Culture method for promoting organogenesis of kidney while retaining kidney formation region

Abstract

The present invention addresses the problem of producing a renal tissue, and produces a renal tissue by co-culturing nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells in a culture medium containing a Wnt signal activator.

Description

A culture method for promoting kidney organogenesis while maintaining the nephrogenic region

[0001] The present invention relates to a method for promoting kidney organogenesis, and more particularly to a renal tissue-like culture having kidney structures, a method for culturing the same, and a method for producing the same.

[0002] The adult mammalian kidney, the metanephros, is formed from multiple embryonic renal progenitor cells, including nephron progenitor cells that give rise to nephron-forming epithelial cells such as glomeruli and tubules, ureteric bud cells that give rise to collecting ducts and the lower urinary tract, and renal interstitial progenitor cells that give rise to renal interstitial cells, mesangial cells, and vascular smooth muscle cells. Nephron progenitor cells, ureteric bud cells, and renal interstitial progenitor cells undergo repeated self-renewal and differentiation, maintaining the kidney-forming region, and gradually undergo organogenesis, developing and growing as organs.

[0003] A culture method has been reported in which nephron progenitor cells, ureteric bud cells, and renal interstitial progenitor cells derived from mouse embryonic kidneys or mouse ES cells are co-cultured in a gel containing bovine serum to promote organogenesis (Non-Patent Document 1). Another culture method has been reported in which renal organoids containing nephron progenitor cells, renal interstitial progenitor cells, etc., prepared from human pluripotent stem cells are separated using a cell dissociation reagent and cultured in a gel containing FGF2, CHIR99021, retinoic acid, Y-27632, and GDNF to derive ureteric bud-like epithelium and promote organogenesis (Non-Patent Document 2). However, to date, there have been no reports of a culture method that promotes organ formation while maintaining the kidney-forming region when co-culturing human-derived nephron progenitor cells, ureteric bud cells, and renal interstitial progenitor cells, such as nephron progenitor cells, ureteric bud cells, and renal interstitial progenitor cells generated from human pluripotent stem cells, such as human induced pluripotent stem (iPS) cells or human embryonic stem (ES) cells.

[0004] Tanigawa S, Tanaka E, Miike K, Ohmori T, Inoue D, Cai CL, et al., Nat Commun. 2022;13: 611.Howden SE, Wilson SB, Groenewegen E, Starks L, Forbes TA, Tan KS, et al., Cell Stem Cell. 2021;28: 671-684.e6.

[0005] An objective of the present invention is to produce renal tissue with a more physiological structure by promoting organogenesis while maintaining the nephron-forming region in cultures of nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells. In particular, an objective of the present invention is to produce renal tissue with a more physiological structure by promoting organogenesis while maintaining the nephron-forming region in cultures of human-derived nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells.

[0006] The present inventors conducted extensive research to solve the above problems and found that co-culturing nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells in the presence of a Wnt signaling activator can promote organogenesis while maintaining the nephrogenic region, thereby enabling the creation of renal tissue-like cultures with kidney structure.

[0007] That is, the present invention has the following features: [1] A method for producing a kidney tissue-like culture, comprising a step of co-culturing nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells in a medium containing a Wnt signaling activator. [2] The method of [1], wherein the co-culturing is performed by suspension culture. [3] The method of [2], wherein the suspension culture is performed in a state where aggregates of nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells are coated with an extracellular matrix. [4] Any of the methods of [1] to [3], wherein the Wnt signaling activator is Wnt3a protein. [5] Any of the methods of [1] to [4], wherein the medium further contains fibroblast growth factor 9 (FGF9). [6] Any of the methods of [1] to [5], wherein the medium further contains retinoic acid (RA) and / or a derivative thereof, and glial cell line-derived neurotrophic factor (GDNF). [7] The method of [6], wherein the medium further contains α-albumin and R-spondin 1 (RSPO1). [8] Any of the methods of [1] to [7], wherein the renal tissue-like culture has one or more of a mesangial structure, a branching ureteric bud structure, an S-shaped body-like structure, and a cap mesenchyme structure. [9] Any of the methods of [1] to [8], wherein the nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells are of human origin.

[10] Any of the methods of [1] to [9], wherein one or more of the nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells are cells induced from pluripotent stem cells.

[11] A renal tissue-like culture, comprising human-derived cells, having a mesangial structure, a branching ureteric bud structure, an S-shaped body-like structure, and a cap mesenchyme structure.

[12] The kidney tissue-like culture of

[11] , wherein the mesangial structure is a structure containing PDGFRB and / or GATA3-positive cells inside a glomerulus-like structure, the branching ureteric bud structure has two or more ureteric buds, the tips of which contain RET-positive cells, the S-shaped body-like structure is a structure containing an S-shaped body-like structure including a proximal region and a distal region, and the cap mesenchyme structure is a structure that surrounds the tips of the ureteric buds and contains SIX2 and / or PAX8-positive cells.

[0008] The methods of the present invention can induce organogenesis in nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells while maintaining the kidney-forming region, thereby producing renal tissue-like cultures with kidney structure. Furthermore, the present invention can provide renal tissue-like cultures with kidney structure, particularly human-derived cultures. Furthermore, the cultures of the present invention can have, in particular, mesangial structures, branching ureteric bud structures, and cap mesenchyme structures. These structures are characteristic of the kidney during organogenesis and are expected to develop and grow more effectively as organs.

[0009] Figure (photograph substitute for drawing) showing the results of immunostaining for SIX2, FOXD1, and CALB1 in tissue-like cultures on day 6 after regeneration of kidney structure under CRFY conditions. Scale bar: 200 μm. Figure (photograph substitute for drawing) showing the results of analyzing gene expression levels by quantitative RT-PCR (qT-PCR) for progenitor cell marker genes in tissue-like cultures on day 6 after regeneration of kidney structure under CRFY conditions, CRFY+G conditions, or RFY+G+WR conditions. Figure (photograph substitute for drawing) showing the results of immunostaining for SIX2, RET, and CALB1 in tissue-like cultures on day 6 after regeneration of kidney structure under RFY+G+WR conditions. Scale bar: 200 μm. Figure (photograph substitute for drawing) showing the results of immunostaining for PAX8, SIX2, FOXD1, and CALB1 in tissue-like cultures on day 6 after regeneration of kidney structure under RFY+G+WR conditions. Scale bar: 200 μm. Immunostaining for BRN1, HNF4α, and CALB1 in tissue-like cultures on day 6 after regeneration of kidney structure under RFY+G+WR conditions (photograph substitute for drawing). Scale bar: 200 μm. Immunostaining for PDGFRB, GATA3, MAFB, and NPHS1 in tissue-like cultures on days 6 and 12 after regeneration of kidney structure under RFY+G+WR conditions (photograph substitute for drawing). Scale bar: 100 μm. Immunostaining for PDFGRB in cell aggregates formed from induced nephron progenitor cells (NPCs) that were cultured for 48 hours under CRFY conditions (photograph substitute for drawing). Immunostaining for ureteric bud marker CK8 and nephron progenitor cell marker SIX2 in tissue-like cultures on day 13 after regeneration of kidney structure under RFY+G+WR conditions (photograph substitute for drawing).

