Mesenchymal cells of ventral hindgut mesoderm and method for producing same

Abstract

This method for producing mesenchymal cells of the ventral hindgut mesoderm comprises: culturing pluripotent stem cells in an induction medium A containing a TGF-β inhibitor, a Wnt agonist, and a fibroblast growth factor to induce differentiation into epiblast-like cells; culturing the epiblast-like cells in an induction medium B containing a Wnt agonist, a fibroblast growth factor, and retinoic acid to induce differentiation into caudal epiblast-like cells; culturing the caudal epiblast-like cells in an induction medium C containing a Wnt agonist, a bone morphogenetic protein, and a TGF-β family member to induce differentiation into posterior primitive streak cells; and culturing the posterior primitive streak cells in an induction medium D containing a Wnt agonist, a fibroblast growth factor, a bone morphogenetic protein, and a hedgehog signaling agonist to induce differentiation into mesenchymal cells of the ventral hindgut mesoderm.

Description

Ventral hindgut mesenchymal cells and method for producing same

[0001] The present invention relates to the field of cell biology.Specifically, the present invention provides ventral hindgut mesodermal mesenchymal cells and the method for producing the same.The present invention also provides mature bladder organoids and the method for producing the same.

[0002] Endodermal organs (e.g., trachea, digestive tract, urinary bladder) share a common structure in which apically organized tubular epithelial cells are covered by basal mesenchymal cells. The interaction between these two cells maintains the structural and functional homeostasis of endodermal organs. Recent organoid research has made it possible to generate various endodermal organ organoids from human pluripotent stem cells. There is growing expectation that these organoids will be used in the development of disease models and drug discovery. However, most endodermal organ organoids are primarily composed of epithelial cells (Non-Patent Document 1).

[0003] Non-Patent Documents 2 and 3 report the induction of differentiation from human pluripotent stem cells into visceral mesenchymal cells. Non-Patent Document 4 reports the induction of differentiation from human pluripotent stem cells into visceral mesenchymal cells.

[0004] Lewis A. , et al. “Self-organization of organoids from endoderm-derived cells.” J Mol Med (Berl). 99(4), 449-462 (2021). Han, L. , et al. “Single cell transcriptomics identifies a signaling network coordinating endoderm and mesoderm diversification during ” Nat Commun 11, 4158 (2020). Kishimoto, K. , et al. “Directed differentiation of human pluripotent stem cells into diverse organ-specific mesenchyme of the digestive and ”Nature protocols, 17(11), 2699-2719 (2022). Kishimoto, K. , et al. “Bidirectional Wnt signaling between endoderm and mesoderm conferences tracheal identity in mouse and human cells.”Nat Commun 11, 4159 (2020).

[0005] Non-Patent Documents 2 and 3 elucidate the specific mechanisms of mesenchymal cells for organs in the visceral mesoderm surrounding the foregut. Non-Patent Document 4 induces differentiation of mesenchymal cells surrounding the respiratory organs, which are tissues derived from the foregut endoderm. However, none of Non-Patent Documents 2 to 4 relates to mesenchymal cells for organs in the hindgut. There is a need in the art for mesenchymal cells suitable for tissues derived from the hindgut mesoderm or endoderm, such as the kidney or bladder.

[0006] One object of the present disclosure is to provide a novel method for producing mesenchymal cells of the ventral hindgut mesoderm. Another object of the present disclosure is to provide a method for producing mature bladder organoids using mesenchymal cells of the ventral hindgut mesoderm produced by the above-mentioned method. The present disclosure provides the following aspects. [Item 1] A method for producing mesenchymal cells of the ventral hindgut mesoderm, the method comprising: culturing pluripotent stem cells in induction medium A containing a TGF-β inhibitor, a Wnt agonist, and a fibroblast growth factor to induce their differentiation into epiblast-like cells; culturing the epiblast-like cells in induction medium B containing a Wnt agonist, a fibroblast growth factor, and retinoic acid to induce their differentiation into caudal epiblast-like cells; culturing the caudal epiblast-like cells in induction medium C containing a Wnt agonist, a bone morphogenetic protein, and a TGF-β family member to induce their differentiation into posterior primitive streak cells; and culturing the posterior primitive streak cells in induction medium D containing a Wnt agonist, a fibroblast growth factor, a bone morphogenetic protein, and a Hedgehog signaling agonist to induce their differentiation into mesenchymal cells of the ventral hindgut mesoderm. [Item 2] The method of Item 1, further comprising culturing the posterior primitive streak cells in a selection medium containing a bone morphogenetic protein and a ROCK inhibitor. [Item 3] The method of Item 1 or 2, wherein the induction medium D further contains retinoic acid. [Item 4] A mesenchymal cell of ventral hindgut mesoderm produced by the method of any of Items 1 to 3, wherein the mesenchymal cell expresses FOXF1, ISL1, TBX4, and HOXD13. [Item 5] The mesenchymal cell of Item 4, further expressing at least one selected from the group consisting of HOXD10, HOXD11, and HOXD12. [Item 6] A pharmaceutical composition for treating injury or disease of the kidney or bladder, or surrounding areas, comprising the mesenchymal cell of Item 4 or 5, and a pharmaceutically acceptable carrier. [Item 7] A method for producing a non-human mammal having mesenchymal cells or muscle cells derived from the mesenchymal cells in the kidney or bladder or a surrounding area thereof, the method comprising introducing the mesenchymal cells according to Item 4 or 5 into the kidney or bladder or a surrounding area thereof.[Item 8] A non-human mammal having the mesenchymal cells or muscle cells derived from the mesenchymal cells according to Item 4 or Item 5 in the kidney or bladder or in a site surrounding the kidney or bladder, or a non-human mammal produced according to the production method according to Item 7. [Item 9] A method for evaluating responsiveness to a test substance, the method comprising contacting the test substance with mesenchymal cells of ventral hindgut mesoderm produced by the method of any one of Items 1 to 3 or mesenchymal cells of ventral hindgut mesoderm described in Item 4 or Item 5, or with mesenchymal cells of ventral hindgut mesoderm produced by the method of any one of Items 1 to 3; mesenchymal cells of ventral hindgut mesoderm described in Item 4 or Item 5; or with a non-human mammal having muscle cells derived from the mesenchymal cells in the kidney or bladder or in a site surrounding the kidney or bladder (for example, the non-human mammal described in Item 8); or with a non-human mammal produced by the method of Item 7, and detecting a change in the test substance or the mesenchymal cells, or a change in the test substance or the mesenchymal cells in the non-human mammal or the muscle cells derived from the mesenchymal cells. [Item 10] A method for producing matured bladder organoids, comprising culturing ventral hindgut organoids or bladder organoids in an induction medium containing retinol and retinoic acid in the presence of the mesenchymal cells according to Item 4 or 5, wherein the matured bladder organoids comprise a superficial cell layer, an intermediate cell layer located laterally relative to the superficial cell layer, a basal cell layer located laterally relative to the intermediate cell layer, and a muscle cell layer located laterally relative to the basal cell layer, wherein the muscle cell layer comprises muscle cells derived from the mesenchymal cells. [Item 11] A matured bladder organoid comprising a superficial cell layer, an intermediate cell layer located laterally relative to the superficial cell layer, a basal cell layer located laterally relative to the intermediate cell layer, and a muscle cell layer located laterally relative to the basal cell layer, wherein the muscle cell layer comprises muscle cells derived from the mesenchymal cells according to Item 4 or 5. [Item 12] A matured bladder organoid produced according to the method of item 10.[Item 13] A method for maturing bladder organoids, comprising culturing ventral hindgut organoids or bladder organoids in an induction medium containing retinol and retinoic acid in the presence of the mesenchymal cells of Item 4 or Item 5. [Item 14] A pharmaceutical composition for treating damage or disease of the bladder or its surrounding area, comprising the matured bladder organoids of Item 11 or Item 12, or cells derived from the bladder organoids, and a pharmaceutically acceptable carrier. [Item 15] A method for producing a non-human mammal having mesenchymal cells or muscle cells derived from the mesenchymal cells in the kidney or bladder or their surrounding area, comprising introducing the matured bladder organoids of Item 11 or Item 12 into the kidney or bladder or their surrounding area. [Item 16] A non-human mammal having the matured bladder organoids of Item 11 or Item 12 in the kidney or bladder or their surrounding area, or a non-human mammal produced according to the method of Item 15. [Item 17] A method for evaluating drug responsiveness to a test substance, comprising contacting the matured bladder organoid according to Item 11 or 12 or the non-human mammal according to Item 16 with the test substance, and detecting changes in the mature bladder organoid in response to the test substance. [Item 18] A method for producing mesenchymal cells of the ventral hindgut mesoderm, comprising inducing differentiation of pluripotent stem cells into epiblast-like cells, inducing differentiation of the epiblast-like cells into caudal epiblast-like cells, inducing differentiation of the caudal epiblast-like cells into posterior primitive streak cells, and inducing differentiation of the posterior primitive streak cells into mesenchymal cells of the ventral hindgut mesoderm. [Item 19] A method for producing epiblast-like cells, comprising culturing pluripotent stem cells in induction medium A containing a TGF-β inhibitor, a Wnt agonist, and a fibroblast growth factor, to induce differentiation into epiblast-like cells.[Item 20] A method for producing caudal epiblast-like cells, comprising culturing pluripotent stem cells in induction medium A containing a TGF-β inhibitor, a Wnt agonist, and a fibroblast growth factor to induce differentiation into epiblast-like cells, and culturing the epiblast-like cells in induction medium B containing a Wnt agonist, a fibroblast growth factor, and retinoic acid to induce differentiation into caudal epiblast-like cells. [Item 21] A method for producing posterior primitive streak cells, comprising: culturing pluripotent stem cells in induction medium A containing a TGF-β inhibitor, a Wnt agonist, and a fibroblast growth factor to induce their differentiation into epiblast-like cells; culturing the epiblast-like cells in induction medium B containing a Wnt agonist, a fibroblast growth factor, and retinoic acid to induce their differentiation into caudal epiblast-like cells; and culturing the caudal epiblast-like cells in induction medium C containing a Wnt agonist, a bone morphogenetic protein, and a TGF-β family member to induce their differentiation into posterior primitive streak cells. [Item 22] A method for treating damage, disease, or disorder of the kidney, bladder, or surrounding areas, comprising introducing the mesenchymal cells of Item 4 or Item 5 or the pharmaceutical composition of Item 6 into the kidney, bladder, or surrounding areas of a mammal in need of treatment. [Item 23] A method for treating damage, disease, or disorder of the bladder or its surrounding area, comprising introducing the bladder organoid according to Item 11 or Item 12, or cells derived from the bladder organoid, or the pharmaceutical composition according to Item 14, into the kidney or bladder or its surrounding area of ​​a mammal in need of treatment.[Item 24] A method for evaluating responsiveness to a test substance, comprising contacting the test substance with mesenchymal cells of ventral hindgut mesoderm, muscle cells, or matured bladder organoids described below, and detecting a change in the test substance, the mesenchymal cells, the muscle cells, or the matured bladder organoids, wherein the mesenchymal cells of the ventral hindgut mesoderm are mesenchymal cells of ventral hindgut mesoderm produced by the method described in any one of Items 1 to 3, the mesenchymal cells of ventral hindgut mesoderm described in Item 4 or Item 5, or the mesenchymal cells of the ventral hindgut mesoderm in the kidney or bladder or a peripheral site thereof of a non-human mammal (for example, a non-human mammal produced according to the production method described in Item 7, or the non-human mammal described in Item 8), The evaluation method, wherein the muscle cells are muscle cells derived from mesenchymal cells of the ventral hindgut mesoderm in the kidney or bladder or a peripheral site thereof of a non-human mammal (e.g., a non-human mammal produced according to the production method described in Paragraph 7, or the non-human mammal described in Paragraph 8), and the matured bladder organoid is a matured bladder organoid produced according to the production method described in Paragraph 10 (e.g., the matured bladder organoid described in Paragraph 12), the matured bladder organoid described in Paragraph 11, or the matured bladder organoid in the kidney or bladder or a peripheral site thereof of a non-human mammal (e.g., a non-human mammal produced according to the production method described in Paragraph 15, or the non-human mammal described in Paragraph 16).

[0007] Figure 1 is a schematic diagram of a mouse embryo at E9.5. The double arrow a indicates the foregut mesoderm region. The double arrow b indicates the midgut mesoderm region. The double arrow c indicates the dorsal hindgut mesoderm region. The double arrow d indicates the ventral hindgut mesoderm region. Figure 2(1) is a schematic diagram showing the differentiation pathway suggested for the development of ventral hindgut mesoderm in vivo. Figure 2(2) is a schematic diagram for inducing mesenchymal cells of the ventral hindgut mesoderm from human pluripotent stem cells (hPSCs). Figure 3A is a brightfield image of human pluripotent stem cells (hPSCs). Figure 3B is a schematic diagram of NANOG ｌｏｗFIG. 3C is a bright-field image of epiblast-like cells. FIG. 3D is a bright-field image of posterior primitive streak cells. FIG. 3E is a bright-field image of mesenchymal cells of the ventral hindgut mesoderm. FIG. 4A shows the medium used for culture from Day 0 to Day 1 in one embodiment. FIG. 4B is a series of fluorescent images of a cell population cultured under the culture conditions used in the embodiment. FIG. 4C is a series of bar graphs showing the percentage of cells expressing NANOG and / or OCT3 / 4 in the cell population. FIG. 4D shows the combination of medium used for culture from Day 0 to Day 2 in one embodiment. FIG. 4E is a series of fluorescent images of a cell population cultured under the culture conditions used in the embodiment. FIG. 4F is a series of bar graphs showing the percentage of cells expressing NANOG and / or OCT3 / 4 in the cell population. FIG. 4G is a series of bar graphs showing the relative expression levels of NKX1-2, CDX1, or CDX2 in the cell population. FIG. 5H shows the medium combination used for culture from Day 0 to Day 3 in one embodiment. FIG. 5I is a series of fluorescent images of a cell population cultured under the culture conditions used in the embodiment. FIG. 5J is a series of bar graphs showing the percentage of cells expressing EOMES or T in the cell population on Day 2 or Day 3. FIG. 6 is a series of line graphs showing the time-dependent relative expression levels of cell markers in a cell population cultured under culture conditions according to one embodiment. Statistical analysis was one-way analysis of variance with Dunnett's multiple comparison test compared to hESCs. FIG. 7A shows the medium combination used for culture from Day 0 to Day 11 in one embodiment. Figure 7B is a series of bar graphs showing the relative expression levels of cell markers in cell populations cultured under the culture conditions used in the embodiment. Statistical analysis was one-way ANOVA with Dunnett's multiple comparison test compared to StemFit culture conditions. Figure 8A is a bar graph showing the relative expression levels of ISL1 in cell populations cultured under culture conditions according to one embodiment.Figure 8B is a bar graph showing the relative expression levels of HOXD13 in the cell population. Figure 8C is a bar graph showing the relative expression levels of TBX4 in the cell population. Figure 8D is a bar graph showing the relative expression levels of FOXF1 in the cell population. Figure 8E is a bar graph showing the relative expression levels of WNT2 in the cell population. Figure 8F is a bar graph showing the relative expression levels of IRX3 in the cell population. Figure 8G is a bar graph showing the relative expression levels of IRX5 in the cell population. Figure 8H is a bar graph showing the relative expression levels of NKX2-3 in the cell population. Figure 8I is a bar graph showing the relative expression levels of NR2F1 in the cell population. Statistical analysis was one-way ANOVA with Dunnett's multiple comparison test compared to the "All" culture condition. Figure 9A is a series of images showing immunofluorescent staining of induced ventral hindgut mesenchymal cells. Figure 9B is an immunostaining image of the mesenchymal cell marker Vimentin in mesenchymal cells of the ventral hindgut mesoderm. Figure 10A is a bar graph showing the relative expression level of FOXF1 in various mesenchymal cells induced to differentiate. Figure 10B is a bar graph showing the relative expression level of HOXA5 in various mesenchymal cells induced to differentiate. Figure 10C is a bar graph showing the relative expression level of ISL1 in various mesenchymal cells induced to differentiate. Figure 10D is a bar graph showing the relative expression level of TBX4 in various mesenchymal cells induced to differentiate. Figure 10E is a bar graph showing the relative expression level of HOXD10 in various mesenchymal cells induced to differentiate. Figure 10F is a bar graph showing the relative expression level of HOXD11 in various mesenchymal cells induced to differentiate. Figure 10G is a bar graph showing the relative expression level of HOXD12 in various mesenchymal cells induced to differentiate. Figure 10H is a bar graph showing the relative expression levels of HOXD13 in various mesenchymal cells induced to differentiate. Figure 11A is a schematic diagram of the co-culture protocol between ventral hindgut endoderm (vHGE) organoids and ventral hindgut mesoderm (vHGM) mesenchymal cells. Figure 11B is a series of bright-field images of vHGE organoids in the presence or absence of vHGM mesenchymal cells.Figure 12A shows images of UPK1 / ΔNP63 / Ecad / DAPI multi-staining of co-cultured vHGM mesenchymal cells and vHGE organoids. Figure 12B shows images of UPK2 / Ecad / DAPI multi-staining of the co-culture. Figure 12C shows images of UPK3 / Ecad / DAPI multi-staining of the co-culture. Figure 12D shows images of GATA3 / CDX2 / Ecad / DAPI multi-staining of the co-culture. Figure 12E shows a series of fluorescent and multi-staining images of SMA / Ecad / FOXF1 of the co-culture. Figure 12F shows images of Ecad / GATA6 / DAPI multi-staining of the co-culture. Figure 12G shows a series of fluorescent and multi-staining images of ALDH1A2 / Ecad / SMA of the co-culture. Figure 13A is a schematic diagram showing the co-culture of vHGE organoids with mesenchymal cells of the ventral hindgut mesoderm (vHGM) or mesenchymal cells of the foregut visceral mesoderm (GM or RM). Figure 13B is a brightfield image of organoids co-cultured with various mesenchymal cells. The "W / O" symbol in the lower right of Figure 13B means "without." Figure 13C is a series of fluorescent images of vHGE organoids co-cultured with various mesenchymal cells. Figure 13D is a series of graphs showing the cell number (far left), cell size (second from the left), number of cells expressing FOXA2 (second from the right), and the ratio of FOXA2-expressing cells to the total number of cells per organoid (far right) of vHGE organoids co-cultured with various mesenchymal cells. Figure 13E is a series of graphs showing the mean fluorescence intensity corresponding to the mean expression levels of FOXF1 (left), SMA (center), and Calponin1 (right) expressed by cell populations within a given range (0-60 μm, 60-120 μm, and 120-180 μm) from co-cultured vHGE organoids. Statistical analysis in Figure 13D was performed by one-way analysis of variance with Dunnett's multiple comparison test for vHGM. Statistical analysis in Figure 13E was performed by two-way analysis of variance with Tukey's multiple comparison test for various mesenchymal cell types. Figure 14A is a schematic diagram of the co-culture protocol for ventral hindgut endoderm organoids and ventral hindgut mesoderm mesenchymal cells.Figure 14B is a series of FOXA2 / CDH1 / DAPI fluorescence images of the organoids co-cultured with the mesenchymal cells in the absence (ctrl) or presence of a hedgehog inhibitor (SANT-1). Figure 14C (left) is a graph showing the number of DAPI-stained cell nuclei per organoid. Figure 14C (center) is a graph showing the diameter of the organoid. Figure 14C (right) is a graph showing the ratio of FOXA2-expressing cells to the total number of cells per organoid. Figure 14D is a series of SMA / Caloponin1 / FOXA2 / CDH1 / DAPI fluorescence images of ventral hindgut endoderm organoids co-cultured in the absence (ctrl) or presence of SANT-1. Figure 14E is a series of graphs showing the expression levels of FOXF1 (left), SMA (center), and Calponin1 (right) in cell populations present at a given distance from ventral hindgut endoderm organoids co-cultured under each condition. Statistical analysis C is a one-way analysis of variance with Dunnett's multiple comparison test for the "ctrl" condition. Statistical analysis E is a two-way analysis of variance with Tukey's multiple comparison test. Bars represent mean values. Figure 15A is a schematic diagram showing the co-culture of E9.5 mouse anterior hindgut endoderm with vHGM mesenchymal cells. Figure 15B is a brightfield image of the anterior hindgut endoderm at 2 days (top) and 21 days (bottom) after co-culture. Figure 15C is a fluorescent image of mouse anterior hindgut endoderm after 21 days of co-culture. FIG. 15D is a series of fluorescent images of the bladder epithelial region in mouse anterior hindgut endoderm after 21 days of co-culture.

