Method for inducing differentiation of intermediate mesodermal cells into renal progenitor cells, and method for inducing differentiation of pluripotent stem cells into renal progenitor cells

Abstract

To provide a method for inducing differentiation of a pluripotent stem cell into a renal precursor cell.SOLUTION: A method includes processes (i) to (vi): (i) culturing a pluripotent stem cell by culture medium containing FGF2, BMP4, GSK-3β inhibitor and retinoic acid or a derivative thereof; (ii) culturing a cell obtained in the previous process by culture medium containing FGF2, GSK-3β inhibitor and BMP7; (iii) culturing a cell obtained in the previous process by culture medium containing FGF2, GSK-3β inhibitor, BMP7 and TGFβ inhibitor; (iv) culturing a cell obtained in the previous process by culture medium containing FGF2, GSK-3β inhibitor, BMP7, activin and ROCK inhibitor; (v) culturing a cell obtained in the previous process by culture medium containing retinoic acid or a derivative thereof and FGF9; and (vi) culturing a cell obtained in the previous process by culture medium containing GSK-3β inhibitor and FGF9, and inducing a renal precursor cell from an intermediate mesodermal cell.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for inducing differentiation of intermediate mesodermal cells into renal progenitor cells. The present invention also relates to a method for inducing differentiation of pluripotent stem cells into renal progenitor cells. [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, more than 300,000 patients with end-stage chronic renal failure 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 kidneys are derived from the intermediate mesoderm, which is tissue in the early embryonic stage. In vertebrates, the intermediate mesoderm forms three kidneys: pronephros, mesonephros, and metanephros. In mammals, the metanephros becomes the adult kidney. It develops through the interaction of two tissues: the ureteric bud, which differentiates into the nephrons and interstitium of the adult kidney, and the ureteric bud, which differentiates into the collecting duct of the adult kidney, the lower renal pelvis, ureter, and part of the bladder. Furthermore, the presence of nephron progenitor cells, which have the multipotency to differentiate into glomeruli that make up nephrons and several types of renal tubular epithelial cells, has been shown in the metanephric mesenchyme (Non-Patent Documents 1 and 2).

[0004] Highly efficient generation of nephron progenitor cells from human induced pluripotent stem (iPS) cells and human embryonic stem (ES) cells If a method for inducing differentiation can be established, it is expected that in the future, three-dimensional kidney reconstruction will be able to solve the shortage of donors for kidney transplants, and that these cells will be used as a source of glomerular and tubular cells for cell therapy. Furthermore, research will be conducted on drug nephrotoxicity evaluation systems using glomerular and tubular cells and kidney tissues containing them, and on the creation of disease models and development of therapeutic drugs using kidney cells and kidney tissues produced from disease-specific iPS cells. It is expected that this will lead to further development in research.

[0005] Several methods have been reported for inducing differentiation of nephron progenitor cells from human iPS cells and human ES cells. However, because embryoid bodies (EBs) are used, the differentiation induction efficiency is low (Non-Patent Documents 3 to 6). (Non-patent Document 3) and it is unclear whether each stage of development is accurately reproduced. This has been a problem (Non-Patent Documents 4 to 6). Furthermore, the present inventors' group has disclosed a method for producing renal progenitor cells from intermediate mesodermal cells, which includes a step of culturing intermediate mesodermal cells in a medium containing a TGFβ signal stimulator and a BMP inhibitor (Patent Document 1). However, there is no method for more efficiently inducing differentiation of renal progenitor cells. It was required. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO2014 / 200115 [Non-patent literature]

[0007] [Non-Patent Document 1] OsafuneK.,etal.,Development2006;133:151-61. [Non-patent document 2] KobayashiA.,etal.,CellStemCell2008;3:169-81. [Non-patent document 3] TaguchiA.,etal.,CellStemCell.2014;14:53-67. [Non-patent document 4] Takasato M.,etal.,NatCellBiol.2014;16:118-26. [Non-Patent Document 5] Takasato M.,etal.,Nature.2015;526:564-568. [Non-patent document 6] MorizaneR.,etal.,Nat.Biotechnol.2015;33:1193-1200. Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a method for efficiently inducing the differentiation of intermediate mesodermal cells into renal progenitor cells, more specifically, a method for inducing the differentiation of intermediate mesodermal cells into renal progenitor cells, the method comprising the step of inducing the differentiation of intermediate mesodermal cells induced from pluripotent stem cells into renal progenitor cells. [Means for solving the problem]

[0009] As a result of extensive research aimed at solving the above problems, the present inventors have found that intermediate mesodermal cells can be differentiated into renal progenitor cells by adhesion culture in a medium containing a GSK-3β inhibitor and FGF9. The present invention was completed based on this finding.

