Step-by-step method for 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] Recently, several groups have reported successful induction of paraxial mesoderm based on different ideas (Non-Patent Documents 6-9). In these reports, cells were treated without Activin / Nodal / TGFβ or with TGFβ inhibitors, and a relatively high concentration of GSK3 inhibitor (WNT signaling activator) was used to induce neural (dorsal) fate. Using these protocols, the induction rate of paraxial mesoderm reached about 70-95% (Non-Patent Documents 7 and 9). This conversion of neural to paraxial mesoderm suggests the existence of common precursors for neural and paraxial mesoderm during 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 paraxial mesoderm (Non-Patent Documents 12 and 12).

[0005] Although induction of paraxial mesoderm and further differentiation has been achieved, there are still some problems to be addressed. During vertebrate development, paraxial mesoderm first forms presegmented mesoderm (PSM) posteriorly and somites (SM) anteriorly. The somites eventually differentiate into dermomyotomes (DM) dorsally and sclerotomes ventrally. The dermomyotomes give rise to dermatomes (D) and myotomes (MYO), precursors of skeletal muscle, and a subpopulation of sclerotomes forms syndesmote, precursors of tendons and ligaments (Non-Patent Document 14). For somite cells induced from pluripotent stem cells to be complete, it is important that the induced somite cells show differentiation potential into dermomyotome, myotome, sclerotome and syndesmote cells. Although the above reports have been successful in inducing myotome and sclerotome cells, the induction protocols for dermomyotome and syndesmote cells have not yet been established. Furthermore, the lateral plate mesoderm is the main source of mesenchymal stromal cells (MSCs) (Non-Patent Document 15), but somite cells can also be a source of mesenchymal stromal cells. However, there have been no reports of inducing mesenchymal stromal cells from pluripotent stem cells via paraxial mesoderm. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Bernardo, AS, Faial, T., Gardner, L., Niakan, KK, Ortmann, D., Senner, CE, Callery, EM, Trotter, MW, Hemberger, M., Smith, JC, 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, SM, Fasano, CA, Papapetrou, EP, 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, CA, Chambers, SM, Lee, G., Tomishima, MJ, and Studer, L. (2010). Efficient derivation of embryonic functional floor plate tissue from humannic 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.

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[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 dermatome cells from somite cells. A further object of the present application is to provide a method for inducing dermatome cells from somite cells. A further object of the present application is to provide a method for inducing mesenchymal stromal cells from somite cells.

[0008] Another object of the present application is to provide a method for inducing myotome cells, dermatome cells, sclerotome cells, ligament node cells and mesenchymal stromal cells from pluripotent stem cells by inducing differentiation of pluripotent stem cells into somite cells, and further inducing differentiation of the somite cells into myotome cells, dermatome cells, sclerotome cells and ligament node cells, and mesenchymal stromal cells, respectively. [Means for solving the problem]

[0009] The entire method of the present application (except for the production of mesenchymal stromal cells) is shown in Figure 1. Somitic cells (SM) were produced in vitro from pluripotent stem cells via presegmented mesoderm (PSM). The obtained somitic cells could be differentiated stepwise in vitro into dermatomes (D), myotomes (MYO), sclerotomes (SCL), and syndesmosomes (SYN), which are known to be further induced to differentiate from somites in vivo. The induction protocols for each were determined. This figure has been modified from a paper (Buckingham et al., 2003).

[0010] The present application relates to a method for producing a pluripotent stem cell, The present invention provides a method for producing somite cells from pluripotent stem cells, the method comprising the step of culturing the pluripotent stem cells in a medium containing a GSK3β inhibitor. In this embodiment, the step of culturing the pluripotent stem cells in a medium containing a GSK3β inhibitor is preferably carried out by: (1) culturing pluripotent stem cells in a medium containing a GSK3β inhibitor to obtain an unarticulated mesoderm cell culture; and (2) The method includes culturing an unsegmented 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, comprising the steps of obtaining somite cells by the above method, and (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, comprising the step (4) of culturing the dermomyotome cells obtained in (3) in a medium containing a GSK3β inhibitor. The myotome cells can be further induced to differentiate by known methods to obtain skeletal muscle cells. The present application also includes a method for producing skeletal muscle cells from such pluripotent stem cells via somite cells and myotome cells.

[0012] The present application also provides a method for producing a method for producing a somite cell comprising the steps of: (5) culturing somite cells in a medium containing a GSK3β inhibitor and BMP; A method for producing dermatome cells from somite cells is provided. In this embodiment, the somite cells may be cells produced from pluripotent stem cells by the above-mentioned method, or may be cells obtained by other methods. That is, this embodiment provides a method for producing dermatome cells from pluripotent stem cells. Dermatome cells can be further induced to differentiate by known methods to obtain dermal cells. The present application also includes 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, comprising the step (6) of obtaining somite cells by the above-mentioned method, and culturing the obtained somite cells in a medium containing a sonic hedgehog activator and a BMP inhibitor. Methods for obtaining bone, cartilage, etc. by further inducing differentiation of sclerotome cells by known methods are known. Methods for obtaining bone, cartilage, etc. from pluripotent stem cells via sclerotome cells are also included in the present application.

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

[0015] The present application further provides a method for producing a method for the preparation of ... The present invention provides a method for producing mesenchymal stromal cells from somite cells, comprising a step of culturing the somite cells in a medium containing FGF. The somite cells in this embodiment may be cells induced from pluripotent stem cells by the method of the present application, or cells provided by other known methods. That is, this embodiment provides a method for producing mesenchymal stromal cells from pluripotent stem cells via somite cells. The mesenchymal stromal cells obtained in this embodiment can be further induced to differentiate by a known method to produce cartilage, bone, or adipocytes. The present application also includes a method of obtaining mesenchymal stromal cells and further inducing the differentiation of the mesenchymal stromal cells to produce cartilage, bone, or adipocytes. Effect of the Invention

[0016] According to the present application, it is possible to efficiently induce differentiation of pluripotent stem cells into somite cells without gene transfer. The somite cells obtained according to the present application can be further induced into dermomyotome cells, myotome cells, sclerotome cells, and ligamentoblast cells. The somite cells obtained according to the present application can also be induced into mesenchymal stromal cells.

[0017] That is, by the method of the present application, it is possible to produce myotome cells, dermomyotome cells, sclerotome cells, ligament node cells, and mesenchymal stromal cells from pluripotent stem cells without gene transfer, via somite cells, and further induce differentiation of 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 defects, bone defects, and tendon rupture. In addition, cells produced by the method of the present application from iPS cells derived from somatic cells of a patient with a genetic disease can be used as a disease model for such diseases. The dermatomes and dermal cells obtained by the method of the present application can be used as models for dermatological research and cosmetic development, or for creating skin wound treatment agents and disease models for skin disease research by cell transplantation.

[0019] The present application also provides a cell transplantation therapy method using cells derived from pluripotent stem cells by the method of the present application. The method of the present application can be used for the treatment of musculoskeletal disorders such as muscular dystrophy, articular cartilage defects, bone defects, and tendon rupture. [Brief description of the drawings]

[0020] [Figure 1] FIG. 1 is a schematic diagram showing the overall method of the present application (excluding the production of mesenchymal stromal cells). [Figure 2A] Schematic diagram of differentiation of human iPS cells into presegmented mesoderm (PSM) and then into somitic cells (SM), followed by stepwise differentiation of SM into sclerotome cells (SCL) and syndesmotic cells (SYN), and finally stepwise differentiation of SM into dermomyotome cells (DM), then into myotome cells (MYO) and dermomyotome cells (D). [Figure 2B] FIG. 1 shows a schematic diagram of a protocol for inducing presegmented mesoderm cells (PSM) from human iPS cells in Example 1. [Figure 2C] The expression patterns of DLL1 and PAX3 during the process of somitic cell (SM) formation from presegmented mesoderm cells (PSM) are shown. [Figure 2D] To determine the optimal protocol for inducing unsegmented mesoderm cells, human iPS cells were cultured under various differentiation induction conditions, and the resulting cells were sorted by FACS for DLL1 and PAX3-GFP. The symbols in the figure indicate the components added to the medium. S: SB431542 10μM, C: CHIR99021 10μM, D: DMH1 2μM, F: FGF2 20ng / ml. [Figure 2E] RT-qPCR analysis results for markers of iPS cells and unsegmented mesoderm cells on day 4 of culture in iPS cell and unsegmented mesoderm cell induction medium are shown. Gene expression was analyzed for cells sorted by DLL1. Error bars are mean ± SE (n = 3). [Figure 2F]The results of investigating the optimal number of days of culture for inducing iPS cells to become unsegmented mesoderm cells. iPS cells were cultured under SCDF conditions (Figure 2D) for 1 to 5 days, and the expression of DLL1 and PAX3 in cultured cells was analyzed by FACS. [Figure 2G] Different iPS cell lines were cultured for 4 days under SCDF conditions (FIG. 2D), and the expression of DLL1 in the cultured cells was analyzed by FACS. [Figure 2H] 1231A3, an iPS cell line maintained under serum-free and feeder cell-free conditions, was cultured for 4 days under SCDF conditions (FIG. 2D), and the expression of DLL1 in cultured cells was analyzed by FACS.

