Use of EPCs derived from induced pluripotent stem cell differentiation in preparation of a treatment for stroke

Abstract

This invention discloses the application of induced pluripotent stem cell (EPC) derived from induced pluripotent stem cell differentiation in the preparation of stroke therapeutic agents. Through intravenous injection of EPCs into an animal model of stroke, this invention demonstrates that this method can treat stroke by inhibiting atherosclerosis, reducing cerebral infarction levels, promoting angiogenesis in lesioned brain tissue, improving inflammatory responses in lesioned brain tissue, and repairing nerve damage and brain injury. The above research results of this invention provide a new treatment method for stroke in clinical practice.

Description

Technical Field

[0001] This invention belongs to the field of cell therapy and relates to the application of EPC derived from induced pluripotent stem cell differentiation in the preparation of stroke therapeutic agents. Background Technology

[0002] Stroke, also known as apoplexy, is a sudden-onset cerebrovascular disorder characterized by high morbidity, mortality, disability, and recurrence rates. According to the 2015 Global Burden of Disease Report, stroke has become the second leading cause of death worldwide. In my country, there are nearly 12.5 million stroke survivors, with a disability rate as high as 75%, and approximately 4.5 million patients experiencing varying degrees of loss of earning capacity or dependence on others for daily living. Ischemic stroke (IS) is the most common type of stroke, accounting for 69.6-70.8% of strokes in my country. About 80% of IS patients have intracranial and extracranial arterial disease, with large artery atherosclerosis posing the greatest risk. Large artery atherosclerosis leads to localized ischemic necrosis and functional impairment of brain tissue due to acute local thrombosis or embolism. Compared to other types of IS, large artery atherosclerotic IS has a higher recurrence, disability, and mortality rate. In Western countries, atherosclerosis in IS patients is mainly extracranial artery, while in my country it is mainly intracranial artery (ICAS).

[0003] Stroke is a complex, multifactorial, and multigenetic disease caused by vascular, environmental, and genetic factors. Cerebral arteriosclerosis (AS) is the main cause of ischemic stroke. AS is a chronic inflammatory pathological process of blood vessels involving multiple cells and cytokines, resulting from damage to vascular endothelial cell function. During the disease process, endothelial damage mediated by monocytes, macrophages, and T cells ultimately leads to the formation of vulnerable atherosclerotic plaques. Vulnerable plaques are characterized by large lipid centers, thin fibrous caps, low levels of smooth muscle cells and collagen, and high levels of inflammatory cells and mediators. Due to their vulnerability, plaques are prone to rupture, and rupture can lead to thrombus formation and vascular occlusion. Therefore, stabilizing plaques is an effective way to prevent and treat ischemic stroke.

[0004] Currently, treatment options for ischemic stroke are very limited. The most effective treatment in the acute phase is thrombolytic therapy, but due to the limited time window, only a small number of patients benefit from it, and most survivors suffer from varying degrees of functional impairment. Other conventional treatments include drug therapy, such as antiplatelet aggregation, statins to lower blood lipids, and control of blood pressure and blood sugar; and surgical treatments, such as intracranial and extracranial vascular stenting and carotid endarterectomy. According to a 2015 study by Wang Yongjun's team, the recurrence rate within one year for patients with acute ischemic stroke treated with aspirin in my country was 14%. A 2016 study by Feng Qin involving 288 patients with aspirin-treated atherosclerotic ischemic stroke showed that the disability rate in the aspirin monotherapy group was still as high as 52%. Therefore, there is an urgent clinical need for new interventions to treat ischemic stroke. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a method for treating stroke, comprising the injection of EPC (endothelial progenitor cells) and EC (endothelial cells) differentiated from iPSCs (induced pluripotent stem cells). The method of this invention can achieve the purpose of treating stroke by inhibiting atherosclerosis, reducing the level of cerebral infarction, promoting angiogenesis in lesioned brain tissue, improving the inflammatory response of lesioned brain tissue, and repairing nerve damage and brain injury.

[0006] According to one aspect of the present invention, the present invention provides the use of iPSC-derived endothelial progenitor cells or endothelial cells in the preparation of cell therapeutic agents that reduce the level of cerebral infarction.

[0007] According to another aspect of the present invention, the present invention provides the use of iPSC-derived endothelial progenitor cells or endothelial cells in the preparation of cell therapeutic agents for repairing neurological functional impairment.

[0008] According to another aspect of the present invention, the present invention provides the use of iPSC-derived endothelial progenitor cells or endothelial cells in the preparation of cell therapeutic agents that increase the number of M2 microglia infiltrating lesion brain tissue.

[0009] According to another aspect of the present invention, the present invention provides the use of iPSC-derived endothelial progenitor cells or endothelial cells in the preparation of cell therapeutic agents for repairing brain injury.

[0010] According to another aspect of the present invention, the present invention provides the use of iPSC-derived endothelial progenitor cells or endothelial cells in the preparation of cell therapeutic agents for the treatment of stroke.

[0011] Furthermore, the stroke includes ischemic stroke and hemorrhagic stroke.

[0012] Furthermore, the ischemic stroke includes transient ischemic attack, atherosclerotic thrombotic stroke, lacunar infarction, and cerebral embolism.

[0013] Furthermore, the hemorrhagic stroke includes cerebral hemorrhage and subarachnoid hemorrhage.

[0014] In a specific embodiment of the present invention, the stroke is ischemic stroke, preferably acute ischemic stroke.

[0015] According to another aspect of the present invention, the present invention provides the use of iPSC-derived endothelial progenitor cells or endothelial cells in the preparation of BDNF.

[0016] According to another aspect of the present invention, the present invention provides the use of iPSC-derived endothelial progenitor cells or endothelial cells in the preparation of cell therapeutic agents that promote the conversion of M1 microglia to M2 microglia.

[0017] According to another aspect of the present invention, the present invention provides the use of iPSC-derived endothelial progenitor cells or endothelial cells in the preparation of cell therapeutic agents that activate microglia.

[0018] Endothelial progenitor cells or endothelial cells derived from iPSC differentiation can be formulated together with a carrier in a suitable morphology, which is a pharmaceutically permissible carrier commonly used in cell therapy. Therefore, the cell therapy agents described above in this invention comprise endothelial progenitor cells or endothelial cells and a pharmaceutically permissible carrier. "Pharmaceutically permissible" refers to a composition that is physiologically permissible and generally does not cause gastrointestinal disturbances, dizziness, or other allergic reactions or similar reactions when administered to humans. Examples of pharmaceutically permissible carriers include, for example, water, suitable oils, physiological saline, water-soluble glucose, and non-oral delivery carriers such as ethylene glycol, and may further include stabilizers and preservatives. Suitable stabilizers are antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives are benzalkonium chloride, methylparaben or propylparaben, and chlorobutanol. For other pharmaceutically permissible carriers, please refer to the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

[0019] The cell therapy agents of the present invention are typically used in the form of non-oral formulations such as injections. Examples of carriers that can be used in non-oral formulations include aqueous carriers such as physiological saline and isotonic solutions containing glucose, D-sorbitol, etc.

