Method for producing neural tissue

Abstract

To provide a method for producing a neural cell or a nerve tissue.SOLUTION: A method includes the following processes (1) to (3). (1) A first process in which a pluripotent stem cell is cultured in a culture medium including 1) a TGF β family signaling pathway inhibitory substance and / or a sonic hedgehog signaling pathway active substance, and 2) an undifferentiated maintaining factor, in the absence of a feeder cell; (2) a second process in which cells obtained in the first process are suspension-cultured to form an aggregate of cells; and (3) a third process in which an aggregate obtained in the second process is suspension-cultured in the presence or in the absence of a differentiation-inducing factor to obtain an aggregate including neural cells or a nerve tissue.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing neural tissue, such as retinal tissue, from pluripotent stem cells. [Background technology]

[0002] As a method for producing neural tissue such as retinal tissue from pluripotent stem cells, a method has been reported in which uniform aggregates of pluripotent stem cells are formed in a serum-free medium, which are then cultured in suspension, followed by culture in a differentiation-inducing culture medium in the presence of appropriate differentiation-inducing factors, etc., to induce differentiation of the pluripotent stem cells into the desired neural cells, thereby producing neural tissue (Patent Document 1 and Non-Patent Document 1). For example, there is a method for obtaining multi-layered retinal tissue from pluripotent stem cells (Non-Patent Document 2 and Patent Document 2), a method for producing neural tissue by culturing uniform aggregates of pluripotent stem cells in a serum-free medium, and a method for producing neural tissue by culturing uniform aggregates of pluripotent stem cells in suspension, which are then cultured in a differentiation-inducing culture medium in the presence of appropriate differentiation-inducing factors, etc. A method for obtaining multilayered retinal tissue is known (Non-Patent Documents 3 and 3), in which retinal cells are formed in a serum-free medium containing a transduction pathway inhibitor, followed by suspension culture in the presence of a basement membrane preparation and then suspension culture in a serum medium. Methods for inducing differentiation of pluripotent stem cells into hypothalamic tissue (Patent Document 4 and Non-Patent Document 4) and methods for inducing differentiation of pluripotent stem cells into neural progenitor cells (Non-Patent Documents 5 and 6) have also been reported. Pluripotent stem cells, which are the starting material for these production methods, have been cultured in the presence of feeder cells and under conditions where factors for maintaining undifferentiation are added, particularly in the case of primate pluripotent stem cells. In recent years, improvements have been made to the culture method for maintaining undifferentiation, and a method has been reported in which primate pluripotent stem cells are cultured in the absence of feeder cells (feeder-free) and under conditions where factors for maintaining undifferentiation are added (Non-Patent Documents 7, 8 and 9). Pluripotent stem cells cultured feeder-free using this method are used as the starting material to produce neural stem cells. There has been a strong demand for a method for stably producing cells or neural tissue. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2009 / 148170 [Patent Document 2] International Publication No. 2011 / 055855 [Patent Document 3] International Publication No. 2013 / 077425 [Patent Document 4] International Publication No. 2013 / 065763 [Non-patent literature]

[0004] [Non-Patent Document 1] Cell Stem Cell, 3, 519-32 (2008) [Non-patent document 2] Nature, 472, 51-56 (2011) [Non-patent document 3] Cell Stem Cell, 10(6), 771-775 (2012) [Non-patent document 4] Nature, 480, 57-62 (2011) [Non-Patent Document 5] Nature Biotechnology, 27(3), 275-80 (2009) [Non-patent document 6] Proc Natl Acad Sci USA, 110(50), 20284-9(2013) [Non-Patent Document 7] Nature Methods, 8, 424-429 (2011) [Non-patent document 8] Scientific Reports, 4, 3594 (2014) [Non-Patent Document 9] In Vitro Cell Dev Biol Anim., 46, 247-58(2010) Summary of the Invention [Problem to be solved by the invention]

[0005] The problem to be solved by the present invention is to provide a method for producing neural tissues such as retinal tissues or nervous system cells from pluripotent stem cells that have been prepared or cultured in an undifferentiated state in the absence of feeder cells. [Means for solving the problem]

[0006] The present inventors have conducted extensive research to solve the above problems, and have found that pluripotent stem cells can be cultured in the absence of feeder cells (under feeder-free conditions) by inhibiting the TGF-β family signaling pathway. We have found that by culturing cells in a medium containing toxic substances and / or substances acting on the Sonic hedgehog signaling pathway, followed by suspension culture, we can efficiently form cell aggregates that are round, have smooth surfaces, are densely packed inside, and maintain undifferentiated state. We have also found that these high-quality cell aggregates can be used to efficiently induce neural tissues such as retinal tissue and neural cells, leading to the completion of the present invention. That is, the present invention relates to the following.

[0007] [1] A method for producing nervous system cells or nervous tissue, comprising the following steps (1) to (3): (1) Pluripotent stem cells were cultured in the absence of feeder cells, and 1) TGFβ family signaling pathway was Pathway inhibitors and / or Sonic Hedgehog signaling pathway agonists, and 2) untreated a first step of culturing in a medium containing a differentiation maintenance factor; (2) a second step of culturing the cells obtained in the first step in suspension to form cell aggregates; and (3) The aggregates obtained in the second step are cultured in suspension in the presence or absence of differentiation-inducing factors. and a third step of obtaining aggregates containing nervous system cells or nervous tissue. [2] In the second step, the cells obtained in the first step are dispersed, and the dispersed cells are cultured in suspension. The manufacturing method described in [1]. [3] The method of producing according to [1] or [2], wherein the factor for maintaining undifferentiation is a substance acting on the FGF signaling pathway. [4] The method according to [3], wherein the substance acting on the FGF signaling pathway is bFGF. [5] In the second step, the cells are treated with a sonic hedgehog signaling pathway agonist. The production method according to any one of [1] to [4], characterized in that the cells are cultured in suspension in a serum-free medium containing the bacterium. [6] In the third step, the aggregates are cultured in suspension in the presence of a differentiation-inducing factor. The manufacturing method according to any one of [1] to [5]. [7] TGFβ family signaling pathway inhibitors inhibit the Nodal / Activin signaling pathway. The method for producing the compound according to any one of [1] to [6], wherein the compound is a toxic substance, a TGFβ signaling pathway inhibitor, or a BMP signaling pathway inhibitor. [8] The method of any one of [1] to [7], wherein the TGFβ family signaling pathway inhibitor is Lefty, SB431542, A-83-01, or LDN193189. [9] The method of any one of [1] to [8], wherein the sonic hedgehog signaling pathway active substance is Shh, SAG, or Purmorphamine.

[10] The method of any one of [1] to [9], wherein the differentiation-inducing factor in the third step is a substance acting on the BMP signaling pathway.

[11] The BMP signaling pathway active substance is selected from the group consisting of BMP2, BMP4, BMP7, and GDF7. The method for producing a protein according to

[10] , wherein the protein is one or more proteins.

[12] The method according to

[10] , wherein the BMP signaling pathway active substance is BMP4.

[13] A manufacturing method described in any one of [1] to

[0012] , wherein the aggregate obtained in the third step is an aggregate containing retinal tissue.

[14] The aggregate obtained in the third step is selected from the group consisting of retinal progenitor cells, neural retinal progenitor cells, photoreceptor progenitor cells, photoreceptor cells, rod photoreceptor cells, cone photoreceptor cells, horizontal cells, amacrine cells, interneuron cells, ganglion cells, retinal pigment epithelial cells, and ciliary marginal cells. The method according to any one of [1] to

[13] , wherein the aggregate contains multiple cells.

[15] The differentiation-inducing factor in the third step is a substance acting on the BMP signaling pathway, and The method according to

[13] or

[14] , wherein the medium used in the process contains a substance acting on the sonic hedgehog signaling pathway.

[16] The pluripotent stem cells are human pluripotent stem cells, and the concentration of the Sonic Hedgehog signaling pathway activator in the medium in the second step is 10 nM to 700 nM SAG.

[15] A manufacturing method described in

[15] , wherein the concentration is equivalent to the signal transmission effect, and the retinal tissue is human retinal tissue.

[17] In the third step, the BMP signaling pathway was activated between day 1 and day 9 after the start of the second step. The method according to

[15] or

[16] , wherein the active substance is added to the culture medium.

[18] A manufacturing method described in

[13] or

[14] , wherein in the third step, the aggregates are cultured in a medium in which the concentration of the Sonic Hedgehog signaling pathway active substance is equal to or lower than the concentration corresponding to the Sonic Hedgehog signaling activity of SAG 700 nM.

[19] The differentiation-inducing factor in the third step is a TGF-β family signaling pathway inhibitor and The method according to any one of [1] to [9], wherein the compound is a compound that inhibits the Wnt signaling pathway and / or a substance that inhibits the Wnt signaling pathway.

[20] The manufacturing method according to

[19] , wherein the TGFβ family signaling pathway inhibitor is Lefty, SB431542, A-83-01 or LDN193189.

[21] The method according to

[19] or

[20] , wherein the Wnt signaling pathway inhibitor is IWR-1-endo. Manufacturing method.

[22] The method of any one of [1] to [9] or

[19] to

[21] , wherein the aggregate obtained in the third step is an aggregate containing cerebral tissue.

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

[22] , wherein the culture time in the first step is 0.5 hours to 144 hours.

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

[23] , wherein the first step is carried out by an adherent culture method.

[25] The method of any one of [1] to

[24] , wherein in the third step, a differentiation inducer is added to the medium between the third and sixth days after the start of the second step.

[26] The pluripotent stem cells are primate pluripotent stem cells, [1]-

[15] and

[17] -

[25] . The manufacturing method according to any one of the preceding claims.

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

[26] , wherein the pluripotent stem cells are human pluripotent stem cells. method.

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

[27] , wherein the pluripotent stem cells are induced pluripotent stem cells. method.

[29] In the second step, a uniform aggregate is formed, as described in any one of [1] to

[28] . Manufacturing method.

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

[29] , wherein the suspension culture is carried out in the absence of a basement membrane preparation. Manufacturing method.

[31] Nervous system cells or nervous tissue produced by the method according to any one of [1] to

[30] . A reagent for evaluating the toxicity and efficacy of a test substance, comprising:

[32] Nervous system cells or neural tissues produced by the method according to any one of [1] to

[30] . A method for evaluating the toxicity and efficacy of a substance, comprising contacting a test substance with a cell or tissue and examining the effect of the substance on the cell or tissue.

[33] Nervous system cells or nervous tissue produced by the method according to any one of [1] to

[30] . A therapeutic agent for a disease caused by a disorder of nervous system cells or nervous tissue, comprising:

[34] The therapeutic agent according to

[33] , wherein the nervous system cells or nervous tissue are retinal progenitor cells, retinal layer-specific neurons, retinal tissue, cerebral nervous system progenitor cells, cerebral layer-specific neurons, or cerebral tissue.

[35] Nervous system cells or nervous tissue produced by the method according to any one of [1] to

[30] . A method for treating a disease caused by a disorder of nervous system cells or nervous tissue, comprising transplanting an effective amount of the compound of formula (I) into a subject in need of transplantation.

[36] For use in the treatment of diseases based on disorders of nervous system cells or nervous tissue,[1] A nervous system cell or nervous tissue produced by the method described in any one of

[30] to

[30] .

[37] Nervous system cells or nervous tissue produced by the method according to any one of [1] to

[30] . A pharmaceutical composition comprising as an active ingredient. [Effects of the Invention]

[0008] According to the present invention, it is possible to efficiently produce high-quality cell aggregates, as well as neural tissues such as retinal tissues and nervous system cells, from pluripotent stem cells cultured in the absence of feeder cells. [Brief explanation of the drawings]

[0009] [Figure 1] 1 shows the culture conditions in Example 1 and the morphology of human iPS cells after culture. [Figure 2] 1 shows the culture conditions of Example 2, and a bright field image of the cells after culture and an immunohistochemical staining image for Oct3 / 4. [Figure 3] Bright field images of the aggregates (A and B) and a graph quantifying the degree of aggregate morphology (C) are shown. [Figure 4] A bright field image of the aggregates is shown. [Figure 5] Bright-field images of the aggregates (A and B) and immunohistochemical staining of the aggregates for neural tissue markers (nestin, TuJ1, PSA-NCAM) (CE) are shown. [Figure 6] 1 shows the culture conditions of Example 7 and immunohistochemical staining images (AD) of aggregates for retinal tissue markers (Chx10, Rx). [Figure 7] 1 shows the culture conditions of Example 8, bright field images of aggregates after culture (AH), and a graph (I) quantifying the degree of aggregate morphology. [Figure 8]Immunohistochemical staining of aggregates for neural tissue markers (nestin, TuJ1, PSA-NCAM) is shown. [Figure 9] 10 shows the culture conditions of Example 9, and immunohistochemical staining images (AH) of aggregates for retinal tissue markers (Chx10, Rx). [Figure 10] Bright field images of aggregates formed under various conditions are shown. [Figure 11] 1 shows the culture conditions of Example 11, and immunohistochemical staining images (AF) of aggregates for neural tissue markers (nestin, TuJ1, PSA-NCAM). [Figure 12] 1 shows the culture conditions of Example 12, and immunohistochemical staining images (AD) of aggregates for retinal tissue markers (Chx10, Rx). [Figure 13] 1 shows the culture conditions of Example 13, and immunohistochemical staining images (AD) of aggregates for Chx10. [Figure 14] 1 shows the culture conditions of Example 14, bright-field images of aggregates, and immunohistochemical staining images of aggregates for retinal tissue markers (Chx10, Rx). [Figure 15] 1 shows the culture conditions of Example 15 and bright field images (AD) of the cells after culture. [Figure 16] 1 shows the culture conditions of Example 16, bright field images of the cells after culture (A, B), and an immunohistochemical staining image of Chx10 aggregates (C). [Figure 17] 1 shows the culture conditions of Example 17 and bright field images (AF) of the cells after culture. [Figure 18] 1 shows the culture conditions of Example 18, a bright field image (AE) of the cells after culture, and an immunohistochemical staining image (FI) of the aggregates for Chx10. [Figure 19] 1 shows the culture conditions of Example 19, bright field images of cells after culture (AD), and immunohistochemical staining images of aggregates for Chx10 (EH). [Figure 20] 1 shows the culture conditions of Example 20 and bright field images (AJ) of the cells after culture. [Figure 21] 1 shows the culture conditions of Example 21 and a bright field image (AL) of the cells after culture. [Figure 22] 1 shows the culture conditions of Example 22, a bright field image (AC) of the cells after culture, and an immunohistochemical staining image (DF) of aggregates for Chx10. [Figure 23] 1 shows the culture conditions of Example 23, bright-field images of cells after culture (A, B), and immunohistochemical staining images of Chx10 aggregates (C, D). [Figure 24] 1 shows the culture conditions of Example 24 and bright field images (AD) of the cells after culture. [Figure 25] 1 shows the culture conditions of Example 25 and bright field images (AD) of cells after culture. [Figure 26] 1 shows the culture conditions of Example 26, a bright field image (AE) of the cells after culture, and an immunohistochemical staining image (FI) of the aggregates for Chx10. [Figure 27] 1 shows the culture conditions of Example 27, a culture image of human ES cells (A), a bright field image of the cells after culture (BD), and fluorescent images of Rx::GFP (E, F). [Figure 28] Immunohistochemical staining images (AF) of aggregates for retinal tissue markers (Rx, Chx10, Crx, Blimp1, Brn3b) and Ki67 are shown. [Figure 29] Immunohistochemical staining images (AH) of the aggregates for retinal tissue markers (calretinin, S-opsin, Rx, Pax6, recoverin, rhodopsin, NRL, calbindin) are shown. [Figure 30] Bright-field images of the cells after culture (A, B) and immunohistochemical staining images of the aggregates for retinal tissue markers (SSEA1, Mitf, Aqp1) (CE) are shown. [Figure 31] 1 shows the culture conditions of Example 31 and bright field images (AC) of the cells after culture. [Figure 32] Immunohistochemical staining of aggregates for cerebral tissue markers (Sox2, FoxG1, Pax6, Tbr1, Ctip2) is shown (AF). [Figure 33] Bright field images (AJ) of cells cultured under various conditions are shown. [Figure 34]1 shows the culture conditions of Example 33, bright-field images of cells after culture (A, B), and immunohistochemical staining images of Chx10 aggregates (C, D). [Figure 35] 1 shows the culture conditions of Example 34, bright field images of cells after culture (AD), and immunohistochemical staining images of aggregates for Chx10 (EH). [Figure 36] 1 shows the culture conditions of Example 35, a bright field image (AE) of the cells after culture, and an immunohistochemical staining image (FI) of the aggregates for Chx10. DETAILED DESCRIPTION OF THE INVENTION

[0010] 1.Definition In the present invention, "stem cells" refer to undifferentiated cells that have differentiation potential and proliferation potential (particularly self-renewal potential) while maintaining differentiation potential. Stem cells include subpopulations such as pluripotent stem cells, multipotent stem cells, and unipotent stem cells, depending on their differentiation potential. Pluripotent stem cells are cells that can be cultured in vitro. Stem cells are stem cells that can be cultivated and have the ability (pluripotency) to differentiate into all cell lineages belonging to the three germ layers (ectoderm, mesoderm, and endoderm) and / or extraembryonic tissues. Multipotent stem cells refer to stem cells that have the ability to differentiate into multiple types of tissues and cells, but not all types. Unipotent stem cells refer to stem cells that have the ability to differentiate into specific tissues and cells.

[0011] Pluripotent stem cells can be derived from fertilized eggs, cloned embryos, germline stem cells, tissue stem cells, somatic cells, etc. Pluripotent stem cells include embryonic stem cells (ES cells). ), Embryonic germ cells (EG cells), induced pluripotent stem cells (iPS cells), etc. Muse cells (Multi-lineage differentiating stress enduring cells) obtained from embryonic stem cells and GS cells produced from germ cells (e.g., testes) are also included in pluripotent stem cells. Embryonic stem cells were first established in 1981, and have been used to produce knockout mice since 1989. Human embryonic stem cells were established in 1998, and are also beginning to be used in regenerative medicine. ES cells can be produced by culturing inner cell mass on feeder cells or in a medium containing LIF. Methods for producing ES cells are described in, for example, WO96 / 22362, WO02 / 101057, US5,843,780, US6,200,806, US6,280,718, etc. Embryonic stem cells can be obtained from designated institutions, and also commercially available products can be purchased. For example, human embryonic stem cells KhES-1, KhES-2, and KhES-3 are available from the Institute for Frontier Medical Sciences, Kyoto University. Human embryonic stem cells Rx::GFP strain (derived from KhES-1) are available from the National Institute of Physical and Chemical Research. In either case, EB5 cells are mouse embryonic stem cells, and the D3 line is from the National Institute of Physical and Chemical Research (RIKEN). It is available from

[0012] Nuclear transfer ES cells (ntES cells), a type of ES cell, can be established from cloned embryos created by transplanting the nucleus of a somatic cell into an egg from which the cell line has been removed.

[0013] EG cells are produced by culturing primordial germ cells in a medium containing mSCF, LIF, and bFGF. (Cell, 70: 841-847, 1992).

[0014] In the present invention, "induced pluripotent stem cells" refer to cells in which pluripotency is induced by reprogramming somatic cells using known methods. Specifically, fibroblasts and peripheral blood mononuclear cells differentiated into somatic cells by activating 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. Examples of such a combination of reprogramming factors include cells in which pluripotency has been induced by reprogramming through the expression of any of the following combinations: (1) Oct3 / 4, Sox2, Klf4, and Myc (c-Myc or L-Myc), and (2) Oct3 / 4, Sox2, Klf4, Lin28, and L-Myc (Stem Cells, 2013;31:458-466). It can be done. In 2006, Yamanaka et al. established induced pluripotent stem cells using mouse cells (Cell, 2006, 126(4), pp.663-676). In 2007, induced pluripotent stem cells were also established using human fibroblasts, and these cells possess the same pluripotency and self-renewal capabilities as embryonic stem cells (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 through gene expression, induced pluripotent stem cells can also be induced from somatic cells by adding chemical compounds (Science, 2013, 341, pp. 651-654). It is also possible to obtain established 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, which are available from Kyoto University and iPS Academia Japan, Inc. Established induced pluripotent stem cell lines, for example, Ff-I01 cells and Ff-I14 cells established at Kyoto University, are available from Kyoto University.

[0015] Somatic cells used in producing induced pluripotent stem cells are not particularly limited, but include tissue-derived fibroblasts, blood cells (e.g., peripheral blood mononuclear cells and T cells), liver cells, pancreatic cells, Examples include intestinal epithelial cells and smooth muscle cells.

[0016] When producing induced pluripotent stem cells, if reprogramming is performed by expressing several types of genes, the means for expressing the genes is not particularly limited. Examples of such means include infection methods using viral vectors (e.g., retroviral vectors, lentiviral vectors, Sendai virus vectors, adenoviral 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), and gene transfer methods using RNA vectors (e.g., calcium phosphate method, lipofection method, retronectin method, electroporation method). Examples of methods include direct injection of proteins, transfection, electroporation, and direct injection of proteins.

[0017] Induced pluripotent stem cells can be produced in the presence or absence of feeder cells (feeder-free). When producing induced pluripotent stem cells in the presence of feeder cells, they can be produced in the presence of undifferentiated maintenance factors by known methods. The medium used to produce induced pluripotent stem cells in the absence of feeder cells is not particularly limited, and known maintenance media for embryonic stem cells and / or induced pluripotent stem cells, or media for establishing induced pluripotent stem cells in a feeder-free environment can be used. Examples of feeder-free establishment media for establishing induced pluripotent stem cells in a feeder-free environment include, for example, Essential 8 medium, TeSR medium, mTeSR medium, mTeSR-E8 medium, StemFit medium, and other feeder-containing medium. When producing induced pluripotent stem cells, for example, induced pluripotent stem cells can be produced by introducing four factors, Oct3 / 4, Sox2, Klf4, and Myc, into somatic cells using a Sendai virus vector in the absence of feeder cells.

[0018] The pluripotent stem cells used in the present invention are preferably ES cells or induced pluripotent stem cells, more preferably induced pluripotent stem cells.

[0019] Examples of multipotent stem cells include tissue stem cells (also called tissue stem cells, tissue-specific stem cells, or somatic stem cells) such as hematopoietic stem cells, neural stem cells, retinal stem cells, and mesenchymal stem cells.

[0020] Genetically modified pluripotent stem cells can be produced, for example, by using homologous recombination techniques. Examples of genes on chromosomes that can be modified include cell marker genes, histocompatibility antigen genes, and disease-related genes due to disorders of nervous system cells. Modification of target genes on chromosomes can be performed using the methods described in "Manipulating the Mouse Embryo, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994)" and "Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993); Biomanual Series 8, Gene targeting, Creation of mutant mice using ES cells, Yodosha (1995), etc. This can be done using the methods described.

[0021] Specifically, for example, genomic DNA containing the target gene to be modified (e.g., a cell marker gene, a gene for a histocompatibility antigen, or a disease-related gene) is isolated, and a target vector for homologous recombination of the target gene is prepared using the isolated genomic DNA. The prepared target vector is introduced into stem cells, and cells in which homologous recombination has occurred between the target gene and the target vector are selected, thereby producing stem cells in which a gene on the chromosome has been modified.

[0022] Methods for isolating genomic DNA containing a target gene include those 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 containing the target gene can be isolated by using a genomic DNA library screening system (Genome Systems) or Universal GenomeWalker Kits (CLONTECH). Instead of genomic DNA, a polynucleotide encoding the target protein can be used. The polynucleotide can be used to isolate the corresponding target protein by PCR. The polynucleotide can be obtained by amplifying the corresponding polynucleotide.

[0023] For details on the construction of target vectors for homologous recombination of target genes and the efficient selection of homologous recombinants, see Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993); Biomanual Series 8, Gene Targeting, Using ES Cells. This can be done according to the method described in "Creation of Mutant Mice with Transformed Genes," Yodosha (1995); etc. Either a replacement or insertion type target vector can be used. Selection methods include positive selection, promoter selection, negative selection, and polyA selection. One method for selecting the desired homologous recombinants from the selected cell lines is to use genomic DNA. Examples of such methods include Southern hybridization and PCR.

[0024] "Mammals" in the present invention include rodents, ungulates, felines, primates, etc. Rodents include mice, rats, hamsters, guinea pigs, etc. Ungulates include pigs, cows, goats, horses, sheep, etc. Felidae include dogs, cats, etc. "Primates" in the present invention refer to mammals belonging to the order Primates, and examples of primates include the suborder Prosimians such as lemurs, lorises, and tree shrews, and the suborder Anthropoidea such as monkeys, apes, and humans.

[0025] The pluripotent stem cells used in the present invention are mammalian pluripotent stem cells, preferably rodent pluripotent stem cells. (e.g., mouse, rat) or primate (e.g., human, monkey) pluripotent stem cells, more preferably human pluripotent stem cells, even more preferably human induced pluripotent stem cells (iPS cells) or human embryos. They are embryonic stem cells (ES cells).

[0026] In the present invention, "suspension culture" or "suspension culture method" refers to culturing cells or cell aggregates while maintaining a state in which they exist suspended in a culture solution, and the method of culturing. In other words, suspension culture is carried out under conditions that do not allow cells or cell aggregates to adhere to cultureware, etc., and culture carried out under conditions that allow cells or cell aggregates to adhere to cultureware, etc. (adhesion culture or adhesion culture method) is not included in the category of suspension culture. In this case, cell adhesion means the formation of strong cell-substratum junctions between the cells or cell aggregates and the cultureware. More specifically, suspension culture refers to culture under conditions that do not allow strong cell-substrate bonds to form between cells or cell aggregates and cultureware, etc., and adhesion culture refers to culture under conditions that do not allow strong cell-substrate bonds to form between cells or cell aggregates and cultureware, etc. Cultivation is carried out under conditions that allow strong cell-substrate bonds to form between the cell aggregates and the cultureware. cormorant.

[0027] In cell aggregates during suspension culture, cells adhere to each other. In cell aggregates during suspension culture, cell-substrate bonds are hardly formed between the cultureware or are poorly formed. In some aspects, in aggregates of cells in suspension culture, endogenous cell-substrate bonds are present inside the aggregates, but cell-substrate bonds are hardly formed between the aggregates and the cultureware, or even if they are formed, their contribution is small.

[0028] Plane attachment between cells means that cells adhere to each other on a surface. More specifically, the surface adhesion between cells means that the proportion of the surface area of ​​a cell that is adhered to the surface of another cell is, for example, 1% or more, preferably 3% or more, and more preferably 5%. The cell surface can be stained with a membrane staining reagent (e.g., DiI) or This can be observed by immunostaining for cell adhesion factors (e.g., E-cadherin and N-cadherin).

[0029] The culture vessel used for suspension culture is not particularly limited as long as it is capable of "suspension culture," and those skilled in the art can appropriately determine the appropriate vessel. Examples of such vessels include flasks, tissue culture flasks, culture 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, spinner flasks, Erlenmeyer flasks, and roller bottles. These culture vessels are preferably non-cell-adhesive to enable suspension culture. Examples of non-cell-adhesive culture vessels that can be used include those whose surfaces have not been artificially treated to improve cell adhesion (e.g., coating with basement membrane preparations, extracellular matrices such as laminin, entactin, collagen, and gelatin, or polymers such as polylysine and polyornithine, or surface treatments such as positive charge treatment). Non-cell-adhesive culture vessels have surfaces that have been artificially treated to reduce cell adhesion (for example, ultra-hydrophilic treatment with MPC polymer, low protein adsorption treatment, etc.). Rotational culture may be performed using a spinner flask, roller bottle, etc. The culture surface of the incubator may be flat or uneven.

[0030] The culture vessel used for adhesion culture is not particularly limited as long as it is capable of "adherent culture," and a person skilled in the art can select an appropriate culture vessel depending on the scale, culture conditions, and culture period of the culture. Examples of such culture vessels include flasks, tissue culture flasks, culture dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, chamber slides, petri dishes, tubes, trays, culture bags, microcarriers, beads, stack plates, spinner flasks, and roller bottles. These culture vessels can be used in a variety of ways. The culture vessel is preferably cell-adhesive to enable adherent culture. Examples of cell-adhesive culture vessels include those whose surfaces have been artificially treated to improve cell adhesion, specifically surface-treated culture vessels or culture vessels whose interiors are coated with a coating agent. Examples of coating agents include laminin [laminin laminin α5β1γ1 (hereinafter referred to as laminin 511), laminin α1β1γ1 (hereinafter referred to as laminin 111), etc., and laminin fragments (such as laminin 511E8)], entactin, collagen, gelatin, vitronectin, Synthemax (Corning), Matrigel, and other extracellular matrices. Examples of the surface-treated culture vessel include culture vessels whose surface has been treated by positive charge treatment or the like.

[0031] In the present invention, the medium used for cell culture can be prepared using a medium commonly used for culturing animal cells as a basal medium. Examples of basal media include BME medium and BGJb medium. CMRL 1066 medium, Glasgow MEM (GMEM) medium, Improved MEM Zinc Option medium, IMDM medium Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, F-12 medium, DMEM / F12 medium, IMDM / F12 medium, Ham's medium, RPMI 1640 medium, Fischer's medium, or a mixture of these mediums, etc. Examples of media include media that can be used for culturing animal cells.

[0032] In the present invention, "serum-free medium" refers to a medium that does not contain unconditioned or unpurified serum. In the present invention, media containing purified blood-derived components or animal tissue-derived components (e.g., growth factors) are also included in the serum-free medium as long as they do not contain unconditioned or unpurified serum.

[0033] The serum-free medium may contain a serum substitute, such as albumin, transferrin, fatty acids, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, or equivalents thereof. Such serum substitutes can be prepared, for example, by the method described in WO98 / 30679. Commercially available serum substitutes may be used. Examples of such commercially available serum substitutes include Knockout TM Serum Replacement (Life Technologies, now ThermoFisher; hereinafter sometimes referred to as KSR), Chemically-defined Lipid concentrated (Life Technologies), Glutamax TM (manufactured by Life Technologies), B27 (manufactured by Life Technologies), and N2 Supplement (manufactured by Life Technologies).

[0034] The serum-free medium used in suspension culture may contain, as appropriate, fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, inorganic salts, etc.

[0035] To avoid the complicated preparation process, we used the commercially available KSR (LifeTechno) serum-free medium. A suitable amount (for example, about 0.5% to about 30%, preferably 10 cells in a serum-free medium (e.g., a 1:1 mixture of F-12 medium and IMDM medium) supplemented with approximately 1% to 20% of the normal serum. Alternatively, a medium containing 100% KSR (chemically-defined lipid concentrated) and 450 μM 1-monothioglycerol may be used. Examples of a medium equivalent to KSR include the medium disclosed in JP-A-2001-508302.

[0036] In the present invention, the term "serum medium" refers to a medium containing unconditioned or unpurified serum. The medium may contain fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, 1-monothioglycerol, pyruvic acid, buffers, inorganic salts, etc. For example, when a basement membrane preparation such as Matrigel is used to induce differentiation of pluripotent stem cells into retinal tissue, etc., a serum medium can be used (Cell Stem Cell, 10(6), 771-775 (2012)). In addition, the neural tissue produced by the present invention can be used in the differentiation of pluripotent stem cells into retinal tissue, etc. A serum-containing medium can be used in the process of maintaining (also called maturation culture) tissues (e.g., retinal tissue, cerebral tissue).

[0037] In the present invention, the culture is preferably carried out under xeno-free conditions. "Xeno-free" refers to conditions under which components derived from organisms different from the organism species of the cells to be cultured are excluded.

[0038] In the present invention, "a medium containing substance X" and "in the presence of substance X" refer to exogenous a medium containing exogenous substance X or a medium containing exogenous substance X, or in the presence of exogenous substance X In other words, if the cells or tissues present in the culture medium express, secrete, or produce the substance X endogenously, the endogenous substance X can be distinguished from exogenous substance X. A medium that does not contain exogenous substance X is considered to contain "substance X" even if it contains endogenous substance X. It is understood that this does not fall under the category of "culture medium."

[0039] For example, the term "medium containing a TGFβ family signaling pathway inhibitor" refers to a medium to which an exogenous TGFβ family signaling pathway inhibitor has been added or an exogenous TGFβ family -A medium containing a signal transduction pathway inhibitor.

