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

Abstract

This culture medium is one for performing the expansion culture of a nephron progenitor cell, and contains at least one component selected from the group consisting of a TBK1 inhibitor, a CSF1R inhibitor, an FLT3 inhibitor, a DYRK1 inhibitor and an STAT6 inhibitor.

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. 2021-02202, filed on January 8, 2021, the contents of which are incorporated herein by reference. [Background technology]

[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] To reduce culture costs and time, it is desirable to develop an expansion culture method that can more efficiently proliferate nephron progenitor cells.

[0007] Therefore, an objective of the present invention is to provide a culture medium for expanding nephron progenitor cells that enables efficient expansion of nephron progenitor cells, 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 culture medium according to one embodiment of the present invention is a culture medium for expanding nephron progenitor cells, and comprises at least one inhibitor selected from the group consisting of a TBK1 inhibitor, a CSF1R inhibitor, an FLT3 inhibitor, a DYRK1 inhibitor, and a STAT6 inhibitor. <2> the above <1> The medium described in 2. above may further contain at least one selected from the group consisting of FGF2, heparin, a ROCK inhibitor, a GSK3β inhibitor, leukemia inhibitory factor (LIF), an ALK inhibitor, a BMP inhibitor, and BMP7. <3> A culture medium according to one embodiment of the present invention is a medium for expanding nephron progenitor cells, and comprises at least one inhibitor selected from the group consisting of a TBK1 inhibitor, a CSF1R inhibitor, an FLT3 inhibitor, a DYRK1 inhibitor, and a STAT6 inhibitor, as well as FGF2, heparin, a ROCK inhibitor, a GSK3β inhibitor, leukemia inhibitory factor (LIF), an ALK inhibitor, a BMP inhibitor, and BMP7. <4> the above <2> or <3> In the medium described above, the ALK inhibitor may be A83-01. <5> the above <2> ~ <4> In the medium according to any one of the above items 1 to 4, the BMP inhibitor may be at least one selected from the group consisting of LDN193189, Dorsomorphin, Noggin, and DMH1. <6> the above <1> The medium described in 1. above may further contain a GSK3β inhibitor, a ROCK inhibitor, and FGF9. <7> the above <2> ~ <6> In any one of the above-described media, the ROCK inhibitor may be Y-27632. <8> the above <2> ~ <7> In any one of the above-described media, the GSK3β inhibitor may be CHIR99021. <9> the above <1> ~ <8> In the medium according to any one of the above items 1 to 4, the TBK1 inhibitor may be MRT67307 or BX795. <10> the above <1> ~ <9> In the medium according to any one of the above items, the CSF1R inhibitor may be GW2580. <11> the above <1> ~ <10> In the medium according to any one of the above items 1 to 4, the FLT3 inhibitor may be ASP2215. <12> the above <1> ~ <11> In the medium according to any one of the above items, the DYRK1 inhibitor may be ID-8. <13> the above <1> ~ <12> In the medium according to any one of the above items 1 to 4, the STAT6 inhibitor may be AS1517499. <14> The method for expanding nephron progenitor cells according to one embodiment of the present invention comprises the steps of: <1> ~ <13> The medium described in any one of the above is used. <15> A method for producing kidney organoids according to one embodiment of the present invention comprises the steps of: <14> 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 efficient expansion of nephron progenitor cells, 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] The figure shows the number of mNPC cells cultured in mNPSR medium (medium containing CHIR99021, Y-27632, FGF-2, LIF, BMP7, heparin, LDN193189, and A83-01) supplemented with MRT67307 at a final concentration of 0 to 1000 nM. [Figure 2] The figure shows the number of mNPC cells cultured in mNPSR medium supplemented with BX795 at final concentrations of 0 to 16 nM. [Figure 3] The figure shows the number of mNPC cells cultured in mNPSR medium supplemented with GW2580 at final concentrations of 0 to 1000 nM. [Figure 4] The figure shows the number of mNPC cells cultured in mNPSR medium supplemented with ASP2215 at final concentrations of 0 to 3.2 nM. [Figure 5] The figure shows the number of mNPC cells cultured in mNPSR medium supplemented with ID-8 at final concentrations of 0 to 30,000 nM. [Figure 6] The figure shows the number of mNPC cells cultured in mNPSR medium supplemented with AS1517499 at final concentrations of 0 to 200 nM. [Figure 7] The figure shows the cell number at each passage number of mNPCs subcultured in mNPSR medium supplemented with DMSO (0.1%) or MRT67307 at a final concentration of 30 nM. MRT67307 was dissolved in DMSO and added to the mNPSR medium. [Figure 8] The cumulative cell number at each passage number of mNPCs subcultured in mNPSR medium supplemented with DMSO (0.1%) or MRT67307 at a final concentration of 30 nM is shown. MRT67307 was dissolved in DMSO and added to the mNPSR medium. [Figure 9] The figure shows the cell number at each passage number of mNPCs subcultured in mNPSR medium supplemented with DMSO (0.1%) or BX795 at a final concentration of 4 nM. BX795 was dissolved in DMSO and added to the mNPSR medium. [Figure 10] The cumulative cell number at each passage number of mNPCs subcultured in mNPSR medium supplemented with DMSO (0.1%) or BX795 at a final concentration of 4 nM is shown. BX795 was dissolved in DMSO and added to the mNPSR medium. [Figure 11] Fluorescence microscopy images showing the results of lectin staining and immunostaining analysis of PODXL (PODOCALYXIN; glomerulus), LTL (Lotus Tetragonolobus Lectin; proximal tubule), and CDH1 (CADHERIN1; distal tubule) in kidney organoids formed from mNPCs after four passages in mNPSR medium supplemented with DMSO (0.1%) or MRT67307 at a final concentration of 30 nM. [Figure 12] These are fluorescence microscopy images showing the results of lectin staining and immunostaining analysis of PODXL (PODOCALYXIN; glomerulus), LTL (Lotus Tetragonolobus Lectin; proximal tubule), and CDH1 (CADHERIN1; distal tubule) in kidney organoids formed from mNPCs after four passages in mNPSR medium supplemented with DMSO (0.1%) or BX795 at a final concentration of 4 nM. [Figure 13]This figure shows the cell count when mNPCs derived from ADPKD (autosomal dominant polycystic kidney disease) model mice were cultured in mNPSR medium supplemented with DMSO (0.1%) or MRT67307 at a final concentration of 30 nM. MRT67307 was dissolved in DMSO and added to the mNPSR medium. [Figure 14] The figure shows the cell counts when mNPCs derived from Alport syndrome model mice were cultured in mNPSR medium supplemented with DMSO (0.1%), AS1517499 (final concentration 100 nM), or ID-8 (final concentration 500 nM). AS1517499 and ID-8 were dissolved in DMSO and added to the mNPSR medium. [Figure 15] 10 shows fluorescence microscopy images showing the results of immunostaining analysis of SIX2 in kidneys removed from mouse fetuses and cultured in FBS medium supplemented with DMSO (0.1%) or MRT67307 at a final concentration of 100 nM. [Figure 16] This figure shows the cell number at each passage number of hiPSC-NPCs derived from the 4A6 lineage, which were subcultured in hNPSR medium supplemented with DMSO (0.1%) or MRT67307 at a final concentration of 30 nM. MRT67307 was dissolved in DMSO and added to the hNPSR medium. [Figure 17] This figure shows the cumulative cell number at each passage number of hiPSC-NPCs derived from the 4A6 lineage, which were subcultured in hNPSR medium supplemented with DMSO (0.1%) or MRT67307 at a final concentration of 30 nM. MRT67307 was dissolved in DMSO and added to the hNPSR medium. [Figure 18] The figure shows the number of hiPSC-NPCs derived from the ASe16 line (a disease-specific iPS cell line derived from a patient with Alport syndrome) at each passage number, which were subcultured in hNPSR medium supplemented with DMSO (0.1%) or MRT67307 at a final concentration of 30 nM. MRT67307 was dissolved in DMSO and added to the hNPSR medium. [Figure 19] This figure shows the cumulative cell number at each passage number of hiPSC-NPCs derived from the ASe16 line, which were subcultured in hNPSR medium supplemented with DMSO (0.1%) or MRT67307 at a final concentration of 30 nM. MRT67307 was dissolved in DMSO and added to the hNPSR medium. [Figure 20] The figure shows the cell counts at each passage number of hiPSC-NPCs subcultured in CFY medium (containing CHIR99021, FGF-9, and Y-27632) supplemented with DMSO (0.1%) or AS1517499 at a final concentration of 100 nM. AS1517499 was dissolved in DMSO and added to the CFY medium. [Figure 21] Fluorescence microscopy images showing the results of lectin staining and immunostaining analysis of PODXL (PODOCALYXIN; glomerulus), LTL (Lotus Tetragonolobus Lectin; proximal tubule), and CDH1 (CADHERIN1; distal tubule) in kidney organoids formed from hiPSC-NPCs after one passage in CFY medium supplemented with AS1517499 at a final concentration of 100 nM (Scale bar: 100 μm). [Figure 22] The figure shows the cell counts at each passage number of c-MET(+)-hiPSC-NPCs subcultured in CFY medium supplemented with DMSO (0.1%) or AS1517499 at a final concentration of 100 nM. AS1517499 was dissolved in DMSO and added to CFY medium. [Figure 23] The cumulative cell number at each passage number of c-MET(+)-hiPSC-NPCs subcultured in CFY medium supplemented with DMSO (0.1%) or AS1517499 at a final concentration of 100 nM is shown. AS1517499 was dissolved in DMSO and added to CFY medium. [Figure 24] Fluorescence microscopy images showing the results of lectin staining and immunostaining analysis of PODXL (podocalyxin; glomerulus), LTL (lotus tetragonolobus lectin; proximal tubule), and CDH1 (cadherin1; distal tubule) in kidney organoids formed from c-MET(+)-hiPSC-NPCs after one passage in CFY medium supplemented with AS1517499 at a final concentration of 100 nM (Scale bar: 100 μm). [Figure 25]The figure shows the cell counts at each passage number of hiPSC-NPCs subcultured in CFY medium supplemented with DMSO (0.1%), MRT67307 (final concentration 30 nM), or TG101348 (final concentration 3 nM). MRT67307 and TG101348 were dissolved in DMSO and added to CFY medium. [Figure 26] Fluorescence microscopy images showing the results of lectin staining and immunostaining analysis of PODXL (PODOCALYXIN; glomerulus), LTL (Lotus Tetragonolobus Lectin; proximal tubule), and CDH1 (CADHERIN1; distal tubule) in kidney organoids formed from hiPSC-NPCs after one passage in CFY medium supplemented with MRT67307 at a final concentration of 30 nM (Scale bar: 100 μm). [Figure 27] Fluorescence microscopy images showing the results of lectin staining and immunostaining analysis of PODXL (PODOCALYXIN; glomerulus), LTL (Lotus Tetragonolobus Lectin; proximal tubule), and CDH1 (CADHERIN1; distal tubule) in kidney organoids formed from hiPSC-NPCs after one passage in CFY medium supplemented with TG101348 at a final concentration of 3 nM (Scale bar: 100 μm). [Figure 28] The figure shows the cell counts at each passage number of c-MET(+)-hiPSC-NPCs subcultured in CFY medium supplemented with DMSO (0.1%), MRT67307 (final concentration 30 nM), or TG101348 (final concentration 3 nM). MRT67307 and TG101348 were dissolved in DMSO and added to CFY medium. [Figure 29] The cumulative cell number at each passage number of c-MET(+)-hiPSC-NPCs was measured in CFY medium supplemented with DMSO (0.1%), MRT67307 (final concentration 30 nM), or TG101348 (final concentration 3 nM). AS1517499 was dissolved in DMSO and added to CFY medium. [Figure 30]Fluorescence microscopy images showing the results of lectin staining and immunostaining analysis of PODXL (podocalyxin; glomerulus), LTL (lotus tetragonolobus lectin; proximal tubule), and CDH1 (cadherin1; distal tubule) in kidney organoids formed from c-MET(+)-hiPSC-NPCs after two passages in CFY medium supplemented with MRT67307 at a final concentration of 30 nM (Scale bar: 100 μm). DETAILED DESCRIPTION OF THE INVENTION

