JP7697646B2 - Co-culture system, kit, and cell culture medium for skeletal muscle cells and motor neurons - Google Patents

Description

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

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 located 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.

It has been reported that suppressing the expression of mutant androgen receptors 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, suppressing the expression of mutant androgen receptors in 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 mutant AR genes were specifically expressed in motor neurons or skeletal muscle cells of adult mice, although motor neurons contributed to motor dysfunction and some muscle pathology, skeletal muscle cells contributed significantly to 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 is on the motor neuron side or on the skeletal muscle side.

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 muscle expressing mutant androgen receptors compared to skeletal muscle expressing wild-type androgen receptors. Then, when iPS cells derived from healthy subjects were co-cultured with motor neurons differentiated from them, 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).

Sahashi K et al., Hum Mol Genet 2015, vol.24 No.21, pp.5985-5994 Lieberman AP et al., Cell Rep 2014, vol.7 pp.774-784 Ramzan et al., J Neurosci 2015, vol.35 No.16, pp.6444-6451 ISSCR Summary June 2018

The present invention aims to provide an improved co-culture system between skeletal muscle cells and motor neurons.

One embodiment of the present invention is a co-culture system of skeletal muscle cells and motor neurons, in which a medium containing agrin, glutamic acid, and testosterone or an analog thereof is used. At least one selected from the group consisting of agrin, glutamic acid, and testosterone or an analog thereof may be exogenously added. The testosterone analog may be a ligand of the androgen receptor, and may have a dissociation constant with the androgen receptor of 10 ^-5 M or less. The testosterone analogue 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.

Another embodiment of the invention is a kit that includes agrin, glutamic acid, and testosterone or an analog thereof.

A further embodiment of the present invention is a cell culture medium containing agrin, glutamic acid, and testosterone or an analog thereof. At least one selected from the group consisting of agrin, glutamic acid, and testosterone or an analog thereof may be exogenously added.

The present invention provides an improved co-culture system between skeletal muscle cells and motor neurons.

FIG. 1 shows experimental results indicating that, in Example 1, 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 before co-culture. FIG. 13 shows experimental results indicating that, in Example 2, when iPS cell-derived skeletal muscle cells and motor neurons are co-cultured to form NMJs, the efficiency of NMJ formation is increased by co-culturing with the addition of three factors, agrin, glutamic acid, and dihydrotestosterone (DHT), to the medium. FIG. 1 shows experimental results indicating that, when skeletal muscle cells and motor neurons are co-cultured to form NMJs in Reference Experimental Example 1, the efficiency of NMJ formation is increased by expressing AChRε in skeletal muscle cells. In Reference Experimental Example 2, it is shown that when skeletal muscle cells having 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.

The preferred embodiment of the present invention will be described in detail below with reference to the attached drawings, but is not necessarily limited thereto. The objectives, features, advantages, and ideas of the present invention will be clear to those skilled in the art from the description in this specification, and those skilled in the art will be able to easily reproduce the present invention from the description in this specification. The embodiment 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 will be clear 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.

==Co-culture system of skeletal muscle cells and motor neurons==
The co-culture of skeletal muscle cells and motor neurons can be performed by a known method, but in the co-culture system of the present disclosure, a medium containing Agrin, glutamate, and testosterone or an analog thereof is used. At least one selected from the group consisting of Agrin, glutamate, and testosterone or an analog thereof may be exogenously 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 an analog thereof is preferably 1-100 nM, more preferably 3-30 nM, and even more preferably 10 nM. By blending these three components in the medium, the efficiency of NMJ formation can be increased. For convenience, a kit containing Agrin, Glutamate, and testosterone or an analog thereof may be manufactured.

As used herein, a testosterone analog refers to a ligand for the androgen receptor, which has a dissociation constant with the androgen receptor under physiological conditions of 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, and even 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.

Both the skeletal muscle cells and motor neurons used in the co-culture system of the present disclosure are described in detail below, but 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.

==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 may be cells isolated from a living body, may be cell lines already established, or may be produced by in vitro differentiation of pluripotent stem cells such as iPS cells and ES cells or skeletal muscle stem cells into skeletal muscle cells. 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. The differentiation of pluripotent stem cells into skeletal muscle cells can be performed using known methods (e.g., Tanaka A et al., PLoS One 2013, e61540).

