Co-culture system

Abstract

To provide an improved coculture system of a skeletal muscle cell differentiated from an iPS cell derived from a patient of spinobulbar muscular atrophy and a motor neuron differentiated from an iPS cell derived from a healthy subject.SOLUTION: The present invention pertains to a coculture system of a skeletal muscle cell differentiated from an iPS cell derived from a patient of spinobulbar muscular atrophy and a motor neuron differentiated from an iPS cell derived from a healthy subject. In the coculture system, used is a medium containing testosterone or an analog thereof. The testosterone or an analog thereof may be one exogenously added to the medium.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a co-culture system. [Background technology]

[0002] Spinal and bulbar muscular atrophy (SBMA) is a neurodegenerative disease of the lower motor neurons that affects adult males. In humans, it is known to be caused by an abnormal expansion of the CAG repeat in the first exon of the androgen receptor (AR) gene on the long arm of the X chromosome (Xq11-12). Men who are hemizygous for a mutant androgen receptor allele with an abnormal expansion of the CAG repeat develop SBMA, which causes neurodegeneration in motor neurons.

[0003] It has been reported that suppressing the expression of mutant androgen receptor in motor neurons of SBMA model mice delays the onset and progression of motor dysfunction, and improves the pathology of motor neurons, neuromuscular junctions (NMJs), and skeletal muscles (Non-Patent Document 1). On the other hand, it has been shown that suppressing the expression of mutant androgen receptor in skeletal muscles of SBMA model mice improves the phenotype, including neurodegeneration of motor neurons (Non-Patent Document 2). In addition, when mutant AR genes were specifically expressed in motor neurons or skeletal muscle cells of adult mice, although motor neurons contributed to motor dysfunction and pathology of some muscles, the contribution of skeletal muscle cells to muscle pathology and changes in muscle gene expression was significant (Non-Patent Document 3). Thus, it has not yet been determined whether the cause of neurodegeneration in SBMA is on the motor neuron side or the skeletal muscle side.

[0004] By the way, when motor neurons and skeletal muscle cells are co-cultured, the motor neurons extend their axon terminals to the skeletal muscle cells to form neuromuscular junctions (NMJs). This co-culture system of motor neurons and skeletal muscle cells was used to elucidate the cause of SBMA (Non-Patent Document 4). First, when a human myoblast cell line (Hu5 / E18) that constitutively expresses wild-type androgen receptors (24 CAG repeats) or mutant androgen receptors (55 or 97 CAG repeats) was induced to differentiate into skeletal muscle, the formation and maturation of myotubes was poor in skeletal muscles expressing mutant androgen receptors compared to skeletal muscles expressing wild-type androgen receptors. Then, when iPS cells derived from healthy subjects were co-cultured with differentiated motor neurons, an increase in neuronal cell death and a decrease in the number of NMJs formed were observed in a CAG repeat number (length of polyglutamine chain)-dependent manner (Non-Patent Document 4). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Sahashi K et al., Hum Mol Genet 2015, vol.24 No.21, pp.5985-5994 [Non-Patent Document 2] Lieberman AP et al., Cell Rep 2014, vol.7 pp.774-784 [Non-Patent Document 3] Ramzan et al., J Neurosci 2015, vol.35 No.16, pp.6444-6451 [Non-Patent Document 4] ISSCR Abstract June 2018 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention aims to provide an improved co-culture system for skeletal muscle cells differentiated from iPS cells derived from patients with spinal and bulbar muscular atrophy and motor neurons differentiated from iPS cells derived from healthy individuals. [Means for solving the problem]

[0007] One embodiment of the present invention is a co-culture system of skeletal muscle cells differentiated from iPS cells derived from a patient with spinal and bulbar muscular atrophy and motor neurons differentiated from iPS cells derived from a healthy individual, in which a medium containing testosterone or an analogue thereof is used. The testosterone or analogue thereof may be exogenously added to the medium. The testosterone analogue is an androgen receptor ligand, and has a dissociation constant with the androgen receptor of 10 or less. -5 The testosterone analog 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. Any of the above co-culture systems may be in vitro model systems for spinal and bulbar muscular atrophy. Effect of the Invention

