WO2025135177A1 - Culture method for promoting organogenesis of kidney while retaining kidney formation region - Google Patents

Abstract

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

Description

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

The present invention relates to a method for promoting kidney organogenesis. More specifically, the present invention relates to a renal tissue-like culture having a kidney structure, a culture method thereof, and a method for producing the same.

The adult mammalian kidney, the metanephros, is formed from multiple embryonic renal progenitor cells, such as nephron progenitor cells that give rise to nephron-forming epithelial cells such as glomeruli and tubules, ureteric bud cells that give rise to collecting ducts and the lower urinary tract, and renal interstitial progenitor cells that give rise to renal interstitial cells, mesangial cells, vascular smooth muscle cells, etc. The metanephros develops and grows as an organ, while the nephron progenitor cells, ureteric bud cells, and renal interstitial progenitor cells maintain the kidney-forming area by repeatedly self-renewing and differentiating.

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

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

The present invention aims to produce renal tissue with a more physiological structure by promoting organ formation while maintaining the kidney-forming region in the culture of nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells. In particular, the present invention aims to produce renal tissue with a more physiological structure by promoting organ formation while maintaining the kidney-forming region in the culture of human-derived nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells.

The present inventors conducted extensive research to solve the above problems. As a result, they discovered that by co-culturing nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells in the presence of a Wnt signal activator, it is possible to promote organ formation while maintaining the kidney-forming region, and to create a renal tissue-like culture with kidney structure.

That is, the present invention has the following features:
[1]
A method for producing a renal tissue-like culture, comprising a step of co-culturing nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells in a medium containing a Wnt signal activator.
[2]
The method according to [1], wherein the co-culture is carried out by suspension culture.
[3]
The method according to [2], wherein the suspension culture is performed in a state in which aggregates of nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells are coated with an extracellular matrix.
[4]
The method according to any one of [1] to [3], wherein the Wnt signal activator is a Wnt3a protein.
[5]
The method according to any one of [1] to [4], wherein the medium further contains fibroblast growth factor 9 (FGF9).
[6]
The method according to any one of [1] to [5], wherein the medium further contains retinoic acid (RA) and/or a derivative thereof, and glial cell line-derived neurotrophic factor (GDNF).
[7]
The method according to [6], wherein the medium further contains alpha-albumin and R-Spondin 1 (RSPO1).
[8]
The method according to any one of [1] to [7], wherein the kidney tissue-like culture has one or more of a mesangial structure, a branching ureteric bud structure, an S-body-like structure, and a cap mesenchyme structure.
[9]
The method according to any one of [1] to [8], wherein the nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells are derived from a human.
[10]
The method according to any one of [1] to [9], wherein one or more of the nephron progenitor cells, the renal interstitial progenitor cells, and the ureteric bud cells are cells induced from pluripotent stem cells.
[11]
1. A kidney tissue-like culture comprising:
Contains cells of human origin,
The cultures contained mesangial structures, branching ureteric bud structures, S-body-like structures, and cap mesenchyme structures.
[12]
The mesangial structure is a structure containing PDGFRB and/or GATA3 positive cells within a glomerulus-like structure,
The branched ureteric bud structure has two or more ureteric buds, and the tip of each ureteric bud contains RET-positive cells.
the S-shaped structure comprises an S-shaped structure comprising a proximal region and a distal region,
The renal tissue-like culture of [11], wherein the cap mesenchyme structure is a structure that surrounds the tip of the ureteric bud and contains SIX2 and/or PAX8 positive cells.

The method of the present invention can promote organogenesis in nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells while maintaining the kidney-forming area, and can create renal tissue-like cultures having kidney structure. The present invention can also provide renal tissue-like cultures having kidney structure, particularly cultures derived from humans. The cultures of the present invention can particularly have a mesangial structure, a branching ureteric bud structure, and a cap mesenchyme structure. These structures are characteristic of the kidney during the organogenesis stage, and are expected to develop and grow into more organs.