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. In skeletal muscle cells isolated from a living body, the acetylcholine receptors are endogenously expressed, so there is no need to express them exogenously, but they may be expressed exogenously to increase the expression level. In the case of differentiation of pluripotent stem cells, the acetylcholine receptors are also expressed, so there is no need to express them exogenously, but they may be expressed exogenously to increase the expression level. In particular, in skeletal muscle cells differentiated from pluripotent stem cells, fetal type (AChRγ) acetylcholine receptors are mainly expressed, but adult type (AChRε) acetylcholine receptors may be expressed exogenously. Details of the case of exogenously expressing the acetylcholine receptor are described below.

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

The skeletal muscle cells used in the co-culture system of the present disclosure may be derived from either males or females. In addition, with respect to the androgen receptor gene, in the case of males, they may be hemizygous with a wild-type allele or hemizygous with a mutant allele, and in the case of females, they 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 heterozygous individual (female) having a wild-type allele and a mutant allele of the androgen receptor gene, a homozygous individual (female) having a mutant allele, or a hemizygous individual (male) having a mutant allele, or may be produced by isolating pluripotent stem cells or skeletal muscle stem cells from an individual having either genotype 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 isolated from a homozygote (female) for a wild-type allele or a hemizygote (male) having a wild-type allele, or into skeletal muscle cells produced by isolating and differentiating pluripotent stem cells or skeletal muscle stem cells from an individual having either genotype, or into skeletal muscle cells obtained by any method regardless of the genotype of the individual from which they were derived. Alternatively, skeletal muscle cells may be isolated from a transgenic animal into which a mutant androgen receptor gene has been introduced, or skeletal muscle cells may be isolated and differentiated into pluripotent stem cells or skeletal muscle stem cells.

In the present disclosure, the wild-type androgen receptor gene refers to an allele of the androgen receptor gene that does not develop spinal-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 is possessed by an individual who develops SBMA 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.

==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 mammals such as humans, mice, rats, and pigs, but is preferably humans. The motor neurons may be cells isolated from a living body, may be cell lines already established, 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 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 motor neurons (e.g., Shimojo et al., Mol.Brain 2015, vol.8 Article No. 79).

The motor neurons used in the co-culture system of the present disclosure may be of male or female origin, and with respect to the androgen receptor gene, in the case of males, they may be hemizygous for a wild-type allele or hemizygous for a mutant allele, and in the case of females, they may be homozygous for a wild-type allele, homozygous for a mutant allele, or heterozygous for a wild-type allele and a mutant allele.

A motor neuron having a mutant androgen receptor gene may be prepared by isolating a motor neuron 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, or by isolating pluripotent stem cells or neural stem cells from an individual having any of these genotypes and differentiating them into a motor neuron. Alternatively, a mutant androgen receptor gene may be exogenously introduced into a motor neuron isolated from a homozygote (female) having a wild-type allele or a hemizygote (male) having a wild-type allele, or into a motor neuron prepared by isolating and differentiating pluripotent stem cells or neural stem cells from an individual having any of these genotypes, or into a motor neuron obtained by any method regardless of the genotype of the individual from which it was derived. Alternatively, a motor neuron having a mutant androgen receptor gene may be isolated from a transgenic animal into which a mutant androgen receptor gene has been introduced, or by isolating pluripotent stem cells or neural stem cells and differentiating them into a motor neuron.

In the above, when there is a step of introducing a gene into a pluripotent cell to express the gene and a step of differentiating the pluripotent cell into a specific cell type, either the gene introduction or the cell differentiation can be performed first.

==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 are as described above. The genotypes of the androgen receptor gene are 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 a skeletal muscle cell that does not have a mutant androgen receptor gene and a motor neuron that does not have a mutant androgen receptor gene, a normal number and shape of NMJs are formed, but in the case of a skeletal muscle cell that has a mutant androgen receptor gene and a motor neuron that does not have a mutant androgen receptor gene, a decrease in the number of NMJs and cell death of the motor neurons are observed. Therefore, under these conditions, the androgen receptor gene of the skeletal muscle cells is considered to be the responsible gene for the decrease in the number of NMJs and cell death of the motor neurons. The latter system is considered to be a model system for SBMA.

