Method for isolating ureteric bud tip cells

Abstract

Provided is a method for isolating ureteric bud tip cells from cells, tissues, or organoids which contain the ureteric bud tip cells, the method comprising: a step for bringing cells, tissue, or organoids which contain the ureteric bud tip cells into contact with a very-low-density lipoprotein receptor (VLDL-R) binding substance; and a step for isolating the ureteric bud tip cells by using said binding substance as an index.

Description

[Technical field]

[0001] This patent application claims priority to Japanese Patent Application No. 2019-181593, the entire contents of which are incorporated herein by reference.

[0002] The present application relates to methods for isolating ureteric bud tip cells. [Background technology]

[0003] Currently, the number of patients with chronic kidney disease (CKD) in Japan is estimated to be approximately 13 million, and it has been called the new national disease. There are few curative treatments for chronic kidney disease, and as the disease progresses, there are more than 330,000 patients with end-stage chronic renal failure who require dialysis therapy, which is a major problem not only medically but also medically and economically. Kidney transplantation is one of the radical treatments for chronic kidney disease, including end-stage chronic renal failure, but due to a serious shortage of donor organs, supply is not keeping up with demand.

[0004] The kidney originates from the intermediate mesoderm, a tissue in the early fetal stage. In vertebrates, three kidneys, the pronephros, mesonephros, and metanephros, are formed from the intermediate mesoderm, and in mammals, the metanephros becomes the adult kidney. The metanephros develops through the interaction of two tissues, the mesenchyme and the ureteric bud (Non-Patent Documents 1 and 2). Furthermore, it has been reported in recent years that the intermediate mesoderm is divided into two parts, anterior and posterior, and that the ureteric bud develops from the anterior intermediate mesoderm, and mesenchyme develops from the posterior intermediate mesoderm (Non-Patent Document 3).

[0005] The ureteric bud is an embryonic precursor tissue that gives rise to the lower urinary tract, from the renal collecting duct to part of the bladder. The ureteric bud is composed of a tip and a stem cell, and the tip is thought to self-renew and act as a stem cell for the ureteric bud, supplying daughter cells of the tip and stem cells.

[0006] If a method for producing ureteric bud cells and tissues derived from human iPS cells or human ES cells with high efficiency can be established, the produced ureteric bud cells and tissues can be used as a source of lower urinary tract cells for cell therapy. In addition, it is expected that in the future, the shortage of donors for kidney transplants can be resolved by reconstructing three-dimensional kidneys. Since many renal diseases occur in the ureteric bud and the collecting duct and lower urinary tract derived from it, it is also useful for creating renal disease models. Furthermore, it is expected that research on drug nephrotoxicity evaluation systems and drug development using collecting duct cells, lower urinary tract cells, or renal tissues containing them will be developed. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO2019 / 098349 [Non-patent literature]

[0008] [Non-Patent Document 1] Osafune K., et al., Development 2006; 133: 151-161. [Non-Patent Document 2] Kobayashi A., et al., Cell Stem Cell 2008; 3: 169-181. [Non-Patent Document 3] Taguchi A., et al., Cell Stem Cell. 2014; 14: 53-67. Summary of the Invention [Problem to be solved by the invention]

[0009] The present application aims to provide a method for isolating ureteric bud tip cells from cells, tissues or organoids containing ureteric bud tip cells. [Means for solving the problem]

[0010] The present application provides a method for isolating ureteric bud tip cells from a cell, tissue, or organoid comprising ureteric bud tip cells, the method comprising: (1-1) contacting a cell, tissue or organoid containing a ureteric bud tip cell with a very low density lipoprotein receptor (VLDL-R) binding substance; and (1-2) isolating ureteric bud tip cells using the binding substance as an indicator The present invention provides a method comprising:

[0011] The present application also relates to isolating ureteric bud tip cells by the method of the present application, and (2) The present invention provides a method for producing ureteric bud tip cell colonies, which comprises the step of culturing the ureteric bud tip cells in a medium containing a glial cell line-derived neurotrophic factor, a fibroblast growth factor, a retinoic acid receptor agonist, a GSK3β inhibitor and a Yes-associated protein (YAP) activity inhibitor.

[0012] The present application also provides: Providing a ureteric bud tip cell colony; and (3) Provided is a method for producing ureteric bud-like organoids, comprising the step of culturing the ureteric bud tip cell colony in a medium containing a Wnt signaling activator, a BMP inhibitor, a fibroblast growth factor, a retinoic acid receptor agonist, and a glial cell line-derived neurotrophic factor to reconstitute ureteric bud-like organoids.

[0013] The present application also provides: Providing a ureteric bud-like organoid; and (4) Provided is a method for producing collecting duct precursor-like organoids, comprising the step of culturing the ureteric bud-like organoids in a medium containing a Wnt signal inhibitor and a TGFβ signal inhibitor.

[0014] The present application also relates to isolating ureteric bud tip cells by the method of the present application, (2') The present invention provides a method for producing collecting duct progenitor cells, the method comprising the step of culturing the ureteric bud tip cells in a medium containing a Wnt signal inhibitor.

[0015] The present application also relates to obtaining ureteric bud tip cell colonies by the method of the present application, and further (3'-1) dissociating the ureteric bud tip cell colony; and (3'-2) The present invention provides a method for producing collecting duct progenitor cells, the method comprising the step of culturing the dissociated cell population in a medium containing a Wnt signal inhibitor.

[0016] The present application also provides: (1-1) contacting a cell, tissue, or organoid containing a ureteric bud tip cell with a VLDL-R binding substance; and (1-2) The present invention provides a method for producing a ureteric bud tip cell population, which comprises a step of isolating ureteric bud tip cells using the binding substance as an indicator.

[0017] The present application also provides: (i) contacting a biological tissue, tissue slice, cultured cells, or cultured tissue that is suspected to contain ureteric bud tip cells with a VLDL-R binding substance; and (ii) providing a method for monitoring ureteric bud tip cells, the method comprising the step of detecting the binding substance.

[0018] The present application also provides a composition for isolating or monitoring ureteric bud tip cells, comprising a VLDL-R binding agent. Effect of the Invention

[0019] The method of the present application makes it possible to efficiently isolate ureteric bud tip cells from cells, tissues, or organoids containing ureteric bud tip cells. [Brief description of the drawings]

[0020] [Figure 1] Schematic diagram summarizing the methods established in this example. We achieved cryopreservation of mesonephric duct leading edge cells (ND leader cells), isolation and proliferation of ureteric bud (UB) tip cells, generation and reconstitution of branching artificial ureteric bud (iUB) organoids, and induction of collecting duct precursors. [Diagram 2]Our previous (a) and new (b) protocols for differentiation of hiPSC / ESC into ureteric bud (UB) cells via the anterior primitive streak. APS, anterior primitive streak; Anterior IM, anterior intermediate mesoderm; ND, mesonephric duct; UB, ureteric bud. A, 100ng / ml Activin A; C3, 3μM CHIR99021; B, 10ng / ml BMP4; F8, 200ng / ml FGF8; TT, 0.1μM TTNPB; A83, 1μM A83-01; LDN, 0.1μM LDN193189; Y, 10μM Y-27632; C1, 1μM CHIR99021; G, 100ng / ml GDNF; F1, 200ng / ml FGF1; E, 50ng / ml EGF; Tzv, 10μM Thiazovivin. [Diagram 3] Immunostaining analysis of stage 4 day 2 cells with ND leader markers GATA3 (green) and RET (red). Scale bar is 100 μm. [Figure 4] Immunostaining analysis of stage 4 cells at days 6, 8 and 10 for E-CADHERIN (green), GATA3 (red) and PAX2 (purple) shows that growth factors used at the mesonephric duct (ND) elongation stage (stage 4) enhance epithelialization of ND leader cells. Scale bar is 100 μm. [Diagram 5] Immunostaining analysis for E-CADHERIN (green), GATA3 (red) and PAX2 (purple) of day 2 aggregates before and after removal of non-ND cells. Scale bar is 100 μm. [Figure 6] No significant difference was found in the number of cells on the second day of ND maturation (stage 5) between those with and without cryopreservation (n=3).

[0021] [Figure 7] Immunostaining analysis for E-CADHERIN (green), GATA3 (red) and PAX2 (purple) of stage 5 day 2 ND aggregates generated from cryopreserved ND leader cells. Scale bar is 100 μm. [Figure 8]Morphological changes of ND aggregates over 4 days with and without low-concentration Matrigel. Scale bar is 100 μm. [Figure 9] Morphology of iUB organoids at day 6. The right panel shows a magnification of the boxed area in the left panel. Scale bar is 300 μm. [Figure 10] Morphology of aggregates cultured with high (50%, left panel) and low (2%, right panel) concentrations of Matrigel on day 7. Scale bar is 100 μm. [Figure 11] Immunostaining analysis of iUB organoids at day 7 for PAX2 (green), EZRIN (red) and LAMININ (purple). Scale bar is 100 μm.