[0020] Specifically, the injectable formulation of this invention comprises injectable solutions such as physiological saline, sodium lactate Ringer's 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, Pulmo-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, and 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, cytidine choline sodium chloride, fat-soluble vitamin II for injection, reduced glutathione for injection, brain protein hydrolysate for injection, sodium deoxynucleotide injection, multiple trace element injection II, mannitol injection, arginine hydrochloride injection, potassium chloride injection, cytidine triphosphate disodium for injection, ornithine aspartate for injection, etc.

[0021] The injection contains an isotonic or hypertonic solution; preferably, the solution is selected from NaCl injection (e.g., 0.9%–2.7% NaCl injection), glucose injection (e.g., 4%–5% glucose injection), sodium lactate Ringer's injection, compound electrolyte injection, HSA injection (e.g., 10%–20% HSA injection), succinogelatin injection (e.g., 4%–5% succinogelatin injection), and any combination thereof.

[0022] In addition to the injection solution, the cell therapy agent of the present invention may also contain one or more injection adjuvants, such as those selected from solubilizers, wetting agents, emulsifiers, buffers, suspending agents, chelating agents, antioxidants, antibacterial agents, local anesthetics, isotonic modifiers, fillers, protectants, and any combination thereof.

[0023] Furthermore, the cell therapy agent of the present invention may also include any one or more of the following functional components:

[0024] 1) Components that maintain the activity of endothelial progenitor cells or endothelial cells;

[0025] 2) Components that promote the proliferation of endothelial progenitor cells or endothelial cells;

[0026] 3) Components that promote the differentiation of endothelial progenitor cells.

[0027] The aforementioned functional components include serum substitutes, non-essential amino acids, glutamine, stabilized dipeptides of L-alanyl-L-glutamine, growth factors, and any combination thereof.

[0028] Furthermore, the functional ingredients include KOSR, MSC serum-free additive, Ultraser™ G, glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, VEGF, bFGF, EGF, TGFβ, PDGF, and any combination thereof.

[0029] As an alternative, the cell therapy agent of the present invention comprises endothelial progenitor cells or secretions of endothelial cells derived from iPSC differentiation.

[0030] The secretion may be the culture supernatant of endothelial progenitor cells or endothelial cells derived from iPSC differentiation, or it may be secretory factors of endothelial progenitor cells or endothelial cells derived from iPSC differentiation contained in purified culture supernatant.

[0031] Cellular therapeutic agents containing secretions can be formulated in any form known in the medical field. For example, they can be in the form of tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injectable solutions and lyophilized powders), etc. The limitations for injections are the same as before and will not be repeated here.

[0032] In a specific embodiment of the present invention, the cell therapy agent is endothelial progenitor cells or a mixture of endothelial cells and physiological saline.

[0033] The cell therapy agent of the present invention can be administered via any conventional route, as long as it can reach the target tissue. Furthermore, the cell therapy agent can be administered via any device capable of delivering the active ingredient to the target cells. When the cell therapy agent of the present invention is a non-oral formulation, examples include local administration such as intravenous (preferably intraperitoneal), intraperitoneal, enteral, subcutaneous, and subcapsular administration (capsule refers to the membrane tissue covering various organs). In a preferred embodiment, the cell therapy agent of the present invention is administered to the organism via intravenous administration.

[0034] The cell therapy agent of the present invention can be locally administered to an organism by being adhered to, in a form attached to a three-dimensional culture carrier, at or near the site of disease occurrence, the site where disease may occur, or the site constituting the cause of disease. In this embodiment, the material of the fibers forming the three-dimensional culture carrier is preferably a biocompatible material, and particularly preferably a biodegradable polymer.

[0035] The cell therapy agents of this invention can be administered at therapeutically effective amounts. As used herein, "therapeutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio suitable for any medical treatment. Effective dose levels can vary due to a variety of factors, including individual type, severity, age, and sex; drug activity; drug sensitivity; timing of administration; route of administration; discharge ratio; treatment duration; co-administered drugs; and other factors known in the medical field.

[0036] Typically, when a disease is treated with a cell therapy agent containing undifferentiated or differentiated cells as the active ingredient, the cell therapy agent is separated according to cell type and then administered to the individual site of injury via a syringe. The rationale for separating the cell therapy agent according to cell type is to prevent a decrease in the effectiveness of the cell therapy agent caused by co-administration of cells and routine injection via syringe.

[0037] In some embodiments, the dosage of the cell therapy agent is not less than 1 × 10⁻⁶. 4 cells / mL (e.g., not less than 1×10⁻⁶) 4 cells / ml, not less than 3 × 10 4 cells / ml, not less than 5 × 10 4 cells / ml, not less than 7 × 10 4 cells / ml, not less than 1×10 5 cells / ml, not less than 3 × 10 5 cells / ml, not less than 5 × 10 5 cells / ml, not less than 7 × 10 5 cells / ml, not less than 1×10 6 cells / ml, not less than 3 × 10 6 cells / ml, not less than 5 × 10 6 cells / ml, not less than 7 × 10 6 cells / ml, not less than 1×10 6 cells / ml, not less than 3 × 10 6 cells / ml, not less than 5 × 10 6 cells / ml, not less than 7 × 10 6 cells / ml, not less than 1×10 6 cells / ml, not less than 3 × 10 6 cells / ml, not less than 5 × 10 6 cells / ml, not less than 7 × 10 6 cells / ml, not less than 1×10 6 cells / ml, not less than 3 × 10 6 cells / ml, not less than 5 × 10 6 cells / ml, not less than 7 × 10 6cells / ml, not less than 1×10 10 cells / ml, not less than 3 × 10 10 cells / ml, not less than 5 × 10 10 1 / ml or not less than 7×10 10 cells / ml, for example 1×10 5 -1×10 8 7×10 5 -7×10 6 1×10 6 -5×10 6 per ml.