[0040] In the present invention, feeder cells refer to cells other than stem cells that are allowed to coexist when the stem cells are cultured. Examples of feeder cells used in the culture of pluripotent stem cells to maintain their undifferentiated state include mouse fibroblasts (MEF, etc.), human fibroblasts, and SNL cells. Feeder cells that have been subjected to growth inhibition treatment are preferred. Growth inhibition treatments include treatment with a growth inhibitor (e.g., mitomycin C), gamma ray irradiation, or UV irradiation. Feeder cells used in the culture of pluripotent stem cells to maintain the undifferentiated state contribute to maintaining the undifferentiated state of pluripotent stem cells by secreting humoral factors (preferably factors for maintaining the undifferentiated state) and by creating a scaffold (extracellular matrix) for cell adhesion.

[0041] In the present invention, "feeder-free" refers to culturing in the absence of feeder cells. Examples of "feeder-free" include conditions in which no feeder cells are added, or conditions in which feeder cells are substantially absent (for example, the ratio of feeder cells to the total number of cells is 3% or less).

[0042] In the present invention, the term "aggregate" refers to a cell aggregate formed by cells dispersed in a medium. A mass formed by the aggregation of cells, in which cells adhere to each other. Cell masses, embryoid bodies, spheres, and spheroids are also included in cell aggregates. Preferably, in the cell aggregate, the cells are in surface adhesion with each other. In some embodiments, the cells are in cell-cell adhesion with each other in part or all of the aggregate. Specifically, the "aggregates" of the present invention include aggregates formed by cells that were dispersed at the start of suspension culture, which are generated in the second step of the present invention [1] above, and / or cell adhesions, such as adherens junctions. Neural cells induced to differentiate from pluripotent stem cells, which are produced in the third step of the present invention [1]. The term "aggregate" includes aggregates containing the above-mentioned granules at the start of the second step of the present invention [1] (as of This includes aggregates that were already formed at the beginning of the suspension culture (i.e., at the start of the suspension culture). The cell aggregates generated in the second step include "embryoid bodies" (EBs).

[0043] In the present invention, "uniform aggregates" means that when multiple aggregates are cultured, the size of each aggregate is constant, and when the size of an aggregate is evaluated by the length of its maximum diameter, a uniform aggregate means that the variance of the length of its maximum diameter is small. More specifically, 75% or more of the aggregates in the entire population of aggregates are within ±100% of the average maximum diameter of the population of aggregates. It preferably means that the value is within the range of the mean value ±50%, and more preferably within the range of the mean value ±20%.

[0044] In the present invention, "forming uniform aggregates" means a process of aggregating cells to form cell aggregates. This refers to the formation of uniformly sized cell aggregates by "rapidly aggregating a certain number of dispersed cells" when forming a suspension culture.

[0045] Dispersion is the process of breaking down cells and tissues into small cell fragments (2-cell fragments) by dispersing them using enzyme treatment, physical treatment, etc. The term "separation" refers to the separation of a cell population to a number of cells (more than 100 cells but less than 100 cells, preferably less than 50 cells) or to a single cell population. A certain number of dispersed cells refers to a collection of a certain number of cell fragments or single cells.

[0046] Methods for dispersing pluripotent stem cells include, for example, mechanical dispersion treatment, cell dispersion treatment, and cell protective agent addition treatment. These treatments may be performed in combination. Preferably, cell dispersion treatment is performed first, followed by mechanical dispersion treatment.

[0047] Methods for mechanical dispersion include pipetting and scraping with a scraper.

[0048] The cell dispersion liquid used for the cell dispersion liquid treatment may contain, for example, enzymes such as trypsin, collagenase, hyaluronidase, elastase, pronase, DNase, or papain, or enzymes such as erythritol, PEG, or PEG-4. Examples of suitable cell dispersion solutions include solutions containing chelating agents such as ethylenediaminetetraacetic acid, etc. Commercially available cell dispersion solutions, such as TrypLESelect (manufactured by Life Technologies) and TrypLEExpress (manufactured by Life Technologies), can also be used.

[0049] When dissociating the pluripotent stem cells, cell death of the pluripotent stem cells may be suppressed by treating them with a cytoprotective agent. Examples of the cytoprotective agent used for the cytoprotective agent treatment include FGF signal transduction inhibitors. These include receptor agonists, heparin, IGF signaling pathway agonists, serum, or serum replacement. Furthermore, to suppress cell death induced by dissociation (particularly cell death of human pluripotent stem cells), a Rho-associated coiled-coil kinase (ROCK) inhibitor or a myosin inhibitor may be added during dissociation. Examples of ROCK inhibitors include Y-27632, Fasudil (HA1077), and H-1152. Examples of myosin inhibitors include Blebbistatin. Preferred cytoprotective agents include ROCK inhibitors.

[0050] For example, as a method for dispersing pluripotent stem cells, a colony of pluripotent stem cells is treated with a cell dispersion solution (TrypLE Select) in the presence of a ROCK inhibitor as a cell protective agent, and then pipetted. For example, dispersion may be performed by coating.

[0051] In the production method of the present invention, it is preferable to rapidly aggregate pluripotent stem cells to form pluripotent stem cell aggregates. When pluripotent stem cell aggregates are formed in this manner, epithelial-like structures can be reproducibly formed in cells induced to differentiate from the formed aggregates. Experimental procedures for forming aggregates include, for example, using a small well plate (for example, a well with a bottom area of ​​0.1 to 2.0 cm2 in terms of a flat bottom). 2 One method is to confine cells in a small space using a micropore or a small centrifuge tube, and to aggregate the cells by centrifugation for a short period of time. 2 48-well plate (approximately 1.0 cm2 in flat-bottom equivalent area) 2 96-well plate (area equivalent to a flat bottom is 0.35 cm 2 Inner diameter: 6-8mm 384-well plate, and preferably 96-well plate. As for the shape of the small well plate, the shape of the bottom when the well is viewed from above may be polygonal, rectangular, elliptical or circular, preferably circular. As for the shape of the small well plate, the shape of the bottom when the well is viewed from the side may be a flat bottom structure or a structure with a high outer periphery and a low concave inner recess. As for the shape of the bottom, for example, a U-bottom, a V-bottom or an M-bottom may be mentioned, preferably a U-bottom or a V-bottom, more preferably a V-bottom. As the small well plate, a cell culture dish (for example, a 60 mm to 150 mm dish, a culture flask, etc.) may be mentioned. Plates with uneven or indented bottoms may also be used. The bottom surface of the plate is preferably a non-cell-adhesive bottom surface, preferably a bottom surface coated with a non-cell-adhesive material.

[0052] The formation and uniformity of aggregates of pluripotent stem cells or cell populations containing pluripotent stem cells can be determined based on the size and cell number of the aggregates, their macroscopic morphology, their microscopic morphology and uniformity determined by histochemical staining analysis, etc. Furthermore, the formation and uniformity of epithelial-like structures in the aggregates can be determined based on the macroscopic morphology of the aggregates, their microscopic morphology and uniformity determined by histochemical staining analysis, the expression and uniformity of differentiated and undifferentiated markers, the control and synchrony of expression of differentiation markers, and the reproducibility of differentiation efficiency between aggregates, etc.

[0053] In the present invention, "tissue" refers to a structure of a cell population having a structure in which one type of cell with uniform morphology and properties, or multiple types of cells with different morphologies and properties, are arranged three-dimensionally in a certain pattern.

[0054] In the present invention, "nervous tissue" refers to tissue composed of nervous system cells, such as the cerebrum, midbrain, cerebellum, spinal cord, retina, peripheral nerves, forebrain, hindbrain, telencephalon, diencephalon, etc., at the developmental or adult stage. Nervous tissue may form a stratified epithelial structure (neuroepithelium), and the amount of neuroepithelium present in cell aggregates can be evaluated by bright-field observation using an optical microscope.

[0055] In the present invention, "neural cells" refer to epidermal cells among ectoderm-derived tissues. The term "neural cells" refers to cells other than neural cells. Specifically, it includes cells such as neural progenitor cells, neurons (nerve cells), glia, neural stem cells, neuronal progenitor cells, and glial progenitor cells. Neural cells also include cells that constitute retinal tissue (retinal cells), retinal progenitor cells, retinal layer-specific neurons, neural retinal cells, and retinal pigment epithelial cells, as described below. Neural cells can be identified using markers such as nestin, TuJ1, PSA-NCAM, and N-cadherin.

[0056] Neurons (neuron / neuronal cells) are functional cells that form neural circuits and contribute to signal transmission. These cells can be identified using the expression of immature neuron markers such as TuJ1, Dcx, and HuC / D, and / or mature neuron markers such as Map2 and NeuN.

[0057] Examples of glia include astrocytes, oligodendrocytes, and Müller glia. Examples of astrocyte markers include GFAP, oligodendrocyte markers include O4, and Müller glia markers include CRALBP.

[0058] Neural stem cells are cells that have the ability to differentiate into neurons and glial cells (pluripotency) and the ability to proliferate while maintaining pluripotency (also known as self-renewal). Markers of neural stem cells include Nestin, Sox2, Musashi, Hes family, and CD133. KA is a marker for all progenitor cells and is not considered to be a neural stem cell-specific marker. The number of neural stem cells can be assessed by neurosphere assays, clonal assays, etc.

[0059] Neuronal progenitor cells are cells that have the ability to proliferate, produce neurons, but do not produce glial cells. Neuronal progenitor cell markers include Tbr2 and Tα1. Alternatively, cells that are positive for immature neuronal markers (TuJ1, Dcx, HuC / D) and proliferation markers (Ki67, pH3, MCM) can be identified as neuronal progenitor cells.

[0060] Glial progenitor cells are cells that have the ability to proliferate and produce glial cells but do not produce nerve cells.

[0061] Neural precursor cells include neural stem cells, neuronal progenitor cells, and Neural progenitor cells are a collection of progenitor cells, including glial progenitor cells, that have the ability to proliferate and produce neurons and glia. Neural progenitor cells can be identified using markers such as Nestin, GLAST, Sox2, Sox1, Musashi, and Pax6. Alternatively, cells that are positive for neural cell markers and proliferation markers (Ki67, pH3, MCM) can also be identified as neural progenitor cells.

[0062] The term "retinal tissue" as used herein refers to photoreceptor cells, photoreceptor precursor cells, rod photoreceptors, cone photoreceptors, interneurons, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells (ganglion cells), retinal pigment epithelial cells (RPE), ciliary marginal cells, and the like, which constitute each retinal layer in a living retina. The term "tissue" refers to a layered, three-dimensional arrangement of one or at least multiple types of cells, such as these progenitor cells or retinal progenitor cells. The retinal layer to which each cell belongs can be determined by known methods, such as the presence or absence of expression of a cell marker or the level of expression.

[0063] In the present invention, the term "retinal layer" refers to each layer constituting the retina, and specific examples thereof include the retinal pigment epithelium layer, photoreceptor layer, outer limiting membrane, outer nuclear layer, outer plexiform layer, inner nuclear layer, inner plexiform layer, ganglion cell layer, nerve fiber layer, and inner limiting membrane.

[0064] In the present invention, the term "retinal progenitor cells" refers to precursor cells that can differentiate into any of mature retinal cells, such as photoreceptors, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells, and retinal pigment epithelial cells.

[0065] In the present invention, "neural retinal progenitor cells" refer to progenitor cells that can differentiate into one or more mature retinal cells, such as photoreceptors, horizontal cells, bipolar cells, amacrine cells, and retinal ganglion cells. Generally, neural retinal progenitor cells do not differentiate into retinal pigment epithelial cells.

[0066] Photoreceptor precursor cells, horizontal cell precursor cells, bipolar cell precursor cells, amacrine cell precursor cells, retinal ganglion cell precursor cells, and retinal pigment epithelial precursor cells refer to precursor cells that are committed to differentiating into photoreceptors, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells, and retinal pigment epithelial cells, respectively.

[0067] In the present invention, the term "retinal layer-specific neurons" refers to neurons that constitute a retinal layer and are specific to that layer. Examples of retinal layer-specific neurons include bipolar cells, retinal ganglion cells, amacrine cells, horizontal cells, photoreceptors, retinal pigment epithelial cells, rod cells, and cone cells.

[0068] Retinal cell markers include Rx (also called Rax), PAX6, and Chx10, which are expressed in retinal progenitor cells; Nkx2.1, which is expressed in hypothalamic neuron progenitor cells but not in retinal progenitor cells; Sox1, which is expressed in the hypothalamic neuroepithelium but not in the retina; and Crx and Blimp1, which are expressed in photoreceptor progenitor cells. Markers of retinal layer-specific neurons include Rx, PAX6, and Chx10, which are expressed in bipolar cells. These include Chx10, PKCα, and L7, which are expressed in retinal ganglion cells; TuJ1 and Brn3, which are expressed in retinal ganglion cells; Calretinin, which is expressed in amacrine cells; Calbindin, which is expressed in horizontal cells; Rhodopsin and Recoverin, which are expressed in mature photoreceptor cells; Nrl and Rhodopsin, which are expressed in rod cells; Rxr-gamma and S-Opsin, which are expressed in cone cells; RPE65 and Mitf, which are expressed in retinal pigment epithelial cells; and Rdh10 and SSEA1, which are expressed in the ciliary body periphery.

[0069] The term "cerebral tissue" as used herein refers to cells constituting the fetal or adult cerebrum (e.g., cortical neural precursor cells, dorsal cerebral nervous system precursor cells, ventral cerebral nervous system precursor cells, cerebral layer structure-specific nerve cells (neurons), layer I neurons, layer II neurons, layer III neurons, layer IV neurons, layer V neurons, layer VI neurons, etc.). The term "cerebrum" refers to a tissue in which one or at least multiple types of cells (e.g., neurocytes, glial cells (astrocytes and oligodendrocytes), and their progenitor cells) are arranged three-dimensionally in layers. The fetal cerebrum is also called the forebrain or telencephalon. The presence of each cell type can be confirmed by known methods, such as the presence or absence of expression of cell markers or the degree of expression.

[0070] In the present invention, the term "cerebral layer" refers to each layer constituting the adult or fetal cerebrum, and specifically includes the molecular layer, external granular layer, external pyramidal cell layer, internal granular layer, neuronal cell layer (inner pyramidal cell layer), polymorphonuclear cell layer, layer I, layer II, layer III, layer IV, layer V, layer VI, cortical zone, intermediate zone, subventricular zone, and ventricular zone.

[0071] Examples of "cerebral nervous system progenitor cells" in the present invention include neuron progenitor cells, layer 1 neuron progenitor cells, layer 2 neuron progenitor cells, layer 3 neuron progenitor cells, layer 4 neuron progenitor cells, layer 5 neuron progenitor cells, layer 6 neuron progenitor cells, astrocyte progenitor cells, oligodendrocyte progenitor cells, etc. Each of these cells is a progenitor cell that is committed to differentiating into layer 1 neurons, layer 2 neurons, layer 3 neurons, layer 4 neurons, layer 5 neurons, layer 6 neurons, astrocytes, and oligodendrocytes. In the present invention, "cerebral nervous system progenitor cells" include multipotent stem cells (multipotent neural stem cells) that have the ability to differentiate (multipotency) into at least several lineages among layer 1 neurons, layer 2 neurons, layer 3 neurons, layer 4 neurons, layer 5 neurons, layer 6 neurons, astrocytes, and oligodendrocytes. nothing.

[0072] In the present invention, "cerebral layer-specific neurons" refer to cells that constitute the cerebral layers and are specific to the cerebral layers. Examples of cerebral layer-specific neurons include layer 1 neurons, layer 2 neurons, layer 3 neurons, layer 4 neurons, layer 5 neurons, layer 6 neurons, cerebral excitatory neurons, and cerebral inhibitory neurons.

[0073] Cerebral cell markers include FoxG1 (also known as Bf1) expressed in cerebral cells, Sox2 and Nestin expressed in cerebral nervous system progenitor cells, Pax6 and Emx2 expressed in dorsal cerebral nervous system progenitor cells, Dlx1, Dlx2 and Nkx2.1 expressed in ventral cerebral nervous system progenitor cells, Tbr2, Nex, Svet1 expressed in neuronal progenitor cells, Tbr1 expressed in layer 6 neurons, Ctip2 expressed in layer 5 neurons, Examples include RORβ, which is expressed in layer 4 neurons, Cux1 or Brn2, which are expressed in layer 3 or layer 2 neurons, and Reelen, which is expressed in layer 1 neurons.

[0074] 2. Method for producing nervous system cells or nervous tissue The production method 1 of the present invention is a method for producing nervous system cells or nervous tissue, comprising the following steps (1) to (3): Here is how: (1) Pluripotent stem cells were cultured in the absence of feeder cells, and 1) TGFβ family signaling pathway was Pathway inhibitors and / or Sonic Hedgehog signaling pathway agonists, and 2) untreated a first step of culturing in a medium containing a differentiation maintenance factor; (2) a second step of culturing the cells obtained in the first step in suspension to form cell aggregates; and (3) The aggregates obtained in the second step are cultured in suspension in the presence or absence of differentiation-inducing factors. and a third step of obtaining aggregates containing nervous system cells or nervous tissue.

[0075] In step (1), pluripotent stem cells are cultured in the absence of feeder cells, 1) in a culture medium containing TGFβ family members. 1) Culture the cells in a medium containing an inhibitor of the Lee signaling pathway and / or an agonist of the Sonic hedgehog signaling pathway, and 2) factors that maintain undifferentiated state.

[0076] Preferred pluripotent stem cells in step (1) include induced pluripotent stem cells or embryonic stem cells (E S cells), and more preferably human induced pluripotent stem cells or human embryonic stem cells (ES cells). can be.

[0077] There are no particular limitations on the method for producing induced pluripotent stem cells, and as described above, they can be produced by methods well known to those skilled in the art. However, it is desirable that the process of producing induced pluripotent stem cells (i.e., the process of initializing somatic cells to establish pluripotent stem cells) is also carried out in a feeder-free manner.

[0078] Here, there are no particular limitations on the method for producing embryonic stem cells (ES cells), and as described above, they can be produced by methods well known to those skilled in the art. However, it is desirable that the process for producing embryonic stem cells (ES cells) is also carried out in a feeder-free manner.

[0079] The maintenance and expansion culture of the pluripotent stem cells used in step (1) can be carried out by those skilled in the art as described above. This can be carried out by well-known methods. Maintenance culture and expansion culture of pluripotent stem cells can be carried out in either adherent culture or suspension culture, but is preferably carried out in adherent culture. Maintenance culture and expansion culture of pluripotent stem cells may be carried out in the presence of feeders or feeder-free, but is preferably carried out feeder-free. Feeder-free maintenance culture and expansion culture of pluripotent stem cells means conditions in which feeder cells are substantially absent (for example, the ratio of feeder cells to the total number of cells is 3% or less). Preferably, maintenance culture and expansion culture of pluripotent stem cells are carried out under feeder-cell-free conditions.

[0080] In step (1), the absence of feeder cells (feeder-free) means the absence of feeder cells. The term "feeder cell culture" refers to conditions in which feeder cells are substantially absent (for example, the ratio of the number of feeder cells to the total number of cells is 3% or less). Preferably, step (1) is carried out under conditions in which feeder cells are not present. The medium used in step (1) is a medium for culturing pluripotent stem cells under feeder-free conditions. There are no particular limitations on the medium as long as it is a medium that allows for the culture of cells to maintain the undifferentiated state (feeder-free medium), but it preferably contains factors for maintaining the undifferentiated state in order to enable the culture to maintain the undifferentiated state.

[0081] The undifferentiated state 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. In the case of primed pluripotent stem cells (e.g., human ES cells and human iPS cells), the undifferentiated state maintenance factor commonly used by those skilled in the art is FGF signaling. Examples of substances acting on the FGF signaling pathway include substances acting on the TGFβ family signaling pathway, insulin, etc. Specific examples of substances acting on the FGF signaling pathway include fibroblast growth factor ( Examples include bFGF, FGF4, and FGF8. In addition, TGFβ family signaling pathways are involved. Examples of the active substances include substances acting on the TGFβ signaling pathway and substances acting on the Nodal / Activin signaling pathway. Examples of the active substances on the TGFβ signaling pathway include substances acting on the TGFβ1 and TGFβ2 signaling pathway. Examples of substances acting on the Nodal / Activin signaling pathway include Nodal, Activin A, and Activin B. When culturing human pluripotent stem cells (human ES cells, human iPS cells), In this case, the medium in step (1) preferably contains bFGF as an undifferentiated state maintenance factor.

[0082] The undifferentiated state maintenance factor used in the present invention is typically a mammalian undifferentiated state maintenance factor. Examples of mammals include those listed above. Since undifferentiated state maintenance factors may be cross-reactive between mammalian species, any mammalian undifferentiated state maintenance factor may be used as long as it maintains the undifferentiated state of the pluripotent stem cells to be cultured. However, it is preferable to use an undifferentiated state maintenance factor from the same mammalian species as the cells to be cultured. For example, human undifferentiated state maintenance factors (e.g., bFGF, FGF4, FGF8, EGF, Nodal, Activin A, Activin B, TGFβ1, TGFβ2, etc.) are used for culturing human pluripotent stem cells. Here, "human protein X" means that protein X has the amino acid sequence of protein X naturally expressed in the human body.

[0083] The undifferentiated maintenance factor used in the present invention is preferably isolated. This means that the protein has been processed to remove components and extracellular factors that are not naturally occurring. Therefore, "isolated protein X" includes any protein produced from the cells or tissues being cultured. It does not include endogenous protein X contained in cells, tissues, or culture media. The purity of the isolated protein X (percentage of the weight of protein X in the total protein weight) is usually 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 99% or more, and even more preferably 100%. Therefore, in one aspect, the present invention provides an isolated undigested protein X. In one embodiment, the medium used in step (1) includes a step of providing a differentiation maintenance factor. The method further comprises a step of exogenously (or exogenously) adding an isolated factor for maintaining undifferentiation to the medium used in step (1). Alternatively, the factor for maintaining undifferentiation may be added in advance to the medium used in step (1).

[0084] The concentration of the undifferentiated maintenance factor in the medium used in step (1) is A concentration at which the undifferentiated state can be maintained can be determined appropriately by a person skilled in the art. For example, specifically, when bFGF is used as the undifferentiated state maintenance factor in the absence of feeder cells, the concentration is usually about 4 ng to 500 ng / mL, preferably about 10 ng to 200 ng / mL, and more preferably about 10 ng to 200 ng / mL. is approximately 30ng to 150ng / mL.

[0085] Many synthetic feeder-free media have been developed and are commercially available, such as Essential 8 medium. Essential 8 medium contains the following additives in DMEM / F12 medium: L-ascorbic acid-2-phosphate magnesium (64 mg / L), sodium selenium (14 μg / L), insulin (19.4 mg / L), NaHCO3 (543 mg / L), transferrin (10.7 mg / L), bFGF (100 ng / mL), and TGFβ family signaling pathway agents (TGFβ1 (2 ng / mL) or Nodal (100 ng / mL)) (Nature Methods, 8, 424-429 (2011)). Commercially available feeder-free media include: Examples include Essential 8 (manufactured by Life Technologies), S-medium (manufactured by DS Pharma Biomedical), StemPro (manufactured by Life Technologies), hESF9 (Proc Natl Acad Sci U.S.A. 2008 Sep 9;105(36):13409-14), mTeSR1 (manufactured by STEMCELL Technologies), mTeSR2 (manufactured by STEMCELL Technologies), and TeSR-E8 (manufactured by STEMCELL Technologies). An example of a feeder-free medium is StemFit (manufactured by Ajinomoto Co., Inc.) By using these in the above step (1), the present invention can be carried out simply and easily.

[0086] The culture of pluripotent stem cells in step (1) is carried out under either suspension culture or adherent culture conditions. However, adherent culture is preferred.

[0087] The culture vessel used for adhesion culture is not particularly limited as long as it is capable of "adhesion culture", but a cell-adhesive culture vessel is preferred. Examples of cell-adhesive culture vessels include culture vessels whose surface has been artificially treated to improve adhesion to cells, and specific examples include the above-mentioned culture vessels whose interior is coated with a coating agent. Examples of coating agents include laminin [laminin α5β1γ1 (hereinafter referred to as laminin 511), laminin laminin α1β1γ1 (hereinafter referred to as laminin 111) and laminin fragments (laminin 511E8, etc.), entactin, collagen, gelatin, vitronectin, Synthemax Examples of suitable materials include extracellular matrices such as Matrigel (Corning Incorporated), and polymers such as polylysine and polyornithine. Surface-treated culture vessels, for example, with a positive charge treatment, can also be used. Laminin is preferred, and laminin 511E-8 is more preferred. Commercially available laminin 511E-8 can be purchased (e.g., iMatrix-511, Nippi).

[0088] The medium used in step (1) contains a TGFβ family signaling pathway inhibitor and / or a sonic hedgehog signaling pathway activator. In the first step, pluripotent stem cells are cultured in a medium containing a TGFβ family signaling pathway inhibitor and / or a sonic hedgehog signaling pathway activator. By treating the cells with a substance that acts on the signaling pathway and then subjecting them to suspension culture in the second step, the state of the pluripotent stem cells is changed, the quality of the aggregates is improved, and they become round, have a smooth surface, and are aggregated. It is possible to produce cell aggregates with high efficiency that maintain a dense, undifferentiated state inside the body.

[0089] The TGFβ family signaling pathway (i.e., the TGFβ superfamily signaling pathway) is a pathway in which TGFβ, Nodal / Activin, or BMP acts as a ligand and mediates the signal transduction of the Smad family within cells. This is a signal transduction pathway that is transmitted by

[0090] A TGFβ family signaling pathway inhibitor refers to a substance that inhibits the TGFβ family signaling pathway, i.e., the signaling pathway transmitted by the Smad family, and specific examples include TGFβ signaling pathway inhibitors, Nodal / Activin signaling pathway inhibitors, and BMP signaling pathway inhibitors.

[0091] The TGFβ signaling pathway inhibitor is not particularly limited as long as it inhibits the signaling pathway caused by TGFβ, and may be any of nucleic acids, proteins, and low-molecular-weight organic compounds. Examples of such substances include substances that directly act on TGFβ (e.g., proteins, antibodies, etc.). aptamers, etc.), substances that suppress the expression of the gene encoding TGFβ (e.g., antisense substances that inhibit the binding of TGFβ receptors to TGFβ; substances that inhibit the physiological activity resulting from signal transduction by TGFβ receptors (e.g., inhibitors of TGFβ receptors) Examples of inhibitors of the TGFβ signaling pathway include inhibitors of TGFβ, such as steroid inhibitors and Smad inhibitors. Known proteins include Lefty and the like. Compounds known to those skilled in the art can be used as TGFβ signaling pathway inhibitors, and specific examples include SB431542, LY-364947, SB-505124, A-83-01, and the like. Here, SB431542 (4-(5-benzo[1,3]dioxane)-2-one) is a compound known to those skilled in the art. A-83-01 (3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbohydrate) Thioamides are inhibitors of TGFβ receptors (ALK5) and activin receptors (ALK4 / 7) (i.e., TGF The TGFβ signaling pathway inhibitor is preferably SB431542 or A-83-01.

[0092] Nodal / Activin signaling pathway inhibitors include those that inhibit signals caused by Nodal or Activin. The substance is not particularly limited as long as it inhibits the signal transduction pathway, and may be any of nucleic acids, proteins, and low-molecular-weight organic compounds. Examples of such substances include substances that act directly on Nodal or Activin (e.g., antibodies, aptamers, etc.), substances that suppress the expression of genes encoding Nodal or Activin (e.g., antisense oligonucleotides, siRNA, etc.), substances that inhibit the binding of Nodal / Activin receptors to Nodal / Activin, and substances that inhibit signal transduction via Nodal / Activin receptors. Examples of substances that inhibit physiological activity resulting from Nodal / Activin signaling pathway include compounds well known to those skilled in the art, such as SB431542 and A-83-01. Proteins known as Nodal / Activin signaling pathway inhibitors (such as Lefty and Cerberus) may also be used. The null signaling pathway inhibitor is preferably SB431542, A-83-01, or Lefty.

[0093] The BMP signaling pathway inhibitor is not particularly limited as long as it inhibits the signaling pathway caused by BMP, and may be any of nucleic acids, proteins, and low molecular weight organic compounds. Here, examples of BMP include BMP2, BMP4, BMP7, and GDF7. Examples of such substances include Examples of the substance that can be used include substances that act directly on BMP (e.g., antibodies, aptamers, etc.), substances that suppress the expression of genes encoding BMP (e.g., antisense oligonucleotides, siRNA, etc.), substances that inhibit the binding of BMP receptors (BMPRs) to BMP, and substances that inhibit physiological activities resulting from signal transduction by BMP receptors. Examples of BMPRs include ALK2 and ALK3. Compounds known to those skilled in the art can be used as BMP signal transduction pathway inhibitors, and specific examples thereof include: Examples of such inhibitors include LDN193189 and Dorsomorphin. LDN193189 (4-[6-(4-piperazin-1-ylphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline) is a known BMPR (ALK2 / 3) inhibitor (hereinafter referred to as a BMPR inhibitor), and is usually commercially available in the form of hydrochloride. Using proteins known as MP signaling pathway inhibitors (Chordin, Noggin, etc.) The BMP signaling pathway inhibitor is preferably LDN193189.

[0094] The TGFβ family signaling pathway inhibitor is preferably Lefty, SB431542, A-83-01, or LDN193189.

[0095] Combining multiple TGFβ family signaling pathway inhibitors with different action points It may also be used. By combining them, it is expected that the effect of improving the quality of aggregates will be enhanced. For example, a combination of a TGFβ signaling pathway inhibitor and a BMP signaling pathway inhibitor, a combination of a TGFβ signaling pathway inhibitor and a Nodal / Activin signaling pathway inhibitor, or a combination of a BMP signaling pathway inhibitor and a Nodal / Activin signaling pathway inhibitor can be mentioned, but preferably a TGFβ signaling pathway inhibitor is used in combination with a BMP signaling pathway inhibitor. A specific preferred combination is the combination of SB431542 and LDN193189.

[0096] Sonic hedgehog signaling (hereinafter sometimes referred to as Shh) transduction pathway activators The term "Shh inhibitors" refers to substances that can enhance signal transduction mediated by Shh. Examples of Shh signal transduction pathway active substances include proteins belonging to the Hedgehog family (e.g., Shh and Ihh), Shh receptors, Shh receptor agonists, PMA (purmorphamine; 9-cyclohexyl-N-[4-(4-methyl-2 ... Examples of the Shh signaling pathway active agent include N-methyl-N'-(3-pyridinylbenzyl)-N'-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4-diaminocyclohexane (SAG; N-methyl-N'-(3-pyridinylbenzyl)-N'-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4-diaminocyclohexane), and the like. The Shh signaling pathway active agent is preferably Shh protein (Genbank accession numbers: NM_000193, NP_000184), SAG, or PMA.

[0097] A TGFβ family signaling pathway inhibitor and a substance acting on the Shh signaling pathway may be used in combination. This combination is expected to enhance the effect of improving the quality of aggregates. Specific examples of such combinations include a combination of a TGFβ family signaling pathway inhibitor selected from the group consisting of Lefty, SB431542, A-83-01, and LDN193189 with a substance acting on the Shh signaling pathway selected from the group consisting of Shh protein, SAG, and PMA. TGFβ family signaling pathway inhibitor When a substance is used in combination with an Shh signaling pathway agonist, cells are cultured in a medium containing both a TGFβ family signaling pathway inhibitor and an Shh signaling pathway agonist. Alternatively, the cells may be treated with either a TGFβ family signaling pathway inhibitor or a substance acting on the Shh signaling pathway, and then subsequently treated with either or both of them.

