Method for inducing differentiation into neuromuscular junctions

Abstract

In a method for inducing differentiation into neuromuscular junctions according to the present disclosure, the neuromuscular junctions are induced by: preculturing human pluripotent stem cells in a medium containing activin, bFGF, a WNT signal inhibitor, a BMP signal inhibitor, and a ROCK signal inhibitor; then culturing the stem cells in a medium containing bFGF, a ROCK signal inhibitor, a WNT signal activator, and a TGFβ signal inhibitor to differentiate into neuromesodermal cells; culturing the neuromesodermal cells in a medium containing a BMP signal inhibitor, a TGFβ signal inhibitor, a retinoic acid activator, and an SHH signal activator to differentiate into skeletal muscle progenitor cells and spinal motor neuron progenitor cells in the same culture dish; and culturing these progenitor cells in a medium for cell maturation containing BDNF, GDNF, AA, and dbcAMP.

Description

Method for inducing differentiation of neuromuscular junctions

[0001] The present invention relates to a method for inducing differentiation of neuromuscular junctions, and more particularly to a method for inducing differentiation of neuromuscular junctions in vitro using the same culture system that mimics in vivo.

[0002] The neuromuscular junction (NMJ) is known as a structure composed of ectoderm-derived spinal motor neurons and Schwann cells and mesoderm-derived skeletal muscles. Conventionally, to construct NMJs ex vivo, motor neurons derived from the brain or spinal cord of mice or rats, or spinal cord-derived motor neurons induced from human pluripotent stem cells (ES cells, iPS cells), have been used as materials. Skeletal muscles derived from mice or rats, pluripotent stem cells, or commercially available muscles have been used. These motor neurons and skeletal muscles were prepared separately and then seeded onto a single culture dish for co-culture to create NMJs.

[0003] It is known that the location to which spinal motor neurons transmit information varies depending on the height of the spinal cord at which they develop. For example, motor neurons in the cervical spinal cord mainly transmit information to the muscles of the upper limbs. It is known that the central nervous system from the cervical spinal cord to the head is derived directly from the neuroectoderm, whereas the spinal cord from the thoracic spinal cord onward differentiates via neuromesoderm, a precursor cell common to the adjacent somitic mesoderm (see, for example, Non-Patent Documents 1 and 2). It is known that when human pluripotent stem cells are induced to differentiate into motor neurons, the result is usually at the cervical spinal cord level in most cases (see, for example, Non-Patent Document 3).

[0004] Curr Opin Genet Dev, Vol 22, Issue 4, August 2012, p. 374-380. PLos Biol, Vol 12, Issue 8, August 2014, e1001938. Cell Syst, Vol 12, Issue 2, February 2021, p. 159-175.

[0005] When constructing an NMJ in a culture dish using motor neurons at the cervical spinal cord level differentiated from the above-mentioned human pluripotent stem cells, skeletal muscles of unspecified locations have been used to construct the NMJ, and in this case, it is unlikely that a physiological NMJ can be reproduced. Therefore, in order to reproduce a physiological NMJ, it is necessary to match the regions where the motor neurons and skeletal muscles originate. For example, when using motor neurons at the cervical spinal cord level, skeletal muscles of the upper limbs must be used. Furthermore, it has become clear in recent years that pluripotent stem cells from non-human animals, such as mice and rats, have different properties from human pluripotent stem cells, and the use of pluripotent stem cells from non-human animals cannot be said to mimic human development.

[0006] The present invention has been made in consideration of the above-mentioned problems, and its purpose is to provide a method for inducing an NMJ that uses human pluripotent stem cells, enabling the induction of an NMJ that mimics human development, and that can construct an NMJ consisting of a motor neuron and skeletal muscle derived from the same spinal cord level.

[0007] In order to achieve the above-mentioned object, the present inventors conducted extensive research and discovered that, under certain conditions, it is possible to induce neuromesoderms, which are common precursor cells that give rise to motor neurons and skeletal muscles posterior to the cervical spinal cord (caudal side), from human pluripotent stem cells, and further, by culturing these neuromesoderms under certain conditions, it is possible to induce motor neurons and skeletal muscles derived from the same spinal cord level in the same culture dish, and to induce NMJs between these motor neurons and skeletal muscles, thereby completing the present invention.

[0008] Specifically, the method for inducing NMJ differentiation according to the present invention includes the steps of preparing human pluripotent stem cells, pre-culturing the pluripotent stem cells in a medium containing Activin A, bFGF (basic fibroblast growth factor), WNT (a portmanteau of Wingless and Int-1) signal inhibitor, BMP (Bone Morphogenetic Protein) signal inhibitor, and ROCK (Rho-associated Protein Kinase) signal inhibitor, and then incubating the pre-cultured pluripotent stem cells with bFGF, a ROCK signal inhibitor, a WNT signal activator, and TGFβ (Transforming growth factor the step of culturing the induced Neuromesoderm in a medium containing a BMP signal inhibitor, a TGFβ signal inhibitor, a retinoic acid activator, and an SHH (Sonic Hedgehog) signal activator to induce skeletal muscle progenitor cells via mesoderm and spinal motor neuron progenitor cells via neuroectodermal cells in the same culture dish; and the step of culturing the induced skeletal muscle progenitor cells and neuroectodermal cells in a cell maturation medium containing BDNF, GDNF, AA, and dbcAMP in a culture dish coated for cell adhesion to induce NMJ.

[0009] According to the method for inducing NMJ differentiation of the present invention, neuromesoderm can be induced by culturing human pluripotent stem cells under the above conditions, and further, by culturing the neuromesoderm under the above conditions, motor neurons and skeletal muscles derived from the same spinal cord level can be induced in the same culture dish, and an NMJ can be induced between the motor neurons and skeletal muscles.

[0010] As the human pluripotent stem cells, human embryonic stem cells (ES cells) or human induced pluripotent stem cells (iPS cells) can be used.

[0011] As the WNT signal inhibitor, an agent containing 4-(2-methyl-4-pyridinyl)-N-[4-(3-pyridinyl)phenyl]-benzeneacetamide can be used.

[0012] As the BMP signal inhibitor, an agent containing 4-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline hydrochloride can be used.

[0013] As the ROCK signal inhibitor, an agent containing (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)cyclohexanecarboxamide dihydrochloride can be used.

[0014] As the WNT signal activator, an agent containing 6-[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)pyrimidin-2-yl]amino]ethylamino]pyridine-3-carbonitrile can be used.

[0015] As the TGFβ signal inhibitor, an agent containing 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide can be used.

[0016] As the retinoic acid activator, an agent containing 4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)ethynyl]benzoic acid can be used.

[0017] As the SHH signal activator, an agent containing 3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-[3-(4-pyridinyl)benzyl]-1-benzothiophene-2-carboxamide can be used.

[0018] The culture dish coated with the cell adhesion coating may be a culture dish coated with ornithine or laminin. In addition to these, fibronectin may also be used for coating.

[0019] According to the method for inducing NMJ differentiation of the present invention, neuromesoderm can be induced by culturing human pluripotent stem cells under the above conditions, and further, by culturing the neuromesoderm under the above conditions, motor neurons and skeletal muscles derived from the same spinal cord level can be induced in the same culture dish, and an NMJ can be induced between the motor neurons and skeletal muscles.

