Method for producing skeletal muscle cells

Abstract

The present invention provides: a method for producing a skeletal muscle cell of which the contraction activity can be induced by an optical stimulation, the method comprising (1) a step for producing a pluripotent stem cell capable of stably expressing channelrhodopsin, (2) a step for differentiating / inducing the cell produced in step (1) into a skeletal muscle cell, and (3) a step for irradiating the cell of which the differentiation / induction is initiated with light; and a method for screening for a substance capable of promoting or suppressing the contraction activity of a skeletal muscle cell, the method comprising (1) a step for differentiating / inducing a pluripotent stem cell capable of stably expressing channelrhodopsin into a skeletal muscle cell, (2) a step for irradiating the cell with light, (3) a step for bringing the cell into contact with each of test substances, (4) a step for measuring the contraction activity of the cell, and (5) a step for selecting a test substance that promotes or suppresses the contraction activity of the cell more effectively compared with the case where the cell is not brought into contact with the test substance.

Description

[Technical Field]

[0001] The present invention relates to a method for producing skeletal muscle cells whose contractile activity is induced by light stimulation, and a method for screening for substances that promote or inhibit the contractile activity of skeletal muscle cells. [Background technology]

[0002] Muscle diseases encompass a wide range of conditions, but the majority of symptoms are muscle atrophy and the resulting loss of muscle strength. Muscle atrophy can be caused by abnormalities in the muscles themselves or in the nerves that control the muscles. The former are called myopathies, and the latter are called neuropathic diseases. Muscular dystrophy is a well-known example of myopathy. Duchenne muscular dystrophy, the most common type of muscular dystrophy, is caused by mutations (point mutations, deletion mutations, duplication mutations, etc.) in the dystrophin gene, which prevents the synthesis of normal dystrophin protein. It is a sex-chromosomal recessive disorder that affects only boys, with an estimated incidence of 3-5 cases per 100,000 people and 1 per 2,000-3,000 male births. There are currently no effective treatments for many myopathies, including Duchenne muscular dystrophy, and the development of a treatment is highly anticipated.

[0003] Developing therapeutic drugs requires in vitro models that reflect human pathologies. The recent development of induced pluripotent stem cells (IPSCs), generated by reprogramming somatic cells, has raised hopes for the use of cells derived from a patient's own cells as pathological models. Skeletal muscle cells are one such type of cell, and various efforts have been made to establish methods for inducing differentiation of induced pluripotent stem cells into skeletal muscle cells. The present inventors have reported that differentiation into skeletal muscle cells can be induced by introducing a tetracycline (Tet)-inducible transcription factor (MyoD or Myf5) into pluripotent stem cells and then continuously adding doxycycline (Dox) from day 1 onward to express the transcription factor in the pluripotent stem cells (Patent Document 1). The present inventors have also improved the above-mentioned method for inducing differentiation into skeletal muscle cells, and have reported that differentiation into skeletal muscle cells can be efficiently and reproducibly induced by continuously adding Dox from day 1 of differentiation induction onwards, thereby expressing exogenous MyoD, and then reseeding the cells in a medium containing 5% knockout serum replacement (KSR) on days 3 to 4 (Non-Patent Document 1). Shoji E et al. have also reported a method for inducing differentiation of pluripotent stem cells into skeletal muscle cells by inducing differentiation in a medium containing 20% ​​knockout serum replacement (KSR), and continuously adding Dox from day 1 of differentiation induction onwards, thereby expressing exogenous MyoD (Non-Patent Document 2).

[0004] Unlike cardiac muscle cells, skeletal muscle cells generally do not contract spontaneously and require external stimuli for contractile responses. Stimulation is particularly necessary for cultured cells to maintain functionality and mature, and electrical stimulation systems are often used (Non-Patent Document 3). Electrical stimulation systems can easily control muscle cell activation, but they are known to emit toxic gases, limiting the strength and duration of electrical stimulation. In recent years, optogenetics technology has emerged as an alternative to electrical stimulation. Optogenetics is a technology that involves expressing a light-activated ion channel protein called channelrhodopsin in cells, which activates them in response to specific light, and this technology is now being used to stimulate muscle cells (Non-Patent Document 4). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2013 / 073246 [Non-patent literature]

[0006] [Non-Patent Document 1] Uchimura T. et al., Stem Cell Research, 25:98-106 (2017) [Non-patent document 2] Shoji E. et al., Science Reports, 5:12831 (2015) [Non-patent document 3] Nedachi et al., Am J Physiol Endocrinol Metab., (2008) [Non-patent document 4] Asano et al., Sie Rep., (2015) Summary of the Invention [Problem to be solved by the invention]

[0007] To develop a screening system that uses the contractile activity of skeletal muscle cells induced to differentiate from pluripotent stem cells as an indicator, it is necessary to stimulate the cells. However, conventional electrical stimulation has the drawback of hydrolyzing the culture medium, generating gases and reactive oxygen species that can damage the cells. Furthermore, it is difficult to create a device that can uniformly apply electrical stimulation to a large number of wells (e.g., a 96- or 384-well plate), making it unsuitable for high-throughput screening.

[0008] Therefore, the present invention aims to provide a method for producing skeletal muscle cells from pluripotent stem cells that have contractile activity equivalent to that achieved using electrical stimulation, and to provide a method for screening candidate substances for therapeutic drugs for muscle diseases using the contractile activity of the skeletal muscle cells obtained as an indicator. [Means for solving the problem]

[0009] The present invention includes the following inventions to solve the above problems. [1] A method for producing skeletal muscle cells in which contractile activity is induced by light stimulation, comprising: (1) a step of producing pluripotent stem cells that stably express channelrhodopsin; (2) inducing differentiation of the cells obtained in step (1) into skeletal muscle cells; and (3) A step of irradiating the cells in which differentiation induction has been initiated with light. A manufacturing method comprising: [2] The manufacturing method according to [1] above, wherein in step (3), light irradiation is initiated before the stage at which cells fuse to form myotubes. [3] The manufacturing method according to [1] or [2] above, wherein in the step (3), the irradiation is continuous from the start to the end of the light irradiation. [4] The method according to any one of [1] to [3] above, wherein the light irradiated onto the cells is blue laser light having a frequency of 0.25 Hz to 0.75 Hz, a pulse width of 2 msec to 100 msec, a voltage of 8 V to 15 V, and a light intensity of 1 mW to 10 mW. [5] The method according to any one of [1] to [4] above, wherein the pluripotent stem cells in step (1) are cells derived from a patient with myopathy or myotonia. [6] The method of any one of [1] to [5] above, wherein the pluripotent stem cells in step (1) are cells that express one or more exogenous skeletal cell-inducing factors selected from MyoD and Myf5. [7] A method for screening for a substance that promotes or inhibits the contractile activity of skeletal muscle cells, comprising: (1) inducing differentiation of pluripotent stem cells stably expressing channelrhodopsin into skeletal muscle cells; (2) irradiating the cells with light; (3) contacting the test substance with the cells; (4) measuring the contractile activity of the cells; and (5) A step of selecting a test substance that promotes or inhibits the contractile activity of cells compared to when the test substance is not contacted. A method comprising: [8] The screening method according to [7] above, wherein a multi-well plate is used. [9] The screening method according to [7] or [8] above, wherein the pluripotent stem cells in step (1) are cells derived from a myopathy or myotonia patient. [Effects of the Invention]

[0010] The present invention provides a method for producing skeletal muscle cells from pluripotent stem cells that have contractile activity equivalent to that achieved by electrical stimulation. The skeletal muscle cells obtained by this method can be used in high-throughput screening of candidate substances for the treatment of muscle diseases, using the contractile activity of skeletal muscle cells as an index. [Brief explanation of the drawings]