[0010] That is, the present invention has the following features: [1] A method for producing renal progenitor cells, comprising the steps of culturing intermediate mesodermal cells in adherent culture in a medium containing a GSK (glycogen synthase kinase)-3β inhibitor and FGF (fibroblast growth factor) 9, and inducing renal progenitor cells from the intermediate mesodermal cells. [2] The method according to [1], wherein the renal progenitor cells are SIX2-positive cells. [3] The method according to [1] or [2], wherein the intermediate mesodermal cells are OSR1-positive cells. [4] The method according to any one of [1] to [3], wherein the GSK-3β inhibitor is CHIR99021. [5] The method according to any one of [1] to [4], wherein the medium further contains a ROCK inhibitor. [6] The method according to any one of [1] to [5], wherein the adherent culture is carried out using a culture vessel coated with an extracellular matrix. [7] The method described in [6], wherein the extracellular matrix is ​​an E8 fragment of laminin-511. [8] The method according to any one of [1] to [7], wherein the intermediate mesodermal cells are intermediate mesodermal cells induced from pluripotent stem cells. [9] The method according to [8], wherein the intermediate mesodermal cells are intermediate mesodermal cells produced by a method comprising the following steps (i) to (v): (i) culturing pluripotent stem cells in a medium containing FGF2, BMP (bone morphogenetic protein) 4, 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 FGF2, a GSK-3β inhibitor, BMP7, and a TGFβ inhibitor; (iv) culturing the cells obtained in step (iii) in a medium containing FGF2, a GSK-3β inhibitor, BMP7, activin and a ROCK inhibitor; and (v) culturing the cells obtained in step (iv) in a medium containing retinoic acid or a derivative thereof and FGF9.

[10] The method according to [9], wherein the medium in step (v) further contains a BMP inhibitor.

[11] The method according to either [8] or

[10] , wherein the pluripotent stem cells are induced pluripotent stem (iPS) cells.

[12] The method according to

[11] , wherein the iPS cells are human iPS cells.

[13] A method for producing renal progenitor cells from pluripotent stem cells, 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 FGF2, a GSK-3β inhibitor, BMP7, and a TGFβ inhibitor; (iv) culturing the cells obtained in step (iii) in a medium containing FGF2, a GSK-3β inhibitor, BMP7, activin, and a ROCK (Rho-kinase) inhibitor; (v) culturing the cells obtained in step (iv) in a medium containing retinoic acid or a derivative thereof and FGF9; and (vi) A step of inducing renal progenitor cells from the intermediate mesodermal cells by culturing the cells obtained in step (v) in a medium containing a GSK-3β inhibitor and FGF9.

[14] The method according to

[13] , wherein the medium in step (v) further contains a BMP inhibitor.

[15] The method according to

[13] or

[14] , wherein the renal progenitor cells are SIX2-positive cells.

[16] The method according to any one of

[13] to

[15] , wherein the cells obtained in the step (v) are OSR1-positive cells.

[17] The method according to any one of

[13] to

[16] , wherein the GSK-3β inhibitor is CHIR99021.

[18] The method according to any one of

[13] to

[17] , wherein the medium in the step (vi) further contains a ROCK inhibitor.

[19] The method according to any one of

[13] to

[18] , wherein the culture is carried out using a culture vessel coated with an extracellular matrix.

[20] The method according to

[19] , wherein the extracellular matrix is ​​an E8 fragment of laminin-511.

[21] The method according to any one of

[13] to

[20] , wherein the pluripotent stem cells are induced pluripotent stem (iPS) cells.

[22] The method according to

[21] , wherein the iPS cells are human iPS cells.

[23] Renal progenitor cells produced by the method described in any one of [1] to

[22] .

[24] Renal organoids obtained using renal progenitor cells produced by any of the methods described in [1] to

[22] .

[25] A pharmaceutical composition comprising renal progenitor cells produced by the method described in any one of [1] to

[22] or renal organoids obtained using the same.

[26] A therapeutic agent for kidney disease, comprising renal progenitor cells produced by the method described in any one of [1] to

[22] or renal organoids obtained using the same. [Effects of the Invention]

[0011] The method of the present invention enables the induction of differentiation of human iPS cells into renal progenitor cells with a high efficiency of over 80% because it allows for culture in adherent culture. Furthermore, because the method accurately reproduces each developmental differentiation stage of the posterior epiblast, late somitic mesoderm, and posterior intermediate mesoderm cells that derive metanephric mesenchymal nephron progenitor cells, it also enables analysis of cells at each stage. The metanephros is one of the fetal renal tissues that form the adult kidney and is an essential component in kidney regeneration. Furthermore, because many renal diseases occur in metanephric nephron progenitor cells and the glomeruli and tubules derived from them, the method of the present invention is also useful for creating renal disease models. Therefore, the method of the present invention is also useful from the perspective of therapeutic methods and drug discovery for renal diseases. [Brief explanation of the drawings]

[0012] [Figure 1] Figure (photo) shows the results of immunocytochemical staining of late posterior epiblast, late primitive streak of the mesodermal lineage, late primitive streak of the metanephric lineage, late posterior intermediate mesoderm, and nephron progenitor cells (kidney progenitor cells) differentiated from human iPS cells. For the late posterior intermediate mesoderm and nephron progenitor cells, the percentage of marker-positive cells was also shown by FACS. [Figure 2] Photographs showing staining results of kidney organoids obtained by co-culturing human iPS cell-derived renal progenitor cells with mouse embryonic kidneys. In the figure, podocalyxin is a marker for glomeruli, LTL is a marker for proximal tubules, CDH1 is a marker for distal tubules, CDH6 is a marker for proximal tubules, and nephrin is a marker for glomeruli. The scale bar indicates 50 μm. [Figure 3]Brightfield and immunostained images of renal organoids obtained by culturing human iPS cell-derived renal progenitor cells. In the image, podocalyxin is a glomerular marker, LTL is a proximal tubule marker, CDH1 and BRN1 are distal tubule and loop of Henle markers, and dolicosbiflorusagglutinin (DBA) is a distal tubule marker. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention will be described in detail below.

