Preparation method of cell mass containing pituitary tissue and the cell mass

Abstract

The present invention aims to provide a method for effectively preparing a cell mass containing pituitary tissue from pluripotent stem cells. The method for preparing a cell mass containing pituitary tissue comprises the following steps (1) and (2): (1) in the first step, pluripotent stem cells are suspended and cultured in the presence of a substance that inhibits the Wnt signal transduction pathway to form cell aggregates; (2) in the second step, the aggregates obtained in the first step are suspended and cultured in the presence of a substance that acts on the BMP signal transduction pathway and a substance that acts on the Sonic hedgehog signal transduction pathway to obtain a cell mass containing pituitary tissue.

Description

Technical Field

[0001] The present invention relates to a method for preparing a cell mass containing pituitary tissue and the cell mass. The present invention further relates to a cell mass containing nervous system cells or nervous tissue, pituitary tissue, and mesenchymal cells in the cell mass. Background Art

[0002] The pituitary gland is an endocrine organ located in the head, and produces various pituitary hormones important for the maintenance and growth of the organism, such as adrenocorticotropic hormone (ACTH) and growth hormone. When the pituitary gland is dysfunctional due to diseases such as pituitary hypoplasia, hypopituitarism, and pituitary adenoma, serious conditions such as developmental disorders, genital-related abnormalities, and abnormalities of the adrenal glands and thyroid glands occur. In general, the damaged pituitary tissue rarely regenerates naturally and recovers its function.

[0003] Patent Document 1 and Non-Patent Documents 1 and 2 report that human pluripotent stem cells were induced to differentiate in the presence of BMP signal transduction pathway inhibitors and even activators, Sonic hedgehog (sometimes referred to as Shh in this specification) signal transduction pathway activators, and TGF-β signal transduction pathway inhibitors, thereby preparing cells derived from the head placode containing pituitary cells. However, the prepared cells are two-dimensionally cultured, and the structure of the complex pituitary tissue of the organism that is important for the performance of the function cannot be reproduced. Therefore, a method for effectively preparing three-dimensional pituitary tissue is urgently desired.

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: Japanese Patent Application Publication No. 2016-538856

[0007] Non-patent literature

[0008] Non-patent literature 1: Dincer et al. Cell Reports 5, 1387-1402, 2013.

[0009] Non-patent literature 2: Zimmer et al. Stem Cell Reports 6, 858-872, 2016 Summary of the invention

[0010] Problems to be solved by the invention

[0011] The object of the present invention is to provide a means for effectively preparing a cell mass comprising pituitary tissue by pluripotent stem cells. Particularly, a means for effectively preparing a cell mass comprising high-quality pituitary tissue containing pituitary tissue stem cells in a tissue is provided. Specifically, a means for effectively preparing a cell mass comprising a pituitary tissue containing high-quality pituitary tissue containing pituitary tissue stem cells is provided by using pluripotent stem cells cultured without feeder cells as starting materials, thereby reducing the amount of expensive recombinant proteins used and preparing more cheaply.

[0012] Solutions to the problem

[0013] The present inventors have repeatedly studied to solve the above problems and found that cell agglomerates containing pituitary tissue can be effectively prepared by adding BMP signal transduction pathway agents and Sonic hedgehog signal transduction pathway agents to suspension culture in the presence of Wnt signal transduction pathway inhibitors. It was further found that the efficiency of preparing cell agglomerates can be improved by culturing pluripotent stem cells in a medium containing 1) TGFβ family signal transduction pathway inhibitors and / or Sonic hedgehog signal transduction pathway agents and 2) undifferentiation maintenance factors before suspension culture in the presence of the Wnt signal transduction pathway inhibitors. In addition, by optimizing the addition conditions of BMP signal transduction pathway agents and identifying the so-called optimal addition period within 72 hours from the start of suspension culture, the induction differentiation of pituitary tissue by BMP4 at a concentration lower than that of conventional methods was successfully performed. That is, the present invention relates to the following.

[0014] [1] A method for preparing a cell mass containing pituitary tissue, comprising the following steps (1) and (2):

[0015] (1) In the first step, pluripotent stem cells are suspended and cultured in the presence of a Wnt signal transduction pathway inhibitor to form cell aggregates;

[0016] (2) In the second step, the aggregates obtained in the first step are suspended and cultured in the presence of a substance acting on the BMP signal transduction pathway and a substance acting on the Sonic hedgehog signal transduction pathway to obtain cell aggregates containing pituitary tissue.

[0017] [2] A method for preparing a cell mass containing pituitary tissue, comprising the following steps (a), (1) and (2):

[0018] (a) step a, culturing the pluripotent stem cells in a medium containing 1) a TGFβ family signal transduction pathway inhibitory substance and / or a Sonic hedgehog signal transduction pathway active substance, and 2) an undifferentiation maintenance factor in the absence of feeder cells;

[0019] (1) In the first step, the pluripotent stem cells cultured in step a are suspended and cultured in the presence of a Wnt signal transduction pathway inhibitory substance to form cell aggregates;

[0020] (2) In the second step, the aggregates obtained in the first step are suspended and cultured in the presence of substances acting on the BMP signal transduction pathway and substances acting on the Sonic hedgehog signal transduction pathway to obtain cell aggregates containing pituitary tissue.

[0021] [3] A method for preparing a cell mass containing pituitary tissue, comprising the following steps (1), (2-1) and (2-2):

[0022] (1) In the first step, pluripotent stem cells are suspended and cultured in the presence of a Wnt signal transduction pathway inhibitor to form cell aggregates;

[0023] (2-1) the second step (1), culturing the aggregates obtained in the first step in suspension in the presence of a substance acting on the BMP signal transduction pathway and a substance acting on the Sonic hedgehog signal transduction pathway;

[0024] (2-2) The second step (2) is to culture the aggregates obtained by suspension culture in the second step (1) in the presence of a BMP signal transduction pathway inhibitory substance to obtain cell aggregates containing pituitary tissue.

[0025] [4] A method for preparing a cell mass containing pituitary tissue, comprising the following steps (a), (1), (2-1) and (2-2):

[0026] (a) step a, culturing the pluripotent stem cells in a medium containing 1) a TGFβ family signal transduction pathway inhibitory substance and / or a Sonic hedgehog signal transduction pathway active substance, and 2) an undifferentiation maintenance factor in the absence of feeder cells;

[0027] (1) In the first step, the pluripotent stem cells cultured in step a are suspended and cultured in the presence of a Wnt signal transduction pathway inhibitory substance to form cell aggregates;

[0028] (2-1) the second step (1), culturing the aggregates obtained in the first step in suspension in the presence of a substance acting on the BMP signal transduction pathway and a substance acting on the Sonic hedgehog signal transduction pathway;

[0029] (2-2) The second step (2) is to culture the aggregates obtained in the second step (1) in suspension in the presence of a BMP signal transduction pathway inhibitory substance to obtain cell aggregates containing pituitary tissue.

[0030] [5] The preparation method according to any one of [1] to [4], wherein the BMP signal transduction pathway activating substance in the above step (2) or step (2-1) is added within 72 hours after 0.5 hours from the start of the suspension culture of pluripotent stem cells in the above step (1).

[0031] [6] A preparation method according to any one of [1] to [4], wherein the BMP signal transduction pathway activating substance in the above step (2) or step (2-1) is added during a period when more than 10% of the cells on the surface of the aggregate formed by the above step (1) form tight junctions.

[0032] [7] The production method according to any one of [1] to [6], wherein the BMP signal transduction pathway acting substance is at least one protein selected from BMP2, BMP4, BMP7 and GDF7.

[0033] [8] The production method according to any one of [1] to [7], wherein the BMP signal transduction pathway acting substance is BMP4.

[0034] [9] The preparation method according to [8], wherein the suspension culture in the above step (2) or step (2-1) is carried out using a culture medium containing the above BMP4 at a concentration of 10 pM to 5 nM.

[0035]

[10] The preparation method according to any one of [3] to [9], wherein the addition of the BMP signal transduction pathway inhibitory substance in the above step (2-2) is carried out within 12 hours and 20 days from the addition of the BMP signal transduction pathway activating substance in the above step (2-1).

[0036]

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

[10] , wherein the Wnt signaling pathway inhibitory substance has an inhibitory activity on the non-canonical Wnt pathway.

[0037]

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

[11] , wherein the Wnt signaling pathway inhibitory substance is a PORCN inhibitor.

[0038]

[13] The preparation method according to

[12] , wherein the PORCN inhibitor is at least one compound selected from the group consisting of IWP-2, IWP-3, IWP-4, IWP-L6, IWP-12, LGK-974, Wnt-C59, ETC-159 and GNF-6231.

[0039]

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

[11] , wherein the Wnt signaling pathway inhibitory substance is KY-02111 or KY-03-I.

[0040]

[15] The production method according to any one of [1] to

[10] , wherein the Wnt signaling pathway inhibitory substance is a TANK inhibitor.

[0041]

[16] The preparation method according to

[15] , wherein the TANK inhibitor is at least one compound selected from IWR1-endo, XAV939 and MN-64.

[0042]

[17] The preparation method according to any one of [1] to

[16] , wherein the suspension culture in the above steps (1), (2), (2-1) and (2-2) is also carried out in the presence of a TGFβ signaling pathway inhibitory substance.

[0043]

[18] The preparation method according to

[17] , wherein the TGFβ signal transduction pathway inhibitory substance is an Alk5 / TGFβR1 inhibitor.

[0044]

[19] The preparation method according to

[18] , wherein the Alk5 / TGFβR1 inhibitor is at least one compound selected from SB431542, SB505124, SB525334, LY2157299, GW788388, LY364947, SD-208, EW-7197, A83-01 and RepSox.

[0045]

[20] The preparation method according to

[17] , wherein the TGFβ signal transduction pathway inhibitory substance is a SMAD3 inhibitor.

[0046]

[21] The preparation method according to

[20] , wherein the SMAD3 inhibitor is SIS3.

[0047]

[22] The preparation method according to any one of [1] to

[21] , wherein the substance acting on the Sonic hedgehog signal transduction pathway is at least one compound selected from SAG, purmorphamine and GSA-10.

[0048]

[23] According to the preparation method described in

[22] , the concentration of the Sonic Hedgehog signal transduction pathway agent contained in the culture medium in the above step (a) is a concentration having a Sonic Hedgehog signal transduction promoting activity equivalent to 10nM to 700nM SAG.

[0049]

[24] The preparation method according to

[22] or

[23] , wherein the concentration of the Sonic hedgehog signal transduction pathway agent contained in the culture medium in the above step (2) or step (2-1) is a concentration having a Sonic hedgehog signal transduction promoting activity equivalent to 100nM to 3μM SAG.

[0050]

[25] The preparation method according to any one of [3] to

[24] , wherein the BMP signal transduction pathway inhibitory substance includes a type I BMP receptor inhibitor.

[0051]

[26] The preparation method according to

[25] , wherein the above-mentioned type I BMP receptor inhibitor includes at least one selected from the group consisting of K02288, Dorsomorphin, LDN-193189, LDN-212854, LDN-214117, ML347, DMH1 and DMH2.

[0052]

[27] The preparation method according to

[25] or

[26] , wherein the type I BMP receptor inhibitor includes K02288, and the step (2-2) is initiated in a culture medium in which the concentration of K02288 is 10 nM to 50 μM.

[0053]

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

[27] , wherein at least one step selected from the above steps (1), (2), (2-1) and (2-2) is carried out in the presence of a substance acting on the FGF signaling pathway.

[0054]

[29] The production method according to any one of [3] to

[27] , wherein the above step (2-2) is carried out in the presence of a substance acting on the FGF signal transduction pathway.

[0055]

[30] The preparation method according to

[28] or

[29] , wherein the FGF signal transduction pathway acting substance comprises at least one selected from the group consisting of FGF2 and FGF8, and their variants.

[0056]

[31] The preparation method according to any one of [1] to

[30] , wherein at least one step selected from the group consisting of the above steps (1), (2), (2-1) and (2-2) is carried out in the presence of a substance acting on the Wnt signaling pathway.

[0057]

[32] The preparation method according to

[31] , wherein the Wnt signal transduction pathway acting substance acts on a signal transduction factor downstream of the action point of the Wnt signal transduction pathway inhibitory substance added in the above step (1).

[0058]

[33] The preparation method according to

[31] or

[32] , wherein the Wnt signal transduction pathway acting substance is a substance that activates the canonical Wnt pathway.

[0059]

[34] The method according to any one of

[31] to

[33] , wherein the substance that activates the canonical Wnt pathway has the effect of inhibiting the degradation of β-catenin and promoting its stabilization.

[0060]

[35] The preparation method according to any one of

[31] to

[34] , wherein the Wnt signal transduction pathway acting substance is a GSK3 inhibitor.

[0061]

[36] The preparation method according to any one of

[31] to

[35] , wherein the Wnt signaling pathway inhibitory substance is a PORCN inhibitor, and the Wnt signaling pathway acting substance is a GSK3 inhibitor.

[0062]

[37] The preparation method according to any one of

[31] to

[36] , wherein the above-mentioned GSK3 inhibitor includes at least one selected from the group consisting of CHIR99021, CHIR98014, TWS119, SB216763, SB415286, BIO, AZD2858, AZD1080, AR-A014418, TDZD-8, LY2090314, IM-12, Indirubin, Bikinin, A 1070722, 3F8, Kenpaullone, 10Z-Hymenialdisine, Indirubin-3'-oxime, NSC 693868, TC-G 24, TCS2002, TCS 21311, CP21R7 and derivatives of these compounds.

[0063]

[38] The preparation method according to any one of

[31] to

[37] , wherein the GSK3 inhibitor includes CHIR99021, and the concentration of the CHIR99021 added in at least one step selected from the group consisting of the above steps (1), (2), (2-1) and (2-2) is 10nM to 50μM.

[0064]

[39] The preparation method according to any one of

[31] to

[34] , wherein the Wnt signaling pathway acting substance comprises at least one selected from the group consisting of BML-284 and SKL2001.

[0065]

[40] The production method according to any one of [1] to

[39] , wherein the suspension culture performed in the above step (1) is a suspension culture using a serum-free medium.

[0066]

[41] The preparation method according to

[40] , wherein the serum-free medium contains a serum substitute.

[0067]

[42] The production method according to any one of [1] to

[41] , wherein the suspension culture performed in the above step (1) is performed using dispersed pluripotent stem cells.

[0068]

[43] The production method according to any one of [1] to

[42] , wherein the suspension culture performed in the above step (1) is performed in the presence of a ROCK inhibitor.

[0069]

[44] The preparation method according to

[43] , wherein the concentration of the ROCK inhibitor added in the above step (1) is a concentration having a cell protection ability equivalent to 1nM to 1mM Y-27632.

[0070]

[45] The preparation method according to any one of [1] to

[44] , wherein the suspension culture in any one or more steps selected from the group consisting of the above steps (1), (2), (2-1) and (2-2) is also carried out in the presence of a Notch signaling inhibitor.

[0071]

[46] The preparation method according to

[45] , wherein the Notch signaling inhibitor is a γ-secretase inhibitor.

[0072]

[47] The preparation method according to

[46] , wherein the γ-secretase inhibitor is DAPT.

[0073]

[48] ​​The preparation method according to any one of [1] to

[47] , wherein the suspension culture in any one or more steps selected from the group consisting of the above steps (1), (2), (2-1) and (2-2) is also carried out in the presence of dexamethasone.

[0074]

[49] The production method according to any one of [1] to

[48] , wherein the pluripotent stem cells are primate pluripotent stem cells.

[0075]

[50] The preparation method according to

[49] , wherein the primate pluripotent stem cells are human pluripotent stem cells.

[0076]

[51] A cell mass comprising pituitary tissue obtained by the preparation method described in any one of [1] to

[50] .

[0077]

[52] A method for preparing pituitary tissue, comprising the following steps (1) to (3):

[0078] (1) In the first step, pluripotent stem cells are suspended and cultured in the presence of a Wnt signal transduction pathway inhibitor to form cell aggregates;

[0079] (2) In the second step, the aggregates obtained in the first step are suspended and cultured in the presence of substances acting on the BMP signal transduction pathway and substances acting on the Sonic hedgehog signal transduction pathway to obtain cell aggregates containing pituitary tissue;

[0080] (3) In the third step, the pituitary tissue is recovered from the cell mass obtained in the second step.

[0081]

[53] A method for preparing pituitary tissue, comprising the following steps (1), (2-1), (2-2) and (3):

[0082] (1) In the first step, pluripotent stem cells are suspended and cultured in the presence of a Wnt signal transduction pathway inhibitor to form cell aggregates;

[0083] (2-1) the second step (1), culturing the aggregates obtained in the first step in suspension in the presence of a substance acting on the BMP signal transduction pathway and a substance acting on the Sonic hedgehog signal transduction pathway;

[0084] (2-2) a second step of culturing the aggregates obtained by suspension culture in the second step (1) in the presence of a substance that inhibits the BMP signal transduction pathway, thereby obtaining a cell mass (2) containing pituitary tissue,

[0085] (3) The third step is to recover the pituitary tissue from the cell mass obtained in the second step (2).

[0086]

[54] Pituitary tissue obtained by the preparation method described in

[52] or

[53] .

[0087]

[55] A cell mass comprising 1) nervous system cells or neural tissue, 2) pituitary tissue, and 3) mesenchymal cells.

[0088]

[56] The cell cluster according to

[55] , wherein the above-mentioned 3) mesenchymal cells express at least one mesenchymal cell marker selected from the group consisting of Nestin, Vimentin, Cadherin-11, Laminin, CD44, CD90 and CD105.

[0089]

[57] A cell mass comprising 1) nervous system cells or neural tissue, 2) pituitary tissue, and 4) neural crest cells or cells derived from the neural crest.

[0090]

[58] The cell cluster according to

[57] , wherein the above-mentioned 4) neural crest cells express at least one neural crest cell marker selected from the group consisting of nestin, Sox10, Slug, Snail, Pax3, Zic1, FoxD3, p75 NTR, HNK-1, CHD7, Numb, and Ascl1.

[0091]

[59] The cell cluster according to any one of

[55] to

[58] , wherein the surface of the cell cluster is covered with non-neuroepithelial tissue, and 2) pituitary tissue is formed in at least a part of the non-neuroepithelial tissue.

[0092]

[60] The cell aggregate according to

[59] , wherein the non-neuroepithelial tissue expresses at least one non-neuroepithelial tissue marker selected from the group consisting of cytokeratin, E-cadherin and EpCAM.

[0093]

[61] The cell cluster according to

[55] or

[56] , wherein the non-neuroepithelial tissue covers at least one of 1) nervous system cells or neural tissue and 3) mesenchymal cells.

[0094]

[62] The cell cluster according to

[57] or

[58] , wherein the non-neuroepithelial tissue covers at least one of 1) nervous system cells or neural tissue and 4) neural crest cells or cells derived from the neural crest.

[0095]

[63] The cell cluster according to

[61] , wherein the 3) mesenchymal cells are present between the non-neuroepithelial tissue covering the surface of the cell cluster and the 1) nervous system cells or neural tissue present inside the cell cluster.

[0096]

[64] The cell cluster according to

[62] , wherein the 4) neural crest cells or cells derived from the neural crest are present between the non-neural epithelial tissue covering the surface of the cell cluster and the 1) nervous system cells or neural tissue present inside the cell cluster.

[0097]

[65] The cell mass according to any one of

[59] to

[64] , wherein the non-neural epithelial tissue is oral epithelium or a precursor tissue thereof.

[0098]

[66] The cell cluster according to any one of

[55] to

[65] , wherein the above-mentioned 2) pituitary tissue contains pituitary hormone-producing cells.

[0099]

[67] The cell cluster according to

[66] , wherein the pituitary hormone-producing cells are at least one selected from the group consisting of growth hormone (GH)-producing cells, prolactin (PRL)-producing cells and adrenocorticotropic hormone (ACTH)-producing cells.

[0100]

[68] The cell cluster according to any one of

[55] to

[67] , wherein the pituitary tissue contains pituitary tissue stem cells.

[0101]

[69] The cell cluster according to

[68] , wherein the pituitary tissue stem cells express at least one pituitary tissue stem cell marker selected from the group consisting of Sox2, Sox9, E-cadherin, nestin, S100β, GFRα2, Prop1, CD133, β-catenin, Klf4, Oct4, Pax6, coxsackievirus-adenovirus co-receptor (CXADR), PRRX1 / 2, Ephrin-B2 and ACE.

[0102]

[70] The cell cluster according to

[68] or

[69] , wherein the pituitary tissue stem cells form a pituitary niche.

[0103]

[71] The cell mass according to any one of

[55] to

[70] , wherein the 1) nervous system cells or nervous tissues are cells or tissues of the ventral diencephalon or hypothalamus.

[0104]

[72] The cell cluster according to

[71] , wherein the 1) nervous system cells or nervous tissues are N-cadherin-positive neuroepithelial tissues.

[0105]

[73] The cell mass according to

[72] , wherein a ventricle-like vacuoles are formed inside the neuroepithelial tissue, and the surface of the neuroepithelial tissue in contact with the vacuoles is a PKC-zeta-positive apical surface.

[0106]

[74] A method for evaluating the toxicity and efficacy of a test substance, comprising the steps of contacting a cell mass prepared by any of the methods described in [1] to

[50] or a pituitary tissue prepared by the method described in claim

[52] with the test substance, and examining the effect of the test substance on the cell mass or pituitary tissue.

[0107]

[75] A reagent for evaluating the toxicity or efficacy of a test substance, comprising a cell mass prepared by the method described in any one of [1] to

[50] or a pituitary tissue prepared by the method described in

[52] .

[0108]

[76] A method for treating a disease in a non-human animal caused by a pituitary disorder, comprising the step of transplanting an effective amount of pituitary tissue prepared by the method described in

[52] to a subject in need of transplantation.

[0109]

[77] A therapeutic drug for a disease caused by a pituitary disorder, comprising a cell mass prepared by the method described in any one of [1] to

[50] or a pituitary tissue prepared by the method described in

[52] .

[0110] Effects of the Invention

[0111] According to the present invention, it is possible to efficiently prepare a cell mass containing pituitary tissue from pluripotent stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0112] [ Figure 1-1 ] Figure 1-1 The upper part is a diagram schematically showing the sequence of preparing a cell mass containing pituitary tissue from human ES cells in Example 1. The lower parts A and B are diagrams showing bright field observation images of the cell mass 28 days after the start of suspension culture in Example 1 under an inverted microscope. The lower parts C to K are diagrams showing the results of examining the expression of each cell marker in the cell mass 28 days after the start of suspension culture by fluorescent immunostaining in Example 1. C to E show Lhx3, Nkx 2.1 and their nuclear staining images, respectively. F to H show Lhx3, Nkx 2.1 and their nuclear staining images, respectively. I to K show aPKC, laminin and their nuclear staining images, respectively.

[0113] [ Figure 1-2 ] Figure 1-2 L to X are figures showing the results of fluorescent immunostaining to examine the expression of each cell marker in the cell mass 28 days after the start of suspension culture in Example 1. L to N respectively show Sox3, EpCAM and their nuclear staining images. O to Q respectively show Tbx3, βIII tubulin (βIII Tub) and their nuclear staining images. S to U respectively show Emx2, nestin and their nuclear staining images. V to X respectively show Pitx1, pan-cytokeratin (Pan-CK) and their nuclear staining images. The lower part Y is a figure schematically showing the structure of a cell mass containing pituitary tissue on the 28th day of culture.

[0114] [ Figure 2 ] Figure 2The upper part is a diagram schematically showing the sequence of making cell clusters containing pituitary tissue from human ES cells in Example 2. The lower parts A and B are diagrams showing bright field observation images of the cell clusters 70 days after the start of suspension culture in Example 2 under an inverted microscope. The lower parts C to J are diagrams showing the results of examining the expression of each cell marker in the cell clusters 70 days or 84 days after the start of suspension culture by fluorescent immunostaining. C and D show ACTH and its nuclear staining images, respectively. E to G show prolactin (PRL), pan-cytokeratin (PanCK) and its nuclear staining images, respectively. H to J show growth hormone (GH), pan-cytokeratin (PanCK) and its nuclear staining images, respectively.

[0115] [ Figure 3 ] Figure 3 The upper part is a diagram schematically showing the sequence of preparing cell aggregates containing pituitary tissue from human ES cells by changing the BMP4 addition period in Reference Example 1. The lower parts A to D are diagrams showing bright field observation images of cell aggregates 10 days after the start of suspension culture in Reference Example 1, taken under an inverted microscope. The diagrams show bright field observation images of cell aggregates formed 10 days after the start of suspension culture, taken under an inverted microscope, respectively: A is a control cell to which BMP4 was not added, and B to D are cells to which BMP4 was added on the 1st, 2nd, and 3rd day from the start of suspension culture, respectively.

[0116] [ Figure 4 ] Figure 4 The upper part is a diagram schematically showing the sequence of making cell aggregates from human ES cells in Example 3. The lower parts A and B are diagrams showing bright field observation images of cell aggregates 2 and 3 days after the start of suspension culture in Example 3, respectively. The lower parts C to H are diagrams showing the results of examining the expression of each cell marker in the cell aggregates 2 days or 3 days after the start of suspension culture by fluorescent immunostaining. C is a staining image of ZO-1 of the cell aggregates 2 days after the start of suspension culture, and F is a staining image of ZO-1 of the cell aggregates 3 days after the start of suspension culture. D is a staining image of Sox2 of the cell aggregates 2 days after the start of suspension culture, and G is a staining image of Sox2 of the cell aggregates 3 days after the start of suspension culture. E is a contrast staining image of the nuclei corresponding to C and D, and H is a contrast staining image of the nuclei corresponding to F and G.

