WO2012108444A1 - Cellule souche pluripotente spécifique pour une myocardiopathie et utilisation de celle-ci - Google Patents

Cellule souche pluripotente spécifique pour une myocardiopathie et utilisation de celle-ci Download PDF

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WO2012108444A1
WO2012108444A1 PCT/JP2012/052788 JP2012052788W WO2012108444A1 WO 2012108444 A1 WO2012108444 A1 WO 2012108444A1 JP 2012052788 W JP2012052788 W JP 2012052788W WO 2012108444 A1 WO2012108444 A1 WO 2012108444A1
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cardiomyopathy
cells
lamin
gene
cell
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Japanese (ja)
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武 牧山
善紀 吉田
木村　剛
伸弥 山中
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国立大学法人京都大学
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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
    • G01N33/5061—Muscle 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • the present invention relates to cardiomyopathy-specific pluripotent stem cells, cardiomyocytes that are induced to differentiate from the stem cells, and express cardiomyopathy traits, and screening for preventive and / or therapeutic agents for cardiomyopathy using the cardiomyocytes Regarding the method. More specifically, the present invention is a pluripotent stem cell having a lamin A / C gene abnormality, in particular an induced pluripotent stem (iPS) cell derived from a patient with lamin A / C cardiomyopathy, induced to differentiate from the stem cell, Cardiomyocytes exhibiting a phenotype characteristic of lamin A / C cardiomyopathy such as abnormal nuclear morphology, nuclear membrane damage, reduced contractile force, etc.
  • the present invention relates to a method for screening a prophylactic and / or therapeutic agent, which comprises selecting a test substance having an improved I as a candidate for a prophylactic and / or therapeutic agent for cardiomyopathy.
  • DCM Dilated cardiomyopathy
  • lamin A / C Abnormalities in the lamin A / C gene are known to cause organ-specific diseases such as cardiomyopathy, muscular dystrophy, peripheral neuropathy, familial lipoatrophy and progeria, and are collectively called laminopathy.
  • organ-specific diseases such as cardiomyopathy, muscular dystrophy, peripheral neuropathy, familial lipoatrophy and progeria
  • laminopathy In the heart, dilated cardiomyopathy (lamin A / C cardiomyopathy) with cardiac conduction disorder is caused (Non-patent Document 3), and the prognosis is poor due to the combination of fatal arrhythmia and severe heart failure, and there is no fundamental cure .
  • DCM beta blockers and ACE inhibitors are used as DCM therapeutics. Recently, attempts have been made to treat heart failure with calcium sensitizers. However, existing drugs have a PDE inhibitory action, which reduces symptoms in the short term, but does not provide a long-term improvement effect. In order to develop effective new DCM therapeutic agents, a disease model that can reproduce the pathology of DCM is required.
  • Non-patent Document 4 As an animal model of DCM, a knock-in mouse (Lmna H226P / H226P ) introduced with a missense mutation (H226P) of a lamin A / C gene identified in a family of Emery-Dreyfs muscular dystrophy is known (Non-patent Document 4). . This mouse develops heart failure at the age of 5-6 months, causes loss of cardiomyocytes and fibrosis of the myocardium and dies by 12-13 months, so it is often used as an animal model for severe DCM, For example, the effect of calcium sensitizers to prevent the onset of heart failure has been studied (Non-patent Document 5). However, it is unclear whether animal models reflect the effects of drugs in humans. From the viewpoint of animal welfare, it is more desirable to use a cultured human cell model for early screening of drug discovery research.
  • iPS cells can be produced from a small amount of somatic cells, and examples of production from skin cells of elderly people have been reported, iPS cells are established from somatic cells of patients with a certain disease, and the pathology of the disease is determined. By differentiating into cell types to be expressed, disease-related cells (disease-relevant cells) having the gene abnormality of the patient can be easily obtained.
  • ALS amyotrophic lateral sclerosis
  • Parkinson's disease has already been reported (Non-patent Documents 6 and 7).
  • ALS amyotrophic lateral sclerosis
  • Parkinson's disease has already been reported (Non-patent Documents 6 and 7).
  • ALS amyotrophic lateral sclerosis
  • Parkinson's disease has already been reported (Non-patent Documents 6 and 7).
  • ALS amyotrophic lateral sclerosis
  • Parkinson's disease has already been reported (Non-patent Documents 6 and 7).