Photograph showing immunostaining of SIX2, FOXD1, and CALB1 in tissue-like cultures on day 6 after reconstitution of renal structure under CRFY conditions. Scale bar, 200 μm. This figure shows the results of an analysis of gene expression levels by quantitative RT-PCR (qT-PCR) of each progenitor cell marker gene in tissue-like cultures on day 6 after reconstitution of kidney structure under CRFY conditions, CRFY+G conditions, or RFY+G+WR conditions. Photograph showing immunostaining of SIX2, RET, and CALB1 in tissue-like cultures on day 6 after reconstitution of renal structure under RFY+G+WR conditions. Scale bar, 200 μm. Photograph showing immunostaining of PAX8, SIX2, FOXD1, and CALB1 in tissue-like cultures on day 6 after reconstitution of renal structure under RFY+G+WR conditions. Scale bar, 200 μm. Photograph showing immunostaining of BRN1, HNF4α, and CALB1 in tissue-like cultures on day 6 after reconstitution of renal structure under RFY+G+WR conditions. Scale bar, 200 μm. Photographs showing immunostaining results for PDGFRB, GATA3, MAFB, and NPHS1 in tissue-like cultures on days 6 and 12 after reconstitution of renal structure under RFY+G+WR conditions. Scale bar, 100 μm. A figure (photograph substituting a drawing) showing the results of immunostaining for PDFGRB in cell masses in which induced nephron progenitor cells (NPCs) were allowed to form aggregates under CRFY conditions and cultured for 48 hours. A figure showing the results of immunostaining for ureteric bud marker CK8 and nephron progenitor cell marker SIX2 in tissue-like cultures on day 13 after reconstitution of kidney structure under RFY+G+WR conditions (photograph in lieu of drawing).

The present invention is explained in detail below.

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

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

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

Specifically, the method for inducing nephron progenitor cells from pluripotent stem cells may be carried out, for example, by first inducing a late primitive streak (PPS) in pluripotent stem cells, then reseeding the cells about 4 days after the start of PPS induction by a three-dimensional culture method, and culturing the cells in a medium containing a GSK-3β (glycogen synthase kinase 3β) inhibitor (e.g., CHIR99021), bFGF, and activin A for about 2 days under agitation, and then (about 6 days after the start of PPS induction) changing the medium to one containing retinoic acid (RA), NOGGIN, and FGF9 and culturing the cells, and on about 8 days after the start of PPS induction, changing the medium to one containing FGF9 and a GSK-3β inhibitor (e.g., CHIR99021) and culturing the cells for about 3 days. In addition, when reseeding the cells by the three-dimensional culture method, a ROCK inhibitor (e.g., Y-27632) may be added to the medium after reseeding.
More specifically, the step of inducing the late primitive streak (PPS) in pluripotent stem cells may be carried out, for example, by seeding pluripotent stem cells on a culture dish preferably coated with an extracellular matrix (specifically, for example, laminin fragments), culturing the cells in a medium containing a GSK-3β inhibitor (e.g., CHIR99021), RA, BMP4, and bFGF for about 1 day (day 0), switching to a medium containing CHIR99021, bFGF, and BMP7 and culturing for another day (day 1), switching to a medium containing a GSK-3β inhibitor (e.g., CHIR99021), BMP7, and a TGFβ inhibitor (e.g., A83-01) and culturing for another day (day 2), switching to a medium containing a GSK-3β inhibitor (e.g., CHIR99021), bFGF, activin A, and a ROCK inhibitor (e.g., Y-27632), culturing for another day (day 3), and recovering the cells on about day 4.

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

Specifically, the method of inducing renal interstitial progenitor cells from pluripotent stem cells may be carried out, for example, by carrying out the above-mentioned step of inducing PPS in pluripotent stem cells, then reseeding the cells on about day 4 after the start of PPS induction to form aggregates (for example, aggregates may be formed by reseeding on a U-bottom plate), culturing the cells for about 2 days in a medium containing a GSK-3β inhibitor (e.g., CHIR99021), bFGF, and activin A, and then (on day 6 after the start of PPS induction) changing the medium to one containing a GSK-3β inhibitor (e.g., CHIR99021), RA, smoothened moiety (SMO) agonist (SAG), and IL-1β but not bFGF, and culturing the cells, and on day 8 changing the medium to one containing a GSK-3β inhibitor (e.g., CHIR99021), RA, SAG, and a TGFβ inhibitor (e.g., SB431542) but not bFGF, and culturing the cells for about 3 days.

