Ciliopathic disease models and their applications

Abstract

Provided is a method for screening or evaluating a therapeutic agent of cilia-related diseases, the method comprising: a step for bringing an agent candidate substance into contact with a renal cell component, such as a renal organoid obtained by differentiation-inducing induced pluripotent stem (iPS) cells obtained from cilia-related disease patient-derived somatic cells or a renal tubule cell included in the renal organoid; and a step for measuring a calcium dynamic and / or a cyst formation in the renal cell component such as the renal organoid or the renal tubule cell.

Description

[Technical field]

[0001] The present invention relates to a cyst formation model obtained from kidney organoids induced from induced pluripotent stem (iPS) cells derived from a patient with a cilia-related disorder, and a method for screening or evaluating therapeutic agents for a cilia-related disorder using the same. [Background technology]

[0002] Autosomal dominant polycystic kidney disease (ADPKD), an intractable genetic disease, progressively forms numerous cysts in the kidneys and progresses to end-stage renal failure after middle age. The causative gene for ADPKD is PKD1 in 85% of cases and PKD2 in 15% of cases. Research has been conducted using experimental animals such as mouse and rat models that have been modified with these genes, but the pathology has not been fully elucidated and no curative treatment has been developed.

[0003] In recent years, active research has been conducted to establish disease-specific iPS cells by introducing causative gene mutations into iPS cells established from somatic cells of patients with intractable diseases or iPS cells derived from healthy individuals, and then to create disease models that reproduce the pathology by inducing differentiation into diseased cell types in vitro, and to perform detailed pathological analysis and therapeutic drug discovery. However, for ADPKD renal cysts, a disease model using iPS cells that can be used for pathological analysis and therapeutic drug discovery has not yet been established.

[0004] Freedman et al.'s group attempted to reproduce renal cysts by inducing differentiation into renal tissue from ADPKD-specific ES cell lines in which homozygous deletion mutations of PKD1 or PKD2 were introduced into healthy human ES cell lines by genome editing (Non-Patent Documents 1-4), but it is unclear whether the kidney development process is accurately reproduced. In addition, administration of forskolin did not result in significant differences in cyst formation compared to healthy controls. Furthermore, in iPS cell lines derived from ADPKD patients (PKD1 heterozygous deletion), differentiation into renal tissue was not stable, and no clear pathological traits were observed compared to healthy iPS cell lines. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] WO 2018 / 216743 [Non-patent literature]

[0006] [Non-Patent Document 1] Freedman BS. et al., J Am Soc Nephrol 2013 [Non-Patent Document 2] Freedman BS. et al., Nat Commun 2015 [Non-Patent Document 3] Cruz NM. et al., Nat Mater 2017 [Non-Patent Document 4] Czerniecki SM. et al., Cell Stem Cell 2018 Summary of the Invention [Problem to be solved by the invention]

[0007] An objective of the present invention is to develop a cyst formation model that can be used for screening and evaluating therapeutic agents for cilia-related diseases such as autosomal dominant polycystic kidney disease (ADPKD). [Means for solving the problem]

[0008] In Patent Document 1, the inventors' group developed a method for producing kidney organoids from iPS cells. In order to solve the above problems, the inventors succeeded in producing a kidney cyst model by establishing iPS cells from fibroblasts of an ADPKD patient and culturing the kidney tissue produced using the differentiation induction method described in Patent Document 1 in the presence of forskolin, blebbistatin, etc. Using this model, the inventors found that it is possible to efficiently screen and evaluate candidate substances for therapeutic agents for cilia-related diseases such as ADPKD by using the shape of the cysts and calcium dynamics as indicators, and thus completed the present invention.

[0009] The gist of the present invention is as follows. [1] A method for screening or evaluating drugs for treating cilia-related diseases, comprising the steps of: contacting renal organoids obtained by inducing differentiation of induced pluripotent stem (iPS) cells obtained from somatic cells derived from a patient with a cilia-related disease, or renal constituent cells contained therein, such as renal tubular cells (including renal tubular epithelial cells; the same applies below), with a drug candidate substance; and measuring calcium dynamics and / or cyst formation in the renal organoids or renal constituent cells, such as renal tubular cells. [2] The method according to [1], wherein the somatic cells harbor a mutant PKD (Polycystic Kidney Disease) gene. [3] The method according to [2], wherein the somatic cells are derived from a patient with a cilia-related disease having a mutation in the PKD1 gene and / or the PKD2 gene. [4] The method according to any one of [1] to [3], wherein the cilia-associated disease is autosomal dominant polycystic kidney disease. [5] The method according to any one of [1] to [4], comprising inducing cyst formation by stimulation with a cyst formation-promoting substance and then measuring cyst formation. [6] The method according to [5], wherein the cyst formation-promoting substance is forskolin, 8-Bromo-cAMP or blebbistatin. [7] The method according to any of [1] to [6], wherein the measurement of cyst formation is measurement of the size, number, or circularity of the cysts. [8] The method according to any of [1] to [7], wherein an mTOR inhibitor or a CFTR inhibitor is used as a positive control. [9] A method according to any of [1] to [8], in which the measurement results are compared with calcium dynamics and / or cyst formation in renal organoids or renal component cells contained therein obtained by inducing differentiation of iPS cells obtained from somatic cells derived from healthy subjects or iPS cells with a repaired gene mutation.

[10] The method according to any of [1] to [9], wherein the renal organoid is obtained by the following steps: (i) culturing pluripotent stem cells in a medium containing FGF (fibroblast growth factor) 2, BMP (bone morphogenetic protein) 4, a GSK (glycogen synthase kinase)-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 TGF (transforming growth factor) β inhibitor; (iv) culturing the cells obtained in step (iii) in a medium containing FGF2, a GSK-3β inhibitor, BMP7, activin and a ROCK inhibitor; (v) culturing the cells obtained in step (iv) in a medium containing retinoic acid or a derivative thereof, a BMP inhibitor and FGF9; (vi) culturing the cells obtained in step (v) in a medium containing a GSK-3β inhibitor and FGF9; and (vii) A step of forming cell masses by non-adherent culture of the cells obtained in step (vi) and culturing the same.