[0022] [Figure 12] Immunostaining analysis of iUB organoids at day 6 for RET (green) and CK8 (red; left panel), LAMININ (white) and EZRIN (red; middle panel), and LAMININ (white) and PAX2 (green; right panel). Scale bars are 100 μm. [Figure 13] Morphology of tips isolated from iUB organoids at day 6. Scale bar is 100 μm. [Figure 14] Morphological changes over the first 7 days of apical organoids isolated from iUB organoids at day 6. Scale bar is 100 μm. [Figure 15] Immunostaining analysis for PAX2 (blue), RET (green) and CK8 (red) of dissociated tip organoids at day 14. Scale bar is 100 μm. [Figure 16] 3D whole mount immunostaining for GATA3 (green), RET (white) and E-CADHERIN (red) of dissociated tip organoids at day 14.

[0023] [Figure 17]Morphological changes of the isolated tip over 7 days. The isolated tip from the reconstituted iUB organoid undergoes branching morphogenesis and reconstitutes the iUB organoid. Scale bar: 100 μm. [Figure 18] Morphology of iUB organoids cultured in organoid medium without EGF and FGF1 or with EGF and FGF1. Scale bar is 100 μm. [Figure 19] Immunostaining analysis for RET (red) and GATA3 (green) of iUB organoids at day 6 cultured in organoid medium containing EGF and FGF1. Scale bar is 100 μm. [Figure 20] Immunostaining analysis for LAMININ (green), EZRIN (white), and PAX2 (red) of iUB organoids at day 6 cultured in organoid medium containing EGF and FGF1. Scale bar is 100 μm. [Figure 21] Schematic showing formation of tip cell colonies from single iUB organoid-derived cells on hydrogel.

[0024] [Figure 22] Morphological changes of single cells dissociated from iUB organoids over 7 days. Scale bar is 100 μm. [Diagram 23] Immunostaining analysis for RET (green) and GATA3 (red) of tip cell colonies at day 7. Scale bar is 100 μm. [Figure 24] Immunostaining analysis for CK8 (green), VLDLR (red) and RET (white) of iUB organoids at day 7. Scale bar is 100 μm. [Diagram 25] Bright-field and fluorescence microscopy images of tip cell colonies, ND aggregates and iUB organoids following DiI-labeled VLDL (DiI-VLDL) uptake. Scale bars are 100 μm. [Figure 26] iUB organoids were sorted by DiI-VLDL using FACS. [Figure 27]Heatmap showing differentially expressed genes between DiI-VLDL+ and DiI-VLDL- cells as determined by RNA sequencing analysis, showing that expression of Tip-associated genes was elevated in DiI-VLDL+ cells, whereas expression of Trunk-associated genes was higher in DiI-VLDL- cells.

[0025] [Figure 28] DiI-VLDL sorting of tip cell colonies by FACS. [Figure 29] Percentage of DiI-VLDL+ cells in iUB organoids and tip cell colonies. *p<0.05 and **p<0.001 by Student's t-test. [Diagram 30] Fluorescence microscopy analysis of DiI-VLDL- and DiI-VLDL+ cells on days 0 and 7. Note that tip cell colonies that take up DiI-VLDL arise not only from DiI+ cells but also from DiI- cells. Scale bar, 100 μm. [Diagram 31] Percentage of DiI-VLDL+ cells in tip cell colonies derived from single DiI-VLDL+ and DiI-VLDL- cells at day 6. *p<0.05 and **p<0.001 by Student's t-test. [Diagram 32] Morphology of iUB organoids derived from tip cell colonies at day 18 after hydrogel culture in organoid medium containing low concentration Matrigel. Scale bar is 300 μm. [Diagram 33] Immunostaining analysis of EZRIN (green; left panel), LAMININ (red; left panel), GATA3 (red; right panel), and RET (green; right panel) of iUB organoids derived from tip cell colonies at day 18. Scale bars are 100 μm.

[0026] [Diagram 34] Morphological changes over 14 days in a tip isolated from a day 14 iUB organoid. Scale bar is 100 μm. [Diagram 35] Morphology of iUB organoids at day 7 reconstituted from tip cell colonies cultured in organoid medium containing CHIR99021 or Wnt3a. Scale bar is 100 μm. [Diagram 36] Immunostaining analysis for GATA3 (green) and AQP2 (red) of dissociated tip cells on day 7 of 2D culture in medium with or without A83-01. Scale bar is 100 μm.

[0027] [Figure 37] Immunostaining analysis of dissociated tip cells on day 7 of 2D culture for AQP2 (red), E-CADHERIN (white) and FOXA1 (green). Scale bar is 100 μm. [Figure 38] Immunostaining analysis of dissociated tip cells on day 7 in 2D culture with the interstitial cell marker CAII (green). Scale bar, 100 μm. [Figure 39] Morphology of collecting duct precursor organoids at day 14. Scale bar is 100 μm. [Diagram 40] Immunostaining analysis of day 14 collecting duct precursor organoids for FOXA1 (white), AQP2 (red), and GATA3 (green).

[0028] [Diagram 41] Schematic diagram showing subculturing of tip cell colonies derived from iUB organoids. [Diagram 42] Morphology of tip cell colonies induced by dissociating iUB organoids into single cells and culturing them in medium further containing A83-01. Scale bar is 100 μm. [Diagram 43] Immunostaining analysis for GATA3 (green), RET (red) and SOX9 (purple) of tip cell colonies induced in medium additionally containing A83-01. [Diagram 44] Morphology of iUB organoids derived from tip cell colonies induced in medium further containing A83-01. [Diagram 45]Immunostaining analysis for GATA3 (green) and RET (red) of iUB organoids derived from tip cell colonies induced in medium further containing A83-01. [Figure 46] Morphology of tip cell colonies after three subcultures. [Figure 47] Immunostaining analysis of tip cell colonies after three passages for GATA3 (green), RET (red) and SOX9 (purple). [Figure 48] Morphology of iUB organoids derived from tip cell colonies after three passages. [Figure 49] Immunostaining analysis of GATA3 (green) and RET (red) in iUB organoids derived from tip cell colonies after three passages. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] In this disclosure, when a numerical value is accompanied by the term "about," it is intended to include a range of ±10% of that value. For example, "about 20" is intended to include "18-22." A range of numerical values ​​includes all values ​​between and at the endpoints. "About" in reference to a range applies to both endpoints of the range. Thus, for example, "about 20-30" is intended to include "18-33."

[0030] In the present specification and claims, the expression "a certain type of cell" means a cell group containing the cell type, unless otherwise specified, and the cell group may contain cells of types other than the specified cell type. For example, "a culture of a certain type of cell" means a culture of a cell group containing the cell type, and may contain cells of types other than the specified cell type. Similarly, the expression "a certain type of cell population" means a cell population containing the cell type, unless otherwise specified, and the cell population may contain cells of types other than the specified cell type.

[0031] In the present application, the medium can be prepared by appropriately adding necessary factors to a basal medium used for culturing animal cells. Examples of the basal medium include MEM Zinc Option medium, IMEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, DMEM / F12 medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof. The basal medium may contain serum (e.g., fetal bovine serum (FBS)) or may be serum-free. If necessary, the medium may contain one or more serum substitutes, such as, for example, albumin, transferrin, KnockOut Serum Replacement (KSR) (a serum substitute for ES cell culture) (Thermo Fisher Scientific), N2 supplement (Thermo Fisher Scientific), B27 supplement (Thermo Fisher Scientific), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, or may contain one or more substances, such as lipids, amino acids, L-glutamine, GlutaMAX (Thermo Fisher Scientific), non-essential amino acids (NEAA), vitamins, growth factors, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, and the like, or one or more substances typically added to animal culture media.

[0032] The basal medium used in the present application is, for example, DMEM / F12 medium or Essential 6, a serum-free medium in which L-ascorbic acid-2-phosphate magnesium, sodium selenium, insulin, NaHCO3, and transferrin are added to DMEM / F12 medium. (商標) The medium may be (Thermo Fisher Scientific).

[0033] In the present specification and claims, "isolation" means that an operation is performed to remove components other than the target component (cells, tissues, organoids, etc.), and the target component is no longer in the state in which it was originally present. In the present application, the isolation or production of the target cell, tissue, or organoid can be confirmed by the expression of their markers, their size and / or shape, etc. The expression of the marker can be confirmed by known methods such as immunostaining, flow cytometry, FACS (fluorescence-activated cell sorting), and MACS (magnetic-activated cell sorting).

[0034] Method for isolating ureteric bud tip cells from cells, tissues or organoids containing ureteric bud tip cells In one embodiment of the present application, there is provided a method for isolating ureteric bud tip cells from a cell, tissue or organoid comprising ureteric bud tip cells, comprising: (1-1) contacting a cell, tissue or organoid containing a ureteric bud tip cell with a very low density lipoprotein receptor (VLDL-R) binding substance; and (1-2) isolating ureteric bud tip cells using the binding substance as an indicator The present invention provides a method comprising:

[0035] "Ureteric bud tip cell" refers to a cell at the tip of the budded or branched region of ureteric bud tissue or ureteric bud-like organoid. The ureteric bud tip cell can be confirmed by the expression of RET or very low density lipoprotein receptor (VLDL-R), or the uptake of very low density lipoprotein (VLDL). The cell, tissue or organoid containing the ureteric bud tip cell can be derived from a mammal, for example, a primate, and is preferably derived from a human.

