JP7274683B2 - METHOD FOR GENERATING KIDNEY CONSTRUCTION WITH DENDRITIC DRANGED COLLECTING ductS FROM PLIPOTENTIAL STEM CELLS - Google Patents

Description

Application of Section 30(2) of the Patent Act November 9, 2017, Cell Stem Cell, 2017 Dec 7;21(6):730-746. e6. doi: 10.1016/j. stem. 2017.11.011. Presented at Epub 2017 Nov 9

The present invention relates to a method for producing Wolffian tube (WD) progenitor cell-like cells, a method for producing ureteroblast-like cells, and a method for producing kidney structures using pluripotent stem cells. The method for producing WD progenitor-like cells includes the step of obtaining CXC chemokine receptor 4 (Cxcr4)-positive and KIT proto-oncogene receptor tyrosine kinase (KIT)-positive cells.

Kidneys are formed from two main groups, three types of progenitor cells and blood vessels. The three types of progenitor cells are (1) nephron progenitor cells, which are the basic cells of the nephron, a functional unit that controls kidney filtration, and (2) nephron progenitor cells that exist around the nephron progenitor cells and promote their formation. (3) ureteric buds, cells that become urine-collecting and excreting tubes made of nephrons, among these: Since (1) and (2) are closely related, they are collectively called metanephric mesenchyme.

The present inventors have so far succeeded in inducing progenitor cells of nephrons, which are functional modules of the kidney, and nephron tissue from pluripotent stem cells, and have reported this for the first time in the world.
On the other hand, there is a report that ureteroblasts, which form the basis of the excretory tract, which is another component of the kidney, were induced (Non-Patent Documents 1 and 2). None of the dendritic branching structures could be reproduced. For example, in Non-Patent Document 2, ureteroblasts and nephron progenitor cells are simultaneously induced, but the ureteroblasts do not have dendrite branching ability.
In order for the kidney to work, the urine that is filtered from the blood by the nephron must be connected to a collecting duct that has one outlet, and many tubes connected to nephrons have one outlet. For this purpose, it is necessary to reproduce dendritic branching in which a single projection called a ureteric bud repeatedly bifurcates at the tip to increase the number of branches. In addition, it is necessary to maintain a progenitor cell niche for regenerated organs equivalent to the fetal period to grow to a sufficient size. Nothing is reported.

Xia Y et al., Nat Cell Biol. 2013 Dec;15(12):1507-15. Takasato M et al., Nature. 2015 Oct 22;526(7574):564-8.

An object of the present invention is to provide a method for producing WD progenitor-like cells and ureteroblast-like cells that can be induced to differentiate into ureteroblast-like cells.

The present inventors have clarified the signals required for the process of ureteric bud formation and identified a cell surface antigen that can sort and purify WD progenitor-like cells that can be induced into ureteric bud-like cells. By doing so, we succeeded in inducing ureteroblast-like cells from mouse ES cells and human iPS cells. By co-culturing the induced ureteroblast-like cells with nephron progenitor cells, we formed a three-dimensional dendritic structure and succeeded in reproducing the higher-order structure of the fetal kidney with progenitor cell niches.
The inventors further conducted extensive studies based on this discovery, and completed the present invention.
Accordingly, the present invention includes the following aspects:
[1] Step A: producing Wolffian tube (WD) progenitor-like cells, including the step of obtaining CXC motif chemokine receptor 4 (Cxcr4)-positive and KIT proto-oncogene receptor tyrosine kinase (KIT)-positive cells How to make.
[2] in the step of obtaining Cxcr4-positive and KIT-positive cells, Cxcr4-positive and KIT-positive cells account for 30% or more, preferably 50% or more, more preferably 70% or more, and still more preferably 80% or more of all cells; More preferably, the method according to [1] above, which is characterized by a step of selecting cells with 90% or more.
[3] A group in which Cxcr4-positive and KIT-positive cells further consist of Paired box (Pax) 2, LIM homeobox (Lhx) 1, empty spirals homeobox (Emx) 2, ret proto-oncogene (RET) and homeobox (HOX) B7 The method of [1], wherein at least two, preferably at least three, and more preferably all selected from are expressed.
[4] Steps B1, B2, C and D below:
Step B1 step of culturing pluripotent stem cells in a medium containing activin A or a tumor growth factor (Tgfb1 or Tgfb2) (preferably activin A),
step B2 culturing the cells obtained in step B1 in a medium containing a Wnt agonist (preferably a Glycogen Synthase Kinase (GSK)-3β inhibitor, more preferably CHIR99021 or SB216763);
Step C The cells obtained by step B2 are treated with retinoic acid (RA) or an RA analogue (preferably RA or AGN193109), fibroblast growth factor (FGF2, FGF4, FGF7, FGF9 or FGF20, preferably FGF9), and TGFβ. culturing in a medium containing a signal pathway inhibitor or Wnt agonist (preferably SB431542 or A83-01);
Step D Cells obtained by Step C are treated with RA or an RA analogue (preferably RA or AGN193109), a Wnt agonist (preferably a GSK-3β inhibitor, more preferably CHIR99021 or SB216763), and a fibroblast growth factor (FGF2 , FGF4, FGF7, FGF9, or FGF20, preferably FGF9 or FGF20, more preferably FGF9).
(However, the components in each step may be the same substance or different substances.)
The method according to any one of [1] to [3], further comprising
[5] The method of [4], wherein step A is performed by selecting Cxcr4-positive and KIT-positive cells from the cells obtained in step D.
[6] The method of [4] or [5], wherein the pluripotent stem cells are embryonic stem cells.
[7] The method of [4] or [5], wherein the pluripotent stem cells are iPS cells.
[8] The method of [4] or [5], wherein the pluripotent stem cells are human iPS cells.
[9] The medium used in step B1 contains 1 ng/mL to 1000 ng/mL, preferably 1 ng/mL to 100 ng/mL, more preferably 3 to 30 ng/mL of activin A, [4] The method according to any one of -[8].
[10] Any of [4] to [9], wherein the medium used in step B1 further contains a BMP signaling pathway agent (preferably BMP2, BMP4, or BMP7, more preferably BMP2 or BMP4, still more preferably BMP4) or the method described in one.
[11] The medium used in step B1 contains 10 ng/mL or less, preferably 0.1 ng/mL to 10 ng/mL, more preferably 0.3 ng/mL to 3 ng/mL, still more preferably about 1 ng/mL of BMP4. The method according to any one of [4] to [9], comprising
[12] The method according to any one of [4] to [11], wherein the Wnt agonist in steps B2 and D is CHIR99021 or SB216763 (preferably CHIR99021).
[13] The medium used in step B2 contains 1 μM to 1000 μM, preferably 1 μM to 200 μM, more preferably 3 μM to 30 μM, even more preferably about 10 μM of CHIR99021, [4] to [11 ] The method according to any one of
[14] Any of [4] to [13], wherein the medium used in step B2 further contains a BMP signaling pathway agonist (preferably BMP2, BMP4, or BMP7, more preferably BMP2 or BMP4, still more preferably BMP4) or the method described in one.
[15] The method of [10] or [14], wherein the BMP signaling pathway agonist is BMP4.
[16] Any of [4] to [13], wherein the medium used in step B2 contains 10 ng/mL or less, preferably 5 ng/mL or less, more preferably 0.3 ng/mL to 3 ng/mL of BMP4. or the method described in one.
[17] The method according to any one of [4] to [16], wherein the culture time in step B2 is about 1 to 2 days.
[18] The method according to any one of [4] to [16], wherein the culture time in step B2 is about 1.5 days.
[19] The method according to any one of [4] to [16], wherein the culture time in step B1 is about 1 day.
[20] The method according to any one of [4] to [16], wherein the culture time in step B1 is about 1 day and the culture time in step B2 is about 1.5 days.
[21] The method according to any one of [4] to [20], wherein the TGFβ signaling pathway inhibitor or Wnt agonist in step C is a TGFβ signaling pathway inhibitor (preferably SB431542 or A83-01).
[22] Any one of [4] to [20], wherein the medium used in step C contains 1 μM to 1000 μM, preferably 3 μM to 500 μM, more preferably 10 μM to 200 μM SB431542 The method described in .
[23] The medium used in step C contains 10 nM to 1 μM, preferably 10 to 500 nM, more preferably 50 nM to 200 nM, still more preferably about 100 nM of retinoic acid, [4] to [22] The method according to any one of
[24] The medium used in step C contains 10 ng to 1 μg/mL, preferably 10 ng to 500 ng/mL, more preferably 50 ng to 200 ng/mL, still more preferably about 100 ng/mL of FGF9. The method according to any one of [4] to [23], including
[25] The method of any one of [4] to [24], wherein the WD progenitor-like cells are human cells, and the medium used in step C further contains a BMP signaling pathway inhibitor.
[26] The method of [25], wherein the BMP signaling pathway inhibitor used in step C is LDN193189 or Noggin.
[27] The medium used in step C contains 1 nM to 1000 nM, preferably 3 nM to 500 nM, more preferably 10 nM to 200 nM, still more preferably about 100 nM of LDN193189. Method.
[28] The method according to any one of [4] to [27], wherein the culture time in step C is about 1 to 3 days, preferably about 1 to 2 days.
[29] The medium used in step D contains 10 nM to 1 μM, preferably 10 to 500 nM, more preferably 50 nM to 200 nM, still more preferably about 100 nM of retinoic acid, [4] to [28 ] The method according to any one of
[30] The medium used in step D contains 0.1 μM to 100 μM, preferably 1 μM to 100 μM, more preferably 1 μM to 10 μM, even more preferably about 3 μM to 5 μM of CHIR99021, [4] The method according to any one of -[29].
[31] The medium used in step D contains 10 ng to 1 μg/mL, preferably 10 ng to 500 ng/mL, more preferably 30 ng to 300 ng/mL, more preferably about 100 ng/mL of FGF9. The method according to any one of [4] to [30].
[32] The method of any one of [4] to [31], wherein the WD progenitor-like cells are human cells, and the medium used in step D further contains a BMP signaling pathway inhibitor.
[33] The method of [32], wherein the BMP signaling pathway inhibitor used in step D is LDN193189 or Noggin.
[34] The medium used in step D contains 1 nM to 500 nM, preferably 10 nM to 100 nM, more preferably 10 nM to 50 nM, still more preferably about 30 nM of LDN193189. Method.
[35] The method according to any one of [4] to [34], wherein the culture time in step D is about 1 to 3 days, preferably about 1.5 to about 2.5 days.
[36] Step (E) Cxcr4-positive and KIT-positive WD progenitor-like cells are treated with RA or RA analog (preferably RA or AGN193109), Wnt agonist (preferably GSK-3β inhibitor or Rspondin1, more preferably , CHIR99021, SB216763 or Rspondin1), a fibroblast growth factor (FGF2, FGF4, FGF7, FGF9 or FGF20, preferably FGF9) and a ROCK inhibitor (preferably Y27632 or Fasudil hydrochloride). A method for producing ureteroblast-like cells, comprising:
[37] The method of [36], wherein the Cxcr4-positive and KIT-positive WD progenitor-like cells are cells obtained by the method of [1] or [2].
[38] The medium used in step (E) has a concentration of 0.1 ng/mL to 100 ng/mL, preferably 0.5 ng/mL to 50 ng/mL, more preferably 2 ng/mL to 10 ng/mL, even more preferably contains about 5 ng/mL FGF9.
[39] The medium used in step (E) contains 10 nM to 1 μM, preferably 10 nM to 500 nM, more preferably 50 nM to 200 nM, even more preferably about 100 nM RA, [36] The method according to any one of [38].
[40] The method of any one of [36] to [39], wherein the ROCK inhibitor is Y27632.
[41] Any of [36] to [39], wherein the medium used in step (E) contains 1 μM to 1000 μM, preferably 1 μM to 100 μM, more preferably 1 μM to 50 μM, further preferably about 10 μM Y27632. The method described in one.
[42] The method of any one of [36] to [41], wherein the Wnt agonist is CHIR99021 or Rspondin1.
[43] The medium used in step (E) contains 0.1 μM to 100 μM, preferably 0.1 μM to 10 μM, more preferably 0.3 μM to 5 μM, even more preferably about 1 μM of CHIR99021, [36]- The method of [41].
[44] The cells for WD progenitor cells are human cells, and the medium used in step (E) further contains FGF1 and BMP signal pathway inhibitors (preferably LDN193189 or Noggin) [36]-[43] The method according to any one of
[45] The medium used in step (E) has a concentration of 10 ng/mL to 1000 ng/mL, preferably 10 ng/mL to 500 ng/mL, more preferably 50 ng/mL to 200 ng/mL, even more preferably contains about 100 ng/mL FGF1.
[46] The method of any one of [44] to [45], wherein the BMP signaling pathway inhibitor is LDN193189.
[47] The medium used in step (E) contains 1 nM to 300 nM, preferably 1 nM to 100 nM, more preferably 1 nM to 20 nM, even more preferably about 10 nM of LDN193189, [44] The method according to any one of [45].
[48] Furthermore,
Step (F) The cells obtained in step (E) are treated with RA or an RA analogue (preferably RA or AGN193109), a Wnt agonist (preferably a GSK-3β inhibitor or Rspondin1, more preferably CHIR99021, SB216763 or Rspondin1) , fibroblast growth factors (FGF2, FGF4, FGF7, FGF9, or FGF20, preferably FGF9), ROCK inhibitors (preferably Y27632 or Fasudil hydrochloride), and glial cell line-derived neurotrophic factor (GDNF) or GDNF analogs (preferably BT18) or FGF10.
[49] The medium used in step (F) has a concentration of 0.1 ng to 100 ng/mL, preferably 0.1 ng to 10 ng/mL, more preferably 0.5 ng to 10 ng/mL, further preferably about 1 ng/mL. The method of any one of [36]-[47], comprising the GDNF of
[50] The medium used in step (F) has a concentration of 0.1 ng/mL to 100 ng/mL, preferably 0.5 ng/mL to 50 ng/mL, more preferably 2 ng/mL to 10 ng/mL, more preferably contains about 5 ng/mL FGF9.
[51] The medium used in step (F) preferably contains 10 nM to 1 μM, preferably 10 nM to 500 nM, more preferably 50 nM to 200 nM, still more preferably about 100 nM of RA, [ 48] The method according to any one of [50].
[52] The method according to any one of [48] to [51], wherein the ROCK inhibitor in step (F) is Y27632.
[53] Any one of [48] to [51], wherein the medium used in step (F) contains 1 μM to 1000 μM, preferably 1 μM to 100 μM, more preferably 1 μM to 50 μM, further preferably about 10 μM Y27632. The method described in one.
[54] The method of any one of [48] to [53], wherein the Wnt agonist in step (F) is CHIR99021.
[55] The medium used in step (F) contains 0.1 μM to 300 μM, preferably 0.3 μM to 100 μM, more preferably 1 μM to 5 μM, even more preferably about 3 μM of CHIR99021, [48] to [53 ] The method according to any one of
[56] of [48] to [55], wherein the WD progenitor-like cells are human cells, and the medium used in step (F) further contains FGF1 and a BMP signaling pathway inhibitor (preferably LDN193189 or Noggin). A method according to any one of the preceding claims.
[57] The medium used in step (F) has a concentration of 10 ng/mL to 1000 ng/mL, preferably 10 ng/mL to 500 ng/mL, more preferably 50 ng/mL to 200 ng/mL, even more preferably contains about 100 ng/mL FGF1.
[58] The method of [56] or [57], wherein the BMP signaling pathway inhibitor is LDN193189.
[59] The medium used in step (F) contains 1 nM to 300 nM, preferably 1 nM to 100 nM, more preferably 5 nM to 20 nM, more preferably about 10 nM of LDN193189, [56] Or the method of [57].
[60] Furthermore,
Step (G) The cells obtained in step (F) are treated with RA or an RA analogue (preferably RA or AGN193109), a Wnt agonist (preferably a GSK-3β inhibitor or Rspondin1, more preferably CHIR99021, SB216763 or Rspondin1) , a ROCK inhibitor (preferably Y27632 or Fasudil hydrochloride), and GDNF or a GDNF analogue (preferably BT18). Method.
[61] The medium used in step (G) contains 0.1 ng to 100 ng/mL, preferably 0.2 ng to 20 ng/mL, more preferably 0.5 ng to 10 ng/mL, more preferably about 2 ng/mL. The method of [60], comprising the GDNF of
[62] The medium used in step (G) contains 10 nM to 1 μM, preferably 10 nM to 500 nM, more preferably 50 nM to 200 nM, even more preferably about 100 nM RA, [60] Or the method of [61].
[63] The method of any one of [60] to [62], wherein the ROCK inhibitor in step (G) is Y27632.
[64] The medium used in step (G) contains 1 μM to 1000 μM, preferably 1 μM to 100 μM, more preferably 1 μM to 50 μM, even more preferably about 10 μM Y27632, [60] The method according to any one of -[62].
[65] The method of any one of [60] to [64], wherein the Wnt agonist in step (G) is CHIR99021.
[66] The medium used in step (G) contains 0.1 μM to 300 μM, preferably 0.3 μM to 100 μM, more preferably 1 μM to 5 μM, even more preferably about 3 μM of CHIR99021, [60] The method according to any one of [64].
[67] of [60] to [66], wherein the WD progenitor-like cells are human cells, and the medium used in step (G) further contains FGF1 and a BMP signaling pathway inhibitor (preferably LDN193189 or Noggin). A method according to any one of the preceding claims.
[68] The medium used in step (G) has a concentration of 10 ng/mL to 1000 ng/mL, preferably 10 ng/mL to 500 ng/mL, more preferably 50 ng/mL to 200 ng/mL, even more preferably contains about 100 ng/mL FGF1.
[69] The method of [67] or [68], wherein the BMP signaling pathway inhibitor is LDN193189.
[70] The medium used in step (G) contains 1 nM to 300 nM, preferably 1 nM to 100 nM, more preferably 5 nM to 20 nM, more preferably about 10 nM of LDN193189, [67] Or the method of [68].
[71] Ureteroblast-like cells induced from WD progenitor-like cells of Cxcr4-positive and KIT-positive cells, nephron progenitor cells, and Platelet Derived Growth Factor Receptor Alpha (Pdgfra)-positive stromal cell populations derived from embryonic kidney A method of producing kidney organoids comprising co-culturing.
[72] The method of [71], wherein the WD progenitor-like cells are produced by the method of any one of [2] to [35].
[73] The method of any one of [36] to [70], wherein the ureteroblast-like cells express Hnf1b, E-Cadherin and CALB1.
[74] The method of any one of [36] to [70], wherein the ureteroblast-like cells express Emx2, Wnt11, Hnf1b, E-Cadherin and CALB1.
[75] medium B1 containing activin A,
Medium B2 containing a Wnt agonist (preferably a GSK-3β inhibitor, more preferably CHIR99021 or SB216763),
Medium C containing RA or an RA analogue, a fibroblast growth factor (either FGF2, FGF9 or FGF20) and a TGFβ signaling pathway inhibitor (preferably SB431542 or A83-01) and
Medium D containing RA or RA analog, Wnt agonist (preferably GSK-3β inhibitor, more preferably CHIR99021 or SB216763) and fibroblast growth factor (either FGF2, FGF9 or FGF20)
A kit for producing WD progenitor-like cells from pluripotent stem cells, comprising:
[76] The kit of [75] and RA or RA analog, Wnt agonist (preferably GSK-3β inhibitor or Rspondin1, more preferably CHIR99021, SB216763 or Rspondin1), fibroblast growth factor (FGF2, FGF9) , or FGF20) and medium E containing a ROCK inhibitor (preferably Y27632 or Fasudil hydrochloride) for producing ureteroblast-like cells from pluripotent stem cells.
[77] The kit of [75] or [76], further comprising a CXCR4 antibody and a KIT antibody.

