JPWO2017073794A1 - Method for producing three-dimensional myocardial tissue from pluripotent stem cells - Google Patents

Abstract

A method for producing myocardial tissue, comprising the following steps;
(A) producing cardiomyocytes from pluripotent stem cells;
(B) producing endothelial cells from pluripotent stem cells,
(C) a step of producing mural cells from pluripotent stem cells,
(D) mixing the wall cells produced in the step (c) with the cardiomyocytes produced in the step (a) and the endothelial cells produced in the step (b) at a ratio of less than 30%; e) A step of culturing the cell mixture obtained in the step (d) in the presence of an extracellular matrix to form a three-dimensional structure.

Description

Field of Invention

  The present invention relates to a method for producing a three-dimensional myocardial tissue from pluripotent stem cells and a therapeutic agent for heart disease containing the obtained myocardial tissue.

  Since adult cardiomyocytes hardly proliferate, cardiomyocytes deficient due to ischemic heart disease or the like cause irreversible damage. Currently, no treatment of any drug used clinically has shown efficacy in replacing myocardial scars with functional contractile tissue. Therefore, a novel treatment for regenerating normal cardiomyocytes is desired, and replacement therapy for administering separately produced cardiomyocytes has been proposed.

  In such replacement therapy, there have been proposed a method of administering cardiomyocytes in a sheet form (Non-patent Document 1) and a method of administering in a string form (Non-patent Document 2) for engraftment in the recipient's heart. Yes.

  Such cells used for replacement therapy have been shown to be more effective when administered not only as cardiac muscle cells but also as tissues containing vascular cells simultaneously (Non-Patent Documents 2, 3, 4). And 5 and patent documents 1 and 2).

  However, in the production of tissue suitable for transplantation, there is no report that mentions the content of vascular cells, particularly wall cells.

WO2012 / 133945 WO2013 / 137491

Shimizu, T, et al, Biomaterials 24, 2309-2316, 2003 S Fujimoto KL et al., Tissue Eng Part A. 2011; 17: 585-96 Shimizu T, et al. FASEB J. 20: 708-10, 2006 Caspi O, et al. Circ Res. 2007 Feb 2; 100 (2): 263-72. Masumoto H et al., Stem Cells. 2012 Jun; 30 (6): 1196-205.

  An object of the present invention relates to a method for producing a strong myocardial tissue containing pluripotent stem cell-derived cardiomyocytes, endothelial cells and mural cells, and a therapeutic agent for heart disease containing the obtained myocardial tissue. Therefore, an object of the present invention is to provide a stronger and functional myocardial tissue by combining cardiomyocytes, endothelial cells and mural cells derived from pluripotent stem cells.

  As a result of intensive studies to solve the above problems, the present inventors have mixed cardiomyocytes, endothelial cells, and mural cells by reducing the content of mural cells derived from pluripotent stem cells to less than 30%. It was found that the function and strength of the myocardial tissue produced in this way are high. Furthermore, it was confirmed that the cardiac function of heart disease model rats can be improved by transplanting this myocardial tissue. The present invention has been completed based on such findings.

That is, the present invention provides the following:
[1] A method for producing myocardial tissue, comprising the following steps;
(A) producing cardiomyocytes from pluripotent stem cells;
(B) producing endothelial cells from pluripotent stem cells,
(C) a step of producing mural cells from pluripotent stem cells,
(D) mixing the wall cells produced in the step (c) with the cardiomyocytes produced in the step (a) and the endothelial cells produced in the step (b) at a ratio of less than 30%; e) A step of culturing the cell mixture obtained in the step (d) in the presence of an extracellular matrix to form a three-dimensional structure.
[2] The method according to [1], wherein the wall cells produced in the step (c) in the step (d) are mixed at a ratio of 5% to 25%.
[3] The method according to [1] or [2], wherein the wall cells produced in the step (c) are mixed at a ratio of 15% in the step (d).
[4] The steps (a) and (b) are simultaneously performed using the same pluripotent stem cells, and cardiomyocytes and endothelial cells are produced from the pluripotent stem cells. The method according to any one of the above.
[5] The method according to [4], wherein the steps (a) and (b) include the following steps;
(I) culturing pluripotent stem cells in the presence of activin A and Wnt3a;
(Ii) a step of culturing the cells obtained in the step (i) in the presence of BMP (Bone Morphogenetic Protein) and bFGF; and (iii) a cell obtained in the step (ii) being VEGF (Vascular Endothelial Growth). Culturing in the presence of Factor).
[6] The method according to any one of [1] to [5], wherein the step (c) includes the following steps;
(I) culturing pluripotent stem cells in the presence of activin A and Wnt3a;
(Ii) culturing the cells obtained in the step (i) in the presence of BMP and bFGF, and (iii) culturing the cells obtained in the step (ii) in the absence of VEGF.
[7] The method according to [5] or [6], wherein the BMP is BMP4.
[8] The method according to any one of [1] to [7], wherein the extracellular matrix is an extracellular matrix containing type I collagen.
[9] The method according to any one of [1] to [8], wherein the three-dimensional structure in the step (e) is a cylindrical structure.
[10] The method according to any one of [1] to [9], wherein the pluripotent stem cell is a human iPS cell.
[11] A therapeutic agent for heart disease comprising the myocardial tissue obtained by the method according to [1] to [10].
[12] A method for treating heart disease, wherein the myocardial tissue obtained by the method according to [1] to [10] is applied to an infarcted site, damaged site, or damaged site of the heart.