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

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

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

ES cells established by known methods can be used. Methods for establishing and maintaining ES cells are described, for example, in US Pat. No. 5,843,780; Thomson JA, et al. (1995), Proc Natl. Acad. Sci. U S A. 92:7844-7848; Thomson JA, et al. (1998), Science. 282:1145-1147; H. Suemori et al. (2006), Biochem. Biophys. Res. Commun., 345:926-932; M. Ueno et al. (2006), Proc. Natl. Acad. Sci. USA, 103:9554-9559; H. Suemori et al. (2001), Dev. Dyn., 222:273-279; H. Kawasaki et al. (2002), Proc. Natl. Acad. Sci. USA, 99:1580-1585; Klimanskaya I, et al. (2006), Nature. 444:481-485. Human ES cell lines, e.g. WA01(H1) and WA09(H9), are available from the WiCell Research Institute, and KhES-1, KhES-2 and KhES-3 are available from the Institute for Frontier Medical Sciences, Kyoto University (Kyoto, Japan).

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

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

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

Co-culture of nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells may preferably be performed in suspension culture.
Here, suspension culture means that cells are cultured in a non-adherent state to a culture substrate, and an example of such a culture mode is one in which cells are cultured using a low cell-adhesive culture vessel.

In addition, in suspension culture, aggregates of nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells may be formed, and the aggregates may be coated. The coating agent is preferably an extracellular matrix, and examples of the coating agent include collagen, proteoglycan, fibronectin, hyaluronic acid, tenascin, entactin, elastin, fibrin, and laminin, or fragments thereof. A method for coating cell aggregates may include, for example, moving the aggregated cell masses to a droplet formed of a medium containing 50% by volume of a coating agent after starting suspension culture (e.g., after 48 hours), coating the aggregates, and then adding additional medium to stop the coating process. The coating time is not particularly limited, but may be, for example, 30 minutes or more.

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

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

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

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

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

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

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

In the present invention, the medium for co-culturing nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells may further contain a ROCK (Rho kinase) inhibitor. It is believed that the addition of a ROCK inhibitor can suppress cell death and efficiently culture, passaging, cell proliferation, and/or differentiation. From the above perspective, the medium of the present invention may contain a ROCK inhibitor, or the medium may contain a ROCK inhibitor at the start of cell culture or at the time of passaging, and then be replaced with a medium that does not contain a ROCK inhibitor. As the ROCK inhibitor, a known one may be used as appropriate, for example, Y-27632. The concentration of the ROCK inhibitor may be, for example, 1 to 50 μM, and preferably 5 to 20 μM. It is preferable to add the ROCK inhibitor to the medium for at least 3 days after the start of cell culture or passaging.

The number of days for co-culturing nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells can be, for example, 2 days or more, 3 days or more, 4 days or more, 5 days or more, or 6 days or more. There is no upper limit to the number of days for culture, but it may be, for example, 30 days or less. The culture conditions are not particularly limited as long as a renal tissue-like culture can be obtained, but the culture temperature is about 30 to 40°C, preferably about 37°C, the oxygen concentration is a normal oxygen concentration (e.g., 15 to 25%, preferably about 20%), and the _CO2 concentration is preferably about 2 to 5%.

