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WO2001048150A1 - Cellules pouvant se differencier en cellules du muscle cardiaque - Google Patents

Cellules pouvant se differencier en cellules du muscle cardiaque Download PDF

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WO2001048150A1
WO2001048150A1 PCT/JP2000/007741 JP0007741W WO0148150A1 WO 2001048150 A1 WO2001048150 A1 WO 2001048150A1 JP 0007741 W JP0007741 W JP 0007741W WO 0148150 A1 WO0148150 A1 WO 0148150A1
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cells
cell
seq
amino acid
acid sequence
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PCT/JP2000/007741
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Japanese (ja)
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Akihiro Umezawa
Jun-Ichi Hata
Keiichi Fukuda
Satoshi Ogawa
Kazuhiro Sakurada
Satoshi Gojo
Yoji Yamada
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Kyowa Hakko Kogyo Co., Ltd.
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Priority claimed from PCT/JP2000/001148 external-priority patent/WO2001048149A1/fr
Application filed by Kyowa Hakko Kogyo Co., Ltd. filed Critical Kyowa Hakko Kogyo Co., Ltd.
Priority to AU10552/01A priority Critical patent/AU1055201A/en
Priority to EP00985950A priority patent/EP1254952A4/fr
Priority to CA002395950A priority patent/CA2395950A1/fr
Priority to AU22281/01A priority patent/AU784618B2/en
Priority to PCT/JP2000/009323 priority patent/WO2001048151A1/fr
Publication of WO2001048150A1 publication Critical patent/WO2001048150A1/fr

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    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0657—Cardiomyocytes; Heart cells
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Definitions

  • the present invention relates to a method for isolating, purifying, culturing, and inducing differentiation of cells having the ability to differentiate into cardiomyocytes.
  • the present invention also relates to a method for growing cells having the ability to bind to cardiomyocytes and a method for controlling the binding to cardiomyocytes using various cytokines and transcription factors.
  • the present invention further provides a method for obtaining a cell-specific surface antigen capable of differentiating into cardiomyocytes,
  • a method for obtaining a gene encoding the surface antigen a method for obtaining an antibody specific to the surface antigen,
  • the present invention relates to a method for obtaining proteins and genes involved in proliferation of cells capable of differentiating into cardiomyocytes and differentiation into cardiomyocytes.
  • the present invention also relates to a therapeutic agent for various heart diseases using cells capable of differentiating into cardiomyocytes.
  • cardiomyocytes Before birth, cardiomyocytes actively divide while autonomously beating. However, at the time of birth, it loses its division ability, does not acquire cell division ability again like hepatocytes, and unlike skeletal muscle cells, it does not have undifferentiated progenitor cells like satellite cells. Therefore, when cardiomyocytes are necrotic due to myocardial infarction, myocarditis, or aging, in vivo, cell enlargement occurs instead of cell division of remaining cardiomyocytes. Cardiac hypertrophy is a physiological adaptation in the early stage, but also leads to a decrease in diastolic function of the heart itself and a decrease in systolic function, coupled with interstitial fibrosis due to the proliferation of coexisting cardiac fibroblasts.
  • heart failure due to myocardial infarction has been focused on symptomatic treatments such as enhancement of cardiac contractility, reduction of cardiac pressure and volume load by vasodilators, and reduction of blood flow by diuretics. .
  • heart transplantation is a fundamental treatment for severe heart failure.However, due to problems such as a shortage of organ donors, difficulty in determining brain death, rejection, and rising medical costs, heart transplantation has become a standard medical treatment. It is not easy to spread. In fact, heart disease is the third leading cause of death in Japan (Health and Welfare White Paper, 1999), and if we can regenerate lost myocardial cells, it will be a major step forward in medical welfare.
  • cell lines that have preserved the properties of cardiomyocytes include AT- cells that have been established from tumors arising in the atrium of transgenic mice produced by recombining the SV40 large T antigen into the atrial natriuretic hormone promoter. 1 cells, and the like [Science, 239; 1029-1038 flight 9 88).
  • AT- cells that have been established from tumors arising in the atrium of transgenic mice produced by recombining the SV40 large T antigen into the atrial natriuretic hormone promoter. 1 cells, and the like [Science, 239; 1029-1038 flight 9 88).
  • 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. To date, although successful examples have been shown for P19 cells, mouse embryonal cancer cells, [Cell Struc. & Func, 21: 101-110 (1996)] No examples have 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 success has been reported so far.
  • a third method is to induce cardiomyocytes from undifferentiated stem cells. Although it has been shown that cardiomyocytes can be derived 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)].
  • ES cells embryonic stem cells
  • hematopoietic stem cells contains mesenchymal stem cells in addition to hematopoietic stem cells and vascular stem cells. It has been reported that stem cells can induce differentiation of bone cells, chondrocytes, tendon cells, ligament cells, skeletal muscle cells, adipocytes, stomatoma cells, and liver oval cells [Science, 284, 143- 147 (1999); Science, 284, 1168-1170 (1999)] 0 Recently, however, it has been found that cardiomyocytes can be induced to differentiate from cells obtained from adult mouse bone marrow [J. Clinical.
  • Antibodies that recognize various surface antigens have been used as a method for obtaining target cells from tissues in a living body.
  • immature hematopoietic stem cells have the characteristics of CD34 + / CD38-HLA-DR- / CD90 (Thy-1) +, and CD38 is expressed as hematopoietic stem cells differentiate. It is known that CD90 (Thy-1) disappears [Protein nucleic acid enzyme Vol. 45, Nol3, 2056–2062 (2000)].
  • Vascular endothelial cells express markers such as CD34, CD31, F Ik-IT ie-2 and E-selectin [Molecular cardiovascular disease V o 1.
  • bone marrow mesenchymal stem cells express markers such as CD90, CD105 and CD140 [Science, 284, 143-147 (1999); Science, 284, 1168-1170 (1999) ⁇ .
  • markers of stem cells that can induce cardiac muscle and vascular endothelial cells have not been clarified.
  • bone marrow cells having the ability to divide into cardiomyocytes from bone marrow cells and controlling the growth or differentiation of bone marrow cells having the ability to divide into cardiomyocytes requires the use of cells derived from bone marrow. It is useful for the development of regenerative therapy for the heart muscles that were affected. To do this, the cells from bone marrow to cardiomyocytes It is necessary to identify cells capable of metabolism and identify cytokines or transcription factors that act on the proliferation or differentiation of the cells. The present inventor has made intensive studies to develop the above problems and obtained the following results. That is, cells derived from mouse bone marrow were first separated to the level of one cell, and a number of cell lines were obtained.
  • each of these cell lines was treated with 5-azacitidine to obtain a plurality of cell lines having cardiomyogenic ability.
  • the selected cell lines are labeled with a retroviral vector expressing GFP (Green Fluorescent Protein), and one cell is traced under a fluorescence microscope to determine the ability to differentiate into cardiomyocytes.
  • GFP Green Fluorescent Protein
  • the cells having the cells are pluripotent stem cells capable of inducing differentiation of at least two different types of cells such as cardiomyocytes and adipocytes.
  • the stem cells can be stochastically administered 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.
  • bone marrow-derived cells found in the present invention differ from the conventionally known hematopoietic stem cells and mesenchymal stem cells in bone marrow, and that all three germ layers of the ectoderm, mesoderm, and endoderm It shows that it has the totipotency to differentiate into.
  • the cells derived from bone marrow found in the present invention are used as surface antigens for hematopoietic cells, CD34, CD117, CD14, CD45, CD90, Sea-1, Ly6c, an antibody that recognizes Ly6g, and blood vessels.
  • Endothelial cell surface antigen F 1k-1 antibody that recognizes CD31, CD105, CD144, mesenchymal cell surface antigen that recognizes CD140, integrin CD49b, CD49d, CD29s
  • the cell is a pluripotent stem cell having at least the ability to divide into cardiomyocytes, adipocytes, skeletal muscle cells, and osteoblasts.
  • the cell is a pluripotent stem cell having the ability to differentiate into at least cardiomyocytes, adipocytes, skeletal muscle cells, osteoblasts, and vascular endothelial cells.
  • the cell is a pluripotent stem cell having at least the ability to differentiate into cardiomyocytes, adipocytes, skeletal muscle cells, vascular endothelial cells, osteoblasts, nervous cells, and hepatocytes.
  • Cardiac progenitor cells that are induced to differentiate only into cardiomyocytes derived from the cells according to any one of (1) to (13).
  • demethylation of chromosomal DNA is at least one selected from the group consisting of demethylase, 5-azacitidine and dimethyl sulfoxide (DMSO). Cells.
  • the factor described in (23), wherein the factor expressed in the fetal heart development region is at least one selected from the group consisting of cytokines, adhesion molecules, bimin, transcription factors and extracellular matrix. ).
  • 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 cytokins, adhesion molecules, vitamins, transcription factors and extracellular matrix.
  • cytokine is platelet-derived growth factor (PDGF).
  • BMP-4 is BMP-4 having an amino acid sequence represented by SEQ ID NO: 70.
  • 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, TEF-5 and MesPl.
  • MEF-2A is MEF-2A having an amino acid sequence represented by SEQ ID NO: 13.
  • MEF-2B is MEF-2B having an amino acid sequence represented by SEQ ID NO: 15.
  • 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.
  • the factor described above, wherein the factor expressed in the fetal heart development region is at least one selected from the group consisting of cytokin, adhesion molecule, bimin, transcription factor and extracellular matrix.
  • At least one kind of factor that acts on differentiation into cardiomyocytes at the stage of fetal cardiac development is at least one member derived from the group consisting of cytokines, adhesion molecules, vitamins, transcription factors and extracellular matrix. The method according to (71) above.
  • cytokine is PDGF.
  • PDGF is PDGF represented by the amino acid sequence of SEQ ID NO: 3 or 5.
  • BMP-4 is BMP-4 having an amino acid sequence represented by SEQ ID NO: 70.
  • the transcription factor is Nkx2.5 / Csx ⁇ GATA4, MEF-2A, MEF-2B, MEF-2C, MEF-2D, The method according to (70) or (72), wherein the method is selected from the group consisting of dHAND, eHAND, TEF-1, TEF-3, TEF-5 and MesPl.
  • Nkx2.5 / Csx is Nkx2.5 / Csx having an amino acid sequence represented by SEQ ID NO: 9.
  • TEF-1 is TEF-1 having an amino 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.
  • a cardiomyogenic agent comprising a chromosomal DNA demethylating agent as an active ingredient.
  • chromosomal DNA demethylating agent is at least one selected from the group consisting of demethylase, 5-azacitidine and DMSO.
  • a cardiomyogenic agent comprising, as an active ingredient, a factor expressed in a fetal heart development region.
