JP2017108705A - Cardiomyocyte production method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide production methods of cardiomyocytes which can maintain the character of a cardiomyocyte over a long period of time.SOLUTION: A cardiomyocyte is produced using a method comprising the following (a) to (d): (a) performing a differentiation inducing process to mesodermal cells for obtained pluripotent stem cells; (b) separating cardiovascular progenitor cells out of the differentiation induced pluripotent stem cells in the step (a);(c) culturing the cardiovascular progenitor cells separated in the step (b) in a culture medium containing a ROCK inhibitor and a Wnt signal inhibitor; and (d) culturing, in a culture medium, the cells cultured in the step (c) to prepare cardiomyocytes, and preferably further comprising the following step (e) and (f): (e) culturing, in a culture medium containing a ROCK inhibitor, the cardiomyocytes prepared in the step (d); and (f) culturing, in a culture medium, the cardiomyocytes cultured in the step (e).SELECTED DRAWING: None

Description

  The present invention relates to a method for efficiently producing (preparing) cardiomyocytes from pluripotent stem cells, a cardiomyocyte obtained by such a production method, a method for producing a myocardial sheet for culturing such cardiomyocytes, and the like.

  Cardiomyocytes are the driving force of the heart's pump function and are extremely important for living organisms to maintain their lives. Attempts have been actively made to artificially produce cardiomyocytes and use them in regenerative medicine for various heart diseases. For example, methods for transplanting sheet-like cardiomyocytes into the heart have been developed. In such regenerative medicine, it is essential that the transplanted cardiomyocytes continue to maintain their functions in vivo.

  In the course of drug development, for the purpose of confirming the safety of a candidate drug, it is required to examine that the drug does not adversely affect the function of the heart, that is, does not have cardiotoxicity. In vitro cardiotoxicity assessment using cardiomyocytes is advantageous compared to methods using animals in that it is simple and can evaluate many candidate drugs. However, in order to correctly evaluate the cardiotoxicity of a candidate drug, it is necessary to stably obtain high-quality cardiomyocytes.

  As a method for obtaining cardiomyocytes, methods for differentiating cardiomyocytes from pluripotent stem cells (embryonic stem cells, induced pluripotent stem cells, etc.) are known (for example, Patent Document 1, Non-Patent Documents 1 to 3). These are methods for inducing differentiation into cardiomyocytes by culturing stem cells as starting materials and culturing in the presence of a protein having cardiomyocyte-inducing ability and a drug that inhibits the Wnt pathway.

International Publication WO2005 / 090558

Uosaki et al.:PLoS One 2011; 6 (8), e23657 Lian et al. : PNAS 2012; 109, E1848-1857 Burridge et al. : Nature Methods 2014; 11,855-860

  The conventional methods for producing cardiomyocytes described above do not provide a method for producing cardiomyocytes capable of maintaining the characteristics of cardiomyocytes over a long period of time. For example, in the cardiomyocytes produced by the method described in Non-Patent Document 3, cardiac troponin T (cardiac troponin T: cTnT or TNNT2) is a specific marker of cardiomyocytes that was 95% or more on the 15th day after the start of differentiation induction. The positive rate decreases to about 60% on the 60th day after the start of differentiation induction.

  From the viewpoint of stable supply of cells and quality control for use in regenerative medicine and drug evaluation, a method for producing cardiomyocytes with more stable traits is required.

  An object of the present invention is to provide a method for producing cardiomyocytes capable of maintaining the characteristics of cardiomyocytes over a long period of time. is there.

  As a result of intensive studies to solve the above problems, the present inventors have performed a step of isolating cardiovascular progenitor cells in the middle of the differentiation induction step from pluripotent stem cells to cardiomyocytes, It has been found for the first time that high-purity cardiomyocytes can be obtained. Furthermore, the present inventors have improved the yield of cardiomyocytes when the ROCK inhibitor and the Wnt signal inhibitor coexist at a specific time in the course of the method, and the obtained cardiomyocytes have long-term characteristics. Found for the first time to maintain. The present invention has been completed based on such knowledge.

That is, the present invention is as follows.
[1] A method for producing cardiomyocytes comprising the following steps (a) to (d).
(A) performing a differentiation-inducing treatment into mesoderm cells on the obtained pluripotent stem cells;
(B) a step of separating cardiovascular progenitor cells from the pluripotent stem cells subjected to differentiation induction in step (a);
(C) culturing the cardiovascular progenitor cells separated in step (b) in a culture solution containing a ROCK inhibitor and a Wnt signal inhibitor;
(D) a step of culturing the cells cultured in step (c) in a culture solution to prepare cardiomyocytes;
[2] The production method of [1] above, wherein the pluripotent stem cell is an induced pluripotent stem cell.
[3] The production method according to [1] or [2] above, wherein the cardiovascular progenitor cells are PDGFRα-positive cells.
[4] The method according to any one of [1] to [3], wherein in step (b), cardiovascular progenitor cells are separated 4 to 6 days after differentiation induction treatment in step (a). Production method.
[5] The production method according to [4] above, wherein in step (c), the cells are cultured for at least 6 to 7 days after differentiation induction treatment in step (a).
[6] The production method of the above-mentioned [5], wherein in the step (d), the cells are cultured until 8 to 22 days after the differentiation induction treatment in the step (a).
[7] The method according to any one of [1] to [6], further comprising the following steps (e) and (f):
(E) culturing the cardiomyocytes prepared in step (d) in a culture solution containing a ROCK inhibitor;
(F) culturing the cardiomyocytes cultured in step (e) in a culture solution;
[8] The production method according to [7] above, wherein in the step (e), the cells are cultured for at least 22 days after the differentiation induction treatment in the step (a).
[9] The production method according to [8] above, wherein in step (f), the cells are cultured from 23 to 91 days after the differentiation induction treatment in step (a).
[10] The production method according to any one of [1] to [9], wherein the ROCK inhibitor is Y-27632.
[11] The production method according to any one of [1] to [10], wherein the Wnt signal inhibitor is XAV939 and / or IWP-4.
[12] Cardiomyocytes prepared using the production method according to any one of [1] to [11] above.
[13] A method for producing a myocardial sheet, comprising culturing the cardiomyocytes according to [12] mixed with endothelial cells and wall cells.
[14] A population of cardiomyocytes capable of maintaining a cTnT positive rate of 80% or more by culturing for at least 70 days.
[15] A kit for inducing a population of cardiomyocytes capable of maintaining a cTnT positive rate of 80% or more by culturing for at least 70 days, comprising a Wnt signal inhibitor.
[16] The kit according to [15] above, wherein the Wnt signal inhibitor is XAV939 and / or IWP-4.

