WO2024171957A1 - Transplantation of pituitary hormone-producing cells - Google Patents

Abstract

The present invention provides a therapeutic agent which is for a disease associated with a pituitary gland disorder and which contains pituitary hormone-producing cells, said therapeutic agent being characterized by being used such that the pituitary hormone-producing cells are transplanted to subcutaneous tissue and/or muscle tissue.

Description

Transplantation of pituitary hormone-producing cells

The present invention relates to a therapeutic agent for diseases caused by disorders of the pituitary gland, which contains pituitary hormone-producing cells and is characterized in that the pituitary hormone-producing cells are used to be transplanted into the subcutaneous tissue and/or muscle tissue of a subject.

The pituitary gland is an endocrine organ located in the head that produces various pituitary hormones, such as adrenocorticotropic hormone (ACTH) and growth hormone, which are important for the maintenance and growth of the body. When pituitary gland malfunctions due to diseases such as pituitary aplasia, hypopituitarism, or pituitary adenoma, serious symptoms such as growth disorders, reproductive organ abnormalities, and abnormalities of the adrenal gland or thyroid gland occur. In general, it is rare for damaged pituitary tissue to regenerate naturally and regain function.

For example, current treatments for hypopituitarism are usually limited to administering the hormone identified as deficient, but adjusting the dosage is difficult. Furthermore, administering such hormones can be problematic, such as forgetting to administer the hormone due to human error. In this regard, if pituitary hormone-producing cells could be deployed clinically, these problems associated with hormone replacement therapy could be resolved. Based on this background, the present inventors have established and improved a method for producing pituitary hormone-producing cells derived from pluripotent stem cells, making it possible to produce high-quality cell populations (Patent Documents 1 to 4, Non-Patent Documents 1 and 2).

　Traditionally, when pituitary hormone-producing cells were transplanted into mice to confirm the hormone secretion effect, etc., they were transplanted under the kidney capsule, but this was highly invasive and placed a heavy burden on the recipient, which was an issue. On the other hand, transplantation into subcutaneous tissue or muscle tissue is considered to be a simple and safe method, as it is less invasive and can be easily removed surgically even if the transplanted cells become tumorous and require removal. However, subcutaneous tissue itself has few blood vessels, making it difficult for transplanted cells to take root, and it was generally thought that subcutaneous tissue was not suitable for transplanting such cells.

International Publication No. 2013/065763 International Publication No. 2016/013669 International Publication No. 2019/103129 PCT/JP2022/36019

Suga et al. Nature. 2011 Nov9; 480(7375): 57-62. Ozone et al. Nature communications 7.10351 (2016): 1-10.

The objective of the present invention is therefore to provide a therapeutic agent for diseases caused by disorders of the pituitary gland, which contains pituitary hormone-producing cells and is characterized by being used for transplantation into a transplantation site (subcutaneous tissue (particularly subcutaneous adipose tissue) and/or muscle tissue) that is less invasive and can be easily surgically removed even if the transplanted cells become tumorous and require removal, in order to realize clinical application of the pituitary hormone-producing cells.

The present inventors have found that pituitary hormone-producing cells induced to differentiate in vitro from pluripotent stem cells can be engrafted when transplanted into subcutaneous tissue, despite the existing belief that subcutaneous tissue is unsuitable for transplantation of cells because of the lack of blood vessels in the subcutaneous tissue itself. That is, as a result of extensive investigations into the pituitary hormone-producing cells derived from pluripotent stem cells produced by the present inventors, they surprisingly found that when the pituitary hormone-producing cells were transplanted into two types of subcutaneous tissue in mice, namely the avascular region (AR) directly beneath the skin of the back and the subcutaneous tissue of the groin rich in white adipose tissue (ISWAT), the cells survived for more than six months and exhibited hormone secretion function. Furthermore, the inventors have found that transplantation into these regions, particularly into the groin, armpits, abdomen, back, groin, thighs, buttocks, upper arms, etc., which are rich in white fat, is more effective, and that angiogenesis is involved in cell engraftment. The inventors have also found that muscle tissue present under the subcutaneous tissue is suitable for transplantation of the pituitary hormone-producing cells, just like subcutaneous tissue. As a result of further research based on these findings, the inventors have completed the present invention, which is characterized in that the pituitary hormone-producing cells are used for transplantation into subcutaneous tissue and/or muscle tissue.

That is, the present invention is as follows.
[1] A therapeutic agent for a disease caused by a disorder of the pituitary gland, comprising pituitary hormone-producing cells, characterized in that the pituitary hormone-producing cells are used to be transplanted into the subcutaneous tissue and/or muscle tissue of a subject.
[2] The therapeutic agent according to [1], wherein the subcutaneous tissue is subcutaneous tissue present in at least one area selected from the groin, armpit, back, abdomen, thigh, buttocks, upper arm, and scalp.
[3] The therapeutic agent described in [1] or [2], wherein the subcutaneous tissue is subcutaneous adipose tissue.
[4] The therapeutic agent described in [3], wherein the adipose tissue is white adipose tissue.
[5] The therapeutic agent described in [1], wherein the muscle tissue is muscle tissue present in at least one area selected from the back, abdomen, thighs, buttocks, and upper arms.
[6] The therapeutic agent according to any one of [1] to [5], wherein the pituitary hormone-producing cells are used so as to be transplanted near blood vessels in the tissue.
[7] The therapeutic agent according to any one of [1] to [6], characterized in that the pituitary hormone-producing cells are used so as to be transplanted into an artificially created space in the tissue.
[8] The therapeutic agent according to any one of [1] to [7], wherein the disease caused by a pituitary disorder is at least one disease selected from hypopituitarism, pituitary dwarfism, adrenal insufficiency, partial hypopituitarism, isolated anterior pituitary hormone deficiency, and craniopharyngioma.
[9] The therapeutic agent according to any one of [1] to [8], wherein the pituitary hormone-producing cells are cell aggregates containing pituitary hormone-producing cells.
[10] The therapeutic agent according to any one of [1] to [9], wherein the pituitary hormone-producing cells are pituitary hormone-producing cells that express an angiogenic factor.
[11] The therapeutic agent described in [10], wherein the angiogenic factor is one or more selected from the group consisting of VEGFA, VEGFB, VEGFC and ANGPT2.
[12] The therapeutic agent according to any one of [1] to [11], wherein the pituitary hormone-producing cells are pituitary hormone-producing cells produced by a method comprising the steps of:
(1) a first step of culturing pluripotent stem cells in the presence of a c-jun N-terminal kinase (JNK) signaling pathway inhibitor and a Wnt signaling pathway inhibitor to obtain a cell population;
(2) A second step of culturing the cell population obtained in the first step in the presence of a substance acting on the BMP signaling pathway and a substance acting on the Sonic Hedgehog signaling pathway to obtain a cell population containing pituitary hormone-producing cells.
[13] The therapeutic agent according to [12], wherein the process further comprises the following step a before the first step:
(a) A step a of culturing pluripotent stem cells in a medium containing 1) a TGFβ family signaling pathway inhibitor and/or a Sonic hedgehog signaling pathway agonist, and 2) an undifferentiated state maintenance factor, in the absence of feeder cells.
[14]
The therapeutic agent according to [12] or [13], wherein the step further comprises the following step after the second step:
A step of selecting and recovering pituitary hormone-producing cells from the cell population containing pituitary hormone-producing cells obtained in the second step.
[15] The therapeutic agent according to any one of [1] to [14], wherein angiogenesis occurs around the transplanted pituitary hormone-producing cells in a human subject after transplantation.
[16] The therapeutic agent according to any one of [1] to [15], which is used in combination with an angiogenesis promoter.
[17] The therapeutic agent according to any one of [1] to [16], wherein the pituitary hormone-producing cells are engrafted in subcutaneous tissue and/or muscle tissue of a human subject after transplantation.
[18] The therapeutic agent according to any one of [1] to [17], characterized in that a pituitary hormone is secreted into the blood in a human subject after transplantation.
[19] A method for treating a disease caused by a pituitary gland disorder, comprising administering pituitary hormone-producing cells to the subcutaneous tissue and/or muscle tissue of a human subject.
[20] A method for producing a non-human animal model bearing human pituitary tissue, which comprises administering human pituitary hormone-producing cells into the subcutaneous tissue and/or muscle tissue of the non-human animal.
[21] A non-human animal model bearing human pituitary gland tissue, which comprises human pituitary hormone-producing cells in subcutaneous tissue and/or muscle tissue, and which is capable of secreting pituitary hormones produced by the human pituitary hormone-producing cells into blood.
[22] A method for evaluating the efficacy and safety of a test substance, comprising administering the test substance to the non-human animal model bearing human pituitary tissue according to [21].

The present invention makes it possible to provide a therapeutic agent for diseases caused by disorders of the pituitary gland, which contains pituitary hormone-producing cells and is characterized by being used for transplantation into subcutaneous tissue and/or muscle tissue that is less invasive and can be easily surgically removed even if the transplanted cells become tumorous and require removal. Furthermore, the therapeutic agent of the present invention makes it possible to realize clinical applications of pituitary hormone-producing cells. Furthermore, the method of the present invention makes it possible to create model animals having human pituitary tissue that maintains the ability to secrete human hormones for a long period of time, which is useful for evaluating the efficacy and safety of compounds.

Figure 1 shows immunofluorescence micrographs of cultured pituitary organoids (PO) 100 days after the start of differentiation induction. (A) Adrenocorticotropic hormone (ACTH), (B) E-cadherin (E-cad), (C) LIM homeobox protein 3 (LHX3), (D, E) Merged images using ACTH, E-cadherin, LHX3, and 4,6-diamidine-2-phenylindole dihydrochloride (DAPI). Scale bars are 100 μm. Figure 2: Inguinal subcutaneous white adipose tissue (ISWAT) implantation of hESC-derived PO. (A) Schematic diagram, mouse processing protocol (hypophysectomy, confirmation of hypopituitarism, PO implantation, and corticotropin-releasing hormone (CRH) challenge). (B) Shaved left groin, mouse in supine position restrained under inhalation anesthesia. (C) Membranous adipose tissue vessels (arrowheads) overlying the femoral vein and artery (arrows) as viewed through a 4 mm vertical skin incision. (D) Pocket created in ISWAT. (E) Placement of PO into pocket via syringe fitted with wide-bore tip. (F) Placed PO. (G) Nylon suture closure of adipose tissue over PO. Scale interval, 1 mm. FIG. 3 is a diagrammatic example of the implantation method. (A, B) A gelatin hydrogel sustained release device (Gel) containing basic fibroblast growth factor with heparin was placed under the skin. After one week, the device was replaced with a PO. The placement site was vascularized and prone to bleeding. (C) Subcutaneous device-less (DL) replacement. The PO was placed in the cavity where the nylon catheter was embedded. (D, E) Inguinal subcutaneous white adipose tissue (ISWAT) implantation method. (F) The PO was placed untreated (NT) in a poorly vascularized area of the back subcutaneous layer of the mouse. (G, H) Comparison of basal and corticotropin-releasing hormone (CRH)-stimulated plasma adrenocorticotropic hormone (ACTH) levels for each method. Figure 4 relates to the evaluation of transplanted mice. All data are presented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. (A) Basal and corticotropin releasing hormone (CRH) stimulated plasma ACTH levels in mice subjected to PO transplantation. Sham-operated (Sham), avascular area (AR), and ISWAT cohorts. Sham, n=6. AR, n=5. ISWAT, n=6. Statistical evaluation, one-way ANOVA with post-hoc Tukey test. (B) Running wheel activity test. Sham, n=3. ISWAT, n=3. Student's t-test. (C) Percentage of body weight change in ISWAT and sham-operated mice. Percentage of body weight change, abscissa; time course, ordinate. Student's t-test. FIG. 5: Macroscopic and histological findings after ISWAT transplantation. (A) Macroscopic appearance, transplantation site, 4 weeks after transplantation. Transplanted PO (arrow), blood vessel associated with the graft (arrowhead). (B) Macroscopic appearance, 21 weeks after transplantation. Transplanted PO (arrow). (C, I) Skin and underlying tissue containing graft. Multiple POs are included. Hematoxylin/eosin (HE). Black box areas D-N. (D-H, J-N) Immunofluorescence micrographs. Counterstained with 4,6-diamidine-2-phenylindole dihydrochloride (DAPI). (D, J) Pituitary progenitor cell markers adrenocorticotropic hormone (ACTH) and LIM homeobox protein 3 (LHX3). (E, K) Pituitary progenitor cell marker adrenocorticotropic hormone (ACTH). (F, L) Human nuclei (hunuclei) and E-cadherin (E-cad), an oral ectoderm marker. (G, M) Human nuclei (hunuclei). (H, N) Smooth muscle actin (SMA), a vascular wall marker. Scale bars are 100 μm. Figure 6 shows histological findings of AR mice. (A) Hematoxylin/eosin (HE) staining. Black boxes B-F. Grey boxes G-K. (B-K) Immunofluorescence micrographs. (B, G). Adrenocorticotropic hormone (ACTH) and LIM homeobox protein 3 (LHX3). Staining in the dermis is a nonspecific reaction. (C, H) Adrenocorticotropic hormone (ACTH). (D, I) Human nuclei (hunuclei) and E-cadherin (E-cad). (E, J) Human nuclei (hunuclei). (F, K) Smooth muscle actin (SMA). Counterstaining with 4,6-diamidine-2-phenylindole dihydrochloride (DAPI), B-K. Scale bar 100 μm. Figure 7: Expression and effects of angiogenic factors in hESC-derived PO. All data are shown as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. Quantitative PCR results for expression of VEGFA (A), VEGFB (B), VEGFC (C), FGF2 (D), and ANGPT2 (E). Graphs show PO without transplantation, transplanted PO ("with transplantation"), PO Tx (-) and PO Tx (+), respectively, and undifferentiated ESCs. Expression was normalized to GAPDH expression. PO Tx (-), n=3. PO Tx (+), n=3. ESC, n=3. Statistical evaluation, one-way ANOVA with post-hoc Tukey's test. (F) Comparison of basal and corticotropin-releasing hormone (CRH)-stimulated plasma ACTH levels following bevacizumab (BEV) or vehicle administration. BEV, n=9. Vehicle, n=9. Student's t-test. Figure 8 relates to the evaluation of transplanted mice. All data are shown as mean ± SEM. *p<0.05. Plasma ACTH levels, basal and corticotropin releasing hormone (CRH) stimulated, in mice subjected to PO transplantation up to 6 months after transplantation. Inguinal (inguinal subcutaneous white adipose tissue transplantation), Axilla (axillary white adipose tissue transplantation), Muscle (intramuscular transplantation). Inguinal, n=2. Axilla, n=3. Muscle, n=4. Statistical evaluation, Criskal-Wallis test. Figure 9 relates to the evaluation of transplanted mice. All data are shown as mean ± SEM. Plasma ACTH levels, basal and corticotropin releasing hormone (CRH) stimulated, in mice subjected to pituitary hormone-producing cell transplantation up to 3 months after transplantation. ISWAT (inguinal white adipose tissue transplantation), n=2. Figure 10 relates to the evaluation of transplanted mice. Survival curve. *p<0.05. Sham, n=3. Axilla, n=5. Inguinal, n=3. Muscle, n=6. Statistical evaluation, log-rank test. Figure 11 relates to the evaluation of transplanted mice. All data are shown as mean ± SEM. Plasma ACTH levels, basal and stimulated with corticotropin releasing hormone (CRH), in mice subjected to pituitary hormone-producing cell transplantation up to one month after transplantation. AR group (back avascular field transplantation group), bFGF group (bFGF + sodium hyaluronate mixture was locally injected subcutaneously into the back), Duragen group (bFGF + sodium hyaluronate mixture was soaked in a Duragen sheet and left subcutaneously on the back for one week, then removed), Surgicel group (bFGF + sodium hyaluronate mixture was soaked in a Surgicel sheet and left subcutaneously on the back for one week). AR group, n=2, bFGF group, n=3, Duragen group, n=3, Surgicel group, n=3. Figure 12 relates to the evaluation of transplanted monkeys. n=1. (A) Baseline plasma ACTH levels in monkeys subjected to PO transplantation up to 6 weeks post-transplant. (B) Baseline Cortisol levels in monkeys subjected to PO transplantation up to 1 month post-transplant. Figure 13 relates to the evaluation of transplanted mice. n=1. (A) Plasma ACTH levels, basal and stimulated with corticotropin releasing hormone (CRH), in monkeys subjected to pituitary hormone-producing cell transplantation up to one month after transplantation. (B) Dexamethasone suppression test, plasma ACTH levels, performed 6 weeks after transplantation.

1. Therapeutic Agent and Cell Transplantation of the Present Invention The present invention relates to a therapeutic agent for diseases due to disorders of the pituitary gland, comprising pituitary hormone-producing cells, characterized in that the pituitary hormone-producing cells are used to be transplanted into subcutaneous tissue, preferably subcutaneous adipose tissue, more preferably subcutaneous white adipose tissue and/or muscle tissue. In this specification, "pituitary hormone-producing cells" refers to cells that produce growth hormone (GH), prolactin (PRL), adrenocorticotropic hormone (ACTH), thyroid stimulating hormone (TSH), follicle stimulating hormone (FSH), luteinizing hormone (LH), melanocyte stimulating hormone (MSH), or the like, and is a general term for cells that produce at least one of these pituitary hormones.

The pituitary hormone-producing cells contained in the therapeutic agent of the present invention are not particularly limited in terms of their origin, production method, etc., as long as they are capable of producing the pituitary hormones described above, but are preferably pituitary hormone-producing cells induced to differentiate from pluripotent stem cells.
In one embodiment, the pituitary hormone-producing cells in the present specification include at least one type selected from the group consisting of adrenocorticotropic hormone (ACTH)-producing cells, growth hormone (GH)-producing cells, and prolactin (PRL)-producing cells.
In one embodiment, the pituitary hormone-producing cells contained in the therapeutic agent of the present invention include at least one type (one, two or three types) selected from the group consisting of adrenocorticotropic hormone (ACTH)-producing cells, growth hormone (GH)-producing cells and prolactin (PRL)-producing cells. Furthermore, in one embodiment, the cell population containing the pituitary hormone-producing cells in the present specification has the following characteristics.

Specifically, the cell population containing the pituitary hormone-producing cells preferably contains cells expressing an angiogenic factor. Here, the angiogenic factor is not particularly limited as long as it is a factor involved in angiogenesis, preferably a factor involved in promoting angiogenesis, and examples thereof include angiogenin, angiopoietin-1, Del-1, fibroblast growth factors: acidic (aFGF) and basic (bFGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), follistatin, granulocyte macrophage-colony stimulating factor (GM-CSF), hepatocyte growth factor (HGF), monocyte chemotactic protein-1 (MCP-1), sphingosine-1-phosphate (S1P), platelet-derived growth factor (PDGF), transforming growth factor α (TGF-α), transforming growth factor β (TGF-β), tumor necrosis factor α (TNF-α), and the like.
In one embodiment, the angiogenic factor is one or more selected from the group consisting of vascular endothelial growth factor A (VEGFA), vascular endothelial growth factor B (VEGFB), vascular endothelial growth factor C (VEGFC), and angiopoietin-2 (ANGPT2). In addition, in the present invention, from the viewpoint of transplanted cells, it has been found that the cells have a characteristic that, by expressing an angiogenic factor, angiogenesis can occur around the pituitary hormone-producing cells contained in the therapeutic agent of the present invention after transplantation. Furthermore, angiogenesis can occur around the pituitary hormone-producing cells, allowing the pituitary hormone-producing cells to be engrafted in subcutaneous tissue or muscle tissue.

Specifically, the cell population containing the pituitary hormone-producing cells may contain cells that can differentiate into pituitary hormone-producing cells. Examples of such cells include pituitary stem cells or pituitary progenitor cells (precursor cells of pituitary hormone-producing cells). That is, the cell population containing the pituitary hormone-producing cells of the present invention includes cell populations containing pituitary hormone-producing cells, and pituitary stem cells and/or pituitary progenitor cells.
That is, the cell population may contain cells expressing a pituitary stem cell marker. Examples of the pituitary stem cell marker include at least one of Sox2, Sox9, E-Cadherin, Nestin, S100β, GFRα2, Prop1, CD133, β-Catenin, Klf4, Oct4, Pax6, coxsackievirus-adenovirus common receptor (CXADR), PRRX1/2, Ephrin-B2, and ACE. In one embodiment, the pituitary stem cell marker may be SOX2 and/or CXADR.

The pituitary hormone-producing cells contained in the therapeutic agent of the present invention may be contained as at least one type of pituitary hormone-producing cells, or may be a cell population containing a single type or multiple types of pituitary hormone-producing cells.
The cell population may be a dispersed cell population, a cell aggregate containing pituitary hormone-producing cells, or a two-dimensional cell sheet.
In one embodiment, the cell population includes a cell aggregate containing pituitary tissue containing pituitary hormone-producing cells and tissue other than pituitary tissue (for example, hypothalamic tissue, etc.).
In one embodiment, the cell population includes pituitary tissue containing pituitary hormone-producing cells, and pituitary tissue recovered from a cell population containing tissue other than pituitary tissue (e.g., hypothalamic tissue, etc.).
As used herein, cell aggregates containing pituitary hormone-producing cells are also referred to as pituitary organoids (PO).
In one embodiment, the cell population is a cell aggregate.

In one aspect, the cell population containing pituitary hormone-producing cells contained in the therapeutic agent of the present invention is preferably a cell population containing pituitary hormone-producing cells at least 5% or more, preferably 10% or more of the total cell number.
The pituitary hormone-producing cells contained in the therapeutic agent of the present invention can be identified using the pituitary hormone-producing markers described below as indicators.

In one embodiment, the pituitary hormone-producing cells contained in the therapeutic agent of the present invention contain at least ACTH-producing cells.
In one embodiment, the cell population containing pituitary hormone-producing cells contained in the therapeutic agent of the present invention is preferably a cell population containing ACTH-positive cells at 3% or more, preferably 5% or more of the total cell number.

In one embodiment, the major axis (or equivalent circle diameter) of the cell aggregate containing pituitary hormone-producing cells contained in the therapeutic agent of the present invention may be, for example, 0.1 to 7 mm, and preferably 0.4 to 5 mm.
The method for measuring the major axis, minor axis and height of the cell aggregate is not particularly limited, and may be measured from an image captured under a microscope. For example, the cell aggregate during culture in a 96-well culture plate can be imaged with a 10x lens of a Keyence inverted microscope, and the height can be measured from the image. Here, the major axis refers to the longest line segment and its length among the line segments connecting the two end points of the cell aggregate in the captured image. The circle equivalent diameter refers to the diameter of a perfect circle, which corresponds to the area of a figure (circular or elliptical) obtained when projected onto a two-dimensional surface. As described later, the aggregate is a spherical aggregate, and the major axis and the circle equivalent diameter are approximate, and the difference between the major axis and the circle equivalent diameter becomes smaller as the aggregate approaches a spherical aggregate close to a perfect sphere.

In one embodiment, the cell population containing pituitary hormone-producing cells contained in the therapeutic agent of the present invention may contain cells of hypothalamic tissue. Such cells include Rx-positive, Pax6-positive and Nkx2.1-negative cells (dorsal hypothalamus) and Rx-positive, Pax6-negative and Nkx2.1-positive cells (ventral hypothalamus).

In one embodiment, the cell population containing pituitary hormone-producing cells included in the therapeutic agent of the present invention may contain pituitary stem cells. The cell population contains at least 1% or more, preferably 2% or more, of the total number of cells that are pituitary stem cells, i.e., cells that are positive for any of the above-mentioned pituitary stem cell markers. Examples of the pituitary stem cell marker include SOX2 and CXADR.

In one embodiment, the cell population containing pituitary hormone-producing cells contained in the therapeutic agent of the present invention may be a cell population from which non-target cells other than pituitary hormone-producing cells generated in the differentiation induction step have been removed. A cell population that does not contain non-target cells or has a reduced proportion of non-target cells can be obtained by selecting pituitary hormone-producing cells using as an index the expression of a marker protein expressed on the cell surface that is expressed in pituitary hormone-producing cells or their intermediates (precursor cells of pituitary hormone-producing cells) and not expressed in non-target cells. Examples of the marker protein include EpCAM (see International Publication No. WO 2021/201175).

The cell population (hereinafter sometimes referred to as "purified body") that does not contain the above-mentioned undesired cells or in which the proportion of undesired cells is reduced can be obtained, for example, by the method described below in "7. Method for producing purified body". In one embodiment, the cell population (purified body) is characterized in that the proportion of EpCAM and/or E-cadherin positive cells in the cell population (cell aggregate) in which the proportion of undesired cells is reduced may be 80% or more, and preferably 85% or more, 90% or more, or 95% or more.

In one embodiment, the proportion of neural cells in a cell population in which the proportion of non-target cells is reduced may be 20% or less, and preferably 15% or less, 10% or less, or 5% or less.

In one embodiment, the proportion of ACTH-positive cells in a cell population in which the proportion of non-target cells is reduced may be 5% or more, and preferably 10% or more. ACTH-positive cells simultaneously express NeuroD1 and Tbx19. Therefore, the proportion of NeuroD1-positive and/or Tbx19-positive cells to the total number of cells present in the cell population may be the same as the proportion of ACTH-positive cells.

In one embodiment, the ACTH secretion ability in a cell population in which the proportion of non-target cells is reduced may be 200 to 1,000,000 pg/ml, 1,000 to 500,000 pg/ml, 5,000 to 300,000 pg/ml, or 10,000 to 100,000 pg/ml.

ACTH secretion ability is affected by the number of ACTH cells and culture conditions. ACTH secretion ability can be expressed as the concentration of ACTH secreted into the medium from one cell population (cell aggregate) under specific culture conditions. For example, the specific culture conditions refer to the following conditions: (i) a medium that can be used for culturing pituitary hormone-producing cells, preferably a serum-free medium containing KSR (e.g., IMDM, DMEM, EMEM, αMEM, GMEM, F-12 medium, DMEM/F12, IMDM/F12, epithelial cell medium (e.g., CnT-Prime epithelial culture medium), or a mixed medium thereof, is used; (ii) no substance that affects ACTH production from the outside is added (e.g., Notch signal inhibitors that increase ACTH production); (iii) the medium volume is set to 0.18 to 20 ml; and (iv) the culture is performed for 3 to 4 days. After culturing under these conditions, the medium is collected and the concentration of ACTH in the medium is measured. Those skilled in the art can measure the concentration of ACTH using known techniques such as ELISA.

In one embodiment, the ratio of the number of pituitary stem cells to the total number of cells present in a cell population in which the proportion of non-target cells is reduced (the proportion of pituitary stem cells present) may be 1% or more, preferably 3% or more or 5% or more. As described above, pituitary stem cells simultaneously express pituitary stem cell markers such as Sox2, Sox9, E-Cadherin, Nestin, S100β, GFRα2, Prop1, CD133, β-Catenin, Klf4, Oct4, Pax6, coxsackievirus-adenovirus common receptor (CXADR), PRRX1/2, Ephrin-B2, and ACE. Therefore, the proportion of pituitary stem cells present can be determined by measuring the proportion of cells positive for the above pituitary stem cell markers (e.g., Sox2, CXADR) to the total number of cells present in the cell population.

In one embodiment, the cell density of the cell population in which the proportion of non-target cells is reduced may be 3,000 to 20,000 cells/mm ² , preferably 5,000 to 10,000 cells/mm ² , and more preferably 6,000 to 9,000 cells/mm ² .

In the present specification, the cell density of a cell population in which the proportion of non-target cells has been reduced can be expressed as the number of cells per unit area on the cut surface of the cell population, for example, at a position within 200 μm from the center. A person skilled in the art can measure the cell density of a cell population using known methods. Specifically, a slice is prepared by cutting the cell population, and the number of cells is measured by a method such as immunostaining using DAPI or anti-E-cadherin antibody, and then divided by the area of the slice.

In one embodiment, the diameter of the cell population in which the proportion of non-target cells is reduced may be 200 to 3,000 μm, preferably 300 to 2,000 μm, and more preferably 400 to 1,500 μm.

In a further embodiment, the cell population in which the proportion of non-target cells is reduced may be a spherical cell aggregate. In this specification, "spherical cell aggregate" refers to a cell aggregate having a three-dimensional shape close to a sphere. A three-dimensional shape close to a sphere is a shape having a three-dimensional structure, and examples of such shapes include a spherical shape that is circular or elliptical when projected onto a two-dimensional surface.

The cell aggregate is preferably a spherical cell aggregate close to a perfect sphere. A spherical cell aggregate close to a perfect sphere means a cell aggregate having a three-dimensional shape close to a perfect sphere. A three-dimensional shape close to a perfect sphere means a spherical shape in which the multiple circles obtained when projected onto a two-dimensional surface from multiple directions all have the same length from the center to the circumference of the circles, or the difference between the longest and shortest line segments is 10% or less, 5% or less, 3% or less, 2% or less, or 1% or less.

In one embodiment, a cell population in which the proportion of non-target cells has been reduced may have, on its surface, EpCAM-positive cells and cells positive for at least one of E-cadherin that adhere to each other to form an epithelial structure.

As used herein, "surface adhesion" means that adjacent cells adhere to each other at their surfaces. More specifically, surface adhesion means that the proportion of the surface area of a cell that is adhered to the surface of another cell is, for example, 1% or more, preferably 3% or more, more preferably 5% or more, and even more preferably 10% or more. In one embodiment, the cell surface means the cell membrane surface. The cell surface can be observed by immunostaining for cell adhesion factors (e.g., E-cadherin, EpCAM, etc.). Point adhesion is an example of a state that is not surface adhesion. Point adhesion means that cells adhere to each other at points.

In this specification, "epithelial structure" refers to a structure that epithelial tissue has characteristically, such as the apical surface or basement membrane. This can be confirmed by immunostaining using markers for the apical surface and basement membrane, which will be described later.

Epithelial tissue is polarized to form an "apical surface" and a "basement membrane." The term "basement membrane" refers to a basement membrane that contains a 50-100 nm basal layer (basement membrane) produced by epithelial cells that is rich in laminin and type IV collagen, which are markers of the basement membrane. The term "apical surface" refers to the surface (superficial surface) that is formed on the opposite side of the "basement membrane." Such apical surfaces can be identified by immunostaining methods well known to those skilled in the art using antibodies against apical surface markers (e.g., atypical-PKC (hereinafter sometimes abbreviated as "aPKC"), E-cadherin, and N-cadherin).

"Epithelial tissue" is tissue formed when cells tightly cover the surface of the body, lumen (such as the digestive tract), and body cavity (such as the pericardial cavity). The cells that form epithelial tissue are called epithelial cells. Epithelial cells have apical-basal polarity. Epithelial cells form strong bonds with each other through adherens junctions and/or tight junctions, and can form cell layers. Epithelial tissue is a tissue formed when one to a dozen or so cell layers are stacked on top of each other.

In one embodiment, the cell population in which the proportion of non-target cells is reduced may have a plurality of island-like structures formed by surface adhesion of EpCAM-positive cells and/or E-cadherin-positive cells to each other. In a further embodiment, the cell population may have a sponge-like structure formed by a collection of a plurality of island-like structures.

The term "islet-like structure" refers to a structure formed by surface adhesion of about 2 to 20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 15, 20) EpCAM-positive cells and/or E-cadherin-positive cells to each other. The number of cells contained in each island-like structure may differ for each island-like structure. Surface adhesion means that adjacent cells are adhered to each other by their surfaces. More specifically, surface adhesion means that the rate at which cells are adhered to each other by their surfaces is, for example, 1% or more, preferably 3% or more, more preferably 5% or more, and even more preferably 10% or more. The major axis of the cells present in the island-like structure may be, for example, 2 to 50 μm, preferably 4 to 30 μm, and the minor axis may be, for example, 0.5 to 20 μm, preferably 1 to 10 μm.

The island structure may contain pituitary hormone-producing cells. Specific examples include ACTH-positive cells, PRL-positive cells, FSH-positive cells, LH-positive cells, GH-positive cells, TSH-positive cells, and MSH-positive cells.

A "sponge-like structure" is a structure formed by a group of multiple island structures with voids between the island structures. It is not necessary that there are voids between all of the island structures. The presence of voids allows nutrients and oxygen from the culture medium to reach the inside of the cell mass, so the proportion of necrotic cells is low even in the center of the cell mass.

"Island-like structures" and "sponge-like structures" are typically not observed before sorting with EpCAM, i.e., in cell aggregates obtained by culturing pluripotent stem cells in suspension. "Island-like structures" and "sponge-like structures" are typically structures that are first identified upon re-aggregation after sorting with EpCAM.

In one embodiment, the GH secretion ability of the cell population in which the proportion of non-target cells is reduced may be 0.05 to 100 ng/ml, and in another embodiment, may be 0.1 to 50 ng/ml, 0.3 to 30 ng/ml, or 0.5 to 10 ng/ml.