[0008] The present invention makes it possible to provide an improved co-culture system of skeletal muscle cells differentiated from iPS cells derived from a patient with spinal and bulbar muscular atrophy and motor neurons differentiated from iPS cells derived from a healthy individual. [Brief description of the drawings]

[0009] [Figure 1] In Example 1, we show that when skeletal muscle cells with mutant androgen receptors are co-cultured with motor neurons, neurodegeneration is promoted by using a medium containing DHT, providing a better model system for SBMA. [Diagram 2] This figure shows the experimental results of Reference Experimental Example 1, which demonstrate that when cell line-derived skeletal muscle cells and motor neurons are co-cultured to form NMJs, the efficiency of NMJ formation is increased by adding three factors, agrin, glutamic acid, and dihydrotestosterone (DHT), to the medium and co-culturing them. [Diagram 3] This figure shows the experimental results of Reference Experimental Example 2, which demonstrate that when iPS cell-derived skeletal muscle cells and motor neurons are co-cultured to form NMJs, the efficiency of NMJ formation is increased by adding three factors, agrin, glutamic acid, and dihydrotestosterone (DHT), to the medium and co-culturing them. [Figure 4] FIG. 13 shows experimental results indicating that when skeletal muscle cells and motor neurons are co-cultured to form NMJs in Reference Experimental Example 3, the efficiency of NMJ formation is increased by expressing AChRε in skeletal muscle cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings, but are 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 do not limit the present invention thereto. It is apparent to those skilled in the art that various modifications and alterations can be made based on the description in this specification within the intent and scope of the present invention disclosed in this specification.

[0011] ==Co-culture system of skeletal muscle cells and motor neurons== The co-culture system of skeletal muscle cells and motor neurons disclosed herein is a co-culture system of skeletal muscle cells differentiated from iPS cells derived from a patient with spinal and bulbar muscular atrophy and motor neurons differentiated from iPS cells derived from a healthy individual. This co-culture can be performed by a known method, but in the co-culture system disclosed herein, a medium containing testosterone or an analog thereof is used. This testosterone or an analog thereof may be exogenously added. Here, the concentration of testosterone or an analog thereof is preferably 1-100 nM, more preferably 3-30 nM, and most preferably 10 nM.

[0012] As used herein, a testosterone analog is a ligand for the androgen receptor, and has a dissociation constant with the androgen receptor of 10 or less under physiological conditions. -5 M or less, preferably 10 -6 M or less, more preferably 10 -7 M or less, more preferably 10 -8 M or less, more preferably 10 -9 M or less. Examples of testosterone analogs 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, with dihydrotestosterone being preferred.

[0013] For both the skeletal muscle cells and motor neurons used in the co-culture system of the present disclosure, the biological 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.

[0014] ==SBMA model system== When skeletal muscle cells and motor neurons are co-cultured, neuromuscular junctions (NMJs) are formed between the skeletal muscle cells and motor neurons regardless of the genotype combination of the androgen receptor gene. However, for example, in the case of skeletal muscle cells without mutant androgen receptor genes and motor neurons without mutant androgen receptor genes, NMJs of normal number and shape are formed, but in the case of skeletal muscle cells with mutant androgen receptor genes and motor neurons without mutant androgen receptor genes, a decrease in the number of NMJs and cell death of motor neurons are observed. Therefore, under these conditions, the androgen receptor gene of skeletal muscle cells is thought to be the responsible gene for the decrease in the number of NMJs and cell death of motor neurons. The latter system is thought to be a model system for SBMA.

[0015] In the co-culture system of the present disclosure, skeletal muscle cells differentiated from iPS cells derived from patients with spinal and bulbar muscular atrophy and motor neurons differentiated from iPS cells derived from healthy subjects are co-cultured. This is a model system for SBMA, but by co-culture using a medium containing testosterone or its analogs, the number of NMJs is reduced, and motor neuron degeneration and cell death are observed compared to when testosterone or its analogs are not added, and a clearer phenotype is reproduced as a model system for SBMA.

