Production method for renal collecting duct cells and pelvic epithelial cells

Abstract

Provided is a production method for renal collecting duct cells, said method including a step for culturing ureteric bud cells or cells that have differentiated from ureteric bud cells in a culture medium that includes a TGFβ inhibitor, a Wnt inhibitor, and a phosphodiesterase inhibitor. Also provided is a production method for a renal collecting duct cell–containing organoid, said method including a step for culturing a ureteric bud organoid or an organoid that has differentiated from a ureteric bud organoid in a culture medium that includes a TGFβ inhibitor, a Wnt inhibitor, and a phosphodiesterase inhibitor.

Description

Method for producing renal collecting duct cells and renal pelvic epithelial cells

[0001] The present application relates to a method for producing renal collecting duct chief cells, renal collecting duct chief cell-containing organoids, renal collecting duct interstitial cells, or renal pelvic epithelial cells.

[0002] The renal collecting duct primarily concentrates and regulates the pH of primary urine that has been filtered by the glomerulus and passed through the renal tubule, maintaining homeostasis of the body's water content and acid-base balance. The renal collecting duct is formed from its main constituent cell types, chief cells, which express water channels such as AQP2 and work to concentrate urine, and interstitial cells, which express carbonic anhydrase and regulate pH. The generation of renal collecting duct cells and renal pelvic epithelial cells is expected to be useful for the creation of models, pathological analysis, and treatment development for ion channel abnormalities and congenital kidney and urinary tract abnormalities, including cystic kidney disease.

[0003] To date, there have been reports of inducing the differentiation of nephron progenitor cells, which are the precursor cells for glomeruli and tubules, and ureteric bud cells, which are the precursor cells for collecting ducts, from human pluripotent stem cells, and then combining and culturing these two types of renal progenitor cells to produce renal tissue containing collecting ducts in addition to glomeruli and tubules (Non-Patent Documents 1 and 2), as well as of purifying ureteric bud lineage cells derived from human pluripotent stem cells using a flow cytometer and producing ureteric bud organoids containing some collecting duct cells from these purified cells (Non-Patent Document 3). However, there have been no reports of a method for selectively inducing the differentiation of renal collecting duct cells and renal pelvic epithelial cells from human pluripotent stem cells.

[0004] Tsujimoto H. et al., Cell Reports 2020; 31(1):107476.Uchimura. et al., Cell Reports 2020; 33(11):108514.Zeng. et al., Nature Communications 2021; 12:3641.

[0005] The present application aims to provide a method for producing renal collecting duct chief cells, renal collecting duct chief cell-containing organoids, renal collecting duct interstitial cells, or renal pelvic epithelial cells.

[0006] The present application provides a method for producing renal collecting duct principal cells, which comprises the step of culturing ureteric bud cells or cells differentiated from ureteric bud cells in a medium containing a TGFβ inhibitor, a Wnt inhibitor, and a phosphodiesterase inhibitor.

[0007] The present application also provides a cell population containing 40% or more renal collecting duct chief cells.

[0008] The present application also provides a method for producing renal collecting duct principal cell-containing organoids, comprising the step of culturing ureteric bud organoids or organoids differentiated from ureteric bud organoids in a medium containing a TGFβ inhibitor, a Wnt inhibitor, and a phosphodiesterase inhibitor.

[0009] The present application also provides a renal collecting duct chief cell-containing organoid containing 40% or more renal collecting duct chief cells.

[0010] The present application also provides a method for producing renal collecting duct intercalated cells, which comprises culturing renal collecting duct principal cells in a medium containing a TGFβ inhibitor, a Wnt inhibitor, a retinoic acid receptor agonist, and a Notch signal inhibitor in the presence of an ETS family protein.

[0011] The present application also provides a cell population containing 30% or more renal collecting duct interstitial cells.

[0012] The present application also provides a method for producing renal pelvic epithelial cells, which includes culturing renal collecting duct progenitor cells in a medium containing a TGFβ inhibitor, a Wnt inhibitor, a retinoic acid receptor agonist, and a Notch signal inhibitor in the presence of an ETS family protein.

[0013] The present application also provides a cell population containing 40% or more renal pelvic epithelial cells.

[0014] The method of the present application makes it possible to produce renal collecting duct chief cells, renal collecting duct chief cell-containing organoids, renal collecting duct intercalated cells, or renal pelvic epithelial cells.

[0015] Figure 1 shows a schematic diagram illustrating the procedure for generating an iPS cell line (Tet-On-ELF5-1231A3) capable of forced expression of ELF5 and for forcing ELF5 expression. Figure 2 shows an example of a protocol for forcing ELF5 expression in Tet-On-ELF5-1231A3 iPS cells. Tet-On-ELF-1231A3 iPS cells were cultured in StemFit® AK02 for one day, then 4 μM doxycycline was added and cultured for another day. Comparison of semi-quantitative PCR results with and without doxycycline (DOX) treatment. Figure 2 shows bright-field and fluorescence microscopy images of Tet-On-ELF5-1231A3 iPS cells treated with doxycycline according to the protocol described in Figure 2. Figure 3 shows an immunostained image of Tet-On-ELF5-1231A3 iPS cells treated with doxycycline according to the protocol described in Figure 2.

[0016] An example of a protocol for inducing mesonephric duct cells from Tet-On-ELF5-1231A3 iPS cells. Anterior PS: anterior primitive streak, Anterior IM: anterior intermediate mesoderm, ND: mesonephric duct, UB: ureteric bud, LDN: LDN193189. Immunostained image of mesonephric duct cells induced according to the protocol described in Figure 6. An example of a protocol for inducing ureteric bud organoids from anterior intermediate mesoderm cells derived from Tet-On-ELF5-1231A3 iPS cells. iUB: ureteric bud. Brightfield image of day 2 ureteric bud organoids induced from anterior intermediate mesoderm cells according to the protocol described in Figure 8.

[0017] An example of a protocol for inducing renal collecting duct progenitor cells from Tet-On-ELF5-1231A3 iPS cells. Immunostained images of cells induced according to the protocol described in Figure 10.

[0018] An example of a protocol for inducing renal collecting duct progenitor cells from Tet-On-ELF5-1231A3 iPS cells. The renal collecting duct progenitor cell induction medium was further supplemented with 4 μM doxycycline and 400 μM IBMX. Immunostained images of renal collecting duct chief cells induced according to the protocol described in Figure 12.

[0019] An example of a protocol for inducing renal collecting duct progenitor cells from Tet-On-ELF5-1231A3 iPS cells. 4 μM doxycycline and various concentrations of IBMX were further added to the renal collecting duct progenitor cell induction medium. Immunostained images of renal collecting duct chief cells induced according to the protocol described in Figure 14.

[0020] An example of a protocol in which renal collecting duct principal cells were cultured in a transwell culture medium from mature mesonephric duct cell aggregates derived from Tet-On-ELF5-1231A3 iPS cells and added with desmopressin (dDAVP). CD: renal collecting duct, PC: principal cells. Schematic diagram of transwell culture. Comparison of AQP2 expression and polarity by immunostaining of renal collecting duct principal cells induced according to the protocol described in Figure 16.

[0021] An example of a protocol for inducing renal collecting duct principal cells from mature mesonephric duct cell aggregates derived from Tet-On-ELF5-1231A3 iPS cells. Renal collecting duct principal cells were induced by plate culture after adding various factors to renal collecting duct principal cell induction medium excluding IBMX. Immunostained images of renal collecting duct principal cells induced according to the protocol described in Figure 19 for E-cadherin (red), AQP2 (white), and AVPR2 (green). AVP: vasopressin acetate; ALD: aldosterone. AQP2-positive cell rate in renal collecting duct principal cells induced according to the protocol described in Figure 19. 2F: aldosterone + K252a.

[0022] An example of a protocol for inducing renal collecting duct principal cells from mature mesonephric duct cell aggregates derived from Tet-On-ELF5-1231A3 iPS cells. The medium for inducing renal collecting duct principal cells was further supplemented with 10 nM aldosterone, 0.1 μM TTNPB, and 100 nM dDAVP. Immunostained images of renal collecting duct principal cells induced according to the protocol described in Figure 22.

[0023] An example of a protocol for inducing renal collecting duct principal cells from ureteric bud organoids derived from Tet-On-ELF5-1231A3 iPS cells. Comparison of the expression levels of renal collecting duct principal cell-related marker genes in collecting duct principal cells induced according to the protocol described in Figure 24. d4AIT: Cells obtained by culturing ureteric bud cells in renal collecting duct progenitor cell induction medium containing TTNPB for 7 days. d4DIAA: Renal collecting duct principal cells induced by culturing ureteric bud cells in renal collecting duct progenitor cell induction medium containing TTNPB for 3 days, followed by a further 4 days in renal collecting duct principal cell induction medium containing TTNPB, dDAVP, and aldosterone.

[0024] An example of a protocol for inducing renal collecting duct principal cell-containing organoids from mature mesonephric duct cell aggregates derived from Tet-On-ELF5-1231A3 iPS cells. Bright-field images of ureteric bud organoids on day 6 and bright-field and fluorescent microscopy images of renal collecting duct principal cell-containing organoids on day 2, induced according to the protocol described in Figure 26. Bright-field images of renal collecting duct principal cell-containing organoids on day 6, induced according to the protocol described in Figure 26. Immunostained images of renal collecting duct principal cell-containing organoids induced according to the protocol described in Figure 26.

[0025] Functional evaluation of renal collecting duct principal cell-containing organoids. On day 6 of induction, renal collecting duct principal cell-containing organoids were further cultured with dDAVP alone or dDAVP and tolvaptan. Bright-field images of renal collecting duct principal cell-containing organoids on days 1 and 3 of culture are shown. Arrowheads indicate areas of cystic changes due to water uptake.

[0026] Figure 32 shows a schematic diagram illustrating the procedure for generating a PKD1 knockout iPS cell line (Tet-On-ELF5-27B6-5) capable of forced expression of ELF5 and for forcing ELF5 expression. Figure 33 shows an example of a protocol for forcing ELF5 expression in Tet-On-ELF5-27B6-5 iPS cells. Tet-On-ELF5-27B6-5 iPS cells were cultured in StemFit® AK02 for one day, followed by the addition of 4 μM doxycycline and further culture for one day. Figure 34 shows bright-field and immunostained images of Tet-On-ELF5-27B6-5 iPS cells treated with doxycycline according to the protocol described in Figure 32. Figure 35 shows immunostained images of Tet-On-ELF5-27B6-5 iPS cells treated with doxycycline according to the protocol described in Figure 32.

[0027] An example of a protocol for inducing ureteric bud organoids from Tet-On-ELF5-27B6-5 iPS cells. Brightfield images of ureteric bud organoids induced according to the protocol described in Figure 35. TEER measurements of renal collecting duct principal cells before (pre) and 24 hours after (24h) benzamil administration. The group without benzamil administration served as a control (Ctl). Immunostained images of renal collecting duct principal cells with and without benzamil administration. Comparison of the ENaC-positive cell rate of renal collecting duct principal cells with and without benzamil (Bz) administration. The group without benzamil administration served as a control (Ctl). Student's t-test was used to test for significance. NS: Not significant. Schematic diagram showing the induction of renal collecting duct intercalated cells (IC) from ureteric bud organoids (iUB) via renal collecting duct progenitor cells (CDP) and renal collecting duct principal cells (PC). DOX: doxycycline. An example of a protocol for inducing renal collecting duct intercalary cells from ureteric bud cells derived from Tet-On-ELF5-1231A3 iPS cells. IC: intercalary cells. Immunostained image of renal collecting duct intercalary cells induced according to the protocol described in Figure 41. Schematic diagram showing the induction of renal pelvic epithelial cells from ureteric bud organoids (iUB) via renal collecting duct progenitor cells (CDP). An example of a protocol for inducing renal pelvic epithelial cells from ureteric bud cells derived from Tet-On-ELF5-1231A3 iPS cells. Immunostained image of renal pelvic epithelial cells induced according to the protocol described in Figure 44. An example of a protocol for inducing renal collecting duct chief cells from ureteric bud cells derived from Tet-On-ELF5-1231A3 iPS cells. Immunostained image of renal collecting duct chief cells induced according to the protocol described in Figure 46.

