Bladder organoids and their manufacturing method

Abstract

To provide ventral hindgut organoids for producing bladder organoids with a layered structure of bladder epithelial cell types similar to those in the bladder.SOLUTION: One aspect of the present invention provides a method for producing ventral hindgut organoids, comprising culturing pluripotent stem cells using induction medium A containing activin A and a GSK3β inhibitor to induce differentiation into definitive endoderm cells, and culturing the definitive endoderm cells using induction medium B that contains a fibroblast growth factor and a GSK3β inhibitor and further may contain a bone morphogenetic protein, and then culturing the cells in induction medium B that contains a fibroblast growth factor and a GSK3β inhibitor and further may contain a bone morphogenetic protein, in the presence of an extracellular matrix, to form ventral hindgut organoids.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to ventral hindgut organoids or bladder organoids, or a method for producing them.The present disclosure also relates to a non-human mammal having ventral hindgut organoids or bladder organoids in its kidney or bladder or their surrounding areas, or a method for producing them.The present disclosure further relates to a method for evaluating drug responsiveness to a test substance in ventral hindgut organoids, bladder organoids, or the non-human mammal.The present disclosure further relates to a regenerative medicine composition comprising ventral hindgut organoids or bladder organoids. [Background technology]

[0002] The urinary bladder is an organ derived from the definitive endoderm and is known to develop from the hindgut, the posteriormost part of the early gastrointestinal tract, through the ventral cloaca. The bladder is a sac-like organ that temporarily stores urine that is delivered from the kidneys via the ureters and excreted through the urethra. The bladder's urine storage and urination functions can be reduced or lost when bladder tissue is damaged by radiation therapy, bladder rupture, diabetes, and other conditions.

[0003] Research into regenerative medicine is being conducted with the aim of regenerating organs that have lost function, treating intractable diseases, and making up for the chronic shortage of organ donors for organ transplants. In the field of regenerative medicine, research is being conducted into the use of organ-like cell aggregates called organoids, which are created in vitro from pluripotent stem cells such as ES cells or iPS cells.

[0004] Non-Patent Document 1 describes the induction of differentiation into bladder epithelium by culturing mouse ES cells in vitro. Non-Patent Documents 2 to 4 describe the induction of differentiation into bladder epithelium by culturing human pluripotent stem cells in vitro. Non-Patent Document 5 describes the induction of differentiation into bladder epithelium by culturing mouse ES cells in a collagen matrix in vitro and then transplanting them under the mouse kidney capsule. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Mauney JR, et. al.,: All-Trans Retinoic Acid Directs Urothelial Specification of Murine Embryonic Stem Cells via GATA4 / 6 Signaling Mechanisms. PLoS One 2010, 5:e11513. [Non-patent document 2] Osborn SL, et. al.,: Induction of human embryonic and induced pluripotent stem cells into urothelium. Stem Cells Transl Med 2014, 3:610-9. [Non-patent document 3] Suzuki K, et. al.,: Directed differentiation of human induced pluripotent stem cells into mature stratified bladder urothelium. Sci Rep 2019, 9:1-13. [Non-patent document 4] Kang M, et. al.,: Generation of Bladder Urothelium from Human Pluripotent Stem Cells under Chemically Defined Serum- and Feeder-Free System. Int J Mol Sci 2014, 15:7139-7157. [Non-patent document 5] Oottamasathien S, et. al.,: Directed differentiation of embryonic stem cells into bladder tissue. Dev Biol 2007, 304:556-566. Summary of the Invention [Problem to be solved by the invention]

[0006] The differentiation induction into bladder epithelium described in Non-Patent Documents 1 to 4 does not mimic the actual bladder development process, nor does it involve differentiation induction in a three-dimensional culture system. The bladder epithelial organoids differentiated in Non-Patent Documents 1 to 4 do not have a layered structure of bladder epithelial cell types like the bladder. It is unclear whether the bladder epithelial organoids differentiated in Non-Patent Document 5 have a three-layered structure like the bladder.

[0007] The present inventors have investigated various conditions for forming bladder organoids from pluripotent stem cells. For example, the present inventors have investigated conditions for constructing a three-dimensional culture system that mimics the actual bladder development process. One objective of the present disclosure is to provide novel ventral hindgut organoids for producing bladder organoids having a layered structure of bladder epithelial cell types, such as the bladder, or to provide a method for producing the same. One objective of the present disclosure is to provide novel bladder organoids having a layered structure of bladder epithelial cell types, such as the bladder, or to provide a method for producing the same. One objective of the present disclosure is to provide novel non-human mammals having ventral hindgut organoids or bladder organoids in their kidneys, bladder, or surrounding areas, or a method for producing the same. One objective of the present disclosure is to provide novel regenerative medicine compositions containing ventral hindgut organoids or bladder organoids. One object of the present disclosure is to provide a novel method for evaluating drug responsiveness to a test substance in ventral hindgut organoids, bladder organoids, or the non-human mammal. [Means for solving the problem]

[0008] The present disclosure provides the following aspects. [Item A1] A method for producing ventral hindgut organoids, comprising: culturing pluripotent stem cells using induction medium A containing activin A and a GSK3β inhibitor to induce differentiation into definitive endoderm cells; culturing the definitive endoderm cells using induction medium B containing a fibroblast growth factor and a GSK3β inhibitor, and then culturing them in an extracellular matrix gel using induction medium B containing a fibroblast growth factor and a GSK3β inhibitor to form hindgut organoids; and culturing the hindgut organoids in an extracellular matrix gel using induction medium b2 containing a fibroblast growth factor, a GSK3β inhibitor, and a bone morphogenetic protein to form ventral hindgut organoids. [Section A2] Ventral hindgut organoids expressing p63, CDX2, HOXA13, and KRT8, and not expressing SOX2. [Section A2-1] Ventral hindgut organoids that express p63, CDX2, HOXA13, and KRT8, and do not substantially express SOX2. [Item A3] The ventral hindgut organoid according to Item A2, wherein the expression level of CDX2 in the ventral hindgut organoid is lower than the expression level of CDX2 in the hindgut organoid, or CDX2 is not expressed in the ventral hindgut organoid. [Item A3-1] The ventral hindgut organoid according to Item A2, wherein the expression level of CDX2 in the ventral hindgut organoid is lower than the expression level of CDX2 in the hindgut organoid, or CDX2 is not substantially expressed in the ventral hindgut organoid. [Item A4] A method for producing bladder organoids, comprising culturing ventral hindgut organoids produced by the method described in Item A1, or the ventral hindgut organoids described in Item A2 or Item A3, in an extracellular matrix gel using induction medium C containing retinoic acid, fibroblast growth factor, and bone morphogenetic protein.

[0009] [Item A5] A bladder organoid comprising: a first cell layer comprising cells that co-express P63 and KRT5 and are located along the outermost periphery of the bladder organoid; a second cell layer comprising cells that co-express P63 and UPK2 and are located inward relative to the first cell layer; and a third cell layer comprising cells that do not express P63 but express UPK2 and are located inward relative to the second cell layer. [Item A5-1] A bladder organoid comprising: a first cell layer containing cells that co-express P63 and KRT5 and are located along the outermost periphery of the bladder organoid; a second cell layer containing cells that co-express P63 and UPK2 and are located inward relative to the first cell layer; and a third cell layer containing cells that do not substantially express P63 but express UPK2 and are located inward relative to the second cell layer. [Item A6] A method for producing bladder organoids, comprising introducing the ventral hindgut organoids produced by the method described in Item A1, or the ventral hindgut organoids described in Item A2 or Item A3, into the kidney or bladder or surrounding areas of a human or non-human mammal. [Item A7] A bladder organoid having a lumen, comprising: a first cell layer comprising cells that co-express P63 and KRT5 and are located along the outermost periphery of the bladder organoid; a second cell layer comprising cells that co-express P63 and UPK2 and are located inward relative to the first cell layer; and a third cell layer comprising cells that do not express P63 but express UPK2 and are located inward relative to the second cell layer and face the lumen. [Item A7-1] A bladder organoid having a lumen, comprising: a first cell layer containing cells that co-express P63 and KRT5 and are located along the outermost periphery of the bladder organoid; a second cell layer containing cells that co-express P63 and UPK2 and are located inward relative to the first cell layer; and a third cell layer containing cells that do not substantially express P63 but express UPK2 and are located inward relative to the second cell layer and face the lumen.

[0010] [Item A8] A method for producing a non-human mammal having bladder organoids in its kidney or bladder or a surrounding area thereof, comprising introducing ventral hindgut organoids produced by the method described in Item A1, or the ventral hindgut organoids described in Item A2, Item A2-1, Item A3 or Item A3-1, or the bladder organoids produced by the method described in Item A4 or Item A6, or the bladder organoids described in Item A5, Item A5-1, Item A7 or Item A7-1 into the kidney or bladder or a surrounding area of ​​the non-human mammal. [Item A9] A non-human mammal having a ventral hindgut organoid produced by the method described in Item A1, or a ventral hindgut organoid described in Item A2, Item A2-1, Item A3 or Item A3-1, or a bladder organoid produced by the method described in Item A4 or Item A6, or a bladder organoid described in Item A5, Item A5-1, Item A7 or Item A7-1 in its kidney or bladder or a surrounding area thereof. [Item A10] A regenerative medicine composition for treating bladder damage or disease, comprising a ventral hindgut organoid produced by the method described in Item A1, or a ventral hindgut organoid described in Item A2, Item A2-1, Item A3, or Item A3-1, or a bladder organoid produced by the method described in Item A4 or Item A6, or a bladder organoid described in Item A5, Item A5-1, Item A7, or Item A7-1. [Item A11] A method for evaluating drug responsiveness to a test substance, comprising contacting a test substance with ventral hindgut organoids produced by the method described in Item A1, or the ventral hindgut organoids described in Item A2, Item A2-1, Item A3 or Item A3-1, or a bladder organoid produced by the method described in Item A4 or Item A6, or the bladder organoids described in Item A5, Item A5-1, Item A7 or Item A7-1, or a non-human mammal described in Item A9; and measuring the drug responsiveness of the ventral hindgut organoids, the bladder organoids, or the non-human mammal to the test substance. [Item A12] A method for treating bladder damage or disease, comprising introducing a ventral hindgut organoid produced by the method described in Item A1, or a ventral hindgut organoid described in Item A2, Item A2-1, Item A3 or Item A3-1, or a bladder organoid produced by the method described in Item A4 or Item A6, or a bladder organoid described in Item A5, Item A5-1, Item A7 or Item A7-1 into the kidney or bladder or a surrounding area of ​​a mammal in need thereof.

[0011] [Section B1] 1. A method for producing ventral hindgut organoids, comprising: Culturing the pluripotent stem cells using induction medium A containing activin A and a GSK3β inhibitor to induce differentiation into definitive endoderm cells; and The method comprises culturing the definitive endoderm cells in induction medium B containing a fibroblast growth factor and a GSK3β inhibitor, and optionally further containing a bone morphogenetic protein, and then culturing the cells in induction medium B containing a fibroblast growth factor and a GSK3β inhibitor, and optionally further containing a bone morphogenetic protein, in the presence of an extracellular matrix, to form ventral hindgut organoids. [Section B2] The formation of the ventral hindgut organoids comprises culturing the definitive endoderm cells in the induction medium B, followed by culturing the cells in the induction medium B in the presence of an extracellular matrix to form hindgut organoids, and culturing the hindgut organoids in the induction medium B in the presence of an extracellular matrix to form ventral hindgut organoids; The method according to paragraph B1, wherein the induction medium B for forming hindgut organoids contains a fibroblast growth factor and a GSK3β inhibitor, and the induction medium B for forming ventral hindgut organoids contains a fibroblast growth factor, a GSK3β inhibitor, and a bone morphogenetic protein. [Section B3] The method described in paragraph B1, wherein induction medium B for forming ventral hindgut organoids contains a fibroblast growth factor, a GSK3β inhibitor, and a bone morphogenetic protein. [Section B4] A ventral hindgut organoid produced by the method according to any one of items B1 to B3.

[0012] [Section B5] 1. A ventral hindgut organoid, comprising: They express the ventral hindgut marker P63, expressing HOXA13 and CK8 / KRT8, and Ventral hindgut organoids that do not substantially express the dorsal hindgut marker SOX2. [Section B5-1] 1. A ventral hindgut organoid, comprising: They express the ventral hindgut marker P63, expressing HOXA13 and CK8 / KRT8, and A ventral hindgut organoid that does not substantially express the dorsal hindgut markers SOX2 and / or CDX2, or even if it expresses SOX2 and / or CDX2, the expression level is lower than the expression level of SOX2 and / or CDX2 in hindgut organoids. [Section B6] A ventral hindgut organoid having a lumen, expressing at least one ventral hindgut marker selected from the group consisting of p63, ΔN63, GATA3, ISL1, and SATB2; expressing at least one marker selected from the group consisting of Phospho-Smad1 / 5 / 8, HOXA13, FOXA2, CK8 / KRT8, and ECAD; and A ventral hindgut organoid that does not substantially express at least one dorsal hindgut marker selected from the group consisting of SOX2, T, and CDX2. [Section B7] A ventral hindgut organoid produced by the method according to any one of items B1 to B3, or a ventral hindgut organoid according to any one of items B4 to B6, Culturing in the presence of an extracellular matrix using induction medium C containing retinoic acid, fibroblast growth factor, and bone morphogenetic protein; into the kidney or bladder or surrounding areas of a human or non-human mammal; or A method for producing bladder organoids, comprising culturing in the presence of mesenchymal stem cells or mesenchymal cells. [Section B8] Bladder organoids produced by the method described in Section B7.

[0013] [Section B9] 1. A bladder organoid, comprising: a first cell layer comprising cells that do not substantially express p63 but express UPK1B and / or UPK2; and a second cell layer comprising cells co-expressing p63 and UPK1B and / or UPK2, the second cell layer being located outward relative to the first cell layer; The bladder organoid may further comprise a third cell layer comprising cells that co-express p63 and KRT5, the third cell layer being located outward relative to the second cell layer. [Section B10] The bladder organoid of paragraph B9, comprising a lumen surrounded by the first cell layer. [Section B11] The bladder organoid of paragraph B9 or paragraph B10, further comprising a fourth cell layer comprising interstitial-like cells located outward relative to the third cell layer, and optionally a fifth cell layer comprising smooth muscle cells located outward relative to the fourth cell layer. [Section B12] A method for producing a non-human mammal having ventral hindgut organoids or bladder organoids in its kidney or bladder or a surrounding area thereof, the method comprising introducing ventral hindgut organoids produced by the method described in any one of paragraphs B1 to B3, ventral hindgut organoids described in any one of paragraphs B4 to B6, bladder organoids produced by the method described in paragraph B7, or bladder organoids described in any one of paragraphs B8 to B11 into the kidney or bladder of the non-human mammal or a surrounding area thereof.

