Method for producing cartilage-like tissue from pluripotent stem cells and method for regulating hardness of said cartilage-like tissue

Abstract

The present invention provides a method for producing a cartilage-like tissue from pluripotent stem cells, the method comprising a step for culturing pluripotent stem cells in a cartilage differentiation culture medium and a step for increasing the ascorbic acid concentration of the cartilage differentiation culture medium. The present invention also provides a method for regulating the hardness of induced cartilage-like tissue differentiated from pluripotent stem cells, the method comprising a step for culturing pluripotent stem cells in a cartilage differentiation culture medium and a step for increasing the ascorbic acid concentration of the cartilage differentiation culture medium.

Description

Method for producing cartilage-like tissue from pluripotent stem cells and method for regulating the stiffness of said cartilage-like tissue

[0001] The present invention relates to a method for producing cartilage-like tissue from pluripotent stem cells and a method for regulating the stiffness of the cartilage-like tissue.

[0002] Articular cartilage covers the ends of bones and ensures smooth joint movement. It can be damaged by trauma and degenerate due to aging and inflammation. Damage and degeneration of articular cartilage can cause pain during movement, a condition known as osteoarthritis, affecting an estimated 20 million people in Japan. Articular cartilage has poor repair capabilities, meaning it cannot heal once damaged or degenerated. Furthermore, no drugs are available to treat the condition. Therefore, regenerative therapies are expected. To date, transplantation of chondrocytes or mesenchymal (stem) cells has been used to treat articular cartilage damage and degeneration, but there is little evidence that the transplanted cells themselves form repair tissue. The repair mechanism is thought to be the effect of repair factors transiently produced by the transplanted cells, which act on the host's (patient's) own cells to promote the induction of repair tissue (known as the cytokine effect, trophic effect, or paracrine effect). However, the effectiveness of these methods has been limited. The host's (patient's) own cells have limited repair capacity, preventing repair with normal cartilage; instead, repair tissue contains fibrous scar tissue.

[0003] The present inventors have reported methods for inducing differentiation of human iPS cells into cartilage (Non-Patent Documents 1-4) and have filed patent applications (Patent Documents 1-3). Furthermore, the present inventors have demonstrated in an animal model that allogeneic iPS cell-derived cartilage can be engrafted into articular cartilage defects and directly form repair tissue (Non-Patent Document 5).

[0004] The present inventors have actually experienced that when human iPS cells are induced to differentiate into cartilage tissue, poor chondrogenesis sometimes occurs. However, the mechanism behind this poor chondrogenesis remains unknown, and no treatment for this problem is known. While iPS cell-derived cartilage is expected to be a regenerative therapy for damaged or degenerated articular cartilage, the instability of its production poses a serious weakness in commercializing it. Therefore, it is necessary to develop a method for stably producing iPS cell-derived cartilage.

[0005] WO2015 / 079117 A1WO2016 / 133208 A1WO2020 / 017575 A1

[0006] Yamashita, A., Yoshitomi, H., Kihara, S., Toguchida, J., and Tsumaki, N., Culture substrate-associated YAP inactivation underlies chondrogenic differentiation of human induced pluripotent stem cells. Stem Cells Transl Med, 10(1): p. 115-127, 2021.Yamashita, A., M. Morioka, Y. Yahara, M. Okada, T. Kobayashi, S. Kuriyama, S. Matsuda, and N. Tsumaki, Generation of Scaffoldless Hyaline Cartilaginous Tissue from Human iPSCs. Stem Cell Reports, 4(3): p. 404-418, 2015.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(7519): p. 507-511, 2014.Chen, X., A. Yamashita, M. Morioka, T. Senba, T. Kamatani, A. Watanabe, A. Kosai, and N. Tsumaki, Integration Capacity of Human Induced Pluripotent Stem Cell-Derived Cartilage. Tissue Light Part A, 25(5-6): p. 437-445, 2019.Abe, K., Yamashita, A., Morioka, M., Horike, N., Takei, Y., Koyamatsu, S. (2019)., Okita, K., Matsuda, S., and Tsumaki, N., Engraftment of allogeneic iPS cell-derived cartilage organoid in a primate model of articular cartilage defect. Nat Commun, 14(1): p. 804, 2023.

[0007] An objective of the present invention is to provide a method for stably producing cartilage-like tissue by suppressing the occurrence of defective chondrogenesis during differentiation induction from pluripotent stem cells into cartilage-like tissue.Another objective of the present invention is to provide a method for adjusting the hardness and surface smoothness of cartilage-like tissue produced from pluripotent stem cells, and to provide said cartilage-like tissue.

[0008] In order to solve the above-mentioned problems, the present invention encompasses the following inventions. [1] A method for producing cartilage-like tissue from pluripotent stem cells, comprising the steps of culturing pluripotent stem cells in a chondrogenic differentiation medium and increasing the ascorbic acid concentration of the chondrogenic differentiation medium. [2] The method according to [1] above, in which the increased ascorbic acid concentration and the timing for increasing the ascorbic acid concentration are determined by the following preliminary studies: (1) confirming the occurrence of fuzzing in the periphery of the cartilage-like tissue after initiating differentiation of the pluripotent stem cells into chondrogenic tissue, (2) identifying the time point at which fuzzing occurs, (3) setting the increased ascorbic acid concentration based on the ascorbic acid concentration in the medium at the time of fuzzing, and (4) setting the time point at which the ascorbic acid concentration is increased to within a range of two weeks before or after the time point at which fuzzing occurs. [3] The method according to [1] or [2] above, in which the ascorbic acid concentration in the culture medium after the increase in ascorbic acid concentration is 150% or more of the ascorbic acid concentration before the increase. [4] A method for regulating the hardness of cartilage-like tissue induced to differentiate from pluripotent stem cells, the method comprising the steps of culturing pluripotent stem cells in a chondrogenesis medium and increasing the ascorbic acid concentration in the chondrogenesis medium. [5] The method according to [4] above, wherein the ascorbic acid concentration in the culture medium is increased at least three weeks after the start of differentiation induction. [6] The method according to [5] above, further comprising suppressing the emergence of non-target cells. [7] The method according to [4] above, further comprising adjusting the smoothness of the surface of the cartilage-like tissue. [8] The method according to [7] above, wherein the ascorbic acid concentration in the culture medium is increased at least seven weeks after the start of differentiation induction. [9] A cartilage-like tissue induced to differentiate from pluripotent stem cells, the cartilage-like tissue being used to treat intervertebral disc injury or articular cartilage injury.

[10] The cartilage-like tissue according to [9] above, wherein the cartilage-like tissue for treating intervertebral disc injury maintains its shape and has a hardness that prevents it from deforming when placed in the space of the nucleus pulposus within the annulus fibrosus of the intervertebral disc.

[11] The cartilage-like tissue for treating intervertebral disc injury described in [9] or

[10] above, which has a hardness score of 2 to 6 as described in Table 1 of the specification.

[12] The cartilage-like tissue for treating articular cartilage damage according to [9] above, which has a hardness such that it is not destroyed when transplanted into the site of articular cartilage damage, or a hardness such that it can be sewn with thread to the tissue at the site of articular cartilage damage.

[13] The cartilage-like tissue for treating articular cartilage damage according to [9] or

[12] above, which has a hardness of a score of 2 to 6 as set forth in Table 1 of the specification.

[14] The cartilage-like tissue according to any of [9] to

[13] above, which has an approximately spherical or lamellar shape.

[0009] The present invention provides a method for suppressing the occurrence of defective chondrogenesis and stably producing cartilage-like tissue during differentiation induction from pluripotent stem cells into cartilage-like tissue. The present invention also provides a method for regulating the hardness and surface smoothness of cartilage-like tissue produced from pluripotent stem cells, and the cartilage-like tissue.

[0010] Figure 1 shows the results of inducing differentiation of human iPS cells into cartilage-like tissue using media with and without antibiotics, and observing the appearance of the cartilage-like tissue 35 and 42 days after the start of differentiation induction. Human iPS cells were induced to differentiate into cartilage-like tissue using two types of media supplemented with different lots of fetal bovine serum (FBS), and shows the appearance 6 weeks after the start of differentiation induction and Safranin O-fast green-iron hematoxylin-stained tissue images 8 weeks after the start of differentiation induction. Figure 2 shows the results of observing the presence of non-target cells 14 weeks after the start of differentiation induction in layered cartilage-like tissue formed by combining cartilage-like tissues obtained by inducing differentiation from human iPS cells. Figure 3 shows the results of measuring the expression of marker genes for non-target cells 14 weeks after the start of differentiation induction in layered cartilage-like tissue formed by combining cartilage-like tissues obtained by inducing differentiation from human iPS cells.

