Co-culture system, kit, and cell culture medium for skeletal muscle cells and motor neurons

Abstract

To provide an improved coculture system of a skeletal muscle cell and a motor neuron.MEANS FOR SOLVING THE PROBLEM: In a coculture system of a skeletal muscle cell and a motor neuron, used is a medium containing Agrin, glutamic acid, and testosterone or an analog thereof. At least one selected from the group consisting of the Agrin, the glutamic acid, and the testosterone or an analog thereof may be an exogenously added one.SELECTED DRAWING: None

Description

Technical Field

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

Background Art

[0002] Spinal and bulbar muscular atrophy (SBMA) is a lower motor neuron neurodegenerative disease that develops in adult men, and 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) in humans. 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] It has been reported that suppressing the expression of the mutant androgen receptor in the motor neurons of SBMA model mice delays the onset and progression of motor dysfunction and also improves the pathology of motor neurons, neuromuscular junctions (NMJs), and skeletal muscles (Non-Patent Document 1). On the other hand, suppressing the expression of the mutant androgen receptor in the skeletal muscles of SBMA model mice has been shown to improve the phenotype, including neurodegeneration of motor neurons (Non-Patent Document 2). In addition, when the mutant AR gene was specifically expressed in the motor neurons or skeletal muscle cells of adult mice, although the motor neurons contributed to motor dysfunction and the pathology of some muscles, the contribution of skeletal muscle cells was prominent with respect to the pathology of muscles 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 co-culturing motor neurons and skeletal muscle cells, the motor neurons extend their 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 (CAG repeat number: 24 times) or a mutant androgen receptor (CAG repeat number: 55 times or 97 times) was induced to differentiate into skeletal muscle, in the skeletal muscle expressing the mutant androgen receptor, the formation and maturation of muscle tubes were poorer compared to 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] An object of the present invention is to provide an improved co-culture system for skeletal muscle cells and motor neurons.

Means for Solving the Problem

[0007] One embodiment according to the present invention is a co - culture system of skeletal muscle cells and motor neurons in which a medium containing Agrin, glutamate, and testosterone or its analog is used. At least one selected from the group consisting of the Agrin, the glutamate, and the testosterone or its analog may be externally added. The analog of testosterone is a ligand of the androgen receptor and may have a dissociation constant with the androgen receptor of 10 -5 M or less. The analog of testosterone may be one or more compounds selected from the group consisting of 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.

[0008] Another embodiment according to the present invention is a kit containing Agrin, glutamate, and testosterone or its analog.

[0009] A further embodiment according to the present invention is a cell culture medium containing Agrin, glutamate, and testosterone or its analog. At least one selected from the group consisting of the Agrin, the glutamate, and the testosterone or its analog may be externally added.

Advantages of the Invention

[0010] The present invention has made it possible to provide an improved co-culture system for skeletal muscle cells and motor neurons.

Brief Description of the Drawings

[0011]

Figure 1

Figure 2

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Figure 4

Modes for Carrying Out the Invention

[0012] 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 are clear 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 illustration or explanation purposes, and the present invention is not limited thereto. It is obvious to those skilled in the art that various modifications and improvements can be made based on the description in this specification within the intention and scope of the present invention disclosed in this specification.

[0013] ==Co - culture system of skeletal muscle cells and motor neurons== Co - culture of skeletal muscle cells and motor neurons can be performed by known methods. However, in the co - culture system of the present disclosure, a medium containing Agrin, glutamate, and testosterone or its analog is 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.

[0014] As used in this specification, 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 Less than M, more preferably less than 10 -9 It refers to those less than M. Examples of analogs 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.

[0015] Both the skeletal muscle cells and motor neurons used in the co-culture system of the present disclosure will be described in detail below. The species from which the skeletal muscle cells are derived and the species from which the motor neurons are derived may be the same or different.

[0016] ==Skeletal Muscle Cells== The animal species from which the skeletal muscle cells used in the co-culture system of the present disclosure are derived is not particularly limited and may be a mammal such as a human, mouse, rat, or pig, but a human is preferred. The skeletal muscle cells may be cells isolated from a living body, an already established cell line, or may be 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 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 the iPS cells are derived is not particularly limited and may be a mammal such as a human, mouse, rat, or pig, but a human is preferred. 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).