[0011] [Figure 1]Figure 1 shows the structures of vectors introduced into iPS cells. (A) shows the structure of the tetracycline-responsive MyoD-forced expression piggyBac vector (pB-EF1α-MyoD-IRES-puro), and (B) shows the structure of the channelrhodopsin-forced expression piggyBac vector (pB-EF1α-ChR2-IRES-puro). [Figure 2] FIG. 1 shows a differentiation induction schedule for one embodiment of the production method of the present invention used in the Examples. [Figure 3] FIG. 1 is a diagram showing a cross section of one well of a 96-well plate used in the Examples. [Figure 4] FIG. 3 shows the results of a flat test in which cells on day 14 of the schedule shown in FIG. 2 were immunostained with an anti-MHC antibody to calculate the skeletal muscle differentiation efficiency. [Figure 5] FIG. 3 shows the results of a flat test in which cells on day 18 of the schedule shown in FIG. 2 were immunostained with an anti-MHC antibody to calculate the skeletal muscle differentiation efficiency. [Figure 6] FIG. 1 shows how to use wells in a 96-well plate when two wells are used for one sample and when four wells are used for one sample. [Figure 7] The contractile force of skeletal muscle cells on day 18 of the schedule shown in Figure 2 was analyzed using a video analyzer, and a flat test was performed on the contraction speed of each sample, with 2 or 4 wells as one sample. (A) shows the results for one sample with 2 wells, and (B) shows the results for one sample with 4 wells. [Figure 8] This figure shows the results of an analysis of the change in contraction speed due to electrical stimulation and light stimulation for skeletal muscle cells on days 18 to 28 of the schedule shown in Figure 2, where (A) shows the results of electrical stimulation and (B) shows the results of light stimulation. [Figure 9] This figure shows the results of an analysis of the change in relaxation rate due to electrical and optical stimulation of skeletal muscle cells on days 18 to 28 of the schedule shown in Figure 2, where (A) shows the results of electrical stimulation and (B) shows the results of optical stimulation. [Figure 10]This figure shows the results of analyzing the changes in acceleration due to electrical stimulation and light stimulation for skeletal muscle cells on days 18 to 28 of the schedule shown in Figure 2, where (A) shows the results of electrical stimulation and (B) shows the results of light stimulation. [Figure 11] This figure shows the results of an analysis of the change in contraction distance due to electrical and optical stimulation of skeletal muscle cells on days 18 to 28 of the schedule shown in Figure 2, where (A) shows the results of electrical stimulation and (B) shows the results of optical stimulation. [Figure 12] FIG. 3 shows the results of analyzing the contractile force of skeletal muscle cells on day 28 of the schedule shown in FIG. 2 using a video analyzer, and conducting a flat test on the contractile speed using one sample in four wells. DETAILED DESCRIPTION OF THE INVENTION

[0012] [Method for producing skeletal muscle cells] The present invention provides a method for producing skeletal muscle cells in which contractile activity is induced by light stimulation (hereinafter referred to as the "production method of the present invention"). The production method of the present invention may include the following steps: (1) a step of producing pluripotent stem cells that stably express channelrhodopsin; (2) inducing differentiation of the cells obtained in step (1) into skeletal muscle cells; and (3) A step of irradiating the cells in which differentiation induction has been initiated with light.

[0013] As used herein, the term "skeletal muscle cell" is a broad concept that encompasses all cells of the skeletal muscle lineage, including skeletal muscle stem cells, myoblasts, myotubes, mature myotubes, and fully mature myotubes. Marker genes for identifying skeletal muscle cells include, for example, myogenin, myosin heavy chain (MHC), MyoD, and Myf5. Skeletal muscle cells may be human skeletal muscle cells or skeletal muscle cells of organisms other than humans. The organisms other than humans are not particularly limited and may be, for example, mammals. Examples of mammals include monkeys, chimpanzees, dogs, cats, cows, horses, pigs, rabbits, mice, and rats.

[0014] The pluripotent stem cells used in the production method of the present invention may be pluripotent stem cells derived from a patient with a muscle disease. Examples of muscle diseases include myopathy and myotonia. Examples of myopathy include muscular dystrophies (Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, limb-girdle muscular dystrophy, facioscapulohumeral muscular dystrophy, oculopharyngeal muscular dystrophy, Emery-Dreifuss muscular dystrophy, congenital muscular dystrophy, distal muscular dystrophy, myotonic dystrophy, etc.), distal myopathies (Miyoshi myopathy, GNE myopathy, oculopharyngeal distal myopathy, etc.), congenital myopathies (nemaline myopathy, central core disease, etc.), glycogen storage disease, periodic paralysis, and mitochondrial myopathy. Myotonia includes grasping myotonia, percussion myotonia, myotonia congenita, paramyotonia congenita, and Schwartz-Jampel syndrome.

[0015] The pluripotent stem cells used in the production method of the present invention may be pluripotent stem cells modified to overexpress a skeletal muscle cell inducing factor. Specific examples include pluripotent stem cells into which an expression vector containing a gene encoding a skeletal muscle cell inducing factor has been introduced. Examples of skeletal muscle cell inducing factors include MyoD, Myf5, and Pax7. Preferably, exogenous MyoD or Myf5 is used. Expression vectors containing genes encoding MyoD or Myf5 can be constructed using known genetic engineering techniques. The resulting expression vector can be introduced into pluripotent stem cells using known gene transfer methods described below.

[0016] The expression vector into which the gene encoding MyoD or Myf5 has been inserted may be a drug-inducible (e.g., tetracycline-inducible) expression vector. The nucleotide sequence of the gene encoding MyoD or Myf5 can be obtained from a publicly known database (e.g., NCBI). For example, the nucleotide sequence of the gene encoding human MyoD (Homo sapiens myogenic differentiation 1 (MYOD1), mRNA) is registered as NCBI Reference Sequence: NM_002478.5. Furthermore, for example, the gene encoding human Myf5 (Homo sapiens myogenic factor 5 (MYF5), mRNA) is registered as NCBI Reference Sequence: NM_005593.3.

[0017] Pluripotent stem cells that can be used in the production methods of the present invention are stem cells that have pluripotency, meaning they can differentiate into all cells present in the body, and also have the ability to proliferate, and include, but are not limited to, embryonic stem (ES) cells, cloned embryo-derived embryonic stem (ntES) cells obtained by nuclear transfer, sperm stem (GS) cells, embryonic germ (EG) cells, induced pluripotent stem (iPS) cells, pluripotent cells derived from cultured fibroblasts or bone marrow stem cells (Muse cells), etc. Preferred pluripotent stem cells are ES cells, ntES cells, and iPS cells.

[0018] (A) Embryonic stem cells ES cells are stem cells that are established from the inner cell mass of early mammalian embryos (for example, blastocysts) such as humans and mice, and have the ability to proliferate through pluripotency and self-renewal.

[0019] ES cells are embryonic stem cells derived from the inner cell mass of a blastocyst, an embryo at the eight-cell stage of a fertilized egg, or after the morula stage. They possess the ability to differentiate into any cell type that makes up an adult, known as pluripotency, and the ability to proliferate through self-renewal. ES cells were discovered in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292:154-156), and subsequently, ES cell lines were established in humans, monkeys, and other primates (JA Thomson et al. (1998), Science 282:1145-1147; JA Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92:7844-7848; JA Thomson et al. (1996), Biol. Reprod., 55:254-259; JA Thomson and VS Marshall (1998), Curr. Top. Dev. Biol., 38:133-165).

[0020] ES cells can be established using methods known in the art. For example, ES cells can be established by isolating the inner cell mass from the blastocyst of a fertilized egg of a target animal and culturing the inner cell mass on a fibroblast feeder. Furthermore, cells can be maintained by subculture in a culture medium supplemented with substances such as leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF). Methods for establishing and maintaining human and monkey ES cells are described, for example, in US Pat. No. 5,843,780; Thomson JA, et al. (1995), Proc. Natl. Acad. Sci. USA 92:7844-7848; Thomson JA, et al. (1998), Science. 282:1145-1147; H. Suemori et al. (2006), Biochem. Biophys. Res. Commun., 345:926-932; M. Ueno et al. (2006), Proc. Natl. Acad. Sci. USA, 103:9554-9559; H. Suemori et al. (2001), Dev. Dyn., 222:273-279; H. Kawasaki et al. (2002), Proc. Natl. Acad. Sci. USA, 99:1580-1585; Klimanskaya I, et al. (2006), Nature. 444:481-485, etc.