FIG. 1a shows a protocol for producing cardiomyocytes (CM) and endothelial cells (EC) from pluripotent stem cells (left figure) and the obtained cell content (right figure). FIG. 1 b shows a protocol for producing mural cells (MC) from pluripotent stem cells (left figure) and the content ratio of the obtained cells (right figure). FIG. 2 was prepared by mixing cardiomyocytes and endothelial cells only (C + E), cardiomyocytes and mural cells only (C + M), or cardiomyocytes, endothelial cells and mural cells (C + E + M). The content rate of each cell in a myocardial tissue is shown. FIG. 3 was prepared by mixing cardiomyocytes and endothelial cells only (C + E), cardiomyocytes and mural cells only (C + M), or cardiomyocytes, endothelial cells and mural cells (C + E + M). The result of having evaluated the function of the myocardial tissue is shown. 3a shows the maximum follow-up frequency in each myocardial tissue, the left figure in FIG. 3b shows the measurement data of contractile force at frequencies of 3 Hz and 3.5 Hz, and the right figure shows each frequency for 2 Hz to 3.5 Hz. The result of having measured the contraction force in is shown. FIG. 4 was prepared by mixing cardiomyocytes and endothelial cells only (C + E), cardiomyocytes and mural cells only (C + M), or cardiomyocytes, endothelial cells and mural cells (C + E + M). The result of having evaluated the function of the myocardial tissue is shown. 4a shows the minimum contraction threshold in each myocardial tissue, the left figure of FIG. 4b shows the measurement data of the contractile force from 5V to 1V, and the right figure shows the result of measuring the contractile force at 3V with respect to 5V. Show. FIG. 5 was prepared by mixing cardiomyocytes and endothelial cells only (C + E), cardiomyocytes and mural cells only (C + M), or cardiomyocytes, endothelial cells and mural cells (C + E + M). The result of measuring the tissue strength of the myocardial tissue is shown. The left figure shows the result of measuring the transition of the passive tension of each myocardial tissue, and the right figure shows the result of calculating the Young's rate from the transition. FIG. 6 shows the results of evaluating the function of myocardial tissue (High MC) prepared by mixing 30% or more mural cells or myocardial tissue (Low MC) prepared by mixing 15% mural cells. Fig. 6a shows the contraction force of the myocardial tissue (left figure) and the contraction force per right number of myocardial cells (right figure) Fig. 6b shows the maximum follow-up frequency of the myocardial tissue (left figure) and the contraction force at 3.5 Hz. Figure 6c shows the minimum contraction threshold for myocardial tissue (left figure) and the contractile force at 3V versus 5V (right figure). FIG. 7 was prepared by mixing cardiomyocytes and endothelial cells only (C + E), cardiomyocytes and mural cells only (C + M), or cardiomyocytes, endothelial cells and mural cells (C + E + M). An electron microscope image of myocardial tissue is shown (photograph). FIG. 8 was prepared by mixing cardiomyocytes and endothelial cells only (C + E), cardiomyocytes and mural cells only (C + M), or cardiomyocytes, endothelial cells and mural cells (C + E + M). Immunostaining image of myocardial tissue against myocardial troponin T (left) (photo), distribution of angle of each myocardial cell axis with respect to tissue long axis direction (middle view), and angle of each myocardial cell axis with respect to tissue long axis direction The result of measuring the dispersion (right figure) is shown. FIG. 9a shows an outline of transplantation and evaluation of myocardial tissue to a myocardial infarction model rat. FIG. 9b shows myocardial tissue before transplantation (left) and myocardial tissue after transplantation (right) (photo). FIG. 9c shows the heart 28 days after transplantation (photo). FIG. 10a shows an echo image on the 28th day after transplantation (photograph) in a group in which the myocardial tissue was not transplanted (left diagram) and a group in which the myocardial tissue was transplanted (right diagram). FIG. 10 b shows the cardiac output per minute (left figure) before transplantation, 14 days after transplantation, and 28 days after transplantation in the group not transplanted with myocardial tissue (Sham) and the group transplanted with myocardial tissue (Tx). ) And the measured cardiac output (right figure). FIG. 11a shows a stained image (left figure: Masson trichrome stain, right figure: cTNT, HNA and DAPI stained images) of the heart transplanted group (photograph). FIG. 11 b shows immunostained images (cTNT, HNA and DAPI) at the transplant site of the group transplanted with myocardial tissue. FIG. 11 c shows immunostained images (vWF (green), HNA (red) and DAPI (blue)) at the transplantation site of the group transplanted with myocardial tissue.

The present invention is described in detail below.
The present invention provides a method for producing myocardial tissue, which comprises the following steps;
(A) producing cardiomyocytes from pluripotent stem cells;
(B) producing endothelial cells from pluripotent stem cells,
(C) a step of producing mural cells from pluripotent stem cells,
(D) mixing the wall cells produced in the step (c) with the cardiomyocytes produced in the step (a) and the endothelial cells produced in the step (b) at a ratio of less than 30%; e) A step of culturing the cell mixture obtained in the step (d) in the presence of an extracellular matrix to form a three-dimensional structure.
<Pluripotent stem cells>
Although a pluripotent stem cell is not specifically limited, For example, the following are mentioned.

(A) Embryonic stem cells Embryonic stem cells (ES cells) are established from the inner cell mass of early embryos (eg, blastocysts) of mammals such as humans and mice, and have the ability to proliferate through self-replication. It has stem cells.

  ES cells are embryonic stem cells derived from the inner cell mass of the blastocyst, the embryo after the morula, in the 8-cell stage of a fertilized egg, and have the ability to differentiate into any cell that constitutes an adult, so-called differentiation. And ability to proliferate by self-replication. ES cells were discovered in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292: 154-156), and then ES cell lines were also established in primates such as humans and monkeys (JA Thomson et al. al. (1998), Science 282: 1145-1147; JA Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92: 7844-7848; JA Thomson et al. (1996), Biol. Reprod ., 55: 254-259; JA Thomson and VS Marshall (1998), Curr. Top. Dev. Biol., 38: 133-165).

  ES cells can be established by removing an inner cell mass from a blastocyst of a fertilized egg of a subject animal and culturing the inner cell mass on a fibroblast feeder. In addition, maintenance of cells by subculture is performed using a culture solution supplemented with substances such as leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF). It can be carried out. For methods of establishing and maintaining human and monkey ES cells, see, for example, USP 5,843,780; Thomson JA, et al. (1995), Proc Natl. Acad. Sci. US A. 92: 7844-7848; Thomson JA, et al. (1998), Science. 282: 1145-1147; H. Suemori et al. (2006), Biochem. Biophys. Res. Commun., 345: 926-932; M. Ueno et al. (2006), Proc Natl. Acad. Sci. USA, 103: 9554-9559; H. Suemori et al. (2001), Dev. Dyn., 222: 273-279; H. Kawasaki et al. (2002), Proc. Natl. Acad. Sci. USA, 99: 1580-1585; Klimanskaya I, et al. (2006), Nature. 444: 481-485.

For example, DMEM / F-12 culture medium supplemented with 0.1 mM 2-mercaptoethanol, 0.1 mM non-essential amino acid, 2 mM L-glutamic acid, 20% KSR and 4 ng / ml bFGF is used as the culture medium for ES cell production. Human ES cells can be maintained in a humid atmosphere at 37 ° C., 5% CO ₂ (H. Suemori et al. (2006), Biochem. Biophys. Res. Commun., 345: 926-932). ES cells also need to be passaged every 3-4 days, where passage is eg 0.25% trypsin and 0.1 mg / ml collagenase IV in PBS containing 1 mM CaCl ₂ and 20% KSR. Can be used.

  In general, ES cells can be selected by Real-Time PCR using the expression of gene markers such as alkaline phosphatase, Oct-3 / 4, Nanog as an index. In particular, in the selection of human ES cells, expression of gene markers such as OCT-3 / 4, NANOG, ECAD and the like can be used as an index (E. Kroon et al. (2008), Nat. Biotechnol., 26: 443 -452).

  Human ES cell lines are obtained from, for example, WA01 (H1) and WA09 (H9) from the WiCell Research Institute, and KhES-1, KhES-2, and KhES-3 from Kyoto University Institute for Regenerative Medicine (Kyoto, Japan). Is possible.

(B) Sperm stem cells Sperm stem cells are testis-derived pluripotent stem cells that are the origin of spermatogenesis. Like ES cells, these cells can be induced to differentiate into various types of cells, and have characteristics such as the ability to create chimeric mice when transplanted into mouse blastocysts (M. Kanatsu-Shinohara et al. ( 2003) Biol. Reprod., 69: 612-616; K. Shinohara et al. (2004), Cell, 119: 1001-1012). It is capable of self-replication in a culture medium containing glial cell line-derived neurotrophic factor (GDNF), and by repeating subculture under the same culture conditions as ES cells, Stem cells can be obtained (Masatake Takebayashi et al. (2008), Experimental Medicine, Vol. 26, No. 5 (extra number), 41-46, Yodosha (Tokyo, Japan)).

(C) Embryonic germ cells Embryonic germ cells are cells that are established from embryonic primordial germ cells and have the same pluripotency as ES cells, such as LIF, bFGF, stem cell factor, etc. It can be established by culturing primordial germ cells in the presence of these substances (Y. Matsui et al. (1992), Cell, 70: 841-847; JL Resnick et al. (1992), Nature, 359: 550 -551).