[0098] TGFβ family signaling pathway inhibitors and sonic hedgehog signaling The concentration of the pathway acting substance can be appropriately set within a range that can achieve the above-mentioned effects. For example, SB431542 is usually used at a concentration of 0.1 to 200 μM, preferably 2 to 50 μM. A-83-01 is usually used at a concentration of 0.05 to 50 μM, preferably 0.5 to 5 μM. LDN193189 is usually used at a concentration of 1 to 2000 nM, preferably 10 to 300 nM. Lefty is usually used at a concentration of 5 to 200 ng / ml, preferably 10 to 300 nM. The concentration of Shh protein is preferably 10 to 50 ng / ml. Preferably, it is used at a concentration of 50 to 300 ng / ml. SAG is usually used at a concentration of 1 to 2000 nM, preferably 10 to 700 nM, more preferably 30 to 600 nM. PMA is usually used at a concentration of 0.002 to 20 μM, preferably 0.02 to 2 μM. In one embodiment, The pathway inhibitor can be used in an amount that has the same inhibitory activity on the TGFβ family signaling pathway as SB43154 at the aforementioned concentration. In one embodiment, the sonic hedgehog signaling pathway agonist can be used in an amount that has the same Shh signaling promoting activity as SAG at the aforementioned concentration.

[0099] The inhibitory activity of SB431542, LDN193189, and the like on the TGF-β family signaling pathway can be determined by methods well known to those skilled in the art, for example, by detecting phosphorylation of Smad by Western blotting (Mol Cancer Ther. (2004) 3, 737-45.). The null signaling promoting activity can be determined by methods well known to those skilled in the art, for example, a reporter gene assay focusing on the expression of the Gli1 gene (Oncogene (2007) 26, 5163-5168).

[0100] The medium used in step (1) may be a serum-containing medium or a serum-free medium. However, from the viewpoint of avoiding contamination with chemically undefined components, a serum-free medium is preferred.

[0101] The medium used in step (1) is selected from the viewpoint of avoiding contamination with chemically undefined components. Alternatively, the medium may be one whose components are chemically determined.

[0102] The pluripotent stem cells in step (1) can be cultured under either suspension culture or adherent culture conditions. The culture may be carried out by adhesion culture, but is preferably carried out by adhesion culture.

[0103] In the step (1), the pluripotent stem cells are cultured under feeder-free conditions using a medium and The feeder-free medium may be used as the feeder-free medium. Examples of the feeder-free medium include Essential 8, S-medium, StemPro, hESF9, mTeSR1, mTeSR2, TeSR-E8, and StemFit. Preferably, Essential8 or StemFit is used.

[0104] In the step (1), pluripotent stem cells are cultured under feeder-free conditions. In order to provide pluripotent stem cells with a scaffold instead of stem cells, an appropriate matrix may be used as a scaffold. Pluripotent stem cells are cultured in an adherent manner in a cell container whose surface is coated with the scaffold matrix.

[0105] Matrices that can be used as scaffolds include laminin (Nat Biotechnol 28,611-615 (2010)), laminin fragments (Nat Commun 3, 1236 (2012)), basement membrane preparations (Nat Biotechnol 19, 971-974 (2001)), gelatin, collagen, heparan sulfate proteoglycan, Examples include entactin and vitronectin.

[0106] Laminin is a heterotrimeric molecule consisting of α, β, and γ chains, and is an extracellular matrix protein that exists in isoforms with different subunit chain compositions. Specifically, laminin is a heterotrimeric combination of five α chains, four β chains, and three γ chains, with approximately 15 isoforms. There are three isoforms: α chain (α1 to α5), β chain (β1 to β4), and γ chain (γ1 The names of laminins are determined by combining the numbers of the α5 chain, β1 chain, and γ1 chain. For example, a laminin made up of a combination of α5, β1, and γ1 chains is called laminin 511. In the present invention, Preferably, laminin 511 is used (NatBiotechnol 28, 611-615 (2010)).

[0107] The laminin used in the present invention is typically mammalian laminin. Examples of mammalian laminins include those listed above. From the perspective of achieving xeno-free conditions, laminin from the same mammalian species as the cells to be cultured is preferably used. For example, human laminin (preferably human laminin 511) is used for culturing human pluripotent stem cells.

[0108] The laminin fragment used in the present invention is not particularly limited as long as it has adhesive properties to pluripotent stem cells and enables the maintenance culture of pluripotent stem cells under feeder-free conditions, but is preferably an E8 fragment. The laminin E8 fragment is a fragment that binds laminin 511 to elastin. Among the fragments obtained by digestion with laminin-511, the E8 fragment was identified as having strong cell adhesion activity (EMBO J., 3:1463-1468, 1984; J. Cell Biol., 105:589-598, 1987). In the present invention, the E8 fragment of laminin-511 is preferably used (Nat Commun 3, 1236 (2012), Scientific Reports 4, 3549 (2014). The laminin E8 fragment does not necessarily have to be a product of laminin elastase digestion, and may be a recombinant product. From the viewpoint of avoiding contamination with unidentified components, recombinant laminin fragments are preferably used in the present invention. The E8 fragment of laminin 511 The material is commercially available and can be purchased from, for example, Nippi Corporation.

[0109] The laminin or laminin fragment used in the present invention is preferably isolated.

[0110] In the present invention, the term "basement membrane preparation" refers to a preparation containing basement membrane components that have the function of regulating epithelial cell-like cell morphology, differentiation, proliferation, motility, and functional expression when desired cells with basement membrane-forming ability are seeded and cultured thereon. For example, when the nervous system cells or neural tissues produced by the present invention are dispersed and further subjected to adhesion culture, they can be cultured in the presence of a basement membrane preparation. Here, "basement membrane components" refer to thin membrane-like extracellular matrix molecules present between the epithelial cell layer and the interstitial cell layer in animal tissues. A basement membrane preparation can be prepared, for example, by removing cells with basement membrane-forming ability that are adhered to a support via a basement membrane from the support using a solution capable of dissolving the lipids of the cells or an alkaline solution. Examples of basement membrane preparations include commercially available products (e.g., Matrigel TM (manufactured by Corning; hereafter referred to as Matrigel) and Geltrex TM (Life Technologies), extracellular matrix molecules known as basement membrane components (e.g. , laminin, type IV collagen, heparan sulfate proteoglycan, entactin, etc.

[0111] Matrigel TMMatrigel is a basement membrane preparation extracted from the EngelbrethHolm Swarn (EHS) mouse sarcoma. TM The main components of Matrigel are type IV collagen, laminin, heparan sulfate proteoglycans, and entactin, along with TGF-β, FGF, tissue plasminogen activator, and growth factors naturally produced by EHS tumors. TM "Growth factor reduced products" are not the same as regular Matrigel TM The concentration of growth factors is lower than that of EGF. <0.5ng / ml, NGF <0.2ng / ml, PDGF <5pg / ml, IGF1 5ng / ml, and TGFβ 1.7ng / ml.

[0112] In order to avoid contamination with unidentified components, isolated laminin or laminin fragments are preferably used in the present invention.

[0113] Preferably, in the culture of pluripotent stem cells under feeder-free conditions in step (1), In this case, pluripotent stem cells are cultured in a cell container whose surface is coated with isolated laminin-511 or the E8 fragment of laminin-511 (more preferably the E8 fragment of laminin-511). The cells are cultured in adherent culture.

[0114] The culture time of the pluripotent stem cells in step (1) is not particularly limited as long as it is within a range that can achieve the effect of improving the quality of the aggregates formed in step (2), but is usually 0.5 to 144 hours. The culture time of the pluripotent stem cells in step (1) is preferably 1 hour or more, 2 hours or more. The culture time of the pluripotent stem cells in step (1) is preferably 96 hours or less, or 72 hours or less. In one aspect, the culture time of the pluripotent stem cells in step (1) is preferably 2 to 96 hours, more preferably 6 to 48 hours, even more preferably 12 to 48 hours, and even more preferably 18 to 28 hours (e.g., 24 hours). That is, 0.5 to 144 hours (preferably 18 to 28 hours) before the start of step (2), After the first step is initiated and step (1) is completed, step (2) is subsequently carried out. In a further embodiment, the range of the culturing time for the pluripotent stem cells in step (1) is preferably 18 to 144 hours, 24 to 144 hours, 24 to 96 hours, or 24 to 72 hours. After treating the cells with either a TGFβ family signaling pathway inhibitor or a Shh signaling pathway agonist, the cells are cultured with the other. When cells are treated successively, the time for each treatment can be set within the above-mentioned culture time range.

[0115] The culture conditions in step (1), such as culture temperature and CO2 concentration, can be set appropriately. The culture temperature is, for example, about 30° C. to about 40° C., preferably about 37° C. The CO2 concentration is, for example, about 1% to about 10%, preferably about 5%.

[0116] In a preferred embodiment, human pluripotent stem cells (e.g., human iPS cells) are cultured on feeder cells. In the absence of erythrocyte proliferation, the cells are cultured in a serum-free medium containing bFGF. The culture is preferably carried out in a cell container whose surface is coated with laminin 511, the E8 fragment of laminin 511, or vitronectin. The culture is preferably carried out in a feeder-free medium such as Essential 8, TeSR medium, mTeSR medium, mTeSR-E8 medium, or StemFit medium, more preferably Typically, this is done using Essential 8 or StemFit medium.

[0117] In a preferred embodiment, human pluripotent stem cells (e.g., human iPS cells) are cultured on feeder cells. In the absence of bFGF, the human pluripotent stem cells are cultured in suspension in a serum-free medium containing bFGF. During the suspension culture, the human pluripotent stem cells may form aggregates of human pluripotent stem cells.

[0118] In a preferred embodiment, the cells obtained by step (1) are cells in which a pluripotent-like state is maintained, and the pluripotent-like state is maintained throughout step (1). Pluripotent-like state refers to a state in which at least some of the traits common to pluripotent stem cells, including pluripotency, are maintained. Strict pluripotency is not required for pluripotent-like state. Specifically, all or some of the markers indicative of a pluripotent state are maintained. A state in which some of these genes are expressed is included in "pluripotency-like properties." Markers of pluripotency-like properties include Oct3 / 4 positivity and alkaline phosphatase positivity. In one embodiment, cells that maintain pluripotency-like properties are Oct3 / 4 positivity. Even if the expression level of Nanog is lower than that of ES cells or iPS cells, the cells still fall under the category of "cells exhibiting pluripotency-like properties."

[0119] In one embodiment, the cells obtained by step (1) are at least neural cells or neural tissue. In one embodiment, the cells obtained by step (1) are stem cells capable of differentiating into at least one of the following: (preferably, retinal tissue, retinal cells, retinal progenitor cells, or retinal layer-specific nerve cells). The stem cells are Oct3 / 4-positive stem cells that have the ability to differentiate into at least neural cells or neural tissue (preferably retinal tissue, retinal cells, retinal progenitor cells, or retinal layer-specific neural cells).

[0120] In a preferred embodiment, human pluripotent stem cells (e.g., iPS cells) are cultured in the absence of feeder cells. TGFβ family signaling pathway inhibitors and / or sonic hedgehog signaling inhibitors The cells are cultured in an adherent state in a serum-free medium containing a signal transduction pathway agent and bFGF.

[0121] The adherent culture is preferably carried out in a cell container whose surface is coated with laminin 511 or the E8 fragment of laminin 511. The inhibitor is preferably a TGFβ signaling pathway inhibitor (e.g., SB431542, A-83-01, Lefty), a Nodal / Activin signaling pathway inhibitor (e.g., Lefty, SB431542, A-83-01), a BMP signaling pathway inhibitor (e.g., LDN193189, Chordin, Noggin), or a combination thereof (e.g., SB431542 and LDN193189). The TGFβ family signaling pathway inhibitor is more preferably Lefty, SB431542, A-83-01, or LDN193189, or a combination thereof (e.g., SB431542 and LDN193189). The sonic hedgehog signaling pathway inhibitor is preferably Shh protein, SAG, or Purmorphamine (PMA), more preferably SAG. TGFβ family signaling pathway inhibitors (e.g., Lefty, SB431542, A-83-01, LDN193189) Sonic hedgehog signaling pathway agonists (e.g., Shh protein, SAG, PMA) The culture time is 0.5 to 144 hours (preferably 18 to 144 hours, 24 to 144 hours, 24 to 96 hours, or 24 to 72 hours (e.g., 18 to 28 hours).

[0122] Step (1) is, for example, a method of culturing pluripotent stem cells in the presence of a factor for maintaining undifferentiated state in the absence of feeder cells. The cells were maintained in a medium containing TGFβ family signaling pathway inhibitors. This is carried out by adding a substance and / or a sonic hedgehog signaling pathway agonist and continuing the culture.

[0123] For example, human pluripotent stem cells (e.g., human iPS cells) are cultured in the presence of bFGF in the absence of feeder cells. The cells are maintained in a serum-free medium containing TGF-β. The maintenance culture is preferably performed by adhesion culture. The adhesion culture is preferably performed in a cell container whose surface is coated with vitronectin, laminin 511, or E8 fragment of laminin 511. Add a TGFβ family signaling pathway inhibitor and / or a sonic hedgehog signaling pathway agonist, and continue culturing. Preferably, TGFβ signaling pathway inhibitors (e.g., SB431542, A-83-01, Lefty), Nodal / Activin signaling pathway inhibitors (e.g., SB431542, A-83-01, Lefty), BMP signaling pathway inhibitors (e.g., SB431542, A-83-01, Lefty), The TGFβ family signaling pathway inhibitor is preferably Lefty, SB431542, A-83-01, or LDN193189, or a combination thereof (e.g., SB431542 and LDN193189). The sonic hedgehog signaling pathway active substance is preferably Shh protein, SAG, or PMA. A TGFβ family signaling pathway inhibitor (e.g., Lefty, SB431542, A-83-01, LDN193189) may be used in combination with a sonic hedgehog signaling pathway active substance (e.g., Shh protein, SAG, PMA). After addition, the cells are incubated for 0.5 to 144 hours (preferably The culture is continued for 18 to 144 hours, 24 to 144 hours, 24 to 96 hours, or 24 to 72 hours (for example, 18 to 28 hours).

[0124] The cells obtained in step (1) are cultured in suspension in a medium to form cell aggregates. Step (2) will now be described.

[0125] The medium used in step (2) is as described in the definition section above. There is no particular limitation on the medium used in step (2). In order to avoid contamination with chemically undefined components, a serum-free medium is preferably used in the present invention. For example, a serum-free medium containing neither a substance acting on the BMP signaling pathway nor a substance inhibiting the Wnt signaling pathway can be used. To avoid the complexity of the preparation, for example, a serum-free medium containing an appropriate amount of a commercially available serum substitute such as KSR (e.g., For example, it is preferable to use a medium in which 10% KSR, 450 μM 1-monothioglycerol, and 1× Chemically Defined Lipid Concentrate are added to a 1:1 mixture of IMDM and F-12, or a medium in which 5% to 20% KSR, NEAA, pyruvic acid, and 2-mercaptoethanol are added to GMEM. The amount of KSR added to serum-free medium is usually about 1% to about 30%, preferably about 2% to about 20%, in the case of human pluripotent stem cells, for example.

[0126] When forming aggregates, first, the cells obtained in step (1) are dispersed by a dispersion operation. The "dispersed cells" obtained by the dispersion procedure are those that are, for example, 70% or more The dispersed cells are preferably in a state where 80% or more of the cells are single cells and 20% or less of the cells are clumps of 2 to 50 cells. Dispersed cells include cells in a state where cell-to-cell adhesion (e.g., surface adhesion) is almost completely eliminated. In some embodiments, dispersed cells include cells in a state where cell-to-cell junctions (e.g., adherens junctions) are almost completely eliminated.

[0127] The dispersion operation of the cells obtained in step (1) can be carried out by the above-mentioned mechanical dispersion treatment, cell dispersion liquid treatment, etc. These treatments may be combined. It is advisable to carry out the cell dispersion treatment simultaneously with the cell protective agent treatment, followed by the mechanical dispersion treatment.

[0128] Cytoprotective agents used in the treatment include FGF signaling pathway activators, hemoglobin receptor activators, and hemoglobin receptor activators. These may include valine, an IGF signaling pathway agent, serum, or serum replacement. Furthermore, a Rho-associated coiled-coil kinase (ROCK) inhibitor or a myosin inhibitor may be added as a cell protective agent to suppress cell death of pluripotent stem cells (particularly human pluripotent stem cells) induced by dissociation. To suppress cell death of pluripotent stem cells (particularly human pluripotent stem cells) induced by dissociation and protect the cells, a Rho-associated coiled-coil kinase (ROCK) inhibitor or a myosin inhibitor may be added from the start of the second culture step. Examples of ROCK inhibitors include Y-27632, Fasudil (HA1077), and H-1152. Examples of myosin inhibitors include Blebbistatin.

[0129] The cell dispersion liquid used for the cell dispersion treatment may contain enzymes such as trypsin, collagenase, hyaluronidase, elastase, pronase, DNase, or papain, or ethylenediamine. Examples of such solutions include solutions containing a chelating agent such as aminetetraacetic acid. Commercially available cell dispersion solutions, such as TrypLESelect (manufactured by Life Technologies) and TrypLEExpress (manufactured by Life Technologies), can also be used.

[0130] Methods for mechanical dispersion include pipetting and scraping with a scraper.

[0131] The dispersed cells are suspended in the above medium.

[0132] Then, a suspension of the dispersed cells is seeded in the culture vessel, and the dispersed cells are cultured under non-adhesive conditions to the culture vessel, thereby causing multiple cells to aggregate and form aggregates.

[0133] In this case, the dispersed cells may be seeded in a relatively large culture vessel such as a 10 cm dish to simultaneously form a plurality of cell aggregates in one culture vessel. Therefore, if a certain number of dispersed stem cells are placed in each well of a multi-well plate (U-bottom, V-bottom) such as a 96-well microplate and then cultured statically, the cells will rapidly aggregate, resulting in one cell in each well. Aggregates are formed, and by collecting these aggregates from multiple wells, a homogeneous population of aggregates can be obtained.

[0134] The cell concentration in step (2) is adjusted to allow cell aggregates to be formed more uniformly and efficiently. For example, when human cells (e.g., cells obtained from human iPS cells in step (1)) are cultured in suspension using a 96-well microwell plate, one well Approximately 1 x 10 3 From approximately 1 x 10 5 cells, preferably about 3 x 10 3 From about 5 x 10 4 cells, more preferably about 4 x 10 3 From about 2 x 10 4 cells, more preferably about 4 x 10 3 to approximately 1.6 x 10 4 cell, Even more preferably about 8 x 10 3 to approximately 1.2 x 10 4 Add the liquid prepared to become cells to the wells. Add the solution and allow the plate to stand to form aggregates.

[0135] The culture conditions in step (2), such as culture temperature and CO2 concentration, can be set appropriately. The culture temperature is, for example, about 30° C. to about 40° C., preferably about 37° C. The CO2 concentration is, for example, about 1% to about 10%, preferably about 5%.

[0136] In step (2), when a medium exchange operation is performed, for example, the original medium is not discarded but a new medium is added. Add the medium (medium addition operation), and add about half the original medium (30-40% of the original medium volume). Examples of such procedures include a half-medium exchange procedure in which approximately 90% of the original medium (e.g., approximately 40-60%) is discarded and approximately half of the new medium (30-90% of the original medium volume, e.g., approximately 40-60%) is added, and a full-medium exchange procedure in which approximately the entire original medium (90% or more of the original medium volume) is discarded and approximately the entire new medium (90% or more of the original medium volume) is added.

[0137] When adding a specific component (e.g., a differentiation-inducing factor) at a certain point in time, for example, after calculating the final concentration, approximately half of the original medium may be discarded and approximately half of a new medium containing the specific component at a concentration higher than the final concentration, specifically 1.5 to 3 times the final concentration, for example, approximately twice the final concentration, may be added (half medium exchange operation, half medium exchange).

[0138] If, at a certain point, the concentration of a particular component contained in the original medium is to be maintained, for example, about half of the original medium may be discarded and about half of a new medium containing the particular component at the same concentration as that contained in the original medium may be added.

[0139] When components contained in the original medium are diluted to reduce their concentrations at a certain point, for example, the medium exchange procedure may be performed multiple times a day, preferably multiple times within an hour (e.g., 2 to 3 times).Furthermore, when components contained in the original medium are diluted to reduce their concentrations at a certain point, the cells or aggregates may be transferred to another culture vessel.

[0140] The tools used for the medium replacement operation are not particularly limited, and examples include pipettors, micropipettes, multichannel micropipettes, repeating dispensers, etc. For example, when a 96-well plate is used as the culture vessel, a multichannel micropipette may be used.

[0141] The suspension culture time required to form cell aggregates can be determined appropriately depending on the cells used so as to uniformly aggregate the cells, but it is desirable that the time be as short as possible to form uniform cell aggregates. The process by which dispersed cells form cell aggregates is divided into a cell aggregation process and a process by which the aggregated cells form cell aggregates. For example, in the case of human cells (e.g., stem cells obtained from human iPS cells in step (1)), the time from the time dispersed cells are seeded (i.e., at the start of suspension culture) to the time cells are aggregated is preferably within about 24 hours, more preferably within about 12 hours. For example, in the case of human pluripotent stem cells (e.g., human iPS cells), the time from the time dispersed cells are seeded (i.e., at the start of suspension culture) to the time cell aggregates are formed is preferably within about 24 hours, more preferably within about 12 hours. The time required for the formation of aggregates is preferably within about 72 hours, more preferably within about 48 hours. The time required for the formation of aggregates can be appropriately adjusted by adjusting the cell aggregation tool, centrifugation conditions, etc.

[0142] The formation of cell aggregates and their uniformity can be determined based on the size and cell number of the aggregates, macroscopic morphology, microscopic morphology and uniformity determined by tissue staining analysis, expression and uniformity of differentiated and undifferentiated markers, control of expression of differentiation markers and their synchronization, and reproducibility of differentiation efficiency between aggregates.

[0143] After the formation of the aggregates, the culture of the aggregates may be continued. The suspension culture time is usually 12 hours to 6 days, and preferably about 12 hours to 48 hours.

[0144] In one embodiment, the medium used in step (2) contains Sonic Hedgehog signaling. In step (1), pluripotent stem cells are treated with a TGFβ family signaling pathway inhibitor and / or a sonic hedgehog signaling pathway activator, and in step (2), the cells obtained in step (1) are subjected to suspension culture in a medium (preferably a serum-free medium) containing a sonic hedgehog signaling pathway activator to form aggregates. By doing so, the quality of the aggregates is further improved, and it is possible to form with high efficiency aggregates of cells that are round, have smooth surfaces, do not lose their shape, are dense inside, and maintain their undifferentiated state.

[0145] The above-mentioned substances can be used as the Sonic Hedgehog signaling pathway active substance. Preferably, the Sonic Hedgehog signaling pathway active substance is SAG, Purmorphamine (PMA), or Shh protein. The concentration of the Sonic Hedgehog signaling pathway active substance in the medium can be appropriately set within a range that can achieve the above-mentioned effects. SAG is usually 1 to 2000 nM, preferably 10 nM to 1000 nM, more preferably 10 nM to 700 nM. It is more preferably used at a concentration of 50 nM to 700 nM, even more preferably 100 nM to 600 nM, even more preferably 100 nM to 500 nM. PMA is usually used at a concentration of 0.002 to 20 μM, preferably 0.02 to 2 μM. Shh protein is usually used at a concentration of 20 to 1000 ng / ml, preferably 50 to 300 ng / ml. Substances acting on the sonic hedgehog signaling pathway other than SAG, PMA, and Shh protein When using SAG, the Sonic Hedgehog signaling promoting activity is equivalent to that of the above SAG concentration. It is desirable to use it at a concentration that shows its activity.

[0146] The timing of adding the Sonic hedgehog signaling pathway active substance to the medium is not particularly limited as long as the above-mentioned effect can be achieved, but the earlier the addition, the greater the effect. The Sonic hedgehog signaling pathway active substance is added to the medium usually within 6 days, preferably within 3 days, more preferably within 1 day, and even more preferably at the start of step (2) from the start of step (2). will be done.

[0147] In a preferred embodiment, in step (2), the human cells obtained in step (1) (e.g., cells obtained from human iPS cells by step (1)) are subjected to suspension culture in a serum-free medium containing a substance acting on the Sonic Hedgehog signaling pathway (e.g., SAG, PMA, Shh protein), The sonic hedgehog signaling pathway activator is preferably included in the medium from the start of suspension culture. The medium may also contain a ROCK inhibitor (e.g., Y-27632). The incubation time is 12 hours to 6 days, preferably 12 hours to 48 hours. The body is preferably a homogeneous aggregate.

[0148] For example, the human cells obtained in step (1) (e.g., human iPS cells obtained in step (1)) The cells were then harvested and cultured in serum-free medium containing a sonic hedgehog signaling pathway agent (e.g., SAG, PMA, Shh protein) until they reached a single cell state or a similar state. The serum-free medium contains a ROCK inhibitor (e.g., Y-27632). A suspension of human pluripotent stem cells (e.g., human iPS cells) is seeded in the above-mentioned culture vessel and separated. The dispersed pluripotent stem cells are cultured in a culture vessel under non-adhesive conditions, whereby a plurality of pluripotent stem cells are aggregated to form aggregates. The culture time is preferably 12 hours to 6 days. The time is 12 to 48 hours. The aggregates formed are preferably uniform aggregates.

[0149] In a preferred embodiment, in step (1), pluripotent stem cells are treated with a TGFβ signaling pathway inhibitor, and in step (2), sonic hedgehog signaling is inhibited. The cells obtained in step (1) are cultured in suspension in a medium containing a pathway-acting substance (e.g., SAG, PMA, Shh protein). SB431542 or A-83-01 can be used.

[0150] In a preferred embodiment, in step (1), pluripotent stem cells are treated with a substance that inhibits the BMP signaling pathway, and in step (2), sonic hedgehog signaling is inhibited. The cells obtained in step (1) are subjected to suspension culture in a medium that does not contain a signaling pathway active substance (e.g., SAG, PMA, Shh protein). Preferably, the medium does not contain a BMP signaling pathway inhibitor. LDN193189 can be used as a quality.

[0151] In a preferred embodiment, in step (1), pluripotent stem cells (e.g., human pluripotent stem cells) ) in combination with a TGFβ family signaling pathway inhibitor (e.g., a TGFβ signaling pathway inhibitor (e.g., Lefty, SB431542, A-83-01), a Nodal / Activin signaling pathway inhibitor (e.g., Lefty, SB431542, A-83-01), a BMP signaling pathway inhibitor (e.g., LDN193189), or combinations (e.g., SB431542 and LDN193189); Sonic hedgehog signaling or TGFβ family signaling pathway inhibitors (e.g., Lefty, SB431542, A-83-01, LDN193189) and sonic hedgehog signaling inhibitors. Treatment with a combination of a signal transduction pathway agent (e.g., Shh protein, SAG, PMA), In step (2), the cells obtained in step (1) are cultured in suspension in a medium containing a substance acting on the sonic hedgehog signaling pathway (e.g., SAG, PMA, Shh protein). can be.

[0152] In another embodiment, in step (1), pluripotent stem cells (e.g., human pluripotent stem cells) are treated with a TGFβ family signaling pathway inhibitor (e.g., a TGFβ signaling pathway inhibitor (e.g., , Lefty, SB431542, A-83-01), Nodal / Activin signaling pathway inhibitors (e.g., Lefty, SB431542, A-83-01), BMP signaling pathway inhibitors (e.g., LDN193189), or combinations thereof Combined (e.g., SB431542 and LDN193189); Sonic Hedgehog signaling pathway agonists (e.g., Shh protein, SAG, PMA); or inhibitors of the TGFβ family signaling pathway (e.g., Lefty, SB431542, A-83-01, LDN193189) and sonic hedgehog signaling Treatment with a combination of a signaling pathway agent (e.g., Shh protein, SAG, PMA) and In step (2), the cells obtained in step (1) are cultured in suspension in a medium that does not contain a sonic hedgehog signaling pathway agent (e.g., SAG, PMA, Shh protein). do.

[0153] In either embodiment, the medium in step (2) preferably contains a ROCK inhibitor (e.g., Y-27632).

[0154] In this way, by carrying out step (2), aggregates of the cells obtained in step (1) or cells derived therefrom are formed. The present invention also provides a method for producing such aggregates. The aggregates obtained in step (2) are obtained by the step (1) of inhibiting TGF-β family signaling. The quality of the aggregates obtained is higher than that obtained without treatment with a substance inhibiting the cell proliferation pathway and / or a substance acting on the Sonic hedgehog signaling pathway. Specifically, a population of aggregates can be obtained that is round, has a smooth surface, is dense inside, and is enriched in aggregates that do not collapse. In one embodiment, when aggregates (e.g., 100 or more) are randomly selected on the sixth day from the start of the second step, the sum of the proportion of aggregates that do not collapse and / or the proportion of aggregates that do not form cysts is, for example, 70% or more, preferably 80% or more.

[0155] The aggregates obtained in step (2) have the ability to differentiate into various differentiated cells and tissues. In one embodiment, the aggregates obtained in step (2) are composed of neural cells or neural tissue (preferably, They have the ability to differentiate into retinal tissue, retinal cells, retinal progenitor cells, or retinal layer-specific neurons.

[0156] In one embodiment, at least the nervous system cells or nervous tissue (preferably In the step (2), stem cells capable of differentiating into at least neural cells or neural tissue (preferably retinal tissue, retinal cells, retinal progenitor cells, or retinal layer-specific nerve cells) (preferably Oct3 / 4-positive stem cells capable of differentiating into at least neural cells or neural tissue (preferably retinal tissue, retinal cells, retinal progenitor cells, or retinal layer-specific nerve cells)) are used. Preferably, aggregates containing stem cells (preferably Oct3 / 4-positive stem cells) capable of differentiating into retinal tissue, retinal cells, retinal progenitor cells, or retinal layer-specific nerve cells can be obtained. The aggregates obtained in step (2) can be cultured under appropriate differentiation conditions to obtain This makes it possible to induce various differentiated cells and tissues with high efficiency.

[0157] In one embodiment, the aggregates obtained in step (2) contain cells corresponding to intermediate-stage cells between cells retaining pluripotency-like properties obtained at the end of step (1) (specifically, expressing Oct3 / 4) and neural cells or neural tissue. The cells express the pluripotency marker Oct3 / 4, ectodermal markers (Sox1, Sox2, N-cadherin, TP63), neuroectodermal markers (Sox1, Sox2, Nestin, N-cadherin, Otx2), or any of the aforementioned neural cell markers. That is, in one embodiment, the aggregates obtained in step (2) contain cells corresponding to intermediate-stage cells between cells retaining pluripotency-like properties obtained at the end of step (1) (specifically, expressing Oct3 / 4) and neural cells or neural tissue. The cells express the pluripotency marker Oct3 / 4, ectodermal markers (Sox1, Sox2, N-cadherin, TP63), neuroectodermal markers (Sox1, Sox2, Nestin, N-cadherin, Otx2), or any of the aforementioned neural cell markers. That is, the aggregate obtained in step (2) contains a mixture of cells containing at least one neuron. The aggregates obtained in step (2) contain stem cells capable of differentiating into neural cells or neural tissue, and / or progenitor cells of neural cells or neural tissue. The progenitor cells are characterized by their competence to express the above-mentioned neural cell markers when cultured under known appropriate culture conditions. Therefore, in one embodiment, the aggregates obtained in step (2) contain at least Oct3 / 4-positive neural cells or neural tissue progenitor cells. The aggregates obtained in step (2) contain stem cells capable of differentiating into neural tissues, and / or neural cells or neural tissue progenitor cells. In one embodiment, the aggregates obtained in step (2) may contain 50% or more, for example 70% or more, of Oct3 / 4 positive cells in all cells.

[0158] In step (2), when a medium exchange operation is performed, for example, the original medium is not discarded but a new medium is added. These include adding medium (medium addition operation), discarding about half of the original medium (about 30-90% of the volume of the original medium, for example, about 40-60%) and adding about half of the new medium (about 30-90% of the volume of the original medium, for example, about 40-60%) (half medium exchange operation), and discarding about the entire volume of the original medium (more than 90% of the volume of the original medium) and adding about the entire volume of new medium (more than 90% of the volume of the original medium) (full medium exchange operation).

[0159] When adding a specific component (e.g., a differentiation-inducing factor) at a certain point, for example, after calculating the final concentration, approximately half of the original medium may be discarded and approximately half of a new medium containing the specific component at a concentration higher than the final concentration (specifically, 1.5 to 3.0 times the final concentration, for example, approximately twice the final concentration) may be added (half medium exchange operation, half medium exchange).

[0160] If, at a certain point, the concentration of a particular component contained in the original medium is to be maintained, for example, about half of the original medium may be discarded and about half of a new medium containing the particular component at the same concentration as that contained in the original medium may be added.

