WO2001048150A1 - Cells capable of differentiating into heart muscle cells - Google Patents

Cells capable of differentiating into heart muscle cells 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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French (fr)
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/en
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 CA002395950A priority patent/CA2395950A1/en
Priority to PCT/JP2000/009323 priority patent/WO2001048151A1/en
Priority to AU22281/01A priority patent/AU784618B2/en
Priority to EP00985950A priority patent/EP1254952A4/en
Publication of WO2001048150A1 publication Critical patent/WO2001048150A1/en

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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.

Abstract

A methods of isolating, purifying and culturing cells capable of differentiating into heart muscle cells and inducing the differentiation thereof; a method of proliferating cells capable of differentiating into heart muscle cells and a method of controlling the differentiation thereof into heart muscle cells with the use of various cytokines, transcription factors, etc.; a method of acquiring a surface antigen specific to cells capable of differentiating into heart muscle cells; a method of acquiring a gene encoding this surface antigen; a method of acquiring an antibody specific to this surface antigen; a method of acquiring a protein and a gene participating in the proliferation of cells capable of differentiating into heart muscle cells and the differentiation thereof into heart muscle cells; remedies for various heart diseases with the use of cells capable of differentiating into heart muscle cells; and a method of inducing the differentiation of various cells and tissues such as nerve cells, liver cells, fat cells, skeleton muscle cells, vascular endothelial cells and ostoblasts by using cells capable of differentiating into heart muscle cells.

Description

心筋細胞への分化能を有する細胞  Cells capable of differentiating into cardiomyocytes
技術分野 Technical field
本発明は、 心筋細胞への分化能を有する細胞の単離、 精製、 培養、 分化誘導法に 関する。 また本発明は、 各種サイトカイン、 転写因子などを用いた、 心筋細胞への 分ィ匕能を有する細胞の増殖方法および心筋細胞への分ィ匕を制御する方法に関する。 本発明はさらに、心筋細胞への分化能を有する細胞に特異的な表面抗原の取得方法、 明  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,
該表面抗原をコ一ドする遺伝子の取得方法、 該表面抗原特異的な抗体の取得方法、 細 A method for obtaining a gene encoding the surface antigen, a method for obtaining an antibody specific to the surface antigen,
心筋細胞への分化能を有する細胞の増殖および心筋細胞への分化に関与する蛋白質 および遺伝子の取得方法に関する。 本発明はまた、 心筋細胞への分化能を有する細 胞を用いた各種心臓疾患の治療薬に関する。 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.
背： Profile:
心筋細胞は、 出生前は自律拍動しながら活発に細胞分裂を行っている。 しかし、 出生と同時にその分裂能は喪失し、 肝細胞のように再び細胞分裂能を獲得すること はなく、 また骨格筋細胞とも異なり衛星細胞といつた未分化な前駆細胞を持つこと もない。 従って、 心筋梗塞、 心筋炎または老化等に伴い心筋細胞が壊死すると、 生 体内では残存心筋細胞の細胞分裂ではなく細胞の肥大がおきる。 心肥大は初期にお いては生理的適応であるが、 また共存する心線維芽細胞の増殖による間質の線維化 と相まつて心臓自体の拡張機能の低下、 さらには収縮機能の低下へと結びつき心不 全を呈するようになる。 心筋梗塞等による心不全のこれまでの治療は心収縮力の増 強、 血管拡張薬による心臓の圧負荷 ·容量負荷の軽減、 利尿薬による血流量の減少 等の対症療法を中心に行われてきた。 これに対し、 心臓移植は重症心不全に対する 根本的な治療法であるが、 臓器提供者の不足、 脳死判定の難しさ、 拒絶反応、 医療 費の高騰等の問題から心臓移植が一般的な医療に普及するのは簡単ではない。実際、 心臓病は我が国の死亡原因の第 3位となっており （厚生白書平成 1 0年） 、 失われ た心筋細胞を再生することができれば医療福祉の大きな前進につながると考えられ る。 現在までに、 心筋細胞の性質を保存した細胞株としては、 心房性ナトリウム利尿 ホルモンのプロモーターに SV40の large T抗原を組み換えて作製したトランスジェ ニヅクマウスの心房に生じた腫瘍から株化された AT - 1細胞があげられる [Science, 239； 1029-1038ひ988)]。 しかしながら、 該細胞は in vivoに移植すると腫瘍を形成す るため、 細胞移植には適さないという問題がある。 そこで、 このような背景のもと、 心筋を再構築するため以下の方法が考えられた。 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. You develop heart failure. Until now, treatment for 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. . On the other hand, 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. To date, 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). However, there is a problem that 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.
1つ目の方法は、 心筋細胞以外の細胞を心筋細胞に変換する方法である。 これは、 線維芽細胞に MyoDを導入すると骨格筋細胞に変換できることから類推された。 こ れまでに、 マウスの胎児性癌細胞である P 1 9細胞での成功例は示されているもの の [Cell Struc. &Func, 21 : 101 - 110 (1996)]、 非ガン細胞での成功例は報告されていな い。  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.
2つ目の方法は、 心筋細胞に再び分裂能を付与する方法である。 これは、 胎児期 に心筋が拍動しながら分裂できる現象に基づいている。 しかしながら、 これまでに 成功例は報告されていない。  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.
3つ目の方法は、 未分化な幹細胞から心筋細胞を誘導する方法である。 すでに、 胚性幹細胞 （ES細胞） から心筋細胞を誘導できることが示されているが、 胚性幹細 胞自身を成体に移植するとカルシノーマを形成すること、 抗原性などの問題が存在 する [Nature Biotechnology, 17, 139 - 142 (1999)]。  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)].
従って、 胚性幹細胞を現実の医療へと応用するためには、 少なくとも心筋前駆細 胞あるいは、 心筋細胞を純粋に精製する技術が不可欠である。 抗原性の問題はクロ —ン化の技術により解決できる可能性は示唆されているが、 煩雑な操作を必要とす ることから一般的な医療への応用は容易ではない。  Therefore, in order to apply embryonic stem cells to real medicine, at least a technique for purifying myocardial progenitor cells or cardiomyocytes is essential. Although it has been suggested that the antigenicity problem can be solved by cloning technology, it is not easy to apply it to general medical care because it requires complicated operations.
中絶胎児から未分化な細胞である心筋前駆細胞を取得して移植に用いる方法も考 えられており、 動物を用いた実験では心筋細胞として有効に機能することが知られ ている [Science, ， 98-101 (1994)]。 しかしながら、 この方法で大量の心筋前駆細胞 を取得することは困難であり、 倫理の観点からも一般的な医療への応用は容易では ない。  A method of obtaining undifferentiated myocardial progenitor cells from aborted fetuses and using them for transplantation has also been considered, and it has been known that they function effectively as cardiomyocytes in experiments using animals [Science, 98-101 (1994)]. However, it is difficult to obtain a large amount of myocardial progenitor cells by this method, and application to general medical care is not easy from an ethical point of view.
成体骨髄には造血系幹細胞および血管幹細胞以外に間葉系幹細胞が存在し、 間葉 系幹細胞からは骨細胞、 軟骨細胞、 腱細胞、 靱帯細胞、 骨格筋細胞、 脂肪細胞、 ス ト口一マ細胞、 肝臓 oval細胞が分化誘導できることが報告されて 、る [Science, 284, 143-147 (1999); Science, 284, 1168 - 1170 (1999)] 0 一方、 最近、 マウス成体の骨髄から 取得した細胞から、 心筋細胞が分化誘導できることを見い出した [J. Clinical Adult bone marrow 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.
Investigation, 103, 10 - 18 (1"9)]。 該報告は患者自身から骨髄液を取得して、 in vitro で細胞培養および薬剤処理を行った後に、 心臓の障害部位へ移植する細胞治療が現 実的な医療として可能になることを示唆している D. Clinical Investigation, 103, 591- 592 (1"9)]。 しかしながら、該報告は、成体マウスの骨髄から樹立した不死化細胞 φ '一部が心筋細胞に分化できることを示したものにすぎない。 また、'成体骨髄中の心 筋細胞に分ィ匕する能力を有する細胞の特性の同定、 該細胞を増殖する方法、 該細胞 から効率的に心筋細胞に分化誘導する方法については明らかでなかった [J. Clinical Investigation, 103, 591-592 (1999)]。 Investigation, 103, 10-18 (1 " 9 )] The report reported that cell therapy involves obtaining bone marrow fluid from the patient himself, performing cell culture and drug treatment in vitro, and then transplanting it to the site of heart failure. D. Clinical Investigation suggesting that enables a realistic medical, 103, 591- 5 92 (1 "9)]. However, the report merely shows that a part of immortalized cells φ ′ established from the bone marrow of adult mice can differentiate into cardiomyocytes. Further, it was not clear how to identify the characteristics of cells having the ability to divide into cardiac muscle cells in adult bone marrow, how to grow the cells, and how to efficiently induce the cells to differentiate into cardiomyocytes. [J. Clinical Investigation, 103, 591-592 (1999)].
生体内の組織から目的の細胞を取得する方法として、 各種表面抗原を認識する抗 体が用いられている。例えば、未熟な造血幹細胞では CD 34 +/CD 38- HLA -DR-/CD 90 (Thy- 1) +の特性を有していること、 また、 造血幹細胞が 分化するに従い、 CD 38が発現し CD 90 (Thy- 1)が消失することが知られて いる [蛋白質核酸酵素 Vol.45, Nol3, 2056— 2062 (2000) ]。血管内皮 細胞では、 CD 34、 CD 31、 F Ik- I T i e— 2、 E—セレクチン等のマーカ —を発現しており [分子心血管病 V o 1. Ι,Νο.3, 294— 302 (2000)]、 骨髄の間葉系幹細胞では CD 90、 CD 105、 CD 140等のマーカ一を発現してい る [Science, 284, 143-147 (1999); Science, 284, 1168 - 1170 (1999)〗。 しかしながら、 心 筋や血管内皮細胞を誘導できる幹細胞の表面マーカ一については明らかにされてい ない。  Antibodies that recognize various surface antigens have been used as a method for obtaining target cells from tissues in a living body. For example, 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. Ι, .ο.3, 294—302 ( 2000)], bone marrow mesenchymal stem cells express markers such as CD90, CD105 and CD140 [Science, 284, 143-147 (1999); Science, 284, 1168-1170 (1999) 〗. However, surface markers of stem cells that can induce cardiac muscle and vascular endothelial cells have not been clarified.
発明の開示 Disclosure of the invention
現在の心疾患治療より安全かつ確実な治療が望まれている。 そこで、 骨髄細胞中 より心筋細胞への分ィヒ能を有する骨髄細胞を選別し、 心筋細胞への分ィ匕能を有する 骨髄細胞の増殖または分化をコントロールすることは、 骨髄由来の細胞を用いた心 筋の再生治療の開発に有用である。 そのために、 骨髄中の細胞から心筋細胞への分 化能を有する細胞を特定して、 該細胞の増殖または分化に働くサイトカインまたは 転写因子を同定することが必要である。 本発明者は上記問題点を開発すべく鋭意研究し、 以下の結果を得た。 すなわち、 マウス骨髄由来の細胞を 1細胞レベルにまず分離を行い、多数の細胞株を取得した。 これら細胞株を一つ一つ、 5—ァザシチジン処理を行うことで心筋形成能を有する 細胞株を複数取得した。 次に選られた細胞株を、 GFP(Green Fluorescent Protein)を発 現するレトロウイルスベクタ一を用いて標識し、 1つの細胞を蛍光顕微 下で追跡 することで、 '心筋細胞への分化能を有する細胞が、 心筋細胞および脂肪細胞の少な くとも 2種類の異なる細胞を分化誘導できる多分化能 (Pumlipotent)を持った幹細胞 であることを見い出した。 さらに、 該幹細胞は通常の培養条件下ではすでに報告さ れている 5—ァザシチジンだけでなく、 DMSO (dimethyl sulfoxide) などの他のゲノ ム DNAの脱メチル化剤の投与によっても、確率的（stochastic) に心筋細胞、脂肪細 胞および骨格筋細胞の系列に分化することを見出し、ゲノム DNAの脱メチル化が骨 髄由来の細胞からの心筋細胞への分化誘導に有効であることを明らかにした。 また F G F - 8 , E T 1 , M i d k i n e , B MP 4の 4種類のサイト力インをそれぞ れ 5—ァザシチジンと組み合わせて添加することで骨髄由来の細胞に心筋特異的な 遺伝子である ANP， cTnlの発現を促進できることを見出した。同様に Nkx2.5， GATA4 の 2種類の転写因子をウィルスベクターを用いて骨髄由来細胞に強制発現を行った 後、 5—ァザシチジン処理を行うことで、 心筋細胞への分化が約 5 0倍促進できる ことを見出した。 また骨髄由来の細胞を心筋細胞の細胞外基質をコ一トした培養皿 で培養することで、骨髄由来の細胞に心筋特異的な遺伝子である ANP, cTnlの発現を 特異的に促進できることを見出した。 さらに、 骨髄由来の細胞を心筋由来の初代培 養細胞と共培養を行うことで骨髄由来の細胞から心筋の形成が約 1 0倍促進するこ とを見出した。 また、 Nkx2.5, GATA4の 2種類の転写因子をウィルスベクタ一を用 いて骨髄由来細胞に強制発現させることと、 心筋細胞との共培養を組み合わせるこ とで、 約 5 0 0倍心筋への分ィヒが促進することを見出した。 A safer and more reliable treatment than the current treatment for heart disease is desired. Therefore, selecting 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. Next, 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. It has been found that 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. In addition, 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. ), And found that genomic DNA demethylation is effective in inducing differentiation of bone marrow-derived cells into cardiomyocytes. . By adding FGF-8, ET1, Midkine, and BMP4 in combination with 5-azacitidine, respectively, ANP and cTnl, which are myocardium-specific genes for bone marrow-derived cells, are added. Was found to be able to promote expression. Similarly, after forcibly expressing two transcription factors, Nkx2.5 and GATA4, in bone marrow-derived cells using a viral vector, and then treating with 5-azacitidine, differentiation into cardiomyocytes is promoted by about 50 times. I found out what I can do. We also found that by culturing bone marrow-derived cells in a culture dish coated with the extracellular matrix of cardiomyocytes, the expression of ANP and cTnl, which are myocardial-specific genes in bone marrow-derived cells, can be specifically promoted. Was. Furthermore, they found that co-culture of bone marrow-derived cells with myocardial-derived primary cultured cells promoted myocardial formation from bone marrow-derived cells by about 10-fold. In addition, by combining the expression of two transcription factors, Nkx2.5 and GATA4, in bone marrow-derived cells using a viral vector and by co-culturing with cardiomyocytes, it is possible to increase the number of We found that Eich promoted.
次に移植実験により、 骨髄由来の細胞の分ィ匕能力を検討した。 まずマウス成体心 臓に移植することで、 骨髄由来の細胞が心筋と血管に分化することを見出した。 さ らに成体マウスの筋肉に移植することで骨格筋を形成できることを見出した。 また マウス胚盤胞に移植すると、 誕生したマウスの中枢神経系、 肝臓、 心臓で移植した 細胞由来の組織が形成された。 これらの結果は、 本発明で見出した骨髄由来の細胞 が今まで知られていた骨髄中の造血幹細胞や間葉系幹細胞とは異なり、 外胚葉系、 中胚葉系ならびに内胚葉系の 3胚葉すべてに分化できる全能性を有していることを 示している。 Next, the ability of the bone marrow-derived cells to divide was examined by transplantation experiments. First mouse adult mind By transplanting it into the kidney, it was discovered that bone marrow-derived cells differentiate into myocardium and blood vessels. Furthermore, they found that skeletal muscle can be formed by transplantation into adult mouse muscle. When transplanted into mouse blastocysts, tissues derived from transplanted cells were formed in the central nervous system, liver, and heart of the born mice. These results show that the 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.
次に本発明で見出した骨髄由来の細胞を造血系細胞の表面抗原 CD 34、 CD 1 17、 CD 14、 CD 45、 CD 90、 S ea— 1、 Ly6 c、 Ly6 gを認識する抗体、 血管. 内皮細胞の表面抗原 F 1 k— 1、 CD 31、 CD 105、 CD 144を認識する抗体、 間 葉系細胞の表面抗原 CD 140を認識する抗体、ィンテグリン CD 49 b、 CD 49 d、 CD 29s CD 41を認識する抗体、 マトリヅクス受容体 CD 54、 CD 102、 CD 10 6、 CD 44を認識する抗体で該幹細胞の表面抗原の発現を解析することで、今まで に知られていない全く新しい発現形態を示していることを見出し、.本発明を完成さ せた。 すなわち、 本発明は以下の （1) 〜 （150) を提供するものである。  Next, 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 By analyzing the expression of the surface antigen of the stem cells with an antibody that recognizes CD41 and an antibody that recognizes the matrix receptors CD54, CD102, CD106, and CD44, a completely new expression that has not been known until now The present inventors have found that the present invention shows an embodiment, and have completed the present invention. That is, the present invention provides the following (1) to (150).
(1) 骨髄または臍帯血から単離され、 心筋細胞に分化する能力を有する細胞。 (1) Cells isolated from bone marrow or cord blood and capable of differentiating into cardiomyocytes.
(2) 細胞が、 少なくとも心筋細胞、 脂肪細胞、 骨格筋細胞、 骨芽細胞に分ィ匕す る能力を有する多分化能幹細胞である、 上記 （1) 記載の細胞。 (2) The cell according to (1), wherein the cell is a pluripotent stem cell having at least the ability to divide into cardiomyocytes, adipocytes, skeletal muscle cells, and osteoblasts.
(3) 細胞が、 少なくとも心筋細胞、 血管内皮細胞に分化する能力を有する多分 化能幹細胞である、 上記 （1) 記載の細胞。  (3) The cell according to the above (1), wherein the cell is a multipotent stem cell having the ability to differentiate into at least cardiomyocytes and vascular endothelial cells.
(4) 細胞が、 少なくとも心筋細胞、 脂肪細胞、 骨格筋細胞、 骨芽細胞、 血管内 皮細胞に分化する能力を有する多分化能幹細胞である、 上記 （1) 記載の細胞。  (4) The cell according to (1) above, wherein 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.
(5) 細胞が、 少なくとも心筋細胞、 脂肪細胞、 骨格筋細胞、 血管内皮細胞、 骨 芽細胞、 神経系細胞、 肝細胞に分化する能力を有する多分化能幹細胞である、 上記 (5) 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.
( 1 ) 記載の細胞。 (1) The cell according to (1).
(6) CD 34陰性、 CD 1 17陰性、 CD 144陰性および CD 140陽性である、 上記 （1) または （2) 記載の細胞。 (6) CD34 negative, CD117 negative, CD144 negative and CD140 positive, The cell according to (1) or (2) above.
(7) CD 34陽性、 CD 1 17陽性および CD 140陽性である、 上記 （1) また は （3) 記載の細胞。  (7) The cell according to (1) or (3) above, which is CD34 positive, CD117 positive, and CD140 positive.
(8) CD 34陽性、 CD 1 17陽性、 CD 144陽性および CD 140陽性である、 上記 （1) または （3) 記載の細胞。  (8) The cell according to (1) or (3), which is CD34 positive, CD117 positive, CD144 positive, and CD140 positive.
(9) CD 34陰性、 CD 1 17陽性、 CD 144陰性および CD 140陽性である、 上記 （1) 、 （4) または （5) 記載の細胞。  (9) The cell according to the above (1), (4) or (5), which is CD34 negative, CD117 positive, CD144 negative and CD140 positive.
(10) CD 1 17陽性および CD 140陽性である、上記（ 1 )、 (4)または（5) 記載の細胞。 ' · · '  (10) The cell according to (1), (4) or (5) above, which is CD117 positive and CD140 positive. '· ·'
(11) CD 34陰性、 CD 117陰性、 CD 14陽性、 CD 45陰性、 CD 90陰性、 Flk- 1陰性、 CD 31陰性、 CD 105陰性、 CD 144陰性、 CD 140 P昜性、 CD 49 b陽性、 CD 49 d陰性、 CD 29陽性、 CD 54陽性、 CD 102陰性、 CD 106 陽性および CD 44陽性である、 上記 （2) 記載の細胞。  (11) CD34 negative, CD117 negative, CD14 positive, CD45 negative, CD90 negative, Flk-1 negative, CD31 negative, CD105 negative, CD144 negative, CD140 positive, CD49b positive The cell according to (2), which is CD49 d-negative, CD29-positive, CD54-positive, CD102-negative, CD106-positive, and CD44-positive.
(12) CD 34陽性、 CD 117陽性、 CD 14陰性、 CD 45陰性、 CD 90陰性、 F Ik— 1陰性、 CD 31陰性、 CD 105陰性、 CD 144陽性、 CD 140陽性、 CD 49 b陰性、 CD 49 d陰性、 CD 29陽性、 CD 54陰性、 CD 102陰性、 CD 106 陰性および CD 44陽性である、 上記 （3) 記載の細胞。  (12) CD34 positive, CD117 positive, CD14 negative, CD45 negative, CD90 negative, FIk-1 negative, CD31 negative, CD105 negative, CD144 positive, CD140 positive, CD140b negative, The cell according to (3), which is CD49 d-negative, CD29-positive, CD54-negative, CD102-negative, CD106-negative, and CD44-positive.
(13) Hoechst 33342を取り込まない、 上記 （1) 記載の細胞。  (13) The cell according to (1), which does not take up Hoechst 33342.
(14) 上記 （1) 〜 （13) のいずれか 1項に記載の細胞から誘導される心筋 細胞のみに分化誘導される心筋前駆細胞。  (14) Cardiac progenitor cells that are induced to differentiate only into cardiomyocytes derived from the cells according to any one of (1) to (13).
(15) 心室筋細胞に分ィ匕する能力を有する、 上記 （1) 〜 （14) のいずれか 1項に記載の細胞。  (15) The cell according to any one of (1) to (14), which has an ability to divide into ventricular myocytes.
(16) 洞結節細胞に分ィ匕する能力を有する、 上記 （1) 〜 （14) のいずれか (16) Any of (1) to (14) above, which has an ability to divide into sinus node cells.
1項に記載の細胞。 ' The cell according to item 1. '
(17) 骨髄または臍帯血がほ乳動物由来のものである、 上記 （1) 〜 （16) のいずれか 1項に記載の細胞。  (17) The cell according to any one of (1) to (16), wherein the bone marrow or cord blood is derived from a mammal.
(18) ほ乳動物がヒト、 ラットおよびマウスから選ばれるものである、上記（1 7 ) 記載の細胞。 (19) 細胞が、 マウス骨髄由来多分化能幹細胞 BMSC(FERM BP - 7043)である、 上記 （ 1 ) に記載の細胞。 (18) The cell according to the above (17), wherein the mammal is selected from human, rat and mouse. (19) The cell according to (1), wherein the cell is a mouse bone marrow-derived multipotent stem cell BMSC (FERM BP-7043).
(20) 染色体 DNAの脱メチル化により心筋細胞に分化する能力を有する、 上記 (1) 〜 （19) のいずれか 1項に記載の細胞。  (20) The cell according to any one of (1) to (19), which has an ability to differentiate into a cardiomyocyte by demethylation of chromosomal DNA.
(21) 染色体 DNAの脱メチル化が、 デメチラーゼ、 5―ァザシチジンおよびジ メチルスルフォキシド（DMSO)からなる群から選ばれる少なくとも 1種によるもの' であることを特徴とする、 上記 （20)記載の細胞。  (21) The method according to (20), wherein the demethylation of chromosomal DNA is at least one selected from the group consisting of demethylase, 5-azacitidine and dimethyl sulfoxide (DMSO). Cells.
(22) デメチラーゼが、 配列番号 1記載で表されるアミノ酸配列を有するデメ チラーゼである、 上記 （21)記載の細胞。  (22) The cell according to (21), wherein the demethylase is a demethylase having an amino acid sequence represented by SEQ ID NO: 1.
(23) 胎児の心臓発生領域で発現している因子により心筋細胞への分化が促進 される上記 （ 1 ) 〜 （19) のいずれか 1項に記載の細胞。  (23) The cell according to any one of the above (1) to (19), wherein differentiation into cardiomyocytes is promoted by a factor expressed in a fetal heart development region.
(24) 胎児の心臓発生領域で発現している因子がサイトカイン、 接着分子、 ビ 夕ミン、 転写因子および細胞外基質からなる群から選ばれる少なくとも 1種である ことを特徴とする、 上記 （23)記載の細胞。 ' (24) 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. ). '
(25) 胎児の心臓発生段階において心筋細胞への分ィヒに働く因子により心筋細 胞への分化が促進される上記 （1) 〜 （19) いずれか 1項に記載の細胞。 (25) The cell according to any one of (1) to (19) above, wherein differentiation into myocardial cells is promoted by a factor that acts on myocardial cells at the stage of fetal heart development.
(26) 胎児の心臓発生段階において心筋細胞への分化に働く因子がサイトカイ ン、 接着分子、 ビタミン、 転写因子および細胞外基質からなる群から選ばれる少な くとも 1種であることを特徴とする、 上記 （25)記載の細胞。  (26) 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. The cell according to (25) above.
(27) サイトカインが血小板由来増殖因子（PDGF) である、 上記（24) また は （26)記載の細胞。  (27) The cell according to (24) or (26), wherein the cytokine is platelet-derived growth factor (PDGF).
(28) PDGFが配列番号 3または 5で表されるァミノ酸配列を有する PDGFで ある、、上記 （27)記載の細胞。  (28) The cell according to (27) above, wherein the PDGF is PDGF having an amino acid sequence represented by SEQ ID NO: 3 or 5.
(29) サイトカインが繊維芽細胞増殖因子 8 (FGF-8) である、 上記 （24) または （26)記載の細胞。  (29) The cell according to (24) or (26), wherein the cytokine is fibroblast growth factor 8 (FGF-8).
(30) FGF-8が配列番号 64で表されるアミノ酸配列を有する FGF-8である、 上記 （ 29 )記載の細胞。  (30) The cell according to (29), wherein FGF-8 is FGF-8 having an amino acid sequence represented by SEQ ID NO: 64.
(31) サイトカインがエンドセリン 1(ET1)である、 上記（24)または（26) 記載の細胞。 (31) The above (24) or (26), wherein the cytokine is endothelin 1 (ET1) A cell as described.
(32) ET1が配列番号 66で表されるアミノ酸配列を有する ETlである、 上記 (31) 記載の細胞。  (32) The cell according to (31), wherein ET1 is ET1 having an amino acid sequence represented by SEQ ID NO: 66.
(33) サイトカインがミドカイン (Midkine)である、 上記 （24) または （26) 記載の細胞。  (33) The cell according to (24) or (26), wherein the cytokine is midkine.
(34) ミドカインが配列番号 68で表されるアミノ酸配列を有するミドカイン である、 上記（33) 記載の細胞。  (34) The cell according to the above (33), wherein the midkine is a midkine having an amino acid sequence represented by SEQ ID NO: 68.
(35) サイトカインが骨形成因子 4 (BMP- 4)である、上記（24)または（26) 記載の細胞。  (35) The cell according to (24) or (26) above, wherein the cytokine is bone morphogenetic factor 4 (BMP-4).
(36) BMP- 4が配列番号 70で表されるァミノ酸配列を有する BMP-4である、 上記 （ 35 ) 記載の細胞。  (36) The cell according to the above (35), wherein BMP-4 is BMP-4 having an amino acid sequence represented by SEQ ID NO: 70.
(37) 接着分子がフイブロネクチンである、 上記 （24) または （26) 記載 の細胞。  (37) The cell according to the above (24) or (26), wherein the adhesion molecule is fibronectin.
(38) ビタミンがレチノイ 酸である、 上記 （24) または （26) 記載の細 胞。  (38) The cell according to (24) or (26), wherein the vitamin is retinoic acid.
(39) 転写因子が、 Nkx2.5/Csx、 GATA4、 MEF- 2A、 MEF - 2B、 MEF_2C、 MEF- 2Dヽ dHAND、 eHAND、 TEF- 1、 TEF- 3、 TEF- 5および MesPlからなる群から選ばれるも のである、 上記 （24) または （26) 記載の細胞。  (39) The transcription factor is selected from the group consisting of Nkx2.5 / Csx, GATA4, MEF-2A, MEF-2B, MEF_2C, MEF-2D ヽ dHAND, eHAND, TEF-1, TEF-3, TEF-5 and MesPl. The cell according to (24) or (26), which is selected.
(40) Nkx2.5/Csxが配列番号 9で表されるアミノ酸配列を有する Nkx2.5/Csx である、 上記 （39) 記載の細胞。  (40) The cell according to (39), wherein Nkx2.5 / Csx is Nkx2.5 / Csx having the amino acid sequence represented by SEQ ID NO: 9.
(41) GATA4が配列番号 11で表されるァミノ酸配列を有する GATA4である、 上記 （ 39 ) 記載の細胞。  (41) The cell according to the above (39), wherein GATA4 is GATA4 having an amino acid sequence represented by SEQ ID NO: 11.
(42) MEF - 2Aが配列番号 13で表されるアミノ酸配列を有する MEF-2Aであ る、 上記 （ 39 ) 記載の細胞。  (42) The cell according to the above (39), wherein MEF-2A is MEF-2A having an amino acid sequence represented by SEQ ID NO: 13.
(43) MEF-2Bが配列番号 15で表されるァミノ酸配列を有する MEF-2Bであ る、 上記 （ 39 ) 記載の細胞。  (43) The cell according to (39), wherein MEF-2B is MEF-2B having an amino acid sequence represented by SEQ ID NO: 15.
(44) MEF - 2Cが配列番号 17で表されるアミノ酸配列を有する MEF-2Cであ る、 上記 （39) 記載の細胞。 (45) MEF-2Dが配列番号 19で表されるアミノ酸配列を有する MEF - 2Dであ る、 上記 （39)記載の細胞。 (44) The cell according to (39), wherein MEF-2C is MEF-2C having an amino acid sequence represented by SEQ ID NO: 17. (45) The cell according to (39) above, wherein MEF-2D is MEF-2D having an amino acid sequence represented by SEQ ID NO: 19.
(46) dHANDが配列番号 21で表されるァミノ酸配列を有する dHANDである、 上記 （ 39 ) 記載の細胞。  (46) The cell according to the above (39), wherein the dHAND is a dHAND having an amino acid sequence represented by SEQ ID NO: 21.
(47) eHANDが配列番号 23で表されるァミノ酸配列を有する eHANDである、 上記 （39.)記載の細胞。  (47) The cell according to the above (39.), wherein the eHAND is an eHAND having an amino acid sequence represented by SEQ ID NO: 23.
(48) TEF-1が配列番号 25で表されるアミノ酸配列を有する TEF-1である、 上記 （39)記載の細胞。 ■  (48) The cell according to (39) above, wherein TEF-1 is TEF-1 having an amino acid sequence represented by SEQ ID NO: 25. ■
(49) TEF-3が配列番号 27で表されるアミノ酸配列を有する TEF-3である、 上記 （ 39 ) 記載の細胞。  (49) The cell according to (39), wherein TEF-3 is TEF-3 having an amino acid sequence represented by SEQ ID NO: 27.
(50) TEF-5が配列番号 29で表されるァミノ酸配列を有する TEF- 5である、 上記 （ 39 ) 記載の細胞。  (50) The cell according to the above (39), wherein TEF-5 is TEF-5 having an amino acid sequence represented by SEQ ID NO: 29.
(51) MesPlが配列番号 62で表されるアミノ酸配列を有する MesPlである、 上記 （ 39 ) 記載の細胞。.  (51) The cell according to (39), wherein MesPl is MesPl having the amino acid sequence represented by SEQ ID NO: 62. .
(52) 細胞外基質が心筋細胞由来の細胞外基質であることを特徴とする上記（2 4) または （26)記載の細胞。  (52) The cell according to the above (24) or (26), wherein the extracellular matrix is a cardiomyocyte-derived extracellular matrix.
(53) 線維芽細胞増殖因子— 2 (FGF-2) により心筋細胞への分化が抑制され る上記 （ 1 ) 記載の細胞。  (53) The cell according to (1), wherein differentiation into cardiomyocytes is suppressed by fibroblast growth factor-2 (FGF-2).
(54) FGF-2が配列番号 7または 8記載のアミノ酸配列を有する FGF-2である、 上記 （ 53 ) 記載の細胞。  (54) The cell according to (53), wherein FGF-2 is FGF-2 having the amino acid sequence of SEQ ID NO: 7 or 8.
(55) 心臓に移植することにより心筋細胞に分ィ匕する能力を有する上記 （1) 〜 （19) のいずれか 1項に記載の細胞。  (55) The cell according to any one of the above (1) to (19), which has an ability to divide into cardiomyocytes by transplanting into a heart.
(56) 心臓に移植することにより血管に分化する能力を有する上記（1)〜（1 9) のいずれか 1項に記載の細胞。  (56) The cell according to any one of the above (1) to (19), which has a capability of differentiating into a blood vessel when transplanted into a heart.
(57) 胚盤胞に移植することで、心筋に分化する能力を有する上記（1)〜（1 9)のいずれか 1項に記載の細胞。  (57) The cell according to any one of the above (1) to (19), which has the ability to differentiate into myocardium by transplantation into a blastocyst.
(58) 心筋細胞と共培養を行うことで、 心筋に分ィ匕する能力を有する上記（ 1 ) ~ (19)のいずれか 1項に記載の細胞。 (59) 核内受容体 PPAR -ァを活性化因子により脂肪細胞に分ィ匕す？)能力を有す る上記 （1) 〜 （19) のいずれか 1項に記載の細胞。 (58) The cell according to any one of the above (1) to (19), which has an ability to divide into a myocardium by co-culturing with a cardiomyocyte. (59) Is nuclear receptor PPAR-a shunted to adipocytes by activator? ) The cell according to any one of the above (1) to (19), which has an ability.
(60) 核内受容体 PPAR-ァの活性化因子がチアゾリジオン骨格を有する化合物 であることを特徴とする上記 （59)記載の細胞。  (60) The cell according to the above (59), wherein the activator of the nuclear receptor PPAR-a is a compound having a thiazolidione skeleton.
(61) チアゾリジオン骨格を有する化合物がトログリ夕ゾン、 ピオグリ夕ゾン、 口ジグリ夕ゾンからなる群から選ばれる少なくとも 1種であることを特徴とする上 記 （ 60 )記載の細胞。  (61) The cell according to the above (60), wherein the compound having a thiazolidione skeleton is at least one selected from the group consisting of troglisuzone, pioglisuzone, and oral diglysazone.
(62) 胚盤胞に移植することで、 神経系細胞に分化する能力を有する上記（ 1) 〜 '（ 19 ) のいずれか 1項に記載の細胞。  (62) The cell according to any one of (1) to (19) above, which has the ability to differentiate into a nervous system cell by transplantation into a blastocyst.
(63) 脳または脊髄に移植することで、 神経系細胞に分化する能力を有する上 記 （1) 〜 （19) のいずれか 1項に記載の細胞。  (63) The cell according to any one of the above (1) to (19), which has a capability of differentiating into a nervous system cell when transplanted into the brain or spinal cord.
(64) 胚盤胞に移植することで、 肝細胞に分化する能力を有する上記 （1) 〜 (19) のいずれか.1項に記載の細胞。  (64) The cell according to any one of (1) to (19) above, which has the ability to differentiate into hepatocytes by transplantation into blastocysts.
(65) 肝臓に移植することで肝細胞に分化する能力を有する上記 （1) 〜 （1 9)のいずれか 1項に記載の細胞。  (65) The cell according to any one of (1) to (19) above, which has a capability of differentiating into a hepatocyte by transplanting into the liver.
(66) 染色体 DNAの脱メチル化剤を用いて、 骨髄由来の細胞から心筋を形成す る方法。  (66) A method of forming myocardium from bone marrow-derived cells using a chromosomal DNA demethylating agent.
(67) 染色体 DNAの脱メチル化剤が、 デメチラ一ゼ、 5—ァザシチジンおよび DMSOからなる群から選ばれる少なくとも 1種であることを特徴とする、 上記 （6 6 )記載の方法。  (67) The method according to the above (66), wherein the demethylating agent for chromosomal DNA is at least one selected from the group consisting of demethylase, 5-azacitidine and DMSO.
(68) デメチラ一ゼが、 配列番号 1記載のアミノ酸配列で表されるデメチラ一 ゼである、 上記 （67)記載の方法。  (68) The method according to (67) above, wherein the demethylase is a demethylase represented by the amino acid sequence of SEQ ID NO: 1.
(69) 胎児の心臓発生領域で発現している因子を用いることを特徴とする、 骨 髄由来の細胞から心筋を形成する方法。  (69) A method for forming myocardium from bone marrow-derived cells, comprising using a factor expressed in a fetal heart development region.
(70) 胎児の心臓発生領域で発現している因子がサイト力イン、 接着分子、 ビ 夕ミン、 転写因子および細胞外基質からなる群から選ばれる少なくとも 1種である ことを特徴とする、 上記 （69)記載の方法。 '  (70) 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. The method according to (69). '
(71) 胎児の心臓発生段階において心筋細胞への分ィ匕に働く因子を用いること を特徴とする、 骨髄由来の細胞から心筋を形成する方法。 (71) Use of factors that act to divide myocardial cells during the fetal heart development stage A method for forming a myocardium from bone marrow-derived cells.