[0028] In this disclosure, when a numerical value is accompanied by the term "about," it is intended to encompass a range of ±10% of that value. For example, "about 20" is intended to include "18 to 22." A range of numerical values ​​includes all values ​​between and at the endpoints. "About" in reference to a range applies to both endpoints of the range. Thus, for example, "about 20 to 30" is intended to include "18 to 33."

[0029] In the present specification and claims, unless otherwise specified, the expression "a certain type of cell" refers to a cell population containing cells of that type, and the cell population may contain cells of types other than the specified type of cell. For example, the expression "a culture of a certain type of cell" refers to a culture of a cell population containing cells of that type, and may contain cells other than the specified type of cell. Similarly, the expression "a certain type of cell population" refers to a cell population containing cells of that type, and the cell population may contain cells other than the specified type of cell, unless otherwise specified.

[0030] In one aspect of the present application, there is provided a method for producing renal collecting duct principal cells, which comprises culturing ureteric bud cells or cells differentiated from ureteric bud cells in a medium containing a TGFβ inhibitor, a Wnt inhibitor, and a phosphodiesterase inhibitor.

[0031] In the present application, renal collecting duct principal cells can be confirmed by the expression of markers such as AQP2, AQP3, AQP4, AVPR2, SCNN1b, ELF5, and KCNJ1. Optionally, it may be confirmed that the cells do not express carbonic anhydrase (CA) II, a marker for renal collecting duct intercalated cells. The expression of renal collecting duct principal cell markers in renal collecting duct principal cells can be confirmed visually under a microscope by immunostaining, or by flow cytometry, such as FACS (fluorescence-activated cell sorting).

[0032] In the present application, the animal from which ureteric bud cells are derived is not limited, and examples include mammals such as mice, rats, hamsters, guinea pigs, cows, horses, pigs, sheep, monkeys, orangutans, chimpanzees, dogs, cats, birds, and humans. Primates are preferred, and humans are more preferred. The identity of ureteric bud cells can be confirmed by the expression of markers such as GATA3, RET, WNT11, LHX1, SOX9, and very-low-density lipoprotein receptor (VLDL-R), or by the uptake of very-low-density lipoprotein (VLDL). In one embodiment, ureteric bud cells are cultured in a medium containing a TGFβ inhibitor, a Wnt inhibitor, and a phosphodiesterase inhibitor.

[0033] Cells differentiated from ureteric bud cells can be any cell that develops into renal collecting duct principal cells, such as renal collecting duct progenitor cells. Renal collecting duct progenitor cells refer to cells that express aquaporin 2 (AQP2) but do not express other collecting duct principal cell marker genes, such as arginine vasopressin receptor 2 (AVPR2). Renal collecting duct progenitor cells can be identified by the expression of AQP2 and the absence of AVPR2 expression. In one embodiment, renal collecting duct progenitor cells differentiated from ureteric bud cells are cultured in a medium containing a TGFβ inhibitor, a Wnt inhibitor, and a phosphodiesterase inhibitor.

[0034] Ureteric bud cells may be differentiated from pluripotent stem cells. For example, ureteric bud cells can be obtained by dissociating ureteric bud organoids differentiated from pluripotent stem cells into single cells. Methods for inducing differentiation of ureteric bud organoids from pluripotent stem cells are known, and any known method can be used. For example, the method described in WO2019 / 098349 or Mae, S.I. & Ryosaka, M. et al. Cell Reports 32, 4, 107943 can be used. Specifically, a method for inducing differentiation of ureteric bud organoids from pluripotent stem cells can be used, comprising the following steps: (i) culturing pluripotent stem cells in the presence of an activator of activin receptor kinase 4, 7 and a GSK3β inhibitor to obtain an anterior primitive streak cell culture; (ii) culturing the anterior primitive streak cell culture in the presence of a fibroblast growth factor, a retinoic acid receptor agonist, a TGFβ inhibitor, and a BMP inhibitor to obtain an anterior intermediate mesoderm cell culture; (iii) culturing the anterior intermediate mesoderm cell culture in the presence of a GSK3β inhibitor, a BMP inhibitor, a fibroblast growth factor, a retinoic acid receptor agonist, and a glial cell line-derived neurotrophic factor to obtain an early mesonephric duct (Wolffian duct) cell culture; (iv) culturing early mesonephric duct (Wolffian duct) cells in the presence of a GSK3β inhibitor, a BMP inhibitor, a fibroblast growth factor, a retinoic acid receptor agonist, and a glial cell line-derived neurotrophic factor to obtain mature mesonephric duct (Wolffian duct) cell aggregates; and (v) culturing mature mesonephric duct (Wolffian duct) cell aggregates in the presence of a GSK3β inhibitor, a BMP inhibitor, a fibroblast growth factor, a retinoic acid receptor agonist, a glial cell line-derived neurotrophic factor, and a cell culture substrate (e.g., Matrigel) to obtain ureteric bud organoids.

[0035] In the present application, "pluripotent stem cells" refer to stem cells that have both pluripotency and proliferation ability, and can differentiate into all cells present in a living body. Examples of such stem cells include embryonic stem (ES) cells (J.A. Thomson et al. (1998), Science 282:1145-1147; J.A. Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92:7844-7848; J.A. Thomson et al. (1996), Biol. Reprod., 55:254-259; J.A. Thomson and V.S. Marshall (1998), Curr. Top. Dev. Biol., 38:133-165), cloned embryonic stem (ntES) cells obtained by nuclear transfer (T. Wakayama et al. (2001), Science, 292:740-743; S. Wakayama et al. (2002), Science, 292:740-743), and cloned embryonic stem (ntES) cells (T. Wakayama et al. (2002), Science, 292:740-743). (2005), Biol. Reprod., 72:932-936; J. Byrne et al. (2007), Nature, 450:497-502), sperm stem cells (“GS cells”) (M. Kanatsu-Shinohara et al. (2003) Biol. Reprod., 69:612-616; K. Shinohara et al. (2004), Cell, 119:1001-1012), embryonic germ cells (“EG cells”) (Y. Matsui et al. (1992), Cell, 70:841-847; JL Resnick et al. (1992), Nature, 359:550-551), induced pluripotent stem (iPS) cells (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, Nat. Biotechnol.26:101-106 (2008); WO2007 / 069666), and pluripotent cells derived from cultured fibroblasts or bone marrow stem cells (Muse cells) (WO2011 / 007900). The animals from which pluripotent stem cells can be derived are not limited, and examples include mammals such as mice, rats, hamsters, guinea pigs, cows, horses, pigs, sheep, monkeys, orangutans, chimpanzees, dogs, cats, birds, and humans. Primates are preferred, and humans are more preferred. Pluripotent stem cells can also be ES cells or iPS cells, such as iPS cells.

[0036] iPS cells can be produced by introducing specific reprogramming factors into somatic cells in the form of DNA or protein (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., Nat. Biotechnol. 26:101-106 (2008); WO2007 / 069666). When iPS cells are used, the iPS cells may be produced from somatic cells by methods known per se, or iPS cells that have already been established and stored may be used. There are no limitations on the somatic cells from which the iPS cells used in the present invention are derived; for example, cells derived from peripheral blood or umbilical cord blood may be used.

[0037] In the present application, the medium can be prepared by appropriately adding necessary factors to a basal medium used for culturing animal cells. Examples of basal media include MEM Zinc Option medium, IMEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, DMEM / F12 medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof. The basal 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) (Thermo Fisher Scientific), N2 supplement (Thermo Fisher Scientific), B27 supplement (Thermo Fisher Scientific), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, etc., or one or more substances, such as lipids, amino acids, L-glutamine, GlutaMAX (Thermo Fisher Scientific), non-essential amino acids (NEAA), vitamins, growth factors, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, and the like, or one or more substances typically added to animal culture media. The basal medium used herein may be, for example, DMEM / F12 medium.

[0038] In one embodiment, ureteric bud cells or cells differentiated from ureteric bud cells are cultured in the presence of an ETS family protein. Examples of ETS family proteins include ELF (ELF1, ELF2, ELF3, ELF4, and ELF5), EHF, GABPA, ERG, FLI1, FEV, ERF, ELK1, ELK3, ELK4, ETS1, ETS2, ETV1, ETV2, ETV3, ETV4, ETV5, ETV6, ETV7, SPDEF, SPI1, SPIB, and SPIC. In the present application, the ETS family gene can be appropriately selected from these known genes, such as ELF, and preferably ELF5. Examples of ELF5 include proteins comprising the amino acid sequence shown in NCBI Reference Sequence: NP_001230009.1, NP_001230010.1, NP_001413.1, NP_938195.1, XP_016872797.1, or XP_016872798.1. In one embodiment, as long as renal collecting duct principal cells can be produced, ELF5 proteins comprising an amino acid sequence in which one or more amino acids have been substituted, deleted, added, and / or inserted in the amino acid sequence shown in NCBI Reference Sequence: NP_001230009.1, NP_001230010.1, NP_001413.1, NP_938195.1, XP_016872797.1, or XP_016872798.1 may be used. "One or more" in this embodiment preferably means 1 to 20, more preferably 1 to 15, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.In other embodiments, as long as renal collecting duct principal cells can be produced, a protein containing an amino acid sequence that has about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the amino acid sequence shown in NCBI Reference Sequence: NP_001230009.1, NP_001230010.1, NP_001413.1, NP_938195.1, XP_016872797.1, or XP_016872798.1 may be used as ELF5.

[0039] The above-mentioned amino acid substitutions may be conservative amino acid substitutions or non-conservative amino acid substitutions.In this specification, conservative amino acid substitutions refer to those that are generally accepted within the range of substitutions that can be made without changing the physiological activity of the resulting molecule, i.e., those that are recognized within the range of conservative substitutions (Watson et al., Molecular Biology of the Gene, etc.).For example, substitutions that occur between amino acids with similar side chains, such as aspartic acid and glutamic acid (acidic amino acids); lysine, arginine and histidine (basic amino acids); alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine and tryptophan (non-polar amino acids); glycine, asparagine, glutamine, cysteine, serine, threonine and tyrosine (uncharged polar amino acids); phenylalanine, tryptophan and tyrosine (aromatic amino acids), can be mentioned. Similarly, they can be classified as glycine, alanine, valine, leucine, isoleucine, serine and threonine (aliphatic amino acids); serine and threonine (aliphatic-hydroxy amino acids); asparagine and glutamine (amide-type amino acids); cysteine ​​and methionine (sulfur-containing amino acids).

[0040] As used herein, "sequence identity" refers to the percentage of identical bases or amino acids at corresponding positions in two or more sequences (nucleotide sequences or amino acid sequences) when the sequences are aligned, taking into account gaps and insertions, to maximize sequence identity. Methods for determining identity are designed to maximize identity between the aligned sequences. Methods for determining identity between two sequences include, but are not limited to, BLASTP, BLASTN, FASTA, etc. Identity between two sequences can also be determined using DNASIS (Hitachi Software Engineering Co., Ltd.) or GENETYX (Genetyx Inc.). Alternatively, for short peptides, identity can be determined simply by comparing the sequences. Those skilled in the art can determine identity between sequences using the methods described above.

[0041] The signal peptide sequence of the ETS family protein used in the present application may be replaced with any other signal peptide sequence. The ETS family protein used in the present application may have a tag sequence such as a FLAG tag, an HA tag, a His tag, a Myc tag, or a V5 tag added. The ETS family protein used in the present application may be a human-derived protein or a protein (i.e., an ortholog) derived from another mammal (e.g., mouse, rat, cow, horse, pig, sheep, monkey, dog, cat, bird, etc.).

[0042] The ETS family protein may be added to the culture medium, or ureteric bud cells or cells differentiated from ureteric bud cells may express the ETS family protein. When the ETS family protein is added to the culture medium, the concentration of the ETS family protein can be appropriately selected by those skilled in the art depending on the ETS family protein used. When the ETS family protein is ELF5, its concentration is, for example, 1 nM to 1 mM.

