Culture method for promoting kidney organogenesis while maintaining the nephrogenic region

Abstract

The research focused on the preparation of kidney tissue by co-culturing nephron precursor cells, renal interstitial precursor cells, and ureteral bud cells in a culture medium containing Wnt signaling activating factors.

Description

Technical Field

[0001] This invention relates to a method for promoting the formation of kidney organs. More specifically, this invention relates to kidney tissue-like cultures having kidney structures, methods for culturing the same, and methods for manufacturing the same. Background Technology

[0002] The metanephron, the adult kidney of mammals, is formed from multiple embryonic renal progenitor cells, including nephron precursor cells that differentiate into epithelial cells of nephrons such as glomeruli and tubules; ureteral bud cells that differentiate into collecting ducts and the lower urinary tract; and renal interstitial precursor cells that differentiate into renal interstitial cells, mesangial cells, and vascular smooth muscle cells. Organogenesis gradually occurs while maintaining the renal formation area, primarily through the repeated self-replication and differentiation of nephron precursor cells, ureteral bud cells, and renal interstitial precursor cells, thus contributing to organogenesis and growth.

[0003] There are reports of a culture method that promotes organogenesis by co-culturing nephron precursor cells, ureteral bud cells, and renal interstitial precursor cells derived from mouse fetal kidneys or mouse ES cells in a gel containing bovine serum (Non-Patent Literature 1). Additionally, there are reports of a culture method that separates kidney organoids, including nephron precursor cells and renal interstitial precursor cells, made from human pluripotent stem cells using a cell dissociation reagent and cultures them in a gel containing FGF2, CHIR99021, retinoic acid, Y-27632, and GDNF, thereby differentiating ureteral bud-like epithelium and promoting organogenesis (Non-Patent Literature 2). However, to date, there are no reports on culture methods that promote organogenesis while maintaining the kidney-forming region when co-culturing human nephron precursor cells, ureteral bud cells, and renal interstitial precursor cells derived from human pluripotent stem cells such as human induced pluripotent stem cells (iPS cells) or human embryonic stem cells (ES cells). [Existing Technical Documents] [Non-patent literature]

[0004] [Non-Patent Literature 1] Tanigawa S, Tanaka E, Miike K, Ohmori T, Inoue D, Cai CL, et al., Nat Commun. 2022;13: 611. [Non-Patent Literature 2] Howden SE, Wilson SB, Groenewegen E, Starks L, ForbesTA, Tan KS, et al., Cell Stem Cell. 2021;28: 671-684.e6. Summary of the Invention [The problem the invention aims to solve]

[0005] The objective of this invention is to produce kidney tissue with a more physiological structure by promoting organogenesis while maintaining the kidney-forming region during the culture of nephron precursor cells, renal interstitial precursor cells, and ureteral bud cells. In particular, this invention aims to produce kidney tissue with a more physiological structure by promoting organogenesis while maintaining the kidney-forming region during the culture of human-derived nephron precursor cells, renal interstitial precursor cells, and ureteral bud cells. [Methods used to solve problems]

[0006] To address the aforementioned issues, the inventors conducted in-depth research. The results showed that by co-culturing nephron precursor cells, renal interstitial precursor cells, and ureteral bud cells in the presence of Wnt signaling activator, organogenesis can be promoted while maintaining the kidney-forming region, and kidney tissue-like cultures with kidney structures can be produced.

[0007] That is, the present invention has the following features: [1] A method for manufacturing a kidney tissue-like culture, comprising a step of co-culturing nephron precursor cells, renal interstitial precursor cells and ureteral bud cells in a culture medium containing Wnt signaling activating factor. [2] According to the method of [1], the co-culture is carried out by suspension culture. [3] According to the method described in [2], the suspension culture is carried out in a state in which an aggregate of nephron precursor cells, renal interstitial precursor cells and ureteral bud cells is coated with an extracellular matrix. [4] The method according to any one of [1] to [3], wherein the Wnt signaling activator is Wnt3a protein. [5] The method according to any one of [1] to [4], wherein the culture medium is a culture medium further comprising fibroblast growth factor 9 (FGF9). [6] The method according to any one of [1] to [5], wherein the culture medium is a culture medium further comprising retinoic acid (RA) and / or its derivatives, and glial cell-derived neurotrophic factor (GDNF). [7] According to the method of [6], wherein the culture medium is a culture medium further comprising α-albumin and R-Spondin 1 (RSPO1). [8] The method according to any one of [1] to [7], wherein the kidney tissue-like culture has one or more of the following: mesangial structure, branched ureteral bud structure, S-shaped structure and cap mesenchyme. [9] The method according to any one of [1] to [8], wherein the nephron precursor cells, renal interstitial precursor cells and ureteral bud cells are derived from humans.

[10] The method according to any one of [1] to [9], wherein one or more of the nephron precursor cells, the renal interstitial precursor cells and the ureteral bud cells are cells induced by pluripotent stem cells.

[11] A kidney tissue-like culture, It contains cells derived from humans. It also has mesangial structure, branched ureteral bud structure, S-shaped structure and cap-shaped mesenchymal structure.

[12] According to the kidney tissue-like culture described in

[11] , wherein, The mesangial structure is a structure containing PDGFRB and / or GATA3-positive cells within a glomerular-like structure. The branched ureteral bud structure is a structure with two or more ureteral buds, and the apical part of the ureteral bud contains RET-positive cells. The S-shaped structure is a structure containing an S-shaped region and a distal region. The cap-shaped mesenchymal structure is a structure that surrounds the tip of the ureteral bud and contains SIX2 and / or PAX8 positive cells. [Invention Effects]

[0008] According to the method of the present invention, nephron precursor cells, renal interstitial precursor cells, and ureteral bud cells can promote organogenesis while maintaining the renal formation region, enabling the production of renal tissue-like cultures with renal structures. Furthermore, according to the present invention, renal tissue-like cultures with renal structures, particularly those derived from humans, can be provided. In particular, the cultures of the present invention can possess mesangial structures, branching ureteral bud structures, and cap-like mesenchymal structures. These structures are characteristic of the renal during organogenesis, and further organogenesis and growth are expected. Attached Figure Description