[0021] Culture methods for preparing ES cells are known in the art. Human ES cells can be maintained at 37°C in a humidified atmosphere of 2% CO₂ / 98% air using DMEM / F-12 medium supplemented with, for example, 0.1 mM 2-mercaptoethanol, 0.1 mM non-essential amino acids, 2 mM L-glutamic acid, 20% KSR (KnockOut Serum Replacement, Invitrogen), and 4 ng / ml bFGF (Fumitaka O. et al. (2008), Nat. Biotechnol., 26:215-224). ES cells may be passaged every 3 to 4 days using, for example, 0.25% trypsin and 0.1 mg / ml collagenase IV in PBS containing 1 mM CaCl₂ and 20% KSR.

[0022] ES cells can generally be selected by real-time PCR using the expression of gene markers such as alkaline phosphatase, Oct-3 / 4, and Nanog as indicators. In particular, human ES cells can be selected using the expression of gene markers such as OCT-3 / 4, NANOG, and ECAD as indicators (E. Kroon et al. (2008), Nat. Biotechnol., 26:443-452).

[0023] Mouse ES cell lines established by inGenious, Inc., RIKEN, and other organizations are available. Human ES cell lines established by the National Institutes of Health (NIH), RIKEN, Kyoto University, and Cellartis are available. Examples of ES cell lines include NIH strains CHB-1 to CHB-12, RUES1, RUES2, and HUES1 to HUES28; WisCell Research Institute strains WA01(H1) and WA09(H9); and RIKEN strains KhES-1, KhES-2, KhES-3, KhES-4, KhES-5, SSES1, SSES2, and SSES3. Furthermore, KhES-1, KhES-2, KhES-3, and KthES11 are available from the Kyoto University Institute for Virus Research and Frontier Medical Sciences (Kyoto, Japan).

[0024] (B) Sperm stem cells Spermatogonial stem cells (SSCs) are pluripotent stem cells derived from the testis and are the source of spermatogenesis. Similar to embryonic stem cells, these cells can be induced to differentiate into various cell lineages. For example, their transplantation into mouse blastocysts can lead to the generation of chimeric mice (M. Kanatsu-Shinohara et al. (2003) Biol. Reprod., 69:612-616; K. Shinohara et al. (2004), Cell, 119:1001-1012). SSCs are capable of self-renewal in culture media containing glial cell line-derived neurotrophic factor (GDNF). Furthermore, SSCs can be obtained by repeated passage under similar culture conditions to those for embryonic stem cells (Takebayashi, M. et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Special Issue), pp. 41-46, Yodosha, Tokyo, Japan).

[0025] (C) Embryonic germ cells Embryonic germ cells are cells established from primordial germ cells during the fetal stage and have pluripotency similar to that of ES cells. They can be established by culturing primordial germ cells in the presence of substances such as LIF, bFGF, and stem cell factor (Y. Matsui et al. (1992), Cell, 70:841-847; J.L. Resnick et al. (1992), Nature, 359:550-551).

[0026] (D) Induced pluripotent stem cells Induced pluripotent stem (iPS) cells are artificial stem cells derived from somatic cells that can be created by introducing specific reprogramming factors into somatic cells in the form of DNA or protein. They have properties similar to those of ES cells, such as pluripotency and the ability to proliferate through self-renewal (K. Takahashi and S. Yamanaka (2006) Cell, 126:663-676; K. Takahashi et al. (2007), Cell, 131:861-872; J. Yu et al. (2007), Science, 318:1917-1920; Nakagawa, M. et al., Nat. Biotechnol.26:101-106 (2008); International Publication WO 2007 / 069666). Reprogramming factors may be composed of genes specifically expressed in ES cells, their gene products or non-coding RNAs, or genes that play an important role in maintaining the undifferentiated state of ES cells, their gene products or non-coding RNAs, or low-molecular-weight compounds. Examples of genes included in reprogramming factors include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, and Glis1. These reprogramming factors may be used alone or in combination. Combinations of reprogramming factors include WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 075119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 / 101407, WO2009 / 102983, WO2009 / 114949, WO2009 / 117439, WO2009 / 126250, WO2009 / 126251, WO2009 / 126655, W O2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 050626, WO 2010 / 056831, WO2010 / 068955, WO2010 / 098419, WO2010 / 102267, WO 2010 / 111409, WO 2010 / 111422, WO2010 / 115050, WO2010 / 124290, WO2010 / 147395, WO2010 / 147612, Huangfu D, et al. (2008), Nat. Biotechnol., 26: 795-797, Shi Y, et al. (2008), Cell Stem Cell, 2: 525-528, Eminli S, et al.(2008), Stem Cells. 26:2467-2474, Huangfu D, et al. (2008), Nat Biotechnol. 26:1269-1275, Shi Y, et al. (2008), Cell Stem Cell, 3, 568-574、Zhao Y, et al. (2008), Cell Stem Cell, 3:475-479、Marson A, (2008), Cell Stem Cell, 3, 132-135、Feng B, et al. (2009), Nat Cell Biol. 11:197-203、RL Judson et al., (2009), Nat. Biotech., 27:459-461、Lyssiotis CA, et al. (2009), Proc Natl Acad Sci US A. 106:8912-8917、Kim JB, et al. (2009), Nature. 461:649-643, Ichida JK, et al. (2009), Cell Stem Cell. 5:491-503、Heng JC, et al. (2010), Cell Stem Cell. 6:167-74、Han J, et al. (2010), Nature. 463:1096-100、Mali P, et al. (2010), Stem Cells. 28:713-720, Maekawa M, et al. (2011), Nature. 474:225-9.

[0027] The reprogramming factors include histone deacetylase (HDAC) inhibitors (e.g., small molecule inhibitors such as valproic acid (VPA), trichostatin A, sodium butyrate, MC 1293, M344, etc., nucleic acid expression inhibitors such as siRNA and shRNA against HDAC (e.g., HDAC1 siRNA Smartpool (Millipore), HuSH 29mer shRNA Constructs against HDAC1 (OriGene)), etc.), MEK inhibitors (e.g., PD184352, PD98059, U0126, SL327, and PD0325901), glycogen synthase kinase-3 inhibitors (e.g., Bio and CHIR99021), DNA methyltransferase inhibitors (e.g., 5-azacytidine), histone methyltransferase inhibitors (e.g., BIX-01294, small molecule inhibitors such as those listed above, nucleic acid expression inhibitors such as siRNA and shRNA against Suv39hl, Suv39h2, SetDBl and G9a, L-channel calcium agonists (e.g., Bayk8644), butyric acid, TGFβ inhibitors or ALK5 inhibitors (e.g., LY364947, SB431542, 616453 and A-83-01), p53 inhibitors (e.g., siRNA and shRNA against p53), ARID3A inhibitors (e.g., siRNA and shRNA against ARID3A), miRNAs such as miR-291-3p, miR-294, miR-295 and mir-302, Wnt signaling inhibitors (e.g., soluble These factors also include factors used to improve establishment efficiency, such as Wnt3a, neuropeptide Y, prostaglandins (e.g., prostaglandin E2 and prostaglandin J2), hTERT, SV40LT, UTF1, IRX6, GLIS1, PITX2, and DMRTBl, and in this specification, these factors used to improve establishment efficiency will not be distinguished from reprogramming factors.

[0028] When the reprogramming factor is in the form of a protein, it may be introduced into somatic cells by techniques such as lipofection, fusion with a cell membrane-permeable peptide (eg, HIV-derived TAT and polyarginine), or microinjection.

[0029] On the other hand, in the case of DNA, for example, vectors such as viruses, plasmids, artificial chromosomes, lipofection, liposomes, microinjection, etc. can be used to introduce them into somatic cells. Examples of viral vectors include retroviral vectors, lentiviral vectors (Cell, 126, pp.663-676, 2006; Cell, 131, pp.861-872, 2007; Science, 318, pp.1917-1920, 2007), adenoviral vectors (Science, 322, 945-949, 2008), adeno-associated virus vectors, and Sendai virus vectors (WO 2010 / 008054). Examples of artificial chromosome vectors include human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), and bacterial artificial chromosomes (BAC, PAC). The plasmid may be a mammalian cell plasmid (Science, 322:949-953, 2008). The vector may contain regulatory sequences such as a promoter, enhancer, ribosome-binding sequence, terminator, and polyadenylation site to enable expression of the nuclear reprogramming substance. Furthermore, if necessary, the vector may contain a drug resistance gene (e.g., a kanamycin resistance gene, an ampicillin resistance gene, a puromycin resistance gene, etc.), a selection marker sequence such as a thymidine kinase gene or a diphtheria toxin gene, or a reporter gene sequence such as green fluorescent protein (GFP) or β-glucuronidase (GUS). Furthermore, the vector may have LoxP sequences before and after the gene encoding the reprogramming factor or the promoter and the gene encoding the reprogramming factor that binds to it, in order to excise both the gene and the promoter after introduction into somatic cells.