(D) Artificial pluripotent stem cellAn artificial pluripotent stem (iPS) cell is an ES cell that can be produced by introducing a specific reprogramming factor into a somatic cell in the form of DNA, RNA or protein. Artificial stem cells derived from somatic cells with almost the same characteristics, such as differentiation pluripotency and proliferation ability by self-replication (K. Takahashi and S. Yamanaka (2006) Cell, 126: 663-676; K. Takahashi et al. (2007), Cell, 131: 861-872; J. Yu et al. (2007), Science, 318: 1917-1920; Nakagawa, M. et al., Nat. Biotechnol. 26: 101-106 (2008 ); International Publication WO 2007/069666). The reprogramming factor is a gene that is specifically expressed in ES cells, its gene product or non-coding RNA, a gene that plays an important role in maintaining undifferentiation of ES cells, its gene product or non-coding RNA, or It may be constituted by a low molecular compound. Examples of genes included in the reprogramming factor include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15 -2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3 or Glis1 etc. are exemplified, and these reprogramming factors may be used alone or in combination. As combinations of reprogramming factors, WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 075119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 / 101407, WO2009 / 102983, WO2009 / 114949, WO2009 / 117439, WO2009 / 126250, WO2009 / 126251, WO2009 / 126655, WO2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 050626, WO 2010/056831, WO2010 / 068955, WO2010 / 098419, WO2010 / 102267, WO 2010/111409, WO 2010/111422, WO2010 / 115050, WO2010 / 124290, WO2010 / 147395, WO2010 / 147612, Huangfu D, et al. 2008), Nat. Biotechnol., 26: 795-797, Shi Y, et al. (2008), Cell Stem Cell, 2: 525-528, Eminli S, et al. (2008), Stem Cells. 26: 2467 -2474, Huangfu D, et al. (2008), Nat Biotechnol. 26: 1269-1275, Shi Y, et al. (2008), Cell Stem Cell, 3, 568-574, Zhao Y, et al. (2008 ), Cell Stem Cell, 3: 475-479, Marson A, (2008), Cell Stem Cell, 3, 132-135, Feng B, et al. (2009), Nat Cell Biol. 11: 197-203, RL Judson et al., (2009), Nat. Biotech., 27: 459-461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci US A. 106: 8912-8917, Kim JB, et al. 2009), Nature. 461: 649-643, Ichida JK, et al. (2009), Cell Stem Cell. 5: 491-503, Heng JC, et al. (2010), Cell Stem Cell. 6: 167-74 , Han J, et al. (2010), Nature. 463: 1096-100, Mali P, et al. (2010), Stem Cells. 28: 713-720, Maekawa M, et al. (2011), Nature. 474: 225-9. Is exemplified.

  The reprogramming factors include histone deacetylase (HDAC) inhibitors [eg small molecule inhibitors such as valproic acid (VPA), trichostatin A, sodium butyrate, MC 1293, M344, siRNA and shRNA against HDAC (eg Nucleic acid expression inhibitors such as HDAC1 siRNA Smartpool (registered trademark) (Millipore), HuSH 29mer shRNA Constructs against HDAC1 (OriGene), etc.], MEK inhibitors (eg, PD184352, PD98059, U0126, SL327 and PD0325901), Glycogen synthase kinase-3 inhibitors (eg, Bio and CHIR99021), DNA methyltransferase inhibitors (eg, 5-azacytidine), histone methyltransferase inhibitors (eg, small molecule inhibitors such as BIX-01294, Suv39hl, Suv39h2, Nucleic acid expression inhibitors such as siRNA and shRNA against SetDBl and G9a), L-channel calcium agonist (eg Bayk8644), butyric acid, TGFβ inhibitor or ALK5 inhibitor (eg LY364947, SB431542) 616453 and A-83-01), p53 inhibitors (eg siRNA and shRNA against p53), ARID3A inhibitors (eg siRNA and shRNA against ARID3A), miR-291-3p, miR-294, miR-295 and mir- MiRNA such as 302, Wnt Signaling (eg soluble Wnt3a), neuropeptide Y, prostaglandins (eg prostaglandin E2 and prostaglandin J2), hTERT, SV40LT, UTF1, IRX6, GLISL, PITX2, DMRTBl, etc. Factors used for the purpose of improving establishment efficiency are also included. In this specification, factors used for the purpose of improving establishment efficiency are not distinguished from initialization factors. .

  In the form of a protein, the reprogramming factor may be introduced into a somatic cell by a technique such as lipofection, fusion with a cell membrane-permeable peptide (for example, TAT and polyarginine derived from HIV), or microinjection.

  On the other hand, in the case of DNA, it can be introduced into somatic cells by techniques such as vectors such as viruses, plasmids, artificial chromosomes, lipofection, liposomes, and microinjection. Examples of viral vectors include retroviral vectors and lentiviral vectors (above, Cell, 126, pp.663-676, 2006; Cell, 131, pp.861-872, 2007; Science, 318, pp.1917-1920, 2007 ), Adenovirus vectors (Science, 322, 945-949, 2008), adeno-associated virus vectors, Sendai virus vectors (WO 2010/008054) and the like. Examples of artificial chromosome vectors include human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), and bacterial artificial chromosomes (BAC, PAC). As a plasmid, a plasmid for mammalian cells can be used (Science, 322: 949-953, 2008). The vector can contain regulatory sequences such as a promoter, enhancer, ribosome binding sequence, terminator, polyadenylation site, etc. so that a nuclear reprogramming substance can be expressed. Selective marker sequences such as kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, thymidine kinase gene, diphtheria toxin gene, reporter gene sequences such as green fluorescent protein (GFP), β-glucuronidase (GUS), FLAG, etc. Can be included. In addition, the above vector has a LoxP sequence before and after the introduction of the gene into a somatic cell in order to excise the gene or promoter encoding the reprogramming factor and the gene encoding the reprogramming factor that binds to it. May be.

  In the case of RNA, it may be introduced into somatic cells by techniques such as lipofection and microinjection, and RNA containing 5-methylcytidine and pseudoridine (TriLink Biotechnologies) is used to suppress degradation. (Warren L, (2010) Cell Stem Cell. 7: 618-630).

  As a culture solution for iPS cell induction, for example, DMEM, DMEM / F12 or DME culture solution containing 10 to 15% FBS (in addition to LIF, penicillin / streptomycin, puromycin, L- Glutamine, non-essential amino acids, β-mercaptoethanol, etc. may be included as appropriate.) Or a commercially available culture medium (for example, a culture medium for mouse ES cell culture (TX-WES culture medium, Thrombo X), primate ES Cell culture medium (primate ES / iPS cell culture medium, Reprocell), serum-free medium (mTeSR, Stemcell Technology)] and the like.

As an example of the culture method, for example, in the presence of 5% CO ₂ at 37 ° C., the somatic cell and the reprogramming factor are brought into contact with DMEM or DMEM / F12 containing 10% FBS for about 4 to 7 days. Then, re-spread the cells on feeder cells (for example, mitomycin C-treated STO cells, SNL cells, etc.), and use bFGF-containing primate ES cell culture medium about 10 days after contact between the somatic cells and the reprogramming factor. Culturing and generating iPS-like colonies about 30 to about 45 days or more after the contact.

Alternatively, 10% FBS-containing DMEM medium (including LIF, penicillin / streptomycin, etc.) on feeder cells (eg, mitomycin C-treated STO cells, SNL cells, etc.) in the presence of 5% CO ₂ at 37 ° C. Can be suitably included with puromycin, L-glutamine, non-essential amino acids, β-mercaptoethanol, etc.) and can generate ES-like colonies after about 25 to about 30 days or more . Desirably, instead of feeder cells, somatic cells to be reprogrammed themselves are used (Takahashi K, et al. (2009), PLoS One. 4: e8067 or WO2010 / 137746), or extracellular matrix (eg, Laminin- 5 (WO2009 / 123349) and Matrigel (BD)) are exemplified.