[0010] The present invention will be described in detail below.

[0011] <1> Method of the Present Invention In one aspect, the present invention relates to a method for producing a renal tissue-like culture, which comprises a step of co-culturing nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells in a medium containing a Wnt signal activator.

[0012] Unless otherwise specified, the "cells" of the present invention are preferably derived from mammals, more preferably from primates or rodents, and even more preferably from humans or mice. Furthermore, unless otherwise specified, the "cells" of the present invention are more preferably derived from primates, and even more preferably from humans. Therefore, in the present invention, the nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells are each preferably derived from mammals, more preferably from primates, and even more preferably from humans. That is, in the present invention, cells used to produce nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells, such as induced pluripotent stem (iPS) cells and embryonic stem (ES) cells, and cells derived from any of the nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells, such as mesangial cells and podocytes, are preferably derived from mammals, more preferably from primates, and even more preferably from humans.

[0013] Nephron progenitor cells (NPCs) are one of the precursor cells for the formation of the metanephros and give rise to nephron-constituting epithelial cells such as glomeruli and renal tubules. In addition to these properties, nephron progenitor cells may also be characterized by the expression of marker genes. Specifically, nephron progenitor cells may be characterized by, for example, the expression of the nephron progenitor cell marker gene SIX2, or the expression of SIX2 and the intermediate mesoderm marker gene OSR1. Nephron progenitor cells are not particularly limited, and may be nephron progenitor cells isolated from a living organism or induced from undifferentiated cells. Examples of nephron progenitor cells isolated from a living organism include nephron progenitor cells isolated from an embryo. Examples of nephron progenitor cells induced from undifferentiated cells include nephron progenitor cells induced from pluripotent stem cells. Pluripotent stem cells are described below. The method for inducing nephron progenitor cells from pluripotent stem cells is not particularly limited, and any known method or a modified version of any known method may be used. Examples of such methods include the method described in Tsujimoto, H., et al., Cell Rep. 31, 107476, 2020, or a modified version of said method. A specific example of a modified version of the method described in Tsujimoto, H., et al., Cell Rep. 31, 107476, 2020 involves reseeding the cells using a three-dimensional culture method on day 4 of culture and performing spinner culture.

[0014] Specifically, the method for inducing nephron progenitor cells from pluripotent stem cells may be performed by, for example, first inducing the late primitive streak (PPS) in pluripotent stem cells, then replated in 3D culture approximately four days after the initiation of PPS induction, and cultured in a medium containing a glycogen synthase kinase 3β (GSK-3β) inhibitor (e.g., CHIR99021), bFGF, and activin A for approximately two days under agitation. Subsequently (approximately six days after the initiation of PPS induction), the medium is changed to one containing retinoic acid (RA), NOGGIN, and FGF9, and cultured. Approximately eight days after the initiation of PPS induction, the medium is further changed to one containing FGF9 and a GSK-3β inhibitor (e.g., CHIR99021), and cultured for approximately three days. Furthermore, when replated in 3D culture, a ROCK inhibitor (e.g., Y-27632) may be added to the medium after replated. More specifically, the step of inducing pluripotent stem cells into the late primitive streak (PPS) may be carried out by, for example, seeding pluripotent stem cells onto a culture dish preferably coated with extracellular matrix (specifically, which may be, for example, laminin fragments), culturing the cells for about 1 day (day 0) in a medium containing a GSK-3β inhibitor (e.g., CHIR99021), RA, BMP4, and bFGF, switching the medium to a medium containing CHIR99021, bFGF, and BMP7 and culturing for another day (day 1), switching the medium to a medium containing a GSK-3β inhibitor (e.g., CHIR99021), BMP7, and a TGFβ inhibitor (e.g., A83-01) and culturing for another day (day 2), switching the medium to a medium containing a GSK-3β inhibitor (e.g., CHIR99021), bFGF, activin A, and a ROCK inhibitor (e.g., Y-27632) and culturing for another day (day 3), and recovering the cells on about day 4.

[0015] Renal interstitial progenitor cells (IPCs) are one type of progenitor cell for the formation of the metanephros and can differentiate into renal interstitial cells, renal erythropoietin-producing cells, mesangial cells, juxtaglomerular cells, and other renin-producing cells. In addition to these properties, renal interstitial progenitor cells may also be characterized by the expression of marker genes. Specifically, renal interstitial progenitor cells may be characterized by, for example, the expression of the renal interstitial progenitor cell marker gene FOXD1 (Forkhead Box D1), or the expression of FOXD1 and OSR1 and / or PDGFRB (platelet-derived growth factor receptor β). Renal interstitial progenitor cells are not particularly limited, and may be renal interstitial progenitor cells isolated from a living organism or induced from undifferentiated cells. Renal interstitial progenitor cells isolated from a living organism include, for example, renal interstitial progenitor cells isolated from an embryo. Renal interstitial progenitor cells induced from undifferentiated cells include, for example, renal interstitial progenitor cells induced from pluripotent stem cells. Pluripotent stem cells are described below. The method for inducing renal interstitial progenitor cells from pluripotent stem cells is not particularly limited, and any known method may be used, or a modified version of any known method may be used. Examples of such methods include the method described in WO 2023 / 017848. Renal interstitial progenitor cells may be by-products of inducing nephron progenitor cells. Examples of such methods include the methods described in JP2020031648A and WO2018216743A1.

[0016] Specifically, a method for inducing renal interstitial progenitor cells from pluripotent stem cells may be carried out, for example, by performing the above-mentioned step of inducing PPS on pluripotent stem cells, then replated to form aggregates on approximately day 4 after the initiation of PPS induction (for example, aggregates may be formed by replated on a U-bottom plate), and culturing the cells for approximately 2 days in a medium containing a GSK-3β inhibitor (e.g., CHIR99021), bFGF, and activin A. Thereafter (on day 6 after the initiation of PPS induction), the medium is changed to a medium containing a GSK-3β inhibitor (e.g., CHIR99021), RA, smoothened monoamine agonist (SAG), and IL-1β but not bFGF, and the cells are cultured. On approximately day 8, the medium is changed to a medium containing a GSK-3β inhibitor (e.g., CHIR99021), RA, SAG, and a TGFβ inhibitor (e.g., SB431542) but not bFGF, and the cells are cultured for approximately 3 days.

