WO2023063187A1 - Production method for organoid - Google Patents

Abstract

The present invention provides a technology for more easily and efficiently producing a neural-muscle organoid. This method for producing a neural-muscle organoid comprises: a step (1a) for obtaining a muscle organoid by, in a culture device provided with at least two pillars extending from the culturing bottom surface toward a culturing vessel, inducing muscle differentiation in, in a state of covering the peripheries of the pillars, an organoid-forming composition containing at least one type of cells selected from the group consisting of pluripotent stem cells and early induction cells obtained through transient expression of MyoD in pluripotent stem cells; and a step (2) for obtaining a neural-muscle organoid by culturing the muscle organoid obtained in the step (1a) in a culture medium containing a neurotrophic factor to induce neural differentiation in same.

Description

Organoid production method

The present invention relates to an organoid production method and the like.

In various neuromuscular diseases, life is threatened by degeneration of motor neurons and skeletal muscle cells, or by abnormalities of the neuromuscular junction (NMJ), which is the junction between motor neurons and skeletal muscle tissue. resulting in progressive motor dysfunction. Known examples of neuromuscular diseases include amyotrophic lateral sclerosis (ALS), spinal and bulbar muscular atrophy (SBMA), and the like.

An evaluation model for in vitro research on the onset mechanism of neuromuscular diseases and screening of therapeutic agents is known. For example, a technique is known in which iPS cells are induced to differentiate into muscle cells, nerve cells, Schwann cells, and the like to form neuromuscular junctions (see Patent Document 1). However, what is obtained by the method described in Patent Document 1 is planar tissue, which is different from neuromuscular tissue (three-dimensional) in a living body.

Patent Document 2 describes a technique in which muscle cells and nerve cells are cultured in separate compartments, and the nerve tissue is connected to the three-dimensional muscle tissue through holes that connect the two compartments. However, this technique requires two types of cells to be prepared and cultured separately.

WO2019/078263 WO 2020/171052

An object of the present invention is to provide a technique for producing neuromuscular organoids more simply and efficiently.

As a result of extensive research in view of the above problems, the present inventors have found that (1a) MyoD is transiently applied to pluripotent stem cells in a culture device having at least two pillars extending from the culture bottom to the culture tank side. and an organoid-forming composition containing at least one type of cell selected from the group consisting of pluripotent stem cells and early-induced cells expressed in the pillars to induce muscle differentiation while covering the periphery of the pillars, and (2) culturing the muscle organoids obtained in step (1a) in a medium containing neurotrophic factors to induce neuronal differentiation to obtain neuro-muscle organoids. We have found that the above problems can be solved by a method for producing organoids. Based on this finding, the inventors have further studied and completed the present invention. That is, the present invention includes the following aspects.

Section 1. (1a) A group consisting of pluripotent stem cells and initial induced cells obtained by transiently expressing MyoD in pluripotent stem cells in a culture device having at least two pillars extending from the bottom of the culture to the culture tank side. The organoid-forming composition containing at least one cell selected from the above is covered with the pillar to induce muscle differentiation to obtain a muscle organoid, and (2) obtained in step (1a) culturing the obtained muscle organoids in a medium containing neurotrophic factors to induce neuronal differentiation to obtain neuro-muscle organoids;
Methods for producing neuro-muscular organoids.

Section 2. Item 1, wherein the induction of muscle differentiation includes transiently expressing MyoD in the cells and/or culturing the organoid-forming composition in a low serum medium or serum-free medium. .

Section 3. The production method according to item 1 or 2, wherein the muscle differentiation induction includes transient expression of MyoD in the cells by expression induction using a drug.

Section 4. Item 3, wherein the drug is doxycycline.

Item 5. Item 4, wherein the concentration of doxycycline in the medium is 0.05 μg/ml or more.

Item 6. Item 6. The production method according to any one of Items 1 to 5, wherein the cell concentration in the organoid-forming composition is 5×10 ⁶ cells/ml or more at the start of muscle differentiation induction in step (1a).

Item 7. The production method according to any one of Items 1 to 6, wherein the culture is performed in a medium containing a lock inhibitor during part or all of the muscle differentiation induction culture period.

Item 8. Item 8. The production method according to any one of items 1 to 7, wherein the amount of medium in step (1a) is 5 to 50 μl per 1×10 ⁴ cells at the start of muscle differentiation induction in step (1a).

Item 9. Items 1 to 8, wherein the organoid-forming composition contains a cell scaffolding material.

Item 10. Items 1 to 8, wherein the cell scaffolding material contains at least one selected from the group consisting of fibrin, matrigel, and collagen.

Item 11. Item 11. The production method according to any one of Items 1 to 10, wherein the neurotrophic factor includes at least one selected from the group consisting of glial cell line-derived Neurotrophic Factor (GDNF) and brain-derived Neurotrophic Factor (BDNF). .

Item 12. Item 12. The production method according to any one of Items 1 to 11, further comprising (3) moving the organoid-forming composition and/or its differentiation inducer to the upper part of the pillar by centrifugation.

Item 13. (1b) Initial induced cells, pluripotent stem cells, and myoblasts obtained by transiently expressing MyoD in pluripotent stem cells in a culture device having at least two pillars extending from the bottom of the culture to the culture tank side a step of inducing muscle differentiation with an organoid-forming composition containing at least one cell selected from the group consisting of cells covering the pillars,
Furthermore, (3) moving the organoid-forming composition and/or its differentiation inducer to the top of the pillar by centrifugation,
Methods of producing organoids.

Item 14. (1b) Initial induced cells, pluripotent stem cells, and myoblasts obtained by transiently expressing MyoD in pluripotent stem cells in a culture device having at least two pillars extending from the bottom of the culture to the culture tank side a step of inducing muscle differentiation with an organoid-forming composition containing at least one cell selected from the group consisting of cells covering the pillars,
The cells are at least one type selected from the group consisting of initial induction cells obtained by transiently expressing MyoD in pluripotent stem cells, and pluripotent stem cells, and one of the culture periods for the muscle differentiation induction. A method for producing organoids, in part or in whole, culturing in a medium containing a lock inhibitor.

Item 15. Item 15. The production method according to item 14, wherein the amount of medium in step (1b) is 5 to 50 μl per 1×10 ⁴ cells at the start of muscle differentiation induction in step (1b).

Item 16. An organoid obtained by the production method according to any one of Items 1 to 15.

Item 17. A kit for producing nerve-muscle organoids, comprising a culture device with at least two pillars extending from the bottom of the culture to the side of the culture tank, and wherein the culture device has one culture tank.

Item 18. A nerve-muscle organoid muscle contraction test kit comprising a culture device having at least two pillars extending from the culture bottom toward the culture tank and nerve-muscle organoids formed around the pillars.

Item 19. (Aa) performing some or all of the steps of the production method according to any one of items 1 to 15 in the presence of a test substance to obtain an organoid, or (Ab) the nerve-muscle organoid according to item 16; contacting with a test substance;
and (B) assessing the function and/or condition of the organoids;
A method of screening for therapeutic agents for neuromuscular diseases, comprising:

According to the present invention, it is possible to provide a technique for producing neuromuscular organoids more simply and efficiently.

1 shows a three-dimensional view of an example of a culture device of the present invention; FIG. 1 shows a plan view of an example of a culture device of the present invention; FIG. 1 shows a cross-sectional view of an example of a culture device of the present invention; FIG. This figure is a cross-sectional view taken along line AA in FIG. 1 shows a three-dimensional view of an example of a culture device of the present invention; FIG. Figure 2 shows the size of the device of the invention obtained in the example. All numerical values are in mm. FIG. 4 shows a schematic diagram of a well of a culture device-mounted microwell plate. An outline of the nerve-muscle organoid production process of Test Example 1 is shown. 1 shows a schematic diagram of a neuromuscular organoid obtained in Test Example 1. FIG. The ribbon-shaped tissue above the pillars is the neuromuscular organoid. 2 shows the measurement results of Test Example 2-1. The vertical axis indicates the amount of displacement of the upper part of the pillar, and the horizontal axis indicates the elapsed time. 2 shows the measurement results of Test Example 2-2. The vertical axis indicates the relative value of the MNX1 gene expression level. The horizontal axis indicates the number of days elapsed since the organization (FIG. 7: D0). FIG. 2 shows an immunostained image obtained in Test Example 2-3. FIG. 4 shows the measurement results of Test Example 2-4. The vertical axis indicates the amount of displacement of the upper part of the pillar, and the horizontal axis indicates the elapsed time from the addition of the chemical. Curare indicates vecuronium bromide and TTX indicates tetrodotoxin. The measurement results of Test Example 3 are shown. The vertical axis indicates the contractile force of the tissue, and the horizontal axis indicates the concentration of Doxycycline added to the muscle induction medium. The measurement results of Test Example 4 are shown. The vertical axis indicates the contractile force of the tissue, and the horizontal axis indicates the presence or absence of the ROCK inhibitor (yes: Y+, no: Y-). 4 shows the measurement results of Test Example 5. FIG. The vertical axis indicates the contractile force of the tissue, and the horizontal axis indicates the cell concentration in the fibrin gel at the start of muscle differentiation induction. 4 shows the measurement results of Test Example 5. FIG. The vertical axis indicates the contractile force of the tissue, and the horizontal axis indicates the number of days elapsed since the organization (FIG. 7: D0). In the legend, "96MNM" indicates the results of culture in a 96-well plate (200 µl of medium), and "48MNM-S" indicates the results of culture in a 48-well plate (700 µl of medium). The measurement results of Test Example 6 are shown. The vertical axis indicates the contractile force of the tissue, and the horizontal axis indicates the number of days elapsed since the organization (FIG. 7: D0). In the legend, "96MNM" indicates the results of culturing in a medium containing neurotrophic factors in a 96-well plate (200 µl of medium), and "96HS" indicates a muscle differentiation medium (2% horse serum, 1% ITS supplement I3146 , and DMEM containing 100 units penicillin). 2 shows the measurement results of contractile force in Test Example 7. FIG. 2 shows the coefficient of variation of contractile force measured in Test Example 7. FIG. The horizontal axis indicates the number of days elapsed after the medium was replaced with the muscle differentiation medium.