[0043] In certain embodiments, ureteric bud cells or cells differentiated from ureteric bud cells express an ETS family protein. In the present application, ureteric bud cells or cells differentiated from ureteric bud cells that express an ETS family protein may express an ETS family protein, such as a cell into which an ETS family gene has been introduced. Gene introduction can be performed using known methods, including infection methods using viral vectors (e.g., retroviral vectors, lentiviral vectors, Sendai virus vectors, adenoviral vectors, or adeno-associated viral vectors), gene introduction methods using plasmid vectors (e.g., plasmid vectors, transposon vectors, or episomal vectors) (e.g., calcium phosphate transfer, lipofection, retronectin transfer, or electroporation), gene introduction methods using RNA vectors (e.g., calcium phosphate transfer, lipofection, or electroporation), and direct protein injection methods (e.g., needle transfer, lipofection, or electroporation). Preferably, the gene is introduced using a method that does not integrate into the cell's genome or that allows for easy removal from the genome. For this purpose, it is preferable to use an episomal vector or a PiggyBac (trademark) vector, which is a transposon vector.

[0044] In the present application, the ETS family gene is not particularly limited as long as it is a gene encoding the above-mentioned ETS family protein, and examples thereof include the ELF5 gene. Examples of the nucleotide sequence encoding the ELF5 gene include the nucleotide sequences of NCBI Reference Sequence: NM_001243080.2, NM_001243081.2, NM_001422.4, NM_198381.2, XM_017017308.1, and XM_017017309.1.

[0045] In one embodiment, ureteric bud cells or cells differentiated from ureteric bud cells that express an ETS family protein can transiently express the ETS family protein. Transient expression of an ETS family protein can be achieved using a system that can control the on / off state of gene expression. Examples of such systems include systems in which expression is reversibly controlled by the presence or absence of an inducer. Examples include the tetracycline (Tet) expression induction system, the Cumate repressor protein CymR system, and the coumermycin / novobiocin regulatory system.

[0046] Examples of tetracycline (Tet) expression induction systems include the Tet-On® / Tet-Off® Gene Expression System (Clontech). The Tet-On® system uses a Tet-On® regulatory plasmid expressing a reverse tetracycline-controlled transactivator (rtTA) and a plasmid encoding a tetracycline response element (TRE) with a tetO repeat sequence. rtTA is a fusion protein composed of a mutant Tet repressor protein (rTetR) and a VP16 activation domain (AD). Adding Dox (doxycycline) to the culture medium binds to the tetracycline response element (TRE) and induces downstream gene expression. The Tet-Off® system induces gene expression in the absence of Dox.

[0047] The Cumate repressor protein CymR system is a system that uses a lentiviral vector to reversibly induce the expression of a target gene. Expression can be induced by introducing a target gene expression vector and a CymR repressor expression vector into cells and adding the expression inducer Cumate. The SparQ Cumate Switch Inducible System (System Biosciences) can be used as the Cumate repressor protein CymR system.

[0048] As a coumermycin / novobiocin regulatory system, the Regulated Mammalian Expression System (Promega) can be used.

[0049] Among the systems capable of controlling the on / off of gene expression, the tetracycline (Tet) expression induction system can be preferably used. In one embodiment, transient expression of an ETS family protein can be controlled by the tetracycline expression induction system, and the medium further contains doxycycline. Doxycycline commercially available from, for example, LKT Labs, can be used. In this embodiment, the concentration of doxycycline can be 4 nM to 400 μmM, 40 nM to 400 μM, or 400 nM to 40 μM, for example, approximately 4 μM.

[0050] The introduced ETS family gene can be removed from the cells when its expression is no longer required. When an episomal vector is used, the gene transfer vector disappears naturally after long-term culture. When a PiggyBac vector is used, the gene once integrated into the genome can be removed using PiggyBac™ transposase.

[0051] In one embodiment, the ureteric bud cells may be derived from pluripotent stem cells that express an ETS family protein. The pluripotent stem cells that express an ETS family protein may be any cells that express an ETS family protein, such as pluripotent stem cells into which an ETS family gene has been introduced. The means for introducing the gene are as described above.

[0052] In one embodiment, pluripotent stem cells expressing an ETS family protein can transiently express the ETS family protein. The transient expression of the ETS family protein can be achieved using the methods described above. In particular, the tetracycline (Tet) gene expression induction system can be suitably used. For example, the transient expression of the ETS family protein can be controlled by the tetracycline expression induction system.

[0053] The "TGFβ inhibitor" is a substance that inhibits signal transduction that continues from binding to the receptor to SMAD, and is not particularly limited as long as it is a substance that inhibits binding to the receptor, ALK family, or a substance that inhibits phosphorylation of SMAD by the ALK family. Examples of such a substance include Lefty-1 (NCBI Accession No.: mouse: NM_010094, human: NM_020997), SB431542, SB202190 (RK Lindemann et al., Mol. Cancer, 2003, 2:20), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276 (Lilly Research Examples include A83-01 (3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide, WO2009146408), ALK5 inhibitor II (2-[3-[6-methylpyridin-2-yl]-1H-pyrazol-4-yl]-1,5-naphthyridine), TGFβRI kinase inhibitor VIII (6-[2-tert-butyl-5-[6-methyl-pyridin-2-yl]-1H-imidazol-4-yl]-quinoxaline), and derivatives thereof. The TGFβ inhibitor used in the present application may be, for example, A83-01. In this embodiment, the concentration of the TGFβ inhibitor can be appropriately selected by those skilled in the art depending on the TGFβ inhibitor used. When the TGFβ inhibitor is A83-01, its concentration can be 1 nM to 100 μM, 10 nM to 100 μM, or 100 nM to 10 μM, for example, about 1 μM.

[0054] The "Wnt inhibitor" is not particularly limited as long as it is a substance that inhibits the Wnt-mediated signaling pathway. Examples include IWR-1, IWP-2, IWP-3, IWP-4, 2-(4-trifluoromethylphenyl)-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-4(3H)-one (XAV939), G-CSF, IGFBP4, Dkk1, Cerberus, anti-Wnt antibodies, Wnt antagonists (Wnt receptor inhibitors), soluble Wnt receptor proteins (e.g., Frzb-1), and dominant negative forms. The Wnt inhibitor used in the present application may be, for example, IWR-1. In this embodiment, the concentration of the Wnt inhibitor can be appropriately selected by those skilled in the art depending on the Wnt inhibitor used. When the Wnt inhibitor is IWR-1, its concentration can be 1 nM to 100 μM, 10 nM to 100 μM, or 100 nM to 10 μM, for example, about 1 μM.

[0055] The term "phosphodiesterase (PDE) inhibitor" is not particularly limited as long as it is a substance that inhibits enzymes that hydrolyze cyclic phosphate diesters such as cAMP and cGMP, and examples thereof include IBMX (3-isobutyl-1-methylxanthine), caffeine, theophylline, theobromine, pentoxifylline, 8-methoxy-3-isobutyl-1-methylxanthine, erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride (EHNA hydrochloride), cilostamide, milrinone, tolexin, etazolate hydrochloride, denbufylline, vardenafil, sildenafil, zaprinast, tadalafil, dipyridamole, 4-{[3',4'-(methylenedioxy)benzyl]amino}-6-methoxyquinazoline, 1-(3-chloroanilino)-4-phenylphthalazine (MY-5445), and derivatives thereof. The PDE inhibitor used in the present application may be, for example, IBMX. The concentration of the PDE inhibitor can be appropriately selected by those skilled in the art depending on the PDE inhibitor used. When the PDE inhibitor is IBMX, the concentration may be 400 nM to 40 mM, 4 μM to 40 mM, or 40 μM to 4 mM, for example, about 400 μM.

[0056] In some embodiments, the medium may further comprise a retinoic acid receptor agonist. A "retinoic acid receptor (RAR) agonist" may be a naturally occurring retinoid, a chemically synthesized retinoid, a retinoic acid receptor agonist compound without a retinoid skeleton, or a natural product with retinoic acid receptor agonist activity. An example of a naturally occurring retinoid with RAR agonist activity is retinoic acid, the stereoisomers of which are all-trans-retinoic acid (all-trans-RA) and 9-cis-retinoic acid (9-cis-RA). Chemically synthesized retinoids are known in the art (e.g., U.S. Pat. No. 5,234,926; U.S. Pat. No. 4,326,055). Examples of retinoic acid receptor agonist compounds without a retinoid skeleton include Am80, AM580 (4-[[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl]carboxamido]benzoic acid), TTNPB (4-[[E]-2-[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl]-1-propenyl]benzoic acid), and AC55649 (4'-octyl-[1,1'-biphenyl]-4-carboxylic acid). Examples of natural products with retinoic acid receptor agonist activity include honokiol and magnolol (Bulletin of the Institute for Biological Function Research 9:55-61, 2009). The RAR agonist used in the present application may be retinoic acid, AM580, TTNPB, or AC55649, for example, TTNPB. In this embodiment, the concentration of the retinoic acid receptor agonist can be appropriately selected by those skilled in the art depending on the retinoic acid receptor agonist used. When the retinoic acid receptor agonist is TTNPB, its concentration may be 0.1 nM to 10 μM, 1 nM to 10 μM, or 10 nM to 1 μM, for example, about 0.1 μM.

[0057] In certain embodiments, the medium may further contain a vasopressin receptor agonist. Examples of vasopressin receptor agonists include, but are not limited to, vasopressin (arginine vasopressin; AVP), desmopressin (dDAVP; 1-desamino-8-D-arginine vasopressin), lypressin, terlipressin, ornipressin, and felypressin. The vasopressin receptor agonist used herein may be vasopressin or desmopressin, such as desmopressin. Commercially available desmopressin, for example, from Sigma, can be used. In this embodiment, the concentration of the vasopressin receptor agonist can be appropriately selected by those skilled in the art depending on the vasopressin receptor agonist used. When the vasopressin receptor agonist is desmopressin, its concentration may be 0.1 nM to 10 μM, 1 nM to 10 μM, or 10 nM to 1 μM, for example, approximately 0.1 μM.

[0058] In one embodiment, the medium may further contain aldosterone. Commercially available aldosterone from, for example, Sigma can be used. In this embodiment, the concentration of aldosterone may be 0.01 nM to 1 μM, 0.1 nM to 1 μM, or 1 nM to 0.1 μM, for example, about 10 nM.

[0059] In certain embodiments, the medium may further contain a calcium calmodulin kinase (CaMK) inhibitor. Examples of CaMK inhibitors include K252a, KN-93, KN-62, AIP, CaM kinase II inhibitor 281-301, lavenderstin C, rottererin, ML-7, ML-9, STO-609, W-7, and W-5. The CaMK inhibitor used in the present application may be, for example, K252a. In this embodiment, the concentration of the CaMK inhibitor can be appropriately selected by those skilled in the art depending on the CaMK inhibitor used. When the CaMK inhibitor is K252a, its concentration may be 0.1 nM to 10 μM, 1 nM to 10 μM, or 10 nM to 1 μM, for example, about 0.1 μM.

[0060] In certain embodiments, ureteric bud cells or cells differentiated from ureteric bud cells can be cultured at an air-liquid interface. Air-liquid interface culture can be performed, for example, by culturing cells on a porous membrane located at the interface between the air and liquid phases. For example, cells can be seeded on the porous membrane of a cell culture insert, with the upper side in the air phase and medium supplied from the lower side through the membrane. Commercially available products such as Transwell (registered trademark) can also be used. Air-liquid interface culture can produce ureteric bud chief cells with maintained polarity.

[0061] In another embodiment, ureteric bud cells or cells differentiated from ureteric bud cells can be cultured in adherent culture. As used herein, "adherent culture" refers to culturing cells in a state where the cells are attached to a culture substrate, such as in a coated culture vessel. Examples of coating agents include Matrigel (BD), Synthemax (Corning), collagen, gelatin, laminin (laminin-511, laminin-111, laminin-411, etc.), heparan sulfate proteoglycan, entactin, fragments thereof, and combinations thereof. In this application, ureteric bud cells can be cultured on cell culture plates coated with extracellular matrix proteins, such as laminin. Commercially available coating agents, such as iMatrix-511 silk, can be used.

[0062] In certain embodiments, the culture period in this step may be 5 to 50 days, 7 to 30 days, or 10 to 21 days, for example, about 14 days. In another embodiment, when ureteric bud cells or cells differentiated from ureteric bud cells are cultured in the presence of an ETS family protein, the culture period in this step may be 1 to 30 days, 2 to 14 days, or 3 to 10 days, for example, about 4 to about 6 days.

