Spinal cord organoid and method for producing same

Abstract

Provided are: a method for producing a spinal cord organoid from a pluripotent stem cell in vitro, the method comprising (1) a step for culturing a human pluripotent stem cell in a culture medium that contains a ROCK inhibitor and a Wnt signal activator but does not substantially contain a TGFβ inhibitor or a BMP signal inhibitor, (2) a step for further culturing the cell obtained in step (1) in a culture medium that contains a retinoic acid receptor agonist and a hedgehog signal activator, and (3) a step for further culturing the cell obtained in step (2) in a culture medium that does not substantially contain a TGFβ inhibitor, a BMP signal inhibitor, a retinoic acid receptor agonist, or a sonic hedgehog stimulator to obtain a spinal cord organoid; and a spinal cord organoid obtained by the method. The spinal cord organoid according to the present application comprises at least a portion of the dorsal side of the spinal cord and a ventral region of the spinal cord.

Description

Spinal cord organoids and methods for producing same

[0001] The present disclosure relates to a method for producing spinal cord organoids from pluripotent stem cells. The present disclosure also relates to spinal cord organoids.

[0002] The spinal cord tissue is composed of well-organized neural circuits that process large amounts of information, enabling organisms to generate motor behavior in response to the environment. During early embryonic development, the spinal cord tissue initially forms a sheet-like structure called the neural plate. The neural plate undergoes morphological changes, such as bending and invaginating toward the dorsal side of the embryo, to form the neural tube, a tubular structure. During the dorsal-ventral patterning of the neural tube, more than 20 different classes of neurons are generated.

[0003] Two organizers play a key role in determining the properties of neural progenitor cells during spinal cord development. The roof plate (RP) is located dorsally and induces the dorsal progenitor domain by producing bone morphogenetic proteins (BMPs) and Wnts. Another organizing center, the floor plate (FP), is located ventrally and induces the ventral progenitor domain by producing Sonic hedgehog (Shh). With the help of these two morphogen-producing organizers, six distinct dorsal progenitor domains and five ventral progenitor domains are generated along the dorsal-ventral axis, as shown in Figure 1. These patterned progenitor domains then give rise to various subclasses of spinal interneurons and motor neurons.

[0004] In recent years, attention has been focused on the efficient generation of spinal motor neurons from human pluripotent stem cells (Non-Patent Documents 1 and 2). There have been several reports on the induction of patterned neural tube-like structures using mouse embryonic stem cells.

[0005] Because the spinal cord functions as an independent functional unit, if we are to analyze the spinal cord as a fully functional organ system, we need to derive organoids that contain not only single spinal motor neurons but also each of the regions that make up the spinal cord tissue.

[0006] A group including some of the inventors has proposed inducing the differentiation of three-dimensional spinal cord tissue by culturing in a medium composition that mimics the developmental process of the spinal cord (Non-Patent Document 3). This method is an improvement over the method for producing spinal cord cells in a specific region by controlling the culture conditions (Non-Patent Document 4).

[0007] In Non-Patent Document 3, organoids containing a specific ventral spinal cord region were successfully created from pluripotent stem cells by changing the concentration of smoothened agonist (SAG) added from the third day of differentiation.

[0008] The method described in Non-Patent Document 3 achieved the separate reproduction of the dorsal, intermediate, and ventral spinal cord regions in a single organoid. However, the living spinal cord is a tissue that contains all of the dorsal, intermediate, and ventral regions in one tissue, and no method for producing spinal cord organoids that simultaneously contain both the dorsal and ventral regions has been known to date.

[0009] Amoroso, M. W., Croft, G. F., Williams, D. J., O'Keeffe, S., Carrasco, M. A., Davis, A. R., Roybon, L., Oakley, D. H., Maniatis, T., Henderson, C. E., et al. (2013). Accelerated high-yield generation of limb-innervating motor neurons from human stem cells. J. Neurosci. 33, 574-586.Du, Z.-W., Chen, H., Liu, H., Lu, J., Qian, K., Huang, C. T.-L., Zhong, X., Fan, F. and Zhang, S.-C. (2015). Generation and expansion of highly pure motor neuron progenitors from human pluripotent stem cells. Nat. Commun. 6, 6626.Takenori Ogura,Hideya Sakaguchi,Susumu Miyamoto, Jun Takahashi: Three-dimensional induction of dorsal, intermediate and ventral spinal cord tissues from human pluripotent stem cells. Development (2018)145(16): dev162214.Maury, Y., Come, J., Piskorowski, R. A., Salah-Mohellibi, N., Chevaleyre, V., Peschanski, M., Martinat, C. and Nedelec, S. (2015). Combinatorial analysis of developmental cues efficiently converts human pluripotent stem cells into multiple neuronal subtypes. Nat.Biotechnol. 33, 89-96.

[0010] In research into the development, formation, and organization of the human spinal cord, applications in regenerative medicine, and screening for treatments for spinal cord-mediated diseases, it is desirable to create spinal cord organoids that contain a wider range of cell types from the spinal cord region, closer to living tissue, within a single organoid. The present disclosure aims to provide a method for producing spinal cord organoids that contain at least a portion of the dorsal and ventral regions of the spinal cord within a single organoid. The present disclosure also aims to provide spinal cord organoids that contain both the dorsal and ventral regions of the spinal cord within a single organoid. Means to solve the problem