[0008] The term "ventral hindgut mesoderm" refers to the ventral region of the hindgut mesoderm that can differentiate into mesenchymal cells and vascular endothelial cells in the hindgut, which develops into the rectum and bladder in a mammalian embryo. The ventral hindgut mesoderm is, for example, the region indicated by the double arrow in Figure 1d of Figure 1.

[0009] The term "mesenchymal cells" refers to cells such as mesenchymal cells derived from the mesenchyme of a multicellular animal during the fetal stage. Mesenchymal cells have the ability to differentiate into, for example, muscle cells, fibrocytes, chondrocytes, osteocytes, blood cells, and adipocytes. Mesenchymal cells of the ventral hindgut mesoderm preferably have the ability to differentiate into muscle cells. Mesenchymal cells of the ventral hindgut mesoderm are, for example, bladder mesenchymal cells. Mesenchymal cells of the ventral hindgut mesoderm are, for example, induced to differentiate from pluripotent stem cells.

[0010] Mesenchymal cells of the ventral hindgut mesoderm express FOXF1, ISL1, TBX4, and HOXD13. The mesenchymal cells further express at least one marker (e.g., a combination of two or more markers, or three or more markers) selected from the group consisting of HOXD10, HOXD11, and HOXD12. The mesenchymal cells express, for example, HOXA5 at a low level.

[0011] The term "progenitor cell" refers to a cell that is in the process of differentiating from a stem cell to a functional cell and has the ability to differentiate into said functional cell. The term "cell population" refers to a collection of multiple cells. A cell population, for example, contains at least two of one cell type. A cell population, for example, contains at least two cell types, with at least one of each cell type.

[0012] The term "pluripotent stem cells" refers to stem cells that can be cultured in vitro and have the ability to differentiate into tissues derived from three germ layers (ectoderm, mesoderm, and endoderm), i.e., pluripotency. Pluripotent stem cells can be established, for example, from fertilized eggs, cloned embryos, germline stem cells, or stem cells in tissues. Pluripotent stem cells are embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells) derived from somatic cells, embryonic tumor cells (EC cells), or embryonic germ stem cells (EG cells). Pluripotent stem cells are preferably ES cells or iPS cells. Pluripotent stem cells are preferably human ES cells or human iPS cells.

[0013] "Epiblast-like cells" can be induced to differentiate by culturing pluripotent stem cells in an induction medium (also referred to as "induction medium A") containing a TGF-β inhibitor, a Wnt agonist, and a fibroblast growth factor. Epiblast-like cells, for example, express low levels of NANOG. In an epiblast-like cell population induced to differentiate by culturing using induction medium A, the proportion of cells co-expressing the undifferentiated markers OCT3 / 4 and NANOG is significantly lower than the proportion of cells co-expressing OCT3 / 4 and NANOG in a cell population (control cell population) obtained by culturing pluripotent stem cells using induction medium A from which a TGF-β inhibitor has been removed. The ratio of the expression level of NANOG to the expression level of OCT3 / 4 in the epiblast-like cell population is significantly lower than the ratio in the control cell population.

[0014] "Caudal epiblast-like cells" can be induced to differentiate by culturing epiblast-like cells in an induction medium (also referred to as "induction medium B") containing a Wnt agonist, fibroblast growth factor, and retinoic acid. Caudal epiblast-like cells express caudal epiblast markers, such as OCT3 / 4. Caudal epiblast-like cells also express, for example, T, but do not express EOMES. Caudal epiblast-like cells preferably also express NANOG at a low level. The caudal epiblast markers are, for example, at least two markers (preferably a combination of three markers) selected from the group consisting of NKX1-2, CDX1, and CDX2.

[0015] "Posterior primitive streak cells" can be induced by culturing caudal epiblast-like cells in an induction medium (also referred to as "induction medium C") containing a Wnt agonist, a bone morphogenetic protein, and a growth differentiation factor. Posterior primitive streak cells express posterior primitive streak markers. Posterior primitive streak markers are, for example, at least two markers (e.g., a combination of three or more markers, four or more markers, preferably five or more markers, and six markers) selected from the group consisting of T, MIXL1, HAND1, ISL1, TBX4, and BMP4. Posterior primitive streak cells do not express, for example, OCT4, NKX1-2, or NANOG. Posterior primitive streak cells express, for example, CDX1 at a lower level than caudal epiblast-like cells.

[0016] The term "induction medium" refers to a medium that can be used to induce or produce differentiation of a specific cell or organoid. The induction medium can be prepared, for example, by adding the additive or differentiation inducer (solid or liquid) according to the present disclosure to a basal medium (liquid).

[0017] The term "selective medium" refers to a medium that favors the growth of specific cells or organoids compared to cells other than specific cells or organoids.Selective medium may, for example, contain a first additive that favors the growth of specific cells or organoids.Selective medium may, for example, contain a second additive that inhibits the growth of cells other than specific cells or organoids or organoids.Selective medium may, for example, contain the first additive and the second additive.

[0018] The "basal medium" may be a cell culture medium that can be prepared according to a known protocol or may be a commercially available cell culture medium. The basal medium may be, for example, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Basal Medium Eagle (BME), Advanced™ DMEM / F-12, a known stem cell medium, or a known medium for differentiating stem cells. The basal medium may be, for example, Advanced™ DMEM / F-12. The basal medium is, for example, a medium for differentiating stem cells. The medium for differentiating stem cells can be prepared, for example, according to Nature Protocols, Vol. 3, No. 5, pp. 768-776, 2008. The basal medium may further contain, for example, a medium supplement (e.g., B-27 supplement without Vitamin A (Thermo Fisher Scientific) or N-2 Supplement (Thermo Fisher Scientific)), a buffer (e.g., 10 mM HEPES), 2 mM L-glutamine, an antibiotic (e.g., penicillin / streptomycin, gentamicin), or an antibiotic-antimycotic mixture (e.g., antibiotic-antimycotic), or a combination thereof.

[0019] "Induction medium A" can be used, for example, to induce differentiation of epiblast-like cells from pluripotent stem cells. The epiblast-like cells, for example, express low levels of NANOG. Induction medium A contains, for example, a TGF-β inhibitor, a Wnt agonist, and a fibroblast growth factor (FGF). Induction medium A may be, for example, a medium supplemented with a TGF-β inhibitor (e.g., A-83-01), a Wnt agonist (CHIR99021), FGF (FGF2), and heparin.

[0020] "Induction medium B" can be used, for example, to induce differentiation of epiblast-like cells into caudal epiblast-like cells. Induction medium B contains, for example, a Wnt agonist, FGF, and retinoic acid. Induction medium B may be, for example, a medium supplemented with a Wnt agonist (e.g., CHIR99021), FGF (e.g., FGF4), retinoic acid (e.g., atRA), and heparin.

[0021] "Induction medium C" can be used, for example, to induce differentiation of posterior primitive streak cells from caudal epiblast-like cells. Induction medium C contains, for example, a Wnt agonist, a bone morphogenetic protein (BMP), and a TGF-β family member. Induction medium C may be, for example, a medium supplemented with a Wnt agonist (e.g., CHIR99021), a BMP (e.g., BMP4), and a TGF-β family member (e.g., growth differentiation factor (GDF) 11).

[0022] "Induction Medium D" can be used, for example, to induce differentiation of posterior primitive streak cells into mesenchymal cells of the ventral hindgut mesoderm. Induction Medium D contains, for example, a Wnt agonist, FGF, BMP, and a hedgehog signaling agonist. Induction Medium D may be a medium supplemented with, for example, a Wnt agonist (e.g., CHIR99021), a BMP (e.g., BMP4), an FGF (e.g., FGF4), a hedgehog signaling agonist (e.g., Hh-Ag1.5), and heparin.

[0023] The term "TGF-β inhibitor" refers to a compound capable of inhibiting transforming growth factor (TGF)-β1. TGF-β signaling is involved in many cellular functions, including cell proliferation, cell fate, and apoptosis. TGF-β inhibitors are, for example, inhibitors of the TGF-β pathway acting via Smad2 and Smad3 and / or via ALK4, ALK5, or ALK7. TGF-β inhibitors are, for example, A83-01, SB-431542, SB-505124, SB-525334, LY364947, SD-208, or SJN 2511, or a combination thereof. The TGF-β inhibitor is preferably A83-01.

[0024] The term "Wnt agonist" refers to any compound that activates Wnt / β-catenin receptor signaling. The Wnt agonist may be, for example, CHIR-99021, SB216763, CHIR-98014, Staurosporine, K252A, WNT (preferably WNT3A), or TWS119, or a combination thereof. The Wnt agonist is preferably CHIR-99021 or WNT (preferably WNT3A).

[0025] The term "fibroblast growth factor (FGF)" refers to a protein with a molecular weight of 16,000 to 20,000 that promotes the proliferation of fibroblasts or endothelial cells. The FGF may be, for example, FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, or FGF23, or a combination thereof. The FGF is preferably FGF2 or FGF4.

[0026] The term "retinoic acid (RA)" generally refers to a compound of the chemical formula C ２０ H ２８ O ２ and is registered under CAS number 302-79-4. The retinoic acid may be, for example, all-trans retinoic acid (atRA), 9-cis-retinoic acid, 11-cis-retinoic acid, or 13-cis-retinoic acid, or a combination thereof. The retinoic acid is preferably atRA or 9-cis-retinoic acid, and more preferably atRA.

[0027] The term "bone morphogenetic protein (BMP)" refers to a protein that belongs to the transforming growth factor β superfamily and induces ectopic bone formation. The BMP may be, for example, BMP2, BMP4, or BMP7, or a combination thereof. The BMP is preferably BMP4.

[0028] "TGF-β family member" refers to a protein structurally similar to activin or bone morphogenetic protein, which acts as a regulatory factor in embryogenesis and organ formation, such as nerve and bone formation. TGF-β family members are synthesized, for example, as precursors with a molecular weight of approximately 40,000, and are cleaved at the C-terminus to form a peptide with a molecular weight of 12,000 to 15,000, which forms an active dimer via a disulfide bond. Examples of TGF-β family members include TGF-β subfamily members, activin subfamily members, and Nodal subfamily members, or growth differentiation factor (GDF) subfamily members, or combinations thereof. Examples of TGF-β family members may be Nodal, activin A, activin B, GDF1, GDF3, GDF8, GDF9, GDF11, or GDF15, or combinations thereof. Preferably, TGF-β family members include GDF subfamily members. The TGF-β family member preferably includes at least one member selected from the group consisting of GDF1, GDF3, GDF8, GDF9, GDF11, and GDF 15. The TGF-β family member is preferably GDF11.

[0029] The term "ROCK inhibitor" refers to a compound that inhibits Rho-associated, coiled-coil containing protein kinase (ROCK). Examples of ROCK inhibitors include N-(4-pyridinyl)-4β-[(R)-1-aminoethyl]cyclohexane-1α-carboxamide (Y-27632), Fasudil (HA1077), (2S)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]hexahydro-1H-1,4-diazepine (H-1152), 4β-[(1R)-1-aminoethyl]-N-(4-pyridyl)benzene-1αcarboxamide (Wf-536), and N-(1H-pyrrolo[2,3-b]pyridin-4-yl)-4β-[(R)-1-aminoethyl]cyclohexane-1α. carboxamide (Y-30141), N-(3-{[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-1H-imidazo[4,5-c]pyridin-6-yl]oxy}phenyl)-4-{[2-(4-morpholinyl)ethyl]-oxy}benzamide (GSK269962A), or N-(6-fluoro-1H-indazol-5-yl)-6-methyl-2-oxo-4-[4-(trifluoromethyl)phenyl]-3,4-dihydro-1H-pyridine-5-carboxamide (GSK429286A), or a combination thereof. The ROCK inhibitor may be, for example, Y-27632 alone or in combination with another ROCK inhibitor. The ROCK inhibitor is preferably Y-27632.

[0030] ROCK is a serine-threonine protein kinase. ROCK phosphorylates, for example, myosin-binding subunit 1 (MYPT1) of myosin light chain phosphatase (MLCP) to inhibit its enzymatic activity. The ROCK inhibitory effect can be measured, for example, based on the amount of phosphorylation of MYPT1 by ROCK in the presence of a compound capable of inhibiting the phosphorylation effect. In one example, the ROCK inhibitory effect can be measured by measuring the amount of phosphorylation of MYPT1 by ROCK, measuring the amount of phosphorylation of MYPT1 by ROCK in the presence of a compound capable of inhibiting the phosphorylation effect, and comparing the amounts of phosphorylation. The ROCK inhibitory activity can be measured using a commercially available kit (for example, a 96-well ROCK Activity Assay Kit (CELL BIOLABS, INC., Catalog No.: STA-416) or a ROCK Activity Immunoblot Kit (STA-416, CELL BIOLABS, INC., Catalog No.: STA-415)).

[0031] The term "hedgehog signaling agonist" refers to a compound that can upregulate the hedgehog signaling system ("HH signaling system"). Examples of HH signaling agonists include parmorphamine (CAS No. 483367-10-8: 2-(1-naphthoxy)-6-(4-morpholinoanilino)-9-cyclohexylpurine 9-cyclohexyl-N-[4-(4-morpholinyl)phenyl]-2-(1-naphthalenyloxy)); SAG (CAS No. 912545-86-9: 3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-[[3-(4-pyridinyl)phenyl]methyl]benzo[b]thiophene-2-carboxamide); HH-Ag1.5 (CAS No. 612542-14-0: The HH signaling agonist may be 3-chloro-4,7-difluoro-N-(4-(methylamino)cyclohexyl)-N-(3-(pyridin-4-yl)benzyl)benzo[b]thiophene-2-carboxamide), or Gli-2, or a combination thereof. The HH signaling agonist is preferably HH-Ag1.5.

[0032] The term "extracellular matrix (ECM)" refers to a collection of extracellular molecules that can provide structural and biochemical support to surrounding cells. The structural and biochemical support can provide anchorage-dependent cells with a scaffold for cell growth. The structural and biochemical support can be in the form of, for example, a gel or a viscous liquid. The ECM includes, for example, water, polysaccharides, elastin, integrins, and glycoproteins. Glycoproteins include, for example, collagen, entactin (nidogen), fibronectin, and laminin.

[0033] ECM can be prepared, for example, by culturing ECM-producing cells (e.g., epithelial cells, endothelial cells, parietal endoderm-like cells, or fibroblasts) in vitro and then removing the ECM-producing cells. The ECM-producing cells may be, for example, chondrocytes, which primarily produce collagen and proteoglycans; fibroblasts, which primarily produce type IV collagen, laminin, interstitial procollagen, and fibronectin; and colonic myofibroblasts, which primarily produce collagen (types I, III, and V), chondroitin sulfate proteoglycans, hyaluronic acid, fibronectin, and tenascin-C.

[0034] ECM is commercially available. Commercially available extracellular matrices may be, for example, extracellular matrix proteins (Invitrogen), basement membrane preparations from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells (e.g., Cultrex® Basement Membrane Extract (Trevigen, Inc.), or Matrigel® (Corning)). The ECM may be a synthetic extracellular matrix (e.g., ProNectin (Sigma Z378666)). The extracellular matrix may be one type or a mixture of two or more types. The ECM is preferably Matrigel.

[0035] The term "organoid" (also referred to as "spheroid") refers to a three-dimensional cell aggregate formed in vitro that resembles a living tissue. The cells that constitute an organoid are, for example, mostly cells that have the ability to differentiate and proliferate. As used herein, an organoid expressing a specific marker refers to an organoid that contains a specific proportion of cells that express the specific marker. The specific proportion may be, for example, 3% or more, 5% or more, 10% or more, or 15% or more of the cells that constitute the organoid.

[0036] "Definitive endoderm cells (DE)" can be prepared from pluripotent stem cells, for example, according to the method disclosed in WO 2022 / 025269 (the entire disclosure of which is incorporated herein by reference). Definitive endoderm cells can be induced to differentiate, for example, by culturing pluripotent stem cells in an induction medium containing activin A (e.g., 100 ng / ml) and a Wnt agonist (e.g., 1-2 μM CHIR99021).

[0037] "Hindgut organoids" can be prepared from definitive endoderm cells (DE) according to the method disclosed in, for example, WO 2022 / 025269 (the entire disclosure of which is incorporated herein by reference). Hindgut organoids can be induced to differentiate by culturing definitive endoderm cells in an induction medium (also referred to as "hindgut induction medium") containing fibroblast growth factor (200 ng / ml FGF4), a Wnt agonist (3 μM CHIR99021), a bone morphogenetic protein (10 ng / ml BMP4), and a ROCK inhibitor (10 μM Y-27632).

[0038] The term "ventral hindgut organoid" refers to an organoid that expresses at least one ventral hindgut marker (P63, ΔNp63, GATA3, ISL1, and SATB2) and does not substantially express at least one dorsal hindgut marker (SOX2, CDX2, and T), or if it does express, the expression level is lower than the expression level in hindgut organoids.

[0039] Ventral hindgut organoids can be prepared from pluripotent stem cells, for example, according to the method disclosed in WO2022 / 025269 (the entire disclosure of which is incorporated herein by reference). Ventral hindgut organoids can be produced, for example, by ventralizing hindgut organoids. Ventralization involves culturing hindgut organoids in the presence of extracellular matrix using a ventralization induction medium. The ventralization induction medium comprises a basal medium and an additive containing fibroblast growth factor (FGF) and a Wnt agonist, and may further contain bone morphogenetic protein (BMP). Ventral hindgut organoids can be differentiated, for example, from definitive endoderm-differentiated cells using a ventralization induction medium. Ventral hindgut organoids can be formed, for example, by culturing definitive endoderm-differentiated cells in a ventralization induction medium to form floating spheroids, and then culturing the floating spheroids in a ventralization induction medium in the presence of an extracellular matrix.

[0040] Ventral hindgut organoids (also referred to as "ventral hindgut endoderm organoids") can be produced, for example, by culturing pluripotent stem cells in an induction medium containing activin A (e.g., 100 ng / ml) and a Wnt agonist (e.g., 1-2 μM CHIR99021) to induce differentiation into definitive endoderm cells (DE); culturing definitive endoderm cells in an induction medium containing fibroblast growth factor (200 ng / ml FGF4), a Wnt agonist (3 μM CHIR99021), bone morphogenetic protein (10 ng / ml hindgut organoids can be differentiated by culturing them in a hindgut induction medium containing 100 ng / ml FGF4, a Wnt agonist (e.g., 3 μM CHIR99021), and a bone morphogenetic protein (10 ng / ml BMP4) in the presence of an extracellular matrix.

