WO2022138593A1

WO2022138593A1 - Method for producing skeletal muscle cell

Info

Publication number: WO2022138593A1
Application number: PCT/JP2021/047144
Authority: WO
Inventors: 英俊櫻井; 智也内村
Original assignee: 国立大学法人京都大学
Priority date: 2020-12-22
Filing date: 2021-12-20
Publication date: 2022-06-30
Also published as: JPWO2022138593A1

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

Method for manufacturing skeletal muscle cells

The present invention relates to a method for producing skeletal muscle cells in which contractile activity is induced by light stimulation and a method for screening a substance that promotes or suppresses contractile activity in skeletal muscle cells.

Muscle diseases include a large number of diseases, but most of the symptoms are muscle atrophy and accompanying muscle weakness. The causes of muscle atrophy include abnormalities in the muscles themselves and abnormalities in the nerves that move the muscles. The former is called myopathy and the latter is called neurogenic disease. Muscular dystrophy is known as a representative of myopathy, and Duchenne muscular dystrophy, which has the largest number of patients among muscular dystrophy, is caused by mutations in the causative gene, the dystrophin gene (point mutation, deletion mutation, duplicate mutation, etc.). , A disease caused by the inability to synthesize normal dystrophin proteins. It is a disease that develops only in boys due to sex chromosome recessive inheritance, and is said to be 3 to 5 per 100,000 population and 1 per 2000 to 3000 born boys. There is still no good treatment for many myopathy, including Duchenne muscular dystrophy, and the development of a treatment is desired.

In developing a therapeutic drug, a model that reflects the pathological condition in humans in vitro is required. In recent years, with the development of induced pluripotent stem cells produced by reprogramming somatic cells, it is expected that cells produced from the patient's own cells will be used as a pathological model. One such cell is skeletal muscle cells, and various efforts have been made to establish a method for inducing differentiation of induced pluripotent stem cells into skeletal muscle cells. As a method for inducing differentiation, the present inventors introduced a tetracycline (Tet) -inducible transcription factor (MyoD or Myf5) into pluripotent stem cells and continued to use Doxycyclin (Dox) from the first day of differentiation induction. It has been reported that the transcription factor can be expressed in pluripotent stem cells by adding)) to induce differentiation into skeletal muscle cells (Patent Document 1). The present inventors also improved the method for inducing skeletal muscle cells and expressed exogenous MyoD by continuously adding Dox after the first day of induction of differentiation, and on the 3rd to 4th days. , 5% Knockout Serum Replacement (KSR) is reported to be able to induce differentiation into skeletal muscle cells efficiently and with high reproducibility by reseeding the cells in a medium containing (KSR) (Non-Patent Document 1). In addition, Shoji E et al. Induced pluripotent stem cells in a medium containing 20% Knockout Serum Replacement (KSR), and continued to add Dox after the first day of differentiation induction to obtain exogenous MyoD. We have reported a method for inducing differentiation of pluripotent stem cells into skeletal muscle cells by expressing them (Non-Patent Document 2).

Unlike cardiomyocytes, skeletal muscle cells basically do not spontaneously contract and require external stimuli for the contraction reaction. Especially in cultured cells, stimulation is required for maintenance of functionality and maturation, and an electrical stimulation system is often used (Non-Patent Document 3). The electrical stimulation system can easily control the activation of muscle cells, but on the other hand, it is known to generate toxic gas, which limits the intensity of electrical stimulation and the length of stimulation time. In recent years, optical genetics (optogenetics) technology has come to be used as an alternative technique to electrical stimulation. It is a mechanism that activates in response to specific light by expressing a photo-driven ion channel protein called channelrhodopsin in cells, and a method of stimulating muscle cells using this technology has been adopted. (Non-Patent Document 4).

International Publication No. 2013/073246

In order to construct a screening system using the contractile activity of skeletal muscle cells induced to differentiate from pluripotent stem cells as an index, it is necessary to stimulate the cells. However, the conventionally used electrical stimulation has a problem that the medium is hydrolyzed to generate gas and active oxygen, which damages cells. Another problem is that it is not suitable for high-throughput screening because it is difficult to fabricate a device that uniformly applies electrical stimulation to a large number of wells (for example, 96-well or 384-well plate).

Therefore, the present invention provides a method for producing skeletal muscle cells having contractile activity equivalent to that when electrical stimulation is used from pluripotent stem cells, and using the obtained skeletal muscle cells, contraction of skeletal muscle cells. The challenge is to provide a method for screening candidate substances for therapeutic agents for muscle diseases using activity as an index.

The present invention includes the following inventions in order to solve the above problems.
[1] A method for producing skeletal muscle cells in which contractile activity is induced by light stimulation.
(1) A step of producing pluripotent stem cells that stably express channelrhodopsin,
(2) A production method including a step of inducing differentiation of the cells obtained in step (1) into skeletal muscle cells, and (3) a step of irradiating the cells that have started differentiation induction with light.
[2] The production method according to the above [1], wherein in the step (3), light irradiation is started before the stage where cells fuse to form a myotube.
[3] The production method according to the above [1] or [2], wherein in the step (3), continuous irradiation is performed from the start of light irradiation to the end of light irradiation.
[4] Any of the above [1] to [3], wherein the light irradiating the cells is a 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 amount of 1 mW to 10 mW. The manufacturing method described in.
[5] The production method according to any one of [1] to [4] above, wherein the pluripotent stem cell in the step (1) is a cell derived from a myopathy patient or a myotonia patient.
[6] The above-mentioned [1] to [5], wherein the pluripotent stem cell of the step (1) is a cell expressing one or more exogenous skeletal cell inducing factors selected from MyoD and Myf5. Production method.
[7] A method for screening a substance that promotes or suppresses contractile activity of skeletal muscle cells.
(1) A step of inducing differentiation of pluripotent stem cells that stably express channelrhodopsin into skeletal muscle cells.
(2) Step of irradiating cells with light,
(3) Step of contacting the test substance with the cells,
A method comprising (4) measuring the contractile activity of cells and (5) selecting a test substance that promotes or suppresses the contractile activity of cells as compared with the case where the test substance is not contacted.
[8] The screening method according to the above [7], which uses a multi-well plate.
[9] The screening method according to the above [7] or [8], wherein the pluripotent stem cells in the step (1) are cells derived from a myopathy patient or a myotonia patient.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a method for producing skeletal muscle cells having contractile activity equivalent to that when electrical stimulation is used from pluripotent stem cells. The skeletal muscle cells obtained by the production method can be used for high-throughput screening of candidate substances for the treatment of muscle diseases using the contractile activity of the skeletal muscle cells as an index.

