Skeletal muscle cells and a co-culture system of skeletal muscle cells and motor neurons

Abstract

To provide a novel skeletal muscle cell, and an improved coculture system of the skeletal muscle cell and a motor neuron.MEANS FOR SOLVING THE PROBLEM: A skeletal muscle cell is a skeletal muscle cell derived from a first pluripotent stem cell in which an acetylcholine receptor ε sub unit is exogenously expressed. Also disclosed is a coculture system that uses the skeletal muscle cell and a motor neuron.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to skeletal muscle cells and a co - culture system of skeletal muscle cells and motor neurons.

Background Art

[0002] Spinal and bulbar muscular atrophy (SBMA) is a lower motor neuron neurodegenerative disease that develops in adult men. In humans, it is known to be caused by an abnormal elongation of the CAG repeat in exon 1 of the Androgen Receptor (AR) gene located on the long arm of the X chromosome (Xq11 - 12). Males who are hemizygous for a mutant androgen receptor allele with an abnormal elongation of the CAG repeat develop SBMA, and neurodegeneration occurs in motor neurons.

[0003] When the expression of the mutant androgen receptor is suppressed in the motor neurons of SBMA model mice, delays in the onset and progression of motor dysfunction are observed, and the pathologies of motor neurons, neuromuscular junctions (NMJs), and skeletal muscles are also improved (Non - Patent Document 1). On the other hand, when the expression of the mutant androgen receptor is suppressed in the skeletal muscles of SBMA model mice, phenotypes are shown to be improved, including neurodegeneration of motor neurons (Non - Patent Document 2). Also, when the mutant AR gene is specifically expressed in the motor neurons or skeletal muscle cells of adult mice, although motor neurons contribute to motor dysfunction and the pathology of some muscles, the contribution of skeletal muscle cells is prominent regarding muscle pathology and changes in muscle gene expression (Non - Patent Document 3). Thus, it has not yet been determined whether the cause of neurodegeneration in SBMA lies on the motor neuron side or the skeletal muscle side.

[0004] Incidentally, when motor neurons and skeletal muscle cells are co-cultured, the motor neurons extend the axon terminals to the skeletal muscle cells to form the neuromuscular junction (NMJ). To elucidate the cause of SBMA, this co-culture system of motor neurons and skeletal muscle cells was used (Non-Patent Document 4). First, when a human myoblast cell line (Hu5 / E18) that constitutively expresses a wild-type androgen receptor (with 24 CAG repeats) or a mutant androgen receptor (with 55 or 97 CAG repeats) is induced to differentiate into skeletal muscle, in the skeletal muscle expressing the mutant androgen receptor, the formation and maturation of myotubes were worse compared to those in the skeletal muscle expressing the wild-type androgen receptor. Then, when co-cultured with motor neurons differentiated from iPS cells derived from healthy individuals, an increase in neuronal cell death and a decrease in the number of NMJ formations were observed in a CAG repeat number (length of polyglutamine chain)-dependent manner (Non-Patent Document 4).

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Summary of the Invention

Problems to be Solved by the Invention

[0006] The present invention aims to provide novel skeletal muscle cells and an improved co-culture system of the skeletal muscle cells and motor neurons.

Means for Solving the Problems

[0007] One embodiment of the present invention is skeletal muscle cells derived from first pluripotent stem cells that externally express the acetylcholine receptor ε subunit. The first pluripotent stem cells may have a mutant androgen receptor gene. The first pluripotent stem cells may be derived from a patient with spinal muscular atrophy.

[0008] Another embodiment of the present invention is a co-culture system of any of the above skeletal muscle cells and motor neurons. The motor neurons may be derived from second pluripotent stem cells. The second pluripotent stem cells may be derived from a healthy subject without a mutant androgen receptor gene.

Effects of the Invention

[0009] The present invention enables the provision of novel skeletal muscle cells and an improved co-culture system of the skeletal muscle cells and motor neurons.