[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 mammals such as humans, mice, rats, and pigs, but is preferably humans. The skeletal muscle cells are produced by in vitro differentiation of pluripotent stem cells such as iPS cells and ES cells into skeletal muscle cells. The pluripotent stem cells are not particularly limited as long as they have pluripotency, and examples of such cells include iPS cells and ES cells. The pluripotent stem cells are preferably iPS cells in that they can be produced from differentiated cells. The animal species from which the iPS cells are derived is not particularly limited, and may be mammals such as humans, mice, rats, and pigs, but is preferably humans. A known method can be used to differentiate pluripotent stem cells into skeletal muscle cells (e.g., 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 acetylcholine receptors (AChR). The acetylcholine receptors may be expressed endogenously or exogenously. When pluripotent stem cells are differentiated, the acetylcholine receptors are expressed, so there is no need to express them exogenously, but they may be expressed exogenously to increase the expression level. In particular, skeletal muscle cells differentiated from pluripotent stem cells mainly express fetal acetylcholine receptors (AChRγ), but adult acetylcholine receptors (AChRε) may be expressed exogenously. Details of the case where the acetylcholine receptor is expressed exogenously are described below.

[0018] There are two types of acetylcholine receptors, nicotinic acetylcholine receptors and muscarinic acetylcholine receptors. Either type may be expressed, but physiologically, nicotinic acetylcholine receptors N M Since the nicotinic acetylcholine receptor N M It is preferable to express the nicotinic acetylcholine receptor N MThe acetylcholine receptor N is composed of four subunits: α, β, δ, and ε. In humans, the subtypes are α1-7, α9-10, β1-4, γ, δ, and ε, which form hetero- or homopentamers and act as acetylcholine receptor N receptors. M Specifically, there are fetal types that have the subunit γ (hereafter referred to as AChRγ) and adult types that have the subunit ε (hereafter 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, the fetal subunit (AChRγ) is mainly expressed, so it is preferable to exogenously express the adult subunit (AChRε). This increases the efficiency of NMJ formation when skeletal muscle cells are co-cultured with motor neurons. M The animal species from which N is derived is not particularly limited, and may be a mammal such as a human, mouse, rat, or pig. M The animal species from which it is derived may be the same as or a different species from the cell in which it is expressed.

[0019] The skeletal muscle cells used in the co-culture system of the present disclosure may be derived from either males or females. With regard to the androgen receptor gene, in the case of males, it is hemizygous with a mutant allele, while in the case of females, it is 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 produced by isolating pluripotent stem cells from a heterozygote (female) having a wild-type allele and a mutant allele 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, skeletal muscle cells having a mutant androgen receptor gene may be produced by exogenously introducing a mutant androgen receptor gene into skeletal muscle cells produced 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, or by exogenously introducing a mutant androgen receptor gene into pluripotent stem cells and then inducing differentiation into skeletal muscle cells. Alternatively, the cells may be produced by isolating pluripotent stem cells from a transgenic animal into which a mutant androgen receptor gene has been introduced 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 the allele as an endogenous gene, and the number of CAG repeats is usually 36 or less, but may be 37. The mutant androgen receptor gene refers to an allele of the androgen receptor gene that an individual who develops SBMA has in a hemizygote (male) having the allele as an endogenous gene, and the number of CAG repeats 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 develops SBMA or may develop it in the future, and a hemizygote (male) having a wild-type allele is a healthy person who does not develop SBMA.

[0021] ==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, and pigs, but is preferably humans. Motor neurons are produced by differentiating pluripotent stem cells such as iPS cells and ES cells into motor neurons. The pluripotent stem cells are not particularly limited as long as they have pluripotency, and examples of such pluripotent stem cells include iPS cells and ES cells. The pluripotent stem cells are preferably iPS cells in that they can be produced from differentiated cells. The animal species from which the iPS cells are derived is not particularly limited, and may be mammals such as humans, mice, rats, and pigs, but is preferably humans. A known method can be used to differentiate pluripotent stem cells into motor neurons (e.g., 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 can be of male or female origin. With respect to the androgen receptor gene, in the case of males, they are hemizygous with wild-type allele, and in the case of females, they are homozygous with wild-type allele. Therefore, the motor neurons that do not have mutant androgen receptor gene are produced by isolating and differentiating pluripotent stem cells from homozygous (female) with wild-type allele or hemizygous (male) with wild-type allele. EXAMPLES

[0023] Example 1 In this example, we demonstrate that the use of a medium containing DHT promotes neurodegeneration when skeletal muscle cells with mutant androgen receptors are co-cultured with motor neurons, providing a better model system for SBMA.