It is a figure showing the structure of the vector introduced into iPS cells, (A) is the structure of tetracycline-responsive MyoD forced expression piggyBac vector (pB-EF1α-MyoD-IRES-puro), and (B) is the channelrhodopsin forced expression piggyBac vector. It is a structure of (pB-EF1α-ChR2-IRES-puro). It is a figure which shows the differentiation induction schedule of one form of the manufacturing method of this invention used in an Example. It is a figure which shows the cross section of one well of a 96-well plate used in an Example. It is a figure which shows the result of performing the flat test by immunostaining the cell of the 14th day of the schedule shown in FIG. 2 with an anti-MHC antibody, calculating the skeletal muscle differentiation efficiency. It is a figure which shows the result of performing the flat test by immunostaining the cell of the 18th day of the schedule shown in FIG. 2 with an anti-MHC antibody, calculating the skeletal muscle differentiation efficiency. It is a figure which shows the usage of the well when 2 well of a 96-well plate is used as 1 sample, and 4 well is used as 1 sample. The figure showing the result of performing a flat test on the contraction rate of each sample by analyzing the contractile force of the skeletal muscle cells on the 18th day of the schedule shown in Fig. 2 with a video analyzer and using 2 wells or 4 wells as one sample. (A) is the result of 1 sample of 2 wells, and (B) is the result of 1 sample of 4 wells. It is a figure which shows the result of having analyzed the change of the contraction rate by the electric stimulus and the light stimulus about the skeletal muscle cell of the 18th to 28th days of the schedule shown in FIG. It is the result of light stimulation. It is a figure which shows the result of having analyzed the change of the relaxation rate by the electric stimulation and the light stimulation about the skeletal muscle cell of the 18th to 28th days of the schedule shown in FIG. It is the result of light stimulation. It is a figure which shows the result of having analyzed the change of the acceleration by the electric stimulus and the light stimulus about the skeletal muscle cell of the 18th to 28th days of the schedule shown in FIG. It is the result of stimulation. It is a figure which shows the result of having analyzed the change of the contraction distance by the electric stimulus and the light stimulus about the skeletal muscle cell of the 18th to 28th days of the schedule shown in FIG. It is the result of light stimulation. It is a figure which shows the result of having analyzed the contraction force of the skeletal muscle cell on the 28th day of the schedule shown in FIG. 2 with the moving image analyzer, and performed the flat test about the contraction rate with one sample of 4 wells.

[Manufacturing method of 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 have the following steps.
(1) A step of producing pluripotent stem cells that stably express channelrhodopsin,
(2) A step of inducing differentiation of the cells obtained in step (1) into skeletal muscle cells, and (3) a step of irradiating the cells that have started differentiation induction with light.

As used herein, "skeletal muscle cell" is a broader concept that includes cells of all skeletal muscle lineages, including skeletal muscle stem cells, myoblasts, myotube cells, mature myotube cells, and fully mature myotube cells. Including all of. Marker genes for identifying skeletal muscle cells include, for example, myogenin, myosin heavy chain (MHC), MyoD, Myf5 and the like. The skeletal muscle cell may be a human skeletal muscle cell or a skeletal muscle cell of a non-human organism. 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, rats and the like.

The pluripotent stem cell used in the production method of the present invention may be a pluripotent stem cell derived from a muscle disease patient. Examples of muscle diseases include myopathy and myotonia. Examples of myopathy include muscular dystrophy (Duschenne muscular dystrophy (DMD), Becker muscular dystrophy, limb muscular dystrophy, facial scapulohumeral muscular dystrophy, ocular throat muscular dystrophy, Emeri-Drefus muscular dystrophy, congenital muscular dystrophy, and distal muscular dystrophy. Muscular dystrophy, muscular dystrophy, etc.), distal myopathy (Miyoshi-type myopathy, GNE myopathy, ophthalmic-pharyngeal type distal myopathy, etc.), congenital myopathy (nemarin myopathy, central core disease, etc.), glycolytic disease, cycle Examples include sexual dystrophy and mitochondrial myopathy. Examples of myotonia include grasping myotonia, tapping myotonia, myotonia syndrome, congenital myotonia, congenital paramyotonia, and Schwartz-Jampel syndrome.

The pluripotent stem cell used in the production method of the present invention may be a pluripotent stem cell modified to overexpress a skeletal muscle cell inducing factor. Specific examples thereof include pluripotent stem cells into which an expression vector into which a gene encoding a skeletal muscle cell inducing factor has been inserted has been introduced. Examples of the skeletal muscle cell inducing factor include MyoD, Myf5, Pax7 and the like. Preferred is extrinsic MyoD or Myf5. Expression vectors into which the gene encoding MyoD or Myf5 has been inserted can be constructed using known gene recombination techniques. The obtained expression vector can be introduced into pluripotent stem cells by using a known gene transfer method described later.

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

The pluripotent stem cells that can be used in the production method of the present invention are stem cells that have pluripotency capable of differentiating into all cells existing in a living body and also have proliferative ability, particularly. Not limited, for example, embryonic stem (ES) cells, cloned embryo-derived embryonic stem (ntES) cells, sperm stem (GS) cells, embryonic reproductive (EG) cells, artificial pluripotent stems obtained by nuclear transplantation. Includes (iPS) cells, cultured fibroblasts and pluripotent cells (Muse cells) derived from bone marrow stem cells. Preferred pluripotent stem cells are ES cells, ntES cells and iPS cells.

(A) Embryonic stem cells ES cells are pluripotent and self-replicating stem cells established from the inner cell mass of early embryos (eg, blastocysts) of mammals such as humans and mice.

ES cells are embryo-derived stem cells derived from the inner cell mass of the scutellum vesicle, which is the embryo after the morula at the 8-cell stage of the fertilized egg, and have the ability to differentiate into all the cells that make up the adult, so-called polymorphism. It has the ability and the ability to proliferate by self-replication. ES cells were discovered in mice in 1981 (M.J. Evans and M.H. Kaufman (1981), Nature 292: 154-156), and then ES cell lines were established in primates such as humans and monkeys (J.A. Thomson et). al. (1998), Science 282: 1145-1147; J.A. Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92: 7844-7848; J.A. Thomson et al. (1996), Biol. ., 55: 254-259; J.A. Thomson and V.S. Marshall (1998), Curr. Top. Dev. Biol., 38: 133-165).

For the establishment of ES cells, a method known in this field is used. For example, it can be established by removing 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. For cell maintenance by subculture, use a culture solution containing substances such as leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF). It can be carried out. For methods of establishing and maintaining ES cells in humans and monkeys, see, for example, USP 5,843,780; Thomson JA, et al. (1995), Proc Natl. Acad. Sci. U S A. 92: 7844-7848; Thomson JA, et al. (1998), Science. 282: 1145-1147; H. Suemori et al. (2006), Biochem. Biophys. Res. Communi., 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.

As a culture method for producing ES cells, a method known in the art is used. As the culture medium, use DMEM / F-12 culture medium supplemented with, for example, 0.1 mM 2-mercaptoethanol, 0.1 mM non-essential amino acid, 2 mM L-glutamic acid, 20% KSR (KnockOut Serum Replacement, Invitrogen) and 4 ng / ml bFGF. However, human ES cells can be maintained in a moist atmosphere of 37 ° C, 2% CO ₂ / 98% air (O. Fumitaka et al. (2008), Nat. Biotechnol., 26: 215-224). ES cells may be passaged every 3-4 days, with passage using, for example, 0.25% trypsin and 0.1 mg / ml collagenase IV in PBS containing 1 mM CaCl2 and 20% KSR. It can be carried out.

ES cells can generally be selected by the Real-Time PCR method using the expression of gene markers such as alkaline phosphatase, Oct-3 / 4, and Nanog as an index. In particular, in the selection of human ES cells, the expression of gene markers such as OCT-3 / 4, NANOG, and ECAD can be used as an index (E. Kroon et al. (2008), Nat. Biotechnol., 26: 443. -452).