[0017] The skeletal muscle cells used in the co-culture system of the present disclosure preferably express an acetylcholine receptor (AChR). The acetylcholine receptor may be expressed endogenously or exogenously. In skeletal muscle cells isolated from a living body, since the acetylcholine receptor is expressed endogenously, it is not necessary to express it exogenously, but it may be expressed exogenously to increase the expression level. Even when pluripotent stem cells are differentiated, they express the acetylcholine receptor, so it is not necessary to express it exogenously, but it may be expressed exogenously to increase the expression level. In particular, in skeletal muscle cells differentiated from pluripotent stem cells, mainly fetal-type (AChRγ) acetylcholine receptors are expressed, but adult-type (AChRε) acetylcholine receptors may be expressed exogenously. Hereinafter, the details of the case of exogenously expressing the acetylcholine receptor will be described.

[0018] There are two types of acetylcholine receptors, nicotinic acetylcholine receptors and muscarinic acetylcholine receptors, and either may 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 is composed 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 become acetylcholine receptor N M . 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), and either may be expressed, but it is preferable to express AChRε. In particular, in the case of skeletal muscle cells obtained by differentiating iPS cells, since mainly fetal-type subunits (AChRγ) are expressed, it is preferable to exogenously express adult-type subunits (AChRε). Thereby, the formation efficiency of the NMJ when the skeletal muscle cells are co-cultured with motor neurons is increased. N MThe animal species from which it is derived is not particularly limited and may be mammals such as humans, mice, rats, pigs, etc. And N M The animal species from which it is derived may be the same species as or different from the cells in which it is to be expressed.

[0019] The skeletal muscle cells used in the co-culture system of the present disclosure may be derived from males or females. Also, with regard to 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 may be isolated 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 skeletal muscle stem cells from an individual having any genotype and differentiating them into skeletal muscle cells. Or, skeletal muscle cells isolated from a homozygote (female) having a wild-type allele or a hemizygote (male) having a wild-type allele, or skeletal muscle cells prepared by isolating and differentiating pluripotent stem cells or skeletal muscle stem cells from an individual having any genotype, or skeletal muscle cells obtained by any method regardless of the genotype of the individual from which they are derived, may be made to have a mutant androgen receptor gene by exogenously introducing a mutant androgen receptor gene. Alternatively, skeletal muscle cells 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 skeletal muscle stem cells and differentiating them into skeletal muscle cells.

[0020] 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.

[0021] ==Motor neuron== 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 a mammal such as a human, mouse, rat, or pig, but is preferably a human. The motor neurons may be cells isolated from a living body, may be an already 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 a mammal such as a human, mouse, rat, or pig, but is preferably a 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).

[0022] 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.

[0023] Motor neurons having a mutant androgen receptor gene may be isolated from heterozygotes (female) having wild - type and mutant alleles of the androgen receptor gene, homozygotes (female) with mutant alleles, or hemizygotes (male) with mutant alleles of the androgen receptor gene. Alternatively, they may be prepared by isolating pluripotent stem cells or neural stem cells from individuals having any of these genotypes and differentiating them into motor neurons. Or, motor neurons isolated from homozygotes (female) with wild - type alleles or hemizygotes (male) with wild - type alleles, or motor neurons prepared by isolating and differentiating pluripotent stem cells or neural stem cells from individuals having any of these genotypes, or motor neurons obtained by any method regardless of the genotype of the originating individual, may be made to have a mutant androgen receptor gene by exogenously introducing the mutant androgen receptor gene. Alternatively, motor neurons may be isolated from transgenic animals into which the 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.

[0024] 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.

[0025] ==SBMA Model System== The co-culture system of the present disclosure is a co-culture system of skeletal muscle cells and motor neurons. The skeletal muscle cells and motor neurons described above are used. Regarding the genotypes of androgen receptor genes, their combinations are not particularly limited, and in any case, a neuromuscular junction (NMJ) is formed between skeletal muscle cells and motor neurons. For example, in the case of skeletal muscle cells without a mutant androgen receptor gene and motor neurons without a mutant androgen receptor gene, NMJs of normal number and shape are formed. However, in the case of skeletal muscle cells with a mutant androgen receptor gene and motor neurons without a mutant androgen receptor gene, a decrease in the number of NMJs and cell death of motor neurons are observed. Therefore, under this condition, it is considered that the androgen receptor gene of skeletal muscle cells is the responsible gene for the decrease in the number of NMJs and cell death of motor neurons. And the latter system is considered to be a model system for SBMA.