GH secretion ability is affected by the number of GH cells and culture conditions. GH secretion ability can be expressed as the concentration of GH secreted into the medium from one cell group (cell aggregate) under specific culture conditions. For example, the specific culture conditions refer to the following conditions: (i) a medium that can be used for culturing pituitary hormone-producing cells, preferably a serum-free medium containing KSR (e.g., DMEM, MEM, GMEM, epithelial cell medium (e.g., CnT-Prime epithelial culture medium) is used, (ii) no substances that affect GH production from the outside are added (e.g., adrenal cortical hormones that increase GH production (e.g., dexamethasone)), (iii) the medium volume is set to 0.18 to 20 ml, and (iv) the culture is performed for 3 to 4 days. The medium after culture under the above conditions is collected, and the GH concentration in the medium is measured. A person skilled in the art can measure the GH concentration using a known method such as ELISA.

　A cell population with a reduced proportion of non-target cells may be cultured in the presence of a specific substance depending on the intended use of the cell population to increase the amount of secretion of a specific pituitary hormone.

For example, in the above method, the GH secretion ability is improved by changing the condition (ii) to a condition of culturing in a medium containing adrenal cortical hormones. Examples of adrenal cortical hormones include artificially synthesized glucocorticoids such as dexamethasone, betamethasone, prednisolone, methylprednisolone, and triamcinolone; and natural glucocorticoids such as hydrocortisone, cortisone acetate, and fludrocortisone acetate. A person skilled in the art can appropriately set the concentration of the adrenal cortical hormones, but in one embodiment, the concentration of dexamethasone may be 4 ng/ml to 40 μg/ml, preferably 40 ng/ml to 4 μg/ml.

In one embodiment, the GH secretion ability of the cell population containing pituitary hormone-producing cells after culturing in the presence of adrenal cortical hormones may be 0.1 to 10,000 ng/ml, 0.5 to 5,000 ng/ml, 5 to 1,000 ng/ml, or 20 to 500 ng/ml. In this case, the ACTH secretion ability is 10 to 500,000 pg/ml, preferably 100 to 100,000 pg/ml, more preferably 500 to 30,000 pg/ml, and even more preferably 1,000 to 10,000 pg/ml.

In this way, the cell population containing pituitary hormone-producing cells after culturing in the presence of corticosteroids contains more pituitary hormone-producing cells than before the culturing. In one embodiment, when cultured in the presence of corticosteroids, the ratio of the number of ACTH-positive (producing) cells to the total number of cells present in the cell population (the proportion of ACTH-positive cells present) may decrease. In that case, the proportion of ACTH-positive cells present is 2% or more, preferably 3% or more, and more preferably 5% or more.

As described above, the cell population (purified product) in which the proportion of undesired cells is reduced may, in one embodiment, have at least one of the following characteristics (i) to (vi).
(i) The proportion of cells positive for at least one of EpCAM and E-cadherin in the cell population is 80% or more.
(ii) The proportion of neural cells in the cell population is 20% or less.
(iii) The proportion of ACTH-positive cells in the cell population is 5% or more.
(iv) The cell density of the cell population is 3,000 to 20,000 cells/ ^mm2 .
(v) the major axis of the cell population is 200 to 3,000 μm.
(vi) On the surface of the cell population, cells positive for at least one of EpCAM and E-cadherin adhere to each other to form an epithelial structure.
(vii) The cell population forms a sponge-like structure.

In a further embodiment, the cell population (purified product) having at least one of the characteristics (i) to (vi) may also have at least one of the characteristics (viii) to (x) below.
(viii) the proportion of pituitary stem cells in the cell population is 3% or more, (ix) the ACTH secretion ability of the cell population is 200 to 1,000,000 pg/ml.
(x) The cell population has a GH secretion ability of 0.05 to 100 ng/ml.

In one embodiment, the cell population (purified body) in which the proportion of non-target cells is reduced may be characterized in that the proportion of EpCAM-positive cells and E-cadherin-positive cells is 80% or more, the proportion of nervous system cells is 20% or less, and the ACTH secretion ability of the cell population is 200 to 1,000,000 pg/ml.

The therapeutic agent of the present invention is used to be implanted into subcutaneous tissue and/or muscle tissue. In this specification, "subcutaneous tissue" refers to the tissue located at the bottom of the three-layer structure of the skin (i.e., on the visceral side) and supporting the epidermis and dermis. "Subcutaneous tissue" can also be said to be tissue sandwiched between the dermis and muscle (including fascia). In this specification, "muscle tissue" refers to tissue located below the subcutaneous tissue and including muscle and fascia. Muscle is broadly divided into skeletal muscle, which is striated muscle, cardiac muscle, and smooth muscle. In this specification, the site "subcutaneous tissue and/or muscle tissue" can be used interchangeably with "site located deeper than the dermis" or "site including from the subcutaneous tissue to the muscle."

The therapeutic agent of the present invention is not particularly limited to the type of subcutaneous tissue or muscle tissue or the site of transplantation, as long as it is used to be transplanted into subcutaneous tissue and/or muscle tissue. When the therapeutic agent of the present invention is used to be transplanted into subcutaneous tissue, it is preferably used in subcutaneous adipose tissue, specifically brown adipose tissue or white adipose tissue, more preferably white adipose tissue. In addition, the subcutaneous adipose tissue is not particularly limited as long as it is a subcutaneous adipose tissue at a site where cells can be surgically transplanted, but is preferably subcutaneous adipose tissue present at at least one site selected from the groin, armpit, back, and abdomen. In one embodiment, the therapeutic agent of the present invention is used to be transplanted into white adipose tissue in the groin. In another embodiment, the therapeutic agent of the present invention is used to be transplanted into white adipose tissue in the armpit.
Since a vascular network is formed in subcutaneous tissue, particularly in subcutaneous adipose tissue, more particularly in white adipose tissue, from the viewpoint of the transplantation site, angiogenesis may occur around the pituitary hormone-producing cells after transplantation of the therapeutic agent of the present invention. In addition, angiogenesis around the pituitary hormone-producing cells may promote the survival of the pituitary hormone-producing cells in the subcutaneous tissue. This survival allows the pituitary hormones to be secreted into the blood vessels. From the viewpoint of survival, in one embodiment, the therapeutic agent of the present invention is used such that the pituitary hormone-producing cells are transplanted near the blood vessels in the subcutaneous tissue.

In addition, when the therapeutic agent of the present invention is used to be transplanted into muscle tissue, it may be used to be transplanted into either striated muscle or smooth muscle, and is not particularly limited, but one embodiment is striated muscle, particularly skeletal muscle. Skeletal muscle is typically composed of muscles (superficial muscles) located in the superficial layer, which have relatively long tendon tissue at both ends and a relatively small attachment area to bone, and muscles (deep muscles) located in the deep layer, which directly attach to bone over a wide area and have little tendon tissue. When the therapeutic agent of the present invention is used to be transplanted into skeletal muscle, it may be transplanted into either superficial muscle or deep muscle, or between deep muscle and superficial muscle. When the therapeutic agent of the present invention is used to be transplanted into muscle tissue, examples of the transplant site include muscle tissue present in areas such as the back (including the lower back), abdomen, thighs, buttocks, upper arms, and chest. Since muscle tissue has a vascular network formed in the same manner as the subcutaneous tissue described above, from the viewpoint of the transplantation site, angiogenesis may occur around the pituitary hormone-producing cells after transplantation of the therapeutic agent of the present invention. Furthermore, angiogenesis around the pituitary hormone-producing cells may promote the engraftment of the pituitary hormone-producing cells into the muscle tissue. This engraftment may cause the pituitary hormones to be secreted into the blood vessels. From the viewpoint of engraftment, in one embodiment, the therapeutic agent of the present invention is used such that the pituitary hormone-producing cells are transplanted near the blood vessels in the muscle tissue.

When the therapeutic agent of the present invention is used to be transplanted into subcutaneous tissue and/or muscle tissue, in one embodiment, the pituitary hormone-producing cells are used to be transplanted into an artificially created space (pocket) in the tissue. The size of the space may be changed appropriately depending on the size and number of cell aggregates (cell populations) contained in the therapeutic agent of the present invention. The size of the artificially created space is typically 0.001 to 50 cm ^3. More specifically, for example, when transplanting five pituitary organoids (e.g., 2 to 5 mm diameter), the size of the space may be 1 to 15 cm ^3. In addition, for example, when transplanting one or more of the above-mentioned purified bodies (e.g., 0.5 to 1 mm diameter), the artificially created space is preferably a space that can accommodate only the desired number of purified bodies, and is also preferably directly under a (small) blood vessel in the subcutaneous tissue or muscle tissue. Furthermore, the therapeutic agent of the present invention may be combined with an angiogenesis promoter (e.g., bFGF, etc.) as described below in "8. Compositions containing pituitary hormone-producing cells and therapeutic methods."

In the present specification, the term "diseases caused by pituitary gland disorders" may refer to animal diseases caused by pituitary gland disorders, or non-human animal diseases caused by pituitary gland disorders. Specific examples of "diseases caused by pituitary gland disorders" include panhypopituitarism, pituitary dwarfism, adult growth hormone deficiency, adrenal insufficiency, partial hypopituitarism, isolated anterior pituitary hormone deficiency, pituitary hypogonadism, autoimmune hypothalamic hypophysitis, and pituitary function and hormone secretion deficiency after surgery for pituitary tumors and other tumors.
The animal is preferably a mammal, and includes rodents, ungulates, felines, lagomorphs, primates, etc. Rodents include mice, rats, hamsters, guinea pigs, etc. Ungulates include pigs, cows, goats, horses, sheep, etc. Felidae include dogs, cats, etc. Lagomorphs include rabbits, etc. Primates refer to mammals belonging to the order Primates, and include prosimians such as lemurs, lorises, and tree shrews, and anthropoids such as monkeys, apes, and humans.

As described above, the pituitary hormone-producing cells, cell aggregates containing pituitary hormone-producing cells, or two-dimensional cell sheets contained in the therapeutic agent of the present invention are not particularly limited in terms of their origin or production method, etc., as long as they are capable of producing the hormones described above. For example, the pituitary hormone-producing cells, cell aggregates containing pituitary hormone-producing cells, and two-dimensional cell sheets described in WO 2013/065763, WO 2016/013669, WO 2019/103129, PCT/JP2022/36019, Ozone C, et al. Functional anterior pituitary generated in self-organizing cult ure of human embryonic stem cells (Nat Commun. 2016; 7: 10351), Kasai T, et al. 4), Suga H, et al. Self-formation of functional ade nohypophysis in three-dimensional culture. Nature (2011; 480: 57-62), Taga S, et al. Cell aggregate including pituitary hormone-producing cell, and method (JP Patent applications 2022-116715), Nakano T, et al. Method for producing cell mass including pituitary tis They can be produced by known methods such as those described in (JP Patent applications 2022-116716), Eiraku M, et al. Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals (Cell Stem Cell. 2008; 3: 519-532), or a combination of these methods.

In one embodiment, the pituitary hormone-producing cells contained in the therapeutic agent of the present invention may be produced by the method described in PCT/JP2022/36019, as described below.

2. Cell Culture In this specification, "stem cells" refer to undifferentiated cells that have differentiation and proliferation capabilities (particularly self-renewal capabilities). Stem cells include pluripotent stem cells, multipotent stem cells, unipotent stem cells, etc., depending on their differentiation capabilities. "Pluripotent stem cells" refer to stem cells that can be cultured in vitro and have the ability to differentiate into all cells that constitute a living body (pluripotency). All cells are cells derived from the three germ layers of ectoderm, mesoderm, and endoderm. "Multipotent stem cells" refer to stem cells that have the ability to differentiate into multiple types of tissues and cells, although not all types. "Unipotent stem cells" refer to stem cells that have the ability to differentiate into specific tissues and cells.

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

Embryonic stem cells were first established in 1981, and have been used to create knockout mice since 1989. Human embryonic stem cells were established in 1998, and are also being used in regenerative medicine. ES cells can be produced by culturing the inner cell population on feeder cells or in a medium containing leukemia inhibitory factor (LIF). Methods for producing ES cells are described in, for example, International Publication No. 96/22362, International Publication No. 02/101057, U.S. Patent No. 5,843,780, U.S. Patent No. 6,200,806, and U.S. Patent No. 6,280,718, etc. Embryonic stem cells are available from designated institutions, and can also be purchased commercially. For example, human embryonic stem cells KhES-1, KhES-2, and KhES-3 are available from the Institute for Frontier Medical Sciences, Kyoto University. Both are mouse embryonic stem cells; EB5 cells are available from the RIKEN Institute, a national research and development agency, and the D3 line is available from the American Type Culture Collection (ATCC). Nuclear transfer ES cells (ntES cells), which are a type of ES cell, can be established from a cloned embryo created by transplanting the nucleus of a somatic cell into an egg from which the nucleus has been removed.

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

"Induced pluripotent stem cells" are cells in which pluripotency has been induced by reprogramming somatic cells using known methods. Specific examples of induced pluripotent stem cells include cells in which pluripotency has been induced by reprogramming somatic cells differentiated into fibroblasts, peripheral blood mononuclear cells, etc., through the expression of multiple genes selected from a group of reprogramming genes including Oct3/4, Sox2, Klf4, Myc (c-Myc, N-Myc, L-Myc), Glis1, Nanog, Sall4, lin28, Esrrb, etc. In 2006, Yamanaka et al. established induced pluripotent stem cells in mouse cells (Cell, 2006, 126(4) pp.663-676). In 2007, artificial pluripotent stem cells were established from human fibroblasts, and like embryonic stem cells, they have pluripotency and the ability to self-replicate (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 producing artificial pluripotent stem cells by direct reprogramming through gene expression, artificial pluripotent stem cells can also be induced from somatic cells by adding compounds (Science, 2013, 341, pp.651-654).

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

When producing induced pluripotent stem cells, if initialization is performed by expression of several types of genes (e.g., four factors: Oct3/4, Sox2, Klf4, and Myc), the means for expressing the genes is not particularly limited. Examples of means for expressing genes include infection methods using viral vectors (e.g., retroviral vectors, lentiviral vectors, Sendai virus vectors, adenoviral vectors, and adeno-associated virus vectors), gene transfer methods using plasmid vectors (e.g., plasmid vectors, episomal vectors) (e.g., calcium phosphate method, lipofection method, retronectin method, and electroporation method), gene transfer methods using RNA vectors (e.g., calcium phosphate method, lipofection method, and electroporation method), and direct protein injection methods.

It is also possible to obtain established induced pluripotent stem cell lines; for example, human induced pluripotent cell lines such as 201B7 cells, 201B7-Ff cells, 253G1 cells, 253G4 cells, 1201C1 cells, 1205D1 cells, 1210B2 cells, and 1231A3 cells established at Kyoto University are available from Kyoto University and iPS Academia Japan Inc. As established induced pluripotent stem cell lines, for example, Ff-I01 cells, Ff-I14 cells, and QHJI01s04 cells established at Kyoto University are available from Kyoto University.

Pluripotent stem cells may be genetically modified. Genetically modified pluripotent stem cells can be produced, for example, by using homologous recombination techniques. Genes on chromosomes that can be modified include, for example, cell marker genes, histocompatibility antigen genes, and disease-related genes based on disorders of nervous system cells. Modification of target genes on chromosomes can be carried out using methods described in Manipulating the Mouse Embryo, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994), Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993), Biomanual Series 8, Gene Targeting, Creation of Mutant Mice Using ES Cells, Yodosha (1995), etc.

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

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

The preparation of a target vector for homologous recombination of a target gene and efficient selection of a homologous recombinant can be performed according to the methods described in Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993), Biomanual Series 8, Gene Targeting, Generation of Mutant Mice Using ES Cells, Yodosha (1995), etc. Either a replacement type or an insertion type target vector can be used. Selection methods that can be used include positive selection, promoter selection, negative selection, and poly A selection. Methods for selecting the desired homologous recombinant from the selected cell lines include Southern hybridization and PCR for genomic DNA.

Pluripotent stem cells that have undergone genome editing can also be used as pluripotent stem cells. "Genome editing" is a technique for intentionally modifying target genes or genomic regions using principles such as site-specific cleavage of genomic DNA strands using nucleases or chemical conversion of bases. Examples of site-specific nucleases include zinc finger nucleases (ZFNs), TALENs, and CRISPR/Cas9. Using genome editing technology, it is possible to create knockout cell lines in which specific genes have been deleted, knock-in cell lines in which a different sequence has been artificially inserted into a specific gene locus, etc.

In one embodiment, the pluripotent stem cells used in the present invention are mammalian pluripotent stem cells, and when producing pituitary hormone-producing cells for use in the therapeutic agent of the present invention, it is preferable that the pluripotent stem cells are the same mammalian pluripotent stem cells as the subject to be transplanted. For example, to produce pituitary hormone-producing cells to be transplanted into a human subject, human pluripotent stem cells are used.
In one embodiment, when a human pituitary tissue-bearing non-human animal model is produced, the pluripotent stem cells used in the present invention are human pluripotent stem cells.
"Cell adhesion" includes adhesion between cells (cell-cell adhesion) and adhesion between cells and extracellular matrix (substrate) (cell-substrate adhesion). Cell adhesion also includes adhesion of cells to culture equipment and the like that occurs in an in vitro artificial culture environment. The bond formed in cell-cell adhesion is a cell-cell junction, and the bond formed in cell-substrate adhesion is a cell-substrate junction. Types of cell adhesion include, for example, anchoring junction, communicating junction, and occluding junction.

Cell-cell junctions include "tight junctions" and "adherence junctions." Tight junctions are relatively strong cell-cell junctions that can occur in epithelial cells. The presence of tight junctions between cells can be detected by techniques such as immunohistochemistry using antibodies against components of tight junctions (anti-claudin antibodies, anti-ZO-1 antibodies, etc.).

"Suspension culture" refers to culturing cells while maintaining a state in which they exist suspended in the culture solution. In other words, suspension culture is carried out under conditions that do not allow cells to adhere to the cultureware and feeder cells, etc. on the cultureware (hereinafter referred to as "cultureware, etc."), and is distinguished from culture carried out under conditions that allow cells to adhere to the cultureware, etc. (adherent culture). More specifically, suspension culture refers to culture under conditions that do not allow strong cell-substrate bonds to be formed between the cells and the cultureware, etc. Those skilled in the art can easily distinguish whether the cultured cells are in a suspension culture state or an adherent culture state, for example, by rocking the cultureware during microscopic observation.

"Adherent culture" refers to culturing cells while maintaining a state in which the cells are attached to culture equipment, etc. Here, cells adhering to culture equipment, etc. refers to, for example, the formation of strong cell-substrate bonds, which is a type of cell adhesion, between the cells and the culture equipment, etc.

In cell aggregates during suspension culture, cells adhere to each other through a plane. In cell aggregates during suspension culture, strong cell-substrate bonds are not formed between the cells and the cultureware, and cell-substrate bonds are hardly formed, or even if they are formed, their contribution is small. Internal cell-substrate bonds may exist inside cell aggregates during suspension culture. "Plane attachment between cells" refers to cells adhering to each other through a plane. More specifically, "plane attachment between cells" refers to the proportion of the surface area of a cell that is attached to the surface of another cell, for example, 1% or more, preferably 3% or more, and more preferably 5% or more. The cell surface can be observed by staining with a membrane-staining reagent (e.g., DiI) or immunostaining with cell adhesion factors (e.g., E-cadherin, N-cadherin, etc.).

The cultureware used for adhesion culture is not particularly limited as long as it is capable of adhesion culture, and a person skilled in the art can appropriately determine the cultureware. Examples of such cultureware include flasks, flasks for tissue culture, dishes, dishes for tissue culture, multi-dishes, microplates, microwell plates, micropores, multi-plates, multi-well plates, chamber slides, petri dishes, tubes, trays, culture bags, and biofunctional chips such as organ-on-chips. Examples of cell-adhesive cultureware that can be used include cultureware whose surface has been artificially treated for the purpose of improving adhesion to cells. Examples of artificial treatments include coating treatments with extracellular matrices, polymers, etc., and surface treatments such as gas plasma treatment and positive charge treatment. Examples of extracellular matrices to which cells are attached include basement membrane preparations, laminin, entactin, collagen, gelatin, etc. Examples of polymers include polylysine, polyornithine, etc. The culture surface of the cultureware may be flat or uneven.

Laminin is a heterotrimeric molecule consisting of α, β, and γ chains, and is an extracellular matrix protein with isoforms that differ in the composition of the subunit chains. Specifically, laminin has approximately 15 isoforms, which are heterotrimeric combinations of five types of α chains, four types of β chains, and three types of γ chains. The names of laminins are determined by combining the numbers of the α chains (α1-α5), β chains (β1-β4), and γ chains (γ1-γ3). For example, a laminin made up of a combination of α5, β1, and γ1 chains is called laminin 511.

The cultureware used for suspension culture is not particularly limited as long as it is capable of suspension culture, and a person skilled in the art can appropriately determine the cultureware. Examples of such cultureware include flasks, flasks for tissue culture, dishes, Petri dishes, dishes for tissue culture, multi-dishes, microplates, microwell plates, micropores, multi-plates, multi-well plates, chamber slides, petri dishes, tubes, trays, culture bags, spinner flasks, and roller bottles. These cultureware are preferably non-adhesive to cells in order to enable suspension culture. As non-adhesive cultureware, those whose surfaces have not been artificially treated to improve adhesion to cells can be used. As non-adhesive cultureware, those whose surfaces have been artificially treated to reduce adhesion to cells can also be used. The culture surface of the cultureware may be flat, U- or V-bottom, or may be uneven. Examples of treatments that reduce adhesion to cells include superhydrophilic treatments using coatings such as 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, Poly(2-hydroxyethyl methacrylate) (Poly-HEMA), and polyethylene glycol (PEG), as well as low protein adsorption treatments.

"Shaking culture" is a culture method in which the culture medium is stirred by shaking the culture equipment, promoting the supply of oxygen to the culture medium and the exchange of materials with the surrounding cells. Agitation culture, flow path culture, etc. can also be performed.

In order to protect the cell aggregates from physical stresses such as shear forces that occur during suspension culture, and to increase the local concentrations of growth factors and cytokines secreted by the cells and promote tissue development, the cell aggregates can be embedded in a gel or encapsulated in a substance-permeable capsule before suspension culture (Nature, 2013, 501.7467:373). The encapsulated cell aggregates may be cultured with shaking. The gel or capsule used for embedding may be made of either a biological or synthetic polymer. Examples of gels or capsules used for such purposes include Matrigel (manufactured by Corning), PuraMatrix (manufactured by 3D Matrix), VitroGel 3D (manufactured by TheWell Bioscience), collagen gel (manufactured by Nitta Gelatin), alginate gel (manufactured by PG Research), and Cell-in-a-Box (manufactured by Austrianova).

The medium used for cell culture can be prepared as a basal medium using a medium commonly used for culturing animal cells. Examples of basal media include Basal Medium Eagle (BME), BGJb medium, CMRL 1066 medium, Glasgow Minimum Essential Medium (Glasgow MEM), Improved MEM Zinc Option, Iscove's Modified Dulbecco's Medium (IMDM), Medium 199, Eagle Minimu Examples of such medium include Eagle MEM, Alpha Modified Eagle Minimum Essential Medium (αMEM), Dulbecco's Modified Eagle Medium (DMEM), F-12 medium, DMEM/F12, IMDM/F12, Ham's medium, RPMI 1640, Fischer's medium, and mixtures of these.

For the maintenance culture of pluripotent stem cells, a medium for pluripotent stem cell culture based on the above-mentioned basal medium, a medium for preferably known embryonic stem cells or induced pluripotent stem cells, a medium for culturing pluripotent stem cells under feeder-free conditions (feeder-free medium), etc. Many synthetic media have been developed and are commercially available as feeder-free media, for example, Essential 8 medium. Essential 8 medium contains, in DMEM/F12 medium, the following additives: L-ascorbic acid-2-phosphate magnesium (64 mg/l), sodium selenium (14 μg/l), insulin (19.4 mg/l), NaHCO ₃ (543 mg/l), transferrin (10.7 mg/l), bFGF (100 ng/mL), and a TGFβ family signaling pathway active substance (TGFβ 1 (2 ng/mL) or Nodal (100 ng/mL)) (Nature Methods, 8, 424-429 (2011)). Commercially available feeder-free media include, for example, Essential 8 (manufactured by Thermo Fisher Scientific), S-medium (manufactured by DS Pharma Biomedical), StemPro (manufactured by Thermo Fisher Scientific), hESF9, mTeSR1 (manufactured by STEMCELL Technologies), mTeSR2 (manufactured by STEMCELL Technologies), TeSR-E8 (manufactured by STEMCELL Technologies), mTeSR Plus (manufactured by STEMCELL Technologies), StemFit (manufactured by Ajinomoto Co., Inc.), and ReproMed iPSC. Examples of such medium include Cellartis DEF-CS Xeno-Free Culture Medium (manufactured by ReproCELL), NutriStem XF (manufactured by Biological Industries), NutriStem V9 (manufactured by Biological Industries), Cellartis DEF-CS Xeno-Free Culture Medium (manufactured by Takara Bio Inc.), Stem-Partner SF (manufactured by Kyokuto Pharmaceutical Co., Ltd.), PluriSTEM Human ES / iPS Cell Medium (manufactured by Merck), and StemSure hPSC Medium Δ (manufactured by Fujifilm Wako Pure Chemical Industries).

The serum-free medium may contain a serum substitute. Examples of serum substitutes include those that contain albumin, transferrin, fatty acids, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, or equivalents thereof, as appropriate. Such serum substitutes can be prepared, for example, by the method described in WO 98/30679. Commercially available products may be used as serum substitutes. Examples of commercially available serum substitutes include Knockout Serum Replacement (manufactured by Thermo Fisher Scientific) (hereinafter sometimes referred to as "KSR"), Chemically-defined Lipid Concentrated (manufactured by Thermo Fisher Scientific), Glutamax (manufactured by Thermo Fisher Scientific), B27 Supplement (manufactured by Thermo Fisher Scientific), and N2 Supplement (manufactured by Thermo Fisher Scientific).

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

To avoid the complication of preparation, a serum-free medium containing an appropriate amount (e.g., about 0.5% to about 30%, preferably about 1% to about 20%) of commercially available KSR (manufactured by Thermo Fisher Scientific) may be used as the serum-free medium (e.g., a medium containing 1x chemically defined lipid concentrated, 5% KSR and 450 μM 1-monothioglycerol added to a 1:1 mixture of F-12 medium and IMDM medium) to avoid the complication of preparation. An example of a KSR equivalent is the medium disclosed in JP2001-508302A.

"Serum medium" refers to a medium containing unconditioned or unpurified serum. The medium may contain fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, 1-monothioglycerol, pyruvic acid, buffers, inorganic salts, etc.

The culture in the present invention is preferably carried out under xeno-free conditions. "Xeno-free" refers to conditions in which components derived from organisms other than the organism of the cells to be cultured are excluded.

The medium used in the present invention is preferably a medium whose components are chemically defined (CDM) in order to avoid contamination with chemically undefined components.

"Basement membrane" refers to a thin membrane-like structure composed of extracellular matrix. In living organisms, basement membranes are formed on the basal side of epithelial cells. Components of basement membranes include type IV collagen, laminin, heparan sulfate proteoglycan (perlecan), entactin/nidogen, cytokines, growth factors, etc. Whether or not basement membrane is present in tissues derived from living organisms and in cell populations produced by the production method of the present invention can be detected by, for example, tissue staining such as PAM staining, and immunohistochemistry using antibodies against components of basement membranes (anti-laminin antibodies, anti-type IV collagen antibodies, etc.).

The term "basement membrane preparation" refers to a preparation containing basement membrane components that have the function of controlling epithelial cell-like cell morphology, differentiation, proliferation, movement, functional expression, etc., when desired cells having basement membrane formation ability are seeded and cultured thereon. In this specification, when cells are cultured for adhesion, they can be cultured in the presence of a basement membrane preparation. Here, "basement membrane components" refers to thin membrane-like extracellular matrix molecules that exist between epithelial cell layers and interstitial cell layers, etc., in animal tissues. A basement membrane preparation can be prepared, for example, by removing cells having basement membrane formation ability that are adhered to a support via a basement membrane from the support using a solution capable of dissolving the lipids of the cells, an alkaline solution, or the like. Examples of basement membrane preparations include products commercially available as basement membrane preparations, such as Matrigel (manufactured by Corning) and Geltrex (manufactured by Thermo Fisher Scientific), and those that contain extracellular matrix molecules known as basement membrane components (e.g., laminin, type IV collagen, heparan sulfate proteoglycan, entactin, etc.).

For cell or tissue culture, basement membrane preparations such as Matrigel (Corning) extracted and solubilized from tissues or cells such as Engelbreth-Holm-Swarm (EHS) mouse sarcoma can be used. Similarly, as basement membrane components for cell culture, human solubilized amniotic membrane (BioResource Application Research Institute), human recombinant laminin produced in HEK293 cells (BioLamina), human recombinant laminin fragments (Nippi), human recombinant vitronectin (Thermo Fisher Scientific), etc. can also be used. From the viewpoint of avoiding contamination with components from different biological species and from the viewpoint of avoiding the risk of infection, it is preferable to use recombinant proteins with known components as basement membrane preparations.

In this specification, "medium containing substance X" and "in the presence of substance X" respectively mean a medium to which exogenous substance X has been added or a medium containing exogenous substance X, and in the presence of exogenous substance X. Exogenous substance X is distinguished from endogenous substance X, for example, in which cells or tissues present in the medium endogenously express, secrete, or produce the substance X. Substance X in the medium may have undergone slight changes in concentration due to decomposition of substance X or evaporation of the medium.

In this specification, the start of culture in a medium in which the concentration of substance X is Y preferably refers to the point in time when the concentration of substance X in the medium becomes uniform at Y, but if the culture vessel is sufficiently small (for example, a 96-well plate or culture in 200 μL or less of culture medium), the start of culture at concentration Y is interpreted as the point in time when the medium addition operation, half-volume medium replacement operation, or full-volume medium replacement operation described below is performed to achieve concentration Y. In addition, when the concentration of substance X in the medium is Y, this includes the case where the average concentration of substance X throughout a certain culture period is Y, the period during which substance X is contained at concentration Y is 50% or more of the culture period, the period during which substance X is contained at concentration Y is longer than the shortest culture period expected in each process, etc.

In this specification, "in the absence of substance X" means a medium to which exogenous substance X has not been added, a medium that does not contain exogenous substance X, or a state in which exogenous substance X is not present.

As used herein, "human protein X" means that protein X has the amino acid sequence of protein X that is naturally expressed in the human body.

"Isolated" means that an operation has been performed to remove the target component or factors other than the cell, and that the protein is no longer in a naturally occurring state. Therefore, "isolated protein X" does not include endogenous protein X that is produced from the cells or tissues to be cultured and is contained in the cells, tissues, and medium. The purity of "isolated protein X" (the percentage of protein X weight in the total protein weight) is usually 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 99% or more, and most preferably 100%.

In this specification, the term "derivative" refers to a group of compounds that are produced by replacing a part of the molecule of a specific compound with another functional group or another atom. In this specification, a "modified form" of a protein refers to a protein that has been mutated by deletion, addition, substitution, or other such amino acid residues to the extent that the properties of the original protein can be maintained. The number of mutated amino acids is not particularly limited, but may be 1-4, 1-3, 1-2, or 1. A "modified form" of a protein may be a protein that has an amino acid sequence that shows at least 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more identity with the original protein. The mutated amino acids in the modified form may be non-natural.

In this specification, "after time A (day A)" includes time A (day A) and refers to anything after time A (day A). "Within time B (day B)" includes time B (day B) and refers to anything before time B (day B).

"Feeder cells" refers to cells other than stem cells that are allowed to coexist when the stem cells are cultured. Examples of feeder cells used in the culture of pluripotent stem cells to maintain their undifferentiated state include mouse fibroblasts (MEF), human fibroblasts, and SNL cells. The feeder cells are preferably subjected to a growth-inhibiting treatment. Examples of the growth-inhibiting treatment include treatment with a growth inhibitor (e.g., mitomycin C) and UV irradiation. The feeder cells used in the culture of pluripotent stem cells to maintain their undifferentiated state contribute to maintaining the undifferentiated state of pluripotent stem cells by secreting humoral factors (preferably factors for maintaining their undifferentiated state) and creating a scaffold (extracellular matrix) for cell adhesion.

In this specification, "in the absence of feeder cells" refers to culturing in the absence of feeder cells. Examples of "in the absence of feeder cells" include conditions where no feeder cells are added, or conditions where feeder cells are substantially not included (for example, the ratio of the number of feeder cells to the total number of cells is 3% or less).