[0009] Figure 1 This is a figure showing the immunostaining results of SIX2, FOXD1, and CALB1 in tissue culture samples on day 6 of renal structural remodeling under CRFY conditions (replacing the photograph in the attached figure). Scale bar: 200 μm. Figure 2This is a graph showing the results of gene expression analysis of various precursor cell marker genes in tissue culture samples on day 6 of renal structure reconstruction under CRFY, CRFY+G, or RFY+G+WR conditions, performed by quantitative RT-PCR (qT-PCR). Figure 3 This is a figure showing the immunostaining results of SIX2, RET, and CALB1 in tissue culture samples on day 6 of renal structure reconstruction under RFY+G+WR conditions (replacing the photograph in the attached figure). Scale bar: 200 μm. Figure 4 This is a figure showing the immunostaining results of PAX8, SIX2, FOXD1, and CALB1 in tissue culture samples on day 6 of renal structure reconstruction under RFY+G+WR conditions (replacing the photograph in the attached figure). Scale bar: 200 μm. Figure 5 This is a figure showing the immunostaining results of BRN1, HNF4α, and CALB1 in tissue culture samples on day 6 of renal structure reconstruction under RFY+G+WR conditions (replacing the photograph in the attached figure). Scale bar: 200 μm. Figure 6 This is a figure showing the immunostaining results of PDGFRB, GATA3, MAFB, and NPHS1 in tissue culture samples on days 6 and 12 of renal structure reconstruction under RFY+G+WR conditions (replacing the photograph in the attached figure). Scale bar is 100 μm. Figure 7 This is a diagram showing the immunostaining results of PDGFRB in cell blocks after inducing nephron precursor cells (NPCs) to form aggregates under CRFY conditions and culturing for 48 hours (instead of the photograph in the accompanying figure). Figure 8 This is a figure showing the immunostaining results of ureteral bud marker CK8 and nephron precursor cell marker SIX2 in tissue-like cultures on day 13 of renal structure reconstruction under RFY+G+WR conditions (instead of the photograph in the attached figure). Detailed Implementation

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

[0011] <1> The method of the present invention As one embodiment, the present invention relates to a method for manufacturing a kidney tissue-like culture, which includes a step of co-culturing nephron precursor cells, renal interstitial precursor cells and ureteral bud cells in a culture medium containing Wnt signaling activating factor.

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

[0013] Nephron precursor cells (NPCs) are among the precursor cells used to form the kidney, producing glomeruli and renal tubules, which are the epithelial cells of the nephron. In addition to this property, nephron precursor cells can also be characterized by the expression of marker genes. Specifically, nephron precursor cells can be characterized, for example, by the expression of the nephron precursor cell marker gene SIX2, or by the expression of SIX2 and the interstitial mesodermal marker gene OSR1. There are no particular limitations on nephron precursor cells; they can be nephron precursor cells isolated from an organism or nephron precursor cells induced from undifferentiated cells. Examples of nephron precursor cells isolated from an organism include, for example, nephron precursor cells isolated from an embryo. Examples of nephron precursor cells induced from undifferentiated cells include, for example, nephron precursor cells induced from pluripotent stem cells. Pluripotent stem cells will be discussed later. There are no particular limitations on the method of inducing nephron precursor cells from pluripotent stem cells; any known method or a modified version of any known method can be used. Examples of such methods include those described in Tsujimoto, H., et al. Cell Rep. 31, 107476, 2020, or methods that are modifications of those methods. Specifically, a method that is a modification of the method described in Tsujimoto, H., et al. Cell Rep. 31, 107476, 2020 could include, for example, reseeding and performing stirred culture using a three-dimensional culture method on the fourth day of cultivation in that method.

[0014] Specifically, the method for inducing nephron progenitor cells from pluripotent stem cells can be implemented as follows: First, pluripotent stem cells undergo a late-stage induction primitive stripe (PPS) process; then, cells approximately 4 days after the start of PPS induction are reseeded using a three-dimensional culture method, and cultured for approximately 2 days in a medium containing a GSK-3β (glycogen synthase kinase 3β) inhibitor (CHIR99021, etc.), bFGF, and activin A; subsequently (approximately 6 days after the start of PPS induction), the medium is changed to a medium containing retinoic acid (RA), NOGGIN, and FGF9; and approximately 8 days after the start of PPS induction, the medium is further changed to a medium containing FGF9 and a GSK-3β inhibitor (e.g., CHIR99021) for approximately 3 days. Additionally, during the reseeding using the three-dimensional culture method, a ROCK inhibitor (e.g., Y-27632) can be added to the reseeded medium. It should be noted that the process of inducing primitive streaks (PPS) in pluripotent stem cells can be implemented more specifically, for example, as follows: Pluripotent stem cells are seeded on a culture dish coated with an extracellular matrix (specifically, for example, laminin fragments), cultured in a medium containing a GSK-3β inhibitor (e.g., CHIR99021), RA, BMP4, and bFGF for about 1 day (Day 0), then cultured in a medium containing CHIR99021, bFGF, and BMP7 for about 1 day (Day 1), then cultured in a medium containing a GSK-3β inhibitor (e.g., CHIR99021), BMP7, and a TGFβ inhibitor (e.g., A83-01) for about 1 day (Day 2), then cultured in a medium containing a GSK-3β inhibitor (e.g., CHIR99021), bFGF, activin A, and a ROCK inhibitor (e.g., Y-27632) for about 1 day (Day 3), and the cells are harvested on about Day 4.

[0015] Renal interstitial progenitor cells (IPCs) are one of the progenitor cells used to form the kidney. They are cells capable of differentiating into renal interstitial cells, erythropoietin-producing cells, mesangial cells, juxtaglomerular cells, and other renin-producing cells. In addition to this property, IPCs can also be characterized by the expression of marker genes. Specifically, IPCs can be characterized, for example, by the expression of FOXD1 (Forkhead Box D1), a marker gene for IPCs, or by the expression of FOXD1 along with OSR1 and / or PDGFRB (platelet-derived growth factor receptor β). There are no particular limitations on the type of IPC; it can be IPCs isolated from an organism or IPCs induced from undifferentiated cells. Examples of IPCs isolated from an organism include embryonic IPCs. Examples of IPCs induced from undifferentiated cells include IPCs induced from pluripotent stem cells. Regarding pluripotent stem cells, as described below. There are no particular limitations on the method for inducing renal interstitial progenitor cells from pluripotent stem cells; any known method or a modification of any known method may be used. Examples of such methods include, for example, the method described in WO 2023 / 017848. Renal interstitial progenitor cells may also be, for example, byproducts of inducing nephron progenitor cells. Examples of such methods include, for example, the methods described in JP2020031648A and WO2018216743A1.