[0036] In one embodiment, the cell, tissue or organoid containing the ureteric bud tip cell is a ureteric bud-like organoid. "Ureteric bud-like organoid" refers to a ureteric bud-like self-organized structure having a branched structure. The size of the ureteric bud-like organoid is, for example, about 10 to about 1000 μm. In addition, one ureteric bud-like organoid branches, for example, 3 to 5 times. The ureteric bud-like organoid can be confirmed by the expression of markers such as GATA3, RET, PAX2, and CALB1. In addition, the branched structure of the ureteric bud-like organoid can be confirmed under a microscope.

[0037] The ureteric bud-like organoid may be one that is differentiated from pluripotent stem cells. Methods for inducing differentiation of ureteric bud-like organoids from pluripotent stem cells are known, and any known method may be used. For example, the method described in WO2019 / 098349 can be used.

[0038] In the present application, the term "pluripotent stem cells" refers to stem cells that have both pluripotency and proliferation ability and can differentiate into all cells present in a living body, and examples of such cells include embryonic stem (ES) cells (JA Thomson et al. (1998), Science 282:1145-1147; JA Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92:7844-7848; JA Thomson et al. (1996), Biol. Reprod., 55:254-259; JA Thomson and VS Marshall (1998), Curr. Top. Dev. Biol., 38:133-165), cloned embryo-derived embryonic stem (ntES) cells obtained by nuclear transfer (T. Wakayama et al. (2001), Science, 292:740-743; S. Wakayama et al. (2005), Biol. Reprod., 72:932-936; J. Byrne et al. (2007), Nature, 450:497-502), spermatogonial stem cells (“GS cells”) (M. Kanatsu-Shinohara et al. (2003) Biol. Reprod., 69:612-616; K. Shinohara et al. (2004), Cell, 119:1001-1012), embryonic germ cells (“EG cells”) (Y. Matsui et al. (1992), Cell, 70:841-847; JL Resnick et al. (1992), Nature, 359:550-551), induced pluripotent stem (iPS) cells (K. Takahashi and S. Yamanaka (2006) Cell, 126:663-676; K. Takahashi et al. (2007), Cell, 131:861-872; J. Yu et al. (2007), Science, 318:1917-1920; Nakagawa, M. et al, Nat. Biotechnol.26:101-106 (2008); WO2007 / 069666), and pluripotent cells derived from cultured fibroblasts or bone marrow stem cells (Muse cells) (WO2011 / 007900). In the present application, the pluripotent stem cells may be derived from mammals, for example, from primates, and preferably from humans. The pluripotent stem cells may be, for example, ES cells or iPS cells.

[0039] iPS cells can be produced by introducing a specific reprogramming factor into somatic cells in the form of DNA or protein (K. Takahashi and S. Yamanaka (2006) Cell, 126:663-676; K. Takahashi et al. (2007), Cell, 131:861-872; J. Yu et al. (2007), Science, 318:1917-1920; Nakagawa, M. et al, Nat. Biotechnol. 26:101-106 (2008); WO2007 / 069666). When using iPS cells, the iPS cells may be produced from somatic cells by a method known per se, or iPS cells that have already been established and stocked may be used. There is no limitation on the somatic cells from which the iPS cells used in the present invention are derived, and for example, cells derived from peripheral blood or cells derived from umbilical cord blood may be used. There are no limitations on the animals from which pluripotent stem cells can be derived, and examples include mammals such as mice, rats, hamsters, guinea pigs, cows, horses, pigs, sheep, monkeys, orangutans, chimpanzees, dogs, cats, birds, and humans, with primates being preferred, and humans being more preferred.

[0040] In step (1-1), cells, tissues or organoids containing ureteric bud tip cells are contacted with a very low density lipoprotein receptor (VLDL-R) binding substance.

[0041] In the present application, the "very low density lipoprotein receptor (VLDL-R) binding substance" is not particularly limited as long as it has the property of binding to VLDL-R. For example, it includes, but is not limited to, compounds, peptides, and proteins that have the property of binding to VLDL-R. For example, proteins that bind to VLDL-R may include VLDL-R ligands, antibodies and antibody fragments that have VLDL-R binding activity, and proteins that contain VLDL-R binding domains. In one embodiment, the VLDL-R binding substance is "very low density lipoprotein (VLDL)". "Very low density lipoprotein (VLDL)" is one of the plasma lipoproteins classified by ultracentrifugation or electrophoresis. The VLDL used herein may also include variants and modified forms of VLDL that have VLDL-R binding activity. In step (1-1), the VLDL-R binding substance is typically added to the culture medium of cells, tissues, or organoids that contain ureteric bud tip cells.

[0042] In one embodiment, the VLDL-R binding substance used in the present invention can be a labeled VLDL-R binding substance, for example, labeled VLDL. Labeled VLDL-R binding substance or VLDL means a VLDL-R binding substance or VLDL that is bound to a label. Labels that can be used include fluorescent labels, magnetic labels, enzymes, coenzymes, chemiluminescent labels, bioluminescent labels, radioactive labels, metals, and other labels known to those skilled in the art. In the present application, the label can be, for example, a fluorescent label or a magnetic label. Examples of fluorescent labels include carbocyanine dyes (DiI, DiO, DiD, DiR, etc.), fluorescein derivatives (fluorescein isothiocyanate (FITC), fluoresceinthiofluorbamyl, etc.), rhodamine derivatives (tetramethylrhodamine, trimethylrhodamine (RITC), Texas Red, rhodamine 110, etc.), Cy dyes (Cy3, Cy5, Cy5.5, Cy7), Cy-chrome, spectrum green, spectrum orange, propidium iodide, allophycocyanin (APC), R-phycoerythrin (R-PE), Alexa Fluor dyes, etc. The labeled VLDL may be a commercially available product, for example, DiI-labeled VLDL (DiI-VLDL; Biomedical Technologies, Inc.). Examples of magnetic labels include magnetic microbeads, etc. The amount of VLDL-R binding substance to be added is not particularly limited as long as it is an amount that can be detected as an indicator. When DiI-VLDL is used, its concentration may be 10 ng / ml to 1 mg / ml, 100 ng / ml to 1 mg / ml, or 1 μg / ml to 100 μg / ml, for example about 10 μg / ml.

[0043] In step (1-2), the ureteric bud tip cells are isolated using the binding substance as an indicator. For example, the binding substance may be labeled as described above, and the label may be used as an indicator. Alternatively, a labeled antibody or the like that recognizes the binding substance may be added, and the label may be used as an indicator. A known method may be appropriately used depending on the type of label to isolate the ureteric bud tip cells using the label as an indicator. For example, when the label is a fluorescent label, the cells can be isolated by flow cytometry or FACS (fluorescence-activated cell sorting). When the label is a magnetic label, the cells can be isolated by MACS (magnetic-activated cell sorting).

[0044] Method for producing ureteric bud tip cell population from cells, tissues or organoids containing ureteric bud tip cells In one embodiment of the present application, (1-1) contacting a cell, tissue, or organoid containing a ureteric bud tip cell with a VLDL-R binding substance; and (1-2) The present invention provides a method for producing a ureteric bud tip cell population, which comprises a step of isolating ureteric bud tip cells using the binding substance as an indicator.

[0045] Steps (1-1) and (1-2) may be carried out in the same manner as steps (1-1) and (1-2) in the method for isolating ureteric bud tip cells from cells, tissues, or organoids containing ureteric bud tip cells described above.

[0046] Method for producing ureteric bud tip cell colonies from isolated ureteric bud tip cells In one embodiment of the present application, the method of the present application comprises isolating ureteric bud tip cells, (2) The present invention provides a method for producing ureteric bud tip cell colonies, which comprises the step of culturing the ureteric bud tip cells in a medium containing a glial cell line-derived neurotrophic factor, a fibroblast growth factor, a retinoic acid receptor agonist, a GSK3β inhibitor and a Yes-associated protein (YAP) activity inhibitor.

[0047] The ureteric bud tip cell colony refers to a cell population proliferated from a single ureteric bud tip cell. The size of the ureteric bud tip cell colony is not particularly limited and can be appropriately determined by a person skilled in the art depending on the purpose of use. The fact that it is a ureteric bud tip cell colony can be confirmed by the expression of RET, GATA3, very low density lipoprotein receptor (VLDLR), or the uptake of very low density lipoprotein (VLDL), etc.

[0048] In step (2), the ureteric bud tip cells isolated by the method of the present application may be seeded on a hydrogel obtained by diluting a three-dimensional scaffold material with a culture medium and cultured three-dimensionally.

[0049] As the three-dimensional scaffold material, various materials for constructing three-dimensional structures of cultured cells are known and commercially available, and are not particularly limited. For example, collagen-based materials, polymer-based materials such as polycaprolactone and polyglycolic acid, or complexes thereof can be used. The form is also not particularly limited, and examples thereof include sponge-like structures. Furthermore, the three-dimensional scaffold material may be made of a sample derived from a living body (e.g., extracellular matrix, basement membrane, etc.). Specifically, Matrigel (商標) (Becton Dickinson), type I collagen gel, and type IV collagen gel are examples. Matrigel (商標) Basement membrane matrix is ​​a soluble basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma that is rich in extracellular matrix proteins, primarily composed of laminin, collagen IV, entactin, and heparan sulfate proteoglycans, and may contain other growth factors such as TGF-β, fibroblast growth factor, tissue plasminogen activator, and EHS.

[0050] In step (2), when Matrigel is used as the three-dimensional scaffold material, the concentration of Matrigel in the hydrogel can be 20% to 80%, 30% to 70%, or 40% to 60%, for example, about 50%.