Example

[0026] (Example 1) In this example, when co-culturing skeletal muscle cells and motor neurons derived from cell lines to form NMJs, it is shown that adding three factors, agrin (Agrin), glutamate, and dihydrotestosterone (DHT), to the medium for co-culture can increase the formation efficiency of NMJs.

[0027] First, a DNA fragment (Gensler S et al., Eur J Biochem 2001, vol.268 pp.2209 - 2217) encoding a fusion protein of the adult 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 a cell line, a human myoblast cell line (Hu5 / E18 - AεG) that constantly expresses the adult acetylcholine receptor was prepared.

[0028] Next, a lentivirus with a reporter (HB9 e438 -b-glo-mRFP) that visualizes motor neurons was produced and introduced into motor neurons differentiated from human iPS cell line 201B7 (provided by Professor Shinya Yamanaka of the Institute for Integrated Cell-Material Sciences, Kyoto University). Note that the lentivirus with HB9 e438 -b-glo-mRFP was created by replacing Venus in the HB9 e438 -b-glo-Venus (HB9 e438 ::Venus) lentivirus (Shimojo et al., Mol.Brain 2015, vol.8 Article No. 79) that expresses the Venus fluorescent protein under the control of a 438-base pair enhancer of HB9 (HB9 e438 ) and the promoter of human beta-globin with mRFP. 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).

[0029] Colonies were detached from iPS cells using 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), 2 mM L - glutamine, 1% NEAA (Sigma - Aldrich, USA), 0.1 mM β - mercaptoethanol (Sigma - Aldrich, USA), 0.5% penicillin - streptomycin) to let feeder cells sink to the bottom. Then, the supernatant was collected and cultured in suspension using a dish for bacterial culture (this day was designated 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.

[0030] The myoblast-derived skeletal muscle cells obtained in this way were co-cultured with the cells obtained by dissociating hEBs, which include motor neurons and motor neuron progenitor cells. 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. After culturing, observation of the motor end plates was performed using a fluorescence microscope with GFP contained in AεG as a marker (Figure 1A). HB9 e438 The neurites of motor neurons labeled with -b-glo-mRFP contact skeletal muscle with nAChR expressed at the motor end plate and labeled with GFP, forming NMJs. Therefore, at the NMJ, clustering of nAChR, which is a marker of the motor end plate, 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 sites where NMJ formation is observed are indicated by white arrows. Also, the number of motor end plates per RFP-positive motor neuron was calculated and graphed (Figure 1B).

[0031] As shown in Figure 1, 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.

[0032] (Example 2) In Example 1, when co-culturing cell line-derived skeletal muscle cells and cells obtained by dissociating hEBs to form NMJs, it was shown that adding three factors, Agrin, glutamate, and dihydrotestosterone (DHT), to the medium for co-culture increases the efficiency of NMJ formation. In this Example 2, it is similarly shown that using iPS cell-derived skeletal muscle cells and these three factors also increases the efficiency of NMJ formation.

[0033] First, 409B2 MyoD-hiPSCs, which are human iPS cells having drug-inducible MyoD, were used to induce differentiation 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 a dish 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% horse serum (Sigma), 10 ng / mL recombinant human insulin-like growth factor-1 (R&D), and 2 mM L-glutamine, and cultured for an additional 3 days to obtain skeletal muscle cells.

[0034] On the other hand, using 201B7, differentiation was induced into motor neurons and motor neuron progenitor cells in the same manner as in Example 1.

[0035] These cells were co-cultured in the same manner as in Example 1. That is, the medium used was MNM, and the medium containing 0.1 pM Agrin, 100 μM glutamate, and 10 nM dihydrotestosterone (DHT) and the medium not containing them were used. After culturing at 5% CO2 and 37°C for 3 days, the number of muscle endplates per RFP-positive motor neuron was calculated and made into a graph.