(72) 胎児の心臓発生段階において心筋細胞への分化に働く因子がサイトカイ ン、 接着分子、 ビタミン、 転写因子および細胞外基質からなる群から達ばれる少な くとも 1種であることを特徴とする、 上記 （71)記載の方法。  (72) 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.
(73) サイトカインが PDGFである、 上記 （70) または （72)記載の方法。 (74) PDGFが配列番号 3または 5記載のアミノ酸配列で表される PDGFであ る、 上記 （63)記載の方法。  (73) The method according to (70) or (72), wherein the cytokine is PDGF. (74) The method according to (63) above, wherein PDGF is PDGF represented by the amino acid sequence of SEQ ID NO: 3 or 5.
(75) サイトカインが繊維芽細胞増殖因子 8 (FGF-8) である、 上記 （70) または （72)記載の方法。  (75) The method according to (70) or (72) above, wherein the cytokine is fibroblast growth factor 8 (FGF-8).
(76) FGF-8が配列番号 64のァミノ酸配列で表される FGF- 8である、上記（ 7 5)記載の方法。  (76) The method according to the above (75), wherein FGF-8 is FGF-8 represented by the amino acid sequence of SEQ ID NO: 64.
(77) サイトカインがェンドセリン 1(ET1)である、上記（70)または（72) 記載の方法。  (77) The method according to the above (70) or (72), wherein the cytokine is endoselin 1 (ET1).
(78) ET1が配列番号 66で表されるアミノ酸配列を有する ET1である、 上記 (78) The ET1 having the amino acid sequence represented by SEQ ID NO: 66, wherein ET1 is
(77)記載の方法。 The method according to (77).
(79) サイトカインがミドカイン (Midkine)である、 上記 （70) または （72) 記載の方法。  (79) The method according to the above (70) or (72), wherein the cytokine is midkine.
(80) ミドカインが配列番号 68で表されるアミノ酸配列を有するミドカイン である、 上記 （79)記載の方法。  (80) The method according to (79) above, wherein the midkine is a midkine having an amino acid sequence represented by SEQ ID NO: 68.
(81) サイトカインが骨形成因子 4 (BMP - 4)である、上記（70)または（72) 記載の方法。  (81) The method according to (70) or (72) above, wherein the cytokine is bone morphogenetic factor 4 (BMP-4).
(82) BMP-4が配列番号 70で表されるァミノ酸配列を有する BMP - 4である、 上記 （ 81 )記載の方法。  (82) The method according to (81), wherein BMP-4 is BMP-4 having an amino acid sequence represented by SEQ ID NO: 70.
(83) 接着分子がフイブロネクチンである、 上記 （70) または （72)記載 の方法。  (83) The method according to (70) or (72) above, wherein the adhesion molecule is fibronectin.
(84) ビタミンがレチノイン酸である、 上記 （70) または （72)記載の方 法。  (84) The method according to the above (70) or (72), wherein the vitamin is retinoic acid.
(85) 転写因子が、 Nkx2.5/Csxヽ GATA4、 MEF - 2A、 MEF- 2B、 MEF - 2C、 MEF- 2D、 dHAND、 eHAND、 TEF- 1、 TEF-3、 TEF- 5および MesPlからなる群から選ばれる、 上記 （70) または （72)記載の方法。 (85) The transcription factor is Nkx2.5 / Csx ヽ 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.
(86) Nkx2.5/Csxが、 配列番号 9で表されるァミノ酸配列を有する Nkx2.5/Csx である、 上記 '(85)記載の方法。  (86) The method according to the above (85), wherein Nkx2.5 / Csx is Nkx2.5 / Csx having an amino acid sequence represented by SEQ ID NO: 9.
(87) GATA4が、 配列番号 11で表されるアミノ酸配列を有する GATA4であ る、 上記 （85)記載の方法。  (87) The method according to (85), wherein GATA4 is GATA4 having the amino acid sequence represented by SEQ ID NO: 11.
(88) MEF-2Aが、 配列番号 13で表されるァミノ酸配列を有する MEF - 2Aで ある、 上記 （85)記載の方法。  (88) The method according to (85) above, wherein MEF-2A is MEF-2A having an amino acid sequence represented by SEQ ID NO: 13.
(89) MEF- 2Bが、 配列番号 15で表されるアミノ酸配列を有する MEF— 2 Bである、 上記 （85)記載の方法。  (89) The method according to (85), wherein MEF-2B is MEF-2B having the amino acid sequence represented by SEQ ID NO: 15.
(90) MEF-2Cが、 配列番号 17で表されるァミノ酸配列を有する MEF - 2Cで ある、 上記 （85)記載の方法。  (90) The method according to the above (85), wherein MEF-2C is MEF-2C having an amino acid sequence represented by SEQ ID NO: 17.
(91) MEF-2Dが、 配列番号 19で表されるァミノ酸配列を有する MEF - 2Dで ある、 上記 （85)記載の方法。  (91) The method according to (85), wherein MEF-2D is MEF-2D having an amino acid sequence represented by SEQ ID NO: 19.
(92) dHANDが、 配列番号 21で表されるァミノ酸配列を有する dHANDであ る、 上記 （85)記載の方法。  (92) The method according to the above (85), wherein the dHAND is a dHAND having an amino acid sequence represented by SEQ ID NO: 21.
(93) eHANDが、 配列番号 23で表されるァミノ酸配列を有する eHANDであ る、 上記 （ 85 )記載の方法。  (93) The method according to the above (85), wherein the eHAND is an eHAND having an amino acid sequence represented by SEQ ID NO: 23.
(94) TEF- 1が、 配列番号 25で表されるァミノ 配列を有する TEF - 1である、 上記 （ 85 ) 記載の方法。  (94) The method according to the above (85), wherein TEF-1 is TEF-1 having an amino sequence represented by SEQ ID NO: 25.
(95) TEF - 3が、 配列番号 27で表されるアミノ酸配列を有する TEF- 3である、 上記 （ 85 ) 記載の方法。  (95) The method according to (85), wherein TEF-3 is TEF-3 having an amino acid sequence represented by SEQ ID NO: 27.
(96) TEF-5が、 配列番号 29で表されるァミノ酸配列を有する TEF- 5である、 上記 （ 85 ) 記載の方法。  (96) The method according to (85), wherein TEF-5 is TEF-5 having an amino acid sequence represented by SEQ ID NO: 29.
(97) MesPlが、配列番号 62で表されるアミノ酸配列を有する MesPlである、 上記 （85)記載の方法。  (97) The method according to (85) above, wherein MesPl is a MesPl having an amino acid sequence represented by SEQ ID NO: 62.
(98) 細胞外基質が心筋細胞由来の細胞外基質であることを特徴とする上記（7 0) または （72)記載の方法。 (99) 核内受容体 PPAR-ァを活性化因子により骨髄由来の細胞から脂肪細胞を 形成する方法。 (98) The method according to the above (70) or (72), wherein the extracellular matrix is a cardiomyocyte-derived extracellular matrix. (99) A method for forming adipocytes from cells derived from bone marrow using an activator of nuclear receptor PPAR-a.
(100) 核内受容体 PPAR-ァの活性化因子がチアゾリジオン骨格を有する化合 物であることを特徴とする上記 （99)記載の方法。  (100) The method according to the above (99), wherein the activator of nuclear receptor PPAR-a is a compound having a thiazolidione skeleton.
(101) チアゾリジオン骨格を有する化合物がトログリ夕ゾン、 ピオグリ夕ゾ ン、 ロジグリタゾンからなる群から選ばれる少なくとも 1種であることを特徴とす る上記 （100)記載の方法。  (101) The method according to the above (100), wherein the compound having a thiazolidione skeleton is at least one selected from the group consisting of troglisuzone, pioglisuzone, and rosiglitazone.
(102) 染色体 DNAの脱メチル化剤を有効成分として含有することを特徴とす る心筋形成剤。  (102) A cardiomyogenic agent comprising a chromosomal DNA demethylating agent as an active ingredient.
(103) 染色体 DNAの脱メチル化剤がデメチラ一ゼ、 5—ァザシチジンおよび DMSOからなる群から選ばれる少なくとも 1種である、 上記 （102)記載の心筋 形成剤。  (103) The cardiomyogenic agent according to (102), wherein the chromosomal DNA demethylating agent is at least one selected from the group consisting of demethylase, 5-azacitidine and DMSO.
(104) デメチラ一ゼが、 配列番号 1記載のアミノ酸配列で表されるデメチラ —ゼである、 上記 （ 103)記載の心筋形成剤。  (104) The cardiomyogenic agent according to (103), wherein the demethylase is a demethylase represented by the amino acid sequence of SEQ ID NO: 1.
(105) 胎児の心臓発生領域で発現している因子を有効成分として含有する心 筋形成剤。  (105) A cardiomyogenic agent comprising, as an active ingredient, a factor expressed in a fetal heart development region.
(106) 胎児の心臓発生領域で発現している因子がサイトカイン、 接着分子、 ビタミン転写因子および細胞外基質からなる群から選ばれる少なくとも 1種である ことを特徴とする、 上記 （ 105)記載の心筋形成剤。  (106) 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.
(107) 胎児の心臓発生段階において心筋細胞への分化に働く因子を有効成分 として含有することを特徴とする心筋形成剤。  (107) A cardiomyogenic agent comprising, as an active ingredient, a factor that acts on differentiation into cardiomyocytes at the stage of fetal heart development.
(108) 胎児の心臓発生段階において心筋細胞への分化に働く因子がサイト力 イン、 接着分子、 ビタミン、 転写因子および細胞外基質からなる群から選ばれる少 なくとも 1種であることを特徴とする、 上記 （107)記載の心筋形成剤。  (108) characterized in that at least one of the factors acting on the differentiation into cardiomyocytes during the fetal heart development stage is selected from the group consisting of cytokins, adhesion molecules, vitamins, transcription factors and extracellular matrix. The cardiomyogenic agent according to (107).
(109) サイトカインが PDGFである、 上記 （106) または （108)記載 の心筋形成剤。  (109) The cardiomyogenic agent according to (106) or (108), wherein the cytokine is PDGF.
(110) PDGFが配列番号 3または 5記載のアミノ酸配列で表される、上記（1 09 )記載の心筋形成剤。 (111) サイトカインが繊維芽細胞増殖因子 8 (FGF-8) である、 上記 （ 10 6) または （108)記載の心筋形成剤。 (110) The cardiomyogenic agent according to (109), wherein PDGF is represented by the amino acid sequence of SEQ ID NO: 3 or 5. (111) The cardiomyogenic agent according to (106) or (108), wherein the cytokine is fibroblast growth factor 8 (FGF-8).
(112) FGF-8が配列番号 64のァミノ酸配列で表される FGF - 8である、 上記 (111)記載の心筋形成剤。  (112) The cardiomyogenic agent according to (111), wherein FGF-8 is FGF-8 represented by the amino acid sequence of SEQ ID NO: 64.
(113.) サイトカインがェンドセリン 1(ET1)である、上言己（106)または（1 08 )記載の心筋形成剤。  (113.) The cardiomyogenic agent according to (106) or (108), wherein the cytokine is endoselin 1 (ET1).
(114) ET1が配列番号 66で表されるアミノ酸配列を有する ET1である、 上 記 （113)記載の心筋形成剤。  (114) The cardiomyogenic agent according to (113), wherein ET1 is an ET1 having an amino acid sequence represented by SEQ ID NO: 66.
(115) サイトカインがミドカイン (Midkine)である、 上記（106)または（1 08 )記載の心筋形成剤。  (115) The cardiomyogen according to (106) or (108), wherein the cytokine is midkine.
(116) ミドカインが配列番号 68で表されるアミノ酸配列を有するミドカイ ンである、 上記 （ 115)記載の心筋形成剤。  (116) The cardiomyogenic agent according to (115), wherein the midkine is a midkine having an amino acid sequence represented by SEQ ID NO: 68.
(117) サイ小カインが骨形成因子 4 (BMP - 4)である、上記（ 106)または（ 1 08 )記載の心筋形成剤。  (117) The cardiomyogenic agent according to (106) or (108), wherein the rhinocerosine is bone morphogenetic factor 4 (BMP-4).
(118) BMP- 4が配列番号 70で表されるアミノ酸配列を有する BMP-4である、 上記 （117)記載の心筋形成剤。  (118) The cardiomyogenic agent according to (117), wherein BMP-4 is BMP-4 having the amino acid sequence represented by SEQ ID NO: 70.
(119) 接着分子がフイブロネクチンである、 上記（106) または （108) 記載の心筋形成剤。  (119) The cardiomyogenic agent according to (106) or (108), wherein the adhesion molecule is fibronectin.
(120) ビタミンがレチノイン酸である、 上記 （106) または （108)記 載の心筋形成剤。  (120) The cardiomyogenic agent according to the above (106) or (108), wherein the vitamin is retinoic acid.
(121) 転写因子が、 Nkx2.5/Csx, GATA4、 MEF- 2A、 MEF- 2Bヽ MEF- 2C、 MEF-2D、 dHAND、 eHAND、 TEF- 1、 TEF - 3、 TEF- 5および MesPlからなる群から選ばれる、 上記 （ 1◦ 6) または （ 108)記載の心筋形成剤。  (121) Transcription factor consists of Nkx2.5 / Csx, GATA4, MEF-2A, MEF-2B ヽ MEF-2C, MEF-2D, dHAND, eHAND, TEF-1, TEF-3, TEF-5 and MesPl The cardiomyogenic agent according to the above (1 6) or (108), which is selected from a group.
(122) Nkx2.5/Csxが、 配列番号 9記載のァミノ酸配列で表される Nkx2.5/Csx である、 上記 （ 121)記載の心筋形成剤。  (122) The cardiomyogenic agent according to (121), wherein Nkx2.5 / Csx is Nkx2.5 / Csx represented by the amino acid sequence of SEQ ID NO: 9.
(123) GATA4が、 配列番号 11記載のァミノ酸配列で表される GATA4であ る、 上記 （ 121 ) 記載の心筋形成剤。  (123) The cardiomyogenic agent according to (121), wherein GATA4 is GATA4 represented by the amino acid sequence of SEQ ID NO: 11.
(124) MEF-2Aが、 配列番号 13記載のァミノ酸配列で表される MEF - 2Aで ある、 上記 （ 121 ) 記載の心筋形成剤。 (124) MEF-2A is MEF-2A represented by the amino acid sequence set forth in SEQ ID NO: 13. The myocardial agent according to (121).
(125) MEF-2Bが、 配列番号 15記載のァミノ酸配列で表される MEF- 2Bで ある、 上記 （ 121 ) 記載の心筋形成剤。  (125) The cardiomyogenic agent according to (121), wherein MEF-2B is MEF-2B represented by the amino acid sequence of SEQ ID NO: 15.
(126) MEF-2Cが、 配列番号 17記載のァミノ酸配列で表される MEF- 2Cで ある、 上記 （ 121 ) 記載の心筋形成剤。  (126) The cardiomyogenic agent according to (121), wherein MEF-2C is MEF-2C represented by the amino acid sequence of SEQ ID NO: 17.
(127) MEF-2Dが、 配列番号 19記載のァミノ酸配列で表される MEF-2Dで ある、 上記 （ 121 ) 記載の心筋形成剤。  (127) The cardiomyogenic agent according to (121), wherein MEF-2D is MEF-2D represented by the amino acid sequence of SEQ ID NO: 19.
(128) dHANDが、 配列番号 21記載のアミノ酸配列で表される dHANDであ る、 上記 （121.)記載の心筋形成剤。  (128) The cardiomyogenic agent according to (121.), wherein the dHAND is a dHAND represented by the amino acid sequence of SEQ ID NO: 21.
(129) eHANDが、 配列番号 23記載のアミノ酸配列で表される eHANDであ る、 上記 （ 121 ) 記載の心筋形成剤。  (129) The cardiomyogenic agent according to (121), wherein eHAND is eHAND represented by the amino acid sequence of SEQ ID NO: 23.
(130) TEF-1が、 配列番号 25記載のアミノ酸配列で表される TEF - 1である、 上記 （ 121 ) 記載の心筋形成剤。  (130) The cardiomyogenic agent according to (121), wherein TEF-1 is TEF-1 represented by the amino acid sequence of SEQ ID NO: 25.
(131) TEF-3が、 配列番号 27記載のアミノ酸配列で表される TEF- 3である、 上記 （ 121 ) 記載の心筋形成剤。  (131) The cardiomyogenic agent according to (121), wherein TEF-3 is TEF-3 represented by the amino acid sequence of SEQ ID NO: 27.
(132) TEF - 5が、 配列番号 29記載のアミノ酸配列で表される TEF- 5である、 上記 （ 121 ) 記載の心筋形成剤。  (132) The cardiomyogen according to (121), wherein TEF-5 is TEF-5 represented by the amino acid sequence of SEQ ID NO: 29.
(133) MesPlが、配列番号 62記載のアミノ酸配列で表される MesPlである、 上記 （ 121 ) 記載の心筋形成剤。  (133) The cardiomyogenic agent according to (121), wherein MesPl is MesPl represented by the amino acid sequence of SEQ ID NO: 62.
(134) 細胞外基質が心筋細胞由来の細胞外基質であることを特徴とする上記 (134) wherein the extracellular matrix is a cardiomyocyte-derived extracellular matrix
( 106 ) または （ 108 )記載の心筋形成剤。 ， (106) or (108). ,
(135) 上記 （1) ~ (65) のいずれか 1項に記載の細胞を用いることを特 徴とする、 心臓疾患により破壊された心臓を再生する方法。  (135) 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).
(136) 上記 （1) 〜 （65) のいずれか 1項に記載の細胞を有効成分とする 心臓再生治療薬。  (136) A therapeutic agent for regenerating heart, comprising the cell according to any one of (1) to (65) as an active ingredient.
(137) 心臓の先天性遺伝子疾患での変異遺伝子に対する野生型遺伝子が導入 された上記 （1)〜 （65) のいずれか 1項に記載の細胞を用いることを特徴とす る、 先天性遺伝子疾患での変異遺伝子に対する野生型遺伝子を心筋へ特異的に輸送 する方法。 (137) a congenital gene characterized by using the cell according to any one of the above (1) to (65), into which a wild-type gene for a mutant gene in a congenital heart disease is introduced. Specific transport of wild-type genes to myocardium for mutant genes in disease how to.
(138) 心臓の先天性遺伝子疾患での変異遺伝子に対する野生型遺伝子が導入 された上記 （1) 〜 （65)のいずれか 1項に記載の細胞を有効成分として含有す る心臓疾患治療薬。  (138) 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.
(139) 上記 （1) 〜 （65) のいずれか 1項に記載の細胞を免疫原として用 いることを特徴とする、 該細胞を特異的に認識する抗体を取得する方法。 ' (139) A method for obtaining an antibody that specifically recognizes the cell according to any one of (1) to (65) above, wherein the cell is used as an immunogen. '
(140) 上記 （139)記載の方法で取得された抗体を用いることを特徴とす る、 ヒト骨髄から心筋細胞への分化能を有する成体骨髄由来細胞を単離 ·精製する 方法。 (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).
(141) 上記 （1) 〜 （65) のいずれか 1項に記載の細胞を用いることを特 徴とする、 該細胞に特異的な表面抗原を取得する方法。  (141) A method for obtaining a surface antigen specific to a cell, characterized by using the cell according to any one of the above (1) to (65).
(1 2) 上記 （1) 〜 （65) のいずれか 1項に記載の細胞を用いることを特 徴とする、 該細胞を増殖する因子をスクリーニングする方法。  (12) A method of screening for a factor that proliferates the cell, characterized by using the cell according to any one of the above (1) to (65).
(143) 上記 （1) 〜 （65) のいずれか 1項に記載の細胞を用いることを特 徴とする、 該細胞の心筋細胞への分化を誘導する因子をスクリーニングする方法。 (143) A method for screening a factor that induces differentiation of the cell into a cardiomyocyte, comprising using the cell according to any one of the above (1) to (65).
(144) 上記 （1) 〜 （65) のいずれか 1項に記載の細胞を用いることを特 徴とする、 該細胞を不死化する因子をスクリーニングする方法。 (144) A method for screening a factor that immortalizes a cell, characterized by using the cell according to any one of the above (1) to (65).
(145) 上記 （1) 〜 （65) のいずれか 1項に記載の細胞にテロメラーゼを 発現させることを特徴とする、 該細胞の不死化方法。  (145) A method for immortalizing a cell according to any one of the above (1) to (65), which comprises expressing telomerase in the cell.
(146) テロメラ一ゼが、 配列番号 31記載で表されるアミノ酸配列を有する テロメラ一ゼである上記 （145)記載の方法。  (146) The method according to (145) above, wherein the telomerase has the amino acid sequence represented by SEQ ID NO: 31.
(147) テロメラ一ゼを発現させることにより、不死化させた上記（ 1)〜（6 5 )のいずれか 1項に記載の細胞を有効成分として含有する心臓疾患治療薬。  (147) 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.
(148) テロメラ一ゼが、 配列番号 31記載で表されるアミノ酸配列を有する テロメラーゼである上記 （147)記載の治療薬。  (148) The therapeutic agent according to the above (147), wherein the telomerase is a telomerase having an amino acid sequence represented by SEQ ID NO: 31.
(1 9) 上記 （ 1 )〜 （ 65 ) のいずれか 1項に記載の細胞を含んだ培養上清。 (150) 上記 （149)記載の培養上清を用いることを特徴とする、 上記（1) 記載の細胞を心筋細胞に分化誘導する方法。 本発明の心筋細胞への分ィ匕能を有する細胞としては、 骨髄、 筋肉、 脳、 滕臓、 肝 臓、腎臓などの成体組織または臍帯血から単離された多分化能幹細胞があげられる。 多分化能幹細胞としては、 心筋細胞とそれ以外の細胞を誘導できる細胞であれば いずれでもよいが、 好ましくは少なくとも心筋細胞、 脂肪細胞、 骨格筋細胞、 骨芽 細胞に分化する能力を有する細胞、 少なくとも心筋細胞、 血管内皮細胞に分ィ匕する. 能力を有する細胞、 少なくとも心筋細胞、 脂肪細胞、 骨格筋細胞、 骨芽細胞、 血管 内皮細胞に分化する能力を有する細胞、 少なくとも心筋細胞、 脂肪細胞、 骨格筋細 ' 胞、 血管内皮細胞、 骨芽細胞、 神経系細胞、 肝細胞に分化する能力を有する細胞な どがあげられる。 (19) A culture supernatant containing the cell according to any one of (1) to (65). (150) A method for inducing differentiation of the cell according to (1) into a cardiomyocyte, comprising using the culture supernatant according to (149). Examples of 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.
成体組織または臍帯血は哺乳類由来であればいかなるものでもよいが、 好ましく はマウス、 ラット、 ヒトなどがあげられる。 ヒトの治療用途にはヒト由来であるこ とが好ましい。  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.
マウス、 ラット、 ヒトなどのほ乳類の成体組織または臍帯血から心筋細胞への分 化能を有する細胞を単離し、 培養した後に、 心筋細胞への分ィヒ能を有する細胞を分 化、 誘導することにより、 心筋細胞を得ることができる。  Isolate cells that have the ability to differentiate into cardiomyocytes from adult human tissues such as mice, rats, and humans or cord blood and culture them, and then divide and induce cells that have the ability to differentiate into cardiomyocytes Thereby, cardiomyocytes can be obtained.
また、 本発明の多分化能幹細胞を用いて、 心筋細胞だけでなく、 血管内皮細胞、 平滑筋、 骨格筋細胞、 脂肪細胞、 骨、 軟骨、 塍内分泌系細胞、 滕外分泌系細胞、 肝 細胞、 腎糸球体細胞、 腎尿細管細胞、 ニューロン、 グリア、 オリゴデンドロサイト などへの分化を誘導することにより、 各種細胞を得ることができる。  Further, using the pluripotent stem cells of the present invention, not only cardiomyocytes, but also vascular endothelial cells, smooth muscle, skeletal muscle cells, fat cells, bone, cartilage, 塍 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.
以下、 本発明を詳細に説明する。  Hereinafter, the present invention will be described in detail.
1 . 心筋細胞への分化能を有する細胞の単離  1. Isolation of cells capable of differentiating into cardiomyocytes
本発明の心筋細胞への分化能を有する細胞は、 成体組織または臍帯血など心筋細 胞への分化能を有する細胞を取得することが可能な組織であればいかなる組織でも よい。 以下に、 骨髄から心筋細胞への分化能を有する骨髄細胞を単離する方法を述 ベる。  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.
( 1 ) 骨髄から心筋細胞への分化能を有する骨髄細胞を単離する方法  (1) Method for isolating bone marrow cells capable of differentiating into cardiomyocytes from bone marrow
ヒトの骨髄より心筋細胞への分化能を有する骨髄細胞を取得する方法としては、 安全かつ効率的に取得される方法であれば特に限定されないが、 S. E. Haynesworth et al. Bone, 13, 81 (1"2)に記載された方法に基づき行うことができる。 As a method for obtaining bone marrow cells capable of differentiating into cardiomyocytes from human bone marrow, 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 ).
胸骨または腸骨から骨髄穿刺を行う。 骨髄穿刺を行う場所の皮膚面を消毒し、 局 所麻酔を行う。 特に骨膜下を充分に麻酔する。 骨髄穿刺針の内筒を抜き、 5000units のへパリンを入れた 10ml注射器を装着して必要量の骨髄液を速やかに吸引する。平 均的には 10ml〜20mlの骨髄液を吸引する。骨髄穿刺針を取り外し、 10分間程圧迫止 血する。 取得した骨髄液を l，000 X gの遠心分離により骨髄細胞を回収した後、 該骨 髄細胞を PBS (Phosphate Buffered Saline)で洗浄する。本ステヅプを 2回繰り返した後、 該骨髄細胞を 10%の FBS (牛胎仔血清） を含む α - MEM (ひ - modified MEM)、 DMEM (Dulbecco's modified MEM)あるいは IMDM (Isocove's modified Dulbecco's medium)等の 細胞培養用培地に再浮遊させることにより骨髄細胞液を得ることができる。  Perform a bone marrow puncture from the sternum or iliac bone. Disinfect the skin surface where the bone marrow puncture is performed and perform local anesthesia. In particular, the subperiosteum is sufficiently anesthetized. Remove the inner tube of the bone marrow puncture needle, attach a 10 ml syringe containing 5000 units of heparin, and quickly aspirate the required amount of bone marrow fluid. On average, aspirate 10 to 20 ml of bone marrow fluid. Remove the bone marrow puncture needle and stop bleeding for about 10 minutes. 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 α-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.
該骨髄細胞液から心筋細胞への分化能を有する骨髄細胞を単離する方法としては、 溶液中に混在する他の細胞、 例えば血球系細胞、 造血幹細胞、 血管幹細胞および線 維芽細胞などを除去できれば特に限定されないが、 'M. F. Pittenger et al. Science, 284, 143 (1999)に記載された方法に基づき骨髄細胞液を密度 1.073g/mlの percollに重層し た後、 1,100 X gで 30分間遠心分離して界面の細胞を回収することにより単離するこ とができる。 また、 該骨髄細胞液に 10 X PBSを加えて 9/10に希釈した percollを同 容量加えて混合した後に、 20，000 X gで 30分間遠心分離し、 密度 1·075~1.060の画 分を回収することにより、 該心筋細胞への分ィ匕能を有する骨髄細胞を含む骨髄細胞 混合物を取得することができる。 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. Although not particularly limited, it is possible to overlay a bone marrow cell solution on a 1.073 g / ml percoll based on the method described in 'MF Pittenger et al. Science, 284, 143 (1999), and then at 1,100 X g for 30 minutes. It can be isolated by collecting cells at the interface by centrifugation. Further, a percoll diluted to 10 X PBS plus 9/10 in bone marrow cell fluid after mixed with equal volume, 20,000 X g at centrifuged for 30 minutes, the field of density 1 · 075 to 1.060 min By recovering, a bone marrow cell mixture containing bone marrow cells having the ability to divide the myocardial cells can be obtained.
" 上記方法により取得した該心筋細胞への分化能を有する骨髄細胞を含む骨髄細胞 混合物は、 ％穴の培養プレートの各穴に 1細胞のみが注入されるように希釈して、 1細胞由来のクローンを多数調製した後、 以下に記載した心筋細胞への分化能を有 する骨髄細胞から心筋細胞を誘導する方法を用いて該クローンを処理し、 自律拍動 する細胞が出現するクローンを選択することにより、 該心筋細胞への分化能を有す る骨髄細胞を得ることができる。  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. After preparing a large number of clones, 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.
ラヅ トやマウスから心筋細胞への分化能を有する骨髄細胞を取得する方法として は、 特に限定されないが以下の手順で取得することができる。 ラットあるいはマウ スを頸椎脱臼により致死させ、 70%エタノールで充分消毒した後、大腿骨の皮膚なら びに大腿四頭筋を切除する。 膝関節の部分にハサミをいれて関節をはずし、 大腿骨 背面の筋肉を除去する。 股関節の部分にハサミを入れて関節を外し、 大腿骨を取り 出す。 大腿骨に付着している筋肉をハサミでできるだけ除去した後、 大腿骨の両端 をハサミで切断する。骨の太さに応じた適当なサイズの針を 2.5mlの注射器に装着し、 10%の FBS (牛胎仔血清） を含む - MEM、 DMEM、 あるいは IMDM等の細胞培養用 培地約 1.5mlを注射器に充填した後、注射針の先端を大腿骨の膝関節側の断端に差し 込む。 注射器内の培養液を骨髄内に注入することで、 股関節側の断端かち骨髄細胞 が押し出される。得られた骨髄細胞はピぺヅティンタにより培養液中に浮遊させる。 該骨髄液からは、 上記のヒト骨髄液からの骨髄細胞の単離と同様の方法により、 心 筋細胞への分化能を有する骨髄細胞を単離することができる。 以上の方法により単. 離した細胞の例としては、 マウス骨髄由来多分化能幹細胞があげられる。 マウス骨 髄由来多分化能幹細胞 BMSCは γ 平成 1 2年 2月 2 2日付けで通商産業省工業技術 院生命工学工業技術研究所 (日本国茨城県つくば巿東 1丁目 1番 3号） に FERM BP - 7043として寄託されている。 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. Remove the joint with scissors in the knee joint and remove the muscles behind the femur. Remove the joint with scissors in the hip joint and remove the femur. After removing the muscle attached to the femur as much as possible with scissors, cut both ends of the femur with scissors. Attach a 2.5 ml syringe with a needle appropriate for the size of the bone and contain 10% FBS (fetal calf serum)-about 1.5 ml of cell culture medium such as MEM, DMEM or IMDM After filling, insert the tip of the injection needle into the stump on the knee joint side of the femur. By injecting the culture solution in the syringe into the bone marrow, bone marrow cells are pushed out from the hip joint stump. The obtained bone marrow cells are suspended in a culture solution using a pigment. From the bone marrow fluid, 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 γ-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.
( 2 ) 骨髄以外の組織から心筋細胞への分化能を有する細胞を単離する方法 後述する 1 2に記載の抗体を用いた分離方法により、 心筋細胞への分化能を有す る細胞を、 骨髄以外の組織からも取得することができる。  (2) Method for isolating cells capable of differentiating into cardiomyocytes from tissues other than bone marrow By the separation method using the antibody described in 12 below, cells capable of differentiating into cardiomyocytes can be obtained by: It can also be obtained from tissues other than bone marrow.
骨髄以外の組織としては、 好ましくは臍帯血があげられる。 具体的には、 以下の 方法で行うことができる。  Tissue other than bone marrow preferably includes cord blood. Specifically, it can be performed by the following method.
まず臍帯から臍帯血を分取し、 ただちに 500units/mlの終濃度になるようにへパ リンを加える。 よく混合した後、 遠心分離して臍帯血から細胞を分取し、 10%の FBS First, 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
(牛胎仔血清） を含むひ- MEM (a- modified MEM)ヽ DMEM (Dulbecco's modified MEM) あるいは IMDM (Isocove's modified Dulbecco's medium)等の細胞培養用培地に再浮遊さ せる。 得られた細胞液から上述した抗体を利用して、 心筋細胞への分化能を有する 細胞を分離することができる。 (Fetal bovine serum), and resuspended in a cell culture medium such as DMEM (Dulbecco's modified MEM) or IMDM (Isocove's modified Dulbecco's medium). Cells having the ability to differentiate into cardiomyocytes can be separated from the obtained cell solution using the above-mentioned antibodies.
2 . 心筋細胞への分ィヒ能を有する細胞の培養  2. Cultivation of cells capable of dividing myocardial cells
上記 1の方法により単離した、 心筋細胞への分ィ匕能を有する細胞を培養するため に用いる培地としては、通常公知（組織培養の技術基礎編 第三版、朝倉書店 1996) の組成の細胞培養用培地を用いることができるが、 好ましくは牛等の血清を 5〜20% 添カロした、 ひ-ΜΕΜ、 DMEMあるいは IMDM等の細胞培養用培地などが用いられる。 培養条件は、細胞が培養可能であればいかなる条件でもよいが、培養温度は 33〜37°C が好ましく、 さらに 5〜10%の二酸化炭素ガスで満たした孵卵器で培養することが好 ましい。 心筋細胞への分化能を有する骨髄細胞は、 通常の組織培養用のプラスチヅ ク製培養皿に接着して増殖することが好ましい。 細胞が培養皿一面に増殖する頃、 培地を除去して、 トリブシン EDTA溶液を加えることで細胞を浮遊させる。 浮遊し た細胞は、 · PBS'あるいは該細胞培養用の培地で洗浄後、 該細胞培養用の培地で 5倍 から 20倍希釈して新しい培養皿に添加することで、 さらに継代培養することができ る。 For culturing cells having the ability to divide myocardial cells, isolated by the above method 1 As the medium used for the culture, a culture medium for cell culture having a composition known in the art (Tissue Culture Technology Basic Edition, Third Edition, Asakura Shoten 1996) can be used. 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. When 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.
3 . 心筋細胞への分化能を有する細胞からの心筋細胞の'誘導  3. Induction of cardiomyocytes from cells capable of differentiating into cardiomyocytes
心筋細胞への分化能を有する細胞より心筋細胞を誘導する方法としては、 ( 1 ) . DNAの脱メチル化剤処理による分ィヒ誘導、 （ 2 ) 胎児の心臓発生領域で発現してい る因子または胎児の心臓発生段階において心筋細胞への分化に働く因子による分化 誘導、 （ 3 ) 心筋細胞への分化能を有する細胞または該細胞から分化した心筋細胞 の培養上清による分化誘導などの方法を挙げることができる。 これらの方法を単独 あるいは組み合わせることにより、 心筋細胞への分化能を有する細胞から心筋細胞 を誘導することができる。  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.
DNAの脱メチル化剤としては、 DNAに対して脱メチル化を引き起こす化合物であ ればいかなるものでもよい。 DNAの脱メチル化剤としては、 染色体 DNA中の GpC 配列中のシトシン残基のメチル化を特異的 阻害する酵素であるデメチラ一ゼ、 5- ァザシチジン （以下 5-aza- Cと略す） 、 DMSO (dimethyl sulfoxide) などがあげられ る。 デメチラーゼとしては、 配列番号 1記載のアミノ酸配列を有するデメチラ一ゼ [Nature, 397, 579-583 (1999)]などがあげられる。 DNAの脱メチル化剤処理による分 化誘導の具体例を以下に示す。  The DNA demethylating agent may be any compound that causes demethylation of DNA. Examples of 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). 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.
3 β mol/1から 10〃 mol/1の間の濃度になるように 5 - aza - Cを心筋細胞への分ィ匕能 を有する細胞を含む培地中に添加し、 24時間上記培養条件下でィンキュぺーシヨン する。 培地を交換することで 5 - aza-Cを除去し、 さらに 2〜3週間培養することで 心筋細胞を取得することができる。 形成される心筋細胞は培養 2〜 3週間目では洞 結節細胞が中心であるが、 培養 4週間目以降心室型心筋細胞を分化誘導することが できる。 3 Add 5-aza-C to a medium containing cells capable of dividing cardiac muscle cells to a concentration between 3 β mol / 1 and 10 μmol / 1 Indication I do. The medium can be replaced to remove 5-aza-C, and cultured for another 2-3 weeks to obtain cardiomyocytes. The formed cardiomyocytes are mainly sinus node cells at 2-3 weeks of culture, but can induce differentiation of ventricular cardiomyocytes after 4 weeks of culture.
胎児の心臓発生領域で発現している因子または胎児の心臓発生段階において心筋 細胞への分化に働く因子としては、 サイトカイン、 ビタミン、 接着分子、 転写因子 などをあげることができる。  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.
サイトカインとしては、 心筋細胞への分化能を有する細胞に、 心臓の発生段階で 心筋細胞への分ィ匕を促進するものであればいかなるサイトカインでもよい。.  As the cytokine, 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. .