[11] A cilia-associated disease model comprising kidney organoids obtained by inducing differentiation of iPS cells obtained from somatic cells derived from a patient with a cilia-associated disease, in which cysts are formed upon stimulation with a cyst formation-promoting substance. Effect of the Invention

[0010] The renal cyst model produced by the present invention reflects the pathology of cilia-related diseases, such as cyst formation and decreased calcium release, in a state closer to that of the living body, and is therefore extremely excellent as an in vitro model of renal cyst formation. By using the renal cyst model of the present invention, it is possible to develop therapeutic agents that suppress renal cyst formation with unprecedented high accuracy. In addition, the pathology model produced by the present invention can be used to elucidate the pathology of cilia-related diseases, such as ADPKD. [Brief description of the drawings]

[0011] [Figure 1] Fluorescence microscopy images showing the results of immunostaining (LTL, ARL13B) for cilia in kidney organoids (10 days of aggregate formation culture) induced to differentiate from iPS cells derived from an ADPKD patient. [Diagram 2] A diagram showing the results of inducing cyst formation when kidney organoids differentiated from iPS cells derived from an ADPKD patient were cultured in the presence of forskolin (10 μM) or blebbistatin (25 μM) (microscope photograph: bright field image). [Diagram 3] Fluorescence microscopy images showing the results of immunostaining (PODXL (glomerular marker), LTL (proximal tubule marker), CDH1 (distal tubule marker)) of kidney organoids containing cysts formed by culturing kidney organoids differentiated from iPS cells derived from an ADPKD patient in the presence of forskolin. The two images are immunostained images of different kidney organoids. [Figure 4] Graph showing the area ratio (cystic area) and circularity of renal cysts when renal organoids (derived from ADPKD patients and healthy individuals) were cultured in the presence of forskolin to induce cyst formation. For each renal organoid, cysts within a certain rank order in terms of size were analyzed, and the horizontal axis shows the number of cysts included in the analysis. [Diagram 5] The photograph shows the results of evaluating the effects of various drugs on forskolin-induced cyst formation. DMSO was used as a control. [Figure 6]Ca flux measurements of proximal tubule cells differentiated from fluorescently labeled human iPS cells (derived from ADPKD patients and healthy individuals). At t=20, 10 μM thapsigargin was administered, and images were taken every 2 seconds until 2 minutes and 40 seconds later (t=180). [Figure 7] A diagram showing the results of inducing cyst formation when renal organoids differentiated from iPS cell lines derived from healthy subjects (Normal Subject-A and B) and from iPS cell lines derived from ADPKD patients (Patient-A and B) were cultured in the presence of forskolin (microscope photograph: bright field image). [Figure 8] A graph showing the results of a statistical test of the renal cyst area ratio (Cystic Area) when renal organoids differentiated from iPS cell lines derived from healthy subjects (Normal Subject-A and B) and from iPS cell lines derived from ADPKD patients (Patient-A and B) were cultured in the presence of forskolin to induce cyst formation. [Figure 9] Renal organoids containing cysts formed by culturing renal organoids differentiated from an iPS cell line derived from an ADPKD patient (Patient-A) in the presence of forskolin were analyzed by immunostaining to show time-dependent changes in expression of markers (same markers as in Figure 3). Fluorescence microscopy images (top row) and corresponding light microscopy images (bottom row) stained with hematoxylin and eosin. [Figure 10] A graph showing the results of a statistical test of the renal cyst area ratio (cystic area) when renal organoids induced to differentiate from an iPS cell line derived from an ADPKD patient (Patient-A) were cultured in the presence of forskolin and various drugs to induce cyst formation. As a control, only forskolin was added. [Figure 11] A diagram showing the results of inducing cyst formation when renal organoids differentiated from iPS cell lines (wild-type) derived from healthy individuals and from mutant iPS cell lines (PKD1 heterozygous line (PKD1+ / -) and PKD1 homozygous line (PKD1- / -)) were cultured in the presence of forskolin (microscope photograph: bright field image). [Figure 12]A graph showing the results of a statistical test of the renal cyst area ratio (Cystic Area) when renal organoids differentiated from iPS cell lines derived from healthy individuals (wild type) and mutant iPS cell lines (PKD1 heterozygous line (PKD1+ / -) and PKD1 homozygous line (PKD1- / -)) were cultured in the presence of forskolin to induce cyst formation. [Figure 13] Fluorescence microscopy images showing the results of immunostaining analysis of marker expression in kidney organoids containing cysts formed by culturing kidney organoids differentiated from a mutant iPS cell line (PKD1 homozygous line (PKD1- / -)) in the presence of forskolin. The two images are immunostained images of the same kidney organoid, and the image on the right is an enlarged image of the image on the left. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The screening or evaluation method of the present invention for a therapeutic agent for cilia-related disease includes the steps of contacting renal organoids obtained by inducing differentiation of iPS cells obtained from somatic cells derived from a patient with cilia-related disease, or renal constituent cells contained therein, such as renal tubular cells, with a drug candidate substance, and measuring calcium dynamics and / or cyst formation in the renal organoids or renal tubular cells.

[0013] Cilia-related disorders include diseases called ciliopathy, which include diseases in which cysts form in the kidneys, and more specifically, autosomal dominant polycystic kidney disease (ADPKD), autosomal recessive polycystic kidney disease (ARPKD), and nephronophthisis.

[0014] These diseases are generally caused by genetic abnormalities. For example, the causative genes for ADPKD include the PKD1 gene and the PKD2 gene. The PKD1 gene encodes a membrane protein "Polycystin (PC)1, transient receptor potential channel interacting" (Ensembl gene ID: ENSG00000008710) (formerly known as polycystic kidney disease 1 (autosomal dominant)). PKD1 can be, for example, a protein having an amino acid sequence registered in the UniProtKB database under accession number P98161. However, since the amino acid sequence of PKD1 varies depending on race, etc., it is not limited to this specific amino acid sequence, and may be an amino acid sequence having 80% or more, 90% or more, 95% or more, or 98% or more identity with the amino acid sequence. The PKD2 gene encodes "polycystin (PC) 2, transient receptor potential cation channel" (Ensembl gene ID: ENSG00000118762) (formerly known as polycystic kidney disease 2 (autosomal dominant)). PKD2 can be, for example, a protein having an amino acid sequence registered in the UniProtKB database under accession number Q13563. However, since the amino acid sequence of PKD2 varies depending on race, etc., it is not limited to this specific amino acid sequence, and may be an amino acid sequence having 80% or more, 90% or more, 95% or more, or 98% or more identity with the amino acid sequence.

[0015] These PKD gene mutations have been reported, for example, in Cell 77: 881-94, 1994 and Science 272: 1339-1342, 1996. In addition, the mutations may be those listed in Table 1 of Ameku et al. Sci Rep 2016;6:30013. PKD1 W429S, S2235L, V3008M, S3404Y, S3405Y, Missense G3818R, Missense E2111K, R2327W, Missense Q3895X Nonsense PKD2 A190T, Missense

[0016] Therefore, it is preferable that patients with cilia-related diseases have mutations in these genes (including missense mutations, nonsense mutations, frameshift mutations, etc.). Heterozygous individuals having a mutation in one of the alleles may be used, but homozygous individuals having mutations in both alleles may also be used. For example, in the case of ADPKD, heterozygous individuals develop the disease and homozygous individuals are not born, so heterozygous individuals are preferred, whereas in the case of ARPKD and nephronophthisis, homozygous individuals develop the disease and homozygous individuals are preferred.

[0017] When producing iPS cells from somatic cells derived from a patient with a cilia-related disorder, the patient's somatic cells may be used as is to produce iPS cells, but it is also possible to reproduce "iPS cells derived from somatic cells derived from a patient with a cilia-related disorder" by introducing a mutant PKD gene derived from a patient with a cilia-related disorder in a heterozygous or homozygous form into iPS cells derived from somatic cells derived from a healthy individual (also called normal iPS cells), and then use the resulting "iPS cells derived from somatic cells derived from a patient with a cilia-related disorder."

