Therapeutic agent for renal disease and method for producing same

Abstract

The present invention provides a method for producing a nephron precursor cell conditioned medium, the method comprising the following steps: (1) a step for subjecting 500 or more cell masses of nephron precursor cells having a diameter of 150-600 μm to suspension culture in the same culture vessel to form a giant cell mass in which the cell masses are fused; and (2) a step for culturing the giant cell mass obtained in the step (1) in a medium in which nephron precursor cells are maintained and amplified to obtain a conditioned medium. The present invention also provides a conditioned medium obtained by said method, and a renal protection factor or the like secreted specifically in the conditioned medium.

Description

Drugs for treating kidney disease and methods for manufacturing the same

[0001] The present invention relates to therapeutic agents for kidney disease and methods for producing the same. More specifically, the present invention relates to large cell aggregates of pluripotent stem cell-derived nephron progenitor cells that secrete renoprotective substances, a culture medium for the same, methods for producing the same, and uses thereof. The present invention also relates to therapeutic agents for kidney disease comprising a protective substance highly secreted in the culture medium or cells that exogenously express the substance.

[0002] The number of patients with chronic kidney disease (CKD) continues to increase, with an estimated 850 million worldwide and over 13 million in Japan. Recently, acute kidney injury (AKI) has attracted attention as one of the causes of CKD. AKI has a sudden onset, and in many cases, kidney function recovers, but recent meta-analyses have revealed that it progresses to CKD and then to end-stage renal failure at a certain frequency. However, apart from kidney transplantation, which suffers from a serious donor shortage, there are few curative treatments, and the development of effective treatments for CKD and AKI is urgently needed.

[0003] Cell transplantation therapy has recently attracted attention as a treatment for CKD and AKI, but clinical trials using mesenchymal stem cells have not shown satisfactory therapeutic effects, and concerns have also been raised about safety due to their multipotency.

[0004] It is possible to create nephron progenitor cells that can differentiate only into kidney-derived cells using human induced pluripotent stem (iPS) cells (for example, Patent Document 1). Transplantation of induced nephron progenitor cells derived from human iPS cells has been reported to reduce renal damage and prevent renal interstitial fibrosis in AKI mice (for example, Non-Patent Document 1), and from a safety standpoint, it is expected to be a novel source for cell therapy for kidney disease. The present inventors have developed an expansion medium and an expansion culture method using the same that can amplify nephron progenitor cells in large quantities while maintaining their differentiation ability into kidney-derived cells, as a means of obtaining a sufficient amount of high-quality nephron progenitor cells necessary for cell therapy (Patent Document 2).

[0005] In Non-Patent Literature 1, induced nephron progenitor cells transplanted subcapsularly did not migrate to the host mouse renal parenchyma and did not contribute to the regeneration of host renal tissue, suggesting that the mechanism of therapeutic effect was a paracrine effect mediated by factors secreted by induced nephron progenitor cells. In cell therapy, it is necessary to prepare nephron progenitor cells derived from an HLA-matched donor for allogeneic transplantation. However, if the therapeutic factors secreted by nephron progenitor cells can be developed as pharmaceuticals, all patients with kidney disease could be targeted. Furthermore, cell therapy requires the use of immunosuppressants, which have side effects such as the development of diabetes, fibrosis of renal tissue, and increased susceptibility to infection. In this sense, developing the therapeutic factors secreted by nephron progenitor cells as pharmaceuticals is also significant. In addition, clinical application of cell therapy to a patient's own kidney is expected to involve expensive, complicated, and highly invasive procedures using robots or endoscopic-guided special devices. Therefore, developing these therapeutic factors as pharmaceuticals would be useful in simplifying the administration method and reducing the burden on both patients and physicians. However, administering the culture supernatant (conditioned medium) obtained when the aforementioned induced nephron progenitor cells were differentiated and spheroids were formed to AKI mice did not show any therapeutic effect on renal impairment.

[0006] International Publication No. 2018 / 216743, International Publication No. 2022 / 149616

[0007] Toyohara T. et al., Stem Cells Transl. Med., 2015; 4: 980-992

[0008] The object of the present invention is to provide a novel renal progenitor cell population suitable for transplantation that is useful for cell therapy of CKD and AKI, that is, a renal progenitor cell population that secretes factors that exert therapeutic effects against CKD and AKI, and a method for producing the same. Another object of the present invention is to provide a novel renal disease treatment agent using a conditioned medium obtained by culturing the renal progenitor cell population, and therapeutically effective components contained in the conditioned medium.

[0009] Non-Patent Document 1 describes how induced nephron progenitor cells were produced according to the method described in Patent Document 1, differentiated to form spheroids (small cell aggregates), and verified the therapeutic effect of these spheroids and the conditioned medium obtained during their culture on AKI. To verify the effect of further enlarging the small cell aggregates used in Non-Patent Document 1, the present inventors cultured 700 small cell aggregates in the same incubator to produce giant cell aggregates. Surprisingly, the resulting giant cell aggregates showed no change in the frequency of apoptosis or cytotoxicity compared to the original small cell aggregates. Even more surprisingly, when the conditioned medium of these giant cell aggregates was administered intraperitoneally to AKI mice, blood urea nitrogen and serum creatinine levels decreased to normal levels, and the survival rate increased significantly.

[0010] Therefore, the inventors performed differential mass spectrometry on the conditioned media for large cell aggregates and small cell aggregates, and succeeded in identifying a group of proteins contained only in the conditioned medium for large cell aggregates, i.e., candidate substances for therapeutic factors. Of these candidate substances, the inventors administered seven proteins, to the best of their knowledge, that had not previously been reported to have any effect on the kidneys, to AKI mice and verified their therapeutic effects. Six of the proteins significantly reduced blood urea nitrogen and serum creatinine levels. Of these, SCUBE3 was further evaluated using another AKI model in which mice did not die, and excellent therapeutic effects were observed even 10 days after drug induction, which corresponds to the transition period from AKI to CKD.

[0011] Based on these findings, the inventors conducted further research and have now completed the present invention. Specifically, the present invention provides the following:

[0012] [Item 1] A method for producing a nephron progenitor cell conditioned medium, comprising the following steps: (1) a step of suspension culturing 500 or more cell aggregates of nephron progenitor cells having a diameter of 150 to 600 μm in the same culture vessel to form a giant cell aggregate by fusing the cell aggregates; and (2) a step of culturing the giant cell aggregate obtained in (1) in a medium that maintains and amplifies nephron progenitor cells to obtain a conditioned medium. [Item 2] The method according to Item 1, wherein the culture in step (1) is carried out for 24 hours or more. [Item 3] The method according to Item 1 or 2, further comprising a step of forming a cell aggregate of 150 to 600 μm in diameter from single-cell nephron progenitor cells before step (1). [Item 4] The method according to any one of Items 1 to 3, wherein the nephron progenitor cells are differentiated and induced from pluripotent stem cells. [Claim 5] The method according to any one of Claims 1 to 4, wherein the culture medium for maintaining and amplifying nephron progenitor cells comprises a GSK-3β inhibitor, a ROCK inhibitor, and FGF9 and / or FGF20. [Claim 6] The method according to any one of Claims 1 to 5, wherein the frequency of apoptosis and / or cytotoxicity does not increase throughout the culture period. [Claim 7] A nephron progenitor cell conditioned medium obtained by the method according to any one of Claims 1 to 6. [Claim 8] A nephron progenitor cell conditioned medium, wherein the nephron progenitor cells are in the form of giant cell aggregates with a minimum diameter of 0.8 mm or more, and the conditioned medium has a nephroprotective effect. [Claim 9] The conditioned medium according to Claim 7 or 8, which has been cryopreserved. [Claim 10] A pharmaceutical composition comprising the conditioned medium according to any one of Claims 7 to 9. [Claim 11] The pharmaceutical composition according to Claim 10, for the treatment of kidney disease. [Claim 11a] A method for treating a kidney disease in a subject, comprising administering a therapeutically effective amount of the conditioned medium described in Clause 7 or 8 or the thawed conditioned medium described in Clause 9 to the subject. [Claim 11b] The conditioned medium described in any one of Clauses 7 to 9 for use in the treatment of kidney disease. [Claim 11c] The use of the conditioned medium described in any one of Clauses 7 to 9 in the manufacture of a therapeutic agent for kidney disease. [Claim 12] The pharmaceutical composition described in Clause 10 or 11 for systemic administration. [Claim 13] A giant cell mass of nephron progenitor cells constructed in vitro, having a minimum diameter of 0.8 mm or more.[Claim 14] A method for producing a giant cell aggregate of nephron progenitor cells, comprising suspension culture of a population of cell aggregates of nephron progenitor cells having a diameter of 150 to 600 μm in the same culture vessel, and fusing the cell aggregates to form a giant cell aggregate with a minimum diameter of 0.8 mm or more. [Claim 15] A giant cell aggregate of nephron progenitor cells obtained by the method of Claim 14. [Claim 16] A renal organoid using the giant cell aggregate described in Claim 13 or 15 as the source of nephron progenitor cells. [Claim 17] A pharmaceutical composition comprising the giant cell aggregate described in Claim 13 or 15 or the renal organoid described in Claim 16. [Claim 18] The pharmaceutical composition described in Claim 17 for the treatment of renal disease. [Claim 18a] A method for treating renal disease in a subject, comprising administering a therapeutically effective amount of the giant cell aggregate described in Claim 13 or 15 or the renal organoid described in Claim 16 to the subject. [Clause 18b] A giant cell aggregate as described in Clause 13 or 15 or a renal organoid as described in Clause 16 for use in the treatment of renal disease. [Clause 18c] Use of a giant cell aggregate as described in Clause 13 or 15 or a renal organoid as described in Clause 16 in the manufacture of a therapeutic agent for renal disease. [Clause 19] A therapeutic agent for renal disease comprising one or more proteins selected from the group consisting of corticotropin-releasing factor-binding protein (CRHBP), SCUBE3 (Signal peptide, CUB and EGF-like domain-containing protein 3), SRPX2 (Sushi repeat-containing protein X-linked 2), complement C7 (C7), carboxypeptidase Q (CPQ), and mucin-5AC (MUC5AC). [Clause 19a] A method for treating renal disease in a subject, comprising administering a therapeutically effective amount to one or more proteins selected from the group consisting of CRHBP, SCUBE3, SRPX2, C7, CPQ, and MUC5AC to the subject. [Clause 19b] One or more proteins selected from the group consisting of CRHBP, SCUBE3, SRPX2, C7, CPQ, and MUC5AC for use in the treatment of renal disease. [Clause 19c] Use of one or more proteins selected from the group consisting of CRHBP, SCUBE3, SRPX2, C7, CPQ, and MUC5AC in the manufacture of a therapeutic agent for renal disease.[Item 20] The agent according to item 19, the method according to item 19a, the protein for use according to item 19b, or the use according to item 19c, wherein the one or more proteins are provided in the form of cells that exogenously secrete and express the nucleic acid encoding the protein.

[0013] According to the large cell mass of the nephron progenitor cells of the present invention, a conditioned medium having a renal protective effect and effective for the treatment of CKD and AKI can be produced. In addition, a factor having a renal protective effect can be identified from the group of proteins specifically secreted from the conditioned medium. The identified renal protective factor can be used as a therapeutic agent for kidney diseases by itself, and cells engineered to highly secrete and express it can be used as a source for cell therapy for the treatment of kidney diseases. Furthermore, the large cell mass itself is useful as a source for the reconstruction of kidney tissue and cell transplantation therapy for kidney diseases.