According to the methods provided in the present invention, in vitro, mouse fetal ureteric buds have commensurate dendritic branching ability, maintain progenitor cell niches at the tips of branches, and connect with individual nephrons. It may be possible to generate ureteroblast-like cells that are capable of forming renal organoids. According to the present invention, by obtaining Cxcr4-positive and KIT-positive cells, it is possible to obtain cells that can differentiate into functional ureteroblast-like cells, and as a result, ureters that can form branched ureteric buds. It may be possible to obtain blast-like cells. In addition, according to the method of the present invention, it is possible to obtain Cxcr4-positive and KIT-positive cells from pluripotent stem cells with high efficiency, and therefore, it is possible to efficiently induce functional ureteroblast-like cells. can be possible.
According to the method for producing WD progenitor-like cells provided in the present invention, (I) growth promoting branching or mixing with metanephric mesenchyme, which could not be derived from pluripotent stem cells so far. When cultured in factor, it undergoes branching; (II) when mixed with metanephric mesenchyme, it has the ability to form a progenitor cell niche that maintains the undifferentiated nature of the nephron progenitor cells therein; (III) When mixed with metanephric mesenchyme, it may allow the production of ureteroblast-like cells characterized by their ability to induce the differentiation of nephron progenitor cells therein into nephrons. The ureteroblast-like cells produced using the method provided by the present invention can form, together with nephron progenitor cells and stromal cells, kidney organoids having a dendritic branching structure of collecting ducts connecting nephrons to each other. . The kidney organoids successfully reconstructed by the present inventors are the first in the world to reproduce the higher-order structure of the kidney, so the present invention will enable the creation of functional artificial kidneys in the future. It can be an indispensable technology for making

An outline of the protocol for inducing ureteric buds of the present invention (upper diagram), and an outline of the protocol for inducing nephron progenitor cells for creating higher-order embryonic kidney structures together with ureteric buds prepared according to the present invention ( Figure below). BRIEF DESCRIPTION OF THE FIGURES Figure 1 outlines the protocol for induction of ureteral buds from mouse embryonic stem cells. A10: 10 ng/mL activin; B0.3: 0.3 ng/mL Bmp4; C5: 5 μM CHIR; C10: 10 μM CHIR; R: 0.1 μM retinoic acid; . Although the concentration and period are described in the drawing as a preferred embodiment of the present invention, the present invention is not limited to this. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 outlines a protocol for induction of ureteric buds from human iPS cells. A10: 10 ng/mL activin; B1: 1 ng/mL Bmp4; C1, C3, C5, C10: 1, 3, 5 or 10 μM CHIR, respectively; R: 0.1 μM retinoic acid; F9-100, F9-5: 100 or 5 ng/mL Fgf9, respectively; SB100: 100 μM SB431542; LDN10, 30, 100: 10, 30 or 100 nM LDN193189, respectively. . Although the concentration and period are described in the drawing as a preferred embodiment of the present invention, the present invention is not limited to this. Fig. 2 shows a schematic diagram of the WD generation process; Portions of WD utilized for reconstruction assays or microarray analysis are outlined by dashed lines. *P<0.05 and **P<0.01 for the number of epithelial tips measured in reconstructed bifurcated ureters. It is the analysis result of gene expression dynamics by qRT-PCR analysis. Gene expression levels at each indicated stage of sorted WD or UB are shown. Relative expression of each transcript to β-actin expression is shown (n=3). Results showing that retinoic acid, Wnt and Fgf/Gdnf signaling mature WD progenitor cells into ureteric buds. Relative expression of each transcript to β-actin is shown as mean±s.e.m. (n=4). Y: Y27632 (Rock inhibitor); R: retinoic acid, C: 3 μM CHIR 99021 (canonical Wnt agonist), C1: 1 μM CHIR 99021, C3: 3 μM CHIR 99021, F: 100 ng/mL Fgf9, F5: 5 ng/mL Fgf9 , G1: 1 ng/ml GDNF, G2: 2 ng/ml GDNF. (Upper) shows the results of two days of in vitro differentiation of the selected E9.5WD. (Lower panel) Results of day 1 in vitro differentiation of selected E8.75WD are shown. It is a bright-field image and a GFP fluorescence image of the induced ureteric bud on the third day of culture (Day 3). Scale bar, 100 μm. This is the result of analyzing the dynamics of marker genes over time. Expression levels in in vivo WD are shown to the left of each marker set. Relative expression of each transcript to β-actin expression is shown as the mean±s.e.m. (n=4). (Upper panel) FACS analysis of WD marker gene expression. Left panel: Analysis of Hoxb7-GFP and Flk1. Hoxb7-GFP+/Flk1-WD progenitor cell fractions are boxed. Right panel: Analysis of Kit/Cxcr4 expression in Hoxb7-GFP+/Flk1-negative WD progenitor cells. The Kit+/Cxcr4+ WD progenitor cell fraction is boxed. (Lower diagram) It is a diagram showing the specificity of Cxcr4 and Kit strongly positive fractions (WD progenitor cell fractions) in the whole fetus as markers. It is the result of FACS analysis of D6.25 by regulation of differentiation factors in Step 4. Results are presented as mean±s.e.m. (n=3). NO: no addition; RC: 0.1 μM retinoic acid + 5 μM CHIR99021; RF: 0.1 μM retinoic acid + 100 ng/mL Fgf9; CF: 5 μM CHIR99021 + 100 ng/mL Fgf9; It is the result of FACS analysis on Day 6.25 (UB) or Day 8.5 (MM) by adjusting the Bmp4 concentration in Step 2. Results are presented as mean±s.e.m. (n=3). B0C10: 0 ng/mL Bmp4 + 10 μM CHIR, B0.3C10: 0.3 ng/mL Bmp4 + 10 μM CHIR, B1C10: 1 ng/mL Bmp4 + 10 μM CHIR. It is the result of FACS analysis on Day 6.25 (UB) or 8.5 (MM) by adjusting the activin A concentration in Step 1. Results are presented as mean±s.e.m. (n=3). A0: 0 ng/mL activin, A1: 1 ng/mL activin, A3: 3 ng/mL activin, A10: 10 ng/mL activin, A30: 30 ng/mL activin. FIG. 14 shows optimization of activin/Bmp concentration at the patterning stage of epiblasts using mouse ES cells. Effect of Activin/Bmp concentration at the patterning stage of the epiblast on UB (left table and graph) versus MM (right table and graph) fate decisions. It shows the dynamics of marker genes over time from immature mouse ES cells at Day 0 to the WD progenitor stage at Day 6.25 (corresponding to WD progenitor cells at E8.75). Expression levels in E8.75 embryonic WD progenitor cells are indicated by triangles. Relative expression of each transcript relative to β-actin expression is shown as mean±s.e.m. (n=3). A summary of the in vitro sorted E8.75WD progenitor cell differentiation protocol is shown. Y: Y27632 (Rock inhibitor); R: 0.1 μM retinoic acid, C1: 1 μM CHIR 99021, C3: 3 μM CHIR 99021, F9-5: 5 ng/mL Fgf9, G1: 1 ng/ml GDNF, G2: 2 ng/ml GDNF . UB at Day 9.25 of differentiation induced from mouse embryonic stem cells. Left panel: Low magnification image of the whole spheroid. A fluorescence image is shown below. Right panel: Magnification of manually isolated induced UB. Fluorescent images are shown on the right. Scale bar, 100 μm. Figure 2 shows the time-course dynamics of marker genes of WD progenitor cells induced from mouse embryonic stem cells. Expression levels in E11.5 embryonic UB are indicated by triangular dots. Relative expression of each transcript to β-actin expression is shown as mean±s.e.m. (n=3). The left figure of FIG. 19 is a time-lapse image of the reconstructed organoid. Each time point from Day 1.5 (D1.5) to Day 6 (D6) is indicated. Arrows and numbers indicate the generation numbers of the indicated bifurcations. Fluorescent images are shown in the lower panel. Scale bar, 100 μm. The right panel shows the total tip number of organoids reconstituted by UB and induced UB as mean±s.e.m. (n=6). P=0.53. The left figure is a 3D projection image of an immunostained 7-day organoid. Upper panel: merged image of CK8 and Six2. Lower panel: Images stained for CK8. Scale bar, 100 μm. The figure on the right is a 3D projection of an immunostained day 7 organoid. Each displayed molecule was stained with a single color (left four panels) or a merged image (merged: rightmost panel). Scale bar, 200 μm. The lower right panel is a section image of an immunostained day 7 organoid. The UB tip area was enlarged. Scale bar, 20 μm. Immunostaining results of mouse ES cell-derived nephron progenitor cells, induced UB and organoids reconstituted by embryonic stromal cells. Left two panels: 3D projections of organoids. Right two panels: organoid sections. It is the result of confirming the influence of each differentiation factor in Step 3 of the method of inducing ureteric buds from human iPS cells. The left figure shows data confirming the expression of specific ureteric bud marker genes. The relative expression of each transcript relative to β-actin expression was analyzed on day 4.5, and the mean value ± s.e.m. (n = 4). Indicated. The figure on the right shows the results of FACS analysis on Day 6.25. The induction rate of the CXCR4-positive/cKIT-positive fraction is shown as mean±s.e.m. (n=3). R: 0.1 μM retinoic acid, F: 100 ng/mL Fgf9, L: LDN 100 nM, S: SB 100 μM. It is the result of confirming the differentiation induction efficiency by adjusting the activin concentration in Step 1 when using human iPS cells. The left figure shows the results of FACS analysis of the ureteric bud induction rate on Day 6.25, and the right figure shows the results of FACS analysis of the nephron progenitor induction rate on Day 12. In the Bmp4 addition condition, 1 ng/mL Bmp4 was added. Results are presented as mean±s.e.m. (n=3). FIG. 10 shows optimization of activin/Bmp concentrations at the patterning stage of epiblasts using human iPS cells. Effect of Activin/Bmp concentration on UB (upper table and graph) versus MM (lower table and graph) fate decisions at the epiblast patterning stage of human iPS cells. FIG. 25 shows the results of FACS analysis on Day 6.25 (UB lineage) or Day 12 (MM lineage) by adjusting the Bmp concentration in Step 2. 1 ng/mL of Bmp4 was added under the Bmp4 addition condition. Results are presented as mean±s.e.m. (n=3). L30C10: 30 nM LDN + 10 μM CHIR, B0C10: 0 ng/mL Bmp4 + 10 μM CHIR, B1C10: 1 ng/mL Bmp4 + 10 μM CHIR. It is the result of FACS analysis of CXCR4/KIT expression on Day 6.25 of human iPS cells induced under optimized conditions. It is the result of analysis of the dynamics of WD marker genes over time in human iPS cells induced under optimized conditions. The relative expression of each transcript to β-actin expression is shown as mean±s.e.m. (n=3). Bright-field image of induced UB branches in 50% Matrigel culture environment. Scale bar, 200 μm. 3D projections of immunostained day 13 organoids are shown. The left figure is an image stained with CK8 and SOX9. The right figure is an image stained with PAX2 and E-cadherin. Scale bar, 200 μm. FIG. 2 shows cell-autonomous requirement for PAX2 in human UB differentiation. Fig. 2 shows bright-field images of aggregates after maturation culture from Day 8.5 to Day 12.5, and aggregates of selected WD progenitor cells on days 3 and 12 of branch culture. FIG. 2 shows the effects on the number of nephrons formed when mouse metanephric mesenchyme is transplanted in combination with fetal spinal cord tissue (left) or mouse ES cell-derived induced ureteric buds (right). The photograph is an image collected 15 days after transplantation of the tissue into an immunodeficient mouse. The punctate structures are glomeruli formed from metanephric mesenchyme. The number of nephrons formed by counting the number of glomeruli was estimated, quantified, and compared in the graph on the right. SC: co-culture with fetal spinal cord, iUB: co-culture with induced ureteral buds.

