+

WO2001048149A1 - Cellule de moelle osseuse adulte capable de se differencier en cellule de muscle cardiaque - Google Patents

Cellule de moelle osseuse adulte capable de se differencier en cellule de muscle cardiaque Download PDF

Info

Publication number
WO2001048149A1
WO2001048149A1 PCT/JP2000/001148 JP0001148W WO0148149A1 WO 2001048149 A1 WO2001048149 A1 WO 2001048149A1 JP 0001148 W JP0001148 W JP 0001148W WO 0148149 A1 WO0148149 A1 WO 0148149A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
amino acid
acid sequence
cell
mef
Prior art date
Application number
PCT/JP2000/001148
Other languages
English (en)
Japanese (ja)
Inventor
Akihiro Umezawa
Jun-Ichi Hata
Keiichi Fukuda
Satoshi Ogawa
Kazuhiro Sakurada
Original Assignee
Kyowa Hakko Kogyo Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kyowa Hakko Kogyo Co., Ltd. filed Critical Kyowa Hakko Kogyo Co., Ltd.
Priority to AU26950/00A priority Critical patent/AU2695000A/en
Priority to PCT/JP2000/007741 priority patent/WO2001048150A1/fr
Priority to AU10552/01A priority patent/AU1055201A/en
Priority to CA002395950A priority patent/CA2395950A1/fr
Priority to EP00985950A priority patent/EP1254952A4/fr
Priority to PCT/JP2000/009323 priority patent/WO2001048151A1/fr
Priority to AU22281/01A priority patent/AU784618B2/en
Priority to US09/749,728 priority patent/US20020142457A1/en
Publication of WO2001048149A1 publication Critical patent/WO2001048149A1/fr

Links

Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P41/00—Drugs used in surgical methods, e.g. surgery adjuvants for preventing adhesion or for vitreum substitution
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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
    • C12N5/0658—Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts
    • 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
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood 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
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/60—Buffer, e.g. pH regulation, osmotic pressure
    • C12N2500/62—DMSO
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/06—Anti-neoplasic drugs, anti-retroviral drugs, e.g. azacytidine, cyclophosphamide
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/135—Platelet-derived growth factor [PDGF]
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/385—Hormones with nuclear receptors of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • 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
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening

Definitions

  • the present invention relates to a method for isolating, purifying, culturing, and inducing differentiation of bone marrow cells having the ability to isolate cardiomyocytes.
  • the present invention also relates to a method for expanding myeloid cells capable of differentiating into cardiomyocytes using various cytokines, transcription factors, and the like, and a method for controlling the division into cardiomyocytes.
  • the present invention further provides a surface antigen specific for bone marrow cells capable of differentiating into cardiomyocytes.
  • Obtaining method obtaining a gene encoding the surface antigen, obtaining an antibody specific to the surface antigen
  • the present invention relates to a method and a method for obtaining proteins and genes involved in proliferation of bone marrow cells capable of differentiating into cardiomyocytes and differentiation into cardiomyocytes.
  • the present invention relates to a therapeutic agent for various heart diseases using bone marrow cells capable of differentiating into cardiomyocytes.
  • cardiomyocytes Before birth, cardiomyocytes actively divide while autonomously beating. However, their division ability is lost at birth, they do not regain cell division ability like hepatocytes, and unlike skeletal muscle cells, they have satellite cells and undifferentiated precursor cells. Absent. Therefore, if cardiomyocytes are necrotic due to myocardial infarction, myocarditis, or aging, in vivo, cell expansion of the remaining cardiomyocytes rather than cell division occurs. Cardiac hypertrophy is initially a physiological adaptation, but it also leads to a decrease in the diastolic function of the heart itself and a decrease in systolic function in conjunction with the fibrosis of the interstitium due to the proliferation of coexisting cardiac fibroblasts, leading to heart failure.
  • heart transplantation is a fundamental treatment for severe heart failure, but heart transplantation is a common medical treatment due to problems such as a shortage of organ donors, difficulty in determining brain death, rejection, and rising medical costs. It is not easy to spread. In fact, heart disease is the third leading cause of death in Japan (Health and Welfare White Paper, Heisei 10), and if we can regenerate lost cardiomyocytes, it will be a major step forward in medical welfare.
  • cell lines that preserve the properties of cardiomyocytes include atrial natriuretic AT-1 cells established from tumors arising in the atrium of transgenic mice produced by recombining the large T antigen of SV40 overnight with the Lemon promoter [Science, 239; 1029-1038 (1988)] ].
  • atrial natriuretic AT-1 cells established from tumors arising in the atrium of transgenic mice produced by recombining the large T antigen of SV40 overnight with the Lemon promoter [Science, 239; 1029-1038 (1988)] ].
  • these cells are not suitable for cell transplantation because they form tumors when transplanted in vivo. Under these circumstances, the following methods were considered to reconstruct the myocardium.
  • the first method is to convert cells other than cardiomyocytes into cardiomyocytes. This was inferred from the fact that introduction of MyoD into fibroblasts could convert them into skeletal muscle cells. Previously, although successful cases have been shown with P19 cells, which are mouse embryonal cancer cells, [Cell Struc. & Func, 21: 101-110 (1996)], successful cases with non-cancer cells Has not been reported.
  • the second method is to give the cardiomyocytes divisional ability again. This is based on the fact that the heart muscle can divide while beating during fetal life. However, no successful cases have been reported so far.
  • the third method is to induce cardiomyocytes from undifferentiated stem cells. Although it has been shown that cardiomyocytes can be induced from embryonic stem cells (ES cells), transplantation of embryonic stem cells themselves into adults has problems such as formation of carcinoma and antigenicity [Nature Biotechnology, 17 , 139-142 (1999)].
  • a method of obtaining undifferentiated myocardial progenitor cells from aborted fetuses and using them for transplantation has also been considered, and it is known that they can function effectively as cardiomyocytes in animal experiments [Science, 264]. , 98-101 (1994)] 0, however, it is difficult to obtain a large amount of cardiac progenitor cells in this way, the applications also to common medical terms of ethical not easy, hematopoiesis in adult bone marrow
  • mesenchymal stem cells in addition to stem cells and vascular stem cells, which include bone cells, chondrocytes, tendon cells, ligament cells, skeletal muscle cells, fat cells, stoma cells, and liver oval cells.
  • a safer and more reliable treatment than the current treatment for heart disease is desired. Therefore, to select bone marrow cells capable of differentiating into cardiomyocytes from bone marrow cells and to control proliferation or sorting of bone marrow cells capable of differentiating into cardiomyocytes, cells using bone marrow-derived cells were used. It is useful for the development of regenerative therapy for myocardium. For this purpose, it is necessary to identify cells capable of dividing bone marrow cells into cardiomyocytes, and to identify cytokines or transcription factors that act on the proliferation or differentiation of the cells. The inventors of the present invention have conducted intensive studies to address the above problems and have obtained the following results.
  • a mouse bone marrow-derived cell line is labeled with a retroviral vector expressing GFP (Green Fluorescent Protein), and by tracking one cell under a fluorescence microscope, it has the ability to differentiate into cardiomyocytes.
  • the cells were found to be pluripotent stem cells capable of inducing differentiation of at least two different types of cells, cardiomyocytes and adipocytes. It was also found that there are myocardial stem cells that are proliferative at the same time and divide only into myocardial cells, and myocardial progenitor cells that have finite cell division ability and differentiate only into myocardial cells.
  • the stem cells can be probabilistically treated by administration of other genomic DNA demethylating agents such as DMSO (dimethyl sulfoxide) as well as 5-azacytidine, which has already been reported under normal culture conditions.
  • DMSO dimethyl sulfoxide
  • 5-azacytidine 5-azacytidine
  • a cytokine such as platelet-derived growth factor (PDGF) or aU-trans retinoic acid, or culturing in a culture dish coated with fibronectin, increased the rate of cardiomyocyte formation.
  • PDGF platelet-derived growth factor
  • FGF- fibroblast growth factor-2
  • the present invention provides the following (1) to (96).
  • the cell according to (1) which is a pluripotent stem cell having at least the ability to differentiate into cardiomyocytes and adipocytes.
  • the cell according to (1) which is a cardiomyocyte that is induced to differentiate only into cardiomyocytes.
  • the factor according to (13) above, wherein the factor expressed in the fetal heart development region is at least one selected from the group consisting of cytokines, adhesion molecules, bimin and transcription factors. cell.
  • the factor according to (15), wherein the factor that acts on differentiation into cardiomyocytes at the stage of fetal heart development is at least one selected from the group consisting of cytokines, adhesion molecules, vitamins and transcription factors. cell.
  • the transcription factor is Nkx2.5 / Csx, GATA4, MEF-2A, MEF-2B, MEF-2C, MEF-
  • Nkx 2. 5 / Csx is Nkx 2. 5 / Csx having the amino acid sequence is represented by SEQ ID NO: 9, the (21), wherein the cell.
  • MEF-2A is MEF-2A having an amino acid sequence represented by SEQ ID NO: 13.
  • MEF-2C is MEF-2C having an amino acid sequence represented by SEQ ID NO: 17, The cell according to the above (21).
  • MEF-2D is MEF-2D having the amino acid sequence represented by SEQ ID NO: 19.
  • TEF-1 is TEF-1 having an amino acid sequence represented by SEQ ID NO: 25.
  • TEF-3 is TEF-3 having an amino acid sequence represented by SEQ ID NO: 27.
  • TEF-5 is TEF-5 having an amino acid sequence represented by SEQ ID NO: 29.
  • FGF-2 is FGF-2 having the amino acid sequence of SEQ ID NO: 7 or 8.
  • demethylating agent for chromosomal DNA is at least one selected from the group consisting of demethylase, 5-azacitidine and DMSO.
  • demethylase is a demethylase represented by the amino acid sequence of SEQ ID NO: 1.
  • a method for forming myocardium from bone marrow-derived cells comprising using a factor expressed in a fetal heart development region.
  • the factor expressed in the fetal heart development region is at least one selected from the group consisting of cytokines, adhesion molecules, bimin, and transcription factors.
  • a method for forming a myocardium from cells derived from bone marrow which comprises using a factor that acts to divide the myocardial cells at the stage of fetal heart development.
  • the factor according to (40), wherein the factor that acts on differentiation into cardiomyocytes during the fetal heart development stage is at least one selected from the group consisting of cytokines, adhesion molecules, vitamins and transcription factors.
  • the PDGF is PDGF represented by the amino acid sequence of SEQ ID NO: 3 or 5, according to (42). the method of.
  • Nkx2.5 / Csx is Nkx2.5 / Csx having the amino acid sequence represented by SEQ ID NO: 9.
  • MEF-2B are, Ru MEF-2 B der with Amino acid sequence represented by SEQ ID NO: 15, the (46) The method according.
  • MEF-2D is MEF-2D having an amino acid sequence represented by SEQ ID NO: 19.
  • dHAND is a dHAND having an amino acid sequence represented by SEQ ID NO: 21, The method according to (46) above.
  • TEF-1 is TEF-1 having the amino acid sequence represented by SEQ ID NO: 25.
  • TEF- 3 is a TEF- 3 having an amino acid sequence represented by SEQ ID NO: 27.
  • a cardiomyogenic agent comprising a chromosomal DNA demethylating agent as an active ingredient.
  • the factor according to the above (61), wherein the factor expressed in the fetal heart development region is at least one selected from the group consisting of cytokines, adhesion molecules, bimin and transcription factors. Cardiomyogen.
  • a cardiomyogenic agent comprising, as an active ingredient, a factor that acts on differentiation into cardiomyocytes at the stage of fetal heart development.
  • the factor described above, wherein the factor that acts on differentiation into cardiomyocytes at the stage of fetal heart development is at least one selected from the group consisting of cytokines, adhesion molecules, vitamins, and transcription factors.
  • PDGF is represented by the amino acid sequence of SEQ ID NO: 3 or 5.
  • the transcription factor is selected from the group consisting of Nkx2.5 / Csx ⁇ GATA4, MEF-2A ⁇ MEF-2B, MEF-2C, MEF-2D, dHAND, eHAND, TEF-1, TEF-3 and TEF-5 Selected, above
  • MEF-2A is a represented by MEF-2 A the amino acid sequence of SEQ ID NO: 13, wherein said (69) myocardial former description.
  • MEF-2D is a MEF_ 2 D represented by the amino acid sequence of SEQ ID NO: 19, wherein said (69) myocardial former description.
  • TEF-1 is TEF-1 represented by the amino acid sequence of SEQ ID NO: 25.
  • TEF-3 is TEF-3 represented by the amino acid sequence of SEQ ID NO: 27, (69) The cardiomyogen according to (69).
  • TEF-5 is TEF-5 represented by the amino acid sequence of SEQ ID NO: 29.
  • a method for regenerating a heart destroyed by a heart disease comprising using the cell according to any one of (1) to (34).
  • a therapeutic agent for regenerating heart comprising the cell according to any one of the above (1) to (34) as an active ingredient.
  • a congenital genetic disease which comprises using the cell according to any one of the above (1) to (34), into which a wild-type gene for a mutant gene in a cardiac congenital genetic disease has been introduced.
  • telomere has the amino acid sequence represented by SEQ ID NO: 31.
  • a therapeutic agent for heart disease comprising as an active ingredient the cell according to any one of the above (1) to (34), which has been immortalized by expressing telomerase.
  • telomerase is a telomerase having an amino acid sequence represented by SEQ ID NO: 31.
  • the bone marrow cells capable of differentiating into cardiomyocytes of the present invention refer to multipotent stem cells, cardiomyocyte stem cells and cardiomyocyte precursor cells isolated from adult bone marrow.
  • Pluripotent stem cells are cells that can induce differentiation of at least two different types of cells, cardiomyocytes and adipocytes.
  • Cardiomyocyte stem cells are cells that are proliferative and have the ability to differentiate only into cardiomyocytes. Induction of myocardial stem cells induces two types of cells, myocardial cells and myocardial stem cells.
  • Cardiac progenitor cells are cells that have a finite cell division ability and have the ability to differentiate only into cardiac myocytes. When differentiation is induced in cardiomyocyte precursor cells, only cardiomyocytes are formed, unlike cardiomyocyte stem cells.
  • the method of obtaining bone marrow cells capable of differentiating into cardiomyocytes from human bone marrow is not particularly limited as long as it is a method that can be safely and efficiently obtained.S.E.Haynesworth et al.
  • the bone marrow cells After collecting the bone marrow cells from the obtained bone marrow fluid by centrifugation at i, ooo xg , the bone marrow cells are washed with PBS (Phosphate Buffered Saline). After repeating this step twice, the bone marrow cells were subjected to 10% FBS (fetal bovine serum) in human MEM (human modified MEM), DMEM
  • a bone marrow cell solution By resuspending the cells in a cell culture medium such as (Dulbecco's modified MEM) or IMDM (Isocove's modified Dulbecco's medium), a bone marrow cell solution can be obtained.
  • a cell culture medium such as (Dulbecco's modified MEM) or IMDM (Isocove's modified Dulbecco's medium)
  • a method for isolating bone marrow cells capable of differentiating into cardiomyocytes from the bone marrow cell solution is to remove other cells mixed in the solution, for example, blood cells, hematopoietic stem cells, vascular stem cells, fibroblasts, and the like.
  • the bone marrow cell fluid was overlaid on percoll having a density of 1.073 g / ml based on the method described in MF Pittenger et al. Science, 284, 143 (1999), and then centrifuged at 1,100 X g for 30 minutes.
  • the cells can be isolated by collecting the cells at the interface.
  • a percoll diluted to 10 X PBS plus 9/10 in bone marrow cell fluid after ⁇ added the same volume, and centrifuged for 30 minutes at 2 0,000 X g, density 1 ⁇ 075 to 1.0
  • a bone marrow cell mixture containing bone marrow cells capable of differentiating into the cardiomyocytes can be obtained.
  • the bone marrow cell 'mixture containing bone marrow cells capable of differentiating into cardiomyocytes obtained by the above method is diluted so that only one cell is injected into each well of a 96-well culture plate, and one cell-derived After preparing a large number of clones, the clones are treated using the method described below for inducing cardiomyocytes from bone marrow cells having the ability to differentiate into cardiomyocytes, and clones in which autonomously pulsating cells appear are selected.