[0016] Specifically, the method for inducing renal interstitial progenitor cells from pluripotent stem cells can be implemented as follows: Perform the above-described PPS induction procedure for pluripotent stem cells; then, reseed the cells from approximately day 4 of PPS induction in an aggregated manner (e.g., by reseeding to a U-plate to form aggregates), and culture them for approximately 2 days in a medium containing a GSK-3β inhibitor (e.g., CHIR99021), bFGF, and activin A; subsequently (day 6 of PPS induction), culture them in a medium containing a GSK-3β inhibitor (e.g., CHIR99021), RA, Smoothened (SMO) agonist (SAG), and IL-1β, but without bFGF; and on approximately day 8, culture them in a medium containing a GSK-3β inhibitor (e.g., CHIR99021), RA, SAG, and a TGFβ inhibitor (e.g., SB431542), but without bFGF, for approximately 3 days.

[0017] Ureteral bud (UB) cells are among the precursor cells for the formation of the metanephron, producing collecting ducts and the lower urinary tract. Ureteral bud cells are also referred to as mesonephric duct (ND) cells depending on their location within the tissue or the stage at which they develop, but these cell types may not be distinguished in vitro. That is, the ureteric bud cells in this invention can also be called ND cells. In addition to this property, ureteric bud cells can be characterized by the expression of marker genes. Specifically, ureteric bud cells can be characterized, for example, by the expression of RET, a marker gene for ureteric bud cells. There are no particular limitations on the ureteric bud cells; they can be ureteric bud cells isolated from an organism or ureteric bud cells induced from undifferentiated cells. Examples of ureteric bud cells isolated from an organism include, for example, ureteric bud cells isolated from an embryo. Examples of ureteric bud cells induced from undifferentiated cells include, for example, ureteric bud cells induced from pluripotent stem cells. Regarding pluripotent stem cells, this will be discussed later. There are no particular limitations on the method for inducing ureteral bud cells from pluripotent stem cells; 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, SI, et al. Cell Rep. 32, 2020. Methods for inducing ureteral bud cells from pluripotent stem cells include, for example, induction via anterior intermediary mesoderm. Specifically, the method for inducing anterior intercalated mesoderm from pluripotent stem cells can be implemented, for example, as follows: pluripotent stem cells are seeded on a culture dish coated with an extracellular matrix (specifically, for example, laminin fragments), and then introduced into Essential... Culture in serum-free medium containing activator A, GSK-3β inhibitor (e.g., CHIR99021), BMP4, and bFGF for approximately 1 day (Day 0) on basal medium such as 6 (E6). Then, culture in medium containing GSK-3β inhibitor (e.g., CHIR99021), bFGF, and BMP7 for approximately 1 day (Day 1). Then, culture in medium containing ALK2 / 3 inhibitor (e.g., LDN193189), TGFβ inhibitor (e.g., A83-01), retinoic acid or its derivatives (e.g., TTNPB), and FGF8 for approximately 2 days (Days 2-3). Finally, culture in medium containing ROCK inhibitor (e.g., Y-27632) in addition to ALK2 / 3 inhibitor (e.g., LDN193189), TGFβ inhibitor (e.g., A83-01), retinoic acid or its derivatives (e.g., TTNPB), and FGF8 for approximately 1 day (Day 4). Specifically, the method for inducing ureteral bud cells from anterior intercalary mesoderm can be implemented as follows: Anterior intercalary mesoderm is cultured for approximately 2 days in E6 medium containing GSK-3β inhibitors (e.g., CHIR99021), ALK2 / 3 inhibitors (e.g., LDN193189), FGF8, GDNF, and retinoic acid or its derivatives (e.g., TTNPB). The cultured cells are then separated into single cells using a cell-splitting agent (e.g., Accutase) and reseeded onto a low-attachment plate. The cells are then cultured for approximately 2 days in E6 medium further containing a ROCK inhibitor (e.g., Y-27632) in addition to GSK-3β inhibitors (e.g., CHIR99021), ALK2 / 3 inhibitors (e.g., LDN193189), FGF8, GDNF, and retinoic acid or its derivatives (e.g., TTNPB) for induction. Alternatively, cells at this stage can be considered as corresponding to ND cells. In addition, the induced cells can be further cultured for about 6 days in E6 medium containing GSK-3β inhibitors (e.g., CHIR99021), ALK2 / 3 inhibitors (e.g., LDN193189), FGF8, GDNF, retinoic acid or its derivatives (e.g., TTNPB), FGF1, EGF, and Matrigel.

[0018] In this invention, nephron precursor cells, renal interstitial precursor cells, and ureteral bud cells can be induced from pluripotent stem cells as described above, or they can be obtained without pluripotent stem cell induction. That is, one or more of these cells can be induced from pluripotent stem cells, or all of these cells can be induced from pluripotent stem cells.

[0019] Pluripotent stem cells (PSCs) are stem cells that possess the ability to differentiate into many cell types present in an organism, and also have proliferative capacity. They include any cell type that can be induced to differentiate into a blastocyst-like structure. There are no particular limitations on PSCs; for example, they include embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, and embryonic germ cells (EG cells). Preferred PSCs are iPS cells and ES cells. Furthermore, PSCs can be juvenile PSCs or primate PSCs; there are no particular limitations. The source of PSCs is preferably from mammals, more preferably from primates, and even more preferably from humans.

[0020] As ES cells, ES cells established by known methods can be used. Methods for establishing and maintaining ES cells are described in, for example, USP5,843,780; Thomson JA, et al. (1995), Proc Natl. Acad. Sci. US A. 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, etc. As human ES cell lines, WA01(H1) and WA09(H9) can be obtained from WiCell Research Institute, and KhES-1, KhES-2 and KhES-3 can be obtained from Kyoto University Institute for Regenerative Medicine Science (Kyoto, Japan).

[0021] Embryonic germ cells are pluripotent cells that are established from primordial germ cells during the embryonic period. 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; JL Resnick et al. (1992), Nature, 359:550-551).