[0011] The present disclosure provides the following: [Item 1] A method for producing spinal cord organoids from pluripotent stem cells in vitro, comprising: (1) culturing human pluripotent stem cells in a medium containing a ROCK inhibitor and a Wnt signaling activator and substantially free of a TGFβ inhibitor and a BMP signaling inhibitor; (2) further culturing the cells obtained in step (1) in a medium containing a retinoic acid receptor agonist and a hedgehog signaling activator; and (3) further culturing the cells obtained in step (2) in a medium substantially free of a TGFβ inhibitor, a BMP signaling inhibitor, a retinoic acid receptor agonist, and a sonic hedgehog stimulator to obtain spinal cord organoids. [Item 2] The method of item 1, wherein step (1) is performed in a medium substantially free of bFGF. [Item 3] The method of item 1, wherein the ROCK inhibitor is Y27632 and / or the Wnt signaling activator is CHIR99021. [Item 4] The method of Item 1, wherein the retinoic acid receptor agonist is retinoic acid, and / or the sonic hedgehog stimulator is a smoothened agonist (SAG). [Item 5] The method of any one of Items 1 to 4, wherein the pluripotent stem cells are iPS cells or ES cells. [Item 6] The method of any one of Items 1 to 5, wherein the spinal cord organoid has a portion expressing PAX7, PAX6, OLIG2, NKX2.2, and FOXA2. [Item 7] A spinal cord organoid obtained by the method of any one of Items 1 to 6. [Item 8] A spinal cord organoid derived from human pluripotent stem cells, having a portion expressing PAX7, PAX6, OLIG2, NKX2.2, and FOXA2. [Item 9] The spinal cord organoid according to Item 8, wherein the portion expressing PAX7 is continuous with the portion expressing one or more of OLIG2, NKX2.2, and FOXA2. [Item 10] A method for obtaining a non-human animal comprising a human spinal cord organoid, comprising the steps of obtaining a spinal cord organoid from human pluripotent stem cells by the method according to any one of Items 1 to 6, and transplanting the spinal cord organoid into a non-human animal. [Item 11] A non-human animal transplanted with the spinal cord organoid according to any one of Items 7 to 9. [Item 12] A regenerative medicine composition for treating spinal cord injury or disease, comprising the spinal cord organoid according to any one of Items 7 to 9.[Item 13] A method for evaluating the effect of a test substance on the spinal cord, comprising the steps of contacting the spinal cord organoid according to any one of Items 7 to 9 with a test substance, and evaluating the effect of the test substance on the spinal cord organoid. [Item 14] A pharmaceutical composition for treating spinal cord injury or disease, comprising as an active ingredient a substance evaluated to have an effect on the spinal cord by the method of Item 13. [Item 15] A method for treating spinal cord injury or disease, comprising administering a substance evaluated to have an effect on the spinal cord by the method of Item 13 to a subject in need of treatment for spinal cord injury or disease.

[0012] Schematic diagram showing the distribution of each differentiation marker within the spinal cord. Schematic diagram of the differentiation protocol of the Example and the Reference Example (Non-Patent Document 3). The results of the Example and the Reference Example (Non-Patent Document 3) are shown. The distribution of each differentiation marker within the spinal cord is shown on the left. Enlarged view of a portion of Figure 3. Representative heat maps (for one test) of intracellular calcium fluctuations over time in cells within organoids of the Example and the Reference Example. The waveforms of the heat map in Figure 5 were analyzed using WaveFinder to measure intercellular synchrony (cross correlation). *p<0.05, 2-way ANOVA Tukey Test (Factor; group & study)

[0013] In this specification and claims, when a numerical value is accompanied by the term "about," it is intended to include a range of ±10% of that value. For example, "about 20" includes "18 to 22." A range of numerical values ​​includes all values ​​between and at the endpoints. "About" in reference to a range applies to both endpoints of the range. Thus, for example, "about 20 to 30" includes "18 to 33."

[0014] In this disclosure, "organoid" refers to a three-dimensional cell aggregate formed in vitro that resembles living tissue. In the context of organoids, "similar to living tissue" or "similar to living tissue" refers to the similarity of the anatomical structure of the organoid to that of living tissue, as well as the respective gene and protein expression patterns. For example, most or all of the cells that make up organoids are derived from cells with differentiation and proliferation capabilities, preferably pluripotent stem cells. The cultured cells that make up organoids can be differentiated into various cell types according to known methods.

[0015] In the present disclosure, "pluripotent stem cells" refers to stem cells that can be cultured in vitro and have the ability to differentiate into tissues derived from three germ layers (ectoderm, mesoderm, and endoderm), i.e., pluripotency. Pluripotent stem cells can be established, for example, from fertilized eggs, cloned embryos, somatic cells, germ stem cells, or intracellular stem cells. Pluripotent stem cells include, for example, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells) derived from somatic cells, embryonic tumor cells (EC cells), or embryonic germ stem cells (EG cells). Pluripotent stem cells are preferably ES cells or iPS cells.

[0016] In the present disclosure, "ES cells" refers to stem cells that have the ability to self-replicate and pluripotency, and are derived from an early embryo. Examples of ES cells include human ES cells.

[0017] In the present disclosure, "iPS cells" refer to pluripotent stem cells induced from somatic cells, and refer to cells artificially endowed with pluripotency similar to that of embryonic stem cells by reprogramming somatic cells. iPS cells can be established by reprogramming differentiated cells such as fibroblasts through the expression of genes such as Oct3 / 4, Sox2, Klf4, and Myc. iPS cells are, for example, human iPS cells established by reprogramming differentiated cells such as human fibroblasts.

[0018] Before being subjected to the method of the present application, the pluripotent stem cells are cultured in a culture vessel coated with a pluripotent stem cell culture coating, such as laminin coating, a commercially available product such as iMatrix Silk (Nippi Corporation), in a commercially available pluripotent cell culture medium, such as StemFit. （登録商標） It is maintained at

[0019] The basic medium used for inducing differentiation of pluripotent stem cells in the method of the present disclosure can be prepared by appropriately adding factors required for each stage to a basal medium used for culturing animal cells. Examples of basal media include MEM Zinc Option medium, IMEM Zinc Option medium, MEM medium, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Glasgow's MEM (G-MEM) medium, DMEM / F12 medium, Ham's F12 medium, RPMI1640 medium, Fischer's medium, and mixtures thereof. The basal medium may contain serum (eg, fetal bovine serum (FBS)) or may be serum-free. If desired, the medium may contain one or more substances such as, for example, albumin, transferrin, serum-free supplements (such as KnockOut Serum Replacement (KSR) (Thermo Fisher Scientific), N2 supplement (Thermo Fisher Scientific), B27 supplement (Thermo Fisher Scientific)), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3′-thiolglycerol, lipids, amino acids, L-glutamine, GlutaMAX (Thermo Fisher Scientific), non-essential amino acids (NEAA), vitamins, growth factors, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, and the like. It may also contain one or more substances known to be added to other animal culture media.