[0041] The term " bladder organoid " refers to an organoid having at least two cell layers. The at least two cell layers include: a first cell layer comprising cells that do not substantially express P63 but express UPK1B and / or UPK2; and a second cell layer that is located outward relative to the first cell layer and comprises cells that co-express P63 and UPK1B and / or UPK2. The bladder organoid having the two cell layers is also referred to as " immature bladder organoid ". For example, immature bladder organoid comprises the first cell layer and the second cell layer that is located outward relative to the first cell layer, and does not comprise the cell layer that comprises cells that express KRT5, a basal cell marker.

[0042] Bladder organoids can be prepared from pluripotent stem cells, for example, according to the method disclosed in WO2022 / 025269 (the entire disclosure of which is incorporated herein by reference). Bladder organoids can be differentiated by culturing ventral hindgut organoids in a bladder induction medium. The bladder induction medium contains a basal medium and an additive containing a relatively low concentration (e.g., 1 nM) of retinoic acid, fibroblast growth factor (FGF), and bone morphogenetic protein (BMP).

[0043] Immature bladder organoids can be differentiated by culturing ventral hindgut organoids in an immature organoid induction medium, which comprises a basal medium and an additive containing a relatively high concentration (e.g., 1 μM) of retinoic acid, fibroblast growth factor (FGF), and bone morphogenetic protein (BMP).

[0044] The term "mature bladder organoid" includes a superficial cell layer (first cell layer), an intermediate cell layer (second cell layer) located outward from the superficial cell layer, and a basal cell layer (third cell layer) located outward from the intermediate cell layer. The "mature bladder organoid" herein may also include a muscle cell layer (fourth cell layer) located outward from the basal cell layer, and the muscle cell layer comprises muscle cells derived from the mesenchymal cells of the present disclosure.

[0045] The matured bladder organoid further comprises, for example, a lumen defined by a superficial cell layer. The matured bladder organoid comprises, for example, a lumen, and comprises a superficial cell layer (first cell layer) facing the lumen, an intermediate cell layer (second cell layer) located outward from the superficial cell layer, a basal cell layer (third cell layer) located outward from the intermediate cell layer, and a muscle cell layer (fourth cell layer) located outward from the basal cell layer, wherein the muscle cell layer comprises muscle cells derived from the mesenchymal cells of the present disclosure.

[0046] The matured bladder organoid further comprises, for example, a mesenchymal cell layer between the basal cell layer and the muscle cell layer.The matured bladder organoid comprises, for example, a superficial cell layer (first cell layer), an intermediate cell layer (second cell layer) located outward relative to the superficial cell layer, a basal cell layer (third cell layer) located outward relative to the intermediate cell layer, a mesenchymal cell layer (fourth cell layer) located outward relative to the basal cell layer, and a muscle cell layer (fifth cell layer) located outward relative to the mesenchymal cell layer, wherein the mesenchymal cell layer comprises the mesenchymal cells of the present disclosure, and the muscle cell layer comprises muscle cells derived from the mesenchymal cells of the present disclosure.

[0047] For example, the matured bladder organoid further comprises a lumen defined by a superficial cell layer, and a mesenchymal cell layer between a basal cell layer and a muscle cell layer.For example, the matured bladder organoid comprises a lumen, and comprises a superficial cell layer (first cell layer) facing the lumen; an intermediate cell layer (second cell layer) located in the outer direction relative to the superficial cell layer; a basal cell layer (third cell layer) located in the outer direction relative to the intermediate cell layer; a mesenchymal cell layer (fourth cell layer) located in the outer direction relative to the basal cell layer; and a muscle cell layer (fifth cell layer) located in the outer direction relative to the mesenchymal cell layer, wherein the mesenchymal cell layer comprises the mesenchymal cells of the present disclosure, and the muscle cell layer comprises muscle cells derived from the mesenchymal cells of the present disclosure.

[0048] For example, in the matured bladder organoid, the surface cell layer and the intermediate cell layer are in direct contact, and the intermediate cell layer and the basal cell layer are in direct contact.For example, in the matured bladder organoid, the intermediate cell layer is interposed between the surface cell layer and the basal cell layer.For example, in the matured bladder organoid, the intermediate cell layer is interposed between the surface cell layer and the basal cell layer, the basal cell layer is interposed between the intermediate cell layer and the mesenchymal cell layer, and / or the mesenchymal cell layer is interposed between the basal cell layer and the muscle cell layer.

[0049] The term "lumen" refers to the space inside a tubular or sac-like cell structure. The lumen may be filled with, for example, a liquid. In matured bladder organoids, the lumen is defined, for example, by a superficial cell layer. The lumen is, for example, the inner space (room) of a sac-like matured bladder organoid composed of a superficial cell layer, an intermediate cell layer, a basal cell layer, and a muscle cell layer. The matured bladder organoid is, for example, a sac-like cell structure composed of a superficial cell layer, an intermediate cell layer, a basal cell layer, and a muscle cell layer, and has a lumen in which there are no cells opposite the superficial cell layer.

[0050] The term "cell layer" refers to an overlap of cell populations that are distinguishable from one another. Examples of cell layers of a given cell type include a cell layer of superficial cells (also referred to as a "surface cell layer"), a cell layer of intermediate cells (also referred to as an "intermediate cell layer"), a cell layer of basal cells (also referred to as a "basal cell layer"), a cell layer of mesenchymal cells (also referred to as a "mesenchymal cell layer"), or a cell layer of muscle cells (also referred to as a "muscle cell layer"). A cell layer of a given cell type is, for example, a cell population in which the given cell type is present at 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more. For example, in a superficial cell layer, superficial cells account for 70% or more of the cell monolayer facing the lumen. For example, in a basal cell layer, basal cells account for 70% or more of the cell monolayer facing mesenchymal cells according to the present disclosure or muscle cells derived from the mesenchymal cells. The intermediate cell layer is, for example, a cell monolayer intermediate between the outermost cell monolayer of the superficial cell layer and the innermost cell monolayer of the basal cell layer, where intermediate cells account for 70% or more. The muscle cell layer is, for example, a cell monolayer intermediate between the outermost cell monolayer of the basal cell layer and the innermost cell monolayer of the basal cell layer, where muscle cells account for 70% or more. When a mesenchymal cell layer is present, for example, a cell monolayer intermediate between the outermost cell monolayer of the basal cell layer and the innermost cell monolayer of the muscle cell layer, where mesenchymal cells account for 70% or more. The specified cells can be identified by detecting cell markers corresponding to the specified cell type (e.g., protein expression and / or gene transcription).

[0051] "Surface layer cells" express uroplakin. Surface layer cells, for example, express uroplakin but do not substantially express P63. Surface layer cells, for example, express uroplakin and further express either CK20 and / or ZO1. Surface layer cells, for example, express uroplakin but do not substantially express P63 and further express either CK20 and / or ZO1.

[0052] "Intermediate cells" co-express P63 and uroplakin. Intermediate cells, for example, co-express P63 and UPK1B and / or UPK2. "Basal cells" co-express P63 and keratin 5 (KRT5). The term "muscle cell" refers to a cell in which many actin filaments and few myosin filaments are present within the cell. Muscle cells, for example, express either or both SMA and Calponin 1. Muscle cells, for example, express SMA and Calponin 1.

[0053] The mesenchymal cells of the ventral hindgut endoderm according to the present disclosure express FOXF1, ISL1, TBX4, and HOXD13. The mesenchymal cells further express at least one marker selected from the group consisting of HOXD10, HOXD11, and HOXD12, for example. The mesenchymal cells do not substantially express WNT2, IRX5, or NKX2-3, for example. The mesenchymal cells further express Vimentin, for example.

[0054] The basal cell layer is, for example, a cell population in which 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more of cells co-express P63 and keratin 5. The intermediate cell layer is, for example, a cell population in which 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more of cells co-express P63 and uroplakin. The superficial cell layer is, for example, a cell population in which 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more of cells express uroplakin but do not substantially express P63. The superficial cell layer is, for example, a cell population in which 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more of cells express uroplakin and either CK20 or ZO1 or both (preferably both)

[0055] The term "OCT3 / 4," also referred to as Octamer-binding transcription factor 4 or POU domain, class 5, transcription factor 1 (POU5F1), is a protein encoded by the POU5F1 gene. Oct-4 is a homeodomain transcription factor with a Pit-Oct-Unc (POU) domain. OCT3 / 4 can be used as a marker for undifferentiated cells. The term "NANOG" refers to a homeobox protein that plays a role in maintaining pluripotency in embryonic stem cells (ESCs) by repressing cellular determinants. NANOG can be used as a marker for undifferentiated cells.

[0056] The term "SOX2," also known as SRY (sex determining region Y)-box2, refers to a transcription factor essential for maintaining self-renewal of undifferentiated ES cells. SOX2 can be used as a dorsal hindgut marker or a lung / stomach lineage marker.

[0057] The term "CDX1" refers to a homeobox protein encoded by the CDX1 gene. The term "CDX2" refers to a homeobox protein encoded by the CDX2 gene. CDX2 can be used as a midgut / hindgut marker or a caudal epiblast marker. The term "NKX1-2" refers to a transcriptional repressor transcription factor that is a key regulator of organ development. NKX1-2 can be used as a caudal epiblast marker.

[0058] The term "T," also known as TBXT (T-box transcription factor T), refers to a transcription factor that binds to a DNA sequence called a palindromic T site via an N-terminal region called a T-box, thereby influencing the transcription of genes required for mesoderm formation and differentiation. T can be used as a dorsal hindgut marker or posterior primitive streak marker. The term "MIXL1" refers to a paired-type homeobox transcription factor consisting of 232 amino acids. MIXL1 preferentially binds to the DNA sequence TAAT on the Mix gene. MIXL1 can be used as a posterior primitive streak marker. The term "Eomesodermin (EOMES)" is also known as T-box brain protein 2 (Tbr2). EOMES refers to a protein with a DNA-binding domain (T-box) encoded by the EOMES gene in humans.

[0059] The term "HAND1" is an abbreviation for Heart- and neural crest derivatives-expressed protein 1, and in humans refers to the protein encoded by HAND1. HAND1 can be used as a posterior primitive streak marker. The term "ISL1" refers to a transcription factor having a LIM homeodomain that acts on the expression regulatory region of the insulin gene. ISL1 can be used as a ventral hindgut marker or a posterior primitive streak marker.

[0060] The term "TBX4," also known as T-box transcription factor, refers to a transcription factor belonging to the T-box gene family. TBX4 can be used as a marker for the posterior primitive streak. The term "BMP4," also known as bone morphogenetic protein 4, refers to a protein encoded by the BMP4 gene in humans. BMP4 can be used as a marker for the posterior primitive streak.

[0061] The term "HOXD10" refers to a protein having a homeobox DNA-binding domain, encoded by the HOXD10 gene in humans. "HOXD11" refers to a protein encoded by the HOXD11 gene in humans. "HOXD12" refers to a protein encoded by the HOXD12 gene in humans. "HOXD13" refers to a homeobox protein encoded by the HOXD13 gene in humans. These can be used as ventral hindgut mesoderm markers.

[0062] The term "GATA6," also known as GATA-binding factor 6, refers to the protein encoded by the GATA6 gene in humans. GATA6 preferentially binds to the consensus binding sequence (A / T / C)GAT(A / T)(A). GATA6 can be used as a marker for perivesaccular mesenchymal cells. The term "ALDH1A2," also known as aldehyde dehydrogenase 1 family, member A2, or ALDH1A2, or retinal aldehyde dehydrogenase 2 (RALDH2), refers to the protein encoded by the ALDH1A2 gene in humans. ALDH1A2 catalyzes the synthesis of retinoic acid (RA) from retinal aldehyde. Retinoic acid, an active derivative of vitamin A (retinol), is a paracrine hormone signaling molecule that functions during development and in adult tissues. ALDH1A2 can be used as a marker for bladder-peristaltic mesenchymal cells. The term "Vimentin" refers to a major cytoskeletal protein in various cells that constitute connective tissue (e.g., fibroblasts, vascular endothelial cells, smooth muscle cells, striated muscle cells, osteocytes, cartilage cells, and nerve sheath cells). Vimentin can be used as a marker for bladder-peristaltic mesenchymal cells.

[0063] The term "FOXF1" is an abbreviation for forkhead box protein F1, which in humans refers to the protein encoded by the FOXF1 gene. FOXF1 can be used as a visceral mesoderm marker.

[0064] The term "CDH1" refers to E-cadherin, a transmembrane glycoprotein. CDH1 is involved in E-cadherin-mediated cell-cell adherens junctions. CDH1 can be used as an epithelial cell marker.

[0065] The term "smooth muscle actin (SMA)," also known as ACTA2, refers to a protein belonging to the actin family. SMA can be used as a muscle cell marker. The term "calponin1" refers to a basic smooth muscle protein that is encoded by the CNN1 gene in humans. Calponin1 can be used as a smooth muscle cell marker.

[0066] The term "TP63" is also called tumor protein p63 or transformation-associated protein 63, and refers to a protein encoded by the TP63 gene in humans. TP63 can be used as a basal and squamous epithelial differentiation marker. The term "ECAD" is also called E-cadherin, and refers to a transmembrane glycoprotein present on the cell surface that acts on cell adhesion. ECAD can be used as an epithelial tissue marker.

[0067] The term "ZO1" refers to a membrane phosphoprotein expressed in the tight junctions of epithelial and endothelial cells. ZO1 can be used as a tight junction marker. ZO1 expression in the cell membrane connecting cells suggests the formation of tight junctions between the cells. The term "cytokeratin 20 (CK20)" refers to a type I acidic low-molecular-weight cytokeratin. CK20 is less acidic than other type I keratins and is known to be expressed in limited tissues. CK20 is present in urothelial cells, gastric and intestinal epithelial cells, and Merkel cells of the skin. CK20 can be used as a surface cell marker.

[0068] The term "SATB2" refers to a DNA-binding protein that binds to AT-rich sequences. SATB2 can be used as a colon or rectum marker or a ventral hindgut marker. The term "FOXA2" is an abbreviation for Forkhead box protein A2 and refers to a transcription factor that plays an important role in development. FOXA2 can be used as an early intestinal epithelial marker or a marker for developing bladder epithelial cells.

[0069] The term "P63" refers to a protein that is a homolog of the tumor suppressor gene P53 and has functions such as differentiation, proliferation, and maintenance in epithelia. P63 can be used as a bladder epithelial cell marker or a ventral hindgut / Cloaca marker. The term "ΔNP63" refers to a p63 isoform lacking the N-terminal transactivation domain (TN). ΔNP63 can be used as a bladder epithelial cell marker or a ventral hindgut marker.

[0070] The term "GATA3" refers to a transcription factor that binds to a target DNA sequence consisting of GATA and controls the on / off of genes. GATA3 can be used as a ventral hindgut marker or a bladder epithelial cell marker.

[0071] The term "uroplakin (UPK)" refers to a membrane glycoprotein that is involved in the formation of surface cells of transitional epithelium that forms the urinary tract epithelium and has the effect of enhancing the permeability and barrier function of surface cells. Uroplakins include uroplakins Ia, Ib, II, and III. UPKs include, for example, UPK1A, UPK1B, UPK2, and UPK3, which can be used as bladder epithelial markers or epithelial tissue markers.

[0072] The term "keratin 5 (KRT5)" refers to a protein encoded by the KRT5 gene that dimerizes with keratin 14 to form intermediate filaments that constitute the cytoskeleton of basal epithelial cells. KRT5 can be used as a bladder epithelial marker.

[0073] The expression level of a marker can be measured, for example, by quantitative PCR or immunostaining. The expression level of a marker measured by quantitative PCR may be, for example, the expression level of mRNA encoding a specific marker. The expression level of a marker measured by immunostaining may be, for example, the intensity of a signal (e.g., fluorescence intensity) derived from a substance (e.g., a fluorescent substance) that can generate a signal when directly or indirectly bound to an antibody that binds to a specific marker. A substance that can generate a signal when indirectly bound to an antibody (primary antibody) that binds to a specific marker may be, for example, a substance that can generate a signal when directly bound to a secondary antibody that can bind to the primary antibody. Markers according to the present disclosure can be measured, for example, using commercially available reagents or the primer sets and antibodies described in the Examples herein.

[0074] In the context of organoids, "not substantially expressing" a certain marker means that the expression level of the certain marker is so low that it cannot be characterized by the marker.An organoid that "not substantially expresses" a certain marker may be an organoid in which the expression level of the particular marker in the organoid is reduced to less than 70%, less than 80%, less than 90%, or less than 95% compared to the expression level of the certain marker in a control organoid.

[0075] The "culturing" of cells or organoids is carried out, for example, under known cell culture conditions. Known cell culture conditions include, for example, 37°C, 5% CO ２ , and 20% O ２ The culture may be, for example, static culture, suspension culture, or rotary culture. In the present disclosure, unless otherwise specified, static culture is intended. As the container used for culture, commercially available cell culture containers (e.g., culture dishes, culture well plates, transwells) can be used appropriately.

[0076] A "pharmaceutical composition" includes mesenchymal cells or matured bladder organoids according to the present disclosure, or cells derived from the bladder organoids. The pharmaceutical composition can be used, for example, to treat kidney or bladder damage or disease in mammals. The term "pharmaceutically acceptable carrier" refers to any component other than the mesenchymal cells or matured bladder organoids according to the present disclosure that is highly safe and has low allergic reactivity in mammals. Pharmaceutically acceptable carriers include, for example, aqueous or non-aqueous solvents, solutions (e.g., saline, basal medium, or cell suspension preservative), cryoprotectants (e.g., glycerol), water-soluble polymers (e.g., dextran), or buffers (e.g., phosphate buffer) suitable for pharmaceutical administration. Pharmaceutical compositions can be appropriately prepared according to known methods. Pharmaceutical compositions according to the present disclosure can be prepared, for example, by combining the mesenchymal cells, matured bladder organoids, or cells derived from the bladder organoids with a pharmaceutically acceptable carrier (e.g., basal medium). The pharmaceutical composition includes, for example, the mesenchymal cells according to the present disclosure and a pharmaceutically acceptable carrier. The pharmaceutical composition comprises, for example, the matured bladder organoids disclosed herein or cells derived from the bladder organoids and a pharmaceutically acceptable carrier.

[0077] A "mammal" is, for example, a human or a non-human mammal. A "non-human mammal" or a "non-human mammal" may be, for example, a rodent such as a mouse, rat, guinea pig, or hamster; a non-human primate such as a chimpanzee; an even-toed ungulate such as a cow, goat, or sheep; a perissodactyl such as a horse; or a companion animal such as a rabbit, dog, or cat. A mammal is a human. A non-human mammal is, for example, a rodent or a non-human primate.

[0078] The term "bladder" refers to a sac-like organ that temporarily stores urine delivered from the kidneys. The bladder may be a normal bladder without any particular damage or disease, or may be an injured bladder or a bladder affected by a bladder disease. The bladder may be, for example, a normal bladder. The bladder may be, for example, an injured bladder or a bladder affected by a bladder disease.

[0079] The term "kidney" refers to an organ of the urinary system that filters and excretes waste products or excess water from the blood to produce urine. The kidney may be a normal kidney without any particular damage or disease, or a damaged kidney or a kidney affected by a kidney disease. The kidney may be, for example, a normal kidney. The kidney may be, for example, a damaged kidney or a kidney affected by a kidney disease.

[0080] "Surrounding area" of the kidney or bladder refers to tissue or area adjacent to or near the urinary system. The urinary system includes the kidney, ureter, bladder, and urethra. The surrounding area of ​​the bladder can be, for example, within the abdominal cavity or the mesentery.

