Stepwise method of producing various types of cells from pluripotent stem cells

Abstract

To provide stepwise methods of producing a somite cell from a pluripotent stem cell, producing a dermatome cell, a myotome cell, a sclerotome cell and a syndetome cell from a somite cell, and producing a mesenchymal stromal cell.SOLUTION: Provided is a method of producing a somite cell from a pluripotent stem cell, comprising the step of culturing a pluripotent stem cell in a medium comprising a GSK3β inhibitor. Provided is a method of producing a dermatome cell from a somite cell, comprising the step of culturing a somite cell in a medium comprising a GSK3β inhibitor and BMP. Provided is a method of producing a syndetome cell from a sclerotome cell, comprising the steps of culturing a sclerotome cell in a medium comprising FGF and then culturing the cell in a medium comprising BMP and TGFβ. Provided is a method of producing a mesenchymal stromal cell from a somite cell, comprising the step of culturing a somite cell in a medium comprising FGF. Provided are methods of producing a myotome cell, a dermatome cell, a sclerotome cell, and a syndetome cell from a pluripotent stem cell through a somite cell by appropriately combining the above methods and known methods.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present application relates to a method for producing somite cells from pluripotent stem cells. The present application also relates to a method for producing myotome cells, dermatome cells, sclerotome cells, ligamentocele cells, and mesenchymal stromal cells from pluripotent stem cells by inducing further differentiation of somite cells produced from pluripotent stem cells. The present application also relates to a method for producing dermatome cells, ligamentocele cells, and mesenchymal stromal cells from somite cells. [Background technology]

[0002] Reproducing the endogenous signaling environment is believed to be important for inducing desired cell types from pluripotent stem cells (PSCs). Based on developmental biology findings, it is known that Activin / Nodal / TGFβ signaling induces differentiation of pluripotent stem cells into mesendoderm, BMP signaling induces differentiation of pluripotent stem cells into mesoderm, and disruption of these signals induces neural cells (Non-Patent Documents 1-4).

[0003] Of note, it has been reported that the mesoderm induced by Activin / Nodal / TGFβ signaling and BMP signaling consists mainly of lateral plate mesoderm (a lateral (ventral) subpopulation of mesoderm) rather than paraxial mesoderm (a subpopulation of mesoderm formed between the neural tube and the lateral plate mesoderm). Although several trials have been performed to induce paraxial mesoderm by modification of Activin / Nodal / TGFβ-based protocols, it has been reported that the rate of induction remained relatively low (about 20%) (Non-Patent Document 5).

[0004] In recent years, several groups have reported success in the induction of axial mesoderm based on different concepts (Non-Patent Documents 6 to 9). In these reports, in order to induce the neural (dorsal) fate, cells were treated without activin / Nodal / TGFβ or with a treatment containing a TGFβ inhibitor, and a relatively high concentration of a GSK3 inhibitor (WNT signaling activator) was used. By using these protocols, the induction rate of axial mesoderm reached approximately 70 to 95% (Non-Patent Documents 7 and 9). This conversion from neural to axial mesoderm suggests that there are common precursors for neural and axial mesoderm in embryogenesis (Non-Patent Documents 10 and 11). These precursors are called neural mesoderm precursors or axial mesoderm. This theory is also supported by the fact that in Wnt3a knockout mice, the formation of ectopic (secondary) neural tubes is observed instead of the disappearance of axial mesoderm (Non-Patent Documents 12 and Non-Patent Document 12).

[0005] Although the induction of axial mesoderm and its subsequent differentiation has been achieved, there are still several problems to be addressed. During the development of vertebrates, axial mesoderm first forms unsegmented mesoderm (PSM) posteriorly and somites (SM) anteriorly. Somites finally differentiate such that the dorsal part becomes the dermomyotome (DM) and the ventral part becomes the sclerotome. The dermomyotome gives rise to the dermatome (D), which is the precursor of the dermis, and the myotome (MYO), which is the precursor of skeletal muscle, and a subpopulation of the sclerotome forms the syndetome, which is the precursor of tendons and ligaments (Non-Patent Document 14). For induced somite cells to be complete, it is important that the induced somite cells show the ability to differentiate into dermatome cells, myotome cells, sclerotome cells, and syndetome cells. The above reports have been successful in inducing myotome cells and sclerotome cells, but the induction protocols for dermatome cells and syndetome cells have not yet been established. Furthermore, the lateral plate mesoderm is a major source of mesenchymal stromal cells (MSC) (Non-Patent Document 15), but somite cells can also be a source of mesenchymal stromal cells. However, there are no reports of inducing mesenchymal stromal cells from pluripotent stem cells via axial mesoderm.

Prior Art Documents

Non-Patent Documents

[0006] [Non-Patent Document 1] Bernardo, A.S., Faial, T., Gardner, L., Niakan, K.K., Ortmann, D., Senner, C.E., Callery, E.M., Trotter, M.W., Hemberger, M., Smith, J.C., et al. (2011). BRACHYURY and CDX2 mediate BMP-induced differentiation of human and mouse pluripotent stem cells into embryonic and extraembryonic lineages. Cell Stem Cell 9, 144-155. [Non-Patent Document 2] Chambers, S.M., Fasano, C.A., Papapetrou, E.P., Tomishima, M., Sadelain, M., and Studer, L. (2009). Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol 27, 275-280. [Non-Patent Document 3] Fasano, C.A., Chambers, S.M., Lee, G., Tomishima, M.J., and Studer, L. (2010). Efficient derivation of functional floor plate tissue from human embryonic stem cells. Cell Stem Cell 6, 336-347. [Non-Patent Document 4] Sumi, T., Tsuneyoshi, N., Nakatsuji, N., and Suemori, H. (2008). Defining early lineage specification of human embryonic stem cells by the orchestrated balance of canonical Wnt / beta-catenin, Activin / Nodal and BMP signaling. Development 135, 2969-2979.

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 14

Non-Patent Document 15

Summary of the Invention

Problems to be Solved by the Invention

[0007] The present application aims to provide a method for producing somite cells from pluripotent stem cells. Another object of the present application is to provide a method for producing dermomyotome cells from somite cells. Yet another object of the present application is to provide a method for inducing syndetome cells from somite cells. Still another object of the present application is to provide a method for inducing mesenchymal stromal cells from somite cells.

[0008] The present application also aims to provide a method for inducing myotome cells, dermomyotome cells, sclerotome cells, syndetome cells, and mesenchymal stromal cells from pluripotent stem cells by inducing the differentiation of pluripotent stem cells into somite cells and further inducing the differentiation of somite cells into myotome cells, dermomyotome cells, sclerotome cells, syndetome cells, and mesenchymal stromal cells, respectively.

Means for Solving the Problems

[0009] The overall process of the method of the present application (excluding the production of mesenchymal stromal cells) is shown in Figure 1. Somite cells (SM) were produced in vitro from pluripotent stem cells via presomitic mesoderm (PSM). The obtained somite cells could be stepwise differentiated in vitro into dermomyotome (D), myotome (MYO), sclerotome (SCL), and syndetome (SYN), respectively, which are known to be further induced to differentiate from somites in vivo. Each induction protocol was determined. Note that this figure is modified from the paper (Buckingham et al., 2003).

[0010] The present application provides a step of providing pluripotent stem cells, and a method for producing somite cells from pluripotent stem cells, which includes a step of culturing pluripotent stem cells in a medium containing a GSK3β inhibitor. In this embodiment, the step of culturing pluripotent stem cells in a medium containing a GSK3β inhibitor preferably (1) includes a step of culturing pluripotent stem cells in a medium containing a GSK3β inhibitor to obtain a presomitic mesoderm cell culture, and (2) includes a step of culturing the presomitic mesoderm cell culture in a medium containing a GSK3β inhibitor to obtain a somite cell culture.

[0011] The present application also provides a method for producing dermomyotome cells from pluripotent stem cells, which includes obtaining somite cells by the above method and further (3) culturing the somite cells in a medium containing a GSK3β inhibitor and BMP. The present application further provides a method for producing myotome cells from pluripotent stem cells, which includes (4) a step of culturing the dermomyotome cells obtained in (3) in a medium containing a GSK3β inhibitor. Myotome cells can be induced to further differentiate by known methods to obtain skeletal muscle cells. A method for producing skeletal muscle cells from such pluripotent stem cells via somite cells and myotome cells is also included in the present application.

[0012] The present application also provides a step of providing somite cells, and Provided is a method for producing dermatome cells from somite cells, which includes a step (5) of culturing somite cells in a medium containing a GSK3β inhibitor and BMP. In this embodiment, the somite cells may be cells produced by the above method from pluripotent stem cells or may be obtained by other methods. That is, according to this embodiment, a method for producing dermatome cells from pluripotent stem cells is provided. The dermatome cells can be further induced to differentiate by known methods to obtain dermal cells. Also included in the present application is a method for producing dermal cells, which includes a step of further inducing differentiation of the dermatome cells obtained in this embodiment.

[0013] The present application also provides a method for producing sclerotome cells from pluripotent stem cells, which includes a step of obtaining somite cells by the above method and a step (6) of culturing the obtained somite cells in a medium containing a sonic hedgehog activator and a BMP inhibitor. Methods for further inducing differentiation of sclerotome cells by known methods to obtain bone, cartilage, etc. are known. Also included in the present application is a method for obtaining bone, cartilage, etc. from pluripotent stem cells via sclerotome cells.

[0014] The present application also includes a step of providing sclerotome cells, (7-1) a step of culturing sclerotome cells in a medium containing FGF, and (7-2) a step of culturing the cell culture obtained in step (7-1) in a medium containing BMP and TGFβ, to provide a method for producing syndetome cells from sclerotome cells. In this embodiment, the sclerotome cells may be those produced from somite cells by the method of the present application, and in this case, the somite cells may be those produced from pluripotent stem cells by the method of the present application. That is, in this embodiment, a method for producing syndetome cells from somite cells and a method for producing syndetome cells from pluripotent stem cells are provided. The syndetome cells can be further induced to differentiate by known methods to produce tendons and ligaments. Also included in the present application is a method for obtaining syndetome cells according to this embodiment and further inducing differentiation of the obtained syndetome cells to produce tendons and ligaments.

[0015] The present application further includes a step of providing somite cells, and Provided is a method for producing mesenchymal stromal cells from somite cells, which includes a step of culturing somite cells in a medium containing FGF. The somite cells in this embodiment may be those induced from pluripotent stem cells by the method of the present application, or cells provided by other known methods. That is, according to this embodiment, a method for producing mesenchymal stromal cells from pluripotent stem cells via somite cells is provided. In this embodiment, the mesenchymal stromal cells obtained can be further induced to differentiate into cartilage, bone, and adipocytes by known methods. A method of obtaining mesenchymal stromal cells and further inducing the differentiation of mesenchymal stromal cells to produce cartilage, bone, and adipocytes is also included in the present application.

Advantages of the Invention

[0016] According to the present application, efficient differentiation induction from pluripotent stem cells to somite cells can be achieved without gene introduction. The somite cells obtained by the present application can be further induced into dermatomyotome cells, myotome cells, sclerotome cells, and syndetome cells. The somite cells obtained by the present application can also be induced into mesenchymal stromal cells.

[0017] That is, by the method of the present application, myotome cells, dermatomyotome cells, sclerotome cells, syndetome cells, and mesenchymal stromal cells can be produced from pluripotent stem cells via somite cells without gene introduction. Further, differentiation induction can be performed from these cells to obtain dermal cells, skeletal muscle cells, bone, cartilage, tendon, and ligament.

[0018] The cells produced by the method of the present application can be applied to cell transplantation therapy and the like. For example, the cells produced by the method of the present application are expected to be used in cell therapy for treating musculoskeletal disorders such as muscular dystrophy, articular cartilage defect, bone defect, and tendon rupture. In addition, cells produced by the method of the present application from iPS cells derived from somatic cells of patients with genetic diseases can be used as disease models for such diseases. The dermal segments and dermal cells obtained by the method of the present application can be used as models for dermatological research and cosmetic development, or for preparing disease models for skin wound treatment agents by cell transplantation and skin disease research.

[0019] This application also provides a cell transplantation therapy method using cells induced from pluripotent stem cells by the method of this application. The method of this application can be used for the treatment of musculoskeletal disorders such as muscular dystrophy, articular cartilage defect, bone defect, tendon rupture, etc.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0028] In the specification and claims of the present application, when a numerical value is referred to as "about", it shall include values up to ±20%, ±15%, ±10%, or ±5% of the indicated numerical value.