[0011] <Culture medium for expanding nephron progenitor cells> A first aspect of the present invention is a culture medium for expanding nephron progenitor cells. In one embodiment, the culture medium of this embodiment contains at least one selected from the group consisting of a TBK1 inhibitor, a CSF1R inhibitor, a FLT3 inhibitor, a DYRK1 inhibitor, and a STAT6 inhibitor. In another embodiment, the culture medium of this embodiment contains at least one selected from the group consisting of a TBK1 inhibitor, a CSF1R inhibitor, a FLT3 inhibitor, a DYRK1 inhibitor, and a STAT6 inhibitor, as well as at least one selected from the group consisting of FGF2, heparin, a ROCK inhibitor, a GSK3β inhibitor, a leukemia inhibitory factor (LIF), an ALK inhibitor, a BMP inhibitor, and BMP7. In another embodiment, the culture medium of this embodiment contains at least one selected from the group consisting of a TBK1 inhibitor, a CSF1R inhibitor, a FLT3 inhibitor, a DYRK1 inhibitor, and a STAT6 inhibitor, as well as FGF2, heparin, a ROCK inhibitor, a GSK3β inhibitor, a leukemia inhibitory factor (LIF), an ALK inhibitor, a BMP inhibitor, and BMP7.

[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] Nephron progenitor cells may be isolated from the metanephric mesenchyme of a living organism, or may be differentiated from pluripotent stem cells (ES cells, iPS cells, etc.).

[0014] 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.

[0015] 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 MET or AGTR2 can be used as an indicator (WO 2020 / 022261).

[0016] 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.

[0017] (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.

[0018] (Culture medium) The medium of this embodiment may be a medium obtained by adding at least one selected from the group consisting of a TBK1 inhibitor, a CSF1R inhibitor, a FLT3 inhibitor, a DYRK1 inhibitor, and a STAT6 inhibitor to a basal medium used for animal culture. Alternatively, the medium may be a medium obtained by adding at least one selected from the group consisting of a TBK1 inhibitor, a CSF1R inhibitor, a FLT3 inhibitor, a DYRK1 inhibitor, and a STAT6 inhibitor, and at least one selected from the group consisting of FGF2, heparin, a ROCK inhibitor, a GSK3β inhibitor, a leukemia inhibitory factor (LIF), an ALK inhibitor, a BMP inhibitor, and BMP7 to a basal medium used for animal culture. Alternatively, the medium may be a medium obtained by adding at least one selected from the group consisting of a TBK1 inhibitor, a CSF1R inhibitor, a FLT3 inhibitor, a DYRK1 inhibitor, and a STAT6 inhibitor, and at least one selected from the group consisting of FGF2, heparin, a ROCK inhibitor, a GSK3β inhibitor, a leukemia inhibitory factor (LIF), an ALK inhibitor, a BMP inhibitor, and BMP7 to a basal medium used for animal culture.

[0019] <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, the medium 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.

[0020] 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.

[0021] The medium of this embodiment contains at least one inhibitor selected from the group consisting of a TBK1 inhibitor, a CSF1R inhibitor, an FLT3 inhibitor, a DYRK1 inhibitor, and a STAT6 inhibitor.

[0022] <TBK1 inhibitor> The TBK1 inhibitor contained in the medium of this embodiment is a substance that inhibits the activity of TANK binding kinase 1 (TBK1). The TBK1 inhibitor is not particularly limited as long as it can inhibit the activity of TANK binding kinase 1. Examples of TBK1 inhibitors include, but are not limited to, MRT67307 (CAS No. 1190378-57-4), BX795 (CAS No. 702675-74-9), TBK1 / IKKε-IN-1 (CAS No. 2058264-32-5), TBK1 / IKKε-IN-2 (CAS No. 1292310-49-6), TBK1 / IKKε-IN-5 (CAS No. 1893397-65-3), GSK8612 (CAS No. 2361659-62-1), Amlexanox (CAS No. 68302-57-8), BAY-985 (CAS No. 2409479-29-2), and GSK319347A (CAS No. 862812-98-4). Among these, the TBK1 inhibitors are preferably MRT67307 and BX 795. One TBK1 inhibitor may be used alone, or two or more TBK1 inhibitors may be used in combination.

[0023] TBK1 inhibitors are not limited to the above-mentioned low molecular weight compounds, but may also be antisense nucleic acids against TBK1, RNA interference-inducing nucleic acids (e.g., siRNA), dominant negative mutants, and expression vectors thereof.