  • ALS amyotrophic lateral sclerosis
  • An object of the present invention is to establish a pluripotent stem cell derived from a cardiomyopathy patient, induce differentiation of a cardiomyocyte expressing a cardiomyopathy trait from the stem cell, and prevent and / or treat cardiomyopathy using the cardiomyocyte It is to provide a method of screening for substances that can be drugs.
  • the present inventors conducted genetic analysis on patients who first developed familial dilated cardiomyopathy and cardiac conduction disorder (DCM-CD).
  • DCM-CD familial dilated cardiomyopathy and cardiac conduction disorder
  • the lamin A / C gene 2 nucleotides (908th to 909th CT) in the coding region (CDS) were deleted, and the 303rd amino acid (Ser) was mutated to Cys, followed by 25 more meaningless amino acids.
  • Ser 303rd amino acid
  • a screening method for a prophylactic and / or therapeutic agent for cardiomyopathy comprising the following steps: (1) a step of bringing a test substance into contact with a cardiomyocyte or tissue derived from a pluripotent stem cell having a lamin A / C gene abnormality; (2) Compared to the case where the test substance is not contacted, the trait different from the normal cardiomyocyte or tissue expressed by the cardiomyocyte or tissue is changed so as to be close to the trait of the normal cardiomyocyte, or A step of selecting a test substance that prevents or delays the expression of a different character as a candidate substance for a prophylactic and / or therapeutic agent for cardiomyopathy.
  • the pluripotent stem cell is an induced pluripotent stem (iPS) cell.
  • the lamin A / C gene abnormality is a frameshift mutation caused by deletion of CT at positions 908 to 909 in the nucleotide sequence shown in SEQ ID NO: 1 or 3.
  • the cardiomyopathy is dilated cardiomyopathy.
  • the cardiomyopathy is lamin A / C cardiomyopathy.
  • Stem cells [15] A cardiomyocyte having an abnormal nuclear morphology and / or a nuclear membrane disorder, which is induced to differentiate from the pluripotent stem cell according to any one of [11] to [14].
  • the myocardial tissue according to [16] wherein the myocardial tissue expresses a trait that reproduces a disease state of cardiomyopathy selected from a decrease in muscle contraction force, muscle fibrosis, and loss of cardiomyocytes.
  • cardiomyopathy preventive and / or therapeutic agents can be screened using cardiomyocytes or tissues induced to differentiate from cardiomyopathy-specific pluripotent stem cells.
  • lamin represents an iPS cell-derived cardiomyocyte established from a Lamin A / C cardiomyopathy patient
  • 201B7 represents a 201B7-derived cardiomyocyte.
  • 2 is an electron micrograph of the nuclear membrane of iPS cell-derived cardiomyocytes established from a patient with lamin A / C cardiomyopathy. The nuclear membrane is damaged at the arrow.
  • the present invention improves or prevents the expression of cardiomyopathy in cardiomyocytes or tissues obtained by inducing differentiation of cardiomyopathy patients, preferably iPS cells, or prevents the expression of the character Alternatively, a method of screening for a substance that delays is provided.
  • the pluripotent stem cell used as a starting material in the present invention has a lamin A / C gene abnormality that causes cardiomyopathy, and has “self-renewal ability” that can proliferate while maintaining an undifferentiated state.
  • Any undifferentiated cell having “differentiated pluripotency” capable of differentiating into all three germ layers can be used.
  • iPS cells, ES cells, and embryonic germ (EG) cells derived from primordial germ cells Examples include mutipotent germline stem (mGS) cells isolated in the process of establishing GS cells from testicular tissue, multipotent adult progenitor cells (MAPC) isolated from bone marrow, and the like.
  • the ES cell may be an ES cell produced by nuclear reprogramming from a somatic cell. Although iPS cells or ES cells are preferred, mGS cells and MAPCs are also preferred in that they can be obtained from individuals after birth. Pluripotent stem cells may have a homozygous lamin A / C gene abnormality, but are preferably heterozygous.
  • the method of the present invention can be applied in any mammal in which any pluripotent stem cell has been established or is capable of being established, such as humans, mice, monkeys, pigs, rats, dogs, etc. Among them, human is preferable.