[0038] In some embodiments, the dosage of the cell therapy agent is not less than 1 × 10⁻⁶. 3 Units / kg (e.g., not less than 1×10) 3 Units / kg, not less than 3×10 3 Units / kg, not less than 5×10 3 Units / kg, not less than 7×10 3 Units / kg, not less than 1×10 4 Units / kg, not less than 3×10 4 Units / kg, not less than 5×10 4 Units / kg, not less than 7×10 4 Units / kg, not less than 1×10 5 Units / kg, not less than 3×10 5 Units / kg, not less than 5×10 5 Units / kg, not less than 7×10 5 Units / kg, not less than 1×10 6 Units / kg, not less than 2×10 6 Units / kg, not less than 5×10 6 Units / kg, not less than 7×10 6 Units / kg, not less than 1×10 7 Units / kg, not less than 3×10 7 Units / kg, not less than 5×10 7 Units / kg, not less than 7×10 7 Units / kg, not less than 1×10 8 Units / kg, not less than 3×10 8 Units / kg, not less than 5×10 8 Units / kg, not less than 7×10 8 Units / kg, not less than 1×10 9 Units / kg, not less than 3×10 9 Units / kg, not less than 5×10 9 Units / kg, not less than 7×10 9Units / kg, not less than 1×10 10 Units / kg, not less than 3×10 10 Units / kg, not less than 5×10 10 Units / kg or not less than 7×10 10 per kg, for example 1×10 5 -1×10 8 7×10 5 -7×10 6 1×10 6 -5×10 6 per kg.

[0039] The cell therapy agent of the present invention can be administered alone or in combination with other therapies. Co-administration of the therapeutic agent of the present invention with other therapies can be performed simultaneously or sequentially. Single or multiple doses are possible. Importantly, all factors should be taken into account when using the minimum possible amount sufficient to achieve maximum efficacy without side effects.

[0040] Other treatments for stroke include medication or surgery. Medication treatment mainly includes thrombolytic agents, neuroprotective agents, calcium channel blockers, and brain metabolism activators.

[0041] Thrombolytic drugs include alteplase, urokinase, streptokinase, recombinant streptokinase, lumbrokinase, and thrombolytic enzymes.

[0042] Neuroprotective drugs include citicoline, brain protein extract, deproteinized calf serum extract, gangliosides, and the antioxidant edaravone.

[0043] Calcium channel blockers include nimodipine, cinnarizine, and flunarizine.

[0044] Brain metabolism activators such as piracetam, piracetam, and piracetam.

[0045] The method of administration of the cell therapy agent of the present invention is as follows:

[0046] 0.1-0.2 ml of cell therapy agent was administered intravenously at a rate of 60 drops per minute.

[0047] Furthermore, the intravenous injection time of the cell therapy agent of the present invention can be 1 day, 2 days, 3 days or more after the onset of stroke.

[0048] According to another aspect of the present invention, the present invention provides a method for preparing BDNF in vitro, the method comprising culturing endothelial progenitor cells or endothelial cells derived from iPSC differentiation.

[0049] Furthermore, the method includes obtaining the culture supernatant of endothelial progenitor cells or endothelial cells from iPSC differentiation.

[0050] Furthermore, the method includes isolating and purifying BDNF from the culture supernatant.

[0051] According to another aspect of the present invention, the present invention provides a method for in vitro promotion of the conversion of M1 microglia into M2 microglia, the method comprising contacting M1 microglia with endothelial progenitor cells or endothelial cells derived from iPSC differentiation, or contacting M1 microglia with secretions from endothelial progenitor cells or endothelial cells derived from iPSC differentiation, or contacting M1 microglia with the aforementioned cell therapy agent.

[0052] According to another aspect of the present invention, the present invention provides a method for treating stroke, the method comprising administering to a patient the iPSC differentiation-derived endothelial progenitor cells or endothelial cells described above, secretions of the iPSC differentiation-derived endothelial progenitor cells or endothelial cells described above, and the cell therapy agent described above.

[0053] In some implementations, the subject is a mammal, such as a mouse or a human.

