Plate-shaped cartilage derived from pluripotent stem cells and method for producing same

Abstract

To provide a plate-shaped cartilage comprising a plurality of pluripotent stem cell-derived cartilaginous particles integrated with one another; and a method for producing the plate-shaped cartilage.SOLUTION: A method comprises a step 1: producing pluripotent stem cell-derived cartilaginous particles, and a step 2: culturing the cartilaginous particles under culture conditions that allow adjacent cartilaginous particles to be in contact with one another, wherein the number of the cartilaginous particles is that required to form the plate-shaped cartilage.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to plate-shaped cartilage derived from pluripotent stem cells and a method for producing the same. [Background technology]

[0002] Articular cartilage covers the ends of bones and provides lubrication between opposing bones during joint movement. Cartilage consists of chondrocytes and the cartilage extracellular matrix (ECM) that embeds the chondrocytes. Chondrocytes and cartilage ECM are interdependent: chondrocytes produce the chondrocyte ECM, and the cartilage ECM provides the environment for chondrocytes to maintain their properties (including the production of cartilage ECM).

[0003] Cartilage injuries rarely heal because of the loss of cartilage ECM. Cells cannot become chondrocytes in the absence of cartilage ECM, and nonchondrocytes do not produce cartilage ECM in the defect of the articular surface created by the injury. Recent regenerative medicine has employed the transplantation of autologous chondrocytes or mesenchymal stem cells into articular cartilage defects, but these cell transplants without cartilage ECM do not regenerate cartilage, as shown by the formation of fibrocartilage repair tissue. To overcome this limitation, transplantation of allogeneic cartilage has been employed. As cartilage is poorly immunogenic, articular cartilage injuries have been treated by transplanting allogeneic cartilage without matching HLA types or using immunosuppressive drugs. Although promising results have been obtained, the lack of donors, the heterogeneous quality of cartilage obtained from different individuals, and the risk of disease transmission make alternative methods desirable. In particular, new materials that can be transplanted into articular cartilage defects as regenerative medicine for joint injuries are desirable.

[0004] The present inventors previously reported a method for creating scaffold-free cartilage tissue from human iPS cells (Patent Documents 1 and 2, Non-Patent Documents 1 and 2). Three-dimensional culture of human iPS cells differentiated into chondrocytes in suspension culture causes these cells to secrete and deposit cartilage ECM to form cartilage. These human iPS cell-derived cartilages are spherical and approximately 1 to 3 mm in diameter. When the obtained human iPS cell-derived cartilage particles were transplanted into articular cartilage defects in minipigs treated with immunosuppressants, they constituted articular cartilage and showed signs of integration with the minipigs' native articular cartilage 4 weeks after transplantation (Non-Patent Document 1). When the obtained human iPS cell-derived cartilage particles were transplanted into articular cartilage defects in immunodeficient rats, they integrated with the adjacent rats' native cartilage on the sides and with bone on the bottom 4 weeks after transplantation (Non-Patent Document 1). Furthermore, the present inventors found that the human iPS cell-derived cartilage particles were hypoimmunogenic like natural cartilage, as shown by the lack of lymphocyte proliferation in a mixed lymphocyte reaction assay (Non-Patent Document 3). These findings indicate that human iPS cell-derived cartilage particles could be a new source for allogeneic transplantation to treat articular cartilage defects. The number of human iPS cell-derived cartilage particles required to fill a defect varies depending on the size of the articular cartilage defect. In clinical settings, a 1 cm 2 They plan to transplant about 25 human iPS cell-derived cartilage particles per defect and fix them with fibrin glue. However, when fixing multiple cartilage particles with fibrin glue, the fixing strength is weak, so there is a possibility that the cartilage particles will fall off after transplantation.

[0005] The inventors have also reported that when two human iPS cell-derived cartilage particles obtained 90 days after the start of differentiation were cultured in contact in a culture medium for 60 days, the two particles merged to form a single particle (Patent Document 1). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO2015 / 064754 [Patent Document 2] WO2016 / 133208 [Non-patent literature]

[0007] [Non-Patent Document 1] Yamashita, A., Morioka, M., Yahara, Y., Okada, M., Kobayashi, T., Kuriyama, S., Matsuda, S., and Tsumaki, N. Generation of Scaffoldless Hyaline Cartilaginous Tissue from Human iPSCs. Stem cell reports 4, 404, 2015. [Non-Patent Document 2] Yamashita, A., Morioka, M., Kishi, H., Kimura, T., Yahara, Y., Okada, M., Fujita, K., Sawai, H., Ikegawa, S., and Tsumaki, N. Statin treatment rescues FGFR3 skeletal dysplasia phenotypes. Nature 513, 507, 2014. [Non-Patent Document 3] Kimura, T., Yamashita, A., Ozono, K., and Tsumaki, N. Limited Immunogenicity of Human Induced Pluripotent Stem Cell-Derived Cartilages. Tissue Eng Part A 22, 1367, 2016. Summary of the Invention [Problem to be solved by the invention]

[0008] An objective of the present invention is to provide plate-shaped cartilage derived from pluripotent stem cells that can be transplanted into an articular cartilage defect site. [Means for solving the problem]

[0009] In order to solve the above problems, the present invention includes the following inventions. [1] Plate-like cartilage formed by the merging of multiple pluripotent stem cell-derived cartilage particles. [2] The plate cartilage described in [1] above, for transplantation into a site of cartilage damage. [3] A method for producing plate cartilage according to [1] or [2] above, comprising the steps of: Step 1: Producing pluripotent stem cell-derived cartilage particles; and Step 2: Cultivating the number of cartilage particles required for forming plate-shaped cartilage in a state in which adjacent cartilage particles can contact each other; The method according to claim 1, further comprising: [4] The method according to [3] above, characterized in that in step 2, the required number of cartilage particles are contained in a frame that can be placed in a culture medium or a container through which liquid can pass. [5] The method according to [4], wherein the container is capable of containing the required number of cartilage particles in a monolayer state. [6] The method according to any one of [3] to [5] above, wherein the cartilage particles are cartilage particles in the process of being formed. [7] The method according to any one of [3] to [6] above, wherein in step 2, the culture is carried out while flowing the culture medium. [8] The method according to any one of [3] to [7] above, wherein in step 2, a culture medium containing an FGF receptor agonist is used. [9] The method according to [8] above, wherein the FGF receptor agonist is FGF18.

[10] A pharmaceutical composition for repairing damaged cartilage, comprising the plate cartilage described in [1] or [2] above.

[0010]

[11] A method for repairing cartilage, comprising the step of transplanting the plate cartilage described in [1] or [2] above into a cartilage defect site.

[12] The plate cartilage described in [1] or [2] above, for use in cartilage repair.

[13] Use of the plate cartilage described in [1] or [2] above for the manufacture of a pharmaceutical composition for repairing damaged cartilage. Effect of the Invention

[0011] According to the present invention, plate-shaped cartilage derived from pluripotent stem cells can be provided. The plate-shaped cartilage of the present invention can be transplanted after being formed or trimmed to a size suitable for the size of the defective site of articular cartilage, and is useful as a pharmaceutical composition for regenerative medicine of joint damage. Furthermore, by combining cartilage particles before transplantation, the possibility of cartilage particles falling off after transplantation, which is a problem in the conventional method, can be reduced. Furthermore, it becomes possible to transplant cartilage tissue derived from pluripotent stem cells suitable for the size of the defective site. As a result, it can be transplanted even for more extensive defects or degeneration of articular cartilage. [Brief description of the drawings]