[0029] The origin of the "cells" described in this specification is from humans and non-human animals (e.g., mice, rats, cows, horses, pigs, sheep, monkeys, dogs, cats, birds, etc.), and although not particularly limited, cells derived from humans are particularly preferred.

[0030] In the method of the present application, for culturing cells, a medium obtained by adding necessary factors to a basal medium for animal cell culture is used. Examples of the basal medium for animal cell culture that can be used in the present application include Iscove's modified Eagle's Medium, Ham's F12 medium, MEM Zinc Option medium, IMEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM), αMEM medium, Dulbecco's modified Eagle's Medium (DMEM), RPMI 1640 medium, Fischer's medium, and mixed media thereof. The basal medium may contain serum (e.g., fetal bovine serum (FBS)) or may be serum-free. If necessary, for example, one or more serum substitutes such as albumin, bovine serum albumin (BSA), transferrin, apotransferrin, KnockOut Serum Replacement (KSR) (serum substitute for ES cell culture) (Invitrogen), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiol glycerol, monothioglycerol may be included, or it may also contain one or more substances such as lipids (e.g., chemically defined lipid concentrate), amino acids, L-glutamine, GlutaMAX (Invitrogen), non-essential amino acids (NEAA), vitamins, growth factors, antibiotics (e.g., penicillin and streptomycin), antioxidants, pyruvic acid, buffers, inorganic salts, and their equivalents.

[0031] In one embodiment, except for the step of inducing mesenchymal stromal cells from the somite cells described below, a serum-free medium or a chemically defined medium (hereinafter "CDM") is preferably used as the basal medium. As the CDM medium, a medium obtained by adding 1x chemically defined lipid concentrate (GIBCO), 15 mg / ml apo-transferrin (Sigma, St. Louis, MO), 450 mM monothioglycerol (Sigma), 5 mg / ml purified BSA (99% purified by crystallization; Sigma), 7 mg / ml insulin (WAKO), and penicillin / streptomycin (Invitrogen, Carlsbad, CA, USA) to Iscove's modified Eagle's Medium / Ham's F12 medium 1:1 (GIBCO, Grand Island, NY, USA) is exemplified. Hereinafter, the medium having this composition is referred to as the "CDM basal medium" in the present specification.

[0032] Also, the culture is preferably performed without using feeder cells, and if necessary, a culture substrate may be used. Examples of the culture substrate include commercially available extracellular matrix Matrigel (BD, Bedford, MA, USA).

[0033] In another embodiment, except for the step of inducing mesenchymal stromal cells from the somite cells described below, it is possible to perform under xeno-free conditions. For example, the steps of inducing unsegmented mesoderm cells from pluripotent stem cells, inducing somite cells from unsegmented mesoderm cells, inducing sclerotome cells from somite cells, and inducing syndetome cells from sclerotome cells can be carried out under xeno-free conditions. "Xeno-free" means a medium or culture conditions that do not contain components derived from organisms different from the species of the cells to be cultured. The xeno-free medium is not particularly limited, but StemFit (登録商標) AK02 medium (Ajinomoto Co., Inc.), StemFit (登録商標) AK03 medium (Ajinomoto Co., Inc.), and CTS (商標)Examples include KnockOut SR XenoFree Medium (Gibco), such as AK03 medium. In xeno-free culture, it is preferable to use a xeno-free culture substrate in combination with the xeno-free medium. Examples of the xeno-free culture substrate include the integrin-binding site (E8) fragment of recombinant human laminin 511. Examples of such xeno-free culture substrates include iMatrix511 (Nippi Inc.) and CTS CELLstart Substrate (Gibco), such as iMatrix511.

[0034] In the method of the present application, cell culture may be performed under general animal cell culture conditions. The culture temperature is not limited to the following, but is about 30 to 40 °C, preferably about 37 °C. The culture is preferably performed in an atmosphere of air containing CO2, and the CO2 concentration is preferably about 2 to 5%.

[0035] In each step, the fact that the target cells are obtained can be confirmed by examining the expression profile of cell surface markers of the obtained cells. The confirmation of the expression profile of cell surface markers may be performed using known methods, such as RT-qPCR, immunocytochemical analysis, FACS (Fluorescence-activated cell sorting), and the like.

[0036] The cell culture obtained in each step may be purified and used when subjected to further differentiation induction. Alternatively, a cell culture containing the target cells may be purified and provided for the target cells. Cell purification can be performed, for example, based on markers on the cell surface. For example, a method of sorting by FACS using an antibody against a cell surface marker that is expressed or not expressed by the target cells is exemplified.

[0037] Production of Somite Cells from Pluripotent Stem Cells The present application provides a step of providing pluripotent stem cells, and a method for producing somite cells from pluripotent stem cells, which includes a step of culturing the pluripotent stem cells in a medium containing a GSK3β inhibitor.

[0038] Pluripotent stem cells are stem cells that have the pluripotency to differentiate into all cells existing in a living body and also possess the ability to proliferate. Examples of such cells include embryonic stem (ES) cells (J.A. Thomson et al. (1998), Science 282:1145-1147; J.A. Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92:7844-7848; J.A. Thomson et al. (1996), Biol. Reprod., 55:254-259; J.A. Thomson and V.S. Marshall (1998), Curr. Top. Dev. Biol., 38:133-165), embryonic stem (ntES) cells derived from cloned embryos obtained by nuclear transfer (T. Wakayama et al. (2001), Science, 292:740-743; S. Wakayama et al. (2005), Biol. Reprod., 72:932-936; J. Byrne et al. (2007), Nature, 450:497-502), spermatogonial stem cells (「GS cells」) (M. Kanatsu-Shinohara et al. (2003) Biol. Reprod., 69:612-616; K. Shinohara et al. (2004), Cell, 119:1001-1012), embryonic germ cells (「EG cells」) (Y. Matsui et al. (1992), Cell, 70:841-847; J.L. Resnick et al. (1992), Nature, 359:550-551), induced pluripotent stem (iPS) cells (K. Takahashi and S. Yamanaka (2006) Cell, 126:663-676; K. Takahashi et al. (2007), Cell, 131:861-872; J. Yu et al. (2007), Science, 318:1917-1920; Nakagawa, M. et al., Nat. Biotechnol.(26:101-106 (2008); WO2007 / 069666), pluripotent cells derived from cultured fibroblasts and bone marrow stem cells (Muse cells) (WO2011 / 007900), and the like. Preferably, the pluripotent stem cells are human pluripotent stem cells, such as ES cells and iPS cells.

[0039] In particular, iPS cells are particularly preferably used as a material for producing cells for treatment and transplantation. When the cells obtained by the present application are used for treatment, from the viewpoint of no rejection reaction occurring, it is desirable to use iPS cells obtained from somatic cells that are identical or substantially identical to the HLA genotype of the recipient individual. Here, "substantially identical" means that the HLA genotypes match to such an extent that the immune reaction can be suppressed by an immunosuppressant against the transplanted cells. For example, somatic cells having an HLA type in which three loci of HLA-A, HLA-B, and HLA-DR or four loci including HLA-C match. Of course, iPS cells induced from the patient's own somatic cells may be used to produce cells for treatment.

[0040] Pluripotent stem cells may be those produced by known methods, commercially available pluripotent stem cells, or pluripotent stem cells stored together with information on the individuals from which they are derived for research or transplantation medicine. In Japan, a project to construct a highly versatile iPS cell bank is currently underway by using humans who are homozygous for frequently occurring HLA haplotypes as donors (CYRANOSKI, Nature vol. 488, 139 (2012)), and pluripotent stem cells obtained from such an iPS cell bank, for example, may be used. As methods for producing iPS cells from human somatic cells, there are reports such as Koyanagi-Aoi et al., 2013; Nakagawa et al., 2014; Okita et al., 2011; Takahashi et al., 2007. A method of inducing iPS cells without using feeder cells under completely xenofree conditions is also known (Nakagawa M, et al. Scientific Reports 4:3594 (2014)). Pluripotent stem cells induced under such xenofree conditions may be used.

[0041] In addition, pluripotent stem cells may be those induced from cells derived from patients with genetic diseases. Somite cells obtained by the method of the present application from such iPS cells derived from genetic diseases, and cells further induced to differentiate from somite cells can be applied to drug discovery research and elucidation of disease mechanisms as disease model cells. For example, it has been reported that iPS cells are induced from somatic cells of patients with fibrodysplasia ossificans progressiva (Matsumoto et al., 2014).

[0042] (1) Inducing Differentiation of Pluripotent Stem Cells (PSC) into Presomitic Mesoderm Cells (PSM) Methods for producing unsegmented mesoderm cells from pluripotent stem cells are known, and any of the known methods may be used. For example, by culturing pluripotent stem cells in a medium containing a relatively high concentration of a GSK3β inhibitor, they can be induced into unsegmented mesoderm cells at a relatively high induction rate (Non-Patent Documents 7 and 9: Loh et al 2016 and Xi 2017).

[0043] GSK3β inhibitors are defined as substances that inhibit the kinase activity of the GSK3β protein (e.g., the ability to phosphorylate β-catenin), and a number of them are already known. GSK3β inhibitors are also known as WNT signaling pathway activators. For example, BIO (also known as GSK-3β inhibitor IX; 6-bromoindirubin 3'-oxime), an indirubin derivative, SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), a maleimide derivative, SB415286 (3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrole-2,5-dione), GSK-3β inhibitor VII (4-dibromoacetophenone), a phenyl α-bromomethyl ketone compound, L803-mts (also known as GSK-3β peptide inhibitor; Myr-N-GKEAPPAPPQSpP-NH2), a cell membrane-permeable phosphorylated peptide, and CHIR99021 (6-[2-[4-(2,4-Dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-ylamino]ethylamino]pyridine-3-carbonitrile) with high selectivity can be mentioned. These compounds are commercially available, for example, from Calbiochem, Biomol, etc., and can be easily used. They may also be obtained from other sources or prepared by oneself.

[0044] When manufacturing unsegmented mesoderm cells from pluripotent stem cells, for example, CHIR99021 can be exemplified as the GSK3β inhibitor used. The concentration of the GSK3β inhibitor can be appropriately determined by those skilled in the art and needs to be relatively high. When using CHIR99021 as the GSK3β inhibitor, it is exemplified that the concentration in the medium is, for example, 0.1 μM to 50 μM, preferably 1 to 20 μM, more preferably about 10 μM.

[0045] The medium used for producing unsegmented mesoderm cells from pluripotent stem cells preferably further contains at least one of a TGFβ inhibitor, a BMP inhibitor, and FGF, and most preferably contains all three.

[0046] As used in the present specification and claims, the TGFβ inhibitor is a substance that inhibits the binding of molecules of the TGFβ family such as TGFβ, Activin, and Nodal to receptors and subsequent signal transduction to downstream SMADs, and is not particularly limited as long as it is a substance that inhibits binding to the ALK family of receptors or a substance that inhibits phosphorylation of SMAD by the ALK family. For example, Lefty-1 (exemplified by mouse: NM_010094, human: NM_020997 as NCBI Accession No.), SB431542, SB202190 (above, R.K. Lindemann et al., Mol. Cancer, 2003, 2:20), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276 (Lilly Research Laboratories), A-83-01 (WO 2009146408) ALK5 inhibitor II (2-[3-[6-methylpyridin-2-yl]-1H-pyrazol-4-yl]-1,5-naphthyridine), TGFβRI kinase inhibitor VIII (6-[2-tert-butyl-5-[6-methyl-pyridin-2-yl]-1H-imidazol-4-yl]-quinoxaline), and derivatives thereof are exemplified.

[0047] Examples of the TGFβ inhibitor used for producing unsegmented mesoderm cells from pluripotent stem cells include SB431542. The concentration of the TGFβ inhibitor may be appropriately determined by those skilled in the art and is not particularly limited. When SB431542 is used as the TGFβ inhibitor, the concentration in the medium is exemplified as, for example, 0.1 μM to 50 μM, preferably 1 to 20 μM, more preferably about 10 μM.

[0048] Examples of BMP inhibitors include proteinaceous inhibitors such as Chordin, Noggin, and Follistatin; Dorsomorphin (6-[4-(2-piperidin-1-yl-ethoxy)phenyl]-3-pyridin-4-yl-pyrazolo[1,5-a]pyrimidine), its derivatives (such as DMH1) (P. B. Yu et al. (2007), Circulation, 116:II_60; P.B. Yu et al. (2008), Nat. Chem. Biol., 4:33-41; J. Hao et al. (2008), PLoS ONE, 3(8):e2904); and LDN-193189 (i.e., 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline).