[0014] Figure 1 shows that the conditioned medium of the large cell mass of human iPSC-derived nephron progenitor cells improves AKI in mice. (A) Schematic diagram of the experimental protocol. (B) Bright-field and fluorescence images showing the morphology of the cell mass and the expression of OSR1 and SIX2 during the formation of the large cell mass from the small cell mass of hiPSC-NPC in (A). Scale bar: 1 mm. (C) Immunostaining analysis of the large cell mass of hiPSC-NPC after 10 days of expansion culture for SIX2, PAX2, SALL1, WT1, which are NPC markers, and Ki67, which is a proliferation marker. Scale bar: 200 μm. (D) Bright-field image of a renal organoid derived from the large cell mass of hiPSC-NPC. Scale bar: 500 μm. (E and F) PODXL ＋ Glomerulus, LTL ＋ CDH1 － Proximal tubule and LTL － CDH1 ＋ Distal tubule (E) and NPHS1 ＋ or WT1 ＋ Glomerulus, JAGEEED1 ＋ CDH1 － Proximal tubule, BRN1 ＋ DBA － Loop of Henle and CDH1 ＋ DBA ＋ or BRN1 ＋DBA ＋Immunostaining analysis of renal organoids derived from giant cell aggregates of hiPSC-NPCs, showing the distal tubule (F). Scale bars: 200 μm in (E), 100 μm in the left and center panels of (F), and 50 μm in the right panel of (F). (G) Immunostaining analysis of giant cell aggregates of hiPSC-NPCs after 10 days of expanded culture for the apoptotic cell marker CLEAVED CASPASE 3 (CC3). Scale bar: 200 μm. (H) LDH assay showing the temporal changes in cytotoxicity during the formation of giant cell aggregates (n=3). (I) BUN and S-Cre levels of cisplatin-induced AKI mice treated as shown (n=11 in each group). (J and K) Quantitative evaluation of HE and PAS staining (J) and histological findings (K) of kidney sections of cisplatin-induced AKI mice (n=15). Scale bar: 100 μm. (L) Kaplan-Meier survival curve of cisplatin-induced AKI mice. The p-value was evaluated by a log-rank test comparing the CM treatment group with the control CFY medium treatment group. CFY is CFY medium, CM is conditioned medium, and HPF is high-power field. Data are expressed as mean ± SD for (H, I, K). **P < 0.01, ***P < 0.001 (Mann-Whitney test for (I), unpaired t-test for (K)). hiPSC-NPC differentiated from the OSR1-GFP / SIX2-tdTomato double knock-in reporter hiPSC strain was used in all experiments. Figure 2 shows that conditioned medium (CM) for cryopreserved hiPSC-NPC giant cell aggregates improves AKI in mice. (A) BUN and S-Cre levels in cisplatin-induced AKI mice (n=5 in each group). (B) Schematic diagram of the experimental protocol. LA plate: low-adhesion plate. (C) BUN and S-Cre levels in wild-type mice and cisplatin-induced AKI mice. (D) Kaplan-Meier survival curves for cisplatin-induced AKI mice. The p-value was evaluated by a log-rank test comparing the cryopreserved CM-treated group with the control cryopreserved CFY-treated group (n=4 in each group). CFY is CFY medium, CM is conditioned medium. Data are expressed as mean ± SD in (A and C). (A) is one-way ANOVA using an unpaired t-test, and (C) is one-way ANOVA using Tukey's test, with ***P < 0.001.hiPSC-NPCs differentiated from the OSR1-GFP / SIX2-tdTomato double knock-in reporter hiPSC strain were used in all experiments. Figure 3 shows the therapeutic effects (BUN and S-Cre levels) of administration of six recombinant proteins (CRHBP, SCUBE3, SRPX2, C7, CPQ, MUC5AC) to cisplatin-induced AKI mice. Phosphate-buffered saline (PBS) was administered as a control. Data are expressed as mean ± SD. Figure 4 shows the therapeutic effects (BUN and S-Cre levels) of SCUBE3 administration to aristolochic acid-induced AKI mice over time. Phosphate-buffered saline (PBS) was administered as a control. Data are expressed as mean ± SD.

[0015] 1. Giant Cell Aggregates of Nephron Progenitor Cells and Method for Producing the Same The present invention provides giant cell aggregates of nephron progenitor cells constructed in vitro, having a minimum diameter of 0.8 mm or more (hereinafter sometimes referred to as "the giant cell aggregates of the present invention"). The giant cell aggregates of the present invention are typically formed by the fusion of 500 or more smaller spherical cell aggregates, and are therefore irregularly shaped cell aggregates as shown in the examples described later (see, for example, Day 4 onwards in Figure 1B), but are characterized by having a minimum diameter of 0.8 mm or more. In conventional methods (for example, Non-Patent Document 1), spherical cell aggregates with a diameter of 600 μm or less are prepared, then maintained culture is performed, and the acclimatization medium is repeatedly collected and the medium is changed, ultimately resulting in medium-sized spherical cell aggregates with a diameter of less than 800 μm. The giant cell aggregates of the present invention have a minimum diameter of 800 μm or more, and a maximum diameter of 5 mm or more.

[0016] The giant cell aggregates of the present invention are a novel cell population that is clearly different from existing in vitro constructs of nephron progenitor cells, in that they secrete factors with renal protective effects in therapeutically effective amounts to subjects with kidney disease, compared to small spherical cell aggregates (diameter 600 μm or less) of nephron progenitor cells produced by conventional methods, and medium-sized spherical cell aggregates (diameter less than 800 μm) obtained by further maintenance culture of the same. Therefore, the giant cell aggregates of the present invention are useful as a source of conditioned medium and its components for therapeutic agents of kidney disease. Moreover, despite having a maximum diameter of 5 mm or more, these giant cell aggregates do not show an increased frequency of apoptosis or cytotoxicity compared to existing smaller spherical cell aggregates. It is known that oxygen supply becomes insufficient when the distance from the surface of the cell aggregate exceeds 200 μm, and the inventors have also confirmed that the proliferation rate of spherical cell aggregates decreases when the seeding density of induced nephron progenitor cells is increased. Therefore, the fact that no significant change in cell survival is observed in such giant cell aggregates is a surprising finding. Therefore, the giant cell aggregates of the present invention are themselves useful as a high-quality source for kidney tissue reconstruction (kidney regeneration) and cell transplantation.

[0017] Nephron progenitor cells are present only in the developing kidney and are precursor cells that differentiate into glomeruli and tubules to form nephrons, the smallest functional units of the kidney. The metanephrocyte in the fetus has a mechanism to maintain and amplify nephron progenitor cells throughout organogenesis, but the self-renewal of nephron progenitor cells is transient, and they are lost immediately after birth, differentiating into glomeruli and tubules. Therefore, it is extremely difficult to isolate a population of undifferentiated human nephron progenitor cells from nature.

[0018] In cell transplantation, two methods can be used: injecting the target cells into the lesion site or attaching a sheet of cells to the lesion site. However, the former method has drawbacks, such as the leakage of injected cells, resulting in low transplantation efficiency and low engraftment rates. On the other hand, if the cell preparation to be transplanted becomes too large, there is a concern that the central part will be susceptible to cytotoxicity and apoptosis due to malnutrition and hypoxia. However, as described above, the giant cell aggregate of the present invention shows no change in the frequency of apoptosis and cytotoxicity compared to existing smaller spherical cell aggregates, and is therefore expected to be an alternative graft in cell transplantation therapy for kidney disease. Furthermore, as revealed in this invention, it is thought that the fusion of smaller spherical cell aggregates to form a giant cell aggregate changes the profile of proteins secreted extracellularly, thereby providing factors with renal protective effects. Therefore, it is also expected that constructing a giant cell aggregate in vitro beforehand will accelerate the manifestation of renal protective effects.

[0019] The giant cell aggregates of the present invention may be manufactured by any method as long as their minimum diameter is 0.8 mm or more and the conditioned medium has a renal protective effect. In one embodiment, however, they can be manufactured by fusing existing small spherical cell aggregates (diameter 600 μm or less) in vitro. In a preferred embodiment, the giant cell aggregates of the present invention can be manufactured by suspension culturing a group of cell aggregates of nephron progenitor cells with a diameter of 150 to 600 μm in the same culture vessel and fusing the cell aggregates.

[0020] Accordingly, the present invention also provides a method for producing a giant cell aggregate of nephron progenitor cells (hereinafter sometimes referred to as "the method for producing a giant cell aggregate of the present invention"), which includes suspension culturing a population of cell aggregates of nephron progenitor cells having a diameter of 150 to 600 μm in the same culture vessel, and fusing the cell aggregates to form a giant cell aggregate with a minimum diameter of 0.8 mm or more.

[0021] The cell aggregates of nephron progenitor cells with a diameter of 150 to 600 μm used in the giant cell aggregate production method of the present invention (hereinafter sometimes referred to as "small cell aggregates") may be provided by any method, as long as they have such a shape and do not secrete renal protective factors to the extent that they themselves have a therapeutic effect against kidney disease, but can secrete renal protective factors when they fuse with each other to form a giant cell aggregate. For example, the small cell aggregates can be provided by suspension culture of single-cell nephron progenitor cells using a culture medium suitable for spheroid formation.

[0022] The nephron progenitor cells used to prepare the small cell aggregate are not particularly limited in their origin, as long as they are progenitor cells capable of forming nephrons by differentiating into glomeruli and tubules. They may be isolated from the metanephrotic mesenchyme of humans or other mammals (e.g., mice, rats, dogs, cats, pigs, cattle, monkeys, etc.), but are preferably induced nephron progenitor cells differentiated from pluripotent stem cells.

[0023] Examples of pluripotent stem cells include iPS cells, embryonic stem cells (ES cells), ES cells derived from cloned embryos obtained by nuclear transfer (ntES cells), pluripotent germ cells (mGS cells), embryonic germ cells (EG cells), and Muse (multi-lineage differentiating stress enduring) cells, but iPS cells are preferred, and human iPS cells are even more preferred. When pluripotent stem cells are ES cells or any cells derived from a human embryo, these cells may be produced by destroying the embryo or without destroying the embryo, but preferably they are cells produced without destroying the embryo.

[0024] iPS cells are artificial stem cells derived from somatic cells that have characteristics almost equivalent to those of ES cells, such as pluripotency and the ability to proliferate by self-renewal, and can be produced by introducing specific reprogramming factors into somatic cells in the form of DNA or protein (Takahashi K. and Yamanaka S. (2006) Cell, 126:663-676; Takahashi K. et al. (2007), Cell, 131:861-872; Yu J. et al., (2007), Science, 318:1917-1920; Nakagawa M. et al. (2008), Nat. Biotechnol., 26:101-106; WO 2007 / 069666).

[0025] "Somatic cells" mean all animal cells (preferably mammalian cells including humans) excluding germline cells or totipotent cells such as eggs, oocytes, and ES cells. Somatic cells include, but are not particularly limited to, somatic cells of fetuses (offspring), newborns (offspring), and mature healthy or diseased somatic cells, and also include any of primary cultured cells, subcultured cells, and established cell lines. Specifically, somatic cells include, for example, (1) tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, dental pulp stem cells, etc., (2) tissue progenitor cells, and (3) differentiated cells such as lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (such as skin cells), hair cells, hepatocytes, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells (such as pancreatic exocrine cells), brain cells, lung cells, kidney cells, and fat cells.