[0024] First, iPS cells were established from SBMA 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).

[0025] Fibroblasts were collected from the dermis of SBMA patients. They were cultured in DMEM containing 10% FBS and diluted to 6 × 10 5Each cell was transfected with 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, Kyoto University) using the Neon transfection system (Thermo Fisher Scientific, USA). After 6 days, the cells were harvested and seeded on SNL fibroblast feeder cells. The next day, the medium was replaced with human ESC 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), and 4 ng / mL recombinant FGF-2 (Peprotech, USA)) and cultured. When the colonies became large enough, they were isolated and expanded.

[0026] The iPS cells obtained in this way were treated with ViaFect. TM A drug-inducible MyoD vector was introduced together with Transposase using Transfection Reagent (Promega). MyoD-hiPSCs, human iPSCs that constitutively express drug-inducible MyoD, were generated by selecting with neomycin (G418) and establishing clones. The MyoD-hiPSCs thus obtained were induced to differentiate into skeletal muscle cells. Specifically, the procedure is as follows. 409B2 MyoD-hiPSCs cultured in StemFit AK02N (Takara, Japan) were dissociated using Accutase, and 2x10 cells were cultured without feeder cells on a dish coated with Matrigel (BD Biosciences) using StemFit+Y27632 medium. 4 pieces / cm 2The cells were seeded at a density of 100 μg / mL (this day was considered as culture day 0). On culture day 1, the hESC medium was replaced with one containing 10 mM Y-27632 (Wako) without FGF-2. On culture day 2, 1 μg / mL doxycycline was added to the medium. On culture day 3, the medium was replaced with αMEM (Nacalai Tesque) containing 10% KSR (Invitrogen), 2% Ultroser G, and 100 mM β-mercaptoethanol. On culture day 7, the obtained cells were infected with lentivirus obtained by inserting a DNA fragment encoding AεG into a lentivirus 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 another 3 days to obtain skeletal muscle cells.

[0027] Next, we used a reporter (HB9 e438 We created a lentivirus carrying mRFP (-b-glo-mRFP) and transduced it into motor neurons differentiated from human iPS cells 201B7 (provided by Professor Shinya Yamanaka, Center for iPS Cell Research and Application, Kyoto University). e438 The lentivirus carrying -b-glo-mRFP expresses the 438 base pair enhancer of HB9 (HB9 e438 ) and HB9 expressing the Venus fluorescent protein driven by the human β-globin promoter. e438 -b-glo-Venus(HB9 e438 The iPSCs were generated by replacing Venus with mRFP in the lentivirus (Shimojo et al., Mol.Brain 2015, vol.8 Article No. 79). The differentiation of iPSCs 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).

[0028] iPS cells were dissociated using a dissociation solution containing 0.25% trypsin, 100 μg / mL collagenase IV, 1 mM CaCl2, and 20% KSR, and transferred to a gelatin-coated dish. The dish was then incubated for 1-2 hours in 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), and 0.5% penicillin-streptomycin) to allow feeder cells to fall to the bottom. The supernatant was then collected and cultured in suspension in a bacterial culture dish (this day was designated day 0 of culture). On day 1, 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 BIO (Sigma-Aldrich, USA). On day 2, 1 μM retinoic acid (Sigma-Aldrich, USA) was added. From day 4 to day 14, the cells were cultured in hEB medium containing 1 μM retinoic acid and 1 μM purmorphamine (Calbiochem, Germany), changing the medium every 2-3 days. On day 14-15, the formed hEBs were dissociated and the resulting cells were plated at 5 × 10 4 -1×10 5 pieces / cm 2The cells were seeded at a density of 100 μM and cultured for 1–4 weeks in media hormone mix (MHM) medium or KBM serum-free medium for neural stem cell culture (Kohjin Bio, Japan) 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), and 200 ng / mL ascorbic acid (Sigma-Aldrich, USA). Half of the medium was replaced with fresh medium every 3–4 days.

