Method for manufacturing multi-system organoids

Abstract

To enable the construction of specific disease models involving multiple cell types and the development of screening systems for specific diseases, we provide a method for producing organoids containing ectoderm and mesoderm cells. [Solution] A method for producing organoids containing ectoderm cells and mesoderm cells, comprising the step (1) of culturing a cell aggregate containing pluripotent stem cells in suspension for at least two days in a culture medium containing a ROCK inhibitor and a TGFβ inhibitor.

Description

[Technical Field]

[0001] The present invention relates to a method for producing organoids containing ectodermal cells and mesodermal cells, comprising the step (1) of culturing a cell aggregate containing pluripotent stem cells in suspension for at least two days in a culture medium containing a ROCK inhibitor and a TGFβ inhibitor. [Background technology]

[0002] In recent years, the use of induced pluripotent stem cells (iPS cells) has been expanding in various research institutions, including applications in regenerative medicine and drug discovery technologies known as drug discovery iPS cells. Differentiation from iPS cells is classified into ectoderm (represented by nerve cells), mesoderm (represented by muscle cells), and endoderm (represented by alveolar cells), and it is known that differentiation into these cell types can occur by exposure to specific compounds under specific culture conditions.

[0003] When using iPS cells in regenerative medicine, it is common practice to differentiate them into a single cell type suitable for transplantation, etc. (Patent Document 1). In drug discovery iPS cells, the application to cell line screening for rare diseases, etc., is generally to induce differentiation into individual target cell types. However, until now, it has been thought difficult to mix cell types that have undergone different differentiation processes, such as ectoderm and mesoderm, within a single organoid (Non-Patent Document 1). [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2021-192627 [Non-patent literature]

[0005] [Non-Patent Document 1] Caoimhe Goldrick et al., Front. Cell Dev. Biol. 2023 Feb 2:11:1083175. doi: 10.3389 / fcell.2023.1083175. [Overview of the project] [Problems that the invention aims to solve]

[0006] Therefore, the object of the present invention is to provide a method for producing an organoid (multiphyletic organoid) containing ectodermal and mesodermal cells, comprising the step (1) of suspension culturing a cell aggregate containing pluripotent stem cells in a medium containing a ROCK inhibitor and a TGFβ inhibitor for at least two days, in order to mix cell types that have undergone different differentiations in a single organoid. [Means for solving the problem]

[0007] The inventors considered that in order to produce multi-system organoids, it is important to expose cell aggregates containing pluripotent stem cells to (multiple) specific compounds at certain timings. As a result of diligent research, they found that multi-system organoids can be induced by first culturing cell aggregates containing pluripotent stem cells in a culture medium containing a ROCK inhibitor and a TGFβ inhibitor in suspension for at least two days, and then continuing the culture thereafter. They also found that serum-free medium is preferable. Based on these findings, the inventors conducted further research and completed the present invention.

[0008] In other words, the present invention is as follows: [1] A method for producing organoids containing ectoderm cells and mesoderm cells, comprising the step (1) of culturing a cell aggregate containing pluripotent stem cells in suspension for at least two days in a medium containing a ROCK inhibitor and a TGFβ inhibitor. [2] The method according to [1], wherein the culture medium comprises osteomorphocytes. [3] The method according to [1] or [2], comprising the step (2) of suspension-culturing cell aggregates cultured in step (1) in a medium containing a ROCK inhibitor and an extracellular matrix. [4] The method according to [3], comprising the step (3) of suspension-culturing cell aggregates cultured in step (2) in a medium containing a ROCK inhibitor. [5] The method according to [4], wherein the medium in step (3) contains retinoic acid. [6] The method according to any one of [1] to [5], wherein the medium in steps (1) to (3) is a serum-free medium. [7] The method according to any one of [1] to [6], wherein the ROCK inhibitor is Y-27632 and the TGFβ inhibitor is SB505124. [8] The method according to any one of [1] to [7], wherein the bone morphogenetic protein is BMP-4. [9] The method according to any one of [1] to [8], wherein the extracellular matrix is a basement membrane preparation.

[10] The method according to any one of [1] to [9], wherein the pluripotent stem cell is a human pluripotent stem cell.

[11] The method according to any one of [1] to

[10] , wherein the pluripotent stem cell is an induced pluripotent stem cell.

[12] An organoid comprising ectodermal cells and mesodermal cells produced by the production method according to any one of [1] to

[11] .

[13] The following: (i) Cells in which at least one gene selected from the group consisting of PAX6, RPE-65, VSX1, VSX2, MITF, MSX2, RCVRN, RGR, and RAX2 is positive, (ii) Cells in which at least one gene selected from the group consisting of FOXG1, OTX1, DLX1, VAX1, SLC1A7, SLC17A6, and SLC17A7 is positive, and (iii) Cells in which at least one gene selected from the group consisting of MYOG, MYH6, MYH7, MYOD1, PAX3, PAX7, TNNT3, and GATA4 is positive An organoid having

[14] An organoid having the following characteristics (1) to (3). (1) Having 1 to 5 layers on the surface containing cells expressing Tuj-1 (2) Having 1 to 50 layers inside the layer of (1) containing cells expressing at least one selected from the group consisting of VSX2, CRX, and Recoverin (3) Having 1 to 3 layers inside the layer of (2) containing cells expressing Myosin

[15] The organoid according to

[14] , wherein the 1 to 50 layers containing cells expressing at least one selected from the group consisting of VSX2, CRX, and Recoverin form a retina-like structure.

[16] The organoid according to

[14] or

[15] , wherein the 1 to 3 layers containing cells expressing Myosin form a muscle fiber-like structure. [Advantages of the Invention]

[0009] According to the present invention, it becomes possible to produce an organoid containing ectodermal cells and mesodermal cells from a cell aggregate containing pluripotent stem cells. In addition, with the organoid containing ectodermal cells and mesodermal cells obtained by the production method of the present invention, the construction of a specific disease model involving a plurality of cell types and the construction of a screening system for a specific disease are expected. Furthermore, by reproducing the in-vivo environment using the organoid containing ectodermal cells and mesodermal cells obtained by the production method of the present invention, it is expected to observe and study how signals are transmitted and interactions are carried out between cells in terms of time and space, and developments such as applications in drug discovery and regenerative medicine are expected. [Brief Description of the Drawings]

[0010] [Figure 1]Figure 1 shows the effects of chemoinhibitors on promoting early cell differentiation into ectoderm and mesoderm. The p-value was determined using Tukey HSD multiple comparison test (n=3, mean ± SEM). [Figure 2] Figure 2 shows the development of organoids derived from multiple human iPS cells. (A) All steps of cell culture and the morphology of organoids at day 0, day 9, and day 27, respectively. (B) Graph showing the change in organoid size from day 0 to day 42. The p-value was determined using Dunnett's test (n=3-6 organoids, mean ± SEM). (C) Characteristics of organoids at day 38. I) H&E staining showing the morphology of a single organoid. II) Immunofluorescence staining showing the expression of Tuj-1 and Ki67. III) Enlarged version of the organoid showing high expression of Tuj-1 in the surface region and Ki67 expression in the internal groove. Scale bar is 100 μm. [Figure 3] Figure 3 shows the optimization of organoid preparation under multiple conditions. (A) Overall cell culture procedure under various conditions. (B) Pie chart showing the occurrence rate of dark RPE spots in organoids from day 38 (n=15-30 organoids). (C) Images of organoids cultured under RA before or after Matrigel embedding. [Figure 4] Figure 4 shows the characteristics of organoids under long-term culture conditions. (A) Organoids prepared by Method A1: (I) shows the appearance of organoids in a culture well with RPE dark spots. (II) H&E staining showing the morphology of organoids on day 52. ​​(III) Immunofluorescence staining showing the expression of Tuj-1 and Ki67. Scale bar is 100 μm. (B) Organoids prepared by Method A2: (I) shows the appearance of organoids in a culture well with RPE dark spots. (II) H&E staining showing the morphology of organoids on day 52. ​​(III) Immunofluorescence staining showing the expression of Tuj-1 and Ki67. Scale bar is 100 μm. (C) Expression of the astrocyte marker GFAP in organoids prepared by (I) Method A1 or (II) Method A2. Scale bar is 100 μm. (D) Bar graph showing the difference in GFAP expression between Method A1 and Method A2. The p-value was determined by a t-test (n=5-6 organoids, mean ± SEM). [Figure 5] Figure 5 shows the characteristics of organoids prepared by Method A3. (A) Immunofluorescence staining of the entire organoid at day 38 showing the expression of PAX6, Tuj-1, and RPE65, using a confocal laser scanning microscope. Scale bar is 500 μm. (B) Characteristics of organoids prepared by Method A3. I) H&E staining of the organoid at day 50 showing the morphology of the organoid including dark areas of RPE cells. II) and III) Immunofluorescence staining of the entire organoid at day 50 showing the distribution of ectoderm-related cells stained with retinal and neuron markers Tuj-1 (neuron marker), Recoverin (retinal photoreceptor marker), and VSX2 (retinal marker). IV) and V) Immunofluorescence staining of the entire organoid at day 50 showing the interphase between ectoderm-related cells (green retinal marker CRX) and mesoderm (red skeletal muscle marker myosin fast chain). Scale bar is 200 μm. [Figure 6] Figure 6 shows RNA-seq analysis illustrating the gene signatures and biological pathways of organoids prepared by Method A3. (A) PCA plot showing gene expression changes between human iPS cells and organoids at day 38 and day 50. (B) Volcano plot representing differentially expressed genes (DEGs) in organoids and human iPS cells (585A1) at day 38(i) or day 50(ii). Genes with a 2-fold change and p-value < 0.05 were applied. Bottom: downregulation genes, Top: upregulation genes. (C) GO pathway of DEGs in organoids at day 38(i) or day 50(ii). In the bubble plot, the z-score was obtained using the following formula: Z-score = (number of upregulated genes - number of downregulated genes) / √total number of genes. [Figure 7] Figure 7 shows RNA-seq analysis of gene expression of specific markers in organoids prepared by Method A3. DEG of pluripotency markers, visual, retinal, forebrain, neuronal (glutamatergic and GABAergic), and muscle markers were analyzed at days 38 and 50. A 2-fold change (2-fold change) and p-value <0.05 were applied. [Modes for carrying out the invention]