[0026] When using iPS cells, these iPS cells may be produced from somatic cells by a known method, or already established and stockpiled iPS cells may be used. Reprogramming factors may consist of genes specifically expressed in ES cells, their gene products or non-coding RNA, or genes that play an important role in maintaining the undifferentiated state of ES cells, their gene products or non-coding RNA, or small molecule compounds. Examples of genes included in the reprogramming factors include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, β-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, or Glis1. These reprogramming factors may be used individually or in combination. The combinations of initialization factors are WO 2007 / 069666, WO 2008 / 118820, WO 2009 / 007852, WO 2009 / 032194, WO 2009 / 058413, WO 2009 / 057831, WO 2009 / 075119, WO 2009 / 079007, WO 2009 / 091659, WO 2009 / 101084, WO 2009 / 101407, WO 2009 / 102983, WO 2009 / 114949, WO 2009 / 117439, WO 2009 / 126250, WO 2009 / 126251, WO 2009 / 126655, WO 2009 / 157593, WO 2010 / 009015, WO 2010 / 033906, WO 2010 / 033920, WO 2010 / 042800, WO 2010 / 050626, WO 2010 / 056831, WO 2010 / 068955, WO 2010 / 098419, WO 2010 / 102267, WO 2010 / 111409, WO 2010 / 111422, WO 2010 / 115050, WO 2010 / 124290, WO 2010 / 147395, WO 2010 / 147612, Huangfu D, et al. (2008), Nat. Biotechnol., 26:795-797, Shi Y, et al.The combinations described in (2008), Cell Stem Cell, 2:525-528, Eminli S, et al. (2008), Stem Cells. 26:2467-2474, Huangfu D, et al. (2008), Nat Biotechnol. 26:1269-1275, Shi Y, et al. (2008), Cell Stem Cell, 3, 568-574, Zhao Y, et al. (2008), Cell Stem Cell, 3:475-479, Marson A, (2008), Cell Stem Cell, 3, 132-135, Feng B, et al. (2009), Nat Cell Biol. 11:197-203, R.L. Judson et al., (2009), Nat. Biotech., 27:459-461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci U S A. 106:8912-8917, Kim JB, et al. (2009), Nature. 461:649-643, Ichida JK, et al. (2009), Cell Stem Cell. 5:491-503, Heng JC, et al. (2010), Cell Stem Cell. 6:167-74, Han J, et al. (2010), Nature. 463:1096-100, Mali P, et al. (2010), Stem Cells. 28:713-720, Maekawa M, et al. (2011), Nature. 474:225-9 are exemplified.

[0027] The above-mentioned reprogramming factors include, for example, histone deacetylase (HDAC) inhibitors [e.g., small molecule inhibitors such as valproic acid (VPA), trichostatin A, sodium butyrate, MC 1293, M344, etc.], and siRNA and shRNA against HDAC [e.g., HDAC1 siRNA Smartpool (Millipore), HuSH 29mer shRNA Constructs against Nucleoside expression inhibitors such as HDAC1 (OriGene), MEK inhibitors (e.g., PD184352, PD98059, U0126, SL327 and PD0325901), glycogen synthase kinase-3 inhibitors (e.g., Bio and CHIR99021), DNA methyltransferase inhibitors (e.g., 5-azacitidine), histone methyltransferase inhibitors (e.g., small molecule inhibitors such as BIX-01294, nucleoside expression inhibitors such as siRNA and shRNA for Suv39hl, Suv39h2, SetDBl and G9a, etc.), L-channel calcium agonists (e.g., Bayk8644), butyrate, TGFβ inhibitors or ALK5 inhibitors (e.g., For example, factors used to enhance the establishment efficiency of p53 inhibitors (e.g., p53-mediated siRNA and shRNA), ARID3A inhibitors (e.g., ARID3A-mediated siRNA and shRNA), miRNAs such as miR-291-3p, miR-294, miR-295, and mir-302, Wnt signaling (e.g., soluble Wnt3a), neuropeptide Y, prostaglandins (e.g., prostaglandin E2 and prostaglandin J2), hTERT, SV40LT, UTF1, IRX6, GLISl, PITX2, DMRTBl, etc. are also included, but are not limited to, these factors. In this specification, factors used to enhance the establishment efficiency of these factors are not distinguished from reprogramming factors.

[0028] Pluripotent stem cells can be maintained in culture by known methods before differentiation into nephron progenitor cells. Examples of basic culture media for maintenance include, but are not limited to, Neurobasal medium, Neuro Progenitor Basal medium, NS-A medium, BME medium, BGJb medium, CMRL 1066 medium, Minimum Essential Medium (MEM), Eagle MEM medium, αMEM medium, Dulbecco's Modified Eagle Medium (DMEM), Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, DMEM / F12 medium, Ham medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof. Alternatively, commercially available culture media for pluripotent stem cells (e.g., primate ES cell medium, mTeSR1, StemFit AK02N, StemFit AK03N, Essential 8, ReproFF2, etc.) may be used.

[0029] The medium can be a serum-containing medium or a serum-free medium. Preferably, a serum-free medium can be used. A serum-free medium (SFM) means a medium that does not contain any untreated or unpurified serum. Thus, media containing purified blood-derived components or animal tissue-derived components (such as growth factors) can be included. The concentration of serum (e.g., fetal bovine serum (FBS), human serum, etc.) can be 0 to 20%, preferably 0 to 5%, more preferably 0 to 2%, and most preferably 0% (i.e., serum-free). The SFM may or may not contain any serum substitute. Examples of serum substitutes include substances appropriately containing albumin (e.g., albumin substitutes such as lipid-rich albumin, recombinant albumin, plant starch, dextran, and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol, or their equivalents. Such serum substitutes can be prepared, for example, by the method described in WO 98 / 30679. Also, for simplicity, commercially available ones can be used. Examples of such commercially available substances include Knockout (trademark) Serum Replacement (KSR), Chemically-defined Lipid concentrated, and Glutamax (Invitrogen).

[0030] The culture medium may also contain other known additives. Examples include growth factors (e.g., insulin), polyamines (e.g., putrescine), minerals (e.g., sodium selenite), sugars (e.g., glucose), organic acids (e.g., pyruvate, lactic acid), amino acids (e.g., non-essential amino acids (NEAA), L-glutamine), reducing agents (e.g., 2-mercaptoethanol), vitamins (e.g., ascorbic acid, d-biotin), steroids (e.g., [beta]-estradiol, progesterone), antibiotics (e.g., streptomycin, penicillin, gentamicin), buffers (e.g., HEPES), and nutritional additives (e.g., B27 supplement, N2 supplement, StemPro-Nutrient Supplement). It is preferable that each additive is included within a concentration range known to the present day.

[0031] Pluripotent stem cells may be cultured in or without feeder cells, but considering clinical application in humans, it is preferable to culture pluripotent stem cells in the absence of feeder cells.

[0032] Culture vessels used for maintaining and culturing pluripotent stem cells are not particularly limited, but may include flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, microslides, chamber slides, petri dishes, tubes, trays, culture bags, and roller bottles. Culture vessels may be cell-adherent. Cell-adherent culture vessels may be coated with any cell adhesion substrate, such as an extracellular matrix (ECM), to improve the adhesion of the culture vessel surface to cells. The cell adhesion substrate may be any substance intended for the adhesion of pluripotent stem cells or feeder cells (if used). Examples of cell adhesion substrates include laminin, collagen, gelatin, poly-L-lysine, poly-D-lysine, poly-L-ornithine, and fibronectin, as well as mixtures thereof, such as Matrigel, and lysed cell membrane preparations (Klimanskaya I et al 2005. Lancet 365:p1636-1641). Commercially available cell adhesion substrates may also be used, such as iMatrix and Synthemax II. These cell adhesion substrates are coated onto culture vessels at concentrations typically used for culturing pluripotent stem cells, depending on their type.

[0033] In this culture, pluripotent stem cells are seeded on the culture vessel, for example, about 10 ４ ~10 ５ cells / cm ２ The cell density is 1-10% CO2. ２ The culture can be performed in an incubator under an atmosphere of 99-90% air at approximately 30-40°C, preferably approximately 37°C.

[0034] In the process of culturing pluripotent stem cells, the culture medium can be changed during the culture period. The culture medium used for the change may have the same components as the medium used before the change, or it may have different components. Preferably, a medium with the same components is used. The timing of the culture medium change is not particularly limited, but it may be performed every 1 day, 2 days, 3 days, 4 days, or 5 days after starting culture with fresh medium.

[0035] The method for differentiating pluripotent stem cells into nephron progenitor cells is not particularly limited, and known methods, such as those described in WO 2014 / 200115, WO 2017 / 043666, WO 2018 / 216743, WO 2022 / 149616, etc., can be used. In one preferred embodiment, the method described in WO 2018 / 216743 or a partially modified version thereof can be used.

[0036] Specifically, first, intermediate mesoderm cells are induced from pluripotent stem cells according to previously reported procedures (Biochem Biophys Res Commun. 393:877-82 (2010), Nat Commun. 4:1367 (2013), WO 2012 / 011610, WO 2014 / 200115), and then nephron progenitor cells are differentiated from the intermediate mesoderm cells. Typically, this method (hereinafter sometimes referred to as "iNPC induction method (1)") includes the following steps. (i) A step of culturing pluripotent stem cells in the presence of FGF2, BMP4, a GSK-3β inhibitor, and retinoic acid or a derivative thereof. (ii) A step of culturing the cells obtained in (i) in the presence of FGF2, a GSK-3β inhibitor, and BMP7. (iii) A step of culturing the cells obtained in (iii) in the presence of FGF2, a GSK-3β inhibitor, BMP7, and a TGFβ inhibitor. (iv) A step of culturing the cells obtained in (iii) in the presence of FGF2, a GSK-3β inhibitor, BMP7, activin, and a Rho-kinase (ROCK) inhibitor. (v) A step of culturing the cells obtained in (iv) in the presence of retinoic acid or a derivative thereof and FGF9. (vi) A step of culturing the cells obtained in (v) in the presence of a GSK-3β inhibitor and FGF9.

[0037] In step (i), late posterior epiblasts are induced from pluripotent stem cells. The culture in this step is preferably carried out by planar adherent culture using the above-mentioned adhesive culture vessel. First, colonies in which pluripotent stem cells in maintenance culture have reached 70% to 80% confluence are picked up, and the cells are dissociated using, for example, a separation solution having protease activity and collagenase activity (e.g., Accutase™ and Accumax™ (Innovative Cell Technologies, Inc.)) or a separation solution having only collagenase activity, and dispersed into single cells by pipetting or the like.

[0038] The culture medium used in step (i) can be prepared by adding FGF2, BMP4, a GSK-3β inhibitor, and retinoic acid or its derivatives to a basic medium. As the basic medium, the same as those exemplified for the maintenance culture of pluripotent stem cells can be used. If necessary, it may also contain serum, serum substitutes, lipids, amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, etc.