[0029] In the co-culture of skeletal muscle cells and motor neurons obtained as described above, the medium was MNM, and the muscle endplates markerred by AChRε-GFP and the motor neurons markerred by mRFP were compared with the presence or absence of DHT. In the presence of DHT, the neurodegeneration image significantly increased, and the number of muscle endplates (indicated by arrows in the figure) per RFP-positive motor neuron significantly decreased (Figure 1A, B). In addition, when staining with an antibody against MHC (myosin heavy chain) to examine the state of skeletal muscle cells, there was no difference between skeletal muscle cells derived from SBMA patients and skeletal muscle cells derived from healthy individuals who did not develop SBMA disease (Figure 1A, C). On the other hand, when the number of degenerated nerves in the neurodegeneration image was quantified, the neurodegeneration image of motor neurons was increased in the presence of DHT compared to the absence of DHT (Figure 1D). In addition, immunostaining with cleaved caspase-3 also showed that cell death of motor neurons was enhanced in the presence of DHT compared to the absence of DHT when patient-derived skeletal muscle was used (Figure 1E). Such a phenotype was not observed when skeletal muscle differentiated from iPS cells without mutant androgen receptor genes was co-cultured with motor neurons with mRFP in this example. Thus, when skeletal muscle cells with mutant androgen receptor genes are co-cultured with motor neurons, neurodegeneration is promoted by using a medium containing DHT, making it a better model system for SBMA.

[0030] (Reference Experimental Example 1) In this experimental example, we show that when cell line-derived skeletal muscle cells and motor neurons are co-cultured to form NMJs, the efficiency of NMJ formation is increased by adding three factors, agrin, glutamate, and dihydrotestosterone (DHT), to the culture medium.

[0031] First, a DNA fragment encoding the fusion protein of the adult acetylcholine receptor subunit AChRε and GFP (AChRε-GFP; AεG) (Gensler S et al., Eur J Biochem 2001, vol.268 pp.2209-2217) 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). A human myoblast cell line (Hu5 / E18-AεG) that constitutively expresses the adult acetylcholine receptor was created by selecting the cells with blasticidin S and establishing a cell line.

[0032] Next, human embryoid bodies (hEBs) formed in the same manner as in Example 1 were dissociated into single cells using TrypLE Select (Thermo Fisher Scientific, USA). These cells include motor neurons and motor neuron progenitor cells.

[0033] Skeletal muscle cells induced from the myoblasts thus obtained were co-cultured with cells obtained by dissociating hEBs, including motor neurons and motor neuron precursor cells. The medium used was MNM, and the cells were cultured for 3 days at 5% CO2 and 37°C in a medium containing or not containing 0.1 pM agrin, 100 μM glutamate, and 10 nM dihydrotestosterone (DHT). After culturing, the muscle endplates were observed under a fluorescent microscope using GFP contained in AεG as a marker (Figure 2A). e438 Motor neuron neurites labeled with -b-glo-mRFP contact skeletal muscle with nAChRs that are expressed at the muscle endplate and labeled with GFP to form the NMJ. Thus, at the NMJ, clustering of nAChRs, a marker of muscle endplates, was observed on the skeletal muscle side, and HB9 e438The neurites expressing -b-glo-mRFP were observed to form NMJs. The white arrows indicate the sites where NMJ formation was observed in Fig. 2A. The number of muscle endplates per RFP-positive motor neuron was also calculated and plotted (Fig. 2B).

[0034] As shown in Figure 2, the efficiency of NMJ formation was significantly higher when these three factors were added to the medium. Thus, when co-culturing skeletal muscle cells and motor neurons, the efficiency of NMJ formation is increased by adding these three factors to the medium.