具体的には、 10〜40ng/mlの血小板由来増殖因子（以下、 PDGFと略記する。 )、 線維芽細胞増殖因子 8 (FGF8)、 エンドセリン 1 (ET1)、 ミドカイン (midkine)、 骨形成 因子 4 (BMP4)などをあげることができる。 PDGFとしては配列番号 3または 5のァ ミノ酸配列で表されるものが、 線維芽細胞増殖因子 8 (FGF8)どしては配列番号 6 4 のアミノ酸配列で表されるものが、 エンドセリン 1 (ΕΤΓ)としては配列番号 6 6のァ ミノ酸配列で表されるものが、ミドカイン (midkine)としては配列番号 6 8のアミノ酸 配列で表されるものが、 骨形成因子 4 (BMP4)としては配列番号 7 0のアミノ酸配列 で表されるものが好ましく用いられる。 Specifically, 10 to 40 ng / ml of platelet-derived growth factor (hereinafter abbreviated as PDGF), fibroblast growth factor 8 (FGF8), endothelin 1 (ET1), midkine, bone formation Factor 4 (BMP4) and the like. PDGF represented by the amino acid sequence of SEQ ID NO: 3 or 5; fibroblast growth factor 8 (FGF8); and that represented by the amino acid sequence of SEQ ID NO: 64, endothelin 1 ( (Ii) is represented by the amino acid sequence of SEQ ID NO: 66, midkine is represented by the amino acid sequence of SEQ ID NO: 68, and sequence is represented by bone morphogenetic factor 4 (BMP4). The one represented by the amino acid sequence of No. 70 is preferably used.
また、 心筋細胞への分ィ匕を抑制するサイトカインに対する阻害剤を用いることに より、 心筋細胞への分化能を有する細胞に、 心臓の発生段階で心筋細胞への分化を 促進することも可能である。  In addition, by using 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.
心筋細胞への分化を抑制するサイトカインとしては、線維芽細胞増殖因子一 2 (以 下、 FGF - 2と略記する。 ）、 具体的には、 配列番号 7または 8で表される FGF-2な どをあげることができる。  Examples of 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.
心筋細胞への分化を抑制するサイトカンに対する阻害剤としては、 サイトカイン の情報伝達を阻害する物質、 例えばサイトカインを中和する抗体、 低分子化合物な どをあげることができる。  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.
ビタミンとしては、 レチノイン酸など心筋細胞への分化能を有する細胞に、 心臓 の発生段階で心筋細胞への分ィ匕を促進するものであればいかなるビタミンでもよい。 具体的には、 10_9Mのレチノイン酸などをあげることができる。 As the vitamin, any vitamin, such as 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. Specifically, and the like retinoic acid 10_ 9 M.
接着分子としては、 フイブロネクチンなど心臓の発生段階で心臓発生領域で発現 していればいかなる接着分子でもよい。 具体的には、 フイブロネクチンをコートし た培養皿で該心筋細胞への分化能を有する細胞を培養することにより心筋細胞への 分ィ匕を促進することができる。  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.
転写因子としては、 ホメォボックス型転写因子 Nkx2.5/CSX (配列番号 9：ァミノ 酸配列、 配列番号 10：塩基配列） 、 GATAファミ リ一に属する Zinc finger型転写因 子 GATA4 (配列番号 11：アミノ酸配列、配列番号 12：塩基配列）、 myocyte enhancer factor- 2(MEF ファミリ一に属する転写因子 MEF- 2Α· (配列番号 13：ァミノ酸配列、 配列番号 14：塩基配列） 、 MEF- 2Β (配列番号 15：アミノ酸配列、 配列番号 16：塩 基配列）、 MEF-2C (配列番号 17：ァミノ酸配列、配列番号 18：塩基配列）と MEF- 2D (配列番号 19：ァミノ酸配列、 配列番号 20：塩基配列) 、 basic helix loop helix型転 写因子に属する dHAND (配列番号 21：アミノ酸配列、 配列番号 22：塩基配列） 、· eHAND (配列番号 23：アミノ酸配列、 配列番号 24：塩基配列） と MesPl (配列番号 61：ァミノ酸配列、 配列番号 62：塩基配列） 、 TEA- DNA結合型転写因子ファミ リ —に属する TEF-1 (配列番号 25：アミノ酸配列、 配列番号 26：塩基配列） 、 TEF- 3 (配列番号 27：アミノ酸配列、 配列番号 28：塩基配列） と TEF - 5 (配列番号 29：ァ ミノ酸配列、 配列番号 30 :塩基配列） などをあげることができる。 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-2Α · belonging to MEF family 1 (SEQ ID NO: 13: amino acid sequence, SEQ ID NO: 14: base sequence), MEF-2Β (sequence 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) belonging to the basic helix loop helix type transcription factor, eHAND (SEQ ID NO: 23: amino acid sequence, SEQ ID NO: 24: base sequence) MesPl (SEQ ID NO: 61: Amino acid sequence, SEQ ID NO: 62: base sequence), TEF-1 belonging to TEA-DNA-binding transcription factor family — TEF-1 (SEQ ID NO: 25: amino acid sequence, SEQ ID NO: 26: base sequence), TEF-3 (SEQ ID NO: 27: amino acid sequence, SEQ ID NO: 28: base sequence) and TEF-5 (SEQ ID NO: 29: amino acid sequence, SEQ ID NO: 30: base sequence).
上述した転写因子は、該因子をコ一ドする DNAを心筋細胞への分化能を有する細 胞中に導入し、 DNAを発現させることにより心筋細胞への分化を誘導させることが できる。  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.
また、 自律拍動する心筋細胞から取得した細胞外基質をコ一トした培養皿を用い ること、 自律拍動する心筋細胞と共培養すること、 自律拍動する心筋細胞の培養上 清を添加することで、 心筋細胞への分ィ匕能を有する細胞を心筋細胞へ分化誘導させ ることができる。  In addition, use a culture dish coated with extracellular matrix obtained from autonomically beating cardiomyocytes, co-culture with autonomically beating cardiomyocytes, and add culture supernatant of autonomically beating cardiomyocytes By doing so, it is possible to induce differentiation of cells having the ability to divide into cardiomyocytes into cardiomyocytes.
また、 4に示す方法で得られる心筋細胞への分化を誘導する因子 （以下、 心筋分 化誘導因子と称する） を用いても、 心筋細胞への分ィ匕能を有する細胞を心筋細胞に 分化誘導することができる。 4 . 心筋分化誘導因子の取得 In addition, even if a factor that induces differentiation into cardiomyocytes obtained by the method shown in 4 (hereinafter referred to as a cardiomyocyte differentiation inducing factor) is used, cells having the ability to divide into cardiomyocytes can be differentiated into cardiomyocytes. Can be guided. 4. Acquisition of cardiac differentiation induction factor
心筋分化誘導因子の取得方法としては、 自律拍動する細胞の培養上清に各種プロ テア一ゼ阻害剤を添加して、 透析、 塩析ならびにクロマトグラフィーなどを組み合 わせることにより取得することができる。  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.
さらにマイクロシ一ケンサ一を用いて、 上記の心筋分化誘導因子の部分ァミノ酸 配列を決定し、該アミノ酸配列に基づき設計した DNAプローブを用いて該自律拍動 する細胞より作製した CDNAラィ.ブラリーをスクリーニングすることにより、 心筋 分化誘導因子の遺伝子を取得することができる。  Furthermore, 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.
5 . · 心筋細胞への分化能を有する細胞を含む心臓再生治療薬または心臓疾患治 本発明の心筋細胞への分ィ匕能を有する細胞は、 心臓再生または心臓疾患の治療薬 として用いることができる。  5. 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.
心臓疾患としては、 .心筋梗塞、 虚血性心疾患、 うつ血性心不全、 不整脈、 肥大型 心筋症、 拡張型心筋症、 心筋炎、 弁膜症などをあげることができる。  Examples of the heart disease include myocardial infarction, ischemic heart disease, congestive heart failure, arrhythmia, hypertrophic cardiomyopathy, dilated cardiomyopathy, myocarditis, and valvular disease.
心臓再生の治療薬としては、 心筋細胞への分ィ匕能を有する細胞を高純度で含み、 心臓の障害部位ならび大きさに応じて、 該心筋細胞への分化能を有する細胞を増殖 させたもの、 好ましくは、 心筋細胞への分化能を有する細胞から'、 心筋内皮細胞  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. , Preferably from cells capable of differentiating into cardiomyocytes, myocardial endothelial cells
(Endocardial endothelial cell), クッション細胞 (Cushion cell), 心室型心筋細胞、 心房型 心筋細胞、 洞結節細胞等の心臓を形成する様々な細胞へ分化誘導できる細胞が用い られる。 (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.
該治療薬は、 心筋梗塞の患者骨髄液中から上 した密度勾配遠心分離法、 後述す る心筋細胞への分化能を有する細胞を特異的に認識する抗体を用いたバニング法 [J. . Immunol., 141(8), 2797-2800 (1988)]あるいは FACS法 [Int. Immunol., 10(3), 275-283 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.
(1"8)]、 または心筋細胞への分化能を有する細胞に特異的な遺伝子のプロモーター を用いたレポ一夕一系を構築する方法により該心筋細胞への分化能を有する細胞の 精製を行うことにより、 製造することができる。 (1 " 8 )] or purifying the cells capable of differentiating into cardiomyocytes by a method of constructing a repo overnight system using a promoter of a gene specific to the cells capable of differentiating into cardiomyocytes. By doing so, it can be manufactured.
また該治療薬には、 後述する心筋形成剤を用いて、 該心筋細胞への分化能を有す る細胞を心筋細胞へ分化誘導させた細胞、高齢者の骨髄から取得した骨髄細胞より、 後述する不死化方法を利用して細胞分裂能を賦活させた心筋細胞への分ィ匕能を有す る細胞も含まれる。 In addition, 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.
上記方法で製造した治療薬は、 上記心筋細胞への分化能を有する細胞を特異的に 認識する抗体と FACS法を組み合わせることで純度を検定することができる。  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.
上記の治療薬を障害部位に輸送する方法としては、 カテーテルを利用する方法等 が用いられる。 以下虚血性心疾患を例に具体的な方法を示す。 虚血性心疾患で障害 を受けた心筋細胞は、 血管狭窄部位の下流に存在する'ことから、 上記の細胞を注入 する前に、 冠動脈造影法 （図説病態内科講座 循環器一 1、 MEDICAL VIEW.1993) により血管の狭窄部位を同定しておく必要がある。器質的狭窄病変は狭窄病態に応' じて求心性狭窄、 偏心性狭窄、 多発性壁不整に分類され、 特に偏心性狭窄はタイプ I およびタイプ Πの 2つのタイプに細分類される。 狭窄形態は狭心症の経過、 予後に 関連することが知られており、 タイプ IIの偏心性狭窄や多発性壁不整は不安定狭心 症例に多く、 心筋梗塞に移行する可能性が高い。 血管が完全に狭窄している場合に は、 注入する細胞が障害部位に到達しない可能性があるので、 事前に経皮的冠動脈 形成術 (PTCA)あるいは血栓溶解療法などにより狭窄部位を再開することが必要であ る。 障害を受けた心筋細胞の部位に応じて、 注入する細胞を心室型や心房型のよう に区別することができる。 カテーテルの挿入法は右上腕動脈より挿入する Sone^ (図説病態内科講座 循環器一 1、 MEDICAL VIEW, 1 9 9 3 ) あるいは大腿動脈よ り挿入する Jundkins法 （図説病態内科講座 循環器一 1、 MEDICAL VIEW, 1993) を 利用することができる。  As a method for transporting the above therapeutic agent to the site of injury, a method using a catheter or the like is used. Hereinafter, 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. In particular, eccentric stenosis is subdivided into two types, type I and type II. It is known that the form of stenosis is related to the course and prognosis of angina. Type II eccentric stenosis and multiple wall irregularities are common in unstable angina patients, and the possibility of transition to myocardial infarction is high. If the blood vessel is completely stenotic, the injected cells may not reach the site of injury, so the stenotic site must be reopened by percutaneous coronary angioplasty (PTCA) or thrombolytic therapy beforehand. is necessary. Depending on the location of the damaged cardiomyocytes, 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.
6 . 心筋形成剤  6. Cardiomyogen
本発明の心筋形成剤は、 染色体 DNAの脱メチル化剤、胎児の心臓発生領域で発現 している因子、 あるいは胎児の心臓発生段階で心筋細胞への分化に働く因子、 心筋 分ィ匕誘導因子の少なくとも一種類を有効成分として含有し、 心筋細胞への分化能を 有する細胞を心筋細胞へ分化誘導させることができる。  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.
当該心筋形成剤としては、 サイトカイン、 ビタミン、 接着分子、 転写因子などを あげることができる。  Examples of the cardiomyogen include cytokines, vitamins, adhesion molecules, transcription factors, and the like.
サイトカインとしては、 心筋細胞への分化能を有する細胞に、 心臓発生段階で心 筋細胞への分化を促進するものであればいかなるサイトカインでもよい。 Cytokines include cells that have the potential to differentiate into cardiomyocytes, Any cytokine that promotes differentiation into muscle cells may be used.
具体的には、 10〜40ng/mlの PDGF、 線維芽細胞増殖因子 8 (FGF8)、 エンドセリン 1 (ET1)、ミドカイン (midkine)、骨形成因子 4 (BMP4)などをあげることができる。 PDGF としては配列番号 3または 5のアミノ酸配列で表されるものが、 線維芽細胞増殖因 子 8 (FGF8)としては配列番号 6 4のァミノ酸配列で表されるものが、 エンドセリン 1 (ET1)としては配列番号 6 6のァミノ酸配列で表されるものが、 ミドカイン  Specific examples include 10 to 40 ng / ml of PDGF, fibroblast growth factor 8 (FGF8), endothelin 1 (ET1), midkine, bone morphogenetic factor 4 (BMP4), and the like. PDGF represented by the amino acid sequence of SEQ ID NO: 3 or 5; fibroblast growth factor 8 (FGF8) represented by the amino acid sequence of SEQ ID NO: 64; endothelin 1 (ET1) Is represented by the amino acid sequence of SEQ ID NO: 66,
(midkine)としては配列番号 6 8のアミノ酸配列で表されるものが、 骨形成因子 4 (BMP4)としては配列番号 7 0のアミノ酸配列で表されるものが好ましく用いられる。 ビタミンとしては、'レチノイン酸など心筋細胞への分化能を有する細胞に、 心臓 発生段階で心筋細胞への分化を促進するものであればいかなるビ夕ミンでもよい。 具体的には、 10_g Mのレチノイン酸などをあげることができる。 As (midkine), those represented by the amino acid sequence of SEQ ID NO: 68, and as bone formation factor 4 (BMP4), those represented by the amino acid sequence of SEQ ID NO: 70 are preferably used. As the vitamin, 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.
接着分子としては、 'フィプロネクチンなど心臓発生段階で心臓発生領域で発現し ていればいかなる接着分子でもよい。 具体的には、 フイブロネクチンをコートした 培養皿で該心筋細胞への分化能を有する細胞を培養することにより心筋細胞への分 化を促進することができる。  As the adhesion molecule, 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. Specifically, the differentiation into cardiomyocytes can be promoted by culturing cells capable of differentiating into cardiomyocytes in a culture dish coated with fibronectin.
転写因子としては、 ホメォボックス型転写因子 Nkx2.5/CSX (配列番号 9：ァミノ 酸配列、 配列番号 10 :塩基配列） 、 GATAファミリ一に属する Zinc finger型転写因 子 GATA4 (配列番号 11：アミノ酸配列、配列番号 12：塩基配列）、 myocyte enhancer factor- 2(MEF- 2)ファミリ一に属する転写因子 MEF- 2A (配列番号 13：ァミノ酸配列、 配列番号 14：塩基配列） 、 MEF- 2B (配列番号 15：アミノ酸配列、 配列番号 16：塩 基配列）、 MEF-2C (配列番号 17：ァミノ酸配列、配列番号 18：塩基配列）と MEF - 2D (配列番号 19：アミノ酸配列、 配列番号 20：塩基配列） 、 basic helix loop helix型転 写因子に属する dHAND (配列番号 21：アミノ酸配列、 配列番号 22：塩基配列） 、 eHAND (配列番号 23：アミノ酸配列、 配列番号 24：塩基配列） と MesPl (配列番号 61 :アミノ酸配列、 配列番号 62 :塩基配列） 、 TEA-DNA結合型転写因子ファミリ —に属する TEF- 1 (配列番号 25：アミノ酸配列、 配列番号 26 :塩基配列） 、 TEF - 3 (配列番号 27：ァミノ酸配列、 配列番号 28：塩基配列） と TEF - 5 (配列番号 29：ァ 'ミノ酸配列、 配列番号 30：塩基酉さ列） などをあげることができる。 該心筋形成剤には心筋分化誘導因子の遺伝子を主成分とするものと、 心筋分化誘 導因子の本体である蛋白質を含むものがある。 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: 23: amino acid sequence, SEQ ID NO: 24: base sequence) belonging to the basic helix loop helix type transcription factor and MesPl (SEQ ID NO: 61: amino acid sequence, SEQ ID NO: 62 : Nucleotide sequence), TEF-1 belonging to the TEA-DNA binding transcription factor family (SEQ ID NO: 25: amino acid sequence, SEQ ID NO: 26: nucleotide sequence), TEF-3 (SEQ ID NO: 27: amino acid sequence, SEQ ID NO: 28) : Nucleotide sequence) and TEF-5 (SEQ ID NO: 29: amino acid sequence; SEQ ID NO: 30: nucleotide sequence). The myocardiogenic agents include those containing a gene for a myocardial differentiation inducing factor as a main component and those containing a protein that is the main body of a myocardial differentiation inducing factor.
( 1 ) 遺伝子を主成分とする心筋形成剤  (1) Gene-based cardiomyogenic agent
以下に本発明の心筋形成剤が心筋分化誘導因子をコードする遺伝子を主成分とす る場合の調製法について述べる。  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.
まず、心筋分ィ匕誘導因子の遺伝子 DNA断片、あるいは全長 CDNAをウィルスべク 夕一プラスミ ド内のプロモ一夕一の下流に揷入することにより.、 組換えウィルスべ クタ一プラスミ ドを造成する。  First, 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.
該組換えウィルスベクタ一プラスミ ドを、 該ウィルスベクタープラスミ ドに適合 したパッケージング細胞に導入する。  The recombinant virus vector-plasmid is introduced into a packaging cell suitable for the virus vector plasmid.
パッケージング細胞としては、 ウィルスのパッケージングに必要なタンパク質を ：コードする遺伝子の少なくとも 1つを欠損している組換えウィルスベクタープラス ミ ドの該欠損する蛋白質を補給できる細胞であればいかなるものも'用いることがで きる。例えばヒト腎臓由来の HEK293細胞、マウス線維芽細胞 MH3T3などを用いる ことができる。  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. For example, human kidney-derived HEK293 cells, mouse fibroblast MH3T3 and the like can be used.
パッケージング細胞で補給する蛋白質としては、 レトロウイルスベクタ一の場合 はマウスレトロウイルス由来の gag、 pol、 envなどの蛋白質、 レンチウィルスベクタ —の場合は HIVウィルス由来の gag、 pol、 env、 vpr、 vpuヽ vif、 tat、 rev、 nefなどの 蛋白質、 アデノウイルスベクタ一の場合はアデノウイルス由来の E1A、 E1Bなどの 蛋白質、 アデノ随伴ウィルスの場合は Rep(p5，P19，p40)、 Vp(Cap)などの蛋白質を用い ることができる。 In the case of a retrovirus vector, 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ヽ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.
ウィルスぺク夕一プラスミ ドとしては上記パッケージング細胞において組換えゥ ィルスが生産でき、 心臓先天性遺伝子疾患の原因遺伝子に対する野生型の遺伝子を 心筋細胞で転写できる位置にプロモーターを含有しているものが用いられる。  Viral DNA plasmid containing a promoter capable of producing a recombinant virus in the above-mentioned packaging cells and capable of transcribing in the cardiomyocyte a wild-type gene corresponding to the gene causing cardiac congenital genetic disease. Is used.
ウィルスベクタープラスミ ドとしては MFG [Proc. Natl. Acad. Sci. USA, 92, 6733 - 6737 (1995)]、 pBabePuro [Nucleic Acids Research, 18, 3587-3596 (1990)]， LL— CG、 CL一 CG、 CS— CG、 CLG [Journal of Virology, 72, 8150 - 8157 (1998)]、 pAdexl [Nucleic Acids Res., 23, 3816-3821 (1995)]等が用いられる。 プロモー夕一としては、 ヒト組織中で発現できるものであればいずれも用いるこ とができ、 例えば、 サイ トメガロウィルス （ヒト CMV) の lEGmmediate early)遺伝子 のプロモーター、 SV40の初期プロモー夕一、 レトロウイルスのプロモーター、 メタ 口チォネインプロモ一夕一、 ヒートショック蛋白質プロモー夕一、 SR ひプロモ一夕 —等をあげることができる。 また、 ヒト CMVの IE遺伝子のェンハンサ一をプロモ —夕一と共に用いてもよい。 また、 Nkx2.5/Csx遺伝子のような心筋細胞特異的な遺 伝子のプロモーターを用いることで、 心筋細胞で特異的に目的の遺伝子を発現させ ることができる。 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. For example, 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. Alternatively, the enhancer of the IE gene of human CMV may be used together with the promoter. Further, by using the cardiomyocyte specific heritage promoter gene such as Nkx 2 .5 / Csx gene can Rukoto specifically to express the gene of interest in cardiomyocytes.
上記組換えウィルスベクタ一プラスミドを上記パッケージング細胞に導入するこ とで組換えウィルスベクターを生産することができる。 上記ノ ヅケ一ジング細胞へ の上記ウィルスベクタープラスミ ドの導入法としては、 例えば、 リン酸カルシウム 法 [特開平 2^227075] 、 リポフエクション法 [Proe. Natl. Acad. Sci. USA, 84, 7413 (1987)] 等をあげることができる。  A recombinant virus vector can be produced by introducing the above-mentioned recombinant virus vector-single plasmid into the above-mentioned packaging cell. Examples of 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)].
上述した組換えウィルスベクタ は、 遺伝子治療剤に用いる基剤と共に調合して 心筋形成剤を製造することができる [Nature Genet., 8, 42 (1994)]。 遺伝子治療剤に用 いる基剤としては、 通常注射剤に用いる基剤であればいかなるものでも用いること ができる。 例えば、 蒸留水、 塩化ナトリゥム又は塩化ナトリゥムと無機塩との混合 物等の塩溶液、 マンニトール、 ラクト一ス、 デキストラン、 グルコース等の溶液、 グリシン、 アルギニン等のアミノ酸溶液、 有機酸溶液又は塩溶液とグルコース溶液 との混合溶液等があげられる。 また常法に従い、 これらの基剤に浸透圧調整剤、 PH 調整剤、 ゴマ油、 ダイズ油等の植物油又はレシチンもしくは非イオン界面活性剤等 の界面活性剤等の助剤を用いて、 溶液、 懸濁液、 分散液として注射剤を調製しても よい。 これらの注射剤を、 粉末化、 凍結乾燥等の操作により用時溶解用製剤として 調製することもできる。 上記の心筋形成剤は、 液体の場合はそのままで、 固体の場 合は治療の直前に必要により滅菌処理をした上記の基剤に溶解して遺伝子治療に使 用することができる。 本発明の心筋形成剤の投与方法は、 患者の治療部位の心筋に 吸収されるように、 カテーテル等を用いて局所的に投与する方法等が用いられる。 上述した組換えウィルスベクターは試験管内で該心筋細胞への分化能を有する細 胞に感染させた後、 上述した心筋形成剤として調製し、 患者に投与することができ る。 または、 組換えウィルスベクタ一を患者の患部に直接投与することもできる。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)]. As the base used for the gene therapy agent, any base that is usually used for injections can be used. For example, distilled water, 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. In addition, according to a conventional method, 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. As 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.
( 2 ) 蛋白質を主成分とする心筋形成剤 (2) Protein-based cardiomyogenic agent
以下に本発明の心筋形成剤が心筋分化誘導因子蛋白質を主成分とする場合の調製 法について述べる。  Hereinafter, a method for preparing the myocardial agent of the present invention in which the myocardial differentiation-inducing factor protein is the main component will be described.
心筋分ィ匕誘導因子蛋白質の完全長 CDNAをもとに、 必要に応じて、 該蛋白質をコ Based on the full-length cDNA of the cardiac inducing factor-inducing protein, the protein may be
―ドする部分を含む適当な長さの DNA断片を調製する。 Prepare a DNA fragment of appropriate length containing the part to be loaded.
該 DNA断片、あるいは完全長 CDNAを発現べクタ一内のプロモ一夕一の下流に揷 入することにより、 該蛋白質の組換発現べク夕一を造成する。  By inserting the DNA fragment or the full-length CDNA downstream of the promoter in the expression vector, a recombinant expression vector of the protein is constructed.
該組換発現べク夕一を、 該発現べク夕一に適合した宿主細胞内に導入する。  The recombinant expression vector is introduced into a host cell compatible with the expression vector.
宿主細胞としては、 目的とする DNAを発現できるものは全て用 、ることができ、 例えば、 ェシエリヒア (Escherichia)属、 セラチア (Serratia)属ヽ コリネバクテリゥム Corynebacterium) 晨、 ブレビノ クテリウム (Brevibacterium) 属、 シュ一ドモナス 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
(Pseudomonas)属、 バチルス (Bacillus)属、 ミクロノ クテリゥム (Microbacterium) 属等に属する細菌、 クルイべ口ミセス (Kluyveromyces) 属、 サヅカロマイセスBacteria belonging to the genera Pseudomonas, Bacillus, Microbacterium, etc., Kluyveromyces, Saccharomyces
( Saccharomyces) 鹛、 シゾサヅカロマイセス ( Shizosaccharomyces)属、 トリコスポ ロン (Trichosporon) 属、 シヮニォミセス (Schwanniomyces) 属等に属する酵母や動 物細胞、 昆虫細胞等を用いることができる。 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.
発現ぺク夕一としては、 上記宿主細胞において自立複製可能なレヽしは染色体中へ の組込みが可能で、心筋分化誘導因子蛋白質の遺伝子 DNAを転写できる位置にプロ モー夕—を含有しているものが用いられる。 細菌を宿主細胞として用いる場合は、 心筋分化誘導因子蛋白質の組換え発現べク 夕一は該細菌中で自立複製可能であると同時に、 プロモ一夕一、 リボソーム結合配 列、心筋分化誘導因子蛋白質をコードする DNAおよび転写終結配列より構成された 組換え発現ぺク夕一であることが好ましい。 プロモー夕一を制御する遺伝子が含ま れていてもよい。  As an expression vector, 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. When a bacterium is used as a host cell, 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.
発現べクタ一としては、 例えば、 pBTrp2、 pBTacl, pBTac2 (いずれもベーリンガ 一マンハイム社より巿販)、 pKK233- 2 (Amersham Pharmacia Biotech社製)、 pSE280 (Imdtrogen社製）、 pGEMEX- 1 (Promega社製）、 pQB- 8 (QIAGEN社製）、 pKYPIO [特開昭 58-110600] 、 p YP200 [Agricultural Biological Chemistry, 48, 669 (1984)]、 pLSAl [Agric. Biol. Chem., 53, 277 (1989)]、 pGELl [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)]、 pBluescript II S (-) (Stratagene社製)、 pGEX (Amersham Pharmacia Biotech 社製） 、 PET- 3 (Novagen社製） 、 pTerm2(USP4686191、 USP4939094、 USP5160735)、 pSupex pUBllOヽ pTP5、 pC194、 pEG400 [J. Bacteriol, 172, 2392 (1990)] 等を例示 することができる。 Expression base Kuta one, for example, pBTrp 2, pBTacl, pBTac2 (巿販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. USA, 82, 4306 (1985)], pBluescript II S (-) (Stratagene), pGEX ( Amersham Pharmacia Biotech), PET-3 (Novagen), pTerm2 (USP4686191, USP4939094, USP5160735), pSupex pUBllO ヽ pTP5, pC194, pEG400 [J. Bacteriol, 172, 2392 (1990)] Can be.
発現べクタ一としては、 リボソーム結合配列であるシャイン一ダルガス (Shine- Dalgarno)配列と開始コドンとの間を適当な距離（例えば 6〜1 8塩基） に調節した ものを用いることが好ましい。  As 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).
プロモ一夕一としては、 宿主細胞中で発現できるものであればいかなるものでも よい。 例えば、 trpプロモ一夕一 （P trp)、 lacプロモータ^" ( P lac)、 PLプロモ一 夕一、 PRプロモー夕一、 T7プロモ一夕一等の大腸菌やファージ等に由来するプロモ 一夕一、 SP01プロモ一夕一、 SP02プロモー夕一、 penPプロモー夕一等をあげるこ とができる。 また P trpを 2つ直列させたプロモ一夕一 （P trp x 2 ) 、 tacプロモ —夕一、 letlプロモー夕一 [Gene, 44, 29 (1986) ] 、 lacT7プロモー夕一のように人 為的に設計改変されたプロモ一夕一等も用いることができる。 Any promoter can be used as long as it can be expressed in a host cell. For example, 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 You can list Yuichi, SP01 Promo Yoichi, SP02 Promo Yuichi, penP Promo Yuichi, etc. Also, 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.
本発明の心筋分化誘導因子蛋白質の遺伝子 DNAの蛋白質をコードする部分の塩基 配列を、 宿主の発現に最適なコドンとなるように、 塩基を置換することにより、 目 的とする蛋白質の生産率を向上させることができる。  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.
本発明の心筋分化誘導因子蛋白質の遺伝子 DNAの発現には転写終結配列は必ずし も必要ではないが、 好適には構造遺伝子直下に転写終結配列を配置することが望ま しい。  Although 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.
宿主細胞としては、 ェシエリヒア属、 セラチア属、 コリネバクテリウム属、 ブレ ビバクテリウム属、 シユードモナス属、 バチルス属、 ミクロバクテリゥム属等に属 する微生物、例えば、 Esch ich— ia coli XL1— Blueヽ 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 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
saccharolvticum ATCC14066、 Corynebacterium glutamicum ATCC13032、 saccharolvticum ATCC14066, Corynebacterium glutamicum ATCC13032,
Corynebacterium glutamicum ATCC14067、 Corynebacterium glutamicum ATCC13869、 Corynebacterium acetoacidophilum ATCC 13870、 Microbacterium ammoniaphilum Corynebacterium glutamicum ATCC14067, Corynebacterium glutamicum ATCC13869, Corynebacterium acetoacidophilum ATCC 13870, Microbacterium ammoniaphilum
ATCC15354、 Pseudomonas sp. D - 0110等をあげることができる。 ATCC15354, Pseudomonas sp. D-0110 and the like.
組換えベクターの導入方法としては、上記宿主細胞へ DNAを導入する方法であれ ばいずれも用いることができ、 例えば、 カルシウムイオンを用いる方法 [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)] \ プロ トプラスト法 （特開昭 63- 248394) 、 または Gene, 17, 107 (1982)や Molecular & General Genetics, 168, 111 (1979)に記載の方法等を あげることができる。  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.For example, 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).
酵母を宿主細胞として用いる場合には、 発現ぺク夕一として、'例えば、 YEpl3 (ATCC37115) 、 YEp24 (ATCC37051) 、 YCp50 (ATCC37419) 、 pHS19、 pHS15 等を例示することができる。  When yeast is used as a host cell, examples of expression vectors include, for example, YEpl3 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, and pHS15.
プロモ一夕一としては、酵母中で発現できるものであればいかなるものでもよく、 例えば、 PH〇5プロモ一夕一、 PGKプロモ一夕一、 GAPプロモー夕一、 ADHプロモ 一夕一、 gal lプロモーター、 gal 10プロモ一夕一、 ヒートショック蛋白質プロモ一 夕一、 MF 1プロモーター、 CUP 1プロモ一夕一等をあげることができる。  Any promoter can be used as long as it can be expressed in yeast. For example, PH〇5 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.
宿主細胞としては、 サヅカロミセス 'セレビシェ (Saccharomyces cerevisiae) 、 シ ソサヅカロミセス ·ポンぺ ί Schizosaccharomvces pombe 、 クリュイベロミセス ·ラ クナス (Kluyveromyces lactis 、 トリコスポロン ·プルフンス (Trichosporon pullulans )、 シュヮニォミセス 'アルビウス (Schwanniomyces alluvius)等をあげることができる。 組換えべクタ一の導入方法としては、酵母に DNAを導入する方法であればいずれ も用いることができ、例えば、エレクトロポレ一シヨン法 [Methods in Enzymol., 194, 182 (1990)] 、 スフエロプラスト法 [Proc. Natl. Acad. Sci. USA, 75, 1929 (1978)] 、 酢 酸リチゥム法 [J. Bacteriol., 153, 163 (1983)、 Proc. Natl. Acad. Sci. USA, -75, 1929 (1978)] 等をあげることができる。  The host cells include Saccharomyces cerevisiae, Schisosaccharomvces pombe, Kluyveromyces lactis, Trichosporon pullionas, etc. As a method for introducing the recombinant vector, 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)].
動物細胞を宿主細胞として用いる場合には、 発現ベクターとして、 例えば、 pCDNAKlnvitrogen社製)、 pCD 8 (Invitrogen社製） 、 pAGE107 [特開平 3-22979； Cytotechnology, 3, 133 (1990)]、 pAS3- 3 (特開平 2-227075)、 pCDM8 [Nature, 329, 840 (1987)]、 pCDNAI/Amp (invitrogen社製） 、 pREP4 (Invitrogen社製）、 pAGE103 [J. Biochem., 101, 1307 (1987)]、 pAGE210等を例示することができる。 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.
プロモー夕一としては、 動物細胞中で発現できるものであればいずれも用いるこ とができ、 例えば、 サイ トメガロウィルス （ヒト CMV) の IE(immediate early)遺伝子 のプロモー夕一、 SV40の初期プロモ一夕一、 レトロウイルスのプロモ一夕一、 メタ 口チォネインプロモ一夕一、 ヒ一トショック蛋白質プロモー夕一、 SR αプロモ一夕 —等をあげることができる。 また、 ヒト CMVの IE遺伝子のェンハンサ^ "をプロモ —夕一と共に用いてもよい。  Any promoter can be used as long as it can be expressed in animal cells. For example, 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. Alternatively, the human CMV IE gene enhancer ^ "may be used with the Promo-Yuichi.
宿主細胞としては、 ヒトの細胞であるナマルバ （Namalwa) 細胞、 サルの細胞であ る COS細胞、 チャイニーズ 'ハムス夕一の細胞である CHO細胞、 HBT5637 [特開 昭 63- 299] 等をあげることができる。  Examples of host cells 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.
組換えべクタ一の導入法としては、動物細胞に DNAを導入できるいかなる方法も 用いることができ、 例えば、 エレクトロポ一レ一シヨン法 [Cytotechnology， ， 133 (1990)]、 リン酸カルシウム法（特開平 2-227075)、 リボフヱクシヨン法 [Proc. Natl. Acad. Sci" USA, 84, 7413 (1987)、 Virology, 52, 456 (1973)] 等を用いることができる。 形質転換体の取得および培養は、 特開平 2-227075号公報あるいは特開平 2-257891 号公報に記載されている方法に準じて行なうことができる。  As a method for introducing the recombinant vector, any method capable of introducing DNA into animal cells can be used. For example, 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.
昆虫細胞を宿主として用いる場合には、 例えばバキュロウィルス *ェクスプレツ シヨン 'ベクタ一ズ, ァ■ラボラトリ一 ·マニュアル [Baculovirus Expression Vectors, A Laboratory Manual, W.H. Freeman and Company, New York (1992)]、 カレント .プロ トコ一ルズ 'イン 'モレキュラー ·バイオロジー サプルメント 1-38(1987-1997)、 Bio/Technology, 6, 47 (1988)等に記載された方法によって、 蛋白質を発現することが できる。  When an insect cell is used as a host, for example, 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.
即ち、 組換え遺伝子導入ベクターおよびバキュロウィルスを昆虫細胞に共導入し て昆虫細胞培養上清中に組換えウィルスを得た後、 さらに組換えウィルスを昆虫細 胞に感染させ、 蛋白質を発現させることができる。 該方法において用いられる遺伝子導入べクタ一としては、 例えば、 pVL1392、 pVL1393、 pBlueBacIII (ともに Invitrogen社製） 等をあげることができる。 That is, after the recombinant gene transfer vector and baculovirus are co-transfected into insect cells to obtain the recombinant virus in the culture supernatant of insect cells, the recombinant virus is further infected into the insect cells to express the protein. Can be. Examples of the gene transfer vector used in the method include pVL1392, pVL1393, pBlueBacIII (all manufactured by Invitrogen) and the like.
バキュロウィルスとしては、 例えば、 夜盗蛾科昆虫に感染するウィルスであるァ ゥトグラファ ·カリフオル二力 ·ヌクレァ一 'ポリへドロシス ·ウィルス (Autographa californica nuclear polyhedrosis virus)等を用いることが、できる。  As the baculovirus, for example, autographa californica nuclear polyhedrosis virus, which is a virus that infects night moth insects, can be used.