  • iPS Artificial pluripotent stem
  • ES cells An artificial stem cell derived from a somatic cell that has almost the same properties as those of the cell line, such as differentiation pluripotency and proliferation ability by self-replication (K. Takahashi and S. Yamanaka (2006) Cell, 126: 663-676; K. Takahashi et al. (2007) Cell, 131: 861-872; J. Yu et al.
  • the nuclear reprogramming substance is not particularly limited as long as it is a gene that is specifically expressed in ES cells, a gene that plays an important role in maintaining undifferentiation of ES cells, or a gene product thereof, such as Oct3 / 4, Klf4, Klf1, Klf2, Klf5, Sox2, Sox1, Sox3, Sox15, Sox17, Sox18, c-Myc, L-Myc, N-Myc, TERT, SV40 Large T antigen, HPV16 E6, HPV16 E7, Bmil, Lin28, Lin28b , Nanog, Esrrb or Esrrg.
  • These reprogramming substances may be used in combination when iPS cells are established. For example, a combination including at least one, two, or three of the above-described initialization substances,
  • nucleotide sequences of mouse and human cDNAs of each of the above nuclear reprogramming substances and the amino acid sequence information of the protein encoded by the cDNA refer to NCBI accession numbers described in WO 2007/069666, and L-Myc, Lin28 , Lin28b, Esrrb and Esrrg mouse and human cDNA and amino acid sequence information can be obtained by referring to the following NCBI accession numbers, respectively.
  • a person skilled in the art can prepare a desired nuclear reprogramming substance by a conventional method based on the cDNA sequence or amino acid sequence information.
  • nuclear reprogramming substances may be introduced into somatic cells in the form of proteins, for example, by lipofection, binding to cell membrane permeable peptides, microinjection, or in the form of DNA, for example, It can be introduced into somatic cells by techniques such as viruses, plasmids, artificial chromosomes, vectors, lipofection, liposomes, and microinjection.
  • Virus vectors include retrovirus vectors, lentivirus vectors (cells, 126, pp.663-676, 2006; Cell, 131, pp.861-872, 2007; Science, 318, pp.1917-1920, 2007 ), Adenovirus vectors (Science, 322, 945-949, 2008), adeno-associated virus vectors, Sendai virus vectors (Proc Jpn Acad Ser B Phys Biol Sci. 85, 348-62, 2009) and the like.
  • artificial chromosome vectors include human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), and bacterial artificial chromosomes (BAC, PAC).
  • a plasmid for mammalian cells can be used (Science, 322: 949-953, 2008).
  • the vector can contain regulatory sequences such as a promoter, enhancer, ribosome binding sequence, terminator, polyadenylation site, etc. so that a nuclear reprogramming substance can be expressed.
  • Selectable marker sequences such as kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, thymidine kinase gene, diphtheria toxin gene, reporter gene sequences such as green fluorescent protein (GFP), ⁇ -glucuronidase (GUS), FLAG, etc. Can be included.
  • the above vector contains a LoxP sequence before and after the gene or promoter encoding the nuclear reprogramming substance and the gene encoding the nuclear reprogramming substance that binds to it. You may have.
  • the vector may also contain EBNA-1 and oriP or Large T and SV40ori sequences so that they are replicated without being incorporated into the chromosome and are present episomally.
  • HDAC histone deacetylase
  • VPA valproic acid
  • MC 1293 sodium butyrate
  • M344 small molecule inhibitors
  • siRNA and shRNA against HDAC eg, HDAC1 siRNA Smartpool O (Millipore)
  • HuSH 29mer shRNA Constructs against HDAC1 Nucleic acid expression inhibitors such as (OriGene) etc.
  • DNA methyltransferase inhibitors eg 5'-azacytidine
  • G9a histone methyltransferase Inhibitors eg, small molecule inhibitors such as BIX-01294 (Cell Stem Cell, 2: 525-528 (2008)), siRNA and shRNA against G9a (eg, G9a siRNA (human) (Santa Cruz Biotechnology), etc.), etc.
  • Nucleic acid expression inhibitors, etc.] L-channel calcium agonist (eg Bayk8644) (Cell Stem Cell, 3, 568-574 (2008)) p53 inhibitors (eg siRNA and shRNA against p53) (Cell Stem Cell, 3, 475-479 (2008)), Wnt Signaling (eg soluble Wnt3a) (Cell Stem Cell, 3, 132-135 (2008)), LIF or cytokines such as bFGF, ALK5 inhibitors (eg SB431542) (Nat Methods, 6: 805-8 (2009)), mitogen-activated protein kinase signaling inhibitors, glycogen synthase kinase-3 inhibitors (PloS Biology, 6 (10 ), 2237-2247 (2008)), miR-291-3p, miR-294, miR-295 and other miRNAs (RL Judson et al., Nat.
