Medium for expanding and culturing nephron progenitor cells, method for expanding and culturing nephron progenitor cells, and method for producing kidney organoids

Abstract

A medium for culturing and expanding nephron progenitor cells, said medium containing a GSK-3β inhibitor, a ROCK inhibitor and at least one fibroblast growth factor selected from the group consisting of FGF9 and FGF20. A method for culturing and expanding nephron progenitor cells using the aforesaid medium. A method for producing renal organoids, said method comprising a step for culturing and expanding nephron progenitor cells by the aforesaid method for culturing and expanding nephron progenitor cells, and a step for differentiating the nephron progenitor cells, which have been thus cultured and expanded, into renal organoids.

Description

[Technical Field]

[0001] The present invention relates to a medium for expanding nephron progenitor cells, a method for expanding nephron progenitor cells, and a method for producing kidney organoids. This application claims priority based on Japanese Patent Application No. 2021-002203, filed on January 8, 2021, the contents of which are incorporated herein by reference.

[0002] Currently, the number of patients with chronic kidney disease (CKD) in Japan is estimated to be approximately 13 million, and it has been called a new national disease. There are few curative treatments for chronic kidney disease, and as the disease progresses, there are more than 340,000 patients with end-stage chronic renal failure who require dialysis therapy, posing a major problem not only medically but also medically and economically. Kidney transplantation is one of the curative 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.

[0003] The kidney originates from the intermediate mesoderm, a tissue found in early embryonic development. In vertebrates, the intermediate mesoderm forms three kidneys: the pronephros, mesonephros, and metanephros. In mammals, the metanephros becomes the adult kidney. The metanephros develops through the interaction of two tissues: the mesenchyme, which differentiates into the nephrons and interstitium of the adult kidney, and the ureteric bud, which differentiates into the collecting ducts of the adult kidney, the lower renal pelvis, ureter, and part of the bladder. Furthermore, the presence of nephron progenitor cells (NPCs), which have the multipotency to differentiate into the glomeruli that make up the nephrons and several types of renal tubular epithelial cells, has been shown within the metanephric mesenchyme (NPLs 1 and 2).

[0004] A method for expanding mouse nephron progenitor cells (mNPCs) has been reported that uses bone morphogenetic protein (BMP) 7. For example, Non-Patent Document 3 reports that mNPCs were expanded for over a year using a medium (NPSR medium) containing BMP7, fibroblast growth factor (FGF) 2, heparin, Y-27632, CHIR99021, leukemia inhibitory factor (LIF), A83-01, and LDN193189. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Osafune K.,et al.,Identification of multipotent progenitors in the embryonic mouse kidney by a novel colony-forming assay.Development 2006;133:151-61. [Non-patent document 2] Kobayashi A., et al.,Six2 defines and regulates a multipotent self-renewing nephron progenitor population throughout mammalian kidney development.Cell Stem Cell 2008;3:169-81. [Non-patent document 3] Z. Li, T. Araoka, JCI Belmonte, Gene Editing in 3D Cultured Nephron Progenitor Cell Lines, in: S. Vainio (Ed.), Kidney Organog Methods Protoc, Humana Press, New York, 2019: pp.151-159. Summary of the Invention [Problem to be solved by the invention]

[0006] In order to utilize expanded nephron progenitor cells in regenerative medicine, etc., it is necessary to maintain the properties of nephron progenitor cells, such as differentiation potential, during expansion culture. Therefore, it is desirable to develop a culture method that enables efficient expansion of nephron progenitor cells while maintaining their properties.

[0007] Therefore, an objective of the present invention is to provide a culture medium for expanding nephron progenitor cells, which enables the expansion of nephron progenitor cells while retaining their differentiation ability, a method for expanding nephron progenitor cells using the culture medium, and a method for producing kidney organoids from nephron progenitor cells obtained by the expansion method. [Means for solving the problem]

[0008] The present invention includes the following aspects. [1] A medium for expanding nephron progenitor cells, comprising a GSK-3β inhibitor, a ROCK inhibitor, and at least one fibroblast growth factor selected from the group consisting of FGF9 and FGF20. [2] A medium for expanding the nephron progenitor cells according to [1], further comprising a JAK inhibitor. [3] The medium described in [2], wherein the JAK inhibitor is a JAK2 inhibitor. [4] The medium described in [3], wherein the JAK2 inhibitor is TG101348. [5] The medium according to any one of [1] to [4], wherein the GSK-3β inhibitor is CHIR99021. [6] The medium according to any one of [1] to [5], wherein the ROCK inhibitor is Y-27632. [7] The medium according to any one of [1] to [6], further comprising 10% by volume of AS401 relative to the total volume of the medium. [8] The medium according to any one of [1] to [7], wherein the nephron progenitor cells are human nephron progenitor cells. [9] The medium according to any one of [1] to [8], wherein the nephron progenitor cells are induced from pluripotent stem cells.

[10] The medium according to [9], wherein the pluripotent stem cells are iPS cells.

[11] A method for expanding nephron progenitor cells, comprising the step of culturing nephron progenitor cells in the medium according to any one of [1] to

[10] .

[12] The method for expanding nephron progenitor cells according to

[11] , wherein the step of culturing the nephron progenitor cells comprises subculturing the nephron progenitor cells.

[13] A method for expanding nephron progenitor cells, comprising: (A) culturing nephron progenitor cells for 30 to 60 hours in the medium described in any one of [1] to

[10] ; and (B) culturing the cells obtained in step (A) in a medium containing a GSK3β inhibitor and at least one fibroblast growth factor selected from the group consisting of FGF9 and FGF20, but not containing a ROCK inhibitor.

[14] (C) A method for expanding nephron progenitor cells according to

[13] , further comprising a step of subculturing the cells obtained in step (B) in a medium according to any one of [1] to

[10] .

[15] The method for expanding nephron progenitor cells described in

[14] , further comprising the steps of: (D) culturing the cells passaged in step (C) in a medium described in any one of [1] to

[10] for 30 to 60 hours; and (E) culturing the cells obtained in step (D) in a medium containing a GSK-3β inhibitor and at least one fibroblast growth factor selected from the group consisting of FGF9 and FGF20, but not containing a ROCK inhibitor.

[16] The method for expanding nephron progenitor cells according to any one of

[11] to

[15] , wherein the nephron progenitor cells are human nephron progenitor cells.

[17] The method for expanding nephron progenitor cells according to any one of

[11] to

[16] , wherein the nephron progenitor cells are induced from pluripotent stem cells.

[18] The method for expanding nephron progenitor cells according to

[17] , wherein the pluripotent stem cells are iPS cells.

[19] The method for expanding nephron progenitor cells according to

[17] or

[18] , wherein the nephron progenitor cells are obtained by a method comprising the following steps (i) to (vi): (i) culturing pluripotent stem cells in a medium containing FGF2, BMP4, a GSK-3β inhibitor, and retinoic acid or a derivative thereof; (ii) culturing the cells obtained in step (i) in a medium containing FGF2, a GSK-3β inhibitor, and BMP7; (iii) culturing the cells obtained in step (ii) in a medium containing a GSK-3β inhibitor, BMP7, and a TGFβ inhibitor. (iv) culturing the cells obtained in step (iii) in a medium containing a cytotoxic agent and not containing FGF2; (v) culturing the cells obtained in step (iv) in a medium containing retinoic acid or a derivative thereof and not containing FGF9; and (vi) culturing the cells obtained in step (v) in a medium containing a GSK-3β inhibitor and at least one fibroblast growth factor selected from the group consisting of FGF9 and FGF20.

[20] The method for expanding nephron progenitor cells according to

[19] , wherein the medium used in step (iv) does not contain BMP7.

[21] At least one of the steps (i) to (vi) is performed on a substrate having a cell adhesion surface of 400 cm 2 The method for expanding nephron progenitor cells according to

[19] or

[20] , which is carried out using the above culture vessel.

[22] A method for producing kidney organoids, comprising the steps of: expanding nephron progenitor cells by the method for expanding nephron progenitor cells described in any one of

[11] to

[21] ; and differentiating the expanded nephron progenitor cells into kidney organoids. [Effects of the Invention]

[0009] The present invention has the effect of providing a culture medium for expanding nephron progenitor cells, which enables the expansion of nephron progenitor cells while retaining their differentiation ability, a method for expanding nephron progenitor cells using the culture medium, and a method for producing kidney organoids from nephron progenitor cells obtained by the expansion method. [Brief explanation of the drawings]

