Kidney organoid and method for producing same

Abstract

One of the purposes of the present invention is to provide a novel method for producing a kidney organoid.　Provided is a method for producing a kidney organoid, the method being characterized by including culturing an initial kidney organoid using a culture medium containing an RXR agonist and a PPAR agonist, and being also characterized by containing a mature proximal convoluted tubule cell.

Description

Renal organoids and their production method

[0001] The present invention relates to renal organoids and methods for producing the same. More specifically, the present invention relates to renal organoids characterized by containing mature proximal tubule cells, and methods for producing the same. The present invention also relates to a composition for regenerative medicine comprising renal organoids characterized by containing mature proximal tubule cells. The present invention also relates to a non-human mammal having renal organoids characterized by containing mature proximal tubule cells, and methods for producing the same. The present invention also relates to a method for evaluating drug responsiveness to a test substance. The present invention also relates to a method for promoting proximal tubule maturation.

[0002] The kidneys concentrate metabolites (e.g., urea, uric acid, and creatinine) produced by the vital processes of cells in the body and excrete them in urine. The kidneys also regulate the volume and physicochemical properties (e.g., electrolyte concentration, osmotic pressure, and pH) of the extracellular fluid, which is the essential living environment for cells in the body.

[0003] The kidney is a collection of nephrons, each of which consists of a renal corpuscle (glomerulus and Bowman's capsule) and a renal tubule as its functional unit. Blood filtered by the glomerulus (primary urine) passes through the renal tubules to become urine. The renal tubules begin with the proximal tubule, which connects to the glomerulus, and then pass through the intermediate tubule, distal tubule, and connecting tubule to connect to the cortical collecting duct.

[0004] Methods for producing kidney organoids from human iPS cells have been reported (Patent Documents 1 and 2).

[0005] Non-Patent Document 1 reports that fenofibrate, a peroxisomal proliferation-activated receptor alpha (PPARA) agonist, increases the expression of proximal tubule genes in mouse proximal tubule cells.

[0006] Special Table 2016-527878 Special Table 2017-537655

[0007] Dhillon P, et. al., Cell Metabolism Volume 33, Issue 2, 2 February 2021, Pages 379-394.e8

[0008] There is a need in the art for renal organoids containing mature proximal tubules. There is a need in the art for a method for evaluating drug responsiveness to a test substance using renal organoids that have increased susceptibility to kidney damage.

[0009] One object of the present disclosure is to provide novel renal organoids characterized by containing mature proximal tubule cells. Another object of the present disclosure is to provide a novel method for producing renal organoids characterized by containing mature proximal tubule cells. Another object of the present disclosure is to provide a novel regenerative medicine composition comprising renal organoids characterized by containing mature proximal tubule cells. Another object of the present disclosure is to provide a novel method for evaluating drug responsiveness to a test substance. Another object of the present disclosure is to provide a method for promoting proximal tubule maturation.

[0010] The present inventors have found that culturing renal organoids in the presence of a combination of a PPARA agonist and an RXR agonist results in the formation of renal organoids containing mature proximal tubule cells, and the formed renal organoids show increased sensitivity to drug response to low concentrations of cisplatin.

[0011] Based on the above findings, the present disclosure provides the invention according to the following aspects. [Item 1] A method for producing renal organoids, comprising culturing early-stage renal organoids in a medium containing an RXR agonist and a PPAR agonist, wherein the renal organoids contain mature proximal tubule cells. [Item 1-1] The method according to Item 1, wherein the PPAR agonist is at least one selected from the group consisting of a PPARA agonist, a PPARG agonist, a PPARD agonist, a PPAR / G agonist, a PPAR / D agonist, a PPARG / D agonist, and a PPAR / G / D agonist. [Item 1-2] The method according to Item 1 or Item 1-1, wherein the PPAR agonist is a PPARA agonist. [Item 2] The method according to any one of Items 1 to 1-2, further comprising inducing the early renal organoids from intermediate mesodermal cells, wherein inducing the early renal organoids comprises culturing the intermediate mesodermal cells in induction medium B containing a fibroblast growth factor and substantially free of an RXR agonist to form intermediate mesodermal spheroids, and culturing the intermediate mesodermal spheroids in induction medium A containing a GSK3β inhibitor, followed by culturing them in induction medium B containing a fibroblast growth factor and substantially free of an RXR agonist. [Item 2-1] The method according to Item 2, wherein the induction medium B substantially free of an RXR agonist further contains substantially no PPAR agonist. [Item 3] The method of Item 2 or 2-1, further comprising inducing the intermediate mesodermal cells from pluripotent stem cells, wherein inducing the intermediate mesodermal cells comprises culturing the pluripotent stem cells in induction medium A containing a GSK3β inhibitor, and then culturing them in induction medium B containing a fibroblast growth factor and substantially free of an RXR agonist. [Item 4] The method of any one of Items 1 to 3, wherein the kidney organoids express a proximal tubule marker, and the proximal tubule marker is at least one selected from the group consisting of UGT2A3, AZGP1, SLC39A4, BHMT, ACE2, AQP6, CUBN, LRP2, and ABCB1.[Item 4-1] The method according to any one of Items 1 to 3, wherein the renal organoids express a proximal tubule marker, and the proximal tubule marker is at least one selected from the group consisting of UGT2A3, AZGP1, SLC39A4, BHMT, ACE2, AQP6, CUBN, LRP2, ABCB1, SERPINA1, APOA1, and SPARCL1. [Item 5] The method according to Item 4, wherein the renal organoids further express a PPARA downstream gene marker, and the PPARA downstream gene marker is at least one selected from the group consisting of APOA1, APOC3, CPT1A, FABP1, CYP27A1, and ACOX1.

[0012] [Item 6] The method of Item 4 or Item 5, wherein the expression level of the at least one proximal tubule marker in the renal organoids is 1.2-fold or more higher than the expression level of the at least one proximal tubule marker in the early-stage renal organoids. [Item 6-1] The method of Item 5, wherein the expression level of the at least one PPARA downstream gene marker in the renal organoids is 1.2-fold or more higher than the expression level of the at least one PPARA downstream gene marker in the early-stage renal organoids. [Item 6-2] The method of Item 5, wherein the expression level of the at least one proximal tubule marker in the renal organoids is 1.5-fold or more higher than the expression level of the at least one proximal tubule marker in the early-stage renal organoids, and the expression level of the at least one PPARA downstream gene marker in the renal organoids is 1.2-fold or more higher than the expression level of the at least one PPARA downstream gene marker in the early-stage renal organoids. [Item 6-3] The method according to Item 6, wherein the number of the at least one proximal tubule marker whose expression level in the kidney organoid is 1.2-fold higher than that in the early-stage kidney organoid is at least 1 (e.g., 1, 2, 3, 4, 5, or 6). [Item 6-4] The method according to Item 6-1, wherein the number of the at least one PPARA downstream gene marker whose expression level in the kidney organoid is 1.2-fold higher than that in the early-stage kidney organoid is at least 1 (e.g., 1, 2, 3, 4, 5, or 6). [Item 6-5] The number of the at least one proximal tubule marker whose expression level in the kidney organoid is 1.2 times higher than that in the early-stage kidney organoid is at least 1 (e.g., 1, 2, 3, 4, 5, or 6), and the number of the at least one PPARA downstream gene marker whose expression level in the kidney organoid is 1.2 times higher than that in the early-stage kidney organoid is at least 1 (e.g., 1, 2, 3, 4, 5, or 6). The method according to Item 6-2.[Item 6-6] The method according to Item 4 or Item 5, wherein the number of the at least one proximal tubule marker whose expression level in the renal organoid is significantly higher than that in the early-stage renal organoid is at least 1 (e.g., 1, 2, 3, 4, 5, or 6). [Item 6-7] The method according to Item 5, wherein the number of the at least one PPARA downstream gene marker whose expression level in the renal organoid is significantly higher than that in the early-stage renal organoid is at least 1 (e.g., 1, 2, 3, 4, 5, or 6). [Item 6-8] The method according to Item 5 or Item 6-7, wherein the number of the at least one proximal tubule marker whose expression level in the renal organoid is significantly higher than that in the early-stage renal organoid is at least 1 (e.g., 1, 2, 3, 4, 5, or 6), and the number of the at least one PPARA downstream gene marker whose expression level in the renal organoid is significantly higher than that in the early-stage renal organoid is at least 1 (e.g., 1, 2, 3, 4, 5, or 6).

[0013] [Item 7] The PPAR agonist is CP775146, pirinixic acid (WY14643), bezafibrate, fenofibrate, gemfibrozil, clofibrate, ciprofibrate, pemafibrate, binifibrate, clinofibrate, clofibric acid, nicofibrate, pirifibrate, plafibrid, lonifibrate, theofibrate, tocofibrate, SR10171, GW6471, fenofibrate, pirinixic acid, GW7647, icariin, eupatilin, gemfibrozil, palmitelaidic acid, NXT629, or saroglitazar. at least one PPARA agonist selected from the group consisting of magnesium, saroglitazar, oleoylethanolamide, BMS-687453, gypenoside XLIX, CUDA, ciprofibrate impurity A, CP-868388 free base, AVE-8134, linoleic acid, linolenic acid, and PPARα-MO-1;Rosiglitazone, troglitazone, pioglitazone, efatutazone, ATx08-001, OMS-405, CHS-131, THR-0921, SER-150-DN, KDT-501, GED-0507-34-Levo, CLC-3001, ALL-4, Rosiglitazone, Rosiglitazone hydrochloride, Troglitazone, T0070907, 5-Aminosalicylic Acid, GW1929, Rosiglitazone maleate, Astaxanthin, Magnolol, Glabridin, Balaglitazone, Mifobate, Inolitazone dihydrochloride, EHP-101, Adelmidrol, Oroxin A, Cefminox sodium, Methyl oleanonate, 15-Deoxy-Δ-12,14-prostaglandin J2, GSK376501A, 4-O-Methyl honokiol, Caulophyllogenin, Darglitazone, Angeloylgomisin at least one PPARG agonist selected from the group consisting of Finadelpar, ASP0367, GW501516, Pparδ agonist 5, MBX8025, GW0742, L165041, HPP-593, and NCP-1046; saroglitazar, aleglitazar, muraglitazar, tesaglitazar, and DSP-8 658; a PPARA / D agonist that is either or both of ELA and T913659; a PPARG / D agonist that is either or both of linoleic acid and T3D-959; or a PPARA / G / D agonist that is at least one selected from the group consisting of IVA337, TTA, bavachinin, GW4148, GW9135, bezafibrate, lobeglitazone, and CS038;or a combination thereof, and / or the RXR agonist is at least one selected from the group consisting of 9-cis retinoic acid (alitretinoin), AGN 195204, TTNPB (arotinoid acid), Adapalene, Bexarotene, Tazarotene, Tamibarotene, CH55, and AM 580. [Item 8] A kidney organoid containing mature proximal tubule cells, produced by the method of any one of Items 1 to 7. [Item 9] A renal organoid, wherein the renal organoid contains mature proximal tubule cells, and the renal organoid expresses a proximal tubule marker, wherein the proximal tubule marker is at least one selected from the group consisting of UGT2A3, AZGP1, SLC39A4, BHMT, ACE2, AQP6, CUBN, LRP2, and ABCB1. [Item 9-1] A renal organoid, comprising mature proximal tubule cells, expressing a proximal tubule marker, wherein the proximal tubule marker is at least one selected from the group consisting of UGT2A3, AZGP1, SLC39A4, BHMT, ACE2, AQP6, CUBN, LRP2, ABCB1, SERPINA1, APOA1, and SPARCL1. [Item 10] The renal organoid according to Item 9, further expressing at least one PPARA downstream gene marker selected from the group consisting of APOA1, APOC3, CPT1A, FABP1, CYP27A1, and ACOX1.

[0014] [Item 11] The renal organoid according to Item 9 or Item 10, wherein the expression level of the at least one proximal tubule marker in the proximal tubule marker is 1.2-fold or more higher than the expression level of the at least one proximal tubule marker in the proximal tubule marker in the early renal organoid. [Item 11-1] The renal organoid according to Item 10, wherein the expression level of the at least one PPARA downstream gene marker in the renal organoid is 1.2-fold or more higher than the expression level of the at least one PPARA downstream gene marker in the early renal organoid. [Item 11-2] The renal organoid according to Item 10, wherein the expression level of the at least one proximal tubule marker in the renal organoid is 1.5-fold or more higher than the expression level of the at least one proximal tubule marker in the early renal organoid, and the expression level of the at least one PPARA downstream gene marker in the renal organoid is 1.2-fold or more higher than the expression level of the at least one PPARA downstream gene marker in the early renal organoid. [Item 11-3] The kidney organoid according to Item 11, wherein the number of the at least one proximal tubule marker whose expression level in the kidney organoid is 1.2-fold higher than that in the early-stage kidney organoid is at least 1 (e.g., 1, 2, 3, 4, 5, or 6). [Item 11-4] The kidney organoid according to Item 11-1, wherein the number of the at least one PPARA downstream gene marker whose expression level in the kidney organoid is 1.2-fold higher than that in the early-stage kidney organoid is at least 1 (e.g., 1, 2, 3, 4, 5, or 6). [Item 11-5] The number of the at least one proximal tubule marker whose expression level in the kidney organoid is 1.2 times higher than that in the early kidney organoid is at least 1 (for example, 1, 2, 3, 4, 5, or 6), and the number of the at least one PPARA downstream gene marker whose expression level in the kidney organoid is 1.2 times higher than that in the early kidney organoid is at least 1 (for example, 1, 2, 3, 4, 5, or 6). The kidney organoid according to Item 11-2.[Item 11-6] The kidney organoid according to Item 9 or Item 10, wherein the number of the at least one proximal tubule marker whose expression level in the kidney organoid is significantly higher than that in the early stage kidney organoid is at least 1 (e.g., 1, 2, 3, 4, 5, or 6). [Item 11-7] The kidney organoid according to Item 10, wherein the number of the at least one PPARA downstream gene marker whose expression level in the kidney organoid is significantly higher than that in the early stage kidney organoid is at least 1 (e.g., 1, 2, 3, 4, 5, or 6). [Item 11-8] The number of the at least one proximal tubule marker whose expression level in the kidney organoid is significantly higher than that in the early stage kidney organoid is at least 1 (for example, 1, 2, 3, 4, 5, or 6), and the number of the at least one PPARA downstream gene marker whose expression level in the kidney organoid is significantly higher than that in the early stage kidney organoid is at least 1 (for example, 1, 2, 3, 4, 5, or 6). The kidney organoid according to Item 10 or 11-7.

[0015] [Item 12] A method for producing a non-human mammal having renal organoids in its kidney or a peripheral region thereof, comprising introducing renal organoids produced by the method of any one of Items 1 to 7, or the renal organoids described in any one of Items 8 to 11-8, into the kidney or a peripheral region of the non-human mammal, wherein the renal organoids contain mature proximal tubule cells. [Item 13] A non-human mammal having renal organoids produced by the method of any one of Items 1 to 7, or the renal organoids described in any one of Items 8 to 11-8, in its kidney or a peripheral region thereof. [Item 14] A regenerative medicine composition for treating kidney damage or disease, comprising renal organoids produced by the method of any one of Items 1 to 7, or the renal organoids described in any one of Items 8 to 11-8. [Item 15] A method for evaluating drug responsiveness to a test substance, comprising contacting a test substance with a renal organoid produced by the method of any one of Items 1 to 7, or the renal organoid of any one of Items 8 to 11-8, a non-human mammal produced by the method of Item 12, or the non-human mammal of Item 13, and measuring the drug responsiveness of the renal organoid or the non-human mammal to the test substance. [Item 15-1] The method of Item 15, further comprising evaluating the nephrotoxicity of the test substance based on the results of the measurement of drug responsiveness. [Item 16] A method for treating kidney damage or disease, comprising introducing a renal organoid produced by the method of any one of Items 1 to 7, or the renal organoid of any one of Items 8 to 11-8, or the medical composition of Item 14 into the kidney or a peripheral site of a mammal in need thereof. [Item 17] A method for promoting maturation of proximal tubules, characterized in that early renal organoids are cultured using a medium containing an RXR agonist and a PPAR agonist.

[0016] Figure 1 is a flowchart outlining one embodiment of the generation of renal organoids containing mature proximal tubule cells and the evaluation of drug nephrotoxicity. Figures 2a-2i are a series of bar graphs showing the expression levels of proximal tubule markers (Figure 2a: UGT2A3, Figure 2b: AZGP1, Figure 2c: SLC39A4, Figure 2d: BHMT, Figure 2e: ACE2, Figure 2f: AQP6, Figure 2g: CUBN, Figure 2h: LRP2, and Figure 2i: ABCB1) in LTL-positive cells derived from renal organoids cultured in the presence of a combination of CP775146 and 9-cis retinoic acid. Figures 3a-3f are a series of bar graphs showing the expression levels of PPARA downstream gene markers (Figure 3a: APOA1, Figure 3b: APOC3, Figure 3c: CPT1A, Figure 3d: FABP1, Figure 3e: CYP27A1, and Figure 3f: ACOX1) in LTL-positive cells derived from kidney organoids cultured in the presence of a combination of CP775146 and 9-cis retinoic acid. Figures 4a-i are a series of bar graphs showing the expression of proximal tubule markers (Figure 4a: UGT2A3, Figure 4b: AZGP1, Figure 4c: SLC39A4, Figure 4d: BHMT, Figure 4e: ACE2, Figure 4f: AQP6, Figure 4g: CUBN, Figure 4h: LRP2, and Figure 4i: ABCB1) in LTL-positive cells derived from kidney organoids cultured in the presence of CP775146 alone, 9-cis retinoic acid alone, or a combination of CP775146 and 9-cis retinoic acid. Figures 5a to 5e are a series of bar graphs showing the expression levels of PPARA downstream genes (Figure 5a: APOA1, Figure 5b: APOC3, Figure 5c: CPT1A, Figure 5d: FABP1, and Figure 5e: ACOX1) in LTL-positive cells derived from kidney organoids cultured in the presence of CP775146 alone, 9-cis retinoic acid alone, or a combination of CP775146 and 9-cis retinoic acid. Figures 6a and 6b are 2D scatter plots of LTL-expressing cells derived from kidney organoids in the control group cultured in the absence (Figure 6a) or presence of 5 μg cisplatin (CDDP) (Figure 6b).6c and 6d are 2D scatter plots of LTL-positive cells derived from renal organoids of the Mature group cultured in the absence of cisplatin (CDDP) (FIG. 6a) or in the presence of 5 μg cisplatin (FIG. 6b).