  • L-channel calcium agonist eg Bayk8644
  • p53 inhibitors eg siRNA and shRNA against p53
  • Examples of the culture medium for iPS cell induction include (1) DMEM, DMEM / F12 or DME medium containing 10 to 15% FBS (these media include LIF, penicillin / streptomycin, puromycin, L-glutamine). , (2) ES cell culture medium containing bFGF or SCF, for example, mouse ES cell culture medium (for example, TX-WES medium, thrombos. X)) or primate ES cell culture medium (eg, primate (human & monkey) ES cell culture medium, Reprocell, Kyoto, Japan).
  • culture methods include, for example, contacting somatic cells with a nuclear reprogramming substance (DNA or protein) on DMEM or DMEM / F12 medium containing 10% FBS in the presence of 5% CO 2 at 37 ° C. Culture for ⁇ 7 days, then re-spread the cells on feeder cells (eg, mitomycin C-treated STO cells, SNL cells, etc.), and bFGF-containing primate ES cells approximately 10 days after contact between the somatic cells and the nuclear reprogramming substance Culturing in a culture medium can produce iPS-like colonies about 30 to about 45 days or more after the contact. Moreover, in order to increase the induction efficiency of iPS cells, the cells may be cultured under conditions of an oxygen concentration as low as 5-10%.
  • a nuclear reprogramming substance DNA or protein
  • FBS-containing DMEM medium including LIF, penicillin / streptomycin, puromycin, L-glutamine, Non-essential amino acids, ⁇ -mercaptoethanol, etc. can be included as appropriate.
  • ES-like colonies can be formed after about 25 to about 30 days or more.
  • the medium is replaced with a fresh medium once a day from the second day after the start of the culture.
  • the number of somatic cells used for nuclear reprogramming is not limited, but ranges from about 5 ⁇ 10 3 to about 5 ⁇ 10 6 cells per 100 cm 2 of culture dish.
  • a marker gene-expressing cell When a gene containing a drug resistance gene is used as a marker gene, a marker gene-expressing cell can be selected by culturing in a medium (selective medium) containing the corresponding drug.
  • the marker gene is a fluorescent protein gene
  • the marker gene-expressing cells can be obtained by observing with a fluorescence microscope, by adding a luminescent substrate in the case of a luminescent enzyme gene, and by adding a chromogenic substrate in the case of a chromogenic enzyme gene. Can be detected.
  • a “somatic cell” may be any cell other than a germ cell derived from a mammal (eg, human, mouse, monkey, pig, rat, etc.), such as an epithelial cell that keratinizes.
  • keratinized epidermal cells mucosal epithelial cells (eg, epithelial cells of the tongue surface), exocrine glandular epithelial cells (eg, mammary cells), hormone-secreting cells (eg, adrenal medullary cells), cells for metabolism and storage (Eg, hepatocytes), luminal epithelial cells that make up the interface (eg, type I alveolar cells), luminal epithelial cells (eg, vascular endothelial cells) in the inner chain, and ciliated cells that are capable of transporting (Eg, airway epithelial cells), extracellular matrix secreting cells (eg, fibroblasts), contractile cells (eg, smooth muscle cells), blood and immune system cells (eg, T lymphocytes), sensory cells (Eg, sputum cells), autonomic nervous system neurons (eg, cholinergic neurons) , Sensory organs and peripheral neuron support cells (eg, companion cells), central nervous system neurons and glial cells
  • undifferentiated progenitor cells including somatic stem cells
  • terminally differentiated mature cells can be used as the source of somatic cells in the invention.
  • undifferentiated progenitor cells include tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells.
  • the mammal individual from which somatic cells are collected is not particularly limited, but is preferably a human. More preferably, a patient with familial lamin A / C cardiomyopathy (preferably dilated cardiomyopathy with cardiac stimulation conduction disorder (DCM-CD)) or unaffected with the same lamin A / C gene mutation as the patient It is desirable to collect somatic cells from humans.
  • gene mutations include lamin A / C gene coding region (CDS) and intron region mutations that cause splicing abnormalities.