[0011] 1. Manufacturing method of the present invention The present invention provides a method for producing organoids containing ectoderm and mesoderm cells (hereinafter referred to as the "production method of the present invention"), comprising the step (1) of culturing a cell aggregate containing pluripotent stem cells in suspension for at least two days in a culture medium containing a ROCK inhibitor and a TGFβ inhibitor. The manufacturing method of the present invention may further include step (2) of suspension culture of the cell aggregates cultured in step (1) in a medium containing a ROCK inhibitor and an extracellular matrix, after step (1). Alternatively, the manufacturing method of the present invention may further include step (3) of suspension culture of the cell aggregates cultured in step (2) in a medium containing a ROCK inhibitor, after step (2). In another embodiment, organoids obtained by the manufacturing method of the present invention are also provided.

[0012] In this specification, "stem cell" means an undifferentiated cell that has the ability to differentiate and proliferate (especially self-renewal). Stem cells include pluripotent stem cells, multipotent stem cells, and unipotent stem cells, depending on their differentiation ability. "Pluripotent stem cell" refers to a stem cell that can be cultured in vitro and has the ability to differentiate into all cells that make up the body (pluripotency). All cells are cells derived from the three germ layers: ectoderm, mesoderm, and endoderm. "Multipotent stem cell" refers to a stem cell that has the ability to differentiate into multiple types of tissues and cells, though not all types. "Unipotent stem cell" refers to a stem cell that has the ability to differentiate into a specific tissue or cell.

[0013] Pluripotent stem cells can be induced from fertilized eggs, cloned embryos, germ cells, tissue-derived stem cells, somatic cells, etc. Examples of pluripotent stem cells include embryonic stem cells (ES cells), embryonic germ cells (EG cells), and induced pluripotent stem cells (iPS cells). Muse cells (Multi-lineage differentiating Stress Enduring cells) obtained from mesenchymal stem cells (MSCs), and GS cells produced from germ cells (e.g., testes) are also included in the category of pluripotent stem cells. Human embryonic stem cells are established from human embryos within 14 days of fertilization.

[0014] Embryonic stem cells were first established in 1981 and have been used in the creation of knockout mice since 1989. Human embryonic stem cells were established in 1998 and are now being used in regenerative medicine. ES cells can be produced by culturing an inner cell population on feeder cells or in a culture medium containing leukemia inhibitory factor (LIF). Methods for producing ES cells are described, for example, in International Publication No. 96 / 22362, International Publication No. 02 / 101057, U.S. Patent No. 5843780, U.S. Patent No. 6200806, and U.S. Patent No. 6280718. Embryonic stem cells can be obtained from designated institutions and can also be purchased commercially. For example, human embryonic stem cells KhES-1, KhES-2, and KhES-3 are available from the Institute for Frontier Medical Sciences, Kyoto University. EB5 cells, both mouse embryonic stem cells, are available from the RIKEN (National Research and Development Institute), and the D3 strain is available from the American Type Culture Collection (ATCC). One type of ES cell, nuclear-transplanted ES cells (ntES cells), can be established from cloned embryos created by transplanting the nucleus of a somatic cell into an egg from which the cell nucleus has been removed.

[0015] EG cells can be produced by culturing primordial germ cells in a medium containing mouse stem cell factor (mSCF), LIF, and basic fibroblast growth factor (bFGF) (Cell, 70:841-847, 1992).

[0016] "Induced pluripotent stem cells" are cells in which pluripotency has been induced by reprogramming somatic cells using known methods. Specifically, induced pluripotent stem cells include somatic cells differentiated into fibroblasts, peripheral blood mononuclear cells, etc., which are reprogrammed to induce pluripotency by expressing multiple genes selected from a group of reprogramming genes including Oct3 / 4, Sox2, Klf4, Myc (c-Myc, N-Myc, L-Myc), Glis1, Nanog, Sall4, lin28, Esrrb, etc. In 2006, induced pluripotent stem cells were established in mouse cells by Yamanaka et al. (Cell, 2006, 126(4)pp. 663-676). Induced pluripotent stem cells were established in human fibroblasts in 2007 and, like embryonic stem cells, possess pluripotency and self-renewal capabilities (Cell, 2007, 131(5)pp. 861-872; Science, 2007, 318(5858)pp. 1917-1920; Nat. Biotechnol., 2008, 26(1)pp. 101-106). In addition to direct reprogramming via gene expression, induced pluripotent stem cells can also be induced from somatic cells by adding compounds (Science, 2013, 341, pp. 651-654).

[0017] There are no particular limitations on the somatic cells used when producing induced pluripotent stem cells, but examples include tissue-derived fibroblasts, hematopoietic cells (e.g., peripheral blood mononuclear cells, T cells, etc.), hepatocytes, pancreatic cells, intestinal epithelial cells, and smooth muscle cells.

[0018] When reprogramming induced pluripotent stem cells by expressing several genes (e.g., four factors: Oct3 / 4, Sox2, Klf4, and Myc), the means of expressing these genes are not particularly limited. Examples of means of expressing genes include infection methods using viral vectors (e.g., retroviral vectors, lentiviral vectors, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors), gene transfer methods using plasmid vectors (e.g., plasmid vectors, episomal vectors) (e.g., calcium phosphate method, lipofection method, retronectin method, electroporation method), gene transfer methods using RNA vectors (e.g., calcium phosphate method, lipofection method, electroporation method), and direct protein injection methods.

[0019] Furthermore, it is possible to obtain induced pluripotent stem cell lines. For example, human induced pluripotent cell lines such as 201B7 cells, 201B7-Ff cells, 253G1 cells, 253G4 cells, 1201C1 cells, 1205D1 cells, 1210B2 cells, and 1231A3 cells, established at Kyoto University, are available from Kyoto University and iPS Academia Japan, Inc. As induced pluripotent stem cell lines, for example, Ff-I01 cells, Ff-I14 cells, and QHJI01s04 cells, established at Kyoto University, are available from Kyoto University. In addition, for example, 585A1 cells are available from RIKEN.

[0020] Pluripotent stem cells may be genetically modified. Genetically modified pluripotent stem cells can be produced, for example, by homologous recombination technology. Examples of chromosomal genes that can be modified include cell marker genes, histocompatibility antigen genes, and disease-related genes based on nerve cell damage. Modification of target genes on chromosomes can be performed using methods described in "Manipulating the Mouse Embryo, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994)", "Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993)", "Biomanual Series 8, Gene Targeting, Production of Mutant Mice Using ES Cells, Yodosha (1995)", etc.

[0021] Specifically, the genomic gene of the target gene to be modified (e.g., cell marker gene, histocompatibility antigen gene, disease-related gene, etc.) is isolated, and a target vector is created using the isolated genomic gene to homologously recombine the target gene. By introducing the created target vector into stem cells and selecting cells that undergo homologous recombination between the target gene and the target vector, stem cells with modified genes on the chromosome can be created.

[0022] Methods for isolating the genomic genes of target genes include known methods described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) and Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997). Genomic DNA library screening systems (Genome Systems) and Universal GenomeWalker Kits (Clontech) can also be used.

[0023] The preparation of target vectors for homologous recombination of target genes and the efficient selection of homologous recombinants can be carried out according to the methods described in Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993), Biomanual Series 8, Gene Targeting, Production of Mutant Mice Using ES Cells, Yodosha (1995), etc. Target vectors can be either replacement or insertion type. Selection methods include positive selection, promoter selection, negative selection, or polyA selection. Methods for selecting the desired homologous recombinant from the selected cell lines include Southern hybridization of genomic DNA and PCR.

[0024] As pluripotent stem cells, genome-edited pluripotent stem cells can also be used. Genome editing is a technique that intentionally modifies target genes or genomic regions using principles such as site-specific cleavage of genomic DNA strands with nucleases or chemical conversion of bases. Examples of site-specific nucleases include zinc finger nucleases (ZFNs), TALENs, and CRISPR / Cas9. By using genome editing technology, it is possible to create knockout cell lines with specific gene deletions, knock-in cell lines with artificially inserted sequences at specific gene loci, and so on.

[0025] As pluripotent stem cells, for example, pluripotent stem cells from warm-blooded animals, preferably mammals, can be used. Examples of mammals include rodents such as mice, rats, hamsters, and guinea pigs, experimental animals such as rabbits, livestock such as pigs, cows, goats, horses, and sheep, pets such as dogs and cats, and primates such as humans, monkeys, orangutans, and chimpanzees. The pluripotent stem cells are preferably pluripotent stem cells from rodents (mice, rats, etc.) or primates (humans, etc.), and more preferably human pluripotent stem cells.

[0026] In this specification, "cell aggregate" refers to a structure formed by the aggregation of cells. For example, embryoid bodies, spheres, and spheroids are also included in the definition of cell aggregate.