[0012] [Figure 1] (A) is an image of a well of a round-bottom 96-well plate containing a pair of human iPS cell-derived cartilage particles, (B) is a schematic diagram of the well in (A), and (C) is a schematic diagram of a serial section of a combined human iPS cell-derived cartilage particle. [Diagram 2] A pair of cartilage particles made from human iPS cell line QHJI was placed in a round-bottom 96-well plate and observed on days 3, 7, 14, 28, 56, and 84. The left column shows the results of macroscopic observation, the second column shows sections stained with Safranin O-Fast Green-Iron Hematoxylin, the third column shows an enlargement of the boxed area in the second column, and the right column shows an enlargement of the boxed area in the second or third column. Scale bar 500 μm. [Diagram 3]A pair of cartilage particles made from human iPS cell line QHJI was placed in a round-bottom 96-well plate, and sections of the combined cartilage particles on days 7, 14, and 84 were immunostained with anti-type I collagen antibody (left column, second column, third column), and a section on day 84 was immunostained with anti-type II collagen antibody (right column). Columns 2 and 3 are enlarged views of the boxed area in the left column. Scale bar: 500 μm. [Figure 4] Cartilage particles were prepared from human iPS cell lines 409B2, 604B1, and 1231A3, and cultured in contact with each other in a culture dish. Sections of coalesced cartilage particles on the 60th day were stained with Safranin O-fast green-iron hematoxylin. [Diagram 5] This figure shows the results of separating the perichondrium-like membrane (upper right) and central cartilage (lower right) from uncoalesced cartilage particles (left), and staining each section with Safranin O-Fast Green-Iron Hematoxylin. [Figure 6] Figure 1. Differences in gene expression in perichondrium and central cartilage. Each point represents one gene. The y-axis shows the fold change of RPKM in perichondrium to that in central cartilage. The x-axis shows log10 of RPKM in perichondrium. The arrow indicates the FGF18 gene. [Figure 7] 1 shows the results of expression analysis by real-time RT-PCR of marker genes in the perichondrium-like membrane and central cartilage, M is the perichondrium-like membrane, and C is the central cartilage, and error bars indicate the mean ± standard deviation. [Figure 8] A pair of cartilage particles made from human iPS cell line QHJI was placed in a round-bottom 96-well plate and cultured in the presence or absence (vehicle) of FGF18 (final concentration, 100 ng / mL), and the results were observed on days 3, 7, and 14. The left column shows images of macroscopic observation, the second column shows sections stained with Safranin O-Fast Green-Iron Hematoxylin, and the third column shows an enlargement of the boxed area in the second column. Scale bar, 500 μm. [Figure 9]A pair of cartilage particles made from human iPS cell line QHJI was placed in a round-bottom 96-well plate and cultured in the presence or absence (vehicle) of FGF inhibitor NVP-BGJ398 (final concentration, 50 nM), and observed on day 14. The left column shows images of macroscopic observation, and the second column shows images of sections stained with Safranin O-Fast Green-Iron Hematoxylin. Scale bar, 500 μm. [Figure 10] Human iPS cell-derived cartilage particles were placed in a mesh bag, which was then placed in a bioreactor containing chondrogenic medium and cultured for 4 weeks with stirring. Figures showing the plate-shaped cartilage obtained (A and B are enlarged front views, and (C) is an enlarged side view). [Figure 11] FIG. 11 is a photograph of the histological section of plate cartilage in FIG. 10 stained with Safranin O-Fast Green-Iron Hematoxylin. [Figure 12] (A) shows star-shaped plate cartilage obtained by placing human iPS cell-derived cartilage particles in a star-shaped frame placed in a culture dish containing chondrogenic medium and culturing for 4 weeks. (B) shows a tissue section of the plate cartilage stained with hematoxylin and eosin, and (C) shows a tissue section of the plate cartilage stained with Safranin O-fast green-iron hematoxylin. [Figure 13] Polycarbonate or polyester transwells were placed in each well of a 24-well plate containing chondrogenic medium, and human iPS cell-derived cartilage particles were placed in the transwells. These figures show the fused cartilage obtained after 4 weeks of culture. (A) shows the results for the polycarbonate transwell, and (B) shows the results for the polyester transwell. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] [Plate cartilage] The present invention provides plate cartilage derived from pluripotent stem cells. The plate cartilage of the present invention is plate cartilage (plate cartilage tissue) formed by the coalescence of a plurality of cartilage particles derived from pluripotent stem cells. The plate cartilage of the present invention may be plate cartilage formed by the coalescence of a plurality of cartilage particles derived from pluripotent stem cells in multiple layers, or may be plate cartilage formed by the coalescence of a plurality of cartilage particles derived from pluripotent stem cells in a single layer.

[0014] The plate cartilage of the present invention may have a thickness equivalent to that found in cartilage in vivo. For example, the thickness of the plate cartilage may be 5 mm or less, 4 mm or less, 3 mm or less, or 2 mm or less. The thickness of the plate cartilage may be about 1.5 to about 4.5 mm, about 2 to about 4 mm, or about 2.5 to about 3.5 mm. The thickness of the plate cartilage may be the average value of thicknesses at multiple points on the plate cartilage, and some parts may exceed the specified thickness.

[0015] The plate cartilage of the present invention can be suitably used for transplantation into a cartilage damage site. The plate cartilage of the present invention can be formed or trimmed and transplanted to fit the shape and size of the cartilage defect at the cartilage damage site, which is very useful. Furthermore, since the thickness of the plate cartilage of the present invention is equivalent to the thickness of articular cartilage, the plate cartilage of the present invention is particularly suitable for transplantation into a damaged articular cartilage site. After transplantation, the plate cartilage of the present invention can be remodeled in vivo. Therefore, its shape, size, and thickness do not have to completely match the site to be transplanted.

[0016] Pluripotent stem cells that can be used to produce cartilage particles are not particularly limited as long as they have pluripotency that allows them to differentiate into all cells present in the living body and also have proliferation ability. Examples of such stem cells include embryonic stem (ES) cells, embryonic stem (ntES) cells derived from cloned embryos obtained by nuclear transfer, sperm stem (GS) cells, embryonic germ (EG) cells, induced pluripotent stem (iPS) cells, cultured fibroblasts, and pluripotent cells derived from bone marrow stem cells (Muse cells). Preferred pluripotent stem cells are ES cells, ntES cells, and iPS cells.

[0017] (A) Embryonic stem cells ES cells are stem cells that are established from the inner cell mass of early mammalian embryos (eg, blastocysts) such as humans and mice, and have pluripotency and the ability to proliferate through self-renewal.

[0018] ES cells are embryo-derived stem cells that originate from the inner cell mass of a blastocyst, which is the embryo at the eight-cell stage of a fertilized egg, or after the morula stage. They have the ability to differentiate into any cell that makes up an adult, known as pluripotency, and the ability to proliferate by self-renewal. ES cells were discovered in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292:154-156), and subsequently ES cell lines were established in humans, monkeys, and other primates (JA Thomson et 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).

[0019] ES cells can be established by extracting the inner cell mass from the blastocyst of a fertilized egg of a target animal and culturing the inner cell mass on a fibroblast feeder. Cells can be maintained by subculture in a culture medium supplemented with substances such as leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF). Methods for establishing and maintaining human and monkey ES cells are described in, for example, US Pat. No. 5,843,780; Thomson JA, et al. (1995), Proc. Natl. Acad. Sci. USA 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.

[0020] As a culture medium for preparing ES cells, for example, DMEM / F-12 culture medium supplemented with 0.1 mM 2-mercaptoethanol, 0.1 mM non-essential amino acids, 2 mM L-glutamic acid, 20% KSR (KnockOut Serum Replacement, Invitrogen) and 4 ng / mL bFGF can be used, and human ES cells can be maintained at 37°C in a humid atmosphere of 2% CO2 / 98% air (O. Fumitaka et al. (2008), Nat. Biotechnol., 26:215-224). In addition, ES cells need to be passaged every 3 to 4 days, and passage can be performed using, for example, 0.25% trypsin and 0.1 mg / mL collagenase IV in PBS containing 1 mM CaCl2 and 20% KSR.

[0021] ES cells can generally be selected by real-time PCR using the expression of gene markers such as alkaline phosphatase, Oct-3 / 4, Nanog, etc. In particular, human ES cells can be selected using the expression of gene markers such as OCT-3 / 4, NANOG, ECAD, etc. (E. Kroon et al. (2008), Nat. Biotechnol., 26:443-452).