  • a mutation that deletes the 908-909th two bases (CT) of CDS (CDS of lamin A ⁇ ⁇ mRNA is shown in SEQ ID NO: 1 and CDS of lamin C mRNA is shown in SEQ ID NO: 3) can be mentioned.
  • CDS of lamin A ⁇ ⁇ mRNA is shown in SEQ ID NO: 1
  • CDS of lamin C mRNA is shown in SEQ ID NO: 3
  • a frame shift occurs to generate a stop codon, and a mutant lamin A / C protein (SEQ ID NO: 6) consisting of 328 amino acids is generated.
  • Lamin A / C gene mutations that lead to DCM-CD include, for example, a missense mutation (R166P) in which the 166th Arg of Lamin A / C protein is replaced with Pro, and the 225th Arg is replaced with another amino acid.
  • R166P missense mutation
  • Missense mutation (R225X), missense mutation (Y259H) in which 259th Tyr is replaced with His, frame shift by replacing 815-818th 4th base of CDS of lamin A / C gene with 6th base of CCAGAC Mutation, mutation in which 9 bases (3 amino acids) are inserted due to splicing abnormality due to A ⁇ G substitution 10 bases upstream from the 3 'end of the third intron of the lamin A / C gene (J Cardiovasc Electrophysiol 2005; 16: 137-45 ), Or natural mutations listed in the sequence annotation information column of human lamin A / C (LMNA_HUMAN) registered as P02545 in UniProtKB.
  • LMNA_HUMAN sequence annotation information column of human lamin A / C
  • an iPS cell having a lamin A / C gene abnormality is a lamin A / C having a gene abnormality that causes cardiomyopathy after the iPS cell is established from a somatic cell having a normal lamin A / C gene by the above-described method.
  • the gene can also be generated by knocking in the lamin A / C locus of iPS cells.
  • PGD pre-implantation diagnosis
  • ES human embryonic stem
  • Examples of ES cell production methods include culturing an inner cell mass at the blastocyst stage of a mammal (see, for example, Manipulating the Mouse Embryo A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994)) , A method of culturing early embryos produced by somatic cell nuclear transfer (Wilmut et al., Nature, 385, 810 (1997); Cibelli et al., Science, 280, 1256 (1998); Akira Iriya et al., Protein nucleic acid enzyme , 44, 892 (1999); Baguisi et al., Nature Biotechnology, 17, 456 (1999); Wakayama et al., Nature, 394, 369 (1998); Wakayama et al., Nature Genetics, 22, 127 (1999) Wakayama et al., Proc.
  • somatic cell nuclear transfer the type of somatic cells and the source from which the somatic cells are collected are the same as in the case of the iPS cells.
  • EG cells can be induced by isolating primordial germ cells according to conventional methods and culturing them in the presence of LIF, bFGF and SCF.
  • mGS cells can be prepared from testis cells according to the method described in WO 2005/100548.
  • Pluripotent adult progenitor cells can be isolated from bone marrow according to the method described in J. Clin. Invest. 109: 337-346 (2002).
  • the cardiomyopathy- specific pluripotent stem cells obtained as described above can be differentiated into cardiomyocytes by any known differentiation induction method.
  • methods for inducing differentiation into cardiomyocytes for example, methods using embryoid body (EB) formation and methods using a directed differentiation approach have been reported (eg, Nature 2008; 453: 524-8, J Clin Invest 2001; 108: 407-14, Circ Res 2002; 91: 501-8, Circ Res 2003; 93: 32-9, Nat Biotechnol 2007; 25: 1015-24, Circulation 2008; 118: 517, Circulation 2008 118: 506, Nat Biotechnol 2005; 23: 611 etc.).
  • EB embryoid body
  • the medium can be used by adding an additive to the basal medium.
  • the basal medium include Neurobasal medium, Neural Progenitor Basal medium, NS-A medium, BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, Improved MEM ⁇ Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, ⁇ MEM medium, DMEM medium, DMEM / F12 medium, Ham medium, RPMI 1640 medium, Fischer's medium, and mixed media for animal cells
  • IMDM medium Neurobasal medium, Neural Progenitor Basal medium, NS-A medium, BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, Improved MEM ⁇ Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, ⁇ MEM medium, DMEM medium, DMEM / F12 medium, Ham medium, RPMI 1640 medium, Fischer's medium, and mixed media for animal cells
  • IMDM medium
  • serum As additives, serum, retinoic acid, ascorbic acid, BMP (BMP2, BMP4), Nodal, TGF ⁇ 1, Activin-A, Dkk1, IGFBP-4, bFGF, EGF, HGF, LIF, amino acids, vitamins, interleukins, Insulin, transferrin, heparin, heparan sulfate, collagen, fibronectin, progesterone, selenite, B27-supplement, N2-supplement, ITS-supplement, antibiotics. It is also preferable to add Nogin as a BMP inhibitor in the early stage of differentiation induction.
