WO2021015245A1 - 繊毛関連疾患モデルおよびその利用 - Google Patents

繊毛関連疾患モデルおよびその利用 Download PDF

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WO2021015245A1
WO2021015245A1 PCT/JP2020/028474 JP2020028474W WO2021015245A1 WO 2021015245 A1 WO2021015245 A1 WO 2021015245A1 JP 2020028474 W JP2020028474 W JP 2020028474W WO 2021015245 A1 WO2021015245 A1 WO 2021015245A1
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cells
renal
inhibitor
ips
cyst formation
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健二 長船
清水　達也
利和 荒岡
伸一 前
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Kyoto University NUC
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

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  • the present invention relates to a cyst formation model obtained from a renal organoid derived from induced pluripotent stem (iPS) cells derived from a patient with a cilia-related disease, and a method for screening or evaluating a therapeutic agent for a cilia-related disease using the cyst formation model.
  • iPS induced pluripotent stem
  • ADPKD Autosomal dominant polycystic kidney disease
  • Non-Patent Documents 1 to 4 it is unclear whether or not the developmental process of the kidney is accurately reproduced.
  • forskolin administration did not show a significant difference in cyst formation compared to healthy controls.
  • the iPS cell line derived from ADPKD patients (PKD1 heterozygous deletion) is not stable in inducing differentiation into renal tissue, and no obvious pathological trait is observed as compared with the healthy iPS cell line.
  • An object of the present invention is to develop a cyst formation model that can be used for screening and evaluation of therapeutic agents for ciliated diseases such as autosomal dominant polycystic kidney disease (ADPKD).
  • ADPKD autosomal dominant polycystic kidney disease
  • Patent Document 1 the group of inventors has developed a method for producing renal organoids from iPS cells. Then, in order to solve the above-mentioned problems, iPS cells were established from fibroblasts of ADPKD patients, and renal tissues prepared by using the differentiation induction method described in Patent Document 1 were present with forscolin, brevisstatin, etc. By culturing underneath, we succeeded in creating a renal cyst model. Then, using this model, it was found that by using the shape of the cyst, the dynamics of calcium, etc. as indicators, it is possible to efficiently screen and evaluate candidate substances for therapeutic agents for ciliary-related diseases such as ADPKD. Was completed.
  • Kidney organoids obtained by inducing differentiation of artificial pluripotent stem (iPS) cells obtained from somatic cells derived from patients with ciliary-related diseases or tubule cells (including tubule epithelial cells) contained therein. Hair-related, including contacting renal constituent cells such as (similarly) with a drug candidate and measuring calcium dynamics and / or cyst formation in renal constituent cells such as the renal organoid or tubule cells.
  • iPS pluripotent stem
  • Pluripotent stem cells are cultured in a medium containing FGF (fibroblast growth factor) 2, BMP (bone morphogenetic protein) 4, GSK (glycogen synthase kinase) -3 ⁇ inhibitor and retinoic acid or a derivative thereof.
  • FGF fibroblast growth factor
  • BMP bone morphogenetic protein
  • GSK glycosogen synthase kinase
  • the renal cyst model produced by the present invention reflects the pathophysiology of ciliary-related diseases such as cyst formation and decreased calcium release in a state closer to the living body, and is extremely excellent as an in vitro model of renal cyst formation.
  • ciliary-related diseases such as cyst formation and decreased calcium release in a state closer to the living body
  • the renal cyst model of the present invention it is possible to develop a therapeutic agent that suppresses renal cyst formation with unprecedented accuracy.
  • the pathological condition of cilia-related diseases such as ADPKD can be elucidated by using the pathological condition model produced by the present invention.
  • Fluorescence micrographs showing the results of immunostaining (LTL, ARL13B) on cilia in renal organoids (aggregate formation culture day 10) induced to differentiate from iPS cells derived from ADPKD patients.
  • the figure which shows the result of having induced the cyst formation by culturing the renal organoid which was induced to differentiate from the iPS cell derived from the ADPKD patient in the presence of forskolin (10 ⁇ M) or brevistatin (25 ⁇ M) (micrograph: bright field image).
  • cysts whose size falls within a certain order of higher rank are targeted for analysis, and the horizontal axis shows the number of cysts to be included in the analysis.
  • DMSO was used as the control.
  • kidney organoids including cysts formed by culturing a kidney organoid derived from an ADPKD patient (Patient-A) -derived iPS cell line in the presence of forskolin. Fluorescence micrograph (upper) showing the results of immunostaining and optical micrograph of the corresponding hematoxylin / eosin staining (lower). Statistics of the area ratio of renal cysts (Cystic Area) when renal organoids induced to differentiate from iPS cell lines derived from ADPKD patients (Patient-A) are cultured in the presence of forskolin and various drugs to induce cyst formation. A graph showing the results of a test. For control, only forskolin was added.
  • Forskolin presents renal organoids induced to differentiate from healthy-derived iPS cell lines (Wild-type) and mutation-introduced iPS cell lines (PKD1 heterozygous line (PKD1 +/- ) and PKD1 homozygous line (PKD1 -/- ))
  • the figure which shows the result which induced the cyst formation by culturing under the (micrograph: bright field image).
  • Forskolin presents renal organoids induced to differentiate from healthy subject-derived iPS cell lines (Wild-type) and mutation-introduced iPS cell lines (PKD1 heterostrain (PKD1 +/- ) and PKD1 homostrains (PKD1 -/- ))
  • the graph which shows the result of the statistical test of the area ratio (Cystic Area) of a renal cyst when it was cultured under the culture and induced cyst formation.