[0161] When components contained in the original medium are diluted to reduce their concentrations at a certain point, for example, the medium exchange procedure may be performed multiple times a day, preferably multiple times within an hour (e.g., 2 to 3 times).Furthermore, when components contained in the original medium are diluted to reduce their concentrations at a certain point, the cells or aggregates may be transferred to another culture vessel.

[0162] The tools used for the medium replacement operation are not particularly limited, and examples include pipettors, micropipettes, multichannel micropipettes, repeating dispensers, etc. For example, when a 96-well plate is used as the culture vessel, a multichannel micropipette may be used.

[0163] Inducing aggregates containing nervous system cells or nervous tissue from the aggregates obtained in step (2). Step (3) will now be described.

[0164] A method for inducing pluripotent stem cell aggregates into neural cells or neural tissue by suspension culture. Many methods have been reported for this purpose. For example, methods described in WO2005 / 123902, WO2009 / 148170, WO2008 / 035110, WO2011 / 055855, Cell Stem Cell, 3, 519-32 (2008), Nature, 472, 51-56 (2011), Cell Stem Cell, 10 (6), 771-775 (2012), Nature Biotechnology, 27 (3), 275-80 (2009), Proc Natl Acad SciUSA, 110 (50), 20284-9 (2013), etc. are known. By applying such various methods for inducing neural cells or neural tissue to the aggregate obtained in step (2) and culturing the aggregate obtained in step (2) under appropriate neuronal differentiation inducing conditions, an aggregate containing neural cells or neural tissue can be produced.

[0165] For example, the aggregates obtained in step (2) can be cultured in the presence or absence (preferably) of a differentiation-inducing factor. The cells are cultured in suspension in a medium containing a neuronal cell or a neural tissue, preferably in the presence of a neurotransmitter, to obtain aggregates containing neural cells or neural tissue.

[0166] A differentiation-inducing factor is a factor that has the activity of inducing differentiation of cells or tissues, such as a factor that has the activity of differentiating stem cells or progenitor cells into a specific lineage (or determining their fate). Examples of differentiation-inducing factors include, but are not limited to, in vivo gene products such as growth factors, low-molecular-weight compounds that control the action of in vivo gene products, hormones, vitamins, and other physiologically active substances. Examples of differentiation-inducing factors commonly used by those skilled in the art include, for example, BMP signaling activators, BMP signaling pathway inhibitors, and Shh signaling pathway inhibitors. agonist, Shh signaling pathway inhibitor, FGF signaling pathway agonist, FGF signal These include Wnt signaling pathway inhibitors, Wnt signaling pathway activators, and Wnt signaling pathway inhibitors. Differentiation-inducing factors are used depending on the cell type and differentiation state (differentiation ability, potential). Differentiation inducers respond differently to different factors, and the effect of differentiation inducers may vary depending on the concentration and timing of addition of the differentiation inducer during the differentiation induction process. It is also known that the optimal concentration of differentiation inducers that exert similar effects varies depending on the animal species. For example, it is generally known that the optimal concentration of differentiation inducers is higher for human cells than for mouse cells (especially for ectoderm and endoderm). Many methods for inducing differentiation of pluripotent stem cells into specific cells or tissues have been reported, and differentiation inducers or differentiation inducement methods suitable for the desired cells or tissues can be selected.

[0167] The differentiation-inducing factor used in the present invention is typically a mammalian differentiation-inducing factor. Examples of mammals include those listed above. Differentiation-inducing factors may have cross-reactivity between mammalian species, so any undifferentiated state maintenance factor from any mammal may be used as long as it is capable of inducing differentiation of the pluripotent stem cells to be cultured. However, it is preferable to use a differentiation-inducing factor from the same mammalian species as the cells to be cultured.

[0168] The differentiation-inducing factor used in the present invention is preferably isolated. Thus, in one embodiment, the present invention includes a step of providing an isolated differentiation-inducing factor. In another embodiment, the present invention includes a step of exogenously adding the isolated differentiation-inducing factor to the medium used in step (3).

[0169] In one embodiment, in the method of the present invention, aggregates containing nervous system cells or nervous tissue can be produced by using a neuronal differentiation inducer as the differentiation inducer.

[0170] In one embodiment, the medium used in step (3) may contain, for example, a differentiation-inducing factor. The medium is a serum-free medium or serum medium (preferably a serum-free medium) containing a basement membrane preparation. A basement membrane preparation may or may not be added to such a medium. As the basement membrane preparation, the above-mentioned preparations can be used. When a basement membrane preparation is added, for example, when Matrigel is used, the concentration is 0.1 to 10% by volume, more preferably 0.5 to 2%. To avoid contamination with chemically unidentified substances, a basement membrane preparation is not added.

[0171] The serum-free medium or serum-free medium used for such a medium is not particularly limited as long as it is as described above. To avoid the complexity of the preparation, for example, a serum substitute such as commercially available KSR may be used. It is preferable to use a serum-free medium containing 10% KSR, 450 μM 1-monothioglycerol, and 1× Chemically Defined Lipid Concentrate in a 1:1 mixture of IMDM and F-12, or a medium containing 5% to 20% KSR, NEAA, pyruvic acid, and 2-mercaptoethanol in GMEM. The amount of KSR added to the serum-free medium is, for example, In this case, it is usually about 1% to about 20%, preferably about 2% to about 20%.

[0172] The medium (preferably serum-free medium) used in step (3) can be the same medium (preferably serum-free medium) used in step (2) as it is, or can be replaced with a new medium (preferably serum-free medium). When the medium is used as is in step (3), a differentiation-inducing factor may be added to the medium.

[0173] In step (3), when a medium exchange operation is performed, for example, the original medium is not discarded but a new medium is added. These include adding medium (medium addition operation), discarding about half of the original medium (about 40-80% of the volume of the original medium) and adding about half of the new medium (40-80% of the volume of the original medium) (half medium exchange operation), and discarding the entire original medium (more than 90% of the volume of the original medium) and adding the entire new medium (more than 90% of the volume of the original medium) (full medium exchange operation).

[0174] When adding a specific component (e.g., a differentiation-inducing factor) at a certain point, for example, after calculating the final concentration, approximately half of the original medium may be discarded and approximately half of a new medium containing the specific component at a concentration higher than the final concentration (specifically, 1.5 to 3.0 times the final concentration, for example, approximately twice the final concentration) may be added (half medium exchange operation, half medium exchange).

[0175] If, at a certain point, the concentration of a particular component contained in the original medium is to be maintained, for example, about half of the original medium may be discarded and about half of a new medium containing the particular component at the same concentration as that contained in the original medium may be added.

[0176] When components contained in the original medium are diluted to reduce their concentrations at a certain point, for example, the medium exchange procedure may be performed multiple times a day, preferably multiple times within an hour (e.g., 2 to 3 times).Furthermore, when components contained in the original medium are diluted to reduce their concentrations at a certain point, the cells or aggregates may be transferred to another culture vessel.

[0177] The tools used for the medium replacement operation are not particularly limited, and examples include pipettors, micropipettes, multichannel micropipettes, repeating dispensers, etc. For example, when a 96-well plate is used as the culture vessel, a multichannel micropipette may be used.

[0178] The differentiation-inducing factor may be added approximately 24 hours or later from the start of the suspension culture in step (2). Alternatively, the differentiation-inducing factor may be added to the medium within several days (for example, within 15 or 18 days) after the initiation of suspension culture. Preferably, the differentiation-inducing factor is added to the medium between days 1 and 18 or between days 1 and 15, more preferably between days 1 and 9, even more preferably between days 3 and 8 or between days 2 and 9, and even more preferably between days 3 and 6 after the initiation of suspension culture.

[0179] In a further embodiment, a differentiation-inducing factor (e.g., a Wnt signaling pathway inhibitor or a TGFβ family signaling pathway inhibitor) is administered simultaneously with the initiation of suspension culture in step (2) or approximately 24 It may be added within a period of time.

[0180] After the differentiation-inducing factor is added to the medium and the differentiation of the aggregates into neural cells is initiated, it is no longer necessary to add the differentiation-inducing factor to the medium, and the medium can be serum-free or serum-free without the differentiation-inducing factor. In one embodiment, after the induction of differentiation of the aggregates into neural cells has begun, the concentration of the differentiation-inducing factor in the medium is gradually or stepwise reduced by 40 to 60% every 2 to 4 days by replacing the medium with a serum-free medium or serum medium that does not contain the differentiation-inducing factor.

[0181] In a specific embodiment, on days 0 to 18, preferably days 1 to 9, more preferably days 2 to 8, and even more preferably days 3 or 4 after the start of suspension culture (i.e., after the start of step (2)), part or all of the medium can be replaced with a medium containing a differentiation-inducing factor, and the cells can be cultured in the presence of the differentiation-inducing factor for approximately 1 to 100 days. Here, part or all of the medium can be replaced with a medium containing the differentiation-inducing factor to maintain the same concentration of the differentiation-inducing factor. Alternatively, as described above, the concentration of the differentiation-inducing factor can be reduced in stages.

[0182] Cells in which differentiation induction into neural cells has begun can be identified, for example, by detecting the expression of neural marker genes in the cells. First, the aggregates formed in step (2) were analyzed to identify cells expressing nervous system marker genes. and a step of culturing the cells in suspension in a serum-free medium or serum medium containing a differentiation-inducing factor at a concentration necessary for inducing neural differentiation until the cells begin to express neural differentiation, thereby obtaining aggregates containing neural cells.

[0183] In one embodiment, a substance acting on the BMP signaling pathway is used as the differentiation inducer. That is, the aggregates obtained in the second step are cultured in suspension in the presence of a substance acting on the BMP signaling pathway. and obtain aggregates containing nervous system cells or nervous tissue.

[0184] A BMP signaling pathway agonist is a substance that can enhance the signaling pathway mediated by BMP. Examples of BMP signaling pathway agonists include BMP2, BMP4, and BMP7. Examples of the BMP protein include BMP proteins such as GDF7, GDF proteins such as GDF2, GDF4, and GDF7, anti-BMP receptor antibodies, and BMP partial peptides. BMP2, BMP4, and BMP7 proteins are available from, for example, R&D Systems, and GDF7 protein is available from, for example, Wako Pure Chemical Industries.

[0185] The concentration of a BMP signaling pathway agent is used to induce the proliferation and differentiation of pluripotent stem cells or stem cells derived therefrom. Any concentration that can induce differentiation of the aggregates into neural cells may be used. For example, in the case of human BMP4, it is added to the medium at a concentration of about 0.01 nM to about 1 μM, preferably about 0.1 nM to about 100 nM, more preferably about 1 nM to about 10 nM, and even more preferably about 1.5 nM (55 ng / mL).

[0186] In one aspect, differentiation into nervous system cells or nervous tissue can also be achieved by carrying out step (3) in a serum-free medium or serum-free medium (preferably a serum-free medium) that does not contain the above-mentioned differentiation-inducing factor, or in a serum-free medium or serum-free medium (preferably a serum-free medium) that does not contain the above-mentioned undifferentiated maintenance factor. For example, BMP signaling pathway agonists (e.g., BMP4) and sonic hedgehog signaling pathway agonists (e.g., SAG, PMA) can be used to induce spontaneous differentiation. The aggregates obtained in step (2) are then dissolved in a serum-free medium or serum-free medium (preferably a serum-free medium) that does not contain (i.e., does not contain any of the above or, if it does contain any of the above, the concentration of the above is below the concentration at which physiological activity is expressed). Aggregates containing nervous system cells or nervous tissue can also be obtained by suspension culture.

[0187] Such a medium is not particularly limited as long as it is as described above. To avoid the complicated preparation, for example, a serum-free medium (e.g., IMDM) containing an appropriate amount of a commercially available serum substitute such as KSR may be used. It is preferable to use a serum-free medium containing a 1:1 mixture of F-12 and 10% KSR, 450 μM 1-monothioglycerol, and 1x Chemically Defined Lipid Concentrate. For human ES cells, the amount of KSR added to the serum-free medium is usually about 1% to about 20%, preferably about 2% to about 20%.

[0188] The medium (preferably a serum-free medium) used in step (3) can be the same medium (preferably a serum-free medium) used in step (2) as is, or can be replaced with a new medium (preferably a serum-free medium).

[0189] In step (3), the culture conditions such as culture temperature and CO2 concentration can be set appropriately. The culture temperature is, for example, about 30° C. to about 40° C., preferably about 37° C. The CO2 concentration is, for example, about 1% to about 10%, preferably about 5%.

[0190] The fact that an aggregate containing neural cells has been obtained can be confirmed, for example, by detecting whether the aggregate contains cells expressing a neural cell marker. Examples of neural cell markers include, but are not limited to, Nestin, TuJ1, PSA-NCAM, etc. In one embodiment, the culture in step (3) is carried out until 20% or more (preferably, 30% or more, 40% or more, 50% or more, or 60% or more) of the cells contained in the aggregate express any one of the neural cell markers selected from the group consisting of Nestin, TuJ1, and PSA-NCAM. will be done.

[0191] The resulting aggregates containing neural cells may be used as they are as reagents for evaluating toxicity and efficacy. Highly purified neural cells can also be obtained by dispersing the aggregates containing neural cells (e.g., with trypsin / EDTA or papain) and then sorting the resulting cells using FACS or MACS.

[0192] In one embodiment, the aggregates containing neural cells obtained in step (3) include neuroepithelial cells. The neuroepithelial structure is present so as to cover the surface of the aggregate, but is also partially formed inside the aggregate. By using the aggregates obtained in step (2), aggregates containing neuroepithelial structures can be induced with high efficiency. Neuroepithelial structures can be identified as epithelial structures positive for nervous system marker genes (e.g., Nestin, TuJ1, PSA-NCAM). The neuroepithelial structures can then be obtained as neural tissue. In one embodiment, the culturing in step (3) is carried out until neuroepithelial structures are formed in the aggregates. In one embodiment of step (3), the culture of the epithelial structures formed in step (2) is carried out. and a step of culturing the resulting aggregates in suspension under neurodifferentiation-inducing conditions (e.g., in a serum-free medium or serum medium containing a differentiation-inducing factor at a concentration required for neurodifferentiation induction) until neuroepithelial structures begin to appear, thereby obtaining aggregates containing neuroepithelial structures.

[0193] In one embodiment, the aggregates obtained in step (2) or (3) may be dispersed and seeded on a cell culture dish for adhesion culture to produce nervous system cells or nervous tissue. Alternatively, in one embodiment, the aggregates obtained in step (2) or (3) may be dispersed, and the dispersed cells may be seeded on a cell culture dish for adhesion culture to produce nervous system cells or nervous tissue. The medium may be the above-mentioned medium, and a differentiation-inducing factor may be added to the medium. The cell culture dish may be the above-mentioned dish for adhesion culture. The cell culture dish may be coated with a cell adhesion factor or the like (e.g., laminin coating). The production of nervous system cells or nervous tissue can be confirmed by immunostaining for nervous system cell or nervous tissue markers.

[0194] In one embodiment, aggregates not containing neural cells obtained during the suspension culture in step (2) or step (3) may be seeded on an adhesion culture dish and cultured in an adhesion culture until differentiation into neural cells. The medium used for adhesion culture is not particularly limited, but may be the medium used in step (3) above. It may also be a culture medium.

[0195] In one embodiment, cells not containing neural cells obtained during the adhesion culture in step (2) or step (3) are detached from the adhesion culture dish and seeded on a suspension culture dish. The medium used for the suspension culture is not particularly limited, and may be the medium used in step (3).

[0196] The neuroepithelial structure present on the surface of the aggregate can also be physically excised from the aggregate using tweezers or the like under a microscope.

[0197] The production method of the present invention enables highly efficient production of neural tissues such as neuroepithelial structures from pluripotent stem cells. Because the neuroepithelial structures obtained by the production method of the present invention contain various neural cells, it is also possible to isolate various neural cells and their progenitor cells by FACS or the like using antibodies against markers of various neural cells.

[0198] The resulting aggregates containing neural tissue such as neuroepithelial structures may be used as reagents for evaluating toxicity and efficacy. Highly purified neural cells can also be obtained by dispersing the aggregates containing neural tissue such as neuroepithelial structures (e.g., with trypsin / EDTA or papain) and then sorting the resulting cells using FACS or MACS.

[0199] 3. Method for producing retinal tissue Production method 2 of the present invention is a method for producing retinal tissue, comprising the following steps (1) to (3): (1) Pluripotent stem cells were cultured in the absence of feeder cells, and 1) TGFβ family signaling pathway was Pathway inhibitors and / or Sonic Hedgehog signaling pathway agonists, and 2) untreated a first step of culturing in a medium containing a differentiation maintenance factor; (2) a second step of culturing the cells obtained in the first step in suspension to form cell aggregates; and (3) A third step in which the aggregates obtained in the second step are cultured in suspension in the presence of a substance acting on the BMP signaling pathway to obtain aggregates containing retinal tissue.

[0200] Step (1) of Production Method 2 of the present invention can be carried out in the same manner as step (1) of Production Method 1 of the present invention.

[0201] Preferably, the cells obtained by step (1) are at least retinal tissue, retinal cells, retinal In one embodiment, the cells obtained by step (1) include at least retinal tissue, retinal cells, retinal progenitor cells, or stem cells capable of differentiating into retinal layer-specific neural cells. In one embodiment, the cells obtained by step (1) contain Oct3 / 4-positive stem cells that are capable of differentiating into retinal layer-specific neurons or retinal layer-specific neurons. Contains more than %.

[0202] Step (2) of Production Method 2 of the present invention can be carried out in the same manner as step (2) of Production Method 1 of the present invention.

[0203] The medium used in step (2) of production method 2 preferably contains a substance that acts on the Sonic Hedgehog signaling pathway. In step (1), pluripotent stem cells are treated with a TGFβ family signaling pathway inhibitor and / or a substance that acts on the Sonic Hedgehog signaling pathway. In step (2), the cells obtained in step (1) are subjected to suspension culture in a medium (preferably a serum-free medium) containing a substance that acts on the Sonic Hedgehog signaling pathway to form aggregates. This further improves the quality of the aggregates and enhances their ability to differentiate into retinal tissue. By using these high-quality aggregates, aggregates containing retinal tissue can be induced with high efficiency.

[0204] As the sonic hedgehog signaling pathway active substance, those mentioned above can be used. Preferably, the sonic hedgehog signaling pathway active substance is an active substance that inhibits the Shh protein. The Sonic Hedgehog signaling pathway agonists in the culture medium are α-glucan, SAG, or PMA. The concentration can be appropriately set within a range that can achieve the above-mentioned effects. SAG is usually used at a concentration of 1 to 2000 nM, preferably 10 nM to 700 nM, and more preferably 30 to 600 nM. PMA is usually used at a concentration of 0.002 to 20 μM, preferably 0.02 to 2 μM. Shh protein is usually used at a concentration of 20 to 1000 ng / ml, preferably 50 to 300 ng / ml. When using a substance acting on the Sonic Hedgehog signaling pathway other than Shh protein, SAG, or PMA, it is desirable to use it at a concentration that exhibits Sonic Hedgehog signaling promoting activity equivalent to the above-mentioned concentration of SAG.

[0205] The concentration of the Sonic hedgehog signaling pathway agent in the medium is For example, at the start of step (2), the amount of Sonic Hedgehog The concentration of the substance acting on a signal transduction pathway may be within the above range, and may be gradually or stepwise reduced at a rate of 40 to 60% reduction per 2 to 4 days.

[0206] The timing of adding the Sonic hedgehog signaling pathway active substance to the medium is not particularly limited as long as the above-mentioned effect can be achieved, but the earlier the addition, the greater the effect. The Sonic hedgehog signaling pathway active substance is added to the medium usually within 6 days, preferably within 3 days, more preferably within 1 day, and even more preferably at the start of step (2) from the start of step (2). will be done.

[0207] In a preferred embodiment, the human cells obtained in step (1) (e.g., cells obtained from human iPS cells in step (1)) are subjected to suspension culture in a serum-free medium containing a substance acting on the Sonic Hedgehog signaling pathway (e.g., SAG, PMA, Shh protein) to form aggregates. The substance acting on the Sonic Hedgehog signaling pathway is preferably contained in the medium from the start of suspension culture. A ROCK inhibitor (e.g., Y-27632) may also be added to the medium. The culture time is 12 hours to 6 days, preferably 12 hours to 48 hours. The formed aggregates are preferably uniform aggregates.

[0208] For example, the human cells obtained in step (1) (e.g., cells obtained from human iPS cells in step (1)) The cells are collected, dispersed to single cells or a state close to single cells, and subjected to suspension culture in a serum-free medium containing a sonic hedgehog signaling pathway activator (e.g., SAG, PMA). The serum-free medium may also contain a ROCK inhibitor (e.g., Y-27632). A suspension of cells (e.g., stem cells derived from human iPS cells) is seeded in the above-mentioned culture vessel and dispersed. The cells are cultured under non-adhesive conditions on the culture vessel, allowing multiple cells to aggregate and form aggregates. The culture time is 12 hours to 6 days (preferably 12 hours to 48 hours). The aggregates formed are preferably uniform aggregates.

[0209] In this way, by carrying out step (2), aggregates of the cells obtained in step (1) or cells derived therefrom are formed. The present invention also provides a method for producing such aggregates. The aggregates obtained in step (2) are obtained by the step (1) of inhibiting TGF-β family signaling. The quality of the resulting aggregates is higher than that of those not treated with a cell proliferation pathway inhibitor and / or a sonic hedgehog signaling pathway agonist. Specifically, a population of aggregates can be obtained that is round, has a smooth surface, is dense inside, and is rich in aggregates that do not lose their shape. In one embodiment, when aggregates (e.g., 100 or more) are randomly selected on the sixth day from the start of the second step, the proportion of aggregates that do not form cysts is, for example, 70% or more, preferably 80% or more.

[0210] The aggregates obtained in step (2) have the ability to differentiate into retinal tissue. The aggregates obtained in step (2) can be differentiated into retinal tissue by culturing the aggregates under the conditions of step (3) below. It is possible to produce aggregates containing

[0211] In one embodiment, at least retinal tissue, retinal cells, retinal progenitor cells obtained in step (1) By using stem cells capable of differentiating into retinal tissue, retinal cells, retinal progenitor cells, or retinal layer-specific nerve cells (preferably Oct3 / 4-positive stem cells capable of differentiating into at least retinal tissue, retinal cells, retinal progenitor cells, or retinal layer-specific nerve cells) in step (2), it is possible to differentiate at least retinal tissue, retinal cells, Aggregates containing stem cells (eg, Oct3 / 4-positive stem cells) that have the potential to differentiate into cytoplasmic cells, retinal progenitor cells, or retinal layer-specific neural cells can be obtained.

[0212] Step (3) of culturing the aggregates formed in step (2) in suspension in the presence of a substance acting on the BMP signaling pathway to obtain aggregates containing retinal tissue will now be described.

[0213] The medium used in step (3) is, for example, a serum-free medium or a serum-based medium (preferably a serum-free medium) supplemented with a substance acting on the BMP signaling pathway.

[0214] The serum-free medium or serum-free medium used for such a medium is not particularly limited as long as it is as described above. To avoid the complexity of the preparation, for example, a serum substitute such as commercially available KSR may be used. Serum-free medium (e.g., a 1:1 mixture of IMDM and F-12 with 10% KSR, 450 μM 1-monophosphate buffer, It is preferable to use a medium supplemented with thioglycerol and 1× Chemically Defined Lipid Concentrate. The amount of KSR to be added to a serum-free medium is, for example, In the case of cells (e.g., iPS cells), it is usually about 1% to about 20%, preferably about 2% to about 20%. is.

[0215] The medium (preferably, serum-free medium) used in step (3) can be the same medium (preferably, serum-free medium) used in step (2) as it is, or can be replaced with a new medium (preferably, serum-free medium). When the medium used in step (2) that does not contain a substance of the BMP signaling pathway is used as it is in step (3), a substance acting on the BMP signaling pathway can be added to the medium.

[0216] Examples of substances acting on the BMP signaling pathway used in step (3) include BMP proteins such as BMP2, BMP4, and BMP7, GDF proteins such as GDF7, anti-BMP receptor antibodies, and BMP partial peptides. BMP2, BMP4, and BMP7 proteins are available from, for example, R&D Systems, and GDF7 protein is available from, for example, Wako Pure Chemical Industries. A preferred example of a substance acting on the BMP signaling pathway is BMP4.

[0217] The concentration of BMP signaling pathway agonists influences the retinal function of cells forming pluripotent stem cell aggregates. For example, in the case of human BMP4, the concentration is from about 0.01 nM to about 100 nM. It is added to the medium at a concentration of about 1 μM, preferably about 0.1 nM to about 100 nM, more preferably about 1 nM to about 10 nM, and even more preferably about 1.5 nM (55 ng / mL). When a substance acting on the signal transduction pathway is used, the BMP signal transduction promoting activity is equivalent to the above concentration of BMP4. It is desirable to use it at a concentration that shows

[0218] The concentration of the substance acting on the BMP signaling pathway in the medium may be varied during step (3). For example, the concentration of the substance acting on the BMP signaling pathway may be set within the above range at the start of step (3), and then gradually or stepwise reduced by 40 to 60% every 2 to 4 days.

[0219] The substance acting on the BMP signaling pathway may be added to the medium at least about 24 hours after the initiation of suspension culture in step (2), or may be added within several days (e.g., within 15 days) after the initiation of suspension culture. Preferably, the substance acting on the BMP signaling pathway is added to the medium between days 1 and 15, more preferably between days 1 and 9, even more preferably between days 3 and 8 or between days 2 and 9, and even more preferably between days 3 and 6 after the initiation of suspension culture.

[0220] In a specific embodiment, on the 1st to 9th day after the start of the suspension culture (i.e., after the start of the step (2)), Preferably, on days 2 to 8, more preferably days 3 or 4, the medium is partially or completely replaced with a medium containing BMP4, and the final BMP4 concentration is adjusted to about 1 to 10 nM. The cells are cultured in the presence of BMP4 for, for example, 1 to 12 days, preferably 2 to 9 days, and more preferably 2 to 5 days. Here, to maintain the same BMP4 concentration, the medium can be partially or completely replaced with a medium containing BMP4 once or twice. Alternatively, the BMP4 concentration can be reduced stepwise, as described above.

[0221] BMP signaling pathway agonists are added to the culture medium and directed to the retinal cells of the cells that form the aggregates. Once differentiation induction has begun, it is no longer necessary to add BMP signaling pathway agents to the culture medium. The medium was replaced with serum-free medium or serum-free medium that did not contain BMP signaling pathway active substances. In one embodiment, after the induction of differentiation into retinal cells, BMP signal transduction may be performed. By replacing the medium with serum-free medium or serum-free medium that does not contain pathway-active substances, BMP in the medium The concentration of the signal transduction pathway active substance is gradually or stepwise reduced by 40 to 60% every 2 to 4 days. Cells in which differentiation induction into retinal cells has begun can be confirmed, for example, by detecting the expression of retinal progenitor cell marker genes (e.g., Rx gene (also known as Rax), Pax6 gene, Chx10 gene) in the cells. Fluorescent reporter protein genes such as GFP can also be detected. Generated by step (2) using pluripotent stem cells in which the gene was knocked into the Rx locus The aggregates were incubated in the presence of a BMP signaling pathway agonist at a concentration required for inducing differentiation into retinal cells. The time when differentiation into retinal cells begins can be confirmed by detecting the fluorescence emitted from the expressed fluorescent reporter protein. In one embodiment, the aggregates formed in step (2) are transfected with a retinal progenitor cell marker gene ( The retina is the stage where cells expressing the Rx gene, Pax6 gene, and Chx10 gene begin to appear. A serum-free medium containing a BMP signaling pathway active substance at a concentration necessary for inducing differentiation into membranous cells, or and a step of culturing the cells in suspension in a serum-containing medium to obtain aggregates containing retinal progenitor cells.

[0222] In step (3), when a medium exchange operation is performed, for example, the original medium is not discarded but a new medium is added. These include adding medium (medium addition operation), discarding about half of the original medium (about 40-80% of the volume of the original medium) and adding about half of the new medium (40-80% of the volume of the original medium) (half medium exchange operation), and discarding the entire original medium (more than 90% of the volume of the original medium) and adding the entire new medium (more than 90% of the volume of the original medium) (full medium exchange operation).

[0223] When adding a specific component (e.g., BMP4) at a certain point, for example, after calculating the final concentration, discard about half of the original medium and add about half of a new medium containing the specific component at a concentration higher than the final concentration (specifically, 1.5 to 3.0 times the final concentration, e.g., about twice the final concentration). (Half-medium exchange operation, half-medium exchange) may be performed.

[0224] If, at a certain point, the concentration of a particular component contained in the original medium is to be maintained, for example, about half of the original medium may be discarded and about half of a new medium containing the particular component at the same concentration as that contained in the original medium may be added.

[0225] When components contained in the original medium are diluted to reduce their concentrations at a certain point, for example, the medium exchange procedure may be performed multiple times a day, preferably multiple times within an hour (e.g., 2 to 3 times).Furthermore, when components contained in the original medium are diluted to reduce their concentrations at a certain point, the cells or aggregates may be transferred to another culture vessel.

[0226] The tools used for the medium exchange operation are not particularly limited, but may include, for example, a pipetter, a micropipettor, Examples of suitable micropipettes include a micropipette, a multichannel micropipette, a repeating dispenser, etc. For example, when a 96-well plate is used as the culture vessel, a multichannel micropipette may be used.

[0227] In one embodiment, the concentration of the Shh signaling pathway active substance added to the medium in step (2) is relatively low (for example, 700 nM or less for SAG, 700 nM or less for other Shh signaling pathway active substances). In this regard, in the case of a concentration that exhibits Shh signaling promoting activity equivalent to or lower than that of SAG at the aforementioned concentration, it is not necessary to change the medium, and a differentiation-inducing factor (e.g., BMP4) can be added to the medium used in step (2). On the other hand, when the concentration of the substance acting on the Shh signaling pathway is relatively high (for example, more than 700 nM or 1000 nM or more for SAG, or a concentration that exhibits Shh signaling-promoting activity equivalent to that of SAG at the above concentrations for other substances acting on the Shh signaling pathway), it is desirable to replace the medium with a fresh medium containing a differentiation-inducing factor (e.g., BMP4) to suppress the effects of any remaining substance acting on the Shh signaling pathway at the time of addition of the differentiation-inducing factor.

[0228] In a preferred embodiment, the concentration of the substance acting on the Shh signaling pathway in the medium used in step (3) is 700 nM or less, preferably 300 nM or less, more preferably 10 nM or less, even more preferably 0.1 nM or less, and even more preferably does not contain any substance acting on the Shh signaling pathway, in terms of the Shh signaling promoting activity of SAG. A medium that is "free of a substance acting on the Shh signaling pathway" refers to a medium that is substantially free of a substance acting on the Shh signaling pathway, for example, a medium that does not contain a substance acting on the Shh signaling pathway at a concentration that adversely affects selective differentiation into retinal progenitor cells and retinal tissue. A medium "free of Shh signaling pathway active substances" also includes a medium that is substantially free of Shh signaling pathway active substances, e.g., a medium that does not contain Shh signaling pathway active substances at concentrations that adversely affect selective differentiation into retinal progenitor cells and retinal tissue. Also included are media with no added nutrients.

[0229] In step (3), the culture conditions such as culture temperature and CO2 concentration can be set appropriately. The culture temperature is, for example, about 30° C. to about 40° C., preferably about 37° C. The CO2 concentration is, for example, about 1% to about 10%, preferably about 5%.

[0230] By such culturing, the cells forming the aggregates obtained in step (2) are transformed into retinal progenitor cells. Differentiation is induced, and aggregates containing retinal progenitor cells can be obtained. The present invention also provides a method for producing such aggregates containing retinal progenitor cells. The fact that aggregates containing retinal progenitor cells have been obtained can be confirmed by, for example, detecting that cells expressing Rx, PAX6, or Chx10, which are markers for retinal progenitor cells, are present. It can be confirmed by detecting that the compound is contained in an aggregate. In one embodiment, the aggregates formed in step (2) are treated by incubation at a temperature where cells expressing the Rx gene appear. The BMP signaling pathway activator was used at a concentration sufficient to induce differentiation into retinal cells until the cells began to differentiate. and a step of suspension culturing the cells in a serum-free medium or serum medium containing the above-mentioned compound to obtain an aggregate containing retinal progenitor cells. In one embodiment, the culture in step (3) is carried out until 20% or more (preferably 30% or more, 40% or more, 50% or more, or 60% or more) of the cells contained in the aggregate express Rx.