[0014] [Section B13] A non-human mammal having a ventral hindgut organoid produced by the method described in any one of paragraphs B1 to B3, a ventral hindgut organoid described in any one of paragraphs B4 to B6, a bladder organoid produced by the method described in paragraph B7, or a bladder organoid described in any one of paragraphs B8 to B11 in its kidney or bladder or surrounding area. [Section B14] A regenerative medicine composition for treating bladder damage or disease, comprising a ventral hindgut organoid produced by the method described in any one of paragraphs B1 to B3, a ventral hindgut organoid described in any one of paragraphs B4 to B6, a bladder organoid produced by the method described in paragraph B7, or a bladder organoid described in any one of paragraphs B8 to B11. [Section B15] A method for evaluating drug responsiveness to a test substance, comprising: Contacting a test substance with a ventral hindgut organoid produced by the method according to any one of Items B1 to B3, a ventral hindgut organoid according to any one of Items B4 to B6, a bladder organoid produced by the method according to Item B7, a bladder organoid according to any one of Items B8 to B11, a non-human mammal produced by the method according to Item B12, or a non-human mammal according to Item B13; and A method comprising measuring drug responsiveness to the test substance in the ventral hindgut organoids, the bladder organoids, or the non-human mammal. [Item B16] A method for treating bladder damage or disease, comprising introducing a ventral hindgut organoid produced by the method described in any one of Items B1 to B3, a ventral hindgut organoid described in any one of Items B4 to B6, a bladder organoid produced by the method described in Item B7, or a bladder organoid described in any one of Items B8 to B11 into the kidney or bladder or a surrounding area of ​​a mammal in need thereof. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 is a flow chart outlining the generation of bladder organoids according to one embodiment. [Figure 2] Figure 2a shows the distribution of SATB2 in intestinal-like organoids. Figure 2b shows the distribution of CDX2 in intestinal-like organoids. Figure 2c shows the distribution of ECAD in intestinal-like organoids. Figure 2d shows DAPI staining in intestinal-like organoids. [Figure 3] Figure 3a is an image showing the distribution of UPK2 in bladder organoids. Note that the white signal observed at the outermost periphery of the bladder organoids is a nonspecific signal derived from Matrigel®. Figure 3b is an image showing the distribution of P63 in bladder organoids. Figure 3c is an image showing the distribution of KRT5 in bladder organoids. Figure 3d is a composite image showing the distribution of UPK2, P63, and KRT5 in bladder organoids. Note that the white signal observed at the outermost periphery of the bladder organoids is a nonspecific signal derived from Matrigel. Figure 3e is an image showing the distribution of ECAD in bladder organoids. Figure 3f is an image of DAPI staining in bladder organoids. [Figure 4] Figure 4a is an image showing the distribution of UPK2 in cell structures induced using induction medium B and induction medium C minus BMP4. The white signal observed at the outermost periphery of the cell structures is a nonspecific signal derived from Matrigel. Figure 4b is an image showing the distribution of P63 in cell structures induced using induction medium B and induction medium C minus BMP4. Figure 4c is an image showing the distribution of ECAD in cell structures induced using induction medium B and induction medium C minus BMP4. Figure 4d is an image of DAPI staining in cell structures induced using induction medium C minus BMP4. [Figure 5]Figure 5a is an image showing the distribution of UPK2 in bladder organoids introduced into non-human mammals. Figure 5b is an image showing the distribution of P63 in bladder organoids introduced into non-human mammals. Figure 5c is an image showing the distribution of KRT5 in bladder organoids introduced into non-human mammals. Figure 5d is an image of DAPI staining in bladder organoids. Figure 5e is a composite image showing the distribution of P63, KRT5, and DAPI staining in bladder organoids introduced into non-human mammals. Figure 5f is an image showing the distribution of UPK2, P63, and DAPI staining in bladder organoids introduced into non-human mammals. Figure 5g is a schematic diagram showing the layered structure of cell types in the in vivo bladder. [Figure 6] FIG. 6 is a flow chart diagram outlining the generation of bladder organoids according to one embodiment. [Figure 7]Figures 7a1-a11 are fluorescent images of hindgut organoids after hindgut differentiation induction in step b1 (day 11 after differentiation induction). Figures 7b1-b11 are fluorescent images of ventral hindgut organoids during ventral hindgut differentiation induction in step b3 (day 11 after differentiation induction). Figures 7a1 and 7b1 are images showing the distribution of the epithelial marker KRT8 in hindgut organoids and ventral hindgut organoids, respectively. Figures 7a2 and 7b2 are images showing the distribution of the ventral marker GATA3 in hindgut organoids and ventral hindgut organoids, respectively. Figures 7a3 and 7b3 are images showing the distribution of the dorsal hindgut marker SOX2 in hindgut organoids and ventral hindgut organoids, respectively. Figures 7a5 and 7b5 are images showing the distribution of the dorsal hindgut marker CDX2 in hindgut organoids and ventral hindgut organoids, respectively. Figures 7a6 and 7b6 show images of the distribution of the dorsal hindgut marker SOX2 in hindgut organoids and ventral hindgut organoids, respectively. Figures 7a7 and 7b7 show images of the distribution of the dorsal hindgut marker T in hindgut organoids and ventral hindgut organoids, respectively. Figures 7a9 and 7b9 show images of the distribution of the intestinal marker FOXA2 in hindgut organoids and ventral hindgut organoids, respectively. Figures 7a10 and 7b10 show fluorescent images of the distribution of the epithelial marker ECAD in hindgut organoids and ventral hindgut organoids, respectively. Figures 7a4, 7a8, and 7a11 show DAPI-stained images in hindgut organoids. Figures 7b4, 7b8, and 7b11 show DAPI-stained images in ventral hindgut organoids. [Figure 8]Figures 8a1 and 8a2 are fluorescence images showing the distribution of dorsal hindgut markers CDX2 and SOX2, respectively, in ventral hindgut organoids undergoing ventral hindgut differentiation (day 14). Figure 8a3 is a fluorescence image showing the distribution of ventral hindgut marker GATA3. Figure 8a4 is a DAPI-stained image of ventral hindgut organoids. Figure 8a5 is a merged image of CDX2, SOX2, GATA3, and DAPI staining. Figure 8b1 is a fluorescence image showing the distribution of early intestinal epithelial marker FOXA2. Figures 8b2 and 8b3 are fluorescence images showing the distribution of ventral hindgut markers ΔNP63 and GATA3, respectively, in ventral hindgut organoids. Figure 8b4 is a DAPI-stained image of ventral hindgut organoids. Figure 8b5 is a merged image of FOXA2, ΔNP63, GATA3, and DAPI staining. Figure 8c1 is a fluorescent image showing the distribution of CK8, an intestinal epithelial marker. Figure 8c2 is a fluorescent image showing the distribution of SATB2, a ventral hindgut marker. Figure 8c3 is a fluorescent image showing the distribution of ZO1, an apical tight junction marker. Figure 8c4 is a DAPI-stained image of ventral hindgut organoids. Figure 8c5 is a merged image of CK8, SATB2, ZO1, and DAPI staining. Figure 8d1 is a fluorescent image showing the distribution of ISL1, a ventral hindgut marker. Figure 8d2 is a fluorescent image showing the distribution of Phospho-Smad1 / 5 / 8, markers for the ventral hindgut and Cloaca region. Figure 8d3 is a fluorescent image showing the distribution of ECAD, an epithelial cell marker. Figure 8d4 is a DAPI-stained image of ventral hindgut organoids. Figure 8d5 is a merged image of ISL1, Phospho-Smad1 / 5 / 8, ECAD, and DAPI staining. Figure 8e1 is a fluorescent image showing the distribution of GATA3, a ventral hindgut marker. Figure 8e2 is a fluorescent image showing the distribution of SOX2, a dorsal hindgut marker. Figure 8e3 is a fluorescent image showing the distribution of UPK2, a bladder epithelial cell marker. Figure 8e4 is a DAPI-stained image of a ventral hindgut organoid. Figure 8e5 is a composite image of GATA3, SOX2, UPK2, and DAPI staining. Figure 8f1 is a fluorescent image showing the distribution of CDX2, a dorsal hindgut marker.Figure 8f2 is a fluorescent image showing the distribution of HOXA13, a posterior intestinal marker. Figure 8f3 is a fluorescent image showing the distribution of KRT5, a bladder epithelial cell marker. Figure 8f4 is a DAPI staining image of the ventral hindgut organoid. Figure 8f5 is a merged image of CDX2, HOXA13, KRT5, and DAPI staining. [Figure 9] Figures 9a to 9d are bright-field images of bladder organoids undergoing bladder epithelial differentiation induction by co-culture of ventral hindgut organoids and bladder mesenchymal cells (day 20 of differentiation induction). [Figure 10] Figures 10a1-a3 are fluorescence images showing the distribution of bladder epithelial cell markers UPK2, ΔNP63, and KRT5 in bladder organoids undergoing differentiation (day 24 of differentiation induction). Figure 10a4 is a DAPI-stained image of the bladder organoids. Figure 10a5 is a superimposed image of UPK2, ΔNP63, KRT5, and DAPI staining. Figure 10b1 is a fluorescence image showing the distribution of ECAD, an epithelial cell marker, in the bladder organoids. Figure 10b2 is a fluorescence image showing the distribution of hLNB1, which indicates human-derived cells, in the bladder organoids. Figure 10b3 is a fluorescence image showing the distribution of VIM, a mesenchymal cell marker, in the bladder organoids. Figure 10b4 is a DAPI-stained image of the bladder organoids. Figure 10b5 is a superimposed image of ECAD, hLNB1, VIM, and DAPI staining. Figure 10c1 is a fluorescence image showing the distribution of ECAD, an epithelial cell marker, in the bladder organoids. Figure 10c2 is a fluorescence image showing the distribution of αSMA, a smooth muscle cell marker, in the bladder organoids. Figure 10c3 is a DAPI-stained image of the bladder organoids. Figure 10c4 is a composite image of ECAD, αSMA, and DAPI staining. Figure 10d1 is a fluorescence image showing the distribution of FOXA2, a marker for developing bladder epithelial cells, in the bladder organoids. Figure 10d2 is a fluorescence image showing the distribution of GATA3, a bladder epithelial cell marker, in the bladder organoids. Figure 10d3 is a DAPI-stained image of the bladder organoids. Figure 10d4 is a composite image of FOXA2, GATA3, and DAPI staining. [Figure 11] Figures 11a1 and 11b1 are fluorescence images showing the distribution of UPK1B and UPK2 in bladder organoids during differentiation (day 42). Figures 11a2 and 11b2 are fluorescence images showing the distribution of ΔNP63 in the bladder organoids. Figures 11a3 and 11b3 are fluorescence images showing the distribution of KRT5 in the bladder organoids. Figures 11a4 and 11b4 are DAPI-stained images of the bladder organoids. Figure 11a5 is a composite image of UPK1B, ΔNP63, KRT5, and DAPI staining. Figure 11b5 is a composite image of UPK2, ΔNP63, KRT5, and DAPI staining. DETAILED DESCRIPTION OF THE INVENTION

[0016] [Ventral hindgut organoids and their manufacturing method] One aspect of the present disclosure provides a method for producing ventral hindgut organoids. Another aspect of the present disclosure provides ventral hindgut organoids.

[0017] The term "organoid" as used herein refers to a three-dimensional cell aggregate similar to living tissue formed in a test tube (in vitro), or a cell aggregate obtained by further culturing the cell aggregate in vivo. For example, most of the cells constituting organoids are cells with differentiation and proliferation capabilities. In this specification, organoids expressing a specific marker refer to organoids containing cells expressing the specific marker at a predetermined ratio. The predetermined ratio may be, for example, 3% or more, 5% or more, 10% or more, or 15% or more of the cells constituting the organoid.

[0018] As used herein, "not substantially expressing" a particular marker means that the expression level of the particular marker is so low that it cannot be characterized by the marker. In one embodiment, organoids that "not substantially express" a particular marker may be organoids 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 particular marker in a comparable organoid. 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 the desired 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 the desired marker. A substance that can generate a signal when indirectly bound to an antibody (primary antibody) that binds to the desired 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.

[0019] In one embodiment, ventral hindgut organoids that do not substantially express SOX2 have a SOX2 expression level that is reduced to less than 70% compared to the SOX2 expression level in the hindgut organoids. In one embodiment, ventral hindgut organoids that do not substantially express at least one (e.g., one, two, or three, preferably three) dorsal hindgut marker selected from the group consisting of SOX2, T, and CDX2 have a dorsal hindgut marker expression level that is reduced to less than 70% compared to the expression level of the corresponding marker in the hindgut organoids. In the embodiment, the hindgut organoids are hindgut organoids formed by carrying out step A and step b1 (e.g., step A and step b1 described in Examples) of the present disclosure using pluripotent stem cells of the same species as the cells that constitute the ventral hindgut organoids.

[0020] The term "ventral hindgut organoid" as used herein refers to an organoid that expresses at least one ventral hindgut marker according to the present disclosure, and does not substantially express at least one dorsal hindgut marker according to the present disclosure, or even if it does, the expression level is lower than that in hindgut organoids. Ventral hindgut organoids can be produced, for example, by the method for producing ventral hindgut organoids according to the present disclosure. In the method for producing ventral hindgut organoids, when hindgut organoids are formed, and then the hindgut organoids are ventralized to form ventral hindgut organoids, these ventral hindgut organoids are sometimes referred to herein as "ventralized hindgut organoids". The major axis of the ventral hindgut organoids may be 80 μ m or more, 100 μ m or more, or 120 μ m or more. Ventral hindgut organoids can be used, for example, to produce bladder organoids, as described below. The ventral hindgut organoids can be used, for example, to evaluate drug responsiveness to a test substance. The ventral hindgut organoids can be used, for example, as an active ingredient in the regenerative medicine composition according to the present disclosure.

[0021] In one embodiment, the ventral hindgut organoid expresses the ventral hindgut marker P63; expresses HOXA13 and CK8 / KRT8; and does not substantially express the dorsal hindgut marker SOX2. In one embodiment, the ventral hindgut organoid expresses the ventral hindgut marker P63; expresses HOXA13 and CK8 / KRT8; does not substantially express the dorsal hindgut marker SOX2; and does not substantially express the dorsal hindgut marker CDX2, or even if CDX2 is expressed, its expression level is lower than that of the CDX2 in the hindgut organoid. According to one embodiment, the ventral hindgut organoids express the ventral hindgut marker P63, HOXA13, and CK8 / KRT8, and do not substantially express the dorsal hindgut markers SOX2 and / or CDX2, or, if they express SOX2 and / or CDX2, the expression levels are lower than those of SOX2 and / or CDX2 in hindgut organoids. According to one embodiment, the ventral hindgut organoids are further characterized in that the expression level of P63 is higher than that of P63 in hindgut organoids. According to one embodiment, the ventral hindgut organoids are further characterized in that the ratio of the expression level of CDX2 to the expression level of P63 is lower than the ratio of the expression level of CDX2 to the expression level of P63 in hindgut organoids. According to one embodiment, the ventral hindgut organoids are further characterized in that HOXA13 is transcribed.

[0022] In one embodiment, the ventral hindgut organoid has a lumen; expresses at least one (for example, one, two, three, or more) ventral hindgut marker selected from the group consisting of P63, ΔN63, GATA3, ISL1 and SATB2; expresses at least one (for example, one, two, three, four, or more) marker selected from the group consisting of Phospho-Smad1 / 5 / 8, HOXA13, FOXA2, CK8 / KRT8 and ECAD; and does not substantially express at least one (for example, one, two, or three, preferably three) selected from the group consisting of SOX2, T and CDX2. In one embodiment, the ventral hindgut organoid further comprises a layer of cells facing the lumen, which co-expresses CK8 / KRT8 and ZO1.

[0023] In one embodiment, the ventral hindgut organoid has lumen: expresses GATA3, which is ventral hindgut marker; expresses at least one (for example, one, two or three) marker selected from the group consisting of FOXA2, CK8 / KRT8 and ECAD; and does not substantially express at least one (for example, one, two or three, preferably three) marker selected from the group consisting of SOX2, T and CDX2.The ventral hindgut organoid is, for example, expresses FOXA2, CK8 / KRT8 and ECAD.

[0024] In one embodiment, the ventral hindgut organoid has a lumen; and comprises a layer of cells facing the lumen, which co-express CK8 / KRT8 and ZO1; and expresses at least one (for example, one, two, or three or more) ventral hindgut marker selected from the group consisting of ΔN63, GATA3, ISL1 and SATB2; and expresses at least one (for example, one, two, three, or four or more) marker selected from the group consisting of Phospho-Smad1 / 5 / 8, HOXA13, CK8 / KRT8, FOXA2 and ECAD; and does not substantially express at least one (for example, one, two, or three, preferably three) selected from the group consisting of SOX2, T and CDX2.The ventral hindgut organoid preferably expresses GATA3.The ventral hindgut organoid further expresses, for example, ΔN63, ISL1 and SATB2. The ventral hindgut organoids further express, for example, Phospho-Smad1 / 5 / 8, HOXA13, CK8 / KRT8, FOXA2, and ECAD.

[0025] The term " lumen " as used herein refers to the space inside tubular or sac-like cell structure.Lumen can be filled with, for example, liquid.In one embodiment, the lumen of bladder organoid is the space inside sac-like cell structure.

[0026] The term "P63" in relation to ventral hindgut organoids refers to a protein that is a homolog of the tumor suppressor gene P53 and has functions such as differentiation, proliferation, and maintenance in epithelium. P63 can be used as a bladder epithelial cell marker or a ventral hindgut / Cloaca marker. P63 expression in cells or organoids can be detected or measured, for example, by immunostaining using an anti-P63 antibody (e.g., anti-P63 rabbit monoclonal antibody (EPR5701)).

[0027] As used herein, 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. ΔNP63 expressed in cells or organoids can be detected or measured, for example, by immunostaining using an anti-ΔNP63 antibody (e.g., Rabbit Anti-ΔNP63 (Cell signaling, #67825)).

[0028] As used herein, the term "GATA3" refers to a transcription factor that binds to target DNA sequences consisting of "GATA" and controls gene on / off. GATA3 can be used as a marker for the ventral hindgut or bladder epithelial cells. GATA3 expression in cells or organoids can be detected or measured, for example, by immunohistochemistry using an anti-GATA3 antibody (e.g., Goat Anti-GATA3 (R&D Systems, #AF2605)).

[0029] As used herein, the term "UPK1B" refers to a membrane glycoprotein that, together with uroplakins Ia, II, and III, is involved in the formation of lining cells of the transitional epithelium that forms the urinary tract epithelium, and has the effect of enhancing the permeability and barrier function of the lining cells. UPK1B can be used as a bladder epithelial marker or epithelial tissue marker. UPK1B expressed in cells or organoids can be detected or measured, for example, by immunostaining using an anti-UPK1B antibody (Mouse Anti-UPK1B monoclonal antibody (clone 1E1) (Sigma-Aldrich, #WH0007348M2)).

[0030] As used herein, the term "ISL1" refers to a transcription factor containing the LIM homeodomain that acts on the expression regulatory region of the insulin gene. ISL1 can be used as a ventral hindgut marker. ISL1 expression in cells or organoids can be detected or measured, for example, by immunostaining using an anti-ISL1 antibody (Mouse Anti-ISL1&2 (DSHB, #39.4D5)).

[0031] The term "SATB2" as used herein 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. SATB2 expression in cells or organoids can be detected or measured, for example, by immunostaining using an anti-SATB2 antibody (Rabbit Anti-SATB2 (CELL MARQUE, #384R-14)).

[0032] As used herein, the term "CDX2" refers to a homeobox protein encoded by the CDX2 gene. CDX2 can be used as a midgut / hindgut marker. CDX2 expression in cells or organoids can be detected or measured, for example, by immunostaining using an anti-CDX2 antibody (e.g., anti-CDX2 mouse monoclonal antibody (CX2-88)).

[0033] As used herein, the term "SOX2," also known as SRY (sex determining region Y)-box 2, refers to a transcription factor essential for the self-renewal of undifferentiated ES cells. SOX2 can be used as a dorsal hindgut marker or a lung / stomach lineage marker. SOX2 expression in cells or organoids can be detected or measured, for example, by immunohistochemistry using an anti-SOX2 antibody (e.g., anti-SOX2 antibody goat polyclonal (R&D Systems, #AF2018)).

[0034] As used herein, 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 the T box, thereby influencing the transcription of genes required for mesoderm formation and differentiation. T can be used as a dorsal hindgut marker. T expression in cells or organoids can be detected or measured, for example, by immunostaining using an anti-T antibody (e.g., Goat Anti-T / Brachyury antibody (R&D Systems, #AF2085)).

[0035] As used herein, 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 cells or organoids can be detected or measured, for example, by immunostaining using an anti-ZO1 antibody (Goat Anti-ZO1 (ThermoFisher, #PA5-19090)).

[0036] As used herein, the term "cytokeratin 8 (KRT8 or CK8)" refers to a subtype of keratin protein, a protein with a molecular weight of approximately 45 kD, expressed in myoepithelial cells and membrane basal cells. CK8 / KRT8 can be used as an intestinal epithelial marker. CK8 / KRT8 expressed in cells or organoids can be detected or measured, for example, by immunostaining using rat monoclonal anti-cytokeratin 8 antibody (TROMA-1).

[0037] As used herein, the term "FOXA2" is an abbreviation for Forkhead box protein A2, a transcription factor that plays an important role in development. FOXA2 can be used as a marker for early intestinal epithelium or developing bladder epithelial cells. FOXA2 can be detected or measured, for example, by immunohistochemistry using an anti-FOXA2 antibody (Mouse Anti-FOXA2 (Santa Cruz Biotechnology, #sc-101060)).

[0038] As used herein, the term "ECAD" refers to a transmembrane glycoprotein present on the cell surface that acts on cell adhesion and is also known as E-cadherin (epithelial cadherin). ECAD can be used as an epithelial tissue marker. ECAD expressed in cells or organoids can be detected or measured, for example, by immunostaining using an anti-ECAD antibody (e.g., Goat Anti-ECAD (R&D SYSTEMS, #AF648)).

[0039] The term "HOXA13" as used herein refers to the homeobox protein encoded by HOXA13 gene in humans.The HOXA13 expressed in cells or organoids can be detected or measured, for example, by quantitative PCR.

[0040] As used herein, the term "Phospho-Smad1 / 5 / 8" refers to a transcription factor that plays an important role in the intracellular TGF-β signaling pathway. Phospho-Smad1 / 5 / 8 can be used as a marker for the ventral hindgut and Cloaca region. Phospho-Smad1 / 5 / 8 expressed in cells or organoids can be detected or measured, for example, by immunostaining using an anti-Smad1 / 5 / 8 antibody (e.g., Rabbit Anti-pSmad1 / 5 / 8 (Cell signaling, #13820)).

[0041] Ventral hindgut organoids according to one embodiment of the present disclosure can be produced by a method comprising: Step A: culturing pluripotent stem cells using induction medium A containing activin A and a GSK3β inhibitor to induce differentiation into definitive endoderm cells; and Step B: culturing the definitive endoderm cells using induction medium B containing a fibroblast growth factor and a GSK3β inhibitor, and optionally further containing a bone morphogenetic protein, and then culturing the cells in induction medium B containing a fibroblast growth factor and a GSK3β inhibitor, and optionally further containing a bone morphogenetic protein, in the presence of an extracellular matrix, to form ventral hindgut organoids.

[0042] In one embodiment, step B comprises: step b1: culture definitive endoderm cells using induction medium B (induction medium b) containing fibroblast growth factor and GSK3β inhibitor, then in the presence of extracellular matrix, culture using the induction medium B (induction medium b1) to form hindgut organoid; and step b2: culture the hindgut organoid using induction medium B (induction medium b2) containing fibroblast growth factor, GSK3β inhibitor and bone morphogenetic protein in the presence of extracellular matrix, to form ventral hindgut organoid. In one embodiment, step B comprises: step b3: culture definitive endoderm cells using induction medium B (induction medium b) containing fibroblast growth factor and GSK3β inhibitor, and may further contain bone morphogenetic protein, then in the presence of extracellular matrix, culture using induction medium B (induction medium b3) containing fibroblast growth factor, GSK3β inhibitor and bone morphogenetic protein to form ventral hindgut organoid.