Brief Description of the Drawings

[0010]

Figure 1

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Figure 4

Modes for Carrying Out the Invention

[0011] Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings, but the present invention is not necessarily limited thereto. The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description in this specification, and those skilled in the art can easily reproduce the present invention from the description in this specification. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention and are shown for illustrative or explanatory purposes, and the present invention is not limited thereto. It will be apparent to those skilled in the art that various modifications and alterations can be made within the spirit and scope of the present invention disclosed in this specification based on the description in this specification.

[0012] ==Skeletal Muscle Cells== The skeletal muscle cells disclosed in this specification are skeletal muscle cells derived from first pluripotent stem cells that externally express the acetylcholine receptor ε subunit.

[0013] The animal species from which the skeletal muscle cells of the present disclosure are derived are not particularly limited and may be mammals such as humans, mice, rats, pigs, etc., but are preferably humans. The skeletal muscle cells of the present disclosure are produced by differentiating pluripotent stem cells such as iPS cells and ES cells or skeletal muscle stem cells into skeletal muscle cells in vitro. The pluripotent stem cells are not particularly limited as long as they have pluripotency, and examples thereof include iPS cells and ES cells. In terms of being able to be produced from differentiated cells, iPS cells are preferred. The animal species from which iPS cells are derived are not particularly limited and may be mammals such as humans, mice, rats, pigs, etc., but are preferably humans. Differentiation of pluripotent stem cells into skeletal muscle cells can be carried out using known methods (for example, Tanaka A et al., PLoS One 2013, e61540).

[0014] The skeletal muscle cells obtained by differentiating pluripotent stem cells express acetylcholine receptors (AChR), but mainly express fetal-type acetylcholine receptors, so adult-type acetylcholine receptors are expressed externally. Hereinafter, the details of externally expressing acetylcholine receptors will be described.

[0015] There are two types of acetylcholine receptors, nicotinic acetylcholine receptors and muscarinic acetylcholine receptors, and either can be expressed. However, in skeletal muscle cells, nicotinic acetylcholine receptor N M is expressed physiologically, so it is preferable to express nicotinic acetylcholine receptor N M . Nicotinic acetylcholine receptor N M consists of four types of subunits, α, β, δ, and ε. In humans, subtypes α1-7, α9-10, β1-4, γ, δ, and ε exist, and these form hetero- or homo-pentamers to form nicotinic acetylcholine receptor N MIt becomes. Specifically, there are a fetal type having γ (hereinafter referred to as AChRγ) as a subunit and an adult type having ε (hereinafter referred to as AChRε). In the case of skeletal muscle cells obtained by differentiating pluripotent stem cells, particularly iPS cells, mainly the fetal type subunit (AChRγ) is expressed, so the adult type subunit (AChRε) is expressed exogenously. Thereby, the formation efficiency of the NMJ when co-culturing skeletal muscle cells with motor neurons is increased. N M The animal species from which N is derived is not particularly limited, and may be a mammal such as a human, mouse, rat, pig, etc. And, N M The animal species from which it is derived may be the same species as the cells to be expressed or a different species. Other subunits do not need to be expressed exogenously, but may be expressed exogenously to increase the expression level.

[0016] The skeletal muscle cells used in the co-culture system of the present disclosure may be derived from males or females. Also, regarding the androgen receptor gene, in the case of males, it may be hemizygous with a wild-type allele or hemizygous with a mutant allele. In the case of females, it may be homozygous with a wild-type allele, homozygous with a mutant allele, or heterozygous with a wild-type allele and a mutant allele. Skeletal muscle cells having a mutant androgen receptor gene are prepared by isolating pluripotent stem cells from a heterozygote (female) having wild-type and mutant alleles of the androgen receptor gene, a homozygote (female) having a mutant allele, or a hemizygote (male) having a mutant allele and differentiating them into skeletal muscle cells. Alternatively, a mutant androgen receptor gene may be introduced exogenously into skeletal muscle cells prepared by isolating and differentiating pluripotent stem cells from a homozygote (female) having a wild-type allele or a hemizygote (male) having a wild-type allele to produce skeletal muscle cells having a mutant androgen receptor gene. Alternatively, it may be prepared by isolating pluripotent stem cells or skeletal muscle stem cells from a transgenic animal into which a mutant androgen receptor gene has been introduced and differentiating them into skeletal muscle cells.