Renal tissue-like culture refers to a culture having a structure similar to that of developing and/or mature kidney tissue, i.e., renal tissue. Here, a structure similar to that of renal tissue may mean not only morphological similarity, but also anatomically and/or functionally similar to that of renal tissue, based on the characteristics of each cell based on marker gene expression, etc., and the characteristics of the structure that these cells constitute. In other words, renal tissue-like culture may be referred to as a renal organoid. In the developing kidney, the pronephros develops through the mesonephros to the metanephros, and the metanephros matures to form an organ that corresponds to the kidney. The period when the organ that corresponds to the kidney is formed may be called the organogenesis period. As described above, nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells are each one of the progenitor cells for the formation of the metanephros. Therefore, in the renal tissue-like culture of the present invention, the developing kidney tissue may be metanephros tissue, or may be kidney tissue at the organogenesis period. In other words, the renal tissue-like culture of the present invention may refer to a culture having a structure similar to that of metanephros tissue, kidney tissue at the organogenesis stage, and/or mature kidney tissue. The renal tissue-like culture of the present invention may maintain kidney tissue at the organogenesis stage. In one embodiment, the renal tissue-like culture of the present invention may maintain a kidney-forming region. In addition, the renal tissue-like culture of the present invention does not exclude having a structure similar to that of mature kidney tissue, but may not have such a structure. In other words, the renal tissue-like culture of the present invention may refer to a culture having a structure similar to that of metanephros tissue and/or kidney tissue at the organogenesis stage.

The renal tissue structures include, but are not limited to, mesangial structures, branched ureteric bud structures, and cap mesenchyme structures. The renal tissue-like culture of the present invention preferably has one or more of mesangial structures, branched ureteric bud structures, S-shaped structures, and cap mesenchyme structures, and more preferably has all of mesangial structures, branched ureteric bud structures, and cap mesenchyme structures. The renal tissue-like culture of the present invention may further maintain a kidney-forming region. For example, the renal tissue-like culture of the present invention may maintain a kidney-forming region, thereby promoting the formation of further mesangial structures, branched ureteric bud structures, S-shaped structures, and/or cap mesenchyme structures.

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

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

The S-shaped structure means a nascent tubule structure formed by epithelialization of nephron progenitor cells. In the present invention, the S-shaped structure preferably includes an S-shaped structure including a proximal region and a distal region. The proximal region may be characterized by, for example, expression of HNF4α. The distal region may be characterized by, for example, expression of BRN1. The distal region may or may not be distinguished from a middle region that is intermediate between the proximal region and the distal region. When the distal region is not distinguished from the middle region, they may be collectively referred to as a middle-distal region. In this case, it may be rephrased that the S-shaped structure preferably includes an S-shaped structure including a proximal region and a middle-distal region. The middle-distal region may also be characterized by, for example, expression of BRN1.

The cap mesenchyme structure refers to a structure of mesenchymal tissue that encases the tip of the ureteric bud. It is generally known that glomerular podocytes and nephron progenitor cells are present in the cap mesenchyme structure. In the present invention, the cap mesenchyme structure preferably contains nephron progenitor cells and/or cells derived from nephron progenitor cells. The nephron progenitor cells and/or cells derived from nephron progenitor cells may be characterized, for example, by the expression of SIX2 and/or PAX8. More specifically, for example, the nephron progenitor cells may be characterized by the expression of SIX2, and the cells derived from nephron progenitor cells may be characterized by the expression of PAX8. That is, the cap mesenchyme structure may be a structure that encases the tip of the ureteric bud and contains SIX2 and/or PAX8 positive cells. The ureteric bud may be characterized by the expression of the CK8 gene. The cells derived from nephron progenitor cells are not particularly limited as long as they are derived from nephron progenitor cells, and may be, for example, cells constituting metanephric vesicles. The cells constituting metanephric vesicles may be characterized, for example, by the expression of PAX8. Furthermore, a region characterized by a cap mesenchyme structure containing at least nephron progenitor cells may be considered as a kidney-forming region. As described above, nephron progenitor cells may be characterized by the expression of SIX2. In other words, the kidney-forming region may be observed as a cap mesenchyme structure containing SIX2-expressing cells. The renal tissue-like culture maintaining the kidney-forming region may mean that the renal tissue-like culture contains a structure having at least the above-mentioned characteristics of the kidney-forming region. In addition, the renal tissue-like culture maintaining the kidney-forming region may mean, as a non-limiting example, that a structure having the above-mentioned characteristics of the kidney-forming region is observed during the production of the culture, and all or part of such a structure is maintained until after the production of the culture.