[0033] Figure 1 shows the results of comparing appearance observation and evaluation of hardness 12 weeks after the start of differentiation induction under differentiation induction conditions in which the ascorbic acid concentration in the chondrogenic differentiation medium was increased to 200% or not increased at different times during the process of forming layered cartilage-like tissue by combining cartilage-like tissue obtained by differentiation induction of human iPS cells. Figure 2 shows the results of appearance observation and evaluation of hardness 16 weeks after the start of differentiation induction when the ascorbic acid concentration in the chondrogenic differentiation medium was increased to 200% 8 to 12 weeks after the start of differentiation induction during the process of forming layered cartilage-like tissue by combining cartilage-like tissue obtained by differentiation induction of human iPS cells. Figure 3 shows the results of time-dependent observation of the process of inducing differentiation of human iPS cells into cartilage-like tissue, showing a case in which the cartilage-like tissue was well formed without fuzzing around the periphery during the differentiation induction process. Fig. 1 shows the results of observing the process of inducing differentiation from human iPS cells into cartilage-like tissue over time, and is a diagram showing a case in which fuzzing occurred around the periphery of the cartilage-like tissue 5 weeks after the start of differentiation induction, and the degree of fuzzing progressed mildly. Fig. 2 shows the results of observing the process of inducing differentiation from human iPS cells into cartilage-like tissue over time, and is a diagram showing a case in which fuzzing occurred around the periphery of the cartilage-like tissue 4 weeks after the start of differentiation induction, and the degree of fuzzing progressed severely.Figure 1 shows the results of measuring the expression of cartilage marker genes 14 weeks after the start of differentiation induction in layered cartilage-like tissue formed by combining cartilage-like tissue obtained by inducing differentiation from human iPS cells. Figure 2 shows the results of measuring the amount of glycosaminoglycan (GAG) 14 weeks after the start of differentiation induction in layered cartilage-like tissue formed by combining cartilage-like tissue obtained by inducing differentiation from human iPS cells. Figure 3 shows images of safranin O-fast green-iron hematoxylin-stained tissue, hematoxylin-eosin-stained images, type I collagen immunostained images, and type II collagen immunostained images of a layered cartilage-like tissue section 14 weeks after the start of differentiation induction in layered cartilage-like tissue formed by combining cartilage-like tissue obtained by inducing differentiation from human iPS cells. Figure 1 shows Safranin O-fast green-iron hematoxylin-stained tissue images, hematoxylin-eosin-stained images, type I collagen immunostained images, and type II collagen immunostained images of layered cartilage-like tissue sections 16 weeks after the start of differentiation induction, when the ascorbic acid concentration in the chondrogenic differentiation medium was increased to 200% 8 to 12 weeks after the start of differentiation induction in the process of forming layered cartilage-like tissue by combining cartilage-like tissues obtained by differentiation induction of human iPS cells. Figure 2 shows the results of appearance observation and evaluation of hardness 12 and 17 weeks after the start of differentiation induction, when the ascorbic acid concentration in the chondrogenic differentiation medium was increased to 200% 8 to 12 weeks after the start of differentiation induction in the process of forming layered cartilage-like tissue by combining cartilage-like tissues obtained by differentiation induction of human iPS cells. Figure 1 shows the results of measuring the expression of cartilage marker genes and non-target cell marker genes 17 weeks after the start of differentiation induction when the ascorbic acid concentration in the chondrogenic differentiation medium was increased to 200% 8 to 12 weeks after the start of differentiation induction during the process of forming layered cartilage-like tissue by combining cartilage-like tissues obtained by differentiation induction of human iPS cells. Figure 2 shows the results of measuring the amount of glycosaminoglycan (GAG) 17 weeks after the start of differentiation induction when the ascorbic acid concentration in the chondrogenic differentiation medium was increased to 200% 8 to 12 weeks after the start of differentiation during the process of forming layered cartilage-like tissue by combining cartilage-like tissues obtained by differentiation induction of human iPS cells.This figure shows Safranin O-fast green-iron hematoxylin-stained tissue images, hematoxylin-eosin-stained images, type I collagen immunostained images, and type II collagen immunostained images of layered cartilage-like tissue sections 17 weeks after the start of differentiation induction when the ascorbic acid concentration in the chondrogenesis medium was increased to 200% 8 to 12 weeks after the start of differentiation induction in the process of forming layered cartilage-like tissue by combining cartilage-like tissues obtained by differentiation induction of human iPS cells. This figure shows the results of observing the cartilage-like tissues of each group 4 weeks after the start of differentiation induction, after human iPS cells were induced to differentiate into cartilage-like tissue under the conditions shown in Table 3. This figure shows Safranin O-stained images of cartilage-like tissue sections of each group 10 weeks after the start of differentiation induction, after human iPS cells were induced to differentiate into cartilage-like tissue under the conditions shown in Table 3. This figure shows HE-stained images of cartilage-like tissue sections of each group 10 weeks after the start of differentiation induction, after human iPS cells were induced to differentiate into cartilage-like tissue under the conditions shown in Table 3. 21 shows immunostained images of type I collagen from cartilage-like tissue sections from each group 10 weeks after the start of differentiation induction of human iPS cells into cartilage-like tissue under the conditions shown in Table 3. 22 shows immunostained images of type II collagen from cartilage-like tissue sections from each group 10 weeks after the start of differentiation induction of human iPS cells under the conditions shown in Table 3. 23 shows the results of cluster analysis using Seurat after single-cell RNA sequencing of two types of cartilage-like tissue formed by differentiation induction up to 10 weeks under the same conditions as Groups A and G in Example 7, and two types of undifferentiated human iPS cells. 24 shows the right panel of Figure 21 divided by sample. 25 shows the percentage (%) of cells belonging to each cluster in each sample. 26 shows the results of examining the expression of marker genes (LIN28A, PAX6, MITF, COL2A1, ACAN, COL1A1, COL1A2) for each cell type using a feature plot. This figure shows the results of gene set enrichment analysis based on differentially expressed genes (DEGs) determined between cluster 2 and undifferentiated iPS cells in clusters 1 and 3.This figure shows the results of gene set enrichment analysis based on differentially expressed genes (DEGs) determined between cluster 0 and undifferentiated iPS cells in clusters 1 and 3.

[0011] The present inventors have discovered that when chondrogenesis defects occur during differentiation induction of pluripotent stem cells into cartilage-like tissue, the peripheral area of ​​the formed immature cartilage-like tissue may appear fuzzy (also appearing as if the cartilage-like tissue has collapsed or ruptured) approximately 5 weeks after the start of differentiation induction. It was found that the degree of fuzziness around the periphery of the cartilage-like tissue increases with continued culture, resulting in chondrogenesis defects. In other words, fuzziness around the periphery of the immature cartilage-like tissue is thought to be one of the causes of chondrogenesis defects during differentiation induction of pluripotent stem cells into cartilage-like tissue. Here, the results of observing the process of differentiation induction of human iPS cells into cartilage-like tissue over time are shown in Figures 7 to 9. Fig. 7 shows the results of observation of a case in which no fuzzing occurred around the periphery of the cartilage-like tissue and good cartilage-like tissue was formed, Fig. 8 shows the results of observation of a case in which fuzzing occurred around the periphery of the cartilage-like tissue 5 weeks after the start of differentiation induction, and the degree of fuzzing subsequently progressed mildly until poor cartilage formation was achieved, and Fig. 9 shows the results of observation of a case in which fuzzing occurred around the periphery of the cartilage-like tissue 4 weeks after the start of differentiation induction, and the degree of fuzzing subsequently progressed severely until poor cartilage formation was achieved. The present inventors have experienced the occurrence of fuzzing around the periphery of immature cartilage-like tissue depending on whether or not an antibiotic was added to the chondrogenesis medium (Example 1) and on the type of fetal bovine serum (FBS) added to the chondrogenesis medium (Reference Example 1), but the occurrence of fuzzing varied with each experiment and was difficult to predict.

[0012] Therefore, the inventors varied the conditions for inducing differentiation of pluripotent stem cells into cartilage-like tissue, and after much trial and error, discovered that by increasing the concentration of ascorbic acid in the cartilage differentiation medium during the differentiation induction process, the fuzziness that had developed could be repaired, and that fuzziness could be prevented, allowing cartilage-like tissue to be produced stably.

[0013] [Method for Producing Cartilage-Like Tissue] The present invention provides a method for producing cartilage-like tissue from pluripotent stem cells (hereinafter referred to as "the production method of the present invention"). The production method of the present invention may include the steps of culturing pluripotent stem cells in a chondrogenesis medium and increasing the ascorbic acid concentration of the chondrogenesis medium. As used herein, "cartilage-like tissue" refers to tissue formed from pluripotent stem cell-derived chondrocytes and an extracellular matrix secreted by the chondrocytes. The production method of the present invention can suppress poor chondrogenesis caused by the occurrence of fuzzing around the periphery of immature cartilage-like tissue during the differentiation induction process, and can stably produce cartilage-like tissue from pluripotent stem cells. Note that fuzzing around the periphery of immature cartilage-like tissue may also be referred to as rupture or collapse of the immature cartilage-like tissue, based on its appearance.

[0014] In the production method of the present invention, the step of culturing pluripotent stem cells in a chondrogenesis medium can be carried out using a known method for inducing differentiation of pluripotent stem cells into cartilage-like tissue, such as those described in Patent Documents 1 to 3 and Non-Patent Documents 1 to 4.

[0015] Pluripotent stem cells that can be used in the production method of the present invention are not particularly limited as long as they have pluripotency, which allows them to differentiate into all cells present in the body, and also have the ability to proliferate. Examples 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, and pluripotent cells derived from cultured fibroblasts or bone marrow stem cells (Muse cells). Preferred pluripotent stem cells are ES cells, ntES cells, and iPS cells.

[0016] (A) Embryonic stem cells ES cells are stem cells that are established from the inner cell mass of early mammalian embryos (e.g., blastocysts) such as humans and mice, and have the ability to proliferate through pluripotency and self-renewal.

[0017] ES cells are embryonic stem cells derived from the inner cell mass of the blastocyst, a post-morula stage embryo (8-cell stage of a fertilized egg). They possess the ability to differentiate into all cell types that constitute an adult, known as pluripotency, and the ability to proliferate through self-renewal. ES cells were discovered in mice in 1981 (MJ Evans and MH Kaufman, Nature (1981) 292:154-156). ES cell lines were subsequently established in humans, monkeys, and other primates (JA Thomson et al., Science (1998) 282:1145-1147; JA Thomson et al., PNAS (1995) 92:7844-7848; JA Thomson et al., Biol Reprod (1996) 55:254-259; JA Thomson and VS Marshall, Curr Top Dev Biol (1998) 38:133-165).

[0018] ES cells can be established using methods known in the art. For example, they can be established by isolating 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. Furthermore, 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, for example, in U.S. Patent No. 5,843,780; J.A. Thomson et al., PNAS (1995) 92:7844-7848; J.A. Thomson et al., Science (1998) 282:1145-1147; H. Suemori et al., BBRC (2006) 345:926-932; M. Ueno et al., PNAS (2006) 103:9554-9559; H. Suemori et al., Dev Dyn (2001) 222:273-279; H. Kawasaki et al., PNAS (2002) 99:1580-1585; I. Klimanskaya, et al., Nature (2006) 444:481-485.

[0019] Culture methods for producing ES cells are known in the art. Human ES cells can be maintained in a culture medium, such as DMEM / F-12 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, at 37°C in a humidified atmosphere of 2% CO2 and 98% air (O ​​Fumitaka et al., Nat Biotechnol (2008) 26:215-224). ES cells may be passaged every 3 to 4 days using, for example, 0.25% trypsin and 0.1 mg / mL collagenase IV in PBS containing 1 mM CaCl2 and 20% KSR.

[0020] 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, and ECAD (E Kroon et al., Nat Biotechnol (2008) 26:443-452).

[0021] Mouse ES cell lines established by Ingenious Targeting Laboratory, Inc., RIKEN, and other institutions are available. Human ES cell lines established by the National Institutes of Health (NIH), RIKEN, Kyoto University, and Cellartis are available. Examples of available ES cell lines include NIH strains CHB-1 to CHB-12, RUES1, RUES2, and HUES1 to HUES28; WiCell Research Institute strains WA01(H1) and WA09(H9); and RIKEN strains KhES-1, KhES-2, KhES-3, KhES-4, KhES-5, SSES1, SSES2, and SSES3. Furthermore, KhES-1, KhES-2, KhES-3, and KthES11 are available from the Institute of Medical Biology, Kyoto University (Kyoto, Japan).

[0022] (B) Spermatogonial stem cells. Spermatogonial stem cells are pluripotent stem cells derived from the testis and are the source of spermatogenesis. Similar to embryonic stem cells, these cells can be induced to differentiate into various cell lineages. For example, when transplanted into mouse blastocysts, chimeric mice can be produced (M Kanatsu-Shinohara et al., Biol Reprod (2003) 69:612-616; K Shinohara et al., Cell (2004) 119:1001-1012). They are capable of self-renewal in culture medium containing glial cell line-derived neurotrophic factor (GDNF). Furthermore, spermatogonial stem cells can be obtained by repeated passage under culture conditions similar to those for embryonic stem cells (Takebayashi M et al., Experimental Medicine, 2008, Vol. 26, No. 5 (Supplementary Issue), pp. 41-46, Yodosha, Tokyo, Japan).

[0023] (C) Embryonic germ cells Embryonic germ cells are cells established from primordial germ cells during the fetal stage 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., Cell (1992) 70:841-847; JL Resnick et al., Nature (1992) 359:550-551).