[0054] In this invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0055] As used in this article, the term "endothelial progenitor cell" refers to a cell whose differentiation is directed toward endothelial cells. Endothelial progenitor cells can be identified by analyzing the expression patterns of transcription factors or cell surface antigens. For example, the expression patterns of transcription factors or cell surface antigens, measured alone or in combination, show that their expression levels are undetectable or low (even if detected before induction of differentiation) and significantly increase after induction of differentiation. Effective markers for identifying endothelial progenitor cells include UBXN11, LRRC75A, MMACHC, FXYD5, VMP1, KRT18, YIPF2, CNP, SPATA21, SAP30L-AS1, SLC12A6, ANP32E, CTNNB1, VIM, CNN3, RPL8, YWHAH, SDC4, HIST1H4I, SRPX2, DNASE1, MYO19, SH3KBP1, ASAP1, RNF24, SPNS2, S100A11, SOX4, RNF214, PLP2, and PIH1D2. , SGPL1, ELK3, F3, UGDH, RBMS2, FMNL3, RPL6, ATP2A1, EFCAB10, SLC12A2, UCKL1-AS1, ARHGAP18, RPE, SFT2D2, CXCL16, PAICS, ZBTB1 1-AS1, CLIC1, CERS5, CCDC85C, DHRS7, CTRL, TYMS, SPCS1, IFITM3, RPS27, RPL34, MZF1-AS1, TRAM1, SUPT7L, NOL9, MFSD11, PLD4, NQO 1, NEAT1, GNAS, LDHA, UNKL, LDHB, CENPK, C1orf174, HSP90B1, TCF7L2, LENG8, IQGAP3, ANKRD13D, NPC1, MYH11, ATP2B4, FANCD2, TNR C6B, DDX17, TIRAP, HERC4, DSTN, FOXP3, ABL2, KDELR2, CCDC50, PARK7, CAPN2, ACAP1, SLC13A4, SEPT11, RAPH1, KRT8, LMO3, CIRBP, CY P20A1, LY6E, FAM69A, PRSS3, MEST, NPB, CLMP, MSN, SUCLA2, PCMTD1, PSMC3, TOMM7, GCHFR, MEIS1, TRIAP1, USP34, SPAG1, RPL39L, PD K1, LARS, MAP4K4, QTRT1, CLK1, TSC22D1-AS1, IL1B, MKRN2OS, ARL16, TSPAN13, HMGCS1, PSAP, SLC44A2, TNFRSF10B, MSC, ENG, TLDC2,TRIM41,CPM,CREB3L2,NSUN6,AHCY,NCBP1,N4BP2L2,RAB31,RNF149,SRPK2 ,SBDS,NAMPT,AES,SERPINF1,RPS27L,HAUS2,AP4B1-AS1,KDM2B,USP6NL,T ONSL,FBLIM1,SLC31A2,ADGRL1,EFHC1,SMAD9,SLC7A11,LPIN1,DDAH2,PID 1,SCAF11,BBS1,PCYOX1,CD55,PMEL,TOR1B,UBQLN1,DUSP5,CUEDC2,FKBP5, LMF2,LYRM7,NSG1,SAT2,LMAN1,NFX1,COMMD1,CCPG1,NINJ1,RNASEH1,SPA TA5,CENPW,DNAJC4,PTTG1,TMEM109,MCTS1,TMEM63A,FBN1,TPM1,MAVS,ADP RHL2,CTSK,INSIG1,C16orf74,MAZ,MEG3,RFTN2,AGAP6,GPC6,PDLIM5,SLC 25A5-AS1,RSL1D1,FAM219B,CFLAR,GNPTAB,PIGU,ITGB3BP,NTM,ZNF106,RP RD1B,ETS1,ITGAE,ECSCR,SND1,TRIO,ANGPTL1,SEC61G,CDR2,CRMP1,PNIS R,OXLD1,PRKACA,NFKBIZ,MORN2,RAB3D,CRKL,MSL1,TMPO,GALNT2,CRELD2 , ETS2, ALKBH4, PHF19, POLH, TRRAP, NET1, ADGRE5, GAS6, VEZF1, BMPR2, SMAD3, GFOD1, RABGAP1L, SLC39A7, TCTN1, CSRNP1, LRRN3, TNRC6A, MFAP2, TMEM9 8,SPATC1L,LINC00476,CENPQ,CSTF3,SLC8A1,E2F7,RPL7L1,ZFHX4,TXNDC 11,STK4,STX16,TMEM11,PTK6,ZFAS1,U2AF2,TRIM16,ZBTB7A,P3H3,TMCO3, C15orf39,ACVRL1,SHQ1,GNS,SPRY1,DIAPH2,DLC1,TCF3,FAM229A,PTGES3 L,RRP7A,MGRN1,GIT2,UBE2T,CSGALNACT2,EFEMP2,CSNK1E,MAGED2,C5AR1,ARHGAP45,CDKN2B-AS1,TMEM234,RNF213,SIDT2,TEX2,MCM3,TMEM67,ZKSC AN1,SLC2A1,MAD2L1,MGLL,NR3C1,PHGDH,SYT1,RERE,KIF1B,NCAPD3,NADK ,FOXRED2,GNB5,TMEM204,USP24,KIF20B,GADD45B,ZNF135,TACC3,FOSB,A COT9,EBF1,MYO1E,PPP1R10,NFKBIB,ROBO4,TPGS1,CFD,KIF22,EIF4A3,RN F14,NPAT,STX11,CDCA7,ST6GALNAC4,RBL1,NIF3L1,GLCE,CDCA7L,RASA3, STK10,EDNRA,ICA1L,DCTN5,GNG2,PTPN2,BST1,BMP1,RPS6KA2,SEC11C,TR MU, H1F0, STXBP5L, AP5B1, STAM, MCM2, ALDH3B1, SLC23A2, TNFAIP3, KLF7, DIP2C, KMT2B, GALNT7, NUAK2, PLXNA4, MRI1, FLNB, MBOAT2, TMEM136, AP3M2, A CSS2,AARS,DUS3L,NGLY1,ZNF274,ICAM1,FAM129A,PPP6R1,TIE1,NUMBL,P LXND1,ENC1,CCDC142,BCAN,PRR15,FLRT2,NUCB2,TARS2,POLB,RAB24,KBT BD6,SLC38A4,MAP2,PMP22,TOP3A,VAMP2,UBTD2,PLK4,MAPK8IP3,APBA2,T BC1D17,ZNF444,PLEKHG4B,MTOR,STIL,GBA,IFT88,B9D1,RAMP1,S1PR3,SHI SA9,MOCS3,CEP104,TIAM1,KIF15,ZNF793,ZNF865,ABCB10,ANKHD1-EIF4E BP3,CMPK2,SYTL3,SHROOM1,SPATA6,PPP2R3B,TNFSF13B,FKBP7,CNTROB,P EX11A,LHFPL2,DOK3,ARMC9,RANBP10,ZNF561,SCAMP5,ARHGAP39,DENND3, ATP9A,DCPS,ZNF302,ARHGAP9,IP6K1,ADAM22,EMCN,CYFIP2,WDR90,PPM1M,C1QTNF6, DACH1, BMF, SETD4, TBC1D9, PIK3IP1, P4HTM, CD101, SCPEP1, TLDC1, GTPBP10, TAP2,

[0056] The isolation, purification, in vitro culture, and characterization of endothelial progenitor cells were described in Hill et al., N. Engl. J. Med.: 593-600 (2003); Assmus et al., Circulation 106: 3009-16 (2002); Wang et al., J. Am. Coll. Cardiol. 4949: 1566-71 (2007); and Kalka et al., PNAS 97: 3422-7 (2000), the contents of which are incorporated herein by reference in their entirety.

[0057] Endothelial progenitor cells (EPCs) and their progeny can be cryopreserved until needed using any method known in the art. (See, for example, U.S. Patent No. 5,071,741, PCT International Patent Applications WO93 / 14191, WO95 / 07611, WO96 / 27287, WO96 / 29862 and WO98 / 14058, Karlsson et al., 65 Biophysical J.2524-2536 (1993)). EPCs can be suspended in an isotonic solution containing a specific cryopreservative, preferably a cell culture medium. Such cryopreservatives include dimethyl sulfoxide (DMSO), glycerol, etc. These cryopreservatives are used at concentrations of 5-15% (e.g., 8-10%). The cells are gradually frozen to temperatures from -10°C to -150°C (e.g., -20°C to -100°C, or -70°C to -80°C).

[0058] The sources of naturally occurring endothelial progenitor cells isolated in the body include peripheral blood, bone marrow, and umbilical cord blood.

[0059] The iPSCs of this invention can be derived from somatic cells and differentiated by induction from somatic cells.

[0060] The somatic cells can be stem cells or mature cells. These cells can be referred to as "donor cells." Adult stem cells are undifferentiated cells distributed throughout the body. They can proliferate through cell division to replenish dead cells and regenerate damaged tissue. Adult or somatic stem cells have been identified in many organs and tissues, including the brain, bone marrow, peripheral blood, blood vessels, skeletal muscle, skin, teeth, heart, intestine, liver, ovarian epithelium, and testes. They are believed to be located in specific regions within each tissue, known as "stem cell nests," and provide a cell source exclusively for that tissue. Types of adult stem cells include hematopoietic stem cells, mesenchymal stem cells, neural stem cells, epithelial stem cells, and skin stem cells.