[0231] In a preferred embodiment of the method for producing retinal cells and / or retinal tissue, in step (1), Human pluripotent stem cells (e.g., human iPS cells) are cultured in a serum-free medium containing a TGFβ signaling pathway inhibitor (e.g., SB431542, A-83-01) and bFGF in the absence of feeder cells. In step (2), the cells are treated with a sonic hedgehog signaling pathway agonist ( In step (3), the aggregates are cultured in suspension in a serum-free medium containing a substance acting on the BMP signaling pathway (e.g., BMP4). do.

[0232] In a preferred embodiment of the method for producing retinal cells and / or retinal tissue, the step (1 ) human pluripotent stem cells (e.g., human iPS cells) were cultured in an adherent manner in a serum-free medium containing a BMP signaling pathway inhibitor (e.g., LDN193189) and bFGF in the absence of feeder cells. In step (2), the cells are cultured in suspension in a serum-free medium containing or not containing a sonic hedgehog signaling pathway active substance (e.g., SAG, PMA), and in step (3), the aggregates are cultured in suspension in a serum-free medium containing a BMP signaling pathway active substance (e.g., BMP4).

[0233] In a preferred embodiment of the method for producing retinal cells and / or retinal tissue, in step (1), Human pluripotent stem cells (e.g., human iPS cells) were cultured in the absence of feeder cells using sonic The cells are cultured in a serum-free medium containing a substance acting on the sonic hedgehog signaling pathway (e.g., SAG, PMA) and bFGF for preferably 1 to 6 days, more preferably 2 to 4 days, as an adherent culture. In step (2), the cells are cultured in a serum-free medium containing a substance acting on the sonic hedgehog signaling pathway (e.g., SAG, PMA). In step (3), the aggregates are cultured in a suspension culture in a serum-free medium containing a substance acting on the sonic hedgehog signaling pathway (e.g., SAG, PMA). The cells are cultured in suspension in a serum-free medium containing an active substance (eg, BMP4).

[0234] In a preferred embodiment of the method for producing retinal cells and / or retinal tissue, in step (1), Human pluripotent stem cells (e.g., human iPS cells) are cultured in the absence of feeder cells. TGFβ family signaling pathway inhibitors (e.g., TGFβ signaling pathway inhibitors (e.g., Lefty, SB431542, A-83-01), Nodal / Activin signaling pathway inhibitors (e.g., Lefty, SB431542, A-83-01), BMP signaling pathway inhibitors (e.g., LDN193189), or combinations thereof (e.g., SB431542 and LDN193189), etc.); Sonic hedgehog signaling pathway agonists (e.g., Shh protein, SAG, PMA); or TGFβ family signaling pathway inhibitors (e.g., Lefty, SB431542, A-83-01, LDN193189) and sonic hedgehog signaling pathway agonists (e.g., Shh protein, SAG, PMA) ) in combination with; and Adherent culture in serum-free medium containing bFGF In step (2), the cells obtained in step (1) are cultured in suspension in a serum-free medium containing a sonic hedgehog signaling pathway activator (e.g., SAG, PMA, Shh protein), and cell aggregation is achieved. Forming a conglomerate, In step (3), the aggregates are cultured in suspension in a serum-free medium containing a substance acting on the BMP signaling pathway (for example, BMP4) to obtain aggregates containing retinal progenitor cells, retinal cells, or retinal tissue. The medium in step (2) preferably contains a ROCK inhibitor (e.g., Y-27632).

[0235] The obtained aggregates containing retinal progenitor cells may be used as they are as reagents for evaluating toxicity and efficacy. Highly purified retinal progenitor cells can also be obtained by dispersing the aggregates containing retinal progenitor cells (e.g., with trypsin / EDTA or papain) and sorting the resulting cells using FACS or MACS.

[0236] Furthermore, by continuing to culture the aggregates containing retinal progenitor cells in serum-free or serum-based medium, the retinal progenitor cells can be further differentiated to produce retinal tissue resembling neuroepithelial structures.

[0237] The serum-free medium or serum medium used for such a medium is not particularly limited as long as it is as described above. For example, a serum medium containing 10% fetal bovine serum, N2 supplement, 100 μM taurine, and 500 nM retinoic acid in DMEM-F12 medium, or a commercially available serum substitute such as KSR, may be used. Examples of such a medium include a serum-free medium supplemented with an appropriate amount of 10% KSR, 450 μM 1-monothioglycerol, and 1× Chemically Defined Lipid Concentrate (for example, a medium prepared by adding 10% KSR, 450 μM 1-monothioglycerol, and 1× Chemically Defined Lipid Concentrate to a 1:1 mixture of IMDM and F-12).

[0238] The culture time for inducing retinal tissue from retinal progenitor cells is determined based on the desired retinal layer-specific neural cells. The incubation period varies depending on the cell, but is, for example, from about 7 days to about 200 days.

[0239] The retinal tissue exists so as to cover the surface of the aggregates. After completion of the suspension culture, the aggregates are fixed using a fixative such as paraformaldehyde solution, frozen sections are prepared, and the formation of retinal tissue with a layered structure can be confirmed by immunostaining or the like. Since the retinal tissue has different retinal progenitor cells (photoreceptors, horizontal cells, bipolar cells, amacrine cells, and retinal ganglion cells) that make up each layer, the formation of a layered structure can be confirmed by immunostaining using antibodies against the above-mentioned markers expressed in these cells. In one aspect, the retinal tissue is an Rx or Chx10-positive neuroepithelial structure.

[0240] It is also possible to physically cut out the retinal tissue present on the surface of the aggregate using tweezers, etc. In this case, since neural tissue other than retinal tissue may be formed on the surface of each aggregate, it is possible to confirm that the tissue is retinal tissue by cutting out a portion of the neural tissue cut out from the aggregate and using the same for confirmation by the immunostaining method described below, etc.

[0241] In one embodiment, the aggregates obtained in step (3) contain retinal tissue and head non-neural ectoderm. In an aggregate that contains retinal tissue and is substantially free of head non-neural ectoderm, for example, in an immunostained image of a frozen section of the aggregate, Rx-positive tissue is observed, and no Rx-negative tissue is observed outside of it.

[0242] In one embodiment of step (3), the aggregates formed in step (2) are treated with a medium necessary for inducing differentiation into retinal cells until cells expressing the Rx gene and / or Chx10 gene begin to appear. and then culture the cells in suspension in a serum-free medium or serum-free medium containing a BMP signaling pathway agent at a concentration suitable for the cell type. and obtaining an aggregate containing retinal progenitor cells, and subsequently culturing the aggregate in suspension in a serum-free medium or a serum medium until retinal tissue is formed, thereby obtaining an aggregate containing retinal tissue. When the aggregate containing retinal progenitor cells is subsequently cultured in suspension in a serum-free medium or a serum medium until retinal tissue is formed, the aggregate containing retinal progenitor cells may be cultured in suspension in a serum-free medium or a serum medium containing a substance acting on the BMP signaling pathway. By replacing the medium with serum-free or serum-free medium, the concentration of BMP signaling pathway active substances contained in the medium to induce retinal progenitor cells was reduced by 40-60% every 2-4 days. In one embodiment, 20% or more (preferably 30% or more, 40% or more, 50% or more, 60% or more) of the cells contained in the aggregate express Chx10. The aggregates containing retinal progenitor cells are subjected to suspension culture until they reach a state where they can be cultured.

[0243] In addition, as one embodiment of step (3), the aggregate obtained in step (2) or the aggregate obtained in step (2) The aggregates obtained in step (1) may be subjected to suspension culture by the above method and then subjected to adhesion culture to form adhesive aggregates. Until the cells begin to appear, the BMP signaling pathway is maintained at a concentration sufficient to induce differentiation into retinal cells. The aggregate containing retinal progenitor cells is cultured in a serum-free medium or serum-containing medium containing a cytotoxic substance to obtain an aggregate containing retinal progenitor cells. The aggregate containing retinal progenitor cells is then cultured in a serum-free medium or serum-containing medium to obtain an aggregate containing retinal tissue. In one embodiment, 10% or more (preferably 20% or more, 30% or more, 40% or more, 50% or more) of the cells express Chx10. The aggregates containing retinal progenitor cells are cultured in an adherent culture until they reach a retinal progenitor cell state.

[0244] Production method 2 of the present invention makes it possible to obtain retinal tissue from pluripotent stem cells with high efficiency. The retinal tissue obtained by the production method 2 of the present invention contains neurons specific to each retinal layer. Since the retinal tissue contains neurons (neurons), it is possible to obtain cells that constitute retinal tissue, such as photoreceptors, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells, or their precursor cells. The identity of the cells obtained from the obtained retinal tissue can be confirmed by a method known per se, for example, by the expression of a cell marker.

[0245] The obtained aggregates containing retinal tissue may be used as they are as a reagent for evaluating toxicity and efficacy. Highly purified retinal tissue-constituting cells, such as highly purified photoreceptors, can also be obtained by dispersing the aggregates containing retinal tissue (e.g., by trypsin / EDTA treatment) and selecting the resulting cells using FACS or MACS.

[0246] A ciliary marginal zone-like structure can be produced from the cell aggregate containing retinal tissue obtained by Production Method 2 of the present invention or the like, by the following steps (A) and (B).

[0247] The ciliary marginal zone-like structure of the present invention refers to a structure similar to the ciliary marginal zone. The "ciliary marginal zone (CMZ)" can be exemplified by a tissue present in the boundary region between retinal tissue (specifically, neural retina) and retinal pigment epithelium in a living retina, and a region containing retinal tissue stem cells (retinal stem cells). The ciliary marginal zone is also called the ciliary margin or retinal margin, and the ciliary marginal zone, ciliary margin, and retinal margin are equivalent tissues. The ciliary marginal zone is known to play an important role in supplying retinal progenitor cells and differentiated cells to retinal tissue, maintaining retinal tissue structure, etc. Marker genes for the ciliary marginal zone include, for example, the Rdh10 gene (positive) and Examples include the Otx1 gene (positive) and the Zic1 gene (positive).

[0248] First, in step (A), a cell aggregate containing retinal tissue obtained by Production Method 2 of the present invention, in which the proportion of Chx10-positive cells in the retinal tissue is 20% or more and 100% or less, is cultured in a serum-free medium or a serum-based medium containing a substance acting on the Wnt signaling pathway and / or a substance inhibiting the FGF signaling pathway, for a period until cells expressing the RPE65 gene appear. Only cultivated in this way.

[0249] Here, a preferred culture for step (A) is suspension culture.

[0250] The serum-free medium used in step (A) may be a serum-free medium in which N2 or KSR is added to a basal medium, more specifically, a serum-free medium in which N2 supplement (Life Technologies) is added to a DMEM / F12 medium. The serum medium may be a serum-free medium in which fetal bovine serum is added to a basal medium.

[0251] The culture conditions in step (A), such as the culture temperature and CO2 concentration, may be set appropriately. The culture temperature may be, for example, in the range of about 30°C to about 40°C. Preferably, it is, for example, about 37°C. The CO2 concentration may be, for example, in the range of about 1% to about 10%, and preferably, for example, about 5%.

[0252] In step (A), the "cell aggregate containing retinal tissue" is cultured in a serum-free medium or a serum-containing medium. During the culture, the substance acting on the Wnt signaling pathway to be contained in the medium is not particularly limited as long as it can enhance the signaling mediated by Wnt. Examples of substances acting on the GSK3β pathway include proteins belonging to the Wnt family (e.g., Wnt1, Wnt3a, Wnt7a), Wnt receptors, Wnt receptor agonists, and GSK3β inhibitors (e.g., 6-Bromoindirubin-3'-oxime (BIO), CHIR99021, Kenpaullone).

[0253] The concentration of the substance acting on the Wnt signaling pathway contained in the serum-free medium or serum-free medium in step (A) is, for example, about 0.1 in the case of a common substance acting on the Wnt signaling pathway such as CHIR99021. The concentration ranges from about 1 μM to about 100 μM. Preferably, the concentration ranges from about 1 μM to about 30 μM. More preferably, the concentration is about 3 μM.

[0254] The "cell aggregates containing retinal tissue" of step (A) are cultured in a serum-free medium or a serum-containing medium. In this case, the FGF signaling pathway inhibitor contained in the medium is not particularly limited as long as it can inhibit signal transduction mediated by FGF. Examples of harmful substances include FGF receptors, FGF receptor inhibitors (e.g., SU-5402, AZD4547, BGJ398), MAP kinase cascade inhibitors (e.g., MEK inhibitors, MAPK inhibitors, ERK inhibitors), and the like. ), PI3 kinase inhibitors, Akt inhibitors, etc.

[0255] The concentration of the FGF signaling pathway inhibitor contained in the serum-free medium or serum-based medium in step (A) may be any concentration that can induce differentiation of the aggregates into periciliary margin-like structures. For example, SU-5402 about 0.1 μM to about 100 μM, preferably about 1 μM to about 30 μM, more preferably about 5 μM Add at a concentration of

[0256] In step (A), "culturing only during the period until cells expressing the RPE65 gene appear" means culturing only during the entire period until cells expressing the RPE65 gene appear or This means culturing only a portion of the retinal tissue. In other words, the period during which the "cell aggregates containing retinal tissue" present in the culture system are composed of cells that do not substantially express the RPE65 gene. It is sufficient to culture all or only a portion of the cells (for any period of time), and by adopting such culture, it is possible to obtain cell aggregates in which cells expressing the RPE65 gene do not appear.

[0257] To set such a specific period, the "cell aggregate containing retinal tissue" is used as a sample, and the presence or absence of expression of the RPE65 gene contained in the sample and its level are determined by conventional genetic engineering techniques. Specifically, for example, a method of immunostaining frozen sections of the "cell aggregates containing retinal tissue" using an antibody against the RPE65 protein may be used. This can be used to examine whether or not the RPE65 gene is expressed, and its level.

[0258] The "period until cells expressing the RPE65 gene appear" in step (A) may be, for example, a period until the proportion of Chx10-positive cells in the retinal tissue increases with the increase in the proportion of the cells expressing the RPE65 gene in a serum-free medium or a serum-free medium containing a substance acting on the Wnt signaling pathway and / or a substance inhibiting the FGF signaling pathway. The period during which the number of cells expressing the RPE65 gene decreases from the time of the start of culturing the retina to within the range of 30% to 0%. List cell aggregates in which the percentage of Chx10-positive cells in the tissue is between 30% and 0%. It is possible.

[0259] The number of days for the "period until cells expressing the RPE65 gene appear" in step (A) is determined based on the Wnt Type of signal transduction pathway active substance and / or FGF signal transduction pathway inhibitor, serum-free medium The period may vary depending on the type of serum medium, other culture conditions, etc., but may be, for example, within 14 days. More specifically, when a serum-free medium (for example, a serum-free medium in which N2 is added to a basal medium) is used, the period may preferably be, for example, within 10 days, and more preferably, for example, within 3 to 6 days. When a serum medium (for example, a serum medium in which fetal bovine serum is added to a basal medium) is used, the period may preferably be, for example, within 12 days, and more preferably, for example, within 6 days. The period can be anywhere from 1 to 9 days.

[0260] Next, in step (B), the "cell aggregates in which cells expressing the RPE65 gene have not appeared" obtained by culturing as described above were cultured in a blood-free medium containing no substance acting on the Wnt signaling pathway. The cells are cultured in a serum-free medium or serum-free medium.

[0261] A preferred culture in step (B) is, for example, suspension culture.

[0262] The serum-free medium in step (B) preferably does not contain an FGF signaling pathway inhibitor.

[0263] The serum-free medium used in step (B) may be a basal medium supplemented with N2 or KSR, and the serum medium may be a basal medium supplemented with fetal bovine serum, more specifically, a serum medium supplemented with DMEM / F12 medium.

[0264] In step (B), the serum-free medium or serum-based medium may contain known growth factors or growth-promoting additives. Examples of known growth factors include EGF, FGF, IGF, and insulin. Examples of additives that promote proliferation include N2 supplement (manufactured by Life Technologies), B27 supplement (manufactured by Life Technologies), and KSR (manufactured by Life Technologies). Examples of chemical substances that promote proliferation include retinoids (for example, retinoic acid) and taurine.

[0265] A preferable culture time in step (B) is, for example, a time period in which the proportion of Chx10-positive cells in the retinal tissue is equal to or greater than that in a serum-free medium or serum-free medium containing no substance acting on the Wnt signaling pathway. The culture time is set to be until the cell aggregates increase to 30% or more compared to the time at the start of culture. This can be done.

[0266] The culture conditions in step (B), such as the culture temperature and CO2 concentration, may be set appropriately. The culture temperature may be, for example, in the range of about 30°C to about 40°C, and preferably, for example, about 37°C. The CO2 concentration may be, for example, in the range of about 1% to about 10%, and preferably, for example, about 5%.

[0267] The above culture is continued until the "cell aggregates containing ciliary margin-like structures" of step (B) are obtained. The number of days varies depending on the type of serum-free or serum-free medium, other culture conditions, etc., but for example, 100 The number of days for culture can be preferably, for example, 20 to 70 days, and more preferably, for example, 30 to 60 days.

[0268] In the "cell aggregate containing ciliary marginal region-like structures" prepared by the above-mentioned steps (A) and (B), the retinal pigment epithelium and retinal tissue (specifically, neural retina) are present adjacent to each ciliary marginal region-like structure within the same cell aggregate. This structure can be confirmed by microscopic observation, etc. Specifically, for example, the presence of the ciliary marginal region-like structures can be confirmed by microscopic observation as an epithelial structure that is thick on the retina side and thin on the retinal pigment epithelium side, formed between the highly transparent retinal tissue and the retinal pigment epithelium where pigmentation is visible. Furthermore, immunostaining of frozen sections of the aggregates has been shown to be Rdh10-positive, Otx1-positive, or Zic1-positive. This allows the presence of a ciliary margin-like structure to be confirmed.

[0269] In a further aspect, the above-mentioned steps (A) and (B) promote differentiation of the retinal tissue (neuroepithelium) contained in the aggregate, and at least one selected from the group consisting of photoreceptor precursor cells, photoreceptors, cone photoreceptors, rod photoreceptors, horizontal cells, and interneurons (amacrine cells, ganglion cells, etc.) is differentiated. It is possible to produce mature retinal tissue and said cells, which preferably contain one or more, more preferably all, of the cells.

[0270] From the cell aggregates containing retinal tissue obtained by Production Method 2 of the present invention, etc., retinal pigment epithelial cells can be produced by the following step (C). A retinal pigment epithelial sheet can be produced by step (D).

[0271] In the present invention, the term "retinal pigment epithelial cells" refers to epithelial cells present outside the neural retinal tissue in the living retina. Those skilled in the art can determine whether a cell is a retinal pigment epithelial cell by, for example, cell microscopy. Marker (RPE65 (mature retinal pigment epithelial cells)), Mitf (immature or mature retinal pigment epithelial cells) It can be confirmed by the expression of melanin, melanin granules, and characteristic polygonal cell morphology.

[0272] First, in step (C), the cell aggregates containing retinal tissue obtained by the manufacturing method 2 of the present invention are cultured in suspension in a serum-free medium or a serum-free medium that does not contain a substance active in the BMP signaling pathway but does contain a substance active in the Wnt signaling pathway, to obtain aggregates containing retinal pigment epithelial cells.

[0273] The serum-free medium used in step (C) may be a serum-free medium in which N2 or KSR is added to a basal medium, and more specifically, a serum-free medium in which N2 supplement (Life Technologies) is added to a DMEM / F12 medium. Examples of such a medium include a serum medium containing fetal bovine serum.

[0274] The serum-free medium used in step (C) may contain, in addition to the aforementioned substance acting on the Wnt signaling pathway, the aforementioned substance acting on the Nodal / Activin signaling pathway and / or the aforementioned substance inhibiting the FGF signaling pathway.

[0275] Here, a preferred culture for step (C) is suspension culture.

[0276] Next, a step of dispersing the aggregates obtained in step (C) of the present invention and culturing the resulting cells in an adherent culture. (D) will be explained.

[0277] Step (D) is carried out within 60 days, preferably within 30 days, more preferably 3 days after the start of step (C).

[0278] The serum-free medium or serum-free medium used for the adherent culture in step (D) may be any of the above-mentioned To avoid the complicated preparation process, commercially available serum substitutes such as KSR can be used. Serum-free medium (e.g., 1:1 mixture of DMEM / F-12 and Neurobasal plus 1 / 2 x N2) supplemented with an appropriate amount of It is preferable to use a serum-free medium supplemented with KSR supplement, 1 / 2 x B27 supplement, and 100 μM 2-mercaptoethanol. For example, in the case of human iPS cell-derived cells, the amount of KSR added to the serum-free medium is usually about 1% to about 20%, preferably about 2% to about 20%.

[0279] In step (D), the cells are cultured in a serum-free medium or serum medium containing a ROCK inhibitor. It is preferable to cultivate

[0280] In step (D), a substance selected from the group consisting of a substance acting on the Wnt signaling pathway, a substance inhibiting the FGF signaling pathway, a substance acting on the Activin signaling pathway, and a substance acting on the BMP signaling pathway is used. It is more preferred to culture the cells in a serum-free medium or a serum-containing medium further containing one or more substances that It's nice.

[0281] The activin signaling pathway active substance is a substance that can enhance signals mediated by activin. Examples of the activin signaling pathway active substance include proteins belonging to the activin family (e.g., activin A, activin B, activin C, activin AB, etc.), activin receptor agonists, and the like. Examples include activin receptor agonists.

[0282] The concentration of the substance acting on the Activin signaling pathway used in step (D) may be any concentration that allows efficient formation of a uniform sheet of retinal pigment epithelial cells. For example, in the case of Recombinant Human / Mouse / Rat Activin A (R&D Systems #338-AC), the concentration is about 1 ng / ml to about 10 μg / ml, preferably about 10 ng / ml to about 1 μg / ml, more preferably about 100 ng / ml. Add. The substance acting on the Activin signaling pathway is added, for example, within 18 days, preferably on the 6th day, from the start of step (D).

[0283] In step (D), it is preferable to carry out adhesion culture on a culture vessel whose surface has been treated with a culture substrate. The culture substrate used for treating the culture vessel in step (D) is preferably a culture medium derived from aggregates. Examples include cell culture substrates that enable cell adhesion culture and the formation of retinal pigment epithelial sheets.

[0284] 4. Method for producing cerebral tissue Production method 3 of the present invention is a method for producing cerebral tissue, comprising the following steps (1) to (3): (1) Pluripotent stem cells were cultured in the absence of feeder cells, and 1) TGFβ family signaling pathway was Pathway inhibitors and / or Sonic Hedgehog signaling pathway agonists, and 2) untreated a first step of culturing in a medium containing a differentiation maintenance factor; (2) a second step of culturing the cells obtained in the first step in suspension to form cell aggregates; and (3) The aggregates obtained in the second step are cultured in suspension in the presence of a TGFβ family signaling pathway inhibitor and / or a Wnt signaling pathway inhibitor to obtain aggregates containing cerebral tissue. Three steps.

[0285] Step (1) of Production Method 3 of the present invention can be carried out in the same manner as step (1) of Production Method 1 of the present invention.

[0286] Step (2) of Production Method 3 of the present invention can be carried out in the same manner as step (2) of Production Method 1 of the present invention.

[0287] The medium used in step (2) of production method 3 may or may not contain a sonic hedgehog signaling pathway active substance.

[0288] The medium used in step (2) may contain a TGFβ family signaling pathway inhibitor and / or a Wnt signaling pathway inhibitor. Add a Wnt signaling pathway inhibitor or a Wnt signaling pathway inhibitor to the culture medium alone. Although they may be added, it is preferable to use both in combination.

[0289] In step (3), a TGFβ family signaling pathway inhibitor or a Wnt signaling pathway inhibitor may be added to the medium alone as a differentiation inducer, or a combination of both may be added. It is preferable to do so.

[0290] The medium used in step (3) is, for example, a serum-free medium or a serum-free medium (preferably a medium containing a TGFβ family signaling pathway inhibitor and / or a Wnt signaling pathway inhibitor) supplemented with the medium. Preferably, it is a serum-free medium.

[0291] The serum-free medium or serum-free medium used for such a medium is not particularly limited as long as it is as described above. To avoid the complexity of the preparation, for example, a serum substitute such as commercially available KSR may be used. It is preferable to use a serum-free medium supplemented with a small amount of KSR (e.g., GMEM medium supplemented with 20% KSR, 0.1 mM 2-mercaptoethanol, non-essential amino acids, and 1 mM pyruvate). For example, in the case of human pluripotent stem cells (e.g., iPS cells), the amount of KSR added to the serum-free medium is usually about 1% to about 30%, and preferably about 2% to about 20%.

[0292] The medium (preferably, serum-free medium) used in step (3) can be the same medium (preferably, serum-free medium) used in step (2) as it is, or can be replaced with a new medium (preferably, serum-free medium). The medium containing no inhibitors of the Wnt signaling pathway and / or inhibitors of the Wnt signaling pathway was When used in step (3), the TGFβ family signaling pathway inhibitor and / or the Wnt signaling pathway inhibitor may be added to the medium. The medium containing the TGFβ family signaling pathway inhibitor and / or the Wnt signaling pathway inhibitor used in step (2) may be used as is. When used in step (3), half of the medium may be replaced with the same medium.

[0293] Examples of the TGFβ family signaling pathway inhibitor used in steps (2) and (3) include a TGFβ signaling pathway inhibitor, a Nodal / Activin signaling pathway inhibitor, and a BMP signaling pathway inhibitor.

[0294] The TGFβ signaling pathway inhibitor is not particularly limited as long as it inhibits the signaling pathway caused by TGFβ, and may be any of nucleic acids, proteins, and low-molecular-weight organic compounds. Examples of such substances include substances that directly act on TGFβ (e.g., proteins, antibodies, etc.). aptamers, etc.), substances that suppress the expression of the gene encoding TGFβ (e.g., antisense substances that inhibit the binding of TGFβ receptors to TGFβ; substances that inhibit the physiological activity resulting from signal transduction by TGFβ receptors (e.g., inhibitors of TGFβ receptors) Examples of inhibitors of the TGFβ signaling pathway include inhibitors of TGFβ, such as steroid inhibitors and Smad inhibitors. Known proteins include Lefty and the like. Compounds well known to those skilled in the art can be used as TGFβ signaling pathway inhibitors, and specific examples include SB431542 (4-(5-benzo[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzamide) and A-83-01 (3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioate). amide)] is an inhibitor of TGFβ receptor (ALK5) and Activin receptor (ALK4 / 7) (i.e., TGFβR The TGFβ signaling pathway inhibitor is preferably SB431542 or A-83-01.

[0295] Nodal / Activin signaling pathway inhibitors include those that inhibit signals caused by Nodal or Activin. The substance is not particularly limited as long as it inhibits the signal transduction pathway, and may be any of nucleic acids, proteins, and low-molecular-weight organic compounds. Examples of such substances include substances that act directly on Nodal or Activin (e.g., antibodies, aptamers, etc.), substances that suppress the expression of genes encoding Nodal or Activin (e.g., antisense oligonucleotides, siRNA, etc.), substances that inhibit the binding of Nodal / Activin receptors to Nodal / Activin, and substances that inhibit signal transduction via Nodal / Activin receptors. Examples of inhibitors of the Nodal / Activin signaling pathway include substances that inhibit physiological activity resulting from signal transduction. Compounds well known to those skilled in the art can be used as inhibitors of the Nodal / Activin signaling pathway, and specific examples include SB431542 and A-83-01. Proteins known as inhibitors of the Nodal / Activin signaling pathway (e.g., Lefty and Cerberus) may also be used.

[0296] The BMP signaling pathway inhibitor is not particularly limited as long as it inhibits the signaling pathway caused by BMP, and may be any of nucleic acids, proteins, and low molecular weight organic compounds. Here, examples of BMP include BMP2, BMP4, BMP7, and GDF7. Examples of such substances include Examples of the substance that can be used include substances that act directly on BMP (e.g., antibodies, aptamers, etc.), substances that suppress the expression of genes encoding BMP (e.g., antisense oligonucleotides, siRNA, etc.), substances that inhibit the binding of BMP receptors (BMPRs) to BMP, and substances that inhibit physiological activities resulting from signal transduction by BMP receptors. Examples of BMPRs include ALK2 and ALK3. Compounds known to those skilled in the art can be used as BMP signal transduction pathway inhibitors, and specific examples thereof include: Examples of such inhibitors include LDN193189 and Dorsomorphin. LDN193189 (4-[6-(4-piperazin-1-ylphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline) is a known BMPR (ALK2 / 3) inhibitor, and is usually commercially available in the form of its hydrochloride salt. Proteins known as BMP signaling pathway inhibitors (Chordin, Noggin, etc.) may also be used. The BMP signaling pathway inhibitor is preferably LDN193189.

[0297] The TGFβ family signaling pathway inhibitor is preferably SB431542, A-83-01, or LDN193189.

[0298] The Wnt signaling pathway inhibitor used in steps (2) and (3) is not particularly limited as long as it can suppress signaling mediated by Wnt, and may be any of proteins, nucleic acids, low molecular weight compounds, etc. Signals mediated by Wnt include Frizzled (Fz) and Wnt signaling pathway inhibitors include substances that act directly on Wnt or Wnt receptors (anti-Wnt antibodies, anti-Wnt receptor antibodies, etc.), ... The expression of the gene encoding the Wnt receptor is suppressed by substances (e.g., antisense oligonucleotides). leotide, siRNA, etc.), substances that inhibit the binding of Wnt receptors and Wnt (soluble Wnt receptors, dominant-negative Wnt receptors, Wnt antagonists, Dkk1, Cerberus protein, etc.), Wnt receptors A substance that inhibits the physiological activity caused by signal transduction by CKI-7 (N-(2-aminoethyl)-5- chloroisoquinoline-8-sulfonamide), D4476 (4-[4-(2,3-dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide), IWR-1-endo (IWR1e) (4-[(3aR,4S,7R,7aS)-1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4 Examples of inhibitors of the Wnt signaling pathway include, but are not limited to, small molecular weight compounds such as N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]acetamide, CKI-7, D4476, IWR-1-endo (IWR1e), IWP-2, etc., which are known Wnt signaling pathway inhibitors, and are commercially available. Examples of inhibitors of the Wnt signaling pathway include, but are not limited to, CKI-7, D4476, IWR-1-endo (IWR1e), IWP-2, etc. Of these, IWR1e is preferred.

[0299] The concentration of Wnt signaling pathway inhibitors influences the cerebral cortex of cells forming pluripotent stem cell aggregates. For example, IWR-1-endo is added to the medium at a concentration of about 0.1 μM to about 100 μM, preferably about 0.3 μM to about 30 μM, more preferably about 1 μM to about 10 μM, and even more preferably about 3 μM. When a substance that inhibits the Wnt signaling pathway is used, it is desirable to use it at a concentration that exhibits Wnt signaling pathway inhibitory activity equivalent to the above-mentioned concentration of IWR1e.

[0300] The TGFβ family signaling pathway inhibitor and / or the Wnt signaling pathway inhibitor may be added to the culture medium simultaneously with the initiation of suspension culture in step (2), or may be added to the culture medium after a certain period of time has elapsed (e.g., after about 24 hours) from the initiation of suspension culture in step (2).

[0301] In step (3), when a medium exchange operation is performed, for example, the original medium is not discarded but a new medium is added. Medium exchange can be performed by adding medium (medium addition operation), discarding about half of the original medium (about 40-80% of the volume of the original medium) and adding about half of the new medium (40-80% of the volume of the original medium) (half medium exchange operation), or discarding about the entire volume of the original medium (more than 90% of the volume of the original medium) and adding about the entire volume of new medium (more than 90% of the volume of the original medium) (full medium exchange operation).