[0024] The concentration of the TBK1 inhibitor in the medium of this embodiment can be appropriately selected depending on the type of TBK1 inhibitor. The TBK1 inhibitor is preferably used at a concentration near IC50, for example. The TBK1 inhibitor can be used at a concentration of, for example, 0.5 nM or more, 1 nM or more, 2 nM or more, 3 nM or more, 4 nM or more, 5 nM or more, 6 nM or more, 7 nM or more, 8 nM or more, 9 nM or more, or 10 nM or more. The upper limit of the TBK1 inhibitor concentration can be, for example, 1000 nM or less, 500 nM or less, 200 nM or less, 100 nM or less, 50 nM or less, or 35 nM or less. When the TBK1 inhibitor is MRT67307, the concentration of MRT67307 in the medium may be, for example, 0.5 to 1000 nM, 5 to 500 nM, 5 to 200 nM, 5 to 100 nM, 5 to 50 nM, 10 to 35 nM, or 20 to 35 nM. When the TBK1 inhibitor is BX795, the concentration of BX795 in the medium may be, for example, 0.5 to 16 nM, 0.5 to 8 nM, 1 to 8 nM, or 2 to 8 nM.

[0025] <CSF1R inhibitor> The CSF1R inhibitor included in this embodiment is a substance that inhibits the activity of colony-stimulating factor 1 receptor (CSF1R). The CSF1R inhibitor is not particularly limited, as long as it can inhibit the activity of colony-stimulating factor 1 receptor. Examples of CSF1R inhibitors include, but are not limited to, GW2580 (CAS No. 870483-87-7), CSF1R-IN-1 (CAS No. 2095849-04-8), AZD7507 (CAS No. 1041852-85-0), PRN1371 (CAS No. 1802929-43-6), cFMS Receptor Inhibitor II (CAS No. 959860-85-6), PLX-3397 hydrochloride (CAS No. 2040295-03-0), Ki20227 (CAS No. 623142-96-1), BLZ945 (CAS No. 953769-46-5), and PLX5622 (CAS No. 1303420-67-8). Among these, GW2580 is a preferred CSF1R inhibitor. One CSF1R inhibitor may be used alone, or two or more may be used in combination.

[0026] CSF1R inhibitors are not limited to the above-mentioned small molecule compounds, but may also be antisense nucleic acids against CSF1R, RNA interference-inducing nucleic acids (e.g., siRNAs), dominant-negative mutants, and expression vectors thereof.

[0027] The concentration of the CSF1R inhibitor in the medium of this embodiment can be selected appropriately depending on the type of CSF1R inhibitor. The CSF1R inhibitor is preferably used at a concentration near IC50. The CSF1R inhibitor can be used at a concentration of, for example, 0.5 nM or more, 1 nM or more, 2 nM or more, 3 nM or more, 4 nM or more, 5 nM or more, 6 nM or more, 7 nM or more, 7.5 nM or more, 8 nM or more, 9 nM or more, 10 nM or more, 50 nM or more, or 100 nM or more. The upper limit of the concentration of the CSF1R inhibitor can be, for example, 5000 nM or less, 3000 nM or less, 2000 nM or less, or 1000 nM or less. When the CSF1R inhibitor is GW2580, the concentration of GW2580 in the medium may be, for example, 0.5 to 5000 nM, 7.5 to 3000 nM, 15 to 2000 nM, 30 to 1000 nM, 60 to 1000 nM, 120 to 1000 nM, 240 to 1000 nM, or 250 to 1000 nM.

[0028] <FLT3 inhibitors> The FLT3 inhibitor included in this embodiment is a substance that inhibits the activity of FMS-like tyrosine kinase 3 (FLT3). The FLT3 inhibitor is not particularly limited, as long as it can inhibit the activity of FMS-like tyrosine kinase 3. Examples of FLT3 inhibitors include, but are not limited to, ASP2215 (CAS No. 1254053-43-4), SKLB4771 (CAS No. 1370256-78-2), CCT241736 (CAS No. 1402709-93-6), and BPR1K871 (CAS No. 2443767-35-7). Of these, ASP2215 (Gilteritinib) is preferred as the FLT3 inhibitor. One FLT3 inhibitor may be used alone, or two or more may be used in combination.

[0029] FLT3 inhibitors are not limited to the above-mentioned low molecular weight compounds, but may also be antisense nucleic acids against FLT3, RNA interference-inducing nucleic acids (eg, siRNA), dominant negative mutants, and expression vectors thereof.

[0030] The concentration of the FLT3 inhibitor in the medium of this embodiment can be appropriately selected depending on the type of FLT3 inhibitor. The FLT3 inhibitor is preferably used at a concentration near IC50, for example. The FLT3 inhibitor can be used at a concentration of, for example, 0.1 nM or more, 0.2 nM or more, 0.3 nM or more, 0.4 nM or more, 0.5 nM or more, 0.6 nM or more, 0.7 nM or more, 0.8 nM or more, 0.9 M or more, 1.0 nM or more, or 1.5 nM or more. The upper limit of the concentration of the FLT3 inhibitor can be, for example, 1000 nM or less, 500 nM or less, 200 nM or less, 100 nM or less, 50 nM or less, 35 nM or less, 20 nM or less, 10 nM or less, or 5 nM or less. When the FLT3 inhibitor is ASP2215, the concentration of ASP2215 in the medium may be, for example, 0.1 to 10 nM, 0.2 to 10 nM, 0.4 to 10 nM, 0.8 to 10 nM, 0.8 to 5.0 nM, or 0.8 to 3.2 nM.

[0031] ≪DYRK1 inhibitor≫ The DYRK1 inhibitor contained in the medium of this embodiment is a substance that inhibits the activity of dual-specificity tyrosine phosphorylation-regulated kinase 1 (DYRK1). The DYRK1 inhibitor is not particularly limited, as long as it can inhibit the activity of dual-specificity tyrosine phosphorylation-regulated kinase 1. The DYRK1 inhibitor is preferably a substance that inhibits the activity of dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) (DYRK1A inhibitor). Examples of DYRK1 inhibitors include, but are not limited to, ID-8 (CAS No. 147591-46-6), INDY (CAS No. 1169755-45-6), Mirk-IN-1 (CAS No. 1386979-55-0), and EHT5372 (CAS No. 1425945-60-3). Among these, ID-8 is preferred as the DYRK1 inhibitor. One DYRK1 inhibitor may be used alone, or two or more may be used in combination.

[0032] DYRK1 inhibitors are not limited to the above-mentioned small molecule compounds, but may also be antisense nucleic acids against DYRK1 (DYRK1A, DYRK1B, etc.), RNA interference-inducing nucleic acids (e.g., siRNA), dominant-negative mutants, and expression vectors thereof.

[0033] The concentration of the DYRK1 inhibitor in the medium of this embodiment can be appropriately selected depending on the type of DYRK1 inhibitor. The DYRK1 inhibitor is preferably used at a concentration near IC50, for example. The DYRK1 inhibitor can be used at a concentration of, for example, 0.5 nM or more, 1 nM or more, 2 nM or more, 3 nM or more, 4 nM or more, 5 nM or more, 6 nM or more, 7 nM or more, 8 nM or more, 9 nM or more, or 10 nM or more. The upper limit of the concentration of the DYRK1 inhibitor can be, for example, 30,000 nM or less, 10,000 nM or less, 1,000 nM or less, or 500 nM or less. When the DYRK1 inhibitor is ID-8, the concentration of ID-8 in the medium is, for example, 0.5 to 30,000 nM, 0.5 to 10,000 nM, 0.5 to 3,000 nM, 0.5 to 1,000 nM, 100 to 1,000 nM, or 250 to 1,000 nM.

[0034] STAT6 inhibitors The STAT6 inhibitor contained in the medium of this embodiment is a substance that inhibits the activity of signal transducer and activator of transcription 6 (STAT6). The STAT6 inhibitor is not particularly limited as long as it can inhibit the activity of signal transducer and activator of transcription 6. Examples of STAT6 inhibitors include, but are not limited to, AS1517499 (CAS No. 919486-40-1). AS1517499 is preferred as the STAT6 inhibitor. One type of STAT6 inhibitor may be used alone, or two or more types may be used in combination.

[0035] STAT6 inhibitors are not limited to the above-mentioned low molecular weight compounds, but may also be antisense nucleic acids against STAT6, RNA interference-inducing nucleic acids (eg, siRNA), dominant negative mutants, and expression vectors thereof.

[0036] The concentration of the STAT6 inhibitor in the medium of this embodiment can be appropriately selected depending on the type of STAT6 inhibitor. The STAT6 inhibitor is preferably used at a concentration near IC50. The STAT6 inhibitor can be used at a concentration of, for example, 1 nM or more, 2 nM or more, 3 nM or more, 4 nM or more, 5 nM or more, 6.25 nM or more, 7 nM or more, 8 nM or more, 9 nM or more, 10 nM or more, 12.5 nM or more, or 25 nM or more. The upper limit of the concentration of the STAT6 inhibitor can be, for example, 1000 nM or less, 500 nM or less, or 200 nM or less. When the STAT6 inhibitor is AS1517499, the concentration of AS1517499 in the medium may be, for example, 1 to 1000 nM, 6.25 to 1000 nM, 12.5 to 1000 nM, 25 to 1000 nM, 25 to 500 nM, or 25 to 200 nM.