[0018] Somatic cells are not particularly limited, but include not only mature somatic cells, but also fetal (baby) and neonatal (baby) 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 mucosa cells, intestinal cells, spleen cells, pancreatic cells (exocrine pancreatic cells, etc.), brain cells, lung cells, kidney cells, and adipocytes.

[0019] The method for producing iPS cells can employ methods known in the art, for example, by introducing reprogramming factors into somatic cells. Here, 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, or Glis1, and 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, WO 2009 / 126655, WO2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 050626, WO2010 / 056831, WO2010 / 0689 55, WO2010 / 098419, WO2010 / 102267, WO2010 / 111409, WO2010 / 111422, WO2010 / 115050, WO2010 / 124290, WO2010 / 147395, WO2010 / 147612, Huangfu D, et al.(2008), Nat.Biotechnol.,26:795-797, Shi Y,et al.(2008),Cell Stem Cell,2:525-528, Eminli S,et al.(2008),Stem Cells.26:2467-2474, Huangfu D,et al. al. (2008), Nat. Biotechnol. 26:1269-1275, Shi Y, etal.(2008),Cell Stem Cell,3,568-574,Zhao Y,et al.(2008),Cell Stem Cell,3:475-479,Marson A,(2008),Cell Stem Cell,3,132-135,Feng B,et al.(2009),Nat.Cell Biol.11:197-203, RLJudson et al.,(2009), Nat.Biotechnol.,27:459-461, Lyssiotis CA,et al.(2009),Proc Natl Acad Sci US A.106:8912-8917, Kim JB,et al. (2009), Nature.461:649-643, Ichida JK, et al. (2009), Cell Stem Examples of the combinations include those described in Cell. 5: 491-503, Heng JC, et al. (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, and Maekawa M, et al. (2011), Nature. 474: 225-9.

[0020] A known method can be used to induce differentiation of iPS cells into renal organoids, but preferably, the method described in Patent Document 1, which includes the following steps (i) to (vi), can be used. (i) culturing iPS 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 inhibitor; (v) culturing the cells obtained in step (iv) in a medium containing retinoic acid or a derivative thereof, a BMP inhibitor and FGF9; (vi) culturing the cells obtained in step (v) in a medium containing a GSK-3β inhibitor and FGF9; and (vii) A step of forming cell masses by non-adherent culture of the cells obtained in step (vi) and culturing them.

[0021] Steps (i) to (vi) are preferably carried out by adhesion culture. Adhesion culture means that cells are cultured in a state where they are attached to a culture substrate, for example, on a coated culture dish. The coating agent is preferably an extracellular matrix, and examples thereof include collagen, proteoglycan, fibronectin, hyaluronic acid, tenascin, entactin, elastin, fibrin, and laminin, or fragments thereof. These extracellular matrices may be used in combination, and may be preparations from cells, such as BD Matrigel (trademark). The extracellular matrix is ​​preferably laminin or a fragment thereof. In the present invention, laminin is a protein having a heterotrimeric structure with one α chain, one β chain, and one γ chain, and is an extracellular matrix protein with isoforms having different subunit chain compositions. Laminin has about 15 isoforms, which are a combination of heterotrimers of five α chains, four β chains, and three γ chains. Examples of the α chain include, but are not limited to, α1, α2, α3, α4, or α5, the β chain includes β1, β2, β3, or β4, and the γ chain includes γ1, γ2, or γ3. More preferably, laminin is laminin 511 consisting of α5, β1, and γ1 (Nat Biotechnol 28, 611-615 (2010)). Laminin may be a fragment, and is not particularly limited as long as it has integrin binding activity, and may be, for example, E8 fragment (laminin 511E8) obtained by digestion with elastase (EMBO J., 3:1463-1468, 1984, J. Cell Biol., 105:589-598, 1987, WO2011 / 043405). Laminin 511E8 is commercially available and can be purchased from, for example, Nippi Corporation.

[0022] The medium used in each step can be prepared by adding cytokines and drugs required in each step 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) 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) (serum replacement for ES cell culture) (Thermo Fisher Scientific), N2 supplement (Thermo Fisher Scientific), B27 supplement (Thermo Fisher Scientific), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, and may also contain one or more substances such as lipids, amino acids, L-glutamine, GlutaMAX (Thermo Fisher Scientific), non-essential amino acids (NEAA), vitamins, growth factors, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, and the like. A medium previously optimized for stem cell culture, such as ReproFF2 (ReproCell), may also be used.

[0023] Each step will be described in further detail below.

[0024] (i) culturing iPS cells in a medium containing FGF2, BMP4, a GSK-3β inhibitor, and retinoic acid or a derivative thereof; In step (i), iPS cells can be separated and cultured by methods known in the art, such as mechanical separation and separation using a separation solution having protease activity and collagenase activity (e.g., Accutase™ and Accumax™ (Innovative Cell Technologies, Inc.)) or a separation solution having only collagenase activity.

[0025] The GSK-3β inhibitor used in step (i) is not particularly limited as long as it can inhibit the function of GSK-3β, for example, kinase activity, and examples thereof include BIO (also known as GSK-3β inhibitor IX; 6-bromoindirubin-3'-oxime), which is an indirubin derivative, SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), which is a maleimide derivative, GSK-3β inhibitor VII (α,4-dibromoacetophenone), which is a phenyl-α-bromomethyl ketone compound, L803-mts (GSK-3β peptide inhibitor), which is a cell membrane-permeable phosphorylated peptide, and CHIR99021 (Nature (2008) 453: 519-523), which has high selectivity. These compounds are available, for example, from Stemgent, Calbiochem, Biomol, etc. An example of the GSK-3β inhibitor 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.

[0026] The FGF2 (basic FGF: bFGF) used in step (i) is preferably human FGF2, and an example of human FGF2 is a protein having an amino acid sequence of NCBI (National Center for Biotechnology Information) accession number: ABO43041.1. FGF2 includes fragments and functional variants 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, more preferably 50 ng / ml to 250 ng / ml.

[0027] The BMP4 used in step (i) is preferably human BMP4, and an example of the human BMP4 is a protein having the amino acid sequence of NCBI accession number: AAH20546.1. BMP4 includes fragments and functional variants as long as it has differentiation-inducing activity. Commercially available BMP4 may be used, or a protein purified from cells or a protein produced by genetic recombination may be used. The concentration of BMP4 used in this step is 0.1 ng / ml to 100 ng / ml, preferably 0.5 ng / ml to 50 ng / ml, more preferably 0.5 ng / ml to 5 ng / ml.

[0028] 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]-benzoic acid (AM580) (Tamura K, et al., Cell Differ. Dev. 32:17-26 (1990)), 4-[(1E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propen-1-yl]-benzoic acid (TTNPB) (Strickland S, et al., Cancer Res. 43:5268-5272 (1983)), and Tanenaga, K. et al., Cancer Res. Res. 40:914-919 (1980), retinol palmitate, retinol, retinal, 3-dehydroretinol, 3-dehydroretinal, and the like. The concentration of retinoic acid or a derivative thereof used in step (i) is, for example, 1 nM to 100 nM, preferably 5 nM to 50 nM, more preferably 5 nM to 25 nM.