[0017] In the present disclosure, the wild-type androgen receptor gene refers to an allele of the androgen receptor gene that does not develop spinal and bulbar muscular atrophy (SBMA) in a hemizygote (male) having that allele as an endogenous gene, and the CAG repeat number is usually 36 or less, but may be 37. The mutant androgen receptor gene refers to an allele of the androgen receptor gene possessed by an individual who develops SBMA in a hemizygote (male) having that allele as an endogenous gene, and the CAG repeat number is usually 38 or more, but may be 37. Therefore, in the case of humans, a hemizygote (male) having a mutant allele of the androgen receptor gene is an SBMA patient who has developed or may develop SBMA in the future, and a hemizygote (male) having a wild-type allele is a healthy person who does not develop SBMA.

[0018] ==Motor neurons== The animal species from which the motor neurons used in the co-culture system of the present disclosure are derived is not particularly limited, and may be mammals such as humans, mice, rats, pigs, etc., but is preferably human. The motor neurons may be cells isolated from a living body, may already be an established cell line, or may be produced by differentiating pluripotent stem cells such as iPS cells and ES cells or neural stem cells into motor neurons. The pluripotent stem cells are not particularly limited as long as they have pluripotency, and examples include iPS cells and ES cells. In terms of being able to be produced from differentiated cells, iPS cells are preferably used. The animal species from which the iPS cells are derived is not particularly limited, and may be mammals such as humans, mice, rats, pigs, etc., but is preferably human. Differentiation of pluripotent stem cells into motor neurons can be carried out using known methods (for example, Shimojo et al., Mol. Brain 2015, vol.8 Article No. 79).

[0019] The motor neurons used in the co-culture system of the present disclosure may be derived from males or females. Regarding the androgen receptor gene, in the case of males, they may be hemizygotes with wild-type alleles or hemizygotes with mutant alleles. In the case of females, they may be homozygotes with wild-type alleles, homozygotes with mutant alleles, or heterozygotes with wild-type and mutant alleles.

[0020] Motor neurons having a mutant androgen receptor gene may be isolated from motor neurons from a heterozygote (female) having wild-type and mutant alleles of the androgen receptor gene, a homozygote (female) having a mutant allele, or a hemizygote (male) having a mutant allele. Alternatively, they may be prepared by isolating pluripotent stem cells or neural stem cells from an individual having any of these genotypes and differentiating them into motor neurons. Or, motor neurons isolated from a homozygote (female) having wild-type alleles or a hemizygote (male) having wild-type alleles, or motor neurons prepared by isolating and differentiating pluripotent stem cells or neural stem cells from an individual having any of these genotypes, or motor neurons obtained by any method regardless of the genotype of the originating individual, may be used to prepare motor neurons having a mutant androgen receptor gene by exogenously introducing a mutant androgen receptor gene. Alternatively, motor neurons may be isolated from a transgenic animal into which a mutant androgen receptor gene has been introduced, or they may be prepared by isolating pluripotent stem cells or neural stem cells and differentiating them into motor neurons.

[0021] In the above, when there are steps of introducing and expressing a gene into pluripotent cells and differentiating the pluripotent cells into a specific cell type, gene introduction and cell differentiation may be performed in either order.

[0022] ==Co-culture system of skeletal muscle cells and motor neurons== The co-culture of skeletal muscle cells and motor neurons can be performed by known methods.

[0023] For example, a medium containing Agrin, glutamate, and testosterone or its analog may be used. At least one selected from the group consisting of Agrin, glutamate, and testosterone or its analog may be externally added. The concentration of Agrin is preferably 0.05 - 0.2 pM, more preferably 0.1 pM. The concentration of glutamate is preferably 50 - 200 μM, more preferably 100 μM. The concentration of testosterone or its analog is preferably 1 - 100 nM, more preferably 3 - 30 nM, and even more preferably 10 nM. By mixing these three components in the medium, the formation efficiency of NMJ can be increased. For convenience, a kit containing Agrin, Glutamate, and testosterone or its analog may be manufactured.

