Cell therapy medicine for treating fatty degeneration related diseases

Abstract

The invention discloses a cell therapy medicine for treating fatty degeneration related diseases. The cell therapeutic drug contains endothelial progenitor cells or endothelial cells derived from iPSC differentiation. The endothelial progenitor cells derived from iPSC differentiation are intravenously injected into a fatty liver disease animal model, and it is found that the mode can achieve the effect of treating the fatty liver disease by relieving fatty degeneration of liver tissue. The research result of the invention provides a new treatment mode for clinically treating fatty degeneration related diseases, especially fatty liver diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular, the present invention relates to a cell therapy drug for treating fatty liver disease. Background Art

[0002] Fatty Liver Disease (FLD) refers to the abnormal accumulation of fat in the liver, which is mainly divided into alcoholic fatty liver disease (AFLD) and non-alcoholic fatty liver disease (NAFLD). NAFLD is one of the most common liver diseases, characterized by fatty degeneration of hepatocytes. During the course of the disease, it is often accompanied by the occurrence of metabolic syndromes such as obesity, type 2 diabetes, hypertension and dyslipidemia. In the absence of a history of continuous or recent heavy drinking, the use of drugs that promote lipogenesis, and the absence of specific genetic diseases, the presence of simple fatty degeneration of hepatocytes in the liver (>5%) can be diagnosed as NAFLD. Its severity can range from simple fat accumulation (simple fatty liver) to steatohepatitis (NASH) accompanied by inflammation, and even develop into cirrhosis and hepatocellular carcinoma.

[0003] Fatty liver disease is widely prevalent worldwide, especially in Western countries. NAFLD is the most common chronic liver disease in Western countries, and its prevalence has increased with the prevalence of obesity and type 2 diabetes. The incidence of fatty liver disease is on the rise worldwide, especially in economically developed countries and regions. The global pooled prevalence of NAFLD is estimated to be 32.4%, with men (39.7%) being higher than women (25.6%). The prevalence has increased year by year, reaching 37.8% after 2016; the pooled prevalence is highest in Latin America (44.4%), followed by the Middle East, North Africa, South Asia, Southeast Asia, North America, East Asia, and lowest in Western Europe (25.1%). In the past 20 years, the aggregate prevalence of NAFLD in adults in my country was 29.6%, with a higher prevalence in men (34.8%) than in women (23.5%). The prevalence of NAFLD in obese and T2DM patients was 66.2% and 51.8%, respectively. It has now replaced viral hepatitis as the number one chronic liver disease in my country (Chinese Society of Hepatology. Guidelines for the Prevention and Treatment of Metabolic-Related (Non-Alcoholic) Fatty Liver Disease (2024 Edition) [J]. Chinese Journal of Hepatology, 2024, 32(5): 418-434.).

[0004] At present, the treatment of fatty liver disease mainly focuses on controlling risk factors and preventing disease progression, including changing lifestyle. Weight loss, increasing physical activity and improving eating habits are the basis for treating NAFLD. Weight loss of 5% can significantly improve hepatic steatosis (EASL-EASD-EASO Clinical Practice Guidelines on the management of metabolic dysfunction-associated steatotic liver disease (MASLD) [J]. J Hepatol, 2024, 81(3): 492-542.); drug treatment. There is currently no approved drug specifically for NAFLD, but some drugs such as vitamin E, insulin sensitizers (such as metformin) and anti-inflammatory drugs (such as corticosteroids) have shown certain effects in clinical trials (Xiang Y, Shen L, Xue Y, et al. Efficacy and safety of diacerein monotherapy in adults with obesity: A randomized, double-blind, placebo-controlled trial [J]. Diabetes Obes Metab, 2024.); surgical treatment. For patients with fatty liver disease caused by severe obesity, if lifestyle intervention and drug treatment are ineffective, weight loss surgery can be considered, which can significantly reduce weight and improve the condition of fatty liver disease (Yang Jialin, Peng Yong. Research progress in weight loss surgery for the treatment of non-alcoholic fatty liver disease [J]. Advances in Modern General Surgery in China, 2021, 24(1): 51-53, 57.). However, lifestyle adjustments require long-term adherence by patients and may be slow to take effect; drug treatment may be accompanied by adverse reactions and must be performed under the guidance of professional medical personnel; surgical treatment involves higher risks and is expensive. Therefore, there is an urgent need to develop new drugs to treat fatty liver disease and provide more treatment options for patients with fatty liver disease. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the inventors of the present invention have conducted extensive and in-depth research and discovered for the first time that endothelial progenitor cells derived from iPSC differentiation have the effect of treating fatty liver disease, especially non-alcoholic fatty liver disease, providing a new treatment for fatty liver disease.