[0063] The culture temperature is, but is not limited to, about 30 to 40°C, for example, about 37°C, and the culture is carried out in an atmosphere of CO2-containing air, with a CO2 concentration of, for example, about 2 to 5%.

[0064] In one embodiment, the method of the present application includes, prior to the above step, a step of culturing ureteric bud cells in a medium containing a TGFβ inhibitor and a Wnt inhibitor. That is, in one embodiment, the method of the present application includes the steps of: (A) culturing ureteric bud cells in a medium containing a TGFβ inhibitor and a Wnt inhibitor; and (B) culturing the ureteric bud cells obtained in (A) or cells differentiated from the ureteric bud cells in a medium containing a TGFβ inhibitor, a Wnt inhibitor, and a phosphodiesterase inhibitor. Here, step (B) corresponds to the above step. In step (A), the ureteric bud cells can be cultured as adherent cells.

[0065] In step (A), the TGFβ inhibitor may be any of the above-mentioned TGFβ inhibitors. The concentration of the TGFβ inhibitor can be appropriately selected by those skilled in the art depending on the TGFβ inhibitor used. When the TGFβ inhibitor is A83-01, its concentration may be 1 nM to 100 μM, 10 nM to 100 μM, or 100 nM to 10 μM, for example, about 1 μM.

[0066] In step (A), the Wnt inhibitor may be any of the above-mentioned Wnt inhibitors. The concentration of the Wnt inhibitor can be appropriately selected by those skilled in the art depending on the Wnt inhibitor used. When the Wnt inhibitor is IWR-1, its concentration may be 1 nM to 100 μM, 10 nM to 100 μM, or 100 nM to 10 μM, for example, about 1 μM.

[0067] In step (A), the medium may further contain a retinoic acid receptor agonist. The above-mentioned retinoic acid receptor agonists can be used as the retinoic acid receptor agonist. The concentration of the retinoic acid receptor agonist can be appropriately selected by those skilled in the art depending on the retinoic acid receptor agonist used. When the retinoic acid receptor agonist is TTNPB, its concentration can be 0.1 nM to 10 μM, 1 nM to 10 μM, or 10 nM to 1 μM, for example, about 0.1 μM.

[0068] The culture period in step (A) is not particularly limited, but is, for example, 1 to 30 days. In one embodiment, the culture period in step (A) may be 1 to 10 days, 1 to 7 days, or 1 to 5 days, for example, about 2 to about 3 days. In another embodiment, the culture period in step (A) may be 2 to 21 days, 3 to 14 days, or 5 to 10 days, for example, about 7 days.

[0069] In step (B), the cells differentiated from ureteric bud cells may be any cells that arise before differentiating into renal collecting duct chief cells, such as renal collecting duct progenitor cells. When the culture period in step (A) is 2 to 21 days, 3 to 14 days, 5 to 10 days, or about 7 days, the cells differentiated from ureteric bud cells may be renal collecting duct progenitor cells.

[0070] The culture temperature in step (A) is, but is not limited to, about 30 to 40°C, for example, about 37°C, and the culture is carried out in an atmosphere of CO2-containing air, with a CO2 concentration of, for example, about 2 to 5%.

[0071] The present application also provides a cell population containing 40% or more renal collecting duct chief cells. A cell population containing 40% or more renal collecting duct chief cells can be produced, for example, by the method of the present application. In certain embodiments, a cell population containing 40% or more renal collecting duct chief cells contains 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more renal collecting duct chief cells. The presence of renal collecting duct chief cells in the cell population of the present application can be confirmed by the expression of the above-mentioned renal collecting duct chief cell markers. The presence of 40% or more renal collecting duct chief cells in the cell population of the present application can also be confirmed using flow cytometry, for example, fluorescence-activated cell sorting (FACS).

[0072] In this embodiment, the animal from which the renal collecting duct principal cells are derived is not limited, and examples include mammals such as mice, rats, hamsters, guinea pigs, cows, horses, pigs, sheep, monkeys, orangutans, chimpanzees, dogs, cats, birds, and humans, with primates being preferred, and humans being more preferred.

[0073] Method for producing renal collecting duct chief cell-containing organoids The present application also provides a method for producing renal collecting duct chief cell-containing organoids, comprising the step of culturing ureteric bud organoids or organoids differentiated from ureteric bud organoids in a medium containing a TGFβ inhibitor, a Wnt inhibitor, and a phosphodiesterase inhibitor.

[0074] As used herein, "renal collecting duct chief cell-containing organoids" refers to self-organized structures containing renal collecting duct chief cells. The size of renal collecting duct chief cell-containing organoids ranges, for example, from about 10 to about 1,000 μm. In certain embodiments, renal collecting duct chief cell-containing organoids contain renal collecting duct chief cells at 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more. The presence of renal collecting duct chief cell-containing organoids in renal collecting duct chief cell-containing organoids can be confirmed by the expression of renal collecting duct chief cell markers such as AQP2, AQP3, AQP4, and AVPR2. The expression of renal collecting duct chief cell markers can be confirmed visually under a microscope by immunostaining, or by using flow cytometry or FACS (fluorescence-activated cell sorting).

[0075] In the present application, the animal from which ureteric bud organoids are derived is not limited, and examples include mammals such as mice, rats, hamsters, guinea pigs, cows, horses, pigs, sheep, monkeys, orangutans, chimpanzees, dogs, cats, birds, and humans. Primates are preferred, and humans are more preferred. The size of ureteric bud organoids ranges from about 10 to about 1,000 μm. Identification of ureteric bud organoids can be confirmed by the expression of markers such as GATA3, RET, WNT11, LHX1, SOX9, and very low-density lipoprotein receptor (VLDL-R), or by the uptake of very low-density lipoprotein (VLDL). In one embodiment, ureteric bud organoids are cultured in a medium containing a TGFβ inhibitor, a Wnt inhibitor, and a phosphodiesterase inhibitor.

[0076] Organoids differentiated from ureteric bud organoids can be any organoid that develops before differentiating into renal collecting duct principal cell-containing organoids, such as renal collecting duct progenitor cell-containing organoids. Renal collecting duct progenitor cell-containing organoids refer to self-organized structures containing renal collecting duct progenitor cells. The size of renal collecting duct progenitor cell-containing organoids is, for example, about 10 to about 1000 μm. The identity of renal collecting duct progenitor cell-containing organoids can be confirmed by the expression of markers such as AQP2. In one embodiment, renal collecting duct principal cell-containing organoids differentiated from ureteric bud organoids are cultured in a medium containing a TGFβ inhibitor, a Wnt inhibitor, and a phosphodiesterase inhibitor.

[0077] Ureteric bud organoid may be differentiated from pluripotent stem cells.The method of inducing differentiation of ureteric bud organoid from pluripotent stem cells is known, and any known method can be used.For example, the above-mentioned method can be used.In some embodiments, pluripotent stem cells are iPS cells.

[0078] In one embodiment, ureteric bud organoids or organoids differentiated from ureteric bud organoids are cultured in the presence of an ETS family protein. In this embodiment, the ETS family protein may be added to the culture medium, and cells contained in the ureteric bud organoids or organoids differentiated from ureteric bud organoids may express the ETS family protein. The ETS family protein may be any of the above-mentioned ETS family proteins. When an ETS family protein is added to the culture medium, the concentration of the ETS family protein can be appropriately selected by those skilled in the art depending on the ETS family protein used. When the ETS family protein is ELF5, its concentration is, for example, 1 nM to 1 mM.

[0079] In one embodiment, the cell contained in ureteric bud organoid or the organoid differentiated from ureteric bud organoid expresses ETS family protein.In the present application, the cell that expresses ETS family protein can be just as long as it expresses ETS family protein, for example, it is the cell that ETS family gene is introduced into.The means of gene introduction is as mentioned above.

[0080] In one embodiment, cells expressing an ETS family protein can transiently express the ETS family protein. The transient expression of the ETS family protein can be achieved using the methods described above. In particular, the tetracycline (Tet) gene expression induction system can be suitably used. In one embodiment, the transient expression of the ETS family protein can be controlled by the tetracycline expression induction system, and the medium further contains doxycycline. In this embodiment, the concentration of doxycycline can be 4 nM to 400 μmM, 40 nM to 400 μM, or 400 nM to 40 μM, e.g., approximately 4 μM.

[0081] The introduced ETS family gene can be removed from the cells when its expression is no longer required. When an episomal vector is used, the gene transfer vector disappears naturally after long-term culture. When a PiggyBac vector is used, the gene once integrated into the genome can be removed using PiggyBac™ transposase.

[0082] In some embodiments, ureteric bud organoid can be derived from the pluripotent stem cell that expresses ETS family protein.The pluripotent stem cell that expresses ETS family protein can be as long as it expresses ETS family protein, for example, it is the pluripotent stem cell that introduces ETS family gene.The means of introducing gene is as mentioned above.

[0083] In one embodiment, pluripotent stem cells expressing an ETS family protein can transiently express the ETS family protein. The transient expression of the ETS family protein can be achieved using the methods described above. In particular, the tetracycline (Tet) gene expression induction system can be suitably used. For example, the transient expression of the ETS family protein can be controlled by the tetracycline expression induction system.

[0084] In this embodiment, the TGFβ inhibitor may be any of the above-mentioned TGFβ inhibitors. The concentration of the TGFβ inhibitor can be appropriately selected by those skilled in the art depending on the TGFβ inhibitor used. When the TGFβ inhibitor is A83-01, its concentration may be 1 nM to 100 μM, 10 nM to 100 μM, or 100 nM to 10 μM, for example, about 1 μM.

[0085] In this embodiment, the Wnt inhibitor may be any of the above-described Wnt inhibitors. The concentration of the Wnt inhibitor can be appropriately selected by those skilled in the art depending on the Wnt inhibitor used. When the Wnt inhibitor is IWR-1, its concentration may be 1 nM to 100 μM, 10 nM to 100 μM, or 100 nM to 10 μM, for example, about 1 μM.

[0086] In this embodiment, the PDE inhibitor may be any of the above-described PDE inhibitors. The concentration of the PDE inhibitor may be appropriately selected by those skilled in the art depending on the PDE inhibitor used. When the PDE inhibitor is IBMX, the concentration may be 400 nM to 40 mM, 4 μM to 40 mM, or 40 μM to 4 mM, for example, about 400 μM.

[0087] In certain embodiments, the medium may further contain a retinoic acid receptor agonist. The retinoic acid receptor agonists described above can be used as the retinoic acid receptor agonist. The concentration of the retinoic acid receptor agonist can be appropriately selected by those skilled in the art depending on the retinoic acid receptor agonist used. When the retinoic acid receptor agonist is TTNPB, its concentration can be 0.1 nM to 10 μM, 1 nM to 10 μM, or 10 nM to 1 μM, for example, about 0.1 μM.

[0088] In some embodiments, the medium may further contain a vasopressin receptor agonist. The above-mentioned vasopressin receptor agonists can be used as the vasopressin receptor agonist. The concentration of the vasopressin receptor agonist can be appropriately selected by those skilled in the art depending on the vasopressin receptor agonist used. When the vasopressin receptor agonist is desmopressin, its concentration can be 0.1 nM to 10 μM, 1 nM to 10 μM, or 10 nM to 1 μM, for example, about 0.1 μM.

[0089] In some embodiments, the medium may further contain aldosterone, the concentration of which may be 0.01 nM to 1 μM, 0.1 nM to 1 μM, or 1 nM to 0.1 μM, for example, about 10 nM.

[0090] In certain embodiments, the medium may further contain a CaMK inhibitor. The CaMK inhibitors described above can be used. The concentration of the CaMK inhibitor can be appropriately selected by those skilled in the art depending on the CaMK inhibitor used. When the CaMK inhibitor is K252a, its concentration can be 0.1 nM to 10 μM, 1 nM to 10 μM, or 10 nM to 1 μM, for example, about 0.1 μM.

[0091] In certain embodiments, the culture period in this step may be 5 to 50 days, 7 to 30 days, or 10 to 21 days, for example, about 14 days. In another embodiment, when ureteric bud organoids or organoids differentiated from ureteric bud organoids are cultured in the presence of an ETS family protein, the culture period in this step may be 1 to 30 days, 2 to 14 days, or 4 to 8 days, for example, about 6 days.