昆虫細胞としては、 Spodoptera frugiperdaの卵巣細胞である Sf9、 Sf21 [Baculovirus Expression Vectors, A Laboratory Manual^ W.H.Freeman.and Company, New YorK3 (1992； I、 Trichoplusia ^の卵巣細胞である High 5 (Invitrogen社製）等を用いることができる。 組換えウィルスを調製するための、 昆虫細胞への上記組換え遺伝子導入ベクター と上記バキュロウィルスの共導入方法としては、 例えば、 リン酸カルシウム法 [特 開平 2-227075] 、 リポフエクション法 [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)] 等をあげることができる。 As insect cells, 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.
遺伝子の発現方法.としては、 直接発現以外に、 モレキュラー 'クロ一ニング 第 2版,' L olecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)] に記載されている方法等に準じて、 分泌生産、 融合蛋白質 発現等を行うことができる。  Examples of 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.
酵母、 動物細胞または昆虫細胞により発現させた場合には、 糖あるいは糖鎖が付 加された蛋白質を得ることができる。  When expressed by yeast, animal cells or insect cells, a sugar or sugar chain-added protein can be obtained.
心筋分化誘導因子をコ一ドする DNAを組み込んだ組換え体 DNAを保有する形質 転換体を培地に培養し、 培養物中に心筋分化誘導因子蛋白質を生成蓄積させ、 該培 養物より該蛋白質を採取することにより、 心筋分化誘導因子蛋白質を製造すること ができる。  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.
心筋分化誘導因子蛋白質を製造する形質転換体を培地に培養する方法は、 宿主の 培養に用いられる通常の方法に従って行うことができる。  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.
大腸菌等の原核生物あるいは酵母等の真核生物を宿主として得られた形質転換体 を培養する培地としては、 該宿主が資化し得る炭素源、 窒素源、 無機物等を含有し、 形質転換体の培養を効率的に行える培地であれば天然培地、 合成培地のいずれでも よい。 炭素源としては、 それぞれの宿主が資化し得るものであればよく、 グルコース、 フラクトース、 スクロース、 これらを含有する糖蜜、 デンプンあるいはデンプン加 水分解物等の炭水化物、 酢酸、 プロピオン酸等の有機酸、 エタノール、 プロパノー ルなどのアルコール類を用いることができる。 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.
窒素源としては、 アンモニア、 塩化アンモニゥム、 硫酸アンモニゥム、 酢酸アン モニゥム、 リン酸アンモニゥム等の各種無機酸若しくは有機酸のアンモニゥム塩、 その他含窒素化合物、 並びに、 ペプトン、 肉エキス、 酵母エキス.、 コ一ンスチープ リカー、 カゼイン加水分解物、 大豆粕および大豆粕加水分解物、 各種発酵菌体およ びその消化物等が用いられる。  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.
無機物としては、 リン酸第一カリウム、 リン酸第二カリウム、 リン酸マグネシゥ ム、 硫酸マグネシウム、 塩化ナトリウム、 硫酸第一鉄、 硫酸マンガン、 硫酸銅、 炭 酸カルシウム等を用いることができる。  As the inorganic substance, 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.
培養は、 振盪培養または深部通気攪拌培養などの好気的条件下で行う。 培養温度 は 15〜40°Cがよく、 培養時間は、 通常 I6時間〜 7日間である。 培養中 PHは、 3.0 〜9.0に保持する。 p Hの調整は、 無機あるいは有機の酸、 アルカリ溶液、 尿素、 炭 酸カルシウム、 アンモニアなどを用いて行う。 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.
また培養中必要に応じて、 アンビシリンゃテトラサイクリン等の抗生物質を培地 に添加してもよい。  If necessary, an antibiotic such as ambicilin / tetracycline may be added to the medium during the culture.
プロモーターとして誘導性のプロモ一夕一を用いた発現ぺク夕一で形質転換した 微生物を培養するときには、必要に応じてインデューサーを培地に添加してもよい。 例えば、 lacプロモータ一を用いた発現べクタ一で形質転換した微生物を培養すると きに イソプロピル一 5— D—チォガラクトビラノシド （IPTG)等を、 trpプロモ一 夕一を用いた発現べク夕一で形質転換した微生物を培養するときにはインド一ルァ クリル酸 （IAA) 等を培地に添カ卩してもよい。  When culturing a microorganism transformed with an expression promoter using an inducible promoter as a promoter, an inducer may be added to the medium, if necessary. For example, when culturing a microorganism transformed with an expression vector using the lac promoter, isopropyl-15-D-thiogalactovyranoside (IPTG) or the like can be used. When culturing the transformed microorganism in the evening, indoleacrylic acid (IAA) or the like may be added to the medium.
動物細胞を宿主細胞と'して得られた形質転換体を培養する培地としては、 一般に 使用されている RPMI1640培地 [The Journal of the American Medical Association, 199, 519 (1967)] 、 Eagleの MEM培地 [Science, 122, 501 (1952)] 、 ダルべヅコ改変 MEM 培地 [Virology, 8, 396 (1959)]、 1 9 9培地 [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)] またはこれら培地に牛胎児血清等を添加した培地等を用いる ことができる。 As 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.
培養は、 通常 pH6〜8、 30〜40°C、 5%C02存在下等の条件下で 1〜7日間行う。 また、 培養中必要に応じて、 カナマイシン、 ペニシリン等の抗生物質を培地に添 加してもよい。 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.
昆虫細胞を宿主細胞として得られた形質転換体を培養する培地としては、 一般に 使用されて 、る TNM- FH培地（Pharmingen社製）、 Sf-900 II SFM培地' (Life Technologies 社製）、 ExCell400、 ExCell405 (いずれも JRH Biosciences社製).、 Grace's Insect Medium [Grace, T.C.C., Nature, 195, 788 (1962)] 等を用いることができる。  As a medium for culturing a transformant obtained by using an insect cell as a host cell, generally used 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.
培養は、 通常 pH6~7、 25〜30°C等の条件下で、 1〜5日間行う。 The cultivation is usually performed under conditions of pH 6 to 7 and 25 to 30 ° C for 1 to 5 days.
また、 培養中必要に応じて、 ゲン夕マイシン等の抗生物質を培地に添カ卩してもよ い。  If necessary, an antibiotic such as genyumycin may be added to the medium during the culture.
上述の形質転換体の培養物から、 心筋分化誘導因子蛋白質を単離精製するには、 通常の蛋白質の単離、 精製法を用いればよい。  In order to isolate and purify the myocardial differentiation-inducing protein from the culture of the above-mentioned transformant, a normal protein isolation and purification method may be used.
例えば、 心筋分化誘導因子蛋白質が、 細胞内に溶解状態で発現した場合には、 培 養終了後、 細胞を遠心分離により回収し水系緩衝液にけん濁後、 超音波破砕機、 フ レンチプレス、 マントンガウリンホモゲナイザー、 ダイノミル等により細胞を破砕 し、 無細胞抽出液を得る。 該無細胞抽出液を遠心分離することにより得られた上清 から、 通常の蛋白質の単離精製法、 即ち、 溶媒抽出法、 硫安等による塩析法、 脱塩 法、有機溶媒による沈殿法、ジェチルアミノエチル（DEAE)—セファロ一ス、 DIAION HPA-75 (三菱化学社製） 等レジンを用いた陰イオン交換クロマトグラフィー法、 S-Sepharose FF(Amersham Pharmacia Biotech社製)等のレジンを用いた陽イオン交換 クロマトグラフィー法、 ブチルセファロ一ス、 フェニルセファロース等のレジンを 用いた疎水性クロマトグラフィー法、 分子篩を用いたゲルろ過法、 ァフィ二ティ一 クロマトグラフィ一法、 クロマトフォ一カシング法、 等電点電気泳動等の電気泳動 法等の手法を単独あるいは組み合わせて用い、 精製標品を得ることができる。  For example, if 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. From the supernatant obtained by centrifuging the 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.
また、 該蛋白質が細胞内に不溶体を形成して発現した場合は、 細胞を回収後破砕 し、 遠心分離することにより、 沈殿画分として蛋白質の不溶体を回収する。 回収した該蛋白質の不溶体を蛋白質変性剤で可溶化する。 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.
該可溶化液を、 希釈あるいは透析により、 該可溶化液中の蛋白質変性剤の濃度を 下げることにより、 該蛋白質の構造を正常な立体構造に戻した後、 上記と同様の単 離精製法により該蛋白質の精製標品を得る。  After reducing the concentration of the protein denaturing agent in the lysate by diluting or dialysis the lysate, 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.
心筋分化誘導因子蛋白質またはその糖修飾体等の誘導体が細胞外に分泌された場 合には、 培養上清から、 該蛋白質またはその糖鎖付加体等の誘導体を回収すること ができる。 即ち、 培養物から遠心分離等の手法により培養上清を回収し、 該培養上 清から、 上記と同様の単離精製法を用いることにより、 精製標品を得るこ'とができ る o  When a derivative such as a myocardial differentiation-inducing factor protein or a modified sugar thereof is secreted extracellularly, 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.o
このようにして取得される蛋白質として、 例えば、 配列番号 5、 6、 10、 12、 14、 16、 18、 20、 22、 24、 26、 28および 30で表されるアミノ酸配列を有する蛋白質等を あげることができる。  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.
また、上記方法により発現させた蛋白質を、 Fmoc法 (フルォレニルメチルォキシカ ルポニル法)、 tBoc法 (t-プチルォキシカルボニル法)等の化学合成法によっても製造 することができる。また、米国 Advanced ChemTech社製、 Perkin- Elmer社製、 Amersham Pnarmacia Biotech社製、米国 Protein Technology Instrument孑ェ製、米国 Synthecell-Vega 社製、 米国 PerSeptive社製、 島津製作所社製等のペプチド合成機を利用して合成す ることもできる。  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). In addition, a peptide synthesizer manufactured by U.S. Advanced ChemTech, Perkin-Elmer, Amersham Pnarmacia Biotech, U.S. Protein Technology Instrument, U.S.A., Synthecell-Vega, U.S.A. It can also be used for synthesis.
心筋細胞への分化を誘導できる蛋白質は、 上記 （1 ) と同様にして心筋形成剤を 形成し使用することができる。  The protein capable of inducing differentiation into cardiomyocytes can be used by forming a cardiomyogen in the same manner as in (1) above.
7 . 先天性遺伝子疾患の治療への利用  7. Use for the treatment of congenital genetic diseases
心不全を来す疾患の中には、 一部であるが単一遺伝子の変異により、 本来必要な 蛋白質が全て欠損するために心不全を来す一群がある。 このような疾患としては、 家族性肥大型心筋症、 Fabri病、 QT延長症候群、 マルファン症候群、 大動脈弁狭窄 症、 ミトコンドリア心筋症、 Duchenne型筋ジストロフィー症等があげられる。 これ らの疾患は、 ミオシン、 トロポニン、 トロポミオシン、 電位依存性 Naチャンネル、 Kチャンネル、 フイブリン、 エラスティン、 ミ トコンドリア、 ジストロフィンなど の遺伝子異常が原因であることが知られている [治療学，^, 1302-1306(1996)] 。 す なわち、 これら患者より本発明の心筋細胞への分化能を有する細胞を取得し、 正常 な遺伝子を導入して心臓に移植することで上記疾患を治療することができる。 正常 な遺伝子は、 上記 6 ( 1 ) で記載した遺伝子治療用のベクタ一を用いることで本発 明の心筋細胞への分化能を有する細胞に導入することができる。 Among the 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. Such 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)]. You That is, 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.
8. 心筋細胞への分化能を有する細胞特異的な表面抗原を特異的に認識する抗体 の取得 ·  8. Obtaining antibodies that specifically recognize cell-specific surface antigens that have the ability to differentiate into cardiomyocytes
以下に、 本発明の心筋細胞への分化能を有する細胞で発現している表面抗原を特 異的に認識する抗体の調製法について述べる。  Hereinafter, a method for preparing an antibody that specifically recognizes a surface antigen expressed on cells capable of differentiating into cardiomyocytes of the present invention will be described.
本発明の心筋細胞への分ィヒ能を有する細胞で特異的に発現している表面抗原を認 識する抗体は、 心筋梗塞などの心臓病の細胞治療を実施する上で必要な心筋細胞へ の分化能を有する細胞の純度検定や精製に用いることができる。  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.
該抗体を取得する方法として、 本発明の心筋細胞への分化能を有する細胞 3〜5 X 105cellS/匹、あるいは該細胞から調製した細胞膜画分 l〜10mg/ 匹程を抗原として、 ゥサギ、 ャギまたは 3〜20週令のラット、 マウスもしくはハムスター等の非ヒトほ 乳動物の皮下、 静 ¾R内または S复腔内に、 適当なアジュバント [例えば、 フロインドの 完全アジュバント（Complete Freund's Adjuvant)または、 水酸化アルミニウムゲル、 百 日咳菌ワクチンなど]とともに投与する。 As a method for obtaining the antibody, 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.].
該抗原の投与は、 1回目の投与の後 1〜2週間おきに 3〜10回行う。各投与後、 3 〜7日目に眼底静脈叢より採血し、 該血清が免疫に用いた抗原と反応するか否かを 酵素免疫測定法 [酵素免疫測定法 （ELISA法） ：医学書院刊 1976年、 Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory, 1988] などで調べる。  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].
免疫に用いた抗原に対し、 その血清が充分な抗体価を示した非ヒトほ乳動物を、 血清または抗体産生細胞の供給源とする。  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.
ポリクローナル抗体は、 該血清を分離、 精製することにより調製することができ 。  A polyclonal antibody can be prepared by separating and purifying the serum.
モノクローナル抗体は、 該抗体産生細胞と非ヒトほ乳動物由来の骨髄腫細胞とを 融合させてハイプリドーマを作製し、 該ハイプリドーマを培養するか、 動物に投与 して該動物を腹水癌化させ、 該培養液または腹水を分離、 精製することにより調製 することができる。 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.
抗体産生細胞としては、 脾細胞、 リンパ節、 末梢血中の抗体産生細胞、 特に脾細 胞が好適に用いられる。  As the antibody-producing cells, spleen cells, lymph nodes, and antibody-producing cells in peripheral blood, particularly splenocytes, are suitably used.
骨髄腫細胞としては、 8—ァザグァニン耐性マウス （BALB/c由来)骨髄腫細胞株 である P3— X63Ag8— U1(P3-U1)株 [Current Topics in Microbiology andlmmunology, 18, 1 (1978)] 、P3— NSl/1— Ag41(NS— 1)株 [European J. Immunology, 6, 511 (1976)] ヽ SP2/0- Agl4(SP— 2)株 [Nature, 276, 269 (1978)] 、 P3— X63_Ag8653(653)株 [J.' Immunology, 123. 1548 (1979)] 、 P3- X63- Ag8(X63)株 [Nature, 256, 495 (1975)]等、 マウス由来の株化 細胞が好適に用いられる。 - ハイプリドーマ細胞は、 以下の方法により作製できる。  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)] ヽ 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.
抗体産生細胞と骨髄腫細胞を混合し、 H A T培地 （正常培地にヒポキサンチン、 チミジンおよびアミノプテリンを加えた培地） に懸濁したのち、 7〜1 4日間培養 する。 培養後、 培養上清の一部をとり酵素免疫測定法などにより、 抗原に反応し、 抗原を含まない蛋白質には反応しないものを選択する。 ついで、 限界希釈法により クローニングを行い、 酵素免疫測定法により安定して高い抗体価の認められたもの をモノクローナル抗体産生ハイプリドーマ細胞として選択する。  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.
ポリクロ一ナル抗体またはモノクローナル抗体を分離、 精製する方法としては、 遠心分離、硫安沈殿、 力プリル酸沈殿、 または DEAE—セファロースカラム、 陰ィォ ン交換カラム、 プロテイン Aまたは G—カラムあるいはゲル濾過カラム等を用いる クロマトグラフィ一等を、 単独または組み合わせて処理する方法があげられる。 上記方法で取得した、 該心筋細胞への分化能を有する細胞で発現している表面抗 原を特異的に認識する抗体を用いて、 検体細胞に対する反応性と造血系幹細胞、 神 経系幹細胞などの対照となる細胞に対する反応性とを比較することで、 検体細胞が 上記特異的表面抗原を発現しているかどうかを容易に検定することができる。  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. Using 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.
9 . 心筋細胞への分化能を有する細胞で発現している表面抗原および該表面杭 原をコードする遺伝子の取得  9. Acquisition of surface antigens expressed on cells capable of differentiating into cardiomyocytes and genes encoding the surface spores
該心筋細胞への分ィ匕能を有する細胞で特異的に発現している表面抗原遺伝子の取 得方法としては、 二つの異なる由来のサンプル間で異なる発現形態を取る遺伝子を 取得する方法であるサブトラクション法 [Proc. Natl. Acad. Sci. USA 85, 5738 - 5742 (1988)]や Representational difference analysis [Nucleic Acids Research, 22, 5640—5648 (1994)]による方法をあげることができる。 As a method for obtaining a surface antigen gene that is specifically expressed in a cell having the ability to divide myocardial cells, a gene that takes a different expression form between samples of two different origins is used. Natl. Acad. Sci. USA 85, 5738-5742 (1988)] and Representational difference analysis [Nucleic Acids Research, 22, 5640-5648 (1994)]. it can.
まず、 心筋細胞への分化能を有する細胞より作製した CDNAライプラリーを、 造 血系幹細胞や神経系幹細胞などの心筋細胞への分ィ匕能を有する細胞以外の対照細胞 より取得した mRNAを用いてサブトラクションを行う。 心筋細胞への分化能を有す る細胞特異的な遺伝子を濃縮した差分ィ匕 CDNAライブラ'.リーを調製した後、 該差分 化 CDNAライブラリ一の挿入 CDNA配列を 5， 側よりランダムに塩基配列解析を行 い、 分泌シグナル配列を持つものだけを選択する。 このようにして得られた CDNA の全長塩基配列を決定することにより、 該 CDNAがコードする蛋白質が分泌蛋白質 か膜蛋白質かを区別することができる。  First, 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.
上記の方法において、 ランダム配列解析の代わりに、 シグナルシーケンストラヅ プ法も用いることもできる [Science, 261, 600-603 (1993); Nature Biotechnology, 17, 487-490 (1999)]。シグナルシーケンストラップ法とは、分泌シグナル配列をもつ遺伝 子を選択的にスクリーニングする方法である。  In the above method, a signal sequence trap method can be used instead of the random sequence analysis [Science, 261, 600-603 (1993); Nature Biotechnology, 17, 487-490 (1999)]. The signal sequence trap method is a method for selectively screening genes having a secretory signal sequence.
効率よく特異的な表面抗原を取得するためには、 シグナルシーケンストラップラ ィブラリ一をサブトラクションが行えるベクタ一を用いて作製し、 心筋細胞への分 化能を有する細胞から作製したシグナルシーケンストラップライブラリ一を造血系 幹細胞や神経系幹細胞などの対照となる細胞より取得した mRNAを用いてサブトラ クシヨンを行う方法が望ましい。 このようにして取得された分泌シグナル配列を含 む DNA断片は全長 CDNAをクローン化するためのプローブとして用いることができ る。  In order to obtain specific surface antigens efficiently, 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.
全長 CDNAはその全長塩基配列を解析することで、 該 CDNAがコ一ドする蛋白質 が分泌蛋白質か膜蛋白質かを区別することができる。  By analyzing the full-length nucleotide sequence of the full-length cDNA, it is possible to distinguish whether the protein encoded by the cDNA is a secretory protein or a membrane protein.
ランダム配列解析あるいはシグナルシーケンストラップ法を用いた場合でも、 得 られたクローンが膜蛋白質をコードする場合は、 塩基配列から類推されるアミノ酸 配列に基づき合成ぺプチドを作製し、 該合成べプチドを抗原として上記方法により 特異的な抗体を取得することができる。 また、 膜蛋白質の場合は、 受容体をコードしているものがあり、 このような受容 体は該心筋細胞への分化能を有する細胞の特異的な増殖または心筋細胞への分化の 調節に働いている可能性があり、 当該受容体のリガンドの探索に用いることができ る。 分泌蛋白質の場合は、 直接心筋細胞への分化能を有する細胞を増殖あるいは分 化させるために用いることができる。 Even when random sequence analysis or signal sequence trapping is used, if the obtained clone encodes 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. In the case of 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. In the case of a secretory protein, it can be used to proliferate or differentiate cells having the ability to directly differentiate into cardiomyocytes.
1 0 . 心筋細胞への分化能を有する細胞の増殖因子および心筋細胞への分ィ匕誘 導因子のスクリーニング  10. Screening of growth factors of cells capable of differentiating into cardiomyocytes and factors inducing differentiation into cardiomyocytes
心筋細胞への分化能を有する細胞の増殖因子および心筋細胞への分化誘導因子の スクリー^ング方法としては、 心筋細胞への分化能を有する細胞を無血清培地中で 培養させる際に、 検体である種々の物質を添加させ、 該細胞が増殖するか、 または 心筋細胞へ分化誘導されるかで調べることにより行うことができる。  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.
検体となる物質としては、 各種サイトカインゃ増殖因子などの分泌蛋白質、 細胞 接着分子などの膜結合蛋白質、 組織抽出液、 合成ペプチド、 合成化合物、 微生物培 養液等などいかなるものでもよい。  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.
増殖能力はコ口ニー形成能や BrdUの取り込みなどで調べることができる。  The proliferative ability can be determined by the ability to form a knee and the uptake of BrdU.
コロニー形成能は、 本発明の心筋細胞への分化能を有する細胞を低密度で播種す ることにより調べることができる。  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の取り込みは、 BrdUを特異的に認識する抗体を用いた免疫染色により調べ ることができる。  BrdU incorporation can be examined by immunostaining using an antibody that specifically recognizes BrdU.
心筋細胞への分化を評価する方法としては、 自律拍動を指標にするかまたは筋細 胞で特異的に発現する遺伝子のプロモ一夕一と GFP(Gleen fluorescent protein) 、 ル シフェラーゼ、 ベ一夕一ガラクトシダ一ゼなどのレポ一夕一遺伝子とを組み合わせ たベクター DNAを該心筋細胞への分化能を有する細胞に導入したレポ一夕一細胞を 用いてレポーター遺伝子の発現を指標にする方法があげられる。  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. Can be
レポ一夕一系の構築には cardiac troponin I(cTNI)のプロモーターを用いる方法があ げられる D. Biological Chemistry, 273, 25371-25380 (1998)]。  One method for constructing the repo overnight system is to use a cardiac troponin I (cTNI) promoter. D. Biological Chemistry, 273, 25371-25380 (1998)].
1 1 . 心筋細胞への分ィヒ能を有する細胞の不死化  1 1. Immortalization of cells that have the ability to isolate cardiomyocytes
心臓疾患の患者、 特に高齢者に本発明の治療薬を投与する場合、 本発明の心筋細 胞への分ィヒ能を有する細胞をガン化させずに細胞分裂の回数を増やすことが望まし い。 When administering the therapeutic agent of the present invention to a patient with a heart disease, particularly the elderly, It is desirable to increase the number of cell divisions without causing cells having the ability to divide cells into cancer.
細胞をガン化させずに細胞分裂の回数を増やす方法としては、 テロメラーゼを本 発明の心筋細胞への分化能を有する細胞に発現させる方法をあげることができる。 例えば、 テロメラーゼの触媒サプュニットである TERT遺伝子、 具体的には配列 番号32で表される DNAを、 レトロウイルスベクタ一に導入した後に心筋細胞への 分ィ匕能を有する細胞に導入する方法、 または心筋細胞への分化能を有する細胞に内 在する TERT遺伝子を誘導発現させる因子を心筋細胞への分化能を有する細胞に投 与する方法、 あるいは TERT遺伝子を誘導発現させる因子をコ一ドする DNAを含む ぺク夕一を心筋細胞への分化能を有する細胞に導入する方法などをあげることがで ぎる。 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. For example, 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.
このような TERT遺伝子を誘導発現させる因子は、 心筋細胞への分化能を有する 細胞に TERT遺伝子プロ乇一夕一と GFP(Green Fluorescent protein)、ルシフェラ一ゼ、 あるいはべ一ターガラクトシダ一ゼを組み合わせたレポ一夕一系を心筋細胞への分 化能を有する細胞に導入することで選別することができる。  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.
1 2 . 心筋細胞への分ィ匕能を有する細胞を抗体を用いて分離する方法  1 2. Method for separating cells having the ability to divide into cardiomyocytes using antibodies
生体内から取り出した各種組織から目的の表面抗原を発現している細胞を取得す る方法としては、 ソーティング機能を有したフロ一サイトメ一夕一を用いる方法、 磁気ビーズを用いる方法があげられる。  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.
フロ一サイトメーターのソ一ティングの方式としては、 水滴荷電方式、 セルキヤ プチヤ一方式などがあげられる （フローサイトメ一夕一自由自在、 p i 4— 2 3、 秀潤社、 1 9 9 9年） 。 どちらの方法も、 細胞の表面に発現している分子に結合し た抗体から発せられる蛍光強度を電気信号に変換することにより抗原の発現量を定 量することができる。 また、 使用する蛍光の種類を組み合わせることで、 複数の表 面抗原を利用して分離することも可能である。 蛍光としては、 FITC(fluorescein isothiocyanate) PE(phycoerythrin APC(Allo-phycocyanin) TR(TexasRed)、 Cy3、 CyChrome、 Red613, Red670, PerCP、 TRI-Color、 QuantumRedなどがあげられる （フ 口一サイトメ一夕一自由自在、 p 3— 1 3、 秀潤社、 1 9 9 9年） 。 生体内から取り出した各種組織、 具体的には骨髄または臍帯血から、 遠心分離な どの方法で細胞を分離したのち、 直接抗体で染色する方法と、 一度適当な培地中で 培養 ·増殖を行った後に抗体で染色する方法が利用できる。 細胞の染色はまず、 表 面抗原を認識する一次抗体と目的の細胞サンプルを混合し、 氷上で 3 0分間〜 1時 間、 インキュベーションする。 一次抗体が蛍光で標識されている場合には、 洗浄後 フローサイ トメ一夕一で分離を行う。 一次抗体が蛍光標識されていない場合には、 洗浄後一次抗体に対して結合活性を有する蛍光標識された二次抗体と一次抗体が反 応した細胞とを混合し、 再び氷上で 3 0分間〜 1時間、 インキュベーションする。 洗浄後、 一次抗体と二次抗体で染色された細胞をアローサイトメ一夕一で分離を行 ラ。 As a sorting method of the flow cytometer, there are a water drop charging method and a self-charging method, etc. (Flow cytometer can be freely adjusted, pi 4-23, Shujunsha, 1999) ). In both methods, 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. Examples of fluorescence 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). 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.
磁気ビーズを用いる方法では、 目的の表面抗原を発現している細胞を大量に分離 することができる。 分離の純度は上述のフロ一サイ トメ一夕一を用いる方法には及 ばないが、 精製を繰り返すことにより、 十分高い細胞純度を確保することができる。 細胞を一次抗体で染色後、 残存する一次抗体を除去し、 一次抗体と特異的に結合 する磁気ビーズを結合させた二次抗体を結合させる。 残存する二次抗体を洗浄除去 した細胞は磁石を設置したスタンドで分離することができる。 これらの操作に必要 な材料および装置は DYNAL社から入手することができる。  In the method using magnetic beads, 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.
磁気ビーズを用いる方法は、 細胞サンプル中より不要な細胞を除去するのにも同 様に利用することができる。 不要な細胞をより効率的に除去するには Stem Cell Technologies Inc(Vancouver, Canada)より販売されている StemSep法を用いること ができる。  The method using magnetic beads can be similarly used to remove unnecessary cells from a cell sample. In order to remove unnecessary cells more efficiently, the StemSep method sold by Stem Cell Technologies Inc (Vancouver, Canada) can be used.
上述の方法で用いられる抗体としては、 前記 8で取得された抗体、 または造血系 細胞の表面抗原 CD34、 CD117, CD14、 CD45、 CD90、 Sca-1、 Ly6c、 Ly を認識する抗 体、 血管内皮細胞の表面抗原 Flk- 1、 CD31s CD105、 CD14 を認識する抗体、 閭葉系 細胞の表面抗原 CD140を認識する抗体、 インテグリン CD49b、 CD49d、 CD29、 CD41を 認識する抗体、 マトリックス受容体 CD54、 CD102、 CD106、 CD44を認識する抗体があ げられる。 これらの抗体を組み合わせることで、 より高い純度で目的の細胞を取得 することができる。 具体的には、 CD34陰性、 CD117陽性、 CD144陰性および CD140陽性の性質を有する 細胞を取得するには、 ヒト骨髄細胞から CD34陽性細胞と CD144陰性細胞を上述した 免疫磁気ビーズの方法などを利用して除去した後、 CD117陽性および CD140陽性の細 胞画分を分取することで目的の細胞を分離することができる。 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. Specifically, to obtain cells with CD34-negative, CD117-positive, CD144-negative, and CD140-positive properties, 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.
1 3 . 心筋特異的な遺伝子のプロモーターレポ一夕一ベクターを用いた心筋前駆 細胞の分離'  1 3. Isolation of myocardial progenitor cells using myocardial-specific gene promoter repo overnight vector '
心筋細胞べの分化能を有する細胞から誘導した心筋細胞や心筋細胞の前駆細胞を 効率的に分離するために、 発光ォワンクラゲの緑色蛍光蛋白質 (green fluorescent protein;GFP)を遺伝子導入のためのレポ一夕一遺伝子の指標として用いることがで きる。  In order to efficiently separate cardiomyocytes and progenitor cells of cardiomyocytes derived from cells capable of differentiating cardiomyocytes, a green fluorescent protein (GFP) of luminescent jellyfish was used for gene transfer. It can be used as an indicator for the Yuichi gene.
具体的には、 心筋で特異的に発現している遺伝子または前記 9項で取得した心筋 細胞への分化能を有する細胞で特異的に発現している遺伝子のプロモー夕一の下流 に GFP遺伝子をづないだぺク夕一を作製し、 心筋細胞への分ィ匕能を有する細胞に導 入する。 このようなレポ一夕一ベクタ一を導入された細胞を薬剤耐性などの指標で 分離後、 心筋細胞へと分化誘導する。 分化誘導した細胞は GFPを発現し、 蛍光を発 生する。 蛍光を発生した心筋細胞ならびに心筋前駆細胞はフローサイトメ一夕一を 用いて容易に分離することができる （フローサイトメ一夕一自由自在、 p 4 4— 5 2、 秀潤社、 1 9 9 9年） 。  Specifically, 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).
心筋で特異的に発現している遺伝子のプロモ一夕一としては MLC2vやトロポニン Iを用いることができる。  MLC2v and troponin I can be used as promoters for genes specifically expressed in the heart muscle.
ベクターとしては、 上述した動物細胞用のプラスミドベクタ一、 アデノウイルス ベクタ一などを用いることができる。  As the vector, the above-described plasmid vector for animal cells, adenovirus vector and the like can be used.
1 4 . 心筋細胞への分化能を有する細胞から各種細胞への分化の誘導  1 4. Induction of differentiation from cardiomyocytes to cells
( 1 ) 心筋細胞への分ィヒ能を有する細胞から脂肪細胞への分ィヒの誘導  (1) Induction of effeciency from adipocytes to cells with effeciency for cardiomyocytes
心筋細胞への分化能を有する細胞から脂肪細胞への分化を誘導する方法としては、 P P ARァ受容体のァゴニストである Pioglitazoneヽ Troglitazoneを 0.4〃Mから 2 μ Μの終濃度となるよう培地中に添加する方法が挙げられる。  As a method for inducing the differentiation of cells having the ability to differentiate into cardiomyocytes into adipocytes, the PAR gamma receptor agonist Pioglitazone ヽ Troglitazone is added to the culture medium to a final concentration of 0.4〃M to 2μΜ. Is added.
または、培養皿一面に密集した細胞の培地中に、終濃度 1 JLLM dexamethasoneN 0.5 mM methyl-isobutylxanthine 0.01 m g/m 1 insulin、 0.2 mM indomethacin となるように、 それぞれすべて添加する方法も挙げられる。 Alternatively, a final concentration of 1 JLLM dexamethasone N 0.5 There is also a method in which each of them is added so that the concentrations thereof are 0.01 mg / m 1 insulin and 0.2 mM indomethacin, respectively.
( 2 ) 心筋細胞への分ィヒ能を有する細胞から軟骨細胞への分化の誘導  (2) Induction of chondrocyte differentiation from cells capable of dividing myocardial cells
心筋細胞への分化能を有する細胞から軟骨細胞への分化を誘導する方法としては、 1 X 105〜3 X 105個の細胞を遠心分離して得られた凝集塊に、終濃度が 0.01〃 g/mlと なるような T G F y5 3を含む培地で培養する方法が挙げられる。 . As a method for inducing the differentiation of cells having the ability to differentiate into cardiomyocytes into chondrocytes, 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. .
( 3 ) 心筋細胞への分化能を有する細胞から骨芽細胞への分ィ匕の誘導 .  (3) Induction of shunting from cells capable of differentiating into cardiomyocytes into osteoblasts.
心筋細胞への分化能を有する細胞から骨芽細胞への分化を誘導する方法としては、 細胞の培地中に終濃度 0. 1 μΜ dexamethasone、 0.05 m M ascorbic acid- 2 - - phosphate、 10 mM β -glycerophosphateとなるように、 それぞれを添加した培地中 で培養する方法が挙げられる。  As a method for inducing the differentiation of cells having the ability to differentiate into cardiomyocytes into osteoblasts, a final concentration of 0.1 μΜ dexamethasone, 0.05 mM M ascorbic acid-2--phosphate, 10 mM β A method of culturing in a medium to which each is added so as to become -glycerophosphate.
以下に実施例をあげて、 本発明を具体的に示す。  Hereinafter, the present invention will be specifically described with reference to examples.
発明を実施するための最良の形態 BEST MODE FOR CARRYING OUT THE INVENTION
実施例 1 . マウス骨髄からの心筋細胞への分ィ匕能を有する骨髄細胞の取得と培養 Example 1. Acquisition and culture of bone marrow cells having the ability to isolate cardiac muscle cells from mouse bone marrow
5週齢の C3H/Heマウス 10匹をエーテルを用い T麻酔し、そのうえで頸椎脱日に より致死させた。 マウスを半側臥位にして、 70%ェ夕ノ一ルを充分かけ消毒した。 次に大腿骨周辺の皮膚を広い範囲にわたり切開し、 大腿骨全面の大腿四頭筋をは さみで切除した。 膝関節の部分に軽くはさみを入れ、 関節を外し、 さらに大腿骨背 面の筋肉を切除した。 股関節の部分にはさみを入れ関節を外し、 大腿骨を取り出し た。 大腿骨に付着している筋肉をはさみで切除し、 大腿骨全体を露出させた。 大腿 骨の両端をはさみで切断後、 テルモ製 23Gの針を装着した 2.5ml注射器に 20%FCS を含有する MDM培地を約 l.5ml入れ、注射針の先端を大腿骨の膝関節側の断端に差 し込み、 試験管の中に培養液を吹き出すことで、 骨髄細胞を押し出した。 取得した 細胞は、 20%FCSヽ 100mg/ml penicillinヽ 250ng/ml streptomycin^ 85mg/ml amphotericin を含有する IMDM培地中で 33°Cで、 5% C02濃度の孵卵機を用いて培養を行った。継 代を続けることで、 細胞は間葉系の細胞へと均一化し、 造血系の細胞は消失した。 約 4ヶ月上記条件で培養を行い、 不死化した細胞を選択した後、 希釈により 192 種類の独立した単一細胞 (single cell)由来の細胞株を樹立した（以下、骨髄由来初代不 死化細胞株と称する） 。 これら独立のクローン由来の細胞にそれぞれに 3 Mの終 濃度になるように 5-aza- Cを添加し 時間培養した後、培地を IMDM培地に代えて さらに 2週間培養することで拍動する細胞を産生するクローンを選択した。 骨髄由 来初代不死化細胞 192個のうち、 心筋細胞への分化能を有する骨髄細胞は 3個であ つた。 このうちの 1つが KUM2である。 以後、 骨髄細胞 KUM2ならびに後述する多 分化能幹細胞 BMSCは特別な指定がない限り、 20%FCS、 lOOmg/ml penicillin, 250ng/ml streptomycin^' 85mg/ml amphotericinを含有する IMDM培地中で 33°Cで、 5% C〇2濃度 の孵卵機を用いて培養を行った ό KUM2細胞は 3 ju Μの終濃度の.5-aza-Cに ¾時間 曝露することで、 · 非特異的に自己拍動する心筋細胞が分化誘導してぐるが、 その頻 度は非常に低かった (10 7 cellに 1つ以下)。 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. After cutting both ends of the femur with scissors, about the MDM medium containing 20% FCS in 2.5ml syringe fitted with a needle manufactured by Terumo 23G l. Put 5 ml, the tip of the injection needle of the femur of the knee joint side Bone marrow cells were extruded by inserting into the stump and blowing the culture into a test tube. Acquired cells, with 20% FCSヽ100 mg / ml penicillinヽ250ng / ml streptomycin ^ 85mg / ml amphotericin in IMDM medium containing 3 3 ° C, and cultured with 5% C0 2 concentration in the incubator machine Was. By continuing the passage, cells became homogenous to mesenchymal cells and hematopoietic cells disappeared. After culturing under the above conditions for about 4 months and selecting immortalized cells, 192 independent cell lines derived from single cells were established by dilution (hereinafter referred to as primary bone marrow-derived primary cells). A dead cell line). Cells pulsating by adding 5- aza-C to each of these independent clone-derived cells to a final concentration of 3 M and culturing for an additional 2 weeks, and replacing the medium with IMDM medium for another 2 weeks Was selected. Of the 192 primary immortalized cells derived from bone marrow, three were capable of differentiating into cardiomyocytes. One of them is KUM2. Thereafter, unless otherwise specified, 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. in 5% C_〇 O Kum2 cells were cultured using 2 concentrations of incubation machine by ¾ 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).