  • bone marrow cells capable of differentiating into the cardiomyocytes can be obtained.
  • Pluripotent stem cells, myocardial stem cells, and myocardial progenitor cells can be selected by performing single cell marking using a reporter gene such as GFP (Green Fluorescent Protein) described below.
  • the method of obtaining bone marrow cells capable of differentiating into cardiomyocytes from rats and mice is not particularly limited, but can be obtained by the following procedure. Rats or mice are sacrificed by cervical prolapse, and after sufficient disinfection with 70% ethanol, the skin of the femur and the quadriceps are removed. Remove the joint with scissors in the knee joint and remove the muscles behind the femur. Remove the joint with scissors in the hip joint and remove the femur. After removing the muscle attached to the femur with scissors as much as possible, scissors both ends of the femur.
  • FBS fetal calf serum
  • the tip of the needle is inserted into the stump of the femur on the knee joint side.
  • the culture solution in the syringe into the bone marrow the bone marrow cells are pushed out from the hip joint stump.
  • the obtained bone marrow cells are suspended in a culture solution by pipetting.
  • bone marrow cells capable of differentiating into cardiomyocytes can be isolated in the same manner as in the above-mentioned method for isolating bone marrow cells from human bone marrow fluid.
  • Examples of cells isolated by the above method include mouse bone marrow-derived stem cells.
  • BMSC a mouse bone marrow-derived stem cell, was sent to the Ministry of International Trade and Industry by the Ministry of International Trade and Industry's Institute of Biotechnology and Industrial Technology (February 22, 2002) at FERM BP-70 (1-3-3 Tsukuba East, Ibaraki, Japan). Deposited as 43 .
  • the medium used for culturing bone marrow cells capable of differentiating into cardiomyocytes isolated by the method 1 described above is usually a known medium (Tissue Culture Technology Basic Edition, Third Edition, Asakura Shoten 1996).
  • a cell culture medium can be used, and preferably, a cell culture medium such as MEM, DMEM, IMDM, or the like supplemented with 5 to 20% of calories from bovine serum or the like is used.
  • the culturing conditions may be any conditions as long as the cells can be cultivated, but the culturing temperature is preferably 33 to 37 ° C, and the culturing is preferably performed in an incubator filled with 5 to 10% carbon dioxide gas.
  • the bone marrow cells having the ability to divide myocardial cells are attached to a normal plastic culture dish for tissue culture and proliferate.
  • the cells grow on the entire surface of the culture dish, remove the medium and suspend cells by adding trypsin EDTA solution.
  • the suspended cells can be further subcultured by washing with PBS or the cell culture medium, and then diluting 5- to 20-fold with the cell culture medium and adding to a new culture dish.
  • Methods for inducing cardiomyocytes from bone marrow cells capable of differentiating into cardiomyocytes include (1) induction of differentiation by treatment with DNA demethylating agent, (2) factors expressed in the fetal heart development region or fetus. (3) Induction of differentiation by bone marrow cells capable of differentiating into cardiomyocytes or culture supernatant of cardiomyocytes differentiated from the bone marrow cells, etc. Can be mentioned. Simply use these methods By themselves or in combination, cardiomyocytes can be derived from bone marrow cells having the ability to differentiate into cardiomyocytes.
  • the DNA demethylating agent may be any compound that causes demethylation of DNA.
  • DNA demethylating agents include demethylase, an enzyme that specifically inhibits methylation of cytosine residues in GpC sequences in chromosomal DNA, and 5-azacytidine (hereinafter abbreviated as 5-aza-C). And DMSO (dimethyl sulfoxide).
  • demethylase examples include a demethylase having the amino acid sequence of SEQ ID NO: 1 [Nature, 397. 579-583 (1999)]. Specific examples of the induction of differentiation by DNA demethylation treatment are shown below.
  • 5-aza-C is added to a medium containing bone marrow cells capable of differentiating into cardiomyocytes to a concentration between 3 mmol / l and 10 mmol / l, and incubated at the above culture conditions for 24 hours. Bastion. The medium is replaced to remove 5-aza-C, and cultured for another 2-3 weeks to obtain cardiomyocytes.
  • the formed cardiomyocytes are mainly sinus node cells at 2-3 weeks of culture, but can induce differentiation of ventricular cardiomyocytes after 4 weeks of culture.
  • Factors expressed in the fetal heart development region or factors that act on cardiomyocyte differentiation during the fetal heart development stage include cytokines, vitamins, adhesion molecules, transcription factors, and the like.
  • any cytokine may be used as long as it promotes differentiation into bone marrow cells having the ability to divide myocardial cells into cardiomyocytes at the stage of cardiac development.
  • PDGF platelet-derived growth factor
  • bone marrow cells capable of dividing into cardiomyocytes are promoted to be divided into cardiomyocytes at the stage of heart development. It is also possible.
  • cytokines that suppress differentiation into cardiomyocytes include fibroblast growth factor-2 (hereinafter abbreviated as FGF-2), specifically, FGF-2 represented by SEQ ID NO: 7 or 8.
  • FGF-2 fibroblast growth factor-2
  • inhibitors for cytokines that suppress differentiation into cardiomyocytes include substances that inhibit cytokine signaling, such as antibodies that neutralize cytokines and low molecular weight compounds.
  • any vitamin can be used as long as it promotes differentiation into bone marrow cells capable of differentiating into cardiomyocytes, such as retinoic acid, and cardiomyocytes during the developmental stage of the heart. Specifically, and the like retinoic acid 10- 9 M.
  • any adhesion molecule such as fibronectin may be used as long as it is expressed in the heart development region at the stage of cardiac development. Specifically, by culturing bone marrow cells capable of differentiating into the cardiomyocytes in a culture dish coated with fibronectin, filtration into the cardiomyocytes can be promoted.
  • transcription factor examples include homebox-type transcription factor Nkx2.5 / Csx (SEQ ID NO: 9: amino acid sequence, SEQ ID NO: 10: base sequence) and Zinc finger-type transcription factor GATA4 belonging to GATA family (SEQ ID NO: 11: amino acid sequence, SEQ ID NO: 12: base sequence), transcription factor MEF-2A belonging to myocyte enhancer factor-2 (MEF-2) family 1 (SEQ ID NO: 13: amino acid sequence, SEQ ID NO: 14: base sequence), MEF-2B (SEQ ID NO: 15: Amino acid sequence, SEQ ID NO: 16: base sequence), MEF-2C (SEQ ID NO: 17: amino acid sequence, SEQ ID NO: 18: base sequence) and MEF-2D (SEQ ID NO: 19: amino acid sequence, SEQ ID NO: 20: base sequence), Basic helix loop dHAND (SEQ ID NO: 21: amino acid sequence, SEQ ID NO: 22: base sequence) and eHAND (SEQ ID NO: 23: amino acid sequence, SEQ ID
  • the above-mentioned transcription factor can induce differentiation into cardiomyocytes by introducing DNA encoding the factor into bone marrow cells capable of differentiating into cardiomyocytes and expressing the DNA.
  • the differentiation into cardiomyocytes can also be induced by adding the factor to a culture solution of bone marrow cells capable of differentiating into cardiomyocytes.
  • the differentiation into cardiomyocytes can be induced by adding the culture supernatant of autonomously beating cardiomyocytes to a bone marrow cell culture medium having the ability to differentiate into cardiomyocytes.
  • a bone marrow-derived cell capable of differentiating into cardiomyocytes can be differentiated into cardiomyocytes by using a cardiomyocyte differentiation-inducing factor (hereinafter referred to as a cardiomyocyte differentiation-inducing factor) obtained by the following method. Can be guided.
  • a cardiomyocyte differentiation-inducing factor hereinafter referred to as a cardiomyocyte differentiation-inducing factor
  • a myocardial differentiation-inducing factor it can be obtained by adding various protease inhibitors to the culture supernatant of autonomously pulsating cells, and combining dialysis, salting out, chromatography and the like.
  • the partial amino acid sequence of the above-described myocardial motility-inducing factor is determined, and a DNA probe designed based on the amino acid sequence is used to prepare the autonomously beating cells.
  • a gene for a myocardial differentiation-inducing factor can be obtained.
  • Cardiac regeneration or heart disease containing bone marrow cells capable of differentiating into cardiomyocytes can be used as therapeutic agents for heart regeneration or heart diseases.
  • heart disease examples include myocardial infarction, ischemic heart disease, congestive heart failure, arrhythmia, hypertrophic cardiomyopathy, dilated cardiomyopathy, myocarditis, valvular disease and the like.
  • bone marrow cells having the ability to differentiate into cardiomyocytes are contained in high purity, and the bone marrow cells having the ability to differentiate into cardiomyocytes are expanded according to the site and size of the damaged heart.
  • Cells preferably from bone marrow cells capable of differentiating into cardiomyocytes, myocardial endothelial cells (Endocardial endothelial cells), cushion cells (Cushion cells), ventricular-type cardiomyocytes, atrial-type cardiac cells, sinus node cells, etc. Cells capable of inducing differentiation into various cells forming the heart are used.
  • the therapeutic agent is a density gradient centrifugation method described above from the bone marrow fluid of a patient with myocardial infarction, a panning method using an antibody that specifically recognizes cells capable of differentiating into cardiomyocytes described below (J. Immunol. 141 (8), 2797-2800 (1988)] or FACS method [Int. Immunol., 10 (3), 275-283 (1998)], or a gene specific to myeloid cells capable of differentiating into cardiomyocytes
  • a bone marrow capable of differentiating into said cardiomyocytes by a method for constructing a repo-one-line system using It can be produced by purifying cells.
  • the therapeutic agent may be a cell obtained by inducing the differentiation of bone marrow cells capable of differentiating into cardiomyocytes into cardiomyocytes using a cardiomyogen described below, a bone marrow cell obtained from bone marrow of an elderly person, Also included are cells capable of differentiating into cardiomyocytes that have been activated to mitosis using the immortalization method described below.
  • the purity of the therapeutic agent produced by the above method can be assayed by combining the antibody specifically recognizing the cells capable of differentiating into cardiomyocytes with the FACS method.
  • ischemic heart disease As a method for transporting the above therapeutic agent to the site of injury, a method using a catheter or the like is used.
  • ischemic heart disease As a method for transporting the above therapeutic agent to the site of injury, a method using a catheter or the like is used.
  • ischemic heart disease As a method for transporting the above therapeutic agent to the site of injury, a method using a catheter or the like is used.
  • ischemic heart disease As a method for transporting the above therapeutic agent to the site of injury, a method using a catheter or the like is used.
  • ischemic heart disease As a method for transporting the above therapeutic agent to the site of injury, a method using a catheter or the like is used.
  • ischemic heart disease As a method for transporting the above therapeutic agent to the site of injury, a method using a catheter or the like is used.
  • ischemic heart disease As a method for transporting the above therapeutic agent to the site of injury, a method using a catheter or the like
  • eccentric stenosis is subdivided into two types, Type I and Evening II. It is known that the form of stenosis is related to the course and prognosis of angina.Type II eccentric stenosis and multiple wall irregularities are common in unstable angina patients, and there is a high possibility of transition to myocardial infarction . If the blood vessel is completely stenotic, the injected cells may not reach the lesion site.Therefore, the stenosis site must be removed by percutaneous coronary angioplasty (PTCA) or thrombolysis beforehand. It is necessary to restart. Depending on the location of the damaged myocardial cells, the cells to be injected can be differentiated into ventricular and atrial types.
  • PTCA percutaneous coronary angioplasty
  • the catheter insertion method is the Sones method (Medical View, 1993), which is inserted from the right upper arm artery, or the Jundkins method (Medical View, 1993), which is inserted through the femoral artery. 1, MEDICAL VIEW, 1993) can be used.
  • the cardiomyogenic agent of the present invention may be a demethylating agent for chromosomal DNA, a factor expressed in the fetal heart development region, or a factor that acts on cardiomyocyte differentiation during the fetal heart development stage.
  • Bone marrow-derived cells can be induced to differentiate into cardiomyocytes by containing at least one factor as an active ingredient.
  • Myocardial differentiation inducing factors include cytokines, vitamins, adhesion molecules, transcription factors, etc. I can give it.
  • any cytokine can be used as long as it promotes the differentiation of bone marrow cells capable of differentiating into cardiomyocytes and the differentiation into cardiomyocytes at the stage of cardiac development.
  • PDGF 10 to 40 ng / ml of PDGF.
  • PDGF those represented by the amino acid sequence of SEQ ID NO: 3 or 5 are preferably used.
  • any vitamin may be used as the vitamin, as long as it promotes the differentiation of bone marrow cells such as retinoic acid into cardiomyocytes having the ability to divide the myocardial cells into cardiomyocytes at the stage of cardiac development. Specifically, and the like retinoic acid 10- 9 M.
  • the adhesion molecule may be any adhesion molecule, such as fibronectin, as long as it is expressed in the heart development region at the stage of cardiac development. Specifically, differentiation into cardiomyocytes can be promoted by culturing bone marrow cells capable of differentiating into cardiomyocytes in a culture dish coated with fibronectin.
  • the transcription factor Homeobokkusu transcription factor Nkx2.5 / C SX (SEQ ID NO: 9: amino acid sequence, SEQ ID NO: 10: nucleotide sequence), GATA family foremost belongs Zinc finger transcription factor GATA4 (SEQ ID NO: 11: amino acid sequence , SEQ ID NO: 12: base sequence), transcription factor MEF-2A belonging to myocyte enhancer factor-2 (MEF-2) family 1 (SEQ ID NO: 13: amino acid sequence, SEQ ID NO: 14: base sequence), MEF-2B (SEQ ID NO: 15: amino acid sequence, SEQ ID NO: 16: base sequence), MEF-2C (SEQ ID NO: 17: amino acid sequence, SEQ ID NO: 18: base sequence) and MEF-2D (SEQ ID NO: 19: amino acid sequence, SEQ ID NO: 20: base sequence) ), Belonging to the basic helix loop helix-type transcription factor.