[0017] 7 is a bar graph showing the results of a cisplatin nephrotoxicity test. FIG. 8a shows an immunostained image of kidney organoids. FIG. 8b is a histogram of cells constituting kidney organoids. FIG. 8c is a bar graph showing the percentage of LTL-positive cells in kidney organoids of the Control group and the Mature group. FIG. 9a is a bar graph showing the expression level of LRP2. FIG. 9b is a bar graph showing the expression level of CUBN. FIG. 10a is a box plot showing the size distribution of endosomes in kidney organoids. FIG. 10b is a box plot showing the ratio of large endosomes (≧0.5 μm) to the total number of endosomes per cell in kidney organoids of the Control group. ２ Fig. 10c is a pie chart showing the ratio of large endosomes (≥ 0.5 μm) to the total number of endosomes per cell in the renal organoids of the Mature group. ２) is a pie chart showing the percentage of maturation markers in the renal organoids. Figure 11 is a box plot showing dextran uptake in renal organoids. Figure 12 is a series of bar graphs showing the expression levels of proximal tubule cell maturation markers in renal organoids generated using PPAPA antagonists. Figure 12a is a bar graph showing the expression levels of UGT2A3. Figure 12b is a bar graph showing the expression levels of AZGP1. Figure 12c is a bar graph showing the expression levels of SLC39A4. Figure 12d is a bar graph showing the expression levels of BHMT. Figure 12e is a bar graph showing the expression levels of ACE2. Figure 12f is a bar graph showing the expression levels of AQP6. Figure 12g is a bar graph showing the expression levels of CUBN. Figure 12h is a bar graph showing the expression levels of LRP2. Figure 12i is a bar graph showing the expression levels of ABCB1. Figure 12j is a bar graph showing the expression levels of APOA1. Figure 12k is a bar graph showing the expression levels of APOC3, Figure 12l is a bar graph showing the expression levels of CYP27A1, and Figure 12m is a bar graph showing the expression levels of FABP1.

[0018] Figure 13 shows a series of bar graphs showing the expression levels of maturation markers of proximal tubule cells in kidney organoids generated using RXR antagonists. Figure 13a is a bar graph showing the expression levels of UGT2A3. Figure 13b is a bar graph showing the expression levels of AZGP1. Figure 13c is a bar graph showing the expression levels of SLC39A4. Figure 13d is a bar graph showing the expression levels of BHMT. Figure 13e is a bar graph showing the expression levels of ACE2. Figure 13f is a bar graph showing the expression levels of AQP6. Figure 13g is a bar graph showing the expression levels of CUBN. Figure 13h is a bar graph showing the expression levels of LRP2. Figure 13i is a bar graph showing the expression levels of ABCB1. Figure 13j is a bar graph showing the expression levels of APOA1. Figure 13k is a bar graph showing the expression levels of APOC3. Figure 13l is a bar graph showing the expression level of CYP27A1. Figure 13m is a bar graph showing the expression level of FABP1. Figure 14 shows a series of bar graphs showing the expression level of proximal tubule cell maturation markers in kidney organoids prepared using pirinixic acid. Figure 14a is a bar graph showing the expression level of UGT2A3. Figure 14b is a bar graph showing the expression level of AZGP1. Figure 14c is a bar graph showing the expression level of SLC39A4. Figure 14d is a bar graph showing the expression level of BHMT. Figure 14e is a bar graph showing the expression level of ACE2. Figure 14f is a bar graph showing the expression level of AQP6. Figure 14g is a bar graph showing the expression level of CUBN. Figure 14h is a bar graph showing the expression level of LRP2. Figure 14i is a bar graph showing the expression level of ABCB1. Figure 14j is a bar graph showing the expression level of APOA1. Figure 14k is a bar graph showing the expression level of APOC3. Figure 14l is a bar graph showing the expression level of CPT1A. Figure 14m is a bar graph showing the expression level of FABP1.

[0019] [Kidney organoid containing mature proximal tubule cells] One aspect of the present disclosure provides the kidney organoid that is characterized by containing mature proximal tubule cells.One aspect of the present disclosure provides the kidney organoid that contains mature proximal tubule cells, expressing proximal tubule marker, and said proximal tubule marker is at least one selected from the group consisting of UGT2A3, AZGP1, SLC39A4, BHMT, ACE2, AQP6, CUBN, LRP2, ABCB1, SERPINA1, APOA1 and SPARCL1. Furthermore, in one aspect of the present disclosure, there is provided a renal organoid containing mature proximal tubule cells, which may further express a PPARA downstream gene marker, wherein the PPARA downstream gene marker is at least one selected from the group consisting of APOA1, APOC3, CPT1A, FABP1, CYP27A1, and ACOX1.

[0020] As used herein, the term "organoid" refers to a three-dimensional cell aggregate formed in vitro that resembles biological tissue. A cell aggregate similar to biological tissue is, for example, a cell aggregate containing a structure observed in the corresponding biological tissue. The structure observed in biological tissue may be, for example, a structure observable under a microscope (e.g., in the case of the kidney, a glomerulus or a renal tubule). Most of the cells that make up organoids are, for example, cells with differentiation and proliferation capabilities. Organoids are characterized, for example, by the expression of marker genes (their transcription products or translation products). Glomeruli can be identified, for example, by the expression of glomerular markers, such as podocalyxin or nephrin, or a combination thereof. Proximal tubules can be identified, for example, by the expression of proximal tubule markers described below.

[0021] As used herein, the term "early stage renal organoid" refers to an organoid having one or more LHX1-positive metanephric vesicles. Early stage renal organoids preferably contain cells expressing the epithelial cell marker EpCAM. The metanephric vesicles are preferably LHX1-positive and JAG1-positive or CDH6-positive. The metanephric vesicles are preferably LHX1-positive, JAG1-positive, and CDH6-positive. Early stage renal organoids can be prepared by known methods, for example, the method described in Takasato M., et al., Nat Cell Biol. 2014;16:118-26 (incorporated herein by reference), or the method described in Takasato M., et al., Nature. 2015;526:564-568. (incorporated herein by reference) can be produced from pluripotent stem cells.Early stage renal organoids can be produced from intermediate mesoderm cells, for example, according to step B of the present disclosure, preferably according to step B described in Examples.Early stage renal organoids can be produced from pluripotent stem cells according to step A of the present disclosure, preferably according to step A described in Examples.Early stage renal organoids can be produced from pluripotent stem cells according to step A and step B of the present disclosure, preferably according to step A and step B described in Examples.

[0022] As used herein, "renal organoids containing mature proximal tubule cells" refers to renal organoids containing cells expressing the proximal tubule markers described below. Renal organoids containing mature proximal tubule cells may have one or more glomeruli and / or tubules. In one example, renal organoids containing mature proximal tubule cells further contain cells expressing a PPARA downstream gene marker (preferably PPARA). Renal organoids containing mature proximal tubule cells include, for example, proximal tubules and glomeruli. As used herein, "renal organoids" include organoids at any maturation stage, from early renal organoids to renal organoids containing mature proximal tubule cells induced by differentiation. Renal organoids also include, for example, known renal organoids or renal organoids produced by known methods.

[0023] As used herein, "mature proximal tubule cells" express the proximal tubule markers described below. In one example, mature proximal tubule cells express the PPARA downstream gene markers described further below. Proximal tubule cells are present, for example, in the vicinity of glomeruli in kidney organoids. In one example, mature proximal tubule cells express the proximal tubule markers and the PPARA downstream gene markers.

[0024] An organoid expressing a specific marker (e.g., a marker gene or marker protein) may be, for example, an organoid in which the average expression level of the specific marker per cell in a specific cell population derived from the organoid is significantly higher than the average expression level of the specific marker per cell in the specific cell population derived from a control organoid. In this context, the term "significantly" means, for example, that the p-value in a t-test is less than 0.05, preferably less than 0.01. The specific cell population is, for example, proximal tubule cells. Proximal tubule cells, which are a specific cell population derived from an organoid, can be prepared, for example, using MACS using an antibody against a specific marker (e.g., LTL). The comparison of expression levels can be performed, for example, using at least three (e.g., three, four, five, six, or seven or eight or more) organoids. In one example, organoids expressing a particular marker have an average expression level of the particular marker per cell in a particular cell population derived from the organoid that is 1.2 times or more (e.g., 1.2 times or more, 1.5 times or more, 1.8 times or more, 2.0 times or more, 2.3 times or more, or 2.5 times or more, or 1.2 times to 2.5 times or 1.5 times to 2.5 times) higher than the average expression level of the particular marker per cell in the particular cell population derived from a control organoid. In one example, the organoid of interest is a kidney organoid containing mature proximal tubule cells according to the present disclosure, and the control organoid is a primary kidney organoid.

[0025] In one embodiment, the renal organoid that contains mature proximal tubule cells has the expression level of at least one proximal tubule marker selected from a group of proximal tubule markers, which is significantly higher than the expression level of at least one proximal tubule marker in early renal organoid.In one embodiment, the renal organoid that contains mature proximal tubule cells has the expression level of at least one PPARA downstream gene marker selected from a group of PPARA downstream gene markers, which is significantly higher than the expression level of at least one PPARA downstream gene marker in early renal organoid. In one embodiment, renal organoids containing mature proximal tubule cells have a significantly higher expression level of at least one proximal tubule marker selected from a group of proximal tubule markers than the expression level of the at least one proximal tubule marker in early renal organoids, and / or a significantly higher expression level of at least one PPARA downstream gene marker selected from a group of PPARA downstream gene markers than the expression level of the at least one PPARA downstream gene marker in early renal organoids.

[0026] In the kidney organoids according to the above-described embodiments, the expression level of the at least one proximal tubule marker in the kidney organoids is higher than the expression level of the at least one proximal tubule marker in the early stage kidney organoids (e.g., 1.2-fold or more, 1.5-fold or more, 1.8-fold or more, 2.0-fold or more, 2.3-fold or more, or 1.2-fold to 2.5-fold or 1.5-fold to 2.5-fold higher), and / or the expression level of the at least one PPARA downstream gene marker in the kidney organoids is higher than the expression level of the at least one PPARA downstream gene marker in the early stage kidney organoids (e.g., 1.2-fold or more, 1.5-fold or more, 1.8-fold or more, 2.0-fold or more, 2.3-fold or more, or 2.5-fold or 1.2-fold to 2.5-fold or 1.5-fold to 2.5-fold higher). In the above examples, the "high expression level" may be "significantly higher expression level".

[0027] The comparison of the expression level of a marker in a target organoid (e.g., a kidney organoid containing mature proximal tubule cells) with the expression level of the marker in a control organoid (e.g., an early stage kidney organoid) can be performed, for example, using a plurality of markers (e.g., at least two). In this example, organoids expressing a specific marker are those in which the average expression level of a predetermined number (e.g., six) or more markers among a plurality of markers (e.g., ten) in the target organoid is higher than the average expression level in the control organoid. The predetermined number may be, for example, the same as the number of markers to be measured (e.g., eight), or may be one or more less (e.g., seven, six, five, four, three, two, or one). For example, when the number of markers to be measured is nine, the predetermined number is three or more, four or more, preferably five or more, and more preferably six or more.

[0028] In the renal organoids according to the above-described embodiment, the number of proximal tubule markers whose expression levels are higher than those in the early renal organoids is a predetermined number (e.g., 1, 2, 3, or 4) or more, and / or the number of PPARA downstream gene markers whose expression levels are higher than those in the early renal organoids is a predetermined number (e.g., 1, 2, 3, or 4) or more. The predetermined number is appropriately set according to the number of markers included in the group of proximal tubule markers and the number of markers included in the group of PPARA downstream gene markers. The predetermined number is set, for example, taking into consideration the type of marker included in the group of markers and the type of marker for which a significant difference or a tendency for an increase in expression level was observed in the examples herein. In one example, when the group of markers includes marker A, marker B, marker C, and marker D, and a significant difference or a tendency for increased expression levels is observed for marker A, marker B, and marker C in the examples, the predetermined number may be set to at least 1 (e.g., 1, 2, or 3), at least 2 (e.g., 2 or 3), or 3. In the example, the "high expression level" may be "significantly high expression level."

[0029] An organoid expressing a specific marker is, for example, an organoid containing cells expressing a specific marker at a predetermined rate or more. In this context, "cells expressing a specific marker" may be mature proximal tubule cells. The predetermined rate may be, for example, 5% or more, 10% or more, 15% or more, 20% or more, or 25% or more of the cell population constituting the organoid. In one example, an organoid expressing a specific marker is an organoid containing 5% or more (e.g., 10% or more, 15% or more, 20% or more, or 25% or more) of cells expressing a specific marker at a predetermined level or more. The predetermined level is, for example, the third quartile of the expression level of a negative control cell population that does not express the specific marker.

[0030] In the above example, the predetermined marker is a proximal tubule marker and / or a PPARA downstream gene marker, as described below. The expression level of a marker may be the amount of its transcription product, the amount of its translation product, or its relative level. When the marker is a gene, the expression level of the marker may be the amount of its transcription product, the amount of its translation product, or its relative level. When the marker is a gene, the expression level of the marker is preferably the amount of its transcription product or its relative level. When the marker is a protein, the expression level of the marker may be the amount of its translation product or its relative level. The relative level refers to the amount of the marker relative to an endogenous control. Examples of endogenous controls that can be used include β-actin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The relative level is, for example, the amount of the marker relative to β-actin. The amount of the transcription product can be measured, for example, by quantitative PCR. The amount of the translation product can be measured, for example, by FACS or MACS. Measurement can be performed by immunofluorescence staining, or by fluorescence-activated cell sorting (FACS) or magnetic affinity cell sorting (MACS). FACS or MACS can be performed using commercially available FACS or MACS machines.

[0031] As used herein, the term "LHX1" is an abbreviation for LIM homeobox 1 and refers to a protein encoded by the LHX1 gene. Cells expressing LHX1 can be detected, for example, by immunostaining using an anti-LHX1 antibody. LHX1-positive metanephric vesicles can be detected in kidney organoids by immunostaining using an anti-LHX1 antibody.

[0032] As used herein, the term "JAG1" is an abbreviation for the Jagged1 protein encoded by the JAG1 gene, and refers to one of five cell surface proteins that interact with four receptors in the Notch signaling pathway. JAG1-expressing cells can be detected, for example, by immunostaining using anti-JAG1 antibodies. JAG1-positive metanephric vesicles can be detected in kidney organoids by immunostaining using anti-JAG1 antibodies.

[0033] As used herein, the term "CDH6" refers to the cadherin 6 protein encoded by the CDH6 gene. CDH6-expressing cells can be detected, for example, by immunostaining using anti-CDH6 antibody. CDH6-positive metanephric vesicles can be detected in kidney organoids by immunostaining using anti-CDH6 antibody.

[0034] As used herein, the term "EpCAM" is an abbreviation for epithelial cell adhesion molecule, which refers to a transmembrane glycoprotein. EpCAM can be used as an epithelial cell marker. Cells expressing EpCAM can be detected, for example, by immunostaining using an anti-EpCAM antibody.

[0035] As used herein, the term "proximal tubule marker" refers to at least one selected from the group consisting of UGT2A3, AZGP1, SLC39A4, BHMT, ACE2, AQP6, CUBN, LRP2, ABCB1, SERPINA1, APOA1, and SPARCL1. As used herein, the proximal tubule marker is also referred to as a proximal tubule maturation marker. The proximal tubule marker is, for example, at least one selected from the group consisting of UGT2A3, AZGP1, SLC39A4, BHMT, ACE2, AQP6, CUBN, LRP2, and ABCB1. The proximal tubule marker is, for example, at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve markers selected from the group of proximal tubule markers. The proximal tubule marker is, for example, at least two markers (e.g., two, three, four, five, six, seven, preferably eight, preferably nine, preferably ten, preferably eleven, or more preferably twelve) selected from the group of proximal tubule markers. The proximal tubule marker is, for example, at least four markers (e.g., four, five, six, seven, preferably eight, preferably nine, preferably ten, preferably eleven, or more preferably twelve) selected from the group of proximal tubule markers. The proximal tubule markers are, for example, at least six (e.g., six, seven, preferably eight, preferably nine, preferably ten, preferably eleven, or more preferably twelve) types selected from the group of proximal tubule markers. The proximal tubule markers are, for example, at least seven (e.g., seven, preferably eight, preferably nine, preferably ten, preferably eleven, or more preferably twelve) types selected from the group of proximal tubule markers. The proximal tubule markers are, for example, eight, nine, ten, eleven, or twelve types selected from the group of proximal tubule markers. The proximal tubule markers are, for example, ten, eleven, or twelve types selected from the group of proximal tubule markers. The proximal tubule markers are, for example, eleven or twelve types selected from the group of proximal tubule markers.

[0036] The proximal tubule marker is, for example, at least one (e.g., one, two, three, four, five, six, seven, eight, nine, or ten) selected from the group consisting of UGT2A3, SLC39A4, ACE2, AQP6, CUBN, LRP2, ABCB1, SERPINA1, APOA1, and SPARCL1. The proximal tubule marker is, for example, at least two (e.g., two, three, four, five, preferably six, preferably seven, preferably eight, preferably nine, or more preferably ten) selected from the group of proximal tubule markers. The proximal tubule marker is, for example, at least four (e.g., four, five, preferably six, preferably seven, preferably eight, preferably nine, or more preferably ten) selected from the group of proximal tubule markers. The proximal tubule markers are, for example, at least 6, 7, 8, 9, or 10 selected from the group of proximal tubule markers. The proximal tubule markers are, for example, at least 8, 9, or 10 selected from the group of proximal tubule markers. The proximal tubule markers are, for example, at least 9 or 10 selected from the group of proximal tubule markers.