  The method of the present invention enables efficient and high yield production of cardiomyocytes capable of maintaining characteristics over a long period of time from pluripotent stem cells. Cardiomyocytes obtained by the method of the present invention can be used as a material for treatment of diseases requiring transplantation of cardiomyocytes, cell models of diseases caused by abnormalities of cardiomyocytes, etc., and safety evaluation of drugs. Can do. A myocardial sheet can be produced by mixing and culturing cardiomyocytes obtained by the method of the present invention with endothelial cells and wall cells. In addition, since cardiomyocytes can be cultured and maintained for a long period (at least 70 days), high-quality cardiomyocytes in regenerative medicine can be provided not only in the area where cardiomyocytes are produced and in the surrounding areas, but also in remote areas.

The cTnT positive rate of the 21st, 31, 41, 51, 61, and 91st day cells after a differentiation induction start is shown. The number of viable cells on days 21, 31, 41, 51, 61, and 91 after initiation of differentiation is shown. The microscope image of the cell of the 9th day when a cell is cultured in the RPMI1640 culture medium which does not contain Y-27632 from the 5th day to the 9th day after the differentiation induction start is shown. When cells were cultured in RPMI1640 medium containing no Wnt inhibitor (XAV939, IWP-4) or cultured in RPMI1640 medium containing XAV939 during the 5th to 9th days after the initiation of differentiation induction, The cTnT positive rate of cells on day 21 when cells were cultured in RPMI 1640 medium containing IWP-4 and when cultured in RPMI 1640 medium containing both XAV939 and IWP-4. The cTnT positive rate of the cells on days 11, 16, 21, 26, and 31 after the start of differentiation induction is shown. On the 61st day (FIG. 6A) and the 91st day (FIG. 6B) when the cells were cultured in a medium containing 10% serum instead of the B27 supplement during the 23rd to 91st days after the initiation of differentiation induction. The cTnT positive rate of a cell is shown. In addition, the vertical axis | shaft of each data shows a side scatter (SSC) level, and a horizontal axis shows the fluorescence level detected with the anti-cTnT-FITC antibody.

  In the method for producing cardiomyocytes of the present invention, the obtained pluripotent stem cell is subjected to differentiation induction treatment into mesoderm cells (a); among the pluripotent stem cells subjected to differentiation induction treatment in step (a) Separating the cardiovascular progenitor cells from the step (b); culturing the cardiovascular progenitor cells separated in the step (b) in a culture medium containing a ROCK inhibitor and a Wnt signal inhibitor (c); A method comprising the steps (a) to (d) of step (d); culturing the cells cultured in (c) in a culture solution to prepare cardiomyocytes (hereinafter referred to as “production method 1 of the present invention”) The prepared cardiomyocytes can be used for a long period (at least 10 days, preferably at least 20 days, more preferably at least 30 days, even more preferably at least 40 days, even more preferably less). When In the case of culturing and maintaining for 50 days, particularly preferably at least 60 days, most preferably at least 70 days), the step of further culturing the cardiomyocytes prepared in step (d) in a culture solution containing a ROCK inhibitor (e ); And the step (e) and the step (f) of the step (f) in which the cardiomyocytes cultured in the step (e) are cultured in a culture medium is preferable.

  In the present invention, the “cardiovascular progenitor cell” means a cell belonging to a mesoderm cell and capable of differentiating into a cardiomyocyte that forms a beating muscle and an electrically conductive tissue and a cell that has the ability to generate vascular smooth muscle. Usually, it is a PDGFRα positive cell, and includes both a CD82 positive cell and a CD82 negative cell. In the present invention, the “cardiomyocyte” means a myocardial cell having the characteristics of self-pulsation, and is usually a cTnT-positive cell.

  When the production method 1 of the present invention is used, a highly pure cardiomyocyte population capable of maintaining a cTnT positive rate of 80% or more can be produced by culturing for at least 70 days (preferably 75 days, more preferably 80 days). That is, in the present invention, “cardiomyocytes (population)” means that the ratio of the number of cardiomyocytes (cTnT positive cells) contained in the cell population is at least 80%, preferably at least 85%, more preferably at least 88%, More preferably, it means a highly pure cardiomyocyte population of at least 90%, even more preferably at least 93%, particularly preferably at least 95%, most preferably at least 98%.

  The cardiovascular progenitor cells include transcription factors such as Nkx2.5 (NK-2 transcription factor related, locus 5), islet1, Tbx5, Tbx20, GATA4, MEF2C (Myocyte Enhancer Factor 2C), MESP1 (Mesoderm Posterior 1), Cardiovascular progenitor markers such as cell surface antigens such as platelet-derived growth factor receptor α (PDGFRα), KDR, Flk1, and CD (cluster of differentiation) 44 are usually expressed. That is, as the cardiovascular progenitor cell, specifically, Nkx2.5 positive, islet1 positive, Tbx5 positive, Tbx20 positive, GATA4 positive, MEF2C positive, MESP1 positive, PDGFRα positive, KDR positive, Flk1 positive, and / or CD44 positive cells can be mentioned, and PDGFRα positive cells are preferred.

  The cardiovascular progenitor cell may be in a state where the cardiovascular progenitor cell is contained in another cell population such as a PDGFRα-negative cell. Here, the cardiovascular progenitor cell contained in the other cell population At least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 98% can be mentioned.

  The biological species of the pluripotent stem cell is not particularly limited, and examples thereof include rodents such as mice, rats, hamsters, and guinea pigs, rabbit eyes such as rabbits, and ungulates such as pigs, cows, goats, horses, and sheep. Primates such as eyes, dogs, cats, humans, monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, chimpanzees, etc. Among these, mice, pigs, or humans are preferred, and cardiomyocytes When used for treatment, humans can be particularly preferably exemplified.

  The pluripotent stem cell cannot be an individual by itself, but may be any cell that has the ability to differentiate into all tissues and cells that constitute a living body. Isolated embryonic stem cells (ES cells) and embryonic germ cells (EG cells) isolated from embryonic primordial germ cells (eg Proc Natl Acad Sci US A. 1998, 95: 13726-31), germline stem cells (GS cells) isolated from testis immediately after birth (see Nature. 2008, 456: 344-349), skin, bone marrow, etc. Pluripotent cells obtained by selecting from mesenchymal tissues (Muse cells [Multi-lineage differentiating Stress Enduring cells]) and embryonic stem (ntES) cells derived from cloned embryos obtained by nuclear transfer (For example, Science. 2001, 292: 740-743; Biol. Reprod. 2005, 72. 932-936; see Nature. 2007, 450: 497-502), pluripotent cells obtained by fusing somatic cells with eggs or ES cells (fusion stem cells), and skin cells. Introducing multiple pluripotent stemcells (iPS) induced by introducing multiple genes into cells to induce dedifferentiation of the subject's own somatic cells and having pluripotency similar to ES cells Among these, iPS cells are preferable, and when the cardiomyocytes obtained by the production method 1 of the present invention are used for the treatment of heart disease, considering the risk of rejection due to transplantation, it is derived from a patient suffering from heart disease. Obtained from iPS cells obtained from human somatic cells or from somatic cells derived from a subject (human) whose human leukocyte antigen (HLA) genotype is the same or substantially the same as that of a patient suffering from heart disease IPS thin It can be preferably exemplified. Here, “the HLA genotype is substantially the same” means that the HLA genotype matches to such an extent that the immune response can be suppressed by the immunosuppressive agent for the transplanted cells. Therefore, as a somatic cell derived from a subject (human) having substantially the same HLA genotype, specifically, three HLA genotypes of HLA-A, HLA-B and HLA-DR, or such HLA Examples include somatic cells derived from a subject (human) having the same HLA genotype in which four HLA genotypes obtained by adding HLA-C to three genotypes are identical.