[0039] FGF2 (basic FGF: bFGF) is not particularly limited as long as it is derived from a mammal, but human FGF2 is preferred. Examples of human FGF2 include the protein having the amino acid sequence registered in the NCBI (National Center for Biotechnology Information) database with accession number ABO43041. FGF2 can be purified from mammalian cells by methods known to the public, or it can be produced by genetic recombination. FGF2 is also commercially available. The concentration of FGF2 used in step (i) is, for example, 1 to 1000 ng / ml, preferably 10 to 500 ng / ml, and more preferably 50 to 250 ng / ml.

[0040] BMP4 (bone morphogenetic protein 4) is not particularly limited as long as it is derived from a mammal, but human BMP4 is preferred. Examples of human BMP4 include the protein having the amino acid sequence registered in the NCBI database with accession number AAH20546. BMP4 can be purified from mammalian cells by methods known to the public, or it can be produced by genetic recombination. BMP4 is also commercially available. The concentration of BMP4 used in step (i) is, for example, 0.1 to 100 ng / ml, preferably 0.5 to 50 ng / ml, and more preferably 0.5 to 5 ng / ml.

[0041] Examples of GSK-3β inhibitors include the indirubin derivative BIO (also known as GSK-3β inhibitor IX; 6-bromoindirubin-3'-oxime), the maleimide derivative SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indole-3-yl)-1H-pyrrole-2,5-dione), the phenyl-α-bromomethyl ketone compound GSK-3β inhibitor VII (α,4-dibromoacetophenone), the cell membrane-permeable phosphorylated peptide L803-mts (also known as GSK-3β peptide inhibitor; Myr-N-GKEAPAPPPQSpP-NH2), and the highly selective CHIR99021 (Nature (2008) 453: 519-523). CHIR99021 is preferred. The concentration of the GSK-3β inhibitor used in step (i) can be appropriately selected by those skilled in the art depending on the GSK-3β inhibitor used, but for example, it is 0.01 to 100 μM, preferably 0.1 to 10 μM, more preferably 0.5 to 3 μM, and particularly preferably 0.5 to 1.5 μM.

[0042] Retinoic acid may be retinoic acid itself, or it may be a derivative of retinoic acid that retains the differentiation-inducing function of natural retinoic acid. Examples of retinoic acid derivatives include 3-dehydroretinoic acid, 4-[[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carbonyl]amino]-Benzoic acid (AM580) (Tamura, K. et al., Cell Differ. Dev. 32: 17-26 (1990)), and 4-[(1E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propen-1-yl]-Benzoic acid (TTNPB) (Strickland, S. et al., Cancer Res. 43: Examples include compounds described in 5268-5272 (1983), Tanenaga, K. et al., Cancer Res. 40: 914-919 (1980), retinyl palmitate, retinol, retinal, 3-dehydroretinol, 3-dehydroretinal, etc. The concentration of retinoic acid or its derivative used in step (i) is, for example, 1 to 100 nM, preferably 5 to 50 nM, more preferably 5 to 25 nM.

[0043] There are no particular restrictions on the seeding density of pluripotent stem cells, but for example, when using a 24-well plate, 10 ３ ~10 ５ Cells / well, preferably 5.0 × 10 ３ ~5.0 x 10 ４ / well, more preferably about 6.0 x 10 ３ Examples include cells / wells. The culture in step (i) can be carried out for 1 to 2 days, preferably 1 day, at a temperature of about 30 to 40°C, preferably about 37°C, in an atmosphere with a CO2 concentration of about 2 to 5%, preferably about 5%.

[0044] In step (ii), the primitive streak of the mesoderm lineage is induced from the late posterior epiblast. The cell population obtained in step (i) may be harvested and re-seeded in a separately prepared adhesive culture vessel, or the cells obtained in step (i) may be cultured as is by changing the culture medium. The culture medium used in step (ii) can be prepared by adding FGF2, a GSK-3β inhibitor, and BMP7 to a basic medium. The same basic medium as in step (i) can be used. The above-mentioned additives may be included as needed.

[0045] The same FGF2 used in step (i) can be used, and its preferred concentration range is also the same.

[0046] The GSK-3β inhibitor used in step (i) can be the same as that used in step (i), and is preferably CHIR99021. The concentration used can be appropriately selected depending on the type of GSK-3β inhibitor, but is, for example, 0.01 to 100 μM, preferably 0.1 to 10 μM, more preferably 1 to 7.5 μM, and particularly preferably 2 to 5 μM. The concentration of the GSK-3β inhibitor used in step (ii) is preferably higher than the concentration used in step (i).

[0047] While there are no particular restrictions on the BMP7 used, as long as it is derived from a mammal, human BMP7 is preferred. Examples of human BMP7 include proteins having an amino acid sequence registered in the NCBI database with accession number NM_001719. BMP7 can be purified from mammalian cells by methods known to the public, or it can be produced by genetic recombination. BMP7 is also commercially available. The concentration of BMP7 used in step (ii) is, for example, 0.1 to 100 ng / ml, preferably 0.5 to 50 ng / ml, and more preferably 0.5 to 5 ng / ml.

[0048] The culture in step (ii) can be carried out for 10 hours to 2 days, preferably 0.5 to 1 day, at a temperature of approximately 30 to 40°C, preferably approximately 37°C, and in an atmosphere with a CO2 concentration of approximately 2 to 5%, preferably approximately 5%.

[0049] In step (iii), the late primitive streak of the mesoderm lineage is induced from the primitive streak of the mesoderm lineage. The cell population obtained in step (ii) may be collected and re-seed in a separately prepared adhesive culture vessel, or the cells obtained in step (ii) may be cultured as is by changing the culture medium. The culture medium used in step (iii) can be prepared by adding FGF2, GSK-3β inhibitor, BMP7, and TGFβ inhibitor to a basic medium. The same basic medium as in step (i) can be used. The above-mentioned additives may be included as needed.

[0050] FGF2, GSK-3β inhibitor, and BMP7 can be the same as in step (ii), and their preferred concentration ranges are also the same.

[0051] In this specification, "TGFβ inhibitor" means a substance that inhibits the signal transduction that follows the binding of TGFβ to its receptor to SMAD, and includes substances that inhibit binding to the ALK family, which are receptors, and substances that inhibit the phosphorylation of SMAD by the ALK family. Examples include Lefty-1 (NCBI Accession No. NM_010094 for mouse, NM_020997 for human), SB431542, SB202190 (from RK Lindemann et al., Mol. Cancer, 2003, 2:20), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276 (Lilly Research Laboratories), A83-01 (WO 2009 / 146408), and their derivatives. The TGFβ inhibitor may preferably be A83-01. The concentration of the TGFβ inhibitor used in step (iii) is, for example, 0.5 to 100 μM, preferably 1 to 50 μM, and more preferably 5 to 25 μM.

[0052] The culture in step (iii) can be carried out for 1 to 3 days, preferably 1.5 to 2 days, at a temperature of approximately 30 to 40°C, preferably approximately 37°C, and in an atmosphere with a CO2 concentration of approximately 2 to 5%, preferably approximately 5%.

[0053] In step (iv), the late primitive streak of the metanephrine lineage is induced from the late primitive streak of the mesoderm lineage. The cell population obtained in step (iii) may be collected and re-seeded in a separately prepared adhesive culture vessel, or the cells obtained in step (iii) may be cultured as is by changing the culture medium. The culture medium used in step (iv) can be prepared by adding FGF2, a GSK-3β inhibitor, BMP7, activin, and a ROCK inhibitor to a basic medium. The same basic medium as in step (i) can be used. The above-mentioned additives may be included as needed.

[0054] FGF2, GSK-3β inhibitor, and BMP7 can be the same as in step (ii), and their preferred concentration ranges are also the same.

[0055] As for the activin, there are no particular restrictions as long as it is derived from a mammal, but human activin is preferred. The activin may be activin A, activin B, or activin AB. Preferably, it is activin A. As for the activin, commercially available products from companies such as R&D Systems can be used. The concentration of the activin used in step (iv) is, for example, 1 to 100 ng / ml, preferably 5 to 50 ng / ml, and more preferably 5 to 25 ng / ml.

[0056] Examples of ROCK inhibitors include Y-27632 (Ishizaki et al., Mol. Pharmacol. 57, 976-983 (2000); Narumiya et al., Methods Enzymol. 325, 273-284 (2000)), Fasudil / HA1077 (Uenata et al., Nature 389: 990-994 (1997)), H-1152 (Sasaki et al., Pharmacol. Ther. 93: 225-232 (2002)), Wf-536 (Nakajima et al., Cancer Chemother Pharmacol. 52(4): 319-324 (2003)) and their derivatives, as well as antisense nucleic acids against ROCK, RNA interference-inducible nucleic acids (e.g., siRNA), dominant-negative mutants, and their expression vectors. In addition, other known small molecule compounds can be used as ROCK inhibitors (e.g., US 2005 / 0209261, US 2005 / 0192304, US 2004 / 0014755, US 2004 / 0002508, US 2004 / 0002507, US 2003 / 0125344, US 2003 / 0087919, WO 2003 / 062227, WO 2003 / 059913, WO 2003 / 062225, WO 2002 / 076976, WO 2004 / 039796). One or more ROCK inhibitors may be used. A preferred ROCK inhibitor is Y-27632. The concentration of the ROCK inhibitor used in step (iv) can be appropriately selected depending on the type of ROCK inhibitor, but for example, it is 0.1 to 100 μM, preferably 1 to 75 μM, and more preferably 5 to 50 μM.

[0057] The culture in step (iv) can be carried out for 1 to 5 days, preferably 3 days, at, for example, about 30 to 40°C, preferably about 37°C, in an atmosphere with a CO2 concentration of about 2 to 5%, preferably about 5%. During this step, the cells can be dissociated and collected, and re-seed into a culture vessel coated with Synthemax II (Corning) (for example, in the case of a 24-well plate, about 2 × 10⁶ cells). ５ Cells / wells can be formed.

[0058] In step (v), the late posterior intermediate mesoderm is induced from the late primitive streak of the metanephrine lineage. The cell population obtained in step (iv) may be harvested and re-seeded in a separately prepared adhesive culture vessel, or the cells obtained in step (iv) may be cultured as is by changing the culture medium. The culture medium used in step (v) can be prepared by adding retinoic acid or its derivatives and FGF9 to a basic medium. The same basic medium as in step (i) can be used. The above-mentioned additives may be included as needed.

[0059] As retinoic acid or its derivatives, the same as in step (i) can be used, and the preferred concentration range is also the same.

[0060] While there are no particular restrictions on the FGF9 used, as long as it is derived from a mammal, human FGF9 is preferred. Examples of human FGF9 include proteins having an amino acid sequence registered in the NCBI database with accession number NP_002001. FGF9 can be purified from mammalian cells by methods known to the public, or it can be produced by genetic recombination. FGF9 is also commercially available. The concentration of FGF9 used in step (v) is, for example, 1 to 500 ng / ml, preferably 1 to 100 ng / ml, more preferably 5 to 50 ng / ml, and particularly preferably 5 to 25 ng / ml.

[0061] The culture in step (v) can be carried out for 1 to 3 days, preferably 2 days, at a temperature of approximately 30 to 40°C, preferably approximately 37°C, and in an atmosphere with a CO2 concentration of approximately 2 to 5%, preferably approximately 5%.

[0062] In step (vi), nephron progenitor cells are induced from the late posterior intermediate mesoderm. The cell population obtained in step (v) may be harvested and re-seeded in a separately prepared adhesive culture vessel, or the cells obtained in step (v) may be cultured as is by changing the culture medium. The culture medium used in step (vi) can be prepared by adding a GSK-3β inhibitor and FGF9 to a basic medium. The same basic medium as in step (i) can be used. The above-mentioned additives may be included as needed.