[0020] FIG. 1 is a diagram showing an experiment of Reference Example 1, where (a) shows an outline of the experimental conditions, and (b) shows the experimental results. FIG. 2 is a diagram showing an experiment of Example 1, where (a) shows an outline of the experimental conditions, and (b) shows the experimental results. FIG. 3 is a diagram showing an experiment of Example 2, where (a) shows an outline of the experimental conditions, and (b) shows the experimental results. FIG. 4 is a diagram showing an experiment of Example 3, where (a) shows an outline of the experimental conditions, and (b) shows the experimental results. FIG. 5 is a diagram showing an experiment of Example 4, where (a) shows an outline of the experimental conditions, and (b) shows the experimental results. FIG. 6 is a diagram showing an experiment of Example 5, where (a) shows an outline of the experimental conditions, and (b) shows the experimental results. FIG. 7 is a diagram showing an experiment of Example 6, where (a) shows an outline of the experimental conditions, and (b) shows the experimental results. FIG. 8 is a diagram showing an experiment of Example 7, where (a) shows an outline of the experimental conditions, and (b) shows the experimental results. FIG. 9 is a diagram showing an experiment of Example 8, where (a) shows an outline of the experimental conditions and (b) shows the experimental results. FIG. 10 is a diagram showing an experiment of Example 9, where (a) shows an outline of the experimental conditions and (b) shows the experimental results. FIG. 11 is a diagram showing the experimental results of Example 10. FIG. 12 is a diagram showing the experimental results of Example 11. FIG. 13 is a diagram showing an experiment of Example 12, where (a) shows an outline of the experimental conditions and (b) shows the experimental results. FIG. 14 is a diagram showing the experimental results of Example 13. FIG. 15 is a diagram showing the experimental results of Example 13. FIG. 16 is a diagram showing the experimental results of Example 13. FIG. 17 is a diagram showing an experiment of Example 14, where (a) shows an outline of the experimental conditions and (b) shows the experimental results. FIG. 18 is a diagram showing an experiment of Example 14, where (a) shows an outline of the experimental conditions and (b) shows the experimental results. FIG. 19 is a diagram showing an experiment of Example 15, where (a) shows an outline of the experimental conditions and (b) shows the experimental results.

[0021] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The following description of preferred embodiments is merely exemplary in nature and is not intended to limit the present invention, its application, or its uses.

[0022] One embodiment of the present invention is a method for inducing differentiation of an NMJ. Specifically, this method involves culturing human pluripotent stem cells under specified conditions, thereby inducing motor neurons and skeletal muscles derived from the same spinal cord level in the same culture dish through induction into neuromesoderms, and inducing an NMJ between the motor neurons and skeletal muscles.

[0023] Specifically, in this embodiment, human pluripotent stem cells are pre-cultured in a medium containing Activin, bFGF, a WNT signaling inhibitor, a BMP signaling inhibitor, and a ROCK signaling inhibitor, and then the pluripotent stem cells are cultured in a medium containing bFGF, a ROCK signaling inhibitor, a WNT signaling activator, and a TGFβ signaling inhibitor to induce neuromesoderm. Subsequently, the neuromesoderm cells are cultured in a medium containing a BMP signaling inhibitor, a TGFβ signaling inhibitor, a retinoic acid activator, and an SHH signaling activator to induce skeletal muscle progenitor cells via mesoderm and spinal motor neuron progenitor cells via neuroectodermal cells in the same culture dish. NMJs are then induced by culturing the skeletal muscle progenitor cells and neuroectodermal cells in a cell maturation medium containing BDNF, GDNF, AA, and dbcAMP in a culture dish coated with a cell adhesion agent.

[0024] In this embodiment, the pre-culture is an adaptation culture for optimizing the human pluripotent stem cells for human developmental processes, and is a necessary pre-treatment for inducing neuromesoderm. It is known that activation of BMP signaling or WNT signaling in epiblast induces extraembryonic cells, so inhibiting these signals results in a state similar to that of human epiblast. Pre-culture is preferably performed for one day or more, for example, for one, two, or three days.

[0025] In this embodiment, the induction of cells into neuromesoderm after preculture is carried out by culturing them in a medium containing bFGF, a ROCK signal inhibitor, a WNT signal activator, and a TGFβ signal inhibitor. Neuromesoderm exhibits properties of both neuroectoderm and mesoderm, which develops posteriorly from the epiblast. Mesoderm develops via mesendoderm, which possesses properties of both mesoderm and endoderm. Therefore, inhibiting differentiation into endoderm is thought to increase the efficiency of differentiation into mesoderm. Therefore, inhibiting the TGFβ signaling required for differentiation from mesendoderm to endoderm can promote differentiation into neuromesoderm. The induction of cells into neuromesoderm after preculture is preferably carried out for two days or more, preferably for two, three, or four days. It is preferable to include the TGFβ signal inhibitor in the medium for only about half a day to one day.

[0026] In this embodiment, to induce skeletal muscle progenitor cells and spinal motor neuron progenitor cells in the same culture dish after induction into Neuromesoderm, the cells are cultured in a medium containing, in addition to the BMP signal inhibitor and TGFβ signal inhibitor, a retinoic acid activator that promotes neural differentiation, and an SHH signal activator, which is a ventralizing signal activator that promotes induction of the spinal cord, the region where spinal motor neurons originate, to the ventral side. The SHH signal activator is preferably added to the medium after the BMP signal inhibitor, TGFβ signal inhibitor, and retinoic acid activator. This promotes induction of Neuromesoderm into skeletal muscle progenitor cells and spinal motor neuron progenitor cells. The induction into skeletal muscle progenitor cells and spinal motor neuron progenitor cells is preferably carried out for 3 days or more, and preferably for 3, 4, 5, 6, 7, 8, 9, or 10 days.

[0027] In this embodiment, skeletal muscle progenitor cells and spinal motor neuron progenitor cells are cultured for a predetermined period in a culture dish coated with a predetermined cell adhesion coating, thereby promoting NMJ induction. NMJ induction is preferably carried out for 15 days or more.

[0028] In this embodiment, for example, human ES cells or iPS cells can be used as human pluripotent stem cells.

[0029] In this embodiment, Activin is contained in the culture medium at a concentration of, for example, 1 ng / ml to 20 ng / ml, preferably at a concentration of 3 ng / ml to 10 ng / ml, and particularly preferably at a concentration of 5 ng / ml.

[0030] In this embodiment, bFGF is contained in the culture medium at a concentration of, for example, 5 ng / ml to 100 ng / ml, preferably 10 ng / ml to 50 ng / ml, and particularly preferably 20 ng / ml to 30 ng / ml.

[0031] In this embodiment, the WNT signaling inhibitor may be, for example, an agent containing 4-(2-methyl-4-pyridinyl)-N-[4-(3-pyridinyl)phenyl]-benzeneacetamide or 2-[4-(trifluoromethyl)phenyl]-7,8-dihydro-5H-thiino[4,3-d]pyrimidin-4-ol. These are commercially available under the product names, for example, Wnt-C59 and XAV939, respectively. The WNT signaling inhibitor, in the case of Wnt-C59, is contained in the medium at a concentration of, for example, 1 nM to 100 nM, preferably at a concentration of 5 nM to 50 nM, and particularly preferably at a concentration of 10 nM. In the case of XAV939, it is contained in the medium at a concentration of, for example, 100 nM to 1000 nM, preferably at a concentration of 300 nM to 500 nM, and particularly preferably at a concentration of 500 nM.

[0032] In this embodiment, the BMP signal inhibitor may be, for example, an agent containing 4-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline hydrochloride. This is commercially available under the product name LDN193189, for example. The BMP signal inhibitor is contained in the medium at a concentration of, for example, 10 nM to 1000 nM, preferably 50 nM to 500 nM, and particularly preferably 100 nM.

[0033] In this embodiment, the ROCK signal inhibitor may be an agent containing (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)cyclohexanecarboxamide dihydrochloride. This is commercially available, for example, under the product name Y-27632. The ROCK signal inhibitor is contained in the medium at a concentration of, for example, 1 μM to 50 μM, preferably 2 μM to 20 μM, and particularly preferably 2 μM to 10 μM.

[0034] In this embodiment, the WNT signal activator can be an agent containing 6-[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)pyrimidin-2-yl]amino]ethylamino]pyridine-3-carbonitrile. This is commercially available under the product name CHIR99021, for example. The WNT signal activator is contained in the medium at a concentration of, for example, 1 μM to 20 μM, preferably 2 μM to 10 μM, and particularly preferably 3 μM to 5 μM.