[0027] ROCK inhibitors (ROCK inhibitors) are not particularly limited as long as they suppress the function of Rho-kinase (ROCK), but examples include Y-27632 ((R)-(+)-trans-4-(1-Aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide,dihydrochloride), Fasudil (HA1077) (1-(5-Isoquinolinylsulfonyl)homopiperazine,hydrochloride), H-1152 (5-[[(2S)-hexahydro-2-methyl-1H-1,4-diazepin-1-yl]sulfonyl]-4-methyl-isoquinoline,dihydrochloride), HA-1100 (Hydroxyfasudil) ([1-(1-Hydroxy-5-isoquinolinesulfonyl)homopiperazine,hydrochloride), Chroman 1((3S)-N-[2-[2-(dimethylamino)ethoxy]-4-(1H-pyrazol-4-yl)phenyl]-6-methoxy-3,4-dihydro-2H-chromene-3-carboxamide), Belumosudil(KD025, 2-[3-[4-[(1H-Indazol-5-yl)amino]qui nazolin-2-yl]phenoxy]-N-isopropylacetamide), HSD1590([2-Methoxy-3-(4,5,10-triazatetracyclo[7.7.0.02,6.012,16]hexadeca-1(9),2(6),3,7,10,12(16)-hexaen-11-yl)phenyl]boronic CRT0066854((S)-3-phenyl-N1-(2-pyridin-4-yl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)propane-1,2-diamine)、RKI1447(1-(3-hydroxybenzyl)-3-(4-(pyridin-4-yl)thiazol-2-yl)urea)、Ripasudil(4-Fluoro-5-[[(2S)-hexahydro-2-methyl-1H-1,4-diazepin-1-yl]sulfonyl]isoquinoline)、GSK269962A(N-[3-[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethylimidazo[4,5-c]pyridin-6-yl]oxyphenyl]-4-(2-morpholin-4-ylethoxy)benzamide)、GSK429286A(N-(6-fluoro-1H-indazol-5-yl)-2-methyl-6-oxo-4-(4-(trifluoromethyl)phenyl)-1,4,5,6-tetrahydropyridine-3-carboxamide)、Y-33075((R)-4-(1-Aminoethyl)-N-1H-pyrrolo[2,3-b]pyridin-4-ylbenzamide)、LX7101(N,N-Dimethylcarbamic acid 3-[[[4-(aminomethyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperidinyl]carbonyl]amino]phenyl ester)、AT13148((alphaS)-alpha-(Aminomethyl)-alpha-(4-chlorophenyl)-4-(1H-pyrazol-4-yl)benzenemethanol)、SAR407899(6-(piperidin-4-yloxy)isoquinolin-1(2H)-one hydrochloride)、GSK180736A(4-(4-fluorophenyl)-N-(1H-indazol-5-yl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide)、Hydroxyfasudil(1-(1-hydroxy-5-isoquinolinesulfonyl)homopiperazine,HCl), bdp5290(4-Chloro-1-(4-piperidinyl)-N-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-1H-pyrazole-3-carboxamide), sr-3677(N-[2-[2-(Dimethylamino)ethoxy]-4-(1H-pyrazol-4-yl) phenyl-2,3-dihydro-1,4-benzodioxin-2-carboxamidehydrochloride), CCG-222740(N-(4-Chlorophenyl)-5,5-difluoro-1-(3-(furan-2-yl)benzoyl)piperidine-3-carboxamide), ROCK inhibitor-2(N-[(1R)-1-(3-methoxyphenyl)ethyl]-4-pyridin-4-ylbenzamide), Rho-Kinase-IN-1(N-[1-[(4-methylsulfanylphenyl)methyl]piperidin-3-yl]-1H-indazol-5-amine), ZINC00881524(N-(4,5-dih ydronaphtho[1,2-d]thiazol-2-yl)-2-(3,4-dimethoxyphenyl)acetamide), SB772077B((3S)-1-[[2-(4-A mino-1,2,5-oxadiazol-3-yl)-1-ethyl-1H-imidazo[4,5-c]pyridin-7-yl]carbonyl]-3-pyrrolidinamine Examples include dihydrochloride, Verosudil (N-(1,2-Dihydro-1-oxo-6-isoquinolinyl)-alpha-(dimethylamino)-3-thiopheneacetamide), GSK-25 (4-(4-chloro-2-fluorophenyl)-2-(2-chloropyridin-4-yl)-1-(6-fluoro-1H-indazol-5-yl)-6-methyl-4H-pyrimidine-5-carboxamide), and their derivatives, among which Y-27632 is preferred. These substances may be used individually or in combination.

[0028] In the manufacturing method of the present invention, the concentration of the ROCK inhibitor in the culture medium can be appropriately set depending on the substance used, within a range in which the above-described effects can be achieved. Typically, the ROCK inhibitor is added to the culture medium at a concentration of about 10 nM to about 10 mM, preferably about 100 nM to about 1 mM, more preferably about 1 μM to about 100 μM, and even more preferably about 10 μM.

[0029] Compounds well known to those skilled in the art can be used as TGFβ inhibitors (TGFβ signaling pathway inhibitors). Specifically, SB431542 (4-[4-(3,4-Methylenedioxyphenyl)-5-(2-pyridyl)-1H-imidazol-2-yl]benzamide) ,SB505124(2-[4-(1,3-Benzodioxol-5-yl)-2-(1,1-dimethylethyl)-1H-imidazol-5-yl]-6-methylpy ridine), SB525334(6-[2-(1,1-Dimethylethyl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-4-yl]quin oxaline), LY2157299(4-[5,6-Dihydro-2-(6-methyl-2-pyridinyl)-4H-pyrrolo[1,2-b]pyrazol-3-yl] -6-quinolinecarboxamide), LY2109761(4-[5,6-dihydro-2-(2-pyridinyl)-4H-pyrrolo[1,2-b]pyraz ol-3-yl]-7-[2-(4-morpholinyl)ethoxy]-quinoline), GW788388(4-{4-[3-(Pyridin-2-yl)-1H-pyraz ol-4-yl]-pyridin-2-yl}-N-(tetrahydro-2H-pyran-4-yl)benzamide), LY364947(4-[3-(2-Pyridinyl )-1H-pyrazol-4-yl]quinoline), SD-208(2-(5-Chloro-2-fluorophenyl)pteridin-4-yl)pyridin-4-yl amine), EW-7197(N-(2-fluorophenyl)-5-(6-methyl-2-pyridinyl)-4-[1,2,4]triazolo[1,5-a]pyridin-6-yl-1H-Imidazole-2-methanamine)、A83-01(3-(6-Methylpyridin-2-yl)-4-(4-quinolyl)-1-phenylthiocarbamoyl-1H-pyrazole)、RepSox(2-[5-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl]-1,5-naphthyridine)、SM16(4-[4-(1,3-Benzodioxol-5-yl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-2-yl]bicyclo[2.2.2]octane-1-carboxamide)、R268712(4-[2-Fluoro-5-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]phenyl]-1H-pyrazole-1-ethanol)、IN1130(3-[[5-(6-Methyl-2-pyridinyl)-4-(6-quinoxalinyl)-1H-imidazol-2-yl]methyl]benzamide)、Galunisertib(4-[5,6-Dihydro-2-(6-methyl-2-pyridinyl)-4H-pyrrolo[1,2-b]pyrazol-3-yl]-6-quinolinecarboxamide)、AZ12799734(4-({4-[(2,6-dimethylpyridin-3-yl)oxy]pyridin-2-yl}amino)benzenesulfonamide)、A77-01(4-[3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl]quinoline)、KRCA 0008(1,1-[(5-Chloro-2,4-pyrimidinediyl)bis[imino(3-methoxy-4,1-phenylene)-4,1-piperazinediyl]]bisethanone)、GSK 1838705(2-[[2-[[1-[(Dimethylamino)ethanoyl]-5-(methyloxy)-2,3-dihydro-1H-indol-6-yl]amino]-7H-pyrrolo[2,3-d]pyrimidin-4-yl]amino]-6-fluoro-N-methylbenzamide)、Crizotinib(3-[(1R)-1-(2,6-Dichloro-3-fluorophenyl)ethoxy]-5-[1-(piperidin-4-yl)-1H-pyrazol-4-yl]-2-aMinopyridine)、Ceritinib(5-Chloro-N2-[2-isopropoxy-5-Methyl-4-(4-piperidyl)phenyl]-N4-(2-isopropylsulfonylphenyl)pyriMidine-2,4-diaMine)、ASP 3026(N2-[2-Methoxy-4-[4-(4-methyl-1-piperazinyl)-1-piperidinyl]phenyl]-N4-[2-[(1-methylethyl)sulfon)、TAE684(5-Chloro-N2-[2-methoxy-4-[4-(4-methyl-1-piperazinyl)-1-piperidinyl]phenyl]-N4-[2-[(1-methylethyl)sulfonyl]phenyl]-2,4-pyrimidinediamine)、AZD3463(N-[4-(4-Amino-1-piperidinyl)-2-methoxyphenyl]-5-chloro-4-(1H-indol-3-yl)-2-pyrimidinamine)、TP0427736(6-[4-(4-methyl-1,3-thiazol-2-yl)-1H-imidazol-5-yl]-1,3-benzothiazole)、TGFBR1-IN-1(5-(1,3-benzothiazol-6-yl)-N-(4-hydroxyphenyl)-1-(6-methylpyridin-2-yl)pyrazole-3-carboxamide)、TEW-7197(2-fluoro-N-[[5-(6-methylpyridin-2-yl)-4-([1,2,4]triazolo[1,LY3200882(2-[4-[[4-[1-cyclopropyl-3-(oxan-4-yl)pyrazol-4-yl]oxypyridin-2-yl]amino ]pyridin-2-yl]propan-2-ol), BIBF-0775((3Z)-N-Ethyl-2,3-dihydro-N-methyl-2-oxo-3-[phenyl[[4-(1-piperidinylmethyl)phenyl]amino]methylen Examples include Alk5 inhibitors such as [e]-1H-indole-6-carboxamide, SMAD3 inhibitors such as SIS3(1-(3,4-dihydro-6,7-dimethoxy-2(1H)-isoquinolinyl)-3-(1-methyl-2-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-2-propen-1-one), receptor degradation promoters such as ITD-1(4-[1,1'-Biphenyl]-4-yl-1,4,5,6,7,8-hexahydro-2,7,7-trimethyl-5-oxo-3-quinolinecarboxylic acid ethyl ester), and derivatives of these compounds. These substances may be used alone or in combination.