<2> Culture of the Present Invention Another aspect of the present invention relates to a kidney tissue-like culture of the present invention.
Specifically, in one aspect, the present invention provides
Contains cells of human origin,
The culture may be a kidney tissue-like culture having mesangial structures, branching ureteric bud structures, S-body-like structures, and cap mesenchyme structures.

The method for obtaining the kidney tissue-like culture of the present invention is not particularly limited, but may be produced by the manufacturing method of the present invention.

The renal tissue-like culture of the present invention may contain cells of human origin. As an example, when the renal tissue-like culture of the present invention is obtained by the culture method of the present invention or produced by the production method of the present invention, one or more cells selected from nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells may be cells of human origin, and it is preferable that all of the nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells are of human origin. In addition, it is preferable that the renal tissue-like culture of the present invention is composed of cells of human origin.

The renal tissue-like culture of the present invention may have a mesangial structure, a branched ureteric bud structure, an S-shaped body-like structure, and a cap mesenchyme structure. Details of the mesangial structure, the branched ureteric bud structure, the S-shaped body-like structure, and the cap mesenchyme structure are as described above.

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

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

The present invention will be specifically explained below based on examples, but the present invention is not limited to the following aspects.

<Late primitive streak (PPS) induction>
Human iPSCs (hiPSCs) were first treated with a 1:1 mixture of TrypLE Select Enzyme (Thermo Fisher Scientific) and 0.5 mM EDTA/PBS for 5 min, then washed with PBS(-). They were then detached using a cell scraper and gently pipetted to dissociate into single cells. One day before the start of differentiation, hiPSCs were seeded at a density of 1.3 × ¹⁰⁴ cells/ ^cm2 in a 24-well plate (Corning) with 0.5 mL of Stem Fit AK02N (Ajinomoto Healthy Supply) supplemented with 10 μM Y-27632 and 2.4 μL/mL iMatrix-511 (Matrixome). After 24 hours (day 0), the cells were cultured in serum-free differentiation medium (hereafter referred to as basal medium) consisting of DMEM/F12 Glutamax (Thermo Fisher Scientific), B27 supplement minus vitamin A (Thermo Fisher Scientific), and 0.5×Penicillin/Streptomycin, supplemented with 5 μM CHIR99021 (Wako), 10 nM retinoic acid (RA) (Sigma), 1 ng/mL BMP4 (Peprotech), and 100 ng/mL bFGF (Wako). After another 24 hours (day 1), the cells were cultured in basal medium containing 5 μM CHIR99021, 100 ng/mL bFGF, and 1 ng/mL BMP7 (R&D Systems). On day 2, the medium was switched to basal medium containing 5 μM CHIR99021, 1 ng/mL BMP7, and 10 μM A83-01 (Wako). On day 3, the medium was switched to basal medium containing 5 μM CHIR99021, 30 ng/mL bFGF, 10 ng/mL activin A (R&D Systems), and 30 μM Y-27632 (Wako). On day 4, cells were washed with PBS(-), treated with Accumax (Innovative Cell Technologies), dissociated into single cells by gentle pipetting, and seeded at a density of 1.05 × ¹⁰⁵ cells/ ^cm2 in 24-well plates (2D culture) or at a density of 2.0 × ¹⁰⁴ cells/ ^cm2 in 96-well low cell attachment plates (Thermo Fisher Scientific) (3D culture). The cells were cultured for 48 hours using basal medium containing 5 μM CHIR99021, 30 ng/mL bFGF, 10 ng/mL activin A, and 30 μM Y-27632 (additionally containing 1.2 μL/well iMatrix-511 for 2D culture).

<Induction of nephron progenitor cells (NPCs)>
On day 6 of PPS induction, the medium was switched to one containing 0.1 μM RA and 25 ng/mL NOGGIN (Peprotech), and on day 8, the medium was switched to one containing 200 ng/mL FGF9 and 1 μM CHIR99021, and the cells were cultured for 72 hours.