[0117] [ Figure 5 ] Figure 5The upper part is a diagram schematically showing the sequence of preparing cell aggregates containing pituitary tissue from human ES cells by changing the added concentration of SAG in Example 4. The lower parts A to H are diagrams showing bright field observation images of cell aggregates 28 days after the start of suspension culture in Example 4 under an inverted microscope. The diagrams respectively show bright field observation images of cell aggregates 28 days after the start of suspension culture formed by the following cells: A is a control cell without the addition of SAG, and B to H are cells to which 100, 300, 500, 700 nM, 1, 2, 5 μM SAG were added on the second day after the start of suspension culture, respectively.

[0118] [ Figure 6 ] Figure 6 The upper part is a diagram schematically showing the sequence of examining the effect of pretreatment with a compound before the start of suspension culture in the preparation of cell aggregates containing pituitary tissue from human ES cells in Example 5. The lower parts A to D are diagrams showing bright field observation images of cell aggregates 28 days after the start of suspension culture in Example 5 using an inverted microscope. A is a cell aggregate not pretreated with a compound, B is a cell aggregate pretreated with 300 nM SAG, C is a cell aggregate pretreated with 5 μM SB431542, and D is a cell aggregate pretreated by adding both SAG and SB431542.

[0119] [ Figure 7 ] Figure 7 The upper part is a diagram schematically showing the order of examining the effects of each Wnt signaling pathway inhibitor in the preparation of cell aggregates containing pituitary tissue from human ES cells in Example 6. The lower parts A to H are diagrams showing bright field observation images of cell aggregates 28 days after the start of suspension culture in Example 6 under an inverted microscope. A is a control showing the condition where no Wnt signaling pathway inhibitor and SB431542 were added, B is a condition where SB431542 was added and no Wnt signaling pathway inhibitor was added, and C to H are diagrams showing bright field observation images of cell aggregates 28 days after the start of suspension culture under an inverted microscope, respectively, where the type of Wnt signaling pathway inhibitor was changed at the start of suspension culture, and then suspension culture was further performed.

[0120] [ Figure 8 ] Figure 8The upper part is a diagram schematically showing the sequence of preparing cell aggregates containing pituitary tissue from human ES cells by changing the added concentration of BMP4 in Example 7. The lower parts A to D are diagrams showing bright field observation images of cell aggregates under an inverted microscope 28 days after the start of suspension culture in Example 7. A is a control showing no addition of BMP4, and B to D are diagrams showing bright field observation images of cell aggregates under an inverted microscope 28 days after the start of suspension culture, respectively, by adding BMP4 at different concentrations (0.5 nM, 1.5 nM, 5 nM) on the second day after the start of suspension culture, and further performing suspension culture.

[0121] [ Fig. 9 ] Fig. 9 The upper part is a diagram schematically showing the order of examining the effects of each TGFβ signaling pathway inhibitor in the preparation of cell aggregates containing pituitary tissue from human ES cells in Example 8. The lower parts A to G are diagrams showing bright field observation images of cell aggregates 28 days after the start of suspension culture in Example 8 under an inverted microscope. A is a control showing the condition where no TGFβ signaling pathway inhibitor and IWP-2 were added, B is a condition where IWP-2 was added and no TGFβ signaling pathway inhibitor was added, and C to G are diagrams showing bright field observation images of cell aggregates 28 days after the start of suspension culture under an inverted microscope, where TGFβ signaling pathway inhibitors were added at different types and concentrations at the start of suspension culture, and then suspension culture was further performed.

[0122] [ Fig.10 ] Fig.10 The upper part is a diagram schematically showing the sequence of making a cell mass containing pituitary tissue from human iPS cells in Example 9. The lower parts A and B are diagrams showing bright field observation images of the cell mass 28 days after the start of suspension culture in Example 9 under an inverted microscope. The lower parts C to R are diagrams showing the results of examining the expression of each cell marker in the cell mass 28 days after the start of suspension culture by fluorescent immunostaining. C to F show the staining images of Lhx3, Nkx2.1, and EpCAM and their nuclear staining images, respectively. G to J show the staining images of Six1, Pitx2, and pan-cytokeratin (PanCK) and their nuclear staining images, respectively. K to N show the staining images of Bf1, Sox2, and E-cadherin and their nuclear staining images, respectively. O to R show the staining images of nestin, βIII tubulin (βIII Tub), and vimentin and their nuclear staining images, respectively.

[0123] [ Fig.11 ] Fig.11The upper part is a diagram schematically showing the sequence of making a cell mass containing neural tissue and non-neural epithelial tissue from human iPS cells in Example 10. The lower parts A to D are diagrams showing bright field observation images of the cell mass 13 days after the start of suspension culture in Example 10 under an inverted microscope. The lower parts E to P are diagrams showing the results of examining the expression of each cell marker in the cell mass 13 days after the start of suspension culture by fluorescent immunostaining. E to G and K to M show the staining images of Dlx5 and N-cadherin and their nuclear staining images, respectively. H to J and N to P show the staining images of Six1 and E-cadherin and their nuclear staining images, respectively.

[0124] [ Fig.12 ] Fig.12 Q~AB is next Fig.11 The figure shows the results of examining the expression of each cell marker in the cell mass 13 days after the start of suspension culture by fluorescent immunostaining in Example 10. Q to S and W to Y show the staining images of Dlx5 and N-cadherin and their nuclear staining images, respectively. T to V and Z to AB show the staining images of Six1 and E-cadherin and their nuclear staining images, respectively.

[0125] [ Fig.13 ] Fig.13 The upper part is a diagram schematically showing the sequence of preparing a cell mass containing pituitary tissue from human iPS cells in Example 11. The lower part A is a diagram showing a bright field observation image of a cell mass 28 days after the start of suspension culture in Example 11 using an inverted microscope. The lower parts B to I are diagrams showing the results of examining the expression of each cell marker in the cell mass 28 days after the start of suspension culture by fluorescent immunostaining. B to E and F to I show staining images of Lhx3, Nkx2.1, and E-cadherin and their nuclear staining images, respectively.

[0126] [ Fig.14 ] Fig.14 J~V stands for next Fig.13 The figures show the results of examining the expression of each cell marker in the cell mass 28 days after the start of suspension culture in Example 11 by fluorescent immunostaining. J to M show the staining images of nestin, Pitx2, and Sox2 and their nuclear staining images, respectively. N to P show the staining images of Six1 and vimentin and their nuclear staining images, respectively. Q to S show the staining images of Rx and N-cadherin and their nuclear staining images, respectively. T to V show the staining images of Tbx3 and pan-cytokeratin and their nuclear staining images, respectively.

[0127] [ Fig.15 ] Fig.15The upper part is a diagram schematically showing the sequence of preparing a cell mass containing pituitary tissue from human iPS cells in Example 12. The lower parts A to D are diagrams showing bright field observation images of the cell mass 28 days after the start of suspension culture in Example 12 under an inverted microscope. The lower parts E to L are diagrams showing the results of examining the expression of each cell marker in the cell mass 28 days after the start of suspension culture by fluorescent immunostaining. E to H and I to L show staining images of Lhx3, Nkx 2.1, and E-cadherin and their nuclear staining images, respectively.

[0128] [ Fig.16 ] Fig.16 M~Y stands for next Fig.15 The figures show the results of the examination of the expression of each cell marker in the cell mass 28 days after the start of suspension culture by fluorescent immunostaining in Example 12. M to P show the staining images of nestin, Pitx2, and Sox2 and their nuclear staining images, respectively. Q to S show the staining images of Six1 and vimentin and their nuclear staining images, respectively. T to V show the staining images of Rx and N-cadherin and their nuclear staining images, respectively. W to Y show the staining images of Tbx3 and pan-cytokeratin and their nuclear staining images, respectively. DETAILED DESCRIPTION

[0129] 1. Definition

[0130] In the present invention, "stem cell" means an undifferentiated cell with differentiation potential and the ability to proliferate (particularly the ability to self-renew) to maintain the differentiation potential. Among stem cells, subgroups such as pluripotent stem cells, multipotent stem cells, and unipotent stem cells are included according to the differentiation potential. Pluripotent stem cells mean stem cells that can be cultured in vitro and have the potential (pluripotency) of differentiating into all cells (derived from tissues of the three germ layers (ectoderm, mesoderm, endoderm) that constitute an organism. Pluripotent stem cells mean stem cells that have the potential to differentiate into various types of tissues or cells, but not all types of tissues or cells. Unipotent stem cells mean stem cells that have the potential to differentiate into specific tissues and cells.

[0131] Pluripotent stem cells can be induced by fertilized eggs, cloned embryos, germ stem cells, stem cells in tissues, somatic cells, etc. As pluripotent stem cells, embryonic stem cells (ES cells: Embryonic stem cell), EG cells (Embryonic germ cell), artificial pluripotent stem cells (iPS cells: induced pluripotent stem cell) etc. can be listed. Muse cells (Multi-lineagedifferentiating Stress Enduring cell) obtained by mesenchymal stem cells (mesenchymal stem cell; MSC), GS cells made by germ cells (such as testis) are also included in pluripotent stem cells. Embryonic stem cells were first established in 1981, and have also been used to produce knockout mice since 1989. In 1998, human embryonic stem cells were established, which are also being used in regenerative medicine. ES cells can be produced by culturing inner cell mass on feeder cells or in a culture medium containing LIF. The preparation method of ES cell is recorded in for example WO 96 / 22362, WO 02 / 101057, US 5,843,780, US 6,200,806, US 6,280,718 etc.Embryonic stem cell can be obtained from given mechanism, or can buy commercially available product.For example, KhES-1, KhES-2 and KhES-3 as human embryonic stem cell can be obtained from Kyoto University Institute of Regenerative Medicine Science.EB5 as any mouse embryonic stem cell can be obtained from National Research and Development Corporation Physical and Chemical Research Institute, and D3 cell line can be obtained from ATCC.As one of ES cell nuclear transplantation ES cell (ntES cell) can be set up from cloned embryo prepared by somatic cell nucleus transplantation to egg cell having removed cell nucleus.

[0132] EG cells can be generated by culturing primordial germ cells in a medium containing mSCF, LIF and bFGF (Cell, 70: 841-847, 1992).

[0133] The "artificial pluripotent stem cells" in the present invention refer to cells induced to have pluripotency by reprogramming somatic cells by a known method. Specifically, the somatic cells differentiated into fibroblasts, peripheral blood mononuclear cells, etc. can be exemplified by reprogramming by expressing any one of a combination of multiple genes in a reprogramming genome selected from a group consisting of Oct3 / 4, Sox2, Klf4, Myc (c-Myc, N-Myc, L-Myc), Glis1, Nanog, Sall4, lin28, Esrrb, etc., thereby inducing cells with pluripotency. In 2006, artificial pluripotent stem cells were established using mouse cells by Yamanaka et al. (Cell, 2006, 126 (4), 663-676). In 2007, artificial pluripotent stem cells were established from human fibroblasts, which have the same pluripotency and self-renewal ability 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 the method of preparing artificial pluripotent stem cells by direct reprogramming of gene expression, artificial pluripotent stem cells can also be induced from somatic cells by adding compounds (Science, 2013, 341 pp. 651654).

[0134] The somatic cells used to obtain artificial pluripotent stem cells are not particularly limited, and examples thereof include tissue-derived fibroblasts, blood lineage cells (eg, peripheral blood mononuclear cells, T cells), hepatocytes, pancreatic cells, intestinal epithelial cells, smooth muscle cells, and the like.

[0135] When artificial pluripotent stem cells are generated by reprogramming by expressing some types of genes (e.g., Oct3 / 4, Sox2, Klf4 and Myc), there is no particular limitation on the method used for gene expression. As the aforementioned method, there can be cited infection methods using viral vectors (e.g., retroviral vectors, lentiviral vectors, Sendai virus vectors, adenoviral vectors, adeno-associated virus vectors), gene introduction methods using plasmid vectors (e.g., plasmid vectors, episomal vectors) (e.g., calcium phosphate method, lipofection method, RetroNectin method, electroporation method), gene introduction methods using RNA vectors (e.g., calcium phosphate method, lipofection method, electroporation method), methods of direct protein injection, and the like.

[0136] The pluripotent stem cells used in the present invention are preferably ES cells or artificial pluripotent stem cells.

[0137] Examples of pluripotent stem cells include tissue stem cells (also referred to as tissue stem cells, tissue-specific stem cells, or somatic stem cells) such as hematopoietic stem cells, neural stem cells, retinal stem cells, pituitary stem cells, and mesenchymal stem cells.

[0138] Can, for example, produce genetically modified pluripotent stem cells by using homologous recombination technology.As the gene on the modified chromosome, for example, can be enumerated: cell marker gene, the gene of histocompatibility antigen, the gene related to the disease caused by the obstacle of nervous system cell etc.The modification of the target gene on chromosome can be carried out using the following methods: Manipulating the Mouse Embryo, A Laboratory Manual (operating mouse embryo, laboratory manual), second edition, Cold Spring Harbor Laboratory Press (1994); Gene Targeting, A Practical Approach (gene targeting, practical method), IRL Press at Oxford University Press (1993); Biomanual Series 8, Gene Targeting, Making of Mutant Mouse using ES cell (gene targeting, using ES cells to produce mutant mice), YODOSHA CO., LTD. (1995); Etc.

[0139] Specifically, for example, the genomic genes of the target gene to be modified (e.g., cell marker genes, tissue compatibility antigen genes, disease-related genes, etc.) are separated, and the separated genomic genes are used to prepare a targeting vector for homologous recombination of the target gene. The generated targeting vector is introduced into stem cells, and cells in which homologous recombination has occurred between the target gene and the targeting vector are selected, thereby preparing the stem cells in which the genes on the chromosomes have been modified.

[0140] As a method for isolating the genomic gene of the target gene, known methods described in the following items can be cited: Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997), etc. For the genomic gene of the target gene, it can also be isolated using a genomic DNA library screening system (manufactured by Genome Systems), a universal genome walker kit (Universal Genome Walker Kit) (manufactured by CLONTECH), etc.

[0141] Preparation of a targeting vector for homologous recombination of a target gene and efficient screening of homologous recombinants can be carried out according to the methods described in the following: Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993); Biomanual Series 8, Gene Targeting, Making of Mutant Mouse using ES cell, YODOSHA CO., LTD. (1995); etc. As the targeting vector, any of a replacement type or an insertion type can be used. As the screening method, methods such as positive selection, promoter selection, negative selection, polyadenylic acid (polyA) selection, etc. can be used.

[0142] Examples of methods for selecting desired homologous recombinants from the selected cell line include Southern hybridization method for genomic DNA and PCR method.

[0143] The "mammals" in the present invention include rodents, ungulates, carnivores, primates, etc. Rodents include mice, rats, hamsters, guinea pigs, etc. Ungulates include pigs, cattle, goats, horses, sheep, etc. In the carnivores, dogs, cats, etc. are included. The "primates" in the present invention refer to mammals belonging to the order Primates, and primates include the suborder Prosimians such as lemurs, lorises, tree shrews, etc., and the suborder Anthropoids such as monkeys, apes, humans, etc.

[0144] The pluripotent stem cells used in the present invention are mammalian pluripotent stem cells, preferably rodent (eg, mouse, rat) or primate (eg, human, monkey) pluripotent stem cells, and most preferably human pluripotent stem cells.

[0145] In the present invention, "signal transduction" refers to the process and mechanism in which receptor proteins present in the cell membrane and the like bind to chemical substances and undergo structural changes, and transmit the substances as stimuli to the cells in sequence, ultimately leading to reactions such as gene expression and channel opening, and indicates the information transmission, amplification, processing, and response mechanism of cells for biochemical stimuli. In the present invention, "downstream of signal transduction" indicates the relative relationship of a certain factor in a series of signal transduction-participating factors to biochemical changes such as phosphorylation in a subsequent process in a temporal and causal manner. For example: If in a series of signal transduction mechanisms, factor A phosphorylates factor B to produce subsequent signal transduction, then factor B is a signal transduction factor downstream of factor A.

[0146] In the present invention, "cell adhesion" refers to the adhesion between cells and the adhesion between cells and the extracellular matrix. The adhesion between cells and culture equipment etc. produced in an artificial culture environment in vitro is also included in cell adhesion. As the types of cell adhesion, anchoring junction, communicating junction and occluding junction can be listed.

[0147] In the present invention, "tight junction" refers to a closed junction seen in vertebrates and chordates in cell-cell adhesion. Tight junctions are formed between epithelial cells. Whether tight junctions exist in tissues derived from organisms and in cell aggregates prepared by the preparation method of the present invention can be detected by immunohistochemistry using, for example, antibodies against the constituent components of tight junctions (anti-claudin antibodies, anti-ZO-1 antibodies, etc.).

[0148] "Suspension culture" or "suspension culture method" in the present invention refers to culturing cells, cell aggregates or cell clumps in a suspended state in a culture medium, and a method for carrying out the culture. That is, suspension culture is carried out under conditions where cells, cell aggregates or cell clumps do not adhere to culture equipment, etc., while culture carried out under conditions where they adhere to culture equipment, etc. (adherent culture, or adherent culture method) is not included in the category of suspension culture. In this case, cell adhesion refers to the formation of a strong cell-substratum junction as a type of cell adhesion between cells, cell aggregates or cell clumps and culture equipment. More specifically, suspension culture means culture under conditions where a strong cell-substratum junction is not formed between cells, cell aggregates or cell clumps and culture equipment, etc., and adherent culture means culture under conditions where a strong cell-substratum junction is formed between cells, cell aggregates or cell clumps and culture equipment, etc.

[0149] In the cell aggregates or cell clumps in suspension culture, cells and cells are surface-adhesive. In the cell aggregates or cell clumps in suspension culture, cell-matrix connections are hardly formed between culture equipment, etc., or even if formed, their contribution is small. In some embodiments, in the cell aggregates or cell clumps in suspension culture, endogenous cell-matrix connections exist inside the aggregates or cell clumps, but cell-matrix connections are hardly formed between culture equipment, etc., or even if formed, their contribution is small.

[0150] Cell-cell surface adhesion (surface connection) means that one cell is connected to another cell by a surface. More specifically, cell-cell surface adhesion means, for example, that the proportion of adhesion to the surface of another cell in the surface area of ​​one cell is, for example, 1% or more, preferably 3% or more, and more preferably 5% or more. The surface of the cell can be detected by staining with a reagent that stains the membrane (e.g., DiI), immunohistochemistry of cell adhesion factors (e.g., E-cadherin, N-cadherin), and the like.

[0151] The culture container used when performing suspension culture is not particularly limited, as long as it can achieve "suspension culture", and those of ordinary skill in the art can appropriately determine such culture containers. As such culture containers, for example: flasks, tissue culture bottles, flat dishes, petri dishes, tissue culture dishes, multiple culture dishes, microplates, microporous plates, micropores, multiple plates, multi-well plates, chamber slides, bowls, tubes, trays, culture bags, rotating flasks or rotating bottles can be cited. In order to achieve suspension culture, these culture containers are preferably non-cell-adhesive. As non-cell-adhesive culture containers, those whose surfaces have not been artificially treated for the purpose of improving adhesion to cells (for example: coating treatment with basement membrane preparations, laminin, entactin, collagen, gelatin and other extracellular matrices, or polymers such as polylysine, polyornithine, etc., or surface processing such as positive charge treatment) can be used. As non-cell-adhesive culture vessels, those whose surfaces have been artificially treated for the purpose of reducing cell adhesion (e.g., super-hydrophilic treatment such as MPC polymer, protein low adsorption treatment, etc.) can be used. Rotary tube culture can be performed using a spinner flask, a shake bottle, etc. The culture surface of the culture vessel may be flat or may have depressions and projections.

[0152] The medium used for culturing cells in the present invention can be prepared by using a medium commonly used for culturing animal cells as a basal medium. Examples of the basal medium include BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, modified 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 mixed medium thereof, which can be used for culturing animal cells.

[0153] In the culture of pluripotent stem cells, a culture medium for pluripotent stem cell culture based on the above-mentioned basal culture medium can be used, preferably a culture medium for known embryonic stem cells and / or artificial pluripotent stem cells, a culture medium for culturing pluripotent stem cells without feeder cells. For example, feeder-free culture mediums such as Essential 8 culture medium, TeSR culture medium, mTeSR culture medium, mTeSR-E8 culture medium, and StemFit culture medium can be cited.

[0154] "Serum-free medium" in the present invention means a medium that does not contain unregulated or unpurified serum. In the present invention, a medium mixed with purified blood-derived components and animal tissue-derived components (e.g., growth factors) is also included in the serum-free medium unless it contains unregulated or unpurified serum.

[0155] The serum-free medium may contain a serum substitute. Examples of serum substitutes include those that appropriately contain, for example, albumin, transferrin, fatty acids, collagen precursors, trace elements, 2-mercaptoethanol or 3'mercaptoglycerol, or their equivalents. Such serum substitutes may be prepared, for example, by the method described in WO98 / 30679. As serum substitutes, commercially available products may also be used. Examples of such commercially available serum substitutes include Knockout TM Serum Replacement (Life Technologies, hereinafter sometimes referred to as KSR), Chemically Defined Lipid Concentrate (manufactured by Life Technologies), Glutamax TM (manufactured by Life Technologies), B27 (manufactured by Life Technologies), and N2 (manufactured by Life Technologies).

[0156] The serum-free medium for suspension culture may contain fatty acids or lipids, amino acids (eg, non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, inorganic salts and the like as appropriate.

[0157] In order to avoid complicated preparation, as such a serum-free medium, a serum-free medium (for example, a medium in which 1× chemically defined lipid concentrate, 5% KSR and 450 μM 1-monothioglycerol are added to a 1:1 mixture of F-12 medium and IMDM medium) to which an appropriate amount of commercially available KSR (manufactured by Life Technology Co., Ltd.) (for example, about 0.5% to about 30%, preferably about 1% to about 20%) can be used. In addition, as a product equivalent to KSR, the medium disclosed in Japanese Patent Publication No. 2001-508302 can be cited.

[0158] The "serum culture medium" in the present invention refers to a culture medium containing unadjusted or unpurified serum. The culture medium may contain fatty acids, 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, a serum culture medium may be used (Cell Stem Cell, 10 (6), 771-775 (2012)).

[0159] In the present invention, the culture is preferably carried out under xeno-free conditions. "Xeno-free" means a condition in which components derived from species different from the species of cells to be cultured are excluded.

[0160] From the viewpoint of avoiding the contamination of chemically undefined components, the culture medium used in the present invention is preferably a culture medium containing chemically defined components (Chemically defined medium; CDM).

[0161] The "basement membrane"-like structure in the present invention refers to a thin membranous structure composed of an extracellular matrix. In an organism, the basement membrane is formed on the basal side of an epithelial cell. As components of the basement membrane, there can be cited: type IV collagen, laminin, heparan sulfate proteoglycan (perlecan), entactin / nidogen, cytokines, growth factors, etc. Whether a basement membrane exists in a tissue derived from an organism and in a cell mass prepared by the preparation method of the present invention, etc., can be detected by using tissue staining such as PAM staining, and immunohistochemistry of antibodies (anti-laminin antibodies, anti-IV collagen antibodies, etc.) against the constituent components of the basement membrane.

[0162] The "basement membrane preparation" in the present invention refers to a preparation containing basement membrane constituents having the following functions: when the desired cells having the ability to form a basement membrane are inoculated and cultured thereon, the cell morphology, differentiation, proliferation, movement, functional expression, etc. of epithelial cells are controlled. For example, when the cells and tissues prepared by the present invention are dispersed and further cultured by adhesion, they can be cultured in the presence of a basement membrane preparation. Here, "basement membrane constituents" refer to the extracellular matrix molecules in the form of a thin film existing between the epithelial cell layer and the interstitial cell layer in animal tissues. The basement membrane preparation can be prepared by, for example, the following operation: cells having the ability to form a basement membrane that are adhered to a support via a basement membrane are removed from the support using a solution, an alkaline solution, etc. having the lipid solubility of the cells. Examples of basement membrane preparations include commercially available products as basement membrane preparations (for example: Matrigel TM (manufactured by Corning Incorporated, hereinafter sometimes referred to as Matrigel)), Geltrex TM (manufactured by Life Technologies Corporation), and those known as basement membrane components, such as extracellular matrix molecules (e.g., laminin, type IV collagen, heparan sulfate proteoglycan, entactin, etc.).

[0163] In the present invention, basement membrane preparations such as matrigel (Corning) extracted from tissues and even cells such as Engelbreth-Holm-Swarm (EHS) mouse sarcoma and made soluble can be used for the culture of cells and tissues. Similarly, as the basement membrane components used in cell culture, human soluble amniotic membrane (Bioresource Application Research Institute), human recombinant laminin (BioLamina) produced in HEK293 cells, human recombinant laminin fragment (Nippi), human recombinant vitronectin (Thermo Fisher) etc. can also be used, but from the viewpoint of avoiding the mixing of components derived from different biological species and the viewpoint of avoiding the risk of infectious diseases, recombinant proteins with clear components are preferred.

[0164] In the present invention, "a medium containing a substance X" and "in the presence of a substance X" refer to a medium to which an exogenous substance X is added or a medium containing an exogenous substance X and in the presence of an exogenous substance X, respectively. An exogenous substance X is distinguished from an endogenous substance X that is endogenously expressed, secreted or produced by cells or tissues present in the culture medium, for example.

[0165] For example, "a medium containing a substance acting on the Sonic hedgehog signal transduction pathway" refers to a medium to which an exogenous substance acting on the Sonic hedgehog signal transduction pathway is added, or a medium containing an exogenous substance acting on the Sonic hedgehog signal transduction pathway.