[0022] Human ES cell lines, for example WA01 (H1) and WA09 (H9), are available from WiCell Research Institute, and KhES-1, KhES-2 and KhES-3 are available from the Institute for Frontier Medical Sciences, Kyoto University (Kyoto, Japan).

[0023] (B) Sperm stem cells Spermatogonial stem cells are pluripotent stem cells derived from the testis, and are the source of spermatogenesis. Like ES cells, these cells can be induced to differentiate into various lineages of cells, and have properties such as the ability to produce 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). They can self-replicate in a culture medium containing glial cell line-derived neurotrophic factor (GDNF), and spermatogonial stem cells can be obtained by repeated passage under the same culture conditions as ES cells (Takebayashi, M. et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Extra Edition), pp. 41-46, Yodosha (Tokyo, Japan)).

[0024] (C) Embryonic germ cells Embryonic germ cells are cells that are established from primordial germ cells during the fetal period and have pluripotency similar to that of ES cells. They can be established by culturing primordial germ cells in the presence of substances such as LIF, bFGF, and stem cell factor (Y. Matsui et al. (1992), Cell, 70:841-847; JL Resnick et al. (1992), Nature, 359:550-551).

[0025] (D) Induced pluripotent stem cells Induced pluripotent stem (iPS) cells can be produced by introducing specific reprogramming factors in the form of DNA or protein into somatic cells. They are artificial stem cells derived from somatic cells and have almost the same characteristics as ES cells, such as pluripotency and the ability to proliferate through self-renewal (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 may be composed of a gene specifically expressed in ES cells, its gene product or non-cording RNA, or a gene that plays an important role in maintaining the undifferentiated state of ES cells, its gene product or non-cording RNA, or a low molecular weight compound. Examples of genes contained 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, and these reprogramming factors may be used alone or in combination. Combinations of reprogramming factors include 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, W O2009 / 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.

[0026] The reprogramming factors include histone deacetylase (HDAC) inhibitors [e.g., small molecule inhibitors such as valproic acid (VPA), trichostatin A, sodium butyrate, MC 1293, M344, and the like, and nucleic acid expression inhibitors such as siRNA and shRNA against HDAC (e.g., HDAC1 siRNA Smartpool (Millipore), HuSH 29mer shRNA Constructs against HDAC1 (OriGene)), and the like], MEK inhibitors (e.g., PD184352, PD98059, U0126, SL327, and PD0325901), glycogen synthase kinase-3 inhibitors (e.g., Bio and CHIR99021), DNA methyltransferase inhibitors (e.g., 5-azacytidine), histone methyltransferase inhibitors (e.g., BIX-01294, small molecule inhibitors such as Suv39hl, Suv39h2, SetDBl and nucleic acid expression inhibitors such as siRNA and shRNA for G9a, L-channel calcium agonists (e.g. Bayk8644), butyric acid, TGFβ inhibitors or ALK5 inhibitors (e.g. LY364947, SB431542, 616453 and A-83-01), p53 inhibitors (e.g. siRNA and shRNA for p53), ARID3A inhibitors (e.g. siRNA and shRNA for ARID3A), miRNAs such as miR-291-3p, miR-294, miR-295 and mir-302, Wnt Signaling (e.g. soluble These factors also include factors used for the purpose of improving the establishment efficiency, such as Wnt3a), neuropeptide Y, prostaglandins (e.g., prostaglandin E2 and prostaglandin J2), hTERT, SV40LT, UTF1, IRX6, GLISl, PITX2, and DMRTBl, and in this specification, factors used for the purpose of improving the establishment efficiency are not particularly distinguished from reprogramming factors.

[0027] When the reprogramming factor is in the form of a protein, it may be introduced into a somatic cell by techniques such as lipofection, fusion with a cell membrane-permeable peptide (eg, HIV-derived TAT and polyarginine), or microinjection.

[0028] On the other hand, in the case of the form of DNA, it can be introduced into somatic cells by, for example, vectors such as viruses, plasmids, and artificial chromosomes, lipofection, liposomes, microinjection, and the like. Examples of viral vectors include retroviral vectors, lentiviral vectors (Cell, 126, pp.663-676, 2006; Cell, 131, pp.861-872, 2007; Science, 318, pp.1917-1920, 2007), adenoviral vectors (Science, 322, 945-949, 2008), adeno-associated virus vectors, and Sendai virus vectors (WO 2010 / 008054). Examples of artificial chromosome vectors include human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), and bacterial artificial chromosomes (BAC, PAC). As the plasmid, a plasmid for mammalian cells can be used (Science, 322:949-953, 2008). The vector can contain control sequences such as a promoter, an enhancer, a ribosome binding sequence, a terminator, and a polyadenylation site so that the nuclear reprogramming substance can be expressed, and further, if necessary, can contain a selection marker sequence such as a drug resistance gene (e.g., a kanamycin resistance gene, an ampicillin resistance gene, a puromycin resistance gene, etc.), a thymidine kinase gene, a diphtheria toxin gene, and a reporter gene sequence such as green fluorescent protein (GFP), β-glucuronidase (GUS), and FLAG. In addition, the above vector may have LoxP sequences before and after a gene encoding a reprogramming factor or a promoter and a gene encoding a reprogramming factor bound thereto in order to excise both of them after introduction into somatic cells.

[0029] In the case of the RNA form, it may be introduced into somatic cells by techniques such as lipofection or microinjection, and RNA incorporating 5-methylcytidine and pseudouridine (TriLink Biotechnologies) may be used to suppress degradation (Warren L, (2010) Cell Stem Cell. 7:618-630).

[0030] Examples of culture media for inducing iPS cells include DMEM, DMEM / F12, or DME culture media containing 10 to 15% FBS (these culture media can further contain LIF, penicillin / streptomycin, puromycin, L-glutamine, nonessential amino acids, β-mercaptoethanol, etc., as appropriate), or commercially available culture media such as culture media for culturing mouse ES cells (TX-WES culture medium, ThromboX), culture media for culturing primate ES cells (culture medium for primate ES / iPS cells, ReproCell), and serum-free pluripotent stem cell maintenance medium (e.g., mTeSR (Stemcell Technology), Essential 8 (Life Technologies), StemFit AK03 (AJINOMOTO)).

[0031] As an example of a culture method, for example, somatic cells are contacted with reprogramming factors in 10% FBS-containing DMEM or DMEM / F12 culture medium at 37°C in the presence of 5% CO2 and cultured for about 4 to 7 days, and then the cells are replated onto feeder cells (e.g., mitomycin C-treated STO cells, SNL cells, etc.) and cultured in bFGF-containing culture medium for primate ES cell culture from about 10 days after contacting the somatic cells with the reprogramming factors, and iPS-like colonies can be generated about 30 to 45 days or more after the contact.

[0032] Alternatively, the cells are cultured on feeder cells (e.g., mitomycin C-treated STO cells, SNL cells, etc.) in a 10% FBS-containing DMEM culture medium (which may further contain LIF, penicillin / streptomycin, puromycin, L-glutamine, non-essential amino acids, β-mercaptoethanol, etc.) at 37°C in the presence of 5% CO2, and ES-like colonies can be generated after about 25 to about 30 days or more. Desirably, somatic cells to be reprogrammed themselves are used instead of feeder cells (Takahashi K, et al. (2009), PLoS One. 4:e8067 or WO2010 / 137746), or extracellular matrices (e.g., Laminin-5 (WO2009 / 123349) and Matrigel (BD)) are used.

[0033] Other examples include a method of culturing using a serum-free medium (Sun N, et al. (2009), Proc Natl Acad Sci US A. 106:15720-15725). Furthermore, to increase the efficiency of establishment, iPS cells may be established under low oxygen 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).

[0034] During the above culture, the culture medium is replaced with fresh medium once a day from the second day of culture onward. The number of somatic cells used for nuclear reprogramming is not limited, but may be up to 100 cells / 100 cm in a culture dish. 2 Approximately 5 x 10 3 ~Approx. 5×10 6 It is the range of cells.