[0037] <FGF2> The medium of this embodiment preferably contains FGF2. Inclusion of FGF2 in addition to at least one inhibitor selected from the group consisting of a TBK1 inhibitor, a CSF1R inhibitor, a FLT3 inhibitor, a DYRK1 inhibitor, and a STAT6 inhibitor improves the proliferation of nephron progenitor cells. Fibroblast growth factor (FGF) 2 is also known as basic FGF (bFGF). The organism from which FGF2 is derived is not particularly limited. For example, human FGF2 can be used. Examples of human FGF2 (NCBI Gene ID: 2247) include a protein having the amino acid sequence of NCBI Accession Number: NP_001348594.1. FGF2 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 FGF2 may be used, or a protein purified from cells or produced by genetic recombination may be used.

[0038] The concentration of FGF2 in the medium is, for example, 1 to 1000 ng / mL, preferably 10 to 500 ng / mL, more preferably 20 to 400 ng / mL, and even more preferably 50 to 350 ng / mL.

[0039] <Heparin> The medium of this embodiment preferably contains heparin. The inclusion of heparin in addition to at least one selected from the group consisting of a TBK1 inhibitor, a CSF1R inhibitor, an FLT3 inhibitor, a DYRK1 inhibitor, and a STAT6 inhibitor improves the proliferation of nephron progenitor cells. 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, with sodium salts being more preferred.

[0040] The concentration of heparin in the medium 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.

[0041] <ROCK inhibitors> The medium of this embodiment preferably contains a ROCK inhibitor. The inclusion of a ROCK inhibitor in addition to at least one inhibitor selected from the group consisting of a TBK1 inhibitor, a CSF1R inhibitor, an FLT3 inhibitor, a DYRK1 inhibitor, and a STAT6 inhibitor improves the proliferation of nephron progenitor cells. The ROCK inhibitor 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)), and 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 (eg, 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).

[0042] 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 the IC50, for example. When the ROCK inhibitor is Y-27632, the concentration of Y-27632 in the medium is, for example, 0.1 to 100 μM, preferably 1 to 75 μM, and more preferably 5 to 50 μM.

[0043] <GSK3β inhibitor> The medium of this embodiment preferably contains a GSK3β inhibitor. The inclusion of a GSK3β inhibitor in addition to at least one inhibitor selected from the group consisting of a TBK1 inhibitor, a CSF1R inhibitor, an FLT3 inhibitor, a DYRK1 inhibitor, and a STAT6 inhibitor improves the proliferation of nephron progenitor cells. The GSK3β inhibitor is a substance that inhibits the function of GSK (Glycogen Synthase Kinase) 3β, for example, its kinase activity (e.g., its 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.

[0044] One GSK3β inhibitor may be used alone, or two or more may be used in combination. A preferred GSK3β inhibitor is CHIR99021. The concentration of the GSK3β inhibitor in the medium can be selected appropriately depending on the type of GSK3β inhibitor. The GSK3β inhibitor is preferably used at a concentration near the IC50, for example. When the GSK3β inhibitor is CHIR99021, the concentration of CHIR99021 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.

[0045] <LIF> The medium of this embodiment preferably contains leukemia inhibitory factor (LIF). The inclusion of LIF in addition to at least one inhibitor selected from the group consisting of a TBK1 inhibitor, a CSF1R inhibitor, a FLT3 inhibitor, a DYRK1 inhibitor, and a STAT6 inhibitor improves the proliferation of nephron progenitor cells. The organism from which LIF is derived is not particularly limited. Examples of LIF that can be used include LIF from humans (Japanese Patent Publication No. 1-502985), mice (Japanese Patent Publication No. 1-502985), sheep (Japanese Patent Publication No. 4-502554), pigs (Japanese Patent Publication No. 4-502554), and cattle (Japanese Patent Publication No. 8-154681). Among these, human or mouse LIF is preferred. Examples of human LIF (NCBI Gene ID: 3976) include proteins having the amino acid sequence of NCBI accession numbers NP_001244064.1 or NP_002300.1. Examples of mouse LIF (NCBI Gene ID: 16878) include proteins having the amino acid sequence of NCBI accession numbers NP_001034626.1 or NP_032527.1. LIF can be selected appropriately depending on the organism from which the nephron progenitor cells are derived. For example, mouse LIF is preferably used when the nephron progenitor cells are derived from mice. Human LIF is preferably used when the nephron progenitor cells are derived from humans. LIF may be a fragment or a functional variant thereof, as long as it has the activity of promoting nephron progenitor cell proliferation. Commercially available LIF may be used, or a protein purified from cells or produced by genetic recombination may be used.

[0046] The concentration of LIF in the medium is, for example, 1 to 1000 ng / mL, preferably 10 to 500 ng / mL, more preferably 50 to 250 ng / mL, and for example, 10 to 5000 units / mL, preferably 100 to 3000 units / mL, more preferably 500 to 2000 units / mL.

[0047] <ALK inhibitors> The medium of this embodiment preferably contains an ALK inhibitor. By including an ALK inhibitor in addition to at least one inhibitor selected from the group consisting of a TBK1 inhibitor, a CSF1R inhibitor, an FLT3 inhibitor, a DYRK1 inhibitor, and a STAT6 inhibitor, the proliferation of nephron progenitor cells is improved. ALK inhibitors are substances that inhibit the function of the ALK (activin receptor-like kinase) family. Examples of ALK inhibitors include substances that inhibit the binding of TGFβ to the ALK family, or substances that inhibit the phosphorylation of SMAD by the ALK family. Examples of ALK inhibitors include inhibitors of ALK4, ALK5, or ALK7. Examples of ALK inhibitors include Lefty-1 (NCBI accession numbers: mouse: NP_034224.1, human: NP_066277.1), SB431542, SB202190 (RK Lindemann et al., Mol. Cancer, 2003, 2:20), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, and LY580276 (Lilly Research Examples of ALK inhibitors include, but are not limited to, A83-01 (3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide, WO2009146408), ALK5 inhibitor II (2-[3-[6-methylpyridin-2-yl]-1H-pyrazol-4-yl]-1,5-naphthyridine), TGFβRI kinase inhibitor VIII (6-[2-tert-butyl-5-[6-methyl-pyridin-2-yl]-1H-imidazol-4-yl]-quinoxaline), and derivatives thereof. The ALK inhibitor may be an antisense nucleic acid against the ALK family, an RNA interference-inducing nucleic acid (e.g., siRNA), a dominant-negative mutant, or an expression vector thereof.

[0048] One ALK inhibitor may be used alone, or two or more may be used in combination. A preferred ALK inhibitor is A83-01. The concentration of the ALK inhibitor in the medium can be selected appropriately depending on the type of ALK inhibitor. The ALK inhibitor is preferably used at a concentration near IC50, for example. When the ALK inhibitor is A83-01, the concentration of A83-01 in the medium is, for example, 0.01 to 1000 nM, preferably 0.1 to 500 nM, more preferably 1 to 100 nM, and particularly preferably 10 to 80 nM.

[0049] <BMP inhibitors> The medium of this embodiment contains a BMP inhibitor. By including a BMP inhibitor in addition to at least one inhibitor selected from the group consisting of a TBK1 inhibitor, a CSF1R inhibitor, an FLT3 inhibitor, a DYRK1 inhibitor, and a STAT6 inhibitor, the proliferation of nephron progenitor cells is improved. The BMP inhibitor is a substance that inhibits BMP (Bone Morphogenetic Protein) signaling. The BMP inhibitor may be, for example, a substance that inhibits the kinase activity of ALK2 or ALK3, which are BMP receptors. Examples of BMP inhibitors include protein inhibitors such as Chordin, Noggin, and Follistatin; dorsomorphin (6-[4-(2-piperidin-1-yl-ethoxy)phenyl]-3-pyridin-4-yl-pyrazolo[1,5-a]pyrimidine) (PB Yu et al. (2007), Circulation, 116:II_60; PB Yu et al. (2008), Nat. Chem. Biol., 4:33-41; J. Hao et al. (2008), PLoS ONE, 3(8):e2904), and LDN193189 (4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline), and DMH1 (dorsomorphin homolog Examples of BMP inhibitors include, but are not limited to, BMP inhibitors such as 1,4-[6-(4-isopropoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline, and derivatives thereof. The BMP inhibitor may be an antisense nucleic acid against BMP, an RNA interference-inducing nucleic acid (e.g., siRNA), a dominant-negative mutant, or an expression vector thereof.