[0051] In step (2), the concentration of the glial cell line-derived neurotrophic factor may be 100 pg / ml to 10 μg / ml, 1 ng / ml to 10 μg / ml, or 10 ng / ml to 1 μg / ml, for example, about 100 ng / ml.

[0052] Known "fibroblast growth factors" include FGF1 to FGF23, and may be appropriately selected from these known factors. In step (2), the concentration of the fibroblast growth factor may be appropriately selected by those skilled in the art depending on the fibroblast growth factor used. When the fibroblast growth factor is FGF1, the concentration may be 200 pg / ml to 20 μg / ml, 2 ng / ml to 20 μg / ml, or 20 ng / ml to 2 μg / ml, for example, about 200 ng / ml.

[0053] A "retinoic acid receptor (RAR) agonist" may be a naturally occurring retinoid, a chemically synthesized retinoid, a retinoic acid receptor agonist compound that does not have a retinoid backbone, or a natural product that has retinoic acid receptor agonist activity. An example of a natural retinoid that has activity as an RAR agonist is retinoic acid (the stereoisomers all-trans-retinoic acid (all-trans RA) and 9-cis-retinoic acid (9-cis RA) are known). Chemically synthesized retinoids are known in the art (e.g., U.S. Pat. No. 5,234,926, U.S. Pat. No. 4,326,055, etc.). Examples of retinoic acid receptor agonist compounds that do not have a retinoid skeleton include Am80, AM580 (4-[[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl]carboxamido]benzoic acid), TTNPB (4-[[E]-2-[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl]-1-propenyl]benzoic acid), and AC55649 (4'-octyl-[1,1'-biphenyl]-4-carboxylic acid). Examples of natural products with retinoic acid receptor agonist activity include honokiol and magnolol (Biofunction Research Institute Bulletin 9:55-61, 2009). The RAR agonist used in the present application can be retinoic acid, AM580, TTNPB, AC55649, for example, TTNPB. In step (2), the concentration of the retinoic acid receptor agonist can be appropriately selected by those skilled in the art according to the retinoic acid receptor agonist used. When the retinoic acid receptor agonist is TTNPB, its concentration can be 0.1nM to 10μM, 1nM to 10μM, or 10nM to 1μM, for example, about 0.1μM.

[0054] The term "GSK3β inhibitor" is defined as a substance that inhibits the kinase activity of GSK3β protein (e.g., the ability to phosphorylate β-catenin). Many of these are already known, including, for example, the indirubin derivative BIO (also known as GSK3β inhibitor IX; 6-bromoindirubin 3'-oxime), the maleimide derivative SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), and SB415286 (3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2 -nitrophenyl)-1H-pyrrole-2,5-dione), GSK3β inhibitor VII (4-dibromoacetophenone), a phenyl α bromomethyl ketone compound, L803-mts (also known as GSK3β peptide inhibitor; Myr-N-GKEAPPAPPQSpP-NH2), a cell membrane permeable phosphorylated peptide, and CHIR99021 (6-[2-[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-ylamino]ethylamino]pyridine-3-carbonitrile), which has high selectivity. These compounds are commercially available from, for example, Calbiochem and Biomol, and can be easily used. The GSK3β inhibitor used in the present application may be, for example, CHIR99021. In step (2), the concentration of the GSK3β inhibitor can be appropriately selected by those skilled in the art depending on the GSK3β inhibitor used. When the GSK3β inhibitor is CHIR99021, its concentration can be 3 nM to 300 μM, 30 nM to 300 μM, or 300 nM to 30 μM, for example, about 3 μM.

[0055] In step (2), the concentration of the YAP activity inhibitor can be appropriately selected by those skilled in the art depending on the YAP activity inhibitor used. As the YAP activity inhibitor, a known one may be appropriately used, for example, Thiazovivin, which has both YAP inhibitory activity and ROCK inhibitory activity. When the YAP activity inhibitor is Thiazovivin, its concentration can be 10nM to 1mM, 100nM to 1mM, or 1μM to 100μM, for example, about 10μM.

[0056] In step (2), the medium may further contain a TGFβ signal inhibitor. The "TGFβ signal inhibitor" is a substance that inhibits signal transduction that continues from the binding of TGFβ to the receptor to SMAD, and is not particularly limited as long as it is a substance that inhibits the binding to the receptor, ALK family, or a substance that inhibits the phosphorylation of SMAD by the ALK family. Examples of such a substance include Lefty-1 (NCBI Accession No. NM_010094 for mouse and NM_020997 for human), SB431542, SB202190 (RKLindemann et al., Mol. Cancer, 2003, 2:20), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276 (Lilly Research Laboratories), A83-01 (3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide, WO2009146408), ALK5 inhibitor II (2-[3-[6-methylpyridin-2-yl]-1H-pyrazol-4-yl]-1,5-naphthyridine), TGFβRI kinase inhibitor VIII (6-[2-tert-butyl-5-[6-methyl-pyridin-2-yl]-1H-imidazol-4-yl]-quinoxaline) and derivatives thereof. The TGFβ signal inhibitor used in the present application may be, for example, A83-01. In step (2), the concentration of the TGFβ signal inhibitor can be appropriately selected by those skilled in the art depending on the TGFβ signal inhibitor used. When the TGFβ signal inhibitor is A83-01, the concentration may be 1 nM to 100 μM, 10 nM to 100 μM, or 100 nM to 10 μM, for example, about 1 μM.

[0057] In the step (2), the culture period may be 2 to 21 days, 3 to 14 days, or 5 to 10 days, for example, about 6 to about 7 days.

[0058] The ureteric bud tip cell colony can be maintained by subculturing every 1 to 12 days, every 5 to 10 days, every 6 to 8 days, or about every 7 days. The ureteric bud tip cell colony can also be maintained by subculturing when it has grown to a size of about 10 to about 1000 μm, about 15 to about 500 μm, about 20 to about 150 μm, or about 80 to about 100 μm. The number of subcultures is not particularly limited, and the colony can be subcultured a desired number of times. For example, the ureteric bud tip cell colony can be subcultured once, twice, three times, four times, five times, or more times. The subculture period is not particularly limited, and the colony can be subcultured for a desired period. For example, the ureteric bud tip colony can be subcultured for 1 day or more, 7 days or more, 14 days or more, 21 days or more, 28 days or more, for example, 70 days or more.

[0059] The culture temperature is, but not limited to, about 30 to about 40° C., for example, about 37° C., and the culture is performed in an atmosphere of CO2-containing air, with a CO2 concentration of, for example, about 2 to about 5%.

[0060] That is, the present invention also provides a method for subculturing ureteric bud tip cell colonies. The substances and their concentrations added to the medium during subculturing are as described above. As an example, a glial cell line-derived neurotrophic factor (e.g., about 100 ng / ml GDNF), a fibroblast growth factor (e.g., about 200 ng / ml FGF1), a retinoic acid receptor agonist (e.g., about 0.1 μM TTNPB), a GSK3β inhibitor (e.g., about 3 μM CHIR99021), a YAP activity inhibitor (e.g., about 10 μM Thiazovivin), and a TGFβ signal inhibitor (e.g., about 1 μM A83-01) are added to the medium. Other culture conditions are also as described above.

[0061] A method for reconstructing ureteric bud-like organoids from ureteric bud tip cell colonies In one embodiment of the present application, (3) A method for producing a ureteric bud-like organoid is provided, which comprises culturing a ureteric bud tip cell colony in a medium containing a Wnt signaling activator, a BMP inhibitor, a fibroblast growth factor, a retinoic acid receptor agonist, and a glial cell line-derived neurotrophic factor to reconstitute a ureteric bud-like organoid. The ureteric bud tip cell colony may be a cell obtained by the method of the present application, or may be a cell obtained by another known method. In this step, the size of the ureteric bud tip cell colony is not particularly limited, but may be, for example, about 10 to about 1000 μm.

[0062] In step (3), the ureteric bud tip cell colony is cultured in a medium containing a Wnt signaling activator, a BMP inhibitor, a fibroblast growth factor, a retinoic acid receptor agonist, and a glial cell line-derived neurotrophic factor. In one embodiment, in step (3), the ureteric bud tip cell colony is cultured in suspension. In the present application, "suspension culture" refers to culturing cells in a non-adherent state on a culture dish. Although not particularly limited, the culture can be performed using a dish that has not been artificially treated (e.g., coated with an extracellular matrix or the like) for the purpose of improving adhesion to the cells, or a dish that has been artificially treated to suppress adhesion (e.g., coated with a polymer of polyhydroxyethyl methacrylate (poly-HEMA) or 2-methacryloyloxyethyl phosphorylcholine (Lipidure)). For example, a commercially available product such as a low-adhesion 35 mm dish (Sumitomo Bakelite) may be used.

[0063] In step (3), the concentration of the Wnt signaling activator can be appropriately selected by those skilled in the art depending on the Wnt signaling activator used. The Wnt signaling activator used in the present application may be, for example, LiCl, Wnt1, Wnt3a, Wnt7a, R-spondin 1, etc. When the Wnt signaling activator is Wnt3a, its concentration may be about 5 to about 20%. A commercially available product such as Afamin / Wnt3a conditioned medium may be used. When the Wnt signaling activator is R-spondin 1, its concentration may be 200 pg / ml to 20 μg / ml, 2 ng / ml to 20 μg / ml, or 20 ng / ml to 2 μg / ml, for example, about 200 ng / ml.