[0063] As the GSK-3β inhibitor, the same one used in step (i) can be used, preferably CHIR99021. The concentration range is also the same as in step (i).

[0064] The same FGF9 used in step (v) can be used, and its concentration range is also the same as in step (v).

[0065] The culture in step (vi) can be carried out for 1 to 5 days, preferably 2 days, at a temperature of approximately 30 to 40°C, preferably approximately 37°C, and in an atmosphere with a CO2 concentration of approximately 2 to 5%, preferably approximately 5%.

[0066] In another preferred embodiment, nephron progenitor cells can be induced from pluripotent stem cells using the method described in WO 2022 / 149616 or a modified version thereof. Typically, such a method (hereinafter sometimes referred to as "iNPC induction method (2)") includes the following steps: (i-2) A step of culturing pluripotent stem cells in the presence of FGF2, BMP4, a GSK-3β inhibitor, and retinoic acid or a derivative thereof. (ii-2) A step of culturing the cells obtained in (i-2) in the presence of FGF2, a GSK-3β inhibitor, and BMP7. (iii-2) A step of culturing the cells obtained in (iii-2) in the presence of a GSK-3β inhibitor, BMP7, and a TGFβ inhibitor, and in the absence of FGF2. (iv-2) A step of culturing the cells obtained in (iii-2) in the presence of FGF2, a GSK-3β inhibitor, activin, and a ROCK inhibitor. (v-2) A step of culturing the cells obtained in (iv-2) in the presence of retinoic acid or a derivative thereof and in the absence of FGF9. (vi-2) A step of culturing the cells obtained in (v-2) in the presence of a GSK-3β inhibitor and FGF9 and / or FGF20.

[0067] Step (i-2) of the iNPC induction method (2) can be carried out in the same manner as step (i) of the iNPC induction method (1). The culture medium used in this step may further contain a ROCK inhibitor. As the ROCK inhibitor, the same one used in step (iv) of the iNPC induction method (1) can be used, and Y-27632 is a preferred example. The concentration of the ROCK inhibitor used in this step can be appropriately selected depending on the type of ROCK inhibitor, but for example, in the case of Y-27632, it is 0.1 to 100 μM, preferably 1 to 75 μM, and more preferably 5 to 50 μM.

[0068] Step (ii-2) can be carried out in the same manner as step (ii) of the iNPC induction method (1).

[0069] Step (iii-2) can be carried out in the same manner as step (iii) of the iNPC induction method (1), except that a culture medium that does not contain FGF2 is used. Since the late primitive streak of the mesoderm lineage is induced even without using FGF2, this method can be carried out at a lower cost than the iNPC induction method (1) which uses a culture medium containing FGF2 in step (iii).

[0070] Step (iv-2) can be carried out in the same manner as step (iv) of the iNPC induction method (1), except that a culture medium that does not contain BMP7 may be used. Since the late primitive streak of the metanephrine lineage is induced even without using BMP7, this method can be carried out at a lower cost than the iNPC induction method (1) in which a culture medium containing BMP7 is used in step (iv).

[0071] Step (v-2) can be carried out in the same manner as step (v) of the iNPC induction method (1), except that a culture medium that does not contain FGF9 is used. Since late posterior intermediate mesoderm is induced even without using FGF9, this method can be carried out at a lower cost than the iNPC induction method (1) in which a culture medium containing FGF9 is used in step (v).

[0072] The culture medium used in step (v-2) may further contain a BMP inhibitor. Examples of BMP inhibitors include protein-based inhibitors such as Chordin, NOGGIN, and Follistatin, and Dorsomorphin (i.e., 6-[4-(2-piperidin-1-yl-ethoxy)phenyl]-3-pyridin-4-yl-pyrazolo[1,5-a]pyrimidine) and its derivatives (PB Yu et al. (2007), Circulation, 116: II_60; PB Yu et al. (2008), Nat. Chem. Biol., 4: 33-41; J. Hao et al. (2008), PLoS ONE, 3(8): Examples include e2904) and LDN193189 (i.e., 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline). NOGGIN is a preferred BMP inhibitor. The concentration of the BMP inhibitor used in step (v-2) can be appropriately selected depending on the type of BMP inhibitor, but when the BMP inhibitor is NOGGIN, for example, it is 1 to 100 ng / ml, preferably 10 to 50 ng / ml.

[0073] Step (vi-2) can be carried out in the same manner as step (vi) of the iNPC induction method (1). In this step, FGF20 can be added to the culture medium instead of or in addition to FGF9. In vivo, it is known that metanephrotic mesenchymal FGF20 works in cooperation with ureteral bud-derived FGF9 to maintain nephron progenitor cells, and that they act similarly during kidney development, and these factors are mutually interchangeable. There are no particular restrictions on the FGF20 as long as it is derived from mammals, but human FGF20 is preferred. Examples of human FGF20 include proteins having an amino acid sequence registered in the NCBI database with accession number NP_062825. FGF20 can be purified from mammalian cells by methods that are known, or it can be produced by genetic recombination. FGF20 is also commercially available. The concentration of FGF20 used in step (vi-2) is, for example, 1 to 500 ng / ml, preferably 10 to 300 ng / ml, and more preferably 50 to 200 ng / ml. When FGF9 and FGF20 are used in combination, the total concentration of both factors can be set within the above range.

[0074] The culture medium used in step (vi-2) may further contain heparin. Heparin is preferably in the form of a salt, such as salts with alkali metals such as lithium, sodium, and potassium; salts with alkaline earth metals such as calcium, barium, and magnesium; salts with metals such as aluminum, zinc, copper, and iron; ammonium salts; salts with organic bases; and salts with amino acids. Among these, alkali metal salts are preferred, and sodium salts are more preferred. The concentration of heparin used in step (vi-2) is, for example, 0.01 to 100 μg / ml, preferably 0.05 to 50 μg / ml, and more preferably 0.1 to 10 μg / ml.

[0075] Nephron progenitor cells obtained as described above, preferably induced nephron progenitor cells derived from pluripotent stem cells, can be cultured in suspension using, for example, the expansion medium described in WO 2022 / 149616 to produce spherical cell aggregates with a diameter of 150 to 600 μm. That is, the method (hereinafter sometimes referred to as the "spheroid induction method") includes culturing nephron progenitor cells in the presence of a GSK-3β inhibitor, a ROCK inhibitor, and FGF9 and / or FGF20.

[0076] For example, when using nephron progenitor cells obtained by the iNPC induction method (1) or (2) as nephron progenitor cells, the colonies of nephron progenitor cells obtained in step (vi) or (vi-2) are dissociated using, for example, a separation solution having both protease activity and collagenase activity (e.g., Accutase™ and Accumax™ (Innovative Cell Technologies, Inc.)) or a separation solution having only collagenase activity, dispersed into single cells by pipetting or the like, and suspended in the medium used in the spheroid induction method (NPC expansion medium). The NPC expansion medium can be prepared by adding a GSK-3β inhibitor, a ROCK inhibitor, and FGF9 and / or FGF20 to a basic medium. The medium may further contain a JAK inhibitor. Preferably, the NPC expansion medium contains a GSK-3β inhibitor, a ROCK inhibitor, and FGF9.

[0077] Examples of basic media include, but are not limited to, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's Modified Eagle's Medium (DMEM) medium, Ham's F12 (F12) medium, RPMI 1640 medium, Fischer's medium, and mixed media thereof. The medium may contain serum (e.g., FBS) or may be serum-free. If necessary, it may also contain one or more serum substitutes, such as albumin, transferrin, KnockOut Serum Replacement (KSR) (Invitrogen), AS401 (StemFit for Difference, Ajinomoto Healthy Supply Co., Ltd.), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, etc. It may also contain one or more substances such as lipids, amino acids, L-glutamine, GlutaMAX (Invitrogen), non-essential amino acids (NEAA), vitamins, growth factors, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, and their equivalents.

[0078] In one preferred embodiment, the basic medium may be a mixed medium of DMEM / F12 medium (for example, a mixed medium of DMEM and F12 in a 1:1 ratio) to which amino acids, non-essential amino acids, serum substitutes, etc., have been added. In a preferred embodiment, a mixed medium of DMEM / F12 medium to which 10% AS401 has been added can be used. In that case, as the basic medium, for example, a mixed medium of DMEM / F12 medium to which 10% AS401 and GlutaMAX-I (Invitrogen) may be added may be used. DMEM / F12 GlutaMAX (Thermo Fisher Scientific Inc.) can also be used as a medium to which GlutaMAX has been added to a mixed medium of DMEM / F12 medium. Using a culture medium supplemented with 10% AS401 improves the proliferation rate of nephron progenitor cells, the shape of cell aggregates, and the expression of nephron progenitor cell markers (e.g., SIX2, OSR1) and renoprotective factors (e.g., VEGFA).

[0079] The NPC expansion medium contains, in addition to the basic medium, a GSK-3β inhibitor, a ROCK inhibitor, and FGF9 and / or FGF20.

[0080] As the GSK-3β inhibitor, those exemplified in the iNPC induction method can be used in the same way, and CHIR99021 is a preferred example. When the GSK-3β inhibitor is CHIR99021, the concentration of the GSK-3β inhibitor in the culture medium is, for example, 0.01 to 100 μM, preferably 0.1 to 10 μM, more preferably 0.5 to 3 μM, and particularly preferably 0.5 to 1.5 μM.

[0081] As the ROCK inhibitor, those exemplified in the iNPC induction method described above can be used, and Y-27632 is preferred. When the ROCK inhibitor is Y-27632, the concentration of the ROCK inhibitor in the culture medium is, for example, 0.1 to 100 μM, preferably 1 to 75 μM, and more preferably 5 to 50 μM.

[0082] As FGF9 and FGF20, those exemplified in the iNPC induction method can be used in the same way. When either FGF9 or FGF20 is used alone, the concentration of FGF9 and FGF20 in the culture medium is, for example, 1 to 800 ng / ml, preferably 5 to 500 ng / ml, more preferably 10 to 400 ng / ml, and particularly preferably 100 to 300 ng / ml. When FGF9 and FGF20 are used in combination, it is preferable that their total concentration be within the above range.

[0083] The NPC expansion medium may further contain a JAK inhibitor. A JAK inhibitor is a substance that inhibits the activity of one or more enzymes of the Janus kinase family (e.g., JAK1, JAK2, JAK3, TYK2). By inhibiting the enzymatic activity of the Janus kinase family, JAK inhibitors inhibit the signaling pathway of the JAK-STAT system. The inclusion of a JAK inhibitor further promotes the proliferation of nephron progenitor cells. The JAK inhibitor is not particularly limited as long as it can inhibit the activity of enzymes of the Janus kinase family, but it is preferable that it inhibits the enzymatic activity of JAK2 or JAK3, and more preferably that it inhibits JAK2. Preferred examples of JAK inhibitors are described in WO 2022 / 149616.