[0050] One BMP inhibitor may be used alone, or two or more may be used in combination. Preferred BMP inhibitors include LDN193189, Dorsomorphin, Noggin, and DMH1, with LDN193189 being more preferred. The concentration of the BMP inhibitor in the medium can be appropriately selected depending on the type of ALK inhibitor. The BMP inhibitor is preferably used at a concentration near IC50, for example. When the BMP inhibitor is LDN193189, the concentration of LDN193189 in the medium is, for example, 0.01 to 1000 nM, preferably 0.1 to 500 nM, more preferably 0.5 to 100 nM, and particularly preferably 1 to 50 nM.

[0051] BMP7 The medium of this embodiment preferably contains BMP7. By including BMP7 in addition to at least one inhibitor selected from the group consisting of a TBK1 inhibitor, a CSF1R inhibitor, a FLT3 inhibitor, a DYRK1 inhibitor, and a STAT6 inhibitor, nephron progenitor cell proliferation is improved. The organism from which BMP7 is derived is not particularly limited. Examples of BMP7 include human BMP7. Examples of human BMP7 (NCBI Gene ID: 655) include a protein having the amino acid sequence of NCBI Accession Number: NP_001710.1. BMP7 may be a fragment or a functional variant thereof, as long as it has the activity of promoting nephron progenitor cell proliferation. Commercially available BMP7 may be used, or a protein purified from cells or produced by genetic recombination may be used.

[0052] The concentration of BMP7 in the medium is, for example, 0.1 to 500 ng / mL, preferably 1 to 300 ng / mL, and more preferably 10 to 100 ng / mL.

[0053] <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.

[0054] 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.

[0055] The medium of this embodiment may contain components other than those described above, such as a JAK inhibitor, as long as the effects of the present invention are not impaired.

[0056] 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 GSK3β inhibitor, a ROCK inhibitor, and FGF9, 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).

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] In one embodiment, the medium of this embodiment comprises at least one selected from the group consisting of a TBK1 inhibitor, a CSF1R inhibitor, a FLT3 inhibitor, a DYRK1 inhibitor, and a STAT6 inhibitor, and a JAK inhibitor, FGF2, heparin, a ROCK inhibitor, a GSK3β inhibitor, a leukemia inhibitory factor (LIF), an ALK inhibitor, a BMP inhibitor, and BMP7. An example of the medium includes a TBK1 inhibitor, FGF2, heparin, a ROCK inhibitor, a GSK3β inhibitor, a leukemia inhibitory factor (LIF), an ALK inhibitor, a BMP inhibitor, and BMP7. An example of the medium of this embodiment includes MRT67307, FGF2, heparin, Y-27632, LIF, CHIR99021, LDN193189, and A83-01.

[0065] In one embodiment, the medium of this embodiment contains at least one inhibitor selected from the group consisting of a TBK1 inhibitor, a CSF1R inhibitor, an FLT3 inhibitor, a DYRK1 inhibitor, and a STAT6 inhibitor, as well as a GSK3β inhibitor, a ROCK inhibitor, and FGF9. The medium of this embodiment may further contain a JAK inhibitor. For example, the medium of this embodiment may contain CHIR99021, Y-27632, and FGF9.

[0066] The medium of this embodiment contains at least one inhibitor selected from the group consisting of a TBK1 inhibitor, a CSF1R inhibitor, an FLT3 inhibitor, a DYRK1 inhibitor, and a STAT6 inhibitor, thereby enabling the proliferation of nephron progenitor cells effectively while maintaining the differentiation ability of the nephron progenitor cells.

[0067] <Method for expanding nephron progenitor cells> A second aspect of the present invention is a method for expanding nephron progenitor cells using the medium of the first aspect.

[0068] 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%.

[0069] The culture period is not particularly limited and can be any period. In the method of this embodiment, the use of the medium of the first embodiment 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 embodiment allows for continued culture of nephron progenitor cells while maintaining their properties. Passage 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.

[0070] The method of this embodiment uses the medium of the first embodiment to culture nephron progenitor cells, allowing for efficient proliferation of nephron progenitor cells. 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. They can also be used to produce renal organoids, as described below.

[0071] <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 nephron progenitor cells (expansion step) by the method of the second aspect, and differentiating the expanded nephron progenitor cells into kidney organoids (differentiation step).

[0072] (Expansion culture process) The expansion culture step is carried out by the method of the second aspect, which allows nephron progenitor cells to be efficiently proliferated to a desired number.

[0073] (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 may 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 may 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, in addition to the GSK-3β inhibitor, FGF9, FGF2, and the like. Examples of the basal medium, GSK-3β inhibitor, and FGF2 are the same as those described above. A preferred basal medium is KSR. For example, the medium reported in Stem Cell, 19, 516-519 (2016) can be used. A preferred GSK3β inhibitor is CHIR99021. A preferred FGF2 is human FGF2. A preferred FGF9 is human FGF9. An example of human FGF9 is a protein having the amino acid sequence of NCBI accession number: NP_002001.1. FGF9 encompasses 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 a protein purified from cells or a protein produced by genetic recombination may be used.

[0074] 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%.

[0075] 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.

[0076] <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.

[0077] 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 transplanting them into the subject's body (e.g., into the peritoneal cavity).

[0078] 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]

[0079] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0080] Example 1 Materials and Methods <Animal experiments> All animal experiments were conducted with the approval of the Kyoto University Animal Experiment Committee. tm3(EGFP / cre / ERT2)Amc B6.Col4a5 / J mice were purchased from Jackson Laboratory Inc. (A. Kobayashi, et al., Cell Stem Cell. 3 (2008) 169-181). tm1Yseg Pkd1 / J mice were purchased from Jackson Laboratory Inc. (M.N. Rheault, et al., J Am Soc Nephrol. 15 (2004) 1466-1474). - / - The (del2-6) mice were kindly provided by Fujita Health University (S Muto, et al., Hum Mol Genet. 11 (2002) 1731-1742.). They were kept under specific pathogen-free (SPF) conditions in the experimental animal facility of the Center for iPS Cell Research and Application, Kyoto University. Mice were provided with food and water ad libitum.

[0081] <Cell culture> Experiments using human iPS cells (hiPSCs) were approved by the Kyoto University Graduate School of Medicine Ethics Committee, in accordance with the Institutional Review Board, and informed consent was obtained from the hiPSC donors. hiPSCs were maintained in feeder-free culture on Synthemax (Corning)-coated cell culture plates in Stem Fit AK02N medium (Takara Bio Inc.). Cells were passaged every 5 days using 0.5 mM EDTA / PBS (Thermo Fisher Scientific) and routinely tested for mycoplasma contamination. Mouse nephron progenitor cell (mNPC) recovery and maintenance were performed as previously described (Z. Li, et al., Cell Stem Cell. 19 (2016) 516-529; Z. Li, et al., Kidney Organog Methods Protoc, Humana Press, New York, 2019: pp. 151-159).

[0082] <Cell proliferation> Cell masses of mNPCs (Z. Li, et al., Cell Stem Cell. 19 (2016) 516-529) expanded in mNPSR 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 tube was incubated at 37°C and 5% CO2 for 10 minutes. After 10 minutes, the tube was diluted with 470 μL of 10% FBS and the cell count was measured using a TC20 (Bio-Rad) tube.

[0083] 2.0x10 per well in a low-attachment 96-well plate (Nunc) 4mNPCs were seeded on 50 μL of mNPSR. TBK1 inhibitors (MRT67307, BX795), CSF1R inhibitor (GW2580), FLT3 inhibitor (ASP2215), DYRK1 inhibitor (ID-8), or STAT6 inhibitor (AS1517499) were added to 50 μL of mNPSR at the following concentration gradients centered around the IC50 (MRT67307: 5 nM, 10 nM, 20 nM, 25 nM, 30 nM, 35 nM, 50 nM, 100 nM, 200 nM, 500 nM, 1000 nM; BX795: 0.5 nM, 1 nM, 2nM, 4nM, 8nM, 16nM; GW2580: 7.5nM, 15nM, 30nM, 60nM, 120nM, 240nM, 250nM, 500nM, 1000nM; ASP2215: 0.1nM, 0.2nM, 0.4nM, 0.8nM, 1.6nM, 3.2nM; ID-8: 250nM, 500nM, 1000nM, 3000nM, 10000nM, 30000nM; AS1517499: 6.25nM, 12.5nM, 25nM, 50nM, 100nM, 200nM). Cells suspended in mNPSR medium alone or mNPSR medium supplemented with TBK1 inhibitors (MRT67307, BX795), CSF1R inhibitor (GW2580), FLT3 inhibitor (ASP2215), DYRK1 inhibitor (ID-8), or STAT6 inhibitor (AS1517499) were seeded in a 96-well plate, centrifuged at 300g for 3 minutes, and then statically cultured in an incubator at 37°C and 5% CO2. Cell clusters formed 6 hours after the start of static culture.