[0064] Examples of "BMP inhibitors" include protein inhibitors such as Chordin, Noggin, and Follistatin, Dorsomorphin 6-[4-(2-piperidin-1-yl-ethoxy)phenyl]-3-pyridin-4-yl-pyrazolo[1,5-a]pyrimidine, its derivatives (PB Yu et al. (2007), Circulation, 116:II_60; PB Yu et al. (2008), Nat. Chem. Biol., 4:33-41; J. Hao et al. (2008), PLoS ONE, 3(8):e2904) and LDN193189 (4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline). The BMP inhibitor used in the present application may be, for example, LDN193189. In step (3), the concentration of the BMP inhibitor can be appropriately selected by those skilled in the art depending on the BMP inhibitor used. When the BMP inhibitor is LDN193189, the concentration can be 0.1 nM to 10 μM, 1 nM to 10 μM, or 10 nM to 1 μM, for example, about 0.1 μM.

[0065] In step (3), the concentration of the fibroblast growth factor can be appropriately selected by those skilled in the art depending on the fibroblast growth factor used. When the fibroblast growth factor is FGF8, the concentration can be 200 pg / ml to 20 μg / ml, 2 ng / ml to 20 μg / ml, or 20 ng / ml to 2 μg / ml, for example, about 200 ng / ml.

[0066] In step (3), the concentration of the retinoic acid receptor agonist can be appropriately selected by those skilled in the art depending on the retinoic acid receptor agonist used. When the retinoic acid receptor agonist is TTNPB, its concentration can be 0.1 nM to 10 μM, 1 nM to 10 μM, or 10 nM to 1 μM, for example, about 0.1 μM.

[0067] In step (3), the concentration of the glial cell line-derived neurotrophic factor may be 100 pg / ml to 10 μg / ml, 1 ng / ml to 10 μg / ml, or 10 ng / ml to 1 μg / ml, for example, about 100 ng / ml.

[0068] In the step (3), the medium may further contain EGF and / or FGF1. EGF is a protein called epidermal growth factor. EGF commercially available from R&D systems, etc. may be used. The concentration of EGF may be 50 pg / ml to 5 μg / ml, 500 pg / ml to 5 μg / ml, or 5 ng / ml to 500 ng / ml, for example, about 50 ng / ml. The concentration of FGF1 may be 200 pg / ml to 20 μg / ml, 2 ng / ml to 20 μg / ml, or 20 ng / ml to 2 μg / ml, for example, about 200 ng / ml.

[0069] In step (3), the medium may further contain Matrigel. The concentration of Matrigel may be 1 to 40%, 2 to 30%, or 5 to 20%, for example, about 10%.

[0070] In step (3), the culture period may be 2 to 30 days, 5 to 20 days, or 8 to 15 days, for example, about 10 days.

[0071] The culture temperature is, but not limited to, about 30 to about 40° C., for example, about 37° C., and the culture is performed in an atmosphere of CO2-containing air, with a CO2 concentration of, for example, about 2 to about 5%.

[0072] Method for producing collecting duct precursor-like organoids from ureteric bud-like organoids In one embodiment of the present application, (4) A method for producing collecting duct precursor-like organoids is provided, comprising culturing ureteric bud-like organoids in a medium containing a Wnt signal inhibitor and a TGFβ signal inhibitor. The ureteric bud-like organoids may be cells produced or reconstituted by the method of the present application, or may be cells obtained by another known method.

[0073] "Collecting duct precursor-like organoid" means a self-organized structure resembling a collecting duct precursor. The size of the collecting duct precursor-like organoid is, for example, about 10 to about 1000 μm. The fact that it is a collecting duct precursor-like organoid can be confirmed by the expression of a marker such as AQP2.

[0074] In step (4), the ureteric bud-like organoid is cultured in a medium that contains a Wnt signal inhibitor and a TGFβ signal inhibitor.In one embodiment, in step (4), the ureteric bud-like organoid is cultured in suspension.

[0075] The "Wnt signal inhibitor" is not particularly limited as long as it is a substance that inhibits the signal transduction pathway mediated by Wnt, and examples thereof include IWR-1, IWP-2, IWP-3, IWP-4, 2-(4-trifluoromethylphenyl)-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-4(3H)-one (XAV939), G-CSF, IGFBP4, Dkk1, Cerberus, anti-Wnt antibodies, Wnt agonists (Wnt receptor inhibitors), soluble Wnt receptor proteins (Frzb-1, etc.), and dominant negative forms. The Wnt signal inhibitor used in the present application may be, for example, IWR-1. In step (4), the concentration of the Wnt signal inhibitor can be appropriately selected by those skilled in the art depending on the Wnt signal inhibitor used. When the Wnt signal inhibitor is IWR-1, the concentration may be 1 nM to 100 μM, 10 nM to 100 μM, or 100 nM to 10 μM, for example, about 1 μM.

[0076] In step (4), the concentration of the TGFβ signal inhibitor can be appropriately selected by those skilled in the art depending on the TGFβ signal inhibitor used. When the TGFβ signal inhibitor is A83-01, the concentration can be 1 nM to 100 μM, 10 nM to 100 μM, or 100 nM to 10 μM, for example, about 1 μM.

[0077] In the step (4), the culture period is 5 to 40 days, 7 to 30 days, or 10 to 20 days, for example, about 14 days.

[0078] The culture temperature is, but not limited to, about 30 to about 40° C., for example, about 37° C., and the culture is performed in an atmosphere of CO2-containing air, with a CO2 concentration of, for example, about 2 to about 5%.

[0079] Method for producing collecting duct progenitor cells from ureteric bud tip cells In one embodiment of the present application, the method of the present application comprises isolating ureteric bud tip cells, (2') The present invention provides a method for producing collecting duct progenitor cells, the method comprising the step of culturing the ureteric bud tip cells in a medium containing a Wnt signal inhibitor.

[0080] "Collecting duct progenitor cells" refers to cells that express aquaporin 2 (AQP2), a marker for collecting duct principal cells, but do not express carbonic anhydrase (CA) II, a marker for interstitial cells. Collecting duct progenitor cells can be confirmed by expression of AQP2. Optionally, it may be confirmed that the cells do not express CAII.

[0081] In step (2'), the ureteric bud tip cells are cultured in a medium containing a Wnt signal inhibitor. In one embodiment, in step (2'), the ureteric bud tip cells are cultured in an adhesion manner. In the present application, "adhesion culture" means that the cells are cultured in a state where they are attached to a culture substrate, for example, in a coated culture dish. Examples of coating agents include laminin (laminin-511, laminin-111, laminin-411, etc.), Matrigel (BD), Synthemax (Corning), collagen, gelatin, heparan sulfate proteoglycan, entactin, fragments thereof, and combinations thereof. In the present application, the single cells can be cultured on a cell culture plate coated with an extracellular matrix protein, for example, laminin. As the coating agent, a commercially available product can be used, for example, iMatrix-511 silk can be used.

[0082] In step (2'), the concentration of the Wnt signal inhibitor can be appropriately selected by those skilled in the art depending on the Wnt signal inhibitor used. When the Wnt signal inhibitor is IWR-1, the concentration can be 1 nM to 100 μM, 10 nM to 100 μM, or 100 nM to 10 μM, for example, about 1 μM.

[0083] In step (2'), the medium may further contain a TGFβ signal inhibitor. The concentration of the TGFβ signal inhibitor can be appropriately selected by those skilled in the art depending on the TGFβ signal inhibitor used. When the TGFβ signal inhibitor is A83-01, the concentration can be 1 nM to 100 μM, 10 nM to 100 μM, or 100 nM to 10 μM, for example, about 1 μM.

[0084] In the step (2'), the culture period may be 2 to 21 days, 3 to 14 days, or 5 to 10 days, for example, about 7 days.

[0085] The culture temperature is, but not limited to, about 30 to about 40° C., for example, about 37° C., and the culture is performed in an atmosphere of CO2-containing air, with a CO2 concentration of, for example, about 2 to about 5%.

[0086] Method for producing collecting duct progenitor cells from ureteric bud tip cell colonies In one embodiment of the present application, a ureteric bud tip cell colony is obtained by the method of the present application, and further (3'-1) dissociating the ureteric bud tip cell colony; and (3'-2) The present invention provides a method for producing collecting duct progenitor cells, the method comprising the step of culturing the dissociated cell population in a medium containing a Wnt signal inhibitor.

[0087] In step (3'-1), the ureteric bud tip cell colony is dissociated. The method for dissociating the cell colony may be appropriately selected from conventionally known methods for dissociating cell aggregates, such as a mechanical dissociation method, or a dissociation method using a dissociation solution having protease activity and collagenase activity (e.g., Accutase and Accumax, etc.) or a dissociation solution having only collagenase activity. For example, a method may be used in which the cell aggregate is dissociated using a dissociation solution having protease activity and collagenase activity (e.g., Accutase), and then mechanically dispersed into fine single cells.

[0088] Step (3'-2) may be carried out in the same manner as step (2') in the above-mentioned method for producing collecting duct precursor cells from ureteric bud tip cells.