[0035] In this embodiment, the TGFβ signaling inhibitor may be an agent containing 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide or 1H-pyrazole-1-carbothioamide, 3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl). These are commercially available under the product names, for example, SB431542 and A83-01. In the case of SB431542, the TGFβ signaling inhibitor is contained in the medium at a concentration of, for example, 1 μM to 20 μM, preferably 2 μM to 10 μM, and particularly preferably 5 μM. In the case of A83-01, it is contained in the medium at a concentration of, for example, 100 nM to 1000 nM, preferably 200 nM to 500 nM, and particularly preferably 500 nM.

[0036] In this embodiment, the retinoic acid activator can be an agent containing 4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)ethynyl]benzoic acid. This is commercially available, for example, under the product name EC23. The retinoic acid activator is contained in the medium at a concentration of, for example, 10 nM to 1000 nM, preferably 50 nM to 500 nM, and particularly preferably 100 nM.

[0037] In this embodiment, the SHH signal activator may be an agent containing 3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-[3-(4-pyridinyl)benzyl]-1-benzothiophene-2-carboxamide. This is commercially available, for example, under the product name SAG. The SHH signal activator is contained in the medium at a concentration of, for example, 5 nM to 1000 nM, preferably 15 nM to 500 nM, and particularly preferably 15 nM to 150 nM.

[0038] The medium used in this embodiment may be, for example, a basal medium consisting of the following components: DMEM / F12 (Fujifilm) supplemented with 1x N2 (Gibco), 1x GlutaMax (Gln; Invitrogen), 1x NEAA (non-essential amino acid; Invitrogen), 250 μM ascorbic acid (AA; Fujifilm), and 0.1x Penicillin & Streptomycin (P&S; Invitrogen) is used as the basal medium NIM.

[0039] In this embodiment, the cell maturation medium used in the step of inducing NMJ is, for example, a medium obtained by adding 1x B27 (without vitamin A, Gibco), 1x Gln, and 0.1x P&S to Neurobasal (Gibco) and mixing it with the basal medium NIM at a ratio of 1:1. The medium may further contain 10 ng / ml of Brain-derived neurotrophic factor (BDNF; R&D), 10 ng / ml of Glial Cell Line-derived neurotrophic factor (GDNF; R&D), 200 μM of ascorbic acid (AA), 1 μM of dibutylyl cyclic amine (DMA), and the like. This medium is prepared by adding dbcAMP (Sigma-Aldrich) and can be used as a medium for long-term culture. The medium is preferably changed, for example, every two days.

[0040] In this embodiment, the culture dish coated with a cell adhesion coating may be a culture dish coated with ornithine and laminin. Coating of these culture dishes can be performed using a method commonly used in the art, and the method is not particularly limited.

[0041] Examples are provided below to explain in detail the method for inducing NMJ differentiation according to the present invention. Specifically, in this example, we used highly differentiated human T-iPS cells (TET1-induced pluripotent stem cells) (see Japanese Patent No. 5987063) to simultaneously induce ectoderm-derived spinal motor neurons and mesoderm-derived skeletal muscle. We investigated the following conditions: 1) conditions for inducing T-iPS cells to a state similar to human epiblast (adaptation culture); 2) conditions for inducing neuromesoderm, which develops in the posterior region of the epiblast (bFGF, Activin, SB431542, LDN193189, CHIR99021); and 3) conditions for inducing motor cortex and skeletal muscle cells at the same spinal cord level from neuromesoderm in the same culture system (SB431542, LDN193189, EC23, SAG). The efficiency of Neuromesoderm induction was determined by measuring the number of cells expressing the markers SOX2 and TBXT, and the efficiency of spinal motor area induction was determined by measuring the number of cells expressing OLIG2. The figures showing each result show the percentage of cells positive for each marker.

[0042] Reference Example 1: Conventional Method for Induction into Spinal Motor Region Before showing examples of the method according to the present invention, we first attempted to induce Neuromesoderm using previously reported conditions for inducing Neuromesoderm from mouse ES / iPS cells to induce spinal motor neurons. An overview of these conditions is shown in Figure 1(a). Neuromesoderm is known to be induced from the posterior epiblast by activating FGF and WNT signals, so bFGF and CHIR99021 were used to activate FGF and WNT signals, respectively.

[0043] Specifically, on the day of induction, a basal medium (NIM) was prepared by adding 1x N2, 1x Gln, 1x NEAA, 250 μM AA, and 0.1x P&S to DMEM / F12. The medium was supplemented with 10 μM Y-27632 (Y; Fujifilm), 20 ng / ml bFGF (Peprotech), and 3 μM CHIR99021 (CHIR; Sigma-Aldrich). T-iPS cells were dissociated with Accutase (Innovative Cell Technologies) and then resuspended in phosphate buffer saline (Ca). ２＋ (-), Mg ２＋ (-)) (PBS; Fujifilm) to a culture dish coated with laminin-511 (Matrixome) diluted to 3.5 μg / ml. ５ cells / cm ２ The T-iPS cells were seeded on the coated culture dish so that the laminin-511 was diluted to 3.5 μg / ml with PBS, and the coated culture dish was then coated with laminin-511 at a concentration of approximately 0.5 to 0.6 μg / cm ２ The cells were prepared by adding 10 μM Y, 20 ng / ml bFGF, and 3 μM CHIR to NIM, and placing the cells in a culture dish at an added amount of 10 μM Y, 20 ng / ml bFGF, and 3 μM CHIR in a 37°C incubator for at least 1 hour. The coated culture dishes used in the examples described later were also prepared in the same manner. On the first and second days, the medium was replaced with a medium from which Y had been removed. On the third day, the cells were detached from the culture dish with Accutase and dissociated into single cells by pipetting. NIM was supplemented with 10 μM Y, 20 ng / ml bFGF, and 3 μM CHIR, and 1.4 × 10 cells were plated on a laminin-511-coated culture dish. ５ cells / cm ２Cells were seeded so that the media was 100%. On day 4, the media was replaced with NIM supplemented with bFGF and CHIR. On day 5, the cells were fixed with 4% formalin (PFA, Fujifilm) and analyzed. The results are shown in Figure 1(b). The analysis was performed as follows. First, a blocking agent "5% (v / v) normal donkey serum (NDS; Millipore) / 0.3% (v / v) Triton X-100 (Nacalai Tesque) / PBS (NDS-TX)" was added to the fixed sample and allowed to react at room temperature for 1 hour. Thereafter, the blocking agent was removed, and a solution of primary antibody diluted with 1% NDS-TX was added, followed by overnight reaction at 4°C. The samples were then washed with a solution (T-PBS) prepared by diluting Tween-20 (Sigma-Aldrich) in PBS to 0.05% (v / v), and a solution of secondary antibody diluted with 1% NDS-TX was added and incubated at room temperature for 2 hours. After washing with T-PBS, the samples were mounted with mounting medium (2.4 g Mowiol (Millipore), 6 ml glycerol (Fujifilm), 6 ml distilled water, 12 ml 200 mM Tris-HCl (Nacalai Tesque), and 0.66 g 1,4-diazabicycle (2.2.2) octane (Sigma-Aldrich)). The enclosed samples were photographed using a Keyence BZ-800. The same analytical method was used in the examples described later.

[0044] As shown in Figure 1(b), under the above conditions, cells were induced that expressed TBXT, a marker for neuromesoderm, but barely expressed SOX2, another marker for neuromesoderm. In other words, under these conditions, it is believed that human iPS cells were not sufficiently induced to become neuromesoderm. The fact that human neuromesoderm was not induced by the conventional induction method of this Reference Example, which was developed based on mouse development, is thought to reflect differences in the initial state and differentiation between mouse ES / iPS cells and human ES / iPS cells.