[0084] Forty-eight hours after the start of static culture, 100 μL of mNPSR medium supplemented with TBK1 inhibitors (MRT67307, BX795), CSF1R inhibitor (GW2580), FLT3 inhibitor (ASP2215), DYRK1 inhibitor (ID-8), or STAT6 inhibitor (AS1517499) at the same concentrations as when the cells were seeded was added to each well. Forty-eight hours after the addition of medium, the cell mass from each well was transferred to a 1.5 mL tube, the supernatant was completely removed, and 30 μL of Accumax (Innovative Cell Technologies, Inc.) was added. The cells were then incubated at 37°C in a 5% CO2 incubator for 10 minutes. After 10 minutes, the cells were suspended in 470 μL of 10% FBS and counted using a TC20 (Bio-Rad) analyzer.

[0085] The composition of the mNPSR medium used in the experiment is shown in Table 1. The mNPSR medium was prepared by adding the reagents shown in Table 1 to the basal medium shown in Table 1.

[0086] [Table 1]

[0087] <Subculture> mNPC cell aggregates cultured in mNPSR medium supplemented with 30 nM of a TBK1 inhibitor (MRT67307, Figures 7 and 8) or DMSO 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 tube was 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 number was measured using a TC20 (Bio-Rad). 2.0 x 10 cells were placed in each well of a low-attachment 96-well plate (Nunc). 4The mNPCs of the cells were suspended and seeded in mNPSR medium supplemented with 30 mM of a TBK1 inhibitor or DMSO. The low-adhesion 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 mNPSR medium supplemented with the TBK1 inhibitor or DMSO at the same concentration as when the cells were seeded was added to each well. 48 hours after the additional addition of the medium, the same operations as above were performed to measure the cell count and passage the cells. Using the same method, the cell count was measured and the cells were passaged for 4 nM of BX795 (Figures 9 and 10) as well.

[0088] <Preparation of kidney organoids> The cell clumps of the passaged mNPCs were placed on Transwell (Corning) in medium supplemented with 30 nM of MRT67307 or 4 nM of BX795 in mNPSR medium, and the surrounding medium was removed. Next, the Transwell with the cell clumps 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, it was fixed with 4% PFA at 4°C for 3 hours while still in the Transwell. After fixation, it was replaced with PBS and left standing at 4°C overnight for immunostaining.

[0089] <Cell culture ex vivo> The FBS medium used in the ex vivo experiment was prepared by adding 10% fetal bovine serum (FBS) and 500 U / ml of PS (Penicillin Strepromycin) to DMEM / F12 Glutamax medium.

[0090] Kidneys were collected from mouse embryos (E12.5), placed on Transwell (Corning), and the surrounding medium was removed. Next, the Transwell with the kidney was set in a culture dish previously filled with FBS medium supplemented with 100 nM of MRT67307. 24 to 72 hours after the start of culture, it was fixed with 4% PFA at 4°C for 3 hours while still on the Transwell. After fixation, it was replaced with PBS and left standing at 4°C overnight for immunostaining.

[0091] [Results] <Promoting effect of TBK1 inhibitor on mNPC proliferation> Cell proliferation assays of mNPC were performed using MRT67307 and BX795 as TBK1 inhibitors. Figure 1 shows the results of the cell proliferation assay in the medium supplemented with MRT67307 in mNPSR medium. From the literature information, the IC50 of MRT67307 is predicted to be around 19 nM. A significant cell proliferation promoting effect was confirmed for MRT67307 at a concentration near the IC50. Figure 2 shows the results of the cell proliferation assay in the medium supplemented with BX795 in mNPSR medium. From the literature information, the IC50 of BX795 is predicted to be around 2 nM. A significant cell proliferation promoting effect was confirmed for BX795 at a concentration near the IC50.

[0092] <Promoting effect of CSF1R inhibitor on mNPC proliferation> Cell proliferation assays of mNPC were performed using GW2580 as a CSF1R inhibitor. Figure 3 shows the results of the cell proliferation assay in the medium supplemented with GW2580 in mNPSR medium. A significant cell proliferation promoting effect was confirmed for GW2580 at 240 nM to 1000 nM.

[0093] <Promoting effect of FLT3 inhibitor on mNPC proliferation> Using ASP2215 as an FLT3 inhibitor, a cell proliferation assay of mNPC was performed. Figure 4 shows the results of the cell proliferation assay in the medium supplemented with ASP2215 in mNPSR medium. From the literature information, the IC50 of ASP2215 is predicted to be around 0.29 nM. A significant cell growth promoting effect was confirmed at around 3.2 nM of ASP2215. This was presumed to be a growth promoting effect due to FLT3 inhibition.

[0094] <Proliferation promoting effect of DYRK1 inhibitor on mNPC> Using ID-8 as a DYRK1 inhibitor, a cell proliferation assay of mNPC was performed. Figure 5 shows the results of the cell proliferation assay in the medium supplemented with ID-8 in mNPSR medium. A significant cell growth promoting effect was confirmed at around 500 nM of ID-8.

[0095] <Proliferation promoting effect of STAT6 inhibitor on mNPC> Using AS1517499 as a STAT6 inhibitor, a cell proliferation assay of mNPC was performed. Figure 6 shows the results of the cell proliferation assay in the medium supplemented with AS1517499 in mNPSR medium. From the literature information, the IC50 of AS1517499 is predicted to be around 21 nM. A significant cell growth promoting effect was confirmed at around 25 - 200 nM of AS1517499.

[0096] <Proliferation promoting effect of TBK1 inhibitor in subculture of mNPC> Using MRT67307 and BX795 as TBK1 inhibitors, subculture of mNPC was performed and the cell number was measured at each passage. [[ID=第十九]] [[ID=第二十]]

[0097] [[ID=第二十一]] Figure 7 shows the change in the number of cells when subcultured in a medium supplemented with 30 nM of MRT67307 in mNPSR medium. mNPCs subcultured in a medium supplemented with 30 nM of MRT67307 in mNPSR medium showed enhanced cell proliferation compared to mNPCs subcultured in a medium supplemented with DMSO in mNPSR medium. Also, by subculturing in a medium supplemented with 30 nM of MRT67307 in mNPSR medium, the cumulative cell number increased compared to when subcultured in a medium supplemented with DMSO in mNPSR medium (Figure 8). The cumulative cell number at P1 can be calculated as follows: Let the number of cells grown at P0 be a, the volume of the culture solution at P0 be b, the volume of the P0 culture solution used for subculture be c, and the number of cells obtained by growing at P1 be d. Then the cumulative cell number at P1 = a × [d / (a × c / b)] × b / c). The cumulative cell number in subsequent subcultures is calculated similarly.

[0098] Figure 9 shows the change in the number of cells when subcultured in a medium supplemented with 4 nM of BX795 in mNPSR medium. mNPCs subcultured in a medium supplemented with 4 nM of BX795 in mNPSR medium showed enhanced cell proliferation compared to mNPCs subcultured in a medium supplemented with DMSO in mNPSR medium. Also, by subculturing in a medium supplemented with 4 nM of BX795 in mNPSR medium, the cumulative cell number increased compared to when subcultured in a medium supplemented with DMSO in mNPSR medium (Figure 10).

[0099] <Effect of TBK1 inhibitor on kidney organoid formation> Kidney organoids were induced to differentiate from mNPCs that had been subcultured 4 times in a medium supplemented with 30 nM of MRT67307 or 4 nM of BX795 in mNPSR medium. As a result, it was confirmed that none of the TBK1 inhibitors affected the formation of kidney organoids (Figures 11 and 12).

[0100] <Proliferation assay using mNPCs derived from ADPKD model mice> As mNPCs, cell proliferation assays were performed in the same manner as above, except that mNPCs extracted from the fetal kidneys of ADPKD (autosomal dominant polycystic kidney disease) model mice were used. As the TBK1 inhibitor, 30 nM of MRT67307 was used. It was confirmed that the addition of MRT67307 also promoted the proliferation of mNPCs derived from ADPKD model mice (Figure 13).

[0101] <Proliferation assay using mNPCs derived from Alport syndrome model mice> As mNPCs, cell proliferation assays were performed in the same manner as above, except that mNPCs extracted from the fetal kidneys of Alport syndrome model mice were used. As the STAT6 inhibitor, 100 nM of AS1517489 was used, and as the DYRK1 inhibitor, 500 nM of ID-8 was used. It was confirmed that the addition of AS1517489 or ID-8 also promoted the proliferation of mNPCs derived from Alport syndrome model mice (Figure 14).