[0089] Methods for monitoring ureteric bud tip cells In one embodiment of the present application, (i) contacting a biological tissue, tissue slice, cultured cells, or cultured tissue that is suspected to contain ureteric bud tip cells with a VLDL-R binding substance; and (ii) providing a method for monitoring ureteric bud tip cells, comprising the step of detecting the binding substance. Examples of VLDL-R binding substances used in this embodiment are as described above. In a preferred embodiment, the VLDL-R binding substance is labeled as described above. In a more preferred embodiment, the VLDL-R binding substance is labeled VLDL.

[0090] The biological tissue, tissue slice, cultured cells, or cultured tissue predicted to contain ureteric bud tip cells may be derived from a mammal. Examples of mammals include humans and non-human animals, such as mice, rats, hamsters, guinea pigs, cows, horses, pigs, sheep, monkeys, orangutans, chimpanzees, dogs, cats, and birds. The biological tissue, tissue slice, cultured cells, or cultured tissue predicted to contain ureteric bud tip cells may be derived from, for example, a mouse or a human.

[0091] In step (i), the VLDL-R binding substance is typically added to a culture medium for a biological tissue, tissue slice, cultured cells or cultured tissue suspected of containing ureteric bud tip cells.

[0092] In step (ii), the means for detecting the binding substance may be a known method appropriately depending on the type of the binding substance. For example, the binding substance may be labeled as described above, and the label may be used as an indicator. Alternatively, a labeled antibody or the like that recognizes the binding substance may be added, and the label may be used as an indicator. The means for detecting the binding substance using the label as an indicator may be a known method appropriately depending on the type of label. For example, when the label is a fluorescent label, the fluorescence derived from the label can be detected using a fluorescent microscope or the like.

[0093] Compositions for isolating or monitoring ureteric bud tip cells In one embodiment of the present application, there is provided a composition for isolating or monitoring ureteric bud tip cells, comprising a VLDL-R binding substance. Examples of VLDL-R binding substances used in this embodiment are as described above.

[0094] The composition of the present application can be prepared as a liquid preparation by adding distilled water, a pH adjuster, a suspending agent, a solubilizing agent, a stabilizer, an isotonicity agent, an antioxidant, a preservative, etc. to the VLDL-R binding substance as necessary. Examples of pH adjusters include hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, sodium monohydrogen phosphate, sodium dihydrogen phosphate, etc. Examples of suspending agents include methylcellulose, polysorbate 80, hydroxyethylcellulose, gum arabic, powdered tragacanth, sodium carboxymethylcellulose, polyoxyethylene sorbitan monolaurate, etc. Examples of solubilizing agents include polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinamide, polyoxyethylene sorbitan monolaurate, etc. Examples of stabilizing agents include sodium sulfite, sodium metasulfite, ether, etc. Examples of isotonicity agents include sodium chloride, glucose, etc. Examples of preservatives include methyl paraoxybenzoate, ethyl paraoxybenzoate, sorbic acid, phenol, cresol, chlorocresol, etc. EXAMPLES

[0095] The present invention will be described in more detail below by showing examples, but the present invention is not limited to these examples in any way. A schematic diagram summarizing the method established in this example is shown in FIG.

[0096] Materials and Methods cell culture Experiments using human induced pluripotent stem cells (hiPSCs) were approved by the ethical committee of the Kyoto University Faculty of Medicine and Graduate School of Medicine. Three hiPSC lines, 585A1, 1231A3 and 1383D2, were cultured in a feeder-free medium using Stem Fit AK02N medium (Ajinomoto) at 0.25 μL / cm 2 Cells were maintained on cell culture plates coated with iMatrix-511 silk (Nippi). Cells were passaged every 4 days using 0.5 mM EDTA / PBS (Thermo Fisher Scientific). Cells were routinely tested for mycoplasma contamination.

[0097] Differentiation induction Cells were induced into the ureteric bud (UB) lineage as previously described with some modifications (Mae, S. I. & Ryosaka, M. et al. Biochem Biophys Res Commun. 495, 954-61 (2018)). Details of the growth factors and small molecules used in this example are shown in Table 1. [Table 1]

[0098] Anterior intermediate mesoderm induction hiPSCs were cultured in 4-well culture plates (Thermo Fisher Scientific) with 10 μM Y-27632 (WAKO) and 0.25 μL / cm 2 5 × 10 cells were cultured in Stem Fit AK02N medium containing iMatrix-511 silk. 4 Cells were seeded at a density of 100 cells / well. After 24 hours, cells were washed with PBS and treated with Essential 6 medium (Thermo Fisher Scientific) containing 100 ng / ml activin A (R&D Systems) and 3 μM CHIR99021 (Stem RD). After 24 hours, cells were washed with PBS and treated with Essential 6 medium containing 0.1 μM LDN193189 (Axon Medchem), 1 μM A83-01 (WAKO), 0.1 μM 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]-benzoic acid (TTNPB; Santa Cruz Biotechnology) and 200 ng / ml fibroblast growth factor (FGF)8 (Peprotech) for 2 days. The cells were then plated at 2 × 10 in Essential 6 medium containing the same four factors and 10 μM Y-27632 in 24-well culture plates coated with Matrigel or Geltrex (Corning). 5The cells were then replated at a density of 1000 cells / well and incubated for an additional 24 hours to induce anterior intermediate mesoderm (AIM).

[0099] Mesonephric duct (ND) guidance AIM cells were treated with Essential 6 medium containing 1 μM CHIR99021, 0.1 μM LDN193189, 200 ng / ml FGF8, 100 ng / ml glial cell line-derived neurotrophic factor (GDNF; R&D Systems) and 0.1 μM TTNPB for 2 days to induce ND leading edge cells (ND leader cells). To enhance epithelialization in 2D culture, AIM cells were treated with the same medium and inducers for 8 days. ND leader cells were treated with Accutase (Innovative Cell Technologies) for 3 min at 37 °C and then dissociated into single cells by pipetting. The cells were plated at 1 × 10 4 Cells were seeded at a density of 100 cells / well and treated with the same medium and factors plus 10 μM Y-27632 to induce mature ND aggregates for 2 days.

[0100] Artificial ureteric bud (iUB) organoid induction Unwanted cells were separated from the ND aggregates by pipetting (Mae, S. I. & Ryosaka, M. et al. Biochem Biophys Res Commun. 495, 954-61 (2018)). The ND aggregates were treated with the same medium and factors containing 2% Matrigel for 6 days to constitute iUB organoids with epithelial polarity and tubular lumen. 50 ng / ml EGF (R&D Systems) and 200 ng / ml FGF1 (R&D Systems) were added to enhance sprouting. The tips were mechanically separated from the iUB organoids and cultured in the same medium and factors containing 2% Matrigel for 6–14 days to reconstitute branched iUB organoids. The tips isolated from the reconstituted iUB organoids were treated with the same medium and factors to repeatedly reconstitute iUB organoids.

[0101] Collecting duct precursor induction For 2D culture, day 7 tip cell colonies detached from the hydrogel were treated with Accutase for 3 min at 37°C and then dissociated into single cells by pipetting. The single cells were cultured in 0.5 μL / cm 2 aliquots of 1 μM IWR-1 (Tocris) with or without 1 μM A83-01. 2 Resuspend the cells in Essential 6 medium containing iMatrix-511 silk and plate 4 x 10 cells in a 96-well plate. 4 For 3D culture, day 14 reconstituted iUB organoids were treated with Essential 6 medium containing 1 μM IWR-1 and 1 μM A83-01 for 14 days.

[0102] ND cryopreservation Dissociated ND leader cells were cultured at 1 × 10 using STEM-CELLBANKER GMP grade (Nippon Zenyaku Kogyo Co., Ltd.). 6 The cells were resuspended at a dilution rate of less than 1000 cells / mL. The cell suspension was distributed into individual cryopreservation tubes. The tubes were frozen at -80°C for 24 hours and transferred to a liquid nitrogen cell storage tank for long-term cryopreservation. To start the culture, the cells were thawed at 37°C using a water bath. The cells were then gently resuspended in Essential 6 medium containing 10 μM Y-27632 and centrifuged at 200g for 5 minutes at room temperature. After removing the supernatant, the cells were resuspended in Essential 6 medium containing 10 μM Y-27632 and ND inducer and plated at 1 × 10 4 Cells were seeded at a density of 1000 cells / well.

[0103] Tip cell proliferation iUB organoids were treated with Accutase for 5 min at 37°C and then dissociated into single cells by pipetting. Cells were resuspended in DMEM / F12 medium (Thermo Fisher Scientific) containing B27 supplement (without vitamin A), 100 ng / ml GDNF, 200 ng / ml FGF1, 0.1 μM TTNPB, 3 μM CHIR99021, and 10 μM Thiazovivin (Santa Cruz Biotechnology). Single cells from 10 iUB organoids were seeded onto one well of a 48-well plate coated with 150 μL hydrogel. The hydrogel consisted of DMEM / F12 medium containing 50% Matrigel and was allowed to solidify for 30 min at 37°C before use. Single cells established tip cell colonies after 6–7 days. Medium was changed every 2 days.