[0061] If mature cells are harvested as donor cells, they can come from any tissue, organ, body fluid, or bodily secretions. Therefore, cells can be skin cells, hair follicle cells, blood cells, cells extracted from urine, or cells collected from any tissue or organ, including but not limited to bone, teeth, dental tissue, heart, lungs, brain, pancreas, liver, kidneys, bladder, uterus, intestines, stomach, gallbladder, muscle, fat, testes, mucous membranes, eyes, foreskin, prostate, spleen, or any other tissue.

[0062] As used herein, the term "culture" refers to the product obtained by culturing cells (e.g., endothelial progenitor cells or endothelial cells of the present invention) in a culture medium.

[0063] As used herein, the term "culture supernatant" refers to a culture medium containing no cells themselves, obtained by culturing cells (e.g., endothelial progenitor cells or endothelial cells of the present invention). Thus, a culture supernatant usable in the present invention can be obtained, for example, by separating and removing cellular components after culturing. This culture supernatant may also undergo other processing, such as centrifugation, concentration, solvent replacement, dialysis, freezing, drying, freeze-drying, dilution, desalting, preservation, etc.

[0064] The term “subject” includes, but is not limited to, various animals, such as mammals, such as bovines, equines, sheep, suidae, canines, felines, rabbits, rodents (e.g., mice or rats), non-human primates (e.g., macaques or cynomolgus monkeys), or humans.

[0065] Advantages and beneficial effects of the present invention:

[0066] The EPCs induced by iPSCs in this invention have a stronger BDNF secretion capacity compared to naturally occurring EPCs in vivo.

[0067] This invention utilizes EPCs induced by iPSC differentiation, which have a stronger therapeutic effect on stroke compared to naturally occurring EPCs in the body. Attached Figure Description

[0068] Figure 1 The experimental results of the balloon injury model are shown, where A: sham surgery group; B: control group; C: treatment group;

[0069] Figure 2 A statistical chart showing CD31 expression;

[0070] Figure 3 A statistical graph showing IgG expression levels;

[0071] Figure 4This figure shows a comparison between EPCs derived from iPSC differentiation and EPCs from natural sources. Detailed Implementation

[0072] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0073] Unless otherwise specified, the experiments and methods described in the examples are generally performed according to conventional methods well known in the art and described in various references. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. Those skilled in the art will understand that the examples describe the invention by way of illustration and are not intended to limit the scope of protection claimed by the invention. All disclosures and other references mentioned herein are incorporated herein by reference in their entirety.

[0074] Example 1: Induced pluripotent stem cells differentiate into endothelial progenitor cells

[0075] The cells described in steps 2-5 were cultured in an anaerobic incubator containing 5% oxygen and 5% carbon dioxide.

[0076] Step 1: Pre-treat induced pluripotent stem cells to obtain a cell resuspension;

[0077] Step 2: Take the cell resuspended solution obtained in Step 1, and mix it at a ratio of 1 cm... 2 Seed 20,000 to 50,000 cells at a ratio of 10,000 to 50,000 cells in the bottom area of ​​the cell culture plate, transfer them into a cell culture plate containing a mixture of TeSR-E8 medium and ROCK inhibitor Y27632, and incubate in a cell culture incubator for 16 to 24 hours;

[0078] Step 3: Remove the old culture medium from the cell culture plate from Step 2, and wash the cell culture plate once with DPBS, adding new culture medium per 10 cm³. 2 The cell culture plate was filled with 4 mL of N2B27 insulin-deficient medium at a ratio of 4 mL to 16-24 hours. The N2B27 insulin-deficient medium was prepared by adding BMP4, CHIR-99021, and Activin A to the N2B27 insulin-deficient medium. The concentration of BMP4 was 20-40 ng / mL, the concentration of CHIR-99021 was 6-12 µM, and the concentration of Activin A was 30-80 ng / mL.

[0079] Step 4: Remove the old culture medium from the cell culture plate from Step 3, and wash the cell culture plate once with DPBS, adding water per 10 cm³. 2The cell culture plate was filled with 6 mL of N2B27 insulin-deficient medium at a ratio of 6 mL to 6 mL. The plate was then incubated in a cell culture incubator for 48 hours to obtain lateral mesodermal cells. The N2B27 insulin-deficient medium was prepared by adding BMP4 and CHIR-99021 to the N2B27 insulin-deficient medium, with the concentration of BMP4 being 20-40 ng / mL and the concentration of CHIR-99021 being 6-12 µM.

[0080] Step 5: Remove the old culture medium from the cell culture plate containing mesodermal cells from Step 4, and wash the cell culture plate once with DPBS, adding new culture medium per 10 cm³. 2 Add 4 mL of StemPro-34 medium to the bottom of the cell culture plate, and then incubate in a cell culture incubator for 16-24 hours; repeat step 5 once to induce endothelial progenitor cells in the cell culture plate; the first StemPro-34 medium is StemPro-34 medium supplemented with VEGFA, Forskolin and SB431542, wherein the concentration of VEGFA is 100-300 ng / mL, the concentration of Forskolin is 1-4 µM, and the concentration of SB431542 is 5-15 µM;

[0081] Step 6: Remove the old culture medium from the cell culture plate containing endothelial progenitor cells from Step 5, and wash the cell culture plate twice with DPBS, at a ratio of 10 cm⁻¹. 2 The cell culture plate was filled with 0.4-0.6 mL of liquid at the bottom area. The second cell enzymatic digestion solution was added for enzymatic digestion. Then, 4 times the volume of DMEM / F12 medium was added to neutralize the digestion solution and the cells were transferred to centrifuge tubes to obtain the enzymatically digested endothelial progenitor cells.

[0082] Step 7: Centrifuge the enzymatically digested endothelial progenitor cells obtained in Step 6, remove the supernatant, and divide them into portions at 10 cm³. 2 The cell culture plate was filled with 1 mL of medium at a ratio of 1 mL to 1 mL of the bottom surface area to obtain a resuspension of endothelial progenitor cells. The second StemPro-34 medium was prepared by adding VEGFA and PluriSIn-I to the StemPro-34 medium, wherein the concentration of VEGFA was 30-80 ng / mL and the concentration of PluriSIn-I was 15-30 µM.

[0083] Step 8: Resuspend the endothelial progenitor cells obtained in Step 7 at a ratio of 1 cm... 2The bottom area of ​​the cell culture plate is seeded with 60,000 to 100,000 cells, which are then transferred into a cell differentiation culture plate containing StemPro-34 medium and cultured in a cell culture incubator for 16 to 24 hours.