[0102] <Proliferation promoting effect of TBK1 inhibitor ex vivo> Kidneys were removed from fetal mice and placed on Transwell for culture. As the TBK1 inhibitor, 100 nM of MRT67307 was used. It was confirmed that the addition of MRT67307 also promoted the proliferation ex vivo (Figure 15).

[0103] (Example 2) [Materials and methods] <Cell proliferation> After inducing differentiation into human iPS cell-derived NPCs on a 24-well plate using the method described in International Publication No. 2018 / 216743 for the 4A6 strain (derived from 201B7), a healthy human-derived hiPS cell line, or the ASe16 strain, an Alport syndrome disease-specific iPS cell line, 100 μL of Accumax was added and left standing in an incubator at 37 °C with 5% CO2 for 30 minutes. After 30 minutes, it was suspended with 900 μL of 10% FBS...6 The cells were suspended in 100 μL of 2% FBS. 10 μL of human-HGFR / cMET antibody (R&D Systems, FAB 3582A) conjugated with the fluorescent substance APC was added to the 100 μL of 2% FBS suspension, and the mixture was left on ice for 30 minutes. After 30 minutes, the mixture was centrifuged at 200 g for 5 minutes, the supernatant was removed, washed with 1 mL of PBS, and centrifuged again at 200 g for 5 minutes, and the supernatant was removed. This procedure was repeated twice. The supernatant-removed cells were suspended in 2% FBS diluted 1:1000 with DAPI, passed through a 40 μm filter, and cMET-positive cells were extracted using flow cytometry. The extracted cells were diluted to 2.0 x 10 per well. 4 The cells were suspended in 50 μL of hNPSR medium alone or hNPSR medium supplemented with 30 nM MRT67307, 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.

[0104] 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.

[0105] The composition of the hNPSR medium used in the experiment is shown in Table 2. The hNPSR medium was prepared by adding the reagents shown in Table 2 to the basal medium shown in Table 2.

[0106] [Table 2]

[0107] <Subculture> Cell aggregates of human iPSC-derived NPCs (hiPSC-NPCs) cultured in hNPSR medium and medium supplemented with 30 nM MRT67307 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 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 iPSC-derived NPCs at 2.0 x 10 4 cells per well were suspended and seeded in a low-attachment 96-well plate (Nunc) with hNPSR medium alone and medium supplemented with 30 nM MRT67307. The low-attachment 96-well plate seeded with 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 hNPSR medium supplemented with the inhibitor at the same concentration as when the cells were seeded was added 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.

[0108] [Results] [Growth promotion effect of TBK1 inhibitor in subculture of hiPSC-NPC] Using MRT67307 as a TBK1 inhibitor, subculture of hiPSC-NPCs was performed, and the cell count was measured at each passage.

[0109] Figure 16 shows the change in the number of cells when subcultured in a medium supplemented with 30 nM of MRT67307 in hNPSR medium. The 4A6 strain-derived hiPSC-NPCs subcultured in a medium supplemented with 30 nM of MRT67307 in hNPSR medium showed enhanced cell proliferation compared to the 4A6 strain-derived hiPSC-NPCs subcultured in a medium supplemented with DMSO in hNPSR medium. Also, by subculturing in a medium supplemented with 30 nM of MRT67307 in hNPSR medium, the cumulative cell number increased compared to when subcultured in a medium supplemented with DMSO in hNPSR medium (Figure 17).

[0110] Figure 18 shows the change in the number of cells when subcultured in a medium supplemented with 30 nM of MRT67307 in hNPSR medium. The ASe16 strain-derived hiPSC-NPCs subcultured in a medium supplemented with 30 nM of MRT67307 in hNPSR medium showed enhanced cell proliferation compared to the ASe16 strain-derived hiPSC-NPCs subcultured in a medium supplemented with DMSO in hNPSR medium. Also, by subculturing in a medium supplemented with 30 nM of MRT67307 in hNPSR medium, the cumulative cell number increased compared to when subcultured in a medium supplemented with DMSO in hNPSR medium (Figure 19).

[0111] (Example 3) (Induction of Nephron Progenitor Cells (hiPSC-NPC) from Human iPS Cells) After inducing differentiation into human iPS cell-derived NPCs on a 24-well plate using the method described in International Publication No. 2018 / 216743 with the 4A6 strain (derived from 201B7), a healthy human-derived hiPS cell line, 100 μL of Accumax was added and 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 it was suspended at a ratio of 100 μL of 2% FBS per 1.0x10 6 cells.

[0112] (Purification of c-MET Positive Cells) To 100 μL of 2% FBS suspension of hiPSC-NPCs induced as described above, 10 μL of human-HGFR / c-MET antibody conjugated with the fluorescent substance APC (R&D Systems, FAB 3582A) was added and incubated on ice for 30 minutes. After 30 minutes, the cells were centrifuged at 200 g for 5 minutes, the supernatant was removed, washed with 1 mL of PBS, and centrifuged again at 200 g for 5 minutes, and the supernatant was removed. This procedure was repeated twice. After removing the supernatant, the cells were suspended in 2% FBS with a 1:1000 dilution of DAPI and passed through a 40 μm filter. c-MET-positive cells were then extracted using flow cytometry.

[0113] Herein, hiPSC-NPCs obtained by purifying c-MET-positive cells may be referred to as "c-MET(+)-hiPSC-NPCs."

[0114] <Expansion culture medium> The test medium for expansion culture was CFY medium supplemented with DMSO, a STAT6 inhibitor dissolved in DMSO (STAT6 inhibitor: AS1517499 (final concentration 100 nM)), a TBK1 inhibitor dissolved in DMSO (TBK1 inhibitor: MRT67307 (final concentration 30 nM)), and a JAK2 inhibitor dissolved in DMSO (JAK2 inhibitor: TG101348 (final concentration 3 nM)).

[0115] The composition of the CFY medium is shown in Table 3. The CFY medium was prepared by adding the reagents shown in Table 3 to the basal medium shown in Table 3.

[0116] [Table 3]

[0117] <Cell proliferation> hiPSC-NPCs were cultured at 2.0x10 per well. 4The 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 with 5% CO2. Cell clusters were formed 6 hours after the start of static culture.

[0118] 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.

[0119] <Subculture> Human iPS cell-derived NPC (hiPSC-NPC) cell aggregates cultured in the test medium 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 mixture was 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 number was measured using a TC20 (Bio-Rad). 2.0 x 10 cells were placed in each well of a low-attachment 96-well plate (Nunc). 4 hiPSC-NPCs from 100 cells were suspended in test medium and seeded. The seeded low-attachment 96-well plate was centrifuged at 300g for 3 minutes and then cultured statically in an incubator at 37°C and 5% CO2. 48 hours after the start of static culture, 100 μL of test medium was added to each well. 48 hours after the addition of medium, the same procedures as above were repeated to count the number of cells and passage them.

[0120] <Creation of kidney organoids> In a test medium, cell clusters of subcultured hiPSC-NPCs were placed on Transwell (Corning), and the surrounding medium was removed. Next, the Transwell with the cell clusters was set in a culture dish previously filled with 5% KSR (Knock-out serum replacement) (Thermo Fisher Scientific) supplemented with 200 ng / mL of FGF2 and 5 μM of CHIR99021. After 48 hours, the medium was changed to 5% KSR without the addition of growth factors and small molecules. Thereafter, every 48 hours, the medium was changed to 5% KSR without the addition of growth factors and small molecules, and on the 10th day of the start of differentiation culture, it was fixed with 4% PFA at 4°C for 3 hours while still in the Transwell. After fixation, it was replaced with PBS and left standing at 4°C overnight for immunostaining.

[0121] <Confirmation of OSR1 and SIX2 expression> hiPS cells were established that had an OSR1-GFP reporter gene with a GFP coding region introduced into the OSR1 locus and a SIX2-tdTomato reporter gene with a tdTomato coding region linked to the SIX2 gene. The expression of OSR1 and SIX2 was confirmed by performing a culture test using the hiPS cells and detecting each fluorescence of GFP and tdTomato with a fluorescence microscope.

[0122] <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.).

[0123] [Results] <Effect of STAT6 inhibitor on promoting the proliferation of hiPSC-NPCs> The STAT6 inhibitor (AS1517499, final concentration 100 nM) was added to the CFY medium, and hiPSC-NPCs were expanded in culture, and the cell numbers were measured (n = 3, t-test ** p<0.01). The cell numbers at each passage 1, 2, and 3 were compared with the case where only DMSO was added to the CFY medium.