  • Immunostaining analyzed the expression of markers in renal organoids including cysts formed by culturing renal organoids induced to differentiate from mutant-introduced iPS cell lines (PKD1 homozygous strain (PKD1 -/- )) in the presence of forskolin. Fluorescence micrograph showing the results. The two images are immunostaining images of the same renal organoid, and the right figure is an enlarged photograph of the left figure.
  • the method for screening or evaluating a therapeutic agent for cilia-related diseases of the present invention is a renal composition such as renal organoids obtained by inducing differentiation of iPS cells obtained from somatic cells derived from patients with cilia-related diseases or tubule cells contained therein. It comprises contacting cells with a drug candidate and measuring calcium kinetics and / or cyst formation in the renal organoid or tubule cells and the like.
  • ciliopathy-related diseases include a disease called ciliopathy (ciliopathy or ciliopathy-related disease), which includes a disease in which cysts are formed in the kidney. More specifically, autosomal dominant polycystic kidney disease (ADPKD) ), Autosomal recessive polycystic kidney disease (ARPKD), and nephronophthisis.
  • ADPKD autosomal dominant polycystic kidney disease
  • ARPKD Autosomal recessive polycystic kidney disease
  • nephronophthisis nephronophthisis
  • ADPKD diseases are generally caused by genetic abnormalities.
  • the causative genes of ADPKD include the PKD1 gene and the PKD2 gene.
  • the PKD1 gene encodes the membrane protein “Polycystin (PC) 1, transient receptor potential channel interacting” (Ensembl gene ID: ENSG00000008710) (formerly known as polycystic kidney disease 1 (autosomal dominant)).
  • Examples of PKD1 include proteins having an amino acid sequence registered in accession number: P98161 of the UniProtKB database. However, since the amino acid sequence of PKD1 differs depending on race, etc., it is not limited to this specific amino acid sequence, and has 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)).
  • Examples of PKD2 include proteins having an amino acid sequence registered in accession number: Q13563 of the UniProtKB database. However, since the amino acid sequence of PKD2 differs depending on race, etc., it is not limited to this specific amino acid sequence, and has 80% or more, 90% or more, 95% or more, or 98% or more identity with the amino acid sequence. It may be an amino acid sequence.
  • patients with ciliary-related diseases preferably have mutations (including missense mutations, nonsense mutations, frameshift mutations, etc.) in these genes. It may be a heterozygous mutant individual having a mutation in one of the alleles, or a homomutant individual having a mutation in both alleles.
  • a heterozygous mutant individual having a mutation in one of the alleles
  • a homomutant individual having a mutation in both alleles.
  • ADPKD it develops in a heterozygous mutant individual, and since a homomutant individual does not give birth, a heterozygous mutant individual is preferable, and in the case of ARPKD and nephronophthisis, it develops in a homomutant individual, so a homomutant individual is preferable.
  • iPS cells may be produced using somatic cells derived from patients as they are, but iPS cells obtained from somatic cells derived from healthy humans (normal).
  • iPS cells obtained from somatic cells derived from healthy humans normal.
  • iPS cells obtained from somatic cells derived from cilia-related disease patients are reproduced and used. You may.
  • the somatic cells are not particularly limited, but include not only mature somatic cells but also fetal (pup) and neonatal (pup) somatic cells, and are any of primary cultured cells, passaged cells, and established cells. Is also included.
  • the somatic cells include, for example, (1) tissue stem cells (somatic stem cells) such as nerve stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells, (2) tissue precursor cells, and (3) blood cells (peripheral).
  • Blood cells umbilical cord blood cells, etc.
  • lymphocytes epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, hepatocytes, gastric mucosal cells, intestinal cells, splenocytes, pancreatic cells (pancreatic exocrine cells, etc.) Etc.
  • differentiated cells such as brain cells, lung cells, renal cells and fat cells are exemplified.
  • the iPS cells can be produced, for example, by introducing a reprogramming factor into somatic cells.
  • the reprogramming factors are, for example, Oct3/4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, Eras, ECAT15.
  • Genes or gene products such as -2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3 or Glis1 are exemplified, and these reprogramming factors may be used alone or in combination. Is also good.
  • 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 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 WO2010 / 056831, WO2010 / 068955, WO2010 / 098419, WO2010 / 102267, WO2010
  • a known method can be used for inducing differentiation of iPS cells into renal organoids, but a method including the following steps (i) to (vi) described in Patent Document 1 can be preferably used.
  • (I) A step of culturing iPS cells in a medium containing FGF2, BMP4, GSK-3 ⁇ inhibitor and retinoic acid or a derivative thereof;
  • (Ii) The step of culturing the cells obtained in step (i) in a medium containing FGF2, GSK-3 ⁇ inhibitor and BMP7;
  • (Iv) The step of culturing the cells obtained in step (iii) in a medium containing FGF2, GSK-3 ⁇ inhibitor, BMP7, activin and ROCK inhibitor;
  • (V) The step of culturing the cells obtained in step (iv) in a medium containing retinoic acid
  • Steps (i) to (vi) are preferably carried out by adhesive culture.
  • Adhesive culture means that cells are cultured in a state of being adhered to a culture substrate, and for example, they are cultured in a coated culture dish.
  • the coating agent is preferably extracellular matrix, and examples thereof include substances such as 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 TM.