[0081] "Bladder damage" or "bladder disease" may be, for example, a bladder damaged by trauma, radiation cystitis, interstitial cystitis, a bladder damaged by diabetes, ischemia, or the like, a bladder damaged by a drug harmful to bladder tissue, cystitis, or bladder cancer. "Kidney damage" or "kidney disease" may be, for example, a kidney damaged by trauma, radiation nephritis, a kidney damaged by ischemia, or the like, a kidney damaged by a drug harmful to kidney tissue, nephritis, or kidney cancer. "Treatment" of kidney or bladder damage or disease includes maintaining, reducing, or eliminating the symptoms or pathology.

[0082] The term "mammal in need of treatment" refers to a mammal having or suspected of having kidney or bladder damage or disease. A mammal having kidney or bladder damage or disease refers to a mammal that has been diagnosed by a medical professional (e.g., a physician) as having kidney or bladder damage or disease according to predetermined diagnostic criteria. A mammal suspected of having kidney or bladder damage or disease may be a mammal suspected of having kidney or bladder damage or disease based on, for example, the mammal's behavioral history (e.g., trauma, radiation therapy, and having or having received drugs harmful to kidney or bladder tissue) or medical history (e.g., having or having suffered from diabetes, ischemia, nephritis, cystitis, or kidney or bladder cancer). The mammal is preferably a human or non-human primate, more preferably a human.

[0083] The term "test substance" may be, for example, a small molecule compound, a protein (e.g., an antibody), DNA, RNA, small interfering RNA, an antisense oligonucleotide, or a bladder organoid (preferably an immature bladder organoid) or a ventral hindgut organoid. The test substance may be, for example, a drug for treating a kidney or bladder disorder or disease, or kidney cancer or bladder cancer, or a candidate substance thereof. The test substance may be, for example, one type, or a mixture of two or more types. The test substance is preferably one type of substance.

[0084] <Mesenchymal cells of ventral hindgut mesoderm and method for producing same> One aspect of the present disclosure provides a method for producing mesenchymal cells of ventral hindgut mesoderm. Another aspect of the present disclosure provides mesenchymal cells of ventral hindgut mesoderm. Another aspect of the present disclosure provides mesenchymal cells of ventral hindgut mesoderm produced according to the production method. The production method includes: (a) culturing pluripotent stem cells in induction medium A containing a TGF-β inhibitor, a Wnt agonist, and a fibroblast growth factor to induce differentiation into epiblast-like cells; (b) culturing the epiblast-like cells in induction medium B containing a Wnt agonist, a fibroblast growth factor, and retinoic acid to induce differentiation into caudal epiblast-like cells; (c) culturing the caudal epiblast-like cells in induction medium C containing a Wnt agonist, a bone morphogenetic protein, and a TGF-β family member to induce differentiation into posterior primitive streak cells; and (d) culturing the posterior primitive streak cells in induction medium D containing a Wnt agonist, a fibroblast growth factor, a bone morphogenetic protein, and a Hedgehog signaling agonist to induce differentiation into mesenchymal cells of the ventral hindgut mesoderm.

[0085] The method for producing mesenchymal cells of the ventral hindgut mesoderm according to this embodiment will be described with reference to Figure 2. In Figure 2b, the period from Day 0 to Day 11 shows the process of producing the mesenchymal cells from human iPS cells. ｌｏｗ Epiblast-like cells were induced to differentiate (Step A: Day 0 to Day 1), and the NANOG ｌｏｗThe method includes inducing differentiation of epiblast-like cells into caudal epiblast-like cells (step B: Day 1 to Day 2), inducing differentiation of the caudal epiblast-like cells into posterior primitive streak cells (step C: Day 2 to Day 5), and inducing differentiation of the posterior primitive streak cells into mesenchymal cells of the ventral hindgut mesoderm (step α and step D: Day 5 to Day 11). The step of inducing differentiation of mesenchymal cells of the ventral hindgut mesoderm includes excluding cells expressing SOX2, SOX3, and FOXD3 from a cell population containing posterior primitive streak cells and selecting cells expressing TBX4, HOXD13, and FOXF1 (step α), and inducing differentiation of the selected cells into mesenchymal cells of the ventral hindgut mesoderm (step D). Each of steps A to D is described in detail below.

[0086] Process A: NANOG from pluripotent stem cells ｌｏｗ Induction of Differentiation into Epiblast-Like Cells Step A comprises culturing pluripotent stem cells in induction medium A containing a TGF-β inhibitor, a Wnt agonist, and a fibroblast growth factor. The culturing induces differentiation into epiblast-like cells. One aspect of the present disclosure provides a method for producing epiblast-like cells, comprising culturing pluripotent cells in induction medium A according to the present disclosure.

[0087] Induction medium A may be, for example, 0.1 to 10 μM, 0.1 to 7.5 μM, 0.1 to 5 μM, 0.1 to 3 μM, 0.1 to 2 μM, 0.1 to 1.5 μM, 0.2 to 10 μM, 0.2 to 7.5 μM, 0.2 to 5 μM, 0.2 to 3 μM, 0.2 to 2 μM, 0.2 to 1.5 μM, 0.3 to 10 μM, 0.3 to 7.5 μM, 0.3 to 5 μM, 0.3 to 3 μM, 0.3 to 2 μM , 0.3 to 1.5 μM, 0.5 to 10 μM, 0.5 to 7.5 μM, 0.5 to 5 μM, 0.5 to 3 μM, 0.5 to 2 μM, 0.5 to 1.5 μM, 0.75 to 10 μM, 0.75 to 7.5 μM, 0.75 to 5 μM, 0.75 to 3 μM, 0.75 to 2 μM, or 0.75 to 1.5 μM, preferably 1 μM, of a TGF-β inhibitor (preferably A83-01).

[0088] Induction medium A may be, for example, 0.1 to 10 μM, 0.1 to 7.5 μM, 0.1 to 5 μM, 0.1 to 3 μM, 0.1 to 2 μM, 0.1 to 1.5 μM, 0.2 to 10 μM, 0.2 to 7.5 μM, 0.2 to 5 μM, 0.2 to 3 μM, 0.2 to 2 μM, 0.2 to 1.5 μM, 0.3 to 10 μM, 0.3 to 7.5 μM, 0.3 to 5 μM, 0.3 to 3 μM, 0.3 to 2 μM, 0.3 to 1.5 μM, 0.5-10 μM, 0.5-7.5 μM, 0.5-5 μM, 0.5-3 μM, 0.5-2 μM, 0.5-1.5 μM, 0.75-10 μM, 0.75-7.5 μM, 0.75-5 μM, 0.75-3 μM, 0.75-2 μM, or 0.75-1.5 μM, preferably 1 μM, of a Wnt agonist (preferably CHIR-99021 or WNT3A).

[0089] Induction medium A may be, for example, 10 to 1000 ng / ml, 10 to 750 ng / ml, 10 to 500 ng / ml, 10 to 300 ng / ml, 10 to 200 ng / ml, 10 to 150 ng / ml, 20 to 1000 ng / ml, 20 to 750 ng / ml, 20 to 500 ng / ml, 20 to 300 ng / ml, 20 to 200 ng / ml, 20 to 150 ng / ml, 30 to 1000 ng / ml, 30 to 750 ng / ml, 30 to 500 ng / ml, 30 to 300 ng / ml, 30 The FGF (preferably FGF2 or FGF4) may be present at up to 200 ng / ml, 30 to 150 ng / ml, 50 to 1000 ng / ml, 50 to 750 ng / ml, 50 to 500 ng / ml, 50 to 300 ng / ml, 50 to 200 ng / ml, 50 to 150 ng / ml, 75 to 1000 ng / ml, 75 to 750 ng / ml, 75 to 500 ng / ml, 75 to 300 ng / ml, 75 to 200 ng / ml, or 75 to 150 ng / ml, preferably 100 ng / ml.

[0090] In step A, pluripotent stem cells are seeded into a culture vessel at about 10 to about 50% confluency, about 10 to about 40% confluency, about 10 to about 30% confluency, about 10 to about 20% confluency, about 20 to about 50% confluency, about 20 to about 40% confluency, or about 20 to about 30% confluency, preferably about 20% confluency. Seeding the pluripotent stem cells so that they reach about 80% confluency after step C (Day 5) described below, or seeding the pluripotent stem cells at about 20% confluency, is convenient because steps A to D can be carried out without detaching the cultured cells from the culture vessel due to the cells having reached confluency. The culture in step A is carried out, for example, for 0.5 to 2 days, 0.5 to 1.5 days, or 0.5 to 1 day, preferably 1 day.

[0091] Process B: NANOG ｌｏｗ Induction of Differentiation of Epiblast-Like Cells into Caudal Epiblast-Like Cells Step B comprises culturing the epiblast-like cells in induction medium B containing a Wnt agonist, a fibroblast growth factor, and retinoic acid. The culturing induces differentiation into caudal epiblast-like cells. One aspect of the present disclosure provides a method for producing caudal epiblast-like cells, comprising culturing pluripotent cells in induction medium A according to the present disclosure to induce differentiation into epiblast-like cells, and culturing the epiblast-like cells in induction medium B according to the present disclosure.

[0092] Induction medium B may be, for example, 0.3 to 30 μM, 0.3 to 20 μM, 0.3 to 10 μM, 0.3 to 7.5 μM, 0.3 to 5 μM, 0.3 to 4 μM, 0.7 to 30 μM, 0.7 to 20 μM, 0.7 to 10 μM, 0.7 to 7.5 μM, 0.7 to 5 μM, 0.7 to 4 μM, 1 to 30 μM, 1 to 20 μM, 1 to 10 μM, 1 to 7.5 μM, 1 to 5 μM, 1 to 4 μM, 1.5 to 30 μM, 1.5 to 20 μM, The Wnt agonist (preferably CHIR-99021 or WNT3A) may be present at 1.5-10 μM, 1.5-7.5 μM, 1.5-5 μM, 1.5-4 μM, 2-30 μM, 2-20 μM, 2-10 μM, 2-7.5 μM, 2-5 μM, 2-4 μM, 2.5-30 μM, 2.5-20 μM, 2.5-10 μM, 2.5-7.5 μM, 2.5-5 μM, or 2.5-4 μM, preferably 3 μM.

[0093] Induction medium B may be, for example, 2 to 200 ng / ml, 2 to 100 ng / ml, 2 to 75 ng / ml, 2 to 50 ng / ml, 2 to 30 ng / ml, 2 to 25 ng / ml, 5 to 200 ng / ml, 5 to 100 ng / ml, 5 to 75 ng / ml, 5 to 50 ng / ml, 5 to 30 ng / ml, 5 to 25 ng / ml, 10 to 200 ng / ml, 10 to 100 ng / ml, 10-75ng / ml, 10-50ng / ml, 10-30ng / ml, 10-25ng / ml, 15-200ng / ml, 15-100ng / ml, 15-75ng / ml, 15-50ng / ml, 15-30ng / ml, or 15-25ng / ml, preferably 20ng / ml, of FGF (preferably FGF2 or FGF4).

[0094] Induction medium B may contain, for example, 10 to 1000 nM, 10 to 750 nM, 10 to 500 nM, 10 to 300 nM, 10 to 200 nM, 10 to 150 nM, 20 to 1000 nM, 20 to 750 nM, 20 to 500 nM, 20 to 300 nM, 20 to 200 nM, 20 to 150 nM, 30 to 1000 nM, 30 to 750 nM, 30 to 500 nM, 30 to 300 nM, 30 retinoic acid (preferably atRA) at up to 200 nM, 30 to 150 nM, 50 to 1000 nM, 50 to 750 nM, 50 to 500 nM, 50 to 300 nM, 50 to 200 nM, 50 to 150 nM, 75 to 1000 nM, 75 to 750 nM, 75 to 500 nM, 75 to 300 nM, 75 to 200 nM, or 75 to 150 nM, preferably 100 nM.

[0095] The culture in step B is carried out for, for example, 0.5 to 2 days, 0.5 to 1.5 days, or 0.5 to 1 day, preferably 1 day.

[0096] Step C: Inducing Differentiation of Caudal Epiblast-Like Cells into Posterior Primitive Streak Cells Step C comprises culturing the caudal epiblast-like cells in induction medium C containing a Wnt agonist, a bone morphogenetic protein, and a TGF-β family member. The culturing induces differentiation into posterior primitive streak cells. One aspect of the present disclosure provides a method for producing posterior primitive streak cells, comprising culturing pluripotent stem cells in induction medium A according to the present disclosure to induce their differentiation into epiblast-like cells; culturing the epiblast-like cells in induction medium B according to the present disclosure to induce their differentiation into caudal epiblast-like cells; and culturing the caudal epiblast-like cells in induction medium C according to the present disclosure.

[0097] Induction medium C contains, for example, 0.6 to 60 μM, 0.6 to 30 μM, 0.6 to 20 μM, 0.6 to 10 μM, 0.6 to 8 μM, 0.6 to 7 μM, 1.5 to 60 μM, 1.5 to 30 μM, 1.5 to 20 μM, 1.5 to 10 μM, 1.5 to 8 μM, 1.5 to 7 μM, 3 to 60 μM, 3 to 30 μM, 3 to 20 μM, 3 to 10 μM, 3 to 8 μM, 3 to 7 μM, 4.5 to 60 μM, 4.5 to 30 μM, 4.5 to 20 μM, 4.5 to 10 μM, 4.5 to 8 μM, or 4.5 to 7 μM, preferably 6 μM, of a Wnt agonist (preferably CHIR-99021 or WNT3A).

[0098] Induction medium C may be, for example, 5 to 500 ng / ml, 5 to 250 ng / ml, 5 to 150 ng / ml, 5 to 100 ng / ml, 5 to 75 ng / ml, 5 to 60 ng / ml, 20 to 500 ng / ml, 20 to 250 ng / ml, 20 to 150 ng / ml, 20 to 100 ng / ml, 20 to 75 ng / ml, 20 to 60 ng / ml, 10 to 500 ng / ml, 30 BMP (preferably BMP4) at up to 250 ng / ml, 30-150 ng / ml, 30-100 ng / ml, 30-75 ng / ml, 30-60 ng / ml, 40-500 ng / ml, 40-250 ng / ml, 40-150 ng / ml, 40-100 ng / ml, 40-75 ng / ml, or 40-60 ng / ml, preferably 50 ng / ml.

[0099] Induction medium C may be, for example, 3 to 300 ng / ml, 3 to 150 ng / ml, 3 to 100 ng / ml, 3 to 75 ng / ml, 3 to 50 ng / ml, 3 to 40 ng / ml, 7 to 300 ng / ml, 7 to 150 ng / ml, 7 to 100 ng / ml, 7 to 75 ng / ml, 7 to 50 ng / ml, 7 to 40 ng / ml, 15 to 200 ng / ml, 15 to 150 ng / ml, 15 to 100 ng / ml, 15-75 ng / ml, 15-50 ng / ml, 15-40 ng / ml, 15-200 ng / ml, 20-150 ng / ml, 20-100 ng / ml, 20-75 ng / ml, 20-50 ng / ml, or 20-40 ng / ml, preferably 30 ng / ml of a TGF-β family member (preferably GDF11, activin A, or Nodal).

[0100] The culture in step C is carried out for, for example, 1 to 7 days, 1 to 6 days, 1 to 5 days, 1 to 4 days, 2 to 7 days, 2 to 6 days, 2 to 5 days, 2 to 4 days, 3 to 7 days, 3 to 6 days, 3 to 5 days, or 3 to 4 days, preferably 3 days.

[0101] Step α: Selection of Cells Expressing TBX4, HOXD13, and FOXF1 Step α involves culturing a cell population containing posterior primitive streak cells in a selection medium α containing BMP4 and a ROCK inhibitor, which eliminates cells expressing SOX2, SOX3, and FOXD3, resulting in the selective proliferation of cells expressing TBX4, HOXD13, and FOXF1.

[0102] The selective medium α may be, for example, 3 to 300 ng / ml, 3 to 150 ng / ml, 3 to 100 ng / ml, 3 to 75 ng / ml, 3 to 50 ng / ml, 3 to 40 ng / ml, 7 to 300 ng / ml, 7 to 150 ng / ml, 7 to 100 ng / ml, 7 to 75 ng / ml, 7 to 50 ng / ml, 7 to 40 ng / ml, 15 to 200 ng / ml, 15 to 15 The composition contains 0 ng / ml, 15-100 ng / ml, 15-75 ng / ml, 15-50 ng / ml, 15-40 ng / ml, 15-200 ng / ml, 20-150 ng / ml, 20-100 ng / ml, 20-75 ng / ml, 20-50 ng / ml, or 20-40 ng / ml, preferably 30 ng / ml of BMP (preferably BMP4).

[0103] The selective medium α may contain, for example, 1 to 100 μM, 1 to 75 μM, 1 to 50 μM, 1 to 30 μM, 1 to 20 μM, 1 to 15 μM, 2 to 100 μM, 2 to 75 μM, 2 to 50 μM, 2 to 30 μM, 2 to 20 μM, 2 to 15 μM, 3 to 100 μM, 3 to 75 μM, 3 to 50 μM, 3 to 30 μM, 3 to 20 μM, 3 to 15 μM, 5 to The compound contains a ROCK inhibitor (preferably Y-27632 or Y-27632) at 100 μM, 5 to 75 μM, 5 to 50 μM, 5 to 30 μM, 5 to 20 μM, 5 to 15 μM, 7.5 to 100 μM, 7.5 to 75 μM, 7.5 to 50 μM, 7.5 to 30 μM, 7.5 to 20 μM, or 7.5 to 15 μM, preferably 10 μM.

[0104] The culture in step α is carried out for, for example, 0.5 to 2 days, 0.5 to 1.5 days, 0.5 to 1 day, preferably 1 day.

[0105] Step D: Inducing differentiation of posterior primitive streak cells into mesenchymal cells of the ventral hindgut mesoderm Step D comprises culturing the posterior primitive streak cells in an induction medium D containing a Wnt agonist, a fibroblast growth factor, a bone morphogenetic protein, and a Hedgehog signaling agonist. The culture induces differentiation into mesenchymal cells of the ventral hindgut mesoderm. Induction medium D may further contain retinoic acid.

[0106] Induction medium D may contain, for example, 0.1 to 10 μM, 0.1 to 7.5 μM, 0.1 to 5 μM, 0.1 to 3 μM, 0.1 to 2 μM, 0.1 to 1.5 μM, 0.2 to 10 μM, 0.2 to 7.5 μM, 0.2 to 5 μM, 0.2 to 3 μM, 0.2 to 2 μM, 0.2 to 1.5 μM, 0.3 to 10 μM, 0.3 to 7.5 μM, 0.3 to 5 μM, 0.3 to 3 μM, 0.3 to 2 μM, 0.3 to 1.5 μM, 0.5-10 μM, 0.5-7.5 μM, 0.5-5 μM, 0.5-3 μM, 0.5-2 μM, 0.5-1.5 μM, 0.75-10 μM, 0.75-7.5 μM, 0.75-5 μM, 0.75-3 μM, 0.75-2 μM, or 0.75-1.5 μM, preferably 1 μM, of a Wnt agonist (preferably CHIR-99021 or WNT3A).

[0107] Induction medium D may be, for example, 1 to 100 ng / ml, 1 to 75 ng / ml, 1 to 50 ng / ml, 1 to 30 ng / ml, 1 to 20 ng / ml, 1 to 15 ng / ml, 2 to 100 ng / ml, 2 to 75 ng / ml, 2 to 50 ng / ml, 2 to 30 ng / ml, 2 to 20 ng / ml, 2 to 15 ng / ml, 3 to 100 ng / ml, 3 to 75 ng / ml, 3 to 50 ng / ml, 3 to 30 ng / ml, 3 to 20 ng / ml , 3 to 15 ng / ml, 5 to 100 ng / ml, 5 to 75 ng / ml, 5 to 50 ng / ml, 5 to 30 ng / ml, 5 to 20 ng / ml, 5 to 15 ng / ml, 7.5 to 100 ng / ml, 7.5 to 75 ng / ml, 7.5 to 50 ng / ml, 7.5 to 30 ng / ml, 7.5 to 20 ng / ml, or 7.5 to 15 ng / ml, preferably 10 ng / ml, of FGF (preferably FGF2 or FGF4).