[0302] When adding a specific component (e.g., IWR-1-endo) at a certain point, for example, after calculating the final concentration, discard about half of the original medium and add about half of a new medium containing the specific component at a concentration higher than the final concentration (specifically, 1.5 to 3.0 times the final concentration, e.g., about twice the final concentration). Alternatively, a procedure similar to the above (half-medium exchange, half-medium exchange) may be performed.

[0303] If, at a certain point, the concentration of a particular component contained in the original medium is to be maintained, for example, about half of the original medium may be discarded and about half of a new medium containing the particular component at the same concentration as that contained in the original medium may be added.

[0304] When components contained in the original medium are diluted to reduce their concentrations at a certain point, for example, the medium exchange procedure may be performed multiple times a day, preferably multiple times within an hour (e.g., 2 to 3 times).Furthermore, when components contained in the original medium are diluted to reduce their concentrations at a certain point, the cells or aggregates may be transferred to another culture vessel.

[0305] The tools used for the medium replacement operation are not particularly limited, and examples include pipettors, micropipettes, multichannel micropipettes, repeating dispensers, etc. For example, when a 96-well plate is used as the culture vessel, a multichannel micropipette may be used.

[0306] In step (3), the culture conditions such as culture temperature and CO2 concentration can be set appropriately. The culture temperature is, for example, about 30° C. to about 40° C., preferably about 37° C. The CO2 concentration is, for example, about 1% to about 10%, preferably about 5%.

[0307] By such culturing, cerebral nervous system progenitor cells are differentiated from the cells forming the aggregates obtained in step (2). The differentiation of the cells into cerebral nervous system progenitor cells is induced, and aggregates containing cerebral nervous system progenitor cells can be obtained. The present invention also provides a method for producing such aggregates containing cerebral nervous system progenitor cells. The acquisition of aggregates containing cerebral nervous system progenitor cells can be confirmed, for example, by detecting that the aggregates contain cells expressing cerebral nervous system progenitor cell markers such as FoxG1, Lhx2, PAX6, and Emx2. In one embodiment of step (3), the aggregates formed in step (2) are cultured in a serum-free medium or a serum-free medium until cells expressing the FoxG1 gene begin to appear. In one embodiment, 20% or more (preferably 30% or more, 40% or more, 50% or more, 60% or more) of the cells contained in the aggregate express FoxG1. The culture in step (3) is carried out until the culture reaches a state where the fermentation product is soluble in water.

[0308] In a preferred embodiment of the method for producing cerebral cells and / or cerebral tissue, in step (1), In step (2), human pluripotent stem cells (e.g., human iPS cells) are cultured in an adherent manner in a serum-free medium containing a TGFβ signaling pathway inhibitor (e.g., SB431542) and bFGF in the absence of feeder cells, and the cells obtained in step (1) are cultured in suspension in a serum-free medium, and in step (3), The aggregates obtained in step (2) are treated with a Wnt signaling pathway inhibitor (IWR-1-endo) and TGFβ The cells are cultured in suspension in a serum-free medium containing a signal transduction pathway inhibitor (e.g., SB431542).

[0309] In a preferred embodiment of the method for producing cerebral cells and / or cerebral tissue, the step (1 ) human pluripotent stem cells (e.g., human iPS cells) are cultured in an adherent culture medium in the absence of feeder cells in a serum-free medium containing a BMP signaling pathway inhibitor (e.g., LDN193189) and bFGF. In step (2), the cells obtained in step (1) are cultured in suspension in a serum-free medium, and in step (3), The aggregates obtained in step (2) are cultured in suspension in a serum-free medium containing a Wnt signaling pathway inhibitor (IWR-1-endo) and a TGFβ signaling pathway inhibitor (e.g., SB431542). .

[0310] In a preferred embodiment of the method for producing cerebral cells and / or cerebral tissue, in step (1), Human pluripotent stem cells (e.g., human iPS cells) are cultured in the absence of feeder cells. TGFβ family signaling pathway inhibitors (e.g., TGFβ signaling pathway inhibitors (e.g., SB431542, A-83-01), Nodal / Activin signaling pathway inhibitors (e.g., Lefty), BMP signaling pathway inhibitors (e.g., LDN193189), or combinations thereof (e.g., SB431542 and LDN193189), etc.); Sonic hedgehog signaling pathway agonists (e.g., Shh protein, SAG, PMA); or TGFβ family signaling pathway inhibitors (e.g., Lefty, SB431542, A-83-01, LDN193189) ) and sonic hedgehog signaling pathway agonists (e.g., Shh protein, SAG, PMA) ) in combination with; and Adherent culture in a serum-free medium containing bFGF, In step (2), the cells obtained in step (1) are TGFβ family signaling pathway inhibitors (e.g., TGFβ signaling pathway inhibitors (e.g., SB431542, A-83-01), Nodal / Activin signaling pathway inhibitors (e.g., Lefty), BMP signaling pathway inhibitors (e.g., LDN193189), or combinations thereof (e.g., SB431542 and LDN193189), etc.); and The cells were cultured in suspension in serum-free medium containing an inhibitor of the Wnt signaling pathway (IWR-1-endo). forming aggregates of In step (3), the aggregates obtained in step (2) are TGFβ family signaling pathway inhibitors (e.g., TGFβ signaling pathway inhibitors (e.g., SB431542, A-83-01), Nodal / Activin signaling pathway inhibitors (e.g., Lefty), BMP signaling pathway inhibitors (e.g., LDN193189), or combinations thereof (e.g., SB431542 and LDN193189), etc.); and The cells were cultured in suspension in serum-free medium containing an inhibitor of the Wnt signaling pathway (IWR-1-endo), and the cerebral cortex was Aggregates containing neural progenitor cells, cerebral cells or cerebral tissue are obtained. The medium in step (2) preferably contains a ROCK inhibitor (e.g., Y-27632).

[0311] More preferably, in step (2), the cells obtained in step (1) are TGFβ family signaling pathway inhibitors (e.g., TGFβ signaling pathway inhibitors (e.g., SB431542, A-83-01), Nodal / Activin signaling pathway inhibitors (e.g., Lefty); Wnt signaling pathway inhibitor (IWR-1-endo); and Cells are cultured in suspension in serum-free medium containing a ROCK inhibitor (e.g., Y-27632) to form cell aggregates. Let, In step (3), the aggregates obtained in step (2) are TGFβ signaling pathway inhibitors (e.g., SB431542, A-83-01) or Nodal / Activin signaling Transduction pathway inhibitors (e.g., Lefty); and The cells were cultured in suspension in serum-free medium containing an inhibitor of the Wnt signaling pathway (IWR-1-endo), and the cerebral cortex was Aggregates containing neural progenitor cells, cerebral cells or cerebral tissue are obtained.

[0312] The resulting aggregates containing cerebral nervous system progenitor cells may be used as they are as reagents for evaluating toxicity and efficacy. Highly purified cerebral nervous system progenitor cells can also be obtained by dispersing the aggregates containing cerebral nervous system progenitor cells (e.g., with trypsin / EDTA or papain) and sorting the resulting cells using FACS or MACS.

[0313] Furthermore, by continuing to culture aggregates containing cerebral nervous system progenitor cells in serum-free medium or serum medium, the cerebral nervous system progenitor cells or cerebral cells can be further differentiated to produce cerebral tissue with a layered structure.

[0314] The serum-free medium or serum medium used for such a medium is not particularly limited as long as it is as described above, and examples thereof include a serum medium obtained by adding 10% fetal bovine serum, an N2 supplement, and retinoic acid to a DMEM-F12 medium, or a serum-free medium to which an appropriate amount of a serum substitute such as a commercially available N2 supplement has been added (e.g., a medium obtained by adding an N2 supplement to a DMEM-F12 medium).

[0315] The culture time for inducing cerebral tissue from cerebral nervous system progenitor cells varies depending on the cerebral layer-specific neurons of interest, but is, for example, from about 7 days to about 200 days.

[0316] The cerebral tissue exists in the aggregates as a neuroepithelial structure. The tissue is fixed with a fixative such as formaldehyde solution, frozen sections are prepared, and the formation of cerebral tissue with a layered structure can be confirmed by immunostaining or the like. Since the cerebral nervous system progenitor cells, ventricular body progenitor cells, and cerebral layer structure-specific neurons that constitute each layer of cerebral tissue are different, the formation of a layered structure can be confirmed by immunostaining using antibodies against the above-mentioned markers expressed in these cells. In one embodiment, the cerebral tissue is a FoxG1-positive neuroepithelial structure.

[0317] It is also possible to physically cut out the cerebral tissue present on the surface of the aggregate using tweezers, etc. In this case, since neural tissue other than cerebral tissue may be formed on the surface of each aggregate, it is possible to confirm that the tissue is cerebral tissue by cutting out a portion of the neural tissue cut out from the aggregate and using the same for confirmation by the immunostaining method described below, etc.

[0318] In one embodiment of step (3), the aggregates formed in step (2) are treated with a nucleotide sequence encoding the FoxG1 gene. Until cells expressing TGFβ begin to appear, the concentration of TGFβ signaling required for differentiation into cerebral cells is maintained. Serum-free medium or blood containing an inhibitor of the Wnt signaling pathway and / or an inhibitor of the Wnt signaling pathway and (c) culturing the aggregates containing cerebral nervous system progenitor cells in a serum-free medium to obtain aggregates containing cerebral nervous system progenitor cells, and subsequently culturing the aggregates containing cerebral nervous system progenitor cells in a serum-free medium or a serum-free medium until cerebral tissue is formed to obtain aggregates containing cerebral tissue. In one embodiment, the suspension culture of the aggregates containing cerebral nervous system progenitor cells is carried out until 20% or more (preferably 30% or more, 40% or more, 50% or more, or 60% or more) of the cells contained in the aggregates express FoxG1. It will be carried out.

[0319] In addition, as one embodiment of step (3), the aggregate obtained in step (2) or the aggregate obtained in step (2) The aggregates obtained in step (1) may be subjected to suspension culture by the above method and then subjected to adhesion culture to form adhesive aggregates. The adhesive aggregates may be cultured until cells expressing the FoxG1 gene begin to appear. Until then, we will be conducting research on TGFβ signaling pathway inhibitors and / or Or, the cells were cultured in a serum-free medium or serum-containing medium containing an inhibitor of the Wnt signaling pathway, and the cells were cultured in a serum-free medium or serum-containing medium containing an inhibitor of the Wnt signaling pathway. An aggregate containing neural progenitor cells is obtained. The aggregate containing cerebral nervous system progenitor cells is subsequently cultured in a serum-free medium or a serum-free medium as an adherent culture until cerebral tissue is formed, thereby obtaining an aggregate containing cerebral tissue. In one embodiment, the aggregate containing cerebral nervous system progenitor cells is cultured in a serum-free medium or a serum-free medium until 10% or more (preferably 20% or more, 30% or more, 40% or more, 50% or more) of the cells express FoxG1. Adherent culture is performed.

[0320] Production method 3 of the present invention makes it possible to obtain cerebral tissue from pluripotent stem cells with high efficiency. The cerebral tissue obtained by the production method 3 of the present invention contains neurons specific to each cerebral layer. Since the cells obtained from the cerebral tissue contain layer-specific neurons (neurons), it is possible to obtain cells that constitute cerebral tissue, such as layer-specific neurons or their progenitor cells. The identity of the cells obtained from the cerebral tissue can be confirmed by a method known per se, for example, by the expression of a cell marker.

[0321] The resulting aggregates containing cerebral tissue may be used as they are as a reagent for evaluating toxicity and efficacy. Highly purified cerebral tissue-constituting cells, such as highly purified cerebral layer-specific neurons, can also be obtained by dispersing the aggregates containing cerebral tissue (e.g., by trypsin / EDTA treatment) and selecting the resulting cells using FACS or MACS.

[0322] 5. Toxicity and efficacy evaluation methods Nervous tissue or nervous system cells (e.g., retinal tissue) produced by Production Method 1, 2 or 3 of the present invention. tissue, retinal progenitor cells, retinal layer-specific neurons, cerebral tissue, cerebral nervous system progenitor cells, cerebral layer-specific neurons) are used for screening of therapeutic drugs for diseases caused by disorders of nervous tissue or nervous system cells. For example, iPS cells can be prepared from human patients with diseases caused by disorders of nervous tissue (e.g., retinal tissue, cerebral tissue), particularly diseases caused by hereditary disorders, and then used as reagents for evaluating the toxicity and efficacy of test substances. These iPS cells can be used to generate neural tissue or neural cells (e.g., retinal tissue, plexus, etc.) by the method of the present invention. The neural tissue or nervous system cells can reproduce in vitro the nervous tissue damage that causes the disease from which the patient is suffering. Thus, the present invention relates to the neural tissue or nervous system cells (e.g., retinal tissue, retinal progenitor cells, retinal layer-specific neural cells, cerebral tissue, cerebral nervous system progenitor cells, cerebral layer-specific neural cells) produced by Production Method 1, 2 or 3 of the present invention. The present invention provides a method for evaluating the toxicity and efficacy of a substance, which comprises contacting a test substance with cells or tissues (e.g., specific neurons, cerebral tissue, cerebral nervous system progenitor cells, cerebral layer-specific neurons) and examining the effect of the substance on the cells or tissues.

[0323] For example, a compound produced by Production Method 1, 2 or 3 of the present invention can be used to treat a specific disorder (e.g., a genetic disorder). Neural tissue or nervous system cells (e.g., retinal tissue, retinal progenitor cells, retinal layer-specific neurons, cerebral tissue, cerebral nervous system progenitor cells, cerebral layer-specific neurons) having a specific disorder (e.g., neuronal damage) are cultured in the presence or absence (negative control) of a test substance. The degree of damage in the neural tissue or nervous system cells treated with the test substance is then compared with that in the negative control. As a result, a test substance that reduces the degree of damage can be selected as a candidate substance for a therapeutic agent for a disease caused by the disorder. For example, a test substance that further improves the physiological activity (e.g., survival promotion or maturation) of nervous system cells produced by the production method of the present invention can be screened as a candidate drug. Alternatively, induced pluripotent stem cells are prepared from somatic cells having a genetic mutation that causes a specific disorder such as a neurological disease, and the test substance is added to the neural system cells produced by inducing differentiation of the cells using the production method of the present invention. Candidate test substances effective as therapeutic or preventive agents for the disorder can be screened using the presence or absence of the disorder as an indicator.

[0324] In the toxicity evaluation, the nervous tissue or nervous system produced by the production method 1, 2 or 3 of the present invention was Neural tissue or nervous system cells (e.g., retinal tissue, retinal progenitor cells, retinal layer-specific neurons, cerebral tissue, cerebral nervous system progenitor cells, cerebral layer-specific neurons) are cultured in the presence or absence (negative control) of a test substance. The degree of toxicity in the nervous tissue or nervous system cells treated with the test substance is then compared with that of the negative control. As a result, test substances that demonstrate toxicity compared with the negative control can be determined to be substances that are toxic to nervous tissue or nervous system cells (e.g., retinal tissue, retinal progenitor cells, retinal layer-specific neurons).

[0325] That is, the present invention encompasses a toxicity evaluation method comprising the following steps: (Step 1) a step of culturing the nervous tissue or nervous system cells produced by Production Method 1, 2 or 3 of the present invention in the presence of a test substance under viable culture conditions for a certain period of time, and then measuring the degree of damage to the cells; (Step 2) culturing the nervous tissue or nervous system cells produced by Production Method 1, 2 or 3 of the present invention under viable culture conditions for a certain period of time in the absence of a test substance or in the presence of a positive control, and then measuring the degree of cell damage; (Step 3) A step of evaluating the toxicity of the test substance in Step 1 based on the difference between the results measured in (Step 1) and (Step 2). Here, "in the absence of a test substance" includes adding only a culture medium or a solvent dissolving the test substance instead of the test substance. Furthermore, "positive control" refers to a known toxic compound. Methods for measuring the degree of cell damage include counting the number of surviving cells, for example, measuring the amount of intracellular ATP, or cell staining (for example, cell Examples include counting the number of viable cells by cytoplasmic staining and morphological observation.

[0326] In step 3, the toxicity of the test substance can be evaluated by, for example, comparing the measured value in step 1 with the measured value of the negative control in step 2, and determining the degree of cell damage in step 1. If the measured value in step 1 is larger than the measured value in step 2, the test substance can be judged to be toxic. In the above cases, the test substance can be determined to be toxic.

[0327] 6. Pharmaceutical Compositions The present invention relates to a method for producing a nervous tissue or nervous system cell (e.g., a cell derived from a nervous system cell) produced by Production Method 1, 2, or 3 of the present invention. The present invention provides a pharmaceutical composition comprising an effective amount of a retinal tissue, retinal progenitor cells, retinal layer-specific neurons, cerebral tissue, cerebral nervous system progenitor cells, or cerebral layer-specific neurons.

[0328] The pharmaceutical composition is a composition containing a nervous tissue or nervous system cell produced by Production Method 1, 2 or 3 of the present invention. The composition comprises an effective amount of cells (e.g., retinal tissue, retinal progenitor cells, retinal layer-specific neurons, cerebral tissue, cerebral nervous system progenitor cells, cerebral layer-specific neurons), and a pharmaceutically acceptable carrier.

[0329] Pharmaceutically acceptable carriers include physiological aqueous solvents (e.g., physiological saline, buffer solutions, serum-free media, etc.) If necessary, commonly used preservatives, stabilizers, reducing agents, isotonicity agents, etc. may be added to pharmaceuticals containing tissues or cells to be transplanted in transplantation medicine.

[0330] The pharmaceutical composition of the present invention is a composition containing nervous tissue or nervous system cells produced by Production Method 1, 2 or 3. can be prepared as a suspension by suspending the above in an appropriate physiological aqueous solvent. If necessary, a cryopreservative may be added, followed by cryopreservation, and the suspension may be thawed at the time of use, washed with a buffer solution, and then used for transplantation therapy.

[0331] The nerve tissue obtained by the production method of the present invention can also be cut into pieces of an appropriate size using tweezers or the like to form a sheet preparation.

[0332] In addition, the cells obtained by the production method of the present invention are subjected to adhesion culture in the differentiation induction step (3). By this method, cells can be formed into a sheet-like form and used as a sheet preparation.

[0333] The pharmaceutical composition of the present invention is useful as a therapeutic agent for diseases caused by disorders of nervous tissue or nervous system cells (e.g., retinal tissue, retinal progenitor cells, retinal layer-specific neurons, cerebral tissue, cerebral nervous system progenitor cells, cerebral layer-specific neurons).

[0334] 7. Therapeutic Drugs Nervous tissue or nervous system cells (e.g., retinal tissue) produced by Production Method 1, 2 or 3 of the present invention The neural tissue or nervous system cells produced by Production Method 1, 2 or 3 of the present invention are useful in transplantation therapy for diseases caused by disorders of the neural tissue or nervous system cells. The present invention provides a therapeutic agent for a disease caused by a disorder of the nervous tissue or nervous system cells, comprising neural tissue or nervous system cells (e.g., retinal tissue, retinal progenitor cells, retinal layer-specific nerve cells, cerebral tissue, cerebral nervous system progenitor cells, cerebral layer-specific nerve cells). The present invention provides a therapeutic agent for a disease caused by a disorder of the nervous tissue or nervous system cells, comprising neural tissue or nervous system cells (e.g., retinal tissue, retinal progenitor cells, retinal layer-specific nerve cells), produced by Production Method 1, 2 or 3 of the present invention, as a therapeutic agent for a disease caused by a disorder of the nervous tissue or nervous system cells, or for replenishing the damaged site in a damaged state of the nervous tissue. The present invention provides a method for transplanting neural stem cells produced by Production Method 1, 2 or 3 of the present invention to patients with diseases caused by disorders of nervous tissue or nervous system cells, or patients with damaged nervous tissue, which require transplantation. By transplanting neural tissue or nervous system cells and replenishing the neural system cells or damaged neural tissue itself, diseases caused by damage to neural tissue or nervous system cells, or damaged states of neural tissue, can be treated. For example, diseases caused by damage to neural tissue or nerve-related cells include neurodegenerative diseases (e.g., cerebral ischemic injury, cerebral infarction, Parkinson's disease, spinal cord injury, Cerebrovascular disease, brain / spinal trauma (e.g., cerebral infarction, head trauma, cerebral contusion (TBI), spinal cord injury, multiple system atrophy), typical neurodegenerative diseases (amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD)) , Parkinson's syndrome, Alzheimer's dementia, Progressive supranuclear palsy (PSP), Huntington's disease, Multiple system atrophy (MSA), Spinocerebellar degeneration (SCD), Demyelinating diseases and neuromuscular diseases (Multiple sclerosis) MS, acute disseminated encephalomyelitis (ADEM), inflammatory diffuse sclerosis (Schilder's disease), subacute sclerotic panencephalitis, progressive multifocal leukoencephalopathy, hypoxic encephalopathy, central pontine myelinopathy, Binswanger's disease, Guillain-Barré syndrome, Fisher's syndrome, chronic inflammatory demyelinating polyradiculoneuropathy, syringomyelia, spinocerebellar degeneration, nigrostriatal degeneration (SND), olivopontocerebellar atrophy (OPCA), Shy- Draeger syndrome (Shy-Drager syndrome), ophthalmological diseases (macular degeneration, age-related macular degeneration, retinitis pigmentosa, cataracts, glaucoma, corneal diseases, retinopathy), intractable epilepsy, progressive supranuclear palsy, syringomyelia, spinal muscular atrophy (SMA), spinal-bulbar muscular atrophy (SBMA), primary lateral sclerosis (PLS), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Huntington's disease (HD), chorea acanthocytosis, syringomyelia, frontotemporal lobar degeneration, Charcot-Marie-Tooth disease Examples of diseases caused by damage to cerebral tissue or cerebral-related cells include neurodegenerative diseases (e.g., cerebral ischemic injury, cerebral infarction, motor neuron disease, ALS, Alzheimer's disease, polyglutaminase), and the like. Examples of diseases caused by damage to retinal tissue or retina-related cells include retinal degeneration, retinitis pigmentosa, age-related macular degeneration, organic mercury poisoning, chloroquine retinopathy, glaucoma, diabetic retinopathy, and neonatal retinopathy. Furthermore, examples of diseases caused by damage to nervous tissue include patients after nerve tissue removal, patients after radiation exposure to intraneuronal tumors, and trauma.

[0335] In transplantation medicine, rejection due to differences in histocompatibility antigens is often a problem, but this problem can be overcome by using pluripotent stem cells established from the somatic cells of the transplant recipient (e.g., induced pluripotent stem cells). That is, in a preferred embodiment, in the method of the present invention, pluripotent stem cells established from the somatic cells of the recipient (e.g., induced pluripotent stem cells) are used as pluripotent stem cells, thereby producing immunologically autologous nervous tissue or nervous system cells for the recipient, which are then transplanted into the recipient.

[0336] Alternatively, allogeneic neural tissue or nervous system cells may be produced from pluripotent stem cells (e.g., induced pluripotent stem cells) established from somatic cells of an individual who is immunocompatible with the recipient (e.g., HLA type or MHC type compatible), and then transplanted into the recipient. [Example]

[0337] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples in any way.

[0338] Example 1: Example of preconditioning of human iPS cells using a TGFβ family signaling pathway inhibitor in step 1 Human iPS cells (1231A3 strain, obtained from Kyoto University) were cultured in a feeder-free medium according to the method described in Scientific Reports, 4, 3594 (2014). StemFit medium (AK03, manufactured by Ajinomoto Co., Inc.) was used as the feeder-free medium, and Laminin511-E8 (manufactured by Nippi Co., Ltd.) was used as the feeder-free scaffold. (manufactured by) was used.

[0339] The specific maintenance culture procedure involves first culturing subconfluent human iPS cells (1231A3 After washing with PBS, the cells were purified to single cells using TrypLE Select (Life Technologies). The human iPS cells dispersed into single cells were then seeded onto a plastic culture dish coated with Laminin511-E8 and cultured in a feeder-free manner in StemFit medium in the presence of Y27632 (a ROCK inhibitor, 10 μM). Well plate (Iwaki, for cell culture, culture area 9.4 cm) 2 ) is used, the single fine The seeding number of dispersed human iPS cells was 6 x 10 3 One day after sowing, Y27632 was added. The medium was then replaced with StemFit medium containing no Y27632. Thereafter, the medium was replaced with StemFit medium containing no Y27632 every 1-2 days. Six days after seeding, the cells were cultured until they became subconfluent (about 60% of the culture area was covered with cells).

[0340] As a specific example of preconditioning in step 1 of the present production method, the following operation was carried out. The lent human iPS cells (1231A3 strain) were washed with PBS and dispersed into single cells using TrypLE Select (Life Technologies). The dispersed human iPS cells were then seeded onto a plastic culture dish (Iwaki) coated with Laminin511-E8 and cultured in a feeder-free medium in StemFit medium in the presence of Y27632 (a ROCK inhibitor, 10 μM). As the plastic culture dish, a 6-well plate (manufactured by Iwaki Co., Ltd., culture surface Total: 9.4 cm 2 ), the seeding number of the dispersed human iPS cells was 6 x 10 3 year The plastic culture dish used was a 60 mm dish (manufactured by Iwaki Co., Ltd., for cell culture, culture area 21 cm). 2 ), the seeding number of the dispersed human iPS cells was 13 x 10 3 One day after seeding, the medium was replaced with StemFit medium without Y27632. The medium was replaced once a day with StemFit medium containing no Y27632. The cells were then cultured until 5 days after seeding, i.e., 1 day before subconfluence (about 50% of the culture area was covered with cells). Similar results were obtained even when the cells were cultured for 6 days after seeding. The cultured human iPS cells, which had reached subconfluence one day prior to the initial stage, were incubated in the presence of SB431542 (TGFβR inhibitor (TGFβRi), 5 μM) (Step 1: Preconditioning treatment, Figure 1 "Precondition TGFβRi 24 hr"). The cells were then incubated for 1 day in the presence or absence of preconditioning (Step 1: No preconditioning treatment, Figure 1 "Control"). The cells were cultured in a feeder-free medium. The cultured cells were observed in bright field using an inverted microscope (Keyence). The results showed that treatment with a TGFβR inhibitor (SB431542) during feeder-free culture did not significantly affect the morphology of the human iPS cells (Figure 1).

[0341] Example 2: Example of preconditioning of human iPS cells using a TGFβ family signaling pathway inhibitor or an Shh agonist in step 1 - 2 Human iPS cells (1231A3 strain) were subcultured in StemFit medium using the method described in Example 1. The human iPS cells were cultured in a feeder-free medium until one day before confluence. The cells were treated with SB431542 (TGFβR inhibitor, 5 μM), LDN193189 (BMPR inhibitor, 100 nM), SAG (Shh The cells were cultured for one day in the presence (Step 1: Preconditioning) or absence (Step 1: No Preconditioning) of the agonist (300 nM), or a TGFβR inhibitor and a BMPR inhibitor (SB431542 at 5 μM and LDN193189 at 100 nM). The cells were fixed with 4% paraformaldehyde and immunostained for Oct3 / 4, a pluripotent stem cell marker. The obtained immunostained cells were visualized using an inverted fluorescence microscope (BIOREVO, Keyence Corporation). Visual field observation and fluorescent image observation were performed. In immunostaining analysis, when determining whether the marker was positive, a luminance value two times or more higher than the background was considered positive. As a result, cells preconditioned with either compound were found to be Oct3 / 4 positive, similar to unpreconditioned cells (Figure 2). This indicates that cells preconditioned under these conditions maintained a pluripotent-like state.

[0342] Example 3: Example of cell aggregate formation from human iPS cells using a TGFβ family signaling pathway inhibitor in step 1 Human iPS cells (1231A3 strain) were subcultured in StemFit medium using the method described in Example 1. The human iPS cells were cultured in a feeder-free medium until one day before confluence. The cells were cultured in the presence of SB431542 (TGFβR inhibitor, 5 μM) (Step 1, Precondition: TGFβRi treatment). ) or in the absence (Step 1: without preconditioning), and the cells were cultured for one day in a feeder-free environment.

[0343] The human iPS cells were treated with TrypLE Select (Life Technologies) to form a cell dispersion. The cells were then further dispersed into single cells by pipetting, and the dispersed human iPS cells were placed in a non-cell-adhesive 96-well culture plate (PrimeSurface 96V bottom plate, Sumitomo Bakelite). 1.2 x 10 per well of Krite 4 The cells were suspended in 100 μl of serum-free medium. The cells were cultured in suspension at 37°C and 5% CO2. The serum-free medium (gfCDM+KSR) used was F-12 medium. A serum-free medium containing a 1:1 mixture of IMDM medium and 10% KSR, 450 μM 1-monothioglycerol, and 1x chemically defined lipid concentrate was used. On day 0 after the start of the incubation (start of step 2), Y27632 (final concentration 20 μM) was added to the serum-free medium. By the second day after the start of the free-floating culture, cell aggregation was observed under both preconditioned and unpreconditioned conditions. The cells were formed (step 2 completed, step 3 started). On the third day after the start of suspension culture, 50 μl of serum-free medium was added.

[0344] The cells thus prepared were observed on the sixth day after the start of suspension culture under an inverted microscope (Keyence ) was used for bright field observation (Figure 3). As a result, in the presence of a TGFβR inhibitor (SB431542), The cells preconditioned with (Fig. 3B) had better morphology of cell aggregates than the cells not preconditioned (Fig. 3A).

[0345] Furthermore, the morphology of the cell aggregates on the sixth day after the start of suspension culture was observed under a bright field inverted microscope. The condition was good (Fig. 3C black bar, round with a smooth surface and dense inside), and the morphology was moderate (Fig. 3 gray bar, round but sac-like). The results showed that without preconditioning, the cell aggregates with poor morphology were approximately 90%, the cell aggregates with medium morphology were approximately 10%, and the cell aggregates with good morphology were approximately 0%. (Fig. 3C “Control”, first bar from the left). In contrast, Under the conditions described above, the percentage of cell aggregates with poor morphology was approximately 0%, the percentage of cell aggregates with moderate morphology was approximately 0%, and the percentage of cell aggregates with good morphology was approximately 100% (Fig. 3C "Precondition (TGFβRi)", third bar from the left). Preconditioning with 1 μM of ATP improved the morphology of cell aggregates in steps 2 and 3. I found out that...

[0346] Example 4: Example of cell aggregate formation from human iPS cells using a TGFβ family signaling pathway inhibitor in step 1 or step 2 Human iPS cells (1231A3 strain) were subcultured in StemFit medium using the method described in Example 1. The human iPS cells were cultured in a feeder-free medium until one day before confluence. The cells were cultured feeder-free for 1 day in the presence (step 1, preconditioning: TGFβRi treatment) or absence (step 1: no preconditioning) of SB431542 (TGFβR inhibitor, 5 μM).

[0347] The human iPS cells were treated with TrypLE Select (Life Technologies) to prepare a cell dispersion solution. The cells were then further dispersed into single cells by pipetting, and the dispersed human iPS cells were then cultured in a non-cell-adhesive 96-well culture plate (PrimeSurface 96V bottom plate, Sumitomo 1.2 x 10 per well of Bakelite 4 Cells were placed in 100 μl serum-free medium. The cells were cultured in suspension at 37°C and 5% CO2 in serum-free medium (gfCDM+KSR). A serum-free medium was used, which was a 1:1 mixture of medium and IMDM medium, supplemented with 10% KSR, 450 μM 1-monothioglycerol, and 1x chemically defined lipid concentrate. On day 0 after the start of culture (start of step 2), Y27632 (final concentration 20 μM) and TGFβR were added to the serum-free medium. An inhibitor (SB431542, 5 μM) was added. Cell aggregates were formed under both the blood-free and blood-free conditions (end of step 2, start of step 3). On the third day after the start of suspension culture, the cells were cultured in a blood-free medium without Y27632 and with the addition of a TGFβR inhibitor (SB431542, 5 μM). 50 μl of supernatant medium was added.

[0348] The cell aggregates thus prepared on day 6 after the start of suspension culture were cultured in the same manner as in Example 3. The results showed that when preconditioning was not performed in step 1 and a TGFβR inhibitor (SB431542) was added in step 2, the cell aggregates with poor morphology accounted for approximately 30%, the cell aggregates with medium morphology accounted for approximately 60%, and the cell aggregates with good morphology accounted for approximately 10% (Figure 3C, 'Control + SB', second bar from the left). In contrast, when preconditioning was performed in step 1, When a TGFβR inhibitor (SB431542) was added in step 2, the percentage of cell aggregates with poor morphology was approximately 0%, the percentage of cell aggregates with moderate morphology was approximately 0%, and the percentage of cell aggregates with good morphology was approximately 100% (Figure 3C, 'Precondition (TGFβRi) +SB', fourth bar from the left). In other words, even when a TGFβR inhibitor (SB431542) was added in steps 2 and 3, preconditioning with a TGFβR inhibitor (SB431542, 5 μM) in step 1 improved the morphology of the cell aggregates compared to when no preconditioning was performed in step 1.