しかし、 KUM2細胞から出現する自己拍動する細胞周辺をク口一ニングシリンジ で採取すると、 増殖能の高い多分化能幹細胞 BMSC(FERM BP-704 と、 限られた回 数のみ増殖し心筋細胞へと分化する細胞 （以下、 単に心筋前駆細胞と称する） の少 なくども 2種類の細胞が観察された。 BMSC細胞は、 グロ一ニングシリンジで回収 した後、 細胞を継代培養し、 不死化する細胞を選別することで、 クローン化を行つ た。 BMSG細胞は、 その親株となる KUM2よりも 100倍以上効率的に分化誘導する ことが観察された。また心筋前駆細胞は再び 5-aza-Cを添加し 24時間培養した後、 培地を IMDM培地に代えてさらに 2~3週間培養することで多くの自律拍動する細胞 が効率的に出現した。 該心筋前駆細胞は、 増殖条件下では、 単核の線維芽細胞様の 形態を呈し、 心筋収縮蛋白質はほとんど発現していない。 しかし 5 - aza - Cにより最 終分化を誘導すると形態は著しく変化した。 However, when you collect the cells peripheral to self-beating emerging from KUM2 cells in a click opening-learning syringe, their ability to grow high multipotent stem cells BMSC (FERM BP- 7 04 and, the heart muscle to grow only a limited number of times 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. After adding -C and culturing for 24 hours, the medium was replaced with IMDM medium and culturing was further performed for 2 to 3 weeks, so that many autonomously pulsating cells appeared efficiently. Has a mononuclear fibroblast-like morphology, Muscle contraction protein was hardly expressed but 5 -. Aza - form to induce a final differentiation was significantly changed by C.
分化誘導 1週間目頃より、 一部の細胞は細胞質が大きくなり円形あるいは棒状を 呈し、 後に自律拍動を開始する細胞となるが、 この時点では自律拍動を行うことは 少なかった。 分化誘導後 2週間になると、 自己拍動を開始した。 この自己拍動した 細胞は互いに連結しあい、縦に連結して筋管細胞様となった。 3週間以後には多くの 細胞が縦に 1列にならび、 同期して収縮した。 分化後 4週間以後には培養皿の上の 直接連結される細胞は、 すべて同期して収縮し心筋組織様になった。 マウスの心臓 は、 毎分 300〜400回程度の心拍数で収縮するが、 これに対してマウス成体骨髄由来 の細胞より分ィ匕した心筋細胞は、培養条件下において毎分 120〜250回の速さで規則 的に収縮した。 From around the first week of differentiation induction, some cells became larger in cytoplasm and exhibited a circular or rod-like shape, and later became cells that started autonomous pulsation. At this point, seldom autonomous pulsation was performed. Two weeks after induction of differentiation, self-pulsation started. The self-pulsating cells were connected to each other and connected vertically to become myotube-like. After three weeks, many cells lined up in a row and contracted synchronously. After 4 weeks post-differentiation, all directly connected cells on the culture dish contracted synchronously and became like myocardial tissue. The mouse heart contracts at a heart rate of about 300 to 400 beats per minute, whereas the adult mouse bone marrow The cardiomyocytes separated from the above cells contracted regularly at a rate of 120 to 250 times per minute under culture conditions.
実施例 2 . マウス骨髄細胞から誘導される心筋細胞の特性  Example 2. Characteristics of cardiomyocytes derived from mouse bone marrow cells
骨髄由来細胞から形成される自律拍動する心筋様細胞が、 実際に心筋細胞の性質 を保有しているかどうかの角军析を行った。  An analysis was performed to determine whether autonomously beating cardiomyocytes formed from bone marrow-derived cells actually possess the properties of cardiomyocytes.
実施例 1で取得した、 骨髄由来初代不死化細胞株、 マウス骨髄由来多分化能幹細 胞 BMSC .および心筋前駆細胞から分ィ匕誘導した心筋細胞から、 それぞれ Trizol Reagents(GIBCO BRL社製)を用いて全 RNAを取得した。次に、該全 RNAを基質とし て Superseiptn reverse transcriptase(GIBCO BRL社製)を用いて First strand CDNAを合 成した。  Using 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. To obtain total RNA. Next, First strand CDNA was synthesized using Superseiptn reverse transcriptase (GIBCO BRL) using the total RNA as a substrate.
次に、 心筋細胞特異的な遺伝子の発現を検討するために、 該 First strand CDNAを 基質どして、 配列番号 33〜58に示した塩基配列を有する合成 DNAを用いて定量的 PCRを.行った。 心筋細胞特異的な遺伝子としては、 ナトリウム利尿ペプチドである ANPおよび BNP、 ミオシン重鎖であるひ -MHCおよび/?- MHC、 ァクチンであるひ -skeletal actinおよび — skeletal actinヽ ミオシン軽鎖である MLC- 2a、 MLC- 2vヽ 心筋 細胞特異的転写因子である Nkx2.5/Csx、 GATA4、 TEF- 1、 MEF- 2C、 MEF- 2D、 MEF-2A を用いた。 Next, in order to examine the expression of a cardiomyocyte-specific gene, quantitative PCR was performed using the first strand cDNA as a substrate and a synthetic DNA having the nucleotide sequence shown in SEQ ID NOS: 33 to 58. Was. 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 ヽ the myosin light chain MLC -. 2a, MLC- 2vヽcardiomyocyte-specific transcription factors Nkx2 5 / Csx, GATA4, TEF- 1, MEF- 2C, MEF- 2D, was used MEF-2A.
ANPの増幅には配列番号 33、 34の塩基配列を有する合成 DNAを、 BNPの増幅に は配列番号 35、 36の塩基配列を有する合成 DNAを、 ひ- MHCの増幅には配列番号 37、 38の塩基配列を有する合成 DNAを、 ?- MHCの増幅には配列番号 39、 40の塩 基配列を有する合成 DNAを、 ひ -skeletal actinの増幅には配列番号 41、 42の塩基配 列を有する合成 DNAを、 skeletal actinの増幅には配列番号 43、 4 の塩基配列を 有する合成 DNAを、 MLC-2aの増幅には配列番号 45、 46の塩基配列を有する合成 DNAを、 MLC- 2vの増幅には配列番号 47、 48の塩基配列を有する合成 DNAを、 Nkx2.5/Csxの増幅には配列番号 49、 50の塩基配列を有する合成 DNAを、 GATA4の 増幅には配列番号 51、 52の塩基配列を有する合成 DNAを、 TEF-1の増幅には配列 番号 53、 54の塩基配列を有する合成 DNAを、 MEF- 2Cの増幅には配列番号 55、 56 の塩基配列を有する合成 DNAを、 MEF- 2Dの増幅には配列番号 57、 58の塩基配列を 有する合成 DNAを、 MEF-2Aの増幅には配列番号 59、 60の塩基配列を有する合成 DNAを用いた。 For the amplification of ANP, a synthetic DNA having the nucleotide sequence of SEQ ID NO: 33 or 34, for the amplification of BNP, a synthetic DNA having the nucleotide sequence of SEQ ID NO: 35 or 36, and for the amplification of MHC, SEQ ID NO: 37 or 38 Synthetic DNA having the nucleotide sequence of SEQ ID NOs: 39 and 40 for the amplification of? -MHC, and 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 The synthetic DNA having the nucleotide sequence of SEQ ID NO: 47 or 48, the synthetic DNA having the nucleotide sequence of SEQ ID NO: 49 or 50 for the amplification of Nkx2.5 / Csx, and the DNA of SEQ ID NO: 51 or 52 for the amplification of GATA4 Synthetic DNA having the nucleotide sequence of SEQ ID NOS: 53 and 54 for amplification of TEF-1 and sequence for amplification of MEF-2C Synthetic DNAs having the nucleotide sequences of Nos. 55 and 56, and the nucleotide sequences of SEQ ID Nos. 57 and 58 for the amplification of MEF-2D For the amplification of MEF-2A, a synthetic DNA having the nucleotide sequences of SEQ ID NOS: 59 and 60 was used.
生体内で分ィヒ誘導する心筋細胞は、 心筋収縮の心拍数またはエネルギー効率に違 いを持たせるために、 胎児期、 新生児期あるいは成熟期によって、 または心房筋あ るいは心室筋の相違によって、 心筋収縮蛋白質のアイソフォームに違いがある。 培養系で心筋細胞に分ィ匕した骨髄細胞の場合、 アイソフオームの発現様式は — ァクチンの場合は骨格筋型のほうが心筋型より多く発現し、 ミオシン重鎖の場合は 型のほうがひ型よりも多く発現していた。 ミオシン軽鎖では 2 V型が発現してい るのに対し、 · 2 a型の発現は観察されなかった。  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. However, there is a difference in 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.
また、 培養系で心筋細胞に分化した骨髄細胞の分化誘導後には、 ナトリウム利尿 ぺプチドである ANPおよび BNPの発現が見られた。以上の心筋収縮蛋白質の発現様 式より判断すると、 ·培養系で心筋細胞に分化した骨髄細胞の表現型は胎児型心室筋' 細胞の形質を有すると考えられる。  In addition, expression of 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.
培養系で心筋細胞に分ィ匕した骨髄細胞では、 Nkx2.5/Csx、 GATA4、 MEF- 2A、 MEF-2C MEF- 2D、 TEF- 1遺伝子の発現が観察された。増殖中の骨髄由来初代不死 化細胞株ではこれらの転写因子の発現は認められなかったが、 増殖中の骨髄由来心 筋前駆細胞では Nkx2.5/Csx、 GATA4および MEF- 2Cの発現が観察され、心筋細胞へ の分化誘導に伴い、 遅れて MEF-2Aおよび MEF- 2Dの発現誘導が観察された。 The expression of 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.
次に、 ガラス微少電極により、 培養系で心筋細胞に分化した骨髄細胞の活動電位 を記録した。 活動電位は、 細胞を 1.49mM CaCl2、 4.23mM KC1、 25mM HEPES(pH7.4) を添加した IMDM培地中で培養し、 Diaphoto - 300実体顕微鏡 (ニコン社製)下、 温度 25°Cで測定した。ガラス電極は電極抵抗を 15~30 Ωに設定して 3M KC1を充填した。 膜電位の測定は MEZ-8300 (日本光電社製）を用いて電流クランプモードで行った。 測定結果は RTA- 1100M (日本光電社製） を用いて熱感紙に記録した。 その結果、 培 養系で心筋細胞に分化した骨髄細胞は、 洞結節細胞型と心室筋細胞型の 2種類が観 察された。 両者に共通する活動電位の特徴は、 ①活動電位持続時間が長いこと、 ② 比較的浅い静止期電位を持つこと、 ③ペースメーカ一細胞にみられる静止期電位の 緩やかな脱分極が認められることであった。 また、 心室筋細胞型では活動電位は Peak&Dome型 （活動電位第 1相を持つ） を呈した。 洞結節細胞型の活動電位持続時 間、 拡張期膜電位、 活動電位振幅は従来ゥサギやラヅ トで報告されている洞結節の 活動電位と近似していた。 心室筋細胞型ではこれに比べて、 静止期膜電位は深く、 活動電位振幅は大きい傾向を示した。 分化誘導後、 2〜3週間の細胞はすべて洞結節 細胞型が記録されたが、 分化誘導後 4週間頃より心室筋細胞型が観察され時間経過 とともに次第に増加した。 Next, 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 Ω. 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). As a result, 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. In the ventricular muscle cell type, 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. In the ventricular myocyte cell type, the resting membrane potential tended to be deeper, and the action potential amplitude tended to be larger. After induction of differentiation, 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.
実施例 3 . サイトカインを用いた心筋細胞への分ィ匕の促進  Example 3. Promotion of shunting to cardiomyocytes using cytokines
心筋細胞への分化能を有するマウス骨髄細胞の心筋分化誘導率を増加させるため、 5- aza-Cで分化誘導をおこなう際に、各種サイトカインを添加して誘導率が増加する かどうか角军析をおこなった。  In order to increase the induction rate of myocardial differentiation in mouse bone marrow cells capable of differentiating into cardiomyocytes, it was analyzed whether addition of various cytokines would increase the induction rate when inducing differentiation with 5-aza-C. Was done.
心筋細胞への分化能を有するマウス骨髄由来多分化能幹細胞 (BMSC)を 2 X 104細胞 /mlとなるように'60mm培養ディヅシュあるいは60mmフィブロネクチン付着ディヅ シュ (fibronectin- coated dish ;Becton Dickinson社製) に蒔き、 33°C！、 5%C02濃度の孵卵 機を用いて培養を行った。 Mouse bone marrow-derived pluripotent stem cells (BMSCs) 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.
翌日、該培養液に 5-aza- Cを終濃度 3〃 Mとなるよう添カ卩した上で、更に、 PDGF のみ添加（培養ディヅシュ A)、 PDGFとレチンイン酸の両方添加（培養ディヅシュ B ) 、 添加なし （培養ディッシュ C ) の 3種類の異なる処理を行い培養を継続した (終濃度は PDGFは 10ng/ml、 レチノィン酸は 10—9M) 。 The next day, 5-aza-C was added to the culture solution to a final concentration of 3 M, and then only PDGF was added (culture dish A), and both PDGF and retinoic acid were added (culture dish B). , without addition culture was continued for 3 different processes (culture dish C) (final concentration PDGF is 10 ng / ml, Rechinoin acid 10- 9 M).
翌日 5-aza - Cを培地から除去するために、 培地を新しいものに交換し、 再び培養 ディッシュ Aには PDGFを終濃度 10ng/mlになるように添加し、 培養デイツシュ B には PDGFを終濃度 lOng/mlとレチノィン酸を終濃度 10_9Mになるように添カ卩した。 それ^ら更に 2日後、 4日後にも同様の培地交換と PDGFあるいはレチノイン酸の添 加を行った。 The next day, in order to remove 5-aza-C from the medium, replace the medium with a fresh medium, add PDGF again to culture dish A to a final concentration of 10 ng / ml, and terminate PDGF in culture dish B. It was添Ka卩concentration LONG / ml and Rechinoin acid to a final concentration of 10_ 9 M. Two or four days later, the same medium exchange and addition of PDGF or retinoic acid were performed.
薬剤を加えてから 4週間後、 細胞の形態を位相差顕微鏡下で観察した。その結果、 5-aza-Cのみを添加した培養ディッシュでは約 3割の細胞が筋管様細胞となるのに 対し、 PDGFを添カ卩すると約 4割、 PDGFとレチノイン酸を同時に添加すると約 5割 の細胞が筋管様細胞となった。 また、 フイブロネクチン付着ディッシュの 3群では、 培養ディッシュの 3群に比べて、 筋管様細胞になる細胞数が約 1割程度ずつ増加し た。 Four weeks after adding the drug, the morphology of the cells was observed under a phase-contrast microscope. As a result, about 30% of the cells in the culture dish containing only 5-aza-C became myotube-like cells, while about 40% were added when PDGF was added, and about 40% when PDGF and retinoic acid were added simultaneously. 50% of cells became myotube-like cells. In addition, the number of cells that become myotube-like cells in the three groups of fibronectin-attached dishes increased by about 10% compared to the three groups of culture dishes. Was.
得られた、 筋管様細胞から RNAを回収して、 該筋管様細胞で発現している遺伝子 を配列番号 7 1〜7 8で示した合成オリゴヌクレオチドを用いて定量的 PCRを解析 したところ、 PDGFあるいはレチノイン酸は骨格筋に関係する MyoD、 fTnl遺伝子の 発現を亢進するが、 心筋に特異的に関係する cTnl, ΑΝΡの発現は誘導しなかった。 次に、 心筋細胞への分化能を有するマウス骨髄由来多分ィ匕能幹細胞 (BMSC)を 2 X 104細胞/ mlとなるように 60mm培養ディヅシュに蒔き、' 33。C、. 5%C02濃度の孵卵機 を用いて培養を行った。 · 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. However, PDGF or retinoic acid enhanced the expression of MyoD and fTnl genes associated with skeletal muscle, but did not induce the expression of cTnl, す る specifically associated with myocardium. Next, 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. ·
翌日、該培養液に 5 - aza-Cを終濃度 3〃Mとなるよう添加した'上で、更に、 FGF-8 を終濃度 10ng/mlになるように添加 （培養ディヅシュ D )、 ET- 1を終濃度 10ng/ml になるように添加 . （培養ディッシュ E )、 Midkineを終濃度 10ng/mlになるように 添カロ （培養ディヅシュ F )、 BMP4を終濃度 10ng/ml.になるように添加 （培養ディ .ヅシユ G ) ， 添加なし （培養デイツシュ H ) の 5種類の異なる処理を行い培養を継 ； Kmした。 On the next day, 5-aza-C was added to the culture solution to a final concentration of 3 M, and FGF-8 was further added to a final concentration of 10 ng / ml (culture dish D), ET -. added 1 to a final concentration of 10 ng / ml (culture dish E), added Caro to a final concentration of 10n g / ml of Midkine (culture Didzushu F), to a final concentration of 10 ng / ml of BMP4. Five different treatments were added as described above (culture dish G) and without addition (culture dish H), and the culture was continued;
翌日 5-aza- Cを培地から除去するために、 培地を新しいものに交換し、 再び培養 ディッシュ Dには FGF- 8を終濃度 10ng/mlになるように添加し、 培養ディヅシュ E には ET- 1を終濃度 10ng/mlになるように添加、 培養ディヅシュ Fには Midkineを終 濃度 10ng/mlになるように添加、培養デイツシュ Gには BMP4を終濃度 10ng/mlにな るように添加して培養を継続した。それから更に 2日後、 4日後にも同様の培地交換 と FGF- 8， ET-1, Midkineあるいは BMP4の添加を行った。 The next day, in order to remove 5-aza-C from the medium, replace the medium with a fresh medium, add FGF-8 again to culture dish D to a final concentration of 10 ng / ml, and add ET to culture dish E. - 1 added to a final concentration of 10 ng / ml, the Midkine the culture Didzushu F added to a final concentration 10n g / ml, in so that such a final concentration 10 ng / ml of BMP4 in culture Deitsushu G The culture was continued with the addition. Two and four days later, the same medium exchange and addition of FGF-8, ET-1, Midkine or BMP4 were performed.
5-aza-Cを加えてから 4週間後、細胞の形態を位相差顕微鏡下で観察した。その結 果、 5 - aza - Cのみを添加した培養ディヅシュでは約 3割の細胞が筋管様細胞となるの に対し、 FGF- 8, ET - 1， Midkineあるいは BMP4を添カロした培養ディヅシュでは約 5 割の細胞が筋管様細胞となった。  Four weeks after the addition of 5-aza-C, cell morphology was observed under a phase contrast microscope. As a result, about 30% of the cells in the culture dish containing only 5-aza-C became myotube-like cells, whereas those in the culture dish supplemented with FGF-8, ET-1, Midkine or BMP4 added to the culture dish. About 50% of the cells became myotube-like cells.
得られた、 筋管様細胞から RNAを回収して、 該筋管様細胞で発現している遺伝子 を配列番号 7 1〜7 8で示した合成オリゴヌクレオチドを用いて定量的 PCR解析を 行ったとところ、 FGF -8， ET-1, Midkineあるいは BMP4は、 それぞれ単独で心筋に特 異的な遺伝子である cTnl, ANPの発現を亢進することが観察された。 実施例 4 . DMSOを用いた骨髄由来幹細胞からの心筋細胞への分化誘導 RNA was collected from the obtained myotube-like cells, and the gene expressed in the myotube-like cells was subjected to quantitative PCR analysis using the synthetic oligonucleotides shown in SEQ ID NOs: 71 to 78. However, it was observed that 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
実施例 1に示した方法により、 取得した心筋細胞への分化能のあるマウス骨髄由 来多分ィ匕能幹細胞 (BMSC)に 3〃 Mの 5- aza- Cの代わりに 10〃 Mの DMSOを添加し 2 4時間培養した後、 培地を IMDM培地に代えて、 さらに 6週間培養を続けた。 その結果、 拍動する心筋細胞が分化誘導されることを見出し、 これらの細胞には Nkx2.5/Csxおよび GATA4遺伝子が発現しており、 5- aza-Cを添加したときと同様の 性質を有した心筋細胞であることが示された。 この解析結果は、 5」aza- Cと DMSO の共通の機能である染色体 DNAの脱メチル化が心筋細胞の分化に必要であることを 示している。 According to the method described in Example 1, 10 μM DMSO was substituted for the obtained mouse bone marrow-derived probably stem cell (BMSC) capable of differentiating into cardiomyocytes instead of 3 μM 5 -aza-C. After adding and culturing for 24 hours, the medium was changed to IMDM medium, and the culture was continued for another 6 weeks. As a result, found that cardiomyocytes beating is induced to differentiate, these cells have been expressed Nkx2.5 / Csx and GATA 4 gene, 5 - properties similar to when the aza-C was added Were shown to be cardiomyocytes having The results of this analysis indicate that chromosomal DNA demethylation, a common function of 5 "aza-C and DMSO, is required for cardiomyocyte differentiation.
実施例 5 . 心筋細胞への分ィヒ能を有するマウス骨髄由来多分化能幹細胞が多分ィ匕 能を有する幹細胞および心筋前駆細胞であることの証明  Example 5. Demonstration that mouse 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
マウス骨髄由来多分化能幹細胞 (BMSC)から分化誘導する拍動細胞が心筋細胞の性 質を保有していることは示されたが、 心筋細胞への分化能を有するマウス骨髄由来 多分ィ匕能幹細胞 (BMSC)に、 心筋前駆細胞が存在しているのか、 もっと未分化で心筋 細胞以外の、 例えば脂肪細胞などに分化可能な幹細胞が存在するかを調べるため、 シングルセル ·マ一キング （Single cell marking)の実験を行った。  Although it was shown that the pulsatile cells induced to differentiate from mouse bone marrow-derived pluripotent stem cells (BMSCs) possess the properties of cardiomyocytes, mouse bone marrow-derived cells with cardiomyocyte differentiation potential 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).
具体的には、 分化誘導を行う前に、 ある 1つの細胞に GFP遺伝子をウィルスべク 夕一を導入して標識し、 その後分化誘導させて標識した細胞がどのような細胞に分 化したかで判断した。  Specifically, before inducing differentiation, 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.
まず、 GFP遺伝子を発現させるレトロウイルスベクタ一プラスミド GAR3- GFPお よび、 Ecotropic遺伝子を発現させる pCMV - Ecoプラスミ ドベクターを、 Molecular し loning, A Laboratory Manual, Second Edition, Cold Spring Haroor Laboratory Press (1989)等に記載のアル力リ中和法および PEG沈殿法を用いて、純度の高い DNAを取 得した。  First, the retroviral vector plasmid GAR3-GFP, which expresses the GFP gene, and the pCMV-Eco plasmid vector, which expresses the Ecotropic gene, are molecularly cloned, A Laboratory Manual, Second Edition, Cold Spring Haroor Laboratory Press (1989) Highly pure DNA was obtained by using the neutralization method and the PEG precipitation method described in the above.
この DNAをトランスフエクシヨンさせる前日に、 コンフルェントになった、 gag および pol遺伝子を保有する 293細胞を 1/5希釈で 10cmディヅシュに継代し、ー晚 37°C、 5%C02濃度の孵卵機を用いて培養をおこなった。 トランスフエクションは以下の通りに行った。 The DNA on the previous day to transflector Ekushi Yong became Konfuruento, passaged 10cm Didzushu 1/5 diluted 293 cells harboring gag and pol genes, over晚37 ° C, 5% C0 2 concentration in the incubation The culture was performed using a machine. Transfection was performed as follows.
GAR3-GFPレトロウイルスベクタープラスミ ド DNA 1 5 gと pCMV- Ecoプラス ミドベクタ一 DNA 5〃 gを 250mM CaCl2 (pH6.95) 0.5mlに加えて溶解させ、その溶液 を 15mlのチューブに入れた 2 X BBS [50mM BES(N，N - bis(2- hydroxyethl) - 2- aminoethanesulfonic acid)s 280mM NaCl、 1.5mM Na2HP〇4(pH6.95)] 0.5mlに滴下して 10 分間室温で静置させた。 その後、 この DNA溶液を、 前日に用意した 293細胞培地中 に滴下させ、 37°C、 5% 02濃度の孵卵機を用いて培養を行った。 翌日、 培地を交換 し、'更に 37°C、 5%C02濃度の孵卵機を用いて培養を行った。 15 g of GAR3-GFP retroviral vector plasmid DNA and 5 μg of pCMV-Eco plasmid vector DNA were added to 0.5 ml of 250 mM CaCl 2 (pH 6.95) and dissolved, and the solution was placed in a 15 ml tube. X BBS [50mM BES (N, N - bis (2- hydroxyethl) - 2- aminoethanesulfonic acid) s 280mM NaCl, 1.5mM Na 2 HP_〇 4 (pH 6.95)] was added dropwise to 0.5ml static at room temperature for 10 minutes Was placed. Thereafter, 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.
培地を交換して 2 ·曰後に、 培養上清を 0·45 mのフィル夕一 （Millipore社製） で ろ過し、 ウィルスベクタ一を含む溶液を回収した。 この溶液を IMDM培地で 10一1、 10一2、 10— 3、 10—4、 10— 5に希釈した。 2 ·曰後Replace the medium, the culture supernatant was filtered through a 0 · 45 m Phil evening one (Millipore Corp.) to recover a solution containing viral vectors scratch. 10 one 1 in the solution IMDM medium, 10 one 2, 10 3, 10 4, and diluted to 10 5.
ウィルスベクタ を導入される側の心筋細胞への分化能を有するマウス骨髄由来 多分化能幹細胞は、 ウィルスをインフエクシヨンさせる前日に 2 X 1 細胞/ゥ iル となるように 6ゥエル ·ディヅシュに蒔いた。  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.
希釈した、 ウィルスベクタ一を含む溶液には、 終濃度 8〃g/mlとなるように、 Hexadimethrine bromide(polybrene) ( S i gm a社製） を添加し、 心筋細胞への分化能 を有するマウス骨髄由来多分化能幹細胞 (BMSC)の培養上清 2 m 1をウィルス液 2ml と置換し、 33°C、5%C02濃度の孵卵機を用いて培養をおこなった。 5時間後、 培養上 清を新しい IMDM培地に交換し、更に 33°C、5%C02濃度の孵卵機を用いて培養を行つ た。 Hexadimethrine bromide (polybrene) (manufactured by Sigma) was added to the diluted solution containing the virus vector to a final concentration of 8 μg / 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.
2日間培養を行った後、 蛍光顕微鏡下で GFPを発現している細胞を観察し、 細胞 1000個あたり 1つの GFP陽性細胞があるような細胞群を得た。  After culturing for 2 days, cells expressing GFP were observed under a fluorescence microscope, and a cell group having one GFP-positive cell per 1000 cells was obtained.
該細胞を 8 X 103細胞/ディヅシュとなるよう、 35mmガラスべ一スディヅシュ（旭 テクノグラス社製） に蒔き、 33°C、5%C02濃度の孵卵機を用いて培養を行った。 So that the cell with 8 X 10 3 cells / Didzushu were plated 35 mm glass base one Sudidzushu (manufactured by Asahi Techno Glass), it was incubated with 33 ° C, 5% C0 2 concentration in the incubator unit.
翌日、 5-aza-C (Sigma社製) 、 PDGF- BB (Peprotech社製） 、 all transレチノイン 酸（Sigma社製） をそれぞれ終濃度 3 z M、 10ng/mls 10— 9Mとなるよう添加し、 添カロ して 2日後および 4日後には、 培地交換を行うとともに、 再度 PDGF— B B (以降 PDGFと略す） 、 all transレチノイン酸を上述と同じ濃度で添加した。 4週間後、 蛍光顕微鏡で GFP陽性細胞がどのように分ィ匕したかを観察すると、 心 筋細胞のみが GFP陽性になっている細胞集団、心筋細胞と未分ィ匕幹細胞が GFP陽性 になっている細胞集団、 ならびに心筋細胞、 脂肪細胞および未分化幹細胞の 3者が GFP陽性になっている細胞集団の 3種類の細胞集団が見られた。 すなわち、 多分化 能幹細胞から心筋前駆細胞が確率的 （stochastic) に分ィ匕誘導してくることが明らか となった。 またこの結果は、 心筋細胞への分化能を有するマウス骨髄細胞には多分 化能をを有する幹細胞が存在することを示した。 The next day, (manufactured by Sigma Co.) 5-aza-C, PDGF- BB ( manufactured by Peprotech, Inc.), final concentration all trans retinoic acid (Sigma Co.), respectively 3 z M, so as to be 10ng / ml s 10- 9 M Two and four days after the addition, the medium was replaced, and PDGF-BB (hereinafter abbreviated as PDGF) and all trans retinoic acid were again added at the same concentrations as described above. Four weeks later, observation of how the GFP-positive cells were separated by fluorescence microscopy revealed that the cell population in which only myocardial cells were GFP-positive, and that the myocardial cells and unseparated stem cells were GFP-positive. Cell population, and three types of cell populations in which cardiomyocytes, adipocytes, and undifferentiated stem cells were GFP-positive. In other words, it became clear that myocardial progenitor cells stochastically induce induction from multipotent stem cells. The results also showed that mouse bone marrow cells capable of differentiating into cardiomyocytes had stem cells capable of multipotency.
実施例 6 . 転写因子の強制発現による心筋細胞分化の促進  Example 6. Promotion of cardiomyocyte differentiation by forced expression of transcription factor
マウス心筋細胞への分化能を有するマウス骨髄由来多分ィ匕能幹細胞 (BMSC).に心筋 細胞分化に関係する転写因子を強制的に発現させることによる心筋細胞への分化に 与える影響を解析した。  The effect of forcibly expressing a transcription factor related to cardiomyocyte differentiation on mouse bone marrow-derived probable stem cell (BMSC), which has the ability to differentiate into mouse cardiomyocytes, on cardiomyocyte differentiation was analyzed.
具体的には、 分化誘導を行う前に、 Nkx2.5/Csxまたは GATA4遺伝子をウィルス ベクターを用いて導入して、 その後分化誘導させて心筋細胞への分ィ匕の効率を f食討 した。  Specifically, before inducing differentiation, 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.
まず、 Nkx2.5/Csxを発現させる目的で、 レトロウイルスベクタ一プラスミド pCLNCX (Imgenex社)に Nkx2.5/Csxを組み込み、 pCLNC - Nkx2.5/Csxを調製した。 また、 GATA4を発現させる目的で、 レトロウイルスぺク夕一プラスミド pCLNCX (Imgenex社)の G^S耐性遺伝子部分をピューロマイシン耐性遺伝子に置換したブラ スミド pCLPCXに、 GATA4を組み込み、 pCLPC- GATA4を調製した。 レトロウィル スぺク夕—プラスミド pCLNC- Nkx2.5/Csxと pCLPC- GATA4および、 Ecotropic遺伝 子を発現させる pCMV- Ecoプラスミドベクタ一（Imgenex社)を、 Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)等に言 3載 のアル力リ中和法および PEG沈殿法を用いて、 純度の高い DNAを取得した。  First, in order to express Nkx2.5 / Csx, Nkx2.5 / Csx was incorporated into a retroviral vector plasmid pCLNCX (Imgenex) to prepare pCLNC-Nkx2.5 / Csx. In addition, for the purpose of expressing GATA4, 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. Retro-Williams-Plasmids pCLNC-Nkx2.5 / Csx and pCLPC-GATA4 and pCMV-Eco plasmid vector (Imgenex) expressing Ecotropic gene Highly pure DNA was obtained using the neutralization method and the PEG precipitation method described in Spring Harbor Laboratory Press (1989).
これらの DNAをトランスフエクシヨンさせる前日に、 コンフルェントになった、 gagおよび pol遺伝子を保有する 293細胞を 1/5希釈で 10cmディヅシュに継代し、 一晩 37°C、 5%C02濃度の孵卵機を用いて培養を行った。 These DNA the day prior to transflector Ekushi Yong became Konfuruento, passaged 10cm Didzushu 1/5 diluted 293 cells harboring gag and pol genes, 37 ° C, 5% C0 2 concentration in the overnight Culture was performed using an incubator.
トランスフエクションは以下の通りにおこなった。  Transfection was performed as follows.
pCLNC- Nkx2.5/Csxあるいは pCLPC— GATA4レトロウィルスベクタ一プラスミド DNA15 pCMV— Ecoプラスミドベクタ一 DNA 5 / gを 250mMCaCl2 (pH6.95) 0.5mlに加えて溶解させ、 その溶液を 15mlのチューブに入れた 2 X BBS [50mM BES(N,N-bis(2-hydroxyethl)-2-aminoethanesulfonic acid)、280mM NaCl、 1.5mM 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
Na2HP04(pH6.95)] 0.5mlに滴下して 10分間室温で静置させた。 その後、 この DNA溶 液を、 前日に用意した 293細胞培地中に滴下させ、 37° 5%C02濃度の孵卵機を用 いて培養を行った。 翌日、 培地を交換し、 更に 37°C、 5%C02濃度の孵卵機を用いて 培養を行った。 Na 2 HP0 4 (pH6.95)] was allowed to stand to room temperature for 10 minutes dropwise to 0.5 ml. Thereafter, the DNA dissolved solution was dropped into 293 in cell culture medium which is prepared on the previous day, it was cultured have use the 37 ° 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.
培地を交換して 2曰後に、 培養上清を 0.45〃 mのフィルター （MilHpore社製） で ろ過し、 ウィルスベクタ を含む溶液を回収した。  After two changes of the medium, the culture supernatant was filtered through a 0.45 μm filter (MilHpore) to recover a solution containing the virus vector.
ウィルスベクターを導入される側の心筋細胞への分化能を有するマウス骨髄由来 多分化能幹細胞 (BMSC)は、 ウィルスをインフエクシヨンさせる前日に 2 X 104細胞/ ゥエルとなるように 6ゥエル ·ディヅシュに蒔いておいた。 Murine bone marrow-derived multipotent stem cells (BMSC), 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.
. 上記で取得したウィルスベクタ一を含む溶液に、 終濃度.8 μ g/mlとなるように、 Hexadimethrine bromide(polybrene)(Sigma社製） を添加し、 心筋細胞への分化能を有す るマウス骨髄由来多分化能幹細胞 (BMSC)の培地と置換し、 33°C、 5%C02濃度の孵卵 機を用いて培養を行った。 5時間後、新しい IMDM培地に交換し、更に 33°C、 5%C02 濃度の脬卵機を用いて培養を行い、 さらに 2日間培養を行った。 Hexadimethrine bromide (polybrene) (manufactured by Sigma) is added to the solution containing the virus vector obtained above to a final concentration of 0.8 μg / 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脬卵machine, it was further cultured for 2 days.
その後、 pCLNC- Nkx2.5と pCMV- Eco導入で産生されたウィルスをインフエクシ ョンした細胞には、 G418を終濃度 300 g/mlになるように添加し、 さらに 7日間 培養した。  Thereafter, 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.
一方、 pCLPC- GATA4と pCMV - Eco導入で産生されたウィルスをインフエクショ ンした細胞には、 ピューロマイシンを終濃度300ng/mlになるように添カ卩し、 さらに 7日間培養した。 On the other hand, PCLPC- GATA4 and pCMV - The cells Infuekusho down the produced viruses with Eco introduction and添Ka卩puromycin to a final concentration of 3 00n g / ml, and cultured for an additional 7 days.
どちらの細胞も、 この間に一部の細胞は死滅して浮遊した。 生き残った細胞をト リブシンで浮遊させ、 新しい培養皿に播種した。  Both cells died and floated during this time. The surviving cells were suspended in triscine and seeded on new culture dishes.