  • the extracellular matrix is preferably laminin or a fragment thereof.
  • laminin is a protein having a heterotrimer structure having one ⁇ chain, one ⁇ chain, and one ⁇ chain, and is an extracellular matrix protein in which isoforms having different subunit chain compositions are present. ..
  • Laminin is a combination of 5 ⁇ -chains, 4 ⁇ -chains and 3 ⁇ -chain heterotrimers and has about 15 isoforms.
  • the ⁇ chain is ⁇ 1, ⁇ 2, ⁇ 3, ⁇ 4 or ⁇ 5
  • the ⁇ chain is ⁇ 1, ⁇ 2, ⁇ 3 or ⁇ 4
  • the ⁇ chain is ⁇ 1, ⁇ 2 or ⁇ 3.
  • Laminin is more preferably laminin 511 consisting of ⁇ 5, ⁇ 1 and ⁇ 1 (Nat Biotechnol 28, 611-615 (2010)).
  • Laminin may be a fragment, and is not particularly limited as long as it has integrin-binding activity.
  • E8 fragment laminin 511E8 (EMBO J.), which is a fragment obtained by digestion with elastase. , 3: 1463-1468, 1984, J. Cell Biol., 105: 589-598, 1987, WO 2011/043405).
  • Laminin 511E8 is commercially available and can be purchased from, for example, Nippi Co., Ltd.
  • the medium used in each step can be prepared by adding cytokines and drugs necessary for each step to the basal medium used for culturing animal cells.
  • the basal medium for example, IMDM medium, Medium 199 medium, Eagle's Minimium Essential Medium (EMEM) medium, ⁇ MEM medium, Dulvecco's Modern Medium (DMEM) medium, Ham's F12 (F12) medium, Ham's F12 (F12) medium , Fisher's medium, and a mixed medium thereof and the like are included.
  • the medium may contain serum (eg, fetal bovine serum (FBS)) or may be serum-free.
  • albumin transferase, KnockOut Serum Replacement (KSR) (serum substitute during ES cell culture) (ThermoFisherScientific), N2 supplement (ThermoFisherScientific), B27 supplement (ThermoFisherScient) , Insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, etc.
  • KSR KnockOut Serum Replacement
  • N2 supplement ThermoFisherScientific
  • B27 supplement ThermoFisherScient
  • Insulin may contain one or more serum substitutes, lipids, amino acids, L-glutamine, GlutaMAX (Thermo Fisher Scientific), It may also contain one or more substances such as non-essential amino acids (NEAAs), vitamins, growth factors, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, and their equivalents.
  • a medium pre-optimized for stem cell culture, such as ReproFF2
  • step (I) The step of culturing iPS cells in a medium containing FGF2, BMP4, GSK-3 ⁇ inhibitor and retinoic acid or a derivative thereof.
  • iPS cells are separated by a method known in the art. And can be cultivated.
  • Methods for separating iPS cells include, for example, mechanical separation, separation solutions having protease activity and collagenase activity (eg, Accutase (TM) and Accumax (TM) (Innovative Cell Technologies, Inc)) or collagenase. Separation using a separation solution having only activity can be mentioned.
  • the GSK-3 ⁇ inhibitor used in step (i) is not particularly limited as long as it can inhibit the function of GSK-3 ⁇ , for example, kinase activity, and is, for example, BIO (also known as GSK-3 ⁇ ) which is an insilvin derivative.
  • Inhibitor IX 6-bromoindylvin-3'-oxym
  • maleimide derivative SB216763 (3- (2,4-dichlorophenyl) -4- (1-methyl-1H-indol-3-yl) -1H- Pyrol-2,5-dione
  • GSK-3 ⁇ inhibitor VII ⁇ ,4-dibromoacetophenone
  • GSK-3 ⁇ L803-mts
  • Peptide inhibitors and CHIR99021 (Nature (2008) 453: 519-523), which have high selectivity.
  • GSK-3 ⁇ inhibitors include CHIR99021.
  • concentration of the GSK-3 ⁇ inhibitor used in this step can be appropriately selected by those skilled in the art depending on the GSK-3 ⁇ inhibitor used, and is, for example, 0.01 ⁇ M to 100 ⁇ M, preferably 0.1 ⁇ M to 10 ⁇ M. More preferably, it is 0.5 ⁇ M to 3 ⁇ M, and particularly preferably 0.5 ⁇ M to 1.5 ⁇ M.
  • the FGF2 (basic FGF: bFGF) used in the step (i) is preferably human FGF2, and the human FGF2 has, for example, the amino acid sequence of NCBI (National Center for Biotechnology Information) accession number: ABO43041.1. Examples include proteins. Fragments and functional variants of FGF2 are included as long as they have differentiation-inducing activity. Commercially available FGF2 may be used, or a protein purified from cells or a protein produced by genetic recombination may be used. You may.
  • the concentration of FGF2 used in this step is from 1 ng / ml to 1000 ng / ml, preferably from 10 ng / ml to 500 ng / ml, more preferably from 50 ng / ml to 250 ng / ml.
  • the BMP4 used in step (i) is preferably human BMP4, and examples of human BMP4 include proteins having the amino acid sequence of NCBI accession number: AAH20546.1. As long as BMP4 has differentiation-inducing activity, its fragments and functional variants are included. BMP4 may be commercially available, or a protein purified from cells or a protein produced by genetic recombination may be used. You may.
  • 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.
  • the retinoic acid used in the step (i) may be the retinoic acid itself or a retinoic acid derivative having the differentiation-inducing function of the natural retinoic acid.