  • the factor according to the above (105), wherein the factor expressed in the fetal heart development region is at least one selected from the group consisting of cytokines, adhesion molecules, vitamin transcription factors and extracellular matrix. Cardiomyogen.
  • a cardiomyogenic agent comprising, as an active ingredient, a factor that acts on differentiation into cardiomyocytes at the stage of fetal heart development.
  • ET1 is an ET1 having an amino acid sequence represented by SEQ ID NO: 66.
  • Transcription factor consists of Nkx2.5 / Csx, GATA4, MEF-2A, MEF-2B ⁇ MEF-2C, MEF-2D, dHAND, eHAND, TEF-1, TEF-3, TEF-5 and MesPl
  • GATA4 is GATA4 represented by the amino acid sequence of SEQ ID NO: 11.
  • MEF-2A is MEF-2A represented by the amino acid sequence set forth in SEQ ID NO: 13.
  • the myocardial agent according to (121) is MEF-2A represented by the amino acid sequence set forth in SEQ ID NO: 13. The myocardial agent according to (121).
  • 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.
  • TEF-5 is TEF-5 represented by the amino acid sequence of SEQ ID NO: 29.
  • extracellular matrix is a cardiomyocyte-derived extracellular matrix
  • a method for regenerating a heart destroyed by a heart disease comprising using the cell according to any one of the above (1) to (65).
  • a therapeutic agent for regenerating heart comprising the cell according to any one of (1) to (65) as an active ingredient.
  • a therapeutic agent for heart disease comprising, as an active ingredient, the cell according to any one of (1) to (65), into which a wild-type gene for a mutant gene in congenital heart disease is introduced.
  • (140) A method for isolating and purifying adult bone marrow-derived cells capable of differentiating human bone marrow into cardiomyocytes, comprising using the antibody obtained by the method according to (139).
  • telomerase 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 (65), which is immortalized by expressing telomerase.
  • telomerase is a telomerase having an amino acid sequence represented by SEQ ID NO: 31.
  • a method for inducing differentiation of the cell according to (1) into a cardiomyocyte comprising using the culture supernatant according to (149).
  • the cells having the ability to differentiate into cardiomyocytes of the present invention include pluripotent stem cells isolated from adult tissues such as bone marrow, muscle, brain, brain, liver, and kidney, or cord blood.
  • the pluripotent stem cells may be any cells capable of inducing cardiomyocytes and other cells, and preferably have at least the ability to differentiate into cardiomyocytes, adipocytes, skeletal muscle cells, osteoblasts, At least cardiomyocytes and vascular endothelial cells.
  • Capable cells at least cardiomyocytes, adipocytes, skeletal muscle cells, osteoblasts, cells capable of differentiating into vascular endothelial cells, at least cardiomyocytes, adipocytes And cells having the ability to differentiate into skeletal muscle cells, vascular endothelial cells, osteoblasts, nervous cells, and hepatocytes.
  • the adult tissue or umbilical cord blood may be of any type as long as it is derived from a mammal, and preferably includes mouse, rat, human and the like. It is preferably of human origin for human therapeutic use.
  • pluripotent stem cells of the present invention not only cardiomyocytes, but also vascular endothelial cells, smooth muscle, skeletal muscle cells, fat cells, bone, cartilage, ⁇ endocrine cells, Teng exocrine cells, hepatocytes, Various cells can be obtained by inducing differentiation into renal glomerular cells, renal tubular cells, neurons, glia, oligodendrocytes, and the like.
  • the cell capable of differentiating into cardiomyocytes of the present invention may be any tissue, such as an adult tissue or cord blood, as long as it can obtain cells capable of differentiating into cardiomyocytes.
  • the method for isolating bone marrow cells having the ability to differentiate into cardiomyocytes from bone marrow is described below.
  • the method is not particularly limited as long as it is a method that can be obtained safely and efficiently, but it can be performed based on the method described in SE Haynesworth et al. Bone, 13, 81 (1 " 2 ).
  • the bone marrow cells After collecting the bone marrow cells from the obtained bone marrow fluid by centrifugation at 1,000 ⁇ g, the bone marrow cells are washed with PBS (Phosphate Buffered Saline). After repeating this step twice, the bone marrow cells were treated with ⁇ -MEM (hy-modified MEM), DMEM (Dulbecco's modified MEM) or IMDM (Isocove's modified Dulbecco's medium) containing 10% FBS (fetal calf serum). By resuspending in a cell culture medium, a bone marrow cell solution can be obtained.
  • ⁇ -MEM hy-modified MEM
  • DMEM Dulbecco's modified MEM
  • 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 includes removing other cells mixed in the solution, such as blood cells, hematopoietic stem cells, vascular stem cells, and fibroblasts.
  • other cells mixed in the solution such as blood cells, hematopoietic stem cells, vascular stem cells, and fibroblasts.
  • a bone marrow cell mixture containing bone marrow cells having the ability to divide the myocardial cells can be obtained.
  • the bone marrow cell mixture containing the 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 culture plate having a% well.
  • the clones are treated using the method described below for inducing cardiomyocytes from bone marrow cells capable of differentiating into cardiomyocytes, and clones in which cells that autonomously beat appear are selected. Thereby, bone marrow cells capable of differentiating into the cardiomyocytes can be obtained.
  • 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.
  • Rat or mau The skull is killed by dislocation of the cervical vertebra and thoroughly disinfected with 70% ethanol, and the skin of the femur and the quadriceps are removed.
  • bone marrow cells capable of differentiating into cardiac muscle cells can be isolated in the same manner as in the above-described method for isolating bone marrow cells from human bone marrow fluid.
  • Examples of cells isolated by the above method include mouse bone marrow-derived pluripotent stem cells.
  • BMSCs derived from mouse bone marrow-derived pluripotent stem cells are available on ⁇ -February 22, 2002 at the Institute of Biotechnology and Industrial Technology, Institute of Industrial Technology, Ministry of International Trade and Industry (1-1-3 Tsukuba East, Ibaraki, Japan) Deposited as FERM BP-7043.
  • Tissue other than bone marrow preferably includes cord blood. Specifically, it can be performed by the following method.
  • umbilical cord blood is collected from the umbilical cord, and heparin is immediately added to a final concentration of 500 units / ml. After mixing well, centrifuge to collect cells from cord blood, and add 10% FBS
  • DMEM Dulbecco's modified MEM
  • IMDM Isocove's modified Dulbecco's medium
  • a culture medium for cell culture having a composition known in the art can be used as the medium used for the culture.
  • a cell culture medium such as HI, DMEM or IMDM is used.
  • Culture conditions may be any conditions as long as the cells can be cultured, but the culture temperature is preferably 33 to 37 ° C, and more preferably in an incubator filled with 5 to 10% carbon dioxide gas. . It is preferable that bone marrow cells capable of differentiating into cardiomyocytes adhere and grow on a plastic culture dish for normal tissue culture.
  • the cells grow on the entire surface of the culture dish, remove the medium and suspend the cells by adding Tribcine EDTA solution.
  • the suspended cells should be further subcultured by washing with PBS 'or the cell culture medium, then diluting 5- to 20-fold with the cell culture medium and adding to a new culture dish. Can be done.
  • Methods for inducing cardiomyocytes from cells capable of differentiating into cardiomyocytes include (1) induction of DNA by treatment with DNA demethylating agent, and (2) factors expressed in the fetal heart development region. Or induction of differentiation by a factor that acts on differentiation into cardiomyocytes during the fetal heart development stage, (3) induction of differentiation by cells having the ability to differentiate into cardiomyocytes or culture supernatants of cardiomyocytes differentiated from the cells. Can be mentioned. By using these methods alone or in combination, cardiomyocytes can be derived from cells capable of differentiating 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 the methylation of cytosine residues in GpC sequences in chromosomal DNA, 5-azacitidine (hereinafter abbreviated as 5-aza-C), DMSO (dimethyl sulfoxide).
  • 5-aza-C 5-azacitidine
  • DMSO dimethyl sulfoxide
  • Examples of the demethylase include demethylase having the amino acid sequence of SEQ ID NO: 1 [Nature, 397, 579-583 (1999)] and the like. Specific examples of the induction of DNA fragmentation by treatment with DNA demethylating agent are shown below.
  • 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 can be used as long as it promotes the differentiation of the cells capable of differentiating into cardiomyocytes into cardiomyocytes at the stage of cardiac development. .
  • PDGF platelet-derived growth factor
  • FGF8 fibroblast growth factor 8
  • ET1 endothelin 1
  • BMP4 bone formation Factor 4
  • an inhibitor for a cytokine that suppresses the differentiation into cardiomyocytes it is possible to promote differentiation into cardiomyocytes during the developmental stage of the heart in cells having the ability to differentiate into cardiomyocytes. is there.
  • cytokines that suppress differentiation into cardiomyocytes include fibroblast growth factor 1-2 (hereinafter abbreviated as FGF-2), and specifically, FGF-2 represented by SEQ ID NO: 7 or 8. Can be given.
  • FGF-2 fibroblast growth factor 1-2
  • Examples of the inhibitor for cytocan that suppresses differentiation into cardiomyocytes include substances that inhibit cytokine signal transduction, such as antibodies that neutralize cytokines and low molecular weight compounds.
  • any vitamin such as retinoic acid
  • retinoic acid may be used as long as it promotes the differentiation of the cells capable of differentiating into cardiomyocytes at the developmental stage of the heart.
  • retinoic acid 10_ 9 M may be used as long as it promotes the differentiation of the cells capable of differentiating into cardiomyocytes at the developmental stage of the heart.
  • 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, by culturing cells capable of differentiating into the cardiomyocytes in a culture dish coated with fibronectin, it is possible to promote the sorting into the cardiomyocytes.
  • the transcription factor Homeobokkusu transcription factor Nkx2.5 / C SX (SEQ ID NO: 9: Amino acid sequence, SEQ ID NO: 10: nucleotide sequence), Zinc finger transcription factor belonging to the GATA family one GATA4 (SEQ ID NO: 11: Amino acid sequence, SEQ ID NO: 12: base sequence), myocyte enhancer factor-2 (transcription factor MEF-2 ⁇ ⁇ belonging to MEF family 1 (SEQ ID NO: 13: amino acid sequence, SEQ ID NO: 14: base sequence), MEF-2 ⁇ (sequence No.
  • the above-mentioned transcription factor can induce differentiation into cardiomyocytes by introducing DNA encoding the factor into cells capable of differentiating into cardiomyocytes and expressing the DNA.
  • cardiomyocyte differentiation inducing factor a factor that induces differentiation into cardiomyocytes obtained by the method shown in 4
  • cells having the ability to divide into cardiomyocytes can be differentiated into cardiomyocytes. Can be guided. 4. Acquisition of cardiac differentiation induction factor
  • Cardiomyocyte differentiation-inducing factors can be obtained by adding various protease inhibitors to the culture supernatant of autonomously pulsating cells and combining dialysis, salting out, chromatography, etc. Can be.