A "cell population" refers to a group of cells composed of two or more cells. A cell population may be composed of one type of cell, or may be composed of multiple types of cells. The cells that compose the cell population may be suspended in a medium, or may be adhered to a culture vessel or the like. Furthermore, the cells that compose the cell population may be single cells, or at least a portion of the cell population may be formed by cell adhesion between the cells.
Here, the term "single cells" refers to, for example, cells that have almost no cell-to-cell adhesion (e.g., surface adhesion). In some embodiments, being dispersed into single cells refers to a state in which cell-to-cell bonds (e.g., adherens junctions) are almost completely lost.
As used herein, "dispersed cells" are preferably single cells, but may also include cell clumps consisting of a small number of cells, for example, between 2 and 100, or may include cell clumps consisting of between 2 and 50 cells. "Dispersed cells" may, for example, include 70% or more single cells and 30% or less cell clumps, and preferably include 80% or more single cells and 20% or less cell clumps.
In one embodiment, the population of dispersed single cells may contain minute aggregates of 5 or fewer cells.
As used herein, a cell population may include cell aggregates and two-dimensional cell sheets.

The term "cell aggregate" refers to a mass formed by the gathering of cells, in which the cells are adhered to each other. Embryoid bodies, spheres, spheroids, and organoids are also included in the cell aggregates. In the cell aggregates, the cells are preferably adhered to each other on their surfaces. In some embodiments, in a part or all of the cell aggregate, the cells are adhered to each other, for example, forming an adhesion junction. In some embodiments, two or more cell aggregates can be further artificially adhered or aggregated to each other. Masses in which cell populations are further adhered or aggregated to each other, and assembloids are also included in the cell aggregates. The shape of the cell aggregates is not limited to spherical, but may be, for example, bisphere-like, bead-like, a collection of spheres, string-like or branched (shapes described in Scientific reports, 11:21421 (2021), Patent Application No. 2021-078154), etc.
As used herein, the term "two-dimensional cell sheet" refers to a monolayer or multilayer structure composed of a single or multiple cells having intercellular junctions in the two-dimensional direction.

"Uniform cell aggregates" means that when multiple cell aggregates are cultured, the size of each cell aggregate is constant, and when the size of a cell aggregate is evaluated by the length of its maximum diameter, uniform cell aggregates means that the variance of the length of the maximum diameter is small. More specifically, this means that the maximum diameter of 75% or more of the multiple cell aggregates is within ±100% of the average maximum diameter of the multiple cell aggregates, preferably within ±50% of the average maximum diameter, and more preferably within ±20% of the average maximum diameter.

"Tissue" refers to a structure made up of a group of cells in which multiple types of cells with different morphologies and properties are arranged three-dimensionally in a specific pattern.

"Ectoderm" refers to the outermost of the three germ layers formed after fertilization of an egg during the early development of an organism. As development progresses, the ectoderm divides into the neuroectoderm and the superficial ectoderm, and the neuroectoderm further divides into the neural tube and neural crest. Various organs of the body are formed from these ectoderm, and organs formed from the ectoderm are said to be derived from the ectoderm. For example, the neural tube forms organs or tissues of the central nervous system, such as the brain and spinal cord. For example, some central nervous system cells, facial bones and cartilage, sensory neurons, autonomic neurons, pigment cells, mesenchymal cells, etc. are formed from the neural crest. The epidermis, inner ear, anterior pituitary gland, upper respiratory tract tissue including olfactory epithelium, etc. are formed from the superficial ectoderm. Placodes and placode-derived tissues are derived from the superficial ectoderm. In the process of differentiating into ectoderm, pluripotent stem cells express genes known as ectoderm markers, such as Pax3, Otx2, and Sox1.

"Endoderm" refers to the innermost of the three germ layers formed after fertilization of an egg during the early development of an organism. For example, the digestive system, urinary tract, pharynx, trachea, bronchi, and lungs are formed from the endoderm. In the process of differentiating into endoderm, pluripotent stem cells express genes known as endoderm markers, such as SOX17, HNF-3β/FoxA2, Klf5, GATA4, GATA6, and PDX-1.
"Mesoderm" refers to the germ layer formed between the ectoderm and endoderm. Organs or tissues such as circulatory system, skeleton, and muscle are formed from the mesoderm. In the process of differentiating into the mesoderm, pluripotent stem cells express genes known as mesoderm markers, such as T/Brachury, SMA, ABCA4, Nkx2.5, and PDGFRα.
"Nervous tissue" refers to tissue composed of nervous system cells such as the developing or adult cerebrum, midbrain, cerebellum, spinal cord, retina, sensory nerves, peripheral nerves, etc. As used herein, "neuroepithelial tissue" refers to nervous tissue that has formed an epithelial structure having a layered structure, and the amount of neuroepithelial tissue in nervous tissue can be evaluated by bright field observation using an optical microscope or the like.

The "central nervous system" refers to the area where nervous tissue is accumulated and which is the center of information processing. In vertebrates, the central nervous system includes the brain and spinal cord. The central nervous system is derived from the ectoderm.

"Neural cells" refers to cells of ectoderm-derived tissues other than epidermal cells. In other words, neural cells include neural progenitor cells, neurons (nerve cells), glial cells, neural stem cells, neuronal progenitor cells, glial progenitor cells, and other cells. Neural cells also include cells that make up retinal tissue (retinal cells), retinal progenitor cells, retinal layer-specific nerve cells, neural retina cells, and retinal pigment epithelial cells, which will be described later. Neural cells can be identified using markers such as nestin, βIII tubulin (Tuj1), PSA-NCAM, and N-cadherin.

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

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

In this specification, the term "ventricle" refers to a cavity formed by central nervous tissue. In living organisms, it is an acellular structure that is usually filled with tissue fluid such as cerebrospinal fluid, with the apical side of the nervous tissue facing the ventricle. The periventricular layer surrounding the ventricle is an area where neural stem cells exist and where cell proliferation and neuronal production occur during development. Whether or not the cell population and tissue produced by the production method of the present invention contains ventricles can be detected by techniques such as immunohistochemistry using central nervous tissue markers (Bf1, Pax6, etc.) and apical surface markers (PKC-zeta, etc.).

In this specification, the term "diencephalon" refers to the neural tissue of the central nervous system adjacent to the third ventricle. The diencephalon includes tissues such as the epithalamus, thalamus, hypothalamus, and pituitary gland.

As used herein, the term "hypothalamus" refers to a region of the diencephalon adjacent to the pituitary gland. The hypothalamus is further regionalized into the dorsal hypothalamus and the ventral hypothalamus. The hypothalamus before regionalization can be identified using markers such as Rx, Vax1, and Six3. The dorsal hypothalamus can be identified using markers such as Otp, Brn2, vasopressin, and Pax6. The ventral hypothalamus can be identified using markers such as Nkx2.1 and SF1.
As used herein, the term "hypothalamic neuroepithelial tissue" refers to neuroepithelial tissue expressing a hypothalamic marker. In one embodiment, the ventral hypothalamic neuroepithelial tissue is Rx-positive, Chx10-negative, and Nkx2.1-positive neuroepithelial tissue. In one embodiment, the dorsal hypothalamic neuroepithelial tissue is Rx-positive, Chx10-negative, and Pax6-positive neuroepithelial tissue.
In this specification, the term "hypothalamus" may include "ependymal cells." Ependymal cells are a type of epithelial cell, and refer to cells localized in the ventricular wall. Among ependymal cells, "elongated ependymal cells (tanycytes)" refer to cells with few cilia that are particularly localized in the third ventricle of the hypothalamus. Elongated ependymal cells can be identified using markers such as Nestin, Vimentin, Lhx2, and Sox2.

As used herein, the term "non-neuroepithelial tissue" refers to tissues having an epithelial structure other than neuroepithelial tissue. Epithelial tissue is formed from any of the germ layers, ectoderm, mesoderm, endoderm, and trophectoderm. Epithelial tissue includes epithelium, mesothelium, and endothelium. Examples of tissues included in non-neuroepithelial tissue include epidermis, corneal epithelium, nasal epithelium, oral epithelium, tracheal epithelium, bronchial epithelium, airway epithelium, kidney epithelium, kidney cortical epithelium, and placental epithelium.
Epithelial tissues are usually connected by various intercellular junctions to form tissues with a single layer or stratified layer structure. The presence or absence of these epithelial tissues and the amount of them present can be confirmed by observation with an optical microscope or by immunohistochemistry using antibodies against epithelial cell markers (anti-E-Cadherin antibody, anti-N-Cadherin antibody, anti-EpCAM antibody, etc.).

As used herein, the term "epithelial polarity" refers to the bias in the distribution of spatially formed components and cell functions within 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 retaining tears on the apical side, and express basal-specific proteins such as α6 integrin and β1 integrin for adhering to the basement membrane on the basal side.
The presence of epithelial cell polarity in epithelial cells and epithelial tissues in tissues derived from a living body and in cell populations produced by the production methods of the present invention can be detected by techniques such as immunohistochemistry using phalloidin, apical markers (anti-MUC-1 antibody, anti-PKC-zeta antibody, etc.), and basal markers (anti-α6 integrin antibody, anti-β1 integrin antibody, etc.).

As used herein, the term "placode" refers to the primordium of an organ that is formed by the thickening of a portion of the epidermal ectoderm, primarily during the developmental process of vertebrates. Examples of tissues derived from the placode include the lens, nose, inner ear, trigeminal nerve, and adenohypophysis. Markers of the placode or its precursor tissue, the preplacode region, include Six1, Six4, Dlx5, Eya2, Emx2, and Bf1.

As used herein, the term "pituitary placode" refers to a thickened structure formed in the epidermal ectoderm region during embryonic development, which expresses pituitary progenitor cell markers. Examples of pituitary progenitor cell markers include Lim3 (Lhx3), Pitx1/2, Islet1/2, etc. The pituitary placode expresses at least one, and preferably all, pituitary progenitor cell markers selected from the group consisting of Lim3, Pitx1/2, and Islet1/2. The pituitary placode invaginates to form Rathke's pouch, a sac-like structure in the developing stage, and as development progresses further, it forms the adenohypophysis.

As used herein, the term "adenohypophysis" refers to tissue that contains at least one type of pituitary cell of the anterior or intermediate lobe. Pituitary cells include pituitary hormone-producing cells that produce hormones that regulate physiological functions, and non-hormone-producing cells. Pituitary hormone-producing cells include cells that make up the anterior lobe, such as 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; and cells that make up the intermediate lobe, such as melanocyte-stimulating hormone (MSH)-producing cells. Non-hormone-producing cells include vascular endothelial cells, pericytes, follicle-stellate cells, pituitary stem cells, and pituitary progenitor cells.

In one embodiment, the adenohypophysis contains at least one, preferably two, more preferably three types of 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 adenohypophysis contains at least one, preferably two or more (2, 3, 4, 5, or 6) types of 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 or not adenohypophysis and pituitary hormone-producing cells are present in tissue derived from a living body and in a cell population produced by the production method of the present invention can be detected by techniques such as immunohistochemistry using pituitary hormone-producing cell markers and ELISA for the secreted hormones.
Pituitary hormone-producing cell markers include 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.), and markers of follicle stimulating hormone (FSH)-producing cells and luteinizing hormone (LH)-producing cells (anti-GATA2 antibody, anti-SF1 antibody, anti-FSH antibody, anti-LH antibody, etc.). These can be detected by immunohistochemistry using these markers, and ELISA for the secreted hormones.

As used herein, "pituitary stem cells" refers to undifferentiated multipotent stem cells or progenitor cells that exist in the pituitary gland and contribute to the regeneration of pituitary tissue and the supply of pituitary hormone-producing cells. Whether or not pituitary stem cells are present in the cell population and tissue produced by the production method of the present invention can be determined by detecting pituitary stem cell markers such as Sox2, Sox9, E-Cadherin, Nestin, S100β, GFRα2, Prop1, CD133, β-Catenin, Klf4, Oct4, Pax6, coxsackievirus-adenovirus common receptor (CXADR), PRRX1/2, Ephrin-B2, and ACE, as well as Ki67, Ribosomal protein markers such as ri ... Detection can be achieved by a variety of techniques, including immunohistochemistry using antibodies against cell proliferation markers such as phosphorylated histone H3 and MCM, proliferative cell labeling assays using nucleic acid analogs such as BrdU, EdU, and IdU, fluorescently labeled dipeptide (β-alanyl-lysyl-N-7-amino-4-methylcoumarin-3-acetic acid) uptake assays, and pituitary sphere formation assays.

In this specification, "oral epithelium" refers to the epithelial tissue and cells that form the oral cavity. Examples of oral epithelium include oral mucosal epithelium, salivary gland epithelium, and odontogenic epithelium. Oral mucosal epithelium is usually a mucosal tissue made of stratified squamous epithelium, and contains basal cells, Merkel cells, melanin-producing cells, etc. on the basement membrane in contact with connective tissue, with spinous cells, granular cells, and a stratum corneum formed on the upper layer. Oral mucosal epithelium can be detected as tissue that is positive for cytokeratin 7, 8, 13, 14, and 19, for example.

In this specification, "niche" or "stem cell niche" refers to a microenvironment involved in the proliferation, differentiation, and maintenance of properties of stem cells. Examples of niches in the body include the hematopoietic stem cell niche, hair follicle stem cell niche, intestinal epithelial stem cell niche, muscle stem cell niche, and pituitary niche. In these niches, tissue-specific stem cells and supporting cells that provide the niche exist, and the stem cells are maintained by cytokines, chemokines, extracellular matrix, cell adhesion factors, intercellular signaling factors, and the like provided by the supporting cells.

As used herein, the term "pituitary niche" refers to the microenvironment involved in 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 cleft) that remains between the anterior and intermediate lobes of the pituitary gland as a vestige of the hollow part of the sac-like Rathke's pouch during development, and the parenchymal niche that is scattered throughout the anterior pituitary gland.

As used herein, "mesenchymal cells" refer to non-epithelial cells that are derived primarily from the mesoderm and neural crest and form connective tissue. Some of these cells are multipotent somatic stem cells known as mesenchymal stem cells. Whether or not mesenchymal cells are present in the cell populations and tissues produced by the production method of the present invention can be detected by techniques such as immunohistochemistry using antibodies against mesenchymal cell markers such as nestin, vimentin, cadherin-11, laminin, and CD44. Whether or not mesenchymal stem cells are present can be detected by techniques such as immunohistochemistry using antibodies against mesenchymal stem cell markers such as CD9, CD13, CD29, CD44, CD55, CD59, CD73, CD105, CD140b, CD166, VCAM-1, STRO-1, c-Kit, Sca-1, Nucleostemin, CDCP1, BMPR2, BMPR1A, and BPMR1B.

3. Method for Producing a Cell Population Comprising Pituitary Tissue In one embodiment, a cell population comprising pituitary hormone-producing cells contained in the therapeutic agent of the present invention can be produced by inducing differentiation using a method comprising the following steps:
(A) A step of culturing pluripotent stem cells in a differentiation-inducing medium to obtain a cell population, and then culturing the cell population in suspension in a medium containing a substance that activates the bone morphogenetic protein signaling pathway and a substance that acts on the Shh signal pathway.

In one embodiment, the cell population is preferably a cell aggregate. Specifically, the method includes the following steps:
(A') A step of suspension-culturing cell aggregates obtained by suspension-culturing pluripotent stem cells in a medium containing an activator of bone morphogenetic protein signaling pathway and a substance acting on the Shh signal pathway.

In the steps (A) and (A'), the bone morphogenetic protein signaling pathway activator and the Shh signal pathway acting substance, their concentrations, and the culture period in their presence can be performed according to the methods described in WO 2016/013669, WO 2019/103129, Ozone C, et al. Functional anterior pituitary generated in self-organizing culture of human embryonic stem cells (Nat Commun. 2016; 7: 10351), etc., or the culture conditions described in step (2) of "4. Method 1 for producing a cell population containing pituitary tissue".

In one embodiment, the culture period of the step (A) or (A') may be any culture condition and culture period that can obtain cell aggregates containing 1) hypothalamic neuroepithelial tissue, and 2) pituitary placode and/or Rathke's pouch. In one embodiment, the step (A) or (A') is performed until 10% or more, preferably 30% or more, more preferably 50% or more of the cell aggregates in the culture contain pituitary placode and/or Rathke's pouch. The culture period of the step (A) or (A') may be, for example, 6 days or more, 8 days or more, 10 days or more, and 20 days or less, 18 days or less, or 16 days or less, and may be, for example, 10 days or more and 16 days or less.
The hypothalamic neuroepithelial tissue contained in the cell aggregate obtained in step (A) or (A') is preferably ventral hypothalamic neuroepithelial tissue.

The concentration of the bone morphogenetic protein signaling pathway activator may be varied during the period of addition; for example, the concentration may be gradually reduced by half every 2 to 4 days.

After completion of step (A) or (A'), the culture may be continued in a medium not containing the bone morphogenetic protein signaling pathway activator.
After completion of step (A) or (A'), the cells may be continuously cultured in a medium that does not contain a substance that activates the bone morphogenetic protein signaling pathway but contains a substance that acts on the Shh signaling pathway.
After completion of step (A) or (A'), the cells may be cultured under high oxygen partial pressure conditions. High oxygen partial pressure conditions refer to conditions in which the oxygen partial pressure exceeds the oxygen partial pressure in air (20%). For example, the oxygen partial pressure is 30 to 60%, preferably 35 to 60%, and more preferably 40 to 60%.

The culture after completion of step (A) or (A') may be performed for a period of time sufficient to induce differentiation of the pituitary placode and/or Rathke's pouch into pituitary hormone-producing cells, for example, until 5% or more, 10% or more, 30% or more, or 50% or more of the cell aggregates being cultured contain pituitary hormone-producing cells. Specifically, the period may be, for example, 10 days or more, 20 days or more, 30 days or more, 40 days or more, and 90 days or less, 80 days or less, 70 days or less, 60 days or less, or 50 days or less.

In one aspect, the cell aggregates obtained by suspension culture of pluripotent stem cells in the step (A') are cell aggregates formed by culturing dispersed pluripotent stem cells under non-adhesive conditions in a differentiable medium.
In one embodiment, the method for producing the cell aggregates includes the SFEBq method (Serum-Free Floating culture of Embryoid Body-like aggregates with quick reaggregation), and the cell aggregates are formed within 12 to 72 hours from the start of differentiation induction (start of floating culture of pluripotent stem cells). The differentiable medium used here can be appropriately selected from the above-mentioned media. The medium may appropriately contain a differentiation inducer suitable for inducing differentiation of pituitary hormone-producing cells, and examples of the differentiation inducer include a ROCK inhibitor, a substance acting on the Shh signaling pathway, a substance inhibiting the JNK signaling pathway, and a substance inhibiting the Wnt signaling pathway. The cell aggregates can also be produced according to the methods described in WO 2016/013669, WO 2019/103129, Ozone C, et al. Functional anterior pituitary generated in self-organizing culture of human embryonic stem cells (Nat Commun. 2016; 7: 10351), WO 2023/054396, etc., or the culture conditions described in step (1) or (1') of "4. Method for producing a cell population containing pituitary tissue 1". In addition, when performing the culture conditions described in step (1) or (1') of "4. Method for producing a cell population containing pituitary tissue 1", the method may be performed in consideration of the method described in the above-mentioned publication (e.g., WO 2023/054396, etc.).

4. Method for producing a cell population containing pituitary tissue 1
One embodiment of a method for producing pituitary hormone-producing cells or a cell population containing said cells included in the therapeutic agent of the present invention is a method for producing a cell population containing pituitary hormone-producing cells, comprising the following steps (1') to (2) (hereinafter also referred to as "production method 1").
(1') a first step of culturing pluripotent stem cells in the presence of a JNK signaling pathway inhibitor to form a cell population;
(2) A second step of culturing the cell population obtained in the first step in the presence of a substance acting on the BMP signaling pathway and a substance acting on the Sonic Hedgehog signaling pathway to obtain a cell population containing pituitary tissue.
In step (1'), preferably, a substance inhibiting the JNK signaling pathway is used in combination with a substance inhibiting the Wnt signaling pathway.
Furthermore, the pituitary tissue in step (2) may contain precursor cells of pituitary hormone-producing cells and/or pituitary hormone-producing cells, depending on the culture period in step (2).
After completion of step (2), the below-described step (3) can be carried out to differentiate the precursor cells generated in step (2) into pituitary hormone-producing cells.

A more preferred embodiment of the production method of the present invention is a method for producing a cell population containing pituitary tissue, comprising the following steps (a) and (1') to (2):
(a) a step a of culturing pluripotent stem cells in a medium containing 1) a TGFβ family signaling pathway inhibitor and/or a Sonic Hedgehog signaling pathway agonist, and 2) an undifferentiated state maintenance factor, in the absence of feeder cells;
(1') a first step of culturing (preferably in suspension) the cells obtained in step a in the presence of a substance inhibiting the JNK signaling pathway;
(2) A second step of culturing (preferably in suspension) the cell population obtained in the first step in the presence of a substance acting on the BMP signaling pathway and a substance acting on the Sonic Hedgehog signaling pathway to obtain a cell population containing pituitary tissue.
Preferably, the first step is a step of forming cell aggregates, and the cell population obtained in the first step that is subjected to the second step may be cell aggregates.
In step (1'), preferably, a substance inhibiting the JNK signaling pathway is used in combination with a substance inhibiting the Wnt signaling pathway.

A more preferred embodiment of the production method of the present invention is a method for producing a cell population containing pituitary tissue, comprising the following step (a) and the following steps (1) to (3):
(a) a step a of culturing pluripotent stem cells in a medium containing 1) a TGFβ family signaling pathway inhibitor and/or a Sonic Hedgehog signaling pathway agonist, and 2) an undifferentiated state maintenance factor, in the absence of feeder cells;
(1) a first step of culturing (preferably in suspension) the cells obtained in step a in the presence of a JNK signaling pathway inhibitor and a Wnt signaling pathway inhibitor;
(2) a second step of culturing (preferably in suspension) the cell population obtained in the first step in the presence of a substance acting on the BMP signaling pathway and a substance acting on the Sonic Hedgehog signaling pathway;
(3) A third step in which the cell population obtained in the second step is cultured (preferably in suspension) in the absence of a substance acting on the Sonic Hedgehog signaling pathway to obtain a cell population containing pituitary hormone-producing cells.
Preferably, the first step is a step of forming cell aggregates, and the cell population obtained in the first step that is subjected to the second step, and the cell population obtained in the second step that is subjected to the third step, may each be a cell aggregate.

<Step (a)>: Step a Step a is described below, in which pluripotent stem cells are cultured in a medium containing 1) a TGFβ family signaling pathway inhibitor and/or a Sonic hedgehog signaling pathway agonist, and 2) an undifferentiated maintenance factor, in the absence of feeder cells.
In step (a), pluripotent stem cells are treated with a TGFβ family signaling pathway inhibitor and/or a Sonic hedgehog signaling pathway agonist, and then cultured (preferably in suspension) in the first step, thereby changing the state of the pluripotent stem cells, improving the efficiency of non-neuroepithelial tissue formation, improving the quality of the resulting cell population (aggregates), making them more likely to differentiate and less likely to suffer cell death, and improving the efficiency of producing pituitary tissue cells.

Step (a) is carried out in the absence of feeder cells.
As used herein, the term "feeder-free" refers to conditions substantially free of feeder cells (for example, the ratio of the number of feeder cells to the total number of cells is 3% or less).

In the method for producing pituitary hormone-producing cells according to the present invention, the pluripotent stem cells are preferably embryonic stem cells or induced pluripotent stem cells. Induced pluripotent stem cells are available from a designated institution, and can also be purchased commercially. For example, human induced pluripotent stem cell lines 201B7, 201B7-Ff, 253G1, 253G4, 1201C1, 1205D1, 1210B2, and 1231A3 are available from Kyoto University and iPS Academia Japan, Inc. As established induced pluripotent stem cell lines, for example, Ff-I01, Ff-I14, and QHJI01s04 cells established at Kyoto University are available from Kyoto University. In addition, HC-6 #10, 1231A3, and 1383D2 lines are available from the National Institute of Physical and Chemical Research.

The TGFβ family signaling pathway (i.e., the TGFβ superfamily signaling pathway) is a signaling pathway that uses transforming growth factor β (TGFβ), Nodal/Activin, or BMP as ligands and is transmitted intracellularly by the Smad family.

The term "TGFβ family signaling pathway inhibitor" refers to a substance that inhibits the TGFβ family signaling pathway, i.e., the signaling pathway transmitted by the Smad family, and specific examples include TGFβ signaling pathway inhibitors, Nodal/Activin signaling pathway inhibitors, and BMP signaling pathway inhibitors. As the TGFβ family signaling pathway inhibitor, TGFβ signaling pathway inhibitors are preferred.

The TGFβ signaling pathway inhibitor is not particularly limited as long as it inhibits the signaling pathway caused by TGFβ, and may be any of nucleic acids, proteins, and low molecular weight organic compounds. Examples of such substances include substances that act directly on TGFβ (e.g., proteins, antibodies, aptamers, etc.), substances that suppress the expression of genes that code for TGFβ (e.g., antisense oligonucleotides, siRNA, etc.), substances that inhibit the binding between TGFβ receptors and TGFβ, and substances that inhibit physiological activities caused by signaling via TGFβ receptors (e.g., TGFβ receptor inhibitors, Smad inhibitors, etc.). An example of a protein known as an inhibitor of the TGFβ signaling pathway is Lefty.

Compounds well known to those skilled in the art can be used as TGFβ signaling pathway inhibitors. Specifically, SB431542 (sometimes abbreviated as "SB431") (4-[4-(3,4-Methylenedioxyphenyl)-5-(2-pyridyl)-1H-imidazol-2-yl]benzoamide), SB505124 (2-[4-(1,3-Benzodioxol-5-yl)-2-(1,1-dimethylethyl)-1H-imidazol-5-yl]-6-methylpyridine), S B525334 (6-[2-(1,1-Dimethylethyl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-4-yl]quinoxaline), LY2157299 (4-[5,6-Dihydro-2-(6 -methyl-2-pyridinyl)-4H-pyrrolo[1,2-b]pyrazol-3-yl]-6-quinolinecarboxamide), LY2109761(4- 5,6-dihydro-2-(2-pyridinyl)-4H-pyrrolo[1,2-b]pyrazol-3-yl]-7-[2-(4-morpholinyl)ethoxy]-quinoline), GW788388 (4-{4-[3- (Pyridin-2-yl)-1H-pyrazol-4-yl]-pyridin-2-yl}-N-(tetrahydro-2H-pyran-4-yl)benzamide), LY364 947 (4-[3-(2-Pyridinyl)-1H-pyrazol-4-yl]quinoline), SD-208 (2-[(5-Chloro-2-fluorophenyl)pteridin-4-yl]pyridin-4-yl-amine ), EW-7197 (N-(2-fluorophenyl)-5-(6-methyl-2-pyridinyl)-4-[1,2,4]triazolo[1,5-a]pyridin-6-y l-1H-Imidazole-2-methanamine), A83-01 (3-(6-Methylpyridin-2-yl)-4-(4-quinolyl)-1-phenylthiocarbamoyl-1H-pyrazole), Rep Sox (2-[5-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl]-1,5-naphthyridine), SM16 (4-[4-(1,3-Benzodi oxol-5-yl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-2-yl]bicyclo[2.2.2]octane-1-carboxamide), R268712(4-[2-Fluoro-5-[3-(6-me thyl-2-pyridinyl)-1H-pyrazol-4-yl]phenyl]-1H-pyrazole-1-ethanol), IN1130(3-[[5-(6-Methyl -2-pyridinyl)-4-(6-quinoxalinyl)-1H-imidazol-2-yl]methyl]benzamide), Galunisertib (4-[5,6-Dihydro-2-(6-methyl-2-pyrid inyl)-4H-pyrrolo[1,2-b]pyrazol-3-yl]-6-quinolinecarboxamide), AZ12799734(4-({4-[(2,6-dimethy lpyridin-3-yl)oxy]pyridin-2-yl}amino)benzenesulfonamide), A77-01(4-[3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl]quino line), KRCA 0008(1,1-[(5-Chloro-2,4-pyrimidinediyl)bis[imino(3-methoxy-4,1-phenylene)-4,1-pipe razinediyl]]bisethanone), GSK 1838705 (2-[[2-[[1-[(Dimethylamino)ethanoyl]-5-(methyloxy)-2,3-dihydro-1H-indol-6-yl]amino] -7H-pyrrolo[2,3-d]pyrimidin-4-yl]amino]-6-fluoro-N-methylbenzamide), Crizotinib (3-[(1R) -1-(2,6-Dichloro-3-fluorophenyl)ethoxy]-5-[1-(piperidin-4-yl)-1H-pyrazol-4-yl]-2-aminopyridine), Ceritinib (5-Chloro-N2-[ 2-isopropoxy-5-methyl-4-(4-piperidyl)phenyl]-N4-(2-isopropylsulfonylphenyl)pyrimidine-2 , 4-diamine), ASP 3026 (N2-[2-Methoxy-4-[4-(4-methyl-1-piperazinyl)-1-piperidinyl]phenyl]-N4-[2-[(1-methylethyl)sulfonyl]p henyl]-1,3,5-triazine-2,4-diamine), TAE684(5-Chloro-N2-[2-methoxy-4-[4-(4-methyl-1-pipe razinyl)-1-piperidinyl]phenyl]-N4-[2-[(1-methylethyl)sulfonyl]phenyl]-2,4-pyrimidinediamine), AZD3463(N-[4-(4-Amino-1- piperidinyl)-2-methoxyphenyl]-5-chloro-4-(1H-indol-3-yl)-2-pyrimidinamine), TP0427736(6-[4 -(4-methyl-1,3-thiazol-2-yl)-1H-imidazol-5-yl]-1,3-benzothiazole), TGFBR1-IN-1(5-(1,3-benzothiazol-6-yl)-N-(4- hydroxyphenyl)-1-(6-methylpyridin-2-yl)pyrazole-3-carboxamide), TEW-7197(2-fluoro-N-[[5-(6-methyl pyridin-2-yl)-4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-1H-imidazol-2-yl]methyl]aniline), LY3200882(2-[4-[[4-[1-cycloprop yl-3-(oxan-4-yl)pyrazol-4-yl]oxypyridin-2-yl]amino]pyridin-2-yl]propan-2-ol), BIBF-0775((3 Z) -N-Ethyl-2,3-dihydro-N-methyl-2-oxo-3-[phenyl[[4-(1-piperidinylmethyl)phenyl]amino]methylene]-1H-indole-6-carboxamide ), SIS3 (1-(3,4-dihydro-6,7-dimethoxy-2(1H)-isoquinolinyl)-3-(1-methyl-2- Examples of such inhibitors include SMAD3 inhibitors such as 4-[1,1'-biphenyl]-4-yl-1,4,5,6,7,8-hexahydro-2,7,7-trimethyl-5-oxo-3-quinolinoleboxylic acid ethyl ester (ITD-1), and derivatives of these compounds. These substances may be used alone or in combination. SB431542 is a compound known as an inhibitor of the TGFβ receptor (ALK5) and Activin receptor (ALK4/7) (i.e., a TGFβR inhibitor). SIS3 is a TGFβ signaling pathway inhibitor that inhibits phosphorylation of SMAD3, an intracellular signaling factor under the control of the TGFβ receptor. ITD-1 is a promoter of proteasomal degradation of the TGF-β type II receptor. Those skilled in the art are aware that the above compounds have activity as TGFβ signaling pathway inhibitors (for example, see Expert Opinion on Investigational Drugs, 2010, 19.1: 77-91, etc.).

The TGFβ signaling pathway inhibitor preferably includes an Alk5/TGFβR1 inhibitor. The Alk5/TGFβR1 inhibitor is preferably SB431542, SB505124, SB525334, LY2157299, GW788388, LY364947, SD-208, EW-7197, A83-01, RepSox, SM16, R268712, IN1130, Galunisertib, AZ1 2799734, A77-01, KRCA 0008, GSK 1838705, Crizotinib, Certinib, ASP 3026, TAE684, AZD3463, TP0427736, and more preferably SB431542 or A83-01.

The concentration of the TGFβ signaling pathway inhibitor in the medium can be set appropriately depending on the substance used within a range that can achieve the above-mentioned effects. When SB431542 is used as the TGFβ signaling pathway inhibitor in step (a), it is usually used at a concentration of about 1 nM to about 100 μM, preferably about 10 nM to about 100 μM, more preferably about 10 nM to about 50 μM, even more preferably about 100 nM to about 50 μM, and particularly preferably about 1 μM to about 10 μM. Furthermore, when a TGFβ signaling pathway inhibitor other than SB431542 is used, it is desirably used at a concentration that exhibits TGFβ signaling pathway inhibitory activity equivalent to that of SB431542 at the above-mentioned concentration. The TGFβ signaling pathway inhibitory activity of SB431542 and the like can be determined by methods well known to those skilled in the art, for example, by detecting phosphorylation of Smads by Western blotting (Mol Cancer Ther. (2004) 3, 737-45.).