In this specification, the expressions "contain" and "include" include the concepts of "contain", "include", "consist essentially of" and "consist only of".

1. In one aspect of the method for producing organoids of the present invention,
(1a) A group consisting of pluripotent stem cells and initial induced cells obtained by transiently expressing MyoD in pluripotent stem cells in a culture device having at least two pillars extending from the bottom of the culture to the culture tank side. The organoid-forming composition containing at least one cell selected from the above is covered with the pillar to induce muscle differentiation to obtain a muscle organoid, and (2) obtained in step (1a) culturing the obtained muscle organoids in a medium containing neurotrophic factors to induce neuronal differentiation to obtain neuro-muscle organoids;
and methods for producing neuro-muscular organoids.

Further, in one aspect of the present invention,
(1b) Initial induced cells, pluripotent stem cells, and myoblasts obtained by transiently expressing MyoD in pluripotent stem cells in a culture device having at least two pillars extending from the bottom of the culture to the culture tank side A step of inducing muscle differentiation with an organoid-forming composition containing at least one cell selected from the group consisting of cells covering the pillars,
and a method for producing organoids.

These will be explained below. Process (1a) and process (1b) are collectively referred to as "process (1)" in some cases. The above manufacturing methods may be collectively referred to as "the manufacturing method of the present invention".

The culture device will be explained with reference to Figures 1 to 4. The culture device comprises a culture bottom 11 and at least two pillars. Typically, culture device 1 includes two pillars, pillar 12a and pillar 12b.

The pillars 12a and 12b extend from the culture bottom surface 11 toward the culture tank 14 side. The pillars 12a, 12b are preferably substantially perpendicular to the bottom surface 11 of culture. For example, the smallest acute angle formed by the pillars 12a, 12b and the culture bottom surface 11 is, for example, 75-90°, preferably 80-90°, more preferably 85-90°.

The shape of the pillars 12a and 12b is not particularly limited, and may be, for example, a cylindrical shape, a prismatic shape, a conical shape, a pyramidal shape, or a combination thereof.

The maximum cross-sectional diameter of the pillars 12a and 12b is not particularly limited, but is, for example, 0.1-2 mm, preferably 0.2-1 mm, and more preferably 0.3-0.7 mm.

Although the height of the pillars 12a and 12b is not particularly limited, it is, for example, 0.5-15 mm, preferably 1-10 mm, and more preferably 2-6 mm.

The distance between the fulcrum 16a of the pillar 12a and the fulcrum 16b of the pillar 12b is not particularly limited as long as a muscle organoid can be formed between the pillars, and is, for example, 0.5-5 mm, preferably 1-4 mm, more preferably 2-3 mm.

The pillars 12a, 12b are preferably provided with barrier structures 13a, 13b at their upper ends. The barrier structures 13a, 13b can more reliably prevent the organoids from slipping out of the upper ends of the pillars 12a, 12b.

The pillars 12a, 12b, if not provided with barrier structures 13a, 13b, are preferably joined at the top to form a pillar link 12 as shown in FIG.

The shape of the barrier structures 13a and 13b is not particularly limited. The barrier structures 13a, 13b can be plate-shaped, for example. The barrier structures 13a, 13b preferably have a side or diameter that is longer than the maximum cross-sectional diameter of the pillars 12a, 12b.

Culture device 1 preferably comprises a wall 15 . The wall 15 allows the shape and area of the culture bottom surface 11 to be adjusted. The shape of the culture bottom surface 11 is not particularly limited, but in a preferred embodiment, it can be dumbbell-shaped. The area of the culture bottom surface 11 is not particularly limited, but is, for example, 1-30 mm ² , preferably 2-20 mm ² , more preferably 3-10 mm ² .

The culture device 1 may include only the culture tank 14 as a culture tank, or may include culture tanks other than the culture tank 14. According to the present invention, the culture device 1 preferably has only one fermenter, as neuro-muscular organoids can be formed in only one fermenter.

There are no particular restrictions on the method of forming the culture device. For example, it may be formed by cutting, formed by a 3D printer, or the like. Alternatively, a template may be produced first using a photolithographic technique and then transferred. In addition, when manufacturing by transferring a template, a plurality of templates may be used as necessary.

There are no particular restrictions on the material of the culture device as long as it can be cut or the template can be transferred. For example, polyethylene, polypropylene, polyvinyl chloride, polyvinylidene chloride, polystyrene, polyvinyl acetate, polytetrafluoroethylene, ABS (acrylonitrile butadiene styrene) resin, AS (acrylonitrile styrene) resin, fluorine resin such as Teflon (registered trademark), Thermoplastic resins such as acrylic resins (PMMA); Thermosetting resins such as phenolic resins, epoxy resins, melamine resins, urea resins, unsaturated polyester resins, alkyd resins, polyurethanes, thermosetting polyimides, and silicone rubbers. In addition, when the skeletal muscle tissue formed in the first unit 2 is to be stained and observed, a resin having optical transparency is desirable. Examples of the resin in that case include cycloolefin polymer (COP), polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polycarbonate (PC), plastics such as rigid polyethylene, and silicon. Also, metal may be used instead of resin.

The culture device is usually used by installing it on the culture dish or the bottom surface of the well of the microwell plate (preferably the bottom surface of the well) as shown in FIG.

Pluripotent stem cells are stem cells that have pluripotency capable of differentiating into many cells existing in the body and also have proliferative ability, and are induced to intermediate mesoderm cells used in the present invention. Any cell that is Examples of pluripotent stem cells include, but are not limited to, embryonic stem (ES) cells, cloned embryo-derived embryonic stem (ntES) cells obtained by nuclear transfer, spermatogonial stem cells (“GS cells”), embryonic These include germ cells (“EG cells”), induced pluripotent stem (iPS) cells, cultured fibroblasts and pluripotent cells derived from bone marrow stem cells (Muse cells). Preferred pluripotent stem cells are iPS cells, more preferably human iPS cells, from the viewpoint that they can be obtained without destroying embryos, ova, etc. in the manufacturing process.

A method for producing iPS cells is known in the art, and can be produced by introducing a reprogramming factor into any somatic cell. Here, the reprogramming factors are, 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 or Glis1 genes or gene products are exemplified, these reprogramming factors may be used alone or in combination Also good. Combinations of initialization factors include WO2007/069666, WO2008/118820, WO2009/007852, WO2009/032194, WO2009/058413, WO2009/057831, WO2009/075119, WO2009/079007, WO2009/091659, WO2009/091659, and WO2009/091659. 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、 WO2010/056831, WO2010/068955, WO2010/098419, WO2010/102267, WO2010/111409, WO2010/111422, WO2010/115050, WO2010/124290, WO2010/147395, WO2010/147, 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, R. L. Judson et al. , (2009), Nat. Biotechnol. , 27:459-461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci USA. 106:8912-8917, Kim JB, et al. (2009), Nature. 461: 649-643, Ichida JK, et al. (2009), Cell Stem Cell. 5:491-503, HengJC, et al. (2010), Cell Stem Cell. 6:167-74, HanJ, 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. The combination described in is exemplified.