[0021] [Figure 3A] Schematic diagram of the protocol for inducing somitic cells from unsegmented mesoderm cells in Example 2. [Figure 3B] The expression of DLL1 and PAX-3GFP in cells cultured for 4 days in media containing various concentrations of C and S was analyzed by FACS. S10: SB431542 10 μM, C1, C5 and C10: CHIR99021 1, 5 and 10 μM, respectively, D2: DMH1 2 μM, F20: FGF2 20 ng / ml. [Figure 3C] In the process of inducing somite cells from unsegmented mesoderm cells, the expression of cell markers before and after induction was analyzed by RT-qPCR. Error bars: Mean ± SE (n = 3). [Figure 3D] The expression of somite cell-specific cell marker genes on day 4 of somite cell induction (day 8 from the start of iPS cell culture) was analyzed by RT-qPCR. Error bars: mean ± SE (n = 3). S10: SB431542 10 μM, I10: IWR1 10 μM, C5: CHIR99021 5 μM. *p < 0.05; ***p < 0.001 (Dunnett's multiple comparison t test). ns: no significant difference.

[0022] [Figure 4A] FIG. 1 is a schematic diagram of the protocol for directional differentiation of somite cells (SM) via dermomyotome cells (DM) to myotome cells (MYO) and dermomyotome cells (D) in Example 3. [Figure 4B] The expression of various marker genes in somite cells (SM) on day 3 of culture in dermomyotome cell induction medium supplemented with various concentrations of CHIR99021 and BMP4 was analyzed by RT-qPCR. ***p<0.001 by Dunnett's multiple comparison t-test compared to the control without supplements (φ, φ). [Figure 4C] The expression of EN1, an indicator of differentiation into dermomyotome cells, was analyzed by FACS using iPS cells as a control. Error bars: mean ± SE (n = 3). [Figure 4D] The progression of differentiation into myotome cells was measured by RT-qPCR to measure the expression levels of the myotome cell markers MYOD, MYOG, and PAX7, and the dermomyotome cell marker ALX4. Error bars: Mean ± SE (n = 3). [Figure 4E] The time-dependent changes in expression levels of marker genes for somite cells (SM), dermomyotome cells (DM), dermomyotome cells (D), myotome cells (MYO) and dermal fibroblasts (DF) were analyzed by RT-qPCR. The vertical axis on the right of the lower left graph (DM / D marker) is an index of the relative expression level of EN1. Error bars: Mean ± SE (n=3). [Figure 4F] Expression of EN1 and PDGFRa on day 9 of dermomyotome (DM) to dermomyotome (D) cell induction was examined by FACS. Mean values ​​± SE from triplicate experiments are shown. iPS cells were used as a control population. Error bars: mean ± SE (n=3).

[0023] [Figure 5A] Schematic diagram of the protocol for inducing somite cells (SM) to sclerotome cells (SCL), and for inducing differentiation of sclerotome cells to synovial cells (SYN) and cartilage. [Figure 5B] The expression levels of sclerotome cell marker genes (PAX1, PAX9, and NKX3.2) on day 4 of induction of differentiation of somite cells (SM) to sclerotome cells (SCL) were examined by RT-qPCR. Mean values ​​± SE of triplicate experiments are shown. [Figure 5C]Relative expression levels of chondrogenic markers on day 21 of three-dimensional chondrogenic induction (3DCI) from sclerotome cells (SCL). The expression level in sclerotome cells was set to 1. Error bars: Mean ± SE (n = 3). [Figure 5D] Relative expression levels of osteogenic markers from sclerotome cells (SCL) on day 18 of two-dimensional osteogenic induction (2DOI). The expression level in sclerotome cells was set to 1. Error bars: Mean ± SE (n = 3). [Figure 5E] Time course of expression of ligament nodal cell marker genes SCX, COLIA1, MKX, and COLIA2 during induction of ligament nodal cells (SYN) from sclerotome cells (SCL) for 21 days. Mean values ​​± SE of triplicate experiments are shown. [Figure 5F] The expression of SCX in cells on day 21 of induction of sclerotome cells (SCL) to synovial cells (SYN) was examined by FACS. iPS cells were used as a control.

[0024] [Figure 6A] Heat map analysis results of the expression levels of each marker gene in cells at each stage of differentiation induced from iPS cells. [Figure 6B] PCA plot showing the stepwise induction of iPS cells into four cell types via unsegmented mesoderm and somitic cells.

[0025] [Figure 7A] Schematic diagram of the somite cell-derived mesenchymal stromal cell (SMMSC) induction protocol from somite cells (SM). [Figure 7B] FACS analysis of somite cells cultured in 10% FBS-containing medium supplemented with FGF2 (4 ng / ml) for 12 days. CD44+, CD73+, CD105+ and CD45- mesenchymal stromal cells were induced. Somite cells were used as a control population. [Figure 7C] Relative expression levels of various cell markers in somite cells and somite cell-derived mesenchymal stromal cells. The higher the expression level, the higher the value. [Figure 7D] Relative expression levels of various cell markers in somite cell-derived sclerotome cells and somite cell-derived mesenchymal stromal cells. The higher expression level is assigned a value of 1. [Figure 8A] The following shows an outline of the protocol in Example 6. Mesenchymal stromal cells (SMMSC) and sclerosing cells (SCL) were induced from FOP-iPS cells and resFOP-iPS cells via somite cells, and each was spotted onto a dish coated with fibronectin, and treated for 5 days under chondrocyte induction conditions in a basal chondrocyte induction medium supplemented with activin A (30 ng / ml). [Figure 8B] Relative expression levels of ACVR1 in induced mesenchymal stromal cells and sclerotome cells. Error bars: mean ± SE (n = 6). The expression levels in resFOP-iPSC-derived MSCs or SCL cells were set to 1, respectively. In Fig. 8B-H, J-L, error bars: mean ± SE (n = 3). *p<0.05; **<0.01; ***<0.001 by Student's t-test. ns, no significant difference; FOP, fibrodysplasia ossificans progressiva; resFOP, rescued FOP clone; CI, chondrogenic induction; R667, R667 10 nM; Rapa, rapamycin 10 nM. [Figure 8C] Mesenchymal stromal cells were obtained from FOP-iPS cells and resFOP-iPS cells via somite cells, and each mesenchymal stromal cell was cultured for 5 days under chondrogenic induction conditions with the addition of activin A. The expression level of resFOP-iPS-derived cells was set to 1. [Figure 8D] GAG / DNA analysis results for the DNA amount of each cell in FIG. 8D. [Figure 8E] Sclerotome cells were obtained from FOP-iPS cells and resFOP-iPS cells via somite cells, and each sclerotome cell was cultured for 5 days under chondrogenic conditions with the addition of activin A. The expression level of resFOP-iPS-derived cells was set to 1. [Figure 8F] GAG / DNA analysis results for each cell in Figure 8E. [Figure 8G]Mesenchymal stromal cells were obtained from FOP-iPS cells via somite cells, and each mesenchymal stromal cell was cultured for 5 days under chondrogenic induction conditions with the addition of activin A in the presence or absence of R667 or rapamycin to determine the relative expression levels of chondrogenic markers in the cells. [Figure 8H] GAG / DNA analysis results of the cells in Figure 8G. [Figure 8I] Mesenchymal stromal cells were induced from FOP-iPS cells via somite cells, and then isolated by FACS into PDGFRα+ / CD31- and PDGFRα- / CD31- populations. [Figure 8J] GAG / DNA analysis results for each cell population in Fig. 8I when cultured for 5 days under chondrocyte induction conditions. [Figure 8K] Relative expression levels of chondrogenic markers in the cells in Figure 8J. The expression level in PDGFRα- / CD31- cells was set to 1. [Figure 8L] Relative expression levels of PAI1 and MMP1 (both surrogate markers of aberrant FOP-ACVR1 signaling) in cells in Figure 8J. Expression levels in PDGFRα- / CD31- cells were set to 1. [Figure 8M] FIG. 13 is a diagram illustrating the results of Example 6.