A substance acting on the Shh signaling pathway is a substance that can enhance signal transduction mediated by Shh. Examples of substances acting on the Shh signaling pathway include proteins belonging to the Hedgehog family (e.g., Shh, Ihh) or fragments or mutants thereof (SHH C25II N-terminus, SHH C24II N-terminus), Shh receptors, Shh receptor agonists, Smo agonists, Purmorphamine (9-cyclohe xyl-N-[4-(morpholinyl)phenyl]-2-(1-naphthalenyloxy)-9H-purin-6-amine), GSA-10(Propyl 4-(1-hexyl-4-hydroxy-2-oxo-1,2-dihydr) oquinoline-3-carboxamide)benzoate), Hh-Ag1.5, 20 (S)-Hydroxycholesterol, SAG (Smoothened Agonist: N-Methyl-N'-(3-pyridinylbenzyl)-N'-(3-chlorobenzo[b]thiophene-2-carbonyl )-1,4-diaminocyclohexane), 20(S)-hydroxy Cholester ol ((3S,8S,9S,10R,13S,14S,17S)-17-[(2R)-2-hydroxy-6-methylheptan-2-yl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol), etc. These substances may be used alone or in combination. Those skilled in the art are aware that the above compounds have activity as Shh signaling pathway acting substances (for example, as described in Molecular BioSystems, 2010, 6.1: 44-54.).

The substance acting on the Shh signaling pathway preferably includes at least one selected from the group consisting of SAG, Purmorphamine, and GSA-10, and more preferably includes SAG. The concentration of the substance acting on the Shh signaling pathway in the medium can be appropriately set depending on the substance used within a range in which the above-mentioned effects can be achieved. In step (a), SAG is usually used at a concentration of about 1 nM to about 2000 nM, preferably about 10 nM to about 1000 nM, more preferably about 10 nM to about 700 nM, even more preferably about 50 nM to about 700 nM, particularly preferably about 100 nM to about 600 nM, and most preferably about 100 nM to about 500 nM. In addition, when a substance acting on the Shh signaling pathway other than SAG is used, it is desirable to use it at a concentration that shows the same Shh signaling promoting activity as SAG at the above-mentioned concentration. The Shh signaling promoting activity can be determined by methods well known to those skilled in the art, for example, a reporter gene assay focusing on the expression of the Gli1 gene (Oncogene (2007) 26, 5163-5168).

The medium used in step (a) contains an undifferentiated state maintenance factor to enable culture to maintain the undifferentiated state. The undifferentiated state maintenance factor is not particularly limited as long as it is a substance that has the effect of suppressing the differentiation of pluripotent stem cells. In the case of primed pluripotent stem cells (e.g., human ES cells, human iPS cells), examples of undifferentiated state maintenance factors commonly used by those skilled in the art include FGF signaling pathway acting substances, TGFβ family signaling pathway acting substances, insulin, etc. Specific examples of FGF signaling pathway acting substances include fibroblast growth factors (e.g., bFGF, FGF4, and FGF8). Examples of TGFβ family signaling pathway acting substances include TGFβ signaling pathway acting substances and Nodal/Activin signaling pathway acting substances. Examples of TGFβ signaling pathway acting substances include TGFβ1 and TGFβ2. Examples of substances acting on the Nodal/Activin signaling pathway include Nodal, Activin A, and Activin B. These substances may be used alone or in combination. When culturing human pluripotent stem cells (e.g., human ES cells, human iPS cells), the medium in step (a) preferably contains bFGF as an undifferentiated maintenance factor.

The undifferentiation maintenance factor is usually a mammalian undifferentiation maintenance factor. Examples of mammals include those mentioned above. Since the undifferentiation maintenance factor may have cross-reactivity between mammalian species, any mammalian undifferentiation maintenance factor may be used as long as it can maintain the undifferentiated state of the pluripotent stem cells to be cultured. The undifferentiation maintenance factor is preferably an undifferentiation maintenance factor of the same mammalian species as the cells to be cultured. For example, human undifferentiation maintenance factors (e.g., bFGF, FGF4, FGF8, EGF, Nodal, Activin A, Activin B, TGFβ1, TGFβ2, etc.) are used for culturing human pluripotent stem cells. The undifferentiation maintenance factor is preferably isolated.

The undifferentiation maintenance factor may be one produced by any host or one artificially synthesized, so long as it has the ability to maintain the undifferentiation state of the pluripotent stem cells to be cultured. The undifferentiation maintenance factor used in the present invention is preferably one that has been modified in the same manner as that occurring in a living body, and more preferably one that has been produced in cells of the same type as the pluripotent stem cells to be cultured under conditions that do not contain any xenogeneic components.
One embodiment of the production method according to the present invention includes a step of providing an isolated factor for maintaining undifferentiation. One embodiment of the production method according to the present invention includes a step of exogenously (or extrinsically) adding the isolated factor for maintaining undifferentiation to the medium used in step (a). The factor for maintaining undifferentiation may be added in advance to the medium used in step (a).

The concentration of the undifferentiation maintenance factor in the medium used in step (a) is a concentration that can maintain the undifferentiated state of the pluripotent stem cells being cultured, and can be appropriately set by a person skilled in the art. For example, when bFGF is used as the undifferentiation maintenance factor in the absence of feeder cells, the concentration is usually 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.

Step (a) is carried out in the absence of feeder cells. The culture of pluripotent stem cells in step (a) may be carried out under either suspension culture or adherent culture conditions, but is preferably carried out by adherent culture. When culturing pluripotent stem cells in the absence of feeder cells, an appropriate matrix may be used as a scaffold to provide the pluripotent stem cells with a scaffold in place of feeder cells. The pluripotent stem cells are cultured in an adherent manner in a culture vessel whose surface is coated with a matrix scaffold.

Matrices that can be used as scaffolds include laminin (Nat Biotechnol. 28, 611-615 (2010)), laminin fragments (Nat Commun 3, 1236 (2012)), basement membrane preparations (Nat Biotechnol 19, 971-974 (2001)), gelatin, collagen, heparan sulfate proteoglycan, entactin, and vitronectin. The matrix used is preferably laminin 511 (Nat Biotechnol 28, 611-615 (2010)).

The laminin fragment is not particularly limited as long as it has adhesive properties to pluripotent stem cells and enables the maintenance culture of pluripotent stem cells under feeder-free conditions, but is preferably an E8 fragment. The laminin E8 fragment was identified as a fragment with strong cell adhesive activity among fragments obtained by digesting laminin 511 with elastase (EMBO J., 3: 1463-1468, 1984; J. Cell Biol., 105: 589-598, 1987). As the laminin fragment, the E8 fragment of laminin 511 is preferably used (Nat Commun 3, 1236 (2012); Scientific Reports 4, 3549 (2014)). The laminin E8 fragment does not need to be a product of elastase digestion of laminin, and may be a recombinant product. It may be produced in a genetically modified animal (such as a silkworm). In order to avoid contamination with unidentified components, recombinant laminin fragments are preferably used. The E8 fragment of laminin 511 is commercially available and can be purchased from, for example, Nippi Corporation.

In order to avoid contamination with unidentified components, the laminin or laminin fragment used in this specification is preferably isolated. In the culture of pluripotent stem cells in the absence of feeder cells in step (a), the pluripotent stem cells are cultured in an adherent manner in a culture vessel whose surface is preferably coated with isolated laminin 511 or the E8 fragment of laminin 511, more preferably with the E8 fragment of laminin 511.

The medium used in step (a) is not particularly limited as long as it is a medium that allows the maintenance of undifferentiated culture of pluripotent stem cells under feeder-free conditions (feeder-free medium).
The medium used in step (a) may be a serum medium or a serum-free medium. From the viewpoint of avoiding contamination with chemically undefined components, the medium used in step (a) is preferably a serum-free medium. The medium may contain a serum substitute.

The culture time of the pluripotent stem cells in step (a) is not particularly limited as long as it is within a range that can achieve the effect of improving the quality of the cell population (aggregates) that can be formed in the subsequent first step, but is usually 0.5 to 144 hours, preferably 2 to 96 hours, more preferably 6 to 48 hours, even more preferably 12 to 48 hours, and particularly preferably 18 to 28 hours, for example 24 hours. In other words, step (a) is started 0.5 to 144 hours, preferably 18 to 28 hours, before the start of the first step, and the first step is continued after step (a) is completed.

In a preferred embodiment of step (a), human pluripotent stem cells are cultured as adherent cells in a serum-free medium containing bFGF in the absence of feeder cells. The adherent culture is preferably performed in a culture vessel whose surface is coated with laminin 511, E8 fragment of laminin 511, or vitronectin. The adherent culture is preferably performed using StemFit as a feeder-free medium. StemFit medium contains bFGF as a component for maintaining undifferentiated state (Scientific Reports (2014) 4, 3594).

In a preferred embodiment of step (a), human pluripotent stem cells are cultured in suspension in a serum-free medium containing bFGF in the absence of feeder cells. In the suspension culture, the human pluripotent stem cells may form aggregates of human pluripotent stem cells.

In step (a) and the steps described below, culture conditions such as culture temperature and _CO2 concentration 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%, when a bicarbonate buffered medium is used.

<Step (1)> and <Step (1')>: First Step In step (1'), pluripotent stem cells are cultured in the presence of an inhibitor of the c-Jun N-terminal kinase (JNK) signaling pathway to obtain a cell population.

JNK is a kinase belonging to the MAPK family and is involved in intracellular signal transduction in response to various environmental stresses, inflammatory cytokines, growth factors, and stimulation by GPCR agonists.

In this specification, the term "JNK signaling pathway inhibitor" refers to any substance that can suppress signal transduction transmitted by JNK, but is not limited thereto. Examples of JNK signaling pathway inhibitors include substances that have the activity of inhibiting signal transduction by mechanisms such as inhibiting factors upstream or downstream of the JNK signaling mechanism, or the enzyme activity, multimerization, and binding to other factors or nucleic acids of JNK itself, and promoting degradation. Examples of JNK signaling pathway inhibitors include, but are not limited to, JNK inhibitors, Rac inhibitors, MKK inhibitors, MEK inhibitors, Src inhibitors, receptor tyrosine kinase (RTK) inhibitors, and ASK inhibitors.

c-Jun N-terminal kinase (JNK) inhibitors include, for example, JNK-IN-8 ((E)-3-(4-(dimethylamino)but-2-enamido)-N-(3-methyl-4-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)benzamide), SP600125 (Anthra[1-9-cd]pyrazol-6(2H)-one), DB07268 (2-[[2-[(3-Hydroxyphenyl)amino]-4-pyrimidinyl]amino]benzamide), and Ta nzisertib (trans-4-[[9-[(3S)-Tetrahydro-3-furanyl]-8-[(2,4,6-trifluorophenyl)amino]-9H-purin-2-yl]amino]cyclohexanol), Be ntamapimod (1,3-Benzothiazol-2-yl)[2-[[4-[(morpholin-4-yl)methyl]benzyl]oxy]pyrimidin-4-yl]acetonitrile, TCS JNK 6o(N -(4-Amino-5-cyano-6-ethoxy-2-py ridinyl)-2,5-dimethoxybenzeneacetamide), SU3327(5-[(5-Nitro-2-thiazolyl)thio]-1,3,4-thiadiazol-2-amine), CEP1347((9S, 10R,12R)-5-16-Bis[(ethylthio)methyl]-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3 ',2',1'-kl]pyrrolo[3,4-i][1, 6] benzodiazocine-10-carboxylic acid methyl ester), c-JUN peptide, AEG3482 (6-Phenylimidazo[2,1-b]-1,3,4-thiadiazole- 2-sulfonamide), TCS JNK 5a (N-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thienyl-2-yl)-1-naphthalenecarboxamide), BI-78D3 (4-(2,3-D ihydro-1,4-benzodioxin-6-yl)-2,4- dihydro-5-[(5-nitro-2-thiazolyl)thio]-3H-1,2,4-triazol-3-one), IQ-3(11H-Indeno[1,2-b]quinoxalin-11-one O-(2-furanylca rbonyl)oxime), SR 3576 (3-[4-[[[(3-Methylphenyl)amino]carbonyl]amino]-1H-pyrazol-1-yl]-N-(3,4,5-trimethoxyphenyl)benzam ide), IQ-1S (11H-Indeno[1,2-b]qui noxalin-11-one oxime sodium salt), JIP-1 (153-163), CC-401 (3-[3-[2-(1-Piperidinyl)ethoxy]phenyl]-5-(1H-1,2,4-triazol-5-yl) -1H-indazole dihydrochloride), BI-87G3 (2-(5-Nitrothiazol-2-ylthio)benzo[d]thiazole), AS601245 (2-(1,3-benzothiazole) ol-2-yl)-2-[2-(2-pyridin-3-ylethylami no. 79-97-4), ER-358063, ER-409903, ER-417258, CC-359 ((4S)-4-(2,4-difluoro-5-pyrimidin-5-ylphenyl)-4-methyl-5,6-dihydro-1,3-thia zin-2-amine), CC-401 (3-[3 -(2-piperidin-1-ylethoxy)phenyl]-5-(1H-1,2,4-triazol-5-yl)-1H-indazole), CC-930 (4-[[9-[(3S)-oxolan-3-yl]-8-(2,4,6-trifluoroanilino)purin-2-yl]amino]cyclohexan-1-ol), SB203580 (4-[4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-1H-imidazol-5-yl]pyridine) and derivatives thereof. These substances may be used alone or in combination. It is known to those skilled in the art that the above compounds have activity as JNK inhibitors (for example, as described in J Enzyme Inhib Med Chem. 2020; 35(1): 574-583).

Rac inhibitors include, for example, EHT1864 (5-(5-(7-(Trifluoromethyl)quinolin-4-ylthio)pentyloxy)-2-(morpholinomethyl)-4H-pyran-4-one dihydrochloride), NSC23766 (N6-[2-[[4-(Diethylamino)-1-methylbutyl]amino]-6-methyl-4-pyrimidinyl nyl]-2-methyl-4,6-quinolinediaminetrihydrochloride), EHop-016(N4-(9-Ethyl-9H-carbazol-3-yl)-N2-[3-(4-morpholinyl)propy l]-2,4-pyrimidinediamine), 1A-116(N-(3,5-Dimethylphenyl)-N'-[2-(trifluoromethyl)phenyl]guan idine), ZCL278 (2-(4-bromo-2-chlorophenoxy)-N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenylcarbamothioyl)acetamide), MBQ-167 (9-ethyl-3-(5-phenyl-1H-1,2,3-triazol-1-yl)-9H-carbazole), KRpep-2d (actinium; [(2S )-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(3R,8R,11S,14S,20S,23S,26S,29S,32S,35S,38S)-8-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-amino- 5-carbamimidamido-1-oxopentan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-5- carbamimidamido-1-oxopentan-2-yl]-5-carbamimidamido-1-oxopentan-2-yl]carbamoyl]-29-[(2R)-butan-2-yl]-20-(carboxymet hyl)-26-(hydroxymethyl)-23,32-bis[(4-hydroxyphenyl)methyl]-35-(2-methylpropyl)-2,10,1 3,19,22,25,28,31,34,37-decaoxo-11-propan-2-yl-5,6-dithia-1,9,12,18,21,24,27,30,33,36-decazatrico[36.3.0.014,18]hente tracontan-3-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-5-carbamimidamido-1-oxop entan-2-yl]amino]-5-carbamimidamide-1-oxopentan-2-yl]azanide), ARS-853 (1-[3-[4 -[2-[[4-Chloro-2-hydroxy-5-(1-methylcyclopropyl)phenyl]amino]acetyl]-1-piperazinyl]-1-a zetidinyl]-2-propen-1-one), Salirasib (2-(((2E,6E)-3,7,11-Trimethyldodeca-2,6,10-trien-1-yl)thio)benzoic acid), ML141 (4-(5-(4-methoxyphenyl)-3-phenyl-4,5-dihydropyrazol-1-yl)benzenesulfonamide) and derivatives thereof. These substances may be used alone or in combination. Those skilled in the art are aware that the above compounds have activity as Rac inhibitors (for example, see Cancer research, 2018, 78.12: 3101-3111).

The timing of addition of the JNK signaling pathway inhibitor in the present invention is not limited as long as the effect of improving the efficiency of production of pituitary tissue from human pluripotent stem cells is exhibited, but it is preferable that it is added already at the time of adding the BMP signaling pathway active substance in step (2) described below, within 72 hours from the start of differentiation induction. The more preferable timing of addition of the JNK inhibitor is simultaneous with the start of differentiation induction.

It is preferable that the medium in step (1') further contains a Wnt signaling pathway inhibitor. Such a first step is referred to as step (1). That is, step (1) is a first step of culturing pluripotent stem cells in the presence of a JNK signaling pathway inhibitor and a Wnt signaling pathway inhibitor.
The Wnt signaling pathway is a signaling pathway that uses Wnt family proteins as ligands and mainly Frizzled as a receptor. Examples of the signaling pathway include the canonical Wnt pathway and the non-canonical Wnt pathway. The canonical Wnt pathway is transmitted by β-Catenin. Examples of the non-canonical Wnt pathway include the Planar Cell Polarity (PCP) pathway, the Wnt/JNK 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, and the Wnt-mTOR pathway. In the non-canonical Wnt pathway, there are common signaling factors that are also activated in signaling pathways other than Wnt. In the present invention, these factors other than the above-mentioned JNK pathway are also considered to be constituent factors of the Wnt signaling pathway, and inhibitors of these factors are also included in Wnt signaling pathway inhibitors.

The Wnt signaling pathway inhibitor is not limited as long as it can suppress signaling induced by Wnt family proteins. The inhibitor may be any of nucleic acids, proteins, and low molecular weight organic compounds. Examples of such substances include substances that inhibit Wnt processing and extracellular secretion, substances that act directly on Wnt (e.g., proteins, antibodies, aptamers, etc.), substances that suppress the expression of genes encoding Wnt (e.g., antisense oligonucleotides, siRNA, CRISPRi, etc.), substances that inhibit the binding between Wnt receptors and Wnt, and substances that inhibit physiological activity resulting from signaling by Wnt receptors.

Proteins known to inhibit the Wnt signaling pathway include 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), APCDD1, APCDD1L, proteins belonging to the Draxin family, IGFBP-4, Notum, and proteins belonging to the SOST/Sclerostin family.

As the Wnt signaling pathway inhibitor, compounds well known to those skilled in the art can be used. Examples of inhibitors of the classical Wnt signaling pathway include Frizzled inhibitors, Dishevelled (Dvl) inhibitors, Tankyrase (TANK) inhibitors, casein kinase 1 inhibitors, catenin-responsive transcription inhibitors, p300 inhibitors, CREB-binding protein (CBP) inhibitors, BCL-9 inhibitors, and TCF degradation inducers (Am J Cancer Res. 2015; 5 (8): 2344-2360). Inhibitors of the non-classical Wnt pathway include, for example, porcupine (PORCN) inhibitors, calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitors, TGF-β-activated kinase 1 (TAK1) inhibitors, Nemo-Like Kinase (NLK) inhibitors, LIM Kinase inhibitors, mammalian target of These include rapamycin (mTOR) inhibitors, Rac inhibitors, c-Jun NH 2-terminal kinase (JNK) inhibitors, protein kinase C (PKC) inhibitors, methionine aminopeptidase 2 (MetAP2) inhibitors, calcineurin inhibitors, and nuclear factor of activated T cells (NFAT) inhibitors. In addition, although the mechanism of action has not been reported, examples of Wnt signaling pathway inhibitors include KY02111 (N-(6-chloro-2-benzothiazolyl)-3,4-dimethoxybenzenepropanamide) and KY03-I (2-(4-(3,4-dimethoxyphenyl)butanamide)-6-iodobenzothiazole). These substances may be used alone or in combination.

PORCN inhibitors include, for example, IWP-2 (N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthioeno[3,2-d]pyrimidin-2-yl)thio]acetamide), IWP-3 (2-[[3-(4-fluorophenyl)-3, IWP-4 (N-(6-methyl-2-b) enzothiazolyl)-2-[[3,4,6,7-tetrahydro-3-(2-methoxy phenyl)-4-oxothieno[3,2-d]pyrimidin-2-yl]thio]acetamide), IWP-L6(N-(5-phenyl-2-pyridinyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-p) henylthieno[3,2-d]pyrimidin-2-yl)thio]acetami de), IWP-12(N-(6-Methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-3,6-dimethyl-4-oxothieno[3,2-d]pyrimidin-2-yl)thio]ac etamide), IWP-O1 (1H-1,2,3-Triazole-1-acetamide, 5-ph enyl-N-(5-phenyl-2-pyridinyl)-4-(4-pyridinyl)-), LGK-974(2-(2',3-Dimethyl-2,4'-bipyridin-5-yl)-N-(5-(pyrazin-2-yl)pyridinyl) n-2-yl) acetamide), Wnt-C59 (2-[4-(2-Methylpyridin) -4-yl)phenyl]-N-[4-(pyridin-3-yl)phenyl]acetamide), ETC-131, ETC-159 (1,2,3,6-Tetrahydro-1,3-dimethyl-2,6-dioxo-N-(6-phenyl- 3-pyridazinyl)-7H-purine-7-acetamide), GNF-1331 (N-(6-methoxy-1,3-benzothiazol-2-yl)-2-[(4-propyl-5-pyridin-4-yl-1,2,4-triazol-3-yl)sulfanyl]acetamide), GNF-6231(N-[ 5-(4-Acetyl-1-piperazinyl)-2-pyridinyl]-2'-fluoro- 3-methyl[2,4'-bipyridine]-5-acetamide), Porcn-IN-1 (N-[[5-fluoro-6-(2-methylpyridin-4-yl)pyridin-3-yl]methyl]-9H-carbazole-2-carboxamide), RXC004, CGX1321, and derivatives thereof. These substances may be used alone or in combination.

The Wnt signaling pathway inhibitor preferably includes at least one selected from the group consisting of a PORCN inhibitor, KY02111, and KY03-I, and more preferably includes a PORCN inhibitor. It is also preferable that the Wnt signaling pathway inhibitor includes a substance having inhibitory activity against the non-classical Wnt pathway of Wnt. More preferably, the Wnt signaling pathway inhibitor includes a substance having inhibitory activity against the Wnt/Planar Cell Polarity (PCP) pathway. The PORCN inhibitor used in the present invention preferably includes at least one 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, more preferably includes IWP-2 or Wnt-C59, and even more preferably includes IWP-2.

The concentration of the Wnt signaling pathway inhibitor in the culture medium can be set appropriately depending on the substance used within a range that can achieve the above-mentioned effects. From the viewpoint of improving the production efficiency of cells that constitute the pituitary gland, for example, when IWP-2, a type of PORCN inhibitor, is used as the Wnt signaling pathway inhibitor, its concentration is usually about 10 nM to about 50 μM, preferably about 10 nM to about 30 μM, more preferably about 100 nM to about 10 μM, and most preferably about 0.5 μM. When Wnt-C59, a type of PORCN inhibitor, is used, its concentration is usually about 10 pM to about 1 μM, preferably about 100 pM to about 500 nM, and more preferably about 50 nM. When KY02111 is used, its concentration is usually about 10 nM to about 50 μM, preferably about 10 nM to about 30 μM, more preferably about 100 nM to about 10 μM, and even more preferably about 5 μM. When using a Wnt signaling pathway inhibitor other than the above, it is desirable to use it at a concentration that shows the same Wnt signaling pathway inhibitory activity as the above concentrations.

It is preferable that a TGFβ signaling pathway inhibitor is further present in the medium in the first step (step (1) or step (1')). The TGFβ signaling pathway inhibitor used in the first step may be the same as that exemplified in step (a). The TGFβ signaling pathway inhibitors in step (a) and the first step may be the same or different, but are preferably the same.

The concentration of the TGFβ signaling pathway inhibitor in the culture medium can be set appropriately depending on the substance used, within a range that can achieve the above-mentioned effects. When SB431542 is used as the TGFβ signaling pathway inhibitor, it is usually used at a concentration of about 1 nM to about 100 μM, preferably about 10 nM to about 100 μM, more preferably about 100 nM to about 50 μM, and even more preferably about 500 nM to about 10 μM. When a TGFβ signaling pathway inhibitor other than SB431542 is used, it is desirable to use it at a concentration that shows the same TGFβ signaling pathway inhibitory activity as SB431542 at the above-mentioned concentration.

In the first and subsequent steps, from the viewpoint of suppressing differentiation into mesendoderm and improving the production efficiency of ectoderm/placode-like tissue, it is also preferable to add an inhibitor of transforming growth factor-β-activated kinase 1 (TAK1). TAK1 is a serine-threonine protein kinase of the MAP kinase kinase kinase (MAPKKK) family that mediates signal transduction activated by TGFβ, bone morphogenetic protein (BMP), interleukin 1 (IL-1), TNF-α, etc.

The TAK1 inhibitor is not limited as long as it can suppress signal transduction mediated by TAK1. It may be any of nucleic acids, proteins, and low molecular weight organic compounds. Examples of such substances include substances that inhibit the binding of TAK1 to a substrate, substances that inhibit the phosphorylation of TAK1, substances that promote the dephosphorylation of TAK1, substances that inhibit the transcription or translation of TAK1, and substances that promote the degradation of TAK1.

As a TAK1 inhibitor, for example, (5Z)-7-Oxozeaenol ((3S, 5Z, 8S, 9S, 11E)-3,4,9,10-tetrahydro-8,9,16-trihydroxy-14-methoxy-3 -methyl-1H-2-benzoxacyclotetradecine-1,7(8H)-dione), N-Des(aminocarbonyl)AZ-TAK1 inhibitor(3-Amino-5-[4-(4-morpholinyl) methyl)phenyl]-2-thiophenecarbox amide), Takinib (N1-(1-Propyl-1H-benzimidazol-2-yl)-1,3-benzenedicarboxamide), NG25 (N-[4-[(4-Ethyl-1-piperazinyl)methyl]-3- (trifluoromethyl)phenyl]-4-methyl-3-(1H-pyrrolo[2,3-b]pyridin-4-yloxy)-benzamide trihydrochloride), Sarsasapo- genin, and their derivatives and analogues. These substances are They may be used alone or in combination.

The TAK1 inhibitor is preferably (5Z)-7-Oxozeaenol. When (5Z)-7-Oxozeaenol is used as the TAK1 inhibitor in the first step, 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 25 μM, and even more preferably about 500 nM to about 10 μM. When a TAK1 inhibitor other than (5Z)-7-Oxozeaenol is used, it is preferably used at a concentration that exhibits TAK1 inhibitory activity equivalent to that of (5Z)-7-Oxozeaenol at the above concentration. TAK1 inhibitory activity can be determined, for example, by a method such as the kinase assay described in Cell chemical biology 24.8 (2017): 1029-1039. From the viewpoint of controlling the proportion of cells contained in the pituitary tissue, the TAK1 inhibitor can be added at any stage of the first step or any subsequent steps and then removed. In a preferred embodiment, the TAK1 inhibitor is added at the start of step (b) described below.

The medium used in the first step is not particularly limited as long as it is as described above. The medium used in the first step may be a serum medium or a serum-free medium. In order to avoid contamination with chemically undefined components, a serum-free medium is preferably used in this specification. To avoid the complexity of the preparation, it is preferable to use a serum-free medium to which an appropriate amount of a serum substitute such as commercially available KSR has been added. The amount of KSR added to the serum-free medium is usually about 1% to about 30%, and preferably about 2% to about 20%, in the case of human ES cells, for example. Examples of serum-free media include a medium in 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 in which 5% to 20% KSR, NEAA, pyruvic acid, and 2-mercaptoethanol are added to GMEM.

At the start of the first step, the cells may be in either an adherent state or a floating state. In a preferred embodiment, the pluripotent stem cells are dispersed into single cells and then re-aggregated to form floating cell aggregates. For this purpose, it is preferable to carry out an operation of dispersing the pluripotent stem cells, for example the pluripotent stem cells obtained in step (a), into single cells before the start of the first step. The "dispersed cells" obtained by the dispersion operation are preferably single cells, but may also include cell clumps consisting of a small number of cells, for example, 2 to 100 cells, or may include cell clumps consisting of 2 to 50 cells. The "dispersed cells" may, for example, include 70% or more single cells and 30% or less cell clumps, and preferably include 80% or more single cells and 20% or less cell clumps.

　Methods for dispersing pluripotent stem cells include mechanical dispersion treatment, cell dispersion treatment, and cell protective agent addition treatment, and these treatments may be performed in combination. A preferred method for dispersing cells is to perform cell dispersion treatment simultaneously with cell protective agent addition treatment, followed by mechanical dispersion treatment.

Cytoprotective agents used in the cytoprotective agent addition treatment include substances acting on the FGF signaling pathway, heparin, Rho-associated protein kinase (ROCK) inhibitors, myosin inhibitors, polyamines, integrated stress response (ISR) inhibitors, caspase inhibitors, cell adhesion promoters, serum, or serum substitutes. A preferred cytoprotective agent is a ROCK inhibitor. In order to suppress cell death of pluripotent stem cells (especially human pluripotent stem cells) induced by dispersion, it is preferable to add a ROCK inhibitor from the start of the culture in the first step. ROCK inhibitors include Y-27632 ((R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide, dihydrochloride), Fasudil (HA1077) (1-(5-isoquinolinylsulfonyl) homopiperazine, hydrochloride), ride), H-1152 (5-[[(2S)-hexahydro-2-methyl-1H-1,4-diazepin-1-yl]sulfonyl]-4-methyl-isoquinoline, dihydrochloride), HA-110 0(Hydroxyfasudil)(1-(1-Hydroxy-5-isoquinolinesulfonyl)h omopiperazine, hydrochloride), Chroman 1 ((3S)-N-[2-[2-(dimethylamino)ethoxy]-4-(1H-pyrazol-4-yl)phenyl]-6-methoxy-3,4-d ihydro-2H-chromene-3-carboxamide), Belumosudil (KD025, 2-[ 3-[4-[(1H-Indazol-5-yl)amino]quinazolin-2-yl]phenoxy]-N-isopropylacetamide), HSD1590([2-Methoxy-3-(4,5,10-triazate tracyclo[7.7.0.02,6.012,16]hexadeca-1(9),2(6),3,7,10,12(16)- hexaen-11-yl) phenyl] boronic acid), CRT0066854 ((S)-3-phenyl-N1-(2-pyridin-4-yl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]p yrimidin-4-yl) propane-1,2-diamine), RKI1447 (1-(3-hydro xybenzyl)-3-(4-(pyridin-4-yl)thiazol-2-yl)urea), Ripasudil(4-Fluoro-5-[[(2S)-hexahydro-2-methyl-1H-1,4-diazepin-1-yl ]sulfonyl]isoquinoline), GSK269962A(N-[3-[2-(4-amino-1,2,5- oxadiazol-3-yl)-1-ethylimidazo[4,5-c]pyridin-6-yl]oxyphenyl]-4-(2-morpholin-4-ylethoxy)benzamide), GSK429286A(N-(6-f luoro-1H-indazol-5-yl)-2-methyl-6-oxo-4-(4-(trifluorometh yl) phenyl)-1,4,5,6-tetrahydropyridine-3-carboxamide), Y-33075((R)-4-(1-Aminoethyl)-N-1H-pyrrolo[2,3-b]pyridin-4-ylbenzam ide), LX7101(N,N-Dimethylcarbamic acid 3-[[[4-(aminomet hyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperidinyl]carbonyl]amino]phenyl ester), AT13148((alphaS)-alpha-(Amin omethyl)-alpha-(4-chlorophenyl)-4-(1H-pyrazol-4-yl) benzenemethanol), SAR407899 (6-(piperidin-4-yloxy)isoquinolin-1(2H)-one hydrochloride), GSK180736A (4-(4-fluorophenyl)-N- (1H-indazol-5-yl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyri midine-5-carboxamide), Hydroxyfasudil (1-(1-hydroxy-5-isoquinoline sulfonyl)homopiperazine, HCl), bdp5290 (4-Chloro-1-(4-pi peridinyl)-N-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-1H-pyraz ole-3-carboxamide), sr-3677(N-[2-[2-(Dimethylamino)ethoxy]-4-(1H-pyrazol-4-yl)phenyl]-2,3-dihydro-1,4-benzodioxin-2-ca rboxamidehydrochloride), CCG-222740 (N-(4-Chlorophenyl)-5, 5-difluoro-1-(3-(furan-2-yl)benzoyl)piperidine-3-carboxamide), ROCK inhibitor-2(N-[(1R)-1-(3-methoxyphenyl)ethyl]-4-pyri din-4-ylbenzamide), Rho-Kinase-IN-1 (N-[1-[(4-methylsul phanylphenyl)methyl]piperidin-3-yl]-1H-indazol-5-amine), ZINC00881524(N-(4,5-dihydronaphtho[1,2-d]thiazol-2-yl)-2-(3, 4-dimethoxyphenyl) acetamide), SB772077B ((3S)-1-[[2-(4-Amino -1,2,5-oxadiazol-3-yl)-1-ethyl-1H-imidazo[4,5-c]pyridin-7-yl]carbonyl]-3-pyrrolidinamine dihydrochloride), Verosudil (N -(1,2-Dihydro-1-oxo-6-isoquinolinyl)-alpha-(dimethylami Examples of the cell adhesion promoter include adhesamine and adhesamine-RGDS derivatives (manufactured by Nagase & Co., Ltd.). As the cell protective agent, a prepared cell protective agent can also be used. Examples of prepared cell protective agents include RevitaCell Supplement (manufactured by Thermo Fisher Scientific), CloneR, and CloneR2 (manufactured by Stemcell Technologies). These substances may be used alone or in combination. In the first step, when the ROCK inhibitor Y-27632 is added as the cell protective agent, it is usually added to the culture environment at a concentration of about 10 nM to about 10 mM, preferably about 100 nM to about 1 mM, and more preferably about 1 μM to about 100 μM. In the first step, when the ROCK inhibitor Chroman 1 is added as the cell protective agent, it is usually added to the culture environment at a concentration of about 10 pM to about 1 mM, preferably about 100 pM to about 100 μM, and more preferably about 1 nM to about 10 μM. The concentration of the ROCK inhibitor in the culture medium may be maintained for the period during which cell death of pluripotent stem cells needs to be suppressed (for example, 1 to 3 days, preferably 1 or 2 days), and may be gradually reduced after the period has elapsed by performing a half-volume medium replacement operation, etc., with a medium that does not contain a ROCK inhibitor, as described below, when performing a medium replacement operation. Also, after the period has elapsed, a full-volume medium replacement operation may be performed with a medium that does not contain a ROCK inhibitor.