  • the partial amino acid sequence of the above-mentioned myocardial differentiation-inducing factor was determined using a microsequencer, and a cDNA library prepared from the autonomously beating cells using a DNA probe designed based on the amino acid sequence. By screening, a gene for a myocardial differentiation-inducing factor can be obtained.
  • Cardiac regeneration or therapeutic agent for heart disease containing cells capable of differentiating into cardiomyocytes The cell of the present invention having the ability to bind to cardiomyocytes can be used as a therapeutic agent for cardiac regeneration or heart disease. it can.
  • heart disease examples include myocardial infarction, ischemic heart disease, congestive heart failure, arrhythmia, hypertrophic cardiomyopathy, dilated cardiomyopathy, myocarditis, and valvular disease.
  • cardiomyocytes As a therapeutic agent for heart regeneration, cells containing cardiomyocytes with the ability to divide the cells were included in high purity, and the cells capable of differentiating into the cardiomyocytes were proliferated according to the damaged area and the size of the heart.
  • myocardial endothelial cells Preferably from cells capable of differentiating into cardiomyocytes, myocardial endothelial cells
  • Endocardial endothelial cells Endocardial endothelial cells
  • cushion cells Cushion cells
  • ventricular-type cardiomyocytes atrial-type cardiomyocytes
  • cells capable of inducing differentiation into various cells forming the heart such as sinus node cells, are used.
  • the therapeutic agent is a density gradient centrifugation method from the bone marrow fluid of a patient with myocardial infarction, and a Banning 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.
  • the therapeutic agent may be selected from cells obtained by inducing the cells capable of differentiating into cardiomyocytes into cardiomyocytes using a cardiomyogen described below and bone marrow cells obtained from the bone marrow of the elderly. Also included are cells having the ability to divide cells into cardiomyocytes in which cell division ability has been activated 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 cell 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 a method using a catheter or the like is used.
  • a specific method will be described using ischemic heart disease as an example.
  • Cardiomyocytes affected by ischemic heart disease are located downstream of vascular stenosis, so coronary angiography (Circulatory organs 1, cardiovascular disease, MEDICAL VIEW. 1993), it is necessary to identify the stenosis site of the blood vessel.
  • Organic stenotic lesions are classified into afferent stenosis, eccentric stenosis, and multiple wall irregularities according to the stenosis pathology.
  • eccentric stenosis is subdivided into two types, type I and type II.
  • 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 the possibility of transition to myocardial infarction is high.
  • PTCA percutaneous coronary angioplasty
  • thrombolytic therapy beforehand. is necessary.
  • the cells to be injected can be distinguished as ventricular or atrial.
  • the catheter insertion method is Sone ⁇ , which is inserted from the right upper arm artery (Medical View, 1993), or Jundkins method, which is inserted from the femoral artery (Illustrated Pathology Course, Cardiology 1, 1). MEDICAL VIEW, 1993) can be used.
  • the cardiomyogenic agent of the present invention includes a demethylating agent for chromosomal DNA, a factor expressed in the fetal heart development region, a factor that acts on differentiation into cardiomyocytes during the fetal heart development stage, Can be induced to differentiate into cells having the ability to differentiate into cardiomyocytes.
  • cardiomyogen examples include cytokines, vitamins, adhesion molecules, transcription factors, and the like.
  • Cytokines include cells that have the potential to differentiate into cardiomyocytes, Any cytokine that promotes differentiation into muscle cells may be used.
  • PDGF fibroblast growth factor 8
  • ET1 endothelin 1
  • BMP4 bone morphogenetic factor 4
  • any bimin can be used as long as it promotes differentiation into cardiomyocytes at the stage of cardiac development, such as retinoic acid and other cells capable of differentiating into cardiomyocytes. Specifically, and the like retinoic acid 10_ g M.
  • any adhesion molecule such as fipronectin may be used as long as it is expressed in the heart development region at the stage of cardiac development.
  • the differentiation into cardiomyocytes can be promoted by culturing 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), Zinc finger transcription factor belonging to the GATA family one GATA4 (SEQ ID NO: 11: amino acid Sequence, SEQ ID NO: 12: nucleotide sequence), transcription factor MEF-2A belonging to myocyte enhancer factor-2 (MEF-2) family (SEQ ID NO: 13: amino acid sequence, SEQ ID NO: 14: nucleotide 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), dHAND (SEQ ID NO: 21: amino acid sequence, SEQ ID NO: 22: base sequence), eHAND (SEQ ID NO:
  • the preparation method in the case where the cardiomyogenic agent of the present invention contains a gene encoding a myocardial differentiation-inducing factor as a main component is described below.
  • a recombinant viral vector plasmid is constructed by introducing a gene DNA fragment of the myocardial motility-inducing factor or a full-length cDNA downstream of the promoter within the viral vector plasmid. I do.
  • the recombinant virus vector-plasmid is introduced into a packaging cell suitable for the virus vector plasmid.
  • the packaging cells include proteins necessary for viral packaging: any cells capable of supplying the protein deficient in a recombinant virus vector plasmid deficient in at least one of the encoding genes. 'Can be used.
  • human kidney-derived HEK293 cells, mouse fibroblast MH3T3 and the like can be used.
  • proteins such as gag, pol, env, etc. derived from mouse retrovirus and in the case of lentivirus vector, gag, pol, env, vpr, etc. derived from HIV virus vpu ⁇ vif, tat, rev, proteins such as nef, E1A from adenovirus in the case of adenovirus vector and foremost, proteins such as E1B, in the case of adeno-associated virus Rep (p5, P 19, p40 ), Vp (Cap ) Can be 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) lEGmmediate early) gene the early promoter of SV40, the retro promoter Viral promoters, meta mouth thynein promoters, heat shock protein promoters, SR hypromo, etc.
  • the enhancer of the IE gene of human CMV may be used together with the promoter.
  • the cardiomyocyte specific heritage promoter gene such as Nkx 2 .5 / Csx gene can Rukoto specifically to express the gene of interest in cardiomyocytes.
  • a recombinant virus vector can be produced by introducing the above-mentioned recombinant virus vector-single plasmid into the above-mentioned packaging cell.
  • a method for introducing the above-mentioned virus vector plasmid into the above-mentioned packaging cells include a calcium phosphate method [Japanese Patent Laid-Open No. 2 ⁇ 227075] and a lipofection method [Proe. Natl. Acad. Sci. USA, 84, 7413 ( 1987)].
  • the above-mentioned recombinant virus vector can be prepared together with a base used for a gene therapy agent to produce a cardiomyogenic agent [Nature Genet., 8, 42 (1994)].
  • a base used for the gene therapy agent any base that is usually used for injections can be used.
  • salt solutions such as sodium chloride or a mixture of sodium chloride and inorganic salts
  • solutions such as mannitol, lactose, dextran, glucose, etc.
  • amino acid solutions such as glycine and arginine
  • organic acid solutions or salt solutions examples thereof include a mixed solution with a glucose solution.
  • an osmotic pressure adjusting agent a pH adjusting agent, a vegetable oil such as sesame oil and soybean oil, or an auxiliary agent such as a surfactant such as lecithin or a nonionic surfactant is used as a solution or suspension for these bases.
  • An injection may be prepared as a suspension or dispersion. These injections can be prepared as preparations for dissolution at the time of use by operations such as powdering and freeze-drying.
  • the above-mentioned myocardial agent can be used for gene therapy by dissolving it in a liquid as it is in a liquid, or dissolving it in the above-mentioned sterilized base immediately before the treatment if necessary.
  • a method for administering the myocardial agent of the present invention a method for local administration using a catheter or the like is used so that it is absorbed into the myocardium at the treatment site of the patient.
  • the above-described recombinant virus vector is a cell capable of differentiating into the cardiomyocyte in a test tube. After infecting the vesicles, they can be prepared as the above-mentioned myocardial agent and administered to patients. Alternatively, the recombinant viral vector can be administered directly to the affected area of the patient.
  • the protein may be any protein that has a full-length cDNA of the cardiac inducing factor-inducing protein. Based on the full-length cDNA of the cardiac inducing factor-inducing protein, the protein may be any protein that has a full-length cDNA of the cardiac inducing factor-inducing protein.
  • a recombinant expression vector of the protein is constructed.
  • the recombinant expression vector is introduced into a host cell compatible with the expression vector.
  • Any host cell that can express the target DNA can be used, and examples thereof include the genus Escherichia, the genus Serratia, the genus Corynebacterium, and the genus Brevibacterium.
  • the Domonas include the genus Escherichia, the genus Serratia, the genus Corynebacterium, and the genus Brevibacterium.
  • Yeasts animal cells, insect cells, etc. belonging to the genus Saccharomyces, the genus Shizosaccharomyces, the genus Trichosporon, the genus Schwanniomyces, and the like can be used.
  • a cell capable of autonomous replication in the above host cell can be integrated into a chromosome, and contains a promoter at a position where gene DNA of a cardiac differentiation induction factor protein can be transcribed. Things are used.
  • the recombinant expression vector of the myocardial differentiation-inducing factor protein is capable of autonomous replication in the bacterium, and at the same time, the promoter, the ribosome binding sequence, the myocardial differentiation-inducing factor protein It is preferable that the recombinant expression vector be composed of a DNA coding for DNA and a transcription termination sequence. A gene controlling the promotion may be included.
  • Expression base Kuta one, for example, pBTrp 2, pBTacl, pBTac2 ( ⁇ from both Beringa one Mannheim), pKK233- 2 (Amersham Pharmacia Biotech Co.), pSE280 (Manufactured by Imdtrogen), pGEMEX-1 (manufactured by Promega), pQB-8 (manufactured by QIAGEN), pKYPIO [JP-A-58-110600], pYP200 [Agricultural Biological Chemistry, 48, 669 (1984)], pLSAl [Agric. Biol. Chem., 53, 277 (1989)], pGELl [Proc. Natl. Acad. Sci.
  • the expression vector it is preferable to use an expression vector in which the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the initiation codon is adjusted to an appropriate distance (for example, 6 to 18 bases).
  • any promoter can be used as long as it can be expressed in a host cell.
  • trp promoter Isseki one P trp
  • lac promoter ⁇ "(P lac) P L promoter one evening one
  • P R promoter evening one promo one derived from Escherichia coli or phage, such as one such T7 promoter Isseki
  • Promote Yoichi Ptrp x 2 with two P trps connected in series, tac Promo — Even 1. Letl Promo Yuichi [Gene, 44, 29 (1986)], lacT7 Promo Yuichi, artificially designed and modified Promo Yoichi, etc. can also be used.