[0017] Ureteric bud (UB) cells are one of the precursor cells for the formation of the metanephros and give rise to the collecting duct and lower urinary tract. Ureteric bud cells are also called mesonephric duct (ND) cells depending on their location in tissue or developmental stage, but these cells do not necessarily need to be distinguished in vitro. That is, the ureteric bud cells of the present invention may also be referred to as ND cells. In addition to the above properties, ureteric bud cells may be characterized by the expression of a marker gene. Specifically, ureteric bud cells may be characterized by the expression of the ureteric bud cell marker gene RET. Ureteric bud cells are not particularly limited, but may be ureteric bud cells isolated from a living organism or ureteric bud cells induced from undifferentiated cells. Examples of ureteric bud cells isolated from a living organism include ureteric bud cells isolated from an embryo. Examples of ureteric bud cells induced from undifferentiated cells include ureteric bud cells induced from pluripotent stem cells. Pluripotent stem cells are described below. The method for inducing ureteric bud cells from pluripotent stem cells is not particularly limited, and any known method or a modified version of any known method may be used. Examples of such methods include those described in Tsujimoto, H., et al. Cell Rep. 31, 107476, 2020 and Mae, S. I., et al. Cell Rep. 32, 2020. Examples of methods for inducing ureteric bud cells from pluripotent stem cells include a method in which they are induced via the anterior intermediate mesoderm.Specifically, the method for inducing anterior intermediate mesoderm from pluripotent stem cells includes, for example, seeding pluripotent stem cells on a culture dish coated with an extracellular matrix (specifically, which may be, for example, a laminin fragment), and then culturing the cells in a manner similar to that described in Essential 6. The cells may be cultured for about 1 day (day 0) in a serum-free medium supplemented with activin A, a GSK-3β inhibitor (e.g., CHIR99021), BMP4, and bFGF in a basal medium such as (E6), then switched to a medium containing a GSK-3β inhibitor (e.g., CHIR99021), bFGF, and BMP7 and cultured for another day (day 1), then switched to a medium containing an ALK2 / 3 inhibitor (e.g., LDN193189), a TGFβ inhibitor (e.g., A83-01), retinoic acid or a derivative thereof (e.g., TTNPB), and FGF8 and cultured for another day (days 2-3), and finally switched to a medium containing a ROCK inhibitor (e.g., Y-27632) in addition to an ALK2 / 3 inhibitor (e.g., LDN193189), a TGFβ inhibitor (e.g., A83-01), retinoic acid or a derivative thereof (e.g., TTNPB), and FGF8 and cultured for another day (day 4). Specifically, ureteric bud cells can be induced from anterior intermediate mesoderm by culturing the anterior intermediate mesoderm in a medium such as E6 containing a GSK-3β inhibitor (e.g., CHIR99021), an ALK2 / 3 inhibitor (e.g., LDN193189), FGF8, GDNF, and retinoic acid or its derivative (e.g., TTNPB) for approximately two days, dissociating the cultured cells into single cells using a cell detachment agent (e.g., Accutase), reseeding them on low-attachment plates, and culturing them for an additional two days in E6 medium containing a GSK-3β inhibitor (e.g., CHIR99021), an ALK2 / 3 inhibitor (e.g., LDN193189), FGF8, GDNF, and retinoic acid or its derivative (e.g., TTNPB) in addition to a ROCK inhibitor (e.g., Y-27632). Cells at this stage may also be considered to correspond to ND cells. In addition, after induction, the cells may be further cultured for approximately 6 days in a medium such as E6 containing a GSK-3β inhibitor (e.g., CHIR99021), an ALK2 / 3 inhibitor (e.g., LDN193189), FGF8, GDNF, retinoic acid or a derivative thereof (e.g., TTNPB), FGF1, EGF, and Matrigel.

[0018] In the present invention, nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells may or may not be derived from pluripotent stem cells, as described above. That is, one or more of the nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells may be derived from pluripotent stem cells, or all of them may be derived from pluripotent stem cells.

[0019] Pluripotent stem cells are stem cells that have the pluripotency to differentiate into many cells present in the body and also have the ability to proliferate, and include any cells that can be induced to differentiate into blastocyst-like structures. Pluripotent stem cells are not particularly limited, but include, for example, embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, embryonic germ cells (EG cells), etc. Preferred pluripotent stem cells are iPS cells and ES cells. Furthermore, pluripotent stem cells may be naive pluripotent stem cells or primed pluripotent stem cells, and are not particularly limited. The origin of pluripotent stem cells is preferably mammalian, more preferably primate, and even more preferably human.

[0020] ES cells that can be used are those established by known methods. Methods for establishing and maintaining ES cells are described, for example, in US Pat. No. 5,843,780; Thomson JA, et al. (1995), Proc. Natl. Acad. Sci. USA 92:7844-7848; Thomson JA, et al. (1998), Science. 282:1145-1147; H. Suemori et al. (2006), Biochem. Biophys. Res. Commun., 345:926-932; M. Ueno et al. (2006), Proc. Natl. Acad. Sci. USA, 103:9554-9559; H. Suemori et al. (2001), Dev. Dyn., 222:273-279; H. Kawasaki et al. (2002), Proc. Natl. Acad. Sci. USA, 99:1580-1585; Klimanskaya I, et al. (2006), Nature. 444:481-485. Human ES cell lines are available, for example, from the WiCell Research Institute (WA01(H1) and WA09(H9)) and from the Institute for Frontier Medical Sciences, Kyoto University (Kyoto, Japan) (Kyoto, Japan).

[0021] Embryonic germ cells are cells established from primordial germ cells during the fetal stage and have pluripotency similar to that of ES cells. They can be established by culturing primordial germ cells in the presence of substances such as LIF, bFGF, and stem cell factor (Y. Matsui et al. (1992), Cell, 70:841-847; J.L. Resnick et al. (1992), Nature, 359:550-551).

[0022] Induced pluripotent stem (iPS) cells are artificial stem cells derived from somatic cells that can be produced by introducing specific reprogramming factors into somatic cells in the form of DNA or protein. They have properties similar to those of ES cells, such as pluripotency and the ability to proliferate through self-renewal (K. Takahashi and S. Yamanaka (2006) Cell, 126:663-676; K. Takahashi et al. (2007) Cell, 131:861-872; J. Yu et al. (2007) Science, 318:1917-1920; Nakagawa, M. et al. (2008) Nat. Biotechnol. 26:101-106; International Publication WO 2007 / 069666). The reprogramming factors may be composed of genes that are specifically expressed in ES cells, their gene products, or non-coding RNAs, or genes that play an important role in maintaining the undifferentiated state of ES cells, their gene products, or non-coding RNAs, or low-molecular-weight compounds with equivalent functions. Numerous genes have been reported as reprogramming factors, and examples include, but are not limited to, Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, and Glis1, and these may be used alone or in combination.The reprogramming factors include histone deacetylase (HDAC) inhibitors (e.g., small molecule inhibitors such as valproic acid (VPA), trichostatin A, sodium butyrate, MC 1293, M344, and the like, nucleic acid expression inhibitors such as siRNA and shRNA against HDAC, etc.), MEK inhibitors (e.g., PD184352, PD98059, U0126, SL327, and PD0325901), glycogen synthase kinase-3 inhibitors (e.g., Bio and CHIR99021), DNA methyltransferase inhibitors (e.g., 5-azacytidine), histone methyltransferase inhibitors (e.g., small molecule inhibitors such as BIX-01294, and nucleic acid expression inhibitors such as siRNA and shRNA against Suv39hl, Suv39h2, SetDBl, and G9a, etc.), L-channel calcium agonists (e.g., Bayk8644), butyric acid, TGFβ inhibitors or ALK5 inhibitors (e.g., LY364947, SB431542, 616453, and A-83-01), p53 inhibitors (e.g., siRNA and shRNA against p53), ARID3A inhibitors (e.g., siRNA and shRNA against ARID3A), miRNAs such as miR-291-3p, miR-294, miR-295, and miR-302, Wnt signaling (e.g., soluble Wnt3a), neuropeptide Y, prostaglandins (e.g., prostaglandin E2 and prostaglandin J2), hTERT, SV40LT, UTF1, IRX6, GLIS1, PITX2, and DMRTBl, which are used to improve the efficiency of iPS cell establishment, can also be used as reprogramming factors. iPS cells can be generated by introducing reprogramming factors into somatic cells as described above, or they can be obtained from cell banks and used.