[0020] In the method of the present disclosure, culturing can be performed using commercially available animal cell culture vessels. In the method of the present disclosure, induction of spinal cord organoids from pluripotent stem cells is performed in suspension culture. "Suspension culture" refers to culturing cells in a non-adherent state to the inner wall of the culture vessel. The culture vessel is not particularly limited, but can be one that has not been treated to artificially improve adhesion to cells, for example, not coated with an extracellular matrix, or one that has been treated to artificially suppress cell adhesion, for example, coated with polyhydroxyethyl methacrylate (poly-HEMA) or a polymer of 2-methacryloyloxyethyl phosphorylcholine (Lipidure).

[0021] The shape of each well of the culture vessel may be any shape that allows cells cultured in suspension to aggregate and consolidate under their own weight, and culture vessels with shapes such as a V-bottom or a U-bottom are preferably used. An example is a V-bottom plate. The size of each well of the culture vessel is not limited, and commercially available culture plates of various sizes can be used. Examples include 6-, 12-, 24-, 48-, and 96-well V- or U-bottom plates, as well as culture vessels with multiple fine wells on the culture surface, such as EZ SPHERE (registered trademark) spheroid formation culture vessels (AGC Technoglass Corporation) and AggreWell (trademark) plates (STEMCELL Technologies).

[0022] The culture may be carried out under general conditions for culturing animal cells, including, but not limited to, a temperature of about 30 to 40°C, for example, about 37°C, in CO ２ The reaction is carried out under an atmosphere containing CO ２ The concentration can be about 0.05-15%, about 3-7%, or about 4-6%, for example about 5%.

[0023] Step (1) In step (1) of the present disclosure, human pluripotent stem cells are cultured in a medium containing a ROCK inhibitor and a Wnt signaling activator, and substantially free of a TGFβ inhibitor and a BMP signaling inhibitor.

[0024] Human pluripotent stem cells, such as ES cells or iPS cells, are usually maintained in an adherent culture state, and before seeding in the medium of step (1), they are detached by a conventional method to form single cells, which are then seeded in the medium of step (1).

[0025] In one embodiment, the basic medium used in step (1) is, for example, a medium based on G-MEM medium to which appropriate additives have been added. Examples of additives include, but are not limited to, KSR, nonessential amino acids, pyruvic acid, 2-mercaptoethanol, and penicillin / streptomycin. The concentration of KSR to be added is not limited, but is, for example, about 10 to 30%, for example, about 20%.

[0026] The medium used in step (1) is a basal medium that contains a ROCK inhibitor and a Wnt signaling activator, and is substantially free of a TGFβ inhibitor and a BMP signaling inhibitor.

[0027] In the present disclosure, the ROCK inhibitor is not particularly limited as long as it can inhibit the function of Rho-kinase (ROCK). Examples include Y-27632, Fasudil / HA1077, SR3677, GSK269962, H-1152, Wf-536, and derivatives thereof, as well as antisense nucleic acids against ROCK, RNA interference-inducing nucleic acids (e.g., siRNA), dominant-negative mutants, and expression vectors thereof. Other known low-molecular-weight compounds can also be used as ROCK inhibitors. When referring to a ROCK inhibitor in this application, one or more ROCK inhibitors may be used. Preferred ROCK inhibitors include Y-27632, Fasudil / HA1077, SR3677, GSK269962, and H-1152, with Y-27632 being particularly preferred.

[0028] The concentration of the ROCK inhibitor in the medium is not particularly limited. When Y-27632 is used, the concentration is, for example, 10 μM to 100 μM, about 50 μM. Alternatively, the concentration of the ROCK inhibitor is, for example, 10 μM to 100 μM, about 50 μM, which is a concentration that exhibits the same level of ROCK inhibitory effect as that of Y-27632.

[0029] In the present disclosure, the Wnt signal activator is not particularly limited as long as it is a substance that can induce activation of the Wnt signal. Examples of the Wnt signal activator include Wnt protein and Wnt1 protein, CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile; CAS Registry Number: 252917-06-9), BIO (6-bromoindirubin-3'-oxime; CAS Registry Number: 667463-62-9), K Examples of GSK3β inhibitors include enpaullone (9-bromo-7,12-dihydroindolo[3,2-d][1]benzazepin-6(5H)-one; CAS Registry Number: 142273-20-9) and IM-16 (3-(4-fluorophenylethylamino)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione; CAS Registry Number: 1129669-05-1). CHIR99021 is particularly preferably used in the methods of the present disclosure.

[0030] The concentration of the Wnt signaling activator in the medium is not particularly limited. When CHIR99021 is used, the concentration is, for example, 1.5 μM to 4.5 μM, or about 3.0 μM. Alternatively, the Wnt signaling activator may be, for example, at a concentration of 1.5 μM to 4.5 μM, or about 3.0 μM, which exhibits the same level of Wnt signaling activation activity as CHIR99021.

[0031] In the method of the present disclosure, the medium in step (1) is substantially free of TGFβ inhibitors and BMP inhibitors.

[0032] In the present disclosure, a TGFβ inhibitor is a substance that inhibits signal transduction that continues from the binding of TGFβ to its receptor to SMAD, and is a substance that inhibits binding to the ALK family of receptors, or a substance that inhibits the phosphorylation of SMAD by the ALK family. Examples of TGFβ inhibitors that are not contained in the medium in step (1) of the present disclosure include, but are not limited to, Lefty-1 (NCBI Accession No.: NM_010094 for mouse, NM_020997 for human), SB431542, SB202190, SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276 (Lilly Research Laboratories), A-83-01 ((3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide)), ALK5 inhibitor II (2-[3-[6-methylpyridin-2-yl]-1H-pyrazol-4-yl]-1,5-naphthyridine), TGFβRI kinase inhibitor VIII (6-[2-tert-butyl-5-[6-methyl-pyridin-2-yl]-1H-imidazol-4-yl]-quinoxaline), and derivatives thereof.

