Kidney organoid and method for producing same

Abstract

Provided are: a chimeric kidney organoid which has a chimeric kidney structure including human cells and cells of a non-human mammal, and in which the non-human mammal is a cetartiodactyl or a non-human primate; and a method for producing chimeric kidney tissue including non-human mammalian embryonic metanephros-derived cells and human nephron progenitor cells or cells derived from human nephron progenitor cells, the method comprising a step (a1) for bringing human nephron progenitor cells and non-human mammalian embryonic metanephros-derived cells into contact with each other to obtain a cell population, and a step (b1) for culturing the cell population obtained in said step (a1) in a chimera-forming culture medium to obtain chimeric kidney tissue, wherein the chimera-forming culture medium is a culture medium containing a Wnt signal activator.

Description

Renal organoids and their production method

[0001] The present invention relates to renal organoids and methods for producing the same. More specifically, the present invention relates to chimeric renal organoids comprising human cells and non-human mammalian cells, methods for producing the same, methods for producing chimeric renal tissue comprising human nephron progenitor cells or cells derived from human nephron progenitor cells and cells derived from non-human mammalian fetal metanephroi, methods for evaluating the ability of human nephron progenitor cells to form chimeric renal structures, porcine renal organoids, and methods for producing renal organoids comprising non-human mammalian cells. This application claims priority based on Japanese Patent Application No. 2024-041743, filed in Japan on March 15, 2024, the contents of which are incorporated herein by reference.

[0002] The kidney is an essential organ for the excretion of waste products, and the nephron is the functional unit responsible for filtration in the kidney. A nephron is composed of the renal corpuscle, which consists of Bowman's capsule and glomerulus, and the renal tubule, which consists of the proximal tubule, the loop of Henle, and the distal tubule.

[0003] During development, the kidney originates from the metanephros and is formed from three types of progenitor cells: nephron progenitor cells, interstitial progenitor cells, and ureteric bud cells, as well as blood vessels. Nephron progenitor cells are the cells that give rise to nephrons and have the ability to differentiate into glomeruli, proximal tubules, and distal tubules. Interstitial progenitor cells surround nephron progenitor cells and support their differentiation and maturation. Ureteric bud cells are progenitor cells that differentiate into the collecting duct and ureter, which constitute the ureteric bud and collect and excrete urine produced by the nephron. The tissue containing nephron progenitor cells and interstitial progenitor cells is also called the metanephric mesenchyme. The kidney is formed through interactions between the ureteric bud and metanephric mesenchyme.

[0004] Patients with kidney failure require dialysis or kidney transplantation, but due to a shortage of donors, most patients with kidney failure are dependent on dialysis. Therefore, research is being conducted to create kidneys for transplantation in vitro.

[0005] For example, Patent Document 1 describes a method for producing an organ for transplantation from human nephron progenitor cells and fetal metanephroi of a non-human mammal.

[0006] Several methods for producing kidney organoids in vitro have also been reported. For example, Non-Patent Document 1 describes the differentiation of nephron progenitor cells from pluripotent stem cells to form organoids with kidney structures.

[0007] Furthermore, for example, Non-Patent Documents 2 and 3 describe the dispersion and reaggregation of human nephron progenitor cells. Furthermore, for example, Non-Patent Document 4 describes the maturation of cell aggregates derived from human nephron progenitor cells.

[0008] Furthermore, Non-Patent Document 5 describes the production of chimeric organoids of mouse fetal metanephroi and human nephron progenitor cells.

[0009] International Publication No. 2018 / 003451 International Publication No. 2022 / 250147

[0010] Taguchi A., et al., Redefining the In Vivo Origin of Metanephric Nephron Progenitors Enables Generation of Complex Kidney Structures from Pluripotent Stem Cells, Cell Stem Cell, 14, 53-67, 2014.Tanigawa S., et al., Activin Is Superior to BMP7 for Efficient Maintenance of Human iPSC-Derived Nephron Progenitors, Stem Cell Reports, 13, 322-337, 2019.Yoshimura, Y., et al., Manipulation of Nephron-Patterning Signals Enables Selective Induction of Podocytes from Human Pluripotent Stem Cells, J Am Soc Nephrol., 30, 304-321, 2019.Morizane R., et al., Nephron organoids derived from human pluripotent stem cells model kidney development and injury, Nat Biotechnol., 33 (11), 1193-1200, 2015.Matsumoto N., et al., Evaluation of the ability of human induced nephron progenitor cells to form chimeric renal organoids using mouse embryonic renal progenitor cells, Biochem Biophys Res Commun., 662, 18-25, 2023.Takamura, T. et al., In vivo Development of Fetal Pig Kidneys in Mature Monkeys Under Clinically Approved Immunosuppressant Drugs, Engineering, 10, 65-73, 2022.

[0011] A technology is being developed to transplant human nephron progenitor cells into the metanephroi of non-human mammals such as pigs to develop kidneys, which can then be transplanted into patients with renal failure. The pig-derived cells are designed to undergo cell death when administered a drug, and ultimately the patient's kidney function is restored from the human nephron progenitor cells.

[0012] This technology requires that human nephron progenitor cells be able to engraft and mature in the porcine metanephroi. That is, human nephron progenitor cells must have the ability to form chimeras with porcine nephron progenitor cells. Therefore, the ability of human nephron progenitor cells to form chimeras with porcine nephron progenitor cells is an important criterion for evaluating the quality of human nephron progenitor cells.

[0013] Furthermore, chimeric kidney organoids formed from human nephron progenitor cells and cells derived from the metanephros of non-human animals, as well as kidney organoids derived from porcine metanephros, can themselves be used for transplantation into patients with renal failure. However, for example, Non-Patent Document 5, mentioned above, shows that a high proportion of chimeric kidney structures were formed by mixing and culturing human nephron progenitor cells and cells derived from mouse fetal metanephros on the second day of culture. However, with continued culture, the proportion of chimeric kidney structures decreased by the sixth day of culture. Therefore, a method for maturing the kidneys to a stage suitable for transplantation while maintaining the chimeric structure has been desired.

[0014] The present invention aims to provide kidney organoid-related technologies.

[0015] The present invention includes the following aspects: [1] A chimeric kidney organoid having a chimeric kidney structure comprising human cells and non-human mammalian cells, wherein the non-human mammal is a cetacean or a non-human primate. [2] The chimeric kidney organoid according to claim 1, wherein the chimeric kidney structure formation rate calculated by the following formula (1) is 30% or higher: Chimeric kidney structure formation rate (%) = number of chimeric kidney structures comprising human cells and non-human mammalian cells / number of total kidney structures × 100 ... (1) [3] The chimeric kidney organoid according to [1] or [2], wherein the chimeric kidney structure comprises one or more cells selected from the group consisting of human-derived nephron progenitor cells, human-derived cells constituting distal tubules, human-derived cells constituting proximal tubules, and human-derived cells constituting glomeruli. [4] The chimeric kidney organoid according to any one of [1] to [3], wherein the chimeric kidney structure comprises one or more cells selected from the group consisting of nephron progenitor cells derived from the non-human mammalian cells, cells constituting glomeruli derived from the non-human mammalian cells, cells constituting distal tubules derived from the non-human mammalian cells, and cells constituting proximal tubules derived from the non-human mammalian cells, and cells constituting ureteric buds and / or collecting ducts derived from the non-human mammalian cells. [5] The chimeric kidney organoid according to any one of [1] to [4], wherein the cetacean is a pig. [6] A method for producing chimeric renal tissue comprising human nephron progenitor cells or cells derived from human nephron progenitor cells, and cells derived from a non-human mammalian fetal metanephroi, the method comprising: a step (a1) of contacting the human nephron progenitor cells with the non-human mammalian fetal metanephroi-derived cells to obtain a cell population; and a step (b1) of culturing the cell population obtained in step (a1) in a chimera-forming medium to obtain chimeric renal tissue, the chimera-forming medium containing a Wnt signaling activator. [7] The method according to [6], wherein the contacting in step (a1) comprises mixing dispersed human nephron progenitor cells with dispersed non-human mammalian fetal metanephroi-derived cells, and the chimeric renal tissue is a chimeric aggregate.[8] The manufacturing method according to [6], wherein the contacting in step (a1) involves injecting human nephron progenitor cells into a non-human mammal fetal metanephroi, and the chimeric kidney tissue is a non-human mammal fetal metanephroi comprising human nephron progenitor cells or cells derived from human nephron progenitor cells. [9] The manufacturing method according to [8], wherein a suicide gene for nephron progenitor cells derived from the non-human mammal is introduced into the non-human mammal fetal metanephroi.

[10] The manufacturing method according to any of [6] to [9], wherein the chimera formation medium further comprises a ROCK inhibitor.

[11] The manufacturing method according to any of [6] to

[10] , wherein the chimera formation medium further comprises an FGF9 signal activator.

[12] The manufacturing method according to any of [6] to

[11] , wherein the chimera formation medium is a serum-free medium.

[13] A method for producing chimeric kidney organoids comprising human cells and non-human mammalian cells, the method comprising: (a2) mixing dispersed human nephron progenitor cells and dispersed non-human mammalian fetal metanephroi-derived cells to obtain a cell population; (b2) culturing the cell population obtained in step (a2) in a chimera-forming medium to obtain chimeric aggregates; and (c2) culturing the chimeric aggregates obtained in step (b2) in a maturation medium to obtain chimeric kidney organoids comprising human cells and non-human mammalian cells, wherein the chimera-forming medium contains a Wnt signaling activator, and the maturation medium does not contain a Wnt signaling activator in at least some steps of step (c2).

[14] The method according to

[13] , wherein step (c2) comprises: (c2-1) culturing the chimeric aggregates obtained in step (b2) in a maturation medium containing a Wnt signaling activator, and (c2-2) culturing the chimeric aggregates obtained in step (b2) in a maturation medium not containing a Wnt signaling activator.

[15] The method of producing according to

[13] or

[14] , wherein the chimera formation medium and / or the maturation medium further comprises an FGF9 signal activator.

[16] The method of producing according to any one of

[13] to

[15] , wherein the chimera formation medium and / or the maturation medium further comprises a ROCK inhibitor.

[17] The method of producing according to any one of

[13] to

[16] , wherein the chimera formation medium and / or the maturation medium is a serum-free medium.

[18] The method according to any one of

[13] to

[17] , wherein the non-human mammal is a rodent, a cetacean, or a non-human primate.

[19] The method according to

[18] , wherein the cetacean is a pig.

[20] A method for evaluating the ability of human nephron progenitor cells to form chimeric renal structures, comprising: a step (a3) ​​of mixing the dispersed human nephron progenitor cells with dispersed cells derived from a non-human mammalian fetal metanephroi to obtain a cell population; a step (b3) of culturing the cell population obtained in step (a3) ​​in a chimera-forming medium to obtain chimeric aggregates; a step (c3) of culturing the chimeric aggregates obtained in step (b3) in a maturation medium to obtain renal organoids containing human cells and non-human mammalian cells; and a step (d3) of determining the ability of human nephron progenitor cells to form chimeric renal structures based on the number of chimeric renal structures and / or the type of cells contained in the chimeric renal structures contained in the renal organoids obtained in step (c3), wherein the chimera-forming medium contains a Wnt signaling activator, and the maturation medium does not contain a Wnt signaling activator in at least a portion of step (c3).

[21] The evaluation method described in

[20] , wherein the step (c3) includes a step (c3-1) of culturing the chimeric aggregate obtained in the step (b3) in a maturation medium containing a Wnt signal activator, and a step (c3-2) of culturing the chimeric aggregate obtained in the step (b3) in a maturation medium not containing a Wnt signal activator.

[22] The evaluation method according to

[20] or

[21] , wherein the step (d3) of determining the ability to form a chimeric renal structure comprises one or more steps selected from the following (i) to (vi): (i) calculating a chimeric renal structure formation rate using the following formula (1), and determining the ability of the human nephron progenitor cells to form a chimeric renal structure based on the calculated chimeric renal structure formation rate: Chimeric renal structure formation rate (%) = number of chimeric renal structures comprising human cells and non-human mammalian cells / number of total renal structures × 100 ... (1); (ii) counting the number of glomerular structures comprising human cells and non-human mammalian cells, and determining the ability of the human nephron progenitor cells to form a chimeric renal structure based on the counted number of glomerular structures; (iii) counting the number of renal tubular structures comprising human cells and non-human mammalian cells, and determining the ability of the human nephron progenitor cells to form a chimeric renal structure based on the counted number of renal tubular structures; (iv) counting the number of nephron segment structures comprising human cells and non-human mammalian cells, and determining the ability of the human nephron progenitor cells to form a chimeric kidney structure based on the counted number of nephron segment structures; (v) counting the number of ureteric bud structures derived from a non-human mammal, comprising human nephron progenitor cells, human distal tubule cells, and / or human proximal tubule cells, and determining the ability of the human nephron progenitor cells to form a chimeric kidney structure based on the counted number of ureteric bud structures; (vi) calculating the proportion of human cells in the kidney structure, and determining the ability of the human nephron progenitor cells to form a chimeric kidney structure based on the calculated proportion of human cells.

[23] The evaluation method described in any of

[20] to

[22] , wherein the chimera-forming medium and / or the maturation medium further comprises an FGF9 signal activator.

[24] The evaluation method described in any of

[20] to

[23] , wherein the chimera-forming medium and / or the maturation medium further comprises a ROCK inhibitor.

[25] The evaluation method according to any one of

[20] to

[24] , wherein the chimera-forming medium and / or the maturation medium is a serum-free medium.

[26] The evaluation method according to any one of

[20] to

[25] , wherein in the step (a3), the ratio of the number of human nephron progenitor cells to the number of non-human mammalian fetal metanephroi-derived cells is 5:1 to 1:1.

[27] The evaluation method according to

[26] , wherein in step (a3), the ratio of the number of human nephron progenitor cells to the number of cells derived from the non-human mammalian fetal metanephroi is about 3:1.

[28] The evaluation method according to any of

[20] to

[27] , wherein the non-human mammal is a rodent, a cetacean, or a non-human primate.

[29] The evaluation method according to

[28] , wherein the cetacean is a pig.

[30] A porcine kidney organoid comprising at least two types of cells selected from the group consisting of nephron component cells or nephron progenitor cells, collecting duct component cells or collecting duct progenitor cells, and renal interstitial cells or renal interstitial progenitor cells, and having the function of kidney component cells or the function of promoting kidney development, and having multiple kidney structures therein.

[31] The porcine kidney organoid according to

[30] , wherein the renal structure comprises cells constituting one or more selected from glomeruli, distal tubules, and proximal tubules, or their precursor cells, and cells constituting collecting ducts, or their precursor cells, and has a CAP structure containing ureteric buds and nephron progenitor cells therein.

[32] A method for producing kidney organoids comprising non-human mammalian cells, the method comprising: (a4) culturing a population of dispersed non-human mammalian fetal metanephroi-derived cells in a reaggregation medium to obtain aggregates; and (b4) culturing the aggregates obtained in (a4) in a maturation medium to obtain kidney organoids comprising non-human mammalian cells, wherein the reaggregation medium contains a Wnt signal activator, and the maturation medium does not contain a Wnt signal activator in at least a part of step (b4).

[33] The production method according to

[32] , wherein the step (b4) comprises a step (b4-1) of culturing the chimeric aggregate obtained in the step (a4) in a maturation medium containing a Wnt signal activator, and a step (b4-2) of culturing the chimeric aggregate in a maturation medium not containing a Wnt signal activator.

[34] The production method according to

[32] or

[33] , wherein the reaggregation medium and / or the maturation medium further comprises an FGF9 signal activator.

[35] The production method according to any of

[32] to

[34] , wherein the reaggregation medium and / or the maturation medium further comprises a ROCK inhibitor.

[36] The production method according to any of

[32] to

[35] , wherein the reaggregation medium and / or the maturation medium is a serum-free medium.

[37] The manufacturing method according to any one of

[32] to

[36] , wherein the non-human mammal is a rodent, a cetacean, or a non-human primate.

[38] The manufacturing method according to

[37] , wherein the non-human mammal is a cetacean or a non-human primate.

[39] The manufacturing method according to

[38] , wherein the cetacean is a pig or a cow.

[40] A transplant material for a patient in need of a kidney transplant, comprising the chimeric kidney organoid according to any one of [1] to [5], chimeric kidney tissue obtained by the manufacturing method according to any one of [6] to

[12] , chimeric kidney organoid obtained by the manufacturing method according to any one of

[13] to

[19] , pig kidney organoid according to

[30] or

[31] , or kidney organoid obtained by the manufacturing method according to

[38] or

[39] .

[41] The transplant material according to

[40] , for treating a patient with kidney damage or injury.

[0016] According to the present invention, kidney organoid-related technologies can be provided.

[0017] Figure 1 shows microscopic images of mouse kidney organoids and human nephron organoids in Experimental Example 1. When reaggregation culture and maturation culture were performed in 10 FBS medium, the reaggregates of mouse metanephric cells formed organoid-like three-dimensional structures and appeared to mature in a culture period-dependent manner, whereas the reaggregates of human nephron progenitor cells (NPCs) could not survive in the culture environment and disappeared.

[0018] Figure 2-1 shows a microscopic image of human nephron organoids in Experimental Example 2. 10FBS medium was used as the reaggregation medium. 10FBS medium, F9 medium, or KR5 medium was used as the maturation medium. When 10FBS medium was used as both the reaggregation medium and the maturation medium, the reaggregates of human NPCs disappeared, as shown in Experimental Example 1. On the other hand, when F9 medium or KR5 medium was used as the maturation medium, organoid-like three-dimensional structures were formed. However, in F9 medium, there were few organoid structures and the overall size was small. In KR5 medium, organoid structures were abundant and of good size, but excessive duct-like structures were observed.

[0019] Figure 2-2 is a microscopic image of another lot of human kidney organoids (human nephron organoids) produced in the same manner as in Experimental Example 2, on day 6 of maturation culture.

[0020] Figure 3-1 shows a microscopic image of mouse kidney organoids in Experimental Example 3. 10FBS medium was used as the reaggregation medium. 10FBS medium, F9 medium, or KR5 medium was used as the maturation medium. Under all culture conditions, organoid-like three-dimensional structures were abundantly observed, and the size was also good. On the other hand, in the group in which KR5 medium was used as the maturation medium, excessive duct-like structures were observed.

[0021] Figure 3-2 is a microscopic image of another lot of mouse kidney organoids produced in the same manner as in Experimental Example 3 on day 6 of maturation culture.

[0022] Figure 4 shows a microscopic image of human-mouse chimeric kidney organoids in Experimental Example 4. 10FBS medium was used as the chimera formation medium. 10FBS medium, F9 medium, or KR5 medium was used as the maturation medium. When 10FBS medium was used for both the chimera formation medium and the maturation medium, a good organoid-like structure was observed on day 2 of culture, but by day 6 of culture, the organoid structure had decreased and the overall size had also decreased (partially disappeared). On the other hand, when F9 medium or KR5 medium was used as the maturation medium, an organoid-like three-dimensional structure was formed. No morphological abnormalities were observed in F9 medium, but excessive duct-like structures were observed in KR5 medium.

[0023] FIG. 5 is a micrograph of another lot of human-mouse chimeric kidney organoids produced in the same manner as in Experimental Example 4.

[0024] Figure 6 shows a microscopic image of human-mouse chimeric kidney organoids in Experimental Example 5. C1F9Y medium was used as the chimera formation medium. 10FBS medium, F9 medium, or KR5 medium was used as the maturation medium. In the group using 10FBS medium as the maturation medium, a good organoid-like structure was confirmed on the second day of culture, but by the sixth day of culture, the organoid structure had decreased and the overall size had also shrunk (partially disappeared). In the group using KR5 medium as the maturation medium, excessive duct-like structures were confirmed. On the other hand, in the group using F9 medium as the maturation medium, abundant organoid-like structures were confirmed and the size was good.

