WO2003074684A1 - Cellules capables de se differencier en cellules de type myocarde - Google Patents

Cellules capables de se differencier en cellules de type myocarde Download PDF

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Publication number
WO2003074684A1
WO2003074684A1 PCT/JP2003/002647 JP0302647W WO03074684A1 WO 2003074684 A1 WO2003074684 A1 WO 2003074684A1 JP 0302647 W JP0302647 W JP 0302647W WO 03074684 A1 WO03074684 A1 WO 03074684A1
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cells
cell
cardiomyocyte
compound
salt
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PCT/JP2003/002647
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English (en)
Japanese (ja)
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Hiroyuki Inuzuka
Masayuki Takizawa
Nobuyuki Koyama
Kenichi Noguchi
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Takeda Chemical Industries, Ltd.
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Priority to AU2003213411A priority Critical patent/AU2003213411A1/en
Publication of WO2003074684A1 publication Critical patent/WO2003074684A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5073—Stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0657—Cardiomyocytes; Heart cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5061—Muscle cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells

Definitions

  • the present invention relates to muscle-derived cells capable of differentiating into cardiomyocyte-like cells and uses thereof. More specifically, the present invention relates to a transplanted animal transplanted with the cells used for searching for a compound that can be a prophylactic or therapeutic agent for heart disease, a screening using the animal, and the like. Background art
  • Heart failure is considered to be contractile dysfunction of the heart muscle, and the mechanism of its onset may be as follows. For example, myocardial infarction, myocarditis, cardiomyopathy or aging, resulting in myocardial damage, mechanical or mechanical failure of the heart pump, and increased blood pressure due to hypertension and pulmonary hypertension; and When the volume load increases due to anemia, acute nephritis, etc., that is, when the heart cannot pump the blood volume according to the demands of the living body, the heart can be used for the sympathetic nervous system, the nervous system, the endocrine system, etc. It tries to maintain the homeostasis of the living body by activating the compensatory mechanism.
  • Compensation mechanisms for heart failure include: 1) increased preload and increased cardiac contractility, resulting in cardiac dilation as a result of increased sarcomere length; and 2) increased myocardial contraction units resulting in increased myocardial hypertrophy. 3) Neural humoral factors are activated to compensate for the inability to excrete the necessary blood throughout the body, and myocardial fibrosis progresses locally. Basically, it is a mechanism that adjusts to the applied load and attempts to adapt, but in some cases, heart failure is further developed due to insufficient expression, and conversely, it operates excessively, resulting in myocardial injury It is understood that it may affect heart failure in some cases.
  • Cardiac hypertrophy results from hypertrophy of cardiomyocytes as a result of activation of the compensatory mechanism.
  • the compensatory mechanism will fail.
  • not enough blood is supplied to the enlarged cardiomyocytes, resulting in ischemia, resulting in myocardial damage such as myocardial contractile dysfunction, a decrease in cardiac output, and organ circulatory disorders. It results in heart failure syndrome with harm, venous congestion, fluid retention, and so on.
  • Treatments include improving cardiomyocyte damage, enhancing cardioprotection, reversing cardiac dysfunction due to myocardial contractility, and suppressing the destructive failure of the living body or improving excessive compensation mechanisms. Required.
  • cardiac glycosides such as digoxin
  • sympathomimetics such as dobuyumin
  • phosphodiesterase inhibitors such as amrinone.
  • Drugs are used, and vasodilators include hydralazine, calcium antagonists, angiotensin I converting enzyme inhibitors, and angiotensin II receptor antagonists.
  • i3 blockers are used for the treatment of other dilated cardiomyopathy.
  • some therapeutic agents cannot recover cardiomyocytes lost by coping therapy.
  • Heart transplant is considered as the current fundamental treatment, but it is widely used as a general treatment due to shortage of organ donors, difficulty in determining brain death, rejection, rising medical costs, etc. I haven't.
  • the present inventors have found that cells derived from adult mouse muscle are cultured without using a differentiation inducer or the like, and differentiate into beating cardiomyocyte-like cells. These cells are used for (a) identification and evaluation of genes involved in myocardial differentiation and regeneration, (b) cell transplantation for various diseases including heart failure such as heart failure and myocardial infarction, and (c) compounds for promoting myocardial differentiation. Search and evaluation, (Search and evaluation of drugs that increase the efficiency of cell transplantation (fixation of transplanted cells to living tissues and promotion of function), (e) Used to create model animals to develop therapeutic agents for cardiac failure The present inventors have further studied and completed the present invention.