  ES cells are cultured in a serum-containing or serum-free culture solution described later on feeder cells such as primary fibroblasts [MEF], STO cells, SNL cells derived from mitomycin C-treated mouse fetuses. Can be manufactured. Methods for producing ES cells are described in, for example, WO96 / 22362, WO02 / 101057, US5,843,780, US6,200,806, US6,280,718 and the like.

  EG cells can be produced by culturing primordial germ cells in a serum-containing or serum-free medium containing mSCF, LIF and bFGF, which will be described later (see, for example, Ce11. 1992, 70: 841-847).

  GS cells can be produced by repeating testicular cell passage under the same culture conditions as ES cells (Takebayashi Masanori et al. (2008), Experimental Medicine, Vol. 26, No. 5 (extra number), 41-46. Page, Yodosha (Tokyo, Japan)).

  Muse cells select stress-resistant cells by applying stress such as trypsin or hypoxia treatment to mesenchymal tissues such as skin and bone marrow, or SSEA-3, which is a surface antigen of pluripotent stem cells. It can be produced by selecting cells using expression as an indicator, and further isolating the cells by overlapping suspension culture in a single cell state (Japanese Patent No. 5185443, Proc Natl Acad Sci US A. 2010). , 107: 8639-8643).

  ntES cells can be produced by combining nuclear transfer technology and ES cell production technology (Kiyaka Wakayama et al. (2008), Experimental Medicine, Vol. 26, No. 5 (extra number), pages 47 to 52). In nuclear transfer, reprogramming can be performed by injecting a somatic cell nucleus into an enucleated unfertilized egg of a mammal and culturing it for several hours.

  Fusion stem cells can be produced by fusing somatic cells and eggs or ES cells (see, for example, Curr. Biol. 2001, 11: 1553-1558; Science. 2005, 26: 1369-1373).

  iPS cells are produced by introducing reprogramming factors such as Oct3 / 4, Sox2 and Klf4 (c-Myc or n-Myc as necessary) into somatic cells (eg, fibroblasts, skin cells, etc.). (Eg, Ce11. 2006, 126: 663-676, Nature. 2007, 448: 313-317, Nat Biotechno 1. 2008, 26; 101-106, Cel1. 2007, 131: 861-872, Science. 2007 , 318: 1917-1920, Ce11 Stem Cells. 2007, 1: 55-70, Nat Biotechnol. 2007, 25: 1177-1181, Nature. 2007, 448: 318-324, Cell Stem Cells. 2008, 2: 10- 12, Nature. 2008, 451: 141-146, Science. 2007, 318: 1917-1920). Stem cells established by culturing early embryos produced by nuclear transfer of somatic cell nuclei are also preferred as pluripotent stem cells (eg, Nature. 1997, 385: 810-813, Science. 1998, 280 : 1256-1258, Nature Biotechnology. 1999, 17: 456-461, Nature. 1998, 394: 369-374, Nature Genetics. 1999, 22: 127-128, Proc. Natl. Acad. Sci. USA. 1999, 96 : 14984-14989), Rideout III et al. (NatureGenetics. 2000, 24: 109-110). Specific examples of pluripotent stem cells include human ES cell H9 strain (WA09), human ES cell H1 (WA01) strain (National Stem Cell bank, WISC Bank), KhES-1, KhES-2 and KhES-3 ( All of them are human ES cells such as the Research Center for Stem Cell Medicine, Kyoto University Regenerative Research Institute), HES3, HES4, and HES6 (all National Stem Cell bank, Monash University), 201B7, 201B7-Ff, 253G1, 253G4, 1201C1, 1205D1 , 1210B2 and 836B3 (all iPS Academia Japan Co., Ltd. or iPS Research Institute, Kyoto University), Tic (JCRB1331 strain), Dotcom (JCRB1327 strain), Squeaky (JCRB1329 strain), and Toe (JCRB1338 strain), Lollipop (JCRB36 strain) (Growth medical care Centers, pharmaceutical Laboratories incurable-disease Resources Research & JCRB cell bank), neither UTA-1 strain and UTA-1-SF-2-2 strain (can be exemplified iPS cells University of Tokyo) and the like.

<Process (a)>
The method for inducing differentiation in the step (a) is not particularly limited as long as it can induce differentiation into at least cardiovascular progenitor cells. In addition to the method for inducing differentiation into cardiovascular progenitor cells, after differentiation induction into cardiovascular progenitor cells Furthermore, a method of preparing mixed cells composed of myocardial progenitor cells, endothelial cells and mural cells by culturing in a culture solution containing VEGF (International Publication No. 2013/137491) is also included.

  Specifically, for example, literature (Yamashita, J. et al., Nature 408: 92-96 (2000), Yamashita, JK et al., FASEB J. 19: 1534-1536 (2005), Narazaki, G. , Circulation 118: 498-506 (2008)), stem cells are cultured on a collagen IV-coated dish (without LIF, without feeder cells), whereby Flk1 (vascular endothelial growth) is a mesodermal progenitor cell. Differentiation induction into positive cells) (also called factor receptor-2 [VEGFR2]), and further induction of differentiation into cardiovascular progenitor cells by culturing Flk1-positive cells in a culture medium containing cyclosporin-A (CSA). And a method for inducing differentiation into cardiovascular progenitor cells by suspension culture of stem cells in a culture solution containing EGF and bFGF (International Publication No. 2006/093276). Pamphlet), a method of inducing differentiation into cardiovascular progenitor cells by adhesion culture or suspension culture in a culture medium containing BMP antagonists such as Noggin and Chordin (International Publication No. 2005/033298 pamphlet), Stem cells are cultured in a culture medium containing Wnt-1, Wnt-3a or Wnt-5a, a GSK3β inhibitor, or a Wnt agonist that is an aminopyrimidine derivative, thereby activating the Wnt signaling pathway and Cardiovascular progenitor cells by inducing differentiation into cells (WO 2007/126077 pamphlet) or by culturing stem cells in a culture solution containing activin A and then in a culture solution containing BMP4 A method for inducing differentiation into a cell (JP 2013-215206 A, Laflamme, MA et al., Nat Biotechnol, 25: 1015-1024 (2007)) or by culturing the stem cells on a culture vessel treated with a coating agent, and then adding the coating agent to coat the whole stem cells. A method of inducing differentiation into cardiovascular progenitor cells by coating (sandwich treatment) with an agent, culturing in a culture solution containing activin A, and then culturing in a culture solution containing BMP4 and bFGF (hereinafter referred to as “this case”) Among these, the preferred differentiation induction treatment method, ie, the step (i) of culturing cells in a culture solution containing activin A, and BMP4 A method of performing differentiation induction treatment by a two-step process (ii) of culturing cells in a culture solution containing. Details of steps (i) and (ii) in the preferred differentiation inducing treatment method will be described below.