[0108] Induction medium D may be, for example, 3 to 300 ng / ml, 3 to 150 ng / ml, 3 to 100 ng / ml, 3 to 75 ng / ml, 3 to 50 ng / ml, 3 to 40 ng / ml, 7 to 300 ng / ml, 7 to 150 ng / ml, 7 to 100 ng / ml, 7 to 75 ng / ml, 7 to 50 ng / ml, 7 to 40 ng / ml, 15 to 200 ng / ml, 15 to 15 The composition contains 0 ng / ml, 15-100 ng / ml, 15-75 ng / ml, 15-50 ng / ml, 15-40 ng / ml, 15-200 ng / ml, 20-150 ng / ml, 20-100 ng / ml, 20-75 ng / ml, 20-50 ng / ml, or 20-40 ng / ml, preferably 30 ng / ml of BMP (preferably BMP4).

[0109] Induction medium D may contain, for example, 0.01 to 1 μM, 0.01 to 0.75 μM, 0.01 to 0.5 μM, 0.01 to 0.3 μM, 0.01 to 0.2 μM, 0.01 to 0.15 μM, 0.02 to 1 μM, 0.02 to 0.75 μM, 0.02 to 0.5 μM, 0.02 to 0.3 μM, 0.02 to 0.2 μM, 0.02 to 0.15 μM, 0.03 to 1 μM, 0.03 to 0.75 μM, 0.03 to 0.5 μM, 0.03 to 0.3 μM, 0.03 to 0. 2 μM, 0.03-0.15 μM, 0.5-1 μM, 0.05-0.75 μM, 0.05-0.5 μM, 0.05-0.3 μM, 0.05-0.2 μM, 0.05-0.15 μM, 0.075-1 μM, 0.075-0.75 μM, 0.075-0.5 μM, 0.075-0.3 μM, 0.075-0.2 μM, or 0.075-0.15 μM, preferably 0.1 μM, of a hedgehog agonist (e.g., Hh-Ag1.5).

[0110] Induction medium D may contain, for example, 10 to 1000 nM, 10 to 750 nM, 10 to 500 nM, 10 to 300 nM, 10 to 200 nM, 10 to 150 nM, 20 to 1000 nM, 20 to 750 nM, 20 to 500 nM, 20 to 300 nM, 20 to 200 nM, 20 to 150 nM, 30 to 1000 nM, 30 to 750 nM, 30 to 500 nM, 30 to 300 nM, 30 to 200 nM,

[0040] The composition may further comprise retinoic acid (preferably atRA) at a concentration of 00 nM, 30-150 nM, 50-1000 nM, 50-750 nM, 50-500 nM, 50-300 nM, 50-200 nM, 50-150 nM, 75-1000 nM, 75-750 nM, 75-500 nM, 75-300 nM, 75-200 nM, or 75-150 nM, preferably 100 nM.

[0111] The culture in step D is carried out for, for example, 3 to 12 days, 3 to 10 days, 3 to 9 days, 3 to 8 days, 3 to 7 days, 4 to 12 days, 4 to 10 days, 4 to 9 days, 4 to 8 days, 4 to 7 days, 5 to 12 days, 5 to 10 days, 5 to 9 days, 5 to 8 days, or 5 to 7 days, preferably 6 days.

[0112] <Pharmaceutical composition comprising mesenchymal cells and method for producing same> One aspect of the present disclosure provides a pharmaceutical composition comprising the mesenchymal cells of the present disclosure. The pharmaceutical composition further comprises a pharmaceutically acceptable carrier. The pharmaceutical composition can be used, for example, to treat damage or disease of the kidney or bladder or surrounding areas in a mammal. The bladder damage or disease is, for example, interstitial cystitis.

[0113] The pharmaceutical composition is administered to a mammal in need thereof, for example, by surgically transplanting it into the kidney, bladder, or a nearby site, or by injecting it into the kidney, bladder, or a nearby site using an instrument such as a syringe. From the viewpoint of reducing graft rejection, the mammal to which the pharmaceutical composition is administered and the animal species of the pluripotent stem cells used to produce the mesenchymal cells contained as an active ingredient in the pharmaceutical composition are preferably the same species, and more preferably the same individual.

[0114] Another aspect of the present disclosure provides a method for producing the pharmaceutical composition. The production method includes, for example, combining mesenchymal cells according to the present disclosure with a pharmaceutically acceptable carrier. The production method includes, for example, mixing mesenchymal cells according to the present disclosure with a pharmaceutically acceptable carrier.

[0115] <Method for treating kidney or bladder disorders or diseases> One aspect of the present disclosure provides a method for treating kidney or bladder damage or disease in a mammal. The treatment method comprises introducing mesenchymal cells according to the present disclosure or a pharmaceutical composition containing the mesenchymal cells into the kidney or bladder or a surrounding area of ​​a mammal in need thereof. From the viewpoint of reducing transplant rejection, it is preferable that the animal species of the pluripotent stem cells used to produce the mesenchymal cells or the mesenchymal cells contained as an active ingredient in the pharmaceutical composition is the same species or the same individual mammal in need thereof. The bladder damage or disease is, for example, interstitial cystitis.

[0116] <Non-human mammal having muscle cells derived from mesenchymal cells, and method for producing same> One aspect of the present disclosure provides a non-human mammal having muscle cells derived from mesenchymal cells according to the present disclosure in the kidney, bladder, or a surrounding area. The non-human mammal, for example, has mesenchymal cells according to the present disclosure and muscle cells derived from the mesenchymal cells in the kidney, bladder, or a surrounding area. The non-human mammal can be used, for example, in a method for evaluating responsiveness to a test substance.

[0117] Another aspect of the present disclosure provides a method for producing the non-human mammal. The production method includes, for example, introducing mesenchymal cells according to the present disclosure into the kidney or bladder of a non-human mammal, or a surrounding area thereof. The production method includes, for example, raising the non-human mammal. Raising the non-human mammal further includes, for example, feeding the non-human mammal with a known diet. In a non-human mammal produced according to the production method, a portion of the mesenchymal cell population introduced into a predetermined area is induced to differentiate into muscle cells. A non-human mammal produced by the production method has, for example, the mesenchymal cells according to the present disclosure and muscle cells derived from the mesenchymal cells in the kidney or bladder, or a surrounding area thereof.

[0118] <Method of evaluation in mesenchymal cells or muscle cells derived from mesenchymal cells> One aspect of the present disclosure provides a method of evaluating the responsiveness of mesenchymal cells according to the present disclosure to a test substance, the method comprising contacting the test substance with mesenchymal cells according to the present disclosure and detecting a change in the test substance or the mesenchymal cells.

[0119] "Contacting" a test substance with the mesenchymal cells means placing the test substance and the mesenchymal cells according to the present disclosure under conditions that allow them to come into contact with each other. Contacting the test substance with the mesenchymal cells may, for example, involve mixing the test substance with a culture medium containing the mesenchymal cells.

[0120] The change in the mesenchymal cells includes, for example, a structural or functional change in the mesenchymal cells. The structural or functional change in the mesenchymal cells includes, for example, differentiation of the mesenchymal cells into muscle cells. The change in the test substance includes, for example, a structural or functional change in the test substance. The structural or functional change in the test substance includes, for example, destruction of structure, binding with other substances, or inhibition or alteration of function. The structural or functional change in the test substance includes, for example, a change in the concentration of the test substance by the mesenchymal cells. When the test substance is a ventral hindgut organoid or a bladder organoid, the change includes maturation into a bladder organoid or further maturation of the bladder organoid. The change in the test substance may include, for example, detecting the presence or absence of a change or the degree of change in the test substance under the same conditions except that the mesenchymal cells according to the present disclosure are not used, and comparing the detection results (changes).

[0121] One aspect of the present disclosure provides a method for evaluating the responsiveness of mesenchymal cells or muscle cells in a non-human mammal according to the present disclosure to a test substance, the method comprising contacting the test substance with a non-human mammal having mesenchymal cells according to the present disclosure or muscle cells derived from the mesenchymal cells in the kidney or bladder or a site thereof, and detecting a change in the test substance or the mesenchymal cells or muscle cells.

[0122] "Contacting" a test substance with a non-human mammal having mesenchymal cells according to the present disclosure or muscle cells derived from the mesenchymal cells means placing the test substance under conditions that allow contact between the mesenchymal cells or muscle cells in the non-human mammal. Contacting the test substance with the mesenchymal cells or muscle cells may be, for example, oral or parenteral administration of the test substance to the non-human mammal.

[0123] The change in the mesenchymal cells or muscle cells may include, for example, a structural or functional change in the mesenchymal cells or muscle cells. The structural or functional change in the mesenchymal cells may include, for example, differentiation of the mesenchymal cells into muscle cells. The change in the test substance may include, for example, a structural or functional change in the test substance. The structural or functional change in the test substance may include, for example, destruction of structure, binding with other substances, or inhibition or alteration of function. The structural or functional change in the test substance may include, for example, a change in the concentration of the test substance by the mesenchymal cells. When the test substance is a ventral hindgut organoid or a bladder organoid, the change may include maturation into a bladder organoid or further maturation of the bladder organoid. The change in the test substance may include, for example, detecting the presence or absence of a change or the degree of change in the test substance under the same conditions except that the mesenchymal cells according to the present disclosure are not used, and comparing the detection results (changes).

[0124] <Method for maturing ventral hindgut organoids or bladder organoids, or method for producing matured bladder organoids> One aspect of the present disclosure provides a method for maturing ventral hindgut organoids or bladder organoids. Another method of the present disclosure provides a method for producing matured bladder organoids. The maturation method and the production method comprise culturing ventral hindgut organoids or bladder organoids in an induction medium containing retinol and retinoic acid in the presence of mesenchymal cells according to the present disclosure.

[0125] The culture of ventral hindgut organoids or bladder organoids in the presence of mesenchymal cells according to the present disclosure preferably includes co-culturing the mesenchymal cells and the organoids so as to achieve stable contact between the mesenchymal cells and the organoids, for example, by co-existing the mesenchymal cells and the organoids in an extracellular matrix.

[0126] The organoids can be cultured in the presence of mesenchymal cells in an extracellular matrix (ECM) gel.The organoids can be cultured in the presence of mesenchymal cells, for example, by forming an extracellular matrix (ECM) gel containing the mesenchymal cells and the organoids.The formation of the ECM gel can be, for example, by forming an ECM gel containing a first ECM gel containing the mesenchymal cells and a second ECM gel containing the organoids.The formation of the ECM gel can be, for example, by preparing a cell suspension containing the mesenchymal cells and the organoids, and forming an ECM gel containing the cell suspension.

[0127] The organoids can be cultured in the presence of mesenchymal cells in a container (e.g., a transwell) containing a second compartment separated from a first compartment by a porous member. The organoids can be cultured in the presence of mesenchymal cells by introducing the mesenchymal cells and the organoids into the first compartment (e.g., dispensing them using a dispensing device such as a pipette) and introducing an induction medium containing retinol (e.g., atROL) and retinoic acid (e.g., atRA) into the second compartment. The mesenchymal cells and organoids introduced into the first compartment may be present in an ECM gel. Culturing the organoids in the presence of the mesenchymal cells includes, for example, introducing an ECM (e.g., 50% Matrigel) containing the mesenchymal cells into a first compartment to form a first ECM gel, introducing an ECM (e.g., 50% Matrigel) containing the organoids onto the first ECM gel in the first compartment to form a second ECM gel, and introducing an induction medium containing retinol (e.g., atROL) and retinoic acid (e.g., atRA) into a second compartment.

[0128] The term "porous member" refers to a member having a large number of pores that can allow a polymer compound to pass through. The polymer compound may be, for example, retinoic acid or retinol. The porous member may have voids that are not in communication with the external space. Examples of the porous member include filter paper, ultrafiltration membranes, nonwoven fabrics, meshes (e.g., gauze-like meshes), porous membranes, and membrane filters. The porous member may be formed from, for example, silicone, nylon, polyvinylidene fluoride, acetyl cellulose, nitrocellulose, polyethylene terephthalate, polycarbonate, polyester (PET), or polytetrafluoroethylene (PTFE).

[0129] Culturing the organoids in the presence of the mesenchymal cells includes, for example, maintaining the mesenchymal cells and the organoids in an induction medium containing retinol (e.g., atROL) and retinoic acid (e.g., atRA). The mesenchymal cells and the organoids are preferably present in an ECM gel.

[0130] The culturing period (also referred to as the "first culturing period") may be, for example, 1 to 5 weeks, 1 to 4 weeks, 1 to 3 weeks, 2 to 5 weeks, 2 to 4 weeks, preferably 2 to 3 weeks. During the first culturing period, the induction culture is preferably replaced with fresh induction medium every 2 to 4 days.

[0131] The induction medium used during the first culture period may contain, for example, 0.1 to 10 μM, 0.1 to 7.5 μM, 0.1 to 5 μM, 0.1 to 3 μM, 0.1 to 2 μM, 0.1 to 1.5 μM, 0.3 to 10 μM, 0.3 to 7.5 μM, 0.3 to 5 μM, 0.3 to 3 μM, 0.3 to 2 μM, 0.3 to 1.5 μM, 0.5 to 10 μM, 0.5 to 7.5 μM, 0.5 to 5 μM, 0.5 to 3 μM, 0.5 to 2 μM, 0.5 to 1.5 μM, 0.7 Retinol (e.g., atROL) at concentrations of up to 10 μM, 0.7-7.5 μM, 0.7-5 μM, 0.7-3 μM, 0.7-2 μM, 0.7-1.5 μM, 0.8-10 μM, 0.8-7.5 μM, 0.8-5 μM, 0.8-3 μM, 0.8-2 μM, 0.8-1.5 μM, 0.9-10 μM, 0.9-7.5 μM, 0.9-5 μM, 0.9-3 μM, 0.9-2 μM, 0.9-1.5 μM, and preferably 1 μM.

[0132] The induction medium used during the first culture period may contain, for example, 0.1 to 10 nM, 0.1 to 7.5 nM, 0.1 to 5 nM, 0.1 to 3 nM, 0.1 to 2 nM, 0.1 to 1.5 nM, 0.3 to 10 nM, 0.3 to 7.5 nM, 0.3 to 5 nM, 0.3 to 3 nM, 0.3 to 2 nM, 0.3 to 1.5 nM, 0.5 to 10 nM, 0.5 to 7.5 nM, 0.5 to 5 nM, 0.5 to 3 nM, 0.5 to 2 nM, 0.5 to 1.5 nM, 0.7 0.9-10 nM, 0.9-7.5 nM, 0.9-5 nM, 0.9-3 nM, 0.9-2 nM, 0.9-1.5 nM, 0.8-10 nM, 0.8-7.5 nM, 0.8-5 nM, 0.8-3 nM, 0.8-2 nM, 0.8-1.5 nM, 0.9-10 nM, 0.9-7.5 nM, 0.9-5 nM, 0.9-3 nM, 0.9-2 nM, 0.9-1.5 nM, preferably 1 nM of retinoic acid (e.g. atRA).

[0133] Culturing the organoids in the presence of the mesenchymal cells further comprises culturing the mesenchymal cells and the organoids in an induction medium containing retinol (e.g., atROL) after the first culture period. The culture period after the first culture period (also referred to as the "second culture period") may be, for example, 0.5 to 4 weeks, 0.5 to 3 weeks, 0.5 to 2 weeks, 1 to 4 weeks, 1 to 3 weeks, or preferably 1 to 2 weeks. During the second culture period, the induction medium is preferably replaced with fresh induction medium every 2 to 4 days.

[0134] The induction medium used in the second culture period may contain, for example, 10 to 1000 nM, 10 to 750 nM, 10 to 500 nM, 10 to 300 nM, 10 to 200 nM, 10 to 150 nM, 30 to 1000 nM, 30 to 750 nM, 30 to 500 nM, 30 to 300 nM, 30 to 200 nM, 30 to 150 nM, 50 to 1000 nM, 50 to 750 nM, 50 to 500 nM, 50 to 300 nM, 50 to 200 nM, 50 to 150 nM, 70 to 1 000 nM, 70-750 nM, 70-500 nM, 70-300 nM, 70-200 nM, 70-150 nM, 80-1000 nM, 80-750 nM, 80-500 nM, 80-300 nM, 80-200 nM, 80-150 nM, 90-1000 nM, 90-750 nM, 90-500 nM, 90-300 nM, 90-200 nM, 90-150 nM, preferably 100 nM of retinol (e.g., atROL).

[0135] When the produced matured bladder organoids are present in ECM gel, they can be removed using, for example, Gentle Cell Dissociation Reagent. The matured bladder organoids in the ECM gel can be removed using, for example, tweezers and a scalpel under a microscope.

[0136] The matured bladder organoid produced according to the production method further comprises, for example, a lumen defined by a superficial cell layer, and a mesenchymal cell layer between a basal cell layer and a muscle cell layer.The matured bladder organoid comprises, for example, a lumen, and comprises a superficial cell layer (first cell layer) facing the lumen; an intermediate cell layer (second cell layer) located in the outer direction relative to the superficial cell layer; a basal cell layer (third cell layer) located in the outer direction relative to the intermediate cell layer; a mesenchymal cell layer (fourth cell layer) located in the outer direction relative to the basal cell layer; and a muscle cell layer (fifth cell layer) located in the outer direction relative to the mesenchymal cell layer, wherein the mesenchymal cell layer comprises the mesenchymal cells of the present disclosure, and the muscle cell layer comprises muscle cells derived from the mesenchymal cells of the present disclosure.

[0137] <Pharmaceutical composition comprising matured bladder organoids and its manufacturing method> One aspect of the present disclosure provides a pharmaceutical composition comprising the matured bladder organoids of the present disclosure or cells derived from the bladder organoids.The pharmaceutical composition further comprises a pharmaceutically acceptable carrier.The pharmaceutical composition can be used to treat bladder injury or disease in mammals.

[0138] The pharmaceutical composition is administered to a mammalian animal in need thereof, for example, by surgically transplanting it into the bladder or its surrounding area, or by injecting it into the bladder or its surrounding area using an instrument such as a syringe. From the viewpoint of reducing graft rejection, the mammalian animal to which the pharmaceutical composition is administered and the animal species of the pluripotent stem cells, ventral hindgut organoids, or bladder organoids (preferably immature bladder organoids) used to produce the matured bladder organoids contained in the pharmaceutical composition as an active ingredient are preferably the same species, and more preferably the same individual.

[0139] The cells derived from the matured bladder organoid of the present disclosure can be prepared, for example, by carrying out the operation of substantially isolating cells on the bladder organoid.The cells derived from the bladder organoid are, for example, a substantially isolated cell population.The term "substantially isolated cells" includes cells that exist individually and are not connected to other cells.The substantially isolated cell population, for example, comprises at least 50% of the cells that exist individually and are not connected to other cells per total cell of the cell population.The substantially isolated cell population does not exclude that all of the progenitor cells of the cell population exist individually and are not connected to other cells.

[0140] The cells derived from the bladder organoids can be prepared, for example, by subjecting the bladder organoids of the present disclosure to a cell dissociation treatment. Subjecting the bladder organoids to a cell dissociation treatment can include, for example, incubating the bladder organoids with a cell dissociation enzyme such as trypsin or TrypLE™ (e.g., TrypLE™ Express or TrypLE™ Select). By subjecting the bladder organoids to a cell dissociation treatment, a substantially isolated cell population can be obtained. The proportion of cells derived from the bladder organoids that exist individually and not linked to other progenitor cells can be adjusted by the time the bladder organoids are treated with a cell detachment agent, the type and concentration of the cell detachment agent, and the extent of the treatment of the bladder organoids treated with the cell detachment agent, such as by pipetting, to separate the cells. The cells derived from the bladder organoids can be prepared, for example, by subjecting the bladder organoids to cell dissociation treatment and then passing them through a cell strainer to remove impurities and cell clumps.