[0026] [Figure 9A] Time course of expression of ligament nodule cell-related marker genes (SCX, MKX, TNMD, TNCC, COL1A1, COL1A2, and FMOD) up to 8 days after induction of ligament nodule cells (SYN) from sclerotome cells (SCL) in a xeno-free environment. The vertical axis represents the expression level in sclerotome cells set at 1. [Figure 9B] Comparison of the expression levels of ligament nodule cell-related marker genes (SCX, TNMD, COL1A1, and COL1A2) from scleroblastoma cells (SCL) to ligament nodule cells (SYN) up to 8 days after induction in a xeno-free environment with the expression levels of each marker gene in human healthy anterior cruciate ligament (hACL) samples. [Figure 9C] Immunostaining image of synovial node cells (SYN) on day 21 of induction.

[0027] [Figure 10A] Overview of the preparation and rearing method of Achilles tendon rupture model rats. [Figure 10B] Footprints of the right hind paw in the transplanted (Trans) and non-transplanted (Ctrl) groups taken weekly after transplantation. [Figure 10C] Achilles Functional Index (AFI) in the transplanted (Trans) and non-transplanted (Ctrl) groups. n=8 on days 0, 7, and 14; n=4 on days 21 and 28. *p<0.05; **p<0.01; ***p<0.001 (Student's t-test). ns: no significant difference. [Figure 10D] Observation of walking function on a treadmill in the transplanted group (Trans) and the non-transplanted group (Ctrl) 2 weeks after transplantation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] In the present specification and claims, the term "about" in reference to a numerical value is intended to encompass values ​​up to ±20%, up to ±15%, up to ±10%, or up to ±5% of the indicated numerical value.

[0029] The origin of the "cells" described herein is human or non-human animals (e.g., mouse, rat, cow, horse, pig, sheep, monkey, dog, cat, bird, etc.), and although not limited thereto, cells of human origin are particularly preferred.

[0030] In the method of the present application, a medium in which necessary factors are added to a basal medium for animal cell culture is used for cell culture. Examples of basal medium for animal cell culture that can be used in the present application include Iscove's modified Eagle's Medium medium, Ham's F12 medium, MEM Zinc Option medium, IMEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, 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. Optionally, the medium may contain one or more serum substitutes, such as, for example, albumin, bovine serum albumin (BSA), transferrin, apotransferrin, KnockOut Serum Replacement (KSR) (serum replacement for ES cell culture) (Invitrogen), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, monothioglycerol, and the like, and 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 the like.

[0031] In one embodiment, except for the step of inducing mesenchymal stromal cells from somite cells described below, a serum-free medium or a synthetic medium (hereinafter referred to as "CDM") is preferably used as the basal medium. An example of the CDM medium is a medium obtained by adding 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) to Iscove's modified Eagle's Medium / Ham's F12 medium 1:1 (GIBCO, Grand Island, NY, USA). Hereinafter, the medium with this composition is referred to as "CDM basal medium" in the present specification.

[0032] The culture is preferably performed without using feeder cells, but may be performed using a culture substrate as necessary, such as Matrigel (BD, Bedford, Mass., USA), a commercially available extracellular matrix.

[0033] In another embodiment, the steps except for the step of inducing mesenchymal stromal cells from somite cells described below can be performed under xeno-free conditions. For example, the steps of inducing unarticulated mesoderm cells from pluripotent stem cells, inducing somite cells from unarticulated mesoderm cells, inducing sclerotome cells from somite cells, and inducing ligament cell from sclerotome cells can be performed under xeno-free conditions. "Xeno-free" refers to a medium or culture conditions that do not contain components derived from an organism different from the organism of the cells to be cultured. Examples of xeno-free medium include, but are not limited to, 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 of such xeno-free culture substrates include KnockOut SR XenoFree Medium (Gibco), such as AK03 medium. In addition, in xeno-free culture, it is preferable to use a xeno-free culture substrate together with the xeno-free medium. Examples of such xeno-free culture substrates include the integrin binding site (E8) fragment of recombinant human laminin 511. Examples of such xeno-free culture substrates include iMatrix511 (Nippi Corporation) and CTS CELLstart Substrate (Gibco), such as iMatrix511.

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

[0035] The fact that the target cells have been obtained in each step can be confirmed by examining the expression profile of cell surface markers of the obtained cells. The expression profile of cell surface markers may be confirmed by a known method, such as RT-qPCR, immunocytochemical analysis, FACS (Fluorescence-activated cell sorting), etc.

[0036] The cell culture obtained in each step may be purified before use when subjected to further differentiation induction. Also, the cell culture containing the target cells may be provided after purification of the target cells. Cell purification can be performed, for example, based on a cell surface marker. 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 relates to a method for producing a pluripotent stem cell, Provided is a method for producing somite cells from pluripotent stem cells, comprising the step of culturing 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 present in the body and also have the ability to proliferate. Examples of pluripotent stem cells include embryonic stem (ES) cells (JA Thomson et al. (1998), Science 282:1145-1147; JA Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92:7844-7848;JA Thomson et al. (1996), Biol. Reprod., 55:254-259; JA Thomson and VS Marshall (1998), Curr. Top. Dev. Biol., 38:133-165), cloned embryo-derived embryonic stem (ntES) cells 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), sperm 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; JL 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), cultured fibroblasts, and pluripotent cells derived from bone marrow stem cells (Muse cells) (WO2011 / 007900). Preferably, the pluripotent stem cells are human pluripotent stem cells, such as ES cells and iPS cells.

[0039] In particular, iPS cells are particularly suitable as a material for producing cells for use in therapy or transplantation. When the cells obtained according to the present application are used for therapy, it is desirable to use iPS cells obtained from somatic cells having the same or substantially the same HLA genotype as the recipient individual, from the viewpoint of preventing rejection. Here, "substantially the same" means that the HLA genotype is identical to the transplanted cells to such an extent that immune reactions can be suppressed with an immunosuppressant, for example, somatic cells having an HLA type that matches the three loci of HLA-A, HLA-B, and HLA-DR, or four loci including HLA-C. Of course, cells for use in therapy may be produced using iPS cells induced from the patient's own somatic cells.

[0040] The pluripotent stem cells may be produced by a known method, commercially available pluripotent stem cells, or pluripotent stem cells stored with information on the individual from which they were derived for research or transplantation medicine. A project to build a versatile iPS cell bank using humans with homozygous HLA haplotypes as donors is currently underway in Japan (CYRANOSKI, Nature vol. 488, 139(2012)), and pluripotent stem cells obtained from such an iPS cell bank may be used. Methods for producing iPS cells from human somatic cells have been reported, for example, in Koyanagi-Aoi et al., 2013; Nakagawa et al., 2014; Okita et al., 2011; Takahashi et al., 2007, etc. A method of inducing iPS cells under completely xeno-free conditions without using feeder cells is also known (Nakagawa M, et al. Scientific Reports 4:3594 (2014)). iPS cells induced under such xeno-free conditions may be used.

[0041] Pluripotent stem cells may be induced from cells derived from a patient with a genetic disease. Somitic cells obtained from iPS cells derived from such genetic disease by the method of the present application, and cells further induced to differentiate from the somitic cells, can be used as disease model cells in drug discovery research and elucidation of disease mechanisms. For example, it has been reported that iPS cells can be induced from somatic cells of a patient with fibrodysplasia ossificans progressiva (Matsumoto et al., 2014).

[0042] (1) Induction of differentiation of pluripotent stem cells (PSCs) into presegmented mesoderm cells (PSMs) Methods for producing unsegmented mesodermal cells from pluripotent stem cells are known, and any known method may be used. For example, pluripotent stem cells can be induced to unsegmented mesodermal cells at a relatively high induction rate by culturing them in a medium containing a relatively high concentration of a GSK3β inhibitor (Non-Patent Documents 7 and 9: Loh et al. 2016 and Xi 2017).