[0024] (D) Induced pluripotent stem cells Induced pluripotent stem (iPS) cells are artificial stem cells derived from somatic cells that can be generated by introducing specific reprogramming factors into somatic cells in the form of DNA or protein. They have properties similar to those of ES cells, such as pluripotency and the ability to proliferate through self-renewal (K Takahashi and S Yamanaka, Cell (2006) 126:663-676; K Takahashi et al., Cell (2007) 131:861-872; J Yu et al., Science (2007) 318:1917-1920; M Nakagawa, M et al., Nat Biotechnol (2008) 26:101-106; International Publication No. WO2007 / 069666). Reprogramming factors may be composed of genes specifically expressed in ES cells, their gene products or non-coding RNA, or genes that play an important role in maintaining the undifferentiated state of ES cells, their gene products or non-coding RNA, or low-molecular-weight compounds. Examples of genes included in reprogramming factors 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, and Glis1. These reprogramming factors may be used alone or in combination. Combinations of reprogramming factors include those described in International Publications 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, and WO2009 / 1262 51, WO2009 / 126655, WO2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 050626, WO2010 / 056831, WO2010 D Huangfu et al., Nat Biotechnol (2008) 26:795-797, Y Shi et al., Cell Stem Cell (2008) 2: 525-528, S Eminli et al., Stem Cells(2008)26:2467-2474、D Huangfu et al., Nat Biotechnol(2008)26:1269-1275、Y Shi et al., Cell Stem Cell(2008)3, 568-574、Y Zhao et al., Cell Stem Cell(2008)3:475-479、A Marson Cell(2008)3, 132-135、B Feng et al., Nat Cell Biol(2009)11:197-203、RL Judson et al., Nat Biotech(2009)27:459-461、CA Lyssiotis et al., PNAS(2009)106:8912-8917、JB Kim et al. Nature(2009)461:649-643、JK Ichida et al., Cell Stem Cell(2009)5:491-503、JC Heng et al., Cell Stem Cell(2010)6:167-74、J Han et al., Nature(2010)463:1096-100、P Mali et al., Stem Cells(2010)28:713-720、M Maekawa et al. Nature(2011)(474:225-9)

[0025] The reprogramming factors include histone deacetylase (HDAC) inhibitors (e.g., small molecule inhibitors such as valproic acid (VPA), trichostatin A, sodium butyrate, MC1293, and M344, 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))), MEK inhibitors (e.g., PD184352, PD98059, U0126, SL327, and PD0325901), glycogen synthase inhibitors (e.g., PD184352, PD98059, U0126, SL327, and PD0325901), and the like. kinase-3 inhibitors (e.g., Bio and CHIR99021), DNA methyltransferase inhibitors (e.g., 5-azacytidine), histone methyltransferase inhibitors (e.g., small molecule inhibitors such as BIX-01294, nucleic acid expression inhibitors such as siRNA and shRNA against Suv39hl, Suv39h2, SetDBl, and 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 against p53), ARID3A inhibitors (e.g., siRNA and shRNA against ARID3A), miRNAs such as miR-291-3p, miR-294, miR-295, and mir-302, Wnt signaling inhibitors (e.g., soluble These factors also include factors used to improve establishment efficiency, such as Wnt3a), neuropeptide Y, prostaglandins (e.g., prostaglandin E2 and prostaglandin J2), hTERT, SV40LT, UTF1, IRX6, GLIS1, PITX2, and DMRTBl, and in this specification, these factors used to improve establishment efficiency will not be distinguished from reprogramming factors.

[0026] When the reprogramming factor is in the form of a protein, it may be introduced into somatic cells by techniques such as lipofection, fusion with a cell membrane-permeable peptide (eg, HIV-derived TAT and polyarginine), or microinjection.

[0027] On the other hand, in the case of DNA, for example, vectors such as viruses, plasmids, artificial chromosomes, lipofection, liposomes, microinjection, etc. can be introduced into somatic cells. Examples of viral vectors include retroviral vectors, lentiviral vectors (Cell (2006) 126: 663-676; Cell (2007) 131: 861-872; Science (2007) 318: 1917-1920), adenoviral vectors (Science (2008) 322: 945-949), adeno-associated virus vectors, Sendai virus vectors (International Publication WO2010 / 008054), etc. Examples of artificial chromosome vectors include human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC, PAC), etc. As plasmids, mammalian cell plasmids can be used (Science (2008) 322: 949-953). The vector may contain regulatory sequences such as a promoter, enhancer, ribosome binding sequence, terminator, polyadenylation site, etc. to enable expression of the nuclear reprogramming substance, and may further contain, as necessary, 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, etc., a reporter gene sequence such as green fluorescent protein (GFP), β-glucuronidase (GUS), etc. Furthermore, the above vector may have LoxP sequences before and after the gene encoding the reprogramming factor or the promoter and the gene encoding the reprogramming factor that binds to it, in order to excise both of them after introduction into somatic cells.

[0028] In addition, when in the form of RNA, 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 (L Warren, Cell Stem Cell (2010) 7:618-630).

[0029] Examples of culture media for inducing iPS cells include DMEM, DMEM / F12, or DME culture media containing 10-15% FBS (these culture media may further contain LIF, penicillin / streptomycin, puromycin, L-glutamine, nonessential amino acids, β-mercaptoethanol, or the like, as appropriate); or commercially available culture media such as a culture medium for mouse ES cells (TX-WES culture medium, Thrombo-X), a culture medium for primate ES cells (Primate ES / iPS cell culture medium, ReproCell), and serum-free pluripotent stem cell maintenance media (e.g., mTeSR (Stemcell Technology), Essential 8 (Life Technologies), StemFit AK03 (AJINOMOTO)).

[0030] As an example of the culture method, for example, somatic cells are contacted with reprogramming factors in DMEM or DMEM / F12 culture medium containing 10% FBS 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 starting about 10 days after contacting the somatic cells with the reprogramming factors, they are cultured in a bFGF-containing culture medium for primate ES cell culture, and iPS-like colonies can be generated about 30 to 45 days or more after the contact.

[0031] Alternatively, ES-like colonies can be generated after about 25 to 30 days or more by culturing on feeder cells (e.g., mitomycin C-treated STO cells, SNL cells, etc.) in 10% FBS-containing DMEM 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. Desirably, somatic cells to be reprogrammed themselves (K. Takahashi et al., PLoS One (2009) 4:e8067 or International Publication WO2010 / 137746) or extracellular matrix (e.g., Laminin-5 (International Publication WO2009 / 123349) and Matrigel (BD)) are used instead of feeder cells.

[0032] Other examples include culturing iPS cells using serum-free media (Sun N, et al., PNAS (2009) 106:15720-15725). Furthermore, to increase the efficiency of establishment, iPS cells may be established under hypoxic conditions (oxygen concentration of 0.1% or more and 15% or less) (Yoshida Y et al., Cell Stem Cell (2009) 5:237-241 or International Publication WO2010 / 013845).

[0033] During the culture, the culture medium is replaced with fresh medium once a day from the second day onwards. The number of somatic cells used for nuclear reprogramming is not limited, but it is recommended to use a 100 cm culture dish. 2 Approximately 5 x 10 3 ~Approx. 5×10 6 It is the range of cells.

[0034] iPS cells can be selected based on the morphology of the colonies they form. On the other hand, if a drug-resistance gene that is expressed in conjunction with a gene expressed during somatic cell reprogramming (e.g., Oct3 / 4 or Nanog) is introduced as a marker gene, established iPS cells can be selected by culturing them in a culture medium containing the corresponding drug (selective culture medium). Furthermore, iPS cells can be selected by observation under a fluorescent microscope if the marker gene is a fluorescent protein gene, by adding a luminescent substrate if the marker gene is a luciferase gene, or by adding a chromogenic substrate if the marker gene is a chromogenic enzyme gene.

[0035] As used herein, the term "somatic cells" 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 (offspring) somatic cells, neonatal (offspring) somatic cells, and mature, healthy or diseased somatic cells. They also include primary culture cells, subcultured 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 (e.g., skin cells), hair cells, hepatocytes, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells (e.g., exocrine pancreatic cells), brain cells, lung cells, kidney cells, and adipocytes.

[0036] Furthermore, when iPS cells and / or cells differentiated therefrom are used 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" HLA type means that the HLA genotype matches to an extent that the transplanted cells can engraft when transplanted, for example, somatic cells with an HLA type that matches the main HLA (three loci: HLA-A, HLA-B, and HLA-DR, or four loci including HLA-C).

[0037] As the induced pluripotent stem cell line, various iPS cell lines established by NIH, RIKEN, Kyoto University, etc. may be used. For example, human iPS cell lines include RIKEN's HiPS-RIKEN-1A line, HiPS-RIKEN-2A line, HiPS-RIKEN-12A line, and Nips-B2 line, and Kyoto University's Ff-WJ-18 line, Ff-I01s01 line, Ff-I01s02 line, Ff-I01s04 line, Ff-I01s06 line, Ff-I14s03 line, and Ff-I14s line. Examples of such cell lines include strains 04, QHJI01s01, QHJI01s04, QHJI14s03, QHJI14s04, AK5, TkDN-Sev2, 692D2, 253G1, 201B7, 409B2, 454E2, 606A1, 610B1, 648A1, 1231A3, 1390D4, and 1390C1. Alternatively, clinical-grade cell lines provided by Kyoto University, Cellular Dynamics International, etc., and research and clinical cell lines prepared using such cell lines may also be used.

[0038] (E) Cloned embryo-derived ES cells obtained by nuclear transfer (ntES cells) ntES cells are ES cells derived from cloned embryos by nuclear transfer technology and have almost the same properties as ES cells derived from fertilized eggs (T Wakayama et al., Science (2001) 292:740-743; S Wakayama et al., Biol Reprod (2005) 72:932-936; J Byrne et al., Nature (2007) 450:497-502). Specifically, ntES (nuclear transfer ES) cells are established from the inner cell mass of blastocysts derived from cloned embryos obtained by replacing the nucleus of an unfertilized egg with that of a somatic cell. To generate ntES cells, nuclear transfer technology (JB Cibelli et al., Nature Biotechnol (1998) 16:642-646) is combined with ES cell generation technology (see above) (Syoka Wakayama et al., Experimental Medicine, 2008, Vol. 26, No. 5 (Supplement), pp. 47-52). In nuclear transfer, the nucleus of a somatic cell is injected into an enucleated unfertilized mammalian egg, followed by incubation for several hours to reprogram the egg.

[0039] (F) Multilineage-differentiating Stress Enduring cells (Muse cells) Muse cells are pluripotent stem cells produced by the method described in International Publication WO2011 / 007900. Specifically, Muse cells are pluripotent cells obtained by prolonged trypsin treatment of fibroblasts or bone marrow stromal cells, preferably for 8 or 16 hours, followed by suspension culture, and are positive for SSEA-3 and CD105.

[0040] In the step of culturing pluripotent stem cells in a chondrogenic differentiation medium in the production method of the present invention, a known method for inducing differentiation of pluripotent stem cells into cartilage-like tissue can be used. The differentiation induction method used in the step of culturing pluripotent stem cells in a chondrogenic differentiation medium in the production method of the present invention is not particularly limited, but the method described below can be suitably used as an example.

[0041] The method for inducing differentiation of pluripotent stem cells into cartilage-like tissue used in the step of culturing pluripotent stem cells in a chondrogenic differentiation medium may be the following method: (i) a step of adhesion culture of pluripotent stem cells 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, and (ii) a step of suspension culture of the cells obtained in step (i) 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.

[0042] The pluripotent stem cells used in (i) are preferably cultured in a three-dimensional suspension culture to form cell masses while maintaining their undifferentiated state. Three-dimensional suspension culture is a method in which cells are cultured in a culture medium under non-adherent conditions with stirring or shaking.