[0043] Ventral hindgut organoids according to one embodiment of the present disclosure can be produced by a method comprising: step A: culturing pluripotent stem cells using the induction medium A to induce differentiation into definitive endoderm cells; step b1: culturing the definitive endoderm cells using an induction medium B (induction medium b) containing a fibroblast growth factor and a GSK3β inhibitor, and then culturing the cells using the induction medium B (induction medium b1) in the presence of an extracellular matrix to form hindgut organoids; and step b2: culturing the hindgut organoids using an induction medium B (induction medium b2) containing a fibroblast growth factor, a GSK3β inhibitor, and a bone morphogenetic protein in the presence of an extracellular matrix to form ventral hindgut organoids.

[0044] Ventral hindgut organoids according to one embodiment of the present disclosure can be produced by a method comprising: step A: culturing pluripotent stem cells using induction medium A containing activin A and a GSK3β inhibitor to induce differentiation into definitive endoderm cells; and step b3: culturing the definitive endoderm cells using induction medium B (induction medium b) containing a fibroblast growth factor and a GSK3β inhibitor, and optionally further containing a bone morphogenetic protein, and then culturing the cells using induction medium B (induction medium b3) containing a fibroblast growth factor, a GSK3β inhibitor, and a bone morphogenetic protein in the presence of an extracellular matrix to form ventral hindgut organoids.

[0045] Step A: Induction of differentiation of definitive endoderm cells Step A involves culturing pluripotent stem cells in induction medium A containing activin A and a GSK3β inhibitor to induce differentiation into definitive endoderm cells.

[0046] As used herein, 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. In one embodiment, the 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.

[0047] As used herein, the term "ES cells" refers to stem cells that have the ability to self-renew and pluripotency, and are derived from an early embryo. In one embodiment, the ES cells are human ES cells.

[0048] As used herein, the term "iPS cells" refers to pluripotent stem cells induced from somatic cells, artificially imparting pluripotency similar to that of embryonic stem cells by reprogramming somatic cells. iPS cells can be established by reprogramming differentiated cells such as fibroblasts through the expression of genes such as Oct3 / 4, Sox2, Klf4, and Myc. In one embodiment, iPS cells are human iPS cells established by reprogramming differentiated cells such as human fibroblasts.

[0049] "Induction medium A" comprises a basal medium and an additive containing activin A and a GSK3β inhibitor. Induction medium A can be prepared, for example, by adding the additive (solid or liquid) to a basal medium (liquid). The concentration of the additive added to induction medium A is appropriately determined by those skilled in the art, taking into account the animal species from which the cells used for culture are derived. 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's (BME), a known stem cell medium, or a known medium for differentiating stem cells. The basal medium is preferably a medium for differentiating stem cells, such as STEMdiff APEL2 medium (STEMCELL Technologies). The medium for differentiating stem cells can be prepared, for example, according to Nature Protocols, Vol. 3, No. 5, pp. 768-776, 2008. Induction medium A may further comprise a protein-free medium (e.g., PFHM-II), an antibiotic (e.g., penicillin / streptomycin, gentamicin), or an antibiotic-antimycotic mixture (e.g., antibiotic-antimycotic), or a combination thereof.

[0050] The term "activin A" as used herein refers to a factor belonging to the TGFβ superfamily and a protein that promotes the secretion of FSH (follicle-stimulating hormone) from the anterior pituitary gland. Activin A exhibits various functions in regulating cell differentiation, proliferation, apoptosis, and carcinogenesis. Induction medium A may contain activin A at, for example, 10 to 500 ng / mL, 30 to 250 ng / mL, or 50 to 150 ng / mL.

[0051] As used herein, the term "GSK3β inhibitor" refers to a compound that inhibits the activity of serine-threonine protein kinase 3β, which is involved in various signaling pathways, including the WNT / β-catenin pathway. Examples of GSK3β inhibitors include CHIR-99021, SB216763, CHIR-98014, staurosporine, K252A, WNT (preferably WNT3A), or TWS119, or a combination thereof. The GSK3β inhibitor is preferably CHIR-99021 or WNT (preferably WNT3A). Induction medium A may contain a GSK3β inhibitor at a concentration that exhibits the same level of GSK3β inhibitory activity as that exhibited by CHIR-99021 at 0.1 to 5 μM, 0.3 to 2.5 μM, or 0.5 to 1.5 μM. The "GSK3β inhibitory activity" can be measured, for example, based on the transcriptional activity due to nuclear translocation of β-catenin in the presence of a predetermined amount of a GSK3β inhibitor. The transcriptional activity due to the nuclear translocation of β-catenin can be measured according to known methods (e.g., luciferase activity measurement). The transcriptional activity due to the nuclear translocation of β-catenin can be measured, for example, using a commercially available kit (e.g., LEADING LIGHT (registered trademark) Wnt Reporter Assay Starter kit). As used herein, the term "similar effect" refers to an effect within ±30%, ±20%, or ±10% of the effect of a control. In one embodiment, the similar effect is an effect within ±30% of the effect of a control.

[0052] Induction medium A may contain a GSK3β inhibitor (preferably CHIR-99021) at a concentration of 0.1 to 5 μM, 0.3 to 2.5 μM, or 0.5 to 1.5 μM. Induction medium A may contain the GSK3β inhibitor at a concentration that exhibits the same level of GSK3β inhibitory activity as that exhibited by CHIR-99021 at a concentration of 0.1 to 5 μM, 0.3 to 2.5 μM, or 0.5 to 1.5 μM. Induction medium A may contain 0.2 to 200 ng / ml, 10 to 100 ng / ml, or 2 to 20 ng / ml of WNT (preferably WNT3A) as a GSK3β inhibitor. When induction medium A contains WNT (preferably WNT3A) as a GSK3β inhibitor, induction medium A may contain WNT (preferably WNT3A) at a concentration of 0.1 to 5 μM, 0.3 to 2.5 μM, or 0.5 to 1.5 μM that exhibits the same GSK3β inhibitory effect as that exhibited by CHIR-99021.

[0053] The induction medium b2 described below may contain a GSK3β inhibitor at a concentration that exhibits the same level of GSK3β inhibitory activity as that exhibited by CHIR-99021 at 1 to 50 μM, 3 to 25 μM, or 5 to 10 μM. In one embodiment, the induction medium b2 may contain 2 to 2000 ng / ml, 100 to 1000 ng / ml, or 20 to 200 ng / ml of WNT (preferably WNT3A). The induction medium b3 described below may contain a GSK3β inhibitor at a concentration that exhibits the same level of GSK3β inhibitory activity as that exhibited by CHIR-99021 at 0.5 to 25 μM, 1 to 15 μM, or 2 to 8 μM. In one embodiment, the induction medium b3 may contain 1 to 1000 ng / ml, 50 to 500 ng / ml, or 10 to 100 ng / ml of WNT (preferably WNT3A).

[0054] Pluripotent stem cells can be cultured using induction medium A under known cell culture conditions. Known cell culture conditions may be maintained at 37°C under 5% CO2. The culture temperature is not limited to 37°C, and any temperature known in the field of cell culture can be used as appropriate. The CO2 concentration is not limited to 5%, and any CO2 concentration known in the field of cell culture can be used as appropriate. Pluripotent stem cells can be cultured for 2 to 5 days, 2 to 4 days, or 3 days.

[0055] "Definitive endoderm cells" can be induced to differentiate by culturing pluripotent stem cells using induction medium A containing activin A and a GSK3β inhibitor.

[0056] Pluripotent stem cells cultured in induction medium A may be pre-cultured using a known medium for stem cells. Such pluripotent stem cells can be pre-cultured, for example, in a cell culture vessel coated with iMatrix-511 using StemFit AK02N medium (REPROCELL). The pre-cultured pluripotent stem cells can be detached from the cell culture vessel using a known method or a commercially available reagent (e.g., TrypLE Select (Thermo Fisher Scientific)). The detached cells can be cultured in a medium (e.g., iMatrix-511 (nippi)) at a final concentration of 0.25 μg / cm. 2 The cells may be suspended in StemFit AK02N (10 μM Y-27632) added so that the concentration is

[0057] In one embodiment, the culture of pluripotent stem cells using induction medium A is carried out by culturing a cell suspension of pre-cultured pluripotent stem cells (e.g., human iPS or ES cells) at a density of 30,000 to 90,000 cells / cm. 2The method includes seeding the cells into a 6-well plate so that the cells are 100 ng / mL Activin A, 1-1.25 μM CHIR99021, 2% PFHM-II, and culturing them at 37°C and 5% CO2 for 1 day. Next, the medium is replaced with induction medium A, STEMdiff APEL2 medium (STEMCELL Technologies) (supplemented with 100 ng / mL Activin A, 1-1.25 μM CHIR99021, 2% PFHM-II, and an antibiotic-antimycotic), and culturing for 3-5 days. This culturing induces differentiation into definitive endoderm. The medium is replaced daily from day 2 onwards. Preferably, the cells are confirmed to express definitive endoderm markers FOXA2 and SOX17. If the definitive endoderm induction period is too long, the proportion of foregut lineage cells (ALB+, AFP+, PDX1+) contaminating the cells during the subsequent hindgut induction (posteriorization) process tends to increase. Therefore, it is preferable to select the definitive endoderm induction period during which FOXA2 and SOX17 expression and the subsequent hindgut induction process are both suppressed to the greatest extent possible. The seeding cell density is preferably selected such that colony-like cell groups spread outward on day 2 of differentiation induction and reach 100% confluence in a sheet-like state on day 3 of differentiation induction. Furthermore, it is preferable to select conditions under which the greatest number of floating spheroids are obtained during hindgut induction. When the cell density is low or high, it tends to be difficult to obtain sufficient floating spheroids.

[0058] Step b1: Hindgut differentiation induction Step b1 includes culturing definitive endoderm cells using induction medium B (induction medium b) containing fibroblast growth factor and GSK3β inhibitor, and then culturing them in the presence of extracellular matrix using induction medium B (induction medium b1) containing fibroblast growth factor and GSK3β inhibitor to form hindgut organoids.

[0059] "Induction medium B" comprises a basal medium and an additive containing a fibroblast growth factor and a GSK3β inhibitor, and may further comprise a bone morphogenetic protein. In one embodiment, induction medium B is used to form spheroids of definitive endoderm cells and comprises a basal medium and an additive containing a fibroblast growth factor and a GSK3β inhibitor. The induction medium B used to form the spheroids is also referred to herein as "induction medium b." In one embodiment, induction medium B is used to form hindgut organoids and comprises a basal medium and an additive containing a fibroblast growth factor and a GSK3β inhibitor. The induction medium B used to form hindgut organoids from the spheroids is also referred to herein as "induction medium b1." The induction medium B used to form ventral hindgut organoids from hindgut organoids, as described below, is also referred to herein as "induction medium b2." Induction medium B used to form ventral hindgut organoids from definitive endoderm cells described below or induction medium B used to form ventral hindgut organoids from the spheroids is also referred to as "induction medium b3" in this specification.

[0060] Induction medium B can be prepared, for example, by adding the additives (solid or liquid) to a basal medium (liquid). The explanation for the basal medium for induction medium A applies as appropriate to the basal medium for induction medium B. The concentrations of the additives added to induction medium B are appropriately determined by those skilled in the art, taking into consideration the animal species from which the cells used for culture are derived. Induction medium B may further contain heparin, a protein-free medium (e.g., PFHM-II), an antibiotic (e.g., penicillin / streptomycin, gentamicin), an antibiotic-antimycotic mixture (e.g., antibiotic-antimycotic), or a combination thereof.

[0061] "Induction medium b" or "induction medium b1" comprises a basal medium and an additive containing FGF and a GSK3β inhibitor. Induction medium b and b1 can be prepared, for example, by adding the additive (solid or liquid) to a basal medium (liquid). The explanation for the basal medium for induction medium A applies to the basal medium for induction medium b and b1, as appropriate. In one embodiment, induction medium b and b1 comprise a fibroblast growth factor and a GSK3β inhibitor and are substantially free of bone morphogenetic protein. In one embodiment, induction medium b and b1 that are substantially free of bone morphogenetic protein are completely free of bone morphogenetic protein. In one embodiment, induction medium b that is substantially free of bone morphogenetic protein may contain bone morphogenetic protein at a concentration that does not inhibit spheroid formation of definitive endoderm cells, for example, less than 1 ng / ml, less than 0.5 ng / ml, or less than 0.1 ng / ml of bone morphogenetic protein. In one embodiment, induction medium b1 that is substantially free of bone morphogenetic protein may contain bone morphogenetic protein at a concentration that does not inhibit hindgut organoid formation from spheroids of definitive endoderm cells, for example, less than 1 ng / ml, less than 0.5 ng / ml, or less than 0.1 ng / ml of bone morphogenetic protein.

[0062] As used herein, 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. FGF (e.g., FGF4) is added, for example, to induction medium B to suppress contamination by cells derived from anterior endoderm. FGF (e.g., FGF4) is added, for example, to induction medium B to form hindgut spheroids. Examples of FGFs include FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23, or a combination thereof. Preferably, FGF is FGF4 or FGF7, or a combination thereof.

[0063] The induction media b and b1 may contain 20 to 1000 ng / mL, 60 to 500 ng / mL, or 100 to 300 ng / mL of FGF (preferably FGF4). The induction media b and b1 may contain 10 to 500 ng / mL, 30 to 250 ng / mL, or 50 to 150 ng / mL of FGF7, or 20 to 1000 ng / mL, 60 to 500 ng / mL, or 100 to 300 ng / mL of FGF4. Induction medium b or b1 may contain FGF at a concentration that exhibits the same level of expression suppression as that of anterior endoderm cell markers (e.g., ALB, a liver lineage marker, PDX1, a pancreatic lineage marker, or SOX2, a lung / gastric lineage marker) in cells or organoids cultured in induction medium b or b1 containing 20-1000 ng / mL, 60-500 ng / mL, or 100-300 ng / mL FGF4. In this context, cells cultured using induction medium b may be definitive endoderm cells induced to differentiate from pluripotent stem cells in step A of the present disclosure. In this context, cells cultured using induction medium b1 may be spheroids formed from the definitive endoderm cells in step b1 of the present disclosure. Expression of anterior endoderm cell markers can be measured, for example, by immunostaining. In immunostaining, expression of anterior endoderm cell markers can be measured, for example, using commercially available antibodies. As used herein, "similar levels of suppression of expression" refer to expression levels within ±30%, ±20%, or ±10% of the expression level of a control. In one embodiment, the similar level of suppression is expression levels within ±30%.

[0064] As used herein, the term "ALB" refers to albumin, a protein consisting of approximately 600 amino acids and having a molecular weight of approximately 66,000. ALB can be used, for example, as a liver lineage marker. ALB expressed in cells or organoids can be detected or measured, for example, by immunostaining using an anti-albumin antibody.

[0065] As used herein, the term "Pancreatic and duodenal homeobox factor-1 (PDX1)" refers to a homeodomain protein that binds to the insulin gene promoter region and is involved in pancreatic β-cell-specific expression of the insulin gene. PDX1 can be used, for example, as a pancreatic lineage marker. PDX1 expression in cells or organoids can be detected or measured, for example, by immunostaining using an anti-PDX1 antibody.

[0066] The induction medium b or b1 may contain FGF at a concentration that exhibits the same level of fibroblast proliferation effect as that exhibited by 20 to 1000 ng / mL, 60 to 500 ng / mL, or 100 to 300 ng / mL of FGF4. The "fibroblast proliferation effect" refers to, for example, the ability of fibroblasts to [ 3 The amount of thymidine taken up into the cells can be measured based on the amount of [H] thymidine taken up from the outside of the cells into the cells. For example, NR6R-3T3 mouse fibroblasts can be used as the fibroblasts. The amount of thymidine taken up from the outside of the cells into the cells can be measured according to known methods (e.g., the method described in *Ethods in Enzymology*, Volume 109, 1985, Pages 749-773).

[0067] In one embodiment, induction medium b or b1 contains FGF at a concentration that exerts an effect similar to the fibroblast proliferation effect exerted by 20 to 1000 ng / mL, 60 to 500 ng / mL, or 100 to 300 ng / mL of FGF4.

[0068] The induction media b and b1 may contain a GSK3β inhibitor (preferably CHIR-99021) at a concentration of 1 to 50 μM, 3 to 25 μM, 5 to 10 μM, 0.5 to 16 μM, 3 to 12 μM, or 6 to 10 μM. The induction media b and b1 may contain a WNT (preferably WNT3A) at a concentration of 2 to 2000 ng / ml, 100 to 1000 ng / ml, or 20 to 200 ng / ml. The induction media b and b1 may contain a GSK3β inhibitor at a concentration that exhibits the same level of GSK3β inhibitory activity as that exhibited by CHIR-99021 at a concentration of 1 to 50 μM, 3 to 25 μM, 5 to 10 μM, 0.5 to 16 μM, 3 to 12 μM, or 6 to 10 μM, or 0.1 to 5 μM, 0.3 to 2.5 μM, or 0.5 to 1.5 μM. When induction medium b or b1 contains WNT (preferably WNT3A) as a GSK3β inhibitor, induction medium b3 contains WNT (preferably WNT3A) at a concentration of 1 to 50 μM, 3 to 25 μM, 5 to 10 μM, 0.5 to 16 μM, 3 to 12 μM, or 6 to 10 μM that exhibits the same GSK3β inhibitory effect as that exhibited by CHIR-99021.

[0069] The induction medium b and the induction medium b1 may be the same or different in composition and / or amount. In one embodiment, the induction medium b and the induction medium b1 are the same in composition and / or amount.

[0070] "Hindgut organoids" can be formed by culturing definitive endoderm cells using, for example, induction medium B according to the present disclosure. In one embodiment, hindgut organoids can be formed by culturing definitive endoderm cells using, for example, induction medium b containing FGF and a GSK3β inhibitor, and then three-dimensionally culturing them in an extracellular matrix gel using induction medium b1 containing FGF and a GSK3β inhibitor. The hindgut organoids in the extracellular matrix gel can be recovered according to known methods or using commercially available reagents. The hindgut organoids in the extracellular matrix gel can be recovered by gently breaking down the extracellular matrix gel, for example, using metalloprotease.

[0071] In one embodiment, hindgut organoid can be formed by using the induction medium b of definitive endoderm cell, for example, that contains FGF and GSK3β inhibitor, and then using the induction medium b1 of FGF and GSK3β inhibitor to culture on extracellular matrix gel.In one embodiment, hindgut organoid can be formed by using the induction medium b of definitive endoderm cell, for example, that contains FGF and GSK3β inhibitor, and then using the induction medium b1 of FGF and GSK3β inhibitor to culture as dispersion component.

[0072] As used herein, the term "extracellular matrix (ECM)" includes, but is not limited to, water, polysaccharides, elastin, integrins, and glycoproteins. Glycoproteins include, for example, collagen, entactin (nidogen), fibronectin, and laminin. ECM can be prepared, for example, by culturing ECM-producing cells (e.g., epithelial cells, endothelial cells, mural endoderm-like cells, or fibroblasts) in vitro and then removing the ECM cells. 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. 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. In one embodiment, the ECM is Matrigel.