[0006] Based on this, the present invention provides the following technical solutions:

[0007] A first aspect of the present invention provides a pharmaceutical composition for treating, preventing, alleviating and / or alleviating diseases associated with steatosis.

[0008] Furthermore, the pharmaceutical composition comprises endothelial progenitor cells or endothelial cells differentiated from iPSCs.

[0009] The term "endothelial progenitor cell" used herein means a cell whose differentiation is directed to an endothelial cell. Endothelial progenitor cells can be confirmed by analyzing the expression pattern of transcription factors or cell surface antigens. For example, the expression pattern of transcription factors or cell surface antigens is measured alone or in combination, wherein its expression level is not detected or low (even if detected before induction of differentiation), and significantly increases after induction of differentiation. The markers effectively used to confirm 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, 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,<h2 style=";text-align:left;direction:ltr">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,<h2 style=";text-align:left;direction:ltr">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. The isolation, purification, ex vivo culture and characteristics of endothelial progenitor cells are 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. ,

[0010] Endothelial progenitor cells and endothelial progenitor cell offspring can be cryopreserved by any method known in the art until they are needed. (See, e.g., U.S. Pat. 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 a concentration of 5-15% (e.g., 8-10%). The cells are gradually frozen to a temperature of -10°C to -150°C (e.g., -20°C to -100°C or -70°C to -80°C).

[0011] In some embodiments, iPSC can be derived from somatic cells, which are induced and differentiated by somatic cells. 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 that are distributed throughout the body. They can proliferate by cell division to supplement dead cells and regenerate damaged tissues. Adult or somatic stem cells have been identified in many organs and tissues, including brain, bone marrow, peripheral blood, blood vessels, skeletal muscle, skin, teeth, heart, intestines, liver, ovarian epithelium and testis. They are considered to be located in a specific area called "stem cell nest" in each tissue and only provide a cell source for that tissue. The type of adult stem cell includes hematopoietic stem cells, mesenchymal stem cells, neural stem cells, epithelial stem cells, skin stem cells. If mature cells are harvested as donor cells, they can come from any tissue, organ, body fluid or body secretion. Thus, the cell can be a skin cell, a hair follicle cell, a blood cell, a cell extracted from urine, a cell collected by biopsy or the like from any tissue or organ including, but not limited to, bone, teeth, dental tissue, heart, lung, brain, pancreas, liver, kidney, bladder, uterus, intestine, stomach, gallbladder, muscle, fat, testicles, mucosa, eye, foreskin, prostate, spleen, or any other tissue.

[0012] In the present invention, any of many standard techniques well known to those skilled in the art can be used to induce iPSCs to differentiate into endothelial progenitor cells. For example, iPSCs can be induced to differentiate into endothelial progenitor cells by adding specific growth factors to the culture medium or adjusting the culture medium components (such as adding vascular endothelial growth factor VEGF or a factor that promotes endothelial cell differentiation, FGF-2, etc.). In a specific embodiment of the present invention, the endothelial progenitor cells derived from iPSC differentiation are derived from Chengnuo Regenerative Medicine Technology (Beijing) Co., Ltd.

[0013] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0014] Endothelial progenitor cells or endothelial cells derived from iPSC differentiation can be formulated in a suitable form together with a carrier, which is a pharmaceutically acceptable carrier commonly used in cell therapy. Therefore, the pharmaceutical composition described above of the present invention comprises endothelial progenitor cells or endothelial cells and a pharmaceutically acceptable carrier. The so-called "pharmaceutically acceptable" refers to a composition that is physiologically permitted and does not usually cause gastrointestinal disorders, dizziness, and other allergic reactions or similar reactions when administered to humans. As a pharmaceutically acceptable carrier, for example, there are: water, suitable oils, physiological saline, water-soluble glucose and ethylene glycol, etc., and non-oral administration carriers, etc., and stabilizers and preservatives may be further included. Suitable stabilizers are antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives are benzalkonium chloride, methyl parahydroxybenzoate or propyl parahydroxybenzoate, chlorobutanol. As other pharmaceutically acceptable carriers, reference may be made to the contents described in the following document (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

[0015] In some embodiments, solid dosage forms for oral administration may include tablets, pills, powders, granules, capsules, etc. Such solid preparations can be prepared by mixing one or more excipients into one or more mixtures, for example, mixed starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition, lubricants such as magnesium stearate and talcum powder can also be used in addition to simple excipients. Liquid preparations for oral administration include suspensions, internal solutions, emulsions, syrups, etc., and in addition to water and liquid paraffin, which are widely used as simple diluents, a variety of excipients such as wetting agents, sweeteners, aromatics, preservatives, etc. can also be included.