  • a method of transplanting the cell according to (1) or the cardiomyocyte according to (2) into an animal comprising: (5) A method for identifying a cell differentiation / regeneration-related gene, comprising using the cell according to (1) or the cardiomyocyte as described in (2).
  • a method for screening a compound or a salt thereof that promotes cell differentiation and regeneration characterized by using the cell according to (1) or the cardiomyocyte according to (2).
  • kits for screening a compound or a salt thereof that promotes cell differentiation and regeneration characterized by containing the cell of (1) or the cardiomyocyte of (2);
  • a method for screening a compound or a salt thereof, which promotes colonization of transplanted cells into a living body comprising using the cell according to (1) or the cardiomyocyte according to (2),
  • a screening kit for a compound or a salt thereof, which promotes the function of established transplanted cells which comprises the cell according to (1) or the cardiomyocyte according to (2),
  • the cells according to (1) above are cultured in a medium containing no differentiation inducer, and the cells according to (1) are induced into the cardiomyocyte-like cells according to (2).
  • (21) a method for preventing or treating heart disease, which comprises administering to a mammal an effective amount of the compound or a salt thereof according to (14) above;
  • cardiomyocyte The “cell isolated from muscle and capable of differentiating into a cardiomyocyte” (hereinafter sometimes abbreviated as “cardiac progenitor cell of the present invention”) of the present invention is isolated from muscle and converted into cardiomyocyte. Any cells may be used as long as they have differentiation ability.
  • muscle examples include skeletal muscles of mammals (eg, human, guinea pig, rat, mouse, egret, pig, hidge, magpie, monkey, etc.), that is, voluntary muscles having a striated structure (eg, vastus lateralis, anterior tibiae) Muscle, brachial muscle, etc.). Preferably, it is the vastus lateralis of a rat or mouse.
  • mammals eg, human, guinea pig, rat, mouse, egret, pig, hidge, magpie, monkey, etc.
  • a striated structure eg, vastus lateralis, anterior tibiae
  • it is the vastus lateralis of a rat or mouse.
  • any method may be used as long as cells including skeletal muscle satellite cells are collected.
  • the method is performed according to the method described in Histochemistry, vol. 87, pp. 27-38, 1987 or a method analogous thereto.
  • myocardial progenitor cells of the present invention When the myocardial progenitor cells of the present invention are induced, they become myocardial-like cells (hereinafter, sometimes abbreviated as the myocardial-like cells of the present invention).
  • a myocardial progenitor cell of the present invention is, for example, a medium containing no site-force-in (eg, bFGF) having a differentiation-inhibiting action; a differentiation-inducing agent (eg, 5-azacytidine, trichostin; , Vitamins!), Hexamethylene bisacetamide, dimethyl acetate, dibutyl cAMP, dimethyl sufoxide, Examples of such methods include culturing in a culture medium that does not contain lysine, hydroxyl peryl, cytosine arabinoside, mitomycin sodenum butyrate, affidicolin, fluorodeoxyperidine, polypropylene, selenium, etc.).
  • a differentiation-inducing agent eg, 5-azacytidine, trichostin; , Vitamins!
  • Hexamethylene bisacetamide dimethyl acetate, dibutyl cAMP, dimethyl sufoxide
  • any medium can be used as long as it is generally used for mammalian cell culture.
  • it is DMEM, Ham's F10 or the like.
  • concentration of fetal bovine serum contained in the medium may be any concentration that is generally used for cell culture. Preferably it is about 10%.
  • various skeletal muscles were collected from mice, nerves, tendons, adipose tissue, etc. were removed, cut into small pieces with scissors, digested in a cell dispersion containing trypsin, and subjected to pibbing, etc. Afterwards, remove undigested tissue residue by passing through a 100 filter. After washing twice, the serum-containing medium was suspended in (for example, 10% fetal bovine serum-containing Ham's F10 medium, etc.) were seeded in about four 10cm culture dishes, 37Tau 2 hours incubation in 5% C0 2 concentration in the incubator machine After that, the non-adherent cells are isolated to obtain the myocardial progenitor cells of the present invention.