[0014] <Induction of differentiation of intermediate mesodermal cells into renal progenitor cells> The present invention provides a method for producing renal progenitor cells, which comprises the step of culturing intermediate mesodermal cells in a medium containing a GSK-3β inhibitor and FGF9 (also referred to as the step of inducing renal progenitor cell differentiation).

[0015] In the present invention, intermediate mesodermal cells refer to any cells that can be induced to become renal progenitor cells by culturing them in a medium containing a GSK-3β inhibitor and FGF9. Methods for obtaining intermediate mesodermal cells include, for example, methods for inducing differentiation of mouse and human pluripotent stem cells into intermediate mesodermal cells (Biochem Biophys Res Commun. 393:877-82 (2010), Nat Commun. 4:1367 (2013), WO2012 / 011610, and Patent Document 1). OSR1 is known as a marker for identifying intermediate mesodermal cells, and examples of intermediate mesodermal cells for use in the methods of the present invention include OSR1-positive intermediate mesodermal cells. For example, pluripotent stem cells carrying a reporter gene (e.g., GFP) introduced under the control of the OSR1 promoter (e.g., OSR1-GFP reporter human iPS cells described in the Examples below) can be cultured, and OSR1-positive intermediate mesodermal cells can be isolated using methods known in the art (e.g., cell sorting) based on the expression of the reporter gene. Expression of OSR1 in intermediate mesodermal cells can also be confirmed by gene expression analysis methods such as quantitative RT-PCR (NatCommun4, 1367, (2013)). In the present invention, OSR1-positive intermediate mesodermal cells include cells expressing the OSR1 protein and cells expressing proteins encoded by genes under the control of the OSR1 promoter. In the present invention, OSR1 includes genes having the nucleotide sequences set forth in the NCBI accession numbers NM_145260.2 for humans and NM_011859.3 for mice, proteins encoded by the genes, and naturally occurring mutants having these functions. Preferably, intermediate mesoderm cells used in the methods of the present invention are SIX2-negative and OSR1-, HOX11-, and WT1-positive.

[0016] In the present invention, renal progenitor cells are considered to be equivalent to nephron progenitor cells and are capable of differentiating in vitro into organ structures such as renal glomerular-like structures and tubular-like structures. The ability to differentiate 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 renal progenitor cells (Cell Stem Cell 3:169-181 (2008)). Examples of renal progenitor cells induced by the method of the present invention include SIX2-positive renal progenitor cells. For example, pluripotent stem cells (e.g., OSR1-GFP / SIX2-tdTomato reporter human iPS cells, described in the Examples below) carrying a reporter gene (e.g., tdTomato) introduced under the control of the SIX2 promoter can be cultured, and SIX2-positive renal progenitor cells can be isolated using methods known in the art (e.g., cell sorting) based on the expression of the reporter gene. Expression of SIX2 in renal progenitor cells can also be confirmed by gene expression analysis methods such as quantitative RT-PCR (NatCommun4, 1367, (2013)). In the present invention, SIX2-positive renal progenitor cells include cells expressing the SIX2 protein and cells expressing proteins encoded by genes under the control of the SIX2 promoter. In the present invention, SIX2 includes a gene having the nucleotide sequence set forth in the NCBI accession numbers NM_016932.4 for humans and NM_011380.2 for mice, as well as proteins encoded by the gene, and naturally occurring mutants having these functions. Preferably, the renal progenitor cells induced by the method of the present invention are cells that are positive for OSR1 and also positive for HOX11, WT1, SIX2, and SALL1.

[0017] In the present invention, intermediate mesodermal cells or renal progenitor cells may be provided as a cell population containing other cell types, or may be a purified population. Preferably, the cell population contains 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10%, 20%, 28%, or 30% or more of the cells. Purification can be performed by methods such as FACS using the above-mentioned markers as indicators.

[0018] In the present invention, "adherent culture" refers to culturing cells in a state where they are attached to a culture substrate, such as culturing cells on a coated culture dish. 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, such as cell-derived preparations such as BDMatrigel™. The extracellular matrix is ​​preferably laminin or a fragment thereof. In the present invention, laminin is a heterotrimeric protein with one α-chain, one β-chain, and one γ-chain. Laminin is an extracellular matrix protein with isoforms that differ in the composition of its 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, α1, α2, α3, α4, or α5 for the α chain, β1, β2, β3, or β4 for the β chain, and γ1, γ2, or γ3 for the γ chain. Laminin is preferably laminin 511, which consists of α5, β1, and γ1 ( NatBiotechnol 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 the E8 fragment (laminin 511E8), which is obtained by digestion with elastase ( EMBO J., 3:1463-1468, 1984 ; J. Cell Biol., 105:589-598, 1987 ; WO 2011 / 043405 ). Laminin 511E8 is commercially available, for example, from Nippi Corporation.

[0019] The medium used in the renal progenitor cell differentiation induction step can be prepared by adding a GSK-3β inhibitor and FGF9 to a basal medium used for culturing animal cells. Examples of basal media include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM), αMEM medium, Dulbecco's Modified Eagle's Medium (DMEM), Ham's F12 (F12), RPMI 1640 medium, Fischer's medium, and mixtures thereof. The medium may contain serum (e.g., fetal bovine serum (FBS)) or may be serum-free. Optionally, the medium may contain one or more serum replacements, such as albumin, transferrin, KnockOutSerumReplacement (KSR) (a serum replacement for ES cell culture) (Invitrogen), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, or one or more substances, such as lipids, amino acids, L-glutamine, GlutaMAX (Invitrogen), non-essential amino acids (NEAA), vitamins, growth factors, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, and the like. Media previously optimized for stem cell culture, such as ReproFF2 (ReproCell), may also be used. Furthermore, the medium used for the renal progenitor cell differentiation induction step may contain a ROCK inhibitor, such as Y-27632, as described below.