[0016] In some embodiments, preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, oils, freeze-dried preparations, suppositories, etc. Non-aqueous solvents and suspending agents can use propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable lipids such as ethyl oleate, etc. The base of the suppository can use witepsol, polyethylene glycol, Tween 61, cocoa butter, lauric acid ester, glycerin, gelatin, etc.

[0017] In some embodiments, the pharmaceutical composition of the present invention can have any dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, suspensions, oils, lyophilized agents and suppositories. The base of the suppository can use witepsol, polyethylene glycol, Tween 61, cocoa butter, lauric acid esters, glycerin, gelatin, etc.

[0018] Furthermore, the diseases associated with steatosis include fatty liver disease, coronary heart disease, myocardial infarction, kidney disease, diabetes, and hyperlipidemia.

[0019] Furthermore, the disease associated with steatosis is selected from fatty liver disease.

[0020] In a specific embodiment of the present invention, the applicant has experimentally demonstrated that endothelial progenitor cells derived from iPSC differentiation can improve the symptoms of fatty liver disease in mice. Therefore, the pharmaceutical composition comprising endothelial progenitor cells or endothelial cells derived from iPSC differentiation of the present invention can treat, prevent, alleviate and / or relieve diseases related to fatty liver disease, including fatty liver disease, coronary heart disease, myocardial infarction, kidney disease, diabetes, hyperlipidemia, wherein the symptom is fatty liver disease.

[0021] Furthermore, the pharmaceutical composition further comprises any one or more of the following functional ingredients:

[0022] 1) Components that maintain the activity of endothelial progenitor cells or endothelial cells;

[0023] 2) ingredients that promote the proliferation of endothelial progenitor cells or endothelial cells;

[0024] 3) Components that promote the differentiation of endothelial progenitor cells.

[0025] Furthermore, the functional ingredients include serum replacement, non-essential amino acids, glutamine, stabilized dipeptide of L-alanyl-L-glutamine, growth factors or any combination thereof.

[0026] Furthermore, the functional ingredients include KOSR, MSC serum-free supplement, UltroserTMG, glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, VEGF, bFGF, EGF, TGFβ, PDGF or any combination thereof.

[0027] As another alternative, the pharmaceutical composition of the present invention comprises secretions of endothelial progenitor cells or endothelial cells derived from iPSC differentiation.

[0028] The secretion may be the culture supernatant of endothelial progenitor cells or endothelial cells differentiated from iPSCs, or may be secretory factors of endothelial progenitor cells or endothelial cells differentiated from iPSCs contained in the purified culture supernatant.

[0029] In the present invention, the term "culture supernatant" refers to a culture fluid obtained by culturing cells (e.g., endothelial progenitor cells or endothelial cells of the present invention) that does not contain the cells themselves. Thus, for example, the culture supernatant that can be used in the present invention can be obtained by separating and removing cell components after culturing. The culture supernatant can also be subjected to other treatments, such as centrifugation, concentration, solvent replacement, dialysis, freezing, drying, freeze drying, dilution, desalting, storage, etc.

[0030] A second aspect of the present invention provides the use of endothelial progenitor cells or endothelial cells derived from iPSC differentiation in the preparation of a drug for treating, preventing, alleviating and / or alleviating diseases associated with steatosis.

[0031] Furthermore, the diseases associated with steatosis include fatty liver disease, coronary heart disease, myocardial infarction, kidney disease, diabetes, and hyperlipidemia.

[0032] Furthermore, the disease associated with steatosis is selected from fatty liver disease.

[0033] Furthermore, the iPSC-derived endothelial progenitor cells or endothelial cells are present in an amount of 1×10 4 ~1×10 6 cells.

[0034] Preferably, the iPSC-derived endothelial progenitor cells or endothelial cells are present in an amount of 1×10 6 cells.

[0035] Furthermore, the drug can be used directly or in the form of a pharmaceutical composition.

[0036] Furthermore, the pharmaceutical composition comprises endothelial progenitor cells or endothelial cells differentiated from iPSCs.

[0037] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0038] Furthermore, the dosage form of the drug includes a parenteral dosage form and / or a parenteral dosage form.

[0039] Furthermore, the non-intestinal administration dosage form includes an injection dosage form, a cavity administration dosage form, a mucosal administration dosage form and / or a skin administration dosage form.