[0033] In the present disclosure, examples of BMP signal inhibitors include, but are not limited to, proteinaceous inhibitors such as Chordin, Noggin, and Follistatin, dorsomorphin (6-[4-2-piperidin-1-yl-ethoxy]-phenyl]-3-pyridin-4-yl-pyrazolo[1,5-a]pyrimidine), [4-2-piperidin-1-yl-ethoxy]phenyl and derivatives thereof, and LDN-193189 (4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline).

[0034] In the method of the present disclosure, the medium in step (1) preferably does not substantially contain bFGF.

[0035] In the present disclosure, the statement that a medium is "substantially free" of a certain component means that a medium to which the component is not added is used. Even if the component is contained in a very small amount in the medium due to the convenience of the reagents and procedures used, for example, up to an amount equivalent to about 10 nM of SB431542 for the TGFβ inhibitor, up to about 0.1 nM of LDN-193189 for the BMP signal inhibitor, or up to about 20 pg / mL of bFGF, the medium is understood to be "substantially free" of the component. In one embodiment, the component is present in the medium at a concentration not exceeding the IC ５０ A medium may be understood to be "substantially free" of a component even if it is present at a concentration less than 1 / 100 of the original value. In the present disclosure, when a medium is described as being "substantially free" of a component, it preferably means that the medium is completely free of that component.

[0036] In the method of the present disclosure, when a 96-well V-bottom culture vessel is used, 1.0 × 10 single-cell pluripotent stem cells are cultured per well. ３ pieces ~ 1.5×10 ４ pieces, for example, about 1.0 × 10 ４ The cells are seeded in a medium containing a ROCK inhibitor and a Wnt signaling activator, and substantially free of a TGFβ inhibitor and a BMP signaling inhibitor, so that the cells are 100% pure. The culture period in step (1) is 2 to 12 days, 2 to 9 days, 2 to 4 days, for example, about 3 days.

[0037] Step (2) is a step of further culturing the cultured cells of step (1) in a medium containing a retinoic acid receptor agonist and a hedgehog signal activator. The basal medium for step (2) may be the same as that used in step (1).

[0038] In the present disclosure, examples of retinoic acid receptor (RAR) agonists include, but are not limited to, retinoic acid (such as the stereoisomers all-trans-trans-retinoic acid (ATRA), EC23, and 9-cis-retinoic acid (9-cisRA)), Am80, AM580 (4-[[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl]carboxamido]benzoic acid), TTNPB (4-[[E]-2-[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl]-1-propenyl]benzoic acid), AC55649 (4'-octyl-[1,1'-biphenyl]-4-carboxylic acid), honokiol, and magnolol. In the present disclosure, retinoic acid (ATRA), AM580, TTNPB, and AC55649 are preferably used as RAR agonists, and for example, retinoic acid (ATRA) is used.

[0039] In step (2), the concentration of the retinoic acid receptor agonist in the medium may be appropriately determined. When retinoic acid (ATRA) is used as the retinoic acid receptor agonist, the concentration in the medium is, for example, 50 nM to 500 nM, or about 100 nM.

[0040] In the present disclosure, examples of hedgehog signal activators include recombinant sonic hedgehog protein, palmorfamine, and 3-chloro-N-[(1r,4r)-4-(methylamino)cyclohexyl]-N-[3-(pyridin-4-yl)benzyl]benzo[b]thiophene-2-carboxamide, which is a smoothened agonist (SAG).

[0041] In step (2), the concentration of the hedgehog signal activator in the medium is not particularly limited. When SAG is used as the hedgehog signal activator, the concentration is, for example, 50 nM to 1 μM, about 500 nM. Alternatively, the hedgehog signal activator may be, for example, a concentration that exhibits the same level of activity as when SAG is used at a concentration of 50 nM to 1 μM, about 500 nM.

[0042] The transition from step (1) to (2) may be carried out by changing the medium for step (2) all at once after the completion of step (1), by providing a culture period using a mixed medium containing the components of both the medium for step (1) and the medium for step (2) at the concentrations used in each step, or by gradually changing the medium from step (1) to the medium for step (2). When providing a culture period using a mixed medium or gradually changing the medium from step (1) to the medium for step (2), there may be an overlapping period in which the cells are cultured in a medium containing the components of both steps, i.e., a medium containing a ROCK inhibitor, a Wnt activator, a hedgehog signal activator, and a retinoic acid receptor agonist. The overlapping period may be, for example, 1, 2, 3, 4, or 5 days.

[0043] In the present disclosure, when there is an overlapping period between step (1) and step (2) or step (2) and step (3), the number of days shown as the culture period for each step refers to the period from the start of culture in a mixed medium of the medium of the previous step and the medium of the step in question to the start of culture in a mixed medium of the medium of the step in question and the medium of the next step.

[0044] The culture in step (2) is carried out for 8 to 19 days, preferably 10 to 18 days, more preferably 13 to 17 days, for example, about 15 days. Preferably, the medium is changed about once every three days.

[0045] Step (3) In step (3), the cells are cultured in a medium substantially free of the substances added in steps (1) and (2). Specifically, the medium used in step (3) is substantially free of TGFβ inhibitors, BMP signal inhibitors, retinoic acid receptor agonists, and sonic hedgehog stimulators. In the method of the present disclosure, the medium used in step (3) is preferably substantially free of bFGF.

[0046] The medium used in step (3) may be, for example, DMEM / F12 (Gibco) supplemented with N2 supplement, Chemically Defined Lipid Concentrate, Fungizone, and Penicillin / Streptomycin.