As mouse ES cells, various mouse ES cell lines established by inGenious, RIKEN (RIKEN), etc. can be used. As human ES cells, various human ES cell lines established by the National Institutes of Health (NIH), RIKEN, Kyoto University, and Cellartis can be used. For example, as ES cell lines, NIH CHB-1 to CHB-12 strains, RUES1 strains, RUES2 strains, HUES1 to HUES28 strains, etc., WisCell Research Institute WA01 (H1) strains, WA09 (H9) strains, RIKEN KhES- One strain, KhES-2 strain, KhES-3 strain, KhES-4 strain, KhES-5 strain, SSES1 strain, SSES2 strain, SSES3 strain and the like can be used. The KhES-1, KhES-2, KhES-3 and KthES11 strains are available from the Institute for Frontier Life and Medical Sciences, Kyoto University (Kyoto, Japan).

(B) Sperm stem cells Sperm stem cells are pluripotent stem cells derived from the testis and are the origin cells for spermatogenesis. Similar to ES cells, these cells can be induced to differentiate into various lineages of cells, and have properties such as the ability to produce chimeric mice when transplanted into mouse blastocysts (M. Kanatsu-Shinohara et al. (M. Kanatsu-Shinohara et al.). 2003) Biol. Reprod., 69: 612-616; K. Shinohara et al. (2004), Cell, 119: 1001-1012). It is self-renewable in a culture medium containing a glial cell line-derived neurotrophic factor (GDNF), and sperm by repeating passage under the same culture conditions as ES cells. Stem cells can be obtained (Masanori Takebayashi et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Special Edition), pp. 41-46, Yodosha (Tokyo, Japan)).

(C) Embryonic germ cells Embryonic germ cells are cells with pluripotency similar to ES cells, which are established from primordial germ cells in the embryonic period, such as LIF, bFGF, and stem cell factor. It can be established by culturing primordial germ cells in the presence of the substance of (Y. Matsui et al. (1992), Cell, 70: 841-847; JL Resnick et al. (1992), Nature, 359: 550. -551).

(D) Induced pluripotent stem cells Induced pluripotent stem (iPS) cells are similar to ES cells, which can be produced by introducing specific reprogramming factors into somatic cells in the form of DNA or protein. 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 release WO 2007/069666). Reprogramming factors are genes that are specifically expressed in ES cells, their gene products or non-cording RNAs, or genes that play an important role in maintaining undifferentiated ES cells, their gene products or non-cording RNAs, or It may be composed of low molecular weight compounds. Genes included in the reprogramming factors include, for example, 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, Glis1, etc. are exemplified, and these initialization factors may be used alone or in combination. The combinations of initialization 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, WO2009 / 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 U S A. 106: 8912-8 917, 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, The combinations described in et al. (2011), Nature. 474: 225-9. Are exemplified.

The reprogramming factors include histone deacetylase (HDAC) inhibitors [eg, small molecule inhibitors such as valproic acid (VPA), tricostatin A, sodium butyrate, MC 1293, M344, siRNA against HDAC and shRNA (eg). , HDAC1 siRNA Smartpool (Millipore), HuSH 29mer shRNA Constructs against HDAC1 (OriGene), etc.), MEK inhibitors (eg PD184352, PD98059, U0126, SL327 and PD0325901), Glycogen synthesis 3 Against small molecule inhibitors such as Bio and CHIR99021, DNA methyltransferase inhibitors (eg 5-azacytidine), histone methyltransferase inhibitors (eg BIX-01294), Suv39hl, Suv39h2, SetDBl and G9a Nucleic acid expression inhibitors such as siRNA and shRNA), L-channel calciumagonist (eg Bayk8644), butyric acid, TGFβ inhibitors or ALK5 inhibitors (eg LY364947, SB431542, 616453 and A-83-01), p53 inhibition Agents (eg siRNA and shRNA for p53), ARID3A inhibitors (eg siRNA and shRNA for ARID3A), miRNAs such as miR-291-3p, miR-294, miR-295 and mir-302, Wnt Signaling (eg soluble Wnt3a) ), Nucleic acid peptide Y, prostaglandins (eg, prostaglandins E2 and prostaglandins J2), hTERT, SV40LT, UTF1, IRX6, GLISl, PITX2, DMRTBl, etc. In this specification, the factors used for the purpose of improving the establishment efficiency are not particularly distinguished from the reprogramming factors.

In the case of protein morphology, the reprogramming factor may be introduced into somatic cells by techniques such as lipofection, fusion with cell membrane permeable peptides (eg, HIV-derived TAT and polyarginine), and microinjection.

On the other hand, in the case of DNA morphology, it can be introduced into somatic cells by methods such as viruses, plasmids, vectors such as artificial chromosomes, lipofection, liposomes, and microinjection. Viral vectors include retro viral vectors and lentiviral vectors (above, Cell, 126, pp.663-676, 2006; Cell, 131, pp.861-872, 2007; Science, 318, pp.1917-1920, 2007. ), Adenovirus vector (Science, 322, 945-949, 2008), adeno-associated virus vector, Sendai virus vector (WO2010 / 008054) and the like. Further, the artificial chromosome vector includes, for example, a human artificial chromosome (HAC), a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC, PAC) and the like. As the plasmid, a plasmid for mammalian cells can be used (Science, 322: 949-953, 2008). The vector can contain regulatory sequences such as promoters, enhancers, ribosome binding sequences, terminators, polyadenylation sites, etc. so that the nuclear reprogramming substance can be expressed, and further, if necessary, a drug resistance gene (drug resistance gene). For example, canamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, etc.), thymidin kinase gene, selection marker sequence such as diphtheriatoxin gene, reporter gene sequence such as green fluorescent protein (GFP), β-glucuronidase (GUS), etc. Can be done. In addition, the above vector has LoxP sequences before and after introduction into somatic cells in order to excise both the gene encoding the reprogramming factor or the promoter and the gene encoding the reprogramming factor that binds to the promoter. You may.

In the case of RNA morphology, it may be introduced into somatic cells by a method such as lipofection or microinjection, and in order to suppress degradation, RNA incorporating 5-methylcytidine and pseudouridine (TriLink Biotechnologies) is used. It may be (Warren L, (2010) Cell Stem Cell. 7: 618-630).

Cultures for iPS cell induction include, for example, DMEM, DMEM / F12 or DME cultures containing 10-15% FBS (these cultures also include LIF, penicillin / streptomycin, puromycin, L-glutamine). , Non-essential amino acids, β-mercaptoethanol, etc. can be appropriately contained.) Or mouse ES cell culture medium (TX-WES culture medium, Thrombo X), primate ES cell culture medium (primates). Commercially available cultures such as ES / iPS cell culture medium, Reprocell), serum-free pluripotent stem cell maintenance medium (for example, mTeSR (Stemcell Technology), Essential 8 (Life Technologies), StemFit AK03 (AJINOMOTO)) Illustrated.

As an example of the culture method, for example, in the presence of 5% CO ₂ at 37 ° C., the somatic cells are brought into contact with the reprogramming factor on a DMEM or DMEM / F12 culture medium containing 10% FBS and cultured for about 4 to 7 days. Then, the cells are re-seeded on feeder cells (for example, mitomycin C-treated STO cells, SNL cells, etc.), and about 10 days after the contact between the somatic cells and the reprogramming factor, the culture medium for bFGF-containing primate ES cell culture is used. It can be cultured and give rise to iPS-like colonies about 30-about 45 days or more after the contact.

Alternatively, in the presence of 5% CO ₂ at 37 ° C., DMEM culture medium containing 10% FBS (for example, LIF, penicillin / streptomycin, etc.) on feeder cells (eg, mitomycin C-treated STO cells, SNL cells, etc.). (Pureomycin, L-glutamine, non-essential amino acids, β-mercaptoethanol, etc. can be appropriately contained), and ES-like colonies can be generated after about 25 to about 30 days or more. Desirably, instead of feeder cells, the reprogrammed somatic cells themselves are used (Takahashi K, et al. (2009), PLoS One. 4: e8067 or WO2010 / 137746), or extracellular matrix (eg, Laminin-). 5 (WO2009 / 123349) and Matrigel (BD)) are exemplified.