[0084] Step 9: Remove the old culture medium from the cell culture plate from Step 8, and wash the cell culture plate twice with DPBS, at a ratio of 10 cm⁻¹. 2 The volume of liquid added to the bottom area of ​​the cell culture plate is 0.4-0.6 mL. The second cell enzymatic digestion solution is added for enzymatic digestion. Then, four times the volume of DMEM / F12 medium is added to neutralize the digestion solution and the mixture is transferred to a centrifuge tube. The endothelial progenitor cell suspension obtained after enzymatic digestion is centrifuged, washed and frozen to obtain the clinical-grade endothelial progenitor cell stock solution.

[0085] Step 10: The frozen endothelial progenitor cell stock solution from Step 9 can be further prepared into an injectable fresh reinfusion formulation for clinical use after thawing, centrifugation, and washing.

[0086] Example 2: Constructing a balloon injury model to study endothelial progenitor cell function

[0087] 1. Method

[0088] Thirty SD rats, half male and half female, were selected and divided into a control group, a sham-operated group, and a low-dose EPC (endothelial progenitor cell) group (0.75 × 10⁻⁶). 6 cell / animal), medium dose group (1.0×10 6 cell / animal), high-dose group (1.5×10 6 (6 rats per group, half male and half female), cyclophosphamide (120 mg / kg) was injected intraperitoneally to establish immunosuppression in rats. Then, a balloon injury model was established using percutaneous transluminal coronary angioplasty (PTCA). EPC derived from iPSC differentiation and an equal amount of solvent were infused in time. After 14 days, the animals were dissected to observe the morphology of the blood vessels, detect the intimal thickness, and assess the colonization of EPC at the injury site.

[0089] Sham surgery group: After anesthetizing the animals, the skin and muscles were incised to expose the external carotid artery without causing damage. The incision was then closed in layers. Physiological saline was injected into the tail vein.

[0090] Control group and treatment group: After anesthetizing the animals, the skin and muscles were incised to expose the external carotid artery. A small transverse incision was made in the distal 1 / 3 of the external carotid artery using microsurgical scissors. From this point, a PTCA catheter was inserted retrogradely through the incision into the common carotid artery 2-2.5 cm. The intima was peeled off by slowly aspirating and retracting three times. The proximal segment of the external carotid artery was ligated (at least 2 mm from the bifurcation). The pulsation of the common carotid artery and internal carotid artery was checked. The subcutaneous tissue and skin were sutured layer by layer.

[0091] Different doses of EPC (cells dissolved in physiological saline, 0.1 ml or 0.2 ml, 60 drops per minute) were infused into the tail vein of the treatment group, while the control group received the same volume of physiological saline. Cyclophosphamide (120 mg / kg) was administered intraperitoneally one day before infusion to establish immunosuppression in rats.

[0092] 2. Results

[0093] Two weeks after carotid artery endothelial injury surgery, light microscopy revealed that the arterial structures of the sham-operated rats were intact, with no reduction in lumen area, smooth intima without hyperplasia, and neatly arranged smooth muscle cells in the media. Figure 1 A). In the control group rats, extensive neointimal formation, significant intimal thickening, and vascular stenosis were observed at the site of injury. Figure 1 B). The treatment group rats showed significantly reduced intimal thickening and luminal stenosis compared to the control group. Figure 1 C). The ratio of intimal thickness to control thickness was 0.22 in the low-dose group, 0.16 in the medium-dose group, and 0.11 in the high-dose group. Human-derived EPCs were observed at the site of endothelial injury.

[0094] The above results indicate that EPCs can be implanted at the injury site, effectively promoting endothelial re-endothelialization at the site of arterial injury, thereby reducing the formation of neointima, maintaining the morphology of the arterial lumen, and preventing the development of atherosclerosis, thus preventing stroke. Furthermore, the number of EPCs transplanted is correlated with the degree of repair of the intimal damaged vessel.

[0095] Example 3: Constructing an MCAO model to study EPC functionality

[0096] 1. Model building methods

[0097] Rats were fasted the night before the experiment and anesthetized with a mixed anesthetic (Shutai 50: Xylazine hydrochloride, 10:1 mixture). They were then fixed in a supine position on a surgical board. A longitudinal incision of approximately 3 cm was made in the neck to expose and bluntly dissect the left common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). A slipknot was tied at the proximal end of the CCA to temporarily block blood supply. A double knot was tied in the external carotid artery (ECA), and a small incision was made in the middle of the double knots using microscissors to insert a silicone-coated nylon suture (with silicone at the tip). The suture is 4.5mm long, with a suture tip diameter of 0.34±0.02mm and a tip length of 30-50mm. The external carotid artery (ECA) is severed between the double knots. The direction of the suture is adjusted to allow it to enter the internal carotid artery (ICA). Using the bifurcation of the ICA and ECA as a reference point, the suture is inserted to the preset mark. Resistance is felt, indicating that the suture head has reached the middle cerebral artery (MCA). The suture is then secured with a knot, and a saline-soaked cotton swab is placed at the surgical incision to keep it moist. One hour after embolization, the suture is removed, the common carotid artery slipknot is loosened, and the incision is sutured shut. The animal is allowed to recover naturally.

[0098] 2. Evaluation metrics for the MCAO model

[0099] 2.1 Neurobehavioral scoring

[0100] (1) Longa 5-point system:

[0101] 0 points: Normal activity, no symptoms of neurological deficit;

[0102] 1 point: When lifting the tail, the paralyzed forelimb is adducted and cannot be fully extended, indicating mild neurological deficit;

[0103] 2 points: When walking, the rat rotates to the paralyzed side, indicating moderate neurological deficit;

[0104] 3 points: When walking, the rats were unsteady on their feet and fell to the paralyzed side, indicating severe neurological deficits;

[0105] 4 points: Unable to walk independently or unconscious;

[0106] (2) mNSS 18-point scoring method:

[0107] The Modified Neurological Severity Score Points (mNSS) includes four components: sensory, motor, balance, and reflexes, with a total score of 18 points. The more severe the neurological impairment, the higher the score.

[0108] Table 1. Scoring Criteria for the mNSS 18-Point Rating Method

[0109]

[0110] 2.2 Brain TTC staining and infarct area calculation

[0111] Principle: TTC (2,3,5-triphenyltetrazolium chloride) reacts with dehydrogenases in normal tissues and turns red, while ischemic tissues, due to tissue necrosis and loss of dehydrogenase activity, cannot react with TTC and therefore appear pale.

[0112] Staining method: Immediately after euthanasia, the animal was rapidly perfused with physiological saline into the left ventricle for 2 minutes. The head was quickly decapitated, the skull was opened, and the intact brain was removed. On ice, the brain was cut into five coronal sections, each approximately 2 mm thick, from front to back. These sections were stained with 2% TTC solution at 37°C in the dark for 30 minutes. Excess dye was washed away, and the sections were fixed with 4% paraformaldehyde before photographing. Normal tissue stained red, while ischemic tissue stained white. The middle brain slice was selected and analyzed using ImageJ image analysis software (1.5e; NIH, Bethesda, MD, USA). Statistical analysis of TTC staining was performed to calculate the pixel values ​​of the infarct area (white area) and the normal brain tissue area (red area). The formula for calculating the proportion of cerebral infarction area was: Cerebral infarction area proportion = Infarct area pixels in the section / Total area pixels.