  • GATA4 Zinc finger transcription factor GATA4
  • MEF-2A belonging to myocyte enhancer factor-2 (MEF
  • DHAND (SEQ ID NO: 21: amino acid sequence, SEQ ID NO: 22: nucleotide sequence) and eHAND (SEQ ID NO: 23: amino acid sequence, SEQ ID NO: 24: nucleotide sequence), TEA- TE belonging to DNA-binding transcription factor family 1 F-1 (SEQ ID NO: 25: amino acid sequence, SEQ ID NO: 26: nucleotide sequence), TEF-3 (SEQ ID NO: 27: amino acid sequence, SEQ ID NO: 28: nucleotide sequence) and TEF-5 (SEQ ID NO: 29: amino acid sequence, sequence) No. 30: base sequence).
  • the myocardial agents include those containing a gene for a myocardial differentiation-inducing factor as a main component and those containing a protein that is the main body of a myocardial differentiation-inducing factor.
  • the cardiomyogenic agent of the present invention contains a gene encoding a myocardial differentiation inducing factor as a main component below. The preparation method in this case will be described.
  • a recombinant virus vector Yuichi Plasmid is constructed by inserting a gene DNA fragment or a full-length cDNA of a cardiomyocyte differentiation-inducing factor into the virus vector downstream of Promote One. .
  • the recombinant virus vector-plasmid is introduced into a packaging cell suitable for the virus vector-plasmid.
  • any cell can be used as long as it can supply the protein deficient in recombinant virus vector plasmid which lacks at least one gene encoding a protein necessary for viral packaging. Those can also be used.
  • HEK293 cells derived from human kidney, mouse fibroblast NIH3T3, and the like can be used.
  • Proteins to be supplied by the packaging cells include gag, pol, and env derived from mouse retrovirus in the case of a retrovirus vector, and gag, po env, vpr, and vpu derived from an HIV virus in the case of a lentivirus vector. , Vif, tat, rev, nef, etc., proteins such as E1A and E1B derived from adenovirus in the case of adenovirus vector, Rep (p5, p19, p40), Vp (Cap ) Can be used.
  • a viral vector plasmid that contains a promoter at a position where recombinant viruses can be produced in the above-mentioned packaging cells and a wild-type gene corresponding to a gene causing cardiac congenital genetic disease can be transcribed in cardiomyocytes is used. Used.
  • Viral vector plasmids include MFG [Proc. Natl. Acad. Sci. USA, 92, 6733-6737 (1995)], pBabePuro [Nucleic Acids Research, 18, 3587-3596 (1990)], LL_CG, CL-CG ⁇ CS-CG, CLG [Journal of Virology, 72, 8150-8157 (1998)], pAdexl [Nucleic Acids Res., 23, 3816-3821 (1995)] and the like are used.
  • any promoter can be used as long as it can be expressed in human tissues.
  • the promoter of the cytomegalovirus (human CMV) IEGmmediate early) gene and the early promoter of SV40 can be used.
  • Retrovirus Promo One, Meta Mouth Choonein Promo One, Heat Shock Protein Promoter, SR Promo One Etc. can be given.
  • the enhancer of the IE gene of human CMV may be used together with the promoter.
  • a cardiomyocyte-specific gene such as the Nkx2.5 / Csx gene, the target gene can be specifically generated in the cardiomyocyte.
  • a recombinant virus vector By introducing the above-mentioned recombinant virus vector plasmid into the above-mentioned packaging cells, a recombinant virus vector can be produced.
  • a method for introducing the viral vector plasmid into the packaging cells include a calcium phosphate method [Japanese Patent Laid-Open No. 2-227075] and a lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)]. Etc. can be given.
  • the above-described recombinant virus vector can be mixed with a base used for a gene therapy agent to produce a cardiomyogenic agent [Nature Genet., 8, 42 (1 "4)].
  • a base used for a gene therapy agent to produce a cardiomyogenic agent
  • Any base can be used as long as it is commonly used for injections, for example, distilled water, salt solutions such as sodium chloride or sodium chloride and a mixture of inorganic salts, mannitol, and lactose.
  • osmotic pressure adjusting agent Using injections as solutions, suspensions, and dispersions, using PH adjusters, vegetable oils such as sesame oil and soybean oil, or auxiliaries such as surfactants such as lecithin or non-ionic surfactants. These injections can also be prepared as preparations for dissolution at the time of use by operations such as pulverization, freeze-drying, etc.
  • the above-mentioned myocardium-forming agent can be used in the form of a solid as it is in the case of a liquid.
  • the myocardium-forming agent of the present invention can be dissolved in the above-mentioned sterilized base immediately before treatment and used for gene therapy if necessary. As described above, a method of local administration using a catheter or the like is used.
  • the above-described recombinant virus vector can be prepared as the above-mentioned cardiomyogen and then administered to a patient after infecting bone marrow cells having the ability to isolate the cardiomyocytes in a test tube. Alternatively, the recombinant viral vector can be administered directly to the affected area of the patient.
  • the cardiomyogenic agent of the present invention contains a cardiac differentiation-inducing factor protein as a main component The law is described.
  • a DNA fragment of an appropriate length containing a portion encoding the protein is prepared.
  • the recombinant expression vector of the protein is constructed by inserting the DNA fragment or the full-length cDNA downstream of the promoter in the expression vector.
  • the recombinant expression vector is introduced into a host cell compatible with the expression vector.
  • any capable of expressing the target DNA can be used, and for example, genus Escherichia, genus Serratia, genus Corynebacterium, genus Brevibacterium , Bacteria belonging to the genus Pseudomonas ill genus Bacillus, genus Microno 'icrobacterium, genus Kluyveromyces, genus Saccharomyces f Saccharomyces, genus Shisosaccharomyces omnibus Yeast, animal cells, insect cells and the like belonging to the genus Ron (Trichosporon), the genus Schwanniomyces and the like can be used.
  • Ron Trichosporon
  • those which can be replicated autonomously in the above-mentioned host cells or can be integrated into the chromosome, and which contain a promoter at a position where the gene DNA of the cardiomyocyte differentiation-inducing factor can be transcribed are used.
  • the recombinant expression vector of the cardiomyocyte differentiation-inducing factor is capable of autonomous replication in the bacterium, and at the same time, induces differentiation into a promoter, a ribosome-binding sequence, and a myocardial cell. It is preferably a recombinant expression vector composed of a DNA encoding a possible protein and a transcription termination sequence. A gene that controls a promoter may be included.
  • Expression base Kuta one, for example, pBTrp2, pBTacl, P BTac2 (both Beringa - ⁇ from Mannheim), pKK233 - 2 (Amersham Pharmacia Biotech Co.), (manufactured by Invitrogen Corp.) pSE280, pGEMEX-1 (Promega PQE-8 (manufactured by QIAGEN), pKYPIO [Japanese Patent Laid-Open No. 58-110600], pYP200 [Agricultural Biological Chemistry, 48, 669 (1984)], pLSAl [Agric. Biol. Chem., 53, 277] (1989)], pGELl [Proc. Natl. Acad. Sci.
  • the expression vector includes the ribosome binding sequence Shine-Dalgarno.
  • (Dalgarno) sequence and the initiation codon are preferably adjusted to an appropriate distance (for example, 6 to 18 bases).
  • the promoter may be any promoter that can be expressed in the host cell.
  • two sets of P trp are connected in series. Promo one night (P trp x 2), tac promo one
  • Improving the production rate of the target protein by substituting the nucleotide sequence of the portion of the gene encoding the protein of the gene for the cardiomyocyte differentiation-inducing factor gene of the present invention so as to be an optimal codon for expression in the host. Can be done.
  • a transcription termination sequence is not always necessary for the expression of the gene DNA of the cardiac differentiation-inducing factor of the present invention, but it is preferable to arrange the transcription termination sequence immediately below the structural gene.
  • host cells include microorganisms belonging to the genus Escherichia, Serratia, Corynebacterium, Plevibacterium, Pseudomonas, Bacillus, Microbacterium, etc., such as Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1 ⁇ Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coli W1485 Escherichia coli JM109 Escherichia coli HB101, Escherichia coli No.49, Escherichia coli W3110, Escherichia coli NY49 N Bacillus subtilis s Bacillus amvloliquefaciens N Brevibacterium am
  • acetoacidophilum ATCC13870 Microbacterium ammoniaphilum ATCC15354 N Pseudomonas sp. D-0110 and the like.
  • Any method for introducing a recombinant vector can be used as long as it is a method for introducing DNA into the above-mentioned host cells.
  • a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 ( 1972)], the protoplast method (JP-A-63-248394), or the method described in Gene, 17, 107 (1982) or Molecular & General Genetics, 168. Ill (1979).
  • examples of expression vectors include YEpl3 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, and pHS15.
  • Any promoter can be used as long as it can be expressed in yeast.
  • PH05 promoter overnight PGK promoter, GAP promoter overnight, ADH promoter overnight, gal promoter, gal promoter 10 Promoter, Heat Shock Protein Promoter-, MF 1 Promoter, CUP 1 Promoter, etc.
  • the host cells include Saccharomyces f Saccharomyces cerevisiae, Schizosaccharomyces pombe ⁇ ⁇ ⁇ ⁇ ⁇ ... ⁇ ⁇ ⁇ ⁇ ⁇ , ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇
  • expression vectors include, for example, pcDNAKlnvitrogen), pcDM8 (Invitrogen), pAGE107 [JP-A-3-22979; Cytotechnology, 3, 133 (1990)], pAS3 -3 (Japanese Patent Laid-Open No. 2-227075), pCDM8 [Nature, 329, 840 (1987)], pcDNAI / Amp (Invitrogen), pREP4 (Invitrogen), pAGE103
  • any promoter that can be expressed in animal cells For example, the promoter of the IE (immediate early) gene of the cytomegalovirus (human CMV), the early promoter of the SV40, the promoter of the retrovirus, the metamouth thionine promoter, the heat shock protein promoter, etc. In the evening, SR promoters can be mentioned.
  • the enhancer of the IE gene of human CMV may be used together with the promoter.
  • Examples of the host cells include Namalwa cells, which are human cells, COS cells, which are monkey cells, CHO cells, which are Chinese hamster cells, and HBT5637 [Japanese Patent Application Laid-Open No. 63-299]. .
  • any method capable of introducing DNA into animal cells can be used.
  • the electroporation method [Cytotechnology,, 133 (1990) 3, calcium phosphate Law (JP 2 - 227 075), lipoic Hue transfection method
  • the transformant can be obtained and cultured according to the method described in JP-A-2-227075 or JP-A-2-257891.
  • insect cells When insect cells are used as hosts, for example, Baculovirus Expression Vectors, A Laboratory, Manual, WH Freeman and Company, New York (1992)], Current ⁇ Protoco — Ruth 'In' Molecular 'Biology Supplement 1-38 (1987-1997),
  • Proteins can be expressed by the method described in Bio / Technology, 6, 47 (1988) and the like.
  • the recombinant gene transfer vector and the baculovirus are co-transfected into insect cells to obtain the recombinant virus in the insect cell culture supernatant, and then the recombinant virus is infected to the insect cells to express the protein. be able to.
  • the gene transfer base Kuta one used in the method can be exemplified P VL1392, P VL1393, pBlueBacIII (both manufactured by Invitrogen Corp.).
  • baculovirus for example, an autographa califomica nuclear polyhedro.sis virus or the like, which is a virus infecting night moth insects, can be used.
  • Insect cells include Sf9 and Sf21 which are ovarian cells of Spodoptera frugiperda [Baculovirus Expression Vectors, A La oratory Manual ⁇ WH Freeman and Company, New York, (1992)] ⁇ High 5 which is an ovarian cell of Trichoplusia (Invitrogen Manufactured) can be used.
  • Examples of a method for co-introducing the above-described recombinant gene introduction vector and the above baculovirus into insect cells for preparing a recombinant virus include a calcium phosphate method [Japanese Patent Laid-Open No. 2-227075] and a lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)].
  • sugar or sugar chain-added protein When expressed by yeast, animal cells or insect cells, a sugar or sugar chain-added protein can be obtained.
  • the transformant having DNA is cultured in a medium to produce and accumulate a protein capable of inducing myocardial differentiation into the cardiomyocyte in the culture, and the protein is collected from the culture to obtain cardiomyocytes.
  • a protein capable of inducing differentiation can be produced.
  • a method for culturing a transformant for producing a protein capable of inducing differentiation into a cardiomyocyte in a medium can be performed according to a usual method used for culturing a host.
  • a culture medium for culturing a transformant obtained by using a prokaryote such as Escherichia coli or a eukaryote such as yeast as a host contains a carbon source, a nitrogen source, an inorganic substance, etc. which can be utilized by the host. Either a natural medium or a synthetic medium can be used as long as the medium can be cultured efficiently.
  • the carbon source may be any one that can be assimilated by each host, such as glucose, fructose, sucrose, molasses containing them, carbohydrates such as starch or starch hydrolysate, and organic acids such as acetic acid and propionic acid. Alcohols such as ethanol, propanol, etc. can be used.
  • Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, and ammonium acetate Ammonium salts of various inorganic or organic acids such as ammonia and ammonium phosphate, and other nitrogen-containing compounds, as well as peptone, meat extract, yeast extract, corn starch, casein hydrolyzate, soybean meal, and soybean meal Hydrolysates, various fermentation cells and digests thereof are used. ,
  • potassium potassium phosphate potassium potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, and the like can be used.
  • Cultivation is performed under aerobic conditions such as shaking culture or deep aeration stirring culture.
  • the culture temperature is
  • the temperature is preferably 15 to 40 ° C, and the culture time is usually 16 hours to 7 days.
  • the pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.
  • an antibiotic such as ampicillin-tetracycline may be added to the medium during the culture.
  • an Indian user may be added to the medium, if necessary.
  • an Indian user may be added to the medium, if necessary.
  • culturing a microorganism transformed with an expression vector using lac promoter overnight use isopropyl-1 /?-D-thiogalactovyranoside (IPTG) or the like, and use trp promoter overnight.
  • IPTG isopropyl-1 /?-D-thiogalactovyranoside
  • trp promoter trp promoter overnight.
  • IAA indoleacrylic acid
  • Culturing is carried out usually pH6 ⁇ 8, 30 ⁇ 40 ° C, 5 % C0 2 present 1 to 7 days under conditions such as lower.