[0029] 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 an atmosphere of CO2-containing air. The CO2 concentration is about 2 to 5%, preferably about 5%. The culture time in step (i) may be any period sufficient for inducing differentiation of late posterior epiblasts, and may be, for example, 1 to 2 days, preferably 1 day.

[0030] (ii) culturing the cells obtained in step (i) in a medium containing FGF2, a GSK-3β inhibitor and BMP7; The FGF2 used in step (ii) is the same as that described in step (i), and the preferred concentration range thereof is also the same.

[0031] The GSK-3β inhibitor used in step (ii) may be any of the GSK-3β inhibitors exemplified in step (i) above, and a preferred example of the 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 50 μM, more preferably 1 μM to 20 μM, and particularly preferably 2 to 10 μM. It is preferable to increase the concentration of the GSK-3β inhibitor used in step (ii) from that in step (i).

[0032] The BMP7 used in step (ii) is preferably human BMP7, and an example of human BMP7 is a protein having the amino acid sequence of NCBI accession number: NM_001719.2. BMP7 includes fragments and functional variants thereof as long as it has differentiation-inducing activity. Commercially available BMP7 may be used, or a protein purified from cells or a protein produced by genetic recombination may be used. The concentration of BMP7 used in this step is 0.1 ng / ml to 100 ng / ml, preferably 0.5 ng / ml to 50 ng / ml, more preferably 0.5 ng / ml to 5 ng / ml.

[0033] In step (ii), 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 (ii) may be any period sufficient for inducing differentiation of the mesodermal lineage primitive streak, and is, for example, 10 hours to 2 days, or 1 to 2 days, preferably 0.5 to 1 day.

[0034] (iii) culturing the cells obtained in step (ii) in a medium containing FGF2, a GSK-3β inhibitor, BMP7 and a TGFβ inhibitor; 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.

[0035] 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 the binding to the ALK family receptor, or substances that inhibit the phosphorylation of SMAD by the ALK family. Examples of such substances include Lefty-1 (NCBI Accession No.: mouse: NM_010094, human: NM_020997), SB431542, SB202190 (RK Lindemann et al., Mol. Cancer, 2003, 2:20), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276 (Lilly Research Laboratories), and A83-01 (WO2009146408). and 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, but is 0.5 μM to 100 μM, preferably 1 μM to 50 μM, and more preferably 5 μM to 25 μM.

[0036] 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 a period sufficient for inducing differentiation of the mesodermal lineage late primitive streak, and is, for example, 0.25 to 3 days, preferably 0.5 to 2 days, and more preferably 0.75 to 1.5 days.

[0037] (iv) culturing the cells obtained in step (iii) in a medium containing FGF2, a GSK-3β inhibitor, BMP7, activin and a ROCK inhibitor; 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.

[0038] The activin used in step (iv) includes activins derived from humans and other animals and their functional variants, but activin A is preferred, and human activin A is more preferred. Examples of human activin A include proteins having the amino acid sequence of NCBI accession number: NP_002183.1 or UniProt accession number P08476.2. For example, commercially available products from R&D systems, etc. can be used. 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.

[0039] The ROCK inhibitor used in step (iv) is not particularly limited as long as it can suppress the function of Rho-kinase (ROCK), and examples thereof include Y-27632 (see, e.g., Ishizaki et al., Mol. Pharmacol. 57, 976-983 (2000); Narumiya et al., Methods Enzymol. 325, 273-284 (2000)), Fasudil / HA1077 (see, e.g., Uenata et al., Nature 389:990-994 (1997)), H-1152 (see, e.g., Sasaki et al., Pharmacol. Ther. 93:225-232 (2002)), Wf-536 (see, e.g., Nakajima et al., Cancer Chemother 1999, 2003), and the like. Pharmacol. 52(4):319-324(2003)) and derivatives thereof, as well as antisense nucleic acids against ROCK, RNA interference-inducing nucleic acids (eg, siRNAs), dominant negative mutants, and expression vectors thereof. 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). 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.

[0040] In step (iv), 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 (iv) may be any period sufficient for inducing differentiation of the metanephric lineage late primitive streak, and may be, for example, 1 to 5 days, preferably 3 days.

[0041] (v) culturing the cells obtained in step (iv) in a medium containing retinoic acid or a derivative thereof, a BMP inhibitor and FGF9; The retinoic acid or derivative thereof used in step (v) is as described in step (i), and its preferred concentration range is, for example, 10 nM to 500 nM, preferably 50 nM to 250 nM.

[0042] The FGF9 used in step (v) is preferably human FGF9, and an example of the human FGF9 is a protein having the amino acid sequence of NCBI accession number: NP_002001.1. FGF9 includes fragments and functional variants thereof as long as they have 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, 10 ng / ml to 500 ng / ml, 50 ng / ml to 300 ng / ml, or 150 ng / ml to 250 ng / ml.

[0043] The medium used in step (v) further comprises a BMP inhibitor. Examples of BMP inhibitors include protein inhibitors such as Chordin, Noggin, and Follistatin, Dorsomorphin (i.e., 6-[4-(2-piperidin-1-yl-ethoxy)phenyl]-3-pyridin-4-yl-pyrazolo [1,5-a]pyrimidine), its derivatives (PB Yu et al. (2007), Circulation, 116:II_60; PB Yu et al. (2008), Nat. Chem. Biol., 4:33-41; J. Hao et al. (2008), PLoS ONE, 3(8):e2904), and LDN193189 (i.e., 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline). The BMP inhibitor is more preferably NOGGIN, and the concentration thereof is, for example, 1 ng / ml to 100 ng / ml, 5 ng / ml to 50 ng / ml, or 10 ng / ml to 30 ng / ml.

[0044] 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 for inducing differentiation of late posterior intermediate mesoderm, and may be, for example, 1 to 3 days, preferably 2 days.

[0045] (vi) culturing the cells obtained in step (v) in a medium containing a GSK-3β inhibitor and FGF9; The GSK-3β inhibitor and FGF9 used in step (vi) are as described in step (i) and step (v), respectively, and their preferred concentration ranges are also the same.

[0046] The number of days for culture in step (vi) is not particularly limited as long as renal progenitor cells can be obtained, and may be, for example, 2 or more days, 3 or more days, 4 or more days, or 5 or more days. 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 the CO2 concentration being preferably about 2 to 5%.

[0047] (vii) forming a cell mass by non-adherent culture of the cells (renal progenitor cells) obtained in step (vi); Methods for obtaining renal organoids from renal progenitor cells, i.e., cell aggregates containing glomeruli, tubules, collecting ducts, blood vessels, and interstitial tissues, include, for example, a method of non-adherently culturing renal progenitor cells obtained by the above method to prepare cell masses, and co-culturing them with feeder cells such as 3T3-Wnt4 cells, mouse fetal spinal cord cells, or mouse fetal kidney cells, or a method of performing semi-aerobic culture using a basal medium containing a GSK-3β inhibitor such as CHIR99021 (Reference Nature, 526, 564-568 (2015)). In addition to the GSK-3β inhibitor, the medium can contain FGF9, FGF2, and even a ROCK inhibitor. The preferred concentrations of FGF9, FGF2, and ROCK inhibitor are the same as those described above. In addition, in some embodiments where semi-aerobic culture is performed, examples of the method include a method including including inclusion of a GSK-3β inhibitor or FGF2 in the medium only for the number of days from the start of the culture until 1 day, from the start of the culture until 2 days, or from the start of the culture until 3 days.