[0035] iPS cells can be selected based on the shape of the colony formed. On the other hand, when a drug resistance gene that is expressed in conjunction with a gene (e.g., Oct3 / 4, Nanog) that is expressed when somatic cells are reprogrammed is introduced as a marker gene, the established iPS cells can be selected by culturing in a culture medium (selection culture medium) containing the corresponding drug. In addition, when the marker gene is a fluorescent protein gene, iPS cells can be selected by observing with a fluorescent microscope, when it is a luminescent enzyme gene, by adding a luminescent substrate, and when it is a chromogenic enzyme gene, by adding a chromogenic substrate.

[0036] The term "somatic cells" as used herein refers to any animal cell (preferably a mammalian cell, including a human cell) excluding germline cells such as eggs, oocytes, and ES cells, or totipotent cells. Somatic cells include, but are not limited to, fetal (baby) somatic cells, neonatal (baby) somatic cells, and mature healthy or diseased somatic cells, as well as primary culture cells, passaged cells, and established cell lines. Specifically, somatic cells include, for example, (1) tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells, (2) tissue progenitor cells, and (3) differentiated cells such as lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, liver cells, gastric mucosa cells, intestinal cells, spleen cells, pancreatic cells (exocrine pancreatic cells, etc.), brain cells, lung cells, kidney cells, and adipocytes.

[0037] Furthermore, when using iPS cells as a source of transplantation cells, it is desirable to use somatic cells with the same or substantially the same HLA genotype as the recipient 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 the immune response can be suppressed with an immunosuppressant, for example, somatic cells with an HLA type that matches the three loci of HLA-A, HLA-B, and HLA-DR, or the four loci including HLA-C.

[0038] (E) ES cells derived from cloned embryos obtained by nuclear transfer. ntES cells are ES cells derived from cloned embryos produced by nuclear transfer technology, and have 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). In other words, ntES (nuclear transfer ES) cells are ES cells established from the inner cell mass of blastocysts derived from cloned embryos obtained by replacing the nucleus of an unfertilized egg with the nucleus of a somatic cell. To generate ntES cells, a combination of nuclear transfer technology (JB Cibelli et al. (1998), Nature Biotechnol., 16:642-646) and ES cell generation technology (mentioned above) is used (Wakayama Sayaka et al. (2008), Experimental Medicine, Vol. 26, No. 5 (special issue), pp. 47-52). In nuclear transfer, the nucleus of a somatic cell is injected into an enucleated unfertilized egg of a mammal, and the egg can be initialized by culturing for several hours.

[0039] (F) Multilineage-differentiating Stress Enduring cells (Muse cells) Muse cells are pluripotent stem cells produced by the method described in WO2011 / 007900. More specifically, Muse cells are pluripotent cells obtained by trypsinizing fibroblasts or bone marrow stromal cells for a long period of time, preferably 8 or 16 hours, followed by culture in suspension; these cells are positive for SSEA-3 and CD105.

[0040] [Method for producing plate-shaped cartilage] The present invention provides a method for producing the above-mentioned plate-shaped cartilage. The production method of the present invention may include the following steps: Step 1: A step of producing cartilage particles derived from pluripotent stem cells Step 2: A step of culturing the necessary number of cartilage particles to form plate-shaped cartilage in a state in which adjacent cartilage particles can come into contact with each other.

[0041] Step 1 is a step of producing cartilage particles derived from pluripotent stem cells. Methods of producing cartilage particles by inducing differentiation of pluripotent stem cells are known, and can be appropriately selected and used from the methods described in, for example, Patent Document 1, Non-Patent Document 1, Non-Patent Document 2, etc. Specifically, the following method can be suitably used. However, the method of producing cartilage particles derived from pluripotent stem cells is not limited to this method.

[0042] The method used in step 1 of the production method of the present invention may be the following method. (i) culturing pluripotent stem cells in an adherent manner in a culture medium containing one or more substances selected from the group consisting of bone morphogenetic protein (BMP) 2, transforming growth factor (TGF) β, and growth differentiation factor (GDF) 5, and a hydroxymethylglutaryl CoA (HMG-CoA) reductase inhibitor; (ii) A step of culturing the cells obtained in the step (i) in a suspension state in a culture medium containing one or more substances selected from the group consisting of BMP2, TGFβ and GDF5, and an HMG-CoA reductase inhibitor.

[0043] The pluripotent stem cells to be subjected to (i) are preferably cultured in a three-dimensional suspension culture while maintaining their undifferentiated state, to form cell masses. Three-dimensional suspension culture is a method in which cells are cultured in a culture medium under non-adhesive conditions with stirring or shaking.

[0044] If the diameter of the cell cluster exceeds 300 μm, differentiation induction or necrosis occurs inside the cell cluster due to the influence of cytokines etc. secreted by the cells, so it is necessary to adjust the diameter of the cell cluster to within 300 μm. Examples of ways to adjust the diameter of the cell cluster include adjusting the size by appropriately adjusting the cell density and stirring speed, or by selecting cell clusters of an appropriate size.

[0045] The culture medium used in step (i) can be prepared by adding one or more substances selected from the group consisting of BMP2, TGFβ, and GDF5, and an HMG-CoA reductase inhibitor to a basal medium used for culturing animal cells. A preferred culture medium used in step (i) is a basal medium to which BMP2, TGFβ, GDF5, and an HMG-CoA reductase inhibitor have been added. Examples of basal media 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. The basal medium may contain, as necessary, substances such as serum (e.g., FBS), albumin, transferrin, KnockOut Serum Replacement (KSR) (a serum substitute for FBS during ES cell culture) (Invitrogen), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, sodium selenite, ethanolamine, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, lipids, amino acids, L-glutamine, GlutaMAX (Invitrogen), non-essential amino acids (NEAA), sodium pyruvate, vitamins, growth factors, small molecule compounds, antibiotics, antioxidants, pyruvic acid, buffers, and inorganic salts. In one embodiment of step (i), the basal medium is DMEM containing insulin, transferrin, sodium selenite, ethanolamine, ascorbic acid, non-essential amino acids, sodium pyruvate, antibiotics, and serum.

[0046] In step (i), BMP2 includes BMP2 derived from humans and other animals, and functional variants thereof, and for example, commercially available products from Osteopharma, Inc. can be used. The concentration of BMP2 used in this step is 0.1 ng / mL to 1000 ng / mL, preferably 1 ng / mL to 100 ng / mL, more preferably 5 ng / mL to 50 ng / mL, 10 ng / mL. In the present invention, BMP2 may be replaced with BMP4.

[0047] In step (i), TGFβ includes TGFβ derived from humans and other animals, as well as functional variants thereof, and can be, for example, commercially available products from PeproTech, Inc. The concentration of TGFβ used in this step is 0.1 ng / mL to 1000 ng / mL, preferably 1 ng / mL to 100 ng / mL, more preferably 5 ng / mL to 50 ng / mL, or 10 ng / mL.

[0048] In step (i), GDF5 includes GDF5 derived from humans and other animals, and functional variants thereof, and for example, commercially available products from PeproTech, Inc. can be used. The concentration of GDF5 used in this step is 0.1 ng / mL to 1000 ng / mL, preferably 1 ng / mL to 100 ng / mL, more preferably 5 ng / mL to 50 ng / mL, 10 ng / mL.

[0049] In step (i), examples of the HMG-CoA reductase inhibitor include mevastatin (compactin) (see USP 3,983,140), pravastatin (see JP-A-57-2240 (USP 4,346,227)), lovastatin (see JP-A-57-163,374 (USP 4,231,938)), simvastatin (see JP-A-56-122,375 (USP 4,444,784)), fluvastatin (see JP-A-56-122,375 (USP 4,444,784)), and the like. Examples of HMG-CoA reductase inhibitors include, but are not limited to, atorvastatin (see JP-A-3-58967 (USP 5273995)), rosuvastatin (see JP-A-5-178841 (USP 5260440)), and pitavastatin (see JP-A-1-279866 (USP 5854259 and USP 5856336)). The HMG-CoA reductase inhibitor in the present invention is preferably a drug selected from the group consisting of mevastatin, atorvastatin, pravastatin, rosuvastatin, fluvastatin, and lovastatin.