[0073] The extracellular matrix gel for hindgut organoid formation is preferably a gel with elasticity comparable to that of a gel formed with a Matrigel solution having a protein concentration of 1 to 8 mg / mL, 1.5 to 6 mg / mL, or 2.5 mg / mL or 5 mg / mL. The extracellular matrix gel for hindgut organoid formation is preferably formed with Matrigel having a protein concentration of 1 to 8 mg / mL, 1.5 to 6 mg / mL, or 1.5 to 3.5 mg / mL or 4 to 6 mg / mL. When forming bladder organoids by performing step c2 described below, the extracellular matrix gel is preferably a gel with elasticity comparable to that of a gel formed with a Matrigel solution having a protein concentration of 1 to 4 mg / mL, 2 to 3 mg / mL, or 2.5 mg / mL. As used herein, the term "similar elasticity" refers to an elasticity within ±30%, ±20%, or ±10% of the target elasticity. In one embodiment, the equivalent elasticity is within ±30% of the target elasticity. The extracellular matrix gel for forming bladder organoids is preferably formed using Matrigel with a protein concentration of 1 to 4 mg / mL, 2 to 3 mg / mL, or 2.5 mg / mL.

[0074] The extracellular matrix as a "dispersed component" is present, for example, in a dispersed or dissolved state in the induction medium. When present as a dispersed component, the extracellular matrix is ​​present, for example, in an amount of 10% v / v or less, 7% v / v or less, 5% v / v or less, e.g., 2.5% v / v per volume of the induction medium. In one embodiment, the induction medium contains the extracellular matrix in an amount of 0.1 to 10% v / v, 0.1 to 7% v / v, 0.1 to 5% v / v, 1 to 10% v / v, 1 to 7% v / v, 1 to 5% v / v, 1 to 2.5% v / v, 2 to 10% v / v, 2 to 7% v / v, 2 to 5% v / v, or 2 to 2.5% v / v. When the extracellular matrix contains a biological product (e.g., a basement membrane preparation from mouse sarcoma cells), the concentration of the extracellular matrix in the induction medium is preferably low in order to prevent or reduce contamination of the organoids being produced.

[0075] Culturing definitive endoderm cells using induction medium B includes, for example, culturing the cells in a state where they are adherent to the surface of a culture vessel. Culture in this state is also referred to as two-dimensional culture herein. In one embodiment, two-dimensional culture of definitive endoderm cells using induction medium B results in the formation of spheroids of the cells, some of which may float in the medium. In one embodiment, static culture of definitive endoderm cells in a low-adhesion culture vessel using induction medium B results in the formation of spheroids of the cells, some of which may float in the medium. Spheroids floating in the medium and spheroids attached to the culture vessel can be recovered by pipetting or the like. Culturing definitive endoderm cells using induction medium B can be carried out, for example, for 3 to 6 days, 3 to 5 days, or 4 days under known cell culture conditions.

[0076] Culturing definitive endoderm cells using induction medium b1 includes culturing spheroids of the definitive endoderm cells in the presence of an extracellular matrix. Culturing the spheroids in the presence of an extracellular matrix includes, for example, culturing the spheroids using induction medium b1 containing an extracellular matrix as a dispersed component. The culture can be performed, for example, by static culture or rotary suspension culture using a low-adhesive culture vessel. Culturing the spheroids in the presence of an extracellular matrix as a dispersed component can form hindgut organoids. Culturing the spheroids in the presence of an extracellular matrix includes, for example, seeding the spheroids on an extracellular matrix gel and culturing them. Culturing the spheroids on an extracellular matrix gel can form hindgut organoids. Culturing the spheroids in the presence of an extracellular matrix includes, for example, culturing the spheroids in an extracellular matrix gel. Culturing under such conditions is also referred to as three-dimensional culture herein. Hindgut organoids can be formed by three-dimensional culture of definitive endoderm cell spheroids. Culture of definitive endoderm cell spheroids using induction medium b1 in the presence of an extracellular matrix can be carried out for, for example, 3 to 6 days, 3 to 5 days, or 4 days under known cell culture conditions.

[0077] In one embodiment, step b1 comprises culturing definitive endoderm cells using induction medium b to form definitive endoderm cell spheroids, forming an extracellular matrix gel containing the definitive endoderm cell spheroids, and then culturing the spheroids in the extracellular matrix gel using induction medium b1 to form hindgut organoids.In one embodiment, step b1 comprises culturing definitive endoderm cells using induction medium b to form definitive endoderm cell spheroids, and then culturing the spheroids on the extracellular matrix gel using induction medium b1 to form hindgut organoids.In one embodiment, step b1 comprises culturing definitive endoderm cells using induction medium b to form definitive endoderm cell spheroids, and then culturing the spheroids in the presence of extracellular matrix as a dispersed component using induction medium b1 to form hindgut organoids.

[0078] In one embodiment, step b1 involves culturing definitive endoderm cells in induction medium b, STEMdiff APEL2 medium (200 ng / mL FGF4, 8 μM CHIR99021, 1 μg / mL Heparin, 2% PFHM-II, Antibiotic-Antimycotic), for 1 to 9 days to form spheroids of definitive endoderm cells. During this spheroid formation period, floating spheroids are collected when they are most floating, and 3D culture is initiated. Shaking culture at 100 rpm is initiated the day before the most floating spheroids are collected. The next day, the floating spheroids are collected into a 35 mm dish. Then, the floating spheroids are pipetted into the remaining spheroids in the well from which they were collected. The collected spheroids are placed on ice. Matrigel Growth Factor Reduced (Corning) was diluted with induction medium b1 (2.5–10 mg / mL) and added at 50 μL / well to a 24-well Cell Culture Insert with a Transparent PET Membrane and 8.0 μm pore size (Corning). The mixture was incubated at 37°C for 30–60 minutes to allow gelation. Next, 30–100 recovered spheroids were added to 50 μL of Matrigel solution, mixed, and the entire volume was added on top of the gelled Matrigel. The mixture was incubated at 37°C under 25% CO2 for 30–60 minutes to allow gelation. Then, 200 μL of induction medium b1 was added to the top of the Cell Culture Insert and 300 μL to the bottom. The mixture was then cultured at 37°C under 25% CO2. Medium changes are preferably performed daily for 2D culture and every two days for 3D culture. During hindgut differentiation induction, it is preferable to confirm the co-expression of the midgut / hindgut markers FOXA2 and CDX2 in the cells. Culture may be continued in induction medium b1 until expression of P63, which is expressed in the hindgut / Cloaca region, is confirmed.

[0079] Step b2: Ventralization of the hindgut Step b2 involves culturing the hindgut organoids in induction medium B (induction medium b2) containing fibroblast growth factor, a GSK3β inhibitor, and bone morphogenetic protein in the presence of an extracellular matrix to form ventral hindgut organoids.

[0080] "Induction medium b2" comprises a basal medium and additives containing FGF, a GSK3β inhibitor, and an osteogenic factor. Induction medium b2 can be prepared, for example, by adding the additives (solid or liquid) to a basal medium (liquid). The explanation for the basal medium for induction medium A applies appropriately to the basal medium for induction medium b2.

[0081] Induction medium b2 may contain FGF (preferably FGF4) at 20 to 1000 ng / mL, 60 to 500 ng / mL, or 100 to 300 ng / mL. Induction medium b2 may contain FGF7 at 10 to 500 ng / mL, 30 to 250 ng / mL, or 50 to 150 ng / mL, or FGF4 at 20 to 1000 ng / mL, 60 to 500 ng / mL, or 100 to 300 ng / mL. Induction medium b2 may contain FGF at a concentration that exhibits the same level of expression suppression of anterior endoderm cell markers (e.g., the liver lineage marker ALB, the pancreatic lineage marker PDX1, or the lung / gastric lineage marker SOX2) as that observed in organoids cultured in induction medium b2 containing 20 to 1000 ng / mL, 60 to 500 ng / mL, or 100 to 300 ng / mL of FGF4. In this context, the organoids cultured using induction medium b2 may be hindgut organoids differentiated from definitive endoderm cells in step b1 of the present disclosure. Induction medium b2 may contain FGF at a concentration that exhibits a fibroblast proliferation effect similar to that exhibited by 20-1000 ng / mL, 60-500 ng / mL, or 100-300 ng / mL of FGF4. In one embodiment, induction medium b2 contains FGF at a concentration that exhibits a fibroblast proliferation effect similar to that exhibited by 20-1000 ng / mL, 60-500 ng / mL, or 100-300 ng / mL of FGF4.

[0082] Induction medium b2 may contain a GSK3β inhibitor (preferably CHIR-99021) at a concentration of 1 to 50 μM, 3 to 25 μM, 5 to 10 μM, 0.5 to 16 μM, 3 to 12 μM, or 6 to 10 μM. Induction medium b2 may contain 2 to 2000 ng / ml, 100 to 1000 ng / ml, or 20 to 200 ng / ml of WNT (preferably WNT3A) as the GSK3β inhibitor. Induction medium b2 may contain the GSK3β inhibitor at a concentration that exhibits the same level of GSK3β inhibitory activity as that exhibited by CHIR-99021 at a concentration of 1 to 50 μM, 3 to 25 μM, or 5 to 10 μM. When induction medium b2 contains WNT (preferably WNT3A) as a GSK3β inhibitor, induction medium b2 may contain WNT (preferably WNT3A) at a concentration of 1 to 50 μM, 3 to 25 μM, or 5 to 10 μM that exhibits the same level of GSK3β inhibitory effect as that exhibited by CHIR-99021.

[0083] As used herein, the term "bone morphogenetic protein (BMP)" refers to a protein belonging to the transforming growth factor β superfamily that induces ectopic bone formation. BMP is added to induction medium B (e.g., induction medium b2 and b3) for the purpose of inducing ventral hindgut organoids. BMP may be, for example, BMP2, BMP4, or BMP7, or a combination thereof. BMP is preferably BMP4. Induction medium b2 may contain BMP (e.g., BMP4) at 3 to 150 ng / mL, 9 to 75 ng / mL, or 15 to 45 ng / mL.

[0084] Induction medium b2 may contain BMP at a concentration of 3 to 150 ng / mL, 9 to 75 ng / mL, or 15 to 45 ng / mL, which has an alkaline phosphatase production-inducing effect similar to that of BMP4. The "alkaline phosphatase production-inducing effect" can be measured, for example, based on the amount of alkaline phosphatase produced in chondrogenic cells in the presence of a predetermined amount of BMP. Mouse chondrogenic cells can be used as chondrogenic cells. The amount of alkaline phosphatase produced can be measured according to known methods (e.g., the method described in Biochemical and Biophysical Research Communications, Volume 315, Issue 2, Pages 272-280).

[0085] Induction medium b2 may contain BMP at a concentration that exhibits expression of ventral hindgut markers (e.g., GATA3, P63, or HOXA13) at a level comparable to that of cells or organoids cultured in induction medium b2 containing 3 to 150 ng / mL, 9 to 75 ng / mL, or 15 to 45 ng / mL of BMP4. In this context, the cells cultured using induction medium b2 may be hindgut organoids induced to differentiate from definitive endoderm cells in step b1 of the present disclosure. The expression of the ventral hindgut markers can be measured, for example, by immunostaining. In immunostaining, the expression of the ventral hindgut markers can be measured using antibodies disclosed herein for each marker. As used herein, the term "expression at a similar level" refers to expression within ±30%, ±20%, or ±10% of the expression level of a control. In one embodiment, expression at a similar level refers to expression within ±30% of the expression level of a control.

[0086] In one embodiment, induction medium b2 contains BMP at a concentration that exhibits an alkaline phosphatase production-inducing effect similar to that exhibited by BMP4 at 3 to 150 ng / mL, 9 to 75 ng / mL, or 15 to 45 ng / mL.

[0087] The induction medium b, the induction medium b1, and the medium obtained by removing BMP from the induction medium b2 may be the same or different in composition and / or dosage. In one embodiment, the induction medium b, the induction medium b1, and the medium obtained by removing BMP from the induction medium b2 have the same composition and / or dosage.

[0088] The culturing of hindgut organoids using induction medium b2 in the presence of an extracellular matrix is ​​carried out as appropriate using the explanation for culturing definitive endoderm cell spheroids using induction medium b1 in the presence of an extracellular matrix. The culturing of hindgut organoids using induction medium b2 in the presence of an extracellular matrix can be carried out, for example, under known cell culture conditions. Known cell culture conditions may be maintained at 37°C under 5% CO2. The culturing temperature is not limited to 37°C, and any temperature known in the field of cell culture can be used as appropriate. The CO2 concentration is not limited to 5%, and any CO2 concentration known in the field of cell culture can be used as appropriate. The culturing of hindgut organoids can be carried out for 3 to 6 days, 3 to 5 days, or 4 days.

[0089] The state of the extracellular matrix (gel state or dispersed component state) in step b1 and step b2 may be the same or different. In one embodiment, the state of the extracellular matrix in step b1 and step b2 is the same. In one embodiment, the extracellular matrix gel containing hindgut organoids may be the extracellular matrix gel formed in step b1, or may be a newly formed extracellular matrix gel containing the recovered hindgut organoids after step b1, after which the hindgut organoids are recovered from the extracellular matrix gel. From the viewpoint of operational simplicity, the extracellular matrix gel containing hindgut organoids in step b2 is the extracellular matrix gel formed in step b1.

[0090] In one embodiment, step b2 comprises that the hindgut organoid in extracellular matrix gel is cultured with induction medium b2 to form ventral hindgut organoid.In one embodiment, step b2 comprises that the hindgut organoid on extracellular matrix gel is cultured with induction medium b2 to form ventral hindgut organoid.In one embodiment, step b2 comprises that the hindgut organoid is cultured with induction medium b2 under the presence of extracellular matrix as dispersion component to form ventral hindgut organoid.

[0091] In one embodiment, culturing hindgut organoids using induction medium b2 involves culturing hindgut organoids in an extracellular matrix gel for 3-6 days using induction medium b2: STEMdiff APEL2 medium (30 ng / mL BMP4, 200 ng / mL FGF4, 8 μM CHIR99021, 1 μg / mL heparin, 2% PFHM-II, antibiotic-antimycotic) or STEMdiff APEL2 medium (30 ng / mL BMP4, 100 ng / mL FGF7, 200 ng / mL FGF4, 8 μM CHIR99021, 1 μg / mL heparin, 2% PFHM-II, antibiotic-antimycotic) to ventralize the hindgut. Induction medium b2 is added in 200 μL on top of the Cell Culture Insert gel and 300 μL on the bottom, and the medium is changed every 2 days. It is preferable to continue ventralization until the expression of dorsal hindgut markers SOX2 and CDX2 is reduced and the ventral hindgut / Cloaca marker P63 is strongly expressed in the cells. If posteriorization and ventralization are insufficient, cells of the midgut and dorsal gut lineages will be mixed in. In this case, the hindgut induction period or the hindgut ventralization period should be extended.

[0092] Step b3: Ventral hindgut organoid formation Step b3 comprises culturing definitive endoderm cells using induction medium B, which contains fibroblast growth factor and GSK3β inhibitor, and may further contain bone morphogenetic protein, and then culturing in the presence of extracellular matrix using induction medium B (induction medium b3) containing fibroblast growth factor, GSK3β inhibitor, and bone morphogenetic protein to form ventral hindgut organoids. In one embodiment, step b3 comprises culturing definitive endoderm cells using the induction medium B to form definitive endoderm cell spheroids, and then culturing in the presence of extracellular matrix using the induction medium b3 to form ventral hindgut organoids. In one embodiment, the induction medium B for forming definitive endoderm cell spheroids may be the same as the induction medium b3 in composition and / or dosage. In this context, step b3 comprises culturing definitive endoderm cells with induction medium B (induction medium b3) containing fibroblast growth factor and GSK3β inhibitor and bone morphogenetic protein, and then culturing with induction medium B (induction medium b3) in the presence of extracellular matrix to form ventral hindgut organoids.

[0093] According to one embodiment of the present disclosure, ventral hindgut organoids can be produced by a method comprising: step A: culturing pluripotent stem cells using induction medium A containing activin A and a GSK3β inhibitor to induce differentiation into definitive endoderm cells; and step b3: culturing the definitive endoderm cells using induction medium B (induction medium b3) which may contain a fibroblast growth factor, a GSK3β inhibitor, and a bone morphogenetic protein, and then culturing the definitive endoderm cells using induction medium B (induction medium b3) in the presence of an extracellular matrix to form ventral hindgut organoids.

[0094] "Induction medium b3" comprises a basal medium and additives containing FGF, a GSK3β inhibitor, and an osteogenic factor. Induction medium b3 can be prepared, for example, by adding the additives (solid or liquid) to a basal medium (liquid). The explanation for the basal medium for induction medium A applies appropriately to the basal medium for induction medium b3.

[0095] Induction medium b3 may contain FGF (preferably FGF4) at 20 to 1000 ng / mL, 60 to 500 ng / mL, or 100 to 300 ng / mL. Induction medium b3 may contain FGF7 at 10 to 500 ng / mL, 30 to 250 ng / mL, or 50 to 150 ng / mL, or FGF4 at 20 to 1000 ng / mL, 60 to 500 ng / mL, or 100 to 300 ng / mL. Induction medium b3 may contain a GSK3β inhibitor (preferably CHIR-99021) at 0.5 to 25 μM, 1 to 15 μM, 2 to 8 μM, 0.1 to 10 μM, 0.5 to 9 μM, 1 to 8 μM, 2 to 6 μM, or 3 to 5 μM. The induction medium b3 may contain an osteogenic factor (preferably BMP4) at a concentration of 0.1 to 100 ng / mL, 0.5 to 80 ng / mL, 1 to 60 ng / mL, 2 to 40 ng / mL, or 5 to 20 ng / mL.

[0096] Induction medium b3 may contain FGF at a concentration that exhibits the same level of expression suppression as that of anterior endoderm cell markers (e.g., ALB, a liver lineage marker, PDX1, a pancreatic lineage marker, or SOX2, a lung / gastric lineage marker) in cells or organoids cultured in induction medium b3 containing 20-1000 ng / mL, 60-500 ng / mL, or 100-300 ng / mL FGF4. In this context, the cells cultured using induction medium b3 may be definitive endoderm cells induced to differentiate from pluripotent stem cells in step A of the present disclosure. In this context, the organoids cultured using induction medium b3 may be hindgut organoids induced to differentiate from the definitive endoderm cells in step b1 of the present disclosure. Induction medium b3 may contain FGF at a concentration that exhibits an effect similar to the fibroblast proliferation effect exhibited by 20 to 1000 ng / mL, 60 to 500 ng / mL, or 100 to 300 ng / mL of FGF4. In one embodiment, induction medium b3 contains FGF at a concentration that exhibits an effect similar to the fibroblast proliferation effect exhibited by 20 to 1000 ng / mL, 60 to 500 ng / mL, or 100 to 300 ng / mL of FGF4.

[0097] Induction medium b3 may contain WNT (preferably WNT3A) as a GSK3β inhibitor at a concentration of 1 to 1000 ng / mL, 50 to 500 ng / mL, or 10 to 100 ng / mL. Induction medium b3 may contain the GSK3β inhibitor at a concentration that exhibits the same level of GSK3β inhibitory activity as that exhibited by CHIR-99021 at a concentration of 0.5 to 25 μM, 1 to 15 μM, or 2 to 8 μM. When induction medium b3 contains WNT (preferably WNT3A) as a GSK3β inhibitor, induction medium b3 may contain WNT (preferably WNT3A) at a concentration that exhibits the same level of GSK3β inhibitory activity as that exhibited by CHIR-99021 at a concentration of 0.5 to 25 μM, 1 to 15 μM, or 2 to 8 μM.