[0022] Induced pluripotent stem cells (iPS cells) are artificial stem cells derived from somatic cells, created by introducing specific reprogramming factors in the form of DNA or proteins into somatic cells. They possess characteristics essentially equivalent to ES cells, such as differentiation pluripotency and the ability to proliferate through self-replication (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). Reprogramming factors can consist of genes specifically expressed in ES cells, their gene products, or non-coding RNAs; genes, their gene products, or non-coding RNAs that play an important role in maintaining the undifferentiated state of ES cells; or small molecule compounds with equivalent functions. Many genes have been reported as potential inclusion factors, and there are no particular limitations. Examples include 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, or Glis1. These can be used alone or in combination. The aforementioned reprogramming factors also include those used to improve iPS cell establishment efficiency, such as histone deacetylase (HDAC) inhibitors (e.g., small molecule inhibitors such as valproic acid (VPA), trichostatin A, sodium butyrate, MC 1293, M344, etc., and nucleic acid expression inhibitors such as siRNA and shRNA targeting HDAC), 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-azacitidine), histone methyltransferase inhibitors (e.g., small molecule inhibitors such as BIX-01294, and nucleic acid expression inhibitors such as siRNA and shRNA targeting Suv39hl, Suv39h2, SetDBl, and G9a, etc.), L-type calcium channel agonists (e.g., Bayk8644), butyrate, TG Fβ inhibitors or ALK5 inhibitors (e.g., LY364947, SB431542, 616453, and A-83-01), p53 inhibitors (e.g., siRNAs and shRNAs targeting p53), ARID3A inhibitors (e.g., siRNAs and shRNAs targeting ARID3A), miRNAs such as miR-291-3p, miR-294, miR-295, and miR-302, Wnt signaling molecules (e.g., soluble Wnt3a), neuropeptide Y, prostaglandins (e.g., prostaglandin E2 and prostaglandin J2), hTERT, SV40LT, UTF1, IRX6, GLISI, PITX2, DMRTB1, etc., can also be used as reprogramming factors. iPS cells can be created and used by introducing reprogramming factors into somatic cells as described above, or they can be obtained from cell banks or other sources.

[0023] The method of this invention includes the step of co-culturing nephron precursor cells, renal interstitial precursor cells, and ureteral bud cells. The term "co-culturing nephron precursor cells, renal interstitial precursor cells, and ureteral bud cells" is not particularly limited as long as these cells are cultured in the same culture medium. For example, it can be carried out by mixing and culturing nephron precursor cells, renal interstitial precursor cells, and ureteral bud cells. The mixing of nephron precursor cells, renal interstitial precursor cells, and ureteral bud cells is not particularly limited as long as a kidney tissue-like culture can be obtained. These cells can be separated into single cells and then mixed, or cell blocks with a certain cell number can be mixed, or they can be combined. Preferably, nephron precursor cells, renal interstitial precursor cells, and ureteral bud cells are separated into single cells and then mixed. The ratio of nephron precursor cells, renal interstitial precursor cells, and ureteral bud cells is not particularly limited as long as a renal tissue-like culture can be obtained. For example, the ratio of cell number can be 25-40:25-40:25-40, preferably 30-35:30-35:30-35, and even more preferably 1:1:1.

[0024] Co-culture of nephron precursor cells, renal interstitial precursor cells, and ureteral bud cells is preferably carried out through suspension culture. Here, suspension culture refers to culturing cells without them adhering to the culture medium, for example, culturing using a culture vessel with low cell adhesion.

[0025] In addition, during suspension culture, aggregates of nephron precursor cells, renal interstitial precursor cells, and ureteral bud cells can be formed, and these aggregates can be coated. The preferred coating agent is an extracellular matrix, such as collagen, proteoglycans, fibronectin, hyaluronic acid, tendinin, nestin, elastin, fibronectin, and laminin, or fragments of these substances. As a method for coating the cell aggregates, for example, after the start of suspension culture (e.g., after 48 hours), the aggregated cell mass can be transferred to droplets formed from a culture medium containing 50% by volume of the coating agent, coated, and then additional culture medium can be added to stop the coating process. The coating time is not particularly limited, and can be, for example, 30 minutes or more.

[0026] The culture medium for co-culturing nephron precursor cells, renal interstitial precursor cells, and ureteral bud cells can be prepared by adding Wnt signaling activator to a basal medium used for animal cell culture, and may further be prepared by adding one or more of GDNF, FGF9, and retinoic acid (RA) and / or its derivatives as needed. Additionally, alpha-albumin (Afamin) and / or R-spondin 1 (RSPO1) may be added as needed. These can be commercially available products. The culture medium for co-culturing nephron precursor cells, renal interstitial precursor cells, and ureteral bud cells is preferably prepared by adding Wnt signaling activator to a basal medium used for animal cell culture, and further adding at least FGF9. The basal culture medium may include, for example, 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. Depending on the requirements, it may contain one or more serum substitutes such as albumin, transferrin, KnockOut Serum Replacement (KSR) (Invitrogen), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, and 3'-thioglycerol. It 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, pyruvate, buffers, inorganic salts, and their equivalents. Media pre-optimized for stem cell culture, such as ReproFF2 (ReproCELL) or Stem Fit AK02N (Ajinomoto Healthy Supply), may also be used.

[0027] In this invention, the culture medium for co-culturing nephron precursor cells, renal interstitial precursor cells, and ureteral bud cells (hereinafter also referred to as the culture medium of this invention) contains Wnt signaling activating factors. There are no particular limitations on the Wnt signaling activating factors; they can include any substance that acts on the Wnt signaling pathway receptor and activates the Wnt signal, such as proteins, peptides, small molecule compounds, organic compounds, antibodies, etc. It should be noted that "Wnt signaling pathway receptors" can include co-receptors of the Wnt signaling pathway, such as LRP. The Wnt signaling activating factors are preferably proteins or peptides. Specifically, examples of Wnt signaling activating factors 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. Among these, Wnt3 or Wnt3a proteins are preferred. In addition, Wnt signaling activators can be, for example, Wnt agonists such as Wnt family protein-like peptides, specifically Foxy5. Preferably, the Wnt signaling activator can be Wnt3a protein or a Wnt3a protein-like peptide, more preferably Wnt3a protein. Wnt3a protein can be obtained from MBL Corporation, for example, or it can be prepared in-house. The method for preparing Wnt3a protein is not particularly limited and can be any known method, such as the culture supernatant obtained by expressing Wnt3a protein in serum-free medium. When using Wnt3a as the Wnt signaling activator, the concentration of Wnt3a in the culture medium of the present invention is not particularly limited as long as it is sufficient to produce kidney tissue-like cultures. For example, it can be 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 less, or less than 10000 ng / mL, 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 combination of these non-contradictory factors. Specifically, the concentration of Wnt3a in the culture medium of the present invention can be, for example, 1 ng / mL to 10000 ng / mL, 10 ng / mL to 1000 ng / mL, 50 ng / mL to 500 ng / mL, 100 ng / mL to 1000 ng / mL, or 100 ng / mL to 500 ng / mL.