In addition to this, a method of culturing using a medium containing no serum is also exemplified (Sun N, et al. (2009), Proc Natl Acad Sci U S A. 106: 15720-15725). Furthermore, in order to improve the establishment efficiency, 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).

During the above culture, the fresh culture solution and the culture solution are exchanged once a day from the second day after the start of the culture. The number of somatic cells used for nuclear reprogramming is not limited, but ranges from about 5 × 10 ³ to about 5 × 10 ⁶ cells per 100 cm ² culture dish.

The iPS cells can be selected according to the shape of the formed colonies. On the other hand, when a drug resistance gene expressed in conjunction with a gene expressed when somatic cells are reprogrammed (for example, Oct3 / 4, Nanog) is introduced as a marker gene, a culture medium containing the corresponding drug (selection). Established iPS cells can be selected by culturing in a culture medium). In addition, iPS cells are selected by observing with a fluorescence microscope when the marker gene is a fluorescent protein gene, by adding a luminescent substrate when it is a luminescent enzyme gene, and by adding a chromogenic substrate when it is a chromogenic enzyme gene. can do.

As used herein, the term "somatic cell" refers to any animal cell (eg, mammalian cell including human) except germline cells such as eggs, egg mother cells, ES cells or totipotent cells. .. Somatic cells include, but are not limited to, fetal (pup) somatic cells, neonatal (pup) somatic cells, and mature healthy or diseased somatic cells, as well as primary cultured cells. , Passed cells, and established cells are all included. Specifically, the somatic cells include, for example, (1) tissue stem cells (somatic stem cells) such as nerve stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells, (2) tissue precursor cells, (3) lymphocytes, and epithelium. Cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, hepatocytes, gastric mucosal cells, intestinal cells, splenocytes, pancreatic cells (pancreatic exocrine cells, etc.), brain cells, lung cells, renal cells And differentiated cells such as fat cells are exemplified.

In addition, when iPS cells and / or cells induced to differentiate from them are used as materials for transplant cells, the HLA genotypes of the transplanted individuals are the same or substantially the same from the viewpoint of not causing rejection. It is desirable to use cells. Here, "substantially the same HLA type" means that the HLA genotypes match to the extent that the transplanted cells can engraft when the cells are transplanted. For example, the main HLA (HLA-). It is a somatic cell having an HLA type that matches the 3 loci of A, HLA-B and HLA-DR or the 4 loci with HLA-C).

As the induced pluripotent stem cell line, various iPS cell lines established by NIH, RIKEN, Kyoto University, etc. may be used. For example, in the case of human iPS cell lines, RIKEN's HiPS-RIKEN-1A strain, HiPS-RIKEN-2A strain, HiPS-RIKEN-12A strain, Nips-B2 strain, etc., Kyoto University's Ff-WJ-18 strain, Ff -I01s01 shares, Ff-I01s02 shares, Ff-I01s04 shares, Ff-I01s06 shares, Ff-I14s03 shares, Ff-I14s04 shares, QHJI01s01 shares, QHJI01s04 shares, QHJI14s03 shares, QHJI14s04 shares, QHJI14s04 shares, AK5 shares, TkDN-S Examples include 253G1 shares, 201B7 shares, 409B2 shares, 454E2 shares, 606A1 shares, 610B1 shares, 648A1 shares, 1231A3 shares, 1390D4 shares and 1390C1 shares. Alternatively, clinical grade cell lines provided by Kyoto University, Cellular Dynamics International, etc., and research and clinical cell lines prepared using these cell lines may be used.

(E) ES cells (ntES cells) derived from cloned embryos obtained by nuclear transplantation
ntES cells are ES cells derived from cloned embryos produced by nuclear transplantation technology and have almost the same characteristics 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). That is, ES cells established from the inner cell mass of blastocysts derived from cloned embryos obtained by replacing the nuclei of unfertilized eggs with the nuclei of somatic cells are ntES (nuclear transfer ES) cells. For the production of ntES cells, a combination of nuclear transplantation technology (JB Cibelli et al. (1998), Nature Biotechnol., 16: 642-646) and ES cell production technology (above) is used (Seika Wakayama). Et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Special Edition), pp. 47-52). In nuclear transplantation, somatic cell nuclei can be injected into unenucleated unfertilized eggs of mammals and cultured for several hours to initialize them.

(F) Multilineage-differentiating Stress Enduring cells (Muse cells)
Muse cells are pluripotent stem cells produced by the method described in WO2011 / 007900, specifically fibroblasts or bone marrow stromal cells treated with long-term trypsin, preferably 8 hours or 16 hours. Pluripotent cells obtained by suspension culture after treatment and positive for SSEA-3 and CD105.

Step (1) is a step of producing pluripotent stem cells that stably express channelrhodopsin. Channelrhodopsin is a photodriven ion channel isolated from algae and has the property of taking up cations by light irradiation. Examples of channelrhodopsin that can be used in the production method of the present invention include channelrhodopsin 1, channelrhodopsin 2, and modified channelrhodopsin (eg, modified channelrhodopsin described in WO2011 / 019081 or WO2020 / 059675). Be done. Channelrhodopsin 2 is preferred. Examples of channelrhodopsin 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 stritata.

Channelrhodopsin 2 is, for example, a protein having the same or substantially the same amino acid sequence as the amino acid sequence shown in SEQ ID NO: 1. The amino acid sequence shown in SEQ ID NO: 1 is registered as GenBank accession number AAM15777.1. An amino acid sequence substantially the same as the amino acid sequence shown in SEQ ID NO: 1 is, for example, an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted or added in the amino acid sequence shown in SEQ ID NO: 1. Can be mentioned. Preferably 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1 amino acid deleted, Amino acid sequence substituted, inserted or added. Alternatively, the amino acid sequence substantially identical to the amino acid sequence shown in SEQ ID NO: 1 has 60% or more identity (for example, 60% or more, 70% or more) with respect to the amino acid sequence shown in SEQ ID NO: 1. An amino acid sequence having 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). Identity can be determined using techniques known as the degree of identity between two sequences. For example, various alignment algorithms and / or programs may be used, including FASTA, BLAST, or ENTREZ. 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 of Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, and Md. The protein having an amino acid sequence substantially the same as 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 photodriven cation channel activity (light wavelength, ion permeability, etc.) is equivalent to the photodriven cation channel activity of channelrhodopsin 2 consisting of the amino acid sequence shown in SEQ ID NO: 1. Is mentioned.

The base sequence of the gene encoding channelrhodopsin 2 is, for example, the base sequence encoding the amino acid sequence shown in SEQ ID NO: 1, but is not particularly limited. For example, it may be the base sequence shown by SEQ ID NO: 2. The basic acid sequence shown in SEQ ID NO: 2 is registered as GenBank accession number AF461397.1. The gene encoding channelrhodopsin may be a humanized codon. Further, the gene encoding channelrhodopsin 2 may be a gene encoding a protein having an amino acid sequence substantially the same as the amino acid sequence shown in SEQ ID NO: 1.