[0037] The proximal tubule marker is, for example, at least one (e.g., one, two, three, four, five, six, or seven) selected from the group consisting of UGT2A3, SLC39A4, ACE2, AQP6, CUBN, LRP2, and ABCB1. The proximal tubule marker is, for example, at least two (e.g., two, three, four, five, preferably six, or more preferably seven) selected from the group of proximal tubule markers. The proximal tubule marker is, for example, at least four (e.g., four, five, preferably six, or more preferably seven) selected from the group of proximal tubule markers. The proximal tubule marker is, for example, at least six or seven selected from the group of proximal tubule markers.

[0038] The proximal tubule marker is, for example, at least one (e.g., one, two, three, four, five, six, or seven) selected from the group consisting of SLC39A4, ACE2, AQP6, CUBN, LRP2, ABCB1, and APOA1. The proximal tubule marker is, for example, at least two (e.g., two, three, four, preferably five, preferably six, or more preferably seven) selected from the group of proximal tubule markers. The proximal tubule marker is, for example, at least four (e.g., four, preferably five, preferably six, or more preferably seven) selected from the group of proximal tubule markers. The proximal tubule marker is, for example, at least five, six, or seven selected from the group of proximal tubule markers. The proximal tubule marker is, for example, at least six or seven selected from the group of proximal tubule markers.

[0039] The proximal tubule marker is, for example, at least one (e.g., one, two, three, four, five, or six) selected from the group consisting of SLC39A4, ACE2, AQP6, CUBN, LRP2, and ABCB1. The proximal tubule marker is, for example, at least two (e.g., two, three, four, preferably five, or more preferably six) selected from the group of proximal tubule markers. The proximal tubule marker is, for example, at least four (e.g., four, preferably five, or more preferably six) selected from the group of proximal tubule markers. The proximal tubule marker is, for example, at least five or six selected from the group of proximal tubule markers.

[0040] The proximal tubule marker is, for example, at least one (e.g., one, two, three, four, five, six, or seven) selected from the group consisting of UGT2A3, SLC39A4, ACE2, CUBN, LRP2, ABCB1, and APOA1. The proximal tubule marker is, for example, at least two (e.g., two, three, four, preferably five, preferably six, or more preferably seven) selected from the group of proximal tubule markers. The proximal tubule marker is, for example, at least four (e.g., four, preferably five, preferably six, or more preferably seven) selected from the group of proximal tubule markers. The proximal tubule marker is, for example, at least five, six, or seven selected from the group of proximal tubule markers. The proximal tubule markers are, for example, at least six or seven selected from the group of proximal tubule markers.

[0041] The proximal tubule marker is, for example, at least one (e.g., one, two, three, four, five, or six) selected from the group consisting of UGT2A3, SLC39A4, ACE2, CUBN, LRP2, and ABCB1. The proximal tubule marker is, for example, at least two (e.g., two, three, four, preferably five, or more preferably six) selected from the group of proximal tubule markers. The proximal tubule marker is, for example, at least four (e.g., four, preferably five, or more preferably six) selected from the group of proximal tubule markers. The proximal tubule marker is, for example, at least five or six selected from the group of proximal tubule markers.

[0042] The term "LTL" is an abbreviation for Lotus Tetragonolobus Lectin and refers to a glycoprotein that can exhibit specificity for α-linked L-fucose containing oligosaccharides. The term "UGT2A3" refers to UDP-glucuronosyltransferase 2A3. The term "AZGP1" is an abbreviation for Zinc-alpha2-glycoprotein. The AZGP1 gene encodes zinc-α2-glycoprotein (ZAG). The term "SLC39A4" refers to Solute Carrier Family 39 Member 4, and the SLC39A4 gene encodes Zinc / Iron-regulated transporter-like Protein 4 (ZIP4). The term "BHMT" is an abbreviation for Betaine-Homocysteine ​​S-Methyltransferase. The BHMT gene encodes an enzyme that utilizes betaine as a methyl donor and catalyzes the remethylation of homocysteine ​​(Hcy).

[0043] The term "ACE2" is an abbreviation for Angiotensin-Converting Enzyme 2. The ACE2 gene encodes an isoform of angiotensin-converting enzyme (ACE). The term "AQP6" is an abbreviation for Aquaporin 6. The AQP6 gene encodes an aquaporin protein that functions as a water channel in cells. The term "CUBN" is an abbreviation for Cubilin. The CUBN gene encodes Cubilin, which functions as an intrinsic factor-vitamin B12 complex. The term "LRP2" is an abbreviation for LDL Receptor Related Protein 2, a protein that belongs to a receptor family with structural similarity to the low-density lipoprotein receptor (LDLR). The LRP2 gene encodes LDL Receptor Related Protein 2. The term "ABCB1" is an abbreviation for ABC transporter subfamily B member 1. The ABCB1 gene encodes P-glycoprotein 1 (also called ABCB1 or MDR1), a member of the ATP-binding cassette (ABC) transporter superfamily.

[0044] The term "SERPINA1" is an abbreviation for serpin family A member 1. The SERPINA1 gene encodes SERPINA1, a prototype member of the serpin superfamily, which are serine protease inhibitors. The term "APOA1" is an abbreviation for Apolipoprotein A-I, which refers to the major protein component of HDL particles in plasma. The APOA1 gene encodes Apolipoprotein A-I. The term "SPARCL1" is an abbreviation for SPARC-like protein 1, which is also known as Hevin, a matrix protein. The SPARCL1 gene encodes the matrix protein SPARCL1.

[0045] As used herein, the term "PPARA downstream gene marker" refers to at least one gene selected from the group consisting of APOA1, APOC3, CPT1A, FABP1, CYP27A1, and ACOX1. The PPARA downstream gene marker is, for example, one, two, three, four, five, or six gene markers selected from the group of PPARA downstream gene markers. The PPARA downstream gene marker is at least two gene markers (e.g., two, three, four, preferably five, or more preferably six) selected from the group of PPARA downstream gene markers. The PPARA downstream gene marker is at least three gene markers (e.g., three, four, preferably five, or more preferably six) selected from the group of PPARA downstream gene markers. The PPARA downstream gene marker is at least four gene markers, preferably five, or more preferably six selected from the group of PPARA downstream gene markers.

[0046] The PPARA downstream gene marker is, for example, at least one (e.g., one, two, three, preferably four, or more preferably five) selected from the group consisting of APOA1, APOC3, CPT1A, FABP1, and CYP27A1. The PPARA downstream gene marker is at least two (e.g., two, three, preferably four, or more preferably five) selected from the group of PPARA downstream gene markers. The PPARA downstream gene marker is four or five selected from the group of PPARA downstream gene markers.

[0047] The PPARA downstream gene marker is, for example, at least one (e.g., one, two, three, preferably four, or more preferably five) gene markers selected from the group consisting of APOA1, CPT1A, FABP1, ACOX1, and CYP27A1. The PPARA downstream gene marker is, for example, at least two (e.g., two, three, preferably four, or more preferably five) gene markers selected from the group of PPARA downstream gene markers. The PPARA downstream gene marker is, for example, four or five gene markers selected from the group of PPARA downstream gene markers.

[0048] The PPARA downstream gene marker is, for example, at least one (e.g., one, two, preferably three, or more preferably four) gene markers selected from the group consisting of APOA1, CPT1A, FABP1, and ACOX1. The PPARA downstream gene marker is, for example, at least two (e.g., two, preferably three, or more preferably four) gene markers selected from the group of PPARA downstream gene markers. The PPARA downstream gene marker is, for example, three or four gene markers selected from the group of PPARA downstream gene markers.

[0049] The PPARA downstream gene marker is, for example, at least one (e.g., one, preferably two, or more preferably three) gene markers selected from the group consisting of APOA1, CPT1A, and FABP1. The PPARA downstream gene marker is, for example, two or three gene markers selected from the group of PPARA downstream gene markers.

[0050] The term "APOA1" is an abbreviation for apolipoprotein A-I, which refers to the major protein component of HDL particles in plasma. The APOA1 gene encodes apolipoprotein A-I. The term "APOC3" is an abbreviation for apolipoprotein C-III, which refers to a relatively small protein composed of 79 amino acids. The APOC3 gene encodes apolipoprotein C-III. The term "CPT1A" is an abbreviation for carnitine palmitoyltransferase IA, which is an enzyme involved in the outer mitochondrial membrane. The CPT1A gene encodes carnitine palmitoyltransferase IA.

[0051] The term "FABP1" is an abbreviation for Fatty Acid Binding Protein 1. The FABP1 gene encodes Fatty Acid Binding Protein 1, also known as liver-type fatty acid binding protein. The term "CYP27A1" refers to a gene encoding a cytochrome P450 oxidase, also known as sterol 27-hydroxylase. The term "ACOX1" is an abbreviation for Acyl-CoA Oxidase 1. The ACOX1 gene encodes peroxisomal acyl-coenzyme A oxidase 1.

[0052] Mature proximal tubule cells express, for example, at least one marker selected from the group of PPARA downstream gene markers disclosed herein. Mature proximal tubule cells express, for example, at least one marker selected from the group of proximal tubule markers disclosed herein. Mature proximal tubule cells express, for example, at least one marker selected from the group of PPARA downstream gene markers disclosed herein and at least one marker selected from the group of proximal tubule markers disclosed herein. Mature proximal tubule cells express, for example, at least three markers (e.g., three, four, preferably five, or more preferably six) selected from the group of PPARA downstream gene markers disclosed herein and at least five markers (e.g., five, six, seven, preferably eight, or more preferably nine) selected from the group of proximal tubule markers disclosed herein.

[0053] Renal organoids containing mature proximal tubule cells express at least one selected from the group of proximal tubule markers of the present disclosure. In one example, renal organoids containing mature proximal tubule cells further express at least one selected from the group of PPARA downstream gene markers of the present disclosure. Renal organoids containing mature proximal tubule cells, for example, express at least one selected from the group of proximal tubule markers of the present disclosure, and also express at least one selected from the group of proximal tubule markers of the present disclosure. The expression of PPARA downstream gene markers in renal organoids containing proximal tubule cells can be detected based on the transcription product of the marker gene or the translation product of the marker gene. In addition, the expression of proximal tubule markers in renal organoids containing proximal tubule cells can be detected based on the transcription product of the marker gene or the translation product of the marker gene. The transcription product of the marker gene can be measured, for example, by quantitative PCR. The translation products of the marker genes can be measured, for example, by fluorescent immunostaining, or by FACS or MACS.

[0054] In the kidney organoid containing mature proximal tubule cells, the expression amount (for example, the amount of transcript of marker gene) of PPARA downstream gene marker can be evaluated as being expressed when it is significantly greater than the expression amount of the corresponding PPARA downstream gene marker in the kidney organoid (control organoid) that is cultured using a medium that is substantially free of either RXR agonist or PPAR agonist or both.Preferably, the expression amount of PPARA downstream gene marker is evaluated as being expressed when it is significantly greater than the expression amount of the corresponding PPARA downstream gene marker in the control organoid that is cultured using a medium that is substantially free of either RXR agonist or PPAR agonist. In addition, the proximal tubule marker in the renal organoid containing mature proximal tubule cells may be evaluated as being expressed when its expression level (for example, the amount of transcription product of the marker gene) is significantly greater than the expression level of the corresponding proximal tubule marker in the renal organoid (control organoid) cultured using a medium that is substantially free of either or both of RXR agonist and PPAR agonist.Preferably, the expression level of the proximal tubule marker is evaluated as being expressed when it is significantly greater than the expression level of the corresponding proximal tubule marker in the control organoid cultured using a medium that is substantially free of either RXR agonist or PPAR agonist.

[0055] The medium that is substantially free of either or both of RXR agonist and PPAR agonist may be, for example, the medium containing RXR agonist and PPAR agonist (for example, the induction medium C described below) used to induce the renal organoid containing the desired mature proximal tubule cells, except that it is substantially free of either or both of RXR agonist and PPAR agonist, and the composition and / or dosage are the same.In this context, "substantially free" does not exclude that it is completely free of RXR agonist or PPAR agonist.In one example, the medium that is substantially free of RXR agonist or PPAR agonist may contain, for example, less than 10 nM, less than 5 nM, or less than 1 nM PPAR agonist, and less than 10 nM, less than 5 nM, or less than 1 nM RXR agonist. In one example, the medium that is substantially free of RXR agonists or PPAR agonists is completely free of RXR agonists or PPAR agonists.

[0056] In another embodiment, the PPARA downstream gene marker in the renal organoid that contains mature proximal tubule cells can be evaluated as being expressed when its expression level (for example, the amount of transcript of marker gene) is compared with the expression level of corresponding PPARA downstream gene marker in early renal organoid (control organoid) significantly higher.In addition, the proximal tubule marker in the renal organoid that contains mature proximal tubule cells can be evaluated as being expressed when its expression level (for example, the amount of transcript of marker gene) is compared with the expression level of corresponding proximal tubule marker in early renal organoid (control organoid) significantly higher.

[0057] [Method for producing renal organoids containing mature proximal tubule cells] One aspect of the present disclosure provides a method for producing renal organoids characterized by containing mature proximal tubule cells, comprising culturing early stage renal organoids using culture medium C containing an RXR agonist and a PPAR agonist.

[0058] One embodiment of the present disclosure provides a method for producing kidney organoids containing mature proximal tubule cells, comprising: culturing intermediate mesodermal cells using induction medium B (induction medium b2) containing a fibroblast growth factor to form intermediate mesodermal spheroids; and culturing the intermediate mesodermal spheroids using induction medium A (induction medium a2) containing a GSK3β inhibitor, followed by culturing them using induction medium B (induction medium b3) containing a fibroblast growth factor to induce early renal organoids (step B); and culturing the early renal organoids using induction medium C containing an RXR agonist and a PPAR agonist (step C).

[0059] One embodiment of the present disclosure provides a method for producing kidney organoids containing mature proximal tubule cells, comprising: culturing pluripotent stem cells to induce intermediate mesodermal cells (Step A); culturing the intermediate mesodermal cells using an induction medium B (induction medium b2) containing a fibroblast growth factor to form intermediate mesodermal spheroids, and culturing the intermediate mesodermal spheroids using an induction medium A (induction medium a2) containing a GSK3β inhibitor, and then using an induction medium B (induction medium b3) containing a fibroblast growth factor to induce early kidney organoids (Step B); and culturing the early kidney organoids using an induction medium C containing an RXR agonist and a PPAR agonist (Step C).

[0060] Step A: Induction of Differentiation into Intermediate Mesodermal Cells Step A comprises culturing pluripotent stem cells to induce intermediate mesodermal cells. More specifically, step A comprises culturing the pluripotent stem cells in an induction medium A (induction medium a1) containing a GSK3β inhibitor, and then culturing the pluripotent stem cells in an induction medium B (induction medium b1) containing a fibroblast growth factor, thereby inducing intermediate mesodermal cells.

[0061] Pluripotent stem cells can be cultured using induction medium a1 under known cell culture conditions, such as 37°C and 5% CO ２ The culture temperature is not limited to 37°C, and any temperature known in the field of cell culture can be used as appropriate.２ The concentration is not limited to 5% and may be any CO concentration known in the field of cell culture. ２ The pluripotent stem cells can be cultured using induction medium A for 2 to 8 days, 3 to 7 days, 4 to 6 days, or 5 days.

[0062] Step A involves culturing pluripotent stem cells using induction medium a1, followed by culturing them using induction medium b1 containing fibroblast growth factors. The culturing using induction medium b1 can be performed, for example, after colonies composed of pluripotent stem cells have spread horizontally and vertically relative to the culture surface and acquired a rounded shape through culturing using induction medium a1. By culturing the rounded cell colonies using induction medium B containing fibroblast growth factors, intermediate mesoderm cells can be induced.

[0063] The rounded cell colonies (pluripotent stem cell cultures using induction medium a1) can be cultured using induction medium b1 under known cell culture conditions, such as 37°C and 5% CO ２ The culture temperature is not limited to 37°C, and any temperature known in the field of cell culture can be used as appropriate. ２ The concentration is not limited to 5% and may be any CO concentration known in the field of cell culture. ２ The rounded cell colonies can be cultured using induction medium B for 2 to 6 days, 2 to 5 days, 2 to 4 days, or 3 days. This culture can change the rounded cell colonies into a sheet-like shape.

[0064] As used herein, the term "pluripotent stem cells" refers to stem cells that can be cultured in vitro and have the ability to differentiate into tissues derived from three germ layers (ectoderm, mesoderm, and endoderm), i.e., pluripotency. Pluripotent stem cells can be established, for example, from fertilized eggs, cloned embryos, germ stem cells, or intracellular stem cells. Pluripotent stem cells include, for example, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells) derived from somatic cells, embryonic tumor cells (EC cells), or embryonic germ stem cells (EG cells). Pluripotent stem cells are preferably ES cells or iPS cells.

[0065] As used herein, the term "ES cells" refers to stem cells that have the ability to self-replicate and pluripotency, and are derived from an early embryo. Examples of ES cells include human ES cells.

[0066] As used herein, the term "iPS cells" refers to pluripotent stem cells induced from somatic cells, which are artificially endowed with pluripotency similar to that of embryonic stem cells by reprogramming somatic cells. iPS cells can be established by reprogramming differentiated cells such as fibroblasts through the expression of genes such as Oct3 / 4, Sox2, Klf4, and Myc. iPS cells are, for example, human iPS cells established by reprogramming differentiated cells such as human fibroblasts.

[0067] As used herein, "induction medium A" includes a basal medium and an additive containing a GSK3β inhibitor. The induction medium A used in step A is also referred to as induction medium a1. The induction medium A used in step B (particularly step b2) is also referred to as induction medium a2. The description of induction medium A in this specification also applies to induction medium a1 and induction medium a2 unless otherwise specified. Induction medium A can be prepared, for example, by adding the additive (solid or liquid) to a basal medium (liquid). The concentration of the additive added to induction medium A is appropriately determined by those skilled in the art, taking into account the animal species from which the cells used for culture are derived. The "basal medium" may be a cell culture medium that can be prepared according to known protocols or may be a commercially available cell culture medium. The basal medium may be, for example, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Basal Medium Eagle's (BME), a known stem cell medium, or a known medium for differentiating stem cells. The basal medium is preferably a medium for differentiating stem cells, and may be, for example, STEMdiff APEL2 medium (STEMCELL Technologies). The medium for differentiating stem cells can be prepared, for example, according to Nature Protocols, Vol. 3, No. 5, pp. 768-776, 2008. Induction medium A may further contain a protein-free medium (e.g., PFHM-II), an antibiotic (e.g., penicillin / streptomycin, gentamicin), an antibiotic-antimycotic mixture (e.g., antibiotic-antimycotic), or a combination thereof.