[0023] The methods of the present invention include a step of co-culturing nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells. Co-culturing nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells is not particularly limited as long as these cells are cultured in the same medium. For example, co-culturing nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells may be performed by mixing and culturing them. The mixing of nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells is not particularly limited as long as a renal tissue-like culture is obtained. These cells may be dissociated into single cells and then mixed, or a certain number of cell clusters may be mixed, or a combination thereof may be used. Preferably, the nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells are each dissociated into single cells and then mixed. The ratio of nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells is not particularly limited as long as a renal tissue-like culture can be obtained. For example, co-culture may be initiated at a ratio of 25-40:25-40:25-40 in terms of cell numbers, preferably 30-35:30-35:30-35, and even more preferably 1:1:1.

[0024] Co-culture of nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells may be preferably performed in suspension culture, where the cells are cultured in a non-adherent state to a culture substrate, such as in a low-cell-adhesion culture vessel.

[0025] Alternatively, in suspension culture, aggregates of nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells may be formed and then coated. The coating agent is preferably an extracellular matrix, such as collagen, proteoglycan, fibronectin, hyaluronic acid, tenascin, entactin, elastin, fibrin, and laminin, or fragments thereof. Coating of cell aggregates may be performed, for example, by transferring the aggregated cell masses to droplets formed with medium containing 50% by volume of the coating agent after initiating suspension culture (e.g., 48 hours later), coating the aggregates, and then adding additional medium to terminate the coating process. The coating time is not particularly limited, but may be, for example, 30 minutes or longer.

[0026] The medium for co-culturing nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells can be prepared by adding a Wnt signaling activator to a basal medium used for animal cell culture. If necessary, it may also be prepared by adding one or more of GDNF, FGF9, and retinoic acid (RA) and / or its derivatives. Furthermore, if necessary, it may also be prepared by adding alpha-albumin (Afamin) and / or R-spondin 1 (RSPO1). Commercially available products can be used. Preferably, the medium for co-culturing nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells is prepared by adding a Wnt signaling activator to a basal medium used for animal cell culture, and further adding at least FGF9. Examples of basal media include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's Modified Eagle's Medium (DMEM) medium, Ham's F12 (F12) medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof. The medium may contain serum (e.g., fetal bovine serum (FBS)) or may be serum-free. If necessary, the medium may contain one or more serum substitutes, such as albumin, transferrin, KnockOut Serum Replacement (KSR) (a serum substitute for ES cell culture) (Invitrogen), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, etc., or may also contain one or more substances, such as lipids, amino acids, L-glutamine, GlutaMAX (Invitrogen), non-essential amino acids (NEAA), vitamins, growth factors, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, and the like. Media previously optimized for stem cell culture, such as ReproFF2 (ReproCell) or Stem Fit AK02N medium (Ajinomoto Healthy Supply), may also be used.

[0027] In the present invention, the medium for co-culturing nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells (hereinafter also referred to as the medium of the present invention) contains a Wnt signaling activator. The Wnt signaling activator is not particularly limited as long as it acts on a receptor of the Wnt signaling pathway and activates Wnt signaling, and may include proteins, peptides, small molecule compounds, organic compounds, antibodies, etc. The "receptor of the Wnt signaling pathway" may include a co-receptor of the Wnt signaling pathway, such as LRP. The Wnt signaling activator is preferably a protein or peptide. Specific examples of Wnt signaling activators include proteins encoded by the Wnt gene family (Wnt family proteins), such as Wnt1, Wnt2, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt6, Wnt7a, Wnt8a, Wnt9a, Wnt10a, Wnt11, and Wnt16. Wnt3 or Wnt3a is preferred as the Wnt family protein. Examples of Wnt signal activators include Wnt agonists such as peptides analogous to Wnt family proteins, specifically Foxy5. In a preferred embodiment, the Wnt signal activator may be Wnt3a protein or a peptide analogous to Wnt3a protein, more preferably Wnt3a protein. Wnt3a protein is available, for example, from MBL, or may be produced in-house. The method for producing Wnt3a protein is not particularly limited and may be any known method, but examples include the culture supernatant obtained by expressing Wnt3a protein in a serum-free medium.When Wnt3a is used as the Wnt signal activator, the concentration of Wnt3a in the medium of the present invention is not particularly limited as long as it enables the production of a kidney tissue-like culture, but may be, for example, 1 ng / mL or more, 5 ng / mL or more, 10 ng / mL or more, 50 ng / mL or more, 100 ng / mL or more, 200 ng / mL or more, 500 ng / mL or more, or 1000 ng / mL or more, or 10000 ng / mL or less, 1000 ng / mL or less, 500 ng / mL or less, 200 ng / mL or less, 100 ng / mL or less, 50 ng / mL or less, or 10 ng / mL or less, or any compatible combination thereof. Specifically, the concentration of Wnt3a in the medium of the present invention may be, for example, 1 ng / mL to 10,000 ng / mL, 10 ng / mL to 1,000 ng / mL, 50 ng / mL to 500 ng / mL, 100 ng / mL to 1,000 ng / mL, or 100 ng / mL to 500 ng / mL.

[0028] In the present invention, the medium for co-culturing nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells may contain α-albumin (Afamin). It is known that Wnt family proteins, such as Wnt3a, are stabilized and prevented from forming aggregates in the presence of α-albumin (Afamin). Therefore, from the above perspective, when the medium of the present invention contains a Wnt family protein, such as Wnt3a, it is preferable that it also contains α-albumin (Afamin). Afamin may be contained, for example, in the medium supernatant obtained by expressing the above-mentioned Wnt3a protein in a serum-free medium. A medium supernatant containing Afamin and Wnt3a, i.e., Afamin / Wnt3a Conditioned Medium, is available, for example, from MBL. When Wnt3a protein is used, the concentration of Wnt3a protein in the medium of the present invention is not particularly limited as long as it allows kidney tissue-like cultures to be obtained. For example, it may be 1% by volume or more, 3% by volume or more, 5% by volume or more, 8% by volume or more, or 10% by volume or more, or 20% by volume or less, 18% by volume or less, 15% by volume or less, 12% by volume or less, or 10% by volume or less, or any compatible combination thereof, calculated as Afamin / Wnt3a Conditioned Medium. Specifically, the concentration of Wnt3a protein in the medium of the present invention may be 1-20% by volume, 3-18% by volume, 5-15% by volume, 8-12% by volume, or 10% by volume, calculated as Afamin / Wnt3a Conditioned Medium. The concentration of Afamin in the medium of the present invention is also not particularly limited as long as it allows kidney tissue-like cultures to be obtained. For example, the above description calculated as Afamin / Wnt3a Conditioned Medium can be used.