[0024] As used herein, an analog of testosterone is a ligand of the androgen receptor, and under physiological conditions, the dissociation constant with the androgen receptor is 10 -5 M or less, preferably 10 -6 M or less, more preferably 10 -7 M or less, even more preferably 10 -8 M or less, even more preferably 10 -9 M or less. Examples of the analog of testosterone include testosterone cypionate, testosterone decanoate, testosterone enanthate, testosterone isocaproate, testosterone phenylpropionate, testosterone propionate, testosterone undecanoate, androstenedione, 4-androstenedione, 5-androstenedione, androstenediol, 5-androstenediol, 4-androstenediol, dehydroepiandrosterone, nandrolone, dihydrotestosterone, etiocholanolone, methyltestosterone, androsterone, epiandrosterone, 17α-ethyltestosterone, and fluoxymesterone. Among them, dihydrotestosterone is preferred.

[0025] For both the skeletal muscle cells and motor neurons used in the co-culture system of the present disclosure, the species from which the skeletal muscle cells are derived and the animal species from which the motor neurons are derived may be the same or different.

[0026] ==SBMA Model System== The co-culture system of the present disclosure is a co-culture system of skeletal muscle cells and motor neurons. The above-described skeletal muscle cells and motor neurons are used. Regarding the genotype of the androgen receptor gene, their combination is not particularly limited, and in any case, a neuromuscular junction (NMJ) is formed between the skeletal muscle cells and the motor neurons. For example, in the case of skeletal muscle cells without the mutant androgen receptor gene and motor neurons without the mutant androgen receptor gene, normal numbers and shapes of NMJs are formed. However, in the case of skeletal muscle cells with the mutant androgen receptor gene and motor neurons without the mutant androgen receptor gene, a decrease in the number of NMJs and cell death of the motor neurons are observed. Therefore, under these conditions, it is considered that the androgen receptor gene of the skeletal muscle cells is the responsible gene. And the latter system is considered to be a model system for SBMA.

Examples

[0027] (Example 1) In this example, when co-culturing skeletal muscle cells and motor neurons to form NMJs, it is shown that by expressing AChRε in the skeletal muscle cells, the formation efficiency of NMJs is increased.

[0028] First, 409B2 MyoD-hiPSCs, which are human iPS cells with drug-inducible MyoD, were induced to differentiate into skeletal muscle cells, and AεG was forcibly expressed. Specifically, it was as follows. 409B2 MyoD-hiPSCs cultured using StemFit AK02N (Takara, Japan) were dissociated using Accutase and seeded onto dishes coated with Matrigel (BD Biosciences) at a density of 2x10 4 cells / cm 2 (this day was designated as day 0 of culture). On day 1 of culture, the medium was replaced with hESC medium containing no FGF-2 and 10 mM Y-27632 (Wako). On day 2 of culture, 1 μg / mL doxycycline was added to the medium. On day 3 of culture, the medium was replaced with αMEM (Nacalai Tesque) containing 10% KSR (Invitrogen), 2% Ultroser G, and 100 mM β-mercaptoethanol. On day 7 of culture, the cells were infected with lentivirus obtained by inserting a DNA fragment encoding AεG into a lentiviral vector to forcibly express AεG. Thereafter, the medium was replaced with DMEM containing 2% fetal bovine serum (Sigma), 10 ng / mL recombinant human insulin-like growth factor-1 (R&D), and 2 mM L-glutamine, and the cells were cultured for an additional 3 days to obtain skeletal muscle cells.

[0029] Next, a lentivirus having a reporter (HB9 e438 -b-glo-mRFP) for visualizing motor neurons was prepared and introduced into motor neurons differentiated from human iPS cells 201B7 (provided by Professor Shinya Yamanaka of the Institute for iPS Cell Research, Kyoto University). The lentivirus having HB9 e438 -b-glo-mRFP expresses Venus fluorescent protein by the enhancer of HB9 of 438 base pairs (HB9 e438 ) and the promoter of human β-globin, HB9 e438 -b-glo-Venus (HB9 e438::Venus was generated by replacing Venus of the lentivirus (Shimojo et al., Mol.Brain 2015, vol.8 Article No. 79) with mRFP. In addition, the differentiation of iPS cells into motor neurons was performed as follows (Shimojo et al., Mol.Brain 2015, vol.8 Article No. 79; Onodera et al., Mol.Brain 2020, vol.13 Article No. 18).