[0124] The results are shown in Fig. 20. Fig. 20 shows the cell numbers in each subculture. As shown in Fig. 20, the growth of hiPSC-NPCs was promoted by the CFY medium supplemented with the STAT6 inhibitor.

[0125] <Generation of kidney organoids from hiPSC-NPCs subcultured in CFY medium supplemented with the STAT6 inhibitor> Subculture of hiPSC-NPCs was performed using the medium supplemented with the STAT6 inhibitor (AS1517499, final concentration 100 nM) in the CFY medium, and an attempt was made to generate kidney organoids from the cells at passage 1.

[0126] The results are shown in Fig. 21 ((PODXL; PODOCALYXIN; glomerulus, LTL; Lotus Tetragonolobus Lectin; proximal renal tubule, CDH1; CADHERIN1; distal renal tubule) (Scale bar; 100 μm)). Kidney organoids were formed from the cells at passage 1 in the medium supplemented with 100 nM of AS1517499 in the CFY medium. From this result, it was confirmed that hiPSC-NPCs can be subcultured while maintaining the ability to form kidney organoids using the medium supplemented with the STAT6 inhibitor in the CFY medium.

[0127] <Proliferation-promoting effect of the STAT6 inhibitor on c-MET(+)-hiPSC-NPCs> The STAT6 inhibitor (AS1517499, final concentration 100 nM) was added to the CFY medium, and c-MET(+)-hiPSC-NPCs were expanded in culture, the cell numbers were measured, and the cumulative cell numbers were calculated. The cell numbers and cumulative cell numbers at each passage 1, 2, and 3 were compared with the case where only DMSO was added to the CFY medium.

[0128] The results are shown in Figs. 22 and 23. Fig. 22 shows the number of cells in each subculture (n = 3, t-test * p < 0.05). Fig. 23 shows the cumulative number of cells in each subculture (n = 3, t-test * p < 0.05, ** p < 0.01). When cultured in a medium with AS1517499 added to the CFY medium, cell growth was promoted in all passages compared to culture in a medium with DMSO added to the CFY medium. From these results, it was confirmed that by adding a STAT6 inhibitor, the growth-promoting effect of c-MET(+)-hiPSC-NPC in the CFY medium was improved.

[0129] <Generation of kidney organoids from c-MET(+)-hiPSC-NPC subcultured in CFY medium supplemented with a STAT6 inhibitor> Subculture of c-MET(+)-hiPSC-NPC was performed using a medium with a STAT6 inhibitor (AS1517499, final concentration 100 nM) added to the CFY medium, and an attempt was made to generate kidney organoids from the cells of the first passage. <0000​​​​​​​The TBK1 inhibitor (MRT67307, final concentration 30 nM) or the JAK2 inhibitor (TG101348, final concentration 3 nM) was added to the CFY medium, and hiPSC-NPCs were expanded in culture, and the cell count was measured (n = 3, One-way ANOVA Tukey test * p < 0.05, ** p < 0.01). The cell count per passage 1 and 2 was compared with the case where only DMSO was added to the CFY medium.

[0132] The results are shown in Fig. 25. Fig. 25 shows the cell count in each subculture. As shown in Fig. 25, the addition of the TBK1 inhibitor or the JAK2 inhibitor to the CFY medium promoted the proliferation of hiPSC-NPCs.

[0133] <Generation of kidney organoids from hiPSC-NPCs subcultured in CFY medium supplemented with a TBK1 inhibitor or a JAK2 inhibitor> Subculture of hiPSC-NPCs was performed using a medium supplemented with the TBK1 inhibitor (MRT67307, final concentration 30 nM) or the JAK2 inhibitor (TG101348, final concentration 3 nM) in the CFY medium, and an attempt was made to generate kidney organoids from the cells of passage 1.

[0134] The results are shown in Figs. 26 and 27 ((PODXL; PODOCALYXIN; glomerulus, LTL; Lotus Tetragonolobus Lectin; proximal renal tubule, CDH1; CADHERIN1; distal renal tubule) (Scale bar; 100 μm)). Fig. 26 shows the results of the TBK1 inhibitor. Fig. 27 shows the results of the JAK2 inhibitor. Kidney organoids were formed from the cells of passage 1 in a medium supplemented with 30 nM MRT67307 or 3 nM TG101348 at the final concentration in the CFY medium. From this result, it was confirmed that hiPSC-NPCs can be subcultured while maintaining the ability to form kidney organoids using a medium supplemented with the TBK1 inhibitor or the JAK2 inhibitor in the CFY medium.

[0135] <c-MET(+)-hiPSC-NPC proliferation promoting effect of the TBK1 inhibitor or the JAK2 inhibitor> The TBK1 inhibitor (MRT67307, final concentration 30 nM) or the JAK2 inhibitor (TG101348, final concentration 3 nM) was added to the CFY medium, and c-MET(+)-hiPSC-NPCs were subcultured. The cell number was measured, and the cumulative cell number was calculated. The cell number and cumulative cell number at each passage 1, 2, and 3 were compared with the case where only DMSO was added to the CFY medium.

[0136] The results are shown in FIGS. 28 and 29. FIG. 28 shows the cell number in each subculture (n = 3, One-way ANOVA Tukey test, * p < 0.05, ** p < 0.01, *** p < 0.001). FIG. 29 shows the cumulative cell number in each subculture (n = 3, One-way ANOVA Tukey test, * p < 0.05, ** p < 0.01, *** p < 0.001). When cultured in the medium supplemented with the TBK1 inhibitor or the JAK2 inhibitor in the CFY medium, cell proliferation was promoted at any passage compared with the culture in the medium supplemented with DMSO in the CFY medium. From this result, it was confirmed that the addition of the TBK1 inhibitor or the JAK2 inhibitor improved the growth promoting effect of c-MET(+)-hiPSC-NPCs in the CFY medium.

[0137] <Production of kidney organoids from c-MET(+)-hiPSC-NPCs subcultured in the CFY medium supplemented with the TBK1 inhibitor> Subculture of c-MET(+)-hiPSC-NPCs was performed using the medium supplemented with the TBK1 inhibitor (MRT67307, final concentration 30 nM) in the CFY medium, and an attempt was made to produce kidney organoids from the cells at passage 2.

[0138] The results are shown in Figure 30 ((PODXL; PODOCALYXIN; glomerulus; LTL; Lotus Tetragonolobus Lectin; proximal tubule; CDH1; CADHERIN1; distal tubule) (Scale bar; 100 μm)). Renal organoids were formed from cells at the second passage in CFY medium supplemented with MRT67307 at a final concentration of 30 nM. These results confirmed that c-MET(+)-hiPSC-NPCs could be subcultured using CFY medium supplemented with a TBK1 inhibitor while maintaining their ability to form kidney organoids. [Industrial Applicability]

[0139] The present invention provides a medium for expanding nephron progenitor cells, which enables efficient expansion of nephron progenitor cells, a method for expanding nephron progenitor cells using the 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 medium for expanding nephron progenitor cells, comprising: at least one selected from the group consisting of a TBK1 inhibitor, a CSF1R inhibitor, a FLT3 inhibitor, a DYRK1 inhibitor, and a STAT6 inhibitor; FGF2, heparin, a ROCK inhibitor, a GSK3β inhibitor, a leukemia inhibitory factor (LIF), an ALK inhibitor, a BMP inhibitor, and BMP7; Including, the TBK1 inhibitor is MRT67307 or BX795; the CSF1R inhibitor is GW2580; the FLT3 inhibitor is ASP2215; the DYRK1 inhibitor is ID-8; the STAT6 inhibitor is AS1517499; the ROCK inhibitor is Y-27632, the GSK3β inhibitor is CHIR99021; the ALK inhibitor is A83-01; The BMP inhibitor is LDN193189; Culture medium.

2. A medium for expanding nephron progenitor cells, comprising: at least one selected from the group consisting of a TBK1 inhibitor, a STAT6 inhibitor, and a JAK2 inhibitor; a GSK3β inhibitor, a ROCK inhibitor, and FGF9; Including, the TBK1 inhibitor is MRT67307; the STAT6 inhibitor is AS1517499; the JAK2 inhibitor is TG101348; the GSK3β inhibitor is CHIR99021; The ROCK inhibitor is Y-27632. Culture medium.

3. A method for expanding nephron progenitor cells using the medium according to claim 1 or 2.

4. a step of expanding and culturing nephron progenitor cells by the method for expanding and culturing nephron progenitor cells according to claim 3; Differentiating the expanded nephron progenitor cells into kidney organoids; A method for producing kidney organoids, comprising:

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