[0010] [Figure 1] hNPCs (bulk) were subcultured using hNPSR medium (see Table 1) or CFY medium (see Table 2), and the expression of SIX2 and OSR1 was confirmed at each subculture. hNPCs were differentiated from hiPSCs using differentiation induction method A. [Figure 2] This figure shows the results of an attempt to generate kidney organoids from hNPCs (bulk) subcultured in hNPSR medium or CFY medium. hNPCs were differentiated from hiPSCs using differentiation induction method A. [Figure 3] hNPCs (bulk) were subcultured in hNPSR medium or CFY medium supplemented with DMSO, and the cumulative cell number was calculated. hNPCs were differentiated from hiPSCs using differentiation induction method A. [Figure 4]hNPCs (bulk) were subcultured in hNPSR medium or CFY medium supplemented with DMSO, and the cell number was measured at each passage. hNPCs were differentiated from hiPSCs using differentiation induction method A. [Figure 5] hNPCs (bulk) were cultured in CFY medium supplemented with DMSO (CFY + DMSO) or CFY medium supplemented with 3 nM TG101348 (CFY + TG(3)), and the cell count was measured. hNPCs were differentiated from hiPSCs using differentiation induction method A. [Figure 6] hNPCs (bulk) were subcultured in CFY medium supplemented with DMSO (CFY+DMSO) or CFY medium supplemented with 3 nM TG101348 (CFY+TG(3)), and the expression of SIX2 and OSR1 was confirmed at each subculture. hNPCs were differentiated from hiPSCs using differentiation method A. [Figure 7] hNPCs (bulk) were subcultured in CFY medium supplemented with DMSO (CFY + DMSO) or CFY medium supplemented with 3 nM TG101348 (CFY + TG(3)), and kidney organoids were generated from the cells at the second passage. hNPCs were differentiated from hiPSCs using differentiation induction method A. [Figure 8] hNPCs (c-MET(+)) were cultured in CFY medium supplemented with DMSO (CFY+DMSO) or CFY medium supplemented with 3 nM TG101348 (CFY+TG(3)). The cell numbers were counted at each passage and the cumulative cell numbers were calculated. hNPCs were differentiated from hiPSCs using differentiation method A. [Figure 9] hNPCs (c-MET(+)) were subcultured in CFY medium supplemented with DMSO (CFY+DMSO) or CFY medium supplemented with 3 nM TG101348 (CFY+TG(3)), and the expression of SIX2 and OSR1 was confirmed at each subculture. hNPCs were differentiated from hiPSCs using differentiation method A. [Figure 10] hNPCs (c-MET(+)) were subcultured in CFY medium supplemented with DMSO (CFY+DMSO) or CFY medium supplemented with 3 nM TG101348 (CFY+TG(3)), and kidney organoids were generated from the first passage of cells. hNPCs were differentiated from hiPSCs using differentiation method A. [Figure 11] Immunodeficient mice (AKI mice) were induced with acute kidney injury (AKI) by cisplatin administration and were then transplanted with hNPC (bulk) cell masses passaged once in CFY medium under the renal capsule. Blood urea nitrogen (BUN) and serum creatinine (S-Cre) were measured. In the figure, "Normal" indicates normal mice, "Saline" indicates AKI mice administered saline under the renal capsule, and "NPC transplant" indicates AKI mice transplanted with hNPC (bulk). hNPCs were differentiated from hiPSCs using differentiation induction method A. [Figure 12] Immunodeficient mice were induced with acute kidney injury (AKI) by cisplatin administration and then transplanted under the renal capsule with hNPC (bulk) cell masses passaged twice in CFY medium. Blood urea nitrogen (BUN) and serum creatinine (S-Cre) were measured. In the figure, "Normal" indicates normal mice, "Saline" indicates AKI mice administered saline under the renal capsule, and "NPC transplant" indicates AKI mice transplanted with hNPC (bulk). hNPCs were differentiated from hiPSCs using differentiation induction method A. [Figure 13] This figure shows the results of confirming the differentiation induction efficiency of SIX2-positive cells when the FGF2 concentration was changed and when heparin was added in step 4 (preparation of the late metanephric lineage primitive streak) in inducing human iPS cells (hiPSCs) to hNPCs using differentiation induction method B (see Example 5). [Figure 14] 1 shows the results of confirming the differentiation induction efficiency of SIX2-positive cells when the FGF2 concentration is changed in step 3 (preparation of the late mesodermal lineage primitive streak) in inducing hiPSCs to hNPCs using differentiation induction method B. [Figure 15]This shows the results of confirming the differentiation induction efficiency of SIX2-positive cells when the FGF9 concentration was changed or when heparin was added in step 5 (preparation of late posterior intermediate mesoderm) during the induction of hiPSCs to hNPCs using differentiation induction method B. [Figure 16] This figure shows the results of investigating the differentiation induction efficiency of SIX2+ cells when inducing hiPSCs into hNPCs using differentiation induction method B, when the additive in the basal medium was changed from B27 (B27 Supplement (50x minus vitamin A, Invitrogen)) to AS401 (StemFit® for Differentiation, Ajinomoto Healthy Supply Co., Ltd.). 10% AS401: basal medium containing 10% AS401 by volume relative to the total volume of the basal medium; 20% AS401: basal medium containing 20% ​​AS401 by volume relative to the total volume of the basal medium; B27: basal medium supplemented with B27. Hereinafter, "medium supplemented with 10% AS401" refers to medium containing 10% AS401 by volume relative to the total volume of the medium. The same applies to medium supplemented with 20% AS401. [Figure 17] This figure shows the results of confirming SIX2 expression when the basal medium additive was changed from B27 to AS401 during differentiation from clinical-grade hiPSCs to hNPCs using differentiation induction method B. iPSCs: clinical-grade hiPSCs; NPCs (B27): NPCs induced using basal medium supplemented with B27; NPCs (AS10): hNPCs induced using basal medium supplemented with 10% AS401. [Figure 18] The conditions for inducing differentiation of hiPSCs into hNPCs (differentiation induction method C) used in Examples 10 to 14 are shown below. Hereinafter, the differentiation induction method on the small scale in Figure 18 will be referred to as "differentiation induction method C (Small)," and the differentiation induction method on the large scale will be referred to as "differentiation induction method C (Large)." In differentiation induction method C, DMEM / F12 Glutamax (Thermo Fisher Scientific Inc.) supplemented with 10% AS401 was used as the basal medium unless otherwise specified. [Figure 19]This shows a fluorescent image of SIX2 expression confirmed by immunostaining in hNPCs induced from an HLA-homologous stock hiPSC (Ff14s04) line using differentiation induction method C (Small). [Figure 20] Photographs of the morphology and SIX2 expression of hNPCs induced to differentiate by differentiation induction method C (Small) or differentiation induction method C (Large) on day 2 of Stage 6, as well as morphology on day 7 after expansion culture, are shown. [Figure 21] The figure shows the cell yield of hNPCs induced to differentiate using differentiation induction method C (Large). [Figure 22] Fluorescence images showing SIX2 expression confirmed by immunostaining in hNPCs differentiated using differentiation induction method C (Large) are shown. [Figure 23] Photographs of the morphology of hNPCs expanded in media with different additives (B27 or AS401) are shown. Scale bar: 300 μm. AS 10%: Modified CFY medium supplemented with 10% AS401 instead of B27 (hereinafter referred to as "CFY medium (-B27, +10% AS401)"); AS 20%: Modified CFY medium supplemented with 20% AS401 instead of B27 (hereinafter referred to as "CFY medium (-B27, +20% AS401)"); B27: CFY medium. [Figure 24] The proliferation rate of hNPCs expanded in media with different additives (B27 or AS401) is shown: AS10%: CFY medium (-B27, +10% AS401); AS20%: CFY medium (-B27, +20% AS401); B27: CFY medium. [Figure 25] hNPC cell clusters expanded in media containing different additives (B27 or AS401) were transplanted under the kidney capsule of AKI mice, and blood urea nitrogen (BUN) was measured. B27: Transplanted hNPC cell clusters expanded in CFY medium; AS10: Transplanted hNPC cell clusters expanded in CFY medium (-B27, +10% AS401); AS20: Transplanted hNPC cell clusters expanded in CFY medium (-B27, +20% AS401); Ctl: No transplant; pre: Before cisplatin administration; post TX: After cisplatin administration. [Figure 26]hNPC cell clusters expanded in media containing different additives (B27 or AS401) were transplanted under the kidney capsule of AKI mice, and serum creatinine (S-Cre) levels were measured. B27: Transplanted hNPC cell clusters expanded in CFY medium; AS10: Transplanted hNPC cell clusters expanded in CFY medium (-B27, +10% AS401); AS20: Transplanted hNPC cell clusters expanded in CFY medium (-B27, +20% AS401); Ctl: No transplant; pre: Before cisplatin administration; post TX: After cisplatin administration. [Figure 27] The results of examining gene expression in hNPCs expanded in media with different additives (B27 or AS401) are shown. B27: CFY medium; AS10: CFY medium (-B27, +10% AS401). [Figure 28] The graph shows the expression of NPC markers in hNPCs expanded in CFY medium (in which FGF9 was replaced with FGF2) (hereinafter also referred to as "CFY medium (-FGF9, +FGF2)") or in CFY medium. hNPCs were differentiated from hiPSCs using differentiation method A (the differentiation induction method described in WO 2018 / 216743). [Figure 29] The results of light microscopic observation of kidney organoids differentiated from hNPCs are shown. hNPCs were expanded in CFY medium (-FGF9, +FGF2) or CFY medium. hNPCs were differentiated from hiPSCs using differentiation method A. [Figure 30] The results of FACS analysis of cells at each passage were shown. The medium was changed to CF medium (CFY medium minus Y-27632) two days after passage, and the method was repeated for three passages. P1: cells at passage 1; P2: cells at passage 2; P3: cells at passage 3. hNPCs were differentiated from hiPSCs using differentiation induction method A. [Figure 31] The medium was replaced with CFY medium after each passaging, and the results of FACS analysis of cells at each passage are shown. P1: cells at passage 1; P2: cells at passage 2; P3: cells at passage 3. hNPCs were differentiated from hiPSCs using differentiation induction method A. [Figure 32] The numbers of cells positive for NPC markers (OSR1(+)SIX2(+), OSR1(+)SIX2(-), c-MET(+)) and total cell counts are shown for the first passage, when the medium was changed to CF medium (CFY → CF) or CFY medium (CFY → CFY) two days after passage. hNPCs were differentiated from hiPSCs using differentiation induction method A. [Figure 33] The numbers of cells positive for NPC markers (OSR1(+)SIX2(+), OSR1(+)SIX2(-), c-MET(+)) and total cell counts are shown after two passages using a passaging method in which the medium was changed to CF medium (CFY → CF) or CFY medium (CFY → CFY) two days after passaging. hNPCs were differentiated from hiPSCs using differentiation induction method A. [Figure 34] The expression of NPC markers is shown after two passages, with the medium being changed to CF medium (CFY → CF) or CFY medium (CFY → CFY) two days after passage. hNPCs were differentiated from hiPSCs using differentiation induction method A. [Figure 35] The results of optical microscopic observation of kidney organoids differentiated from hNPCs are shown. hNPCs were used after one passage, using a medium change to CF medium (CFY → CF) or CFY medium (CFY → CFY) two days after passage. hNPCs were used after differentiation induction from hiPSCs using differentiation induction method A. DETAILED DESCRIPTION OF THE INVENTION

[0011] <Culture medium for expanding nephron progenitor cells> A first aspect of the present invention is a medium for expanding nephron progenitor cells. The medium of this aspect comprises a GSK-3β inhibitor, a ROCK inhibitor, and at least a fibroblast growth factor selected from the group consisting of FGF9 and FGF20. In one embodiment, the medium of this aspect further comprises a JAK inhibitor.

[0012] (Nephron progenitor cells: NPCs) NPCs are cells that can differentiate in vitro into organ structures such as glomerular-like structures and tubule-like structures in the kidney. The differentiation potential of nephron progenitor cells into organ structures can be evaluated, for example, by the method described in Osafune K, et al. (2006), Development 133:151-61. SIX2 is known as a characteristic factor for maintaining the state of nephron progenitor cells (Cell Stem Cell 3:169-181 (2008)). Examples of nephron progenitor cells that can be cultured using the method of this embodiment include SIX2-positive nephron progenitor cells. For example, pluripotent stem cells (e.g., OSR1-GFP / SIX2-tdTomato reporter human iPS cells described in the Examples below) containing a reporter gene (e.g., tdTomato) introduced under the control of the SIX2 promoter can be cultured, and SIX2-positive nephron progenitor cells can be isolated using methods known in the art (e.g., cell sorting) based on the expression of the reporter gene. Alternatively, SIX2 expression in nephron progenitor cells can be confirmed by methods for analyzing gene expression, such as quantitative RT-PCR (NatCommun 4, 1367, (2013)). As used herein, SIX2-positive nephron progenitor cells encompass cells expressing the SIX2 protein and cells expressing proteins encoded by genes under the control of the SIX2 promoter. Examples of the human SIX2 gene (NCBI Gene ID: 10736) include the gene having the nucleotide sequence registered under NCBI accession number NM_016932.5, and examples of the mouse SIX2 gene (NCBI Gene ID: 20472) include the gene having the nucleotide sequence registered under NCBI accession number NM_011380.2, but are not limited thereto. Nephron progenitor cells cultured in the medium of this embodiment are preferably positive for OSR1, as well as HOX11, WT1, SIX2, and SALL1.

[0013] The organism from which the nephron progenitor cells are derived is not particularly limited, and may be any animal having kidneys. The nephron progenitor cells are preferably derived from mammals, and more preferably from humans. That is, human nephron progenitor cells are preferred.

[0014] Nephron progenitor cells may be isolated from metanephric mesenchyme in vivo, or may be differentiated and induced from pluripotent stem cells (ES cells, iPS cells, etc.). The nephron progenitor cells expanded in the medium of this embodiment are preferably induced from pluripotent stem cells, and more preferably induced from iPS cells.

[0015] Differentiation of nephron progenitor cells from pluripotent stem cells can be induced by known methods, such as those described in International Publication Nos. 2014 / 200115, 2017 / 043666, and 2018 / 216743.

[0016] Nephron progenitor cells can be isolated from a cell population collected from metanephric mesenchyme or a cell population induced to differentiate from pluripotent stem cells using, for example, the expression of nephron progenitor cell markers OSR1, HOX11, WT1, SIX2, or SALL1 as an indicator. Alternatively, the expression of c-MET or AGTR2 can be used as an indicator (WO 2020 / 022261).

[0017] Nephron progenitor cells expanded in the medium of this embodiment may be provided as a cell population containing other cell types, or as a cell population of purified nephron progenitor cells. In the case of a cell population containing nephron progenitor cells and other cell types, the proportion of nephron progenitor cells relative to the total number of cells (100%) is preferably 30%, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more.

[0018] (Expansion culture) "Expansion culture" refers to culture in which nephron progenitor cells are proliferated while maintaining their properties. That is, expanded nephron progenitor cells can differentiate in vitro into organ structures such as kidney glomerulus-like structures and renal tubule-like structures. The medium of this embodiment enables expansion culture of nephron progenitor cells while maintaining their properties, even through repeated passages.

[0019] (Culture medium) The medium of this embodiment may be a medium obtained by adding a GSK-3β inhibitor, a ROCK inhibitor, and at least a fibroblast growth factor selected from the group consisting of FGF9 and FGF20 to a basal medium used for animal culture, or may be a medium obtained by adding a JAK inhibitor to a basal medium used for animal culture in addition to a GSK-3β inhibitor, a ROCK inhibitor, and at least a fibroblast growth factor selected from the group consisting of FGF9 and FGF20.

[0020] <Basal medium> The basal medium is not particularly limited, and any medium commonly used in animal culture can be used without particular limitation. Examples of basal media include, but are not limited to, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's Modified Eagle's Medium (DMEM) medium, Ham's F12 (F12) medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof. The medium may contain serum (e.g., fetal bovine serum (FBS)) or may be serum-free. If necessary, it may contain one or more serum substitutes, such as albumin, transferrin, KnockOut Serum Replacement (KSR) (a serum substitute for ES cell culture) (Invitrogen), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, and 3'-thiolglycerol. It may also contain one or more substances such as lipids, amino acids, L-glutamine, GlutaMAX (Invitrogen), non-essential amino acids (NEAA), vitamins, growth factors, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, and the like.

[0021] The basal medium may be, for example, a mixed medium of DMEM / F12 medium (e.g., a mixed medium in which DMEM:F12 is mixed at a ratio of 1:1) to which amino acids, non-essential amino acids, serum substitutes, etc. have been added.

[0022] Supplements may be added to the basal medium as appropriate. Examples of supplements include AS401 (StemFit for Differentiation, Ajinomoto Healthy Supply Co., Ltd.) and B27 (B-27 Supplement, minus vitamin A, Invitrogen). The basal medium preferably contains 10% by volume of AS401 relative to the total volume of the basal medium. In this specification, a medium containing 10% by volume of AS401 relative to the total volume of the medium may be referred to as a medium supplemented with 10% AS401. The basal medium may be, for example, a DMEM / F12 mixed medium supplemented with 10% AS401. The basal medium may be, for example, a DMEM / F12 mixed medium supplemented with 10% AS401 and GlutaMAX-I (Invitrogen). DMEM / F12 Glutamax (Thermo Fisher Scientific Inc.) may be used as a DMEM / F12 mixed medium supplemented with GlutaMAX. The basal medium may be DMEM / F12 Glutamax supplemented with 10% AS401. The use of a basal medium supplemented with 10% AS401 improves the proliferation rate of nephron progenitor cells, the shape of cell clusters, and the expression of NPC markers (SIX2, OSR1) and renal protective factor (VEGFA).

[0023] The medium of this embodiment contains a GSK-3β inhibitor, a ROCK inhibitor, and at least one fibroblast growth factor selected from the group consisting of FGF9 and FGF20.

[0024] <GSK-3β inhibitor> The medium of this embodiment contains a GSK-3β inhibitor. A GSK-3β inhibitor is a substance that inhibits the function of GSK (Glycogen Synthase Kinase) 3β, for example, kinase activity (for example, the ability to phosphorylate β-catenin). Examples of GSK-3β inhibitors include the indirubin derivative BIO (also known as GSK-3β inhibitor IX; 6-bromoindirubin 3'-oxime), the maleimide derivatives SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), SB415286 (3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrole-2,5-dione), and phenyl α-bromo Examples of such inhibitors include the methyl ketone compound GSK-3β inhibitor VII (4-dibromoacetophenone), the cell membrane-permeable phosphorylated peptide L803-mts (also known as GSK-3β peptide inhibitor; Myr-N-GKEAPPAPPQSpP-NH2), and CHIR99021 (6-[2-[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-ylamino]ethylamino]pyridine-3-carbonitrile), as well as derivatives thereof. These compounds are available from, for example, Stemgent, Calbiochem, Biomol, etc., or may be prepared in-house. GSK-3β inhibitors may also be antisense nucleic acids, RNA interference-inducing nucleic acids (e.g., siRNA), dominant-negative mutants, and expression vectors thereof against GSK-3β.