  • a retinoic acid derivative for example, 3-dehydroretinoic acid, 4-[[(5,6,7,8-terahydro-5,5,8,8-tetramethyl-2-naphthalenyl) carbonyl] amino] -Benzoic acid ( AM580) (Tamura K, et al., Cell Differ. Dev.
  • retinoic acid or a derivative thereof used in step (i) is, for example, 1 nM to 100 nM, preferably 5 nM to 50 nM, and more preferably 5 nM to 25 nM.
  • the culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C., and the culture is carried out in an atmosphere of CO 2 containing air.
  • the CO 2 concentration is about 2-5%, preferably about 5%.
  • the culture time in step (i) may be a period sufficient for the late posterior epiblast to be induced to differentiate, but is, for example, 1 to 2 days of culture, preferably 1 day.
  • step (Ii) The step of culturing the cells obtained in step (i) in a medium containing FGF2, GSK-3 ⁇ inhibitor and BMP7.
  • FGF2 used in step (ii) is described in step (i). The same is true for the above, and the preferred concentration range is also the same.
  • the GSK-3 ⁇ inhibitor used in the step (ii) As the GSK-3 ⁇ inhibitor used in the step (ii), the GSK-3 ⁇ inhibitor exemplified in the above-mentioned step (i) can be used, and CHIR99021 is mentioned as a preferable GSK-3 ⁇ inhibitor.
  • the concentration of the GSK-3 ⁇ inhibitor used in step (ii) can be appropriately selected by those skilled in the art depending on the GSK-3 ⁇ inhibitor used, and is, for example, from 0.01 ⁇ M to 100 ⁇ M, preferably from 0.1 ⁇ M. It is 50 ⁇ M, more preferably 1 ⁇ M to 20 ⁇ M, and particularly preferably 2 to 10 ⁇ M.
  • the concentration of the GSK-3 ⁇ inhibitor used in step (ii) is preferably higher than the concentration in step (i).
  • the BMP7 used in step (ii) is preferably human BMP7, and examples of human BMP7 include proteins having the amino acid sequence of NCBI accession number: NM_001719.2. As long as BMP7 has differentiation-inducing activity, fragments and functional variants thereof are included. BMP7 may be commercially available, or a protein purified from cells or a protein produced by genetic recombination may be used. You may.
  • 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.
  • the culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C., and the culture is carried out in an atmosphere of CO 2 containing air.
  • the CO 2 concentration is about 2-5%, preferably about 5%.
  • the culture time of step (ii) may be a period sufficient for the mesoderm lineage primitive streak to be induced to differentiate, but is, for example, 10 hours to 2 days, or 1 to 2 days, preferably. 0.5 to 1 day.
  • step (Iii) The step of culturing the cells obtained in step (iii) in a medium containing FGF2, GSK-3 ⁇ inhibitor, BMP7 and TGF ⁇ inhibitor FGF2, GSK- used in step (iii).
  • the 3 ⁇ inhibitor, BMP7 is similar to step (ii), and the preferred concentration range thereof is also the same, but the concentration range of the GSK-3 ⁇ inhibitor is 0.01 ⁇ M to 100 ⁇ M, preferably 0.1 ⁇ M to 10 ⁇ M, and further. It is preferably 1 ⁇ M to 7.5 ⁇ M, and particularly preferably 2 to 5 ⁇ M.
  • the TGF ⁇ inhibitor used in step (iii) is a substance that inhibits the signal transduction of TGF ⁇ from binding to the receptor to SMAD, and is a substance that inhibits the binding to the ALK family of receptors, or ALK.
  • substances that inhibit the phosphorylation of SMAD by the family include Lefty-1 (NCBI Accession No. includes mouse: NM_010094 and human: NM_020997), SB431542, SB202190 (above, RKLindemann et al., Mol.
  • the TGF ⁇ inhibitor can preferably be A83-01.
  • the concentration of the TGF ⁇ inhibitor in the culture medium is not particularly limited as long as it inhibits ALK, but is 0.5 ⁇ M to 100 ⁇ M, preferably 1 ⁇ M to 50 ⁇ M, and more preferably 5 ⁇ M to 25 ⁇ M.
  • the culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C., and the culture is carried out in an atmosphere of CO 2 containing air.
  • the CO 2 concentration is about 2-5%, preferably about 5%.
  • the culture time of the step (iii) may be a period sufficient for inducing differentiation of the late mesoderm lineage primitive streak, but is, for example, 0.25 to 3 days, preferably 0.5 to 2. It is a day, more preferably 0.75 to 1.5 days.
  • the GSK-3 ⁇ inhibitor, BMP7 is the same as in step (ii), and the preferred concentration range thereof is also the same, but the concentration range of the GSK-3 ⁇ inhibitor is 0.01 ⁇ M to 100 ⁇ M, preferably 0.1 ⁇ M to 10 ⁇ M. More preferably, it is 1 ⁇ M to 7.5 ⁇ M, and particularly preferably 2 to 5 ⁇ M.
  • Activins used in step (iv) include activins from humans and other animals as well as functional variants thereof, with activin A being preferred and human activin A being more preferred.
  • human activin A include proteins having the amino acid sequence of NCBI accession number: NP_002183.1 or UniProt accession number P08476.2.
  • the concentration of activin used in the 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.