Pluripotent stem cells that stably express channelrhodopsin can be prepared by introducing an expression vector into which a gene encoding channelrhodopsin is inserted into pluripotent stem cells and selecting clones that stably express channelrhodopsin. .. The gene encoding channelrhodopsin can be obtained by PCR or the like based on the sequence information. It can also be chemically synthesized.

As the expression vector, for example, a vector such as a virus, a plasmid, or an artificial chromosome can be used. Examples of the virus vector include a retrovirus vector, a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, a Sendai virus vector and the like. Examples of the artificial chromosome vector include a human artificial chromosome (HAC), a yeast artificial chromosome (YAC), and a bacterial artificial chromosome (BAC, PAC). Examples of the plasmid include a plasmid for mammalian cells. The vector can contain regulatory sequences such as promoters, enhancers, ribosome binding sequences, terminators, polyadenylation sites, etc. so that the DNA encoding channel rhodopsin can be expressed, as well as drug resistance genes (as needed). For example, a canamycin resistance gene, an ampicillin resistance gene, a puromycin resistance gene, etc.), a selection marker sequence such as a thymidine kinase gene, a diphtheriatoxin gene, a fluorescent protein, a reporter gene sequence such as β-glucuronidase (GUS), and the like can be included. As promoters, 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, CAG Promoters and the like can be mentioned.

The vector contains the expression cassette (gene containing promoter, gene sequence and terminator) for inserting the nucleic acid encoding channel rhodopsin into the chromosome or, if necessary, excising the nucleic acid inserted into the chromosome. It may have a transposon sequence before and after the expression unit). The transposon sequence is not particularly limited, but piggyBac is exemplified. In order to introduce an expression cassette into a chromosome using a transposon, it is desirable to introduce the transposase into the cell together with a vector having the expression cassette. In the present invention, in order to introduce a transposase, the above-mentioned vector may contain a nucleic acid encoding the transposase, or another vector may contain a nucleic acid encoding the transposase, and the nucleic acid may be introduced into a cell at the same time. You may. In addition, the gene product encoding the transposase may be directly introduced. In the present invention, the preferred transposase is the transposase corresponding to the transposon sequence described above, preferably the piggyBac transposase.

When a viral vector is used, the gene encoding channelrhodopsin is transferred to the pluripotent stem cell by introducing the DNA or RNA encoding channelrhodopsin into the above-exemplified virus and infecting the pluripotent stem cell with this recombinant virus. Can be introduced. When using a non-viral vector, a method of introduction using liposomes (liposomal method, HVJ-liposome method, cationic liposome method, lipofection method, lipofectamine method, etc.), microinjection method, calcium phosphate method, electroporation method , A method of transferring into a cell together with a carrier (metal particle) with a gene gun (GeneGun) can be used.

A method for selecting pluripotent stem cells that stably express channel rhodopsin is, for example, a method for selecting surviving colonies by culturing cells in a drug-containing medium after transfection using an expression vector containing a drug resistance gene. Can be mentioned.

Step (2) is a step of inducing differentiation of the cells obtained in step (1) into skeletal muscle cells. The method for inducing the differentiation of pluripotent stem cells that stably express channel rhodopsin into skeletal muscle cells is not particularly limited, and known pluripotent stem cells can be appropriately selected from the methods for inducing differentiation into skeletal muscle cells.

The method of inducing the differentiation of pluripotent stem cells into skeletal muscle cells may be a method of overexpressing the skeletal muscle cell inducing factor in the pluripotent stem cells. Examples of such a differentiation-inducing method include the method described in the international publication WO2013 / 073246 A1, the method described in the international publication WO2020 / 090836 A1, and Uchimura et al. (Stem Cell Research, 25: 98-106 (2017)). Examples include the method described and the method described in Shoji et al. (Science Reports, 5: 12831 (2015)). In addition, the method of inducing the differentiation of pluripotent stem cells into skeletal muscle cells is a method of mimicking the developmental process of the fetus without using a transgene (via paraxial mesoderm cells, segment cells, and cutaneous muscle cells). It may be a method of inducing differentiation of skeletal muscle lineage cells). Examples of such differentiation induction methods include the method described in Zhao et al. (Stem Cell Reports, Vol. 15 1-15 July 14, 2020), the method described in the international publication WO2016 / 108288 A1, and Hicks et al. (Nat Cell Biol. The method described in 2018 Jan; 20 (1): 46-57) can be mentioned.

The skeletal muscle cells whose contractile activity is induced by photostimulation produced by the production method of the present invention have very low variation in skeletal muscle differentiation efficiency between wells when seeded on a multi-well plate and induced to differentiate. It has also been confirmed that the variation in contractile force of skeletal muscle cells between wells is very low. Therefore, in step (2), pluripotent stem cells may be seeded on a multi-well plate to induce differentiation. Examples of the multi-well plate include a 24-well plate, a 48-well plate, a 96-well plate, and a 384-well plate. A 96-well plate or a 384-well plate is preferable. In the production method of the present invention, the skeletal muscle cells produced using the multi-well plate can be suitably used, for example, for high-throughput screening of candidate substances for therapeutic agents for muscle diseases.

The multi-well plate is not particularly limited, but it is preferable to use a plate having a culture surface in which the contractile activity of cells is not limited. Examples of such a plate include a plate whose culture surface is hydrogel. Examples of the hydrogel include gelatin hydrogel, collagen hydrogel, starch hydrogel, pectin hydrogel, hyaluronic acid hydrogel, chitin hydrogel, chitosan hydrogel, and alginate hydrogel. Of these, collagen hydrogel or gelatin hydrogel is preferable. The gel hardness (elastic modulus) of the hydrogel may be 10 kPa or more (10 kPa, 11 kPa, 12 kPa, 13 kPa, 14 kPa, 15 kPa or more) and 25 kPa or less (25 kPa, 20 kPa, 19 kPa, 18 kPa, 17 kPa, 16 kPa, 15 kPa). The following) may be used. It is preferably 12 kPa. The plate in which the culture surface is hydrogel may be a plate in which the hydrogel is coated on the culture surface, or a plate in which the recess of the plate having the recess for placing the hydrogel on the culture surface is filled with hydrogel. May be there.

Step (3) is a step of irradiating the cells that have started differentiation induction with light. Differentiation induction is initiated when the pluripotent stem cell medium is replaced with a differentiation medium, or when a differentiation induction method for introducing a skeletal muscle cell inducing factor expression vector into pluripotent stem cells is used, the skeletal muscle cell inducing factor. Whichever comes first, the time when the onset of expression begins or the time when the medium is replaced with a differentiation medium.

In the step (3), the time to start the light irradiation is not particularly limited, and the light irradiation may be started at the same time as the start of the differentiation induction, or may be started after an arbitrary period from the start of the differentiation induction. It is preferable to start light irradiation before the stage where cells fuse to form a myotube, and light irradiation should be started within 4 days, 3 days, 2 days, and 1 day after the start of differentiation induction. Is preferable. It can be confirmed by observing the morphology of the cells under a microscope that the cells do not fuse to form a myotube.

Although the end time of light irradiation is not particularly validated, it is preferable to continue light irradiation until the differentiation-induced skeletal muscle cells start contractile activity and the test using contractile activity as an index is completed. For example, when skeletal muscle cells produced by the production method of the present invention are used for screening a candidate substance for a therapeutic agent for muscle disease, 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, but may be, for example, 0.02 to 0.9, 0.05 to 0.5, or 0.05 to 0.1. Continuous irradiation is preferable.