[0045] Example 1: Investigation of the Timing of Addition of Adaptation Culture Reagents Based on the results of the above Reference Example, we conceived the need to induce neuromesoderm after subjecting T-iPS cells cultured in an artificial environment to adaptive culture to optimize their developmental state. Experiments using monkeys (Sasaki et al., Developmental Cell, 2016) and our previous research have shown that activating BMP and WNT signals in the epiblast induces extraembryonic cells. Therefore, we attempted to induce neuromesoderm by inhibiting these signals to simulate a state similar to that of the human epiblast. The conditions are outlined in Figure 2(a). To bring human iPS cells into a state close to Epiblast, we investigated the timing of adding bFGF, Activin, and the WNT signal inhibitor WNT-C59 (C59; Cellagen Technology) and the BMP signal inhibitor LDN193189 (LDN; Sigma-Aldrich) to the culture medium.

[0046] Specifically, on the day of induction, 1 × 10 cells were cultured in a culture dish coated with laminin-511 diluted to 3.5 μg / ml with PBS using a medium containing 10 μM Y, 10 nM C59, 5 ng / ml Activin, 100 nM LDN, and 20 ng / ml bFGF in basal medium NIM. ５ cells / cm ２T-iPS cells were seeded so that the total volume of the cells was 100 μm. The seeded cells were then divided into three groups and cultured under the following three conditions, Conditions 1 to 3, from Day 1 to Day 4 after seeding. (Condition 1) On Day 1, the medium was replaced with NIM supplemented with bFGF. From Day 2, the medium was replaced with bFGF and 3 μM CHIR. (Condition 2) On Day 1, the medium was replaced with NIM supplemented with C59, Activin, LDN, and bFGF. On Day 2, the medium was replaced with NIM supplemented with bFGF. From Day 3, the medium was replaced with bFGF and CHIR. (Condition 3) On Day 1, the medium was replaced with NIM supplemented with C59, Activin, LDN, and bFGF, and this medium was used again on Day 2. On Day 3, the medium was replaced with NIM supplemented with bFGF. From the fourth day onwards, the medium was replaced with one supplemented with bFGF and CHIR.

[0047] On day 4 under each condition, cells were detached from the culture dish with Accutase and dissociated into single cells by pipetting. NIM was supplemented with bFGF, CHIR, and 10 μM Y, and 1.2 × 10 cells were plated on a culture dish coated with laminin-511. ５ cells / cm ２ The cells were seeded so that the media was 100% NIM. On day 5, the media was replaced with NIM supplemented with bFGF and CHIR. On day 6, the cells were fixed with 4% PFA and analyzed. The results are shown in Figure 2(b).

[0048] As shown in Figure 2(b), cells co-expressing the Neuromesoderm markers SOX2 and TBXT were induced under all of the above conditions 1 to 3, but it is believed that condition 2 is particularly effective in inducing Neuromesoderm.

[0049] Example 2: Examination of the Timing of Addition of Adapted Culture Reagents and TGFβ Signaling Inhibitor SB431542 (1) Neuromesoderm is derived from epiblast and exhibits properties of both neuroectoderm and mesoderm. Because mesoderm is derived via mesencoderm, which possesses properties of both mesoderm and endoderm, it is thought that inhibiting differentiation into endoderm may increase the efficiency of mesoderm differentiation. Therefore, we investigated the addition of SB431542 (SB; Sigma-Aldrich), which inhibits TGFβ signaling, necessary for differentiation of mesencoderm into endoderm. Specifically, we investigated the timing of addition of adapted culture reagents (Y, C59, Activin, LDN) and the TGFβ signaling inhibitor SB, which inhibits differentiation into endoderm. An overview of the conditions is shown in Figure 3(a).

[0050] Specifically, on the day of induction, 1 × 10 cells were cultured in a culture dish coated with laminin-511 diluted to 3.5 μg / ml with PBS using a medium containing 10 μM Y, 10 nM C59, 5 ng / ml Activin, 100 nM LDN, and 100 ng / ml bFGF in basal medium NIM. ５ cells / cm ２ iPS cells were seeded onto the medium so that the total volume of the medium was 100 μM. On day 1, the medium was replaced with NIM supplemented with 2 μM Y, 10 nM C59, 5 ng / ml Activin, 100 nM LDN, and 100 ng / ml bFGF. The seeded cells were then divided into two groups, and on days 2 and 3 after seeding, they were cultured under the following two conditions, Conditions 1 and 2. (Condition 1) On day 2, the medium was replaced with NIM supplemented with 3 μM CHIR, 100 ng / ml bFGF, and 5 μM SB. On day 3, the medium was replaced with NIM supplemented with 3 μM CHIR and 100 ng / ml bFGF. (Condition 2) On day 2, the medium was replaced with NIM supplemented with 2 μM Y, 10 nM C59, 5 ng / ml Activin, 100 nM LDN, and 100 ng / ml bFGF. On day 3, the medium was replaced with NIM supplemented with 3 μM CHIR, 100 ng / ml bFGF, and 5 μM SB.

[0051] On day 4 under both conditions, cells were detached from the culture dish with Accutase and dissociated into single cells by pipetting. NIM was supplemented with bFGF, CHIR, and 10 μM Y, and 1.2 × 10 cells were plated on a culture dish coated with laminin-511. ５ cells / cm ２ The cells were seeded so that the media was 100% NIM. On day 5, the media was replaced with NIM supplemented with bFGF and CHIR. On day 6, the cells were fixed with 4% PFA and analyzed. The results are shown in Figure 3(b).

[0052] As shown in FIG. 3(b), both conditions showed similar Neuromesoderm induction efficiency, demonstrating that Neuromesoderm could be induced more efficiently than the condition of Example 1 in which SB was not added.

[0053] Example 3: Examination of the timing of addition of adaptive culture reagents and TGFβ signal inhibitor SB (2) Early development progresses moment by moment, and there is an optimal timing for external induction factors to function. Therefore, in order to more efficiently induce Neuromesoderm, the conditions for the timing of reagent addition were carefully examined. Specifically, the timing of addition of adaptive culture reagents and TGFβ signal inhibitor SB431542 was carefully examined. An overview of the conditions is shown in Figure 4(a).

[0054] Specifically, on the day of induction, 1 × 10 cells were cultured in a culture dish coated with laminin-511 diluted to 3.5 μg / ml with PBS using a medium containing 10 μM Y, 10 nM C59, 5 ng / ml Activin, 100 nM LDN, and 20 ng / ml bFGF in basal medium NIM. ５ cells / cm ２iPS cells were seeded so that the cells were 100% pure. The seeded cells were then divided into four groups, and on days 1 to 3 after seeding, they were cultured under the following four conditions, Conditions 1 to 4. (Condition 1) On the morning of Day 1, the medium was replaced with NIM supplemented with 3 μM CHIR, 5 μM SB, and 20 ng / ml bFGF. 12 hours later, the medium was replaced with NIM supplemented with 3 μM CHIR and 20 ng / ml bFGF. From Day 2 onwards, the medium was replaced with NIM supplemented with 3 μM CHIR and 20 ng / ml bFGF. (Condition 2) On the morning of Day 1, the medium was replaced with NIM supplemented with 2 μM Y, 10 nM C59, 5 ng / ml Activin, 100 nM LDN, and 20 ng / ml bFGF. After 12 hours, the medium was replaced with NIM supplemented with 3 μM CHIR, 5 μM SB, and 20 ng / ml bFGF. From day 2 onwards, the medium was replaced with NIM supplemented with 3 μM CHIR and 20 ng / ml bFGF. (Condition 3) On the morning of day 1, the medium was replaced with NIM supplemented with 2 μM Y, 10 nM C59, 5 ng / ml Activin, 100 nM LDN, and 20 ng / ml bFGF. On the morning of day 2, the medium was replaced with NIM supplemented with 3 μM CHIR, 5 μM SB, and 20 ng / ml bFGF. After 12 hours, the medium was replaced with NIM supplemented with 3 μM CHIR and 20 ng / ml bFGF. From day 3 onwards, the medium was replaced with NIM supplemented with 3 μM CHIR and 20 ng / ml bFGF. (Condition 4) On the morning of day 1, the medium was replaced with NIM supplemented with 2 μM Y, 10 nM C59, 5 ng / ml Activin, 100 nM LDN, and 20 ng / ml bFGF. On the morning of day 2, the medium was replaced with NIM supplemented with 2 μM Y, 10 nM C59, 5 ng / ml Activin, 100 nM LDN, and 20 ng / ml bFGF. 12 hours later, the medium was replaced with NIM supplemented with 3 μM CHIR, 5 μM SB, and 20 ng / ml bFGF. From day 3 onwards, the medium was replaced with NIM supplemented with 3 μM CHIR and 20 ng / ml bFGF.