[0049] For example, DMH1 is used as a BMP inhibitor when producing unsegmented mesoderm cells from pluripotent stem cells. The concentration of the BMP inhibitor may be appropriately determined by those skilled in the art and is not particularly limited. When DMH1 is used as the BMP inhibitor, the concentration in the medium is exemplified as, for example, 0.1 μM to 20 μM, preferably 1 to 5 μM, and more preferably about 2 μM.

[0050] Examples of FGF (fibroblast growth factor) include FGF2, FGF7, FGF8, and FGF10. For example, FGF2 is used as the FGF when producing unsegmented mesoderm cells from pluripotent stem cells. The concentration of FGF may be appropriately determined by those skilled in the art and is not particularly limited. When FGF2 is used as the FGF, the concentration in the medium is exemplified as, for example, 0.1 ng / ml to 100 ng / ml, preferably 1 to 50 ng / ml, and more preferably about 20 ng / ml.

[0051] The number of days of culture when producing unsegmented mesoderm cells from pluripotent stem cells may be appropriately determined by those skilled in the art and is not particularly limited. For example, it may be 1 to 7 days, preferably 3 to 5 days, and more preferably about 4 days.

[0052] The unsegmented mesoderm cells can be identified as, for example, cells expressing DLL1. When using iPS cells as pluripotent stem cells, confirmation that unsegmented mesoderm cells have been produced from the iPS cells can be achieved by the fact that markers specific to iPS cells, such as NANOG, OCT3 / 4, and SOX2, are not expressed, and by the expression of any one or a combination of BRACHYURY, DLL1, TBX6, MSGN1, and WNT3A, which are markers of unsegmented mesoderm cells.

[0053] The unsegmented mesoderm cell culture obtained in this step may be purified and then used in subsequent steps, or used as it is. For the purification of specific types of cells in the cell culture, for example, FACS using an antibody against a cell surface marker that is expressed or not expressed by unsegmented mesoderm cells is exemplified. As an example, sorting by FACS using the expression of DLL1 on the cell surface as an indicator can be mentioned.

[0054] (2) Inducing Differentiation of Presomitic Mesoderm Cells (PSM) into Somite Cells (SM) Somite cells are transient stem cells that give rise to multiple cell types (such as dermomyotome (D), myotome (MYO), sclerotome (SCL), and syndetome (SYN)), and are also cells that serve as the origin of mesenchymal stromal cells (MSC) that give rise to bone, cartilage, and fat after birth.

[0055] The unsegmented mesoderm cells are cultured in a medium containing a GSK3β inhibitor to induce somite cells. The GSK3β inhibitor used when producing somite cells from unsegmented mesoderm cells can be the same as those described above, and can be, for example, CHIR99021. The concentration of the GSK3β inhibitor can be appropriately determined by those skilled in the art and is not particularly limited. When using CHIR99021 as the GSK3β inhibitor, its concentration in the medium is exemplified to be, for example, 0.1 μM to 50 μM, preferably 1 to 20 μM, more preferably about 5 μM.

[0056] When manufacturing somite cells from unsegmented mesoderm cells, the medium used may further contain a TGFβ inhibitor. As the TGFβ inhibitor, those similar to those described above can be used, and for example, it can be SB431542. The concentration of the TGFβ inhibitor may be appropriately determined by those skilled in the art and is not limited. When using SB431542 as the TGFβ inhibitor, its concentration in the medium is exemplified to be, for example, 0.1 μM to 50 μM, preferably 1 to 20 μM, more preferably about 10 μM.

[0057] The number of days of culture when manufacturing somite cells from unsegmented mesoderm cells is not particularly limited, and for example, it may be 1 to 7 days, preferably 3 to 5 days, more preferably about 4 days.

[0058] The generation of somite cells can be confirmed by appropriately combining the expression of one or more of somite cell markers, such as MEOX1 and PARAXIS and the transcription factor PAX3, and the disappearance of the expression of one or more of unsegmented mesoderm cell markers.

[0059] Stepwise Inducing Differentiation of Somite Cells into Various Cells (3) Inducing Differentiation of Somite Cells (SM) into Dermomyotome Cells (DM) The dermomyotome is formed by the differentiation of the dorsal side of the somite, giving rise to the dermatome, which is the precursor of the dermis, and the myotome, which is the precursor of the skeletal muscle.

[0060] Methods for manufacturing dermomyotome cells from somite cells are known, and any known method may be used. Dermomyotome cells can be manufactured, for example, by culturing in a medium containing a GSK3 inhibitor and BMP.

[0061] When manufacturing dermomyotome cells from somite cells, the somite cells used as the starting material may be somite cells manufactured from pluripotent stem cells through the above steps (1) and (2), or somite cells obtained by other methods. Somite cells may also be obtained from the living body of an animal.

[0062] When producing dermomyotome cells from somite cells, GSK3 inhibitors used can be the same as those described above, and can be, for example, CHIR99021. The concentration of the GSK3β inhibitor is not particularly limited and can be appropriately determined by those skilled in the art. When using CHIR99021 as the GSK3β inhibitor, its concentration in the medium is exemplified to be, for example, 0.1 μM to 50 μM, preferably 1 to 20 μM, more preferably about 5 μM.

[0063] BMPs used when producing dermomyotome cells from somite cells include, for example, BMP2, BMP4, BMP7, etc., and can be, for example, BMP4. The concentration of BMP is not particularly limited and can be appropriately determined by those skilled in the art. When using BMP4 as the BMP, its concentration in the medium is exemplified to be, for example, 0.1 ng / ml to 100 ng / ml, preferably 1 to 50 ng / ml, more preferably about 10 ng / ml.

[0064] The number of days of culture when producing dermomyotome cells from somite cells is not particularly limited and can be appropriately determined by those skilled in the art. For example, it can be 1 to 5 days, preferably 2 to 4 days, more preferably about 3 days. The medium can be appropriately replaced with a new one during the culture.

[0065] ALX4, EN1, NOGGIN, etc. are known as markers for dermomyotome cells. Confirmation that dermomyotome cells have been produced from somite cells can be appropriately combined and confirmed by the maintenance of PAX3, a transcription factor common to somite cells, the expression of one or more dermomyotome cell markers, and the disappearance of the expression of one or more somite cell markers.

[0066] The dermomyotome cell culture produced from somite cells can be directly used for the production of myotome cells or dermal segment cells, or can be used for the production of dermal segment cells after purifying the dermomyotome cells. Purification of dermal myotome cells is exemplified to be performed by FACS using antibodies against markers known to be expressed and / or not expressed by dermal myotome cells.

[0067] (4) Inducing Differentiation of Dermomyotome Cells (DM) into Myotome Cells (MYO) As one aspect of the invention of the present application, a method is provided for obtaining dermomyotome cells from pluripotent stem cells through the above steps (1), (2), and (3), and further obtaining myotome cells from the dermomyotome cells. Myotome cells are precursor cells of skeletal muscle cells. Myotome cells can be produced, for example, by culturing dermomyotome cells in a medium containing a GSK3β inhibitor.

[0068] As the GSK3 inhibitor used for producing myotome cells from dermomyotome cells, those similar to the above-mentioned ones can be used, and it can be, for example, CHIR99021. The concentration of the GSK3β inhibitor is not particularly limited and can be appropriately determined by those skilled in the art. When using CHIR99021 as the GSK3β inhibitor, its concentration in the medium can be exemplified as, for example, 0.1 μM to 50 μM, preferably 1 to 20 μM, more preferably about 5 μM.

[0069] The number of days of culture when producing myotome cells from dermomyotome cells is not particularly limited and can be appropriately determined by those skilled in the art. For example, it can be 20 days to 45 days, 25 days to 40 days, or about 30 days. The medium is appropriately replaced with a new one during the culture. The medium can be replaced, for example, every about 2 to 3 days.

[0070] As myotome cell markers, MYOD, MYOG, PAX7, etc. are known. The generation of myotome cells can be confirmed by the expression of one or more of these known markers and / or the non-expression of one or more dermomyotome cell markers. Also, the obtained myotome cell culture can be purified by FACS using antibodies against markers known to be expressed and / or not expressed by myotome cells.

[0071] The obtained myotome cells can be further differentiated to produce skeletal muscle cells. The method for inducing differentiation from myotome cells to skeletal muscle cells is known.

[0072] (5) Inducing Differentiation of Dermomyotome Cells (DM) into Dermatome Cells (D) The dermatome is induced from the somite and becomes the precursor of the dorsal dermis. Dermatome cells can be produced by culturing somite cells in a medium containing a GSK3β inhibitor and BMP. When producing dermatome cells from somite cells, the somite cells used as starting materials can be somite cells produced from pluripotent stem cells through the above steps (1) to (2) and then produced by step (3), or somite cells produced from somite cells obtained by other methods, or dermatome cells obtained by other methods. The somite cells may also be those obtained from the living body of an animal.

[0073] The GSK3 inhibitor used when producing dermatome cells from somite cells can be the same as those described above, and can be, for example, CHIR99021. The concentration of the GSK3β inhibitor is not particularly limited and can be appropriately determined by those skilled in the art. When using CHIR99021 as the GSK3β inhibitor, its concentration in the medium is exemplified to be, for example, 0.1 μM to 50 μM, preferably 1 to 20 μM, more preferably about 5 μM.

[0074] The BMP used when producing dermatome cells from somite cells includes, for example, BMP2, BMP4, BMP7, etc., and is, for example, BMP4. The concentration of BMP is not particularly limited and can be appropriately determined by those skilled in the art. When using BMP4 as the BMP, its concentration in the medium is exemplified to be, for example, 0.1 ng / ml to 100 ng / ml, preferably 1 to 50 ng / ml, more preferably about 10 ng / ml.

[0075] The number of days of culture when producing dermatome cells from somite cells is not particularly limited and can be appropriately determined by those skilled in the art. In one aspect, for example, it can be 8 days to 15 days, about 9 days.

[0076] As markers for dermomyotome cells, PDGFRα, EN1, ALX4, MSX1, COLIA2 and the like are known. The generation of dermomyotome cells can be confirmed by appropriately combining one or more of the expression of these known markers and the disappearance of the expression of dermomyotome cell markers. Further, the obtained dermomyotome cell culture may be purified by FACS using antibodies against markers that are known to be expressed and / or not expressed by dermomyotome cells. Purification of dermomyotome cells by FACS using an anti-PDGFRα antibody is exemplified.

[0077] The obtained dermomyotome cells can be further differentiated to produce dermal cells.

[0078] (6) Inducing Differentiation of Somite Cells (SM) into Sclerotome Cells (SCL) The sclerotome is formed by the differentiation of the ventral side of the somite, giving rise to the syndetome, which is a precursor of tendons and ligaments. As one aspect of the present invention, there is provided a method for producing sclerotome cells from somite cells through the above steps (1) and (2) from pluripotent stem cells.

[0079] Methods for producing sclerotome cells from somite cells are known (Zhao et al., 2014), and any known method may be used for induction from the obtained somite cells. In one aspect, sclerotome cells are produced by culturing somite cells in a medium containing a sonic hedgehog activator (SHH activator) and a BMP inhibitor.

[0080] Examples of sonic hedgehog activators include proteins belonging to the hedgehog family (e.g., Shh, Shh-N), Shh receptors, and Shh receptor agonists (Purmorphamine, SAG), and for example, SAG can be used. The concentration of the SHH activator is not particularly limited and may be appropriately determined by those skilled in the art. When SAG is used as the SHH activator, the concentration in the medium is exemplified to be, for example, 1 nM to 1 μM, preferably 10 to 500 nM, more preferably about 100 nM.

[0081] When manufacturing sclerotome cells from somite cells, BMP inhibitors similar to those described above can be used, and for example, it can be LDN193189. The concentration of the BMP inhibitor is not particularly limited and can be appropriately determined by those skilled in the art. When using LDN193189 as the BMP inhibitor, its concentration in the medium is exemplified to be, for example, 0.01 μM to 10 μM, preferably 0.1 μM to 1 μM, and more preferably about 0.6 μM.

[0082] The number of days of culture when manufacturing sclerotome cells from somite cells is not particularly limited and can be appropriately determined by those skilled in the art. For example, it can be 1 to 5 days, preferably 2 to 4 days, and more preferably about 3 days.

[0083] As markers for sclerotome cells, PAX1, PAX9, NKX3.2, etc. are known. The generation of sclerotome cells can be confirmed by appropriately combining one or more of the expression of these known markers and the disappearance of the expression of markers specific to somite cells. Also, the obtained sclerotome cell culture can be purified by FACS using antibodies against markers that are known to be expressed and / or not expressed by sclerotome cells.