[0029] In the present invention, the medium for co-culturing nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells may further contain R-spondin 1 (RSPO1). RSPO1 is known to promote Wnt signaling. Therefore, in light of the above, it is preferable that a medium containing a Wnt signaling activator, particularly a medium containing a Wnt family protein or a Wnt agonist, further contains RSPO1. RSPO1 is available, for example, from R&D, or it may be prepared in-house. The concentration of RSPO1 in the medium of the present invention is not particularly limited, as long as a kidney tissue-like culture is obtained. For example, the concentration may be 10 ng / mL or more, 20 ng / mL or more, 50 ng / mL or more, 80 ng / mL or more, 100 ng / mL or more, 150 ng / mL or more, 180 ng / mL or more, or 200 ng / mL or more; or 1000 ng / mL or less, 500 ng / mL or less, 400 ng / mL or less, 300 ng / mL or less, 250 ng / mL or less, 220 ng / mL or less, or 200 ng / mL or less; or any compatible combination thereof. Specifically, the concentration of RSPO1 in the medium of the present invention may be, for example, 10 ng / mL to 1000 ng / mL, 20 ng / mL to 500 ng / mL, 50 ng / mL to 400 ng / mL, 100 ng / mL to 300 ng / mL, 150 ng / mL to 250 ng / mL, 180 ng / mL to 220 ng / mL, or 200 ng / mL.

[0030] In the present invention, the medium for co-culturing nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells may further contain glial cell line-derived neurotrophic factor (GDNF). GDNF is available, for example, from R&D, or may be prepared in-house. The concentration of GDNF in the medium of the present invention is not particularly limited as long as a renal tissue-like culture is obtained. For example, the concentration may be 10 ng / mL or more, 20 ng / mL or more, 50 ng / mL or more, 80 ng / mL or more, 100 ng / mL or more, 150 ng / mL or more, 180 ng / mL or more, or 200 ng / mL or less, or 1000 ng / mL or less, 500 ng / mL or less, 400 ng / mL or less, 300 ng / mL or less, 250 ng / mL or less, 220 ng / mL or less, or 200 ng / mL or less, or any compatible combination thereof. The concentration of GDNF in the medium of the present invention may be, for example, 10 ng / mL to 1000 ng / mL, 20 ng / mL to 500 ng / mL, 50 ng / mL to 400 ng / mL, 100 ng / mL to 300 ng / mL, 150 ng / mL to 250 ng / mL, 180 ng / mL to 220 ng / mL, or 200 ng / mL.

[0031] In the present invention, the medium for co-culturing nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells may further contain FGF9. From the viewpoint of producing a stable renal tissue-like culture, the medium for co-culturing nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells preferably further contains FGF9. FGF9 is available, for example, from Peprotech, Inc., or may be prepared in-house. The concentration of FGF9 in the culture medium is not particularly limited as long as a kidney tissue-like culture can be obtained, but may be, for example, 5 ng / mL or more, 10 ng / mL or more, 25 ng / mL or more, 30 ng / mL or more, 40 ng / mL or more, 50 ng / mL or more, 80 ng / mL or more, or 100 ng / mL or more, or 1000 ng / mL or less, 500 ng / mL or less, 300 ng / mL or less, 200 ng / mL or less, 150 ng / mL or less, 120 ng / mL or less, or 100 ng / mL or less, or any compatible combination thereof. The concentration of FGF9 in the medium may be, for example, 5 ng / mL to 1000 ng / mL, 10 ng / mL to 500 ng / mL, 25 ng / mL to 300 ng / mL, 30 ng / mL to 200 ng / mL, 50 ng / mL to 150 ng / mL, 80 ng / mL to 120 ng / mL, or 100 ng / mL.

[0032] In the present invention, the medium for co-culturing nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells may further contain retinoic acid (RA) and / or its derivatives. RA is available from, for example, Sigma, or may be prepared in-house. The concentration of RA and / or its derivatives in the medium is not particularly limited as long as a renal tissue-like culture is obtained. For example, the concentration may be 0.005 μM or more, 0.01 μM or more, 0.025 μM or more, 0.03 μM or more, 0.04 μM or more, 0.05 μM or more, 0.08 μM or more, or 0.1 μM or more, or 1.0 μM or less, 0.5 μM or less, 0.3 μM or less, 0.2 μM or less, 0.15 μM or less, 0.12 μM or less, or 0.1 μM or less, or any compatible combination thereof. The concentration of RA and / or its derivatives in the medium may be, for example, 0.005 μM to 1.0 μM, 0.01 μM to 0.5 μM, 0.025 μM to 0.3 μM, 0.03 μM to 0.2 μM, 0.05 μM to 0.15 μM, 0.08 μM to 0.12 μM, or 0.1 μM. Examples of RA derivatives include 3-dehydroretinoic acid, 4-[[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carbonyl]amino]-benzoic acid (AM580) (Tamura K, et al., Cell Differ. Dev. 32:17-26 (1990)), 4-[(1E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propen-1-yl]-benzoic acid (TTNPB) (Strickland Examples of the compounds include those described in S., et al., Cancer Res. 43:5268-5272 (1983) and Tanenaga, K. et al., Cancer Res. 40:914-919 (1980), retinol palmitate, retinol, retinal, 3-dehydroretinol, and 3-dehydroretinal.

[0033] In the present invention, the medium for co-culturing nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells may further contain a ROCK (Rho kinase) inhibitor. It is believed that the addition of a ROCK inhibitor suppresses cell death and enables efficient culture, passage, cell proliferation, and / or differentiation. From the above perspective, the medium of the present invention may contain a ROCK inhibitor. Alternatively, a ROCK inhibitor may be added to the medium at the initiation or passaging of cell culture, and then replaced with a medium that does not contain a ROCK inhibitor. Known ROCK inhibitors can be used as appropriate, such as Y-27632. The concentration of the ROCK inhibitor may be, for example, 1 to 50 μM, preferably 5 to 20 μM. The ROCK inhibitor is preferably added to the medium for at least 3 days after the initiation or passaging of cell culture.

[0034] The number of days for co-culturing nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells can be, for example, 2 or more days, 3 or more days, 4 or more days, 5 or more days, or 6 or more days. There is no upper limit to the number of days for culture, but it may be, for example, 30 days or less. The culture conditions are not particularly limited as long as a kidney tissue-like culture is obtained, but the culture temperature is about 30 to 40°C, preferably about 37°C, the oxygen concentration is a normal oxygen concentration (e.g., 15 to 25%, preferably about 20%), and the CO2 concentration is preferably about 2 to 5%.