  • the gene of the myocardial differentiation-inducing factor protein of the present invention is obtained by substituting the base sequence of the DNA-encoding portion of the DNA so as to be an optimal codon for expression in the host, thereby increasing the production rate of the target protein. Can be improved.
  • a transcription termination sequence is not necessarily required for expression of the gene DNA of the myocardial differentiation-inducing protein of the present invention, it is desirable to arrange the transcription termination sequence immediately below the structural gene.
  • Examples of the host cell include microorganisms belonging to the genus Escherichia, Serratia, Corynebacterium, Brevibacterium, Pseudomonas, Bacillus, Microbacterium, etc., for example, Eschich-ia coli XL1-Blue Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coji W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No.49, Escherichia coli W3110, Escherichia coli NY49, Bacillus subtilis, Bacillus amvloliquefaciens ⁇ Brevibacterium ammoniagenes, Brevibacterium immariophilum ATCC14068, Brevibacterium
  • Any method for introducing a recombinant vector can be used as long as it is a method for introducing DNA into the above host cells.
  • a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 ( 1972)] ⁇ Protoplast method (JP-A-63-248394) or the method described in Gene, 17, 107 (1982) or Molecular & General Genetics, 168, 111 (1979).
  • yeast When yeast is used as a host cell, examples of expression vectors include, for example, YEpl3 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, and pHS15.
  • Any promoter can be used as long as it can be expressed in yeast.
  • PH ⁇ 5 Promo overnight PGK Promo overnight, GAP Promo evening, ADH Promo overnight
  • gal l Promoters gal 10 promoters, heat shock protein promoters, MF 1 promoters, CUP 1 promoters, etc.
  • the host cells include Saccharomyces cerevisiae, Schisosaccharomvces pombe, Kluyveromyces lactis, Trichosporon pullionas, etc.
  • any method can be used as long as it introduces DNA into yeast, for example, an electroporation method [Methods in Enzymol., 194, 182 (1990)]. USA), 75, 1929 (1978)], the lithium acetate method [J. Bacteriol., 153, 163 (1983), Proc. Natl. Acad. Sci. USA, -75, 1929 (1978)].
  • pCDNAKlnvitrogen When animal cells are used as host cells, as an expression vector, for example, pCDNAKlnvitrogen, pCD8 (Invitrogen), pAGE107 [JP-A-3-22979; Cytotechnology, 3, 133 (1990)], pAS3--3 (JP-A-2-227075), pCDM8 [Nature, 329, 840 ( 1987)], pCDNAI / Amp (manufactured by Invitrogen), pREP4 (manufactured by Invitrogen), pAGE103 [J. Biochem., 101, 1307 (1987)], pAGE210, and the like.
  • pCDNAKlnvitrogen When animal cells are used as host cells, as an expression vector, for example, pCDNAKlnvitrogen, pCD8 (Invitrogen), pAGE107 [JP-A-3-22979; Cytotechnology, 3, 133 (1990)], pAS3-
  • any promoter can be used as long as it can be expressed in animal cells.
  • the promoter of the IE (immediate early) gene of cytomegalovirus (human CMV) and the early promoter of SV40 can be used. Overnight, retrovirus promo overnight, meta-mouth choonein promo overnight, heat shock protein promo overnight, SR alpha promo overnight, etc.
  • the human CMV IE gene enhancer ⁇ may be used with the Promo-Yuichi.
  • host cells examples include Namalwa cells, which are human cells, COS cells, which are monkey cells, CHO cells, which are Chinese 'Hamus cells, and HBT5637 [Japanese Patent Application Laid-Open No. 63-299]. Can be.
  • any method capable of introducing DNA into animal cells can be used.
  • an electroporation method [Cytotechnology,, 133 (1990)]
  • a calcium phosphate method Japanese Unexamined Patent Application Publication No. Natl. Acad. Sci "USA, 84, 7413 (1987), Virology, 52, 456 (1973)] and the like. It can be carried out according to the method described in JP-A-2-227075 or JP-A-2-257891.
  • baculovirus * expression 'vectors a laboratory manual [Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company, New York (1992)], current. Proteins can be expressed by the method described in Protocols “In” Molecular Biology Supplements 1-38 (1987-1997), Bio / Technology, 6, 47 (1988) and the like.
  • the recombinant virus is further infected into the insect cells to express the protein.
  • the gene transfer vector used in the method include pVL1392, pVL1393, pBlueBacIII (all manufactured by Invitrogen) and the like.
  • baculovirus for example, autographa californica nuclear polyhedrosis virus, which is a virus that infects night moth insects, can be used.
  • Sf9 and Sf21 which are ovary cells of Spodoptera frugiperda [Baculovirus Expression Vectors, A Laboratory Manual ⁇ WH Freeman.and Company, New YorK 3 (1992; I, ovarian cells of Trichoplusia ⁇ High 5 (manufactured by Invitrogen
  • Examples of a method for co-transferring the above-mentioned recombinant gene transfer 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], Natl. Acad. Sci. USA, 84, 7413 (1987)] and the like.
  • gene expression methods include, in addition to direct expression, methods described in Molecular 'Cloning 2nd edition,' Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)] Secretory production, fusion protein expression, and the like can be performed according to the method described above.
  • 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.
  • a transformant harboring the recombinant DNA into which the DNA encoding the cardiomyocyte differentiation-inducing factor has been incorporated is cultured in a medium, and the cardiomyocyte differentiation-inducing factor protein is produced and accumulated in the culture. By collecting, a myocardial differentiation-inducing factor protein can be produced.
  • the method of culturing the transformant producing the cardiac differentiation-inducing factor protein 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 these, carbohydrates such as starch or hydrolyzed starch, organic acids such as acetic acid and propionic acid, Alcohols such as ethanol and propanol can be used.
  • Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate and other inorganic or organic acid ammonium salts, other nitrogen-containing compounds, and peptone, meat extract, yeast extract. Use is made of steak liquor, casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and their digests.
  • potassium phosphate monobasic, potassium phosphate dibasic, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like can be used.
  • the culture is performed under aerobic conditions such as shaking culture or deep aeration stirring culture.
  • the culture temperature is preferably 15 to 40 ° C, and the culture time is usually 6 hours to 7 days.
  • In the culture PH is maintained at 3.0 to 9.0.
  • the pH is adjusted by using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.
  • an antibiotic such as ambicilin / tetracycline may be added to the medium during the culture.
  • an inducer may be added to the medium, if necessary.
  • an inducer may be added to the medium, if necessary.
  • IPTG isopropyl-15-D-thiogalactovyranoside
  • IAA indoleacrylic acid
  • a medium for culturing the transformant obtained by using animal cells as host cells commonly used RPMI1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], Eagle's MEM medium [Science, 122, 501 (1952)], Dulbecco's modified MEM medium [Virology, 8, 396 (1959)], 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)], or a medium obtained by adding fetal bovine serum or the like to such a medium.
  • Culture is carried out usually pH6 ⁇ 8, 30 ⁇ 40 ° C, 5% C0 2 present 1 to 7 days under conditions such as lower. If necessary, an antibiotic such as kanamycin or penicillin may be added to the medium during the culture.
  • TNM-FH medium manufactured by Pharmingen
  • Sf-900 II SFM medium manufactured by Life Technologies
  • ExCell400 And ExCell405 all manufactured by JRH Biosciences
  • Grace's Insect Medium [Grace, TCC, Nature, 195, 788 (1962)] and the like can be used.
  • the cultivation is usually performed under conditions of pH 6 to 7 and 25 to 30 ° C for 1 to 5 days.
  • an antibiotic such as genyumycin may be added to the medium during the culture.
  • a normal protein isolation and purification method may be used.
  • the myocardial differentiation-inducing protein is expressed in a lysed state in the cells
  • the cells are recovered by centrifugation after suspension of the culture, suspended in an aqueous buffer, and then sonicated with a sonicator, French press, The cells are disrupted using a Manton Gaulin 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 (Mitsubishi Chemical), and a resin such as S-Sepharose FF (Amersham Pharmacia Biotech) Cation exchange chromatography, hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, etc.
  • a purified sample can be obtained by using techniques such as electrophoresis such as electrofocusing 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 recover the insoluble form of the protein 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, and then the same purification method as described above is used. A purified sample of the protein is obtained.
  • the protein or a derivative such as a sugar chain adduct thereof can be recovered from the culture supernatant. That is, a purified sample can be obtained by collecting the culture supernatant from the culture by a technique such as centrifugation and using the same isolation and purification method as described above from the culture supernatant.
  • 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 and the tBoc method (t-butyloxycarbonyl method).
  • a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl) method and the tBoc method (t-butyloxycarbonyl method).
  • the protein capable of inducing differentiation into cardiomyocytes can be used by forming 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 essential proteins due to mutation of a single gene.
  • diseases include familial hypertrophic cardiomyopathy, Fabri disease, long QT syndrome, Marfan syndrome, aortic stenosis, mitochondrial cardiomyopathy, Duchenne muscular dystrophy and the like.
  • These diseases are known to be caused by genetic abnormalities in myosin, troponin, tropomyosin, voltage-gated Na channels, K channels, fibrin, elastin, mitochondria, dystrophin, etc. [Therapeutics, ⁇ , 1302 -1306 (1996)].
  • the above-mentioned disease can be treated by obtaining cells capable of differentiating into the cardiomyocytes of the present invention from these patients, introducing a normal gene, and transplanting the cells into the heart.
  • a normal gene can be introduced into a cell capable of differentiating into a cardiomyocyte according to the present invention by using the vector for gene therapy described in 6 (1) above.
  • the antibody of the present invention which recognizes a surface antigen specifically expressed in a cell having the ability to divide myocardial cells, can be used for cardiomyocytes necessary for performing cell therapy for heart diseases such as myocardial infarction. It can be used for purity assay and purification of cells having differentiation ability.
  • a cell having the ability to differentiate into cardiomyocytes of the present invention 3 to 5 ⁇ 10 5 cells S / mouse, or a cell membrane fraction prepared from the cells 1 to 10 mg / mouse as an antigen
  • a suitable adjuvant e.g., Freund's Complete Adjuvant
  • subcutaneously, intravenously or intracellularly in non-human mammals such as rats, mice or hamsters such as rabbits, goats or 3-20 weeks old.
  • Adjuvant or aluminum hydroxide gel, B. 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.
  • Enzyme-linked immunosorbent assay [Enzyme-linked immunosorbent assay (ELISA): published by Medical Shoin 1976 Year, Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory, 1988].
  • 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.
  • the monoclonal antibody is prepared by fusing the antibody-producing cells with myeloma cells 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 tumor. Prepared by separating and purifying the culture or ascites can do.