[0068] As used herein, the term "GSK3β inhibitor" refers to a compound that inhibits the action of serine / threonine protein kinase 3β, which is involved in various signal transduction pathways, including the WNT / β-catenin pathway. Examples of GSK3β inhibitors include CHIR-99021, SB216763, CHIR-98014, staurosporine, K252A, WNT (preferably WNT3A), or TWS119, or a combination thereof. The GSK3β inhibitor is preferably CHIR-99021 or WNT (preferably WNT3A). Induction medium a1 may contain a GSK3β inhibitor other than CHIR-99021 at a concentration that exhibits the same level of GSK3β inhibitory activity as that exhibited by CHIR-99021 at 1 to 50 μM, 2 to 25 μM, 5 to 15 μM, 5 to 8 μM, or 8 μM. Induction medium a2 may contain a GSK3β inhibitor other than CHIR-99021 at a concentration that exhibits an effect similar to the GSK3β inhibitory effect exhibited by CHIR-99021 at 1 to 50 μM, 2 to 25 μM, 5 to 15 μM, 5 to 10 μM, or 10 μM.

[0069] "GSK3β inhibitory effect" can be measured, for example, based on the transcription activity caused by the nuclear translocation of β-catenin in the presence of a predetermined amount of a GSK3β inhibitor. The transcription activity caused by the nuclear translocation of β-catenin can be measured according to known methods (e.g., luciferase activity measurement). The transcription activity caused by the nuclear translocation of β-catenin can be measured, for example, using a commercially available kit (e.g., LEADING LIGHT (registered trademark) Wnt Reporter Assay Starter kit). As used herein, the term "similar effect" refers to an effect within ±30%, ±20%, or ±10% of the effect of a control. In one embodiment, the similar effect is an effect within ±30% of the effect of a control.

[0070] Induction medium A may contain a GSK3β inhibitor (preferably CHIR-99021) at 1 to 50 μM, 2 to 25 μM, or 5 to 15 μM. Induction medium A may contain the GSK3β inhibitor at a concentration that exhibits the same level of GSK3β inhibitory effect as that exhibited by CHIR-99021 at 1 to 50 μM, 2 to 25 μM, or 5 to 15 μM. Induction medium A may contain 1 to 2000 ng / ml, 2 to 1000 ng / ml, or 5 to 400 ng / ml of WNT (preferably WNT3A) as a GSK3β inhibitor. When induction medium A contains WNT (preferably WNT3A) as a GSK3β inhibitor, induction medium a1 may contain WNT (preferably WNT3A) at a concentration that exhibits the same level of GSK3β inhibitory effect as that exhibited by CHIR-99021 at 1 to 50 μM, 2 to 25 μM, 5 to 15 μM, or 8 μM. Induction medium a2 may contain WNT (preferably WNT3A) at a concentration that exhibits the same level of GSK3β inhibitory effect as that exhibited by CHIR-99021 at 1 to 50 μM, 2 to 25 μM, 5 to 15 μM, or 10 μM.

[0071] As used herein, "induction medium B" comprises a basal medium and an additive containing a fibroblast growth factor and substantially free of an RXR agonist. The induction medium B used in step A is also referred to as induction medium b1. The induction medium B used in step b1 is also referred to as induction medium b2. The induction medium B used in step b3 is also referred to as induction medium b3. In this specification, the description of induction medium B also applies to induction medium b1, induction medium b2, and induction medium b3, unless otherwise specified. In one example, the additive of induction medium B further contains substantially no PPAR agonist. The additive of induction medium B contains, for example, a fibroblast growth factor and contains substantially no RXR agonist or PPAR agonist. Induction medium B can be prepared, for example, by adding the additive (solid or liquid) to a basal medium (liquid). The description of the basal medium for induction medium A applies, as appropriate, to the basal medium for induction medium B. The concentrations of the additives added to induction medium B can be appropriately determined by those skilled in the art, taking into consideration the animal species from which the cells used for culture have been given. Induction medium B may further contain heparin, a protein-free medium (e.g., PFHM-II), an antibiotic (e.g., penicillin / streptomycin, gentamicin), an antibiotic-antimycotic mixture (e.g., antibiotic-antimycotic), or a combination thereof. Induction medium b2, which is used to form spheroids from intermediate mesodermal cells, may further contain a ROCK inhibitor (e.g., Y-27632).

[0072] In the context of induction medium B, the term "substantially free of RXR agonists" means that the RXR agonist is contained in an amount sufficient to induce differentiation of organoids having the same level of maturity as the early renal organoids formed in step B using a medium having the same composition and / or dosage as the early renal organoids formed in step B, except that induction medium B is completely free of RXR agonists. "Organoids having the same level of maturity as early renal organoids" have one or more LHX1-positive metanephric vesicles and contain less than 5% (preferably less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5%) of Forff's duct cells relative to the total cells derived from the organoid. Metanephric vesicles are preferably LHX1-positive and JAG1-positive or CDH6-positive. Metanephric vesicles are preferably LHX1-positive, JAG1-positive, and CDH6-positive. "Forff's duct cells" are CDH1-positive, PAX2-positive, and GATA3-positive cells. The proportion of Forf duct cells to the total cells derived from organoids can be measured, for example, by single-cell proteome analysis (e.g., single-cell RNA sequencing). Substantially free of RXR agonists does not exclude complete absence of RXR agonists. In one example, induction medium B substantially free of RXR agonists contains less than 10 pM, less than 5 pM, less than 1 pM, less than 0.5 pM, or less than 0.1 pM of RXR agonists (e.g., 9cis retinoic acid). Preferably, induction medium B substantially free of RXR agonists is completely free of RXR agonists (e.g., 9cis retinoic acid).

[0073] In another example, induction medium B that is substantially free of RXR agonists is substantially free of PPAR agonists (e.g., at least one selected from the group consisting of PPARA agonists, PPARG agonists, PPARD agonists, PPARA / G agonists, PPARA / D agonists, PPARG / D agonists, and PPARA / G / D agonists). The term "substantially free of RXR agonists and PPAR agonists" in the context of induction medium B means that the medium contains an RXR agonist and / or PPAR agonist in an amount such that organoids having the same level of maturity as the primary renal organoids formed in step B using a medium having the same composition and / or dosage, except that induction medium B is completely free of RXR agonists and PPAR agonists, are induced to differentiate. In one example, induction medium B that is substantially free of RXR agonists and PPAR agonists contains less than 10 pM, preferably less than 5 pM, less than 1 pM, less than 0.5 pM, or less than 0.1 pM of RXR agonists and / or PPAR agonists. Induction medium B that is substantially free of RXR agonists and PPAR agonists is preferably completely free of RXR agonists (e.g., 9cis retinoic acid) and PPAR agonists (e.g., PPARA agonists).

[0074] As used herein, the term "CDH1" refers to a gene encoding the cadherin 1 protein. CDH1-positive cells are cells in which CDH1 transcripts are produced. CDH1-positive cells can be detected by quantitative PCR using reverse transcriptase or single-cell proteome analysis.

[0075] As used herein, the term "PAX2" is an abbreviation for paired box gene 2, which refers to a gene encoding a homeobox transcription factor. PAX2-positive cells are cells in which PAX2 transcripts are produced. PAX2-positive cells can be detected by quantitative PCR using reverse transcriptase or single-cell proteome analysis.

[0076] As used herein, the term "GATA3" refers to a gene encoding a GATA3 transcription factor. GATA3-positive cells are cells in which GATA3 transcripts are produced. GATA3-positive cells can be detected by quantitative PCR using reverse transcriptase or single-cell proteome analysis.

[0077] CDH1-positive, PAX2-positive, and GATA3-positive cells are cells in which CDH1 transcripts, PAX2 transcripts, and GATA3 transcripts are produced, and such cells can be detected by single-cell proteome analysis.

[0078] As used herein, the term "fibroblast growth factor (FGF)" refers to a protein with a molecular weight of 16,000 to 20,000 that promotes the proliferation of fibroblasts or endothelial cells. The FGF may be, for example, FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, or FGF23, or a combination thereof. The FGF may be, for example, FGF9 alone or a combination of FGF9 and another FGF (e.g., FGF4). The FGF is preferably FGF9 alone. Induction medium B may contain 20 to 1000 ng / mL, 60 to 500 ng / mL, or 100 to 300 ng / mL of FGF (preferably FGF9). Induction medium B may contain an FGF other than FGF9 at a concentration that exhibits a fibroblast proliferation effect similar to that exhibited by 20 to 1000 ng / mL, 60 to 500 ng / mL, or 100 to 300 ng / mL of FGF9.

[0079] As used herein, the term "intermediate mesoderm" refers to a sheet-shaped cell colony formed by culturing pluripotent stem cells in induction medium A (induction medium a1) to form rounded cell colonies, which are then cultured in induction medium B (induction medium b1). As used herein, the term "intermediate mesoderm cells" can be produced by culturing pluripotent stem cells in induction medium A (induction medium a1) and then culturing them in induction medium B (induction medium b1). Intermediate mesoderm cells can be produced, for example, by culturing pluripotent stem cells in induction medium A (induction medium a1) for 2 to 8 days, 3 to 7 days, 4 to 6 days, or 5 days, and then culturing them in induction medium B (induction medium b1) for 2 to 6 days, 2 to 5 days, 2 to 4 days, or 3 days.

[0080] Step A can be carried out, for example, according to the following procedure: Human iPS or ES cells cultured on an iMatrix-511 coating using StemFit AK02N medium (REPROCELL) are detached using TrypLE Select (Thermo Fisher Scientific). The detached cells are suspended in StemFit AK02N (10 μM Y-27632), and iMatrix-511 (nippi) is added to the cell suspension at a final concentration of 0.25 μg / cm. ２ The cell suspension is added so that the density is 10,000 to 15,000 cells / cm. ２ The cells were seeded in a 12-well plate so that the total number of cells was 100, and the cells were incubated at 37°C and CO ２The cells are cultured for 1 day under 5% CO₂. The seeded cells are then cultured for 3-5 days in induction medium A (induction medium a1), STEMdiff APEL2 medium (STEMCELL Technologies) (8 μM CHIR99021, 1% PFHM II, antibiotic-antimycotic; additives to the basal medium are listed in parentheses below), to induce the primitive streak. The medium is changed daily from day 4 onwards. A 5-day culture period allows for an increased proportion of posterior intermediate mesoderm cells, thereby enabling the formation of numerous proximal tubules. After culturing in induction medium A, the cells are cultured for 3 to 5 days using induction medium B (induction medium b1), STEMdiff APEL2 medium (200 ng / mL FGF9, 1 μg / mL Heparin, 1% PFHM II, antibiotic-antimycotic), to induce intermediate mesoderm cells.

[0081] Step B: Spheroid formation and formation of early renal organoids Step B includes culturing the intermediate mesodermal cells formed in step A using a fibroblast growth factor induction medium B (induction medium b2) to form intermediate mesodermal spheroids (step b1), and culturing the intermediate mesodermal spheroids using an induction medium A containing a GSK3β inhibitor (induction medium a2) (step b2), followed by culturing them using an induction medium B containing a fibroblast growth factor (induction medium b3) (step b3) to induce early renal organoids.

[0082] (Step b1) Step b1 comprises culturing the intermediate mesodermal cells formed in step A using an induction medium b2 containing fibroblast growth factors to form intermediate mesodermal spheroids. Step b1 may further comprise detaching the intermediate mesodermal cells formed in step A from the culture plate using a cell detachment solution. The treatment with the cell detachment solution separates the intermediate mesodermal cells to obtain a cell suspension. Therefore, step b1 may further comprise separating the intermediate mesodermal cells formed in step A to obtain a cell suspension. The "cell detachment solution" refers to a solution containing an enzyme that degrades cell adhesion molecules. The cell detachment solution contains, for example, trypsin, preferably recombinant trypsin (e.g., TrypLE Select (Thermo Fisher Scientific)). The treatment with the cell detachment solution comprises, for example, incubation at 37°C for 3 to 10 minutes, or 5 to 10 minutes.

[0083] Step b1 involves culturing the cell suspension in an induction medium b2 containing a fibroblast growth factor. Treatment with this induction medium b2 can result in the formation of spheroids. From the viewpoint of efficient spheroid formation, induction medium b2 preferably contains a ROCK inhibitor. The culturing in step b1 can be carried out under known cell culture conditions. Known cell culture conditions include 37°C and 5% CO ２ The culture temperature is not limited to 37°C, and any temperature known in the field of cell culture can be used as appropriate. ２ The concentration is not limited to 5% and may be any CO concentration known in the field of cell culture. ２Concentrations can be used appropriately. The culture in step b1 using induction medium b2 containing a ROCK inhibitor can be carried out for 0.5 to 2 days, 0.5 days, 1 day, or 1.5 days, preferably 1 day. If induction medium b2 is substantially free of a ROCK inhibitor, the culture in step b1 may include an additional day of culture. In the context of induction medium B (particularly induction medium b2), the term "substantially free" means containing a ROCK inhibitor at a concentration of less than 100 nM, less than 50 nM, less than 10 nM, less than 5 nM, or less than 1 nM. "Substantially free" does not exclude complete absence of a ROCK inhibitor. The culture in step b1 forms spheroids (also referred to herein as "intermediate mesoderm spheroids") composed of a collection of intermediate mesoderm cells. As used herein, the term "spheroid" refers to a three-dimensional cell aggregate formed in a test tube.

[0084] As used herein, the term "ROCK inhibitor" refers to a compound that inhibits Rho kinase (Rho-associated, coiled-coil containing protein kinase: ROCK). Examples of ROCK inhibitors include N-(4-pyridinyl)-4β-[(R)-1-aminoethyl]cyclohexane-1α-carboxamide (Y-27632), Fasudil (HA1077), (2S)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]hexahydro-1H-1,4-diazepine (H-1152), 4β-[(1R)-1-aminoethyl]-N-(4-pyridyl)benzene-1αcarboxamide (Wf-536), and N-(1H-pyrrolo[2,3-b]pyridin-4-yl)-4β-[(R)-1-aminoethyl]cyclohexane-1α. carboxamide (Y-30141), N-(3-{[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-1H-imidazo[4,5-c]pyridin-6-yl]oxy}phenyl)-4-{[2-(4-morpholinyl)ethyl]-oxy}benzamide (GSK269962A), or N-(6-fluoro-1H-indazol-5-yl)-6-methyl-2-oxo-4-[4-(trifluoromethyl)phenyl]-3,4-dihydro-1H-pyridine-5-carboxamide (GSK429286A), or a combination thereof. The ROCK inhibitor may be, for example, Y-27632 alone or in combination with another ROCK inhibitor. The ROCK inhibitor is preferably Y-27632. Induction medium B (particularly induction medium b2) contains a ROCK inhibitor at 1 to 50 μM, 2 to 25 μM, 5 to 15 μM, or 10 μM. Induction medium B (particularly induction medium b2) may contain a ROCK inhibitor other than Y-27632 at a concentration that exhibits the same level of ROCK inhibitory effect as that exhibited by Y-27632 at 1 to 50 μM, 2 to 25 μM, 5 to 15 μM, or 10 μM.

[0085] ROCK is a serine-threonine protein kinase. ROCK phosphorylates, for example, myosin-binding subunit 1 (MYPT1) of myosin light chain phosphatase (MLCP), thereby inhibiting its enzymatic activity. The "ROCK inhibitory effect" can be measured, for example, based on the amount of phosphorylation of MYPT1 by ROCK in the presence of a compound capable of inhibiting the phosphorylation effect. In one example, the ROCK inhibitory effect can be measured by measuring the amount of phosphorylation of MYPT1 by ROCK, measuring the amount of phosphorylation of MYPT1 by ROCK in the presence of a compound capable of inhibiting the phosphorylation effect, and comparing the amounts of phosphorylation. The ROCK inhibitory activity can be measured using a commercially available kit (for example, a 96-well ROCK Activity Assay Kit (CELL BIOLABS, INC., Catalog No.: STA-416) or a ROCK Activity Immunoblot Kit (STA-416, CELL BIOLABS, INC., Catalog No.: STA-415)).

[0086] The induction medium b2 used in step b1 may be the same as or different from the induction medium b1 used in step A in terms of composition and / or dosage. The induction medium b2 is preferably the same as the induction medium b1 in terms of composition and / or dosage. The induction medium b2 is, for example, the same as the induction medium b1 in terms of composition and / or dosage, except that it contains a ROCK inhibitor.

[0087] (Step b2) Step b2 involves culturing the intermediate mesodermal spheroids formed in step b1 using induction medium A (induction medium a2) containing a GSK3β inhibitor. The culture in step b2 is carried out, for example, at an air-liquid interface or in a liquid medium. The culture in step b2 is preferably carried out at an air-liquid interface.

[0088] Air-liquid interface culture can be performed, for example, using a culture device equipped with a culture well insert capable of accommodating cells and having a liquid-permeable membrane on the bottom, and a culture base plate onto which the culture well insert can be placed and which can be filled with culture medium. Such a culture device is, for example, commercially available, and may be, for example, Cell Culture Insert Transparent PET membrane (Corning). The culture in step b2 can be performed under known cell culture conditions. Known cell culture conditions include incubation at 37°C and 5% CO ２ The culture temperature is not limited to 37°C, and any temperature known in the field of cell culture can be used as appropriate. ２ The concentration is not limited to 5% and may be any CO concentration known in the field of cell culture. ２ The culture can be carried out for 1 to 24 hours, 1 to 12 hours, 1 to 8 hours, 1 to 5 hours, 2 to 4 hours, or 3 hours.