  • Culture media for transformants obtained using insect cells as host cells include commonly used TNM-FH media (Pharmingen), Sf-900II SFM media (Life Technologies), ExCell400, ExCell405 (all manufactured by JRH Biosciences), Grace's Insect Medium [Grace, TCC, Nature, 195, 788 (1962)] and the like can be used.
  • Cultivation is usually carried out for 1 to 5 days under conditions such as ⁇ to 7, 25 to 30 ° C.
  • an antibiotic such as genyumycin may be added to the medium during the culture.
  • a normal protein isolation and purification method may be used.
  • a protein capable of inducing shunting into cardiomyocytes is expressed in a dissolved state in the cells
  • the cells are recovered by centrifugation after suspension of the culture, suspended in an aqueous buffer, and then sonicated.
  • the cells are disrupted using a crusher, French press, Mentongaulin homogenizer, Dynomill, etc. to obtain a cell-free extract.
  • a normal protein isolation and purification method that is, a solvent extraction method, a salting out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Anion exchange chromatography using a resin such as getylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Chemical Corporation), and a cation using a resin such as S-Sepharose FF (manufactured by Amersham Pharmacia Biotech) Exchange mouth chromatography, hydrophobic chromatography using resins such as butyl sepharose and phenylsepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, isoelectric point
  • a purified sample can be obtained by using an electrophoresis method such as electrophoresis alone or in combination.
  • the cells When the protein is expressed by forming an insoluble form in the cells, the cells are collected, crushed, and centrifuged to collect the protein insoluble form as a precipitate fraction.
  • the recovered insoluble form of the protein is solubilized with a protein denaturant.
  • the structure of the protein is returned to a normal three-dimensional structure.
  • a purified sample of the protein is obtained by the production method.
  • a derivative such as the protein or a sugar chain-adduct thereof is obtained from the culture supernatant.
  • the culture supernatant is collected from the culture by a technique such as centrifugation, and a purified sample can be obtained from the culture supernatant by using the same isolation and purification method as described above.
  • Examples of the protein thus obtained include, for example, proteins having the amino acid sequences represented by SEQ ID NOs: 5, 6, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30. I can give it.
  • the protein expressed by the above method can also be produced by a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl) method, the tBoc method (t-butyloxycarbonyl method), and the like. .
  • a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl) method, the tBoc method (t-butyloxycarbonyl method), and the like.
  • a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl) method, the tBoc method (t-butyloxycarbonyl method), and the like.
  • a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl) method, the tBoc method (t-butyloxycarbonyl method), and the like.
  • a protein capable of inducing differentiation into cardiomyocytes can be used to form and use a cardiomyogen in the same manner as in (1) above.
  • diseases that cause heart failure there is a group that causes heart failure due to partial deletion of all necessary proteins due to mutation of a single gene.
  • diseases include familial hypertrophic cardiomyopathy, Fabri disease, long QT syndrome, Marfan syndrome, aortic stenosis, mitochondrial cardiomyopathy, and Duchenne muscular dystrophy.
  • diseases are known to be caused by genetic abnormalities such as myosin, troponin, tropomyosin, voltage-gated Na channel, K channel, fibrin, elastin, mitochondria, and dystrophin [Therapeutics, ⁇ , O 2 -.
  • the antibody of the present invention which recognizes a surface antigen specifically expressed on bone marrow cells capable of differentiating into cardiomyocytes, is capable of differentiation into cardiomyocytes necessary for performing cell therapy for heart diseases such as myocardial infarction. It can be used for purity test and purification of bone marrow cells having the ability.
  • bone marrow cells capable of differentiating into cardiomyocytes of the present invention 3 to 5 ⁇ 10 5 cell S / animal, or a cell membrane fraction l to 10 mg / animal prepared from the cells as an antigen
  • a suitable adjuvant subcutaneously, intravenously or intraperitoneally in non-human mammals such as rabbits, goats, or 3-20 week old rats, mice or hams, [Complete Freund's Adjuvant) or aluminum hydroxide gel, pertussis vaccine, etc.].
  • the administration of the antigen is performed 3 to 10 times every 1 to 2 weeks after the first administration. Blood is collected from the fundus venous plexus 3 to 7 days after each administration, and it is determined whether the serum reacts with the antigen used for immunization by enzyme immunoassay [enzyme immunoassay (ELISA): published by Medical Shoin. I 976 , Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory, 1988].
  • enzyme immunoassay enzyme immunoassay
  • a non-human mammal whose serum shows a sufficient antibody titer against the antigen used for immunization is used as a source of serum or antibody-producing cells.
  • a polyclonal antibody can be prepared by separating and purifying the serum.
  • a monoclonal antibody is prepared by fusing the antibody-producing cells with myeloma derived from a non-human mammal to produce a hybridoma, and culturing the hybridoma or administering it to an animal to cause the animal to develop ascites carcinoma. It can be prepared by separating and purifying the culture solution or ascites fluid.
  • spleen cells As antibody-producing cells, spleen cells, lymph nodes, and antibody-producing cells in peripheral blood, particularly splenocytes, are suitably used.
  • Myeloma cells include 8-azaguanine-resistant mouse (derived from BALB) myeloma cell line P3-X63Ag8-U1 (P3-U1) strain [Current Topics in Microbiology andlmmunology, 18, 1 (1978)], P3- NSl / 1—Ag41 (NS-1) strain [European J. Immunology, 6, 511 (1976)] ⁇ SP2 / 0-Agl4 (SP-2) strain [Nature, 276, 269 (1978)], P3-X63 -Ag8653 (653) strain [J. Immunology. 123, 1548 (1979)], and mouse-derived cell lines such as P3- ⁇ 3-Ag8 (X6 strain [Nature, 256, 495 (1975)]) are preferably used.
  • P3-X63Ag8-U1 P3-U1 strain [Current Topics in Microbiology andlmmunology, 18, 1 (1978)]
  • Hybridoma cells can be prepared by the following method.
  • Antibody-producing cells and myeloma cells are mixed, suspended in HAT medium (medium containing hypoxanthine, thymidine, and aminopterin in normal medium), and cultured for 7 to 14 days.
  • HAT medium medium containing hypoxanthine, thymidine, and aminopterin in normal medium
  • a portion of the protein is selected from those that react with the antigen but do not react with the protein that does not contain the antigen by enzyme immunoassay or the like.
  • the cells are cloned by the limiting dilution method, and those having a stable and high antibody titer determined by the enzyme immunoassay are selected as monoclonal antibody-producing hybridoma cells.
  • Methods for separating and purifying polyclonal or monoclonal antibodies include centrifugation, ammonium sulfate precipitation, caprylic acid precipitation, or DEAE-Sepharose column, anion exchange column, protein A or G-column or gel filtration column. Chromatography, etc. using these methods alone or in combination.
  • the antibody obtained by the above method and specifically recognizing a surface antigen expressed on bone marrow cells capable of differentiating into cardiomyocytes, the reactivity to sample cells and the hematopoietic stem cells, neural stem cells, etc. By comparing the reactivity with the control cells, it can be easily determined whether or not the sample cells express the above-mentioned specific surface antigen.
  • a subtraction method which is a method for obtaining genes having different expression forms between two samples of different origins, Proc. Natl. Acad. Sci. USA 85, 5738-5742 (1988)] and Representational difference analysis [Nucleic Acids Research, 22, 5640-5648 (1994)].
  • subtraction is performed using a cDNA library prepared from bone marrow cells having the ability to divide into cardiomyocytes using mRNA obtained from control cells other than bone marrow cells such as hematopoietic stem cells and neural stem cells.
  • a differential cDNA library enriched for bone marrow cell-specific genes capable of differentiating into cardiomyocytes
  • the ⁇ C DNA sequence randomly from 5 5 side performs the sequence analysis, selecting only those with the secretion signal sequence. By determining the full-length nucleotide sequence of the cDNA thus obtained, it is possible to distinguish whether the protein encoded by the cDNA is a secretory protein or a membrane protein.
  • the signal sequence trap method is a method for selectively screening genes having a secretory signal sequence.
  • a signal sequence trap library was prepared using a vector capable of performing subtraction, and a signal sequence prepared from bone marrow cells having the ability to divide myocardial cells.
  • a preferred method is to perform trap subtraction using mRNA obtained from control cells such as hematopoietic stem cells and neural stem cells.
  • the DNA fragment containing the secretory signal sequence thus obtained can be used as a probe for cloning full-length cDNA.
  • a synthetic peptide is prepared based on the amino acid sequence inferred from the nucleotide sequence, and the synthetic peptide is synthesized.
  • a specific antibody can be obtained by the above method.
  • a membrane protein some of them encode a receptor, and such a receptor is used to regulate the specific proliferation of bone marrow cells capable of differentiating into the cardiomyocytes or the regulation of differentiation into cardiomyocytes. It may be working and can be used to search for ligands for the receptor.
  • a secretory protein it can be used for directly proliferating or differentiating bone marrow cells capable of differentiating into cardiomyocytes.
  • Bone marrow cell growth factor and cardiomyocyte differentiation inducer capable of cardiomyocyte differentiation As a screening method, when culturing bone marrow cells capable of differentiating into cardiomyocytes in a serum-free medium, various substances as specimens are added, and the cells proliferate or differentiate into cardiomyocytes. It can be done by examining whether it is induced.
  • the substance to be the specimen may be any substance such as secreted proteins such as various cytokines and growth factors, membrane-bound proteins such as cell-adhered molecules, tissue extracts, synthetic peptides, synthetic compounds, and microbial culture solutions.
  • the proliferative ability can be examined based on the ability to form the ostium and the uptake of BrdU.
  • the colony forming ability can be examined by seeding the bone marrow cells of the present invention at a low density.
  • BrdU incorporation can be examined by immunostaining using an antibody that specifically recognizes BrdU.
  • Cardiomyocyte differentiation can be evaluated by using autonomic pulsation as an index or by promoting genes specifically expressed in muscle cells and GFP (Gleen fluorescent protein), Luciferase, A method in which the expression of a reporter gene is used as an index by using a reporter cell in which a DNA combined with a reporter gene such as galactosidase is introduced into bone marrow cells capable of differentiating the cardiomyocytes. Is raised.
  • GFP Green fluorescent protein
  • Luciferase Green fluorescent protein
  • TERT gene is a catalytic Sapuyunitto of Teromera Ichize, DNA specifically represented by SEQ ID NO 3 2, the bone marrow Hosohi having minute I spoon ability to cardiomyocytes after introducing a retroviral vector Transfection method, or into bone marrow cells that have the potential to differentiate into cardiomyocytes
  • a method of administering a factor capable of inducing and expressing an endogenous TERT gene to bone marrow cells capable of differentiating into cardiomyocytes or a method of administering a vector containing DNA encoding a factor capable of inducing and expressing TERT gene to bone marrow capable of differentiating into cardiomyocytes
  • Examples of the method include introduction into cells.
  • the factor that induces and expresses the TERT gene is obtained by combining TERT gene promoter and GFP (Green Fluorescent protein), luciferase or galactosidase in bone marrow cells that have the potential to differentiate into cardiomyocytes. Selection can be achieved by introducing the repo overnight system into bone marrow cells capable of differentiating into cardiomyocytes.
  • GFP Green Fluorescent protein
  • Example 1 Obtaining and culturing bone marrow cells capable of differentiating into cardiomyocytes from mouse bone marrow
  • mice Ten 5-week-old C3H / He mice were anesthetized with ether and then killed by cervical dislocation. The mice were placed in a semi-lateral position and disinfected with 70% ethanol. Next, the skin around the femur was incised over a wide area, and the quadriceps femoris over the entire femur was removed with scissors. Light scissors were inserted into the knee joint, the joint was removed, and the muscles behind the femur were resected. A scissor was placed in the hip joint, the joint was removed, and the femur was removed. Muscles attached to the femur were excised with scissors to expose the entire femur.
  • bone marrow-derived primary immortalized cells After culturing under the above conditions for about 4 months and selecting immortalized cells, dilution was used to establish 192 independent single cell-derived cell lines (hereinafter referred to as bone marrow-derived primary immortalized cells). Stocks). After culturing for 24 hours after adding 5-aza-C to each of these independent clone-derived cells to a final concentration of 3 mM, culturing is continued for another 2 weeks by replacing the medium with IMDM medium. A cell producing clone was selected. Primary bone marrow-derived Of the 19 2 immortalized cells, 3 were bone marrow cells capable of differentiating into cardiomyocytes.
  • the percentage of autologous beating cells that non-specifically induce differentiation from pluripotent stem cells is very small.
  • the surrounding area of the self-pulsating cells was collected with a closing syringe, and 5-aza-C was added again to the cultured cells (hereinafter simply referred to as myocardial progenitor cells) and cultured for 24 hours. More autonomously pulsating cells were obtained by further culturing for 2 to 3 weeks instead of IMDM medium.
  • the bone marrow stem cells capable of differentiating into cardiomyocytes exhibit a mononuclear fibroblast-like morphology under proliferating conditions, and hardly express cardiac contractile proteins.
  • 5 - aZaC the morphology changed significantly.
  • RNA was obtained from each of the bone marrow-derived primary immortalized cell lines obtained in Example 1, and possibly cardiomyocytes differentiated from immature stem cells and cardiac progenitor cells, using Trizol Reagents (GIBCO BRL). Next, using the total RNA as a substrate, Supersciptll reverse