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. , passaged cells, and cell lines are all included. 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) blood cells (peripheral stem cells). blood cells, umbilical cord blood cells, etc.), lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, hepatocytes, gastric mucosa cells, enterocytes, splenocytes, pancreatic cells (pancreatic exocrine cells) etc.), differentiated cells such as brain cells, lung cells, kidney cells and adipocytes.

Early-stage induced cells are cells at the early stage of muscle differentiation obtained by transiently expressing MyoD in pluripotent stem cells, and are not particularly limited in this respect. Early induced cells are cells at a stage of differentiation prior to differentiation into myoblasts.

MyoD used in the present invention includes human myogenic differentiation 1 (MYOD1) consisting of the amino acid sequence represented by SEQ ID NO: 2, its orthologs in other mammals, and transcriptional and splicing variants thereof. . Alternatively, it has 90% or more, preferably 95% or more, more preferably 97% or more amino acid identity with any of the above proteins, and has a function equivalent to the protein (e.g., activation of transcription of a muscle-specific promoter) etc.).

Nucleic acids encoding MyoD include human myogenic differentiation 1 (MYOD1) cDNA consisting of the nucleotide sequence represented by nucleotide numbers 213 to 1175 in SEQ ID NO: 1, its orthologues in other mammals, transcriptional variants thereof, splicing Examples include mutants. Alternatively, it has 90% or more, preferably 95% or more, more preferably 97% or more nucleotide identity with any of the above nucleic acids, and has a function equivalent to the protein encoded by the nucleic acid (e.g., muscle-specific It may be a nucleic acid encoding a protein having a transcriptional activation of a promoter, etc.). Alternatively, it may have a sense strand having such a degree of complementarity that it can hybridize with the complementary strand of any of the above nucleic acids under stringent conditions. Here, stringent conditions are binding complexes or probes as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego CA). It can be determined based on the melting temperature (Tm) of the nucleic acid. For example, washing conditions after hybridization include conditions of "0.1×SSC, 0.1% SDS, 60° C.", and it is preferable that the hybridized state is maintained even after washing under such conditions. . 

The nucleic acid encoding MyoD may be DNA, RNA, or a DNA/RNA chimera. Also, the nucleic acid may be single-stranded, double-stranded DNA, double-stranded RNA, or a DNA:RNA hybrid. Double-stranded DNA or single-stranded RNA is preferred. The RNA may be RNA incorporated with 5-methylcytidine and pseudouridine (TriLink Biotechnologies) to suppress degradation, or modified RNA by phosphatase treatment. 

Although the method for transiently expressing MyoD in pluripotent stem cells is not particularly limited, for example, the following method can be used. Here, the term "expression" means that, in the case of a nucleic acid encoding MyoD, the MyoD protein is generated by transcription and translation from the nucleic acid in the cell, and in the case of the MyoD protein, It is synonymous with introducing the protein into the cell.

When MyoD is in the form of DNA, vectors such as viruses, plasmids, and artificial chromosomes can be introduced into pluripotent stem cells by methods such as lipofection, liposomes, and microinjection. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, Sendai viral vectors and the like. Examples of artificial chromosome vectors include human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC, PAC), and the like. Examples of plasmids include plasmids for mammalian cells.

The vector can contain regulatory sequences such as a promoter, an enhancer, a ribosome binding sequence, a terminator, and a polyadenylation site so that the DNA encoding MyoD can be expressed. (e.g., kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, etc.), selectable marker sequences such as thymidine kinase gene, diphtheria toxin gene, etc., reporter gene sequences such as fluorescent protein, β-glucuronidase (GUS), FLAG, etc. can be done. Promoters include 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 A promoter and a TRE promoter (a CMV minimal promoter with a Tet response element consisting of 7 consecutive tetO sequences) are exemplified.

In order to transiently express MyoD, the MyoD gene may be introduced into pluripotent stem cells using a transient expression vector. It is preferred to use Examples of inducible expression systems include drug-induced expression systems using tetracycline and its derivatives (e.g., doxycycline). It is preferable to use pluripotent stem cells into which a gene construct capable of drug-induced expression of MyoD has been previously introduced. .

For example, when using the above TRE promoter, it is desirable to simultaneously express a fusion protein with tetR and VP16AD or a fusion protein with reverse tetR (rtetR) and VP16AD (rtTA) in the same cell. In this Tet-On system, the fusion protein does not bind to the TRE and transcription does not occur in the absence of the drug. MyoD can be expressed transiently during the addition of

In addition, the above vector incorporates an expression cassette consisting of a DNA encoding a promoter and MyoD linked thereto into the chromosome of the pluripotent stem cell, and, if necessary, excises it. may have Examples of transposon sequences include, but are not limited to, piggyBac. As another embodiment, the expression cassette may be preceded and followed by LoxP sequences for the purpose of removing the expression cassette.

Also, when MyoD is in the form of RNA, it may be introduced into pluripotent stem cells by techniques such as electroporation, lipofection, and microinjection. When MyoD is in protein form, it may be introduced into pluripotent stem cells by techniques such as lipofection, fusion with cell membrane penetrating peptides (eg, TAT and polyarginine from HIV), microinjection, and the like.

Early-induced cells directly control the expression of MyoD itself (for example, introducing a nucleic acid encoding MyoD, expressing it using an agent that controls the promoter activity of an artificially introduced MyoD expression cassette, etc.) , Indirectly controlling the expression of MyoD, such as controlling the expression of transcription factors (e.g., Pax7, etc.) upstream of MyoD, and finally expressing MyoD by controlling the concentration of compounds such as cytokines can be obtained by

The period during which MyoD is transiently expressed in pluripotent stem cells may be a period during which cells in the early stage of muscle differentiation can be obtained, and can be appropriately changed depending on the type and properties of the pluripotent stem cells used. 12-48 hours, preferably 18-30 hours. For example, when using the drug-inducible expression system described above, it is preferable to add the drug and culture for this period. In another embodiment, when a vector having a transposon sequence is used, expression is stopped by introducing a transposase into the cell after the above-mentioned period of time has passed, and when a vector having a LoxP sequence is used, a desired period of time has passed. Later, a method of terminating expression by introducing Cre into cells is exemplified.

On the other hand, when MyoD is RNA or protein, introduction may be performed multiple times so that MyoD is present in the cell during the above period.

When generating early-induced cells, it is preferable to culture the pluripotent stem cells in the presence of a lock inhibitor before transiently expressing MyoD. Lock inhibitors are not particularly limited and include, for example, Y-27632, CloneR ^™ , RevitaCell, SMC4, CEPT and the like.

The culture conditions in the preparation of initial induced cells are preferably adherent culture conditions. For example, cell adhesion molecules such as extracellular matrices, specifically matrigel (BD), type I collagen, type IV collagen, gelatin, laminin, heparan sulfate proteoglycans, or entactin, and combinations thereof. It is preferable to culture in a culture dish with the addition of serum or a serum substitute, using a medium used for culturing animal cells as a basal medium. Here, as the basal medium, for example, GMEM (Glasgow Minimum Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), 199 medium, Eagle's Minimum Essential Medium ( EMEM), αMEM medium, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixed media thereof. In addition, as serum substitutes, albumin, transferrin, fatty acids, insulin, collagen precursors, trace elements, Knockout Serum Replacement (KSR) (serum substitute for FBS during ES cell culture), ITS-supplements and mixtures thereof, etc. subsumed.

The culture temperature is not particularly limited, but is about 30 to 40°C, preferably about 37°C, culture is performed in an atmosphere of air containing CO ₂ , and the CO ₂ concentration is preferably about 2 to 5%. .

Myoblasts are preferably skeletal myoblasts and are characterized by the expression of marker genes such as CD56, MyoD or myogenin. Myoblasts may be, for example, isolated cells or established cell lines obtained by subculturing them. Myoblasts are preferably of mammalian origin, more preferably of human origin.

The organoid-forming composition contains at least one type of cell selected from the group consisting of early induced cells, pluripotent stem cells, and myoblasts. The organoid-forming composition preferably comprises early-derived cells and may also comprise pluripotent stem cells.

The organoid-forming composition preferably contains a cell scaffolding material.

The cell scaffold material is not particularly limited as long as it is a material that serves as a scaffold for cells. Examples of cell scaffold materials include fibrin, collagen, matrigel, gelatin, elastin, keratin, laminin, entactin, fibronectin, heparan sulfate proteoglycan, hyaluronic acid, chitosan, alginic acid, agarose, polyethylene glycol hydrogel, peptide gel, DNA gel, ultra Molecular gel, polylactic acid, polyethylene glycol, silica gel, cellulose and the like. Among these, fibrin, Matrigel, collagen and the like are particularly preferred.

The organoid-forming composition is gel-like or sol-like, preferably gel-like.