[0166] In the present invention, "feeder cells" refer to cells other than stem cells that coexist when culturing stem cells. Feeder cells used to culture pluripotent stem cells while maintaining an undifferentiated state include, for example, mouse fibroblasts (MEF), human fibroblasts, or SNL cells. For feeder cells, feeder cells that have undergone proliferation inhibition treatment are preferred. As proliferation inhibition treatment, treatment with a proliferation inhibitor (for example, mitomycin C) or UV irradiation can be cited. For feeder cells used to culture pluripotent stem cells while maintaining an undifferentiated state, it helps to maintain the undifferentiation of pluripotent stem cells by secreting humoral factors (preferably undifferentiated maintenance factors) or producing a framework (extracellular matrix) for cell adhesion.

[0167] In the present invention, the "aggregate" of cells refers to a mass formed by a collection of cells dispersed in a culture medium, in which cells adhere to each other. The aggregate of cells also includes: cell mass, embryoid body, sphere, spheroid, and organoid. Preferably, in the aggregate of cells, cells are adhered to each other by their surfaces. In some embodiments, in part or all of the aggregate, cells sometimes form cell-cell junctions and / or cell adhesions such as adhesion junctions. As the "aggregate" in the present invention, specifically, the aggregate formed by cells that are dispersed at the beginning of suspension culture and generated in the first step of the following "2. Method for preparing a cell mass containing pituitary tissue" can be cited.

[0168] In the present invention, "uniform aggregates" means that when a plurality of aggregates are cultured, the size of each aggregate is uniform, and when the size of the aggregate is evaluated by the length of the maximum diameter, as a uniform aggregate, the length of the maximum diameter varies little. More specifically, it means that more than 75% of the aggregates in the population of all aggregates are within the range of the average value ±100%, preferably the average value ±50%, and more preferably the average value ±20% of the maximum diameter in the population of the aggregates.

[0169] In the present invention, "forming uniform aggregates" means that when cells aggregate to form aggregates of cells for suspension culture, a given number of dispersed cells are rapidly aggregated to thereby form aggregates of cells of uniform size.

[0170] "Dispersion" means separating cells or tissues into small cell masses (more than 2 cells and less than 100 cells, preferably less than 50 cells) or single cells by dispersing treatment such as enzyme treatment or physical treatment. A given number of dispersed cells refers to a collection of cell masses or a specific number of single cells.

[0171] As a method for dispersing pluripotent stem cells, mechanical dispersion treatment, cell dispersion solution treatment, and treatment with addition of a cell protective agent can be cited. These treatments can be performed in combination. Preferably, the cell dispersion solution treatment is performed, followed by the mechanical dispersion treatment.

[0172] Examples of the mechanical dispersion treatment method include a blowing treatment and a scraping operation using a spatula.

[0173] Regarding the cell dispersion liquid used for the cell dispersion liquid treatment, for example, a solution containing any one of trypsin, collagenase, hyaluronidase, elastase, pronase, DNA enzyme, papain, etc. and a chelating agent such as ethylenediaminetetraacetic acid can be cited. Commercially available cell dispersion liquids can also be used, for example: Accumax (manufactured by Innovative Cell Technologies) and TrypLE Select (manufactured by Life Technologies).

[0174] Examples of the cytoprotective agent used in the treatment of adding a cytoprotective agent include substances acting on the FGF signal transduction pathway, heparin, ROCK inhibitory substances, serum, or serum substitutes. Preferred cytoprotective agents include ROCK inhibitory substances.

[0175] For example, as a method for dispersing pluripotent stem cells, there is a method in which a colony of pluripotent stem cells is treated with a cell dispersion solution (Accumax) in the presence of a ROCK inhibitory substance as a cell protectant, and then dispersed by pipetting.

[0176] In the preparation method of the present invention, it is preferred to form pluripotent stem cell aggregates by rapidly gathering pluripotent stem cells. When the aggregates of pluripotent stem cells are formed in this way, epithelial-like structures can be formed in cells induced and differentiated by the formed aggregates with good reproducibility. As experimental operations for forming aggregates, for example, a plate with small holes (for example, the bottom area of ​​the hole is 0.1 to 2.0 cm when converted with a flat bottom) can be cited. 2A method of controlling cells in a small space by using a small centrifuge tube and centrifuging for a short time to aggregate cells. Examples of small-hole plates include 24-well plates (area 1.88 cm when converted to a flat bottom) and micro-well plates. 2 ), 48-well plate (the area is 1.0cm when converted with a flat bottom 2 About), 96-well plate (the area is 0.35cm when converted with flat bottom 2 About 6 to 8 mm in inner diameter), 384-well plate. Preferably, a 96-well plate can be cited. Regarding the shape of the plate with small holes, the shape of the bottom surface when the hole is viewed from above can be listed as polygonal, rectangular, elliptical, and circular, preferably circular. As the shape of the plate with small holes, the shape of the bottom surface when the hole is viewed from the side is preferably a concave structure with a high outer periphery and a low inner concave part, which can be listed as, for example: U-shaped bottom, V-shaped bottom, M-shaped bottom, preferably U-shaped bottom or V-shaped bottom, and most preferably V-shaped bottom. As the plate with small holes, those with concave-convex or serrated bottom surfaces of cell culture dishes (for example: 60 mm to 150 mm flat dishes, culture bottles) can also be used. The bottom surface of the plate with small holes preferably uses a non-cell-adhesive bottom surface, preferably a bottom surface coated with the aforementioned non-cell-adhesive coating.

[0177] The formation of aggregates of pluripotent stem cells and the formation of epithelial-like structures in each cell forming the aggregate can be judged based on the size and cell number of the aggregates, the macroscopic morphology, the microscopic morphology and its uniformity analyzed by tissue staining, the expression and uniformity of differentiation and undifferentiation markers, the expression control and synchronization of differentiation markers, the reproducibility of differentiation efficiency between aggregates, etc.

[0178] The "tissue" in the present invention means a structure of a cell group having a structure in which a plurality of types of cells different in morphology and properties are spatially arranged in a given pattern.

[0179] In the present invention, "nervous tissue" refers to tissue composed of nervous system cells, including the cerebrum, midbrain, diencephalon, cerebellum, spinal cord, retina, peripheral nerves, etc. in the developmental stage or adult stage. The nervous tissue sometimes forms an epithelial structure (neuroepithelium) having a layer structure, and the amount of neuroepithelium in the nervous tissue can be evaluated by bright field observation using an optical microscope.

[0180] In the present invention, "diencephalon" refers to the neural tissue of the central nervous system connected to the third ventricle. The diencephalon includes tissues such as the epithalamus, thalamus, hypothalamus, and pituitary gland.

[0181] In the present invention, "hypothalamus" refers to a region of the diencephalon connected to the pituitary gland. The hypothalamus is further divided into the dorsal hypothalamus and the ventral hypothalamus. The hypothalamus can be identified using markers such as Rx, Vax1, and Six3. The dorsal hypothalamus can be identified using markers such as Otp, Brn2, and vasopressin. The ventral hypothalamus can be identified using markers such as Nkx 2.1 and SF1.

[0182] In the present invention, "cerebral ventricle" refers to a cavity formed by central nervous tissue. In an organism, it is a cell-free structure that is usually filled with tissue fluid such as cerebrospinal fluid, and the apical surface of the nervous tissue faces the ventricle. The periventricular layer surrounding the ventricle is a region where neural stem cells exist and cells proliferate and neurons are produced during the developmental stage. Whether the cell mass and tissue prepared by the preparation method of the present invention contain ventricles can be detected by, for example, immunohistochemistry using central nervous tissue markers (Bf1, Pax6, etc.) and apical surface markers (PKC-zeta, etc.).

[0183] In the present invention, "neural cell" refers to cells other than epidermal lineage cells in tissues derived from the ectoderm. That is, it includes cells such as nervous system precursor cells, neurons (neural cells), glial cells, neural stem cells, neuronal precursor cells and glial precursor cells. Nervous system cells also include cells constituting the retinal tissue mentioned below (retinal cells), retinal progenitor cells, retinal layer-specific neural cells, neural retinal cells and retinal pigment epithelial cells. Nervous system cells can be identified using nestin, TuJ1, PSA-NCAM, N-cadherin, etc. as markers.

[0184] Neurons are functional cells that form neural circuits and contribute to signal transduction, and can be identified by using the expression of immature neural cell markers such as TuJ1, Dcx, HuC / D, and / or mature neural cell markers such as Map2 and NeuN as indicators.

[0185] Examples of glial cells include astrocytes, oligodendrocytes, and Muller glial cells. Examples of markers for astrocytes include GFAP, examples of markers for oligodendrocytes include o4, and examples of markers for Muller glial cells include CRALBP.

[0186] Neural stem cells are cells with differentiation potential (multipotentiality) to neurons and glial cells and the ability to proliferate (sometimes referred to as self-renewal ability) to maintain multipotentiality. Regarding neural stem cell markers, nestin, Sox2, Musashi, Hes family, CD133, etc. can be listed; however, these markers are markers for all progenitor cells / precursor cells and are not considered to be markers specific to neural stem cells. The number of neural stem cells can be assessed by neurosphere assays, clonal assays, etc.

[0187] Neuronal precursor cells are cells with proliferation ability, which produce neurons and do not produce glial cells. As markers of neuronal precursor cells, Tbr2, Tα1, etc. can be listed. Alternatively, cells that are positive for immature neuronal cell markers (TuJ1, Dcx, HuC / D) and proliferation markers (Ki67, pH3, MCM) can also be identified as neuronal precursor cells.

[0188] Glial progenitor cells are cells that have the ability to proliferate, produce glial cells, and do not produce neurons.

[0189] Nervous system precursor cells (Neural Precursor cell) are aggregates of precursor cells including neural stem cells, neuronal precursor cells and glial precursor cells, with proliferation ability and neuron and glial cell production ability. Nervous system precursor cells can be identified with nestin, GLAST, Sox2, Soxl, Musashi, Pax6, etc. as markers. Alternatively, cells that are positive for nervous system cell markers and growth markers (Ki67, pH3, MCM) can also be identified as nervous system precursor cells.

[0190] In the present invention, "non-neuroepithelial tissue" refers to tissues other than neuroepithelial tissues in tissues having an epithelial structure. Epithelial tissues can be formed by any germ layer of ectoderm, mesoderm, endoderm, and trophectoderm. Epithelial tissues include epithelium, mesoderm, and endothelium. Examples of tissues included in non-neuroepithelial tissues include epidermis, corneal epithelium, nasal epithelium, oral epithelium, tracheal epithelium, bronchial epithelium, tracheal epithelium, renal epithelium, renal cortical epithelium, placental epithelium, etc.

[0191] Epithelial tissues are usually connected by various cell-cell bonds to form a single-layer or multi-layered layer structure. The presence or absence of these epithelial tissues and the quantification of their presence can be performed by observation using an optical microscope or immunohistochemistry using antibodies against epithelial cell markers (anti-E-cadherin antibodies, anti-N-cadherin antibodies, anti-EpCAM antibodies, etc.).

[0192] In the present invention, "epithelial polarity" refers to the spatial distribution bias of components and cell functions in epithelial cells. For example, corneal epithelial cells are localized in the outermost layer of the eyeball, and express apical-specific proteins such as membrane-bound mucins (MUC-1, 4, 16) for tear maintenance on the apical side, and express basal-specific proteins such as α6 integrin and β1 integrin for adhesion to the basement membrane on the basal side.

[0193] Whether epithelial cell polarity exists in epithelial cells and epithelial tissues in tissues derived from organisms and in cell aggregates prepared by the preparation method of the present invention can be detected by immunohistochemistry using phalloidin, apical markers (anti-MUC-1 antibodies, anti-PKC-zeta antibodies, etc.) and basal markers (anti-α6 integrin antibodies, anti-β1 integrin antibodies, etc.).

[0194] In the present invention, "placode" mainly refers to the primordial organ of an organ formed by hypertrophy of a part of the epidermal ectoderm during the development of vertebrates. Examples of tissues derived from the placode include the lens, nose, inner ear, trigeminal nerve, and adenohypophysis. Examples of markers for the placode or the preplacode region of its precursor tissue include Six1, Six4, D1x5, Eya2, Emx2, and Bf1.

[0195] In the present invention, "pituitary placode" refers to a hypertrophic structure formed in the region of the epidermal ectoderm during embryonic development, which expresses pituitary precursor cell markers. Examples of pituitary precursor cell markers include Lim3, Pitx1 / 2, Islet1 / 2, etc. The pituitary placode expresses at least one, preferably all, pituitary precursor cell markers selected from the group consisting of Lim3, Pitx1 and Islet1 / 2. The pituitary placode sinks into the Rathke'spouch, a bag-like structure during development, and further development progresses to form the glandular pituitary.

[0196] In the present invention, "adenohypophysis" refers to a pituitary cell tissue comprising at least one anterior lobe or middle lobe. Among the pituitary cells, pituitary hormone-producing cells and non-hormone-producing cells that produce hormones that regulate physiological functions are included. As pituitary hormone-producing cells, there can be listed: growth hormone (GH) producing cells, prolactin (PRL) producing cells, adrenocorticotropic hormone (ACTH) producing cells, thyroid stimulating hormone (TSH) producing cells, follicle stimulating hormone (FSH) producing cells, luteinizing hormone (LH) producing cells and other cells that constitute the anterior lobe; melanocyte stimulating hormone (MSH) producing cells and other cells that constitute the middle lobe. As non-hormone-producing cells, they include: vascular endothelial cells, pericytes, follicular astrocytes, pituitary stem cells, pituitary precursor cells.

[0197] In one embodiment, the glandular pituitary gland comprises at least one, preferably two, and more preferably three pituitary hormone-producing cells selected from the group consisting of growth hormone (GH)-producing cells, prolactin (PRL)-producing cells, and adrenocorticotropic hormone (ACTH)-producing cells. In a further embodiment, the glandular pituitary gland comprises at least one, preferably two or more (2, 3, 4, 5 or 6) pituitary hormone-producing cells selected from the group consisting of growth hormone (GH)-producing cells, prolactin (PRL)-producing cells, adrenocorticotropic hormone (ACTH)-producing cells, thyroid stimulating hormone (TSH)-producing cells, follicle stimulating hormone (FSH)-producing cells, and luteinizing hormone (LH)-producing cells. Whether the glandular pituitary gland and pituitary hormone-producing cells are contained in the tissue derived from the organism and in the cell mass prepared by the preparation method of the present invention can be detected by immunohistochemistry using growth hormone (GH) producing cell markers (anti-Pit1 antibody, anti-GH antibody, etc.), prolactin (PRL) producing cell markers (anti-Pit1 antibody, anti-PRL antibody, etc.), adrenocorticotropic hormone (ACTH) producing cell markers (anti-T-Pit antibody, anti-NeuroD1 antibody, anti-ACTH antibody, etc.), thyroid stimulating hormone (TSH) producing cell markers (anti-GATA2 antibody, anti-ACTH antibody, etc.), follicle stimulating hormone (FSH) producing cells, and luteinizing hormone (LH) producing cell markers (anti-GATA2 antibody, anti-SF1 antibody, anti-FSH antibody, anti-LH antibody, etc.) and ELISA for secreted hormones.

[0198] The "pituitary stem cells" in the present invention refer to undifferentiated pluripotent stem cells or precursor cells that exist in the pituitary gland and promote the regeneration of pituitary tissue and the supply of pituitary hormone-producing cells. Whether the cell aggregates and tissues prepared by the preparation method of the present invention contain pituitary stem cells can be detected by, for example, immunohistochemistry using antibodies against pituitary stem cell markers such as Sox2, Sox9, E-cadherin, nestin, S100β, GFRα2, Prop1, CD133, β-catenin, Klf4, Oct4, Pax6, coxsackievirus-adenovirus co-receptor (CXADR), PRRX1 / 2, Ephrin-B2, ACE, YAP1, TAZ, and cell proliferation markers such as Ki67, phosphorylated histone H3, and MCM, proliferating cell labeling analysis using nucleic acid analogs such as BrdU, EdU, and IdU, an uptake test of fluorescently labeled dipeptide (β-alanyl-lysyl-N-7-amino-4-methylcoumarin-3-acetic acid), a pituisphere formation test, and the like.

[0199] In the present invention, "niche" or "stem cell niche" refers to a microenvironment related to the proliferation, differentiation, maintenance of properties, etc. of stem cells. Examples of microhabitats in organisms include: hematopoietic stem cell niche, hair follicle stem cell niche, intestinal epithelial stem cell niche, muscle stem cell niche, pituitary niche, etc. In these niches, there are stem cells specific to each tissue and supporting cells that provide the niches. Stem cells are maintained by cytokines, chemokines, extracellular matrix, cell adhesion factors, intercellular signal transduction factors, etc. provided by the supporting cells.

[0200] In the present invention, "pituitary niche" refers to a microenvironment related to the proliferation, differentiation, and maintenance of properties of pituitary stem cells. Examples of pituitary niches include the marginal cell layer (MCL) niche that exists around the residual cavity (Rathke's fissure) that remains between the anterior and middle pituitary lobes as a trace of the hollow part of the bag-like Rathke's capsule in the developmental stage, and the parenchymal niche that is scattered in the anterior pituitary.

[0201] In the present invention, "neural crest" refers to a structure formed between the epidermal ectoderm and the neural plate in the early development of vertebrates, and "neural crest cells or cells derived from the neural crest" refer to cells present in the neural crest and even cells derived from the neural crest that migrate and differentiate. Cells derived from the neural crest include, for example, nerve cells of the peripheral nervous system, supporting cells, pigment cells, endocrine cells, smooth muscles, head bone cells, mesenchymal cells, etc. Whether the cell agglomerates and tissues prepared by the preparation method of the present invention contain neural crest cells and cells derived from the neural crest can be detected by immunohistochemistry using antibodies against neural crest cell markers such as nestin, Sox10, Sox9, Slug, Snail, Twist, FoxD3, TFAP2, Pax3, Pax7, HNK-1, p75NTR, etc.

[0202] "Mesenchymal cells" in the present invention refer to non-epithelial cells that are mainly derived from the mesoderm and neural crest and form connective tissue. Some of these cells are multipotent somatic stem cells called mesenchymal stem cells. Whether the cell agglomerates and tissues prepared by the preparation method of the present invention contain mesenchymal cells can be detected by immunohistochemistry using antibodies for mesenchymal cell markers such as nestin, vimentin, cadherin-11, laminin, CD44, etc. Whether mesenchymal stem cells are contained can be detected by immunohistochemistry using antibodies for mesenchymal stem cell markers such as CD9, CD13, CD29, CD44, CD55, CD59, CD73, CD105, CD140b, CD166, VCAM-1, STRO-1, c-Kit, Sca-1, nuclear stem cell factor (Nucleostemin), CDCP1, BMPR2, BMPR1A and BPMR1B.

[0203] 2. Preparation of cell pellets containing pituitary tissue

[0204] The present invention provides a cell mass containing pituitary tissue and a method for preparing the same, which is hereinafter also referred to as the preparation method of the present invention.

[0205] One embodiment of the preparation method of the present invention is a method for preparing a cell mass containing pituitary tissue, comprising the following steps (1) and (2):

[0206] (1) In the first step, pluripotent stem cells are suspended and cultured in the presence of a Wnt signal transduction pathway inhibitor to form cell aggregates;

[0207] (2) In the second step, the aggregates obtained in the first step are suspended and cultured in the presence of a substance acting on the BMP signal transduction pathway and a substance acting on the Sonic hedgehog signal transduction pathway to obtain cell aggregates containing pituitary tissue.

[0208] A preferred embodiment of the preparation method of the present invention is a method for preparing a cell mass containing pituitary tissue, comprising the following steps (a), (1) and (2):

[0209] (a) step a, culturing the pluripotent stem cells in a medium containing 1) a TGFβ family signal transduction pathway inhibitory substance and / or a Sonic hedgehog signal transduction pathway active substance, and 2) an undifferentiation maintenance factor in the absence of feeder cells;

[0210] (1) In the first step, the cells cultured in step a are suspended and cultured in the presence of a Wnt signal transduction pathway inhibitory substance to form cell aggregates;

[0211] (2) In the second step, the aggregates obtained in the first step are suspended and cultured in the presence of substances acting on the BMP signal transduction pathway and substances acting on the Sonic hedgehog signal transduction pathway to obtain cell aggregates containing pituitary tissue.

[0212] A more preferred embodiment of the preparation method of the present invention is a method for preparing a cell mass containing pituitary tissue, comprising the following steps (1), (2-1) and (2-2):

[0213] (1) In the first step, pluripotent stem cells are suspended and cultured in the presence of a Wnt signal transduction pathway inhibitor to form cell aggregates;

[0214] (2-1) the second step (1), culturing the aggregates obtained in the first step in suspension in the presence of a substance acting on the BMP signal transduction pathway and a substance acting on the Sonic hedgehog signal transduction pathway;

[0215] (2-2) The second step (2) is to culture the aggregates obtained by suspension culture in the second step (1) in the presence of a BMP signal transduction pathway inhibitory substance to obtain cell aggregates containing pituitary tissue.

[0216] A further embodiment of the preparation method of the present invention is a method for preparing a cell mass containing pituitary tissue, comprising the following steps (a), (1), (2-1) and (2-2):

[0217] (a) step a, culturing the pluripotent stem cells in a medium containing 1) a TGFβ family signal transduction pathway inhibitory substance and / or a Sonic hedgehog signal transduction pathway active substance, and 2) an undifferentiation maintenance factor in the absence of feeder cells;

[0218] (1) In the first step, the cells cultured in step a are suspended and cultured in the presence of a Wnt signal transduction pathway inhibitory substance to form cell aggregates;

[0219] (2-1) the second step (1), culturing the aggregates obtained in the first step in suspension in the presence of a substance acting on the BMP signal transduction pathway and a substance acting on the Sonic hedgehog signal transduction pathway;

[0220] (2-2) The second step (2) is to culture the aggregates obtained by suspension culture in the second step in the presence of a BMP signal transduction pathway inhibitory substance to obtain a cell mass containing pituitary tissue.

[0221] <Step (a)>

[0222] Step a of culturing pluripotent stem cells in the absence of feeder cells in a medium containing 1) a TGFβ family signaling pathway inhibitory substance and / or a Sonic hedgehog signaling pathway activating substance and 2) an undifferentiation maintaining factor will be described.

[0223] In step (a), the pluripotent stem cells are treated with TGFβ family signal transduction pathway inhibitors and / or Sonichedgehog signal transduction pathway activating substances, and then suspended cultured in the first step, thereby changing the state of the pluripotent stem cells, improving the formation efficiency of non-neuroepithelial tissues, improving the quality of aggregates, making it easier to differentiate and less likely to cause cell death, and efficiently preparing cell aggregates that are densely packed inside and maintain undifferentiated properties.

[0224] Step (a) is preferably performed in the absence of feeder cells.

[0225] The condition in which feeder cells are absent (feeder cell-free) in the present invention refers to a condition in which feeder cells are substantially not contained (for example, the ratio of the number of feeder cells to the total number of cells is 3% or less).

[0226] The culture medium used in step (a) is not particularly limited as long as it is a culture medium that can achieve undifferentiation maintenance culture of pluripotent stem cells under feeder cell-free conditions (feeder cell-free culture medium). Examples thereof include a culture medium containing an undifferentiation maintenance factor and a TGFβ family signal transduction pathway inhibitory substance and / or a Sonic hedgehog signal transduction pathway activating substance.

[0227] The culture medium used in step (a) contains undifferentiation maintenance factors in order to achieve undifferentiated maintenance culture. There is no particular limitation on the factors used to maintain undifferentiation, as long as they are substances that inhibit the differentiation of pluripotent stem cells. As undifferentiation maintenance factors widely used by ordinary technicians in the field, in the case of primed pluripotent stem cells (e.g., human ES cells, human iPS cells), they include: FGF signal transduction pathway acting substances, TGFβ family signal transduction pathway acting substances, insulin, etc. As FGF signal transduction pathway acting substances, specifically, fibroblast growth factors (e.g., bFGF, FGF4, FGF8) can be listed. In addition, as TGFβ family signal transduction pathway acting substances, they include: TGFβ signal transduction pathway acting substances, Nodal / activator protein signal transduction pathway acting substances. As TGFβ signal transduction pathway acting substances, for example, TGFβ1 and TGFβ2 can be listed. As Nodal / activator protein signal transduction pathway acting substances, for example, Nodal, activator protein A, and activator protein B can be listed. When culturing human pluripotent stem cells (human ES cells, human iPS cells), the medium in step (a) preferably contains bFGF as an undifferentiation-maintaining factor.

[0228] The undifferentiation maintenance factor used in the present invention is generally a factor that maintains the undifferentiated state of mammals. Regarding mammals, those mentioned above can be cited. Since the undifferentiated maintenance factor can have cross-reactivity between mammalian species, factors for maintaining the undifferentiated state of any mammal can also be used, as long as the undifferentiated state of the pluripotent stem cells to be cultured can be maintained. Preferably, an undifferentiated maintenance factor of a mammal of the same species as the cells to be cultured is used. For example, for the culture of human pluripotent stem cells, human undifferentiated maintenance factors (e.g., bFGF, FGF4, FGF8, EGF, Nodal, activator protein A, activator protein B, TGFβ1, TGFβ2, etc.) are used. Here, "human protein X" refers to protein X (undifferentiated maintenance factor, etc.) having the amino acid sequence of protein X naturally expressed in the human organism.

[0229] The undifferentiation-maintaining factor used in the present invention is preferably isolated.

[0230] "Isolated" means removing the target component or factors other than cells and leaving the naturally existing state. Therefore, "isolated protein X" does not include endogenous protein X contained in cells or tissues produced by the cultured object, tissues, and culture medium. The purity of "isolated protein X" (the percentage of the weight of protein X to the weight of total proteins) is usually 70% or more, preferably 80% or more, more preferably 90% or more, further preferably 99% or more, and most preferably 100%.