  • the incubator can be coated with a coating agent such as collagen, gelatin, matrigel, poly-L-lysine, poly-D-lysine, fibronectin, laminin and the like.
  • a coating agent such as collagen, gelatin, matrigel, poly-L-lysine, poly-D-lysine, fibronectin, laminin and the like.
  • the concentration of pluripotent stem cells at the start of culture can be appropriately set so as to efficiently form cardiomyocytes.
  • the concentration of pluripotent stem cells at the start of culture is not particularly limited.
  • the concentration is about 1 ⁇ 10 3 to about 1 ⁇ 10 6 cells / ml, preferably about 1 ⁇ 10 4 to about 5 ⁇ 10 5 cells / ml. It is.
  • Other culture conditions such as culture temperature and CO 2 concentration can be set as appropriate.
  • the culture temperature is not particularly limited, but is, for example, about 30 to 40 ° C., preferably about 37 ° C.
  • the CO 2 concentration is, for example, about 1 to 10%, preferably about 5%.
  • the cardiomyocytes obtained as described above express traits characteristic of lamin A / C cardiomyopathy as typified by abnormal nuclear morphology and / or nuclear membrane damage.
  • As a prophylactic and / or therapeutic drug for cardiomyopathy, particularly lamin A / C cardiomyopathy, can be screened.
  • cardiomyocytes can be purified from an ascorbic acid-treated embryoid body with a Percoll gradient, and myocardial tissue can be constructed in the presence of type I collagen and matrigel (Circulation 2006; 113: 2237).
  • pathological conditions characteristic to cardiomyopathy such as a decrease in muscle contraction force, loss of cardiomyocytes, and fibrosis of the myocardial tissue are reproduced. Therefore, screening for prophylactic and / or therapeutic drugs for cardiomyopathy, especially dilated cardiomyopathy, including lamin A / C cardiomyopathy, using as an index the improvement of these traits or the inhibition or delay of the expression of these traits be able to.
  • the present invention comprises contacting a cardiomyocyte or tissue derived from cardiomyopathy-specific pluripotent stem cells obtained as described above with a test substance. Using as an index whether the test substance changes a trait different from the normal cardiomyocyte or tissue expressed by the cardiomyocyte or tissue so that it approximates the trait of the normal cardiomyocyte, or prevents or delays the expression of the different trait.
  • the present invention provides a method for screening a candidate substance for a prophylactic and / or therapeutic agent for cardiomyopathy, preferably dilated cardiomyopathy, particularly lamin A / C cardiomyopathy.
  • test substance in the present invention may be any known compound and novel compound, for example, nucleic acid, carbohydrate, lipid, protein, peptide, organic low molecular weight compound, compound library prepared using combinatorial chemistry technology And random peptide libraries prepared by solid-phase synthesis or phage display methods, or natural components derived from microorganisms, animals and plants, marine organisms, and the like.
  • the cardiomyocytes when not in contact with a test substance and the form and / or degree of damage to the nucleus of the cardiomyocytes when contacted with the test substance are compared,
  • a method for selecting a test substance as a candidate for a prophylactic and / or therapeutic drug for cardiomyopathy when abnormalities in nuclear morphology and / or nuclear membrane damage at the time of contact are milder than those at the time of non-contact Is mentioned.
  • Nuclear morphology and nuclear membrane damage can be assayed by observing cardiomyocytes with an electron microscope in a conventional manner.
  • the test substance is a candidate substance for a prophylactic and / or therapeutic agent for cardiomyopathy The method of selecting as is mentioned.
  • the substance selected by the above screening method can be used as a prophylactic and / or therapeutic agent for cardiomyopathy.
  • the cardiomyopathy preventive / therapeutic agent in the present invention is prepared by mixing the selected substance as an active ingredient alone or with a pharmacologically acceptable carrier, excipient, diluent, etc. It can be administered orally or parenterally as a pharmaceutical composition.
  • compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), and syrups. Agents, emulsions, suspensions and the like.
  • a composition for parenteral administration for example, injections, suppositories and the like are used, and injections include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, infusions, and the like. Dosage forms may be included.
  • excipients eg sugar derivatives such as lactose, sucrose, sucrose, mannitol, sorbitol; starch derivatives such as corn starch, potato starch, alpha starch, dextrin; cellulose derivatives such as crystalline cellulose; Gum arabic; dextran; organic excipients such as pullulan; and silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, magnesium magnesium aluminosilicate; phosphates such as calcium hydrogen phosphate; Carbonates such as calcium; inorganic excipients such as sulfates such as calcium sulfate), lubricants (eg, stearic acid metal salts such as stearic acid, calcium stearate, magnesium stearate; talc; Colloidal silica; wax like beeswax and gay wax Borax; adipic acid; sulfate such as sodium sulfate; glycol; fuma
  • the dose of the active ingredient of the cardiomyopathy preventive / therapeutic agent in the present invention can vary depending on various conditions such as the patient's symptoms, age, weight and the like.
  • the dose varies depending on symptoms, age, etc.
  • the lower limit is 0.1 mg (preferably 0.5 mg) and the upper limit is 1000 mg (preferably 500 mg).
  • the dose may be increased or decreased depending on the symptoms.
  • cardiomyopathy preventive / therapeutic agent in the present invention may be used in combination with other drugs such as ⁇ blocker, ACE inhibitor, calcium sensitizer and the like.
  • the agent for preventing / treating cardiomyopathy and these other agents in the present invention can be administered simultaneously, sequentially or separately.
  • lamin A / C gene in iPS cells was quantified by q-PCR. Specifically, total lamin A and C mRNA were quantified with the N-terminal primer, and wild-type allele lamin A or lamin C was quantified with the C-terminal primer. The result is shown in figure 2. Compared with control iPS cells (201B7), iPS cells derived from lamin A / C cardiomyopathy patients did not show a decrease in the expression level of lamin A / C gene and expressed mutant allele-derived mRNA.
  • iPS cells are aggregated to form embryoid bodies, which are differentiated into cardiomyocytes by treatment with Activin A, BMP4, bFGF for 3 days and VEGF, DKK1 for 4 days in suspension culture.
  • Activin A BMP4
  • VEGF vascular endothelial growth factor
  • DKK1 DKK1 for 4 days in suspension culture.
  • a beating embryoid body YangYet al, Nature. 2008 May 22; 453 (7194): 524-8.
  • iPS cells cultured on Matrigel-coated plates are treated with Activin A and BMP4 for 5 days, and then cultured for 2-3 weeks, resulting in the appearance of beating cardiomyocytes. (Laflamme et al, Nat Biotechnol. 2007 Sep; 25 (9): 1015-24.).
  • iPS cells were prepared from skin fibroblasts of lamin A / C-related cardiomyopathy patients, and the disease could be reproduced in differentiated myocardium.
  • Lamin A / C-related cardiomyopathy is an intractable disease for which no fundamental treatment has been established, and it is expected that this model will be useful for elucidating the pathophysiology and evaluating drug efficacy.

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Abstract

La présente invention concerne un procédé pour cribler un agent prophylactique et/ou thérapeutique pour une myocardiopathie, ledit procédé comprenant : (1) une étape pour mettre en contact une substance d'essai avec des cellules ou du tissu myocardiques dérivés de cellules souches pluripotentes avec mutation du gène de lamine A/C ; et (2) une étape pour sélectionner, en tant que candidat pour un agent prophylactique et/ou thérapeutique pour une myocardiopathie, une substance d'essai qui modifie un caractère exprimé par les cellules ou le tissu myocardiques mentionnés ci-dessus, ledit caractère étant différent d'un caractère original exprimé par des cellules ou un tissu myocardiques normaux lorsque aucune substance d'essai n'est mise en contact avec ceux-ci, de manière à rapprocher le caractère différent du caractère original, ou une substance d'essai qui inhibe ou retarde l'expression du caractère ci-dessus différent du caractère original.