[0349] Furthermore, by comparing Example 3 and Example 4, the condition in which preconditioning was performed with a TGFβR inhibitor (SB431542) in step 1 and no TGFβR inhibitor (SB431542) was added in steps 2 and 3 was ( Figure 3C "Precondition (TGFβRi)", third bar from the left) Compared with the condition in which no treatment was performed and a TGFβR inhibitor (SB431542) was added in steps 2 and 3 (Figure 3C In the "Control + SB" (second bar from the left), the proportion of cell aggregates with good shape was high. In other words, it was found that adding the TGFβR inhibitor (SB431542) during step 1 (preconditioning) is the best timing.

[0350] Example 5: Example of cell aggregate formation from human iPS cells using a TGFβ family signaling pathway inhibitor in step 1 Human iPS cells (1231A3 strain) were subcultured in StemFit medium using the method described in Example 1. The human iPS cells were cultured in a feeder-free medium until one day before confluence. The cells were cultured in the presence of SB431542 (TGFβR inhibitor, 5 μM) or LDN193189 (BMPR inhibitor, 100 nM). Step 1: Preconditioning treatment) or its absence (Step 1: No Preconditioning) The cells were cultured feeder-free for 1 day.

[0351] The human iPS cells were treated with TrypLE Select (Life Technologies) to prepare a cell dispersion solution. The cells were then further dispersed into single cells by pipetting, and the dispersed human iPS cells were then cultured in a non-cell-adhesive 96-well culture plate (PrimeSurface 96V bottom plate, Sumitomo 1.2 x 10 per well of Bakelite 4 Cells were placed in 100 μl serum-free medium. The cells were cultured in suspension at 37°C and 5% CO2 in serum-free medium (gfCDM+KSR). A serum-free medium was used, which was a 1:1 mixture of medium and IMDM medium, supplemented with 10% KSR, 450 μM 1-monothioglycerol, and 1x chemically defined lipid concentrate. On day 0 after the start of culture (start of step 2), Y27632 (final concentration 20 μM) was added to the serum-free medium. By the second day after the start of suspension culture, cells were able to grow in both preconditioned and unpreconditioned conditions. Aggregates were formed (end of step 2, start of step 3). On the third day after the start of suspension culture, After the sixth day from the start of suspension culture, 50 to 70% of the medium volume was replaced with the serum-free medium containing no Y27632 every two to four days.

[0352] The cell aggregates thus prepared on the 9th day after the start of suspension culture were observed under an inverted microscope (Key As a result, similar to Example 3, cells preconditioned in the presence of a TGFβR inhibitor (SB431542) in step 1 (FIG. 4B) showed a higher percentage of cells than untreated cells. The shape of the cell aggregates in step 3 was found to be better than that in the untreated group (Figure 4A). Furthermore, the shape of the cell aggregates in step 3 was also found to be better than that in the untreated group (Figure 4C), even in cells preconditioned in the presence of a BMPR inhibitor (SB431542) in step 1 (Figure 4A). In other words, the use of either the TGFβR inhibitor (SB431542) or the BMPR inhibitor (LDN193189), which are inhibitors of the TGFβ family signaling pathway, in preconditioning treatment (step 1) was found to have the effect of improving the shape of the cell aggregates.

[0353] Example 6: Preconditioning was performed using a TGFβ family signaling pathway inhibitor in step 1 , Example of neural tissue formation from human iPS cells Human iPS cells (1231A3 strain) were subcultured in StemFit medium using the method described in Example 1. The human iPS cells were cultured in a feeder-free medium until one day before confluence. The cells were cultured feeder-free for 1 day in the presence (step 1: preconditioning treatment) or absence (step 1: no preconditioning) of LDN193189 (BMPR inhibitor, 100 nM).

[0354] The human iPS cells were treated with TrypLE Select (Life Technologies) to prepare a cell dispersion solution. The cells were then further dispersed into single cells by pipetting, and the dispersed human iPS cells were then cultured in a non-cell-adhesive 96-well culture plate (PrimeSurface 96V bottom plate, Sumitomo 1.2 x 10 per well of Bakelite 4 Cells were placed in 100 μl serum-free medium. The cells were cultured in suspension at 37°C and 5% CO2 in serum-free medium (gfCDM+KSR). A serum-free medium was used, which was a 1:1 mixture of medium and IMDM medium, supplemented with 10% KSR, 450 μM 1-monothioglycerol, and 1x chemically defined lipid concentrate. On day 0 after the start of culture (start of step 2), Y27632 (final concentration 20 μM) was added to the serum-free medium. By the second day after the start of suspension culture, cells were able to grow in both preconditioned and unpreconditioned conditions. Aggregates were formed (end of step 2, start of step 3). On the third day after the start of suspension culture, After 6 days from the start of suspension culture, half of the medium was replaced with the serum-free medium containing no Y27632 every 2 to 4 days. For half-medium replacement, half the volume of the medium in the incubator, i.e., 75 μl, was discarded, and 75 μl of fresh serum-free medium was added, bringing the total medium volume to 150 μl.

[0355] The cells prepared in this way on the 23rd day after the start of suspension culture were subjected to bright field observation using an inverted microscope (Keyence) (Figure 5A, B). If this step is not performed, the cell aggregates will collapse in step 3 and no neural tissue will be formed. On the other hand, in the cells preconditioned in the presence of the BMPR inhibitor (LDN193189) in step 1, the shape of the cell aggregates in step 3 was good, and neural tissue with a neuroepithelial structure was observed (Figure 5A). Furthermore, the morphology of the cells on the 23rd day after the start of suspension culture was observed by bright field observation using an inverted microscope (Keyence). The following cell aggregates were quantified: "cell aggregates with poor morphology," "cell aggregates with less than 10% neural tissue (cell aggregates with moderate morphology)," and "cell aggregates with less than 10% neural tissue (cell aggregates containing neural tissue)." The results showed that under conditions where preconditioning was not performed in step 1, the cell aggregates with poor morphology were approximately 100%, the cell aggregates with moderate morphology were approximately 0%, and the cell aggregates containing neural tissue were approximately 0%. In contrast, under conditions where preconditioning was performed in step 1, the cell aggregates with poor morphology were approximately 0%, the cell aggregates with moderate morphology were approximately 0%, and the cell aggregates containing neural tissue were approximately 100%.

[0356] Furthermore, the suspension culture medium prepared using the preconditioned iPS cells as a starting material was The cell aggregates (conditions in Figure 5B) on day 23 after the start of culture were fixed with 4% paraformaldehyde and frozen sections were prepared. These frozen sections were analyzed for the expression of neural tissue markers (neural progenitor cells). Nestin (anti-Nestin antibody, Millipore, mouse), a neuronal tissue marker (Neuro TuJ1 (anti-βIII-tubulin antibody, Promega, mouse), one of the βIII-tubulin antibodies, or neural tissue mice PSA-NCAM (anti-PSA-NCAM antibody, Millipore, mouse IgM), one of the markers, was immunoreactive. These immunostained sections were observed using an inverted fluorescence microscope. As a result, the cell aggregates prepared under conditions preconditioned with the BMPR inhibitor (LDN193189) showed no significant changes. The proportion of Nestin-positive cells was approximately 20%, the proportion of TuJ1-positive cells was approximately 70%, and the proportion of PSA-NCAM-positive cells was approximately 70% (Fig. 5C-E). Furthermore, analysis of serial sections confirmed that the areas that could be identified as neural tissue by morphological observation under bright field in an inverted microscope were Nestin-positive, TuJ1-positive, and PSA-NCAM-positive (Fig. 5C-E). These results indicate that by using feeder-free cultured human iPS cells as the starting material and preconditioning them with a TGFβ family signaling pathway inhibitor in step 1, neural tissue can be efficiently produced in step 3.

[0357] Example 7: Preconditioning with a TGFβ family signaling pathway inhibitor in step 1 In step 3, a BMP signaling pathway activator was used as a differentiation inducer to differentiate the retinal cells from human iPS cells. Example of membrane formation Human iPS cells (1231A3 strain) were subcultured in StemFit medium using the method described in Example 1. The human iPS cells were cultured in a feeder-free medium until one day before confluence. The cells were incubated in the presence of LDN193189 (BMPR inhibitor, 100 nM) (Step 1: Preconditioning treatment) for 1 day. The cells were cultured feeder-free.

[0358] The human iPS cells were treated with TrypLE Select (Life Technologies) to prepare a cell dispersion solution. The cells were then further dispersed into single cells by pipetting, and the dispersed human iPS cells were then cultured in a non-cell-adhesive 96-well culture plate (PrimeSurface 96V bottom plate, Sumitomo 1.2 x 10 per well of Bakelite 4 Cells were placed in 100 μl serum-free medium. The cells were cultured in suspension at 37°C and 5% CO2 in serum-free medium (gfCDM+KSR). A serum-free medium was used, which was a 1:1 mixture of medium and IMDM medium, supplemented with 10% KSR, 450 μM 1-monothioglycerol, and 1x chemically defined lipid concentrate. On day 0 after the start of culture (start of step 2), Y27632 (final concentration 20 μM) was added to the serum-free medium. By the second day after the start of suspension culture, cell aggregates were formed under preconditioned conditions (Fig. (Step 2 completed, step 3 started) On the third day after the start of suspension culture, 50 μl of serum-free medium not containing Y27632 was added.

[0359] In step 3, culture was performed under the following conditions 1 and 2. For condition 1 (+BMP), on day 6 after the start of suspension culture, the medium was replaced with a medium containing human recombinant BMP4 (R&D) without Y27632 so that the final concentration of exogenous human recombinant BMP4 was 1.5 nM (55 ng / ml) (Fig. 6B, D). For condition 2 (-BMP), on day 6 after the start of suspension culture, the medium was replaced with a medium containing human recombinant BMP4 (R&D) without Y27632 so that the final concentration of exogenous human recombinant BMP4 was 1.5 nM (55 ng / ml) (Fig. 6B, D). Half of the medium was replaced with medium containing no pathway-acting substances, without the addition of exogenous human recombinant BMP4 (Fig. 6A, C).

[0360] From the sixth day onwards, the suspension culture was incubated with Y27632 and human recombinant BMP4-free solution every two to four days. On the 23rd day after the start of suspension culture, the morphology was observed with an inverted microscope. It was found that the aggregates were maintained and neural tissue was formed.

[0361] The cell aggregates prepared in this way using the iPS cells preconditioned in step 1 as starting material on day 23 of suspension culture were fixed with 4% paraformaldehyde and frozen sections were prepared. These frozen sections were probed for Chx10 (anti-Chx10 antibody, Exalpha, sheep), a retinal tissue marker, or Rx (anti-Rx antibody), a retinal tissue marker. Immunostaining was performed on the cells (Takara, guinea pig). These immunostained sections were observed using an inverted fluorescence microscope. As a result, in the cell aggregates prepared under the conditions of preconditioning with a BMPR inhibitor (LDN193189) in step 1 and not adding BMP4 in step 3, all The proportion of Rx-positive cells in the cells was less than 10% (Rx-strongly positive (corresponding to the retina) was less than 3%, and Rx-weakly positive ( The percentage of Chx10-positive cells was less than 3%. On the other hand, in the cell aggregates prepared under the conditions of preconditioning with the BMPR inhibitor (LDN193189) in step 1 and adding BMP4 in step 3, the percentage of Rx-positive cells among all cells was The percentage of Chx10-positive cells was approximately 60%, and the percentage of Chx10-positive cells among all cells was also approximately 60% (Fig. 6B, D Furthermore, analysis of serial sections revealed that the proportion of Chx10-positive cells was high (approximately 95%) in neural tissue. These results suggest that Rx was also strongly positive (Fig. 6B, D). Under these conditions, neural tissue is formed, and differentiation inducers are then produced during suspension culture in step 3. By adding BMP signaling pathway agonists as a substrate, feeder-free cultured human It has been found that retinal tissue can be efficiently produced from human iPS cells (hereinafter sometimes referred to as feeder-free human iPS cells).

[0362] Example 8: Example of neural tissue formation from feeder-free human iPS cells using a TGFβ family signaling pathway inhibitor or an Shh signaling pathway agonist in step 1, and an Shh signaling pathway agonist in step 2 Human iPS cells (1231A3 strain) were subcultured in StemFit medium using the method described in Example 1. The human iPS cells were cultured in a feeder-free medium until one day before confluence. The cells were cultured in the presence (preconditioning) or absence of SB431542 (TGFβR inhibitor, 5 μM), LDN193189 (BMPR inhibitor, 100 nM), or SAG (Shh signaling pathway agonist, 300 nM). (Step 1: No preconditioning) and then cultured in a feeder-free medium for 1 day.

[0363] The human iPS cells were treated with TrypLE Select (Life Technologies) to form a cell dispersion. The cells were then further dispersed into single cells by pipetting, and the dispersed human iPS cells were placed in a non-cell-adhesive 96-well culture plate (PrimeSurface 96V bottom plate, Sumitomo Bakelite). 1.2 x 10 per well of Krite 4 The cells were suspended in 100 μl of serum-free medium. The cells were cultured in suspension at 37°C and 5% CO2. The serum-free medium (gfCDM+KSR) used was F-12 medium. A serum-free medium was used, which was a 1:1 mixture of IMDM medium and 10% KSR, 450 μM 1-monothioglycerol, and 1x chemically defined lipid concentrate.

[0364] At the start of suspension culture (day 0 after suspension culture was started, starting step 2), culture was carried out under the following two conditions: Conditions 1 and 2. As condition 1 (+SAG), Y27632 (final concentration 20 μM) and SAG (a substance acting on the Shh signaling pathway, 300 nM) were added to the serum-free medium at the start of suspension culture (Fig. 7B-D, FH, '+SAG'). As condition 2 (-SAG), Y27632 (final concentration 20 μM) was added to the serum-free medium at the start of suspension culture, but SAG was not added (Fig. 7A, E, '-'). By the second day after the start of suspension culture, Under all conditions, cell aggregates were formed (step 2 completed, step 3 initiated). 50 μl of serum-free medium without Y27632 or SAG was added to the eyes.

[0365] On the sixth day after the start of suspension culture, the cells were cultured without Y27632 or SAG, but with human recombinant BMP4 (R&D). The media containing or not containing exogenous human recombinant BMP4 at a final concentration of 1.5 nM (Fig. 7E-H) or without BMP signaling pathway agonists (Fig. 7A-D) were used. From day 6 onwards after the start of suspension culture, half of the medium was replaced with the serum-free medium containing no Y27632, SAG or human recombinant BMP4 every 2 to 4 days.

[0366] The cells prepared in this way were subjected to bright field observation using an inverted microscope (Keyence) on the 23rd day after the start of suspension culture (Figure 7). Morphology was quantified using the method described in Example 6 (Fig. 7I). As a result, it was found that when preconditioning was not performed in step 1 and SAG was not added in step 2, cell aggregates collapsed and neural tissue was not formed (Fig. 7A, E). On the other hand, when preconditioning was performed with a TGFβR inhibitor (SB431542), a BMPR inhibitor (LDN193189), or an agent acting on the Shh signaling pathway in step 1 and SAG was not added in step 2, the cells were not aggregated and neural tissue was not formed (Fig. 7A, E). When a substance acting on the signaling pathway was added, cell aggregates were formed, and neural tissue was efficiently formed (Figure 7B-D, FI).

[0367] Furthermore, the preconditioning with the TGFβR inhibitor (SB431542) or the BMPR inhibitor (LDN193189) Cell aggregates produced from treated iPS cells on the 23rd day after the start of suspension culture The tissues were fixed with 4% paraformaldehyde and frozen sections were prepared. These frozen sections were analyzed for Nestin (anti-Nestin antibody, Millipore, mouse), a neural tissue marker (neuron), and TuJ1 (anti-βIII-tubulin antibody, Pr Immunostaining was performed for PSA-NCAM (anti-PSA-NCAM antibody, Millipore, mouse IgM), a neural tissue marker. These immunostained sections were observed using an inverted fluorescence microscope. As a result, in cell aggregates prepared under conditions preconditioned with a TGFβR inhibitor (SB431542) or a BMPR inhibitor (LDN193189), nestin-positive cells in all cells were detected. The proportion of neural cells was approximately 20%, the proportion of TuJ1-positive cells among all cells was approximately 70%, and the proportion of PSA-NCAM-positive cells was approximately 70% (Fig. 8A-F). Furthermore, analysis of serial sections confirmed that the areas distinguishable as neural tissue by morphological observation in bright-field images were positive for Nestin, TuJ1, and PSA-NCAM (Fig. 8A-F).

[0368] From these results, it is possible to detect the presence of a TGFβ family signaling pathway inhibitor or Shh signaling inhibitor in step 1. When preconditioning was performed with a substance acting on the neural signaling pathway and then a substance acting on the Shh signaling pathway was added in step 2, neural tissue could be efficiently produced from feeder-free human iPS cells, regardless of whether a substance acting on the BMP signaling pathway was added in step 3. It was found that...

[0369] Example 9: In step 1, a TGFβ family signaling pathway inhibitor is used, and in step 2, Shh signaling is inhibited. Example of retinal tissue formation from feeder-free human iPS cells using a substance acting on the signal transduction pathway and a substance acting on the BMP signaling pathway as a differentiation inducer in step 3. Human iPS cells were cultured using StemFit medium as a feeder-free medium according to the method described in Example 8. Using this as a starting material, cell aggregates were prepared using a TGFβR inhibitor (SB431542) or a BMPR inhibitor (LDN193189) in step 1, a substance acting on the Shh signaling pathway in step 2, and with or without the addition of a substance acting on the BMP signaling pathway in step 3. Neural tissue was formed in the cell aggregates formed under all of these conditions on day 23 after the start of suspension culture. The cell aggregates were fixed with 4% paraformaldehyde and frozen sections were prepared. These frozen sections were analyzed for Chx10 (anti-Chx10 antibody, Exalpha, sheep), a retinal tissue marker, or Rx (anti-Rx antibody, Takara, guinea pig), a retinal tissue marker. Immunostaining was performed on the cells and observed under a fluorescent microscope. As a result, in the cell aggregates prepared under the conditions of preconditioning with a TGFβR inhibitor (SB431542) or a BMPR inhibitor (LDN193189) in step 1, adding an agent acting on the Shh signaling pathway in step 2, and not adding BMP4 in step 3, all cells were The proportion of Rx-positive cells in the retina was less than 10% (Rx-strongly positive (corresponding to the retina) was less than 3%), and Rx-weakly positive (corresponding to the retina) was less than 3%. The percentage of cells positive for Chx10 was less than 3%. On the other hand, in step 1, the TGFβR inhibitor (SB431542) was used for preconditioning. In step 2, a substance acting on the Shh signaling pathway was added, and in step 3, BMP4 was added. In the prepared cell aggregates, the percentage of Rx-positive cells among all cells was 80% or more, and the percentage of Chx10-positive cells among all cells was 80% or more. The percentage of viable cells was also found to be over 70% (Fig. 9B, F). In addition, the cells were preconditioned with a BMPR inhibitor (LDN193189) in step 1, added with an Shh signaling pathway agonist in step 2, and then cultured in step 3. In cell aggregates prepared under conditions in which BMP4 was added, the proportion of Rx-positive cells among all cells was found to be over 50%, and the proportion of Chx10-positive cells among all cells was also found to be over 40% (Figure 9D, H). Furthermore, analysis of serial sections revealed that Chx10 was also expressed in neural tissue with a high percentage of positive cells (over 95%). In this case, the cells were found to be strongly Rx positive (Fig. 9B, D, F, H).

[0370] Based on these results, in step 1, preconditioning was performed with a TGFβ family signaling pathway inhibitor, in step 2, an Shh signaling pathway activator was added, and in step 3, differentiation was performed during suspension culture. It was found that retinal tissue can be efficiently produced from feeder-free human iPS cells by adding a substance that acts on the BMP signaling pathway as an inducer.

[0371] Example 10: Example of cell aggregate formation using human iPS cells grown in Essential 8 medium as a feeder-free medium as a starting material and a TGFβ family signaling pathway inhibitor in step 1 Human iPS cells (1231A3 strain, obtained from Kyoto University) were cultured as described in Scientific Reports, 4, 3594 (20 Feeder-free culture was performed according to the method described in "14" (2011). Essential 8 medium (Life Technologies, Nature Methods, 8, 424-429 (2011)) was used as the feeder-free medium, and Laminin511-E8 (Nippi) was used as the feeder-free scaffold.

[0372] As a specific maintenance culture procedure, first, the cells are grown to subconfluent (60% of the culture area is covered with cells). Human iPS cells (1231A3 strain) that had reached a size where they could be cultured (enough to be cultured) were washed with PBS and dispersed into single cells using TrypLE Select (Life Technologies). The dispersed human iPS cells were then seeded onto a plastic culture dish (Iwaki) coated with Laminin511-E8 and cultured in feeder-free Essential 8 medium in the presence of Y27632 (10 μM, a ROCK inhibitor). The plastic culture dish was a 6-well plate (manufactured by Iwaki Co., Ltd., Cultivation area 9.4 cm 2 ), the seeding number of the dispersed human iPS cells was 6 x 103 One day after seeding, the medium was changed to Essential 8 medium without Y27632. Six days after seeding, the cells were cultured until they became subconfluent (about 60% of the culture area was covered with cells).

[0373] As a preconditioning procedure, first, subconfluent human iPS cells (1231A3 strain) After washing with PBS, the cells were dispersed into single cells using TrypLE Select (Life Technologies). The dispersed human iPS cells were then seeded onto a plastic culture dish (Iwaki) coated with Laminin511-E8 and cultured in a feeder-free manner in Essential 8 medium in the presence of Y27632 (10 μM, a ROCK pathway inhibitor). A 6-well plate (Iwaki, for cell culture, culture area 9.4 cm) was used as a support. 2 ), the seeding number of the dispersed human iPS cells was 6 x 10 3 One day after sowing Afterwards, the medium was changed to Essential 8 medium without Y27632. Five days after seeding, the cells were cultured until they reached one day before subconfluence (about 50% of the culture area was covered with cells). The feeder-free cultured human iPS cells that reached one day before subconfluence were cultured feeder-free for one day in the presence (Step 1: Preconditioning) or absence (Step 1: No Preconditioning, control) of SB431542 (TGFβR inhibitor, 5 μM), LDN193189 (BMPR inhibitor, 100 nM), or dual inhibition of TGFβR and BMPR (SB431542 at 5 μM and LDN193189 at 100 nM). When the cells were observed under bright field conditions using an inverted microscope (Keyence), it was found that treatment with a TGFβ family signaling pathway inhibitor during feeder-free culture did not significantly affect the morphology of human iPS cells.

[0374] The human iPS cells were treated with TrypLE Select (Life Technologies) to form a cell dispersion. The cells were then further dispersed into single cells by pipetting, and the dispersed human iPS cells were placed in a non-cell-adhesive 96-well culture plate (PrimeSurface 96V bottom plate, Sumitomo Bakelite). 1.2 x 10 per well of Krite 4 The cells were suspended in 100 μl of serum-free medium. The cells were cultured in suspension at 37°C and 5% CO2. The serum-free medium (gfCDM+KSR) used was F-12 medium. A serum-free medium was used, which was a 1:1 mixture of IMDM medium and 10% KSR, 450 μM 1-monothioglycerol, and 1x chemically defined lipid concentrate.

[0375] At the start of suspension culture (day 0 after suspension culture was started, starting step 2), culture was carried out under the following two conditions: Conditions 1 and 2. In condition 1 (+SAG), Y27632 (final concentration 20 μM) and SAG (a substance acting on the Shh signaling pathway, 300 nM) were added to the serum-free medium at the start of suspension culture (Fig. 10C, F, H, J, L, N). Condition 2 (-SAG) was achieved by adding Y27632 (final concentration 20 μM) to the serum-free medium at the start of suspension culture, without adding SAG (Fig. 10A, B, D, E, G, I, K, M "-"). Cell aggregates were formed under all conditions by the second day after the start of suspension culture (end of step 2, step 3). On the third day after the start of suspension culture, 50 μl of serum-free medium containing neither Y27632 nor SAG was added.

[0376] On the sixth day after the start of suspension culture, the cells were cultured without Y27632 or SAG, but with human recombinant BMP4 (R&D). Cultures were cultured in media containing or without exogenous human recombinant BMP4 at a final concentration of 1.5 nM. (Fig. 10D-F), or medium without BMP signaling pathway agonists (Fig. 10K-N). From day 6 onwards after the start of suspension culture, half of the medium was replaced with the serum-free medium containing no Y27632, SAG or human recombinant BMP4 every 2 to 4 days.

[0377] The cells prepared in this way were observed under an inverted microscope (Keyence) on the 7th day after the start of suspension culture. Bright-field observation was performed using the 100% ATP-dependent ATPase inhibitor (Figure 10). As a result, it was found that when preconditioning was not performed in step 1 and SAG was not added in step 2, the cell aggregates collapsed and neural tissue was not formed. It was found that under the conditions of preconditioning in step 1, cell aggregates were formed both when no Shh signaling pathway acting substance was added in step 2 and when an Shh signaling pathway acting substance was added in step 2 (Fig. 10). That is, even when Essential 8 medium was used as the feeder-free medium, the TGF-β family in step 1 was not affected. Preconditioning with signaling pathway inhibitors enhances the differentiation of cells formed from human iPS cells. It was found that the morphology of the cell aggregates improved.

[0378] Example 11: Example of neural tissue formation using human iPS cells grown in Essential 8 medium as a feeder-free medium as a starting material, a TGFβ family signaling pathway inhibitor in step 1, and an Shh signaling pathway activator in step 2 Using the method described in Example 10, human iPS cells were used as a starting material in feeder-free culture using Essential 8 medium, and a TGFβR inhibitor (SB431542) was used in step 1, and an Shh signal inhibitor was used in step 2. Cell aggregates were prepared using substances acting on the neural signaling pathway. The cell aggregates were fixed with 4% paraformaldehyde on day 18 of suspension culture and cryosectioned. These cryosections were immunostained for Nestin (anti-Nestin antibody, Millipore, mouse), a neural tissue marker (neuron), TuJ1 (anti-βIII-tubulin antibody, Promega, mouse), a neural tissue marker (neuron), or PSA-NCAM (anti-PSA-NCAM antibody, Millipore, mouse IgM), a neural tissue marker, and observed under a fluorescence microscope. The results showed that the percentage of Nestin-positive cells in the cell aggregates was approximately 30%, the percentage of TuJ1-positive cells was approximately 70%, and the percentage of PSA-NCAM-positive cells was approximately 70% (Figure 11). Furthermore, analysis of serial sections confirmed that the areas that could be identified as neural tissue by morphological observation in bright-field images were Nestin-positive, TuJ1-positive, and PSA-NCAM-positive (Figure 11). That is, under the conditions where the cells were preconditioned with a TGFβR inhibitor in step 1 and SAG was added in step 2, It was found that neural tissue was efficiently formed in the cell aggregates prepared under these conditions.

[0379] Example 12: Example of retinal tissue formation using human iPS cells cultured in Essential 8 medium as a starting material for feeder-free culture, with a TGFβ family signaling pathway inhibitor used in step 1, a substance acting on the Shh signaling pathway used in step 2, and a substance acting on the BMP signaling pathway used in step 3 Using the method described in Example 10, human iPS cells were used as a starting material in feeder-free culture using Essential 8 medium, and a TGFβR inhibitor (SB431542) was used in step 1, and an Shh signal inhibitor was used in step 2. Cell aggregates were prepared using a substance acting on the basal transduction pathway, with or without the addition of a substance acting on the BMP signaling pathway in step 3. The cell aggregates on day 18 after the start of suspension culture were fixed with 4% paraformaldehyde and frozen sections were prepared. These frozen sections were immunolabeled for Chx10 (anti-Chx10 antibody, Exalpha, sheep) or Rx (anti-Rx antibody, Takara, guinea pig), a retinal tissue marker. Immunostaining was performed and observed under a fluorescent microscope. As a result, in step 1, the cells were preconditioned with a TGFβR inhibitor (SB431542), in step 2, a substance acting on the Shh signaling pathway was added, and in step 3, BMP4 was added. In the cell aggregates prepared under the condition where no addition of HCl was made, the proportion of Rx-positive cells among all cells was less than 10% (Rx-strongly positive (corresponding to the retina) was less than 3%, and Rx-weakly positive (corresponding to neural tissue other than the retina) was less than 7%), and the proportion of Chx10-positive cells was also less than 3% (Figure 12). Then, the cells were preconditioned with a TGFβR inhibitor (SB431542), and in step 2, an agent acting on the Shh signaling pathway was added. In the cell aggregates prepared under the conditions where BMP4 was added in step 3, the Rx strength in all cells was The percentage of positive cells was 30% or more, and the percentage of Chx10-positive cells among all cells was 20% or more. Furthermore, analysis of serial sections revealed that Chx10 also showed a high percentage of positive cells (over 95%). From these results, we have found that the neural tissues of the 100-kDa mice (top) were strongly Rx positive. Based on these results, we have used human iPS cells grown in Essential 8 medium for feeder-free culture as the starting material, and in step 1 we used a TGFβR inhibitor, in step 2 we used an Shh signaling pathway activator, and in step 3 we used a BMP signaling activator. It was found that retinal tissue could be produced under conditions in which substances acting on the neural signaling pathway were added.

[0380] Example 13: Human iPS cells grown in StemFit as a feeder-free medium were used as a starting material, and in step 1, a TGFβ signaling pathway inhibitor and an Shh signaling pathway activator were used. Example of retinal tissue formation using a substance acting on the BMP signaling pathway in step 3 Human iPS cells (1231A3 strain) were subcultured in StemFit medium using the method described in Example 1. The human iPS cells were cultured in a feeder-free medium until they reached subconfluency one day prior to the day of confluence. ...fluency. The human iPS cells were cultured in a feeder-free medium until they reached subconfluency one day prior to the day of con The cells were cultured in the presence of TGFβRi and SAG for 1 day (Step 1, Precondition: TGFβRi+SAG treatment).

[0381] The human iPS cells were treated with TrypLE Select (Life Technologies) to form a cell dispersion. The cells were then further dispersed into single cells by pipetting, and the dispersed human iPS cells were placed in a non-cell-adhesive 96-well culture plate (PrimeSurface 96V bottom plate, Sumitomo Bakelite). 1.2 x 10 per well of Krite 4 The cells were suspended in 100 μl of serum-free medium. The cells were cultured in suspension at 37°C and 5% CO2. The serum-free medium used was either F-12 medium or IMDM medium. A serum-free medium containing 10% KSR, 450 μM 1-monothioglycerol, and 1 x chemically defined lipid concentrate was used. In step 3 (step 2, step 2 initiation), Y27632 (final concentration 20 μM) and SAG (a substance acting on the Shh signaling pathway, 300 nM) were added to the serum-free medium. Cell aggregates were formed within one day, up to the second day after the start of suspension culture (step 2 completion, step 3 initiation). Thereafter, in step 3, the cells were cultured under the following conditions 1 to 3.