The organoid-forming composition can be obtained by mixing cells with a cell scaffold material. For example, when fibrin is employed as a cell scaffolding material, cells, fibrinogen, and thrombin are mixed in a liquid so that thrombin acts on fibrinogen to form fibrin, thereby forming a gel-like organoid-forming composition. can be obtained. The organoid-forming composition can preferably be a fibrin gel containing cells.

At the start of muscle differentiation induction in step (1), the cell concentration in the organoid-forming composition is not particularly limited as long as muscle organoids can be formed from the organoid-forming composition. The cell concentration is, for example, 1×10 ⁶ cells/ml or more, preferably 2×10 ⁶ cells/ml or more, more preferably 3×10 ⁶ cells/ml or more, still more preferably 4×10 ⁶ cells/ml or more, More preferably, it is 5×10 ⁶ cells/ml or more. The cell concentration is preferably 6×10 ⁶ cells/ml or higher, more preferably 7×10 6 cells/ml or higher, and even more preferably 7.5× ^{10 6 cells} /ml or higher, within the above concentration range. The upper limits of the cell concentration are, for example, 40×10 ⁶ cells/ml, 20×10 ⁶ cells/ml, 15×10 ⁶ cells/ml and 10×10 ⁶ cells/ml. By setting the cell concentration as described above, the muscle contractile force of the resulting muscle organoids, as well as neuro-muscle organoids, can be increased, making these organoids more suitable for drug screening.

In step (1), muscle differentiation is induced in a culture device with the organoid-forming composition covering the periphery of the pillars. The method for covering the perimeter of the pillars with the organoid-forming composition is not particularly limited, but for example, the precursor solution of the organoid-forming composition is placed on the culture bottom surface of the culture device and spread over the entire culture bottom surface. A method of gelling or solifying the precursor solution in a spread state may be mentioned.

The method for inducing muscle differentiation in step (1) is not particularly limited as long as it is a method capable of forming muscle organoids from the organoid-forming composition. Induction of muscle differentiation is preferably performed by transiently expressing MyoD in cells, and/or adding the organoid-forming composition to a low serum medium (serum concentration is, for example, 8% or less, preferably 5% or less, more preferably 3% or less medium) or serum-free medium.

In step (1), the method for transiently expressing MyoD is the same as the method for producing the initial induced cells described above, except for the following.

In step (1), the period during which MyoD is transiently expressed in cells to induce muscle cells should be sufficient for muscle differentiation, and can be changed as appropriate depending on the type and properties of the cells used. The period is, for example, about 3 to 12 days, preferably about 4 to 10 days, more preferably about 5 to 8 days. For example, when using the drug-inducible expression system described above, it is preferable to add the drug and culture for this period. In another embodiment, when a vector having a transposon sequence is used, expression is stopped by introducing a transposase into the cell after the above-mentioned period of time has passed, and when a vector having a LoxP sequence is used, a desired period of time has passed. Later, a method of terminating expression by introducing Cre into cells is exemplified.

In step (1), the induction of muscle differentiation preferably includes transient expression of MyoD in the cells by induction of expression using a drug. The drug preferably includes doxycycline. In this case, culture is performed in a medium containing doxycycline to transiently express MyoD. The concentration of doxycycline in the medium is, for example, 0.01 μg/ml or more, preferably 0.02 μg/ml or more, more preferably 0.05 μg/ml or more, still more preferably 0.07 μg/ml or more, and even more preferably 0.09 μg/ml or more. , particularly preferably 0.1 μg/ml or more. The upper limit of the concentration is, for example, 5 μg/ml, 4 μg/ml, 3 μg/ml, or 2 μg/ml. By setting the concentration of doxycycline in the medium as described above, the muscle contractile force of the resulting muscle organoids, as well as the neuro-muscle organoids, can be increased, making these organoids more suitable for drug screening. can be done.

In one aspect of the present invention, it is preferable to culture in a lock inhibitor-containing medium during part or all of the culture period for inducing muscle differentiation in step (1). This can lead to higher muscle contractility of the resulting muscle organoids, and even neuro-muscle organoids, making these organoids more suitable for drug screening. As lock inhibitors, those described above can be used. The concentration of the lock inhibitor in the medium is, for example, 1-50 μM, preferably 2-40 μM, more preferably 5-20 μM. The period of culture in the lock inhibitor-containing medium is, for example, 1 to 4 days, preferably 1 to 3 days, more preferably 1 to 2 days.

The medium amount in step (1) is not particularly limited. The amount of medium in step (1) is, for example, 5 to 200 μl per 1×10 ⁴ of the cells (cells in the organoid-forming composition) at the start of muscle differentiation induction in step (1a). The volume of the medium is preferably 5-100 μl, more preferably 5-50 μl, even more preferably 10-40 μl, still more preferably 10-30 μl. By adjusting the medium amount to the above range, the muscle contractile force of the obtained muscle organoids, and furthermore, the muscle contractility of the nerve-muscle organoids can be increased, making these organoids more suitable for drug screening.

Muscle organoids are obtained from the organoid-forming composition in step (1). The availability of muscle organoids can be confirmed by the presence of muscle markers such as MHC and MEF2c. It should be noted that the muscle organoids produced in this manner may be a cell population containing several types of cells rather than a single cell population. Muscle organoids can be contracted by electrical stimulation. The contractile force can be measured according to or according to the method described in Test Example 3 below.

In step (2), the muscle organoids obtained in step (1) are cultured in a medium containing neurotrophic factors to induce neuronal differentiation to obtain nerve-muscle organoids.

Neurotrophic factors are ligands to membrane receptors that play an important role in the survival and maintenance of motor neuron function. NT-3), Neurotrophin 4/5 (NT-4/5), Neurotrophin 6 (NT-6), basic FGF, acidic FGF, FGF-5, Epidermal Growth Factor (EGF), Hepatocyte Growth Factor (HGF), Insulin , Insulin Like Growth Factor 1 (IGF 1), Insulin Like Growth Factor 2 (IGF 2), Glia cell line-derived Neurotrophic Factor (GDNF), TGF-b2, TGF-b3, Interleukin 6 (IL-6), Ciliary Neurotrophic Factor (CNTF) and LIF. Preferred neurotrophic factors in the present invention are GDNF and BDNF. Neurotrophic factors are commercially available from, for example, Wako, R&D systems, etc. and can be easily used, but may be obtained by forced expression in cells by methods known to those skilled in the art.

The concentration of GDNF in the culture medium can be, for example, 0.1 ng/mL to 100 ng/mL, preferably 1 ng/mL to 50 ng/mL, more preferably 5 ng/mL to 20 ng/mL.

The concentration of BDNF in the culture medium can be, for example, 0.1 ng/mL to 100 ng/mL, preferably 1 ng/mL to 50 ng/mL, more preferably 5 ng/mL to 20 ng/mL.

In the present invention, the culture medium used in step (2) can be prepared using a medium used for culturing animal cells as a basal medium. Examples of basal media include Glasgow's Minimal Essential Medium (GMEM) medium, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, and RPMI 1640. Medium, Fischer's medium, Neurobasal Medium (Life Technologies) and mixed medium of these are included. The medium may contain serum or may be serum-free. If necessary, the medium contains, for example, albumin, transferrin, Knockout Serum Replacement (KSR) (serum replacement for FBS during ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen May contain one or more serum replacements such as precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, It may also contain one or more substances such as growth factors, small molecules, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, and the like. Preferred media are media containing non-essential amino acids, B27 supplement, cAMP, GDNF, BDNF, IGF, retinoic acid, purmorphamine, and ascorbic acid.

After inducing muscle differentiation in step (1), neural differentiation can be induced by stopping MyoD expression and changing the medium to a medium for neural differentiation.

Regarding the culture conditions, the culture temperature is not particularly limited, but _is about 30 to 40°C, preferably about 37°C _. 5%.

The culture period is not particularly limited as long as motor neurons and Schwann cells appear, but step (2) is preferably performed for at least 3 days. It is more preferably 4 days or more, still more preferably 5 days or more. The upper limit of the culture period is not particularly limited, and is, for example, 30 days, 50 days, 100 days, or 200 days. Longer periods of culture can yield more mature organoids. However, from the viewpoint of cost and convenience, the upper limit of the culture period is preferably 20 days, more preferably 12 days, and even more preferably 10 days.

As a result, artificial neuromuscular junctions, that is, neuromuscular organoids containing motor neurons, muscle cells, Schwann cells, etc., can be obtained.