[0104] Reconstitution of iUB organoids from single tip cells The hydrogel was dissolved with Cell Recovery Solution (BD Biosciences) for 1 h at 4 °C to isolate the day 7 tip cell colonies. After further washing with Cell Recovery Solution, the tip cell colonies were centrifuged at 500 g for 5 min at room temperature. Essential 6 medium containing 0.1 μM LDN193189, 100 ng / ml GDNF, 0.1 μM TTNPB, 200 ng / ml FGF8, 1 μM CHIR99021 or 10% Afamin / Wnt3a conditioned medium (MBL), 200 ng / ml R-spondin 1 (R&D systems), 50 ng / ml EGF, 200 ng / ml FGF1 and 10% Matrigel was added to the tube containing the tip cell colonies. After gentle pipetting with a wide-mouth micropipette (BMBio) to avoid disrupting the colonies, the suspension was distributed at 2 mL / dish into low-attachment 35 mm dishes (Sumitomo Bakelite). The medium was changed every 2 days. On day 4, each colony was placed in the same medium on one well of a low-attachment 96-well plate. After another 6 days, the apical region was separated from the reconstituted iUB organoids and treated with the same medium for 35 days.

[0105] Maintenance culture of tip cell colonies iUB organoids were treated with Accutase at 37°C for 5 min and then dissociated into single cells by pipetting. Cells were suspended in DMEM / F12 medium containing B27 supplement (without vitamin A), 100ng / ml GDNF, 200ng / ml FGF1, 0.1μM TTNPB, 3μM CHIR99021, 10μM Thiazovivin and 1μM A83-01. Cells were seeded onto one well of a 48-well plate coated with 150μL hydrogel. The hydrogel consisted of DMEM / F12 medium containing 50% Matrigel and was allowed to solidify for 1 h at 37°C before use. Tip cell colonies were generated by culturing single cells at 37°C and 5% CO2 for 7 days. The medium was changed every 2–3 days. The generated tip cell colonies were subcultured in the same medium every 7 days. iUB organoids were reconstituted from tip cell colonies that had been generated or subcultured three times using the same method as described above.

[0106] Monitoring of leading edge cells using DiI-labeled VLDL (DiI-VLDL) iUB organoids, tip cell colonies and ND aggregates were treated with 10 μg / ml DiI-labeled VLDL (Biomedical Technologies, Inc.) for 2 hours at 37°C.

[0107] Flow cytometry analysis iUB organoids and tip cell colonies treated with DiI-VLDL were treated with Accutase for 3 min at 37°C and then dissociated into single cells by pipetting. After washing with DMEM / 10% FBS, cells were resuspended in PBS / 2% FBS. Single cells were analyzed and sorted using a FACS Aria II (BD). Cells from iUB organoids and tip cell colonies not treated with DiI-VLDL were used as negative controls for gating.

[0108] RNA sequencing analysis Day 6 iUB organoids were treated with DiI-VLDL for 2 hours. Flow cytometry analysis was performed to determine the effect of DiI-VLDL on iUB organoids. +Cells and DiI-VLDL - The cells were sorted. Sorted DiI-VLDL + Cells and DiI-VLDL - Total cellular RNA was isolated using NucleoSpin RNA XS (Takara). Samples were stored at -80°C and subjected to RNA sequencing analysis by DNAFORM Co., Ltd.

[0109] immunostaining Immunostaining analysis was performed as previously described with some modifications (Mae, S. I. & Ryosaka, M. et al. Biochem Biophys Res Commun. 495, 954-61 (2018)). For 2D cultures, cells were fixed with 4% paraformaldehyde (PFA) / PBS for 20 min at 4 °C. After washing twice with PBS, cells were blocked with 1% normal donkey serum (MERCK) and 3% bovine serum albumin (BSA; Nacalai Tesque) / PBST (PBS / 0.25% Triton X-100, Nacalai Tesque) for 1 h at room temperature. Primary antibodies were diluted 1:500 in blocking solution and incubated with the samples overnight at 4 °C. After washing twice with PBST, secondary antibodies diluted 1:500 in blocking solution were incubated for 1 h at room temperature.

[0110] For immunostaining analysis of frozen sections, samples were fixed with 4% PFA / PBS for 1 h at 4°C. Fixed samples were treated with 30% sucrose / PBS, frozen in OCT compound (Tissue-Tek), and cryosectioned by cryosectioning. Frozen sections were washed with distilled water and incubated with blocking solution for 1 h at room temperature. Primary antibodies were diluted 1:500 in blocking solution and incubated with samples overnight at room temperature. After washing twice with distilled water, cells were incubated with secondary antibodies diluted 1:500 in blocking solution for 1 h at room temperature.

[0111] For three-dimensional image analysis, Clear, Unobstructed Brain / Body Imaging Cocktails and Computational analysis (CUBIC) was applied (Susaki EA. et al. Nat protoc. 10, 1709-27 (2015); Nojima S. et al. Sci Rep. 24, 9269 (2017)). After fixation with 4% PFA / PBS for 24 hours at 4°C, the samples were treated with CUBIC1 solution diluted 1:2 with distilled water on a seesaw shaker set at 30 rpm for 8 hours at room temperature for tissue clearing. The samples were then treated with CUBIC1 solution on a seesaw shaker set at 30 rpm for 24 hours at room temperature. After washing with PBS, the samples were incubated with blocking solution on a seesaw shaker set at 30 rpm for 2 hours at 4°C. Primary antibodies were diluted in blocking solution and incubated with the samples on a seesaw shaker set at 30 rpm for 24 hours at 4°C. After washing with blocking solution, cells were incubated with secondary antibodies for 24 hours at 4°C on a seesaw shaker set at 30 rpm. After washing twice with PBS, samples were treated with CUBIC2 solution diluted 1:2 in PBS for 8 hours at room temperature on a seesaw shaker set at 30 rpm. Samples were then treated with CUBIC2 solution for 24 hours at room temperature on a seesaw shaker set at 30 rpm. They were then analyzed using a Zeiss LSM710 (Zeiss). Details of the primary antibodies used in this example are shown in Table 2. [Table 2] Details of the secondary antibodies (Thermo Fisher Scientific) used in this example are shown in Table 3. In this example, the secondary antibodies listed in Table 3 were used in appropriate combination. [Table 3] CUBIC1 solution is a mixture of urea (final concentration 10%, Nacalai Tesque), Quadrol (final concentration 25%, Tokyo Chemical Industry), Triton X-100 (final concentration 5%), 5M NaCl, and distilled water. CUBIC2 solution is a mixture of urea (final concentration 25%), sucrose (final concentration 50%, Nacalai Tesque), triethanolamine (final concentration 10%, Wako), and distilled water. Hoechst33342 (Thermo Fisher Scientific, Catalog Number: H1399) was used as a nuclear stain.

[0112] statistical analysis Flow cytometry and cell count analysis data are expressed as mean values ​​± SE. Statistical analysis of differences between two groups was performed using Student's t-test. Differences were considered significant when p<0.05.

[0113] [result] First, because retinoic acid (RA) signaling plays an important role in mesonephric duct (ND) development (Stewart, K. & Bouchard, M. Semin Cell Dev Biol. 36, 13-20 (2014)), we modified our previously reported ND induction method by adding the RA agonist TTNPB to the stage 4 treatment (Figures 2 and 3). We also found that epithelialization of ND cells was induced by extending the treatment with the ND elongation factors used in stage 4 (Figure 4). Therefore, we generated ND epithelial aggregates using the same inducer as in stage 4. Unnecessary cells were removed by pipetting (Figure 5).

[0114] Because cells preserved using commercially available cryopreservation reagents maintained the ability to form ND epithelial aggregates to the same extent as non-cryopreserved ND leading edge cells (ND leader cells) (Figures 6 and 7), commercially available cryopreservation reagents are useful for preserving stage 4 ND leader cells.

[0115] It has been reported that the addition of low concentrations of Matrigel to the culture medium promotes the organization of continuous epithelial tissue (Koehler, KR et al. Nat Biotechnol. 35, 583-9 (2017)). Therefore, we added a low concentration (2%) of Matrigel to the ND aggregate culture medium. As a result, UB-like structures were efficiently induced (Figures 8 to 10). Immunostaining analysis showed that these UB-like structures expressed the apical marker EZRIN and the basal marker LAMININ. This result suggests that these structures are involved in the formation of RET + Tip and CK8 + In addition to the formation of stem differentiation domains, the organelles were shown to have apical-basal polarity and a tubular lumen. Structures with these characteristics are referred to as iUB organoids (Figures 11 and 12).

[0116] Next, the tips were manually separated from the iUB organoids and cultured in the same medium (hereafter referred to as organoid medium) containing a low concentration of Matrigel (Figure 13). The separated tips formed branched structures with tip and stem regions and epithelial polarity similar to those of the original iUB organoids (Figures 13-16). This suggests the reconstitution of iUB organoids. Furthermore, the tips separated from the reconstituted iUB organoids again reconstituted iUB organoids with branching morphogenesis. It was found that this reconstitution method could be repeated at least three times, and the total number of branches was about 20 times (Figure 17). Furthermore, it was found that the addition of EGF and FGF1 to the organoid medium promoted sprouting with tip and stem structures and epithelial polarity (Figures 18-20).