[0092] In this embodiment, ureteric bud organoids or organoids differentiated from ureteric bud organoids can be cultured in suspension. In this application, "suspension culture" refers to culturing cells in a non-adherent state on a culture dish. While not particularly limited, culture can be performed using cultures that have not been artificially treated to improve cell adhesion (e.g., coated with extracellular matrix, etc.), or that have been artificially treated to suppress adhesion (e.g., coated with polyhydroxyethyl methacrylate (poly-HEMA) or 2-methacryloyloxyethyl phosphorylcholine polymer (Lipidure)). For example, commercially available products such as 96-well low-attachment plates (Sumitomo Bakelite) and 35 mm low-attachment dishes (Sumitomo Bakelite) can be used.

[0093] The culture temperature is, but is not limited to, about 30 to 40°C, for example, about 37°C, and the culture is carried out in an atmosphere of CO2-containing air, with a CO2 concentration of, for example, about 2 to 5%.

[0094] In one embodiment, the method of the present application comprises, before the above step, culturing the ureteric bud organoid in a medium containing a TGFβ inhibitor and a Wnt inhibitor. That is, in one embodiment, the method of the present application comprises: (A) culturing the ureteric bud organoid in a medium containing a TGFβ inhibitor and a Wnt inhibitor; and (B) culturing the ureteric bud organoid obtained in (A) or the organoid differentiated from the ureteric bud organoid in a medium containing a TGFβ inhibitor, a Wnt inhibitor and a phosphodiesterase inhibitor. Here, step (B) corresponds to the above step. In step (A), the ureteric bud organoid can be cultured in suspension.

[0095] In step (A), the TGFβ inhibitor may be any of the above-mentioned TGFβ inhibitors. The concentration of the TGFβ inhibitor can be appropriately selected by those skilled in the art depending on the TGFβ inhibitor used. When the TGFβ inhibitor is A83-01, its concentration may be 1 nM to 100 μM, 10 nM to 100 μM, or 100 nM to 10 μM, for example, about 1 μM.

[0096] In step (A), the Wnt inhibitor may be any of the above-mentioned Wnt inhibitors. The concentration of the Wnt inhibitor can be appropriately selected by those skilled in the art depending on the Wnt inhibitor used. When the Wnt inhibitor is IWR-1, its concentration may be 1 nM to 100 μM, 10 nM to 100 μM, or 100 nM to 10 μM, for example, about 1 μM.

[0097] In step (A), the medium may further contain a retinoic acid receptor agonist. The above-mentioned retinoic acid receptor agonists can be used as the retinoic acid receptor agonist. The concentration of the retinoic acid receptor agonist can be appropriately selected by those skilled in the art depending on the retinoic acid receptor agonist used. When the retinoic acid receptor agonist is TTNPB, its concentration can be 0.1 nM to 10 μM, 1 nM to 10 μM, or 10 nM to 1 μM, for example, about 0.1 μM.

[0098] The culture period in step (A) is not particularly limited, but is, for example, 1 to 50 days. In one embodiment, the culture period in step (A) may be 1 to 10 days, 1 to 7 days, or 2 to 4 days, for example, about 3 days. In another embodiment, the culture period in step (A) may be 5 to 40 days, 7 to 30 days, or 10 to 20 days, for example, about 14 days.

[0099] In step (B), the organoids differentiated from the ureteric bud organoids may be any organoids that arise before differentiating into renal collecting duct chief cell-containing organoids, for example, renal collecting duct progenitor cell-containing organoids. When the culture period in step (A) is 5 to 40 days, 7 to 30 days, 10 to 20 days, or about 14 days, the organoids differentiated from the ureteric bud organoids may be renal collecting duct progenitor cell-containing organoids.

[0100] The culture temperature in step (A) is, but is not limited to, about 30 to 40°C, for example, about 37°C, and the culture is carried out in an atmosphere of CO2-containing air, with a CO2 concentration of, for example, about 2 to 5%.

[0101] Renal collecting duct chief cell-containing organoids The present application also provides renal collecting duct chief cell-containing organoids containing 40% or more renal collecting duct chief cells. Renal collecting duct chief cell-containing organoids containing 40% or more renal collecting duct chief cells can be produced, for example, by the method of the present application. In some embodiments, renal collecting duct chief cell-containing organoids containing 40% or more renal collecting duct chief cells contain 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more renal collecting duct chief cells. The presence of renal collecting duct chief cell-containing organoids of the present application in renal collecting duct chief cell can be confirmed by the expression of the above-mentioned renal collecting duct chief cell markers. The presence of 40% or more renal collecting duct chief cells in renal collecting duct chief cell-containing organoids of the present application can be confirmed by flow cytometry, for example, FACS (fluorescence-activated cell sorting).

[0102] In this embodiment, the animal from which the renal collecting duct principal cells are derived is not limited, and examples include mammals such as mice, rats, hamsters, guinea pigs, cows, horses, pigs, sheep, monkeys, orangutans, chimpanzees, dogs, cats, birds, and humans, with primates being preferred, and humans being more preferred.

[0103] Autosomal dominant polycystic kidney disease (ADPKD)-specific ureteric bud cells expressing ETS family proteins The present application also provides autosomal dominant polycystic kidney disease (ADPKD)-specific ureteric bud cells expressing ETS family proteins.

[0104] Autosomal dominant polycystic kidney disease (ADPKD)-specific cells refer to cells derived from an ADPKD patient or cells with an ADPKD-associated gene mutation. An ADPKD-associated gene mutation refers to a mutation that can directly or indirectly cause ADPKD by causing an ADPKD-associated gene to malfunction. Examples of ADPKD-associated genes include the PKD1 and PKD2 genes, which are causative genes of ADPKD, and genes related to them, such as the PKD1 gene.

[0105] In one embodiment, cells having a genetic mutation associated with ADPKD are cells in which an ADPKD-associated gene has been knocked out or knocked down. Methods for knocking out or knocking down genes are known and are not particularly limited. Examples of gene knockout methods include homologous recombination technology and genome editing techniques using the CRISPR system, TALEN, ZFN, etc. Examples of gene knockdown methods include the antisense method, in which RNA corresponding to the antisense strand of mRNA is introduced into cells, and the RNAi method, which uses siRNA, shRNA, microRNA, etc.

[0106] The ADPKD-specific ureteric bud cells are ureteric bud cells derived from an ADPKD patient or ureteric bud cells having a genetic mutation associated with ADPKD. In one embodiment, the ADPKD-specific ureteric bud cells are ureteric bud cells in which the PKD1 gene has been knocked out. The ureteric bud cells having a genetic mutation associated with ADPKD may be produced by genetically modifying ureteric bud cells or may be derived from cells having a genetic mutation associated with ADPKD.

[0107] ADPKD-specific ureteric bud cells can also be differentiated from pluripotent stem cells.For example, ureteric bud cells can be obtained by dissociating ureteric bud organoids differentiated from pluripotent stem cells into single cells.The method of inducing ureteric bud organoids differentiation from pluripotent stem cells is known, and any known method can be used.For example, the above-mentioned method can be used.In one embodiment, pluripotent stem cells can be ADPKD-specific pluripotent stem cells, for example, pluripotent stem cells that have PKD1 gene knocked out.In one embodiment, pluripotent stem cells are iPS cells.

[0108] In this embodiment, the autosomal dominant polycystic kidney disease (ADPKD)-specific ureteric bud cells expressing an ETS family protein may be any cells that express an ETS family protein, such as ADPKD-specific ureteric bud cells into which an ETS family protein gene has been introduced. The gene introduction method is as described above. The ETS family protein may be any of the ETS family proteins described above.

[0109] In some embodiments, autosomal dominant polycystic kidney disease (ADPKD)-specific ureteric bud cells expressing an ETS family protein can transiently express the ETS family protein. Transient expression of the ETS family protein can be achieved using the methods described above. In particular, the tetracycline (Tet) gene expression induction system can be suitably used.

[0110] Autosomal dominant polycystic kidney disease (ADPKD)-specific ureteric bud organoids expressing ETS family proteins The present application also provides autosomal dominant polycystic kidney disease (ADPKD)-specific ureteric bud organoids expressing ETS family proteins.

[0111] ADPKD specific ureteric bud organoid is the ureteric bud organoid from ADPKD patients, or the ureteric bud organoid has the gene mutation associated with ADPKD.In some embodiments, ADPKD specific ureteric bud organoid is the ureteric bud organoid that PKD1 gene is knocked out.The ureteric bud organoid that has the gene mutation associated with ADPKD can be produced by genetically modifying ureteric bud organoid, or can be derived from the cell that has the gene mutation associated with ADPKD.

[0112] ADPKD-specific ureteric bud organoid can be differentiated from pluripotent stem cells.The method of inducing differentiation of ureteric bud organoid from pluripotent stem cells is known, and any known method can be used.For example, the above-mentioned method can be used.In some embodiments, pluripotent stem cells can be ADPKD-specific pluripotent stem cells, for example, pluripotent stem cells that have PKD1 gene knocked out.In some embodiments, pluripotent stem cells are iPS cells.

[0113] In this embodiment, the autosomal dominant polycystic kidney disease (ADPKD) specific ureteric bud organoid that expresses ETS family protein can be as long as it expresses ETS family protein, for example, it is the ADPKD specific ureteric bud organoid that is introduced with ETS family gene.The means of gene introduction is as described above.As ETS family protein, the above-mentioned ETS family protein can be used.

[0114] In some embodiments, the ureteric bud organoids expressing ETS family protein can be transiently expressed ETS family protein.The transient expression of ETS family protein can be carried out by the above-mentioned method.In particular, the tetracycline (Tet) gene expression induction system can be preferably used.

[0115]

[0013] In one aspect of the present application, there is provided a method for producing renal collecting duct intercalated cells, comprising culturing renal collecting duct principal cells in a medium containing a TGFβ inhibitor, a Wnt inhibitor, a retinoic acid receptor agonist, and a Notch signaling inhibitor in the presence of an ETS family protein. In one embodiment, the renal collecting duct principal cells can be cultured at an air-liquid interface or on a plate, preferably at an air-liquid interface.

[0116] In the present application, renal collecting duct intercalated cells can be confirmed by the expression of markers such as carbonic anhydrase (CA) II, E-cadherin, and ATP6V1B. Optionally, it may be confirmed that the cells do not express the renal pelvic epithelial cell marker UPK2. The expression of renal collecting duct intercalated cells by renal collecting duct intercalated cell markers can be confirmed visually under a microscope by immunostaining, or by flow cytometry, such as FACS (fluorescence-activated cell sorting).

[0117] The renal collecting duct main cells may be produced by the method of the present application. That is, in certain embodiments, this aspect further comprises the step of producing renal collecting duct main cells by the method of the present application. Furthermore, the renal collecting duct main cells may be induced from pluripotent stem cells. In certain embodiments, the pluripotent stem cells are iPS cells.

[0118] In this embodiment, the ETS family protein may be added to the culture medium, or the renal collecting duct principal cells may express the ETS family protein. The ETS family proteins described above can be used as the ETS family protein. When the ETS family protein is added to the culture medium, the concentration of the ETS family protein can be appropriately selected by those skilled in the art depending on the ETS family protein used. When the ETS family protein is ELF5, its concentration is, for example, 1 nM to 1 mM.

[0119] In one embodiment, renal collecting duct chief cells express an ETS family protein. In the present application, the cells expressing an ETS family protein are any cells that express an ETS family protein, for example, cells into which an ETS family gene has been introduced. The means for introducing the gene are as described above.

[0120] In one embodiment, cells expressing an ETS family protein can transiently express the ETS family protein. The transient expression of the ETS family protein can be achieved using the methods described above. In particular, the tetracycline (Tet) gene expression induction system can be suitably used. In one embodiment, the transient expression of the ETS family protein can be controlled by the tetracycline expression induction system, and the medium further contains doxycycline. In this embodiment, the concentration of doxycycline can be 4 nM to 400 μmM, 40 nM to 400 μM, or 400 nM to 40 μM, e.g., approximately 4 μM.