<Induction of renal interstitial progenitor cells (IPCs)>
On day 4 of PPS induction, dissociated cells were seeded at a density of 2.0 × 104 cells/well in 96-well U-bottom plates (Sumitomo Bakelite or Thermo Fisher Scientific) with basal medium containing 5 μM CHIR99021, 30 ng/mL bFGF, ¹⁰ ng/mL activin A, and 30 μM Y-27632 to form aggregates and cultured for 48 h. On day 6, cells were cultured in AK02N minus bFGF medium or basal medium containing 1 μM CHIR99021, 0.1 μM RA, 500 nM smoothened agonist (SAG) (Selleck), and 10 ng/mL IL-1β (Wako). On day 8, the cells were cultured for 72 hours in AK02N minus bFGF medium or basal medium containing 1 μM CHIR99021, 0.1 μM RA, 500 nM SAG, and 10 μM SB431542.

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

<Reconstruction of kidney structure>
The NPC, IPC and ND cells induced as described above were incubated with Accumax at 37° C. for 10 minutes and then gently pipetted to dissociate them into single cells. Dissociated single cells were mixed and resuspended in basal medium containing 1 μM CHIR99021, 0.1 μM RA, 100 ng/mL FGF9, and 10 μM Y-27632 (CRFY), CRFY plus 200 ng/mL GDNF (CRFY+G), or basal medium containing 0.1 μM RA, 100 ng/mL FGF9, and 10 μM Y-27632, 200 ng/mL GDNF, 10% Afamin/Wnt3a Conditioned Medium (MBL; J-ORMW301R) and 200 ng/mL R-spondin1 protein (RSPO1) (R&D) (RFY+G+WR) and plated in 96-well low cell-binding U-bottom plates at a density of 1.7 × 10 ⁴ cells/well for NPCs, 1.7 × 10 6 cells/well for 1 h. IPCs were seeded at a density of ⁴ cells/well and ND cells at a density of 1.7 × ¹⁰⁴ cells/well to form aggregates. After 48 hours, the mixed aggregates were transferred to a 24-well plate and incubated at 37°C for 30 minutes with the same medium as above supplemented with 20 μL of 50% Matrigel. After some solidification, 180 μL of the same medium as above was added. Half of the medium was then replaced every 2 days.

Renal structure was reconstructed under CRFY conditions, and immunohistochemical analysis of the tissue-like cultures on day 6 revealed that CALB1(+) epithelial structures were reconstructed, and SIX2(+) NPCs and FOXD1(+) IPCs surrounded the ureteric bud (UB)-like epithelial structures (Figure 1). However, no RET expression was observed in the UB tip-like structures.
In our previous report (Mae, SI, et al. Cell Rep. 32. 2020), the medium used for cell proliferation at the tip of the ureteric bud contained GDNF. Therefore, renal structure was reconstructed under CRFY+G conditions, in which GDNF was added to CRFY. In tissue-like cultures on day 6 under these conditions, quantitative RT-PCR (qT-PCR) analysis showed that RET expression was slightly increased, while other progenitor cell markers, such as SIX2 and FOXD1, were unaffected (Figure 2).

On the other hand, in tissue-like cultures on the 6th day after reconstitution of kidney structure under RFY+G+WR conditions, in which GDNF was added to CRFY and Wnt3a and RSPO1 were used instead of CHIR99021, qRT-PCR analysis showed that RET expression was further increased compared to those under CRFY+G conditions (Figure 2). Furthermore, immunostaining analysis of the cultures showed that the budded UB tips were RET positive (Figure 3), and nephrogenic region-like structures were formed, such as SIX2(+) or PAX8(+) cap mesenchyme structures (Figure 4) and S-shaped structures consisting of the HNF4α(+) proximal region and the BRN1(+) mid-distal region (Figure 5).

Mesangial cells originate from Foxd1(+) IPCs and migrate through the glomerular tuft during development. In the glomeruli of embryonic kidneys at the capillary loop stage, podocytes surround Pdgfrb(+) mesangial cells to form mesangial structures. Consistent with this, renal structure reconstruction under RFY+G+WR conditions revealed glomerular-like structures with PDGFRB(+) or GATA3(+) mesangial-like cells, i.e., mesangial structures, in tissues at the capillary loop stage on days 6 and 12 and tissues at the mature stage on day 12 (Fig. 6). When IPCs constitutively expressing enhanced green fluorescent protein (EGFP) and non-fluorescent NPCs were cultured under RFY+G+WR conditions, EGFP(+)PDGFRB(+) mesangial-like structures were observed, confirming that the mesangial-like cells were derived from IPCs.