[0117] Next, we aimed to obtain a large number of iUB organoids. We found that single iUB organoid-derived cells seeded on soft hydrogels successfully proliferated to form colonies resembling the UB tip expressing RET and GATA3 (Figures 21-23). ​​To clarify the origin of the tip cell colonies, we attempted to develop a method to monitor tip cell development. By immunostaining analysis, we found that the very low density lipoprotein (VLDL) receptor was expressed in the tip region (Figure 24). This is consistent with previous reports (Yuri, S. et al. Stem Cell Reports. 8, 401-16 (2017);Rutledge, EA, Benazet, JD & McMahon, AP Development. 144, 3177-88 (2017)). Therefore, we hypothesized that tip cells might specifically take up VLDL and examined the uptake of DiI-labeled VLDL (DiI-VLDL) by tip cell colonies, ND epithelial aggregates and iUB organoids. As expected, DiI-VLDL was taken up by the entire tip cell colony and the tip region of iUB organoids, but not by ND aggregates (Figure 25). Furthermore, DiI purified by flow cytometry was also found to be effective in treating endothelial cell colonies. + Cells and DiI - RNA sequencing analysis of DiI cells revealed that expression of apical-associated genes was significantly increased in + We found that DiI expression was elevated in cells and that expression of stem-related genes was increased in DiI cells. - The results showed that the percentage of cells at the tip was higher than that of cells at the tip (Fig. 26, Fig. 27). This technique for monitoring the tip using DiI-VLDL showed that the percentage of cells at the tip increased from 54.0±2.1% to 91.8±2.8% by our hydrogel culture (n=3, Fig. 26, Fig. 28, Fig. 29). Next, the isolated single DiI + Cells and DiI - Cells were cultured on the hydrogel. Interestingly, after 7 days, the leading edge cell colonies that took up DiI-VLDL were the ones that were most sensitive to DiI. +Not only cells, but also DiI - It was also produced from cells (Figure 30). - The percentage of tip cells arising from DiI cells was high (80.5 ± 1.6%). + This was significantly lower than the percentage of tip cells that arose from UB stem cells (88.9±0.5%) (Figure 31). These results are consistent with previous findings that UB stem cells can regenerate tip cells in vitro (Yuri, S. et al. Stem Cell Reports. 8, 401-16 (2017)).

[0118] Next, the tip cell colonies were cultured in organoid medium. This resulted in the reconstitution of branched iUB organoids with tip and stem regions and epithelial polarity (Figure 32, Figure 33). Furthermore, the tips isolated from the elongated iUB organoids were found to exhibit branching morphogenesis, demonstrating the establishment of a simplified propagation method of developmentally competent tip cells that give rise to iUB organoids (Figure 34). However, this reconstitution of iUB organoids was only observed in tip cell colonies derived from 585A1 iPS cells. To establish a robust reconstitution method from tip cell colonies to iUB organoids, we focused on the Wnt signaling pathway. In the organoid medium, CHIR99021 was replaced with Wnt3a conditioned medium and R-spondin 1. As a result, it was found that iUB organoids could be reconstituted from tip cell colonies derived from any iPS cell line (Figure 35).

[0119] Next, because Wnt / β-catenin signaling maintains stem cells in the UB tip region (Rutledge, EA, Benazet, JD & McMahon, AP Development. 144, 3177-88 (2017)), we recovered tip cell colonies from the hydrogel and cultured the tip cell colonies with IWR-1, a WNT signaling inhibitor, to differentiate them into stem cells. We found that IWR-1 treatment promoted the differentiation of dissociated tip cell colony cells into cells that express aquaporin (AQP) 2, a principal cell marker of the collecting duct, but do not express carbonic anhydrase (CA) II, an interstitial cell marker, and that the combination of IWR-1 and A83-01, a TGFβ signaling inhibitor, improved the induction rate of these cells (Figures 36 to 38). Furthermore, we found that the same treatment with the same factors increased the induction rate of AQP2 in the reconstituted iUB organoids derived from the tip cell colonies. + We successfully induced collecting duct precursor organoids (Figure 39, Figure 40). These collecting duct precursors may resemble collecting ducts in embryos at about 7 weeks gestation (GW), which express only principal cell markers (Wang, P. et al. Cell Rep. 24, 3554-67 e3 (2018)).

[0120] iUB organoids were dissociated into single cells by Accutase treatment. Single cells were cultured on the hydrogel in medium further containing A83-01, and GATA3 + , RET + , SOX9 + Ureteric bud tip cell colonies were generated (Figures 42 and 43). The generated ureteric bud tip cell colonies were cultured in organoid medium, resulting in the generation of iUB organoids (Figure 44). These iUB organoids express GATA3 + , RET + Apical region and GATA3 +The ureteric bud tip cell colony had a stem region (Figure 45). The ureteric bud tip cell colony was subcultured three times in the same medium (Figures 46 and 47). After three subcultures, the ureteric bud tip cell colony was cultured in organoid medium, resulting in the generation of iUB organoids (Figure 48). These iUB organoids express GATA3 + , RET + Apical region and GATA3 + It contained the stem region (Figure 49).

Claims

1. 1. A method for isolating ureteric bud tip cells from a human-derived cell, tissue or organoid comprising ureteric bud tip cells, comprising: (1-1) contacting a cell, tissue, or organoid containing a ureteric bud tip cell with very low density lipoprotein (VLDL); and (1-2) Isolating ureteric bud tip cells using VLDL as an indicator The method includes:

2. The method of claim 1, wherein the VLDL is labeled VLDL.

3. The method of claim 2, wherein the label is a fluorescent label.

4. The method according to any one of claims 1 to 3, wherein the cell, tissue or organoid comprising a ureteric bud tip cell is a ureteric bud-like organoid.

5. The method according to any one of claims 1 to 4, wherein the cells, tissues or organoids comprising ureteric bud tip cells are induced to differentiate from pluripotent stem cells.

6. The method according to claim 5 , wherein the pluripotent stem cells are iPS cells.

7. Isolating ureteric bud tip cells by the method according to any one of claims 1 to 6, and further (2) A method for producing ureteric bud tip cell colonies, comprising the step of culturing the ureteric bud tip cells in a medium containing a glial cell line-derived neurotrophic factor, a fibroblast growth factor, a retinoic acid receptor agonist, a GSK3β inhibitor and a Yes-associated protein (YAP) activity inhibitor.

8. The method according to claim 7, wherein in step (2), the fibroblast growth factor is FGF1, the retinoic acid receptor agonist is TTNPB, the GSK3β inhibitor is CHIR99021, and the Yes-associated protein (YAP) activity inhibitor is Thiazovivin.

9. The method according to claim 7 or 8, wherein in step (2), the medium further contains a TGFβ signal inhibitor.

10. The method according to claim 9, wherein the TGFβ signal inhibitor is A83-01.

11. The method according to any one of claims 7 to 10, further comprising a step of subculturing the obtained ureteric bud tip cell colony after step (2).

12. Obtaining a ureteric bud tip cell colony by the method according to any one of claims 7 to 11, and further (3) A method for producing ureteric bud-like organoids, comprising the step of culturing the ureteric bud tip cell colony in a medium containing a Wnt signaling activator, a BMP inhibitor, a fibroblast growth factor, a retinoic acid receptor agonist, and a glial cell line-derived neurotrophic factor to reconstitute a ureteric bud-like organoid.

13. The method according to claim 12, wherein in step (3), the Wnt signaling activator is Wnt3a and / or R-spondin 1, the BMP inhibitor is LDN193189, the fibroblast growth factor is FGF8, and the retinoic acid receptor agonist is TTNPB.

14. The method according to claim 12 or 13, wherein in step (3), the medium further contains EGF and FGF1.

15. A method for obtaining a ureteric bud-like organoid by the method according to any one of claims 12 to 14, further comprising the steps of: (4) A method for producing collecting duct precursor-like organoids, comprising the step of culturing the ureteric bud-like organoids in a medium containing a Wnt signal inhibitor and a TGFβ signal inhibitor.

16. The method according to claim 15, wherein in step (4), the Wnt signal inhibitor is IWR-1 and the TGFβ signal inhibitor is A83-01.

17. Isolating ureteric bud tip cells by the method according to any one of claims 1 to 6, and further (2') A method for producing collecting duct precursor cells, comprising the step of culturing the ureteric bud tip cells in a medium containing a Wnt signal inhibitor.

18. Obtaining a ureteric bud tip cell colony by the method according to any one of claims 7 to 11, and further (3'-1) dissociating the ureteric bud tip cell colony; and (3'-2) A method for producing collecting duct progenitor cells, comprising the step of culturing the dissociated cell population in a medium containing a Wnt signal inhibitor.

19. The method according to claim 17 or 18, wherein in step (2') or (3'-2), the Wnt signal inhibitor is IWR-1.

20. The method according to any one of claims 17 to 19, wherein in step (2') or (3'-2), the medium further contains a TGFβ signaling inhibitor.

21. The method according to claim 20, wherein in step (2') or (3'-2), the TGFβ signal inhibitor is A83-01.

22. (1-1) contacting a human-derived cell, tissue, or organoid containing a ureteric bud tip cell with VLDL; and (1-2) A method for producing a ureteric bud tip cell population, comprising a step of isolating ureteric bud tip cells using VLDL as an indicator.

23. (i) contacting a human-derived biological tissue, tissue slice, cultured cell, or cultured tissue that is predicted to contain ureteric bud tip cells with VLDL; and (ii) A method for monitoring ureteric bud tip cells, comprising the step of detecting VLDL.

24. A composition for isolating or monitoring human-derived ureteric bud tip cells, comprising VLDL.

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