[0141] Another aspect of the present disclosure provides a method for producing the pharmaceutical composition. The method comprises combining the matured bladder organoids of the present disclosure or cells derived from the bladder organoids with a pharmaceutically acceptable carrier. The method comprises, for example, mixing the mesenchymal cells of the present disclosure with a pharmaceutically acceptable carrier.

[0142] <Method for treating bladder damage or disease> One aspect of the present disclosure provides a method for treating bladder damage or disease in a mammal. The treatment method comprises introducing the matured bladder organoids of the present disclosure, cells derived from the bladder organoids, or the pharmaceutical composition into the bladder or its surrounding area of ​​a mammal in need thereof. From the viewpoint of reducing transplant rejection, it is preferable that the animal species of the pluripotent stem cells, ventral hindgut organoids, or bladder organoids used to produce the matured bladder organoids, cells derived from the bladder organoids, or the pharmaceutical composition is the same species or individual as the mammal in need thereof.

[0143] <Non-human mammal having matured bladder organoids and method for producing the same> One aspect of the present disclosure provides a non-human mammal having the matured bladder organoids of the present disclosure in the bladder or its surrounding area. The non-human mammal can be used, for example, in a method for evaluating the matured bladder organoids in response to a test substance.

[0144] Another aspect of the present disclosure provides a method for producing the non-human mammal. The method comprises introducing the matured bladder organoids of the present disclosure into the bladder of a non-human mammal or a site surrounding the bladder. The method further comprises, for example, raising the non-human mammal. Raising the non-human mammal comprises, for example, feeding the non-human mammal with a known diet.

[0145] In one example, by mixing tumor cells or tumor fragments into the matured bladder organoids according to the present disclosure when producing them, matured bladder organoids containing the tumor cells or tumor fragments can be produced. By introducing the matured bladder organoids into the bladder or its surrounding area of ​​a non-human mammal, a non-human mammal having bladder epithelial tissue in its bladder or its surrounding area as a bladder epithelial cancer model can be produced. A non-human mammal having bladder epithelial tissue in its bladder or its surrounding area as a bladder epithelial cancer model can be used in a method for evaluating the effectiveness of a candidate therapeutic agent for bladder epithelial cancer, which includes contacting the non-human mammal with a candidate therapeutic agent for bladder epithelial cancer and evaluating the effectiveness of the candidate therapeutic agent.

[0146] <Method for evaluating the responsiveness of matured bladder organoids> One aspect of the present disclosure provides a method for evaluating the responsiveness of matured bladder organoids of the present disclosure to test substance.Method includes, for example, contacting the matured bladder organoids of the present disclosure with test substance, and detecting the change in the matured bladder organoids in response to the test substance.Method includes, for example, contacting the non-human mammal with the matured bladder organoids of the present disclosure with test substance, and detecting the change in the matured bladder organoids in response to the test substance.

[0147] "Contacting" the matured bladder organoids or the non-human mammal with a test substance means placing the matured bladder organoids or the non-human mammal under conditions that allow contact between the test substance and the matured bladder organoids or the non-human mammal. Contacting the matured bladder organoids with a test substance may, for example, be mixing the test substance with a culture medium containing the matured bladder organoids. Contacting the non-human mammal with a test substance may, for example, be orally or parenterally administering the test substance to the non-human mammal. The change in the matured bladder organoids in response to the test substance includes, for example, structural or functional changes in the matured bladder organoids. The change includes, for example, a change in the concentration of the test substance caused by the matured bladder organoids.

[0148] <Method for evaluating responsiveness in mesenchymal cells of ventral hindgut mesoderm, muscle cells, and matured bladder organoids according to the present disclosure> As described above, one aspect of the present disclosure provides a method for evaluating responsiveness in mesenchymal cells of ventral hindgut mesoderm, muscle cells, and matured bladder organoids according to the present disclosure. Specifically, this aspect provides a method for evaluating responsiveness to a test substance, comprising contacting the test substance with mesenchymal cells of ventral hindgut mesoderm, muscle cells, or matured bladder organoids according to the present disclosure, and detecting changes in the test substance, the mesenchymal cells, the muscle cells, or the matured bladder organoids.

[0149] The mesenchymal cells of the ventral hindgut mesoderm are produced, for example, according to the method for producing mesenchymal cells disclosed herein. The mesenchymal cells of the ventral hindgut mesoderm are identified, for example, by the expression of a predetermined marker disclosed herein (e.g., a combination of FOXF1, ISL1, TBX4, and HOXD13, and optionally at least one selected from the group consisting of HOXD10, HOXD11, and HOXD12). The mesenchymal cells of the ventral hindgut mesoderm are present, for example, in the kidney or bladder of a non-human mammal disclosed herein, or in areas surrounding these. The non-human mammal is produced, for example, according to the method for producing the non-human mammal disclosed herein. Contacting the test substance with the mesenchymal cells of the ventral hindgut mesoderm includes placing the test substance under conditions that allow contact between the test substance and the mesenchymal cells. For example, when the mesenchymal cells are present in the kidney or bladder of a non-human mammal or in a site surrounding the kidney or bladder, contacting the mesenchymal cells with the test substance includes orally or parenterally administering the test substance to the non-human mammal. In this context, a change in the test substance includes, for example, a structural or functional change in the test substance caused by contact with the mesenchymal cells.

[0150] The muscle cells are, for example, derived from mesenchymal cells of the ventral hindgut mesoderm according to the present disclosure. The muscle cells are, for example, produced according to the method for producing a non-human mammal having muscle cells according to the present disclosure. The muscle cells are, for example, present in the kidney or bladder of a non-human mammal according to the present disclosure, or in a region surrounding these, and are derived from mesenchymal cells of the ventral hindgut mesoderm. Contacting a test substance with muscle cells includes placing the test substance under conditions that allow contact between the muscle cells. For example, when the muscle cells are present in the kidney or bladder of a non-human mammal, or in a region surrounding these, contacting the muscle cells with the test substance includes orally or parenterally administering the test substance to the non-human mammal. In this context, a change in the test substance includes, for example, a structural or functional change in the test substance caused by contact with the muscle cells.

[0151] The matured bladder organoids are produced, for example, according to the method for maturing ventral hindgut organoids or bladder organoids disclosed herein.The matured bladder organoids, for example, comprise predetermined cell layers (e.g., a superficial cell layer, an intermediate cell layer located outward relative to the superficial cell layer, a basal cell layer located outward relative to the intermediate cell layer, and a muscle cell layer located outward relative to the basal cell layer) disclosed herein.The matured bladder organoids are, for example, present in the kidney or bladder of a non-human mammal or their surrounding areas disclosed herein.Contacting the matured bladder organoids with a test substance comprises placing the test substance and the matured bladder organoids under conditions that allow them to come into contact with each other. For example, when the matured bladder organoid is present in the kidney or bladder or their surrounding areas of non-human mammal, the contact between the test substance and the matured bladder organoid comprises administering the test substance orally or parenterally to the non-human mammal.In this context, the change in the test substance comprises, for example, the structural or functional change of the test substance that is caused by contact with the matured bladder organoid.

[0152] In this specification, ordinal numbers such as first and second are used for the purpose of conveniently distinguishing between substances and spaces having the same or similar structures or properties. The ordinal numbers do not in any way limit the structures or properties.

[0153] The term "comprising" means that the recited elements and / or steps are present, and that other elements and / or steps may be added. The term "consisting of" means that the recited elements and / or steps are present, and that other elements and / or steps are excluded. The term "consisting essentially of" means that the recited elements and / or steps are present, and that other elements and / or steps may be added to the extent that they do not adversely affect the technical features of the present disclosure. As used herein, the term "substantially free" does not exclude "completely free."

[0154] The terms and descriptions referred to in this disclosure for a particular aspect or embodiment also apply to other aspects or embodiments as appropriate, unless expressly stated otherwise.

[0155] Specific examples will be described below, but they are intended to illustrate preferred embodiments of the present invention and are not intended to limit the invention described in the appended claims in any way.

[0156] (Materials and Methods) Maintenance of human embryonic stem cells (hESCs) hESCs (KthES15 strain) were obtained from the Human Embryonic Stem Cell Research Center, Kyoto University. hESCs were maintained on iMatrix-511 silk (Nippi Corporation) in StemFit AK02N (Ajinomoto Co., Inc.). hESCs were detached with 5 mM EDTA / PBS and seeded on iMatrix-511 silk in StemFit AK02N supplemented with 10 μM Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.) on the first day. Every 6–7 days, the hESC population was divided into several subpopulations. The medium was changed every 1–2 days.

[0157] Immunofluorescence staining (IF) Cells cultured on plates were fixed with 4% paraformaldehyde (PFA) for 15 minutes at room temperature (RT), washed with PBS, and stored in PBS at 4°C until use. Fixed cells were blocked for 30 minutes at room temperature in a blocking buffer containing 10% donkey serum and 0.1% Triton X in PBS. The cells were incubated with primary antibodies in blocking buffer for 3 hours at room temperature, washed with PBS, and then incubated with secondary antibodies and DAPI in blocking buffer for 1 hour at room temperature. After washing with PBS, the cells were imaged using a CQ1 (Yokogawa Electric Corporation) and analyzed using CellPathfinder (Yokogawa Electric Corporation).

[0158] Immunofluorescence staining (IF) of frozen sections For frozen sections, samples were fixed in 4% PFA overnight at 4°C, washed with PBS, and then stored in PBS at 4°C. Fixed samples were either immersed in 30% sucrose / PBS at 4°C overnight and embedded in frozen tissue embedding medium (OCT compound), or immersed in 15% sucrose / PBS at 4°C overnight, immersed in 7% gelatin / 15% sucrose / PBS at 37°C, and embedded in 7% gelatin / 15% sucrose / PBS.

[0159] Reverse transcription and quantitative polymerase chain reaction (RT-qPCR). Total RNA was extracted using NucleoSpin RNA (MACHEREY-NAGEL), and cDNA was synthesized using PrimeScript™ RT Master Mix (Takara) according to the manufacturer's instructions. Real-time qPCR was performed using TB Green Premix Ex TaqII (Takara) in QuantStudio 5 (Applied Biosystems), and data were analyzed using Design and Analysis Software (Applied Biosystems). The primers used for qPCR are listed in Table S1 below. The undetected Ct value was arbitrarily set to 40.

[0160]

[0161] 7-10 μm cryosections were obtained from frozen samples and stored at -80°C until use. The cryosections were incubated in HistVT One (Nacalai) at 70°C for 20 minutes for antigen retrieval and then blocked in blocking buffer at room temperature for 30 minutes. The sections were incubated with primary antibodies in blocking buffer at room temperature for 3 hours, washed with PBS, and then incubated with secondary antibodies and DAPI in blocking buffer at room temperature for 1 hour. The antibodies used for IF are listed in Table 2 below. After staining, the samples were washed with PBS and distilled water, mounted in FluorSave reagent (Merck), and images were taken with an LSM900 (Zeiss).

[0162]

[0163] The reagents used in the examples are shown in Table S3 below.

[0164] Statistical Analysis Statistical analysis was performed using Prism 9 (GraphPad).

[0165] (Procedure for Inducing Ventral Hindgut Mesoderm (vHGM) Mesenchymal Cells) Prior to describing the method for inducing ventral hindgut mesoderm (vHGM) mesenchymal cells described below, we will explain the differentiation pathway suggested for the development of ventral hindgut mesoderm in vivo ( FIG. 2 (1)). In vivo, ventral hindgut mesoderm does not differentiate directly from epiblast. It has been suggested that the in vivo differentiation pathway of ventral hindgut mesoderm involves differentiation of the epiblast into a caudal epiblast, which then differentiates into a posterior primitive streak, and then further differentiates into ventral hindgut mesoderm, thereby forming ventral hindgut mesoderm. It has been suggested that this differentiation pathway, unlike anterior tissues, follows a differentiation pathway that does not express EOMES.

[0166] The following method for inducing mesenchymal cells from the ventral hindgut mesoderm (vHGM) is described with reference to Figure 2 (2). In step A of Figure 2 (2), human pluripotent stem cells (hPSCs) are cultured in the presence of low concentrations of a WNT agonist (CHIR), FGF2, and a TGF-β inhibitor (A83-01) to induce epiblast-like cells (NANOG) that express low levels of NANOG. ｌｏｗIn step B, caudal epiblast-like cells are induced by culturing the NANOG-low-expressing epiblast-like cells in the presence of moderate concentrations of CHIR, FGF4, and RA (Day 2). In step C, caudal epiblast-like cells are induced by culturing the caudal epiblast-like cells in the presence of high concentrations of CHIR, BMP4, and GDF11 (Day 5). In step α, the caudal epiblast-like cells are maintained in the presence of BMP4 and a ROCK inhibitor (Y-27632), thereby reducing or eliminating the number of ectodermal cells expressing SOX2, SOX3, and FOXD3, while selectively expanding cells capable of differentiating into ventral hindgut mesenchymal cells expressing TBX4, HOXD13, and FOXF1. In step D, the selected cells of interest are cultured in the presence of BMP4, FGF4, CHIR, and an HH agonist (HH-Ag1.5) to induce mesenchymal cells of the ventral hindgut mesoderm (Day 11).

[0167] Example 1: (1) Induction of differentiation of mesenchymal cells from ventral hindgut mesoderm Step A: NANOG from pluripotent stem cells ｌｏｗ Induction of Differentiation into Epiblast-Like Cells hESCs were cultured on iMatrix-511 in StemFit AK02N supplemented with 10 μM Y-27632 in a CellBIND plate (Corning) at a cell density of approximately 10,000 cells / cm. ２ On Day 0, the medium was replaced with N2B27AdvDF ("Induction Medium A") supplemented with 1 μM A-83-01 (Fujifilm Wako Pure Chemical Industries, Ltd.), 1 μM CHIR99021 (Tocris), 100 ng / ml FGF2 (Nacalai Tesque, Inc.), and 1 μg / ml heparin (Sigma-Aldrich) (Figure 3A). Epiblast-like cells with low NANOG expression were induced by culturing hESCs. N2B27AdvDF contained Advanced™ DMEM / F-12, 0.5X B27, 0.5X N2, 10 mM HEPES, and 2 mM L-glutamine (all Thermo Fisher Scientific).

[0168] Process B: NANOG ｌｏｗ Induction of differentiation of epiblast-like cells into caudal epiblast-like cells. On the first day of culture (Day 1), NANOG ｌｏｗ Caudal epiblast-like cells (Figure 3B) were induced by culturing the epiblast-like cells in N2B27AdvDF (induction medium B) supplemented with 3 μM CHIR99021, 20 ng / ml FGF4 (R&D Systems), 1 μg / ml heparin, and 100 nM all-trans retinoic acid (atRA, Sigma-Aldrich) for 1 day.

[0169] Step C: Induction of differentiation of caudal epiblast-like cells into posterior primitive streak cells. On day 2 of culture (Day 2), caudal epiblast-like cells (FIG. 3C) were cultured for 3 days in N2B27AdvDF ("induction medium C") supplemented with 6 μM CHIR99021, 50 ng / ml BMP4 (R&D Systems), and 30 ng / ml GDF11 (Peprotech). On day 4 of culture, the medium was replaced with fresh induction medium C to induce posterior primitive streak cells.

[0170] Step α: Selective Expansion of Cells Expressing TBX4, HOXD13, and FOXF1 On day 5 of culture (Day 5), posterior primitive streak cells (FIG. 3D) were detached with Accutase (Thermo Fisher Scientific) and cultured at a cell density of approximately 100,000 cells / cm in StemPro-34 SFM (Thermo Fisher Scientific) supplemented with 30 ng / ml BMP4 and 10 μM Y-27632. ２ The cells were seeded onto 2% Matrigel-coated CellBIND plates at a concentration of 1000 μg / ml and cultured for 1 day. This culture eliminated cells expressing SOX2, SOX3, and FOXD3, and selectively proliferated cells expressing TBX4, HOXD13, and FOXF1.

[0171] Step D: Induction of differentiation of posterior primitive streak cells into mesenchymal cells of the ventral hindgut mesoderm. On day 6 of culture, the medium was replaced with StemPro-34 SFM supplemented with 30 ng / ml BMP4, 10 ng / ml FGF4, 1 μg / ml heparin, 0.1 μM Hh-Ag1.5 (Collagen Technology), and 1 μM CHIR99021 ("Induction Medium D"), and the target cell population was cultured to induce mesenchymal cells of the ventral hindgut mesoderm ( FIG. 3E ). The medium was replaced with fresh Induction Medium D every 1 to 2 days until day 11 of culture (Day 11).

[0172] (2) Cell analysis in mesenchymal cell induction from ventral hindgut mesoderm (2-1) Cell analysis in step A (Day 0-1) In step A, NANOG cells were derived from pluripotent stem cells using induction medium A “CHIR / FGF2 / A83-01” described in Example 1(1). ｌｏｗ As a comparison, StemFit medium or CHIR / FGF2 medium (A83-01), which was the same as induction medium A except that it did not contain A83-01, was used. － ) was used to culture the pluripotent stem cell population. The abbreviation "CHIR" stands for CHIR99021.

[0173] The expression of NANOG and OCT3 / 4 was examined in cells cultured in each medium ( FIG. 4B ). Cells expressing OCT3 / 4 were observed in cell populations induced to differentiate in all three media used (center column in FIG. 4B ). The proportion of cells expressing OCT3 / 4 was not significantly different among the three medium conditions used (left graph in FIG. 4C ). Statistical analysis in Example 1 was performed using one-way analysis of variance with Dunnett's multiple comparison test compared with the StemFit condition.

[0174] NANOG-expressing cells were observed in the cell populations induced to differentiate using all three media (left column in Figure 4B). To examine the presence of cells that co-expressed OCT3 / 4 and NANOG, the percentage of NANOG-expressing cells among OCT3 / 4-expressing cells was calculated (center graph in Figure 4C). The center graph in Figure 4C shows the percentage of cells that co-expressed OCT3 / 4 and NANOG under culture conditions using StemFit medium and CHIR / FGF2 medium (A83-01). － ) and the culture conditions using induction medium A (A83-01). ＋ The results show that the proportion of these cells was significantly reduced in the cell population cultured with induction medium A (A83-01) compared to the cell population cultured with StemFit medium (P<0.001). ＋ ) compared to cell populations cultured using StemFit medium. ＋ This suggested that the proportion of cells expressing NANOG in the cell population was reduced.

[0175] To investigate these suggestions, we measured the mean fluorescence intensity (MFI) of NANOG expression in cells expressing OCT3 / 4 in cells cultured in each medium (right graph in Figure 4C). The right graph in Figure 4C shows the results of culture conditions using StemFit medium and CHIR / FGF2 medium (A83-01). － ) and the culture conditions using induction medium A (A83-01) (P=0.0618). ＋ The MFI of the cell population cultured with induction medium A (A83-01) was significantly smaller than that of the cell population cultured with StemFit medium (P<0.01). ＋ The results of Example 1 (2-1) show that under the culture conditions using induction medium A containing a TGFβ signal inhibitor (A83-01), differentiation into epiblast-like cells with low NANOG expression was achieved, similar to epiblasts in vivo that express low levels of NANOG.