  • the ROCK inhibitor used in the step (iv) is not particularly limited as long as it can suppress the function of Rho-kinase (ROCK), and is, for example, Y-27632 (eg, Ishizaki et al., Mol. Pharmacol. 57,976-983 (2000); see Narumiya et al., Methods Enzymol. 325,273-284 (2000)), Fasudil / HA1077 (eg, see Uenata et al., Nature 389: 990-994 (1997)), H-1152. (See, eg, Sasaki et al., Pharmacol. Ther.
  • Y-27632 eg, Ishizaki et al., Mol. Pharmacol. 57,976-983 (2000); see Narumiya et al., Methods Enzymol. 325,273-284 (2000)
  • Fasudil / HA1077 eg, see Uenata et al.,
  • Wf-536 eg, Nakajima et al., Cancer Chemother, Pharmacol. 52 (4): 319-324 (2003).
  • Wf-536 eg, Nakajima et al., Cancer Chemother, Pharmacol. 52 (4): 319-324 (2003).
  • antisense nucleic acids against ROCK eg, siRNA
  • RNA interference-inducing nucleic acids eg, siRNA
  • dominant negative variants eg, and their expression vectors.
  • other known low molecular weight compounds can also be used as the ROCK inhibitor (for example, US Patent Application Publication Nos. 2005/0209261, 2005/0192304, 2004/0014755, 2004/0002508).
  • ROCK inhibitors include Y-27632.
  • concentration of the ROCK inhibitor used in the step (iv) can be appropriately selected by those skilled in the art depending on the ROCK inhibitor used, and is, for example, 0.1 ⁇ M to 100 ⁇ M, preferably 1 ⁇ M to 75 ⁇ M, and more preferably. Is from 5 ⁇ M to 50 ⁇ M.
  • the culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C., and the culture is carried out in an atmosphere of CO 2 containing air.
  • the CO 2 concentration is about 2-5%, preferably about 5%.
  • the culture time of the step (iv) may be a period sufficient for the late renal lineage late primitive streak to be induced to differentiate, but is, for example, 1 to 5 days, preferably 3 days.
  • step (V) The step of 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 a derivative thereof used in step (v) is As described in step (i), the preferred concentration range thereof is, for example, 10 nM to 500 nM, preferably 50 nM to 250 nM.
  • the FGF9 used in step (v) is preferably human FGF9, and examples of human FGF9 include proteins having the amino acid sequence of NCBI accession number: NP_002001.1. Fragments and functional variants of FGF9 are included 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.
  • the medium used in step (v) further comprises a BMP inhibitor.
  • BMP inhibitors include proteinin inhibitors such as Chordin, Noggin, Follistatin, Dorsomorphin (ie, 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.
  • LDN 193189 ie 4- (6- (4- (piperazin-1-yl) phenyl) pyrazolo [1,5-a ] Pyrimidin-3-yl) quinoline) is illustrated. More preferably, it is NOGGIN as a BMP inhibitor, the concentration thereof being, for example, 1 ng / ml to 100 ng / ml, 5 ng / ml to 50 ng / ml, 10 ng / ml to 30 ng / ml.
  • the culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C., and the culture is carried out in an atmosphere of CO 2 containing air.
  • the CO 2 concentration is about 2-5%, preferably about 5%.
  • the culture time may be a period sufficient for inducing differentiation of the late posterior intermediate mesoderm, but is, for example, 1 to 3 days, preferably 2 days.
  • step (Vi) The step of culturing the cells obtained in step (v) in a medium containing a GSK-3 ⁇ inhibitor and FGF9.
  • the GSK-3 ⁇ inhibitor and FGF9 used in step (vi) are each step.
  • the preferred concentration range thereof is also the same.
  • the number of culture days in the step (vi) is not particularly limited as long as renal progenitor cells can be obtained, and examples thereof include 2 days or more, 3 days or more, 4 days or more, and 5 days or more.
  • the culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C., and the culture is carried out in an atmosphere of CO 2 containing air, and the CO 2 concentration is preferably about 2 to 5%. is there.
  • step (Vii) A step of forming a cell mass by non-adhesive culture of cells (renal progenitor cells) obtained in step (vi). From renal progenitor cells to renal organoids, that is, glomerules, tubules, and collecting tubes.
  • renal progenitor cells obtained by the above method are non-adhesively cultured to prepare a cell mass, which is then used as 3T3-Wnt4 cells or the like.
  • a method of co-culturing with feeder cells, mouse fetal spinal cells, or mouse fetal kidney cells, or a method of semi-gastric culture using a basal medium containing a GSK-3 ⁇ inhibitor such as CHIR99021 (Reference Nature, 526, 564-568 (2015)) can be mentioned.
  • the medium can contain FGF9, FGF2, and even a ROCK inhibitor in addition to the GSK-3 ⁇ inhibitor.
  • the preferred concentrations of FGF9, FGF2, and ROCK inhibitor are the same as above.
  • GSK-3 ⁇ inhibitor or FGF2 is used, for example, from the start of the culture to 1 day after the start of the culture, to 2 days after the start of the culture, and to 3 days after the start of the culture.
  • a method including containing only the number of days in the medium can be mentioned.
  • the number of days of culturing in the step (vii) is not particularly limited as long as renal organoids are formed, and examples thereof include 5 days or more, 6 days or more, 7 days or more, and 8 days or more.
  • the culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C., and the culture is carried out in an atmosphere of CO 2 containing air, and the CO 2 concentration is determined. It is preferably about 2-5%.