[0030] Furthermore, when in the form of RNA, it may be introduced into somatic cells by techniques such as lipofection or microinjection, and RNA incorporating 5-methylcytidine and pseudouridine (TriLink Biotechnologies) may be used to suppress degradation (Warren L, (2010) Cell Stem Cell. 7:618-630).

[0031] Examples of culture media for inducing iPS cells include DMEM, DMEM / F12, or DME culture media containing 10-15% FBS (these culture media may further contain LIF, penicillin / streptomycin, puromycin, L-glutamine, non-essential amino acids, β-mercaptoethanol, etc., as appropriate), as well as commercially available culture media such as culture media for mouse ES cells (TX-WES culture medium, Thrombo-X), culture media for primate ES cells (culture medium for primate ES / iPS cells, ReproCell), and serum-free pluripotent stem cell maintenance media (e.g., mTeSR (Stemcell Technology), Essential 8 (Life Technologies), StemFit AK03 (AJINOMOTO)).

[0032] As an example of a culture method, for example, somatic cells are contacted with reprogramming factors in DMEM or DMEM / F12 culture medium containing 10% FBS at 37°C in the presence of 5% CO2 and cultured for approximately 4 to 7 days, and then the cells are plated on feeder cells (e.g., mitomycin C-treated STO cells, SNL cells, etc.) and cultured in a bFGF-containing culture medium for primate ES cell culture from approximately 10 days after contacting the somatic cells with the reprogramming factors, and iPS-like colonies can be generated approximately 30 to 45 days or more after the contact.

[0033] Alternatively, ES-like colonies can be generated after approximately 25 to 30 days or more of culture on feeder cells (e.g., mitomycin C-treated STO cells, SNL cells, etc.) in 10% FBS-containing DMEM medium (which may further contain LIF, penicillin / streptomycin, puromycin, L-glutamine, non-essential amino acids, β-mercaptoethanol, etc.) at 37°C in the presence of 5% CO2. Desirably, somatic cells to be reprogrammed themselves can be used instead of feeder cells (Takahashi K, et al. (2009), PLoS One. 4:e8067 or WO2010 / 137746), or extracellular matrix (e.g., Laminin-5 (WO2009 / 123349) and Matrigel (BD)).

[0034] Another example is a method of culturing iPS cells using a serum-free medium (Sun N, et al. (2009), Proc Natl Acad Sci USA. 106:15720-15725). Furthermore, to increase the efficiency of establishment, iPS cells may be established under hypoxic conditions (oxygen concentration of 0.1% or more and 15% or less) (Yoshida Y, et al. (2009), Cell Stem Cell. 5:237-241 or WO2010 / 013845).

[0035] During the above culture, the culture medium is replaced with fresh medium once a day from the second day onwards. The number of somatic cells used for nuclear reprogramming is not limited, but it is recommended to use a 100cm culture dish. 2 Approximately 5 x 10 3 ~Approx. 5×10 6 It is the range of cells.

[0036] iPS cells can be selected based on the morphology of the colonies they form. On the other hand, if a drug-resistance gene that is expressed in conjunction with a gene expressed when somatic cells are reprogrammed (e.g., Oct3 / 4 or Nanog) is introduced as a marker gene, established iPS cells can be selected by culturing them in a culture medium containing the corresponding drug (selective culture medium). Furthermore, if the marker gene is a fluorescent protein gene, iPS cells can be selected by observing them under a fluorescent microscope; if the marker gene is a luciferase gene, by adding a luminescent substrate; and if the marker gene is a chromogenic enzyme gene, by adding a chromogenic substrate.

[0037] As used herein, the term "somatic cells" refers to any animal cell (e.g., a mammalian cell, including a human cell) excluding germline cells such as eggs, oocytes, and ES cells, or totipotent cells. Somatic cells include, but are not limited to, fetal (offspring) somatic cells, neonatal (offspring) somatic cells, and mature, healthy or diseased somatic cells. They also include primary culture cells, passaged cells, and established cell lines. Specifically, somatic cells include, for example, (1) tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells; (2) tissue progenitor cells; and (3) differentiated cells such as lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (e.g., skin cells), hair cells, hepatocytes, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells (e.g., exocrine pancreatic cells), brain cells, lung cells, kidney cells, and adipocytes.

[0038] Furthermore, when iPS cells and / or cells differentiated therefrom are used as a source of transplant cells, it is desirable to use somatic cells with the same or substantially the same HLA genotype as the recipient individual, from the viewpoint of preventing rejection. Here, "substantially the same" HLA type means that the HLA genotype matches to an extent that the transplanted cells can engraft when transplanted, for example, somatic cells with an HLA type that matches the main HLA (three loci: HLA-A, HLA-B, and HLA-DR, or four loci including HLA-C).

[0039] As the induced pluripotent stem cell line, various iPS cell lines established by NIH, RIKEN, Kyoto University, etc. may be used. For example, human iPS cell lines include RIKEN's HiPS-RIKEN-1A line, HiPS-RIKEN-2A line, HiPS-RIKEN-12A line, and Nips-B2 line, and Kyoto University's Ff-WJ-18 line, Ff-I01s01 line, Ff-I01s02 line, Ff-I01s04 line, Ff-I01s06 line, Ff-I14s03 line, and Ff-I14s line. Examples of such cell lines include strains 04, QHJI01s01, QHJI01s04, QHJI14s03, QHJI14s04, AK5, TkDN-Sev2, 692D2, 253G1, 201B7, 409B2, 454E2, 606A1, 610B1, 648A1, 1231A3, 1390D4, and 1390C1. Alternatively, clinical-grade cell lines provided by Kyoto University, Cellular Dynamics International, etc., and research and clinical cell lines prepared using such cell lines may also be used.

[0040] (E) ES cells derived from cloned embryos (ntES cells) obtained by nuclear transfer. ntES cells are ES cells derived from cloned embryos produced by nuclear transfer technology and have almost the same properties as ES cells derived from fertilized eggs (T. Wakayama et al. (2001), Science, 292:740-743; S. Wakayama et al. (2005), Biol. Reprod., 72:932-936; J. Byrne et al. (2007), Nature, 450:497-502). Specifically, ntES (nuclear transfer ES) cells are established from the inner cell mass of blastocysts derived from cloned embryos obtained by replacing the nucleus of an unfertilized egg with that of a somatic cell. To generate ntES cells, a combination of nuclear transfer technology (JB Cibelli et al. (1998), Nature Biotechnol., 16:642-646) and ES cell generation technology (mentioned above) is used (Wakayama Sayaka et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Special Issue), pp. 47-52). In nuclear transfer, the nucleus of a somatic cell is injected into an enucleated unfertilized mammalian egg, and the egg can be reprogrammed by culturing for several hours.

[0041] (F) Multilineage-differentiating Stress Enduring cells (Muse cells) Muse cells are pluripotent stem cells produced by the method described in WO2011 / 007900. Specifically, they are pluripotent cells obtained by treating fibroblasts or bone marrow stromal cells with trypsin for a long period of time, preferably 8 or 16 hours, followed by suspension culture, and are positive for SSEA-3 and CD105.

[0042] Step (1) is a step of preparing pluripotent stem cells stably expressing channelrhodopsin. Channelrhodopsin is a light-activated ion channel isolated from algae and has the property of taking up cations upon light irradiation. Examples of channelrhodopsins that can be used in the production method of the present invention include channelrhodopsin 1, channelrhodopsin 2, and modified channelrhodopsins (e.g., modified channelrhodopsins described in WO2011 / 019081 or WO2020 / 059675). Channelrhodopsin 2 is preferred. Examples of channelrhodopsins that can be used in the production method of the present invention include, but are not limited to, those derived from Chlamydomonas reinhardtii, Volvox carteri, Tetraselmis subcordiformis, and Tetraselmis striata.