[0020] The GSK-3β inhibitor used in the renal progenitor cell differentiation induction step is not particularly limited as long as it can inhibit the function of GSK-3β, for example, kinase activity, and examples thereof include the indirubin derivative BIO (also known as GSK-3β inhibitor IX; 6-bromoindirubin-3'-oxime), the maleimide derivative SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), the phenyl-α-bromomethyl ketone compound GSK-3β inhibitor VII (α,4-dibromoacetophenone), the cell membrane-permeable phosphorylated peptide L803-mts (also known as GSK-3β peptide inhibitor; Myr-N-GKEAPPAPPQSpP-NH2), and the highly selective CHIR99021 (Nature (2008) 453: 519-523). These compounds are available from, for example, Stemgent, Calbiochem, Biomol, etc., or may be prepared in-house. A preferred GSK-3β inhibitor for use in this step is CHIR99021. The concentration of the GSK-3β inhibitor used in this step can be appropriately selected by those skilled in the art depending on the GSK-3β inhibitor used, and is, for example, 0.01 μM to 100 μM, preferably 0.1 μM to 10 μM, more preferably 0.5 μM to 3 μM, and particularly preferably 0.5 μM to 1.5 μM.

[0021] The FGF9 used in the renal progenitor cell differentiation induction step is preferably human FGF9. For example, human FGF9 can be obtained from the National Center for Biotechnology Information (NCBI). An example is a protein having the amino acid sequence of accession number NP_002001.1. FGF9 encompasses its fragments and functional variants as long as it has differentiation-inducing activity. Commercially available FGF9 may be used, or a protein purified from cells or a protein produced by genetic recombination may be used. The concentration of FGF9 used in this step is, for example, 1 ng / ml to 500 ng / ml, 1 ng / ml to 100 ng / ml, 5 ng / ml to 50 ng / ml, or 5 ng / ml to 25 ng / ml.

[0022] The number of days for culture in the renal progenitor cell differentiation induction step has no upper limit because long-term culture does not particularly affect the production efficiency of renal progenitor cells, and examples include 2 or more days, 3 or more days, 4 or more days, and 5 or more days. In the renal progenitor cell differentiation induction step, the culture temperature is, but is not limited to, about 30 to 40°C, preferably about 37°C, and culture is performed in an atmosphere of CO2-containing air, with a CO2 concentration of preferably about 2 to 5%.

[0023] In one embodiment of the present invention, the intermediate mesodermal cells are intermediate mesodermal cells induced from pluripotent stem cells. In this case, the induced intermediate mesodermal cells may be isolated and then induced into renal progenitor cells by the culture process of the present invention. Alternatively, intermediate mesodermal cells may be induced from pluripotent stem cells and then directly subjected to the culture process of the present invention without isolation, thereby being induced into renal progenitor cells.

[0024] When isolating intermediate mesodermal cells, pluripotent stem cells having a reporter gene whose expression is controlled by the endogenous OSR1 promoter may be used. Methods for introducing a reporter gene into pluripotent stem cells under the control of the OSR1 promoter include, for example, homologous recombination using a BAC vector, and are described in WO2012 / 011610, etc. Furthermore, to isolate induced renal progenitor cells, pluripotent stem cells having a reporter gene whose expression is controlled by the SIX2 promoter may be used, and methods similar to those described above may be used. Reporter genes can be produced using a variety of methods. Examples of reporter genes that can be used include genes encoding known reporter proteins such as β-galactosidase, β-gluconidase, luciferase, green fluorescent protein (GFP), tdTomato, and cell surface proteins. Intermediate mesoderm cells or renal progenitor cells induced from these pluripotent stem cells can be isolated using methods known in the art, such as a method using a cell sorter with the expression of the reporter protein as an indicator, a method using magnetic beads to magnetically select cells with an antibody against the cell surface protein (e.g., MACS), or a method using a carrier on which the antibody or the like is immobilized (e.g., a cell concentration column).

[0025] In the present invention, pluripotent stem cells are stem cells that have the pluripotency to differentiate into many cells present in the body and also have the ability to proliferate, and include any cells that can be induced to become the intermediate mesoderm cells used in the present invention. Pluripotent stem cells include, but are not limited to, embryonic stem (ES) cells, embryonic stem cells derived from cloned embryos obtained by nuclear transfer (ntES) cells, spermatogonial stem cells ("GS cells"), embryonic germ cells ("EG cells"), induced pluripotent stem (iPS) cells, and pluripotent cells derived from cultured fibroblasts or bone marrow stem cells (Muse cells). Pluripotent stem cells are preferably iPS cells, more preferably human iPS cells, because they can be obtained without destroying embryos, eggs, etc. during the production process.