In steps (1) and (2), an organoid-forming composition or a differentiation inducer thereof (a differentiation inducer in the middle of differentiation from an organoid-forming composition to a muscle organoid, a muscle organoid, a muscle organoid to a nerve-muscle Differentiation inducers in the middle stages of differentiation into organoids, or neuro-muscle organoids) tend to migrate toward the top of the pillar as the contractile force increases. However, due to various factors, there are samples that do not move toward the upper end of the pillar, or that move only a little. In order to measure the contractile force, it is important that the organoids are present above the pillars. It is preferable to carry out the operation of pulling up the composition for forming an organoid or its differentiation inducer to the upper part of the pillar (preferably to the upper end) a plurality of times. The method of this lifting operation is not particularly limited, and for example, a centrifugal operation, a suction operation, or the like can be employed. In order to reduce sample-to-sample variability in muscle contractility, this lifting operation is preferably performed by centrifugation.

From the above viewpoint, the production method of the present invention preferably further includes a step (3) of moving the organoid-forming composition and/or its differentiation inducer to the upper part of the pillar by centrifugation.

The centrifugation operation is not particularly limited as long as the composition for forming an organoid and/or its differentiation inducer can move to the upper part of the pillar. Typically, a culture dish or microwell plate equipped with a culture device is placed in a centrifuge with a lid so that the medium does not spill out, and the pillar top faces in the direction opposite to the center of centrifugation, By centrifuging, the organoid-forming composition and/or its differentiation inducer can be moved to the top of the pillars.

The centrifugal force can be appropriately set according to the length of the pillar, the shape of the pillar, the structure of the upper end of the pillar, etc. The centrifugal force is, for example, 50-500×g, preferably 80-300×g, more preferably 100-250×g.

The centrifugation time can be set as appropriate according to the length of the pillar, etc. The centrifugation time is, for example, 1 second to 1 minute, preferably 3 to 30 seconds, more preferably 5 to 15 seconds.

The number of times of step (3) is not particularly limited. The number of times can be, for example, 1 to 10 times or 1 to 5 times from the viewpoint of cost and convenience.

2. kit 1
In one aspect of the present invention, a kit for producing nerve-muscle organoids, comprising a culture device having at least two pillars extending from the bottom surface of the culture to the side of the culture tank, and the culture device has one culture tank, Regarding. This will be explained below.

The culture device is as explained above.

The kit of the present invention is preferably a kit for use in the production method of the present invention.

The kit of the present invention preferably contains the cells, culture medium, additives, etc. used in steps (1) and (2) described above. For example, one or more reagents selected from the group consisting of pluripotent stem cells introduced in a MyoD-inducible state, drugs such as tetracycline or derivatives thereof, neurotrophic factors, culture dishes, microwell plates, and basal medium A kit containing The kit of the present invention may further include a document or an instruction describing the procedure of the manufacturing process.

3. Screening method In one aspect of the present invention,
(Aa) a step of performing some or all of the steps of the production method of the present invention in the presence of a test substance to obtain an organoid, or (Ab) a neuro-muscle organoid obtained by the production method of the present invention and a test substance contacting;
and (B) assessing the function and/or condition of the organoids;
and screening methods for therapeutic agents for neuromuscular diseases, including This will be explained below.

There are no particular restrictions on neuromuscular diseases as long as they are diseases related to signal transmission between motor neurons and skeletal muscle tissue. Examples include genetic diseases caused by gene mutations such as amyotrophic lateral sclerosis (ALS) and spinobulbar muscular atrophy (SBMA). In addition to genetic diseases, for example, muscle atrophy due to aging or bedridden, brachial plexus injury, facial nerve paralysis, etc. Axons extending from the cell body of motor nerve cells are degenerated by physical impact or neurotoxic substances. diseases caused by 

Test substances are, for example, cell extracts, cell culture supernatants, microbial fermentation products, extracts from marine organisms, plant extracts, purified or crude proteins, peptides, non-peptide compounds, synthetic low-molecular-weight compounds, and natural compounds. are exemplified. 

Test substances can also be used in (1) biological libraries, (2) synthetic library methods using deconvolution, (3) "one-bead one-compound" library methods, and (4) can be obtained using any of the many approaches in combinatorial library methods known in the art, including synthetic library methods using affinity chromatography selection; Biological library methods using affinity chromatography sorting are limited to peptide libraries, whereas the other four approaches can be applied to small compound libraries of peptides, non-peptide oligomers, or compounds (Lam (1997 ) Anticancer Drug Des. 12: 145-67). Examples of methods for synthesizing molecular libraries can be found in the art (DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6909-13; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91: 11422-6; Zuckermann et al. (1994) J. Med. Chem. 37: 2678-85; Cho et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2061; 37: 1233-51). Compound libraries can be in solution (see Houghten (1992) Bio/Techniques 13: 412-21) or beads (Lam (1991) Nature 354: 82-4), chips (Fodor (1993) Nature 364: 555-4), 6), bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698, 5,403,484, and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1865-9) or phage (Scott and Smith (1990) Science 249: 386-90; Devlin (1990) Science 249: 404-6; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87 Felici (1991) J. Mol. Biol. 222: 301-10; U.S. Patent Application No. 2002103360).

Step (Aa) can be carried out, for example, by adding the test substance to the culture medium of step (1) and/or step (2). Step (Ab) can be carried out, for example, by adding the test substance to the medium containing the neuro-muscle organoids. The concentration of the test substance in the medium can be appropriately set according to the type of the test substance.

In steps (Aa) and (Ab), a substance that induces or exacerbates neuromuscular disease can be used together with the test substance. Further, in step (Aa) and step (Ab), as cells in the organoid-forming composition, cells having gene mutations that induce or exacerbate neuromuscular diseases are used, or obtained using the cells Organoids can also be used.

The period of contact with the test substance is not particularly limited, but can be, for example, 1 hour to 14 days.

The evaluation target in step (B) is the function and/or state of the organoid. Organoid functions include, but are not limited to, contractile force. The contractile force can be measured, for example, according to or according to the methods described in Test Examples 2-4 and 3 below. The state of the organoid is not particularly limited, but includes, for example, the shape and size of the organoid, and the expression level and location of genes such as muscle cell markers and nerve cell markers in the organoid. These can be measured by, for example, PCR, Western blotting, immunostaining, etc., according to or in accordance with conventionally known methods.

If the results of the evaluation in step (B) yield an evaluation that indicates a therapeutic effect on a neuromuscular disease in comparison with the evaluation results of a control sample under the same conditions except that the test substance is not used (e.g., organoid morphology) normalization, increase in contractile force, increase in expression level of marker gene, etc.), the test substance can be selected as a therapeutic drug (or a candidate substance thereof) for neuromuscular diseases.

Four. kit 2
In one aspect of the present invention, a nerve-muscle organoid muscle contraction test kit comprising a culture device having at least two pillars extending from the bottom surface of the culture to the culture tank side and a nerve-muscle organoid formed around the pillars , concerning.

The culture device, etc. are as explained above.

The kit of the present invention is preferably a kit for use in the screening method of the present invention.

The kit of the present invention preferably contains the cells, culture medium, additives, etc. used in the screening method of the present invention. Examples include kits containing one or more reagents selected from the group consisting of test substances, culture dishes, microwell plates, and basal media. The kit of the present invention may further include written documents or instructions describing procedures such as a muscle contraction test, screening steps, and the like.

The present invention will be described in detail below based on examples, but the present invention is not limited by these examples.

Reference example 1. Preparation of Culture Device A culture device shown in FIGS. 1 to 3 was prepared. The size of the culture device is shown in FIG. The structure of the culture device will be described with reference to FIGS. The culture device 1 includes a culture bottom surface 11 and two pillars (pillar 12a, pillar 12b). The two pillars extend from the culture bottom surface 11 toward the culture tank 14 side. The culture device 1 has a wall 15 so that the culture bottom surface 11 has a dumbbell shape. The pillars 12a, 12b are provided with semicircular barrier structures 13a, 13b at their upper ends (ends opposite to the culture bottom surface 11).

The manufacturing method of the culture device is as follows. The culture device was fabricated using PDMS (Polydimethylsiloxane) (SILPOT 184, DuPont Toray Spacialty Materials, Tokyo, Japan). PDMS was poured into a mold made of Teflon and solidified by heating at 70°C for 1 hour. After removing from the mold, a disk made of thin film PDMS was placed on top of the pillar. A thin film disc was prepared as follows: a few drops of PDMS were dropped on a Teflon sheet, the rotation speed was increased to 1000 rpm for 30 seconds, spin-coated for 30 seconds, and then heated at 70°C for 1 hour. After solidification, a few drops of PDMS were added, the rotation speed was increased to 2000 rpm in 30 seconds, and spin coating was performed for 30 seconds. The thin film disk was placed on the top of the pillars of the culture device and fixed to the top of the pillars by heating at 70°C for 1 hour. The thin film disk was then cut in half to give a semicircular shape.