[0040] Furthermore, the dosage forms for administration via the gastrointestinal tract include tablets, granules, capsules, solutions, powders, sustained-release preparations, emulsions, suspensions, syrups and / or drops.

[0041] Preferably, the dosage form of the drug is selected from an injection dosage form.

[0042] In some embodiments, the dosage form of the pharmaceutical composition of the present invention is a dosage form that is prepared by conventional methods and is conducive to administration, including but not limited to: parenteral dosage forms, gastrointestinal dosage forms, and specific examples include but are not limited to: aqueous solution injection, powder injection, pills, powders, tablets, patches, suppositories, emulsions, creams, gels, granules, capsules, aerosols, sprays, powder sprays, sustained-release agents and controlled-release agents, etc.

[0043] In some embodiments, the injection dosage forms include, but are not limited to, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, intracavity injections and other injections.

[0044] In some embodiments, the cavity administration dosage form includes, but is not limited to, suppositories, aerosols, effervescent tablets, drops, pills, etc., and is used in the rectum, vagina, urethra, nasal cavity, ear canal, etc.

[0045] In some embodiments, the mucosal administration dosage form includes, but is not limited to, eye drops, nasal drops, eye ointments, gargles, sublingual tablets, adhesive tablets, patches, and the like.

[0046] In some embodiments, the skin administration dosage form includes, but is not limited to, topical solutions, lotions, liniments, ointments, plasters, pastes, patches, and the like.

[0047] The third aspect of the present invention provides an in vitro method for reducing and / or alleviating fatty degeneration of hepatocytes, cardiomyocytes or renal tubular epithelial cells.

[0048] Furthermore, the method comprises contacting hepatocytes, cardiomyocytes or renal tubular epithelial cells with endothelial progenitor cells or endothelial cells differentiated from iPSCs, or contacting hepatocytes, cardiomyocytes or renal tubular epithelial cells with secretions of endothelial progenitor cells or endothelial cells differentiated from iPSCs.

[0049] In some embodiments, fatty degeneration mostly occurs in hepatocytes, cardiomyocytes or renal tubular cells. In a specific embodiment of the present invention, the inventors have experimentally demonstrated that endothelial progenitor cells or endothelial cells derived from iPSC differentiation can reduce fatty degeneration of liver tissue; therefore, the endothelial progenitor cells or endothelial cells derived from iPSC differentiation of the present invention or their secretions can reduce and / or alleviate fatty degeneration of hepatocytes, cardiomyocytes or renal tubular epithelial cells.

[0050] The fourth aspect of the present invention provides a method for evaluating whether a drug to be screened has the effect of treating, preventing, alleviating and / or alleviating diseases related to steatosis.

[0051] Furthermore, the method includes using endothelial progenitor cells or endothelial cells derived from iPSC differentiation as positive drugs to evaluate the effect of the drug to be screened.

[0052] Furthermore, the diseases associated with steatosis include fatty liver disease, coronary heart disease, myocardial infarction, kidney disease, diabetes, and hyperlipidemia.

[0053] Preferably, the disease associated with steatosis is selected from fatty liver disease.

[0054] A fifth aspect of the present invention provides a method for treating a disease associated with steatosis.

[0055] Furthermore, the method comprises administering to the subject an effective amount of the iPSC-derived endothelial progenitor cells or endothelial cells, the secretions of the iPSC-derived endothelial progenitor cells or endothelial cells, or the pharmaceutical composition described above.

[0056] Furthermore, the diseases associated with steatosis include fatty liver disease, coronary heart disease, myocardial infarction, kidney disease, diabetes, and hyperlipidemia.

[0057] Preferably, the disease associated with steatosis is selected from fatty liver disease.

[0058] A "therapeutically effective amount" would be the amount of an agent that is capable of preventing or at least slowing down (mitigating) a medical condition such as steatosis. The dosage and administration of the disclosed cells or pharmaceutical compositions can be determined by a person of ordinary skill in the field of clinical pharmacology or pharmacokinetics. The effective amount of an agent of the invention used therapeutically will depend, for example, on the therapeutic goal, the route of administration, and the patient's condition. Accordingly, it may be necessary for the therapist to adjust the dosage and change the route of administration as needed to obtain the best therapeutic effect.

[0059] The terms "subject" or "patient" can be used interchangeably. The term includes, but is not limited to, humans, non-human animals, for example, non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic subjects such as dogs and cats; laboratory animals, including rodents such as mice, rats and guinea pigs; and the like. The term does not indicate a specific age or gender. Therefore, it is intended to cover adult and newborn subjects and fetuses, whether male or female. The term "subject" also includes living organisms susceptible to disorders or disease states as generally disclosed throughout this specification (but not limited thereto). Examples of subjects include humans, dogs, cats, cattle, goats and mice, including transgenic species.