[0025] One GSK-3β inhibitor may be used alone, or two or more may be used in combination. A preferred GSK-3β inhibitor is CHIR99021. The concentration of the GSK-3β inhibitor in the medium can be selected appropriately depending on the type of GSK-3β inhibitor. The GSK-3β inhibitor is preferably used at a concentration near the IC50, for example. When the GSK-3β is CHIR99021, the concentration of GSK-3β in the medium is, for example, 0.01 to 100 μM, preferably 0.1 to 10 μM, more preferably 0.5 to 3 μM, and particularly preferably 0.5 to 1.5 μM.

[0026] <ROCK inhibitors> The medium of this embodiment contains a ROCK inhibitor, which is a substance that inhibits the function of Rho-kinase (ROCK). Examples of ROCK inhibitors include Y-27632 (see, e.g., Ishizaki et al., Mol. Pharmacol. 57, 976-983 (2000); Narumiya et al., Methods Enzymol. 325, 273-284 (2000)), Fasudil / HA1077 (see, e.g., Uenata et al., Nature 389:990-994 (1997)), H-1152 (see, e.g., Sasaki et al., Pharmacol. Ther. 93:225-232 (2002)), Wf-536 (see, e.g., Nakajima et al., Cancer Chemother Pharmacol. 52(4):319-324 (2003)) and derivatives thereof, as well as antisense nucleic acids against ROCK, RNA interference-inducing nucleic acids (e.g., siRNA), dominant-negative mutants, and expression vectors thereof. Other known small molecule compounds can also be used as ROCK inhibitors (see, for example, U.S. Patent Application Publication Nos. 2005 / 0209261, 2005 / 0192304, 2004 / 0014755, 2004 / 0002508, 2004 / 0002507, 2003 / 0125344, 2003 / 0087919, and International Publication Nos. 2003 / 062227, 2003 / 059913, 2003 / 062225, 2002 / 076976, and 2004 / 039796).

[0027] One ROCK inhibitor may be used alone, or two or more may be used in combination. A preferred ROCK inhibitor is Y-27632. The concentration of the ROCK inhibitor in the medium can be selected appropriately depending on the type of ROCK inhibitor. The ROCK inhibitor is preferably used at a concentration near IC50, for example. When the ROCK inhibitor is Y-27632, the concentration of the ROCK inhibitor in the medium is, for example, 0.1 to 100 μM, preferably 1 to 75 μM, and more preferably 5 to 50 μM.

[0028] The medium of this embodiment contains at least one fibroblast growth factor selected from the group consisting of FGF9 and FGF20.

[0029] <FGF9> The medium of this embodiment may contain fibroblast growth factor (FGF)9. The organism from which FGF9 is derived is not particularly limited. For example, human FGF9 can be used. An example of human FGF9 (NCBI Gene ID: 2254) is a protein having the amino acid sequence of NCBI accession number: NP_002001.1. FGF9 may be a fragment or a functional variant thereof, as long as it has the activity of promoting the proliferation of nephron progenitor cells. Commercially available FGF9 may be used, or a protein purified from cells or a protein produced by genetic recombination may be used. Furthermore, FGF20 belongs to the same subfamily as FGF9 and is thought to be able to replace FGF9, based on findings that it has overlapping effects with FGF9 in the developing mouse kidney (Barak, H. et al. FGF9 and FGF20 Maintain the Stemness of Nephron Progenitors in Mice and Man. Dev. Cell 22, 1191-1207 (2012).).

[0030] The concentration of FGF9 in the medium is, for example, 1 to 800 ng / ml, preferably 5 to 500 ng / ml, more preferably 10 to 400 ng / ml, and even more preferably 100 to 300 ng / ml.

[0031] <FGF20> The medium of this embodiment may contain FGF20 instead of or in addition to FGF9. As described above, FGF20 and FGF9 are known to act similarly during renal development and are mutually substitutable. The organism from which FGF20 is derived is not particularly limited. For example, human FGF20 can be used. Examples of human FGF20 (NCBI Gene ID: 26281) include a protein having the amino acid sequence of NCBI Accession Number: NP_062825.1. FGF20 may be a fragment or a functional variant thereof, as long as it has the activity of promoting the proliferation of nephron progenitor cells. Commercially available FGF20 may be used, or a protein purified from cells or a protein produced by genetic recombination may be used.

[0032] The concentration of FGF20 in the medium is, for example, 1 to 800 ng / ml, preferably 5 to 500 ng / ml, more preferably 10 to 400 ng / ml, and even more preferably 100 to 300 ng / ml.

[0033] The medium of this embodiment may contain either one or both of FGF9 and FGF20. When the medium of this embodiment contains both FGF9 and FGF20, the total concentration of FGF9 and FGF20 is, for example, 1 to 800 ng / ml, preferably 5 to 500 ng / ml, more preferably 10 to 400 ng / ml, and even more preferably 100 to 300 ng / ml.

[0034] Other ingredients In addition to the above components, the medium of this embodiment may contain other components, such as a JAK inhibitor.

[0035] JAK inhibitors The medium of this embodiment may contain a JAK inhibitor. A JAK inhibitor is a substance that inhibits the activity of one or more enzymes in the Janus kinase family (e.g., JAK1, JAK2, JAK3, TYK2). A JAK inhibitor inhibits the activity of Janus kinase family enzymes, thereby inhibiting signal transduction of the JAK-STAT system. When the medium contains a JAK inhibitor in addition to a GSK-3β inhibitor, a ROCK inhibitor, and at least a fibroblast growth factor selected from the group consisting of an FGF9 inhibitor and FGF20, the proliferation of nephron progenitor cells is further promoted. The JAK inhibitor is not particularly limited as long as it can inhibit the activity of an enzyme in the Janus kinase family. The JAK inhibitor is preferably one that inhibits the activity of at least one enzyme selected from the group consisting of JAK1, JAK2, and JAK3, and more preferably one that inhibits the activity of JAK2 (JAK2 inhibitor).

[0036] The JAK3 inhibitor is not particularly limited as long as it can inhibit the activity of JAK3. Examples of the JAK3 inhibitor include TCS21311 (CAS No. 1260181-14-3), WHI-P154 (CAS No. 211555-04-3), PF-06651600 (CAS No. 1792180-81-4), FM-381 (CAS No. 2226521-65-7), CP690550 (CAS No. 540737-29-9), 7H-pyrrolo[2,3-d]pyrimidine-5-carboxylic acid, 4-[3-[(2-methyl-1-oxo-2-propen-1-yl)amino]phenyl]-, ethyl ester, and JAK3 INHIBITOR IV (CAS No. 58753-54-1), ZM449829 (CAS No. 4452-06-6), AZD1480 (CAS No. 935666-88-9), and Selective JAK3 inhibitor 1 (CAS No. 1443235-95-7), as well as derivatives thereof, etc. Among these, the JAK3 inhibitor is preferably TCS21311.

[0037] The JAK2 inhibitor is not particularly limited as long as it can inhibit the activity of JAK2. Examples of JAK2 inhibitors include, but are not limited to, NVP-BSK805 2HCl (CAS No. 1942919-79-0), TG101209 (CAS No. 936091-14-4), TG101348 (CAS No. 936091-26-8), 1,2,3,4,5,6-hexabromocyclohexane (CAS No. 1837-91-8), CEP-33779 (CAS No. 1257704-57-6), and NSC33994 (CAS No. 82058-16-0), and derivatives thereof. Among them, TG101348 is preferred as the JAK2 inhibitor.

[0038] The JAK inhibitor may be a JAK2 / 3 inhibitor. The JAK2 / 3 inhibitor is an inhibitor that inhibits JAK2 and JAK3. Examples of JAK2 / 3 inhibitors include, but are not limited to, AG490 (CAS No. 133550-30-8), AT9283 (CAS No. 896466-04-9), and LY3009104 (CAS No. 1187594-09-7), and derivatives thereof.

[0039] The JAK inhibitor may be a JAK1 / 2 inhibitor. The JAK1 / 2 inhibitor is an inhibitor that inhibits JAK1 and JAK2. Examples of JAK1 / 2 inhibitors include, but are not limited to, CYT387 (CAS No. 1056634-68-4), S-Ruxolitinib (INCB018424) (CAS No. 941685-37-6), and LY2784544 (CAS No. 1229236-86-5), and derivatives thereof.

[0040] JAK inhibitors are not limited to the small molecule compounds described above, but may also be antisense nucleic acids against the Janus kinase family (JAK1, JAK2, JAK3, TYK2), RNA interference-inducing nucleic acids (e.g., siRNA), dominant-negative mutants, and expression vectors thereof.

[0041] The JAK inhibitor may be used alone or in combination of two or more. The JAK inhibitor is preferably a JAK2 inhibitor, more preferably TG101348.

[0042] The concentration of the JAK inhibitor in the medium of this embodiment can be appropriately selected depending on the type of JAK inhibitor. The JAK inhibitor is preferably used, for example, at a concentration near IC50. The JAK inhibitor can be used, for example, at a concentration of 0.01 nM or more, 0.05 nM or more, 0.1 nM or more, 0.5 nM or more, or 1 nM or more. The upper limit of the concentration of the JAK inhibitor can be, for example, 100 nM or less, 50 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, or 5 nM or less. When the JAK inhibitor is TG101348, the concentration of TG101348 in the medium can be, for example, 0.5 to 50 nM, 1 to 30 nM, 1 to 20 nM, 1 to 10 nM, or 1 to 5 nM.

[0043] The medium of this embodiment may contain components other than those described above, provided that the effects of the present invention are not impaired. Preferably, the medium of this embodiment does not contain signal transduction inhibitors, enzyme inhibitors, cytokines, growth factors, etc., other than those described above.

[0044] In one embodiment, the medium of this embodiment comprises a GSK-3β inhibitor, a ROCK inhibitor, and at least a fibroblast growth factor selected from the group consisting of FGF9 and FGF20. In one embodiment, the medium comprises a GSK-3β inhibitor, a ROCK inhibitor, FGF9, and a JAK inhibitor. In one embodiment, the medium comprises a GSK-3β inhibitor, a ROCK inhibitor, FGF9, and a JAK2 inhibitor. In one embodiment, the medium of this embodiment comprises CHIR99021, Y-27632, and FGF9. In one embodiment, the medium of this embodiment comprises CHIR99021, Y-27632, FGF9, and TG101348. FGF9 may be substituted with FGF20.

[0045] The medium of this embodiment contains a combination of a GSK-3β inhibitor, a ROCK inhibitor, and at least a fibroblast growth factor selected from the group consisting of FGF9 and FGF20, thereby enabling the proliferation of nephron progenitor cells while maintaining their differentiation potential.The medium of this embodiment contains a JAK inhibitor in addition to the above components, thereby further improving the proliferation of nephron progenitor cells.

[0046] By using the medium of this embodiment, nephron progenitor cells can be successfully proliferated while maintaining their differentiation potential, even after repeated passage of the cells. Therefore, the medium of this embodiment can also be said to be a medium for subculture of nephron progenitor cells. The medium of this embodiment can also be said to be a maintenance medium for nephron progenitor cells.

[0047] <Method for expanding nephron progenitor cells> A second aspect of the present invention is a method for expanding nephron progenitor cells, comprising the step of culturing nephron progenitor cells in the medium of the first aspect.

[0048] Nephron progenitor cells to be cultured by the method of this embodiment include those listed above in the section (Nephrony progenitor cells: NPCs) under <Culture medium for expansion of nephron progenitor cells>. The nephron progenitor cells are preferably human nephron progenitor cells. The nephron progenitor cells are preferably induced from pluripotent stem cells, and more preferably from iPS cells. In one embodiment, the nephron progenitor cells are induced from human iPS cells.

[0049] The culture method of this embodiment is not particularly limited, as long as the medium of the first embodiment is used. Nephron progenitor cells can be cultured under culture conditions typically used for culturing animal cells. The culture temperature is not particularly limited as long as it allows nephron progenitor cells to proliferate, but it is typically 25 to 40°C, preferably 30 to 40°C. A specific example of the culture temperature is approximately 37°C. Nephron progenitor cells can typically be cultured in an atmosphere of CO2-containing air. The CO2 concentration is typically approximately 0.3 to 5%, preferably approximately 2 to 5%. A specific example of the CO2 concentration is approximately 5%. The culture may be either an adherent culture or a suspension culture, with suspension culture being preferred.

[0050] The culture period is not particularly limited and can be any period. In the method of this aspect, the use of the medium of the first aspect enables long-term culture while maintaining the properties of nephron progenitor cells. For long-term culture, it is preferable to passage the cells appropriately. Repeated passaging using the medium of the first aspect allows for continued culture of nephron progenitor cells while maintaining their properties. Passaging can be performed by collecting cell clusters of nephron progenitor cells from the culture medium and seeding them in a new medium. During passaging, the cell clusters may be dispersed using a cell dispersion solution containing enzymes such as protease, collagenase, and DNase, and then seeded in a new medium. The interval between passagings is not particularly limited and can be, for example, about 2 to 10 days, or about 3 to 7 days. In one embodiment, the method of this aspect includes passaging the nephron progenitor cells.

[0051] The method for expanding nephron progenitor cells of this embodiment may include the following steps (A) and (B): (A) culturing nephron progenitor cells in the medium of the first embodiment for 30 to 60 hours; and (B) Culturing the cells obtained in step (A) in a medium containing a GSK-3β inhibitor and at least one fibroblast growth factor selected from the group consisting of FGF9 and FGF20, but not containing a ROCK inhibitor.

[0052] In step (A), nephron progenitor cells are cultured in the medium of the first embodiment for 30 to 60 hours. The culture time in the medium of the first embodiment may be about 2 days. Examples of culture times in the medium of the first embodiment include 35 to 55 hours and 40 to 50 hours. The culture time is measured from the time the nephron progenitor cells are seeded in the medium of the first embodiment.