When using pluripotent stem cells that stably express channelrhodopsin 2, a blue laser is used as the light to irradiate. 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 8V to 15V, preferably 10V. The amount of light is 1 mW to 10 mW, preferably 2 mW to 3 mW. As the light irradiation device, it is preferable to use a device that can uniformly irradiate all the wells of the multi-well plate used. For example, a device that uniformly irradiates the entire bottom surface of the multi-well plate with light can be used. Examples of such a device include an LED array system for optogenetics (BRC Bioresearch Center).

For the skeletal muscle cells obtained by the method of the present invention to perform contractile activity by photostimulation, a commercially available imaging system capable of analyzing moving images of cells (for example, Sony's live cell imaging system SI8000, etc.) can be used. Can be confirmed using.

[Screening method]
The present invention provides a method for screening a substance that promotes or suppresses 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 have the following steps.
(1) A step of inducing differentiation of pluripotent stem cells that stably express channelrhodopsin into skeletal muscle cells.
(2) Step of irradiating cells with light,
(3) A step of contacting the test substance with the cells, and (4) a step of selecting a test substance that promotes or suppresses the contractile activity of skeletal muscle cells as compared with the case where the test substance is not contacted.

The steps (1) and (2) in the screening method of the present invention can be carried out in the same manner as the steps (2) and (3) of the manufacturing method of the present invention. That is, the screening method of the present invention can be rephrased as a screening method using skeletal muscle cells produced by the production method of the present invention.

The screening method of the present invention can perform high-throughput screening using a multi-well plate, preferably a 96-well plate or a 384-well plate. Further, in the screening method of the present invention, a desired test substance can be selected using the contractile activity of skeletal muscle cells as an index. When using a 96-well plate or a 384-well plate, 2 wells may be evaluated as 1 sample and 4 wells may be evaluated as 1 sample. By using a plurality of wells as one sample, the variation between the samples can be reduced.

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

Step (3) is a step of bringing the test substance into contact with the cells. The test substance is not particularly limited, and for example, nucleic acid, peptide, protein, non-peptidic compound, synthetic compound, fermentation product, cell extract, cell culture supernatant, plant extract, mammalian tissue extract, plasma and the like. Can be used. The test substance may be a novel substance or a known substance. These test substances may form salts. As the salt of the test substance, a salt with a physiologically acceptable acid or base is used.

The time when the test substance is brought into contact with the cells may be before the cells start contractile activity or after the cells start contractile activity. Contact between the cells and the test substance can be performed by adding the test substance to the medium. When multiple wells are used as one sample, the same test substance is added to the multiple wells. Specifically, for example, when 2 wells are 1 sample, the same test substance is added to 2 wells, and when 4 wells are 1 sample, the same test substance is added to 4 wells.

Step (4) is a step of measuring the contractile activity of cells. The contractile activity of cells can be measured using a commercially available imaging system (for example, Sony's live cell imaging system SI8000 or the like) capable of analyzing moving images of cells. Measurement items include contraction speed, relaxation speed, acceleration, contraction distance and the like.

In the screening method of the present invention, when a test substance that promotes cell contractile activity is selected, skeletal muscle cells induced to differentiate from pluripotent stem cells derived from myopathy patients are usually used. It has been found that skeletal muscle cells induced to differentiate from pluripotent stem cells derived from myopathy patients have reduced contractile force from the time when maximum contractile force is observed (see Examples). Therefore, when using skeletal muscle cells differentiated from pluripotent stem cells derived from myopathy patients, the test substance is preferably contacted immediately before or on the day of the observation of maximum contractile force, and measurement of cell contractile activity. Is preferably performed at a time when the contractile force is reduced. The measurement of cell contractile activity may be performed twice, at the time when the maximum contractile force is observed and at the time when the contractile force decreases. By measuring twice, the rate of decrease in contractile force can be evaluated.

In the screening method of the present invention, when a test substance that suppresses cell contractile activity is selected, usually, pluripotent stem cells derived from healthy subjects or skeletal muscle cells induced to differentiate from pluripotent stem cells derived from myotonia patients are used. used. In this case, it is preferable that the test substance is brought into contact with the test substance immediately before or on the day when the maximum contractile force is observed, and the cell contraction activity may be measured multiple times over time immediately after the test substance is brought into contact with the test substance. preferable. By measuring multiple times over time, it is possible to evaluate how the effect of suppressing contractile activity appears in the long term.

In the screening method of the present invention, cells that are not in contact with the test substance are used as controls, and in step (4), the contractile activity of the control cells is similarly measured.

Step (5) is a step of selecting a test substance that promotes or suppresses the contractile activity of cells as compared with the case where the test substance is not contacted.
When selecting a test substance that promotes cell contraction activity, for example, when the contraction rate is used as an index, the contraction rate of control cells that have not been in contact with the test substance is 50% or more, 60% or more, 70% or more, 80. A test substance that recovers to% or more and 90% or more may be selected. At the same time, skeletal muscle cells induced to differentiate from pluripotent stem cells derived from healthy subjects may be used as healthy control cells, and a test substance that restores the contraction rate to a contraction rate close to the contraction rate level of healthy control may be selected.

When selecting a test substance that suppresses cell contraction activity, for example, when the contraction rate is used as an index, the contraction rate of control cells that have not been in contact with the test substance is 10% or less, 20% or less, 30% or less, 40. A test substance that suppresses% or less and 50% or less may be selected.

The test substance selected as a substance that promotes the contractile activity of cells is useful as a candidate substance for an active ingredient of a prophylactic or therapeutic drug for myopathy. In addition, the test substance selected as a substance that suppresses the contractile activity of cells is useful as a candidate substance for an active ingredient of a prophylactic or therapeutic agent for myotonia.

Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited thereto.

〔experimental method〕
1. 1. Cultured human iPS cell DMD (Duchenne muscular dystrophy) iPS cell line (clone ID: CiRA00111, hereinafter referred to as "DMDΔ44") is episodic from skin fibroblasts of DMD patients lacking the dystrophin gene exxon 44. It was established by the Maruvector system (Okita et al., Stem Cells, 31; 458-466, 2012). The maintenance culture of human iPS cells was performed in a feeder-free state. As the maintenance medium, 500 mL of StemFit (Ajinomoto Co., Inc.) plus 50 mU / L penicillin / 50 μg / L streptomycin (Invitrogen Co., Ltd.) was used. For the maintenance culture of human iPS cells after introduction of the Tet vector (described later) and ChR2 vector (described later), a medium supplemented with 100 μg / mL puromycin and Blasticidin (Invitrogen) was used. Cell passage was performed when the cell colonies were 80-90% confluent. Peel the cells with the cell dissociation solution Accutase (Funakoshi), then collect the cells with a scraper, and place them on a newly laminin-coated (Nippi) plate with the ROCK inhibitor Y-27632 (Nakalitesk) (hereinafter, [ROCK inhibitor]. (Y]) The seeds were sown in the added medium and cultured in an incubator in an environment of 37 ° C, 5% CO ₂ , and 100% humidity.

2. 2. Preparation of Tetracycline Responsive Gene Forced Expression Vector (Tet Vector) The tetracycline responsive gene forced expression piggyBac vector was developed by Woltjen et al. Alternatively, KW879 (see Addgene plasmid # 80478, Induced Pluripotent Stem (iPS) Cells pp111-131) was used. This vector incorporates both a reverse tetracycline transactivator (rtTA) and a tetracycline responsive region (TRE). KW879 can be selected by the puromycin resistance gene. These vectors are mixed with the pENTR / D-TOPO-MyoD (or Myf5) entry vector and subjected to a recombination reaction using LR chronase (Invitrogen). A vector (pB-EF1α-MyoD-IRES-puro, hereinafter referred to as “pB-Tet-MyoD”) was prepared. The pENTR / D-TOPO-MyoD entry vector and the pENTR / D-TOPO-Myf5 entry vector are entry vectors in which the cDNA of MyoD or Myf5 is incorporated into pENTR / D-TOPO (Thermo Fisher Scientific, catalog number K240020), respectively. be.