  • serum-containing medium for example, 10% fetal bovine serum-containing Ham's F10 medium, etc.
  • Cardiac progenitor cells were cultured in Ham's F10 medium containing 20% fetal bovine serum containing FGF-2 for 7 days, changed to DMEM containing 10% fetal bovine serum without FGF-2, and further cultured for 5 days. A beating cardiomyocyte of the present invention is obtained.
  • transplanted animal into which the cardiomyocyte of the present invention or the cardiomyocyte of the present invention has been transplanted examples include, for example, mammals (eg, guinea pig, rat, mouse) , Egrets, pigs, sheep, sheep, squirrels, monkeys, etc.).
  • a method for transplanting the myocardial progenitor cells of the present invention or the myocardial-like cells of the present invention methods for injecting into cardiac muscle, for example, Journa lof Clini cal Inves ti gation, 96, 2034-2042, The method described in 1995 can be used.
  • the myocardial progenitor cells of the present invention, the myocardial-like cells of the present invention, and the transplanted animal of the present invention include (a) identification and evaluation of genes involved in cell differentiation and regeneration, and (b) search and evaluation of compounds that promote cell differentiation. (C) Search and evaluation of compounds that promote the transplantation of transplanted cells to the living body, (d) Search and evaluation of compounds that promote the function of transplanted cells that have been transplanted, (e) Cell transplantation of various diseases such as heart disease Used for etc.
  • the use of the myocardial progenitor cell of the present invention, the myocardial-like cell of the present invention and the transplanted animal of the present invention will be described in detail below.
  • the expression level of each gene can be comprehensively measured using known methods, for example, using a macroarray, microarray, DNA chip, or Northern blotting.
  • a gene whose expression level in the above (ii) or (iii) is increased by about 2 times or more, preferably about 5 times or more as compared with the case of (i) promotes cell differentiation and regeneration Gene to be selected. It is possible to select a gene whose expression level in the case (ii) or (iii) is reduced by about 2 times or more, preferably about 5 times or more as compared with the case (i), as a gene that suppresses cell differentiation and regeneration. it can.
  • the cardiomyocyte of the present invention in which the candidate gene has been expressed can be obtained by introducing the candidate gene into the cardiomyocyte of the present invention using, for example, a known gene transfer method (eg, calcium phosphate method). Obtainable.
  • the gene library can be obtained by expressing the gene library in the myocardial progenitor cells of the present invention and selecting only the cells into which the gene has been introduced using, for example, a gene introduction marker (eg, a drug resistance marker).
  • Cardiomyocyte marker-tamper Perform immunostaining of proteins (eg, ANP, Nkx2.5, GATA4, etc.) to measure and compare the number of positive cells.
  • Marker protein of cardiomyocytes in culture supernatant or cell extract (E.g., ANP in culture supernatant, Nkx2.5 or GATA4 in cell extract) Measure the expression level (e.g., using ELISA, Western blotting, etc.) and compare
  • the number of pulsating cells, the number of positive cells, the myocardial primary protein or the cardiomyocyte marker gene expression level in (V) above is approximately 20% lower than that in () above. Or more, preferably 30% or more, more preferably about 5%
  • Candidate genes that promote 0% or more can be selected as genes that promote cell differentiation and regeneration.Candidate genes that suppress about 20% or more, preferably 30% or more, and more preferably about 50% or more ⁇ Can be selected as a gene that suppresses regeneration.
  • the cardiac muscle progenitor cell of the present invention in which the expression of a candidate gene has been suppressed can be obtained by a known method, for example, an antisense RNA, a hybrid lipozyme, or the like.
  • the myocardial progenitor cells of the present invention, in which the function of the candidate gene has been suppressed can be obtained by known methods, for example, dominant negative gene expression, peptide abmers, and the like.
  • the number of beating cells in (vi) and (vii) are measured and compared, (2) marker proteins of cardiomyocytes (eg, ANP, Nkx2.5, (3) Marker genes for cardiomyocytes in culture supernatant or cell extract (eg, ANP in culture supernatant, cell extract) Expression level of Nkx2.5 or GATA4)
  • marker proteins of cardiomyocytes eg, ANP, Nkx2.5
  • Marker genes for cardiomyocytes in culture supernatant or cell extract eg, ANP in culture supernatant, cell extract
  • cardiomyocyte marker genes e.g., ANP, Nkx2.5, GATA4, etc.