[0084] (7) Inducing Differentiation of Sclerotome Cells (SCL) into Syndetome Cells (SYN) The syndetome differentiates from the inner part of the sclerotome and gives rise to tendons and ligaments. As one aspect of the present application, a method for manufacturing syndetome cells from sclerotome cells including the following steps is provided: (7-1) A step of culturing sclerotome cells in a medium containing FGF, and (7-2) A step of culturing the cells obtained in step 7-1 in a medium containing BMP and TGFβ.

[0085] The sclerotome cells used as the starting material may be cells induced by known methods from other cell types such as somite cells. When manufacturing sclerotome cells from somite cells, the somite cells may be those manufactured from pluripotent stem cells by the method of the present application.

[0086] Examples of FGF used for producing ligament nodule cells from sclerotome cells include FGF2, FGF7, FGF8, and FGF10, such as FGF8 for example. The concentration of FGF is not particularly limited and may be appropriately determined by those skilled in the art. When using FGF8 as FGF, the concentration in the medium is exemplified to be, for example, 0.1 ng / ml to 100 ng / ml, preferably 1 to 50 ng / ml, more preferably about 20 ng / ml.

[0087] Examples of BMP used for producing ligament nodule cells from sclerotome cells include BMP2, BMP4, and BMP7, such as BMP7 for example. The concentration of BMP is not particularly limited and may be appropriately determined by those skilled in the art. When using BMP7 as BMP, the concentration in the medium is exemplified to be, for example, 0.1 ng / ml to 100 ng / ml, preferably 1 to 50 ng / ml, more preferably about 10 ng / ml.

[0088] In step 7-1, the medium may further contain TGFβ. Examples of TGFβ used for producing ligament nodule cells from sclerotome cells include TGFβ1, TGFβ2, and TGFβ3, such as TGFβ3 for example. The concentration of TGFβ is not particularly limited and may be appropriately determined by those skilled in the art. When using TGFβ3 as TGF, the concentration in the medium is exemplified to be, for example, 0.1 ng / ml to 100 ng / ml, preferably 1 to 50 ng / ml, more preferably about 10 ng / ml.

[0089] In a certain aspect, sclerotome cells are once taken out from the sclerotome cell culture, seeded into the medium for ligament nodule culture, and the culture is started. The number of culture days for each of steps (7-1) and (7-2) is not particularly limited and may be appropriately determined by those skilled in the art. When the medium does not contain TGFβ in step (7-1), the number of culture days for step (7-1) is exemplified to be, for example, 1 to 7 days, preferably about 3 days, and the number of days for step (7-2) is about 15 to 25 days, preferably about 18 days. When the medium contains TGFβ in step (7-1), the number of culture days for step (7-1) is exemplified to be, for example, 1 to 5 days, preferably about 2 days, and the number of days for step (7-2) is, for example, about 4 to 8 days, preferably about 6 days.

[0090] As markers for ligament articular cells, SCX, MKX, COL1A1, COL1A2, etc. are known. The generation of ligament articular cells can be confirmed by appropriately combining one or more of the expression of these known markers and the disappearance of scleroblast markers. In addition, the obtained ligament articular cell culture may be purified by FACS using antibodies against markers that are known to be expressed and / or not expressed by ligament articular cells.

[0091] (8) Inducing Differentiation of Somite Cells (SM) into Somite-derived Mesenchymal Stromal Cells (SMMSC) Mesenchymal stroma is known to differentiate into bone, cartilage, and fat, and mesenchymal stromal cells are pluripotent cells that can be obtained from multiple sites in the body, such as bone marrow, adipose tissue, or blood. As one aspect of the present application, there is provided a method for producing mesenchymal stromal cells (somite-derived mesenchymal stromal cells) including a step of culturing somite cells in a medium containing FGF. The somite cells may be cells isolated from a living body or cells produced from other cell types. In one aspect, the somite cells used are somite cells induced from pluripotent stem cells by the above steps (1) and (2).

[0092] The FGF used when producing mesenchymal stromal cells from somite cells can be the same as those described above, and can be, for example, FGF2. The concentration of FGF is not particularly limited and may be appropriately determined by those skilled in the art. When FGF2 is used as FGF, the concentration in the medium is exemplified to be, for example, 0.4 ng / ml to 40 ng / ml, preferably 1 to 10 ng / ml, more preferably about 4 ng / ml. The medium used when producing mesenchymal stromal cells from somite cells is exemplified by αMEM medium. The medium used in this step preferably contains serum. As the serum, fetal bovine serum (FBS) may be used, or sera of other animal species may be used, such as human serum in the case of inducing differentiation of human cells. The concentration of FBS is not particularly limited and may be appropriately determined by those skilled in the art. For example, it may be about 10%.

[0093] When producing mesenchymal stromal cells from somite cells, the number of culture days is not particularly limited and may be appropriately determined by those skilled in the art. For example, it may be 4 to 30 days, preferably 8 to 18 days, more preferably 10 to 15 days, and even more preferably about 12 days.

[0094] As markers for mesenchymal stromal cells, CD44, CD73, CD105, CD90, etc. are known. The generation of mesenchymal stromal cells can be confirmed by appropriately combining one or more of the expression of these known markers and the disappearance of somite cell markers. In addition, the obtained mesenchymal stromal cell culture may be purified by FACS using antibodies against markers that are known to be expressed and / or not expressed by mesenchymal stromal cells.

[0095] As described in detail above, the present application provides a method for obtaining somite cells from pluripotent stem cells. The somite cells obtained by the method of the present application can be further stepwise induced to differentiate into dermal segment cells and myotome cells via dermatome cells, and into ligament segment cells via sclerotome cells. In addition, the somite cells obtained by the method of the present application can be further induced to differentiate into mesenchymal stromal cells.

[0096] That is, the present application provides a method for inducing differentiation from pluripotent stem cells including the following into myotome cells: Inducing presomitic mesoderm cells (PSM) from pluripotent stem cells (PSC) according to (1) above, Inducing somite cells (SM) from the induced presomitic mesoderm cells (PSM) according to (2) above, Inducing dermomyotome cells (DM) from the induced somite cells (SM) according to (3) above, and Inducing myotome cells (MYO) from the induced dermomyotome cells (DM) according to (4) above.

[0097] The present application also provides a method for inducing differentiation from pluripotent stem cells including the following into dermal segment cells: Inducing presomitic mesoderm cells (PSM) from pluripotent stem cells (PSC) according to (1) above, Inducing somite cells (SM) from the induced presomitic mesoderm cells (PSM) according to (2) above, Inducing dermomyotome cells (DM) from the induced somite cells (SM) according to (3) above, and Inducing dermatome cells (D) from the induced dermomyotome cells (DM) according to (5) above.

[0098] The present application further provides a method for inducing differentiation from pluripotent stem cells including the following to sclerotome cells: Inducing presomitic mesoderm cells (PSM) from pluripotent stem cells (PSC) according to (1) above, Inducing somite cells (SM) from the induced presomitic mesoderm cells (PSM) according to (2) above, and Inducing sclerotome cells (SCL) from the induced somite cells (SM) according to (6) above.

[0099] The present application also further provides a method for inducing differentiation from pluripotent stem cells including the following to syndetome cells: Inducing presomitic mesoderm cells (PSM) from pluripotent stem cells (PSC) according to (1) above, Inducing somite cells (SM) from the induced presomitic mesoderm cells (PSM) according to (2) above, Inducing sclerotome cells (SCL) from the induced somite cells (SM) according to (6) above, and Inducing syndetome cells (SYN) from the induced sclerotome cells (SCL) according to (7) above.

[0100] The present application also further provides a method for inducing differentiation from pluripotent stem cells including the following to somite-derived mesenchymal stromal cells: Inducing presomitic mesoderm cells (PSM) from pluripotent stem cells (PSC) according to (1) above, Inducing somite cells (SM) from the induced presomitic mesoderm cells (PSM) according to (2) above, and Inducing somite-derived mesenchymal stromal cells (SMMSC) from the induced somite cells (SM) according to (8) above.

[0101] This application also provides a cell transplantation therapy method using cells derived from pluripotent stem cells by the method of this application. For example, various iPS cell-derived somite-derived cells obtained in this application can be used for the treatment of musculoskeletal disorders such as muscular dystrophy and tendon rupture. Specifically, it is exemplified that ligament nodule cells can be used for the treatment of tendon and ligament-related diseases such as ossification of the posterior longitudinal ligament and progressive fibrodysplasia ossificans and tendon rupture.

[0102] When using iPS cell-derived ligament nodule cells obtained by the method of this application for the treatment of tendon and ligament-related diseases and tendon rupture, etc., the ligament nodule cells may be dispersed in a biocompatible base or the like and then injected into the disease / injury site. As the biocompatible base material, known ones can be appropriately adopted, and Matrigel is exemplified. The number of cells to be injected and the injection site may be appropriately determined according to the disease / injury to be treated, and are not particularly limited. For example, 10 12 ~10 6 cells or 10 11 ~10 7 cells, or 10 10 ~10 8 iPS cell-derived ligament nodule cells are injected. If necessary, surgical procedures such as suturing of the disease / injury site are also carried out.

Examples

[0103] Examples are shown below for more detailed description, but the present invention is not limited by the examples at all. Cell Culture Human iPS cells were prepared and maintained using the method of Takahashi et al., 2007. Specifically, human iPS cells were maintained on SNL feeder cells in a primate ES cell medium (ReproCELL, Tokyo, Japan) supplemented with 4 ng / ml FGF2 (WAKO, Osaka, Japan). Unless otherwise specified, in all experiments of Examples 1 to 5, 201B7-PAX3-GFP iPS cells in which one allele of the PAX3 coding sequence of exon 1 was replaced with EGFP were used. PAX3 having the same knock-in design GFP / +The heterozygous mice are viable and fertile. GFP expression means the expression of the endogenous Pax3 in the mice (Lagha, M. et al., 2010). Also, in Example 1, the reproducibility due to differences in iPS cell lines was examined using various iPS cell lines (201B7, TIG118-4f, 414C2, 409B2, and 1231A3) (cells prepared according to Koyanagi-Aoi et al., 2013; Nakagawa et al., 2014; Okita et al., 2011; Takahashi et al., 2007).

[0104] Culture Medium The composition of the CDM basal medium used in the examples is shown below: 1x chemically defined lipid concentrate (GIBCO), 15 mg / ml apotransferrin (Sigma, St. Louis, MO), 450 mM monothioglycerol (Sigma), 5 mg / ml purified BSA (99% purified by crystallization; Sigma), 7 mg / ml insulin (WAKO), and penicillin / streptomycin (Invitrogen, Carlsbad, CA, USA) were added to Iscove's modified Eagle's Medium / Ham's F12 medium 1:1 (GIBCO, Grand Island, NY, USA).

[0105] RT-qPCR Analysis Total RNA was purified using an RNeasy kit (Qiagen, Valencia, CA) and treated with a DNase-one kit (Qiagen) to remove genomic DNA. Reverse transcription was performed using 1 μg of total RNA and Superscript III reverse transcriptase (Invitrogen) according to the manufacturer's instructions. RT-qPCR was performed using Thunderbird SYBR qPCR Mix (TOYOBO, Osaka, Japan) and analyzed using a QunatStudio12K Flex PCR system (Applied Biosystems, Forester City, CA) or a StepOne real-time PCR system (Applied Biosystems).

[0106] Immunocytochemistry, Immunohistochemistry, Histological Analysis Before immunocytochemistry with antibodies, the cells on the plate were fixed with 2% paraformaldehyde at 4°C for 10 minutes, washed twice with PBS, incubated with 0.2% MtOH (Nacalai Tesque) or 0.2% tween20 (sigma) / PBS as a surfactant for permeabilization at 4°C for 15 minutes, treated with Blocking One (Nacalai Tesque) or 1% BSA / PBS at 4°C for 1 hour, and treated with the primary antibody at 4°C overnight. Next, the samples were washed several times with 0.2% tween20 / PBS and incubated with the secondary antibody at room temperature for 1 hour. The nuclei were counterstained using DAPI (1:5000; Sigma). Immunohistochemistry for anti-type II collagen antibody and histological analysis (HE staining, Alcian blue staining, and Safranin O staining) of the induced 3DCI pellets were performed at the Center for Anatomical, Pathological and Forensic Medical Researches, Graduate School of Medicine, Kyoto University. Observation and evaluation of the samples were performed using a BZ-X700 (Keyence, Osaka, Japan). For immunocytochemistry of MHC, photographs were taken using the optical sectioning system of the BZ-X700.