The present invention will now be described in detail by way of illustrative embodiments, but the present invention is not limited to the embodiments described below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition, any materials and methods equivalent or similar to those described herein can likewise be used in the practice of the invention.
Also, all publications and patents cited herein in connection with the invention described herein, for example, as indicative of methods, materials, etc., which may be used in the invention, are hereby incorporated by reference. It constitutes a part.
As used herein, "and/or" is used in the sense of including either one or both. As used herein, "about" is used in the sense of allowing ±10%.
As used herein, the term kidney organoids refers to differentiated nephrons consisting of glomeruli and renal tubules, nephron progenitor cells, interstitial cells, and higher-order dendritic structures of collecting ducts that connect the differentiated nephrons to each other. It means structured kidney-like tissue.

The details of the present invention are described below.
FIG. 1 shows an overview of the method for producing Wolffian tube (WD) progenitor cell-like cells of the present invention and the protocol for inducing ureteric buds using the same (upper diagram), and an embryo together with the ureteric buds prepared according to the present invention. FIG. 2 is a diagram showing a schematic (lower diagram) of a protocol for induction of nephron progenitor cells to create a higher-order structure of the sex kidney. Each step will be described below.

A method for producing Wolffian tube (WD) progenitor-like cells, comprising a step A of obtaining CXC chemokine receptor 4 (Cxcr4)-positive and KIT proto-oncogene receptor tyrosine kinase (KIT)-positive cells. A method for producing Wolffian tube (WD) progenitor-like cells, comprising the step A of obtaining CXC chemokine receptor 4 (Cxcr4)-positive and KIT proto-oncogene receptor tyrosine kinase (KIT)-positive cells (herein (also referred to as method 1 of the present invention).
When the WD progenitor-like cells produced by the method of the present invention are produced as a cell population, Cxcr4-positive and KIT-positive cells account for 30% or more, preferably 50% or more, more preferably 70% of the total cells. Above, more preferably 80% or more, still more preferably 90% or more, is produced.

As used herein, the term "WD progenitor-like cells" means cells that are destined to differentiate into ureteroblasts with branching ability when given developmentally appropriate stimuli, and are WD progenitors. Cytoid cells are cells that express CXC chemokine receptor 4 (Cxcr4) and KIT proto-oncogene receptor tyrosine kinase (KIT).
A developmentally appropriate stimulus means a stimulus that differentiates WD progenitor cells into ureteroblast-like cells by a method according to the method described in the Examples. The fact that ureteroblast-like cells have the ability to branch means that ureteric buds formed by aggregation of ureteroblast-like cells branch in a dendritic manner.

WD progenitor-like cells, in addition to Cxcr4 and KIT, preferably further comprise Paired box (Pax) 2, LIM homeobox (Lhx) 1, empty spirals homeobox (Emx) 2, ret proto-oncogene (RET) and homeobox (HOX ) express at least two, more preferably at least three, and even more preferably all of B7.
WD progenitor-like cells are preferably FLK1-negative (ie, vascular endothelial growth factor receptor 2 (VEGFR2)-negative) cells.

An example of the above-described developmentally appropriate stimulation includes co-culturing nephron progenitor cells and an embryonic kidney-derived Pdgfra+ stromal cell population by a method according to the method described in the Examples.

Cells (or cell populations) that are positive for a specific marker (e.g., Cxcr4, KIT, etc.) can be identified by, but not limited to, flow cytometry, ie FACS (fluorescence activated cell sorting). It can be separated and obtained. For example, Cxcr4-positive cells are treated with anti-Cxcr4 antibodies (e.g., APC anti-human CD184 (CXCR4) Antibody, Clone 12G5, BioLegend, APC anti-mouse CD184 (CXCR4) Antibody, Clone L276F12, BioLegend). WD progenitor-like cell populations that are Cxcr4 positive can also be separated by a cell sorter based on binding strength to reagents and based on other parameters such as cell size and light scattering. In addition, anti-KIT antibody (e.g., PE anti-human CD117 (c-kit) Antibody, Clone 104D2, BioLegend, CD117 (c-Kit) Monoclonal Antibody (2B8), PE , eBioscience company) can also be used.

Separation of cells (or cell populations) that are positive for a marker can be performed, for example, by FACS using an antibody specific for the marker and an isotype-matched control antibody. A cell can be determined to be positive for the marker if the intensity of staining of the cell with an antibody specific for the marker exceeds the intensity of staining of the cell (or cell population) with an isotype-matched control antibody. . Also, when there is no difference in the intensity of staining of cells with an antibody specific for the marker and the intensity of staining of cells (or cell populations) with an isotype-matched control antibody, the cells are negative for the marker. can be determined to be

Cells positive for a particular marker can also be enriched, depleted, separated, sorted, and/or purified using conventional affinity or antibody techniques. For example, to the ligand and/or antibody, a label, such as a magnetic bead; biotin, which binds with high affinity to avidin or streptavidin; a fluorescent dye that can be used in a fluorescence-activated cell sorter; a hapten; Conjugation of likes and the like can also facilitate isolation of specific cell types.

In one aspect of the present invention, the method of the present invention comprises the step of sorting Cxcr4-positive and KIT-positive cells with a cell sorter.

Method for Inducing WD Progenitor-Like Cells from Pluripotent Stem Cells Further, the present invention provides a method for inducing WD progenitor-like cells from pluripotent stem cells (also referred to as the WD induction method of the present invention).
Specifically, the present invention provides
Step B1 step of culturing pluripotent stem cells in a medium containing activin or a tumor growth factor (Tgfb1 or Tgfb2),
Step B2 step of culturing the cells obtained in step B1 in a medium containing a Wnt agonist (preferably a GSK-3β inhibitor);
Step C Retinoic acid (RA) or RA analogues (AGN193109, AM580, AM80, BMS453, BMS195614, AC 261066, AC55649, Isotretinoin), fibroblast growth factors (FGF2, FGF4, FGF7, FGF9) , or FGF20) and a TGFβ signaling pathway inhibitor or a Wnt agonist (preferably, a GSK-3β inhibitor).
Step D Cells obtained by Step C are treated with RA or RA analogs (AGN193109, AM580, AM80, BMS453, BMS195614, AC 261066, AC55649, Isotretinoin), Wnt agonists (preferably GSK-3β inhibitors), and fibroblasts. A step of inducing WD progenitor-like cells from pluripotent stem cells may be included, including culturing in a medium containing growth factors (FGF2, FGF4, FGF7, FGF9, or FGF20).

Process B1
As used herein, the term "pluripotent stem cells (PSCs)" refers to "self-renewal" that enables proliferation while maintaining an undifferentiated state, and differentiation into all three primary germ layers of the embryo. It can be any undifferentiated cell that retains the "pluripotency" that allows it. Pluripotent stem cells used in the present invention are preferably embryonic stem cells (ES) or induced pluripotent stem cells (iPS cells), more preferably iPS cells.

ES cells are stem cells that can be established from the inner cell mass of early embryos (eg, blastocysts) and have pluripotent and self-renewal proliferative potential. ES cells can be established by removing the inner cell mass from the blastocyst of a fertilized egg and culturing the inner cell mass on fibroblast feeder cells. Methods for establishing and maintaining ES cells are known.

Induced pluripotent stem (iPS) cells are artificial stem cells derived from somatic cells and can be produced by introducing specific reprogramming factors into somatic cells in the form of DNA or protein. , exhibit properties (e.g., proliferation ability based on differentiation pluripotency and self-renewal) similar to ES 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. WO2007/069666). The reprogramming factor is a gene that is specifically expressed in ES cells, its gene product or its non-coding RNA, a gene that plays an important role in maintaining undifferentiated ES cells, its gene product or its non-coding RNA, or It may be composed of low molecular weight compounds. Examples of genes involved in reprogramming factors 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, Glis1 and the like. These reprogramming factors may be used alone or in combination. When the c-MYC gene is used as a reprogramming factor by introducing it into somatic cells, an introduction method is used in which the introduced c-MYC gene is unlikely to be integrated into the chromosome of the target cell after the iPS cells are created. is preferred, and examples thereof include, but are not limited to, introduction using Sendai virus vectors and episomal vectors.

The method for producing ES cells or iPS cells, the method for culturing them, the method for maintaining an undifferentiated state, etc. are known per se, and for example, they are produced and cultured according to the methods described in the literatures exemplified above or the methods described in the examples. be able to.

In the present invention, although not particularly limited, the culture temperature is usually about 30 to 40°C, preferably about 37°C, culture is performed in an atmosphere of CO2-containing air, and the CO2 concentration is about 2-5%, preferably 5%.

The medium used in the present invention can be prepared using a medium used for culturing animal cells as a basal medium. Examples of the basal medium are not limited as long as the desired cells can be obtained. Dulbecco's medium), αMEM (Eagle's minimum essential medium α-modified), etc., and mixtures thereof.

The medium used in the present invention may contain serum or may be serum-free. The medium used in the present invention may optionally include, for example, albumin, N-2 supplement (Thermo Fisher Scientific), B-27 (registered trademark) supplement minus vitamin A (Thermo Fisher Scientific), 2-mercaptoethanol, 1- At least one or more media additives such as thioglycerol, amino acids, L-glutamine, non-essential amino acids, ascorbic acid, etc. may also be included.

Activin A means a homodimer of two inhibin βA chains, and in the present invention, the N-terminal peptide of the inhibin βA chain (for example, NCBI accession number: NP_002183), which is an active form in which the N-terminal peptide is cleaved. It is preferable to use a homodimer in which a Gly311-Ser426 fragment having been cleaved is disulfide-linked. Such activin A can be purchased, for example, from R&D Systems, Inc. (R&D).
The concentration of activin A in the medium used in step B1 of the present invention (also referred to as medium B1 herein) varies depending on the cells used, the culture time, the amount of Wnt agonist used in step B2, etc. Although it is not particularly limited as long as cells can be obtained, it is usually 1 ng/mL to 1000 ng/mL, preferably 1 ng/mL to 100 ng/mL, more preferably 3 ng/mL to 30 ng/mL , more preferably about 10 ng/mL. In step B1, instead of activin A, a tumor growth factor, Tgfb1 or Tgfb2 can be used. As for the concentration of Tgfb1 or Tfgb2 to be used, the concentration of Tgfb1 or Tfgb2 that exhibits an equivalent effect can be set with activin A as a reference.

The medium in step B1 may further contain a ROCK inhibitor. The ROCK inhibitor is not particularly limited as long as it can suppress the function of Rho kinase (ROCK). HA 1100 hydrochloride can be used, preferably Y27632 or Fasudil hydrochloride, more preferably Y27632. When Y27632 is used, the concentration is 1 μM to 1000 μM, preferably 1 μM to 100 μM, more preferably 1 μM to 100 μM, more preferably about 10 μM. In step B1, when other Rock inhibitors are used instead of Y27632, the concentrations showing equivalent effects can be set with reference to Y27632.

The culture time in step B1 is, for example, culture for 5 days or less, preferably 0.5 to 3 days, more preferably about 1 day.

In step B1, about 100 to 100,000 cells can be aggregated to form aggregates and subjected to suspension culture. Although not limited to this, for example, about 1,000 mouse cells and about 10,000 human cells can be used.
Suspension culture refers to culturing cells in a non-adhesive state to an incubator. Although not particularly limited, it can be carried out by a method using a material that has not been artificially treated (for example, coating treatment with an extracellular matrix or the like) for the purpose of improving adhesion to cells. Although not particularly limited, examples of such cell non-adhesive incubators include V-bottom 96-well low cell binding plates (Sumitomo Bakelite).

When using human pluripotent stem cells (e.g., human iPS cells), medium B1 further contains a BMP signaling pathway agent (preferably preferably contains BMP2, BMP4 or BMP7, more preferably BMP2 or BMP4, even more preferably BMP4). The concentration of BMP4 in the medium B1 of the present invention varies depending on the cells used, the culture time, the amount of the BMP signaling pathway agonist used in step C, the amount of the Wnt agonist, etc., and is particularly limited as long as the desired cells can be obtained. However, for example, it is 10 ng/mL or less, preferably 0.1 ng/mL to 10 ng/mL, more preferably 0.3 ng/mL to 3 ng/mL, further preferably about 1 ng/mL be. When other BMP signaling pathway agonists are used, the concentration can be appropriately selected so as to exhibit the same effects as those obtained when BMP4 is used.

When using mouse pluripotent stem cells (e.g., mouse ES cells), dissociate the cells with Accutase (ESGRO) or the like, aggregate them at about 1,000 cells per aggregate, and then aggregate the cells except that activin A is not included. It is preferable to culture for about 2 days in a medium having the same components as medium B1 of the invention, and subject the resulting culture to step B1 of the invention.

Process B2
Immature mesodermal cells can be obtained by culturing the culture result obtained by the culture in step B1 above in a medium containing a Wnt agonist (preferably a GSK-3β inhibitor).

A Wnt agonist is defined as an agent that activates TCF/LEF-mediated transcription in cells. Wnt agonists are therefore selected from bona fide Wnt agonists that bind to and activate members of the Frizzled receptor family, including any and all Wnt family proteins, inhibitors of intracellular β-catenin degradation and activators of TCF/LEF. be. Wnt agonists also include Wnt signaling pathway inhibitors, GSK-3β inhibitors, Dkk1 antagonists, and the like.
The GSK-3β inhibitor is defined as a substance that inhibits the kinase activity of the Glycogen Synthase Kinase (GSK)-3β protein (for example, the ability to phosphorylate β-catenin). ), BIO (CAS number: 667463-62-9), SB216763 (CAS number: 280744-09-4), and many others are already known.
The Wnt agonist used in the present invention is preferably CHIR99021, SB216763, BIO, A 1070722, Lithium carbonate, 3F8, SB 415286, TDZD 8, TWS 119, TCS 2002, Wnt3, Wnt3a, more preferably CHIR99021, or SB216763, more preferably CHIR99021.

When CHIR99021 is used as a Wnt agonist in step B2, the concentration of CHIR99021 in the medium used in step B2 (also referred to herein as medium B2) depends on the cells used, the culture time, and the amount of activin A used in step B1. Although it is not particularly limited as long as the desired cells can be obtained, it is usually 1 μM to 1000 μM, preferably 1 μM to 200 μM, more preferably 3 μM to 30 μM, more preferably about 10 μM. μM. When another Wnt agonist is used in place of CHIR99021 in step B2, the concentration that exhibits an equivalent effect can be set with reference to CHIR99021.

The culture time in step B2 is preferably about 1 to 2 days, more preferably about 1.5 days, from the viewpoint of increasing the proportion of both Cxcr4- and KIT-positive cells in the cell population obtained in step D.

Step B2 can be performed, for example, by replacing medium B1 with medium B2 in step 2 after culturing in step B1.

Medium B2 may further comprise a BMP signaling pathway agonist (preferably BMP2, BMP4 or BMP7, more preferably BMP2 or BMP4, even more preferably BMP4). The concentration of the BMP signaling pathway agonist contained in medium B2 varies depending on the amount of activin A and the like used in step B1, and the Cxcr4- and KIT-positive WD progenitor-like cells in the cell population obtained in step D. For example, it is 10 ng/mL or less, preferably 5 ng/mL or less, more preferably 0.3 ng/mL to 3 ng/mL. When other BMP signaling pathway agonists are used, the concentration can be appropriately selected so as to exhibit the same effects as those obtained when BMP4 is used.

Process C
In one aspect of the present invention, the culture result obtained by the culture in step B2 above is treated with RA or an RA analog, a fibroblast growth factor (FGF2, Fgf4, Fgf7, FGF9, or FGF20), and a TGFβ signaling pathway inhibitor. Cultivate in medium containing a substance or Wnt agonist (preferably a GSK-3β inhibitor).