The cell dispersion liquid used in the cell dispersion treatment may be a solution containing at least one of an enzyme such as trypsin, collagenase, hyaluronidase, elastase, pronase, DNase, papain, etc., and a chelating agent such as ethylenediaminetetraacetic acid. Commercially available cell dispersion liquids, such as TripLE Select (Thermo Fisher Scientific), TripLE Express (Thermo Fisher Scientific), and Accumax (Innovative Cell Technologies), may also be used. A preferred cell dispersion liquid for treating the pluripotent stem cells obtained after step (a) is TrypLE Select, but is not limited to this.

Mechanical dispersion methods include pipetting or scraping with a scraper. The dispersed cells are suspended in the above-mentioned medium.

One method for dispersing pluripotent stem cells is, for example, treating a colony of pluripotent stem cells with ethylenediaminetetraacetic acid or Accumax in the presence of a ROCK inhibitor, and then dispersing the colony by pipetting.

If suspension culture is performed in the first step, a suspension of dispersed pluripotent stem cells is seeded into a non-adhesive cultureware. If the cultureware is non-adhesive, the cells are cultured in suspension, and multiple pluripotent stem cells gather together to form cell aggregates.

In suspension culture, dispersed pluripotent stem cells may be seeded in a relatively large culture vessel such as a 10 cm dish to simultaneously form multiple cell aggregates in one culture vessel. However, from the viewpoint of preventing variation in size of each cell aggregate, it is preferable to seed a certain number of dispersed pluripotent stem cells in each well of a multi-well plate (U-bottom, V-bottom) such as a non-cell-adhesive 96-well microplate. When this is cultured statically, the cells rapidly aggregate to form one cell aggregate in each well (Serum-free culture of Embryoid Body-like aggregates with quick reaggregation; SFEBq method). For example, the culture vessel can be made non-cell-adhesive by processing such as coating the surface of the culture vessel with a superhydrophilic polymer. An example of a non-cell-adhesive multi-well plate is the PrimeSurface 96V-bottom plate (MS-9096V, manufactured by Sumitomo Bakelite Co., Ltd.). Centrifugation may be performed to form cell aggregates more quickly. A uniform population of cell aggregates can be obtained by collecting the cell aggregates formed in each well from multiple wells. If the cell aggregates are uniform, the production efficiency for each well and each repeated experiment can be more stable in the subsequent steps, and cells that make up the pituitary gland can be produced with greater reproducibility.

As another aspect for forming cell aggregates from dispersed pluripotent stem cells, a culture vessel in which one well is divided into a plurality of microwells and two or more cell aggregates are formed can be used. In other words, in one aspect of this embodiment, the suspension culture of any one or more of the first step, the second step, the b step, and the third step can be performed in a culture vessel in which at least one well is formed, and the well is divided into a plurality of microwells, and the suspension culture is performed so that one cell aggregate is formed in each of the microwells, and the number of cell aggregates corresponding to the number of divided microwells can be prepared for each well. As the microwell, a culture vessel in which a plurality of mortars, downward pyramids, concaves, grids, protuberances, etc. are formed on the bottom surface, which allows cells to settle in one place and promotes the formation of aggregates, or a culture vessel in which only a part of the bottom surface is processed to allow cells to adhere to the bottom surface so that aggregates can be easily formed can be used. The culture area per well of a culture device having microwells is not particularly limited, but from the viewpoint of efficient production of cell masses, the bottom area is preferably greater than 1 ^cm2 (equivalent to a 48-well plate), more preferably greater than ² cm2 (equivalent to a 24-well plate), and even more preferably greater than 4 ^cm2 (equivalent to a 12-well plate). Examples of the culture equipment include, but are not limited to, the embryoid body formation plate AggreWell (manufactured by StemCell Technologies), PAMCELL (manufactured by ANK), spheroid microplate (manufactured by Corning), NanoCulture Plate/Dish (manufactured by Organogenix), Cell-able (manufactured by Toyo Gosei), EZSPHERE (manufactured by AGC Technoglass), SPHERICALPLATE 5D (manufactured by Mito Kogyo Co., Ltd.), TASCL (manufactured by Sims Bio), and microwell bag (e.g., those described in Scientific reports, 2022, 12.1: 1-11.).

As the culture equipment, it is also preferable to use a three-dimensional cell culture vessel that allows the culture medium to be replaced for the entire plate at once while the cell aggregates remain in each well. An example of such a three-dimensional cell culture vessel is the PrimeSurface 96 slit well plate (manufactured by Sumitomo Bakelite Co., Ltd.). This plate has narrow openings (slits) at the top of each of the 96 wells through which the culture medium can enter and exit. The slits are set to a width that makes it difficult for cell aggregates to pass through, so the culture medium for the entire plate can be replaced at once while preventing adhesion between the cell aggregates, improving the efficiency of the operation and the quality of the cell aggregates.

The concentration of pluripotent stem cells in the first step can be appropriately set so as to form cell aggregates more uniformly and efficiently. For example, when human pluripotent stem cells (e.g., human iPS cells obtained from step (a)) are cultured in suspension using a 96-well microwell plate, a liquid prepared so as to obtain about 1 x 10 ³ to about 1 x 10 ⁵ cells, preferably about 3 x 10 ³ to about 5 x 10 ⁴ cells, more preferably about 4 x 10 ³ to about 2 x 10 ⁴ cells, even more preferably about 4 x 10 ³ to about 1.6 x 10 ⁴ cells, and particularly preferably about 8 x 10 ³ to about 1.2 x 10 ⁴ cells per well is added to each well, and the plate is left to form cell aggregates. For example, when human pluripotent stem cells (e.g., human iPS cells obtained in step (a)) are cultured in suspension using EZSPHERE SP Dish 35 mm Type 905, a cultureware having about 260 microwells per dish, a liquid prepared so that typically about 1 x ¹⁰⁴ to about 1 x ¹⁰⁸ cells, preferably about 3 x ¹⁰⁴ to about 5 x ¹⁰⁷ cells, more preferably about 4 x ¹⁰⁴ to about 2 x ¹⁰⁷ cells, even more preferably about 4 x ¹⁰⁴ to about 1.6 x ¹⁰⁷ cells, and particularly preferably about 8 x ¹⁰⁴ to about 1.2 x ¹⁰⁷ cells per dish is added to the dish, and the dish is allowed to stand to form cell aggregates. For example, when human pluripotent stem cells (e.g., human iPS cells obtained from step (a)) are cultured in suspension using AggreWell 800, 6-well plate, a cultureware having about 1800 microwells per well, a solution prepared so that each well contains usually about 1×10 ⁴ to about 1×10 ⁸ cells, preferably about 3×10 ⁴ to about 5×10 ⁷ cells, more preferably about 4×10 ⁴ to about 2×10 ⁷ cells, even more preferably about 4×10 ⁴ to about 1.6×10 ⁷ cells, and particularly preferably about 8×10 ⁴ to about 1.2×10 ⁷ cells is added to each well, and the plate is centrifuged to form cell aggregates. The number of cells can be determined by counting with a hemocytometer.

The time required for suspension culture to form cell aggregates can be appropriately determined depending on the pluripotent stem cells used, but it is desirable that the time be as short as possible in order to form uniform cell aggregates. The process from the dispersed cells until they form cell aggregates can be divided into a process in which the cells gather and a process in which the gathered cells form aggregates. For example, in the case of human pluripotent stem cells (human iPS cells, etc.), the time from the time the dispersed cells are seeded (i.e., the time when suspension culture begins) until the cells gather is preferably within about 24 hours, more preferably within about 12 hours. For example, in the case of human pluripotent stem cells (human iPS cells, etc.), the time from the time the dispersed cells are seeded (i.e., the time when suspension culture begins) until the cell aggregates are formed is preferably within about 72 hours, more preferably within about 48 hours. The time until the cell aggregates are formed can be appropriately adjusted by adjusting the tool for aggregating the cells, the centrifugation conditions, etc.

When pluripotent stem cells are rapidly aggregated to form cell aggregates, epithelial-like structures can be reproducibly formed in cells induced to differentiate from the formed aggregates. Examples of experimental procedures for forming cell aggregates include a method of confining cells in a small space using a small well plate (e.g., a plate with a well bottom area of about 0.1 to ^2.0 cm2 calculated as a flat bottom) or a micropore, and a method of aggregating cells by centrifuging for a short time using a small centrifuge tube. Examples of small well plates include 24-well plates (area of about 1.88 ^cm2 calculated as a flat bottom), 48-well plates (area of about 1.0 ^cm2 calculated as a flat bottom), 96-well plates (area of about 0.35 ^cm2 calculated as a flat bottom, inner diameter of about 6 to 8 mm), and 384-well plates. A preferred example is a 96-well plate. Examples of the shape of the small well plate, when viewed from above, include polygonal, rectangular, elliptical, and perfect circle as the shape of the bottom of the well. A preferred example is a perfect circle. As for the shape of the small well plate, the shape of the bottom of the well when viewed from the side is preferably a structure with a high outer periphery and a low inner recess, for example, a U-bottom, a V-bottom, or an M-bottom, preferably a U-bottom or a V-bottom, and most preferably a V-bottom. As the small well plate, a cell culture dish (e.g., a 60 mm to 150 mm dish, culture flask) having an uneven or recessed bottom may be used. The bottom of the small well plate is preferably a non-cell-adhesive bottom, preferably a bottom coated with a non-cell-adhesive material.

As another method for forming cell aggregates, it is also preferable to use a three-dimensional printing machine or 3D printer. A cell population of the desired shape can be prepared by suspending a dispersed single cell or a spheroid composed of multiple cells in a biocompatible ink (bioink) and printing it out using a bio 3D printer (e.g., Celllink's BIO X, etc.), or by piercing a cell population with a needle and stacking it (Cyfuse's Spike, etc.).

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

At the start of the first step, in a preferred embodiment, adherent culture is performed. The pluripotent stem cells on the culture vessel after step (a) may be used as they are in the first step, or the pluripotent stem cells may be dispersed into single cells and then seeded again on the adherent culture vessel. When reseeding the pluripotent stem cells after dispersing them into single cells, an appropriate extracellular matrix or synthetic cell adhesion molecule may be used as a scaffold. The scaffold allows adherent culture of the pluripotent stem cells in the culture vessel whose surface is coated. The extracellular matrix is preferably matrigel or laminin. Examples of synthetic cell adhesion molecules include synthetic peptides containing a cell adhesive domain such as poly-D-lysine and RGD sequence. The number of cells seeded is not particularly limited as long as differentiation into the pituitary gland occurs, but from the viewpoint of reproducing adhesion and interaction between cells, it is also preferable that the cell density reaches semi-confluence, which corresponds to more than 60% of the culture space of the vessel, within 72 hours after seeding on the culture vessel.

It is also preferable to use a cultureware with a micropattern as the adhesive cultureware. The micropattern on the cultureware can be composed of a cell adhesive region and a cell non-adhesive region, and it is preferable that cells are cultured in the cell adhesive region. The shape of the cell adhesive region and the cell non-adhesive region is not limited as long as they can be developed on the cultureware. The cell adhesive region and the cell non-adhesive region may be formed as a single region on one cultureware, or multiple regions may be formed. It is preferable that the cell adhesive region is artificially treated for the purpose of improving adhesion. Examples of cultureware with a micropattern include CYTOOChip (manufactured by CYTOO), ibidi Micropatterning (manufactured by ibidi), etc. The cultureware can also be prepared using a PDMS mold and a matrix, etc. Alternatively, a cultureware coated with an extracellular matrix or a substrate that promotes cell adhesion may be processed with a laser or the like using, for example, a cell processing device (Model: CPD-017, Kataoka Seisakusho Co., Ltd.) to create cell adhesive and non-cell adhesive regions of any shape. When culturing the pluripotent stem cells obtained in step (a) on a cultureware with a micropattern, this can be carried out by referring to, for example, a previously reported method (Nature protocols, 11(11), 2223-2232.).

It is also preferable that the culture equipment has a flow path (micro-flow path) for perfusing the medium, and cells may be cultured in a perfusion environment in the first step and subsequent steps. Such a culture equipment is also called a microfluidic chip. The culture equipment (e.g., microfluidic chip) may be connected by a flow path to other culture equipment (e.g., microfluidic chip) for culturing cells or tissues other than the cells to be cultured in the manufacturing method of the present invention. This makes it possible to reproduce the interaction between the pituitary gland and other cells or tissues. Examples of other cells or tissues to be co-cultured with the pituitary gland include, but are not limited to, tissues that are regulated by hormones secreted from the pituitary gland, and tissues that promote the growth, differentiation, maturation, and survival of the pituitary gland, such as cells or tissues of the brain, blood vessels, bone, muscle, fat, thyroid gland, liver, adrenal gland, testis, ovary, and breast. Examples of methods for perfusing the medium include, but are not limited to, the use of a magnetic stirrer, a peristaltic pump, etc.

The culture equipment may have a membrane that is permeable to oxygen or culture medium. The culture equipment may be capable of forming a concentration gradient of compounds, growth factors, etc. The membrane is, for example, a porous membrane. In culture equipment having such a membrane, cells can be cultured by the manufacturing method of the present invention on one side separated by the membrane, and other cells or tissues, feeder cells, etc. can be cultured on the other side. This makes it possible to culture cells that constitute the pituitary gland or their precursor cells and cell populations containing these without contamination with other cells or tissues.

In the first and subsequent steps, when a medium exchange operation is performed, examples include an operation in which new medium is added without discarding the original medium (medium addition operation), an operation in which about half the original medium (about 30-90% of the volume of the original medium, for example about 40-60%) is discarded and about half the new medium (about 30-90% of the volume of the original medium, for example about 40-60%) is added (half medium exchange operation), and an operation in which about the entire amount of the original medium (90% or more of the volume of the original medium) is discarded and about the entire amount of new medium (90% or more of the volume of the original medium) is added (full medium exchange operation).

When adding a specific component at a certain point in time, for example, after calculating the final concentration, an operation may be performed in which about half of the original medium is discarded and about half of a new medium containing the specific component at a concentration higher than the final concentration is added (half medium exchange operation). When diluting a component contained in the original medium to reduce the concentration at a certain point in time, for example, the medium exchange operation may be performed multiple times a day, preferably multiple times within an hour (e.g., 2 to 3 times). Also, when diluting a component contained in the original medium to reduce the concentration at a certain point in time, the cells or cell aggregates may be transferred to another culture vessel. The tools used for the medium exchange operation are not particularly limited, but examples include a pipetter, Pipetman (registered trademark), a multichannel pipette, and a continuous dispenser. For example, when a 96-well plate is used as the culture equipment, a multichannel pipette may be used.

The culture time in the first step is usually about 8 hours to 6 days, preferably about 12 hours to 60 hours.

In the first and subsequent steps, it is also preferable to add a compound that promotes differentiation into the placode region in order to improve the efficiency of pituitary production. Examples of compounds that have the above-mentioned effect include BRL-54443, phenanthrolin, and parthenolide, which are described in US Patent US20160326491A1. When BRL-54443 is used as a compound that promotes differentiation into the placode region, it is usually used at a concentration of about 10 nM to about 100 μM, when phenanthrolin is used, it is usually used at a concentration of about 10 nM to about 100 μM, and when parthenolide is used, it is usually used at a concentration of about 10 nM to about 100 μM.

In the first step, from the viewpoint of improving the efficiency of inducing differentiation into pituitary gland, the culture can be performed in the presence of a substance acting on the Sonic Hedgehog signaling pathway. As the substance acting on the Shh signaling pathway used in the first step, the same substances as those exemplified in step (a) can be used. The substances acting on the Shh signaling pathway in step (a) and the first step may be the same or different, but are preferably the same, and are preferably SAG.
The concentration of the substance acting on the Shh signaling pathway in the medium can be appropriately set depending on the substance used within a range that can achieve the above-mentioned effects. When SAG is used as the substance acting on the Shh signaling pathway in the first step, it is usually used at a concentration of about 1 nM to about 3 μM, preferably about 10 nM to about 2 μM, more preferably about 30 nM to about 1 μM, and even more preferably about 50 nM to about 500 nM.

<Step (2)>: Second Step In step (2), the cell population obtained in the first step is cultured in the presence of a substance acting on the BMP signaling pathway and a substance acting on the Sonic Hedgehog signaling pathway. If the cells are cultured in suspension in the first step, the formed cell aggregates may be continuously cultured in suspension in step (2). If the cells are cultured in adhesion in the first step, the cells may be continuously cultured in adhesion in step (2). After the cells are cultured in suspension in the first step, they may be cultured in adhesion in step (2).

A substance acting on the BMP signaling pathway is a substance that can enhance the signaling pathway mediated by BMP. Examples of substances that can enhance the signaling pathway mediated by BMP include substances that stabilize BMP ligands in a culture environment and improve their potency, substances that bind to type I BMP receptors ALK-1, ALK-2, ALK-3, and ALK-6 and activate and induce intracellular signaling downstream of the receptor, substances that induce phosphorylation of Smad-1, Smad-5, Smad-8, and Smad-9 involved in intracellular BMP signaling, and substances that induce and enhance functions such as activation and inhibition of gene transcription by Smad-1/5/8/9. Examples of substances acting on the BMP signaling pathway include BMP proteins such as BMP2, BMP4, and BMP7, GDF proteins such as GDF5, 6, and 7, anti-BMP receptor antibodies, and BMP partial peptides. These substances may be used alone or in combination. In terms of biological activity, a substance acting on the BMP signal transduction pathway can be defined as a substance that has the ability to induce differentiation into osteoblast-like cells and the ability to induce alkaline phosphatase production in cells such as mouse precursor chondrocyte cell line ATDC5, mouse calvaria-derived cell line MC3T3-E1, and mouse striated muscle-derived cell line C2C12. Examples of substances having the above activities include BMP2, BMP4, BMP5, BMP6, BMP7, BMP9, BMP10, BMP13/GDF6, BMP14/GDF5, GDF7, etc.

BMP2 protein and BMP4 protein are available, for example, from R&D Systems, BMP7 protein is available, for example, from Biolegend, GDF5 protein is available, for example, from Peprotech, GDF6 protein is available, for example, from Primegene, and GDF7 protein is available, for example, from Fujifilm Wako Pure Chemical Industries, Ltd. The BMP signaling pathway active substance preferably includes at least one protein selected from the group consisting of BMP2, BMP4, BMP7, BMP13, and GDF7, and more preferably includes BMP4.

The concentration of the substance acting on the BMP signaling pathway in the culture medium can be set appropriately depending on the substance used within a range that can achieve the above-mentioned effects. From the viewpoint of improving the production efficiency of cells that constitute the pituitary gland, when BMP4 is used as the substance acting on the BMP signaling pathway, it is usually used at a concentration of about 1 pM to about 100 nM, preferably about 10 pM to about 50 nM, more preferably about 25 pM to about 25 nM, even more preferably about 25 pM to about 5 nM, particularly preferably about 100 pM to about 5 nM, and most preferably about 500 pM to about 2 nM. Furthermore, when a substance acting on the BMP signaling pathway other than BMP4 is used, it is desirably used at a concentration that exhibits the same BMP signaling pathway promoting activity as BMP4 at the above-mentioned concentration. When using, for example, a commercially available recombinant BMP protein as a substance acting on the BMP signaling pathway, a person skilled in the art can easily determine the concentration of the substance acting on the BMP signaling pathway to be added by comparing the activity described in the product insert, such as the ED50 value of the ability to induce alkaline phosphatase production in the mouse precursor chondrocyte cell line ATDC5, with the concentration and activity of the above-mentioned BMP4.

Compounds well known to those skilled in the art can also be used as BMP signaling pathway active substances. Examples of BMP signaling pathway active substances include Smurf1 inhibitors, Chk1 inhibitors, and phosphorylated Smad stabilizers. Examples of compounds having the above-mentioned activity include A-01 ([4-[[4-Chloro-3-(trifluoromethyl)phenyl]sulfonyl]-1-piperazinyl][4-(5-methyl-1H-pyrazol-1-yl)phenyl]methane), PD 407824 (9-Hydroxy-4-phenyl-pyr Examples include rolo[3,4-c]carbazole-1,3(2H,6H)-dione, SB4 (2-[[(4-bromophenyl)methyl]thio]benzoxazole), SJ000291942 (2-(4-ethylphenoxy)-N-(4-fluoro-3-nitrophenyl)-acetamide) and their derivatives.

The substance acting on the Shh signaling pathway used in step (2) may be the same as those exemplified in step (a). The substances acting on the Shh signaling pathway in steps (a) and (2), and optionally the substance acting on the Shh signaling pathway in step (1), may be the same or different, but are preferably the same, and are preferably SAGs.
The concentration of the substance acting on the Shh signaling pathway in the medium can be appropriately set depending on the substance used within a range that can achieve the above-mentioned effects. When SAG is used as the substance acting on the Shh signaling pathway in step (2), 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.

The medium used in step (2) is not particularly limited as long as it contains a substance acting on the Shh signaling pathway and a substance acting on the BMP signaling pathway. Examples of the medium used in step (2) include the medium listed in the first step.

From the viewpoint of improving the efficiency of pituitary gland production, the start time of step (2) is preferably 0.5 hours to 6 days after the start of the culture in the first step, more preferably 0.5 hours to 72 hours, and even more preferably 24 hours to 60 hours. When performing suspension culture, if step (2) is started during the above period in the presence of a Wnt signal pathway inhibitor, non-neuroepithelial-like tissue is formed on the surface of the cell aggregates, and pituitary glands are formed extremely efficiently.

When suspension culture is performed in the first step, from the viewpoint of improving the production efficiency of cells constituting the pituitary gland, the start time of step (2) is preferably the time when at least 10% of the cells on the surface layer of the cell aggregate formed in the first step, more preferably at least 30%, and even more preferably at least 50% of the cells form tight junctions with each other. Those skilled in the art can easily determine whether tight junctions are formed in the cell aggregate by, for example, observation under a microscope or immunostaining using an anti-ZO-1 antibody.

In step (2), the start of culture in the presence of a substance acting on the BMP signaling pathway may be performed by carrying out the above-mentioned medium replacement operation (e.g., medium addition operation, half-medium replacement operation, full-medium replacement operation, etc.) using the culture vessel in which the first step was carried out, or the cells may be transferred to a different culture vessel.

The period of culture in the medium containing the substance acting on the BMP signaling pathway in step (2) can be appropriately set. The period of culture in step (2) may be any period during which the concentration of the substance acting on the BMP signaling pathway in the culture medium is maintained for a period necessary for inducing pituitary tissue (specifically, a cell population containing pituitary progenitor cells), and is usually 8 hours or more, preferably 10 hours or more, more preferably 12 hours or more, even more preferably 14 hours or more, and most preferably 16 hours or more.
When the medium replacement operation is performed after the lapse of the period, the concentration of the substance acting on the BMP signaling pathway may be gradually reduced after the lapse of the period by performing the above-mentioned half-volume medium replacement operation with a medium not containing a substance acting on the BMP signaling pathway. Also, after the lapse of the period, a full-volume medium replacement operation may be performed with a medium not containing a substance acting on the BMP signaling pathway. Even in this case, as long as the culture is continued in a medium containing a substance acting on the Shh signaling pathway, it is included as part of step (2) in the present specification.

The period of culture in the medium containing the substance acting on the Shh signaling pathway in step (2) can be set appropriately. When the suspension culture in step (1) is further performed in the presence of a substance acting on the Shh signaling pathway, from the viewpoint of improving the ability to secrete pituitary hormones (particularly ACTH), the culture period in the presence of the substance acting on the Shh signaling pathway in steps (1) and (2) is 20 to 40 days, preferably 25 to 35 days, and more preferably about 30 days.

In order to promote differentiation into pituitary placodes in step (2) and the subsequent steps, it is also preferable to add an FGF signaling pathway active substance to the culture environment. The FGF signaling pathway active substance is not particularly limited as long as it is a substance that can enhance the signaling pathway mediated by FGF (fibroblast growth factor). Examples of FGF signaling pathway active substances include FGF proteins such as FGF1, FGF2 (also called bFGF), FGF3, FGF8, and FGF10, anti-FGF receptor antibodies, and FGF partial peptides. These substances may be used alone or in combination.

FGF2 protein and FGF8 protein are available, for example, from Fujifilm Wako Pure Chemical Industries, Ltd. The FGF signaling pathway active substance preferably includes at least one selected from the group consisting of FGF2, FGF3, FGF8, FGF10, and modified forms thereof, more preferably includes FGF2, and even more preferably includes recombinant human FGF2.

The concentration of the FGF signaling pathway active substance in the medium can be set appropriately depending on the substance used within a range that can achieve the above-mentioned effects. From the viewpoint of differentiation into cells that constitute the pituitary gland and promotion of cell survival and proliferation, when FGF2 is used as the FGF signaling pathway active substance, it is usually used at a concentration of about 1 pg/ml to about 100 μg/ml, preferably about 10 pg/ml to about 50 μg/ml, more preferably about 100 pg/ml to about 10 μg/ml, even more preferably about 500 pg/ml to about 1 μg/ml, and most preferably about 1 ng/ml to about 200 ng/ml. When using an FGF signaling pathway active substance other than FGF2, it is desirable to use it at a concentration that shows the same FGF signaling pathway promoting activity as FGF2 at the above concentration. The FGF signaling pathway promoting activity of the added substance can be measured, for example, by a method such as a cell proliferation test using 3T3 cells.

In order to maintain the activity of the FGF protein in the medium, it is also preferable to add heparin or heparan sulfate to the medium containing the FGF protein. Heparin is available as a sodium salt, for example from Fujifilm Wako Pure Chemical Industries, Ltd. The concentration of heparin or heparan sulfate in the medium can be appropriately set within a range that can achieve the above-mentioned effects. The concentration of heparin sodium in the 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, even more 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 preferable that the concentration has the same FGF protein protection activity as the above-mentioned concentration of heparin. For the purpose of maintaining the activity of FGF protein in a cell culture environment at 37°C or the like, it is also preferable to use modified FGF such as Thermostable FGF2 described in US Patent No. US8772460B2, or FGF2 sustained-release beads such as StemBeads FGF2 in which FGF2 is bound to a biodegradable polymer. Thermostable FGF2 is available, for example, from HumanZyme. StemBeads FGF2 is available, for example, from StemCulture.

The timing of addition of the substance acting on the FGF signaling pathway in step (2) and the subsequent steps can be set as appropriate. In a preferred embodiment, the substance acting on the FGF signaling pathway is added 6 hours or more, more preferably 12 hours or more, and even more preferably 18 hours or more after the addition of the substance acting on the BMP signaling pathway in step (2).

In step (2), it is also preferable to continue adding the additives used in step (a) or the first step, such as a JNK signaling pathway inhibitor, a Wnt signaling pathway inhibitor, a TGFβ signaling pathway inhibitor, a TAK1 inhibitor, etc. The JNK signaling pathway inhibitor, Wnt signaling pathway inhibitor, or TGFβ signaling pathway inhibitor added in step (2) may be different from the substance used in the previous step, but is preferably the same. The concentration and type of additives can be adjusted as appropriate. The timing of addition of these substances may be simultaneous with the start of step (2) or may be different.

<Step (b)>: Step b In step (b), the cell population obtained in step (2) is cultured under conditions in which a substance inhibiting the BMP signaling pathway is added. If the cells are cultured in suspension in step (2), the cell aggregates formed may be cultured in suspension in step (b). If the cells are cultured in adhesion in step (2), the cells may be cultured in adhesion in step (b).

The BMP signaling pathway inhibitor is not limited as long as it can suppress signaling induced by BMP family proteins. It may be any of nucleic acids, proteins, and low molecular weight organic compounds. Examples of such substances include substances that inhibit BMP processing and extracellular secretion, substances that act directly on BMP (e.g., proteins, antibodies, aptamers, etc.), substances that suppress the expression of genes encoding BMP (e.g., antisense oligonucleotides, siRNA, etc.), substances that inhibit the binding of BMP receptors to BMP, and substances that inhibit physiological activity resulting from signaling by BMP receptors. There are type I BMP receptors and type II BMP receptors, and BMPR1A, BMPR1B, and ACVR are known as type I BMP receptors, while TGF-beta R-II, ActR-II, ActR-IIB, BMPR2, and MISR-II are known as type II BMP receptors.

Proteins known as BMP signaling pathway inhibitors include, for example, Noggin, Chordin, Follistatin, Gremlin, Inhibin, Twisted Gastrulation, Coco, and secreted proteins belonging to the DAN family. Since a substance acting on the BMP signaling pathway is added to the culture medium in step (2) above, from the viewpoint of more effectively inhibiting the subsequent BMP signaling pathway, it is preferable that the BMP signaling pathway inhibitor in step (b) includes a substance that inhibits the signaling pathway subsequent to the secretion of BMP outside the cell, such as a substance that inhibits the binding between BMP receptors and BMP, or a substance that inhibits physiological activity resulting from signaling by BMP receptors, and more preferably includes an inhibitor of type I BMP receptor.

Compounds well known to those skilled in the art can also be used as BMP signaling pathway inhibitors. Examples of BMP signaling pathway inhibitors include inhibitors of type I BMP receptors. Compounds having the above-mentioned activity include, for example, K02288 (3-[(6-Amino-5-(3,4,5-trimethoxyphenyl)-3-pyridinyl]phenol), Dorsomorphin (6-[4-[2-(1-Piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)pyrazolo[1,5-a]pyrimid), and ine), 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]quin oline), 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-Isopr opoxyphenyl) pyrazolo[1,5-a]pyrimidin-3-yl)quinoline), DMH2(4-[6-[4-[2-(4-Morpholinyl)ethoxy]phenyl]pyrazolo[1,5-a]py rimidin-3-yl]-quinoline), Compound 1(3-(1,2,3-benzothiadiazol-6-y l)-1-[2-(cyclohex-1-en-1-yl)ethyl]urea), VU0465350(7-(4-isopropoxyphenyl)-3-(1H-pyrazol-4-yl)imidazo[1,2-a]pyridine), V U0469381 (5-(6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl) quinolone), OD36(4-chloro-7,10-dioxa-13,17,18,21-tetrazatetracyclo[12.5.2.12,6.017,20]docosa-1(20),2(22),3,5,14(21),15 , 18-heptaene), OD52, E6201 ((3S, 4R, 5Z, 8S, 9S, 11E)-14-(ethylamino) -8,9,16-trihydroxy-3,4-dimethyl-3,4,9,10-tetrahydro-1H-benzo[c][1]oxacyclotetradecine-1,7(8H)-dione), Saracatinib (N-(5-ch loro-1,3-benzodioxol-4-yl)-7-[2-(4-methylpiperazin-1-yl)et hoxy]-5-(oxan-4-yloxy)quinazolin-4-amine, BYL719 ((2S)-1-N-[4-methyl-5-[2-(1,1,1-trifluoro-2-methylpropan-2-yl)pyridin-4-yl]-1,3-thiazol-2-yl]pyrrolidine-1,2-dicarboxamide), etc. These substances may be used alone or in combination.

The BMP signaling pathway inhibitor is preferably a type I BMP receptor inhibitor, more preferably at least one selected from the group consisting of K02288, Dorsomorphin, LDN-193189, LDN-212854, LDN-214117, ML347, DMH1 and DMH2, and even more preferably K02288 to LDN-193189.