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

[0044] An example of a GSK3β inhibitor used in producing unsegmented mesoderm cells from pluripotent stem cells is CHIR99021. The concentration of the GSK3β inhibitor can be appropriately determined by those skilled in the art, and should be relatively high. When using CHIR99021 as a GSK3β inhibitor, 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 in producing unarticulated mesodermal 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] The TGFβ inhibitor used in the present specification and claims is a substance that inhibits signal transmission that continues to downstream SMADs when molecules of the TGFβ family, such as TGFβ, Activin, and Nodal, bind to their receptors, and is not particularly limited as long as it is a substance that inhibits binding to the receptor, the ALK family, or a substance that inhibits phosphorylation of SMADs by the ALK family. For example, Lefty-1 (NCBI Accession No.: mouse: NM_010094, human: NM_020997) is exemplified, SB431542, SB202190 (RK 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) Examples include 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.

[0047] Examples of TGFβ inhibitors used in producing unarticulated mesoderm cells from pluripotent stem cells include SB431542. The concentration of the TGFβ inhibitor can be determined appropriately 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, for example, 0.1 μM to 50 μM, preferably 1 to 20 μM, more preferably about 10 μM.

[0048] Examples of BMP inhibitors include protein 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) and its derivatives (DMH1, etc.) (PB Yu et al. (2007), Circulation, 116:II_60; PB 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] As the BMP inhibitor used in producing unarticulated mesoderm cells from pluripotent stem cells, for example, DMH1 is used. The concentration of the BMP inhibitor can 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, for example, 0.1 μM to 20 μM, preferably 1 to 5 μM, more preferably about 2 μM.

[0050] Examples of FGF (fibroblast growth factor) include FGF2, FGF7, FGF8, and FGF10. For example, FGF2 is used as FGF for producing unarticulated mesoderm cells from pluripotent stem cells. The concentration of FGF can be appropriately determined by those skilled in the art and is not particularly limited. When FGF2 is used as FGF, its concentration in the medium is, 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 for culture when producing unarticulated mesodermal cells from pluripotent stem cells can be determined appropriately 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. Just do that.

[0052] Unsegmented mesodermal cells can be identified as cells expressing, for example, DLL1. When using iPS cells as pluripotent stem cells, the confirmation that unsegmented mesodermal cells are produced from iPS cells can be confirmed by the absence of expression of markers specific to iPS cells, such as NANOG, OCT3 / 4, and SOX2, and the expression of any one or a combination of markers of unsegmented mesodermal cells, such as BRACHYURY, DLL1, TBX6, MSGN1, and WNT3A.

[0053] The unarticulated mesodermal cell culture obtained in this step may be purified and then subjected to a subsequent step, or may be used as is. A specific type of cell in the cell culture may be purified, for example, by FACS using an antibody against a cell surface marker that is expressed or not expressed by unarticulated mesodermal cells. One example is sorting by FACS using the expression of DLL1 on the cell surface as an index.

[0054] (2) Induction of differentiation of presegmented mesodermal cells (PSM) into somitic cells (SM) Somite cells are transient stem cells that give rise to multiple cell types (e.g., dermatome (D), myotome (MYO), sclerotome (SCL) and synovial (SYN)) and are also the source of mesenchymal stromal cells (MSCs) that give rise to bone, cartilage and fat after birth.

[0055] Unsegmented mesodermal cells are cultured in a medium containing a GSK3β inhibitor to induce somitic cells. The GSK3β inhibitor used in producing somitic cells from unsegmented mesodermal cells can be the same as those described above, 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, the concentration in the medium can be, for example, 0.1 μM to 50 μM, preferably 1 to 20 μM, more preferably about 5 μM.

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

[0057] The number of days for culture when producing somitic cells from unarticulated mesodermal cells is not particularly limited, and may be, for example, 1 to 7 days, preferably 3 to 5 days, and more preferably about 4 days.

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

[0059] Stepwise induction of differentiation of somite cells into various cell types (3) Induction of differentiation of somite cells (SM) into dermomyotome cells (DM) The dermomyotome is formed by differentiation of the dorsal side of the somite and gives rise to the dermatome, the precursor of the dermis, and the myotome, the precursor of skeletal muscle.

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

[0061] In producing dermomyotome cells from somite cells, the somite cells used as starting materials may be somite cells produced from pluripotent stem cells via 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] The GSK3 inhibitor used in producing dermomyotome cells from somite cells can be the same as those described above, for example, CHIR99021. The concentration of the GSK3β inhibitor is not particularly limited and may be appropriately determined by those skilled in the art. When CHIR99021 is used as the GSK3β inhibitor, the concentration in the medium is, for example, 0.1 μM to 50 μM, preferably 1 to 20 μM, more preferably about 5 μM.

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

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

[0065] Known markers for dermomyotome cells include ALX4, EN1, and NOGGIN. Production of dermomyotome cells from somite cells can be confirmed by an appropriate combination of the maintenance of PAX3, a transcription factor common to somite cells, the expression of one or more dermomyotome cell markers, and the loss of expression of one or more somite cell markers.

[0066] The dermomyotome cell cultures produced from somite cells may be used directly to produce myotome cells or dermomyotome cells, or the dermomyotome cells may be purified before being used to produce dermomyotome cells. For example, the purification of dermomyotome muscle cells may be performed by FACS using antibodies against markers known to be expressed and / or not expressed by dermomyotome muscle cells.

[0067] (4) Induction of differentiation of dermomyotome cells (DM) into myotome cells (MYO) In one embodiment of the present invention, 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] The GSK3 inhibitor used in producing myotome cells from dermomyotome cells can be the same as those described above, for example, CHIR99021. The concentration of the GSK3β inhibitor is not particularly limited and may be appropriately determined by those skilled in the art. When using CHIR99021 as the GSK3β inhibitor, the concentration in the medium is, for example, 0.1 μM to 50 μM, preferably 1 to 20 μM, more preferably about 5 μM.

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

[0070] Known sarcolemmal cell markers include MYOD, MYOG, and PAX7. The generation of myotome cells can be confirmed by the expression of one or more of these known markers and / or the absence of expression of one or more dermomyotome cell markers. The resulting myotome cell culture may also be purified by FACS using antibodies against markers known to be expressed and / or not expressed by dermomyotome myocytes.

[0071] The resulting myotome cells can be further differentiated to produce skeletal muscle cells. Methods for inducing differentiation of myotome cells into skeletal muscle cells are known.

[0072] (5) Induction of differentiation of dermomyotome cells (DM) into dermomyotome cells (D) Dermatomes are induced from dermomyotomes and become precursors of the dorsal dermis. Dermomyotome cells can be produced by culturing dermomyotome cells in a medium containing a GSK3β inhibitor and BMP. In producing dermomyotome cells from dermomyotome cells, the dermomyotome cells used as a starting material may be dermomyotome cells produced by step (3) from somite cells produced from pluripotent stem cells via steps (1) and (2) above, or may be produced from somite cells obtained by other methods, or may be dermomyotome cells obtained by other methods. The dermomyotome cells may also be obtained from a living animal.

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

[0074] Examples of BMP used in producing dermomyotome cells from dermomyotome cells include BMP2, BMP4, BMP7, and the like, for example, BMP4. The concentration of BMP is not particularly limited and may be appropriately determined by those skilled in the art. When BMP4 is used as BMP, the concentration in the medium is, for example, 0.1 ng / ml to 100 ng / ml, preferably 1 to 50 ng / ml, and more preferably about 10 ng / ml.

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

[0076] Known markers for dermatome cells include PDGFRα, EN1, ALX4, MSX1, and COLIA2. The generation of dermatome cells can be confirmed by an appropriate combination of one or more of the expression of these known markers and the disappearance of the expression of dermatome cell markers. The obtained dermatome cell culture may also be purified by FACS using an antibody against a marker known to be expressed and / or not expressed by dermatome cells. An example of the purification of dermatome cells by FACS is the use of an anti-PDGFRα antibody.

[0077] The resulting dermatome cells can be further differentiated to produce dermal cells.

[0078] (6) Induction of differentiation of somite cells (SM) into sclerotome cells (SCL) The sclerotome is formed by differentiation of the ventral side of the somite, giving rise to the ligamentum, which is the precursor of tendons and ligaments. In one embodiment of the present invention, a method is provided for obtaining somite cells from pluripotent stem cells through the above steps (1) and (2), and further for producing sclerotome cells from the somite cells.

[0079] Methods for producing sclerotome cells from somite cells are known (Zhao et al., 2014), and sclerotome cells may be derived from the obtained somite cells using any known method. In one embodiment, 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, for example, 1 nM to 1 μM, preferably 10 to 500 nM, and more preferably about 100 nM.