Retinoic acid that can be used in the present invention is exemplified by all-trans retinoic acid (ATRA), which can be purchased from Sigma-Aldrich or the like. Also, artificially modified retinoic acid can be used while retaining the functions of natural retinoic acid.
When ATRA is used as retinoic acid in step C, the concentration of ATRA in medium C varies depending on the culture conditions, etc., and is not particularly limited as long as the desired cells can be obtained. It is preferably between 10 and 500 nM, more preferably between 50 nM and 200 nM, even more preferably about 100 nM.

In step C, retinoic acid analogs can be used in place of retinoic acid. Examples of retinoic acid analogues include AGN193109, AM580, AM80, BMS453, BMS195614, AC261066, AC55649 and Isotretinoin, with AGN193109 being particularly preferred. When an RA analogue is used in place of RA in step C, the concentration showing an equivalent effect can be set with reference to RA.

Fibroblast growth factor (FGF2, FG4, FGF7, FGF9, or FGF20) used in medium C may be prepared by referring to a method known per se based on known amino acid sequence information, and assembled from R & D Systems etc. Purchased recombinant human FGF protein can also be used. The FGF to be used is preferably FGF9 or FGF20, more preferably FGF9. The concentration of FGF9 protein in medium C varies depending on the culture conditions and the like, but is, for example, 10 ng to 1 μg/mL, preferably 10 ng to 500 ng/mL, more preferably 50 ng to 200 ng/mL, and further Preferably about 100 ng/mL. In step C, when other FGFs are used instead of FGF9, concentrations that exhibit equivalent effects can be set with reference to FGF9.

A TGFβ signaling pathway inhibitor is defined as a substance that inhibits signal transduction from binding of TGFβ to its receptor to SMAD. A number of substances have been reported, including substances that inhibit the phosphorylation of SMAD by .
The TGFβ signaling pathway inhibitor or Wnt agonist used in Step C of the present invention is not particularly limited as long as the desired cells can be obtained in Step D. -41-9) or A83-01 (CAS number: 909910-43-6), D4476, GW788388, LY364947, R268712, RepSox, SB505124, SB525334, or SD208, preferably SB431542 or A83-01, SB431542 is more preferred.
When SB431542 is used as a TGFβ signaling pathway inhibitor or Wnt agonist in step C, the concentration of SB431542 in the medium used in step C (herein also referred to as medium C) varies depending on the culture conditions, etc., and the desired cells Although it is not particularly limited as long as the is obtained, it is usually 1 μM to 1000 μM, preferably 3 μM to 500 μM, more preferably 10 μM to 200 μM, and mouse pluripotent stem cells in step B1 When used, it is more preferably about 10 μM, and when human pluripotent stem cells are used in step B1, it is more preferably about 100 μM. In step C, when other components are used instead of SB431542, the concentrations showing equivalent effects can be set with reference to SB431542.

Medium C may be substantially free of GSK-3β inhibitors. Although it is not particularly limited as long as the desired cells can be obtained, specifically, it is preferably about 5 μM or less, more preferably about 3 μM or less.

The culture time in step C is not particularly limited as long as the proportion of WD progenitor-like cells in the cell population obtained in step D does not decrease, and is, for example, about 1 to 3 days, more preferably About 1-2 days.

Step C can be performed, for example, by replacing medium B2 with medium C after culturing in step B2.

When human pluripotent stem cells (preferably human iPS cells) are used in step B1, medium C further contains BMP signal pathway inhibitors (preferably LDN193189, Noggin, Gremlin, DMH-1, DMH2, Dorsomorphin dihydrochloride, K 02288, LDN 212854 or ML 347, more preferably LDN193189 or Noggin, still more preferably LDN193189).
When mouse pluripotent stem cells (preferably mouse ES cells) are used in step B1, medium C preferably contains neither a BMP signaling pathway inhibitor nor a BMP signaling pathway agonist.
When medium C contains LDN193189, the concentration of LDN193189 in medium C is preferably 1 nM to 1000 nM, more preferably 3 nM to 500 nM, still more preferably 10 nM to 200 nM, even more preferably about 100 nM. nM. In step C, when other substances are used in place of LDN193189, the concentrations showing equivalent effects can be set with reference to LDN193189.

Process D
In one aspect of the present invention, the culture result obtained by the culture in step C above is combined with RA or an RA analog, a Wnt agonist (preferably a GSK-3β inhibitor), and a fibroblast growth factor (FGF2, FGF4 , FGF7, FGF9, or FGF20).

When ATRA is used as retinoic acid in step D, the concentration of ATRA in the medium used in step D (herein also referred to as medium D) varies depending on culture conditions, etc., and is particularly limited as long as desired cells can be obtained. However, it is usually 10 nM to 1 μM, preferably 10 nM to 500 nM, more preferably 50 nM to 200 nM, still more preferably about 100 nM.

In step D, retinoic acid analogs can be used in place of retinoic acid. Examples of retinoic acid analogs include the compounds listed in Step C, with AGN193109 being preferred. When an RA analogue is used in place of RA in step D, the concentration showing an equivalent effect can be set with reference to RA.

The FGF that can be used in medium D is the same as in step C, preferably FGF9 or FGF20, more preferably FGF9. The concentration of FGF9 protein in medium D varies depending on the culture conditions and the like, but is, for example, 10 ng to 1 μg/mL, preferably 10 ng to 500 ng/mL, more preferably 30 ng to 300 ng/mL, and Preferably about 100 ng/mL. In step D, when other FGFs are used in place of FGF9, concentrations that exhibit equivalent effects can be set using FGF9 as a reference.

The Wnt agonist used in step D can use the components listed in step B2, preferably CHIR99021 or SB216763, more preferably CHIR99021. When CHIR99021 is used in step D, the concentration of CHIR99021 in medium D is not particularly limited as long as the desired cells can be obtained. More preferably 1 μM to 10 μM, still more preferably about 3 μM to about 5 μM. In step D, when other Wnt agonists are used instead of CHIR99021, CHIR99021 can be used as a reference to set a concentration that exhibits an equivalent effect.

The culture time in step D is not particularly limited as long as the proportion of WD progenitor-like cells in the cell population obtained in step D does not decrease, for example, about 1 to 3 days, preferably about 1.5 days to 2.5 days.

Step D can be carried out, for example, by replacing medium C with medium D after culturing in step C.

When using human pluripotent stem cells (preferably human iPS cells) in step B1, medium D may further contain a BMP signaling pathway inhibitor. BMP signaling pathway inhibitors that can be used in step D include the substances listed in step C, preferably LDN193189 or Noggin, more preferably LDN193189. When medium D contains LDN193189, the concentration of LDN193189 in medium D is preferably 1 nM to 500 nM, more preferably 10 nM to 100 nM, even more preferably 10 nM to 50 nM, even more preferably about 30 nM. nM. In step D, when other substances are used in place of LDN193189, concentrations that exhibit equivalent effects can be set with reference to LDN193189.

A cell population containing WD progenitor-like cells can be obtained as a product of the culture in step D.

The method for producing WD progenitor-like cells of the present invention preferably comprises
Step A: Wolff tube (WD ) A method for producing progenitor-like cells, comprising:
Steps B1, B2, C and D below:
Step B1 Pluripotent stem cells (preferably ES cells or iPS, more preferably human iPS cells or mouse ES cells) are treated with activin A (1 ng/mL to 1000 ng/mL, preferably 1 ng/mL to 100 ng /mL, more preferably 3 to 30 ng/mL activin A) (when culturing with human iPS cells, in addition to activin A, preferably 10 ng/mL or less, preferably 0.1 ng/mL to 10 ng/mL, more preferably 0.3 ng/mL to 3 ng/mL, more preferably about 1 ng/mL of BMP4, or a BMP signaling pathway agonist at a concentration capable of exhibiting a similar effect) culture in medium B1 (preferably performing suspension culture; the culture period is 5 days or less, preferably 0.5 to 3 days, more preferably about 1 day);
Step B2 The cells obtained from step B1 are treated with a Wnt agonist, preferably a GSK-3β inhibitor (preferably CHIR99021, 1 μM to 1000 μM, preferably 1 μM to 200 μM, more preferably 3 μM to 30 μM, more preferably about 10 μM CHIR99021) (preferably in addition to the Wnt agonist, 10 ng/mL or less, preferably 5 ng/mL or less, more preferably 0.3 ng/mL to 3 ng/mL BMP4; Alternatively, it is cultured in medium B2 (preferably, suspension culture is performed; the culture period is preferably about 1 to 2 days, and more preferably about 1.5 days) step,
Step C The cells obtained by step B2 are treated with retinoic acid (preferably ATRA, 10 nM to 1 μM, preferably 10 to 500 nM, more preferably 50 nM to 200 nM, more preferably about 100 nM ATRA), FGF9 (10 ng to 1 μg/mL, preferably 10 ng to 500 ng/mL, more preferably 50 ng to 200 ng/mL, even more preferably about 100 ng/mL of FGF9), and a TGFβ signaling pathway inhibitor ( Preferably SB431542, 1 μM to 1000 μM, preferably 3 μM to 500 μM, more preferably 10 μM to 200 μM SB431542, about 10 μM when mouse pluripotent stem cells are used in step B1, step When human pluripotent stem cells are used in B1, about 100 μM SB431542) (when human pluripotent stem cells (preferably human iPS cells) are used in step B1, further BMP signal pathway inhibitors (preferably LDN193189 and cultured in medium C (preferably about 1-2 days, more preferably about 1 day) step,
Step D Cells obtained by Step C are treated with RA (preferably ATRA, 10 nM to 1 μM, preferably 10 to 500 nM, more preferably 50 nM to 200 nM, even more preferably about 100 nM ATRA), FGF9 (10 ng to 1 μg/mL, preferably 10 ng to 500 ng/mL, more preferably 30 ng to 300 ng/mL, even more preferably about 100 ng/mL of FGF9), and a GSK-3β inhibitor ( preferably CHIR99021, comprising 0.1 μM to 100 μM, preferably 1 μM to 100 μM, more preferably 1 μM to 10 μM, even more preferably about 3 μM to 5 μM CHIR99021 (human pluripotent When using sex stem cells (preferably human iPS cells), a BMP signal pathway inhibitor (preferably LDN193189, 1 nM to 500 nM, preferably 10 nM to 100 nM, more preferably 10 nM to 50 nM, and further culturing in medium D (preferably containing about 30 nM LDN193189) (preferably about 1-3 days, more preferably about 1.5-2.5 days).

The present invention further includes any one or more media selected from the group consisting of the above media A, B, C and D, or two or more, more preferably three or more, still more preferably four media selected from the group. A combinatorial kit for generating WD progenitor-like cells from pluripotent stem cells is provided.
The medium can be provided as a liquid medium or a powder medium, or can be provided as a medium additive that can be added to a commercially available basal medium to provide the medium of the present invention.
The kit may contain an anti-Cxcr4 antibody and/or an anti-KIT antibody in addition to the medium or medium additive. The antibody can be provided conjugated to a labeling molecule, such as a fluorescent molecule, for sorting of cells.
In addition to the above kit, the present invention further includes any one or more media selected from the group consisting of media E, F, and G described later, or two or more, more preferably three media selected from the group. A kit for generating ureteroblast-like cells from pluripotent stem cells, comprising:

Process E
The WD progenitor-like cells of the present invention can be subjected to further maturation culture and differentiated into ureteroblast-like cells. Further, in the present invention, as step E, Cxcr4-positive and KIT-positive WD progenitor-like cells are treated with RA or RA analog, Wnt agonist (preferably GSK-3β inhibitor or Rspondin1), fibroblast growth factor (FGF2, FGF4, FGF7, FGF9, or FGF20) and a ROCK inhibitor (also referred to herein as medium E).

When ATRA is used as retinoic acid in step E, the concentration of ATRA in medium E varies depending on the culture conditions, etc., and is not particularly limited as long as the desired cells can be obtained. It is preferably 10 nM to 500 nM, more preferably 50 nM to 200 nM, still more preferably about 100 nM.

In step E, retinoic acid analogs can be used in place of retinoic acid. Examples of retinoic acid analogs include the compounds listed in Step C, with AGN193109 being preferred. In step E, when an RA analog is used instead of RA, the concentration showing an equivalent effect can be set with reference to RA.

Fibroblast growth factors that can be used in medium E are the same as in step C, preferably FGF9 or FGF20, more preferably FGF9. Although not particularly limited as long as the desired ureteroblast-like cells are obtained, the concentration of FGF9 protein in medium E is, for example, 0.1 ng/mL to 100 ng/mL, preferably 0.5 ng/mL to 50 ng/mL, more preferably 2 ng/mL to 10 ng/mL, even more preferably about 5 ng/mL. In step E, when other FGFs are used in place of FGF9, the concentrations showing equivalent effects can be set with reference to FGF9.

The Wnt agonist used in step E can use the components listed in step B2 and additionally Rspondin1, preferably CHIR99021, SB216763 or Rspondin1, more preferably CHIR99021. When using CHIR99021 as a Wnit agonist in step E, the concentration of CHIR99021 in medium E is not particularly limited as long as the desired cells can be obtained. It is preferably 0.3 μM to 5 μM, more preferably about 1 μM. When another Wnt agonist is used in place of CHIR99021 in step E, the concentration that exhibits an equivalent effect can be set with reference to CHIR99021.

Medium E may further contain a ROCK inhibitor with a view to enhancing cell viability. The ROCK inhibitor is not particularly limited as long as it can inhibit the function of Rho kinase (ROCK). Specifically, the substances listed in step B1 can be mentioned, preferably Y27632 or Fasudil hydrochloride. , more preferably Y27632. When Y27632 is used, the concentration is 1 μM to 1000 μM, preferably 1 μM to 100 μM, more preferably 1 μM to 50 μM, more preferably about 10 μM. In step E, when other Rock inhibitors are used instead of Y27632, the concentration showing equivalent effects can be set with reference to Y27632.

In a preferred embodiment, medium E may further comprise a culture support, such as, but not limited to, growth factor-reduced tomato trigel, collagen, laminin. When medium E contains growth factor-reduced tomato rigel, its concentration in medium E is, for example, 5% to 50%, preferably 5% to 20%, more preferably 10% to 20%, more preferably about 10%. %.

When the WD progenitor-like cells are derived from human pluripotent stem cells, medium E contains fibroblast growth factors (FGF1, FGF2, FGF4, FGF5, FGF6, FGF7, FGF10, or FGF20, preferably FGF1 or FGF2, more preferably FGF1).
The FGF (e.g., FGF1) used in medium E may be prepared by referring to a method known per se based on known amino acid sequence information, or a recombinant human FGF protein purchased from R & D Systems, etc. can also be used. The concentration of FGF1 protein in medium E varies depending on the culture conditions and the like, but is, for example, 10 ng to 1 μg/mL, preferably 10 ng to 500 ng/mL, more preferably 50 ng to 200 ng/mL, and even more preferably. is approximately 100 ng/mL. In step E, when other FGFs are used in place of FGF1, concentrations that exhibit equivalent effects can be set using FGF1 as a reference.

Combinations of FGFs essential for step E and FGFs for culturing WD progenitor-like cells derived from human pluripotent stem cells include, but are not limited to, for example, FGF9 or FGF20, and FGF1 or FGF2. A combination can be mentioned, preferably a combination of FGF9 and FGF1.

If the WD progenitor-like cells are derived from human pluripotent stem cells, medium E may further contain a BMP signaling pathway inhibitor. BMP signaling pathway inhibitors that can be used in step E include the substances listed in step C, preferably LDN193189 or Noggin, more preferably LDN193189.
When medium E contains LDN193189, the medium concentration is preferably 1 nM to 300 nM, more preferably 1 nM to 100 nM, still more preferably 1 nM to 20 nM, and even more preferably about 10 nM. In step E, when other substances are used instead of LDN193189, the concentrations showing equivalent effects can be set with reference to LDN193189.