[0121] The introduced ETS family gene can be removed from the cells when its expression is no longer required. When an episomal vector is used, the gene transfer vector disappears naturally after long-term culture. When a PiggyBac vector is used, the gene once integrated into the genome can be removed using PiggyBac™ transposase.

[0122] In one embodiment, renal collecting duct chief cells can be derived from pluripotent stem cells that express an ETS family protein. The pluripotent stem cells that express an ETS family protein may be any cells that express an ETS family protein, such as pluripotent stem cells into which an ETS family gene has been introduced. The means for introducing the gene are as described above.

[0123] In one embodiment, pluripotent stem cells expressing an ETS family protein can transiently express the ETS family protein. The transient expression of the ETS family protein can be achieved using the methods described above. In particular, the tetracycline (Tet) gene expression induction system can be suitably used. For example, the transient expression of the ETS family protein can be controlled by the tetracycline expression induction system.

[0124] In this embodiment, the TGFβ inhibitor may be any of the above-mentioned TGFβ inhibitors. The concentration of the TGFβ inhibitor can be appropriately selected by those skilled in the art depending on the TGFβ inhibitor used. When the TGFβ inhibitor is A83-01, its concentration may be 1 nM to 100 μM, 10 nM to 100 μM, or 100 nM to 10 μM, for example, about 1 μM.

[0125] In this embodiment, the Wnt inhibitor may be any of the above-described Wnt inhibitors. The concentration of the Wnt inhibitor can be appropriately selected by those skilled in the art depending on the Wnt inhibitor used. When the Wnt inhibitor is IWR-1, its concentration may be 1 nM to 100 μM, 10 nM to 100 μM, or 100 nM to 10 μM, for example, about 1 μM.

[0126] In this embodiment, the retinoic acid receptor agonist may be any of the above-mentioned retinoic acid receptor agonists. The concentration of the retinoic acid receptor agonist can be appropriately selected by those skilled in the art depending on the retinoic acid receptor agonist used. When the retinoic acid receptor agonist is TTNPB, its concentration may be 0.1 nM to 10 μM, 1 nM to 10 μM, or 10 nM to 1 μM, for example, about 0.1 μM.

[0127] The Notch signal inhibitor is not particularly limited as long as it can suppress Notch-mediated signal transduction. Examples of Notch signal inhibitors include DAPT (GSI-IX), DBZ (Dibenzazepine, YO-01027), RO4929097, Semagacestat (LY450139), LY411575, IMR-1, FLI-06, Crenigacestat (LY3039478), and derivatives thereof. The Notch signal inhibitor used in the present application is, for example, DAPT. In this embodiment, the concentration of the Notch signal inhibitor can be appropriately selected by those skilled in the art depending on the Notch signal inhibitor used. When the Notch signal inhibitor is DAPT, its concentration can be 5 nM to 500 μM, 50 nM to 500 μM, or 500 nM to 50 μM, for example, about 5 μM.

[0128] The culture period in this step may be 1 to 30 days, 2 to 14 days, or 5 to 10 days, for example, about 7 days.

[0129] The culture temperature is, but is not limited to, about 30 to 40°C, for example, about 37°C, and the culture is carried out in an atmosphere of CO2-containing air, with a CO2 concentration of, for example, about 2 to 5%.

[0130] The present application also provides a cell population containing 30% or more renal collecting duct intercalated cells. A cell population containing 30% or more renal collecting duct intercalated cells can be produced, for example, by the methods of the present application. In certain embodiments, a cell population containing 30% or more renal collecting duct intercalated cells contains 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more renal collecting duct intercalated cells. The presence of renal collecting duct intercalated cells in the cell population of the present application can be confirmed by the expression of the above-mentioned renal collecting duct intercalated cell markers. The presence of 30% or more renal collecting duct intercalated cells in the cell population of the present application can also be confirmed using flow cytometry, for example, fluorescence-activated cell sorting (FACS).

[0131] In this embodiment, the animal from which the renal collecting duct intercalated cells are derived is not limited, and examples include mammals such as mice, rats, hamsters, guinea pigs, cows, horses, pigs, sheep, monkeys, orangutans, chimpanzees, dogs, cats, birds, and humans, with primates being preferred, and humans being more preferred.

[0132]

[0003] In one aspect of the present application, there is provided a method for producing renal pelvic epithelial cells, comprising culturing renal collecting duct progenitor cells in a medium containing a TGFβ inhibitor, a Wnt inhibitor, a retinoic acid receptor agonist, and a Notch signaling inhibitor in the presence of an ETS family protein. In one embodiment, the renal collecting duct progenitor cells can be cultured at an air-liquid interface or on a plate, preferably at an air-liquid interface.

[0133] In the present application, the identity of renal pelvic epithelial cells can be confirmed by the expression of markers such as UPK2, E-cadherin, and CK8. Optionally, it may be confirmed that the cells do not express ATP6V1B, a marker for renal collecting duct interstitial cells. The expression of renal pelvic epithelial cell markers in renal pelvic epithelial cells can be confirmed visually under a microscope by immunostaining, or by flow cytometry, such as FACS (fluorescence-activated cell sorting).

[0134] Renal collecting duct progenitor cells may be produced by the above-mentioned method. For example, renal collecting duct progenitor cells can be induced by a method comprising culturing ureteric bud cells in a medium containing a TGFβ inhibitor and a Wnt inhibitor. Alternatively, renal collecting duct progenitor cells can be induced from pluripotent stem cells. In one embodiment, the pluripotent stem cells are iPS cells.

[0135] In this embodiment, the ETS family protein may be added to the culture medium, or the renal collecting duct progenitor cells may express the ETS family protein. The ETS family proteins described above can be used as the ETS family protein. When the ETS family protein is added to the culture medium, the concentration of the ETS family protein can be appropriately selected by those skilled in the art depending on the ETS family protein used. When the ETS family protein is ELF5, its concentration is, for example, 1 nM to 1 mM.

[0136] In one embodiment, renal collecting duct progenitor cells express an ETS family protein. In the present application, the cells expressing an ETS family protein are any cells that express an ETS family protein, such as cells into which an ETS family gene has been introduced. The means for introducing the gene are as described above.

[0137] In one embodiment, cells expressing an ETS family protein can transiently express the ETS family protein. The transient expression of the ETS family protein can be achieved using the methods described above. In particular, the tetracycline (Tet) gene expression induction system can be suitably used. In one embodiment, the transient expression of the ETS family protein can be controlled by the tetracycline expression induction system, and the medium further contains doxycycline. In this embodiment, the concentration of doxycycline can be 4 nM to 400 μmM, 40 nM to 400 μM, or 400 nM to 40 μM, e.g., approximately 4 μM.

[0138] The introduced ETS family gene can be removed from the cells when its expression is no longer required. When an episomal vector is used, the gene transfer vector disappears naturally after long-term culture. When a PiggyBac vector is used, the gene once integrated into the genome can be removed using PiggyBac™ transposase.

[0139] In one embodiment, renal collecting duct progenitor cells can be derived from pluripotent stem cells that express an ETS family protein. The pluripotent stem cells that express an ETS family protein may be any stem cells that express an ETS family protein, such as pluripotent stem cells into which an ETS family gene has been introduced. The gene introduction method is as described above.

[0140] In one embodiment, pluripotent stem cells expressing an ETS family protein can transiently express the ETS family protein. The transient expression of the ETS family protein can be achieved using the methods described above. In particular, the tetracycline (Tet) gene expression induction system can be suitably used. For example, the transient expression of the ETS family protein can be controlled by the tetracycline expression induction system.

[0141] In this embodiment, the TGFβ inhibitor may be any of the above-mentioned TGFβ inhibitors. The concentration of the TGFβ inhibitor can be appropriately selected by those skilled in the art depending on the TGFβ inhibitor used. When the TGFβ inhibitor is A83-01, its concentration may be 1 nM to 100 μM, 10 nM to 100 μM, or 100 nM to 10 μM, for example, about 1 μM.

[0142] In this embodiment, the Wnt inhibitor may be any of the above-described Wnt inhibitors. The concentration of the Wnt inhibitor can be appropriately selected by those skilled in the art depending on the Wnt inhibitor used. When the Wnt inhibitor is IWR-1, its concentration may be 1 nM to 100 μM, 10 nM to 100 μM, or 100 nM to 10 μM, for example, about 1 μM.

[0143] In this embodiment, the retinoic acid receptor agonist may be any of the above-mentioned retinoic acid receptor agonists. The concentration of the retinoic acid receptor agonist can be appropriately selected by those skilled in the art depending on the retinoic acid receptor agonist used. When the retinoic acid receptor agonist is TTNPB, its concentration may be 0.1 nM to 10 μM, 1 nM to 10 μM, or 10 nM to 1 μM, for example, about 0.1 μM.

[0144] In this embodiment, the Notch signal inhibitor can be any of the above-mentioned Notch signal inhibitors. The concentration of the Notch signal inhibitor can be appropriately selected by those skilled in the art depending on the Notch signal inhibitor used. When the Notch signal inhibitor is DAPT, its concentration can be 5 nM to 500 μM, 50 nM to 500 μM, or 500 nM to 50 μM, for example, about 5 μM.

[0145] The culture period in this step may be 1 to 30 days, 2 to 14 days, or 5 to 10 days, for example, about 7 days.

[0146] The culture temperature is, but is not limited to, about 30 to 40°C, for example, about 37°C, and the culture is carried out in an atmosphere of CO2-containing air, with a CO2 concentration of, for example, about 2 to 5%.

[0147] The present application also provides a cell population containing 40% or more renal pelvic epithelial cells. A cell population containing 40% or more renal pelvic epithelial cells can be produced, for example, by the method of the present application. In certain embodiments, a cell population containing 40% or more renal pelvic epithelial cells contains 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more renal pelvic epithelial cells. The presence of renal pelvic epithelial cells in the cell population of the present application can be confirmed by the expression of the above-mentioned renal pelvic epithelial cell markers. The presence of 40% or more renal pelvic epithelial cells in the cell population of the present application can also be confirmed using flow cytometry, for example, fluorescence-activated cell sorting (FACS).

[0148] In this embodiment, there are no limitations on the animal from which the renal pelvic epithelial cells are derived, and examples include mammals such as mice, rats, hamsters, guinea pigs, cows, horses, pigs, sheep, monkeys, orangutans, chimpanzees, dogs, cats, birds, and humans, with primates being preferred, and humans being more preferred.

[0149] The present invention will be explained in more detail below by showing examples, but the present invention is not limited to these examples in any way.

[0150] [Materials and Methods] Details of the reagents used in this example are shown in Table 1.

[0151] Method for generating Tet-On-ELF5-1231A3 iPS cells: The ELF5 sequence was cloned from human fetal kidney tissue cDNA. An entry vector was created using the pENTR™ / D-TOPO™ Cloning Kit (Thermo Fisher Scientific). The ELF5 sequence was inserted into an all-in-one PiggyBac vector (S. Kim et al. (2015) Methods in Molecular Biology) using Gateway™ LR Clonase™ II Enzyme Mix (Thermo Fisher Scientific). The vector was introduced into 1231A3 undifferentiated iPS cells by electroporation, and the resulting cells were selected with ampicillin and used as Tet-On-ELF5-1231A3 iPS cells.

[0152] Renal collecting duct principal cell induction: Plate culture. Day 6 ureteric bud organoids were generated from Tet-On-ELF5-1231A3 iPS cells using a method previously described (WO2021 / 066076). Ten ureteric bud organoids were collected in a 1.5 mL tube and washed twice with 500 μL of PBS. 100 μL of Accutase was added and incubated for 3 minutes at 37°C and 5% CO2. The ureteric bud organoids were completely disaggregated into single cells by pipetting, and 900 μL of DMEM / 10% FBS was added. The cells were then counted.

[0153] 0.25-1.0 x 10 per 24-well plate (6-, 12-, 48-, or 96-well plates, or 6-, 12-, or 24-well transwell plates can also be used). 5 cells / cm 2 The required amount of suspension was collected and centrifuged at 300 G for 5 minutes so that the cell count was preferably 1 × 10 5 cells / cm 2 After removing the supernatant, the cells were cultured in DMEM / F12 GlutaMAX+B-27 Supplement minus vitamin A, 10% FBS, 1 μM A83-01, and 1 μM IWR-1 (renal collecting duct progenitor cell induction medium) at 0.125 μg / cm 2 The cells were resuspended in a medium supplemented with iMatrix-511 silk and 10 μM Y-27632, adjusted to a suspension volume of 500 μL / well, and seeded. The medium may also be supplemented with 0.1 μM TTNPB.