  In addition, a method of culturing using a medium not containing serum is also exemplified (Sun N, et al. (2009), Proc Natl Acad Sci USA 106.15720-15725). Furthermore, in order to increase establishment efficiency, iPS cells may be established under hypoxic conditions (oxygen concentration of 0.1% or more and 15% or less) (Yoshida Y, et al. (2009), Cell Stem Cell. 5: 237 -241 or WO2010 / 013845).

During the culture, the culture medium is exchanged with a fresh culture medium once a day from the second day onward. The number of somatic cells used for nuclear reprogramming is not limited, but ranges from about 5 × 10 ³ to about 5 × 10 ⁶ cells per 100 cm ^{2 of} culture dish.

  iPS cells can be selected according to the shape of the formed colonies. On the other hand, when a drug resistance gene that is expressed in conjunction with a gene that is expressed when somatic cells are initialized (for example, Oct3 / 4, Nanog) is introduced as a marker gene, a culture solution containing the corresponding drug (selection The established iPS cells can be selected by culturing with the culture medium. In addition, if the marker gene is a fluorescent protein gene, iPS cells are selected by observing with a fluorescence microscope, in the case of a luminescent enzyme gene, by adding a luminescent substrate, and in the case of a chromogenic enzyme gene, by adding a chromogenic substrate can do.

  As used herein, the term “somatic cell” refers to any animal cell (preferably, a mammalian cell including a human) except a germ line cell such as an egg, oocyte, ES cell, or totipotent cell. Say. Somatic cells include, but are not limited to, fetal (pup) somatic cells, neonatal (pup) somatic cells, and mature healthy or diseased somatic cells. , Passage cells, and established cell lines. Specifically, somatic cells include, for example, (1) neural stem cells, hematopoietic stem cells, mesenchymal stem cells, tissue stem cells such as dental pulp stem cells (somatic stem cells), (2) tissue progenitor cells, (3) lymphocytes, epithelium Cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, hepatocytes, gastric mucosal cells, enterocytes, spleen cells, pancreatic cells (exocrine pancreas cells, etc.), brain cells, lung cells, kidney cells Examples thereof include differentiated cells such as fat cells.

  When iPS cells are used as a material for transplantation cells, it is desirable to use somatic cells having the same or substantially the same HLA genotype of the transplant destination individual from the viewpoint of preventing rejection. Here, “substantially the same” means that the HLA genotype matches the transplanted cells to such an extent that an immune response can be suppressed by an immunosuppressive agent. For example, HLA-A, HLA-B And somatic cells having an HLA type in which 3 loci of HLA-DR or 4 loci plus HLA-C are matched.

(E) ES cell derived from a cloned embryo obtained by nuclear transfer An ES cell derived from a cloned embryo obtained by nuclear transfer (nt ES cell) is an ES cell derived from a cloned embryo produced by nuclear transfer technology, It has almost the same properties as ES cells derived from fertilized eggs (T. Wakayama et al. (2001), Science, 292: 740-743; S. Wakayama et al. (2005), Biol. Reprod., 72 : 932-936; J. Byrne et al. (2007), Nature, 450: 497-502). That is, an ES cell established from an inner cell mass of a clonal embryo-derived blastocyst obtained by replacing the nucleus of an unfertilized egg with the nucleus of a somatic cell is an nt ES (nuclear transfer ES) cell. For the production of nt ES cells, a combination of nuclear transfer technology (JB Cibelli et al. (1998), Nature Biotechnol., 16: 642-646) and ES cell production technology is used (Kiyaka Wakayama et al. ( 2008), Experimental Medicine, 26, No. 5 (extra number), 47-52). Nuclear transfer can be initialized by injecting a somatic cell nucleus into a mammal's enucleated unfertilized egg and culturing for several hours.

(F) Multilineage-differentiating Stress Enduring cells
Multilineage-differentiating Stress Enduring cells (Muse cells) are pluripotent stem cells produced by the method described in WO2011 / 007900. Specifically, fibroblasts or bone marrow stromal cells are treated with trypsin for a long time. Preferably, it is a pluripotent cell obtained by trypsin treatment for 8 hours or 16 hours and then suspension culture, and is positive for SSEA-3 and CD105.

  In the present invention, preferred pluripotent stem cells are human iPS cells.

<Cardiomyocyte production method>
In the present invention, cardiomyocytes mean cells expressing at least cardiac troponin (cTnT) or αMHC. cTnT is exemplified by NCBI accession number NM_000364 for humans and NM_001130174 for mice. αMHC is exemplified by NCBI accession number NM_002471 for humans, and NM_001164171 for mice.

The method for inducing cardiomyocytes from pluripotent stem cells is not particularly limited as long as it is a known method. For example, (1) a method performed in the absence of feeder cells, and (2) in the presence of feeder cells The method of performing is illustrated.
In the present invention, (1) as a method for inducing cardiomyocytes from pluripotent stem cells in the absence of feeder cells, (i) a step of culturing induced pluripotent stem cells in a medium containing Activin A, and (ii) ) After step (i), a step of further culturing in a medium containing BMP4 and bFGF is exemplified.

(1) A method for inducing cardiomyocytes from pluripotent stem cells in the absence of feeder cells
(i) Step of culturing in medium containing Activin A In this step, pluripotent stem cells may be isolated by any method and cultured by suspension culture, or adherently cultured using a coated culture dish. Also good. Preferably, it is an adhesion culture. Here, as a separation method, a separation solution having mechanical, protease activity and collagenase activity (for example, Accutase (TM) and Accumax (TM)) or a separation solution having only collagenase activity may be used. . Preferably, it is a method of dissociating using a separation solution having only collagenase activity and separating finely mechanically. Here, as the pluripotent stem cell to be used, it is preferable to use a colony cultured until it becomes about 80% confluent with respect to the used dish.

  Suspension culture means culturing cells in a non-adherent state on a culture dish, and is not particularly limited, but is artificially treated (for example, coated with an extracellular matrix or the like) for the purpose of improving adhesion to cells. It can be carried out using a material that has not been treated) or a material that has been artificially inhibited from adhesion (for example, a coating treatment with polyhydroxyethyl methacrylic acid (poly-HEMA)).

  Adhesion culture is a culture method performed in an arbitrary medium in a coated culture dish. Examples of the coating agent include matrigel (BD), collagen, gelatin, laminin, heparan sulfate proteoglycan, entactin, and combinations thereof. Matrigel is preferable. More preferably, in the adhesion culture by the Matrigel sandwich method in which the pluripotent stem cells are coated with Matrigel by adding the Matrigel to the culture dish coated with Matrigel and then adding Matrigel into the medium. is there.

  The medium in this step (i) can be prepared by adding Activin A to a basal medium that is used for culturing animal cells.

  Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Doulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixed media thereof. Etc. are included. Preferably, RPMI 1640 medium. The basal medium may contain serum or may be serum-free. Serum substitutes such as albumin, transferrin, Knockout Serum Replacement (KSR) (FBS serum substitute for ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, It may contain one or more serum replacements such as collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins It may also contain one or more substances such as antibiotics, antioxidants, pyruvate, buffers, inorganic salts. As a preferred basal medium in this step (i), RPMI medium containing L-glutamine and B27 supplement is exemplified.

  In addition to Activin A, the medium in this step (i) is a growth factor comprising Wnt1, Wnt3, Wnt3a, Wnt4, Wnt7a, TGF-β, Nodal, BMP2, BMP4, BMP6, BMP7, GDF, bFGF and VEGF One or more may be added to the basal medium. A preferred growth factor is Wnt3a.

  The concentration of Activin A added to the medium is, for example, 10 ng / mL, 25 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 110 ng / mL, 120 ng / mL, 130 ng / mL, 140 ng / mL, 150 ng / mL, 175 ng / mL or 200 ng / mL, but not limited thereto. Preferably, the concentration of Activin A added to the medium is 100 ng / mL.