[0030] The TGFβ inhibitor is preferably an ALK5 inhibitor. Examples of ALK5 inhibitors include SB431542, SB505124, SB525334, LY2157299, GW788388, LY364947, SD-208, EW-7197, A83-01, RepSox, SM16, R268712, IN1130, Galunisertib, AZ12799734, A77-01, KRCA 0008, GSK 1838705, Crizotinib, Ceritinib, ASP 3026, TAE684, AZD3463, TP0427736, etc., with SB505124 being preferred.

[0031] In the manufacturing method of the present invention, the concentration of the TGFβ inhibitor (TGFβ signaling pathway inhibitor) in the culture medium can be appropriately set depending on the substance used, within a range in which the above-described effects can be achieved. Typically, the TGFβ inhibitor is added to the culture medium at a concentration of about 1 nM to about 100 μM, preferably about 10 nM to about 100 μM, more preferably about 10 nM to about 50 μM, even more preferably about 100 nM to about 50 μM, even more preferably about 1 μM to about 10 μM, and most preferably about 10 μM.

[0032] Bone morphogenetic protein (BMP) may be added to the culture medium used in step (1) of the manufacturing method of the present invention. Bone morphogenetic protein is a secreted signaling molecule belonging to the TGF-β superfamily. Examples of bone morphogenetic proteins used in step (1) of the manufacturing method of the present invention include BMP2, BMP3, BMP3b, BMP4, BMP5, BMP6, BMP7, BMP8, BMP9, BMP10, BMP11, BMP12, BMP13, BMP14, BMP15, BMP16, BMP17, and BMP18, with BMP4 being preferred. Two or more types of BMP may be used in combination. The concentration of BMP in the culture medium is adjusted as appropriate depending on the type of BMP added, but is typically 1 to 100 ng / ml, preferably 2 to 30 ng / ml.

[0033] Examples of extracellular matrix used in step (2) of the manufacturing method of the present invention include basement membrane preparations, laminin or its fragments, entactin, collagen, gelatin, etc. In one embodiment, the extracellular matrix used in step (2) of the manufacturing method of the present invention is a basement membrane preparation such as Matrigel.

[0034] In this specification, "basement membrane preparation" refers to a preparation containing basement membrane components that, when desired cells capable of forming a basement membrane are seeded and cultured on it, have the function of controlling epithelial cell-like cell morphology, differentiation, proliferation, motility, and functional expression. Here, "basement membrane components" refers to thin, membrane-like extracellular matrix molecules present between the epithelial cell layer and the stromal cell layer in animal tissue. A basement membrane preparation can be prepared, for example, by removing cells capable of forming a basement membrane that are adhered to a support via the basement membrane from the support using a solution or alkaline solution that has lipid-solving ability for the cells. Examples of basement membrane preparations include commercially available basement membrane preparations such as Matrigel (manufactured by Corning), Geltrex (manufactured by Thermo Fisher Scientific), and preparations containing extracellular matrix molecules known as basement membrane components (e.g., laminin, type IV collagen, heparan sulfate proteoglycan, entactin, etc.) or fragments thereof, with Matrigel being preferred.

[0035] In this specification, "laminin or its fragment" is not particularly limited as long as it has high affinity for at least the integrin α3β1 complex, but for example, laminin-111 or a fragment containing its E8 region, laminin-211 or a fragment containing its E8 region (e.g., iMatrix-211), laminin-121 or a fragment containing its E8 region, laminin-221 or a fragment containing its E8 region, laminin-332 or a fragment containing its E8 region, laminin-3A11 or a fragment containing its E8 region, laminin-411 or a fragment containing its E8 region (e.g., iMatrix-411), laminin-421 or a fragment containing its E8 region, laminin-511 or a fragment containing its E8 region (e.g., iMatrix-511, iMatrix-511) Examples include fragments containing laminin-521 or its E8 region, laminin-213 or its E8 region, laminin-423 or its E8 region, laminin-523 or its E8 region, laminin-212 / 222 or its E8 region, laminin-522 or its E8 region, and the like.

[0036] In step (2) of the present invention, the concentration of the extracellular matrix contained in the culture medium can be appropriately set to form the desired organoid. The concentration of the extracellular matrix in the culture medium is appropriately adjusted depending on the type of extracellular matrix added, but typically, when using Matrigel, a basement membrane preparation, it can be added to the culture medium to concentrations such as 0.05% (v / v) to 20% (v / v), 0.1% (v / v) to 5% (v / v), or 0.5% (v / v) to 2% (v / v).

[0037] Retinoic acid may be added to the culture medium used in step (3) of the manufacturing method of the present invention at a predetermined timing as described later. The concentration of retinoic acid in the culture medium is typically 10 nM to 10 μM, preferably 100 nM to 1 μM, and more preferably 200 nM to 500 nM.

[0038] In step (3) of the manufacturing method of the present invention, neurotrophic factors may be added to the culture medium at a predetermined timing as described later. Examples of neurotrophic factors include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), neurotrophin-3 (NT-3), and neurotrophin-4 / 5 (NT-4 / 5). Among these, BDNF and GDNF are preferred. Two or more types of neurotrophic factors may be used in combination. The concentration of neurotrophic factors in the culture medium is appropriately adjusted depending on the type of neurotrophic factor added, but typically, when using BDNF or GDNF, it is 0.1 ng / ml to 1 mg / ml, preferably 1 ng / ml to 100 ng / ml, and more preferably 10 ng / ml to 50 ng / ml.

[0039] In at least one of steps (1), (2), and (3) of the manufacturing method of the present invention, cells may be cultured under feeder-free and / or xeno-free conditions. In this specification, "feeder-free" means a culture medium or culture conditions that do not contain other cell types (i.e., feeder cells) that play an auxiliary role in preparing the culture conditions for the cells to be cultured. Also, "xeno-free" means a culture medium or culture conditions that do not contain components of a different biological origin from the species of the cells to be cultured.

[0040] As the culture medium, a medium for culturing pluripotent stem cells based on a basal medium (e.g., Basal Medium Eagle (BME), BGJb medium, CMRL 1066 medium, Glasgow Minimum Essential Medium (Glasgow MEM), Improved MEM Zinc Option, Iscove's Modified Dulbecco's Medium (IMDM), Medium 199, Eagle Minimum Essential Medium (Eagle MEM), Alpha Modified Eagle Minimum Essential Medium (αMEM), Dulbecco's Modified Eagle Medium (DMEM), F-12 medium, DMEM / F12, IMDM / F12, Ham medium, RPMI 1640, Fischer's medium, or a mixture thereof) can be used, preferably a known medium for embryonic stem cells or induced pluripotent stem cells, or a medium for culturing pluripotent stem cells under feeder-free conditions (feeder-free medium). The above-mentioned mediums may contain serum, but serum-free mediums are preferred.

[0041] Many synthetic culture media have been developed and are commercially available as feeder-free media. Examples include StemFit® (manufactured by Ajinomoto Co., Inc.), Essential 6 (E6) (manufactured by Thermo Fisher Scientific), Essential 8 (manufactured by Thermo Fisher Scientific), S-medium (manufactured by DS Pharma Biomedical), StemPro (manufactured by Thermo Fisher Scientific), hESF9, mTeSR1 (manufactured by STEMCELL Technologies), mTeSR2 (manufactured by STEMCELL Technologies), TeSR-E8 (manufactured by STEMCELL Technologies), mTeSR Plus (manufactured by STEMCELL Technologies), ReproMed iPSC Medium (manufactured by Reprocell), NutriStem XF (manufactured by Biological Industries), NutriStem V9 (manufactured by Biological Industries), Cellartis DEF-CS Xeno-Free Culture Medium (manufactured by Takara Bio Inc.), and Stem-Partner. Examples include SF (manufactured by Kyokuto Pharmaceutical Co., Ltd.), PluriSTEM Human ES / iPS Cell Medium (manufactured by Merck Ltd.), StemSure hPSC MediumΔ (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and preferably Essential 6 and mTeSR Plus.

[0042] The medium may contain a serum substitute. Examples of the serum substitute include those appropriately containing albumin, transferrin, fatty acids, collagen precursors, trace elements, 2-mercaptoethanol or 3'-thiol glycerol, or equivalents thereof. Such a serum substitute can be prepared, for example, by the method described in WO 98 / 30679. Commercially available products may be used as the serum substitute. Examples of commercially available serum substitutes include Knockout Serum Replacement (KSR) (manufactured by Thermo Fisher Scientific), Chemically-defined Lipid concentrated (manufactured by Thermo Fisher Scientific), Glutamax (manufactured by Thermo Fisher Scientific), B27 Supplement (manufactured by Thermo Fisher Scientific), N2 Supplement (manufactured by Thermo Fisher Scientific), and the like.

[0043] The medium may appropriately contain fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, inorganic salts, and the like.