[0048] The number of days for culture in step (vii) is not particularly limited as long as kidney organoids are formed, but may be, for example, 5 days or more, 6 days or more, 7 days or more, or 8 days or more. 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 under an atmosphere of CO2-containing air, and the CO2 concentration is preferably about 2 to 5%.

[0049] The kidney organoids obtained as described above can be used to screen and evaluate drugs for treating cilia-related diseases.

[0050] In the screening method of the present invention, any test substance can be used, for example, cell extracts, cell culture supernatants, microbial fermentation products, extracts from marine organisms, plant extracts, purified or crude proteins, peptides, non-peptide compounds, synthetic small molecule compounds, and natural compounds. In the present invention, the test substance can also be obtained using any of the many approaches in combinatorial library methods known in the art, including (1) biological library methods, (2) synthetic library methods using deconvolution, (3) "one-bead one-compound" library methods, and (4) synthetic library methods using affinity chromatography selection. The biological library methods using affinity chromatography selection are limited to peptide libraries, while the other four approaches can be applied to peptide, non-peptide oligomer, or small molecule compound libraries of compounds (Lam (1997) Anticancer Drug Des. 12: 145-67). Examples of methods for the synthesis of molecular libraries can be found in the art (DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6909-13; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91: 11422-6; Zuckermann et al. (1994) J. Med. Chem. 37: 2678-85; Cho et al. (1993) Science 261: 1303-5; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2061; Gallop et al. (1994) J. Med. Chem. 37:1233-51).Compound libraries may be prepared in solution (see Houghten (1992) Bio / Techniques 13: 412-21) or on beads (Lam (1991) Nature 354: 82-4), chips (Fodor (1993) Nature 364: 555-6), bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698, 5,403,484, and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1865-9), or phages (Scott and Smith (1990) Science 249: 386-90; Devlin (1990) Science 249: 404-6; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87: 6378-82; Felici (1991) J. Mol. Biol. 222: 301-10; U.S. Patent Application No. 2002103360).

[0051] By administering a cyst formation-promoting substance to the renal organoid obtained as described above for a certain period of time, for example, 1 to 30 days, preferably 5 to 15 days, renal cyst formation can be induced, and this can be used as a cilia-related disease model.

[0052] Examples of cyst formation promoters include cAMP pathway activators such as forskolin and 8-Bromo-cAMP, and myosin II inhibitors such as blebbistatin. The concentration of forskolin is preferably 1 to 100 μM, more preferably 5 to 20 μM. The concentration of 8-Bromo-cAMP is preferably 10 to 1000 μM, more preferably 50 to 200 μM. The concentration of blebbistatin is preferably 1 to 100 μM, more preferably 10 to 50 μM.

[0053] When screening or evaluating drugs using cyst formation as an index, for example, when renal organoids are cultured in the presence of a cyst formation promoter such as forskolin, and / or before and after, a test substance is contacted and cultured for a certain period of time (for example, 1 hour to 72 hours), and then cyst formation is measured and compared with the case where the test substance is not contacted. Measurement of cyst formation includes not only measurement of cyst size and number, but also measurement of cyst shape such as roundness. Cyst size can be measured, for example, by the area ratio described below.

[0054] When renal organoids induced to differentiate from patient-derived iPS cells are cultured in the presence of a cyst formation promoter such as forskolin, the number of cysts increases, the size increases, and the circularity decreases significantly compared to renal organoids induced to differentiate from iPS cells derived from healthy subjects. Here, when the number of cysts decreases, the size decreases, or the circularity improves when the test substance is contacted, the test substance can be evaluated as having the ability to reduce or inhibit cyst formation, or can be selected as a candidate substance for a cilia-related disease treatment drug.

[0055] The effect of the test substance can also be evaluated by comparing it with the effect of a positive control having a cyst formation inhibitory effect, which can be an mTOR inhibitor such as rapamycin or everolimus, or a CFTR inhibitor such as CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) inhibitor II or CFTR inhibitor 172.

[0056] On the other hand, drugs can be screened and evaluated by measuring intracellular calcium dynamics using kidney organoids. For measuring intracellular calcium dynamics, it is preferable to isolate and use renal constituent cells such as tubular epithelial cells and proximal tubular cells from kidney organoids. Isolation of renal tubular epithelial cells from kidney organoids can be performed, for example, by sorting using an antibody against a renal tubular epithelial cell marker such as CD326 (EpCAM). Isolation of proximal tubular cells from kidney organoids can be performed, for example, by sorting using an antibody against a proximal tubular cell marker such as CD10 or CD13 (PLoS ONE 8: e66750.2013), or a specific lectin against proximal tubular cells such as LTL (Lotus tetragonolobus Lectin). Alternatively, intracellular calcium dynamics can be measured in the state of kidney organoids. By using kidney organoids that endogenously express Ca indicator in specific kidney constituent cells, intracellular calcium dynamics can also be measured using the change in fluorescence intensity in specific kidney constituent cells as an index.

[0057] The intracellular calcium kinetics can be measured according to a conventional method. Specifically, calcium-sensitive dyes (calcium indicators) such as Fura-2 (Dojindo Laboratories), Fluo4, Fluo3, Fura2, Indo1, Rhod2, Quin2, Fura-PE3, Fura Red, calcium green1, calcium crimson, Oregon green 488, BAPTA-1, fluo-3FF, fluo-5N, mag-fura-5, mag-indo-1, rhod-5N, Calbryte-590, and Cal-520 (RY Tsien, Methods Cell Biol., 1989, 30, 127) are loaded into cells, and the intracellular calcium dynamics is measured using the change in fluorescence intensity as an index with a measuring device such as FDSS (Hamamatsu Photonics). It is preferable to measure both the process of measuring calcium release by a drug and the process of measuring calcium influx from outside the cell afterwards.

[0058] In renal component cells such as tubular epithelial cells and proximal tubule cells obtained from renal organoids prepared by inducing differentiation from iPS cells derived from disease patients, calcium release by Thapsigargin was reduced compared to renal component cells such as tubular epithelial cells and proximal tubule cells obtained from renal organoids prepared by inducing differentiation from iPS cells derived from healthy subjects.

[0059] Renal constituent cells, such as tubular epithelial cells and proximal tubular cells, obtained from kidney organoids prepared by inducing differentiation from iPS cells derived from a patient with a disease are incubated with a test substance for a certain period of time (e.g., 1 hour to 72 hours), and then the intracellular calcium concentration is measured. If the calcium concentration increases compared to when not incubated with the test substance or when incubated with a negative control, the test substance can be evaluated as having the ability to increase the intracellular calcium concentration, and can be selected as a candidate substance for a therapeutic drug for cilia-related diseases.