(I) A step of culturing cells using a culture solution containing activin A In step (i), pluripotent stem cells obtained by any method are suspended or cultured in a culture solution containing activin A ( Preferably, adhesion culture is performed. The culture vessel may be of any material and any shape as long as it does not inhibit the maintenance, survival, differentiation, maturation, and self-replication of pluripotent stem cells. And a synthetic resin including a nonwoven fabric, a natural resin, a metal, and the like. In addition, the shape of the culture vessel may be a polygonal prism such as a triangular prism, a cube or a rectangular parallelepiped, a polygonal pyramid such as a triangular pyramid or a quadrangular pyramid, an arbitrary shape such as a gourd, a sphere, a hemisphere, a circle, an ellipse, a semicircle, etc. Can be mentioned. As the incubator, a commercially available culture flask, culture dish (culture dish), culture bag, or the like can also be used.

  The above "suspension culture" means culturing cells in a non-adherent state in a culture container, and suspension culture is usually a culture container that is not coated with a cell adhesive substance (for example, an extracellular matrix) This is performed using a culture vessel that has been artificially treated to suppress adhesion (for example, coating with polyhydroxyethyl methacrylic acid [poly-HEMA]).

  Adhesion culture is usually performed using a culture vessel treated with a coating agent. Examples of such coating agents include Matrigel, type I collagen, type IV collagen, polylysine, gelatin, laminin, heparan sulfate proteoglycan or entactin, temperature-responsive polymer (PIPAAm), and combinations thereof. preferable.

  As a method for separating (peeling) the pluripotent stem cells cultured in an adhesion culture, a physical method, a method using a chelating agent, a cell separation solution having protease activity and / or collagenase activity (for example, trypsin, lysyl endopeptidase, (Pronase, pepsin, elastase, collagenase, hyaluronidase, etc.) and a combination thereof can be mentioned, and a treatment method using a cell separation solution can be preferably exemplified. Pluripotent stem cells are usually subcultured and maintained with about 80% confluence as a guide for the culture vessel.

  The culture solution in step (i) may be a serum-containing culture solution or a serum-free culture solution. Examples of such serum-containing cultures include animal cells containing 0.1 to 30 (v / v)% serum (Fetal bovine serum; FBS, calf bovine serum; CS, etc.). Examples of the culture medium for culture (DMEM, EMEM, IMDM, RPMI1640, αMEM, F-12, F-10, M-199, AIM-V, etc.) can also be mentioned. Serum substitutes such as B27 supplement (-insulin) (manufactured by Life Technologies), N2 supplement (manufactured by Life Technologies), B27 supplement (manufactured by Life Technologies), Knockout Serum Replacement (manufactured by Invitrogen), etc. ˜30%) and the above-mentioned culture medium for animal cell culture.

  Examples of the culture solution in step (i) include a reducing agent (for example, 2-mercaptoethanol, dithiothreitol (DTT), etc.), an iron source (for example, transferrin, etc.), a mineral (for example, sodium selenite). ), Amino acids (eg, non-essential amino acids such as L-glutamine, alanine, asparagine, serine, aspartic acid, cysteine, glutamic acid, glycine, proline, tyrosine), vitamins (eg, choline chloride, pantothenic acid, folic acid, nicotinamide) , Pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, ascorbic acid, biotin, inositol etc.), saccharides (eg glucose etc.), organic amines (eg putrescine etc.), steroids (eg progesterone, β-estradiol etc.), antibiotics (For example, Nicilin, streptomycin, etc.), interleukins (eg, IL-1, IL-2, IL-3, IL-6, etc.), adhesion factors (eg, heparin, heparan sulfate, collagen, fibronectin, etc.), organic acids (eg, , Pyruvic acid, succinic acid, lactic acid, etc.) or a salt thereof, a buffering agent (eg, HEPES, etc.) and a combination thereof may be appropriately supplemented as necessary.

  The concentration of activin A contained in the culture solution in step (i) is usually within the range of 5 to 2000 ng / mL, preferably 10 to 1000 ng / mL, more preferably 25 to 500 ng / mL, and further preferably 50. ~ 200 ng / mL.

  The culture solution in step (i) may contain BMP4 or may not contain BMP4. However, when BMP4 is contained, it affects the differentiation-inducing treatment into mesoderm cells. Those containing BMP4 at a low concentration are preferred. The concentration of BMP4 that does not affect the differentiation induction treatment into mesoderm cells is 2 ng / mL or less, preferably 1 ng / mL or less, more preferably 0.5 ng / mL or less.

The cell culture temperature in the step (i) is usually within a range of 30 to 40 ° C, preferably 37 ° C. In addition, the CO ₂ concentration during culture is usually in the range of about 1 to 10%, preferably about 5%. Moreover, the humidity at the time of culture | cultivation is in the range of about 70-100% normally, Preferably it exists in the range of about 95-100%. Moreover, the culture | cultivation time in process (i) is in the range of 6 hours-5 days normally, Preferably it is 12 to 48 hours.

(Ii) A step of culturing cells using a culture solution containing BMP4 Step (ii) is a step of culturing the cells after culturing in step (i) in a suspension culture or adhesion culture in a culture solution containing BMP4 (preferably, Adhesive culture). From the viewpoint of simplicity, it is usually performed by exchanging the culture solution containing activin A with a culture solution containing BMP4.

  The culture medium in step (ii) may be the above-mentioned serum-containing culture medium or the above-mentioned serum-free culture medium, and further contains basic fibroblast growth factor (bFGF). Is preferred. As a density | concentration of bFGF contained in a culture solution, it is 0.5-200 ng / mL normally, Preferably it is 1-100 ng / mL, More preferably, it is 2-50 ng / mL. In addition, the above-mentioned additive may be appropriately supplemented to the culture solution as necessary.

  As a density | concentration of BMP4 contained in the culture solution of a process (ii), it is in the range of 0.5-200 ng / mL normally, Preferably it is 1-100 ng / mL, More preferably, it is 2-50 ng / mL.

  The culture solution in step (ii) may contain activin A or may not contain activin A. However, when activin A is contained, it is used for differentiation induction treatment to mesoderm cells. Those containing activin A at a concentration that does not affect are preferred. The concentration of activin A that does not affect the differentiation induction treatment to mesoderm cells is 50 ng / mL or less, preferably 25 ng / mL or less, more preferably 10 ng / mL or less, and even more preferably. Is 5 ng / mL or less.