[0050] In step (i), when rosuvastatin is used as the HMG-CoA reductase inhibitor, the concentration is 0.01 μM to 100 μM, preferably 0.1 μM to 10 μM, more preferably 0.5 μM to 5 μM, or 1 μM.

[0051] In step (i), bFGF may be further added to the basal medium, and bFGF includes bFGF derived from humans and other animals, as well as functional variants thereof, and may be, for example, commercially available products from WAKO Co., Ltd. The concentration of bFGF used in this step is 0.1 ng / mL to 1000 ng / mL, preferably 1 ng / mL to 100 ng / mL, more preferably 5 ng / mL to 50 ng / mL, or 10 ng / mL.

[0052] In step (i), a pterosin derivative may be further added to the basal medium, and examples of the pterosin derivative include those described in 14 / 315,809, and more preferably pterosin B. The concentration of pterosin B used in this step is 10 μM to 1000 μM, preferably 100 μM to 1000 μM.

[0053] Adhesion culture refers to culturing cells in a state where the cells are adhered to a culture vessel. The culture vessel to be used is not particularly limited as long as it is a culture vessel capable of adhesion culture of cultured cells. Examples include flasks, flasks for tissue culture, dishes, Petri dishes, dishes for tissue culture, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, chamber slides, culture slides, and petri dishes. The culture vessel may be one that has been subjected to a surface treatment suitable for cell adhesion, or one that has not been subjected to such a surface treatment (non-coated). A commercially available culture vessel having a surface treatment suitable for cell adhesion can be used, and an example of such a culture vessel is a tissue culture dish by IWAKI. In another embodiment, the culture may be performed by culturing the cells using a culture vessel that has been coated with an extracellular matrix. The coating treatment can be performed by putting a solution containing an extracellular matrix into the culture vessel and then appropriately removing the solution.

[0054] The extracellular matrix used for coating may be naturally derived or artificial (recombinant). For example, polylysine, polyornithine, collagen, proteoglycan, fibronectin, hyaluronic acid, tenascin, entactin, elastin, fibrillin, laminin, and fragments thereof may be used. These extracellular matrices may be used in appropriate combination.

[0055] In step (i), the culture temperature is not particularly limited, but is about 30 to 40°C, preferably about 37°C, and the culture is performed under an atmosphere of CO2-containing air. The CO2 concentration is about 2 to 5%, preferably about 5%. The culture period in step (i) is not particularly limited, so long as it is longer than the period during which the seeded cell masses adhere to the culture vessel and form nodules. The culture period in step (i) may be 7 days or more, 14 days or more, 21 days or more, or 28 days or more. The culture period in step (i) may be 35 days or less, 28 days or less, 21 days or less, or 14 days or less. It is preferably 14 days.

[0056] In step (i), the cells that have formed nodules may naturally detach and float during the culture period, or may remain attached to the culture vessel until the end of the culture period. The nodules (cell clumps) that have naturally detached and floated are directly subjected to suspension culture in step (ii). The nodules (cell clumps) that are attached to the culture vessel are detached from the culture vessel and subjected to suspension culture in step (ii). The method of detaching the nodules (cell clumps) from the culture vessel is preferably performed by a mechanical separation method (e.g., a method using pipetting or a scraper, etc.), and is preferably a method that does not use a separation solution having protease activity and / or collagenase activity (e.g., solutions containing trypsin and collagenase, such as Accutase(TM) and Accumax(TM) (Innovative Cell Technologies, Inc.)).

[0057] In step (ii), the cells obtained in step (i) are cultured in suspension. Suspension culture means culturing the cells in a non-adherent state to a culture vessel, and is not particularly limited, but is preferably performed using a culture vessel (e.g., a Petri dish) that has not been artificially treated (e.g., coated with an extracellular matrix or the like) for the purpose of improving adhesion to the cells, or a culture vessel that has been artificially treated to suppress adhesion (e.g., coated with polyhydroxyethyl methacrylate (poly-HEMA)).

[0058] In step (ii), the same culture medium as in step (i) can be used.

[0059] In step (ii), the culture temperature is not particularly limited, but is about 30 to 40°C, preferably about 37°C, and the culture is performed under an atmosphere of CO2-containing air. The CO2 concentration is about 2 to 5%, preferably about 5%. The culture period is not particularly limited, and the culture may be performed until the desired cartilage particles are obtained. The culture period in step (ii) may be 7 days or more, 14 days or more, 21 days or more, 28 days or more, 35 days or more, 42 days or more, 49 days or more, 56 days or more, 63 days or more, or 70 days or more. The culture period in step (ii) may be 140 days or less, 133 days or less, 126 days or less, 119 days or less, 112 days or less, 105 days or less, 98 days or less, 91 days or less, 84 days or less, 77 days or less, 70 days or less, 63 days or less, 56 days or less, 49 days or less, 42 days or less, 35 days or less, 28 days or less, 21 days or less, or 14 days or less. The production of cartilage particles can be confirmed by taking some particles from the culture and staining them with Safranin O.

[0060] Step 2 is a step of culturing the number of cartilage particles required for the formation of plate-shaped cartilage under a condition in which adjacent cartilage particles can come into contact with each other. The cartilage particles used in step 2 are the cartilage particles produced in step 1, and the inventors have confirmed that the size of the cartilage particles varies depending on the number of days from the start of differentiation induction in step 1 (the day the medium was replaced with a cartilage differentiation medium). Specifically, the inventors have confirmed that the size (diameter) of the cartilage particles 4 weeks after the start of differentiation induction is about 1 mm, the size (diameter) of the cartilage particles 12 weeks after the start of differentiation induction is about 2 to 3 mm, and that even if the culture is continued thereafter, the growth of the cartilage particles stops at a diameter of about 3 to 4 mm. The cartilage particles to be subjected to step 2 may be cartilage particles less than 4 weeks (28 days) from the start of differentiation induction in step 1, cartilage particles aged 4 weeks (28 days) to 6 weeks (42 days), cartilage particles aged 6 weeks (42 days) to 8 weeks (56 days), cartilage particles aged 8 weeks (56 days) to 10 weeks (70 days), cartilage particles aged 10 weeks (70 days) to 12 weeks (84 days), cartilage particles aged 12 weeks (84 days) to 14 weeks (98 days), or cartilage particles aged more than 14 weeks (98 days). Cartilage particles in the middle of formation are preferred. As cartilage particles in the middle of formation, cartilage particles aged 6 weeks (42 days) or less, 5 weeks (35 days) or less, or 4 weeks (28 days) or less from the start of differentiation induction are preferred, and cartilage particles about 4 weeks (25 to 32 days) are more preferred.

[0061] The required number of cartilage particles can be appropriately determined depending on the size of the plate-shaped cartilage to be produced. When the cartilage particles to be subjected to step 2 are cartilage particles in the process of being formed, it is preferable to adjust the required number of cartilage particles taking into consideration the increase of the cartilage particles themselves during the culture period in step 2.

[0062] In step 2, in order to culture the cartilage particles in a state in which they can come into contact with each other, a frame that can be placed in the culture solution can be suitably used. For example, a culture medium can be placed in a culture dish, and an appropriate frame can be placed in it. Specifically, a smaller-sized culture dish, a transwell for cell migration tests, or the like can be used as the frame. The cartilage particles produced in step 1 are placed in this frame. The cartilage particles are preferably filled in the frame so that they come into contact with adjacent cartilage particles, and are preferably filled in a single layer.

[0063] In step 2, in order to culture the cartilage particles in a state where adjacent cartilage particles can contact each other, a container through which liquid can pass can be suitably used. Examples of containers through which liquid can pass include mesh bags for storing biopsy samples. The size of the mesh bag may be selected according to the size of the plate-shaped cartilage to be produced. The cartilage particles produced in step 1 are contained in the mesh bag and placed in a culture solution. The required number of cartilage particles to be contained in the mesh bag is not particularly limited, and may be the number that can be packed closely in a single layer in the mesh bag, or may be about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, or about 30% of the number of cartilage particles required for close packing in a single layer. When the cartilage particles to be subjected to step 2 are cartilage particles in the middle of formation, it is preferable to adjust the required number of cartilage particles taking into consideration the growth of the cartilage particles themselves during the culture period of step 2.