The concentration of the BMP signaling pathway inhibitor in the culture medium can be set appropriately depending on the substance used within a range that can achieve the above-mentioned effects. From the viewpoint of the efficiency of pituitary tissue formation, when K02288 is used as the BMP signaling pathway inhibitor in step (b), 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 500 nM to about 25 μM. When LDN-193189 is used as the BMP signaling 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 1 μM, and even more preferably about 100 nM to about 500 nM. When LDN-212854 is used as the BMP signaling 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 ML347 is used as the BMP signaling 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 the BMP signaling 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. In addition, when using a BMP signaling pathway inhibitor other than K02288, it is desirable to use it at a concentration that exhibits the same BMP signaling pathway inhibitory activity as K02288 at the above concentration.

The timing for starting step (b) after carrying out step (2) can be set appropriately. The timing for starting step (b) is usually at least 8 hours and within 15 days, preferably at least 10 hours and within 12 days, more preferably at least 12 hours and within 9 days, even more preferably at least 14 hours and within 8 days, and most preferably at least 16 hours and within 7 days after starting step (2).

In step (2) and the subsequent steps, the cell population may be treated with corticosteroids by adding corticosteroids to the medium. Treatment with corticosteroids promotes differentiation of the pituitary placode and/or Rathke's pouch 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.). Examples of corticosteroids include, but are not limited to, natural glucocorticoids such as hydrocortisone, cortisone acetate, and fludrocortisone acetate; and artificially synthesized glucocorticoids such as dexamethasone, betamethasone, prednisolone, methylprednisolone, and triamcinolone.

The concentration of the adrenal cortical hormones in the medium is not particularly limited as long as it can promote differentiation from the pituitary placode and/or Rathke's pouch into pituitary hormone-producing cells (excluding ACTH-producing cells), and can be appropriately set depending on the type of adrenal cortical hormone. For example, in the case of hydrocortisone, it is usually 100 ng/ml or more, preferably 1 μg/ml or more. As long as there is no adverse effect on differentiation into pituitary hormone-producing cells (excluding ACTH-producing cells), there is no particular upper limit for the hydrocortisone concentration, but from the viewpoint of culture costs, it is usually 1000 μg/ml or less, preferably 100 μg/ml or less. In one embodiment, the hydrocortisone concentration in the medium is usually about 100 ng/ml to about 1000 μg/ml, preferably about 1 to about 100 μg/ml. When dexamethasone is used as the adrenal cortical hormone, its concentration in the medium can be about 1/25 of that of hydrocortisone.

In step (2) and the subsequent steps, the timing of adding the corticosteroids to the medium is not particularly limited as long as it can promote differentiation of the pituitary placode and/or Rathke's pouch into pituitary hormone-producing cells (excluding ACTH-producing cells). The corticosteroids may be added to the medium from the start of the second step, or the corticosteroids may be added to the medium after a certain period of culture in a medium without corticosteroids after the start of the second step. Preferably, the corticosteroids are added to the medium when the appearance of ACTH-producing cells is confirmed in the cell population after the start of the second step. That is, until the appearance of ACTH-producing cells is confirmed in the cell aggregate, the cell aggregate is cultured in a medium without corticosteroids, and after the appearance of ACTH-producing cells is confirmed, the second step and the subsequent steps are continued in a medium containing corticosteroids. The appearance of ACTH-producing cells can be confirmed by immunohistological staining using an antibody against ACTH. When human pluripotent stem cells are used, the appearance of ACTH-producing cells can generally be expected 30 days or more after the start of the first step, so in one embodiment, corticosteroids are added to the medium 30 days or more after the start of the first step.

The period for treating the cell aggregate with the corticosteroids is not particularly limited as long as it is possible to promote differentiation of the pituitary placode and/or Rathke's pouch into pituitary hormone-producing cells (excluding ACTH-producing cells), but the cell aggregate is usually treated with the corticosteroids until promotion of differentiation into pituitary hormone-producing cells (excluding ACTH-producing cells) is confirmed in the corticosteroid-treated group compared to the corticosteroid-untreated group. The treatment period is usually 7 days or more, preferably 12 days or more. The upper limit of the treatment period is not particularly limited, but the corticosteroids may be removed from the medium at the stage when promotion of differentiation into pituitary hormone-producing cells (excluding ACTH-producing cells) is confirmed in the corticosteroid-treated group compared to the corticosteroid-untreated group.

From the viewpoint of promoting differentiation into pituitary gland and growth hormone-producing cells, it is also preferable to carry out step (2) and the subsequent steps in the presence of a substance that acts on the retinoic acid signaling pathway. Examples of substances that act on the retinoic acid signaling pathway include substances that bind to retinoic acid receptors (RAR) or retinoid X receptors (RXR) and activate downstream transcription. Examples of compounds that have the above-mentioned effects include all-trans retinoic acid, isotretinoin, 9-cis retinoic acid, TTNPB (4-[(E)-2-[(5,5,8,8-Tetramethyl-5,6,7,8-tetrahydronaphthalene)-2-yl]-1-propenyl]benzoic acid), Ch55 (4-[(E)-3-(3,5 -di-tert-butylphenyl)-3-oxo-1-propenyl]benzoic acid), EC19(3-[2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)e thynyl]benzoic acid), EC23(4-[2-(5,6,7,8-Tetrahydro-5,5,8 , 8-tetramethyl-2-naphthalenyl)ethynyl]-benzoicacid), Fenretinide (4-hydroxyphenylretinamide), Acitretin ((all-e)-9-(4-metho xy-2,3,6-trimethylphenyl)-3,7-dimethyl-2,4,6,8-nonatetra en), Trifarotene, Adapalene, AC 261066 (4-[4-(2-Butoxyethoxy-)-5-methyl-2-thiazolyl]-2-fluorobenzoic acid), AC 55649 (4-N-Oc tylbiphenyl-4-carboxylic acid), AM 580(4-[(5,6,7,8-Tetrahydr o-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamide]benzoic acid), AM80(4-[(5,5,8,8-Tetramethyl-6,7-dihydronaphthalenyl-2-yl) carbamoyl]benzoic acid), BMS 753 (4-[[(2,3-Dihydro-1,1,3,3 -tetramethyl-2-oxo-1H-inden-5-yl)carbonyl]amino]benzoicacid), BMS 961 (3-Fluoro-4-[(r)-2-hydroxy-2-(5,5,8,8-tetramethyl-5 ,6,7,8-tetrahydro-naphthalen-2-yl)-acetylamino]-benzoic a cid), CD1530 (4-(6-Hydroxy-7-tricyclo[3.3.1.13,7]dec-1-yl-2-naphthalenyl)benzoicacid), CD2314 (5-(5,6,7,8-Tetrahydro-5,5,8, 8-tetramethyl-2-anthracenyl)-3-thiophenecarboxylic acid) , CD437 (2-naphthalenecarboxylic acid, 6-(4-hydroxy-3-triclo(3.3.1.1(3,7))dec-1-ylphen)), CD271 (6-[3-(1-adamantyl)-4-methoxyphenyl]-2-naphthalene carboxylic acid) and derivatives thereof. These substances may be used alone or in combination.

The retinoic acid pathway acting substance in step (2) and the subsequent steps preferably includes all-trans retinoic acid or EC23. The concentration of the retinoic acid pathway acting substance in the medium is not particularly limited as long as it is within a range in which the above-mentioned effects can be achieved, but when EC23 is used as the retinoic acid pathway acting substance, the concentration of EC23 is, for example, about 10 pM to about 30 μM, preferably about 100 pM to about 20 μM, more preferably about 10 nM to about 10 μM, and even more preferably about 100 nM to about 5 μM. When a retinoic acid pathway acting substance other than EC23 is used, it is desirable to use it at a concentration that exhibits retinoic acid pathway acting activity equivalent to that of EC23 at the above-mentioned concentration.

From the viewpoint of regulating differentiation tendency, it is also preferable to carry out step (2) and the subsequent steps in the presence of a Notch signaling pathway inhibitor. In this specification, the Notch signaling pathway refers to a signaling pathway that is 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 to which the Notch signal is transmitted, the Notch protein is processed in stages, and the intracellular domain cut out on the membrane is transported into the nucleus to control the expression of downstream genes.

The Notch signaling pathway inhibitor is not particularly limited as long as it can suppress signaling mediated by Notch. It may be any of nucleic acids, proteins, and low molecular weight organic compounds. Examples of such substances include functionally defective Notch receptors and ligands, substances that inhibit Notch processing (S1 cleavage), substances that inhibit glycosylation of Notch and Notch ligands, substances that inhibit cell membrane translocation, substances that inhibit processing (S2 cleavage, S3 cleavage) of the intracellular domain of Notch (NICD) (gamma secretase inhibitors), substances that degrade NICD, and substances that inhibit NICD-dependent transcription.

Compounds well known to those skilled in the art can also be used as Notch signaling pathway inhibitors. Examples of compounds having activity as Notch signaling pathway inhibitors include DAPT (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester), DBZ ((2S)-2-[[2-(3,5-difluorophenyl)acetyl]amino]-N-[( 7S)-5-methyl-6-oxo-7H-benzo[d][1]benzazepin-7-yl]propanamide), MDL28170 (benzyl N-[(2S)-3-methyl-1-oxo-1-[[(2S)-1-o xo-3-phenylpropan-2-yl]amino]butan-2-yl]carbamate), FLI- 06 (cyclohexyl 2,7,7-trimethyl-4-(4-nitrophenyl)-5-oxo-1,4,6,8-tetrahydroquinoline-3-carboxylate), L-685,458 (tert-butyl N -[6-[[1-[(1-amino-1-oxo-3-phenylpropan-2-yl)amino ]-4-methyl-1-oxopentan-2-yl]amino]-5-benzyl-3-hydroxy-6-oxo-1-phenylhexan-2-yl]carbamate), CB-103 (6-(4-tert-butylphenoxy)pyridin-3-amine) and derivatives thereof, as well as substances described in Onco Targets Ther. 2013; 6: 943-955. The Notch signaling pathway inhibitor preferably includes DAPT.

The concentration of the Notch signaling pathway inhibitor in the medium is not particularly limited as long as it is within a range in which the above-mentioned effects can be achieved, but for example, when DAPT is used as the Notch signaling pathway inhibitor, the concentration of DAPT is, for example, about 100 pM to about 50 μM, preferably about 1 nM to about 30 μM, more preferably about 100 nM to about 20 μM, and even more preferably about 1 μM to about 10 μM. When using a Notch signaling pathway inhibitor other than DAPT, it is desirable to use it at a concentration that exhibits Notch signaling pathway inhibitory activity equivalent to that of DAPT at the above-mentioned concentration.

<Step (3)>, <Step (3')>: Third Step In the third step, the cell population cultured in step (2) or step (b) is cultured under conditions in which a substance acting on the Sonic Hedgehog signaling (Shh) pathway is not added. The third step of culturing the cell population obtained in the second step in the absence of a substance acting on the Shh signaling pathway to obtain a cell population containing pituitary tissue is called step (3), and the third step of culturing the cell population obtained in step b in the absence of a substance acting on the Shh signaling pathway to obtain a cell population containing pituitary tissue is called step (3'). When the cells are cultured in suspension in step (2) or step (b), the formed cell aggregates may be continuously cultured in suspension in the third step. When the cells are cultured in adhesion in step (2) or step (b), the cells may be continuously cultured in adhesion in the third step. After the cells are cultured in suspension in step (2) or step (b), they may be cultured in adhesion in the third step.

The medium used in the third step is not particularly limited as long as it does not contain a substance acting on the Shh signaling pathway. The condition of not adding a substance acting on the Shh signaling pathway in this step refers to a condition in which a substance acting on the Shh signaling pathway is not intentionally added to the culture environment of the cell population, and also includes a case in which a substance acting on the Shh signaling pathway is unintentionally included in the culture environment due to autocretion by the cell population, etc. Examples of the medium used in the third step include the medium listed in the first step and gfCDM medium containing 10% to 20% KSR.

The third and subsequent steps may include a step of embedding the cell aggregates in a gel and culturing them in order to promote the survival and differentiation/maturation of the cells that make up the pituitary gland. Examples of gels include gels made of agarose, methylcellulose, collagen, matrigel, etc., and it is preferable to use matrigel.

In the third and subsequent steps of culturing cells by embedding them in a gel, the cell aggregates may be embedded as they are, or the cells may be seeded in the gel after being dispersed and isolated. Specific cell types such as basal cells may be separated using a cell sorter or the like and then seeded. As a further embodiment of the culture method of embedding cells in a gel, co-culture with cells other than the pituitary gland, such as fibroblasts, mesenchymal cells, and vascular cells, may also be performed. The gel-embedded culture described above may be performed by referring to, for example, Nature 501, 373-379 (2013), Nature, 499, 481-484 (2013), Nat Protoc 14, 518-540 (2019), Genes 2020, 11, 603, etc.

In the third and subsequent steps, it is also preferable to carry out a culture method in which the cells are physically shaken in order to improve the supply of nutrients and oxygen to the cells and to improve material exchange. Such culture methods include methods other than stationary culture, such as shaking culture, rotary culture, and agitation culture. The means for carrying out shaking culture, rotary culture, agitation culture, and the like are not particularly limited, but for example, shaking culture, rotary culture, agitation culture, and the like can be carried out by placing the culture equipment in which the cells are cultured on a rotator, shaker, or the like, or placing the cells in an environment in which a stirrer or the like is rotating. Those skilled in the art can appropriately set parameters such as the speed of shaking culture, rotary culture, and agitation culture within a range that does not cause damage to the cells. For example, when shaking culture is carried out using a 3D shaker with a wave-type agitation (e.g., Mini-Shaker 3D, manufactured by Biosan), the shaking speed range can be set, for example, to 5 to 60 rpm, preferably 5 to 40 rpm, and more preferably 5 to 20 rpm. When shaking culture is performed using a reciprocating shaker (e.g., NS-LR, manufactured by AS ONE), the shaking speed range can be set, for example, in the range of 15 to 60 rpm, preferably 15 to 50 rpm, and more preferably 15 to 45 rpm. When shaking culture is performed using a seesaw shaker (e.g., NS-S, manufactured by AS ONE), the shaking speed range can be set, for example, in the range of 5 to 50 rpm, preferably 5 to 40 rpm, and more preferably 5 to 30 rpm. When culturing cell aggregates by stirring culture or rotary culture, for example, a spinner flask (e.g., 3152, manufactured by Corning) can be placed on a magnetic stirrer and culture can be performed at a rotation speed at which the cell aggregates do not settle with the naked eye. Culture can also be performed using a three-dimensional rotary suspension culture device (e.g., CellPet CUBE, manufactured by J-Tech; Clinostar, manufactured by Celvivo). From the viewpoint of suppressing physical damage to the cells, such as friction, it is also preferable to subject the cell aggregates embedded in the gel to shaking culture, rotation culture, or stirring culture.

In the third and subsequent steps, it is also preferable to culture in a high-oxygen atmosphere from the viewpoint of suppressing cell death and promoting cell proliferation. High-oxygen conditions during the culture process can be achieved, for example, by connecting an oxygen tank to the incubator in which the cells are cultured and artificially supplying oxygen. The oxygen concentration for this purpose is usually 25% to 80%, and more preferably 30% to 60%.

In the third and subsequent steps, culture equipment with high gas exchange efficiency can be used to increase the amount of oxygen supplied to the medium in which the cell aggregates are cultured. Examples of such culture equipment include cell culture dishes, Lumox dishes (manufactured by Sarstedt Co., Ltd.) with a gas-permeable film on the bottom of the plate, and VECELL 96-well plates (manufactured by Vecel Co., Ltd.). It is also preferable to use this in combination with the culture under high oxygen concentration conditions described above.

In the third and subsequent steps, a cell protective agent may be added to the medium from the viewpoint of maintaining the structure of the non-neuroepithelial tissue in the cell aggregate. Examples of cell protective agents used in the third and subsequent steps include the above-mentioned FGF signaling pathway active substances, heparin, ROCK inhibitors, basement membrane preparations, myosin inhibitors, polyamines, ISR inhibitors, caspase inhibitors, serum, or serum substitutes. Examples of myosin inhibitors include blebbistatin, which is an inhibitor of nonmuscle myosin II ATPase, and ML-7, ML-9, W-7, MLCK inhibitor peptide 18, and derivatives thereof, which are inhibitors of myosin light chain kinase (MLCK). The cell protective agent added may be different from that added in the first step, but is preferably the same. A preferred cell protective agent is a ROCK inhibitor. In the third and subsequent steps, when the ROCK inhibitor Y-27632 is added as a cell protective agent, it is usually added to the culture environment at a concentration of about 10 nM to about 10 mM, preferably about 100 nM to about 1 mM, and more preferably about 1 μM to about 100 μM. When the ROCK inhibitor Chroman 1 is added, it is usually added to the culture environment at a concentration of about 10 pM to about 1 mM, preferably about 100 pM to about 100 μM, and more preferably about 1 nM to about 10 μM. When the nonmuscle myosin II ATPase inhibitor Blebbistatin is added as a cell protective agent, it is usually added to the culture environment at a concentration of about 10 nM to about 10 mM, preferably about 100 nM to about 1 mM, and more preferably about 1 μM to about 100 μM.

In the third step and subsequent steps, a substance other than a cytoprotectant that has the effect of maintaining the structure of non-neuroepithelial tissue can also be added. Examples of such substances include substances that promote cell adhesion, substances that promote the synthesis of basement membrane components, and substances that inhibit the decomposition of basement membrane components. The substance that promotes cell adhesion may promote cell-cell adhesion, cell-basement membrane adhesion, or cell-culture equipment adhesion, or may promote the production of factors involved in cell adhesion. Examples of substances that promote cell adhesion include adhesamine, adhesamine-RGDS derivatives, pyrintegrin, biotin tripeptide-1, acetyl tetrapeptide-3, RGDS peptide, and derivatives thereof. Examples of substances that promote the synthesis of basement membrane components include ascorbic acid derivatives. Examples of ascorbic acid derivatives include sodium ascorbyl phosphate, magnesium ascorbyl phosphate, ascorbyl 2-glucoside, 3-O-ethyl ascorbic acid, ascorbyl tetrahexyldecanoate, ascorbyl palmitate, ascorbyl stearate, ascorbyl 2-phosphate-6 palmitate, and glyceryl octyl ascorbic acid. Examples of substances that inhibit the decomposition of basement membrane components include inhibitors of matrix metalloproteases and serine proteases. When ascorbic acid 2-phosphate, which is a type of ascorbic acid derivative that promotes the synthesis of basement membrane components, is added, it is usually added to the culture environment at a concentration of 10 μg/ml or more and 1000 μg/ml or less, preferably 30 μg/ml or more and 500 μg/ml or less, and more preferably 50 μg/ml or more and 300 μg/ml or less. When adding other ascorbic acid or ascorbic acid derivatives, etc., add them so that the molar equivalents are approximately the same as the above concentrations.

In the third and subsequent steps, from the viewpoint of promoting the survival of pituitary cells, it is also preferable to add a substance that has the effect of reducing oxidative stress. Examples of substances having such activity include antioxidants, substances with free radical scavenging action, NADPH oxidase inhibitors, cyclooxygenase inhibitors, lipoxygenase (LOX) inhibitors, superoxide dismutase (SOD)-like substances, Nrf2 activators, etc. Examples of substances having the above-mentioned activity include ascorbic acid, N-acetyl-L-cysteine, (±)-α-tocopherol acetate, apocynin (4'-hydroxy-3'-methoxyacetophenone), nicotinamide, taurine (2-aminoethanesulfonic acid), IM-93 (1-isopropyl-3-(1-methyl-1H-indole-3-yl)-4-(N,N-dimethyl-1,3-pr Celastrol (3-Hydroxy-24-nor-2-oxo-1 (10), 3,5, 7-friedelatetraen-29-oic Acid; Tripterin), Ebselen (2-Phenyl-1, 2-benzisoselenazol-3 (2H)-one), (-)-Epigallocatechin Gallate ((2R,3R)-2-(3,4,5-Trihydroxyphenyl)-3,4-dihydro-1[2H]-benzopyran-3,5,7-triol-3-(3, 4,5-trihydroxybenzoate)), EUK-8(N,N'-Bis(salicylideneamino)ethane-manganese(II)), E Examples of such antioxidants include, but are not limited to, daravonone (3-methyl-1-phenyl-2-pyrazolin-5-one), MnTBAP (Mn(III) tetrakis(4-benzoic acid) porphyrin chloride), Nordihydroguaiaretic Acid, Resveratrol (trans-3,4,5-Trihydroxystilbene) and their derivatives. Reagents already prepared for cell culture (e.g., antioxidant supplements, Sigma Aldrich, A1345) can also be used. The substance having the effect of reducing oxidative stress used in the present invention preferably includes at least one selected from the group consisting of ascorbic acid, N-acetyl-L-cysteine and their derivatives. Ascorbic acid can be added to the medium, for example, in the form of its derivative ascorbic acid 2-phosphate, at a concentration of about 1 nM to about 1 M, preferably about 10 nM to about 100 mM, more preferably about 100 nM to about 10 mM, and even more preferably about 1 μM to about 3 mM, and N-acetyl-L-cysteine can be added to the medium at a concentration of, for example, about 1 nM to about 1 M, preferably about 10 nM to about 100 mM, more preferably about 100 nM to about 10 mM, and even more preferably about 1 μM to about 5 mM.

In the third and subsequent steps, from the viewpoint of promoting survival of pituitary cells, it is also preferable to add an inhibitor of the stress-responsive signaling pathway (a substance that inhibits the intracellular signaling mechanism in response to stress). The stress-responsive MAP kinase pathway (stress-activated protein kinase: SAPK) is one of the main intracellular signaling mechanisms in response to stress. Examples of inhibitors of the stress-responsive MAP kinase pathway include MAP3K inhibitors, MAP2K inhibitors, ASK inhibitors, MEK inhibitors, Akt inhibitors, Rho family kinase inhibitors, JNK inhibitors, p38 inhibitors, MSK inhibitors, STAT inhibitors, NF-κB inhibitors, and CAMK inhibitors.

Examples of MEK inhibitors include Selumetinib (AZD6244, 6-(4-bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide), Mirdametinib (PD0325901, N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-(2-fluoro-4-iodoanilino)benzamide), Trametinib (GSK1120212, N -[3-[3-cyclopropyl-5-(2-fluoro-4-iodoanilino)-6,8-dimethyl-2,4,7-trioxopyrido[4,3-d]pyrimidin-1-yl]phenyl]acetamide), U012 6(1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene), PD184352(CI-1040,2-(2-chloro-4-iodoanilino)-N-(cyclopropyl methoxy)-3,4-dif luorobenzamide), PD98059 (2-(2-amino-3-methoxyphenyl)chromen-4-one), BIX 02189 (3-[N-[3-[(dimethylamino)methyl]phenyl]-C-ph enylcarbonimidoyl]-2-hydroxy-N,N-dimethyl-1H-indole-6-carboxamide), Pimasertib (AS-703026, N-[(2S)-2,3-dihydroxypropyl]-3-(2 -fluoro-4-iodoanil ino)pyridine-4-carboxamide), Pelitinib (EKB-569, (E)-N-[4-(3-chloro-4-fluoroanilino)-3-cyano-7-ethoxyquinolin-6-yl]-4-(dim) but-2-enamide), BIX 02188 (3-[N-[3-[(dimethylamino)methyl]phenyl]-C-phenylcarbonimidoyl]-2-hydroxy-1H-indole-6-c arboxamide), TAK-73 3(3-[(2R)-2,3-dihydroxypropyl]-6-fluoro-5-(2-fluoro-4-iodoanilino)-8-methylpyrido[2,3-d]pyrimidine-4,7-dione), AZD8330(2 -(2-fluoro-4-iodoanilino)-N-(2-hydroxyethoxy)-1,5-dimethyl-6-oxopyridine-3-carboxamide), Binimetinib (MEK162,6-(4-bromo-2-f luoroanilino)-7-f luoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide), SL-327((Z)-3-amino-3-(4-aminophenyl)sulfanyl-2-[2-(t rifluoromethyl) phenyl]prop-2-enenitrile), Refametinib (RDEA119,N-[3,4-difluoro-2-(2-fluoro-4-iodoanilino)-6-methoxyphenyl]- 1-[(2S)-2,3-dihydroxyp [ropyl]cyclopropane-1-sulfonamide), GDC-0623 (5-(2-fluoro-4-iodoanilino)-N-(2-hydroxyethoxy)imidazo[1,5-a]pyridine-6-carbo xamide), BI-847325 (3-[3-[N-[4-[(dimethylamino)methyl]phenyl]-C-phenylcarbonimidoyl]-2-hydroxy-1H-indol-6-yl]-N-ethylprop-2 -ynamide), RO512676 6 (CH5126766, 3-[[3-fluoro-2-(methylsulfamoylamino)pyridin-4-yl]methyl]-4-methyl-7-pyrimidin-2-yloxychromen-2-one), cobimetinib (GDC-0973, [3,4-difluoro-2-(2-fluoro-4-iodoanilino)phenyl]-[3-hydroxy-3-[(2S)-piperidin-2-yl]azetidin-1-yl]methaneone) and their derivatives.

Examples of p38 inhibitors include SB203580 (4-[4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-1H-imidazol-5-yl]pyridine), Doramapimod (BIRB 796, 1-[5-tert-butyl-2-(4-methylphenyl)pyrazo l-3-yl]-3-[4-(2-morpholin-4-ylethoxy)naphthalen-1-yl]urea), SB202190 (FHPI,4-[4-(4-fluorophenyl)-5-pyridin-4-yl-1H-imidaz ol-2-yl]phenol), Ralimetinib dimesylate (5-[2-t ert-butyl-4-(4-fluorophenyl)-1H-imidazol-5-yl]-3-(2,2-dimethylpropyl)imidazo[4,5-b]pyridin-2-amine; methanesulfonic ac id), VX-702 (6-(N-carbamoyl-2,6-difluoroanilino) -2-(2,4-difluorophenyl)pyridine-3-carboxamide), PH-797804 (3-[3-bromo-4-[(2,4-difluorophenyl)methoxy]-6-methyl-2-oxopyridin -1-yl]-N,4-dimethylbenzamide), Neflamapimod ( VX-745,5-(2,6-dichlorophenyl)-2-(2,4-difluorophenyl)sulfanylpyrimido[1,6-b]pyridazin-6-one), TAK-715(N-[4-[2-ethyl-4-(3- methylphenyl)-1,3-thiazol-5-l]pyridin-2-yl]b TA-02 (4-[2-(2-fluorophenyl)-4- (4-fluorophenyl)-1H-imidazol-5-yl]pyridine), SD 0006 (1-[4-[3-(4-chlorophenyl)-4-pyrimidin-4-yl-1H-pyrazol-5-yl]piperidin-1-yl]-2-hydroxyethanone), Pamapimod (6-(2,4-difl) urophenoxy)-2-(1,5-dihydroxypentan-3-ylamino )-8-methylpyrido[2,3-d]pyrimidin-7-one), BMS-582949 (4-[5-(cyclopropylcarbamoyl)-2-methylanilino]-5-methyl-N-propylpyrrolo[ 2,1-f][1,2,4]triazine-6-carboxamide), SB2390 63 (4-[4-(4-fluorophenyl)-5-(2-methoxypyrimidin-4-yl)imidazol-1-yl]cyclohexan-1-ol), Skepinone-L (13-(2,4-difluoroanilino) -5-[(2R)-2,3-dihydroxypropoxy]tricyclo[9.4.0 ．． 03,8] pentadeca-1(11),3(8),4,6,12,14-hexaen-2-one), DBM 1285(N-cyclopropyl-4-[4-(4-fluorophenyl)-2-piperidin-4-yl-1,3-thia zol-5-yl]pyrimidin-2-amine; dihydrochloride) , SB 706504 (1-cyano-2-[2-[[8-(2,6-difluorophenyl)-4-(4-fluoro-2-methylphenyl)-7-oxopyrido[2,3-d]pyrimidin-2-yl]amino]ethyl ]guanidine), SCIO 469 (2-[6-chloro-5-[(2R,5S )-4-[(4-fluorophenyl)methyl]-2,5-dimethylpiperazine-1-carbonyl]-1-methylindol-3-yl]-N,N-dimethyl-2-oxoacetamide), Pexmet inib(1-[5-tert-butyl-2-(4-methylphenyl)pyrazo l-3-yl]-3-[[5-fluoro-2-[1-(2-hydroxyethyl)indazol-5-yl]oxyphenyl]methyl]urea), UM-164(2-[[6-[4-(2-hydroxyethyl)piperaz in-1-yl]-2-methylpyrimidin-4-yl]amino]-N-[2-me thyl-5-[[3-(trifluoromethyl)benzoyl]amino]phenyl]-1,3-thiazole-5-carboxamide), p38 MAPK Inhibitor (4-(2,4-difluorophenyl) l)-8-(2-methylphenyl)-7-oxido-1,7-naphthyridin- 7-ium), p38 MAP Kinase Inhibitor III (4-[5-(4-fluorophenyl)-2-methylsulfanyl-1H-imidazol-4-yl]-N-(1-phenylethyl)pyridin-2 -amine), p38 MAP Kinase Inhibitor IV (3,4,6-tri Examples include chloro-2-(2,3,5-trichloro-6-hydroxyphenyl)sulfonylphenol, CAY105571 (4-[5-(4-fluorophenyl)-2-[4-(methylsulfonyl)phenyl]-1H-imidazol-4-yl]-pyridine) and their derivatives.

Examples of JNK inhibitors include those described in the first step. The substance used in the present invention that inhibits the intracellular signal transduction mechanism in response to stress is preferably one or more selected from the group consisting of MEK inhibitors, p38 inhibitors, and JNK inhibitors. When SB203580 is used as the p38 inhibitor, it can be added to the medium at a concentration of usually about 1 nM to about 1 mM, preferably about 10 nM to about 100 μM, more preferably about 100 nM to about 10 μM, and even more preferably about 500 nM to about 5 μM. When PD0325901 is used as the MEK inhibitor, it can be added to the medium at a concentration of usually about 1 nM to about 1 mM, preferably about 10 nM to about 100 μM, more preferably about 100 nM to about 10 μM, and even more preferably about 500 nM to about 5 μM. When JNK-IN-8 is used as the JNK inhibitor, it can be added to the medium at a concentration similar to that described in the first step. When using other MEK inhibitors, p38 inhibitors, or JNK inhibitors, it is preferable to add them at a concentration that has the same inhibitory activity as the above inhibitors.

5. Cell Populations Comprising Pituitary Tissue The cell populations comprising pituitary hormone-producing cells of the present invention may be cell aggregates comprising pituitary hormone-producing cells.
The cell aggregate may be a cell aggregate containing pituitary hormone-producing cells throughout, and in one embodiment, containing them evenly.The cell aggregate may be a cell aggregate containing both a partial structure containing many pituitary hormone-producing cells (also referred to as pituitary tissue in this specification) and a partial structure containing a small amount of pituitary hormone-producing cells or no pituitary hormone-producing cells (also referred to as a cell population containing pituitary tissue and tissue other than the pituitary gland).
The therapeutic agent of the present invention may contain any of the cell aggregates described above.
In one embodiment, the cell population containing pituitary tissue contained in the therapeutic agent of the present invention may be a cell aggregate containing pituitary tissue and not containing any tissue other than the pituitary gland. The cells contained in the cell aggregate are as described in 1 above.
In one embodiment, the cell population comprising pituitary tissue contained in the therapeutic agent of the present invention may be a cell population comprising both pituitary tissue and tissue other than the pituitary gland, and the cell population may be a cell population comprising 1) nervous cells or nervous tissue and 2) pituitary tissue.
The cell population can be obtained by the production method 1 described above.

In one embodiment, the 1) nervous system cells or nervous tissue in the cell population of the present invention obtained by the above-mentioned production method 1 are cells or tissues of the central nervous system, or precursor tissues thereof. Examples of the cells or tissues of the central nervous system include the diencephalon (specifically the hypothalamus) and cells (including precursor cells) contained in these tissues, more preferably the hypothalamus or its precursor tissues, or cells contained therein, and even more preferably the hypothalamus having a ventricle-like structure in the tissue, or its precursor tissues, or cells contained therein. 1) The nervous system cells or nervous tissue is, for example, N-Cadherin-positive neuroepithelial tissue.

In one embodiment, 2) pituitary tissue in the cell population of the present invention obtained by the above-mentioned Production Method 1 is formed continuously with non-neuroepithelial tissue, and it is further preferred that the non-neuroepithelial tissue and pituitary tissue cover 1) nervous cells or nervous tissue.
In one embodiment, the non-neural epithelial tissue obtained by the above-mentioned production method 1 is oral epithelium or a precursor tissue thereof. The pituitary tissue preferably contains pituitary hormone-producing cells or their precursor cells, that is, pituitary precursor cells, and may contain pituitary stem cells or folliculostellate cells, or may contain all of pituitary hormone-producing cells, pituitary precursor cells, pituitary stem cells, and folliculostellate cells.
It is also preferable that a pituitary niche is formed in the pituitary tissue, and it is also preferable that the pituitary niche is an MCL niche-like structure around the residual cavity remaining between the anterior and intermediate lobes of the pituitary gland, and it is also preferable that the pituitary niche is a parenchymal layer niche-like structure, and it is even more preferable that it contains both an MCL niche-like structure and a parenchymal layer niche-like structure.