[0025] Figure 7 shows a microscopic image of mouse kidney organoids in Experimental Example 6. C1F9Y medium was used as the reaggregation medium. 10FBS medium, F9 medium, or KR5 medium was used as the maturation medium. Under all culture conditions, organoid-like three-dimensional structures were abundantly observed, and the size was also good. On the other hand, in the group using KR5 medium as the maturation medium, excessive duct-like structures were observed.

[0026] Figure 8-1 shows a microscopic image of human nephron organoids in Experimental Example 6. C1F9Y medium was used as the reaggregation medium. 10FBS medium, F9 medium, or KR5 medium was used as the maturation medium. In the group using 10FBS medium as the maturation medium, a good organoid-like structure was observed on the second day of culture, but by the sixth day of culture, the organoid structure had decreased and the overall size had also shrunk (partially disappeared). In the group using KR5 medium as the maturation medium, excessive duct-like structures were observed. On the other hand, in the group using F9 medium as the maturation medium, abundant organoid-like structures were observed and the size was good.

[0027] Figure 8-2 shows the results of quantifying the size (area) of mouse kidney organoids, human kidney organoids (human nephron organoids), and human-mouse chimeric kidney organoids based on microscopic images on day 6 of culture in Figures 2-2, 3-2, 5, 6, 7, and 8-1. ImageJ software was used for quantification. The average values ​​were calculated for each of the three types of organoids (mouse, human, and chimera) under six culture conditions. Subsequently, the organoid size (relative value) under each culture condition was calculated, normalized by the calculated average value. As a result, it became possible to quantitatively identify culture conditions that can induce large-sized organoids for the three types of organoids (mouse, human, and chimera). When C1F9Y medium was used as the chimera formation medium and F9 medium or KR5 medium was used as the maturation medium, larger organoid sizes were observed for all three types of organoids (mouse, human, and chimera) compared to other culture conditions.

[0028] 9 is a fluorescence microscope image showing the results of immunochemical staining of a section of a human-mouse chimeric kidney organoid in Experimental Example 7. A Ku80 antibody was used to label human cell nuclei. Compared to the group using 10% FBS medium as the chimera formation medium, the group using C1F9Y medium as the chimera formation medium showed a large number of Ku80-positive human cells.

[0029] Figure 10 shows fluorescence microscopy images of the sections in which 10% FBS medium was used as the chimera formation medium, in which ECAD-positive cells and CK8-positive cells were detected in addition to Ku80-positive cells. Enlarged images of the areas where chimera structures were formed are also shown.

[0030] Figure 11 shows fluorescence microscopy images of the sections in Figure 9 where C1F9Y medium was used as the chimera formation medium, in which ECAD-positive cells and CK8-positive cells were detected in addition to Ku80-positive cells. Enlarged images of the areas where chimeric structures were formed are also shown. The four figures at the bottom are enlarged images of the dashed line area in the center image in the second row from the top.

[0031] FIG. 12 shows fluorescence microscopy images of WT1-positive cells, CK8-positive cells, HuNu-positive cells, and DAPI-positive cells detected in separate sections of each sample in which 10% FBS medium was used as the chimera formation medium.

[0032] Figure 13 shows fluorescence microscopy images of WT1-positive cells, CK8-positive cells, HuNu-positive cells, and DAPI-positive cells detected in separate sections of each sample in which C1F9Y medium was used as the chimera formation medium. Enlarged images of the areas where chimera structures were formed are also shown. The four figures at the bottom are enlarged images of the dashed-lined areas in the center images in the second row from the top.

[0033] 14 is a graph showing the calculated values ​​of the proportion of human cells (Ku80-positive cells) in human-mouse chimeric kidney organoids based on the results of FIG. 9. "*" indicates a significant difference at p<0.05. "**" indicates a significant difference at p<0.01. Compared to the group using 10FBS medium as the chimera formation medium, it was quantitatively confirmed that there were many Ku80-positive human cells in the group using C1F9Y medium as the chimera formation medium. This tendency was particularly noticeable in the groups using F9 medium or KR5 medium as the maturation medium.

[0034] Figure 15 shows the results of quantitative analysis of the proportions of constituent cells based on the results of Figures 9, 10, and 11. "*" indicates a significant difference at p<0.05. "**" indicates a significant difference at p<0.01. The abundance ratios of CK8-positive cells, ECAD-positive cells, and WT1-positive cells relative to total cells were calculated as area ratios in the stained images and shown in graphs. The graph on the bottom right shows the proportions of nephron segments. NPCs are known to differentiate into one of the nephron-constituting nephron segments: glomeruli (WT1), proximal tubules, or distal tubules (ECAD). It is considered desirable for NPCs to differentiate without a significant bias toward a specific segment. In this study, proximal tubules were not stained, so only glomeruli (WT1) and distal tubules (ECAD) were evaluated. "C1F9Y→F9" showed good results without bias.

[0035] FIG. 16 is a fluorescence microscope image showing the results of evaluation based on distal tubules as kidney structures in Experimental Example 8.

[0036] Figure 17 is a graph quantitatively showing the results of Figure 16. "**" indicates a significant difference at p<0.01. The vertical axis represents the chimeric kidney structure formation rate (%). In groups using C1F9Y medium as the chimera formation medium and 10FBS medium or F9 medium as the maturation medium, the chimeric kidney structure formation rate was significantly increased compared to the control group. The highest value was observed in the combination of C1F9Y chimera formation medium → F9 maturation medium.

[0037] FIG. 18 is a fluorescence microscope image showing the results of evaluation based on glomeruli as kidney structures in Experimental Example 8.

[0038] Figure 19 is a graph quantitatively showing the results of Figure 18. "**" indicates a significant difference at p<0.01. The vertical axis represents the rate of chimeric kidney structure formation (%). In groups using C1F9Y medium as the chimera formation medium and 10FBS medium or F9 medium as the maturation medium, the rate of chimeric kidney structure formation was significantly increased compared to the control group. The highest value was observed for the combination of C1F9Y chimera formation medium and F9 maturation medium.

[0039] Figure 20 shows an immunostained image of a human-mouse chimeric organoid on day 2 of culture (Day 2), cultured using C1F9Y medium as the chimera formation medium and F9 medium as the maturation medium. Staining was performed with anti-WT1 antibody, anti-CK8 antibody, anti-HuNu antibody, and DAPI. The arrowhead in the lower right image indicates the CAP structure, the arrow in the lower right image indicates the S-shaped body, the arrowhead in the upper right image indicates the RV, and the arrow in the upper right image indicates the C-shaped body. The scale bar is 200 μm. Chimera formation was observed in structures characteristic of early nephron development, such as the CAP structure, RV, C-body, and S-body, and a mosaic nephron was formed.

[0040] Figure 21 shows an image of the same sample as in Figure 20, immunostained with a different marker. The sample was stained with anti-ECAD antibody, anti-CK8 antibody, anti-Ku80 antibody, and DAPI. The scale bar is 200 μm.

[0041] Figure 22 shows a microscopic image of porcine kidney organoids in Experimental Example 9. 10FBS medium was used as the reaggregation medium. 10FBS medium, F9 medium, or KR5 medium was used as the maturation medium. Under all conditions, an organoid-like three-dimensional structure was formed. However, in the group using 10FBS medium for both the reaggregation medium and maturation medium (10FBS → 10FBS), a reduction in size was observed on the sixth day of culture compared to the second day of culture. Specifically, 2 x 10 ５ When cells were seeded, the size (long diameter) of the aggregates on day 6 of culture was 405 μm for 10FBS → 10FBS, 814 μm for 10FBS → F9, and 862 μm for 10FBS → KR5. In addition, in the case of 10FBS → KR5, excessive formation of tubular structures was observed.

[0042] Figure 23 is a microscopic image of pig kidney organoids in Experimental Example 9. C1F9Y medium was used as the reaggregation medium, and the organoids were cultured for 1 day (24 hours). Next, 10FBS medium, F9 medium, or KR5 medium was used as the maturation medium. Under all conditions, organoid-like three-dimensional structures were formed. In the groups using F9 medium or KR5 medium as the maturation medium, larger aggregate sizes were observed on the sixth day of maturation culture compared to the group using 10FBS medium as the maturation medium. Specifically, 2 x 10 ５ When cells were seeded, the size (longest diameter) of the aggregates on day 6 of culture was: C1F9Y→10FBS: 577 μm, C1F9Y→F9: 1041 μm, C1F9Y→KR5: 1202 μm. In addition, in the case of 10FBS→KR5, excessive formation of tubular structures was observed.

[0043] Figure 24 is a fluorescence microscope image showing the results of immunochemical staining of a section of a pig kidney organoid in Experimental Example 10. Staining was performed with an anti-ECAD antibody, an anti-CK8 antibody, an anti-Ku80 antibody, and DAPI. ECAD-positive distal tubule structures and CK8-positive ureteric bud structures were confirmed. Abundant ECAD-positive distal tubules were observed under all culture conditions. Furthermore, no staining was observed with Ku80, confirming that the Ku80 antibody used is specific to human cells.

[0044] FIG. 25 is a fluorescent microscope image showing an enlargement of a part of the central bottom panel of FIG.

[0045] Figure 26 is a graph showing the results of calculating the ratio of the area of ​​the ECAD-positive region to the area of ​​the renal organoid section based on the results of Figure 24. The presence of abundant ECAD-positive distal tubules was quantitatively confirmed under all culture conditions. The combination of reaggregation medium C1F9Y medium and maturation medium F9 medium showed the highest positive rate.

[0046] Figure 27 is a fluorescence microscope image showing the results of immunochemical staining of a section of pig kidney organoid in Experimental Example 10. Staining was performed with anti-WT1 antibody, anti-CK8 antibody, anti-HuNu antibody, and DAPI. WT1-positive glomerular structures and CK8-positive ureteric bud structures were confirmed. The presence of a small number of WT1-positive glomerular structures was observed under all culture conditions, and the formation rate of glomerular structures was particularly high when C1F9Y→F9 medium was used. Furthermore, Ku80 did not stain, confirming that the Ku80 antibody used is specific to human cells.

[0047] FIG. 28 is a fluorescent microscope image showing an enlargement of a part of the central bottom panel of FIG.

[0048] Figure 29-1 is a graph showing the results of calculating the ratio of the area of ​​the WT1-positive area to the area of ​​the section of the porcine kidney organoid based on the results of Figure 27. The presence of a small number of WT1-positive glomeruli was quantitatively confirmed under all culture conditions. The combination of reaggregation medium C1F9Y medium and maturation medium F9 medium showed the highest positive rate.

[0049] Figure 29-2 shows the results of quantifying the ECAD positive rate and WT1 positive rate based on the area ratio of the ECAD positive area of ​​the porcine kidney organoid shown in Figure 26 and the area ratio of the WT1 positive area of ​​the porcine kidney organoid shown in Figure 29-1. For each culture condition, the area ratio of the ECAD positive area was added up to calculate the average value, and a standardized value based on that average value was calculated for each culture condition. In addition, for each culture condition, the area ratio of the WT1 positive area was added up to calculate the average value, and a standardized value based on that average value was calculated for each culture condition. As a result, it became possible to quantitatively show culture conditions that can induce high ECAD positive area rate and WT1 positive area rate in porcine kidney organoids. When C1F9Y medium was used as the reaggregation medium and F9 medium was used as the maturation medium, porcine kidney organoids with the highest ECAD positive area rate and WT1 positive area rate could be induced.

[0050] Figure 30 shows a microscopic image of pig-human chimeric kidney tissue in Experimental Example 11. When human NPCs and pig metanephric cells were mixed at ratios of 1:1, 2:1, and 3:1, it was confirmed that chimeric kidney tissue was formed at all ratios. C1F9Y medium was used as the chimera formation medium.

[0051] Figure 31 shows a microscopic image of the pig-human chimeric kidney organoids in Experimental Example 11. All of the aggregates, in which the mixing ratios of human NPCs and pig metanephric cells were 1:1, 2:1, and 3:1, respectively, formed organoid-like three-dimensional structures. C1F9Y medium was used as the chimera formation medium, and F9 medium was used as the maturation medium. 2 x 10 ５ When cells were seeded, the sizes (long diameters) of the aggregates on the sixth day of culture (reaggregation was carried out for 24 hours, followed by maturation culture up to the sixth day) were as follows: C1F9Y→F9 (human:pig=1:1) = 1103 μm, C1F9Y→F9 (human:pig=2:1) ​​= 1038 μm, C1F9Y→F9 (human:pig=3:1) = 1023 μm.

[0052] Figure 32 is a fluorescence microscope image showing the results of immunochemical staining of a section of a human-pig chimeric kidney organoid in Experimental Example 12. In all groups where the mixing ratio was changed, ECAD-positive distal tubule structures, CK8-positive ureteric bud structures, and Ku80-positive human cell nuclei were observed. It was observed that the higher the human mixing ratio, the higher the proportion of human cells in the formed organoids.

[0053] Figure 33 is a fluorescence microscope image showing the results of staining with anti-human Ku80 antibody and DAPI in the same field of view as Figure 32. It was quantitatively confirmed that the higher the human mixture ratio, the higher the proportion of human cells in the formed organoids.

[0054] Figure 34 shows representative microscopic images showing the presence of chimeric kidney structures in Experimental Example 12. Cells were stained with anti-ECAD antibody, anti-CK8 antibody, anti-Ku80 antibody, and DAPI. Multiple chimeric kidney structures were confirmed in all groups with different mixing ratios.

[0055] Figure 35 is an enlarged view of the upper left image in Figure 34 (the upper left image in the "human:pig = 1:1" comparison). The dashed line on the left in the second image from the left indicates the distal tubule tissue, and the dashed line on the right indicates the ureteric bud. The upper dashed area in the rightmost image indicates human-derived tissue, and the lower dashed area indicates pig-derived tissue.

[0056] Figure 36 shows images of human-pig chimeric organoids, similar to those shown in Figures 32 to 34, stained with an antibody against a different marker protein. It can be seen that WT1, CK8, and Ku80-positive cells are present in a mosaic pattern at all mixing ratios of human-derived cells and pig-derived cells. The three images on the right of Hu(3):Pig(1) are enlarged partial images, showing the formation of WT1-positive glomerular tissue.

[0057] Figure 37-1 is a graph showing the results of evaluating the chimeric kidney structure formation rate for one section of human-pig chimeric kidney organoids in Experimental Example 12. It was quantitatively confirmed that the higher the human mixing ratio, the higher the chimeric kidney structure formation rate in the formed organoids.

[0058] Figure 37-2 shows the results of quantifying the size, positive area rate of various markers, and chimeric kidney structure formation rate of human-pig chimeric kidney organoids when the mixing ratio of human-derived cells and pig-derived cells was assigned. The area of ​​the organoids was quantified based on the bright field image on day 6 shown in Figure 31. ImageJ software was used for quantification. The area of ​​the organoids was standardized based on the area of ​​human:pig = 1:1 organoids, and the relative size (area) of human-pig chimeric kidney organoids at each mixing ratio was calculated (organoid size). In addition, the positive area rate of each marker, ECAD, WT1, CK8, and Ku80, was calculated based on Figures 32, 33, and 36. The positive area rate was expressed as a percentage (%) of the DAPI-positive area. The positive area rate of each marker in human: pig = 1: 1 organoids was standardized as a standard, and the relative value of the positive area rate (constituent cell ratio) of human-pig chimeric kidney organoids at each mixing ratio was calculated (Cell population). Furthermore, based on Figures 32 and 36, the chimeric kidney structure formation rate of the ECAD-positive area indicating distal tubules and the WT1-positive area indicating glomerular tissue was quantified. The chimeric kidney structure formation rate of each human: pig = 1: 1 organoid was standardized as a standard, and the relative value of the chimeric kidney structure formation rate of human-pig chimeric kidney organoids at each mixing ratio was calculated (Chimeric rate). High-magnification photographs of representative chimeric kidney structures are shown in Figures 34, 35, and 36. From the above results, it was quantitatively shown that there was no significant difference in organoid size or ECAD / WT1 / CK8 positive area ratio (constituent cell ratio) even when the mixing ratio was different, but the human-pig chimeric kidney organoid with the highest composition ratio of human cells (Ku80 positive cells) was the human:pig = 3:1 human:pig chimeric kidney organoid, and that the human:pig = 3:1 human:pig chimeric kidney organoid also had the highest chimeric kidney structure formation rate.

[0059] Figure 37-3 is a fluorescence microscope image showing the results of staining sections of a 3:1 human:pig chimeric kidney organoid (day 6) with multiple glomerular markers, proximal tubule markers, and distal tubule markers. The scale bar is 100 μm. WT1, NEPHRIN, and PODOCALYXIN were used as glomerular markers. JAGGED1 (JAG1), LTL, CDH6, AQP1, and MEGALIN were used as proximal tubule markers. DBA, ECAD, and NCC were used as distal tubule markers. As a result, it was confirmed that various nephron segments could be reliably induced and that a certain degree of maturity was also exhibited.

[0060] Figure 37-4 is a fluorescence microscope image showing the results of staining sections of porcine kidney organoids (day 6) with multiple glomerular markers, proximal tubule markers, and distal tubule markers. The scale bar is 100 μm. WT1, NEPHRIN, and PODOCALYXIN were used as glomerular markers. JAGGED1 (JAG1), LTL, CDH6, AQP1, and MEGALIN were used as proximal tubule markers. DBA, ECAD, and NCC were used as distal tubule markers. As a result, it was confirmed that various nephron segments could be reliably induced and that a certain degree of maturity was also exhibited.

[0061] Figure 37-5 shows fluorescence microscopy images of sections of human kidney organoids (human nephron organoids) (day 6) stained with multiple glomerular markers, proximal tubule markers, and distal tubule markers. The scale bar is 100 μm. WT1, nephrin, and podocalyxin were used as glomerular markers. JAGGED1 (JAG1), LTL, CDH6, AQP1, and megalin were used as proximal tubule markers. DBA, ECAD, and NCC were used as distal tubule markers. The results confirmed that various nephron segments were successfully induced and showed a certain degree of maturation.

[0062] Figure 37-6 is a graph showing the results of comparing the ratios of constituent cells between human:mouse = 3:1 human-mouse chimeric kidney organoids (day 6) and human:pig = 3:1 human-pig chimeric kidney organoids (day 6). "*" indicates a significant difference at p<0.05. "**" indicates a significant difference at p<0.01. The ratios of constituent cells in the "C1F9Y→F9" human-mouse chimeric kidney organoids shown in Figures 14 and 15 were compared with the ratios of constituent cells in human-pig chimeric kidney organoids (N=3) quantified in the same manner as in Figures 14 and 15. As a result, differences were observed in the ratios of ECAD-positive cells and WT1-positive cells, but no significant differences were observed in the ratios of other constituent cells.

[0063] Figure 37-7 is a graph showing the results of comparing the chimeric kidney structure formation rate of human: mouse = 3: 1 human-mouse chimeric kidney organoids (day 6) and human: pig = 3: 1 human-pig chimeric kidney organoids (day 6). The chimeric kidney structure formation rate of the "C1F9Y → F9" human-mouse chimeric kidney organoids shown in Figures 17 and 19 was compared with the chimeric kidney structure formation rate of human-pig chimeric kidney organoids quantified in the same manner as in Figures 17 and 19 (N = 3). As a result, no significant differences were observed in either the chimeric kidney structure formation rate of ECAD-positive cells or the chimeric kidney structure formation rate of WT1-positive cells.

[0064] 38 shows fluorescence microscopy images showing the results of immunochemical staining of the mouse metanephroi injected with human nephron progenitor cells in Experimental Example 13. Human nephron progenitor cells were engrafted in a layer just below the capsule of the mouse metanephroi, and partial chimera formation was confirmed.

[0065] Figure 39 shows stereomicroscopic images of a mouse metanephroi injected with iPS cell-derived human nephron progenitor cells fluorescently labeled with GFP at the start of culture (Day 0). The image on the left is a bright-field image, and the image on the right is a fluorescent image in which GFP was detected. The scale bar is 750 μm. It was confirmed that the injected human nephron progenitor cells were present in sufficient quantities at the target location.