[0053] In step (B), the cells obtained in step (A) are cultured in a medium containing a GSK-3β inhibitor and at least one fibroblast growth factor selected from the group consisting of FGF9 and FGF20, but not containing a ROCK inhibitor (hereinafter referred to as a "ROCK inhibitor-free medium"). Examples of the medium used in step (B) include the medium of the first embodiment, from which the ROCK inhibitor has been removed. The culture in step (B) can be initiated by replacing the medium used in the culture in step (A) with a ROCK inhibitor-free medium. In step (B), the medium may be replaced at intervals of about once every two days. The medium used for the medium replacement can be a ROCK inhibitor-free medium. The medium replacement in step (B) can be performed about 1 to 5 times, about 2 to 4 times, twice, or three times. Since the medium used in step (B) does not contain a ROCK inhibitor, it can be performed more inexpensively than when a medium containing a ROCK inhibitor is used.

[0054] The method of this embodiment may further include the following step (C) after the step (B). (C) Passaging the cells obtained in step (B) into the medium of the first embodiment.

[0055] The method of this embodiment may further include the following steps (D) and (E) after the step (C). (D) culturing the cells passaged in step (C) in the medium of the first embodiment for 30 to 60 hours; and (E) Culturing the cells obtained in step (D) in a medium containing a GSK-3β inhibitor and at least one fibroblast growth factor selected from the group consisting of FGF9 and FGF20, but not containing a ROCK inhibitor.

[0056] In step (D), the cells can be cultured for 30 to 60 hours in the medium used in step (C). The culture time in step (D) may be about 2 days. Examples of the culture time in step (D) include 35 to 55 hours and 40 to 50 hours. The culture time is the time from the time the nephron progenitor cells are passaged.

[0057] In step (E), the cells obtained in step (D) are cultured in a ROCK inhibitor-free medium. The same ROCK inhibitor-free medium as in step (B) can be used in step (E). The culture in step (E) can be carried out in the same manner as in step (B).

[0058] The nephron progenitor cells used in the method of this embodiment may be differentiated from pluripotent stem cells (e.g., iPS cells) or may be obtained by the differentiation method described in WO 2018 / 216743.

[0059] Furthermore, the nephron progenitor cells used in the method of this embodiment may be obtained, for example, by a method comprising the following steps (i) to (vi). (i) culturing pluripotent stem cells in a medium containing FGF2, BMP4, a GSK-3β inhibitor, and retinoic acid or a derivative thereof; (ii) culturing the cells obtained in step (i) in a medium containing FGF2, a GSK-3β inhibitor, and BMP7; (iii) culturing the cells obtained in the step (ii) in a medium containing a GSK-3β inhibitor, BMP7, and a TGFβ inhibitor, but not containing FGF2; (iv) culturing the cells obtained in the step (iii) in a medium containing FGF2, a GSK-3β inhibitor, an activin, and a ROCK inhibitor; (v) culturing the cells obtained in the step (iv) in a medium containing retinoic acid or a derivative thereof and not containing FGF9; and (vi) culturing the cells obtained in step (v) in a medium containing a GSK-3β inhibitor and at least one fibroblast growth factor selected from the group consisting of FGF9 and FGF20, thereby inducing nephron progenitor cells from the intermediate mesodermal cells.

[0060] (Step (i)) In this process, late posterior epiblasts are induced from pluripotent stem cells. Late posterior epiblasts are characterized as cells that are positive for at least one of the following markers: CDX1, OCT4, NANOG, and E-CADHERIN (CDH1), and preferably all of these markers. Late posterior epiblasts are also preferably negative for EOMES and BRACHYURY.

[0061] In step (i), pluripotent stem cells are isolated by any method known in the art and preferably cultured by adherent culture. Examples of methods for isolating pluripotent stem cells include mechanical separation, or separation using a separation solution having protease and collagenase activity (e.g., Accutase™ and Accumax™ (Innovative Cell Technologies, Inc.)), or a separation solution having only collagenase activity. A preferred method involves dissociating the cells using a separation solution having protease and collagenase activity, followed by mechanical dispersion into fine single cells. The human pluripotent stem cells used in step (i) are preferably colonies cultured to 70% to 80% confluence in the dish used.

[0062] The medium used in step (i) can be prepared by adding FGF2, BMP4, a GSK-3β inhibitor, and retinoic acid or a derivative thereof to a basal medium used for culturing animal cells. The medium used in step (i) may also contain a ROCK inhibitor. In this case, the medium used in step (i) can be prepared by adding the ROCK inhibitor to the basal medium in addition to the above components. The basal medium can be the basal medium described above, and may contain serum or may be serum-free. If necessary, it may contain serum substitutes, lipids, amino acids, vitamins, growth factors, low-molecular-weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, etc.

[0063] The ROCK inhibitor may be the same as that described above. A preferred example of the ROCK inhibitor is Y-27632. The concentration of the ROCK inhibitor used in step (i) can be appropriately selected by those skilled in the art depending on the ROCK inhibitor used. When the ROCK inhibitor is Y-27632, the concentration of the ROCK inhibitor is, for example, 0.1 to 100 μM, preferably 1 to 75 μM, and more preferably 5 to 50 μM.

[0064] The GSK-3β inhibitor may be the same as that described above. A preferred GSK-3β inhibitor is CHIR99021. The concentration of the GSK-3β inhibitor used in step (i) can be appropriately selected by those skilled in the art depending on the GSK-3β inhibitor used. When the GSK-3β inhibitor is CHIR99021, the concentration of the GSK-3β inhibitor is, for example, 0.01 to 100 μM, preferably 0.1 to 10 μM, more preferably 0.5 to 3 μM, and particularly preferably 0.5 to 1.5 μM.

[0065] The FGF2 (basic FGF: bFGF) is preferably human FGF2. An example of human FGF2 is a protein having the amino acid sequence of NCBI (National Center for Biotechnology Information) accession number ABO43041.1. FGF2 includes fragments and functional variants thereof, as long as they have differentiation-inducing activity. Commercially available FGF2 may be used, or a protein purified from cells or a protein produced by genetic recombination may be used. The concentration of FGF2 used in this step is, for example, 1 to 1000 ng / ml, preferably 10 to 500 ng / ml, and more preferably 50 to 250 ng / ml.

[0066] The BMP4 is preferably human BMP4. An example of human BMP4 is a protein having the amino acid sequence of NCBI (National Center for Biotechnology Information) accession number AAH20546.1. BMP4 includes fragments and functional variants thereof, as long as they have differentiation-inducing activity. Commercially available BMP4 may be used, or a protein purified from cells or a protein produced by genetic recombination may be used. The concentration of BMP4 used in this step is, for example, 0.1 to 100 ng / ml, preferably 0.5 to 50 ng / ml, and more preferably 0.5 to 5 ng / ml.

[0067] The retinoic acid may be retinoic acid itself, or a retinoic acid derivative that retains the differentiation-inducing function of natural retinoic acid. Examples of retinoic acid derivatives include 3-dehydroretinoic acid, 4-[[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carbonyl]amino]-benzoic acid (AM580) (Tamura K, et al., Cell Differ. Dev. 32:17-26(1990)), 4-[(1E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propen-1-yl]-benzoic acid (TTNPB) (Strickland S, et al., Cancer Res. 43:5268-5272(1983)), and Tanenaga, K. et al., Cancer Res. 40:914-919 (1980), retinol palmitate, retinol, retinal, 3-dehydroretinol, 3-dehydroretinal, and the like. The concentration of retinoic acid or a derivative thereof used in step (i) is, for example, 1 to 100 nM, preferably 5 to 50 nM, and more preferably 5 to 25 nM.

[0068] In step (i), the culture temperature is not particularly limited, but is about 30 to 40°C, preferably about 37°C. The culture is carried out in an atmosphere of CO2-containing air. The CO2 concentration is about 2 to 5%, preferably about 5%. The culture time in step (i) may be any time period sufficient to induce differentiation of late posterior epiblasts, but is, for example, 1 to 2 days, preferably 1 day.

[0069] (Step (ii)) In this process, the mesodermal lineage primitive streak is induced from the late posterior epiblast. The mesodermal lineage primitive streak is characterized as cells that are positive for CDX1 and BRACHYURY. The mesodermal lineage primitive streak is also preferably negative for OCT4, NANOG, and CDH1.

[0070] In step (ii), the cell population obtained in step (i) above may be isolated and cultured as adherent cells in a separately prepared coated culture vessel, or the cells obtained by adherent culture in step (i) may be cultured as is by replacing the culture medium.

[0071] The medium used in step (ii) can be prepared by adding FGF2, a GSK-3β inhibitor, and BMP7 to a basal medium used for culturing animal cells. The basal medium can be the same as those described above, and may contain serum or may be serum-free. If necessary, the medium may contain serum substitutes, lipids, amino acids, vitamins, growth factors, low-molecular-weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, and the like.

[0072] The FGF2 used in step (ii) may be the same as that used in step (i).

[0073] The GSK-3β inhibitor may be the same as that described above. A preferred GSK-3β inhibitor is CHIR99021. The concentration of the GSK-3β inhibitor used in step (ii) can be appropriately selected by those skilled in the art depending on the GSK-3β inhibitor used, and may be, for example, 0.01 to 100 μM, preferably 0.1 to 10 μM, more preferably 1 to 7.5 μM, and particularly preferably 2 to 5 μM. The concentration of the GSK-3β inhibitor used in step (ii) is preferably higher than the concentration in step (i).

[0074] The BMP7 is preferably human BMP7. An example of human BMP7 is a protein having the amino acid sequence of NCBI (National Center for Biotechnology Information) accession number NM_001719.2. BMP7 encompasses fragments and functional variants thereof, so long as they have differentiation-inducing activity. Commercially available BMP7 may be used, or a protein purified from cells or a protein produced by genetic recombination may be used. The concentration of BMP7 used in step (ii) is, for example, 0.1 to 100 ng / ml, preferably 0.5 to 50 ng / ml, and more preferably 0.5 to 5 ng / ml.

[0075] In step (ii), the culture temperature is not particularly limited, but is about 30 to 40°C, preferably about 37°C. The culture is carried out in an atmosphere of CO2-containing air. The CO2 concentration is about 2 to 5%, preferably about 5%. The culture time in step (ii) may be any period sufficient to induce differentiation of the mesodermal lineage primitive streak, and may be, for example, 10 hours to 2 days, or 1 to 2 days, preferably 0.5 to 1 day.

[0076] (Step (iii)) In this process, the mesodermal lineage primitive streak is induced into the mesodermal lineage late primitive streak, which is characterized as cells that are positive for CDX2 and BRACHYURY.

[0077] In step (iii), the cell population obtained in the aforementioned step (ii) may be isolated and cultured as adherent cells in a separately prepared coated culture vessel, or the cells obtained by adherent culture in step (ii) may be cultured as is by replacing the culture medium.

[0078] The medium used in step (iii) can be prepared by adding a GSK-3β inhibitor, a BMP7 inhibitor, and a TGFβ inhibitor to a basal medium used for culturing animal cells. The basal medium can be the basal medium described above, and may contain serum or may be serum-free. If necessary, the medium may contain serum substitutes, lipids, amino acids, vitamins, growth factors, low-molecular-weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, etc.

[0079] The GSK-3β inhibitor may be the same as that described above. The concentration of the GSK-3β inhibitor used in step (iii) may be the same as that used in step (ii). The concentration of the GSK-3β inhibitor is, for example, 0.01 to 100 μM, preferably 0.1 to 10 μM, more preferably 1 to 7.5 μM, and particularly preferably 2 to 5 μM.

[0080] The BMP7 used in step (iii) can be the same as that used in step (ii).

[0081] TGFβ inhibitors are substances that inhibit the signal transduction that follows from the binding of TGFβ to its receptor to SMAD. Examples of TGFβ inhibitors include substances that inhibit binding to the ALK family of TGFβ receptors or substances that inhibit phosphorylation of SMAD by the ALK family, such as Lefty-1 (NCBI Accession No.: mouse: NM_010094, human: NM_020997), SB431542, SB202190 (RK Lindemann et al., Mol. Cancer, 2003, 2:20), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276 (Lilly Research Laboratories), A83-01 (WO2009146408), and derivatives thereof. The TGFβ inhibitor may preferably be A83-01. The concentration of the TGFβ inhibitor used in step (iii) is not particularly limited as long as it is a concentration that inhibits ALK. When the TGFβ inhibitor is A83-01, the concentration of the TGFβ inhibitor is, for example, 0.5 to 100 μM, preferably 1 to 50 μM, and more preferably 5 to 25 μM.

[0082] The medium used in step (iii) does not contain FGF2. Because the mesodermal lineage late primitive streak can be induced without the addition of FGF2, this method can be performed more cheaply than a method using a medium containing FGF2.

[0083] In step (iii), the culture temperature is not particularly limited, but is about 30 to 40°C, preferably about 37°C. The culture is carried out in an atmosphere of CO2-containing air. The CO2 concentration is about 2 to 5%, preferably about 5%. The culture time in step (iii) may be any period sufficient to induce differentiation of the mesodermal lineage late primitive streak, and is, for example, 1 to 3 days, preferably 1 to 2 days.

[0084] (Step (iv)) During this process, the late mesodermal primitive streak is induced into the late metanephric lineage primitive streak, which is characterized as cells positive for HOX11 and BRACHYURY.

[0085] In step (iv), the cell population obtained in the aforementioned step (iii) may be isolated and cultured as adherent cells in a separately prepared coated culture vessel, or the cells obtained by adherent culture in step (iii) may be cultured as is by replacing the medium.

[0086] The medium used in step (iv) can be prepared by adding FGF2, a GSK-3β inhibitor, an activin, and a ROCK inhibitor to a basal medium used for culturing animal cells. The basal medium can be the same as those described above, and may contain serum or may be serum-free. If necessary, the medium may contain serum substitutes, lipids, amino acids, vitamins, growth factors, low-molecular-weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, and the like.