[0043] Channelrhodopsin 2 is a protein consisting of an amino acid sequence identical or substantially identical to the amino acid sequence shown in SEQ ID NO: 1, for example. The amino acid sequence shown in SEQ ID NO: 1 has been registered in GenBank under accession number AAM15777.1. An example of an amino acid sequence substantially identical to the amino acid sequence shown in SEQ ID NO: 1 is an amino acid sequence in which 1 to 10 amino acids have been deleted, substituted, inserted, or added in the amino acid sequence shown in SEQ ID NO: 1. Preferably, the amino acid sequence is an amino acid sequence in which 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid has been deleted, substituted, inserted, or added. Alternatively, an amino acid sequence substantially identical to the amino acid sequence set forth in SEQ ID NO: 1 is an amino acid sequence having 60% or more identity (e.g., 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity) to the amino acid sequence set forth in SEQ ID NO: 1. Identity can be determined using known methods as the degree of identity between two sequences. For example, various alignment algorithms and / or programs, including FASTA, BLAST, or ENTREZ, may be used. FASTA and BLAST are available as part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.) and can be used, for example, with default settings. ENTREZ is available through the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD. A protein consisting of an amino acid sequence substantially identical to the amino acid sequence shown in SEQ ID NO: 1 is preferably a protein having substantially the same activity as channelrhodopsin 2 consisting of the amino acid sequence shown in SEQ ID NO: 1. Specifically, the light-driven cation channel activity (light wavelength, ion permeability, etc.) is equivalent to the light-driven cation channel activity of channelrhodopsin 2 consisting of the amino acid sequence shown in SEQ ID NO: 1.

[0044] The nucleotide sequence of the gene encoding channelrhodopsin 2 is, for example, a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1, but is not particularly limited thereto. For example, it may be the nucleotide sequence shown in SEQ ID NO: 2. The nucleotide sequence shown in SEQ ID NO: 2 is registered in GenBank under accession number AF461397.1. The gene encoding channelrhodopsin may have humanized codons. Alternatively, the gene encoding channelrhodopsin 2 may be a gene encoding a protein consisting of an amino acid sequence substantially identical to the amino acid sequence shown in SEQ ID NO: 1.

[0045] Pluripotent stem cells stably expressing channelrhodopsin can be prepared by introducing an expression vector into which a gene encoding channelrhodopsin has been inserted, and then selecting clones that stably express channelrhodopsin. The gene encoding channelrhodopsin can be obtained by PCR or other methods based on its sequence information. It can also be chemically synthesized.

[0046] Examples of expression vectors that can be used include viruses, plasmids, and artificial chromosomes. Examples of viral vectors include retrovirus vectors, lentivirus vectors, adenovirus vectors, adeno-associated virus vectors, and Sendai virus vectors. Examples of artificial chromosome vectors include human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), and bacterial artificial chromosomes (BACs and PACs). Examples of plasmids include plasmids for mammalian cells. The vector can contain regulatory sequences such as promoters, enhancers, ribosome binding sequences, terminators, and polyadenylation sites to enable expression of DNA encoding channelrhodopsin. If necessary, the vector can further contain selectable marker sequences such as drug resistance genes (e.g., kanamycin resistance genes, ampicillin resistance genes, and puromycin resistance genes), thymidine kinase genes, and diphtheria toxin genes, as well as reporter gene sequences such as fluorescent proteins and β-glucuronidase (GUS). Examples of promoters include the SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney mouse leukemia virus) LTR, HSV-TK (herpes simplex virus thymidine kinase) promoter, EF-α promoter, and CAG promoter.

[0047] The above-mentioned vector may have transposon sequences before and after the expression cassette (a gene expression unit including a promoter, a gene sequence, and a terminator) for inserting a nucleic acid encoding a channelrhodopsin into a chromosome or for excising the nucleic acid inserted into a chromosome as needed. Examples of transposon sequences include, but are not limited to, piggyBac. To introduce an expression cassette into a chromosome using a transposon, it is desirable to introduce a transposase into the same cell as a vector carrying the expression cassette. In the present invention, to introduce a transposase, the nucleic acid encoding the transposase may be contained in the aforementioned vector, or a nucleic acid encoding the transposase may be contained in another vector and introduced into the cell simultaneously. Furthermore, a gene product encoding the transposase may be directly introduced. In the present invention, a preferred transposase is a transposase corresponding to the above-mentioned transposon sequence, preferably piggyBac transposase.

[0048] When a viral vector is used, DNA or RNA encoding channelrhodopsin is introduced into the above-mentioned virus, and the pluripotent stem cells are infected with this recombinant virus, thereby introducing a gene encoding channelrhodopsin into the pluripotent stem cells. When a non-viral vector is used, methods of introduction using liposomes (liposome method, HVJ-liposome method, cationic liposome method, lipofection method, lipofectamine method, etc.), microinjection method, calcium phosphate method, electroporation method, and method of transferring into cells together with a carrier (metal particles) using a gene gun can be used.

[0049] Methods for selecting pluripotent stem cells that stably express channelrhodopsin include, for example, transfection with an expression vector containing a drug resistance gene, followed by culturing the cells in a drug-containing medium and selecting surviving colonies.

[0050] Step (2) is a step of inducing the differentiation of the cells obtained in step (1) into skeletal muscle cells. The method for inducing the differentiation of pluripotent stem cells stably expressing channelrhodopsin into skeletal muscle cells is not particularly limited, and any method appropriately selected from known methods for inducing the differentiation of pluripotent stem cells into skeletal muscle cells can be used.

[0051] The method for inducing differentiation of pluripotent stem cells into skeletal muscle cells may involve overexpressing a skeletal muscle cell-inducing factor in the pluripotent stem cells. Examples of such differentiation induction methods include the methods described in International Publication WO 2013 / 073246 A1, International Publication WO 2020 / 090836 A1, Uchimura et al. (Stem Cell Research, 25:98-106 (2017)), and Shoji et al. (Science Reports, 5:12831 (2015)). Furthermore, the method for inducing differentiation of pluripotent stem cells into skeletal muscle cells may be a method that mimics the fetal developmental process without using a transgene (a method for inducing differentiation of skeletal muscle lineage cells via paraxial mesoderm cells, somite cells, and dermomyotome cells). Examples of such differentiation induction methods include the method described by Zhao et al. (Stem Cell Reports, Vol. 15 1-15 July 14, 2020), the method described in International Publication WO2016 / 108288 A1, and the method described by Hicks et al. (Nat Cell Biol. 2018 Jan;20(1):46-57).

[0052] It has been confirmed that when skeletal muscle cells whose contractile activity is induced by light stimulation, produced by the production method of the present invention, are seeded in a multiwell plate and induced to differentiate, the variability in skeletal muscle differentiation efficiency between wells is very low, and the variability in the contractile force of skeletal muscle cells between wells is also very low. Therefore, in step (2), pluripotent stem cells may be seeded in a multiwell plate to induce differentiation. Examples of multiwell plates include 24-well plates, 48-well plates, 96-well plates, and 384-well plates. A 96-well plate or a 384-well plate is preferred. Skeletal muscle cells produced using the production method of the present invention using a multiwell plate can be suitably used, for example, for high-throughput screening of candidate substances for therapeutic agents for muscle diseases.

[0053] Although the multiwell plate is not particularly limited, it is preferable to use a plate with a culture surface that does not restrict cell contractile activity. Examples of such plates include plates with a hydrogel culture surface. Examples of hydrogels include gelatin hydrogel, collagen hydrogel, starch hydrogel, pectin hydrogel, hyaluronic acid hydrogel, chitin hydrogel, chitosan hydrogel, and alginate hydrogel. Among these, collagen hydrogel or gelatin hydrogel is preferred. The gel hardness (elastic modulus) of the hydrogel may be 10 kPa or more (10 kPa, 11 kPa, 12 kPa, 13 kPa, 14 kPa, or 15 kPa or more) or 25 kPa or less (25 kPa, 20 kPa, 19 kPa, 18 kPa, 17 kPa, 16 kPa, or 15 kPa or less). 12 kPa is preferred. A plate with a hydrogel culture surface may be a plate whose culture surface is coated with hydrogel, or a plate whose culture surface has recesses for placing hydrogel, and the recesses are filled with hydrogel.