[0231] In one embodiment, the present invention includes the step of providing an isolated undifferentiation-maintaining factor. In addition, in one embodiment, the present invention includes the step of exogenously (or exogenously) adding the isolated undifferentiation-maintaining factor to the culture medium used in step (a). Alternatively, the undifferentiation-maintaining factor may be pre-added to the culture medium used in step (a).

[0232] The concentration of the undifferentiation maintenance factor in the culture medium used in step (a) is a concentration that can maintain the undifferentiated state of the cultured pluripotent stem cells, and can be appropriately set by those skilled in the art. For example, when bFGF is used as an undifferentiation maintenance factor in the absence of feeder cells, its concentration is generally about 4 ng / mL to about 500 ng / mL, preferably about 10 ng / mL to about 200 ng / mL, and more preferably about 30 ng / mL to about 150 ng / mL.

[0233] Regarding feeder-free culture media, various synthetic culture media have been developed and are commercially available, for example, Essential 8 medium can be cited. Essential 8 medium contains the following as additives in DMEM / F12 medium: L-ascorbic acid-2-phosphate magnesium (64 mg / L), sodium selenate (14 μg / L), insulin (19.4 mg / L), NaHCO3 (543 mg / L), transferrin (10.7 mg / L), bFGF (100 ng / mL), and TGFβ family signal transduction pathway activating substances (TGFβ1 (2 ng / mL) or Nodal (100 ng / mL)) (Nature Methods, 8, 424-429 (2011)). Commercially available feeder-free cell culture media include, for example, Essential 8 (manufactured by Life Technologies), S-medium (manufactured by DS Pharma Biomedical), StemPro (manufactured by Life Technologies), hESF9, mTeSR1 (manufactured by STEMCELL Technologies), mTeSR2 (manufactured by STEMCELL Technologies), and TeSR-E8 (manufactured by STEMCELL Technologies). In addition to these, StemFit (manufactured by Ajinomoto Co., Inc.) can be cited as a feeder-free cell culture medium. The present invention can be easily implemented by using them in the above step (a). As described in Scientific Reports (2014), 4, 3594., the StemFit culture medium contains bFGF as an undifferentiated maintenance component.

[0234] The medium used in step (a) may be a serum medium or a serum-free medium, but is preferably a serum-free medium from the viewpoint of avoiding contamination of chemically undefined components.

[0235] From the viewpoint of avoiding the contamination of chemically undefined components, the medium used in step (a) is preferably a medium containing chemically defined components.

[0236] The culturing of pluripotent stem cells in step (a) may be carried out under any conditions of suspension culture or adherent culture, and is preferably carried out under adherent culture.

[0237] In the culture of pluripotent stem cells under feeder cell-free conditions in step (a), the above-mentioned feeder cell-free medium can be used as the culture medium.

[0238] In the cultivation of pluripotent stem cells without feeder cell conditions in step (a), an appropriate matrix can be used as a framework to provide a framework for pluripotent stem cells to replace feeder cells.Pluripotent stem cells can be cultured by adhering to the cell container coated on the surface using the matrix as the framework.

[0239] Matrices that can be used as the framework 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, entactin, and vitronectin.

[0240] "Laminin" refers to a heterotrimeric molecule containing α, β, and γ chains, and is an extracellular matrix protein with different subtypes of subunit chain composition. Specifically, there are about 15 subtypes of laminin, which are combinations of heterotrimers of 5 α chains, 4 β chains, and 3 γ chains. The name of laminin is determined by combining the number of α chains (α1-α5), β chains (β1-β4), and γ chains (γ1-γ4). For example, laminin with a combination of α5 chain, β1 chain, and γ1 chain is called laminin 511. In the present invention, laminin 511 is preferably used (Nat Biotechnol 28, 611-615 (2010)).

[0241] The laminin fragment used in the present invention is not particularly limited as long as it has adhesion to pluripotent stem cells and allows the maintenance and culture of pluripotent stem cells under feeder cell-free conditions, and it is preferably an E8 fragment. Among the multiple fragments obtained by digesting laminin 511 with elastase, the laminin E8 fragment was identified as a fragment 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 used in the present invention does not need to be an elastase digestion product of laminin, and may be a recombinant. Or it may be those produced in genetically recombinant animals (silkworms, etc.). In order to avoid the mixing of chemically undefined components, it is preferred to use a recombinant laminin fragment in the present invention. The E8 fragment of laminin 511 is commercially available and can be purchased from, for example, Nippi, Inc., etc.

[0242] From the viewpoint of avoiding the contamination of chemically undefined components, the laminin or laminin fragment used in the present invention is preferably isolated.

[0243] Preferably, in the culture of pluripotent stem cells under feeder cell-free conditions in step (a), the human pluripotent stem cells are adherently cultured in a cell container whose surface is coated with isolated laminin 511 or E8 fragment of laminin 511 (most preferably, E8 fragment of laminin 511).

[0244] The culture time of the pluripotent stem cells in step (a) is not particularly limited, and can be within the range that can achieve the effect of continuing to improve the quality of the aggregates formed in step (1), and is generally 0.5 to 144 hours, preferably 2 to 96 hours, more preferably 6 to 48 hours, further preferably 12 to 48 hours, and further preferably 18 to 28 hours (for example, 24 hours). That is, step (a) starts 0.5 to 144 hours (preferably 18 to 28 hours) before the start of step (1), and step (1) is continued after step (a) is completed.

[0245] The culture conditions such as the culture temperature and CO2 concentration in step (a) can be appropriately set. The culture temperature is, for example, about 30° C. to about 40° C., preferably about 37° C. In addition, the CO2 concentration is, for example, about 1% to about 10%, preferably about 5%.

[0246] In a preferred embodiment, human pluripotent stem cells (e.g., human iPS cells) are adherently cultured in the absence of feeder cells and in a serum-free medium containing bFGF. The adherent 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 adherent culture is preferably carried out using StemFit as a feeder-free culture medium.

[0247] In a preferred embodiment, human pluripotent stem cells (eg, human iPS cells) are cultured in suspension in the absence of feeder cells and in a serum-free medium containing bFGF. In the suspension culture, the human pluripotent stem cells can form human pluripotent stem cell aggregates.

[0248] Sonic hedgehog (Shh) signal transduction pathway acting substance refers to a substance that can enhance the signal transduction mediated by Shh. Examples of Shh signal transduction pathway acting substance include proteins belonging to the Hedgehog family (e.g., Shh, Ihh), Shh receptors, Shh receptor agonists, Smo agonists, purmorphamine, GSA-10, Hh-Ag 1.5, 20(S)-hydroxycholesterol, or SAG (Smoothened Agonist; N-methyl-N′-(3-pyridylbenzyl)-N′-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4-diaminocyclohexane). The Shh signal transduction pathway acting substance is preferably SAG. The concentration of the Shh signal transduction pathway acting substance in the culture medium can be appropriately set within the range that can achieve the above-mentioned effect. SAG is usually used in step (a) at a concentration of about 1 nM to about 2000 nM, preferably about 10 nM to about 1000 nM, particularly preferably about 10 nM to about 700 nM, more preferably about 100 nM to about 600 nM, and further preferably about 100 nM to about 500 nM. When a substance acting on the Shh signal transduction pathway other than SAG is used, it is preferably used at a concentration that imparts Shh signal transduction promoting activity equivalent to the activity of SAG at the above-mentioned concentration. The Shh signal transduction promoting activity of SAG or the like can be determined by a method 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). For example, as a substance acting on the Shh signaling pathway having an activity to promote Shh signaling equivalent to 10 nM to 700 nM of SAG, there can be mentioned 20 nM to 2 μM of purmorphamine, 20 nM to 3 μM of GSA-10, and 10 nM to 1 μM of Hh-Ag 1.5.

[0249] The TGFβ family signal transduction pathway (ie, the TGFβ superfamily signal transduction pathway) refers to a signal transduction pathway that uses TGFβ, Nodal / activator protein or BMP as ligands and is transduced by the Smad family in cells.

[0250] The TGFβ family signal transduction pathway inhibitory substance refers to a substance that inhibits the GFβ family signal transduction pathway, that is, the signal transduction pathway transduced by the Smad family, and specifically includes: TGFβ signal transduction pathway inhibitory substance, Nodal / activin signal transduction pathway inhibitory substance and BMP signal transduction pathway inhibitory substance. As the TGFβ family signal transduction pathway inhibitory substance, the TGFβ signal transduction pathway inhibitory substance is preferred.

[0251] There is no particular limitation on the substance that inhibits the signal transduction pathway caused by TGFβ, as long as it is a substance that inhibits the signal transduction pathway caused by TGFβ, and it can be any one of nucleic acids, proteins, and low molecular organic compounds. As such substances, for example, substances that directly act on TGFβ (for example, proteins, antibodies, aptamers, etc.), substances that inhibit the expression of genes encoding TGFβ (for example, antisense oligonucleotides, siRNA, etc.), substances that inhibit the binding of TGFβ receptors to TGFβ, and substances that inhibit the physiological activity caused by the signal transduction of TGFβ receptors (for example, inhibitors of TGFβ receptors, inhibitors of Smad, etc.). Proteins known as TGFβ signal transduction pathway inhibitors include Lefty, etc.

[0252] As TGFβ signal transduction pathway inhibitors, compounds well known to those skilled in the art can be used. Specifically, Alk5 / TGFβR1 inhibitors such as SB431542 (sometimes abbreviated as SB431 in this specification), SB505124, SB525334, LY2157299, LY2109761, GW788388, LY364947, SD-208, EW-7197, A83-01, and RepSox, and SMAD3 inhibitors such as SIS3 can be mentioned. Here, SB431542 (4-(5-benzo[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazole-2-yl)-benzamide) and A 83-01 (3-(6-methyl-2-pyridine)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carboxythioamide) are compounds known as inhibitors of TGFβ receptor (ALK5) and activin receptor (ALK4 / 7) (i.e., TGFβR inhibitors). SIS3 is a TGFβ signaling pathway inhibitory substance that inhibits the phosphorylation of SMAD3, an intracellular signaling factor under the control of the TGFβ receptor. The TGFβ signaling pathway inhibitory substance used in the present invention is preferably SB431542 or A-83-01.

[0253] The concentration of the TGFβ signaling pathway inhibitor in the culture medium can be appropriately set within the range that can achieve the above-mentioned effect. When SB431542 is used as the TGFβ signaling pathway inhibitor in step (a), it is usually used at a concentration of about 1nM to about 100μM, preferably about 10nM to about 50μM, more preferably about 100nM to about 50μM, and further preferably about 1μM to about 10μM. In addition, when a TGFβ signaling pathway inhibitor other than SB431542 is used, it is preferably used at a concentration that imparts TGFβ signaling pathway inhibitory activity equivalent to the above-mentioned concentration of SB431542.

[0254] <Step (1)>

[0255] The first step of forming cell aggregates by culturing pluripotent stem cells maintained under undifferentiated conditions, preferably pluripotent stem cells obtained in step (a), in suspension in the presence of a Wnt signaling pathway inhibitory substance will be described.

[0256] The Wnt signal transduction pathway refers to a signal transduction pathway with Wnt family and protein as ligands and Frizzled as the main receptor. As this signal pathway, in addition to the canonical Wnt pathway transduced by β-catenin, the non-canonical Wnt pathway (Non-CanonicalWnt pathway) independent of β-catenin (β-Catenin) can be listed. As non-canonical Wnt pathways, the planar cell polarity (PCP) pathway, Wnt / Calcium pathway, Wnt-RAP1 pathway, Wnt-Ror2 pathway, Wnt-PKA pathway, Wnt-GSK3MT pathway, Wnt-aPKC pathway, Wnt-RYK pathway, Wnt-mTOR pathway, etc. can be listed. In the non-classical Wnt pathway, there are common signal transduction factors that are also activated in signal transduction pathways other than Wnt, but in the present invention, these factors are also regarded as components of the Wnt signal transduction pathway, and inhibitory substances against these factors are also included in the Wnt signal transduction pathway inhibitory substances.

[0257] In the present invention, there is no limitation on the Wnt signal transduction pathway inhibitory substance, as long as it can inhibit the signal transduction induced by the Wnt family and proteins. It can be any one of nucleic acids, proteins, and low molecular organic compounds. As such substances, for example, substances that inhibit the processing and extracellular secretion of Wnt, substances that directly act on Wnt (for example, proteins, antibodies, aptamers, etc.), substances that inhibit the expression of genes encoding Wnt (for example, antisense oligonucleotides, siRNA, etc.), substances that inhibit the binding of Wnt receptors and Wnt, and substances that inhibit physiological activity caused by signal transduction via Wnt receptors. For proteins known as Wnt signal transduction pathway inhibitors, proteins belonging to the secreted Frizzled Related Protein (sFRP) class (sFRP1~5, Wnt Inhibitory Factor-1 (WIF-1), Cerberus), proteins belonging to the Dickkopf (Dkk) class (Dkk1~4, Kremen), etc. can be listed.

[0258] As Wnt signal transduction pathway inhibitors, compounds well known to those skilled in the art can be used. As Wnt signal transduction pathway inhibitors, for example, Porcupine inhibitors (also known as PORCN inhibitors), DKK inhibitors, Frizzled inhibitors, Dvl inhibitors, Tankyrase inhibitors (also known as TANK inhibitors), casein kinase 1 inhibitors, beta-catenin responsive transcription inhibitors, p300 inhibitors, CBP inhibitors, BCL-9 inhibitors (Am J Cancer Res. 2015; 5 (8): 2344-2360), etc. In addition, although the mechanism of action has not been reported, KY 02111 and KY-03-I can be listed as Wnt signal transduction pathway inhibitors. As PORCN inhibitors, for example, IWP-2, IWP-3, IWP-4, IWP-L6, IWP-12, LGK-974, ETC-159, GNF-6231, Wnt-C59, etc. can be listed. As TANK inhibitors, for example, IWR1-endo, XAV939, MN-64, WIKI4, TC-E 5001, JW 55, AZ6102, etc. The Wnt signaling pathway inhibitor used in the present invention is preferably a PORCN inhibitor, a TANK inhibitor or KY-02111, and more preferably a PORCN inhibitor. The PORCN inhibitor used in the present invention is preferably IWP-2 or Wnt-C59. The TANK inhibitor used in the present invention is preferably XAV939.

[0259] The Wnt signaling pathway inhibitory substance used in the present invention also preferably has an inhibitory activity against the non-canonical Wnt pathway. Examples of Wnt signaling pathway inhibitory substances having an inhibitory activity against the non-canonical Wnt pathway include PORCN inhibitors, anti-Frizzled antibodies, and Box5 peptides. Porcupine is known to be involved in lipid modification of Wnt5a, Wnt5b, and Wnt11, which are ligands of the non-canonical Wnt pathway, in addition to Wnt1 and Wnt3a, which are ligands of the canonical Wnt pathway (Dev Biol. 2012 Jan 15; 361(2): 392-402., Biochem J. 2007 Mar 15; 402(Pt 3): 515-523.), and PORCN inhibitors inhibit both the canonical Wnt pathway and the non-canonical Wnt pathway.

[0260] The concentration of the Wnt signaling pathway inhibitor in the culture medium can be appropriately set within the range that can achieve the above-mentioned effects. For example, when IWP-2, a type of PORCN inhibitor, is used as a Wnt signaling pathway inhibitor, its concentration is usually about 0.01 μM to about 30 μM, preferably about 0.1 μM to about 30 μM, and more preferably about 2 μM. When Wnt-C59, a type of PORCN inhibitor, is used, its concentration is usually about 1 pM to about 30 μM, preferably about 100 pM to about 30 μM, and more preferably about 1 nM to about 1 μM. When XAV939, a type of TANK inhibitor, is used, its concentration is usually about 0.01 μM to about 30 μM, preferably about 0.1 μM to about 30 μM, and more preferably about 1 μM. When KY 02111 is used, the concentration thereof is generally about 0.01 μM to about 30 μM, preferably about 0.1 μM to about 30 μM, and more preferably about 5 μM.

[0261] From the viewpoint of forming mesenchymal cells, it is preferred that the suspension culture be performed in the presence of a Wnt signaling pathway inhibitory substance and a TGFβ signaling pathway inhibitory substance in step (1).

[0262] As the TGFβ signaling pathway inhibitory substance used in step (1), the same substances as those exemplified in step (a) can be used. The TGFβ signaling pathway inhibitory substances in step (a) and step (1) may be the same or different, but are preferably the same.

[0263] The concentration of the TGFβ signaling pathway inhibitor in the culture medium can be appropriately set within the range that can achieve the above-mentioned effect. When SB431542 is used as the TGFβ signaling pathway inhibitor in step (1), it is usually used at a concentration of about 1nM to about 100μM, preferably about 10nM to about 50μM, more preferably about 100nM to about 50μM, and further preferably about 500nM to about 10μM. In addition, when a TGFβ signaling pathway inhibitor other than SB431542 is used, it is preferably used at a concentration that imparts TGFβ signaling pathway inhibitory activity equivalent to the above-mentioned concentration of SB431542.

[0264] The culture medium used in step (1) is not particularly limited as long as it is those described in the definition section mentioned above. The culture medium used in step (1) may be a serum-containing medium or a serum-free medium. From the viewpoint of avoiding the mixing of chemically undefined components, a serum-free medium is preferably used in the present invention. In order to avoid complicated preparation, for example, it is preferred to use a serum-free medium to which a commercially available serum substitute such as KSR is added in an appropriate amount (for example, a medium to which 5% KSR, 450 μM 1-monothioglycerol and 1x chemically defined lipid concentrate are added to a 1:1 mixture of IMDM and F-12, or a medium to which 5% to 20% KSR, NEAA, pyruvic acid, and 2-mercaptoethanol are added to GMEM). Regarding the amount of KSR added to the serum-free medium, for example, in the case of human ES cells, it is generally about 1% to about 30%, preferably about 2% to about 20%.

[0265] When forming aggregates in step (1), it is preferred to first perform a dispersion operation of the pluripotent stem cells obtained in step (a). The "dispersed cells" obtained by the dispersion operation may be, for example, a state in which more than 70% are single cells and less than 30% are aggregates of 2 to 50 cells.

[0266] As dispersed cells, preferably, a state in which 80% or more are single cells and 20% or less are cell aggregates of 2 to 50 cells can be cited. Regarding dispersed cells, a state in which adhesion (e.g., surface adhesion) between cells is almost gone can be cited. In some embodiments, dispersed cells refer to a state in which cell-cell connections (e.g., adhesion connections) are almost gone.

[0267] The dispersion operation of the pluripotent stem cells obtained in step (a) may include the mechanical dispersion treatment, cell dispersion liquid treatment, and treatment with the addition of a cell protectant as described above. These treatments may be performed in combination. Preferably, the cell dispersion liquid treatment may be performed while the cell protectant is added, and then the mechanical dispersion treatment is performed.

[0268] As the cell protectant used for the treatment of adding the cell protectant, there can be listed: FGF signal transduction pathway acting substance, heparin, ROCK inhibitory substance, myosin inhibitory substance, serum, or serum substitute. As for the preferred cell protectant, there can be listed ROCK inhibitory substance. In order to inhibit the cell death of pluripotent stem cells (especially human pluripotent stem cells) induced by dispersion, an inhibitor of Rho-associated coiled-coil kinase (ROCK) can also be added from the beginning of the first step of culture. As ROCK inhibitory substances, there can be listed: Y-27632, Fasudil (HA1077), H-1152, HA-100, etc. Alternatively, a prepared cell protectant can also be used. As a prepared cell protectant, for example: RevitaCell Supplement (manufactured by Thermo Fisher Scientific), CloneR (manufactured by Stemcell Technologies).

[0269] Regarding the cell dispersion liquid used for cell dispersion treatment, there can be cited any solution containing enzymes such as trypsin, collagenase, hyaluronidase, elastase, pronase, DNA enzyme, papain, and chelating agents such as ethylenediaminetetraacetic acid. Commercially available cell dispersion liquids can also be used, for example: TrypLE Select (manufactured by Thermo Fisher Scientific), TrypLE Express (manufactured by Thermo Fisher Scientific), Accumax (manufactured by Innovative Cell Technologies).

[0270] Examples of the mechanical dispersion treatment method include a blowing treatment and a scraping operation using a spatula.

[0271] The dispersed cells were suspended in the above-mentioned medium.

[0272] Then, the suspension of dispersed pluripotent stem cells is seeded in the above-mentioned culture container, and the dispersed pluripotent stem cells are cultured under non-adhesive conditions with respect to the culture container, thereby allowing a plurality of cells to aggregate to form aggregates.

[0273] At this time, the dispersed pluripotent stem cells can be inoculated in a relatively large culture vessel such as a 10 cm plate, thereby forming the aggregate of multiple cells in one culture vessel at the same time, but from the viewpoint of the deviation of the size of each aggregate that is not easy to produce, preferably in each well of such a multi-well plate (U bottom, V bottom) of a 96-well microtiter plate, a certain amount of dispersed pluripotent stem cells are added, and when statically cultured, the cells rapidly condense, thereby forming an aggregate in each well. Aggregates can be recovered from multiple wells, thereby obtaining a uniform aggregate group.

[0274] In step (1), in order to avoid the complicated preparation of aggregates, a three-dimensional cell culture container can be used in which the culture medium of the entire plate can be replaced at one time while the aggregates are placed in each well. Examples of three-dimensional cell culture containers include PrimeSurface96 slotted well plates (manufactured by Sumitomo BakeLite Co., Ltd.).

[0275] The concentration of the pluripotent stem cells in step (1) can be appropriately set to be more uniform and to effectively form cell aggregates. For example, when a 96-well microplate is used to culture human pluripotent stem cells (such as human iPS cells obtained in step (a)) in suspension, the concentration of the pluripotent stem cells in each well is generally about 1×10 3 ~about 1×10 5 cells, preferably about 3×10 3 ~About 5×10 4 cells, more preferably about 4×10 3 ~about 2×10 4 cells, more preferably about 4×10 3 ~about 1.6×10 4 cells, particularly preferably about 8×10 3 ~About 12×10 4 The cell suspension was added to the wells and the plate was left to stand to form aggregates.

[0276] The culture conditions such as the culture temperature and CO2 concentration in step (1) can be appropriately set. 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%.

[0277] In step (1), when performing a culture medium replacement operation, examples include: an operation in which a new culture medium is added without discarding the original culture medium (culture medium addition operation), an operation in which about half the amount (about 30 to 90% of the volume of the original culture medium, for example, about 40 to 60%) of the original culture medium is discarded and about half the amount (30 to 90% of the volume of the original culture medium, for example, about 40 to 60%) of the new culture medium is added (half-amount culture medium replacement operation), and an operation in which about the entire amount (more than 90% of the volume of the original culture medium) of the original culture medium is discarded and about the entire amount (more than 90% of the volume of the original culture medium) of the new culture medium is added (full-amount culture medium replacement operation).

[0278] When adding a specific component (for example, a differentiation-inducing factor) at a specific time point, an operation can be performed, for example, by discarding about half of the original culture medium based on the calculated final concentration and adding about half of a new culture medium containing the specific component at a higher concentration than the final concentration (half-volume culture medium replacement operation).

[0279] When the concentration of the components contained in the original culture medium is reduced by dilution at a specific time point, for example, the medium replacement operation can be performed multiple times a day, preferably multiple times (e.g., 2-3 times) within 1 hour. In addition, when the concentration of the components contained in the original culture medium is reduced by dilution at a specific time point, the cells or aggregates can be transferred to another culture container.

[0280] The tool used in the medium replacement operation is not particularly limited, and examples thereof include a pipette, a micropipette (PIPETMAN), a multichannel micropipette (MULTICHANNEL PIPETMAN), a continuous dispenser, etc. For example, when a 96-well plate is used as a culture container, a multichannel micropipette can be used.

[0281] For the time of suspension culture required for forming cell aggregates, it can be appropriately determined according to the pluripotent stem cells used in a manner that allows the cells to aggregate uniformly, but in order to form uniform cell aggregates, it is preferably as short a time as possible. The step from dispersed cells to forming cell aggregates can be divided into: a step in which cells aggregate; and a step in which the aggregated cells form cell aggregates. From the moment of inoculating dispersed cells (i.e., when suspension culture begins) to the moment of cell aggregation, for example, in the case of human pluripotent stem cells (e.g., human iPS cells), it is preferably within about 24 hours, and more preferably within about 12 hours to form aggregated cells. In the step from the moment of inoculating dispersed cells (i.e., when suspension culture begins) to the step of forming cell aggregates, for example, in the case of human pluripotent stem cells (e.g., iPS cells), it is preferably within about 72 hours, and more preferably within about 48 hours to form aggregates. The time to form aggregates can be appropriately adjusted by adjusting the apparatus for cell aggregation, centrifugation conditions, etc.

[0282] The formation of cell aggregates can be judged based on the size and cell number of the aggregates, the macroscopic morphology, the microscopic morphology and its uniformity analyzed by tissue staining, the expression and uniformity of differentiation and undifferentiation markers, the expression control and synchronization of differentiation markers, the reproducibility of differentiation efficiency between aggregates, etc.

[0283] After the aggregates are formed, the aggregates can also be directly cultured. The suspension culture in step (1) is usually carried out for about 8 hours to 6 days, preferably for about 12 hours to 48 hours.

[0284] <Step (2)> and <Step (2-1)>

[0285] The second step of suspending and culturing the cell aggregates obtained in step (1) in the presence of substances acting on the BMP signal transduction pathway and substances acting on the Sonic hedgehog signal transduction pathway to obtain cell aggregates containing pituitary tissue, or the second step (1) of suspending and culturing the cell aggregates obtained in step (1) in the presence of substances acting on the BMP signal transduction pathway and substances acting on the Sonic hedgehog signal transduction pathway is described.