[0087] The FGF2 and GSK-3β inhibitors that can be used are the same as those described above. The concentration ranges of the FGF2 and GSK-3β inhibitors used in step (iv) can be the same as those in step (ii). The concentration of the GSK-3β inhibitor used in step (iv) is, for example, 0.01 to 100 μM, preferably 0.1 to 10 μM, more preferably 1 to 7.5 μM, and particularly preferably 2 to 5 μM. The concentration of FGF2 used in step (iv) is, for example, 1 ng / ml to 1000 ng / ml, preferably 30 ng / ml or more, and more preferably 30 to 100 ng / ml.

[0088] The activin may be human-derived activin, activin derived from other animals, or a functional variant of these activins. The activin may be activin A, activin B, or activin AB. Activins commercially available from, for example, R&D Systems, Inc., can be used. A preferred activin is activin A. The concentration of activin used in step (iv) is, for example, 1 to 100 ng / ml, preferably 5 to 50 ng / ml, and more preferably 5 to 25 ng / ml.

[0089] The ROCK inhibitor may be the same as that described above. A preferred example of the ROCK inhibitor is Y-27632. The concentration of the ROCK inhibitor used in step (iv) can be appropriately selected by those skilled in the art depending on the ROCK inhibitor used. When the ROCK inhibitor is Y-27632, the concentration of the ROCK inhibitor is, for example, 0.1 to 100 μM, preferably 1 to 75 μM, and more preferably 5 to 50 μM.

[0090] The medium used in step (iv) may not contain BMP7. By using a medium that does not contain BMP7, step (iv) can be carried out more inexpensively.

[0091] In step (iv), the culture temperature is not particularly limited, but is about 30 to 40°C, preferably about 37°C. The culture is carried out in an atmosphere of CO2-containing air. The CO2 concentration is about 2 to 5%, preferably about 5%. The culture time in step (iv) may be any period sufficient to induce differentiation of the metanephric lineage late primitive streak, but is, for example, 1 to 5 days, preferably 3 days.

[0092] (Process (v)) During this process, the late metanephric lineage primitive streak induces the late posterior intermediate mesoderm, which is characterized by cells positive for OSR1, HOX11, and WT1.

[0093] In step (v), the cell population obtained in the aforementioned step (iv) may be isolated and cultured as adherent cells in a separately prepared coated culture vessel, or the cells obtained by adherent culture in step (iv) may be cultured as is by replacing the medium.

[0094] The medium used in step (v) can be prepared by adding retinoic acid or a derivative thereof to a basal medium used for culturing animal cells. The medium used in step (v) may contain a BMP inhibitor. In this case, the medium used in step (v) can be prepared by adding the BMP inhibitor to the basal medium in addition to the above components. The basal medium can be the basal medium described above, and may contain serum or may be serum-free. If necessary, it may contain serum substitutes, lipids, amino acids, vitamins, growth factors, low-molecular-weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, etc.

[0095] BMP inhibitors are substances that inhibit the function of BMPs. Examples of BMP inhibitors include protein inhibitors such as chordin, noggin, and follistatin, as well as dorsomorphin (i.e., 6-[4-(2-piperidin-1-yl-ethoxy)phenyl]-3-pyridin-4-yl-pyrazolo[1,5-a]pyrimidine), its derivatives (PBYu et al. (2007), Circulation, 116:II_60; PBYu et al. (2008), Nat. Chem. Biol., 4:33-41; J. Hao et al. (2008), PLoS ONE, 3(8):e2904), and LDN193189 (i.e., 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline). A preferred example of the BMP inhibitor is NOGGIN. When the BMP inhibitor is NOGGIN, the concentration of the BMP inhibitor used in step (v) is, for example, 1 to 100 ng / ml, and preferably 10 to 50 ng / ml.

[0096] The medium used in step (v) does not contain FGF9. Since late posterior intermediate mesoderm can be induced even without FGF9, this method can be performed more inexpensively than a method using a medium containing FGF9. The medium used in step (v) does not need to contain FGF20.

[0097] In step (v), the culture temperature is not particularly limited, but is about 30 to 40°C, preferably about 37°C. The culture is carried out in an atmosphere of CO2-containing air. The CO2 concentration is about 2 to 5%, preferably about 5%. The culture time in step (v) may be any period sufficient to induce differentiation of late posterior intermediate mesoderm, but is, for example, 1 to 3 days, preferably 2 days.

[0098] (Process (vi) During this process, nephron progenitor cells are induced from the late posterior intermediate mesoderm and are positive for OSR1, HOX11, WT1, SIX2, and SALL1.

[0099] In step (vi), the cell population obtained in the aforementioned step (v) may be isolated and cultured as adherent cells in a separately prepared coated culture vessel, or the cells obtained by adherent culture in step (v) may be cultured as is by replacing the medium.

[0100] The medium used in step (vi) can be prepared by adding a GSK-3β inhibitor and at least one fibroblast growth factor selected from the group consisting of FGF9 and FGF20 to a basal medium used for culturing animal cells. The medium used in step (vi) may contain heparin. In this case, the medium used in step (vi) can be prepared by adding heparin to the basal medium in addition to the above components. The basal medium can be any of the above-mentioned basal media, and may contain serum or may be serum-free. If necessary, the medium may contain serum substitutes, lipids, amino acids, vitamins, growth factors, low-molecular-weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, etc.

[0101] Heparin is preferably in the form of a salt. Examples of heparin salts include salts with alkali metals such as lithium, sodium, and potassium; salts with alkaline earth metals such as calcium, barium, and magnesium; salts with metals such as aluminum, zinc, copper, and iron; ammonium salts; salts with organic bases; and salts with amino acids. Among these, alkali metal salts of heparin are preferred, and sodium salts are more preferred. The concentration of heparin used in step (vi) is, for example, 0.01 to 100 μg / ml, preferably 0.05 to 50 μg / ml, and more preferably 0.1 to 10 μg / ml.

[0102] The GSK-3β inhibitor, FGF9, and FGF20 may be the same as those described above. The concentration of the GSK-3β inhibitor is, for example, 0.01 to 100 μM, preferably 0.1 to 10 μM, more preferably 0.5 to 3 μM, and particularly preferably 0.5 to 1.5 μM. The concentration of FGF9 or FGF20 (or the total concentration of FGF9 and FGF20) is, for example, 1 to 500 ng / ml, such as 10 to 300 ng / ml or 50 to 200 ng / ml.

[0103] In step (vi), the culture temperature is not particularly limited, but is approximately 30 to 40°C, preferably approximately 37°C. Culture is carried out in an atmosphere of CO2-containing air. The CO2 concentration is approximately 2 to 5%, preferably approximately 5%. The culture time in step (vi) may be any period sufficient to induce differentiation of nephron progenitor cells, but is, for example, 1 to 5 days, preferably 2 days.

[0104] The basal medium used in steps (i) to (vi) above can be the same as that described above in "Culture Medium for Expansion of Nephron Progenitor Cells." For example, a basal medium can be used that contains a DMEM / F12 mixed medium supplemented with B27, AS401, GlutaMAX-I (Invitrogen), or the like. Specific examples of basal media include DMEM / F12 Glutamax supplemented with B27, 10% AS401, or 20% AS401, with DMEM / F12 Glutamax supplemented with 10% AS401 being preferred.

[0105] The culture vessel used in the above steps (i) to (vi) is not particularly limited. Examples of the culture vessel include a 24-well plate, a 12-well plate, a petri dish, a culture flask, a bioreactor, and a cell culture bag. It is preferable to use a culture vessel that allows adhesion culture. The area of ​​the cell adhesion surface of the culture vessel (for example, the bottom surface of the culture vessel) is, for example, 1 to 1000 cm. 2 The culture vessel has a cell adhesion surface of 400 cm 2 The area of ​​the cell adhesion surface of the culture vessel may be, for example, 400 to 800 cm. 2 , 400~700cm 2 , and 400-600cm 2In the differentiation induction method including steps (i) to (vi), differentiation of nephron progenitor cells can be induced even using a large-capacity culture vessel as described above. The use of a large-capacity culture vessel can increase the cell yield of nephron progenitor cells. In steps (i) to (vi), the culture vessel may be changed at the timing of medium replacement or cell seeding. For example, in steps (i) to (iii), a medium-capacity culture vessel (e.g., a vessel with a cell adhesion surface of 50 to 200 cm) may be used. 2 ) was used in steps (iv) to (vi), and a large-capacity culture vessel (cell adhesion surface 400 cm 2 (above) may also be used.

[0106] Adherent culture refers to culturing cells in a state where they are attached to a culture substrate, for example, in a coated culture vessel. The coating agent is preferably an extracellular matrix, such as collagen, proteoglycan, fibronectin, hyaluronic acid, tenascin, entactin, elastin, fibrin, and laminin, or fragments thereof. These extracellular matrices may be used in combination, or may be cell-derived preparations such as BD Matrigel™. The extracellular matrix is ​​preferably laminin or a fragment thereof. In the present invention, laminin is a protein with a heterotrimeric structure containing one α-chain, one β-chain, and one γ-chain, and is an extracellular matrix protein with isoforms that differ in the composition of the subunit chains. Laminin has approximately 15 isoforms, consisting of a heterotrimeric combination of five α-chains, four β-chains, and three γ-chains. Examples of laminin include, but are not limited to, α chains such as α1, α2, α3, α4, or α5; β chains such as β1, β2, β3, or β4; and γ chains such as γ1, γ2, or γ3. Laminin is preferably laminin 511, which consists of α5, β1, and γ1 ( Nat Biotechnol 28, 611-615 (2010)). Laminin may be a fragment, and is not particularly limited as long as it has integrin-binding activity. For example, it may be an E8 fragment (laminin 511E8) obtained by digestion with elastase ( EMBO J., 3:1463-1468, 1984; J. Cell Biol., 105:589-598, 1987; WO2011 / 043405). Laminin 511E8 is commercially available, for example, from Nippi Corporation.

[0107] The nephron progenitor cells obtained in steps (i) to (vi) can be expanded by culturing them in the medium of the first embodiment. Alternatively, the nephron progenitor cells obtained in steps (i) to (vi) can be expanded by subjecting them to a method comprising steps (A) and (B) and, optionally, steps (C) to (E). In steps (A) to (E), the same culture vessels as those described in steps (i) to (vi) above can be used.

[0108] The method of this embodiment cultures nephron progenitor cells using the medium of the first embodiment, allowing for good proliferation of nephron progenitor cells while maintaining their differentiation potential. Furthermore, because the differentiation potential of nephron progenitor cells is maintained even after subculture, nephron progenitor cells can be maintained for a long period of time. Nephron progenitor cells expanded by the method of this embodiment can be administered to a subject with renal disease to treat the renal disease. They can also be used as a pharmaceutical composition for treating or preventing renal disease. Furthermore, because nephron progenitor cells cultured by the method of this embodiment maintain their ability to form renal organoids, they can be used to produce renal organoids, as described below.

[0109] <Method for producing kidney organoids> The third aspect of the present invention is a method for producing kidney organoids. This method for producing kidney organoids comprises the steps of expanding a nephron progenitor culture by the method of the second aspect (expansion step), and differentiating the expanded nephron progenitor cells into kidney organoids (differentiation step).

[0110] (Expansion culture process) The expansion culture step is carried out by the method of the second aspect, which allows nephron progenitor cells to be successfully proliferated to a desired number while maintaining their differentiation potential.

[0111] (differentiation process) Nephron progenitor cells can be differentiated into renal organoids using known methods. For example, the method reported in Nature, 526, 564-568 (2015) can be used. For example, the cell mass of nephron progenitor cells obtained in the expansion culture step can be co-cultured with feeder cells such as 3T3-Wnt4 cells, mouse fetal spinal cord cells, or mouse fetal kidney cells. Alternatively, the cell mass of nephron progenitor cells can be cultured in a semi-gas phase culture (preferably, gas-liquid phase culture; see Nature, 526, 564-568 (2015)) using a basal medium containing a GSK-3β inhibitor. The medium used in the semi-gas phase culture may contain FGF9, FGF2, and the like in addition to the GSK-3β inhibitor. Examples of the basal medium, GSK-3β inhibitor, and FGF2 are the same as those described above. A preferred basal medium is KSR. A preferred GSK-3β inhibitor is CHIR99021. Preferred FGF2 includes human FGF2. Preferred FGF9 includes human FGF9. Examples of human FGF9 include a protein having the amino acid sequence of NCBI accession number: NP_002001.1. FGF9 includes fragments and functional variants thereof as long as they have the activity of inducing differentiation into kidney organoids. Commercially available FGF9 may be used, or proteins purified from cells or proteins produced by genetic recombination may be used. In the above, FGF9 may be replaced with FGF20. Alternatively, FGF9 and FGF20 may be used in combination.

[0112] Culture conditions for the differentiation step can be those typically used for culturing animal cells. The culture temperature is not particularly limited as long as it can induce differentiation into renal organoids, but is typically 25 to 40°C, preferably 30 to 40°C. A specific example of the culture temperature is about 37°C. The CO2 concentration is typically about 0.3 to 5%, preferably about 2 to 5%. A specific example of the CO2 concentration is about 5%.

[0113] The renal organoids obtained by the production method of this embodiment can be administered to a subject with renal disease to treat the renal disease, and can also be used as a pharmaceutical composition for treating or preventing renal disease.

[0114] (Other processes) The production method of this embodiment may include other steps in addition to the steps described above. Examples of other steps include a step of inducing nephron progenitor cells from pluripotent stem cells prior to the expansion step. Induction of nephron progenitor cells from pluripotent stem cells can be performed by known methods.