  • cardiomyocyte marker genes e.g., ANP, Nkx2.5, GATA4, etc.
  • Use Northern blotting, TaqMan PCR, etc. to compare and select genes related to cell differentiation and regeneration.
  • the number of pulsating cells, the number of positive cells, the marker protein of cardiomyocytes, or the amount of marker gene expression in cardiomyocytes in (vii) can be determined by
  • a candidate gene that promotes about 20% or more, preferably 30% or more, more preferably about 50% or more of the case of (vi) can be selected as a gene that suppresses cell differentiation and regeneration.
  • Candidate genes that suppress 20% or more, preferably 30% or more, and more preferably about 50% or more can be selected as genes that promote cell differentiation and regeneration.
  • a compound or a salt thereof that promotes cell differentiation / regeneration is screened by comparing the cardiomyocyte precursor cells of the present invention or the cardiomyocyte-like cells of the present invention and a test compound with those obtained by culturing.
  • cardiomyocyte marker genes eg, ANP in culture supernatant, Nkx2.5 or GATA4 in cell extract
  • culture supernatant or cell extract eg, ELISA method
  • marker genes eg, ANP, Nkx2.5, GATA4, etc.
  • cardiac muscle cells eg, Northern blotting, TaqMan PCR, etc.
  • Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. Or a known compound Good.
  • the cell respiratory activity, number of pulsating cells, number of positive cells, cardiomyocyte marker protein amount, or cardiomyocyte marker gene expression amount in the case of the above (ix) are determined in the case of the above (viii).
  • a test compound that promotes about 20% or more, preferably 30% or more, and more preferably about 50% or more can be selected as a compound that promotes cell differentiation and regeneration or a salt thereof.
  • Cardiomyocyte marker Transplantation by measuring the expression level of genes (eg, ANP, Nkx2.5, GATA4, etc.) (eg, using Northern blotting, TaqManPCR, etc.) A compound or a compound that promotes colonization of cells To select the salt.
  • the primary cardiomyocytes used in the above screening method for example, cells obtained by the method described in Jouranl of Clinical Investigation 99, 2898-2905, 1997 and the like are used.
  • Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. Or a known compound.
  • the cell respiratory activity, the number of pulsatile cells, the number of positive cells, the marker protein of cardiomyocytes, or the expression level of marker gene of cardiomyocytes in the case of (xi) above Test compounds that promote at least about 20%, preferably at least about 30%, more preferably at least about 50% of the test compound, as a compound or a salt thereof that promotes the transplantation of transplanted cells into a living body. it can.
  • the selected compound or a salt thereof has an immunosuppressive effect.
  • the presence or absence of an immunosuppressive effect is determined by a known method, for example, by measuring cell respiratory activity using mouse spleen cells by the MTT method.
  • the hearts of the animals to which the cells have been administered are excised, and the rate of colonization of the transplanted cells is calculated and compared to screen for a compound or salt thereof that promotes the transplantation of the transplanted cells to the living body.
  • a heart tissue section is prepared in the above (xi i) and (xiii), and the number of nuclei at the transplanted cell (graft) site is calculated by nuclear staining and compared.
  • a compound or a salt thereof that promotes transplantation of the transplanted cells to the living body is selected.
  • Animals used in the above screening method include normal animals, myocardial infarction model animals, cardiomyopathy model animals, and the like.
  • Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. Or a known compound.
  • the administration time of the test compound is not particularly limited, and may be any time before, during, or after transplantation. Specifically, about 1 minute to 24 hours before transplantation, between the start of transplantation and the end of transplantation, about 1 hour to 3 years after transplantation, and the like.
  • the number of nuclei at the transplanted cell (graft) site in the case of (xi ii) is about 20% or more, preferably 30% or more, more preferably, as compared with the case of the above (xii).
  • a test compound that promotes about 50% or more promotes colonization of transplanted cells in living organisms It can be selected as a compound or a salt thereof.
  • the selected compound or a salt thereof has an immunosuppressive effect.
  • the presence or absence of an immunosuppressive effect is determined by a known method, for example, by observing the infiltration of lymphocytes using a tissue section and examining the presence or absence of inflammation.
  • the primary cardiomyocytes used in the above screening method for example, cells obtained by the method described in Jouranl of Clinical Investigation 99, 2898-2905, 1997, and the like are used.
  • Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. Or a known compound. .
  • the cell respiratory activity, number of pulsating cells, number of positive cells, cardiomyocyte marker protein, cardiomyocyte marker gene expression, etc. in the case of (XV) above are compared with those in the above (xiv).
  • a test compound that preferably promotes about 50% or more can be selected as a compound or a salt thereof that promotes the function of a transplanted cell.
  • an animal into which the cardiomyocyte precursor of the present invention or the cardiomyocyte of the present invention is transplanted and (xvii) a cardiomyocyte precursor of the present invention or the cardiomyocyte of the present invention transplanted, and further administering a test compound
  • the heart function eg, cardiac contractile action, etc.
  • the heart function of the isolated animal is measured and compared to screen for a compound or salt thereof that promotes the function of the transplanted cells.
  • cardiac function is measured by a cardiac catheterization method according to the method described in, for example, Circulation Research, Vol. 69, pp. 370-377, 1991.
  • cardiac function is measured non-invasively using an echocardiography diagnostic apparatus (Cell, Vol. 97, pp. 189-198, 1998).
  • cardiac function is measured by a known Langendorff-perfused heart using the extracted heart.
  • Examples of the animals used in the above screening method include normal animals, heart disease model animals (e.g., myocardial infarction model animals, myocarditis model animals, paging heart failure model animals, chest or abdominal aortic stenosis cardiac hypertrophy model animals, hypertension Model animals (eg, SHR, etc.).
  • heart disease model animal a known animal may be used.
  • Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. Or a known compound.
  • the administration time of the test compound is not particularly limited, and may be any time before, during, or after transplantation. Specifically, about 1 minute to 24 hours before transplantation, between the start of transplantation and the end of transplantation, about 1 hour to 3 years after transplantation, and the like.
  • a test that promotes cardiac function in the case of the above (xvi i) by about 20% or more, preferably 30% or more, more preferably about 50% or more in comparison with the case of the above (xvi i) can be selected as a compound that promotes the function of the transplanted cells or a salt thereof.
  • the screening kit of the present invention contains the cardiomyocyte precursor of the present invention, the cardiomyocyte of the present invention, the transplanted animal of the present invention, and the like.
  • the compound or a salt thereof obtained by the screening method or the screening kit according to any one of the above (1) to (4) has a function of promoting cell differentiation, a function of promoting the transplantation of cells into the living body, or a function of established transplanted cells.
  • Prevention and treatment of heart disease eg, depressive cardiomyopathy, hypertrophic obstructive cardiomyopathy, hypertrophic non-obstructive cardiomyopathy, idiopathic cardiomyopathy, myocardial infarction, chronic heart failure, arrhythmia, etc.
  • heart disease eg, depressive cardiomyopathy, hypertrophic obstructive cardiomyopathy, hypertrophic non-obstructive cardiomyopathy, idiopathic cardiomyopathy, myocardial infarction, chronic heart failure, arrhythmia, etc.
  • It can be used as an agent.
  • a salt with a physiologically acceptable acid eg, an inorganic acid, an organic acid
  • a base eg, an alkali metal
  • the salt include a salt with an inorganic acid (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or an organic acid
  • salts with acetic acid formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid
  • acetic acid formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid
  • the above compound or a salt thereof is orally acceptable, for example, as a sugar-coated tablet, capsule, elixir, microcapsule or the like, or water or other pharmaceutically acceptable, if necessary. It can be used parenterally in the form of a sterile solution with liquid, or as an injection, such as a suspension.
  • the compound or its salt can be combined with a physiologically acceptable carrier, flavoring agent, excipient, vehicle, preservative, stabilizer, binder, etc., in a unit dosage required for generally accepted formulation practice. It can be manufactured by mixing in the form. The amount of active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.
  • Additives that can be mixed with tablets, capsules and IJ include, for example, binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin Swelling agents such as alginic acid, lubricating agents such as magnesium stearate, sweetening agents such as sucrose, lactose or saccharine, and flavoring agents such as peppermint, cocoa oil or cellulose.
  • the unit dosage form is a capsule
  • the above type of material can further contain a liquid carrier such as an oil or fat.
  • Sterile composition for injection Can be formulated according to the usual practice of dissolving or suspending an active substance in a vehicle such as water for injection, or a naturally occurring vegetable oil such as sesame oil or coconut oil.