[0035] Renal tissue-like cultures refer to cultures having a structure similar to that of nascent and / or mature kidney tissue, i.e., renal tissue. Here, a structure similar to that of renal tissue may refer not only to morphological similarity but also to a structure anatomically and / or functionally similar to that of renal tissue, based on the characteristics of each cell based on marker gene expression, etc., and the characteristics of the structure constituted by those cells. In other words, renal tissue-like cultures may also be referred to as renal organoids. The nascent kidney develops from the pronephros through the mesonephros to form the metanephros, and the metanephros matures to form the kidney. The period during which the kidney organ is formed may also be referred to as the organogenesis period. As described above, nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells are each one of the precursor cells for the formation of the metanephros. Therefore, in the renal tissue-like cultures of the present invention, the nascent kidney tissue may be either metanephros tissue or kidney tissue during organogenesis. In other words, the renal tissue-like culture of the present invention may refer to a culture having a structure similar to that of metanephros tissue, kidney tissue at the organogenesis stage, and / or mature kidney tissue. The renal tissue-like culture of the present invention may maintain kidney tissue at the organogenesis stage. In one embodiment, the renal tissue-like culture of the present invention may maintain a nephrogenic region. Furthermore, the renal tissue-like culture of the present invention does not exclude having a structure similar to that of mature kidney tissue, but may not have such a structure. In other words, the renal tissue-like culture of the present invention may refer to a culture having a structure similar to that of metanephros tissue and / or kidney tissue at the organogenesis stage.

[0036] Examples of renal tissue structures include, but are not limited to, mesangial structures, branching ureteric bud structures, and cap mesenchyme structures. The renal tissue-like cultures of the present invention preferably have one or more of mesangial structures, branching ureteric bud structures, S-shaped body-like structures, and cap mesenchyme structures, and more preferably have all of mesangial structures, branching ureteric bud structures, and cap mesenchyme structures. The renal tissue-like cultures of the present invention may further maintain a nephrogenic region. For example, by maintaining a nephrogenic region, the renal tissue-like cultures of the present invention may promote the formation of additional mesangial structures, branching ureteric bud structures, S-shaped body-like structures, and / or cap mesenchyme structures.

[0037] The mesangial structure refers to a structure formed by podocytes surrounding mesangial cells in the glomeruli of the metanephros or kidneys during organogenesis. The mesangial structure may be characterized, for example, by a structure having mesangial cells within a glomerular-like structure. The mesangial cells may be characterized, for example, by the expression of mesangial cell marker genes. Examples of mesangial cell marker genes include PDGFRB and GATA3. That is, the mesangial structure may be, for example, a structure containing PDGFRB- and / or GATA3-positive cells within a glomerular-like structure.

[0038] The branch ureteric bud structure refers to a structure derived from the ureteric bud formed by branching and elongation of the ureteric bud. In the present invention, the branch ureteric bud structure preferably has a ureteric bud, the tip of which contains RET-positive cells. More preferably, the branch ureteric bud structure may have two or more ureteric buds, the tip of which contains RET-positive cells. Furthermore, the ureteric bud may be characterized by expression of the CK8 gene.

[0039] The term "S-shaped structure" refers to a nascent renal tubule structure formed by epithelialization of nephron progenitor cells. In the present invention, the term "S-shaped structure" preferably includes a proximal region and a distal region. The proximal region may be characterized by, for example, expression of HNF4α. The distal region may be characterized by, for example, expression of BRN1. The distal region may or may not be distinguished from a mid-region intermediate between the proximal and distal regions. When the distal region is not distinguishable from the mid-region, they may be collectively referred to as the mid-distal region. In this case, the term "S-shaped structure" may be rephrased as preferably including a proximal region and a mid-distal region. The mid-distal region may also be characterized by, for example, expression of BRN1.

[0040] The term "cap mesenchyme structure" refers to the mesenchymal tissue structure surrounding the tip of the ureteric bud. Glomerular podocytes and nephron progenitor cells are generally known to reside in the cap mesenchyme structure. In the present invention, the cap mesenchyme structure preferably contains nephron progenitor cells and / or cells derived from nephron progenitor cells. Nephron progenitor cells and / or cells derived from nephron progenitor cells may be characterized by, for example, expression of SIX2 and / or PAX8. More specifically, nephron progenitor cells may be characterized by expression of SIX2, and cells derived from nephron progenitor cells may be characterized by expression of PAX8. That is, the cap mesenchyme structure may be a structure surrounding the tip of the ureteric bud and containing SIX2- and / or PAX8-positive cells. The ureteric bud may be characterized by expression of the CK8 gene. Nephron progenitor cell-derived cells are not particularly limited as long as they are derived from nephron progenitor cells, but may also be, for example, cells that constitute metanephric vesicles. Cells constituting metanephric vesicles may be characterized by, for example, expression of PAX8. Furthermore, a region characterized by a cap mesenchyme structure containing at least nephron progenitor cells may be considered a nephrogenic region. As described above, nephron progenitor cells may be characterized by expression of SIX2. In other words, the nephrogenic region may be observed as a cap mesenchyme structure containing SIX2-expressing cells. "Maintaining a nephrogenic region" in a renal tissue-like culture may mean that the renal tissue-like culture contains at least a structure exhibiting the characteristics of the nephrogenic region described above. Furthermore, "maintaining a nephrogenic region" in a renal tissue-like culture may mean, by a non-limiting example, that a structure exhibiting the characteristics of the nephrogenic region described above is observed during the production of the culture, and that all or part of such a structure is maintained even after the production of the culture.

[0041] <2> Culture of the Present Invention Another embodiment of the present invention relates to a kidney tissue-like culture of the present invention. Specifically, one embodiment of the present invention may be a kidney tissue-like culture containing human-derived cells and having a mesangial structure, a branching ureteric bud structure, an S-shaped body-like structure, and a cap mesenchyme structure.

[0042] The method for obtaining the kidney tissue-like culture of the present invention is not particularly limited, and the kidney tissue-like culture may be produced by the production method of the present invention.

[0043] The renal tissue-like culture of the present invention may comprise cells of human origin. For example, when the renal tissue-like culture of the present invention is obtained by the culture method of the present invention or produced by the production method of the present invention, one or more cells selected from nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells may be human-derived cells, and it is preferable that all of the nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells are human-derived. Furthermore, it is preferable that the renal tissue-like culture of the present invention is composed of cells of human origin.

[0044] The kidney tissue-like culture of the present invention may have a mesangial structure, a branching ureteric bud structure, an S-shaped body-like structure, and a cap mesenchyme structure. The details of the mesangial structure, the branching ureteric bud structure, the S-shaped body-like structure, and the cap mesenchyme structure are as described above.

[0045] The kidney tissue-like culture of the present invention may be cultured under culture conditions such as the medium in the method of the present invention.

[0046] The renal tissue-like culture of the present invention has a structure similar to that of renal tissue and is expected to gradually undergo organogenesis and develop and grow as an organ. Therefore, the renal tissue-like culture of the present invention, which may contain or consist of human cells, is expected to be useful in a variety of applications, including renal tissue and kidney reconstruction, regenerative medicine, renal disease models, drug discovery screening, and drug toxicity evaluation systems.

[0047] The present invention will be specifically described below based on examples, but the present invention is not limited to the following embodiments.