As described above, the cell culture temperature in the step (ii) is usually in the range of 30 to 40 ° C, preferably 37 ° C. Further, as described above, the CO ₂ concentration at the time of culture is usually in the range of about 1 to 10%, preferably about 5%. Moreover, the humidity at the time of culture is usually in the range of about 70 to 100%, preferably in the range of about 95 to 100%, as described above. Moreover, the culture | cultivation time in process (ii) is in the range of 1-10 days normally, Preferably it is 2-6 days, More preferably, it is 3-5 days.

<Step (b)>
In the step (b), cardiovascular progenitor cells are separated from the pluripotent stem cells subjected to differentiation induction treatment. Here, “separation” means physically removing cardiovascular progenitor cells from the culture vessel. When the cardiovascular progenitor cells are adherently cultured, the cardiovascular progenitor cells are detached from the culture vessel by performing the treatment with the cell dispersion under a temperature condition in the range of 20 to 37 ° C. The culture solution containing the progenitor cells can be separated into another container by decantation, or separated into another container using a pipette or pipette man, and the cardiovascular progenitor cells can be suspended in the culture solution. If so, the culture solution containing the cardiovascular progenitor cells can be separated from the culture container into another container by decantation, or separated into another container using Pipetman.

  The presence of cardiovascular progenitor cells in the separated cell population can be confirmed by positive of at least one (preferably PDGFRα) selected from the cardiovascular progenitor markers. Expression of the above-mentioned cardiovascular progenitor marker can be carried out by analyzing mRNA expression such as quantitative RT-PCR method, RT-PCR method, Southern blotting method, Western blotting method, flow cytometry, ELISA method, EIA method, RIA This can be confirmed by detection using a method for analyzing protein expression such as a method.

  When preparing high-purity cardiomyocytes using such separated cardiovascular progenitor cells, it is preferable to use cardiovascular progenitor cells that have been isolated as high-purity cardiovascular progenitor cells. Isolation of cardiovascular progenitor cells can be accomplished by FACS using an antibody against the cardiovascular progenitor marker labeled with a fluorescent substance, an antibody against the cardiovascular progenitor marker labeled with a labeling substance such as a fluorescent substance, biotin, or avidin. It can be carried out by an automatic magnetic cell separator (autoMACS) using an antibody against such a labeled substance and a conjugate antibody of MACS beads (magnetic beads). Examples of the fluorescent material include APC, PE, FITC, Alexa Fluor 488, Alexa Fluor 647, Alexa Fluor 700, PE-Texas Red, PE-Cy5, and PE-Cy7.

  When pluripotent stem cells are induced to differentiate into cardiovascular progenitor cells, the method of analyzing the expression of mRNA of the cardiovascular progenitor marker gene from the cell sample at each time after differentiation induction, or the cardiovascular progenitor marker protein This can be confirmed by detecting the above-mentioned cardiovascular progenitor marker positive cells using a method for analyzing the expression of.

  The timing for isolating cardiovascular progenitor cells from the pluripotent stem cells subjected to differentiation induction treatment varies depending on the differentiation induction treatment method and culture conditions, and thus cannot be generally specified, but is usually 1 to 14 after differentiation induction treatment. It is the day, preferably the 3rd to 11th day, more preferably the 4th to 8th day, and still more preferably the 4th to 6th day.

<Step (c)>
In the step (c), the cardiovascular progenitor cells are cultured in a culture solution containing a Rho-associated protein kinase (ROCK) inhibitor and a Wnt signal inhibitor. Such a culture solution may be the serum-containing culture solution or the serum-free culture solution. In addition to the above-mentioned additives, the culture solution contains cell growth factors (eg, insulin, epidermal growth factor [EGF], insulin-like growth factor [IGF], bFGF, platelets). Platelet-derived growth factor (PDGF), vascular endothelial growth factor [VEGF; Vesicular endothelial growth factor], hepatocyte growth factor [HGF], FGF9, bone morphogenetic protein 4 [BMP4; bone morphogenetic protein 4], BMP5, activin A, stem cell factor [SCF], Dkk1 [Dickkopf-1], leukemia inhibitory factor [LIF; Leukemia Inhibitory Factor], TGF-β, etc.) may be supplemented as necessary. May be.

  The ROCK inhibitor in the step (c) is not particularly limited as long as it can inhibit (suppress) the function of ROCK, that is, a protein kinase controlled by Rho, which is a low molecular weight GTP-binding protein. Include low molecular weight compounds such as Y-27632, Fasudil (also referred to as HA1077), H-1152, Wf-536, Y-30141, and derivatives thereof, antisense nucleic acids against the ROCK gene, RNA interference-inducing nucleic acids (for example, siRNA), competitive peptides for ROCK, antagonist peptides, inhibitory antibodies, antibody-ScFV fragments, dominant negative mutants, and expression vectors thereof, ROCK inhibitors that are low molecular weight compounds are preferred, Y-27632 It can illustrate suitably. In the present invention, two or more ROCK inhibitors may be used in combination.

  The concentration of the ROCK inhibitor contained in the culture solution can be appropriately selected according to the type of the ROCK inhibitor. For example, when Y-27632 is used, it is usually in the range of 1 to 100 μM, preferably 2 to 2. 50 μM, more preferably 10 to 40 μM.

  The Wnt signal inhibitor in the above step (c) is not particularly limited as long as it can suppress the Wnt signal pathway involved in gene expression and cytoskeleton control. Specifically, XAV939 (tankyase inhibitor), Low molecular weight compounds such as IWP-1, IWP-2, IWP-3, IWP-4, IWR-1, 53AH (above porcupine inhibitor), KY02111 and their derivatives, and proteins such as IGFBP4, DKK1, and Wnt-C59 And antisense nucleic acids, RNA interference-inducing nucleic acids (eg, siRNA), competitive peptides, antagonist peptides, inhibitory antibodies, antibody-ScFV fragments, dominant negative mutants that suppress the expression or function of proteins constituting the Wnt and Wnt signaling pathways And their expression vectors Rukoto can, Wnt signal inhibitor is a low molecular compounds are preferred, it can be preferably exemplified a XAV939 and IWP-4. In the present invention, two or more Wnt signal inhibitors may be used in combination, and XAV939 and IWP-4 are particularly preferably used in combination.

  The concentration of the Wnt signal inhibitor contained in the culture solution can be appropriately selected according to the type of the Wnt signal inhibitor. For example, when XAV939 is used, it is usually in the range of 0.025 to 100 μM, preferably 0.075-50 μM, more preferably 0.25-10 μM. When IWP-4 is used, it is usually in the range of 0.0125-50 μM, preferably 0.0725-15 μM, more preferably 0.125. ~ 5 μM.

