Method for manufacturing collecting duct of mature kidney

Abstract

The purpose of the present invention is to provide a method for manufacturing a collecting duct of a mature kidney. The purpose of the present invention is also to provide a method for manufacturing a collecting duct organoid of a mature kidney derived from iPSC. According to the present invention, provided is a method for manufacturing a collecting duct of a kidney, the method comprising culturing a ureteric bud in a medium having an isotonic osmotic pressure (iso-osmotic pressure) and then culturing the bud in a medium having one or more osmotic pressures exceeding the iso-osmotic pressure, wherein the medium having at least one osmotic pressure exceeding the iso-osmotic pressure has an osmotic pressure of at least 600 mOsm / kg or greater.

Description

Method for manufacturing mature kidney collecting ducts

[0001] This invention relates to a method for producing mature kidney collecting tubules.

[0002] The kidneys play a vital role in maintaining homeostasis by producing urine in the body. They also function as important endocrine organs, regulating blood pressure and fluid balance through renin production and the renin-angiotensin-aldosterone system (RAAS). When kidney function is lost, regeneration is virtually impossible, leading to the need for dialysis. In Japan, the number of dialysis patients exceeds several hundred thousand, contributing to soaring medical costs. However, kidney transplants, which would replace dialysis, suffer from a chronic shortage of donors, and groundbreaking new treatments and regenerative therapies are urgently needed.

[0003] The kidney is constructed through the interaction of three progenitor cells: nephron progenitor cells, ureteric buds, and interstitial progenitor cells, and is formed through a complex process different from that of other major organs. For an artificially reconstructed kidney to function, it is essential to construct the kidney's characteristic higher-order structure, in which the nephron, the main functional unit consisting of glomeruli and tubules, is located at the periphery of the kidney and connected to numerous branched collecting ducts.

[0004] Several groups, including the present inventors, have recently developed methods for inducing kidney organoids from iPS cells. For example, the present inventors have reported a method for inducing nephron progenitor cells and ureteriblasts from iPS cells, and from these, inducing nephron organoids with glomeruli and tubules, and immature collecting duct organoids (Non-Patent Documents 1 and 2, Patent Document 1). In this method, it was reported that ureteriblasts induced from human iPS cells were differentiated into immature collecting duct organoids branched within 50% Matrigel. In addition, other groups have reported ureteriblast and collecting duct organoids induced from human iPS cells (Non-Patent Documents 3 and 4). However, the kidney organoids reported to date correspond to the fetal kidney in the second stage of pregnancy, and their degree of organ maturity is insufficient, so further improvement is desired.

[0005] Furthermore, devices and culture methods that combine liquid flow have been reported (Non-Patent Literature 5). These reports describe a method for two-dimensional culture of ureteral bud organoids induced from human stem cells on a device (Epi-MAP) while maintaining liquid flow of the culture medium. It has been reported that collecting tubules cultured with this device exhibited improved functions specific to collecting tubules compared to static culture without flow.

[0006] Furthermore, in terms of the maturation of the collecting ducts of the kidney, the contributions of two hormones, vasopressin and aldosterone, have been reported (Non-Patent Documents 3, 4, 6, and 7).

[0007] WO2020 / 095423

[0008] Taguchi et al., Cell Stem Cell 14(1):53-67 (2014)Taguchi et al., Cell Stem Cell 21(6):730-746 (2017)Zeng et al. Nat. Commun. 15, 3641 (2021)Shi et al. Nat. Biotechnol. 41, 252-261 (2023)Soongweon et. al., Journal of the American Society of Nephrology 34(11S): p 722, SA-PO001 November 2023.Uchimura et al. Cell Reports 33, 108514, 2020Howden et al. Cell Stem Cell. 28, 671-684 (2021)Boselt, I. et al. Nephrol. Dial. Transplant 27, 1521-1528, 2012

[0009] An object of the present invention is to provide a method for producing the collecting ducts of the kidney. Another object of the present invention is to provide a method for producing kidney collecting duct organoids derived from iPS cells.