[0043] The chondrogenic differentiation medium used 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 chondrogenic differentiation medium is a basal medium supplemented with BMP2, TGFβ, GDF5, and an HMG-CoA reductase inhibitor. Examples of basal media include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM), αMEM medium, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof. The basal medium may optionally contain substances such as serum (e.g., FBS), albumin, transferrin, KnockOut Serum Replacement (KSR) (a serum substitute for FBS used in 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 molecules, antibiotics, antioxidants, pyruvic acid, buffers, and inorganic salts. A preferred basal medium is DMEM containing insulin, transferrin, sodium selenite, ethanolamine, ascorbic acid, non-essential amino acids, sodium pyruvate, and serum.

[0044] BMP2 to be added to the chondrogenesis medium includes BMP2 derived from humans and other animals, as well as functionally modified forms thereof. For example, commercially available BMP2 from Osteopharma, Inc. can be used. The concentration of BMP2 in the chondrogenesis medium 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. BMP2 may be replaced with BMP4.

[0045] The TGFβ added to the chondrogenesis medium includes TGFβ derived from humans and other animals, as well as functionally modified forms thereof, and can be, for example, commercially available products from PeproTech, Inc. The concentration of TGFβ in the chondrogenesis medium 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.

[0046] The GDF5 added to the chondrogenesis medium includes GDF5 derived from humans and other animals, as well as functionally modified forms thereof, and may be commercially available, for example, from PeproTech, Inc. The concentration of GDF5 in the chondrogenesis medium 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.

[0047] Examples of HMG-CoA reductase inhibitors added to the chondrogenic differentiation medium include mevastatin (compactin) (see US Pat. No. 3,983,140), pravastatin (see Japanese Patent Application Laid-Open No. 57-2240 (US Pat. No. 4,346,227)), lovastatin (see Japanese Patent Application Laid-Open No. 57-163,374 (US Pat. No. 4,231,938)), simvastatin (see Japanese Patent Application Laid-Open No. 56-122,375 (US Pat. No. 4,444,784)), and fluvastatin (see Japanese Patent Application Laid-Open No. 56-122,375 (US Pat. No. 4,444,784)). Examples of HMG-CoA reductase inhibitors include, but are not limited to, atorvastatin (see JP 60-500015 A (USP 4739073)), atorvastatin (see JP 3-58967 A (USP 5273995)), rosuvastatin (see JP 5-178841 A (USP 5260440)), and pitavastatin (see JP 1-279866 A (USP 5854259 and USP 5856336)). The HMG-CoA reductase inhibitor is preferably a drug selected from the group consisting of mevastatin, atorvastatin, pravastatin, rosuvastatin, fluvastatin, and lovastatin. 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.

[0048] In the chondrogenesis medium, bFGF may be further added to the basal medium. bFGF includes bFGF derived from humans and other animals, as well as functionally modified forms thereof. For example, commercially available products from WAKO Co., Ltd. can be used. The concentration of bFGF in the chondrogenesis medium 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.

[0049] The basal medium for chondrogenesis may further contain bFGF. Examples of pterosin derivatives include those described in JP 2015-028005 A, and more preferably pterosin B. The concentration of pterosin B in the chondrogenesis medium is 10 μM to 1000 μM, preferably 100 μM to 1000 μM.

[0050] Adhesion culture refers to culturing cells in a state where they are attached to the culture vessel. The culture vessel used is not particularly limited as long as it is capable of supporting the adherent culture of cultured cells. Examples include flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, chamber slides, culture slides, and petri dishes. The culture vessel may be surface-treated for cell adhesion, or may not be surface-treated (non-coated). Commercially available culture vessels with surface treatments for cell adhesion can be used, such as IWAKI tissue culture dishes. Alternatively, culture may be performed using a culture vessel coated with an extracellular matrix. Coating can be performed by adding a solution containing the extracellular matrix to the culture vessel and then appropriately removing the solution.

[0051] The extracellular matrix used for coating may be naturally occurring or artificial (recombinant). Examples include polylysine, polyornithine, collagen, proteoglycan, fibronectin, hyaluronic acid, tenascin, entactin, elastin, fibrillin, laminin, and fragments thereof. These extracellular matrices may be used in combination as appropriate.

[0052] 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 in 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, as long as it is longer than the period required for the seeded cell masses to adhere to the culture vessel and form nodules. The culture period in step (i) may be 3 days or more, 7 days or more, 10 days or more, or 14 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. Preferably, it is 14 days.

[0053] In step (i), the cells that formed nodules may spontaneously detach and float during the culture period, or may remain attached to the culture vessel until the end of the culture period. The naturally detached and floating nodules are directly subjected to suspension culture in step (ii). The nodules that adhere to the culture vessel are detached from the culture vessel and subjected to suspension culture in step (ii). The method for detaching nodules from the culture vessel is preferably a mechanical separation method (e.g., pipetting or a method using a scraper), and preferably does not use a separation solution having protease activity and / or collagenase activity.

[0054] In step (ii), the cells obtained in step (i) are cultured in suspension. Suspension culture refers to culturing cells in a non-adherent state to a culture vessel, and is not particularly limited. Preferably, the culture vessel is a culture vessel (e.g., a Petri dish) that has not been artificially treated to improve cell adhesion (e.g., coated with an extracellular matrix) or a culture vessel that has been artificially treated to suppress adhesion (e.g., coated with polyhydroxyethyl methacrylate (poly-HEMA)).

[0055] In step (ii), the same culture medium as in step (i) can be used.

[0056] In step (ii), the culture temperature is not particularly limited, but is preferably about 30-40°C, and is preferably about 37°C. Culture is performed in a CO2-containing air atmosphere. The CO2 concentration is about 2-5%, preferably about 5%. The culture period is not particularly limited; it may be performed until the desired cartilage-like tissue is 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 formation of cartilage-like tissue can be confirmed by collecting a portion of the cartilage-like tissue from the culture and staining it with safranin O.

[0057] The cartilage-like tissue formed in step (ii) is roughly spherical, and it has been confirmed that its size varies depending on the number of days since the start of differentiation induction. Specifically, it has been confirmed that the size (diameter) of the cartilage-like tissue is approximately 1 mm four weeks after the start of differentiation induction, approximately 2-3 mm after 12 weeks, and that even with continued culture thereafter, the growth of the cartilage particles stops at a diameter of approximately 3-4 mm. Therefore, the production method of the present invention can produce roughly spherical cartilage-like tissue with a maximum diameter of approximately 4 mm.

[0058] The cartilage-like tissue produced by the production method of the present invention may be a layered cartilage-like tissue formed by combining a plurality of the above-mentioned approximately spherical cartilage-like tissues. The layered cartilage-like tissue can be produced by using a plurality of the above-mentioned approximately spherical cartilage-like tissues and culturing them in a state where adjacent approximately spherical cartilage-like tissues can come into contact with each other.

[0059] The approximately spherical cartilage-like tissue used to form lamellar cartilage-like tissue may be approximately spherical cartilage-like tissue less than 4 weeks (28 days) after the start of differentiation induction, approximately spherical cartilage-like tissue 4 weeks (28 days) to 6 weeks (42 days), approximately spherical cartilage-like tissue 6 weeks (42 days) to 8 weeks (56 days), approximately spherical cartilage-like tissue 8 weeks (56 days) to 10 weeks (70 days), approximately spherical cartilage-like tissue 10 weeks (70 days) to 12 weeks (84 days), approximately spherical cartilage-like tissue 12 weeks (84 days) to 14 weeks (98 days), or approximately spherical cartilage-like tissue more than 14 weeks (98 days). Preferably, the approximately spherical cartilage-like tissue is approximately 6 weeks (42 days) or less, 5 weeks (35 days) or less, or 4 weeks (28 days) or less after the start of differentiation induction, and more preferably approximately 4 weeks (25 to 32 days) after the start of differentiation induction.

[0060] The required number of approximately spherical cartilage-like tissues can be determined appropriately depending on the size (area) of the lamellar cartilage-like tissue to be produced. In producing lamellar cartilage-like tissue, a frame that can be placed in a culture medium can be used to culture adjacent cartilage particles in a state where they can come into contact with each other. For example, a culture medium can be placed in a culture dish, and an appropriate frame can be placed inside it. Specifically, a smaller-sized culture dish, a transwell for cell migration tests, or the like can be used as a frame. The approximately spherical cartilage-like tissue is placed inside this frame. It is preferable that the approximately spherical cartilage-like tissue be filled into the frame so that it comes into contact with adjacent cartilage particles, and it is preferable that it be filled in a single layer.

[0061] In producing layered cartilage-like tissue, a liquid-permeable container can be preferably used to culture adjacent cartilage particles in a state where they can come into contact with each other. Examples of liquid-permeable containers include mesh bags used for storing biopsy samples. The size of the mesh bag can be selected depending on the size (area) of the plate-shaped cartilage to be produced. Approximately spherical cartilage-like tissues are placed in the mesh bag and then placed in a culture medium. The required number of approximately spherical cartilage-like tissues to be placed in the mesh bag is not particularly limited, and may be the number required to form a single layer of close-packed cartilage 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 a single layer of close-packed cartilage.

[0062] In producing lamellar cartilage-like tissue, when cartilage particles are placed in a liquid-permeable container and cultured, it is preferable to culture the tissue while the culture medium is flowing. Examples of methods for culturing the tissue while the culture medium is flowing include a method using a bioreactor. Specifically, a method in which a liquid-permeable container containing cartilage particles is placed in the culture medium in a bioreactor and the culture medium is flowed is exemplified. The bioreactor used is not particularly limited, and for example, a bioreactor from Able Corporation equipped with a magnetic stirrer can be suitably used. Flowing the culture medium can promote the formation of lamellar cartilage-like tissue. Culture may be performed while the culture medium is flowing, and after the formation of lamellar cartilage-like tissue, the culture medium may be returned to static culture. Alternatively, the formed lamellar cartilage-like tissue may be sliced ​​into two pieces and cultured while the culture medium is flowing. The period for culturing the tissue while the culture medium is flowing is not particularly limited and can be determined appropriately based on the appearance of the lamellar cartilage-like tissue, etc. The period of culture with flowing culture medium may be 4 weeks or more, 5 weeks or more, 6 weeks or more, 7 weeks or more, 8 weeks or more, or 14 weeks or less, 13 weeks or less, 12 weeks or less, 11 weeks or less, or 10 weeks or less.

[0063] In the production method of the present invention, the step of increasing the ascorbic acid concentration in the chondrogenic medium can be carried out by increasing the ascorbic acid concentration in the chondrogenic medium at a specific time point in the step of culturing pluripotent stem cells in the chondrogenic medium. By increasing the ascorbic acid concentration in the chondrogenic medium, poor chondrogenesis caused by the occurrence of fuzzing around the periphery of immature chondroid tissue during the differentiation induction process can be suppressed, and chondroid tissue can be stably produced from pluripotent stem cells.

[0064] The method for increasing the ascorbic acid concentration in the chondrogenic differentiation medium is not particularly limited, and examples include adding ascorbic acid to the chondrogenic differentiation medium during the differentiation induction process from pluripotent stem cells to cartilage-like tissue, or replacing the culture with a new chondrogenic differentiation medium containing an increased ascorbic acid concentration compared to the ascorbic acid concentration contained in the chondrogenic differentiation medium during the culture. The ascorbic acid concentration in the chondrogenic differentiation medium may be increased in stages. The increased ascorbic acid concentration is usually maintained at the same concentration until the desired cartilage-like tissue is recovered, but the ascorbic acid concentration in the chondrogenic differentiation medium may be changed (increased or decreased) after increasing the ascorbic acid concentration to prevent fuzzing or after repairing fuzzing that has occurred.