[0066] Figure 40-1 shows stereomicroscopic images of mouse metanephroi injected with iPS cell-derived human nephron progenitor cells fluorescently labeled with GFP and cultured for four days (Day 4). The image on the left is a bright-field image, and the image on the right is a fluorescent image in which GFP was detected. The scale bar is 750 μm. It was confirmed that the injected human nephron progenitor cells remained in the target location and were present in sufficient quantities even after four days of culture.

[0067] Figure 40-2 is a graph showing the change in size during culture of mouse metanephros injected with human nephron progenitor cells (NPCs). "**" indicates a significant difference at p<0.01. Images of human NPC-injected mouse metanephros, i.e., two-dimensional projections of mouse metanephros, are shown in Figures 39 and 40-1. The area corresponding to the mouse kidney and the area corresponding to human NPCs are quantified. "Area Mouse Kidney" is the value quantified using ImageJ software for the mouse kidney portion in the bright-field image. "Area Human NPC" is the value quantified using ImageJ software for the GFP-positive portion in the GFP fluorescent image. The results demonstrated that both the mouse metanephros and the human NPCs injected therein expanded in size after 4 days of culture.

[0068] Figure 41 is a fluorescence microscope image showing the results of immunochemical staining of a mouse metanephroi injected with human nephron progenitor cells after culturing for 4 days in Experimental Example 13, and is another image of the same tissue as Figure 38.

[0069] Figure 42 is an enlarged image of the part indicated by the arrow in Figure 38. It was confirmed that human cells had adhered to the tip of the mouse ureteric bud and formed a chimeric structure.

[0070] Figure 43 is a fluorescence microscopy image showing the results of immunochemical staining of mouse metanephroi injected with human nephron progenitor cells after 4 days of culture in Experimental Example 13, which is another image of the same tissue as in Figure 42. The scale bar is 100 μm. The dashed line in the center image indicates the outer edge of the CAP structure (Cap Mesenchyme), and in the rightmost image, the outer dashed line indicates the region of human-derived tissue, and the inner dashed line indicates the region of mouse-derived tissue.

[0071] Figure 44 shows fluorescent microscopic images showing the results of immunochemical staining of a mouse metanephroi injected with GFP-introduced human nephron progenitor cells in Experimental Example 13. The results showed that the GFP-expressing cells and HuNu-stained cell nuclei completely overlapped, further confirming the formation of chimeras between the human cells and the mouse metanephroi. Furthermore, the formation of a human-mouse Cap structure was also confirmed.

[0072] Figure 45 is an enlarged image of the boundary between the human cells and mouse cells in Figure 44. As a result, it was confirmed that the human cells had adhered to the mouse cells and formed a single structure.

[0073] FIG. 46 is a schematic diagram showing the experimental scheme of Experimental Example 14.

[0074] Figure 47 shows images of the state of the metanephroi of a mouse injected with human nephron progenitor cells during air-liquid interface culture in Experimental Example 14. As a result, even on day 3 after the start of culture, survival and engraftment of human nephron progenitor cells were confirmed to be at the same level as at the start of culture.

[0075] The upper panel of Figure 48 shows a bright-field image and a GFP fluorescence image showing the state of the human nephron progenitor cell-injected mouse metanephroi on day 3 from the start of air-liquid interface culture. The lower panel of Figure 48 shows a bright-field image and a GFP fluorescence image showing the state of the human nephron progenitor cell-injected mouse metanephroi after transplantation into a recipient (adult mouse).

[0076] The upper part of Figure 49 shows a bright-field image of the vicinity of the recipient's aorta 7 days after transplantation, an image obtained by observing GFP fluorescence, and a merged image of these. The presence of human cells was confirmed by the presence of GFP fluorescence. The lower part of Figure 49 shows a bright-field image of the graft excised from the recipient 7 days after transplantation, an image obtained by observing GFP fluorescence, and a merged image of these. The GFP fluorescence confirmed the engraftment of approximately the same amount of human cells as before transplantation.

[0077] Figure 50 shows fluorescent microscopic images of the graft excised from the recipient 7 days after transplantation, showing the results of immunochemical staining. It was confirmed that human nephron progenitor cells engrafted and differentiated into distal tubules, forming a chimeric kidney structure with mouse metanephroi-derived distal tubules.

[0078] [Notation of Gene Names and Protein Names] In this specification, human genes and human proteins are represented by capital letters. Mouse genes are represented by an initial capital letter followed by lowercase letters. Mouse proteins are represented by capital letters. However, in some cases, human genes, mouse genes, genes of other species, human proteins, mouse proteins, and proteins of other species may be represented without strict distinction.

[0079] Chimeric Renal Organoids In one embodiment, the present invention provides chimeric renal organoids having chimeric kidney structures comprising human cells and non-human mammalian cells, wherein the non-human mammal is a cetacean or a non-human primate.

[0080] Organoid is the cell aggregate that is formed in vitro and comprises the three-dimensional structure similar to organ or tissue.In this specification, kidney organoid means the cell aggregate that comprises the kidney structure that is formed in vitro.Chimeric kidney organoid means the kidney organoid that comprises the chimeric kidney structure described below.

[0081] In one embodiment, the chimeric kidney organoid of this embodiment has a chimeric kidney structure formation rate of 30% or more, as calculated by the following formula (1):

[0082] Chimeric kidney structure formation rate (%) = number of chimeric kidney structures containing human cells and non-human mammalian cells / number of total kidney structures × 100 (1)

[0083] As described below in the Examples, the present inventors have succeeded in producing chimeric kidney organoids with a higher chimeric kidney structure formation rate than conventional chimeric kidney organoids. In the kidney organoids of this embodiment, the chimeric kidney structure formation rate calculated by the above formula (1) is 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, or 90% or more. In addition, the chimeric kidney structure formation rate can be 30% to 90% or 40% to 80%.

[0084] As used herein, the term "renal structure" refers to a cell population containing the metanephros and its developmental and differentiation products, and refers to a cell population formed by the aggregation of cells that constitute the kidney, such as glomeruli, proximal tubules, distal tubules, and collecting ducts (ureteric buds), or their progenitor cells. The renal structure may also contain interstitial progenitor cells present between the renal structures.

[0085] The renal structure expresses a marker protein of cells constituting a glomerulus, a proximal tubule, a distal tubule, or a ureteric bud. In one embodiment, the renal structure includes a glomerulus, a proximal tubule, a distal tubule, or a precursor tissue thereof, and a collecting duct, a ureteric bud, or a precursor tissue thereof. The renal structure may include nephron progenitor cells.

[0086] Furthermore, a chimeric kidney structure refers to a kidney structure containing human cells and non-human mammalian cells within a single kidney structure. The human cells include nephron progenitor cells and / or cells derived from nephron progenitor cells. The nephron progenitor cell-derived cells include cells that constitute glomeruli, proximal tubules, distal tubules, etc., or their progenitor cells.

[0087] Specific examples of chimeric kidney structures include chimeric kidney structures that contain human cells that constitute at least glomeruli, proximal tubules, or distal tubules, or their precursor tissues, and also contain non-human mammalian cells that constitute at least glomeruli, proximal tubules, distal tubules, collecting ducts, or ureteric buds, or their precursor tissues.

[0088] The chimeric kidney structure preferably includes a chimeric kidney structure comprising human cells that constitute glomeruli, proximal tubules, and distal tubules, and non-human mammalian cells that constitute glomeruli, proximal tubules, distal tubules, and collecting ducts or ureteric buds.

[0089] As used herein, human nephron progenitor cells used in kidney organoids include nephron progenitor cells derived from pluripotent stem cells and nephron progenitor cells derived from human (including fetal) kidneys. Examples of pluripotent stem cells include embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells).

[0090] As used herein, non-human mammals include rodents, cetaceans, non-human primates, etc. Rodents include mice, rats, guinea pigs, etc. Cetaceans include pigs, cows, etc. Non-human primates include monkeys, baboons, etc.

[0091] In this specification, the non-human mammalian cells used for kidney organoids include cells derived from the metanephros of non-human mammalian fetuses. Non-human mammalian fetuses include mice, rats, guinea pigs, pigs, etc. The metanephros-derived cells used here are not particularly limited, as long as they are cells from the developmental stage at which the ureteric bud invades the metanephric mesenchyme or later.

[0092] In the renal organoid of this embodiment, the chimeric renal structure formation rate is the ratio of the number of chimeric renal structures containing human cells and non-human mammalian cells to the total number of all renal structures, i.e., the number of renal structures containing only human cells, the number of renal structures containing only non-human mammalian cells, and the number of chimeric renal structures containing human cells and non-human mammalian cells.

[0093] Here, kidney structures containing only human cells, kidney structures containing only non-human mammalian cells, and chimeric kidney structures containing both human and non-human mammalian cells can be distinguished by immunochemically staining sections of kidney organoids and observing them under a microscope or imaging analyzer. For immunochemical staining, for example, proteins expressed in cells constituting the kidney structure can be used as markers, and staining can be performed with antibodies against these markers. The markers used here are not particularly limited, and markers well known to those skilled in the art can be used in appropriate combinations.

[0094] For example, a section of a kidney organoid can be stained with an antibody against one or more markers expressed in glomeruli, such as Wilms Tumor 1 (WT1) protein, NEPHRIN protein, PODOCIN protein, PODOCALYXIN protein, and SYNAPTOPODIN protein, and the marker-positive area can be determined to be a glomerulus.

[0095] Furthermore, sections of kidney organoids can be stained with antibodies against one or more markers selected from markers expressed in the distal tubules, such as E-cadherin protein (ECAD), DBA protein, BRN1 protein, NCC protein, UROMODULIN protein, and SLC12A3 protein, and marker-positive areas can be determined to be distal tubules.

[0096] Furthermore, sections of kidney organoids can be stained with antibodies against one or more markers selected from markers expressed in the proximal tubules, such as JAGGED1 protein, LTL protein, SLC3A1 protein, CUBILIN protein, MEGALIN protein, AQP1 protein, and CDH6 protein, and marker-positive areas can be determined to be proximal tubules.

[0097] Furthermore, sections of kidney organoids can be stained with a marker expressed in collecting ducts (ureteric buds), such as an antibody against cytokeratin 8 (CK8) protein, and marker-positive areas can be determined to be collecting ducts (ureteric buds).

[0098] Furthermore, staining for species-specific antigens can determine whether a renal structure contains human cells or non-human mammalian cells. Examples of species-specific antigens include antigens present in either human or non-human mammalian cells, or antibodies that recognize partial amino acid sequences of the same antigen that differ between species. Markers well known to those skilled in the art can be used in combination as appropriate. For example, cells stained with an anti-CK8 antibody that stains only mouse cytokeratin 8 protein can be determined to be mouse cells. Furthermore, cells stained with an anti-HuNu antibody that stains the human-specific nuclear antigen HuNu, an anti-human Ku80 antibody that stains human Ku80 protein, an anti-STEM121 antibody that stains STEM121, an anti-HLA antibody that stains HLA, and the like can be determined to be human cells.

[0099] The chimeric kidney structure formation rate calculated by the above formula (1) may be calculated based on the results of immunochemical staining per section of a kidney organoid, or may be calculated based on the results of immunochemical staining of multiple sections derived from one or more kidney organoids.

[0100] In one aspect, the diameter of the chimeric kidney organoid of the present invention is 500 μm to 3,000 μm, preferably 700 μm to 2,000 μm, more preferably 800 μm to 2,000 μm, 800 μm to 1,500 μm, or 900 μm to 1,200 μm.

[0101] In this specification, the diameter of a chimeric kidney organoid can be measured, for example, from an image captured under a microscope, and the length (major axis) of the longest line segment connecting the two endpoints in the captured frontal image can be considered to be the diameter.

[0102] Furthermore, the chimeric kidney organoids of the present invention are characterized by the absence of excessive duct-like structures, which can be determined by the area occupied by duct-like structures in images taken under a microscope.

[0103] In one aspect, the invention encompasses chimeric kidney organoids comprising human cells and non-human mammalian cells, wherein the non-human mammal is a cetacean or a non-human primate, preferably a pig.

[0104] [Method for producing chimeric renal tissue] In one embodiment, the present invention provides a method for producing chimeric renal tissue comprising human nephron progenitor cells or cells derived from human nephron progenitor cells, and cells derived from a non-human mammalian fetal metanephroi, the method comprising the steps of: (a1) contacting the human nephron progenitor cells with the non-human mammalian fetal metanephroi-derived cells to obtain a cell population; and (b1) culturing the cell population obtained in step (a1) in a chimera-forming medium to obtain chimeric renal tissue, wherein the chimera-forming medium contains a Wnt signaling activator.

[0105] As used herein, the term "chimeric kidney tissue" is not particularly limited as long as it is a tissue containing a mixture of human nephron progenitor cells or cells derived from human nephron progenitor cells and cells derived from a non-human mammalian fetal metanephroi, and may be a chimeric cell aggregate (also referred to as a chimeric cell aggregate) or a non-human mammalian fetal metanephroi containing human nephron progenitor cells or cells derived from human nephron progenitor cells. The non-human mammalian fetal metanephroi may be a renal primordium with a bladder, which includes a ureter and a bladder. A renal primordium with a bladder is also called a cloaca.

[0106] The metanephros of a non-human mammalian fetus used herein is not particularly limited, as long as it is a metanephros at a developmental stage that contains nephron progenitor cells and ureteric bud cells or their precursor cells. For example, in the case of a mouse, metanephros from embryonic day 10 to postnatal day 5, preferably embryonic day 10 to 20, more preferably embryonic day 10 to 17, and even more preferably embryonic day 13 to 14 can be used. In the case of a pig, metanephros from embryonic day 15 to postnatal day 25, preferably embryonic day 15 to 60, more preferably embryonic day 20 to 32, and even more preferably embryonic day 28 to 30 can be used.

[0107] As used herein, the term "chimeric aggregate" is not particularly limited as long as it is a cell aggregate containing human nephron progenitor cells and cells derived from a non-human mammalian fetal metanephroi, and may be a concept that includes both cell aggregates that do not contain chimeric renal structures and chimeric renal organoids.

[0108] The human nephron progenitor cells used in step (a1) can be obtained by inducing differentiation of human pluripotent stem cells using methods known to those skilled in the art. Examples of pluripotent stem cells include embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells).

[0109] For example, during the process of culturing pluripotent stem cells, differentiation into nephron progenitor cells can be induced in a culture dish by culturing them in a medium containing factors appropriate for the appropriate differentiation stage. For example, human pluripotent stem cells can be induced to differentiate into human nephron progenitor cells by the following method.

[0110] Specifically, differentiation of pluripotent stem cells into nephron progenitor cells can be induced by contacting them with an appropriate combination of differentiation inducers selected from activin receptor kinase 4 and 7 signal activators such as activin A, fibroblast growth factors such as bFGF, FGF8, and FGF9, BMP signal activators such as BMP4 and BMP7, Wnt signal activators (including GSK3β inhibitors) such as CHIR99021, retinoic acids such as all-trans retinoic acid, ROCK inhibitors such as Y-27632, BMP signal inhibitors, and TGFβ inhibitors in an appropriate order and at an appropriate time.

[0111] The differentiation induction method includes at least (1) the step of culturing in a medium containing a Wnt signaling activator, and (2) the step of culturing in a medium containing an FGF9 signaling activator, but before or after each step, or during each step, the cells may be cultured in a medium containing another differentiation inducer. Specific differentiation induction methods can be appropriately selected from methods well known to those skilled in the art, and the following methods can be exemplified.

[0112] [Method 1 for Inducing Differentiation of Human Nephron Progenitor Cells] For example, human nephron progenitor cells can be induced to differentiate according to the methods described in the following publications (Taguchi A., et al., “Redefining the In Vivo Origin of Metanephric Nephron Progenitors Enables Generation of Complex Kidney Structures from Pluripotent Stem Cells,” Cell Stem Cell, 14, 53-67, 2014 (Non-Patent Document 1) and Taguchi A., et al., “Higher-Order Kidney Organogenesis from Pluripotent Stem Cells,” Cell Stem Cell, 21, 730-746, 2017)). Specifically, the method includes the following steps: (Step 1) culturing pluripotent stem cells in a medium containing bFGF and an activin receptor kinase 4,7 signal activator (e.g., activin A), and optionally a BMP signal activator (e.g., BMP4), (Step 2) culturing the cell population obtained in Step 1 in a medium containing a Wnt signal activator (e.g., a GSK3β inhibitor), (Step 3) culturing the cell population obtained in Step 2 in a medium containing a Wnt signal activator (e.g., a GSK3β inhibitor), an activin receptor kinase 4,7 signal activator (e.g., activin A), a BMP signal activator (e.g., BMP4), and retinoic acid, and (Step 4) culturing the cell population obtained in Step 3 in a medium containing a Wnt signal activator (e.g., a GSK3β inhibitor) and FGF9. The media in Steps 1 to 4 above may independently further contain a ROCK inhibitor.

[0113] [Method 2 for inducing differentiation of human nephron progenitor cells] For example, human nephron progenitor cells can also be induced to differentiate according to the method described in the following literature (Tsujimoto H., et al., A Modular Differentiation System Maps Multiple Human Kidney Lineages from Pluripotent Stem Cells, Cell Reports, 31, 107476, 2020). Specifically, the methods include the following steps: (Step 1) culturing pluripotent stem cells in a medium containing bFGF and a ROCK inhibitor; (Step 2) culturing the cell population obtained in Step 1 in a medium containing a Wnt signal activator (e.g., a GSK3β inhibitor), a BMP signal activator (e.g., BMP4), bFGF, and retinoic acid; (Step 3) culturing the cell population obtained in Step 2 in a medium containing a Wnt signal activator (e.g., a GSK3β inhibitor), a BMP signal activator (e.g., BMP7), and bFGF; (Step 4) culturing the cell population obtained in Step 3 in a medium containing a Wnt signal activator (e.g., a GSK3β inhibitor), a BMP signal activator (e.g., BMP7), bFGF, and a TGFβ signal inhibitor; (Step 5) culturing the cell population obtained in Step 4 in a medium containing FGF9, retinoic acid, and a BMP signal inhibitor (e.g., Noggin or LDN193189); and (Step 6) culturing the cell population obtained in Step 5 in a medium containing FGF9 and a Wnt signal activator (e.g., a GSK3β inhibitor).

[0114] [Method 3 for inducing differentiation of human nephron progenitor cells] Human nephron progenitor cells can also be induced to differentiate according to the method described in the following document (WO 2020 / 213734). Specifically, the method includes the following steps: (Step 1) culturing pluripotent stem cells in a medium containing an activin receptor kinase 4, 7 signal activator (e.g., activin A) and a Wnt signal activator (e.g., a GSK3β inhibitor); (Step 2) culturing the cell population obtained in Step 1 in a medium containing a Wnt signal activator (e.g., a GSK3β inhibitor), a BMP signal inhibitor (e.g., Noggin or LDN193189), and a TGFβ signal inhibitor, and optionally containing FGF8; (Step 3) culturing the cell population obtained in Step 2 in a medium containing a Wnt signal activator (e.g., a GSK3β inhibitor), a BMP signal inhibitor (e.g., Noggin or LDN193189), and an activin receptor kinase 4, 7 signal activator (e.g., activin A); (Step 4) culturing the cell population obtained in Step 3 in a medium containing FGF8 and an activin receptor kinase 4, 7 signal activator (e.g., activin A); and (Step 5) culturing the cell population obtained in Step 4 in a medium containing FGF9, a Wnt signal activator (e.g., a GSK3β inhibitor), a BMP signal inhibitor (e.g., Noggin or LDN193189), and a ROCK inhibitor.

[0115] [Method 4 for inducing differentiation of human nephron progenitor cells] Differentiation of human nephron progenitor cells can also be induced according to the method described in the following literature (Morizane, R. et al., Nephron organoids derived from human pluripotent stem cells model kidney development and injury, Nat. Biotechnol., 33 (11), 1193-1200, 2015. (Non-Patent Document 4)). Specifically, the steps include the following: (Step 1) culturing pluripotent stem cells in a medium containing bFGF and a ROCK inhibitor; (Step 2) culturing the cell population obtained in Step 1 in a medium containing a Wnt signal activator (e.g., a GSK3β inhibitor) and, optionally, a BMP signal inhibitor (e.g., Noggin or LDN193189); (Step 3) culturing the cell population obtained in Step 2 in a medium containing an activin receptor kinase 4, 7 signal activator (e.g., activin A); and (Step 4) culturing the cell population obtained in Step 3 in a medium containing FGF9.