The culture time in step E is not particularly limited, but is, for example, about 1 to 5 days, more preferably about 1 to 3 days, and the WD progenitor-like cells are human pluripotent stem cells. About 2 to 3 days is more preferred if it is more induced.

In step E, about 100 to 100,000 cells (for example, about 10,000 cells) of WD progenitor-like cells can be aggregated to form aggregates and subjected to suspension culture, which is not particularly limited, but culture As a vessel, a V-bottom 96-well low cell binding plate (Sumitomo Bakelite) or the like can be used.

WD progenitor-like cells of Cxcr4-positive and KIT-positive cells (preferably WD progenitor-like cells prepared by the above steps B1, B2, C, D and A; preferably 100 to 100,000 cells, more preferably 1000 to 50,000 cells) with retinoic acid (preferably ATRA, 10 nM to 1 μM, preferably 10 nM to 500 nM, more preferably 50 nM to 200 nM, even more preferably about 100 nM ATRA), a Wnt agonist, Preferably GSK-3β inhibitor or Rspondin1 (preferably CHIR99021, such as 0.1 μM to 100 μM, preferably 0.1 μM to 10 μM, more preferably 0.3 μM to 5 μM, more preferably about 1 μM CHIR99021), FGF9 (0.1 ng/mL to 100 ng/mL, preferably 0.5 ng/mL to 50 ng/mL, more preferably 2 ng/mL to 10 ng/mL, even more preferably about 5 ng/mL of FGF9) and ROCK inhibition substance (preferably Y27632, 1 μM to 1000 μM, preferably 1 μM to 100 μM, more preferably 1 μM to 50 μM, even more preferably about 10 μM Y27632) (preferably further containing growth factor reduced tomatorigel, 5% to 50%, preferably 5% to 20%, more preferably 10% to 20%, more preferably about 10% growth factor reduced tomato ligel) in medium E (preferably floating clump culture, more Preferably cultured as floating clumps on a low-adherence incubator; preferably cultured for about 1-5 days, or about 2 days if the WD progenitor-like cells are derived from human pluripotent stem cells. up to 3 days, or about 1 to 3 days if derived from mouse pluripotent stem cells).

In a preferred embodiment, the cells obtained in step E are Emx2, Ret positive and Hnf1b, Wnt9b, Calb1, E-cadherin positive.

Process F
In the present invention, as step (F), the cells obtained in step (E) are further treated with RA or RA analogues, Wnt agonists (preferably GSK-3β inhibitors or Rspondin1), fibroblast growth factors (FGF2, FGF4 , FGF7, FGF9, or FGF20), a ROCK inhibitor, and glial cell line-derived neurotrophic factor (GDNF) or a GDNF analog (BT18, or SIB4035) or FGF10 (also referred to herein as medium F ), a method for producing ureteroblast-like cells is provided.

When ATRA is used as retinoic acid in step F, the concentration of ATRA in the medium varies depending on the culture conditions, etc., and is not particularly limited as long as the desired cells can be obtained. nM to 500 nM, more preferably 50 nM to 200 nM, more preferably about 100 nM.

In step F, retinoic acid analogues can be used in place of retinoic acid. Examples of retinoic acid analogs include the compounds listed in Step C, with AGN193109 being preferred. In step F, when an RA analog is used instead of RA, the concentration showing an equivalent effect can be set with reference to RA.

Fibroblast growth factors that can be used in medium F are the same as in step C, preferably FGF9 or FGF20, more preferably FGF9. Although not particularly limited as long as the desired ureteroblast-like cells are obtained, the concentration of FGF9 protein in medium F is, for example, 0.1 ng to 100 ng/mL, preferably 0.5 ng to 50 ng/mL, More preferably 2 ng to 10 ng/mL, even more preferably about 5 ng/mL. In step F, when other FGFs are used in place of FGF9, concentrations that exhibit equivalent effects can be set using FGF9 as a reference.

Wnt agonists that can be used in step F can use the components listed in step B2 and additionally Rspondin1, preferably CHIR99021, SB216763 or Rspondin1, more preferably CHIR99021. When CHIR99021 is used as a Wnt agonist in step F, the concentration of CHIR99021 in medium F is not particularly limited as long as the desired cells can be obtained, usually 0.1 μM to 300 μM, preferably 0.3 μM to 100 μM. , more preferably 1 μM to 5 μM, more preferably about 3 μM. In step F, when other Wnt agonists are used instead of CHIR99021, CHIR99021 can be used as a reference to set a concentration that exhibits an equivalent effect.

The glial cell line-derived neurotrophic factor (GDNF) used in medium F may be prepared by referring to a method known per se based on known amino acid sequence information, or a recombinant human GDNF protein from R & D Systems, etc. can also be used. The concentration of GDNF protein in medium F varies depending on the culture conditions and the like, but is, for example, 0.1 ng to 100 ng/mL, preferably 0.1 ng to 10 ng/mL, more preferably 0.5 ng to 10 ng/mL, and further Preferably about 1 ng/mL.

In medium F, GDNF analogues or FGF10 can be used instead of GDNF. GDNF analogs include BT18 or SIB4035, with BT18 being preferred. In step F, when a GDNF analog or FGF10 is used instead of GDNF, a concentration showing an equivalent effect can be set with reference to GDNF.

Medium F may further contain a ROCK inhibitor with a view to enhancing cell viability. The ROCK inhibitor is not particularly limited as long as it can inhibit the function of Rho kinase (ROCK). Specifically, the substances listed in step B1 can be mentioned, preferably Y27632 or Fasudil hydrochloride. , more preferably Y27632. When medium F contains Y27632, the concentration in the medium is, for example, 1 μM to 1000 μM, preferably 1 μM to 100 μM, more preferably 1 μM to 50 μM, still more preferably about 10 μM. When other Rock inhibitors are used in place of Y27632 in medium F, concentrations that exhibit equivalent effects can be set with reference to Y27632.

In a preferred embodiment, medium F may further comprise growth factor-reduced tomato rigel. When medium F contains growth factor-reduced tomato rigel, its concentration in medium F is, for example, 5% to 50%, preferably 5% to 20%, more preferably 10% to 20%, more preferably about 10%. %.

When the WD progenitor-like cells are derived from human pluripotent stem cells, the medium F contains the above FGF and further fibroblast growth factors (FGF1, FGF2, FGF4, FGF5, FGF6, FGF7, FGF10, or FGF20, preferably FGF1 or FGF2, more preferably FGF1).
The FGF (e.g., FGF1) used in medium F may be prepared by referring to a method known per se based on known amino acid sequence information, or a recombinant human FGF protein purchased from R & D Systems, etc. can also be used. The concentration of FGF1 protein in medium F varies depending on the culture conditions and the like, but is, for example, 10 ng to 1 μg/mL, preferably 10 ng to 500 ng/mL, more preferably 50 ng to 200 ng/mL, and Preferably about 100 ng/mL. In step F, when other FGFs are used instead of FGF1, the concentrations showing equivalent effects can be set with reference to FGF1.

Combinations of FGFs essential for step F and FGFs for culturing WD progenitor-like cells derived from human pluripotent stem cells include, but are not limited to, FGF9 or FGF20, and FGF1 or FGF2. A combination can be mentioned, preferably a combination of FGF9 and FGF1.

If the WD progenitor-like cells are derived from human pluripotent stem cells, medium F may further contain a BMP signaling pathway inhibitor. BMP signaling pathway inhibitors that can be used in step F include the substances listed in step C, preferably LDN193189 or Noggin, more preferably LDN193189. When medium F contains LDN193189, the concentration in the medium is preferably 1 nM to 300 nM, more preferably 1 nM to 100 nM, even more preferably 5 nM to 20 nM, even more preferably about 10 nM. . In step F, when other substances are used in place of LDN193189, concentrations that exhibit equivalent effects can be set with reference to LDN193189.

The culture time in step F is not particularly limited, but is, for example, about 1 to 5 days, more preferably about 1 to 3 days. It is more preferably about 1 day when the WD progenitor-like cells used in step (E) are derived from mouse pluripotent stem cells, and the WD progenitor-like cells used in step (E) are human In the case of pluripotent stem cells, it takes about 2 days.

In a preferred embodiment, step F comprises treating the cells (preferably cell clumps) obtained by step (E) with retinoic acid (preferably ATRA, 10 nM to 1 μM, preferably 10 nM to 500 nM, more preferably 50 nM to 200 nM, more preferably about 100 nM ATRA), Wnt agonist (preferably CHIR99021, 0.1 μM to 300 μM, preferably 0.3 μM to 100 μM, more preferably 1 μM to 5 μM, more preferably CHIR99021 at about 3 μM), a ROCK inhibitor (preferably Y27632, 1 μM to 1000 μM, preferably 1 μM to 100 μM, more preferably 1 μM to 50 μM, even more preferably about 10 μM Y27632) and GDNF (0.1 ng to 100 ng/ mL, preferably 0.1 ng to 10 ng/mL, more preferably 0.5 ng to 10 ng/mL, even more preferably about 1 ng/mL GDNF) (preferably further containing growth factor reduced tomato trigel, 5% ~50%, preferably 5% to 20%, more preferably 10% to 20%, even more preferably about 10% growth factor reduced tomatorigel; When derived from potential stem cells, preferably 10 ng to 1 μg/mL, more preferably 10 ng to 500 ng/mL, even more preferably 50 ng to 200 ng/mL, even more preferably further comprising about 100 ng/mL of FGF1; when the WD progenitor-like cells used in step (E) are derived from human pluripotent stem cells, preferably 1 nM to 300 nM, more preferably 1 nM to 100 nM, more preferably 5 nM to 20 nM, even more preferably about 10 nM of LDN193189 is cultured in medium (preferably floating clump culture, more preferably low adherence culture vessel) culturing as floating clumps; preferably culturing for about 1 to 5 days, more preferably culturing for about 1 to 3 days).

Process G
In the present invention, as step (G), the cells obtained by step F are further treated with RA or RA analogs, Wnt agonists (preferably GSK-3β inhibitors or Rspondin1), ROCK inhibitors, and GDNF or GDNF analogs ( BT18, or SIB4035) or FGF10 (also referred to herein as medium G).
When ATRA is used as retinoic acid in step G, the concentration of ATRA in the medium varies depending on the culture conditions, etc., and is not particularly limited as long as the desired cells can be obtained. nM to 500 nM, more preferably 50 nM to 200 nM, more preferably about 100 nM.

In step G, retinoic acid analogs can be used in place of retinoic acid. Examples of retinoic acid analogs include the compounds listed in Step C, with AGN193109 being preferred. In step E, when an RA analog is used instead of RA, the concentration showing an equivalent effect can be set with reference to RA.

Wnt agonists that can be used in step G can use the components listed in step B2 and additionally Rspondin1, preferably CHIR99021, SB216763 or Rspondin1, more preferably CHIR99021. When using CHIR99021 as a Wnt agonist in step G, the concentration of CHIR99021 in medium G is not particularly limited as long as the desired cells can be obtained, but usually 0.1 μM to 300 μM, preferably 0.3 μM to 100 μM. , more preferably 1 μM to 5 μM, more preferably about 3 μM. When another Wnt agonist is used in place of CHIR99021 in step G, a concentration that exhibits an equivalent effect can be set with reference to CHIR99021.

Although not particularly limited as long as the desired ureteroblast-like cells are obtained, the concentration of the GDNF protein in the medium G is, for example, 0.1 ng to 100 ng/mL, preferably 0.2 ng to 20 ng/mL, More preferably 0.5 ng to 10 ng/mL, even more preferably about 2 ng/mL.

In medium G, GDNF analogues or FGF10 can be used instead of GDNF. GDNF analogs include BT18 or SIB4035, with BT18 being preferred. In step G, when using a GDNF analogue or FGF10 instead of GDNF, a concentration showing an equivalent effect can be set with reference to GDNF.

Medium G may further contain a ROCK inhibitor with a view to enhancing cell viability. The ROCK inhibitor is not particularly limited as long as it can inhibit the function of Rho kinase (ROCK). Specifically, the substances listed in step B1 can be mentioned, preferably Y27632 or Fasudil hydrochloride. , more preferably Y27632. When medium G contains Y27632, the concentration in the medium is 1 μM to 1000 μM, preferably 1 μM to 100 μM, more preferably 1 μM to 50 μM, even more preferably about 10 μM. When other Rock inhibitors are used instead of Y27632 in medium G, the concentrations showing equivalent effects can be set with reference to Y27632.

In a preferred embodiment, medium G may further comprise growth factor-reduced tomato rigel. When medium G contains growth factor-reduced tomato ligel, its concentration in medium G is, for example, 5% to 50%, preferably 5% to 20%, more preferably 10% to 20%, more preferably about 10%. %.

When the WD progenitor-like cells used in step (E) are derived from human pluripotent stem cells, medium G further contains fibroblast growth factors (FGF1, FGF2, FGF4, FGF5, FGF6, FGF7, FGF10 , or FGF20, preferably FGF1 or FGF2, more preferably FGF1). The concentration of FGF1 protein in medium G is, for example, 10 ng to 1 μg/mL, preferably 10 ng to 500 ng/mL, more preferably 50 ng to 200 ng/mL, more preferably about 100 ng/mL. . In step G, when other FGFs are used instead of FGF1, the concentrations showing equivalent effects can be set with reference to FGF1.

When the WD progenitor-like cells used in step (E) are derived from human pluripotent stem cells, medium G may further contain a BMP signaling pathway inhibitor. BMP signaling pathway inhibitors that can be used in step G include the substances listed in step C, preferably LDN193189 or Noggin, more preferably LDN193189.
When medium G contains LDN193189, the concentration of LDN193189 in medium G is preferably 1 nM to 300 nM, more preferably 1 nM to 100 nM, even more preferably 5 nM to 20 nM, even more preferably about 10 nM. In step F, when other substances are used in place of LDN193189, concentrations that exhibit equivalent effects can be set with reference to LDN193189.

The culture time in step G is not particularly limited, but is, for example, about 0.5 to 5 days, more preferably about 1 to 3 days. It is more preferably about 1 day when the WD progenitor-like cells used in step (E) are derived from mouse pluripotent stem cells, and the WD progenitor-like cells used in step (E) are human In the case of pluripotent stem cells, it takes about 2 days.

In a preferred embodiment, step G comprises treating the cells (preferably cell clumps) obtained by step (F) with retinoic acid (preferably ATRA, 10 nM to 1 μM, preferably 10 nM to 500 nM, more preferably 50 nM to 200 nM, more preferably about 100 nM ATRA), Wnt agonist (preferably CHIR99021, 0.1 μM to 300 μM, preferably 0.3 μM to 100 μM, more preferably 1 μM to 5 μM, more preferably CHIR99021 at about 3 μM), a ROCK inhibitor (preferably Y27632, between 1 μM and 1000 μM, preferably between 1 μM and 100 μM, more preferably between 1 μM and 50 μM, more preferably between about 10 μM Y27632) and GDNF (0.1 ng to 100 ng/mL, preferably 0.2 ng to 20 ng/mL, more preferably 0.5 ng to 10 ng/mL, even more preferably about 2 ng/mL GDNF) (preferably also growth factor 5% to 50%, preferably 5% to 20%, more preferably 10% to 20%, even more preferably about 10% growth factor reduced tomatorigel; used in step (E); When the WD progenitor-like cells are derived from human pluripotent stem cells, 10 ng to 1 μg / mL, preferably 10 ng to 500 ng / mL, more preferably 50 ng to 200 ng / mL, more preferably about 100 ng / mL of FGF1; when the WD progenitor-like cells used in step (E) are derived from human pluripotent stem cells, a BMP signaling pathway inhibitor ( It is preferably LDN193189 and is cultured in a medium (preferably Cultivating floating clumps, more preferably culturing as floating clumps on a low-adherence incubator; preferably culturing for about 1 to 5 days, more preferably culturing for about 1 to 3 days).