  • Aqueous liquids for injection include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.).
  • Agents for example, alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol, etc.), nonionic surfactants (eg, Polysorbate 80 TM , HC 150, etc.), etc. You may use together.
  • the oily liquid include sesame oil and soybean oil, which may be used in combination with a solubilizing agent such as benzyl benzoate or benzyl alcohol.
  • buffers eg, phosphate buffer, sodium acetate buffer, etc.
  • soothing agents eg, benzalkonium chloride, proactive hydrochloride, etc.
  • stabilizers eg, human serum albumin, polyethylene glycol, etc.
  • preservatives eg, benzyl alcohol, phenol, etc.
  • the preparations obtained in this way are safe and have low toxicity, for example, mammals (eg, humans, rats, mice, guinea pigs, egrets, sheep, pigs, pigs, dogs, cats, dogs, monkeys) , Etc.).
  • mammals eg, humans, rats, mice, guinea pigs, egrets, sheep, pigs, pigs, dogs, cats, dogs, monkeys
  • the dose of the above compound or its salt varies depending on the target disease, the administration subject, and the like.
  • the compound or its salt when administered as a therapeutic agent for myocardial infarction, it is generally used in adults (body weight). 60 kg), the compound or a salt thereof is administered in an amount of about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day,
  • the cardiomyocyte of the present invention may be used, for example, for heart diseases such as a cardiomyogen, a heart activator, a pulsatility regulator, a cardiac function enhancer, and a heart regenerating agent (eg, congestive cardiomyopathy, hypertrophic obstructive cardiomyopathy, fertilizer) It is used safely as a treatment for large non-obstructive cardiomyopathy, idiopathic cardiomyopathy, myocardial infarction, chronic heart failure, arrhythmia, etc.).
  • heart diseases such as a cardiomyogen, a heart activator, a pulsatility regulator, a cardiac function enhancer, and a heart regenerating agent (eg, congestive cardiomyopathy, hypertrophic obstructive cardiomyopathy, fertilizer) It is used safely as a treatment for large non-obstructive cardiomyopathy, idiopathic cardiomyopathy, myocardial infarction, chronic heart failure, arrhythmia, etc.).
  • the myocardium of the present invention The like cells are used as a cell suspension.
  • the agents obtained in this way are safe and have low toxicity, for example, mammals (e.g., humans, rats, mice, guinea pigs, egrets, higgs, bushus, dogs, dogs, cats, dogs, Monkeys, etc.) can be administered by catheter injection, direct injection into the heart, intravenous injection, or biopsy.
  • mammals e.g., humans, rats, mice, guinea pigs, egrets, higgs, bushus, dogs, dogs, cats, dogs, Monkeys, etc.
  • sequence numbers in the sequence listing in the present specification indicate the following sequences.
  • Fig. 3 shows the nucleotide sequence of primer 11 used in Example 3 below.
  • mice Three 1-week-old C57BL / 6J cl mice were anesthetized with ether and then killed by vertebral dislocation. After the lower body of the mouse was thoroughly disinfected with 70% ethanol, the skin on both feet was cut open over a wide area, and the quadriceps femoris, biceps femoris, large * small adductor, and prestriated muscle were collected. Nerve, tendon, adipose tissue, etc. were removed from the collected muscular tissue, and then cut with scissors and placed in a 60 mL cell suspension (0.2% collagenase, 0.1% trypsin / Hexans balanced salt solution). Digested for 1 hour at ° C. 0.25 mL 50 mg / mL DNase 1 was added, pipetting was performed, and the mixture was passed through a lOOim filter. Contains 10% fetal bovine serum
  • Non-adherent cells were harvested, re-suspended in growth medium (20% fetal bovine serum and 2.5 ng / mL bFGF-containing Ham s FIO medium) were seeded in 6-well of 6-well culture dishes, 5% C0 2 concentration in the incubation Incubated at 37 ° C. On the first day of culture, non-adherent cells were collected and cultured.
  • non-adherent cells 1 day of culture
  • the non-adherent cells were recovered again on the second day of culture and cultured, and on day 5, the medium was replaced with a differentiation medium (DMEM containing 10% fetal calf serum), and the culture was continued.