このようにして、 取得した Nkx2.5/Csxあるいは GATA4の安定形質転換細胞につ いて、 上記実施例 3の方法により分化誘導を行い、 心筋細胞への分化の効率を検定 した。 Nkx2.5/Csxを強制発現した心筋細胞への分化能を有する骨髄細胞 (BMSC - Nkx2.5) と GATA4を強制発現した心筋細胞への分化能を有する骨髄細胞 (BMSC-GATA4)を 2 X 104細胞/ mlとなるように 60mm培養ディッシュに蒔き、 33°C、 5%C02濃度の孵卵 機を用いて培養を行った。 翌日、 該培養液に 5-aza- Cを終濃度 3〃Mとなるよう添 加した。 さらに 24時間、 33°C；、 5%C02濃度の孵卵機を用いて培養を行った後に培地 を新しいものに交換することで 5- aza - Cを除去し、 さらに4週間培養を続けた。 位 相差顕微鏡での筋管様細胞の数は Nkx2.5/Csxあるいは GATA4の強制発現によって は大きく変化しなかった。 次に得られた、 筋管様細胞から RNAを回収して、 該筋管 様細胞で発現している遺伝子を配列番号 7 1〜7 8で示した合成オリゴヌクレオチ ドを用いて定量的 PCR解析を行った。 その結果、 Nkx2.5/Csxあるいは GATA4の強 制発現により心筋に特異的な遺伝子である cTnl, ANPの発現を亢進することが観察 された。 In this way, it obtained Nkx 2. 5 / Csx or in have stably transformed cells Nitsu of GATA 4, performs a differentiation induced by the method of Example 3 and assayed for efficiency of differentiation into cardiomyocytes. 2x bone marrow cells capable of differentiating into cardiomyocytes expressing Nkx2.5 / Csx (BMSC-Nkx2.5) and bone marrow cells capable of differentiating into cardiomyocytes expressing GATA4 forcedly (BMSC-GATA4) 10 4 plated 60mm culture dish so that the cells / ml, were incubated with 33 ° C, 5% C0 2 concentration in the incubator unit. The next day, 5-aza-C was added to the culture solution to a final concentration of 3 M. Further 2 4 hours, 3 3 ° C ;, 5% C0 2 by concentration using incubation machine is replaced with a new medium after the culture 5-aza - C is removed and the further 4 weeks in culture Continued. The number of myotube-like cells in the phase difference microscope was not changed significantly depending Nkx 2. 5 / Forced expression of Csx or GATA4. Next, RNA was recovered from the obtained myotube-like cells, and the gene expressed in the myotube-like cells was analyzed by quantitative PCR using the synthetic oligonucleotides shown in SEQ ID NOs: 71 to 78. Was done. As a result, it was observed that the forced expression of Nkx2.5 / Csx or GATA4 enhanced the expression of cTnl and ANP, which are genes specific to myocardium.
次にまず、 Nkx2.5/CSxと GATA4の両遺伝子を同時に心筋細胞への分ィ匕能を有す る骨髄細胞に発現させる目的で、レトロウィルスベクタ一プラスミド pCLPC - GATA4 を、 上述した方法に従い、 組み換えウィルスを生産し、 Nkx2.5/Csxを強制発現させ た心筋細胞への分ィヒ能を有する骨髄細胞 (BMSC-NkX2.5)に感染させた後、 300ng/ml の終濃度になるようにピューロマイシンを添加し、 薬剤耐性クローン (BMSC - Nkx2.5— GATA4)を取得した。 Next First, the purpose of expressing the myeloid cells that have a simultaneous partial I匕能into cardiomyocytes both genes of Nkx2.5 / C S x and GATA4, retroviral vectors one plasmid PCLPC - the GATA4, described above according to the method, to produce recombinant viruses, after infecting bone marrow cells with a minute it inhibits the ability of Nkx2.5 / Csx into cardiac cells forced to express (BMSC-Nk X 2.5), 300n g / ml final Puromycin was added to a concentration to obtain drug-resistant clones (BMSC-Nkx2.5-GATA4).
Nkx2.5/Csxと GATA4の両遺伝子を強制発現した心筋細胞への分化能を有する骨 髄細胞 (BMSC - Nkx2.5 - GATA4)を 2 X 104細胞/ mlとなるように 60mm培養デイツシュ に蒔き、 33°C、 5%C02濃度の孵卵機を用いて培養を行った。 Nkx2.5 / Csx and bone marrow cells capable of differentiating into cardiomyocytes forced expression of both genes GATA4 (BMSC - Nkx 2 5 - . GATA 4) so as to have a 2 X 10 4 cells / ml 6 0 mm plated in culture Deitsushu were cultured using 33 ° C, 5% C0 2 concentration in the incubator unit.
翌日、 該培養液に 5- aza - Cを終濃度 3 Mとなるよう添加した。さらに 24時間、 33°C 5%C02濃度の孵卵機を用いて培養を行った後に培地を新しいものに交換する ことで 5 - aza - Cを除去し、 さらに 4週間培養を続けた。 位相差顕微鏡での筋管様細 胞の数は Nkx2.5/Csxと GATA4の両遺伝子の強制発現によっては大きく変化しなか つたが、 拍動する心筋細胞の数は両遺伝子を強制発現していない心筋細胞への分ィ匕 能を有する骨髄細胞と比較して 50倍以上増加した。次に得られた、 筋管様細胞から RNAを回収して、 該筋管様細胞で発現している遺伝子を配列番号 7 1〜7 8で示し た合成ォリゴヌクレオチドを用いて定量的 PCR解析を行った。その結果、 Nkx2.5/Csx と GATA4の強制発現により心筋に特異的な遺伝子である cTnl, ANPの発現を亢進す ることも観察された。 実施例 8 . 転写因子の強制発現とサイ トカインの組み合わせによる心筋細胞分化 の促進 The next day, 5-aza-C was added to the culture solution to a final concentration of 3M. Additional 24 hours, 33 ° C 5% C0 2 concentration by replacing with a new culture medium after the culture by using the incubator unit 5 - aza - C was removed and further continued for 4 weeks in culture. The number of myotube-like cells by phase contrast microscopy did not change significantly with the forced expression of both Nkx2.5 / Csx and GATA4 genes, but the number of beating cardiomyocytes was forced to express both genes. It increased 5 0 times as compared to bone marrow cells with a minute I spoon ability to no cardiomyocytes. Next, 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
上述した心筋分化'促進能のある転写因子 (Nkx2.5/CsX， GATA4)とサイト力イン (FGF-8, ET-1, Midkine, BMP4)を組み合わせることによる、 心筋細胞分ィ匕に及ぼす影. 響を解析した。 By combining a transcription factor with the above-mentioned myocardial differentiation 'promoting capacity (Nkx2.5 / Cs X, GATA4) and site force in (FGF-8, ET-1 , Midkine, BMP4), on cardiomyocyte fraction I spoon The effects were analyzed.
Nkx2.5/Csxと GATA4の両遺伝子を強制発現した心筋細胞への分化能を有する骨 髄細胞 (BMSC- Nkx2.5- GATA4)を 2 X 104細胞/ mlとなるように 60mm培養デイツシュ に蒔き、 33°C；、 5%C02濃度の孵卵機を用いて培養を行った。 Nkx2.5 / Csx and bone marrow cells capable of differentiating into cardiomyocytes forced expression of both genes GATA4 (BMSC- Nkx 2 5 -. 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 .
Nkx2.5/Csxと GATA4の両遺伝子を強制発現した心筋細胞への分化能を有する骨 髄細胞 (BMSC- Nkx2.5- GATA4)を'2 X 104細胞/ mlとなるように 60mm培養ディヅシュ に蒔き、 33°C、 5%C02濃度の孵卵機を用いて培養を行った。翌日、該培養液に 5- aza - C を終濃度 3〃Mとなるよう添加した上で、 更に、 FGF - 8を終濃度 lOng/mlになるよ うに添加 （培養ディッシュ I )、 ET- 1を終濃度 lOng/mlになるように添加 （培養 ディヅシュ J )、 Midkineを終濃度 10ng/mlになるように添カロ （培養ディヅシュ K )、 BMP4を終濃度 10ng/mlになるように添加 （培養ディッシュ L ) , 添加なし （培養 ディッシュ M) の 5種類の異なる処理を行い培養を継続した。 Nkx2.5 / Csx and bone marrow cells capable of differentiating into cardiomyocytes forced expression of both genes GATA4 (BMSC- Nkx 2 5 -. GATA4) a 'such that 2 X 10 4 cells / ml 6 0 mm plated in culture Didzushu were cultured using 33 ° C, 5% C0 2 concentration in the incubator unit. The next day, 5-aza-C was added to the culture solution to a final concentration of 3〃M, and FGF-8 was further added to a final concentration of lOng / ml (culture dish I), ET-1 added to a final concentration of LONG / ml (culture Didzushu J), added Caro to a final concentration of 10 ng / ml of Midkine (culture Didzushu K), added BMP4 to a final concentration of 10n g / ml ( Culture was continued by performing five different treatments: culture dish L) and no addition (culture dish M).
翌日 5_aZa- Cを培地から除去するために、 培地を新しいものに交換し、 再び培養 ディッシュ Iには FGF- 8を終濃度 lOng/mlになるように添加し、 培養デイツシュ J には ET-1を終濃度 10ng/mlになるように添加、 培養ディヅシュ Kには Midkineを終 濃度 10ng/mlになるように添加、培養ディヅシュ Lには BMP4を終濃度 lOng/mlにな るように添加して培養を継続した。それから更に 2日後、 4日後にも同様の培地交換 と FGF— 8, ET - 1， Midkineあるいは BMP4の添加を行った。 The next day, to remove 5_aZa -C from the medium, replace the medium with a fresh medium, add FGF- 8 again to culture dish I to a final concentration of lOng / ml, and add ET- 1 to a final concentration of 10 ng / ml, add Midkine to culture dish K to a final concentration of 10 ng / ml, and add BMP4 to culture dish L to a final concentration of lOng / ml. The culture was continued. Two and four days later, the same medium exchange and addition of FGF-8, ET-1, Midkine or BMP4 were performed.
5-aza-Cを加えてから 4週間後、細胞の形態を位相差顕微鏡下で観察した。その結 果、 5 - aza - Cのみを添加した培養ディッシュでは約 3割の細胞が筋管様細胞となるの に対し、 FGF - 8， ET-1, Midkineあるいは BMP4を添加した培養ディヅシュでは約 5 割の細胞が筋管様細胞となった。 一方、 拍動する心筋の数は FGF -8, ET- l， Midkine あるいは BMP4の添加により増加しなかった。 Four weeks after the addition of 5-aza-C, cell morphology was observed under a phase contrast microscope. As a result, about 30% of cells in a culture dish containing only 5 -aza-C become myotube-like cells. In contrast, about 50% of the cells in the culture dish supplemented with FGF-8, ET-1, Midkine or BMP4 became myotube-like cells. On the other hand, the number of beating myocardium did not increase by the addition of FGF- 8 , ET-1, Midkine or BMP4.
次に得られた、 筋管様細胞から RNAを回収して、 該筋管様細胞で発現している遺 伝子を配列番号 7 1〜 7 8で示した合成ォリゴヌクレオチドを用いて定量的 PCR解 析を行った。その結果、 FGF - 8， ET - 1, Midkineあるいは' BMP4は Nkx2.5/Cs文と GATA4 の強制発現により促進される cTnl, ANPの発現をさらに亢進することはなかった。 実施例 9 . 心筋細胞への分化能を有するマウス骨髄細胞の'心臓への移植  Next, 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
心筋細胞への分化能を有する骨髄細胞が生体内で心筋に分化し心臓に定着するか どうかを明らかにするために、実施例 5で作製した、 GFPで標識した心筋細胞への分 化能を有する骨髄細胞 (BMSC- GFP)を、 マウスへ移植するためのドナ 細胞とした。 具体的には、 以下の方法を実施した。 GFPで標識した BMSC細胞を予め 5-aza- Cで 2 4時間処理した後、 l x l08cells/mlとなるよう PBSに懸濁し、 移植直前まで氷上で 保存した。なお、 BMSC細胞は 0.05%エリス口シン染色により 95%程度生存しているこ とを確認、している。 To determine whether bone marrow cells capable of differentiating into cardiomyocytes differentiate into myocardium in vivo and colonize the heart, the ability to differentiate into GFP-labeled cardiomyocytes prepared in Example 5 was evaluated. The bone marrow cells (BMSC-GFP) 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.
一方、 レシピエントの C3H/Heマウス （日本チャールズリバ一社製） は、 ェ一テル を用いて麻酔の導入を行い、 テルモ製のテルモシリンジ （1ml) を用いてチォペン夕 —ル 30mgの腹腔内投与することで麻酔の維持を行った。マウスの四肢をテープでコ ルク板に固定し、 さらに首が反り返るように上顎をゴムでコルク板に固定した。 こ の時点で左右の上肢及び右下肢に心電図電極を刺入し心電図のモニタリングを行つ た。 続いて、 メ一ョ剪刀 (NONAKA RIKAKI CO.，LTD NK- 174-14)で頸部を気管にそつ て 1 cmほど切開し、 白十字社のベビー綿棒で甲状腺を左右に剥離をし、気管周囲の 筋肉をマイクロ剪刀 (NONAKA RIKAKI CO ·， LTD NY - 334-08)で切開し気管を露出した。 ついでマイクロフエザ一 （メス） で気管を lmmほど切開しここから J型に変型させ たテルモ製サ一フロ一フラッシュ （22G)の針を揷入し口腔から外に出し、 この針を ガイドにサ一フロ一フラッシュ （20G)の外筒を気管内に挿入した。 この外筒にレス ピレ一夕（シナノ製作所製の MODEL SN- 480- 7)をつなぎ 100パーセント酸素を 1ml/ 分で流し、 一回換気量は lml、 呼吸回数は 120/分で人工呼吸を開始した。 このとき にガイド針を挿入した穴からエアーがもれるので気管周囲の皮膚をモスキート鉗子 (NONAKA RIKA I C〇.，LTD) を用いて気管をおおうようにして閉鎖した。 つぎに、 胸骨柄より頸部に向かい 2cmほどメ一ョ剪刀で切開、 ついで胸骨を 2cmほど胸骨柄 から頸部に向かい切開をした。 出血をバイポーラの電気メスで止血し、 テルモ製の テルモシリンジ （1ml) にジーエルサイエンス社製の 30Gの針 （メタルハプ交換針 N730) をつけて心尖部に,ドナ一細胞を PBSに浮遊した液体を 0.1ml注入した。 つい で ETHICON社製の 4- 0 ETHIBOND X761を用いて胸骨の閉鎖、 皮膚の閉鎖を行い、 同じ針糸で頸部の皮膚の閉鎖をした。 自発呼吸の出現を確認しレスピレ一夕をはず しィンファントウォーマ一を 37°Cに加熱しこの中で覚醒を待った。 なお本実験の操 作は DESIGN FOR VISON 4.5 X SURGICAL TELESCOPESを用いて行った On the other hand, 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. At this time, ECG electrodes were inserted into the left and right upper limb and right lower limb to monitor the ECG. Then, a 1 cm incision was made along the trachea along the trachea with a macho scissor (NONAKA RIKAKI CO., LTD NK-174-14), and the thyroid gland was stripped left and right with a White Cross baby swab. The surrounding muscles were incised with a micro-scissor (NONAKA RIKAKI CO., LTD NY-334-08) to expose the trachea. Then, the trachea was incised with a micro feather (female) to about lmm, and the needle of Terumo saflo flash (22G) transformed into J-type was inserted from here and taken out of the oral cavity, and this needle was used as a guide. An outer cylinder of flow flash (20G) was inserted into the trachea. To this outer cylinder, connect Respire Isuzu (MODEL SN-480-7 manufactured by Shinano Seisakusho) and supply 100% oxygen 1ml / The artificial respiration was started at a tidal volume of 1 ml and a respiratory rate of 120 / min. At this time, since air leaked from the hole into which the guide needle was inserted, the skin around the trachea was closed using mosquito forceps (NONAKA RIKA IC〇., LTD) to cover the trachea. Next, an incision was made about 2 cm from the sternum handle toward the neck with a scalpel, and an incision was made about 2 cm from the sternum handle toward the neck. The bleeding hemostasis bipolar electrocautery was suspended in apical with a GL Sciences Inc. of 3 0G needle (Metaruhapu exchange needle N730) to Terumo Terumo syringe (1 ml), Donna single cell in PBS liquid 0.1 ml was injected. With ETHICON, Inc. of 4 - using a 0 ETHIBOND X761 closure of the sternum, it performs the closure of the skin, was the closure of the skin of the neck at the same needle thread. After confirming the appearance of spontaneous breathing, the respire was removed and the infant warmer was heated to 37 ° C and awakened in this. The operation of this experiment was performed using DESIGN FOR VISON 4.5 X SURGICAL TELESCOPES.
移植しで 7 7日後のマウスから組織を摘出し、 10%ホルマリンで固定し、 パラフィ ンで包埋した。 包埋した組織サンプルをミクロトームで 6〃mの厚さに薄切し、 予 め poly- L- lysineでコーティングしておいたスライドグラス上に貼り付けた。 1 0 0 %キシレンに浸して脱パラフィンをした後、 ェ夕ノ一ルで洗浄し、 更に 0.3%¾02 溶液に 30分間浸して抗体反応の前処理をおこなった。 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 μm 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% ¾0 2 solution for 30 minutes.
その後、 PBSで洗浄したサンプルに対し、 5 %正常ブタ血清溶液を 30分間反応さ せ、 プロヅキングをおこなった。 ブロッキング後、 PBSで洗浄し、 PBSで 100倍に希 釈したマウス抗 GFPモノクローナル抗体 （CL0NTECH社製） で 4 °Cに一晩置き、 抗体 反応をおこなった。 P B Sで洗浄後、 パ一ォキシダ一ゼ標識デキストラン結合ャギ 抗マウスィムノグロブリン抗体（DAC0社製） を室温で 30分間反応させた。更に PBS で洗浄後、 発色液〔10〃g/ml 3, 35 -Diaminobenzidine(DAB) Tetrahydrochloride)^ 0.01°/o ¾0Z, 0.05M Tris-HCl(pH6.7) を添加して 1 0分間程度反応をおこない、 P B Sで洗浄して反応を停止させた。 更に、 そのスライドグラスに対して、 メチルグリ —ン染色もおこなった。 Thereafter, the sample washed with PBS was reacted with a 5% normal pig serum solution for 30 minutes to perform blocking. After blocking, the plate was washed with PBS, and then put at 4 ° C overnight with a mouse anti-GFP monoclonal antibody (manufactured by CL0NTECH) diluted 100-fold with PBS to perform an antibody reaction. After washing with PBS, a oxidase-conjugated goat anti-mouse immunoglobulin antibody (manufactured by DAC0) labeled with oxidase was reacted for 30 minutes at room temperature. After further washing with PBS, color development solution [10〃G / ml 3, 3 5 -Diaminobenzidine ( DAB) Tetrahydrochloride) ^ 0.01 ° / o ¾0 Z, 0.05M Tris-HCl (pH6.7) added to 1 0 minutes After the reaction, the reaction was stopped by washing with PBS. In addition, the slide glass was also stained with methyl green.
一方、 組織切片の形態を明らかにするため、 連続切片の一部をへマトキシリン ' ェォジンで染色した。  On the other hand, to clarify the morphology of the tissue section, a part of the serial section was stained with hematoxylin and eosin.
その結果、心筋細胞および血管内皮細胞において、 GFP抗体陽性細胞が見られた。 従って、 マウス骨髄細胞は、 移植により心筋細胞および血管内皮細胞に分化したと いえる。 実施例 1 0 . 培養心筋細胞由来の因子による骨髄細胞の心筋分化促進 As a result, GFP antibody-positive cells were found in cardiomyocytes and vascular endothelial cells. Therefore, it can be said that mouse bone marrow cells differentiated into cardiomyocytes and vascular endothelial cells by transplantation. Example 10: Promotion of cardiomyocyte differentiation of bone marrow cells by factors derived from cultured cardiomyocytes
実施例 9で示したように、 心筋細胞への分ィ匕能を有する骨髄細胞 (BMSC)を心臓に 移植することで心筋への分化が観察された。 この結果は、 心筋細胞自身が骨髄細胞 を心筋細胞へ分ィ匕誘導する因子を発現している可能性を示唆している。 この仮説を 検証する目的で妊娠 16日目の C3H/Heマウスから胎児心臓を摘出し、公知の方法（心 臓血管研究方法の開発 b 江橋節朗編集、 学会出版センダ一発行、 1 9 8 3 ) に従つ て、 心筋細胞の初代培養細胞を樹立した（以後、 培養心筋細胞と称する）。 As shown in Example 9, differentiation into myocardium was observed by transplanting bone marrow cells (BMSC) having the ability to isolate myocardial cells into the heart. This result suggests that cardiomyocytes themselves may express a factor that induces bone marrow cells into cardiomyocytes. It was excised fetal heart from gestation day 16 of C3H / He mice in order to test this hypothesis, a known method (development of heart臓血tube research methods b Ebashi Setsuro editing, Society of Publication sender one issue, 1 9 8 3) Accordingly, primary cultured cells of cardiomyocytes were established (hereinafter referred to as cultured cardiomyocytes).
まず、 培養心筋細胞から分泌される因子に心臓分ィ匕を促進させる活性があるかど うかを検証するために、 培養心筋細胞を 6cmの培養ディヅシュに 5 X 106 cellsを 72 時間培養した後、 培養上清を 0.45 ^ mのフィルタ一 . (Millipore.社製） でろ過し、 ろ 過した培養上清と等量の培地を加えて、 培養心筋細胞から分泌される因子を含む培 養液 （以後コンディションド · ミィディアムと称する） を調整した。 First, in order to verify whether the factors secreted from cultured cardiomyocytes have the activity to promote cardiac arrest, 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").
あらかじめ心筋紬胞への分化能を有する骨髄細胞 (BMSC)あるいは Nkx2.5と GATA4 の両遺伝子を強制発現した心筋細胞への分化能を有する骨髄細胞 (BMSC-Nkx2.5 - GATA4)を 6cmの培養ディヅシュに 1 X 105細胞となるよう培養し、その後コンデイシ ヨンド ·ミィディアムと培地を置換した。 このとき同時に5-&∑&-( を終濃度3 ¾[ になるように添カロした。 翌日、 培地を新しいコンディションド · ミィディアムに交 換し、 さらに 4週間培養を続けた。 この間、 3日に一度培地を新しいコンディション ド ·ミィディアムと交換した。 その結果、 コンディションド · ミィディアムの添カロ により、 心筋細胞への分化能を有する骨髄幹細胞 (BMSC)からの筋管様細胞の増加は 観察されなかったが、 ANP, cTnIの二つの心筋特異的な遺伝子の発現が亢進すること が観察された。 一方、 Nkx2.5と GATA4の両遺伝子を強制発現した心筋細胞への分ィ匕 能を有する骨髄細胞 (BMSC - Nkx2.5- GATA4)はコンディシヨンド ' ミィディアムの添 加により、 筋管細胞の数は増加せず、 ANP，cTnIの二つの心筋特異的な遺伝子の発現 は Nkx2.5と GATA4以上による発現亢進と同じレベルであり、促進効果は観察されな かった。 Bone marrow cells (BMSC) that have the ability to differentiate into cardiac muscle cells (BMSC) or bone marrow cells (BMSC-Nkx2.5-GATA4) that have the ability to differentiate into 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 3¾ [. The next day, the medium was replaced with a new conditioned medium, and the culture was continued for another 4 weeks. The medium was replaced with a new conditioned medium once every day, and as a result, no increase in myotube-like cells from bone marrow stem cells (BMSC) capable of differentiating into cardiomyocytes was observed due to the addition of conditioned medium. However, it was observed that the expression of two myocardium-specific genes, ANP and cTnI, was enhanced, whereas bone marrow that had the ability to divert to cardiomyocytes that forcibly expressed both Nkx2.5 and GATA4 genes was observed. cells (BMSC - Nkx 2 5 -. 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.
次に、心筋細胞が発現している細胞外基質 (ECM)に心筋分化促進活性があるかどう かを検証するために、心筋細胞を培養した培養ディヅシュから 0.45%のトリプシン · EDTAを 30分間程度処理することで心筋細胞を除去し、培養心筋細胞の細胞外基質を コートした培養ディッシュ （以後 ECMコート 'ディッシュと称する） を作製した。 次に、'この 6cm.の ECMコ一ト ·ディヅシュ上に心筋細胞への分化能を有する骨髄細 胞 （BMSC)あるいは Nkx2.5と GATA4の両遺伝子を強制発現した心筋細胞への分化能 を有する骨髄細胞 (BMSC- Nkx2.5「GATA4)を 1 X 105細胞となるよう培養し、'その後 5-aza-Cを終濃度 3〃 Mになるように添加した。翌日、 5-aza-Cを除去するために新 しい培地に交換し、 さらに 4週間培養を続けた。 この間、 3日に 1回程度、培地を新 しいものに交換した。心筋細胞への分化能を有する骨髄細胞（BMSC)は ECMコート · ディヅシュにより筋管様細胞の数は増加しなかったが、 A P，cTnIの二つの心筋特異 的な遺伝子の発現が亢進するこどが観察された。 」方、 Nkx2.5.と ' GATA4の両遺伝子 を強制発現した心筋細胞への分化能を有する骨髄細胞 (BMSC-Nkx2.5- GATA4)は ECM コート 'ディッシュにより、 筋管細胞の数は増加せず、 A P, cTnIの二つの心筋特異 的な遺伝子の発現は Nkx2.5と GATA4以上による発現亢進と同じレベルであり、促進 効果は観察されなかった。 Next, in order to verify whether the extracellular matrix (ECM) expressing cardiomyocytes has cardiomyocyte differentiation-promoting activity, 0.45% trypsin / EDTA was cultured for 30 minutes from the culture dish in which cardiomyocytes were cultured. The cardiomyocytes were removed by the treatment, and a culture dish coated with the extracellular matrix of the cultured cardiomyocytes (hereinafter referred to as ECM coated 'dish') was prepared. Next, the ability to differentiate into bone marrow cells (BMSC) that have the ability to differentiate into cardiomyocytes or cardiomyocytes that express both the Nkx2.5 and GATA4 genes on the 6 cm. 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 μM. 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. and 'Bone marrow cells (BMSC-Nkx2.5-GATA4) that have the ability to differentiate into cardiomyocytes that express both GATA4 genes are In addition, the number of myotube cells did not increase, and the expression of two myocardial-specific genes, AP and cTnI, was at the same level as that of Nkx2.5 and GATA4 or higher, and no promoting effect was observed.
次に、 2 X 104個の培養心筋細胞と、 8 X 104個の心筋細胞への分ィ匕能を有する骨髄 細胞 (BMSC)または 8 X 104個の Nkx2.5と GATA4の両遺伝子を強制発現した心筋細胞へ の分ィ匕能を有する骨髄細胞 (BMSC - Nkx2.5- GATA4)とを 6cmの培養ディッシュで共培 養を行った。 培養心筋細胞と骨髄細胞を識別するために、 2種類の骨髄細胞 (BMSC と BMSC- Nkx2.5- GATA4) は実施例 5で示した方法により GFPで標識したものを利 用した。 共培養を開始した翌日に 5-aza-Cを終濃度 3〃Μになるように添加し、 そ の翌日に 5- aza-Cを除去するために新しい培地に交換し、 さらに 4週間培養を続け た。 この間、 3曰に 1回程度、 培地を新しいものに交換した。 その結果、 BMSCまた は BMSC- Nkx2.5 - GATA4を単独で培養したときと比較して、約 10倍拍動する心筋の 数が増加した。この結果、 Nkx2.5と GATA4遺伝子の強制発現と心筋細胞との共培養 を組み合わせることで、 心筋分化効率は 5 0 0倍以上上昇することが明らかになつ た o 実施例 1 1 . KUM2細胞と BMSC細胞の表面抗原の解析 Next, 2 x 10 4 cultured cardiomyocytes and 8 x 10 4 bone marrow cells (BMSC) capable of dividing into cardiomyocytes or 8 x 10 4 Nkx2.5 and GATA4 genes Was co-cultured with a bone marrow cell (BMSC-Nkx2.5-GATA4) having the ability to divide into myocardial cells in which was forcibly expressed in a 6 cm culture dish. In order to distinguish cultured cardiomyocytes from bone marrow cells, two types of bone marrow cells (BMSC and BMSC-Nkx2.5-GATA4) labeled with GFP according to the method described in Example 5 were used. The day after the start of the co-culture, 5-aza-C was added to a final concentration of 3〃Μ, and the next day, the medium was replaced with a fresh medium to remove 5-aza-C, and the culture was continued for another 4 weeks. Continued. During this time, the medium was replaced with a new one about three times. As a result, BMSC or BMSC- Nkx 2 .5 - GATA4 as compared to when cultured alone, the number of heart muscle moving about 10 times beats increases. As a result, it was clarified that the combination of forced expression of Nkx2.5 and GATA4 gene and co-culture of cardiomyocytes increased myocardial differentiation efficiency by more than 500 times. O Example 1 1. Analysis of surface antigens of KUM2 cells and BMSC cells
KUM2細胞と BMSC細胞の異同を明らかにすること、骨髄中から効率的に心筋形成能 を有する単離'精製する方法を開発する目的で、 KUM2細胞と BMSC細胞の表面抗原の 解析を行った d Analysis of the surface antigens of KUM2 cells and BMSC cells was performed to clarify the difference between KUM2 cells and BMSC cells and to develop a method for efficiently isolating and purifying myocardium from bone marrow d
解析に用いたのは、 血管内皮細胞の表面抗原として知られている CD105、 Flk-lv CD31、 CD144、 造血系細胞の表面抗原として知られている CD34、 GDI 17, CD14、 CM5、. CD90、 Sea- 1、 Ly6c、 Ly6gヽ 間葉系細胞の表面抗原として知られている CD140、 'イン テグリン CD49b、 CD49d、 CD29マトリックス受容体 CD54、 C囊、 CD106、 CD44の 20 種類である。  CD105, Flk-lv CD31, CD144, known as surface antigens on vascular endothelial cells, CD34, GDI 17, CD14, CM5, .CD90, known as surface antigens on hematopoietic cells, were used for analysis. Sea-1, Ly6c, Ly6g ヽ There are 20 kinds of CD140, 'integrin CD49b, CD49d, CD29 matrix receptor CD54, C 囊, CD106, CD44 known as surface antigens of mesenchymal cells.
まず KUM2細胞 l x lO4個を 9 '6ゥヱル U字プレートに分注した。公知の方法 [酵素 抗体法：'学際企画刊（1985) ]でピオチン標識した抗: γウス CD105抗体. (Pharmingen 社製） を FACS用緩衝液 （1¾BSA-PBS、 0.02%EDTA、 0.05%NaN3、 ΡΉ7.4) に加えゥエル に添加し、 氷中で 30分間反応させた。 陰性対象としては、 ラヅト IgG2a、 精製抗 体 （Pharmingen社製） を用いた。 緩衝液で 2回洗浄後、 ストレプトアビジン一 P EFirst, 4 KUM2 cells lx lO were dispensed into a 9'6 μl U-shaped plate. A known method [enzyme antibody method: 'Interdisciplinary publication (1985)]' was labeled with a biotin-labeled anti: γus CD105 antibody (Pharmingen) using a buffer for FACS (1¾BSA-PBS, 0.02% EDTA, 0.05% NaN 3). was added to Ueru addition to Ρ Ή7.4), it was reacted in ice for 30 minutes. As a negative control, rat IgG2a and a purified antibody (Pharmingen) were used. After washing twice with buffer, streptavidin-PE
(日本べクトン .ディッキンソン社製） を 20 1加えた。遮光し氷中で 3 0分間反 応後、 緩衝液で 3回洗浄し、 最終的に 50Q β 1に懸濁して、 フロ一サイトメ一夕一 で蛍光強度を測定し、 抗体の添加により蛍光強度が増加するか否かで抗体の発現の 有無を調べた。 その結果、 KUM2細胞は CD105陰性であった。 (Nippon Becton Dickinson) was added. After reacting on ice for 30 minutes in the dark, wash with buffer 3 times, and finally suspend in 50Q β1, measure the fluorescence intensity throughout the flow cytometer, and add the antibody to the fluorescence intensity. The presence or absence of the expression of the antibody was examined based on whether the expression increased. As a result, KUM2 cells were CD105 negative.
Flkl抗原の発現についても、同様にピオチン化した抗マウス Flkl抗体 (Pharmingen 社製； PM-28181D) を用いて抗体反応をおこない、 フローサイトメ一夕一で測定した。 その結果、 KUM2細胞は Flkl陰性細胞であつた。  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. As a result, the KUM2 cells were Flkl negative cells.
CD31 抗原の発現の有無については、 FITC 標識された抗マウス CD31 抗体 For expression of CD31 antigen, use FITC-labeled anti-mouse CD31 antibody.
(Pharmingen社製； PM-01954D)を用いて抗体反応をおこない、 フローサイトメ一夕 —で測定した。 その結果、 KUM2細胞は CD31陰性であった。 (Pharmingen; PM-01954D) to carry out an antibody reaction, and measurement was carried out using a flow cytometer. As a result, KUM2 cells were CD31 negative.
CD144抗原の発現については、 ピオチン化した抗マウス CD144抗体 (Pharmingen 社製； PM- 28091D) を用いて抗体反応を行い、 フロ一サイトメ一夕一で測定した。 そ の結果、 KUM2細胞は CD144陰性細胞であつた。 For 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.
CD34抗原の発現の有無については、 FITC標識された抗マウス CD34 抗体 (Pharmingen社製； PM-09434D)を用いて抗体反応を行い、 フロ一サイトメ一夕一で 測定した。 その結果、 KUM2細胞は、 CD34陰性細胞であった。  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) 抗原の発現については、 FITC標識された抗マウス CD117抗体 For expression of CD117 (c-kit) antigen, FITC-labeled anti-mouse CD117 antibody
(Pharmingen社製； PM-01904D)を用いて抗体反応を行い、 フローサイトメ一夕一で 測定した。 その結果、 KUM2細胞は、 CD117陰性細胞であった。 (Pharmingen; PM-01904D) to carry out an antibody reaction, and measurement was performed throughout the flow cytometer. As a result, the KUM2 cells were CD117 negative cells.
CD14抗原の発現について-は、 FITC標識した抗マウス CD' 1 4抗体 （Pharmingen社 製； PM-09474) を用いて抗体反応を行い、 フロ一サイ卜メーターで測定した。 その 結果、 KUM2細胞は、 CD14陽性細胞であった。  Regarding the expression of the CD14 antigen, 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. As a result, the KUM2 cells were CD14 positive cells.
CD45抗原の発現については、 FITC標識した抗マウス CD45抗体（Pharmingen社製； PM-01114) を用い'て抗体反応を行い、 フローサイトメ一夕一で測定した。 その結果、 KUM2細胞は、 CD45陰性細胞であった。 ノ  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
CD90抗原の'発現については、 FITC標識した抗マウス CD90抗体（Pharmingen社製； PM-22214) を用いて抗体反応を行い、 フロ一サイトメ一夕一で測定した。 その結果、 KUM2細胞は、 CD90陰性細胞であった。  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-l)抗原の発現については、 FITC標識した抗マウス Ly6A/E(Sca- 1)抗体 For expression of Ly6A / E (Sca-1) antigen, FITC-labeled anti-mouse Ly6A / E (Sca-1) antibody
(Pharmingen社製； PM-01164A)を用いて抗体反応を行い、 フローサイトメ一夕一で 測定した。 その結果、 KUM2細胞は、 Ly6A/E(Sca-l )陽性細胞であった。 (Pharmingen; PM-01164A) was used to carry out an antibody reaction, and measurement was performed all over the flow cytometer. As a result, the KUM2 cells were Ly6A / E (Sca-1) positive cells.
Ly6c抗原の発現については、 FITC標識した抗マウス Ly6c抗体（Pharmingen社製； PM-01152) を用いて抗体反応を行い、 フロ一サイトメ一夕一で測定した。 その結果、 KUM2細胞は、 Ly6c陽性細胞であった。  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抗原の発現については、 FITC標識した抗マウス Ly6g抗体（Pharmingen社製； PM - 01214) を用いて抗体反応を行い、 フローサイトメ一夕一で測定した。 その結果、 KUM2細胞は、 Ly6g陰性細胞であった。  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.
CD140抗原の発現については、 ピオチン化した抗マウス CD140抗体 (Pharmingen 社製； PM- 28011A) を用いて抗体反応を行い、 フローサイトメ一夕一で測定した。 そ の結果、 KUM2細胞は、 CD140陽性細胞であった。 CD49b抗原の発現については、 FITC標識した抗マウス CD49b抗体 （Pharmingen社 製； PM- 09794) を用いて抗体反応を行い、 フローサイトメ一夕一で測定した。 その 結果、 KUM2細胞は、 CD49b陽性細胞であった。 For 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. For the expression of the CD49b antigen, 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.
CD49d抗原の発現については、 FITC標識した抗マウス CD49d抗体 （Pharmingen社 製； PM- 01274) を用いて抗体反応を行い、 フローサイトメ一夕一で測定した。 その 結果、 KUM2細胞は、 CD49d陰性細胞であった。  For 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.
CD29抗原の発現については、 FITC標識した抗マウス CD29抗体（Pharmingen社製； PM- 22634) を用いて抗体反応を行い、 フローサイトメ一夕一で測定した。その結果、 KUM2細胞は、 CD29陽性細胞であつ'た。  For 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.
CD54抗原の発現については、 FITC標識した抗マウス CD54抗体（Pharmingen社製； PM-01544) を用いて抗体反応を行い、 フローサイトメ一夕一で測定した。 その結果、 KUM2細胞は、 CD54陽性細胞であった。  For 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.