[0026] Methods for producing iPS cells are known in the art, and iPS cells can be produced by introducing reprogramming factors into any somatic cells. Examples of reprogramming factors include genes or gene products such as Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, and Glis1. These reprogramming factors may be used alone or in combination. Combinations of reprogramming factors include WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 075119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 / 101407, WO2009 / 102983, WO2009 / 114949, WO2009 / 117439, WO2009 / 126250, WO2009 / 126251, WO2009 / 126655, WO2009 / 1575 93, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 050626, WO2010 / 056831, WO 2010 / 068955, WO2010 / 098419, WO2010 / 102267, WO2010 / 111409, WO2010 / 111422, WO2010 / 115050, WO2010 / 124290, WO2010 / 147395, WO2010 / 147612, HuangfuD, etal.(2008), Nat.Biotechnol.,26:795-797, ShiY, etal.(2008),CellStemCell,2:525-528,EminliS,etal.(2008),StemCells.26:2467-2474,HuangfuD,etal. al.(2008), Nat.Biotechnol.26:1269-1275, ShiY,etal.(2008),CellStemCell,3,568-574, ZhaoY,etal.(2008),CellStemCell,3:475-479,MarsonA,(2008),CellStemCell,3,132-135,FengB,etal.(2009),Nat.CellBiol.11:197-203,RLJudsonetal.,(2009),Nat.Biotechnol., 27:459-461, LyssiotisCA,etal.(2009),ProcNatlAcadSciUSA.106:8912-8917,KimJB,etal.(2009),Nature.461:649-643,IchidaJK,etal.(2009),CellStemCell.5:491-5. 03, HengJC,etal.( 2010), Cell Stem Cell. 6: 167-74, Han J, et al. (2010), Nature. 463: 1096-100, Mali P, et al. (2010), Stem Cells. 28: 713-720, Maekawa M, et al. (2011), Nature. 474: 225-9.

[0027] Somatic cells include, but are not limited to, fetal (offspring) somatic cells, neonatal (offspring) somatic cells, and mature healthy or diseased somatic cells, as well as primary culture cells, passaged cells, and established cell lines. Specific examples of somatic cells include (1) tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells, (2) tissue progenitor cells, and (3) differentiated cells such as blood cells (peripheral blood cells, umbilical cord blood cells, etc.), lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, liver cells, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells (exocrine pancreatic cells, etc.), brain cells, lung cells, kidney cells, and adipocytes.

[0028] Furthermore, when iPS cells are used as a source of transplantation cells, it is desirable to use somatic cells with an HLA genotype identical or substantially identical to that of the recipient individual, from the viewpoint of preventing rejection. Here, "substantially identical" means that the HLA genotype matches the transplanted cells to an extent that an immune response can be suppressed with an immunosuppressant, for example, somatic cells with an HLA type that matches the three loci of HLA-A, HLA-B, and HLA-DR, or four loci including HLA-C.

[0029] <Induction of differentiation from pluripotent stem cells to intermediate mesoderm cells> In the present invention, a method comprising the following steps can be used to induce differentiation of pluripotent stem cells into intermediate mesodermal cells. (i) culturing pluripotent stem cells in a medium containing FGF2, BMP (bone morphogenetic protein) 4, 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 FGF2, a GSK-3β inhibitor, BMP7, and a TGFβ inhibitor; (iv) culturing the cells obtained in step (iii) in a medium containing FGF2, a GSK-3β inhibitor, BMP7, activin and a ROCK inhibitor; and (v) culturing the cells obtained in step (iv) in a medium containing retinoic acid or a derivative thereof and FGF9.

[0030] Each step will be further explained below.

[0031] (i) culturing pluripotent stem cells in a medium containing FGF2, BMP4, a GSK-3β inhibitor, and retinoic acid or a derivative thereof; 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-CDH (CDH1), and preferably all of these markers. Late posterior epiblasts are also preferably negative for EOMES and BRACHYURY.

[0032] In step (i), pluripotent stem cells are isolated by any method known in the art and cultured, preferably by adherent culture. Methods for isolating pluripotent stem cells include, for example, mechanical separation and separation solutions having protease and collagenase activity (e.g., Accutase™ and Accumax). Examples of methods include separation using a separation solution containing only collagenase activity (e.g., ELISA (TM) (Innovative Cell Technologies, Inc.)) or a separation solution containing only collagenase activity. Preferably, the method involves dissociating the cells using a separation solution containing both protease and collagenase activity, followed by mechanically dispersing the cells 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.

[0033] The medium used in step (i) can be prepared by adding FGF2, BMP4, a GSK-3β inhibitor, and retinoic acid or its derivatives 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 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.

[0034] The GSK-3β inhibitor used in step (i) can be any of the GSK-3β inhibitors exemplified in the renal progenitor cell differentiation induction step described above, and a preferred GSK-3β inhibitor is CHIR 99021. 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, and is, for example, 0.01 μM to 100 μM, preferably 0.1 μM to 10 μM, more preferably 0.5 μM to 3 μM, and particularly preferably 0.5 μM to 1.5 μM.

[0035] The FGF2 (basic FGF: bFGF) used in step (i) is preferably human FGF2, and examples of human FGF2 include 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 it has 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 1 ng / ml to 1000 ng / ml, preferably 10 ng / ml to 500 ng / ml, and more preferably 50 ng / ml to 250 ng / ml.

[0036] The BMP4 used in step (i) is preferably human BMP4. Examples of human BMP4 include those listed under NCBI (National Center for Biotechnology Information) accession number NCBI-10001 ... Examples of BMP4 include proteins having the amino acid sequence AAH20546.1. BMP4 includes fragments and functional variants thereof as long as they have differentiation-inducing activity. Commercially available BMP4 may be used, or proteins purified from cells or produced by genetic recombination may be used. The concentration of BMP4 used in this step is 0.1 ng / ml to 100 ng / ml, preferably 0.5 ng / ml to 50 ng / ml, and more preferably 0.5 ng / ml to 5 ng / ml.