[0044] In the production method of the present invention, the culture density of the cells is not particularly limited as long as the cells can grow. Typically, it is 1.0×10 1 ~1.0×10 9 cells / ml, preferably 1.0×10 2 ~1.0×10 8 cells / ml, more preferably 1.0×10 3 ~1.0×10 7 cells / ml, even more preferably 1.0×10 3 ~1.0×10 6 cells / ml (e.g., 1.0×10 4 cells / ml, 5.0×10 4 cells / ml, etc.).

[0045] In this specification, "suspension culture" means culture carried out under conditions that maintain a state in which cells or cell aggregates are suspended in the culture medium, that is, culture under conditions that do not allow strong cell-substrate junctions to form between the cells or cell aggregates and the culture vessel. In suspension culture, cells typically exist in the form of cell aggregates before and after suspension culture.

[0046] The culture vessels used when performing suspension culture are not particularly limited as long as they are capable of "suspension culture," and can be appropriately determined by those skilled in the art. Examples of such culture vessels include flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, micropores, multi-plates, multi-well plates, chamber slides, petri dishes, tubes, trays, culture bags, and roller bottles. Furthermore, bioreactors are exemplified as containers for suspension culture. These culture vessels are preferably cell-non-adherent in order to enable suspension culture. As cell-non-adherent culture vessels, those whose surfaces have not been artificially treated (e.g., coated with extracellular matrix, etc.) for the purpose of improving adhesion to cells can be used. The well bottom shape of these culture vessels is not particularly limited, and examples include flat bottoms, U-shaped, and V-shaped. In this specification, "cell-non-adherent" also includes low cell adhesion and very low cell adhesion.

[0047] The culture temperature is not particularly limited, but is approximately 30-40°C, preferably approximately 37°C, and the culture is carried out in an atmosphere of CO2-containing air, with a CO2 concentration of preferably approximately 2-5%.

[0048] The incubation period in step (1) of the manufacturing method of the present invention is typically 2 days or more, preferably 2 to 7 days, more preferably 2 to 5 days, and even more preferably 3 to 4 days.

[0049] The incubation period in step (2) of the manufacturing method of the present invention is typically 2 days or more, preferably 2 to 7 days, more preferably 2 to 5 days, and even more preferably 2 to 3 days.

[0050] The culture period in step (3) of the manufacturing method of the present invention is typically 7 days or more, preferably 7 to 100 days, preferably 14 to 60 days, and more preferably 20 to 38 days. In step (3), retinoic acid is typically present in the culture medium for 1 to 30 days from the start of step (3), preferably 3 to 14 days, and more preferably 7 to 12 days. In step (3), neurotrophic factors (e.g., BDNF, GDNF, etc.) are typically present in the culture medium from 3 days after the start of step (3), preferably from 7 days after. In addition, neurotrophic factors (e.g., BDNF, GDNF, etc.) are typically present in the culture medium for 10 days or more, preferably 10 to 97 days, and more preferably 15 to 38 days.

[0051] The manufacturing method of the present invention may include a step (1') prior to step (1) in which pluripotent stem cells are cultured in suspension in a medium containing a ROCK inhibitor for at least one day. Step (1') can be used to prepare cell aggregates containing pluripotent stem cells for use in the manufacturing method of the present invention.

[0052] Prior to step (1') above, a procedure may be performed to disperse pluripotent stem cells into single cells. The "dispersed cells" obtained by the dispersion procedure are preferably single cells, but may also include cell clusters consisting of a small number of cells, for example, 2 to 100, or cell clusters consisting of 2 to 50. The "dispersed cells" may contain, for example, 70% or more single cells and 30% or less cell clusters, preferably 80% or more single cells and 20% or less cell clusters.

[0053] Methods for dispersing pluripotent stem cells include mechanical dispersion, cell dispersion treatment, and cytoprotective agent addition, and these treatments may be combined. As a method for dispersing cells, for example, cytoprotective agent addition and cell dispersion treatment may be performed simultaneously, followed by mechanical dispersion.

[0054] Examples of cytoprotective agents used in the cytoprotective agent addition treatment include FGF signaling pathway agonists, heparin, ROCK inhibitors, myosin inhibitors, polyamines, integrated stress response (ISR) inhibitors, caspase inhibitors, serum, and serum substitutes. ROCK inhibitors, as mentioned above, are preferred cytoprotective agents. Pre-prepared cytoprotective agents can also be used. Examples of pre-prepared cytoprotective agents include RevitaCell Supplement (Thermo Fisher Scientific) and CloneR (Stemcell Technologies). These substances may be used individually or in combination.

[0055] Examples of cell dispersions used in cell dispersion treatment include solutions containing at least one enzyme such as trypsin, collagenase, hyaluronidase, elastase, pronase, DNase, and papain, and a chelating agent such as ethylenediaminetetraacetic acid. Commercially available cell dispersions, such as TripLE Select (Thermo Fisher Scientific), TripLE Express (Thermo Fisher Scientific), and Accumax (Innovative Cell Technologies), can also be used. Preferred cell dispersions for the treatment of pluripotent stem cells are TrypLE Select or phosphate-buffered water (PBS) with 5 mM EDTA, but are not limited to these.

[0056] Mechanical dispersion methods include pipetting or scraping with a scraper. The dispersed cells are then suspended in a culture medium.

[0057] Therefore, one method for dispersing pluripotent stem cells is to treat pluripotent stem cell colonies with TrypLE Select, ethylenediaminetetraacetic acid, or Accumax in the presence of a ROCK inhibitor, and then disperse them in serum-free medium by pipetting.

[0058] The suspension culture in step (1') may be carried out under the same conditions (e.g., cell culture density, culture medium, etc.) and methods as in steps (1) to (3) above. Furthermore, for purposes such as protecting cell aggregates from physical stresses like shear forces generated during suspension culture, and increasing the local concentration of growth factors and cytokines secreted by cells to promote tissue development, the cell aggregates may be embedded in a gel or encapsulated in a permeable capsule before suspension culture (Lancaster MA et al., Nature, 2013, 501. 7467:373-379). The encapsulated cell aggregates may also be subjected to shaking culture. In this specification, "shaking culture" refers to a culture method in which the culture medium is agitated by shaking the culture equipment, thereby promoting oxygen supply to the culture medium and exchange of substances with the surrounding cells. Agitation culture, flow channel culture, etc., can also be performed. The gel or capsule used for embedding may be of biological origin or made of synthetic polymer. Examples of gels or capsules used for this purpose include Matrigel (manufactured by Corning), PuraMatrix (manufactured by 3D Matrix), VitroGel 3D (manufactured by TheWell Bioscience), collagen gel (manufactured by Nitta Gelatin Co., Ltd.), alginate gel (manufactured by PG Research), and Cell-in-a-Box (manufactured by Austrianova).

[0059] More specifically, in the manufacturing method of the present invention, when performing suspension culture, dispersed pluripotent stem cells may be seeded into a relatively large culture compartment such as a 10 cm dish to simultaneously form multiple cell aggregates in one culture compartment. However, from the viewpoint of reducing variations in the size of each cell aggregate, for example, a certain number of dispersed pluripotent stem cells may be seeded in each well of a multi-well plate (U-bottom, V-bottom), such as a cell-non-adherent 96-well microplate. When this is cultured statically, the cells aggregate rapidly, forming one cell aggregate in each culture compartment (Serum-free culture of Embryoid Body-like aggregates with quick reaggregation; SFEBq method).

[0060] The culture medium used in step (1') above may contain, for example, an undifferentiated maintenance factor. The undifferentiated maintenance factor is not particularly limited as long as it is a substance that has the effect of suppressing the differentiation of pluripotent stem cells. Examples of undifferentiated maintenance factors commonly used by those skilled in the art include FGF signaling pathway activators, TGFβ family signaling pathway activators, and insulin in the case of primed pluripotent stem cells (e.g., human ES cells, human iPS cells). Specifically, examples of FGF signaling pathway activators include fibroblast growth factors (e.g., bFGF, FGF4, and FGF8). Examples of TGFβ family signaling pathway activators include TGFβ signaling pathway activators and Nodal / Activin signaling pathway activators. Examples of TGFβ signaling pathway activators include TGFβ1 and TGFβ2. Examples of Nodal / Activin signaling pathway activators include Nodal, Activin A, and Activin B. These substances may be used individually or in combination.

[0061] Undifferentiated maintenance factors can be those produced by any host or artificially synthesized, as long as they have the ability to maintain the undifferentiated state of the pluripotent stem cells being cultured. The undifferentiated maintenance factors used in this invention are preferably those that have undergone modifications similar to those that occur in vivo, and are even more preferably those produced in cells of the same type as the pluripotent stem cells being cultured under conditions that do not contain heterologous components.

[0062] In step (1'), the concentration of the undifferentiated maintenance factor in the culture medium is a concentration capable of maintaining the undifferentiated state of the pluripotent stem cells being cultured, and can be appropriately set by those skilled in the art. For example, when bFGF is used as the undifferentiated maintenance factor in the absence of feeder cells, its concentration is typically about 4 ng / ml to about 500 ng / ml, preferably about 10 ng / ml to about 200 ng / ml, and more preferably about 30 ng / ml to about 150 ng / ml.

[0063] In step (1'), the suspension culture time required to form cell aggregates can be appropriately determined depending on the pluripotent stem cells used, but it is desirable to keep it as short as possible in order to form uniform cell aggregates. The culture period in step (1') is typically within 48 hours, preferably within 36 hours, and more preferably within 24 hours.