CD102抗原の発現については、 FITC標識した抗マウス CD102抗体 （Pharmingen社 製； PM- 01804) を用いて抗体反応を行い、 フロ一サイトメ一夕一で測定した。 その 結果、 KUM2細胞は、 CD102陰性細胞であった。  For 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.
CD106抗原の発現については、 FITC標識した抗マウス CD106抗体 （Pharmingen社 製； PM- 01814) を用いて抗体反応を行い、 フローサイトメ一夕一で測定した。 その 結果、 KUM2細胞は、 CD106陽性細胞であった。  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.
CD4 抗原の発現については、 FITC標識した抗マウス CD44抗体 (Pharmingen社製； PM-28154) を用いて抗体反応を行い、 フロ一サイトメ一夕一で測定した。 その結果、 KUM2細胞は、 CD44陽性細胞であった。  For 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.
上述したのと同様の方法により、 BMSC細胞で発現している表面抗原を解析した結 果、 CD34S CD117、 Ly6c、 Ly6A/E( Sca- 1 )、 CD140、 CD29、 CD44の 7種類の抗原につい て陽性であった。 また Flklヽ CD31、 CD105, CD144、 CD14、 CD45、 C廳ヽ Ly6g、 CD49bヽ CD49d、 CD54、 CD10Z, CD106の 1 3種類の抗原に関しては陰性であった。表 1にフロ 一サイトメ一夕一で測定した解析結果をまとめた。 表 1 As a result of analyzing surface antigens expressed on BMSC cells by the same method as described above, seven types of antigens, CD34 S CD117, Ly6c, Ly6A / E (Sca-1), CD140, CD29, and CD44, were analyzed. Was positive. In addition, 13 types of antigens, Flkl ヽ CD31, CD105, CD144, CD14, CD45, Cca ヽ Ly6g, CD49b ヽ CD49d, CD54, CD10Z and CD106, were negative. Table 1 summarizes the analysis results measured throughout the flow cytometer. table 1
KUM2 BMSC  KUM2 BMSC
Hemato  Hemato
CD34  CD34
CD117(c- kit)  CD117 (c- kit)
CD14  CD14
CD45  CD45
CD90(Thyl)  CD90 (Thyl)
Ly-6a/e(Scal)  Ly-6a / e (Scal)
Ly6c  Ly6c
Ly6g  Ly6g
. Enodthelial . Enodthelial
Flk-1  Flk-1
CD31  CD31
CD105  CD105
CD144  CD144
Mesenchyaml Mesenchyaml
CD140 (PDGFR)  CD140 (PDGFR)
Integrin Integrin
CD49b( a2)  CD49b (a2)
Matrix Matrix
CD54(ICAM-1)  CD54 (ICAM-1)
CD102(ICA -2)  CD102 (ICA -2)
CD106(VCAM-1)  CD106 (VCAM-1)
CD44(Hyaluronate)―  CD44 (Hyaluronate)
実施例 1 2 . マウス MLC2vプロモ一夕一を利用した分化前駆細胞の濃縮 Example 1 2. Enrichment of differentiated progenitor cells using mouse MLC2v promoter overnight
心筋細胞への分化を有するマウス骨髄由来細胞から心筋に分ィヒする細胞を効率よ く取得するため、 心筋細胞に特異的に発現するマウス MLC2v (myosin light chain- In order to efficiently obtain cells that differentiate into the myocardium from mouse bone marrow-derived cells that have differentiated into cardiomyocytes, mouse MLC2v (myosin light chain-
2v) 遺伝子のプロモーター発現系を構築した。 具体的には、 マウス MLC2v遺伝子の プロモーター配列下に EGFP遺伝子 （CL0NTECH社製） をつなぎ、 neomycin耐性遺伝 子の発現ュニヅト含んだ pMLC- 2- EGFPプラスミドを構築した。このプラスミドの DNA を、 Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press(1989)等に記載のアル力リ中和法により取得した。 2v) 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.
上記 DNA 2〃 gを、予め 6穴プレートに 1 X 105個となるように培養しておいた KUM2 細胞に対し、 リポフエクトァミン （LIFE TECHNOLOGY社製） を用いて遺伝子導入をお こなった。具体的な方法は製品の添付プロトコルに従った。遺伝子導入して 48時間 後に G418 (Sigma社製） を終濃度 lmg/mlとなるよう添加し、 生存している遺伝子導 入細胞だけを選択した。 2 導入 g of the above DNA was transfected into lipofectamine (manufactured by LIFE TECHNOLOGY) into KUM2 cells that had been cultured in a 6-well plate at 1 x 10 5 cells in advance. Was. The specific method followed the protocol attached to the product. 48 hours after the gene transfer, G418 (manufactured by Sigma) was added to a final concentration of lmg / ml, and only viable transgenic cells were selected.
遺伝子を導入して 14日目の細胞に対し、 5-aza-Cを終濃度 3 / Mとなるように添 加し、 24時間後に培地を交換して、 分化誘導をおこなった。 分化誘導後、 3日目よ り GFP陽性細胞が観察された。分化誘導後 4日目の細胞うち、 I X 104個の細胞を FACS Caliber (Becton Dickinson社製）で GFP陽性細胞のみを分取し更に培養を続けた。 その結果、 9割以上の細胞が筋管様構造を有する細胞に分ィ匕しており、効率的に分化 する細胞を濃縮できたといえる。 この GFP陽性細胞は FACSで分取後、 実施例 11の 方法に従い、 移植を行うと血管内皮への分化は認められず、 骨格筋や心筋などの筋 肉系組織への分化が特異的に観察された。 実施例 1 3 . 心筋細胞への分化能を有するマウス骨髄細胞からの脂肪細胞の誘導 心筋細胞への分化能を有する骨髄細胞（BMSC)は心筋細胞以外に脂肪細胞に分化 誘導することができる。 この^肪細胞への分化を制御する目的で分化誘導条件の検 討を行つ^。 まず、 PPARァ受容体の発現を定量的 PCR法により解析を行った結果、 BMSC細胞は PPARァ 1受容体は発現しているが、 PPAR γ 2受容体は発現していな いことが観察された。 次に、 PPAR ァ受容体のァゴニストである Pioglitazone、 troglitazoneを、様々な濃度で心筋細胞への分ィ匕能を有する骨髄細胞（BMSC)に添カロ したところ、 濃度依存的に脂肪細胞分化が促進され、 0.4〃？^1で約50%、 2〃Mでは ほぼ 100%の細胞が脂肪細胞へと分化した。 実施例 1 4 . 心筋細胞への分化能を有するマウス骨髄細胞を胚盤胞への移植による 神経系細胞、 肝細胞、 心筋細胞への分化誘導 On day 14 after the introduction of the gene, 5-aza-C was added to a final concentration of 3 / M, and the medium was changed 24 hours later to induce differentiation. GFP-positive cells were observed from day 3 after the induction of differentiation. Among differentiation induction after 4 days cells were continued preparative further cultured only GFP positive cells IX 10 4 cells in FACS Caliber (Becton Dickinson Co.). As a result, 90% or more of the cells were divided into cells having a myotube-like structure, and it can be said that cells that efficiently differentiated could be concentrated. After the GFP-positive cells were sorted by FACS and transplanted according to the method of Example 11, differentiation into vascular endothelium was not observed, and differentiation into musculoskeletal tissues such as skeletal muscle and cardiac muscle was specifically observed. Was done. 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 PPARγ2 receptor. Was. Next, PPAR receptor agonists, Pioglitazone and troglitazone, were added to bone marrow cells (BMSC), which have the ability to divide myocardial cells at various concentrations, and 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 4. Induction of differentiation into nervous system cells, hepatocytes, and cardiomyocytes by transplanting mouse bone marrow cells capable of differentiating into cardiomyocytes into blastocysts
はじめに、心筋細胞への分化能を有する骨髄細胞 (BMSC)を GFPで標識した安定形 質転換細胞を得るため、 以下の方法で遺伝子導入をおこなった。  First, in order to obtain stable transformed cells in which bone marrow cells (BMSC) capable of differentiating into cardiomyocytes were labeled with GFP, gene transfer was carried out by the following method.
まず、 レトロウイルスベクタ一プラスミド pCLNCX (Imgenex社)に GFPを組み込 み、 pCLNC- GFPを調製した。 レトロウイルスベクタープラスミド pCLNC-GFPと Ecotropic遺伝子を発現ざせる pCMV- Ecoプラスミドベクタ一 （Imgeriex社)を、 Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laborator Press (1989)等に記載のアル力リ中和法および PEG沈殿法を用いて、純度の高い DNA を取得した。  First, 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.
これらの DNAをトランスフエクシヨンさせる前日に、 コンフルェントになった、 gagおよび pol遺伝子を保有する 293細胞を 1/5希釈で 10cmディッシュに継代し、 —晚 37°C、 5%C02濃度の孵卵機を用いて培養を行った。 ' These DNA the day prior to transflector Ekushi Yong became Konfuruento, passaged gag and pol genes 293 cells harboring the 10cm dishes at 1/5 dilution, -晚37 ° C, 5% C0 2 concentration Culture was performed using an incubator. '
トランスフエクシヨンは以下の通りにおこなった。  The transfection was performed as follows.
pCLNC-GFPレトロウィルスベクタープラスミド DNA15 // gと pCMV- Ecoプラス ミドぺク夕一 DNA 5〃 gを 250mMCaCl2 (pH6.95) 0.5mlに加えて溶解させ、 その溶液 を 15mlのチュ一ブに入れた 2 X BBS [50mM BES(N,N- bis(2- hydroxyethl)- 2 - aminoethanesulfonic acid), 280mM NaCl、 1.5mM Na2HP04(pH6.95)] 0.5mlに滴下して 10 分間室温で静置させた。 その後、 この DNA溶液を、 前日に用意した 293細胞培地中 に滴下させ、 37°C、 5%C02濃度の孵卵機を用いて培養を行った。 翌日、 培地を交換 し、 更に 37°C、 5%C02濃度の孵卵機を用いて培養を行った。 pCLNC-GFP retroviral vector plasmid DNA 15 // g and pCMV-Eco plus Midopeku evening one DNA 5〃 g dissolved in addition to 250mMCaCl 2 (pH6.95) 0.5ml, the solution to 15ml of Ju part brewed 2 X BBS [50mM BES (N , N- bis (2- hydroxyethl) - 2 - aminoethanesulfonic acid), 280mM NaCl, 1.5mM Na 2 HP0 4 (pH6.95)] at room temperature was added dropwise to 0.5 ml 10 min At rest. Thereafter, 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.
培地を交換して 2日後に、 培養上清を 0.45〃mのフィル夕一 （Millipore社製） で ろ過し、 ウィルスベクタ一を含む溶液を回収した。 Replace the medium after 2 days, the culture supernatant was filtered through a 0.4 5 〃M Phil evening one (Millipore Corp.) to recover a solution containing viral vectors scratch.
ウィルスベクターを導入される側の心筋細胞への分化能を有するマウス骨髄細胞 (BMSC)は、 ウィルスをインフエクシヨンさせる前日に 2 X 104細胞 ゥエルとなるよ うに 6ゥエル 'ディヅシュに蒔いておいた。 Mouse bone marrow cells (BMSC), which have the ability to differentiate into cardiomyocytes into which the viral vector is introduced, are seeded on a 6-well dish so as to have 2 x 10 4 cells per day before infection of the virus. Was.
上記で取得したウィルスベクターを含む溶液に、 終濃度 8〃g/mlとなるように、 Hexadimethrine bromide(polybrene)(Sigma社製） を添加し、 心筋細胞への分化能を有す るマウス骨髄細胞 (BMSC)の培地と置換し、 33°C、 5%C02濃度の孵卵機を用いて培養 を行った。 5時間後、 新しい IMDM培地に交換し、 更に 33°C；、 5%C02濃度の孵卵機 を用いて培養を行った。 Hexadimethrine bromide (polybrene) (manufactured by Sigma) is added to the solution containing the virus vector obtained above to a final concentration of 8 μg / 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.
2日間培養を行つた後、 G418を終濃度 300 ju g/mlになるように添加し、 さらに 7 日間培養した。 この間に一部の細胞は死滅して浮遊した。 生き残った細胞をトリプ シンで浮遊させ、 新しい培養皿に播種した。 After having conducted a two-day culture, the addition of G 4 1 8 to a final concentration of 3 00 ju g / ml, and cultured for an additional 7 days. During this time, some cells died and floated. The surviving cells were suspended in trypsin and seeded on new culture dishes.
このようにして取得した、 GFP標識された心筋細胞への分ィ匕能を有する骨髄細胞 を、 6cmの培養ディヅシュで増殖させ、 培地を除去後、 0.5mlの 0.25%のトリプシン EDTAを添加して 1分間処理した後、 l.5mlの新しい培地を添加して、 細胞を懸濁し たところに、 ゥシ胎児血清 (Lexicon Genetics社製) ¾加えて混合し、 該細胞懸濁液を マウス胚盤胞への注入に用いた。 マウス胚盤胞は過排卵処理を施した雌の C57B1/6J マウスを同系の雄マウスと自然交配させ、 4日後に摘出した子宮の内部を M15培地 で灌流することにより取得した。 これらを 37°C、 5% C02条件下で胚盤胞腔が十分に 膨らむまで放置した後、約 4 °Cに冷却した 20mMの HEPESを含む M15培地中に移し、 マイクロインジェクター （成茂科学社製） 及びマイクロマニピュレータ一 （成茂科 学社製） を装備した倒立顕微鏡 （ニコン社製） 下で観察しながら、 注入針を操作し 10〜15個の BMSC細胞を胚盤胞腔内へ顕微注入した。該胚盤胞を 3 7 °C、 5%じ〇2条 件下で胚盤胞腔が膨らむまで放置した後、 Manipulating the Mouse Embryo A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994)に言己載の 方法に従い、偽妊娠の雌 MCH系統のマウスの卵管側子宮部分に移植後、着床させた。 偽妊娠の雌 M C H系統のマウスは、 10週以降の精管結さつ雄 M C H系統マウスと 移植 3日前の Π:00に 1:1で同居、 交配させ、翌朝 9:00に膣栓確認を行い、 2日後に 上記の目的で使用した。 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. After blastocyst alveolar spaces were left to swell sufficiently in these 37 ° C, 5% C0 2 conditions, transferred into M15 medium containing 20mM HEPES, cooled to approximately 4 ° C, microinjector (NaruShigeru Science Company Ltd.) and an inverted microscope (Nikon equipped with micromanipulator one (manufactured by NaruShigeruka Gakusha)) while observing under, by operating the injection needle 10 to 1 5 of BMSC cells in blastocyst alveolar space Was microinjected. 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 Π00, 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.
誕生したマウスを解剖して、 臓器を摘出し、 GFPの発現を観察した。 その結果、 脳内ならびに肝臓で GFPの発現が観察され BMSCが神経系ならびに肝臓に分ィ匕する ことが示された。 また、 別の個体から取得した心臓より、 ゲノム DNAを取得し、 配 列番号79、 δθのプライマ一を用いて PCRを行った結果、 BMSCが心臓にも取り込ま れことが確認された。 これらの結果は、 BMSC が、 神経、 心臓、 肝臓の 3胚葉すべ てに分化できる全能性を有していることを示した。 The born 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. In addition, genomic DNA was obtained from a heart obtained from another individual, and PCR was performed using primers of SEQ ID NOs: 79 and δθ. 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
実施例 1 5 . 心筋細胞への分化能を有するマウス骨髄細胞でのテロメラ一ゼ活性 心筋細胞への分ィ匕能を有するマウス骨髄細胞のテロメラ一ゼ活性は Telomeric Repeat Amplification Protocol(TRAP)法により検討した ( Oncor社製 TRAPeze Telomerase Detection Kit)。テロメラ一ゼ活性の測定は原則的に添付されていたプロ トコ一ルに従ったが、 具体的には以下の通りに行った。 まず、 6cm径の培養皿上で 培養した心筋細胞への分化能を有するマウス骨髄細胞'（およそ 106個).を PBSで洗 浄した後 ·、 200 lの 1 X CHAPS液を加え、 氷上で SO分間静置した。 その後、 溶液 と共に細胞を l.5ml容遠沈管に回収し、 14，000rpmで 20分間遠心分離 (4°C；、 HITACHI Example 15 5. 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
-、.  -,.
社製 himacCFl5)し、 上清を細胞抽出液として回収した。 Protein assay (BioRad社製） を用いて蛋白質含有量を測定したところ、 上記条件で取得した心筋細胞への分化能 を有するマウス骨髄細胞の細胞抽出液はおよそ lmg/mlであった。 ' 次にこの細胞抽出液を用いて、 プロトコ一ルに従ってテロメァ伸長反応及び PCR増幅を行った。 Taqポリメラ一ゼは EX Taq polymerase (宝酒造製） を用いた。 反応終了後の試料は 10 X染色液 （0.25%bromophenol blue, 0.25%Xylene cyanol FF, 30% glycerol) を 1/10量添加し、 12.5%ポリアクリルアミドゲル （TRAPeze Telomerase Detection Kitのプロトコ一ルに記載されている通り調製） に載せ、 250mV定電圧下 で泳動した。 泳動後、 ゲルをサイバーグリーン （FMC社製） で染色し、 蛍光色素分 析装置、 FluoroImagerOvlolecular Dynamics社製)を用レ、て解析した。 その結果、 細胞抽 出液の終濃度が 0.4〜4 ju g/ralの試料でテロメラ一ゼ活性が検出された。 WasacCFl 5 ), and the supernatant was collected as a cell extract. When the protein content was measured using a protein assay (manufactured by BioRad), the cell extract of mouse bone marrow cells capable of differentiating into cardiomyocytes obtained under the above conditions was approximately 1 mg / ml. 'Next, using this cell extract, telomere extension reaction and PCR amplification were performed according to the protocol. As Taq polymerase, EX Taq polymerase (Takara Shuzo) was used. After the reaction is completed, add 1/10 volume of 10X staining solution (0.25% bromophenol blue, 0.25% Xylene cyanol FF, 30% glycerol) to 12.5% polyacrylamide gel (described in the protocol of TRAPeze Telomerase Detection Kit). And electrophoresed under a constant voltage of 250 mV. After the electrophoresis, the gel was stained with Cyber Green (manufactured by FMC) and analyzed using a fluorescent dye analyzer (manufactured by FluoroImager Ovlolecular Dynamics). As a result, telomerase activity was detected in a sample in which the final concentration of the cell extract was 0.4 to 4 jug / ral.
実施例 1 6 . ラヅ卜骨髄からの心筋細胞への分化能を有する骨髄細胞の取得と培養 5週齢の Wistar rat (日本 SLC株式会社)雌 6匹を頸椎脱臼した後、 70%ェ夕ノ一ル を充分かけ消毒した。 次に足部の皮膚を広範囲に渡り切開し、 大腿骨や脛骨を覆う 筋肉を切除しながら、 大腿骨と脛骨を取り出した。 取り出した大腿骨と脛骨は PBS (GibcoBRL社製）の入った 10cm径培養皿 (岩城硝子社製)に移し、筋肉及び関節を完 全に切除した。 続いてこれらの骨の両端をハサミで切り、 20G注射針を付けた 10ml 用注射器 （テルモ社製） を用いて、 培養液 （D-PBS、 GibclBRL社製） の水流で骨髄 中の内容物を押し出した。 取得した細胞塊はさらに注射器を通して一様になるよう にほぐした。このようにして得た細胞浮遊液は 50ml容遠沈管（BECTON DICKINSON 社製） に回収し、 l,500rpmで 10分間遠心分離し （TOMY社製低速遠心機）、 沈殿し た細胞を 6mlの D-PBS中に懸濁した。 改良ノィバウエル型血球計算盤にて細胞数を 計測したところ、 回収した細胞は合計 2.6 X 109個であった。 大腿骨または脛骨 1本 当たりから 1 X 108個の細胞を回収したことになる。 回収した細胞は lml当たり 1.3 X 108個の濃度になるよう希釈し、 50ml容遠沈管に入った 1.073g/mlに調製された PercolKAiiiersham Pharmacia Biotech社製)/ D-PBS液 (25ml) 上に 5ml重層した後、 室 温で 3， lOOrpmで；30分間遠心分離した。 遠心分離後、 Percoll液と細胞浮遊液との界 面より細胞を回収し、 D - PBSで 4倍に希釈した後、 2300rpmで 10分間遠心分離し、 分画した細胞集団を回収した。 回収した細胞は 20%FCS、 100 β g/ml penicillin, 250 ng/ml streptomycin, 85 JLL g/ml amphotericin (GibcoBRL社製) を含む IMDM培地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. Then cut both ends of these bones with scissors and attach 20G syringe needle to 10ml The contents of the bone marrow were extruded with a culture solution (D-PBS, GibclBRL) using a syringe for injection (Termo). The obtained cell mass was further loosened uniformly through a syringe. Thus obtained cell suspension was collected in 5 0 ml volumes centrifuge tube (BECTON DICKINSON Co., Ltd.), l, 5 were centrifuged for 10 minutes at rpm (TOMY Co. slow centrifuge), the precipitated cells 6ml In D-PBS. When the number of cells was counted using an improved Neubauer hemocytometer, the total number of recovered cells was 2.6 × 10 9 . This means that 1 × 10 8 cells were collected from each femur or tibia. 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. After centrifugation, cells were collected from the interface between the Percoll solution and the cell suspension, diluted 4-fold with D-PBS, and centrifuged at 2300 rpm for 10 minutes to collect a fractionated cell population. The recovered cells were IMDM medium containing 20% FCS, 100 β g / ml penicillin, 250 ng / ml streptomycin, 85 JLL g / ml amphotericin (GibcoBRL)
(GibcoBRL社製） に懸濁した。 この時点で再度細胞数を計測したところ、 回収した 骨髄由来細胞は合計 4.7 X 個あり、処理前の細胞の約 2%相当を回収したことにな る。 このようにして分画した骨髄由来細胞は 2〜5 X 105個ん m2になるように 10cm径 の動物細胞用の培養皿 （岩城硝子社製、 以下 10cm培養皿と略す） 3枚に撒き、 C02 培養器（夕バイ社製） にて 33°C、 5%C02濃度で培養を開始した。培地は M時間後、 72時間後にそれぞれ半分交換した。 その 3〜4日後に培地を半分交換した。 15日経 過し、 コロニーが密集してきたので、 細胞をトリプシン EDTA処理ではがし、 2/3 は4 mlの保存液 （10%DMSO、 50%の骨髄由来細胞培養上清、 40%の未使用上記培地） に懸濁し、 2ml容チューブ（住友べ一クライト社製） に 1本当たり lml分注して凍結 保存し、 残り 1/3は 10cm培養皿 2枚に蒔き直し継代した。 実施例 1 7 . ラヅト骨髄由来細胞の心筋細胞への分化能の検討 (Manufactured by GibcoBRL). At this point, the number of cells was measured again, and the total number of bone marrow-derived cells recovered was 4.7 X, which means that about 2% of the cells before treatment were recovered. In this way, 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. After 15 days, colonies have grown dense and the cells were detached by trypsin EDTA treatment. 2/3 of the cells were 4 ml of a stock solution (10% DMSO, 50% bone marrow-derived cell culture supernatant, 40% unused The above medium was suspended in a 2 ml tube (manufactured by Sumitomo Beilkrite Co., Ltd.), and the mixture was frozen and preserved. The remaining 1/3 was replated on two 10 cm culture dishes and subcultured. Example 17 7. Examination of differentiation ability of rat bone marrow-derived cells into cardiomyocytes
上記で継代したラヅト骨髄由来細胞は密集したところを再度トリプシン EDTA処 理ではがし、 6ゥエルプレート （BECTON DICKINSON社製） には 1ゥエル当たり 5 X 104個になるように、 またヒトフイブロネクチンをコートした 6cm径の培養皿 (BECTON DICKINSON社製 Biocoaり には l.3 X 105個になるように細胞を蒔き直し た。 1日後に5 -ァザシチジン （Sigma社製、 終濃度 10〃M) のみを加えたものと、 5 -ァザシチジン、 PDGF- BB (Pepro Tech EC LTD.社製、 終濃度 lOng/ml)、 all-trans レチノイン酸（ A、 Sigma社製、終濃度 10—9M)を加えた二種類の異なる培養条件培養 を行い、 2日間培養した後に培地を交換した（後者の場合は培地交換時に再度 PDGF、 all- transレチノイン酸を加え、 2曰後と 4日後にさらに加えた） 。 その 3〜4日後に、 培地を交換し、 3週間培養した。 その結果 5-ァザシチジン、 PDGF - BB、 レチノ.イン 酸を加えたもので筋管様細胞の分化が観察された。 The 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 μl 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 (. Pepro Tech EC LTD company Ltd., final concentration of lOng / ml), all-trans retinoic acid (a, Sigma Co., final concentration 10- 9 M) the two different culture conditions was added After culturing and culturing for 2 days, the medium was changed (in the latter case, PDGF and all-trans retinoic acid were added again when the medium was changed, and further added 2 and 4 days later). After changing the medium, the cells were cultured for 3 weeks, and as a result, differentiation of myotube-like cells was observed with 5-azacytidine, PDGF-BB, and retinoic acid.
産業上の利用可能性 Industrial applicability
本発明によれば、 心筋細胞の破壊ならびに変性を伴う心疾患の治療ならびに治療 薬の探索に有効な骨髄細胞、 増殖因子、 ビタミン、 接着分子、 及びこれらの利用法 が提供される。 ·  According to the present invention, 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. ·
「配列表フリーテキスト」  "Sequence List Free Text"
配列番号 3 3 -人工配列の説明:合成 DNA SEQ ID NO: 33-Description of artificial sequence: Synthetic DNA
配列番号 3 4—人工配列の説明:合成 DNA SEQ ID NO: 34—Description of artificial sequence: Synthetic DNA
配列番号 3 5—人工配列の説明:合成 DNA SEQ ID NO: 35—Description of artificial sequence: Synthetic DNA
配列番号 3 6—人工配列の説明:合成 DNA SEQ ID NO: 36—Description of artificial sequence: Synthetic DNA
配列番号 3 7—人工配列の説明:合成 DNA SEQ ID NO: 37—Description of artificial sequence: Synthetic DNA
配列番号 3 8—人工配列の説明:合成 DNA SEQ ID NO: 38—Description of artificial sequence: Synthetic DNA
配列番号 3 9一人工配列の説明:合成 DNA SEQ ID NO: 39 Description of artificial sequence: Synthetic DNA
配列番号 4 0 -人工配列の説明:合成 DNA SEQ ID NO: 40-Description of artificial sequence: Synthetic DNA
配列番号 4 1 -人工配列の説明:合成 DNA SEQ ID NO: 41-Description of artificial sequence: Synthetic DNA
配列番号 4 2一人工配列の説明:合成 DNA SEQ ID NO: 42 Description of artificial sequence: Synthetic DNA
配列番号 4 3—人工配列の説明:合成 DNA SEQ ID NO: 4 3—Description of artificial sequence: synthetic DNA
配列番号 4 4—人工配列の説明:合成 DNA SEQ ID NO: 4 4—Description of artificial sequence: synthetic DNA
配列番号 4 5 -人工配列の説明:合成 DNA SEQ ID NO: 45-Description of Artificial Sequence: Synthetic DNA
配列番号 4 6—人工配列の説明:合成 DNA SEQ ID NO: 46—Description of artificial sequence: Synthetic DNA
配列番号 4 7一人工配列の説明:合成 DNA 配列番号 4 8—人工配列の説明:合成 DNA 配列番号 4 9一人工配列の説明:合成 DNA 配列番号 5 0一人工配列の説明:合成 DNA 配列番号 5 1一人工配列の説明:合成 DNA 配列番号 5 2—人工配列の説明:合成 DNA 配列番号 5 3一人工配列の説明:合成 DNA 配列番号 5 4—人工配列の説明:合成 DNA 配列番号 5 5一人工配列の説明:合成 DNA 配列番号 5 6—人工配列の説明:合成 DNA 配列番号 5 7一人工配列の説明:合成 DNA 配列番号 5 8—人工配列の説明:合成 DNA 配列番号 5 9—人工配列の説明:合成 DNA 配列番号 6 0一人工配列の説明:合成 DNA 配列番号 6 1一人工配列の説明:合成 DNA 配列番号 6 2一人工配列の説明:合成 DNA 配列番号 6 3一人工配列の説明:合成 DNA 配列番号 6 4一人工配列の説明:合成 DNA 配列番号 6 5一人工配列の説明:合成 DNA 配列番号 6 6—人工配列の説明:合成 DNA 配列番号 6 7—人工配列の説明:合成 DNA 配列番号 6 8—人工配列の説明:合成 DNA 配列番号 6 9一人工配列の説明:合成 DNA 配列番号 7 0一人工配列の説明:合成 DNA 配列番号 7 1—人工配列の説明:合成 DNA 配列番号 7 2一人工配列の説明:合成 DNA 配列番号 7 3一人工配列の説明:合成 DNA 配列番号 7 4—人工配列の説明:合成 DNA 配列番号 7 5一人工配列の説明:合成 DNA 配列番号 7 6—人工配列の説明:合成 DNA 配列番号 7 7 -人工配列の説明:合成 DNA 配列番号 7 8—人工配列の説明:合成 DNA 配列番号 7 9—人工配列の説明:合成 DNA 配列番号 8 0—人工配列の説明:合成 DNA 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. 5 8—Description of Artificial Sequence: Synthetic DNA SEQ ID No. 5 9—Description of Artificial Sequence: Synthetic DNA SEQ ID NO. Description of Sequence: Synthetic DNA SEQ ID No. 6 1 Description of Artificial Sequence: Synthetic DNA SEQ ID No. 6 2 Description of Artificial Sequence: Synthetic DNA SEQ ID No. 6 3 Description of Artificial Sequence: Synthetic DNA SEQ ID No. 6 4 Artificial Sequence Description: Synthetic DNA SEQ ID No. 6 5 Description of Artificial Sequence: Synthetic DNA SEQ ID No. 6 6—Description of Artificial Sequence: Synthetic DNA SEQ ID No. 6 7—Description of Artificial Sequence: Synthetic DNA SEQ ID No. 6 8—Description of Artificial Sequence: Synthetic DNA SEQ ID No. 6 9 1 Description of Artificial Sequence: Synthetic DNA SEQ ID No. 70 0 Description of Artificial Sequence: Synthetic DNA SEQ ID No. 7 1 — Description of Artificial Sequence: Synthetic DNA SEQ ID No. 7 2 Description of Artificial Sequence: Synthetic DNA SEQ ID No. 7 3 — Description of Artificial Sequence: Synthetic DNA SEQ ID No. 7 4—Description of Artificial Sequence: Synthetic DNA SEQ ID No. 7 5 — Artificial Sequence Sequence description: synthetic DNA SEQ ID No. 7 6—Description of Artificial Sequence: Synthetic DNA SEQ ID No. 7 7-Description of Artificial Sequence: Synthetic DNA SEQ ID No. 7 8—Description of Artificial Sequence: Synthetic DNA SEQ ID No. 7 9—Description of Artificial Sequence: Synthetic DNA SEQ ID No. 8 0—Description of Artificial Sequence: Synthetic DNA

Claims

請求の範囲 The scope of the claims
1. 骨髄または臍帯血から単離され、 心筋細胞に分化する能力を有する細胞。  1. Cells isolated from bone marrow or cord blood and capable of differentiating into cardiomyocytes.
2. 細胞が、 少なくとも心筋細胞、 脂肪細胞、 骨格筋細胞、 骨芽細胞に分化する能 力を有する多分化能幹細胞である、 請求項 1記載の細胞。  2. The cell according to claim 1, wherein the cell is a pluripotent stem cell capable of differentiating into at least a cardiomyocyte, an adipocyte, a skeletal muscle cell, and an osteoblast.
3. 細胞が、 少なくとも心筋細胞、血管内皮細胞に分化する能力を有する多分化能 幹細胞である、 請求項 1記載の細胞。  3. The cell according to claim 1, wherein the cell is a pluripotent stem cell having at least the ability to differentiate into a cardiomyocyte and a vascular endothelial cell.
4. 細胞が、 少なくとも心筋細胞、 脂肪細胞、 骨格筋細胞、 骨芽細胞、 血管内皮細 胞に分化する能力を有する多分化能幹細胞である、 請求項 1記載の細胞。  4. The cell according to claim 1, wherein the cell is at least a multipotent stem cell capable of differentiating into a cardiomyocyte, an adipocyte, a skeletal muscle cell, an osteoblast, and a vascular endothelial cell.
5. 細胞が、 少なくとも心筋細胞、 脂肪細胞、 骨格筋細胞、 血管内皮細胞、 骨芽細 胞、 神経系細胞、 肝細胞に分化する能力を有する多分化能幹細胞である、 請求項 1 記載の細胞。  5. The cell according to claim 1, wherein the cell is at least a multipotent stem cell capable of differentiating into a cardiomyocyte, an adipocyte, a skeletal muscle cell, a vascular endothelial cell, an osteoblast, a nervous system cell, and a hepatocyte. .
6. CD34陰性、 CD117陰性、 CD144陰性および CD140陽性である、 請求項 1または 6. Claim 1 or CD34 negative, CD117 negative, CD144 negative and CD140 positive
2記載の細胞。 2. The cell according to 2.
7. CD34陽性、 CD117陽性および CD140陽性である、 請求項 1または 3記載の細胞。 7. The cell according to claim 1, which is positive for CD34, positive for CD117, and positive for CD140.
8. CD34陽性、 CD117陽性、 CD144陽性および CD140陽性である、 請求項 1または 3記載の細胞。 8. The cell according to claim 1, which is positive for CD34, CD117, CD144, and CD140.
9. CD34陰性、 CD117陽性、 CD144陰性および CD140陽性である、 請求項 1、 4ま たは 5記載の細胞。  9. The cell according to claim 1, 4 or 5, which is CD34 negative, CD117 positive, CD144 negative and CD140 positive.
10. CD117陽性および CD140陽性である、 請求項 1、 4または 5記載の細胞。 ' 10. The cell according to claim 1, 4 or 5, which is positive for CD117 and positive for CD140. '
11. CD34陰性、 CD117陰性、 CD14陽性、 CD45陰性、 CD90陰性、 Flk- 1陰性、 CD31 陰性、 CD105陰性、 CD144陰性、 CD 140陽性、 CD49b陽性、 CD49d陰性、 CD29陽性、 CD54陽性、 CD102陰性、 CD106陽性および CD44陽性である、 請求項 2記載の細胞。11.CD34 negative, CD117 negative, CD14 positive, CD45 negative, CD90 negative, Flk-1 negative, CD31 negative, CD105 negative, CD144 negative, CD140 positive, CD49b positive, CD49d negative, CD29 positive, CD54 positive, CD102 negative, 3. The cell according to claim 2, which is positive for CD106 and positive for CD44.
12. CD 3 4陽性、 CD117陽性、 CD14陰性、 CD45陰性、 CD90陰性、 Flk- 1陰性、 CD31 陰性、 CD105陰性、 CD144陽性、 CD140陽性、 CD49b陰性、 CD49d陰性、 CD29陽性、 CD54 P貪性、 CD102陰性、 CD106陰性および CD44陽性である、 請求項 3記載の細胞。12.CD34 positive, CD117 positive, CD14 negative, CD45 negative, CD90 negative, Flk-1 negative, CD31 negative, CD105 negative, CD144 positive, CD140 positive, CD49b negative, CD49d negative, CD29 positive, CD54 P tropism, 4. The cell according to claim 3, which is CD102 negative, CD106 negative and CD44 positive.
13. Hoechst33342を取り込まない、 請求項 1記載の細胞。 13. The cell of claim 1, which does not take up Hoechst33342.
14. 請求項 1〜 1 3のいずれか 1項に記載の細胞から誘導される心筋細胞のみに 分化誘導される心筋前駆細胞。 14. A cardiomyocyte that is induced to differentiate only into a cardiomyocyte derived from the cell according to any one of claims 1 to 13.
15. 心室筋細胞に分化する能力を有する、請求項 1〜1 4のいずれか 1項に記載の 細胞。 15. The cell according to any one of claims 1 to 14, which has an ability to differentiate into a ventricular myocyte.
16. 洞結節細胞に分化する能力を有する、請求項 1〜1 4のいずれか 1項に記載の 細胞。  16. The cell according to any one of claims 1 to 14, which has an ability to differentiate into a sinus node cell.
17. 骨髄または臍帯血がほ乳動物由来のものである、請求項 1〜1 6のいずれか 1 項に記載の細胞。  17. The cell of any one of claims 1-16, wherein the bone marrow or cord blood is from a mammal.
18. ほ乳動物がヒト、 ラットおよびマウスから選ばれるものである、請求項 1 7記 載の細胞。  18. The cell according to claim 17, wherein the mammal is selected from human, rat and mouse.
19. 細胞が、 マウス骨髄由来多分化能幹細胞 BMSC'(FERM BP- 7043)である、請求項 1に記載の細胞。  19. The cell according to claim 1, wherein the cell is a mouse bone marrow-derived multipotent stem cell BMSC '(FERM BP-7043).
20. 染色体 DNAの脱メチル化により心筋細胞に分化する^力を有する、 請求項 1 〜 1 9のいずれか 1項に記載の細胞。  20. The cell according to any one of claims 1 to 19, which has a power to differentiate into a cardiomyocyte by demethylation of chromosomal DNA.