[0060] When used for treatment, the "dosage regimen" may vary according to a variety of factors, including the activity of the particular composition used, the patient's age, weight, general health, sex and diet, administration time, route of administration, excretion rate, drug combination, and the severity of the particular disease to be prevented or treated. Although the dosage of the pharmaceutical composition can vary according to the patient's condition and weight, the severity of the disease, the form of the drug, and the route and duration of administration, it can be appropriately selected by those skilled in the art, and administration can be performed once a day or several times a day. This dosage is not meant to limit the scope of the present invention in any way. The pharmaceutical composition of the present invention can be formulated in the form of pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, or suspensions.

[0061] In some embodiments, the dosage of the iPSC-derived endothelial progenitor cells or endothelial cells is no less than 1×10 4 / mL (e.g. not less than 1×10 4 / ml, not less than 3×10 4 / ml, not less than 5×10 4 / ml, no less than 7×10 4 / ml, not less than 1×10 5 / ml, not less than 3×10 5 / ml, not less than 5×10 5 / ml, no less than 7×10 5 / ml, not less than 1×10 6 / ml, not less than 3×10 6 / ml, not less than 5×10 6 / ml, no less than 7×10 6 / ml, not less than 1×10 6 / ml, not less than 3×10 6 / ml, not less than 5×10 6 / ml, no less than 7×10 6 / ml, not less than 1×10 6 / ml, not less than 3×10 6 / ml, not less than 5×10 6 / ml, no less than 7×10 6 / ml, not less than 1×10 6 / ml, not less than 3×10 6 / ml, not less than 5×10 6 / ml, no less than 7×10 6 / ml, not less than 1×10 10 / ml, not less than 3×10 10 / ml, not less than 5×1010 / ml or not less than 7×10 10 / ml, for example 1×10 5 -1×10 8 ,7×10 5 -7×10 6 , 1×10 6 -5×10 6 Pieces / ml.

[0062] In other embodiments, the dosage of the iPSC-derived endothelial progenitor cells or endothelial cells is no less than 1×10 3 / kg (e.g. not less than 1×10 3 / kg, not less than 3×10 3 / kg, not less than 5×10 3 / kg, not less than 7×10 3 / kg, not less than 1×10 4 / kg, not less than 3×10 4 / kg, not less than 5×10 4 / kg, not less than 7×10 4 / kg, not less than 1×10 5 / kg, not less than 3×10 5 / kg, not less than 5×10 5 / kg, not less than 7×10 5 / kg, not less than 1×10 6 / kg, not less than 2×10 6 / kg, not less than 5×10 6 / kg, not less than 7×10 6 / kg, not less than 1×10 7 / kg, not less than 3×10 7 / kg, not less than 5×10 7 / kg, not less than 7×10 7 / kg, not less than 1×10 8 / kg, not less than 3×10 8 / kg, not less than 5×10 8 / kg, not less than 7×10 8 / kg, not less than 1×10 9 / kg, not less than 3×10 9 / kg, not less than 5×10 9 / kg, not less than 7×10 9 / kg, not less than 1×10 10 / kg, not less than 3×10 10 / kg, not less than 5×10 10 pieces / kg or not less than 7×10 10 / kg, for example 1×10 5 -1×10 8 ,7×10 5 -7×10 6 , 1×10 6 -5×10 6 Pieces / kg.

[0063] The endothelial progenitor cells or endothelial cells derived from iPSC differentiation of the present invention can be administered alone or in combination with other therapies. The co-administration of the cells of the present invention and other therapies can be performed simultaneously or sequentially. Single or multiple doses are possible. It is important to use the minimum possible amount sufficient to obtain the maximum therapeutic effect without side effects, taking all factors into consideration.