[0089] The induction medium a2 used in step b2 may be the same as or different from the induction medium a1 used in step A in terms of composition and / or dosage. The induction medium a2 is preferably the same as the induction medium a1 in terms of composition and / or dosage. The induction medium a2 is the same as the induction medium a1 in terms of composition and / or dosage, except for the difference in the concentration of GSK3β, for example.

[0090] (Step b3) Step b3 involves culturing the intermediate mesodermal spheroids cultured in step b2 using induction medium B (induction medium b3) containing fibroblast growth factors. By culturing in step b3, primary renal organoids can be formed. The culturing in step b3 can be carried out under known cell culture conditions. Known cell culture conditions include 37°C and 5% CO ２ The culture temperature is not limited to 37°C, and any temperature known in the field of cell culture can be used as appropriate. ２ The concentration is not limited to 5% and may be any CO concentration known in the field of cell culture. ２The concentration can be appropriately used. The culture can be carried out for 4 to 12 days, 4 to 10 days, 5 to 8 days, 6 days, 7 days, or 8 days, preferably 6 days. The culture in step b3 is carried out, for example, until the formation of nephrons containing proximal tubules is confirmed in the cultured spheroids. Spheroids in which the formation of nephrons containing proximal tubules is confirmed in step b3 are also referred to as early renal organoids.

[0091] Induction medium b3 may contain 20 to 1000 ng / mL, 60 to 500 ng / mL, or 100 to 300 ng / mL of FGF (preferably FGF9). The composition and / or dosage of induction medium b3 may be the same as or different from those of induction medium b1 or b2. Preferably, induction medium b3 has the same composition and / or dosage as induction medium b1. For example, induction medium b3 has the same composition and / or dosage as induction medium b2. Preferably, induction medium b3 has the same composition and / or dosage as induction medium b2, except that it contains a ROCK inhibitor.

[0092] Step B can be performed, for example, according to the following procedure: After intermediate mesoderm cell induction in step A, the cells are detached using TrypLE Select (Thermo Fisher Scientific). The detached cells are suspended in induction medium B (induction medium b2), STEMdiff APEL2 medium (200 ng / mL FGF9, 1 μg / mL Heparin, 1% PFHM II, Antibiotic-Antimycotic, 10 μM Y-27632), and the resulting cell suspension is seeded onto a PrimeSurface (registered trademark) 96M plate (Sumitomo Bakelite) at a density of 100,000 cells / well. The next day, the spheroids formed in the plate were placed on a Cell Culture Insert Transparent PET membrane 6-well 0.4 μm pore size (Corning) Cell Culture Insert at a density of 3 to 6 spheroids per well. The Cell Culture Insert containing the spheroids was then placed in the corresponding well of a culture plate. STEMdiff APEL2 medium (10 μM CHIR99021, 1% PFHM II, antibiotic-antimycotic), which is induction medium A (induction medium a2), was added between the well of the culture plate and the underside of the transwell of the Cell Culture Insert, and the spheroids were incubated at the air-liquid interface at 37°C under CO ２ After culturing for 3 hours, the medium was replaced with STEMdiff APEL2 medium (200 ng / mL FGF9, 1 μg / mL Heparin, 1% PFHM II, antibiotic-antimycotic), which is induction medium B (induction medium b3), and the cells were further cultured.

[0093] Step C: Proximal Tubule Maturation Step C involves culturing early stage renal organoids in a medium containing an RXR agonist and a PPAR agonist (also referred to as induction medium C). The early stage renal organoids used in step C may be early stage renal organoids produced from pluripotent stem cells by carrying out steps A and B of the present disclosure, or early stage renal organoids produced by a known method. Examples of known methods include the method described in Takasato M., et al., Nat Cell Biol. 2014;16:118-26 (incorporated herein by reference) or the method described in Takasato M., et al., Nature. 2015;526:564-568 (incorporated herein by reference). In one embodiment, the primary renal organoids used in step C are primary renal organoids produced from pluripotent stem cells by carrying out steps A and B of the present disclosure.

[0094] The early stage renal organoids used for culturing in step C contain early stage proximal tubules. By culturing the early stage renal organoids in step C, renal organoids containing mature proximal tubule cells can be formed. Renal organoids containing mature proximal tubule cells are characterized by the expression of markers according to the present disclosure.

[0095] The culture in step C can be carried out under known cell culture conditions, such as at 37°C and 5% CO ２ The culture temperature is not limited to 37°C, and any temperature known in the field of cell culture can be used as appropriate. ２ The concentration is not limited to 5% and may be any CO concentration known in the field of cell culture. ２ The culture is carried out for 5 to 20 days, 6 to 18 days, 7 to 16 days, 7 to 14 days, 7 to 12 days, 7 to 10 days, 7 to 9 days, or 7 to 8 days.

[0096] The renal organoids containing mature proximal tubule cells formed in step C can be used as an active ingredient of a composition for regenerative medicine. The renal organoids containing mature proximal tubule cells can be used in a method for evaluating renal damage caused by a test substance or a method for evaluating drug responsiveness to a test substance.

[0097] As used herein, "induction medium C" comprises a basal medium and an additive containing an RXR agonist and a PPAR agonist. Induction medium C can be prepared, for example, by adding the additive (solid or liquid) to a basal medium (liquid). The explanation for the basal medium for induction medium A applies to the basal medium for induction medium C, as appropriate. The basal medium may contain polyunsaturated fatty acids, preferably linoleic acid and / or linolenic acid. When the basal medium contains polyunsaturated fatty acids (e.g., linoleic acid and / or linolenic acid) at a concentration that does not affect the effects of the present invention, the additives, RXR agonist and PPAR agonist, may be added at the concentrations disclosed herein, regardless of the concentration of the polyunsaturated fatty acids in the basal medium. When the basal medium contains polyunsaturated fatty acids (e.g., linoleic acid and / or linolenic acid) at concentrations that can affect the effects of the present invention, the additives, RXR agonist and PPAR agonist, are added taking into consideration the concentration of the polyunsaturated fatty acids in the basal medium. The concentrations of the additives added to induction medium C can be appropriately determined by those skilled in the art, taking into consideration the animal species from which the cells used for culture are derived. Induction medium C may further contain a protein-free medium (e.g., PFHM-II), an antibiotic (e.g., penicillin / streptomycin, gentamicin), an antibiotic-antimycotic mixture (e.g., antibiotic-antimycotic), or a combination thereof.

[0098] The term "RXR" is an abbreviation for Retinoid X Receptor and refers to a protein that is a member of the steroid / thyroid hormone superfamily of nuclear receptors. RXR forms heterodimers with nuclear receptors such as PPARs or retinoic acid receptors (RARs) and regulates transcriptional activity.

[0099] As used herein, the term "RXR agonist" refers to a compound or composition that, when combined with a retinoid X receptor (RXR), increases gene transcriptional regulatory activity. Examples of RXR agonists include 9-cis retinoic acid (alitretinoin, CAS No.: 5300-03-8), AGN195204 (CAS No.: 220619-73-8), TTNPB (arotinoid acid, CAS No.: 71441-28-6), Adapalene (CAS No.: 106685-40-9), Bexarotene (CAS No.: 153559-49-0), Tazarotene (CAS No.: 118292-40-3), Tamibarotene (CAS No.: 94497-51-5), CH55 (CAS No. No.: 110368-33-7), and AM580 (CAS No.: 102121-60-8). An example of the RXR agonist is 9-cis retinoic acid or AGN 195204. An example of the RXR agonist is 9-cis retinoic acid.

[0100] The amount of RXR agonist (e.g., 9-cis retinoic acid) added to induction medium C can be 0.1 to 10 μM, 0.5 to 5 μM, 0.5 to 3 μM, 0.5 to 2 μM, or 1 μM. Induction medium C may contain an RXR agonist other than 9-cis retinoic acid at a concentration that exhibits an RXR agonistic effect similar to that exhibited by 0.1 to 10 μM, 0.5 to 5 μM, 0.5 to 3 μM, 0.5 to 2 μM, or 1 μM 9-cis retinoic acid. The amount of RXR agonist added can be adjusted appropriately depending on the amount of the component having RXR agonistic effect contained in the basal medium used for induction medium C.

[0101] The "RXR agonist effect" can be measured, for example, based on the expression level of a luciferase gene located downstream of a promoter region to which RXR and PPAR form a heterodimer in the presence of a compound capable of activating RXR, where the heterodimer binds. In one example, the RXR agonist effect can be measured by measuring the expression level of luciferase induced by the heterodimer of RXR and PPAR formed in the presence of 9-cis retinoic acid, measuring the expression level of luciferase induced by the heterodimer of RXR and PPAR in the presence of a compound capable of activating RXR, and comparing the results. The RXR agonist effect can be measured using a commercially available assay (INDIGO, Nuclear Receptor Luciferase Reporter Assay).

[0102] As used herein, "PPAR" is an abbreviation for Peroxisome Proliferator-Activated Receptor, and belongs to the nuclear receptor superfamily. There are three subtypes of PPAR: PPAR alpha (A), PPAR gamma (G), and PPAR delta (D).

[0103] As used herein, the term "PPAR agonist" refers to a compound or composition that activates PPAR, such as PPARA, PPARG, or PPARD, or a combination thereof. The PPAR agonist is at least one selected from the group consisting of a PPARA agonist, a PPARG agonist, a PPARD agonist, a PPARA / G agonist, a PPARA / D agonist, a PPARG / D agonist, and a PPARA / G / D agonist. The PPAR agonist is preferably a PPARA agonist, a PPARG agonist, or a PPARD agonist. The PPAR agonist is preferably a PPARA agonist or a PPARG agonist. The PPAR agonist is more preferably a PPARA agonist.

[0104] As used herein, the term "PPARA agonist" refers to, for example, a compound or composition that activates PPARA. PPARA agonists are commercially available (see, for example, https: / / www.medchemexpress.com / Targets / PPAR.html, incorporated herein by reference). Examples of PPARA agonists include CP775146 (CAS No.: 702680-17-9), pirinixic acid (WY14643, CAS No.: 50892-23-4), bezafibrate (CAS No.: 41859-67-0), fenofibrate (CAS No.: 49562-28-9), gemfibrozil (CAS No.: 25812-30-0), clofibrate (CAS No.: 637-07-0), ciprofibrate (CAS No.: 52214-84-3), pemafibrate (CAS No.: 848259-27-8), and binifibrate (CAS No.: No.: 69047-39-8), clinofibrate (CAS No.: 30299-08-2), clofibric acid (CAS No.: 882-09-7), nicofibrate (CAS No.: 31980-29-7), pirifibrate (CAS No.: 55285-45-5), plafibride (CAS No.: 63394-05-8), lonifibrate (CAS No.: 42597-57-9), theofibrate (CAS No.: 49562-28-9), tocofibrate (CAS No.: 50465-39-9), SR10171, GW6471 (CAS No. : 880635-03-0), Fenofibrate (CAS NO.: 49562-28-9), Pirinixic acid (CAS NO.: 50892-23-4), GW7647 (CAS NO.: 265129-71-3), Icariin (CAS NO.: 489-32-7), Eupatilin (CAS NO.: 22368-21-4), Gemfibrozil (CAS NO.: 25812-30-0), Palmitelaidic Acid (CAS NO.: 10030-73-6), NXT629 (CAS No. : 1454925-59-7), Saroglitazar Magnesium (CAS NO.: 1639792-20-3),Saroglitazar (CAS NO.: 495399-09-2), Oleoylethanolamide (CAS NO.: 111-58-0), BMS-687453 (CAS NO.: 1000998-59-3), Gypenoside XLIX (CAS NO.: 94987-08-3), CUDA (CAS NO.: 479413-68-8), Ciprofibrate impurity A (CAS NO.: 1474058-89-3), CP-868388 free base (CAS NO.: 702681-67-2), AVE-8134 (CAS No.: 304025-09-0), linoleic acid, linolenic acid, and PPARα-MO-1 (CAS No.: 810677-36-2). The PPARA agonist is preferably CP775146, pirinixic acid (WY14643), bezafibrate, fenofibrate, gemfibrozil, clofibrate, ciprofibrate, linoleic acid, linolenic acid, or a combination thereof. The PPARA agonist is more preferably CP775146 or pirinixic acid (WY14643). The PPARA agonist is more preferably CP775146.

[0105] As used herein, the term "PPARG agonist" refers to a compound or composition that activates PPARG. PPARG agonists are commercially available (see, for example, https: / / www.medchemexpress.com / Targets / PPAR.html, incorporated herein by reference). Examples of PPARG agonists include rosiglitazone (CAS No.: 122320-73-4), troglitazone (CAS No.: 97322-87-7), pioglitazone (CAS No.: 111025-46-8) (which may be deuterated), efatutazone (CAS No.: 223132-37-4), ATx08-001 (CAS No.: 193012-35-0), OMS-405, CHS-131 (CAS No.: 315224-26-1), THR-0921 (CAS No.: 606932-81-4), SER-150-DN, KDT-501 (CAS No. No. : 1374259-84-3), GED-0507-34-Levo, CLC-3001, ALL-4, Rosiglitazone (CAS NO.: 122320-73-4), Rosiglitazone hydrochloride (CAS NO.: 302543-62-0), Troglitazone (CAS NO.: 97322-87-7), T0070907 (CAS NO.: 313516-66-4), 5-Aminosalicylic Acid (CAS NO.: 89-57-6), GW1929 (CAS NO.: 196808-24-9), Rosiglitazone maleate (CAS NO.: 155141-29-0), Astaxanthin (CAS NO.: 472-61-7), Magnolol (CAS NO.: 528-43-8), Glabridin (CAS No.: 59870-68-7), Balaglitazone (CAS No.: 199113-98-9), Mifobate (CAS No.: 76541-72-5), Inolitazone dihydrochloride (CAS NO.: 223132-38-5), EHP-101 (CAS NO.: 1818428-24-8), Adelmidrol (CAS No. :1675-66-7),Oroxin A (CAS NO.: 57396-78-8), Cefminox sodium (CAS NO.: 75498-96-3), Methyl oleanonate (CAS NO.: 1721-58-0), 15-Deoxy-Δ-12,14-prostaglandin J2 (CAS NO.: 87893-55-8), GSK376501A (CAS NO.: 1010412-80-2), 4-O-Methyl honokiol (CAS NO.: 68592-15-4), Caulophyllogenin (CAS No.: 52936-64-8), Darglitazone (CAS No.: 141200-24-0), Angeloylgomisin H (CAS No.: 66056-22-2), DS-6930 (CAS No.: 1242328-82-0), Inolitazone (CAS No.: 223132-37-4), and Arhalofenate (CAS No.: 24136-23-0). The PPARG agonist is preferably rosiglitazone or troglitazone, or a combination thereof.

[0106] As used herein, the term "PPARD agonist" refers to a compound or composition that activates PPARD. PPARD agonists are commercially available (see, for example, https: / / www.medchemexpress.com / Targets / PPAR.html, incorporated herein by reference). PPARD agonists include, for example, Finadelpar (CAS No.: 515138-06-4), ASP0367, GW501516 (Endurabol), Pparδ agonist 5 (molecular formula: C ２３ H ２１ F ３ N ２ O ２S), MBX8025 (Seladelpar, CAS No.: 851528-79-5), GW0742 (CAS No.: 317318-84-6), L165041 (CAS No.: 79558-09-1), HPP-593 (CAS No.: 1604815-32-8), and NCP-1046. The PPARD agonist is preferably finadelpar or ASP0367, or a combination thereof.

[0107] As used herein, the term "PPARA / G agonist" refers to a compound or composition that activates PPARA / G. PPARA / G agonists are commercially available. Examples of PPARA / G agonists include at least one selected from the group consisting of saroglitazar (CAS No.: 495399-09-2), aleglitazar (CAS No.: 475479-34-6), muraglitazar (CAS No.: 331741-94-7), tesaglitazar (CAS No.: 251565-85-2), and DSP-8658.

[0108] As used herein, the term "PPARA / D agonist" refers to a compound or composition that activates PPARA / D. PPARA / D agonists are commercially available. Examples of PPARA / D agonists include ELA and / or T913659.

[0109] As used herein, the term "PPARG / D agonist" refers to a compound or composition that activates PPARG / D. PPARG / D agonists are commercially available. Examples of PPARG / D agonists include linoleic acid (CAS No.: 60-33-3) and / or T3D-959 (CAS No.: 1258076-66-2).

[0110] As used herein, the term "PPARA / G / D agonist" refers to a compound or composition that activates PPARA / G / D. PPARA / G / D agonists are commercially available. The PPARA / G / D agonist is, for example, at least one selected from the group consisting of IVA337 (lanifibranor, CAS No.: 927961-18-0), TTA (tetradecylthioacetic acid, CAS No.: 2921-20-2), bavachinin (CAS No.: 19879-30-2), GW4148, GW9135, bezafibrate (CAS No.: 41859-67-0), lobeglitazone (CAS No.: 607723-33-1), and CS038 (CAS No.: 743438-45-1).

[0111] The amount of PPAR agonist (e.g., CP775146) added to induction medium C can be 0.05 to 5 μM, 0.1 to 3 μM, 0.1 to 2 μM, 0.2 to 1.5 μM, 0.2 to 1 μM, or 0.3 to 1 μM. Induction medium C may also contain a PPAR agonist other than CP775146 at a concentration that exhibits the same level of PPAR agonistic activity as CP775146 at 0.05 to 5 μM, 0.1 to 3 μM, 0.1 to 2 μM, 0.2 to 1.5 μM, 0.2 to 1 μM, or 0.3 to 1 μM. The amount of PPAR agonist added can be adjusted appropriately depending on the amount of components having PPAR agonistic activity contained in the basal medium used for induction medium C. PPAR agonist activity can be measured in the same manner as RXR agonist activity described herein. In one example, PPAR agonist activity can be measured by measuring the expression level of luciferase induced by the heterodimer of RXR and PPAR formed in the presence of CP775146, measuring the expression level of luciferase induced by the heterodimer of RXR and PPAR in the presence of a compound that can activate PPAR, and comparing them. PPAR agonist activity can be measured using a commercially available assay (INDIGO, Nuclear Receptor Luciferase Reporter Assay).