[0286] BMP signal transduction pathway action substances refer to substances that can enhance signal transduction mediated by BMP. As BMP signal transduction pathway action substances, for example: BMP2, BMP4 or BMP7 and other BMP proteins, GDF7 and other GDF proteins, anti-BMP receptor antibodies or BMP partial peptides, etc. BMP2 protein and BMP4 protein can be obtained from, for example, R&D Systems, BMP7 protein can be obtained from Biolegend, and GDF7 protein can be obtained from, for example, Wako Pure Chemical Industries, Ltd. The BMP signal transduction pathway action substance is preferably BMP4.

[0287] The concentration of the BMP signal transduction pathway acting substance in the culture medium can be appropriately set within the range that can achieve the above-mentioned effect. When BMP4 is used as the BMP signal transduction pathway acting substance, it is usually used at a concentration of about 1pM to about 100nM, preferably about 10pM to about 50nM, more preferably about 10pM to about 25nM, and further preferably about 10pM to about 5nM. In addition, when a BMP signal transduction pathway acting substance other than BMP4 is used, it is preferably used at a concentration that imparts the same BMP signal transduction pathway enhancing activity as the above-mentioned concentration of BMP4.

[0288] As the Shh signal transduction pathway acting substance used in step (2) or step (2-1), the same ones as exemplified in step (a) can be used. The Shh signal transduction pathway acting substances in step (a) and step (2) or step (2-1) may be the same or different, but are preferably the same.

[0289] The concentration of the Shh signal transduction pathway acting substance in the culture medium can be appropriately set within the range that can achieve the above-mentioned effect. When SAG is used as the Shh signal transduction pathway acting substance in step (2) or step (2-1), it is usually used at a concentration of about 1 nM to about 5 μM, preferably about 10 nM to about 4.5 μM, more preferably about 50 nM to about 4 μM, and even more preferably about 100 nM to about 3 μM.

[0290] The medium used in step (2) or step (2-1) is not particularly limited as long as it contains a substance acting on the BMP signal transduction pathway and a substance acting on the Shh signal transduction pathway. The medium used in step (2) or step (2-1) may be a serum-containing medium or a serum-free medium. From the viewpoint of avoiding the mixing of chemically undefined components, a serum-free medium is preferably used in the present invention. In order to avoid complicated preparation, for example, it is preferred to use a serum-free medium to which a commercially available serum substitute such as KSR is added in an appropriate amount (for example, a medium to which 5% KSR, 450 μM 1-monothioglycerol and 1x chemically defined lipid concentrate are added to a 1:1 mixture of IMDM and F-12, or a medium to which 5% to 20% KSR, NEAA, pyruvic acid, and 2-mercaptoethanol are added to GMEM). Regarding the amount of KSR added to the serum-free medium, for example, in the case of human ES cells, it is generally about 1% to about 30%, preferably about 2% to about 20%. In the second step, by suspending and culturing the aggregates of pluripotent stem cells obtained in the first step in a culture medium (preferably, a serum-free culture medium) containing BMP signal transduction pathway activating substances and Shh signal transduction pathway activating substances to form cell agglomerates, the quality of the cell agglomerates is improved, and cell agglomerates containing pituitary tissue can be efficiently produced.

[0291] In step (2) or step (2-1), the addition period of the BMP signal transduction pathway acting substance and the Shh signal transduction pathway acting substance may be the same or different. Preferably, the Shh signal transduction pathway acting substance is added at the same period or after the addition of the BMP signal transduction pathway acting substance. The addition period of the Shh signal transduction pathway acting substance is within 12 days, preferably within 10 days, and more preferably within 6 days from the addition of the BMP signal transduction pathway acting substance. In the case of an embodiment in which step (2-2) is implemented following step (2-1), the addition of the Shh signal transduction pathway acting substance may also be carried out simultaneously with the start of step (2-2) described later.

[0292] <Step (2-2)>

[0293] The second step (2) of further performing suspension culture on the cell aggregates obtained in step (2-1) in a medium containing a BMP signaling pathway inhibitory substance to obtain a cell mass containing pituitary tissue is described.

[0294] In the present invention, there is no limitation on the substance that inhibits the BMP signal transduction pathway, as long as it can inhibit the signal transduction induced by the BMP family and proteins. It can be any one of nucleic acids, proteins, and low molecular organic compounds. Examples of such substances include: substances that inhibit processing and secretion to the extracellular space, substances that directly act on BMP (e.g., protein antibodies, aptamers, etc.), substances that inhibit the expression of genes encoding BMP (e.g., antisense oligonucleotides, siRNA, etc.), substances that inhibit the binding of BMP receptors and BMPs, and substances that inhibit physiological activity caused by signal transduction via BMP receptors. Among BMP receptors, there are type I BMP receptors and type II BMP receptors. As type I BMP receptors, BMPR1A, BMPR1B, and ACVR are known, and as type II BMP receptors, TGF-beta R-II, ActR-II, ActR-IIB, BMPR2, and MISR-II are known.

[0295] Proteins known as BMP signal transduction pathway inhibitors include, for example, Noggin, Chordin, Follistatin, Gremlin, Inhibin, Twisted Gastrulation, Coco, secretory proteins belonging to the DAN family, etc. From the viewpoint of adding a BMP signal transduction pathway-acting substance to the culture medium in the above step (2-1) to more effectively inhibit the subsequent BMP signal transduction pathway, the BMP signal transduction pathway inhibitor in step (2-2) preferably contains a substance of the downstream signal transduction pathway that inhibits the secretion of extracellular BMP, such as a substance that inhibits the binding of a BMP receptor to BMP, a substance that inhibits the physiological activity caused by signal transduction using a BMP receptor, etc., and more preferably contains an inhibitor of type I BMP receptor.

[0296] As a BMP signal transduction pathway inhibitor, a compound well known to those skilled in the art can also be used. Examples of BMP signal transduction pathway inhibitors include K02288 (3-[(6-amino-5-(3,4,5-trimethoxyphenyl)-3-pyridyl]phenol, 3-[6-amino-5-(3,4,5-trimethoxyphenyl)-3-pyridyl]-phenol), Dorsomorphin (6-[4-[2-(1-piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)pyrazolo[1,5-a]pyrimidine), LDN-193189 (4-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline dihydrochloride), LDN-212854 (5 -[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline), LDN-214117 (1-(4-(6-methyl-5-(3,4,5-trimethoxyphenyl)pyridin-3-yl)phenyl)piperazine), ML347 (5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline)), DMH1 (4-(6-(4-isopropoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline), DMH2 (4-[6-[4-[2-(4-morpholinyl)ethoxy]phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline), etc. These substances can be used alone or in combination.

[0297] The BMP signaling pathway inhibitor used in the present invention is preferably a type I BMP receptor inhibitor, more preferably comprises at least one selected from the group consisting of K02288, Dorsomorphin, LDN-193189, LDN-212854, LDN-214117, ML347, DMH1 and DMH2, and further preferably comprises K02288.

[0298] The concentration of the BMP signal transduction pathway inhibitor in the culture medium can be appropriately set within the range that can achieve the above-mentioned effects. From the perspective of the formation efficiency of pituitary tissue, when K02288 is used as the BMP signal transduction pathway inhibitor in step (2-2), it is usually used at a concentration of about 1nM to about 100μM, preferably about 10nM to about 50μM, more preferably about 100nM to about 50μM, and further preferably about 500nM to about 25μM. When LDN-193189 is used as the BMP signal transduction pathway inhibitor, it is usually used at a concentration of about 1nM to about 100μM, preferably about 10nM to about 10μM, more preferably about 25nM to about 1μM, and further preferably about 100nM to about 500nM. When LDN-212854 is used as a BMP signal transduction pathway inhibitor, it is usually used at a concentration of about 1 nM to about 100 μM, preferably about 10 nM to about 10 μM, more preferably about 25 nM to about 5 μM, and even more preferably about 250 nM to about 3 μM. When ML-347 is used as a BMP signal transduction pathway inhibitor, it is usually used at a concentration of about 1 nM to about 100 μM, preferably about 10 nM to about 50 μM, more preferably about 100 nM to about 50 μM, and even more preferably about 1 μM to about 25 μM. When DMH2 is used as a BMP signal transduction pathway inhibitor, it is usually used at a concentration of about 1 nM to about 100 μM, preferably about 10 nM to about 10 μM, more preferably about 25 nM to about 5 μM, and even more preferably about 50 nM to about 3 μM. When a BMP signaling pathway inhibitory substance other than K02288 is used, it is preferably used at a concentration that imparts a BMP signaling pathway inhibitory activity equivalent to that of K02288 at the above concentration.

[0299] The medium used in step (2-2) is not particularly limited as long as it contains a BMP signal transduction pathway inhibitor. The medium used in step (2-2) is also preferably a medium common to step (1) or the entire step (2-1). The medium used in step (2-2) may be a serum-containing medium or a serum-free medium. From the viewpoint of avoiding the mixing of chemically undefined components, a serum-free medium is preferably used in the present invention. In order to avoid complicated preparation, for example, it is preferred to use a serum-free medium to which a commercially available serum substitute such as KSR is added in an appropriate amount (for example, a medium to which 5% KSR, 450 μM 1-monothioglycerol and 1x chemically defined lipid concentrate are added to a 1:1 mixture of IMDM and F-12, or a medium to which 5% to 20% KSR, NEAA, pyruvic acid, and 2-mercaptoethanol are added to GMEM). Regarding the amount of KSR added to the serum-free medium, for example, in the case of human ES cells, it is generally about 1% to about 30%, preferably about 2% to about 20%.

[0300] In step (2-2), it is also preferred to add a Shh signal transduction pathway agent to the culture medium following step (2-1). As the Shh signal transduction pathway agent, the same ones as those exemplified in step (a) can be used. The Shh signal transduction pathway agents in steps (a), (2-1) and (2-2) may be the same or different, but are preferably the same.

[0301] The concentration of the Shh signal transduction pathway agent in the culture medium can be appropriately set within the range that can achieve the above-mentioned effects. When SAG is used as the Shh signal transduction pathway agent in step (2-2), it is usually used at a concentration of about 1nM to about 5μM, preferably about 10nM to about 4.5μM, more preferably about 50nM to about 4μM, and further preferably about 100nM to about 3μM. When the Shh signal transduction pathway agent is added after the BMP signal transduction pathway agent is added in step (2-1), the addition of the Shh signal transduction pathway agent can also be carried out simultaneously with the start of step (2-2).

[0302] In steps (2), (2-1) and (2-2), the suspension culture is preferably further performed in the presence of a TGFβ signal transduction pathway inhibitory substance.

[0303] As the TGFβ signaling pathway inhibitory substances used in steps (2), (2-1) and (2-2), the same substances as those exemplified in step (a) can be used. The TGFβ signaling pathway inhibitory substances in steps (a), (1), (2), (2-1) and (2-2) may be the same or different, but are preferably the same.

[0304] The concentration of the TGFβ signaling pathway inhibitor in the culture medium can be appropriately set within the range that can achieve the above-mentioned effect. When SB431542 is used as the TGFβ signaling pathway inhibitor in steps (2), (2-1) and (2-2), it is usually used at a concentration of about 1 nM to about 100 μM, preferably about 10 nM to about 50 μM, more preferably about 100 nM to about 50 μM, and further preferably about 500 nM to about 10 μM. In addition, when a TGFβ signaling pathway inhibitor other than SB431542 is used, it is preferably used at a concentration that imparts TGFβ signaling pathway inhibitory activity equivalent to that of SB431542 at the above-mentioned concentration.

[0305] In the present invention, it is preferred that any one or more of steps (1), (2), (2-1) and (2-2) be performed in the presence of a substance acting on the FGF signal transduction pathway. The substance acting on the FGF signal transduction pathway is not particularly limited, as long as it is a substance that can enhance the signal transduction pathway mediated by FGF (fibroblast growth factor). Examples of substances acting on the FGF signal transduction pathway include FGF proteins such as FGF1, FGF2 (also called bFGF), and FGF8, anti-FGF receptor antibodies, and FGF partial peptides. These substances can be used alone or in combination.

[0306] FGF2 protein and FGF8 protein can be obtained from, for example, Fujifilm Wako Pure Chemical Industries, Ltd. The substance acting on the FGF signal transduction pathway preferably comprises at least one selected from the group consisting of FGF2 and FGF8, and variants thereof, more preferably comprises FGF2, and further preferably comprises recombinant human FGF2.

[0307] The concentration of the FGF signal transduction pathway acting substance in the culture medium can be appropriately set within the range that can achieve the above-mentioned effect. From the viewpoint of promoting the formation of the epithelial structure of the nerve tissue part, differentiation to the basement, and the survival and proliferation of cells, when FGF2 is used as the FGF signal transduction pathway acting substance, it is usually used at a concentration of about 1pg / ml to about 100μg / ml, preferably about 10pg / ml to about 50μg / ml, more preferably about 100pg / ml to about 10μg / ml, further preferably about 500pg / ml to about 1μg / ml, and most preferably about 1ng / ml to about 200ng / ml. In addition, when using an FGF signal transduction pathway acting substance other than FGF2, it is preferably used at a concentration that imparts an FGF signal transduction pathway enhancing activity equivalent to the above-mentioned concentration of FGF2.

[0308] In order to maintain the activity of the FGF protein in the culture medium, it is also preferred to add heparin or heparan sulfate to the culture medium containing the FGF protein. Heparin can be obtained as a sodium salt from, for example, Fujifilm Wako Pure Chemical Industries, Ltd. The concentration of heparin or heparan sulfate in the culture medium can be appropriately set within the range that can achieve the above-mentioned effect. The concentration of heparin sodium in the culture medium is usually about 1 ng / ml to about 100 mg / ml, preferably about 10 ng / ml to about 50 mg / ml, more preferably about 100 ng / ml to about 10 mg / ml, further preferably about 500 ng / ml to about 1 mg / ml, and most preferably about 1 μg / ml to about 200 μg / ml. When heparan sulfate is used, it is preferably a concentration that has the same activity of protecting the FGF protein as the above-mentioned concentration of heparin. For the purpose of maintaining the activity of FGF protein in a cell culture environment such as 37°C, it is also preferred to use FGF2 sustained-release beads such as FGF variants such as thermostable FGF2 described in U.S. Pat. No. 8,772,460B2, or StemBeads FGF2 in which FGF2 is bound to a biodegradable polymer. Thermostable FGF2 can be obtained from, for example, HumanZyme. StemBeads FGF2 can be obtained from, for example, StemCulture.

[0309] In the present invention, from the viewpoint of promoting the survival and proliferation of nervous system cells or tissues in the aggregate, any one or more of steps (1), (2), (2-1) and (2-2) may be performed in the presence of a substance acting on the Wnt signal transduction pathway. As described above, examples of the Wnt signal transduction pathway include the canonical Wnt pathway, the non-canonical Wnt pathway, and the like. Examples of the non-canonical Wnt pathway include the Planar Cell Polarity (PCP) pathway, the Wnt / Calcium pathway, the Wnt-RAP1 pathway, the Wnt-Ror2 pathway, the Wnt-PKA pathway, the Wnt-GSK3MT pathway, the Wnt-aPKC pathway, the Wnt-RYK pathway, the Wnt-mTOR pathway, and the like.

[0310] In the present invention, the Wnt signal transduction pathway acting substance is not limited, as long as it can activate the signal transduction induced by the Wnt family and protein. It can be any one of nucleic acids, proteins, and low molecular organic compounds. As the substance, for example, substances that promote the autosecretion of Wnt, substances that stabilize Wnt and inhibit decomposition, recombinant proteins of Wnt, partial sequence peptides of Wnt and their derivatives, derivatives, substances that activate Wnt receptors, substances that activate the intracellular signal transduction mechanism of Wnt, intracellular signal transduction factors of Wnt and their variants (β-catenin S33Y, etc.), substances that activate gene expression downstream of the Wnt response sequence, etc. Proteins known as Wnt signal transduction pathway acting substances include Wnt, R-Spondin, etc.

[0311] As the Wnt signal transduction pathway acting substance, a compound well known to those skilled in the art can be used. Examples of compounds having activity as a Wnt signal transduction pathway acting substance include lithium chloride, AMBMP hydrochloride, SGC AAK1 1, Foxy 5, CHIR99021, CHIR98014, TWS119, SB216763, SB415286, BIO, AZD2858, AZD1080, AR-A014418, TDZD-8, LY2090314, IM-12, Indirubin, Bikinin, A1070722, 3F8, Kenpakone, 10Z-Hymenialdisine, Indirubin-3′-oxime, NSC 693868, TC-G24, TCS 2002, TCS 21311, CP21R7, BML-284, SKL2001, WAY262611, IIIC3a, methyl vanillate, IQ-1 and their derivatives, etc.

[0312] In the present invention, although a Wnt signal transduction pathway inhibitory substance is added in step (1), a Wnt signal transduction pathway acting substance and a Wnt signal transduction pathway inhibitory substance having different action points can be used in combination, thereby activating or inhibiting only a specific pathway among the above-mentioned multiple Wnt signal transduction pathways. In the present invention, the Wnt signal transduction pathway acting substance is preferably a substance that acts on a factor downstream of the Wnt signal transduction pathway inhibitory substance added by step (1). In the present invention, the added Wnt signal transduction pathway acting substance is also preferably a substance that activates the canonical Wnt pathway, and is further preferably a substance that activates the Wnt signal transduction pathway by inhibiting and stabilizing the degradation of β-catenin, a Wnt signal transduction factor in the cell. As substances that have an inhibitory and stabilizing effect on the degradation of β-catenin, for example, GSK3 inhibitors, BML-284, SKL2001, etc. can be cited. The added Wnt signal transduction pathway acting substance is also preferably a substance that promotes and activates β-catenin responsive transcription (β-catenin responsive transcription: CRT).

[0313] As a preferred combination of Wnt signal transduction pathway acting substances and Wnt signal transduction pathway inhibitory substances in the present invention, GSK3 inhibitors and PORCN inhibitors can be cited. When a GSK3 inhibitor and a PORCN inhibitor are used in combination, the canonical Wnt pathway is activated and the non-canonical Wnt pathway is inhibited. When a GSK3 inhibitor and a PORCN inhibitor are used in combination, the preferred GSK3 inhibitor in the present invention includes at least one selected from the group consisting of CHIR99021, CHIR98014, SB216763, SB415286, and BIO, and more preferably includes CHIR99021. When a GSK3 inhibitor and a PORCN inhibitor are used in combination, the preferred PORCN inhibitor in the present invention includes at least one selected from the group consisting of IWP-2, IWP-3, IWP-4, IWP-L6, IWP-12, LGK-974, ETC-159, GNF-6231, and Wnt-C59, and more preferably includes IWP-2. In addition, GSK3 inhibitors and KY02111 and its derivatives can be used in combination. For GSK3 inhibitors, substances that have different mechanisms of action and have inhibitory and stabilizing effects on the degradation of β-catenin include BML-284 and SKL2001. These compounds and their derivatives can also be used in combination with PORCN inhibitors, KY02111 and its derivatives.

[0314] The concentration of the Wnt signal transduction pathway agent in the culture medium can be appropriately set within the range that can achieve the above-mentioned effect. From the viewpoint of promoting the survival and proliferation of nervous system cells or tissues in the aggregate, when CHIR99021 is used as a Wnt signal transduction pathway agent, it is usually used at a concentration of about 10pM to about 10mM, preferably about 100pM to about 1mM, more preferably about 1nM to about 100μM, further preferably about 10nM to about 30μM, and most preferably about 100nM to about 3μM. In addition, when using a Wnt signal transduction pathway agent other than CHIR99021, it is preferably used at a concentration that imparts Wnt signal transduction pathway enhancing activity equivalent to the above-mentioned concentration of CHIR99021.

[0315] In the present invention, in any one or more of steps (1), (2), (2-1) and (2-2), from the viewpoint of inhibiting cell death and promoting cell proliferation, it is also preferred to further add a growth factor to the culture medium. The type of growth factor added is not particularly limited as long as the above-mentioned purpose can be achieved. Examples of growth factors for the above-mentioned purpose include epidermal growth factor (EGF), insulin-like growth factor (IGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3, neurotrophin 4 / 5, ciliary neurotrophic factor (CNTF), vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), hepatocyte growth factor (HGF), etc. These growth factors may be added at concentrations that can achieve the above-mentioned purpose.

[0316] In any one or more of steps (1), (2), (2-1) and (2-2) of the present invention, from the viewpoint of inhibiting cell death and promoting cell proliferation, it is also preferred to add a platelet-derived growth factor (PDGF) or other platelet-derived growth factor receptor-acting substance to the culture medium. There are four genes of A, B, C, and D in PDGF, which form homodimers of a homodimer or a specific combination of AA, AB, BB, CC, and DD and function as a ligand. There are two genes of α and β in PDGF receptor, which form homodimers of a homodimer or a specific combination of αα, αβ, and ββ and function as a receptor. Among them, PDGFRβ is usually expressed in the non-neural ectoderm containing the basement membrane, and the platelet-derived growth factor receptor-acting substance used in the present invention preferably has an effect on PDGFRββ or PDGFRαβ, and more preferably comprises any one of PDGF-AB, PDGF-BB, PDGF-CC, and PDGF-DD, and even more preferably comprises any one of PDGF-BB and PDGF-CC. PDGF-AB, PDGF-BB, PDGF-CC, and PDGF-DD can be obtained from R&D Systems, GenScript, and the like in the form of recombinant proteins.

[0317] In any one or more of steps (1), (2), (2-1) and (2-2) of the present invention, from the viewpoint of improving the preparation efficiency of pituitary tissue, it is also preferred to add a compound that promotes differentiation to the substrate area. As compounds having the above-mentioned effects, for example, BRL-54443, phenanthroline, and Parthenolide described in U.S. Patent No. US20160326491A1 can be cited. When BRL-54443 is used as a compound that promotes differentiation to the substrate area, it is usually used at a concentration of 10nM to 100μM, when phenanthroline is used, it is usually used at a concentration of 10nM to 100μM, and when Parthenolide is used, it is usually used at a concentration of 10nM to 100μM.

[0318] By culturing the prepared cell mass for a long period of time in any of steps (2) and (2-2) of the present invention, a more mature cell mass containing cells that have progressed in differentiation can also be obtained. Such a culture is also referred to as a maturation culture. In the maturation culture process of steps (2) and (2-2), a serum-free medium can be preferably used from the viewpoint of avoiding the mixing of chemically undefined components. In the maturation culture of steps (2) and (2-2), the culture medium used can also be the same as that used in steps (2) and (2-2). In addition, a known culture medium used for culturing neural cells, etc. can also be used, for example, a culture medium (N2B27 culture medium) in which 0.5% N2 supplement and 1% B27 supplement are added to a 1:1 mixture of DMEM / F-12 culture medium and Neurobasal culture medium, a culture medium (StemPro NSC SFM culture medium) in which StemProNSC SFM supplement is added to Knockout DMEM / F-12 culture medium, etc. When performing the maturation culture, the culture can also be continued in the culture vessel used at the beginning of the suspension culture. Alternatively, the cell mass may be transferred to a new culture container, such as a suspension culture dish or a suspension culture flask, and culture may be performed.

[0319] In any of step (2), step (2-1) and step (2-2) of the present invention, it is also preferred to culture in a high oxygen atmosphere from the viewpoint of inhibiting cell death and promoting cell proliferation. The high oxygen condition during the culture process can be achieved by, for example, connecting an incubator for culturing cells to an oxygen pump and artificially supplying oxygen. The oxygen concentration used for the target is generally 25% to 80%, more preferably 30% to 60%.

[0320] In any of the steps (2), (2-1) and (2-2) of the present invention, from the viewpoint of increasing the oxygen supply in the culture medium for culturing the cell mass, a culture medium with high gas exchange efficiency may be used. Examples of such culture medium include a cell culture dish having a gas permeable membrane on the bottom surface of the plate, a Lumox plate (manufactured by SARSTEDT Co., Ltd.), a VECELL 96-well plate (manufactured by Vessel Co., Ltd.), and the like.

[0321] In any of the steps (2), (2-1) and (2-2) of the present invention, it is also preferred to treat the cell mass with adrenocortical hormones by adding adrenocortical hormones to the culture medium. Adrenocortical hormones can also be added in step (1). Treatment with adrenocortical hormones can promote differentiation from the pituitary placode and / or Rathke's capsule into pituitary hormone-producing cells other than ACTH-producing cells (i.e., GH-producing cells, PRL-producing cells, TSH-producing cells, LH-producing cells, FSH-producing cells, etc.). As adrenocortical hormones, natural glucocorticoids such as hydroxycortisone, cortisone acetate, and fludrocortisone acetate; artificially synthesized glucocorticoids such as dexamethasone, betamethasone, prednisolone, methylprednisolone, and triamcinolone, etc., but are not limited to these.

[0322] The concentration of adrenocortical hormones in the culture medium is not particularly limited, as long as it can promote differentiation from the pituitary placode and / or Rathke's capsule into pituitary hormone-producing cells (but ACTH-producing cells are excluded), and in addition, it can be appropriately set according to the type of adrenocortical hormones. For example, in the case of hydroxycortisone, it is usually more than 100ng / ml, preferably more than 1μg / ml. There is no adverse effect on the differentiation of pituitary hormone-producing cells (but ACTH-producing cells are excluded), and there is no specific upper limit for the concentration of hydroxycortisone. From the perspective of culture cost, it is usually less than 1000μg / ml, preferably less than 100μg / ml. In one embodiment, the concentration of hydroxycortisone in the culture medium is usually 100ng / ml~1000μg / ml, preferably 1~100μg / ml. When dexamethasone is used as adrenocortical hormones, its concentration in the culture medium can be about 1 / 25 of hydroxycortisone.