  • spleen cells As the antibody-producing cells, spleen cells, lymph nodes, and antibody-producing cells in peripheral blood, particularly splenocytes, are suitably used.
  • the myeloma cells include 8-azaguanine-resistant mouse (derived from BALB / c) 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)], P3-X63-Ag8 (X63) strain [Nature, 256, 495 (1975)], etc.
  • Can be -Hybridoma cells can be prepared by the following method.
  • the 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. After cultivation, a portion of the culture supernatant is removed, and those that react with the antigen but do not react with the protein containing no antigen are selected by enzyme immunoassay or the like. Next, cloning is performed by the limiting dilution method, and those with a stable and high antibody titer determined by the enzyme immunoassay are selected as monoclonal antibody-producing hybridoma cells.
  • HAT medium medium containing hypoxanthine, thymidine and aminopterin in normal medium
  • Methods for separating and purifying polyclonal or monoclonal antibodies include centrifugation, ammonium sulfate precipitation, caprylic acid precipitation, or DEAE-Sepharose columns, anion exchange columns, protein A or G columns, or gel filtration columns. And the like, which may be used alone or in combination.
  • the antibody obtained by the above method and specifically recognizing a surface antigen expressed on the cell capable of differentiating into cardiomyocytes the reactivity with the 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 specific surface antigen.
  • a cDNA library prepared from cells capable of differentiating into cardiomyocytes was prepared by using mRNA obtained from control cells other than cells capable of dividing into cardiomyocytes such as hematopoietic stem cells and neural stem cells. Perform subtraction. After preparing a differential cDNA library enriched for a cell-specific gene capable of differentiating into cardiomyocytes, the inserted cDNA sequence of the differentiated cDNA library was randomly sequenced from the 5th and 5th sides. Perform an analysis and select only those with a secretory 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 subtraction, and a signal sequence trap library was prepared from cells capable of differentiating into cardiomyocytes. It is desirable to perform 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.
  • the protein encoded by the cDNA is a secretory protein or a membrane protein.
  • a synthetic peptide is prepared based on the amino acid sequence deduced from the nucleotide sequence, and the synthetic peptide is used as an antigen.
  • a specific antibody can be obtained by the above method.
  • a membrane protein some of them encode a receptor, and such a receptor acts to regulate the specific proliferation of cells capable of differentiating into the cardiomyocytes or the differentiation into cardiomyocytes. Can be used to search for the ligand of the receptor.
  • a secretory protein it can be used to proliferate or differentiate cells having the ability to directly differentiate into cardiomyocytes.
  • the screening method for the growth factor of cells capable of differentiating into cardiomyocytes and the factor that induces differentiation into cardiomyocytes is as follows. It can be performed by adding certain substances and examining whether the cells proliferate or are induced to differentiate into cardiomyocytes.
  • the substance to be a specimen may be any substance such as secreted proteins such as various cytokines and growth factors, membrane-bound proteins such as cell adhesion molecules, tissue extracts, synthetic peptides, synthetic compounds, and microbial culture solutions.
  • secreted proteins such as various cytokines and growth factors
  • membrane-bound proteins such as cell adhesion molecules, tissue extracts, synthetic peptides, synthetic compounds, and microbial culture solutions.
  • the proliferative ability can be determined by the ability to form a knee and the uptake of BrdU.
  • the ability to form colonies can be examined by seeding cells of the present invention having the ability to differentiate into cardiomyocytes 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 using promoters for genes specifically expressed in muscle cells and GFP (Gleen fluorescent protein), luciferase, A method using the reporter gene expression as an index using a repo overnight cell in which vector DNA combined with a repo overnight gene such as 1-galactosidase is introduced into a cell capable of differentiating the cardiomyocyte.
  • GFP Green fluorescent protein
  • luciferase luciferase
  • Examples of a method for increasing the number of cell divisions without causing the cells to become cancerous include a method for expressing telomerase in cells capable of differentiating into cardiomyocytes of the present invention.
  • a method of introducing the TERT gene, which is a catalytic supplement of telomerase, specifically, the DNA represented by SEQ ID NO: 32 into a retrovirus vector and then into a cell having the ability to divide into cardiomyocytes, or
  • a method in which a factor capable of inducing and expressing a TERT gene in a cell capable of differentiating into a cardiomyocyte is administered to a cell capable of inducing differentiation into a cardiomyocyte, or a DNA encoding a factor capable of inducing and expressing a TERT gene A method of introducing a protein containing the above into a cell capable of differentiating into a cardiomyocyte can be mentioned.
  • TERT Factors that induce and express the TERT gene include TERT gene protein and GFP (Green Fluorescent protein), luciferase, or beta-galactosidase in cells that have the potential to differentiate into cardiomyocytes. Selection can be achieved by introducing the combined repo overnight system into cells capable of differentiating into cardiomyocytes.
  • GFP Green Fluorescent protein
  • luciferase luciferase
  • beta-galactosidase in cells that have the potential to differentiate into cardiomyocytes. Selection can be achieved by introducing the combined repo overnight system into cells capable of differentiating into cardiomyocytes.
  • Methods for obtaining cells expressing the target surface antigen from various tissues taken out of the living body include a method using a flow cytometer having a sorting function and a method using magnetic beads.
  • the expression level of the antigen can be determined by converting the fluorescence intensity emitted from the antibody bound to the molecule expressed on the cell surface into an electric signal. Also, by combining the types of fluorescence used, it is possible to separate using multiple surface antigens.
  • fluorescence examples include FITC (fluorescein isothiocyanate) PE (phycoerythrin APC (Allo-phycocyanin) TR (TexasRed), Cy3, CyChrome, Red613, Red670, PerCP, TRI-Color, QuantumRed, etc. Freedom, p3—13, Shujunsha, 199).
  • FITC fluorescein isothiocyanate
  • PE phycoerythrin APC (Allo-phycocyanin) TR (TexasRed)
  • Cells were separated from various tissues taken out of the body, specifically bone marrow or cord blood, by centrifugation or other methods, and then directly stained with an antibody, and then cultured and expanded once in an appropriate medium. A method of staining with an antibody later can be used.
  • For cell staining first mix the primary antibody that recognizes the surface antigen with the cell sample of interest, and incubate on ice for 30 minutes to 1 hour. If the primary antibody is fluorescently labeled, separate the cells after washing by flow cytometry. If the primary antibody is not fluorescently labeled, after washing, mix the fluorescently labeled secondary antibody, which has binding activity to the primary antibody, with the cells that have reacted with the primary antibody, and again on ice for 30 minutes. Incubate for 1 hour. After washing, the cells stained with the primary and secondary antibodies were separated by arrow cytometry.
  • cells expressing the target surface antigen can be separated in large quantities. Although the purity of the separation is inferior to the method using the above-mentioned flow cytometry, a sufficiently high cell purity can be ensured by repeating the purification. After staining the cells with the primary antibody, the remaining primary antibody is removed, and the secondary antibody bound to magnetic beads that specifically binds to the primary antibody is bound. Cells from which residual secondary antibodies have been washed away can be separated on a stand with a magnet. The materials and equipment needed for these operations are available from DYNAL.
  • the method using magnetic beads can be similarly used to remove unnecessary cells from a cell sample.
  • the StemSep method sold by Stem Cell Technologies Inc can be used.
  • Examples of the antibody used in the above method include the antibody obtained in the above item 8, or an antibody recognizing hematopoietic cell surface antigens CD34, CD117, CD14, CD45, CD90, Sca-1, Ly6c, Ly, and vascular endothelium.
  • Antibody that recognizes cell surface antigens Flk-1, CD31s CD105 and CD14, Antibody that recognizes Leuja cell surface antigen CD140, Antibody that recognizes integrin CD49b, CD49d, CD29 and CD41, Matrix receptor CD54, CD102, Antibodies that recognize CD106 and CD44 are raised. By combining these antibodies, target cells can be obtained with higher purity.
  • CD34-positive cells and CD144-negative cells are extracted from human bone marrow cells using the immunomagnetic bead method described above. After removal, the target cells can be separated by collecting the CD117-positive and CD140-positive cell fractions.
  • GFP green fluorescent protein
  • the GFP gene is placed downstream of the gene specifically expressed in cardiac muscle or the gene specifically expressed in cells capable of differentiating into cardiomyocytes obtained in section 9 above.
  • An inducible protein is prepared and introduced into cells having the ability to divide myocardial cells.
  • the cells into which such a repo overnight vector has been introduced are separated by an index such as drug resistance, and then differentiated into cardiomyocytes.
  • Differentiated cells express GFP and fluoresce. Fluorescent myocardial cells and myocardial progenitor cells can be easily separated using flow cytometry (Flow cytometry overnight, p44-52, Shujunsha, 19). 9 9 years).
  • MLC2v and troponin I can be used as promoters for genes specifically expressed in the heart muscle.
  • the above-described plasmid vector for animal cells, adenovirus vector and the like can be used as the vector.
  • the PAR gamma receptor agonist Pioglitazone ⁇ Troglitazone is added to the culture medium to a final concentration of 0.4 ⁇ M to 2 ⁇ . Is added.
  • an aggregate obtained by centrifuging 1 ⁇ 10 5 to 3 ⁇ 10 5 cells has a final concentration of 0.01%.
  • a method of culturing the cells in a medium containing TGFy53 so that the concentration becomes ⁇ g / ml is exemplified. .
  • a final concentration of 0.1 ⁇ dexamethasone, 0.05 mM M ascorbic acid-2--phosphate, 10 mM ⁇ A method of culturing in a medium to which each is added so as to become -glycerophosphate.
  • Example 1 Acquisition and culture of bone marrow cells having the ability to isolate cardiac muscle cells from mouse bone marrow
  • mice Ten 5-week-old C3H / He mice were T-anesthetized with ether and then killed on the day of cervical spine prolapse. The mice were placed in a semi-lateral position and disinfected by applying sufficient 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. The knee joint was lightly scissored, the joint was removed, and the muscles on the back of the femur were resected. A scissor was placed in the hip joint, the joint was removed, and the femur was removed. The muscle attached to the femur was excised with scissors to expose the entire femur.
  • bone marrow cells KUM2 and pluripotent stem cells BMSC described below are 33 ° C in IMDM medium containing 20% FCS, 100 mg / ml penicillin, 250 ng / ml streptomycin ⁇ '85 mg / ml amphotericin, unless otherwise specified.
  • 5% C_ ⁇ O Kum2 cells were cultured using 2 concentrations of incubation machine by 3 ⁇ 4 time exposed to .5-aza-C a final concentration of 3 ju Micromax, ⁇ nonspecifically self beat Migrating cardiomyocytes induce differentiation, but their frequency is very low (less than 1 in 10 7 cells).