Reference example 2. Preparation of culture device-mounted microwell plate The prepared culture device was immersed in 70% ethanol, sterilized, and dried thoroughly before use. Uncured PDMS was dropped on the bottom of the microwell plate, and the culture device was put on it and oriented. It was fixed by heating on a hot plate set at 70°C for 30 minutes to obtain a culture device-mounted microwell plate. FIG. 6 shows a schematic diagram of the wells of the culture device-mounted microwell plate. The bottom of FIG. 6 is the bottom of the well. The prepared plates were sterilized by UV light with the lid open for at least 30 minutes. In order to prevent attachment of muscle tissue, the culture device was filled with a 2% Pluronic (registered trademark) solution the day before the start of culture and allowed to stand at 4°C. Pluronic solution was removed immediately before use.

Test example 1. Production of nerve-muscle organoids Induced cells were prepared by transiently expressing MyoD in pluripotent stem cells (Test Examples 1-1 and 1-2) and cultured (Test Example 1-3). After inducing muscle differentiation to obtain a muscle organoid while covering the two pillars of the device (Test Example 1-4), the obtained muscle organoid was kept in contact with the two pillars. The cells were cultured in a medium containing neurotrophic factors to induce neuronal differentiation to obtain nerve-muscle organoids (Test Example 1-5). An outline of the manufacturing process is shown in FIG. Details of the materials and methods used are as follows.

<Test Example 1-1. Drug-dependent MyoD overexpressing human iPS cell line 409B2 ^MyoD ＞
Undifferentiated human iPS cell line 409B2 ^MyoD (Uchimura, T., et al., A human iPS cell myogenic differentiation system permitting high-throughput drug screening, Stem Cell res. 25: 98-106 (2017)) is Kyoto University iPS cell Provided by the laboratory. 409B2 ^MyoD cells are induced to overexpress exogenous MyoD by Doxycycline, and iPS cells can be directly induced to become skeletal muscle cells. In addition, 409B2 ^MyoD cells have been introduced with puromycin resistance, and by adding puromycin to the medium, non-transfected cells can be removed.

<Test example 1-2. Undifferentiated culture of iPS cells>
409B2 ^MyoD, human iPS cells, were cultured according to the following procedure. Media was removed from the growing T25 flask and washed once with PBS, followed by TrypLE ^™ Select CTS ^™ (A12859-01, Life Technologies, California, USA) and UltraPure ^™ 0.5 M diluted 1:1000 in PBS. 350 µl of a trypsin solution mixed with an equal volume of EDTA pH 8.0 (15575-038, Life Technologies, California, USA) was added and incubated in a 37°C incubator for 4 minutes. After removing the trypsin solution and washing once with PBS, the lock inhibitor Y-27632 (08945-71, Nacalai Tesque, Japan) was added to 10 μM StemFit ^TM (AK02N, Ajinomoto, Tokyo, Japan). was added, and the cells were harvested with a cell scraper (99002, Techno Plastic Products, Trasadingen, Switzerland). After suspending the recovered cells 10 times with a 1 ml pipetman, the cell count was calculated using a fully automatic cell counter (TC20 ^TM , Bio-Rad Laboratories, California, USA), and the cells were added to a T25 flask at 25,000 cells/ml. 3 ml of AK02N containing 10 μM of Y-27632 and 10 μl of iMatrix-511 silk were cultured together in an incubator at 37° C. and 5% CO ₂ . After 48 hours, the medium was replaced with AK02N without Y-27632 and iMatrix-511 Silk, and then after 72 hours, the medium was replaced. After an additional 48 hours, subconfluent cells were passaged again.

<Test example 1-3. Early differentiation induction of human iPS cells into skeletal muscle cells>
Corning (registered trademark) Matrigel (registered trademark) Basement Membrane Matrix (354234, Corning. USA) was added to a 6-well plate (TR5000, Trueline, Japan) in serum-free DMEM/F-12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F- 12) Matrigel solution diluted 100-fold with (042-30555, Wako, Japan) was added at 1 ml/well and allowed to stand in a 37°C incubator for 1 hour. After that, the matrigel solution is removed, and the human iPS cells obtained by the same procedure as at the time of passage are suspended in StemFit containing 10 μM Y-27632, and 1 ml per well is 1.5*10 ⁵ cells/ml. and cultured in a 37°C, 5% CO ₂ incubator. 48 hours later Human ES cell medium (DMEM/F-12 + 20% Knockout Serum Replacement (KSR, 10828028, Sigma-Aldrich), 1% MEM Non-Essential Amino Acids solution (NEAA, 11140050, Gibco ^TM , USA), 2 Replace the medium with 10 μM Y-27632 added to mM L-glutamine, 100 μM 2-Mercaptoethanol (2-ME, 21438-82, Wako, Japan) and incubate at 37°C in a 5% CO ₂ incubator for 24 hours. cultured for hours. Thereafter, the medium was replaced with a human ES cell medium supplemented with 1 μg/ml Doxycycline Hyclate (Dox, D4116, Tokyo Chemical Industry), and cultured at 37° C. in a 5% CO ₂ incubator for 24 hours.

<Test Example 1-4. Preparation and induction of human iPS cell skeletal muscle tissue>
The medium was removed from the 6-well plate in which the human iPS cells were initially differentiated, and the plate was washed with PBS three times. 350 µl of Accutase was added, and the mixture was allowed to stand in a 37°C incubator for 4 minutes. Add 1 ml of muscle induction medium (αMEM + 10% KSR, 2% UltroserG, 200 μM 2-ME, 0.5% penicillin/streptomycin) supplemented with Y-27632 to 10 μM, pipette 10 times, Collected in a centrifuge tube. It was centrifuged at 800 rpm for 5 minutes and the supernatant was removed. The cells were counted and suspended in a growth medium to a concentration of 16.67*10 ⁶ cells/ml. 48.4% cell suspension, 20% DMEM (2X), 20% Fibrinogen from bovine plasma (10 mg/ml) (F8630, Sigma-Aldrich, Massachusetts, USA), 10% Matrigel® Basement Membrane Matrix (354234) , Corning. USA) and 1.6% Thrombin (2%) (T4648, Sigma-Aldrich, Massachusetts, USA). Solidified by incubating. Then, 200 μl/well of muscle induction medium containing 10 μM Y-27632 and 1 μg/ml Dox plus 1 mg/ml TA to prevent gel degradation was added at 37°C, 5% CO. ₂ cultured in an incubator. Every 24 hours thereafter, the medium was changed to myogenic induction medium without Y-27632 and supplemented with Dox, TA.

<Test Example 1-5. Induction of human iPS cell neuromuscular organoids>
After fibrin gel preparation and muscle induction for 6 days (Fig. 7: D6), the tissue was lifted to the top of the pillars, and the medium was changed to neural induction medium (KBM Neural stem cell (16050100, Kohjin Bio)) containing 1.0 mg/ml TA. 2% B-27 ^TM supplement, serum free (17504-44, Gibco ^TM , USA), 1% NEAA, 1 μM N6,2′-O-Dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt (cAMP, D0260, Sigma-Aldrich), 10 ng/ml Brain Derived Neurotrophic Factor (BDNF, 248-BD-025, R&D Systems, Minesota, USA), 10 ng/ml Glial cell Derived Neurotrophic Factor (GDNF, 212- GD-025, R&D Systems), 10 ng/ml Insulin like Growth Factor-1 (IGF-1, 291-G1-050, R&D Systems), 50 nM Retinoic Acid (RA, 180-01114, Wako), 500 nM Purmorphamine (540220, Calbiochem, California, USA), medium supplemented with Ascorbic Acid (AA, 012-04802, Wako)) to induce nerve cells. The cells were cultured in a 37°C, 5% CO ₂ incubator and replaced with a fresh neural induction medium containing TA once every two days. A schematic diagram of the obtained neuromuscular organoid is shown in FIG.

Test example 2. Evaluation of nerve-muscle organoids <Test Example 2-1. Acquisition of Shrinkage Characteristics>
On the 14th day after organization (Fig. 7: D0), electrical stimulation with a voltage of 4 V/mm, a frequency of 30 Hz, and a pulse duration of 2 ms was applied to the neuromuscular organoids for 3 seconds. A digital camera for microscopes (ORCA-Spark, Hamamatsu Photonics, Hamamatsu, Japan) incorporated in Japan) was used to shoot movies at a frame rate of 60 Hz. Motion tracking was performed on the captured video using PV Studio 2D (OA Science, Miyazaki, Japan), and the state of contraction was quantified.

The results are shown in Figure 9. Neuromuscular organoids were found to contract in response to electrical stimulation.