[0113] 3. In vivo drug efficacy plan

[0114] Twenty-four hours after surgery in rats with the MCAO model, neurological function was assessed using the mNSS and Longa scales. Rats with a Longa 5 score of 2 and an mNSS 18 score between 7 and 10 were selected and divided into three groups: G1-G3, with 12 animals in each group. Group G1 included 3 animals as a sham-operated group. Group G2 received a tail vein injection of physiological saline containing 3% albumin, and Group G3 received a tail vein injection of 2 x 10^9 EPC cells derived from iPSC differentiation. 6 Cells / kg, EPC injection method as before).

[0115] 4. Results

[0116] 1) EPC therapy derived from iPSC differentiation reduces the level of cerebral infarction.

[0117] When the EPC administration window was 2 days (2 days after successful animal model establishment and 2 days after stroke onset), the proportions of cerebral infarction on day 1 and day 7 of treatment were 0.38±0.09 and 0.20±0.03, respectively, and 0.07±0.03 and 0.09±0.04 after treatment, respectively. In the solvent control group, the proportions were 0.49±0.03 and 0.24±0.04, respectively. The proportion of cerebral infarction on day 1 after treatment was significantly lower than that in the solvent control group at the same time point (0.07±0.03 vs 0.49±0.03, p<0.001); the proportion of cerebral infarction on day 7 after treatment was also significantly lower than that in the solvent control group at the same time point (0.09±0.04 vs 0.24±0.04, p<0.05). These results indicate that EPC treatment can significantly reduce the proportion of cerebral infarction in rats with MCAO and shows a certain dose-response trend.

[0118] 2) Level of improvement in neurobehavioral behavior (Longa 5-point scale and mNSS score)

[0119] When the administration window for EPC derived from iPSC differentiation was 1 day (1 day after successful animal modeling and 1 day after stroke onset), at 3 and 7 days post-administration, the Longa5 score in the solvent control group was 2.0±0.0, while in the treatment group it was 1.5±0.2 and 1.2±0.2, respectively. Compared with the solvent control group, the Longa5 score in the treatment group was significantly lower, showing a significant decrease at 7 days post-administration (p<0.01). At 7 days post-administration, the mNSS score in the treatment group was significantly higher than that in the control group (5.7±2.2 vs 7.7±0.2, p<0.01). These results indicate that administration can improve neurological function impairment in the model group.

[0120] Seven days after treatment with iPSC-derived EPCs, the mNSS scores at dosing windows of days 1, 2, and 3 were 4.8±0.5, 5.7±0.2, and 6.2±0.5, respectively, all significantly lower than those in the solvent control group at the same time point (7.7±0.2, G4, G5, p<0.01; G6, p<0.05). These results indicate that dosing at different time windows can reduce the neurological function scores of rats.

[0121] 3) EPCs derived from iPSC differentiation promote angiogenesis in brain tissue of MCAO rat lesions.

[0122] CD31 expression was increased in the brain tissue of MCAO rats at the lesion site on both 1 and 7 days after EPC treatment, with CD31 expression on 7 days post-treatment significantly higher than that in the solvent control group (p<0.05). Figure 2 Furthermore, VEGF mRNA expression in the brain lesions was significantly higher than that in the solvent control group one day after EPC treatment (p<0.05). The results indicate that EPC treatment promotes angiogenesis in the brain lesions.

[0123] 4) EPC reduces inflammation and brain damage in the infarcted brain of MCAO rats.

[0124] One day after treatment with EPC derived from iPSC differentiation, the mRNA expression of IL-6 and TNF-α in the brain tissue of MCAO rat lesions was significantly lower than that in the solvent control group (p<0.05, p<0.01). In addition, CD68 expression in the brain tissue of lesions showed a decreasing trend at 1 day and 7 days after EPC treatment.

[0125] 5) Effects of transplantation of iPSC-derived EPCs / ECs on microglia / macrophages

[0126] In the pathological process of stroke, the neurological pathological response initially manifests as demyelination, followed by axonal injury and neuronal necrosis as the disease progresses. Microglia have two main phenotypes, M1 and M2, both of which influence myelin regeneration. Effective myelin repair after injury requires a pro-regenerative response from microglia (M2 phenotype). To assess the alteration of microglia phenotype by iPSC-derived ECs, Arg1 double-labeled microglia / macrophages (CD11b+) were used in the lesion area. Immunohistochemistry showed that after transplantation of iPSC-derived ECs, the density of Arg1+CD11b+ double-labeled cells in the lesion area significantly increased (control: 27.28 / mm). 2 EC of iPSC divergence source: 57.54 / mm 2 The proportion of Arg1+ cells marked with Arg1 was also increased (48.38% vs 65.88%, p<0.05). These data suggest that ECs derived from iPSC differentiation regulate microglia / macrophage activation and promote phenotype M1 to M2 switching, which is beneficial for OPC differentiation and maturation.

[0127] Regarding the reduction in the number of M2 type microglia and the reduction in infarct area, naturally derived EPCs only showed improvement at extremely high doses (10). 7 Only 1 / 3 of the efficacy of EPCs with 2 million cells / kg as the source of iPSC differentiation is achieved (p < 0.05%).

[0128] 6) EPCs derived from iPSC differentiation reduce blood-brain barrier permeability in MCAO rats.

[0129] Following treatment with EPC derived from iPSC differentiation, IgG expression in the brain tissue of MCAO rat lesions decreased at both 1 and 7 days. At 7 days post-treatment, IgG levels were significantly lower than in the solvent control group (p<0.05). This result indicates that EPC treatment can reduce blood-brain barrier permeability in MCAO rats. Figure 3 ).

[0130] Example 4: Comparison of the efficacy of EPC derived from iPSC differentiation and EPC derived from natural sources.

[0131] 1. Collect EPC from natural sources

[0132] EPC derived from umbilical cord blood was used. The isolation and culture methods of EPC are as described in the literature (Hypertension 201259(5):1037-43).

[0133] 2. Detect the amount of BDNF secreted by EPC.

[0134] Procedure: EPCs derived from iPSC differentiation and EPCs derived from nature were fixed with 4% paraformaldehyde for 20 minutes, then blocked with BSA for 30 minutes. Next, BDNF mouse primary antibody was added and incubated overnight at 4°C. After washing, the mice were incubated with anti-mouse secondary antibody carrying green fluorescence for 1 hour at room temperature. After washing, the fluorescence intensity was observed and photographed using a confocal fluorescence microscope.