[0064] In step 2, when culturing by containing cartilage particles in a container through which liquid can pass, it is preferable to culture while flowing the culture liquid. An example of a method for culturing while flowing the culture liquid is a method using a bioreactor. Specifically, a method of placing a container through which liquid can pass and containing cartilage particles into the culture liquid in a bioreactor and flowing the culture liquid is exemplified. The bioreactor used is not particularly limited, and for example, a bioreactor manufactured by Able Co., Ltd. equipped with a magnetic stirrer can be suitably used.

[0065] The culture medium used in step 2 can be the culture medium used in step 1(i) from which the HMG-CoA reductase inhibitor has been removed.

[0066] The culture medium used in step 2 may contain a fibroblast growth factor (FGF) receptor agonist. The FGF receptor (FGFR) agonist may be a factor that binds to FGFR and activates the intracellular signal transduction system. The FGFR agonist may be an agonist of FGFR1, FGFR2, FGFR3 or FGFR4. FGFR agonists include FGFs derived from humans and other animals, as well as functional variants thereof. FGF18 is preferred, and human FGF18 is more preferred. The concentration of the FGFR agonist is 1 ng / mL to 1000 ng / mL, preferably 5 ng / mL to 500 ng / mL, more preferably 10 ng / mL to 200 ng / mL, 100 ng / mL. By using an FGF receptor agonist culture medium, it is possible to promote the merging of adjacent cartilage particles, and as a result, it is possible to promote the formation of plate-shaped cartilage.

[0067] The coalescence of cartilage particles is first achieved by coalescence of the perichondrium-like membrane around the cartilage particle, followed by coalescence of the cartilage at the center of the cartilage particle. Since the inventors have confirmed that FGF signaling promotes the coalescence of the early perichondrium-like membrane, the medium containing the FGFR agonist may be used at an early stage of step 2, and does not need to be used throughout the entire period of step 2. For example, the medium containing the FGFR agonist may be used within 28 days, within 14 days, within 12 days, within 10 days, within 9 days, within 8 days, or within 7 days from the start of step 2.

[0068] In step 2, the culture temperature is not particularly limited, but is about 30 to 40°C, preferably about 37°C, and culture is performed under an atmosphere of CO2-containing air. The CO2 concentration is about 2 to 5%, preferably about 5%. The culture period in step 2 is not particularly limited, so long as it is longer than the period during which adjacent cartilage particles combine with each other to form plate-shaped cartilage. The culture period in step 2 may be 7 days or more, 14 days or more, 21 days or more, 28 days or more, 35 days or more, 42 days or more, 49 days or more, 56 days or more, 63 days or more, or 70 days or more. The culture period in step 2 may be 140 days or less, 133 days or less, 126 days or less, 119 days or less, 112 days or less, 105 days or less, 98 days or less, 91 days or less, 84 days or less, 77 days or less, 70 days or less, 63 days or less, 56 days or less, 49 days or less, 42 days or less, 35 days or less, 28 days or less, 21 days or less, or 14 days or less. The formation of plate-shaped cartilage can be confirmed by confirming with the naked eye that adjacent cartilage particles are united with each other.

[0069] Pharmaceutical Compositions The present invention provides a pharmaceutical composition for repairing damaged cartilage, comprising the plate cartilage of the present invention. The pharmaceutical composition of the present invention may also comprise an effective amount of the plate cartilage of the present invention. Here, the pharmaceutical composition may include medicines, medical devices, and regenerative medicine products. The method of administering the pharmaceutical composition of the present invention includes a method of administering (transplanting) the plate cartilage to a cartilage defect in a damaged cartilage. The plate cartilage to be administered is preferably shaped or trimmed to fit the shape and size of the cartilage defect before administration. The contact area between the cartilage defect and the plate cartilage may be coated with fibrin glue, gelatin gel, collagen gel, hyaluronic acid gel, etc. to prevent the plate cartilage from peeling off. The plate cartilage administered (transplanted) to the cartilage defect may also be fixed with periosteum, etc. The cartilage plate may also be attached to the surrounding cartilage or bone by threading. The subject to which the pharmaceutical composition of the present invention is administered is, for example, a mammal (e.g., mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey, human), preferably a human.

[0070] The diseases to be treated by the pharmaceutical composition of the present invention include, for example, defects of facial cartilage such as nasal cartilage and auricular cartilage, defects of articular cartilage, degenerated or ruptured meniscus, and degenerated intervertebral disc or nucleus pulposus. Defects of articular cartilage include cartilage damage caused by trauma and osteoarthritis. The pharmaceutical composition of the present invention may also be used for the purpose of preventing these diseases.

[0071] Furthermore, the present invention also includes the following inventions in accordance with the above-mentioned pharmaceutical composition of the present invention. (i) A method for repairing cartilage, comprising the step of transplanting the plate cartilage of the present invention into a cartilage defect site. (ii) The plate cartilage of the present invention, for use in cartilage repair. (iii) Use of the plate cartilage of the present invention for producing a pharmaceutical composition for repairing damaged cartilage.

[0072] The present invention further includes the following inventions. (1) A method for promoting fusion of pluripotent stem cell-derived cartilages with each other and / or between pluripotent stem cell-derived cartilage and adult cartilage, the method being characterized by contacting pluripotent stem cell-derived cartilages with each other and / or between pluripotent stem cell-derived cartilage and adult cartilage in the presence of an FGF receptor agonist. (2) The method according to (1) above, wherein the FGF receptor agonist is FGF18. (3) A method for producing a cartilage mass for transplantation, comprising a step of culturing a plurality of cartilage derived from pluripotent stem cells while in contact with each other using a medium containing an FGF receptor agonist. (4) The method according to (3) above, wherein the FGF receptor agonist is FGF18. (5) A cartilage mass for transplantation formed by integrating multiple pluripotent stem cell-derived cartilages. (6) A method for repairing cartilage, comprising the steps of transplanting pluripotent stem cell-derived cartilage into a cartilage defect site, and supplementing the pluripotent stem cell-derived cartilage site with a GF receptor agonist. (7) The method according to (6) above, wherein the FGF receptor agonist is FGF18. (8) The method according to (6) or (7) above for repairing an articular cartilage defect site. (9) The method according to (8) above for repairing articular cartilage defects caused by osteoarthritis. EXAMPLES

[0073] The present invention will be described in detail below with reference to examples, but the present invention is not limited thereto. All experiments were approved by the Institutional Review Board, Institutional Animal Committee (as necessary), and Institutional Biosafety Committee of Kyoto University.

[0074] Example 1: Integration of human iPS cell-derived cartilage particles using a 96-well round-bottom plate 1. Materials and Methods (1)Cell Four human iPS cell lines (409B2, 604B1, 1231A3 and QHJI) provided by Drs. Okita, Nakagawa and Yamanaka of the Center for iPS Cell Research and Application, Kyoto University, were used as human iPS cells. 409B2 was generated from human skin fibroblasts, and 604B1, 1231A3 and QHJI were generated from human peripheral mononuclear cells. Episomal plasmid vectors (pCXLE-hOCT3 / 4-shp53-F, hSK, hUL, EBNA1) were electroporated, and all cells were negative for genomic integration.

[0075] (2) Preparation of human iPS cell-derived cartilage particles Human iPS cell-derived cartilage particles (hereinafter referred to as "cartilage particles") were produced by a partially modified method of the method described in Non-Patent Document 1. Specifically, the cartilage particles were produced by the following method.