  • the renal organoids obtained as described above can be used for screening and evaluation of therapeutic agents for cilia-related diseases.
  • test substance can be used, for example, cell extract, cell culture supernatant, microbial fermentation product, marine organism-derived extract, plant extract, purified protein or crude protein, and the like. Peptides, non-peptide compounds, synthetic low molecular weight compounds, and natural compounds are exemplified.
  • the test substance is also (1) a biological library method, (2) a synthetic library method using deconvolution, (3) a "one-bead one-compound” library method, And (4) can be obtained using any of the many approaches in combinatorial library methods known in the art, including synthetic library methods using affinity chromatography sorting.
  • Biological library methods using affinity chromatography sorting are limited to peptide libraries, but the other four approaches are applicable to small molecule libraries of peptides, non-peptide oligomers, or compounds (Lam (1997) Anticancer Drug Des). . 12: 145-67).
  • Examples of methods for synthesizing 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.
  • Compound libraries include solutions (see Houghten (1992) Bio / Techniques 13: 412-21) or beads (Lam (1991) Nature 354: 82-4), chips (Fodor (1993) Nature 364: 555-6). ), Bacteria (US Pat. No. 5,223,409), Spores (US Pat. Nos.
  • renal organoid obtained as described above a cyst formation promoting substance for a certain period of time, for example, 1 to 30 days, preferably 5 to 15 days, renal cyst formation can be induced, and cilia can be induced. It can be a related disease model.
  • Cyst formation promoters include cAMP pathway activators such as forskolin and 8-Bromo-cAMP and myosin II inhibitors such as brevisstatin.
  • concentration of forskolin is preferably 1 to 100 ⁇ M, more preferably 5 to 20 ⁇ M.
  • concentration of 8-Bromo-cAMP is preferably 10 to 1000 ⁇ M, more preferably 50 to 200 ⁇ M.
  • concentration of blevisstatin is preferably 1 to 100 ⁇ M, more preferably 10 to 50 ⁇ M.
  • cyst formation When screening or evaluating drugs using cyst formation as an index, for example, when culturing renal organoids in the presence of a cyst formation-promoting substance such as forskolin and / or before and after that, the test substance is contacted for a certain period of time. After culturing (for example, 1 hour to 72 hours), 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. The size of the cyst can be measured, for example, by the area ratio described below.
  • cysts are significantly compared to the case of renal organoids induced to differentiate from healthy subjects-derived iPS cells.
  • the number of cysts increases, the size increases, and the roundness decreases.
  • the test substance when the test substance is brought into contact with the test substance, if the number of cysts is reduced, the size is reduced, or the roundness is improved, the test substance has the ability to reduce or inhibit cyst formation. It can be evaluated as having or selected as a candidate substance for a therapeutic agent for ciliary-related diseases.
  • 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.
  • mTOR inhibitors such as rapamycin and everolimus
  • CFTR inhibitors such as CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) inhibitor II and CFTR inhibitor 172 can be used.
  • drug screening and evaluation can also be performed by measuring intracellular calcium dynamics using renal organoids.
  • intracellular calcium dynamics measurement it is preferable to isolate and use renal constituent cells such as tubular epithelial cells and proximal tubular cells from renal organoids. Isolation of tubular epithelial cells from renal organoids can be performed, for example, by sorting with an antibody against a tubular epithelial cell marker such as CD326 (EpCAM).
  • EpCAM tubular epithelial cell marker
  • proximal tubular cells from renal organoids is performed, for example, by antibodies against proximal tubular cell markers such as CD10, CD13 (PLoS ONE 8: e66750.2013), or proximal urine such as LTL (Lotus tetragonolobus lectin). This can be done by sorting with a specific lectin for tubular cells.
  • intracellular calcium dynamics can be measured in the state of renal organoids. By using a renal organoid that endogenously expresses Ca indicator in a specific renal constituent cell, it is possible to measure intracellular calcium dynamics using a change in fluorescence intensity in the specific renal constituent cell as an index.
  • the intracellular calcium dynamics measurement can be performed according to a conventional method. Specifically, Fura-2 (Dojin Kagaku Kenkyusho), Fluo4, Fluo3, Fura2, Indo1, Rhod2, Quin2, Fura-PE3, Fura Red, calcium green1, calcium crimson, Oregon green 488 BAPTA-1, fluo-3FF , Fluoro-5N, mag-fura-5, mag-indo-1, rhod-5N, Calbryte-590, Cal-520, etc.
  • Renal constituent cells such as tubule epithelial cells and proximal tubule cells obtained from renal organoids prepared by inducing differentiation from iPS cells derived from diseased patients are prepared by inducing differentiation from iPS cells derived from healthy subjects.
  • Thapsigargin has reduced calcium release.
  • Renal constituent cells such as tubular epithelial cells and proximal tubular cells obtained from renal organoids prepared by inducing differentiation from iPS cells derived from diseased patients were used as test substances for a certain period of time (for example, 1 to 72 hours). After incubation, the intracellular calcium concentration is measured, and as a result, if the calcium concentration is increased compared to when it is not incubated with the test substance or when it is incubated with the negative control, the test substance has the ability to increase the intracellular calcium concentration. It can be evaluated as having or selected as a candidate substance for a therapeutic agent for ciliary-related diseases.
  • the effect of the test substance can also be evaluated by comparing it with the effect of a positive control having an effect of increasing the calcium concentration in the cytoplasm.