[0060] The effect of the test substance can also be evaluated by comparing it with the effect of a positive control that has the effect of increasing intracytoplasmic calcium concentration.

[0061] In the present invention, it is preferable to compare the measurement results of kidney organoids or kidney constituent cells such as tubular epithelial cells and proximal tubular cells induced to differentiate from iPS cells derived from a disease patient with the measurement results of kidney organoids or kidney constituent cells such as tubular epithelial cells and proximal tubular cells induced to differentiate from gene mutation repaired iPS cells. It is also preferable to compare the measurement results of kidney organoids or kidney constituent cells such as tubular epithelial cells and proximal tubular cells induced to differentiate from a strain in which a PKD gene mutation is introduced into normal iPS cells with the measurement results of kidney organoids or kidney constituent cells such as tubular epithelial cells and proximal tubular cells induced to differentiate from normal iPS cells, which are the parent strain.

[0062] The above-mentioned gene mutation repaired iPS cells refer to iPS cells in which the mutated site of the causative gene of a patient with a cilia-related disease has been replaced with a normal sequence, and iPS cells have the same genetic information as the patient with a cilia-related disease, except that the mutated site has been replaced with a normal sequence. By comparing with kidney organoids or kidney constituent cells such as tubular epithelial cells and proximal tubular cells from such gene repaired iPS cells, the effect of a drug on a cilia-related disease can be evaluated more accurately. Gene mutation repaired iPS cells can be prepared by known gene repair techniques. For example, this can be done by replacing the causative gene of a cilia-related disease in a patient-derived iPS cell with a normal type using genome editing techniques such as CRISPR / Cas9, TALEN, and ZFN. Specifically, in the case of CRISPR / Cas9, this can be done by introducing an oligonucleotide having a normal type sequence into a patient-derived iPS cell together with Cas9 and gRNA, and replacing the mutant sequence. EXAMPLES

[0063] The present invention will be specifically described below by way of examples, but the aspects of the present invention are not limited to the following examples.

[0064] Example 1 <Experimental Procedure> Induction of renal progenitor cells from iPS cells iPS cells were generated by introducing OCT4, SOX2, KLF4, and c-MYC genes into somatic cells of an ADPKD patient with a mutation in the PKD1 gene using a retroviral vector (CiRA00009 strain, CiRA00007 strain). The obtained iPS cells were cultured by the method described in the Examples of WO 2018 / 216743 to obtain kidney (nephron) progenitor cells. As a control, kidney (nephron) progenitor cells were obtained in the same manner from iPS cells derived from a healthy individual. The CiRA00009 strain has a Q3895X nonsense mutation, and the CiRA00007 strain has a G3818R missense mutation (Ameku et al. Sci Rep 2016;6:30013).

[0065] In addition, kidney (nephron) progenitor cells were induced in the same manner from the following iPS cells. 1) iPS cells generated by introducing OCT4, SOX2, KLF4, and c-MYC genes into somatic cells of an ADPKD patient with a mutation in the PKD2 gene using a retroviral vector (Ameku et al. Sci Rep 2016;6:30013). 2) iPS cells generated by introducing OCT4, SOX2, KLF4, L-MYC, LIN28, shp53 or p53DD into somatic cells of ADPKD patients with mutations in the PKD1 or PKD2 genes using episomal vectors. 3) iPS cells generated by introducing mutant PKD1 and PKD2 genes into iPS cells derived from healthy human cells.

[0066] Renal organoid generation and drug-induced kidney organoid cyst formation assay (Experimental Method) 1. As described above, nephron progenitor cells were produced from undifferentiated human iPS cells (derived from patients or healthy individuals) using the differentiation induction system described in the Examples of WO 2018 / 216743, and then used to produce renal organoids by the following procedure. 2. Day 11-12: Dissociate nephron progenitor cells into single cells using Accumax. Add 100 μL of Accumax per well of a 24-well plate and incubate at 37℃, 5% CO2 for 10-15 minutes. Add 900 μL of 10% FBS to stop the reaction, then gently pipette with a P-1000 pipetman to dissociate into single cells. 3. Measure the cell count of the cell suspension. 4. Serum-free medium consisting of DMEM / F12 Glutamax medium (Thermo Fisher Scientific), vitamin A-free B27 supplement (Thermo Fisher Scientific), and 500 U / ml PS was supplemented with 200 ng / ml FGF9 and 1 μM CHIR99021. Add Y-27632 to a final concentration of 10 μM to prepare the medium for cell aggregate preparation. 5. 1.0-1.5 x 10 per cell aggregate 5 Transfer the cell suspension to an Eppendorf tube and centrifuge at 300 G for 3 min. 6. Discard the supernatant and resuspend the cells using the medium prepared in step 4 so that each cell aggregate will have 50-100 μL of medium. 7. Add 50-100 μL of cell suspension to a U-bottom 96 well plate (non-adhesive) and centrifuge at 300 G for 3 min. 8. After 1-2 days of culture, the formed cell aggregates are transferred onto the inserts of 24-well Transwells and cultured at the air-liquid interface (day 0: organoid culture begins). The medium is KR5 medium (DMEM / F12 Glutamax containing 0.1 mM non-essential amino acids, 500 U / ml PS, 55 μM 2-mercaptoethanol and 5% KSR) supplemented with 200 ng / ml FGF2 and 5 μM CHIR99021 (medium is replaced at 120 μL per well). After 9.48 hours, the medium was replaced with KR5 medium (day 2), and the medium was replaced with KR5 medium every 2 days thereafter. By day 8, the cells were differentiated into nephron organoids with glomerular and tubule-like structures. 10. Cyst formation induction and cyst measurement were performed as follows. From day 8, change the medium to KR5 medium supplemented with 10 μM forskolin (or 100 μM 8-Br-cAMP) (medium change every 2 days). Use the same concentration of DMSO as a control. 11. Cultivation was continued until day 15, and phase contrast images were taken using a KEYENCE BZ-X700 to obtain image data. 12. Image data is analyzed using the IN Cell Developer Toolbox (GE Healthcare). The cyst area ratio to the entire organoid and the circularity of the cyst are used as indicators of cysts. A protocol that limits the analysis to only those cysts that fall within a certain rank order in terms of size is useful. 13. The effects of CFTR inhibitors (Yang B et al. JASN 2008) and mTOR inhibitors (Shillingford J et al. Proc Natl Acad Sci USA 2006) on cyst formation were examined. Three hours before starting forskolin on day 8, 100 μM CFTR inhibitor 172, 50 μM CFTR inhibitor II, 10 μM rapamycin or 10 μM everolimus were preincubated. After that, the medium was changed to contain 10 μM forskolin along with these drugs, and the medium was changed every two days. Using the protocol described above, the area ratio and circularity of the cyst part on day 15 were measured.