[0048] <Late primitive streak (PPS) induction> Human iPS cells (hiPSCs) were first treated with a 1:1 mixture of TrypLE Select Enzyme (Thermo Fisher Scientific) and 0.5 mM EDTA / PBS for 5 minutes and then washed with PBS(-). They were then detached using a cell scraper and gently pipetted to dissociate into single cells. One day before the start of differentiation, 1.3 × 10 cells were plated in a 24-well plate (Corning) with 0.5 mL of Stem Fit AK02N (Ajinomoto Healthy Supply) supplemented with 10 μM Y-27632 and 2.4 μL / mL iMatrix-511 (Matrixome). 4 cells / cm 2 Cells were seeded at a density of 1000 μM. After 24 hours (day 0), culture was initiated in serum-free differentiation medium (basal medium) consisting of DMEM / F12 Glutamax (Thermo Fisher Scientific), B27 supplement minus vitamin A (Thermo Fisher Scientific), and 0.5x penicillin / streptomycin. The medium was supplemented with 5 μM CHIR99021 (Wako), 10 nM retinoic acid (RA) (Sigma), 1 ng / mL BMP4 (Peprotech), and 100 ng / mL bFGF (Wako). After another 24 hours (day 1), the cells were cultured in basal medium containing 5 μM CHIR99021, 100 ng / mL bFGF, and 1 ng / mL BMP7 (R&D Systems). On day 2, the medium was switched to basal medium containing 5 μM CHIR99021, 1 ng / mL BMP7, and 10 μM A83-01 (Wako). On day 3, the medium was switched to basal medium containing 5 μM CHIR99021, 30 ng / mL bFGF, 10 ng / mL activin A (R&D Systems), and 30 μM Y-27632 (Wako). On day 4, the cells were washed with PBS(-), treated with Accumax (Innovative Cell Technologies), and gently pipetted to dissociate into single cells. 1.05 × 10 cells were plated onto a 24-well plate. 5 cells / cm 2(2D culture) or 2.0 x 10 cells in 96-well low cell attachment plates (Thermo Fisher Scientific). 4 cells / cm 2 The cells were seeded at a density of 1000 (3D culture) and cultured for 48 hours in basal medium containing 5 μM CHIR99021, 30 ng / mL bFGF, 10 ng / mL activin A, and 30 μM Y-27632 (for 2D culture, 1.2 μL / well of iMatrix-511 was also added).

[0049] Nephron progenitor cell (NPC) induction: On day 6 of PPS induction, the medium was switched to one containing 0.1 μM RA and 25 ng / mL NOGGIN (Peprotech). On day 8, the medium was switched to one containing 200 ng / mL FGF9 and 1 μM CHIR99021, and the cells were cultured for 72 hours.

[0050] <Induction of renal interstitial progenitor cells (IPCs)> On day 4 of PPS induction, dissociated cells were plated at 2.0 × 10 cells per well in a 96-well U-bottom plate (Sumitomo Bakelite or Thermo Fisher Scientific) with basal medium containing 5 μM CHIR99021, 30 ng / mL bFGF, 10 ng / mL activin A, and 30 μM Y-27632. 4 Cells were seeded at a density of 100 cells / well to form aggregates and cultured for 48 hours. On day 6, cells were cultured in AK02N minus bFGF medium or basal medium containing 1 μM CHIR99021, 0.1 μM RA, 500 nM Smoothened Monoamine Activator (SMO) agonist (SAG) (Selleck), and 10 ng / mL IL-1β (Wako). On day 8, cells were cultured in AK02N minus bFGF medium or basal medium containing 1 μM CHIR99021, 0.1 μM RA, 500 nM SAG, and 10 μM SB431542 for 72 hours.

[0051] <Induction of Mesonephric Duct (ND) Cells (Ureteric Bud Cells)> First, anterior intermediate mesoderm was induced from hiPSCs using the following procedure. hiPSCs were treated with a 1:1 mixture of TrypLE Select Enzyme and 0.5 mM EDTA / PBS for 5 minutes and then washed with PBS(-). Next, cells were detached using a cell scraper and dissociated into single cells by gentle pipetting. One day before the start of differentiation, cells were seeded into 10-cm dishes (Falcon) at a density of 4.0 × 10^5 cells / dish using Stem Fit AK02N (supplemented with 10 μM Y-27632 and 20 μL / dish of iMatrix-511). After 24 hours (day 0), cells were cultured in serum-free differentiation medium (E6; Thermo Fisher Scientific) supplemented with 50 ng / mL activin A, 5 μM CHIR99021, 25 ng / mL BMP4, and 25 ng / mL bFGF. Approximately 22 hours later (day 1), cells were cultured in E6 medium supplemented with 100 nM LDN193189, 1 μM A83-01, 0.1 μM TTNPB (Santa Cruz Biotechnology), and 200 ng / mL FGF8 (Peprotech) for 2 days. Cells were then replated at a density of 8.7 × 10^6 cells / dish in E6 medium supplemented with the same four factors and 10 μM Y-27632 in 10-cm dishes and cultured for an additional 24 hours. Next, ND cell induction from the anterior intermediate mesoderm was performed as follows. Anterior intermediate mesoderm was treated with E6 medium containing 1 μM CHIR99021, 100 nM LDN193189, 200 ng / mL FGF8, 100 ng / mL GDNF, and 0.1 μM TTNPB for 2 days. After treatment with Accutase (Nacalai tesque) for 3 minutes at 37°C, single cells were dissociated by pipetting and seeded at a density of 1.0 × 10^4 cells / well into low-attachment 96-well plates (Sumitomo Bakelite). ND cell differentiation was then induced for 2 days in the same medium and factors plus 10 μM Y-27632.

[0052] <Reconstruction of Renal Structure> The NPC, IPC, and ND cells induced as described above were left to stand with Accumax at 37°C for 10 minutes, and then gently pipetted to dissociate them into single cells. Dissociated single cells were mixed and resuspended in basal medium containing 1 μM CHIR99021, 0.1 μM RA, 100 ng / mL FGF9, and 10 μM Y-27632 (CRFY), CRFY plus 200 ng / mL GDNF (CRFY+G), or basal medium containing 0.1 μM RA, 100 ng / mL FGF9, and 10 μM Y-27632, 200 ng / mL GDNF, 10% Afamin / Wnt3a Conditioned Medium (MBL; J-ORMW301R), and 200 ng / mL R-spondin1 protein (RSPO1) (R&D) (RFY+G+WR), and plated at 1.7 × 10 cells per well in a 96-well low-cell-binding U-bottom plate. 4 NPC density: 1.7 x 10 cells / well 4 IPC, and 1.7 x 10 cells / well density 4 ND cells were seeded at a density of 100 cells / well to form aggregates. After 48 hours, the mixed aggregates were transferred to a 24-well plate and incubated with the same medium as above supplemented with 20 μL of 50% Matrigel at 37°C for 30 minutes. After some solidification, 180 μL of the same medium as above was added. Half of the medium was then replaced every two days.

[0053] Renal structures were reconstituted under CRFY conditions, and immunohistochemistry of the tissue-like cultures at day 6 revealed the reconstitution of CALB1(+) epithelial structures, and SIX2(+) NPCs and FOXD1(+) IPCs surrounding ureteric bud (UB)-like epithelial structures (Figure 1). However, RET expression was not observed in the UB tip-like structures. In a previous study (Mae, SI, et al. Cell Rep. 32. 2020), the medium used for cell proliferation at the ureteric bud tip contained GDNF. Therefore, renal structures were reconstituted under CRFY+G conditions, in which GDNF was added to CRFY. Quantitative RT-PCR (qT-PCR) analysis of tissue-like cultures at day 6 under these conditions revealed a slight increase in RET expression, while other progenitor cell markers, such as SIX2 and FOXD1, were unaffected (Figure 2).