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

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

[0083] Known markers for sclerotome cells include PAX1, PAX9, and NKX3.2. The generation of sclerotome cells can be confirmed by an appropriate combination of one or more of the expression of these known markers and the loss of expression of markers specific to somite cells. The resulting sclerotome cell culture may also be purified by FACS using antibodies against markers known to be expressed and / or not expressed by sclerotome cells.

[0084] (7) Induction of differentiation of sclerotome cells (SCL) into synovial cells (SYN) The syndesmote differentiates from the medial portion of the sclerotome and gives rise to tendons and ligaments. In one aspect, the present application provides a method for producing syndesmote cells from sclerotome cells, comprising the steps of: (7-1) culturing sclerotome cells in a medium containing FGF; and (7-2) Culturing the cells obtained in step 7-1 in a medium containing BMP and TGFβ.

[0085] The sclerotome cells used as starting material may be cells derived from other cell types, such as somite cells, by known methods. When sclerotome cells are produced from somite cells, the somite cells may be produced from pluripotent stem cells by the methods of the present application.

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

[0087] Examples of BMP used in producing ligament node cells from sclerotome cells include BMP2, BMP4, and BMP7, such as BMP7. The concentration of BMP is not particularly limited and may be appropriately determined by those skilled in the art. When BMP7 is used as BMP, the concentration in the medium is, 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 in producing ligament node cells from sclerotome cells include TGFβ1, TGFβ2, and TGFβ3, for example, TGFβ3. The concentration of TGFβ is not particularly limited and may be appropriately determined by those skilled in the art. When TGFβ3 is used as TGF, the concentration in the medium is, for example, 0.1 ng / ml to 100 ng / ml, preferably 1 to 50 ng / ml, and more preferably about 10 ng / ml.

[0089] In one embodiment, sclerotome cells are first removed from the sclerotome cell culture and seeded in a ligament node culture medium to initiate the culture. The number of days for each of the steps (7-1) and (7-2) is not particularly limited and may be appropriately determined by a person skilled in the art. When the medium does not contain TGFβ in the step (7-1), the number of days for the culture in the step (7-1) is, for example, 1 to 7 days, preferably about 3 days, and the number of days for the culture in the step (7-2) is, for example, about 15 to 25 days, preferably about 18 days. When the medium contains TGFβ in the step (7-1), the number of days for the culture in the step (7-1) is, for example, 1 to 5 days, preferably about 2 days, and the number of days for the culture in the step (7-2) is, for example, about 4 to 8 days, preferably about 6 days.

[0090] Known markers for ligament node cells include SCX, MKX, COL1A1, and COL1A2. The generation of ligament node cells can be confirmed by an appropriate combination of one or more of the expression of these known markers and the loss of expression of sclerotome cell markers. The obtained ligament node cell culture may also be purified by FACS using antibodies against markers known to be expressed and / or not expressed by ligament node cells.

[0091] (8) Induction of differentiation of somite cells (SM) into somite cell-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. In one embodiment of the present application, a method for producing mesenchymal stromal cells (mesenchymal stromal cells derived from somite cells) is provided, which comprises 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 embodiment, the somite cells used are somite cells induced from pluripotent stem cells by the above steps (1) and (2).

[0092] The FGF used in producing mesenchymal stromal cells from somite cells can be the same as those mentioned above, 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, its concentration in the medium is, 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 in producing mesenchymal stromal cells from somite cells is, for example, αMEM medium. The medium used in this step preferably contains serum. As the serum, fetal bovine serum (FBS) may be used, or serum from other animal species may be used, such as human serum in the case of differentiation induction 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] The number of days for culture when producing mesenchymal stromal cells from somite cells is not particularly limited and may be appropriately determined by those skilled in the art, for example, 4 to 30 days, preferably 8 to 18 days, more preferably 10 to 15 days, and further preferably about 12 days.

[0094] Known markers for mesenchymal stromal cells include CD44, CD73, CD105, and CD90. The generation of mesenchymal stromal cells can be confirmed by an appropriate combination of one or more of the expression of these known markers and the disappearance of somite cell markers. The obtained mesenchymal stromal cell culture may also be purified by FACS using antibodies against markers known to be expressed and / or not expressed by mesenchymal stromal cells.

[0095] As described in detail, 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 induced to differentiate stepwise from dermatome cells to dermatome cells, myotome cells, and sclerotome cells to ligamentophone cells. The somite cells obtained by the method of the present application can also be further induced to differentiate into mesenchymal stromal cells.

[0096] That is, the present application provides a method for inducing differentiation of pluripotent stem cells into myotome cells, comprising: Inducing presegmented mesoderm cells (PSM) from pluripotent stem cells (PSCs) according to (1) above; Induce somitic cells (SM) from the induced presegmented mesoderm cells (PSM) according to (2) above; Induce dermomyotome cells (DM) from the induced somite cells (SM) according to (3) above, and Myotome cells (MYO) are induced from the induced dermomyotome cells (DM) according to the procedure described above (4).

[0097] The present application also provides a method for inducing differentiation of pluripotent stem cells into dermatome cells, comprising: Inducing presegmented mesoderm cells (PSM) from pluripotent stem cells (PSCs) according to (1) above; Induce somitic cells (SM) from the induced presegmented mesoderm cells (PSM) according to (2) above; Induce dermomyotome cells (DM) from the induced somite cells (SM) according to (3) above, and Dermatome cells (D) are induced from the induced dermomyotome cells (DM) according to (5) above.

[0098] The present application further provides a method for inducing differentiation of pluripotent stem cells into sclerotome cells, comprising: Inducing presegmented mesoderm cells (PSM) from pluripotent stem cells (PSCs) according to (1) above; Inducing somitic cells (SM) from the induced presegmented mesoderm cells (PSM) according to (2) above; and Sclerotome cells (SCL) are induced from the induced somite cells (SM) as described above (6).

[0099] The present application also provides a method for inducing differentiation of pluripotent stem cells into ligament node cells, comprising: Inducing presegmented mesoderm cells (PSM) from pluripotent stem cells (PSCs) according to (1) above; Induce somitic cells (SM) from the induced presegmented mesoderm cells (PSM) according to (2) above; Induce sclerotome cells (SCL) from the induced somite cells (SM) according to (6) above, and Syndesmotic nodes cells (SYN) are induced from the induced sclerotome cells (SCL) as described above (7).

[0100] The present application also provides a method for inducing differentiation of pluripotent stem cells into somite-derived mesenchymal stromal cells, comprising: Inducing presegmented mesoderm cells (PSM) from pluripotent stem cells (PSCs) according to (1) above; Inducing somitic cells (SM) from the induced presegmented mesoderm cells (PSM) according to (2) above; and Somite cell-derived mesenchymal stromal cells (SMMSCs) are induced from the induced somite cells (SM) as described above (8).

[0101] The present application also provides a cell transplantation therapy method using cells induced from pluripotent stem cells by the method of the present application. For example, various iPS cell-derived somite-derived cells obtained in the present application can be used for the treatment of musculoskeletal disorders such as muscular dystrophy and tendon rupture. Specifically, ligamentum cells can be used for the treatment of tendon-ligament-related diseases such as ossification of the posterior longitudinal ligament and fibrodysplasia ossificans progressiva, as well as tendon rupture.

[0102] When using iPS cell-derived ligament node cells obtained by the method of the present application for the treatment of tendon / ligament-related diseases, tendon rupture, etc., the ligament node cells may be dispersed in a biocompatible base or the like and then injected into the diseased / injured site. Any known biocompatible base material may be appropriately used, and Matrigel is an example. The number of cells to be injected and the injection site may be appropriately determined depending on the disease / injury to be treated, and are not particularly limited. For example, 10 cells may be injected into one injection site. 12 ~10 6 pcs or 10 11 ~10 7 Pieces or 10 10 ~10 8 Inject iPSC-derived ligament node cells into the affected area. If necessary, perform surgical procedures such as suturing the diseased / injured area. EXAMPLES

[0103] The present invention will be described in more detail below by showing examples, but the present invention is not limited to these examples in any way. 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 primate ES cell medium (ReproCELL, Tokyo, Japan) supplemented with 4 ng / ml FGF2 (WAKO, Osaka, Japan). Unless otherwise noted, 201B7-PAX3-GFP iPS cells, in which one allele of the PAX3 coding sequence in exon 1 was replaced with EGFP, were used in all experiments in Examples 1 to 5. PAX3 with the same knock-in design was also used. GFP / +Heterozygous mice are viable and fertile. GFP expression indicates endogenous Pax3 expression in mice (Lagha, M. et al., 2010). In Example 1, reproducibility due to differences in iPS cell lines was examined using various iPS cell lines (201B7, TIG118-4f, 414C2, 409B2, and 1231A3) (Koyanagi-Aoi et al., 2013; Nakagawa et al., 2014; Okita et al., 2011; cells produced according to Takahashi et al., 2007).