3. 3. Preparation of channelrhodopsin (ChR2) forced expression vector (ChR2 vector) In order to prepare a channelrhodopsin forced expression piggyBac vector, first, the gene sequence of ChR2 was added to the pENTR / D-TOPO entry vector (Thermo Fisher Scientific, catalog number K240020). A pENTR / D-TOPO-ChR2 vector was prepared by inserting by cloning using PCR. Then, the piggyBac-EF1α-IRES-blastcidin vector (SEQ ID NO: 3) and the pENTR / D-TOPO-ChR2 vector (entry vector) were mixed and subjected to a recombination reaction using LR chronase (Invitrogen). The ChR2 forced expression piggyBac vector (pB-EF1α-ChR2-IRES-Bsr, hereinafter referred to as “pB-ChR2”) shown in B) was prepared. This vector can be drug-selected by the blastidin resistance gene.

4. Vector introduction into iPS cells and selection of transformed cells For iPS cell clones (DMD-Δ44) derived from DMD patients, cells for one 10 cm dish were prepared. From the day before the introduction of the vector, the cells were cultured in a medium containing the ROCK inhibitor Y, and then transfection of the seeded cells and the vector by the electroporation method was performed in the same manner as in the maintenance culture. 5 μg each of a vector (EF1α-PBase) incorporating Transposase downstream of the pB-Tet-MyoD, pB-ChR, and EF1α-promoter was prepared and dissolved in 100 μl of Opti-MEM (Invitrogen). 1.0 × 10 ⁶ cells were suspended in Opti-MEM containing the vector and the vector was transfected using the NEPA21 electroporator (Nepagene) under the conditions shown in Table 1. Transfected cells were seeded on 6-well plates at 1.0 × 10 ³ to 5.0 × 10 ⁴ cells / well. After 48 hours, the medium was replaced with 100 μg / ml blastsaidin and puromycin (Nacalai Tesque) -containing medium. After that, the medium was replaced with a drug-containing medium every two days, and cells transformed into drug resistance were selected.

5. Selection of Transformed Cell Clone The obtained clones were seeded on 6-well plates coated with Matrigel (Invitrogen) diluted 100-fold in medium. The number of seeded cells was 1.0 × 10 ³ to 5.0 × 10 ⁴ cells / well. After 48 hours, doxycycline (Dox; LKT Laboratories) was added to the medium at 1 μg / ml. Four days after Dox addition, among the cells induced in skeletal muscle cells, clones with good differentiation efficiency are selected based on the state of cells having multiple nuclei and elongated morphology, which is a characteristic of myotube cells. did.

6. Induction of differentiation of Tet-MyoD iPS cells into skeletal muscle cells (see Fig. 2)
The clones (Tet-MyoD iPS cells) selected in 5 above were seeded on Matrigel-coated plates using StemFit medium containing the ROCK inhibitor Y (day 0). The number of seeded cells was 1.0 × 10 ³ to 5.0 × 10 ⁴ cells / well. On the first day, the medium was replaced with PECM medium (Reprocell) supplemented with ROCK inhibitor Y, and on the second day, the medium was replaced with PECM medium supplemented with Dox from 0.4 μg / mL to 1.5 μg / mL. On the 4th day, 48 hours later, reseeding was performed on a plate of interest suitable for the assay (eg, μ-Plate Angiogenesis 96 Well (Ibidi, Catalog No. 89646) described below). In that case, αMEM (Nakalitesk), 2% (v / v) horse serum (HS: Invitrogen), 100 μM 2-mercaptoethanol, insulin, SB431542 (Wako Pure Chemical Industries), glucose (Invitrogen), A medium supplemented with 50 mU / L penicillin / 50 μg / L streptomycin was used. After reseeding, the cells were cultured in Dox-free medium for 2 days, and then 1 μg / mL Dox was re-added. Immunostaining was performed between the 14th and 28th days of culture to confirm whether differentiation into skeletal muscle cells was induced.

7. At the time of re-seeding on the 4th day (Day 4) of culturing to promote maturation by photostimulation of differentiated skeletal muscle cells, the inside of μ-Plate Angiogenesis 96 wells was coated with collagen gel (Nippi) and then re-seeded. From the 10th day, the promotion of maturation by light stimulation was started (see Fig. 3), and light stimulation was continuously applied until each test was performed. The number of cells reseeded was 1.0 × 10 ³ to 5.0 × 10 ⁴ cells / well. The light stimulator used a blue LED array system for optogenetics (BRC Bioresearch Center) to continuously apply stimulation with a frequency of 0.5 Hz, a pulse width of 50 msec, and a voltage of 10 V for 24 hours every day during the period. The medium was changed at least once every two days.

8. Immunostaining of differentiated skeletal muscle cells The differentiated cells were fixed with 2% paraformaldehyde (Nakalitesk) / PBS at 4 ° C for 10 minutes, washed with PBS for 5 minutes three times, and then methanol (Nakalitesk). ) Was decolorized with a solution containing 1% hydrogen peroxide (Wako) for 15 minutes. Washing with PBS at 4 ° C for 5 minutes was performed again 3 times. Blocking was performed at 4 ° C for 15 minutes at BlockingOne (Nacalai Tesque). For the primary antibody, anti-MHC (Mouse Monoclonal. R & D, 1: 400 dilution) was diluted in the above blocking solution and used. After reacting at 4 ° C for 16 to 18 hours and washing 3 times with 0.2% Triton X-100-added PBS (PBST), anti-Mouse IgG-Alexa644 (Molecular Probes) was used as the secondary antibody at 1: 500 PBST. And reacted at 4 ° C. for 16-18 hours. Then, in order to stain the cell nuclei, 5 μg / ml DAPI (Sigma) was diluted 5000 times with PBST, reacted at room temperature for 5 minutes, washed 3 times with PBS, and then Opera Phenix high-throughput high-content imaging. Observed with the system (PerkinElmer). Then, the efficiency of skeletal muscle differentiation was calculated by the following formula: (number of nuclei stained with DAPI staining on MHC-positive cells / total number of nuclei stained with DAPI staining). A flat test was performed by calculating the skeletal muscle differentiation efficiency of each well and calculating the mean (Mean), standard deviation (SD) and coefficient of variation (CV).

9. SI8000 video analyzer A live cell imaging system SI8000 (SONY) was used to analyze the contractile activity of differentiated skeletal muscle cells. The contractile activity was stimulated with a frequency of 0.5 Hz, a pulse width of 2 msec, and a voltage of 10 V using a C-Pace EP and a C-Dish system (IonOptics). Using SI8000, we photographed at 27 frames / sec for a total of 270 frames for 10 seconds, and analyzed the velocity and distance during contraction activity with SI8000 software.

