothelial cells containing scavenger receptors that specifically recognize modified LDL, generating foam-like cells. Ulex europaeus agglutinin (UEA) is a protein extracted from Ulex europaeus. Lectins play an important role in cell recognition and adhesion reactions, mainly promoting cell adhesion. Lectins have more than one sugar-binding site and can thus participate in cell recognition and adhesion, linking different cells together. It can be seen from the figure that acetylated low-density lipoprotein (Ac-LDL) is rarely or even not taken up by early EPCs; late EPCs can efficiently take up acetylated low-density lipoprotein (Ac-LDL).

[0286] Conclusion: There are significant differences in the phagocytic ability of early EPCs and late EPCs for acetylated low-density lipoprotein (Ac-LDL). The phagocytic ability of late EPCs is stronger, which can be used as a functional test to distinguish early EPCs and late EPC cells.

[0287] Example 7: Pharmacodynamic Study of EPCs in the Treatment of Lower Limb Ischemia in Mice

[0288] Eighteen male Balb / c nude mice were selected and divided into 3 groups, namely the model control group, the early EPC treatment group, and the late EPC treatment group, with 6 animals in each group. The animals were established with a lower limb ischemia model by double ligation and transection of the femoral artery. After modeling, the treatment groups were given the test article endothelial progenitor cells EPCs (1×10 7 cells / kg) by intravenous injection; the model control group was only modeled without medication. Fourteen days after administration, lower limb scoring and laser speckle blood flow detection were performed, and the lower limb ischemia condition and function were judged based on the detection results to evaluate the therapeutic effect of the test article on lower limb ischemia in animals.

[0289] The results are as Figure 6 and 7 shown. After treatment with early EPCs, the lower limb ischemia rate of the animals in each treatment group showed a decreasing trend to varying degrees 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 the animals in each treatment group showed a significant decrease compared with the model control group, with statistical difference, indicating that late EPCs had a therapeutic effect on lower limb ischemia.

[0290] The description of the above embodiments is only for understanding the method of the present invention and its core idea. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

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

2. The use according to claim 1, characterized in that: The late EPC also has at least one of the following expression profiles: CD31 高 / + 、CD34 高 / + and / or KDR 高 / + .

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

4. The use according to claim 1, characterized in that: The late EPCs were identified by: The method comprises determining the expression of biomarkers in a cell, wherein the biomarkers include CD144, vWF, and CD133; and when the cell is determined to have the following expression profile, the EPC is a late EPC: CD144 高 / + 、vWF 高 / + and CD 133 低 / - .

5. The use according to claim 4, characterized in that: The method further comprises the step of identifying the cell as an EPC.

6. The use according to claim 4, characterized in that: Determining the expression of a biomarker in a cell comprises contacting the cell with a binding agent for the biomarker.

7. The use according to claim 1, characterized in that: The late EPCs are identified by the following method: the method comprises detecting the phagocytic ability of the cells.

8. The use according to claim 7, characterized in that: Compared with the early EPC subset, the late EPC subset has stronger phagocytic ability.

9. The use according to claim 7, characterized in that: The method detects the phagocytic ability of cells by detecting the uptake ability of acetylated LDL.

10. The use according to claim 1, characterized in that: The late EPCs are sorted, separated or enriched from the cell population by the following method: cells with the following expression profiles are selected: CD144 高 / + 、vWF 高 / + and CD 133 低 / - .

11. The use according to claim 10, characterized in that: The method further comprises selecting cells having at least one of the following expression profiles: CD31 高 / + 、CD34 高 / + and / or KDR 高 / + .

12. The use according to claim 11, characterized in that: The method comprises selecting cells having the following expression profile: CD144 高 / + 、vWF 高 / + and / or CD 133 低 / - 、CD31 高 / + 、CD34 高 / + and KDR 高 / + .

13. The use according to claim 10, characterized in that: The method further comprises culturing the late EPCs.

14. The use according to claim 1, characterized in that: The late EPCs are quantified by the following method, comprising: The expression level of at least one biomarker of cells in the target sample is detected, wherein the biomarker is selected from CD144 and vWF, and the content of late EPC is determined according to the expression level of the biomarker.

15. A pharmaceutical composition for treating ischemic diseases, characterized in that: The pharmaceutical composition comprises late stage EPCs having the following expression profile: CD144 高 / + 、vWF 高 / + and CD 133 低 / - The late EPC is derived from iPSC, and the dosage form of the pharmaceutical composition is an infusion type.

16. The pharmaceutical composition according to claim 15, characterized in that The late EPC also has at least one of the following expression profiles: CD31 高 / + 、CD34 高 / + and / or KDR 高 / + .

17. The pharmaceutical composition according to claim 16, characterized in that The late EPCs have the following expression profile: CD144 高 / + 、vWF 高 / + 、CD 133 低 / - 、CD31 高 / + 、CD34 高 / + and KDR 高 / + .

18. The pharmaceutical composition according to any one of claims 15 to 17, characterized in that The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

19. The pharmaceutical composition according to claim 15, characterized in that The ischemic disease is selected from lower limb ischemia.

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