[0084] The culture vessel used in the spheroid induction method is not particularly limited as long as it is non-adhesive or low-adhesive and suitable for suspension culture, as long as it can produce spherical cell aggregates with a diameter of 150 to 600 μm. In a preferred embodiment, however, a culture vessel can be used that has a non-adhesive or low-adhesive culture surface as its inner bottom surface, wherein a plurality of identical recesses (microwells) are densely arranged adjacent to each other on the culture surface, and each recess has an inner wall surface that is a funnel-shaped slope and a bottom surface that is a concave curved surface smoothly connected to the inner wall surface. Here, "identical to each other" means that the size (opening diameter, depth, etc.) and shape of the plurality of recesses are similar to each other to the extent that when cells are seeded in each recess at the same density, the spherical cell aggregates formed in each recess will have a uniform size and number of cells. They do not need to be strictly identical. In the present invention, when the plurality of recesses are said to be "the same size" or "the same shape," it means that their size or shape is "identical" in the same sense as above. "Densely arranged" means a state in which the recesses are arranged as close together as possible and adjacent to each other, such as in a square matrix arrangement or a dense (honeycomb) arrangement, so that the total opening area of ​​the recesses occupies a larger proportion of the culture surface (i.e., the proportion occupied by the partitions between recesses occupies a smaller proportion). "Funnel-shaped slope" refers to a slope such as the inner slope of a mortar, where the opening diameter decreases from the opening towards the bottom. "Concave curved surface" refers to a curved surface that is concave in a way that can promote cell aggregation, such as a hemispherical concave surface or a parabolic concave surface.

[0085] Examples of culture vessel shapes include, but are not limited to, dishes (e.g., 10 mm, 35 mm, 100 mm, etc.) and multi-well plates (e.g., 6-well, 96-well, etc.). Examples of culture vessel materials include plastics and polymer materials such as polystyrene. The dimensions of the microwells are such that the opening diameter is about 200 to 2000 μm and the depth (length from the opening to the bottom) is about 100 to 900 μm, but it is desirable to have an opening diameter and depth such that the diameter of the spherical cell aggregate after suspension culture for 24 to 60 hours is 150 to 600 μm, preferably 200 to 500 μm, and more preferably 300 to 400 μm.

[0086] The culture surface of the culture vessel is non-adhesive or low-adhesive so that cells do not adhere to the culture surface. Such a culture surface can be formed by surface treatment using a cell adhesion inhibitor (low-protein adhesive). Examples of cell adhesion inhibitors include phospholipid polymers (such as 2-methacryloyloxyethyl phosphorylcholine), polyhydroxyethyl methacrylate, fluorine-containing compounds, or polyethylene glycol. In addition to inhibiting cell adhesion with a coating layer, the culture vessel may also be molded with a resin that has a cell adhesion inhibitory effect, such as silicone resin.

[0087] Because the culture surface is non-adherent or has low adhesion, and the microwells have a funnel-shaped sloping inner wall and a smoothly connected concave curved bottom surface, nephron precursor cells seeded as single cells can settle evenly into each microwell, and without adhering to the bottom surface, adjacent cells can bind together to form uniformly sized spherical cell aggregates in a short time. Preferably, the inner walls of adjacent recesses are connected so as to be smooth and continuous with each other, so that there are no flat surfaces between adjacent recesses. As a result, most of the seeded cells settle into the wells, and spherical cell aggregates can be formed without cell loss.

[0088] The culture vessel can be manufactured, for example, by the method described in WO 2017 / 047735. Alternatively, commercially available culture vessels can be used (e.g., SPERICALPLATE 5D from KUGELMEIERS, EZSPHERE® from AGC Technoglass, AggreWell from StemCell Technologies). ＴＭ You can also use Elplasia (registered trademark) manufactured by Kuraray Co., Ltd.

[0089] The density of nephron progenitor cells seeded in the culture vessel should be such that the number of cells per microwell is 5,000 to 15,000, preferably 7,000 to 12,000, and more preferably about 10,000. For example, when using SPERICALPLATE 5D, there are about 700 microwells per well, so for example, about 3.5 × 10⁶ cells per well. ６ ~1 x 10 ７ Cells, preferably 5 × 10 ６ ~8.5 x 10 ６ Cells, more preferably about 7 × 10 ６ Cells can be seeded.

[0090] In the spheroid induction method, the culture is carried out, for example, at about 30-40°C, preferably about 37°C, in an atmosphere with a CO2 concentration of about 2-5%, preferably about 5%. The culture period is not particularly limited as long as spherical cell aggregates with a diameter of 150-600 μm are obtained, but for example, it is 30-60 hours, preferably 40-55 hours, more preferably about 48 hours.

[0091] The obtained spherical cell aggregates (small cell aggregates) are collected, suspended in NPC expansion medium, and seeded in a non-adhesive or low-adhesive culture vessel (e.g., a 24-well plate). The seeding density is not particularly limited as long as adjacent small cell aggregates fuse together approximately 24 hours after seeding. For example, when using a 24-well plate, seeding can be done at a density of 500 cells / well or more, preferably 600 to 800 cells / well, and more preferably about 700 cells / well.

[0092] In the method for producing giant cell aggregates of the present invention, the culture is carried out by suspension culture at, for example, about 30 to 40°C, preferably about 37°C, in an atmosphere with a CO2 concentration of about 2 to 5%, preferably about 5%. The culture period is not particularly limited as long as giant cell aggregates with a minimum diameter of 0.8 mm or more are obtained, but for example, it is 24 hours or more, preferably 30 to 60 hours, more preferably 40 to 55 hours, and particularly preferably about 48 hours.

[0093] The resulting giant cell aggregates can be maintained in NPC expanded medium, for example, by changing the culture medium every 1 to 3 days, preferably every 2 days. The duration of maintenance culture is not particularly limited, as long as the giant cell aggregates do not experience an increased frequency of apoptosis and / or cytotoxicity throughout the culture period and / or secrete renal protective factors to a degree that provides a therapeutic effect against kidney diseases (e.g., CKD, AKI), but may be, for example, 1 day or more, preferably 2 days or more, more preferably 3 days or more, and even more preferably 4 days or more. Alternatively, the duration of maintenance culture may be 1 to 14 days, preferably 2 to 14 days, more preferably 3 to 12 days, and even more preferably 4 to 10 days.

[0094] The nephron progenitor cells of the giant cell aggregate of the present invention retain the ability to differentiate into cells of the renal lineage, such as glomeruli and tubules; therefore, renal organoids can be produced by differentiating the giant cell aggregate. Differentiation into renal organoids can be carried out by known methods. For example, the method described in Nature, 526, 564-568 (2015) or a similar method can be used. For example, the giant cell aggregate of the present invention can be co-cultured with feeder cells such as 3T3-Wnt4 cells, mouse fetal spinal cord cells, or mouse fetal kidney cells. Alternatively, the giant cell aggregate may be cultured in a semi-gastric culture (preferably a gas-liquid interface culture; see Nature, 526, 564-568 (2015)) using a basic medium containing a GSK-3β inhibitor. The medium used in the semi-gastric culture may contain FGF9 and FGF2 in addition to the GSK-3β inhibitor. Examples of the basic culture medium, GSK-3β inhibitor, and FGF2 are the same as those described above. A preferred GSK-3β inhibitor is CHIR99021. In the above, FGF9 may be substituted with FGF20. Alternatively, FGF9 and FGF20 may be used in combination.

[0095] The culture in the differentiation process is carried out at, for example, 30-40°C, preferably about 37°C, CO2 ２ The culture can be carried out in an atmosphere with a concentration of 2-5%, preferably about 5%. The culture period is not particularly limited, as long as it is long enough for kidney organoids to form.

[0096] 2. The present invention provides a method for producing a nephron progenitor cell conditioned medium, comprising the following steps: (1) a step of suspension culturing 500 or more cell aggregates of nephron progenitor cells having a diameter of 150 to 600 μm in the same culture vessel to form a giant cell aggregate by fusing the cell aggregates; and (2) a step of culturing the giant cell aggregate obtained in (1) in a medium that maintains and amplifies nephron progenitor cells to obtain a conditioned medium (hereinafter sometimes referred to as "the present invention's method for producing a conditioned medium").

[0097] Step (1) of the method for producing conditioned culture medium of the present invention can be carried out in the same manner as the method for producing large cell aggregates of the present invention.

[0098] Step (2) of the method for producing conditioned medium according to the present invention can be carried out, for example, in the same manner as the maintenance culture of the giant cell aggregate according to the present invention. The NPC expansion medium can be cited as a medium in which nephron progenitor cells are maintained and amplified. That is, in step (1), when small cell aggregates are cultured in suspension in the same culture vessel using NPC expansion medium, the small cell aggregates fuse together to form a giant cell aggregate after about 24 hours, and by continuing the culture, renoprotective factors are secreted into the medium. After that, only the culture supernatant is collected to obtain conditioned medium. Preferably, the collected culture supernatant is passed through a filter to remove suspended matter so as not to include cells. Fresh NPC expansion medium is added to the remaining giant cell aggregate, and the culture is continued. By repeating the above operation when changing the medium during maintenance culture, conditioned medium can be obtained multiple times. The conditioned medium collected multiple times may be mixed and pooled.

[0099] The resulting conditioned medium contains renal protective factors secreted by the giant cell aggregates, thereby exhibiting renal protective effects and therapeutic effects against kidney diseases (e.g., CKD, AKI, etc.). Examples of renal protective factors include one or more proteins selected from those present in the conditioned medium for the giant cell aggregates of the present invention, but not in the conditioned medium for the small cell aggregates (150-600 μm in diameter) or the medium-sized spherical cell aggregates (less than 800 μm in diameter) obtained by further maintenance culture of the small cell aggregates. Here, "not present" means that the protein is below the detection limit by mass spectrometry. Examples of secreted factors specific to the conditioned medium of the present invention include the proteins listed in Table 2 below.

[0100] The conditioned medium can be cryopreserved and exhibits renal protective effects even after freezing and thawing. The cryopreservation method for the conditioned medium is not particularly limited, and known methods can be used. The cryopreserved conditioned medium can be stored at -80°C or below, for example, using a deep freezer. Accordingly, the present invention also provides a cryopreserved conditioned medium for large cell aggregates of the present invention. The cryopreserved conditioned medium can be thawed by conventional methods and formulated as a pharmaceutical composition.

[0101] 3. Renal Protective Factors The present invention also provides renal protective factors contained in the conditioned medium of the present invention. Examples of renal protective factors of the present invention include one or more proteins selected from the group of proteins listed in Table 2. In one preferred embodiment, the renal protective factor is corticotropin-releasing factor-binding protein (CRHBP), SCUBE3 (Signal peptide, CUB and EGF-like domain-containing protein 3), SRPX2 (Sushi repeat-containing protein X-linked) 2) The protein may be selected from the group consisting of complement C7 (C7), carboxypeptidase Q (CPQ), progonadriverin-1 (GNRH1), laminin subunit α2 (LAMA2), mucin-5AC (MUC5AC), neuroserpin (SERPINI1), thyrotropin-releasing hormone (TRH), angiogenin (ANG), endothelial cell-specific molecule 1 (ESM1), plasminogen activator inhibitor 2 (SERPINB2), and vascular endothelial growth factor D (VEGFD), or a combination of two or more of these. Preferably, the renal protective factor may be a protein selected from the group consisting of CRHBP, SCUBE3, SRPX2, C7, CPQ, GNRH1, LAMA2, MUC5AC, SERPINI1, and TRH, more preferably CRHBP, SCUBE3, SRPX2, C7, CPQ, and MUC5AC, or a combination of two or more of these. Also preferably, the renal protective factor may be CRHBP and / or SCUBE3 and / or SRPX2.