[0055] On day 3 under all conditions, cells were detached from the culture dish with Accutase and dissociated into single cells by pipetting. NIM was supplemented with bFGF, CHIR, and 10 μM Y, and 1.4 × 10 cells were plated on a culture dish coated with laminin-511. ５ cells / cm ２ The cells were seeded so that the media was 100%. On day 4, the media was replaced with NIM supplemented with bFGF and CHIR. On day 5, the cells were fixed with 4% PFA and analyzed. The results are shown in Figure 4(b).

[0056] As shown in FIG. 4(b), among the above conditions 1 to 4, condition 4 was able to induce Neuromesoderm most efficiently.

[0057] Example 4: Concentration study of CHIR, a small molecule compound that activates WNT signaling. Because each differentiation-inducing factor has an optimal activation state, it is necessary to determine the optimal concentration of each compound or recombinant protein used. Therefore, we investigated the concentration of CHIR, a small molecule compound that activates WNT signaling. Figure 5(a) shows an overview of the conditions.

[0058] Specifically, on the first day of induction, a medium containing 1x B27, 10 μM Y, 10 nM C59, 100 nM LDN, 3 ng / ml Activin, and 100 ng / ml bFGF was used. 1x10 cells were placed on a culture dish coated with laminin-511 diluted to 3.5 μg / ml with PBS. ５ cells / cm ２ iPS cells were seeded at 1.5 x 10 cells / well. The next day, the medium was replaced with NIM supplemented with bFGF. On day 2, the cells were divided into three groups, and CHIR was added to the medium of each group at concentrations of 1 μM, 2 μM, or 3 μM. On day 3, the cells were detached from the culture dish with Accutase and dissociated into single cells by pipetting. NIM was supplemented with 100 ng / ml bFGF, 10 μM Y, and various concentrations of CHIR, and 1.5 x 10 cells / well were plated on a laminin-511-coated culture dish. ５ cells / cm ２On day 4, the medium was replaced with NIM supplemented with bFGF and various concentrations of CHIR. On day 5, the cells were fixed with 4% PFA and analyzed. The results are shown in Figure 5(b).

[0059] As shown in FIG. 5(b), the induction efficiency was highest under the condition using 3 μM CHIR.

[0060] Example 5: Study on bFGF concentration Next, the optimum concentration of bFGF was studied. Figure 6(a) shows an outline of the conditions.

[0061] Specifically, on the first day of induction, a medium containing 10 μM Y, 10 nM C59, and 3 ng / ml Activin was used. bFGF was added to this medium to a concentration of 25 ng / ml, 50 ng / ml, or 100 ng / ml. 1.2 × 10 cells were placed on a culture dish coated with laminin-511 diluted to 3.5 μg / ml with PBS. ５ cells / cm ２ iPS cells were seeded at a density of 1.5 × 10 cells / well. The next day, the medium was replaced with NIM supplemented with various concentrations of bFGF. From the second day, the medium was replaced with 3 μM CHIR supplemented. On the third day, the cells were detached from the culture dish with Accutase and dissociated into single cells by pipetting. NIM was supplemented with 3 μM CHIR, 10 μM Y, and various concentrations of bFGF, and the cells were plated at a density of 1.5 × 10 cells / well on a laminin-511-coated culture dish. ５ cells / cm ２ On day 4, the medium was replaced with NIM supplemented with CHIR and various concentrations of bFGF. On day 5, the cells were fixed with 4% PFA and analyzed. The results are shown in Figure 6(b).

[0062] As shown in FIG. 6(b), the induction efficiency was highest when 25 ng / ml of bFGF was used.

[0063] Example 6: Examination of Activin concentration Next, the optimum concentration of Activin was examined. Figure 7(a) shows an outline of the conditions.

[0064] Specifically, on the day of induction, Activin was added to the basal medium NIM supplemented with 10 μM Y, 10 nM C59, 100 nM LDN, and 20 ng / ml bFGF at 0, 3, or 5 ng / ml. 1.0 × 10 cells were placed on a culture dish coated with laminin-511 diluted to 3.5 μg / ml with PBS. ５ cells / cm ２ iPS cells were seeded at 100 μM. The next day, 2 nM Y, 10 nM C59, 100 nM LDN, and 20 ng / ml bFGF were added to NIM, and the medium was replaced with one containing various concentrations of Activin. On the morning of the third day, the medium was replaced with the medium from day 2 minus Y, and 12 hours later, the medium was replaced with NIM supplemented with 10 nM C59, 20 ng / ml bFGF, 3 μM CHIR, and 5 μM SB. On day 3, the cells were detached from the culture dish with Accutase and dissociated into single cells by pipetting. 3 μM CHIR, 10 μM Y, and 20 ng / ml bFGF were added to NIM, and 1.5 x 10 cells were plated on a laminin-511-coated culture dish. ５ cells / cm ２ On day 4, the medium was replaced with NIM supplemented with CHIR and bFGF. On day 5, the cells were fixed with 4% PFA and analyzed. The results are shown in Figure 7(b).

[0065] As shown in FIG. 7(b), the induction efficiency was highest under the condition of using 5 ng / ml of Activin.

[0066] Example 7: Study of WNT signaling inhibitors and TGFβ signaling inhibitors Next, to examine whether there is a difference in induction efficiency depending on the agent used for induction, the induction efficiency was evaluated when XAV939 or C59 was used as the WNT signaling inhibitor and A83-01 was used as the TGFβ signaling inhibitor. An outline of the conditions is shown in Figure 8(a).

[0067] Specifically, on the day of induction, 1 × 10 cells were cultured in a culture dish coated with laminin-511 diluted to 3.5 μg / ml with PBS using a medium containing 10 μM Y, 10 nM C59 or 500 nM XAV939 (Tocris), 5 ng / ml Activin, 100 nM LDN, and 20 ng / ml bFGF. ５ cells / cm ２ iPS cells were seeded onto the plate at 100°C. On day 1, the medium was replaced with NIM supplemented with 2 μM Y, 10 nM C59 or 500 nM XAV939, 5 ng / ml Activin, 100 nM LDN, and 20 ng / ml bFGF. On the morning of day 2, the medium was replaced with NIM supplemented with 2 μM Y, 10 nM C59 or 500 nM XAV939, 5 ng / ml Activin, 100 nM LDN, and 20 ng / ml bFGF. 12 hours later, the medium was replaced with NIM supplemented with 3 μM CHIR, 500 nM A83-01, and 20 ng / ml bFGF. On day 3, the cells were detached from the culture dish using Accutase and dissociated into single cells by pipetting. NIM was supplemented with bFGF, CHIR, and 10 μM Y, and 1.4 × 10 cells were cultured on a laminin-511-coated culture dish. ５ cells / cm ２ The cells were seeded so that the media was 100% NIM. On day 4, the media was replaced with NIM supplemented with bFGF and CHIR. On day 5, the cells were fixed with 4% PFA and analyzed. The results are shown in Figure 8(b).

[0068] As shown in FIG. 8(b), Neuromesoderm could be efficiently induced even when XAV939 was used as the WNT signal inhibitor and A83-01 was used as the TGFβ signal inhibitor.