3) Mesenchymal cells contained in the cell population of the present invention obtained by the above-mentioned Production Method 1 express at least one mesenchymal cell marker selected from the group consisting of, for example, nestin, vimentin, cadherin-11, laminin, CD44, CD90, and CD105.
Non-neuroepithelial tissues that can be included in the present invention express at least one non-neuroepithelial tissue marker selected from the group consisting of, for example, cytokeratin, E-Cadherin, and EpCAM.
Pituitary stem cells that may be contained in the cell population contained in the therapeutic agent 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 common receptor (CXADR), PRRX1/2, Ephrin-B2, and ACE. A preferred embodiment of the cell population of the present invention contains pituitary stem cells that are positive for a pituitary stem cell marker (e.g., CXADR). The proportion of pituitary stem cells in the cell population (proportion of pituitary stem cells present) may be 1% or more, preferably 3% or more or 5% or more.

6. Method for Producing Pituitary Tissue In the method for producing the pituitary tissue contained in the therapeutic agent of the present invention, the pituitary tissue may be collected from the cell population containing pituitary tissue obtained by the above-mentioned "2. Method for Producing a Cell Population Containing Pituitary Tissue". One embodiment includes the following steps (1), (2) and (4).
(1) a first step of culturing pluripotent stem cells in the presence of a JNK signaling pathway inhibitor and a Wnt signaling pathway inhibitor to obtain a cell population;
(2) a second step of culturing (preferably in suspension) the cell population obtained in the first step in the presence of a substance acting on the BMP signaling pathway and a substance acting on the Sonic Hedgehog signaling pathway to obtain a cell population containing pituitary tissue;
(4) A fourth step of recovering pituitary tissue from the cell population obtained in the second step.
The first and second steps can be carried out in the same manner as the first and second steps in "2. Method for producing a cell population containing pituitary tissue" above. If desired, step a may be carried out before step 1. If desired, step 3 may be carried out between steps 2 and 4.
In the fourth step of recovering pituitary tissue from a cell population containing pituitary tissue, when the formed cell population is a planar tissue obtained by adhesion culture or the like, the pituitary tissue can be recovered by a method such as physically peeling off the pituitary tissue using a needle under a microscope. When the formed cell population is a three-dimensional tissue such as a cell mass, the pituitary tissue formed on the outside of the cell mass (Rathke's pouch portion) is peeled off and recovered using tweezers under a microscope. The pituitary tissue can be identified as a semi-transparent thin epithelium on the surface of the obtained cell mass, as described in Nature communications, 2016, 7. In the fourth step, freezing and thawing, preferably slow freezing, can also be used as a method for recovering pituitary tissue from a cell population (cell mass). In this method, the outer pituitary tissue is peeled off from the cell mass without physical treatment by freezing and thawing a cell mass having pituitary tissue on the outside and mesenchymal nerve or neuroepithelial tissue on the inside.

In one embodiment, the pituitary hormone-producing cells, cell populations containing pituitary hormone-producing cells, or cell aggregates containing pituitary hormone-producing cells, cell populations containing pituitary tissue, or pituitary tissue recovered from cell populations containing pituitary tissue, contained in the therapeutic agent of the present invention, can be produced by the production method described above.

7. Method for producing purified product In order to obtain a cell population (purified product) that does not contain unintended cells (cells other than pituitary hormone-producing cells) or has a reduced proportion of unintended cells, as described in the above "1. Therapeutic drug and cell transplantation of the present invention", the following steps may be performed. Specifically, after the second step (step (2)) described in the above "4. Method for producing a cell population containing pituitary tissue 1", the following step may be performed: selecting and recovering pituitary hormone-producing cells from the cell population containing pituitary hormone-producing cells obtained in the second step. This step may be performed with reference to a method known per se (e.g., International Publication No. WO 2023/054395).

In this step, cells expressing EpCAM (EpCAM-positive cell population) are separated from the cell population containing pituitary tissue obtained in the second step. Prior to carrying out this separation step, a step of dispersing the cell population containing pituitary tissue obtained in the second step may be carried out.

In the dispersion step, the obtained cell population containing pituitary tissue may first be pretreated with a ROCK inhibitor. In order to suppress cell death induced by the subsequent dispersion of the cell population containing pituitary tissue, it is preferable to add the ROCK inhibitor before the start of the pretreatment. The ROCK inhibitor is added, for example, at least 24 hours, at least 12 hours, at least 6 hours, at least 3 hours, at least 2 hours, or at least 1 hour before the start of the treatment. Examples of ROCK inhibitors include Y-27632 ((R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide, dihydrochloride). The concentration of the ROCK inhibitor used in this treatment is a concentration that can suppress cell death induced by the subsequent dispersion of the cell population containing the pituitary tissue. For example, for Y-27632, such a concentration is usually about 0.1 to 200 μM, preferably about 2 to 50 μM. The concentration of the ROCK inhibitor may be varied during the period of addition, for example, the concentration may be reduced by half in the latter half of the period. The medium used in step (2) can be used as the medium for carrying out this pretreatment. Note that if the ROCK inhibitor has already been added to the medium at the desired concentration, there is no need to carry out this pretreatment step.

Next, the cell population containing the pituitary tissue that has been pretreated as described above is dispersed by enzyme treatment. Specifically, the cell population that has been pretreated as described above is first transferred to an incubator containing PBS and washed with the same medium. The enzyme for dispersion is not particularly limited as long as it can disperse the cells, but examples include enzymes such as papain, EDTA, trypsin, collagenase (collagenase types I to VII), metalloprotease, hyaluronidase, elastase, dispase, deoxyribonuclease, and mixtures thereof. A preferred enzyme is papain. The conditions for the enzyme treatment (temperature, time, etc.) can be appropriately set depending on the enzyme used, etc. In addition, in order to promote the enzyme treatment, a step of physically cutting the cell population containing the pituitary tissue (e.g., scalpel, scissors, etc.) into small pieces or physically cutting the cell population (e.g., scalpel, scissors, etc.) may be performed before the treatment.

After the enzyme treatment, the floating cells may be collected and the enzyme treatment may be carried out again. This may be repeated multiple times. The enzyme treatment may be carried out using the enzymes described above, and preferred enzymes include EDTA; trypsin, collagenase (collagenase types I to VIII), metalloprotease, hyaluronidase, elastase, dispase, deoxyribonuclease, and mixtures thereof. Preferred enzymes include collagenase, more preferably collagenase type I. The conditions for the enzyme treatment (temperature, time, etc.) may be set appropriately depending on the enzyme used.

After the enzyme treatment, the floating cells are collected and the enzyme treatment is carried out again. The enzyme treatment can be carried out with the enzymes mentioned above, and preferred enzymes include EDTA; trypsin, and more preferably EDTA; trypsin and deoxyribonuclease. Commercially available products such as TrypLE (Invitrogen) may be used in place of EDTA; trypsin. The enzyme treatment conditions (temperature, time, etc.) can be appropriately set depending on the enzymes used. A single-cell suspension can be prepared by the above series of enzyme treatments. When preparing a single-cell suspension, dead cells may be removed by a method known per se.

In this step, cells expressing EpCAM are separated from the dispersed cell population obtained as described above. Methods for separating desired cells expressing EpCAM from dispersed cell populations include methods using flow cytometry or mass cytometry, magnetic cell separation methods, etc., and these methods can be performed using methods known per se. For example, cells expressing EpCAM can be separated by a method including a step of contacting the cells with a substance (e.g., antibody, etc.) that specifically binds to EpCAM molecules. The above-mentioned substances include those that have a detectable label (e.g., GFP, PE) attached thereto, and those that have no label attached thereto. When the above-mentioned substances have no label attached thereto, the separation can be achieved by further using a substance that has a detectable label attached thereto that directly or indirectly recognizes the substance. For example, when the substance is an antibody, a fluorescent dye, a metal isotope, or beads (e.g., magnetic beads) can be directly or indirectly supported on the antibody, thereby labeling a marker on the cell surface, and the cells can be separated based on the label. In this case, only one type of antibody may be used, or two or more types of antibodies may be used. After carrying out this step, the above-mentioned step (3) may be carried out.
The timing of carrying out this step is not limited to after the second step (step (2)). In one embodiment, step (b) may be carried out after step (2) and then this step may be carried out. In another embodiment, step (b) may be carried out after step (2), and then step (3) may be carried out, and then this step may be carried out. In these embodiments, the above-mentioned step (3) may be carried out after carrying out this step.

8. Compositions Comprising Pituitary Hormone-Producing Cells and Treatment Methods The pituitary hormone-producing cells contained in the therapeutic agent of the present invention may be administered in a therapeutically effective amount for a disease based on a pituitary disorder for which cell transplantation therapy is desired, and may vary depending on factors such as the age, weight, and severity of the disease of the transplantation subject, and are not particularly limited, and may be, for example, 10 x ¹⁰ cells or more, or about 10 x ¹⁰ cells to 10 x ¹⁰ cells, preferably 10 x ¹⁰ cells or more, or about 10 x ¹⁰ cells to 10 x ¹⁰ cells, and more preferably ¹⁰ x ¹⁰ cells or more, or about 10 x 10 cells to 10 x ¹⁰ cells. Furthermore, when the therapeutic agent of the present invention contains pituitary hormone-producing cells as a cell population containing pituitary hormone-producing cells, a cell aggregate containing pituitary tissue, or pituitary tissue recovered from a cell population containing pituitary tissue, it is sufficient to administer a therapeutically effective amount for a disease due to a pituitary disorder for which cell transplantation therapy is desired, and this can be varied appropriately depending on factors such as the age, weight, and severity of the disease of the transplantation subject.

When the pituitary hormone-producing cells or cell aggregates containing pituitary hormone-producing cells contained in the therapeutic agent of the present invention are used in cell transplantation therapy, it is desirable to use cells derived from iPS cells established from somatic cells with the same or substantially the same HLA genotype of the recipient individual, from the viewpoint of preventing rejection reactions. Here, "substantially the same" means that the HLA genotype matches the transplanted cells to such an extent that the immune response can be suppressed by an immunosuppressant, for example, somatic cells with HLA types matching the three loci of HLA-A, HLA-B, and HLA-DR, or four loci including HLA-C, or six loci including HLA-DP and HLA-DQ. If sufficient cells cannot be obtained due to age, constitution, etc., they can also be transplanted in a state where they are embedded in a capsule or porous container made of polyethylene glycol or silicone to avoid rejection reactions.

The therapeutic agent of the present invention can be prepared by mixing with a medicamentously acceptable aqueous liquid, for example. Thus, in one embodiment, there is also provided a method for producing a therapeutic agent for a disease due to a disorder of the pituitary gland, which contains pituitary hormone-producing cells or cell aggregates containing pituitary hormone-producing cells, and includes a step of formulating the pituitary hormone-producing cells or cell aggregates containing pituitary hormone-producing cells, of the present invention. Such a production method may include a step of preparing the pituitary hormone-producing cells or cell aggregates containing pituitary hormone-producing cells, of the present invention. It may also include a step of preserving the pituitary hormone-producing cells or cell aggregates containing pituitary hormone-producing cells, of the present invention.

The pharma- ceutically acceptable aqueous liquid that may be contained in the therapeutic agent of the present invention may contain, for example, an appropriate selection of a buffer, an isotonicity agent, a pH adjuster, an antioxidant, a chelating agent, etc., within a range that does not affect the viability and physiological activity of the transplanted pituitary hormone-producing cells.
Examples of the buffer include phosphate buffer, borate buffer, citrate buffer, tartrate buffer, acetate buffer, amino acid, epsilon-aminocaproic acid, etc. Examples of the isotonicity agent include sugars such as D-sorbitol, D-glucose, D-mannitol, etc., polyhydric alcohols such as glycerin, propylene glycol, etc., salts such as sodium chloride, boric acid, etc.
Chelating agents include sodium edetate and citric acid.
Examples of pH adjusters include sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, boric acid or a salt thereof (borax), hydrochloric acid, citric acid or a salt thereof (sodium citrate, sodium dihydrogen citrate, etc.), phosphoric acid or a salt thereof (disodium hydrogen phosphate, potassium dihydrogen phosphate, etc.), acetic acid or a salt thereof (sodium acetate, ammonium acetate, etc.), tartaric acid or a salt thereof (sodium tartrate, etc.), and the like.
Examples of antioxidants include ascorbic acid, glutathione, sodium hydrogen sulfite, dry sodium sulfite, sodium pyrosulfite, and tocopherol.
Specific examples of the "pharmaceutical acceptable aqueous liquid" include aqueous liquids such as physiological saline, isotonic solutions containing glucose and other auxiliary drugs (eg, D-sorbitol, D-mannitol, sodium chloride, etc.).
The therapeutic agent of the present invention may be compounded with, for example, a soothing agent (eg, benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (eg, human serum albumin, polyethylene glycol, etc.), a preservative, an antioxidant, and the like.

The therapeutic agent of the present invention may be combined with an angiogenesis promoter to form a combination drug (combination drug). The angiogenesis promoter is not particularly limited as long as it is medicamentically acceptable, and examples thereof include vascular endothelial growth factor (VEGF), fibroblast growth factor: acidic (aFGF) and basic (bFGF), epidermal growth factor (EGF), granulocyte macrophage-colony stimulating factor (GM-CSF), hepatocyte growth factor (HGF), and sphingosine-1-phosphate (S1P). When used as a combination drug, the active ingredients may be formulated separately, or each active ingredient may be formulated as a single agent. When formulated as a single agent, the combination drug of the present invention may contain one or more of each formulation.
When the agent is formulated separately, there is no particular limitation, but the agent may be contained in a biocompatible gel (particularly, a hydrogel, etc.), film, etc., with the intention of sustained release of the angiogenesis promoter, etc. The biocompatible gel, film, etc. may be bioabsorbable. The above-mentioned gel, film, etc., are not particularly limited, but examples thereof include amniotic membrane, polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE), polyurethane, polypropylene, polyester, vinyl chloride, polycarbonate, acrylic, silicone, MPC (2-methacryloyloxyethyl phosphorylcholine), polylactic acid, polyglycolic acid, lactic acid-glycolic acid copolymer (PLGA), fibrin, gelatin, collagen, collagen sodium alginate, poly(N-isopropyl acrylate), ... The gel may be a temperature-responsive gel in which polyacrylamide (PIPAAm) is crosslinked with polyethylene glycol (PEG), hyaluronic acid, glycosaminoglycan, proteoglycan, chondroitin, cellulose, agarose, carboxymethylcellulose, chitin, chitosan, gelatin, atelocollagen, elastin, fibronectin, pronectin, laminin, tenascin, fibroin, entactin, thrombospondin, retronectin, dextrin, trehalose, etc. Examples of commercially available biocompatible gels and films include Duragen (Integra Japan), Surgicel (Johnson & Johnson), and Spongel (LTL Pharma).

When each active ingredient in the combination drug of the present invention is formulated separately, each formulation can be administered (transplanted) to a subject simultaneously or at different times, via the same route or via different routes. The administration (transplantation) amount and number of administrations (transplants) of each formulation can be appropriately determined depending on various conditions such as the type of angiogenesis promoter, the symptoms, age, weight, and drug tolerance of the subject to be administered.

One example is a method in which a gelatin hydrogel sustained release device containing basic fibroblast growth factor (FGF) is applied to the transplant site in advance, and the therapeutic agent of the present invention is transplanted after a certain period of time has passed.

The therapeutic agent of the present invention is provided in a frozen state under conditions normally used for cryopreservation of cells, and can be thawed when needed. In this case, it may further contain serum or a substitute thereof, an organic solvent (e.g., DMSO), etc. In this case, the concentration of serum or a substitute thereof is not particularly limited, but may be about 1 to about 30% (v/v), preferably about 5 to about 20% (v/v). The concentration of the organic solvent is not particularly limited, but may be 0 to about 50% (v/v), preferably about 5 to about 20% (v/v).

Furthermore, as described above, cell populations such as pituitary hormone-producing cells or cell aggregates containing pituitary hormone-producing cells contained in the therapeutic agent of the present invention can be transplanted into the subcutaneous tissue and/or muscle tissue of a subject to treat diseases caused by disorders of the pituitary gland that contain pituitary hormone-producing cells. Therefore, the present invention also provides a treatment method characterized by treating diseases caused by disorders of the pituitary gland by administering a therapeutically effective amount of pituitary hormone-producing cells or cell aggregates containing pituitary hormone-producing cells to the subcutaneous tissue and/or muscle tissue.

The pituitary hormone-producing cells, cell populations containing pituitary hormone-producing cells, cell aggregates containing pituitary hormone-producing cells, cell populations containing pituitary tissue, or pituitary tissue recovered from cell populations containing pituitary tissue contained in the therapeutic agent of the present invention may be produced using pluripotent stem cells whose genes have been manipulated by genome editing as a raw material. Genes that are the subject of genome editing include, but are not limited to, genes involved in the differentiation of pituitary tissue by the above-mentioned method for producing pituitary hormone-producing cells, etc. of the present invention, genes involved in the differentiation into unintended cells other than the pituitary gland that are by-produced by the production method, hormone-related genes secreted from the pituitary gland, genes involved in the infection of diseases, etc.

9. Model Animals An embodiment of the present invention includes a method for producing a non-human animal model bearing human pituitary tissue, which comprises administering human pituitary hormone-producing cells into the subcutaneous tissue and/or muscle tissue of the non-human animal.
In one embodiment, the non-human animal is not particularly limited as long as it is a mammal other than a human, and specifically includes a mammal used for evaluating the efficacy or safety of a substance, such as a mouse, a rat, a hamster or a guinea pig, a dog, a rabbit, or a lemur, a loris, a tree shrew, or a monkey.
The human pituitary hormone-producing cells include a cell population containing any of the above-mentioned human pituitary hormone-producing cells. The subcutaneous tissue and muscle tissue into which the human pituitary hormone-producing cells are transplanted are as described above in 1. The human pituitary hormone-producing cells may be transplanted into a non-human animal in accordance with the Examples of the present specification.
In one embodiment, the model non-human animal of the present invention may be an animal in which part or all of the pituitary gland has been surgically removed.

The present invention includes a non-human animal model bearing human pituitary tissue obtained by the above-mentioned production method.
The present invention encompasses a method for evaluating the efficacy or safety of a test substance, which comprises administering the test substance to the non-human animal model bearing human pituitary tissue.
In one embodiment, the method of administering the test substance to the non-human animal is not particularly limited, and the substance can be administered orally or parenterally (including, but not limited to, intravenous administration, subcutaneous administration, intradermal administration, etc.).
In one aspect, the effect of the test substance on the secretion or in vivo action of a pituitary hormone can be examined by administering the test substance to the non-human animal and measuring the concentration of the pituitary hormone in the non-human animal (e.g., blood concentration).
That is, in one embodiment, the evaluation method of the present invention may comprise the following steps:
(i) administering a test substance to the non-human animal model bearing human pituitary gland tissue;
(ii) examining the amount of pituitary hormone or activity of pituitary hormone in a non-human animal administered the test substance; and (iii) evaluating the effect of the test substance on the secretion or activity of the pituitary hormone based on the results of (ii).

In one embodiment, the evaluation method of the present invention is a method for screening for a preventive or therapeutic agent for a disease caused by a pituitary gland disorder, and may comprise the following steps:
(i) administering a test substance to the non-human animal model bearing human pituitary gland tissue;
(ii) examining the amount or activity of the pituitary hormone in the non-human animal administered the test substance; and (iii) selecting the test substance in which the amount or activity of the pituitary hormone is increased/decreased as a candidate for a preventive or therapeutic drug for a disease based on a disorder of the pituitary gland.

The test substances used in the screening method of the present invention include, for example, cell extracts, cell culture supernatants, microbial fermentation products, extracts from marine organisms, plant extracts, purified or crude proteins, peptides, non-peptide compounds, synthetic small molecule compounds, and natural compounds. The test substances can also be obtained using any of the many approaches in combinatorial library methods known in the art, including (1) biological libraries, (2) synthetic library methods using deconvolution, (3) "one-bead one-compound" library methods, and (4) synthetic library methods using affinity chromatography selection. While the biological library method using affinity chromatography selection is limited to peptide libraries, the other four approaches can be applied to small molecule compound libraries of peptides, non-peptide oligomers, or compounds (Lam (1997) Anticancer Drug Des. 12:145-67). Examples of methods for the synthesis of molecular libraries can be found in the art (DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90:6909-13; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422-6; Zuckermann et al. (1994) J. Med. Chem. 37:2678-85; Cho et al. (1993) Science 261:1303-5; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; Gallop et al. al. (1994) J. Med. Chem. 37:1233-51). Chemical libraries have been developed in solution (see Houghten (1992) Bio/Techniques 13:412-21) or on beads (Lam (1991) Nature 354:82-4), on chips (Fodor (1993) Nature 364:555-6), on bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698, 5,403,484, and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Ac. 1999, 10:131-132), and on spores (U.S. Pat. Nos. 5,571,698, 5,403,484, and 5,223,409). ad. Sci. USA 89:1865-9) or as phages (Scott and Smith (1990) Science 249:386-90; Devlin (1990) Science 249:404-6; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87:6378-82; Felici (1991) J. Mol. Biol. 222:301-10; U.S. Patent Application No. 2002103360).

The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these in any way.

Example 1: Examination of transplantation sites of pituitary hormone-producing cells <Materials and methods>
(1) Maintenance and differentiation culture of human embryonic stem cells (hESC) (pituitary organoids)
hESCs (KhES-1) were obtained from the RIKEN BioResource Research Center and used in accordance with the Japanese government's hESC research guidelines. All experimental protocols and procedures were approved by the Ethics Committee of the Nagoya University Graduate School of Medicine (approval ES-001). hESC maintenance and differentiation cultures were cultured under feeder-free conditions according to the method described in Scientific Reports, 4, 3594 (2014). StemFit medium (AK03, Ajinomoto Co., Inc.) was used as the feeder-free medium, and Laminin511-E8 (Nippi Co., Ltd.) was used as the feeder-free scaffold.
As a specific maintenance culture operation, first, subconfluent hESC (KhES-1 strain) was washed with PBS and dispersed into single cells using TrypLE Select (Life Technologies). Then, the human ES cells dispersed into the single cells were seeded on a plastic culture dish coated with Laminin511-E8 and cultured feeder-free in StemFit medium in the presence of Y27632 (ROCK inhibitor, 10 μM). When a 6-well plate (Iwaki, for cell culture, culture area 9.4 cm ² ) was used as the plastic culture dish, the seeded cell number of the human ES cells dispersed into the single cells was 2.4×10 ^4. One day after seeding, the entire medium was replaced with StemFit medium not containing Y27632. Thereafter, the entire medium was replaced with StemFit medium not containing Y27632 once every 1 to 2 days, and then the cells were cultured until they became subconfluent (about 60% of the culture area was covered with cells) 7 days after seeding.

As a differentiation induction operation, human ES cells (KhES-1 strain) were cultured for 24 hours using StemFit medium, 6 days after seeding, with the addition of SB431542 (5 μM) and SAG (300 nM) at the same time as changing the medium to StemFit medium. The prepared subconfluent human ES cells were treated with cell dispersion liquid using TrypLE Select (Life Technologies), and further dispersed into single cells by pipetting. Thereafter, the human ES cells dispersed into the single cells were suspended in 100 μL of serum-free medium so that the number of cells was 1.0 × 10 ⁴ per well of a non-cell-adhesive 96-well culture plate (PrimeSurface 96V bottom plate, MS-9096V, Sumitomo Bakelite Co., Ltd.), and the suspension culture was performed under the conditions of 37 ° C. and 5% CO ₂ . The serum-free medium (gfCDM+KSR) used was a 1:1 mixture of F-12 medium and IMDM medium, to which 5% KSR, 450 μM 1-monothioglycerol, and 1x chemically defined lipid concentrate had been added. At the start of suspension culture (day 0 after the start of differentiation induction), Y27632 (final concentration 20 μM), IWP-2 (0.5 μM), SB431542 (1 μM), JNK-IN-8 (1 μM), and SAG (100 nM) were added to the serum-free medium.

On the second day after the start of differentiation induction, 100 μL of serum-free medium containing IWP-2 (0.5 μM), SB431542 (1 μM), JNK-IN-8 (1 μM), BMP4 (final concentration 0.5 nM), and SAG (final concentration 700 nM) was added per well. Thereafter, on the 6th, 9th, 12th, 15th, 19th, 22nd, and 26th days after the start of differentiation induction, half of the medium was replaced using serum-free medium containing IWP-2 (0.5 μM), SB431542 (1 μM), JNK-IN-8 (1 μM), and SAG, without Y27632 and BMP4. From the 19th day onwards, the oxygen partial pressure during culture was set to 40%.
On the 29th day after the start of differentiation induction, the cell aggregates were transferred to 10 cm suspension culture dishes at 60 cell aggregates per dish, and suspension culture was continued using serum-free medium under conditions of 40% O2 and 5% CO2. From the 29th to 50th day after the start of differentiation induction, serum-free medium supplemented with 10% KSR was used for culture, and from the 50th day after the start of differentiation induction, serum-free medium supplemented with 20% KSR was used for culture.

The cell aggregates on the 100th day after the start of differentiation induction were fixed with 4% paraformaldehyde and frozen sections were prepared. These frozen sections were immunostained using adrenocorticotropic hormone (ACTH) (anti-ACTH antibody, Fitzgerald, mouse), which is a pituitary hormone-producing cell marker, E-cadherin (anti-E-cadherin antibody, TAKARA, rat), which is an epithelial cell marker, and Lhx3 (anti-Lhx3 antibody, homemade (Non-Patent Documents 1 and 2), rabbit), which is a pituitary precursor marker. The cell nuclei were stained with DAPI. These stained sections were observed using a confocal laser scanning microscope (Olympus), and immunostained images were obtained. As a result, it was confirmed that ACTH-positive pituitary hormone-producing cells were present in a part of the E-cadherin-positive epithelial tissue in the cell aggregates on the 100th day after the start of differentiation induction induced by the above-mentioned differentiation induction method (Figure 1). The scale bar at the bottom right indicates 100 μm. Pituitary organoids (PO) harvested 100 to 200 days after differentiation were used for transplantation into mice.

(2) Mouse hypophysectomy All animal experiments were approved by the Animal Care and Use Committee of the Nagoya University Graduate School of Medicine and were performed in accordance with the institutional guidelines for the care and use of animals. Male severe combined immunodeficiency (SCID) mice (CB-17/Icr-Hsd-Prkdc ^scid , Japan SLC Co., Ltd., Shizuoka, Japan) aged 8-9 weeks were subjected to transhypopituitary glandectomy. Mice were anesthetized with an intraperitoneal (i.p.) injection of a mixture of three drugs (medetomidine 0.75 mg/kg, midazolam 4 mg/kg, butorphanol 5 mg/kg) (Kawai S, et al. Effect of three types of mixed anesthetic agents alternate to ketamine in mice. Exp Anim. 2011; 60: 481-487), and pituitary tissue was aspirated from the sella turcica via the ear canal using a needle (KN-390, Natsume Seisakusho Co., Ltd., Tokyo, Japan) attached to a 1 ml syringe containing 0.2 ml saline. After the procedure, mice were injected with the medetomidine antagonist atipamezole (0.75 mg/kg, i.p.).

(3) Blood sampling and ACTH measurement ACTH levels were evaluated as a biomarker of pituitary function. Blood samples were collected by tail snip and sampled before and 1 h after administration of human CRH (2 μg/kg, i.p., Tanabe, Nipro ES Pharma Co., Ltd., Osaka, Japan). Plasma was separated from the blood samples by centrifugation at 1000×g for 15 min at 4°C. Plasma ACTH assay was performed using an ACTH ELISA kit (MD bioproducts, Oakdale, MN), and absorbance (ACTH concentration) was read using a Cytation 5 (Biotek, Winooski, VT). CRH loading tests were performed 1 week after hypophysectomy and 1/4 weeks after PO transplantation treatment, including sham-operated mice, up to 6 months later (Figure 2A). Mice with plasma ACTH levels below 10 pg/ml after CRH stimulation were classified as hypopituitaric and used as subjects.

(4) Measurement of spontaneous ACTH secretion in PO in vitro Five POs were cultured in 2.5 ml of cell culture medium (Iscove's modified Dulbecco's medium (Sigma-Aldrich, St. Louis, MO); Ham's F12 (Thermo Fisher Scientific, Waltham, MA) (1:1); 1% GlutaMAX (Thermo Fisher Scientific); 1% Chemically Defined Lipid Concentrate (Thermo Fisher Scientific); 450 μM 1-thioglycerol (Sigma-Aldrich); and 20% The cells were incubated in KnockOut Serum Replacement (Thermo Fisher Scientific) at 37°C for 72 hours. The culture supernatant was collected. The ACTH concentration in the supernatant was determined using an electrochemiluminescence immunoassay (ECLIA) kit (SRC Co., Ltd., Tokyo, Japan) that is used clinically in Japan.

(5) Transplantation Method Mice were anesthetized with isoflurane and placed in a supine position. For subcutaneous white adipose tissue (ISWAT) transplantation in the groin, the left groin was shaved. A 4 mm vertical skin incision was made, a pocket was created in the subcutaneous adipose tissue, and five POs harvested from cell culture were placed in the pocket using a wide-bore tip under a microscope. Skin closure was performed following nylon suture closure on the PO (Fig. 2B-G). In the sham operation group, a vertical skin incision was made in the left groin, a small pocket was created in the subcutaneous fat, and surgical wound closure was performed without PO transplantation. In the avascular area (AR) transplantation group, a vertical skin incision was made in the left dorsal skin, and five POs were placed. All hypopituitarized mice were subjected to an intramuscular injection of dexamethasone at 0.2 mg/0.61 ml/mouse to prevent adrenal crisis.

(6) Evaluation of PO-implanted mice Body weight was monitored and the rate of weight loss was evaluated. Running wheel activity test was performed using a running wheel device (ENV-044; Med Associates, Georgia, VT).

(7) Histological Evaluation: Cell aggregates, skin, and fat from SCID mice were fixed in 10% formalin, dehydrated for paraffin infiltration, and sectioned on a slide microtome. Five-μm sections were stained with hematoxylin and eosin (H&E) or subjected to immunofluorescence microscopy for various antigens with nuclear 4',6-diamidino-2-phenylindole counterstaining. Antigen targets include ACTH (mouse, 1:200, 10C-CR1096M1; Fitzgerald), LHX3 (LIM homeobox protein 3, rabbit, 1:3000, in-house (Non-Patent Documents 1 and 2)), human nucleus (mouse, 1:1000, MAB4383; Millipore), E-cadherin (rat, 1:50, M108; Takara Bio Inc.), and SMA (smooth muscle actin, mouse, 1:200, M0851; DAKO).

(8) RNA extraction and cDNA synthesis from PO and undifferentiated hESC RNA was extracted from PO and undifferentiated hESC using RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. RNA quality was assessed using TapeStation 4150 (Agilent Technologies, Santa Clara, CA). cDNA was synthesized using ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan).

(9) Quantitative PCR
Quantitative PCR (qPCR) was performed on five POs and five transplanted POs (separated from 10,000 ESCs/sample) using a LightCycler 480 system (Roche Diagnostics, Rotkreuz, Switzerland). Data were normalized to that of GAPDH as an endogenous control and relative quantification based on a standard curve was determined. The primers used are shown in Table 1 below.

(10) Statistical Analysis All data were analyzed using SPSS statistical software (version 28.0.0.0, IBM, Armonk, NY). Data are presented as mean ± standard error. Comparison between two groups was performed using Student's t-test. Comparison between multiple groups was performed by one-way analysis of variance (ANOVA) with post-hoc Tukey's test. P values of <0.05 ( ^* ), <0.01 ( ^** ), and <0.001 ( ^*** ) were considered significant.

<Results>
(1) Evaluation of subcutaneous implantation methods The following four methods:
(i) a gelatin hydrogel sustained release device containing basic fibroblast growth factor (FGF) with heparin (Uematsu SS, et al. The optimization of the prevascularization procedures for improving subcutaneous islet engraftment. Transplantation. 2018; 102: 387-395) (Fig. 3A,B: GEL) or (ii) a medically approved vascular access catheter (Pepper AR, et al. A prevascularized subcutaneous device-less site for islet and cellular transplantation. Nat Biotechnol. 2015; 33: 518-523) (Fig. 3C, device-less, DL);
(iii) graft placement in ISWAT (Yasunami Y, et al. A novel subcutaneous site of islet transplantation superior to the liver. Transplantation. 2018; 102: 945-952) (Fig. 3D, E); and (iv) graft placement in the relative AR just under the skin on the back (Fig. 3F, no treatment: NT). In subcutaneous transplantation, we hypothesized that blood supply determines the fate of the graft. Placement in the AR serves as a control for the other three options. Although adipose tissue is inherently highly vascularized, the GEL and DL pre-vascularization methods increase blood supply to the transplantation site. However, these methods require invasive manipulations 2 weeks or 1 month before transplantation, respectively, which may be stressful for hypopituitarized mice. The plasma ACTH level one month after transplantation of hESC-derived PO was the highest in the ISWAT cohort mice (ACTH level in PO culture medium: 30-100 pg/ml; all POs were transplanted from the same lot). Therefore, ISWAT transplantation was considered to be the best (Figures 3G, H). In other words, it was found that inguinal transplantation and/or subcutaneous white fat transplantation are good methods for administering the pituitary gland.