[0098] Induction medium b3 may contain BMP at a concentration that exhibits an alkaline phosphatase production-inducing effect similar to that exhibited by BMP4 at 0.1 to 100 ng / mL, 0.5 to 80 ng / mL, 1 to 60 ng / mL, 2 to 40 ng / mL, or 5 to 20 ng / mL. Induction medium b3 may contain BMP at a concentration that exhibits an expression similar to that of ventral hindgut markers (e.g., GATA3, p63, or HOXA13) in cells or organoids cultured in induction medium b3 containing 0.1 to 100 ng / mL, 0.5 to 80 ng / mL, 1 to 60 ng / mL, 2 to 40 ng / mL, or 5 to 20 ng / mL BMP4. In one embodiment, induction medium b3 contains BMP at a concentration of 0.1 to 100 ng / mL, 0.5 to 80 ng / mL, 1 to 60 ng / mL, 2 to 40 ng / mL, or 5 to 20 ng / mL that exhibits the same alkaline phosphatase production-inducing effect as that exhibited by BMP4.

[0099] In one embodiment, step b3 comprises culturing definitive endoderm cells using induction medium B (preferably induction medium b3) to form definitive endoderm cell spheroids, forming an extracellular matrix gel containing the definitive endoderm cell spheroids, and then culturing the spheroids in the extracellular matrix gel using induction medium b3 to form ventral hindgut organoids.In one embodiment, step b3 comprises culturing definitive endoderm cells using induction medium B (preferably induction medium b3) to form definitive endoderm cell spheroids, and then culturing the spheroids on the extracellular matrix gel using induction medium b3 to form ventral hindgut organoids.In one embodiment, step b3 comprises culturing definitive endoderm cells using induction medium B (preferably induction medium b3) to form definitive endoderm cell spheroids, and then culturing the spheroids in the presence of extracellular matrix as a dispersed component using induction medium b3 to form ventral hindgut organoids.

[0100] In one embodiment, step b3 involves culturing definitive endoderm cells in induction medium b3, STEMdiff APEL2 medium (10-30 ng / mL BMP4, 200 ng / mL FGF4, 4 μM CHIR99021, 1 μg / mL heparin, 2% PFHM-II, antibiotic-antimycotic), for 8-11 days to form spheroids of definitive endoderm cells. During this spheroid formation period, floating spheroids are collected when they are most floating, and 3D culture is initiated. Shaking culture at 100 rpm is initiated the day before the most floating spheroids are obtained. The following day, the floating spheroids are collected into a 35 mm dish. Next, medium is sprayed onto the spheroids remaining in the well from which the floating spheroids were collected, and the spheroids are suspended and collected. The collected spheroids are placed on ice. Matrigel Growth Factor Reduced (Corning) was diluted with induction medium b3 (25–100% concentration) and added 50 μL / well to a 24-well Cell Culture Insert Transparent PET Membrane with 8.0 μm pore size (Corning). The mixture was incubated at 37°C for 30–60 minutes to allow gelation. Next, 30–100 recovered spheroids were added to 50 μL of Matrigel solution, mixed, and the entire volume was added on top of the gelled Matrigel. The mixture was incubated at 37°C and 5% CO2 for 30–60 minutes to allow gelation. Then, 200 μL of induction medium b3 was added to the top of the Cell Culture Insert and 300 μL to the bottom. The mixture was then cultured at 37°C and 5% CO2. It is recommended that the medium be changed daily for 2D culture and every two days for 3D culture. During ventral hindgut differentiation, cells are cultured in induction medium b3 until the expression of dorsal midgut / hindgut markers CDX2, SOX2, and T is reduced or absent, and the expression of ventral hindgut / Cloaca markers GATA3 and p63 is observed. The resulting ventral hindgut organoids are rounded and possess a luminal structure. In organoids that are not sufficiently posteriorly or ventrally differentiated, midgut and dorsal gut lineage cells are observed.In this case, the culture in induction medium b3 is extended or the concentration of BMP4 in induction medium b3 is increased.

[0101] The explanations of each term and the explanations of the embodiments, including the culture conditions, in each step of inducing differentiation of definitive endoderm cells (step A) and forming ventral hindgut organoids (step B) apply, as appropriate, to the corresponding terms and embodiments described in those steps.

[0102] [Bladder organoids and their manufacturing method] One aspect of the present disclosure provides a method for producing bladder organoids. Another aspect of the present disclosure provides bladder organoids.

[0103] The term " bladder organoid " as used herein refers to the organoid that has at least two cell layers.These at least two cell layers include: a first cell layer that comprises the cell that does not substantially express P63 but expresses UPK1B and / or UPK2; and a second cell layer that comprises the cell that co-expresses P63 and UPK1B and / or UPK2, and is located outward relative to the first cell layer.In one embodiment, the bladder organoid comprises: a first cell layer that comprises the cell that does not substantially express P63 but expresses UPK1B and / or UPK2; and a second cell layer that comprises the cell that co-expresses P63 and UPK1B and / or UPK2, and is located outward relative to the first cell layer; and a third cell layer that comprises the cell that co-expresses P63 and KRT5, and is located outward relative to the second cell layer. For example, the bladder organoid may be specified as comprising: a first cell layer, which is located along its outermost periphery and comprises cells that co-express P63 and KRT5; a second cell layer, which is located inward relative to the first cell layer and comprises cells that co-express P63 and UPK1B and / or UPK2; and a third cell layer, which is located inward relative to the second cell layer and comprises cells that do not substantially express P63 but express UPK1B and / or UPK2.In this specification, the first cell layer is also referred to as a basal cell layer, the second cell layer is also referred to as an intermediate cell layer, and the third cell layer is also referred to as a superficial cell layer.

[0104] Bladder organoids can be produced, for example, by the method for producing bladder organoids according to the present disclosure. In the method for producing bladder organoids, when ventral hindgut organoids are formed, and then bladder organoids are formed from the ventral hindgut organoids by in vitro culture, the bladder organoids are sometimes referred to herein as "bladder-like organoids." The major axis of the bladder organoids may be 80 μm or more, 100 μm or more, 120 μm or more, 150 μm or more, or 200 μm or more. Bladder organoids can be used, for example, to evaluate drug responsiveness to a test substance. Bladder organoids can be used, for example, as an active ingredient in the regenerative medicine composition according to the present disclosure.

[0105] In one embodiment, the bladder organoid does not have a lumen structure; comprises a first cell layer that does not substantially express P63 but expresses UPK1B and / or UPK2; 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; and a third cell layer that is located outward relative to the second cell layer and comprises cells that co-express P63 and KRT5.In one embodiment, the bladder organoid comprises a fourth cell layer that is located outward relative to the third cell layer and comprises interstitial-like cells.In one embodiment, the bladder organoid further comprises a fifth cell layer that is located outward relative to the fourth cell layer and comprises smooth muscle cells.

[0106] In one embodiment, the bladder organoid has a lumen; and comprises a first cell layer facing the lumen, 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 first cell layer surrounds the lumen or faces the lumen.In one embodiment, the bladder organoid has a lumen; and comprises a first cell layer facing the lumen, comprising cells that do not substantially express P63 but express UPK1B and / or UPK2; 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; and a third cell layer that is located outward relative to the second cell layer and comprises cells that co-express P63 and KRT5. Said first cell layer surrounds said lumen or faces said lumen.In one embodiment, said bladder organoid comprises a fourth cell layer, and said fourth cell layer comprises interstitial-like cells, and said third cell layer is located in the outer direction.In one embodiment, said bladder organoid further comprises a fifth cell layer, and said fifth cell layer comprises smooth muscle cells, and said fourth cell layer is located in the outer direction.

[0107] Bladder organoid is further characterized by, for example, expressing at least one (for example, one, two, or three or more) ventral hindgut marker selected from the group consisting of ΔN63, GATA3, ISL1 and SATB2; expressing at least one (for example, one, two, three, four, five or six or more) marker selected from the group consisting of Smad1 / 5 / 8, HOXA13, FOXA2, CK8 / KRT8, ECAD and UPK1B; and not substantially expressing at least one (for example, one, two or three, preferably three) selected from the group consisting of SOX2, T and CDX2.In one embodiment, bladder organoid further expresses either one or both of ΔNP63 and GATA3. The bladder organoid expresses at least one (for example, one, two, or three or more) marker selected from the group consisting of Smad1 / 5 / 8, FOXA2, ECAD, and UPK1B.The bladder organoid does not substantially express any of the dorsal hindgut markers SOX2 and CDX2.

[0108] As used herein, the term "uroplakin 2 (UPK2)" refers to a membrane glycoprotein with a molecular weight of approximately 15 kDa that is involved in the formation of lining cells of the transitional epithelium that forms the urinary tract epithelium together with uroplakins Ia, Ib, and III, and that acts to enhance the permeability and barrier function of lining cells. UPK2 can be used as a bladder epithelium marker. UPK2 expressed in cells or organoids can be detected or measured, for example, by immunostaining using an anti-UPK2 antibody (e.g., Mouse Anti-Uroplakin II (BIOCARE MEDICAL, #ACR3051C)).

[0109] The term "P63" in relation to bladder organoids can be used as a bladder epithelial marker. P63 expression in cells or organoids can be detected or measured, for example, by immunostaining using an anti-P63 antibody (e.g., Rabbit Anti-P63 (Abcam, #ab124762)).

[0110] As used herein, 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. KRT5 expression in cells or organoids can be detected or measured, for example, by immunostaining using an anti-KRT5 antibody (e.g., Chicken Anti-Keratin 5 (BioLegend, #905903)).

[0111] The term "cell layer" as used herein refers to a cell population overlap in which a particular cell type is predominant. A cell layer may be, for example, a cell population overlap in which a particular cell type is present at 70% or more, 75%, 80%, 85%, or 90% or more. In one example, the first, second, or third cell layer may be a region of a cell population in which a particular cell type is predominantly present, and which can be distinguished from other cell populations.

[0112] In one example, the cell layer contains 70% or more, 75% or more, 80% or more, preferably 85% or more, and more preferably 90% or more cells that do not substantially express P63 but express UPK1B and / or UPK2. In one example, the cell layer essentially consists of cells that do not substantially express P63 but express UPK1B and / or UPK2. In one example, a cell layer comprising or essentially consisting of cells that do not substantially express P63 but express UPK1B and / or UPK2 has a sac-like structure and has a lumen. In the example, the lumen is surrounded by the cell layer. In the example, the cell layer faces the lumen. In one example, a cell layer comprising or essentially consisting of cells that do not substantially express P63 but express UPK1B and / or UPK2 faces the lumen and overlaps inwardly with a cell layer comprising or essentially consisting of cells that co-express P63 and UPK1B and / or UPK2, as described below.

[0113] In one example, the cell layer contains 70% or more, 75% or more, 80% or more, preferably 85% or more, and more preferably 90% or more cells co-expressing P63 and UPK1B and / or UPK2. In one example, the cell layer essentially consists of cells co-expressing P63 and UPK1B and / or UPK2. In one example, the cell layer comprising or essentially consisting of cells co-expressing P63 and UPK1B and / or UPK2 has a pouch-like structure. In one example, the cell layer is overlaid outwardly on a cell layer comprising or essentially consisting of cells that do not substantially express P63 but express UPK1B and / or UPK2. In the example, the cells co-expressing P63 and UPK1B and / or UPK2 are located outwardly of the cell layer comprising or essentially consisting of cells expressing UPK1B and / or UPK2. In one example, a cell layer comprising or essentially consisting of cells co-expressing P63 and UPK1B and / or UPK2 is present between a cell layer comprising or essentially consisting of cells that do not substantially express P63 but express UPK1B and / or UPK2, and a cell layer comprising or essentially consisting of cells co-expressing P63 and KRT5, as described below.

[0114] In one example, the cell layer contains 70% or more, 75% or more, 80% or more, preferably 85% or more, and more preferably 90% or more cells co-expressing P63 and KRT5. In one example, the cell layer essentially consists of cells co-expressing P63 and KRT5. In one example, the cell layer comprising or essentially consisting of cells co-expressing P63 and KRT5 has a pouch-like structure. In one example, the cell layer is superimposed outwardly on a cell layer comprising or essentially consisting of cells co-expressing P63 and UPK1B and / or UPK2. In the example, the cells co-expressing P63 and KRT5 are located outwardly of the cell layer comprising or essentially consisting of cells co-expressing P63 and UPK1B and / or UPK2. In one example, a cell layer comprising or essentially consisting of cells co-expressing P63 and KRT5 is present between a cell layer comprising or essentially consisting of cells co-expressing P63 and UPK1B and / or UPK2 and a cell layer comprising or essentially consisting of stromal-like cells, as described below.

[0115] In one example, the cell layer comprises 70% or more, 75% or more, 80% or more, preferably 85% or more, and more preferably 90% or more interstitial-like cells. In one example, the cell layer essentially consists of interstitial-like cells. In one example, the cell layer comprising or consisting essentially of interstitial-like cells has a pouch-like structure. In one example, the cell layer overlaps outward with a cell layer comprising or consisting essentially of cells co-expressing P63 and KRT5. In the example, the interstitial-like cells are located outward with respect to a cell layer comprising or consisting essentially of cells co-expressing P63 and KRT5. In one example, the cell layer comprising or consisting essentially of interstitial-like cells is located between a cell layer comprising or consisting essentially of cells co-expressing P63 and KRT5 and a cell layer comprising or consisting essentially of smooth muscle cells, as described below.

[0116] In one example, the cell layer comprises 70% or more, 75% or more, 80% or more, preferably 85% or more, and more preferably 90% or more smooth muscle cells. In one example, the cell layer consists essentially of smooth muscle cells. In one example, the cell layer comprising or consisting essentially of smooth muscle cells has a pouch-like structure. In one example, the cell layer overlaps outwardly with a cell layer comprising or consisting essentially of interstitial-like cells. In the above example, the smooth muscle cells are located outwardly with respect to a cell layer comprising or consisting essentially of interstitial-like cells.

[0117] As used herein, the term "stromal-like cells" refers to cells that constitute the support tissue of epithelial cells. Stromal-like cells include, for example, fibroblasts. In one embodiment, the cell layer containing stroma-like cells mainly contains cells that express vimentin (VIM). In one embodiment, the cell layer containing stroma-like cells is a cell layer located in a bladder organoid between a cell layer containing cells that co-express P63 and KRT5 and a cell layer containing smooth muscle cells that express αSMA, and the cells mainly contained in the cell layer express VIM.

[0118] As used herein, the term "VIM" refers to intermediate filaments specific to mesenchymal cells. VIM can be used as a mesenchymal stem cell marker. VIM expressed in cells or organoids can be detected or measured, for example, by immunostaining using an anti-VIM antibody (e.g., Chicken Anti-Vimentin (NOVUS, #NB300-223)).

[0119] As used herein, the term "smooth muscle cells" refers to elongated, spindle-shaped, mononuclear cells containing many actin filaments and a few myosin filaments. In one embodiment, a cell layer containing smooth muscle cells predominantly contains cells that express αSMA. In one embodiment, a cell layer containing smooth muscle cells predominantly contains cells that co-express αSMA and VIM.

[0120] As used herein, the term "α-smooth muscle actin (αSMA)" refers to a protein belonging to the actin family, also known as ACTA2. αSMA can be used as a smooth muscle cell marker. αSMA expression in cells or organoids can be detected or measured, for example, by immunostaining using an anti-αSMA antibody (Rabbit Anti-αSMA (Cell signaling, #19245T)).

[0121] Bladder organoids can be produced by a method comprising: step A: culturing pluripotent stem cells using induction medium A to induce differentiation into definitive endoderm cells; step B: culturing the definitive endoderm cells using induction medium B, and then culturing them using induction medium B in the presence of an extracellular matrix to form ventral hindgut organoids; and step c1: culturing using induction medium C containing retinoic acid, fibroblast growth factor, and bone morphogenetic protein in the presence of an extracellular matrix; step c2: introducing the organoids into the kidney or bladder or a site surrounding the kidney or bladder of a human or non-human mammal; or step c3: culturing the organoids in the presence of mesenchymal stem cells or mesenchymal cells.

[0122] Step c1: Induction of differentiation of bladder epithelium (in vitro culture) Step c1 involves culturing the ventral hindgut organoids using induction medium C in the presence of an extracellular matrix.

[0123] "Induction medium C" comprises a basal medium and additives containing retinoic acid, fibroblast growth factor, and bone morphogenetic protein. Induction medium C can be prepared, for example, by adding the additives (solid or liquid) to a basal medium (liquid). The concentration of the additives added to induction medium C is appropriately determined by those skilled in the art, taking into consideration the animal species from which the cells used for culture are derived. The explanation for the basal medium for induction medium A applies, as appropriate, to the basal medium for induction medium C. Induction medium C may further comprise heparin, a protein-free medium (e.g., PFHM-II), an antibiotic (e.g., penicillin / streptomycin, gentamicin), an antibiotic-antimycotic mixture (e.g., antibiotic-antimycotic), or a combination thereof.

[0124] Induction medium C may contain FGF (preferably FGF7) at 10 to 500 ng / mL, 30 to 250 ng / mL, or 50 to 150 ng / mL. Induction medium C may contain FGF7 at 10 to 500 ng / mL, 30 to 250 ng / mL, or 50 to 150 ng / mL, or FGF4 at 20 to 1000 ng / mL, 60 to 500 ng / mL, or 100 to 300 ng / mL. Induction medium C may contain FGF at a concentration that exhibits a fibroblast proliferation effect similar to that exhibited by 10 to 500 ng / mL, 30 to 250 ng / mL, or 50 to 150 ng / mL of FGF7.

[0125] Induction medium C may contain bone morphogenetic protein (preferably BMP4) at a concentration of 3 to 150 ng / mL, 9 to 75 ng / mL, or 15 to 45 ng / mL. Induction medium C may contain BMP at a concentration that exhibits the same alkaline phosphatase production-inducing activity as that exhibited by 3 to 150 ng / mL, 9 to 75 ng / mL, or 15 to 45 ng / mL BMP4. Induction medium C may contain BMP at a concentration that exhibits the same level of expression of ventral hindgut markers (e.g., GATA3, p63, or HOXA13) as that exhibited by organoids cultured in induction medium C containing 3 to 150 ng / mL, 9 to 75 ng / mL, or 15 to 45 ng / mL BMP4. In this context, the organoids cultured using induction medium C may be bladder organoids formed in step b2 or b3 of the present disclosure. The expression of the ventral hindgut markers can be measured, for example, by immunostaining. In immunostaining, the expression of ventral hindgut markers can be measured using the antibodies for each marker described in the present disclosure. In one embodiment, induction medium C contains BMP at a concentration that exhibits the same alkaline phosphatase production-inducing effect as that exhibited by BMP4 at 3 to 150 ng / mL, 9 to 75 ng / mL, or 15 to 45 ng / mL.

[0126] Induction medium C may contain retinoic acid (preferably all-trans retinoic acid) at a concentration of 0 to 1 μM, 10 nM to 500 nM, 30 nM to 300 nM, or 50 nM to 150 nM.

[0127] The culturing of ventral hindgut organoids using induction medium C in the presence of an extracellular matrix is ​​similar to the culturing of definitive endoderm cell spheroids using induction medium b1 in the presence of an extracellular matrix. The culturing of ventral hindgut organoids using induction medium C in the presence of an extracellular matrix can be carried out, for example, under known cell culture conditions. Known cell culture conditions may include maintaining the cells at 37°C under 5% CO2. The culturing temperature is not limited to 37°C, and any temperature known in the field of cell culture can be used as appropriate. The CO2 concentration is not limited to 5%, and any CO2 concentration known in the field of cell culture can be used as appropriate. The culturing of ventral hindgut organoids can be carried out for 5 to 30 days, 5 to 25 days, 5 to 20 days, 5 to 15 days, or 5 to 10 days.