[0112] In the present invention, the molar concentration ratio of the RXR agonist to the PPAR agonist added to the basal medium is in the range of 0.3 to 10, preferably 0.5 to 5, and more preferably 0.5 to 4, when the PPAR agonist is taken as 1. When the basal medium contains either or both of the agonists, the ratio can be adjusted as appropriate, but it is desirable that the total amount is within the above range.

[0113] Step C can be performed, for example, according to the following procedure: Renal organoids are cultured in induction medium C, STEMdiff APEL2 medium (0.3 μM CP775146, 1 μM 9-cis retinoic acid, 1% PFHM II, antibiotic-antimycotic), to promote maturation of proximal tubules.

[0114] Induction medium C may contain a PPAR agonist (e.g., pirinixic acid) at a concentration of 0.1 to 30 μM, 0.5 to 25 μM, 1 to 20 μM, 2 to 18 μM, 5 to 15 μM, 7 to 12 μM, or 10 μM. Step C can be performed, for example, according to the following procedure. Renal organoids are cultured in induction medium C, STEMdiff APEL2 medium (10 μM pirinixic acid, 1 μM 9-cis retinoic acid, 1% PFHM II, antibiotic-antimycotic), to promote maturation of proximal tubules.

[0115] [Non-human mammal having renal organoids containing mature proximal tubule cells, and a method for producing the same] One aspect of the present disclosure provides a non-human mammal having renal organoids in the kidney or its surrounding area, characterized by containing mature proximal tubule cells. One aspect of the present disclosure provides a method for producing a non-human mammal having renal organoids in the kidney or its surrounding area, characterized by containing mature proximal tubule cells.

[0116] A non-human mammal having renal organoids containing mature proximal tubule cells can be produced by a method comprising introducing renal organoids containing mature proximal tubule cells into the kidney or its surrounding area of ​​a non-human mammal. The non-human mammal can be produced by a method comprising introducing renal organoids containing mature proximal tubule cells into the kidney or its surrounding area of ​​a non-human mammal, and raising the non-human mammal. Raising the non-human mammal includes, for example, feeding a known diet for non-human mammals. From the viewpoint of reducing graft rejection, the non-human mammal and the animal species of the pluripotent stem cells, intermediate mesoderm cells, or early renal organoids used to produce the renal organoids containing mature proximal tubule cells are preferably the same species, and more preferably the same individual.

[0117] In one example, tumor cells or tumor fragments are introduced into the renal organoids, and the renal organoids are then introduced into the kidney or a peripheral region of a non-human mammal, thereby producing a non-human mammal having the renal organoids in its kidney or a peripheral region as a renal cancer model. The non-human mammal as a renal cancer model can be used in a method for evaluating the effects of candidate therapeutic substances for renal cancer. From the perspective of reducing transplant rejection, the non-human mammal as a renal cancer model and the animal species of the pluripotent stem cells, intermediate mesoderm cells, or early renal organoids used to produce the renal organoids into which tumor cells or tumor fragments have been introduced are preferably the same species, and even more preferably the same individual.

[0118] As used herein, the term "non-human mammal" may refer to, for example, rodents such as mice, rats, guinea pigs, hamsters, etc.; non-human primates such as chimpanzees; even-toed ungulates such as cows, goats, sheep, etc.; perissodactyls such as horses, etc.; or pet animals such as rabbits, dogs, cats, etc. The non-human mammal is preferably a rodent or a non-human primate.

[0119] [Composition for regenerative medicine] One aspect of the present disclosure provides a composition for regenerative medicine comprising a renal organoid characterized by containing mature proximal tubule cells according to the present disclosure.

[0120] As used herein, "regenerative medicine compositions" include renal organoids containing mature proximal tubule cells according to the present disclosure. The regenerative medicine compositions according to the present disclosure can be used to treat kidney damage or disease in mammals. The regenerative medicine compositions may, for example, appropriately contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein refers to any component other than the renal organoids that is highly safe and has low allergic reactivity in mammals. Pharmaceutically acceptable carriers include, for example, aqueous or non-aqueous solvents, solutions (e.g., saline, basal medium, or cell suspension preservative), cryoprotectants (e.g., glycerol), water-soluble polymers (e.g., dextran), or buffers (e.g., phosphate buffer) suitable for pharmaceutical administration. Regenerative medicine compositions can be appropriately prepared according to known methods. In one example, the regenerative medicine compositions according to the present disclosure can be prepared by combining the renal organoids with a pharmaceutically acceptable carrier (e.g., basal medium).

[0121] The regenerative medicine composition is administered to a mammal in need thereof, for example, by surgically transplanting it into a predetermined site of the kidney, or by injecting it into a predetermined site of the kidney using an instrument such as a syringe. From the viewpoint of reducing graft rejection, the mammal to which the regenerative medicine composition is administered and the animal species of the pluripotent stem cells, intermediate mesoderm cells, or early renal organoids used to produce renal organoids containing mature proximal tubule cells are preferably the same species, and more preferably the same individual.

[0122] The term "mammal" in relation to the regenerative medicine composition includes, for example, humans and non-human mammals. Non-human mammals may be, for example, rodents such as mice, rats, guinea pigs, and hamsters, non-human primates such as chimpanzees, even-toed ungulates such as cows, goats, and sheep, perissodactyls such as horses, and pets such as rabbits, dogs, and cats. In one embodiment, the mammal is a human.

[0123] As used herein, "kidney damage" or "kidney disease" may be, for example, a kidney damaged by trauma, a kidney disease (e.g., atrophic kidney, renovascular hypertension, amyloid kidney, hyperuricemic nephropathy, and renal tubular acidosis), a chronic kidney disease (e.g., chronic glomerulonephritis, chronic tubulointerstitial nephritis, chronic pyelonephritis, and chronic renal failure), or a chronic syndrome (e.g., Gitelman syndrome, Bartter syndrome, Fanconi syndrome, Lowe syndrome, nephrotic syndrome), or kidney cancer.

[0124] The regenerative medicine composition according to the present disclosure can be used in a method for treating kidney damage or disease in a mammal. One embodiment provides a method for treating kidney damage or disease, comprising administering to a mammal in need thereof a regenerative medicine composition comprising a kidney organoid containing mature proximal tubule cells according to the present disclosure.

[0125] Methods of Treating Kidney Damage or Disease One aspect of the present disclosure provides methods of treating kidney damage or disease in a mammal.

[0126] A method for treating kidney damage or disease in a mammal comprises introducing renal organoids containing mature proximal tubule cells or a regenerative medicine composition into the kidney or its surrounding area of ​​a mammal in need thereof. The renal organoids introduced into a mammal may be, for example, in the form of organoids, or may be either single cells or cell masses composed of multiple cells, or both. In one example, a method for treating kidney damage or disease in a mammal comprises injecting a group of cells derived from renal organoids containing mature proximal tubule cells into the kidney or its surrounding area of ​​a mammal in need thereof.

[0127] As used herein, the term "mammal in need thereof" refers to a mammal having or suspected of having kidney damage or disease. A mammal having kidney damage or disease refers to a mammal diagnosed by a medical professional (e.g., a physician) as having kidney damage or disease according to predetermined diagnostic criteria. A mammal suspected of having kidney damage or disease may be a mammal suspected of having kidney damage or disease based on, for example, the mammal's behavioral history (e.g., trauma and / or exposure to drugs harmful to kidney tissue) or medical history (e.g., suffering from or having suffered from a kidney disease, chronic kidney disease, kidney-related syndrome, or kidney cancer according to the present disclosure). The mammal according to this embodiment is, for example, a human or a non-human mammal. The non-human mammal may be, for example, a non-human primate such as a chimpanzee; an artiodactyla such as a cow, goat, or sheep; an odd-toed ungulate such as a horse; or a pet animal such as a rabbit, dog, or cat. The mammal according to this embodiment is preferably a human or a non-human primate, more preferably a human.

[0128] "Treating" kidney damage or disease includes maintaining, reducing, or eliminating the symptoms or pathology. Treating kidney damage or disease includes curing kidney damage or disease.

[0129] From the viewpoint of reducing graft rejection, it is preferable that the animal species of the pluripotent stem cells, intermediate mesodermal cells, or early renal organoids used to produce renal organoids or regenerative medicine compositions containing mature proximal tubule cells to be introduced into a mammal in need thereof is the same species or the same individual as the mammal in need thereof.

[0130] [Evaluation Method] One aspect of the present disclosure provides a method for evaluating drug responsiveness to a test substance, comprising contacting a renal organoid containing mature proximal tubule cells according to the present disclosure or a non-human mammal having the renal organoid with a test substance, and measuring the drug responsiveness of the renal organoid or the non-human mammal to the test substance.

[0131] As used herein, a "test substance" may be, for example, a small molecule compound, a protein (e.g., an antibody), DNA, RNA, a small interfering RNA, or an antisense oligonucleotide. The test substance may be, for example, a drug for treating a disease in a mammal (e.g., a disease in an organ such as the kidney, bladder, stomach, or intestine) or cancer (e.g., kidney cancer, bladder cancer, stomach cancer, or intestinal cancer), or a candidate substance thereof. The test substance may be, for example, a substance known to cause or potentially cause kidney damage. The test substance may be, for example, one type, or a mixture of two or more types. The test substance is preferably one type of substance.

[0132] "Contacting" a test substance with a renal organoid containing mature proximal tubule cells or a non-human mammal having the renal organoid means placing the renal organoid or the non-human mammal under conditions that allow contact between the test substance and the renal organoid.Contacting the renal organoid with the test substance may, for example, be by mixing the test substance with a culture medium containing the renal organoid.Contacting the non-human mammal with the test substance may, for example, be by orally or parenterally administering the test substance to the non-human mammal.

[0133] The drug responsiveness includes, for example, changes in the structural or functional properties of renal organoids containing mature proximal tubule cells due to the test substance. In this example, measuring the drug responsiveness includes measuring the structural or functional properties of the renal organoids. The structural property may be, for example, the proportion of mature proximal tubule cells in the renal organoids. The functional property may be, for example, the amount of dextran uptake in the renal organoids. Dextran uptake by renal organoids can be measured, for example, according to the method described in Cell Stem Cell 25, 373-387, September 5, 2019 (incorporated herein by reference) or Journal of Biological Methods, 2021, Vol. 8(2) e150.

[0134] The change in structural characteristics may involve, for example, comparing the proportion of mature proximal tubule cells in renal organoids containing mature proximal tubule cells that have not been contacted with a test substance (hereinafter referred to as the "first proportion") with the proportion of mature proximal tubule cells in renal organoids containing mature proximal tubule cells that have been contacted with a test substance (hereinafter referred to as the "second proportion"). The change in structural characteristics may be, for example, the ratio of the second proportion to the first proportion (= [second proportion] / [first proportion]). A ratio of 0.7 suggests that contact with the test substance has resulted in a 30% decrease in mature proximal tubule cells. This decrease in mature proximal tubule cells is thought to be due, for example, to cell death, such as apoptosis, induced by contact between the test substance and the cells. This decrease in mature proximal tubule cells suggests that the test substance has nephrotoxicity. Therefore, the evaluation method according to the present disclosure includes evaluating the nephrotoxicity of the test substance based on the results of measuring drug responsiveness. In another example, the evaluation method according to the present disclosure includes evaluating the nephrotoxicity of a test substance on a renal organoid containing mature proximal tubule cells or a non-human mammal having the renal organoid, based on the results of measuring drug responsiveness. Here, examples of nephrotoxic test substances include, but are not limited to, antibiotics and antibacterial drugs (aminoglycosides, vancomycin, penicillin, etc.), nonsteroidal anti-inflammatory drugs (ibuprofen, indomethacin, fenoprofen, etc.), anticancer drugs (cisplatin, etc.), contrast agents, and immunosuppressants (cyclosporine, etc.).

[0135] The proportion of mature proximal tubule cells in renal organoids according to the present disclosure can be calculated, for example, by separating the cells constituting the renal organoids into single cells and dividing the number of mature proximal tubule cells in the separated cell population by the total number of cells in the separated cell population. Methods for separating single cells from renal organoids containing mature proximal tubule cells can be performed using known methods for separating individual cells from cell masses (e.g., spheroids or organoids). Individual cells from the renal organoids can be obtained, for example, by performing the "preparation of single cells derived from renal organoids" according to the present disclosure. The number of mature proximal tubule cells, the total number of cells, or the proportion in the separated cell population can be measured, for example, by FACS. Measurement of mature proximal tubule cells by FACS can be performed, for example, using a lectin (e.g., LTL) or antibody that specifically binds to proximal tubules according to the present disclosure. The antibody may be labeled with a known fluorescent substance.

[0136] The change in functional characteristics includes, for example, comparing the amount of dextran uptake in renal organoids containing mature proximal tubule cells that have not been contacted with a test substance (hereinafter referred to as the "first uptake amount") with the amount of dextran uptake in renal organoids containing mature proximal tubule cells that have been contacted with a test substance (hereinafter referred to as the "second uptake amount"). The change in functional characteristics may be, for example, the ratio of the second uptake amount to the first uptake amount (= [second uptake amount] / [first uptake amount]). The first uptake amount and the second uptake amount are preferably measured using the same area of ​​the proximal tubule. If the ratio is less than 1, this suggests that the functional characteristics of renal organoids containing mature proximal tubule cells have been reduced by contact with the test substance. Therefore, the evaluation method according to the present disclosure includes evaluating the reduction in renal function caused by the test substance based on the results of measuring drug responsiveness. In another example, the evaluation method of the present disclosure includes evaluating the decline in renal function of a renal organoid containing mature proximal tubule cells or a non-human mammal having said renal organoid caused by a test substance based on the measurement results of drug responsiveness.

[0137] The amount of dextran uptake in renal organoids containing mature proximal tubule cells can be measured by, for example, culturing the renal organoids in a medium containing fluorescently labeled dextran for a predetermined time (for example, 24 hours), and then detecting the fluorescence emitted from the renal organoids.The fluorescence can be measured, for example, using a fluorescence microscope.

[0138] In one example, measuring the drug responsiveness comprises: measuring the structural or functional characteristics of the renal organoid that contains mature proximal tubule cells that are not contacted with the test substance to obtain a first measurement result; measuring the structural or functional characteristics of the renal organoid that contains mature proximal tubule cells that are contacted with the test substance to obtain a second measurement result; and comparing the first measurement result with the second measurement result.In one example, the evaluation method of the present disclosure comprises evaluating the drug responsiveness (for example, the presence or absence or level of renal function) of the renal organoid that contains mature proximal tubule cells or the non-human mammal that has the renal organoid by the test substance based on the measurement result of the drug responsiveness.

[0139] The drug responsiveness may be, for example, an average value of results measured from a renal organoid containing at least three (e.g., three, four, five, six, or seven or eight or more) mature proximal tubule cells or a non-human mammal having the renal organoid.

[0140] [Method for promoting maturation of proximal tubules] One aspect of the present disclosure provides a method for promoting maturation of proximal tubules.The method comprises culturing early stage renal organoids with a medium containing RXR agonist and PPAR agonist.The method for promoting maturation of proximal tubules is carried out in vitro.

[0141] As used herein, the term "proximal tubule" refers to the region in kidney organoids where LTL-binding cells are clustered. Proximal tubules have, for example, a brush border. In one example, if the expression level of a proximal tubule marker in kidney organoids (organoids of interest) obtained by culturing early-stage kidney organoids using a medium containing an RXR agonist and a PPAR agonist is significantly greater than the expression level of the proximal tubule marker in kidney organoids (control organoids) obtained by culturing early-stage kidney organoids using a medium substantially free of either or both of an RXR agonist and a PPAR agonist, it can be evaluated that maturation of the proximal tubules has been promoted. In another example, if the number of endosomes of a predetermined size or larger in the organoids of interest is significantly greater than the number of endosomes of a predetermined size or larger in the target organoids, it can be evaluated that maturation of the proximal tubules has been promoted. The predetermined size in the context of endosomes is, for example, 0.5 μm ２ In another example, when the amount of dextran taken up in the target organoid is significantly greater than the amount of dextran taken up in the target organoid, it can be evaluated that the maturation of proximal tubules is promoted.

[0142] As used herein, the term "comprising" means that the recited elements and / or steps are present, and that other elements and / or steps may be added. As used herein, the term "consisting of" means that the recited elements and / or steps are present, and that other elements and / or steps are excluded. As used herein, the term "essentially consisting of" means that the recited elements and / or steps are present, and that other elements and / or steps may be added to the extent that they do not affect the novel technical features of the cell layer, organoid, composition, and method. As used herein, the term "substantially free" does not exclude "completely free."

[0143] The descriptions of terms, aspects, and embodiments provided by the present disclosure apply appropriately between corresponding terms, aspects, and embodiments provided by the present disclosure, unless otherwise specified.

[0144] Specific examples will be described below, but they are intended to illustrate preferred embodiments of the present invention and are not intended to limit the invention described in the appended claims in any way.

[0145] Materials and Methods The following growth factors and compounds were used in Examples 2 and 3 below: CHIR99021 (TOCRIS, #4423), Recombinant Human FGF9 (R&D SYSTEMS, #273-F9-025), Heparin (Sigma, #H4784-250MG), CP775146 (TOCRIS, #4190), and 9-cis retinoic acid (Abcam, #ab141023).

[0146] (Preparation of single cells derived from kidney organoids) The kidney organoids were transferred to a centrifuge tube, and a cell detachment solution (DPBS(+), 5 mg / mL Bacillus Licheniformis Protease (Sigma), 125 U / mL DNase (Roche)) was added to the tube. Up to 12 organoids were placed per centrifuge tube. The centrifuge tube was incubated at 6°C for 30 minutes. During the 30-minute incubation, the tube was pipetted every 5 minutes to prepare a cell suspension containing cells detached from the organoids. 4 mL of ice-cold diluent A (DPBS(-), 0.5% FBS, 2 mM EDTA, 125 U / mL DNase) was added to 1 mL of the resulting cell suspension. The centrifuge tube containing the diluted cell suspension was centrifuged at 300 rcf at 4°C for 10 minutes, and the supernatant was removed. The precipitate was resuspended in ice-cold dilution solution A. The cell suspension was centrifuged again, and the precipitate was resuspended in dilution solution A. The resulting cell suspension was transferred to a tube equipped with a 35 μm filter (Falcon) and passed through the filter to prepare a cell suspension containing single cells.