　From the above, by co-culturing NPC cells, IPC cells and ND cells under RFY+G+WR conditions, appropriate UB structures, kidney-forming areas and mesangial structures could be formed. In other words, by co-culturing NPC cells, IPC cells and ND cells (ureteric bud cells) under culture conditions containing Wnt3a, a Wnt signal activator, renal tissue-like cultures with the structure of kidneys at the organogenesis stage were created.

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

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

<Reconstruction of kidney structure using ureteric bud organoids and NPCs>
The NPCs induced as described above were left to stand with Accumax for 10 minutes at 37°C and gently pipetted to dissociate into single cells. The dissociated single-cell NPCs were resuspended in basal medium (RFY+G+WR) containing 0.1 μM RA, 100 ng/mL FGF9, and 10 μM Y-27632, 200 ng/mL GDNF, 10% Afamin/Wnt3a Conditioned Medium, and 200 ng/mL R-spondin1 protein (RSPO1). The suspension of NPCs was seeded on the ureteric bud organoids induced as described above at a density of 5 × ¹⁰⁴ cells/well to form aggregates. After 48 hours, the mixed aggregates were transferred to a 24-well plate and left to stand for 30 minutes at 37°C with the same medium as above plus 20 μL of 50% Matrigel. After some solidification, 180 μL of the same medium as above was added. Then, half of the medium was replaced with basal medium every two days.

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

From the above, by co-culturing NPC cells (including some IPC cells) with ureteric bud organoids under RFY+G+WR conditions, appropriate UB structures and kidney-forming regions could be formed. In other words, by co-culturing NPC cells, IPC cells, and ureteric bud organoids (ureteric bud cells) under culture conditions containing Wnt3a, a Wnt signal activator, renal tissue-like cultures with the structure of kidneys at the organogenesis stage were created.

Claims (12)

A method for producing a renal tissue-like culture, comprising the step of co-culturing nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells in a medium containing a Wnt signal activator. The method according to claim 1, wherein the co-culture is performed by suspension culture. The method according to claim 2, wherein the suspension culture is performed in a state where aggregates of nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells are coated with an extracellular matrix. The method of claim 1, wherein the Wnt signal activator is a Wnt3a protein. The method according to claim 1, wherein the medium further contains fibroblast growth factor 9 (FGF9). The method according to any one of claims 1 to 5, wherein the medium further contains retinoic acid (RA) and/or a derivative thereof, and glial cell line-derived neurotrophic factor (GDNF). The method according to claim 6, wherein the medium further contains α-albumin and R-Spondin 1 (RSPO1). The method according to any one of claims 1 to 5, wherein the kidney tissue-like culture has one or more of a mesangial structure, a branching ureteric bud structure, an S-shaped body-like structure, and a cap mesenchyme structure. The method according to any one of claims 1 to 5, wherein the nephron progenitor cells, renal interstitial progenitor cells, and ureteric bud cells are derived from a human. The method according to any one of claims 1 to 5, wherein one or more of the nephron progenitor cells, the renal interstitial progenitor cells, and the ureteric bud cells are cells induced from pluripotent stem cells. 1. A kidney tissue-like culture comprising:
Contains cells of human origin,
The cultures contained mesangial structures, branching ureteric bud structures, S-body-like structures, and cap mesenchyme structures. The mesangial structure is a structure containing PDGFRB and/or GATA3 positive cells within a glomerulus-like structure,
The branched ureteric bud structure has two or more ureteric buds, and the tips of the ureteric buds contain RET-positive cells;
the S-shaped structure comprises an S-shaped structure comprising a proximal region and a distal region,
The renal tissue-like culture of claim 11, wherein the cap mesenchyme structure is a structure that surrounds the tip of the ureteric bud and contains SIX2 and/or PAX8 positive cells.

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