[0128] The state of the extracellular matrix in step c1 (gel state or state of dispersed components) may be the same as or different from those in steps b1 and b2. In one embodiment, the state of the extracellular matrix in step c1 is the same as those in steps b1 and b2. In one embodiment, the extracellular matrix gel containing the ventral hindgut organoids may be the extracellular matrix gel formed in steps b1 and b2, or may be a newly formed extracellular matrix gel containing the recovered ventral hindgut organoids after step b2, after which the ventral hindgut organoids are recovered from the extracellular matrix gel. From the viewpoint of operational simplicity, the extracellular matrix gel containing the ventral hindgut organoids in step c1 is the extracellular matrix gel formed in steps b1 and b2.

[0129] In one embodiment, step c1 comprises culturing the ventral hindgut organoid in extracellular matrix gel with induction medium C to form bladder organoid.In one embodiment, step c1 comprises culturing the ventral hindgut organoid on extracellular matrix gel with induction medium C to form bladder organoid.In one embodiment, step c1 comprises culturing the ventral hindgut organoid in the presence of extracellular matrix as dispersion component with induction medium C to form bladder organoid.

[0130] In one embodiment, culturing ventral hindgut organoids using induction medium C involves culturing the ventral hindgut organoids for 6 days or more in induction medium C, STEMdiff APEL2 medium (10 or 30 ng / mL BMP4, 0.1 to 1 μM all-trans retinoic acid, 100 ng / mL FGF7 or FGF9, 1 μg / mL heparin, 2% PFHM-II, antibiotic-antimycotic) to induce differentiation into bladder epithelium. Induction medium C is added in an amount of 200 to 400 μL on top of the Cell Culture Insert gel and 300 to 600 μL on the bottom, and the medium is changed every two days. Preferably, culturing is continued until the expression of bladder epithelial markers UPK2, P63, and KRT5 can be confirmed. Next, PPARγ agonist (0.1 to 10 μM rosiglitazone) is added to induction medium C to mature bladder epithelial cells, and the cells are cultured.

[0131] In one embodiment, bladder organoids can be produced by a method comprising: step A: culturing pluripotent stem cells using induction medium A to induce differentiation into definitive endoderm cells; step b1: culturing the definitive endoderm cells using induction medium B (induction medium b) containing a fibroblast growth factor and a GSK3β inhibitor, and then culturing the definitive endoderm cells using induction medium B (induction medium b1) in the presence of an extracellular matrix (preferably in an extracellular matrix gel) to form hindgut organoids; and step b2: culturing the hindgut organoids using induction medium B (induction medium b2) in the presence of an extracellular matrix (preferably in an extracellular matrix gel) to form ventral hindgut organoids; and step c1: culturing the ventral hindgut organoids using induction medium C containing retinoic acid, fibroblast growth factor, and bone morphogenetic protein in the presence of an extracellular matrix.

[0132] In one embodiment, bladder organoids can be produced by a method comprising: step A: culturing pluripotent stem cells using induction medium A to induce differentiation into definitive endoderm cells; step b3: culturing the definitive endoderm cells using induction medium B (induction medium b) containing a fibroblast growth factor and a GSK3β inhibitor, and optionally further containing bone morphogenetic protein, and then culturing the cells using induction medium B (induction medium b3) containing a fibroblast growth factor, a GSK3β inhibitor, and bone morphogenetic protein in the presence of an extracellular matrix (preferably, an extracellular matrix as a dispersed component) to form ventral hindgut organoids; and step c1: culturing the ventral hindgut organoids using induction medium C containing retinoic acid, fibroblast growth factor, and bone morphogenetic protein in the presence of an extracellular matrix.

[0133] Step c2: Introduction into the kidney or bladder or surrounding areas of a mammal Step c2 comprises introducing ventral hindgut organoid into the kidney or bladder or their surrounding area of ​​human or non-human mammal, and forming bladder organoid.In one embodiment, step c2 comprises introducing ventral hindgut organoid into the kidney or bladder or their surrounding area of ​​non-human mammal, and forming bladder organoid.When ventral hindgut organoid exists in extracellular matrix gel, step c2 can further comprise removing ventral hindgut organoid from extracellular matrix gel before being introduced into human or non-human mammal.

[0134] The term "non-human mammal" as used herein may refer to, for example, rodents such as mice, rats, guinea pigs, hamsters, etc.; non-human primates such as chimpanzees; even-toed ungulates such as cows, goats, sheep, etc.; perissodactyls such as horses, etc.; or companion animals such as rabbits, dogs, cats, etc. In one embodiment, the non-human mammal is a rodent or a non-human primate.

[0135] As used herein, 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. As used herein, 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. In one embodiment, the kidney is a normal kidney. In one embodiment, the kidney is a damaged kidney or a kidney affected by a kidney disease.

[0136] The term "bladder" as used herein refers to a sac-like organ that temporarily stores urine delivered from the kidneys. As used herein, 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. In one embodiment, the bladder is a normal bladder. In one embodiment, the bladder is an injured bladder or a bladder affected by a bladder disease.

[0137] "Area surrounding" the kidney or bladder refers to tissue or an area adjacent to or near the urinary system. The urinary system includes the kidney, ureter, bladder, and urethra. The area surrounding the kidney or bladder can be, for example, within the peritoneal cavity or the mesentery.

[0138] " Introducing " ventral hindgut organoid into kidney or bladder or their surrounding area means the operation of placing ventral hindgut organoid into kidney or bladder or their surrounding area.The introduction of ventral hindgut organoid into kidney or bladder or their surrounding area includes, for example, transplanting the ventral hindgut organoid in extracellular matrix gel into kidney or bladder or their surrounding area by surgical operation.The introduction of ventral hindgut organoid into kidney or bladder or their surrounding area includes, for example, using a device such as a syringe to inject the ventral hindgut organoid in solution into kidney or bladder or their surrounding area.

[0139] For example, ventral hindgut organoid can be produced from the pluripotent stem cells of the animal species different from that of human or non-human mammal that is introduced into its kidney or bladder or their surrounding area, or can be produced from the pluripotent stem cells of the same animal species or the same individual.In one embodiment, the method for producing bladder organoid comprises introducing the ventral hindgut organoid that is produced from human ES cell or human iPS cell into the kidney or bladder or their surrounding area of ​​human or non-human mammal.

[0140] In one embodiment, the introduction of ventral hindgut organoids into the kidney involves transplanting ventral hindgut spheroids under the renal capsule of a NOD-SCID mouse. The transplanted mouse is then housed for four weeks, and the transplanted ventral hindgut organoids are then cultured in vivo for four weeks. This in vivo culture can result in the formation of bladder organoids with a sac-like structure. In one embodiment, the in vivo culture can be carried out for one to six weeks, two to five weeks, or three to five weeks.

[0141] In one embodiment, bladder organoids can be produced by a method comprising: step A: culturing pluripotent stem cells using induction medium A to induce differentiation into definitive endoderm cells; step b1: culturing the definitive endoderm cells using induction medium B (induction medium b) containing a fibroblast growth factor and a GSK3β inhibitor, and then culturing the cells in the presence of an extracellular matrix (preferably in an extracellular matrix gel) using the induction medium B (induction medium b1) to form hindgut organoids; and step b2: culturing the hindgut organoids in the presence of an extracellular matrix (preferably in an extracellular matrix gel) using induction medium B (induction medium b2) containing a fibroblast growth factor, a GSK3β inhibitor, and a bone morphogenetic protein to form ventral hindgut organoids; and step c2: introducing the organoids into the kidney or bladder or a site surrounding the kidney or bladder of a human or non-human mammal.

[0142] In one embodiment, bladder organoids can be produced by a method comprising: step A: culturing pluripotent stem cells using induction medium A to induce differentiation into definitive endoderm cells; step b3: culturing the definitive endoderm cells using induction medium B (induction medium b) containing a fibroblast growth factor and a GSK3β inhibitor, and optionally further containing a bone morphogenetic protein, and then culturing the definitive endoderm cells using induction medium B (induction medium b3) containing a fibroblast growth factor, a GSK3β inhibitor, and a bone morphogenetic protein in the presence of an extracellular matrix (preferably, an extracellular matrix as a dispersed component) to form ventral hindgut organoids; and step c2: introducing the organoids into the kidney or bladder or a site surrounding the kidney or bladder of a human or non-human mammal.

[0143] Step c3: Co-culturing in the presence of mesenchymal stem cells or mesenchymal cells Step c3 comprises culturing ventral hindgut organoid in the presence of mesenchymal stem cell or mesenchymal cell to form bladder organoid.When ventral hindgut organoid exists in extracellular matrix gel, step c3 can further comprise before co-cultivation, extracting said ventral hindgut organoid from said extracellular matrix gel.

[0144] As used herein, the term "mesenchymal cells" refers to cells derived from the mesenchyme of a multicellular animal during its fetal stage. Mesenchymal cells have the ability to differentiate into, for example, supportive tissue, connective tissue, bone cells, chondrocytes, and adipocytes. In one embodiment, the mesenchymal cells are bladder mesenchymal cells. Bladder mesenchymal cells can be prepared, for example, from non-human mammals according to known methods or methods described in the Examples herein. Mesenchymal cells prepared from non-human mammals include, for example, embryonic fibroblasts. In one embodiment, the embryonic fibroblasts are mouse embryonic fibroblasts (MEFs). Mesenchymal cells can be prepared, for example, from ES cells or iPS cells by inducing differentiation according to known methods. Mesenchymal cells can be obtained, for example, commercially.

[0145] As used herein, the term "mesenchymal stem cells" refers to cells that have the ability to self-renew and differentiate into cells that constitute non-epithelial mesenchyme, such as connective tissue, bone cells, chondrocytes, and adipocytes. Mesenchymal stem cells can be prepared, for example, from ES cells or iPS cells by inducing differentiation according to known methods. Mesenchymal stem cells can be obtained, for example, commercially. Mesenchymal stem cells can be recovered, for example, from living organisms according to known methods. Mesenchymal stem cells are known to exist in living organisms, for example, in dental pulp or bone marrow fluid.

[0146] In one embodiment, step c3 comprises culturing the ventral hindgut organoids in a medium in the presence of mesenchymal stem cells or mesenchymal cells to form bladder organoids. The medium used in step c3 may be, for example, a basal medium, and may contain the additives described in the present disclosure. In one embodiment, the medium is characterized in that no extracellular matrix is ​​added. In one embodiment, the medium is STEMdiff APEL2 medium (STEMCELL Technologies) (supplemented with 2% PFHM-II and antibiotic-antimycotic) or STEMdiff APEL2 medium (STEMCELL Technologies) (supplemented with 2% PFHM-II, 2% FBS, and antibiotic-antimycotic).

[0147] In one embodiment, step c3 comprises statically culturing ventral hindgut organoids in the presence of mesenchymal stem cells or mesenchymal cells, followed by rotational suspension culture of the ventral hindgut organoids and mesenchymal stem cells or mesenchymal cells to form bladder organoids. Rotary suspension culture can be performed, for example, using a 3D rotary suspension culture device, CellPet 3D-iP (J-Tech Corporation). The rotation speed in rotational suspension culture can be appropriately set by those skilled in the art so that cell clusters do not fall by gravity and touch the culture vessel. The rotation speed may be, for example, 1 to 50 rpm, 1 to 30 rpm, 1 to 15 rpm, 1 to 10 rpm, 2 to 50 rpm, 2 to 30 rpm, 2 to 15 rpm, 2 to 10 rpm, 3 to 50 rpm, 3 to 30 rpm, 3 to 15 rpm, or 3 to 10 rpm.

[0148] In one embodiment, step c3 is to carry out a rotary suspension culture of ventral hindgut organoids or bladder organoids together with E12.5 mouse bladder mesenchymal cells for 2 weeks or more to form bladder organoids that contain bladder epithelial cells, interstitial cells, and smooth muscle cells, and that exhibit a sac-like structure. The culture in step c3 can be carried out for, for example, 1 week to 6 weeks, 2 weeks to 5 weeks, or 3 weeks to 5 weeks.

[0149] In one embodiment, bladder organoids can be produced by a method comprising: step A: culturing pluripotent stem cells using induction medium A to induce differentiation into definitive endoderm cells; step b1: culturing the definitive endoderm cells using induction medium B (induction medium b) containing a fibroblast growth factor and a GSK3β inhibitor, and then culturing the definitive endoderm cells using induction medium B (induction medium b1) in the presence of an extracellular matrix (preferably in an extracellular matrix gel) to form hindgut organoids; and step b2: culturing the hindgut organoids using induction medium B (induction medium b2) containing a fibroblast growth factor, a GSK3β inhibitor, and a bone morphogenetic protein in the presence of an extracellular matrix (preferably in an extracellular matrix gel) to form ventral hindgut organoids; and step c3: culturing in the presence of mesenchymal stem cells or mesenchymal cells.

[0150] In one embodiment, bladder organoids can be produced by a method comprising: step A: culturing pluripotent stem cells using induction medium A to induce differentiation into definitive endoderm cells; step b3: culturing the definitive endoderm cells using induction medium B (induction medium b) containing a fibroblast growth factor and a GSK3β inhibitor, and optionally further containing a bone morphogenetic protein, and then culturing the cells using induction medium B (induction medium b3) containing a fibroblast growth factor, a GSK3β inhibitor, and a bone morphogenetic protein in the presence of an extracellular matrix (preferably, an extracellular matrix as a dispersed component) to form ventral hindgut organoids; and step c3: culturing in the presence of mesenchymal stem cells or mesenchymal cells.

[0151] The explanations of each term and the explanations of the embodiments, including the culture conditions, in each step of inducing differentiation of definitive endoderm cells (step A), forming ventral hindgut organoids (step B), and forming bladder organoids (step C) apply, as appropriate, to the corresponding terms and embodiments described in those steps.

[0152] [Non-human mammals having ventral hindgut organoids or bladder organoids, and methods for producing the same] One aspect of the present disclosure provides a method for producing non-human mammalian organoids, which have ventral hindgut organoids or bladder organoids in kidney or bladder or their surrounding area.Another aspect of the present disclosure provides a non-human mammalian organoids, which have ventral hindgut organoids or bladder organoids in kidney or bladder or their surrounding area.

[0153] A non-human mammal having ventral hindgut organoids in the kidney or bladder or their surrounding areas can be produced by a method comprising introducing the ventral hindgut organoids of the present disclosure into the kidney or bladder or their surrounding areas of a non-human mammal. A non-human mammal having bladder organoids in the kidney or bladder or their surrounding areas can be produced by a method comprising introducing the ventral hindgut organoids of the present disclosure into the kidney or bladder or their surrounding areas of a non-human mammal, and raising the non-human mammal into which the ventral hindgut organoids of the present disclosure have been introduced. Raising the non-human mammal includes, for example, feeding a known diet for non-human mammals.

[0154] In one example, tumor cells or tumor fragments are introduced into the ventral hindgut organoids of the present disclosure, and then the ventral hindgut organoids containing the tumor cells or tumor fragments are introduced into the kidney, bladder, or surrounding area of ​​a non-human mammal, thereby producing a non-human mammal having bladder organoids as a kidney or bladder cancer model in its kidney or bladder, or surrounding area. A non-human mammal having bladder organoids as a kidney or bladder cancer model in its kidney or bladder, or surrounding area, can be used in a method for evaluating the effectiveness of a candidate therapeutic substance for kidney cancer or bladder cancer, which includes contacting the non-human mammal with a candidate therapeutic substance for kidney cancer and evaluating the effectiveness of the candidate therapeutic substance.

[0155] [Method for assessing drug response to test substance] A method for evaluating drug responsiveness to a test substance includes contacting a test substance with a ventral hindgut organoid, a bladder organoid, or a non-human mammal having a ventral hindgut organoid or a bladder organoid according to the present disclosure, and measuring the drug responsiveness of the ventral hindgut organoid, the bladder organoid, or the non-human mammal to the test substance.

[0156] The term "test substance" as used herein may be, for example, a small molecule compound, a protein (e.g., an antibody), DNA, RNA, small interfering RNA, or an antisense oligonucleotide. 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.

[0157] "Contacting" a test substance with ventral hindgut organoids, bladder organoids or non-human mammals means placing the ventral hindgut organoids, bladder organoids or non-human mammals under conditions that allow contact between the test substance and the ventral hindgut organoids, bladder organoids or non-human mammals.Contacting a test substance with ventral hindgut organoids or bladder organoids may, for example, be mixing the test substance into a culture medium containing ventral hindgut organoids or bladder organoids.Contacting a test substance with a non-human mammal having ventral hindgut organoids or bladder organoids in the kidney or bladder or their surrounding areas may, for example, be administering the test substance orally or parenterally to the non-human mammal.

[0158] Drug responsiveness includes, for example, structural or functional changes in the ventral hindgut organoids caused by a test substance. Drug responsiveness includes, for example, changes in the concentration of a test substance caused by the ventral hindgut organoids.

[0159] [Composition for regenerative medicine or method for producing the same] One aspect of the present disclosure provides a regenerative medicine composition for treating bladder injury or bladder disease.

[0160] The term "regenerative medicine composition" as used herein includes ventral hindgut organoids or bladder organoids according to the present disclosure. The regenerative medicine composition according to the present disclosure can be used to treat bladder damage or bladder disease in mammals. The regenerative medicine composition may, for example, appropriately contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein refers to any component other than the ventral hindgut organoids or 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. The regenerative medicine composition can be appropriately manufactured according to known methods. In one example, the regenerative medicine composition according to the present disclosure can be manufactured by combining the ventral hindgut organoids or bladder organoids according to the present disclosure with a pharmaceutically acceptable carrier (e.g., basal medium).

[0161] The regenerative medicine composition is administered to a mammal in need thereof, for example, by surgically transplanting it into a predetermined site in the bladder, or by injecting it into a predetermined site in the bladder using an instrument such as a syringe. From the viewpoint of reducing graft rejection, the mammal to which the regenerative medicine composition is administered and the animal species of the pluripotent stem cells used to produce the ventral hindgut organoids or bladder organoids are preferably the same species, and more preferably the same individual.

[0162] The term "mammal" in relation to the regenerative medicine composition refers to, for example, humans and non-human mammals. Non-human mammals may be, for example, rodents such as mice, rats, guinea pigs, and hamsters; non-human primates such as chimpanzees; even-toed ungulates such as cows, goats, and sheep; perissodactyls such as horses; and pet animals such as rabbits, dogs, and cats. In one embodiment, the mammal is a human.

[0163] The term "bladder injury" or "bladder disease" as used herein may refer to, for example, a bladder damaged by trauma, radiation cystitis, a bladder damaged by diabetes or ischemia, a bladder damaged by a drug harmful to bladder tissue, cystitis, or bladder cancer.

[0164] The regenerative medicine composition of the present disclosure can be used in a method for treating bladder damage or disease in a mammal. One embodiment provides a method for treating bladder damage or disease, comprising administering a regenerative medicine composition comprising the ventral hindgut organoid or bladder organoid of the present disclosure to a mammal in need thereof.