[0154] After 2 or 3 days, the cells were washed with 500 μL of PBS and then replaced with DMEM / F12 GlutaMAX+B-27 Supplement minus vitamin A medium (renal collecting duct chief cell induction medium) containing 10% FBS, 1 μM A83-01, 1 μM IWR-1, 4 μM doxycycline, and 400 μM IBMX. The medium may also contain 0.1 μM TTNPB, 100 nM desmopressin (dDAVP), 10 nM aldosterone, and 100 nM K252a. A medium containing the triple combination of TTNPB, dDAVP, and aldosterone is preferred. The medium was then replaced every two days, and induction into chief cells was complete by day 4.

[0155] Renal collecting duct principal cell induction: Transwell culture. Day 6 ureteric bud organoids were generated from Tet-On-ELF5-1231A3 iPS cells using a method previously described (WO2021 / 066076). Ten ureteric bud organoids were collected in a 1.5 mL tube and washed twice with 500 μL of PBS. 100 μL of Accutase was added and incubated for 3 minutes at 37°C and 5% CO2. The ureteric bud organoids were completely disaggregated into single cells by pipetting, and 900 μL of DMEM / 10% FBS was added. The cells were then counted.

[0156] To assess apical-basal polarity, cells were seeded in Transwell 12-well plates (CORNING, 3460). 5 cells / cm 2 The required amount of suspension was collected and centrifuged at 300 G for 5 minutes so that the cell count was preferably 1 × 10 5 cells / cm 2 After removing the supernatant, 0.125 μg / cm was added to the renal collecting duct progenitor cell induction medium. 2 The cells were resuspended in medium supplemented with iMatrix-511 silk and 10 μM Y-27632, and the suspension was adjusted to fill 300 μL / well. The cells were seeded in the upper chamber. 0.1 μM TTNPB may also be added to the medium.

[0157] After two or three days, the medium in the upper chamber was removed, and 300 μL of renal collecting duct principal cell induction medium was added to the lower chamber. The medium may further contain 0.1 μM TTNPB, 100 nM dDAVP, 10 nM aldosterone, or 100 nM K252a. A triple combination of TTNPB, dDAVP, and aldosterone is preferred. The medium was then replaced every two days, and induction of apical-basal polarized principal cells was complete by day four.

[0158] Generation of renal collecting duct chief cell-containing organoids. Day 6 ureteric bud organoids were generated from Tet-On-ELF5-1231A3 iPS cells using a method previously described (WO2021 / 066076). The medium was removed and replaced with 100 μL / well of renal collecting duct progenitor cell induction medium. 0.1 μM TTNPB may also be added to the medium.

[0159] After 3 days, the medium was replaced with 100 μL / well of renal collecting duct principal cell induction medium. The medium may further contain 0.1 μM TTNPB, 100 nM dDAVP, 10 nM aldosterone, or 100 nM K252a. A medium containing the triple combination of TTNPB, dDAVP, and aldosterone is preferred. The medium was replaced every two days, and the generation of chief cell-containing organoids was completed by day 6.

[0160] Renal collecting duct intercalated cell induction. Renal collecting duct principal cells were induced from Tet-On-ELF5-1231A3 iPS cells using the transwell culture method described above. For renal collecting duct principal cells on days 7-10, the medium in the lower chamber was aspirated and washed with 300 μL of PBS. 300 μL of renal collecting duct progenitor cell induction medium containing 0.1 μM TTNPB, 5 μM DAPT, and 4 μM doxycycline was added. The medium was changed every two days, and the generation of renal collecting duct intercalated cells was completed by day 7.

[0161] Renal pelvic epithelial cell induction: Day 6 ureteric bud organoids were generated from Tet-On-ELF5-1231A3 iPS cells using a method previously described (WO2021 / 066076). Ten ureteric bud organoids were collected in a 1.5 mL tube and washed twice with 500 μL of PBS. 100 μL of Accutase was added and incubated for 3 minutes at 37°C and 5% CO2. The ureteric bud organoids were completely disaggregated into single cells by pipetting, and 900 μL of DMEM / 10% FBS was added. The cells were then counted.

[0162] To seed cells into a Transwell 12-well plate (CORNING, 3460), 0.5-2 × 10 5 cells / cm 2The required amount of suspension was collected and centrifuged at 300 G for 5 minutes so that the cell count was preferably 1 × 10 5 cells / cm 2 After removing the supernatant, 0.125 μg / cm was added to the renal collecting duct progenitor cell induction medium. 2 The cells were resuspended in medium containing iMatrix-511 silk and 10 μM Y-27632, and the suspension was adjusted to 300 μL / well and seeded in the upper chamber. The medium may also contain 0.1 μM TTNPB.

[0163] On day 2 or 3, the medium in the upper chamber was aspirated and washed with 300 μL of PBS. 300 μL of renal collecting duct progenitor cell induction medium containing 0.1 μM TTNPB, 5 μM DAPT, and 4 μM doxycycline was added to the lower chamber. The medium was changed every two days, and the generation of renal pelvic epithelial cells was completed on day 7.

[0164] Renal collecting duct principal cell induction without ELF5 forced expression. Day 6 ureteric bud organoids were generated from Tet-On-ELF5-1231A3 iPS cells using a method previously described (WO2021 / 066076). Ten ureteric bud organoids were collected in a 1.5 mL tube and washed twice with 500 μL of PBS. 100 μL of Accutase was added and incubated for 3 minutes at 37°C and 5% CO2. The ureteric bud organoids were completely disaggregated into single cells by pipetting, and 900 μL of DMEM / 10% FBS was added. The cells were then counted.

[0165] To seed cells into a Transwell 12-well plate (CORNING, 3460), 0.5-2 × 10 5 cells / cm 2 The required amount of suspension was collected and centrifuged at 300 G for 5 minutes so that the cell count was preferably 1 × 10 5 cells / cm 2 After removing the supernatant, 0.125 μg / cm was added to the renal collecting duct progenitor cell induction medium. 2The cells were resuspended in medium containing iMatrix-511 silk and 10 μM Y-27632, and the suspension was adjusted to 300 μL / well. The cells were seeded in the upper chamber. 0.1 μM TTNPB may also be added to the medium.

[0166] After two or three days, the medium in the upper chamber was removed, and 300 μL of renal collecting duct progenitor cell induction medium containing 400 μM IBMX was added to the lower chamber. The medium may further contain 0.1 μM TTNPB, 100 nM dDAVP, 10 nM aldosterone, or 100 nM K252a. A medium containing the triple combination of TTNPB, dDAVP, and aldosterone is preferred. The medium was then replaced every two days, and induction into renal collecting duct principal cells was completed after 10–21 days of culture, preferably 14 days.

[0167] Immunostaining: Cultured cells were washed twice with PBS and then fixed with 4% PFA for 20 minutes at 4°C. After washing twice with PBS, they were blocked for 1 hour at room temperature using 2% normal donkey serum (MERCK) / PBT (PBS / 0.25% Triton X-100, Nacalai tesque) (blocking solution). Each primary antibody was diluted 1:200 in the blocking solution and left to stand with the sample for 24 hours at 4°C. After washing twice with 0.25% PBT, each secondary antibody was diluted 1:500 in the blocking solution and left to stand with the sample for 2 hours at room temperature. Cells were observed using a microscope such as a BZX-700 (Keyence).

[0168] mRNA measurement method: RNA was extracted from cultured cells using the RNeasy mini kit (QIAGEN), and cDNA was synthesized using ReverTra Ace (TOYOBO). Real-time PCR was performed using SYBR Premix Ex Taq II (TaKaRa). Measurement values ​​were normalized by the expression level of β-actin.

[0169] Measurement of transepithelial electrical resistance (TEER) in renal collecting duct principal cells. Day 7 renal collecting duct principal cells were generated from Tet-On-ELF5-1231A3 iPS cells using the transwell culture method described above. The medium in the lower chamber was aspirated, and 300 μL of new medium containing 0.1 μM TTNPB, 100 nM dDAVP, and 10 nM aldosterone was added. 100 μL of DMEM / F12 GlutaMAX + B-27 Supplement minus vitamin A + 10% FBS was added to the upper chamber. TEER was measured using a Millicell ERS2 (Millipore) with the sensor tip in contact with the medium in both the upper and lower chambers. Three measurements were taken, and the average value was used as the 0-h value. For the benzamil-treated group, 1 μM benzamil was added to the lower chamber. The cells were incubated for 24 hours at 37°C and 5% CO2. TEER was measured using a Millicell ERS2 with the sensor tip in contact with the medium in the upper and lower chambers. Three measurements were taken, and the average value was used as the 24-h value.

[0170] [Results] We generated an iPS cell line (Tet-On-ELF5-1231A3) capable of overexpressing ELF5, an upstream gene of renal collecting duct principal cells (Figure 1). Tet-On-ELF5-1231A3 iPS cells were cultured in StemFit® AK02 for 1 day, then 4 μM doxycycline was added and cultured for an additional day (Figure 2). PCR and immunostaining confirmed that doxycycline-treated Tet-On-ELF5-1231A3 iPS cells expressed ELF5 and mCherry (Figures 3-5).

[0171] Next, mesonephric duct cells were induced from Tet-On-ELF5-1231A3 iPS cells according to a previously described method (WO2021 / 066076, Figure 6). On day 3 of mesonephric duct induction, 4 μM doxycycline was added and the cells were cultured for an additional day. Immunostaining revealed that doxycycline administration did not affect the expression of mesonephric duct markers (Figure 7). Furthermore, ureteric bud organoids were induced according to the protocol described in Figure 8 (Figure 8). The established Tet-On-ELF5-1231A3 line was confirmed to differentiate into ureteric bud organoids (Figure 9).

[0172] Ureteric bud organoids derived from Tet-On-ELF5-1231A3 iPS cells were dissociated into single cells as described above and cultured in renal collecting duct progenitor cell induction medium (Figure 10). The addition of 4 μM doxycycline to the renal collecting duct progenitor cell induction medium resulted in the expression of ELF5 and mCherry in the cultured cells (Figure 11). The addition of doxycycline did not contribute to the early appearance of AQP2-positive cells.

[0173] Next, 4 μM doxycycline and 400 μM IBMX were added to the renal collecting duct progenitor cell induction medium (Fig. 12). After culture, the cells expressed the renal collecting duct marker AVPR2 and early expressed AQP2 (Fig. 13). This suggests that the addition of IBMX to the renal collecting duct progenitor cell induction medium promotes the generation of renal collecting duct principal cells.

[0174] We investigated the concentration of IBMX added to the renal collecting duct progenitor cell induction medium (Fig. 14). At concentrations up to 400 μM, the number of AQP2-expressing cells increased in a concentration-dependent manner, but the addition of 4000 μM IBMX caused significant cell detachment (Fig. 15).

[0175] Previously, we reported that administration of desmopressin (dDAVP) promoted AQP2 expression and apical polarization in mouse renal collecting duct principal cell lines (Ando F. et al., Nat Commun. 2018 Apr 12;9(1):1411). Therefore, to assess apical-basal polarization, renal collecting duct principal cells were cultured in transwells (Figures 16 and 17). Adding 100 nM dDAVP to the culture medium in the lower chamber increased AQP2 expression polarity toward the contralateral side (Figure 18).

[0176] Next, we investigated factors effective in inducing renal collecting duct principal cells. Various factors were added to IBMX-free renal collecting duct principal cell induction medium, and renal collecting duct principal cells were induced by plate culture (Fig. 19). Addition of vasopressin acetate (AVP), aldosterone (ALD), or the calcium calmodulin kinase 2 (CaMK2) inhibitor K252a to the medium did not promote AQP2 expression (Figs. 20 and 21). On the other hand, addition of IBMX and dDAVP to the medium, or addition of IBMX, AVP, or dDAVP, ALD, and K252a to the medium, promoted AQP2 expression. These findings suggest that the addition of dDAVP, AVP, ALD, and K252a acts additively with IBMX.