[0065] The increased ascorbic acid concentration and the timing for increasing the ascorbic acid concentration are preferably determined through preliminary studies. The content of the preliminary study is not particularly limited, but may include, for example, the following (1) to (4): (1) confirming the occurrence of fuzzing around the cartilage-like tissue after initiating differentiation induction from pluripotent stem cells to cartilage-like tissue; (2) identifying the time point at which fuzzing occurs; (3) setting the increased ascorbic acid concentration based on the ascorbic acid concentration in the medium at the time of occurrence; and (4) setting the time for increasing the ascorbic acid concentration to be within a range of two weeks before or after the time point at which fuzzing occurs.

[0066] The timing for increasing the ascorbic acid concentration is preferably set within a range of two weeks before or after the occurrence of the fuzzing. For example, if, in a preliminary study, fuzzing occurred around the periphery of the cartilage-like tissue five weeks after the start of differentiation induction, it is preferable to set the timing for increasing the ascorbic acid concentration between three and seven weeks after the start of differentiation induction in the same differentiation induction system as in the preliminary study. If the purpose is to prevent fuzzing, it is preferable to increase the ascorbic acid concentration three to four weeks after the start of differentiation induction. Furthermore, by increasing the ascorbic acid concentration three weeks after the start of differentiation induction and before fuzzing occurs, it is possible to prevent fuzzing while suppressing the appearance of cells other than chondrocytes (non-target cells). On the other hand, if the ascorbic acid concentration is not increased before fuzzing around the periphery of the cartilage-like tissue and the purpose is to repair the fuzzing that has occurred, the ascorbic acid concentration may be increased immediately after the occurrence of fuzzing is confirmed, one day later, two days later, or between the third day and two weeks after the start of differentiation induction.

[0067] If no preliminary study is conducted on the timing of increasing the ascorbic acid concentration, it is preferable to increase the ascorbic acid concentration after confirming the occurrence of fluffing around the cartilage-like tissue, preferably on the same day, one day, two days, or three days after confirming the occurrence of fluffing.

[0068] The ascorbic acid concentration in the culture medium after expansion can be set based on the ascorbic acid concentration before expansion. Because the chondrogenic differentiation medium used at the start of differentiation induction typically contains a low concentration of ascorbic acid, the ascorbic acid concentration of the chondrogenic differentiation medium at the start of differentiation induction can be used as a reference. The ascorbic acid concentration of the chondrogenic differentiation medium at the start of differentiation induction may be selected from the ranges of 0-30 μg / mL, 0-20 μg / mL, or 0-10 μg / mL. The ascorbic acid concentration of the chondrogenic differentiation medium at the start of differentiation induction may be set to, for example, 20 μg / mL, 15 μg / mL, 10 μg / mL, 5 μg / mL, 3 μg / mL, 2 μg / mL, or 1 μg / mL. The ascorbic acid concentration in the culture medium after the increase may be 150% or more, 200% or more, 250% or more, 300% or more, 350% or more, 400% or more, 500% or more, 600% or more, 700% or more, 800% or more, 900% or more, or 1000% or more of the ascorbic acid concentration before the increase, preferably 200% or more. If the chondrogenic differentiation medium does not contain ascorbic acid or the ascorbic acid concentration is unknown when fuzzing occurs around the cartilage-like tissue, the ascorbic acid concentration in the culture medium after the increase may be set to 40 μg / mL or more, 50 μg / mL or more, 60 μg / mL or more, 70 μg / mL or more, 80 μg / mL or more, 90 μg / mL or more, 100 μg / mL or more, 150 μg / mL or more, or 200 μg / mL or more, preferably 100 μg / mL or more.

[0069] The culture period after increasing the ascorbic acid concentration in the culture medium is not particularly limited, and the culture may be terminated when the desired cartilage-like tissue is formed, which may be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 20 weeks or more after the start of differentiation induction.

[0070] [Method for regulating the stiffness of cartilage-like tissue] The present invention provides a method for regulating the stiffness of cartilage-like tissue induced to differentiate from pluripotent stem cells (hereinafter referred to as the "stiffness regulation method of the present invention"). The stiffness regulation method of the present invention may include the steps of culturing pluripotent stem cells in a chondrogenesis medium and increasing the ascorbic acid concentration in the chondrogenesis medium.

[0071] In the stiffness regulation method of the present invention, the step of culturing pluripotent stem cells in a cartilage differentiation medium can be carried out in the same manner as in the above-mentioned production method of the present invention. That is, it can be carried out using a known method for inducing differentiation of pluripotent stem cells into cartilage-like tissue, and the same method as the method for inducing differentiation of pluripotent stem cells into cartilage-like tissue explained in the above-mentioned production method of the present invention can be preferably used. However, the present invention is not limited to this method.

[0072] In the stiffness adjustment method of the present invention, the step of increasing the ascorbic acid concentration in the chondrocyte differentiation medium can be carried out by increasing the ascorbic acid concentration in the chondrocyte differentiation medium at a specific time point in the step of culturing pluripotent stem cells in the chondrocyte differentiation medium. The method for increasing the ascorbic acid concentration in the chondrocyte differentiation medium is not particularly limited, and the same method as in the production method of the present invention can be used. By increasing the ascorbic acid concentration in the chondrocyte differentiation medium, the cartilage-like tissue produced by the production method of the present invention can be made stiffer than when the ascorbic acid concentration in the chondrocyte differentiation medium is not increased.

[0073] The method for evaluating hardness is not particularly limited, but a score defining a desired hardness may be created and used for evaluation. Alternatively, cartilage-like tissue corresponding to a score defining the desired hardness may be measured using a commercially available hardness meter, and the score may correspond to the measured value of the hardness meter. The inventors defined "soft," "normal," and "hard," and created and used a six-level score system as shown in Table 1 of Example 2. The definitions of hardness and the scores listed in Table 1 are as follows: "soft": When pinched with tweezers, it breaks and cannot be lifted. "normal": When pinched with tweezers, the surface sinks, but it can be lifted without breaking. "hard": When pinched with tweezers, the surface does not sink, but when pinched with tweezers, lifted, and pressed against a dish, it bends. Score 1: Overall soft Score 2: Soft and normal parts are mixed Score 3: Overall normal Score 4: Normal and hard parts are mixed Score 5: Overall hard Score 6: Extremely hard, so hard that it does not bend even when pinched with tweezers, lifted, and pressed against a dish

[0074] In the stiffness-regulating method of the present invention, when increasing the stiffness of cartilage-like tissue, it is preferable to increase the ascorbic acid concentration in the chondrocyte differentiation medium after 3 weeks from the start of differentiation induction. The inventors have confirmed that increasing the ascorbic acid concentration in the chondrocyte differentiation medium before 3 weeks from the start of differentiation induction may induce differentiation of cells other than chondrocytes (non-target cells). Therefore, in the stiffness-regulating method of the present invention, increasing the ascorbic acid concentration in the chondrocyte differentiation medium after 3 weeks from the start of differentiation induction can simultaneously regulate the stiffness of the cartilage-like tissue and suppress differentiation into cells other than chondrocytes (the appearance of non-target cells). The timing of increasing the ascorbic acid concentration in the chondrocyte differentiation medium is not particularly limited as long as it is after 3 weeks from the start of differentiation induction, but it may also be after 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks. Increasing the ascorbic acid concentration 3 to 4 weeks from the start of differentiation induction increases the stiffness of the cartilage-like tissue and prevents fuzzing around the cartilage-like tissue.

[0075] The ascorbic acid concentration in the culture medium after the expansion is not particularly limited, but can be the same as that in the production method of the present invention. Specifically, the ascorbic acid concentration in the culture medium after the expansion may be 150% or more, 200% or more, 250% or more, 300% or more, 350% or more, 400% or more, 500% or more, 600% or more, 700% or more, 800% or more, 900% or more, or 1000% or more of the ascorbic acid concentration before the expansion. Furthermore, the ascorbic acid concentration in the culture medium after the expansion may be 40 μg / mL or more, 50 μg / mL or more, 60 μg / mL or more, 70 μg / mL or more, 80 μg / mL or more, 90 μg / mL or more, 100 μg / mL or more, 150 μg / mL or more, or 200 μg / mL or more.

[0076] The culture period after increasing the ascorbic acid concentration in the culture medium is not particularly limited, and the culture may be terminated as appropriate when the hardness of the cartilage-like tissue reaches a desired hardness. The present inventors have confirmed that the hardness of the cartilage-like tissue increases depending on the length of the culture period after increasing the ascorbic acid concentration.

[0077] The method for adjusting the stiffness of the present invention may be a method for simultaneously adjusting the stiffness of the cartilage-like tissue and the smoothness of the surface of the cartilage-like tissue. The smoothness of the surface of the cartilage-like tissue can be evaluated visually or by touch.

[0078] In the method of adjusting stiffness of the present invention, if the surface smoothness of the cartilage-like tissue is also adjusted, the ascorbic acid concentration of the chondrogenic medium is preferably increased 7 weeks or later after the start of differentiation induction. The inventors have confirmed that if the ascorbic acid concentration of the chondrogenic medium is increased before 7 weeks after the start of differentiation induction, the hardness of the cartilage-like tissue increases, but the surface smoothness of the cartilage-like tissue may be insufficient. Therefore, if both increased hardness and surface smoothness of the cartilage-like tissue are required, the ascorbic acid concentration of the chondrogenic medium may be increased 7 weeks or later, or may be increased 8 weeks or later, 9 weeks or later, or 10 weeks or later after the start of differentiation induction.

[0079] Furthermore, in the method of adjusting stiffness of the present invention, when the surface smoothness of the cartilage-like tissue is also adjusted, the period during which the ascorbic acid concentration in the chondrogenic differentiation medium is increased may be limited, and the ascorbic acid concentration may then be decreased. The ascorbic acid concentration after the decrease is not particularly limited, and may be returned to the concentration before the increase, decreased to a higher concentration than the concentration before the increase, or decreased to a lower concentration than the concentration before the increase. The present inventors have confirmed that cartilage-like tissue with the desired stiffness and smoothness was obtained when the ascorbic acid concentration in the chondrogenic differentiation medium was increased for a certain period of time after 7 weeks from the start of differentiation induction, and then the concentration was decreased to the original level and the culture was continued.

[0080] The period for which the ascorbic acid concentration of the chondrocyte differentiation medium is increased is not particularly limited, and may be 1 week or more, 2 weeks or more, 3 weeks or more, or 4 weeks or more. If cartilage-like tissue with the desired hardness and smoothness is obtained during the period for which the ascorbic acid concentration of the chondrocyte differentiation medium is increased, the culture period may be terminated without reducing the ascorbic acid concentration. When culturing is continued after increasing the ascorbic acid concentration of the chondrocyte differentiation medium for a certain period, and then reducing the ascorbic acid concentration, the culture period after reducing the ascorbic acid concentration is not particularly limited, and may be 1 week or more, 2 weeks or more, 3 weeks or more, or 4 weeks or more. The culture period may be terminated appropriately when cartilage-like tissue with the desired hardness and smoothness is obtained.