<Test example 2-2. Acquisition of gene expression changes>
The expression of the nerve cell marker gene (MNX1) in the organoids on the 6th and 16th days after the organization (Fig. 7: D0) was analyzed. RNA extraction was performed using Nucleo Spin® RNA XS (740902.50, MACHEREY-NAGEL, Germany). The tissue on the device was washed with PBS three times, removed from the culture device, the PBS was wiped off, and the three samples were collectively collected in a microtube. After that, RNA was extracted according to the protocol of the kit. RNA quantification was performed using Qubit 3.0 Fluorometer (Invitrogen, USA) and Qubit RNA HS assay (Q32852, Invirogen). Reverse transcription to cDNA was performed using ReverTra Ace qPCR RT Master Mix with gDNA Remover (FSQ-301, TOYOBO, Japan). Samples were diluted with Nuclease-free Water so that the RNA concentration was 16.7 ng/μl, and the protocol was followed. The generated cDNA was stored at -80°C. RT-PCR was detected with StepOne real-time PCR System (Applied Biosystems, USA) using THUNDERBIRD SYBR qPCR Mix (QPS201, TOYOBO). First, the cDNA was denatured by heating at 95°C for 1 minute, followed by 40 cycles of annealing and extension at 60°C for 1 minute. The sequences of the primers used are shown below. Relative changes in gene expression were calculated by the comparative Ct (ΔΔCt) method and normalized to GAPDH as an endogenous control gene.

The results are shown in Fig. 10. In neuromuscular organoids (16 days after organization (FIG. 7: D0)), it was found that the expression of the neuronal cell marker gene (MNX1) is enhanced.

<Test Example 2-3. Acquisition of Immunofluorescence Image>
After the cultured tissue was washed with PBS three times, it was fixed by reacting with 4% paraformaldehyde/phosphate buffer (163-20145, Wako) for 1 hour on a culture device. The fixative was removed, the culture device was removed from the well plate, and the tissue was removed from the culture device and collected in a microtube. The cells were washed with PBS for 10 minutes three times, added with a 3% Triton X-100 (T8787, Sigma-Aldrich) solution diluted with PBS, and treated at room temperature for 1 hour to permeabilize the membrane. After washing with PBS three times, a base solution (PBS + 10% Goat serum (S-1000, Vector Laboratories, USA), 0.01% Triton X-100) was added and allowed to react at room temperature for 1 hour for blocking. A primary antibody was added to the base solution and allowed to react overnight at room temperature. The primary antibodies used were anti-α-actinin mouse IgG antibody (A7811-2ML, Sigma-Aldrich) and anti-tubulin β-3 rabbit IgG antibody (802001, Biolegend, USA), respectively. After washing with PBS three times, a secondary antibody and DAPI (340-07971, Dojindo) were added to the base solution and allowed to react at room temperature for 2 hours. The secondary antibodies used were CF488A ^TM Goat anti rabbit IgG (H+L) (2 mg/ml, 20012, biotium) and CF555 ^TM Goat anti mouse IgG (H+L) (2 mg/ml, 20231, biotium). be. After washing with PBS three times, the tissue was placed on a slide glass, and a few drops of mounting medium for fluorescent staining (H-1400, Vector Laboratories) were added. Osaka, Japan) was used to acquire fluorescence images.

The results are shown in Fig. 11. Expression of both α-actinin, a muscle cell marker, and tubulin β-3, a nerve cell marker, were confirmed.

<Test Example 2-4. Functional Evaluation of Neuromuscular Junction>
In order to functionally evaluate the formation of the neuromuscular junction, we confirmed the contraction of skeletal muscle by single stimulation of motor neurons in the constructed neuromuscular organoids. Glutamic acid (070-00502, Wako), an agonist of NMDA receptors, was used to stimulate motor neurons. Glutamate is known to be a neurotransmitter that can act on lower motor neurons to promote neuronal firing. First, glutamic acid was added to a final concentration of 400 μM, and the appearance of the tissue was observed with a microscope digital camera (ORCA-Spark, Hamamatsu Photonics, Hamamatsu, Japan) incorporated into an upright microscope (BX53F, OLYMPUS, Japan). The video was shot at a frame rate of 60Hz. Next, in order to confirm contraction due to stimulation of motor neurons, tetrodotoxin was added to a final concentration of 2 μM, and the state of the tissue was observed. Tetrodotoxin can inhibit neuronal firing by selectively blocking voltage-gated sodium channels in the excitatory membrane of motor neurons. In addition, in order to confirm that nerve cell stimulation causes skeletal muscle contraction via the neuromuscular junction, we added glutamic acid, observed the state of the tissue, and then added vecuronium bromide (Curare , 223-01811, Wako) was added and the state of the tissue was observed. Contractions were quantified by motion tracking using PV Studio 2D.

The results are shown in Fig. 12. It was found that motor nerves and NMJs (neuromuscular junctions) were formed in the produced neuromuscular organoids.

Test example 3. A skeletal muscle tissue was constructed in the same manner as the production of human iPS cell skeletal muscle tissue with optimized Doxycycline concentration (Test Examples 1-1 to 1-4). In this test example, the concentration of Doxycycline added to the muscle induction medium during culture was set to 0, 0.01, 0.1, and 1 μg/ml, and culture was performed for 6 days (up to D6 in FIG. 7). The tissue was lifted and the muscle contraction force was measured. The details of the method for measuring muscle contractility are as follows.

<Production of electrical stimulator for measuring contractile force>
Electrodes were fabricated using a 3D printer and platinum electrodes in order to measure the contractile force by applying electrical stimulation to muscle tissue. Chemically stable platinum was used as the electrode, and it was designed as a cylindrical electrode with a diameter of 0.5 mm and a distance between the electrodes of 5 mm. The 3D-printed electrode base has a window at the top of each well, allowing observation of the muscle tissue. Each electrode is controlled by a selector switch, enabling electrical stimulation per well.

<Measurement of muscle contraction force>
Electrical stimulation to muscle tissue was generated by C-PACE (C-PACE 100, IonOptix, Massachusetts, USA). Electrical stimulation is generated by C-PACE and distributed to each well by the fabricated electrodes. A voltage of 4 V/mm, a frequency of 30 Hz, and a pulse duration of 2 msec were used for the measurements, using a microscope digital camera (ORCA-Spark, Hamamatsu Photonics, Hamamatsu, Japan) incorporated in an upright microscope (BX53F, OLYMPUS, Japan). The movement of muscle tissue was imaged by The micropost displacement was calculated using image analysis software ImageJ from photographs of muscle tissue before and after electrical stimulation. The measured micropost displacement was converted to contractile force using the following equation. E (modulus of elasticity of PDMS) is 1.7 MN/m ² , R (radius of cylinder) is 0.25 mm, L (length of cylinder) is 4 mm, and δ is the distance contracted by each muscle tissue. Formula: F= ^3πER4δ / ^4L3
The results are shown in FIG. It was found that the contractile force increased when the concentration of doxycycline was above a certain level.

Test example 4. Analysis of Effect of ROCK Inhibitor Skeletal muscle tissue was constructed in the same manner as in the preparation of human iPS cell skeletal muscle tissue (Test Examples 1-1 to 1-4). In this test example, the cells were cultured under the condition that Y-27632 (ROCK inhibitor) was added/not added (PBS only) to the muscle induction medium during the initial two days of culture (Fig. 7: D0 to 1). After culturing for 6 days (up to D6 in FIG. 7), lifting operation was performed, and muscle contraction force was measured in the same manner as in Test Example 3.

The results are shown in FIG. It was found that the addition of a lock inhibitor during the induction of muscle differentiation increased the contractile force.

Test example 5. Optimization of Cell Density The early differentiation-induced cells obtained in Test Example 1-3 were collected with Accutase, and the number of cells was counted. In order to examine the tissue cell density, cell suspensions were prepared at concentrations of 16.67, 12.5, 8.33 and 4.17*10 ⁶ cells/ml, and fibrin gels were prepared as described in Test Example 1-4. The cell densities in the fibrin gel are 8, 6, 4, 2*10 ⁶ cells/ml respectively. Thereafter, culture was performed as in Test Example 1-4, and after culturing for 6 days (up to D6 in FIG. 7), lifting operation was performed, and muscle contraction force was measured in the same manner as in Test Example 3.

The results are shown in FIG. It was found that when the cell concentration in the fibrin gel at the start of muscle differentiation induction was above a certain level, the contractile force became stronger.

Test example 6. Comparison of medium amount Culture devices were fixed to 96-well plates and 48-well plates. The fixing method is the same as in Reference Example 2. Using the obtained well plate, skeletal muscle tissue was constructed in the same manner as in the preparation of human iPS cell skeletal muscle tissue (Test Examples 1-1 to 1-4). Medium volume is 200 μl for 96-well plates and 700 μl for 48-well plates. After culturing for 6 days (up to D6 in FIG. 7), muscle contractility was measured over time in the same manner as in Test Example 3.