[0047] After completion of the culture in step (2), the medium is replaced with the medium for step (3). After completion of the culture in step (2), the spheroids formed in each well may be transferred to another culture vessel containing the medium for step (3) and step (3) may be initiated, or the medium for step (2) may be replaced with the medium for step (3) in the same culture vessel.

[0048] The transition from step (2) to (3) may be carried out by exchanging the medium for step (3) all at once after the completion of step (2), by providing a period of culture in a mixed medium in which the components of step (2) are added to the basic medium for step (3), or by gradually exchanging from step (2) to the medium for step (3). When a culture period is provided in a mixed medium in which the components of step (2) are added to the basic medium for step (3), or when the medium is gradually exchanged from step (2) to the medium for step (3), an overlap period occurs in which the cells are cultured in a mixed medium containing a retinoic acid receptor agonist and a hedgehog signal activator for step (2). For example, this overlap period may be 1, 2, 3, 4, or 5 days. When an overlap period is included in step (3), the cells are cultured in a medium substantially free of a TGFβ inhibitor, a BMP signal inhibitor, a retinoic acid receptor agonist, and a sonic hedgehog stimulant after the overlap period has ended.

[0049] As described above, in this disclosure, the statement that a medium is "substantially free" of a certain component means that a medium to which the component is not added is used. Even if the component is contained in a very small amount in the medium due to the convenience of the reagents and procedures used, for example, a TGFβ inhibitor in an amount equivalent to about 10 nM of SB431542, a BMP signal inhibitor in an amount equivalent to about 0.1 nM of LDN-193189, bFGF in an amount equivalent to about 20 pg / mL, a retinoic acid receptor agonist in an amount equivalent to 0.1 nM of all-trans retinoic acid (ATRA), and a hedgehog signal activator in an amount equivalent to 0.05 nM of SAG, the medium is understood to be "substantially free" of the component. In one embodiment, the component is present in the medium at a concentration of less than the IC ５０ Value or EC ５０A medium may be understood to be "substantially free" of a component even if it is present at a concentration below 1 / 100 of the value. In the present disclosure, when a medium is described as being "substantially free" of a component, it preferably means that the medium is completely free of that component.

[0050] Step (3) can be performed until one or more or all of the differentiation markers selected from PAX7 (pd2-pd6 marker), PAX6 (pd1-pMN marker), OLIG2 (pMN marker), NKX2.2 (P3 marker), and FOXA2 (FP marker) for each region of the spinal cord shown in Figure 1 are expressed. The culture period may be 3 days or more, 5 days or more, 7 days or more, for example, 3 to 12 days, 5 to 12 days, or 7 to 12 days. Preferably, the medium is changed approximately once every 3 days. The upper limit of the culture period is not particularly limited, and after obtaining organoids expressing differentiation markers, culture may be continued under the conditions of step (3) to maintain them.

[0051] Spinal cord organoids of the present disclosure are obtained in step (3). Organoids obtained by the method of the present disclosure express PAX7 (pd2-pd6 marker), as well as one or more selected from OLIG2 (pMN marker), NKX2.2 (P3 marker), and FOXA2 (FP marker). In one embodiment, organoids obtained by the method of the present disclosure express all of the differentiation markers for each region of the spinal cord shown in FIG. 1 : PAX7 (pd2-pd6 marker), PAX6 (pd1-pMN marker), OLIG2 (pMN marker), NKX2.2 (P3 marker), and FOXA2 (FP marker). Preferably, the portion expressing PAX7 is contiguous with the portion expressing one or more of OLIG2, NKX2.2, and FOXA2.

[0052] In the present disclosure, the expression "express all" of the listed markers means that the organoid contains regions that express one or more of the listed markers, and the organoid as a whole expresses all of the listed markers. The present disclosure provides spinal cord organoids obtained by the method of the present disclosure.

[0053] The expression of each marker in the spinal organoid of the present disclosure can be confirmed by known methods. For example, the obtained organoid can be fixed, frozen, and then immunostained using antibodies specific to each marker.

[0054] The spinal cord organoids provided by the present disclosure can be used as a useful experimental tool for analyzing human spinal cord development and for studying the formation and organization of the spinal cord.The spinal cord organoids provided by the present disclosure can also be used to evaluate the effects of drugs on the human spinal cord.

[0055] One aspect of the present disclosure provides a method for evaluating the effect of a test substance on the spinal cord, comprising contacting a spinal cord organoid provided by the present disclosure with a test substance, and measuring the effect of the test substance on the spinal cord organoid.

[0056] As used herein, a "test substance" may be, for example, a small molecule compound, a protein (e.g., an antibody), DNA, RNA, small interfering RNA, or an antisense oligonucleotide. The test substance may be a drug for treating a disease or cancer in the spinal cord, or a candidate substance for such a drug. The test substance may be, for example, a substance known to cause damage to the spinal cord or a substance that has the potential to do so. The test substance may be, for example, one type, or a mixture of two or more types. The test substance is preferably one type of substance.

[0057] The present disclosure provides that "contact" spinal cord organoid and test substance means that the spinal cord organoid and test substance are placed under the condition that they can contact each other.Contacting the spinal cord organoid and test substance can be, for example, by mixing the test substance into the culture solution that contains the spinal cord organoid.

[0058] The evaluation of the effect of test substance on spinal cord includes, for example, the evaluation of the structural or functional characteristics of spinal cord organoids caused by test substance.In this example, the evaluation includes the measurement of the structural or functional characteristics of spinal cord organoids.Specifically, for example, the change in the composition of cells that constitute spinal cord organoids, the rate of cell death in the cells that constitute spinal cord organoids, the activity of cells that constitute spinal cord organoids, such as neural firing, calcium dynamics, etc. can be measured or evaluated.

[0059] The effect of the test substance on the spinal cord may be evaluated, for example, as the average value of the results measured from at least three (e.g., three, four, five, six, or seven or eight or more) spinal cord organoids or non-human mammals having the spinal cord organoids.