[0010] The present inventors, after diligently studying methods for producing renal collecting ducts, discovered that mature collecting ducts can be produced by culturing ureteral buds under hyperosmolarity, and thus completed the present invention. The present invention includes the following: [1] A method for producing renal collecting ducts, comprising the step of culturing ureteral buds in an isotonic (isoosmolarity) medium, and then culturing them in one or more mediums having an osmolarity exceeding isosmolarity (hereinafter referred to as "hyperosmolarity medium"), wherein at least one hyperosmolarity medium is a medium having an osmolarity of at least about 600 mOsm / kg or more. [2] The production method according to [1] above, wherein the step of culturing in the hyperosmolarity medium comprises the step of culturing in a hyperosmolarity medium having an osmolarity of at least about 700 mOsm / kg or more. [3] The production method according to [1] or [2] above, wherein the step of culturing in the hyperosmolarity medium comprises the step of culturing in at least two or more hyperosmolarity mediums having different osmolarity, and the culturing is carried out sequentially and stepwise in media having increasingly higher osmolarity. [4] The method for producing cells according to [3], wherein the step of culturing in the hyperosmolar medium is the step of culturing in a hyperosmolar medium having at least three or more (preferably four or more, more preferably five or more, and even more preferably six or more) different osmotic pressures. [5] The method for producing cells according to any one of [1] to [4], wherein the osmotic pressure of the hyperosmolar medium is a medium prepared by adding a cell membrane impermeable molecule (for example, sodium chloride, glucose, sucrose, mannitol, preferably sodium chloride) to the medium. [6] The method for producing cells according to any one of [1] to [5], wherein the hyperosmolar medium is a medium to which forskolin has been added. [7] The method for producing cells according to any one of [1] to [6], wherein the hyperosmolar medium is a medium to which a hormone that acts on the renal tubules (for example, vasopressin, desmopressin, aldosterone, preferably vasopressin and / or aldosterone) has been added. [8] The manufacturing method according to any one of [1] to [7] above, wherein the culture step is carried out in a culture medium containing about 10% or more (preferably about 20% or more, more preferably about 30% or more, even more preferably about 40% or more, and even more preferably about 50% or more) of Matrigel.[9] The manufacturing method according to any one of [1] to [8] above, wherein the ureteric bud is a ureteric bud induced from iPS cells.

[10] The manufacturing method according to [9] above, wherein the renal collecting duct is a renal collecting duct organoid induced from iPS cells.

[11] The manufacturing method according to [9] or

[10] above, wherein the iPS cells are human-derived iPS cells.

[12] The human-derived iPS cells are genetically modified to produce AVPR2 in the vasopressin type 2 receptor gene. Ｑ１７４Ｒ A method for producing human iPS cells into which the mutation has been introduced, as described in

[11] above.

[13] A method for producing human iPS cells into which the NFAT5 gene has been knocked out by genetic manipulation, as described in

[11] above.

[14] A collecting duct organoid produced by any one of the methods described in [1] to

[13] above.

[15] A collecting duct organoid produced by the method described in

[10] above, which is used as a disease model (for example, a congenital nephrogenic diabetes insipidus model, an ADPKD disease model).

[16] A collecting duct organoid produced by the method described in

[13] above, which is used as a salt-sensitive hypertension model.

[0011] The present invention allows for the production of collecting ducts from ureteral buds. In particular, collecting duct organoids can be produced from ureteral bud organoids differentiated from iPS cells.