  • BMSC multipotent stem cells
  • cardiac progenitor cells At least two types of cells that differentiate into BMSC cells (hereinafter simply referred to as cardiac progenitor cells) were observed, and BMSC cells were subcultured after being recovered with a growing syringe and immortalized. BMSG cells were observed to induce differentiation more than 100 times more efficiently than their parental strain, KUM2.
  • Trizol Reagents (GIBCO BRL) from the bone marrow-derived primary immortalized cell line, mouse bone marrow-derived multipotent stem cells BMSC, and cardiomyocytes derived from cardiomyocyte progenitor cells obtained in Example 1, respectively.
  • BMSC mouse bone marrow-derived multipotent stem cells
  • cardiomyocytes derived from cardiomyocyte progenitor cells obtained in Example 1, respectively.
  • RNA total RNA.
  • First strand CDNA was synthesized using Superseiptn reverse transcriptase (GIBCO BRL) using the total RNA as a substrate.
  • Cardiomyocyte-specific genes include the natriuretic peptides ANP and BNP, the myosin heavy chain sp -MHC and /?-MHC, the actin sp -skeletal actin and — skeletal actin ⁇ the myosin light chain MLC -. 2a, MLC- 2v ⁇ cardiomyocyte-specific transcription factors Nkx2 5 / Csx, GATA4, TEF- 1, MEF- 2C, MEF- 2D, was used MEF-2A.
  • nucleotide sequences of SEQ ID NOs: 41 and 42 for the amplification of the -skeletal actin Amplification of synthetic DNA, skeletal actin amplification of a synthetic DNA having the nucleotide sequence of SEQ ID NO: 43 or 4, MLC-2a amplification of a synthetic DNA having the nucleotide sequence of SEQ ID NO: 45 or 46, and amplification of MLC-2v
  • Cardiomyocytes that are induced in vivo may differ in fetal, neonatal, or maturation phases, or in atrial or ventricular muscles, to alter heart rate or energy efficiency of myocardial contraction.
  • isoforms of myocardial contractile protein In the case of bone marrow cells, which are divided into cardiomyocytes in the culture system, the isoform is expressed in the following manner. Was also frequently expressed. In myosin light chain, type 2V was expressed, whereas ⁇ type 2a was not observed.
  • the natriuretic peptides ANP and BNP was observed after the induction of bone marrow cells differentiated into cardiomyocytes in the culture system. Judging from the above expression of 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 muscle cells.
  • Nkx2.5 / Csx, GATA4, MEF-2A, MEF-2C, MEF-2D, and TEF-1 gene was observed in bone marrow cells separated from cardiomyocytes in the culture system. Expression of these transcription factors was not observed in proliferating bone marrow-derived primary immortalized cell lines, but expression of Nkx2.5 / Csx, GATA4 and MEF-2C was observed in proliferating bone marrow-derived cardiomyocyte precursor cells. with the induction of differentiation into cardiac myocytes, induction of the expression of late MEF-2 a and MEF-2 D were observed.
  • the action potential of bone marrow cells differentiated into cardiomyocytes in the culture system was recorded using 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 KC1 and 25 mM HEPES (pH 7.4). did.
  • the glass electrode was filled with 3M KC1 with the electrode resistance set at 15 to 30 ⁇ .
  • the measurement of the membrane potential was performed 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).
  • RTA-1100M manufactured by Nihon Kohden Corporation.
  • two types of bone marrow cells 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 a pacemaker cell. there were.
  • the action potential 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, egrets and rats.
  • the resting membrane potential tended to be deeper, and the action potential amplitude tended to be larger.
  • the cells of the sinus node cell type were recorded in all the cells for 2 to 3 weeks, but the ventricular myocyte cell type was observed from about 4 weeks after the induction of differentiation, and gradually increased over time.
  • BMSCs Mouse bone marrow-derived pluripotent stem cells capable of differentiating into cardiomyocytes were cultured in 60 mm culture dishes or 60 mm fibronectin-coated dishes (Becton Dickinson) at a concentration of 2 ⁇ 10 4 cells / ml. 33 ° C! They were cultured using 5% C0 2 concentration in the incubator unit.
  • RNA was recovered from the obtained myotube-like cells, and the genes expressed in the myotube-like cells were analyzed by quantitative PCR using the synthetic oligonucleotides shown in SEQ ID NOs: 71 to 78.
  • PDGF or retinoic acid enhanced the expression of MyoD and fTnl genes associated with skeletal muscle, but did not induce the expression of cTnl, ⁇ ⁇ specifically associated with myocardium.
  • mouse bone marrow-derived probable stem cells BMSC having the ability to differentiate into cardiomyocytes were seeded on a 60 mm culture dish at 2 ⁇ 10 4 cells / ml, and '33. It was incubated with C ,. 5% C0 2 concentration in the incubator unit.
  • BMSC mouse bone marrow-derived probable stem cells
  • FGF- 8 , ET-1, Midkine or BMP4 alone increased the expression of myocardial specific genes cTnl and ANP.
  • Example 4 Induction of differentiation of bone marrow-derived stem cells into cardiomyocytes using DMSO
  • BMSC mouse bone marrow-derived probably stem cell
  • mice bone marrow-derived multipotent stem cells having the ability to differentiate into cardiomyocytes are probably stem cells and cardiac progenitor cells having the ability to inhibit cardiac muscle cells
  • mouse bone marrow-derived pluripotent stem cells possess the properties of cardiomyocytes
  • mouse bone marrow-derived cells with cardiomyocyte differentiation potential Single cell masking Single cell masking (Single) was performed to determine whether stem cells (BMSCs) contain cardiomyocyte precursor cells or whether there are more undifferentiated stem cells other than cardiomyocytes, such as adipocytes. cell marking).
  • a certain cell was labeled by introducing the virus vector into the GFP gene, and then differentiation was induced to differentiate the labeled cells into cells. Judged by.
  • the retroviral vector plasmid GAR3-GFP which expresses the GFP gene
  • the pCMV-Eco plasmid vector which expresses the Ecotropic gene
  • the DNA solution was added dropwise to 293 in cell culture medium which is prepared the day before, were incubated with 37 ° C, 5% 0 2 concentration of incubation machine. The next day, the medium was changed, were cultured using the 'further 3 7 ° C, 5% C0 2 concentration in the incubator unit.
  • Murine bone marrow-derived pluripotent stem cells capable of differentiating into the cardiomyocyte into which the viral vector is to be introduced were placed on a 6-well dish to reach 2 x 1 cells / well on the day before virus influx. Sowed.
  • Hexadimethrine bromide (polybrene) (manufactured by Sigma) was added to the diluted solution containing the virus vector to a final concentration of 8 ⁇ g / ml, and mice with the ability to differentiate into cardiomyocytes were added.
  • culture supernatants 2 m 1 of bone marrow-derived multipotent stem cells (BMSC) and replaced with a virus solution 2 ml was subjected to culturing using a 33 ° C, 5% C0 2 concentration in the incubator unit. After 5 hours, replacing the culture supernatant to a new IMDM medium, having conducted a cultured with further 33 ° C, 5% C0 2 concentration in the incubator unit.
  • BMSC bone marrow-derived multipotent stem cells
  • BMSC bone marrow-derived probable stem cell
  • Nkx2.5 / Csx or GATA4 gene was introduced using a viral vector, and then the differentiation was induced to examine the efficiency of screening for cardiomyocytes.
  • Nkx2.5 / Csx was incorporated into a retroviral vector plasmid pCLNCX (Imgenex) to prepare pCLNC-Nkx2.5 / Csx.
  • pCLPC-GATA4 was prepared by incorporating GATA4 into a plasmid pCLPCX in which the G ⁇ S resistance gene of the retrovirus vector plasmid pCLNCX (Imgenex) was replaced with a puromycin resistance gene. did.
  • pCLNC- Nkx2.5 / Csx or pCLPC—GATA4 retrovirus vector-plasmid DNA15 pCMV—Eco plasmid vector DNA 5 / g was added to and dissolved in 0.5 ml of 250 mM CaCl 2 (pH6.95), and the solution was placed in a 15 ml tube of 2 ⁇ BBS [50 mM BES (N, N-bis (2 -hydroxyethl) -2-aminoethanesulfonic acid), 280mM NaCl, 1.5mM
  • the culture supernatant was filtered through a 0.45 ⁇ m filter (MilHpore) to recover a solution containing the virus vector.
  • Murine bone marrow-derived multipotent stem cells which have the ability to differentiate into cardiomyocytes into which the viral vector is introduced, are converted to 2 x 10 4 cells / well on the day before virus infection. We sowed in dish.
  • Hexadimethrine bromide (polybrene) (manufactured by Sigma) is added to the solution containing the virus vector obtained above to a final concentration of 0.8 ⁇ g / ml, which has the ability to differentiate into cardiomyocytes. and replaced with medium mouse bone marrow-derived multipotent stem cells (BMSC), it was incubated with 33 ° C, 5% C0 2 concentration in the incubator unit. After 5 h, replaced with fresh IMDM medium, further subjected to culturing using a 33 ° C, 5% C0 2 concentration ⁇ machine, it was further cultured for 2 days.
  • BMSC medium mouse bone marrow-derived multipotent stem cells
  • G418 was added to cells in which the virus produced by the introduction of pCLNC-Nkx2.5 and pCMV-Eco was added to a final concentration of 300 g / ml, and the cells were further cultured for 7 days.
  • PCLPC- GATA4 and pCMV - The cells Infuekusho down the produced viruses with Eco introduction and ⁇ Ka ⁇ puromycin to a final concentration of 3 00n g / ml, and cultured for an additional 7 days.
  • RNA obtained from the myotube-like cells obtained was collected, and the genes expressed in the myotube-like cells are shown in SEQ ID NOs: 71 to 78. Quantitative PCR analysis was performed using the synthesized oligonucleotides. As a result, it was also observed that forced expression of Nkx2.5 / Csx and GATA4 enhanced the expression of myocardium-specific genes cTnl and ANP.
  • Example 8 Combination of forced expression of transcription factors and cytokines promotes cardiomyocyte differentiation
  • Nkx2.5 / Csx and bone marrow cells capable of differentiating into cardiomyocytes forced expression of both genes GATA4 becomes a 2 X 10 4 cells / ml as 60mm culture Deitsushu And cultured at 33 ° C. using an incubator having a concentration of 5% CO 2 .
  • GATA4 BMSC- Nkx 2 5 -. GATA4
  • RNA was recovered from the obtained myotube-like cells, and the genes expressed in the myotube-like cells were quantitatively analyzed using the synthetic oligonucleotides shown in SEQ ID NOs: 71 to 78. PCR analysis was performed. As a result, FGF-8, ET-1, Midkine or 'BMP4 did not further enhance the expression of cTnl and ANP, which were promoted by the forced expression of Nkx2.5 / Cs and GATA4.