[0037] The retinoic acid used in step (i) 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]-Benzoicacid (AM580) (Tamura K, et al., CellDiffer. Dev. 32:17-26 (1990)), 4-[(1E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carbonyl]amino]-Benzoicacid (AM580) (Tamura K, et al., CellDiffer. Dev. 32:17-26 (1990)), and 4-[(1E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carbonyl]amino]-Benzoicacid (AM580). Examples of such compounds include [(2-hydroxybenzoic acid)-2-naphthalenyl)-1-propen-1-yl]-benzoicacid (TTNPB) (Strickland S, et al., Cancer Res. 43:5268-5272 (1983)), and the compounds described in Tanenaga K. et al., Cancer Res. 40:914-919 (1980), retinol palmitate, retinol, retinal, 3-dehydroretinol, and 3-dehydroretinal. The concentration of retinoic acid or a derivative thereof used in step (i) is, for example, 1 nM to 100 nM, preferably 5 nM to 50 nM, more preferably 5 nM to 25 nM.

[0038] In step (i), the culture temperature is, but not limited to, about 30 to 40°C, preferably about 37°C, and the culture is performed in a CO2-containing air atmosphere. 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.

[0039] (ii) culturing the cells obtained in step (i) in a medium containing FGF2, a GSK-3β inhibitor, and BMP7; 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 E-CDH.

[0040] 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 dish, or the cells obtained by adherent culture in step (i) may be cultured as is by replacing the medium.

[0041] 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.

[0042] The FGF2 used in step (ii) is the same as that described in step (i), and its preferred concentration range is also the same.

[0043] The GSK-3β inhibitor used in step (ii) can be any of the GSK-3β inhibitors exemplified in step (i) above, and 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 is, for example, 0.01 μM to 100 μM, preferably 0.1 μM to 10 μM, more preferably 1 μM to 7.5 μM, and particularly preferably 2 to 5 μM. The concentration of the GSK-3β inhibitor used in step (ii) is preferably increased from the concentration in step (i).

[0044] The BMP7 used in step (ii) is preferably human BMP7. Examples of human BMP7 include those listed under NCBI (National Center for Biotechnology Information) accession number 11111111. The protein with the amino acid sequence No. NM_001719.2 is BMP7. As long as BMP7 has activity, its fragments and functional variants may be commercially available, or a protein purified from cells or a protein produced by genetic recombination may be used. The concentration of BMP7 used in this step is 0.1 ng / ml to 100 ng / ml, preferably 0.5 ng / ml to 50 ng / ml, and more preferably 0.5 ng / ml to 5 ng / ml.

[0045] In step (ii), the culture temperature is, but is not limited to, about 30 to 40°C, preferably The incubation temperature is about 37°C, and the incubation is carried out in a CO2-containing atmosphere. The CO2 concentration is about 2 to 5%, preferably about 5%. The incubation 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.

[0046] (iii) culturing the cells obtained in step (ii) in a medium containing FGF2, a GSK-3β inhibitor, BMP7, and a TGFβ inhibitor; During this process, the mesodermal lineage primitive streak is induced into the late mesodermal lineage primitive streak, which is characterized as cells positive for CDX2 and BRACHYURY.

[0047] 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 dish, or the cells obtained by adherent culture in step (ii) may be cultured as is by replacing the medium.

[0048] The medium used in step (iii) can be prepared by adding FGF2, a GSK-3β inhibitor, BMP7, and a TGFβ 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, etc.

[0049] The FGF2, GSK-3β inhibitor, and BMP7 used in step (iii) are the same as those in step (ii), and their preferred concentration ranges are also the same, but the concentration range of the GSK-3β inhibitor is 0.01 μM to 100 μM, preferably 0.1 μM to 10 μM, more preferably 1 μM to 7.5 μM, and particularly preferably 2 to 5 μM.

[0050] The TGFβ inhibitor used in step (iii) is a substance that inhibits signal transduction that continues from the binding of TGFβ to the receptor to SMAD, and examples thereof include substances that inhibit binding to the ALK family receptor, or substances that inhibit phosphorylation of SMAD by the ALK family, and examples thereof include Lefty-1 (NCBI Accession No.: mouse: NM_010094, human: NM_020997), SB431542, SB202190 (RK Lindemannetal., Mol. Cancer, 2003, 2:20), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276 (Lilly Research Laboratories), A83-01 (WO2009146408), and Examples include derivatives thereof. The TGFβ inhibitor may preferably be A83-01. The concentration of the TGFβ inhibitor in the culture medium is not particularly limited as long as it is a concentration that inhibits ALK. However, it is 0.5 μM to 100 μM, preferably 1 μM to 50 μM, and more preferably 5 μM to 25 μM.

[0051] In step (iii), the culture temperature is, but not limited to, about 30 to 40°C, preferably about 37°C, and the culture is performed 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 time period sufficient to induce differentiation of the mesodermal lineage late primitive streak, and is, for example, 1 to 3 days, preferably 1.5 to 2 days.

[0052] (iv) culturing the cells obtained in step (iii) in a medium containing FGF2, a GSK-3β inhibitor, BMP7, activin, and a ROCK inhibitor; 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.