[0012] The figure shows the process of gradually increasing the osmotic pressure of the culture medium by adding NaCl. The figure shows bright-field images of collecting tubules at 300 and 750 mOsm / kg. The lower panel is a high-magnification image of the area enclosed by the black rectangle in the upper panel. Scale bar = 200 μm. The figure shows the results of measurements of lumen diameter, cytoplasmic area, and cell height of collecting tubules at 300 and 750 mOsm / kg. This is the result of gene expression analysis in cultured mouse collecting tubules (mCD) matured at 300 and 750 mOsm / kg. The figure shows bright-field images of collecting tubules at 300 mOsm / kg (Ctrl) and 750 mOsm / kg (urea, sucrose, NaCl). The lower panel is a high-magnification image of the area enclosed by the black rectangle in the upper panel. Scale bar = 200 μm. The figure shows the results of H&E staining. This is the result of analyzing gene expression in mCDs matured at 300 mOsm / kg (Ctrl) and 750 mOsm / kg (urea, sucrose, NaCl). This is the result of confirming the effect of arginine vasopressin (AVP) in the mCD culture process. After adding 100 nM AVP for 3 days from day 6 to day 9 and culturing, the results were immunostained with KRT8 (cytoskeleton: red) and AQP2 (water channel: green) on day 9. The left figure shows the result of immunostaining with KRT8 (cytoskeleton: red), the right figure shows the result of immunostaining with AQP2 (water channel: green), and the center figure shows the combined result. AQP2 expression was observed at the point indicated by the arrow (apical end). These are bright-field images of CDs matured at normal osmotic pressure (300 mOsm / kg) and hyperosmotic pressure (750 mOsm / kg) after being exposed to 450 mM urea and cultured for one day. The bottom image is a magnified view of the area enclosed by the rectangle. These are bright-field images of ureteral buds (UBs) isolated from Nfat5 KO mice and cultured (matured). Control is UBs cultured from wild-type mice. Scale bar = 400 μm. This shows an outline of the maturation protocol for producing collecting duct organoids by maturing ureteral bud organoids induced from human iPS cells. These are bright-field images of collecting duct organoids cultured using the maturation protocol in Figure 11.The left figure shows the results of culturing at 300 mOsm / kg with the addition of forskolin (FSK), and the right figure shows the results of culturing with the addition of FSK and the tonicity gradually increased to 900 mOsm / kg. The bottom figure is a magnified view of the square area. The scale bar is 200 μm. This shows the results of confirming the expression of marker genes in CD organoids cultured under hormone (AVP and aldosterone), forskolin, and forskolin + high tonic conditions. This shows the results of confirming the effects of AVP, aldosterone, and forskolin in the culture process. After culturing with 100 nM AVP and 100 nM aldosterone added from day 6, the results of immunostaining with CDH1 (cell adhesion molecule: white) and AQP2 (water channel: green) on day 20 are shown. AQP2 expression was observed at the point indicated by the arrow (apical end). "Normal osmotic pressure" and "Hyperosmotic pressure" refer to the results of culturing at normal or hyperosmotic pressure with the addition of FSK, respectively. "Hormone" refers to the results of culturing at normal osmotic pressure with the addition of vasopressin and aldosterone. These are bright-field images of mature CD organoids cultured at 900 mOsm / kg for control (UB derived from normal iPSCs) and KO (UB derived from NFAT5 KO iPS). Scale bar = 200 μm. These are the results of immunostaining with AQP2 (water channel: green) on CD organoids induced from NFAT5 KO iPS cells using a method combining hypertonic stimulation and hormones. Ctr and KO are the results for CD organoids induced from normal iPS cells or NFAT5 KO iPS cells, respectively. The arrows indicate AQP2 expression (localization). These are the results of confirming the expression of marker genes in CD organoids. Ctr and KO are the same as in Figure 16. The SCNN1A and SCNN1G genes were elevated only under hypertonic stimulation in knockout (KO) induced organoids. The results show immunostaining of CD organoids induced from iPS cells derived from healthy individuals and NDI patients using a method combining hypertonic stimulation and hormones, with AQP2 (water channel: green). AQP2 expression was observed on the luminal side (apical side) in healthy individuals, whereas it was not observed in patients. The arrows in the figure in healthy individuals indicate the expression (localization) of AQP2.This is the result of maturing collecting tubule organoids by gradually increasing the osmotic pressure by 100 mOsm / kg each day. The figure shows the morphology of the organoids observed under a microscope when the osmotic pressure reached 700 mOsm / kg.

[0013] The present invention will be described below, with illustrative embodiments as examples, along with preferred methods and materials that may be used in carrying out the invention, but the present invention is not limited to the embodiments described below. Unless otherwise specified herein, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Any materials and methods equivalent to or similar to those described herein may be used in carrying out the present invention. Furthermore, all publications and patents cited herein in connection with the present invention are cited herein and constitute part of this specification, for example, as indicating methods, materials, and other matters that may be used in the present invention.

[0014] In this specification, the notation "A to B" indicating a numerical range means a numerical range that includes the endpoints A and B. The same applies to "A to B". In this specification, "approximately" is used to mean allowing a tolerance of ±10%, meaning that when the lower limit is intended, a tolerance of -10% is allowed, and when the upper limit is intended, a tolerance of +10% is allowed.

[0015] In this specification, "isotonic osmotic pressure," "isotonic pressure," or "standard osmotic pressure" means a state in which the osmotic pressure inside and outside the cell is equal under normal cell culture or maintenance conditions. In this specification, "isotonic osmotic pressure," "isotonic pressure," and "standard osmotic pressure" are used interchangeably. In the manufacturing method of the present invention, isotonic osmotic pressure means an osmotic pressure within a range that is physiologically suitable for mammalian animal cells, particularly mouse cells or human cells, for example, an osmotic pressure of about 280 to about 320 mOsm / kg.