  • First strand cDNA was synthesized using transcriptase (GIBCO BRL).
  • Cardiomyocyte-specific genes include ANP, a natriuretic peptide And BNP, myosin heavy chain -MHC and /?-MHC, actin -skeletal actin and /?-Skeletal actin, myosin light chain MLC-2a, MLC-2v, cardiomyocyte-specific transcription factor
  • ANP a natriuretic peptide And BNP
  • myosin heavy chain -MHC and /?-MHC actin -skeletal actin and /?-Skeletal actin
  • myosin light chain MLC-2a MLC-2v
  • cardiomyocyte-specific transcription factor Some Nkx2.5 / Csx, GATA4, TEF-1, MEF-2C, MEF-2D, MEF-2A were used.
  • MEF-2D for amplification of the synthetic DNAs having the nucleotide sequences of SEQ ID Nos. 57 and 58
  • MEF-2A for the amplification of the nucleotide sequences of SEQ ID Nos. 59 and 60 was used.
  • Cardiomyocytes that are induced to differentiate in vivo may contract in fetal, neonatal, or maturation phases, or in atrial or ventricular muscles to create differences in heart rate or energy efficiency of myocardial contraction. There are differences in protein isoforms.
  • natriuretic peptides ANP and BNP
  • myocardial contractile protein the phenotype of bone marrow cells differentiated into cardiomyocytes in the culture system is considered to have the characteristics of fetal ventricular myocytes.
  • the bone marrow cells were separated I spoon into cardiomyocytes in culture systems, Nkx 2. 5 / Csx, GATA4, MEF-2A, MEF-2C, Expression of MEF-2D and TEF-1 genes was observed. Expression of these transcription factors in bone marrow-derived primary immortalized cell lines in proliferation was not observed, but the expression of Nkx2.5 / Csx GATA 4 and MEF-2 C in bone marrow-derived myocardial progenitor cells in proliferation It was observed, with the induction of differentiation into cardiac myocytes, induction of the expression of late MEF-2 a and MEF-2D were observed.
  • the action potential of the bone marrow cells separated into the cardiomyocytes in the culture system was recorded by a glass microelectrode.
  • the action potential is measured at 25 ° C under a Diaphoto-300 stereo microscope (Nikon) by culturing cells in IMDM medium supplemented with 1.49 mM CaCl 2 , 4.23 mM KC, 25 mM HEPES (pH 7.4). did.
  • the glass electrode was filled with 3M KC1 with the electrode resistance set to 15 to 30 ⁇ .
  • the membrane potential was measured in a current clamp mode using MEZ-8300 (manufactured by Nihon Kohden Corporation). The measurement results were recorded on thermal paper using RTA-1100M (manufactured by Nihon Kohden Corporation).
  • sinus node cell type two types differentiated into cardiomyocytes in the culture system were observed: sinus node cell type and ventricular myocyte type.
  • the characteristics of action potentials common to both are: (1) a long action potential duration, (2) a relatively shallow resting potential, and (3) a gradual depolarization of the resting potential seen in pacemaker cells. there were.
  • the action potential In the ventricular myocyte cell type, the action potential exhibited a peak & dome type (having the first phase of action potential).
  • the action potential duration, diastolic membrane potential, and action potential amplitude of the sinus node cell type were similar to those of the sinus node reported in the past.
  • the resting membrane potential tended to be deep and the action potential amplitude tended to be large.
  • the cells of the sinus node cell type were recorded in all the cells for 2 to 3 weeks, but the ventricular myocyte type was observed from about 4 weeks after the induction of differentiation, and gradually increased over time.
  • Mouse bone marrow cells having the ability to divide into cardiomyocytes were seeded at 2 ⁇ 10 4 cells / ml on eOmm culture dish or 60 mm fibronectin-coated dish (Fibronectin-coated dish: Becton Dickinson), and 33 ° C, having conducted a cultured using 5% C0 2 concentration in the incubator unit.
  • the medium was exchanged again, and the same amount as the first group was added to each of the three groups without PDGF alone, both PDGF and retinoic acid, and no addition.
  • the morphology of the cells was observed under a phase-contrast microscope.
  • about 30% of cells in cultured tissue containing only 5-aza-C became myotube-like cells, whereas about 40% when PDGF was added, and about 50% when PDGF and retinoic acid were added simultaneously.
  • Some cells became myotube-like cells.
  • the number of cells that became myotube-like cells increased by about 10% in the three groups with the fibronectin-attached dish compared to the three groups with the culture dish.
  • Example 4 Induction of differentiation of bone marrow-derived stem cells into cardiomyocytes using DMSQ
  • 10 ⁇ M DMSO was added to the obtained bone marrow stem cells capable of differentiating into cardiomyocytes instead of 3 ⁇ M 5 -aza-C, and the cells were cultured for 24 hours. Was replaced with IMDM medium, and the culture was continued for another 6 weeks.
  • beating cardiomyocytes were induced to differentiate, and these cells expressed Nkx2.5 / Csx and GATA4 genes, and exhibited the same properties as when 5-aza-C was added. It was shown to be a cardiomyocyte that had.
  • the results of this analysis indicate that chromosomal DNA demethylation, a common function of 5-aza-C and DMSO, is required for cardiomyocyte differentiation.
  • Example 5 Demonstration that mouse bone marrow cells capable of dividing into cardiomyocytes are multipotent stem cells and cardiomyocyte precursor cells.
  • one cell is labeled by introducing the GFP gene into the virus vector, and then differentiation is induced to determine the type of cells that the labeled cell has differentiated into. It was judged.
  • a retrovirus vector plasmid expressing the GFP gene, GAR3-GFP, and a pCMV-Eco plasmid vector expressing the Ecotropic gene were obtained from Molecular Cionmg, A Laboratory Manual, second Edition, Cold Spring Harbor Laooratory Press (1989), etc. Highly pure DNA was obtained using the neutralization method and the PEG precipitation method described in Section c. The day before transfection of the DNA, the confluent gag and pol genes were retained. The cells were subcultured to a 10 cm dish at a dilution of 1/5, and cultured using an incubator having a concentration of 37 ° C. and 5% CO 2 .
  • the transfection was conducted as follows.
  • GAR3-GFP retroviral vector one plasmid DNA 1 5 ⁇ g and pCMV-Eco plus Midobekuta one DNA 5 ⁇ g dissolved in addition to 250mM CaCl 2 (pH6.95) 0.5ml, was charged the solution to 15ml tubes 2 X BBS [50mM BES (N , N- bis (2- hydroxyethl) - 2 - aminoethanesulfonic acid), 280mM NaCl ⁇ 1.5mM Na 2 HP0 4 (pH6.95) ] was added dropwise to 0.5ml static at room temperature for 10 minutes Was placed.
  • BBS 50mM BES (N , N- bis (2- hydroxyethl) - 2 - aminoethanesulfonic acid), 280mM NaCl ⁇ 1.5mM Na 2 HP0 4 (pH6.95)
  • the DNA solution was added dropwise to 293 in cell culture medium were prepared the day before, were incubated with 37 ° C, '5% C0 2 concentration in the incubator unit. The next day, the medium was changed, were cultured further using a 37 ° C, 5% C0 2 concentration in the incubator unit.
  • IMDM medium 10-10 The solution in IMDM medium 10-10 one 2,
  • Mouse bone marrow cells capable of differentiating into cardiomyocytes to which the virus is introduced are seeded on a 6-well dish at 2 ⁇ 10 4 cells / well on the day before virus influx.
  • diluted solution containing the virus vector For the diluted solution containing the virus vector, add a final concentration of 8 ⁇ g / ml.
  • the Nkx2.5 / Csx or GATA4 gene was introduced using a viral vector, and then differentiation was induced to examine the efficiency of differentiation into cardiomyocytes.
  • Retroviral vector one plasmid P CLNC-Nkx2.5 / Csx and PCLNC- GATA4 and, pCMV-Eco plasmid base click evening one to express Ecotropic gene (Imgenex Fe) 3 ⁇ 4, Molecular Cloning, A Laboratory Manual, Second Edition, Highly pure DNA was obtained using the neutralization method and the PEG precipitation method described in Cold Spring Harbor Laboratory Press (1989) and the like.
  • pCLNC—Nkx2.5 / Csx or pCLNC—GATA4 15 ⁇ g of retrovirus vector DNA and 5 ⁇ g of pCMV-Eco plasmid vector DNA are added to 0.5 ml of 250 mM CaCl 2 (pH6.95) and dissolved.
  • 2 X BBS 50m BES (N , N- bis (2-hydroxyethl) -2-aminoethanesulfonic acid), 280mM NaCl, 1.5mM Na 2 HP0 4 (pH6.95)] were placed in a tube was dropped into 0.5ml It was left at room temperature for 10 minutes.
  • the DNA solution was added dropwise to 2 ⁇ 3 in cell medium were prepared the day before, were incubated with 37 ° C, 5% C0 2 concentration in the incubator unit. The next day, the medium was changed, were cultured with further 3 7 ° C, 5% C0 2 concentration in the incubator unit.
  • Hexadimethrine bromide (polybrene) (Sigma) was added to the solution containing the virus vector obtained above to a final concentration of 8 ⁇ g / ml, and mouse bone marrow cells capable of differentiating into cardiomyocytes were added. And the culture was performed using an incubator at 33 ° C. and 5% CO 2 concentration. After 5 hours, and replaced with a new MDM medium was cultured 3 ⁇ 4 ⁇ Ryo' with further 3 3 ° C, 5% C_ ⁇ 2 concentration of incubation machine.
  • CS CS was added to a final concentration of 300 g / ml, and culturing was further performed for 3 days. During this time, some cells died and floated. The surviving cells were suspended in trypsin and seeded on new culture dishes.
  • Nkx2.5 / Csx or GATA4 stably transformed cells were induced to differentiate by the method of Example 3 above, and the efficiency of cardiomyocyte differentiation was assayed. As a result, it was found that the forced expression of these transcription factors promoted the efficiency of differentiation into cardiomyocytes.
  • Example 7 Telomerase activity in mouse bone marrow cells capable of differentiating into cardiomyocytes Teguchi-merase activity of mouse bone marrow cells capable of dividing into cardiomyocytes was examined by Telomeric Repeat Amplification Protocol (TRAP) method (Oncor's TRAPeze Telomerase Detection Kit). The measurement of telomerase activity basically followed the attached protocol, and was specifically performed as follows. First, mouse bone marrow cells (approximately 10 6 ) capable of differentiating into cardiomyocytes cultured on a 6 cm diameter culture dish were washed with PBS, added with 1X CHAPS solution, and allowed to stand on ice for 30 minutes. Was placed. Thereafter, the cells in a solution and the co-harvested l.
  • TRAP Telomeric Repeat Amplification Protocol
  • the cell extract of mouse bone marrow cells capable of differentiating into cardiomyocytes obtained under the above conditions was approximately 1 mg / ml.
  • telomere extension reaction was performed according to the protocol.
  • Taq polymerase used was EX Taq polymerase (Takara Shuzo). After the reaction is completed, 1/10 volume of 10X staining solution (0.25% bromophenol blue, 0.25% Xylene cyanol FF, 30% glycerol) is added, and 12.5% polyacrylamide gel (TRAPeze Telomerase Detection Kit protocol) And electrophoresed under a constant voltage of 250 mV. After swimming, the gel was stained with Cyber Green (manufactured by FMC).
  • telomerase activity was detected in a sample having a final concentration of the cell extract of 0.4 to 4 jug / ml.
  • Example 8 Transplantation of mouse bone marrow cells capable of differentiating into cardiomyocytes into the heart.
  • anesthesia was first introduced into C3H / He mice (Charles River Japan, Inc.) using ether and Terumo Anesthesia was maintained by intraperitoneal administration of 30 mg of thiopental using a thermosyringe (1 ml).
  • the limbs of the mouse were fixed to the cork board with tape, and the upper jaw was fixed to the cork board with rubber so that the neck would bend back.
  • electrocardiogram electrodes were inserted into the left and right upper limbs and the right lower limb to monitor the electrocardiogram.
  • the trachea was incised about 1 mm with a microfeather (female), and a Terumo surfro-flash (22G) needle transformed into a J-shape was inserted out of the mouth and taken out of the oral cavity.
  • the outer tube of the flash (G) was inserted into the trachea.
  • a respire overnight (model SN-480-7 manufactured by Shinano Seisakusho) was connected to this outer cylinder, and 100% oxygen was flowed at 1 ml / min.
  • the tidal volume was 1 ml, and the respiration rate was 120 / min. Started.
  • mouse bone marrow cells capable of dividing the transplanted cardiomyocytes were established in the myocardium by extracting the heart of the transplanted mouse, preparing a tissue section, and staining with a GFP-recognizing antibody. .
  • Example 9 Acquisition and culture of bone marrow cells capable of differentiating into cardiomyocytes from rat bone marrow After removing 6 female 5-week-old Wistar rats (Japan SLC Co., Ltd.) from the cervical spine, 70% ethanol was removed. It was disinfected enough. Next, a large incision was made in the skin of the foot, and the femur and tibia were removed while removing the muscle covering the femur and tibia. The removed femur and tibia were transferred to a 10 cm diameter culture dish (manufactured by Iwaki Glass) containing PBS (manufactured by GibcoBRL), and the muscles and joints were completely resected.
  • a 10 cm diameter culture dish manufactured by Iwaki Glass
  • PBS manufactured by GibcoBRL
  • the total number of recovered cells was 2.6 ⁇ 10 9 .
  • 1 ⁇ 10 8 cells were collected from one femur or tibia.
  • the collected cells were diluted to a concentration of 1.3 ⁇ 10 8 cells / ml, and Percoll (manufactured by Amersham Pharmacia Biotech) / D-PBS solution (ml) adjusted to 1.073 g / ml in a 50 ml centrifuge tube After overlaying on top, centrifuged at room temperature at 3,100 rpm; for 30 minutes. After centrifugation, the cells were collected from the interface between the Percoll solution and cell suspension.
  • the bone marrow-derived cells thus fractionated into 3 culture dishes for animal cells with a diameter of 10 cm (Iwaki Glass, hereinafter abbreviated as 10 cm culture dishes) so as to have a size of 2 to 5 x 10 5 cells / cm 2.
  • 10 cm culture dishes The bone marrow-derived cells thus fractionated into 3 culture dishes for animal cells with a diameter of 10 cm (Iwaki Glass, hereinafter abbreviated as 10 cm culture dishes) so as to have a size of 2 to 5 x 10 5 cells / cm 2.
  • 10 cm culture dishes Iwaki Glass, hereinafter abbreviated as 10 cm culture dishes
  • Example 10 Examination of differentiation ability of rat bone marrow-derived cells into cardiomyocytes
  • the confluent rat bone marrow-derived cells were separated again by trypsin EDTA treatment, and the cells were transferred to a 6-well plate (BECTON DICKINSON) in such a manner that 5 x 10 4 cells per 1-well were obtained.
  • the culture dish 6cm diameter coated with Hitofu Eve Lone cutin was reseeded cells to become 1.'3 X 10 5 cells.
  • a bone marrow cell a growth factor, a vitamin, an adhesion molecule, and a method of using the same that are effective for treating a heart disease associated with destruction and degeneration of cardiomyocytes and for searching for a therapeutic agent are provided.
  • SEQ ID NO: 4 1 Description of artificial sequence: Synthetic DNA
  • SEQ ID NO: 46 Description of artificial sequence: Synthetic DNA