[0069] Example 8: Examination of the Nervous System and Timing of Ventralization Induction Based on the results of each of the above experiments, we investigated the induction of ventralization, i.e., induction into the spinal motor region, using conditions that provided good induction efficiency of Neuromesoderm (SOX2-positive and TBXT-positive) on day 5 of induction. Figure 9(a) shows an overview of these conditions. In particular, we investigated the timing of addition of reagents for inducing Neuromesoderm into the nervous system (TGFβ signal inhibitor SB, BMP signal inhibitor LDN, and small molecule compound EC23 (Sigma-Aldrich) with retinoic acid activity) and the timing of addition of a reagent for inducing ventralization (SHH signal-activating small molecule compound SAG (ENZO Life Sciences)). To confirm whether induction had occurred, we used OLIG2 (red), a marker for the region from which motor neurons in the spinal cord originate, and SOX2 (green), a marker for the nervous system.

[0070] On the day of induction, 1 × 10 cells were cultured in a culture dish coated with laminin-511 diluted to 3.5 μg / ml with PBS using a medium containing 10 μM Y, 10 nM C59, 5 ng / ml Activin, 100 nM LDN, and 20 ng / ml bFGF in basal medium NIM. ５ cells / cm ２ iPS cells were seeded at 100 μM. On day 1, the medium was replaced with NIM supplemented with 2 μM Y, 10 nM C59, 5 ng / ml Activin, 100 nM LDN, and 20 ng / ml bFGF. On the morning of day 2, the medium was replaced with NIM supplemented with 10 nM C59, 5 ng / ml Activin, 100 nM LDN, and 20 ng / ml bFGF. 12 hours later, the medium was replaced with NIM supplemented with 20 ng / ml bFGF, 3 μM CHIR, and 5 μM SB. On day 3, the cells were detached from the culture dish with Accutase and dissociated by pipetting to form single cells. NIM supplemented with bFGF, CHIR, and 10 μM Y was plated at 1.4 x 10 ５ cells / cm ２On days 4 and 5, the medium was replaced with NIM supplemented with bFGF and CHIR. On day 6, the cells were detached from the culture dish with Accutase and dissociated into single cells by pipetting. NIM supplemented with bFGF, CHIR, and 10 μM Y were plated at 1.5 × 10 cells / well on a laminin-511-coated culture dish. ５ cells / cm ２ Cells were seeded so that the total volume of the cells was 100 μm. The seeded cells were then divided into four groups, and from day 7 onwards, they were cultured under the following four conditions, Conditions 1 to 4. (Condition 1) From day 7 onwards, the medium was replaced with NIM supplemented with 5 μM SB, 100 nM LDN, and 100 nM EC23. On day 10, the medium was further replaced with 150 nM SAG. (Condition 2) On day 7, the medium was replaced with NIM supplemented with 20 ng / ml bFGF and 3 μM CHIR. On day 8, the medium was replaced with NIM supplemented with 5 μM SB, 100 nM LDN, and 100 nM EC23. On day 11, the medium was further replaced with 150 nM SAG. (Condition 3) On days 7 and 8, the medium was replaced with NIM supplemented with 20 ng / ml bFGF and 3 μM CHIR. On day 9, the medium was replaced with NIM supplemented with 5 μM SB, 100 nM LDN, and 100 nM EC23. On day 12, the medium was replaced with NIM supplemented with 150 nM SAG. (Condition 4) From days 7 to 10, the medium was replaced with NIM supplemented with 20 ng / ml bFGF and 3 μM CHIR. On day 11, the medium was replaced with NIM supplemented with 5 μM SB, 100 nM LDN, and 100 nM EC23. On day 12, the medium was replaced with NIM supplemented with 150 nM SAG.

[0071] On day 13 under all conditions 1 to 4, the cells were detached from the culture dish with Accutase and dissociated into single cells by pipetting. ４Cells were seeded at 100 cells / well. The medium used was NIM supplemented with 5 μM SB, 100 nM LDN, 100 nM EC23, and 150 nM SAG. On day 18, the cells were fixed with 4% PFA and analyzed. The results are shown in Figure 9(b).

[0072] Figure 9(b) shows OLIG2 (red), a marker of the area where motor neurons develop (spinal motor area), and SOX2 (green), a marker of the nervous system. As shown in Figure 9(b), OLIG2 was efficiently induced under all conditions 1 to 4, with no significant differences observed.

[0073] Example 9: Study on concentration of ventralization signal Next, the optimum concentration of SAG for activating the ventralization signal was studied. Figure 10(a) shows an outline of the conditions.

[0074] On the day of induction, 1 × 10 cells were cultured in a culture dish coated with laminin-511 diluted to 3.5 μg / ml with PBS using a medium containing 10 μM Y, 10 nM C59, 5 ng / ml Activin, 100 nM LDN, and 20 ng / ml bFGF in basal medium NIM. ５ cells / cm ２ iPS cells were seeded at 100 μM. On day 1, the medium was replaced with NIM supplemented with 2 μM Y, 10 nM C59, 5 ng / ml Activin, 100 nM LDN, and 20 ng / ml bFGF. On the morning of day 2, the medium was replaced with NIM supplemented with 10 nM C59, 5 ng / ml Activin, 100 nM LDN, and 20 ng / ml bFGF. 12 hours later, the medium was replaced with NIM supplemented with 20 ng / ml bFGF, 3 μM CHIR, and 5 μM SB. On day 3, the cells were detached from the culture dish with Accutase and dissociated by pipetting to form single cells. NIM supplemented with bFGF, CHIR, and 10 μM Y was plated at 1.4 x 10 ５ cells / cm ２On days 4 and 5, the medium was replaced with NIM supplemented with bFGF and CHIR. On day 6, the cells were detached from the culture dish with Accutase and dissociated into single cells by pipetting. NIM supplemented with bFGF, CHIR, and 10 μM Y were plated at 1.5 × 10 cells / well on a laminin-511-coated culture dish. ５ cells / cm ２ On days 7 and 8, the medium was replaced with NIM supplemented with 20 ng / ml bFGF and 3 μM CHIR. On day 9, the medium was replaced with NIM supplemented with 5 μM SB, 100 nM LDN, and 100 nM EC23. On day 12, the medium was replaced with medium supplemented with 0 nM, 10 nM, 150 nM, and 500 nM SAG. On day 13, the cells were detached from the culture dish with Accutase and dissociated into single cells by pipetting. 1 x 10 cells were placed in a 96-well round-bottom plate. ４ Cells were seeded at 100 cells / well. The medium used was NIM supplemented with 5 μM SB, 100 nM LDN, 100 nM EC23, and various concentrations of SAG. On day 18, the cells were fixed with 4% PFA and analyzed. The results are shown in Figure 10(b).

[0075] As shown in Figure 10(b), OLIG2-expressing cells were observed under conditions other than 0 nM SAG. Furthermore, expression of HOXC9, a marker for the thoracic spinal cord region, was confirmed.

[0076] Example 10: Long-term culture On the 18th day of the differentiation induction performed in Example 9 above, the cell aggregates induced at each SAG concentration were cultured at 0.5 μg / cm ２ ornithine (Sigma-Aldrich) and 0.4 μg / cm ２Long-term culture was performed by adhering the cells to culture dishes coated with laminin (Matrixome). The coated culture dishes were prepared by diluting ornithine with Milli-Q water to a 15 μg / mL ornithine solution, adding 300 μl per well to a 24-well plate, and incubating at room temperature for one day. After washing the wells three times with PBS, the laminin solution was added and the dish was then incubated at 37°C for one day. Neurobasal (Gibco) was supplemented with 1x B27 (without vitamin A, Gibco), 1x Gln, and 0.1x P&S, and mixed 1:1 with basal medium NIM to prepare a medium. This medium was supplemented with 10 ng / mL BDNF, 10 ng / mL GDNF, 200 μM AA, and 1 μM dbcAMP, and used as the medium for long-term culture. The medium was changed every two days. The resulting cell masses were then observed under a microscope on day 36 after the start of differentiation induction (day 18 after the start of adhesion culture on the coated culture dish). The results are shown in Figure 11.

[0077] As a result of the observation, no cells exhibiting muscle contraction were observed in the cell masses induced with 0 nM SAG, whereas moving cells exhibiting muscle contraction were observed under other conditions.