[0177] Next, we cultured renal collecting duct chief cells in the medium containing aldosterone, TTNPB, and dDAVP (Fig. 22). Immunostaining confirmed that renal collecting duct chief cell-like cells expressing sodium channels were generated (Fig. 23).

[0178] Renal collecting duct principal cells were induced by culturing ureteric bud cells in renal collecting duct progenitor cell induction medium containing TTNPB for 3 days, followed by culturing them in renal collecting duct principal cell induction medium containing TTNPB, dDAVP, and aldosterone for 4 days (Fig. 24). Gene expression profiles of the induced renal collecting duct principal cells were compared with those obtained by culturing ureteric bud cells in renal collecting duct progenitor cell induction medium for 7 days (Fig. 25). Renal collecting duct principal cells showed significantly elevated expression of renal collecting duct principal cell-associated marker genes (AQP2, AQP3, AVPR2, and SCNN1b). Therefore, the induced renal collecting duct principal cells also demonstrated renal collecting duct principal cell properties at the mRNA level.

[0179] Next, we induced renal collecting duct chief cell-containing organoids from Tet-On-ELF5-1231A3 iPS cells (Figs. 26-28). Immunostaining revealed that the renal collecting duct chief cell-containing organoids expressed the renal collecting duct chief cell markers AQP2, E-cadherin, and AVPR2 (Fig. 29).

[0180] Functional evaluation of renal collecting duct principal cell-containing organoids was performed. On day 6 of induction, the medium for renal collecting duct principal cell-containing organoids was replaced with medium containing 1 μM dDAVP or 1 μM dDAVP and 10 μM tolvaptan.

[0181] When dDAVP was added to the culture medium, water uptake was promoted and cyst-like structures formed the following day (Figure 30). When tolvaptan, a vasopressin V2 receptor (V2R) blocker, was added to the culture medium together with dDAVP, the cyst-like structures were inhibited. Therefore, it was suggested that renal collecting duct chief cell-containing organoids exhibit responsiveness to existing drugs.

[0182] We generated PKD1 knockout iPS cell lines (Tet-On-ELF5 27B6-5-1 to -5) capable of overexpressing ELF5 using PKD1 knockout iPS cells generated by a previously reported method (Ishida K. et al., Sci Rep. 8:310 (2018); Shimizu T. et al., Biochem Biophys Res Commun. 529, 1186-1194 (2020)). After culturing the cells in StemFit® AK02 for 1 day, 4 μM doxycycline was added and the cells were cultured for an additional day (Figure 32). Immunostaining confirmed that at least two cell lines (Tet-On-ELF5 27B6-5-1 and Tet-On-ELF5 27B6-5-2) expressed ELF5 and gRNA (Figures 33 and 34). Figures 33 and 34 show the results of experiments with the Tet-On-ELF5 27B6-5-1 strain.

[0183] Ureteric bud organoids were derived from Tet-On-ELF5 27G6 cells according to a previously described method ( WO2021 / 066076 , Figure 35). Sprouting transformation was confirmed in day 2 ureteric bud organoids induced in 2% Matrigel ( Figure 36 ). Thus, ureteric bud organoids derived from Tet-On-ELF5 27G6 cells exhibited branching similar to that of ureteric bud organoids derived from healthy iPS cell lines.

[0184] Next, we evaluated the function of renal collecting duct principal cells prepared using the present method. It is known that the addition of sodium channel blockers (amiloride, benzamil, etc.) increases the transepithelial electrical resistance (TEER) of renal collecting duct principal cells. Therefore, we evaluated the Na channel function of renal collecting duct principal cells prepared using the present method using a transwell by measuring TEER.

[0185] Renal collecting duct chief cells generated by the present method showed an increase in TEER with benzamil administration under continuous aldosterone administration (Fig. 37). Furthermore, the percentage of ENaC-positive cells did not change with benzamil administration, confirming that this change in TEER was not due to a change in differentiation state (Figs. 38 and 39).

[0186] We successfully induced the differentiation of renal collecting duct chief cells into renal collecting duct interstitial cells (Figs. 40 and 41). Immunostaining revealed that the induced renal collecting duct interstitial cells showed a decrease in AQP2-positive cells and were positive for the renal collecting duct interstitial cell marker ATP6V1B (Fig. 42). Furthermore, the induced renal collecting duct interstitial cells were negative for the renal pelvic epithelial cell marker UPK2.

[0187] Furthermore, we succeeded in inducing differentiation of renal collecting duct progenitor cells into renal pelvic epithelial cells (Figs. 43 and 44). Immunostaining revealed that the renal pelvic epithelial cell marker UPK2 was positive (Fig. 45).

[0188] We also succeeded in inducing renal collecting duct chief cells without forced ELF5 expression by extending the culture period. When renal collecting duct progenitor cells were cultured in a transwell culture medium containing 10% FBS, 1 μM A83-01, 1 μM IWR-1, and 400 μM IBMX (excluding DOX) for 14 days, ELF5 expression was observed even without DOX administration, demonstrating that renal collecting duct chief cell induction is possible without forced ELF5 expression by DOX administration (Figs. 46 and 47).

Claims

1. A method for producing renal collecting duct chief cells, comprising the step of culturing ureteriblasts or cells differentiated from ureteriblasts in a medium containing a TGFβ inhibitor, a Wnt inhibitor, and a phosphodiesterase inhibitor.

2. The method according to claim 1, wherein ureteroblasts are cultured in a medium containing a TGFβ inhibitor, a Wnt inhibitor, and a phosphodiesterase inhibitor.

3. The method according to claim 1, wherein renal collecting duct progenitor cells differentiated from ureteroblasts are cultured in a medium containing a TGFβ inhibitor, a Wnt inhibitor, and a phosphodiesterase inhibitor.

4. (A) A step of culturing ureteroblasts in a medium containing a TGFβ inhibitor and a Wnt inhibitor, and (B) The method according to claim 1, comprising the step of culturing ureteroblasts obtained in (A) or cells differentiated from ureteroblasts in a medium containing a TGFβ inhibitor, a Wnt inhibitor, and a phosphodiesterase inhibitor.

5. The method according to claim 1, wherein ureteroblasts or cells differentiated from ureteroblasts are cultured in the presence of ETS family proteins.

6. The method according to claim 5, wherein ureteroblasts or cells differentiated from ureteroblasts that can transiently express ETS family proteins are cultured in a medium containing a TGFβ inhibitor, a Wnt inhibitor, and a phosphodiesterase inhibitor under conditions in which ETS family proteins are expressed.

7. The method according to claim 5, wherein the ETS family protein is ELF5.

8. The method according to claim 1, wherein the TGFβ inhibitor is A83-01, the Wnt inhibitor is IWR-1, and the phosphodiesterase inhibitor is IBMX.

9. The method according to claim 1, wherein the culture medium further comprises a retinoic acid receptor agonist, a vasopressin receptor agonist, a CaMK inhibitor, and aldosterone.

10. The method according to claim 9, wherein the retinoic acid receptor agonist is TTNPB, the vasopressin receptor agonist is desmopressin, and the CaMK inhibitor is K252a.

11. The method according to claim 1, wherein the ureteroblasts are derived from pluripotent stem cells.

12. The method according to claim 11, wherein the pluripotent stem cells are iPS cells.

13. The method according to claim 1, wherein the renal collecting duct chief cells are obtained as a cell population containing 40% or more renal collecting duct chief cells.

14. A method for producing renal collecting duct chief cell-containing organoids, comprising the step of culturing ureteral bud organoids or organoids differentiated from ureteral bud organoids in a medium containing a TGFβ inhibitor, a Wnt inhibitor, and a phosphodiesterase inhibitor.

15. The method according to claim 14, wherein ureteral bud organoids are cultured in a medium containing a TGFβ inhibitor, a Wnt inhibitor, and a phosphodiesterase inhibitor.

16. The method according to claim 14, wherein organoids containing renal collecting duct progenitor cells differentiated from ureteral bud organoids are cultured in a medium containing a TGFβ inhibitor, a Wnt inhibitor, and a phosphodiesterase inhibitor.

17. (A) A step of culturing ureteral bud organoids in a medium containing a TGFβ inhibitor and a Wnt inhibitor, and The method according to claim 14, further comprising the step of culturing the ureteric bud organoid obtained in (A) or an organoid differentiated from the ureteric bud organoid in a medium containing a TGFβ inhibitor, a Wnt inhibitor, and a phosphodiesterase inhibitor.

18. The method according to claim 14, wherein cells contained in a ureteric bud organoid or an organoid differentiated from a ureteric bud organoid are cultured in the presence of an ETS family protein.

19. The method according to claim 18, wherein ureteral bud organoids or organoids differentiated from ureteral bud organoids containing cells capable of transiently expressing ETS family proteins are cultured in a medium containing a TGFβ inhibitor, a Wnt inhibitor, and a phosphodiesterase inhibitor under conditions in which ETS family proteins are expressed.

20. The method according to claim 18, wherein the ETS family protein is ELF5.

21. The method according to claim 14, wherein the TGFβ inhibitor is A83-01, the Wnt inhibitor is IWR-1, and the phosphodiesterase inhibitor is IBMX.

22. The method according to claim 14, wherein the culture medium further comprises a retinoic acid receptor agonist, a vasopressin receptor agonist, a CaMK inhibitor, and aldosterone.

23. The method according to claim 22, wherein the retinoic acid receptor agonist is TTNPB, the vasopressin receptor agonist is desmopressin, and the CaMK inhibitor is K252a.

24. The method according to claim 14, wherein the ureteric bud organoid is derived from pluripotent stem cells.

25. The method according to claim 24, wherein the pluripotent stem cells are iPS cells.

26. The method according to claim 14, wherein a renal collecting duct chief cell-containing organoid is obtained that contains 40% or more renal collecting duct chief cells as the renal collecting duct chief cell-containing organoid.

27. A method for producing interstitial cells of the renal collecting duct, comprising the step of culturing renal collecting duct chief cells in a medium containing a TGFβ inhibitor, a Wnt inhibitor, a retinoic acid receptor agonist, and a Notch signaling inhibitor in the presence of ETS family proteins.

28. The method according to claim 27, wherein renal collecting duct chief cells express ETS family proteins.

29. The method according to claim 28, wherein renal collecting duct chief cells capable of transiently expressing ETS family proteins are cultured under conditions in which ETS family proteins are expressed.

30. The method according to claim 27, wherein the ETS family protein is ELF5, the TGFβ inhibitor is A83-01, the Wnt inhibitor is IWR-1, the retinoic acid receptor agonist is TTNPB, and the Notch signaling inhibitor is DAPT.

31. The method of claim 27, further comprising the step of producing renal collecting duct chief cells by the method of any one of claims 1 to 12.

32. The method according to claim 27, wherein the renal collecting duct chief cells are derived from pluripotent stem cells.

33. The method according to claim 32, wherein the pluripotent stem cells are iPS cells.

34. The method according to claim 27, wherein the renal collecting duct interstitial cells are obtained as a cell population containing 30% or more renal collecting duct interstitial cells.

35. A method for producing renal pelvic epithelial cells, comprising the step of culturing renal collecting duct progenitor cells in a medium containing a TGFβ inhibitor, a Wnt inhibitor, a retinoic acid receptor agonist, and a Notch signaling inhibitor in the presence of ETS family proteins.

36. The method according to claim 35, wherein renal collecting duct progenitor cells express ETS family proteins.

37. The method according to claim 36, wherein renal collecting duct progenitor cells capable of transiently expressing ETS family proteins are cultured under conditions in which ETS family proteins are expressed.

38. The method according to claim 35, wherein the ETS family protein is ELF5, the TGFβ inhibitor is A83-01, the Wnt inhibitor is IWR-1, the retinoic acid receptor agonist is TTNPB, and the Notch signaling inhibitor is DAPT.

39. The method according to claim 35, wherein the renal collecting duct progenitor cells are derived from pluripotent stem cells.

40. The method according to claim 39, wherein the pluripotent stem cells are iPS cells.

41. The method according to claim 39, wherein the renal pelvis epithelial cells are obtained as a cell population containing 40% or more renal pelvis epithelial cells.