[0054] On the other hand, in the RFY+G+WR condition, in which GDNF was added to CRFY and Wnt3a and RSPO1 were used instead of CHIR99021, qRT-PCR analysis of the tissue-like cultures on day 6 showed further enhanced RET expression compared to those under the CRFY+G condition (Fig. 2). Furthermore, immunohistochemistry of the cultures revealed that the tip of the budding UB was RET-positive (Fig. 3), and nephrogenic region-like structures were formed, such as SIX2(+) or PAX8(+) cap mesenchyme structures (Fig. 4) and S-shaped structures consisting of the HNF4α(+) proximal region and the BRN1(+) mid-distal region (Fig. 5).

[0055] Mesangial cells originate from Foxd1(+) IPCs and migrate through the glomerular tuft during development. In the glomerulus of the embryonic kidney at the capillary loop stage, podocytes surround Pdgfrb(+) mesangial cells to form mesangial structures. Consistent with this finding, renal architecture reconstituted under RFY+G+WR conditions revealed glomerular-like structures containing PDGFRB(+) or GATA3(+) mesangial-like cells, i.e., mesangial structures, in capillary loop-stage tissue at days 6 and 12, as well as in mature tissue at day 12 (Fig. 6). Furthermore, when IPCs constitutively expressing EGFP and non-fluorescent NPCs were cultured under RFY+G+WR conditions, EGFP(+)PDGFRB(+) mesangial-like structures were observed, confirming that the mesangial-like cells originated from IPCs.

[0056] These results suggest that co-culturing NPC, IPC, and ND cells under RFY+G+WR conditions resulted in the formation of appropriate UB structures, nephrogenic regions, and mesangial structures. Specifically, co-culturing NPC, IPC, and ND (ureteric bud) cells under conditions containing the Wnt signaling activator Wnt3a produced renal tissue-like cultures with the structure of organogenic kidneys.

[0057] Evaluation of Nephron Progenitor Cells (NPCs) NPCs induced as described above were incubated with Accumax at 37°C for 10 minutes and gently pipetted to dissociate into single cells. The dissociated cells were mixed and resuspended in basal medium (CRFY) containing 1 μM CHIR99021, 200 ng / mL FGF9, and 10 μM Y-27632. They were then plated at a density of 4 × 10^4 cells / well in a 96-well low-cell-binding U-bottom plate to form aggregates. After 48 hours, the cell aggregates were harvested, cryosectioned, and immunofluorescently stained for stromal progenitor cell marker genes, revealing that some of the cells contained stromal progenitor cells, as indicated by PDGFRB(+) expression (Figure 7).

[0058] <Induction of ureteric bud organoids> Next, we induced ureteric bud organoids from mesonephric duct (ND) cells (ureteric bud cells) using the following procedure: After washing with PBS, mesonephric duct (ND) cells (ureteric bud cells) were treated with E6 medium containing 1 μM CHIR99021, 100 nM LDN193189, 200 ng / mL FGF8, 100 ng / mL GDNF, 0.1 μM TTNPB, 200 ng / mL FGF1, 50 ng / mL EGF, and 2% Matrigel, and cultured for 7 days to induce ureteric bud organoids.

[0059] <Reconstruction of kidney structure using ureteric bud organoids and NPCs> The NPCs induced as described above were incubated with Accumax at 37°C for 10 minutes and gently pipetted to dissociate into single cells. The dissociated single-cell NPCs were resuspended in basal medium (RFY+G+WR) containing 0.1 μM RA, 100 ng / mL FGF9, 10 μM Y-27632, 200 ng / mL GDNF, 10% Afamin / Wnt3a Conditioned Medium, and 200 ng / mL R-spondin1 protein (RSPO1). 5 × 10 NPCs were sprinkled on top of the ureteric bud organoids induced as described above. 4 NPC suspensions were seeded at a density of 100 cells / well to form aggregates. After 48 hours, the mixed aggregates were transferred to a 24-well plate and incubated with the same medium as above supplemented with 20 μL of 50% Matrigel at 37°C for 30 minutes. After some solidification, 180 μL of the same medium as above was added. Half of the medium was then replaced with basal medium every two days.

[0060] Renal structure was reconstructed under RFY+G+WR conditions, and the tissue-like cultures on day 13 were analyzed by immunostaining. Results showed that renal tissue maintained the cap mesenchyme structure of SIX2(+) nephron progenitor cells, maintained the nephrogenic region, and had a CK8(+) branching ureteric bud structure (Fig. 8).

[0061] These results suggest that co-culturing NPC cells (including some IPC cells) with ureteric bud organoids under RFY+G+WR conditions resulted in the formation of appropriate UB structures and nephrogenic regions. Specifically, co-culturing NPC cells, IPC cells, and ureteric bud organoids (ureteric bud cells) under conditions containing the Wnt signaling activator Wnt3a produced renal tissue-like cultures with the structure of organogenic kidneys.

Claims

1. A method for producing a renal tissue-like culture, comprising the step of co-culturing nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells in a medium containing a Wnt signal activator.

2. The method according to claim 1, wherein the co-culture is carried out by suspension culture.

3. The method according to claim 2, wherein the suspension culture is performed in a state in which aggregates of nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells are coated with an extracellular matrix.

4. The method of claim 1, wherein the Wnt signal activator is Wnt3a protein.

5. The method according to claim 1, wherein the medium further contains fibroblast growth factor 9 (FGF9).

6. The method according to any one of claims 1 to 5, wherein the medium further contains retinoic acid (RA) and / or a derivative thereof, and glial cell line-derived neurotrophic factor (GDNF).

7. The method according to claim 6, wherein the medium further contains alpha albumin and R-Spondin 1 (RSPO1).

8. The method of any one of claims 1 to 5, wherein the kidney tissue-like culture has one or more of mesangial structures, branching ureteric bud structures, S-body-like structures, and cap mesenchyme structures.

9. The method according to any one of claims 1 to 5, wherein the nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells are of human origin.

10. The method according to any one of claims 1 to 5, wherein one or more of the nephron progenitor cells, the renal interstitial progenitor cells, and the ureteric bud cells are cells induced from pluripotent stem cells.

11. A renal tissue-like culture comprising cells of human origin and having mesangial structures, branching ureteric bud structures, S-body-like structures, and cap mesenchyme structures.

12. A renal tissue-like culture as described in claim 11, wherein the mesangial structure is a structure containing PDGFRB and / or GATA3 positive cells inside a glomerulus-like structure, the branched ureteric bud structure is a structure having two or more ureteric buds, the tip of which contains RET positive cells, the S-shaped body-like structure is a structure containing an S-shaped body-like structure including a proximal region and a distal region, and the cap mesenchyme structure is a structure that surrounds the tip of the ureteric bud and contains SIX2 and / or PAX8 positive cells.

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