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Rheumatology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Surgery (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Cardiology (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne : des procédés permettant d'isoler, purifier, cultiver des cellules de moelle osseuse capables de se différencier en cellules de muscle cardiaque et d'induire cette différenciation ; un procédé permettant de multiplier les cellules de moelle osseuse capables de se différencier en cellules de muscle cardiaque à l'aide de diverses cytokines, facteurs de transcription, etc. ; un procédé permettant de réguler la différenciation en cellules de muscle cardiaque ; un procédé permettant d'obtenir un antigène de surface spécifique des cellules de moelle osseuse capables de se différencier en cellules de muscle cardiaque ; un procédé permettant d'obtenir un gène codant l'antigène de surface précité ; un procédé permettant d'obtenir un anticorps spécifique de l'antigène de surface précité ; un procédé permettant d'obtenir une protéine et un gène participant à la prolifération et différenciation en cellules de muscle cardiaque des cellules de moelle osseuse capables de se différencier en cellules de muscle cardiaque ; et des remèdes contre diverses maladies cardiaques faisant appel aux cellules de moelle osseuse capables de se différencier en cellules de muscle cardiaque de l'invention.
PCT/JP2000/001148 1999-12-28 2000-02-28 Cellule de moelle osseuse adulte capable de se differencier en cellule de muscle cardiaque WO2001048149A1 (fr)