  In the step (c), the time for culturing the cells differs depending on the differentiation induction treatment method and the culture conditions, and thus cannot be specified in general. However, it is usually the time when the cells express PDGFRa, Specifically, in the step (b), when the cardiovascular progenitor cells are separated on the 4th to 6th days after the differentiation induction treatment in the step (a), at least 6-7 after the differentiation induction treatment in the step (a). Preferably between days, more preferably between at least 5-8 days.

As described above, the cell culture temperature in the step (c) is usually in the range of 30 to 40 ° C, preferably 37 ° C. Further, as described above, the CO ₂ concentration at the time of culture is usually in the range of about 1 to 10%, preferably about 5%. Moreover, the humidity at the time of culture is usually in the range of about 70 to 100%, preferably in the range of about 95 to 100%, as described above. Moreover, the culture | cultivation time in the said process (c) is in the range of 2-6 days normally, Preferably it is 2-4 days, More preferably, it is 3-4 days.

<Step (d)>
In the step (d), the cells cultured in the step (c) are cultured in a culture solution to prepare cardiomyocytes. Such a culture solution may be the serum-containing culture solution or the serum-free culture solution. In addition, such a culture solution may or may not be supplemented with the above-mentioned additives and cell growth factors as necessary. In addition, such a culture solution may or may not contain a ROCK inhibitor and a Wnt signal inhibitor. However, when it contains a ROCK inhibitor and a Wnt signal inhibitor, the maturation of cardiomyocytes Those containing a ROCK inhibitor and a Wnt signal inhibitor at a concentration that does not affect the conversion are preferred. As used herein, “mature cardiomyocytes” refers to cells that exhibit the same properties as adult fully cardiomyocytes. Mature cardiomyocytes are usually MLC2v positive. MLC2v is one of the isoforms of Myosin Light Chain 2 and is strongly expressed in ventricular cardiomyocytes in the adult heart.

  In the step (d), the time for culturing the cells varies depending on the differentiation induction treatment method and the culture conditions, and thus cannot be specified in general. However, in the step (b), the cardiovascular progenitor cells are separated in the step (d). When the cells are cultured for 4 to 6 days after differentiation induction treatment in (a) and cells are cultured for at least 6 to 7 days after differentiation induction treatment in step (a), It is preferably from 8 to 22 days after differentiation induction treatment in a), more preferably from 9 to 21 days.

As described above, the cell culture temperature in the step (d) is usually in the range of 30 to 40 ° C, preferably 37 ° C. Further, as described above, the CO ₂ concentration at the time of culture is usually in the range of about 1 to 10%, preferably about 5%. Moreover, the humidity at the time of culture is usually in the range of about 70 to 100%, preferably in the range of about 95 to 100%, as described above. Moreover, the culture | cultivation time in the said process (d) is in the range of 5-13 days normally, Preferably it is 7-13 days, More preferably, it is 9-13 days.

<Process (e)>
In the step (e), the cardiomyocytes prepared in the step (d) are cultured in a culture solution containing a ROCK inhibitor. Such a culture solution may be the serum-containing culture solution or the serum-free culture solution. In addition, such a culture solution may or may not be supplemented with the above-mentioned additives and cell growth factors as necessary.

  As described above, the concentration of the ROCK inhibitor contained in the culture solution can be appropriately selected according to the type of the ROCK inhibitor. For example, when Y-27632 is used, it is usually in the range of 1 to 100 μM. , Preferably 2 to 50 μM, more preferably 10 to 40 μM.

  In the step (e), the time for culturing the cells differs depending on the differentiation induction treatment method and the culture conditions, and thus cannot be specified in general. However, in the step (b), the cardiovascular progenitor cells are separated in the step (e). (A) 4 to 6 days after differentiation induction treatment, and in step (c), cells are cultured for at least 6 to 7 days after differentiation induction treatment in step (a), and step (d) ), The cells are cultured until 8 to 22 days after differentiation induction treatment in step (a), and cardiomyocytes are prepared, preferably during at least 22 days after differentiation induction treatment in step (a), More preferably between the 21st and 22nd days.

As described above, the cell culture temperature in the step (e) is usually in the range of 30 to 40 ° C, preferably 37 ° C. Further, as described above, the CO ₂ concentration at the time of culture is usually in the range of about 1 to 10%, preferably about 5%. Moreover, the humidity at the time of culture is usually in the range of about 70 to 100%, preferably in the range of about 95 to 100%, as described above. Moreover, the culture | cultivation time in the said process (e) is in the range of 1-4 days normally, Preferably it is 1-3 days.

<Step (f)>
In the step (f), the cardiomyocytes cultured in the step (e) are cultured in a culture solution. Such a culture solution may be the serum-containing culture solution or the serum-free culture solution. In addition, such a culture solution may or may not be supplemented with the above-mentioned additives and cell growth factors as necessary. In addition, such a culture solution may or may not contain a ROCK inhibitor and a Wnt signal inhibitor. However, when it contains a ROCK inhibitor and a Wnt signal inhibitor, the maturation of cardiomyocytes Those containing a ROCK inhibitor and a Wnt signal inhibitor at a concentration that does not affect the maintenance of conversion.

  In the step (f), the time for culturing the cells varies depending on the differentiation induction treatment method and the culture conditions, and thus cannot be specified in general. However, in the step (b), the cardiovascular progenitor cells are separated in the step (A) 4 to 6 days after differentiation induction treatment, and in step (c), cells are cultured for at least 6 to 7 days after differentiation induction treatment in step (a), and in step (d) The cells are cultured until 8 to 22 days after differentiation induction treatment in step (a) to prepare cardiomyocytes, and in step (e), the cells are at least 22 after differentiation induction treatment in step (a). Considering the possibility that if the culture period of cardiomyocytes is too long when the culture is performed for the second day, the survival and properties of the cells are adversely affected, it is usually up to 150 days, preferably up to 130 days, more preferably 120 days. Even better until day Until day 110, even more preferably up to 100 days, particularly preferably up to 91 days. Therefore, the storage period is usually within the range of 23 to 150 days, preferably within the range of 23 to 130 days, and more preferably within the range of 23 to 120 days. More preferably, it is within the range from the 23rd to the 110th day, even more preferably within the range from the 23rd to the 100th day, and particularly preferably within the range from the 23rd to the 91st day.

As described above, the cell culture temperature in the step (f) is usually in the range of 30 to 40 ° C., preferably 37 ° C. Further, as described above, the CO ₂ concentration at the time of culture is usually in the range of about 1 to 10%, preferably about 5%. Moreover, the humidity at the time of culture is usually in the range of about 70 to 100%, preferably in the range of about 95 to 100%, as described above. Moreover, the culture time in the said process (f) is in the range of 69-128 days normally, Preferably it is 69-80 days.