[0147] (Acquisition of LTL-positive proximal tubule cells using the MACS method) LTL-biotin (1:50) was added to the prepared cell suspension containing single cells, and the mixture was incubated at 4°C for 15 minutes. The cell suspension was then centrifuged at 300 rcf at 4°C for 5 minutes, and the supernatant was removed. Ice-cold Dilution Solution A was added to the precipitate and the mixture was resuspended. The procedure of centrifuging the cell suspension and obtaining a cell suspension from the precipitate was repeated two more times. Anti-biotin microBeads Ultra Pure were added to the obtained cell suspension in Dilution Solution A, and the mixture was incubated at 4°C for 15 minutes. The cell suspension was then centrifuged at 300 rcf at 4°C for 5 minutes, and the supernatant was removed. Ice-cold Dilution Solution A was added to the precipitate and the mixture was resuspended. This procedure was repeated two more times. Cells to which LTL binds (also referred to as LTL-binding cells or LTL-positive cells) were obtained by the MACS method from the obtained cell suspension in diluted solution A. More specifically, the cell suspension was applied to an MS column (Miltenyi) immobilized in a magnetic field, and LTL-positive proximal tubule cells were obtained as a positive fraction.

[0148] (Analysis of gene expression levels in LTL-positive cells) Total RNA was extracted from the cell suspension containing the obtained LTL-positive proximal tubule cells using the Purelink RNA micro kit (Thermofisher Scientific). Using the obtained total RNA as a template, cDNA synthesis was performed using PrimeScript™ RTmastermix (Perfect Real Time) (TaKaRa). The synthesized cDNA was quantified by real-time PCR using TB Green™ Premix Ex Taq™ II (Tli RNase H Plus) (TaKaRa). Expression level analysis was performed using this quantitative PCR.

[0149] Example 1: Single-cell RNA sequencing of renal organoid-derived cell populations Example 1 was conducted with the aim of identifying factors involved in the maturation of renal organoids. The inventors performed single-cell RNA sequencing on organoids produced by carrying out the steps A and B described below, and performed cell clustering after each differentiation induction day. The results showed that over 31 days of differentiation induction, the organoids differentiated into diverse cell populations, including interstitial cell populations, muscle cell populations, neural cell populations, glomerular cell populations, nephron progenitor cell populations, and tubular cell populations, and that the number of days required to reach each cell population varied. Furthermore, the results of single-cell RNA sequencing were analyzed using a pseudotime axis. As a result, it was found that in organoids produced over a short period of time, cells of a specific cell type exhibiting a specific pseudotime exist, i.e., cells that mature quickly, and that in organoids produced over a long period of time, cells of the same cell type exhibiting the same pseudotime exist, i.e., cells that mature slowly. These results suggest that by comparing cell populations derived from renal organoids with the same pseudotime but different manufacturing dates, it may be possible to identify genes related to the maturation of a particular cell type. Therefore, we performed gene expression analysis of proximal tubule cells derived from renal organoids with the same pseudotime but different manufacturing dates. In this gene analysis, we selected genes with increased expression levels and low p-values ​​among upstream regulatory factors.

[0150] The results showed that PPARA-related genes, PPARG-related genes, and RXR-related genes were activated. Specifically, among PPARA-related genes, PPARA (2.6-fold, p=2.0E-12), pirinixic acid (3.1-fold, p=4.3E-11), bezafibrate (2.6-fold, p=4.8E-9), fenofibrate (2.4-fold, p=1.4E-7), gemfibrozil (2.8-fold, p=7.2E-7), and clofibrate (2.8-fold, p=9.0E-6) were activated. Furthermore, PPARG-related genes resiglitazone (3.0-fold, p=1.2E-5) and PPARG (2.6-fold, p=1.3E-5) were activated, and RXR-related genes alitretinoin (2.4-fold, p=5.9E-3) and AGN194204 (2.0-fold, p=7.4E-3) were activated.

[0151] The results of Example 1 suggest that mature cells can be induced in renal organoid culture by using a PPARA agonist, a PPARG agonist, or an RXR agonist, or a combination thereof.

[0152] Example 2: Preparation of renal organoids containing mature proximal tubule cells In Example 2, the maturation-promoting effect of a PPARA agonist, a PPARG agonist, an RXR agonist, or a combination thereof, as suggested in Example 1, was examined. FIG. 1 is a flowchart showing an overview of the preparation of renal organoids containing mature proximal tubule cells according to one embodiment. FIG. 1 shows step A of inducing differentiation from human iPS cells into intermediate mesoderm, step B of inducing differentiation from intermediate mesoderm into early renal organoids, and step C of inducing differentiation from early renal organoids into renal organoids containing mature proximal tubule cells. FIG. 1 also shows the step of evaluating nephrotoxicity, which will be described later. In Example 2, steps A to C shown in FIG. 1 are carried out to produce renal organoids containing mature proximal tubule cells. In step C of Example 2, the kidney organoids are cultured using induction medium C containing a combination of CP775146, a PPARA agonist, and 9cis retinoic acid, an RXR agonist.

[0153] Pre-culture of cells for differentiation induction: Human iPS cells (1502.3 strain, donated by Dr. Melissa Little, Murdoch Children's Research Institute) were maintained and cultured in StemFit AK02N medium (REPROCELL) on the surface of a culture plate coated with iMatrix-511 (Nippi). The cells were detached by incubation at 37°C for 5 minutes using TrypLE Select (Thermo Fisher Scientific) to obtain a cell suspension. The cell suspension was centrifuged at 200 rcf for 5 minutes at room temperature, the supernatant was removed, and the precipitate was resuspended in StemFit AK02N (10 μM Y-27632). The cell suspension was treated with iMatrix-511 (Nippi) at 0.25 μg / cm ２ The amount of added water was 15,000 cells / cm ２ The cells were seeded in a 12-well plate (Corning) so that the total number of cells was 100, and the cells were incubated at 37°C and CO ２ The mixture was cultured under 5% CO₂ for 1 day.

[0154] Step A: Induction of intermediate mesoderm differentiation Next, the culture medium used in the above culture was replaced with induction medium A, STEMdiff APEL2 medium (STEMCELL Technologies) (8 μM CHIR99021, 1% PFHM II, antibiotic-antimycotic), and the cells were cultured for 5 days to induce primitive streaks from the iPS cells. The medium was replaced daily from day 4 onwards. On day 1 of differentiation induction, the iPS cells aggregated to form colonies, and on day 2 of differentiation induction, the cell colonies began to spread outwards. On day 3 of differentiation induction, proliferated cells began to layer near the center of the cell colony, and on day 4 of differentiation induction and later, the colonies became rounded. On the fifth day of differentiation induction, induction medium A was changed to induction medium B, STEMdiff APEL2 medium (200 ng / mL FGF9, 1 μg / mL Heparin, 1% PFHM II, antibiotic-antimycotic).

[0155] The cell colonies were further cultured for 3 days in induction medium B to induce differentiation from the primitive streak to intermediate mesoderm. During this culture, the shape of the colonies gradually changed from a rounded state to a sheet-like state.

[0156] Step B: Spheroid formation and early renal organoid formation (Step b1) After intermediate mesoderm induction (day 8 of differentiation induction), the cells were detached using TrypLE Select (Thermo Fisher Scientific) by incubation at 37 ° C for 7 minutes to obtain a cell suspension. The cell suspension was centrifuged at 400 rcf and room temperature for 3 minutes, the supernatant was removed, and the precipitate was resuspended in induction medium B, STEMdiff APEL2 medium (200 ng / mL FGF9, 1 μg / mL Heparin, 1% PFHM II, Antibiotic-Antimycotic, 10 μM Y-27632). The cell suspension was seeded onto a PrimeSurface (registered trademark) 96M plate (Sumitomo Bakelite) at 100,000 cells / well. The next day (day 9 after differentiation induction), spheroids were formed in the wells.

[0157] (Step b2) The formed spheroids were removed and placed on a Cell Culture Insert Transparent PET membrane 6-well 0.4 μm pore size (Corning) Cell Culture Insert so that there were six spheroids per well. The Cell Culture Insert containing the spheroids was placed in the corresponding well of a culture plate. Induction medium A, STEMdiff APEL2 medium (10 μM CHIR99021, 1% PFHM II, Antibiotic-Antimycotic), was added between the well of the culture plate and the underside of the transwell of the Cell Culture Insert, and the spheroids were incubated at the air-liquid interface at 37°C in CO. ２ The mixture was incubated at 5% for 3 hours.

[0158] (Step b3) After the 3 hours of culture, induction medium A was replaced with induction medium B, STEMdiff APEL2 medium (200 ng / mL FGF9, 1 μg / mL Heparin, 1% PFHM II, Antibiotic-Antimycotic), and the spheroids were cultured for an additional 6 days (until day 15 of differentiation induction) to induce early renal organoids. On days 11 and 12 of differentiation induction, the formation of nephrons including proximal tubules could not be confirmed in the spheroids. On day 15 of differentiation induction, a nephron-like structure including early proximal tubules was confirmed.

[0159] Step C: Proximal tubule maturation On day 15 of differentiation induction, the medium was replaced with induction medium C, STEMdiff APEL2 medium (0.3 μM CP775146, 1 μM 9-cis retinoic acid, 1% PFHM II, Antibiotic-Antimycotic), and the resulting primary renal organoids were cultured for a further 8 days (until day 23 of differentiation induction).

[0160] In step C, early renal organoids in which nephron-like structures including early proximal tubules have been confirmed are used. It is believed that when spheroids in which the formation of nephrons including proximal tubules cannot be confirmed are cultured in a medium containing an RXR agonist (e.g., 9-cis retinoic acid), they proceed in a different direction of differentiation, making it impossible to efficiently produce renal organoids containing mature proximal tubules. For example, in step B, when intermediate mesoderm cells are cultured in an induction medium containing an RXR agonist (e.g., 9-cis retinoic acid), organoids are formed in which Wolffian ducts (derived from the anterior IM) are predominantly induced. As a result, organoids are formed in which nephron progenitor cells (derived from the posterior IM) are reduced, making it impossible to efficiently produce renal organoids containing mature proximal tubule cells.

[0161] On day 23 of differentiation induction, the maturation of proximal tubules was examined based on the expression level of maturation markers of proximal tubule cells in kidney organoids determined by the MACS method using LTLs. In the group cultured in induction medium C between 15 and 23 days after differentiation induction (CP0.3_9cisRA1 group or Mature group), CP775146 and 9-cis retinoic acid were removed from induction medium C. In comparison with the group cultured in STEMdiff APEL2 medium (0.2% DMSO, 1% PFHM II, Antibiotic-Antimycotic) (Control group), the expression levels of proximal tubule marker genes (Figure 2a: UGT2A3, Figure 2b: AZGP1, Figure 2c: SLC39A4, Figure 2d: BHMT, Figure 2e: ACE2, Figure 2f: AQP6, Figure 2g: CUBN, Figure 2h: LRP2, and Figure 2i: ABCB1) were significantly increased. These results indicate that culturing renal organoids in the presence of a combination of a PPARA agonist (e.g., CP775146) and an RXR agonist (e.g., 9-cis retinoic acid) can induce mature proximal tubule cells in a shorter period than conventional methods.

[0162] The expression levels of PPARA downstream genes (Figure 3a: APOA1, Figure 3b: APOC3, Figure 3c: CPT1A, Figure 3d: FABP1, Figure 3e: CYP27A1, and Figure 3f: ACOX1) were also significantly elevated in the CP0.3_9cisRA1 group compared to the control group. These results indicate that culturing renal organoids in the presence of a combination of a PPARA agonist (e.g., CP775146) and an RXR agonist (e.g., 9-cis retinoic acid) can induce mature proximal tubule cells.

[0163] [Example 3] Modification of Step C In Example 3, renal organoids are cultured using media containing the PPARA agonist CP775146 alone, the RXR agonist 9cis retinoic acid alone, or a combination thereof in Step C. The PPARA concentration (1 μM) in the agonist combination used in Example 3 is different from the PPARA concentration (0.3 μM) in the agonist combination used in Example 2.

[0164] In step C, kidney organoids on the 15th day of differentiation induction were cultured for 8 days in STEMdiff APEL2 medium (1 μM CP775146, 1% PFHM II, Antibiotic-Antimycotic), which is a medium obtained by removing retinoic acid from induction medium C. CP1 group was prepared. Renal organoids on the 15th day of differentiation induction were cultured for 8 days in STEMdiff APEL2 medium (1 μM 9-cis retinoic acid, 1% PFHM II, Antibiotic-Antimycotic), which is a medium obtained by removing CP775146 from induction medium C. 9cisRA1 group was cultured, and CP1_9cisRA1 group (Example 3) was cultured in induction medium C with a concentration of CP775146 of 1 μM.

[0165] As in Example 2, the expression levels of the proximal tubule marker genes in each group (Figure 4a: UGT2A3, Figure 4b: AZGP1, Figure 4c: SLC39A4, Figure 4d: BHMT, Figure 4e: ACE2, Figure 4f: AQP6, Figure 4g: CUBN, Figure 4h: LRP2, and Figure 4i: ABCB1) were measured. Compared to the control group, in the CP1_9cisRA1 group cultured using induction medium C, a tendency for the expression levels to increase in a number of proximal tubule marker genes (Figure 4a: UGT2A3, Figure 4c: SLC39A4, Figure 4e: ACE2, Figure 4g: CUBN, Figure 4h: LRP2, and Figure 4i: ABCB1) was observed. No significant differences in expression levels were observed for almost all proximal tubule marker genes between the control group, the CP1 group, and the 9cisRA1 group. These results indicate that proximal tubule cells can be induced by culturing renal organoids in the presence of a combination of a PPARA agonist (e.g., CP775146) and an RXR agonist (e.g., 9-cis retinoic acid). These results also indicate that culturing renal organoids in the presence of a PPARA agonist alone or an RXR agonist alone does not efficiently induce proximal tubule cells.

[0166] As in Example 2, the expression levels of PPARA downstream genes in each group (Figure 5a: APOA1, Figure 5b: APOC3, Figure 5c: CPT1A, Figure 5d: FABP1, and Figure 5e: ACOX1) were measured. Compared to the control group, a tendency for increased expression was observed in many PPARA downstream genes (Figure 5a: APOA1, Figure 5c: CPT1A, Figure 5d: FABP1, and Figure 5e: ACOX1). Between the control group, the CP1 group, and the 9cisRA1 group, no significant differences were observed in the expression levels of almost all PPARA downstream genes. These results indicate that mature proximal tubule cells can be induced by culturing renal organoids in the presence of a combination of a PPARA agonist (e.g., CP775146) and an RXR agonist (e.g., 9-cis retinoic acid). These results also indicate that culturing kidney organoids in the presence of a PPARA agonist alone or an RXR agonist alone does not efficiently induce mature proximal tubule cells.

[0167] Example 4: Evaluation of nephrotoxicity against cisplatin In Example 4, steps A to C shown in Figure 1 were performed, and nephrotoxicity evaluation was also performed. Specifically, as in Example 1, human iPS cells were induced to differentiate into intermediate mesoderm (step A), the intermediate mesoderm was induced to differentiate into early renal organoids (step B), and the early renal organoids were further induced to differentiate into renal organoids containing mature proximal tubule cells (step C). Renal organoids containing mature proximal tubule cells on day 23 of differentiation induction were subjected to the cisplatin nephrotoxicity test described below.

[0168] Cisplatin nephrotoxicity test in renal organoids (cisplatin treatment) Renal organoids from the Mature group (Example 2) on day 23 of differentiation induction were cultured for 6 days in cisplatin-free induction medium C (CDDP 0 μM) or cisplatin-containing STEMdiff APEL2 medium (cisplatin intravenous infusion 10 mg "Marco" (Nichi-Iko Pharma) 5 μM, 0.3 μM CP775146, 1 μM 9-cis retinoic acid, 1% PFHM II, Antibiotic-Antimycotic). Medium changes were performed once every two days.

[0169] Control kidney organoids on day 23 of differentiation induction were cultured for 6 days in STEMdiff APEL2 medium (CDDP 0 μM, 1 μg / mL Heparin, 1% PFHM II, Antibiotic-Antimycotic) without cisplatin or STEMdiff APEL2 medium (CDDP 5 μM, 1 μg / mL Heparin, 1% PFHM II, Antibiotic-Antimycotic). Medium changes were performed every 2 days.

[0170] (Evaluation of the proportion of proximal tubule cells by flow cytometry) According to the above-mentioned "Preparation of single cells derived from kidney organoids," a cell suspension containing single cells was prepared from kidney organoids after cisplatin treatment. The cells in the suspension were suspended in 4% PFA / PBS(-), and the resulting cell suspension was incubated at 37°C for 10 minutes. The cell suspension was then centrifuged at 300 rcf and 4°C for 5 minutes, and the supernatant was removed. The precipitate was resuspended in 100 μL of DPBS(-), and 900 μL of cold methanol was added and incubated on ice for 30 minutes. The cell number was adjusted to 10^6 cells / 100 μL using FACS buffer (0.5% FBS, 2 mM EDTA DPBS(-)). LTL-FITC (Vector Laboratory) was added to the resulting cell suspension at a concentration of 1:100, and the mixture was rotated and stirred at 4°C for 30 minutes. The cell suspension was then centrifuged at 300 rcf for 5 minutes at 4°C, and the supernatant was removed. The precipitate was resuspended in FACS buffer (0.5% FBS, 2 mM EDTA DPBS(-)), and the cell suspension was passed through a cell strainer. The resulting cell suspension was analyzed using a flow cytometer (Agilent, NovoCyte).

[0171] (Results) In the control group of renal organoids cultured in cisplatin-free STEMdiff APEL2 medium (CDDP 0 μM), the cell population strongly stained with LTL accounted for 26.5% of the total cells (FIG. 6a). In the mature group of renal organoids cultured in cisplatin-free induction medium C (CDDP 0 μM), the cell population highly expressing LTL accounted for 28.1% of the total cells (FIG. 6c). These results indicate that the proportion of the cell population strongly stained with LTL induced in the control group of renal organoids was almost equal to the proportion of the cell population strongly stained with LTL induced in the mature group of renal organoids. As shown in Examples 2 and 3, mature proximal tubule cells were induced in renal organoids cultured using induction medium C. Considering the results of Examples 2 and 3 together, it is speculated that the cell population strongly stained with LTL shown in Figure 6c contains a higher proportion of mature proximal tubule cells than the cell population strongly stained with LTL shown in Figure 6a.