[0078] Example 11: Study of coating conditions for culture dish Next, the coating conditions for culture dish for adhering cell clusters were studied. Cell clusters induced with 150 nM SAG on the 18th day of differentiation induction in Example 9 above were used, and 0.5 μg / cm ２ of ornithine and 0.4 μg / cm ２ The culture dishes were coated with 0.5 μg / cm laminin and 0.5 μg / cm ２ of ornithine and 0.4 μg / cm ２ of laminin and 150 μg / cm ２ Two conditions were examined: one in which the culture dish was coated with fibronectin, and the other in which the culture dish was coated with fibronectin. The results of microscopic observation 42 days or later from the start of induction are shown in FIG.

[0079] As a result of the observations, under both conditions, cell clusters were able to adhere and be cultured, and cells that contracted into muscles were observed.

[0080] Example 12: Investigation of Conditions for Posteriorization Next, we investigated the conditions for guiding the spinal cord to the lumbar or sacral spinal cord. It is known that there is a concentration gradient of FGF in the posterior part of the anterior-posterior axis of the spinal cord, with a higher gradient in the posterior part. Therefore, we attempted to induce the spinal cord by varying the concentration of FGF. Figure 13(a) shows an overview of the conditions.

[0081] On the day of induction, 1 × 10 cells were cultured in a culture dish coated with laminin-511 diluted to 3.5 μg / ml with PBS using a medium containing 10 μM Y, 10 nM C59, 5 ng / ml Activin, 100 nM LDN, and 20 ng / ml bFGF in basal medium NIM. ５ cells / cm ２ iPS cells were seeded at 100 μM. On day 1, the medium was replaced with NIM supplemented with 2 μM Y, 10 nM C59, 5 ng / ml Activin, 100 nM LDN, and 20 ng / ml bFGF. On the morning of day 2, the medium was replaced with NIM supplemented with 10 nM C59, 5 ng / ml Activin, 100 nM LDN, and 20 ng / ml bFGF. 12 hours later, the medium was replaced with NIM supplemented with 20 ng / ml bFGF, 3 μM CHIR, and 5 μM SB. On day 3, the cells were detached from the culture dish with Accutase and dissociated by pipetting to form single cells. NIM supplemented with bFGF, CHIR, and 10 μM Y was plated at 1.4 x 10 ５ cells / cm ２ On days 4 and 5, the medium was replaced with NIM supplemented with bFGF and CHIR. On day 6, the cells were detached from the culture dish with Accutase and dissociated into single cells by pipetting. NIM supplemented with bFGF, CHIR, and 10 μM Y were plated at 1.5 × 10 cells / well on a laminin-511-coated culture dish. ５ cells / cm ２Cells were seeded at 1000 x g / cm². On days 7 and 8, the medium was replaced with NIM supplemented with 20 ng / ml bFGF and 3 μM CHIR. From day 9, 3 μM CHIR and 40 ng / ml Growth Differentiation Factor 11 (GDF11; R&D) were added to NIM, and 20 ng / ml, 100 ng / ml, or 500 ng / ml bFGF was added, respectively, and the medium was replaced daily. Sampling was performed every other day, and the expression level of the HOX gene was examined by quantitative PCR. HOXA11, HOXC11, and HOXD11, as well as HOXC12 and HOXD12, are lumbar spinal cord markers, and HOXA13, HOXB13, HOXC13, and HOXD13 are sacral spinal cord markers. The results are shown in FIG.

[0082] As shown in the respective graphs in FIG. 13(b), it was confirmed that increasing the concentration of bFGF increased the expression at the lumbar spinal cord level (HOX11-12) and the sacral spinal cord level (HOX13).

[0083] Example 13: Confirmation of Skeletal Muscle Induction In the examples described above, although the fluorescence intensity varied by varying the concentration and timing of the addition of each reagent, we were successful in inducing cells co-expressing SOX2 and TBXT, markers of neuromesoderm. Furthermore, in order to induce the motor region, where spinal motor neurons originate, from the neuromesoderm, we investigated the conditions for ventralization. As a result, the number of OLIG2-positive cells, a marker of the motor region where motor neurons originate, varied depending on the conditions, and at this stage, we have obtained conditions that allow induction with an efficiency of over 90%. Furthermore, by varying the concentration of bFGF, we obtained conditions that increased the expression of HOX11-13, a marker for the region posterior to the cervical spinal cord. When these cells were cultured for a long period under ventralization conditions, elongated cell morphology and multinucleated cells believed to be skeletal muscle, as well as moving cells believed to be undergoing muscle contraction, were observed. Specifically, under the 150 nM SAG conditions shown in Figure 10, 0.5 μg / cm ２ of ornithine and 0.4 μg / cm ２ of laminin and 150 μg / cm ２A single cell cluster was placed in a 24-well plate coated with fibronectin (Figure 14). Long-term culture was performed. The medium used from day 18 onward was the long-term culture medium described in Example 10. Medium was replaced every two days. On day 50, cells were fixed with 4% PFA and analyzed. Cells stained with myosin heavy chain (MHC), a skeletal muscle marker, were observed (Figures 14, 15, and 16). Some cells also exhibited morphological striations characteristic of skeletal muscle (Figure 16). It was confirmed that these cells expressed acetylcholine receptors (Figure 14, Bungarotoxin), and that movement ceased when exposed to tubocurarine, a drug that paralyzes skeletal muscle (not shown). It was confirmed that the cells in this culture system expressed the neuronal marker Tubulin beta III (TUJ1) and ISL LIM Homeobox 1 (ISL1), which is a property of motor neurons (FIG. 15).

[0084] Example 14: Induction of NMJ using other iPS cell lines 1 Next, we investigated the induction of NMJ when using cell lines different from the iPS cell lines initially used in the previous Examples (6T9 line (produced from female fibroblasts under the same conditions as the T-iPS cells described above) and 409B2 line (obtained from the RIKEN cell bank)). Compared to the cell lines used in the induction investigations in the above Examples, the cell size and cell proliferation after the initiation of differentiation were different, so the number of cells seeded on the first day was changed. Furthermore, because the responsiveness to small molecule compounds was different, induction was performed by changing the concentration of CHIR. An overview of the conditions used is shown in Figure 17(a) (6T9 line) and Figure 18(a) (409B2 line).

[0085] On the day of induction, 6-8 × 10 cells were cultured in a culture dish coated with laminin-511 diluted to 3.5 μg / ml with PBS using a medium containing 10 μM Y, 10 nM C59, 5 ng / ml Activin, 100 nM LDN, and 20 ng / ml bFGF. ４ cells / cm ２iPS cells were seeded at 100 μM. On day 1, the medium was replaced with NIM supplemented with 2 μM Y, 10 nM C59, 5 ng / ml Activin, 100 nM LDN, and 20 ng / ml bFGF. On the morning of day 2, the medium was replaced with NIM supplemented with 10 nM C59, 5 ng / ml Activin, 100 nM LDN, and 20 ng / ml bFGF. 12 hours later, the medium was replaced with NIM supplemented with 20 ng / ml bFGF, 3-5 μM CHIR, and 5 μM SB. On day 3, the cells were detached from the culture dish with Accutase and dissociated into single cells by pipetting. NIM was supplemented with 20 ng / ml bFGF, 3-5 μM CHIR, and 10 μM Y, and 1.2 × 10 cells were cultured on a laminin-511-coated culture dish. ５ cells / cm ２ On days 4 and 5, the medium was replaced with NIM supplemented with 20 ng / ml bFGF and 3-5 μM CHIR. On day 6, the cells were detached from the culture dish with Accutase and dissociated into single cells by pipetting. NIM supplemented with 20 ng / ml bFGF, 3-5 μM CHIR, and 10 μM Y was used to seed the cells at a density of 1.5 × 10 cells onto a laminin-511-coated culture dish. ５ cells / cm ２ On days 7 and 8, the medium was replaced with NIM supplemented with 20 ng / ml bFGF and 3-5 μM CHIR. On day 9, the medium was replaced with NIM supplemented with 5 μM SB, 100 nM LDN, and 100 nM EC23. On day 12, the medium was replaced with 15 nM SAG. On day 13, the cells were detached from the culture dish with Accutase and dissociated into single cells by pipetting. 1 x 10 cells were placed in a 96-well round-bottom plate. ４Cells were seeded at 100 cells / well. The medium used was NIM supplemented with 5 μM SB, 100 nM LDN, 100 nM EC23, 150 nM SAG, and 10 μM Y. On day 15, the medium was replaced with NIM supplemented with 100 nM LDN, 100 nM EC23, and 150 nM SAG. On day 18, the cells were fixed with 4% PFA and analyzed. The results are shown in Figure 17(b) and Figure 18(b).