[0165] [Method of treating bladder injury or disease] One aspect of the present disclosure provides a method for treating bladder damage or disease in a mammal.The method for treating bladder damage or disease in a mammal comprises introducing the ventral hindgut organoid, bladder organoid, or regenerative medicine composition of the present disclosure into the kidney or bladder or their surrounding area of ​​a mammal in need thereof.In one embodiment, the method for treating bladder damage or disease in a mammal comprises introducing the regenerative medicine composition of the present disclosure into the kidney or bladder or their surrounding area of ​​a mammal in need thereof.

[0166] The term "mammal in need thereof" as used herein refers to a mammal having or suspected of having a bladder injury or disease. A mammal having a bladder injury or disease refers to a mammal diagnosed by a medical professional (e.g., a physician) as having a bladder injury or disease according to predetermined diagnostic criteria. A mammal suspected of having a bladder injury or disease may be a mammal suspected of having a bladder injury or disease based on, for example, the mammal's behavioral history (e.g., trauma, radiation therapy, and having received or having received drugs harmful to bladder tissue) or medical history (e.g., having or having suffered from diabetes, ischemia, cystitis, or bladder cancer). The mammal according to this embodiment is preferably a human or a non-human primate, more preferably a human.

[0167] "Treating" a bladder injury or disease includes maintaining, reducing, or eliminating the symptoms or pathology. Treating a bladder injury or disease includes curing the bladder injury or disease.

[0168] From the viewpoint of reducing transplant rejection, it is preferable that the animal species of the pluripotent stem cells used to produce the ventral hindgut organoids, bladder organoids, or regenerative medicine composition to be introduced into a mammal in need thereof and the mammal in need thereof are the same species or the same individual.

[0169] As used herein, the term "comprises" means that the listed elements and / or steps are present, and that other elements and / or steps may be added. As used herein, the term "consisting of" means that the listed elements and / or steps are present, and that other elements and / or steps are excluded. As used herein, the term "essentially consisting of" means that the listed elements and / or steps are present, and that other elements and / or steps may be added to the extent that they do not affect the novel technical features of the cell layer, organoid, composition, and method. As used herein, the term "substantially free" does not exclude "completely free."

[0170] The terms and embodiment descriptions provided by this disclosure apply appropriately among the aspects and embodiments provided by this disclosure unless otherwise specified.

[0171] 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. [Example]

[0172] material and method The following growth factors and compounds were prepared: Recombinant Human / Mouse / Rat Activin A(R&D SYSTEMS, #338-AC), Recombinant Human FGF4(R&D SYSTEMS, #7460-F4), Recombinant Human BMP4(R&D SYSTEMS, #314-BP), Recombinant Human KGF / FGF7(R&D SYSTEMS, #251-KG), CHIR99021 (TOCRIS, #4423), All-trans retinoic acid (SIGMA, R2625).

[0173] Immunofluorescence staining For frozen sectioning, bladder organoids or explants were incubated overnight in 4% PFA with shaking at 4°C. They were then washed three times with PBS(-) and replaced with sucrose solution. Sucrose was prepared in 10%, 20%, and 30% PBS(-) solutions, and incubated at 4°C until the samples sank. For bladder organoids, the surrounding Matrigel was removed with tweezers and the tissue was embedded in OCT compound to produce frozen sections. Sections were prepared at 10 μm and blocked for 1 hour at room temperature in PBS(-) supplemented with 10% donkey serum and 0.3% Triton-X (hereinafter referred to as "Blocking Buffer"). Then, sections were incubated overnight at 4°C with primary antibodies diluted in Blocking Buffer. Next, sections were washed three times with PBS(-) and incubated overnight at 4°C with secondary antibodies diluted in PBS(-). Nuclei were stained with DAPI. Then, the sections were washed three times with PBS(-), mounted with FluorSave Reagent (Millipore, #345789), and observed under a confocal microscope (ZEISS, LSM800).

[0174] Example 1: Production of ventral hindgut organoids Figure 1 is a flow chart showing an outline of the preparation of bladder organoids according to one embodiment. Figure 1 shows the steps of: step A of inducing differentiation from human iPS cells into definitive endoderm cells; step b1 of inducing differentiation from definitive endoderm cells into hindgut organoids; step b2 of inducing differentiation from hindgut organoids into ventral hindgut organoids; and step c1 of inducing differentiation from ventral hindgut organoids into bladder-like organoids. In step B, which includes steps b1 and b2, the ventral hindgut is induced to differentiate. In Example 1, step A, step b1 and step b2 are carried out to produce ventral hindgut organoids.

[0175] Pre-cultivation of human iPS cells Human iPS cells (1502.3 strain, donated by Dr. Melissa Little, Murdoch Children's Research Institute) were maintained and cultured in StemFit AK02N medium (REPROCELL) on a culture plate surface coated with iMatrix-511 (nippi). The cells were detached using TrypLE Select (Thermo Fisher Scientific) to obtain a cell suspension. The cell suspension was centrifuged at 200 rcf and room temperature for 5 minutes, the supernatant was removed, and the precipitate was resuspended in StemFit AK02N (10 μM Y-27632). iMatrix-511 (nippi) was added to the suspension at 0.25 μg / cm. 2 The amount of added water was 60,000 cells / cm 2 The cells were seeded onto a 6-well plate (Corning) so that the total volume was 1000 μg / well, and cultured at 37°C under 5% CO2 for 1 day.

[0176] Step A: Induction of differentiation of definitive endoderm cells The culture medium was then replaced with induction medium A, STEMdiff APEL2 medium (STEMCELL Technologies) (supplemented with 100 ng / mL Activin A, 1 μM CHIR99021, 2% PFHM-II, and an antibiotic-antimycotic), and the cells were cultured for three days to induce differentiation into definitive endoderm cells. The medium was replaced daily from day two onwards. Human iPS cells gathered in colonies on day one of differentiation induction, but by day two, the cells began to spread outside the colonies. By day three of differentiation induction, the cells had proliferated in sheets, reaching 100% confluence. Paving stone-like cells typical of definitive endoderm were induced.

[0177] Step b1: Induction of hindgut differentiation After definitive endoderm induction (day 3 of differentiation induction), induction medium A was replaced with induction medium b1, STEMdiff APEL2 medium (supplemented with 200 ng / mL FGF4, 8 μM CHIR99021, 1 μg / mL Heparin, 2% PFHM-II, and antibiotic-antimycotic), and the differentiated definitive endoderm cells were cultured for 4 days.

[0178] On day 7 of differentiation induction, the floating spheroids were collected into a 35 mm dish. Next, the medium was pipetted onto the spheroids remaining in the well from which the floating spheroids were collected, causing them to float, and the spheroids were collected into the same 35 mm dish. The collected spheroids were placed on ice.

[0179] Matrigel Growth Factor Reduced (Corning) (protein concentration: 10 mg / mL) was diluted to 50% with induction medium b1 and added 50 μL / well to a 24-well Cell Culture Insert Transparent PET Membrane with an 8.0 μm pore size (Corning). The mixture was then added to 50 μL of 50% Matrigel solution and mixed. The entire mixture was added on top of the gelled 50% Matrigel and incubated at 37°C and 5% CO2 for 30 minutes to form a gel. Cell Culture Insert gels were obtained. Induction medium b1 was added to the top and bottom of the Cell Culture Insert gels (200 μL and 300 μL, respectively). The cells were cultured at 37°C and 5% CO2 for 4 days. Medium changes were performed daily for 2D culture and every two days for 3D culture. Spheroids in 50% Matrigel were grown by culturing in induction medium b1, forming slightly rounded, tube-shaped cell structures (hindgut organoids).

[0180] Step b2: Ventralization of the hindgut After hindgut induction (day 11 of differentiation induction), induction medium b1 was replaced with induction medium b2 (STEMdiff APEL2 medium supplemented with 30 ng / mL BMP4, 200 ng / mL FGF4, 8 μM CHIR99021, 1 μg / mL heparin, 2% PFHM-II, and antibiotic-antimycotic). The induced hindgut was cultured in the Cell Culture Insert gel for 3 days to ventralize the hindgut. 200 μL of induction medium b2 was added to the top and 300 μL to the bottom of the Cell Culture Insert gel, and the medium was changed every 2 days. When cultured in the ventral hindgut medium, the growth of hindgut organoids slowed and they transformed into spherical cell structures (ventral hindgut organoids).

[0181] The ventral hindgut organoids (14 days after differentiation induction) were subjected to immunofluorescence staining using the antibodies listed in the table below. [Table 1]

[0182] Ventral hindgut organoids expressed the ventral hindgut marker p63, weakly expressed the dorsal hindgut markers CDX2 and SOX2, respectively, and expressed the intestinal epithelial marker KRT8. Furthermore, RT-PCR confirmed increased transcription of HOXA13, a marker of the posterior intestine and cloaca region. These results are summarized in the table below. [Table 2] +++: Strongly expressed ++: Expressed or strongly expressed in some cells +: Weakly expressed or expressed in some cells -: No increase in expression or no expression

[0183] [Comparative Example 1] Differentiation of intestinal-like organoids (SATB2 positive) In step b2 of Example 1, the hindgut organoids in the Cell Culture Insert gel obtained in step b1 were cultured for 21 days using STEMdiff APEL2 medium (supplemented with 100 ng / mL BMP2, 100 ng / mL EGF, 500 ng / mL RSPO1, and 0.1 μM retinoic acid) to induce differentiation into intestinal-like organoids (SATB2 positive).

[0184] The intestinal-like organoids expressed the colon or rectum marker SATB2 (Fig. 2a). The intestinal organoids partially expressed the midgut / hindgut marker CDX2 and also expressed the epithelial tissue marker ECAD (Fig. 2b and 2c). Fluorescent images of SATB2, CDX2, ECAD, and the nuclear marker DAPI (Fig. 2d) did not reveal a cell layer structure in which specific cell types were regularly localized.

[0185] The intestinal-like organoids obtained in Comparative Example 1 also expressed the ventral hindgut / Cloaca marker P63, the bladder epithelial marker UPK2, and the bladder epithelial marker KRT5. The results of P63, UPK2, and KRT5 also did not indicate that the intestinal-like organoids obtained in Comparative Example 1 had a cell layer structure in which specific cell types were regularly localized.

[0186] In Comparative Example 1, a different combination of additives (BMP2, EGF, RSPO1, and retinoic acid) was used than the combination of additives (BMP4, FGF4, and CHIR99021) used in step b2 of Example 1. The cell structure obtained in Comparative Example 1 did not have a cell layer structure in which specific cell types were regularly localized, as in the ventral hindgut organoids obtained in Example 1.

[0187] The cell structures obtained using other combinations of additives different from the combination of additives used in step b2 of Example 1 (BMP4, FGF4, and CHIR99021) (a combination of EGF, RSPO1, and retinoic acid; a combination of EGF, RSPO1, retinoic acid, and Noggin; a combination of EGF, RSPO1, retinoic acid, Noggin, and FGF7; and a combination of EGF, RSPO1, retinoic acid, BMP2, and FGF7) also did not have a cell layer structure in which specific cell types were regularly localized, like the ventral hindgut organoids obtained in Example 1.

[0188] [Example 2] Production of bladder-like organoids Step c1: Induction of bladder epithelial differentiation After ventralization of the hindgut (day 14 of differentiation induction), induction medium b2 was replaced with induction medium C, STEMdiff APEL2 medium (supplemented with 30 ng / mL BMP4, 100 nM all-trans retinoic acid, 100 ng / mL FGF7, 1 μg / mL heparin, 2% PFHM-II, and antibiotic-antimycotic), and the spherical cell structures were cultured for 6 days to induce bladder epithelial cells. Induction medium C was added in 200 μL on top of the Cell Culture Insert gel and 300 μL on the bottom, and the medium was changed every 2 days. The induced bladder-like organoids were spherical.

[0189] The bladder-like organoids were subjected to immunofluorescence staining. The following antibodies were used in the immunofluorescence staining in Example 2 and the following Example 3. The primary antibodies used for immunofluorescence staining were Mouse Anti-Uroplakin II (1:100, BIOCARE MEDICAL, #ACR3051C), Rabbit Anti-P63 (1:100, abcam, #ab124762), Chicken Anti-Keratin 5 (1:300, BioLegend, #905903), and Goat Anti-ECAD (1:300, R&D SYSTEMS, #AF648). The secondary antibodies used for immunofluorescence staining were Alexa Fluor 488 Donkey Anti-Mouse IgG (1:400, Life Technologies, #A21202), Alexa Fluor 568 Donkey Anti-Rabbit IgG (1:400, Life Technologies, #A10042), Alexa Fluor 647 Donkey Anti-Goat IgG (1:400, Life Technologies, #A21447), and Alexa Fluor 647 Donkey Anti-Chicken IgY (1:400, Jackson ImmunoResearch, #703-606-155).

[0190] Bladder-like organoids expressed the epithelial tissue marker ECAD (Figure 3e). Bladder-like organoids also expressed the bladder epithelial markers UPK2 (Figure 3a), P63 (Figure 3b), and KRT5 (Figure 3c). In Figures 3a and 3d, the signals observed at the outermost periphery of the bladder-like organoids are nonspecific signals derived from Matrigel. Figure 3d, which overlays fluorescent images of UPK2, P63, and KRT5, shows that basal cell-like cells co-expressing P63 and KRT5 are located along the outermost periphery of the bladder-like organoids. Toward the medial side of the region containing the KRT5-basal cell-like cells, intermediate cell-like cells co-expressing P63 and UPK2 are located. Toward the medial side of the region containing the intermediate cell-like cells, superficial cell-like cells expressing UPK2 but not P63 are located. The layered structure of these cell types in the bladder-like organoids resembles that of the developing bladder epithelium in vivo (Fig. 5g). Although the number of KRT5-expressing cells was low in these bladder-like organoids, KRT5-basal cells, which express KRT5, are a cell type that appears later in bladder development, suggesting that the induced bladder-like organoids are at an immature stage.

[0191] Comparative Example 2 The hindgut organoids were induced to differentiate into bladder epithelium in the same manner as in Example 2, except that induction medium b2 and the induction medium C, which was obtained by removing BMP4, were used. The cell structures formed in Comparative Example 2 were subjected to immunofluorescence staining in the same manner as in Example 2. The cell structures formed in Comparative Example 2 expressed the bladder epithelial marker P63 (FIG. 4b) and the epithelial tissue marker ECAD (FIG. 4c), similar to the bladder-like organoids formed in Example 2. However, the cell structures formed in Comparative Example 2 did not express the bladder epithelial marker UPK2, as observed in the bladder-like organoids formed in Example 2 (FIGS. 3a and 4a). In FIG. 4a, the signal observed at the outermost periphery of the bladder-like organoids is a nonspecific signal derived from Matrigel. Thus, the cell structures formed in Comparative Example 2 did not have the cell layer structure observed in the bladder-like organoids formed in Example 2.

[0192] Example 3: In vivo production of bladder organoids Pre-cultivation of human iPS cells 90,000 cells / cm in a 6-well plate 2 Human iPS cells were precultured in the same manner as in Example 1, except that the cells were seeded so that the total number of cells was 100. Step A: Induction of differentiation into definitive endoderm cells In the same manner as in Example 1, human iPS cells were induced to differentiate into definitive endoderm cells. Step b1: Hindgut differentiation induction Hindgut differentiation induction was carried out essentially in the same manner as in Example 1, except that the Cell Culture Insert gel was prepared by diluting Matrigel Growth Factor Reduced (Corning) (protein concentration 10 mg / mL) with induction medium b1 to a concentration of 2.5 mg / mL and gelling it, and then containing 90 to 100 spheroids formed by two-dimensional culture of definitive endoderm cells inside the gel.

[0193] Step b2: Ventralization of the hindgut After hindgut induction (day 11 of differentiation induction), induction medium b1 was replaced with induction medium b2 (STEMdiff APEL2 medium supplemented with 30 ng / mL BMP4, 100 ng / mL FGF7, 200 ng / mL FGF4, 8 μM CHIR99021, 1 μg / mL heparin, 2% PFHM-II, and antibiotic-antimycotic agents), and the Cell Culture Insert gel was cultured for 3 days to ventralize the hindgut. 500 μL of induction medium b2 was added below the Cell Culture Insert gel, and the medium was replaced every 2 days.

[0194] Step c2: Transplantation into the renal capsule of immunodeficient mice After ventralization of the hindgut (day 14 of differentiation induction), the Cell Culture Insert gel was cut from the Cell Culture Insert Transparent PET Membrane membrane and placed on a 35 mm dish. Next, the ventral hindgut organoids were removed from the 2.5 mg / mL Matrigel using tweezers under a stereomicroscope.

[0195] Under isoflurane inhalation anesthesia (1.3-1.4% isoflurane, 190-200 mL / min), the renal capsule of 4-week-old male NOD SCID JAX mice (Oriental Yeast Co., Ltd.) was incised with a razor blade, and the ventral hindgut organoids were transplanted between the renal capsule and the renal parenchyma. Four weeks after transplantation, the area containing the transplant was harvested and fixed overnight in 4% PFA at 4°C. The fixed transplants were frozen and cryosectioned. The cryosections were subjected to immunofluorescence staining and H&E staining.

[0196] Four weeks after transplantation, the transplanted ventral hindgut organoids formed sac-like cell structures (bladder organoids) with a lumen, similar to the in vivo bladder. Bladder organoids contained cells expressing the bladder epithelial markers UPK2 (Figure 5a), P63 (Figure 5b), and KRT5 (Figure 5c), resembling the layered structure of the mature bladder in vivo (Figure 5g). Specifically, in the bladder organoids, Krt5-basal cell-like cells co-expressing P63 and KRT5 were localized at the outermost periphery of the sac-like cell structures (Figure 5e). Furthermore, a small number of intermediate cell-like cells co-expressing UPK2 and P63 were present inward of the region containing the KRT5-basal cell-like cells (Figure 5f). Furthermore, superficial cell-like cells that expressed UPK2 but did not express P63 or KRT5 were localized inward of the area where intermediate cell-like cells were localized, facing the lumen of the bladder organoid (Fig. 5e and f).

[0197] Examples 2 and 3 showed that multiple types of bladder epithelial cells can be induced, and that the cell structure composed of these multiple types of bladder epithelial cells has a layered structure. Examples 2 and 3 showed that a bladder-like three-dimensional structure composed of multiple types of bladder epithelial cells can be formed. When comparing the ventral hindgut organoids in the in vitro differentiation induction system of Example 2 with the in vivo differentiation induction system of Example 3, it was shown that in order to form and mature a bladder-like sac-like structure from the ventral hindgut organoids, it is preferable to combine them with an in vivo differentiation induction system that allows long-term culture or in which mesenchyme is present.

[0198] [Example 4] Production of ventral hindgut organoids Figure 6 is a flow chart showing the culture of bladder organoid production. Figure 6 shows step A of inducing differentiation from human iPS cells into definitive endoderm cells, step b3 of inducing differentiation from definitive endoderm cells into ventral hindgut organoids, and step c3 of inducing differentiation from ventral hindgut organoids into bladder organoids by co-culturing with mesenchymal cells or mesenchymal stem cells. The number of days listed at the top of Figure 6 is the number of days required for exemplary differentiation induction according to one embodiment. In Example 4, step A and step b3 are carried out to produce ventral hindgut organoids.

[0199] Pre-cultivation of human iPS cells 45,000 cells / cm in a 6-well plate 2 Human iPS cells were precultured in the same manner as in Example 1, except that the cells were seeded so that the total number of cells was 100. Step A: Induction of differentiation into definitive endoderm cells Differentiation induction of human iPS cells into definitive endoderm cells was carried out in the same manner as in Example 1, except that the concentration of the additive CHIR99021 contained in induction medium A, STEMdiff APEL2 medium, was changed from 1 μM to 1.25 μM.