[0107] FACS (Fluorescence-activated cell sorting) and Analysis Fluorescence-activated cell sorting (FACS) was performed using an Aria II (BD) according to the manufacturer's protocol. Intracellular flow cytometry analysis was also performed using an Aria II (BD) according to the manufacturer's protocol. Briefly, cells were fixed and permeabilized prior to antibody staining. The expression ratio of each differentiation marker was calculated by comparing with iPSCs or induced somite cells.

[0108] GAG (Glucosaminoglycan) Assay The GAG content in the pellet was quantified using a Blyscan Glycosaminoglycan Assay Kit (Biocolor Ltd., Belfast, UK). The DNA content was quantified using a PicoGreen dsDNA Quantitation Kit (Invitrogen).

[0109] Microarray Analysis Total RNA was prepared using the RNeasy Mini Kit (Qiagen). cDNA was synthesized using the GeneChip WT (Whole Transcript) Sense Target Labeling and Control Reagents Kit as described by the manufacturer (Affymetrix, Santa Clara, CA). Hybridization, washing, and scanning with the GeneChip Human Gene 1.0 ST expression array were performed according to the manufacturer's (Affymetrix) protocol. Expression values were calculated using the RMA summarization method, and the obtained data were analyzed with GeneSpring GX 14.5 (Agilent Technologies, Santa Clara, CA, USA) for heat map and principal component analysis (PCA). PCA analysis was performed at expression values (with 2-fold higher, with statistical significance). Statistical analysis was performed using one-way ANOVA with Benjamini and Hochberg False Discovery Rate (BH-FDR ≤ 0.01) multiple test correction, followed by Tukey's HSD post hoc test (GeneSpring GX).

[0110] Statistics The statistical significance of all experiments was calculated using GraphPad Prism7 (GraphPad Software, inc., La Jolla, CA, USA). P values less than 0.05 were considered statistically significant.

[0111] Examples 1-4 An overview of Examples 1-4 is shown in FIG. 2A.

Example

[0112] (1) Inducing Differentiation of Human iPS Cells into Presomitic Mesoderm Cells (PSM) To minimize the influence of growth factors secreted from feeder cells and growth factors contained in the medium, SNL feeder cells were removed from the iPS cell culture, and human iPS cells were seeded onto dishes coated with Matrigel (BD, Bedford, MA, USA) (1.3x10 6 cells / 10 cm dish). The cells were cultured for 3 days under feeder-free conditions containing mTeSR1 medium (STEMCELL Technology, Vancouver, Canada). Thereafter, the iPS cells were cultured for 4 days in CDM basal medium supplemented with each of the four factors one by one and in combinations as appropriate (Figure 2B). The added factors are as follows. When using a combination of two or more factors, the symbols are shown side by side: S: 10 μM SB431542 (TGFβ inhibitor; Sigma) C: 10 μM CHIR99021 (GSK3β inhibitor; WAKO) D: 2 μM DMH1 (BMP inhibitor; Tocris, Bristol, UK) F: 20 ng / ml FGF2

[0113] The medium was changed on day 3. To detect the induction efficiency of unsegmented mesoderm cells, a cell population that was positive for DLL1 (a surface marker for unsegmented mesoderm cells and the posterior part of somites) and negative for PAX3-GFP (indicating newly formed and segmented somite cells) (Figure 2C) was detected by FACS. Since PAX3 is a transcription factor, PAX3-GFP knock-in iPS cells (PAX3-GFP iPS cells) were used for the detection of PAX3-positive cells. The results are shown in Figure 2D.

[0114] The effects of the four factors (SB431542, CHIR99021, DMH1, and FGF2) were analyzed one by one. CHIR99021 (C) was the most effective in increasing the DLL1 + / PAX3-GFP -Cells were efficiently induced (56.3±3.1%). Such results are consistent with previous reports (Chal et al., 2015; Loh et al., 2016; Sudheer et al., 2016; Umeda et al., 2012; Xi et al., 2017). Next, combinations of two factors were evaluated, and CHIR99021 and SB431542 or DMH1 (SC or CD) were found to induce DLL1 + / PAX3-GFP - cells more efficiently (80.5±1.7% and 80.6±1.2%, respectively). However, the combination of CHIR99021 and FGF2 (CF) conversely suppressed efficiency (42.7±1.1%). The combination of DMH1, SB431542, and CHIR99021 (SCD) induced DLL1 + / PAX3-GFP - cells almost maximally (83.8±1.1%), but DLL1 - / PAX3-GFP + cells also appeared. This suggests that cells differentiate into PAX3 + somite cells and / or neural cells under this condition.

[0115] Next, when all four molecules were added (SCDF) and cultured, under this condition, it was found that DLL1 + cells without PAX3-GFP + / PAX3-GFP - were induced from PAX3-GFP iPS cells (85.4±0.4%). Regarding the induction efficiency of DLL1 + cells, there was no obvious difference among SCD, SCF, and SCDF. However, since SCDF reproduced the exact endogenous signaling environment, the SCDF condition was used in further analysis.

[0116] The induction of unsegmented mesoderm cells under the SCDF condition was also confirmed by immunohistochemistry using anti-TBX6, BRACHYURY, and CDX2 antibodies (data not shown).

[0117] Furthermore, the relative expression levels of iPS cell markers (NANOG, OCT3 / 4, SOX2) and presomitic mesoderm cell markers (BRACHYURY, DLL1, TBX6, MSGN1, and WNT3A) in cells before and after culturing for 4 days under SCDF conditions were examined by RT-qPCR. The results are shown in Fig. 2E. When the culture period under SCDF conditions was set from 1 to 5 days, the induction efficiency of DLL1 + / PAX3-GFP - cells peaked on day 4 (Fig. 2F).

[0118] To confirm the robustness of the protocol, the induction efficiency of DLL1 + cells from other iPS cell clones (201B7, 409B2, 414C2, and TIG118-4f, as well as 1231A3) was examined. Each iPS cell clone was cultured for 4 days under SCDF conditions and analyzed by FACS. The results are shown in Figs. 2G and 2F. DLL11 + cells were obtained with higher efficiency from all iPS cell clones. In addition, DLL11 + cell induction was also observed from 1231A3, an iPS cell line maintained in feeder cell-free, serum-free medium.

Example

[0119] (2) Inducing Differentiation of Presomitic Mesoderm Cells (PSM) into Somite Cells (SM) 1.0 x 10 5 all DLL1 + presomitic mesoderm cells sorted by FACS after culturing iPS cells for 4 days under the SCDF conditions of Example 1 were seeded onto one well of a 12-well plate coated with Matrigel to induce somite cells (Fig. 3A). Somite cell induction was performed for 4 days in CDM basal medium supplemented with SB431542 and / or 5 μM CHIR99021. The medium was changed on the third day of somite cell induction. The expression of PAX3-GFP was used as a marker for somite cells.

[0120] The added factors and their concentrations are as follows: S10: SB431542 10 μM C1: CHIR99021 1 μM C5: CHIR99021 5 μM C10: CHIR99021 10 μM D2: DMH1 2 μM F20: FGF2 20 ng / ml I10: IWR1 10 μM

[0121] The results are shown in Fig. 3B. In the 4-day culture, both 10 μM SB431542 (S10) and 5 μM CHIR99021 (C5) efficiently induced PAX3-GFP + cells (52.1 ± 0.8% and 70.7 ± 0.1% respectively). Treatment with both of them (S10C5) resulted in the maximum induction of somite cells (74.7 ± 0.5%). On the other hand, a higher amount of CHIR99021 (10 μM) conversely failed to induce PAX3-GFP + cells (0.3 ± 0.0% in C10 and 0.7 ± 0.1% in S10C10). These results suggest that excessive WNT signaling suppresses the induction of somite cells. Furthermore, the effects of FGF and BMP inhibition were also confirmed, and it was confirmed that 10 μM SB431542 and 5 μM CHIR99021 (S10C5) most efficiently induced PAX3-GFP + cells from unsegmented mesoderm cells. Immunohistochemistry using an anti-PARAXIS (TCF15) antibody also showed, as in the results obtained using FACS shown in Fig. 3B, that PARAXIS-expressing cells indicating somite cells (SM) were observed in C4, S10C1, S10C5, and S10C5D2, and it was confirmed that the proportion was the highest in the S10C5 group.

[0122] The expression levels of unsegmented mesoderm cell markers (TBX6, MSGN1, and WNT3A) and somite cell markers (MEOX1, PARAXIS, and PAX3) before and after induction under the S10C5 condition were examined by RT-qPCR. The results are shown in Fig. 3C. The markers of somite cells also peaked on the 4th day of somite cell induction (the 8th day from iPS cells). When the expression of anti-TBX6, PARAXIS, and MEOX1 antibodies before and after induction of cells induced under the same conditions was examined by immunohistochemistry, and the expression of PAX3-GFP was examined by fluorescence, results similar to those in Fig. 3C were obtained.

[0123] Unsegmented mesoderm cells were cultured for 4 days under the conditions of S10I10, S10, and S10C5. The expression levels of PARAXIS and MEOX1, which are somite markers, in the obtained cells were examined by RT-qPCR. The results are shown in Fig. 3D. The expression levels of PARAXIS and MEOX1 were high when CHIR99021 was included. When the expression of CDH11 (a marker of epithelial somite cells) was examined by immunohistochemistry, the expression of CDH11 was observed at cell-cell junctions only under the condition containing CHIR99021 (S10C5). PAX3 after FACS sorting + Since the viable cell count was low, the obtained cells were used in Example 3 without sorting.

Example

[0124] Inducing Myotome Cells (MYO) and Dermatome Cells (D) from Somite (SM) Cells via Dermomyotome Cells (DM) A schematic is shown in Fig. 4A.

[0125] (3) Inducing Dermomyotome Cells (DM) from Somite Cells (SM) The medium used for somite cell induction was replaced with the medium for dermomyotome cell induction, and the culture was continued. For inhibition - activation of WNT signaling, 10 μM of IWR1 (Cayman chemical, Michigan, USA) (I10) or CHIR99021 (0, 0.1, 1, and 5 μM) (C0, C0.1, C1, and C5) was used. For control of BMP activity, BMP4 (R&D, Minneapolis, KA, USA) (0, 0.1, 1, and 10 ng / ml) (B0, B0.1, B1, and B10) and 10 μM of DMH1 (Tocris) (D10) were used.

[0126] The medium of the somite cell culture obtained in Example 2 was replaced with a medium in which factors for controlling WNT signaling and factors for controlling BMP activity were added to the CDM basal medium, and the culture was further continued to induce dermomyotome cells. The culture was carried out for 3 days, and the medium was replaced on the second day.

[0127] For the cells after 3 - day culture, the relative expression levels of the dermomyotome cell markers ALX4, EN1, and NOGGIN were examined by RT - qPCR. The results are shown in Figure 4B. It was found that these dermomyotome cell markers were most induced when both the GSK3β inhibitor concentration, which is a WNT signal activator, and the BMP activator concentration were high. For the same obtained cells, immunocytochemistry using anti - ALX4 and anti - EN1 antibodies and PAX3 - GFP expressed in both somite cells and dermomyotome cells also confirmed the generation of dermomyotome cells. Furthermore, FACS using anti - EN1 antibody (Figure 4C) also confirmed the generation of dermomyotome cells.

[0128] (4) Inducing Myotome Cells (MYO) from Dermomyotome Cells (DM) An attempt was made to directly induce myotome cells from the dermomyotome cells obtained in (3) above. The medium of the dermomyotome cell culture obtained in (3) was replaced with a medium in which 5 μM of CHIR99021 was added to the CDM basal medium, and further cultured. The medium was changed every 3 days and cultured for 30 days. A small amount of cultured cells was collected every 6 days, and the expression of myogenic markers MYOD, MYOG, and PAX7 over time was examined. Induction of myogenic markers was observed 18 to 30 days after medium replacement. On the other hand, the expression of ALX4, a dermomyotome cell marker, decreased over time (Figure 4D). In addition, the expression of MYOD and MYOG was examined by immunocytochemistry. When the induction efficiency from dermatome cells to myotome cells was calculated based on the number of MYOD-positive cells and MYOG-positive cells, it was approximately 22%.