In a preferred embodiment, the cells obtained in step G are PAX2, Emx2, Ret positive and Hnf1b, Wnt9b and Calb1 positive.

Production of Kidney Organoids The invention also includes a method of producing kidney organoids comprising co-culturing ureteroblast-like cells produced using the methods of the invention with nephron progenitor cell and stromal progenitor cell populations. As nephron progenitor cells, both embryonic nephron progenitor cells isolated from embryos and nephron progenitor cells induced from pluripotent stem cells (eg, ES cells and iPS cells) can be used. A method for inducing nephron progenitor cells from pluripotent stem cells can be prepared, for example, with reference to a report by the present inventors (A. Taguchi et al., Cell Stem Cell 14, 53-67, 2014). As the stromal progenitor cell population, for example, a stromal progenitor cell population isolated from an embryo can be used, and without being limited thereto, a stromal cell population selected from an embryonic kidney can be used. Pdgfra+ stromal cell populations are preferred.

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples.
The following animal experiments were performed according to protocols approved by the Animal Care and Use Committee of the Kumamoto University School of Medicine.

(Material and method)
mouse
The Hoxb7-GFP mouse strain was maintained on an outbred background (Jcl: ICR, Clea Japan, Inc.). Mice were fed CE-2 radiation-sterilized chow and housed in plastic cages on a 12 hour light cycle. All analyzes were performed using a minimum of 3 litters. For the embryonic stage analysis, day 0.5 of the embryonic period (E) was considered to be noon, the day on which a vaginal plug was confirmed in a mated female. Hoxb7-GFP, which expresses GFP under the control of a fragment of the Hoxb7 promoter, was purchased from Jackson laboratory.

Mouse ES cell culture medium Mouse ES cell line (Osr1-GFP) (Taguchi et al., Cell stem cell 14, 53-67, 2014) contains 15% FBS, 1% (v/v) non-essential amino acids (NEAA). , 0.1 mM 2-mercaptoethanol (2-ME) and 1,000 U/mL leukemia inhibitory factor (LIF: Millipore) maintained on mitotically inactivated mouse embryonic fibroblasts (MEF) in DMEM . Prior to initiation of differentiation, ES cells were injected with 15% FBS, 1% (v/v) non-essential amino acids, 0.1 mM 2-ME, 1× penicillin/streptomycin (P/S), 1,000 U/mL LIF, 3 μM CHIR99021 ( Axon) and 1 μM PD0325901 (Wako) were added in DMEM for one passage on a feeder cell-free gelatin-coated culture dish.
The established mouse ES cell line (Hoxb7-GFP) is 14% KSR, 1% FBS, 1% (v/v) NEAA, 1% (v/v) sodium pyruvate, 0.1 mM 2-ME, 1,000 U/ Mitotically inactivated MEFs were maintained in GMEM supplemented with mL LIF, 1.5 μM CHIR99021 and 0.5 μM PD0325901. All mouse ES cell lines were cultured in a humidified atmosphere at 37°C and 5% CO2. Cells were passaged every other day.

Mouse ES cell differentiation medium The medium is a mixed medium of 75% Iscove's modified Dulbecco's medium (IMDM) and 25% Ham's F12 medium, 0.5 × N2 (Thermo Fisher), 0.5 × B27 without retinoic acid (Thermo Fisher), 0.5 × P/S, supplemented with 0.05% BSA, 2 mM L-glutamine, 0.5 mM ascorbic acid and 4.5×10 −4 M 1-thioglycerol were used.

Human iPS Cell Culture Medium Human iPS cells (201B7) were maintained on iMatrix-511 (Nippi Co., Ltd.) in StemFit AK03N medium (Ajinomoto Co., Inc.). Human iPS cells were cultured at 37°C in a 5% CO2 humidified atmosphere. Cells were passaged every 6 days.

Human iPS cell differentiation medium
2% (v/v) B27 (without retinoic acid), 2 mM L-glutamine, 1% (v/v) ITS, 1% (v/v) NEAA (without retinoic acid), 90 μM 2-ME and Serum-free differentiation medium containing DMEM/F12 (Invitrogen) supplemented with 0.5×P/S was used.

Establishment of Mouse ES Cells Female 129/sv mice with superstimulated ovaries were mated with male Hoxb7-GFP mice to obtain fertilized 8-cell stage eggs. Embryos collected in M2 medium (ARK Resource) were cultured for 24 hours. Embryos that developed to the blastocyst stage were transferred to 0.1% gelatin-coated plastic dishes containing mouse ES cell maintenance medium. After 6 days, expanded cells were passaged onto mitotically inactivated mouse embryonic fibroblasts (MEF) with ES cell maintenance medium. The established ES cells were further proliferated, and early-passage cells were used for differentiation experiments.

Kidney Reconstitution Assay Metanephros were removed by manual dissection from ICR mouse embryos at E11.5. Intact ureteric buds (UBs) were isolated by incubating kidneys for 4 min at 37° C. in DMEM/10% FBS containing 1 mg/mL Type XI collagenase (SIGMA). UB was manually isolated from the metanephric mesenchyme (MM) using a 30G needle. Harvested MMs were washed once in PBS and dissociated by incubation with 0.05% trypsin/EDTA for 5 minutes at 37°C. The dissociated MM cells were resuspended in mouse ES cell differentiation medium at 70,000 cells/100 μL, seeded on a low cell-binding U-bottom plate (Thermo), and centrifuged (1,000 rpm, 3 minutes) to remove MM cells. was allowed to settle. Isolated UBs or Wolffian tubes (WD), or induced UBs, were placed on the deposited sheet-like MM cells. MM cells spontaneously aggregated, encased the UB and finally formed spheroids after 24 hours of culture. Reaggregated spheroids were transferred to transwell inserts (Corning) containing 50% Matrigel (50 μL) in DMEM/F12 medium (10% FBS and P/S), then transwells were transferred to DMEM/F12 medium (10 μL). % FBS and penicillin/streptomycin) medium.

Culture of selected embryonic cells
For embryonic tissue culture at E9.5, metanephric regions were harvested from the forelimbs of 22-26 somite stage embryos. Harvested tissues were incubated in DMEM/10% FBS containing 1 mg/mL Type XI collagenase for 6 minutes at 37°C, followed by treatment with DNase I and 0.25% trypsin for 6 minutes at 37°C. Cells were dissociated. Cell surface marker (Flk1) staining was performed after blocking with normal mouse serum. FACS-sorted Hoxb7-GFP+/Flk1- cells were resuspended in mouse ES cell differentiation medium and plated in V-bottom 96-well low cell binding plates (Sumitomo Bakelite, Cat#MS-2) at approximately 1,600 cells per well. 9096V). After centrifugation (210 G, 4 min), the supernatant medium was mixed with 10 μM Y27632 (Wako), 0.1 μM retinoic acid, 3 μM CHIR99021, 5 ng/mL human Fgf9 (R&D), 1 ng/mL human GDNF (R&D). and medium containing 10% growth factor reduced tomato rigel (BD) (“Step 6 medium”). After 24 hours, aggregated spheroids were transferred to medium containing 10 μM Y27632, 0.1 μM retinoic acid, 3 μM CHIR99021, 2 ng/mL human GDNF and 10% growth factor reduced tomatorigel (“Step 7 medium”). .

For embryonic tissue culture at E8.75, the region caudal to the cardiac primordium of 12-15 somite stage embryos was harvested and Hoxb7-GFP+/Flk1- cells were sorted by FACS. Sorted cells were clumped in V-bottom 96-well low cell binding plates at approximately 1,200 cells per clump. The first 24 hours were cultured using medium containing 10 μM Y27632, 0.1 μM retinoic acid, 1 μM CHIR99021, 5 ng/mL human Fgf9 and 10% growth factor reduced tomatorigel (“Step 5 medium”), followed by Then, the spheroids were transferred to "Step 6 medium" and "Step 7 medium" and cultured for 24 hours respectively for differentiation.

Induction of UB Lineage from Mouse ES Cells ES cells were differentiated in a serum-free medium as follows. ES cells were dissociated using Accutase™ (ESGRO) and cultured in serum-free mouse ES cell differentiation medium. Harvested cells were aggregated in 96-well U-bottom low cell binding plates at 1,000 cells per clump to form embryoid bodies (EBs). After 48 hours (Day 2), EBs were dissociated with Accutase and reaggregated in serum-free differentiation medium supplemented with 10 ng/mL human activin A (R&D) (Step 1). After 24 hours (Day 3), the medium was replaced with medium containing 0.3 ng/mL human Bmp4 (R&D) and 10 μM CHIR 99021 (Step 2). After 36 hours (Day 4.5), the medium was replaced with Step 3 medium containing 0.1 µM retinoic acid, 100 ng/mL human Fgf9, and 10 µM SB431542 (Wako). On Day 5.5 (24 hours later), the medium was changed to medium containing 0.1 μM retinoic acid, 100 ng/mL human Fgf9 and 5 μM CHIR99021 (“Step 4 medium”). Differentiation factors for the Hoxb7-GFP ES cell line are shown in the table below along with those for the Osr-GFP ES cell line.

Maturation culture of mouse ES cell-derived Wolffian duct (WD) progenitor cells
On Day 6.25 (Day 6.26), induced spheroids were harvested and cells were dissociated by incubating in 0.25% trypsin/EDTA for 6 minutes at 37°C. After blocking with normal mouse serum, cell surface marker (Cxcr4/Kit) staining was performed in buffer containing 1% BSA, 1 x HBSS and 0.035% NaHCO3. 3,000 FACS-sorted Hoxb7-GFP+/Cxcr4+/Kit+ cells were aggregated in V-bottom 96-well low cell binding plates to form spheroids. They were then cultured using the E8.75 embryonic tissue culture conditions described above.

Mouse ES cell-derived single ureteric bud branch culture
On Day 9.25, mouse ES cell-derived induced ureteral buds (UBs) were isolated manually with a sharpened tungsten needle. Isolated UBs were placed in 150 μL of branching medium in 24-well transwell inserts. Branching medium was DMEM/F12 containing 50% Matrigel, 10% FBS, 0.1 μM retinoic acid, 100 ng/mL human Rspondin 1 (R&D), 2 ng/mL human GDNF and 100 ng/mL mouse Fgf1 (R&D). media (Life Technologies). Transwell inserts were cultured in 500 μL branching medium without Matrigel.

Induction of MM Lineage from Mouse ES Cells A previously reported protocol for MM lineage induction of mouse ES cells (Taguchi, supra, 2014) was used with minimal modifications. Embryoid bodies (EBs) were allowed to form clumps of 1,000 cells in 96-well U-bottom low cell binding plates. After 48 hours (Day 2), embryoid bodies (EBs) were dissociated using Accutase™ and then reaggregated in serum-free differentiation medium supplemented with 1 ng/mL human activin A (R&D). . After 24 hours (Day 3), the medium was replaced with medium containing 10 μM CHIR. After 36 hours (Day 4.5), the medium was replaced with fresh medium containing 10 µM Y27632 and 10 µM CHIR. On Day 5.5, medium was changed to medium containing 10 ng/mL activin A, 3 ng/mL Bmp4, 3 μM CHIR, 0.1 μM retinoic acid and 10 μM Y27632. On Day 6.5, the medium was changed to medium containing 1 μM CHIR, 5 ng/mL human Fgf9 and 10 μM Y27632.

Induction of ureteric bud (UB) lineages from human iPS cells
Cells were repopulated at 10,000 cells per clump in V-bottom 96-well low cell binding plates using media containing 10 μM Y27632 and 10 ng/mL human activin A and 1 ng/mL human Bmp4 (“Step 1 media”). Aggregated to form embryoid bodies (EBs). Twenty-four hours later (Day 1), clumps were transferred to U-bottom 96-well low cell binding plates containing medium containing 10 μM CHIR and 1 ng/mL human Bmp4 (“Step 2 medium”). After 36 hours (Day 2.5), the medium was replaced with a medium containing 0.1 µM retinoic acid, 100 ng/mL human Fgf9, 100 nM LDN193189, and 100 µM SB431542 ("Step 3 medium"). On Day 4.5, the medium was replaced with medium containing 0.1 μM retinoic acid, 5 μM CHIR, 100 ng/mL human Fgf9 and 30 nM LDN193189 (“Step 4 medium”).

Maturation culture of human iPS cell-derived Wolffian duct (WD) progenitor cells
On day 6.25, induced spheroids were harvested and cells were dissociated by incubating in 0.25% trypsin/EDTA for 6 minutes at 37°C. After blocking with normal mouse serum, cell surface marker (CXCR4/KIT) staining was performed in buffer containing 1% BSA, 1 x HBSS and 0.035% NaHCO3. 5,000 FACS-sorted CXCR4+/KIT+ cells were plated in V-bottom 96-well low cell binding plates and pelleted by centrifugation (210 G for 4 minutes). The supernatant was added to medium containing 10 μM Y27632, 0.1 μM retinoic acid, 1 μM CHIR, 5 ng/mL human Fgf9, 100 ng/mL human Fgf1, 10 nM LDN193189 and 10% growth factor reduced tomatorigel (“Step 5 medium” ). On day 8.5, spheroids were treated with 10 μM Y27632, 0.1 μM retinoic acid, 3 μM CHIR, 5 ng/mL human Fgf9, 1 ng/mL human GDNF, 100 ng/mL human Fgf1, 10 nM LDN193189 and 10 Transferred to medium containing % growth factor reduced tomatorigel ("Step 6 medium"). On day 10.5, spheroids were treated with 10 μM Y27632, 0.1 μM retinoic acid, 3 μM CHIR, 2 ng/mL human GDNF, 100 ng/mL human Fgf1, 10 nM LDN193189 and 10% growth factor reduced tomatorigel. was transferred to a medium containing ("Step 7 medium").

Branch culture of human iPS cell-derived ureteric buds
On Day 12.5, human iPS cell-derived induced ureteroblast spheroids were placed in 150 μL of branching medium in 24-well transwell inserts. Branching medium is 50% Matrigel, 10% FBS, 0.1 μM retinoic acid, 100 ng/mL human Rspondin 1 (R&D), 2 ng/mL human GDNF, 100 ng/mL human Fgf1, 30 ng/mL human Fgf7 and 10 DMEM/F12 medium containing nM LDN193189. Transwell inserts were cultured in 500 μL branching medium without Matrigel.

Induction of MM Lineage from Human iPSCs A previously reported protocol (Taguchi 2014, supra) was used with modifications. Cells were reaggregated at 10,000 cells per clump to form embryoid bodies (EBs) in V-bottom 96-well low cell binding plates in the presence of 10 μM Y27632 and 1 ng/mL human activin A. After 24 hours (Day 1), clumps were transferred to U-bottom 96-well low cell binding plates containing mesoderm induction medium containing 10 μM CHIR. Subsequently, half of the culture medium was replaced with fresh medium every other day (Day 3 and Day 5). On Day 7, the medium was changed to ABC3R medium containing 10 ng/mL human activin A, 3 ng/mL human Bmp4, 3 μM CHIR and 0.1 μM retinoic acid. On Day 9, the medium was changed to C1F medium containing 1 μM CHIR and 5 ng/mL human Fgf9.