[0200] Step b3: Induction of ventral hindgut differentiation After definitive endoderm induction (day 3 of differentiation induction), induction medium A was replaced with induction medium b3, STEMdiff APEL2 medium (supplemented with 10 ng / mL BMP4, 200 ng / mL FGF4, 4 μM CHIR99021, 1 μg / mL Heparin, 2% PFHM-II, and antibiotic-antimycotic), and the differentiated definitive endoderm cells were cultured for 3 days.

[0201] On day 5 of differentiation induction, shaking culture was started at 100 rpm. On day 6 of differentiation induction, the floating spheroids were collected into a 35 mm dish. Next, medium was sprayed onto the spheroids remaining in the well from which the floating spheroids were collected, using a pipette, to suspend the spheroids, which were then collected into the same 35 mm dish. The collected spheroids were placed on ice.

[0202] Matrigel Growth Factor Reduced (Corning) (protein concentration: 10 mg / mL) was diluted to 50% with induction medium b3 and added 50 μL / well to a 24-well Cell Culture Insert Transparent PET Membrane with an 8.0 μm pore size (Corning). The mixture was then added to 50 μL of 50% Matrigel solution and mixed. The entire mixture was added on top of the gelled 50% Matrigel and incubated at 37°C and 5% CO2 for 30 minutes to form a gel. Cell Culture Insert gel was obtained. Induction medium b3 was added to the top and bottom of the Cell Culture Insert gels (200 μL and 300 μL, respectively). The cells were cultured at 37°C and 5% CO2 for an additional 8 days (until day 14 of differentiation induction). Medium changes were performed daily for 2D culture and every two days for 3D culture. Spheroids in 50% Matrigel were grown in induction medium b3 and formed rounded cellular structures with luminal structures (ventral hindgut organoids).

[0203] Immunofluorescence staining was performed on the ventral hindgut organoids on days 11 and 14 after differentiation induction. The following antibodies were used in the immunofluorescence staining in Examples 4 and 5. Private labels were Mouse Anti-CDX2(1:100, BioGenex, #MU392-UC), Mouse Anti-FOXA2(1:100, Santa Cruz). Biotechnology、#sc-101060)、Rabbit Anti-GATA3(1:100、Cell Signaling、#5852S)、Goat Anti-GATA3(1:100、R&D Systems、#AF2605) Mouse Anti-ISL1&2(1:100、DSHB、#39.4D5)、Goat Anti-SOX2(1:100、R&D Systems、#AF2018)、Rabbit Anti-SATB2(1:100、CELL MARKER、#384R-14) Rabbit Anti-HOXA13(1:200、abcam、#ab106503)、Goat Anti-ZO1(1:100、ThermoFisher、#PA5-19090) Rabbit Anti-pSmad1 / 5 / 8(1:100、Cell signaling、#13820) Rat Anti-CK8(1:200、DSHB、#TROMA-1)、Mouse Anti-Uroplakin II(1:100、BIOCARE MEDICAL、#ACR3051C)、Rabbit Anti-ΔNP63(1:100、Cell signaling、#67825) Rabbit Anti-P63(1:100;abcam、#ab124762)、Chicken Anti-Keratin 5(1:300、BioLegend、#905903)、Mouse Anti-ECAD(1:200、BD Transduction Laboratories、#610181) Goat Anti-ECAD(1:300; R&D SYSTEMS, #AF648), Chicken Anti-Vimentin (1:300, NOVUS, #NB300-223), Rabbit Anti-αSMA (1:100, Cell Signaling, #19245T).

[0204] The secondary antibodies used for immunofluorescence staining were Alexa Fluor 488 Donkey Anti-Mouse IgG (1:400, Life Technologies, #A21202), Alexa Fluor 568 Donkey Anti-Rabbit IgG (1:400, Life Technologies, #A10042), Alexa Fluor 647 Donkey Anti-Goat IgG (1:400, Life Technologies, #A21447), and Alexa Fluor 647 Donkey Anti-Chicken IgY (1:400, Jackson ImmunoResearch, #703-606-155).

[0205] Hindgut organoids after hindgut differentiation induction in step b1 of Example 1 (day 11 of differentiation induction) did not have a luminal structure (Figure 7a). Ventral hindgut organoids after ventral hindgut differentiation induction in step b3 of Example 4 (day 11 of differentiation induction) had a luminal structure (Figure 7b). Both hindgut organoids and ventral hindgut organoids expressed the epithelial markers KRT8 and ECAD (Figures 7a1, a10, b1, and b10). Hindgut organoids weakly expressed the ventral hindgut marker GATA3 (Figure 7a2), whereas ventral hindgut organoids strongly expressed it (Figure 7b2). Hindgut organoids expressed dorsal hindgut markers SOX2, CDX2, and T (Figures 7a3 and a5-a7), whereas ventral hindgut organoids did not (Figures 7b3 and b5-b7). The hindgut organoids did not express the intestinal marker FOXA2 (Fig. 7a9), but the ventral hindgut organoids did (Fig. 7b9). Thus, the hindgut organoids before ventralization of the hindgut in step b2 in Example 1 did not have a luminal structure and expressed dorsal hindgut markers (SOX2, CDX2, and T), while the ventral hindgut marker (GATA3) and intestinal marker (FOXA2) were only weakly expressed or not expressed (Fig. 7a). In contrast, the ventral hindgut organoids during ventral hindgut differentiation induction in step b3 in Example 4 had a luminal structure and did not express the dorsal hindgut marker, but expressed the ventral hindgut marker and intestinal marker (Fig. 7b).

[0206] Ventral hindgut organoids on day 8 of ventral hindgut differentiation induction in step b3 of Example 4 (day 14 of differentiation induction) were observed under a microscope using immunofluorescence staining (Figure 8). The ventral hindgut organoids expressed the epithelial tissue marker ECAD (Figure 8d3) and the tight junction marker ZO1 (Figure 8c3). In addition, the ventral hindgut organoids expressed early intestinal epithelial markers FOXA2 (Figure 8b1) and CK8 (Figure 8c1), and ventral hindgut markers GATA3 (Figures 8a3, b3, and e1), ISL1 (Figure 8d1), SATB2 (Figure 8c2), and ΔNP63 (Figure 8b2). Furthermore, the ventral hindgut organoids expressed the ventral Cloaca region marker Phospho-Smad1 / 5 / 8 (Figure 8d2) and the posterior gut and Cloaca region marker HOXA13 (Figure 8f2). On the other hand, the ventral hindgut organoids barely expressed the dorsal hindgut markers CDX2 (Figures 8a1 and f1) and SOX2 (Figures 8a2 and e2). Furthermore, the ventral hindgut organoids did not express the bladder epithelial progenitor cell and bladder epithelial cell markers UPK2 (Figure 8e3) and KRT5 (Figure 8f3). These results suggest that 8 days of culture in induction medium b3 (14 days of differentiation induction) resulted in the formation of ventral hindgut organoids with luminal structures. Furthermore, these results suggest the formation of ventral hindgut / Cloaca-like cells prior to the development of bladder epithelial progenitor cells.

[0207] Example 5: In vitro production of bladder organoids In Example 5, the steps A, b3 and c3 shown in Figure 6 are carried out to produce bladder organoids in vitro. In Example 5, step C, the ventral hindgut organoids formed in step b3 are cultured in vitro with bladder mesenchymal cells derived from E12.5 mice (step c3).

[0208] Pre-cultivation of human iPS cells Human iPS cells were precultured in the same manner as in Example 4. Step A: Induction of differentiation into definitive endoderm cells In the same manner as in Example 4, human iPS cells were induced to differentiate into definitive endoderm cells. Step b3: Hindgut differentiation induction In the same manner as in Example 4, differentiation induction from definitive endoderm cells to ventral hindgut organoids was carried out.

[0209] Step c3: Induction of bladder epithelial differentiation by co-culture Preparation of E12.5 mouse bladder mesenchymal cells ICR mice on day 12 of pregnancy were euthanized by spinal dislocation. The gestational sac containing the fetus was removed from the ICR mouse and placed in ice-cold 10% FBS / PBS(-). The fetus was removed from the gestational sac using tweezers, and the placenta was dissected away from the fetus. Next, the upper half of the fetus was removed with tweezers to expose the bladder, and the region from the bladder to the urethra was collected. The collected bladder and urethral region was placed in 200 μL of Dissociation Buffer (Dispase: Corning #354235, 1 mg / mL DNase I: Sigma-Aldrich #11284932001) and incubated at 37°C for 1 minute. The cells were then diluted twice with 3 mL of ice-cold M2 medium and left on ice for 10 minutes. Using tweezers and a glass needle, only the bladder mesenchymal cells above the ureteral insertion site were collected and collected in a 1.5 mL tube. Next, 200 μL of 37°C-warmed TrypLE Select (Thermo Fisher Scientific) was added to the tube and incubated at 37°C for 3 minutes, followed by pipetting until the tissue was isolated into single cells. The enzyme reaction was stopped by adding 1 mL of DMEM (supplemented with 10% FBS, GultaMAX-I, and antibiotics / antimycotics). The tube was centrifuged at 300 rcf for 3 minutes, and the supernatant was removed. The pellet was resuspended in 250 μL of STEM-CELLBAMKER GMP grade (ZENOQ #CB045). The resulting cell suspension was transferred to a cryopreservation tube and stored at -80°C.

[0210] Co-culture with E12.5 mouse bladder mesenchymal cells The ventral hindgut organoids in the extracellular matrix (50% Matrigel) gel obtained in step b3 were detached from the gel by pipetting and placed on ice.

[0211] The cryopreserved E12.5 mouse bladder mesenchymal cells were thawed in a 37°C water bath and then suspended in 1 mL of STEMdiff APEL2 medium. The resulting cell suspension was centrifuged at 300 rcf for 3 minutes, and the supernatant was removed. The pellet was resuspended in 200 μL of STEMdiff APEL2 medium. The resulting cell suspension was counted and added to a PrimeSurface® plate 96V (Sumitomo Bakelite #MS-9096V) at 25,000 cells / well. The plate was then centrifuged at 200 rcf for 5 minutes.

[0212] The ventral hindgut organoids detached from Matrigel were placed on the plate at one cell per well and allowed to settle. The cell suspension was then added to each well at 25,000 cells per well, and the plate was centrifuged at 200 rcf for 5 minutes. The ventral hindgut organoids and bladder mesenchymal cells were then cultured at 37°C and 5% CO2 for 1 day. The combined cell masses were then transferred from the plate wells to a container, and the container was placed in a 3D rotary suspension culture device, CellPet 3D-iP (J-Tech Corporation). The combined cell masses were cultured in STEMdiff APEL2 medium (STEMCELL Technologies) (supplemented with 2% PFHM-II and antibiotic-antimycotic) or STEMdiff APEL2 medium (STEMCELL Technologies) (supplemented with 2% PFHM-II, 2% FBS, and antibiotic-antimycotic) CellPet 3D-iP at 37°C under 5% CO2 for 11 days to induce bladder organoids (day 24 of differentiation induction).

[0213] Bladder organoids on day 7 of co-culture of ventral hindgut organoids and bladder mesenchymal cells in step c3 (day 20 of differentiation induction) were observed under a bright field microscope (Figure 9). A double structure consisting of epithelial and luminal structures was observed in the bladder organoids (Figures 9a-c). Even when the double structure was difficult to observe under bright field (Figure 9d), it was observed when observed using fluorescent staining.

[0214] Bladder organoids grown on day 11 of co-culture in step c3 (day 24 of differentiation induction) were spherical and had a luminal structure. The bladder organoids were observed under a microscope using immunofluorescence staining (Figure 10). The bladder organoids had a lumen and a layer of cells expressing the epithelial cell marker ECAD, which faced the lumen (Figure 10b1). A layer of cells expressing the mesenchymal cell marker VIM was located outside the ECAD-expressing cell layer (Figure 10b3 and b5). The bladder organoids contained a DAPI-stained cell layer (Figure 10c4) outside the ECAD-expressing cell layer (Figure 10c1), and a layer of cells expressing the smooth muscle cell marker αSMA was located outside the DAPI-stained cell layer (Figure 10c2 and c4). These results show that the bladder organoids have a lumen; an epithelial cell layer expressing ECAD facing the lumen; a stromal-like cell layer expressing VIM outside the epithelial cell layer; and a smooth muscle cell layer expressing αSMA outside the stromal-like cell layer.

[0215] Cells in the ECAD cell layer of the bladder organoids co-expressed human lamin B1 (hLNB) (Figure 10b2 and b). This result indicates that the epithelial cells of the bladder organoids are derived from human iPS cells. Therefore, the bladder organoids have a lumen; an epithelial cell layer derived from human iPS cells facing the lumen; a stromal-like cell layer derived from mouse bladder mesenchymal cells outside the epithelial cell layer; and a smooth muscle cell layer derived from mouse mesenchymal cells outside the stromal-like cell layer.

[0216] Contractile movements were observed in the bladder organoids, suggesting that the smooth muscle cell layer of the bladder organoids was functional.

[0217] The epithelial cell layer of the bladder organoids contained an innermost layer of UPK2-expressing cells (Figure 10a1), and an outer layer of ΔNP63-expressing cells (Figures 10a2 and a5). These results indicate that the bladder organoids resemble the structure of the bladder epithelium during bladder development. No cells expressing the bladder epithelial marker KRT5 were observed in the epithelial cell layer of the bladder organoids (Figure 10a3). Basal cells expressing KRT5 are known to appear in the late stages of bladder development. These results suggest that the bladder organoids on day 24 of differentiation induction in Example 5 are at a stage of bladder development. The epithelial cell layer of the bladder organoids expressed the bladder epithelial cell marker GATA3 (Figure 10d2), but expressed the developing bladder epithelial cell marker FOXA2 (Figure 10d1). These results also suggest that the bladder organoids on day 24 of differentiation induction in Example 5 are at a stage in the process of bladder development.

[0218] The bladder organoids on the 29th day of co-culture in step c3 (42nd day of differentiation induction) were observed under a microscope using immunofluorescence staining (Fig. 11).The bladder organoids had a luminal structure.The epithelial cell layer of the bladder organoids faces the lumen and has a layer of cells expressing UPK1B (Fig. 11a1) and UPK2 (Fig. 11b1);Outside of this cell layer, there is a layer of cells expressing ΔNP63 (Fig. 11a2 and b2);And outside of this cell layer, there is a layer of cells expressing KRT5 (Fig. 11a3 and b3) (Fig. 11a5 and b5).Since it is known that basal cells expressing KRT5 appear at a late stage of bladder development, these results suggest that the bladder organoids on the 42nd day of differentiation induction in Example 5 are at a late stage of bladder development.

Claims

1. 1. A method for producing ventral hindgut organoids, comprising: Culturing the pluripotent stem cells using induction medium A containing activin A and a GSK3β inhibitor to induce differentiation into definitive endoderm cells; and The method comprises culturing the definitive endoderm cells in an induction medium B containing a fibroblast growth factor and a GSK3β inhibitor, and optionally further containing a bone morphogenetic protein, and then culturing the cells in an induction medium B containing a fibroblast growth factor and a GSK3β inhibitor, and optionally further containing a bone morphogenetic protein, in the presence of an extracellular matrix, to form ventral hindgut organoids.

2. The formation of the ventral hindgut organoids comprises culturing the definitive endoderm cells in the induction medium B, followed by culturing the cells in the induction medium B in the presence of an extracellular matrix to form hindgut organoids, and culturing the hindgut organoids in the induction medium B in the presence of an extracellular matrix to form ventral hindgut organoids; The method of claim 1, wherein the induction medium B for forming hindgut organoids comprises a fibroblast growth factor and a GSK3β inhibitor, and the induction medium B for forming ventral hindgut organoids comprises a fibroblast growth factor, a GSK3β inhibitor, and a bone morphogenetic protein.

3. The method of claim 1, wherein the induction medium B for forming ventral hindgut organoids comprises fibroblast growth factor, GSK3β inhibitor and bone morphogenetic protein.

4. A ventral hindgut organoid produced by the method according to any one of claims 1 to 3.

5. 1. A ventral hindgut organoid, comprising: They express the ventral hindgut marker P63, expresses HOXA13 and CK8 / KRT8, and Ventral hindgut organoids that do not substantially express the dorsal hindgut marker SOX2.

6. A ventral hindgut organoid having a lumen, expressing at least one ventral hindgut marker selected from the group consisting of P63, ΔN63, GATA3, ISL1, and SATB2; expressing at least one marker selected from the group consisting of Phospho-Smad1 / 5 / 8, HOXA13, FOXA2, CK8 / KRT8, and ECAD; and A ventral hindgut organoid that does not substantially express at least one dorsal hindgut marker selected from the group consisting of SOX2, T, and CDX2.

7. A ventral hindgut organoid produced by the method according to any one of claims 1 to 3, or a ventral hindgut organoid according to any one of claims 4 to 6, Culturing the cells in the presence of an extracellular matrix using induction medium C containing retinoic acid, fibroblast growth factor, and bone morphogenetic protein; into the kidney or bladder or surrounding areas of a human or non-human mammal; or A method for producing bladder organoids, comprising culturing in the presence of mesenchymal stem cells or mesenchymal cells.

8. Bladder organoids produced by the method of claim 7.

9. 1. A bladder organoid, comprising: a first cell layer comprising cells that do not substantially express p63 but express UPK1B and / or UPK2; and a second cell layer comprising cells co-expressing P63 and UPK1B and / or UPK2, the second cell layer being located laterally relative to the first cell layer; The bladder organoid may further comprise a third cell layer comprising cells that co-express P63 and KRT5, the third cell layer being located laterally relative to the second cell layer.

10. The bladder organoid of claim 9, comprising a lumen surrounded by the first cell layer.

11. The bladder organoid of claim 9 or 10, comprising a fourth cell layer comprising interstitial-like cells located outward relative to the third cell layer, and optionally further comprising a fifth cell layer comprising smooth muscle cells located outward relative to the fourth cell layer.

12. A method for producing a non-human mammal having ventral hindgut organoids or bladder organoids in its kidney or bladder or a peripheral site thereof, comprising introducing ventral hindgut organoids produced by the method according to any one of claims 1 to 3, ventral hindgut organoids according to any one of claims 4 to 6, bladder organoids produced by the method according to claim 7, or bladder organoids according to any one of claims 8 to 11 into the kidney or bladder or a peripheral site thereof of the non-human mammal.

13. A non-human mammal having a ventral hindgut organoid produced by the method according to any one of claims 1 to 3, a ventral hindgut organoid according to any one of claims 4 to 6, a bladder organoid produced by the method according to claim 7, or a bladder organoid according to any one of claims 8 to 11 in its kidney or bladder or a surrounding area thereof.

14. A regenerative medicine composition for treating bladder damage or disease, comprising a ventral hindgut organoid produced by the method of any one of claims 1 to 3, a ventral hindgut organoid described in any one of claims 4 to 6, a bladder organoid produced by the method of claim 7, or a bladder organoid described in any one of claims 8 to 11.

15. A method for evaluating drug responsiveness to a test substance, comprising: Contacting a test substance with a ventral hindgut organoid produced by the method according to any one of claims 1 to 3, a ventral hindgut organoid according to any one of claims 4 to 6, a bladder organoid produced by the method according to claim 7, a bladder organoid according to any one of claims 8 to 11, a non-human mammal produced by the method according to claim 12, or a non-human mammal according to claim 13; and A method comprising measuring drug responsiveness to the test substance in the ventral hindgut organoids, the bladder organoids, or the non-human mammal.

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