[0030] Colonies of iPS cells were detached with a dissociation solution containing 0.25% trypsin - 100 μg / mL collagenase IV - 1 mM CaCl2 - 20% KSR, transferred to a gelatin - coated dish, and incubated for 1 - 2 hours using hESC medium (DMEM / F12 containing 20% KSR (Thermo Fisher Scientific, USA), 0.1 mM β - mercaptoethanol (Sigma - Aldrich, USA), 0.5% penicillin - streptomycin) to let feeder cells fall to the bottom. Then, the supernatant was collected and cultured in suspension using a dish for bacterial culture (designating this day as day 0 of culture). On day 1 of culture, the medium was replaced with hEB medium (DMEM / F - 12 containing 5% KSR - 2 mM L - glutamine - 1% NEAA - 0.1 mM β - mercaptoethanol - 300 nM LDN193189 (Sigma - Aldrich, USA) - 3 μM SB431542 (Santa Cruz, USA) - 3 μM CHIR99021 (Sigma - Aldrich, USA)). On day 2 of culture, 1 μM retinoic acid (Sigma - Aldrich, USA) was further added for culture. From day 4 to day 14 of culture, hEB medium containing 1 μM retinoic acid and 1 μM purmorphamine (Calbiochem, Germany) was used, and the culture was carried out with medium change once every 2 - 3 days. On days 14 - 15 of culture, the formed hEB (human embryoid body) was dissociated into single cells using TrypLE Select (Thermo Fisher Scientific, USA). This cell population contains motor neurons and motor neuron progenitor cells.

[0031] The above - mentioned skeletal muscle cells derived from iPS cells were co - cultured with the obtained motor neurons and motor neuron progenitor cells derived from iPS cells. MNM was used as the medium, and the co - culture was carried out at 5% CO2 and 37 °C. After 3 days, HB9 e438Motor neurons labeled with -b-glo-mRFP were stained with an anti-RFP antibody (MBL), Alexa488-conjugated αBTX was bound, and observations were made using a fluorescence microscope (Figure 1A). In addition, the number of NMJs per RFP-positive motor neuron was represented in the graph of Figure 1B.

[0032] As shown in Figure 1, co-culture using skeletal muscle cells expressing AεG had approximately 4-fold higher NMJ formation efficiency. Thus, when co-culturing iPS cell-derived skeletal muscle cells with motor neurons, expressing AChRε in the skeletal muscle cells increases the NMJ formation efficiency.

[0033] (Reference Experimental Example 1) In this example, when co-culturing cell line-derived skeletal muscle cells and motor neurons to form NMJs, it is shown that adding three factors, agrin, glutamate, and dihydrotestosterone (DHT), to the medium for co-culture increases the NMJ formation efficiency.

[0034] First, a DNA fragment (Gensler S et al., Eur J Biochem 2001, vol.268 pp.2209-2217) encoding a fusion protein of the adult-type acetylcholine receptor subunit AChRε and GFP (AChRε-GFP; AεG) was inserted into a PiggyBac vector, and the resulting expression vector was introduced into a human myoblast cell line (Hu5 / E18) together with Transposase using Gene Juice (Merck Millipore). By selecting with blasticidin S and establishing cell lines, a human myoblast cell line (Hu5 / E18-AεG) that constantly expresses the adult-type acetylcholine receptor was prepared.