[0060] Another example of evaluating the effect of a test substance on the spinal cord is to obtain spinal cord organoids, disperse the organoids into single cells, and then culture them to measure or evaluate the degree of axonal extension, the rate of cell death of the dispersed cultured cells, and functional evaluation of the dispersed cultured cells, such as neuronal firing and calcium dynamics.

[0061] The method of evaluating the effect of a test substance on the spinal cord of the present disclosure can be used for screening and evaluating the toxicity of drugs for treating spinal cord injuries or diseases. The present disclosure includes a pharmaceutical composition for treating spinal cord injuries or diseases, which comprises, as an active ingredient, a substance evaluated by such a method to have an effect on the spinal cord.

[0062] In another aspect of the present disclosure, the spinal cord organoids of the present disclosure can be used as a regenerative medicine composition. The regenerative medicine composition of the present disclosure can be used to treat spinal cord injuries or diseases.

[0063] The regenerative medicine composition of the present disclosure may contain, for example, a pharmaceutically acceptable carrier together with the spinal cord organoid of the present disclosure. In the present disclosure, the term "pharmaceutically acceptable carrier" refers to any component other than the spinal cord organoid that is highly safe and has low allergic reactivity. Pharmaceutically acceptable carriers include, for example, aqueous or non-aqueous solvents, solutions (e.g., saline, basal medium, or cell suspension preservative solution) suitable for pharmaceutical administration, cryoprotectants (e.g., glycerol), water-soluble polymers (e.g., dextran), or buffers (e.g., phosphate buffer). The regenerative medicine composition can be appropriately manufactured according to known methods. In one example, the regenerative medicine composition according to the present disclosure can be manufactured by combining the spinal cord organoid with a pharmaceutically acceptable carrier (e.g., basal medium).

[0064] The regenerative medicine composition according to the present disclosure can be used in a method for treating spinal cord injury or disease. The present disclosure provides a method for treating spinal cord injury or disease, comprising administering a regenerative medicine composition comprising the spinal cord organoid according to the present disclosure to a patient in need thereof.

[0065] In the present disclosure, a "spinal cord injury" or a "spinal cord disease" may be, for example, a spinal cord damaged by trauma, a spinal cord disease such as, but not limited to, amyotrophic lateral sclerosis, spinal muscular atrophy, spinocerebellar degeneration, spinal and bulbar muscular atrophy, spinal cord infarction, syringomyelia, or spinal cord cancer.

[0066] The method for treating spinal cord injury or disease of the present disclosure comprises introducing the regenerative medicine composition of the present disclosure into the spinal cord or a surrounding area of ​​a human subject in need thereof. The introduced spinal cord organoid may be, for example, in the form of an organoid, or may be either or both of single cells and cell masses composed of multiple cells. In one example, the method for treating spinal cord injury or disease comprises surgically transplanting or injecting a group of cells derived from the spinal cord organoid of the present disclosure into the spinal cord or a surrounding area of ​​a human subject in need thereof using a device such as a syringe.

[0067] As used herein, the term "human subject in need thereof" refers to a human subject who has or is suspected of having a spinal cord injury or disease. A human subject with a spinal cord injury or disease refers to a person who has been diagnosed by a medical professional (e.g., a physician) as having a spinal cord injury or disease according to predetermined diagnostic criteria. Examples of a human subject suspected of having a spinal cord injury or disease include subjects who are suspected of having a spinal cord injury or disease based on, for example, behavioral history (e.g., trauma and having received or been receiving drugs that are harmful to spinal cord tissue) or medical history.

[0068] For example, when administering a regenerative medicine composition to a patient, an immunosuppressant can be used to control graft rejection. From the viewpoint of reducing graft rejection, for example, the patient to whom the regenerative medicine composition is administered and the pluripotent stem cells used to produce the spinal organoids of the present disclosure can be selected so that their HLA matches to a certain degree or more. Alternatively, for example, pluripotent stem cells can be used that have at least a portion of HLA knocked out.

[0069] "Treating" a spinal cord injury or disease includes maintaining, reducing, or eliminating the symptoms or pathology. Treating a spinal cord injury or disease includes curing the spinal cord injury or disease.

[0070] In another aspect, the present disclosure provides a non-human mammal having a human spinal cord organoid, which is obtained by transplanting the spinal cord organoid provided by the present disclosure into a non-human animal. Examples of non-human animals into which the spinal cord organoid of the present disclosure can be transplanted include immunodeficient animals such as nude mice, model animals such as mice, rats, rabbits, dogs, marmosets, and macaques administered with immunosuppressants, and humanized animals transplanted with human genes.

[0071] In one example, tumor cells or tumor fragments are introduced into the spinal organoids, and the spinal organoids are then introduced into a non-human mammal to produce a non-human mammal having spinal organoids as a spinal cancer model. The non-human mammal as a spinal cancer model can be used to evaluate the effects of candidate therapeutic agents for spinal cancer.

[0072] The present disclosure will be explained in more detail below using examples and reference examples. The present disclosure is not limited to these examples. The media and reagents used in the examples and reference examples are shown below.

[0073] In the Examples and Reference Examples, the culture was carried out at 37°C and 5% CO ２ The experiment was carried out under the following conditions.

[0074] A schematic diagram of the method for producing spinal cord organoids in this example is shown in Figure 2. In this example, the following medium A and medium B were used as basic media.

[0075]

[0076]

[0077] Human iPS cells (1231A3 strain, provided by the Center for iPS Cell Research and Application, Kyoto University) were used for seeding pluripotent stem cells. Human iPS cells maintained and cultured in StemFit® AK02N medium (REPROCELL) on an iMatrix-511 coating were detached from the coating by incubation in TrypLE Select (Thermo Fisher Scientific) at 37°C for approximately 5 minutes (4-8 minutes). The culture vessel was then centrifuged at 200G for 5 minutes at room temperature, the supernatant was removed, and the cells were suspended in StemFit® AK02N (supplemented with 10 μM Y-27632).