[0104] Culture medium The composition of the CDM basal medium used in the examples is shown below: Iscove's modified Eagle's Medium / Ham's F12 medium 1:1 (GIBCO, Grand Island, NY, USA) was supplemented with 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).

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

[0106] Immunocytochemistry, immunohistochemistry, histological analysis Prior to immunocytochemistry with antibodies, cells on plates were fixed with 2% paraformaldehyde for 10 min at 4°C, washed twice with PBS, incubated with 0.2% MtOH (Nacalai Tesque) or 0.2% tween20 (Sigma) / PBS as a detergent for permeabilization for 15 min at 4°C, treated with Blocking One (Nacalai Tesque) or 1% BSA / PBS for 1 h at 4°C, and treated with primary antibodies overnight at 4°C. Samples were then washed several times with 0.2% tween20 / PBS and incubated with secondary antibodies for 1 h at room temperature. Nuclei were counterstained with DAPI (1:5000; Sigma). Immunohistochemistry with anti-type II collagen antibodies and histological analysis of induced 3DCI pellets (HE staining, Alcian blue staining, and Safranin O staining) 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 MHC immunocytochemistry, photographs were taken using the optical sectioning system of the BZ-X700.

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

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

[0109] Microarray analysis Total RNA was prepared using RNeasy Mini Kit (Qiagen). cDNA was synthesized using GeneChip WT (Whole Transcript) Sense Target Labeling and Control Reagents Kit as described by the manufacturer (Affymetrix, Santa Clara, CA). Hybridization to GeneChip Human Gene 1.0 ST expression arrays, washing, and scanning were performed according to the manufacturer's protocol (Affymetrix). Expression values ​​were calculated using the RMA summary method, and the resulting data were analyzed with GeneSpring GX 14.5 (Agilent Technologies, Santa Clara, CA, USA) for heatmaps and principal component analysis (PCA). PCA analysis was performed on expression values ​​(2-fold higher with statistical significance). Statistical analysis was performed using one-way ANOVA with Benjamini and Hochberg False Discovery Rate (BH-FDR 50.01) multiple testing correction, followed by Tukey's HSD post-hoc test (GeneSpring GX).

[0110] statistics 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 to 4 FIG. 2A shows an overview of Examples 1 to 4. EXAMPLES

[0112] (1) Induction of differentiation of human iPS cells into presegmented mesoderm (PSM) cells To minimize the influence of growth factors secreted from the feeder cells and contained in the medium, SNL feeder cells were removed from the iPS cell cultures, and human iPS cells were seeded (1.3 × 10 6 iPS cells were cultured for 3 days under feeder-free conditions containing mTeSR1 medium (STEMCELL Technology, Vancouver, Canada). The iPS cells were then cultured for 4 days in CDM basal medium supplemented with one of four factors or an appropriate combination of factors (Figure 2B). The factors added are as follows. When two or more factors are used in combination, 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: 20ng / ml FGF2

[0113] The medium was changed on day 3. To detect the induction efficiency of unsegmented mesoderm cells, the 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 divided somites) (Figure 2C) was detected by FACS. Because PAX3 is a transcription factor, PAX3-GFP knock-in iPS cells (PAX3-GFP iPS cells) were used to detect PAX3-positive cells. The results are shown in Figure 2D.

[0114] The effects of four factors (SB431542, CHIR99021, DMH1, and FGF2) were analyzed one by one. CHIR99021 (C) inhibited DLL1 + / PAX3-GFP -The results were consistent with previous reports (Chal et al., 2015; Loh et al., 2016; Sudheer et al., 2016; Umeda et al., 2012; Xi et al., 2017). We next evaluated the combination of two factors and found that CHIR99021 and SB431542 or DMH1 (SC or CD) effectively induced DLL1 + / PAX3-GFP - The combination of DMH1 with SB431542 and CHIR99021 (SCD) induced DLL1 expression at almost the maximum level. + / PAX3-GFP - cells (83.8 ± 1.1%), but DLL1 - / PAX3-GFP + The cells also appeared because, under these conditions, the cells express PAX3 + These results suggest that the blastocysts differentiate into somitocytes and / or neurons.

[0115] Next, when all four molecules were added (SCDF) and cultured, PAX3-GFP was detected under these conditions. + DLL1-free cells + / PAX3-GFP - The cells were found to be derived from PAX3-GFP iPS cells (85.4±0.4%). + Although there was no obvious difference between SCD, SCF and SCDF in terms of cell induction efficiency, SCDF conditions were used in further analysis because SCDF reproduces the exact endogenous signaling environment.

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

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

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

[0119] (2) Induction of differentiation of presegmented mesodermal cells (PSM) into somitic cells (SM) iPS cells were cultured for 4 days under the SCDF conditions of Example 1, and a total of 1.0 x 10 5 DLL1 + Unsegmented mesoderm cells were seeded onto one well of a 12-well plate coated with Matrigel for induction of somitic cells (Figure 3A). Somitic cell induction was performed in CDM basal medium supplemented with SB431542 and / or 5 μM CHIR99021 for 4 days. The medium was changed on the third day of somitic cell induction. Expression of PAX3-GFP was used as a marker for somitic cells.

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

[0121] The results are shown in Figure 3B. After 4 days of culture, 10 μM SB431542 (S10) and 5 μM CHIR99021 (C5) both suppressed the PAX3-GFP + Treatment with both CHIR99021 and CHIR99021 (10 μM) efficiently induced somite cells (52.1 ± 0.8% and 70.7 ± 0.1%, respectively). Treatment with both CHIR99021 (S10C5) induced the greatest number of somite cells (74.7 ± 0.5%), whereas treatment with a higher dose of CHIR99021 (10 μM) induced the opposite: PAX3-GFP cells. + We were unable to induce somite cell induction (0.3±0.0% in C10 and 0.7±0.1% in S10C10), suggesting that excessive WNT signaling suppresses somite cell induction. We further examined the effects of FGF and BMP inhibition, finding that 10 μM SB431542 and 5 μM CHIR99021 (S10C5) significantly inhibited PAX3-GFP induction from unsegmented mesoderm cells. + It was confirmed that this induces cells most efficiently. Immunohistochemistry using anti-PARAXIS (TCF15) antibody also revealed that PARAXIS-expressing cells, indicating that they were somitic cells (SM), were identified in C4, S10C1, S10C5, and S10C5D2, similar to the results obtained using FACS shown in Figure 3B, with the proportion of PARAXIS-expressing cells being highest in the S10C5 group.

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

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

[0124] Induction of myotome (MYO) and dermatome (D) cells from somite (SM) cells via dermomyotome (DM) cells A schematic diagram is shown in FIG. 4A.

[0125] (3) Induction of dermomyotome cells (DM) from somite cells (SM) The medium used for somite cell induction was replaced with medium for dermomyotome cell induction, and the culture was continued. To inhibit or activate WNT signaling, 10 μM IWR1 (Cayman Chemical, Michigan, USA) (I10) or CHIR99021 (0, 0.1, 1, and 5 μM) (C0, C0.1, C1, and C5) were used. To control 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 DMH1 (Tocris) (D10) were used.

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

[0127] After 3 days of culture, the relative expression levels of 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 the concentrations of GSK3β inhibitor, a WNT signal activator, and BMP activator were both high. The generation of dermomyotome cells was also confirmed by immunocytochemistry using anti-ALX4 and EN1 antibodies and PAX3-GFP, which is expressed in both somite cells and dermomyotome cells. The generation of dermomyotome cells was also confirmed by FACS using anti-EN1 antibody (Figure 4C).

[0128] (4) Induction of myotome cells (MYO) from dermomyotome cells (DM) We attempted 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 CDM basal medium supplemented with 5 μM CHIR99021, and further cultured. The medium was changed every 3 days and cultured for 30 days. Every 6 days, a small amount of cultured cells was taken and the time-dependent expression of myogenic markers MYOD, MYOG, and PAX7 was examined. Induction of myogenic markers was observed 18 to 30 days after medium change. 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. The induction efficiency of myotome cells from dermomyotome cells was calculated based on the number of MYOD-positive cells and MYOG-positive cells, and was approximately 22%.