[0035] The skeletal muscle cells derived from the myoblasts obtained in this way were co-cultured with the cells obtained by dissociating hEBs, which included the motor neurons and motor neuron progenitor cells prepared in Example 1. The medium used was MNM, and the cells were cultured at 5% CO2 and 37 °C for 3 days using a medium containing 0.1 pM Agrin, 100 μM glutamate, and 10 nM dihydrotestosterone (DHT) and a medium not containing these components. Then, using GFP contained in AεG as a marker, the neuromuscular junctions were observed under a fluorescence microscope (Figure 2A). HB9 e438 The neurites of the motor neurons labeled with -b-glo-mRFP contact the skeletal muscle through the nAChR expressed at the neuromuscular junction and labeled with GFP, forming the NMJ. Therefore, at the NMJ, clustering of nAChR, which is a marker of the neuromuscular junction, is observed on the skeletal muscle side, and the point where the neurites expressing HB9 e438 -b-glo-mRFP connect is observed. In Figure 1A, the site where NMJ formation is observed is indicated by a white arrow. Also, the number of neuromuscular junctions per RFP-positive motor neuron was calculated and graphed (Figure 2B).

[0036] As shown in Figure 2, the efficiency of NMJ formation was significantly higher when these three factors were added to the medium for co-culture. Thus, when co-culturing skeletal muscle cells and motor neurons, adding these three factors to the medium for co-culture increases the efficiency of NMJ formation.

[0037] (Reference Experimental Example 2) In Reference Experimental Example 1, it was shown that when co-culturing skeletal muscle cells derived from cell lines and cells obtained by dissociating hEBs to form NMJs, adding three factors, Agrin, glutamate, and dihydrotestosterone (DHT), to the medium for co-culture increases the efficiency of NMJ formation. In this Reference Experimental Example 2, it is similarly shown that using skeletal muscle cells derived from iPS cells and using these three factors also increases the efficiency of NMJ formation.

[0038] First, using human iPS cells 409B2 MyoD-hiPSCs having drug-inducible MyoD, differentiation induction into skeletal muscle cells was carried out in the same manner as in Example 1, and AεG was forcibly expressed.

[0039] On the other hand, using 201B7, differentiation induction into motor neurons and motor neuron progenitor cells was carried out in the same manner as in Reference Experimental Example 1.

[0040] These cells were co-cultured in the same manner as in Reference Experimental Example 1. That is, the medium used was MNM, and a medium containing 0.1 pM agrin, 100 μM glutamate, and 10 nM dihydrotestosterone (DHT) and a medium not containing them were used. After culturing at 5% CO2 and 37 °C for 3 days, the number of muscle end plates per RFP-positive motor neuron was calculated and made into a graph.

[0041] As shown in FIG. 3, similar to Reference Experimental Example 1, even when using an iPS cell-derived skeletal muscle cell line, the formation efficiency of NMJ was significantly higher when co-cultured with these three factors added to the medium. Thus, when co-culturing skeletal muscle cells and motor neurons, the formation efficiency of NMJ can be increased by adding these three factors to the medium and co-culturing.

[0042] (Reference Experimental Example 3) In this experimental example, it is shown that when co-culturing skeletal muscle cells having a mutant androgen receptor with motor neurons, using a medium containing DHT promotes neurodegeneration and results in a better model system for SBMA.

[0043] First, iPS cells were established from SBMA disease patients as follows (Shimojo et al., Mol. Brain 2015, vol. 8 Article No. 79; Onodera et al., Mol. Brain 2020, vol. 13 Article No. 18).

[0044] Fibroblasts were collected from the dermis of SBMA disease patients. Cultured in DMEM containing 10% FBS, 6×105 For each cell, 1 μg each of pCXLE-hOCT3 / 4-shp53 (OCT4 and shTP53), pCXLE-hSK (SOX2 and KLF4), and pCXLE-hUL (L-MYC and LIN28; provided by Professor Shinya Yamanaka of Kyoto University) were transfected using the Neon transfection system (Thermo Fisher Scientific, USA). Six days later, the cells were harvested and seeded onto feeder cells of SNL fibroblasts. The next day, the medium was replaced with a medium for human ESCs (DMEM / F12 containing 20% KSR (Thermo Fisher Scientific, USA), 2 mM L-glutamine, 1% NEAA (Sigma-Aldrich, USA), 0.1 mM β-mercaptoethanol (Sigma-Aldrich, USA), 4 ng / mL recombinant FGF-2 (Peprotech, USA)), and the culture was continued. When the colonies became large enough, the colonies were isolated and expanded.