  • Example 9 Transplantation of mouse bone marrow cells capable of differentiating into cardiomyocytes into the heart
  • the bone marrow cells were used as donor cells for transplantation into mice. Specifically, the following method was implemented. After 2 4 hours the labeled BMSC cells previously with 5-aza- C with GFP, were suspended in PBS to be a lx l0 8 cells / ml, and stored on ice until immediately before implantation. The BMSC cells were confirmed to have survived about 95% by 0.05% erythrocyte oral staining.
  • the recipient C3H / He mouse (Charles River Japan, Inc.) introduced anesthesia using a ether, and then used a Terumo thermosyringe (1 ml) to inject 30 mg of the abdominal cavity of Teropena using a thermosyringe (1 ml).
  • Intravenous administration maintained anesthesia.
  • 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.
  • ECG electrodes were inserted into the left and right upper limb and right lower limb to monitor the ECG.
  • An outer cylinder of flow flash (20G) was inserted into the trachea.
  • connect Respire Isuzu MODEL SN-480-7 manufactured by Shinano Seisakusho
  • the artificial respiration was started at a tidal volume of 1 ml and a respiratory rate of 120 / min.
  • the skin around the trachea was closed using mosquito forceps (NONAKA RIKA IC ⁇ ., LTD) to cover the trachea.
  • Tissues were removed from the mice 77 days after transplantation, fixed with 10% formalin, and embedded with paraffin.
  • the embedded tissue sample was sliced to a thickness of 6 ⁇ m with a microtome, and attached to a slide glass previously coated with poly-L-lysine. After the soaking deparaffinized 1 0 0% xylene, washed with E evening Bruno Ichiru, it was subjected to pretreatment antibody response immersed further 0.3% 3 ⁇ 40 2 solution for 30 minutes.
  • tissue section a part of the serial section was stained with hematoxylin and eosin.
  • BMSC bone marrow cells
  • cultured cardiomyocytes were cultured for 5 hours at 5 ⁇ 10 6 cells in a 6 cm culture dish.
  • the culture supernatant is filtered through a 0.45 ⁇ m filter (Millipore.), An equal amount of medium is added to the filtered culture supernatant, and a culture solution containing a factor secreted from cultured cardiomyocytes ( (Hereinafter called "conditioned medium").
  • BMSC Bone marrow cells
  • BMSC-Nkx2.5-GATA4 bone marrow cells
  • cardiomyocytes that express both Nkx2.5 and GATA4 genes in advance
  • the cells were cultured in a culture dish so as to have 1 ⁇ 10 5 cells, and then the medium was replaced with a medium. At the same time, 5- & ⁇ &-(was added to the culture so that the final concentration was 33 ⁇ 4 [. The next day, the medium was replaced with a new conditioned medium, and the culture was continued for another 4 weeks.
  • BMSC bone marrow stem cells
  • GATA4 by hydrogenation pressure of conditionality Chillon de 'Myidiamu, the number of myotubes is not increased, ANP, expression of two cardiac specific genes of cTnI is Nkx2.5 and GATA4 Increased expression due to the above The same level, promoting effect Do observed won.
  • ECM coated 'dish' a culture dish coated with the extracellular matrix of the cultured cardiomyocytes
  • the cultured bone marrow cells (BMSC-Nkx 2.5 “GATA4”) were cultured to 1 ⁇ 10 5 cells, and then 5-aza-C was added to a final concentration of 3 ⁇ M. The medium was replaced with a fresh medium to remove -C, and the culture was continued for another 4 weeks, during which time the medium was replaced about once every three days. (BMSC) did not increase the number of myotube-like cells due to the ECM coat dish, but it was observed that the expression of two myocardium-specific genes, AP and cTnI, was enhanced. ”Nkx2. 5.
  • BMSC-Nkx2.5-GATA4 'Bone marrow cells
  • AP and cTnI two myocardial-specific genes
  • BMSC bone marrow cells
  • BMSC-Nkx2.5-GATA4 bone marrow cells
  • Sea-1, Ly6c, Ly6g ⁇ There are 20 kinds of CD140, 'integrin CD49b, CD49d, CD29 matrix receptor CD54, C ⁇ , CD106, CD44 known as surface antigens of mesenchymal cells.
  • the expression of the Flkl antigen was also determined by performing an antibody reaction using an anti-mouse Flkl antibody (Pharmingen; PM-28181D), which was also biotinylated, and measured throughout the flow cytometer.
  • the KUM2 cells were Flkl negative cells.
  • CD31 antigen For expression of CD31 antigen, use FITC-labeled anti-mouse CD31 antibody.
  • KUM2 cells were CD31 negative.
  • the expression of the CD144 antigen an antibody reaction was performed using a biotinylated anti-mouse CD144 antibody (Pharmingen; PM-28091D), and the reaction was measured throughout the flow cytometer. So As a result, the KUM2 cells were CD144 negative cells.
  • the presence or absence of expression of the CD34 antigen was determined by performing an antibody reaction using a FITC-labeled anti-mouse CD34 antibody (manufactured by Pharmingen; PM-09434D). As a result, the KUM2 cells were CD34 negative cells.
  • CD117 (c-kit) antigen For expression of CD117 (c-kit) antigen, FITC-labeled anti-mouse CD117 antibody
  • the KUM2 cells were CD117 negative cells.
  • an antibody reaction was performed using a FITC-labeled anti-mouse CD'14 antibody (manufactured by Pharmingen; PM-09474), and measurement was performed with a flow cytometer.
  • the KUM2 cells were CD14 positive cells.
  • the expression of the CD45 antigen was determined by performing an antibody reaction using a FITC-labeled anti-mouse CD45 antibody (manufactured by Pharmingen; PM-01114) and measuring it throughout the flow cytometer. As a result, the KUM2 cells were CD45 negative cells. No
  • the expression of the CD90 antigen was determined by performing an antibody reaction using a FITC-labeled anti-mouse CD90 antibody (Pharmingen; PM-22214) and measuring it throughout the flow cytometer. As a result, the KUM2 cells were CD90 negative cells.
  • Ly6A / E (Sca-1) antigen FITC-labeled anti-mouse Ly6A / E (Sca-1) antibody
  • the KUM2 cells were Ly6A / E (Sca-1) positive cells.
  • Ly6c antigen For the expression of Ly6c antigen, an antibody reaction was performed using a FITC-labeled anti-mouse Ly6c antibody (Pharmingen; PM-01152), and the reaction was measured throughout the flow cytometer. As a result, the KUM2 cells were Ly6c positive cells.
  • Ly6g antigen For the expression of Ly6g antigen, an antibody reaction was performed using a FITC-labeled anti-mouse Ly6g antibody (Pharmingen; PM-01214), and the reaction was measured throughout the flow cytometer. As a result, the KUM2 cells were Ly6g negative cells.
  • the expression of the CD140 antigen an antibody reaction was performed using a biotinylated anti-mouse CD140 antibody (Pharmingen; PM-28011A), and the reaction was measured throughout the flow cytometer. As a result, the KUM2 cells were CD140-positive cells.
  • an antibody reaction was performed using a FITC-labeled anti-mouse CD49b antibody (manufactured by Pharmingen; PM-09794), and the reaction was measured throughout the flow cytometer. As a result, the KUM2 cells were CD49b-positive cells.
  • the expression of the CD49d antigen an antibody reaction was performed using a FITC-labeled anti-mouse CD49d antibody (manufactured by Pharmingen; PM-01274), and the reaction was measured throughout the flow cytometer. As a result, the KUM2 cells were CD49d negative cells.
  • the expression of the CD29 antigen an antibody reaction was carried out using a FITC-labeled anti-mouse CD29 antibody (manufactured by Pharmingen; PM-22634), and the reaction was measured throughout the flow cytometer. As a result, the KUM2 cells were CD29-positive cells.
  • the expression of the CD54 antigen an antibody reaction was performed using a FITC-labeled anti-mouse CD54 antibody (manufactured by Pharmingen; PM-01544), and the reaction was measured throughout the flow cytometer. As a result, the KUM2 cells were CD54-positive cells.
  • the expression of the CD102 antigen an antibody reaction was performed using a FITC-labeled anti-mouse CD102 antibody (Pharmingen; PM-01804), and measurement was carried out throughout the flow cytometer. As a result, the KUM2 cells were CD102 negative cells.
  • the expression of the CD106 antigen was determined by performing an antibody reaction using a FITC-labeled anti-mouse CD106 antibody (manufactured by Pharmingen; PM-01814) throughout the flow cytometer. As a result, the KUM2 cells were CD106 positive cells.
  • the expression of the CD4 antigen an antibody reaction was carried out using a FITC-labeled anti-mouse CD44 antibody (manufactured by Pharmingen; PM-28154), and the reaction was measured throughout the flow cytometer. As a result, the KUM2 cells were CD44 positive cells.
  • CD140 (PDGFR)
  • CD102 (ICA -2)
  • Example 1 Enrichment of differentiated progenitor cells using mouse MLC2v promoter overnight
  • mouse MLC2v myosin light chain
  • a gene promoter expression system was constructed. Specifically, mouse MLC2v gene By connecting an EGFP gene (manufactured by CL0NTECH) under the promoter sequence, a pMLC-2-EGFP plasmid containing an expression unit of a neomycin resistance gene was constructed. The DNA of this plasmid was obtained by the method of neutralization described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) and the like.
  • Example 13 Induction of adipocytes from mouse bone marrow cells capable of differentiating into cardiomyocytes Bone marrow cells (BMSC) capable of differentiating into cardiomyocytes can be induced to differentiate into adipocytes in addition to cardiomyocytes. In order to control the differentiation into adipocytes, we will investigate the conditions for inducing differentiation. First, analysis of PPARa receptor expression by quantitative PCR revealed that BMSC cells express PPARa1 receptor but do not express PPAR ⁇ 2 receptor. Was.
  • PPAR receptor agonists Pioglitazone and troglitazone
  • BMSC bone marrow cells
  • adipocyte differentiation was promoted in a concentration-dependent manner. Is 0.4 ⁇ ? ⁇ About 50% at 1, almost 100% of the cells in 2 ⁇ M have differentiated into adipocytes.
  • Example 14 Induction of differentiation into nervous system cells, hepatocytes, and cardiomyocytes by transplanting mouse bone marrow cells capable of differentiating into cardiomyocytes into blastocysts
  • BMSC bone marrow cells
  • GFP was integrated into a retrovirus vector-plasmid pCLNCX (Imgenex) to prepare pCLNC-GFP.
  • the retroviral vector plasmid pCLNC-GFP and the pCMV-Eco plasmid vector for expressing the Ecotropic gene (Imgeriex) were purchased from Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laborator Press (1989), etc. Highly pure DNA was obtained using the re-neutralization method and the PEG precipitation method.