[0323] In steps (2), (2-1) and (2-2) of the present invention, the time for adding adrenocortical hormones to the culture medium is not particularly limited, as long as differentiation from the pituitary placode and / or Rathke's capsule into pituitary hormone-producing cells (but excluding ACTH-producing cells) can be promoted. Adrenocortical hormones can be added to the culture medium at the beginning of the culture step of steps (2), (2-1) and (2-2) of the present invention, or adrenocortical hormones can be added to the culture medium after culturing in a culture medium without adrenocortical hormones for a certain period of time after the culture step starts. Preferably, adrenocortical hormones are added to the culture medium at a stage when the appearance of ACTH-producing cells is confirmed in the cell aggregates after starting steps (2), (2-1) and (2-2) of the present invention. That is, before the appearance of ACTH-producing cells is confirmed in the cell aggregates, the cell aggregates are cultured in a culture medium without adrenocortical hormones, and after the appearance of ACTH-producing cells is confirmed, the third culture step is continued in a culture medium containing adrenocortical hormones. The appearance of ACTH-producing cells can be confirmed by immunohistological staining using antibodies against ACTH. When human pluripotent stem cells are used, the appearance of ACTH-producing cells can be expected as long as it is 37 days after the culture steps of steps (2), (2-1) and (2-2) of the present invention. Therefore, in one embodiment, adrenocortical hormones are added to the culture medium 37 days after the culture steps of steps (2), (2-1) and (2-2) of the present invention.

[0324] There is no particular limitation on the period when the cell aggregation mass is treated with adrenocortical hormones, as long as differentiation into pituitary hormone producing cells (but ACTH producing cells are excluded) can be promoted from pituitary base plate and / or Rathke capsule, usually, compared with the adrenocortical hormone non-treated group, after confirming that differentiation into pituitary hormone producing cells (but ACTH producing cells are excluded) is promoted in the adrenocortical hormone treated group, the cell aggregation mass is treated in adrenocortical hormones. The treatment period is usually more than 7 days, preferably more than 12 days. The upper limit of the treatment period is not particularly limited, compared with the adrenocortical hormone non-treated group, in the adrenocortical hormone treated group, adrenocortical hormones can also be removed from the culture medium at the stage of promotion confirmed to differentiate into pituitary hormone producing cells (but ACTH producing cells are excluded).

[0325] Furthermore, the addition of adrenocortical hormones to the culture medium acts inhibitorily on the differentiation induction of ACTH-producing cells through feedback inhibition.

[0326] In the present invention, the Notch signal transduction pathway refers to a signal transduction pathway activated by direct interaction between the Notch protein, which is a receptor expressed on the cell membrane, and the Notch ligand (Delta, Jagged, etc.) expressed on the membrane of an adjacent cell. In cells with Notch signaling, the Notch protein is gradually processed, and the intracellular domain cut out on the membrane is transported to the nucleus to control the expression of downstream genes.

[0327] The culture medium used in steps (2), (2-1) and (2-2) of the present invention preferably contains a Notch signal transduction pathway inhibitor (Notch signal inhibitor). The Notch signal inhibitor promotes differentiation from the pituitary placode and / or Rathke's capsule into pituitary hormone-producing cells (particularly ACTH-producing cells). Using the Notch signal inhibitor, the expression of the transcription factor Tbx19, which controls the upstream production of ACTH, is increased.

[0328] A Notch signaling inhibitor may also be contained in the culture medium used in step (1).

[0329] Notch signal inhibitors are not particularly limited as long as they can inhibit signal transduction mediated by Notch. It can be any of nucleic acids, proteins, and low molecular weight organic compounds. As the substance, for example: functionally deficient Notch receptors and ligands, substances that inhibit Notch processing (S1 cleavage), substances that inhibit sugar chain modification of Notch and Notch ligands, substances that inhibit cell membrane transfer, substances that inhibit processing (S2 cleavage, S3 cleavage) of the intracellular domain (NICD) of Notch (γ secretase inhibitors), substances that decompose NICD, substances that inhibit NICD-dependent transcription, etc.

[0330] As Notch signal inhibitors, compounds well known to those skilled in the art can also be used. As compounds having activity as Notch signal inhibitors, for example, DAPT (N-[N-(3,5-difluorophenylacetyl)-l-alanyl]-S-phenylglycine tert-butyl ester), DBZ, MDL28170, etc., or substances described in Onco Targets Ther. 2013; 6: 943-955., etc. Among them, DAPT is preferred.

[0331] The concentration of the Notch signaling inhibitor in the culture medium is not particularly limited, as long as it is a concentration that can promote differentiation from the pituitary placode and / or Rathke's capsule into pituitary hormone-producing cells (especially ACTH-producing cells). For example, in the case of DAPT, it is usually 0.1 μM or more, preferably 1 μM or more. There is no specific upper limit for the DAPT concentration, as long as it has no adverse effect on differentiation into pituitary hormone-producing cells. From the perspective of culture cost, it is usually 1000 μM or less, preferably 100 μM or less. In one embodiment, the DAPT concentration in the culture medium is usually 0.1 to 1000 μM, preferably 1 to 100 μM (for example, 10 μM).

[0332] 3. Cell clumps containing pituitary tissue

[0333] The present invention provides a cell mass comprising 1) nervous system cells or neural tissue, 2) pituitary tissue, and 3) mesenchymal cells. Alternatively, a cell mass comprising 1) nervous system cells or neural tissue, 2) pituitary tissue, and 4) neural crest cells or cells derived from the neural crest is provided. Hereinafter, it is also referred to as the cell mass of the present invention. The cell mass of the present invention can be preferably prepared by the preparation method of the present invention described above.

[0334] 1) The nervous system cells or neural tissues in the cell mass of the present invention are preferably cells or tissues of the central nervous system, or precursor tissues thereof, and examples of the cells or tissues of the central nervous system include retina, cerebral cortex, diencephalon (e.g., hypothalamus), and cells derived from these tissues, more preferably diencephalon or precursor tissues thereof, and further preferably diencephalon or precursor tissues thereof having a ventricle-like structure in the tissue. 1) The nervous system cells or neural tissues are, for example, neuroepithelial tissues positive for N-cadherin.

[0335] In the cell mass of the present invention, 2) pituitary tissue is preferably formed continuously with non-neuroepithelial tissue, and more preferably, at least one of 1) nervous system cells or neural tissue and 3) mesenchymal cells is covered by non-neuroepithelial tissue and pituitary tissue, or at least one of 1) nervous system cells or neural tissue and 4) neural crest cells or cells derived from neural crest is covered by non-neuroepithelial tissue and pituitary tissue.

[0336] The above-mentioned non-neuroepithelial tissue is also preferably oral epithelium or its precursor tissue. The pituitary tissue preferably comprises pituitary hormone producing cells, preferably comprises pituitary tissue stem cells, preferably comprises follicular stellate cells, and further preferably comprises all of pituitary hormone producing cells, pituitary tissue stem cells, and follicular stellate cells. As pituitary hormone producing cells, at least one of the group consisting of, for example, growth hormone (GH) producing cells, prolactin (PRL) producing cells, and adrenocorticotropic hormone (ACTH) producing cells can be cited.

[0337] It is also preferred that a pituitary microhabitat is formed in the pituitary tissue, and the pituitary microhabitat is also preferably an MCL microhabitat-like structure around the residual cavity remaining between the anterior lobe and the middle lobe of the pituitary, and the pituitary microhabitat is also preferably a parenchymal layer microhabitat-like structure, and it is further preferred to include both the MCL microhabitat-like structure and the parenchymal layer microhabitat-like structure.

[0338] 3) Mesenchymal cells in the cell aggregate of the present invention are preferably head mesenchymal cells.

[0339] 3) Mesenchymal cells are preferably present between the non-neuroepithelial tissue covering the surface of the cell mass and 1) nervous system cells or neural tissue present inside the cell mass.

[0340] 4) Neural crest cells or cells derived from the neural crest in the cell mass of the present invention are preferably head neural crest cells or cells derived from the head neural crest.

[0341] 4) Neural crest cells or cells derived from neural crest preferably exist between non-neural epithelial tissue covering the surface of the cell mass and 1) nervous system cells or neural tissue present inside the cell mass. 4) Neural crest cells or cells derived from neural crest are also preferably 3) mesenchymal cells.

[0342] The cell mass of the present invention forms, for example, ventricle-like vacuoles inside 1) the neuroepithelial tissue, and the surface of 1) the neuroepithelial tissue in contact with the vacuoles is the apical surface positive for PKC-zeta.

[0343] 3) The mesenchymal cells contained in the cell aggregate of the present invention express at least one mesenchymal cell marker selected from the group consisting of, for example, nestin, vimentin, cadherin-11, laminin, CD44, CD90 and CD105.

[0344] 3) The neural crest cells or cells derived from the neural crest contained in the cell mass of the present invention express at least one neural crest cell marker selected from the group consisting of, for example, Nestin, Sox10, Slug, Snail, Pax3, Zic1, FoxD3, p75 NTR, HNK-1, CHD7, Numb, and Ascll.

[0345] The non-neuroepithelial tissue that may be included in the present invention expresses at least one non-neuroepithelial tissue marker selected from the group consisting of, for example, cytokeratin, E-cadherin and EpCAM.

[0346] The pituitary tissue stem cells that may be contained in the cell mass of the present invention express at least one pituitary stem cell marker selected from the group consisting of, for example, Sox2, Sox9, E-cadherin, nestin, S100β, GFRα2, Prop1, CD133, β-catenin, Klf4, Oct4, Pax6, coxsackievirus-adenovirus co-receptor (CXADR), PRRX1 / 2, Ephrin-B2 and ACE.

[0347] 4. Preparation of Pituitary Tissue

[0348] The present invention provides a method for preparing pituitary tissue, which comprises the following steps (1) to (3).

[0349] (1) In the first step, pluripotent stem cells are suspended and cultured in the presence of a Wnt signal transduction pathway inhibitor to form cell aggregates;

[0350] (2) In the second step, the aggregates obtained in the first step are suspended and cultured in the presence of substances acting on the BMP signal transduction pathway and substances acting on the Sonic hedgehog signal transduction pathway to obtain cell aggregates containing pituitary tissue;

[0351] (3) In the third step, the pituitary tissue is recovered from the cell mass obtained in the second step.

[0352] The first step and the second step can be carried out in the same manner as the first step and the second step of the above-mentioned "2. Method for preparing a cell mass containing pituitary tissue". In addition, step a can be carried out before the first step as needed. Alternatively, the second step (1) and the second step (2) can be carried out instead of the second step as needed.

[0353] The third step of recovering the pituitary tissue from the cell mass obtained in the second step or the second step (2) is usually performed by peeling off and recovering the pituitary tissue formed on the outside (Rathke's capsule part) of the cell mass using tweezers or the like under microscopic observation. The pituitary tissue can be identified as a thin translucent epithelium present on the surface of the obtained cell mass, as described, for example, in Nature communications, 2016, 7. As a method for recovering the pituitary tissue in the third step, freeze-thaw, preferably slow freezing, can also be used. In this method, a cell mass having pituitary tissue on the outside and mesenchymal nerves and neuroepithelial tissue on the inside is frozen and thawed, thereby peeling off the outer pituitary tissue from the cell mass without performing physical treatment.

[0354] 5. Toxicity and efficacy evaluation reagents and toxicity and efficacy evaluation methods

[0355] The cell mass of the present invention, the cell mass prepared by the preparation method of the present invention, or the tissue recovered from the cell mass can be pituitary tissue. Therefore, a reagent for evaluating the toxicity and efficacy of a test substance can be provided, which contains the cell mass of the present invention, the cell mass prepared by the preparation method of the present invention, or the pituitary tissue recovered from the cell mass.

[0356] In addition, the present invention can provide a method for evaluating toxicity and drug efficacy using the above-mentioned cell aggregate or pituitary tissue collected from the cell aggregate.

[0357] Examples of methods for evaluating the toxicity and efficacy of a test substance include the steps of contacting a cell mass or a pituitary tissue recovered from the cell mass with a test substance, and

[0358] The step of examining the effect of the test substance on the cell mass or pituitary tissue.

[0359] 6. Therapeutic drugs and treatment methods of diseases

[0360] A therapeutic drug for a disease caused by a pituitary disorder can be provided, which contains the cell aggregate of the present invention, the cell aggregate produced by the production method of the present invention, or the pituitary tissue recovered from the cell aggregate.

[0361] Examples of therapeutic agents for diseases caused by pituitary disorders include transplants containing a suspension comprising the cell aggregate of the present invention or the cell aggregate produced by the production method of the present invention.

[0362] The suspension includes, for example, a suspension of cell aggregates in artificial tears or physiological saline. The suspension may also contain non-neuroepithelial cells separated from the cell aggregates, and may contain factors that promote cell adhesion, such as extracellular matrix, hyaluronic acid, and the like.

[0363] Instead of the cell mass, pituitary tissue recovered from the cell mass may be used.

[0364] Furthermore, a method for treating a disease caused by a pituitary disorder can be provided, comprising the step of transplanting an effective amount of pituitary tissue from the cell aggregate of the present invention or the cell aggregate prepared by the preparation method of the present invention to a subject in need of transplantation.

[0365] The above-mentioned disease caused by pituitary disorder can be a disease of an animal caused by pituitary disorder, or a disease of a non-human animal caused by pituitary disorder. Specifically, the disease caused by pituitary disorder includes panhypopituitarism, pituitary dwarfism, adrenocortical insufficiency, partial hypopituitarism, simple anterior pituitary hormone deficiency, etc.

[0366] The present invention is further described in detail below with reference to the examples, but the scope of the present invention is not limited thereto. In addition, the reagents and materials used are not particularly limited, as long as they can be obtained commercially.

[0367] Example

[0368] Example 1: Cell Aggregates Containing Pituitary Tissue Prepared from Human ES Cells

[0369] Human ES cells (KhES-1 strain, obtained from Kyoto University) were cultured under feeder-free conditions according to the method described in Scientific Reports, 4, 3594 (2014). StemFit medium was used as a feeder-free medium, and laminin 511-E8 was used as a feeder-free framework.

[0370] As a specific maintenance culture operation, the sub-confluent human ES cells (KhES-1 strain) were first washed with PBS, and then treated with Accumax (manufactured by Innovative Cell Technologies), and then StemFit medium was added and the cells were peeled off from the surface of the culture dish using a cell scraper, and dispersed into single cells by blowing. After that, the human ES cells dispersed into single cells were inoculated in a plastic culture dish coated with laminin 511-E8, and feeder-free cell culture was carried out using StemFit medium in the presence of Y27632 (ROCK inhibitor, manufactured by Wako Pure Chemical Industries, Ltd., 10 μM). As the above-mentioned plastic culture dish, a 6-well plate (manufactured by Corning, for cell culture, culture area 9.5 cm) was used. 2 ), the number of inoculated cells of the human ES cells dispersed into single cells was set to 1.2×10 4 . One day after inoculation, the total amount of culture medium was replaced with StemFit medium that does not contain Y27632. Then, the total amount of culture medium was replaced with StemFit medium that does not contain Y27632 once every 1-2 days. Thereafter, culture was continued until subconfluence (60% of the culture area is covered with cells) was reached 7 days after inoculation. When the cultured cells were used to induce differentiation, SB-431542 (TGF-β signaling pathway inhibitor, manufactured by Wako Pure Chemical Industries, Ltd., 5 μM) and SAG (Shh signaling pathway activator, manufactured by Enzo Life Sciences, 300 nM) were added at the same time as the medium was replaced with Stemfit medium 6 days after inoculation.

[0371] The subconfluent human ES cells prepared as described above were washed with PBS, treated with an enzyme using Accumax, and then serum-free medium for differentiation induction was added and cells were peeled off from the surface of the culture dish using a cell scraper and dispersed into single cells by blowing. Subsequently, the human ES cells dispersed into single cells were plated in a non-cell-adhesive 96-well culture plate (PrimeSurface96V bottom plate, manufactured by Sumitomo BakeLite Co., Ltd.) at a density of 1×10 4 The cells were suspended in 100 μl of serum-free medium and cultured in suspension at 37°C and 5% CO2. The serum-free medium (gfCDM+KSR) used at this time was a 1:1 mixture of F-12+Glutamax medium (Thermo Fisher Scientific) and IMDM+Glutamax medium (Thermo Fisher Scientific) supplemented with 5% Knockout serum replacement (Thermo Fisher Scientific), 450 μM 1-monothioglycerol (Wako Pure Chemical Industries, Ltd.), 1x chemically defined lipid concentrate (Thermo Fisher Scientific), and 50 units / ml penicillin-50 μg / ml streptomycin (Nacalai Tesque). At the start of suspension culture (day 0 after the start of suspension culture, start of step 1), Y27632 (final concentration 20 μM), IWP-2 (Wnt signaling pathway inhibitor, manufactured by Tocris Bioscience, 2 μM), and SB-431542 (TGFβ signaling pathway inhibitor, manufactured by Wako Pure Chemical Industries, Ltd., 1 μM) were added to the above-mentioned serum-free medium.

[0372] On the second day after the start of suspension culture, 100 μl of serum-free medium containing IWP-2, SB-431542, BMP4 (BMP signal transduction pathway activator), and SAG without Y27632 was added to each well. Regarding BMP4, 3 nM was added to the added medium so that the final concentration in the well was 1.5 nM. Regarding SAG, 4 μM was added to the added medium so that the final concentration in the well was 2 μM. Thereafter, on the 6th, 10th, 13th, 17th, 21st, and 24th days after the start of suspension culture, the medium was replaced by half with serum-free medium containing IWP-2, SB-431542, and SAG without Y27632 and BMP4.

[0373] On the 28th day after the start of suspension culture, the cell mass was recovered into a dish and bright field observation was performed using an inverted microscope (manufactured by Keyence, BIOREVO) (Figure 1A-B). The scale bar on the lower right of Figure 1A represents 1000 μm, and the scale bar on the lower right of Figure 1B represents 200 μm. As a result, by the 28th day of suspension culture of human ES cells by the above-mentioned induced differentiation method, a cell mass with a diameter of about 1 mm containing neural tissue and non-neural epithelial tissue was formed. An epithelial-like structure with a thickness of about 30 μm was formed on the surface of the cell mass.

[0374] The cell aggregates on day 28 after the start of the suspension culture were fixed with 4% paraformaldehyde at room temperature for 15 minutes, immersed in 20% sucrose / PBS at 4°C overnight, cryoprotected, and then prepared into frozen sections. These frozen sections were analyzed using an anti-Lhx3 antibody (Merck Millipore, rabbit) as an early marker of the pituitary, an anti-Emx2 antibody (R&D Systems, sheep) as a marker of the basal plate, an anti-Pitx1 antibody (Santa Cruz Biotechnology, goat) as a marker of the oral epithelium and pituitary basal plate, an anti-Nkx 2.1 antibody (Merck Millipore, mouse) as a marker of the ventral diencephalon, an anti-Tbx3 antibody (R&D Systems, goat), an anti-Sox3 antibody (R&D Systems, goat), an anti-CD326 / EpCAM antibody (Acris Antibodies, mouse) as a marker of non-neuroepithelial tissues, an anti-pan-cytokeratin antibody (Sigma Aldrich, mouse), an anti-nestin antibody (Spring Bioscience, rabbit) as a marker of undifferentiated nerves, neural crest and mesenchymal cells, and an anti-laminin antibody (Kyowa Pharma Chemical Co., Ltd., mouse) as a marker of the basement membrane. Co., Ltd., mouse), anti-aPKC antibody (Santa Cruz Biotechnology, Inc., rabbit) for apical surface labeling, and anti-βIII tubulin (Tuj1) antibody (Sigma Aldrich, Inc., mouse) for neuronal cell labeling were used for fluorescent immunostaining. As fluorescent labeled secondary antibodies, Alexa488-labeled donkey anti-rabbit antibody (Thermo Fisher Scientific, Inc.), CF555-labeled donkey anti-mouse antibody (Biotium, Inc.), CF555-labeled donkey anti-goat antibody, CF543-labeled donkey anti-sheep antibody, and Alexa647-labeled donkey anti-mouse antibody were used for multiple staining, and Hoechst33342 (SigmaAldrich, Inc.) was used for contrast staining of nuclei. FIG. 1E is a comparative staining image of the nucleus with respect to FIG. 1C and D, FIG. 1H with FIG. 1F and G, FIG. 1K with FIG. 1I and J, FIG. 1N with FIG. 1L and M, FIG. 1Q with FIG. 1O and P, FIG. 1U with FIG. 1S and T, and FIG. 1X with FIG. 1V and W. An upright fluorescence microscope Axio Imager M2 (manufactured by Zeiss) and attached software Axio Vision were used for observation of the stained sections and acquisition of images. In FIG. 1C to FIG. 1E, the scale bar at the lower right of FIG. 1F indicates 200 μm, the scale bar at the lower right of FIG. 1I, FIG. 1L, FIG. 1O, and FIG. 1S indicates 100 μm, and the scale bar at the lower right of FIG. 1V indicates 50 μm.

[0375] The results showed that on the 28th day after the suspension culture induced by the above-mentioned differentiation induction method was started, there were neuroepithelial tissues of the ventral diencephalon and hypothalamus (Figure 1D, G, L, O, P) positive for Nkx 2.1, Tbx3, Sox3, and βIII tubulin on the inner side of the cell mass, and non-neuroepithelial tissues positive for EpCAM and pan-cytokeratin on the outer side (Figure 1M, W). It can be seen that the inner side of the inner neuroepithelial tissue is further aPKC-positive apical surface, forming a ventricular-like tissue with vacuoles without cells (Figure 1I, *). A basement membrane-like structure formed by laminin and the like was formed on the inner side of the outer non-neuroepithelial tissue (Figure 1J). It can be further seen that a part of the non-neuroepithelial tissue was positive for Lhx3, Emx2, and Pitx1, forming a Rathke's cyst-like structure and a pituitary placode where the non-neuroepithelial tissue was trapped (Figure 1C, F, S, V). In addition, between the non-neuroepithelial tissue on the outside and the neuroepithelial tissue on the inside, there are head mesenchymal cells that are nestin-positive, pan-cytokeratin, weakly laminin-positive, and negative for βIII tubulin. According to the above results, the preparation method according to the present invention can prepare the following cell agglomerates: it contains non-neuroepithelial, neuroepithelial, and mesenchymal cells, and the outside is covered with non-neuroepithelial, and the inside is formed with a ventral diencephalon with a ventricle-like structure and a hypothalamus-like neuroepithelial tissue, and there are head mesenchymal cells between the non-neuroepithelial and neuroepithelial, and a part of the non-neuroepithelial on the outside is a pituitary placode. The pattern diagram of the cell agglomerate on the 28th day after the start of suspension culture is shown in Figure 1Y.

[0376] Example 2: Maturation of cell aggregates containing pituitary tissue prepared from human ES cells by long-term culture

[0377] Human ES cells (KhES-1 strain, obtained from Kyoto University) were cultured under feeder-free conditions according to the method described in Scientific Reports, 4, 3594 (2014). StemFit medium was used as a feeder-free medium, and laminin 511-E8 was used as a feeder-free framework.

[0378] The specific maintenance culture operation was carried out in the same manner as in Example 1.

[0379] Thereafter, suspension culture was carried out under the same conditions as in Example 1 using a 96-well plate.

[0380] On the 28th day after the start of the suspension culture, the cell clumps were transferred to a 10 cm suspension culture dish (manufactured by Sumitomo BakeLite) at a rate of 48 cell clumps per dish, and the suspension culture was continued until the 84th day using a serum-free medium. The serum-free medium (gfCDM + KSR) at this time was a 1:1 mixture of F-12 + Glutamax medium (manufactured by Thermo Fisher Scientific) and IMDM + Glutamax medium (manufactured by Thermo Fisher Scientific) supplemented with 10% Knockout serum replacement (manufactured by Thermo Fisher Scientific), 450 μM 1-monothioglycerol (manufactured by Wako Pure Chemical Industries, Ltd.), 1x chemically defined lipid concentrate (manufactured by Thermo Fisher Scientific), 50 units / ml penicillin-50 μg / ml streptomycin (manufactured by Nacalai Tesque), and 2 μM SAG, with 15 ml per dish, and the medium was replaced by half every 3 to 4 days.

[0381] The cell aggregates on the 70th day after the start of the suspension culture were observed by bright field using an inverted microscope (manufactured by Keyence, BIOREVO). Figure 2 A, B). Figure 2 The scale bar in the lower right corner of A represents 1000 μm. Figure 2 The scale bar in the lower right of B represents 200 μm. As a result, on the 70th day after the start of suspension culture, a cell mass covered with epithelial tissue of about 100 μm in thickness was formed. A part of the above-mentioned cell mass was fixed in 4% paraformaldehyde, and the remaining cell mass was further suspended and cultured. Regarding the culture medium at this time, a culture medium in which 10 μM dexamethasone (made by SigmaAldrich, mouse) was further added to the above-mentioned serum-free culture medium was used, and the culture was cultured to the 84th day after the start of suspension culture, and then fixed with 4% paraformaldehyde. Frozen sections were prepared from the cell aggregates cultured on the 70th and 84th days in the same manner as in Example 1, and fluorescent immunostaining and observation were performed in the same manner as in Example 1 using anti-adrenocorticotropic hormone (ACTH) antibody (manufactured by Lab Vision, mouse), anti-prolactin (PRL) antibody (manufactured by Cell Marque, rabbit), anti-growth hormone antibody (manufactured by R&D Systems, goat) for marking hormone-producing cells of the pituitary, and anti-pan-cytokeratin antibody for marking non-neuroepithelial tissue. Figure 2 D is Figure 2 C, Figure 2 G is Figure 2 E and F, Figure 2 J is Figure 2 H and I are contrast stained images of cell nuclei. Figure 2 C, Figure 2 The scale bar in the lower right corner of E represents 100 μm. Figure 2 The scale bar in the lower right of H represents 50 μm.