[0064] Steps (1') to (3) of the manufacturing method of the present invention may be carried out in the same culture vessel, in separate culture vessels, or some steps may be carried out in the same culture vessel. For example, if each step is carried out in a separate culture vessel, the culture may be collected after the completion of each step and the collected culture may be transferred to a separate culture vessel in which the next step will be carried out. Furthermore, the cell aggregate containing pluripotent stem cells used in step (1) may be obtained by carrying out step (1') as described above, or it may be prepared by another known method.

[0065] The manufacturing method of the present invention may include a step of recovering the obtained target organoid. The recovered organoid may be cryopreserved, for example, using a cell cryopreservation solution.

[0066] In the cell production method of the present invention, undifferentiated cells may be removed as appropriate. The method for removing undifferentiated cells is not particularly limited as long as it can remove cells other than those contained in the organoids produced by the production method of the present invention, and can be carried out by adding known undifferentiated cell removal agents to the culture medium (for example, Di Mao., et al. Angewandte Chemie International Edition; 9 January 2017, Ben-David, U., et al. Cell Stem Cell, 12, 167 (2013), WO2019 / 187918, JP 2016-93178, Yoshiki Nakashima, et. al., Molecular Therapy Vol. 26 No 7 July 2018, etc.).

[0067] Quality inspections may be performed as appropriate to determine whether the organoids obtained by the manufacturing method of the present invention are as desired. The items for quality inspection are not particularly limited, but examples include basic tests such as organoid morphology, presence or absence of cell surface marker expression, sterility tests, endotoxin tests, and evaluation of cell viability, and testing equipment appropriate for each item can be used.

[0068] 2. Organoids obtained by the manufacturing method of the present invention The manufacturing method of the present invention produces organoids containing ectoderm and mesoderm cells (hereinafter sometimes referred to as "organoids of the present invention"). In this specification, "organoid" means a structure formed by the accumulation of cells, and typically has a structure and function similar to that of tissues in living organisms. Whether a structure is an organoid can be confirmed, for example, by examining a sample that has been stained as necessary (e.g., alizarin red staining, HE staining, Azan staining, etc.) under a microscope to check for the presence or absence of layered structure formation, or by examining the expression of marker proteins.

[0069] In one embodiment, the organoid of the present invention has the following features (1) to (3). (1) The surface has 1 to 5 layers containing Tuj-1 expressing cells (i.e., the number of layers containing Tuj-1 expressing cells is 1 to 5; the same applies hereinafter). (2) Inside the layer of (1), there are 1 to 50 layers containing cells that express at least one selected from the group consisting of VSX2, CRX, and Recoverin. (3) Inside the layer of (2), there are 1 to 3 layers containing cells that express Myosin. The cells mentioned in (2) above specifically include cells expressing VSX2, cells expressing CRX, cells expressing Recoverin, cells expressing VSX2 and CRX, cells expressing VSX2 and Recoverin, cells expressing CRX and Recoverin, and cells expressing VSX2, CRX, and Recoverin.

[0070] In one embodiment, layers 1 to 50 containing cells expressing at least one selected from the group consisting of VSX2, CRX, and Recoverin as described in (2) above form a retinal-like structure.

[0071] In one embodiment, layers 1 to 3 containing cells expressing the Myosin described in (3) above form a muscle fiber-like structure.

[0072] The organoids of the present invention typically have a major axis (or equivalent circle diameter) of, for example, 0.5 to 50 mm. The method for measuring the major axis (or equivalent circle diameter) of the organoid is not particularly limited and can be measured, for example, from an image taken under a microscope. For example, cell aggregates cultured in a 96-well culture plate can be imaged with a Keyence inverted microscope 10x lens and measured from the image. Here, the major axis refers to the longest line segment and its length among the line segments connecting the two endpoints of the cell aggregate in the imaged image. The equivalent circle diameter refers to the diameter of a perfect circle, which corresponds to the area of ​​the figure (circular or elliptical) obtained when projected onto a two-dimensional surface.

[0073] The cells contained in the organoids obtained by the manufacturing method of the present invention may be undifferentiated cells such as stem cells or progenitor cells, or they may be terminally differentiated cells. Hereinafter, the term "differentiated cells" may be used to encompass both undifferentiated cells and terminally differentiated cells. In this specification, "undifferentiated cells" means cells that have not reached terminal differentiation in the cell lineage, and examples of undifferentiated cells include stem cells other than pluripotent stem cells, progenitor cells, etc. Examples of stem cells or progenitor cells include ectodermal cells such as neural crest cells, neural stem cells, neural progenitor cells, glial progenitor cells, retinal stem cells, retinal progenitor cells, corneal stem cells, keratinocyte epidermal stem cells, melanocyte stem cells, mammary gland stem cells, mesodermal cells such as hematopoietic progenitor cells, myeloid stem cells, lymphoid stem cells, B progenitor cells, T progenitor cells, mesenchymal stem cells, cardiac stem cells, cardiac progenitor cells, vascular endothelial progenitor cells, vascular pericytes, platelet progenitor cells, skeletal muscle stem cells, adipose stem cells, renal progenitor cells, and endodermal cells such as hepatic stem cells, hepatic progenitor cells, intestinal stem cells, and airway stem cells.

[0074] In this specification, "terminally differentiated cells" means cells that have reached terminal differentiation in a cell lineage. Terminally differentiated cells are not particularly limited, but examples include ectodermal cells such as nerve cells, retinal pigment epithelial cells, astrocytes, oligodendrocytes, melanocytes, and corneal endothelial cells; mesodermal cells such as cardiomyocytes, skeletal muscle cells, smooth muscle cells, osteocytes, chondrocytes, and adipocytes; and endodermal cells such as hepatocytes.

[0075] The organoids obtained by the manufacturing method of the present invention include cells in which the expression of at least one gene marker related to ectoderm cells and at least one gene marker related to mesoderm cells is upregulated compared to cell aggregates containing pluripotent stem cells used in culture in step (1). Examples of gene markers related to ectoderm cells include visual gene markers (retinal markers) (e.g., PAX6, RPE-65, VSX1, VSX2, MITF, MSX2, RCVRN, RGR, RAX2, etc.), photoreceptor-related gene markers (e.g., RCVRN (rods), RGR, RAX2, etc.), forebrain-related gene markers (e.g., FOXG1, OTX1, DLX1, VAX1, etc.), and glutamatergic neuron gene markers (e.g., SLC1A7, SLC17A6, SLC17A7, etc.). Genetic markers related to mesodermal cells include skeletal muscle and cardiomyocyte-related gene markers (e.g., MYOG, MYH6, MYH7, MYOD1, PAX3, PAX7, TNNT3, GATA4, etc.).

[0076] In one embodiment, the organoid of the present invention has the following features: (i) Cells that are positive for at least one gene selected from the group consisting of PAX6, RPE-65, VSX1, VSX2, MITF, MSX2, RCVRN, RGR, and RAX2, (ii) Cells that are positive for at least one gene selected from the group consisting of FOXG1, OTX1, DLX1, VAX1, SLC1A7, SLC17A6, and SLC17A7, and (iii) Cells that are positive for at least one gene selected from the group consisting of MYOG, MYH6, MYH7, MYOD1, PAX3, PAX7, TNNT3, and GATA4. It has.

[0077] The organoids obtained by the manufacturing method of the present invention are organoids containing at least ectodermal cells and mesodermal cells, and can therefore be used to construct specific disease models involving multiple cell types or to construct screening systems for specific diseases. Examples of such diseases include amyotrophic lateral sclerosis (ALS), Parkinson's disease, age-related macular degeneration (AMD), diabetic retinopathy, spinal muscular atrophy (SMA), and sarcopenia.

[0078] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. [Examples]

[0079] Example 1: Investigation of a method for manufacturing multi-system organoids (1) Materials and Methods <Culture of human induced pluripotent stem cells> Human induced pluripotent stem cells (hiPSCs), specifically 585A1 and 201B7f, were obtained from the RIKEN Cell Bank and handled in accordance with the guidelines established by the Ritsumeikan University Ethics Committee (Approval Number 2021-004-3). Culture dishes were pre-coated overnight at 37°C with imatrix-511 (Nippi Corporation). mTeSR was treated with 10 μM Y27632. TM 1 × 10⁶ cells in plus medium (manufactured by STEMCELL Technologies) 4 The viable cells were seeded into a 6-well plate. The next day, the cell culture medium was changed to mTeSR™ plus only. The culture medium was replaced with fresh mTeSR™ every two days. TM The medium was changed to plus medium. For cell subculturing, the cells were washed with PBS (Fujifilm Wako Pure Chemical Industries) and treated with 0.5 TryPLE Select (Thermo Fisher Scientific) at 37°C for 4 minutes.

[0080] Next, the cell suspension was transferred to a 15 ml tube and centrifuged at 160 × g for 5 minutes. After centrifugation, the supernatant was discarded and the cells were added to mTeSR containing 10 μM Y27632 (Fujifilm Wako Pure Chemical Industries, Ltd.). TMThe cells were then resuspended in plus medium (STEMCELL Technologies). Subsequently, 1 × 10⁶ cells were placed per well. 4 Cells were seeded at a specified density and cultured in a humidified incubator at 37°C and 5% CO2 for 24 hours. The culture medium was replaced with fresh mTeSR every two days. TM The medium was replaced with plus medium.

[0081] <Screening of chemical substances (additives)> When hiPSC_585A1 reached 30-40% confluence, the culture medium was changed to E6 basal medium (Thermo Fisher Scientific) containing a chemical inhibitor and bFGF growth factor (Table 1), and the cells were cultured until day 4. After that, they were cultured in E6 until day 7, and then the cells were harvested and gene expression analysis was performed.