[0045] The drug-inducible MyoD vector was introduced together with Transposase into the iPS cells thus obtained using ViaFect TM Transfection Reagent (Promega). MyoD-hiPSCs, which are human iPS cells that constantly express drug-inducible MyoD, were generated by selection with neomycin (G418) and establishment of clones. After making them feeder-free, MyoD-hiPSCs were induced to differentiate into skeletal muscle cells in the same manner as in Example 1. The obtained skeletal muscle cells were infected with a lentivirus obtained by inserting a DNA fragment encoding AεG into a lentiviral vector to forcibly express AεG.

[0046] Next, the obtained skeletal muscle cells were co-cultured with motor neurons having mRFP. The motor neurons were obtained by dissociating hEBs obtained in the same manner as in Example 1 and plating the cells at 5×10 4 -1×10 5 cells / cm2 Cells were seeded at a density of and cultured in a motor neuron medium (MNM) consisting of media hormone mix (MHM) medium supplemented with 2% B27 supplement (Thermo Fisher Scientific, USA), 1% NEAA, 50 nM RA, 500 nM purmorphamine, 10 μM cyclic AMP (cAMP) (Sigma-Aldrich, USA), 10 ng / mL recombinant BDNF (R&D systems, USA), 10 ng / mL recombinant GDNF (R&D systems, USA), 10 ng / mL recombinant human IGF-1 (R&D systems, USA), 200 ng / mL ascorbic acid (Sigma-Aldrich, USA) or serum-free medium for culturing KBM neural stem cells (Kohjin Bio, Japan) for 1-4 weeks. In addition, half of the medium was replaced with fresh medium once every 3-4 days.

[0047] In co-culture, MNM was used as the medium, and the neuromuscular junctions marked with AChRε-GFP and the motor neurons marked with mRFP were compared with or without DHT. As a result, in the presence of DHT, the neurodegenerative phenotype was significantly increased, and the number of neuromuscular junctions per RFP-positive motor neuron (indicated by arrows in the figure) was significantly decreased (Figure 4A, B). In addition, when stained with an antibody against MHC (myosin heavy chain) to examine the state of skeletal muscle cells, the skeletal muscle cells derived from SBMA patients showed no change compared with those derived from healthy individuals who did not develop SBMA disease (Figure 4A, C). On the other hand, when quantifying the number of degenerated nerves in the neurodegenerative phenotype, the neurodegenerative phenotype of motor neurons was increased in the presence of DHT compared with that in the absence (Figure 4D). Also, in immunostaining with Cleaved Caspase-3, when using patient-derived skeletal muscle, cell death of motor neurons was enhanced in the presence of DHT compared with that in the absence (Figure 4E). Such phenotypes were not observed when co-culturing skeletal muscle differentiated from iPS cells without the mutant androgen receptor gene with the motor neurons having mRFP prepared in Example 1. Thus, when co-culturing skeletal muscle cells having the mutant androgen receptor gene with motor neurons, using a medium containing DHT promotes neurodegeneration, resulting in a better model system for SBMA.

Claims

Claim 1 A skeletal muscle cell derived from a first pluripotent stem cell that externally expresses an acetylcholine receptor ε subunit. Claim 2 The skeletal muscle cell according to claim 1, wherein the first pluripotent stem cell has a mutant androgen receptor gene with a CAG repeat number of 37 or more. Claim 3 The skeletal muscle cell according to claim 2, wherein the first pluripotent stem cell is derived from a patient with spinal muscular atrophy. Claim 4 A co-culture system of the skeletal muscle cell according to any one of claims 1 to 3 and a motor neuron. Claim 5 The co-culture system according to claim 4, wherein the motor neuron is derived from a second pluripotent stem cell. Claim 6 The co-culture system according to claim 5, wherein the second pluripotent stem cell is derived from a healthy subject without a mutant androgen receptor gene.

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