[0115] <Other aspects> The present invention also provides a pharmaceutical composition comprising nephron progenitor cells obtained by the method of the second aspect or renal organoids obtained by the production method of the third aspect. The present invention also provides a renal disease therapeutic agent comprising the nephron progenitor cells or the renal organoids. The present invention also provides a method for treating or preventing renal disease, comprising administering a therapeutically effective amount of the nephron progenitor cells or the renal organoids to a subject with or at risk of renal disease. The present invention also provides a method for producing cells for treating renal disease, comprising culturing nephron progenitor cells in the medium of the first aspect.

[0116] Methods for administering the nephron progenitor cells to a subject in need of treatment or prevention of renal disease include, for example, forming a sheet of the nephron progenitor cells and attaching it to the subject's kidney; transplanting a cell suspension of the nephron progenitor cells in physiological saline or the like into the subject's kidney; three-dimensionally culturing the nephron progenitor cells (e.g., Dev Cell. Sep 11, 2012; 23(3):637-651) and directly transplanting the resulting cell mass into the subject's kidney; and three-dimensionally culturing the nephron progenitor cells on a scaffold composed of Matrigel or the like and transplanting the resulting cell mass into the subject's kidney. The transplantation site is not particularly limited as long as it is within the kidney, but is preferably under the renal capsule. Methods for administering the renal organoids to a subject in need of treatment or prevention of renal disease include transplantation into the subject's body (e.g., intraperitoneally).

[0117] The renal disease to be treated or prevented is not particularly limited, and examples include acute kidney injury, chronic renal failure, chronic kidney disease that does not progress to chronic renal failure, etc. The number of the nephron progenitor cells to be transplanted or the size of the renal organoids is not particularly limited as long as they can survive after transplantation, and can be appropriately adjusted depending on the size of the diseased site, the body size, age, sex, etc. of the recipient. [Example]

[0118] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0119] Materials and Methods <Animal experiments> All animal experiments were performed with the approval of the Kyoto University Animal Care and Use Committee. scid / J mice were purchased from Charles River Japan. They were housed under specific pathogen-free (SPF) conditions in the experimental animal facility at the Center for iPS Cell Research and Application, Kyoto University. Mice were provided with food and water ad libitum.

[0120] <Induction of nephron progenitor cells (hNPCs) from human iPS cells> The 4A6 strain (derived from 201B7), a human iPS cell line derived from a healthy individual, was induced to differentiate into NPCs derived from human iPS cells on a 24-well plate using the method described in International Publication No. 2018 / 216743. Then, 100 μL of Accumax was added and the mixture was left standing in an incubator at 37 °C with 5% CO2 for 30 minutes. After 30 minutes, it was suspended in 900 μL of 10% FBS. The suspended cells were counted using TC20, and the cells required for flow cytometry were transferred to a 1.5 mL tube and centrifuged at 200 g for 5 minutes. After centrifugation, the supernatant was removed, and the cells were suspended in 100 μL of 2% FBS at a ratio of 1.0x10 6 cells.

[0121] <Purification of c-MET positive cells> To 100 μL of 2% FBS in which the hNPCs induced as described above were suspended, 10 μL of a human-HGFR / c-MET antibody (R&D systems, FAB 3582A) conjugated with the fluorescent substance APC was added, and the mixture was left standing on ice for 30 minutes. After 30 minutes, it was centrifuged at 200 g for 5 minutes. After removing the supernatant, it was washed with 1 mL of PBS, and then centrifuged again at 200 g for 5 minutes to remove the supernatant. This operation was performed twice. The cells with the supernatant removed were suspended in 2% FBS in which DAPI was diluted 1,000-fold, passed through a 40-μm filter, and then c-MET positive cells were extracted using flow cytometry.

[0122] In this specification, hNPCs in which purification of c-MET positive cells has not been performed are referred to as "hNPC (Bulk)". hNPCs in which purification of c-MET positive cells has been performed may be referred to as "hNPC (c-MET(+))".

[0123] <Medium for expansion culture> As a test medium for expansion culture, hNPSR medium, CFY medium, and a medium obtained by adding DMSO or a JAK inhibitor (JAK2 inhibitor: TG101348 (final concentration 3 nM) dissolved in DMSO) to CFY medium were used.

[0124] The composition of hNPSR medium is shown in Table 1. hNPSR medium was prepared by adding the reagents shown in Table 1 to the basal medium shown in Table 1. The composition of CFY medium is shown in Table 2. CFY medium was prepared by adding the reagents shown in Table 2 to the basal medium shown in Table 2.

[0125] [Table 1]

[0126] [Table 2]

[0127] <Cell proliferation> hNPCs were cultured at 2.0x10 per well. 4 The cells were suspended in 50 μL of test medium, seeded onto a low-adhesion 96-well plate (Nunc), centrifuged at 300 g for 3 minutes, and then statically cultured in an incubator at 37°C and 5% CO2. Cell clusters were formed 6 hours after the start of static culture.

[0128] Forty-eight hours after the start of static culture, 100 μL of the same medium used in seeding the cells was added to each well. Forty-eight hours after the addition of medium, 100 μL of supernatant was removed from each well, and 100 μL of new medium was added. This procedure was repeated every 48 hours thereafter. Seven days after the start of suspension culture, the cell masses from each well were transferred to a 1.5 ml tube, the supernatant was completely removed, 30 μL of Accumax (Innovative Cell Technologies, Inc.) was added, and the cells were left to stand in an incubator at 37°C and 5% CO2 for 10 minutes. After 10 minutes, the cells were suspended in 470 μL of 10% FBS, and the cell count was measured using a TC20 (Bio-Rad) instrument.

[0129] <Subculture> Cell aggregates of human iPS cell-derived NPCs (hNPCs) cultured in a test medium were transferred to a 1.5 mL tube, and the supernatant was completely removed. 30 μL of Accumax (Innovative Cell Technologies, Inc.) was added, and the mixture was left standing in an incubator at 37 °C with 5% CO2 for 10 minutes. After 10 minutes, the cells were resuspended in 470 μL of 10% FBS, and the cell count was measured using a TC20 (Bio-Rad). Human iPS cell-derived NPCs at 2.0x10 4 cells per well were suspended and seeded in a low-attachment 96-well plate (Nunc) with the test medium. The low-attachment 96-well plate seeded with the cells was centrifuged at 300 g for 3 minutes and then statically cultured in an incubator at 37 °C with 5% CO2. 48 hours after the start of static culture, 100 μL of the test medium was additionally administered to each well. 48 hours after the additional administration of the medium, the same operations as above were performed to measure the cell count and passage the cells.

[0130] <Generation of kidney organoids> Cell aggregates of subcultured hNPCs were placed on a Transwell (Corning) in the test medium, and the surrounding medium was removed. Next, the Transwell with the cell aggregates was set in a culture dish filled with 5% KSR (Knock-out serum replacement) (Thermo Fisher Scientific) supplemented with 200 ng / mL of FGF2 and 5 μM of CHIR99021 in advance. 48 hours later, the medium was changed to 5% KSR without the addition of growth factors and small molecule compounds. Thereafter, the medium was changed every 48 hours to 5% KSR without the addition of growth factors and small molecule compounds, and on the 10th day of the start of differentiation culture, the cells were fixed with 4% PFA at 4 °C for 3 hours while still on the Transwell. After fixation, the cells were replaced with PBS and left standing at 4 °C overnight for immunostaining.

[0131] <Confirmation of OSR1 and SIX2 expression> Human iPS cells were established that had an OSR1-GFP reporter gene with the GFP coding region introduced into the OSR1 locus and a SIX2-tdTomato reporter gene with the tdTomato coding region linked to the SIX2 gene. The expression of OSR1 and SIX2 was confirmed by performing a culture test using the human iPS cells and detecting each fluorescence of GFP and tdTomato with a fluorescence microscope.

[0132] <Imaging of kidney organoids> Bright-field images of kidney organoids were taken with a KEYENCE BZ-X700 or a confocal microscope (Zeiss LSM710). Image analysis was performed using Image J version 1.51j8 (National Institutes of Health) (C.A. Schneider, et al., Nat Methods. 9(2012)671 - 675.).

[0133] <Administration test of hNPC><f Male NOD.CB17-Prkdc scid / J mice (hereinafter referred to as "NOD-SCID mice") were subcutaneously injected with cisplatin adjusted to 1 mg / mL with physiological saline at a dose of 21 mg / kg to prepare a cisplatin-induced acute kidney injury (AKI) model. Twenty-four hours after cisplatin administration, the left lower dorsal part of NOD-SCID mice under general anesthesia with isoflurane was incised about 1 cm in the skin, and then the retroperitoneum was incised about 1 cm to expose the left kidney outside the body. The exposed kidney was punctured with a 24-gauge Surflo indwelling needle, and the outer cylinder was indwelt under the renal capsule. The renal capsule and renal parenchyma were separated by blowing air or injecting physiological saline with a syringe through the outer cylinder. Subsequently, the outer cylinder of the 24-gauge Surflo indwelling needle was removed, and a cell mass (about 3.0 x 10 6 cells equivalent) of hNPC (Bulk) subcultured once or twice in CFY medium was aspirated, and the outer cylinder of the aspirated 22-gauge Surflo indwelling needle was inserted, and the cells were slowly injected (cell transplantation). In the raw food group, 15 μL of physiological saline was injected under the renal capsule instead of hNPC (Bulk). After injecting the cells, the outer cylinder of the 22-gauge surflo catheter was removed. After confirming that there was no outflow of cells or bleeding from the puncture site, the exposed kidney was returned into the retroperitoneum, and the retroperitoneum and skin were sutured with 4-0 blade silk suture. Ninety-six hours after cisplatin administration (72 hours after cell transplantation), 100 - 200 μL of blood was collected from the inferior ophthalmic vein plexus and centrifuged to obtain serum. Subsequently, using the serum obtained by centrifugation, serum creatinine (S-Cre) and blood urea nitrogen (BUN) were measured with Fuji Drychem NX500.

[0134] (Example 1) Subculture of hNPC (Bulk) was performed using CFY medium and hNPSR medium, and for the cultured cells, cell proliferation, expression of NPC markers, and the ability to form kidney organoids were confirmed.

[0135] <Maintenance culture of hNPC (Bulk) with CFY medium> Subculture of hNPC (Bulk) was performed using hNPSR medium or CFY medium, and in each subculture, it was confirmed whether the expression of SIX2 and OSR1, which are NPC markers, was maintained.

[0136] The results are shown in Figure 1. For hNPC (Bulk) subcultured in hNPSR medium, the expression of OSR1 was detected up to the third passage, but the expression of SIX2 was hardly detected from the second passage. On the other hand, for hNPC (Bulk) subcultured in CFY medium, the expression of both OSR1 and SIX2 was detected in any of the first to third passages. From this result, it was confirmed that subculture with CFY medium is likely to maintain the expression of NPC markers. That is, it was confirmed that hNPC (Bulk) can be subcultured while maintaining the properties as NPCs by using CFY medium.

[0137] <Preparation of kidney organoids from hNPC (Bulk) subcultured with CFY medium> An attempt was made to prepare kidney organoids from hNPC (Bulk) subcultured using hNPSR medium or CFY medium.

[0138] The results are shown in Fig. 2. When subcultured in hNPSR medium, kidney organoids were formed from the first passage of hNPC (Bulk). However, kidney organoids were not formed from the second and third passages of hNPC (Bulk). On the other hand, when subcultured in CFY medium, kidney organoids were formed from hNPC (Bulk) at any passage from passage 1 to passage 3. From these results, it was confirmed that by using CFY medium, hNPC (Bulk) can be subcultured while maintaining the ability to form kidney organoids.

[0139] <Cell proliferation in subculture of hNPC (Bulk) using CFY medium> Subculture of hNPC (Bulk) was performed using a medium supplemented with DMSO in hNPSR medium or CFY medium, and the cell number was measured.

[0140] The results are shown in Figs. 3 and 4. Fig. 3 shows the cumulative cell number calculated from the cells in each subculture. The cumulative cell number at passage 1 can be calculated as follows: Let the cell number grown at passage 0 be a, the volume of the culture solution at passage 0 be b, the volume of the culture solution of P0 used for subculture be c, and the cell number obtained by growing at P1 be d. Then the cumulative cell number at passage 1 = a × [d / (a × c / b)] × b / c. The cumulative cell number in subsequent subcultures is calculated similarly.

[0141] As shown in Figs. 3 and 4, the growth of hNPC (Bulk) was promoted regardless of whether hNPSR medium or CFY medium was used. The growth promoting effect of hNPC (Bulk) was higher in hNPSR medium than in CFY medium.

[0142] (Example 2) Subculture of hNPC (Bulk) was performed using a medium supplemented with a JAK inhibitor in CFY medium, and for the cultured cells, cell proliferation, expression of NPC markers, and the ability to form kidney organoids were confirmed.

[0143] <Cell growth promoting effect by JAK inhibitor>[ hNPC (Bulk) was cultured using a medium obtained by adding DMSO to the CFY medium or a medium obtained by adding 3 nM of TG101348 (JAK2 inhibitor) to the CFY medium, and the cell count was measured.

[0144] The results are shown in Fig. 5. When cultured in a medium obtained by adding TG101348 to the CFY medium (CFY+TG(3)), cell proliferation was promoted compared to culturing in a medium obtained by adding DMSO to the CFY medium (CFY+DMSO). From these results, it was confirmed that by adding a JAK inhibitor, the growth promoting effect of hNPC (Bulk) in the CFY medium was improved.

[0145] <Maintenance culture of hNPC (Bulk) with JAK inhibitor-added CFY medium> Subculture of hNPC (Bulk) was performed using a medium obtained by adding DMSO to the CFY medium or a medium obtained by adding 3 nM of TG101348 (JAK2 inhibitor) to the CFY medium, and in each subculture, it was confirmed whether the expression of OSR1 and SIX2, which are NPC markers, was maintained.