[0053] 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 dish, or the cells obtained by adherent culture in step (iii) may be cultured as is by replacing the medium.

[0054] The medium used in step (iv) can be prepared by adding FGF2, a GSK-3β inhibitor, BMP7, 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 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.

[0055] The FGF2, GSK-3β inhibitor, and BMP7 used in step (iv) are the same as those in step (ii), and their preferred concentration ranges are also the same, but the concentration range of the GSK-3β inhibitor is 0.01 μM to 100 μM, preferably 0.1 μM to 10 μM, more preferably 1 μM to 7.5 μM, and particularly preferably 2 to 5 μM.

[0056] The activin used in step (iv) includes activins derived from humans and other animals, as well as functional variants thereof, and may be commercially available, for example, from R&D Systems, Inc. The concentration of activin used in step (iv) is 1 ng / ml to 100 ng / ml, preferably 5 ng / ml to 50 ng / ml, more preferably 5 ng / ml to 25 ng / ml.

[0057] The ROCK inhibitor used in step (iv) is not particularly limited as long as it can inhibit the function of Rho-kinase (ROCK). Examples of the ROCK inhibitor 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 (1999)), and the like. 997), 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 their derivatives, as well as antisense nucleic acids, RNA interference-inducing nucleic acids (e.g., siRNA), dominant-negative mutants, and expression vectors thereof against ROCK. Other known low molecular weight 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). In the present invention, one or more ROCK inhibitors can be used. A preferred 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, and is, for example, 0.1 μM to 100 μM, preferably 1 μM to 75 μM, and more preferably 5 μM to 50 μM.

[0058] In step (iv), the culture temperature is, but not limited to, about 30 to 40°C, preferably about 37°C, and 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 time period sufficient to induce differentiation of the metanephric lineage late primitive streak, but may be, for example, 1 to 5 days, preferably 3 days. is.

[0059] (v) culturing the cells obtained in step (iv) in a medium containing retinoic acid or a derivative thereof and FGF9; 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.

[0060] 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 dish, or the cells obtained by adherent culture in step (iv) may be cultured as is by replacing the medium.

[0061] The medium used in step (v) can be prepared by adding retinoic acid or its derivatives and FGF9 to a basal medium used for culturing animal cells. The basal medium can be any of the above-mentioned basal media, which 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.

[0062] The retinoic acid or its derivative and FGF9 used in step (v) are as described in step (i) and the renal progenitor cell induction step, respectively, and the preferred concentration ranges thereof are also the same.

[0063] The medium used in step (v) may further contain a BMP inhibitor. 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 (PBYuet al. (2007), Circulation, 116:II_60; PBYuet al. (2008), Nat. Chem. Biol., 4:33-41; J. Haoet al. (2008), PLoSONE, 3(8):e2904), and LDN193189 (i.e., 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline). The more preferred BMP inhibitor is NOGGIN, and its concentration can be, for example, 1 to 100 ng / ml.

[0064] In step (v), the culture temperature is, but not limited to, about 30 to 40°C, preferably about 37°C, and the culture is performed 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, and is, for example, 1 to 3 days, preferably 2 days.

[0065] Renal progenitor cells can be induced by culturing the intermediate mesodermal cells obtained in step (v) in a medium containing a GSK-3β inhibitor and FGF9, as described in the renal progenitor cell induction step.

[0066] The present invention also provides renal organoids obtained using renal progenitor cells obtained by the above-mentioned method. Renal organoids derived from iPS cells are described, for example, in Nature, 526, 564-568 (2015). In the present invention, for example, renal progenitor cells obtained by the above method are cultured to prepare cell clusters, which are then co-cultured with feeder cells such as 3T3-Wnt4 cells, mouse fetal spinal cord cells, or mouse fetal kidney cells, or with a GSK-3β inhibitor such as CHIR99021. This can be achieved by semi-aerobic culture using a basal medium containing GSK-3β inhibitors (Nature, 526, 564-568 (2015)). The medium contains FGF9 and FGF2 in addition to GSK-3β inhibitors. It is possible.

[0067] The present invention provides a pharmaceutical composition containing renal progenitor cells obtained by the above-described method or renal organoids obtained using the same, a renal disease therapeutic agent containing the renal progenitor cells or renal organoids obtained using the same, and a method for treating renal disease comprising administering a therapeutically effective amount of the renal progenitor cells or renal organoids obtained using the same. Methods for administering therapeutic agents to patients in need of treatment include, for example, forming a sheet of the obtained renal progenitor cells into the patient's kidney, suspending the obtained renal progenitor cells in physiological saline or the like, or three-dimensionally culturing the obtained cell mass (e.g., DevCell. Sep 11, 2012; 23(3): 637-651), and directly transplanting the resulting cell mass into the patient's kidney, or three-dimensionally culturing the obtained renal progenitor cells on a scaffold composed of Matrigel or the like, and transplanting the resulting renal progenitor cell mass. The transplant site is not particularly limited as long as it is within the kidney, but is preferably under the renal capsule. Renal diseases include acute kidney injury, chronic renal failure, and chronic kidney disease that does not progress to chronic renal failure. In the present invention, the number of renal progenitor cells contained in the renal disease therapeutic agent is not particularly limited as long as the graft can survive after administration, and may be adjusted to an appropriate number depending on the size of the affected area and the size of the body. [Example]

[0068] The present invention will be specifically described below based on examples, but the present invention is not limited to the following embodiments.