[0116] [Cells derived from non-human mammal fetal metanephroi] Cells derived from non-human mammal fetal metanephroi can be obtained by removing the metanephroi of a non-human mammal fetus and recovering cells from the metanephroi. Here, metanephroi that has been removed and cryopreserved in advance can also be used as the metanephroi of a non-human mammal fetus. Dispersed cells derived from non-human mammal fetal metanephroi can be obtained by dissociating the metanephroi of a non-human mammal fetus into one to several cells, preferably into single cells. Dispersion of non-human mammal fetal metanephroi can be performed in the same manner as described above, by performing an enzyme treatment with trypsin, TrypLE Select (Thermo Fisher Scientific), Accutase (Thermo Fisher Scientific), or the like, which is typically used for cell dissociation, followed by pipetting, etc.

[0117] The metanephros of a non-human mammalian fetus used herein is not particularly limited, as long as it is a metanephros at a developmental stage that contains nephron progenitor cells and ureteric bud cells or their precursor cells. For example, in the case of a mouse, metanephros from embryonic day 10 to postnatal day 5, preferably embryonic day 10 to 20, more preferably embryonic day 10 to 17, and even more preferably embryonic day 13 to 14 can be used. In the case of a pig, metanephros from embryonic day 15 to postnatal day 25, preferably embryonic day 15 to 60, more preferably embryonic day 20 to 32, and even more preferably embryonic day 28 to 30 can be used.

[0118] [Regarding Step (a1)] When the chimeric kidney tissue is a chimeric aggregate, dispersed human nephron progenitor cells and dispersed non-human mammalian fetal metanephroi-derived cells are mixed in step (a1).

[0119] When the chimeric kidney tissue is a non-human mammal fetal metanephroi comprising human nephron progenitor cells or cells derived from human nephron progenitor cells, in step (a1), the human nephron progenitor cells are injected into the non-human mammal fetal metanephroi.

[0120] The non-human mammal fetal metanephroi may be introduced with a suicide gene targeting specific cells derived from the non-human mammal, such as nephron progenitor cells. Examples of suicide genes include genes introduced to eliminate cells upon contact with a specific drug using a drug-induced cell elimination system. More specific suicide genes include the diphtheria toxin receptor (Yamanaka S, et al., "Generation of interspecies-limited chimeric nephrons using a conditional nephron progenitor cell replacement system," Nature Communications 8, Article number 1719, 2017; Fukunaga S et al., "Optimal route of diphtheria toxin administration to eliminate native nephron progenitor cells in vivo for kidney regeneration," Biochemical and Biophysical Research Communications, 496 (4), 1176-1182, 2018).

[0121] Dispersing human nephron progenitor cells means dissociating human nephron progenitor cells that form an adhesive cell population (such as a layered cell population or a cell mass with a three-dimensional structure) into one to several cells, preferably into individual cells. Dispersion of human nephron progenitor cells can be performed by treating the cells with an enzyme typically used for cell dissociation, such as trypsin, TrypLE Select (Thermo Fisher Scientific), or Accutase (Thermo Fisher Scientific), followed by pipetting.

[0122] [Regarding Step (b1)] When the chimeric renal tissue is a cell aggregate containing human nephron progenitor cells and cells derived from a non-human mammalian fetal metanephroi, in step (b1), the cell population obtained in step (a1) is cultured in suspension in a chimera-forming medium to obtain the chimeric renal tissue as a chimeric aggregate. That is, the cell population obtained in step (a1) can be seeded in a low-adhesion culture vessel and cultured to obtain a chimeric aggregate. The culture vessel is not particularly limited as long as the cells do not adhere to the vessel and aggregate formation is not hindered, and U-bottom well plates, V-bottom well plates, etc. can be used. The culture period in step (b1) is preferably at least 12 hours, preferably 12 to 36 hours, more preferably 18 to 30 hours, for example, about 24 hours (e.g., 24±2 hours). In one embodiment, the size of the chimeric aggregate obtained by the production method of the present invention is 500 μm or more in diameter.

[0123] When the chimeric renal tissue is a non-human mammalian fetal metanephroi containing human nephron progenitor cells or cells derived from human nephron progenitor cells, in step (b1), the non-human mammalian fetal metanephroi obtained in step (a1) is cultured in a chimera-forming medium. There are no particular limitations on the culture vessel used, as long as it can accommodate the non-human mammalian fetal metanephroi. The culture time in step (b1) is at least 3 hours, preferably 6 hours to 7 days, and more preferably 12 hours to 3 days.

[0124] The non-human mammalian fetal metanephroi containing human nephron progenitor cells or cells derived from human nephron progenitor cells obtained by the production method of the present invention is superior to transplantation into a donor animal without performing step (b1) in that the human nephron cells derived from human nephron progenitor cells engraft and survive and are maintained after transplantation.

[0125] As the chimera formation medium, a medium obtained by adding a Wnt signal activator to a basal medium can be used. The Wnt signal activator is not particularly limited as long as it can enhance signal transduction mediated by Wnt. Specific examples of the Wnt signal activator include proteins belonging to the Wnt family (e.g., Wnt-1, Wnt-2, Wnt-2b, Wnt-3, Wnt-3a, Wnt-4, Wnt-5a, Wnt-5b, Wnt-6, Wnt-7a, Wnt-7b, Wnt-8, Wnt-8a, Wnt-8b, Wnt-9a, Wnt-9b, Examples of inhibitors include Wnt-10a, Wnt-10b, Wnt-11, Wnt-16, and Wnt-16b), Wnt receptor agonists, and GSK3β inhibitors (e.g., 6-bromoindirubin-3′-oxime (BIO), CHIR99021 (CAS number: 252917-06-9), Kenpaullone, and AZD2858). The concentration of the Wnt signal activator contained in the chimera formation medium is preferably a concentration that exhibits GSK3β inhibitory activity or Wnt signal activity equivalent to 0.1 to 5 μM, preferably 0.5 to 3 μM, when CHIR99021 is used, more preferably 0.5 to 2.5 μM, even more preferably 0.5 to 2 μM, particularly preferably 0.5 to 1.5 μM, and most preferably about 1 μM, specifically 1±0.2 μM, when CHIR99021 is used.

[0126] Examples of basal media include α-MEM medium, DMEM medium, F12 medium, DMEM / F12 medium, and mixed media thereof. The chimera formation medium may further contain medium supplements. Examples of medium supplements include glutamic acid, glutamate salts, glutamic acid-containing supplements such as GlutaMax (Thermo Fisher Scientific product name), aqueous amino acid solutions such as Non-Essential Amino Acids (Thermo Fisher Scientific), 2-mercaptoethanol, serum substitutes such as KnockOut Serum Replacement (KSR) (Thermo Fisher Scientific), and serum such as fetal bovine serum (FBS).

[0127] The chimera formation medium preferably further contains a fibroblast growth factor 9 (FGF9) signal activator. Examples of FGF9 signal activators include FGF9. The concentration of the FGF9 signal activator contained in the chimera formation medium is 1 to 20 ng / mL, preferably 3 to 15 ng / mL, and more preferably 5 to 10 ng / mL.

[0128] It is preferable that the chimera formation medium further contains a Rho-kinase (ROCK) inhibitor. Examples of ROCK inhibitors include Y-27632 (CAS No.: 129830-38-2), HA1077 (Fasudil; CAS No.: 103745-39-7), H-1152 (CAS No.: 871543-07-6), thiazovivin, GSK429286, AR13324 (Netarsudil), Ripasudil, RKI-1447, and Chroman 1. The concentration of the ROCK inhibitor contained in the chimera formation medium is 1 to 50 μM, preferably 5 to 30 μM, and more preferably 5 to 20 μM.

[0129] The chimera formation medium is preferably a serum-free medium. The serum-free medium need only contain no serum, and may contain a serum substitute. Examples of serum substitutes include KnockOut Serum Replacement. The chimera formation medium is preferably a medium that does not contain non-human animal-derived products (zeno-free medium). Furthermore, producing chimeric kidney tissue in a medium that does not contain animal-derived products facilitates medical applications.

[0130] [Method for producing chimeric kidney organoids] In one embodiment, the present invention provides a method for producing chimeric kidney organoids comprising human cells and non-human mammalian cells, the method comprising: a step (a2) of mixing dispersed human nephron progenitor cells and dispersed non-human mammalian fetal metanephroi-derived cells to obtain a cell population; a step (b2) of culturing the cell population obtained in step (a2) in a chimera-forming medium to obtain chimeric aggregates; and a step (c2) of culturing the chimeric aggregates obtained in step (b2) in a maturation medium to obtain chimeric kidney organoids comprising human cells and non-human mammalian cells, wherein the chimera-forming medium contains a Wnt signaling activator, and the maturation medium does not contain a Wnt signaling activator in at least a portion of step (c2).

[0131] As will be described later in the Examples, the production method of this embodiment makes it possible to produce chimeric renal organoids with a higher chimeric renal structure formation rate than conventional chimeric renal organoids.

[0132] In step (a2), the dispersed human nephron progenitor cells and dispersed non-human mammal fetal metanephroi-derived cells are mixed to obtain a cell population. Here, the human nephron progenitor cells, non-human mammal fetal metanephroi-derived cells, and their dispersion are similar to those in step (a1) described above.

[0133] When dispersed human nephron progenitor cells and dispersed non-human mammal fetal metanephroi-derived cells are mixed, the ratio of the number of human nephron progenitor cells to the number of non-human mammal fetal metanephroi-derived cells is preferably 5:1 to 1:1, more preferably about 3:1, specifically 3±0.3:1. As described below in the Examples, mixing cells within the above range improves the survival rate of human-derived cells, and as a result, chimeric kidney organoids with a high chimeric kidney structure formation rate (composition ratio of human-derived cells) tend to be obtained more easily.

[0134] Subsequently, in step (b2), the cell population obtained in step (a2) is cultured in a chimera formation medium to obtain chimeric aggregates. Here, the chimera formation medium described in the preceding section "Regarding step (b1)" can be used. That is, for chimera formation, a medium containing a Wnt signal activator added to a basal medium, preferably a medium containing a Wnt signal activator and an FGF9 signal activator, can be used. As described below in the Examples, by using a medium containing a Wnt signal activator and an FGF9 signal activator as the chimera formation medium, chimeric kidney organoids with a high chimeric kidney structure formation rate can be produced. Here, the chimera formation medium may contain other substances to the extent that they do not adversely affect the formation of chimeric kidney structures. In one aspect, the medium used in step (b2) can be a medium that does not contain signal activators or inhibitors such as activin, TGFβ, BMP, and sonic hedgehog.

[0135] The Wnt signal activator, FGF9 signal activator, basal medium, medium supplements, etc. are the same as those described above. The cell population obtained in step (a2) can be seeded and cultured in a low-adhesion culture vessel to obtain chimeric aggregates. The culture vessel is not particularly limited as long as the cells do not adhere to the vessel and aggregate formation is not hindered, but U-bottom well plates, V-bottom well plates, etc. can be used. Alternatively, a 2.5-dimensional culture vessel having a cell adhesion region only in a portion of the culture vessel can also be used.

[0136] The culture period in step (b2) is preferably at least 12 hours, preferably 12 to 36 hours, more preferably 18 to 30 hours, for example, about 24 hours, specifically 24±0.5 hours.

[0137] In one embodiment, suspension culture is performed in step (b2). The method of suspension culture is not particularly limited, and for example, the Serum-free Floating Culture of Embryoid Body-like Aggregates with Quick Reaggregation (SFEBq) method (Eiraku, M. et al., Self-organized formation of polarized cortical tissues from ESCs and their active manipulation by extrinsic signals, Cell Stem Cell, 3, 519-532, 2008) can be used.

[0138] Subsequently, in step (c2), the chimeric aggregate obtained in step (b2) is cultured in a maturation medium to obtain chimeric kidney organoids containing human cells and non-human mammalian cells. The maturation medium is a medium capable of differentiating the chimeric aggregate obtained in step (b2) into kidney organoids containing chimeric kidney structures, and a maturation medium not containing a Wnt signal activator is used in at least a part of step (c2).

[0139] For example, step (c2) may include a step (c2-1) of culturing the chimeric aggregate obtained in step (b2) in a maturation medium containing a Wnt signal activator, and a step (c2-2) of culturing the chimeric aggregate obtained in step (b2) in a maturation medium not containing a Wnt signal activator.

[0140] The duration of step (c2-1) is not particularly limited, and the cells may be cultured in the medium until cell aggregates having a three-dimensional structure become visible. Alternatively, the cells may be cultured in the medium until cells positive for markers such as WT1, LTL, or ECAD, which are expressed in the early stages of nephron development, appear. More specifically, for example, the cells may be cultured in a maturation medium containing a Wnt signaling activator for the first 24 to 72 hours, preferably 36 to 60 hours, and more preferably about 2 days, specifically 48±4 hours, of step (c2), and then cultured in a maturation medium not containing a Wnt signaling activator.

[0141] Here, the "maturation medium containing a Wnt signaling activator" in step (c2-1) can be appropriately selected from the chimera formation media described above, and may be the same as or different from the chimera formation medium used in step (b2). Specifically, a medium in which a Wnt signaling activator is added to a basal medium can be used.

[0142] In one embodiment, the "maturation medium containing a Wnt signal activator" in step (c2-1) may contain FGF9.

[0143] The "maturation medium free of Wnt signaling activator" in step (c2) or step (c2-2) may be a medium substantially free of Wnt signaling activator, and means that no Wnt signaling activator is intentionally added to the maturation medium. Therefore, the "medium free of Wnt signaling activator" is acceptable even if a trace amount (e.g., an amount equivalent to a concentration of about 20 nM or less) of Wnt signaling activator is inevitably mixed in due to carryover from the chimera formation medium during medium exchange, or if the Wnt signaling activator is detected as a result of expression and secretion by the cells themselves.

[0144] The maturation medium not containing a Wnt signaling activator may be the basal medium described above, or a medium obtained by adding the medium supplement described above to the basal medium described above, so long as it does not contain a Wnt signaling activator. The maturation medium may further contain the FGF9 signaling activator described above. The concentration of the FGF9 signaling activator contained in the maturation medium is 1 to 20 ng / mL, preferably 3 to 15 ng / mL, and more preferably 5 to 10 ng / mL. The maturation medium may further contain the Rho-kinase (ROCK) inhibitor described above. The concentration of the ROCK inhibitor contained in the maturation medium is 1 to 50 μM, preferably 5 to 30 μM, and more preferably 5 to 20 μM.

[0145] As described below in the Examples, by using a medium containing an FGF9 signal activator as the maturation medium, chimeric kidney organoids with a high chimeric kidney structure formation rate can be produced.

[0146] Step (c2) is a step of culturing the chimeric aggregates obtained in step (b2) in a maturation medium. The culture method is not particularly limited, and either adherent culture or suspension culture can be used. The culture in step (c2) is preferably adherent culture, and any culture vessel known to those skilled in the art may be used. Examples of culture vessels include well plates, flasks, and membranes (filters). Step (c2) is preferably performed by air-liquid interface culture, specifically, by culturing cells adherent to a filter immersed in a culture medium. For example, the cells can be seeded and cultured in a Transwell insert mounted in a well plate. The culture period in step (c2) can be at least the period until a chimeric kidney structure is formed, specifically, at least the period until WT1, LTL, ECAD (CDH1), or the like is detected. It is desirable to continue the culture in step (c2) until a glomerular marker such as nephrin is detected. The culture period in step (c2) is specifically 2 to 10 days, preferably 5 to 8 days, and more preferably 6 to 7 days.

[0147] In the production method of this embodiment, the chimera formation medium and / or maturation medium may be serum-free. More specifically, the chimera formation medium may be a serum-containing medium and the maturation medium may be serum-free, or the chimera formation medium may be serum-free and the maturation medium may be serum-containing, or both the chimera formation medium and the maturation medium may be serum-free. Here, the serum-free medium may contain a serum substitute.

[0148] In the production method of this embodiment, the non-human mammals are the same as those described above, and include rodents, cetaceans, non-human primates, etc. Rodents include mice and rats. Cetaceans include pigs and cows. Non-human primates include monkeys and baboons.

[0149] The production method of the present invention is useful for obtaining high-quality chimeric kidney organoids. For example, chimeric kidney organoids having a diameter of 500 μm to 3,000 μm, preferably 700 μm to 2,000 μm, more preferably 800 μm to 2,000 μm, 800 μm to 1,500 μm, or 900 μm to 1,200 μm can be obtained.

[0150] In one embodiment, 2 x 10 ５ When cells are seeded, chimeric kidney organoids of 900 μm to 1,200 μm can be obtained.

[0151] Here, the diameter of the chimeric kidney organoid can be measured, for example, from an image taken under a microscope, and the length of the longest line segment connecting the two endpoints in the captured frontal image can be considered to be the diameter.

[0152] Furthermore, the manufacturing method of the present invention can suppress the excessive generation of tubular structures, which can be determined by the area occupied by tubular structures in an image captured under a microscope.

[0153] In other words, the production method of the present invention makes it possible to obtain high-quality chimeric kidney organoids that maintain the above-mentioned size and have suppressed duct-like structures.

[0154] [Method for Evaluating the Ability of Human Nephron Progenitor Cells to Form Chimeric Kidney Structures] In one embodiment, the present invention provides a method for evaluating the ability of human nephron progenitor cells to form chimeric kidney structures, comprising: (a3) ​​mixing dispersed human nephron progenitor cells and dispersed non-human mammalian fetal metanephroi-derived cells to obtain a cell population; (b3) culturing the cell population obtained in step (a3) ​​in a chimera-forming medium to obtain chimeric aggregates; (c3) culturing the chimeric aggregates obtained in step (b3) in a maturation medium to obtain renal organoids comprising human cells and non-human mammalian cells; and (d3) determining the ability of the human nephron progenitor cells to form chimeric kidney structures based on the number of chimeric kidney structures and / or the type of cells contained in the renal organoids obtained in step (c3); wherein the chimera-forming medium in step (b3) contains a Wnt signaling activator, and the maturation medium does not contain a Wnt signaling activator in at least a portion of step (c3).

[0155] As described above, when producing human-non-human mammal chimeric kidney organoids, it is necessary for human nephron progenitor cells to have a high ability to form chimeras with non-human mammalian nephron progenitor cells, and for the chimeric kidney structure to be continuously matured to an appropriate stage based on this ability. The evaluation method of this embodiment allows the ability of human nephron progenitor cells to form chimeric kidney structures to be evaluated.

[0156] In step (a3), the dispersed human nephron progenitor cells and dispersed non-human mammal fetal metanephroi-derived cells are mixed to obtain a cell population. Here, the human nephron progenitor cells, non-human mammal fetal metanephroi-derived cells, and their dispersion are similar to those in step (a1) described above.

[0157] When dissociated human nephron progenitor cells and dissociated non-human mammal fetal metanephroi-derived cells are mixed, the ratio of the number of human nephron progenitor cells to the number of non-human mammal fetal metanephroi-derived cells is preferably 5:1 to 1:1, and more preferably about 3:1. As used herein, "about 3:1" refers to a ratio that allows for a variation of 3:1 of ±10% or less, more preferably ±5% or less. As described below in the Examples, mixing cells within the above range tends to facilitate the production of chimeric kidney organoids with a high rate of chimeric kidney structure formation.

[0158] Subsequently, in step (b3), the cell population obtained in step (a3) ​​is cultured in a chimera formation medium to obtain chimeric aggregates. Here, the chimera formation medium described in "Regarding step (b1)" can be used. That is, for chimera formation, a medium containing a basal medium supplemented with a Wnt signal activator can be used. Preferably, a medium containing a Wnt signal activator and an FGF9 signal activator can be used. The Wnt signal activator, FGF9 signal activator, basal medium, medium supplement, culture vessel, culture period, etc. are the same as those in step (b2) described above. Preferably, the chimera formation medium further contains a ROCK inhibitor.