  The concentration of Wnt3a added to the medium is, for example, 10 mg / mL, 25 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 110 mg / mL, 120 mg / mL. It is not limited to mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 175 mg / mL or 200 mg / mL. Preferably, the concentration of Wnt3a added to the medium is 100 mg / mL.

The culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C. The culture is performed in an atmosphere of CO ₂ -containing air, and the CO ₂ concentration is preferably about 2 to 5%. is there. The culture time is, for example, 1 day to 5 days, preferably 1 day.

(ii) Step of culturing in a medium containing BMP and bFGF In this step (ii), when the previous step was carried out in suspension culture, the obtained cell population was directly coated in a culture dish coated with any medium. You may culture in. Examples of the coating agent include matrigel (BD), collagen, gelatin, laminin, heparan sulfate proteoglycan, entactin, and combinations thereof. Matrigel is preferable. Alternatively, when the previous step is performed by adhesion culture, the culture may be continued by exchanging the medium.

The medium used in this step (ii) can be prepared by adding BMP and bFGF to a basal medium that is used for culturing animal cells.
As the basal medium, the same medium as in step (i) described above can be used.

  As the BMP used in this step (ii), BMP belonging to the TGFβ superfamily is preferable, and BMP2, BMP4, and BMP7 are exemplified. A preferred BMP is BMP4.

  The concentration of BMP4 added to the medium is, for example, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 2.5 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL , 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 17.5 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL or 50 ng / mL. It is not limited. Preferably, the concentration of BMP4 added to the medium is 10 ng / mL.

  The concentration of bFGF added to the medium is, for example, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 2.5 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL , 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 17.5 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL or 50 ng / mL. It is not limited. Preferably, the concentration of bFGF added to the medium is 10 ng / mL.

The culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C. The culture is performed in an atmosphere of CO ₂ -containing air, and the CO ₂ concentration is preferably about 2 to 5%. is there. The culture time is, for example, 1 day to 10 days, preferably 4 days.

(2) Method Performed in the Presence of Feeder Cells In the present invention, as a method for inducing cardiomyocytes from 2) pluripotent stem cells in the presence of feeder cells, OP9 cells (Nishikawa, SI et al, Development 125, 1747- 1757 (1998)) or END-2 cells (Mummery C, et al, Circulation. 107: 2733-40 (2003)) and pluripotent stem cells or Fllk1-positive cells derived from pluripotent stem cells Is done.

  A medium for co-culture with feeder cells can be prepared by appropriately adding additives using a medium used for culturing animal cells as a basal medium.

  Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Doulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixed media thereof. Etc. are included. Preferably, RPMI 1640 medium. The basal medium may contain serum or may be serum-free. Serum substitutes such as albumin, transferrin, Knockout Serum Replacement (KSR) (FBS serum substitute for ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, It may contain one or more serum replacements such as collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins It may also contain one or more substances such as antibiotics, antioxidants, pyruvate, buffers, inorganic salts. Preferred basal media include αMEM medium containing 10% FBS, αMEM medium containing 10% FBS, and DMEM medium containing 10% FBS.

  Examples of additives to the basal medium in co-culture with feeder cells include 1 to 3 μg / mL of cyclosporin A, activin A and BMP4.

The culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C. The culture is performed in an atmosphere of CO ₂ -containing air, and the CO ₂ concentration is preferably about 2 to 5%. is there. The culture time is the number of days required for cardiac troponin and / or αMHC to be expressed, for example, 10 to 20 days.

  As a preferable condition, after culturing in αMEM medium containing 10% FBS for 4 days, Flk1-positive cells are isolated and co-cultured with OP9 cells for 6 days using αMEM medium containing 3 μg / mL cyclosporin A and 10% FBS. Or a condition of co-culturing with END-2 cells for 16 days using a DMEM medium containing 10% FBS.

  For use in producing the myocardial tissue of the present invention, the obtained cardiomyocytes may be further cultured in a basal medium supplemented with VEGF.

  Basal media to which VEGF is added include, for example, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Doulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and These mixed media are included. Preferably, RPMI 1640 medium. The basal medium may contain serum or may be serum-free. Serum substitutes such as albumin, transferrin, Knockout Serum Replacement (KSR) (FBS serum substitute for ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, It may contain one or more serum replacements such as collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins It may also contain one or more substances such as antibiotics, antioxidants, pyruvate, buffers, inorganic salts. As a preferred basal medium in this step (i), RPMI medium containing L-glutamine and B27 supplement is exemplified.

  The concentration of VEGF added to the medium is, for example, 10 ng / mL to 500 ng / mL, 25 ng / mL to 300 ng / mL, 40 ng / mL to 200 ng / mL, 50 ng / mL to 100 ng / mL, 60 ng / mL to 90 ng / It can be in the range of mL or 65 ng / mL to 85 ng / mL. Preferably, the concentration of VEGF added to the medium is 50 ng / mL to 100 ng / mL. The concentration of VEGF added to the medium is 10 ng / mL, 25 ng / mL, 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / It may be, but is not limited to, mL, 90 ng / mL, 95 ng / mL, 100 ng / mL, 110 ng / mL, 120 ng / mL, 130 ng / mL, 140 ng / mL, 150 ng / mL or 200 ng / mL. Preferably, the concentration of VEGF added to the medium is 75 ng / mL.

The culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C. The culture is performed in an atmosphere of CO ₂ -containing air, and the CO ₂ concentration is preferably about 2 to 5%. is there. The culture time is, for example, 4 days to 20 days, preferably 10 days.

<Endothelial cell production method>
In the present invention, endothelial cells mean cells expressing any one of PE-CAM, VE-cadherin and von Willebrand factor (vWF). The PE-CAM is exemplified by NCBI accession number NM_000442 for humans, and NM_001032378 for mice. VE-cadherin is exemplified by NCBI accession number NM_001795 for humans and NM_009868 for mice. As for vWF, NCBI accession number NM_000552 is exemplified for humans, and NM_011708 is exemplified for mice.

  The method for inducing endothelial cells from pluripotent stem cells is not particularly limited as long as it is a known method.For example, (a) a step of culturing pluripotent stem cells in a medium containing Activin A and Wnt3a, (b ) A method comprising culturing the cells obtained in step (a) in a medium containing BMP and bFGF, (c) culturing the cells obtained in step (b) in a medium containing VEGF. Illustrated.

  In steps (a), (b) and (c), the same method as the method for inducing cardiomyocytes described above may be used. Therefore, in the present invention, a method for simultaneously inducing cardiomyocytes and endothelial cells including the steps (a), (b) and (c) can be used.

  In the present invention, cAMP for inducing endothelial cells may be further added in step (c). The concentration of cAMP is, for example, in the range of more than 0.5 mM and less than 2 mM, for example, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, Although it is 1.6mM, 1.7mM, 1.8mM, 1.9mM, it is not limited to these. Preferably, it is 1 mM. The period for adding cAMP is not particularly limited, but is preferably 1 to 5 days, and particularly preferably 3 days.

<Wall cell manufacturing method>
In the present invention, the mural cell means a cell expressing Smooth muscle actin (SMA) and / or PDGFRB. SMA is exemplified by NCBI accession number NM_001141945 for humans and NM_007392 for mice. PDGFRB is exemplified by NCBI accession number NM_002609 for humans, and NM_001146268 for mice.