[0129] (5) Induction of dermomyotome cells (DM) into dermomyotome cells (D) In vivo, dermomyotome cells also differentiate into dermomyotome cells, and dermomyotome cells differentiate into the dermis of the back, but no protocol has been established for inducing dermomyotome cells to dermis via dermomyotome cells in vitro. The cells were cultured in the dermomyotome cell induction medium (C5B10) described in (3) above without subculturing from day 11 (day 3 from SM) onwards even after dermomyotome cells were generated. The expression of various cell markers was examined by RT-qPCR, and the expression levels of PAX3 and PARAXIS (dermomyotome cell and somite cell markers: SM / DM markers) and PAX7 and NCAD (dermomyotome cell and myotome cell markers: DM / MYO markers) decreased 6 days after DM generation (Figure 4E, top). Therefore, the induction of dermomyotome cells from dermomyotome cells was performed by continuing to culture the dermomyotome cells in the dermomyotome cell induction medium (medium described in (3) above). After dermomyotome cells were generated, the cells were cultured for 9 days and the medium was changed every 3 days. It is known that PDGFRα is expressed in dermatome cells (D) and dermal fibroblasts (DF) (Orr-Urtreger et al., 1992), and EN1 is expressed in dermatome cells (D) and dermomyotome cells (DM) (Ahmed et al., 2006). Expression of PDGFRα and EN1 was examined by RT-qPCR (Fig. 4E, bottom) and immunocytochemistry (data not shown), and these markers were found to be predominantly expressed on day 9 of the dermatome cell induction process. Expression of ALX4 and MSX1 (DM / D markers) and COL1A2 (D / DF markers) were also elevated on day 9 of the dermatome cell induction process (Fig. 4E, bottom). FACS analysis with anti-PDGFRα and anti-EN1 revealed that 69.5 ± 1.4% of the dermatome cell cultures expressed PDGFRα. + and PDGFRα + 92.7±0.4% of cells were EN1 + It was confirmed that dermatome cells (D) were induced (FIG. 4F). EXAMPLES

[0130] Induction of differentiation of somite cells (SM) through sclerotome cells (SCL) into chondrocytes, osteocytes, and synovial cells (SYN) A schematic diagram is shown in FIG. 5A.

[0131] (6) Induction of differentiation of somite cells into sclerotome cells Somite cells were induced to sclerotome cells as previously described (Zhao et al., 2014). Specifically, the somite cell induction medium was replaced with 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)) for 3 days. The medium was replaced on the second day.

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

[0133] Three-dimensional chondrogenesis induction (3DCI) from scleroblast cells (SCL) Total 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 changed every 3 days.

[0134] The cultured sclerotome cells were harvested, centrifuged to form a pellet, and further cultured in basal chondrogenic medium.Cartilage was confirmed to have been generated on day 21 of cartilage 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] Two-dimensional osteogenic induction from sclerotome cells (SCL) (2DOI) Total 4.0x10 5 The induced sclerotome cells were seeded on 12-well plates coated with Matrigel and then induced to differentiate into osteocytes in 2D using MSC go rapid osteogenic medium (Biological Industries, Kibbutz Beit-Haemek, Israel). The expression of PAX1, RUNX2, COL1A1, OSX, and OPN in the cells on day 18 of osteocyte induction (day 29 from iPS cells) (Figure 5D) and Alizarin Red staining of the cells (data not shown) confirmed that the sclerotome cells had differentiated into osteocytes.

[0136] (7) Induction of synovial cells (SYN) from sclerotome cells (SCL) The dorsal portion of the sclerotome is defined as the syndesmote, which is the anlage of tendons and ligaments (Brent et al., 2003). The protocol for inducing sclerotome cells into syndesmote was unknown prior to this application. Induced sclerotome cells were removed from the dish with 0.25% trypsin-EDTA (GIBCO) and a total of 5.0 x 10 4 The cells were seeded onto one well of a 24-well plate coated with Matrigel, followed by ligament node cell induction.

[0137] Sclerotome cells were cultured in ligament node cell induction medium A (CDM basal medium supplemented with 20 ng / ml FGF8 (Peprotech, Rocky Hill, NJ, USA)) for 3 days (step 7-1). Thereafter, without subculturing, the medium was replaced with ligament node 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 replaced every 3 days.

[0138] In the later stage of induction into ligament node cells, the expression of ligament node cell markers SCX, MKX, COL1A1, and COL1A2 increased over time (Figure 5E). In addition, the protein expression of each factor was confirmed by immunocytochemistry on day 21 of ligament node cell induction, and the results were similar to those in Figure 5E. SCX-expressing cells were confirmed by FACS, and the induction rate into ligament node cells was confirmed to be 68.0 ± 2.4% (Figure 5F).

[0139] In the present example, pluripotent stem cells were induced to differentiate into presegmented mesoderm (PSM) and then into somite cells (SM). The SM cells were then further induced to differentiate into dermatome cells (DM), myotome cells (MYO), sclerotome cells (SCL) and syndesmote cells (SYN). Gene expression profiles of each induced cell line were examined, and the heat map analysis and PCA plots are shown in Figure 6A and Figure 6B, respectively. These figures show preferential and stepwise differentiation into each stage of cells, supporting the rationale of each approach. EXAMPLES

[0140] (8) Induction of mesenchymal stromal cells (MSCs) from somite cells (SMs) Somite cells can be precursors of mesenchymal stromal cells, but there have been no reports 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 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). Cultured cells were detached from the vessel using 0.25% trypsin-EDTA (GIBCO) and subcultured every 4 days. Cells were cultured at 2x10 4 cells / cm 3 The cells were seeded onto tissue culture dishes at a density of 100-200 μg / ml.

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

[0142] The obtained mesenchymal stromal cells were subjected to osteogenic induction (OI), chondrogenic induction (CI), and adipogenic induction (AI) by the above-mentioned known methods, and the differentiation potential of the somite cell-derived mesenchymal stromal cells (SMMSCs) was confirmed. CI, OI, AI, and their assays (Alizarin Red staining, Alcian Blue staining, Oil Red O staining) were performed according to a previous report (Fukuta et al., 2014), and the generation of cartilage, bone, and adipocytes was confirmed.

[0143] Bone formation and chondrogenesis can be induced from sclerotome cells derived from somite cells as well as from mesenchymal stromal cells. To distinguish between 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 in each cell by RT-qPCR. The induced mesenchymal stromal cells were positive for mesenchymal stromal cell markers (CD44, CD73, CD90, and CD105), but not for somite or sclerotome cell markers (Figures 7C and 7D). These results indicated that the cells induced by (8) above were mesenchymal stromal cells. EXAMPLES

[0144] Differences in chondrogenesis between mesenchymal stromal cells derived from somite cells and sclerotome cells derived from somite cells One of the promising applications 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 somite cell-derived mesenchymal stromal cells. This protocol was applied to iPS cells generated from somatic cells of patients with fibrodysplasia ossificans progressiva (FOP), which is caused by a genetic mutation (FOP-iPS cells). FOP is an intractable rare disease characterized mainly by endochondral ossification in the soft tissues of patients after birth, and is known to be caused by a mutation in ACVR1 that causes it to overactivate. A research group including the present inventors has previously reported that mesenchymal stromal cells induced from neural crest cells derived from FOP-iPS cells were used to demonstrate enhanced chondrogenesis (Hino et al., 2015; Matsumoto et al., 2015). Similarly, chondrocytes derived from mesenchymal stromal cells induced from somite cells derived from FOP-iPS cells are predicted to have enhanced chondrogenesis, whereas embryonic chondrocytes derived from sclerotome cells are predicted to have no enhanced chondrogenesis.

[0145] The study outline is shown in Figure 8A. Somite cells were derived 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 in Examples 1 and 2 above. Somite cell-derived mesenchymal stromal cells and sclerotome cells were derived according to the protocols in Examples 4 and 5.

[0146] The obtained mesenchymal stromal cells and sclerocytes were subjected to induction of two-dimensional chondrogenesis using chondrogenic medium ( Hino et al., 2015 ) supplemented with or without activin A (a promoter of ACVR1 mutants).

[0147] 2D cartilage formation induction (2DCI) Total 1.5x10 5 Each induced mesenchymal stromal or sclerotoma cell was 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 1 h of incubation at 37°C in 5% CO2, the cells formed a micromass.