[0054] Step (3) is a step of irradiating the cells after initiating differentiation induction with light. The initiation of differentiation induction is when the medium for the pluripotent stem cells is replaced with a differentiation medium, or, in the case of a differentiation induction method in which a skeletal muscle cell induction factor expression vector is introduced into the pluripotent stem cells, when the expression of the skeletal muscle cell induction factor begins or when the medium is replaced with a differentiation medium, whichever occurs first.

[0055] In step (3), the timing of starting light irradiation is not particularly limited, and light irradiation may be started simultaneously with the initiation of differentiation induction or any time after the initiation of differentiation induction. Light irradiation is preferably started before the stage at which cells fuse to form myotubes, and preferably within 4 days, 3 days, 2 days, or 1 day after the initiation of differentiation induction. Whether the cells have not fused to form myotubes can be confirmed by observing the cell morphology under a microscope.

[0056] The end time of light irradiation is not particularly determined, but it is preferable to continue light irradiation until the differentiation-induced skeletal muscle cells begin contractile activity and the test using contractile activity as an indicator is completed. For example, when skeletal muscle cells produced by the production method of the present invention are used to screen candidate substances for muscle disease therapeutic agents, it is preferable to continue light irradiation until the screening is completed. During the light irradiation period, light irradiation may be performed intermittently or continuously. When light irradiation is performed intermittently, the on / off ratio is not particularly limited, and may be, for example, 0.02 to 0.9, 0.05 to 0.5, or 0.05 to 0.1. Continuous irradiation is preferred.

[0057] When pluripotent stem cells stably expressing channelrhodopsin 2 are used, a blue laser is used as the light to be irradiated. The frequency of the irradiated light is 0.25 Hz to 0.75 Hz, preferably 0.4 Hz to 0.6 Hz, and more preferably 0.5 Hz. The pulse width of the irradiated light is 2 msec to 100 msec, preferably 5 msec to 50 msec. The voltage is 8 V to 15 V, preferably 10 V. The light intensity is 1 mW to 10 mW, preferably 2 mW to 3 mW. It is preferable to use a light irradiation device that can uniformly irradiate all wells of the multiwell plate used. For example, a device that can uniformly irradiate the entire bottom surface of the multiwell plate with light can be used. An example of such a device is the Optogenetics LED Array System (BRC Bioresearch Center).

[0058] The fact that skeletal muscle cells obtained by the method of the present invention undergo contractile activity in response to light stimulation can be confirmed using a commercially available imaging system (e.g., Sony's Live Cell Imaging System SI8000, etc.) that is capable of analyzing dynamic images of cells.

[0059] [Screening method] The present invention provides a method for screening for a substance that promotes or inhibits the contractile activity of skeletal muscle cells (hereinafter referred to as the "screening method of the present invention"). The screening method of the present invention may comprise the following steps: (1) inducing differentiation of pluripotent stem cells stably expressing channelrhodopsin into skeletal muscle cells; (2) irradiating the cells with light; (3) contacting the cells with a test substance; and (4) A step of selecting a test substance that promotes or inhibits the contractile activity of skeletal muscle cells compared to when the test substance is not contacted.

[0060] Steps (1) and (2) in the screening method of the present invention can be carried out in the same manner as steps (2) and (3) in the production method of the present invention. In other words, the screening method of the present invention can be said to be a screening method that uses skeletal muscle cells produced by the production method of the present invention.

[0061] The screening method of the present invention can be used for high-throughput screening using a multiwell plate, preferably a 96-well plate or a 384-well plate. Furthermore, the screening method of the present invention can select a desired test substance using the contractile activity of skeletal muscle cells as an index. When using a 96-well or 384-well plate, two or four wells may be evaluated as one sample. By using multiple wells as one sample, variability between samples can be reduced.

[0062] The pluripotent stem cells in step (1) may be pluripotent stem cells derived from a healthy individual or from a patient with a muscle disease. The muscle disease may be myopathy or myotonia. Skeletal muscle cells obtained by inducing differentiation from pluripotent stem cells derived from a myopathy patient are suitable for screening methods for substances that promote the contractile activity of skeletal muscle cells, and skeletal muscle cells obtained by inducing differentiation from pluripotent stem cells derived from a healthy individual or a patient with myotonia are suitable for screening methods for substances that promote the contractile activity of skeletal muscle cells.

[0063] Step (3) is a step of contacting a test substance with cells. The test substance is not particularly limited, and examples thereof include nucleic acids, peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, cell culture supernatants, plant extracts, mammalian tissue extracts, and plasma. The test substance may be a novel substance or a known substance. These test substances may form salts. Salts of test substances with physiologically acceptable acids or bases are used.

[0064] The timing of contacting the cells with the test substance may be before or after the cells have begun to contract. Contact between the cells and the test substance can be achieved by adding the test substance to the culture medium. When multiple wells are used as one sample, the same test substance is added to multiple wells. Specifically, for example, when two wells are used as one sample, the same test substance is added to two wells, and when four wells are used as one sample, the same test substance is added to four wells.

[0065] Step (4) is a step of measuring the contractile activity of the cells. The contractile activity of the cells can be measured using a commercially available imaging system (e.g., Sony's Live Cell Imaging System SI8000) that can analyze dynamic images of cells. Measurement items include contraction velocity, relaxation velocity, acceleration, and contraction distance.

[0066] In the screening method of the present invention, when selecting a test substance that promotes cellular contractile activity, skeletal muscle cells induced to differentiate from pluripotent stem cells derived from a myopathy patient are typically used. It has been shown that skeletal muscle cells induced to differentiate from pluripotent stem cells derived from a myopathy patient experience a decrease in contractile force once the maximum contractile force is observed (see Examples). Therefore, when using skeletal muscle cells induced to differentiate from pluripotent stem cells derived from a myopathy patient, it is preferable to contact the cells with the test substance immediately before or on the day the maximum contractile force is observed, and it is preferable to measure the contractile activity of the cells once the contractile force has decreased. Cell contractile activity may be measured twice: once when the maximum contractile force is observed and once when the contractile force has decreased. By measuring twice, the rate of decrease in contractile force can be evaluated.

[0067] In the screening method of the present invention, when selecting a test substance that inhibits the contractile activity of cells, skeletal muscle cells differentiated from pluripotent stem cells derived from healthy individuals or pluripotent stem cells derived from myotonia patients are typically used. In this case, the test substance is preferably contacted immediately before or on the day when the maximum contractile force is observed, and the contractile activity of the cells is preferably measured multiple times over time starting immediately after contact with the test substance. By measuring multiple times over time, it is possible to evaluate how the effect of inhibiting contractile activity manifests itself over the long term.

[0068] In the screening method of the present invention, cells that have not been contacted with a test substance are used as a control, and in step (4), the contractile activity of the control cells is also measured in the same manner.

[0069] Step (5) is a step of selecting a test substance that promotes or inhibits the contractile activity of cells compared to when the test substance is not contacted. When selecting a test substance that promotes the contractile activity of cells, for example, if contraction rate is used as an index, a test substance that restores the contraction rate of control cells that have not been contacted with the test substance to 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more may be selected. Alternatively, skeletal muscle cells induced to differentiate from pluripotent stem cells derived from healthy individuals may be used as healthy control cells, and a test substance that restores the contraction rate to a level close to that of the healthy control may be selected.

[0070] When selecting a test substance that inhibits the contractile activity of cells, for example, if contraction rate is used as an indicator, a test substance that inhibits the contraction rate of control cells that have not been contacted with the test substance by 10% or less, 20% or less, 30% or less, 40% or less, or 50% or less may be selected.