  The method of inducing endothelial cells from pluripotent stem cells is not particularly limited as long as it is a known method. For example, (I) a step of culturing pluripotent stem cells in a medium containing Activin A, step (II) Examples include a step of culturing the cells obtained in (I) in a medium containing BMP and bFGF, and (III) culturing the cells obtained in step (II) in a medium not containing VEGF.

(I) Step of culturing in a medium containing Activin A In this step, pluripotent stem cells may be isolated by any method and cultured by suspension culture, or adherently cultured using a coated culture dish. Also good. Preferably, it is an adhesion culture. Here, as a separation method, a separation solution having mechanical, protease activity and collagenase activity (for example, Accutase (TM) and Accumax (TM)) or a separation solution having only collagenase activity may be used. . Preferably, it is a method of dissociating using a separation solution having only collagenase activity and separating finely mechanically. Here, the pluripotent stem cell used is preferably a colony cultured until it becomes 80% confluent with respect to the used dish.

  Suspension culture means culturing cells in a non-adherent state on a culture dish, and is not particularly limited, but is artificially treated (for example, coated with an extracellular matrix or the like) for the purpose of improving adhesion to cells. It can be carried out using a material that has not been treated) or a material that has been artificially inhibited from adhesion (for example, a coating treatment with polyhydroxyethyl methacrylic acid (poly-HEMA)).

  Adhesion culture is a culture method performed in an arbitrary medium in a coated culture dish. Examples of the coating agent include matrigel (BD), collagen, gelatin, laminin, heparan sulfate proteoglycan, entactin, and combinations thereof. Matrigel is preferable. More preferably, in the adhesion culture by the Matrigel sandwich method in which the pluripotent stem cells are coated with Matrigel by adding the Matrigel to the culture dish coated with Matrigel and then adding Matrigel into the medium. is there.

  The medium in this step (I) can be prepared by adding Activin A to a basal medium that is used for culturing animal cells.

  Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixed media thereof. Etc. are included. Preferably, RPMI 1640 medium. The basal medium may contain serum or may be serum-free. Serum substitutes such as albumin, transferrin, Knockout Serum Replacement (KSR) (FBS serum substitute for ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, It may contain one or more serum replacements such as collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins It may also contain one or more substances such as antibiotics, antioxidants, pyruvate, buffers, inorganic salts. A preferable basal medium in this step (I) is exemplified by RPMI medium containing L-glutamine and B27 supplement.

  In addition to Activin A, the medium in this step (I) is a growth factor comprising Wnt1, Wnt3, Wnt3a, Wnt4, Wnt7a, TGF-β, Nodal, BMP2, BMP4, BMP6, BMP7, GDF, bFGF and VEGF One or more may be added to the basal medium. A preferred growth factor is Wnt3a.

  The concentration of Activin A added to the medium is, for example, 10 ng / mL, 25 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 110 ng / mL, 120 ng / mL, 130 ng / mL, 140 ng / mL, 150 ng / mL, 175 ng / mL or 200 ng / mL, but not limited thereto. Preferably, the concentration of Activin A added to the medium is 100 ng / mL.

  The concentration of Wnt3a added to the medium is, for example, 10 mg / mL, 25 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 110 mg / mL, 120 mg / mL. It is not limited to mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 175 mg / mL or 200 mg / mL. Preferably, the concentration of Wnt3a added to the medium is 100 mg / mL.

The culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C. The culture is performed in an atmosphere of CO ₂ -containing air, and the CO ₂ concentration is preferably about 2 to 5%. is there. The culture time is, for example, 1 day to 5 days, preferably 1 day.

(II) Step of culturing in a medium containing BMP and bFGF In this step (II), if the previous step was carried out in suspension culture, the obtained cell population was used as it was in a culture dish coated with any medium. You may culture in. Examples of the coating agent include matrigel (BD), collagen, gelatin, laminin, heparan sulfate proteoglycan, entactin, and combinations thereof. Matrigel is preferable. Alternatively, when the previous step is performed by adhesion culture, the culture may be continued by exchanging the medium.

The medium used in this step (II) can be prepared by adding BMP and bFGF to a basal medium that is used for culturing animal cells.
As the basal medium, the same medium as in step (I) described above can be used.

  As the BMP used in this step (ii), BMP belonging to the TGFβ superfamily is preferable, and BMP2, BMP4, and BMP7 are exemplified. A preferred BMP is BMP4.

  The concentration of BMP4 added to the medium is, for example, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 2.5 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL , 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 17.5 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL or 50 ng / mL. It is not limited. Preferably, the concentration of BMP4 added to the medium is 10 ng / mL.

  The concentration of bFGF added to the medium is, for example, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 2.5 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL , 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 17.5 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL or 50 ng / mL. It is not limited. Preferably, the concentration of bFGF added to the medium is 10 ng / mL.

The culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C. The culture is performed in an atmosphere of CO ₂ -containing air, and the CO ₂ concentration is preferably about 2 to 5%. is there. The culture time is, for example, 1 day to 10 days, preferably 2 days.

(III) A step of culturing in a medium not containing VEGF The cells obtained in the above step (II) are cultured in a basal medium as a medium used for culturing animal cells not containing VEGF. The medium that does not contain VEGF may be a medium that does not substantially contain VEGF. For example, the medium has a VEGF concentration of less than 1 ng / mL, preferably less than 0.1 ng / mL, and more preferably 0.

  The basal medium used in this step (III) is, for example, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Doulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium , And mixed media thereof. Preferably, RPMI 1640 medium. The basal medium may contain serum or may be serum-free. Serum substitutes such as albumin, transferrin, Knockout Serum Replacement (KSR) (FBS serum substitute for ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, It may contain one or more serum replacements such as collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins It may also contain one or more substances such as antibiotics, antioxidants, pyruvate, buffers, inorganic salts. As a preferred basal medium used in this step (III), an RPMI medium containing 10% FBS is exemplified.

<Method of forming myocardial tissue>
In the present invention, the myocardial tissue means a three-dimensional structure including cardiomyocytes, endothelial cells, and wall cells, and the three-dimensional structure is a structure that repeatedly contracts and expands.

  In the present invention, the myocardial tissue can be obtained by mixing the above-described cardiomyocytes, endothelial cells and wall cells and culturing them in the presence of an extracellular matrix to form a three-dimensional structure. It is preferred that the wall cells are mixed at a content of less than 30%, more preferably the wall cells are mixed in the range of 5% to 25%, more preferably the wall cells are from 5%. Mix at 15%, most preferably the wall cells are mixed at 15%.

  In the present invention, the extracellular matrix means a protein secreted extracellularly, and examples of the extracellular matrix include collagen, gelatin, laminin, heparan sulfate proteoglycan, entactin, fragments thereof, and combinations thereof. Is done. The extracellular matrix used in the formation of the myocardial tissue is not particularly limited, but is an extracellular matrix containing at least type I collagen, and more preferably Matrigel containing type I collagen (available for purchase from BD).

  The medium used for the formation of myocardial tissue is, for example, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Doulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, And mixed media thereof and the like. Preferably, it is a DMEM medium. The basal medium may contain serum or may be serum-free. Serum substitutes such as albumin, transferrin, Knockout Serum Replacement (KSR) (FBS serum substitute for ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, It may contain one or more serum replacements such as collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins It may also contain one or more substances such as antibiotics, antioxidants, pyruvate, buffers, inorganic salts. A preferable medium used for the formation of myocardial tissue is exemplified by a DMEM medium containing 20% FBS.

  Formation of the myocardial tissue can be performed by mixing the above-described culture medium in which the mixed cells are suspended and the extracellular matrix, placing the mixture in a container having a desired shape, and allowing a certain period of time to pass. Since the container is easy to handle myocardial tissue, it is desirable that the contact area with the cell mixture is covered with a silicone film coated with type I collagen. Such containers are purchased from Flexcell International. it can. The shape of the container is preferably a columnar shape, and more preferably a cylindrical shape, from the viewpoint of ease of handling the formed myocardial tissue.