[0078] Step (1) In step (1), a medium prepared by adding 50 μM Y-27632 and 3 μM CHIR99021 to Medium A (hereinafter referred to as Medium A-1) was used. 1.0 × 10 cells were suspended in StemFit® AK02N (supplemented with 10 μM Y-27632). ５ The cells were suspended in medium A-1 to a concentration of 1000 cells / mL.

[0079] 100 μL of the cell suspension was seeded onto a primeSurface (registered trademark) 96V plate (Sumitomo Bakelite). Hereinafter, the seeding day is designated as differentiation day 0. The cells were cultured for 3 days.

[0080] Step (2) In step (2), a medium (medium A-2) prepared by adding 0.1 μM retinoic acid and 0.5 μM SAG to medium A was used. On day 3 of differentiation, 100 μL of medium A-2 was added to each well. On day 6 of differentiation, 125 μL of medium was removed from each well and 75 μL of medium A-2 was added. On days 10 and 14 of differentiation, 75 μL was removed from each well and 75 μL of medium A-2 was added, and the cells were cultured until day 18 of differentiation.

[0081] Step (3) In step (3), medium B was used. On day 18 of differentiation (day 15 of step (2)), 100 μL was removed from each well and 150 μL of medium B was added. Then, 150 μL was removed from each well again and 150 μL of medium B was added. Thereafter, 150 μL of medium was removed from each well and 150 μL of medium B was added every 3 or 4 days to perform a medium change. On day 25 of differentiation (day 7 of step (3)), a portion of the spinal cord organoids obtained was fixed, and a portion was subjected to calcium imaging.

[0082] On day 25 of differentiation, spinal cord organoids were collected in a test tube and washed once with 1 mL of PBS. After removing the PBS, the organoids were immersed in 4% paraformaldehyde for 15 minutes for fixation. After fixation, the paraformaldehyde was removed and the organoids were washed twice with 1 mL of PBS. The PBS was replaced with 18% sucrose solution and allowed to stand overnight at 4°C.

[0083] Preparation of frozen sections of spinal cord organoids: Frozen sections of sucrose-substituted organoids were prepared using a commercially available embedding dish for frozen section preparation, Cryomold No. 2 (Sakura Finetech Japan Co., Ltd.). At least six organoids per condition were placed on one embedding dish.

[0084] Excess sucrose solution was completely aspirated from the organoids, and OCT Compound (Sakura Finetech Japan Co., Ltd.), an embedding medium for preparing frozen tissue sections, was added and allowed to settle for 20 minutes. The embedding dish was then placed on an aluminum block chilled with dry ice and rapidly frozen. The frozen, embedded organoids were sliced ​​to a thickness of 10 μm using a cryostat (Leica Biosystems) and attached to glass slides (MAS-GP, 15 x 26, Matsunami Glass Industrial Co., Ltd., S099010-100) to obtain frozen sections.

[0085] Evaluation of differentiation markers by immunostaining of frozen sections The following antibodies were used.

[0086] The slides were thoroughly thawed and dried. Approximately 1 mL of PBS was dropped onto the slide to thoroughly remove the OCT compound (approximately 15 minutes). A sufficient amount of 0.3% Triton X 100 solution (prepared in PBS) was added three times at 15-minute intervals for permeabilization. Blocking was performed for 1 hour using up to 250 μL of 10% NDS solution (prepared in 0.3% Triton X 100 solution) per slide. After blocking, primary antibodies diluted to the desired concentration in 0.05% Tween 20 solution (prepared in PBS) were added to each slide at up to 250 μL per slide and incubated overnight at 4°C.

[0087] The next day, the primary antibody was removed, and the slides were washed three times with a sufficient amount of 0.05% Tween 20 solution at 15-20 minute intervals. Secondary antibodies were diluted to the desired concentration with a solution prepared by diluting DAPI solution 500-fold with 0.05% Tween 20 solution and filtered through a Millex-GV Filter Unit 0.22 μm (Millipore). 250 μL of the filtered secondary antibody solution was applied to the slides and incubated for 2 hours in the dark. After incubation, the secondary antibody solution was removed, and the slides were washed three times with a sufficient amount of 0.05% Tween 20 solution at 15-20 minute intervals. Finally, the slides were mounted with a cover glass using ProLong Glass Antifade, dried, and then imaged using a confocal microscope FV3000 (Olympus). The results are shown in Figure 3. Reference example

[0088] As a reference example, we show the results of inducing 3D spinal cord organoids from iPS cells under the 3-DiSC conditions described in Non-Patent Document 3. The following experiment was performed using human iPS cells from the 1231A3 human iPS cell line (gift from the Center for iPS Cell Research and Application, Kyoto University) as single cells in the same manner as in the Examples.

[0089] An outline of the spinal cord organoid induction method is shown in Figure 2. For culture, medium B, the same as in Example 1, and medium C, described below, were used as basic media. A 96-well U-bottom cell culture plate was used as the culture vessel.

[0090]

[0091] A medium (medium C-1) was used, which was medium C supplemented with 10 μM SB431542, 50 μM Y-286832, 20 ng / mL bFGF, and 3 μM CHIR99021. iPS cells dissociated into single cells were seeded into 100 μL of medium C-1 at 9,000 cells / well and cultured for 3 days. The day of seeding was designated day 0.

[0092] On the third day, the medium was replaced with a medium (medium C-2) supplemented with 10 μM SB431542, 100 nM retinoic acid, and 500 nM SAG, and the cells were further cultured for 3 days.

[0093] On the sixth day, the medium was replaced with medium C supplemented with 100 nM retinoic acid and 500 nM SAG (medium C-3), and the cells were cultured for another 9 days.

[0094] On the 18th day of differentiation (step (2) day 15), 100 μL was removed from each well and 150 μL of medium B was added. Then, 150 μL was removed from each well again and 150 μL of medium B was added. Thereafter, 150 μL of medium was removed from each well and 150 μL of medium B was added every 3 or 4 days to perform a medium change. On the 25th day of differentiation (step (3) day 7), the organoids formed in each well were collected and immobilized.