[0146] The results are shown in Fig. 6. In both the medium obtained by adding DMSO to the CFY medium (+DMSO) and the medium obtained by adding 3 nM of TG101348 to the CFY medium (+TG(3)), the expression of both OSR1 and SIX2 was maintained up to the third passage. From these results, it was confirmed that using a medium obtained by adding a JAK inhibitor to the CFY medium, hNPC (Bulk) can be subcultured while maintaining the properties as NPC.

[0147] <Generation of kidney organoids from hNPC (Bulk) subcultured with JAK inhibitor-added CFY medium> Subculture of hNPC (Bulk) was performed using a medium obtained by adding DMSO to the CFY medium or a medium obtained by adding 3 nM of TG101348 (JAK2 inhibitor) to the CFY medium, and an attempt was made to generate kidney organoids from the cells of the second passage.

[0148] The results are shown in Fig. 7. Kidney organoids were formed from the cells of the second passage in both the medium obtained by adding DMSO to the CFY medium (+DMSO) and the medium obtained by adding 3 nM of TG101348 to the CFY medium (+TG(3)). From these results, it was confirmed that hNPC (Bulk) can be subcultured while maintaining the ability to form kidney organoids using the medium obtained by adding a JAK inhibitor to the CFY medium.

[0149] (Example 3) Subculture of hNPC (c-MET(+)) was performed using the medium obtained by adding a JAK inhibitor to the CFY medium, and for the cultured cells, cell proliferation, expression of NPC markers, and the ability to form kidney organoids were confirmed.

[0150] <Growth promoting effect of JAK inhibitor in subculture of hNPC (c-MET(+))> Subculture of hNPC (c-MET(+)) was performed using the medium obtained by adding DMSO to the CFY medium or the medium obtained by adding 3 nM of TG101348 (JAK2 inhibitor) to the CFY medium, and the cell number was measured to calculate the cumulative cell number.

[0151] The results are shown in Fig. 8. When cultured in the medium obtained by adding TG101348 to the CFY medium (CFY+TG(3)), cell proliferation was promoted in all passages compared to the culture in the medium obtained by adding DMSO to the CFY medium (CFY+DMSO). From these results, it was confirmed that the growth promoting effect of hNPC (c-MET(+)) in the CFY medium is improved by adding a JAK inhibitor.

[0152] <Maintenance culture of hNPC (c-MET(+)) with JAK inhibitor-added CFY medium> Subculture of hNPC (c-MET(+)) was performed using the medium obtained by adding DMSO to the CFY medium or the medium obtained by adding 3 nM of TG101348 (JAK2 inhibitor) to the CFY medium, and in each subculture, it was confirmed whether the expression of OSRl and SIX2, which are NPC markers, was maintained.

[0153] The results are shown in Fig. 9. In both the medium with DMSO added to the CFY medium (+DMSO) and the medium with 3 nM of TG101348 added to the CFY medium (+TG(3)), the expression of both OSR and SIX2 was maintained until the 3rd passage. From these results, it was confirmed that hNPC (c-MET(+)) can be subcultured while maintaining the properties as NPCs using the medium with a JAK inhibitor added to the CFY medium.

[0154] <Generation of kidney organoids from hNPC (c-MET(+)) subcultured in CFY medium supplemented with JAK inhibitor> Subculture of hNPC (c-MET(+)) was performed using the medium with DMSO added to the CFY medium or the medium with 3 nM of TG101348 (JAK2 inhibitor) added to the CFY medium, and an attempt was made to generate kidney organoids from the cells of the 1st passage.

[0155] The results are shown in Fig. 10. In both the medium with DMSO added to the CFY medium (+DMSO) and the medium with 3 nM of TG101348 added to the CFY medium (+TG(3)), kidney organoids were formed from the cells of the 1st passage. From these results, it was confirmed that hNPC (c-MET(+)) can be subcultured while maintaining the ability to form kidney organoids using the medium with a JAK inhibitor added to the CFY medium.

[0156] (Example 4) Cell clumps of hNPC (Bulk) expanded in CFY medium were transplanted under the renal capsule of AKI mice to confirm the therapeutic effect of hNPC (Bulk).

[0157] The results of transplantation of hNPC cell masses after 1st and 2nd passages are shown in Figures 11 and 12, respectively. In Figures 11 and 12, "Normal" indicates normal mice, "Saline" indicates AKI mice administered saline under the renal capsule, and "NPC Transplant" indicates AKI mice transplanted with hNPC (Bulk) cell masses under the renal capsule. The saline-administered AKI mice had significantly higher BUN and S-Cre levels than the normal mice, confirming renal dysfunction. On the other hand, the hNPC (Bulk) cell mass transplanted mice had significantly lower BUN and S-Cre levels than the saline-administered mice, indicating improved renal function, for both passage 1 and passage 2 hNPCs. These results confirmed that transplantation of hNPCs expanded in CFY medium can improve renal dysfunction.

[0158] In the above-mentioned Examples 1 to 4, NPCs were used that had been induced to differentiate by the following differentiation induction method A based on the method described in WO 2018 / 216743. <Differentiation induction method A> 1. Undifferentiated hiPSC cells were denatured by accutase treatment and then suspended in 500 μL of AK02N medium (Ajinomoto) supplemented with 10 μM Y-27632 and 1.25 μL of iMatrix (Nippi). 1.0 × 10 cells were plated onto a 24-well plate. 4 cells / well~5.0×10 4 Cells were seeded at a density of 1000 cells / well and incubated at 37°C for 24 hours. 2. After 24 hours (day 1), the medium was replaced with DMEM / F12 Glutamax (Thermo Fisher Scientific Inc.) supplemented with B27 (B-27 Supplement, minus vitamin A, Invitrogen) and medium supplemented with 1 μM CHIR99021, 10 nM retinoic acid, 1 ng / ml BMP4, and 100 ng / ml FGF2. (Generation of late posterior epiblasts ··· 1) 3. On Day 2, the medium was changed to a medium supplemented with 3 μM CHIR99021, 1 ng / ml BMP7, and 100 ng / ml FGF2 in the same basal medium (Preparation of mesodermal lineage primitive streak... 2) 4. On Day 3, the medium was changed to a medium supplemented with 3 μM CHIR99021, 1 ng / ml BMP7, 100 ng / ml FGF2, and 10 μM A83-01 in the same basal medium (Preparation of late mesodermal lineage primitive streak... 3) 5. On Day 4, Day 5, and Day 6, the medium was changed to a medium supplemented with 3 μM CHIR99021, 1 ng / ml BMP7, 100 ng / ml FGF2, 10 ng / ml activin, and 30 μM Y-27632 in the same basal medium (Preparation of late primitive streak of metanephric lineage... 4) 6. On Day 7 and Day 8, the medium was changed to a medium supplemented with 200 ng / ml FGF9, 100 nM retinoic acid, and 25 ng / ml NOGGIN in the same basal medium (Preparation of late posterior intermediate mesoderm... 5) 7. On Day 9, Day 10, and Day 11, the medium was changed to a medium supplemented with 200 ng / ml FGF9 and 1 μM CHIR99021 in the same basal medium (Preparation of renal progenitor cells (nephron progenitor cells)... 6)

[0159] (Example 5) It was examined whether hNPC could be induced to differentiate from human iPS cells (hiPSC) according to the following protocol. The hiPSC used was OSR1-GFP / SIX2-tdTomato reporter human iPS cells derived from 201B7.

[0160] <Method for inducing differentiation of hNPC from hiPSC (Differentiation induction method B)>[[]]END]] 1. After dissociating undifferentiated hiPSC into single cells by TrypLE Select Enzyme (Gibco) treatment, DMEM / F12 Glutamax (Invitrogen) supplemented with B27 was used as the basal medium, and in a medium supplemented with 1 μM CHIR99021, 10 nM retinoic acid, 1 ng / ml BMP, 4 4, 100 ng / ml FGF2, 10 μM Y-27632, and iMatrix-511 (Nippi), 1.0 × 104 Seed cells at a density of 1000 cells / well and incubate at 37°C for 24 hours (Generation of late posterior epiblasts: Step 1). 2. On Day 1, the medium was replaced with the same basal medium supplemented with 5 μM CHIR99021, 1 ng / ml BMP7, and 100 ng / ml FGF2 (preparation of mesodermal lineage primitive streak: step 2). 3. On day 2, the medium was replaced with the same basal medium supplemented with 5 μM CHIR99021, 1 ng / ml BMP7, 100 ng / ml FGF2, and 10 μM A83-01 (preparation of late mesodermal lineage primitive streak: step 3). 4. On day 3, the medium was replaced with the same basal medium supplemented with 5 μM CHIR99021, 100 ng / ml FGF2, 1 ng / ml BMP7, 10 ng / ml activin A, and 30 μM Y-27632 (preparation of the metanephric lineage late primitive streak: step 4). 5. On day 6, the medium was replaced with the same basal medium supplemented with 100 nM retinoic acid, 200 ng / ml FGF9, and 25 ng / ml Noggin (preparation of late posterior intermediate mesoderm: step 5). 6. On day 8, the medium was replaced with the same basal medium supplemented with 1 μM CHIR99021 and 200 ng / ml FGF9 (hNPC preparation: step 6).

[0161] In addition, in step 4 of differentiation induction method B (preparation of the late metanephric lineage primitive streak: step 4), hiPSCs were differentiated into hNPCs using a method similar to differentiation induction method B, except that 100 ng / ml FGF2 was replaced with 30 ng / ml FGF2; 30 ng / ml FGF2 and 0.18 U / ml (1 μg / mL) heparin; 10 ng / ml FGF2; 10 ng / ml FGF2 and 0.18 U / ml (1 μg / mL) heparin; or 0 ng / ml FGF2.

[0162] The efficiency of differentiation induction of SIX2-positive cells from differentiation-induced hNPCs was confirmed and is shown in Figure 13. The percentage of SIX2-positive cells among live cells that were not DAPI-stained is shown. In step 4 above (preparation of the late metanephric lineage primitive streak: step 4), when the FGF2 concentration was 10 ng / mL or less, the efficiency of differentiation into hNPCs decreased. The presence of heparin did not affect the efficiency of differentiation into hNPCs.

[0163] Example 6 According to the protocol of the differentiation induction method B, differentiation of hiPSCs into hNPCs was induced. In addition, differentiation of hiPSCs to hNPCs was induced in the same manner as in differentiation induction method B, except that in step 3 of differentiation induction method B (preparation of the late mesodermal lineage primitive streak: step 3), 100 ng / ml FGF2 was replaced with 30 ng / ml FGF2 or 10 ng / ml FGF2, and in step 4 of differentiation induction method B (preparation of the late metanephric lineage primitive streak: step 4), 100 ng / ml FGF2 was replaced with 30 ng / ml FGF2.

[0164] The efficiency of differentiation induction of SIX2-positive cells from differentiation-induced hNPCs was confirmed and the results are shown in Figure 14. The percentage of SIX2-positive cells among live cells that were not DAPI-stained is shown. In step 3 above (preparation of late mesodermal lineage primitive streak: step 3), the concentration of FGF2 did not affect the efficiency of differentiation into hNPCs.

[0165] Example 7 According to the protocol of the differentiation induction method B, differentiation of hiPSCs into hNPCs was induced. In addition, differentiation of hiPSCs to hNPCs was induced in the same manner as in differentiation induction method B, except that in step 5 of differentiation induction method B (preparation of late posterior intermediate mesoderm: step 5), 200 ng / ml FGF9 was replaced with 100 ng / ml FGF9; 100 ng / ml FGF9 and 0.18 U / ml (1 μg / ml) heparin; 25 ng / ml FGF9; 25 ng / ml FGF9 and 0.18 U / ml (1 μg / ml) heparin; 10 ng / ml FGF9; 10 ng / ml FGF9 and 0.18 U / ml (1 μg / ml) heparin; or 0 ng / ml FGF9, and further, in step 4 of differentiation induction method B (preparation of late metanephric lineage primitive streak: step 4), 100 ng / ml FGF2 was replaced with 30 ng / ml FGF2.

[0166] The efficiency of differentiation induction of SIX2-positive cells was confirmed for differentiation-induced hNPCs, and the results are shown in Figure 15. The percentage of SIX2-positive cells among live cells that were not DAPI-stained is shown. In step 5 (preparation of late posterior intermediate mesoderm: step 5), the concentration of FGF9 did not affect the efficiency of differentiation into hNPCs. The presence of heparin did not affect the efficiency of differentiation into hNPCs.

[0167] Example 8 According to the protocol of the differentiation induction method B, differentiation of hiPSCs into hNPCs was induced. Furthermore, in the differentiation induction method B-2 to B-6, DMEM / F12 Glutamax supplemented with 10% or 20% AS401 (StemFit (registered trademark) for Differentiation, Ajinomoto Healthy Supply Co., Ltd.) was used as the basal medium, and further, in the differentiation induction method B-3 (preparation of the late mesodermal lineage primitive streak: step 3), 100 ng / ml FGF2 was replaced with 0 ng / ml FGF2, further, in the differentiation induction method B-4 (preparation of the late metanephric lineage primitive streak: step 4), 100 ng / ml FGF2 was replaced with 30 ng / ml FGF2, further, in the differentiation induction method B-5 (preparation of the late posterior intermediate mesoderm: step 5), 200 ng / ml FGF9 was replaced with 0 ng / ml FGF9, and further, in the differentiation induction method B-6 (preparation of hNPC: step 6), 200 ng / ml FGF9 was replaced with 100 ng / ml FGF9. Differentiation of hiPSCs into hNPCs was induced in the same manner as in differentiation induction method B, except that FGF9 and 0.18 U / ml (1 μg / mL) heparin were used.

[0168] The results of examining the efficiency of differentiation induction of SIX2-positive cells for differentiation-induced hNPCs are shown in Figure 16. Figure 16 shows the percentage of SIX2-positive cells among live cells that were not DAPI-stained. A tendency for improved hNPC induction efficiency was confirmed when basal medium supplemented with 10% AS401 was used.