[0076] (2-1) Cultivation of human iPS cells 0.5x TrypLE Select was added to the cultured human iPS cells, and after incubation, the cells were detached using a cell scraper. The cells were counted and the number of cells was 0.5-1.0x10 7The cells were transferred to a 100 mL bioreactor (BWV-S10A, Able), and 100 mL of StemFit AK03N (Ajinomoto) supplemented with 10 nM Y-27632 (Wako) was added. The cells were rotated at 60 rpm on a magnetic stirrer (BWS-S03NOS-6, Able) and cultured for 4-7 days at 37°C and 5% CO2. As a result, iPS cell clusters with diameters of 50 μm to 300 μm were obtained.

[0077] (2-2) Chondrocyte induction The iPS cell clusters obtained by the above method were collected and seeded into 10 cm suspension culture dishes (Sumitomo) 4-12 dishes containing 5 mL of chondrocyte differentiation medium. The chondrocyte differentiation medium contained 0.2% FBS (Invitrogen), 1% ITS-X (Invitrogen), 50 μg / mL ascorbic acid (Nakalai), 1 × 10 -4 DMEM (SIGMA) supplemented with M nonessential amino acids (Invitrogen), 1 mM sodium pyruvate (Invitrogen), 10 ng / mL BMP2 (PeproTech), 10 ng / mL TGF-β3 (Wako), 10 ng / mL GDF5 (Biovision), and 1 μM rosuvastatin (Biovision) was used. After seeding, the cells were cultured at 37°C and 5% CO2. After 1-3 days, the medium was replaced with new chondrocyte differentiation medium, and the medium was replaced every 2-5 days and cultured for 2-3 weeks. The iPS cell aggregates gradually adhered to the dish and formed nodules. The resulting nodules either naturally detached from the dish and floated, or were peeled off with a cell scraper and transferred to a 6 cm suspension culture dish (Sumitomo) and cultured at 37°C and 5% CO2. After 1-5 days, the medium was replaced with new chondrocyte differentiation medium. Thereafter, the medium was changed every 2-7 days. When changing the medium, any nodules attached to the dish were removed with a cell scraper and floated. Cartilage particles 12 weeks after the start of differentiation induction were used in the following experiments.

[0078] (3) In vitro cartilage particle coalescence Two cartilage particles were placed in a 96-well round-bottom plate (see Fig. 1(A) and (B)) and cultured in chondrogenic medium (DMEM (Sigma) containing: 1% ITS, 1% FBS, 2 mM L-glutamine (Thermo), 1 x 10 -4 The cells were cultured at 37°C under 5% CO2 with M nonessential amino acids (Thermo), 1 mM sodium pyruvate (Thermo), 50 units penicillin, 50 mg / mL streptomycin, 50 μg / mL ascorbic acid (Nacalai), 10 ng / mL BMP2 (Peprotech), 10 ng / mL TGFβ1 (Peprotech), and 10 ng / mL GDF5 (PTT). A total of 84 wells containing one pair of cartilage particles were prepared. The wells were divided into seven groups (12 pairs per group), and macroscopic and histological analyses were performed on days 0, 3, 7, 14, 28, and 56 after the start of the experiment. The medium was gently changed every day, taking care not to displace the cartilage particles.

[0079] (4) Stop-motion image of the combination Cartilage particles were generated from human iPS cell line 201B7 carrying either CAG-EGFP (317-12) or CAG-mCherry (511-5B) transgenes targeting the AAVS1 locus. The coalescence of EGFP-expressing cartilage particles and mCherry-expressing cartilage particles was observed in a time-lapse manner using a multiphoton laser microscope (Nikon A1R MP+) and analysis software (Nikon NIS Elements). Fluorescent images were recorded every hour for 11.5 consecutive days. Each image in the movie represents 100 ms. Thus, 24 hours corresponds to 2.4 s. The time-lapse images were interrupted several times when the iPS-Cart moved and went out of the field of view.

[0080] (5) Investigation of FGF signaling in the coalescence of cartilage particles Recombinant human FGF18 (PeproTech) was dissolved in phosphate-buffered saline (PBS) to prepare a stock solution (100 μg / mL). A total of 90 pairs of cartilage particles were cultured under the conditions described in (3) above. One pair of cartilage particles was cultured in 0.3 mL of medium per well. 45 pairs of cartilage particles were cultured in medium supplemented with solvent (0.3 μl of PBS), and the other 45 pairs were cultured in medium supplemented with 0.3 μl of FGF18 stock solution (final concentration, 100 ng / mL). The 45 pairs from each group were further divided into three groups and subjected to histological analysis (3, 7, or 14 days after the start of the experiment).

[0081] To further investigate the effect of FGF on coalescence, NVP-BGJ398 (ChemScene LLC), an FGF inhibitor, was used in the culture. NVP-BGJ398 was dissolved in DMSO to prepare a 50 μM stock solution. A total of 30 pairs of cartilage particles were cultured under the above condition (3). Fifteen pairs of cartilage particles were cultured in medium supplemented with solvent (0.3 μl of DMSO), and the other 15 pairs were cultured in medium supplemented with 0.3 μl of NVP-BGJ398 stock solution (final concentration, 50 nM). After 14 days of culture, they were subjected to histological analysis.

[0082] (6)Histological analysis Pairs of cartilage particles were fixed in 4% paraformaldehyde, processed, and embedded in paraffin. To ensure sections covering the area of ​​greatest contact between the two cartilage particles, 20–150 serial sections were made around the junction of the samples (see Fig. 1(C)). The serial section of the greatest contact area was selected and used for further analysis. Sections were stained with hematoxylin-eosin and Safranin O-fast green-iron hematoxylin. Sections were also immunostained with goat anti-type I collagen antibody (Souther Biotech) and anti-type II collagen antibody (Thermo).

[0083] (7) RNA extraction To separate the perichondrium-like membrane of cartilage particles made from human iPS cell line QHJI from the central cartilage, the perichondrium-like membrane was peeled off using tweezers under a stereomicroscope. RNA was extracted separately from the perichondrium-like membrane and central cartilage. The samples were frozen in liquid nitrogen and crushed with a Multi Beads Shocker (Yasui Kikai), and total RNA was extracted using ISOGEN (registered trademark) (Nippon Gene) and purified with RNeasy (registered trademark) (Qiagen).

[0084] (8) RNA sequence analysis The quality of the extracted RNA was assessed with Bioanalyzer 2100 (Agilent Technologies). 1 μg of total RNA was used to generate libraries using TruSeq® Stranded mRNA Library Prep Kit (Illumina) according to the manufacturer's instructions. The quality and quantity of the constructed libraries were assessed with Bioanalyzer 2100 and Qubit® dsDNA HS assay kit (Thermo). Libraries were sequenced with NextSeq® 500 (Illumina) in Single-Read mode for 75 cycles. All read sequences were extracted in FASTQ format using BCL2FASTQ Conversion Software (v2.17.1.14). Adapters, polyA sequences, and low-quality bases at the 3' read ends were trimmed by cutadapt-1.12. Untrimmed and trimmed read sequences were mapped to the human genome hg38 using TopHat-2.1.1. We utilized human gene annotations from GENCODE release v25. For gene expression analysis, expression levels of each gene were normalized to reads per kb of exon per million read sequences (RPKM) using Cufflinks-2.1.1.

[0085] (9) Expression analysis by real-time RT-PCR Total RNA was extracted separately from three perichondrium-like membrane samples, three central cartilage samples, and three whole cartilage particles. 500 ng of total RNA was reverse transcribed into first-strand cDNA using ReverTra Ace® (Toyobo, Tokyo, Japan) and oligo(dT)20 primer. PCR amplification was performed using KAPA PROBE FAST qPCR kit or KAPA SYBR® FAST qPCR kit Master Mix ABI prism (KAPA Biosystems, MA, USA). The PCR primers used are shown in Table 1. RNA expression levels were normalized to the level of GAPDH expression. The amplified products were used to derive a standard curve for real-time quantitative RT-PCR.

[0086] [Table 1]

[0087] (10) Statistical analysis Data are presented as mean and standard deviation. Two-tailed Student's t-test or Steel-Dwass test was used in this study. P values ​​less than 0.05 were considered statistically significant.