Whole Mount Immunohistochemistry Organoids were fixed in PBS containing 4% PFA for 60 minutes, washed 3 times in PBS containing 0.1% Tryton X-100, 10% goat serum, 1% Tryton X-100, 2 Blocking was performed twice for 1 hour each with PBS containing % skim milk. Tissues were incubated with primary antibody and then with Alexa Fluor 488, 568, 594, 633 or 647 conjugated secondary antibody. After immunostaining, tissues were cleared (Klingberg et al., J. Am Soc Nephrol 28, 452-459, 2017). Specimens were dehydrated with ethanol and exchanged with ethyl cinnamate. 3D fluorescence images were taken with a two-photon microscope (FV1000-MPE; Olympus) or a confocal microscope (TSCSP8; Leica) and reconstructed by software (Imaris; Bitplane or LASX; Leica).

Sectional Immunohistochemistry Samples were fixed with PBS containing 4% PFA for 60 minutes, washed with PBS, dehydrated with PBS containing sucrose, embedded in OCT compound (TissueTek), and plated at a thickness of 10 μm. Frozen sections were made. For fluorescence immunohistochemistry analysis, sections were incubated with primary antibody followed by Alexa Fluor 488, 568, 594, 633 or 647 conjugated secondary antibody. Nuclei were counterstained with DAPI. Fluorescent images were taken with a confocal microscope (TSCSP8; Leica).

RNA extraction, reverse transcription and quantitative RT-PCR
The collected spheroids or cells were homogenized, total RNA was isolated using RNeasy Plus Micro Kit (Qiagen), and reverse transcription reaction was performed using random primers and Superscript III (Invitrogen). Quantitative PCR was performed using the Real-Time PCR system (Takara Bio) and Thunderbird SYBR qPCR Mix (Toyobo). Relative mRNA expression levels were analyzed, normalized by the β-actin gene. .

Flow cytometric analysis using immunostaining Cell aggregates derived from embryonic tissue or mouse ES cells/human iPS cells were dissociated, blocked with normal mouse serum, 1% BSA, 1 x HBSS and 0.035% NaHCO3. Cell surface marker staining was performed in a buffer containing Data analysis was performed using FlowJo software (Treestar).

Microarray analysis
Microarray analysis was performed using Agilent SurePrint G3 mouse gene expression (8 x 60K) microarrays. Data were normalized by gene SpringGX software (Agilent).

Quantification and Statistical Analysis All data analyzes were performed in three independent experiments unless otherwise stated. Values are expressed as mean±SE. Student's t-test was applied for statistical analysis of the two groups.

(result)
Example 1:
It was confirmed as follows that robust branching ability can be acquired by maturation of Wolffian tube (WD) development.
To assess the functional maturation process of early-stage WD, we first utilized a Hoxb7-GFP transgenic mouse line (Srinivas et al., Dev Genet 24, 241-151, 1999), and renal A reconstruction assay system was created. FIG. 4 shows a schematic diagram of the generation process of WD. Portions of WD used for reconstruction assays or microarray analysis are indicated by dashed lines. At E8.75, WD progenitor cells committed to WD become "committed WD progenitors". Isolated E11.5 metanephric mesenchyme (containing nephron progenitor and stromal cells) was dissociated into single cells and isolated from E9.5, E10.5 and E11.5 stage embryos. Reaggregated with UB. The number of branched tips of isolated UBs or WDs was counted on day 7 of organ culture, when each organoid stopped branching. UB or WD isolated from E11.5 embryos showed robust branch formation. On the other hand, WD isolated from E10.5 and E9.5 stage embryos showed a lower number of branches. Results are shown in FIG. Final branch numbers were not statistically different between tail and snout WD of E10.5 or E11.5 embryos. These results indicate that developmental progression acquires a branching capacity that is preserved regardless of the anteroposterior position of the WD.

Gene expression array analysis was performed. To identify markers that can monitor the developmental maturation process, gene expression array analysis was performed at each stage of UB, WD and their progenitor cells from E8.75-E11.5. The results are shown in FIG. Since the Hoxb7-GFP transgenic line leaked GFP fluorescence into a portion of the vascular endothelial population, WD progenitor cells in the Hoxb7-GFP+/Flk1- fraction were sorted by flow cytometry. Non-biased clustering analysis and similar entity analysis for representative UB marker genes identified several groups exhibiting different gene expression dynamics. Many of the key transcription factors involved in early WD development (Pax2, Lhx1, Emx2, Sim1, Gata3) were already expressed in WD progenitors at E8.75 and maintained regardless of developmental stage or location. rice field. Another group was the ureteral tip marker genes (eg, En2, Wnt11, Ret), which showed higher expression in the leading tip legion of WD or UB. Conversely, the expression of a series of genes (eg E-cadherin, Wnt9b, Hnf1b) increases with developmental progression, which may be useful for monitoring maturation.

Example 2:
We next confirmed that retinoic acid, Wnt and Fgf/Gdnf signaling matured WD progenitor cells into ureteroblast-like cells as follows.
The inventors have established a protocol for making UBs by a reverse induction approach. As a first step, we found factors that cause E9.5WD to mature into E11.5UB-like cells as follows. Microarray analysis identified repeated expression of retinoic acid synthase (Raldh3), Wnt co-receptor (Lgr5) and Fgf receptor/target genes from the beginning of early development in WD (data not shown). Therefore, WD were sorted from dissociated E9.5 mouse embryos and allowed to reaggregate in the presence of a combination of these growth factors. A Rho kinase inhibitor (10 μM Y27632) and 10% growth factor reducing tomatorigel were included to support epithelial cell survival.

Combination of RA, Wnt agonist (3 μM CHIR99021) and Fgf9 synergistically maintained lineage markers (Pax2, Emx2), apical markers (Ret) and induced mature WD markers (Hnf1b, Wnt9b and Calb1) (Fig. 7). However, spheres did not maintain Wnt11 expression and morphologically failed to undergo sprout formation. Therefore, we attempted to utilize Gdnf, a potent inducer of ureteric buds and a well-known upstream inducer of Wnt11. As expected, optimized conditions utilizing Gdnf successfully induced Wnt11 expression and bud-like structure formation (FIG. 5). These results are consistent with previous genetic loss-of-function studies demonstrating a requirement for each growth factor signal, including Fgf, canonical Wnt and retinoic acid signals, during WD development.

As a second step, we found the factor that matures E8.75WD to E9.5WD as follows. We investigated maturation triggers in the early stages of WD progenitor cells from the embryonic E8.75 to E9.5 stage. At the embryonic E8.75 to E9.5 stage, Hoxb7-GFP-positive WD progenitor cells are first clearly detectable in the anterior trunk (8-10 somite level) of the embryo. Similar to induction from E9.5 to E11.5 stage, sorted Hoxb7-GFP-positive progenitor cells maintained Emx2 and Ret expression in the presence of retinoic acid (RA), Wnt agonists and Fgf9. In contrast, the same concentrations of Wnt agonist and Fgf9 (3 μM and 100 ng/ml, respectively) suppressed the expression of maturation marker genes (Hnf1b, Wnt9b, Calb1, E-cadherin) at this stage. worked (Fig. 7). It could therefore be concluded that the combination of RA and low concentrations of Fgf9 (5 ng/ml) and Wnt agonist (1 μM) is optimal for induction at this stage. At this stage, Wnt11 expression could be maintained by Fgf9 without using Gdnf. These results suggest that somewhat different modes of gene control circuitry exist during the early and late developmental stages of the WD maturation process.

Finally, a 3-day induction protocol that combined the above factors to mature E8.75WD into E11.5UB-like cells was sequentially applied to the sorted E8.75WD progenitor cells. A schematic is shown in FIG. At day 3 of induction, E11.5 embryos showed formation of ureteric bud-like structures (Fig. 8) and gene expression levels quantitatively equivalent to embryonic UB (Fig. 9). Quantitative RT-PCR analysis confirmed gene expression kinetics during ex vivo maturation cultures similar to those in vivo.

Example 3:
Using the E8.75WD progenitor-like population from mouse embryonic stem cells, we searched for inducers of WD progenitor cells as follows.
First, to specifically and quantitatively assess the WD progenitor cell induction efficiency, we explored combinations of cell surface molecules specifically expressed in WD progenitor cells at E8.75. Wolff progenitors and ureteroblasts (E8.75WD, E8.75WD, E9.5WD_C, E10.5WD_R, E10.5WD_C, E11.5UB) and metanephric mesenchymal progenitors (E9.5IM_R, E9.5WD_C, E11.5UB) by developmental stage E9.5IM_C), focusing on Cxcr4, Icam2, Itgb3, and Kit as molecules expressed from the earliest Wolffian tube at E8.75 without expression in the metanephric mesenchymal lineage. made it
Mouse E8.75 fetuses expressing GFP in Wolff's tubes were dissociated into single cells and analyzed by flow cytometry for surface molecules thought to be expressed in the WD. FACS analysis using Itgb3 revealed that most of the Hoxb7-GFP+ metanephric mesenchymal progenitor cells were Itgb3-negative. In addition, FACS analysis using Icam2 revealed that Icam2 is strongly expressed in the vascular endothelium, but is only weakly expressed in a small part in WD. FACS analysis using Cxcr4 and cKIT revealed that cKIT is slightly weakly expressed in vascular endothelium, but Cxcr4 is expressed only in WD, and more than 99% of CXCR4-positive/Hoxb7-GFP-positive WD are Kit-positive. there were. The majority of Hoxb7-GFP-positive/Flk1-negative WD progenitors at E8.75 were positive for Cxcr4 and Kit, indicating that the population of WD progenitors among the total cells contained in E8.75 embryos and Cxcr4 strong The positive and cKit strongly positive populations were consistent, confirming specificity as markers for Cxcr4 and cKIT (circles in FIG. 10).

Example 4:
We investigated the induction of the UB lineage (AIM) from the T-positive immature mesoderm state.
For the derivation of WD progenitor cells from mouse embryonic stem cells, we first investigated the process of AP pattern formation in the intermediate mesoderm in vivo. The UB and MM lineage segregation model already constructed by the present inventors (Taguchi, 2014 above) shows that the UB lineage differentiates earlier than the MM lineage from the T-positive immature mesoderm state. , the immature state is maintained by strong Wnt signaling. Therefore, first, we tentatively shortened the incubation period with high concentrations of Wnt agonists to 1.5 days for UB induction versus 2.5 days for MM induction (step 2 in FIG. 2).

Next, genes that are minimally conserved between the anterior intermediate mesoderm (AIM) (Osr1+, Pax2+, Pax8+, Emx2+, Lhx1+, Gata3+) and the posterior intermediate mesoderm (PIM) (Osr1+, Wt1+, Hox11+) Based on the expression profile, we hypothesized that the signaling of the AIM differentiation stage was partially different from that of PIM (Step 3 in FIG. 2).
First, retinoic acid was raised as a common inducer for both AIM and PIM induction. Endogenous FGF signals were sufficient for PIM induction, but high concentrations of Fgf9 further enhanced AIM markers. In contrast to PIM induction, addition of activin A and Bmp4 was suppressive to the induction of AIM markers. Suppression of the smad2/3 pathway by SB431542 (SB) enhanced AIM marker induction. This suggests a major role for activin/Tgfb signaling in AIM versus PIM fate determination. On the other hand, either addition or inhibition of Bmp signal acted suppressively on AIM induction. This means that AIM specification requires an optimal level of Bmp signal.

Next, we examined factors that specify AIM to WD progenitor cells with Cxcr4 + / Kit + (Fig. 2:
step 4). The results are shown in FIG. At this stage, we found a synergistic effect of RA, Wnt agonist and Fgf9. In particular, withdrawal of the Wnt agonist dramatically reduced induction of the Cxcr4+/KIT+ population. This suggests that Wnt agonists play a crucial role in the induction of WD progenitor cells.

To further fine-tune the conditions and understand the UB lineage differentiation process, we repeated the experiment and varied the duration of incubation with Wnts, thereby confirming the optimal timing for efficient differentiation of early mesoderm into AIMs. (Figure 2: step 2). The allowed time window for AIM induction was approximately Day 4.5 (36 h of Wnt treatment). At day 4 (step 2 duration of 1 day) or day 5 (step 2 duration of 2 days) timing, the efficiency decreased dramatically. In vivo, this is a very narrow anterior-posterior mesoderm domain (pronephros primordia) that first appears at E8.5 within a 2-somite width (somite levels 8-10) of the intermediate mesoderm. may reflect

From Day 2 to Day 3 (Step 1) and from Day 3 to Day 4.5 (Step 2) of differentiation, concentration-dependent pattern formation by activin/Bmp signaling was observed, so epiblast and primitive streak/early mesoderm We investigated fate-specific signals within the stage. In mesoderm formation/patterning (Step 2), UB induction was maximal at higher Bmp4 concentrations compared to MM (Fig. 12). At the epiblast patterning stage (Step 1), UB induction favored higher concentrations of activin compared to those in MM (Fig. 13). These two-step combinatorial analyzes showed reciprocal patterns of optimal concentration ranges for induction to UB and MM (Fig. 14). These results suggest that UB and MM cell fate patterning begins before and during the formation of immature mesoderm. By these optimizations (optimization of step 1 and step 2), an average Cxcr4+/KIT+ population of 35.6% could be obtained on Day 6.25 of mouse ES cell differentiation.

Gene expression dynamics from immature ES cells at Day 0 to the WD progenitor stage at Day 6.25 (corresponding to WD progenitor cells at E8.75) were analyzed. The results are shown in FIG. The induced spheroids exhibited gene expression levels that were quantitatively equivalent to E8.75 WD progenitors and lacked expression of PIM or metanephric nephron progenitor markers (Hoxd11, Wt1 and Six2). This indicates successful selective induction into the UB lineage.

Example 5:
Using the induced UB, reconstruction of the higher-order structure of the embryonic kidney was carried out as follows.
Next, we investigated WD maturation factors for the induced WD progenitor cells. To visualize branching morphogenesis, mouse embryonic stem cells were established from Hoxb7-GFP transgenic mice. Minimal refinement of the initial induction step successfully induced Hoxb7-GFP+/Cxcr4+/KIT+ WD progenitor cells at day 6.25 of differentiation. The sorted GFP+/Cxcr4+/KIT+ cell population was reaggregated and cultured under WD maturation conditions established by the E8.75 WD culture experiment shown in FIG. Aggregates formed ureteroblast-like structures at day 9.25 of induction (Fig. 17) and expressed UB markers comparable to UB levels at E11.5 (Fig. 18). To create a single outlet and confirm branching capacity, single linear or bi-branching buds were manually isolated from the spheroids and reaggregated with the isolated E11.5 MM. In the presence of MM, induced UBs produced 6 to 7 bifurcated branch formations (once/day) in 6 to 7 days of organ culture (Fig. 19, left panel). The final tip number from a single bud averaged 141±12 (n=6), which was comparable to UB from E11.5 embryos (Fig. 19, right panel). The branching ability of induced UB was evaluated in cell-free branching culture conditions by modifying the method described previously (Rosines et al., Hum Mol Genet 20, 1143-1153, 2007). As a result, the optimized medium contained RA, Fgf1, Wnt agonist (Rspo1), Gdnf and 10% FBS in the presence of 50% Matrigel.

To further validate the functionality of the induced UB, the progenitor niche maintenance and differentiation capacity was analyzed by whole-mount staining of day 7 reconstituted organoids.

Induced UBs maintain Six2-positive nephrogenic progenitor cells at each UB edge at the organoid periphery, which corresponds to the nephrogenic zone of the embryonic kidney (Fig. 20, left panel). . In contrast, inside the organoids, differentiated nephrons were found that contained a series of E-Cadherin-positive distal tubular segments, LTL-positive proximal tubular segments and Nephrin-positive glomerular structures. This confirmed the ability of the induced UB to induce nephrons (the right figure in FIG. 20). The distal end of each nephron is connected to the tip of the ureter, which is essential for interconnection with the nephron for urine excretion (FIG. 20, bottom right).