21. 染色体 DNAの脱メチル化が、 デメチラーゼ、 5—ァザシチジンおよびジメチ ルスルフォキシド（DMSO)からなる群から選ばれる少なくとも 1種によるものであ ることを特徴とする、 請求項 2 0記載の細胞。  21. The cell according to claim 20, wherein the demethylation of chromosomal DNA is at least one selected from the group consisting of demethylase, 5-azacitidine and dimethyl sulfoxide (DMSO).
22. デメチラーゼが、配列番号 1記載で表されるアミノ酸配列を有するデメチラ一 ゼである、 請求項 2 1記載の細胞。  22. The cell according to claim 21, wherein the demethylase is a demethylase having an amino acid sequence represented by SEQ ID NO: 1.
23. 胎児の心臓発生領域で発現している因子により心筋細胞への分化が促進され る請求項 1〜 1 9のいずれか 1項に記載の細胞。  23. The cell according to any one of claims 1 to 19, wherein differentiation into cardiomyocytes is promoted by a factor expressed in a fetal heart development region.
24. 胎児の心臓発生領域で発現している因子がサイトカイン、接着分子、 ビ夕ミン、 転写因子および細胞外基質からなる群から選ばれる少なくとも 1種であることを特 徴とする、 請求項 2 3記載の細胞。  24. The factor which is characterized in that 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. 3. The cell according to 3.
25. 胎児の心臓発生段階において心筋細胞への分化に働く因子により心筋細胞へ の分化が促進される請求項 1〜 1 9いずれか 1項に記載の細胞。  25. The cell according to any one of claims 1 to 19, wherein differentiation into cardiomyocytes is promoted by a factor that acts on cardiomyocyte differentiation at the stage of fetal heart development.
26. 胎児の心臓発生段階において心筋細胞への分化に働く因子がサイトカイン、接 着分子、 ビタミン、 転写因子および細胞外基質からなる群から選ばれる少なくとも 1種であることを特徴とする、 請求項 2 5記載の細胞。  26. The factor that acts on differentiation into cardiomyocytes during the fetal heart development stage is at least one selected from the group consisting of cytokines, adhesion molecules, vitamins, transcription factors and extracellular matrix. 25. The cell according to 5.
27. サイトカインが血小板由来増殖因子 （PDGF) である、 請求項 2 4または 2 6 記載の細胞。 27. The method of claim 24 or 26, wherein the cytokine is platelet-derived growth factor (PDGF). A cell as described.
28. PDGFが配列番号 3または 5で表されるアミノ酸配列を有する PDGFである、 請求項 2 7記載の細胞。  28. The cell according to claim 27, wherein the PDGF is PDGF having the amino acid sequence represented by SEQ ID NO: 3 or 5.
29. サイ ト力インが繊維芽細胞増殖因子 8 (FGF-S) である、 請求項 2 4または 2 6記載の細胞。  29. The cell of claim 24 or 26, wherein the site force-in is fibroblast growth factor 8 (FGF-S).
30. FGF-8が配列番号 6 で表されるアミノ酸配列を有する FGP-8である、 請求 項 2 9記載の細胞。  30. The cell according to claim 29, wherein FGF-8 is FGP-8 having the amino acid sequence represented by SEQ ID NO: 6.
31. サイ トカインがェンドセリン 1 (ET1)である、請求項 2 4または 2 6記載の細胞。 31. The cell of claim 24 or 26, wherein the cytokine is endoselin 1 (ET1).
32. ET1が配列番号 6 6で表されるアミノ酸配列を有する ET1である、請求項 3 1 記載の細胞。 32. The cell according to claim 31, wherein the ET1 is an ET1 having an amino acid sequence represented by SEQ ID NO: 66.
33. サイ トカインがミ ドカイン (Midkine)である、 請求項 2 4または 2 6記載の細胞。 33. The cell according to claim 24 or 26, wherein the cytokine is Midkine.
34. Midkineが配列番号 6 8で表されるァミノ酸配列を有する Midkineである'、請求 項 3 3記載の細胞。 34. The cell according to claim 33, wherein the Midkine is a Midkine having an amino acid sequence represented by SEQ ID NO: 68.
35. サイ トカインが骨形成因子 4 (BMP- 4)である、：請求項 2 4または 2 6記載の細 胞。 35. The cell of claim 24 or 26, wherein the cytokine is bone morphogenetic factor 4 (BMP- 4 ).
36. BMP - 4が配列番号 7 0で表されるアミノ酸配列を有する BMP- 4である、 請求 項 3 5記載の細胞。  36. The cell according to claim 35, wherein BMP-4 is BMP-4 having the amino acid sequence represented by SEQ ID NO: 70.
37. 接着分子がフイブロネクチンである、 請求項 2 4または 2 6記載の細胞。  37. The cell of claim 24 or 26, wherein the adhesion molecule is fibronectin.
38. ビタミンがレチノイン酸である、 請求項 2 4または 2 6記載の細胞。  38. The cell according to claim 24 or 26, wherein the vitamin is retinoic acid.
39. 転写因子が、 Nkx2.5/Csxヽ GATA4、 MEF - 2A、 MEF- 2B、 MEF- 2C、 MEF- 2D、 dHAND、 eHAND、 TEF- 1、 TEF- 3、 TEF - 5および MesPlからなる群から選ばれるも のである、 請求項 2 4または 2 6記載の細胞。  39. The transcription factor is a group consisting of Nkx2.5 / Csx ヽ GATA4, MEF-2A, MEF-2B, MEF-2C, MEF-2D, dHAND, eHAND, TEF-1, TEF-3, TEF-5, and MesPl. The cell according to claim 24 or 26, which is selected from the group consisting of:
40. Nkx2.5/Csxが配列番号 9で表されるァミノ酸配列を有する Nkx2.5/Csxである、 請求項 3 9記載の細胞。  40. The cell according to claim 39, wherein Nkx2.5 / Csx is Nkx2.5 / Csx having an amino acid sequence represented by SEQ ID NO: 9.
41. GATA4が配列番号 1 1で表されるァミノ酸配列を有する GATA4である、請求 項 3 9記載の細胞。  41. The cell according to claim 39, wherein the GATA4 is a GATA4 having an amino acid sequence represented by SEQ ID NO: 11.
42. MEF- 2Aが配列番号 1 3で表されるアミノ酸配列を有する MEF- 2Aである、請 求項 3 9記載の細胞。 42. The cell according to claim 39, wherein MEF-2A is MEF-2A having an amino acid sequence represented by SEQ ID NO: 13.
43. MEF-2Bが配列番号 1 5で表されるアミノ酸配列を有する MEF - 2Bである、請 求項 3 9記載の細胞。 43. The cell according to claim 39, wherein MEF-2B is MEF-2B having the amino acid sequence represented by SEQ ID NO: 15.
44. MEF-2Cが配列番号 1 7で表されるアミノ酸配列を有する MEF-2Cである、請 求項 3 9記載の細胞。  44. The cell according to claim 39, wherein MEF-2C is MEF-2C having the amino acid sequence represented by SEQ ID NO: 17.
45. MEF-2Dが配列番号 ί 9で表されるアミノ酸配列を有する MEF-2Dである、請 求項 3 9記載の細胞。  45. The cell according to claim 39, wherein MEF-2D is MEF-2D having the amino acid sequence represented by SEQ ID NO: 9.
46. dHANDが配列番号 2 1で表されるアミノ酸配列を有する dHANDである、請求 項 3 9記載の細胞。  46. The cell according to claim 39, wherein the dHAND is a dHAND having the amino acid sequence represented by SEQ ID NO: 21.
47. ' eHANDが配列番号 2 3で表されるァミノ酸配列を有する eHANDである、請求 項 3 9記載の細胞。  47. The cell according to claim 39, wherein the 'eHAND is an eHAND having an amino acid sequence represented by SEQ ID NO: 23.
48·· TEF-1が配列番号 2 5で表されるアミノ酸配列を有する TEF-1である、 請求 項 3 9記載の細胞。  48. The cell according to claim 39, wherein TEF-1 is TEF-1 having an amino acid sequence represented by SEQ ID NO: 25.
49. TEF- 3が配列番号 2 7で表されるアミノ酸配列を有する TEF- 3である、 請求 項 3 9記載の細胞。  49. The cell according to claim 39, wherein TEF-3 is TEF-3 having an amino acid sequence represented by SEQ ID NO: 27.
50. TEF- 5が配列番号 2 9で表されるアミノ酸配列を有する TEF- 5である、 請求 項 3 9記載の細胞。  50. The cell according to claim 39, wherein the TEF-5 is TEF-5 having an amino acid sequence represented by SEQ ID NO: 29.
51. MesPlが配列番号 6 2で表されるアミノ酸配列を有する MesPlである、請求項 3 9記載の細胞。  51. The cell according to claim 39, wherein MesPl is MesPl having the amino acid sequence represented by SEQ ID NO: 62.
52. 細胞外基質が心筋細胞由来の細胞外基質であることを特徴とする請求項 2 4 または 2 6記載の細胞。  52. The cell according to claim 24 or 26, wherein the extracellular matrix is a cardiomyocyte-derived extracellular matrix.
53. 線維芽細胞増殖因子— 2 (FGF-2) により心筋細胞への分化が抑制される請求 項 1記載の細胞。 '  53. The cell of claim 1, wherein fibroblast growth factor-2 (FGF-2) suppresses differentiation into cardiomyocytes. '
54. FGF -2が配列番号 7または 8記載のアミノ酸配列を有する FGF- 2である、 請 求項 5 3記載の細胞。 54. The cell of claim 53, wherein FGF- 2 is FGF- 2 having the amino acid sequence of SEQ ID NO: 7 or 8.
55. 心臓に移植することにより心筋細胞に分化する能力を有する請求項 1〜 1 9 のいずれか 1項に記載の細胞。  55. The cell according to any one of claims 1 to 19, which has the ability to differentiate into cardiomyocytes by transplantation into the heart.
56. 心臓に移植することにより血管に分化する能力を有する請求項 1〜1 9のい ずれか 1項に記載の細胞。 56. The cell of any one of claims 1 to 19, which has the ability to differentiate into blood vessels when transplanted into the heart.
57. 胚盤胞に移植することで、心筋に分ィ匕する能力を有する請求項 1〜1 9のいず れか 1項に記載の細胞。 57. The cell according to any one of claims 1 to 19, which has an ability to divide into a myocardium by transplantation into a blastocyst.
58. 心筋細胞と共培養を行うことで、心筋に分化する能力を有する請求項 1〜 1 9 のいずれか 1項に記載の細胞。  58. The cell according to any one of claims 1 to 19, which has an ability to differentiate into a myocardium by co-culturing with a myocardial cell.
59. 核内受容体 PPAR -ァを活性化因子により脂肪細胞に分化する能力を有する請 求項 1〜 1 9のいずれか 1項に記載の細胞。  59. The cell according to any one of claims 1 to 19, wherein the cell has an ability to differentiate a nuclear receptor PPAR-a into an adipocyte by an activator.
60. 核内受容体 PPAR-ァの活性化因子がチアゾリジオン骨格を有する化合物であ ることを特徴とする請求項 5 9記載の細胞。  60. The cell according to claim 59, wherein the activator of the nuclear receptor PPAR-a is a compound having a thiazolidione skeleton.
61. チアゾリジオン骨格を有する化合物がトログリ夕ゾン、 ピオグリ夕ゾン、 ロジ グリ夕ゾンからなる群から選ばれる少なくとも 1種であることを特徴とする請求項 6 0記載の細胞。  61. The cell according to claim 60, wherein the compound having a thiazolidione skeleton is at least one selected from the group consisting of troglisuzone, pioglisuzone, and rosiglisone.
62. 胚盤胞に移植することで、神経系細胞に分化する能力を有する請求項 1〜Ί 9 いずれか 1項に記載の細胞。  62. The cell according to any one of claims 1 to 9, wherein the cell has the ability to differentiate into a nervous system cell when transplanted into a blastocyst.
63. 脳または脊髄に移植することで、神経系細胞に分化する能力を有する請求項 1 〜 1 9いずれか 1項に記載の細胞。  63. The cell according to any one of claims 1 to 19, which has an ability to differentiate into a nervous system cell when transplanted into the brain or spinal cord.
64. 胚盤胞に移植することで、肝細胞に分化する能力を有する請求項 1〜1 9のい ずれか 1項に記載の細胞。  64. The cell according to any one of claims 1 to 19, which has the ability to differentiate into hepatocytes by transplantation into blastocysts.
65. 肝臓に移植することで肝細胞に分化する能力を有する請求項 1〜1 9のいず れか 1項に記載の細胞。  65. The cell according to any one of claims 1 to 19, which has the ability to differentiate into hepatocytes by transplantation into the liver.
66. 染色体 DNAの脱メチル化剤を用いて、 骨髄由来の細胞から心筋を形成する方 法。  66. A method of forming myocardium from cells derived from bone marrow using a chromosomal DNA demethylating agent.
67. 染色体 DNAの脱メチル化剤が、デメチラ一ゼ、 5—ァザシチジンおよび DMS〇 からなる群から選ばれる少なくとも 1種であることを特徴とする、 請求項 6 6記載 の方法。  67. The method according to claim 66, wherein the demethylating agent for chromosomal DNA is at least one selected from the group consisting of demethylase, 5-azacitidine and DMS〇.
68. デメチラーゼが、配列番号 1記載のアミノ酸配列で表されるデメチラ一ゼであ る、 請求項 6 7記載の方法。  68. The method according to claim 67, wherein the demethylase is a demethylase represented by the amino acid sequence of SEQ ID NO: 1.
69. 胎児の心臓発生領域で発現している因子を用いることを特徴とする、骨髄由来 の細胞から心筋を形成する方法。 69. A method for forming myocardium from bone marrow-derived cells, comprising using a factor expressed in a fetal heart development region.
70. 胎児の心臓発生領域で発現している因子がサイ トカイン、 接着分子、 ビ夕ミン、 転写因子および細胞外基質からなる群から選ばれる少なくとも 1種であることを特 徴とする、 請求項 6 9記載の方法。 70. The factor characterized in that 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. 6 Method according to 9.
71. 胎児の心臓発生段階において心筋細胞への分化に働く因子を用いることを特 徴とする、 骨髄由来の細胞から心筋を形成する方法。  71. A method for forming a myocardium from bone marrow-derived cells, which comprises using a factor that acts on cardiomyocyte differentiation during the fetal heart development stage.
72. 胎児の心臓発生段階において心筋細胞への分化に働く因子がサイトカイン、接 着分子、 ビタミン、 転写因子および細胞外基質からなる群かち選ばれる少なぐとも 1種であることを特徴とする、 請求項 7 1記載の方法。  72. The factor that acts on cardiomyocyte differentiation during the fetal heart development stage is at least one member selected from the group consisting of cytokines, adhesion molecules, vitamins, transcription factors and extracellular matrix. 71. The method of claim 71.
73. サイ トカインが PDGFである、 請求項 7 0または 7 2記載の方法。  73. The method of claim 70 or 72, wherein the cytokine is PDGF.
74. PDGFが配列番号 3または 5記載のァミノ酸配列で表される PDGFである、 請 求項 6 3記載の方法。  74. The method according to claim 63, wherein the PDGF is PDGF represented by the amino acid sequence of SEQ ID NO: 3 or 5.
75. サイ トカインが繊維芽細胞増殖因子 8 (FGF-8) である、 請求填 7 0まだは 7 2記載の方法。  75. The method according to claim 70 or 72, wherein said cytokine is fibroblast growth factor 8 (FGF-8).
76. FGF-8が配列番号 6 4のァミノ酸配列で表される FGF - 8である、 請求項 7 5 記載の方法。  76. The method of claim 75, wherein FGF-8 is FGF-8 represented by the amino acid sequence of SEQ ID NO: 64.
77. サイ トカインがェンドセリン 1 (ET1)である、請求項 7 0または 7 2記載の方法 c 77. The method c of claim 70 or 72, wherein the site cytokine is endoselin 1 (ET1).
78. ET1が配列番号 6 6で表されるアミノ酸配列を有する ET1である、請求項 7 7 言 3載の方法。 78. The method of claim 77, wherein ET1 is an ET1 having the amino acid sequence represented by SEQ ID NO: 66.
79. サイ トカインがミ ドカイン (Midkine)である、 請求項 7 0または 7 2記載の方法 c 79. The method c of claim 70 or 72, wherein the sitekine is Midkine.
80. Midkineが配列番号 6 8で表されるァミノ酸配列を有する Midkineである、請求 項 7 9記載の方法。 80. The method according to claim 79, wherein the Midkine is a Midkine having an amino acid sequence represented by SEQ ID NO: 68.
81. サイ トカインが骨形成因子 4 (BMP- 4)である、 請求項 7 0または 7 2記載の方 法。  81. The method of claim 70 or 72, wherein the cytokine is bone morphogenetic factor 4 (BMP-4).
82. BMP-4が配列番号 7 0で表されるアミノ酸配列を有する BMP - 4である、 請求 項 8 1記載の方法。  82. The method of claim 81, wherein BMP-4 is BMP-4 having the amino acid sequence represented by SEQ ID NO: 70.
83. 接着分子がフイブロネクチンである、 請求項 7 0または 7 2記載の方法。  83. The method of claim 70 or 72, wherein the adhesion molecule is fibronectin.
84. ビタミンがレチノイン酸である、 請求項 7 0または 7 2記載の方法。  84. The method of claim 70 or 72, wherein the vitamin is retinoic acid.
85. 転写因子が、 Nkx2.5/Csx、 GATA4、 MEF - 2A、 MEF - 2B、 MEF - 2C、 MEF - 2Dヽ dHAND、 eHAND、 TEF- 1、 TEF- 3、 TEF-5および MesPlからなる群から選ばれる、 請求項 7 0または Ί 2記載の方法。 85. Transcription factors are Nkx2.5 / Csx, GATA4, MEF-2A, MEF-2B, MEF-2C, MEF-2D ヽ The method according to claim 70 or claim 2, wherein the method is selected from the group consisting of dHAND, eHAND, TEF-1, TEF-3, TEF-5 and MesPl.
86. Nkx2.5/Csxが、 配列番号 9で表されるアミノ酸配列を有する Nkx2.5/Csxであ る、 請求項 8 5記載の方法。  86. The method according to claim 85, wherein Nkx2.5 / Csx is Nkx2.5 / Csx having the amino acid sequence represented by SEQ ID NO: 9.
87. GATA4が、配列番号 1 1で表されるァミノ酸配列を有する GATA4である、 請 求項 8 5記載の方法。 ■  87. The method according to claim 85, wherein GATA4 is GATA4 having an amino acid sequence represented by SEQ ID NO: 11. ■
88. MEF-2Aが、配列番号 1 3で表されるァミノ酸配列を有する MEF- 2Aである、 請求項 8 5記載の方法。  88. The method according to claim 85, wherein MEF-2A is MEF-2A having an amino acid sequence represented by SEQ ID NO: 13.
89. : MEF-2Bが、 '配列番号 1 5で表されるアミノ酸配列を有する M E F— 2 Bであ る、 請求項 8 5記載の方法。 89 .: The method according to claim 85, wherein MEF-2B is' MEF-2B having the amino acid sequence represented by SEQ ID NO : 15.
90. MEF-2Cが、配列番号 1 7で表されるアミノ酸配列を有する MEF- 2Cである、 請求項 8 5記載の方法。  90. The method according to claim 85, wherein MEF-2C is MEF-2C having the amino acid sequence represented by SEQ ID NO: 17.
91. MEF-2Dが、配列番号 1 9で表されるァミノ酸配列を有する MEF- 2Dである、 請求項 8 5記載の方法。 ' .  91. The method according to claim 85, wherein MEF-2D is MEF-2D having an amino acid sequence represented by SEQ ID NO: 19. '.
92. dHANDが、配列番号 2 1で表されるアミノ酸配列を有する dHANDである、請 求項 8 5記載の方法。  92. The method according to claim 85, wherein the dHAND is a dHAND having the amino acid sequence represented by SEQ ID NO: 21.
93. eHANDが、配列番号 2 3で表されるァミノ酸配列を有する eHANDである、請 求項 8 5記載の方法。  93. The method according to claim 85, wherein the eHAND is an eHAND having an amino acid sequence represented by SEQ ID NO: 23.
94. TEF-1が、 配列番号 2 5で表されるァミノ酸配列を有する TEF-1である、 請 求項 8 5記載の方法。  94. The method according to claim 85, wherein TEF-1 is TEF-1 having an amino acid sequence represented by SEQ ID NO: 25.
95. TEF-3が、 配列番号 2 7で表されるァミノ酸配列を有する TEF - 3である、 請 求項 8 5記載の方法。  95. The method according to claim 85, wherein TEF-3 is TEF-3 having an amino acid sequence represented by SEQ ID NO: 27.
96. TEF-5が、 配列番号 2 9で表されるァミノ酸配列を有する TEF- 5である、 請 求項 8 5記載の方法。 '  96. The method according to claim 85, wherein TEF-5 is TEF-5 having an amino acid sequence represented by SEQ ID NO: 29. '
97. MesPlが、配列番号 6 2で表されるアミノ酸配列を有する MesPlである、請求 項 8 5記載の方法。  97. The method according to claim 85, wherein MesPl is a MesPl having an amino acid sequence represented by SEQ ID NO: 62.
98. 細胞外基質が心筋細胞由来の細胞外基質であることを特徴とする請求項 7 0 または 7 2記載の方法。 ' 98. The method of claim 70 or 72, wherein the extracellular matrix is a cardiomyocyte-derived extracellular matrix. '
99· 核内受容体 PPAR-7を活性化因子により骨髄由来の細胞から脂肪細胞を形成 する方法。 99 · A method of forming adipocytes from bone marrow-derived cells by activating the nuclear receptor PPAR-7.
100. 核内受容体 PPAR -ァの活性ィ匕因子がチアゾリジオン骨格を有する化合物であ ることを特徴とする請求項 9 9記載の方法。  100. The method according to claim 99, wherein the activator of the nuclear receptor PPAR-a is a compound having a thiazolidione skeleton.
101. チアゾリジオン骨格を有する化合物がトログリ夕ゾ'ン、 ピオグリ夕ゾン、ロジ グリ夕ゾンからなる群から選ばれる少なくとも 1種であることを特徴とする請求項 1 0 0記載の方法。  101. The method according to claim 100, wherein the compound having a thiazolidione skeleton is at least one member selected from the group consisting of troglizozone, pioglizozone, and rosiglyzone.
102. 染色体 DNAの脱メチル化剤を有効成分として含有することを特 f (とする心筋 形成剤。  102. A cardiomyogenic agent characterized by containing a chromosomal DNA demethylating agent as an active ingredient.
103. 染色体 DNAの脱メチル化剤がデメチラーゼ、 5—ァザシチジンおよび DMSO からなる群から選ばれる少なくとも 1種である、 請求項 1 0 2記載の心筋形成剤。 103. The cardiomyogenic agent according to claim 102, wherein the demethylating agent for chromosomal DNA is at least one member selected from the group consisting of demethylase, 5-azacitidine and DMSO.
104. デメチラーゼが、配列番号 1記載のアミノ酸配列で表されるデメチラ一ゼであ る、 請求項 1 0 3記載の心筋形成剤。 104. The cardiomyogenic agent according to claim 103, wherein the demethylase is a demethylase represented by the amino acid sequence set forth in SEQ ID NO: 1.
105. 胎児の心臓発生領域で発現している因子を有効成分として含有する心筋形成 剤。  105. A cardiomyogenic agent containing, as an active ingredient, a factor expressed in a fetal heart development region.
106. 胎児の心臓発生領域で発現している因子がサイトカイン、接着分子、 ビ夕ミン 転写因子および細胞外基質からなる群から選ばれる少なくとも 1種であることを特 徴とする、 請求項 1 0 5記載の心筋形成剤。  106. The factor characterized in that the factor expressed in the fetal heart development region is at least one member selected from the group consisting of a cytokine, an adhesion molecule, a bimin transcription factor and an extracellular matrix. 5. The cardiomyogen according to 5.
107. 胎児の心臓発生段階において心筋細胞への分化に働く因子を有効成分として 含有することを特徴とする心筋形成剤。  107. A cardiomyogenic agent comprising, as an active ingredient, a factor that acts on differentiation into cardiomyocytes at the stage of fetal heart development.
108. 胎児の心臓発生段階において心筋細胞への分化に働く因子がサイトカイン、接 着分子、 ビタミン、 転写因子および細胞外基質からなる群から選ばれる少なくとも 1種であることを特徴とする、 請求項 1 0 7記載の心筋形成剤。  108. The factor that acts on cardiomyocyte differentiation during the fetal heart development stage is at least one selected from the group consisting of cytokines, adhesion molecules, vitamins, transcription factors and extracellular matrix. 107. The cardiomyogen according to 107.
109. サイトカインが PDGFである、 請求項 1 0 6または 1 0 8記載の心筋形成剤。 109. The cardiomyogenic agent according to claim 106 or 108, wherein the cytokine is PDGF.
110. PDGFが配列番号 3または 5記載のアミノ酸配列で表される、請求項 1 0 9記 載の心筋形成剤。 110. The cardiomyogenic agent according to claim 109, wherein PDGF is represented by the amino acid sequence of SEQ ID NO: 3 or 5.
111. サイトカインが繊維芽細胞増殖因子 8 (FGF-8)である、請求項 1 0 6または 1 0 8記載の心筋形成剤。 111. The cardiomyogenic agent according to claim 106 or 108, wherein the cytokine is fibroblast growth factor 8 (FGF-8).
112. FGF-8が配列番号 6 4のアミノ酸配列で表される FGF - 8である、 請求項 1 1 1記載の心筋形成剤。 112. The cardiomyogenic agent according to claim 11, wherein FGF-8 is FGF-8 represented by the amino acid sequence of SEQ ID NO: 64.
113. サイトカインがェンドセリン 1 (ET1)である、 請求項 1 0 6または 1 0 8記載 の心筋形成剤。  113. The cardiomyogenic agent according to claim 106 or 108, wherein the cytokine is endoselin 1 (ET1).
114. ET1が配列番号 6 6で表されるアミノ酸配列を有する BT1である、 請求項 1 1 3記載の心筋形成剤。  114. The cardiomyogenic agent according to claim 113, wherein ET1 is BT1 having the amino acid sequence represented by SEQ ID NO: 66.
115. サイト力インがミドカイン (Midkine)である、請求項 1 0 6または 1 0 8記載の 心筋形成剤。  115. The cardiomyogenic agent according to claim 106 or 108, wherein the cytodynamic force is Midkine.
116. Midkineが配列番号 6 8で表されるァミノ酸配列を有する Midkin0である、 請 求項 1 1 5記載の心筋形成剤。  116. The cardiomyogenic agent according to claim 115, wherein Midkine is Midkin0 having an amino acid sequence represented by SEQ ID NO: 68.
117. サイトカインが骨形成因子 4 (BMP- である、 請求項 1 0 6または 1 0 8記載 の心筋形成剤。  117. The cardiomyogenic agent according to claim 106 or 108, wherein the cytokine is bone morphogenetic factor 4 (BMP-).
118. BMP-4が配列番号 Ί 0で表されるアミノ酸配列を有する BMP- 4である、請求 項 1 1 7記載の心筋形成剤。  118. The cardiomyogenic agent according to claim 117, wherein BMP-4 is BMP-4 having the amino acid sequence represented by SEQ ID NO: 0.
119. 接着分子がフイブロネクチンである、請求項 1 0 6または 1 0 8記載の心筋形 成剤。  119. The cardiomyogenic agent of claim 106 or 108, wherein the adhesion molecule is fibronectin.
120. ビ夕ミンがレチノイン酸である、請求項 1 0 6または 1 0 8記載の心筋形成剤 c 120. The cardiomyogenic agent c of claim 106 or 108, wherein bimin is retinoic acid.
121. 転写因子が、 Nkx2.5/Csxく GATA4、 MEF- 2A、 MEF - 2B、 MEF— 2C、 MEF- 2Dヽ dHAND、 eHAND、 TEF - 1、 TEF- 3、 TEF-5および MesPlからなる群から選ばれる、 請求項 1 0 6または 1 0 8記載の心筋形成剤。 121. The transcription factor is a group consisting of Nkx2.5 / Csx and GATA4, MEF-2A, MEF-2B, MEF-2C, MEF-2D ヽ dHAND, eHAND, TEF-1, TEF-3, TEF-5 and MesPl. The myocardial agent according to claim 106 or 108, which is selected from:
122. Nkx2.5/Csxが、配列番号 9記載のアミノ酸配列で表される Nkx2.5/Csxである、 請求項 1 2 1記載の心筋形成剤。  122. The cardiomyogenic agent according to claim 121, wherein Nkx2.5 / Csx is Nkx2.5 / Csx represented by the amino acid sequence of SEQ ID NO: 9.
123. GATA4が、 配列番号 1 1記載のアミノ酸配列で表される GATA4である、 請 求項 1 2 1記載の心筋形成剤。  123. The cardiomyogenic agent according to claim 122, wherein GATA4 is GATA4 represented by the amino acid sequence set forth in SEQ ID NO: 11.
124. MEF-2Aが、 配列番号 1 3記載のァミノ酸配列で表される MEF- 2Aである、 請求項 1 2 1記載の心筋形成剤。  124. The cardiomyogenic agent according to claim 121, wherein MEF-2A is MEF-2A represented by the amino acid sequence set forth in SEQ ID NO: 13.
125. MEF-2Bが、配列番号 1 5記載のアミノ酸配列で表される MEF- 2Bである、請 求項 1 2 1記載の心筋形成剤。 125. The cardiomyogenic agent according to claim 122, wherein MEF-2B is MEF-2B represented by the amino acid sequence set forth in SEQ ID NO: 15.
126. MEF-2Cが、 配列番号 1 7記載のアミノ酸配列で表される MEF- 2Cである、 請求項 1 2 1記載の心筋形成剤。 126. The cardiomyogenic agent according to claim 121, wherein MEF-2C is MEF-2C represented by the amino acid sequence of SEQ ID NO: 17.
127. MEF-2Dが、 配列番号 1 9記載のァミノ酸配列で表される MEF- 2Dである、 請求項 1 2 1記載の心筋形成剤。  127. The cardiomyogenic agent according to claim 121, wherein MEF-2D is MEF-2D represented by the amino acid sequence set forth in SEQ ID NO: 19.
128. dHANDが、 配列番号 2 1記載のァミノ酸配列で表される dHANDである、 請 求項 1 2 1記載の心筋形成剤。  128. The cardiomyogenic agent according to claim 122, wherein the dHAND is a dHAND represented by the amino acid sequence set forth in SEQ ID NO: 21.
129. eHANDが、 配列番号 2 3.記載のァミノ酸配列で表される eHANDである、 請 求項 1 2 1記載の心筋形成剤。  129. The cardiomyogenic agent according to claim 122, wherein the eHAND is an eHAND represented by the amino acid sequence described in SEQ ID NO: 23.
130. TEF-1が、 ·配列番号 2■ 5記載のァミノ酸配列で表される TEF - 1である'、 請求 項 1 2 1記載の心筋形成剤。  130. The cardiomyogenic agent according to claim 121, wherein TEF-1 is a TEF-1 represented by the amino acid sequence of SEQ ID NOS: 2-5.
131. TEF-3が、 配列番号 2 7記載のアミノ酸配列で表される TEF- 3である、 請求 項 1 2 1記載の心筋形成剤。  131. The cardiomyogenic agent according to claim 121, wherein TEF-3 is TEF-3 represented by the amino acid sequence of SEQ ID NO: 27.
132. TEF二 5が、 配列番号 2 9記載のアミノ酸配列で表される TEF-5である、 請求 項 1 2 1記載の心筋形成剤。  132. The cardiomyogenic agent according to claim 121, wherein TEF25 is TEF-5 represented by the amino acid sequence of SEQ ID NO: 29.
133. MesPlが、 配列番号 6 2記載のアミノ酸配列で表ざれる MesPlである、 請求 項 1 2 1記載の心筋形成剤。  133. The cardiomyogenic agent according to claim 121, wherein MesPl is MesPl represented by the amino acid sequence of SEQ ID NO: 62.
134. 細胞外基質が心筋細胞由来の細胞外基質であることを特徴とする請求項 1 0 6または 1 0 8記載の心筋形成剤。  134. The cardiomyogenic agent according to claim 106 or 108, wherein the extracellular matrix is a cardiomyocyte-derived extracellular matrix.
135. 請求項 1〜6 5のいずれか 1項に記載の細胞を用いることを特徴とする、心臓 疾患により破壊された心臓を再生する方法。 .  135. A method for regenerating a heart destroyed by a heart disease, comprising using the cell according to any one of claims 1 to 65. .
136. 請求項 1〜 6 5のいずれか 1項に記載の細胞を有効成分とする心臓再生治療 薬。  136. An agent for regenerating a heart, comprising the cell according to any one of claims 1 to 65 as an active ingredient.
137. 心臓の先天性遺伝子疾患での変異遺伝子に対する野生型遺伝子が導入された 請求項 1〜6 5のいずれか 1項に記載の細胞を用いることを特徴とする、 先天性遺 伝子疾患での変異遺伝子に対する野生型遺伝子を心筋へ特異的に輸送する方法。 137. In a congenital genetic disease, wherein the cell according to any one of claims 1 to 65 has been introduced with a wild-type gene for a mutant gene in a congenital genetic disease of the heart. A method for specifically transporting a wild-type gene corresponding to a mutant gene to myocardium.
138. 心臓の先天性遺伝子疾患での変異遺伝子に対する野生型遺伝子が導入された 請求項 1〜 6 5のいずれか 1項に記載の細胞を有効成分として含有する心臓疾患治 療薬。 138. A therapeutic drug for heart disease, comprising the cell according to any one of claims 1 to 65, into which a wild-type gene for a mutant gene in congenital heart disease has been introduced.
139. 請求項 1〜6 5のいずれか 1項に記載の細胞を免疫原として用いることを特 徴とする、 該細胞を特異的に認識する抗体を取得する方法 139. A method for obtaining an antibody that specifically recognizes a cell according to any one of claims 1 to 65, which comprises using the cell according to any one of claims 1 to 65 as an immunogen.
140. 請求項 1 3 9記載の方法で取得された抗体を用いることを特徴とする、ヒト骨 髄から心筋細胞への分化能を有する成体骨髄由来細胞を単離 ·精製する方法。 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 claim 13.
141. 請求項 1〜6 5のいずれか 1項に記載の細胞を用いることを特徴とする、該細 胞に特異的な表面抗原を取得する方法。 141. A method for obtaining a cell-specific surface antigen, comprising using the cell according to any one of claims 1 to 65.
142. 請求項 1〜6 5のいずれか 1項に記載の細胞を用いることを特徴とする、該細 胞を増殖する因子をスクリーニングする方法。  142. A method for screening for a factor that proliferates the cell, comprising using the cell according to any one of claims 1 to 65.
143. 請求項 1〜6 5のいずれか 1項に記載の細胞を用いることを特徴とする、該細 胞の心筋細胞への分化を誘導する因子をスクリーニングする方法。  143. A method for screening for a factor that induces differentiation of a cell into a cardiomyocyte, comprising using the cell according to any one of claims 1 to 65.
144. 請求項 1〜6 5のいずれか 1項に記載の細胞を用いることを特徴とする、該細 胞を不死化する因子をスクリ一ニングする方法。  144. A method for screening a factor for immortalizing a cell, comprising using the cell according to any one of claims 1 to 65.
145. 請求項 1〜6 5のいずれか 1項に記載の細胞に,テロメラ一ゼを発現させるこ とを特徴とする、 該細胞の不死化方法。  145. A method for immortalizing a cell according to any one of claims 1 to 65, characterized by expressing telomerase in the cell.
146. テロメラーゼが、配列番号 3 1記載で表されるアミノ酸配列を有するテロメラ —ゼである請求項 1 4 5記載の方法。  146. The method according to claim 144, wherein the telomerase is a telomerase having an amino acid sequence represented by SEQ ID NO: 31.
147. テロメラ一ゼを発現させることにより、不死化させた請求項 1〜6 5のいずれ か 1項に記載の細胞を有効成分として含有する心臓疾患治療薬。  147. A therapeutic agent for heart disease comprising the cell according to any one of claims 1 to 65 immortalized by expressing telomerase, as an active ingredient.
148. テロメラーゼが、配列番号 3 1記載で表されるアミノ酸配列を有するテロメラ ーゼである請求項 1 4 7記載の治療薬。  148. The therapeutic agent according to claim 147, wherein the telomerase is a telomerase having an amino acid sequence represented by SEQ ID NO: 31.
149. 請求項 1〜 6 5のいずれか 1項に記載の細胞を含んだ培養上清。  149. A culture supernatant containing the cell according to any one of claims 1 to 65.
150. 請求項 1 4 9記載の培養上清を用いることを特徴とする、請求項 1記載の細胞 を心筋細胞に分化誘導する方法。  150. A method for inducing differentiation of the cells according to claim 1 into cardiomyocytes, using the culture supernatant according to claim 1449.
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