[0172] In the control group of kidney organoids cultured in STEMdiff APEL2 medium (CDDP 5 μM) containing cisplatin, the percentage of cells strongly stained with LTL was 22.5% (FIG. 6b). Considering this together with the percentage of the cell population strongly stained with LTL (26.5%) shown in FIG. 6a, Example 4 shows that in the cell population strongly stained with LTL induced in the control group of kidney organoids, approximately 15% (= (1-22.5 / 26.5) × 100) of cells were sensitive to cisplatin.

[0173] In the Mature group of kidney organoids cultured in induction medium C (CDDP 5 μM) containing cisplatin, the percentage of cells strongly stained with LTL was 13.6% (Fig. 6d). Considering this together with the percentage of the cell population strongly stained with LTL (28.1%) shown in Fig. 6c, Example 4 shows that in the cell population strongly stained with LTL induced in the Mature group of kidney organoids, approximately 52% (= (1 - 13.6 / 28.1) × 100) of cells were sensitive to cisplatin.

[0174] Example 4 showed that the percentage of cells sensitive to cisplatin was approximately 15% in the renal organoids of the control group, while it was approximately 52% in the renal organoids of the mature group. This result indicates that the renal organoids of the mature group in Examples 2 and 3 are more sensitive to low concentrations of cisplatin (e.g., 5 μM) than the renal organoids of the control group. Cisplatin is known to cause kidney damage. It is known that kidney damage caused by cisplatin is mainly due to damage to the proximal tubules. Considering these facts, it is suggested that the renal organoids containing mature proximal tubule cells provided by the present disclosure can detect kidney damage caused by cisplatin with high sensitivity. It is also suggested that the renal organoids containing mature proximal tubule cells provided by the present disclosure can provide a method for evaluating drug-induced kidney damage that may occur in the proximal tubules.

[0175] (Additional Test) An additional cisplatin nephrotoxicity test was conducted on the above kidney organoids (n = 5). In the control group kidney organoids cultured in the presence of cisplatin (CDDP 5 μM), the percentage of cells strongly stained with LTL was 18.1%, while in the mature group kidney organoids cultured in the presence of cisplatin, it was 15.4%. A similar test was performed five times. As a result, the sensitivity of proximal tubule cells in kidney organoids to low concentrations of cisplatin was shown to be significantly higher in the mature group kidney organoids than in the control group kidney organoids (e.g., 5 μM) (Figure 7).

[0176] Example 5: Promotion of maturation in proximal tubule cells Renal organoids were prepared according to the method for preparing kidney organoids described in Example 2. Renal organoids on day 23 of differentiation induction were immunostained and photographed (Figure 8a). Immunostaining and clearing (IF staining and clearing) Renal organoids were treated with 4% paraformaldehyde at 4°C for 30 minutes, then 0.5x CUBIC-L (1M NaCl:CUBIC-L = 1:1) (Tokyo Chemical Industry Co., Ltd., T3740) was added and incubated overnight at room temperature. Subsequently, the kidney organoids were added with 0.5% PBTX (0.5% TritonX100 in DPBS(-)) and incubated for 15 minutes, followed by membrane permeabilization. The kidney organoids were washed three times with DPBS(-). Renal organoids were treated with blocking buffer (10% donkey serum + 0.3% Triton X in DPBS(-)) at room temperature for 3 hours, then primary antibody was added and incubated overnight at 4 ° C. After that, they were washed five times with 0.3% PBTX, and secondary antibody was added and incubated overnight at 4 ° C. After washing three times with DPBS(-), they were incubated overnight at 4 ° C with 4% PFA, and then washed three times with DPBS(-). After further treatment at room temperature for 15 minutes with 0.5x CUBIC-R (Tokyo Chemical Industry Co., Ltd., T3983) (5 mg / mL DAPI 1:1000), the solution was replaced with CUBIC-R, and images were taken with a confocal laser microscope (LSM800 Carl Zeiss).

[0177] No differences were observed between control kidney organoids (Fig. 8a, upper panel) and mature kidney organoids (Fig. 8a, lower panel) in the staining pattern of LTL binding to proximal tubule markers, the expression pattern of the distal tubule marker ECAD, and the expression pattern of the glomerular marker NPHS1. Cells constituting kidney organoids on day 29 of differentiation induction were subjected to a flow cytometer, and the proportion of proximal tubule cells (PT cells) among all cells was measured (Fig. 8b). The proportion of proximal tubule cells in control kidney organoids was 26.5%, and the proportion of proximal tubule cells in mature kidney organoids was 28.1%. Similar analyses were performed five times. As a result, there was no significant difference between the proportion of proximal tubule cells in control kidney organoids and the proportion of proximal tubule cells in mature kidney organoids (Fig. 8c, unpaired t-test).

[0178] Example 5 shows that the increased expression of maturation marker genes in the renal organoids of the Mature group shown in Examples 2 and 3 is not the result of promoting differentiation of nephron progenitor cells into proximal tubule cells, but rather the result of promoting maturation of proximal tubule cells. The Examples herein show that the combination of a PPARA agonist and an RXR agonist promotes the maturation of proximal tubule cells in the production of renal organoids.

[0179] [Example 6] Expression levels of LRP2 and CUBN involved in protein reabsorption The function of proximal tubules includes protein reabsorption.For example, albumin (protein) in primary urine is taken up into the endosomes of proximal tubules.The albumin taken up into endosomes is released into the blood vessels and recycled, or is degraded into amino acids in lysosomes, and these amino acids are reused.LRP2 and CUBN are involved in protein reabsorption.

[0180] Control and Mature renal organoids were prepared as described in Example 2. The expression levels of LRP2 and CUBN were measured in organoids on days 20, 23, 26, and 29 of culture, respectively (Figures 9a and 9b). The expression levels of LRP2 and CUBN were normalized to the expression level of β-actin (ACTB), respectively. Expression levels of these proteins were increased in Mature renal organoids on day 26 of differentiation induction compared with Control renal organoids (Figures 9a and 9b). Similar results were obtained for UGT2A3, AZGP1, SLC39A4, BHMT, ACE2, AQP6, and ABCB1. These results indicate that Mature renal organoids mature more quickly than Control renal organoids with respect to proximal tubule protein reabsorption.

[0181] Example 7 Number of Endosomes in Renal Organoids (Quantification of Endosome Number by TEM) Tissue samples for imaging with a transmission electron microscope (TEM) were prepared as follows. Renal organoids on day 26 of culture were fixed using phosphate-buffered 2% glutaraldehyde, and then fixed using 2% osmium tetroxide in an ice bath for 3 hours. The fixed organoids were dehydrated using an ethanol solution and embedded in epoxy resin. Sections of the embedded organoids were prepared using an ultramicrotome method. The sections were stained with uranyl acetate for 10 minutes, followed by lead staining for 5 minutes. The stained sections were subjected to TEM observation (HITACHI H-7600 at 100 kV). The acquired images were analyzed using Image J Fiji to measure the area of ​​the cytoplasm (μm ) of proximal tubule cells. ２ ), number and size of endosomes (μm ２ ) was measured and quantified.

[0182] (Result) Cytoplasm (μm ２ The number of endosomes per 0.5 μm was significantly higher in the renal organoids of the Mature group than in the renal organoids of the Control group (Fig. 10a). ２The percentage of cells with 0% or more endosomes was 65.4% in the kidney organoids of the control group, but was only 38.5% in the kidney organoids of the mature group (Fig. 10b and Fig. 10c). ２ The percentage of cells with endosomes of 1 to 20% or more was 15.4% in the renal organoids of the control group, whereas it was 34.6% in the renal organoids of the mature group (Fig. 10b and Fig. 10c). ２ The percentage of cells with endosomes of 41-60% or more was 3.8% in the control kidney organoids, whereas it was 7.7% in the mature kidney organoids (Fig. 10b and Fig. 10c). These results suggest that the mature kidney organoids had significantly more endosomes relative to the cytoplasmic area than the control kidney organoids, and that the number of endosomes was significantly higher than that of the control kidney organoids. ２ This indicates that the proportion of endosomes above this level increases.

[0183] In fetal rats, as development progresses, the number of small and large vacuoles increases in the proximal tubules (Okada T, et al., Ann Anat. 1993 Feb;175(1):89-94, Journal of Ultrastructure Research 53, 254-270(1975)). In the literature, small vacuoles refer to vacuoles with a diameter of less than 0.5 μm. Therefore, Example 7 shows that renal organoids in the Mature group contain more mature cells than renal organoids in the Control group.

[0184] Example 8 Dextran Uptake by Renal Organoids Dextran Uptake Assay pHrodo™ Red Dextran (10,000 MW) for Endocytosis (Invitrogen, P10361) was added to a medium containing renal organoids on day 26 cultured in the same manner as in Example 2, to a concentration of 10 μg / mL, and the medium was incubated at 37° C., 5% CO ２The kidney organoids were cultured under ice-cold conditions for 24 hours. They were then washed three times with ice-cold DPBS(-) and maintained in DPBS(-) containing LTL-FITC (Vector Laboratories, FL-1321) (1:100) for 2 hours at 4°C to stain the proximal tubules with LTL. 2x2 tile images of the stained kidney organoids were acquired using a CV8000 (Yokokawa, X20 lens). Fluorescence images were acquired every 5 μm over a 100 μm Z-axis (thickness). The CV8000 was capable of irradiating with 488 nm and 561 nm light and was equipped with BP525 / 50 and BP600 / 37 filters. Two-dimensional images of LTL were created from the three-dimensional images using Average Image Projection (AveIP), and two-dimensional images of Dextran were created using Sum Image Projection (SumIP). Using the created images, the fluorescence intensity of Dextran in the LTL-binding region was measured and quantified.

[0185] (Results) The fluorescence intensity of dextran was significantly stronger in the renal organoids of the Mature group than in the renal organoids of the Control group (Figure 11, **p<0.01, unpaired t-test). This result indicates that the renal organoids of the Mature group have an improved function of taking up extracellular substances compared to the renal organoids of the Control group. Example 8 shows that the renal organoids of the Mature group contain mature proximal tubule cells.

[0186] [Comparative Example 1] Expression level of maturation markers of proximal tubule cells in the presence of a PPARA antagonist In step C of Example 2 (day 15 of differentiation induction), instead of the PPARA agonist and RXR agonist, the PPARA antagonist GW6471 was used, except that kidney organoids were produced according to the same method as the kidney organoid production method described in Example 2. The expression level of proximal tubule marker genes in the produced kidney organoids was measured. In step C (day 15 of differentiation induction), a group cultured in STEMdiff APEL2 medium (0.2% DMSO, 1% PFHM II, Antibiotic-Antimycotic) without GW6471 was used as a control.

[0187] (Results) Using a given concentration of GW6471, the expression levels of UGT2A3 (Fig. 12a), AZGP1 (Fig. 12b), BHMT (Fig. 12d), ACE2 (Fig. 12e), AQP6 (Fig. 12f), CUBN (Fig. 12g), LRP2 (Fig. 12h), ABCB1 (Fig. 12i), APOA1 (Fig. 12j), APOC3 (Fig. 12k), and FABP1 (Fig. 12n), which are markers of proximal tubule cell maturation, were significantly reduced. There was also a tendency for the expression levels of SLC39A4 (Fig. 12c) and CYP27A1 (Fig. 12l) to decrease. These results indicate that PPARA antagonists can prevent early differentiation into mature proximal tubule cells.

[0188] [Comparative Example 2] Expression levels of maturation markers of proximal tubule cells in the presence of an RXR antagonist In step C (day 15 of differentiation induction) of Example 2, instead of the PPARA agonist and RXR agonist, the RXR antagonist HX531 was used, except that kidney organoids were produced according to substantially the same method as the kidney organoid production method described in Example 2. The expression levels of proximal tubule marker genes in the produced kidney organoids were measured. In step C (day 15 of differentiation induction), a group cultured in STEMdiff APEL2 medium (0.2% DMSO, 1% PFHM II, Antibiotic-Antimycotic) without HX531 was used as a control.

[0189] (Results) The expression levels of UGT2A3 (Fig. 13a), AZGP1 (Fig. 13b), SLC39A4 (Fig. 13c), BHMT (Fig. 13d), ACE2 (Fig. 13e), AQP6 (Fig. 13f), CUBN (Fig. 13g), LRP2 (Fig. 13h), ABCB1 (Fig. 13i), APOA1 (Fig. 13j), APOC3 (Fig. 13k), CYP27A1 (Fig. 13l), and FABP1 (Fig. 13n), which are markers of proximal tubule cell maturation, were significantly reduced by the administration of GW6471 at a given concentration. These results indicate that RXR antagonists can prevent early differentiation into mature proximal tubule cells.

[0190] As described above, the results of Comparative Examples 1 and 2 show that PPARA antagonist or RXR antagonist can prevent early differentiation into mature proximal tubule cells.These results support that the combination of PPARA agonist and RXR agonist promotes the differentiation into mature proximal tubule cells in kidney organoids.

[0191] [Example 9] Preparation of renal organoids in the presence of pirinixic acid (WY14643) In step C of Example 2 (15th day of differentiation induction), except that instead of the combination of PPARA agonist (CP775146) and RXR agonist (9cis retinoic acid), PPAPA agonist (pirinixic acid) was used, renal organoids were prepared according to substantially the same method as the method for preparing renal organoids described in Example 2. The expression level of proximal tubule marker gene group in the prepared renal organoids was measured (n=3). Figure 14 shows that pirinixic acid alone, a PPAPA agonist, increased the expression of proximal tubule markers.

[0192] Therefore, it is expected that combining a PPAPA agonist (pirinixic acid) with an RXR agonist will promote the maturation of proximal tubule cells in the production of renal organoids.

Claims

1. 1. A method for producing kidney organoids, comprising: Culturing primary renal organoids using a medium containing an RXR agonist and a PPAR agonist; The method, wherein the kidney organoids contain mature proximal tubule cells.

2. Further comprising deriving said early renal organoids from intermediate mesodermal cells; Inducing the early renal organoids comprises culturing the intermediate mesoderm cells in induction medium B containing fibroblast growth factor and substantially free of RXR agonist to form intermediate mesoderm spheroids; The method of claim 1, comprising culturing the intermediate mesodermal spheroids using induction medium A containing a GSK3β inhibitor, and then culturing them using induction medium B containing a fibroblast growth factor and substantially free of an RXR agonist.

3. further comprising deriving said intermediate mesoderm cells from pluripotent stem cells; 3. The method of claim 2, wherein inducing the intermediate mesoderm cells comprises culturing the pluripotent stem cells in induction medium A containing a GSK3β inhibitor, and then culturing the pluripotent stem cells in induction medium B containing a fibroblast growth factor and substantially free of an RXR agonist.

4. the kidney organoid expresses a proximal tubule marker; 2. The method of claim 1, wherein the proximal tubule marker is at least one selected from the group consisting of UGT2A3, AZGP1, SLC39A4, BHMT, ACE2, AQP6, CUBN, LRP2, ABCB1, SERPINA1, APOA1, and SPARCL1.

5. A renal organoid containing mature proximal tubule cells, produced by the method according to any one of claims 1 to 4.

6. A kidney organoid, the kidney organoids contain mature proximal tubule cells; The kidney organoids express proximal tubule markers; The renal organoid, wherein the proximal tubule marker is at least one selected from the group consisting of UGT2A3, AZGP1, SLC39A4, BHMT, ACE2, AQP6, CUBN, LRP2, ABCB1, SERPINA1, APOA1, and SPARCL1.

7. A method for producing a non-human mammal having a renal organoid in the kidney or a peripheral region thereof, comprising introducing the renal organoid according to claim 5 into the kidney or a peripheral region of the non-human mammal, The method, wherein the kidney organoids contain mature proximal tubule cells.

8. A method for producing a non-human mammal having a renal organoid in the kidney or a peripheral region thereof, comprising introducing the renal organoid according to claim 6 into the kidney or a peripheral region of the non-human mammal, The method, wherein the kidney organoids contain mature proximal tubule cells.

9. A non-human mammal having the renal organoid described in claim 5 in the kidney or its surrounding area.

10. A non-human mammal having the renal organoid described in claim 6 in the kidney or its surrounding area.

11. A regenerative medicine composition for treating kidney damage or disease, comprising the renal organoid described in claim 5.

12. A regenerative medicine composition for treating kidney damage or disease, comprising the renal organoid described in claim 6.

13. A method for evaluating drug responsiveness to a test substance, comprising: Contacting the renal organoid of claim 5 with a test substance; and A method comprising measuring drug responsiveness in the renal organoid or the non-human mammal to the test substance.

14. A method for evaluating drug responsiveness to a test substance, comprising: Contacting the renal organoid of claim 6 with a test substance; and A method comprising measuring drug responsiveness in the renal organoid or the non-human mammal to the test substance.

15. A method for evaluating drug responsiveness to a test substance, comprising: contacting a non-human mammal produced by the method of claim 7 with a test substance; and A method comprising measuring drug responsiveness in the renal organoid or the non-human mammal to the test substance.

16. A method for evaluating drug responsiveness to a test substance, comprising: contacting a non-human mammal produced by the method of claim 8 with a test substance; and A method comprising measuring drug responsiveness in the renal organoid or the non-human mammal to the test substance.

17. A method for evaluating drug responsiveness to a test substance, comprising: Contacting the non-human mammal of claim 9 with a test substance; and A method comprising measuring drug responsiveness in the renal organoid or the non-human mammal to the test substance.

18. A method for evaluating drug responsiveness to a test substance, comprising: contacting the non-human mammal of claim 10 with a test substance; and A method comprising measuring drug responsiveness in the renal organoid or the non-human mammal to the test substance.

19. A method for promoting maturation of proximal tubules, comprising culturing early renal organoids using a medium containing an RXR agonist and a PPAR agonist.