  As a method for producing a myocardial sheet of the present invention, a method in which cardiomyocytes prepared using the production method 1 of the present invention are mixed with endothelial cells and mural cells (hereinafter referred to as “production method 2 of the present invention”). If there is, there is no particular limitation.

<Process for producing endothelial cells and mural cells>
In the production method 2 of the present invention, the endothelial cells and mural cells to be used can be produced using FLK positive cells. First, FLK-positive cells are obtained by culturing pluripotent stem cells by any method, for example, the method described in Yamashita et al. (2000), Nature, 408: 92-96. Next, mixed cells of endothelial cells and mural cells are obtained by culturing FLK positive cells based on the description in the same document. Here, the endothelial cell means a cell exhibiting the same properties as a vascular endothelial cell (one cell inside the blood vessel) or a progenitor cell thereof. The wall cell means a cell exhibiting the same properties as a blood vessel wall cell (a cell surrounding a vascular endothelial cell from the outside) or a progenitor cell thereof.

<Process for manufacturing myocardial sheet>
In the production method 2 of the present invention, a myocardial sheet is produced by mixing and culturing cardiomyocytes, endothelial cells and wall cells. For example, after culturing FLK-positive cells for 1 to 7 days (for example, 3 days) on a temperature-sensitive culture vessel (for example, CellCell, Inc., UpCell), mixed cells of endothelial cells and wall cells, and an appropriate amount of cardiomyocytes Add to culture vessel. By culturing this cell mixture in a medium containing vascular endothelial growth factor (VEGF) for 1 to 10 days (for example, 4 days), a sheet-like cell structure is generated. The myocardial sheet thus formed can be separated from the culture container and collected by placing the culture container at room temperature. Further, the manufactured myocardial sheets are laminated as desired.

  The myocardial sheet thus produced can be used for the treatment of heart diseases such as heart failure, myocardial infarction, ischemic heart disease, myocarditis, various cardiomyopathy, and other applications. For example, regarding a heart disease such as myocardial infarction, a myocardial sheet is disposed so as to cover a desired part of the heart, and treatment is performed. The myocardial sheet is engrafted in the heart tissue and promotes functional recovery of the heart.

  The kit of the present invention includes a Wnt signal inhibitor and is particularly a kit that has a limited use such as “to induce a population of cardiomyocytes capable of maintaining a cTnT positive rate of 80% or more by culturing for at least 70 days”. Without being limited, it may further include a package insert, a pipette, a centrifuge tube and the like on which a ROCK inhibitor, a culture solution, and a method for inducing a cardiomyocyte population are described. The definitions of the terms “ROCK inhibitor”, “Wnt signal inhibitor”, and “culture medium” are as described in the above item <Step (c)>.

  EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

1. Method for Inducing Differentiation into Cardiomyocytes Human iPS cells (1201C1 and 253G1 strains) were received from Prof. Yamanaka of Kyoto University and reported previously (Uosaki H. et al. PLoS One 2011; 6: e23657) Maintenance culture of human iPS cells was performed using the same method. Details are as follows. Human iPS cells were seeded in a culture vessel coated with matrigel (growth factor reduced, 1:60 dilution, Corning), and conditioned medium (hereinafter referred to as MEF-CM) of mouse embryonic fibroblasts (MEF). And a medium supplemented with human basic fibroblast growth factor (hbFGF, manufactured by WAKO) so as to be 4 ng / mL. MEF-CM is 20% KSR (manufactured by GIBCO), 1 mM L-glutamine (manufactured by GIBCO), 0.1 mM 2-mercaptoethanol (manufactured by GIBCO), 1% Non-Essential Amino Acids (manufactured by GIBCO), Knockout DMEM (GIBCO) containing 4 ng / mL hbFGF was used as a basal medium, and prepared using MEF treated with mitomycin C (WAKO) for 2.5 hours. In the culture of human iPS cells using MEF-CM, a CTK solution (phosphate buffered saline containing 0.1% collagenase IV, 0.25% trypsin, 20% KSR and 1 mM CaCl ₂ is used every 4 to 6 days. The human iPS cell colonies were detached using water), subcultured after being made into a small cell mass with a cell scraper.

Subsequently, in order to efficiently induce differentiation into mesoderm cells, the following process (Matrigel sandwich method) was performed. Versene (manufactured by Invitrogen) was added to a culture vessel for human iPS cells subcultured by the above method, and incubated at 37 ° C. for 3 to 5 minutes. Versene was removed, MEF-CM was added, and the cells were completely detached from the culture vessel. Further, pipetting was performed to prepare a suspension in which the cell mass was dispersed, and then the number of cells was counted. Next, cells were seeded on a matrigel-coated culture vessel at a density of 70,000 to 105,000 cells / cm ² and cultured in MEF-CM medium containing 4 ng / mL hbFGF for 1-2 days. Thereafter, the medium was changed to a MEF-CM medium containing 1:60 diluted Matrigel and 4 ng / mL hbFGF, and further cultured for 16 to 24 hours to cover the upper layer of the cells with Matrigel.

  The resulting Matrigel-coated cells were induced to differentiate into mesoderm cells by the following procedure. First, the medium was changed to RPMI1640 medium (insulin-free, manufactured by GIBCO) containing 100 ng / mL activin A, 2 mM L-glutamine, and B27 supplement (manufactured by GIBCO). And cultured for 24 hours (Day 1). Next, the medium was changed to RPMI 1640 medium (without insulin) containing 10 ng / mL human bone morphogenetic protein 4 (hBMP4, manufactured by R & D), 10 ng / mL hbFGF, 2 mM L-glutamine, and B27 supplement, and cultured for 4 days. (Day 5).

PDGFRα-positive cells were separated from the culture on the fifth day after the start of differentiation induction (Day 5) using a magnetic cell separator (MACS, manufactured by Miltenyi biotech) to obtain cardiovascular progenitor cells. In separating PDGFRα-positive cells, an anti-PDGFRα antibody bound with an APC fluorescent label was used as a primary antibody for labeling cells, and the cells labeled with the primary antibody were collected using magnetic beads embedded with the anti-APC antibody. . When the ratio of PDGFRα positive cells (PDGFRα positive rate) present in cells separated by MACS was measured by flow cytometry, it was 89 to 99%. The obtained PDGFRα-positive cells were replated at a cell density of 300,000 to 420,000 cells / cm ² , 10% FBS, 50 units / mL penicillin, 50 μg / mL streptomycin, 2 mM L-glutamine, 20 μM Y-27632 ( ROMI inhibitor), RPMI1640 medium containing 10 μM XAV939 (Wnt inhibitor), 5 μM IWP-4 (Wnt inhibitor) was cultured in a CO ₂ incubator at 37 ° C. for 2 days.

  On day 7 after the start of differentiation induction (Day 7), the medium was replaced with RPMI 1640 medium containing 2 mM L-glutamine, B27 supplement, 20 μM Y-27632, 0.25 μM XAV939, 0.125 μM IWP-4, and cultured for 2 days. .