〔Experimental result〕
1. Skeletal muscle differentiation efficiency of cells on day 14 The cells on day 14 were fixed and immunostained with an anti-MHC antibody to calculate the skeletal muscle differentiation efficiency, and the results of a flat test are shown in FIG. The upper row is a heat map of the skeletal muscle differentiation efficiency (%) of each well of the 96-well plate, and the lower row is a table showing the mean value (Mean), standard deviation (SD) and coefficient of variation (CV). The cells on the 14th day are skeletal muscle cells that have not yet started contractile activity. Since the average value of the skeletal muscle differentiation efficiency of each well was 98.02% and the coefficient of variation was 2.84, the skeletal muscle cells obtained by using the production method of the present invention sufficiently satisfy the screening quality. Shown. If the coefficient of variation is less than 10, it is considered that the quality of screening is satisfied (Iversen et al. Assay Guidance Manual. 2004.).

2. Skeletal muscle differentiation efficiency of cells on day 18 The cells on day 18 were fixed and immunostained with an anti-MHC antibody to calculate the skeletal muscle differentiation efficiency, and the results of a flat test are shown in FIG. The upper row is a heat map of the skeletal muscle differentiation efficiency (%) of each well of the 96-well plate, and the lower row is its mean (Mean), standard deviation (SD) and coefficient of variation (CV). The cells on the 18th day are skeletal muscle cells that have already started contractile activity, and are considered to be the time when they exert the most contractile force. Since the average value of the skeletal muscle differentiation efficiency of each well was 93.67% and the coefficient of variation was 3.91, the skeletal muscle cells obtained by using the production method of the present invention sufficiently satisfy the screening quality. Was shown.

3. 3. Examination of screening method to evaluate the contractile force of skeletal muscle cells The contractile force of skeletal muscle cells on the 18th day of culture was analyzed with a moving image analyzer and a flat test was performed. In order to determine how many wells of data should be analyzed as one sample to maintain the accuracy of screening, we tried the method of analyzing 2 wells or 4 wells as 1 sample (Fig. 6). reference). The results of evaluating the contraction velocity are shown in Fig. 7. (A) is the result of 1 sample of 2 wells, and (B) is the result of 1 sample of 4 wells. The coefficient of variation (CV) is lower in the 4-well 1 sample, but since the CV value of both the 2-well and 4-well was below the standard 10, it is appropriate to analyze at least 2 wells as 1 data. It was considered.

4. Changes in contractile force of skeletal muscle cells due to electrical and light stimulation Changes in contractile force of skeletal muscle cells on

days

18, 21, and 28 of culture were compared with those given electrical stimulation. Electrical stimulation was started from the 6th day of culture using C-Pace EM (manufactured by IonOptics) and C-Dish 6-well plate (manufactured by IonOptics) at a frequency of 0.5 Hz and a pulse width of 2 msec. The number of seeded cells was 1.0 × 10 ⁵ to 5.0 × 10 ⁵ cells / well. The voltage was gradually increased and finally stimulated at 20V. Specifically, it was continuously stimulated with 2V for the first 48 hours, 5V for the next 48 hours, 10V for the next 72 hours, 15V for the next 72 hours, and then 20V until the 28th day of culture. Electrical stimulation was evaluated with 1 sample of 3 wells, and light stimulation was evaluated with 1 sample of 4 wells on a 96-well plate.

The results of comparing electrical stimulation and light stimulation are shown in FIGS. 8 to 11. FIG. 8 shows the result of contraction velocity, FIG. 9 shows the result of relaxation velocity, FIG. 10 shows the result of acceleration, and FIG. 11 shows the result of contraction distance. In each figure, (A) is the result of electrical stimulation, and (B) is the result of light stimulation. Changes in contractile force due to electrical stimulation were evaluated using skeletal muscle cells on

days

19, 21, and 27 of culture. As shown in FIGS. 8 to 11, the maximum contractile force was observed on the 19th day of culture (electrical stimulation) and the 18th day of culture (light stimulation), and the maximum contractile force of light stimulation was the maximum contractile force of electrical stimulation. It was shown to be equivalent. After that, on the 27th day of culture (electrical stimulation) and the 28th day of culture (light stimulation), the contractile force decreased to less than half of the maximum value, and it was shown that the degree of decrease was equivalent between electrical stimulation and light stimulation. rice field. That is, skeletal muscle cells having the same contractile force can be produced by using light stimulation instead of electrical stimulation, and skeletal muscle cells that do not express dystrophin such as DMDΔ44 can be either electrical stimulation or light stimulation. Even when given, it was shown that the contractile force decreased from the time when the maximum contractile force was observed.

In order to confirm that the skeletal muscle cells with reduced contractility meet the screening quality, the contractile force of the light-stimulated skeletal muscle cells on the 28th day of culture was analyzed with a video analyzer and a flat test was performed. rice field. Figure 12 shows the results of evaluating the contraction velocity. It was shown that the cells on the 28th day of culture with reduced contractile force sufficiently satisfied the screening quality when screening for a test substance that promotes the contractile activity of skeletal muscle cells.

5. At the time of re-seeding on the 4th day (Day 4) of culturing to promote maturation by photostimulation of differentiated skeletal muscle cells , collagen gel (Nippi) was spread inside the CellCarrier-Ultra 384 well, coated with Matrigel, and then re-seeded. Then, from the 10th day, the promotion of maturation by light stimulation is started, and light stimulation is continuously given until each test is performed. The light stimulator uses a blue LED array system for optogenetics (BRC Bio Research Center) to continuously apply stimulation with a frequency of 0.5 Hz, a pulse width of 50 msec, and a voltage of 10 V for 24 hours every day during the period. Change the medium at least once every two days. A light diffusing sheet is sandwiched between the array system and the plate so that even light stimuli are applied to each well. Evaluate the minimum number of wells to maintain muscle differentiation efficiency, contractile movement, and screening accuracy performed using 96-well plates.

The present invention is not limited to the above-described embodiments and examples, and various modifications can be made within the scope of the claims, and the technical means disclosed in the different embodiments may be appropriately combined. The obtained embodiments are also included in the technical scope of the present invention. In addition, all of the academic and patent documents described in this specification are incorporated herein by reference.

Claims

A method for producing skeletal muscle cells in which contractile activity is induced by light stimulation.
(1) A step of producing pluripotent stem cells that stably express channelrhodopsin,
(2) A production method including a step of inducing differentiation of the cells obtained in step (1) into skeletal muscle cells, and (3) a step of irradiating the cells that have started differentiation induction with light.
The production method according to claim 1, wherein in the step (3), light irradiation is started before the stage where cells fuse to form a myotube.
The manufacturing method according to claim 1 or 2, wherein in the step (3), continuous irradiation is performed from the start of light irradiation to the end of light irradiation.
13. Production method.
The production method according to any one of claims 1 to 4, wherein the pluripotent stem cell in the step (1) is a cell derived from a myopathy patient or a myotonia patient.
The production method according to any one of claims 1 to 5, wherein the pluripotent stem cell in the step (1) is a cell expressing one or more exogenous skeletal cell inducing factors selected from MyoD and Myf5.
A screening method for substances that promote or suppress the contractile activity of skeletal muscle cells.
(1) A step of inducing differentiation of pluripotent stem cells that stably express channelrhodopsin into skeletal muscle cells.
(2) Step of irradiating cells with light,
(3) Step of contacting the test substance with the cells,
A method comprising (4) measuring the contractile activity of cells and (5) selecting a test substance that promotes or suppresses the contractile activity of cells as compared with the case where the test substance is not contacted.
The screening method according to claim 7, using a multi-well plate.
The screening method according to claim 7 or 8, wherein the pluripotent stem cells in the step (1) are cells derived from a myopathy patient or a myotonia patient.