[0082] [Table 1]

[0083] <Organoid fabrication> After 5-6 days, when hiPSCs reached 60-70% confluence, cells were harvested using 0.5 TryPLE Select and counted using a Countess automated cell counter (Thermo Fisher Scientific). Subsequently, 0.1 ml of mTeSR containing 10 μM Y27632 was used. TM 5 × 10 in plus medium 4 Individual living cells were transferred to a 96-well U-bottom ultra-low adhesion plate (manufactured by Sumitomo Bakelite Co., Ltd.) to form spheroids.

[0084] The next day, mTeSR TM The plus medium was replaced with a chemically derived medium consisting of fresh E6 medium (Thermo Fisher Scientific) supplemented with 10 μM TGF-β kinase / activin receptor-like kinase inhibitor (SB-505124) and 10 μM Y27632 or BMP4 (20 ng / ml) (Table 2).

[0085] [Table 2]

[0086] These conditions were maintained for 3 days. After that, the cells were treated with Geltrix. TM The organoids were cultured for 2 days in E6 medium supplemented with hESC-qaulified (44:1 v / v) (Thermo Fisher Scientific) and 10 μM Y27632. The medium was then switched to E6 medium supplemented with 10 μM Y27632. To promote the formation of eye primordial field / retinal pigment epithelial cells, 500 nM retinoic acid was added at this stage and maintained for 7 days. On day 14, the organoids were transferred to a 6-well plate with an ultra-low adhesion surface (Corning) and cultured in E6 medium supplemented with only 10 μM Y27632. To evaluate the maturation of the organoids into brain-derived cells, recombinant human GDNF (BioLegend) and recombinant human BDNF (BioLegend) were added from day 20.

[0087] <Measurement of retinal epithelial formation efficiency> Regarding the formation of dark spots, organoids were visually inspected from day 38 onward. The RPE formation efficiency was as follows: RPE formation efficiency (%) = (Number of organoids with dark spots / Total number of organoids) × 100 It was calculated as follows.

[0088] <Immunofluorescence staining of paraffin blocks, H&E, and organoids> The organoids were fixed overnight at 4°C in PBS containing 4% paraformaldehyde, and then processed to create paraffin blocks. Subsequently, 2-5 μm sections were collected and stored at 4°C. For hematoxylin and eosin (H&E) staining, samples were deparaffinized before staining. For immunofluorescence staining, samples were antigen-retrieved using sodium citrate buffer (pH 6.0), boiled in a microwave for 10–15 minutes, and then permeabilized overnight at 4°C in PBS containing 0.5% Triton X-100. Subsequently, cells were blocked at room temperature for 90 minutes with blocking buffer (5% bovine serum albumin, 0.1% Tween-20), followed by incubation overnight at 4°C with primary antibody diluted in blocking buffer (Table 3).

[0089] [Table 3]

[0090] After washing, the cells were incubated with appropriate secondary antibodies (Alexa Fluor 555 goat anti-mouse IgG, Alexa Fluor 488 goat anti-rabbit IgG, 1:500 v / v, Thermo Fisher Scientific) at room temperature for 60 minutes. Finally, the cells were mounted at 25°C for 1 hour in a bleed prevention solution containing the nuclear stain DAPI Fluoro-KEEPER (Nacalai Tesque). Imaging was performed using a fluorescence microscope (Keyence).

[0091] <Immunofluorescence staining of the entire organoid> The organoids were fixed overnight at 4°C with PBS containing 4% paraformaldehyde. Subsequently, tissue clearing was performed using a Scalview (registered trademark) kit (manufactured by Fujifilm Wako Pure Chemical Corporation) according to the manufacturer's recommendations. Next, the organoids were permeabilized overnight at 4°C with PBS containing 0.5% Triton X-100. After permeabilization, blocking was carried out overnight at 4°C with blocking buffer (5% bovine serum albumin, 0.1% Tween-20). Then, the organoids were incubated with the primary antibody diluted in the blocking buffer at 4°C for 2 days. After washing several times, the cells were incubated with the appropriate secondary antibody at 4°C for 24 hours. Imaging was performed using a confocal laser scanning microscope FV3000 (manufactured by OLYMPUS).

[0092] <RNA Extraction and Gene Expression Analysis> RNA extraction was performed using a Sepasol (registered trademark)-RNAI Super G kit (manufactured by Nacalai Tesque) according to the manufacturer's protocol. Reverse transcription for real-time PCR (RT-PCR) was carried out using a ReveTra Ace (registered trademark) qPCR RT kit (manufactured by Toyobo). The obtained cDNA was quantified using PowerUp SYBR Green Master Mix (manufactured by Thermo Fisher Scientific) and a StepOne Real-Time PCR System (manufactured by Thermo Fisher Scientific). The target mRNA expression level was normalized to the mRNA expression level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using the ΔΔCT method. The primers used are shown in Table 4.

[0093]

Table 4

[0094] In RNA-seq analysis, the quality of the samples was evaluated using a Bioanalyzer (manufactured by Agilent Technologies), and it was confirmed that the RNA integrity number (RIN) values of all samples were 7 or higher. Subsequently, next-generation sequencing (manufactured by Relixa) was performed on the samples, and libraries were constructed using the NEBNext® Poly(A) mRNA Magnetic Isolation Module and the NEBNext® Ultra II Directional RNA Library Prep Kit. Subsequently, sequencing was performed on the NovaSeq 6000 Illumina system (PE150 150 bp × 2 paired-end) at 4 G bases and 26.7 M reads (13.3 M pairs) per sample.

[0095] <RNA-seq Data Mining and GO Enrichment Analysis> Quality control of the sequenced raw reads was performed by calculating read quality, total base count, total number of reads, GC (%), and basic statistics (FastQC v0.11.7). To reduce bias in the analysis, low-quality reads, adapter sequences, contaminating DNA, or artifacts such as PCR duplicates were removed (Trimmomatic 0.38) (Anthony M Bolger et al., Bioinformatics, 2014 30(15), 2114-2120). The trimmed reads were mapped to the reference genome using the splice-aware aligner HISAT2 (HISAT2 version 2.1.0, Bowtie2 2.3.5.1) (Daehwan Kim et al., Nature methods, 2015 12(4), 357-360). Transcripts were assembled using aligned reads with featureCounts (featureCounts version 1.6.3) (Heng Li et al., Bioinformatics. 2009 25(16), 2078-2079, Yang Liao et al., Bioinformatics. 2014 30(7), 923-30). This process provides information on known, novel, and alternative splicing transcripts. Expression profiles are expressed as read counts and normalized values ​​based on transcript length and coverage depth. Raw read counts normalized by FPKM (fragments per kilobase of transcript per million mapped reads) or TPM (transcripts per million) were used as normalized values. Differentially expressed genes or transcripts were excluded by statistical hypothesis testing in groups under different conditions.

[0096] Principal component analysis (PCA) was performed on normalized counts, and each sample was projected onto a 2D plane with the first and second PCA axes using the stats (version 3.6.1) and gplots (version 3.0.1.1) R packages. Raw read counts were normalized by relative log normalization (RLE), and differential expression analysis was performed using DESeq2 (version 1.24.0). Differentially expressed genes (DEGs) were detected with |log2FC (Fold Change)| > 1 and adjusted p-values ​​< 0.05 by the Benjamini and Hochberg (BH) methods. Gene ontology (GO) enrichment analysis of DEGs was performed using GOATOOLS (version 1.1.6).

[0097] The p-values ​​were corrected using the Benjamini-Hochberg method for multiple test calibration. In the bubble plot, the z-score was calculated using the following formula: Z-score = (Number of genes upregulated - Number of genes downregulated) / √Total number of genes It was obtained like this.

[0098] <Statistical Analysis and Data Visualization> Statistical analysis was performed using Dunnett's test, paired t-test, and Tukey's comparative test via GraphPad Prism 8 (GraphPad Software). Data visualization was performed using GraphPad Prism 8 and R Studio (with ggplot library) or Python Jupyter notebook 6.1.4 (with Pandas and Biinfinite packages). Data visualization was also performed using Python 3 (with matplotlib and seaborn packages).

[0099] (2) Results <The role of chemoinhibitors in promoting cell differentiation toward ectoderm and mesodermal system formation> To investigate the effects of the chemical inhibitors SB505124 and IWP2 on early cell differentiation into the ectoderm and mesoderm, with or without bFGF, we improved our previous method (https: / / doi.org / 10.1007 / s13577-022-00713-5). Specifically, cells were treated for 7 days with a combination of Xenofree medium E6 and specific factors. Gene expression analysis revealed a significant decrease in the pluripotency marker POU5F1B. In contrast, the eye development marker PAX6 was significantly increased in all treatments, with a particularly pronounced increase observed in cells treated with SB505124 alone or in combination with bFGF. Similarly, a significant increase in the forebrain / retinal system marker OTX2 was observed with SB505124 alone or in combination with bFGF. Interestingly, treatment with SB505124 alone, or in combination with bFGF, also upregulated GATA, a marker gene associated with mesodermal lineages (Figure 1).

[0100] <Development and optimization of multi-lineage human iPS cell-derived organoids> In a further development of the previous differentiation method, hiPSCs were cultured as spheroids under 3D conditions. First, spheroids on day 0 were treated with SB505124 and Y27632 in E6 medium for 3 days, followed by the addition of Matrigel ECM, and then the treatment was extended with Y27632 alone in E6 medium (essential method, method A1). By day 9, the morphology of the spheroids had changed, showing a neuroepithelial-like structure. Between days 20 and 26, dark pigment spots appeared in the organoids, indicating the formation of RPE regions (Figure 2A). During this period, the size of the organoids also significantly increased (Figure 2B). Furthermore, H&E staining showed the formation of neural grooves in the organoids. In addition, immunofluorescence staining on day 38 revealed the expression of the neuron (nerve cell) marker Tuj-1 along the margins and the expression of the neural progenitor stem cell marker Ki67 in the inner grooves (Figure 2C). To optimize these conditions, considering the efficiency of RPE region formation, organoids were subjected to a wide range of conditions following chemical induction and Matrigel treatment (Figure 3A).