[0159] Subsequently, in step (c3), the chimeric aggregates obtained in step (b3) are cultured in a maturation medium to obtain kidney organoids containing human cells and non-human mammalian cells. Here, the maturation medium, culture vessel, culture period, etc. are the same as those in step (c2) described above. In at least some steps of step (c3), a maturation medium not containing a Wnt signaling activator is used. For example, step (c3) may include a step (c3-1) of culturing the chimeric aggregates obtained in step (b3) in a maturation medium containing a Wnt signaling activator, and a step (c3-2) of culturing them in a maturation medium not containing a Wnt signaling activator. The period of culture in the medium containing a Wnt signaling activator is not particularly limited, and the aggregates may be cultured in the medium until a stage where cell aggregates having a three-dimensional structure can be visually recognized. Alternatively, the aggregates may be cultured in the medium until a stage where cells positive for markers such as WT1, LTL, or ECAD, which are expressed in the early stages of nephron development, appear. More specifically, for example, the cells can be cultured in a maturation medium containing a Wnt signaling activator for the first approximately two days of step (c3), and then cultured in a maturation medium not containing a Wnt signaling activator. Here, the "maturation medium containing a Wnt signaling activator" in step (c3-1) can be appropriately selected from the chimera formation media described above and may be the same as or different from the chimera formation medium used in step (b3). Specific examples include a medium in which a Wnt signaling activator is added to a basal medium.

[0160] The "maturation medium free of Wnt signaling activator" in step (c3) or step (c3-2) may be a medium substantially free of Wnt signaling activator, and means that no Wnt signaling activator is intentionally added to the maturation medium. Therefore, the "medium free of Wnt signaling activator" is acceptable even if a trace amount (e.g., an amount equivalent to a concentration of about 20 nM or less) of Wnt signaling activator is inevitably mixed in due to carryover from the chimera formation medium during medium exchange, or if the Wnt signaling activator is detected as a result of expression and secretion by the cells themselves.

[0161] Next, in step (d3), the ability of the human nephron progenitor cells to form chimeric renal structures is determined based on the number of chimeric renal structures and / or the type of cells contained in the chimeric renal structures contained in the renal organoids obtained in step (c3).

[0162] In one embodiment, if a chimeric renal structure is present in the renal organoid obtained in step (c3), it can be determined that the human nephron progenitor cells have the ability to form a chimeric renal structure.

[0163] In one embodiment, the chimeric kidney structure formation rate in the kidney organoids obtained in step (c3) is calculated using the following formula (1), and the ability of human nephron progenitor cells to form chimeric kidney structures can be evaluated.

[0164] Chimeric kidney structure formation rate (%) = number of chimeric kidney structures containing human cells and non-human mammalian cells / number of total kidney structures × 100 (1)

[0165] The calculated chimeric renal structure formation rate corresponds to the ability of human nephron progenitor cells to form chimeric renal structures. Therefore, the ability of human nephron progenitor cells to form chimeric renal structures can be evaluated or determined based on the calculated chimeric renal structure formation rate. The kidney structures and chimeric renal structure formation rate are the same as those described above.

[0166] In one embodiment, the suitability of human nephron progenitor cells as a transplant material, or a raw material or intermediate for its production, can be determined based on the numerical value of the chimeric renal structure formation rate. Here, a threshold value for determining suitability can be set appropriately depending on the purpose. For example, when the chimeric renal structure formation rate calculated in step (c3) is, for example, 30% or more, 40% or more, or 50% or more, it may be determined that the human nephron progenitor cells used in the evaluation method are usable as a transplant material, or a raw material or intermediate for its production. The chimeric renal structure formation rate used as the judgment criterion in the evaluation method may be set appropriately depending on the purpose. Furthermore, the higher the numerical value of the chimeric renal structure formation rate, the higher the quality of the human nephron progenitor cells may be evaluated or determined.

[0167] In one embodiment, the suitability of the method for producing a chimeric renal structure, specifically the suitability of each culture condition, medium component, and medium component concentration, can be evaluated or determined based on the numerical value of the chimeric renal structure formation rate. Furthermore, the higher the numerical value of the chimeric renal structure formation rate, the higher the suitability of the method for producing a chimeric renal structure can be evaluated or determined.

[0168] In one embodiment, the ability to form a chimeric kidney structure can be evaluated or determined by one or more indicators selected from the following: (1) the number of glomerular structures containing human cells and non-human mammalian cells, (2) the number of tubular structures containing human cells and non-human mammalian cells, (3) the number of nephron segment structures containing human cells and non-human mammalian cells, (4) the number of ureteric bud structures derived from a non-human mammal containing human nephron progenitor cells, human distal tubule constituent cells, and / or human proximal tubule constituent cells, and (5) the proportion of human cells in the kidney structure.

[0169] Based on the above-mentioned indicators, it may be determined that the human nephron progenitor cells used in the evaluation method can be used as a transplant material, or as a raw material or intermediate for the production thereof. Furthermore, it may be evaluated or determined that the more of the above-mentioned indicators that are satisfied, the higher the quality of the human nephron progenitor cells.

[0170] In one aspect, when determining the ability to form a chimeric kidney structure based on the above (1) to (4), the ability to form a chimeric kidney structure may be evaluated in absolute terms, or may be evaluated or determined as a percentage of (1) the total number of glomerular structures, (2) the total number of tubular structures, (3) the total number of nephron segment structures, or (4) the total number of ureteric bud structures. In this case, by setting a reference value, it is possible to evaluate or determine that the quality of human nephron progenitor cells is sufficient when the reference value is met or exceeded.

[0171] In one embodiment, the ability to form a chimeric kidney structure may be determined based on (5). For example, the ratio of the number of human cells to the total number of cells contained in the kidney structure can be evaluated as the proportion of human cells. Alternatively, the proportion of human cells can be evaluated as the area ratio of the human cell region to the cross-sectional area of ​​any cross section of the kidney structure, preferably a cross section including the center (the section with the largest area) of the kidney structure.

[0172] The number of glomerular structures can be determined by detecting marker proteins or genes expressed by cells constituting the glomeruli, such as WT1, nephrin, podocalyxin, and podocin.

[0173] The number of tubular structures can be determined by detecting marker proteins or genes expressed by cells constituting the tubules. Here, the tubules are classified into distal tubules and proximal tubules. Markers expressed in cells constituting the distal tubules include ECAD, JAGGED1, BRN1, UROMODULIN, and NCC. Markers expressed in cells constituting the proximal tubules include LTL, MEGALIN, AQP1, and CDH6.

[0174] The number of ureteric bud structures derived from a non-human mammal, which contain human nephron progenitor cells and / or human distal tubule constituent cells, can be detected by detecting marker proteins or genes expressed by human nephron progenitor cells.

[0175] Human distal tubules can be detected using markers expressed in the cells that make up the distal tubules. Specifically, they can be distinguished using a marker specific to human-derived cells, such as Ku80, in addition to a distal tubule marker.

[0176] The number of ureteric bud structures can be determined by detecting marker proteins or genes expressed by cells that constitute the ureteric bud, such as CK8, GATA3, and AQP2.

[0177] Furthermore, ureteric buds derived from non-human mammals that contain human nephron progenitor cells can be identified by the presence of a CAP structure, which will be described later. In this case, the presence of a CAP structure derived from a non-human mammal allows the identification of human-derived nephron progenitor cells among the nephron progenitor cells.

[0178] Furthermore, ureteric buds derived from non-human mammals containing human distal tubule-forming cells can be identified by the chimeric structure of the junction between the ureteric bud and the ureteric tubule, which can be distinguished by the presence of both a region containing Ku80-positive cells and a region containing CK8-positive cells in the structure of ECAD-positive cells, which are markers of both the ureteric bud and the distal tubule.

[0179] As used herein, "nephron segment" refers to a cell type (a differentiation product derived from a nephron progenitor cell) that constitutes a nephron and generally refers to a collection of glomeruli, proximal tubules, and distal tubules. As used herein, "nephron segment structure" refers to a structure that includes glomerular structure and / or tubular structure, and the presence or absence or number of such structures can be determined by detecting markers expressed in cells that constitute the glomeruli or tubules.

[0180] The proportion of human cells can be determined by detecting a marker protein or gene that is specifically expressed in human-derived cells and calculating the proportion of the total number of cells, including cells derived from non-human animals. Alternatively, the proportion of human cells can be determined by calculating the area ratio of the region expressing the marker protein or gene, instead of the cell number. Examples of markers specifically expressed in human-derived cells include HuNu, Ku80, STEM121, and HLA.

[0181] In the evaluation method of this embodiment, the chimera formation medium and / or maturation medium may be serum-free. More specifically, the chimera formation medium may be a serum-containing medium and the maturation medium may be serum-free, or the chimera formation medium may be a serum-free medium and the maturation medium may be serum-containing, or both the chimera formation medium and the maturation medium may be serum-free.

[0182] In the evaluation method of this embodiment, non-human mammals are the same as those described above, and include rodents, cetaceans, non-human primates, etc. Rodents include mice and rats. Cetaceans include pigs and cows. Non-human primates include monkeys and baboons.

[0183] [Pig kidney organoid] In one embodiment, the present invention provides a pig kidney organoid comprising at least two types of cells selected from the group consisting of nephron constituent cells or nephron progenitor cells, collecting duct (ureter, ureteric bud) constituent cells or collecting duct progenitor cells, and renal interstitial cells or renal interstitial progenitor cells, and having the function of kidney constituent cells or the function of progressing kidney development, and having multiple kidney structures inside.

[0184] As will be described later in the Examples, there have been no reports of the production of pig kidney organoids, and the inventors are the first to succeed in producing pig kidney organoids. The pig kidney organoids of this embodiment are pig kidney organoids that contain glomeruli, distal tubules, and collecting ducts, which are adjacent to each other and exhibit the structures of glomeruli, tubules, and ureteric buds.

[0185] The porcine kidney organoid of this embodiment has multiple renal structures therein. In one embodiment, the renal structures are not localized to a specific location within the organoid, but exist randomly. In one embodiment, the porcine kidney organoid of this embodiment has a CAP structure, which is characteristic of tissue in the middle of nephron development, including a ureteric bud, nephron progenitor cells, and interstitial progenitor cells, and in which a layer of nephron progenitor cells surrounds the tip of the ureteric bud. The CAP structure does not have a specific orientation, and the boundary between the outer (cortex) and the inner (medulla) is not clear. One or more CAP structures are randomly scattered throughout the organoid, and glomeruli, tubules, and collecting ducts are present not only on the surface of the organoid but also inside the organoid. In this respect, it is clearly different from the structure of fetal metanephric tissue, in which CAP structures are arranged in a specific direction on the outside of the kidney (directly below the kidney capsule) and are aggregated into collecting ducts.

[0186] A normal adult kidney has a layered structure consisting of an outer cortex made up of renal corpuscles (including glomeruli and Bowman's capsule) and some tubules, and an inner medulla made up of proximal tubules, loops of Henle, distal tubules, and collecting ducts, and each layer can be clearly distinguished by visual inspection.

[0187] The porcine kidney organoids of this embodiment are characterized by a small imbalance in the number of glomerular cells and distal tubule cells, and a constituent cell ratio that is close to that of a living kidney.

[0188] In one aspect, the diameter of the porcine kidney organoid of the present invention is 500 μm to 3,000 μm, preferably 700 μm to 2,000 μm, more preferably 800 μm to 2,000 μm, 800 μm to 1,500 μm, or 900 μm to 1,200 μm.

[0189] Here, the diameter of the porcine kidney organoid can be measured, for example, from an image captured under a microscope, and the length of the longest line segment connecting the two endpoints in the captured frontal image can be considered to be the diameter.

[0190] Furthermore, the porcine kidney organoids of the present invention are characterized by the absence of excessive duct-like structures, which can be evaluated by the area occupied by duct-like structures in images taken under a microscope.

[0191] [Method for producing kidney organoids comprising non-human mammalian cells] In one embodiment, the present invention provides a method for producing kidney organoids comprising non-human mammalian cells, comprising the steps of: (a4) culturing a population of dispersed cells derived from a non-human mammalian fetal metanephroi in a reaggregation medium to obtain aggregates; and (b4) culturing the aggregates obtained in step (a4) in a maturation medium to obtain kidney organoids comprising non-human mammalian cells, wherein the reaggregation medium in step (a4) contains a Wnt signal activator; and in at least a portion of step (b4), the maturation medium does not contain a Wnt signal activator.

[0192] As will be described later in the Examples, the production method of this embodiment makes it possible to produce kidney organoids containing non-human mammalian cells.

[0193] In step (a4), the dispersed population of non-human mammal fetal metanephroi-derived cells is cultured in a reaggregation medium to obtain aggregates. Here, the non-human mammal fetal metanephroi-derived cells and the dispersion of non-human mammal fetal metanephroi-derived cells are similar to those in step (a1) described above.

[0194] The reaggregation medium can be the chimera formation medium described in "Regarding step (b1)." That is, a medium obtained by adding a Wnt signal activator, preferably a Wnt signal activator and an FGF9 signal activator, to a basal medium can be used as the reaggregation medium. The Wnt signal activator, FGF9 signal activator, basal medium, medium supplement, culture vessel, culture period, etc. are the same as those for the chimera formation medium in step (b2) described above. Preferably, the reaggregation medium further contains a ROCK inhibitor.

[0195] Subsequently, in step (b4), the aggregates obtained in step (a4) are cultured in a maturation medium to obtain kidney organoids containing non-human mammalian cells, where the maturation medium, culture vessel, culture period, etc. are the same as those in step (c2) described above.

[0196] That is, the maturation medium is a medium capable of differentiating the aggregates obtained in step (a4) into renal organoids containing renal structures, and a maturation medium not containing a Wnt signaling activator is used in at least a portion of step (b4). For example, step (b4) may include a step (b4-1) of culturing the chimeric aggregates obtained in step (a4) in a maturation medium containing a Wnt signaling activator, and a step (b4-2) of culturing them in a maturation medium not containing a Wnt signaling activator. The duration of step (b4-1) is not particularly limited, and the aggregates may be cultured in the medium until a stage at which cell aggregates having a three-dimensional structure can be visually recognized. Alternatively, the aggregates may be cultured in the medium until a stage at which cells positive for markers such as WT1, LTL, or ECAD, which are expressed in the early stages of nephron development, appear. More specifically, for example, in step (b4), the cells can be cultured in a maturation medium containing a Wnt signaling activator for the first 24 to 72 hours, preferably 36 to 60 hours, and more preferably about 2 days, specifically 48±4 hours, and then cultured in a maturation medium not containing a Wnt signaling activator.

[0197] Here, the "maturation medium containing a Wnt signaling activator" in step (b4-1) can be appropriately selected from reaggregation media, and may be the same as or different from the reaggregation medium used in step (a4). Specific examples include a medium in which a Wnt signaling activator is added to a basal medium.

[0198] In one embodiment, the "maturation medium containing a Wnt signaling activator" in step (b4-1) may contain FGF9.

[0199] The "maturation medium free of Wnt signaling activator" in step (b4) or step (b4-2) may be a medium substantially free of Wnt signaling activator, and means that no Wnt signaling activator is intentionally added to the maturation medium. Therefore, the "medium free of Wnt signaling activator" is acceptable even if a trace amount (e.g., an amount equivalent to a concentration of about 20 nM or less) of Wnt signaling activator is inevitably mixed in due to carryover from the reaggregation medium during medium exchange, or if a Wnt signaling activator is detected as a result of expression and secretion by the cells themselves.

[0200] The maturation medium that does not contain a Wnt signaling activator may be the above-mentioned basal medium, or a medium obtained by adding the above-mentioned medium supplement to the above-mentioned basal medium, as long as it does not contain a Wnt signaling activator.

[0201] The maturation medium may contain the above-mentioned FGF9 signal activator. The concentration of the FGF9 signal activator contained in the maturation medium may be 1 to 20 ng / mL, preferably 3 to 15 ng / mL, and more preferably 5 to 10 ng / mL. The maturation medium may further contain the above-mentioned Rho-kinase (ROCK) inhibitor. The concentration of the ROCK inhibitor contained in the maturation medium may be 1 to 50 μM, preferably 5 to 30 μM, and more preferably 5 to 20 μM.

[0202] The culture period in step (b4) may be at least the period until a kidney structure is formed, specifically at least the period until WT1, LTL, ECAD (CDH1), etc. are detected. The culture in step (b4) is preferably continued until a glomerular marker such as nephrin is detected. The culture period in step (b2) may be specifically 2 to 10 days, preferably 5 to 8 days, and more preferably 6 to 7 days.

[0203] In the production method of this embodiment, the reaggregation medium and / or the maturation medium may be a serum-free medium. More specifically, the reaggregation medium may be a serum-containing medium and the maturation medium may be a serum-free medium, or the reaggregation medium may be a serum-free medium and the maturation medium may be a serum-containing medium, or both the reaggregation medium and the maturation medium may be serum-free medium. Preferably, both the reaggregation medium and the maturation medium are serum-free medium.

[0204] In the production method of this embodiment, the non-human mammals are the same as those described above, and include rodents, cetaceans, non-human primates, etc. Rodents include mice and rats, etc. Cetaceans include pigs and cows, and non-human primates include monkeys and baboons.

[0205] [Pharmaceutical Compositions, Therapeutic Drugs, and Transplantation Materials] In one embodiment, the present invention provides a renal organoid (hereinafter sometimes referred to as the "chimeric renal organoid of the present invention") having a chimeric renal structure comprising a human and a non-human mammal, wherein the non-human mammal is a cetacean or a non-human primate, or a pharmaceutical composition comprising the above-mentioned pig renal organoid.

[0206] The pharmaceutical composition comprises the chimeric kidney organoids or porcine kidney organoids of the present invention and a pharmaceutically acceptable carrier.

[0207] As a pharmaceutically acceptable carrier, a physiological aqueous solvent (e.g., physiological saline, buffer solution, serum-free medium, etc.) can be used. If necessary, the pharmaceutical composition may contain preservatives, stabilizers, reducing agents, isotonicity agents, etc.

[0208] The pharmaceutical composition can be used to treat diseases caused by kidney damage or injury, such as renal failure, renal malformations such as hypoplastic kidneys, and urinary tract malformations.

[0209] In one embodiment, the present invention provides a therapeutic agent for diseases caused by kidney damage or injury, comprising the chimeric kidney organoid or pig kidney organoid of the present invention.

[0210] In this embodiment, the chimeric kidney organoids or pig kidney organoids of the present invention can be used as transplantation materials for patients in need of kidney transplantation.

[0211] There is no particular limitation on the number of cells used for each transplantation, but the number of transplanted cells is 1.0 × 10 ３ pieces ~ 2.5×10 ９ The number of cells in each organoid (aggregate) can be 1 to 500. There is no particular limitation on the number of cells contained in one organoid, but typically 1.0 × 10 cells per organoid. ３ pieces ~ 5.0×10 ６ The cell may contain cells.

[0212] The method for transplanting the chimeric kidney organoids or porcine kidney organoids of the present invention is not particularly limited, but they can usually be transplanted near the renal artery, near the aorta, under the renal capsule, subcutaneously, etc. Specifically, a depression (pocket) may be formed in the site or tissue to be transplanted, and the chimeric kidney organoids or porcine kidney organoids of the present invention may be placed in the pocket. After placement, the pocket may be covered with surrounding tissue and closed.

[0213] In this embodiment, when administering the chimeric kidney organoid or pig kidney organoid of the present invention to a recipient, an immunosuppressant can be used in combination. For example, the immunosuppressant described in Patent Document 2 or Non-Patent Document 6 can be used.