[0129] (5) Inducing Dermatome Cells (D) from Dermomyotome Cells (DM) In vivo, dermatomyotome cells also differentiate into dermal segment cells, and dermal segment cells differentiate into the dorsal dermis. However, a protocol for inducing differentiation from dermatomyotome cells to dermal segment cells and then to the dermis in vitro has not been established. In the dermatomyotome cell induction medium (C5B10) of (3) above, after the 11th day (3 days from SM) without subculture, the culture was continued even after dermatomyotome cells appeared. As a result of examining the expression of various cell markers by RT-qPCR, the expression levels of PAX3 and PARAXIS (dermatomyotome cell and somite cell markers: SM / DM markers) as well as PAX7 and NCAD (dermatomyotome cell and myotome cell markers: DM / MYO markers) decreased 6 days after DM generation (upper part of Fig. 4E). Therefore, the induction of dermatomyotome cells to dermal segment cells was carried out by continuously culturing the dermatomyotome cells in the dermatomyotome cell induction medium (the medium of (3) above). After the appearance of dermatomyotome cells, the culture was continued for 9 days, and the medium was changed every 3 days. PDGFRα is expressed in dermal segment cells (D) and skin fibroblasts (DF) (Orr-Urtreger et al., 1992), and EN1 is known to be expressed in dermal segment cells (D) and dermatomyotome cells (DM) (Ahmed et al., 2006). The expression of PDGFRα and EN1 was examined by RT-qPCR (lower part of Fig. 4E) and immunocytochemistry (data not shown), and it was found that these markers were mainly expressed on the 9th day of the dermal segment cell induction process. The expression of ALX4 and MSX1 (dermatomyotome cell and dermal segment cell markers: DM / D markers) as well as COL1A2 (dermal segment cell and skin fiber cell markers: D / DF markers) also increased on the 9th day of the dermal segment cell induction process (lower part of Fig. 4E). FACS analysis was performed with anti-PDGFRα and anti-EN1, and it was confirmed that 69.5 ± 1.4% of the dermal segment cell culture was PDGFRα + and that 92.7 ± 0.4% of the PDGFRα + cells were EN1 + (Fig. 4F). From such results, it was confirmed that dermal segment cells (D) were induced.

Example

[0130] Inducing Differentiation of Somite Cells (SM) into Chondrocytes, Osteocytes and Syndetome Cells (SYN) via Sclerotome Cells (SCL) It is schematically shown in Fig. 5A.

[0131] (6) Inducing Differentiation of Somite Cells into Sclerotome Cells Induction from somite cells to sclerotome cells was performed by a known method (Zhao et al., 2014). Specifically, the somite cell induction medium was replaced with a sclerotome cell induction medium (CDM basal medium containing 100 nM SAG (SHH activator; Calbiochem, La Jolla, CA, USA) and 0.6 μM LDN193189 (BMP inhibitor; Stemgent, Cambridge, MA, USA)), and the cells were cultured for 3 days. The medium was replaced on the second day.

[0132] The expression of PAX1, PAX9, and NKX3.2 was confirmed in cells obtained by culturing somite cells for 3 days (day 11 from iPS cells) (Fig. 5B). The induction rate from somite cells to sclerotome cells, calculated based on immunocytochemistry with anti-PAX1 and anti-PAX9 antibodies, was approximately 45%.

[0133] Inducing 3D Cartilage Cell Formation (3DCI) from Sclerotome Cells (SCL) A total of 1.0x10 6 induced sclerotome cells (SCL) were suspended in 0.5 ml of chondrogenic basal medium (DMEM:F12 (Invitrogen) containing 1% (v / v) ITS+Premix (BD Biosciences, San Jose, CA, USA), 0.1 μM dexamethasone (WAKO), 0.17 mM L-ascorbic acid 2-phosphate sesquimagnesium hydrate (Sigma), 0.35 mM proline (Sigma), 0.15% (v / v) glucose (Sigma), 1 mM sodium pyruvate, 2 mM GlutaMax (Invitrogen), 1% (v / v) FBS) supplemented with 10 ng / ml BMP7 (R&D) and 10 ng / ml TGFβ3 (R&D), transferred into a 15 ml tube (Corning Inc., Corning, NY, USA), centrifuged to form a pellet, and incubated at 37 °C in 5% CO2. The medium was replaced every 3 days.

[0134] The cultured sclerotome cells were collected, centrifuged to form a pellet, and further cultured in chondrogenic basal medium. Cartilage formation was confirmed on day 21 of chondrogenic induction from sclerotome cells (day 32 from iPS cells) by Alcian blue staining, Safranin O staining, immunocytochemistry with anti-type II collagen antibody (data not shown), and RT-qPCR of chondrogenic markers (Figure 5C).

[0135] Inducing 2D Bone Formation (2DOI) from Sclerotome Cells (SCL) A total of 4.0x10 5 induced sclerotome cells were seeded onto a 12-well plate coated with Matrigel, and then MSC go rapid osteogenic medium (Biological Industries, Kibbutz Beit-Haemek, Israel) was used to induce differentiation into osteocytes in two dimensions. Differentiation of sclerotome cells into osteocytes was confirmed by the expression of PAX1, RUNX2, COL1A1, OSX, and OPN (Figure 5D) and alizarin red staining of the cells (data not shown) on day 18 of osteogenic induction (day 29 from iPS cells).

[0136] (7) Inducing Syndetome Cells (SYN) from Sclerotome Cells (SCL) The dorsal part of the sclerotome is defined as the ligamentous sclerotome, which is the anlage of tendons and ligaments (Brent et al., 2003). A protocol for inducing ligamentous sclerotome from sclerotome cells was not known prior to this application. The induced sclerotome cells were removed from the dish with 0.25% trypsin-EDTA (GIBCO), and a total of 5.0x10 4 cells were seeded onto one well of a 24-well plate coated with Matrigel, and then ligamentous sclerotome cell induction was performed.

[0137] The sclerotome cells were cultured in the ligamentous sclerotome cell induction medium A (CDM basal medium supplemented with 20 ng / ml FGF8 (Peprotech, Rocky Hill, NJ, USA)) for 3 days (step 7-1). Then, without subculturing, the medium was replaced with the ligamentous sclerotome cell induction medium B (CDM basal medium containing 10 ng / ml BMP7 (R&D) and 10 ng / ml TGFβ3 (R&D)) and cultured for 18 days (step 7-2). The medium was changed every 3 days.

[0138] In the late stage of induction into ligamentous sclerotome cells, the expression of SCX, MKX, COL1A1, and COL1A2, which are ligamentous sclerotome cell markers, increased over time (Figure 5E). Also, the protein expression of each factor was confirmed by immunocytochemistry on the 21st day of ligamentous sclerotome cell induction, and the same results as in Figure 5E were confirmed. SCX-expressing cells were confirmed by FACS, and it was confirmed that the induction rate into ligamentous sclerotome cells was 68.0 ± 2.4% (Figure 5F).

[0139] In the examples of the present application, somite cells (SM) were induced from pluripotent stem cells via presomitic mesoderm (PSM). The somite cells (SM) were further induced to differentiate into dermomyotome cells (DM), myotome cells (MYO), sclerotome cells (SCL), and ligamentous sclerotome cells (SYN). The gene expression profiles of each induced cell were examined. The heatmap analysis diagram is shown in Figure 6A, and the PCA plot is shown in Figure 6B. From these figures, preferential and stepwise differentiation into cells at each stage is shown, and this result supports the theoretical basis of each method.

Examples

[0140] (8) Induction of mesenchymal stromal cells (MSCs) from somite cells (SM) Somite cells can be precursors of mesenchymal stromal cells, but there has been no report of inducing mesenchymal stromal cells from human pluripotent stem cells via somite cells. To induce mesenchymal stromal cells from somite cells, the somite cell induction medium was replaced with a somite cell-derived mesenchymal stromal cell induction medium (αMEM (Nacalai Tesque, Kyoto, Japan) containing 10% fetal bovine serum (Nichirei Inc., Tokyo, Japan) and 4 ng / ml FGF2 (WAKO)) and further cultured (Figure 7A). The cultured cells were detached from the container using 0.25% trypsin-EDTA (GIBCO) and passaged every 4 days. The cells were seeded on tissue culture dishes at a density of 2x10 4 cells / cm 3 .

[0141] Somite cell-derived mesenchymal stromal cell induction was performed for 12 days, and the expression levels of each marker on day 12 were analyzed by FACS. The cell morphology changed to fibroblast-like. The cells were positive for the mesenchymal stromal cell markers CD44, CD73, and CD105 (Figure 7B), confirming the generation of mesenchymal stromal cells.

[0142] Osteogenic induction (OI), chondrogenic induction (CI), and adipogenic induction (AI) were performed from the obtained mesenchymal stromal cells by the above-known methods to confirm the differentiation ability of mesenchymal stromal cells induced from somite cells (SMMSC). When CI, OI, AI, and these assays (Alizarin red staining, Alcian blue staining, Oil red O staining) were performed according to a previous report (Fukuta et al., 2014), the generation of chondrocytes, osteocytes, and adipocytes was confirmed.

[0143] Both sclerotome cells induced from somite cells and mesenchymal stromal cells can induce bone formation and chondrogenesis. To distinguish somite cells, sclerotome cells, and mesenchymal stromal cells, the expression of somite cell markers (PAX3, PARAXIS, MEOX1), mesenchymal stromal cell markers (CD44, CD73, CD90, and CD105), and sclerotome cell markers (PAX1, PAX9, NKX3.2) was analyzed by RT-qPCR in each cell. The induced mesenchymal stromal cells were positive for mesenchymal stromal cell markers (CD44, CD73, CD90, and CD105), but not positive for somite cell or sclerotome cell markers (Figure 7C and Figure 7D). These results indicated that the cells induced by the above (8) were mesenchymal stromal cells.

Example

[0144] Differences in chondrogenesis between mesenchymal stromal cells induced from somite cells and sclerotome cells induced from somite cells One promising use of iPS cells is disease modeling with patient-specific iPS cells. In Examples 4 and 5, we successfully induced two different types of chondrocytes: chondrocytes derived from sclerotome cells and chondrocytes derived from mesenchymal stromal cells from somite cells. This protocol was applied to iPS cells generated from somatic cells of patients with fibrodysplasia ossificans progressiva (FOP), a rare and intractable disease caused by mutations in ACVR1 that lead to hyperactivity and characterized mainly by endochondral ossification in the soft tissues of postnatal patients. A research group including the present inventors previously reported that enhanced chondrogenesis was observed from mesenchymal stromal cells induced from FOP-iPS-derived neural crest cells (Hino et al., 2015; Matsumoto et al., 2015). Similarly, it is predicted that chondrogenesis is enhanced in chondrocytes derived from mesenchymal stromal cells induced from FOP-iPS cell-derived somite cells, but not in embryonic chondrocytes derived from sclerotome cells.

[0145] The outline of the experiment is shown in Fig. 8A. Somite cells were induced from FOP-iPS cells (Matsumoto et al., 2013) and gene-repaired (rescued) FOP-iPS cells (resFOP-iPS cells) (Hino et al., 2015; Matsumoto et al., 2015) according to the protocols of Examples 1 and 2 above. Mesenchymal stromal cells and sclerotome cells derived from somite cells were induced according to the protocols of Examples 4 and 5.

[0146] The obtained mesenchymal stromal cells and sclerotome cells were subjected to two-dimensional chondrogenic induction using chondrogenic medium (Hino et al., 2015) with or without activin A (an accelerator of ACVR1 mutants) added.

[0147] Two-dimensional chondrogenesis induction (2DCI) A total of 1.5x10 5 induced mesenchymal stromal cells or sclerotome cells were suspended in 5 μL of chondrogenic basal medium (DMEM:F12 (Invitrogen), 1% (v / v) ITS + Premix (BD Biosciences, San Jose, CA, USA), 0.1 μM dexamethasone (WAKO), 0.17 mM L-ascorbic acid 2-phosphate sesquimagnesium hydrate (Sigma), 0.35 mM proline (Sigma), 0.15% (v / v) glucose (Sigma), 1 mM sodium pyruvate, 2 mM GlutaMax (Invitrogen), 1% (v / v) FBS), and then transferred to one well of a 24-well plate coated with fibronectin (BD Biosciences). After incubation for 1 hour at 37 °C in 5% CO2, the cells formed micromasses.

[0148] Thereafter, the micromasses were cultured for 5 days at 37 °C in 5% CO2 in 1 mL of chondrogenic basal medium with or without 30 ng / mL of activin A (R&D) added and with 10 ng / ml of BMP7 (R&D) and 10 ng / ml of TGFβ3 (R&D) added.

[0149] Furthermore, microaggregates derived from mesenchymal stromal cells derived from somite cells induced from FOP-iPS were similarly cultured in 1 mL of chondrogenic basal medium containing 30 ng / mL of activin A (R&D), with or without 10 nM of R667 (Toronto Research Chemicals, Toronto, ON, Canada) or 10 nM of rapamycin (MedChem Express, Monmouth Junction, NJ, USA).