[0169] Example 9 hNPCs were induced to differentiate as in Example 8 (differentiation induction method B), except that clinical hiPSC stock lines were used as hiPSCs. In this example, B27 or DMEM / F12 Glutamax supplemented with 10% AS401 was used as the basal medium.

[0170] The results of confirming SIX2 expression in differentiation-induced hNPCs are shown in Figure 17. A tendency for hNPC induction efficiency to improve was confirmed when basal medium supplemented with 10% AS401 was used.

[0171] Example 10 hNPCs were differentiated from hiPSCs under the conditions shown in Figure 18 (hereinafter referred to as "differentiation induction method C"). The basal medium was DMEM / F12 Glutamax supplemented with 10% AS401. For small-scale iPSCs, iPSCs were seeded in a 24-well plate (greiner bio-one, 662160) at Stage 1, and reseeded on the 24-well plate after the first day of Stage 4. After the second day of Stage 6, hNPCs were collected and expanded in CFY medium using a low-attachment 96-well plate (PrimeSurface plate 96U (Sumitomo Bakelite Co., Ltd., MS-9096U)). For large-scale iPSCs, iPSCs were seeded in a T150 flask or T75 flask (Iwaki) at Stage 1, and reseeded on the 512 cm plate after the first day of Stage 4. 2 The cells were replated onto plates (Sumitomo Bakelite, Peel-off Culture Vessel 512, MS-28500, for adherent cell culture). After the second day of Stage 6, hNPCs were collected and expanded in CFY medium using low-adhesion 96-well plates.

[0172] FIG. 19 shows the results of immunostaining to confirm SIX2 expression in hNPCs induced from an HLA-homologous stock hiPSC (Ff14s04) line by a small-scale differentiation induction method (differentiation induction method C (Small)).

[0173] Figure 20 shows morphological photographs and SIX2 expression levels of cells that underwent differentiation induction using either small-scale (differentiation induction method C (Small)) or large-scale (differentiation induction method C (Large)) in Figure 18. Morphological photographs and SIX2 expression levels at Stage 6 confirmed that hNPCs were induced to differentiate in both small-scale and large-scale cultures. Morphological photographs taken 7 days after expansion confirmed that hNPCs induced to differentiate in both small-scale and large-scale cultures could be expanded in CFY medium.

[0174] Example 11 hNPCs were differentiated from hiPSCs using differentiation induction method C (Large). The basal medium used was DMEM / F12 Glutamax supplemented with 10% AS401.

[0175] The cell yield after Stage 6 in differentiation induction method C (Large) is shown in Figure 21. 8 It was confirmed that large-scale hNPCs can be produced. FIG. 22 shows fluorescent images confirming SIX2 expression in hNPCs after Stage 6 by immunostaining.

[0176] Example 12 To compare the effects of additives on expansion culture, CFY medium supplemented with 10% AS401 instead of B27 (CFY medium (-B27, +10% AS401)), or CFY medium supplemented with 20% AS401 instead of B27 (CFY medium (-B27, +20% AS401)), and CFY medium were prepared.

[0177] hNPCs were differentiated from hiPSCs using differentiation induction method C (Small). The basal medium was DMEM / F12 Glutamax supplemented with 10% AS401. hNPCs were collected after Stage 6 and expanded in low-attachment 96-well plates in CFY medium, CFY medium (-B27, +10% AS401), or CFY medium (-B27, +20% AS401).

[0178] Figure 23 shows photographs of the morphology of hNPCs during expansion. When CFY medium or CFY medium (-B27, +20% AS401) was used, the morphology of the hNPC clusters changed to an oval shape. On the other hand, when CFY medium (-B27, +10% AS401) was used, the morphology of the hNPC clusters remained round. The proliferation rate is shown in Figure 24. hNPCs showed the best proliferation rate in CFY medium (-B27, +10% AS401). These results suggest that CFY medium (-B27, +10% AS401) is the most suitable for the expansion of hNPCs.

[0179] Example 13 hNPCs were differentiated from hiPSCs using differentiation induction method C (Small). The basal medium was DMEM / F12 Glutamax supplemented with 10% AS401. hNPCs were collected after Stage 6 and expanded in low-attachment 96-well plates in CFY medium, CFY medium (-B27, +10% AS401), or CFY medium (-B27, +20% AS401). 3 x 10 hNPC cell clumps on day 7 of expansion culture 6 The equivalent number of hNPC cells were transplanted under the kidney capsule of AKI mice, and the therapeutic effect of hNPC was confirmed.

[0180] The results of measuring blood urea nitrogen (BUN) and serum creatinine (S-Cre) are shown in Figures 25 and 26, respectively. No significant differences were detected among the four groups, including the Ctl group, but a tendency for improvement was observed in both BUN and S-Cre in the transplant group.

[0181] Example 14 hNPCs were differentiated from hiPSCs using differentiation induction method C (Small). The basal medium was DMEM / F12 Glutamax supplemented with 10% AS401. hNPCs were collected after Stage 6 and expanded in CFY medium or CFY medium (-B27, +10% AS401) in low-attachment 96-well plates.

[0182] The results of examining gene expression in hNPC cell clusters on day 7 of expansion culture are shown in Figure 27. Expansion culture in CFY medium (-B27, +10% AS401) tended to result in higher expression of NPC markers and angiogenesis-related genes.

[0183] Example 15 To confirm the effect of FGF9 in CFY medium, a medium was prepared by adding 300 ng / ml of FGF2 instead of FGF9 (CFY medium (-FGF9, +FGF2)). hNPCs were differentiated from hiPSCs using differentiation method A. The basal medium was DMEM / F12 Glutamax supplemented with B27 (B-27 Supplement, minus vitamin A, Invitrogen). hNPCs were collected after Stage 6 and expanded in CFY medium or CFY medium (minus FGF9, plus FGF2) in low-attachment 96-well plates.

[0184] On day 7 of expansion, the cell clusters were observed under an optical microscope and a fluorescent microscope, and the morphology observed under an optical microscope was compared with the expression of NPC markers (OSR1, SIX2) observed under a fluorescent microscope. The results are shown in Figure 28. The expression of NPC markers was higher in cells expanded in a medium containing FGF9.

[0185] Example 16 hNPCs expanded in CFY medium or CFY medium (-FGF9, +FGF2) were induced to differentiate into kidney organoids. The results are shown in Figure 29. When expanded in a medium containing FGF9, the area of ​​the kidney organoids (lightly colored areas) increased, indicating better kidney organoid formation.

[0186] Example 17 To confirm the effect of the ROCK inhibitor (Y-27632) in CFY medium, a medium (CF medium) was prepared by removing the ROCK inhibitor from CFY medium. hNPCs maintained in CFY medium were passaged into CFY medium. Two days after passage, the medium was changed to CFY or CF medium (first medium change). Then, two days after the first medium change, the medium was changed again to CFY or CF medium (second medium change). Two days after the second medium change, the cells were passaged into CFY medium. This cycle of subculture was repeated up to three passages.

[0187] Cells were collected at the time of passage and analyzed by flow cytometry for NPC marker-positive cells (OSR1, SIX2). The results are shown in Figures 30 and 31. Whether the medium used two days or more after passaging was CF medium (CFY → CF) or CFY medium (CFY → CFY) did not affect the expression of NPC markers.

[0188] Example 18 hNPCs maintained in CFY medium were passaged into CFY medium. Two days after passage, the medium was changed to CFY or CF medium (first medium change). Then, two days later, the medium was changed again to CFY or CF medium (second medium change). Two days after the second medium change, the cells were passaged into CFY medium. This cycle of subculture was repeated up to three passages.

[0189] Cells were collected at the time of passage, and the proportion of NPC marker (OSR1, SIX2)-positive cells and c-MET (a cell surface antigen protein specifically expressed in NPCs)-positive cells among live cells that did not stain with DAPI was analyzed by flow cytometry. The results are shown in Figure 32 (passage 1) and Figure 33 (passage 2). The cell counts are calculated based on the proportion of each cell group (OSR1(+)SIX2(+), OSR1(+)SIX2(-), c-MET(+)) measured by flow cytometry out of the total number of viable cells that did not stain with DAPI. The change of medium from 2 days after passaging to either CF medium (CFY → CF) or CFY medium (CFY → CFY) did not affect the expression of NPC markers.

[0190] Example 19 hNPCs maintained in CFY medium were passaged into CFY medium. Two days after passage, the medium was changed to CFY or CF medium (first medium change). Then, two days later, the medium was changed again to CFY or CF medium (second medium change). Two days after the second medium change, the cells were passaged into CFY medium. This cycle of subculture was continued for up to two passages.

[0191] After two passages, the cell aggregates were observed under an optical microscope and a fluorescent microscope, and the morphology observed under an optical microscope was compared with the expression of NPC markers (OSR1, SIX2) observed under a fluorescent microscope. The results are shown in Figure 34. After two days of passage, whether the medium used was CF medium (CFY → CF) or CFY medium (CFY → CFY) did not affect the expression of NPC markers.

[0192] Example 20 hNPCs maintained in CFY medium were passaged into CFY medium. Two days after passage, the medium was changed to CFY or CF medium (first medium change). Then, two days later, the medium was changed again to CFY or CF medium (second medium change). Two days after the second medium change, the cells were passaged into CFY medium. This cycle of subculture was continued until the first passage.

[0193] After one passage, hNPCs were induced to differentiate into renal organoids. The morphology of the renal organoids was observed using an optical microscope. The results are shown in Figure 35. Renal organoids were formed regardless of whether the medium used was CF medium (CFY → CF) or CFY medium (CFY → CFY) from two days after passage. The formation of renal organoids was equivalent when the medium used was CF medium (CFY → CF) from two days after passage to when CFY medium was used (CFY → CFY). [Industrial Applicability]

[0194] The present invention provides a culture medium for expanding nephron progenitor cells, which allows expansion of nephron progenitor cells while maintaining differentiation potential, a method for expanding NPCs using the culture medium, and a method for producing renal organoids from nephron progenitor cells obtained by the expansion method. The nephron progenitor cells and renal organoids obtained by the method of the present invention can be used to treat or prevent kidney diseases.

Claims

1. A method for treating a fibroblast growth factor comprising: CHIR99021, Y-27632, and at least one fibroblast growth factor selected from the group consisting of FGF9 and FGF20, does not contain any growth factors other than the fibroblast growth factor; A medium for expanding nephron progenitor cells.

2. The medium of claim 1 , wherein the nephron progenitor cells are human nephron progenitor cells.

3. The medium according to claim 1 or 2, wherein the nephron progenitor cells are induced from pluripotent stem cells.

4. The medium according to claim 3 , wherein the pluripotent stem cells are iPS cells.

5. A method for expanding nephron progenitor cells, comprising the step of culturing nephron progenitor cells in the medium according to any one of claims 1 to 4.

6. The method for expanding nephron progenitor cells according to claim 5 , wherein the step of culturing the nephron progenitor cells comprises subculturing the nephron progenitor cells.

7. (A) culturing nephron progenitor cells in the medium according to any one of claims 1 to 4 for 30 to 60 hours; (B) culturing the cells obtained in the step (A) in a medium containing a GSK-3β inhibitor and at least one fibroblast growth factor selected from the group consisting of FGF9 and FGF20, and not containing a ROCK inhibitor; A method for expanding and culturing nephron progenitor cells, comprising:

8. The method for expanding nephron progenitor cells according to claim 7, further comprising: (C) a step of subculturing the cells obtained in step (B) in the medium according to any one of claims 1 to 4.

9. (D) culturing the cells passaged in the step (C) in the medium according to any one of claims 1 to 4 for 30 to 60 hours; (E) culturing the cells obtained in the step (D) in a medium containing a GSK3β inhibitor and at least one fibroblast growth factor selected from the group consisting of FGF9 and FGF20, and not containing a ROCK inhibitor; The method for expanding nephron progenitor cells according to claim 8, further comprising:

10. The method for expanding nephron progenitor cells according to any one of claims 5 to 9, wherein the nephron progenitor cells are human nephron progenitor cells.

11. The method for expanding nephron progenitor cells according to any one of claims 5 to 9, wherein the nephron progenitor cells are induced from pluripotent stem cells.

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

13. The method for expanding nephron progenitor cells according to claim 11 or 12, wherein the nephron progenitor cells are obtained by a method comprising the following steps (i) to (vi): (i) culturing pluripotent stem cells in a medium containing FGF2, BMP4, a GSK-3β inhibitor, and retinoic acid or a derivative thereof; (ii) culturing the cells obtained in the step (i) in a medium containing FGF2, a GSK-3β inhibitor, and BMP7; (iii) culturing the cells obtained in the step (ii) in a medium containing a GSK-3β inhibitor, BMP7, and a TGFβ inhibitor, but not containing FGF2; (iv) culturing the cells obtained in the step (iii) in a medium containing FGF2, a GSK-3β inhibitor, activin, and a ROCK inhibitor; (v) culturing the cells obtained in the step (iv) in a medium containing retinoic acid or a derivative thereof and not containing FGF9; and (vi) culturing the cells obtained in step (v) in a medium containing a GSK-3β inhibitor and at least one fibroblast growth factor selected from the group consisting of FGF9 and FGF20.

14. The method for expanding nephron progenitor cells according to claim 13, wherein the medium used in step (iv) does not contain BMP7.

15. At least one of the steps (i) to (vi) is performed on a substrate having a cell adhesion surface of 400 cm ２ A method for expanding nephron progenitor cells according to claim 13 or 14, carried out using the above culture vessel.

16. A step of expanding nephron progenitor cells by the method for expanding nephron progenitor cells according to any one of claims 5 to 15; Differentiating the expanded nephron progenitor cells into kidney organoids; A method for producing kidney organoids, comprising:

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