[0067] Ca flux measurement in human iPS cell-derived renal tubular epithelial cells (Experimental Method) 1. Prepare kidney organoids on Transwell plates according to steps 1-9 above. By continuing to replace the medium with KR5 medium, kidney organoids can be maintained until day 12. 2. Collect kidney organoids on day 8-12 into an Eppendorf tube. Using a P-1000 pipette, spray PBS(-) at moderate strength to detach the organoids adhering to the insert, and collect approximately 10 organoids per 1.5 mL Eppendorf tube. 3. Discard the PBS(-) supernatant. 4. Add 500 μL of TrypLE select (Thermo Fisher Scientific) and incubate for 30 minutes at 37℃, 5% CO2. At approximately 15 minutes, perform gentle pipetting using a P-1000 pipette. 5.Add 500 μL of 10% FBS and dissociate into single cells by gentle pipetting. 6. After passing through a cell strainer (35 μm), the number of cells is counted. 7. EpCAM CD326 (EpCAM) MicroBeads (Miltenyi, 130-061-101) were used. + Enrich renal tubular epithelial cells using MACS (Forbes T et al. Am J Hum Genet 2018). While following the manufacturer's protocol, adjust the amount of buffer appropriately depending on the number of cells. As previously reported, EpCAM + It is possible to obtain sufficient purification of cells (98.5%). 8. Add Y-27632 to Renal Epithelial Cell Growth Medium 2, RECGM2 (PromoCell) to a final concentration of 50 μM, and then add 5.0 × 10 4 Prepare a cell suspension at 50 μL per cell. Add 5.0 × 10 cells per well to a clear-bottom, half-area 96-well plate (Greiner, 675090). 4 EpCAM with cells (50μL) + Cells are seeded and incubated overnight at 37° C., 5% CO2. 9. The next day, the cells will be confluent, so measure Ca flux using the FDSS-μCELL. Add the Ca indicator Cal-520 (AAT Bioquest, 21131) to 4 μM and water soluble probenecid (Thermo Fisher Scientific, P36400) to RECGM2 to make the final concentration 2.5 mM. Add 50 μL of this RECGM2 mixture to each well (final concentrations are Cal-520 2 μM and probenecid 1.25 mM). Incubate at 37℃, 5% CO2 for 30 minutes. 10. Immediately before measurement, wash each well with 200 μL of HBSS (free of Ca, Mg, and phenol red). Then, add 80 μL of HBSS (free of Ca, Mg, and phenol red) to each well. At this time, you may add 1.25 mM water-soluble probenecid and 0.05 to 0.5 mg / mL Acid Red 27 (Tokyo Kasei, A0583) as a quencher. 11. Set the FDSS-μCELL imaging protocol to measure Ca influx after Ca release in two stages of drug solution injection (20 μL each). 12. In the first step, drugs that induce Ca release, such as thapsigargin (final concentration 10 μM), Angiotensin II, human (final concentration 10 μM), and [Arg8]-Vasopressin (final concentration 10 μM), are dissolved in Ca-free HBSS and injected. In the second step, store-operated Ca influx is measured by injecting CaCl2 at a final concentration of 2 mM (measure the ratio of fluorescence intensity to baseline). 13. As a positive control, a final concentration of 5 μM ionomycin was used in the first step, and a final concentration of 30 mM EGTA was used in the second step to demonstrate that changes in intracellular Ca concentration could be measured. 14. Analyze the peak value, AUC (area under the curve) value of the first phase (Ca release), and the peak value, AUC value, and plateau value of the second phase (Ca influx).

[0068] Measurement of Ca flux in fluorescently labeled human iPS cell-derived proximal tubule cells (Experimental Method) 1. Prepare cells from nephron organoids on days 8-12 in the same manner as steps 1-6 above. 2. Add Y-27632 to Renal Epithelial Cell Growth Medium 2, RECGM2 (PromoCell) to a final concentration of 50 μM, and then dilute 1.0 × 10 5Prepare a cell suspension at 100 μL per cell. Add 1.0 × 10 cells per well of a 96-well plate with clear bottom. 5 Seed cells with cell (100 μL) and incubate overnight at 37℃, 5% CO2. Coating of the 96-well plate is not necessary. 3. The next day, the cells will be confluent and Ca flux will be measured using an IN Cell Analyzer 6000 (GE Healthcare). 4. To label proximal tubule cells, add 20 μM 6-carboxyfluorescein (6-CF) to RECGM2 and add 100 μL to each well (final concentration 10 μM). Incubate at 37℃, 5% CO2 for 1 hour. After 1 hour, add 4 μM of the Ca indicator Calbryte-590 (AAT Bioquest, 20700) and 2.5 mM of water soluble probenecid (Thermo Fisher Scientific, P36400) to RECGM2, and replace the medium with 100 μL per well. Incubate at 37℃, 5% CO2 for 1 hour. 6. Immediately before measurement, wash each well with 200 μL of HBSS (without Ca, Mg, or phenol red), then add 100 μL of HBSS (without Ca, Mg, or phenol red) to each well. 7. Measure Ca flux by injecting two drug solutions (100 μL each) using the Liquid Handling function of the IN Cell Analyzer 6000. After the first step of imaging is completed, reduce the amount of solution in the well to 100 μL using a pipette. 8. By limiting the measurement of Ca flux to cells fluorescently labeled with 6-CF, it is possible to measure Ca flux in proximal tubule cells. Measurements are performed in the same manner as in 12-13 above.

[0069] <Result> As shown in Figure 1, renal organoids containing renal tubules could be generated from iPS cell lines derived from ADPKD patients, and primary cilia were confirmed in the renal tubules based on the expression of the proximal tubule marker LTL and the cilia marker ARL13B. When forskolin or blebbistatin was added on the 8th day after the start of organoid culture and the culture was continued, the formation of renal cysts was observed on the 15th day (Figure 2). When confirmed by marker staining, the glomerular-derived cysts were hardly noticeable, and the tubular-derived cysts were confirmed (Figure 3). Note that by treating with forskolin, the increase in cysts in nephron organoids was actually observed until around day 30-40.

[0070] The area ratio and circularity of the renal cysts were measured using an image analyzer. The area ratio was calculated as follows. Renal cyst area ratio (%) = total renal cyst area / total organoid area × 100 The results are shown in Figure 4. As described above, the area ratio of renal cysts was significantly increased in patient-derived renal organoids, and the circularity was lower (cysts were irregular) than in healthy subjects, demonstrating that quantitative evaluation of renal cyst formation and drug effects is possible.

[0071] Next, we investigated the effects of drugs on forskolin-induced cyst formation. As a result, as shown in Figure 5, several drugs, especially mTOR inhibitors and CFTR inhibitors, suppressed forskolin-induced renal cyst formation .

[0072] It has also been reported that calcium influx into cells is reduced in kidney cells from patients with the disease compared to those from healthy individuals. Using the above-mentioned calcium influx measurement system, we can reproduce the reduced calcium influx in tubular cells derived from ADPKD patient iPS cells, and by adding drugs to these cells, we can evaluate the effects of drugs on the reduced calcium influx. The results in Figure 6 show that calcium release from the endoplasmic reticulum by thapsigargin is reduced and the time to peak tends to be slower in the proximal tubular cells and tubular epithelial cells of kidney organoids induced from ADPKD patient iPS cell lines compared to the proximal tubular cells and tubular epithelial cells of kidney organoids induced from ADPKD patient iPS cell lines.