[0102] The amino acid sequences of the above-mentioned proteins and the coding sequences of their genes are both publicly known, and those skilled in the art can isolate and purify these proteins from nature or produce them by genetic engineering based on this sequence information.

[0103] The aforementioned renal protective factors can also be used in combination with other renal protective factors, such as vascular endothelial growth factor A (VEGFA).

[0104] In one embodiment, the renal protective factor may be provided as a source for cell transplantation therapy for kidney disease in the form of cells that exogenously secrete and express the nucleic acid encoding the factor. Preferred such cells are nephron progenitor cells, particularly induced nephron progenitor cells derived from pluripotent stem cells. For example, cells that stably secrete and express the factor (recombinant or gene-edited cells) can be produced by introducing the nucleic acid encoding the renal protective factor into pluripotent stem cells using a conventional method, and then differentiate these cells into nephron progenitor cells using one of the differentiation induction methods described above to obtain cells that highly secrete and express the desired renal protective factor.

[0105] 4. Pharmaceutical Compositions The present invention also provides pharmaceutical compositions containing the conditioned medium of the present invention or a renal protective factor contained in the conditioned medium. The conditioned medium or renal protective factor of the present invention can be formulated as a pharmaceutical composition as is or together with a pharmacologically acceptable carrier, diluent or excipient. The pharmaceutical composition is provided in a dosage form suitable for oral or parenteral administration, preferably parenteral administration. If the renal protective factor is provided in the form of cells that exogenously secrete and express the nucleic acid encoding the factor, it can be formulated in the same manner as described later for "pharmaceutical compositions containing a giant cell aggregate or a renal organoid containing the giant cell aggregate as a source of nephron progenitor cells of the present invention."

[0106] For parenteral administration, for example, injectable preparations, suppositories, etc., can be used, and injectable preparations may include dosage forms such as intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, intraperitoneal injection, and drip infusion injection. Such injectable preparations can be prepared according to known methods. As a method for preparing injectable preparations, for example, the conditioned medium or renoprotective factor of the present invention can be dissolved, suspended, or emulsified in a sterile aqueous solution or oily solution commonly used for injectable preparations. As aqueous solutions for injection, for example, physiological saline, isotonic solutions containing glucose or other adjuvants can be used, and may be used in combination with appropriate solubilizers, such as alcohol (e.g., ethanol), polyalcohol (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduc of hydrogenated castor oil)]. As oily solutions, for example, sesame oil, soybean oil, etc., can be used, and may be used in combination with solubilizers such as benzyl benzoate or benzyl alcohol. The prepared injection solution is preferably filled into a suitable ampoule. Suppositories used for rectal administration may be prepared by mixing the conditioned medium or renal protective factor of the present invention with a conventional suppository base.

[0107] The parenteral or oral pharmaceutical compositions described above are preferably prepared in dosage forms of drug units that are suitable for the dosage of the active ingredient. Examples of such dosage forms of drug units include tablets, pills, capsules, injections (ampoules), and suppositories. The renal protective factor secreted from the conditioned medium of the present invention is preferably contained in an amount of 0.1 to 500 mg per drug unit dosage form, particularly 5 to 100 mg for injections and 10 to 250 mg for other dosage forms.

[0108] The dosage of the above pharmaceutical composition varies depending on the target patient, target disease, symptoms, and route of administration. However, when the conditioned medium of the present invention is used as the active ingredient, for example, 0.1 to 100 mL, preferably 1 to 50 mL, of the conditioned medium is used per dose. When a renal protective factor is used as the active ingredient, for example, a single dose of approximately 0.0001 to 20 mg / kg body weight can be administered parenterally 1 to 5 times a day. Systemic administration such as intravenous, subcutaneous, intradermal, intramuscular, or intraperitoneal administration is preferred. Alternatively, local injection into the renal parenchyma or implantation of a sustained-release formulation containing the renal protective factor in a hydrogel or the like under the renal capsule is also possible. In cases of severe symptoms, the dosage may be increased according to the symptoms.

[0109] The pharmaceutical compositions containing the conditioned medium or renal protective factor of the present invention are intended for use in kidney diseases, such as CKD, AKI, and chronic kidney disease that does not progress to CKD. They can also be used as protective agents for transplanted kidneys in kidney transplantation.

[0110] The present invention also provides a pharmaceutical composition containing a giant cell aggregate or a renal organoid containing the giant cell aggregate as a source of nephron progenitor cells. The pharmaceutical composition may be in a known form suitable for parenteral administration, such as an injection or infusion. The pharmaceutical composition may optionally contain pharmacologically acceptable excipients. The pharmaceutical composition may also contain physiological saline, phosphate-buffered saline (PBS), culture medium, etc., to maintain the cells stably. Furthermore, the pharmaceutical composition may contain pharmaceutically acceptable carriers (e.g., human serum albumin), preservatives, etc., for stabilization purposes.

[0111] The target animal (preferably a human) should ideally be an individual whose HLA genotype is substantially identical to that of the cells constituting the giant cell mass or renal organoid. Here, "substantially identical" means that the HLA genotype matches to the extent that the immune response to the transplanted cells can be suppressed by an immunosuppressant. For example, it is desirable that the treatment be administered to a subject with an HLA type in which three gene loci (HLA-A, HLA-B, and HLA-DR) or four gene loci (including HLA-C) match.

[0112] Methods for administering the above-mentioned pharmaceutical composition include, for example, forming the giant cell aggregate or renal organoid of the present invention into a sheet and attaching it to the target kidney, or transplanting a suspension of the giant cell aggregate or renal organoid of the present invention in physiological saline or the like into the target kidney. The transplantation site is not particularly limited as long as it is within the kidney, but is preferably subcapsular. Nephron progenitor cells transplanted subcapsularly do not migrate to the renal parenchyma and act paracrinely (or, nephron progenitor cells transplanted to one kidney act endocrynically on the other kidney) via secreted renal protective factors, so administration to the renal parenchyma is not necessarily required. Furthermore, the renal organoids to the target are not limited to the kidney, but can be ectopically transplanted into the target's body (for example, intraperitoneal).

[0113] The pharmaceutical composition containing the giant cell aggregate or renal organoid of the present invention is intended for use in kidney diseases, such as CKD, AKI, and chronic renal impairment that does not progress to CKD. It can also be used as a protective agent for transplanted kidneys in kidney transplantation.

[0114] The giant cell aggregates of the present invention, and the renal organoids that use them as a source of nephron progenitor cells, are useful not only as a source for cell transplantation therapy but also as material for renal reconstruction (kidney regeneration). Furthermore, these giant cell aggregates or renal organoids can be induced from iPS cells specific to kidney disease and used in the search for therapeutic agents for said disease, or they can be used as a tool to evaluate the cytotoxicity of renal disease therapeutic agents.

[0115] The present invention will be described in more detail below with reference to examples, but these are merely illustrative and the present invention is not limited to these examples.

[0116] Example 1 <Method> (1) Differentiation induction from human iPS cells to nephron progenitor cells OSR1-GFP / SIX2-tdTomato double knock-in human iPS cell (hiPSC) strain 4A6C3-10 (Stem Cells Transl Med 4, 980-992 (2015)) was maintained feeder-free on a 6-well cell culture plate (CellBIND #3335, Corning Corporation) coated with iMatrix-511 silk (Nippi Corporation) using Stem Fit AK02 N medium (Ajinomoto Co., Inc.). Cells were treated with 0.5 mM EDTA / PBS (Thermo Fischer Scientific) at 37°C for 4 minutes every 4–6 days, then gently pipettered and subcultured. Periodically, the cells were tested for mycoplasma contamination. Optimized seeding density (6 × 10⁻⁶) ３ hiPSCs (cells / well) were maintained for 2 days in a 24-well cell culture plate coated with iMatrix (CellBIND #3337, Corning), and then differentiated into nephron progenitor cells (NPCs) and kidney organoids by changing the concentration of CHIR99021 from 3 μM to 5 μM, as previously reported (Cell Rep 31, 107476 (2020)). However, during the culture corresponding to step (iv) of the iNPC induction method, 48 hours after the start (Day 5), the cells were detached from the plate using accumax and placed in a 24-well plate coated with Synthemax® II (Corning) for 24 hours, with 2 × 10⁶ cells added. ５ The cells were reseeded to achieve a cell / well ratio. Synthemax® II showed stronger cell adhesion than iMatrix.

[0117] (2) Induction of large cell aggregates of hiPSC-NPC and preparation of acclimatization medium After removing the culture supernatant from the NPCs induced in the 24-well plate in (1) above, Accumax was added at a rate of 100 μL / well and the mixture was left to stand in a 37°C incubator for 20 minutes. After adding 900 μL of 10% FBS, the cells were gently pipettered to detach them from the plate and separated into single cells, and the number of cells was measured. Next, the mixture was centrifuged at 300 g for 3 minutes and the supernatant was removed. The iNPC pellet was placed in one well (24-well format) of a spheroid preparation plate SPERICALPLATE 5D (KUGELMEIERS, Switzerland) with iNPC 7 × 10 ６ To ensure the cells could be contained, they were suspended in 1 mL of CFY medium (CHIR99021 1 μM, FGF9 200 ng / mL, Y-23647 10 μM) and seeded (Day 0). Suspension culture was then performed in a 37°C incubator (the SPERICALPLATE 5D has 700 microwells, so 1 × 10⁶ microwells were used per microwell). ４ Cells were seeded. 48 hours after cell seeding (Day 2), approximately 700 spherical small cell aggregates with a diameter of approximately 300-400 μm were formed. These small cell aggregates, along with the culture supernatant, were transferred to a 1.5 mL tube and centrifuged at 300 g for 3 minutes. After removing the supernatant, 1 mL of fresh CFY medium was added, and the small cell aggregate suspension was seeded into one well of a low-adhesion 24-well plate, followed by suspension culture in a 37°C incubator. After 48 hours of suspension culture (Day 4), the small cell aggregates had fused to form a giant cell aggregate. Care was taken not to remove the giant cell aggregate, and only the culture supernatant was collected, filtered through a 40 μm filter, and stored at -80°C. 1 mL of fresh CFY medium was added to the giant cell aggregate from which the supernatant had been removed, and suspension culture was again performed in a 37°C incubator. The procedure of collecting the culture supernatant and replacing it with fresh medium was repeated on days 6, 8, 10, and 12. Approximately 4-5 mL of conditioned medium was obtained by collecting the supernatant on day 12.

[0118] (3) Preparation of control acclimatization medium For comparison, the iNPC pellets that were separated into single cells in (2) above, centrifuged, and the supernatant removed were placed in one well of a low-adhesion 96-well plate, 2 × 10５ To ensure the cells are present, they were suspended in 50 μL of CFY medium and then seeded (35 wells, total 7 × 10). ６ Cells were cultured in suspension in a 37°C incubator. 48 hours after cell seeding (Day 2), one cell cluster approximately 500-600 μm in diameter was formed in each well. The culture supernatant was removed, 100 μL of fresh CFY medium was added, and suspension culture was continued in a 37°C incubator. Following the same procedure as in (2) above, the culture supernatant was collected on Days 4, 6, 8, 10, and 12, passed through a 40 μm filter, and the combined culture supernatants from all 35 wells were stored at -80°C to obtain acclimatized medium.