[0382] Condition 1: On the third day after the start of suspension culture, 50 μl of serum-free medium that does not contain Y27632, SAG, or BMP signaling pathway active substances was added. From the sixth day after the start of suspension culture, half of the medium was replaced (half the volume) with the serum-free medium that does not contain Y27632, SAG, or BMP signaling pathway active substances every three days. An aliquot, i.e., 75 μl, was discarded and the remaining aliquot was diluted with 100 μl of PBS containing no Y27632, SAG, or BMP signaling pathway agents. Then, 75 μl of the serum-free medium was added. Condition 2: On the third day after the start of suspension culture, human recombinant BMP4 was added to a final concentration of 1.5 ml without Y27632 or SAG. After the sixth day from the start of suspension culture, half of the medium was replaced (half the volume, i.e., 1 / 2 of the volume) with the serum-free medium containing no Y27632, SAG, or BMP acting substance every three days. 75 μl was discarded, and the blood-free solution containing Y27632, SAG, and BMP signaling pathway active substances was 75 μl of supernatant medium was added). Condition 3: On the third day after the start of suspension culture, 50 μl of serum-free medium that did not contain Y27632, SAG, or substances acting on the BMP signaling pathway was added. On the sixth day after the start of suspension culture, 50 μl of serum-free medium that did not contain Y27632 or SAG and that did not contain human Serum-free medium was added with recombinant BMP4 at a final concentration of 1.5 nM. After the sixth day of suspension culture, Once every three days, half of the medium was replaced with the serum-free medium containing no Y27632, SAG, or BMP acting substances. Half the volume (i.e., 75 μl) was discarded and analyzed to determine whether Y27632, SAG, or BMP signaling pathways were affected. (75 μl of the serum-free medium containing no substance was added).

[0383] When cultured under the above conditions 1 to 3, cell aggregates were formed under all conditions on the 26th day after the start of suspension culture, and neural tissue was formed. The cell aggregates on the 26th day after the start of suspension culture were fixed with 4% paraformaldehyde and frozen sections were prepared. These frozen sections were immunostained for Chx10 (anti-Chx10 antibody, Exalpha, sheep), a retinal tissue marker. As a result, the cell aggregates under condition 1 had a Chx10-positive cell ratio of less than 3% of the total cells. On the other hand, the cell aggregates under conditions 2 and 3 showed that Chx1 in the whole cells From these results, it was found that the proportion of 0-positive cells was 60% or more (Fig. 13, middle, right). It was found that retinal tissue could be produced by using a substance acting on the nucleoside signaling pathway and adding a substance acting on the BMP signaling pathway in step 3.

[0384] Example 14: Examination of the number of cells seeded in step 2 (human iPS cells grown in StemFit as a feeder-free medium were used as the starting material, and a TGFβ signaling pathway inhibitor and an Shh signaling pathway agonist were used in step 1) Human iPS cells (1231A3 strain) were subcultured in StemFit medium using the method described in Example 1. The cells were cultured feeder-free until one day before confluence. The cells were cultured in a feeder-free environment for 1 day in the presence of SB431542 (TGFβR inhibitor, 5 μM) and SAG (Shh signaling pathway agonist, 300 nM) (Step 1, Precondition: TGFβRi + SAG treatment). ).

[0385] The human iPS cells were treated with TrypLE Select (Life Technologies) to form a cell dispersion. The human iPS cells were then dispersed into single cells by pipetting. The dispersed human iPS cells were then placed in a non-cell-adhesive 96-well culture plate (PrimeSurface 96V bottom plate, Sumitomo Chemical Co., Ltd.). The following four conditions were used per well of a plate (manufactured by Bakelite Co., Ltd.): 0.4 x 10 4 , 0.8 x 10 4 , 1.2 x 10 4 , or 1.6 x 10 4 The cells were suspended in 100 μl of serum-free medium and cultured at 37°C and 5% CO2. The serum-free medium (gfCDM+KSR) used at this time consisted of F-12 medium and IMDM medium. A 1:1 mixture of 10% KSR, 450 μM 1-monothioglycerol, and 1 x Chemically defined A serum-free medium containing lipid concentrate was used. On the second day after the start of suspension culture (day 1, start of step 2), Y27632 (final concentration 20 μM) and SAG (a substance acting on the Shh signaling pathway, 300 nM) were added to the serum-free medium. Cell aggregates were formed within one day, up to day 2 after the start of suspension culture (end of step 2, start of step 3). Thereafter, on day 3 after the start of suspension culture, serum-free medium was added without Y27632 or SAG, and human recombinant BMP4 was added to a final concentration of 1.5 nM. From the sixth day onwards, the cells were cultured in a medium containing no Y27632, SAG or BMP-active substances every two to four days. Half of the medium was replaced with the serum-free medium described above.

[0386] On the 18th day after the start of the suspension culture, cell aggregates were formed under all conditions, and neural tissue was formed (Figure 14, upper panel). The cell aggregates on the 18th day after the start of the suspension culture were fixed with 4% paraformaldehyde and frozen sections were prepared. These frozen sections were analyzed for the presence of a retinal tissue marker, α-receptor β-glucan. Immunostaining was performed for Chx10 (anti-Chx10 antibody, Exalpha, sheep) or Rx (anti-Rx antibody, Takara, guinea pig), a retinal tissue marker, and the cells were observed under a fluorescent microscope. 4 , 0.8 x 10 4 , 1.2 x 10 4 , or 1.6 x 10 4 In the case of Chx10-positive cells, the percentage of Chx10-positive cells among all cells was found to be 20% or more. (Fig. 14, middle, right). In particular, when the seeding cell number was 0.8 x 10 4 , or 1.2 x 10 4 The cell conditions resulted in a particularly high percentage of Chx10-positive cells. The percentage of Rx-positive cells was also similar to that of Chx10. From these results, it can be concluded that the number of cells seeded in step 2 was 0.4 x 10 4 ~1.6 x 10 4 Cellular This demonstrated that retinal tissue could be produced.

[0387] Example 15: In step 1, Lefty, a TGFβ family signaling pathway inhibitor, was used to Example of neural tissue formation from feeder-free human iPS cells using SAG as an agent acting on the Shh signaling pathway in Step 2 Human iPS cells (1231A3 strain, obtained from Kyoto University) were cultured in a feeder-free environment according to the method described in Scientific Reports, 4, 3594 (2014). The feeder-free medium used was Stem Fit medium (AK03; manufactured by Ajinomoto Co., Inc.), and the feeder-free scaffold used was Laminin 511-E8 (manufactured by Nippi Co., Ltd.). was used.

[0388] Human iPS cells (1231A3 strain) were subcultured using StemFit medium according to the method described in Example 1. The cells were cultured in a feeder-free environment for one day before incubation. Lee cultured. Condition 1: Human recombinant Lefty-A (Nodal / Activin signaling pathway inhibitor, manufactured by R&D, Lefty-A C-terminus, 20 μg / ml) Condition 2: Human recombinant Lefty-A (Nodal / Activin signaling pathway inhibitor, R&D Lefty-A C-terminus, 20 μg / ml) and SAG (Shh signaling pathway agonist, 300 nM) Condition 3: Exogenous TGFβ family signaling pathway inhibitors and Shh signaling pathway Absence of active substance (no preconditioning)

[0389] The human iPS cells prepared under conditions 1-3 were treated with TrypLE Select (Life Technologies) to form a cell dispersion solution, and then dispersed into single cells by pipetting. The dispersed human iPS cells were then plated into a non-cell-adhesive 96-well culture plate (PrimeSurface 96V bottom plate, Sumitomo Bakelite Co., Ltd.) at 1.2 x 10 cells per well. 4 Into cells The cells were suspended in 100 μl of serum-free medium and cultured at 37°C and 5% CO2. The serum-free medium (gfCDM+KSR) contained 10% KSR, 450 μM 1-monothiocyanate, and 1:1 mixture of F-12 medium and IMDM medium. A serum-free medium supplemented with glycerol and 1× chemically defined lipid concentrate was used.

[0390] At the start of suspension culture (day 0 after suspension culture was started, starting step 2), culture was carried out under the following two conditions: Conditions 1 and 2. As condition 1 (+SAG), Y27632 (final concentration 20 μM) and SAG (a substance acting on the Shh signaling pathway, 300 nM) were added to the serum-free medium at the start of suspension culture. As condition 2 (-SAG), At the start of suspension culture, Y27632 (final concentration 20 μM) was added to the serum-free medium, but SAG was not added. Cell aggregates were formed under all conditions by the second day after the start of suspension culture (end of step 2, start of step 3). On the third day after the start of suspension culture, 50 μl of medium containing human recombinant BMP4 (manufactured by R&D Co., Ltd.) but not Y27632 or SAG was added so that the final concentration of exogenous human recombinant BMP4 was 1.5 nM (55 ng / ml). On the sixth day after the start of suspension culture, Y27632, SAG, and human recombinant BMP4 were added. After that, half of the medium was replaced with serum-free medium containing no Y27632, SAG, or ATP every 2 to 4 days. Half of the medium was replaced with the serum-free medium containing no human recombinant BMP4.

[0391] The cells prepared in this way were subjected to bright field observation using an inverted microscope (Keyence Corporation) on the 18th day after the start of suspension culture (Figure 15). As a result, cells that were not preconditioned in step 1 were observed in bright field. When SAG was not added in step 2, the cell aggregates collapsed and neural tissue was not formed (Fig. 15, A). On the other hand, when the Nodal / Activin signaling pathway inhibitor, Alternatively, cells were preconditioned with a Nodal / Activin signaling pathway inhibitor and an Shh signaling pathway agonist, and then in step 2, cell aggregates were formed, with or without the addition of an Shh signaling pathway agonist, and neural tissue was efficiently formed (Figure 15, B, C, D).

[0392] These results suggest that even under conditions where the cells were preconditioned using Lefty, an inhibitor of the Nodal / Activin signaling pathway, as an inhibitor of the TGFβ family signaling pathway in step 1, High-quality aggregates were formed from leader-free human iPS cells, and the efficiency of neuroepithelial production was improved. It was found that

[0393] Example 16: Example of formation of neural tissue and retinal tissue from feeder-free human iPS cells using A83-01, a TGFβ family signaling pathway inhibitor, in step 1 and SAG, an Shh signaling pathway activator, in step 2 Human iPS cells (1231A3 strain, obtained from Kyoto University) were cultured in a feeder-free environment according to the method described in Scientific Reports, 4, 3594 (2014). The feeder-free medium used was Stem Fit medium (AK03; manufactured by Ajinomoto Co., Inc.), and the feeder-free scaffold used was Laminin 511-E8 (manufactured by Nippi Co., Ltd.). was used.

[0394] Human iPS cells that had reached subconfluence one day prior to the initial stage, as prepared by the method described in Example 15, were cultured in a feeder-free medium for one day in the presence (preconditioning) or absence (step 1: no preconditioning) of A83-01 (Wako, TGFβR inhibitor, 0.5 μM).

[0395] The human iPS cells were treated with TrypLE Select (Life Technologies) to prepare a cell dispersion solution. The cells were then further dispersed into single cells by pipetting, and the dispersed human iPS cells were then cultured in a non-cell-adhesive 96-well culture plate (PrimeSurface 96V bottom plate, Sumitomo Bakelite) per well, 1.0 x 10 4 The cells were suspended in 100 μl of serum-free medium and cultured at 37°C and 5% CO2. The serum-free medium (gfCDM+KSR) used was F-12 medium. A 1:1 mixture of IMDM medium and 10% KSR, 450 μM 1-monothioglycerol, 1 x Chemically Serum-free medium supplemented with a defined lipid concentrate was used.

[0396] At the start of suspension culture (day 0 after suspension culture was started, starting step 2), culture was carried out under the following two conditions: Conditions 1 and 2. As condition 1 (+SAG), Y27632 (final concentration 20 μM) and SAG (a substance acting on the Shh signaling pathway, final concentration 30 nM) were added to the serum-free medium at the start of suspension culture. As condition 2 (-SAG), Y27632 (final concentration 20 μM) was added to the serum-free medium at the start of suspension culture, but SAG was not added. Cell aggregates were formed under all conditions by the second day after the start of suspension culture ( (Step 2 completed, Step 3 started). On the third day after the start of suspension culture, the final concentration of exogenous human recombinant BMP4 was 50 μl of medium containing human recombinant BMP4 (R&D) but not Y27632 or SAG was added to the suspension so that the concentration of BMP4 was 1.5 nM (55 ng / ml). Half of the medium was replaced with serum-free medium without BMP4, and then every 2 to 4 days, half of the medium was replaced with the serum-free medium without Y27632, SAG, and human recombinant BMP4.

[0397] The cells prepared in this manner were subjected to bright-field observation using an inverted microscope (Keyence Corporation) on day 20 after the start of suspension culture (Figure 16, AB). The results showed that when preconditioning was not performed in step 1 and SAG was not added in step 2, cell aggregates collapsed and neural tissue was not formed (Figure 16, A). On the other hand, when preconditioning was performed with A83-01 in step 1 and SAG was added in step 2, cell aggregates were formed and neural tissue was efficiently formed (Figure 16, B).

[0398] The cell aggregates on day 20 of suspension culture were fixed with 4% paraformaldehyde and frozen sections were prepared. These frozen sections were analyzed by immunoblotting using Chx10 (anti-Chx10 antibody), a retinal tissue marker. Immunostaining was performed on sheep (Exalpha, sheep) and observed using a fluorescent microscope. As a result, it was found that when preconditioning was performed with A83-01 in step 1 and SAG was added in step 2, the proportion of Chx10-positive cells among all cells was approximately 20% (FIG. 16, C). These results demonstrated that retinal tissue can be produced by preconditioning with the TGFβR inhibitor A83-01 as an inhibitor of the TGFβ family signaling pathway in step 1, and adding an Shh signaling pathway active substance in step 2.

[0399] Example 17: In step 1, A83-01, a TGFβ family signaling pathway inhibitor, or TGF A combination of a β family signaling pathway inhibitor (A83-01) and an Shh signaling pathway agonist (either SAG, Purmorphamine, or human recombinant Shh) was used to generate neural tissue from feeder-free human iPS cells using an Shh signaling pathway agonist in step 2. Formation example Human iPS cells (1231A3 strain, obtained from Kyoto University) were cultured in a feeder-free environment according to the method described in Scientific Reports, 4, 3594 (2014). The feeder-free medium used was Stem Fit medium (AK03; manufactured by Ajinomoto Co., Inc.), and the feeder-free scaffold used was Laminin 511-E8 (manufactured by Nippi Co., Ltd.). was used.

[0400] Human iPS cells one day before subconfluent, prepared by the method described in Example 15, were cultured under the following six conditions: The cells were cultured in a feeder-free medium for 1 day. Condition 1. A83-01 (Wako, TGFβR inhibitor, 0.5 μM) Condition 2: A83-01 (Wako, TGFβR inhibitor, 0.5 μM) and SAG (Enzo, Shh signal inhibitor) transduction pathway agonist, 300 nM) Condition 3: A83-01 (Wako, TGFβR inhibitor, 0.5 μM) and Purmorphamine (Wako, Shh signaling pathway activator, 0.2 μM) Condition 4. A83-01 (Wako, TGFβR inhibitor, 0.5 μM) and human recombinant Shh (R&D, Shh signaling pathway activator, 50 ng / ml) Condition 5. A83-01 (Wako, TGFβR inhibitor 0.5 μM) and human recombinant Shh (R&D, Shh signaling pathway agonists, 300ng / ml) Condition 6: Exogenous TGFβ family signaling pathway inhibitors and Shh signaling pathway Absence of active substance (no preconditioning)

[0401] The human iPS cells were treated with TrypLE Select (Life Technologies) to prepare a cell dispersion solution. The cells were then further dispersed into single cells by pipetting, and the dispersed human iPS cells were then cultured in a non-cell-adhesive 96-well culture plate (PrimeSurface 96V bottom plate, Sumitomo Bakelite) per well, 1.0 x 10 4 The cells were suspended in 100 μl of serum-free medium and cultured at 37°C and 5% CO2. The serum-free medium (gfCDM+KSR) used was F-12 medium. A 1:1 mixture of IMDM medium and 10% KSR, 450 μM 1-monothioglycerol, 1 x Chemically Serum-free medium supplemented with a defined lipid concentrate was used.

[0402] At the start of suspension culture (day 0 after suspension culture was started, starting step 2), the following two conditions (A) and (B) were used. Condition (A) (+SAG) was added to the serum-free medium at the start of suspension culture. Condition (B) (-SAG ) At the start of suspension culture, Y27632 (final concentration 20 μM) was added to the serum-free medium, but no SAG was added.

[0403] By the second day after the start of suspension culture, cell aggregates were formed under all conditions (end of step 2, start of step 3). On the third day after the start of suspension culture, when the final concentration of exogenous human recombinant BMP4 was 1.5 nM (55 ng / ml) and does not contain Y27632, SAG, Purmorphamine, or human recombinant Shh. 50 μl of medium containing recombinant BMP4 (R&D) was added. On day 6 after the initiation of suspension culture, half of the medium was replaced with serum-free medium that did not contain Y27632, SAG, Purmorphamine, human recombinant Shh, or human recombinant BMP4. Thereafter, half of the medium was replaced every 2–4 days with the same serum-free medium that did not contain Y27632, SAG, Purmorphamine, human recombinant Shh, or human recombinant BMP4.

[0404] The cells thus prepared were subjected to bright field observation using an inverted microscope (Keyence Corporation) on the 20th day after the start of suspension culture (Figure 17). As a result, under the condition 6 described above, where preconditioning was not performed in step 1 and SAG was not added in step 2, the cell aggregates collapsed and neural tissue was formed. On the other hand, under the condition 1-5 in which A83-01 and Shh signaling pathway acting substances (SAG, PMA, human recombinant Shh 50 ng / ml, human recombinant Shh 300 ng / ml) were applied in step 1 and SAG was added in step 2 (condition (A) (+SAG)), no tissue was formed. It was found that cell aggregates were formed and neural tissue was efficiently formed (Figure 17, BF). . From these results, it was found that in step 1, preconditioning was performed using a TGFβ family signaling pathway inhibitor (A83-01), or a TGFβ family signaling pathway inhibitor (A83-01) and an Shh signaling pathway agonist (SAG, PMA, or human recombinant Shh). It was discovered that neural tissue can be efficiently produced from human iPS cells cultured in a feeder-free environment. Ta.

[0405] Example 18: In step 1, TGFβ family signaling pathway inhibitor and Shh signaling pathway Example of retinal tissue formation from feeder-free human iPS cells using an agent acting on the Shh signaling pathway (either SAG, Purmorphamine, or human recombinant Shh) in step 2. Human iPS cells (1231A3 strain, obtained from Kyoto University) were cultured in a feeder-free environment according to the method described in Scientific Reports, 4, 3594 (2014). The feeder-free medium used was Stem Fit medium (AK03; manufactured by Ajinomoto Co., Inc.), and the feeder-free scaffold used was Laminin 511-E8 (manufactured by Nippi Co., Ltd.). was used.

[0406] Human iPS cells that had reached subconfluence one day prior to the initial stage, as prepared by the method described in Example 15, were cultured in a feeder-free medium for one day in the presence (preconditioning) or absence (step 1: no preconditioning) of A83-01 (Wako, TGFβR inhibitor, 0.5 μM) and SAG (Shh signaling pathway active substance, 300 nM).

[0407] The human iPS cells were treated with TrypLE Select (Life Technologies) to form a cell dispersion. The cells were then further dispersed into single cells by pipetting, and the dispersed human iPS cells were placed in a non-cell-adhesive 96-well culture plate (PrimeSurface 96V bottom plate, Sumitomo Bakelite). 1.0 x 10 per well of Kryte 4 The cells were suspended in 100 μl of serum-free medium and cultured at 37°C and 5% CO. The serum-free medium (gfCDM+KSR) used was a 1:1 mixture of F-12 medium and IMDM medium supplemented with 10% KSR, 450 μM 1-monothioglycerol, and 1x chemically defined lipid concentrate.

[0408] Using the cells obtained in step 1, suspension culture was initiated under the following conditions in step 2. In all conditions, the serum-free medium supplemented with Y27632 (final concentration 20 μM) was used. Condition 1: SAG (Enzo, Shh signaling pathway activator, 30 nM) was added. Condition 2: Purmorphamine (Wako, Shh signaling pathway activator, 0.2 μM) was added. Condition 3: Add human recombinant Shh (R&D, Shh signaling pathway activator, 300ng / ml) did. Condition 4: No exogenous Shh signaling pathway agonists were added at the start of suspension culture.

[0409] By the second day after the start of suspension culture, cell aggregates were formed under all conditions (end of step 2, start of step 3). On the third day after the start of suspension culture, when the final concentration of exogenous human recombinant BMP4 was 1.5 nM (55 ng / ml) and does not contain Y27632, SAG, Purmorphamine, or human recombinant Shh. 50 μl of medium containing recombinant BMP4 (R&D) was added. On the sixth day after the start of suspension culture, half of the medium was replaced with serum-free medium containing Y27632, SAG, Purmorphamine, human recombinant Shh, and human recombinant BMP4. Thereafter, the medium was replaced with serum-free medium containing Y27632, SAG, and human recombinant BMP4 every 2 to 4 days. Half of the medium was replaced with the serum-free medium.

[0410] The cells prepared in this manner were subjected to bright-field observation using an inverted microscope (Keyence) on day 20 after the start of suspension culture (Fig. 18, A-E). As a result, it was found that under the condition of not preconditioning in step 1 and not adding an Shh signaling pathway active substance in step 2 (condition 4 above), cell aggregates collapsed and neural tissue was not formed (Fig. 18, A). On the other hand, under the condition of not preconditioning with A83-01 and SAG in step 1 and not adding an exogenous Shh signaling pathway active substance in step 2, cell aggregates collapsed and neural tissue was not formed (Fig. 18, A). Cell aggregates were formed efficiently in the absence of any of the Shh signaling pathway activators, SAG, Purmorphamine (PMA), or human recombinant Shh. It was found that neural tissue was formed (Fig. 18, B-E).

[0411] From these results, it was found that in step 1, the TGFβ family signaling pathway inhibitor and Shh signal Preconditioning with a substance acting on the Shh signaling pathway and then preconditioning with a substance acting on the Shh signaling pathway at the start of suspension culture. The feed was performed under the conditions where either SAG, Purmorphamine, or human recombinant Shh was added as a substrate. It has been found that neural tissue can be efficiently produced from dark-free human iPS cells.

[0412] The cell aggregates on day 20 of suspension culture were fixed with 4% paraformaldehyde and frozen sections were prepared. These frozen sections were analyzed by immunoblotting using Chx10 (anti-Chx10 antibody), a retinal tissue marker. Immunostaining was performed on sheep (Exalpha, sheep) and observed using a fluorescent microscope. As a result, in step 1, preconditioning was performed with A83-01 and SAG, and in step 2, exogenous Shh signaling was initiated. When no pathway-active substances were added, the percentage of Chx10-positive cells among all cells was approximately 40%. On the other hand, in step 1, the cells were preconditioned with A83-01 and SAG, and at the start of suspension culture, When SAG, Purmorphamine, or human recombinant Shh was added as an agent acting on the Shh signaling pathway, the proportion of Chx10-positive cells among all cells was found to be approximately 90% (FIG. 18, GI).

[0413] From these results, it was found that in step 1, the TGFβ family signaling pathway inhibitor and Shh signal In the condition where the cells were preconditioned with a substance acting on the Shh signaling pathway and then SAG, Purmorphamine, or human recombinant Shh was added as a substance acting on the Shh signaling pathway in step 2, the feeder follicles It was found that retinal tissue can be efficiently produced from human iPS cells.

[0414] Example 19: In step 1, SB431542 and Shh were used as TGFβ family signaling pathway inhibitors. Purmorphamine was used as a signal transduction pathway activator, and in step 2, the Shh signal transduction pathway was activated. Example of retinal tissue formation from feeder-free human iPS cells using Purmorphamine as a target substance Human iPS cells (1231A3 strain, obtained from Kyoto University) were cultured in a feeder-free environment according to the method described in Scientific Reports, 4, 3594 (2014). The feeder-free medium used was Stem Fit medium (AK03; manufactured by Ajinomoto Co., Inc.), and the feeder-free scaffold used was Laminin 511-E8 (manufactured by Nippi Co., Ltd.). was used.

[0415] Human iPS cells that had reached subconfluence one day prior to the initial stage, as prepared by the method described in Example 15, were cultured in a feeder-free medium for one day in the presence of SB431542 (a TGFβR inhibitor, 5 μM) and purmorphamine (PMA; an Shh signaling pathway agent, 0.02 μM or 0.2 μM) (preconditioning treatment).

[0416] The human iPS cells were treated with TrypLE Select (Life Technologies) to form a cell dispersion. The cells were then further dispersed into single cells by pipetting, and the dispersed human iPS cells were placed in a non-cell-adhesive 96-well culture plate (PrimeSurface 96V bottom plate, Sumitomo Bakelite). 1.0 x 10 per well of Kryte 4 The cells were suspended in 100 μl of serum-free medium and cultured at 37°C and 5% CO. The serum-free medium (gfCDM+KSR) used was a 1:1 mixture of F-12 medium and IMDM medium supplemented with 10% KSR, 450 μM 1-monothioglycerol, and 1x chemically defined lipid concentrate.

[0417] At the start of suspension culture (day 0 after the start of suspension culture, the start of step 2), Y27632 (20 μM) and Purmorphamine (manufactured by Wako, an Shh signaling pathway active substance, 0.2 μM or 2 μM) were added to the serum-free medium. By the second day after the start of suspension culture, cell aggregates were formed (end of step 2, start of step 3). On the third day after the start of suspension culture, exogenous human recombinant BMP4 was added at a final concentration of 1.5 nM (55 50 μl of medium containing human recombinant BMP4 (R&D) but not containing Y27632 or Purmorphamine was added to the suspension to achieve a concentration of 100 ng / ml. Half of the medium was replaced with serum-free medium not containing human recombinant BMP4. Thereafter, half of the medium was replaced with the serum-free medium not containing Y27632, Purmorphamine, and human recombinant BMP4 every 2 to 4 days. We swapped locations.

[0418] The cells prepared in this way were observed under an inverted microscope (Keyence) on the 17th day after the start of suspension culture. Bright-field observation was performed using a microscope (manufactured by the manufacturer) (Figure 19, AD). As a result, in step 1, the cells were preconditioned with SB431542 and Purmorphamine (0.02 μM or 0.2 μM), and in step 2, the cells were preconditioned with Purmorphamine (0.2 μM). It was found that under conditions in which 1 μM or 2 μM of Purmorphamine was added, cell aggregates were formed at all Purmorphamine concentrations, and neural tissue was efficiently formed (FIG. 19, AD).

[0419] The cell aggregates on day 17 after the start of suspension culture were fixed with 4% paraformaldehyde and frozen sections were prepared. These frozen sections were analyzed by immunoblotting using Chx10 (anti-Chx10 antibody), a retinal tissue marker. Immunostaining was performed on sheep (Exalpha, sheep). These immunostained sections were The cells were observed using an inverted fluorescence microscope. As a result, in step 1, the cells were preconditioned with SB431542 and Purmorphamine (0.02 μM or 0.2 μM), and in step 2, the cells were preconditioned with Purmorphamine (0.2 μM or 2 μM). It was found that under the conditions where Purmorphamine was added, the proportion of Chx10-positive cells among all cells was approximately 60% at all Purmorphamine concentrations (Fig. 19, EH).

[0420] These results demonstrate that retinal tissue can be efficiently produced from feeder-free human iPS cells by preconditioning in step 1 using SB431542 as an inhibitor of the TGFβ family signaling pathway and Purmorphamine as an agent acting on the Shh signaling pathway, and then adding the agent acting on the Shh signaling pathway (Purmorphamine) at the start of suspension culture.

[0421] Example 20: In step 1, an Shh signaling pathway active substance or a TGFβ family signaling pathway active substance is used. Examples of neural tissue formation from feeder-free human iPS cells preconditioned for 1, 2, or 3 days with a combination of a pathway inhibitor and an Shh signaling pathway agonist. Human iPS cells (1231A3 strain, obtained from Kyoto University) were cultured in a feeder-free environment according to the method described in Scientific Reports, 4, 3594 (2014). The feeder-free medium used was Stem Fit medium (AK03; manufactured by Ajinomoto Co., Inc.), and the feeder-free scaffold used was Laminin 511-E8 (manufactured by Nippi Co., Ltd.). was used.

[0422] The human iPS cells were cultured in a feeder-free manner under the following six conditions. Condition 1: SAG (Enzo, Shh signaling pathway agonist, 300 nM) for 48 hours Condition 2: SAG (Enzo, Shh signaling pathway agonist, 300 nM) for 72 hours Condition 3. A83-01 (Wako, TGFβR inhibitor, 0.5 μM) and SAG (Enzo, Shh signal inhibitor) transduction pathway agonist, 300 nM) for 24 hours Condition 4: A83-01 (Wako, TGFβR inhibitor, 0.5 μM) and SAG (Enzo, Shh signaling pathway activator, 300 nM) were administered for 48 hours. Condition 5. A83-01 (Wako, TGFβR inhibitor, 0.5 μM) and SAG (Enzo, Shh signal inhibitor) transduction pathway agonist, 300 nM) for 72 hours Condition 6: Exogenous TGFβ family signaling pathway inhibitors and Shh signaling pathway Absence of active substance (no preconditioning)

[0423] The human iPS cells were treated with TrypLE Select (Life Technologies) to form a cell dispersion. The cells were then dispersed into single cells by pipetting, and the dispersed human iPS cells were placed in a non-cell-adhesive 96-well culture plate (PrimeSurface 96V bottom plate, Sumitomo Bakelite). 1.0 x 10 per well of Kryte 4 The cells were suspended in 100 μl of serum-free medium and cultured at 37°C and 5% CO. The serum-free medium (gfCDM+KSR) used was a 1:1 mixture of F-12 medium and IMDM medium supplemented with 10% KSR, 450 μM 1-monothioglycerol, and 1x chemically defined lipid concentrate.

[0424] At the start of suspension culture (day 0 after suspension culture was started, starting step 2), the following two conditions (A) and (B) were used. Under condition (A), Y27632 (final concentration 20 μM) and SAG (a substance acting on the Shh signaling pathway, 30 nM) were added to the serum-free medium at the start of suspension culture. Under condition (B), Y27632 (final concentration 20 μM) was added to the serum-free medium at the start of suspension culture, but SAG was not added. By the second day after the start of the floating culture, cell aggregates were formed under all conditions (end of step 2, start of step 3). On the third day after the start of the suspension culture, 50 μl of medium containing human recombinant BMP4 (m...

Claims

1. A population of cell aggregates comprising cells differentiated from pluripotent stem cells, (1) each cell aggregate comprises Oct3 / 4-positive cells and cells expressing one or more selected from the group consisting of Sox2, Sox1, Nestin, and Otx2; (2) The number of cells contained in each cell aggregate is 1 × 10 3 ~1×10 5 cells, (3) epithelial structures are formed in each cell aggregate; (4) 75% or more of the cell aggregates in the entire cell aggregate population are within ±20% of the average maximum diameter of the aggregate population, (5) The proportion of non-cystic cell aggregates in the population is 80% or more. A population of cell aggregates characterized by:

2. The population of cell aggregates according to claim 1 , wherein the pluripotent stem cells are induced pluripotent stem cells.

3. The population of cell aggregates described in claim 1 or 2, wherein the pluripotent stem cells are pluripotent stem cells that have been maintained and / or expanded in a medium containing an undifferentiated maintenance factor in the absence of feeder cells.

4. The population of cell aggregates described in claim 3, wherein the undifferentiated maintenance factor is an FGF signaling pathway active substance.

5. The population of cell aggregates described in claim 4, wherein the FGF signaling pathway active substance is bFGF.

6. A population of cell aggregates according to any one of claims 1 to 5, which has the ability to differentiate into neural cells or neural tissue.

7. A population of cell aggregates according to any one of claims 1 to 6, wherein cells form cell-cell bonds and / or cell adhesions in a portion of each cell aggregate.

8. A culture comprising a population of cell aggregates according to any one of claims 1 to 7 and a medium.

9. The culture described in claim 8, wherein the medium is a serum-free medium.

10. 10. An in vitro culture system comprising the culture according to claim 8 or 9, and a 24-well plate, a 48-well plate, a 96-well plate or a 384-well plate as culture equipment for said culture.

11. A method for producing nervous system cells or nervous tissue, comprising: culturing a population of cell aggregates described in any one of claims 1 to 7 in suspension in the presence or absence of a differentiation-inducing factor to obtain aggregates containing nervous system cells or nervous tissue.

12. The manufacturing method described in claim 11, wherein the aggregate containing nervous system cells or neural tissue is an aggregate containing retinal tissue.

13. The manufacturing method described in claim 11, wherein the nervous system cells or nervous tissue are retinal progenitor cells, retinal layer-specific neurons, retinal tissue, cerebral nervous system progenitor cells, cerebral layer-specific neurons, or cerebral tissue.

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