[0064] Advantages and beneficial effects of the present invention:

[0065] The present invention discovered for the first time that endothelial progenitor cells derived from iPSC differentiation can be used to treat fatty liver disease, especially non-alcoholic fatty liver disease, and provides a new treatment method for fatty liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 It is a graph of the weight changes of each group of mice during the experiment;

[0067] Figure 2 is the HE staining picture of the liver of mice in each group;

[0068] Figure 3 This is a graph showing the scoring results of fatty degeneration, inflammatory cell infiltration, macrophage proliferation and necrosis in each group of mice. DETAILED DESCRIPTION

[0069] The present invention will be further described in detail below in conjunction with the accompanying drawings and examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the examples where specific conditions are not specified are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0070] Example 1 Study on the therapeutic effect of iPSC-EPCs on non-alcoholic fatty liver disease in db / db mice

[0071] 1. Experimental Materials

[0072] (1) Test and reference substances

[0073] Test product

[0074] Name: iPSC-EPCs (marked with DIR)

[0075] Batch number: 2024412

[0076] Specification: 100μL / tube

[0077] Concentration: 1×10 8 cells / mL

[0078] Storage conditions: 2~8℃

[0079] Validity period: Use within 4 hours

[0080] Storage conditions and validity period after opening: one-time use

[0081] Provided by: Chengnuo Regenerative Medicine Technology (Beijing) Co., Ltd.

[0082] Manufacturer: Chengnuo Regenerative Medicine Technology (Beijing) Co., Ltd.

[0083] Reference substance / solvent

[0084] Name: Normal saline containing 3% human albumin

[0085] Specification: 100μL / tube

[0086] Storage conditions: 2~8℃

[0087] Validity period: Use within 4 hours

[0088] Storage conditions and validity period after opening: one-time use

[0089] Provided by: Chengnuo Regenerative Medicine Technology (Beijing) Co., Ltd.

[0090] Manufacturer: Chengnuo Regenerative Medicine Technology (Beijing) Co., Ltd.

[0091] (2) Experimental materials and equipment

[0092] The reagents and instruments used in the in vivo efficacy experiments are shown in Tables 1 and 2.

[0093] Table 1 Reagents used in in vivo experiments

[0094]

[0095] Table 2 Instruments and equipment used in in vivo experiments

[0096]

[0097]

[0098]

[0099] (3) Experimental animals and husbandry

[0100] animal

[0101] Species & strain: db / db mice; db / m mice

[0102] Grade: SPF grade

[0103] Number of animals and sex: A total of 16 male db / db mice and 5 db / m mice were ordered.

[0104] Supplier: Hangzhou Ziyuan Experimental Animal Technology Co., Ltd.

[0105] Production license number: SCXK (Zhejiang) 2019-0004

[0106] Animal quality certificate number: 20240311Abbb0105000608.

[0107] Rearing conditions

[0108] Animals were kept in intelligent independent ventilation animal cages (IVCs) with cage specifications of 362 mm × 154 mm × 135 mm, with no more than 5 animals per box. Artificial lighting, light: dark = 12h:12h. The animals were allowed to eat and move freely during the entire breeding process.

[0109] feed

[0110] SPF rat growth and breeding feed, produced by Beijing Keao Xieli Feed Co., Ltd. The batch number is 200663313, the production license number is SCXK (Beijing) 2019-0003, and the quality certificate number is 1112622000023997. Fully supplement feed 1 to 2 times a week. Fast for about 4 to 5 hours before slaughter.

[0111] Drinking water

[0112] Watsons drinking water, change the water bottle 1 to 2 times a week.

[0113] Litter

[0114] Wood shavings bedding, Dachang Hui Autonomous County Chenfu Eden Garden Bedding Processing Factory. Batch number is 20200622, production license number is SCXK (Hebei) 2017-001. Replace once a week, and replace at any time if there is any abnormality such as leakage or contamination in the cage box.

[0115] Identification of experimental animals

[0116] The animals were marked with ear numbers and the ear numbers of the animals in the cage were displayed on the cage cards.

[0117] Experimental cage number

[0118] Experimental cage labeling: Before grouping, use temporary cage cards to mark the animal cages before grouping. After grouping, write the test number, cage number, dose, animal group, breed, sex, animal number, experiment leader, experiment start and end dates, etc. on cage cards of different colors.

[0119] 2. Experimental methods

[0120] 3. (1) Model building and grouping

[0121] The db / db mouse has a leptin receptor gene defect and is a typical diabetic rodent model that manifests itself as overeating, obesity, hyperlipidemia, insulin resistance, and hepatic steatosis. It is a suitable animal model for simulating NAFLD associated with human metabolic syndrome.

[0122] Grouping method: All animals were weighed and their blood sugar was tested in a clean bench. Sexual behavior tests were performed to record the number of erections. Body weight and blood sugar were used as the basis for grouping. The random number method was used for grouping. The final grouping was the one with the smallest difference in the average values ​​of each group. The specific dosing regimen is shown in Table 3.