[0382] As a result, ACTH-positive cells, which are a marker for hormone-producing cells of the pituitary gland, were present in the cell mass on the 70th day after the start of suspension culture ( Figure 2 C).

[0383] Furthermore, in the cell mass on the 84th day after the start of suspension culture in which dexamethasone was added for 14 days, PRL-positive cells and GH-positive cells, which are markers for hormone-producing cells of the pituitary gland, were present ( Figure 2 E, H). These results indicate that the above-mentioned differentiation induction method can be used to induce differentiation of pituitary hormone-producing cells from human pluripotent stem cells.

[0384] Reference Example 1: Study on the timing of adding BMP4 in the preparation of cell aggregates containing pituitary tissue derived from human ES cells (1)

[0385] Human ES cells (KhES-1 strain, obtained from Kyoto University) were cultured under feeder-free conditions according to the method described in Scientific Reports, 4, 3594 (2014). StemFit medium was used as a feeder-free medium, and laminin 511-E8 was used as a feeder-free framework.

[0386] The specific maintenance and culture operation was carried out in the same manner as in Example 1.

[0387] Thereafter, under the same conditions as in Example 1, suspension culture using a 96-well plate was started.

[0388] The condition without adding BMP signal transduction pathway-affecting substances during the suspension culture period was used as a control (-BMP4 condition, Figure 3 A). Then, on the 1st, 2nd or 3rd day after the start of suspension culture, a condition in which a substance acting on the BMP signal transduction pathway is added (1 to 3 days + BMP4 condition, Figure 3B to D), 100 μl of serum-free medium containing IWP-2, SB-431542, and BMP4 but not Y27632 was added to each well. Regarding BMP4, 3 nM was added to the added medium to make the final concentration in the well 1.5 nM. On the 6th day after the start of suspension culture, half of the medium was replaced with serum-free medium containing IWP-2 and SB-431542 but not Y27632 and BMP4. On the 10th day after the start of suspension culture, bright field observation was performed using an inverted microscope (Keyence, BIOREVO) ( Figure 3 AD). Figure 3 The scale bar in the lower right of AD represents 200 μm.

[0389] The results showed that, without the addition of BMP4, smooth embryoid bodies were formed, and no non-neuroepithelial-like structures were confirmed on the surface of the embryoid bodies ( Figure 3 A). Under the condition of adding BMP4 on the 1st and 2nd day after differentiation induction (1, 2 days + BMP4), a cell mass containing neural tissue and non-neuroepithelial tissue was formed, which had a two-layer structure with a central part with a neural epithelial-like structure covered by a non-neuroepithelial-like layer ( Figure 3 B, C). When the two conditions were compared, the addition of BMP4 on day 2 resulted in the formation of embryoid bodies compared to the condition in which BMP4 was added on day 1 ( Figure 3 B) Larger embryoid bodies ( Figure 3 C) When BMP4 was added on the third day after differentiation induction, no non-neuroepithelial-like tissue was formed on the outside, and only an embryoid body consisting of smooth-surfaced neuroepithelial-like tissue was formed ( Figure 3 D) The above results show that the addition of a substance acting on the BMP signaling pathway within 72 hours after the start of suspension culture is effective in the formation of a cell mass covered with non-neuroepithelial tissue, which is the basis of future pituitary tissue, from human pluripotent stem cells.

[0390] Example 3: Study on the timing of adding BMP4 in the preparation of cell aggregates containing pituitary tissue derived from human ES cells (2)

[0391] Human ES cells (KhES-1 strain, obtained from Kyoto University) were cultured under feeder-free conditions according to the method described in Scientific Reports, 4, 3594 (2014). StemFit medium was used as a feeder-free medium, and laminin 511-E8 was used as a feeder-free framework.

[0392] The specific maintenance and culture operation was carried out in the same manner as in Example 1.

[0393] Thereafter, under the same conditions as in Example 1, suspension culture using a 96-well plate was started.

[0394] Bright field observation of aggregates was performed using an inverted microscope on the second and third days after the start of suspension culture without adding a medium. Figure 4 The scale bar in the lower right corner of A indicates 200 μm. The results of observation showed that the aggregates on the second day after the start of suspension culture, which is the optimal period for the addition of BMP4, had uneven surfaces and a deformed shape ( Figure 4 A). On the other hand, in the aggregates on the third day after the start of suspension culture, the unevenness observed on the second day was reduced and the shape was close to a sphere ( Figure 4 B).

[0395] The aggregates on the second and third days after the start of the above suspension culture were fixed by the method described in Example 1 and prepared into frozen sections. For these frozen sections, fluorescent immunostaining was performed using anti-ZO-1 antibody (manufactured by Thermo Fisher Scientific, rabbit) as a tight junction marker and anti-Sox2 antibody (manufactured by Santa Cruz Biotechnology, goat) as a neural stem cell and pluripotent stem cell marker. The fluorescently labeled secondary antibodies and nuclear contrast staining used those described in Example 1. Figure 4 The scale bar in the lower right corner of C represents 100 μm. As a result, in the aggregates on the second day after the start of suspension culture, some of the cells on the outermost layer of the aggregates were ZO-1 positive and formed tight junctions ( Figure 4 C) On the other hand, in the aggregates on the third day after the start of suspension culture, ZO-1-positive cells were taken into the interior of the aggregates and were not observed locally in the outermost layer ( Figure 4 F). In addition, in the aggregates on the third day after the start of suspension culture, the two-layer structure of the outer layer where cells are more closely packed and the inner layer where cells are loose and strongly positive for Sox2 can be more clearly confirmed ( Figure 4 D, G) The above results show that detection of tight junctions in the outermost cells of the aggregate can be used as a means for determining the timing of adding BMP4 during the preparation of a cell aggregate containing neural tissue and non-neuroepithelial tissue.

[0396] Example 4: Study on the conditions for adding substances acting on the Sonic hedgehog signal transduction pathway in the preparation of cell aggregates containing pituitary tissue from human ES cells

[0397] Human ES cells (KhES-1 strain, obtained from Kyoto University) were cultured under feeder-free conditions according to the method described in Scientific Reports, 4, 3594 (2014). StemFit medium was used as a feeder-free medium, and laminin 511-E8 was used as a feeder-free framework.

[0398] The specific maintenance and culture operation was carried out in the same manner as in Example 1.

[0399] Thereafter, under the same conditions as in Example 1, suspension culture using a 96-well plate was started.

[0400] On the second day after the start of suspension culture, 100 μl of serum-free medium containing IWP-2, SB-431542, BMP4, and SAG but not Y27632 was added to each well. BMP4 was added to the added medium at 3 nM to a final concentration of 1.5 nM in the well, and SAG, a substance acting on the Sonic hedgehog signal transduction pathway, was added at concentrations of 100, 300, 500, 700 nM, and 1, 2, and 5 μM. The condition in which no substance acting on the Sonic hedgehog signal transduction pathway was added during the suspension culture period was used as a control (-SAG condition, Figure 5 A).

[0401] On days 6, 10, 13, 17, 21, and 24 after the start of suspension culture, the medium was replaced by half with a serum-free medium containing IWP-2, SB-431542, and various concentrations of SAG but not containing Y27632 and BMP4.

[0402] On the 28th day after the start of suspension culture, bright field observation was performed using an inverted microscope (manufactured by Keyence, BIOREVO) ( Figure 5 AH). Figure 5 The scale bar at the lower right of A represents 1000 μm.

[0403] The results showed that, when SAG was not added to the suspension culture, a nearly spherical tissue with a non-neuroepithelial-like structure was formed on the surface of the cell aggregate ( Figure 5 A). On the other hand, under the condition of adding various concentrations of SAG, larger cell clusters were formed than under the condition of no addition, the non-neuroepithelial tissue on the surface was sunken into Rathke's cysts, and there were neuroepithelial-like tissues and mesenchymal cell clusters inside the cell clusters ( Figure 5BH). The above results show that in the preparation of cell aggregates with pituitary tissue, the SAG concentration in suspension culture is appropriately 100 nM to 5 μM. In particular, when SAG is added on the second day of culture in such a manner that the final concentration is 100 nM, 300 nM, 500 nM, 700 nM, 1 μM or 2 μM ( Figure 5 BG) and 5 μM addition conditions ( Figure 5 H), the size of the cell aggregates was larger than that of the control group, and the pituitary tissue on the surface of the cell aggregates was well formed.

[0404] Example 5: Effect of compound treatment before differentiation induction in the preparation of cell aggregates containing pituitary tissue derived from human ES cells

[0405] Human ES cells (KhES-1 strain) were cultured under feeder-free conditions based on the method described in Scientific Reports, 4, 3594 (2014). StemFit medium was used as a feeder-free medium, and laminin 511-E8 was used as a feeder-free framework. The specific maintenance and culture operation was performed in the same manner as in Example 1.

[0406] As a pretreatment for inducing differentiation of cultured cells, cells were maintained and cultured under the following four conditions: a condition in which nothing was added (control), a condition in which only 300 nM SAG was added (+300 nM SAG), a condition in which only 5 μM SB-431542 was added (+5 μM SB-431542), and a condition in which both SAG and SB431542 were added (+SAG / SB) at the same time when the medium was replaced with Stemfit medium 6 days after inoculation.

[0407] On the day following the above treatment, human ES cells of the four subconfluent conditions were induced to differentiate into cell masses containing pituitary tissues by suspension culture using the method described in Example 1.

[0408] On the 28th day after the start of suspension culture, bright field observation was performed using an inverted microscope ( Figure 6 AD). Figure 6 The scale bar in the lower right corner of A represents 1000 μm. The results showed that when no compound (SAG and / or SB431542) was added before suspension culture, the cell aggregates formed were small, and no mesenchymal cells were confirmed between the outer non-neuroepithelial tissue and the inner neuroepithelial tissue ( Figure 6 A). When treated with 300 nM SAG alone, the cell aggregates became larger than those without addition, and the presence of mesenchymal cells was also confirmed ( Figure 6B). When only 5 μM SB-431542 was added and when both SAG and SB431542 were added, larger cell aggregates containing pituitary tissue were formed ( Figure 6 C, D).

[0409] The above results show that compound treatment before suspension culture improves the efficiency of forming cell aggregates containing pituitary tissue, and further preferred conditions are the condition of adding only 5 μM SB-431542 (+5 μM SB-431542) and the condition of adding both SAG and SB431542 (+SAG / SB).

[0410] Example 6: Study on the conditions for adding Wnt signaling pathway inhibitory substances in the preparation of cell aggregates containing pituitary tissue derived from human ES cells

[0411] Human ES cells (KhES-1 strain) were cultured under feeder-free conditions according to the method described in Scientific Reports, 4, 3594 (2014). StemFit medium was used as a feeder-free medium, and laminin 511-E8 was used as a feeder-free framework.

[0412] The specific maintenance culture operation was carried out in the same manner as in Example 1.

[0413] Thereafter, suspension culture using a 96-well plate was performed under the same conditions as in Example 1 except that the type or presence of the Wnt signaling pathway inhibitor added at the start of the suspension culture was changed. At this time, as the Wnt signaling pathway inhibitor, IWP-2, C-59 (manufactured by Cayman Chemicals, 500 nM), LGK974 (manufactured by Cayman Chemicals, 5 μM) which are PORCN inhibitors, XAV939 (manufactured by Cayman Chemicals, 1 μM) which are TANK inhibitors, IWR1-endo (manufactured by Merck Millipore, 1 μM) and KY02111 (manufactured by Cayman Chemicals, 5 μM) which has no reported mechanism of action were added at the start of the suspension culture, and as a control without the addition of the Wnt signaling pathway inhibitor, only DMSO was added.

[0414] On the second day after the start of suspension culture, 100 μl of serum-free medium containing each Wnt signaling pathway inhibitor, SB-431542, BMP4, and SAG was added to each well. BMP4 was added to the added medium at 3 nM to a final concentration of 1.5 nM in the well. SAG was added to the added medium at 4 μM to a final concentration of 2 μM in the well.

[0415] On days 6, 10, 13, 17, 21, and 24 after the start of suspension culture, the medium was replaced by half using a serum-free medium that did not contain Y27632 and BMP4 but contained each Wnt signaling pathway inhibitor, SB-431542, and SAG. In addition to the above conditions, conditions were also implemented in which neither SB431542 nor Wnt signaling pathway inhibitors were added to the suspension culture.

[0416] On the 28th day after the start of suspension culture, bright field observation was performed using an inverted microscope (manufactured by Keyence, BIOREVO) ( Figure 7 A~H). Figure 7 The scale bar at the lower right of A represents 1000 μm.

[0417] The results showed that no non-neural epithelial tissue was formed on the surface of the cell mass under the condition where neither SB431542 nor the Wnt signaling pathway inhibitor was added, and under the condition where SB431542 was added but the Wnt signaling pathway inhibitor was not added. Figure 7 On the other hand, under the conditions of adding IWP-2, KY02111, C59, LGK974, XAV939, and IWR1-endo, which are Wnt signaling pathway inhibitors, cell clusters with non-neuroepithelial structures on the surface were formed ( Figure 7 In particular, when 2μM IWP-2, 5μM KY02111, 1μM XAV939, and 1μM IWR1e were added, large, well-maintained cell aggregates were formed ( Figure 7 C, D, G, H). The above results indicate that inhibition of the Wnt signal transduction pathway using PORCN inhibitors, TANK inhibitors, etc. can promote the formation of non-neuroepithelial tissue on the surface of cell clusters containing pituitary tissues by BMP signals.

[0418] Example 7: Study on the concentration of BMP4 added in the preparation of cell aggregates containing pituitary tissue derived from human ES cells

[0419] Human ES cells (KhES-1 strain, obtained from Kyoto University) were cultured under feeder-free conditions according to the method described in Scientific Reports, 4, 3594 (2014). StemFit medium was used as a feeder-free medium, and laminin 511-E8 was used as a feeder-free framework.

[0420] The specific maintenance culture operation was carried out in the same manner as in Example 1.

[0421] Thereafter, under the same conditions as in Example 1, suspension culture using a 96-well plate was started.

[0422] On the second day after the start of suspension culture, 100 μl of serum-free medium containing IWP-2, SB-431542, BMP4, and SAG but not containing Y27632 was added to each well. At that time, four conditions were set: the amount of BMP4 added was 0.5 nM, 1.5 nM, 5 nM, and a non-added control. BMP4 was added to the added medium at a concentration twice that of the set condition, so that the final concentration in the well reached the set condition. Regarding SAG, 4 μM was added to the added medium, so that the final concentration in the well was 2 μM.

[0423] On days 6, 10, 13, 17, 21, and 24 after the start of suspension culture, half of the medium was replaced with a serum-free medium containing IWP-2, SB-431542, and SAG but not containing Y27632 and BMP4.

[0424] On the 28th day after the start of suspension culture, bright field observation was performed using an inverted microscope ( Figure 8 A~D). Figure 8 The scale bar at the lower right of A represents 1000 μm.

[0425] The results showed that no non-neuroepithelial structures were formed on the surface of the cell aggregates without the addition of BMP4. Figure 8 A). On the other hand, when 0.5nM, 1.5nM, and 5nM BMP4 were added, non-neuroepithelial structures were formed on the surface of these cell aggregates ( Figure 8 BD). The above results indicate that the single addition of BMP4 on the second day of suspension culture resulted in the formation of non-neuroepithelial tissue on the surface of cell aggregates containing pituitary tissue.

[0426] Example 8: Study on TGFβ signaling pathway inhibition conditions in the preparation of cell pellets containing pituitary tissue from human ES cells

[0427] Human ES cells (KhES-1 strain) were cultured under feeder-free conditions according to the method described in Scientific Reports, 4, 3594 (2014). StemFit medium was used as a feeder-free medium, and laminin 511-E8 was used as a feeder-free framework.

[0428] The specific maintenance culture operation was carried out in the same manner as in Example 1.

[0429] Thereafter, suspension culture using a 96-well plate was performed under the same conditions as in Example 1 except that the concentration and type of the TGFβ signaling pathway inhibitor added at the start of the suspension culture were changed. As the TGFβ signaling pathway inhibitor at this time, SB431542 (1 or 10 μM), A 83-01 (manufactured by Cayman Chemicals, 1 or 10 μM), and RepSox (manufactured by Cayman Chemicals, 1 μM) were added at various concentration conditions.

[0430] Then, on the second day after the start of suspension culture, a serum-free medium containing IWP-2, each TGFβ signaling pathway inhibitor, BMP4, and SAG but not Y27632 was added to each well at 100 μl. BMP4 was added to the added medium at 3 nM to a final concentration of 1.5 nM in the well. SAG was added to the added medium at 4 μM to a final concentration of 2 μM in the well.

[0431] On days 6, 10, 13, 17, 21, and 24 after the start of suspension culture, a serum-free medium containing IWP-2, each TGFβ signaling pathway inhibitor, and SAG but not Y27632 or BMP4 was used for half-medium replacement. In addition to the above conditions, conditions in which neither the TGFβ signaling pathway inhibitor nor IWP-2 was added to the suspension culture, and conditions in which IWP-2 was added but the TGFβ signaling pathway inhibitor was not added were also implemented.

[0432] The results showed that, when neither the TGFβ signal transduction pathway inhibitor nor IWP-2 was added in step (1), no non-neuroepithelial structure was formed on the surface of the cell aggregates on day 28 after the start of suspension culture ( Fig. 9 A). When IWP-2 was added but TGFβ signaling pathway inhibitors were not added, non-neuroepithelial structures were formed on the surface of the cell clusters. The cell clusters formed were small, and no mesenchymal cells were confirmed between the non-neuroepithelial tissue on the outside and the neuroepithelial tissue on the inside ( Fig. 9B) When various TGFβ signal transduction pathway inhibitors were added, a large cell mass containing pituitary tissue was formed with mesenchymal cells between the outer non-neuroepithelial tissue and the inner neuroepithelial tissue ( Fig. 9 CG). The above results show that by adding a TGFβ signal transduction pathway inhibitory substance to the suspension culture, a large cell mass containing pituitary tissue in which mesenchymal cells exist between the outer non-neuroepithelial tissue and the inner neuroepithelial tissue can be prepared.

[0433] Example 9: Cell Aggregates Containing Neural Tissue and Non-Neuroepithelial Tissue Prepared from Human iPS Cells

[0434] Human iPS cells (201B7 strain, obtained from iPS Academia Japan, Inc.) were cultured under feeder-free conditions according to the method described in Scientific Reports, 4, 3594 (2014). StemFit medium was used as a feeder-free medium, and laminin 511-E8 was used as a feeder-free framework.

[0435] As a specific maintenance culture operation, the same method as in Example 1 was carried out.

[0436] Thereafter, suspension culture was carried out under the same conditions as in Example 1 using a 96-well plate.

[0437] On the second day after the start of suspension culture, 100 μl of serum-free medium containing IWP-2, SB-431542, SAG, and BMP4 but not Y27632 was added to each well. BMP4 was added to the added medium at 3 nM to a final concentration of 1.5 nM in the well. SAG was added to the added medium at 4 μM to a final concentration of 2 μM in the well.

[0438] On days 6, 10, 13, 17, 21, and 24 after the start of suspension culture, a half-medium replacement was performed using a serum-free medium containing IWP-2, SB-431542, and SAG but not containing Y27632 and BMP4.

[0439] On the 28th day after the start of suspension culture, bright field observation was performed using an inverted microscope ( Fig.10 A, B). Fig.10 The scale bar in the lower right corner of A represents 1000 μm. Fig.10 The scale bar in the lower right of B indicates 200 μm. As a result, it was found that, similar to the case of preparation from the human ES cell line KhES-1 in Example 1, a neural tissue cell mass with non-neuroepithelial tissue surrounding the central part was formed from the human iPS cell line 201B7.

[0440] The cell mass on day 28 of culture was fixed by the same method as in Example 1, and frozen sections were prepared. For these frozen sections, anti-Lhx3 antibody as an early marker of the pituitary, anti-Pitx2 antibody as a marker of oral epithelium and pituitary placode (R&D Systems, sheep), anti-Six1 antibody as a marker of non-neural epithelium and placode (Atlas Antibodies, rabbit), anti-Nkx 2.1 antibody as a marker of ventral diencephalon, Sox2 antibody as a marker of undifferentiated nerves and placode, Bfl antibody as a marker of telencephalon and placode (TAKARA Bio, rabbit), anti-CD326 / EpCAM antibody as a marker of non-neural epithelial tissue (R&D Systems, goat), anti-pan-cytokeratin antibody, anti-E-cadherin antibody (Santa Cruz Biotechnology, mouse), anti-nestin antibody as a marker of undifferentiated nerves, neural crest and mesenchymal cells, anti-βIII tubulin (Tuj1) antibody as a marker of nerve cells, and anti-vimentin antibody as a marker of mesenchymal cells (R&D Systems, goat). Fluorescent immunostaining was performed using Alexa647-labeled donkey anti-goat antibody (manufactured by Thermo Fisher Scientific) as fluorescently labeled secondary antibodies in addition to those used in Example 1, and multiple staining was performed using Hoechst 33342 for contrast staining of nuclei. Fig.10 F is Fig.10 C, D, E, Fig.10 J is Fig.10 G, H, I, Fig.10 N is Fig.10 K, L, M, Fig.10 R is Fig.10 Contrast staining images of nuclei corresponding to O, P, and Q. An upright fluorescence microscope Axio Imager M2 (manufactured by Zeiss) and attached software Axio Vision were used for observation of the stained sections and acquisition of images. Fig.10 The scale bar in the lower right corner of C represents 200 μm. Fig.10 O The scale bar at the lower right represents 100 μm.

[0441] As a result, it can be seen that on the 28th day after the suspension culture induced from the human iPS cell line 201B7 by the above-mentioned differentiation induction method, there are neuroepithelial tissues of the ventral diencephalon and hypothalamus that are positive for Nkx 2.1, Sox2, Bf1, and βIII tubulin on the inside of the cell mass, and non-neuroepithelial tissues of the oral cavity that are positive for EpCAM, pan-cytokeratin, E-cadherin, and Pitx2 on the outside. Further, it can be seen that a part of the non-neuroepithelial tissue is positive for Lhx3, Bf1, and Sox2, and the non-neuroepithelial tissue is sunken to form a Rathke's cyst-like structure and a pituitary basement. In addition, between the non-neuroepithelial tissue on the outside of the nucleus and the neuroepithelial tissue on the inside, there are head mesenchymal cells that are positive for nestin and vimentin, weakly positive for pan-cytokeratin and laminin, and negative for βIII tubulin. According to the above results, the preparation method of the present invention can prepare the following cell agglomerates from human iPS cells in the same way as human ES cells: the agglomerates contain non-neural epithelium, neural epithelium, and mesenchymal cells, and the non-neural epithelium is covered on the outside, and the ventral diencephalon and hypothalamus-like neural epithelial tissue with a ventricle-like structure are formed on the inside, head mesenchymal cells are present between the non-neural epithelium and the neural epithelium, and a part of the non-neural epithelium on the outside is the pituitary placode.

[0442] Example 10: Cell Aggregates Containing Neural Tissue and Non-Neuroepithelial Tissue Prepared from Human iPS Cells Using a Combination of Wnit Signaling Pathway Acting Substances and Inhibiting Substances

[0443] Human iPS cells (HC-6#10 strain, obtained from RIKEN) were cultured under feeder-free conditions according to the method described in Scientific Reports, 4, 3594 (2014). StemFit medium was used as a feeder-free medium, and laminin 511-E8 was used as a feeder-free framework.

[0444] As a specific maintenance culture operation, the same method as in Example 1 was carried out.

[0445] The subconfluent human iPS cells prepared as described above were washed with PBS, treated with an enzyme using Accumax, and then serum-free medium for differentiation induction was added and cells were peeled off from the surface of the culture dish using a cell scraper and dispersed into single cells by blowing. Subsequently, the human ES cells dispersed into single cells were plated in a non-cell-adhesive 96-well culture plate (PrimeSurface96V bottom plate, manufactured by Sumitomo BakeLite Co., Ltd.) with 9×10 cells per well. 3The cells were suspended in 100 μl of serum-free medium and cultured in suspension at 37°C and 5% CO2. The serum-free medium (gfCDM+KSR) used here was a 1:1 mixture of F-12+Glutamax medium (Thermo Fisher Scientific) and IMDM+Glutamax medium (Thermo Fisher Scientific) supplemented with 5% Knockout serum replacement (Thermo Fisher Scientific), 450 μM 1-monothioglycerol (Wako Pure Chemical Industries, Ltd.), 1x chemically defined lipid concentrate (Thermo Fisher Scientific), and 50 units / ml penicillin-50 μg / ml streptomycin (Nacalai Tesque). At the start of suspension culture (day 0 after the start of suspension culture, step 1), Y27632 (final concentration 20 μM), SB-431542 (TGFβ signal transduction pathway inhibitor, Wako Pure Chemical Industries, Ltd., 1 μM) and IWP-2 (Wnt signal transduction pathway inhibitor, Tocris Bioscience, Inc., 2 μM) were added to the above serum-free medium. In addition, the following four conditions were set: conditions of adding CHIR99021 (Cayman Chemical Co., Ltd.) 300 nM, 1 μM, and 3 μM as a Wnt signal transduction pathway agent, and conditions of adding only DMSO as a control without addition. In addition, CHIR99021 is sometimes referred to as CHIR in this example.