Priority Applications (8)

Application Number Priority Date Filing Date Title
AU26950/00A AU2695000A (en) 1999-12-28 2000-02-28 Adult bone marrow-origin cell capable of differentiating into heart muscle cell
PCT/JP2000/007741 WO2001048150A1 (fr) 1999-12-28 2000-11-02 Cellules pouvant se differencier en cellules du muscle cardiaque
AU10552/01A AU1055201A (en) 1999-12-28 2000-11-02 Cells capable of differentiating into heart muscle cells
CA002395950A CA2395950A1 (fr) 1999-12-28 2000-12-27 Cellules pouvant induire une differenciation dans des cellules du muscle cardiaque
EP00985950A EP1254952A4 (fr) 1999-12-28 2000-12-27 Cellules pouvant induire une differenciation dans des cellules du muscle cardiaque
PCT/JP2000/009323 WO2001048151A1 (fr) 1999-12-28 2000-12-27 Cellules pouvant induire une differenciation dans des cellules du muscle cardiaque
AU22281/01A AU784618B2 (en) 1999-12-28 2000-12-27 Cells capable of differentiating into heart muscle cells
US09/749,728 US20020142457A1 (en) 1999-12-28 2000-12-28 Cell having the potentiality of differentiation into cardiomyocytes

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP37282699 1999-12-28
JP11/372826 1999-12-28

Publications (1)

Publication Number Publication Date
WO2001048149A1 true WO2001048149A1 (fr) 2001-07-05

Family

ID=18501111

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2000/001148 WO2001048149A1 (fr) 1999-12-28 2000-02-28 Cellule de moelle osseuse adulte capable de se differencier en cellule de muscle cardiaque

Country Status (2)

Country Link
AU (1) AU2695000A (fr)
WO (1) WO2001048149A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002036772A1 (fr) * 2000-10-30 2002-05-10 Takeda Chemical Industries, Ltd. Nouveau gene associe a une maladie et son utilisation
WO2004000993A3 (fr) * 2002-06-21 2004-04-29 Univ Quebec Montreal Oxytocine utilisee pour induire la cardiomyogenese et utilisations
WO2005082933A1 (fr) * 2004-02-26 2005-09-09 Bml, Inc. Nouvelle protéine chimère, gène codant celle-ci et moyen d'évaluation d'une leucémie utilisant le gène et la protéine

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000006701A1 (fr) * 1998-07-31 2000-02-10 Genzyme Corporation Amelioration du fonctionnement cardiaque par transplantation de cellules souches mesenchymateuses

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000006701A1 (fr) * 1998-07-31 2000-02-10 Genzyme Corporation Amelioration du fonctionnement cardiaque par transplantation de cellules souches mesenchymateuses

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JEFFREY M. LEIDEN.: "Beating the odds: a cardiomyocyte cell line at last", JOURNAL OF CLINICAL INVESTIGATION,, vol. 103, no. 5, March 1999 (1999-03-01), pages 591 - 592, XP002927081 *
TOMITA SHINJI ET AL.: "Autologous transplantation of bone marrow cells improves damaged heart fuction, Biosis No. 200000032267", CIRCULATION,, vol. 110, no. 19, November 1999 (1999-11-01), pages II247 - II256 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002036772A1 (fr) * 2000-10-30 2002-05-10 Takeda Chemical Industries, Ltd. Nouveau gene associe a une maladie et son utilisation
WO2004000993A3 (fr) * 2002-06-21 2004-04-29 Univ Quebec Montreal Oxytocine utilisee pour induire la cardiomyogenese et utilisations
WO2005082933A1 (fr) * 2004-02-26 2005-09-09 Bml, Inc. Nouvelle protéine chimère, gène codant celle-ci et moyen d'évaluation d'une leucémie utilisant le gène et la protéine

Also Published As

Publication number Publication date
AU2695000A (en) 2001-07-09

Similar Documents

Publication Publication Date Title
AU784618B2 (en) Cells capable of differentiating into heart muscle cells
US20020142457A1 (en) Cell having the potentiality of differentiation into cardiomyocytes
WO2006078034A1 (fr) Cellules capables de differenciation en cellules de muscle cardiaque
WO2003087349A1 (fr) Methode de formation de cellules pancreatiques $g(b) a partir de cellules mesenchymales
EP1712616B1 (fr) Technique pour induire la differenciation de cellules de moelle osseuse de mammiferes ou de cellules de sang de cordon ombilical en cellules myocardiques au moyen de tissu adipeux
JP2022024047A (ja) 線維芽細胞治療活性を増強する方法
Jiang et al. Transcriptional profiling of young and old mesenchymal stem cells in response to oxygen deprivation and reparability of the infarcted myocardium
AU757816B2 (en) Conditional immortalisation of cells
US20220401518A1 (en) Mesenchymal stem cell expressing hepatocyte growth factor, and use thereof
US20220401491A1 (en) Mesenchymal stem cell expressing trail and cd, and use thereof
Baccini et al. Role of p21Cip1/Waf1 in cell-cycle exit of endomitotic megakaryocytes
CA2862661A1 (fr) Methodes et compositions permettant l'obtention, a des fins cliniques, d'une cellule allogene et ses utilisations therapeutiques
KR20150104049A (ko) 체세포로부터 혈관 전구 세포로의 직접교차분화 유도용 조성물 및 이의 용도
Huang et al. The migration and differentiation of hUC-MSCsCXCR4/GFP encapsulated in BDNF/chitosan scaffolds for brain tissue engineering
KR102793810B1 (ko) 간세포성장인자를 발현하는 중간엽줄기세포를 유효성분으로 포함하는 혈관계 질환 예방 또는 치료용 약학적 조성물
US20070282456A1 (en) Compositions and Methods for Myogenesis of Fat-Derived Stem Cells Expressing Telomerase and Myocardin
WO2001048150A1 (fr) Cellules pouvant se differencier en cellules du muscle cardiaque
WO2001048149A1 (fr) Cellule de moelle osseuse adulte capable de se differencier en cellule de muscle cardiaque
WO2006116678A2 (fr) Ciblage tissulaire de cellules souches
JP7630132B2 (ja) 巨核球前駆細胞および巨核球細胞の製造方法並びに得られた巨核球前駆細胞および巨核球細胞
JP7144829B2 (ja) 安全性と抗炎症作用を高めた間葉系幹細胞
JP2022516187A (ja) 視力を改善するための方法
CN110551692A (zh) hFGF9基因修饰型间充质干细胞及其制备方法和用途
JP2020058333A (ja) 脳卒中のための神経幹細胞治療
JP2005506090A (ja) 一時的な不死化

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

NENP Non-entry into the national phase

Ref country code: JP

Ref document number: 2001548662

Format of ref document f/p: F

122 Ep: pct application non-entry in european phase
点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载