[0086] As shown in Figure 17(b) and Figure 18(b), it was confirmed that OLIG2 in the motor neuron region and HOXC9, a marker at the thoracic spinal cord level, were expressed in all cell lines.

[0087] On the 18th day after differentiation induction, the induced cell mass was cultured at 0.5 μg / cm ２ of ornithine and 0.4 μg / cm ２ of laminin and 150 μg / cm ２ The morphology of cell clusters was examined when cells were cultured on fibronectin-coated culture dishes. Neurobasal was supplemented with 1x B27, 1x Gln, and 0.1x P&S, and mixed 1:1 with NIM basal medium to prepare a medium. This medium was supplemented with 10 ng / ml BDNF, 10 ng / ml GDNF, 200 μM AA, and 1 μM dbcAMP for long-term culture. Medium changes were performed every two days. Moving cells exhibiting muscle contractions were observed, but the number of moving cells varied depending on the strain.

[0088] Example 15: Induction of NMJ using other iPS cell lines 2 Next, we investigated the induction of NMJ when using a cell line (6C6 line (produced using only Yamanaka factors from the same female fibroblasts as 6T9)) that is different from the iPS cell lines initially used in the previous Examples and the cell line used in Example 14 above. An overview of the conditions is shown in Figure 19(a).

[0089] On the day of induction, 1 × 10 cells were cultured in a culture dish coated with laminin-511 diluted to 3.5 μg / ml with PBS using a medium containing 10 μM Y, 10 nM C59, 5 ng / ml Activin, 100 nM LDN, and 20 ng / ml bFGF. ５ cells / cm２ iPS cells were seeded at 100 μM. On day 1, the medium was replaced with NIM supplemented with 2 μM Y, 10 nM C59, 5 ng / ml Activin, 100 nM LDN, and 20 ng / ml bFGF. On the morning of day 2, the medium was replaced with NIM supplemented with 10 nM C59, 5 ng / ml Activin, 100 nM LDN, and 20 ng / ml bFGF. 12 hours later, the medium was replaced with NIM supplemented with 20 ng / ml bFGF, 3-5 μM CHIR, and 5 μM SB. On day 3, the cells were detached from the culture dish with Accutase and dissociated into single cells by pipetting. NIM was supplemented with 20 ng / ml bFGF, 3-5 μM CHIR, and 10 μM Y, and 1.4 × 10 cells were cultured on a laminin-511-coated culture dish. ５ cells / cm ２ On days 4 and 5, the medium was replaced with NIM supplemented with 20 ng / ml bFGF and 3-5 μM CHIR. On day 6, the cells were detached from the culture dish with Accutase and dissociated into single cells by pipetting. NIM supplemented with 20 ng / ml bFGF, 3-5 μM CHIR, and 10 μM Y was used to seed the cells at a density of 1.5 × 10 cells onto a laminin-511-coated culture dish. ５ cells / cm ２ On days 7 and 8, the medium was replaced with NIM supplemented with 20 ng / ml bFGF and 3-5 μM CHIR. On day 9, the medium was replaced with NIM supplemented with 5 μM SB, 100 nM LDN, and 100 nM EC23. On day 12, the medium was replaced with 15 nM SAG. On day 13, the cells were detached from the culture dish with Accutase and dissociated into single cells by pipetting. 1 x 10 cells were placed in a 96-well round-bottom plate. ４Cells were seeded at 100 cells / well. The medium used was NIM supplemented with 5 μM SB, 100 nM LDN, 100 nM EC23, 150 nM SAG, and 10 μM Y. On day 15, the medium was replaced with NIM supplemented with 100 nM LDN, 100 nM EC23, and 150 nM SAG. On day 18, the cells were fixed with 4% PFA and analyzed. The results are shown in Figure 19(b).

[0090] As shown in FIG. 19(b), it was confirmed that OLIG2 in the motor neuron region and HOXC9, a marker at the thoracic spinal cord level, were also expressed in the 6C6 line.

[0091] On the 18th day after differentiation induction, the induced cell mass was cultured at 0.5 μg / cm ２ of ornithine and 0.4 μg / cm ２ of laminin and 150 μg / cm ２ The morphology of cell clusters was observed when cells were cultured on a fibronectin-coated culture dish. Neurobasal was supplemented with 1x B27, 1x Gln, and 0.1x P&S, and mixed 1:1 with basal medium NIM to prepare a medium. This medium was supplemented with 10 ng / ml BDNF, 10 ng / ml GDNF, 200 μM AA, and 1 μM dbcAMP for use as a long-term culture medium. Medium changes were performed every two days. Moving cells exhibiting muscle contractions were observed.

[0092] As described above, according to the method for inducing NMJ differentiation of the present invention, by culturing human pluripotent stem cells under the above conditions, it is possible to induce neuromesoderms, and further by culturing the neuromesoderms under the above conditions, it is possible to induce motor neurons and skeletal muscles derived from the same spinal cord level in the same culture dish, and it is possible to induce NMJs between the motor neurons and skeletal muscles.

Claims

1. A method for inducing differentiation of a neuromuscular junction, comprising the steps of: preparing human pluripotent stem cells and pre-culturing the pluripotent stem cells in a medium containing activin, bFGF, a WNT signaling inhibitor, a BMP signaling inhibitor, and a ROCK signaling inhibitor; culturing the pre-cultured pluripotent stem cells in a medium containing bFGF, a ROCK signaling inhibitor, a WNT signaling activator, and a TGFβ signaling inhibitor to induce them into neuromesodermal cells; culturing the induced neuromesodermal cells in a cell maturation medium containing a BMP signaling inhibitor, a TGFβ signaling inhibitor, a retinoic acid activator, and an SHH signaling activator to induce skeletal muscle progenitor cells via the mesoderm and spinal motor neuron progenitor cells via the neuroectodermal cells in the same culture dish; and culturing the induced skeletal muscle progenitor cells and neuroectodermal cells in a medium containing BDNF, GDNF, AA, and dbcAMP in a culture dish coated with a cell adhesion material to induce neuromuscular junctions.

2. The method of claim 1, wherein the human pluripotent stem cells are human embryonic stem cells (ES cells) or human induced pluripotent stem cells (iPS cells).

3. The method of claim 1, wherein the WNT signaling inhibitor comprises 4-(2-methyl-4-pyridinyl)-N-[4-(3-pyridinyl)phenyl]-benzeneacetamide.

4. The method of claim 1, wherein the BMP signal inhibitor comprises 4-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline hydrochloride.

5. The method of claim 1, wherein the ROCK signal inhibitor comprises (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)cyclohexanecarboxamide dihydrochloride.

6. The method of claim 1, wherein the WNT signal activator comprises 6-[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)pyrimidin-2-yl]amino]ethylamino]pyridine-3-carbonitrile.

7. The method of claim 1, wherein the TGFβ signaling inhibitor comprises 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide.

8. The method of claim 1, wherein the retinoic acid activator comprises 4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)ethynyl]benzoic acid.

9. The method of claim 1, wherein the SHH signal activator comprises 3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-[3-(4-pyridinyl)benzyl]-1-benzothiophene-2-carboxamide.

10. The method according to claim 1, wherein the culture dish coated for cell adhesion is a culture dish coated with ornithine and laminin.

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