[0081] [Cartilage-like tissue] The present invention provides cartilage-like tissue with adjusted hardness, or cartilage-like tissue with adjusted hardness and surface smoothness (hereinafter referred to as "cartilage-like tissue of the present invention"). The cartilage-like tissue of the present invention is pluripotent stem cell-derived cartilage-like tissue produced by inducing differentiation from pluripotent stem cells, and can be provided as cartilage-like tissue having a substantially spherical shape (substantially spherical cartilage-like tissue) or cartilage-like tissue having a thin layer shape (also expressed as plate-like) (layered cartilage-like tissue). The cartilage-like tissue of the present invention is useful for treating intervertebral disc injury and articular cartilage injury.

[0082] The cartilage-like tissue for treating intervertebral disc injury can be suitably used for transplantation into an injured intervertebral disc site. Examples of diseases associated with intervertebral disc injury include spondylosis degenerativeis, lumbar disc herniation, lumbar degenerative spondylolisthesis, lumbar spondylolysis, and scoliosis. The cartilage-like tissue for treating intervertebral disc injury preferably has a hardness that allows it to maintain its shape and not dislodge when placed in the space surrounding the nucleus pulposus within the annulus fibrosus of the intervertebral disc. The hardness that allows it to maintain its shape and not dislodge when placed in the space surrounding the nucleus pulposus within the annulus fibrosus of the intervertebral disc is intended to avoid the risk of leakage from the implantation site, since a jelly-like structure similar to that of the nucleus pulposus poses a risk of leakage from the implantation site. Such hardness may be within the range of 2 to 6 on the score scale listed in Table 1. Preferably, the hardness is within the range of 3 to 5 on the score scale listed in Table 1. While both roughly spherical cartilage-like tissue and lamellar cartilage-like tissue can be used as the cartilage-like tissue for treating intervertebral disc injury, roughly spherical cartilage-like tissue is preferred.

[0083] Cartilage-like tissue for treating articular cartilage damage can be suitably used for transplantation into the site of articular cartilage damage. Examples of diseases associated with articular cartilage damage include articular cartilage damage, osteoarthritis, osteochondritis dissecans, osteonecrosis, and rheumatoid arthritis. Cartilage-like tissue for treating articular cartilage damage preferably has a hardness that prevents it from being destroyed when transplanted into the site of articular cartilage damage, or that allows it to be sewn to the tissue at the site of articular cartilage damage with thread. A hardness that allows it to be sewn to the tissue at the site of articular cartilage damage with thread is understood to mean a hardness that prevents the cartilage-like tissue from being cut like cheese slices with a suture. Such a hardness may be within a score range of 2 to 6 as shown in Table 1. A hardness that is within a score range of 4 to 5 as shown in Table 1 is preferred. Although either approximately spherical cartilage-like tissue or lamellar cartilage-like tissue can be used as the cartilage-like tissue for treating articular cartilage damage, lamellar cartilage-like tissue is preferred.

[0084] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0085] Example 1: Development and repair of fuzziness around cartilage-like tissue during differentiation induction of human iPS cells into cartilage-like tissue. 1. Materials and methods (1) Culturing human iPS cells. Accutase (trade name, Nacalai) was added to the cultured human iPS cells. After incubation, the cells were detached using a cell scraper. The cells were counted and approximately 2 × 10 6 The cells were transferred to a 100 mL bioreactor (BWV-S10A, Able), and 100 mL of StemFit AK03N (Ajinomoto) supplemented with 10 μM Y-27632 (Nacalai) was added. The cells were rotated at 60 rpm on a magnetic stirrer (BWS-S03NOS-6, Able) and cultured at 37°C and 5% CO2 for 4–7 days. As a result, iPS cell clusters with diameters ranging from 50 μm to 300 μm were obtained.

[0086] (2) Induction of differentiation of human iPS cells into cartilage-like tissue (2-1) Culture medium The following culture media were used: * Cartilage differentiation medium: DMEM (SIGMA) supplemented with 0.2% FBS (Gibco), 1% ITSE (Invitria), 50 μg / mL ascorbic acid (Nacalai), 1% nonessential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), 10 ng / mL BMP2 (PeproTech), 10 ng / mL TGF-β3 (Wako), 10 ng / mL GDF5 (Biovision), and 1 μM rosuvastatin (Biovision) * Cartilage differentiation medium PS: Cartilage differentiation medium supplemented with 1% penicillin streptomycin (SIGMA).

[0087] (2-2) Differentiation Induction The iPS cell clumps obtained by the method in (1) above were collected and seeded onto 8-10 6 cm suspension culture dishes (Sumitomo) containing 2.5 mL of chondrogenic differentiation medium. Differentiation induction was initiated by culturing at 37°C and 5% CO2. During week 0 of differentiation (days 1-7), medium was added twice (2.5 mL each time) every 2-3 days, and during week 1 of differentiation (days 8-14), medium was replaced (5 mL each time) every 2-3 days. The iPS cell clumps gradually adhered to the dish, forming nodules. Some nodules spontaneously detached from the dish and floated away. On day 14, the attached nodules were detached with a cell scraper and transferred, along with the floating nodules, to a 9 cm suspension culture dish (Sumitomo) containing 10 mL of chondrogenic differentiation medium (antibiotic-free group) or chondrogenic differentiation medium PS (antibiotic-containing group) and cultured at 37°C in 5% CO2. After 3–5 days, the medium was replaced with fresh medium. During the 3rd week of differentiation (days 22–28), medium was added twice (5 mL each time) with 2–4 day intervals. From week 4 of differentiation onward (day 29 onward), medium was replaced (20 mL each time) every 2–7 days. From day 14 onward, the growth and appearance of the nodules were observed daily under a phase-contrast microscope.

[0088] 2. Results The results are shown in Figure 1. (A) shows the results for the antibiotic-free group, and (B) for the antibiotic-containing group. In both groups, after day 14 of differentiation, the nodules gradually accumulated extracellular matrix within them, growing into roughly spherical cartilage-like tissue. As shown in (A), fuzziness around the cartilage-like tissue was observed in the antibiotic-free group on day 35 [(A) top row]. Therefore, both groups were further divided into two groups, and on day 37, ascorbic acid was added to one of the two groups, increasing the ascorbic acid concentration to 100 μg / mL. When culture was continued in antibiotic-free medium without ascorbic acid, the degree of fuzziness around the cartilage-like tissue increased by day 42 [(A) bottom left]. When the ascorbic acid concentration in the antibiotic-free medium was increased to 200% on day 37, the fuzziness around the cartilage-like tissue at day 42 almost disappeared [(A) bottom right]. On the other hand, as shown in (B), in the antibiotic-containing group on day 35, no fuzzing around the cartilage-like tissue was observed [(B) upper panel], and the cartilage-like tissue grew normally until day 42 [(B) lower panel left]. When the ascorbic acid concentration in the medium was increased to 200% on day 37, the cartilage-like tissue grew into a roughly spherical shape on day 42, similar to when the ascorbic acid concentration was not increased [(B) lower panel right].

[0089] Reference Example 1: Development of fuzziness around cartilage-like tissue during differentiation induction of human iPS cells into cartilage-like tissue. 1. Materials and Methods. Human iPS cells were differentiated into cartilage-like tissue using the same method as the antibiotic-containing group in Example 1. Two groups, Group A and Group B, were established using two types of medium supplemented with different lots of FBS. From day 14 of differentiation onward, the growth and appearance of nodules were observed daily using a phase-contrast microscope. After fuzziness around the cartilage-like tissue was observed in one group, a tissue specimen of the cartilage-like tissue was prepared according to standard procedures, stained with Safranin O-fast green-iron hematoxylin, and observed under a microscope.

[0090] 2. Results The results are shown in Figure 2. (A) shows the results for Group A, and (B) for Group B. The top row shows phase-contrast micrographs taken 6 weeks after the start of differentiation induction. Fuzziness was observed around the cartilage-like tissue in Group B, but no fuzziness was observed in Group A. The bottom row shows tissue images stained with Safranin O-fast green-iron hematoxylin 8 weeks after the start of differentiation induction. In Group B, where fuzziness was observed around the cartilage-like tissue, the extracellular matrix around the edge of the cartilage-like tissue appears to be frayed.

[0091] Example 2: Formation of Layered Cartilage-Like Tissue from Human iPS Cells, Evaluation of Stiffness, and Detection of Nontarget Cells 1. Materials and Methods (1) Formation of Layered Cartilage-Like Tissue Differentiation of human iPS cells into cartilage-like tissue was induced using the same method as for the antibiotic-free group in Example 1. The amount and timing of ascorbic acid concentration increases were as shown in the group composition in (2) below. Four weeks after the start of differentiation induction (day 28), cartilage-like tissue was collected, and 0.18 g to 0.28 g of cartilage-like tissue was placed in the central pocket (15 mm x 15 mm) of a mesh bag (SEFAR, Product Name: FLAT BAGS MADE OF SEFAR MEDIFAB 07-105 / 52 US CUT / 2 SEAMS BOTTOM FOLDED LENGTH: 54 mm FLAT WIDTH: 42 mm, Product Number: 3071-1000-466-M1, 54 mm x 42 mm). The mesh bag containing the cartilage-like tissue was placed in a bioreactor (BWV-S03A, Able) containing 100 mL of chondrogenic differentiation medium, and cultured for 4 weeks at 37°C with 5% CO2 with agitation. The cartilage-like tissue within the mesh consisted of roughly spherical cartilage-like tissues that coalesced into a single layer, forming layered cartilage-like tissue. The resulting layered cartilage-like tissue (8 weeks after the start of differentiation induction) was transferred to a larger mesh bag (45 mm x 75 mm) and cultured with agitation. The thicker cartilage-like tissue was sliced ​​into two pieces, each of which was placed in a separate mesh bag and cultured with agitation. 12 weeks after the start of differentiation induction, agitation was discontinued, and the cells were transferred to static culture and cultured.

[0092] (2) Group Composition: Three groups were established: a control group, a 6W group with increased ascorbic acid concentration (6W group), and a 2W group with increased ascorbic acid concentration (2W group). In the control group, the ascorbic acid concentration (50 μg / mL) was not changed during the culture period, and the lamellar cartilage-like tissue was sliced ​​into two pieces 10 weeks after the start of differentiation induction. In the 6W group, the ascorbic acid concentration was increased from 50 μg / mL to 100 μg / mL from 6 weeks after the start of differentiation induction, and the lamellar cartilage-like tissue was sliced ​​into two pieces 10 weeks after the start of differentiation induction. In the 2W group, the ascorbic acid concentration was increased from 50 μg / mL to 100 μg / mL from 2 weeks after the start of differentiation induction, and the lamellar cartilage-like tissue was sliced ​​into two pieces 8 weeks after the start of differentiation induction.

[0093] (3) Hardness Evaluation The lamellar cartilage-like tissue in each group was evaluated for hardness based on the scores listed in Table 1, with the following definitions: "soft," "normal," and "hard." For the control and 6W groups, evaluation was performed three times when the mesh bag was changed: 10 weeks (10W), 12 weeks (12W), and 14 weeks (14W) after the start of differentiation induction. For the 2W group, evaluation was performed a total of four times, including when the mesh bag was changed 8 weeks (8W) after the start of differentiation induction. Soft: When pinched with tweezers, the tissue broke and could not be lifted. Normal: When pinched with tweezers, the surface sank, but could be lifted without breaking. Hard: The surface did not sink when pinched with tweezers, but it bent when pinched with tweezers, lifted, and pressed against a dish.

[0094]

[0095] (4) Detection of Unintended Cells (4-1) Observation of the Appearance of the Lamellar Cartilage-Like Tissue When the hardness evaluation in (3) above was carried out, the appearance of the lamellar cartilage-like tissue in each group was observed with the naked eye to confirm the presence or absence of unintended cells.