[0176] (2-2) Cell Analysis in Step B (Day 1-2) In Step B, NANOG was analyzed using the induction medium B “CHIR / FGF4 / RA” described in Example 1(1). ｌｏｗ Caudal epiblast-like cells were induced from epiblast-like cells (cultured in induction medium A "CHIR / FGF2 / A83-01" in step A) (FIG. 4D). As a comparison, StemFit medium or CHIR / FGF4 (RA) medium, which was the same as induction medium B except that it did not contain retinoic acid (RA), was used. － ) using NANOG ｌｏｗ The epiblast-like cell population was cultured. Figure 4D shows five combinations of the medium used in step A (Day 0-1) and the medium used in step B (Day 1-2).

[0177] The expression of NANOG (Fig. 4E, left column) and OCT3 / 4 (Fig. 4E, center column) in the cell populations cultured in each medium combination was examined (Fig. 4E, right column: merged images). ＋ ) was used, and on Day 1-2, the medium contained RA (RA ＋ ) (i.e., the culture conditions for Day 0-1 and Day 1-2 are A83-01 ＋ _RA ＋ In the case of (1), the expression level of OCT4 was equivalent to that under other culture conditions, but the expression level of NANOG was observed to be reduced.

[0178] The percentage of cells expressing specific markers in specific cell populations was examined (Figure 4F). Even on day 2 of culture, the percentage of cells expressing the undifferentiated marker OCT3 / 4 exceeded 70% in each cell population cultured in each medium combination (upper graph in Figure 4F). The culture conditions for Day 0-1 and Day 1-2 were A83-01. ＋ _RA ＋ or R.A. － In the case of Day 0-1 and Day 1-2, the proportion of cells expressing NANOG in the cell population expressing OCT3 / 4 was significantly smaller than that in the cell population cultured for 2 days using StemFit medium (center graph in Figure 4F). ＋ _RA＋ or R.A. － In the case of A83-01, the MFI for NANOG expression in cells expressing OCT3 / 4 was significantly smaller than that in the cell population cultured for 2 days using StemFit medium (lower graph in Figure 4F). ＋ _RA ＋ In the case of , the MFI was the smallest.

[0179] The expression levels of caudal epiblast markers, NKX1-2, CDX1, and CDX2, were examined relative to TATA-binding protein (TBp) (Fig. 4G). The upper graph in Fig. 4G shows the results of the culture conditions of Day 0-1 and Day 1-2 under A83-01. ＋ _RA ＋ or R.A. － The relative expression level of NKX1-2 is higher than that in the cell populations cultured in other medium combinations. The middle graph in Figure 4G shows that the culture conditions of Day 0-1_Day 1-2 were A83-01 ＋ or A83-01 － _RA ＋ The lower graph in Figure 4G shows that the relative expression level of CDX1 was higher than that in the cell populations cultured in other medium combinations. ＋ _RA ＋ ) indicates that the relative expression level of CDX2 is higher than the relative expression level of CDX2 in cell populations cultured in other medium combinations, thereby inducing differentiation of the cell population.

[0180] The results of Figures 4F and 4G show that the culture conditions on Day 0-1 and Day 1-2 were A83-01 ＋ ＿ R.A. ＋ In this case, differentiation of a cell population that strongly expresses the undifferentiated marker OCT3 / 4, while expressing NANOG at low levels, and strongly expresses caudal epiblast markers (NKX1-2, CDX1 and CDX2) is induced.

[0181] Figure 5I, Day 2 (top row) and Figure 5J, left column show that under the culture conditions using StemFit medium (Day 0-2), few cells expressing EOMES or T were observed. Figure 5I, Day 2 (remaining rows) and Figure 5J, left column show that under the culture conditions (Day 0-2), induction medium A (A83-01) was used on Day 0-1. ＋ ) was used, and induction medium B (RA ＋ ) showed the lowest percentage of cells expressing EOMES and T.

[0182] The results of Example 1 (2-2) (Figs. 4F, 4G, 5I, and 5J) were obtained by incubating the cells with induction medium A (A83-01) on Day 0-1. ＋ ) was used, and induction medium B (RA ＋ We show that culture conditions using the α-glucanase inhibitor α-glucanase (α-glucanase) enable differentiation into caudal epiblast-like cells that express the undifferentiated marker OCT3 / 4, caudal epiblast markers (NKX1-2, CDX1, and CDX2), and the posterior primitive streak marker T, while expressing low levels of NANOG and not EOMES.

[0183] (2-3) Cell Analysis in Step C (Day 2-5) In Step C, posterior primary streak cells were induced from the caudal epiblast-like cells using the induction medium C "CHIR / BMP4 / GDF11" described in Example 1(1) (FIG. 5H). As a comparative example, a CHIR / BMP medium (GDF11) was used, which was the same as induction medium C except that it did not contain GDF11. － ) was used to culture a caudal epiblast-like cell population.

[0184] Figure 5I, Day 3 (top row) and Figure 5J, right column show that cells expressing EOMES or T were observed under the culture conditions using StemFit medium on Day 0-2 and CHIR / BMP4 medium on Day 3. Figure 5I, Day 3 (remaining rows) and Figure 5J, right column show that among the culture conditions (Day 0-3) used, cells expressing EOMES or T were observed under the culture conditions using induction medium A (A83-01) on Day 0-1. ＋ ) was used, and induction medium B (RA ＋ ) and on Day 2-3, induction medium C (GDF11 ＋) shows that no EOMES-expressing cells were observed and the proportion of T-expressing cells was the smallest.

[0185] On Day 0-1, induction medium A (A83-01 ＋ ) was used, and induction medium B (RA ＋ ) and induction medium C (GDF11 ＋ ) culture conditions using the α-glucanase inhibitor ...

[0186] (2-4) Cell analysis in step D (Day 6-11) In step D, mesenchymal cells of the ventral hindgut mesoderm were induced from posterior primitive streak cells using the "BMP4 / FGF4 / CHIR / Hh-Ag1.5 / RA" medium, which further contained 100 nM atRA in addition to the induction medium D described in Example 1(1) (FIG. 7A). The two graphs on the left side of FIG. 7B show the results of the induction medium A (A83-01) on Day 0-1. ＋ The second graph from the right in Figure 7B shows that the relative expression levels of ventral hindgut mesoderm markers ISL1 and HOXD13 were significantly higher under the culture conditions using Induction Medium A (A83-01) than under the StemFit culture conditions. ＋ )_Induction medium B (RA ＋ The rightmost graph in Figure 7B shows that the relative expression level of HOXD12, a posterior tissue marker, was higher under the culture conditions of Day 0-1, Day 1-2, Day 2-5, and Day 6-11 in induction medium A (A83-01). ＋)_Induction medium B (RA ＋ )_Induction medium C (GDF11 ＋ )_This shows that no significant difference was observed in the relative expression level of the visceral mesoderm marker FOXF1 expressed by peribladder mesenchymal cells under the culture condition of "BMP4 / FGF4 / CHIR / Hh-Ag1.5 / RA" medium compared to the culture condition of StemFit.

[0187] As shown by "All" in Figure 8C, the cell population cultured under the above culture conditions expressed TBX4, which is highly expressed in the ventral hindgut mesoderm. The results for ISL1, HOXD13, and TBX4 in Example 1 (2-4) were similar to those in the induction medium A (A83-01). ＋ )_Induction medium B (RA ＋ )_Induction medium C (GDF11 ＋ )_This shows that the culture conditions of "BMP4 / FGF4 / CHIR / Hh-Ag1.5 / RA" medium enable the induction of differentiation of ventral hindgut mesoderm into mesenchymal cells that express ventral hindgut mesoderm markers.

[0188] (2-5) Identification of differentiation-inducing factors important for the induction of ventral hindgut mesodermal mesenchymal cells (Step D) Among the differentiation-inducing factors added to the "BMP4 / FGF4 / CHIR / Hh-Ag1.5 / RA" medium (BMP4 / FGF4 / CHIR / Hh-Ag1.5 / RA is also referred to as "All-inducing factors"), factors important for the induction of ventral hindgut mesodermal mesenchymal cells were identified. Specifically, the above-mentioned Step D (Day 6-11) was carried out using a medium containing All-inducing factors but omitting one of the differentiation-inducing factors. Therefore, in some culture conditions, induction medium D supplemented with 100 nM atRA was used. Ventral hindgut mesodermal mesenchymal cells were induced according to the method described in Example 1(1).

[0189] 8A shows that no significant difference was observed in the relative expression level of ISL1 when cultured using "BMP4 / FGF4 / CHIR / Hh-Ag1.5 / RA" medium (also referred to as "All medium") without atRA (-atRA medium) or without CHIR (-CHIR99021 medium) compared to cultured using All medium (also referred to as "All culture condition"). Cultured using All medium without BMP4 (-BMP4 medium), FGF4, or heparin (-FGF4 medium), or without a hedgehog agonist (e.g., Hh-Ag1.5) (-Hh-Ag1.5 medium), compared to All culture condition. Figure 8B shows that no significant difference in the relative expression level of HOXD13 was observed under the culture condition using -atRA medium compared to the All culture condition. The relative expression level of HOXD13 was significantly reduced under the culture conditions using media that contained differentiation-inducing factors other than atRA (-BMP4 medium, -FGF4 medium, -Hh-Ag1.5 medium, or -CHIR99021 medium) compared to the All culture condition.

[0190] Figure 8C shows that no significant difference was observed in the relative expression level of TBX4 when cultured using -atRA medium, -BMP4 medium, or -FGF4 medium compared to the All culture condition. When cultured using -Hh-Ag1.5 medium or -CHIR99021 medium, the relative expression level of TBX4 was significantly reduced compared to the All culture condition. Figure 8D shows that no significant difference was observed in the relative expression level of FOXF1, a visceral mesoderm marker expressed by bladder peripheral mesenchymal cells, when cultured using -atRA medium, -FGF4 medium, or -CHIR99021 medium compared to the All culture condition. When cultured using -BMP4 medium or -Hh-Ag1.5 medium, the relative expression level of FOXF1 was significantly reduced compared to the All culture condition.

[0191] Figures 8A to 8D show that in the induction of ventral hindgut mesodermal mesenchymal cells (step D), removal of BMP (e.g., BMP4), FGF (e.g., FGF4), Wnt agonist (e.g., CHIR), or hedgehog agonist (e.g., Hh-Ag1.5) significantly reduces ventral hindgut mesoderm markers, indicating that these four differentiation-inducing factors are important for the induction of the desired ventral hindgut mesenchymal cells.

[0192] Figure 8E shows that a significant increase in the relative expression level of the extraembryonic mesoderm marker WNT2 was observed in the culture condition using -FGF4 medium compared to the All culture condition. Figure 8F shows that a significant increase in the relative expression level of the lateral plate mesoderm marker IRX3 was observed in the culture condition using -BMP4 medium compared to the All culture condition. Figure 8G shows that a significant increase in the relative expression level of the lateral plate mesoderm marker IRX5 was observed in the culture condition using -BMP4 medium or -HhAg1.5 compared to the All culture condition. Figure 8H shows that a significant increase in the relative expression level of the dorsal mesoderm marker NKX2-3 was observed in the culture condition using -BMP4 medium or -CHIR99021 medium compared to the All culture condition. Figure 8I shows that a significant increase in the relative expression level of NR2F1, a dorsal mesoderm marker, was observed under culture conditions using -BMP4 medium or -CHIR99021 medium compared to the All culture condition. Figures 8E to 8I suggest that in the induction of ventral hindgut mesenchymal cells (step D), removal of BMP (e.g., BMP4), FGF (e.g., FGF4), Wnt agonist (e.g., CHIR), or hedgehog agonist (e.g., Hh-Ag1.5) may induce cells other than the desired ventral hindgut mesenchymal cells.

[0193] FIG. 8 shows that in the induction of ventral hindgut mesenchymal cells (step D), BMP (e.g., BMP4), FGF (e.g., FGF4), Wnt agonist (e.g., CHIR), and hedgehog agonist (e.g., Hh-Ag1.5) are important for the induction of desired ventral hindgut mesenchymal cells without inducing non-desired cells.

[0194] (3) Ventral hindgut mesodermal mesenchymal cells Ventral hindgut mesodermal mesenchymal cells were induced according to substantially the same method as that described in Example 1(1). The induced ventral hindgut mesodermal mesenchymal cells were subjected to immunofluorescence staining (IF). Figure 9A shows that 80% or more of the induced ventral hindgut mesodermal mesenchymal cells were cells expressing ISL1 and FOXF1. Figure 9B shows that the induced ventral hindgut mesodermal mesenchymal cells also expressed Vimentin, a mesenchymal cell marker. The results of Example 1(3) demonstrate that ventral hindgut mesodermal mesenchymal cells, more specifically, bladder-perimesenchymal cells, can be induced from pluripotent stem cells by the method described in Example 1(1).

[0195] (4) Comparison with Foregut Splanchnic Mesoderm (FG-SpM) (Induction of Mesenchymal Cells from Foregut Splanchnic Mesoderm) Mesenchymal cells from foregut splanchnic mesoderm (STM, GM, and RM) were induced according to the method described by Kishimoto et al. (Nature Protocols, 2022) with some modifications. Note that STM (septum transversum mesenchyme) refers to phrenic mesenchyme, GM (gastric mesenchyme) refers to gastrointestinal mesenchyme, and RM (respiratory mesenchyme) refers to respiratory mesenchyme. In this specification, mesenchyme and mesenchyme may be used synonymously. Specifically, hESCs were plated on iMatrix-511 silk in CellBIND plates at a cell density of approximately 20,000-30,000 cells / cm. ２On Day 0, cells were cultured in N2B27AdvDF supplemented with 6 μM CHIR99021, 40 ng / ml BMP4, 30 ng / ml Activin A, 20 ng / ml FGF2, and 100 nM PIK90 (Selleck). From Day 1 to Day 2, cells were cultured in N2B27AdvDF supplemented with 30 ng / ml BMP4, 1 μM A-83-01, 2 μM IWR1 (Selleck), and 2 μM atRA. From Day 2 to Day 4, cells were cultured in N2B27AdvDF supplemented with 20 ng / ml FGF2. The medium used from day 1 to day 4 of culture was also supplemented with 0.1 μM Hh-Ag1.5 for anterior FG-SpM induction.

[0196] On day 4 of culture, to induce GM from posterior FG-SpM, the cells were cultured in N2B27AdvDF supplemented with 0.5 μM Hh-Ag1.5 and 2 μM atRA until day 7. On day 7 of culture, 500 nM LDN193189 (Tocris) was added to the medium.

[0197] To induce STM, posterior FG-SpM were treated with 30 ng / ml BMP4 and 2 μM atRA from culture day 4 to 7. To induce RM from anterior FG-SpM, the cells were cultured in N2B27AdvDF supplemented with 0.5 μM Hh-Ag1.5, 30 ng / ml BMP4, and 2 μM atRA, with 1 μM CHIR99021 added for the final day.

[0198] (Mesenchymal Cells of Ventral Hindgut Mesoderm) Ventral hindgut mesoderm mesenchymal cells were produced in substantially the same manner as described in Example 1(1).

[0199] The relative expression levels of cell markers in the prepared foregut visceral mesoderm mesenchymal cells (STM, GM, and RM) and ventral hindgut mesoderm (vHGM) mesenchymal cells were examined ( Figure 10 ). Figure 10A shows that the expression levels of the visceral mesoderm marker FOXF1 in ventral hindgut mesoderm mesenchymal cells were lower than those in GM and RM, which highly express FOXF1, but significantly higher than those in STM, which lowly express FOXF1. These results suggest that the cells induced to differentiate in this example possess the properties of FOXF1+ visceral mesoderm.

[0200] Figures 10C and 10D show that mesenchymal cells of the ventral hindgut mesoderm, distinct from the mesenchymal cells STM, GM, and RM of the foregut splanchnic mesoderm, significantly express the ventral hindgut mesoderm markers ISL1 and TBX4, respectively.

[0201] The HOX genes are classified into groups Hox1 to Hox13. The expression of HOX genes varies from anterior to posterior in the embryo; generally, lower-numbered groups are expressed anteriorly, whereas higher-numbered groups are expressed posteriorly. Compared with the mesenchymal cells of the foregut visceral mesoderm (STM, GM, and RM), the mesenchymal cells of the ventral hindgut mesoderm exhibited significantly lower relative expression levels of HOXA5 (Figure 10B) and significantly expressed HOXD10 to HOXD13 (Figures 10E-10H). Compared with the mesenchymal cells of the ventral hindgut mesoderm (STM, GM, and RM), the mesenchymal cells of the foregut visceral mesoderm exhibited significantly higher relative expression levels of HOXA5 (Figure 10B) and did not express HOXD10 to HOXD13 (Figures 10E-10H). These results indicate that the cells induced to differentiate in this example are posterior-like cells that express HOXA5 at low levels and highly express posterior HOXD10-13.

[0202] The results of Example 1(4) ( FIG. 10 ) confirm that the ventral hindgut mesoderm cells produced by the method described in Example 1(1) are mesenchymal cells of the ventral hindgut mesoderm, different from mesenchymal cells of the foregut visceral mesoderm.

[0203] Example 2: Referring to Figure 11A, the co-culture of ventral hindgut endoderm (vHGE) organoids and ventral hindgut mesoderm (vHGM) mesenchymal cells is described below. Endoderm differentiation is induced (Figure 11A(a)) to induce vHGE organoids from human pluripotent stem cells (hPSCs). Mesoderm differentiation is induced (Figure 11A(b)) to induce vHGM mesenchymal cells from hPSCs. The induced vHGE organoids and vHGM mesenchymal cells are co-cultured in the presence of an extracellular matrix (Figure 11A(c)). After a certain period of co-culture (Figure 11A(d)), the culture is observed under a microscope (Figure 11A(e)). (1) Preparation of Ventral Hindgut Endoderm (vHGE) Organoids. Ventral hindgut endoderm organoids were generated according to the method described in International Publication WO 2022-025269 A1, with some modifications. Specifically, hESCs were seeded onto iMatrix-511 silk in a 12-well CellBIND plate at a cell density of approximately 320,000-400,000 cells / well. On Day 0, the cells were treated with 2 μM CHIR99021 and 100 ng / ml Activin A in RPMI 1640 supplemented with 1× non-essential amino acids (NEAA, Thermo Fisher Scientific). The cells were then cultured in RPMI1640 supplemented with 100 ng / ml Activin A, NEAA and B27 for 2 days (0.05X on day 1 of culture and 0.5X on day 2 of culture) to induce definitive endoderm (DE).

[0204] On day 3 of culture, the DE cells were detached from the culture plate using Accutase, frozen in a STEM-CELLBANKER, and stored at -150°C until use. To generate vHGE organoids, the DE cells were thawed and seeded onto a 6-well EZSphere plate (IWAKI) at a cell density of 540,000-810,000 cells per well (approximately 200-300 cells per microwell) in SFD medium supplemented with 3 μM CHIR99021, 200 ng / ml FGF4, 1 μg / ml heparin, 10 ng / ml BMP4, and 10 μM Y-27632. SFD medium contained 75% IMDM, 25% F-12, 0.5X B27, 0.5X N2, 50 μg / ml L-ascorbic acid 2-phosphate (Sigma), 0.05% AlbuMAX II (Thermo Fisher Scientific), and 50 μg / ml 1-thioglycerol (Sigma).

[0205] After one day, the generated organoids were harvested, passed through a 100 μm cell strainer, and embedded in 100% Matrigel droplets at a density of approximately 200 organoids per droplet. After the Matrigel droplets solidified, the cells were cultured in SFD medium supplemented with 3 μM CHIR99021, 200 ng / ml FGF4, 1 μg / ml heparin, and 10 ng / ml BMP4 until day 13. The medium was changed every three days.