[0035] (Reference Experimental Example 2) In Reference Experimental Example 1, when NMJs were formed by co-culturing skeletal muscle cells derived from a cell line with cells obtained by dissociating hEBs, it was shown that the efficiency of NMJ formation was increased by adding three factors, agrin, glutamate, and dihydrotestosterone (DHT), to the culture medium.In Reference Experimental Example 2, it was shown that the efficiency of NMJ formation was also increased by using these three factors when skeletal muscle cells derived from iPS cells were used.

[0036] First, human iPS cells 409B2 MyoD-hiPSCs were induced to differentiate into skeletal muscle cells by the same method as in Example 1. The resulting skeletal muscle cells were infected with a lentivirus obtained by inserting a DNA fragment encoding AεG into a lentivirus vector to forcibly express AεG.

[0037] On the other hand, 201B7 was induced to differentiate into motor neurons and motor neuron precursor cells in the same manner as in Reference Experimental Example 1.

[0038] These cells were co-cultured in the same manner as in Reference Experimental Example 1. That is, the cells were cultured for 3 days at 5% CO2 and 37°C using MNM medium containing or not containing 0.1 pM agrin, 100 μM glutamic acid, and 10 nM dihydrotestosterone (DHT), and the number of muscle endplates per RFP-positive motor neuron was calculated and graphed.

[0039] As shown in Figure 3, the efficiency of NMJ formation was significantly higher when these three factors were added to the medium and co-cultured, even when using a skeletal muscle cell line derived from iPS cells, as in Reference Experimental Example 1. Thus, when co-culturing skeletal muscle cells and motor neurons, the efficiency of NMJ formation is increased by adding these three factors to the medium.

[0040] (Reference Experimental Example 3) In this experimental example, it is shown that when NMJs are formed by co-culturing skeletal muscle cells and motor neurons, the efficiency of NMJ formation is increased by expressing AChRε in skeletal muscle cells.

[0041] First, 409B2 MyoD-hiPSCs, which are human iPS cells having drug-inducible MyoD, were induced to differentiate into skeletal muscle cells in the same manner as in Example 1, and AεG was forcibly expressed.

[0042] Next, these iPS cell-derived skeletal muscle cells were co-cultured with the iPS cell-derived motor neurons and motor neuron precursor cells prepared in Reference Experimental Example 1. The medium used was MNM, and the co-culture was carried out at 5% CO2 and 37°C. After 3 days, the cells were cultured in HB9 e438 The motor neurons labeled with -b-glo-mRFP were stained with anti-RFP antibody (MBL) and conjugated with Alexa488-conjugated αBTX, and observed under a fluorescent microscope (Figure 4A). The number of NMJs per RFP-positive motor neuron is shown in the graph in Figure 4B.

[0043] As shown in Figure 4, the efficiency of NMJ formation was about four times higher when co-cultured with skeletal muscle cells expressing AεG. Thus, when iPS cell-derived skeletal muscle cells are co-cultured with motor neurons, the efficiency of NMJ formation is increased by expressing AChRε in the skeletal muscle cells.

Claims

1. A co-culture system comprising skeletal muscle cells differentiated from iPS cells derived from a patient with spinal and bulbar muscular atrophy, the cells being co-cultured to form a neuromuscular junction, and motor neurons differentiated from iPS cells, A medium containing testosterone or an analog thereof, A co-culture system, wherein the analog is a ligand for the androgen receptor and has a dissociation constant with the androgen receptor of 10 −5 M or less.

2. The co-culture system described in claim 1, wherein the motor neurons are differentiated iPS cells derived from healthy individuals.

3. The co-culture system described in claim 1, wherein the motor neurons are differentiated iPS cells derived from patients with spinal and bulbar muscular atrophy.

4. The co-culture system according to any one of claims 1 to 3, wherein the testosterone or an analog thereof is exogenously added to the medium.

5. The testosterone analog is 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. The co-culture system according to any one of claims 1 to 4.

6. A co-culture system described in any one of claims 1 to 5, which is an in vitro model system of spinal and bulbar muscular atrophy, wherein the skeletal muscle cells have a mutant androgen receptor gene.

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