[0028] In this invention, the culture medium for co-culturing nephron precursor cells, renal interstitial precursor cells, and ureteral bud cells may contain α-albumin (Afamin). It is known that Wnt family proteins such as Wnt3a are stabilized and prevent aggregate formation in the presence of α-albumin (Afamin). Therefore, from the above perspective, when the culture medium of this invention contains Wnt family proteins such as Wnt3a, it is preferable to also contain α-albumin (Afamin). Afamin can be included, for example, in the culture supernatant obtained by expressing Wnt3a protein in the above-mentioned serum-free culture medium. The culture supernatant containing Afamin and Wnt3a, i.e., Afamin / Wnt3a conditioned medium, can be obtained, for example, from MBL Corporation. When using Wnt3a protein, the concentration of Wnt3a protein in the culture medium of the present invention is not particularly limited as long as it can produce kidney tissue-like cultures. For example, when converted to Afamin / Wnt3a conditioned medium, it can be 1% or more, 3% or more, 5% or more, 8% or more, or 10% or more, or it can be less than 20% or less, 18% or less, 15% or less, 12% or less, or 10% or less, or any combination of these non-contradictory values. Specifically, when converted to Afamin / Wnt3a conditioned medium, the concentration of Wnt3a protein in the culture medium of the present invention can be 1-20% by volume, 3-18% by volume, 5-15% by volume, 8-12% by volume, or 10% by volume. The concentration of Afamin in the culture medium of the present invention is not particularly limited as long as it can produce kidney tissue-like cultures; for example, the above description when converted to Afamin / Wnt3a conditioned medium can be cited.

[0029] In this invention, the culture medium for co-culturing nephron precursor cells, renal interstitial precursor cells, and ureteral bud cells may further contain R-spondin 1 (RSPO1). RSPO1 is known to promote Wnt signaling. Therefore, from the above viewpoint, culture media containing Wnt signaling activators, especially those containing Wnt family proteins or Wnt agonists, preferably further contain RSPO1. RSPO1 can be obtained, for example, from an R&D company or can be prepared in-house. The concentration of RSPO1 in the culture medium of the present invention is not particularly limited as long as it can produce kidney tissue-like cultures. For example, it can 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. It can also be 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. Any combination of these non-contradictory concentrations is also possible. Specifically, the concentration of RSPO1 in the culture medium of the present invention can 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 this invention, the culture medium for co-culturing nephron precursor cells, renal interstitial precursor cells, and ureteral bud cells may further contain glial cell-derived neurotrophic factor (GDNF). GDNF can be obtained from R&D companies or prepared in-house. The concentration of GDNF in the culture medium of this invention is not particularly limited as long as it yields a kidney tissue-like culture; for example, it can 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 less than 1000 ng / mL, 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 any combination of these non-contradictory elements. Specifically, the concentration of GDNF in the culture medium of the present invention can 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 this invention, the culture medium for co-culturing nephron precursor cells, renal interstitial precursor cells, and ureteral bud cells may further contain FGF9. From the viewpoint of stably producing kidney tissue-like cultures, the culture medium for co-culturing nephron precursor cells, renal interstitial precursor cells, and ureteral bud cells preferably further contains FGF9. FGF9 can be obtained, for example, from Peprotech, or it can be prepared in-house. There are no particular restrictions on the concentration of FGF9 in the culture medium as long as it can produce kidney tissue-like cultures. For example, it can be above 5 ng / mL, above 10 ng / mL, above 25 ng / mL, above 30 ng / mL, above 40 ng / mL, above 50 ng / mL, above 80 ng / mL, or above 100 ng / mL. It can also be below 1000 ng / mL, below 500 ng / mL, below 300 ng / mL, below 200 ng / mL, below 150 ng / mL, below 120 ng / mL, or below 100 ng / mL. Any combination of these non-contradictory concentrations is also acceptable. The specific concentration of FGF9 in the culture medium can 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 this invention, the culture medium for co-culturing nephron precursor cells, renal interstitial precursor cells, and ureteral bud cells may further contain retinoic acid (RA) and / or its derivatives. RA can be obtained, for example, from Sigma-Aldrich or can be prepared in-house. The concentration of RA and / or its derivatives in the culture medium is not particularly limited as long as it yields a kidney tissue-like culture; for example, it can be ≥0.005 μM, ≥0.01 μM, ≥0.025 μM, ≥0.03 μM, ≥0.04 μM, ≥0.05 μM, ≥0.08 μM, or ≥0.1 μM, or it can be <1.0 μM, <0.5 μM, <0.3 μM, <0.2 μM, <0.15 μM, <0.12 μM, or <0.1 μM, or any combination thereof without contradiction. The concentration of RA and / or its derivatives in the culture 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 (4-[[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carbonyl]amino]benzoic acid (AM580) (Tamura K, etal., Cell Differ. Dev. 32:17-26 (1990)), and 4-[(1E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl-1-yl]- Benzoic acid (4-[(1E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propen-1-yl]-Benzoic acid) (TTNPB) (Strickland S, et al., Cancer Res. 43:5268-5272 (1983)), and compounds described in Tanenaga, K. et al., Cancer Res. 40:914-919 (1980), retinyl palmitate, retinol, retinaldehyde, 3-dehydroretinol, 3-dehydroretinaldehyde, etc.

[0033] In this invention, the culture medium for co-culturing nephron precursor cells, renal interstitial precursor cells, and ureteral bud cells may further contain a ROCK (Rho kinase) inhibitor. It is believed that by adding a ROCK inhibitor, cell death can be suppressed, and efficient culture, passage, cell proliferation, and / or differentiation can be achieved. Based on the above viewpoint, the culture medium of this invention may contain a ROCK inhibitor. Alternatively, the ROCK inhibitor may be included in the culture medium at the beginning of cell culture or during passage, and then replaced with a ROCK inhibitor-free medium. Known substances can be appropriately used as ROCK inhibitors, such as Y-27632. The concentration of the ROCK inhibitor may be, for example, 1–50 μM, preferably 5–20 μM. The ROCK inhibitor is preferably added to the culture medium at least 3 days after the start of cell culture or passage.

[0034] The culture time for co-culturing nephron precursor cells, renal interstitial precursor cells, and ureteral bud cells can be, for example, more than 2 days, more than 3 days, more than 4 days, more than 5 days, or more than 6 days. There is no upper limit to the culture time; for example, it can be less than 30 days. Culture conditions are not particularly limited as long as a kidney tissue-like culture can be obtained. The culture temperature is approximately 30–40°C, preferably approximately 37°C, the oxygen concentration is a typical oxygen concentration (e.g., 15–25%, preferably approximately 20%), and the CO2 concentration is preferably approximately 2–5%.