Example 1
In this example, we show that when NMJs are formed by co-culturing cell line-derived skeletal muscle cells and motor neurons, the efficiency of NMJ formation is increased by adding three factors, agrin, glutamic acid, and dihydrotestosterone (DHT), to the medium during co-culturing.

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 to obtain an expression vector, which was then 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.

Next, a lentivirus carrying 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, Institute for iPS Cell Research and Application, Kyoto University). The lentivirus carrying HB9 ^e438 -b-glo-mRFP was prepared by replacing Venus with mRFP in HB9 ^e438 -b-glo-Venus (HB9 ^e438 ::Venus) lentivirus (Shimojo et al., Mol.Brain 2015, vol.8 Article No. 79), which expresses Venus fluorescent protein using the 438 base pair HB9 enhancer (HB9 ^e438 ) and human β-globin promoter. In addition, differentiation of iPS cells into motor neurons was carried out as follows (Shimojo et al., Mol.Brain 2015, vol.8 Article No. 79; Onodera et al., Mol.Brain 2020, vol.13 Article No. 18).

iPS cell colonies were detached using a dissociation solution containing 0.25% trypsin, 100 μg/mL collagenase IV, 1 mM CaCl ₂ , 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 using a bacterial culture dish (this day was designated as day 0 of culture). On the first day 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 the second day of culture, 1 μM retinoic acid (Sigma-Aldrich, USA) was further added and cultured. From the fourth to the 14th day of culture, the cells were cultured in hEB medium containing 1 μM retinoic acid and 1 μM purmorphamine (Calbiochem, Germany), with the medium being replaced once every 2-3 days. On day 14-15 of culture, the formed human embryoid bodies (hEBs) were dissociated into single cells using TrypLE Select (Thermo Fisher Scientific, USA), which contain motor neurons and motor neuron progenitor cells.

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 at 5% CO ₂ and 37°C for 3 days using a medium containing 0.1 pM agrin, 100 μM glutamic acid, and 10 nM dihydrotestosterone (DHT) or not. The muscle endplate was then observed under a fluorescent microscope using GFP contained in AεG as a marker (Figure 1A). The neurites of motor neurons labeled with ^{HB9 e438} -b-glo-mRFP contact skeletal muscle with nAChRs expressed at the muscle endplate and labeled with GFP, forming NMJs. Thus, at the NMJ, clustering of nAChR, a marker of muscle endplates, was observed on the skeletal muscle side, where neurites expressing HB9 ^e438 -b-glo-mRFP were observed to connect. In Figure 1A, the site where NMJ formation was observed is indicated by a white arrow. In addition, the number of muscle endplates per RFP-positive motor neuron was calculated and graphed (Figure 1B).

As shown in Figure 1, the efficiency of NMJ formation was significantly higher when these three factors were added to the medium and co-cultured. Thus, when co-cultured with skeletal muscle cells and motor neurons, the efficiency of NMJ formation is increased by adding these three factors to the medium.

Example 2
In Example 1, when NMJs are 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 can be increased by adding three factors, agrin, glutamic acid, and dihydrotestosterone (DHT), to the medium when co-culturing. In this Example 2, it is shown that the efficiency of NMJ formation can also be increased by using iPS cell-derived skeletal muscle cells by using these three factors.

First, 409B2 MyoD-hiPSCs, which are human iPSCs with drug-inducible MyoD, were used to induce differentiation into skeletal muscle cells, and AεG was forced to be expressed. Specifically, the procedure is as follows. 409B2 MyoD-hiPSCs cultured using StemFit AK02N (Takara, Japan) were dissociated using Accutase, and the cells were seeded on a dish coated with Matrigel (BD Biosciences) at a density of ^2x104 cells/ ^cm2 without feeder cells using StemFit+Y27632 medium (this day is considered to be 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 the third day of culture, the medium was replaced with αMEM (Nacalai Tesque) containing 10% KSR (Invitrogen), 2% Ultroser G, and 100 mM β-mercaptoethanol. On the seventh day of culture, the obtained cells were infected with a 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 the cells were cultured for another 3 days to obtain skeletal muscle cells.

On the other hand, 201B7 was used to induce differentiation into motor neurons and motor neuron progenitor cells in the same manner as in Example 1.

These cells were co-cultured in the same manner as in Example 1. That is, the cells were cultured for 3 days at 37°C with 5% CO ₂ 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.