[0016] In this specification, "high osmotic pressure" or "high tonicity" means an osmotic pressure exceeding isotonic pressure. In this specification, "high osmotic pressure" and "high tonicity" are interchangeable terms. In this specification, culturing under high osmotic pressure or high tonicity means culturing in a medium having an osmotic pressure exceeding isotonic pressure. In this specification, "high osmotic medium" or "high tonicity medium" means "a medium having an osmotic pressure exceeding isotonic pressure."

[0017] In one embodiment, the present invention is a method for producing collecting ducts (CDs) by culturing ureteric buds (UBs) under high osmotic pressure or high tonicity. The present invention is a culturing method (also referred to as a "maturation method") for producing collecting ducts from ureteric buds. Therefore, in this specification, the step of producing collecting ducts from ureteric buds by the culturing method of the present invention may also be referred to as a step of maturing ureteric buds or collecting ducts, and these terms are used interchangeably.

[0018] In one embodiment, the present invention provides a method for producing collecting ducts by maturing ureteral buds isolated from living organisms, for example, ureteral buds isolated from mouse fetal kidneys, by culturing them under hyperosmolarity or hypertonicity.

[0019] In one embodiment, the present invention provides a method for producing collecting duct organoids by maturing ureteral bud organoids differentiated from pluripotent stem cells under hyperosmolarity or hypertonic conditions. Pluripotent stem cells can be, for example, ES cells or iPS cells, and are preferably iPS cells. iPS cells can be, for example, human-derived iPS cells or mouse-derived iPS cells, and are preferably human-derived iPS cells.

[0020] In this specification, a method for producing collecting ducts or collecting duct organoids by culturing ureteric buds or ureteric bud organoids to mature them may be referred to as the culture method, production method, or maturation method of the present invention, and in this sense, "culture," "production," and "maturation" are used as interchangeable terms. Accordingly, in this specification, a method for culturing ureteric buds (organoids) or collecting ducts (organoids) under high osmotic pressure or high tonic pressure is also a method for maturing ureteric buds (organoids) or collecting ducts (organoids).

[0021] In this specification, when we refer to normal (or standard) osmotic pressure or normal (or standard) tonicity, we mean the osmotic pressure or tonicity of a normal ureteral bud differentiation medium, for example, an osmotic pressure of approximately 280 to approximately 320 mOsm / kg. Therefore, in this specification, when we refer to normal osmotic pressure or normal tonicity, we mean a medium that, when measured using an osmometer, shows, for example, approximately 280, approximately 290, approximately 300, approximately 310, or approximately 320 mOsm / kg. In this specification, for convenience, the osmotic pressure of a medium with normal osmotic pressure or normal tonicity will be expressed as 300 mOsm / kg.

[0022] The term "osmotic pressure" is used to express the total molecular concentration per kilogram of solvent in a solution. When referring to osmotic pressure, it includes both solutes that can and cannot pass through the cell membrane. Osmotic pressure is calculated even for substances that can pass through the membrane, while tonicity is an indicator of the effect of a solution on cells, relating to the direction of water movement in cells (whether water flows into or out of the cell) and defined based on molecules that cannot pass through the cell membrane (impermeable molecules). Some molecules, such as urea, diffuse freely across the cell membrane, and their concentration gradient does not induce water movement between the intracellular and extracellular compartments. On the other hand, other molecules, such as NaCl, glucose, and sucrose, do not easily permeate the cell membrane, resulting in water movement across the cell boundary. The concentration of these molecules that cannot pass through the cell membrane (cell membrane impermeable molecules) is defined as the effective osmotic pressure, also known as tonicity. Therefore, in this specification, when referring to cell membrane impermeable molecules, "osmotic pressure" and "tonicity" are synonymous and used as interchangeable terms. For example, in this specification, when NaCl is added to the differentiation medium, "hyperosmotic pressure" and "hypertonicity" are used as interchangeable terms.

[0023] In this specification, "cell membrane impermeable molecule" does not need to be completely impermeable to the cell membrane, but is not particularly limited as long as it is a molecule that can be used in culture and can adjust the osmotic pressure of the culture medium when added to it. Examples include sodium chloride, glucose, sucrose, mannitol, dextran, polyethylene glycol, and serum albumin (e.g., BSA and HSA). Preferably, it is sodium chloride, sucrose, or mannitol.

[0024] Method for maturing collecting ducts: The following describes a method for maturing ureteric buds by culturing them under hyperosmolarity or hypertonicity. Unless the context makes it clear whether a specific ureteric bud, such as one isolated from an embryo (especially an embryonic kidney) or a ureteric bud organoid differentiated from iPS cells, is being referred to, the term should be understood to encompass both. Similarly, unless the context makes it clear whether the ureteric bud organoid is derived from mouse or human iPS cells, the term should be understood to encompass both.