[0135] 3. Balloon injury model

[0136] The naturally sourced EPC was applied to the experiment in accordance with the method of Example 2.

[0137] 4. MCAO Model

[0138] The naturally sourced EPC was applied to the experiment in accordance with the method of Example 3.

[0139] 5. Results

[0140] In a balloon injury model, EPCs derived from iPSC differentiation and those of natural origin showed similar results in repairing and promoting microvascular regeneration after stroke (p>0.05%). However, the BDNF secretion of naturally derived EPCs was only 0.7 times that of iPSC-derived EPCs (p<0.05%). Regarding the reduction in the number of M2 microglia and the infarct area, naturally derived EPCs showed significant differences only at very high doses (e.g., 1010). 7 Only 2 million cells / kg of EPC, which are the source of iPSC differentiation, can exert 1 / 3 of the efficacy (p<0.05%). The above comparison results are shown in [reference needed]. Figure 4 In the figure, iPS-EPC represents EPC derived from iPSC (induced pluripotent stem cells) differentiation, while EPC refers to EPC of natural origin.

[0141] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. Application of iPSC-derived endothelial progenitor cells in the preparation of cell therapy agents that increase the number of M2 microglia infiltrating lesion brain tissue; The endothelial progenitor cells are differentiated from iPSCs under hypoxic conditions, and the specific steps are as follows: Step 1: Pre-treat induced pluripotent stem cells to obtain a cell resuspension; Step 2: Take the cell resuspended solution obtained in Step 1, and mix it at a ratio of 1 cm... 2 Seed 20,000 to 50,000 cells at a ratio of 10,000 to 50,000 cells in the bottom area of ​​the cell culture plate, transfer them into a cell culture plate containing a mixture of TeSR-E8 medium and ROCK inhibitor Y27632, and incubate in a cell culture incubator for 16 to 24 hours; Step 3: Remove the old culture medium from the cell culture plate from Step 2, and wash the cell culture plate once with DPBS, adding new culture medium per 10 cm³. 2 The cell culture plate was filled with 4 mL of N2B27 insulin-deficient medium at a ratio of 4 mL to 16 mL, and then incubated in a cell culture incubator for 16-24 hours. The first N2B27 insulin-deficient medium was prepared by adding BMP4 and CHIR to N2B27 insulin-deficient medium. CHIR-99021 and ActivinA, wherein the concentration of BMP4 is 20-40 ng / mL, the concentration of CHIR-99021 is 6-12 μM, and the concentration of ActivinA is 30-80 ng / mL; Step 4: Remove the old culture medium from the cell culture plate from Step 3, and wash the cell culture plate once with DPBS, adding water per 10 cm³. 2 The cell culture plate was filled with 6 mL of N2B27 insulin-deficient medium at a ratio of 6 mL to 6 mL. The plate was then incubated in a cell culture incubator for 48 hours to obtain lateral mesodermal cells. The N2B27 insulin-deficient medium was prepared by adding BMP4 and CHIR-99021 to the N2B27 insulin-deficient medium, with the concentration of BMP4 being 20-40 ng / mL and the concentration of CHIR-99021 being 6-12 μM. Step 5: Remove the old culture medium from the cell culture plate containing mesodermal cells from Step 4, and wash the cell culture plate once with DPBS, adding new culture medium per 10 cm³. 2 Add 4 mL of StemPro-34 medium to the bottom of the cell culture plate, and then incubate in a cell culture incubator for 16-24 hours; repeat step 5 once to induce endothelial progenitor cells in the cell culture plate; the first StemPro-34 medium is StemPro-34 medium supplemented with VEGFA, Forskolin and SB431542, wherein the concentration of VEGFA is 100-300 ng / mL, the concentration of Forskolin is 1-4 μM, and the concentration of SB431542 is 5-15 μM; Step 6: Remove the old culture medium from the cell culture plate containing endothelial progenitor cells from Step 5, and wash the cell culture plate twice with DPBS, at a ratio of 10 cm⁻¹. 2 The volume of liquid added to the bottom area of ​​the cell culture plate was 0.

4. Add 0.6 mL of cell digestion solution to perform enzymatic digestion, then add 4 times the volume of DMEM / F12 medium to neutralize, and transfer to a centrifuge tube to obtain enzymatically digested endothelial progenitor cells; Step 7: Centrifuge the enzymatically digested endothelial progenitor cells obtained in Step 6, remove the supernatant, and divide them into portions at 10 cm³. 2 The cell culture plate was filled with 1 mL of a second StemPro-34 medium to obtain a resuspension of endothelial progenitor cells. The second StemPro-34 medium was prepared by adding VEGFA and PluriSIn-I to StemPro-34 medium. The concentration of VEGFA was 30-80 ng / mL, and the concentration of PluriSIn-I was 15-30 μM; Step 8: Resuspend the endothelial progenitor cells obtained in Step 7 at a ratio of 1 cm... 2 The bottom area of ​​the cell culture plate is seeded with 60,000 to 100,000 cells, which are then transferred into a cell differentiation culture plate containing StemPro-34 medium and cultured in a cell culture incubator for 16 to 24 hours. Step 9: Remove the old culture medium from the cell culture plate from Step 8, and wash the cell culture plate twice with DPBS, at a ratio of 10 cm⁻¹. 2 The cell culture plate was filled with 0.4-0.6 mL of cell digestion solution at a ratio of 0.4-0.6 mL. The cell digestion solution was then added for enzymatic digestion. Subsequently, 4 times the volume of DMEM / F12 medium was added for neutralization, and the mixture was transferred to a centrifuge tube. The endothelial progenitor cell suspension obtained after enzymatic digestion was centrifuged, washed, and frozen to obtain the clinical-grade endothelial progenitor cell stock solution. The cells described in steps 2-5 were cultured in an anaerobic incubator containing 5% oxygen and 5% carbon dioxide.

2. The application according to claim 1, characterized in that, The method for preparing the cell therapy agent includes mixing iPSC-derived endothelial progenitor cells with a pharmaceutically permissible carrier.

3. The application according to claim 2, characterized in that, The cell therapy agent is prepared by mixing iPSC-derived endothelial progenitor cells with physiological saline.

4. The application according to claim 1, characterized in that, The cell therapy agent is administered via intravenous injection.

5. The application according to claim 1, characterized in that, The method of administration of the therapeutic agent is as follows: 0.1-0.2 ml of cell therapy agent was administered intravenously at a rate of 60 drops per minute.

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