  • the transfection was performed as follows.
  • the DNA solution was added dropwise to 293 in cell culture medium were prepared the day before, were incubated with 3 7 ° 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.
  • the culture supernatant was filtered through a 0.4 5 ⁇ M Phil evening one (Millipore Corp.) to recover a solution containing viral vectors scratch.
  • BMSC Mouse bone marrow cells
  • Hexadimethrine bromide (polybrene) (manufactured by Sigma) is added to the solution containing the virus vector obtained above to a final concentration of 8 ⁇ g / ml, which has the ability to differentiate into cardiomyocytes.
  • the culture medium was replaced with a mouse bone marrow cell (BMSC) medium and incubated at 33 ° C. using an incubator at a concentration of 5% CO 2 . After 5 h, replaced with fresh IMDM medium were incubated with further 3 3 ° C ;, 5% C0 2 concentration in the incubator unit.
  • BMSC mouse bone marrow cell
  • the thus obtained bone marrow cells capable of dividing GFP-labeled cardiomyocytes are grown in a 6 cm culture dish, the medium is removed, and 0.5 ml of 0.25% trypsin EDTA is added. After treating for 1 minute, l. 5 ml of fresh medium was added, and the cells were suspended. Then, fetal serum (manufactured by Lexicon Genetics) was added and mixed. Used for injection into blastocysts. Mouse blastocysts were obtained by spontaneous mating of superovulated female C57B1 / 6J mice with syngeneic male mice, and after 4 days, the inside of the removed uterus was perfused with M15 medium.
  • the blastocysts were allowed to stand at 37 ° C and 5% ju under two conditions until the blastocyst cavity swelled, and then transferred to the Manipulating the Mouse Embryo A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994). According to the method described in the statement, the mice were transplanted into the oviduct-side uterus of pseudo-pregnant female MCH strain mice and then implanted. Pseudopregnant female MCH strain mice were bred together with male MCH strain mice after 10 weeks at 1: 1 at ⁇ 00, 3 days before transplantation, and the vaginal plug was confirmed at 9:00 the following morning. And 2 days later used for the above purpose.
  • mice were dissected, the organs were excised, and the expression of GFP was observed. As a result, the expression of GFP was observed in the brain and liver, indicating that BMSCs were distributed to the nervous system and liver.
  • genomic DNA was obtained from a heart obtained from another individual, and PCR was performed using primers of SEQ ID NOs: 79 and ⁇ . As a result, it was confirmed that BMSC was also taken into the heart. These results indicate that BMSCs were able to identify all three germ layers in the nerve, heart, and liver. It was shown to have totipotency to differentiate into
  • Example 15 Telomerase activity in mouse bone marrow cells capable of differentiating into cardiomyocytes Telomerase activity in mouse bone marrow cells capable of dividing into cardiomyocytes was determined by the Telomeric Repeat Amplification Protocol (TRAP) method. (Oncor's TRAPeze Telomerase Detection Kit). The measurement of telomerase activity was carried out in principle according to the attached protocol. Specifically, the measurement was carried out as follows. First, mouse bone marrow cells (approximately 106 cells) capable of differentiating into cardiomyocytes cultured on a 6 cm diameter culture dish (approximately 10 6 ) were washed with PBS, and 200 l of 1X CHAPS solution was added. For SO minutes. Thereafter, cells were harvested in l. 5 ml volume centrifuge tube with the solution, 2 0 min centrifugation at 14,000rpm (4 ° C ;, HITACHI
  • telomerase activity was detected in a sample in which the final concentration of the cell extract was 0.4 to 4 jug / ral.
  • Example 16 Acquisition and culture of bone marrow cells capable of differentiating into cardiomyocytes from rat bone marrow Six 6-week-old Wistar rats (Japan SLC Co., Ltd.) were dislocated to 6 Disinfection was performed by applying sufficient amount of oil. Next, a large incision was made in the skin of the foot, and the muscles covering the femur and tibia were excised while removing 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 muscle and joint were completely resected.
  • Iwaki Glass 10 cm diameter culture dish
  • PBS manufactured by GibcoBRL
  • the total number of recovered cells was 2.6 ⁇ 10 9 .
  • the recovered cells were diluted to 1.3 X 10 8 cells of density per lml, 1.0 73 g / PercolKAiiiersham Pharmacia Biotech Inc. were prepared ml entering the 50ml volume centrifuge tube) / D-PBS solution (25 ml) over was centrifuged at room temperature for 3 minutes at 100 rpm; for 30 minutes.
  • the recovered cells were IMDM medium containing 20% FCS, 100 ⁇ g / ml penicillin, 250 ng / ml streptomycin, 85 JLL g / ml amphotericin (GibcoBRL)
  • the fractionated bone marrow-derived cells 2 to 5 X 10 5 cells N culture dishes for animal cells of 10cm diameter such that m 2 (manufactured by Iwaki Glass, hereinafter abbreviated as 10cm dish) to three plated, and culturing was started at 33 ° C, 5% C0 2 concentration in C0 2 incubator (evening by Co.). The medium was changed half after M hours and after 72 hours. Three to four days later, the medium was changed half.
  • rat bone marrow-derived cells that have been passaged as described above are again detached from the confluence by trypsin EDTA treatment, and 5 ⁇ 10 4 cells per 1 ⁇ l are added to a 6-well plate (manufactured by BECTON DICKINSON). 6cm diameter culture dish coated with fibronectin (Becton Dickinson Biocoa cells were re-seeded to l. 3 x 10 5 cells. One day later, only 5 -azacitidine (Sigma, final concentration 10 ⁇ M) was added. - Azashichijin, PDGF- BB (.
  • 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 searching for a therapeutic agent are provided. ⁇
  • SEQ ID NO: 47 Description of artificial sequence: Synthetic DNA SEQ ID No. 4 8—Description of Artificial Sequence: Synthetic DNA SEQ ID No. 49 Description of Artificial Sequence: Synthetic DNA SEQ ID No. 50 Description of Artificial Sequence: Synthetic DNA SEQ ID No. 5 1 Description of Artificial Sequence: Synthetic DNA SEQ ID No. 5 2—Description of Artificial Sequence: Synthetic DNA SEQ ID No. 5 3 Description of Artificial Sequence: Synthetic DNA SEQ ID No. 5 4—Description of Artificial Sequence: Synthetic DNA SEQ ID No. 5 5 Description of Artificial Sequence: Synthetic DNA SEQ ID No. 5 6 —Description of Artificial Sequence: Synthetic DNA SEQ ID No. 5 7—Description of Artificial Sequence: Synthetic DNA SEQ ID No.

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Abstract

L'invention concerne des méthodes d'ioslation, de purification et de culture de cellules pouvant se différencier en cellules du muscle cardiaque et induire la différenciation desdites cellules. Elle concerne une méthode de prolifération de cellules pouvant se différencier en cellules du muscle cardiaque et une méthode mettant en oeuvre divers cytokines, facteurs de transcription, etc. pour réguler la différenciation desdites cellules en cellules du muscle cardiaque. L'invention concerne également une méthode d'acquisition d'un antigène de surface spécifique aux cellules pouvant se différencier en cellules du muscle cardiaque; une méthode d'acquisition d'un gène codant ledit antigène de surface; une méthode d'acquisition d'un anticorps spécifique audit antigène de surface; une méthode d'acquisition d'une protéine et d'un gène intervenant dans la prolifération de cellules pouvant se différencier en cellules du muscle cardiaque et induire la différenciation desdites cellules en cellules du muscle cardiaque; des remèdes contre diverses maladies cardiaques utilisant des cellules pouvant se différencier en cellules du muscle cardiaque; et une méthode pour induire la différenciation de diverses cellules et tissus (cellules nerveuses, cellules hépatiques, cellules adipeuses, cellules du muscle squelettique, cellules endothéliales et ostoblastes, par exemple) en utilisant des cellules pouvant se différencier en cellules du muscle cardiaque.
PCT/JP2000/007741 1999-12-28 2000-11-02 Cellules pouvant se differencier en cellules du muscle cardiaque WO2001048150A1 (fr)

Priority Applications (5)

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AU10552/01A AU1055201A (en) 1999-12-28 2000-11-02 Cells capable of differentiating into heart muscle cells
EP00985950A EP1254952A4 (fr) 1999-12-28 2000-12-27 Cellules pouvant induire une differenciation dans des cellules du muscle cardiaque
CA002395950A CA2395950A1 (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
PCT/JP2000/009323 WO2001048151A1 (fr) 1999-12-28 2000-12-27 Cellules pouvant induire une differenciation dans des cellules du muscle cardiaque

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JP11/372826 1999-12-28
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JPPCT/JP00/01148 2000-02-28
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

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WO2002036772A1 (fr) * 2000-10-30 2002-05-10 Takeda Chemical Industries, Ltd. Nouveau gene associe a une maladie et son utilisation
WO2005063967A1 (fr) * 2003-12-25 2005-07-14 Kanazawa University Technology Licensing Organization Ltd. 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
WO2014006228A1 (fr) * 2012-07-06 2014-01-09 Katholieke Universiteit Leuven Cellules endothéliales spécifiques du lit vasculaire
US9068991B2 (en) 2009-06-08 2015-06-30 Singulex, Inc. Highly sensitive biomarker panels
US9182405B2 (en) 2006-04-04 2015-11-10 Singulex, Inc. Highly sensitive system and method for analysis of troponin
US9494598B2 (en) 2006-04-04 2016-11-15 Singulex, Inc. Highly sensitive system and method for analysis of troponin

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Cited By (9)

* 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
WO2005063967A1 (fr) * 2003-12-25 2005-07-14 Kanazawa University Technology Licensing Organization Ltd. 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
US8093047B2 (en) 2003-12-25 2012-01-10 Japan Science And Technology Agency Induction of myocardial cell from mammalian bone marrow cell or cord blood-derived cell and fat tissue
US9182405B2 (en) 2006-04-04 2015-11-10 Singulex, Inc. Highly sensitive system and method for analysis of troponin
US9494598B2 (en) 2006-04-04 2016-11-15 Singulex, Inc. Highly sensitive system and method for analysis of troponin
US9719999B2 (en) 2006-04-04 2017-08-01 Singulex, Inc. Highly sensitive system and method for analysis of troponin
US9977031B2 (en) 2006-04-04 2018-05-22 Singulex, Inc. Highly sensitive system and method for analysis of troponin
US9068991B2 (en) 2009-06-08 2015-06-30 Singulex, Inc. Highly sensitive biomarker panels
WO2014006228A1 (fr) * 2012-07-06 2014-01-09 Katholieke Universiteit Leuven Cellules endothéliales spécifiques du lit vasculaire

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