As shown in Figure 2, as in Example 1, even when using a skeletal muscle cell line derived from iPS cells, the efficiency of NMJ formation was significantly higher when co-cultured with these three factors added to the medium. Thus, when co-cultured skeletal muscle cells and motor neurons, the efficiency of NMJ formation is increased by adding these three factors to the medium.

(Reference Experimental Example 1)
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.

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

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. The medium used was MNM, and the cells were co-cultured at 5% CO ₂ and 37°C. After 3 days, the motor neurons labeled with HB9 ^e438 -b-glo-mRFP were stained with anti-RFP antibody (MBL), bound with Alexa488-conjugated αBTX, and observed under a fluorescent microscope (Figure 3A). The number of NMJs per RFP-positive motor neuron is shown in the graph in Figure 3B.

As shown in Figure 3, the efficiency of NMJ formation was approximately 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.

(Reference Experimental Example 2)
In this experimental example, we show that when skeletal muscle cells with mutant androgen receptors are co-cultured with motor neurons, the use of a medium containing DHT promotes neurodegeneration, providing a better model system for SBMA.

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).

Fibroblasts were harvested from the dermis of SBMA patients. They were cultured in DMEM containing 10% FBS, and 6 × ¹⁰⁵ cells were 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 culture was continued. When the colonies became large enough, they were isolated and expanded.

The drug-inducible MyoD vector was introduced into the iPS cells thus obtained together with Transposase using ViaFect ^TM Transfection Reagent (Promega). Human iPS cells that constitutively express drug-inducible MyoD, MyoD-hiPSCs, were produced by selecting with neomycin (G418) and establishing clones. After making the cells feeder-free, the 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 lentivirus vector, and AεG was forcibly expressed.

The resulting skeletal muscle cells were then co-cultured with motor neurons bearing mRFP. Motor neurons were prepared by seeding dissociated hEBs obtained in the same manner as in Example 1 at a density of 5×10 ⁴ -1×10 ⁵ cells/cm ² on a dish coated with mouse laminin (Thermo Fisher Scientific, USA) and culturing the cells in a media hormone mix 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). The cells were cultured for 1-4 weeks in motor neuron medium (MNM) consisting of MHM medium or serum-free medium for KBM neural stem cell culture (Kohjin Bio, Japan). Half of the medium was replaced with fresh medium every 3-4 days.

In the co-culture, MNM medium was used, and muscle endplates marked by AChRε-GFP and motor neurons marked by mRFP were compared with and without DHT. In the presence of DHT, neurodegeneration images were significantly increased, and the number of muscle endplates (indicated by arrows in the figure) per RFP-positive motor neuron was significantly decreased (Figure 4A, B). In addition, when staining with an antibody against MHC (myosin heavy chain) to examine the state of skeletal muscle cells, skeletal muscle cells derived from SBMA patients did not differ from skeletal muscle cells derived from healthy subjects without SBMA disease (Figure 4A, C). On the other hand, when the number of degenerated nerves in the neurodegeneration images was quantified, the neurodegeneration images of motor neurons were increased in the presence of DHT compared to the absence of DHT (Figure 4D). 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 4E). Such a phenotype was not observed when skeletal muscle differentiated from iPS cells not having a mutant androgen receptor gene was co-cultured with motor neurons having mRFP, as prepared in Example 1. Thus, when skeletal muscle cells having a mutant androgen receptor gene are co-cultured with motor neurons, neurodegeneration is promoted by using a medium containing DHT, providing a better model system for SBMA.

Claims (3)

A medium additive kit for co-culture of skeletal muscle cells and motor neurons, comprising Agrin, glutamic acid, and testosterone or an analog thereof,
The co-culture medium additive kit , wherein the testosterone analog is a ligand for an androgen receptor and has a dissociation constant with the androgen receptor of 10 ⁻⁵ M or less. A medium for co-culturing skeletal muscle cells and motor neurons, comprising Agrin, glutamic acid, and testosterone or an analog thereof ,
A co-culture medium , wherein the testosterone analog is a ligand for an androgen receptor and has a dissociation constant with the androgen receptor of 10 ⁻⁵ M or less. 3. The medium for co-culturing skeletal muscle cells and motor neurons according to claim 2 , wherein at least one selected from the group consisting of Agrin, glutamic acid, and testosterone or an analog thereof is exogenously added.

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