[0446] On the second day after the start of suspension culture, 100 μl of serum-free medium containing SB-431542, IWP-2, BMP4 (BMP signal transduction pathway agent) and CHIR99021 of each set condition was added to each well. Regarding BMP4, 3 nM was added to the added medium so that the final concentration in the well was 1.5 nM. As a means for inducing cell clusters containing non-neural epithelium and placodes, half of the medium was replaced on the third day after the start of suspension culture using a serum-free medium containing IWP-2, SB-431542, K02288 (BMP signal transduction pathway inhibitor, manufactured by AdooQ Bioscience), FGF2-TS (FGF signal transduction pathway agent, manufactured by HumanZyme) and sodium heparin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). Regarding K02288, FGF2 and heparin sodium were added to the medium at 2 μM, 40 ng / ml, and 20 μg / ml, respectively, so that the final concentrations in the wells were 1 μM, 20 ng / ml, and 10 μg / ml. Half of the medium was replaced on the 6th and 10th days after the start of suspension culture.

[0447] On the 13th day after the start of suspension culture, bright field observation was performed using an inverted microscope ( Fig.11 A~D). Fig.11 The scale bar on the lower right of A represents 1000 μm. As a result, it can be seen that, as in the case of making the human ES cell line KhES-1 using Example 1, a neural tissue cell mass surrounded by non-neural epithelial tissue in the central part was formed by the human iPS cell line HC-6#10 strain. In addition, it can be seen that even if BMP signal transduction pathway inhibitors and FGF signal transduction pathway agents were added on the third day after the start of suspension culture, non-neural epithelial tissue was formed on the surface. Further, it can be seen that in the presence of IWP-2, which is a Wnt signal transduction pathway inhibitor, CHIR99021, which is a Wnt signal transduction pathway agent, also plays a role, and the cell mass becomes larger in a concentration-dependent manner. On the other hand, under the condition of adding 3 μM CHIR99021, the formation of non-neural epithelial tissue on the surface of the cell mass is inhibited.

[0448] The cell mass on the 13th day of culture was fixed by the same method as in Example 1, and frozen sections were prepared. For these frozen sections, fluorescent immunostaining was performed using anti-Six1 antibody for non-neural epithelium and placode markers, anti-Dlx5 antibody (Atlas Antibodies, rabbit) for central nervous system and placode markers, anti-E-cadherin antibody (R&D Systems, goat), and anti-N-cadherin antibody (BD Biosciences, mouse) ( Fig.11E~P, Fig.12 Q to AB). The stained sections were observed and images were obtained using an upright fluorescence microscope Axio Imager M2 (manufactured by Zeiss) and the attached software Axio Vision. Fig.11 E, H, K, N, Fig.12 The scale bar at the lower right of Q, T, W, and Z represents 100 μm.

[0449] As a result, in the cell aggregates on the 13th day after the suspension culture of the human iPS cell line HC-6#10 induced by the above-mentioned differentiation induction method, the formation of non-neural epithelial and placode precursor tissues positive for D1x5, Six1, E-cadherin, and N-cadherin was observed under the conditions of no CHIR addition and the conditions of adding 300nM and 1μM CHIR ( Fig.11 E~P, Fig.12 Q~V). When 3μM CHIR was added, no non-neural epithelial formation was observed, suggesting that differentiation to other tissues occurred ( Fig.12 W~AB). In addition, when comparing the conditions without CHIR addition and the conditions with addition of 300nM and 1μM CHIR, a larger cell mass was formed under the condition with addition of 300nM CHIR relative to the condition without CHIR addition, and the survival and epithelialization of the internal neural tissue were promoted. On the other hand, under the condition with addition of 1μM CHIR, the hypertrophy and placode formation of the non-neural epithelium on the outside were inhibited. According to the above results, in the presence of IWP-2, which is a Wnt signal transduction pathway inhibitor, a certain degree (less than 3μM CHIR99021) of β-catenin-dependent classical Wnt pathway activation is effective for the growth of the entire cell mass, the survival of the internal neural tissue and the promotion of epithelialization.

[0450] Example 11: Cell Agglomerates Containing Neural Tissue and Pituitary Tissue Prepared from Human iPS Cells Using a Combination of a Substance Acting on and Inhibiting the Wnt Signaling Pathway

[0451] Human iPS cells (HC-6#10 strain, obtained from RIKEN) were cultured under feeder-free conditions according to the method described in Scientific Reports, 4, 3594 (2014). StemFit medium was used as a feeder-free medium, and laminin 511-E8 was used as a feeder-free framework.

[0452] As a specific maintenance culture operation, the same method as in Example 1 was carried out.

[0453] The subconfluent human iPS cells prepared as described above were washed with PBS, treated with an enzyme using Accumax, and then serum-free medium for differentiation induction was added and cells were peeled off from the surface of the culture dish using a cell scraper and dispersed into single cells by blowing. Subsequently, the human ES cells dispersed into single cells were plated in a non-cell-adhesive 96-well culture plate (PrimeSurface96V bottom plate, manufactured by Sumitomo BakeLite Co., Ltd.) with 9×10 cells per well. 3 The cells were suspended in 100 μl of serum-free medium and cultured in suspension at 37°C and 5% CO2. The serum-free medium (gfCDM+KSR) used here was a 1:1 mixture of F-12+Glutamax medium (Thermo Fisher Scientific) and IMDM+Glutamax medium (Thermo Fisher Scientific) supplemented with 5% Knockout serum replacement (Thermo Fisher Scientific), 450 μM 1-monothioglycerol (Wako Pure Chemical Industries, Ltd.), 1x chemically defined lipid concentrate (Thermo Fisher Scientific), and 50 units / ml penicillin-50 μg / ml streptomycin (Nacalai Tesque). At the start of suspension culture (day 0 after the start of suspension culture, step 1), Y27632 (final concentration 20 μM), SB-431542 (TGFβ signaling pathway inhibitor, Wako Pure Chemical Industries, Ltd., 1 μM) and IWP-2 (Wnt signaling pathway inhibitor, Tocris Bioscience, Inc., 2 μM) were added to the serum-free medium. In addition, 300 nM of CHIR99021 (Cayman Chemical Co., Ltd.) was added as a Wnt signaling pathway agent.

[0454] On the second day after the suspension culture was started, a serum-free medium containing SB-431542, IWP-2, CHIR99021, BMP4 (BMP signaling pathway agent), and SAG (Shh signaling pathway agent) was added to each well at 100 μl. BMP4 was added to the added medium at 3 nM to a final concentration of 1.5 nM in the well. SAG was added to the added medium at 1.4 μM to a final concentration of 700 nM in the well.

[0455] On days 6, 10, and 13 after the start of suspension culture, the medium was replaced by half with a serum-free medium containing SAG, SB-431542, IWP-2, and CHIR99021 at the same concentrations but without Y27632 or BMP4. On days 17, 21, and 24 after the start of suspension culture, the medium was replaced by half with a serum-free medium containing FGF2-TS (FGF signal transduction pathway activating substance, manufactured by HumanZyme, 20 ng / ml) and sodium heparin (FGF stabilizer, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., 10 μg / ml) in addition to SAG, SB-431542, IWP-2, and CHIR99021 for the purpose of further growth of the formed placode precursor tissue.

[0456] On the 28th day after the start of suspension culture, bright field observation was performed using an inverted microscope ( Fig.13 A). Fig.11 The scale bar in the lower right of A indicates 1000 μm. The results showed that, similar to the case of preparation from human ES cell line KhES-1 in Example 1, under the condition of adding SAG, the human iPS cell line HC-6#10 formed a cell mass in which non-neural epithelial tissue surrounded the central part of the neural tissue.

[0457] The cell mass on day 28 of culture was fixed and frozen sections were prepared in the same manner as in Example 1. These frozen sections were fluorescently immunostained in the same manner as in Example 1 using antibodies against Lhx3, Nkx 2.1, E-cadherin, nestin, Pitx2, Sox2, Six1, vimentin, Rx, N-cadherin, Tbx3, and pan-cytokeratin ( Fig.13 B~I, Fig.14 J~V). Fig.13 E.I. Fig.14 M, P, S, and V are contrast staining images of cell nuclei with respect to the immunostaining images performed simultaneously.

[0458] As a result, the cell mass on the 28th day after the suspension culture induced by the human iPS cell line HC-6#10 strain using the above-mentioned induction differentiation method with the addition of Wnt signal transduction pathway agent and FGF signal transduction pathway agent was started, there were Nkx 2.1, Sox2, Tbx3, Rx, N-cadherin-positive ventral diencephalon, hypothalamus neuroepithelial tissue, and pan-cytokeratin, E-cadherin, Pitx2-positive oral non-neuroepithelial tissue on the outside. Further, a part of the non-neuroepithelial tissue was Lhx3, Six1, Sox2-positive, and it was known that a Latke's capsule-like structure and a pituitary base plate were formed in which the non-neuroepithelial tissue was trapped. In addition, between the non-neuroepithelial tissue on the outside and the neuroepithelial tissue on the inside, there were nestin, vimentin-positive, and weakly pan-cytokeratin-positive head mesenchymal cells. According to the above results, the following cell mass can be prepared by adding Wnt signal transduction pathway activating substances and FGF signal transduction pathway activating substances to human iPS cells through the induced differentiation conditions: it contains non-neural epithelium, neuroepithelium, and mesenchymal cells, and the outside is covered with non-neural epithelium, and the inside is formed with the ventral diencephalon and hypothalamus-like neuroepithelial tissue with a ventricle-like structure, head mesenchymal cells are present between the non-neural epithelium and the neuroepithelium, and a part of the outer non-neural epithelium is the pituitary placode.

[0459] Example 12: Cell Agglomerates Comprising Neural Tissue and Non-Neuroepithelial Tissue Prepared from Human iPS Cells After Addition of BMP Signaling Pathway Acting Substances and Use of BMP Signaling Pathway Inhibiting Substances After a Certain Period of Time

[0460] Human iPS cells (HC-6#10 strain, obtained from RIKEN) were cultured under feeder-free conditions according to the method described in Scientific Reports, 4, 3594 (2014). StemFit medium was used as a feeder-free medium, and laminin 511-E8 was used as a feeder-free framework.

[0461] As a specific maintenance culture operation, the same method as in Example 1 was carried out.

[0462] After washing the sub-confluent human iPS cells prepared as described above with PBS, the cells were treated with enzymes using Accumax, and then serum-free medium for differentiation induction was added and cells were peeled off from the surface of the culture dish using a cell scraper and dispersed into single cells by blowing. After that, the human ES cells dispersed into single cells were suspended in 100 μl of serum-free medium in a non-cell-adhesive 96-well culture plate (PrimeSurface96V bottom plate, manufactured by Sumitomo BakeLite) with 9×103 cells per well, and suspended and cultured at 37°C, 5% CO2. The serum-free medium (gfCDM+KSR) at this time was a 1:1 mixture of F-12+Glutamax medium (manufactured by Thermo Fisher Scientific) and IMDM+Glutamax medium (manufactured by Thermo Fisher Scientific) supplemented with 5% Knockout Serum Replacement (manufactured by Thermo Fisher Scientific), 450 μM 1-monothioglycerol (manufactured by Wako Pure Chemical Industries, Ltd.), 1x chemically defined lipid concentrate (manufactured by Thermo Fisher Scientific), and 50 units / ml penicillin-50 μg / ml streptomycin (manufactured by Nacalai Tesque). At the beginning of suspension culture (day 0 after the start of suspension culture, start of step 1), Y27632 (final concentration 20 μM), SB-431542 (TGFβ signaling pathway inhibitor, manufactured by Wako Pure Chemical Industries, Ltd., 1 μM), IWP-2 (Wnt signaling pathway inhibitor, manufactured by Tocris Bioscience, 2 μM), and CHIR99021 (Wnt signaling pathway activator, manufactured by Cayman Chemical, 300 nM) were added to the above-mentioned serum-free medium.

[0463] On the second day after the start of suspension culture, a serum-free medium containing SB-431542, IWP-2, CHIR99021, BMP4 (BMP signaling pathway agent), and SAG (Shh signaling pathway agent) was added to each well at 100 μl. Regarding BMP4, 3 nM was added to the added medium so that the final concentration in the well was 1.5 nM. Regarding SAG, 1.4 μM was added to the added medium so that the final concentration in the well was 700 nM. On the third, sixth, tenth, and thirteenth days after the start of suspension culture, the medium was replaced by half using a serum-free medium containing SAG, SB-431542, IWP-2, and CHIR99021 at the same concentrations but without Y27632 or BMP4. In addition, the following four conditions were set: a condition in which K02288 (BMP signaling pathway inhibitor, manufactured by Adooq BioScience, 300 nM) was added when half of the medium was replaced at any time on the 3rd, 6th, and 10th days after the start of suspension culture, and a condition in which DMSO was added as a control solvent. Regarding K02288, 600 nM was added to the added medium so that the final concentration in the well was 300 nM. On the 17th, 21st, and 24th days after the start of suspension culture, in addition to SAG, SB-431542, IWP-2, CHIR99021, and K02288, half of the medium was replaced with a serum-free medium having a final concentration of 20 ng / ml FGF2-TS and 10 μg / ml sodium heparin for the purpose of further growing the formed placode precursor tissue. Bright field observation was performed using an inverted microscope on the 28th day after the start of suspension culture ( Fig.15 A~D). Fig.15 The scale bar on the lower right of A represents 1000 μm. The results showed that, except for the control without K02288 addition, even when K02288 was added at any time point on days 3, 6, and 10 after the start of suspension culture, a cell mass in which non-neuroepithelial tissue surrounded the central part of the neural tissue was formed.

[0464] Furthermore, the cell mass on the 28th day of culture induced to differentiate by adding K02288 on the 6th day after the start of suspension culture was fixed by the same method as in Example 1, and frozen sections were prepared. For these frozen sections, fluorescent immunostaining was performed in the same method as in Example 1 using antibodies against Lhx3, Nkx 2.1, E-cadherin, nestin, Pitx2, Sox2, Six1, vimentin, Rx, N-cadherin, Tbx3, and pan-cytokeratin ( Fig.15 E~L, Fig.16 M~Y). Fig.15 H, L, Fig.16P, S, V, and Y are contrast staining images of cell nuclei relative to the immunostaining images performed simultaneously.

[0465] As a result, it can be seen that the inner side of the cell mass on the 28th day after the suspension culture started by the human iPS cell line HC-6#10 strain using the induction differentiation method of adding the above-mentioned BMP signal transduction pathway acting substance and adding the BMP signal transduction pathway inhibitor after a certain period of time, there are Nkx2.1, Sox2, Tbx3, Rx, N-cadherin-positive ventral diencephalon and hypothalamus neuroepithelial tissue, and the outer side is pan-cytokeratin, E-cadherin, Pitx2-positive oral non-neuroepithelial tissue. Further, part of the non-neuroepithelial tissue is Lhx3, Six1, Sox2-positive, and it can be seen that a Rathke's cyst-like structure and a pituitary base plate in which the non-neuroepithelial tissue is trapped are formed. In addition, between the non-neuroepithelial tissue on the outside and the neuroepithelial tissue on the inside, there are head mesenchymal cells that are positive for nestin, vimentin, and weakly positive for pan-cytokeratin. Furthermore, by adding a BMP signaling pathway inhibitor, cell aggregates were obtained in which the proportion of Lhx3-positive cells, a pituitary placode marker, on the surface of the cell aggregates was extremely high.

[0466] These results indicate that the proportion of pituitary placodes formed in a non-neural epithelial part can be increased by inducing differentiation of human iPS cells using a BMP signaling pathway activating substance and, after a certain period of time, a BMP signaling pathway inhibitor.

[0467] Industrial Applicability

[0468] According to the present invention, a cell mass containing central nervous system tissues such as the ventral diencephalon, head mesenchymal cells and pituitary tissues can be efficiently prepared from pluripotent stem cells at low cost.

[0469] This application is based on Japanese Patent Application No. 2017-226312 (filing date: November 24, 2017) filed in Japan, the entire contents of which are incorporated herein by reference.

Claims

1. A method for preparing a cell mass containing pituitary tissue, comprising the following steps (a), (1) and (2): (a) step a, culturing pluripotent stem cells in a medium containing 1) a substance inhibiting the TGFβ family signal transduction pathway and / or a substance acting on the Sonic hedgehog signal transduction pathway, and 2) an undifferentiation maintenance factor in the absence of feeder cells; (1) In the first step, the pluripotent stem cells cultured in step a are suspended and cultured in the presence of a Wnt signal transduction pathway inhibitory substance to form cell aggregates; (2) In the second step, the aggregates obtained in the first step are suspended and cultured in the presence of a substance acting on the BMP signal transduction pathway and a substance acting on the Sonic hedgehog signal transduction pathway to obtain cell aggregates containing pituitary tissue, wherein the concentration of the substance acting on the Sonic hedgehog signal transduction pathway is a concentration having a Sonic hedgehog signal transduction promoting activity equivalent to 100 nM to 3 μM of SAG, in, The BMP signal transduction pathway acting substance in step (2) is added within 72 hours after 0.5 hours from the start of suspension culture of pluripotent stem cells in step (1), The TGFβ family signal transduction pathway inhibitory substance is at least one substance selected from SB431542, A83-01, RepSox, SB505124, SB525334, LY2157299, LY2109761, GW788388, LY364947, SD-208 and EW-7197, The Wnt signal transduction pathway inhibitory substance is at least one substance selected from IWP-2, IWP-3, IWP-4, IWP-L6, IWP-12, LGK-974, ETC-159, GNF-6231, Wnt-C59, IWR1-endo, XAV939, MN-64, WIKI4, TC-E5001, JW55, AZ6102 and KY-02111, The BMP signal transduction pathway agent is BMP2 or BMP4, The substance acting on the Sonic hedgehog signal transduction pathway is at least one substance selected from Shh, purmorphamine, GSA-10, Hh-Ag1.5 and SAG.

2. A method for preparing a cell mass containing pituitary tissue, comprising the following steps (a), (1), (2-1) and (2-2): (a) step a, culturing pluripotent stem cells in a medium containing 1) a substance inhibiting the TGFβ family signal transduction pathway and / or a substance acting on the Sonic hedgehog signal transduction pathway, and 2) an undifferentiation maintenance factor in the absence of feeder cells; (1) In the first step, the pluripotent stem cells cultured in step a are suspended and cultured in the presence of a Wnt signal transduction pathway inhibitory substance to form cell aggregates; (2-1) the second step (1), culturing the aggregates obtained in the first step in suspension in the presence of a substance acting on the BMP signal transduction pathway and a substance acting on the Sonic hedgehog signal transduction pathway, wherein the concentration of the substance acting on the Sonic hedgehog signal transduction pathway is a concentration having a Sonic hedgehog signal transduction promoting activity equivalent to 100 nM to 3 μM of SAG; in, The BMP signal transduction pathway acting substance in step (2-1) is added within 72 hours after 0.5 hours from the start of suspension culture of pluripotent stem cells in step (1), (2-2) The second step (2) is to culture the aggregates obtained by suspension culture in the second step (1) in the presence of a substance that inhibits the BMP signal transduction pathway, thereby obtaining a cell mass containing pituitary tissue. in, The TGFβ family signal transduction pathway inhibitory substance is at least one substance selected from SB431542, A83-01, RepSox, SB505124, SB525334, LY2157299, LY2109761, GW788388, LY364947, SD-208 and EW-7197, The Wnt signal transduction pathway inhibitory substance is at least one substance selected from IWP-2, IWP-3, IWP-4, IWP-L6, IWP-12, LGK-974, ETC-159, GNF-6231, Wnt-C59, IWR1-endo, XAV939, MN-64, WIKI4, TC-E5001, JW55, AZ6102 and KY-02111, The BMP signal transduction pathway agent is BMP2 or BMP4, The substance acting on the Sonic hedgehog signal transduction pathway is at least one substance selected from Shh, purmorphamine, GSA-10, Hh-Ag1.5 and SAG, The BMP signal transduction pathway inhibitory substance is at least one substance selected from K02288, Dorsomorphin, LDN-193189, LDN-212854, LDN-214117, ML347, DMH1 and DMH2.

3. The preparation method according to claim 1 or 2, wherein The Wnt signal transduction pathway inhibitory substance is at least one substance selected from IWP-2, IWP-3, IWP-4, IWP-L6, IWP-12, LGK-974, ETC-159, GNF-6231, and Wnt-C59.

4. The preparation method according to claim 1 or 2, wherein: The suspension culture in steps (1), (2), (2-1) and (2-2) is also carried out in the presence of a TGFβ signal transduction pathway inhibitory substance, wherein the TGFβ signal transduction pathway inhibitory substance is at least one substance selected from SB431542, A83-01, RepSox, SB505124, SB525334, LY2157299, LY2109761, GW788388, LY364947, SD-208 and EW-7197.

5. The preparation method according to claim 1 or 2, wherein: At least one step selected from steps (1), (2), (2-1) and (2-2) is performed in the presence of a substance acting on the FGF signal transduction pathway, wherein the substance acting on the FGF signal transduction pathway is FGF2.

6. The preparation method according to claim 1 or 2, wherein: At least one step selected from steps (1), (2), (2-1) and (2-2) is implemented in the presence of a substance acting on the FGF signaling pathway, wherein the substance acting on the FGF signaling pathway is at least one substance selected from thermostable FGF2 and FGF2 sustained-release beads.

7. The preparation method according to claim 1 or 2, wherein: At least one step selected from steps (1), (2), (2-1) and (2-2) is carried out in the presence of a substance acting on the Wnt signaling pathway, wherein the substance acting on the Wnt signaling pathway is at least one substance selected from CHIR99021, CHIR98014, SB216763, SB415286, BIO, BML-284, and SKL2001.

8. The preparation method according to claim 1 or 2, wherein: The suspension culture in any one or more steps selected from steps (1), (2), (2-1) and (2-2) is further performed in the presence of dexamethasone.

9. A method for preparing pituitary tissue, comprising the following steps (a), (1), (2) and (3): (a) step a, culturing pluripotent stem cells in a medium containing 1) a substance inhibiting the TGFβ family signal transduction pathway and / or a substance acting on the Sonic hedgehog signal transduction pathway, and 2) an undifferentiation maintenance factor in the absence of feeder cells; (1) In the first step, pluripotent stem cells are cultured in suspension in the presence of a Wnt signal transduction pathway inhibitor to form cell aggregates; (2) a second step of culturing the aggregates obtained in the first step in suspension in the presence of a substance acting on the BMP signal transduction pathway and a substance acting on the Sonic hedgehog signal transduction pathway to obtain a cell mass containing pituitary tissue, wherein the concentration of the substance acting on the Sonic hedgehog signal transduction pathway is a concentration having a Sonic hedgehog signal transduction promoting activity equivalent to 100 nM to 3 μM of SAG; in, The BMP signal transduction pathway acting substance in step (2) is added within 72 hours after 0.5 hours from the start of suspension culture of pluripotent stem cells in step (1), (3) In the third step, the pituitary tissue is recovered from the cell mass obtained in the second step. in, The TGFβ family signal transduction pathway inhibitory substance is at least one substance selected from SB431542, A83-01, RepSox, SB505124, SB525334, LY2157299, LY2109761, GW788388, LY364947, SD-208 and EW-7197, The Wnt signal transduction pathway inhibitory substance is at least one substance selected from IWP-2, IWP-3, IWP-4, IWP-L6, IWP-12, LGK-974, ETC-159, GNF-6231, Wnt-C59, IWR1-endo, XAV939, MN-64, WIKI4, TC-E5001, JW55, AZ6102 and KY-02111, The BMP signal transduction pathway agent is BMP2 or BMP4, The substance acting on the Sonic hedgehog signal transduction pathway is at least one substance selected from Shh, purmorphamine, GSA-10, Hh-Ag1.5 and SAG.

10. A method for preparing pituitary tissue, comprising the following steps (a), (1), (2-1), (2-2) and (3): (a) step a, culturing pluripotent stem cells in a medium containing 1) a substance inhibiting the TGFβ family signal transduction pathway and / or a substance acting on the Sonic hedgehog signal transduction pathway, and 2) an undifferentiation maintenance factor in the absence of feeder cells; (1) In the first step, pluripotent stem cells are cultured in suspension in the presence of a Wnt signal transduction pathway inhibitor to form cell aggregates; (2-1) the second step (1), culturing the aggregates obtained in the first step in suspension in the presence of a substance acting on the BMP signal transduction pathway and a substance acting on the Sonic hedgehog signal transduction pathway, wherein the concentration of the substance acting on the Sonic hedgehog signal transduction pathway is a concentration having a Sonic hedgehog signal transduction promoting activity equivalent to 100 nM to 3 μM of SAG; in, The BMP signal transduction pathway acting substance in step (2-1) is added within 72 hours after 0.5 hours from the start of suspension culture of pluripotent stem cells in step (1), (2-2) the second step (2), culturing the aggregates obtained by suspension culture in step (1) in the presence of a BMP signal transduction pathway inhibitory substance to obtain cell aggregates containing pituitary tissue; (3) In the third step, the pituitary tissue is recovered from the cell mass obtained in the second step (2), in, The TGFβ family signal transduction pathway inhibitory substance is at least one substance selected from SB431542, A83-01, RepSox, SB505124, SB525334, LY2157299, LY2109761, GW788388, LY364947, SD-208 and EW-7197, The Wnt signal transduction pathway inhibitory substance is at least one substance selected from IWP-2, IWP-3, IWP-4, IWP-L6, IWP-12, LGK-974, ETC-159, GNF-6231, Wnt-C59, IWR1-endo, XAV939, MN-64, WIKI4, TC-E5001, JW55, AZ6102 and KY-02111, The BMP signal transduction pathway agent is BMP2 or BMP4, The substance acting on the Sonic hedgehog signal transduction pathway is at least one substance selected from Shh, purmorphamine, GSA-10, Hh-Ag1.5 and SAG, The BMP signal transduction pathway inhibitory substance is at least one substance selected from K02288, Dorsomorphin, LDN-193189, LDN-212854, LDN-214117, ML347, DMH1 and DMH2.

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