[0150] The differentiation potential of the obtained cells was analyzed by RT-qPCR analysis, glycosaminoglycan (GAG) quantification, and alcian blue staining. Briefly, the induced cells were fixed with 4% paraformaldehyde (WAKO) for 30 minutes, rinsed with PBS, and then stained overnight with an alcian blue solution (1% alcian blue, pH 1) (MUTO PURE CHEMICAL CO., LTD, Tokyo, Japan).

[0151] The results are shown in Figures 8B - D. The expression levels of ACVR1 in chondrocytes derived from somite cell-derived mesenchymal stromal cells and sclerotome cells were not different regardless of the cell type of origin and the presence or absence of ACVR1 mutation repair (Figure 8B).

[0152] In the absence of activin A stimulation, the expression levels of chondrogenic markers were not different regardless of the cell type of origin and the presence or absence of ACVR1 mutation repair (data not shown).

[0153] When somite cell-derived mesenchymal stromal cells induced from FOP-iPS were induced into cartilage under activin stimulation, enhanced chondrogenesis was shown in terms of the expression level of chondrogenic markers and the production amount of glycosaminoglycan (GAG) compared to the cells induced from resFOB-iPS cells (Figs. 8C and D). Enhanced chondrogenesis was also observed in somite cell-derived mesenchymal stromal cells induced from FOB-iPS cells in Alcian blue staining (data not shown). On the other hand, when chondrocytes were induced from sclerotome cells derived from somite cells stimulated with activin A, there were no differences in the expression of chondrogenic markers, GAG quantification, and Alcian blue staining results between FOB-iPS-derived and resFOB-iPS-derived cells (Figs. 8E and F).

[0154] When R667 or rapamycin was added during the activin-stimulated cartilage induction of somite cell-derived mesenchymal stromal cells induced from FOP-iPS, the enhancement of the expression of chondrogenic markers and the production amount of glycosaminoglycan (GAG) was significantly suppressed (Figs. 8G, H). In addition, it was confirmed by Alcian blue staining that the enhancement of chondrogenesis was suppressed. Both R667 (retinoic acid receptor γ agonist) and rapamycin (mTOR inhibitor) have been reported to be potent inhibitors of heterotopic ossification (Hino et al., 2017).

[0155] In recent years, as one of the origin cells of FOP lesions, PDGFRα + / CD31 - cells have been proposed (Dey et al., 2016). FOP-SMMSCs were sorted separately by FACS to obtain PDGFRα + / CD31 - cells and PDGFRα - / CD31 - cells (Fig. 8I). 2D chondrogenic differentiation was performed on each cell according to the above protocol. There was no difference in the expression level of ACVR1 in the isolated cells (data not shown). As expected, PDGFRα + / CD31 - cells were PDGFRα - / CD31- showed enhanced chondrogenesis compared to cells (Figure 8J and 6K). Interestingly, PAI1 and MMP1 (indicator genes for ACVR1 mutant activation) were significantly higher in PDGFRα + / CD31 - cells (Figure 8L). These results are summarized in Figure 8M. It shows cell type specificity of the FOP phenotype and indicates that our protocol can be used for disease modeling, phenotype analysis, and drug discovery.

Example

[0156] Xeno-free differentiation induction from sclerotome cells (SCL) to syndetome cells (SYN) Sclerotome cells were induced from iPS cells induced under xeno-free conditions in a xeno-free environment. Specifically, 1231A3 induced under xeno-free conditions was used as the iPS cells. First, the iPS cells were cultured for 4 days under the SCDF conditions of Example 1 to induce unspecified mesoderm cells. StemFit (登録商標) AK03 (C-free) medium (Ajinomoto Co., Inc., hereinafter referred to as AK03(-C) medium) was used. Next, for somite cell induction, a total of 1.0x10 5 individual DLL1 + unspecified mesoderm cells were seeded into each well of a 12-well plate coated with iMatrix511 (nippi, Inc.) containing AK03(-C) medium supplemented with 10 μM SB431542 and 5 μM CHIR99021 and cultured for 4 days. The medium was changed on the 3rd day of somite cell induction. For sclerotome cell induction, the medium was changed to AK03(-C) medium containing 100 nM SAG and 0.6 μM LDN193189 and cultured for an additional 3 days. The medium was changed on the 2nd day of sclerotome cell induction.

[0157] Plates coated with iMatrix511 were prepared the day before starting the induction of ligamentous sclerotome cells. To prepare 24-well plates coated with iMatrix511, 0.5 mL of iMatrix511 solution was added to each well at 4°C and left overnight.

[0158] The medium of the induced nodule cell culture solution was aspirated and washed with PBS. 0.2 mL of the cell detachment reagent Accutase 登録商標 (Innovative Cell Technologies, Inc.) was added to each well and left at room temperature for 3 minutes. Next, 0.8 mL of AK03(-C) medium was added to each well, and all the cells were scraped off and collected in a 15 mL conical tube. Then, centrifugation was performed at 280×g for 3 minutes. The supernatant was carefully aspirated and resuspended in 1 mL of ligament nodule cell induction medium A’ (AK03(-C) medium supplemented with 20 ng / mL FGF8 and 10 ng / mL TGFβ3). The cell count was measured using an automatic cell counter.

[0159] 5.0×10 4 cells were seeded into each well of a 24-well plate coated with iMatrix511 containing 1 mL of ligament nodule cell induction medium A’ and incubated at 37 °C in 5% CO2 for 2 days (Step 7-1). On the second day of ligament nodule cell induction, the medium was replaced with ligament nodule cell induction medium B’ (AK03(-C) medium supplemented with 10 ng / mL BMP7 and 10 ng / mL TGFβ3). Incubation was carried out at 37 °C in 5% CO2 for 6 days until the eighth day (Step 7-2). The medium was replaced every two days.

[0160] In Step 7-1, in Example 4, a medium supplemented with 20 ng / ml FGF8 (ligament nodule cell induction medium A) was used, but in this example, a medium supplemented with 20 ng / mL FGF8 and 10 ng / mL TGFβ3 (ligament nodule cell induction medium A’) was used. As a result, the culture period in Step 7-1 was shortened to 2 days, and the culture period in Step 7-2 was shortened to 6 days.

[0161] From the start of ligament enthesis cell induction until the 8th day, the expression of seven ligament enthesis cell-related markers (SCX, MKX, TNMD, TNCC, COL1A1, COL1A2, and FMOD) increased over time (Figure 9A). Also, the mRNA expression levels of ligament enthesis cell-related markers (SCX, TNMD, COL1A1, and COL1A2) on the 8th day of induction were examined. For comparison, the expression levels of each marker in human healthy anterior cruciate ligament samples (CDD-H-6800-N-R, Articular Engineering) were examined. The mRNA expression levels of each marker in both were comparable (Figure 9B). Furthermore, as a result of immunostaining on the 21st day of induction, the protein expression of each marker (SCX, TNMD, COL1A1, and COL1A2) was confirmed (Figure 9C).

Example

[0162] Transplantation therapeutic effect of iPS cell-derived syndetome cells The cells on the 8th day of ligament enthesis cell induction in Example 7 were transplanted into Achilles tendon rupture model rats, and the transplantation treatment effect was observed for 4 weeks.

[0163] An overview of the preparation and breeding of Achilles tendon rupture model rats is shown in Figure 10A. To prepare Achilles tendon rupture model rats, the left hind limb of 8-week-old F344 / NSlc male rats was incised (upper left in Figure 10A), and the Achilles tendon site 5 mm from the calcaneus was transected (upper right in Figure 10A). Then, the incision was sutured, and 3×10 6 individual iPS cell-derived ligament enthesis cells / 50 μL Matrigel:DMEM / F12 = 1:1 solution was injected (lower left in Figure 10A). The rats were bred with their tails suspended for 1 week (lower right in Figure 10A), and then observed for 3 weeks.

[0164] Every week after transplantation, footprint images of the left hind limbs of the transplantation group (Trans) and the non-transplantation group (Ctrl) were obtained (Figure 10B). Healthy rats walk with their heels raised (see Pre-OP in Figure 10B). In the transplantation group, significant recovery was observed 2 weeks after transplantation compared to the non-transplantation group.

[0165] In addition, the verification of the transplantation treatment effect was performed based on the Achilles Functional Index (AFI; Murrell et al., 2014) (Figure 10C). In the transplantation group, significant recovery was observed two weeks after transplantation compared to the non-transplanted group.

[0166] Furthermore, the walking function on a treadmill was observed four weeks after transplantation (Figure 10D). In the transplantation group, significant recovery in the height from the floor to the heel and the ankle angle was shown compared to the non-transplanted group.