[0071] Test substances selected as substances that promote cellular contractile activity are useful as candidate active ingredients for preventive or therapeutic drugs for myopathy, and test substances selected as substances that inhibit cellular contractile activity are useful as candidate active ingredients for preventive or therapeutic drugs for myotonia. [Example]

[0072] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0073] [Experimental Method] 1. Culturing human iPS cells The DMD (Duchenne muscular dystrophy) iPS cell line (clone ID: CiRA00111, hereafter referred to as "DMDΔ44") was established using an episomal vector system from skin fibroblasts of a DMD patient with a deletion of exon 44 of the dystrophin gene (Okita et al., Stem Cells, 31; 458-466, 2012). Human iPS cells were maintained in a feeder-free environment. The maintenance medium used was 500 mL of StemFit (Ajinomoto Co.) supplemented with 50 mU / L penicillin and 50 μg / L streptomycin (Invitrogen). For the maintenance of human iPS cells after transfection with the Tet vector (described below) or ChR2 vector (described below), medium supplemented with 100 μg / mL puromycin and blasticidin (Invitrogen) was used. Cells were passaged when the cell colonies reached 80-90% confluence. Cells were detached using the cell dissociation solution Accutase (Funakoshi) and then collected with a scraper. They were then seeded onto newly laminin-coated plates (Nippi) in medium supplemented with ROCK inhibitor Y-27632 (Nacalai Tesque) (hereafter referred to as "ROCK inhibitor Y") and cultured in an incubator at 37°C, 5% CO2, and 100% humidity.

[0074] 2. Construction of tetracycline-responsive gene expression vector (Tet vector) The piggyBac vectors used for forced expression of tetracycline-responsive genes were KW111 (Addgene Plasmid #80475) or KW879 (Addgene Plasmid #80478, see Induced Pluripotent Stem (iPS) Cells pp. 111-131), developed by Woltjen et al. (Woltjen K. et al., Nature 458, 766, 2009). These vectors incorporate both a reverse tetracycline transactivator (rtTA) and a tetracycline-responsive element (TRE). KW879 allows drug selection via a puromycin resistance gene. These vectors were mixed with the pENTR / D-TOPO-MyoD (or Myf5) entry vector, and recombination using LR clonase (Invitrogen) was performed to generate the tetracycline-responsive MyoD-expressing piggyBac vector (pB-EF1α-MyoD-IRES-puro, hereafter referred to as "pB-Tet-MyoD") shown in Figure 1(A). The pENTR / D-TOPO-MyoD entry vector and pENTR / D-TOPO-Myf5 entry vector are entry vectors in which MyoD or Myf5 cDNA has been inserted, respectively, into pENTR / D-TOPO (Thermo Fisher Scientific, catalog number K240020).

[0075] 3. Construction of channelrhodopsin (ChR2) forced expression vector (ChR2 vector) To construct the channelrhodopsin-expressing piggyBac vector, the ChR2 gene sequence was inserted into the pENTR / D-TOPO entry vector (Thermo Fisher Scientific, catalog number K240020) by PCR cloning to create the pENTR / D-TOPO-ChR2 vector. The piggyBac-EF1α-IRES-blastcidin vector (SEQ ID NO: 3) and the pENTR / D-TOPO-ChR2 vector (entry vector) were then mixed and recombined using LR clonase (Invitrogen) to generate the ChR2-expressing piggyBac vector (pB-EF1α-ChR2-IRES-Bsr, hereafter referred to as "pB-ChR2") shown in Figure 1(B). This vector allows for drug selection via the blasticidin resistance gene.

[0076] 4. Vector introduction into iPS cells and selection of transformed cells A 10-cm dish of iPS cell clones (DMD-Δ44) derived from a DMD patient was prepared. The cells were cultured in a medium containing ROCK inhibitor Y starting the day before vector transfection. The plated cells and vector were then transfected by electroporation, similar to the maintenance culture. Five micrograms each of pB-Tet-MyoD, pB-ChR, and a vector incorporating transposase downstream of the EF1α promoter (EF1α-PBase) were prepared and dissolved in 100 μl of Opti-MEM (Invitrogen). 1.0 × 10 6 The cells were suspended in Opti-MEM containing the vector and transfected with the vector using a NEPA21 electroporator (Nepagene) under the conditions shown in Table 1. The transfected cells were diluted to 1.0 × 10 3 ~5.0×10 4 The cells were seeded onto a 6-well plate at 100 cells / well. After 48 hours, the medium was changed to one containing 100 μg / ml blasticidin and puromycin (Nacalai Tesque). Thereafter, the medium was changed to drug-containing medium every two days, and drug-resistant transformed cells were selected.

[0077] [Table 1]

[0078] 5. Selection of transformed cell clones The obtained clones were seeded onto a 6-well plate coated with Matrigel (Invitrogen) diluted 100-fold with medium. The number of cells seeded was 1.0 × 10 3 ~5.0×10 4 The cells were cultured at 1 μg / ml per well. After 48 hours, doxycycline (Dox; LKT Laboratories) was added to the medium at 1 μg / ml. Four days after Dox addition, clones with high differentiation efficiency were selected from the cells induced to skeletal muscle cells based on the state of cells with multiple nuclei and an elongated morphology, characteristic of myotubes.

[0079] 6. Tet-MyoD induces differentiation of iPS cells into skeletal muscle cells (see Figure 2) The clones (Tet-MyoD iPS cells) selected in step 5 above were seeded onto Matrigel-coated plates using StemFit medium containing ROCK inhibitor Y (day 0). The number of cells seeded was 1.0 × 10 3 ~5.0×10 4The media was PECM (Reprocell) supplemented with ROCK inhibitor Y on day 1, and then PECM supplemented with 0.4 μg / mL to 1.5 μg / mL Dox on day 2. 48 hours later, on day 4, the cells were reseeded into assay-compatible plates (e.g., μ-Plate Angiogenesis 96 Well (Ibidi, catalog number 89646) described below). The media used was αMEM (Nacalai Tesque) supplemented with 2% (v / v) horse serum (HS: Invitrogen), 100 μM 2-mercaptoethanol, insulin, SB431542 (all from Wako Pure Chemical Industries), glucose (Invitrogen), and 50 mU / L penicillin / 50 μg / L streptomycin. After reseeding, the cells were cultured in Dox-free media for 2 days, after which 1 μg / mL Dox was re-supplemented. Immunostaining was performed between days 14 and 28 of culture to confirm whether differentiation into skeletal muscle cells had been induced.

[0080] 7. Light-stimulated maturation of differentiated skeletal muscle cells On Day 4, cells were reseeded onto a 96-well μ-Plate Angiogenesis plate coated with collagen gel (Nippi Co., Ltd.). From Day 10, maturation promotion by light stimulation began (see Figure 3). Light stimulation was continued until each test was performed. The number of reseeded cells was 1.0 × 10 3 ~5.0×10 4 The cells were cultured at 100 cells / well. A blue LED array system for optogenetics (BRC Bioresearch Center) was used as a light stimulator, and stimulation was performed at a frequency of 0.5 Hz, a pulse width of 50 msec, and a voltage of 10 V for 24 hours every day. The culture medium was changed at least once every two days.

[0081] 8. Immunostaining of Differentiated Skeletal Muscle Cells Differentiated cells were fixed with 2% paraformaldehyde (Nacalai Tesque) in PBS for 10 minutes at 4°C, washed three times with PBS for 5 minutes, and then destained for 15 minutes with methanol (Nacalai Tesque) in 1% hydrogen peroxide (Wako). They were then washed three times with PBS for 5 minutes at 4°C. Blocking was performed with BlockingOne (Nacalai Tesque) for 15 minutes at 4°C. The primary antibody used was anti-MHC (mouse monoclonal, R&D, diluted 1:400) in the blocking solution. The cells were incubated for 16–18 hours at 4°C, washed three times with PBS containing 0.2% Triton X-100 (PBST), and then incubated for 16–18 hours with anti-mouse IgG-Alexa644 (Molecular Probes) diluted 1:500 in PBST as the secondary antibody. To stain the cell nuclei, 5 μg / ml DAPI (Sigma) was diluted 5000-fold in PBST and incubated at room temperature for 5 minutes. After washing three times with PBS, the cells were observed using an Opera Phenix high-throughput, high-content imaging system (PerkinElmer). The skeletal muscle differentiation efficiency was calculated using the following formula: (number of nuclei stained with DAPI on MHC-positive cells / total number of nuclei stained with DAPI). The skeletal muscle differentiation efficiency for each well was calculated, and a flat test was performed to calculate the mean, standard deviation (SD), and coefficient of variation (CV).