[0214] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0215] [Materials and Methods] (Chimera formation medium and reaggregation medium) The following media were used as chimera formation medium and reaggregation medium. (1) Basal medium containing 10% fetal bovine serum (FBS) and 10 μM Y-27632 (hereinafter, sometimes referred to as "10FBS medium"). DMEM / F12 was used as the basal medium. (2) 1 μM CHIR99021 (CAS number: 252917-06-9), 5 ng / mL recombinant human FGF9, 10 μM Y-27632, 2% (vol / vol) B27 supplement (Thermo Fisher Scientific, B-27 ＴＭThe basal medium used was DMEM / F12, which contained 2 mM L-glutamine, 1% (vol / vol) ITS (insulin, transferrin, and sodium selenite supplement; PeproTech, ITS (Insulin-Transferrin-Selenium) (100x)), 1% (vol / vol) non-essential amino acid (Thermo Fisher Scientific, MEM Non-Essential Amino Acids Solution (100x)), and 90 μM 2-mercaptoethanol.

[0216] In this specification, when chimera formation is carried out using this medium, this medium is sometimes referred to as a chimera formation medium, and when chimera formation is not carried out using this medium, this medium is sometimes referred to as a reaggregation medium.

[0217] (Maturation medium) The following media were used as maturation media. (1) Basal medium containing 10% FBS (10FBS medium). DMEM / F12 was used as the basal medium. (2) Basal medium containing 1 μM CHIR99021, 10 ng / mL recombinant human FGF9, 2% (vol / vol) B27 supplement, 2 mM L-glutamine, 1% (vol / vol) ITS, 1% (vol / vol) Nonesential amino acid, and 90 μM 2-mercaptoethanol. However, from day 3 of maturation culture, the medium with the above composition without CHIR99021 was used (hereinafter sometimes referred to as "F9 medium"). DMEM / F12 was used as the basal medium. (3) 4.5 μM CHIR99021, 200 ng / mL recombinant human FGF2, 5% KSR (KnockOut ＴＭ DMEM / F12 medium containing 1x Non-Essential Amino Acids, 0.1 μM 2-mercaptoethanol, and GlutaMAX (hereinafter sometimes referred to as "KR5 medium").

[0218] (Preparation of Mouse Embryonic Metanephroi-Derived Cells) Metanephroi were excised from embryonic day 13.5 mice (C57BL / 6J) and collected in a 1.5 mL tube containing basal medium. Subsequently, the mixture was centrifuged at 700 × g for 3 minutes, and the supernatant was discarded. MEMα was used as the basal medium. Next, 1 mL of Accutase (Thermo Fisher Scientific) was added and mixed using a vortex mixer. After incubation for 5 minutes, the mixture was pipetted, mixed again using a vortex mixer, and centrifuged at 300 × g for 5 minutes. The precipitated cells were dissociated into single cells and suspended in medium and used in the following experiments.

[0219] (Preparation of fetal porcine metanephroi-derived cells) Metanephroi were excised from 30-day-old pigs (microminipigs) and collected in a 1.5 mL tube containing basal medium. Subsequently, the mixture was centrifuged at 700 × g for 3 minutes, and the supernatant was discarded. MEMα was used as the basal medium. Next, 1 mL of Accutase (Thermo Fisher Scientific) was added and mixed using a vortex mixer. After incubation for 5 minutes, the mixture was pipetted, mixed again using a vortex mixer, and centrifuged at 300 × g for 5 minutes. The precipitated cells were dissociated into single cells and suspended in medium and used in the following experiments.

[0220] (Preparation of human nephron progenitor cells) Human iPS cells (iPS cell line established by the inventors) were cultured at 1.0 × 10 ４ The cells were seeded at 100 cells / well in a U-bottom 96-well plate (slit-well) and induced to differentiate according to the method described in Taguchi, A et al., Higher-Order Kidney Organogenesis from Pluripotent Stem Cells, Cell Stem Cell, 21, 730-746, 2017, to obtain human nephron progenitor cells.

[0221] Specifically, on the day of induction initiation (day 0), 10,000 iPSCs were seeded per well into a PrimeSurface 96-slit well plate (Sumitomo Bakelite) using a medium containing 3 ng / mL human activin A (R&D Systems), 1 ng / mL BMP4 (R&D Systems), 20 ng / mL human basic fibroblast growth factor (b-FGF) (R&D Systems), and 10 μM Y27632 (Fujifilm Wako Pure Chemical Industries) in a basal medium for human nephron progenitor cells (NPC) (see below).

[0222] The NPC basal medium was prepared using 2% (vol / vol) B27 supplement (Thermo Fisher Scientific, B-27 ＴＭ DMEM / F12 medium containing 1% (vol / vol) ITS (insulin, transferrin, and sodium selenite supplement (50X, minus vitamin A) was used), 2 mM L-glutamine, 1% (vol / vol) ITS (insulin-transferrin-selenium) (100X) from PeproTech was used), 1% (vol / vol) non-essential amino acid (MEM Non-Essential Amino Acids Solution (100X) from Thermo Fisher Scientific was used), and 90 μM 2-mercaptoethanol was used.

[0223] After 24 hours of incubation (day 1), the medium was replaced with NPC basal medium containing 10 μM CHIR99021 (Fujifilm Wako Pure Chemical Industries, Ltd.). Every other day thereafter (days 3 and 5), half of the medium was replaced with NPC basal medium containing 10 μM CHIR99021 (Fujifilm Wako Pure Chemical Industries, Ltd.) and 10 μM Y27632 (Fujifilm Wako Pure Chemical Industries, Ltd.).

[0224] On day 7, the medium was replaced with NPC basal medium supplemented with 10 ng / mL human activin A (R&D Systems), 5 ng / mL human BMP4 (R&D Systems), 3 μM CHIR99021, 0.1 μM retinoic acid (Sigma-Aldrich), and 10 μM Y27632 (Fujifilm Wako Pure Chemical Industries).

[0225] On day 10, the medium was replaced with NPC basal medium supplemented with 1 μM CHIR99021 (Fujifilm Wako Pure Chemicals), 5 ng / mL fibroblast growth factor-9 (FGF-9) (Abcam), and 10 μM Y27632 (Fujifilm Wako Pure Chemicals).

[0226] Spheres were harvested on day 13 and used for experiments. NPCs for injection into mouse fetal kidneys were derived according to a previously reported method (K. Matsui et al., "Long-term viable chimeric nephrons generated from progenitor cells are a reliable model in cisplatin-induced toxicity," Commun Biol. 6, 1097, 2023).

[0227] The resulting human nephron progenitor cells were collected in a 15 mL tube, and 1 mL of 0.5-fold diluted TryPLE SELECT was added. The cells were incubated for 10 minutes and then dissociated into single cells by pipetting. Subsequently, the cells were labeled with ITGA8 antibody and purified by magnetic-activated cell sorting (MACS) to select only ITGA8-positive human nephron progenitor cells. The sorted cells were dissociated into single cells by pipetting, suspended in medium, and used in the following experiments.

[0228] Experimental Example 1 (Preparation of Renal Organoids 1: Human Renal Organoids) Human kidney organoids (human nephron organoids) were prepared. For comparison, mouse kidney organoids were also prepared. Human nephron progenitor cells dissociated into single cells were suspended in a reaggregation medium and collected at 2.0 × 10 ５The cells were seeded at a cell density of 100 cells / well on a low-adhesion 96-well plate to form aggregates. The reaggregation medium used was the 10 FBS medium described above. Furthermore, mouse fetal metanephroi-derived cells dissociated into single cells were suspended in the reaggregation medium and resuspended at 2.0 × 10 cells / well. ５ The cells were seeded at a cell density of 10 cells / well onto a low-adhesion 96-well plate to form aggregates. The above-mentioned 10 FBS medium was used as the reaggregation medium.

[0229] After 24 hours, the aggregates were seeded into Transwell inserts mounted in a 96-well plate and cultured at the air-liquid interface in maturation medium, which was the 10% FBS medium described above.

[0230] Figure 1 shows microscopic images of kidney organoids on days 0 (24 hours after the start of reaggregation), 2, and 6 of maturation culture. As a result, mouse kidney organoids were formed. On the other hand, human nephron progenitor cells underwent cell death, and human kidney organoids (human nephron organoids) could not be formed. These results demonstrate that known culture methods are not suitable for culturing human nephron progenitor cells.

[0231] [Experimental Example 2] (Preparation of Renal Organoids 2: Human Renal Organoids) To investigate a medium suitable for aggregating human nephron progenitor cells and maturing them into organoids, human kidney organoids (human nephron organoids) were prepared in the same manner as in Experimental Example 1, except that the maturation medium was changed. As in Experimental Example 1, 10FBS medium was used as the reaggregation medium. The above-mentioned F9 medium and KR5 medium were used as the maturation medium. For comparison, an experiment was also conducted using 10FBS medium as the maturation medium.

[0232] Figure 2-1 shows microscopic images of kidney organoids on days 0, 2, and 6 of maturation culture. In Figure 2-1, "10FBS → 10FBS" indicates the results when 10FBS medium was used as the reaggregation medium and 10FBS medium was used as the maturation medium, and the same applies below. As a result, when 10FBS medium was used as both the reaggregation medium and the maturation medium, the reaggregates of human NPCs disappeared, as shown in Experimental Example 1. On the other hand, by using F9 medium or KR5 medium as the maturation medium, human kidney organoids (human nephron organoids) containing organoid-like three-dimensional structures were formed. However, in F9 medium, the organoid structure was scarce and the overall size was small. Specifically, the diameter of the human kidney organoids (human nephron organoids) was less than 1,000 μm. In KR5 medium, the organoid structure was abundant and of good size, but excessive duct-like structures were observed.

[0233] As described above, we were unable to form high-quality human kidney organoids (human nephron organoids).

[0234] Figure 2-2 is a microscopic image of another lot of human kidney organoids (human nephron organoids) produced in the same manner as in Experimental Example 2, on day 6 of maturation culture.

[0235] [Experimental Example 3] (Preparation of Renal Organoids 3: Mouse Renal Organoids) Mouse kidney organoids were prepared in the same manner as in Experimental Example 1, except that the maturation medium was changed. 10FBS medium was used as the reaggregation medium. The above-mentioned F9 medium or KR5 medium was used as the maturation medium. For comparison, an experiment was also conducted using 10FBS medium as the maturation medium.

[0236] Figure 3-1 shows microscopic images of kidney organoids on days 0, 2, and 6 of maturation culture. As a result, in the case of mouse embryonic metanephric cells, abundant organoid-like three-dimensional structures were observed, and kidney organoids of good size were formed regardless of the maturation medium used. However, on the other hand, excessive duct-like structures were observed in the group using KR5 medium as the maturation medium, and kidney organoids of better quality were formed when F9 medium was used as the maturation medium than when KR5 medium was used.

[0237] Figure 3-2 is a microscopic image of another lot of mouse kidney organoids produced in the same manner as in Experimental Example 3 on day 6 of maturation culture.

[0238] [Experimental Example 4] (Preparation of Renal Organoids 4: Human-Mouse Chimeric Renal Organoids) Human-mouse chimeric kidney organoids were prepared. 1.5 x 10 human nephron progenitor cells dissociated into single cells were cultured. ５ 0.5 × 10 cells / well and 0.5 × 10 cells / well ５ The cells were mixed at a concentration of 10 cells / well, suspended in a chimera formation medium, and seeded onto a low-adhesion 96-well plate to form aggregates. The chimera formation medium used was the 10 FBS medium described above.

[0239] After 24 hours, the aggregates were seeded into Transwell inserts mounted in a 96-well plate and cultured at the air-liquid interface in maturation medium, which was the above-mentioned 10FBS medium, F9 medium, or KR5 medium.

[0240] Figure 4 shows microscopic images of renal organoids on days 0, 2, and 6 of culture. The results show that cell aggregates with organoid-like three-dimensional structures were formed regardless of the medium used as the maturation medium. Figure 5 shows microscopic photographs of a different lot of human-mouse chimeric renal organoids produced in the same manner as in this experimental example.

[0241] When 10% FBS medium was used for both the chimera formation medium and maturation medium, good organoid-like structures were observed on the second day of culture, but by the sixth day of culture, the organoid structure had decreased and the overall size had also shrunk (partially disappeared). On the other hand, when F9 medium or KR5 medium was used as the maturation medium, organoid-like three-dimensional structures were formed. No morphological abnormalities were observed in F9 medium, but excessive duct-like structures were observed in KR5 medium. In other words, particularly by using F9 medium as the maturation medium, high-quality kidney organoids could be formed.

[0242] [Experimental Example 5] (Preparation of Renal Organoids 5: Human-Mouse Chimeric Renal Organoids) Human-mouse chimeric kidney organoids were prepared. 1.5 x 10 dissociated human nephron progenitor cells were cultured in a 2000-well plate. ５ 0.5 × 10 cells / well and 0.5 × 10 cells / well ５ The cells were mixed at a concentration of 1000 / well, suspended in a chimera formation medium, and plated onto a low-adhesion 96-well plate to form aggregates. The chimera formation medium used was the C1F9Y medium described above.

[0243] After 24 hours, the aggregates were seeded into Transwell inserts mounted in a 96-well plate and cultured at the air-liquid interface in maturation medium, which was the above-mentioned 10FBS medium, F9 medium, or KR5 medium.

[0244] Figure 6 shows microscopic images of kidney organoids on days 0, 2, and 6 of maturation culture. The results show that cell aggregates with organoid-like three-dimensional structures were formed regardless of the medium used as the maturation medium.

[0245] In the group using 10FBS medium as the maturation medium, good organoid-like structures were observed on the second day of culture, but by the sixth day of culture, the organoid structure had decreased and the overall size had also shrunk (partially disappeared). In the group using KR5 medium as the maturation medium, excessive duct-like structures were observed. On the other hand, in the group using F9 medium as the maturation medium, abundant organoid-like structures were observed and the size was good. In other words, particularly by using F9 medium as the maturation medium, high-quality kidney organoids could be formed.

[0246] [Experimental Example 6] (Preparation of Renal Organoids 6: Human and Mouse Renal Organoids) Mouse kidney organoids and human kidney organoids (human nephron organoids) were prepared in the same manner as in Experimental Example 1, except that the above-mentioned C1F9Y medium was used as the reaggregation medium. The above-mentioned 10FBS medium, F9 medium, or KR5 medium was used as the maturation medium.

[0247] 7 shows microscopic images of mouse kidney organoids on days 0, 2, and 6 of maturation culture. As a result, compared to the results of Experimental Example 3, when C1F9Y medium was used as the reaggregation medium, better kidney organoids tended to be formed than when 10FBS medium was used as the reaggregation medium. That is, under all culture conditions, abundant organoid-like three-dimensional structures were observed, with diameters of the three-dimensional structures being 1,000 μm or more and good sizes. On the other hand, in the group using KR5 medium as the maturation medium, excessive duct-like structures were observed, and F9 medium was found to be the most excellent maturation medium.

[0248] 8-1 shows microscopic images of human kidney organoids (human nephron organoids) on days 0, 2, and 6 of maturation culture. As a result, compared with the results of Experimental Example 2, it was observed that the use of C1F9Y medium as the reaggregation medium tended to result in the formation of better quality kidney organoids than the use of 10FBS medium as the reaggregation medium.

[0249] In the group using 10FBS medium as the maturation medium, good organoid-like structures were observed on the second day of culture, but by the sixth day of culture, the organoid structures had decreased and the overall size had also shrunk (partially disappeared). In the group using KR5 medium as the maturation medium, excessive duct-like structures were observed. On the other hand, in the group using F9 medium as the maturation medium, abundant organoid-like structures were observed, and the diameter of the three-dimensional structures was 1,000 μm or more, and the size was also good.

[0250] Figure 8-2 shows the results of quantifying the size (area) of mouse kidney organoids, human kidney organoids (human nephron organoids), and human-mouse chimeric kidney organoids based on microscopic images on day 6 of culture in Figures 2-2, 3-2, 5, 6, 7, and 8-1. ImageJ software was used for quantification. The average values ​​were calculated for each of the three types of organoids (mouse, human, and chimera) under six culture conditions. Subsequently, the organoid size (relative value) under each culture condition was calculated, normalized by the calculated average value. As a result, it became possible to quantitatively identify culture conditions that can induce large-sized organoids for the three types of organoids (mouse, human, and chimera). When C1F9Y medium was used as the chimera formation medium and F9 medium or KR5 medium was used as the maturation medium, larger organoid sizes were observed for all three types of organoids (mouse, human, and chimera) compared to other culture conditions.

[0251] [Experimental Example 7] (Evaluation of Renal Organoids 1) The human-mouse chimeric kidney organoids prepared in Experimental Examples 4 and 5 on day 6 of culture were fixed with paraformaldehyde, cryo-embedded, and sliced ​​to prepare sections, which were then evaluated by immunochemical staining.

[0252] Figure 9 shows fluorescent microscopic images of sections of human-mouse chimeric kidney organoids stained with immunochemical staining. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Human Ku80 protein was stained with an anti-human Ku80 antibody. Compared to the group using 10% FBS medium as the chimera formation medium, the group using C1F9Y medium as the chimera formation medium showed a large number of Ku80-positive human cells.

[0253] Figure 10 shows fluorescence microscopy images of the sections in which 10% FBS medium was used as the chimera formation medium, in which ECAD-positive cells and CK8-positive cells were detected in addition to Ku80-positive cells. Enlarged images of the areas where chimera structures were formed are also shown.

[0254] Figure 11 shows fluorescence microscopy images of the sections in Figure 9 where C1F9Y medium was used as the chimera formation medium, in which ECAD-positive cells and CK8-positive cells were detected in addition to Ku80-positive cells. Enlarged images of the areas where chimeric structures were formed are also shown. The four figures at the bottom are enlarged images of the dashed line area in the center image in the second row from the top.

[0255] FIG. 12 shows fluorescence microscopy images of WT1-positive cells, CK8-positive cells, HuNu-positive cells, and DAPI-positive cells detected in separate sections of each sample in which 10% FBS medium was used as the chimera formation medium.

[0256] Figure 13 shows fluorescence microscopy images of WT1-positive cells, CK8-positive cells, HuNu-positive cells, and DAPI-positive cells detected in separate sections of each sample in which C1F9Y medium was used as the chimera formation medium. Enlarged images of the areas where chimera structures were formed are also shown. The four figures at the bottom are enlarged images of the dashed-lined areas in the center images in the second row from the top.

[0257] Figure 14 is a graph showing the values ​​calculated using the following formula (A) based on the results of Figure 9, representing the proportion of human cells. The results are shown per section. In Figure 14, for example, "10FBS → F9" indicates that 10FBS medium was used as the chimera formation medium and F9 medium was used as the maturation medium, and for example, "C1F9Y → KR5" indicates that C1F9Y medium was used as the chimera formation medium and KR5 medium was used as the maturation medium, and so on. "*" indicates a significant difference at p<0.05. "**" indicates a significant difference at p<0.01.

[0258] Percentage of human cells (%) = area of ​​human Ku80-positive area / area of ​​DAPI-positive area × 100 (A)

[0259] As a result, the use of C1F9Y medium as the chimera formation medium increased the percentage of human cells compared to the use of 10FBS medium as the chimera formation medium. Furthermore, the use of F9 medium or KR5 medium as the maturation medium enabled the formation of human-mouse chimeric kidney organoids with a higher percentage of human cells (50% or more).

[0260] Figure 15 shows the results of quantitative analysis of the proportions of constituent cells based on the results of Figures 9, 10, and 11. "*" indicates a significant difference at p<0.05. "**" indicates a significant difference at p<0.01. The abundance ratios of CK8-positive cells, ECAD-positive cells, and WT1-positive cells relative to total cells were calculated as area ratios in the stained images and shown in graphs. The graph on the bottom right shows the proportions of nephron segments. NPCs are known to differentiate into one of the nephron-constituting nephron segments: glomeruli (WT1), proximal tubules, or distal tubules (ECAD). It is considered desirable for NPCs to differentiate without a significant bias toward a specific segment. In this study, proximal tubules were not stained, so only glomeruli (WT1) and distal tubules (ECAD) were evaluated. "C1F9Y→F9" showed good results without bias.