[0035] A kidney tissue-like culture refers to a culture having a structure similar to that of a developing and / or mature kidney, i.e., a kidney tissue structure. Here, a structure similar to a kidney tissue structure is not limited to morphological similarity, but can also refer to a structure that is anatomically and / or functionally similar to a kidney tissue structure based on characteristics of individual cells, such as those based on marker gene expression, and the characteristics of the structures composed of these cells. That is, a kidney tissue-like culture can also be called a kidney organoid. During the developmental period, the anterior kidney develops into the metanephron via the mesonephron, and the metanephron matures to form an organ equivalent to a kidney. The period during which this organ equivalent to a kidney is formed can also be called the organogenesis period. As mentioned above, nephron precursor cells, renal interstitial precursor cells, and ureteral bud cells are each of the precursor cells used to form the metanephron. Therefore, in the kidney tissue-like culture of the present invention, the developing kidney tissue can be either metanephron tissue or organogenesis-stage kidney tissue. In other words, the kidney tissue-like culture of the present invention can refer to a culture having a structure similar to metanephron tissue, organogenesis-stage kidney tissue, and / or mature kidney tissue. The kidney tissue-like culture of the present invention can maintain organogenesis-stage kidney tissue. In one embodiment, the kidney tissue-like culture of the present invention can maintain the kidney-forming region. Furthermore, the kidney tissue-like culture of the present invention does not exclude having a structure similar to mature kidney tissue, but it may not also lack such a structure. That is, the kidney tissue-like culture of the present invention can refer to a culture having a structure similar to metanephrotic tissue and / or kidney tissue during organogenesis.

[0036] Examples of renal tissue structures include, but are not limited to, mesangial structures, branching ureteral bud structures, and cap-shaped mesenchymal structures. The renal tissue-like culture of the present invention preferably has one or more of the following: mesangial structures, branching ureteral bud structures, S-shaped structures, and cap-shaped mesenchymal structures; more preferably, it has all of these structures. The renal tissue-like culture of the present invention can also maintain the renal formation region. For example, the renal tissue-like culture of the present invention can promote the further formation of mesangial structures, branching ureteral bud structures, S-shaped structures, and / or cap-shaped mesenchymal structures by maintaining the renal formation region.

[0037] Mesangial structures refer to the structures formed by podocytes surrounding mesangial cells within the glomeruli of the metanephron or organogenesis-stage kidney. Mesangial structures can be characterized, for example, by the presence of mesangial cells within glomerular-like structures. Mesangial cells can be characterized, for example, by the expression of mesangial cell marker genes. Examples of mesangial cell marker genes include PDGFRB and GATA3. That is, as an example, a mesangial structure can be a structure containing PDGFRB and / or GATA3-positive cells within a glomerular-like structure.

[0038] A branched ureteric bud structure refers to a structure derived from a ureteric bud formed through the branching and elongation of a ureteric bud. In this invention, the branched ureteric bud structure preferably has a ureteric bud, and the apical portion of the ureteric bud contains RET-positive cells. More preferably, the branched ureteric bud structure may have two or more ureteric buds, and the apical portion of each ureteric bud contains RET-positive cells. Furthermore, the ureteric bud can also be characterized by the expression of the CK8 gene.

[0039] S-shaped structures refer to developing renal tubular structures formed through epithelialization of nephron precursor cells. In this invention, the S-shaped structure preferably comprises an S-shaped structure containing a proximal region and a distal region. The proximal region can be characterized, for example, by the expression of HNF4α. The distal region can be characterized, for example, by the expression of BRN1. It should be noted that the distal region may or may not be distinguished from the intermediate region located between the proximal and distal regions. When the distal region is not distinguished from the intermediate region, it can be collectively referred to as the mid-distal region. In this case, it can also be stated that the S-shaped structure preferably comprises an S-shaped structure containing a proximal region and a mid-distal region. The mid-distal region can also be characterized, for example, by the expression of BRN1.

[0040] A cap-shaped mesenchymal structure refers to the mesenchymal tissue surrounding the tip of the ureteral bud. It is generally known that glomerular podocytes and nephron precursor cells are present in the cap-shaped mesenchymal structure. In this invention, the cap-shaped mesenchymal structure preferably contains nephron precursor cells and / or cells derived from nephron precursor cells. Nephron precursor cells and / or cells derived from nephron precursor cells can be characterized, for example, by the expression of SIX2 and / or PAX8. More specifically, for example, nephron precursor cells can be characterized by the expression of SIX2, and cells derived from nephron precursor cells can be characterized by the expression of PAX8. That is, the cap-shaped mesenchymal structure can be a structure surrounding the tip of the ureteral bud, and is a structure containing SIX2 and / or PAX8 positive cells. It should be noted that the ureteral bud can also be characterized by the expression of the CK8 gene. Cells derived from nephron precursor cells are not particularly limited as long as they originate from nephron precursor cells; for example, they can be cells constituting the metanephron vesicles. Cells constituting the metanephron vesicles can be characterized, for example, by the expression of PAX8. Furthermore, a region characterized by a cap-shaped mesenchymal structure containing at least nephron precursor cells can be considered a kidney-forming region. As mentioned above, nephron precursor cells can also be characterized by SIX2 expression. In other words, a kidney-forming region can also be observed as a cap-shaped mesenchymal structure containing SIX2-expressing cells. The maintenance of a kidney-forming region by a kidney tissue-like culture can mean that the kidney tissue-like culture contains at least the structures exhibiting the aforementioned kidney-forming region characteristics. Alternatively, the maintenance of a kidney-forming region by a kidney tissue-like culture, as a non-limiting example, can mean that the structures exhibiting the aforementioned kidney-forming region characteristics are visible during the culture manufacturing process, and that all or part of such structures are maintained after culture manufacturing.

[0041] <2> The culture of the present invention As another aspect of the present invention, the present invention relates to kidney tissue-like cultures. Specifically, as one approach, the present invention can be: A kidney tissue-like culture, It contains cells derived from humans. It also has mesangial structure, branched ureteral bud structure, S-shaped structure and cap-shaped mesenchymal structure.

[0042] There are no particular limitations on the method for obtaining the kidney tissue-like culture of the present invention, and it can be prepared by the manufacturing method of the present invention.

[0043] The kidney tissue-like culture of the present invention may contain human-derived cells. As an example, when the kidney tissue-like culture of the present invention is obtained by the culture method of the present invention or manufactured by the manufacturing method of the present invention, one or more types of cells selected from nephron precursor cells, renal interstitial precursor cells, and ureteral bud cells may be human-derived cells; preferably, all cells of the nephron precursor cells, renal interstitial precursor cells, and ureteral bud cells are human-derived. Furthermore, the kidney tissue-like culture of the present invention is preferably composed of human-derived cells.

[0044] The kidney tissue-like culture of the present invention can have mesangial structures, branching ureteral bud structures, S-shaped body-like structures, and cap-shaped mesenchymal structures. Details of the mesangial structures, branching ureteral bud structures, S-shaped body-like structures, and cap-shaped mesenchymal structures are as described above.