[0096] (4-2) Gene Expression Analysis: Markers of Nontarget Cells. The expression of MITF (melanocyte-inducing transcription factor) and PAX6 (paired box 6) was measured by RT-PCR as markers of nontarget cells. MITF is a melanocyte marker, and PAX6 is a neuroectoderm marker. At 14 weeks (14W) after the start of differentiation induction, lamellar cartilage-like tissue from each group was collected, frozen in liquid nitrogen, and pulverized using a Multi Beads Shocker (Yasui Kikai). Total RNA was extracted using Qiazol® and the miRNeasy Mini Kit (Qiagen). Genomic DNA was removed from the total RNA by DNase treatment, and 400 ng of total RNA was reverse-transcribed using the ReverTra Ace® qPCR RT Master (Toyobo) to prepare cDNA. PCR was performed using THUNDERBIRD Probe qPCR Mix (Toyobo). The TaqMan IDs of the primers used are listed below. The RNA expression levels were normalized to the GAPDH level, and the ratio of the test sample RNA expression level to the iPS cell expression level (test sample / iPS cells) was calculated. PAX6: Hs01088114_m1 MITF: Hs01117294_m1 GAPDH: Hs03929097_g1

[0097] 2. Results (1) Evaluation of hardness The results are shown in Table 2. In the 6W and 2W groups, increasing the concentration of ascorbic acid (AA) halfway through the culture period increased the score for hardness of the cartilage-like tissue, indicating that the cartilage-like tissue became harder.

[0098]

[0099] (2) Visual observation of lamellar cartilage-like tissue 14 weeks after the start of differentiation induction (14W) The results of visual observation of the 14W lamellar cartilage-like tissue in the 2W group are shown in Figure 3. The left shows the inner appearance, and the right shows the outer appearance. Black spots were observed all over the surface of both surfaces. However, no such black spots were observed in the 14W lamellar cartilage-like tissue in the control group or the 6W group.

[0100] (3) Gene expression analysis The results are shown in Figure 4. (A) shows the results for MITF, and (B) shows the results for PAX6. Both of the non-target cell marker genes were shown to be highly expressed in the 2W group at 14 weeks.

[0101] (4) Visual Observation of the Lamellar Cartilage-Like Tissue 12 Weeks After the Start of Differentiation Induction (12W) The results of visual observation of the 12W lamellar cartilage-like tissue in each group are shown in Figure 5. (A) shows the appearance of the control group, (B) that of the 6W group, and (C) that of the 2W group. Holes and cracks were observed on the surface of the 6W and 2W groups, and smoothness was lost. On the other hand, the surface of the control group was smooth, but the hardness was insufficient.

[0102] Example 3: Investigation of differentiation induction conditions for achieving desired hardness and surface smoothness in layered cartilage-like tissue formed from human iPS cells. 1. Materials and Methods (1) Formation of layered cartilage-like tissue. Human iPS cells were induced to differentiate into cartilage-like tissue using the same method as in the antibiotic-free group in Example 1. Four weeks after the start of differentiation induction (day 28), cartilage-like tissue was collected. 0.26 g of cartilage-like tissue was placed in the central pocket (15 mm × 15 mm) of a mesh bag (54 mm × 42 mm) as in Example 2. The mesh bag containing the cartilage-like tissue was placed in a bioreactor containing 100 mL of chondrogenic differentiation medium and cultured at 37°C and 5% CO2 for 4 weeks with stirring. The cartilage-like tissue within the mesh consisted of roughly spherical cartilage-like tissues coalescing into a single layer to form layered cartilage-like tissue. The resulting layered cartilage-like tissue (8 weeks after the start of differentiation induction) was transferred to a large mesh bag (45 mm x 75 mm), and the culture medium was changed to one with an increased ascorbic acid concentration from 50 μg / mL to 100 μg / mL, and agitated culture was continued. Ten weeks after the start of differentiation induction, the layered cartilage-like tissue was sliced ​​into two pieces and transferred to static culture, where culture was continued. Twelve weeks after the start of differentiation induction, the ascorbic acid concentration in the culture medium was reduced to 50 μg / mL, and static culture was continued.

[0103] (2) Evaluation of Appearance and Hardness Sixteen weeks after the start of differentiation induction, the appearance of the lamellar cartilage-like tissue was observed with the naked eye, and the hardness of the lamellar cartilage-like tissue was evaluated based on the scores shown in Table 1.

[0104] 2. Results The results are shown in Figure 6. The left is the outside appearance, and the right is the inside appearance. The surface of the lamellar cartilage-like tissue was smooth, with no holes or cracks observed. No appearance of non-target cells (black dots) was observed. The tissue also had sufficient hardness (score 5 in Table 1).

[0105] Example 4: Analysis of cartilage marker gene expression, glycosaminoglycan amount measurement, and histological evaluation of the lamellar cartilage-like tissue of Example 2 1. Experimental method The lamellar cartilage-like tissue formed by differentiation induction from human iPS cells in Example 2, 14 weeks (14W) after the start of differentiation induction, was used. (1) Gene expression analysis The expression of cartilage marker genes COL2A1 (type II collagen) and ACAN (aggrecan) was measured by RT-PCR. The experimental method was the same as that described in (4-2) of Example 2. The TaqMan IDs of the primers used are shown below. COL2A1: Hs00264051_m1 ACAN: Hs00153936_m1 GAPDH: Hs03929097_g1

[0106] (2) Measurement of glycosaminoglycan (GAG) content. The layered cartilage-like tissue was minced and dried in a freeze-dryer (Tokyo Rikakikai, FDS-1000) for at least 3 hours. The dry weight was measured using an analytical balance. The freeze-dried cartilage-like tissue was disrupted using a bead shocker (Yasui Kikai, MNX1001(S)). 4.5 U papain solution (4.5 U papain, 2 mM acetylcysteine, 50 mM phosphate buffer, 2 mM EDTA) was added and the tissue was treated with enzymes at 65°C for 16–24 hours. GAGs were eluted from the supernatant using the Blyscan Sulfated Glycosaminoglycan Assay (Biocolor, B3000). The concentration was measured using a plate reader (PerkinElmer, Enspire), and the total GAG content (mg / g) per dry weight was calculated.

[0107] (3) Histological Evaluation: The lamellar cartilage-like tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. The sections were stained with hematoxylin and eosin (hereafter referred to as "HE staining") and Safranin O-fast green-iron hematoxylin (hereafter referred to as "Safranin O staining"). The sections were also immunostained with an anti-type I collagen antibody (clone COL-1, ascites fluid) (Sigma, C2456) and an anti-type II collagen antibody (Collagen II Ab-2, clone 2B1.5) (Thermo, MS-235-PO).

[0108] 2. Results (1) Gene expression analysis The results are shown in Figure 10. (A) shows the results for COL2A1, and (B) for ACAN. In the 6W group, both cartilage marker genes were highly expressed, but in the 2W group, both cartilage marker genes were low in expression.

[0109] (2) Total GAG content per dry weight The results are shown in Figure 11. The GAG ​​content per dry weight of living cartilage is said to be 100-200 mg / g, but the total GAG content of the 6W group was 338 mg / g and the total GAG content of the 2W group was 277 mg / g, indicating that they contained larger amounts of GAG than living cartilage.

[0110] (3) Histological evaluation The results are shown in Figure 12. The lamellar cartilage-like tissue in all groups exhibited a cartilage histological image with cells scattered in the cartilage ECM stained with Safranin O. Furthermore, type I collagen was expressed at the margins of the lamellar cartilage-like tissue, and type II collagen was expressed throughout the lamellar cartilage-like tissue.

[0111] Example 5: Histological evaluation of the lamellar cartilage-like tissue of Example 3 1. Experimental method For the lamellar cartilage-like tissue formed by inducing differentiation from human iPS cells in Example 3, 16 weeks after the start of differentiation induction (see Figure 6), tissue sections were prepared in the same manner as in Example 4 and subjected to HE staining and Safranin O staining. Immunostaining for type I collagen and type II collagen was also performed.

[0112] 2. Results The results are shown in Figure 13. (A) shows the results of Safranin O staining, (B) shows the results of HE staining, (C) shows the results of type I collagen immunostaining, and (D) shows the results of type II collagen immunostaining. The lamellar cartilage-like tissue formed in Example 3 exhibited the histological image of cartilage with cells scattered in cartilage ECM stained with Safranin O, with type I collagen expressed at the periphery of the lamellar cartilage-like tissue and type II collagen expressed throughout the lamellar cartilage-like tissue.

[0113] Example 6: Investigation of the timing at which desired hardness is achieved in lamellar cartilage-like tissue formed from human iPS cells. 1. Materials and Methods (1) Formation of lamellar cartilage-like tissue. Lamellar cartilage-like tissue was prepared from human iPS cells using the same method as in Example 3. (2) Evaluation of Appearance and Hardness. The appearance of the lamellar cartilage-like tissue was observed with the naked eye at 12, 14, 16, and 17 weeks after the start of differentiation induction, and the hardness of the lamellar cartilage-like tissue was evaluated based on the scores listed in Table 1. (3) Gene Expression Analysis. Using the same method as in Examples 2 and 4, the expression of unintended cell marker genes (MITF, PAX6) and cartilage marker genes (COL2A1, ACAN) in the lamellar cartilage-like tissue 17 weeks after the start of differentiation induction was measured by RT-PCR. (4) Total GAG Content per Dry Weight. Using the same method as in Example 4, the total GAG content per dry weight of the lamellar cartilage-like tissue 17 weeks after the start of differentiation induction was measured. (5) Histological Evaluation Using the same method as in Example 4, sections of the lamellar cartilage-like tissue were prepared 17 weeks after the start of differentiation induction, and were subjected to HE staining, Safranin O staining, immunostaining for type I collagen, and immunostaining for type II collagen.

[0114] 2. Results (1) Appearance and Hardness Evaluation The results are shown in Figure 14. (A) shows the outer and inner appearances of the lamellar cartilage-like tissue at 12 weeks and (B) at 17 weeks. In both cases, the surface of the lamellar cartilage-like tissue was smooth, with no observed holes or cracks, and no appearance of non-target cells (black dots). The hardness score was 5 at both 12 and 17 weeks, demonstrating that lamellar cartilage-like tissue with a size, quality, and hardness suitable for transplantation had been formed at 12 weeks. Continuing culture after 12 weeks increased the size of the cartilage-like tissue, but the lamellar cartilage-like tissue at 17 weeks also maintained a size, quality, and hardness suitable for transplantation.

[0115] (2) Gene expression analysis The results are shown in Figure 15. (A) shows the results for COL2A1, (B) for ACAN, (C) for PAX6, and (D) for MITF. (A) and (B) show that cartilage marker genes were expressed at much higher levels than in iPS cells. (C) and (D) show that unintended cell marker genes were expressed at lower levels than in iPS cells, indicating that unintended cells did not appear.

[0116] (3) Total GAG content per dry weight The results are shown in Figure 16. The total GAG content exceeded 200 mg / g, indicating that the lamellar cartilage-like tissue contained sufficient GAG.