  • the measurement results of renal organoids induced to differentiate from iPS cells derived from disease patients or renal constituent cells such as tubule epithelial cells and proximal tubule cells were induced to differentiate from gene mutation repair iPS cells. It is preferable to compare with the measurement results of renal organoids or renal constituent cells such as tubule epithelial cells and proximal tubule cells.
  • the measurement results of renal organoids induced to differentiate from a strain in which a PKD gene mutation was introduced into normal iPS cells or renal constituent cells such as tubule epithelial cells and proximal tubule cells were differentiated from the parent strain of normal iPS cells. It is also preferable to compare the results with the induced renal organoids or renal constituent cells such as tubule epithelial cells and proximal tubule cells.
  • the above-mentioned gene mutation repair iPS cells mean iPS cells in which the mutation site of the causative gene of a patient with a cilia-related disease is normally replaced, and the person with the cilia-related disease except that the mutation site is replaced with a normal sequence. Means iPS cells having the same genetic information as. By comparing with renal organoids from such gene-repaired iPS cells or renal constituent cells such as tubular epithelial cells and proximal tubular cells, the effect of the drug on ciliary-related diseases can be evaluated more accurately. Gene mutation repair iPS cells can be prepared by known gene repair techniques.
  • genome editing techniques such as CRISPR / Cas9, TALEN, and ZFN can be used to replace the genes responsible for ciliary-related diseases in patient-derived iPS cells with normal forms.
  • CRISPR / Cas9 it can be carried out by introducing an oligonucleotide having a normal sequence together with Cas9 and gRNA into patient-derived iPS cells and substituting the mutant sequence.
  • Example 1 Induction of renal progenitor cells from iPS cells IPS cells were prepared by introducing the OCT4, SOX2, KLF4, and c-MYC genes into somatic cells of ADPKD patients with mutations 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 renal (nephron) progenitor cells.
  • renal (nephron) progenitor cells were obtained from healthy human-derived iPS cells in the same manner.
  • the CiRA00009 strain has a Q3895X, Nonsense mutation
  • the CiRA00007 strain has a G3818R, Missense mutation (Ameku et al. Sci Rep 2016; 6: 30013).
  • renal (nephron) progenitor cells were also induced from the following iPS cells in the same manner.
  • iPS cells prepared by introducing the OCT4, SOX2, KLF4, and c-MYC genes into somatic cells of ADPKD patients with mutations in the PKD2 gene using a retroviral vector Ameku et al. Sci Rep 2016; 6: 30013.
  • Kidney organoid cyst formation assay by kidney organoid preparation and drug treatment (experimental method) 1.
  • nephron progenitor cells were prepared from undifferentiated human iPS cells (derived from patients or healthy individuals) using the differentiation-inducing system described in the examples of WO 2018/216743, and the nephron progenitor cells were prepared using the cells according to the following procedure. Kidney organoids were made.
  • the nephron progenitor cells on day 11-12 are dissociated into single cells using Accumax. Add 100 ⁇ L Accumax per well on a 24-well plate and incubate at 37 ° C. for 10-15 minutes at 5% CO 2 .
  • step 4 Discard the supernatant and use the medium prepared in step 4 to resuspend the cells to 50-100 ⁇ L per cell aggregate. 7. Take 50-100 ⁇ L of cell suspension on a 96 well plate (non-adherent) at the bottom of the U and centrifuge at 300 G x 3 min. 8. After culturing for 1-2 days, the formed cell aggregates are transferred onto the insert of 24-well transwell and used for gas-phase liquid-phase interfacial culture (day 0: organoid culture started).
  • KR5 medium (DMEM / F12 Glutamax containing 0.1 mM non-essential amino acids, 500 U / ml PS, 55 ⁇ M 2-mercaptoethanol and 5% KSR) with 200 ng / ml FGF2 and 5 ⁇ M CHIR99021 added to the medium. (Change medium at 120 ⁇ L per well). 9. After 48 hours, change the medium to KR5 medium (day 2), and then change the medium to KR5 medium every 2 days thereafter. On day 8, it differentiates into nephron organoids with glomerular and tubular structures. 10. Induction of cyst formation and measurement of cysts were performed according to the following procedure.
  • Y-27632 to Renal Epithelial Cell Growth Medium 2, RECGM2 (PromoCell) to a final concentration of 50 ⁇ M, and prepare a cell suspension to a final concentration of 50 ⁇ L per 5.0 ⁇ 10 4 cells.
  • EpCAM + cells are seeded in a clear-bottomed half-area 96-well plate (Greiner, 675090) at 5.0 ⁇ 10 4 cells (50 ⁇ L) per well and incubated overnight at 37 ° C. and 5% CO 2 . 9. Since it will be confluent the next day, Ca flux measurement by FDSS- ⁇ CELL will be performed.
  • Acid Red 27 (Tokyo Kasei, A0583) of water soluble probenecid 1.25 mM and 0.05 to 0.5 mg / mL as a quencher may be added.
  • 11. Set the FDSS- ⁇ CELL imaging protocol to measure Ca influx after Ca release with two-step chemical injection (20 ⁇ L each). 12.
  • drugs that cause Ca release such as Thapsigargin (final concentration 10 ⁇ M), Angiotensin II, human (final concentration 10 ⁇ M), and [Arg8] -Vasopressin (final concentration 10 ⁇ M), were dissolved in Ca-free HBSS. Inject something.
  • the second step CaCl 2 at a final concentration of 2 mM is injected to measure store-operated Ca influx (measure the ratio of fluorescence intensity to baseline). 13.