[0082] 9.SI8000 video analysis device The contractile activity of differentiated skeletal muscle cells was analyzed using the SI8000 live cell imaging system (SONY). Contractile activity was measured using the C-PaceEP and C-Dish systems (IonOptics) with a frequency of 0.5 Hz, a pulse width of 2 msec, and a voltage of 10 V. Images were captured for 10 seconds using the SI8000 at 27 frames / sec for a total of 270 frames, and the speed and distance of contractile activity were analyzed using the SI8000 software.

[0083] [Experimental results] 1. Skeletal muscle differentiation efficiency of cells on day 14 The cells on day 14 were fixed and immunostained with anti-MHC antibodies to calculate the skeletal muscle differentiation efficiency. The results of the flat test are shown in Figure 4. The top panel is a heat map of the skeletal muscle differentiation efficiency (%) for each well of a 96-well plate, and the bottom panel is a table showing the mean, standard deviation (SD), and coefficient of variation (CV). The cells on day 14 were skeletal muscle cells that had not yet begun contractile activity. The mean skeletal muscle differentiation efficiency for each well was 98.02%, and the coefficient of variation was 2.84, demonstrating that the skeletal muscle cells obtained using the production method of the present invention fully met the screening quality standard. Note that a coefficient of variation of less than 10 is considered to meet the screening quality standard (Iversen et al., Assay Guidance Manual, 2004).

[0084] 2. Skeletal muscle differentiation efficiency of cells on day 18 The cells on day 18 were fixed and immunostained with anti-MHC antibodies to calculate the skeletal muscle differentiation efficiency, and the results of a flat test are shown in Figure 5. The top row is a heat map of the skeletal muscle differentiation efficiency (%) for each well of a 96-well plate, and the bottom row is the mean, standard deviation (SD), and coefficient of variation (CV). Cells on day 18 are skeletal muscle cells that have already begun contractile activity and are thought to be at the stage when they exert the most contractile force. The mean skeletal muscle differentiation efficiency for each well was 93.67%, and the coefficient of variation was 3.91, demonstrating that the skeletal muscle cells obtained using the production method of the present invention fully met the quality requirements for screening.

[0085] 3. Screening methods for evaluating the contractile force of skeletal muscle cells A flat test was performed by analyzing the contractile force of skeletal muscle cells on day 18 of culture using a video analyzer. To determine the minimum number of wells required for analysis as one sample to maintain screening accuracy, we tried analyzing two or four wells as one sample (see Figure 6). Figure 7 shows the results of evaluating contraction velocity. (A) shows the results for one sample from two wells, and (B) shows the results for one sample from four wells. Although the coefficient of variation (CV) was lower for one sample from four wells, the CV values ​​for both two and four wells were below the standard of 10, suggesting that analyzing at least two wells as one data point is appropriate.

[0086] 4. Changes in contractile force of skeletal muscle cells due to electrical and optical stimulation The changes in contractile force of skeletal muscle cells on days 18, 21, and 28 of culture were compared with those observed when electrical stimulation was applied. Electrical stimulation was initiated on day 6 of culture using a C-Pace EM (IonOptics) and a C-Dish 6-well plate (IonOptics) at a frequency of 0.5 Hz with a pulse width of 2 msec. The number of cells seeded was 1.0 × 10 5 ~5.0×10 5 The voltage was gradually increased, ultimately reaching 20 V. Specifically, stimulation was at 2 V for the first 48 hours, 5 V for the next 48 hours, 10 V for the next 72 hours, and 15 V for the next 72 hours, and then continuously at 20 V until day 28 of culture. Electrical stimulation was evaluated in one sample of three wells, and light stimulation was evaluated in one sample of four wells of a 96-well plate.

[0087] The results of comparing electrical and optical stimulation are shown in Figures 8 to 11. Figure 8 shows the results for contraction velocity, Figure 9 shows the results for relaxation velocity, Figure 10 shows the results for acceleration, and Figure 11 shows the results for contraction distance. In each figure, (A) shows the results for electrical stimulation, and (B) shows the results for optical stimulation. The change in contractile force due to electrical stimulation was evaluated using skeletal muscle cells on days 19, 21, and 27 of culture. As shown in Figures 8 to 11, maximum contractile force was observed on day 19 of culture (electrical stimulation) and day 18 of culture (optical stimulation), indicating that the maximum contractile force with optical stimulation was equivalent to that with electrical stimulation. Subsequently, on day 27 of culture (electrical stimulation) and day 28 of culture (optical stimulation), the contractile force decreased to less than half of its maximum value, indicating that the degree of decrease was equivalent between electrical and optical stimulation. In other words, skeletal muscle cells with equivalent contractile force could be produced using light stimulation instead of electrical stimulation, and it was shown that skeletal muscle cells that do not express dystrophin, such as DMDΔ44, showed a decrease in contractile force after the maximum contractile force was observed, regardless of whether they were stimulated electrically or by light stimulation.

[0088] To confirm that skeletal muscle cells with reduced contractile force meet the screening quality standard, a flat test was performed in which the contractile force of skeletal muscle cells exposed to light stimulation on day 28 of culture was analyzed using a video analyzer. The results of evaluating contraction velocity are shown in Figure 12. Cells on day 28 of culture with reduced contractile force were shown to fully meet the screening quality standard when screening for test substances that promote the contractile activity of skeletal muscle cells.

[0089] 5. Light-stimulated maturation of differentiated skeletal muscle cells On Day 4, cells were reseeded onto a CellCarrier-Ultra 384 well, lined with collagen gel (Nippi) and coated with Matrigel. Light stimulation to promote maturation began on Day 10, and light stimulation was continued until each test was performed. An optogenetics blue LED array system (BRC BioResearch Center) was used as the light stimulation device, providing stimulation at a frequency of 0.5 Hz, a pulse width of 50 msec, and a voltage of 10 V for 24 hours, every day. The medium was changed at least every two days. A light diffusion sheet was placed between the array system and the plate to ensure uniform light stimulation to each well. Myogenic differentiation efficiency, contractile activity, and the minimum number of wells required to maintain screening accuracy were evaluated using a 96-well plate.

[0090] The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope of the claims. The technical scope of the present invention also includes embodiments obtained by appropriately combining the technical means disclosed in different embodiments. Furthermore, all academic literature and patent documents described in this specification are incorporated herein by reference.

Claims

1. A method for producing skeletal muscle cells in which contractile activity is induced by light stimulation, comprising: (1) preparing pluripotent stem cells that stably express channelrhodopsin; (2) inducing the differentiation of the cells obtained in step (1) into skeletal muscle cells; and (3) A step of irradiating the cells in which differentiation induction has been initiated with light wherein the light irradiated onto the cells is blue laser light having a frequency of 0.25Hz to 0.75Hz, a pulse width of 2msec to 100msec, a voltage of 8V to 15V, and a light intensity of 1mW to 10mW.

2. The method according to claim 1 , wherein in step (3), light irradiation is initiated before the stage in which the cells fuse to form myotubes.

3. The method according to claim 1 or 2, wherein in the step (3), the light irradiation is continuous from the start to the end of the light irradiation.

4. The method according to any one of claims 1 to 3, wherein the pluripotent stem cells in step (1) are cells derived from a patient with myopathy or myotonia.

5. The method according to any one of claims 1 to 4, wherein the pluripotent stem cells in step (1) are cells that express one or more exogenous skeletal cell-inducing factors selected from MyoD and Myf5.

6. A method for screening for a substance that promotes or inhibits the contractile activity of skeletal muscle cells, comprising: (1) inducing differentiation of pluripotent stem cells stably expressing channelrhodopsin into skeletal muscle cells; (2) irradiating the cells with light; (3) contacting the cells with a test substance; (4) measuring the contractile activity of the cells; and (5) A step of selecting a test substance that promotes or inhibits the contractile activity of cells compared to when the test substance is not contacted. wherein the light irradiated onto the cells is blue laser light having a frequency of 0.25 Hz to 0.75 Hz, a pulse width of 2 msec to 100 msec, a voltage of 8 V to 15 V, and a light intensity of 1 mW to 10 mW.

7. The screening method according to claim 6, wherein a multi-well plate is used.

8. The screening method according to claim 6 or 7, wherein the pluripotent stem cells in step (1) are cells derived from a myopathy or myotonia patient.

Images