[0167] List of cited references Ahmed, M.U., Cheng, L., and Dietrich, S. (2006). Establishment of the epaxial-hypaxial boundary in the avian myotome. Dev Dyn 235, 1884-1894. Benazeraf, B., and Pourquie, O. (2013). Formation and segmentation of the vertebrate body axis. Annual review of cell and developmental biology 29, 1-26. Bernardo, A.S., Faial, T., Gardner, L., Niakan, K.K., Ortmann, D., Senner, C.E., Callery, E.M., Trotter, M.W., Hemberger, M., Smith, J.C., et al. (2011). BRACHYURY and CDX2 mediate BMP-induced differentiation of human and mouse pluripotent stem cells into embryonic and extraembryonic lineages. Cell Stem Cell 9, 144-155 . Brent, A.E., Schweitzer, R., and Tabin, C.J. (2003). A somitic compartment of tendon progenitors. Cell 113, 235-248. Brent, A.E., and Tabin, C.J. (2002). Developmental regulation of somite derivatives: muscle, cartilage and tendon. Current opinion in genetics & development 12, 548-557. Buckingham, M., Bajard, L., Chang, T., Daubas, P., Hadchouel, J., Meilhac, S., Montarras, D., Rocancourt, D., and Relaix, F. (2003). The formation of skeletal muscle: from somite to limb. Journal of anatomy 202, 59-68. Chal, J., Oginuma, M., Al Tanoury, Z., Gobert, B., Sumara, O., Hick, A., Bousson, F., Zidouni, Y., Mursch, C., Moncuquet, P., et al. (2015). Differentiation of pluripotent stem cells to muscle fiber to model Duchenne muscular dystrophy. Nat Biotechnol 33, 962-969. Chambers, S.M., Fasano, C.A., Papapetrou, E.P., Tomishima, M., Sadelain, M., and Studer, L. (2009). Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol 27, 275-280. Chapman, S.C., Brown, R., Lees, L., Schoenwolf, G.C., and Lumsden, A. (2004). Expression analysis of chick Wnt and frizzled genes and selected inhibitors in early chick patterning. Dev Dyn 229, 668-676. Chapman, S.C., Schubert, F.R., Schoenwolf, G.C., and Lumsden, A. (2002). Analysis of spatial and temporal gene expression patterns in blastula and gastrula stage chick embryos. Developmental biology 245, 187-199. Chong, J.J., Yang, X., Don, C.W., Minami, E., Liu, Y.W., Weyers, J.J., Mahoney, W.M., Van Biber, B., Cook, S.M., Palpant, N.J., et al. (2014). Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts. Nature 510, 273-277. Dey, D., Bagarova, J., Hatsell, S.J., Armstrong, K.A., Huang, L., Ermann, J., Vonner, A.J., Shen, Y., Mohedas, A.H., Lee, A., et al. (2016). Two tissue-resident progenitor lineages drive distinct phenotypes of heterotopic ossification. Science translational medicine 8, 366ra163. Fasano, C.A., Chambers, S.M., Lee, G., Tomishima, M.J., and Studer, L. (2010). Efficient derivation of functional floor plate tissue from human embryonic stem cells. Cell Stem Cell 6, 336-347. Faure, S., de Santa Barbara, P., Roberts, D.J., and Whitman, M. (2002). Endogenous patterns of BMP signaling during early chick development. Developmental biology 244, 44-65. Fomenou, M.D., Scaal, M., Stockdale, F.E., Christ, B., and Huang, R. (2005). Cells of all somitic compartments are determined with respect to segmental identity. Dev Dyn 233, 1386-1393. Fukuta, M., Nakai, Y., Kirino, K., Nakagawa, M., Sekiguchi, K., Nagata, S., Matsumoto, Y., Yamamoto, T., Umeda, K., Heike, T., et al. (2014). Derivation of mesenchymal stromal cells from pluripotent stem cells through a neural crest lineage using small molecule compounds with defined media. PLoS One 9, e112291. Galli, L.M., Willert, K., Nusse, R., Yablonka-Reuveni, Z., Nohno, T., Denetclaw, W., and Burrus, L.W. (2004). A proliferative role for Wnt-3a in chick somites. Developmental biology 269, 489-504. Gouti, M., Delile, J., Stamataki, D., Wymeersch, F.J., Huang, Y., Kleinjung, J., Wilson, V., and Briscoe, J. (2017). A Gene Regulatory Network Balances Neural and Mesoderm Specification during Vertebrate Trunk Development. Developmental cell 41, 243-261 e247. Hardy, K.M., Yatskievych, T.A., Konieczka, J., Bobbs, A.S., and Antin, P.B. (2011). FGF signalling through RAS / MAPK and PI3K pathways regulates cell movement and gene expression in the chicken primitive streak without affecting E-cadherin expression. BMC developmental biology 11, 20. Hino, K., Horigome, K., Nishio, M., Komura, S., Nagata, S., Zhao, C., Jin, Y., Kawakami, K., Yamada, Y., Ohta, A., et al. (2017). Activin-A enhances mTOR signaling to promote aberrant chondrogenesis in fibrodysplasia ossificans progressiva. The Journal of clinical investigation 127, 3339-3352. Hino, K., Ikeya, M., Horigome, K., Matsumoto, Y., Ebise, H., Nishio, M., Sekiguchi, K., Shibata, M., Nagata, S., Matsuda, S., et al. (2015). Neofunction of ACVR1 in fibrodysplasia ossificans progressiva. Proc Natl Acad Sci U S A 112, 15438-15443. Hirsinger, E., Duprez, D., Jouve, C., Malapert, P., Cooke, J., and Pourquie, O. (1997). Noggin acts downstream of Wnt and Sonic Hedgehog to antagonize BMP4 in avian somite patterning. Development 124, 4605-4614. Hubaud, A., and Pourquie, O. (2014). Signalling dynamics in vertebrate segmentation. Nature reviews Molecular cell biology 15, 709-721. Iimura, T., Yang, X., Weijer, C.J., and Pourquie, O. (2007). Dual mode of paraxial mesoderm formation during chick gastrulation. Proc Natl Acad Sci U S A 104, 2744-2749. Ikeya, M., and Takada, S. (1998). Wnt signaling from the dorsal neural tube is required for the formation of the medial dermomyotome. Development 125, 4969-4976. Jiang, Y.J., Aerne, B.L., Smithers, L., Haddon, C., Ish-Horowicz, D., and Lewis,J. (2000). Notch signalling and the synchronization of the somite segmentation clock. Nature 408, 475-479. Jouve, C., Iimura, T., and Pourquie, O. (2002). Onset of the segmentation clock in the chick embryo: evidence for oscillations in the somite precursors in the primitive streak. Development 129, 1107-1117. Kam, R.K., Deng, Y., Chen, Y., and Zhao, H. (2012). Retinoic acid synthesis and functions in early embryonic development. Cell & bioscience 2, 11. Koyanagi-Aoi, M., Ohnuki, M., Takahashi, K., Okita, K., Noma, H., Sawamura, Y., Teramoto, I., Narita, M., Sato, Y., Ichisaka, T., et al. (2013). Differentiation-defective phenotypes revealed by large-scale analyses of human pluripotent stem cells. Proc Natl Acad Sci U S A 110, 20569-20574. Lagha, M., Sato, T., Regnault, B., Cumano, A., Zuniga, A., Licht, J., Relaix, F., and Buckingham, M. (2010). Transcriptome analyses based on genetic screens for Pax3 myogenic targets in the mouse embryo. BMC genomics 11, 696. Lee, J.Y., Zhou, Z., Taub, P.J., Ramcharan, M., Li, Y., Akinbiyi, T., Maharam, E.R., Leong, D.J., Laudier, D.M., Ruike, T., et al. (2011). BMP-12 treatment of adult mesenchymal stem cells in vitro augments tendon-like tissue formation and defect repair in vivo. PLoS One 6, e17531. Loh, K.M., Chen, A., Koh, P.W., Deng, T.Z., Sinha, R., Tsai, J.M., Barkal, A.A., Shen, K.Y., Jain, R., Morganti, R.M., et al. (2016). Mapping the Pairwise Choices Leading from Pluripotency to Human Bone, Heart, and Other Mesoderm Cell Types. Cell 166, 451-467. Marcelle, C., Stark, M.R., and Bronner-Fraser, M. (1997). Coordinate actions of BMPs, Wnts, Shh and noggin mediate patterning of the dorsal somite. Development 124, 3955-3963. Maretto, S., Cordenonsi, M., Dupont, S., Braghetta, P., Broccoli, V., Hassan, A.B., Volpin, D., Bressan, G.M., and Piccolo, S. (2003). Mapping Wnt / beta-catenin signaling during mouse development and in colorectal tumors. Proc Natl Acad Sci U S A 100, 3299-3304. Matsumoto, Y., Hayashi, Y., Schlieve, C.R., Ikeya, M., Kim, H., Nguyen, T.D., Sami, S., Baba, S., Barruet, E., Nasu, A., et al. (2013). Induced pluripotent stem cells from patients with human fibrodysplasia ossificans progressiva show increased mineralization and cartilage formation. Orphanet journal of rare diseases 8, 190. Matsumoto, Y., Ikeya, M., Hino, K., Horigome, K., Fukuta, M., Watanabe, M., Nagata, S., Yamamoto, T., Otsuka, T., and Toguchida, J. (2015). New Protocol to Optimize iPS Cells for Genome Analysis of Fibrodysplasia Ossificans Progressiva. Stem Cells 33, 1730-1742. Moriyama, A., Kii, I., Sunabori, T., Kurihara, S., Takayama, I., Shimazaki, M., Tanabe, H., Oginuma, M., Fukayama, M., Matsuzaki, Y., et al. (2007). GFP transgenic mice reveal active canonical Wnt signal in neonatal brain and in adult liver and spleen. Genesis 45, 90-100. Nakagawa, M., Taniguchi, Y., Senda, S., Takizawa, N., Ichisaka, T., Asano, K., Morizane, A., Doi, D., Takahashi, J., Nishizawa, M., et al. (2014). A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells. Sci Rep 4, 3594. Nimmagadda, S., Geetha Loganathan, P., Huang, R., Scaal, M., Schmidt, C., and Christ, B. (2005). BMP4 and noggin control embryonic blood vessel formation by antagonistic regulation of VEGFR-2 (Quek1) expression. Developmental biology 280, 100-110. Okita, K., Matsumura, Y., Sato, Y., Okada, A., Morizane, A., Okamoto, S., Hong, H., Nakagawa, M., Tanabe, K., Tezuka, K., et al. (2011). A more efficient method to generate integration-free human iPS cells. Nature methods 8, 409-412. Orr-Urtreger, A., Bedford, M.T., Do, M.S., Eisenbach, L., and Lonai, P. (1992). Developmental expression of the alpha receptor for platelet-derived growth factor, which is deleted in the embryonic lethal Patch mutation. Development 115, 289-303. Patwardhan, V., Gokhale, M., and Ghaskadbi, S. (2004). Acceleration of early chick embryo morphogenesis by insulin is associated with altered expression of embryonic genes. Int J Dev Biol 48, 319-326. Pryce, B.A., Watson, S.S., Murchison, N.D., Staverosky, J.A., Dunker, N., and Schweitzer, R. (2009). Recruitment and maintenance of tendon progenitors by TGFbeta signaling are essential for tendon formation. Development 136, 1351-1361. Rhinn, M., and Dolle, P. (2012). Retinoic acid signalling during development. Development 139, 843-858. Sakurai, H., Inami, Y., Tamamura, Y., Yoshikai, T., Sehara-Fujisawa, A., and Isobe, K. (2009). Bidirectional induction toward paraxial mesodermal derivatives from mouse ES cells in chemically defined medium. Stem cell research 3, 157-169. Sakurai, H., Sakaguchi, Y., Shoji, E., Nishino, T., Maki, I., Sakai, H., Hanaoka, K., Kakizuka, A., and Sehara-Fujisawa, A. (2012). In vitro modeling of paraxial mesodermal progenitors derived from induced pluripotent stem cells. PLoS One 7, e47078. Schwarting, T., Lechler, P., Struewer, J., Ambrock, M., Frangen, T.M., Ruchholtz, S., Ziring, E., and Frink, M. (2015). Bone morphogenetic protein 7 (BMP-7) influences tendon-bone integration in vitro. PLoS One 10, e0116833. Sheng, G. (2015). The developmental basis of mesenchymal stem / stromal cells (MSCs). BMC developmental biology 15, 44. Streit, A., and Stern, C.D. (1999). Establishment and maintenance of the border of the neural plate in the chick: involvement of FGF and BMP activity. Mechanisms of development 82, 51-66. Sudheer, S., Liu, J., Marks, M., Koch, F., Anurin, A., Scholze, M., Senft, A.D., Wittler, L., Macura, K., Grote, P., et al. (2016). Different Concentrations of FGF Ligands, FGF2 or FGF8 Determine Distinct States of WNT-Induced Presomitic Mesoderm. Stem Cells 34, 1790-1800. Sumi, T., Tsuneyoshi, N., Nakatsuji, N., and Suemori, H. (2008). Defining early lineage specification of human embryonic stem cells by the orchestrated balance of canonical Wnt / beta-catenin, Activin / Nodal and BMP signaling. Development 135, 2969-2979. Tajbakhsh, S., Borello, U., Vivarelli, E., Kelly, R., Papkoff, J., Duprez, D., Buckingham, M., and Cossu, G. (1998). Differential activation of Myf5 and MyoD by different Wnts in explants of mouse paraxial mesoderm and the later activation of myogenesis in the absence of Myf5. Development 125, 4155-4162. Takada, S., Stark, K.L., Shea, M.J., Vassileva, G., McMahon, J.A., and McMahon, A.P. (1994). Wnt-3a regulates somite and tailbud formation in the mouse embryo. Genes & development 8, 174-189. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861-872. Takemoto, T., Uchikawa, M., Yoshida, M., Bell, D.M., Lovell-Badge, R., Papaioannou, V.E., and Kondoh, H. (2011). Tbx6-dependent Sox2 regulation determines neural or mesodermal fate in axial stem cells. Nature 470, 394-398. Tanaka, A., Woltjen, K., Miyake, K., Hotta, A., Ikeya, M., Yamamoto, T., Nishino, T., Shoji, E., Sehara-Fujisawa, A., Manabe, Y., et al. (2013). Efficient and reproducible myogenic differentiation from human iPS cells: prospects for modeling Miyoshi Myopathy in vitro. PLoS One 8, e61540. Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S., and Jones, J.M. (1998). Embryonic stem cell lines derived from human blastocysts. Science (New York, NY) 282, 1145-1147. Umeda, K., Zhao, J., Simmons, P., Stanley, E., Elefanty, A., and Nakayama, N. (2012). Human chondrogenic paraxial mesoderm, directed specification and prospective isolation from pluripotent stem cells. Sci Rep 2, 455. Xi, H., Fujiwara, W., Gonzalez, K., Jan, M., Liebscher, S., Van Handel, B., Schenke-Layland, K., and Pyle, A.D. (2017). In Vivo Human Somitogenesis Guides Somite Development from hPSCs. Cell Rep 18, 1573-1585. Yoshikawa, Y., Fujimori, T., McMahon, A.P., and Takada, S. (1997). Evidence that absence of Wnt-3a signaling promotes neuralization instead of paraxial mesoderm development in the mouse. Developmental biology 183, 234-242. Zhao, J., Li, S., Trilok, S., Tanaka, M., Jokubaitis-Jameson, V., Wang, B., Niwa, H., and Nakayama, N. (2014). Small molecule-directed specification of sclerotome-like chondroprogenitors and induction of a somitic chondrogenesis program from embryonic stem cells. Development 141, 3848-3858.

Claims

Step of providing somite cells, and A method for producing dermomyotome cells from somite cells, comprising the step of culturing somite cells in a medium containing a GSK3β inhibitor and BMP. The method according to claim 1, further comprising the step of purifying the obtained dermomyotome cell culture by fluorescence-activated cell sorting. The method according to claim 1 or 2, wherein the GSK3β inhibitor is CHIR99021. The method according to any one of claims 1 to 3, wherein the BMP is BMP4. Step of providing somite cells, and A method for producing mesenchymal stromal cells from somite cells, comprising the step of culturing somite cells in a medium containing FGF. The method according to claim 5, wherein the somite cells are obtained by a method comprising the step of culturing pluripotent stem cells in a medium containing a GSK3β inhibitor. The method according to claim 5 or 6, wherein the FGF is FGF2.

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