[0123] Table 3 Dosage schedule

[0124]

[0125] (2) Detection indicators

[0126] General observation: After grouping until the end of the experiment, observations were conducted twice a week, including the site of administration, animal mental state, diet, exercise, etc.

[0127] Body weight measurement: After grouping until the end of the experiment, the mice were weighed twice a week using an electronic balance.

[0128] Sample collection: Before euthanasia, 0.5-1.0 mL of blood was collected through the abdominal aorta, centrifuged at 4000 r for 10 min, and the serum was separated and stored at -80 °C for later use.

[0129] (3) Euthanasia of experimental animals

[0130] At the end of the experiment or at the humane end point, animals were euthanized using CO2.

[0131] Gross anatomy

[0132] Gross observation: All animals found dead, euthanized on the verge of death, and euthanized as planned on D17 in this experiment were systematically dissected, and liver tissue was obtained for subsequent operation and fixation.

[0133] During the gross dissection, observe whether there are any abnormalities in the animal's body surface, body orifices, cranial cavity, thoracic cavity, abdominal cavity, etc. and their contents. Record the gross dissection and injury results and take photos.

[0134] (4) Histopathological examination

[0135] Organ weight: The final body weight and liver weight of all animals scheduled for euthanasia were measured after fasting, and the organ coefficient was calculated.

[0136] Histopathology: The liver tissue is fixed, dehydrated, and embedded to make a wax block. The surface of the wax block is trimmed with a rotary slicer to fully expose the tissue sample, and then the slices are sliced ​​at a thickness of 3-5 μm. After slicing, the slices are placed in a 65°C constant temperature oven for 30-60 minutes. HE staining: The sliced ​​tissue and transplant samples are stained with hematoxylin and eosin dye, and the tissue pathology is observed under a microscope.

[0137] (5) Statistical analysis

[0138] The measurement data such as body weight, organ weight and coefficient were calculated according to the following method:

[0139] The Kolmogorov-Smirnov method should be used for normality test, the Levene median method should be used for variance homogeneity test, and one-way ANOVA method should be performed. If the normality and variance homogeneity tests fail, the non-parametric Kruskal-Wallis test should be performed.

[0140] If the result of the variance analysis test was significant (P ≤ 0.05), the Dunnett t test was used for multiple comparisons; if the result of the variance analysis was not significant (P > 0.05), the statistics were terminated.

[0141] If the result of the Kruskal-Walis test was significant (P ≤ 0.05), the Mann-Whitney test was used for multiple comparisons; if the result of the Kruskal-Walis test was not significant (P > 0.05), the statistics were terminated.

[0142] 3. Experimental results

[0143] (1) General observation

[0144] General observation results showed that no abnormalities were observed in the mice of each group during the experimental observation period.

[0145] (2)Weight

[0146] The results of weight monitoring showed that the weights of the wild-type control group mice (db / m) were 30.32±1.24g and 29.48±1.11g on D0 and D17, respectively, which were relatively stable; the weights of the model control group were 60.80±3.95g and 62.18±2.54g on D0 and D17, respectively, with a steady increase in weight; the weights of the iPSC-EPCs group were 60.28±7.23g and 56.89±7.33g on D0 and D17, respectively, with a gradual downward trend. The weight changes of the animals in each group are shown in Figure 2 Figure 1 .

[0147] (3) Gross anatomical observation and organ coefficients

[0148] The gross anatomical organs were observed with the naked eye. The organs of the wild-type control group mice (db / m) had no visible abnormalities. The liver tissues of all db / db mice were abnormally enlarged, and other organs had no visible abnormalities. Compared with the model control group, the liver surface of the iPSC-EPCs group was smoother and reddish, and other organs had no visible changes compared with the model control group.

[0149] The results of liver weight showed that compared with the wild-type control group (db / m), the liver weight of the model control group (db / db) mice was significantly increased (P<0.0001). Compared with the model control group, the liver weight of the iPSC-EPCs group was significantly reduced (P<0.05).

[0150] The results of liver coefficient showed that compared with the wild-type control group (db / m), the liver coefficient of the model control group (db / db) mice was significantly increased (P<0.0001). Compared with the model control group, the liver coefficient of the iPSC-EPCs group was significantly decreased (P<0.05).

[0151] Table 4 Liver weight and liver coefficient of each group of animal test endpoint (D17)

[0152]

[0153] Note: **** indicates that the model control group was compared with the wild-type control group, P<0.0001; # indicates that the iPSC-EPCs group was compared with the model control group, P<0.05.