[0095] The spinal cord organoids obtained in the Reference Example were fixed in the same manner as in the Examples, and frozen sections were prepared and immunostained to examine the expression of each marker. The results are shown in Figures 3 and 4.

[0096] The results of evaluation of each differentiation marker in the organoids obtained in the Examples and Reference Examples are shown. "-" indicates that no positive cells were observed, and the number of "+" indicates the percentage of positive cells.

[0097] As shown in Table 6 and Figures 3 and 4, in the organoids obtained by the method of the Examples, all of the markers PAX7, PAX6, OLIG2, NKX2.2, and FOXA2 were expressed in some part of the organoid, confirming that all progenitor cell domains from the ventral to dorsal sides were formed within a single organoid. Furthermore, it was confirmed that PAX7, a marker for the dorsal region, was expressed adjacent to a region expressing any of the ventral region markers OLIG2, NKX2.2, or FOXA2.

[0098] The protocol of the Reference Example mainly induces domains in the ventral region of the spinal cord, and expression of ventral region markers was observed, but expression of PAX7, a marker for the dorsal region, was not observed.

[0099] Acquisition of calcium imaging data from spinal cord organoids In the Examples and Reference Examples, on day 25 of differentiation, 150 μL of medium was removed from each well, 150 μL of Neurobasal Plus medium was added, and further culture was performed. During culture, 150 μL of medium was removed from each well and 150 μL of Neurobasal Plus medium was added once every 3 or 4 days to perform a medium change.

[0100] On day 32 of differentiation, three wells' worth of organoids were obtained. Neurobasal Plus medium supplemented with Ca indicator: Cal-520 5 μM and Pluronic 0.01% was added to each well of a 96-well flat-bottom plate, and one organoid was placed in each well. 37°C, 5% CO ２ After the reaction, 30 to 100 cells that responded to the Ca indicator were selected from the organoids using a Cell Voyager 6000 (CV6000, Yokogawa Electric Corporation), a high-content analysis system, and 300 consecutive measurements were taken with an imaging time of 120 seconds, an imaging interval of 400 msec, and an exposure time of 400 msec.

[0101] After capturing the image, the high-content analysis software CellPathfinder (Yokogawa Electric Corporation) was used to calculate the relative brightness value of the image using the brightness value of Timepoint 1 as the reference using the following formula: Relative brightness value = I ｔ / L １ (In the formula, I ｔ is the luminance at any timepoint, I １ The time-dependent changes in relative brightness indicated by (where ≠ luminance at Timepoint 1) were obtained. The obtained waveform patterns were analyzed using waveform analysis software WaveFinder (Revvity). The results are shown in Figure 5.

[0102] It was confirmed that the organoids of the Example tended to exhibit more synchronous firing than the organoids of the Reference Example. Therefore, a cross-correlation analysis was performed to quantify the synchrony of the waveform patterns.

[0103] The obtained results were subjected to statistical analysis (two-way analysis of variance) with the differences in differentiation induction conditions and test times as factors, but no significant interaction was observed between the differences in differentiation induction conditions and each test time (p = 0.46). Next, statistical analysis was performed on the results of the three tests together, and it was shown that the synchrony between cells in the Example was significantly higher than in the Reference Example (p < 0.02). The results of the analysis of the three tests together are shown in Figure 6.

Claims

1. A method for producing spinal cord organoids from pluripotent stem cells in vitro, comprising the steps of: (1) culturing human pluripotent stem cells in a medium containing a ROCK inhibitor and a Wnt signal activator, and substantially free of a TGFβ inhibitor and a BMP signal inhibitor; (2) further culturing the cells obtained in step (1) in a medium containing a retinoic acid receptor agonist and a hedgehog signal activator; and (3) further culturing the cells obtained in step (2) in a medium substantially free of a TGFβ inhibitor, a BMP signal inhibitor, a retinoic acid receptor agonist, and a sonic hedgehog stimulator to obtain spinal cord organoids.

2. The method according to claim 1, wherein step (1) is carried out in a medium substantially free of bFGF.

3. The method according to claim 1, wherein the ROCK inhibitor is Y27632 and / or the Wnt signal activator is CHIR99021.

4. The method of claim 1, wherein the retinoic acid receptor agonist is retinoic acid and / or the sonic hedgehog stimulator is a smoothened agonist (SAG).

5. The method according to any one of claims 1 to 4, wherein the pluripotent stem cells are iPS cells or ES cells.

6. The method of any one of claims 1 to 5, wherein the spinal cord organoid has portions that express PAX7, PAX6, OLIG2, NKX2.2, and FOXA2.

7. A spinal cord organoid obtained by the method according to any one of claims 1 to 6.

8. Human pluripotent stem cell-derived spinal cord organoids with sections expressing PAX7, PAX6, OLIG2, NKX2.2 and FOXA2.

9. The spinal cord organoid according to claim 8, wherein the portion expressing PAX7 is continuous with the portion expressing one or more of OLIG2, NKX2.2 and FOXA2.

10. A method for obtaining a non-human animal containing a human spinal organoid, comprising the steps of obtaining a spinal organoid from human pluripotent stem cells by the method of any one of claims 1 to 6, and transplanting the spinal organoid into the non-human animal.

11. A non-human animal into which the spinal cord organoid described in any one of claims 7 to 9 has been transplanted.

12. A regenerative medicine composition for treating spinal cord injury or disease, comprising the spinal cord organoid of any one of claims 7 to 9.

13. A method for evaluating the effect of a test substance on the spinal cord, comprising the steps of contacting the spinal cord organoid described in any one of claims 7 to 9 with a test substance, and evaluating the effect of the test substance on the spinal cord organoid.

14. A pharmaceutical composition for treating spinal cord injury or disease, comprising as an active ingredient a substance evaluated to have an effect on the spinal cord by the method of claim 13.

15. A method for treating spinal cord injury or disease, comprising administering a substance evaluated to have an effect on the spinal cord by the method of claim 13 to a subject in need of treatment for spinal cord injury or disease.

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