[0036] As shown in Fig. 2, similar to Example 1, even when using iPS cell-derived skeletal muscle cell lines, the formation efficiency of NMJs 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 can increase the formation efficiency of NMJs.

[0037] (Reference Experimental Example 1) In this experimental example, it is shown that when co-culturing skeletal muscle cells and motor neurons to form NMJs, the formation efficiency of NMJs can be increased by expressing AChRε in skeletal muscle cells.

[0038] First, 409B2 MyoD-hiPSCs, which are human iPS cells with drug-inducible MyoD, were induced to differentiate into skeletal muscle cells in the same manner as in Example 2, and AεG was forcibly expressed.

[0039] Next, these iPS cell-derived skeletal muscle cells were co-cultured with the iPS cell-derived motor neurons and motor neuron progenitor cells prepared in Example 1. MNM was used as the medium, and co-culture was performed at 5% CO2 and 37 °C. After 3 days, e438 The motor neurons labeled with HB9-b-glo-mRFP were stained with an anti-RFP antibody (MBL), Alexa488-conjugated αBTX was bound, and observation was performed with a fluorescence microscope (Fig. 3A). Also, the number of NMJs per RFP-positive motor neuron was represented in the graph of Fig. 3B.

[0040] As shown in Fig. 3, the formation efficiency of NMJs was approximately 4 times higher when co-cultured using skeletal muscle cells expressing AεG. Thus, when co-culturing iPS cell-derived skeletal muscle cells with motor neurons, the formation efficiency of NMJs can be increased by expressing AChRε in skeletal muscle cells.

[0041] (Reference Experimental Example 2) In this experimental example, it is shown that when co-culturing skeletal muscle cells with mutant androgen receptors with motor neurons, using a medium containing DHT can promote neurodegeneration and result in a better model system for SBMA.

[0042] 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).

[0043] Fibroblasts were collected from the dermis of SBMA disease patients. The cells were cultured in DMEM containing 10% FBS, and 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 into 6×10 5 cells using the Neon transfection system (Thermo Fisher Scientific, USA). Six days later, the cells were collected 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.

[0044] The iPS cells thus obtained were transfected with ViaFect TMThe drug-inducible MyoD vector was introduced together with transposase using Transfection Reagent (Promega). By selecting with neomycin (G418) and establishing clones, MyoD-hiPSCs, which are human iPS cells that constantly express drug-inducible MyoD, were generated. After making them feeder-free, MyoD-hiPSCs were induced to differentiate into skeletal muscle cells in the same manner as in Reference Experimental 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.

[0045] 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 a density of 5×10 4 -1×10 5 cells / cm 2 onto a dish coated with mouse laminin (Thermo Fisher Scientific, USA), and culturing them for 1 - 4 weeks 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). In addition, half of the medium was replaced with fresh medium once every 3 - 4 days.

[0046] 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 images were significantly increased, and the number of neuromuscular junctions per RFP-positive motor neuron (indicated by arrows in the figure) was significantly decreased (Figures 4A and 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 (Figures 4A and C). On the other hand, when the number of degenerated nerves in the neurodegenerative images was quantified, the neurodegenerative images of motor neurons were increased in the presence of DHT compared with those 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, by using a medium containing DHT, neurodegeneration is promoted, resulting in a better model system for SBMA.

Claims

1. An additive kit for a co - culture medium of skeletal muscle cells and motor neurons, comprising Agrin, glutamic acid, and testosterone or an analog thereof, wherein the analog of testosterone is a ligand of the androgen receptor and has a dissociation constant with the androgen receptor of 10−5 M or less. An additive kit for a co - culture medium.

2. A co - culture medium of skeletal muscle cells and motor neurons, containing Agrin, glutamic acid, and testosterone or an analog thereof, wherein the analog of testosterone is a ligand of the androgen receptor and has a dissociation constant with the androgen receptor of 10−5 M or less. A co - culture medium.

3. The co - culture medium for skeletal muscle cells and motor neurons according to claim 2, wherein at least one selected from the group consisting of the Agrin, the glutamic acid, and the testosterone or an analog thereof is externally added.

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