Sox9, a typical UB tip marker, was expressed at the outer edge (data not shown). Cytokeratin 8, on the other hand, showed stronger expression in the medullary region of the kidney, similar to that in the E14.5 embryonic kidney. This indicates that proper tip-stalk patterning is occurring. The ubiquitous expression of Calb1 and Gata3 in the entire ureteral epithelium further confirmed the ureteral lineage-specific characteristics of this branched epithelium (data not shown).

Taken together, the induced UB fulfilled the functional criteria of UB, including the ability to develop branching morphogenesis, the ability to maintain nephron progenitor cells and the ability to differentiate the nephron. This indicates that reconstruction of the higher-order structure of the embryonic kidney with metanephric mesenchyme has become possible.

Example 6:
We investigated the use of induced nephron progenitor cells instead of embryonic MM.
Since we have already established conditions under which nephron progenitor cells can be induced, we considered replacing embryonic MM with induced nephron progenitor cells. First, we examined the necessity of each of the populations that constitute MM; that is, nephron progenitor cells and stromal progenitor cells. As previously demonstrated, most nephron progenitor cells at E11.5 reside in the Itga8+/Pdgfra− fraction, while stromal progenitor cells present Pdgfra (Taguchi et al., Cell stem cell 14 , 53-67, 2014). Therefore, each single fraction was sorted and reaggregated with induced UB. Stromal cells did not support UB branching in the absence of nephron progenitor cells. On the other hand, nephron progenitor cells, even in the absence of a stromal cell population, induce irregular branch formation, exhibiting a polygonal shape rather than the typical biantennary or triantennary UB. tips were formed (data not shown). In addition, an irregularly thickened layer of nephron progenitor cells was observed at each site at the tip of the UB (data not shown). This is reminiscent of previously reported knockout mouse phenotypes lacking functional stromal populations. These indicate that both stromal and nephron progenitor cell populations are required for proper organization of ureteric bud branching morphogenesis. Therefore, we decided to reconstruct kidney organoids using induced nephron progenitor cells, induced UB, and a Pdgfra+ stromal cell population sorted from embryonic kidney at E11.5. Similar to reconstruction using whole embryonic MM, reconstructed kidney tissue exhibited robust arborization with a nephron progenitor cell niche and differentiated nephron components. The functionality of both mouse ES cell-derived nephron progenitor cells and ureteric buds was demonstrated, indicating that they are capable of interacting to form kidney organoid conformations (FIG. 21). To confirm the selective contribution of each population, we utilized CAG-delta-Tomato mouse embryos for the Pdgfra+ stromal cell population. The embryonic cells were found to be a limited contribution to the stromal populations and medullary regions surrounding the organoid nephron progenitor cell niche (data not shown).

Example 7:
Induction of WD from human iPS cells was performed as follows. With reference to experiments using mouse ES cells, early mesoderm was first induced by activin followed by a high-concentration Wnt agonist (FIG. 3, Steps 1 and 2). AIM inducers and WD inducers were then made at each Step (Step 3 and Step 4) by combining RA, Fgf9 and Tgfb inhibitors or RA, Wnt agonists and Fgf9. In contrast to mouse ES cell induction, addition of the Bmp inhibitor LDN further enhanced both of these stages of differentiation. In particular, administration of LDN in the AIM induction stage (Step 3) greatly enhanced AIM marker gene expression and induction of CXCR4+/KIT+ WD progenitor cells. This was confirmed by quantitative analysis on Day 4.5 and Day 6.25, respectively (Figure 22).

Next, we examined the optimal time window to induce the anterior or posterior intermediate mesoderm from the early mesoderm. Since early mesoderm populations were maintained at high concentrations of Wnt agonists, we varied the incubation period with Wnt agonists and applied conditions specific to the UB or metanephric nephron progenitor lineage for subsequent differentiation. The induction efficiency of the CXCR4+/KIT+ WD progenitor cell fraction or the ITGA8+/PDGFRA- nephron progenitor cell fraction was examined on Day 6.25 or Day 12, respectively. Similar to the mouse ES cell experiments, the human UB lineage also required an inflexible time window for AIM induction, optimal at Day 2.5 (1.5 days of CHIR treatment). On the other hand, the induction efficiency of the nephron progenitor cell lineage peaked with administration of the PIM inducer on Day 7 (6 days of CHIR treatment), confirming our previous report. (Taguchi 2014, supra). Compared to the permissive time window of CHIR treatment (between 5 and 7 days) for nephron progenitor cell induction, the UB lineage was not induced beyond the optimal time window. Thus, the UB lineage was not induced in the timeframe of nephron progenitor cell differentiation.

Experiments with mouse ES cells showed that premature patterning during the epiblast to early mesoderm stage affected the fate decisions of UB versus nephron progenitor cells. Optimization of activin/Bmp concentrations in stages was investigated. At the epiblast patterning stage (differentiation days 0-1), UB favored higher activin signals, consistent with the mouse ES cell results, and addition of low concentrations of Bmp4 at this stage further reduced WD progenitor cell induction was further enhanced and peaked with the combination of 10 ng/ml activin A and 1 ng/ml Bmp4 (Fig. 23 left panel, Fig. 24). In contrast, in the absence of Bmp4, lower concentrations of activin enhanced the induction of nephron progenitor cells, maximizing at 1 ng/ml activin A (Fig. 23 right panel, Fig. 24).
The effect of co-administered Bmp signals was examined during the subsequent mesoderm induction/patterning stage by CHIR treatment (Days 1-2.5 for UB induction and Days 1-7 for MM induction). UB progenitor cells were most efficiently induced by the addition of 1 ng/ml Bmp4, whereas nephron progenitor cells were most efficiently induced in the absence of Bmp ligand or antagonist (Figure 25). Taken together, these results strongly suggest that there is an early, mutually independent process of lineage specification between the UB and metanephric nephron progenitor lineages. Under the final determined conditions, approximately 51.2% of CXCR4+/KIT+ WD progenitor cells were induced on day 6.25 of induction (Fig. 26), and kinetic analysis from day 0 to day 6.25 revealed that the WD marker set was efficient. It was found to be well induced (Fig. 27).

Next, on Day 6.25, the CXCR4+/KIT+ WD progenitor cell fraction was sorted and reaggregated in the presence of 10% Matrigel and WD maturation factors. In the differentiation of human WD progenitor cells, continuous administration of Fgf1 and LDN in addition to the mouse WD maturation cocktail further enhanced mature WD marker gene expression (data not shown). Optimized culture conditions induced mature UB markers including Hnf1b, E-Cadherin and CALB1 (data not shown), resulting in multiple bud formation on day 6 of culture (12 days of induction overall). (Fig. 28).
Induced UB showed branching ability in gel culture environment. Under these culture conditions, the first branch could be observed at approximately day 7 of culture, and the second formation of bifurcations was confirmed at the end of the second week (Fig. 28). This is considerably slower compared to the mouse UB branch, but comparable to human development in vivo. Branched UB organoids expressed Sox9 in the apical region of the ureteral epithelium and CK8 in the stalk region (FIG. 29, left panel). Structurally, this indicates tip-stalk patterning. In the apical region, typical bifurcated branches stained by E-cadherin and PAX2 were identified (Fig. 29, right panel). This represents the first evidence of reconstitution of human UB branching morphogenesis in vitro.

Example 8:
Since the method of selective induction of MM and UB developed by the present inventors has enabled the analysis of the lineage-specific roles of developmental genes, this method was used to identify the PAX2 gene in the MM and UB lineages. We tried to examine the cell-autonomous role.

Deletion of PAX2 from the MM lineage showed no gross abnormalities, at least in nephron progenitor induction and mesenchymal-epithelial transition to nephrons. Conversely, in the UB lineage, we observed a phenotype of incomplete UB differentiation. At day 6.25 of differentiation, a slight decrease in induction efficiency of CXCR4+/KIT+ WD progenitor cells was observed in knockout clones compared to controls (data not shown). Nonetheless, results of quantitative RT-PCR analysis showed genetically equivalent WD marker gene expression profiles, with the exception of PAX2, and a significant proportion of CXCR4+/KIT+ WD progenitors compared to control and PAX2. It was also obtained from one of the knockout clones (data not shown).

The selected CXCR4+/KIT+ WD progenitor cell population was further cultured under WD maturation conditions, but on Day 8.5 (second day of maturation culture), macroscopic morphological differences were observed between control and knockout clones. I couldn't. On day 10.5 (4th day of maturation culture), active cellularprotrusion formation reminiscent of migrating WD tips (Soofi et al., 2012) was observed in control clones, but knockout clones showed fewer protrusions and moderately decreased expression of PAX2 target genes, including LHX1, GATA3 and RET (data not shown).

At Day 12.5 (6th day of maturation culture), the control spheroids developed into giant ureteric bud-like structures, while the knockout clones showed an undulating surface with no apparent bud formation (Fig. 30). Expression levels of E-cadherin in knockout clones were significantly lower than in control clones. suggesting failure of proper mesenchymal epithelial transition in knockout clones. When the spheroids on Day 12.5 were analyzed by immunostaining of whole-mount specimens, E-cadherin signaling was clearly accumulated in the extracellular membrane of the basal side of the ureteric bud in the control, whereas in the knockout clone E-cadherin did not localize to the membrane and showed weak cytoplasmic expression.
This implies that loss of PAX2 in the UB lineage leads to failure to induce epithelial-mesenchymal transition at the maturation stage.

Example 9:
Regarding the effect on the induction of nephron differentiation from nephron progenitor cells, ureteroblast-like cells prepared using the method of the present invention were used together with conventional fetal spinal cord tissue (without ureteral buds). A comparison was made using the culture method.
Nephrons were induced as follows using ureteroblast-like cells prepared according to the method described in Examples. Metanephric mesenchyme collected from mouse embryos in which glomerular epithelial cells fluoresce with GFP were aggregated with induced ureteroblast-like cells, and after organ culture for 7 days, they were transplanted into immunodeficient mice. 15 days after transplantation, the number of glomeruli confirmed as GFP-positive globular structures was counted and the total number of nephrons finally formed was estimated.
For comparison, organ culture was carried out by the method of co-cultivating fetal spinal cord tissue and metanephric mesenchyme described in a report by the present inventors (A. Taguchi et al., Cell Stem Cell 14, 53-67, 2014), and transplantation was performed. gone.
The results are shown in FIG. It was found that maintaining nephron progenitor cells using ureteroblast-like cells produced by the method of the present invention markedly increases the number of ultimately formed nephrons.

According to the methods provided in the present invention, in vitro, mouse fetal ureteric buds have commensurate dendritic branching ability, maintain progenitor cell niches at the tips of branches, and connect with individual nephrons. It may be possible to generate ureteroblast-like cells that are capable of forming renal organoids.
In the method for producing WD progenitor-like cells provided in the present invention, by obtaining Cxcr4-positive and KIT-positive cells, (I) mixing with metanephric mesenchymal cells or growth factors promoting branching When cultured, it undergoes branching, (II) when mixed with metanephric mesenchyme, has the ability to form a progenitor cell niche that maintains the undifferentiated nature of nephron progenitor cells therein; Production of ureteroblast-like cells that have the ability to induce differentiation of nephron progenitor cells therein into nephrons when mixed with renal mesenchyme, and production of WD progenitor-like cells that serve as progenitor cells can allow
The kidney organoids reconstructed according to the present invention are the world's first reproduction of the higher-order structure of the kidney, and may be an indispensable technology for enabling the creation of functional artificial kidneys in the future. .

Claims (13)

The following steps:
Step B: a step of culturing pluripotent stem cells to obtain early mesoderm (Nascent Mesoderm) in a step consisting of the following steps B1 and B2;
Step B1: a step of culturing pluripotent stem cells in a medium containing (i) activin A or (ii) Tgfb1 and/or Tgfb2 for 0.5 to 3 days to obtain epiblasts;
Step B2: Step of culturing the epiblast obtained from step B1 for 1-2 days in a medium containing a Wnt agonist to obtain early mesoderm;
Step C: early mesoderm obtained by step B in a medium containing (i) retinoic acid or a retinoic acid analogue, (ii) fibroblast growth factor and (iii) a TGFβ signaling pathway inhibitor or Wnt agonist obtaining an Anterior Intermediate Mesoderm by culturing for 1-3 days;
Step D: The anterior intermediate mesoderm obtained from Step C is cultured for 1-3 days in a medium containing (i) retinoic acid or a retinoic acid analogue, (ii) a Wnt agonist, and (iii) fibroblast growth factor. obtaining a cell population containing Wolffian Duct Progenitor cells ,
and,
Step A: From the cell population obtained in Step D, CXC chemokine receptor 4 (Cxcr4)-positive and KIT proto-oncogene receptor tyrosine kinase (KIT)-positive Wolff duct progenitor cells are sorted or by magnetic beads. Including the step of sorting,
A method for producing Wolffian tube progenitor cells. The method according to claim 1, wherein the step A is a step of selecting cells in which Cxcr4-positive and KIT-positive cells account for 80% or more of all cells. The Cxcr4-positive and KIT-positive cells are further selected from the group consisting of Paired box (Pax) 2, LIM homeobox (Lhx) 1, empty spirals homeobox (Emx) 2, ret proto-oncogene (Ret) and homeobox (Hox) B7 2. The method of claim 1, wherein at least two of the The Wnt agonist in step B2, C or step D is each independently CHIR99021 or SB216763, the TGFβ signaling pathway inhibitor in step C is SB431542 or A83-01, and the retinoin in step C or step D The acid or retinoic acid analogue is each independently retinoic acid or ANG193109, and the fibroblast growth factor in step C or step D is each independently FGF2, FGF4, FGF7, FGF9 or FGF20. 4. The method according to any one of 1 to 3 . The method according to any one of claims 1 to 4, wherein said pluripotent stem cells are human iPS cells. 6. The method according to claim 5, wherein the medium used in step C further contains a BMP signaling pathway inhibitor. The method according to any one of claims 1 to 6, wherein the medium used in step B1 contains 1-1000 ng/ml activin A and the medium used in step B2 contains 1-1000 μM CHIR99021. A method of making a ureteroblast, comprising:
A step of producing Wolff duct progenitor cells by the method according to any one of claims 1 to 7 ; , (ii) a Wnt agonist, (iii) FGF9, and (iv) culturing in a medium containing a ROCK inhibitor. moreover,
Step (F): the cells obtained by step (E) are treated with (i) retinoic acid or a retinoic acid analogue, (ii) Wnt agonist, (iii) fibroblast growth factor, (iv) ROCK inhibitor, and (v 9. The method of claim 8, comprising culturing in a medium comprising glial cell line-derived neurotrophic factor (GDNF) or a GDNF analogue or FGF10. moreover,
Step (G): treating the cells obtained by step (F) in a medium containing (i) retinoic acid or a retinoic acid analog, (ii) a Wnt agonist, (iii) a ROCK inhibitor, and (iv) GDNF or a GDNF analog 10. The method of claim 9, comprising culturing with. The retinoic acid or retinoic acid analog in said Step E or Step F or Step G is each independently retinoic acid or ANG193109 and the Wnt agonist in said Step E or Step F or Step G is each independently CHIR99021 or SB216763 or Rspondin1, the ROCK inhibitor in step E or step F or step G is independently Y27632 or Fasudil hydrochlorid, and the fibroblast growth factor in step F is FGF2, FGF4, FGF7, FGF9 or 11. The method of claim 10, wherein FGF20 and the glial cell line-derived neurotrophic factor (GDNF) or GDNF analog in step F or step G is BT18. A method for producing kidney organoids, the step of producing ureteroblasts by the method according to any one of claims 8 to 11, and combining said ureteroblasts with nephron progenitor cells and embryonic kidney co-culturing with Platelet Derived Growth Factor Receptor Alpha (Pdgfra)-positive stromal cells from . The method of any one of claims 8-11, wherein the ureteroblasts express Hnf1b, E-Cadherin and CALB1.

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