The results are shown in Figures 16 and 17. It was found that the contractile force was strengthened by adjusting the amount of medium for a certain number of cells.

Test example 7. We investigated the method of lifting the tissue in the improvement of the culture method and the induction of muscle differentiation. Details of the materials and methods used are as follows.

<Test Example 7-1. Subculture of skeletal muscle cells>
Hu5/KD3 (transferred from the National Center for Geriatrics and Gerontology) was used as skeletal muscle cells and cultured according to the following procedure. Collagen coating of the flask was performed first. A collagen solution was prepared by dissolving collagen (Cellmatrix type I-C: Nitta Gelatin Co., Ltd., Osaka, Japan) in 0.02N acetic acid at a concentration of 50 μg/ml. 5 ml of the prepared collagen solution was added to a T75 flask and allowed to stand at room temperature for 1 hour. After that, the collagen solution was removed and washed twice with 5 ml of PBS. The culture medium was removed from the cultured T75 flask, washed twice with PBS, added with 1 ml of 0.05% trypsin, and incubated in a 37° C. incubator for 2 minutes. muscle growth medium (DMEM (20% FBS, 2mM L-glutamine (073-05391, Wako, Tokyo, Japan), 0.5% penicillin/streptomycin, 2% UltroserG (15950-017, Sartorius, Gottingen, Deutschland))) ml and centrifuged at 1000 rpm for 5 minutes to collect the cell pellet. The resulting pellet was suspended in 2 ml of muscle growth medium, the number of cells was calculated using a fully automatic cell counter (TC20 ^™ , Bio-Rad Laboratories, California, USA), and 2.0*10 ⁵ cells were added to a coated flask. Add to 10 ml of muscle growth medium. The cells were seeded on T75 and cultured in an incubator at 37°C, 10% CO ₂ , 5% O ₂ . After 72 hours, the subconfluent cells were subcultured again.

<Test Example 7-2. Preparation of skeletal muscle tissue and lifting of muscle tissue by centrifugal operation>
The cells cultured in the T75 flask were collected by the same method as the subculture operation, the number of cells was counted, and the cells were suspended in a growth medium to a concentration of 4.17*10 ⁶ cells/ml. 48.4% cell suspension, 20% DMEM (2X), 20% Fibrinogen from bovine plasma (10 mg/ml) (F8630, Sigma-Aldrich, Massachusetts, USA), 10% Matrigel® Basement Membrane Matrix (354234) , Corning. USA), 1.6% Thrombin (2%) (T4648, Sigma-Aldrich, Massachusetts, USA), 12 μl of the cell-mixed hydrogel solution was seeded in a culture device and incubated at 37°C for 30 minutes. Solidified by incubating. After that, 2.0 mg/ml 6-Amino caproic Acid (6-AA, A2504, Sigma-Aldrich, Massachusetts, USA) and 1 mg/ml trans-4- (Aminomethyl) were added to muscle growth medium to prevent gel degradation. A mixture containing cyclohexanecarboxylic Acid (TA, A0236, Tokyo Chemical Industry, Tokyo, Japan) was added at 200 μl/well and cultured at 37° C. in a 5% CO ₂ incubator. After 48 hours, medium was removed and muscle tissue was lifted.

A conventional aspiration operation and a new centrifugation operation were used for lifting. In the suction operation, a gel loading tip (Q-010, QSP) was set on an aspirator, and muscle tissue was grasped by suction and lifted to the top of the pillar. For centrifugation, the plate was placed upside down in a tabletop centrifuge (5010, KUBOTA, Japan) and 200 xg for 10 seconds to lift the muscle tissue upwards. After that, 200 μl/well of muscle differentiation medium (DMEM (+ 2% FBS, 1% penicillin/streptomycin, 1% ITS supplement (I3146, Sigma-Aldrich, Massachusetts, USA))) containing 6-AA and TA was added. It was cultured in a 5% CO ₂ incubator at 37° C. After 48 hours, it was lifted again by the same operation, and a muscle differentiation medium was added.After that, the medium was replaced with a fresh muscle differentiation medium every 48 hours. The contractile force of the muscle tissue was measured in the same manner as in 3.

The results are shown in Figures 18 and 19. It was found that the sample-to-sample variability in muscle contractility was reduced by centrifuging tissue lifting.

Claims (19)

1. (1a) A group consisting of pluripotent stem cells and initial induced cells obtained by transiently expressing MyoD in pluripotent stem cells in a culture device having at least two pillars extending from the bottom of the culture to the culture tank side. The organoid-forming composition containing at least one cell selected from the above is covered with the pillar to induce muscle differentiation to obtain a muscle organoid, and (2) obtained in step (1a) culturing the obtained muscle organoids in a medium containing neurotrophic factors to induce neuronal differentiation to obtain neuro-muscle organoids;
   Methods for producing neuro-muscular organoids.
2. The muscle differentiation induction comprises transiently expressing MyoD in the cells and/or culturing the organoid-forming composition in a low serum medium or serum-free medium, The production according to claim 1 Method.
3. The production method according to claim 1 or 2, wherein the induction of muscle differentiation includes transiently expressing MyoD in the cells by expression induction using a drug.
4. 4. The manufacturing method according to claim 3, wherein the drug is doxycycline.
5. 5. The production method according to claim 4, wherein the concentration of doxycycline in the medium is 0.05 μg/ml or more.
6. The production method according to any one of claims 1 to 5, wherein the cell concentration in the organoid-forming composition is 5 × 10 ⁶ cells/ml or more at the start of muscle differentiation induction in step (1a).
7. The production method according to any one of claims 1 to 6, wherein the cell is cultured in a lock inhibitor-containing medium during part or all of the muscle differentiation-inducing culture period.
8. 8. The production method according to any one of claims 1 to 7, wherein the amount of medium in step (1a) is 5 to 50 μl per 1×10 ⁴ cells at the start of muscle differentiation induction in step (1a).
9. The production method according to any one of claims 1 to 8, wherein the organoid-forming composition contains a cell scaffolding material.
10. The production method according to any one of claims 1 to 8, wherein the cell scaffolding material contains at least one selected from the group consisting of fibrin, matrigel, and collagen.
11. The production according to any one of claims 1 to 10, wherein the neurotrophic factor comprises at least one selected from the group consisting of glial cell line-derived Neurotrophic Factor (GDNF) and brain-derived Neurotrophic Factor (BDNF). Method.
12. The production method according to any one of claims 1 to 11, further comprising (3) moving the organoid-forming composition and/or its differentiation inducer to the upper part of the pillar by centrifugation.
13. (1b) Initial induced cells, pluripotent stem cells, and myoblasts obtained by transiently expressing MyoD in pluripotent stem cells in a culture device having at least two pillars extending from the bottom of the culture to the culture tank side a step of inducing muscle differentiation with an organoid-forming composition containing at least one cell selected from the group consisting of cells covering the pillars,
    Furthermore, (3) moving the organoid-forming composition and/or its differentiation inducer to the top of the pillar by centrifugation,
    Methods of producing organoids.
14. (1b) Initial induced cells, pluripotent stem cells, and myoblasts obtained by transiently expressing MyoD in pluripotent stem cells in a culture device having at least two pillars extending from the bottom of the culture to the culture tank side a step of inducing muscle differentiation with an organoid-forming composition containing at least one cell selected from the group consisting of cells covering the pillars,
    The cells are at least one type selected from the group consisting of initial induction cells obtained by transiently expressing MyoD in pluripotent stem cells, and pluripotent stem cells, and one of the culture periods for the muscle differentiation induction. A method for producing organoids, in part or in whole, culturing in a medium containing a lock inhibitor.
15. 15. The production method according to claim 14, wherein the amount of medium in step (1b) is 5 to 50 μl per 1×10 ⁴ cells at the start of muscle differentiation induction in step (1b).
16. An organoid obtained by the production method according to any one of claims 1 to 15.
17. A kit for producing nerve-muscle organoids, comprising a culture device having at least two pillars extending from the bottom of the culture to the side of the culture tank, and wherein the culture device has one culture tank.
18. A nerve-muscle organoid muscle contraction test kit, comprising a culture device having at least two pillars extending from the bottom surface of the culture to the culture tank side, and nerve-muscle organoids formed around the pillars.
19. (Aa) performing some or all of the steps of the production method according to any one of claims 1 to 15 in the presence of a test substance to obtain an organoid, or (Ab) the nerve-muscle according to claim 16. contacting the organoid with the test substance;
    and (B) assessing the function and/or condition of the organoids;
    A method of screening for therapeutic agents for neuromuscular diseases, comprising:

Images