PCT/JP2012/052788 2011-02-07 2012-02-07 Cellule souche pluripotente spécifique pour une myocardiopathie et utilisation de celle-ci WO2012108444A1 (fr)

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WO2014071323A3 (fr) * 2012-11-02 2014-10-09 Duke University Inhibition de l'histone méthyltransférase pour la re-programmation cardiaque
WO2014185358A1 (fr) * 2013-05-14 2014-11-20 国立大学法人京都大学 Procédé efficace d'induction de cellules myocardiques
WO2014189144A1 (fr) * 2013-05-23 2014-11-27 国立大学法人京都大学 Méthode de criblage de médicaments pour le traitement/la prévention du syndrome myélodysplasique, etc.
WO2015025957A1 (fr) * 2013-08-23 2015-02-26 国立大学法人大阪大学 Procédé de fabrication d'une puce tissulaire de muscle cardiaque utilisée pour le criblage d'un composé candidat médicamenteux
WO2015133147A1 (fr) * 2014-03-05 2015-09-11 公益財団法人先端医療振興財団 Modèle animal humanisé pathologique et tissu de tératome
WO2018164098A1 (fr) * 2017-03-06 2018-09-13 国立大学法人大阪大学 Population de cellules à utiliser en tant que modèle de fibrage de cœur, procédé pour sa production, procédé de criblage l'utilisant, procédé d'évaluation de modèle de fibrage de cœur l'utilisant et système d'évaluation de modèle de fibrage de cœur
JPWO2017175876A1 (ja) * 2016-04-05 2019-03-14 学校法人自治医科大学 幹細胞を再樹立する方法
JPWO2018164098A1 (ja) * 2017-03-16 2020-01-16 国立大学法人大阪大学 心臓線維化モデルとして用いられる細胞集団、その製造方法、それを利用したスクリーニング方法及び心臓線維化モデルの評価方法、並びに心臓線維化モデルの評価システム

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Cited By (13)

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Publication number Priority date Publication date Assignee Title
WO2014071323A3 (fr) * 2012-11-02 2014-10-09 Duke University Inhibition de l'histone méthyltransférase pour la re-programmation cardiaque
US10130637B2 (en) 2012-11-02 2018-11-20 Duke University Inhibition of histone methyltransferase for cardiac reprogramming
US9822342B2 (en) 2013-05-14 2017-11-21 Kyoto University Method of efficiently inducing cardiomyocytes
WO2014185358A1 (fr) * 2013-05-14 2014-11-20 国立大学法人京都大学 Procédé efficace d'induction de cellules myocardiques
JPWO2014185358A1 (ja) * 2013-05-14 2017-02-23 国立大学法人京都大学 効率的な心筋細胞の誘導方法
WO2014189144A1 (fr) * 2013-05-23 2014-11-27 国立大学法人京都大学 Méthode de criblage de médicaments pour le traitement/la prévention du syndrome myélodysplasique, etc.
JPWO2014189144A1 (ja) * 2013-05-23 2017-02-23 国立大学法人京都大学 骨髄異形成症候群等の治療／予防薬のスクリーニング方法
WO2015025957A1 (fr) * 2013-08-23 2015-02-26 国立大学法人大阪大学 Procédé de fabrication d'une puce tissulaire de muscle cardiaque utilisée pour le criblage d'un composé candidat médicamenteux
JPWO2015025957A1 (ja) * 2013-08-23 2017-03-02 国立大学法人大阪大学 薬剤候補化合物のスクリーニングに用いる心筋組織チップの製造方法
WO2015133147A1 (fr) * 2014-03-05 2015-09-11 公益財団法人先端医療振興財団 Modèle animal humanisé pathologique et tissu de tératome
JPWO2017175876A1 (ja) * 2016-04-05 2019-03-14 学校法人自治医科大学 幹細胞を再樹立する方法
WO2018164098A1 (fr) * 2017-03-06 2018-09-13 国立大学法人大阪大学 Population de cellules à utiliser en tant que modèle de fibrage de cœur, procédé pour sa production, procédé de criblage l'utilisant, procédé d'évaluation de modèle de fibrage de cœur l'utilisant et système d'évaluation de modèle de fibrage de cœur
JPWO2018164098A1 (ja) * 2017-03-16 2020-01-16 国立大学法人大阪大学 心臓線維化モデルとして用いられる細胞集団、その製造方法、それを利用したスクリーニング方法及び心臓線維化モデルの評価方法、並びに心臓線維化モデルの評価システム

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