[0261] [Experimental Example 8] (Evaluation of Renal Organoids 2) Human-mouse chimeric kidney organoids prepared in Experimental Examples 4 and 5 on day 6 of culture were fixed with paraformaldehyde, cryo-embedded, and sectioned. Subsequently, immunochemical staining was performed to evaluate the chimeric kidney structure formation rate. The chimeric kidney structure formation rate was calculated using the following formula (B). When both human cells and mouse cells were contained within a single distal tubule structure, the distal tubule structure was counted as a kidney structure containing both human and mouse cells.

[0262] Chimeric kidney structure formation rate (%) = number of chimeric kidney structures containing human cells and mouse cells / number of total kidney structures × 100 (B)

[0263] Figure 16 is a fluorescence microscope image showing the results of an evaluation based on distal tubules as kidney structures. Immunochemical staining was performed using an anti-ECAD antibody, which stains both human and mouse distal tubules; an anti-CK8 antibody, which stains only mouse cytokeratin 8 protein; an anti-human Ku80 antibody, which stains only human Ku80 protein; and DAPI, which stains nuclei. Figure 17 is a graph quantitatively representing the results of Figure 16. "**" indicates a significant difference at p<0.01. The vertical axis represents the chimeric kidney structure formation rate (%).

[0264] The results showed that changing the chimera-forming medium and maturation medium altered the formation rate of chimeric kidney structures containing ECAD-positive distal tubules. Specifically, when 10% FBS was used as the chimera-forming medium, the chimeric kidney structure formation rate was approximately 20% or less for both maturation media. On the other hand, when C1F9Y medium was used as the chimera-forming medium and 10% FBS medium or F9 medium was used as the maturation medium, the chimeric kidney structure formation rate was significantly increased compared to the control group. The highest value was observed with the combination of C1F9Y chimera-forming medium → F9 maturation medium, and the chimeric kidney structure formation rate was approximately 80%.

[0265] 18 shows fluorescence microscopy images showing the results of evaluation based on glomeruli as kidney structures. Immunochemical staining was performed using an anti-WT1 antibody that stains both human and mouse glomeruli, an anti-CK8 antibody that stains only mouse cytokeratin 8 protein, an anti-HuNu antibody that stains HuNu, a human-specific nuclear antigen, and DAPI that stains nuclei.

[0266] Figure 19 is a graph quantitatively showing the results of Figure 18. "**" indicates a significant difference at p<0.01. The vertical axis represents the rate (%) of chimeric kidney structure formation. When a single glomerular structure contained both human and mouse cells, the glomerular structure was counted as a kidney structure containing both human and mouse cells.

[0267] The results showed that changing the chimera-forming medium and maturation medium altered the rate of chimeric kidney structure formation containing WT1-positive glomeruli. Specifically, when 10% FBS was used as the chimera-forming medium, the rate of chimeric kidney structure formation was approximately 20% or less for both maturation media. On the other hand, when C1F9Y medium was used as the chimera-forming medium and F9 medium was used as the maturation medium, the rate of chimeric kidney structure formation was significantly increased compared to the control group. The highest value was observed with the combination of C1F9Y medium → F9 medium, and the rate of chimeric kidney structure formation was approximately 80%.

[0268] Figure 20 shows an immunostained image of a human-mouse chimeric organoid on Day 2 of culture, cultured in a combination of C1F9Y medium and F9 medium, as in Experimental Example 5. Staining was performed with anti-WT1 antibody, anti-CK8 antibody, anti-HuNu antibody, and DAPI. The arrowhead in the lower right image indicates the CAP structure, the arrow in the lower right image indicates the S-shaped body, the arrowhead in the upper right image indicates the RV, and the arrow in the upper right image indicates the C-shaped body. The scale bar is 200 μm. Chimera formation was observed in structures characteristic of early nephron development, such as the CAP structure, RV, C-body, and S-body, and a mosaic nephron was formed.

[0269] Figure 21 shows an image of the same sample as in Figure 20, immunostained with a different marker. The sample was stained with anti-ECAD antibody, anti-CK8 antibody, anti-Ku80 antibody, and DAPI. The scale bar is 200 μm.

[0270] The above results were similar when the distal tubules and glomeruli were evaluated as chimeric kidney structures.

[0271] [Experimental Example 9] (Preparation of Renal Organoids 7: Pig Kidney Organoids) Pig kidney organoids were prepared. Single cells dissociated from porcine fetal metanephroi were suspended in a reaggregation medium and collected at a concentration of 2.0 × 10 ５ The cells were seeded at a cell density of 10 cells / well onto a low-adhesion 96-well plate to form aggregates. The reaggregation medium used was the 10 FBS medium or C1F9Y medium described above.

[0272] After 24 hours, the aggregates were seeded into Transwell inserts mounted in a 96-well plate and cultured at the air-liquid interface in maturation medium, which was the above-mentioned 10FBS medium, F9 medium, or KR5 medium.

[0273] Figure 22 shows microscopic images of kidney organoids on days 0, 2, and 6 of maturation culture when 10 FBS medium was used as the reaggregation medium. In Figure 22, "10 FBS → 10 FBS" indicates the results when 10 FBS medium was used as the reaggregation medium and 10 FBS medium was used as the maturation medium, and the same applies below. Figure 23 shows microscopic images of kidney organoids on days 0, 2, and 6 of maturation culture when C1F9Y medium was used as the reaggregation medium.

[0274] As a result, pig kidney organoids containing organoid-like three-dimensional structures were formed regardless of whether the reaggregation medium or maturation medium was used. In the group using 10FBS medium for both the reaggregation medium and maturation medium, the diameter of the cell aggregates was less than 405 μm on day 6 of culture compared to day 2 of culture, and a reduction in size was observed. On the other hand, when F9 medium or KR5 medium was used as the maturation medium, larger aggregate sizes were observed on day 6 of culture compared to the group using 10FBS medium as the maturation medium. When C1F9Y was used as the reaggregation medium and F9 was used as the maturation medium, the diameter was approximately 1,000 μm, a three-dimensional structure was confirmed, and the three-dimensional structure was not accompanied by excessive ductal formation, allowing the formation of pig kidney organoids of consistent quality. Previously, there had been no reported cases of pig kidney organoids being produced, and the inventors have now succeeded in producing pig kidney organoids for the first time.

[0275] [Experimental Example 10] (Evaluation of renal organoids 3) Sections of porcine renal organoids were prepared and evaluated by immunochemical staining. The porcine renal organoids prepared in Experimental Example 9 on the 6th day of culture were fixed with paraformaldehyde, frozen and embedded to prepare sections, and evaluated by immunochemical staining.

[0276] Figure 24 is a fluorescent microscope image showing the results of immunochemical staining of a section of pig kidney organoid. Staining was performed with an anti-ECAD antibody, an anti-CK8 antibody that stains cytokeratin 8 protein, an anti-Ku80 antibody that stains Ku80 protein, and DAPI that stains nuclei. Figure 25 is a fluorescent microscope image enlarged from a portion of the central lower view of Figure 24.

[0277] As a result, under all culture conditions, the presence of ECAD-positive regions, which indicate distal tubules, and ECAD-positive CK8-positive regions, which indicate ureteric buds, was confirmed, confirming that the pig kidney organoids were functionally differentiated and mature. Furthermore, the presence of abundant ECAD-positive distal tubules was observed under all culture conditions.

[0278] Figure 26 is a graph showing the results of calculating the ratio of the area of ​​the ECAD-positive region to the area of ​​a section of a pig kidney organoid based on the results of Figure 24. For comparison, Figure 26 also shows the results of evaluating mouse kidney organoids on day 6 of culture prepared in Experimental Example 3 by paraformaldehyde fixation, cryo-embedding, and sectioning, followed by immunochemical staining. As a result, the area ratio of the ECAD-positive region in mouse kidney organoids was approximately 30-40%, while the area ratio of the ECAD-positive region in pig kidney organoids was approximately 60% or more. The abundant presence of ECAD-positive distal tubules was quantitatively confirmed under all culture conditions. In particular, when C1F9Y was used as the reaggregation medium and F9 was used as the maturation medium, the ratio was approximately 80%, indicating a higher tendency.

[0279] Figure 27 is a fluorescence microscope image showing the results of immunochemical staining of a section of pig kidney organoid. Staining was performed with an anti-WT1 antibody to stain glomeruli, an anti-CK8 antibody to stain cytokeratin 8 protein, an anti-Ku80 antibody to stain Ku80 protein, and DAPI to stain nuclei. Figure 28 is a fluorescence microscope image enlarged from a portion of the central lower panel of Figure 27.

[0280] As a result, the proportion of WT1-positive areas in pig kidney organoids was lower than that in mouse kidney organoids, but the presence of WT1-positive areas indicating glomeruli was confirmed under all culture conditions. Figure 29-1 is a graph showing the results of calculating the ratio of the area of ​​WT1-positive areas to the area of ​​kidney organoid sections based on the results of Figure 27. For comparison, Figure 29-1 also shows the results of mouse kidney organoids prepared in Experimental Example 3 on day 6 of culture, fixed with paraformaldehyde, cryo-embedded, and sectioned, and evaluated by immunochemical staining. When C1F9Y was used as the reaggregation medium and F9 was used as the maturation medium, the proportion was approximately 5%, the highest.

[0281] Figure 29-2 shows the results of quantifying the ECAD positive rate and WT1 positive rate based on the area ratio of the ECAD positive area of ​​the porcine kidney organoid shown in Figure 26 and the area ratio of the WT1 positive area of ​​the porcine kidney organoid shown in Figure 29-1. For each culture condition, the area ratio of the ECAD positive area was added up to calculate the average value, and a standardized value based on that average value was calculated for each culture condition. In addition, for each culture condition, the area ratio of the WT1 positive area was added up to calculate the average value, and a standardized value based on that average value was calculated for each culture condition. As a result, it became possible to quantitatively show culture conditions that can induce high ECAD positive area rate and WT1 positive area rate in porcine kidney organoids. When C1F9Y medium was used as the reaggregation medium and F9 medium was used as the maturation medium, porcine kidney organoids with the highest ECAD positive area rate and WT1 positive area rate could be induced.

[0282] [Experimental Example 11] (Preparation of Renal Organoids 8: Human-Pig Chimeric Renal Organoids) Human-pig chimeric kidney organoids were prepared. 1.0 × 10 dissociated human nephron progenitor cells were cultured in a 2000-well culture medium. ５ 1.0 x 10 cells / well and 1.0 x 10 cells / well dissociated into single cells from fetal porcine metanephroi. ５ The cells / well were mixed and suspended in chimera formation medium, and seeded onto a low-attachment 96-well plate to form aggregates (human:pig = 1:1). The chimera formation medium used was the C1F9Y medium described above. Also, 1.33 × 10 dissociated human nephron progenitor cells were used. ５ 0.67 x 10 cells / well and 0.67 x 10 cells / well ５ The cells / well were mixed and suspended in chimera formation medium, and then seeded onto a low-attachment 96-well plate to form aggregates (human:pig = 2:1). The chimera formation medium used was the C1F9Y medium described above.

[0283] In addition, 1.5 × 10 human nephron progenitor cells dissociated into single cells were ５ 0.5 × 10 cells / well and 0.5 × 10 cells / well ５The cells / well were mixed and suspended in chimera formation medium, and then seeded onto a low-attachment 96-well plate to form aggregates (human:pig = 3:1). The chimera formation medium used was the C1F9Y medium described above.

[0284] Figure 30 shows microscopic images of aggregates on day 1 of culture. As a result, the formation of pig-human chimeric aggregates was confirmed under all conditions.

[0285] Subsequently, after 24 hours, the aggregates were seeded into Transwell inserts mounted in a 96-well plate and cultured at the air-liquid interface in maturation medium, which was the F9 medium described above.

[0286] Figure 31 shows microscopic images of kidney organoids on days 0, 2, and 6 of maturation culture. As a result, human-pig chimeric kidney organoids were formed under all conditions. There have been no reports of the creation of human-pig chimeric kidney organoids, and the inventors have now succeeded in creating human-pig chimeric kidney organoids for the first time.

[0287] Furthermore, it was also possible to produce human-pig chimeric kidney organoids by thawing frozen pig fetal metanephros and using pig fetal metanephros-derived cells that had been dissociated into single cells as the starting material.

[0288] [Experimental Example 12] (Evaluation of Renal Organoids 4) The human-pig chimeric kidney organoids prepared in Experimental Example 11 on day 6 of culture were fixed with paraformaldehyde, cryo-embedded, and sliced, and evaluated by immunochemical staining.

[0289] Figure 32 shows fluorescent microscopic images of sections of human-pig chimeric kidney organoids subjected to immunochemical staining. Brightfield microscopic images of each kidney organoid are shown in the upper left corner of each image in Figure 32. Immunochemical staining was performed using an anti-ECAD antibody that stains distal tubules, an anti-CK8 antibody that stains cytokeratin 8 protein, an anti-human Ku80 antibody that stains only human Ku80 protein, and DAPI that stains nuclei.

[0290] As a result, under all conditions, the presence of ECAD-positive regions indicating distal tubules and ECAD-positive and CK8-positive regions indicating ureteric buds was confirmed, confirming that the human-pig chimeric kidney organoids were functionally differentiated and mature. Furthermore, the presence of human cells in the kidney organoids was confirmed.

[0291] Figure 33 is a fluorescence microscope image showing the results of staining with anti-human Ku80 antibody and DAPI in the same field of view as Figure 32. In Figure 32, the numbers at the bottom right of each image indicate the percentage of human cells calculated using the following formula (A). In other words, human-pig chimeric kidney organoids were obtained in which the percentage of human cells was 30% to 35% of the total area.

[0292] Percentage of human cells (%) = area of ​​human Ku80-positive area / area of ​​DAPI-positive area × 100 (A)

[0293] Figure 34 shows representative microscopic images demonstrating the presence of chimeric kidney constructs. Cells were stained with anti-ECAD, anti-CK8, and anti-Ku80 antibodies and DAPI. In Figure 34, the arrowhead indicates a chimeric kidney construct containing both human and porcine cells.

[0294] Figure 35 is an enlarged view of the upper left image in Figure 34 (the upper left image in the "human:pig = 1:1" ratio). The dashed line on the left in the second image from the left indicates the distal tubule tissue, and the dashed line on the right indicates the ureteric bud. The upper dashed area in the rightmost image indicates human-derived tissue, and the lower dashed area indicates pig-derived tissue.

[0295] Figure 36 shows images of human-pig chimeric organoids stained with antibodies against a different marker protein, using the same samples as in Figures 32 to 34. It can be seen that WT1, CK8, and Ku80-positive cells are present in a mosaic pattern at all mixing ratios of human-derived cells and pig-derived cells. In Figure 36, "Hu(1):Pig(1)" indicates a mixture of human and pig cells at a human:pig ratio of 1:1, "Hu(2):Pig(1)" indicates a mixture of human and pig cells at a human:pig ratio of 2:1, and "Hu(3):Pig(1)" indicates a mixture of human and pig cells at a human:pig ratio of 3:1. The three images to the right of "Hu(3):Pig(1)" are enlarged images of a portion, showing the formation of WT1-positive glomerular tissue.

[0296] 37-1 is a graph showing the results of evaluating the chimeric kidney structure formation rate for one section of a human-pig chimeric kidney organoid. The chimeric kidney structure formation rate was calculated using the following formula (C).

[0297] Chimeric kidney structure formation rate (%) = number of chimeric kidney structures containing human cells and pig cells / number of total kidney structures × 100 (C)

[0298] When both human and porcine cells were contained within a distal tubule structure, the distal tubule structure was counted as a renal structure containing both human and porcine cells.

[0299] The results showed that the higher the proportion of human nephron progenitor cells mixed, the higher the rate of chimeric kidney structure formation. At a human:pig ratio of 3:1, the rate of chimeric kidney structure formation was 80% or higher.

[0300] Figure 37-2 shows the results of quantifying the size, positive area rate of various markers, and chimeric kidney structure formation rate of human-pig chimeric kidney organoids when the mixing ratio of human-derived cells and pig-derived cells was varied.

[0301] The area of ​​the organoids was quantified based on the bright-field image on day 6 shown in Figure 31. ImageJ software was used for quantification. The area of ​​organoids was standardized using the area of ​​organoids with a human:pig ratio of 1:1 as a reference, and the relative size (area) of human-pig chimeric kidney organoids at each mixture ratio was calculated (organoid size).

[0302] In addition, based on Figures 32, 33, and 36, the positive area rate of each marker, ECAD, WT1, CK8, and Ku80, was calculated. The positive area rate was expressed as a percentage (%) of the DAPI-positive area. The positive area rate of each marker in human:pig = 1:1 organoids was used as a standard, and the relative value of the positive area rate (constituent cell ratio) of human-pig chimeric kidney organoids at each mixture ratio was calculated (cell population).

[0303] Furthermore, based on Figures 32 and 36, the chimeric kidney structure formation rates of the ECAD-positive region indicating the distal tubules and the WT1-positive region indicating the glomerular tissue were quantified. The chimeric kidney structure formation rates of the 1:1 human:pig organoids were used as a standard, and the relative chimeric kidney structure formation rates of the human-pig chimeric kidney organoids at each mixture ratio were calculated (chimeric rate). High-magnification photographs of representative chimeric kidney structures are shown in Figures 34, 35, and 36.

[0304] From the above results, it was quantitatively shown that there was no significant difference in organoid size or ECAD / WT1 / CK8 positive area ratio (constituent cell ratio) even when the mixing ratio was different, but the human-pig chimeric kidney organoid with the highest composition ratio of human cells (Ku80 positive cells) was the human:pig = 3:1 human:pig chimeric kidney organoid, and that the human:pig = 3:1 human:pig chimeric kidney organoid also had the highest chimeric kidney structure formation rate.

[0305] Figure 37-3 shows fluorescence microscopy images of sections of human:pig = 3:1 chimeric kidney organoids (day 6) stained with multiple glomerular markers, proximal tubule markers, and distal tubule markers. The scale bar is 100 μm.

[0306] Figure 37-4 shows fluorescence microscopy images of sections of porcine kidney organoids (day 6) stained with multiple glomerular markers, proximal tubule markers, and distal tubule markers. The scale bar is 100 μm.

[0307] Figure 37-5 shows fluorescence microscopy images of sections of human kidney organoids (human nephron organoids) (day 6) stained with multiple glomerular markers, proximal tubule markers, and distal tubule markers. The scale bar is 100 μm.

[0308] In Figures 37-3, 37-4, and 37-5, WT1, NEPHRIN, and PODOCALYXIN were used as glomerular markers, JAGGED1 (JAG1), LTL, CDH6, AQP1, and MEGALIN were used as proximal tubule markers, and DBA, ECAD, and NCC were used as distal tubule markers.

[0309] As a result, it was confirmed that human-pig chimeric kidney organoids, pig kidney organoids, and human kidney organoids (human nephron organoids) were all successfully induced into various nephron segments and showed a certain degree of maturation.

[0310] Figure 37-6 is a graph showing the results of comparing the ratios of constituent cells between human:mouse = 3:1 human-mouse chimeric kidney organoids (day 6) and human:pig = 3:1 human-pig chimeric kidney organoids (day 6). "*" indicates a significant difference at p<0.05. "**" indicates a significant difference at p<0.01. The ratios of constituent cells in the "C1F9Y→F9" human-mouse chimeric kidney organoids shown in Figures 14 and 15 were compared with the ratios of constituent cells in human-pig chimeric kidney organoids (N=3) quantified in the same manner as in Figures 14 and 15. As a result, differences were observed in the ratios of ECAD-positive cells and WT1-positive cells, but no significant differences were observed in the ratios of other constituent cells.

[0311] Figure 37-7 is a graph showing the results of comparing the chimeric kidney structure formation rate of human: mouse = 3: 1 human-mouse chimeric kidney organoids (day 6) and human: pig = 3: 1 human-pig chimeric kidney organoids (day 6). The chimeric kidney structure formation rate of the "C1F9Y → F9" human-mouse chimeric kidney organoids shown in Figures 17 and 19 was compared with the chimeric kidney structure formation rate of human-pig chimeric kidney organoids quantified in the same manner as in Figures 17 and 19 (N = 3). As a result, no significant differences were observed in either the chimeric kidney structure formation rate of ECAD-positive cells or the chimeric kidney structure formation rate of WT1-positive cells.