[0073] Example 2 In the same manner as in Example 1, iPS cells were produced from ADPKD patients (Patient-A, B). It should be noted that Patient-A and Patient-B correspond to CiRA00009 and CiRA00007 in Example 1, respectively. Using the prepared iPS cells, kidney organoids were induced from iPS cells derived from ADPKD patients (Patient-A, B) according to the same procedure as in Example 1. Renal organoids were also induced in the same manner from healthy subjects (Normal Subjects-A and B).

[0074] From day 10 of organoid culture, 10 μM forskolin was added, and cyst formation was evaluated on day 17. The results are shown in Figure 7. It was confirmed that forskolin caused cyst formation in kidney organoids differentiated from iPS cell lines derived from all patients.

[0075] The results of the statistical test of the renal cyst area ratio (Cystic Area) are shown in Figure 8. Renal organoids differentiated from iPS cell lines derived from both Patient-A (nonsense) and Patient-B (missense) showed a statistically significant increase in cysts compared to renal organoids differentiated from iPS cell lines derived from healthy subjects.

[0076] In addition, we evaluated the time course of cyst formation when 10 μM forskolin was added to renal organoids differentiated from an iPS cell line derived from an ADPKD patient (Patient-A). The results are shown in Figure 9. These results show that in renal organoids differentiated from an iPS cell line derived from an ADPKD patient, tubule expansion began just 1 hour after the start of forskolin administration. After 6 hours, the difference between non-dilated tubules and tubules with dilated and flattened epithelium became clear.

[0077] Next, to evaluate the effect of various drugs on cyst formation by forskolin, renal organoids were induced from an iPS cell line derived from an ADPKD patient (Patient-A), and various drugs were added together with 10 μM forskolin from the 10th day of organoid culture, and the renal cyst area ratio (cystic area) was measured at the 17th day. The results are shown in Figure 10. Tolvaptan (a clinically used drug to suppress cyst growth in ADPKD) did not show a statistically significant difference in cyst size compared to the control (forskolin alone), but CFTR inhibitor 172 and everolimus statistically significantly suppressed cyst growth.

[0078] Example 3 A mutant PKD1 gene was introduced into an iPS cell line derived from a healthy individual using genome editing technology using CRISPR / Cas9. The mutant PKD1 gene was introduced into the iPS cell line derived from a healthy individual using genome editing technology using CRISPR / Cas9. + / - ) and PKD1 homozygous strain (PKD1 - / - )) was prepared and analyzed.

[0079] Disease-specific iPS cell lines were established using CRISPR / Cas9 genome editing technology as follows. Using the method described by Ishida et al. (Site-specific randomization of the endogenous genome by a regulatable CRISPR-Cas9 piggyBac system in human cells, Sci. Rep. 8 (2018) 310.), a mutation was introduced into the splicing acceptor site of exon 34 of the PKD1 gene by non-homologous end joining (NHEJ). Specifically, the 585A1 strain, a human iPS cell line derived from a healthy individual described in Okita et al. (An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells, Stem Cells 31 (2013) 458-466.), was used as the wild-type strain, and a gRNA expression sequence targeting the splicing acceptor site of exon 34 of the TetO-Cas9-GR gene and the PKD1 gene was introduced into this wild-type strain using a piggyBac transposon vector (Addgene ID 100596, Addgene ID 100598, and a PiggyBac Transposase expression vector). Cas9 activity was then inducibly induced by drug administration to cause genome editing by nonhomologous end joining (NHEJ). After genome editing, the iPS cells were isolated into single colonies by flow cytometry, and then sequence analysis was performed to confirm that a frameshift mutation was present in one allele, determining them as PKD1 heterozygous strains, and those that contained frameshift mutations in both alleles as PKD1 homozygous strains.

[0080] iPS cell lines derived from healthy subjects (wild type) and disease-specific iPS cell lines (PKD1 heterozygous line (PKD1 + / - ) and PKD1 homozygous strain (PKD1- / - )) were induced into renal organoids in the same manner as in Example 2, and 10 μM forskolin was added from the 10th day of organoid culture, and cyst formation was evaluated at the 17th day. The results are shown in FIG. 11. In renal organoids induced to differentiate from iPS cell lines derived from healthy subjects, cysts were not formed by the addition of forskolin, but in renal organoids induced to differentiate from iPS cell lines introduced with mutations, cyst formation was promoted by the addition of forskolin, and the degree of this was remarkable in the PKD1 homozygous strain. This was also confirmed by a statistical test of the renal cyst area ratio (Cystic Area) (FIG. 12).

[0081] In addition, iPS cell lines derived from healthy individuals or disease-specific iPS cell lines (PKD1 homozygous line (PKD1 - / - Renal organoids induced to differentiate from the 10-kDa ovarian tumor cells were cultured in the presence of forskolin, and the renal organoids containing the formed cysts were analyzed by immunostaining. The results are shown in Figure 13. The glomerular cysts were barely noticeable, but the tubular cysts were confirmed.

[0082] Patents, published applications and other publications, GenBank accession numbers and associated sequence information and other data available through databases such as the National Center for Biotechnology Information (NCBI), referred to herein are incorporated by reference in whole or in part.

Claims

1. A method for screening or evaluating drugs for treating autosomal dominant polycystic kidney disease, comprising the steps of: contacting renal organoids obtained by inducing differentiation of induced pluripotent stem (iPS) cells obtained from somatic cells derived from a patient with autosomal dominant polycystic kidney disease having mutations in the PKD1 gene and / or PKD2 gene with a candidate drug substance; and measuring cyst formation in the renal organoids, wherein the step of measuring cyst formation includes inducing cyst formation by stimulation with a myosin II inhibitor and then measuring cyst formation.

2. The method of claim 1, wherein the myosin II inhibitor is blebbistatin.

3. The method according to any one of claims 1 to 2, wherein the measurement of cyst formation is a measurement of the size, number or circularity of the cysts.

4. The method according to any one of claims 1 to 3, wherein an mTOR inhibitor or a CFTR inhibitor is used as a positive control.

5. The method according to any one of claims 1 to 4, wherein the measurement results are compared with cyst formation in renal organoids obtained by inducing differentiation of iPS cells or gene mutation repaired iPS cells obtained from somatic cells derived from a healthy individual.

6. The method according to any one of claims 1 to 5, wherein the renal organoid is obtained by the following steps: (i) culturing pluripotent stem cells in a medium containing FGF (fibroblast growth factor) 2, BMP (bone morphogenetic protein) 4, GSK (glycogen synthase kinase)-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 TGF (transforming growth factor) β inhibitor; (iv) culturing the cells obtained in step (iii) in a medium containing FGF2, a GSK-3β inhibitor, BMP7, activin and a ROCK inhibitor; (v) culturing the cells obtained in step (iv) in a medium containing retinoic acid or a derivative thereof, a BMP inhibitor and FGF9; (vi) culturing the cells obtained in step (v) in a medium containing a GSK-3β inhibitor and FGF9; and (vii) A step of forming cell masses by non-adherent culture of the cells obtained in step (vi) and culturing the same.

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