[0206] (2) Preparation of vHGM Mesenchymal Cells Ventral hindgut mesoderm (vHGM) mesenchymal cells were prepared from hESCs in substantially the same manner as described in Example 1(1).

[0207] (3) Co-culture of vHGE organoids with vHGM mesenchymal cells. The prepared vHGM mesenchymal cells on day 11 of culture were detached from the culture plate using Accutase and resuspended in 50% Matrigel / SFD supplemented with 10 μM Y-27632 to a cell density of 2,000,000 cells / ml. vHGE organoids were recovered using Gentle Cell Dissociation Reagent (Stem cell technologies) at 4 °C for 30 minutes and resuspended in 50% Matrigel containing vHGM mesenchymal cells.

[0208] Co-culture was performed by seeding 150 μl of 50% Matrigel containing vHGM mesenchymal cells onto 12-well inserts (Falcon, 0.4 μm pores), followed by overlaying 100 μl of 50% Matrigel containing both vHGE organoids and vHGM mesenchymal cells. The lower chamber was replenished with SFD medium supplemented with 1 nM atRA, 1 μM all-trans retinol (atROL, Abcam), and 0.1X Antibiotic-Antimycotic (Thermo Fisher Scientific). The medium was changed every 2–4 days. After 2–3 weeks, RA was removed from the medium. The cells were cultured for an additional 1–2 weeks in medium containing 0.1 μM atROL.

[0209] (4) Microscopic observation of vHGE organoids and vHGM mesenchymal cells after co-culture. Figure 11B shows that co-culture of vHGE organoids with vHGM mesenchymal cells (+vHGM) increased their size over time. The increased size of vHGE organoids had a multilayered epithelial cell layer. On the other hand, when vHGE organoids were cultured in the absence of vHGM mesenchymal cells (-vHGM), their size remained almost unchanged from 1 to 4 weeks of co-culture.

[0210] Figure 12 shows a series of multi-stained images of vHGE organoids co-cultured with vHGM mesenchymal cells. vHGE organoids co-cultured with vHGM mesenchymal cells expressed the bladder epithelial cell markers UPK1A (Figure 12A), UPK2 (Figure 12B), UPK3 (Figure 12C), ΔNP63 (Figure 12A), and GATA3 (Figure 12D). The vHGE organoids contained at least two cell layers: one expressing the epithelial cell marker Ecad and the other expressing the bladder epithelial cell markers UPK1A, UPK2, UPK3, and GATA3 (Figures 12A-12D). FIG. 12A shows that the vHGE organoids comprise three cell layers, including a cell layer expressing the bladder epithelial cell marker ΔNP63, a cell layer expressing the epithelial cell marker Ecad, and a cell layer expressing the bladder epithelial cell marker UPK1A.

[0211] Figure 12E shows that vHGM mesenchymal cells cocultured with vHGE organoids express the smooth muscle cell marker SMA and FOXF1, which is expressed by perivesical mesenchymal cells. Figures 12F and 12G show that they express GATA6 and ALCH1A2, respectively, which are expressed by perivesical mesenchymal cells. The results in Figure 12A suggest that coculture with vHGM mesenchymal cells can differentiate vHGE organoids into cell cultures with at least three epithelial cell layers. Furthermore, vHGM mesenchymal cells cocultured with vHGE organoids expressed perivesical mesenchymal cell markers (Figures 12E-12G), suggesting that they are perivesical mesenchymal cells expressing the smooth muscle cell marker SMA. SMA-expressing cells formed a muscle cell layer around the vHGE organoids, which contained a cell layer expressing Ecad. The results of Example 2(4) show that co-culture of vHGM mesenchymal cells and vHGE organoids results in the formation of bladder-like structures.

[0212] (5) Contraction of muscle layer in co-culture vHGE organoids and vHGM mesenchymal cells were co-cultured for 4 weeks. After 4 weeks, the co-culture contained a multilayered epithelial cell layer around the vHGE organoids, and muscle cells thought to be derived from vHGM mesenchymal cells were formed. When the co-culture was observed under a microscope, contraction of the muscle cell layer was observed. The results of Example 2 (5) show that vHGM mesenchymal cells induced from pluripotent stem cells (PSCs) can be differentiated into muscle cells using substantially the same method as that described in Example 1 (1).

[0213] (6) Comparison of vHGE organoids co-cultured with mesenchymal cells of the foregut visceral mesoderm (digestive mesenchyme or respiratory mesenchyme; GM or RM) or mesenchymal cells of the ventral hindgut mesoderm (vHGM) vHGE organoids were co-cultured with vHGE organoids in substantially the same manner as described in Examples 2 (3) and (4), except that GM or RM was used instead of the mesenchymal cells of vHGM co-cultured with vHGE organoids (Fig. 13A). The size (long axis length) of the vHGE organoids co-cultured with vHGM mesenchymal cells (Fig. 13B, upper left) was significantly larger than that of the vHGE organoids co-cultured with GM or RM (Fig. 13B, upper right and lower left) (Fig. 13B and Fig. 13D, second from the left).

[0214] vHGE organoids co-cultured with various mesenchymal cells were fluorescently stained (Fig. 13C). The number of cells constituting a single organoid was significantly higher in vHGE organoids co-cultured with vHGM mesenchymal cells than in vHGE organoids co-cultured with GM or RM (Fig. 13D, far left: Count of DAPI per spheroid).

[0215] Figure 13C shows that in vHGE organoids co-cultured with any mesenchymal cells, expression of KRT5 (a marker of more mature epithelial cells) was not observed (top image), and expression of FOXA2 (a marker of bladder epithelial progenitor cells) was observed (second image from the top). These results suggest that the co-cultured vHGE organoids are organoids composed of immature epithelial progenitor cells.

[0216] The number and percentage of cells expressing FOXA2 (a marker for early intestinal epithelium or developing bladder epithelial cells) were higher in vHGE organoids co-cultured with vHGM mesenchymal cells than in vHGE organoids co-cultured with GM or RM (Fig. 13D, right two graphs). These results suggest that vHGM mesenchymal cells are more suitable for maintaining the epithelial cell stage and highly proliferative bladder epithelial cells than GM and RM, resulting in the formation of vHGE organoids with a larger cell number and larger size (long axis length).

[0217] In vHGE organoids cocultured with various mesenchymal cells, cells expressing strongly FOXF1 clustered near (0-60 μm) cells expressing epithelial cell markers (Figure 13C, center and Figure 13E, left). Figure 13E, center, shows that in vHGE organoids cocultured with vHGM mesenchymal cells, cells expressing strongly SMA, a muscle cell marker, were more abundant at distances of 60 μm or more than at distances of 0-60 μm. Figure 13E, right, also shows that cells expressing strongly Calponin1, a smooth muscle cell marker, were more abundant at distances of 60 μm or more than at distances of 0-60 μm. These results indicate that an intermediate cell layer composed of cells with high FOXF1 expression and low SMA and Calponin1 expression was formed near (0-60 μm) vHGE organoids co-cultured with vHGM mesenchymal cells, whereas a smooth muscle cell layer composed of cells with low FOXF1 expression and high SMA and Calponin1 expression was formed in areas 60 μm or more away (60-120 μm and 120-180 μm).

[0218] In vHGE organoids cocultured with GM or RM, cells with high SMA expression were observed in the 0-60 μm range (Fig. 13E, center). Cells with high Calponin1 expression were not observed in any range (0-180 μm). These results indicate that when cocultured with GM or RM, the bilayer structure of mesenchymal cells, as observed in cocultures with vHGM mesenchymal cells, was not formed around the vHGE organoids.

[0219] The results of Example 2(6) show that, unlike GM and RM, vHGM mesenchymal cells can increase the size and cell number of organoids by maintaining bladder epithelial progenitor cells in vHGE organoids, and can form a two-layer structure of an intermediate cell layer and a smooth muscle cell layer around the organoids.

[0220] (7) Interaction between epithelial cells and mesenchymal cells via Hedgehog signaling In the bladder, the HH signaling derived from epithelial cells acts on mesenchymal cells, resulting in the formation of the intermediate layer (SMA / Calponin1) just below the epithelial cells. － ) and the outer muscle layer (SMA / Calponin1 ＋ ) is formed. Homeostasis of bladder epithelial cells is maintained by feedback from mesenchymal cells that receive HH signals.

[0221] Ventral hindgut endoderm organoids were co-cultured with ventral hindgut mesoderm (vHGM) mesenchymal cells prepared according to substantially the same method as described in Example 1 (1) (Figure 14A). The co-culture resulted in the presence of many FOXA2-expressing cells near the organoids (Figure 14B "ctrl"). The presence of the hedgehog signal inhibitor SANT-1 (250 nM or 500 nM) during co-culture resulted in a concentration-dependent decrease in the number of FOXA2-expressing cells present around the organoids (Figure 14B "+250 nM SANT-1" and "+500 nM SANT-1"). Furthermore, the hedgehog signal inhibitor (SANT-1) during co-culture concentration-dependently reduced the number of cells constituting the organoids and the size of the organoids (Figure 14C left and center). Similarly, expression of FOXA2, a marker of bladder epithelial progenitor cells, was decreased in a concentration-dependent manner by the Hedgehog signaling inhibitor (SANT-1) (Figure 14C, right). These results suggest that vHGM mesenchymal cells interact with epithelial cells constituting organoids through HH signaling and are suitable for maintaining highly proliferative bladder epithelial progenitor cells.

[0222] The expression distribution of SMA and Calponin1 was examined for organoids co-cultured under the above-mentioned culture conditions (Figure 14D). Furthermore, the expression levels of FOXF1 (left), SMA (center), and Calponin1 (right) in cell populations at predetermined distances from the organoids were examined (Figure 14E). Numerous FOXF1-expressing cells were observed around organoids co-cultured under Ctrl conditions (Figure 14D, top left). Figure 14E, left (blue dots) shows that the mean fluorescence intensity (MFI) of FOXF1 decreased with increasing distance from the organoids, from 0 μm to 60 μm, 60 μm to 120 μm, and 120 μm to 180 μm. The MFI of SMA was significantly higher at distances of 60 μm to 120 μm from the organoid compared to the MFI in other ranges (Figure 14E, middle (blue dots)), and the MFI of Calponin1 was significantly higher at distances of 60 μm to 180 μm compared to the distance of 0 μm to 60 μm from the organoid (Figure 14E, right (blue dots)). These results suggest that the co-culture formed an intermediate cell layer with strong FOXF1 expression and low SMA / Calponin1 expression near the epithelial cells of the organoid (0-60 μm range), and that smooth muscle cell layers with low FOXF1 expression and high SMA and Calponin1 expression or high Calponin1 expression were formed at distances of 60-120 μm and 120-180 μm. Furthermore, a layer of cells with relatively high expression levels of Calponin 1 and relatively low expression levels of FOXF1 and SMA was formed relatively distally (120 μm to 180 μm) from the organoids. These results and the fluorescence image (Figure 14D, left) suggest that the co-culture resulted in the formation of an intermediate layer of cells expressing FOXF1 and SMA covering the organoids, and a muscle layer of cells expressing FOXF1, SMA, and Calponin 1 covering the intermediate layer.

[0223] When ventral hindgut endoderm organoids were co-cultured in the presence of a low concentration (250 nM) of HH inhibitor, the MFI of FOXF1 decreased with increasing distance from the organoid, similar to that of Ctrl (Figure 14E, left, orange dots). Unlike Ctrl, the MFI of SMA was relatively high near the organoid (0 μm to 60 μm) and decreased with increasing distance from the organoid (from the 60 μm to 120 μm range to the 120 μm to 180 μm range) (Figure 14E, middle, orange dots). Unlike Ctrl, the MFI of Calponin1 was also relatively high near the organoid (0 μm to 120 μm) and significantly decreased relatively distally (120 μm to 180 μm range) (Figure 14E, middle, orange dots). These results and the fluorescence image (Fig. 14D, middle) suggest that the co-culture in the presence of low concentrations of HH inhibitors inhibited the formation of an intermediate layer and resulted in the formation of a muscle layer of cells expressing FOXF1, SMA, and Calponin1 overlying the organoids.

[0224] When ventral hindgut endoderm organoids were co-cultured in the presence of a high concentration (500 nM) of HH inhibitor, few FOXF1-expressing cells were observed around the organoids compared to Ctrl (Figure 14D, upper right). Similarly, cells expressing SMA or Calponin1 were not observed around the organoids (0 μm to 180 μm) (Figure 14D, middle right, bottom right; Figure 14E (red dots)). These results indicate that co-culture in the presence of a high concentration of HH inhibitor inhibited the aggregation of co-cultured mesenchymal cells around the epithelial cells of the organoids. Example 2(7) suggests that epithelial and mesenchymal cell interactions mediated by HH signaling are occurring between the epithelial and mesenchymal cells of the organoids, similar to those in the bladder in vivo.

[0225] (8) Maturation of E9.5 Mouse Anterior Hindgut Endoderm into Bladder Epithelial Cells As shown in Figure 15A, the anterior hindgut endoderm of E9.5 mouse embryos, which are induced to differentiate into bladder epithelial cells and intestinal epithelial cells, was co-cultured with vHGM mesenchymal cells derived from human pluripotent stem cells. Specifically, the co-culture was performed as follows. <Preparation of E9.5 Mouse Anterior Hindgut Endoderm> A portion containing the hindgut was excised from an E9.5 mouse embryo and treated with dispase / DNase (1 mg / ml) at 37°C for 1 minute. Subsequently, cells surrounding the anterior hindgut endoderm were manually removed, and the anterior hindgut, including the ventral hindgut, was excised to prepare E9.5 mouse anterior hindgut endoderm.

[0226] Co-culture of E9.5 Mouse Anterior Hindgut Endoderm and Human Mesenchymal Cells from vHGM Human mesenchymal cells from the ventral hindgut mesoderm (vHGM) were derived from human ES cells (KthES15) using essentially the same method as described in Example 1(1). The human mesenchymal cells were suspended in SFD medium containing 10 μM Y-27632 and seeded onto a low-adhesion 96-well U-bottom plate at 100,000 cells / well. The plate was centrifuged at 300 x g for 3 minutes to allow the cells to settle to the bottom of the plate. Next, the prepared E9.5 mouse anterior hindgut endoderm was added to the sedimented vHGM. 100,000 vHGM mesenchymal cells were added to cover the deposited E9.5 mouse anterior hindgut endoderm. The plate was centrifuged at 300 xg for 3 minutes, and then medium containing 20% ​​Matrigel was added, so that the medium in the wells contained a final concentration of 2% Matrigel.

[0227] After culturing the mouse anterior hindgut endoderm for one day, the organoids covered with vHGM human mesenchymal cells were removed from the wells and transferred to a 12-well culture insert so that one organoid was present per well. The mouse anterior hindgut endoderm and the mesenchymal cells were cultured at an air-liquid interface. For culture, SFD medium containing B27 supplement (containing vitamin A) was used with the addition of 0.5X Anti-Anti. The medium was changed every 2-3 days.

[0228] As a result of co-culture, the bladder epithelial cells (GATA3) were differentiated in the anterior hindgut endoderm derived from mouse.＋ / CDH1 ＋ ) and intestinal epithelial cells (CDX2 ＋ / CDH1 ＋ ) (Figures 15B and 15C). In the bladder epithelial region, a three-layer structure consisting of a layer of UPK3-expressing cells, a layer of P63 (TRP63 / ΔNP63)-expressing cells, and a layer of KRT5-expressing cells was observed, similar to mature mouse bladder epithelial cells (Figure 15D, left two panels). This result suggests that vHGM mesenchymal cells are mesenchymal cells capable of inducing a mature bladder epithelial cell layer.

[0229] The cell population surrounding the epithelial cells in the mouse anterior hindgut endoderm expressed human-specific Human LaminB1 (Figure 15D, right, two panels). This cell population also expressed the muscle cell marker SMA (Figure 15D, far right). These results suggest that the cell population surrounding the epithelial cells in the mouse anterior hindgut endoderm is not muscle cells differentiated from mouse-derived mesenchymal cells, but rather muscle cells differentiated from the human mesenchymal cells of the vHGM used in this co-culture. Furthermore, these results suggest that maturation of the bladder epithelial region of the mouse anterior hindgut endoderm is brought about by interactions between human mesenchymal cells of the vHGM and epithelial cells of the anterior hindgut endoderm.

Claims

1. A method for producing mesenchymal cells of the ventral hindgut mesoderm, comprising: culturing pluripotent stem cells in induction medium A containing a TGF-β inhibitor, a Wnt agonist, and a fibroblast growth factor to induce differentiation into epiblast-like cells; culturing the epiblast-like cells in induction medium B containing a Wnt agonist, a fibroblast growth factor, and retinoic acid to induce differentiation into caudal epiblast-like cells; culturing the caudal epiblast-like cells in induction medium C containing a Wnt agonist, a bone morphogenetic protein, and a TGF-β family member to induce differentiation into posterior primitive streak cells; and culturing the posterior primitive streak cells in induction medium D containing a Wnt agonist, a fibroblast growth factor, a bone morphogenetic protein, and a Hedgehog signaling agonist to induce differentiation into mesenchymal cells of the ventral hindgut mesoderm.

2. The method of claim 1, further comprising culturing the posterior primitive streak cells in a selective medium containing bone morphogenetic protein and a ROCK inhibitor.

3. The method of claim 1, wherein said induction medium D further contains retinoic acid.

4. A mesenchymal cell of the ventral hindgut mesoderm produced by the method of any one of claims 1 to 3, wherein the mesenchymal cell expresses FOXF1, ISL1, TBX4 and HOXD13.

5. The mesenchymal cell according to claim 4, which further expresses at least one selected from the group consisting of HOXD10, HOXD11, and HOXD12.

6. A pharmaceutical composition for treating damage or disease of the kidney or bladder or surrounding areas, comprising the mesenchymal cells of claim 4 and a pharmaceutically acceptable carrier.

7. A method for producing a non-human mammal having mesenchymal cells or muscle cells derived from said mesenchymal cells in the kidney or bladder or surrounding areas thereof, comprising introducing the mesenchymal cells according to claim 4 into the kidney or bladder or surrounding areas thereof.

8. A non-human mammal having the mesenchymal cells according to claim 4 or muscle cells derived from said mesenchymal cells in the kidney or bladder or in the surrounding area thereof.

9. A method for producing matured bladder organoids, comprising culturing ventral hindgut organoids or bladder organoids in an induction medium containing retinol and retinoic acid in the presence of the mesenchymal cells of claim 4, wherein the matured bladder organoids comprise a superficial cell layer, an intermediate cell layer located laterally relative to the superficial cell layer, a basal cell layer located laterally relative to the intermediate cell layer, and a muscle cell layer located laterally relative to the basal cell layer, and the muscle cell layer comprises muscle cells derived from the mesenchymal cells.

10. A method for evaluating responsiveness to a test substance, comprising contacting the test substance with mesenchymal cells of the ventral hindgut mesoderm, muscle cells, or matured bladder organoids described below, and detecting changes in the test substance, the mesenchymal cells, the muscle cells, or the matured bladder organoids, wherein the mesenchymal cells of the ventral hindgut mesoderm are mesenchymal cells of the ventral hindgut mesoderm produced by the method of any one of claims 1 to 3, the mesenchymal cells of the ventral hindgut mesoderm described in claim 4 or 5, or mesenchymal cells of the ventral hindgut mesoderm in the kidney or bladder or a peripheral site thereof of a non-human mammal, and the muscle cells are muscle cells derived from the mesenchymal cells of the ventral hindgut mesoderm in the kidney or bladder or a peripheral site thereof of a non-human mammal, The evaluation method, wherein the matured bladder organoid is a matured bladder organoid produced according to the production method described in claim 9, or the matured bladder organoid in the kidney or bladder or a surrounding area thereof of a non-human mammal.

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