[0312] From the results of Figures 37-6 and 37-7, it was considered that the human-mouse chimeric kidney organoids and the human-pig chimeric kidney organoids were roughly equivalent in the ratio of constituent cells and the rate of chimeric kidney structure formation.

[0313] Experimental Example 13 (Production of Chimeric Kidney Tissue) Human nephron progenitor cells were injected into mouse fetal metanephroi and cultured to produce chimeric kidney tissue.

[0314] Pregnant C57BL / 6 mice were purchased, and mouse fetuses were removed at embryonic day 13.5 to collect only the metanephros of the mouse fetuses. The collected metanephros was placed in a 1.5 mL tube containing MEMα and stored on ice.

[0315] Preparation of human nephron progenitor cells Human iPS cells (201B7 cell line or EGFP-transfected 317-12-Ff cell line) were induced to differentiate into human nephron progenitor cells by the method described above. However, ITGA8-positive human nephron progenitor cells were not selected after differentiation induction.

[0316] On the day of injection into the mouse metanephroi, the differentiation-induced human nephron progenitor cells were dissociated into single cells, placed in a 1.5 mL tube, centrifuged, and the supernatant was removed to prepare pelleted human nephron progenitor cells.

[0317] <<Injection of human nephron progenitor cells into mouse metanephroi>> The prepared human nephron progenitor cells were injected into the mouse metanephroi just below the renal capsule using a glass needle. The injection amount was approximately 1 × 10 per metanephroi. ３ pieces ~ 1×10６ There were 100 pieces.

[0318] Subsequently, the mouse metanephroi injected with human nephron progenitor cells were cultured at the air-liquid interface for 4 days using chimera formation medium (C1F9Y medium). However, after 24 hours of culture, a medium was used in which only Y27632 had been removed from C1F9Y medium.

[0319] On day 4 of culture, the human nephron progenitor cell-injected mouse metanephroi were fixed with paraformaldehyde, cryo-embedded, sectioned, and evaluated by immunochemical staining.

[0320] Figure 38 shows fluorescence microscopy images showing the results of immunochemical staining of the metanephroi of a mouse injected with human nephron progenitor cells (using the 201B7 strain of iPS cells). The left and right images were taken at different cross sections (heights).

[0321] Staining was performed with anti-HuNu antibody (HNA) that stains HuNu, a human-specific nuclear antigen, anti-Ecad antibody that stains distal tubules, anti-CK8 antibody that stains cytokeratin 8 protein, and DAPI that stains nuclei.

[0322] As a result, human nephron progenitor cells were confirmed to have engrafted in layers just beneath the capsule of the mouse metanephros, partially forming chimeric kidney structures. In Figure 38, the arrow indicates the morphology of a CAP structure. Human-mouse CAP structure formation (adhesion) was clearly observed, indicating improved human cell survival and chimera formation rate. These results demonstrate the usefulness of C1F9Y medium as a chimera formation medium.

[0323] Figure 39 shows stereomicroscopic images of a mouse metanephroi injected with iPS cell-derived human nephron progenitor cells fluorescently labeled with GFP at the start of culture (Day 0). The image on the left is a bright-field image, and the image on the right is a fluorescent image in which GFP was detected. The scale bar is 750 μm. It was confirmed that the injected human nephron progenitor cells were present in sufficient quantities at the target location.

[0324] Figure 40-1 shows stereomicroscopic images of mouse metanephroi injected with iPS cell-derived human nephron progenitor cells fluorescently labeled with GFP and cultured for four days (Day 4). The image on the left is a bright-field image, and the image on the right is a fluorescent image in which GFP was detected. The scale bar is 750 μm. It was confirmed that the injected human nephron progenitor cells remained in the target location and were present in sufficient quantities even after four days of culture.

[0325] Figure 40-2 is a graph showing the change in size of mouse metanephros injected with human nephron progenitor cells (NPCs) during culture. "**" indicates a significant difference at p<0.01. Images of human NPC-injected mouse metanephros, i.e., two-dimensional projections of mouse metanephros, are shown in Figures 39 and 40-1. The area corresponding to the mouse kidney and the area corresponding to human NPCs are quantified. "Area Mouse Kidney" is the value quantified using ImageJ software for the mouse kidney portion in the bright-field image. "Area Human NPC" is the value quantified using ImageJ software for the GFP-positive portion in the GFP fluorescent image. The results demonstrated that both the mouse metanephros and the human NPCs injected therein expanded in size after 4 days of culture.

[0326] FIG. 41 is a fluorescence microscopy image showing the results of immunochemical staining of mouse metanephroi injected with human nephron progenitor cells after 4 days of culture, and is another image of the same tissue as in FIG.

[0327] Figure 42 is an enlarged image of the area indicated by the arrow in Figure 38. Human cells were confirmed to have adhered to the tip of the mouse ureteric bud, forming a chimeric kidney structure. Based on structural and morphological characteristics, this is thought to be a CAP structure from the early stages of kidney development, and it was observed that most of the tip of the ureteric bud, where mouse nephron progenitor cells normally reside, had been replaced by human cells.

[0328] Figure 43 shows fluorescence microscopy images of the same tissue as in Figure 42, showing the results of immunochemical staining of mouse metanephroi injected with human nephron progenitor cells after 4 days of culture. The scale bar is 100 μm. The dashed line in the center image indicates the outer edge of the CAP structure (Cap Mesenchyme), and the outer dashed line in the rightmost image indicates the region of human-derived tissue, while the inner dashed line indicates the region of mouse-derived tissue.

[0329] Figure 44 shows fluorescence microscopy images showing the results of immunochemical staining of mouse metanephroi injected with human nephron progenitor cells using the EGFP-introduced 317-12-Ff iPS cell line. The left and right images were taken at different cross sections (heights). Staining was performed with an anti-HuNu antibody (HNA) that stains HuNu, a human-specific nuclear antigen; an anti-Ecad antibody that stains distal tubules; an anti-CK8 antibody that stains cytokeratin 8 protein; and DAPI that stains nuclei.

[0330] As a result, the GFP-expressing cells and the HuNu-stained cell nuclei all overlapped, clearly confirming that the human cells had formed a chimeric tissue with the mouse metanephroi. The formation of a human-mouse CAP structure was also confirmed.

[0331] Figure 45 is an enlarged image of the boundary between the human cells and mouse cells in Figure 44. As a result, it was confirmed that the human cells had adhered to the mouse cells and formed a single structure.

[0332] [Experimental Example 14] (Transplantation of chimeric kidney tissue into mice) Chimeric kidney tissue was transplanted into adult mice (C57BL / 6). Figure 46 is a schematic diagram showing the experimental scheme of this experiment.

[0333] A chimeric tissue was prepared in the same manner as in Experimental Example 13, except that the metanephroi of the mouse injected with human nephron progenitor cells was cultured at the air-liquid interface for 3 days. The tissue was then transplanted into the para-aortic cavity of an adult mouse, and the tissue was harvested 7 days after transplantation.

[0334] Figure 47 shows images of the state of the mouse metanephroi injected with human nephron progenitor cells during air-liquid interface culture. Because human nephron progenitor cells were induced to differentiate from the GFP-introduced 201B7 cell line, the injected human nephron progenitor cells emitted green fluorescence.

[0335] As a result, even on day 3 after the start of culture, survival and engraftment of human nephron progenitor cells were confirmed to be at the same level as at the start of culture. Furthermore, the three-dimensional morphology suggested that the injected human nephron progenitor cells were differentiating (maturing) normally. These results further support the usefulness of C1F9Y medium as a chimera formation medium.

[0336] The upper panel of Figure 48 shows a bright-field image and an image of GFP fluorescence showing the state of the human nephron progenitor cell-injected mouse metanephroi on day 3 after the start of air-liquid interface culture. The lower panel of Figure 48 shows a bright-field image and an image of GFP fluorescence showing the state of the human nephron progenitor cell-injected mouse metanephroi after culturing for 3 days in a space created by peeling off the tissue capsule near the aorta of the recipient (adult mouse). The green fluorescence of the human cells was confirmed even after transplantation.

[0337] The top row of Figure 49 shows a bright-field image of the vicinity of the recipient's aorta 7 days after transplantation, an image obtained by observing GFP fluorescence, and a merged image of these. The presence of human cells was confirmed by the presence of GFP fluorescence. The bottom row of Figure 49 shows a bright-field image of the graft removed from the recipient 7 days after transplantation, an image obtained by observing GFP fluorescence, and a merged image of these. The GFP fluorescence confirmed the engraftment of approximately the same amount of human cells as before transplantation. The graft exhibited a three-dimensional structure and appeared to be further maturing.

[0338] Figure 50 shows a fluorescence microscopy image showing the results of immunochemical staining of a graft excised from a recipient 7 days after transplantation. It was confirmed that human nephron progenitor cells engrafted and differentiated into distal tubules, forming a chimeric renal structure with mouse metanephroi-derived distal tubules. Compared to previous reports, the engraftment rate (survival rate) of human cells and the rate of chimeric renal structure formation were significantly improved.

[0339] In the past, when mouse-derived metanephros were injected with human nephron progenitor cells without dispersing them and then transplanted into mice, the survival rate of human nephron progenitor cells was low. In response to this issue, we demonstrated that human nephron progenitor cells were maintained and nephrons were formed by culturing mouse metanephros injected with human nephron progenitor cells using C1F9Y medium as a chimera-forming medium and then transplanting the metanephros into mice.

[0340] According to the present invention, kidney organoid-related technologies can be provided.

Claims

1. A chimeric kidney organoid, comprising a chimeric kidney structure comprising human cells and non-human mammalian cells, wherein the non-human mammal is a cetacean or a non-human primate.

2. The chimeric kidney organoid according to claim 1, wherein the chimeric kidney structure formation rate calculated by the following formula (1) is 30% or more: Chimeric kidney structure formation rate (%) = number of chimeric kidney structures containing human cells and non-human mammalian cells / number of all kidney structures × 100 ... (1) 3. The chimeric kidney organoid of claim 1 or 2, wherein the chimeric kidney structure comprises one or more cells selected from the group consisting of human-derived nephron progenitor cells, human-derived cells constituting distal tubules, human-derived cells constituting proximal tubules, and human-derived cells constituting glomeruli.

4. The chimeric kidney organoid according to any one of claims 1 to 3, wherein the chimeric kidney structure comprises one or more cells selected from the group consisting of nephron progenitor cells derived from the non-human mammalian cells, cells constituting glomeruli derived from the non-human mammalian cells, cells constituting distal tubules derived from the non-human mammalian cells, and cells constituting proximal tubules derived from the non-human mammalian cells, and cells constituting ureteric buds and / or collecting ducts derived from the non-human mammalian cells.

5. A chimeric kidney organoid described in any one of claims 1 to 4, wherein the cetacean is a pig.

6. A method for producing chimeric renal tissue comprising human nephron progenitor cells or cells derived from human nephron progenitor cells, and cells derived from the fetal metanephroi of a non-human mammal, the method comprising: a step (a1) of contacting human nephron progenitor cells with cells derived from the fetal metanephroi of a non-human mammal to obtain a cell population; and a step (b1) of culturing the cell population obtained in step (a1) in a chimera-forming medium to obtain chimeric renal tissue, wherein the chimera-forming medium contains a Wnt signal activator.

7. The method of claim 6, wherein the contacting in step (a1) comprises mixing dispersed human nephron progenitor cells and dispersed non-human mammalian fetal metanephroi-derived cells, and the chimeric renal tissue is a chimeric aggregate.

8. The manufacturing method described in claim 6, wherein the contacting in step (a1) is injecting human nephron progenitor cells into the non-human mammal fetal metanephroi, and the chimeric kidney tissue is a non-human mammal fetal metanephroi containing human nephron progenitor cells or cells derived from human nephron progenitor cells.

9. The method of claim 8, wherein a suicide gene for nephron progenitor cells derived from the non-human mammal is introduced into the non-human mammal fetal metanephroi.

10. The method of any one of claims 6 to 9, wherein the chimera-forming medium further contains a ROCK inhibitor.

11. The production method according to any one of claims 6 to 10, wherein the chimera-forming medium further contains an FGF9 signal activator.

12. The method of any one of claims 6 to 11, wherein the chimera-forming medium is a serum-free medium.

13. A method for producing chimeric kidney organoids comprising human cells and non-human mammalian cells, comprising: a step (a2) of mixing dispersed human nephron progenitor cells and dispersed cells derived from a non-human mammalian fetal metanephroi to obtain a cell population; a step (b2) of culturing the cell population obtained in step (a2) in a chimera-forming medium to obtain chimeric aggregates; and a step (c2) of culturing the chimeric aggregates obtained in step (b2) in a maturation medium to obtain chimeric kidney organoids comprising human cells and non-human mammalian cells, wherein the chimera-forming medium contains a Wnt signal activator, and wherein the maturation medium does not contain a Wnt signal activator in at least a portion of step (c2).

14. The manufacturing method described in claim 13, wherein step (c2) comprises step (c2-1) of culturing the chimeric aggregate obtained in step (b2) in a maturation medium containing a Wnt signal activator, and step (c2-2) of culturing the chimeric aggregate obtained in step (b2) in a maturation medium not containing a Wnt signal activator.

15. The production method described in claim 13 or 14, wherein the chimera formation medium and / or the maturation medium further contain an FGF9 signal activator.

16. The production method according to any one of claims 13 to 15, wherein the chimera formation medium and / or the maturation medium further contain a ROCK inhibitor.

17. A method for production according to any one of claims 13 to 16, wherein the chimera formation medium and / or the maturation medium is a serum-free medium.

18. The method according to any one of claims 13 to 17, wherein the non-human mammal is a rodent, a cetacean, or a non-human primate.

19. The method of claim 18, wherein the cetacean is a pig.

20. A method for evaluating the ability of human nephron progenitor cells to form chimeric renal structures, comprising: a step (a3) ​​of mixing the dispersed human nephron progenitor cells with dispersed cells derived from a non-human mammalian fetal metanephroi to obtain a cell population; a step (b3) of culturing the cell population obtained in step (a3) ​​in a chimera-forming medium to obtain chimeric aggregates; a step (c3) of culturing the chimeric aggregates obtained in step (b3) in a maturation medium to obtain renal organoids comprising human cells and non-human mammalian cells; and a step (d3) of determining the ability of human nephron progenitor cells to form chimeric renal structures based on the number of chimeric renal structures and / or the type of cells contained in the chimeric renal structures contained in the renal organoids obtained in step (c3), wherein the chimera-forming medium contains a Wnt signaling activator; and wherein the maturation medium does not contain a Wnt signaling activator in at least a portion of step (c3).

21. The evaluation method described in claim 20, wherein step (c3) comprises steps (c3-1) of culturing the chimeric aggregate obtained in step (b3) in a maturation medium containing a Wnt signal activator, and (c3-2) of culturing the chimeric aggregate obtained in step (b3) in a maturation medium not containing a Wnt signal activator.

22. The evaluation method according to claim 20 or 21, wherein the step (d3) of determining the ability to form a chimeric renal structure comprises one or more steps selected from the following (i) to (vi): (i) calculating a chimeric renal structure formation rate using the following formula (1), and determining the ability of the human nephron progenitor cells to form a chimeric renal structure based on the calculated chimeric renal structure formation rate: Chimeric renal structure formation rate (%) = number of chimeric renal structures comprising human cells and non-human mammalian cells / number of total renal structures × 100 ... (1); (ii) counting the number of glomerular structures comprising human cells and non-human mammalian cells, where the counted number of glomerular structures corresponds to the ability of the human nephron progenitor cells to form a chimeric renal structure; (iii) counting the number of renal tubular structures comprising human cells and non-human mammalian cells, and determining the ability of the human nephron progenitor cells to form a chimeric renal structure based on the counted number of renal tubular structures; (iv) counting the number of nephron segment structures comprising human cells and non-human mammalian cells, and determining the ability of the human nephron progenitor cells to form chimeric renal structures based on the counted number of nephron segment structures; (v) counting the number of ureteric bud structures derived from a non-human mammal comprising human nephron progenitor cells, human distal tubule constituent cells and / or human proximal tubule constituent cells, and determining the ability of the human nephron progenitor cells to form chimeric renal structures based on the counted number of ureteric bud structures; (vi) calculating the proportion of human cells in the renal structures, and determining the ability of the human nephron progenitor cells to form chimeric renal structures based on the calculated proportion of human cells.

23. The evaluation method according to any one of claims 20 to 22, wherein the chimera formation medium and / or the maturation medium further contain an FGF9 signal activator.

24. The evaluation method according to any one of claims 20 to 23, wherein the chimera formation medium and / or the maturation medium further contain a ROCK inhibitor.

25. The evaluation method according to any one of claims 20 to 24, wherein the chimera formation medium and / or the maturation medium is a serum-free medium.

26. An evaluation method described in any one of claims 20 to 25, wherein in step (a3), the ratio of the number of human nephron progenitor cells to the number of cells derived from the non-human mammalian fetal metanephroi is 5:1 to 1:

1.

27. The evaluation method described in claim 26, wherein in step (a3), the ratio of the number of human nephron progenitor cells to the number of cells derived from the non-human mammalian fetal metanephroi is approximately 3:

1.

28. The evaluation method according to any one of claims 20 to 27, wherein the non-human mammal is a rodent, a cetacean, or a non-human primate.

29. The evaluation method according to claim 28, wherein the cetacean is a pig.

30. A porcine kidney organoid comprising at least two types of cells selected from the group consisting of nephron component cells or nephron progenitor cells, collecting duct component cells or collecting duct progenitor cells, and renal interstitial cells or renal interstitial progenitor cells, which has the function of kidney component cells or the function of promoting kidney development, and which has multiple kidney structures within it.

31. The pig kidney organoid described in claim 30, wherein the renal structure comprises cells constituting one or more selected from glomeruli, distal tubules, and proximal tubules, or their precursor cells, and cells constituting collecting ducts, or their precursor cells, and has a CAP structure therein containing ureteric buds and nephron progenitor cells.

32. A method for producing renal organoids comprising non-human mammalian cells, comprising: a step (a4) of culturing a population of dispersed cells derived from a non-human mammalian fetal metanephroi in a reaggregation medium to obtain aggregates; and a step (b4) of culturing the aggregates obtained in step (a4) in a maturation medium to obtain renal organoids comprising non-human mammalian cells, wherein the reaggregation medium contains a Wnt signal activator, and wherein the maturation medium does not contain a Wnt signal activator in at least a portion of step (b4).

33. The manufacturing method described in claim 32, wherein step (b4) comprises step (b4-1) of culturing the chimeric aggregate obtained in step (a4) in a maturation medium containing a Wnt signal activator, and step (b4-2) of culturing the chimeric aggregate obtained in step (a4) in a maturation medium not containing a Wnt signal activator.

34. The production method described in claim 32 or 33, wherein the reaggregation medium and / or the maturation medium further contain an FGF9 signal activator.

35. The production method according to any one of claims 32 to 34, wherein the reaggregation medium and / or the maturation medium further comprises a ROCK inhibitor.

36. The method of any one of claims 32 to 35, wherein the reaggregation medium and / or the maturation medium is a serum-free medium.

37. The method of any one of claims 32 to 36, wherein the non-human mammal is a rodent, a cetacean, or a non-human primate.

38. The method of claim 37, wherein the non-human mammal is a cetacean or a non-human primate.

39. The method of claim 38, wherein the cetacean is a pig or a cow.

40. A transplant material for a patient in need of a kidney transplant, comprising the chimeric kidney organoid according to any one of claims 1 to 5, chimeric kidney tissue obtained by the manufacturing method according to any one of claims 6 to 12, chimeric kidney organoid obtained by the manufacturing method according to any one of claims 13 to 19, porcine kidney organoid according to claim 30 or 31, or kidney organoid obtained by the manufacturing method according to claim 38 or 39.

41. The implantable material of claim 40 for treating patients with kidney damage or injury.

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