  • DMEM fetal calf serum
  • the appearance of autonomously pulsating cells was confirmed, and on the 11th day, autonomic pulsation of myotube-like cells was confirmed.
  • RNA PCR Kit AMV
  • Ver2.1 Takara Shuzo
  • Cardiac progenitor cells and cardiomyocyte-like cells of the present invention can be obtained from muscle without using embryonic stem cells (ES cells) and bone marrow cells.
  • A Identification and evaluation of genes involved in cardiac differentiation and regeneration;
  • B Cell transplants such as heart disease (eg, depressive cardiomyopathy, hypertrophic obstructive cardiomyopathy, hypertrophic non-obstructive cardiomyopathy, idiopathic cardiomyopathy, myocardial infarction, chronic heart failure, arrhythmia, etc.),
  • Heart disease eg, Depressive cardiomyopathy, hypertrophic obstructive cardiomyopathy, hypertrophic non-obstructive cardiomyopathy, idiopathic cardiomyopathy, myocardial infarction, chronic heart failure, arrhythmia, etc.
  • Heart disease eg, Prevention of depressive cardiomyopathy, hypertrophic obstructive cardiomyopathy, hypertrophic non-obstructive cardiomyopathy, idiopathic cardiomyopathy, myocardial infar
  • cardiomyocyte of the present invention may be used for safe heart disease (eg, depressive cardiomyopathy, hypertrophic obstructive cardiomyopathy, hypertrophic non-obstructive cardiomyopathy, idiopathic cardiomyopathy, myocardial infarction, chronic heart failure, arrhythmia, etc.) It is used as a preventive and therapeutic agent.
  • safe heart disease eg, depressive cardiomyopathy, hypertrophic obstructive cardiomyopathy, hypertrophic non-obstructive cardiomyopathy, idiopathic cardiomyopathy, myocardial infarction, chronic heart failure, arrhythmia, etc.

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Abstract

Cette invention se rapporte à des cellules précurseurs du myocarde et à des cellules de type myocarde, qui peuvent être obtenues à partir de muscles sans l'aide de cellules souches embryonnaires ou de cellules de la moelle épinière et qui sont utilisables: (a) pour identifier et évaluer un gène relatif à la différenciation et à la régénération du myocarde; (b) pour réaliser une transplantation de cellules en cas d'affection cardiaque; (c) pour rechercher et évaluer un composé favorisant la différenciation du myocarde; (d) pour rechercher et évaluer un médicament augmentant l'efficacité de la transplantation cellulaire (fixation de cellules greffées dans un tissu biologique et stimulation de sa fonction); et (e) pour construire un modèle animal destiné à mettre au point un agent prophylactique/thérapeutique contre une maladie cardiaque. Les composés obtenus par cette recherche sont utilisables comme agents prophylactiques et thérapeutiques surs contre les maladies cardiaques.
PCT/JP2003/002647 2002-03-07 2003-03-06 Cellules capables de se differencier en cellules de type myocarde WO2003074684A1 (fr)

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AU2003213411A AU2003213411A1 (en) 2002-03-07 2003-03-06 Cells capable of differentiating into myocardium-like cells

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000017326A1 (fr) * 1998-09-21 2000-03-30 Musc Foundation For Research Development Cellules non hematopoietiques, y compris les cardiomyocytes et les cellules de muscle squelettique, que l'on derive de cellules souches hematopoietiques, et procedes relatifs a leur elaboration et a leur utilisation
WO2001011011A2 (fr) * 1999-08-05 2001-02-15 Mcl Llc Cellules souches adultes toutes-puissantes et procede d'isolement
WO2001021767A2 (fr) * 1999-09-24 2001-03-29 Morphogen Pharmaceuticals, Inc. Cellules souches embryonnaires totipotentes

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000017326A1 (fr) * 1998-09-21 2000-03-30 Musc Foundation For Research Development Cellules non hematopoietiques, y compris les cardiomyocytes et les cellules de muscle squelettique, que l'on derive de cellules souches hematopoietiques, et procedes relatifs a leur elaboration et a leur utilisation
WO2001011011A2 (fr) * 1999-08-05 2001-02-15 Mcl Llc Cellules souches adultes toutes-puissantes et procede d'isolement
WO2001021767A2 (fr) * 1999-09-24 2001-03-29 Morphogen Pharmaceuticals, Inc. Cellules souches embryonnaires totipotentes

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