  From day 9 to day 21 (Day 9 to 21) after the start of differentiation induction (12 days), RPMI1640 medium containing 2 mM L-glutamine and B27 supplement was used, and culture was continued while the medium was changed every 2 days. did. The purity of the cardiomyocytes (cTnT positive rate) was measured for the cells 21 days after the start of differentiation induction (Day 21) by using flow cytometry using an anti-cTnT antibody, and it was 96 to 99%.

  Tables 1 and 2 show the results of cell analysis in Day 5 and Day 21 for 9 cardiomyocyte productions performed using the 1201C strain and 2 cardiomyocyte productions performed using the 253G1 strain, respectively. In both tables, 1201C1 and 253G1 in the experiment numbers indicate human iPS cell lines used in each experiment. As is apparent from Table 2, high purity cardiomyocytes were obtained in all the production examples.

2. Long-term culture of cardiomyocytes Cardiomyocytes cultured from the human iPS cells (836B3 strain) received from Professor Yamanaka of Kyoto University by the method described in Example 1 until the 21st day after the start of differentiation induction were prepared. The cardiomyocytes recovered from the culture vessel using 0.25% trypsin-EDTA, phenol red (GIBCO) were polylysine (5 μg / mL, SIGMA) at a density of about 200,000 cells / cm ^2. And then seeded on a culture vessel coated with gelatin (0.5%). The cardiomyocytes were cultured in αMEM containing 10% serum, 50 units / mL penicillin, 50 μg / mL streptomycin, 50 μM 2-mercaptoethanol, and 20 μM Y-27632 for 2 days in a CO ₂ incubator at 37 ° C. The medium was replaced with RPMI 1640 medium containing 2 mM L-glutamine and B27 supplement (23 days after initiation of differentiation induction). Thereafter, the culture was continued while changing the medium every two days.

Cardiomyocytes were sampled on days 21, 31, 41, 51, 61, and 91 after the initiation of differentiation, respectively, and flow cytometry using an anti-cTnT antibody was performed to measure the purity of the cardiomyocytes. As shown in FIG. 1, the cardiomyocytes prepared by the method described in Example 1 maintained a cTnT positive rate exceeding 95% until the 91st day after initiation of differentiation induction. Moreover, the result of having measured the number of living cells on a culture container is shown in FIG. No decrease in the number of cells was observed after 21 days from the start of differentiation induction.
On the other hand, when cells were cultured in RPMI 1640 medium not containing Y-27632 (ROCK inhibitor) for 5 to 9 days after initiation of differentiation induction, almost no living cells (adherent cells) were observed ( In addition, when RPMI1640 medium containing XAV939 or IWP-4 alone was used for 5-9 days after the start of differentiation induction, the ratio of cTnT positive cells (cardiomyocytes) was 74.9. % And 72.2% (refer to the second and third graphs from the left in FIG. 4). In addition, when RPMI1640 medium containing no Wnt inhibitors (XAV939 and IWP-4) was used, the ratio of cTnT positive cells (cardiomyocytes) was significantly reduced to 7.1% (from the left in FIG. 4). See the second graph).
Further, at least 11 days after the initiation of differentiation induction, the ratio of cTnT positive cells (cardiomyocytes) exceeded 90% (see FIG. 5).
Furthermore, from the 23rd day after the start of differentiation induction, a high cTnT positive rate was maintained when serum was used instead of B27 supplement (see FIG. 6).

  The above results show that when cardiovascular progenitor cells (PDGFRα-positive cells) are cultured in the presence of ROCK inhibitor and Wnt inhibitor at a specific time (5-9 days after differentiation induction treatment to mesoderm cells), It shows that differentiation can be efficiently induced into cardiomyocytes, and that cardiomyocytes are cultured and maintained for a long period of time (at least 80 days from 11 to 91 days after initiation of differentiation).

  The method of the present invention enables efficient and high yield production of cardiomyocytes capable of maintaining characteristics over a long period of time from pluripotent stem cells. Cardiomyocytes obtained by the method of the present invention can be used as a material for treatment of diseases requiring transplantation of cardiomyocytes, cell models of diseases caused by abnormalities of cardiomyocytes, etc., and safety evaluation of drugs. Can do. A myocardial sheet can be produced by mixing and culturing cardiomyocytes obtained by the method of the present invention with endothelial cells and wall cells. These myocardial cells and myocardial sheets are particularly useful in the field of regenerative medicine.

Claims (16)

A method for producing cardiomyocytes comprising the following steps (a) to (d):
(A) performing a differentiation-inducing treatment into mesoderm cells on the obtained pluripotent stem cells;
(B) a step of separating cardiovascular progenitor cells from the pluripotent stem cells subjected to differentiation induction in step (a);
(C) culturing the cardiovascular progenitor cells separated in step (b) in a culture solution containing a ROCK inhibitor and a Wnt signal inhibitor;
(D) a step of culturing the cells cultured in step (c) in a culture solution to prepare cardiomyocytes;   The production method according to claim 1, wherein the pluripotent stem cell is an induced pluripotent stem cell.   The production method according to claim 1 or 2, wherein the cardiovascular progenitor cells are PDGFRα-positive cells.   The method according to any one of claims 1 to 3, wherein in step (b), cardiovascular progenitor cells are separated on the 4th to 6th days after the differentiation induction treatment in step (a).   In the step (c), the cell is cultured for at least 6 to 7 days after the differentiation induction treatment in the step (a).   In the step (d), the cells are cultured until 8 to 22 days after the differentiation induction treatment in the step (a). Furthermore, the manufacturing method in any one of Claims 1-6 provided with the following processes (e) and (f).
(E) culturing the cardiomyocytes prepared in step (d) in a culture solution containing a ROCK inhibitor;
(F) culturing the cardiomyocytes cultured in step (e) in a culture solution;   In the step (e), the cell is cultured for at least 22 days after the differentiation induction treatment in the step (a).   In the step (f), the cells are cultured until 23 to 91 days after the differentiation induction treatment in the step (a).   The method according to any one of claims 1 to 9, wherein the ROCK inhibitor is Y-27632.   The production method according to any one of claims 1 to 10, wherein the Wnt signal inhibitor is XAV939 and / or IWP-4.   A cardiomyocyte prepared using the production method according to claim 1.   A method for producing a myocardial sheet, comprising mixing and culturing the cardiomyocytes according to claim 12 with endothelial cells and wall cells.   A population of cardiomyocytes capable of maintaining a cTnT positive rate of 80% or more by culturing for at least 70 days.   A kit for inducing a population of cardiomyocytes capable of maintaining a cTnT positive rate of 80% or more by culturing for at least 70 days, comprising a Wnt signal inhibitor.   The kit according to claim 15, wherein the Wnt signal inhibitor is XAV939 and / or IWP-4.