[0069] <1> Induction of differentiation of iPS cells into renal progenitor cells Renal progenitor cells were differentiated from iPS cells using the following protocol. We used OSR1-GFP / SIX2-tdTomato reporter human iPS cells derived from the mouse.

[0070] 1. Undifferentiated iPS cells were denatured by accutase treatment and then suspended in ReproFF2 medium (ReproCell) supplemented with 10 μM Y27632. 1.0 × 10 cells were plated onto a well coated with Matrigel (BD Biosciences). 4 / well ~ 5.0 × 10 4 100 cells / well and incubate at 37°C for 24 hours. 2. 24 hours later (day 1), vitamin A free B27 supplement (ThermoFisher Scientific Inc.) was administered. The basal medium was DMEM / F12 Glutamax (ThermoFisher Scientific Inc.) supplemented with 1 μM CHIR99021, 10 nM Retinoic acid, 1 ng / ml BMP4, and 100 ng / ml FGF2. (Late posterior) Preparation of epiblasts 1) 3. On day 2, the same basal medium was supplemented with 3 μM CHIR99021, 1 ng / ml BMP7, and 100 ng / ml FGF2. Replacement (Preparation of the Mesoderm Lineage Primitive Streak 2) 4. On days 3 and 4, the medium was replaced with the same basal medium supplemented with 3 μM CHIR99021, 1 ng / ml BMP7, 100 ng / ml FGF2, and 10 μM A83-01 (preparation of the late mesodermal lineage primitive streak 3). 5. On days 5, 6, and 7, the medium was replaced with the same basal medium supplemented with 3 μM CHIR99021, 1 ng / ml BMP7, 100 ng / ml FGF2, 10 ng / ml ACTIVIN, and 30 μM Y27632 (preparation of the metanephric lineage late primitive streak···4). 6. On days 8 and 9, the medium was replaced with the same basal medium supplemented with 100 ng / ml FGF9 and 100 nM Retinoic acid. Melting (Preparation of late posterior intermediate mesoderm 5) 7. On days 10, 11, and 12, the medium was replaced with the same basal medium supplemented with 10 ng / ml FGF9 and 1 μM CHIR99021 (Generation of Renal Progenitor Cells 6).

[0071] The progress of differentiation induction at each stage was confirmed by the expression of the markers listed in Table 1. We confirmed that negative markers were not expressed. Figure 1 shows the marker staining results for cells at each stage. For late posterior intermediate mesoderm and renal progenitor cells, we were able to obtain marker-positive cells in more than 80% of cells. Similar results were obtained when the medium in step 5 above was changed to 200 ng / ml FGF9, 100 nM Retinoic acid, and 25 ng / ml NOGGIN.

[0072] [Table 1]

[0073] These results demonstrate that renal progenitor cells could be differentiated from iPS cells with a significantly high differentiation efficiency of over 80%.

[0074] <2> Generation of renal organoids from renal progenitor cells by co-culture with mouse fetal kidney cells <1> 1.0 × 10 renal progenitor cells obtained in 5 The cells were cultured for 1 day in a basal medium supplemented with 1 μM CHIR99021 and 10 ng / ml FGF9. As a result, glomeruli and tubules were confirmed, as shown in Figure 2, and kidney organoids were successfully produced.

[0075] <3> Generation of renal organoids by single-cell culture of renal progenitor cells <1> 1.0 × 10 renal progenitor cells obtained in 5 The cell aggregates were cultured for 1-2 days in a basal medium supplemented with 1 μM CHIR99021 and 200 ng / ml FGF9. The cells were cultured in a semi-aerial phase for 2 days in a basal medium supplemented with 200 ng / ml FGF2, and then cultured in the basal medium alone. The cells were cultured in a semi-aerobic phase for 8 days. As a result, glomeruli and tubules were confirmed, as shown in Figure 3, and kidney organoids were successfully produced. Furthermore, BRN1(+)CDH1(+)DBA(-) loops of Henle were observed.

Claims

1. A method for producing renal progenitor cells, comprising the steps of: (i) Pluripotent stem cell-derived, late metanephric lineage primitive streak cells positive for HOX11 and BRACHYURY were cultured in retinoic acid or 3-dehydroretinoic acid, 4-[[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carbonyl]amino]-Benzoic acid ( AM580), 4-[(1E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propen-1-yl]-benzoic acid (TTNPB), retinol palmitate, Inducing OSR1-positive intermediate mesoderm cells by culturing in a medium containing a retinoic acid derivative selected from retinol, retinal, 3-dehydroretinol, and 3-dehydroretinal and FGF9; and (ii) A step of culturing the OSR1-positive intermediate mesodermal cells obtained in step (i) in an adhesion culture medium containing a GSK-3β inhibitor and FGF9 to induce SIX2-positive renal progenitor cells.

2. 2. The method of claim 1, wherein the GSK-3β inhibitor is CHIR99021.

3. The method according to any one of claims 1 to 2, wherein the medium further comprises a ROCK inhibitor.

4. The method according to any one of claims 1 to 3, wherein the adherent culture is carried out using a culture vessel coated with an extracellular matrix.

5. The method of claim 4, wherein the extracellular matrix is ​​an E8 fragment of laminin-511.

6. The method according to any one of claims 1 to 5, wherein the pluripotent stem cells are induced pluripotent stem (iPS) cells.

7. The method of claim 6, wherein the iPS cells are human iPS cells.

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