(2) Comparison between ISWAT, sham, and AR groups Plasma ACTH levels were followed up for 6 months in the ISWAT group (n=6), sham group (n=6), and AR group (n=5). The AR group served as a control for the study of the highly vascularized ISWAT, as described above. Pre-implant basal and CRH-stimulated plasma ACTH levels were not significantly different among the three groups (ISWAT vs. sham vs. AR, basal; 4.6±1.8pg/ml vs. 2.3±1.6pg/ml vs. 2.5±1.1pg/ml, p=0.27-0.92; stimulated; 1.7±0.8pg/ml vs. 1.7±2.3pg/ml vs. 7.2±2.1pg/ml, p=0.06-0.72).

After transplantation, basal plasma ACTH levels ("basal", Figure 4A) were consistently higher in the ISWAT group than in the sham group. At 2, 4, 8, and 17 weeks after transplantation, ACTH values were significantly different between groups (p<0.001-0.05, Figure 4A). CRH-stimulated plasma ACTH levels ("stimulated", Figure 4A) were also higher in the ISWAT group than in the sham group, with statistically significant differences between groups at 2, 4, 8, 21, and 26 weeks (p<0.001-0.005, Figure 4A).

In vitro pre-transplant ACTH secretion, assessed by ACTH levels in PO culture medium, was not significantly different between the ISWAT and AR groups (ISWAT vs. AR, 44,416 ± 8,435 pg/ml vs. 45,800 ± 17,291 pg/ml, p = 0.876). After transplantation, basal and CRH-stimulated plasma ACTH levels were higher in the AR group than in the sham-operated group and lower than in the ISWAT group, basal plasma ACTH levels in the ISWAT group were significantly higher than those in the AR group at 2 weeks (p = 0.009), and CRH-stimulated plasma ACTH levels in the ISWAT group were significantly higher than those in the AR group at 2, 8, and 21 weeks (p < 0.001-0.013) (Figure 4A). These results showed that when PO was implanted in a vascularized site such as adipose tissue, it released ACTH more efficiently in the subcutaneous tissue than in a site with poor blood flow.

In the running wheel activity test, the ISWAT group showed greater activity than the sham group (Figure 4B). The weight loss rate in the ISWAT group was smaller than that in the sham group (Figure 4C).

(3) Macroscopic and histological findings after subcutaneous transplantation Macroscopic examination 4 weeks after ISWAT transplantation revealed graft vascularization (Fig. 5A, transplanted cell aggregates: arrows in the figure, blood vessels associated with the graft: arrowheads in the figure). At 21 weeks after transplantation, no adipose tissue was observed macroscopically, but the grafts appeared intact (Fig. 5B, arrows in the figure). Microscopic examination of the skin and underlying grafts harvested en bloc at 21 weeks after transplantation revealed that the transplanted cell aggregates were located within the subcutaneous tissue (Fig. 5C, I, black boxes in the figure; Fig. 5D-N). Immunofluorescence microscopy revealed that the grafts expressed ACTH, E-cadherin (a marker for oral ectoderm), and LHX3 (a marker for pituitary progenitor cells) (Fig. 5D, E, F, J, K, L). Co-expression of human nuclei indicated that the cells were transplanted hESC-derived pituitary cells (Fig. 5F, G, L, M). Furthermore, the blood vessel wall marker SMA was expressed in clusters around and within the grafts (Fig. 5H,N), indicative of neovascularization. These observations indicate that hESC-derived POs can engraft and function in vivo. In contrast, immunostaining of the progeny of AR grafts showed engraftment, but with fewer viable areas and less SMA expression than ISWAT grafts (Fig. 6; Fig. 6A upper right box: Fig. 6B-F; Fig. 6A lower left box: Fig. 6G-K). Scale bars in Fig. 5 and Fig. 6 lower right indicate 100 μm.

(4) Promotion of angiogenesis of hESC-derived PO Having observed that hESC-derived PO functions after subcutaneous transplantation, the question arose as to how angiogenesis affects the engraftment of subcutaneously transplanted PO. Vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF2), and angiopoietin 2 (ANGPT2) are involved in the early stages of angiogenesis (Carmeliet P, et al. Molecular mechanisms and clinical applications of angiogenesis. Nature. 2011; 473: 298-307, Ortega S, et al. Neuronal defects and delayed wound healing in mice lacking fibroblast growth factor 2. Proc Natl Acad Sci U S A. 1998; 95: 5672-5677, and Nag S, et al. Angiopoietins are expressed in the normal rat pituitary gland. Endocr Pathol. 2005; 16: 67-73). The expression of these genes in PO was evaluated by qPCR. PO before transplantation, PO harvested 12 hours after transplantation, and undifferentiated hESCs as a control were analyzed. The expression levels of VEGFA, VEGFB, VEGFC, and ANGPT2 in PO were significantly higher than those in undifferentiated hESCs. In particular, the expression levels of VEGFC and ANGPT2 increased after transplantation (Figures 7A-E). These results suggested that PO itself may express angiogenic factors and promote engraftment into subcutaneous tissue.

Finally, to further evaluate the importance of angiogenesis in the survival of subcutaneous PO grafts, we tested PO function in vivo by administering the VEGF inhibitor bevacizumab (2 mg/kg, i.p., 2x/week for 2 weeks (Lin Y, et al. A strategy of vascular-targeted therapy for liver fibrosis. Hepatology. 2022; 76: 660-675); Selleck, Houston, TX). Hypopituitaric SCID mice after ISWAT transplantation were divided into bevacizumab-treated (n=9) and vehicle-treated (n=9) groups. Although ACTH levels in the culture medium of POs used for transplantation were not significantly different between groups (bevacizumab vs. vehicle, 19445±6774 pg/ml vs. 20295±6610 pg/ml, p=0.93), plasma ACTH levels after CRH stimulation were significantly lower in the bevacizumab group than in the vehicle group (4.6±3.0 pg/ml vs. 34.5±11.7 pg/ml, p=0.035). These results suggest that angiogenesis is important for ISWAT engraftment PO function (Figure 7F).

In this example, we demonstrate that hESC-derived POs can be transplanted into the subcutaneous tissue of hypopituitaric SCID mice and are functional. Transplanted mice responded to CRH with the release of ACTH into the circulation, indicating that the injected CRH stimulated the transplanted ACTH-producing cells. Comparison of ISWAT and AR transplants demonstrated that the vascularized subcutaneous adipose tissue was superior to the relatively vascularized space under the skin on the back, resulting in more sustained ACTH secretion. PSC-derived PO transplantation improves physical activity levels and body weight (Ozone C, et al. Functional anterior pituitary generated in self-organizing culture of human embryonic stem cells. Nat Commun. 2016; 7: 10351; and Suga H, et al. Self-formation of functional adenohypophysis in three-dimensional culture. Nature. 2011; 480: 57-62). This was confirmed in the present study. Furthermore, hESC-derived PO expressed angiogenic factors such as VEGF and ANGPT2, suggesting that PO autonomously promotes angiogenesis and engraftment. The levels of VEGFC and ANGPT2 expression were increased in PO after transplantation. This is likely due to the contribution of adiponectin, a cytokine released by adipocytes (Sakata N, et al. Mechanism of transplanted islet engraftment in visceral white adipose tissue. Transplantation. 2020; 104: 2516-2527). Finally, bevacizumab administration reduced ACTH secretion, indicating that angiogenesis is important, at least in subcutaneous transplantation. These results support that intraadipose tissue engraftment of hESC-derived POs may be achievable if the subcutaneous site is selected.

The demonstration of functionality in subcutaneously transplanted PSC-derived POs in this example is an important step in regenerative medicine technology. Subrenal capsule transplantation and subcutaneous transplantation are significantly different. PO recipient patients suffer from hypopituitarism and are therefore sensitive to stresses such as invasive procedures. Both subrenal capsule transplantation and subcutaneous transplantation require general anesthesia, but in humans, subcutaneous transplantation can be performed under local anesthesia, making it significantly less invasive than the alternative methods. Subrenal capsule transplantation can cause damage to normal kidneys. In contrast, the risk of collateral damage during subcutaneous transplantation is low. The surgery itself is simple and can be performed quickly in both mice and humans, likely allowing for outpatient work. A key aspect of transplantation of PSC-derived cells is the removal of the graft in the event of tumor development. Subcutaneous transplantation as employed in this invention allows for relatively simple and non-invasive removal. As demonstrated in this example, transplantation into adipose tissue is more effective than transplantation into an avascular site (AR), which is important for clinical application.

Example 2: Further investigation of transplantation sites of pituitary hormone-producing cells (inguinal subcutaneous, axillary subcutaneous, muscle)
Materials and Methods
hESC-derived PO was prepared using the same differentiation induction method as in Example 1. PO harvested 90-100 days after differentiation was used for transplantation into mice.

(1) Transplantation Method As in Example 1, mice were hypophysectomized, and blood samples were taken and ACTH was measured. As in Example 1, mice were subjected to inguinal ISWAT transplantation (Inguinal). For axillary subcutaneous white adipose tissue transplantation (Axilla), mice were placed in a supine position and the left axillary region was shaved under isoflurane inhalation anesthesia. A 4 mm vertical skin incision was made, a pocket was made in the subcutaneous adipose tissue, and five POs taken from cell culture medium were placed in the pocket using a wide-bore tip under a microscope. Nylon suture closure on the PO was followed by skin closure. For gluteal intramuscular transplantation (Muscle), mice were placed in a prone position and the left gluteal region was shaved under isoflurane inhalation anesthesia. A 4 mm vertical skin incision was made, a pocket was made in the gluteal muscle, and five POs taken from cell culture medium were placed in the pocket using a wide-bore tip under a microscope. Skin closure was performed followed by nylon suture closure over the PO. All hypopituitaric mice were subjected to an intramuscular injection of dexamethasone at 0.2 mg/0.61 ml/mouse to prevent adrenal crisis.

(2) Statistical Analysis All data were analyzed using R (Version 4.3.0). Data are presented as mean ± standard error. Comparisons between multiple groups were performed using the Criskall-Wallis test. A P value of <0.05 ( ^* ) was considered significant.

<Results>
To evaluate suitable transplantation sites in addition to the ISWAT in the groin, plasma ACTH levels were followed up for 6 months in the Inguinal (n=2), Axilla (n=3), and Muscle (n=4) groups (Figure 8). Pre-transplant basal and CRH-stimulated plasma ACTH levels were not significantly different among the three groups (inguinal white adipose tissue vs. axillary white adipose tissue vs. gluteal intramuscular, basal: 27.81±13.20 pg/ml vs. 14.58±4.52 pg/ml vs. 23.00±8.93 pg/ml, p=0.717; stimulated: 61.33±23.91 pg/ml vs. 44.40±14.35 pg/ml vs. 53.58±14.85 pg/ml, p=0.76). The axillary subcutaneous white adipose tissue transplantation group and intramuscular transplantation group were comparable when compared with the ISWAT group. In other words, in addition to ISWAT, axillary subcutaneous white adipose tissue transplantation and intramuscular transplantation are also suitable transplantation sites.

Then, the n number was increased to confirm reproducibility. Two animals were added to the Axilla group, one to the Inguinal group, and two to the Muscle group. Plasma ACTH levels in the Axilla group (n=5), Inguinal group (n=3), and Muscle group (n=6) were followed up for 6 months. There were no significant differences between the basal plasma ACTH levels before transplantation and CRH-stimulated plasma ACTH levels among the three groups, even when the n number was increased. (Axillary white adipose tissue vs. inguinal white adipose tissue vs. gluteal intramuscular tissue, basal: 15.17±3.58 pg/ml vs. 19.05±9.39 pg/ml vs. 32.23±9.41 pg/ml, p=0.6771, stimulated: 38.83±9.11 pg/ml vs. 48.87±17.97 pg/ml vs. 83.50±21.93 pg/ml, p=0.6269).

Example 3: Examination of transplantation of purified cell aggregates (purified bodies) (subcutaneously in the groin)
Materials and Methods
(1) Purification of pituitary hormone-producing cells (purified bodies) from hESC-derived PO Cell sorting (purification) using anti-EpCAM antibody was carried out on hESC-derived PO on days 64-76 after the start of differentiation induction, which was obtained by the same differentiation induction method as in Example 1. The cell sorting was carried out in three steps: 1. pretreatment, 2. dispersion of cell aggregates, and 3. separation of EpCAM-positive cells.

1. Pretreatment with Y27632 To suppress cell death due to dispersion treatment, Y27632 (20 μM) was added to the medium on the day before the operation to pretreat the cell aggregates. In the subsequent operations before cell sorting, 20 μM Y27632 was added to all solutions to which the cells were exposed.

2. Dispersion of cell aggregates To promote dispersion by enzyme treatment, the cell aggregates were chopped or cut with a scalpel. Next, the cell aggregates were washed with PBS, and then the dispersion liquid of the nerve cell dispersion kit (manufactured by Wako) was added, and the enzyme treatment was performed at 37°C for 30 minutes, and the cell aggregates were further loosened by pipetting. After the reaction was completed, the cells were washed with DMEM/F12 (manufactured by Wako), and a collagenase solution was added and the cells were swirled and shaken (140-150 rpm) at 37°C for 30 minutes. The composition of the collagenase solution was the above-mentioned DMEM/F12 to which 0.2% collagenase type I (manufactured by Wako) and 0.1% BSA (manufactured by Thermofisher) were added. After collagenase treatment, the cells were washed with PBS, and 10x TrypLE Select solution (Thermofisher) + 0.2 mg/ml DNase I (Roche) was added and treated with the enzyme at 37°C for 10 minutes, after which the cells were dispersed into single cells by pipetting. The cells were suspended and neutralized in serum-free medium containing 20% KSR, centrifuged, and then resuspended in serum-free medium containing 10 μg/ml DNase I and 20% KSR. The single cells were passed through a 70 μm cell strainer to remove aggregates.

3. Separation of EpCAM-positive cells by FACS The single cells obtained above were suspended in a DMEM/F-12 solution containing 1 mM EDTA and 1% FBS after centrifugation. PE-labeled anti-EpCAM antibody (Miltenyi) was added, incubated at 4°C for 10 minutes, and fluorescence-activated cell sorting (FACS) was performed to separate and collect EpCAM-positive cells. A BD FACS Aria Fusion, a Stream-In-Air type cell sorter, was used as the cell sorter for FACS.
The purified EpCAM-positive cells were reaggregated in 200 μL of serum-free medium supplemented with 20% KSR and 20 μM Y27632 to give 10×10 ⁴ cells per well of a non-adhesive 96-well culture plate, and cultured in suspension at 37° C., 40% O ₂ , and 5% CO _2. From the third day after the start of suspension culture, serum-free medium supplemented with 20% KSR without Y27632 was used, and half of the medium was replaced every 3 to 4 days for long-term culture.

(2) Transplantation Method Purified pituitary hormone-producing cells harvested 137-144 days after differentiation were used for transplantation into mice. Mice were hypophysectomized as in Example 1, and blood samples were collected and ACTH was measured. ISWAT transplantation into the groin was performed as in Example 1. All hypopituitarism mice were given an intramuscular injection of dexamethasone at 0.2 mg/0.61 ml/mouse to prevent adrenal crisis.

<Results>
Purified pituitary hormone-producing cells were transplanted into ISWAT (n=2), and plasma ACTH levels were followed up for 3 months (Figure 9). After transplantation, the basal plasma ACTH concentration was improved compared to before transplantation. In addition, CRH-stimulated plasma ACTH levels ("stimulated") were also improved, as were the basal levels.

Example 4: Further investigation of transplantation sites of pituitary hormone-producing cells (inguinal subcutaneous, axillary subcutaneous, muscle)
Materials and Methods
hESC-derived PO was prepared using the same differentiation induction method as in Example 1. PO harvested 90-100 days after differentiation was used for transplantation into mice.

(1) Transplantation Method Mice were hypophysectomized in the same manner as in Example 2, and then transplanted with ISWAT in the groin (Inguinal), subcutaneous white adipose tissue in the axilla (Axilla), or intramuscular transplantation in the gluteal region (Muscle).

(2) Statistical Analysis For survival time analysis, the log-rank test was subjected to Bonferroni correction. A P value of <0.05 (*) was considered significant.

Results
To evaluate survival rates, the non-transplant group (sham, n = 3), the Axilla group (n = 5), the Inguinal group (n = 3), and the Muscle group (n = 6) were followed up for 336 days ( FIG. 10 ). The axillary white adipose tissue/axillary white adipose tissue/gluteal intramuscular transplant group showed improved survival rates compared to the sham surgery group (p = 0.0002367).

Example 5: Examination of the transplantation effect by angiogenesis induction <Materials and methods>
hESC-derived PO was prepared using the same differentiation induction method as in Example 1. PO harvested 90-100 days after differentiation was used for transplantation into mice.

(1) Transplantation Method Mixtures prepared by mixing 25 mg of Artz Dispo joint injection and basic fibroblast growth factor (FGF) containing heparin were soaked in Duragen artificial dura mater (Integra Japan Co., Ltd.)/Surgicel SNOW (Johnson & Johnson Co., Ltd.) to prepare sheets. Mice were anesthetized with isoflurane and placed in a prone position. One week before cell transplantation, a 4 mm linear skin incision was made in the subcutaneous adipose tissue in the midline of the back, a pocket was made in the subcutaneous adipose tissue, and a Duragen sheet containing the mixture (Duragen group) and a Surgicel sheet containing the mixture (Surgicel group) were placed in the subcutaneous pocket for one week. The mixture was also locally injected using a syringe for one week (bFGF group). For the Duragen group, the sheet placed during cell transplantation was removed using the skin incision during pretreatment, and five POs taken from the cell culture medium were placed in the pocket using a wide-bore tip under a microscope. Skin closure was performed following nylon suture closure on the PO. For the Surgicel group, the skin incision during pretreatment was used, and five POs taken from the cell culture medium were placed in the pocket using a wide-bore tip under a microscope, since Surgicel is bioabsorbable and does not need to be removed. Skin closure was performed following nylon suture closure on the PO. For the bFGF group, the skin incision during pretreatment was used, and five POs taken from the cell culture medium were placed in the pocket using a wide-bore tip under a microscope. Skin closure was performed following nylon suture closure on the PO. For the avascular area (AR) transplantation group, a linear skin incision was made in the midline skin of the back, and five POs were placed. All hypopituitaric mice were subjected to an intramuscular injection of dexamethasone at 0.2 mg/0.61 ml/mouse to prevent adrenal crisis.

Results
In preparation for cell transplantation into a subcutaneous transplantation site with poor blood flow, in order to promote blood flow around the transplant cavity and increase cell engraftment, plasma ACTH levels were followed up for one month in the AR group (n=2), bFGF group (n=3), Duragen group (n=3), and Surgicel group (n=3) ( FIG. 11 ). After transplantation, the basal plasma ACTH level ("basal") tended to improve the most in the Duragen group. In addition, the CRH-stimulated plasma ACTH level ("stimulated") also tended to improve the most in the Duragen group, similar to the basal plasma ACTH level. (AR vs. bFGF vs. Duragen vs. Surgicel, Basal; 14.7±20.05 pg/ml vs. 17.12±16.37 pg/ml vs. 37.17±34.52 pg/ml vs. 2.39±2.66 pg/ml, Stimulated; 42.54±20.05 pg/ml vs. 60.77±16.37 pg/ml vs. 109.22±34.52 pg/ml vs. 6.29±2.66 pg/ml).

Example 6: Subcutaneous transplantation of human ES cell-derived pituitary organoids into primates under immunosuppressive drug conditions
Materials and Methods
hESC-derived PO was prepared using the same differentiation induction method as in Example 1. PO harvested 103 to 117 days after differentiation was used for transplantation into monkeys.

(2) Monkeys and hypophysectomy All animal experiments were approved by the Animal Care and Use Committee of the Shiga University of Medical Science and were performed in accordance with the institutional guidelines for the care and use of animals. Healthy adult male cynomolgus monkeys weighing 6.22 kg were used in this study.
Preoperative blood tests and CT scans for intraoperative navigation were performed before surgery while the monkeys were sedated. Serum concentrations of ACTH, cortisol, growth hormone (GH), and IGF-1 were collected before surgery to evaluate anterior pituitary function (SRL Co., Ltd., Tokyo, Japan). Similarly, arginine vasopressin (AVP) levels were measured to evaluate posterior pituitary function.
Anesthesia was induced by intramuscular administration of atropine (50 μg/kg) as preoperative medication. Ketamine (5 mg/kg) and xylazine (1 mg/kg) were administered intramuscularly. A tracheal tube was inserted and fixed to the left side of the monkey's mouth. Dexamethasone (120 μg), thyroxine (10 μg/body), desmopressin (0.5 μg), and ceftriaxone (20 mg/kg) were administered intravenously immediately before surgery as steroid replacement and antibiotic therapy, respectively. Anesthesia during surgery was administered by inhalation of isoflurane at a concentration of 1-3%.
An endoscopic transoral transsphenoidal approach was used for sphenoidal hypophysectomy. The exact direction of the pituitary gland was confirmed by an intraoperative navigation system. The monkey was placed prone and the head was rotated slightly toward the surgeon. The maxillary canines were pulled upward with a thread to open the monkey's mouth as wide as possible. The soft palate was incised linearly in the midline, and then the soft palate was retracted laterally with a thread. The mucosa in front of the sphenoid bone was incised linearly in the midline to expose the sphenoid bone. A hole was drilled in the sphenoid bone with a high-speed drill with a diamond burr. Because there are no surgical landmarks inside the petrous sphenoid sinus, the surgical direction was confirmed many times during this operation by the navigation system. After removing the sphenoid bone and the floor of the sella, the dural sella was cut with scissors. The pituitary gland and stalk were removed, and the pathological specimens were frozen and preserved. Close examination with an angled endoscope confirmed that no pituitary gland remained. The saddle was reconstructed with a fat graft taken from the abdominal subcutaneous fat layer, and fibrin glue was used to prevent postoperative cerebrospinal fluid (CSF) leakage. No bleeding or CSF leakage was observed after reconstruction. The soft palate was sutured to complete the operation. The infusion was discontinued immediately after the monkey regained consciousness. After awakening, the monkey was able to eat food and drink water freely. Ceftriaxone (20 mg/kg) was administered intramuscularly for 7 days after surgery. In the event of postoperative pain, intramuscular administration of buprenorphine (4 μg/kg) was considered.
For hypopituitarism, hormone replacement was administered intramuscularly daily. The doses of dexamethasone, thyroxine, and vasopressin were determined based on the clinical doses for hypopituitarism in humans. Dexamethasone was gradually tapered from 120 μg/day to 40 μg/day, and then changed to oral administration of 1.5 mg of cortryl from 66 days after surgery. Thyroxine (10 μg/day) was administered intramuscularly immediately after surgery. Diabetes insipidus (DI) was treated with vasopressin (0.5 μg/day) and free drinking of water. The monkeys' water balance was evaluated by body weight and serum sodium (Na) value, because it was difficult to accurately measure water intake and urine volume. Water and electrolyte imbalance was evaluated by blood tests. Infection was evaluated by the surgical wound, body temperature, and food intake. A hormone stimulation test was performed under sedation on the 17th day after surgery. Anterior pituitary function was measured after intravenous administration of corticotropin-releasing hormone (CRH) (1.5 μg/kg) and growth hormone-releasing factor (GRF) (1 μg/kg). Blood was collected 60 minutes after injection and posterior pituitary function was evaluated based on the presence or absence of DI and blood AVP concentration.

(3) Immunosuppressant therapy A six-month follow-up period was set to confirm that there were no side effects from long-term administration of immunosuppressants to monkeys. In the case of Tac, the dose was started at 0.3 mg/day, with a target trough of 15-20 ng/mL. MMF was started at 100 mg/day, and adjusted to 100-400 mg while monitoring side effects. Etanercept was subcutaneously injected at 2.5 mg one hour before transplantation and on the 3rd, 7th, and 10th days after transplantation. rATG was administered at an initial dose of 1.5 mg/kg over 12 hours starting 12 hours before transplantation, and then at 1.5 mg/kg over 12 hours from the start of transplantation. After that, it was administered twice after a 12-hour break. In other words, 1.5 mg/kg was administered once and 1.5 mg/kg three times, for a total dose of 6 mg/kg.

(4) Blood collection and ACTH measurement ACTH and Cortisol levels were evaluated as biomarkers of pituitary function. Blood samples were collected via the dorsal vein of the hand and sampled before and 1 hour after administration of human CRH (2 μg/kg, intraperitoneally, Tanabe, Nipro ES Pharma Co., Ltd., Osaka, Japan). After blood collection, the blood was heparinized and centrifuged to collect plasma. After storing at −80° C. in 1 mL aliquots, the ACTH/Cortisol concentrations in the blood supernatant were determined using an electrochemiluminescence immunoassay (ECLIA) kit (SRL Co., Ltd., Tokyo, Japan) that is used clinically in Japan. ACTH/Cortisol concentrations were measured weekly until one month after transplantation treatment. Thereafter, ACTH/Cortisol concentrations were measured every two weeks.
CRH loading tests were performed 17 days after hypophysectomy and 4 weeks after PO implantation treatment.
The negative feedback of the graft on cortisol was evaluated. Blood samples were collected via the dorsal vein of the hand before and 2 hours after administration of 1 mg dexamethasone. After blood collection, the blood was heparinized and centrifuged to obtain plasma. After storage at -80°C in 1 mL aliquots, ACTH concentrations in the blood supernatant were determined using an electrochemiluminescence immunoassay (ECLIA) kit (SRL Co., Ltd., Tokyo, Japan), which is used clinically in Japan. A dexamethasone suppression test was performed 6 weeks after PO transplantation treatment.

(5) Measurement of Spontaneous ACTH Secretion from PO in Vitro Using the same method as in Example 1, spontaneous ACTH secretion from PO in vitro was measured.

(6) Transplantation Method A mixture of 25 mg of Artz Dispo joint injection and basic fibroblast growth factor (FGF) containing heparin was soaked in Duragen artificial dura mater (Integra Japan Co., Ltd.) to prepare a sheet. Monkeys were anesthetized in the same manner as in hypophysectomy and placed in a prone position. One week before cell transplantation, a 4 mm linear skin incision was made in the subcutaneous adipose tissue in the midline of the back, a pocket was made in the subcutaneous adipose tissue, and a Duragen sheet containing the mixture was placed in the subcutaneous pocket for one week. During cell transplantation, the sheet placed during cell transplantation was removed using the skin incision made during pretreatment, and 1440 POs (5 pieces/20 g) washed with HBSS under visual observation were placed in the pocket. Following nylon suture closure on the PO, skin closure was performed. To prevent postoperative adrenal crisis, the patient was given an intravenous injection of 4.5 mg hydrocortone.

<Results>
Pre-transplant ACTH secretion in vitro was evaluated by ACTH levels in PO culture medium, with a mean ACTH concentration of 13123 pg/ml. After transplantation, basal plasma ACTH levels and CRH-stimulated plasma ACTH levels remained higher than before transplantation (Figures 12A and 13A). Basal Cortisol levels were also comparable to those observed when oral Cortisol was administered preoperatively (Figure 12B). In a dexamethasone suppression test performed 6 weeks after transplantation, plasma ACTH levels 2 hours after dexamethasone administration were lower than those observed before administration (Figure 13B).

The present invention is useful because it makes it possible to provide a therapeutic drug for diseases caused by disorders of the pituitary gland, which contains pituitary hormone-producing cells and is characterized by being used to transplant into a transplantation site (subcutaneous tissue (particularly subcutaneous adipose tissue) and/or muscle tissue) that is less invasive and easy to resect when the transplanted cells become tumorous. The therapeutic drug of the present invention is also useful because it makes it possible to realize clinical applications of pituitary hormone-producing cells. The method of the present invention is also useful because it makes it possible to create a model animal having human pituitary tissue that maintains hormone secretion ability for a long period of time, which is useful for evaluating the efficacy and safety of compounds.

This application is based on patent application No. 2023-021207 (filing date: February 14, 2023) and patent application No. 2023-176873 (filing date: October 12, 2023) filed in Japan, the contents of which are incorporated in their entirety into this specification.

Claims (22)

A therapeutic agent for diseases caused by disorders of the pituitary gland, comprising pituitary hormone-producing cells, the pituitary hormone-producing cells being used to be transplanted into the subcutaneous tissue and/or muscle tissue of a subject. The therapeutic agent according to claim 1, wherein the subcutaneous tissue is present in at least one site selected from the groin, armpit, back, abdomen, thigh, buttocks, upper arm, and scalp. The therapeutic agent according to claim 1 or 2, wherein the subcutaneous tissue is subcutaneous adipose tissue. The therapeutic agent according to claim 3, wherein the adipose tissue is white adipose tissue. The therapeutic agent according to claim 1, wherein the muscle tissue is muscle tissue present in at least one region selected from the back, abdomen, thighs, buttocks, and upper arms. The therapeutic agent according to any one of claims 1 to 5, characterized in that the pituitary hormone-producing cells are used to be transplanted near blood vessels in the tissue. The therapeutic agent according to any one of claims 1 to 6, characterized in that the pituitary hormone-producing cells are used by being transplanted into an artificially created space in the tissue. The therapeutic agent according to any one of claims 1 to 7, wherein the disease caused by a pituitary disorder is at least one disease selected from hypopituitarism, pituitary dwarfism, adrenal insufficiency, partial hypopituitarism, isolated anterior pituitary hormone deficiency, and craniopharyngioma. The therapeutic agent according to any one of claims 1 to 8, wherein the pituitary hormone-producing cells are cell aggregates containing pituitary hormone-producing cells. The therapeutic agent according to any one of claims 1 to 9, wherein the pituitary hormone-producing cells are pituitary hormone-producing cells that express an angiogenic factor. The therapeutic agent according to claim 10, wherein the angiogenic factor is one or more selected from the group consisting of VEGFA, VEGFB, VEGFC, and ANGPT2. The therapeutic agent according to any one of claims 1 to 11, wherein the pituitary hormone-producing cells are pituitary hormone-producing cells produced by a method comprising the steps of:
(1) a first step of culturing pluripotent stem cells in the presence of a c-jun N-terminal kinase (JNK) signaling pathway inhibitor and a Wnt signaling pathway inhibitor to obtain a cell population;
(2) A second step of culturing the cell population obtained in the first step in the presence of a substance acting on the BMP signaling pathway and a substance acting on the Sonic Hedgehog signaling pathway to obtain a cell population containing pituitary hormone-producing cells. The therapeutic agent according to claim 12, characterized in that the process further comprises the following step a before the first step:
(a) A step a of culturing pluripotent stem cells in a medium containing 1) a TGFβ family signaling pathway inhibitor and/or a Sonic hedgehog signaling pathway agonist, and 2) an undifferentiated state maintenance factor, in the absence of feeder cells. The therapeutic agent according to claim 12 or 13, characterized in that the process further comprises the following step after the second step:
A step of selecting and recovering pituitary hormone-producing cells from the cell population containing pituitary hormone-producing cells obtained in the second step. The therapeutic agent according to any one of claims 1 to 14, which induces angiogenesis around the transplanted pituitary hormone-producing cells in a human subject after transplantation. The therapeutic agent according to any one of claims 1 to 15, which is used in combination with an angiogenesis promoter. The therapeutic agent according to any one of claims 1 to 16, wherein the pituitary hormone-producing cells are engrafted in subcutaneous tissue and/or muscle tissue in a human subject after transplantation. The therapeutic agent according to any one of claims 1 to 17, characterized in that a pituitary hormone is secreted into the blood in a human subject after transplantation. A method for treating a disease caused by a disorder of the pituitary gland, comprising administering pituitary hormone-producing cells to the subcutaneous tissue and/or muscle tissue of a human subject. A method for producing a non-human animal model bearing human pituitary tissue, comprising administering human pituitary hormone-producing cells to the subcutaneous tissue and/or muscle tissue of the non-human animal. A non-human animal model bearing human pituitary tissue, which contains human pituitary hormone-producing cells in subcutaneous tissue and/or muscle tissue, and which is capable of secreting pituitary hormones produced by the human pituitary hormone-producing cells into the blood. A method for evaluating the efficacy and safety of a test substance, comprising administering the test substance to a non-human animal bearing human pituitary tissue model according to claim 21.

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