  • store-operated Ca influx measure the ratio of fluorescence intensity to baseline. 13
  • the change in intracellular Ca concentration can be measured using the final concentration of 5 ⁇ M ionomycin in the first step and the final concentration of 30 mM EGTA in the second step. 14.
  • the peak value of the first stage (Ca release), the AUC (area under the curve) value, the peak value of the second stage (Ca influx), the AUC value, and the plateau value are analyzed.
  • renal organoids containing renal tubules could be prepared from iPS cell lines derived from ADPKD patients, and primary cilia were confirmed in the renal tubules by the expression of the proximal tubule marker LTL and the ciliary marker ARL13B.
  • forskolin or blevisstatin was added on the 8th day from the start of the organoid culture and the culture was continued, the formation of renal cysts was observed on the 15th day (Fig. 2).
  • marker staining When confirmed by marker staining, glomerular-derived cysts were hardly conspicuous, and renal tubular-derived cysts were confirmed (Fig. 3).
  • cysts of nephron organoids actually increase up to about 30-40 days.
  • the area ratio (Cystic Area) and roundness of renal cysts were measured using an image analyzer.
  • the area ratio was calculated as follows.
  • Area ratio of renal cyst (%) total area of renal cyst / total area of organoid ⁇ 100
  • the results are shown in Fig. 4.
  • the area ratio of renal cysts is remarkably increased in patient-derived renal organoids, and the roundness is lower than that of healthy subjects-derived renal organoids (the cyst morphology is irregular).
  • Renal cyst formation and quantitative evaluation of the effect of the drug were found to be possible.
  • the intracellular Ca influx of renal cells of patients with the same disease is lower than that of healthy subjects.
  • the above Ca inflow measurement system it is possible to reproduce the decrease in Ca inflow in tubular cells derived from iPS cells of ADPKD patients, and by adding a drug here, the effect of the drug on the decrease in Ca inflow can be evaluated. ..
  • the proximal tubule cells and renal tubules of the renal organoid derived from the iPS cell line derived from ADPKD patients were compared with the renal organoid proximal tubule cells and tubular epithelial cells derived from the healthy iPS cell line. It was found that in epithelial cells, the release of calcium from the endoplasmic reticulum by Thapsigargin was reduced, and the time to peak tended to be slow.
  • Example 2 IPS cells were prepared from ADPKD patients (Patient-A, B) in the same manner as in Example 1.
  • Patient-A and B correspond to CiRA00009 and CiRA00007 of Example 1, respectively.
  • renal 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 from healthy subjects (Normal Subject-A and B) in the same manner.
  • Forskolin 10 ⁇ M was added from the 10th day after the start of organoid culture, and cyst formation was evaluated at the 17th day. The results are shown in FIG. It was confirmed that forskolin formed cysts in renal organoids that were induced to differentiate from iPS cell lines derived from any patient.
  • Example 3 Mutant-introduced iPS cell lines (PKD1 heterogeneous strain (PKD1 +/- ) and PKD1 homozygous strain (PKD1 -/- )) in which the mutant PKD1 gene was introduced into a healthy person-derived iPS cell line using genome editing technology using CRISPR / Cas9 It was prepared and analyzed.
  • a disease-specific iPS cell line was established using CRISPR / Cas9 genome editing technology according to the following procedure. Using the method described in Ishida et al. (Site-specific randomization of the endogenous genome by a regulatable CRISPR-Cas9 piggyBac system in human cells, Sci. Rep. 8 (2016) 310.), Exxon 34 of the PKD1 gene Mutations were introduced into the splicing acceptor site by non-homologous end joining (NHEJ). Specifically, healthy subjects 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.).
  • TetO-Cas9- using the 585A1 strain which is a human iPS cell line of origin, as a wild-type strain and using piggyBac transposon vectors (Addgene ID 100596 and Addgene ID 100598 and PiggyBac Transposase expression vector) for this wild-type strain.
  • piggyBac transposon vectors Additional genes that are involved in the splicing receptor site of Exxon 34 of the GR gene and PKD1 gene was introduced, and Cas9 activity was induced inducibly by drug administration to cause genome editing by non-homologous end binding (NHEJ).
  • Genome-edited iPS cells were isolated into single colonies by flow cytometry, and then sequence analysis was performed to confirm frameshift mutations in one allele and PKD1 heterogeneous strains, and frameshift mutations in both alleles.
  • the PKD1 homozygous strain was used.
  • Organoids were induced, forskolin 10 ⁇ M was added from the 10th day of organoid culture, and cyst formation was evaluated at the 17th day. The results are shown in FIG. Cyst formation was not formed by forskolin addition in renal organoids induced to differentiate from healthy iPS cell lines, but cyst formation was promoted by addition of forskolin in renal organoids differentiated from mutant-introduced iPS cell lines. The degree was remarkable in the PKD1 homozygous strain. This was also confirmed by a statistical test (Fig. 12) of the area ratio (Cystic Area) of renal cysts.
  • renal organoids induced to differentiate from healthy subject-derived iPS cell lines or disease-specific iPS cell lines were cultured in the presence of forskolin, and renal organoids containing cysts formed were formed.
  • PPD1 homozygous strain PPD1 -/-
  • renal organoids containing cysts formed were formed.
  • GenBank accession numbers and related sequence information and other data available through databases such as patents, publications and other publications, the National Center for Biotechnology Information (NCBI) referred to herein are one of them. Part or whole is incorporated by reference.

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