  The formed myocardial tissue can be stored in any medium. Examples of such a medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Doulbecco's modified Eagle's Medium ( DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixed media thereof. Preferably, it is a DMEM medium. The basal medium may contain serum or may be serum-free. Serum substitutes such as albumin, transferrin, Knockout Serum Replacement (KSR) (FBS serum substitute for ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, It may contain one or more serum replacements such as collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins It may also contain one or more substances such as antibiotics, antioxidants, pyruvate, buffers, inorganic salts. A preferable medium for culturing myocardial tissue is exemplified by αMEM medium containing 10% FBS, 2-mercaptoethanol and antibiotics.

  The myocardial tissue is preferably used after 7 days or more have passed since it is formed, and more preferably used after 14 days or more have passed.

<Health disease treatment agent>
The present invention provides a therapeutic agent for heart disease comprising the above-described myocardial tissue. The heart disease to which the present invention is applied is not particularly limited as long as it is a disease state in which cardiomyocytes or the like are deficient. Examples include hypertrophic cardiomyopathy and dilated cardiomyopathy.

  The myocardial tissue of the present invention may be used by directly sewing the myocardial tissue to the defective part, infarcted part, damaged part or damaged part of the myocardial cell in the formed shape, for adhesion of organs such as fibrin glue. You may transplant using the biological paste to be used.

  The myocardial tissue used for treating heart disease may be prepared by appropriately increasing or decreasing according to the size of the affected part or the size of the body.

  The present invention will be described more specifically with reference to the following examples. However, the scope of the present invention is not limited to these examples.

Human iPS cell culture method Human iPS cells (literature and establishment literature) were obtained from the iPS Cell Research Institute of Kyoto University and used.

Human iPS cell culture is treated with Mitomycin-C (MMC) (WAKO) in basal medium for 2.5 hours in a FALCON culture dish (10 cm) coated with Matrigel (growth factor reduced, 1:60 dilution, Corning). Incubation was performed using a medium in which 4 ng / mL recombinant human bFGF (rhbFGF, WAKO) was added to a supernatant (hereinafter referred to as MEF-CM) in which mouse embryonic fibroblasts (MEF) were cultured. Basal medium is Knockout DMEM (Life technologies) 471 mL, Knockout serum replacement (KSR) (Life technologies) 120 mL, NEAA (Sigma-Aldrich) 6 mL, 200 mM L-Glutamine (Life technologies) 3 mL and 55 mM 2-ME (mercaptoethanol) Made by mixing (Life technologies). Human iPS cells were passaged by small clump every 4-6 days. Passage is CTK solution [0.1% Collagenase IV (Life technologies), 0.25% Trypsin (Life technologies), 20% KSR, and 1 mM CaCl ₂ (Sigma-Aldrich) in Phosphate buffered saline (PBS) (Life technologies)] After peeling around the cell colony, the cell strainer was used to form a small clump.

Cardiomyocyte / vascular endothelial cell simultaneous induction method (Figure 1a)
Confluent human iPS cells were detached by incubation at 37 ° C. for 3-5 minutes with Versene (Life technologies). After aspirating Versene, pipetting with MEF-CM, collecting with single cells, and then centrifuging to count the number of cells. About 3 × 10 ⁶ human iPS cells were recovered per 10 cm dish. The matrigel coat was rewound at about 1 × 10 ⁵ cells / cm ² . When 6 well plate was used, 1 × 10 ⁶ cells were seeded per well. A medium in which 4 ng / mL rhbFGF was added to the above-mentioned MEF-CM was used.

  After culturing for 2-3 days, the medium was changed with Matrigel (1:60 dilution) and MEF-CM containing 4 ng / mL rhbFGF (also called Matrigel sandwich) (Day -1) .

  After 24 hours, RPMI + B27 medium [RPMI1640 (Life technologies), 2 mM L-glutamine, x1 B27 supplement without insulin (Life technologies)] with 100 ng / mL of Activin A (ActA; R & D) and 100 mg / mL of rhWnt3a ( The medium was replaced with a medium supplemented with recombinant human Wnt3a (R & D) (Day 0).

  After 24 hours, the medium was replaced with a medium (RPMI + B27 medium) containing 10 ng / mL human bone morphogenetic protein 4 (BMP4; R & D) and 10 ng / mL rhbFGF (Day 1). Thereafter, the culture was continued for 4 days without changing the medium.

  Replaced with a medium (RPMI + B27 medium) containing 50 ng / mL of VEGF (rhVEGF, Miltenyi). Thereafter, the medium was replaced with the same medium every two days (Day 5). The pulsation was observed from Day9-11.

  Cells were collected (Day 15) and a portion was used for FACS analysis. The result of FACS analysis is shown in FIG. The cells were detached and collected using AccuMax (Innovative Cell Technologies). The collected cells were used for the production of bioengineering cardiac tissue (referred to as ECT) described later.

Vascular wall cell differentiation induction method (Fig. 1b)
Confluent human iPS cells were detached by incubation at 37 ° C. for 3-5 minutes with Versene (Life technologies). After aspirating Versene, pipetting with MEF-CM, collecting with single cells, and then centrifuging to count the number of cells. About 3 × 10 ⁶ human iPS cells were recovered per 10 cm dish. The matrigel coat was rewound at about 1 × 10 ⁵ cells / cm ² . When 6 well plate was used, 1 × 10 ⁶ cells were seeded per well. A medium in which 4 ng / mL rhbFGF was added to the above-mentioned MEF-CM was used.

  After culturing for 2-3 days, the medium was changed with Matrigel (1:60 dilution) and MEF-CM containing 4 ng / mL rhbFGF (also called Matrigel sandwich) (Day -1) .

  After 24 hours, RPMI + B27 medium [RPMI1640 (Life technologies), 2 mM L-glutamine, x1 B27 supplement without insulin (Life technologies)] with 100 ng / mL of Activin A (ActA; R & D) and 100 mg / mL of rhWnt3a ( R & D) was added to the medium added (Day 0).

  After 24 hours, the medium was replaced with a medium (RPMI + B27 medium) containing 10 ng / mL human bone morphogenetic protein 4 (BMP4; R & D) and 10 ng / mL rhbFGF (Day 1). Thereafter, the culture was continued for 2 days without changing the medium.

  The medium was replaced with a medium containing 10% FBS (RPMI + 10% FBS) (Day 3). Thereafter, the medium was replaced with the same medium every two days.

  Cells were collected (Day 15) and a portion was used for FACS analysis. The result of FACS analysis is shown in FIG. The cells were detached and collected using AccuMax (Innovative Cell Technologies). The collected cells were used for the preparation of ECT described later.

ECT preparation method 15% of the cell group containing MC and CM without purifying the cardiomyocytes / vascular endothelial cells (hereinafter referred to as CM + EC) and vascular wall cells (hereinafter referred to as MC) obtained by the above-described method. Cells containing + EC were mixed at a ratio of 85% (MC was mixed with 4.5 × 10 ⁵ cells and CM + EC was mixed with 2.55 × 10 ⁶ cells), and a total of 3 × 10 ⁶ mixed cells were added to the medium (high glucose- Modified Dulbecco's essential medium (Life technologies), 20% FBS (Life technologies)) was suspended (referred to as cell suspension).