[0154] (4) Histopathological examination

[0155] The liver tissues of mice in each group were stained with HE to observe pathology. The results are as follows: Figure 2As shown in the figure, no pathological changes were observed in the wild-type control group (db / m). The livers of the model control group (db / db) mice showed severe fatty degeneration, with a small amount of inflammatory cell infiltration and macrophage proliferation, and occasional punctate necrosis. Compared with the model control group, the iPSC-EPCs group had milder fatty degeneration, and no significant difference in the degree of inflammatory cell infiltration, macrophage proliferation, and punctate necrosis.

[0156] The degree of fatty degeneration, inflammatory cell infiltration, macrophage proliferation and necrosis of each group of animals was scored. Figure 3 As shown, the fatty degeneration score of the model control group was significantly higher than that of the wild-type control group (P<0.0001); the fatty degeneration score of the iPSC-EPCs group was significantly lower than that of the model control group (P<0.01).

[0157] In summary, under the conditions of this experiment, iPSC-EPCs (1×10 6 A single intravenous injection of iPSC-EPCs into db / db mice reduced the body weight, liver weight, liver coefficient and the degree of liver fatty degeneration in mice, indicating that iPSC-EPCs have the potential to treat fatty liver disease.

[0158] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A pharmaceutical composition for treating, preventing, alleviating and / or alleviating diseases associated with steatosis, characterized in that: The pharmaceutical composition comprises endothelial progenitor cells or endothelial cells derived from iPSC differentiation; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier; Preferably, the diseases associated with steatosis include fatty liver disease, coronary heart disease, myocardial infarction, kidney disease, diabetes, and hyperlipidemia; Preferably, the disease associated with steatosis is selected from fatty liver disease.

2. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutical composition further comprises any one or more of the following functional ingredients: 1) Components that maintain the activity of endothelial progenitor cells or endothelial cells; 2) ingredients that promote the proliferation of endothelial progenitor cells or endothelial cells; 3) Components that promote the differentiation of endothelial progenitor cells.

3. The pharmaceutical composition according to claim 2, characterized in that The functional ingredients include serum replacement, non-essential amino acids, glutamine, stabilized dipeptide of L-alanyl-L-glutamine, growth factors or any combination thereof.

4. Use of endothelial progenitor cells or endothelial cells differentiated from iPSCs in the preparation of drugs for treating, preventing, alleviating and / or alleviating diseases associated with fatty degeneration.

5. The use according to claim 4, characterized in that: The diseases related to steatosis include fatty liver disease, coronary heart disease, myocardial infarction, kidney disease, diabetes, and hyperlipidemia; Preferably, the disease associated with steatosis is selected from fatty liver disease.

6. The use according to claim 4, characterized in that: The iPSC-derived endothelial progenitor cells or endothelial cells are present in a unit dosage form in an amount of 1×10 4 ~1×10 6 cells; Preferably, the iPSC-derived endothelial progenitor cells or endothelial cells are present in an amount of 1×10 6 cells; Preferably, the drug can be used directly or in the form of a pharmaceutical composition; Preferably, the pharmaceutical composition comprises endothelial progenitor cells or endothelial cells derived from iPSC differentiation; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

7. The use according to claim 4, characterized in that: The dosage form of the drug includes a parenteral dosage form and / or a parenteral dosage form; Preferably, the parenteral dosage form includes an injection dosage form, a cavity dosage form, a mucosal dosage form and / or a skin dosage form; Preferably, the dosage form for administration via the gastrointestinal tract includes tablets, granules, capsules, solutions, powders, sustained-release preparations, emulsions, suspensions, syrups and / or drops; Preferably, the dosage form of the drug is selected from an injection dosage form.

8. A method for reducing and / or alleviating fatty degeneration of hepatocytes, cardiomyocytes or renal tubular epithelial cells in vitro, characterized in that: The method comprises contacting hepatocytes, cardiomyocytes or renal tubular epithelial cells with endothelial progenitor cells or endothelial cells differentiated from iPSCs, or contacting hepatocytes, cardiomyocytes or renal tubular epithelial cells with secretions of endothelial progenitor cells or endothelial cells differentiated from iPSCs.

9. A method for evaluating whether a drug to be screened has the effect of treating, preventing, alleviating and / or alleviating a disease associated with steatosis, characterized in that: The method comprises taking endothelial progenitor cells or endothelial cells derived from iPSC differentiation as positive drugs to evaluate the effect of the drug to be screened.

10. The method according to claim 9, characterized in that The diseases related to steatosis include fatty liver disease, coronary heart disease, myocardial infarction, kidney disease, diabetes, and hyperlipidemia; Preferably, the disease associated with steatosis is selected from fatty liver disease.

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