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

[0149] In addition, microaccumulations derived from mesenchymal stromal cells derived from FOP-iPS-derived somite cells were similarly cultured in 1 mL of chondrogenic basal medium containing 30 ng / mL activin A (R&D) with or without 10 nM R667 (Toronto Research Chemicals, Toronto, ON, Canada) or 10 nM 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 min, rinsed with PBS, and then stained with Alcian blue solution (1% Alcian blue, pH 1) (MUTO PURE CHEMICAL CO., LTD, Tokyo, Japan) overnight.

[0151] The results are shown in Figures 8B to D. There was no difference in the ACVR1 expression levels in chondrocytes derived from somite cell-derived mesenchymal stromal cells and sclerotome cells, regardless of the cell type of origin or whether ACVR1 mutation had been repaired (Figure 8B).

[0152] In the absence of activin A stimulation, there was no difference in the expression levels of chondrogenic markers regardless of the cell type of origin or whether or not ACVR1 mutation had been repaired (data not shown).

[0153] When somite cell-derived mesenchymal stromal cells induced from FOP-iPS cells were induced to cartilage under activin stimulation, the expression of chondrogenic markers and the amount of glycosaminoglycan (GAG) production showed enhanced chondrogenesis compared to cells induced from resFOB-iPS cells (Fig. 8C and D). Enhanced chondrogenesis was also observed in somite cell-derived mesenchymal stromal cells induced from FOB-iPS cells by 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 was no difference between FOB-iPS-derived and resFOB-iPS-derived cells in the expression of chondrogenic markers, glycosaminoglycan (GAG) quantification, or Alcian blue staining results (Fig. 8E and F).

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

[0155] In recent years, PDGFRα has been identified as one of the cells of origin for FOP lesions. + / CD31 - FOP-SM MSCs were separately sorted by FACS and expressed PDGFRα + / CD31 - Cells and PDGFRα - / CD31 - Cells were obtained (Figure 8I). Each cell was subjected to 2D chondrogenic differentiation using 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 expressing PDGFRα - / CD31- The ACVR1 mutant cells showed enhanced chondrogenesis compared to the control cells (Figures 8J and 6K). Interestingly, PAI1 and MMP1 (indicator genes of ACVR1 mutant activation) were upregulated by PDGFRα + / CD31 - It was significantly higher in cells (Fig. 8L). These results are summarized in Figure 8M, demonstrate the cell type specificity of the FOP phenotype, and demonstrate that our protocol can be used for disease modeling, phenotypic analysis, and drug discovery. EXAMPLES

[0156] Xeno-free induction of differentiation from sclerotome cells (SCL) to synovial cells (SYN) Sclerotome cells were induced in a xeno-free environment from iPS cells induced under xeno-free conditions. Specifically, 1231A3 cells induced under xeno-free conditions were used as iPS cells. First, iPS cells were cultured for 4 days under the SCDF conditions of Example 1 to induce unsegmented mesoderm cells. The medium was StemFit (登録商標) AK03 (C solution-free) medium (Ajinomoto Co., Inc., hereafter referred to as AK03(-C) medium) was used. Next, to induce somite cells, a total of 1.0 x 10 5 DLL1 + Unsegmented mesoderm cells were seeded into each well of a 12-well plate coated with iMatrix511 (Nippi Corporation) containing AK03(-C) medium supplemented with 10 μM SB431542 and 5 μM CHIR99021 and cultured for 4 days. The medium was changed on the third 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 second day of sclerotome cell induction.

[0157] The day before initiating the induction of ligament node cells, a plate coated with iMatrix511 was prepared. To prepare a 24-well plate 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 sclerotome cell cultures was aspirated and washed with PBS. 0.2 mL of the cell detachment reagent Accutase was added to each well. 登録商標 (Innovative Cell Technologies, Inc.) was added and left at room temperature for 3 minutes. Then, 0.8 mL of AK03(-C) medium was added to each well, and all cells were scraped and collected in a 15 mL conical tube. Then, the wells were centrifuged at 280 × g for 3 minutes. The supernatant was carefully aspirated and resuspended in 1 mL of ligament node cell induction medium A' (AK03(-C) medium supplemented with 20 ng / mL FGF8 and 10 ng / mL TGFβ3). The number of cells was counted using an automated cell counter.

[0159] 5.0×10 4 The cells were seeded into each well of a 24-well plate coated with iMatrix511 containing 1 mL of ligament node cell induction medium A' and incubated for 2 days at 37°C in 5% CO2 (step 7-1). On the second day of ligament node cell induction, the medium was replaced with ligament node cell induction medium B' (AK03(-C) medium supplemented with 10 ng / mL BMP7 and 10 ng / mL TGFβ3). The cells were incubated for 6 days at 37°C in 5% CO2 until the 8th day (step 7-2). The medium was replaced every 2 days.

[0160] In step 7-1, a medium supplemented with 20 ng / mL FGF8 (ligament node cell induction medium A) was used in Example 4, whereas a medium supplemented with 20 ng / mL FGF8 and 10 ng / mL TGFβ3 (ligament node cell induction medium A') was used in this example. As a result, the number of days for culture in step 7-1 was shortened to 2 days, and the number of days for culture in step 7-2 was shortened to 6 days.

[0161] From the start of ligament node cell induction to day 8, the expression of seven ligament node cell-related markers (SCX, MKX, TNMD, TNCC, COL1A1, COL1A2, and FMOD) increased over time (Figure 9A). In addition, the mRNA expression levels of ligament node cell-related markers (SCX, TNMD, COL1A1, and COL1A2) on day 8 of induction were examined. For comparison, the expression levels of each marker in a human healthy anterior cruciate ligament sample (CDD-H-6800-NR, Articular Engineering) were examined. The mRNA expression levels of each marker in both samples were comparable (Figure 9B). Furthermore, immunostaining on day 21 of induction confirmed the protein expression of each marker (SCX, TNMD, COL1A1, and COL1A2) (Figure 9C). EXAMPLES

[0162] Therapeutic effect of transplanting iPS cell-derived ligament node cells The cells on the 8th day after induction of ligament node cells in Example 7 were transplanted into a rat model of Achilles tendon rupture, and the therapeutic effect of transplantation was observed for 4 weeks.

[0163] An outline of the preparation and rearing of Achilles tendon rupture model rats is shown in Figure 10A. To prepare Achilles tendon rupture model rats, the left hind leg of 8-week-old F344 / NSlc male rats was incised (Figure 10A, upper left), and the Achilles tendon was cut 5 mm from the calcaneus (Figure 10A, upper right). The incision was then sutured, and 3 × 10 6 A solution of 10 iPS cell-derived ligament node cells / 50 μL Matrigel:DMEM / F12 = 1:1 was injected (Figure 10A, bottom left). The rats were kept with their tails suspended for one week (Figure 10A, bottom right), and then observed for three weeks.

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

[0165] We also verified the efficacy of transplantation therapy based on the Achilles Functional Index (AFI; Murrell et al., 2014) (Figure ​(Figure1C).10C). Two weeks after transplantation, the transplanted group showed significant recovery compared to the non-transplanted group.

[0166] Furthermore, 4 weeks after transplantation, walking function was observed on a treadmill (Figure 10D).The transplanted group showed significant recovery in heel height from the floor and ankle angle compared to the non-transplanted group.

[0167] References Ahmed, MU, 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, AS, Faial, T., Gardner, L., Niakan, KK, Ortmann, D., Senner, CE, Callery, EM, Trotter, MW, Hemberger, M., Smith, JC, 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-s tem-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

1. Providing dermomyotome cells; and A method for producing myotome cells from dermomyotome cells, comprising the step of culturing the dermomyotome cells in a medium containing a GSK3β inhibitor.

2. The method according to claim 1 , wherein the dermomyotome cells are obtained by a method comprising the step of culturing somite cells in a medium containing a GSK3β inhibitor.

3. The method according to claim 2 , wherein the somite cells are obtained by a method comprising the step of culturing pluripotent stem cells in a medium containing a GSK3β inhibitor.

4. Providing dermomyotome cells; and A method for producing dermomyotome cells from dermomyotome cells, comprising the step of culturing the dermomyotome cells in a medium containing a GSK3β inhibitor and BMP.

5. The method according to claim 4 , wherein the dermomyotome cells are obtained by a method comprising the step of culturing somite cells in a medium containing a GSK3β inhibitor.

6. The method of claim 4 or 5, further comprising the step of purifying the resulting dermatome cell culture by FACS.

7. The method of any one of claims 4 to 6, wherein the GSK3β inhibitor is CHIR99021.

8. The method according to any one of claims 4 to 7, wherein the BMP is BMP4.

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