[0119] (4) Immunostaining Immunostaining of hiPSC-NPC giant cell aggregates for NPC markers SIX2, PAX2, SALL1, WT1 and the proliferation marker Ki67, as well as immunostaining of renal organoids derived from hiPSC-NPC giant cell aggregates for various differentiation markers of the renal lineage, was performed using the antibodies shown in Table 1.

[0120]

[0121] (5) LDH Leakage Assay The cytotoxicity induced by long-term culture of giant cell aggregates was evaluated by the leakage of lactate dehydrogenase (LDH) into the culture medium. The LDH activity in the conditioned medium obtained from giant cell aggregates was measured using a commercially available assay kit (Cytotoxicity LDH Assay Kit #347-91751, Dojindo Molecular Technologies, Inc.). The conditioned medium and reagents were mixed in a 96-well plate (Falcon), and the absorbance was recorded using an EnVision 2104 Multilabel Reader (PerkinElmer). The results were analyzed using EnVision Manager (version 1.14.3049.528).

[0122] (6) Cisplatin-induced acute kidney injury (AKI) model mouse C57B6 / J mice (purchased from Shimizu Laboratory Materials Co., Ltd.) were intraperitoneally administered 16 mg / kg of cisplatin, and 24 hours later, 500 μL of acclimatization medium was administered intraperitoneally every 8 hours for 3 consecutive days. CFY medium was used as a control. Blood and kidney tissue samples were collected 4 days after cisplatin administration. All mice were housed in the experimental animal housing facility of the Center for iPS Cell Research and Application (CiRA) at Kyoto University under specific pathogen-free (SPF) conditions. The room temperature (21-25°C), humidity (45-55%), and light (12-hour light-dark cycle) were controlled, and the mice had free access to food and water.

[0123] (7) Differential mass spectrometry Differential mass spectrometry was performed on the proteins contained in the acclimatization medium for the large cell aggregates prepared in (2) and the control acclimatization medium prepared in (3). Each acclimatization medium was collected as a sample, and an equal volume of 2×PTS buffer containing a protease inhibitor mixture (Cat#P8340, Sigma-Aldrich), 24 mM sodium deoxycholate (SDC, #190-08313), 24 mM sodium lauroyl sarcosinate (SLS, #192-10382), and 200 mM Tris-HCl (pH 9.0) was added to prepare the protein sample. These protein samples were treated with diaPASEF (PMID: 33257825, Meier et al., 2020) and DIA-NN (PMID: 31768060, Demichev et al., 2020), as in a previously reported study (PMID: 38996472), and analyzed using a Bruker timsTOFpro2 system.

[0124] <Results> In preliminary experiments in which hiPSC-NPCs were transplanted subcapsulate of AKI and CKD mice, the inventors found that the transplanted hiPSC-NPCs engrafted for a long period of time while expressing NPC markers, but migration to the host renal parenchyma and integration were not observed (data not shown). Furthermore, since therapeutic effects were obtained in both kidneys of mice in which hiPSC-NPCs were transplanted subcapsulate of one kidney, it was considered possible that factors secreted by hiPSC-NPCs exert renoprotective effects through paracrine and endocrine effects. Therefore, a test was conducted in which 500 μL of conditioned medium taken from a 96-well plate in which hiPSC-NPCs were cultured was administered three times a day to cisplatin-induced AKI mice, but no therapeutic effect was observed (Figure 2A). Next, a giant cell aggregate was created by fusing approximately 700 hiPSC-NPC small cell aggregates with a diameter of 300-400 μm (Figure 1, A and B). These giant cell aggregates expressed NPC markers and proliferation markers and retained the ability to differentiate into kidney organoids even after long-term culture (Figure 1, B-F). However, it was thought that they would induce apoptosis and cytotoxicity due to poor oxygen and nutrient supply. Contrary to expectations, however, these giant cell aggregates did not show a significant increase in the expression of CLEAVED CASPASE 3 (CC3), an indicator of apoptosis, or in the release of LDH, an indicator of cytotoxicity (Figure 1, G and H). Furthermore, when administered to cisplatin-induced AKI mice, the conditioned medium of the giant cell aggregates showed a therapeutic effect, lowering blood urea nitrogen (BUN) and serum creatinine (S-Cre) levels to those of normal mice and improving the survival rate of cisplatin-induced AKI mice (Figure 1, I-L). Moreover, it was confirmed that the therapeutic effect was maintained even when the conditioned medium, frozen at -80°C, was thawed and administered (Figure 2, B-D). These results suggest that renal protective factors play an important role in the therapeutic effect of hiPSC-NPC.

[0125] Identification of secreted proteins specific to giant cell aggregates Mass spectrometry was performed on the conditioned medium of the giant cell aggregates obtained in (2) of the <Methods> section and the control conditioned medium obtained in (3), and the proteins contained in each were identified. 288 types of proteins were identified from the conditioned medium of the giant cell aggregates, and 212 types of proteins were identified from the control conditioned medium. The 112 types of proteins found only in the conditioned medium of the giant cell aggregates are shown in Table 2 (each protein is indicated by the abbreviation of the gene encoding it).

[0126]

[0127] Example 2 Identification of Renal Protective Factors (1) From the proteins shown in Table 2, the gene products of CRHBP, SCUBE3, SRPX2, C7, CPQ, GNRH1, LAMA2, MUC5AC, SERPINI1, TRH, ANG, ESM1, SERPINB2, and VEGFD were purchased as commercially available recombinant proteins and dissolved in phosphate-buffered saline (PBS) at concentrations of 100 ng / mL to 10 μg / mL. These were administered intraperitoneally to cisplatin-induced AKI mice in acclimatization medium according to the method described above. Blood samples were collected 4 days after cisplatin administration, and BUN and S-Cre levels were measured. The cisplatin-induced AKI mice were kept alive after candidate protein administration, and their survival rate was measured. Compared to the PBS-administered group, proteins that improved blood tests and / or survival rate were identified as renal protective factors.

[0128] Example 3 Identification of Renal Protective Factors (2) (i) Evaluation using cisplatin-induced AKI mice C57B6 / J mice were intraperitoneally administered 15 mg / kg of cisplatin, and from 24 hours later, solutions of each test protein (CRHBP, SCUBE3, SRPX2, C7, CPQ, MUC5AC, or SERPINI1) dissolved in 500 μL of PBS at a concentration of 1 or 10 μg / mL were administered intraperitoneally every 8 hours for 3 consecutive days. The group administered only 500 μL of PBS was used as a control. Blood samples were collected 4 days after cisplatin administration, and BUN and S-Cre levels were measured. As a result, the groups administered CRHBP, SCUBE3, SRPX2, C7, CPQ, and MUC5AC showed a tendency for lower BUN and S-Cre levels compared to the control group (Figure 3).

[0129] (ii) Evaluation using aristolochic acid-induced AKI mice Cisplatin-induced AKI mice allow for drug efficacy evaluation in a short period of 4 days and are useful as primary screening, but they die after about 7 days. Therefore, aristolochic acid-induced AKI mice, which do not die and allow for evaluation not only of AKI but also of the transition period from AKI to CKD, were used to further evaluate the effect of SCUBE3, whose therapeutic effect was confirmed in cisplatin-induced AKI mice. C57B6 / J mice were intraperitoneally administered 6 mg / kg of aristolochic acid, and from 24 hours later, a solution of SCUBE3 dissolved in 500 μL of PBS at a concentration of 1 or 10 μg / mL was administered intraperitoneally every 8 hours for 3 consecutive days. Administration of 500 μL of PBS alone was used as a control. Blood samples were collected 4, 7, and 10 days after aristolochic acid administration, and BUN and S-Cre levels were measured. As a result, the SCUBE3 administration group showed a sustained downward trend in BUN and S-Cre levels, not only in the acute phase but also during the transition from AKI to CKD, compared to the control group (Figure 4).

[0130] The conditioned medium for the giant cell aggregates of the present invention has nephroprotective properties and exhibits therapeutic effects against CKD and AKI, making it useful as a therapeutic agent for kidney diseases. Furthermore, the giant cell aggregates themselves can be used in kidney regeneration, cell transplantation therapy, and other applications, making them useful. Moreover, the nephroprotective factors specifically secreted by these giant cell aggregates may be useful as a precursor for novel therapeutic agents for kidney diseases.

[0131] This application is based on Japanese Patent Application No. 2024-187809, filed in Japan on 24 October 2024, the contents of which are incorporated herein by reference in their entirety.

Claims

1. A method for producing a nephron progenitor cell conditioned medium, comprising the following steps: (1) a step of suspension culturing 500 or more cell aggregates of nephron progenitor cells having a diameter of 150 to 600 μm in the same culture vessel to form a giant cell aggregate by fusing the cell aggregates; and (2) a step of culturing the giant cell aggregate obtained in (1) in a medium that maintains and amplifies nephron progenitor cells to obtain a conditioned medium.

2. The method according to claim 1, wherein the culture in step (1) is carried out for 24 hours or more.

3. The method according to claim 1, further comprising the step of forming a cell aggregate with a diameter of 150 to 600 μm from single-cell nephron progenitor cells before step (1).

4. The method according to claim 1, wherein the nephron progenitor cells are differentiated and induced from pluripotent stem cells.

5. The method according to claim 1, wherein the culture medium for maintaining and amplifying nephron progenitor cells comprises a GSK-3β inhibitor, a ROCK inhibitor, and FGF9.

6. The method according to claim 1, wherein the frequency of apoptosis and / or cell injury does not increase throughout the culture period.

7. A nephron progenitor cell acclimatization medium obtained by the method described in claim 1.

8. A conditioned medium for nephron progenitor cells, wherein the nephron progenitor cells are in the form of large cell aggregates with a minimum diameter of 0.8 mm or more, and the conditioned medium has a renal protective effect.

9. A frozen culture medium according to claim 7 or 8.

10. A pharmaceutical composition comprising the conditioned medium described in claim 7 or 8, or the conditioned medium that has been cryopreserved.

11. The pharmaceutical composition according to claim 10, for the treatment of kidney disease.

12. The pharmaceutical composition according to claim 10, which is administered systemically.

13. A giant cell mass of nephron progenitor cells constructed in vitro, with a minimum diameter of 0.8 mm or more.

14. A method for producing a giant cell aggregate of nephron precursor cells, comprising: culturing a group of cell aggregates of nephron precursor cells with a diameter of 150 to 600 μm in suspension in the same culture vessel; and fusing the cell aggregates to form a giant cell aggregate with a minimum diameter of 0.8 mm or more.

15. A large cell mass of nephron progenitor cells obtained by the method described in claim 14.

16. A renal organoid in which a giant cell aggregate of nephron progenitor cells constructed in vitro having a minimum diameter of 0.8 mm or more, or the giant cell aggregate described in claim 15, is used as the source of nephron progenitor cells.

17. A pharmaceutical composition comprising a large cell mass according to claim 13 or 15 or a renal organoid according to claim 16.

18. The pharmaceutical composition according to claim 17, for the treatment of kidney disease.

19. A therapeutic agent for renal disease containing one or more proteins selected from the group consisting of corticotropin-releasing factor-binding protein (CRHBP), SCUBE3 (Signal peptide, CUB and EGF-like domain-containing protein 3), SRPX2 (Sushi repeat-containing protein X-linked 2), complement C7 (C7), carboxypeptidase Q (CPQ), and mucin-5AC (MUC5AC).

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