[0117] (4) Histological evaluation The results are shown in Figure 17. (A) shows the results of Safranin O staining, (B) shows the results of HE staining, (C) shows the results of type I collagen immunostaining, and (D) shows the results of type II collagen immunostaining. Seventeen weeks after the start of differentiation induction, the lamellar cartilage-like tissue exhibited the histological appearance of cartilage with cells scattered in cartilage ECM stained with Safranin O, with type I collagen expressed at the periphery of the lamellar cartilage-like tissue and type II collagen expressed throughout the lamellar cartilage-like tissue.

[0118] Example 7: Investigation of the initial concentration of ascorbic acid and the timing of increasing the concentration The present inventors conducted a preliminary study and confirmed that the use of a medium containing a high concentration of ascorbic acid from the beginning of differentiation induction increases the occurrence of black spots, that the use of a medium not containing ascorbic acid from the beginning of differentiation induction causes fuzzing around the periphery of the cartilage-like tissue 4 to 5 weeks after the start of differentiation induction, and that the use of a medium containing ascorbic acid from week 5 onwards reduces fuzzing but reduces the yield. Based on the results of these preliminary studies, the initial concentration of ascorbic acid and the timing of increasing the concentration were investigated.

[0119] 1. Materials and Methods (1) Induction of Differentiation from Human iPS Cells to Cartilage-Like Tissue Human iPS cells were cultured in the same manner as in Example 1 to obtain iPS cell clusters. The resulting iPS cell clusters were cultured in chondrogenesis medium to initiate differentiation induction. The chondrogenesis medium used was the antibiotic-free chondrogenesis medium of Example 1, but with various ascorbic acid concentrations. As in Example 1, iPS cell clusters were seeded in 6 cm suspension culture dishes (sumitomo) and cultured at 37°C and 5% CO2. On day 14, nodules were collected and transferred to 9 cm suspension culture dishes (sumitomo), where they were cultured at 37°C and 5% CO2. The growth and appearance of nodules were observed daily using a phase-contrast microscope from the start of differentiation induction.

[0120] (2) Group composition Seven groups were established as shown in Table 3. Five groups (C to G) used medium containing ascorbic acid at concentrations of 0 to 10 μg / mL for three weeks after the start of differentiation induction, and from three weeks onward used medium containing 50 μg / mL of ascorbic acid, the same concentration as the control group (Group A).

[0121] (3) Evaluation Items The following items were evaluated. (3-1) Fuzziness around the Cartilage-like Tissue The appearance of the cartilage-like tissue was observed over time from the start of differentiation induction until 10 weeks later. (3-2) Occurrence of Unintended Cells (Black Spots) Six weeks after the start of differentiation induction, the presence or absence of black spots in the cartilage-like tissue (nodules) was observed, and the ratio of the number of cartilage-like tissues with black spots to the number of cartilage-like tissues observed was calculated. (3-3) Yield of Cartilage-like Tissue The yield of cartilage-like tissue was determined by measuring the wet weight of the cartilage-like tissue 10 weeks after the start of differentiation induction, the number of cells per gram of wet weight, and the amount of glycosaminoglycan (GAG) per gram of dry weight. (3-4) Histological Evaluation Sections of the cartilage-like tissue 10 weeks after the start of differentiation induction were prepared using the same method as in Example 4 and subjected to HE staining, Safranin O staining, immunostaining for type I collagen, and immunostaining for type II collagen.

[0122] 2. Results (1) Fuzziness around the cartilage-like tissue Figure 18 shows the results of observing the cartilage-like tissue in each group 4 weeks after the start of differentiation induction. Fuzziness around the cartilage-like tissue was observed 4 weeks after the start of differentiation induction only in Group B, which used a medium without ascorbic acid. No fuzziness around the cartilage-like tissue was observed in Group C, which was cultured in a medium without ascorbic acid from 0 to 3 weeks and then cultured in a medium containing 50 μg / mL of ascorbic acid. These results suggest that using a medium containing ascorbic acid from 3 weeks after the start of differentiation induction is effective in preventing fuzziness around the cartilage-like tissue 4 weeks after the start of differentiation induction.

[0123] (2) Non-target cells and yield (wet weight, cell number, glycosaminoglycan content). The results are shown in Table 4. Six weeks after the start of differentiation induction, the occurrence of non-target cells (black dots) was observed only in groups A and E, with the highest occurrence in group A. These results suggest that lowering the ascorbic acid concentration in the medium at the start of differentiation induction to less than 50 μg / mL is effective in preventing the occurrence of non-target cells. Ten weeks after the start of differentiation induction, the wet weight, cell number, and glycosaminoglycan content of the cartilage-like tissue were nearly equivalent in all groups, except for group B, which used a medium without ascorbic acid.

[0124]

[0125] (3) Histological Evaluation The results of Safranin O staining are shown in Figure 19, the results of HE staining in Figure 20, the results of immunostaining for type I collagen in Figure 21, and the results of immunostaining for type II collagen in Figure 22. Except for group B, which used a medium without ascorbic acid, all groups exhibited a cartilage histology stained with Safranin O, with cells scattered in the cartilage ECM, and type I collagen was expressed at the periphery of the lamellar cartilage-like tissue, and type II collagen was expressed throughout the lamellar cartilage-like tissue.

[0126] Example 8: Single-cell RNA sequencing analysis 1. Materials and methods (1) Test samples Two types of cartilage-like tissue were used as test samples: one was formed under the same conditions as Group A in Example 7, i.e., an initial ascorbic acid concentration of 50 μg / mL, and differentiation was induced for 10 weeks without changing the concentration (hereafter referred to as "AAx1_10w"); the other was formed under the same conditions as Group G in Example 7, i.e., an initial ascorbic acid concentration of 10 μg / mL, increased to 50 μg / mL from week 3 onwards, and differentiation was induced for 10 weeks (hereafter referred to as "AAx0.2_10w"). Two types of undifferentiated human iPS cells (hereafter referred to as "hiPSC.1" and "hiPSC.2") were used as controls.

[0127] (2) Single-cell RNA sequencing This was performed according to the method previously described by the present inventors (Kamatani, T. et al., Biomaterials 284 (2022) 121491).

[0128] 2. Results (1) Cluster Analysis. Data from AAx1_10w and AAx0.2_10w were merged with data from hiPSC.1 and hiPSC.2 and analyzed using Seurat. After principal component analysis, clustering was performed, and dimensionality reduction was performed using Uniform Manifold Approximation and Projection (UMAP) to plot each cell in two dimensions. The results are shown in Figure 23. As shown in the left panel, cells were divided into nine clusters. The right panel of Figure 21 is divided into each sample and shown in Figure 24. Undifferentiated iPS cells were enriched in clusters 1, 3, 5, and 8, while cartilage-like tissue at 10 weeks after differentiation induction was enriched in clusters 0, 2, 4, 6, and 7, regardless of ascorbic acid concentration.

[0129] The percentage of cells belonging to each cluster in each sample is shown in Figure 25. Cluster 2 showed a difference in the degree of enrichment between AAx0.2_10w and AAx1_10w, and was more enriched in AAx1_10w than in AAx0.2_10w. These results indicate that the appearance of Cluster 2 can be suppressed by lowering the ascorbic acid concentration in the medium at the start of differentiation induction.

[0130] (2) Marker Gene Expression Figure 26 shows the results of examining the expression of marker genes for each cell type using a feature plot. The non-target cell markers PAX6 and MITF were barely expressed in any of the clusters. LIN28A was highly expressed in clusters 1, 3, 5, and 8, which were enriched for undifferentiated iPS cells. Of the clusters enriched for cells at 10 weeks of chondrocyte differentiation, clusters 0, 4, 6, and 7, excluding cluster 2, showed high expression of COL2A1 and ACAN, suggesting that these cells correspond to chondrocytes. COL1A1 and COL1A2 were highly expressed in cluster 2, which was enriched for AAx1_10w cells.

[0131] (3) Analysis of Cluster 2 Cells To identify the cell type of Cluster 2, we determined the differentially expressed genes (DEGs) between Cluster 2 and undifferentiated iPS cells, Clusters 1 and 3, and performed gene set enrichment analysis based on the results. The results are shown in Figure 27. We found that Cluster 2 corresponds to stromal cells or fibroblasts.

[0132] (4) Analysis of Cluster 0 Cells To identify the cell type of Cluster 0, we determined the differentially expressed genes (DEGs) between Cluster 0 and undifferentiated iPS cells, Clusters 1 and 3, and performed gene set enrichment analysis based on the results. The results are shown in Figure 28. We found that Cluster 0 corresponds to chondrocytes.

[0133] (5) Conclusion It was revealed that by lowering the ascorbic acid concentration in the medium at the start of differentiation induction, the ratio of stromal cells / fibroblasts was reduced and the purity of chondrocytes was improved.

[0134] The present invention is not limited to the above-described 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 different embodiments. Furthermore, all academic literature and patent documents described in this specification are incorporated herein by reference.

Claims

1. A method for producing cartilage-like tissue from pluripotent stem cells, comprising the steps of culturing pluripotent stem cells in a chondrogenic differentiation medium and increasing the ascorbic acid concentration of the chondrogenic differentiation medium.

2. The method of claim 1, wherein the increased ascorbic acid concentration and the timing for increasing the ascorbic acid concentration are set by the following preliminary studies: (1) confirming the occurrence of fluffing around the cartilage-like tissue after initiating differentiation induction from pluripotent stem cells to cartilage-like tissue; (2) identifying the time point at which the fluffing occurs; (3) setting the increased ascorbic acid concentration based on the ascorbic acid concentration in the culture medium at the time point at which the fluffing occurs; and (4) setting the time point at which the ascorbic acid concentration is increased to within a range of two weeks before or after the time point at which the fluffing occurs.

3. The method according to claim 1 or 2, wherein the ascorbic acid concentration in the culture medium after the increase in ascorbic acid concentration is 150% or more of the ascorbic acid concentration before the increase.

4. A method for regulating the stiffness of cartilage-like tissue induced to differentiate from pluripotent stem cells, comprising the steps of culturing pluripotent stem cells in a cartilage differentiation medium and increasing the ascorbic acid concentration in the cartilage differentiation medium.

5. The method according to claim 4, wherein the concentration of ascorbic acid in the culture medium is increased at least three weeks after the start of differentiation induction.

6. The method according to claim 5, further comprising suppressing the appearance of non-target cells.

7. The method of claim 4, further comprising adjusting the surface smoothness of the cartilage-like tissue.

8. The method according to claim 7, wherein the concentration of ascorbic acid in the culture medium is increased at least 7 weeks after the start of differentiation induction.

9. A cartilage-like tissue obtained by inducing differentiation from pluripotent stem cells, which is used to treat intervertebral disc damage or articular cartilage damage.

10. The cartilage-like tissue for treating intervertebral disc damage according to claim 9, which has a hardness that allows it to maintain its shape and not to deform when placed in the space of the nucleus pulposus within the annulus fibrosus of the intervertebral disc.

11. The cartilage-like tissue according to claim 10, which is for treating intervertebral disc damage and has a hardness score of 2 to 6 as set forth in Table 1 of the specification.

12. The cartilage-like tissue described in claim 9, wherein the cartilage-like tissue for treating articular cartilage damage has a hardness such that it is not destroyed when transplanted into the site of articular cartilage damage, or a hardness such that it can be sewn with thread to the tissue at the site of articular cartilage damage.

13. The cartilage-like tissue according to claim 12, which is for treating articular cartilage damage and has a hardness score of 2 to 6 as set forth in Table 1 of the specification.

14. The cartilage-like tissue according to any one of claims 9 to 13, wherein the shape of the cartilage-like tissue is approximately spherical or lamellar.

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