[0088] 2.Results (1) Analysis of the in vitro fusion process between cartilage particles generated from the human iPS cell line QHJI The two cartilage particles began to bond after 7 days, and then began to form a firm bond (Figure 2, left column). Histologically, one cartilage particle consisted of a central cartilage and a perichondrium-like membrane tissue surrounding the cartilage (Figure 2, second and third columns, right columns). On day 7, when the cartilage particles started to coalesce, only the perichondrium-like membrane had coalesced, and the cartilage of the cartilage particles was separated by a membrane. A portion of the perichondrium-like membrane at the coalescence site thickened on days 14, 28, and 56 (Figure 2, right column). This suggests that cells in the perichondrium-like membrane had proliferated. On day 14, parts of the cartilage of the two cartilage particles were found to be in contact. The contact became substantial on day 28, and the cartilage coalesced by days 56 and 84 (Figure 2, second and third columns).

[0089] The perichondrium-like membrane of the cartilage particles expressed type I collagen (Figure 3), whereas the central cartilage expressed type II collagen (Figure 3, right column). At day 7, a clear line indicating type I collagen expression was detected at the interface between the two cartilage particles (Figure 3, second column). This indicates that type I collagen expression was at the interface between the two cartilage particles and that the contact was between the perichondrium-like membranes. At day 14, the line of type I collagen expression was disappearing, and by day 84, it had completely disappeared, indicating that the two cartilage particles had merged into cartilage. Type II collagen was expressed at the junction with the same intensity as in the central cartilage (Figure 3, right column).

[0090] (2) Statistical analysis of the coalescence process of cartilage particles For statistical analysis, the degree of coalescence of cartilage particles at each time point was graded. No coalescence was graded 0, coalescence of perichondrium-like membrane was graded 1, and coalescence of cartilage was graded 2 (Table 2). Because the grades are nonparametric data, the Steel-Dwass test was used for statistical analysis. All samples were graded 0 (no coalescence) on day 0 and grade 2 (coalescence of cartilage) on day 56. Grades at time points after day 7 were significantly different from the grades at day 0 (Table 3). Grades at time points before day 14 were significantly different from the grades at day 56 (Table 3).

[0091] [Table 2]

[0092] [Table 3]

[0093] (3) Combination of cartilage particles produced from different human iPS cell lines To confirm that cartilage particles produced from human iPS cell lines other than the human iPS cell line QHJI also coalesce in the same way, cartilage particles were produced from human iPS cell lines 409B2, 604B1, and 1231A3. When two cartilage particles were cultured in contact with each other in a culture dish, they gradually coalesced spontaneously, and the coalescence was also confirmed histologically in Safranin O-Fast Green-Iron Hematoxylin stained specimens of tissue sections on the 60th day (Figure 4).

[0094] (4) Time-lapse images of cartilage particle fusion The time-lapse images of cartilage particle coalescence showed that cells from the perichondrium-like membrane migrated to the perichondrium-like membrane of other cartilage particles, suggesting that the perichondrium-like membrane plays a substantial role in coalescence.

[0095] (5) mRNA expression analysis of perichondrium-like membrane of cartilage particles Histological analysis of cartilage particles stripped of the perichondrium-like membrane confirmed the separation of the perichondrium-like membrane and the central cartilage (Figure 5). RNA was extracted separately from the perichondrium-like membrane and central cartilage and subjected to RNA-seq analysis. The RPKM values ​​of COL2A1 and SOX9 were higher in the central cartilage than in the perichondrium-like membrane, whereas the RPKM value of COL1A1 was the opposite (Table 4). This result indicates that the sampling and RNA-seq experiments were successfully performed. Among the differentially expressed genes, FGF18 mRNA was more highly expressed in the perichondrium-like membrane than in the central cartilage (Tables 4, 5, Figure 6). Real-time RT-PCR expression analysis confirmed that the expression of these genes was different between the perichondrium-like membrane and the central cartilage (Figure 7).

[0096] [Table 4]

[0097] [Table 5]

[0098] (6) Regulation of cartilage particle coalescence by FGF18 Addition of FGF18 to the medium promoted the coalescence of cartilage particles. On day 14, the vehicle group showed minimal coalescence of cartilage particles, whereas the FGF18 group showed substantial coalescence (Figure 8). Histologically, the FGF18 group showed thick adhesion by a perichondrium-like membrane. On day 14, a significant difference was observed in the coalescence grade of cartilage particles between the vehicle group and the FGF18 group (Table 6).

[0099] [Table 6]

[0100] Addition of the FGFR inhibitor NVP-BGJ398 to the culture medium inhibited the coalescence of cartilage particles. On day 14, 11 out of 15 pairs of cartilage particles in the vehicle group (Vehicle) coalesced, whereas no cartilage particles in the NVP-BGJ398 group coalesced (Figure 9, Table 7).

[0101] [Table 7]

[0102] Example 2: Combination of human iPS cell-derived cartilage particles using a mesh bag Cartilage particles were prepared from human iPS cell line QHJI using the same method as in the materials and methods (2) of Example 1, and cartilage particles 4 weeks after the start of differentiation induction were used. Cartilage particles were placed in a mesh bag for storing biopsy samples (Eiken Chemical, product name: Sample Bag, product number: KA1000, 45 mm x 74 mm). The amount of cartilage particles was set to 30% to 40% of the area of ​​the mesh bag (30 mm x 50 mm) when the cartilage particles were moved to one corner of the mesh bag. The mesh bag containing the cartilage particles was placed in a bioreactor (BWV-S03A, Able) containing the chondrogenic medium described in the materials and methods (3) of Example 1, and cultured for 4 weeks under conditions of 37 °C and 5% CO2 while stirring the culture solution. When the cartilage particles were removed from the mesh bag, cartilage that had been united into a plate shape was formed (Figure 10). Figure 11 shows a tissue section of the obtained plate-shaped cartilage stained with Safranin O-Fast Green-Iron Hematoxylin. The tissue image of the cartilage was that of cells scattered in the cartilage ECM stained with Safranin O.

[0103] Example 3: Integration of human iPS cell-derived cartilage particles using a frame Cartilage particles were prepared from human iPS cell line 409B2 using the same method as in Materials and Methods (2) of Example 1. However, in this Example, rosuvastatin was not added. Cartilage particles were used 8 weeks after the start of differentiation induction. The chondrogenic medium described in Materials and Methods (3) of Example 1 was placed in a 3.5 cm culture dish, a star-shaped frame was placed in it, cartilage particles were placed tightly in the frame, and the culture dish was left to stand for 16 weeks at 37°C and 5% CO2. The cartilage particles in the frame coalesced to form star-shaped plate-like cartilage (Figure 12).

[0104] Example 4: Integration of human iPS cell-derived cartilage particles using a transwell Cartilage particles were prepared from the human iPS cell line QHJI using the same method as in the materials and methods (2) of Example 1, and the cartilage particles were used 4 weeks after the start of differentiation induction. Polycarbonate transwells and polyester transwells were used. The chondrogenic medium described in the materials and methods (3) of Example 1 was placed in each well of a 24-well plate (Corning), a transwell (membrane diameter 6.5 mm) was placed in each well, cartilage particles were placed therein without any gaps, and the plate was left to stand, and culture was performed for 5 weeks under conditions of 37°C and 5% CO2. The cartilage particles within the frame were united to form plate-shaped cartilage (Figure 13).

[0105] The present invention is not limited to the above-mentioned embodiments and examples, and various modifications are possible within the scope of the claims. The technical scope of the present invention also includes embodiments obtained by appropriately combining the technical means disclosed in the different embodiments. In addition, all academic documents and patent documents described in this specification are incorporated herein by reference.

Claims

[Claim 1] A method for producing plate-shaped cartilage formed by combining a plurality of cartilage particles derived from pluripotent stem cells, comprising the steps of: Step 1: A step of producing cartilage particles by inducing differentiation of pluripotent stem cells; and Step 2: Cultivating the number of cartilage particles required for forming plate-shaped cartilage in a state in which adjacent cartilage particles can contact each other; wherein in step 2, a required number of cartilage particles are placed in a container through which liquid can pass, and the particles are cultured while the culture medium is flowing.

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