[0101] As shown below, the optimization was performed under various factors. Method A1: From day 0 to day 3, chemical induction was performed using SB505124 and Y27632 in E6 medium, and from day 3 onwards, Matrigel was added to the E6 medium + Y27632. Method A2: Similar to Method A1, but from day 30, E6 medium + Y27632 was combined with BDNF and GDNF. Method A3: Similar to Method A1, but E6 medium + Y27632 was combined with retinoic acid (RA) from day 6 to day 12. Method A4: Similar to Method A1, but in the chemical induction step, BMP4 was added to E6 medium + Y27632 along with SB505124 from day 0 to day 3, and RA was added to E6 medium + Y27632 (A4) from day 6 to day 12.

[0102] On day 38, the appearance of dark-colored, pigmented RPE-like spots was counted using each of the above methods (Figure 3B). Adding RA after Matrigel in Method A3 significantly accelerated the formation of pigmented organoids (in contrast to before addition, Figure 3C), with over 90% of organoids showing pigmentation. In contrast, Methods A1 and A2 yielded 68% and 66% pigmented organoids, respectively. In particular, the addition of BMP4 (Method A4) significantly reduced the number of pigmented organoids, with only 50% showing pigmentation. Furthermore, culturing organoids from Methods A1 and A2 for more than 50 days revealed significant morphological changes as represented by H&E staining and immunofluorescence staining images. In Method A1, cells formed a slit in the center of the organoid.

[0103] Immunofluorescence of the neuron marker Tuj-1 was observed along the edges, while the neural progenitor stem cell marker Ki67 was expressed in the internal grooves, and astrocyte marker expression was barely detectable (Figure 4A, Figure 4C-I). In contrast, in Method A2, the addition of growth factors necessary for brain organoid maturation significantly altered the organoid morphology, mimicking brain organoid-like structures and reducing internal voids. Furthermore, immunofluorescence of the neuron marker Tuj-1 was strongly expressed along the edges compared to organoids in Method A1, but the neural progenitor stem cell marker Ki67 was almost completely absent, indicating that neural progenitor cells had fully differentiated into neurons under maturation conditions. A notable difference from Method A1 was that the organoids in Method A2 also contained a considerable number of astrocytes (Figure 4B, Figure 4C-II, Figure 4D).

[0104] Method A3 yielded a high proportion of RPE cells. Adding RA after Matrigel was the key to efficiently forming dark spots of high proportion of RPE cells. Next, the characteristics of the organoids produced by this method were investigated. Significant expression of RPE-65, an RPE cell marker, was observed in these organoids. This indicates that neurons formed a network on the surface of the organoid, and Tuj-1 expression was shown in the 3D projection of the entire organoid at day 38 (Figure 5A). The organoids could be cultured for up to day 50 and remained viable, and significant morphological changes were observed when comparing Method A1 and Method A2 in H&E staining.

[0105] Specifically, RPE dark spots and neural grooves, such as those in the retina and forebrain grooves in vivo, were clearly detected (Figure 5B; I), and the formation of mesoderm-associated cell populations, confirmed by immunofluorescence staining of mesoderm and ectoderm protein markers, was also clearly demonstrated. The overall structure of the organoid of the present invention is as follows. Ectoderm-associated cells are distributed in the following two patterns: (1) and (2). (1) Based on the neuronal marker Tuj-1 (red), nerve cells are distributed in 1 to 5 layers on the surface of the organoid (Figure 5B; II). (2) Based on retinal markers (VSX2, CRX, Recoverin), the remaining ectoderm-related cells are located in the neural grooves, which form retinal-like structures consisting of 1 to 50 layers of overlapping cells (Figure 5B; III). Furthermore, mesoderm-related cells are distributed in the central part of the organoid of the present invention, based on the muscle cell marker (fast myosin), and form a structure resembling 1 to 3 layers of muscle fibers (Figure 5B; IV and V).

[0106] <RNA-seq analysis of organoids prepared using Method A3> To investigate the transient transcriptome signatures of biological pathways activated during differentiation based on Method A3, we selected the 585A1 human iPS cell line and performed RNA-seq analysis on organoids generated at day 38 (D38) and day 50 (D50). Comparing the organoids generated at D38 and D50 with iPS cells, clear separation was observed by PCA, indicating distinct differences in cell differentiation compared to iPS cells. Furthermore, differentially expressed genes (DEGs) identified by volcano plots showed high levels of upregulation of genes related to ectoderm and mesoderm lineages (Figure 6A, B).

[0107] Further annotation of cell fate within organoids via gene ontology (GO) in both D38 and D50 cells revealed significant upregulation of genes with GO pathways, such as vision, followed by pathways associated with nervous system development, neuronal differentiation, and axon guidance, and finally pathways associated with muscle organ development and cardiac development (Figure 6C). To investigate marker genes related to the above cell lineages, the expression of specific gene markers—pluripotency markers, visual (retinal) gene markers, brain-related gene markers, and muscle-related gene markers—was examined (log2 difference ≥ 1, p-value < 0.05). Pluripotency markers such as TDGF1, NANOG, SOX2, DNMT3B, OCT4 (POU5F1), and GDF3 were significantly reduced in both D38 and D50 cells. Among visual gene markers (retinal markers), PAX6, RPE-65, VSX1, MITF, and MSX2 were significantly upregulated in both D38 and D50 cells. Furthermore, photoreceptor-related marker genes such as RCVRN (rod), RGR, and RAX2 were also significantly upregulated. Among the gene markers related to brain development, forebrain-related genes, such as FOXG1, OTX1, DLX1, and VAX1, were found to be specifically upregulated in both D38 and D50. In addition, compared to GABA-related neuron gene markers, high upregulation was found in glutamate neuron-related gene markers (SLC1A7, SLC17A6, SLC17A7, etc.). Regarding muscle cell-related gene markers, significant upregulation was confirmed in both D38 and D50 for genes related to both skeletal muscle and cardiomyocytes, such as MYOG, MYH6, MYH7, MYOD1, PAX3, PAX7, TNNT3, and GATA4 (Figure 7). From the above results, it was shown that organoids containing ectoderm and mesoderm cells can be effectively produced by the manufacturing method of the present invention. [Industrial applicability]

[0108] The present invention is useful because it makes it possible to produce organoids containing ectodermal and mesodermal cells from cell aggregates containing pluripotent stem cells. Furthermore, the organoids containing ectodermal and mesodermal cells obtained by the production method of the present invention are expected to be useful for constructing specific disease models involving multiple cell types and for constructing screening systems for specific diseases. Moreover, by reproducing the in vivo environment using organoids containing ectodermal and mesodermal cells obtained by the production method of the present invention, it is expected that it will be possible to observe and study how signals are transmitted and interactions occur between cells in terms of time and space, which is expected to be useful for drug discovery and the development of regenerative medicine.

Claims

1. A method for producing organoids containing ectoderm cells and mesoderm cells, comprising the step (1) of culturing a cell aggregate containing pluripotent stem cells in suspension for at least two days in a medium containing a ROCK inhibitor and a TGFβ inhibitor.

2. The method according to claim 1, wherein the culture medium contains bone morphogenetic protein.

3. The method according to claim 1 or 2, further comprising step (2) of suspension culturing the cell aggregates cultured in step (1) in a medium containing a ROCK inhibitor and an extracellular matrix.

4. The method according to claim 3, further comprising step (3) of suspension culturing the cell aggregates cultured in step (2) in a medium containing a ROCK inhibitor.

5. The method according to claim 4, wherein the culture medium in step (3) contains retinoic acid.

6. The method according to any one of claims 1 to 5, wherein the culture medium in steps (1) to (3) is a serum-free culture medium.

7. The method according to any one of claims 1 to 6, wherein the ROCK inhibitor is Y-27632 and the TGFβ inhibitor is SB505124.

8. The method according to any one of claims 1 to 7, wherein the bone morphogenetic protein is BMP-4.

9. The method according to any one of claims 1 to 8, wherein the extracellular matrix is ​​a basement membrane preparation.

10. The method according to any one of claims 1 to 9, wherein the pluripotent stem cells are human pluripotent stem cells.

11. The method according to any one of claims 1 to 10, wherein the pluripotent stem cells are induced pluripotent stem cells.

12. An organoid comprising ectoderm cells and mesoderm cells produced by the manufacturing method described in any one of claims 1 to 11.

13. below: (i) Cells that are positive for at least one gene selected from the group consisting of PAX6, RPE-65, VSX1, VSX2, MITF, MSX2, RCVRN, RGR, and RAX2, (ii) Cells that are positive for at least one gene selected from the group consisting of FOXG1, OTX1, DLX1, VAX1, SLC1A7, SLC17A6, and SLC17A7, and (iii) Cells that are positive for at least one gene selected from the group consisting of MYOG, MYH6, MYH7, MYOD1, PAX3, PAX7, TNNT3, and GATA4. An organoid having [a certain characteristic].

14. An organoid having the following characteristics (1) to (3). (1) Having 1 to 5 layers on its surface containing cells expressing Tuj-1 (2) Inside the layer of (1), there are 1 to 50 layers containing cells that express at least one selected from the group consisting of VSX2, CRX, and Recoveryin. (3) Inside the layers of (2), there are 1 to 3 layers containing cells that express Myosin.

Images