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

[0046] The kidney tissue-like culture of the present invention has a structure similar to that of kidney tissue and is expected to gradually undergo organogenesis and further develop and grow as an organ. Therefore, the kidney tissue-like culture of the present invention, which may contain human cells or be composed of human cells, is expected to be applied to various uses such as reconstruction of kidney tissue and kidney, regenerative medicine, kidney disease models, drug discovery and screening, and drug toxicity evaluation systems.

Example

[0047] The present invention will now be described in detail based on embodiments, but the present invention is not limited to the following methods.

[0048] <Late-stage primary stripe (PPS) induction> First, human iPS cells (hiPSCs) were enzymatically treated for 5 minutes with a 1:1 mixture of TrypLE Select Enzyme (Thermo Fisher Scientific) and 0.5 mM EDTA / PBS, followed by washing with PBS(-). Subsequently, cells were dissected using a cell scraper and gently pipetted to separate into single cells. One day before differentiation began, these cells were added to 0.5 mL of Stem Fit AK02N (Ajinomoto Healthy Supply) containing 10 μM Y-27632 and 2.4 μL / mL iMatrix-511 (Matrixome) at a concentration of 1.3 × 10⁻⁶. 4 cells / cm 2Seeds were sown at a density of 5 μM in 24-well plates (Corning). After 24 hours (Day 0), the culture was initiated in a serum-free differentiation medium (hereinafter referred to as basal medium) consisting of DMEM / F12 Glutamax (Thermo Fisher Scientific), B27 supplement (without Vitamin A) (Thermo Fisher Scientific), and 0.5× penicillin / streptomycin, 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 culture was continued 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 replaced with basal medium containing 5 μM CHIR99021, 1 ng / mL BMP7, and 10 μM A83-01 (Wako). On day 3, the culture medium was replaced with 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, cells were washed with PBS(-), treated with Accumax (InnovativeCell Technologies), and gently dissociated into single cells by pipetting. Cells were then cultured at 1.05 × 10⁻⁶ cells per cell. 5 cells / cm 2 Seed at a density of 2.0 × 10⁻⁶ wells in 24-well plates (2D culture), or at 2.0 × 10⁻⁶ wells. 4 cells / cm 2 The cells were seeded at a density of 100% in 96-well low-gravity plates (Thermo Fisher Scientific) for 3D culture. The culture medium consisted of 5 μM CHIR99021, 30 ng / mL bFGF, 10 ng / mL activin A, and 30 μM Y-27632 (for 2D culture, the medium was further supplemented with 1.2 μL / well iMatrix-511), and cultured for 48 hours.

[0049] <Induction of Nephron Precursor Cells (NPCs)> On day 6 of PPS induction, the medium was replaced with basal medium containing 0.1 μM RA and 25 ng / mL NOGGIN (Peprotech). On day 8, the medium was replaced with basal medium containing 200 ng / mL FGF9 and 1 μM CHIR99021, and cultured for 72 hours.

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

[0051] <Induction of mesonephric duct (ND) cells (ureteral bud cells)> First, the anterior interstitial mesoderm is induced by hiPSC using the following method. hiPSCs were enzymatically treated for 5 minutes using a 1:1 mixture of TrypLE Select Enzyme and 0.5 mM EDTA / PBS, followed by washing with PBS(-). Cells were then dissected using a cell scraper and separated into single cells by gentle pipetting. One day before differentiation began, cells were cultured at 4.0 × 10⁻⁶. 5Cells were seeded at a density of 10-cm culture dishes (Falcon) using Stem Fit AK02N (with 10 μM Y-27632 and 20 μL / dish iMatrix-511). After 24 hours (day 0), cells were cultured in serum-free differentiation medium (E6; Thermo Fisher Scientific) supplemented with 50 ng / mL activator A, 5 μM CHIR99021, 25 ng / mL BMP4, and 25 ng / mL bFGF. Approximately 22 hours later (day 1), E6 medium was supplemented with 100 nM LDN193189, 1 μM A83-01, 0.1 μM TTNPB (Santa Cruz Biotechnology), and 200 ng / mL FGF8 (Peprotech), and cultured for 2 days. Subsequently, the cells were subjected to a treatment containing the same four factors and 10 μM Y-27632 at a concentration of 8.7 × 10⁻⁶. 6 Cells were reseeded at a density of 10-cm culture dishes and cultured for another 24 hours. Next, ND cells were induced from the anterior intermediary mesoderm using the following method. The anterior intercellular mesoderm was treated for 2 days with E6 medium containing 1 μM CHIR99021, 100 nM LDN193189, 200 ng / mL FGF8, 100 ng / mL GDNF, and 0.1 μM TTNPB. Following treatment with Accutase (Nacalai tesque) at 37°C for 3 minutes, the cells were separated into single cells by pipetting and cultured at 1.0 × 10⁻⁶ cells / mL. 4 Cells were seeded at a low-attachment 96-well plate (Sumitomo Bakelite) at a density of 10 μM. Then, the cells were cultured for 2 days in the same medium and with additional 10 μM Y-27632 to induce ND cells.

[0052] <Reconstruction of Kidney Structure> The NPC, IPC, and ND cells induced as described above were incubated with Accumax at 37°C for 10 minutes and gently dissociated into single cells using a pipette. The dissociated single cells were mixed and resuspended in basal medium (CRFY) containing 1 μM CHIR99021, 0.1 μM RA, 100 ng / mL FGF9, and 10 μM Y-27632; medium (CRFY+G) further containing 200 ng / mL GDNF; or 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 (MBL; J-ORMW301R), and 200 ng / mL R-spondin1 protein (RSPO1) (R&D). The culture was then incubated at 1.7 × 10⁻⁶ cells / mL. 4 NPC of cell / pore density, 1.7 × 10⁻⁶ 4 IPC of cell / pore density and 1.7 × 10 4 ND cells at a cell / well density were seeded into 96-well low-cell-attachment U-plates to form aggregates. After 48 hours, the aggregates were transferred to 24-well plates, and 20 μL of 50% Matrigel and the same medium as described above were added. The plates were then incubated at 37°C for 30 minutes. After some solidification, 180 μL of the same medium as described above was added. Half of the medium was then replaced every two days.

[0053] On day 6 of renal structure remodeling under CRFY conditions, immunostaining analysis showed that CALB1(+) epithelial structures were remodeled, and SIX2(+) NPC and FOXD1(+) IPC surrounded ureteral bud (UB)-like epithelial structures. Figure 1 However, RET expression was not observed in the apical structure of UB. The culture medium used for proliferation of ureteral bud apical cells in a previous report (Mae, SI, et al. Cell Rep. 32. 2020) contained GDNF. Therefore, renal structure remodeling was performed under CRFY+G conditions (CRFY with added GDNF). On day 6, tissue-like cultures under these conditions showed a slight increase in RET expression via quantitative RT-PCR (qT-PCR), while other progenitor cell markers such as SIX2 and FOXD1 remained unaffected. Figure 2 ).