Neutralize acid-soluble rat-tail collagen type I (Sigma-Aldrich) with alkali buffer (0.2M NaHCO ₃ , 0.2M HEPES, and 0.1M NaOH) on ice, and add Matrigel (15% of total volume). Mixed (referred to as gel matrix). The cell suspension and gel matrix were mixed so that the collagen type I concentration was 0.67 mg / mL (referred to as cell / matrix mixture).

Cylindrical loading post (FX-4000TT; Flexcell International) was mounted on BioFlex culture plate (Flexcell International) according to the manufacturer's manual. Using a vacuum, indent the center of the silicon plate on the culture plate with a length of 20 mm and a width of 2 mm, pour 200 μL of the cell / matrix mixture, and incubate for 120 minutes in a CO ₂ incubator (37 ° C, 5% CO ₂ ). A cylindrical structure was produced (FIG. 2). Pre-culture medium [alpha minimum essential medium (aMEM; Life technologies), 10% FBS, 5 × 10 ⁻⁵ M 2-mercaptoethanol, 100 U / mL Penicillin-Streptomycin (Life technologies)] was added and cultured. The obtained ECT was subjected to medium exchange every day, cultured for 14 days, and used for the following evaluation (the ECT is referred to as C + E + M).

  As a control, CM + EC alone (the ECT is called C + E), cells mixed by the method of Uosaki, PLoS One 2011 so that the CM and MC content is 15% (the ECT is called C + M) ECT was prepared in the same manner.

Tissue Tension Measurement The tissue tension of the ECT obtained by the above method was measured using a tissue tension measurement system of Aurora Scientific (Aurora, Canada). Specifically, room temperature (25 ° C) and oxygenated Tyrode solution [(in mM) 119.8 NaCl, 5.4 KCl, 2.5 CaCl ₂ , 1.05 MgCl ₂ , 22.6 NaHCO ₃ , 0.42 NaH ₂ PO ₄ , 0.05 Na ₂ EDTA , 0.28 ascorbic acid, 5.0 glucose, 30 2,3-butanedione monoxime (BDM)], and one end was fixed to a force transducer (model 403A, Aurora Scientific) using 10-0 nylon. The other end was fixed to a high-speed length controller (model 322C, Aurora Scientific) connected to a micromanipulator. The perfusion chamber where the ECT was fixed was filled with BDM-free warmed Tyrode solution (37 ° C), field-stimulation (2 Hz / 5V) was performed for 20 minutes, and the tension was measured at each voltage and frequency.

  As a result, the maximum follow-up frequency (the maximum frequency that can be followed when the external stimulation frequency (frequency) was increased) was significantly maximum at C + E + M. Furthermore, the decrease in contractile force with increasing frequency was significantly less with C + E + M. This indicates that it can follow the various host heartbeats after transplantation more efficiently with a strong contractile force (FIG. 3).

  In addition, the minimum contraction threshold (the minimum voltage required for external contraction induction) was the smallest for C + E + M. Furthermore, C + M or C + E + M containing MC was able to maintain a significantly higher contractile force against voltage drop than C + E containing no MC. This indicates that inclusion of MC can follow a lower host voltage with a strong contractile force (FIG. 4).

  Subsequently, Young's modulus in Hook's law was measured by measuring the transition of the passive tension caused by gradually extending the ECT from the loose state. As a result, it was shown that C + M or C + E + M containing MC had higher tissue strength than C + E containing no MC (FIG. 5).

  Furthermore, in order to investigate whether the content rate of MC enhances these tissue characteristics, ECT (high MC) containing 30% or more of MC was prepared and compared with MC (low MC) obtained by the above method. In all items, high MC was shown to be inferior to low MC (FIG. 6). This indicates that the MC content is preferably at least less than 30% from the viewpoint of myocardial tissue function.

Electron microscopy
The most mature sarcomere structure was found in the C + E + M ECT. This seems to be one factor explaining the superior characteristics in the tissue tension test described above (FIG. 7).

Cardiomyocyte array
In the ECT containing MC, the array of cardiomyocytes in the tissue was more aligned. This also seems to be one factor explaining the excellent characteristics in the tissue tension test described above (FIG. 8).

Transplantation into a rat myocardial infarction model The ECT obtained by the above-described method was transplanted into a rat myocardial infarction model, and its therapeutic effect and myocardial regeneration effect were confirmed. Specifically, the rat myocardial infarction model was prepared by ligating the anterior descending branch of an immunodeficient nude rat (NTac: NIH- ^Foxn1rnu , Taconic Biosciences) with 7-0 silk thread to cause myocardial infarction.

  Transplantation was performed by re-opening the rat myocardial infarction model one week after the ligation and sewing three hiPSC-ECT (C + E + M) to the infarct with 7-0 silk (FIG. 9).

  Cardiac function evaluation was performed with an echo [Vevo2100 system (VisualSonics), 21-MHz imaging transducer (MS250; VisualSonics)] on the day before transplantation and 2-4 weeks after transplantation.

  As a result, it was confirmed that transplantation improved both cardiac output and stroke volume per minute, and significantly increased compared to the sham treatment (Sham) group. (FIG. 10).

  Histological examination was performed by sacrifice at 4 weeks after transplantation and removal of the heart. As a result, at 4 weeks after transplantation, a regenerated myocardium (HNA; positive for human nuclear antigen) derived from hiPSC-ECT (graft) having a thickness exceeding 1/2 of the infarct wall was observed. The engrafted myocardium showed a clear sarcomere structure. Furthermore, in the regenerated myocardium, host-derived and partly graft-derived vascular networks (vWF positive) were observed (FIG. 11).

Claims (12)

A method for producing myocardial tissue, comprising the following steps;
(A) producing cardiomyocytes from pluripotent stem cells;
(B) producing endothelial cells from pluripotent stem cells,
(C) a step of producing mural cells from pluripotent stem cells,
(D) mixing the wall cells produced in the step (c) with the cardiomyocytes produced in the step (a) and the endothelial cells produced in the step (b) at a ratio of less than 30%; e) A step of culturing the cell mixture obtained in the step (d) in the presence of an extracellular matrix to form a three-dimensional structure.   The method according to claim 1, wherein the wall cells produced in the step (c) are mixed in the step (d) at a ratio of 5% or more and 25% or less.   The method according to claim 1 or 2, wherein the wall cells produced in the step (c) are mixed at a ratio of 15% in the step (d).   The steps (a) and (b) are simultaneously performed using the same pluripotent stem cells, and cardiomyocytes and endothelial cells are produced from the pluripotent stem cells. The method described. The method according to claim 4, wherein the steps (a) and (b) include the following steps;
(I) culturing pluripotent stem cells in the presence of activin A and Wnt3a;
(Ii) culturing the cells obtained in the step (i) in the presence of BMP and bFGF, and (iii) culturing the cells obtained in the step (ii) in the presence of VEGF. The method according to any one of claims 1 to 5, wherein the step (c) includes the following steps;
(I) culturing pluripotent stem cells in the presence of activin A and Wnt3a;
(Ii) culturing the cells obtained in the step (i) in the presence of BMP and bFGF, and (iii) culturing the cells obtained in the step (ii) in the absence of VEGF.   The method according to claim 5 or 6, wherein the BMP is BMP4.   The method according to any one of claims 1 to 7, wherein the extracellular matrix is an extracellular matrix containing type I collagen. The method according to any one of claims 1 to 8, wherein the three-dimensional structure in the step (e) is a cylindrical structure. The method according to any one of claims 1 to 9, wherein the pluripotent stem cell is a human iPS cell.   The therapeutic agent for heart diseases containing the myocardial tissue obtained by the method of Claims 1-10.   A cardiac disease treatment method, wherein the myocardial tissue obtained by the method according to claim 1 is applied to an infarcted site, a damaged site or a damaged site of the heart.