[0054] On the other hand, in RFY+G+WR conditions where GDNF was added to CRFY and Wnt3a and RSPO1 were used instead of CHIR99021, tissue culture samples from kidney structure remodeling on day 6 showed, according to qRT-PCR analysis, that RET expression was further increased compared to tissue culture samples from CRFY+G conditions. Figure 2 Furthermore, immunostaining analysis of the culture showed that the tips of the budding UB were RET positive. Figure 3 This forms kidney-forming region-like structures, such as cap-shaped mesenchymal structures of SIX2(+) or PAX8(+). Figure 4 ) and the S-shaped structure formed by the proximal region of HNF4α(+) and the mid-distal region of BRN1(+) ( Figure 5 ).

[0055] Mesangial cells originate from Foxd1(+) IPCs and migrate to the glomerular capillary loops during development. In the glomeruli of embryonic kidneys at the capillary loop stage, podocytes surround Pdgfrb(+) mesangial cells to form mesangial structures. Consistent with this, glomerular-like structures with PDGFRB(+) or GATA3(+) mesangial-like cells, i.e., mesangial structures, were observed in tissues corresponding to the capillary loop stage on days 6 and 12, and in tissues corresponding to the mature stage on day 12, under RFY+G+WR conditions for renal structural remodeling. Figure 6 It should be noted that when IPCs constitutively expressing green fluorescent protein (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] In summary, co-culturing NPC cells, IPC cells, and ND cells under RFY+G+WR conditions enabled the formation of appropriate UB structures, kidney-forming regions, and mesangial structures. Specifically, by co-culturing NPC cells, IPC cells, and ND cells (ureteral bud cells) under conditions containing Wnt3a, a Wnt signaling activator, a kidney tissue-like culture with organogenesis-stage kidney structures was created.

[0057] <Evaluation of Nephron Precursor Cells (NPCs)> The NPCs induced by the above-described <Induction of Nephron Progenitor Cells (NPCs)> were incubated with Accumax at 37°C for 10 minutes and then gently dissociated into single cells using a pipette. The dissociated single cells were mixed and resuspended in basal medium (CRFY) containing 1 μM CHIR99021, 200 ng / mL FGF9, and 10 μM Y-27632, at a growth rate of 4 × 10⁻⁶. 4Cells were seeded at a density of 96-well low-cell-attachment U-shaped substrate to form aggregates. After 48 hours, cell blocks were harvested, frozen sections were prepared, and fluorescent immunostaining was performed on marker genes for mesenchymal precursor cells. The results showed that a portion of these sections contained mesenchymal precursor cells indicated by PDGFRB(+). Figure 7 ).

[0058] <Induction of Ureteral Twig Organoids> Next, ureteral bud organoids were induced from mesonephric duct (ND) cells (ureteral bud cells) using the following method. After washing the mesonephric duct (ND) cells (ureteral bud cells) with PBS, they 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 ureteral bud organoids.

[0059] <Kidney Structural Reconstruction via Ureter Bulgomorphs and NPCs> The NPCs induced above were incubated with Accumax at 37°C for 10 minutes and then gently dissociated into single cells using a pipette. The dissociated NPC cells 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). The NPCs were then sown from above onto the induced ureteral buds at a rate of 5 × 10⁻⁶ cells / cm². 4 The NPC suspension was seeded at a cell / well density to form aggregates. After 48 hours, the aggregates were transferred to 24-well plates, and 20 μL of 50% Matrigel and the same medium as described above were added. The plates were then incubated at 37°C for 30 minutes. After some solidification, 180 μL of the same medium as described above was added. Subsequently, half of the medium was replaced with basal medium every two days.

[0060] On day 13 of renal structure reconstruction under RFY+G+WR conditions, immunostaining analysis of tissue-like cultures showed that the cap-like mesenchymal structure of SIX2(+) nephron precursor cells was maintained, and renal tissue with CK8(+) branched ureteral bud structure was created while maintaining the kidney-forming region. Figure 8 ).

[0061] In summary, by co-culturing NPC cells (containing some IPC cells) with ureteral bud organoids under RFY+G+WR conditions, appropriate UB structures and kidney-forming regions can be formed. Specifically, by co-culturing NPC cells, IPC cells, and ureteral bud organoids (ureteral bud cells) under conditions containing Wnt3a, a Wnt signaling activator, a kidney tissue-like culture with organogenesis-stage kidney structures was created.

Claims

1. A method for manufacturing a kidney tissue-like culture, comprising a step of co-culturing nephron precursor cells, renal interstitial precursor cells, and ureteral bud cells in a culture medium containing Wnt signaling activating factor.

2. The method of claim 1, wherein, The co-culture was carried out via suspension culture.

3. The method of claim 2, wherein, The suspension culture was performed with aggregates of nephron precursor cells, renal interstitial precursor cells, and ureteral bud cells coated with an extracellular matrix.

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

5. The method of claim 1, wherein, The culture medium is a culture medium that further contains fibroblast growth factor 9 (FGF9).

6. The method according to any one of claims 1 to 5, wherein, The culture medium is a medium that further contains retinoic acid (RA) and / or its derivatives, as well as glial cell-derived neurotrophic factor (GDNF).

7. The method of claim 6, wherein, The culture medium is a medium further containing α-albumin and R-Spondin 1 (RSPO1).

8. The method according to any one of claims 1 to 5, wherein, The kidney tissue-like culture has one or more of the following: mesangial structure, branching ureter bud structure, S-shaped structure, and cap-shaped mesenchymal structure.

9. The method according to any one of claims 1 to 5, wherein, The nephron precursor cells, renal interstitial precursor cells, and ureteral bud cells are derived from humans.

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

11. A kidney tissue-like culture, It contains cells derived from humans. It also has mesangial structure, branched ureteral bud structure, S-shaped structure and cap-shaped mesenchymal structure.

12. The kidney tissue-like culture according to claim 11, wherein, The mesangial structure is a structure containing PDGFRB and / or GATA3-positive cells within a glomerular-like structure. The branched ureteral bud structure is a structure with two or more ureteral buds, and the apical part of the ureteral bud contains RET-positive cells. The S-shaped structure is a structure containing an S-shaped region and a distal region. The cap-shaped mesenchymal structure is a structure that surrounds the tip of the ureteral bud and contains SIX2 and / or PAX8 positive cells.

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