[0025] The present invention provides a method for producing collecting ducts, characterized by comprising the step of culturing ureteral buds in a hyperosmolar medium, wherein the step includes culturing in a medium having an osmotic pressure of at least 500 mOsm / kg, preferably at least about 600 mOsm / kg, and more preferably about 700 mOsm / kg. The step of culturing in a hyperosmolar medium in the present invention includes culturing in one or more, preferably two or more, more preferably three or more, even more preferably four or more, even more preferably five or more, and most preferably six or more, different media having osmotic pressures higher than isotonic, wherein the culturing includes steps of culturing in media having progressively higher osmotic pressures. The number of media with progressively higher osmotic pressures used for culturing is not particularly limited and can be arbitrarily selected based on the type of ureteral bud, the degree of maturation of the collecting duct, and other factors. For example, but not limited to this, when culturing in media with three stages of high osmotic pressure, one example is to cultivate under isotonic conditions, then in a medium with a higher osmotic pressure (stage 1), then in a medium with an even higher osmotic pressure (stage 2), and finally in a medium with an even higher osmotic pressure (stage 3). Furthermore, there are no particular restrictions on the duration of cultivation at each stage, including the period of cultivation under isotonic conditions, and these can be arbitrarily selected based on the type of ureteral bud, the degree of maturation of the collecting duct, and other factors.

[0026] While not limited to this, in the case of mouse ureteral buds, for example, one method is to culture them in two or three stages of high-osmotic medium. While not limited to these, for example, in a two-stage process, the culture can be performed under isotonic conditions (approximately 280 to 320 mOsm / kg), then under a first hyperosmolar condition (e.g., approximately 400 to 500 mOsm / kg), and then under a second hyperosmolar condition (e.g., approximately 600 to 800 mOsm / kg). Similarly, in a three-stage process, the culture can be performed under isotonic conditions (approximately 280 to 320 mOsm / kg), then under a first hyperosmolar condition (e.g., approximately 400 to 500 mOsm / kg), then under a second hyperosmolar condition (e.g., approximately 550 to 650 mOsm / kg), and finally under a third hyperosmolar condition (e.g., approximately 700 to 800 mOsm / kg). There are no particular restrictions on the incubation period under each osmotic pressure, but for example, in the case of a two-stage incubation, the incubation period can be 5-6 days under isotonic pressure, about 1-2 days under the first hyperosmolar pressure, and about 1-2 days under the second hyperosmolar pressure. For example, in the case of a three-stage incubation, the incubation period can be 5-6 days under isotonic pressure, about 1 day under the first hyperosmolar pressure, about 1 day under the second hyperosmolar pressure, and about 1 day under the third hyperosmolar pressure.

[0027] Furthermore, although not limited to this, in the case of human iPS-derived ureteral bud organoids, for example, methods of culturing under hyperosmolarity in 3, 4, 5, or 6 stages can be mentioned. While not limited to these, for example, in the case of four stages, the culture can be performed under isotonic conditions (approximately 280 to 320 mOsm / kg), then under a first hyperosmolarity (e.g., approximately 350 to 450 mOsm / kg), then under a second hyperosmolarity (e.g., approximately 500 to 550 mOsm / kg), then under a third hyperosmolarity (e.g., approximately 600 to 700 mOsm / kg), and finally under a fourth hyperosmolarity (e.g., approximately 750 to 950 mOsm / kg). For example, in the case of five stages, the culture can be performed under isotonic conditions (approximately 280 to 320 mOsm / kg), then under a first hyperosmolarity (e.g., approximately 350 to 400 mOsm / kg), then under a second hyperosmolarity (e.g., approximately 400 to 450 mOsm / kg). The culture can be performed under a high osmotic pressure of mOsm / kg, then under a third high osmotic pressure (e.g., about 500 to about 550 mOsm / kg), then under a fourth high osmotic pressure (e.g., about 600 to about 700 mOsm / kg), and finally under a fifth high osmotic pressure (e.g., about 750 to about 950 mOsm / kg). The stepwise increase in osmotic pressure during culture under stepwise high osmotic pressures is preferably less than 100 mOsm / kg per day, more preferably 80 mOsm / kg or less, and even more preferably 70 mOsm / kg or less, from isotonic to a high osmotic pressure of 600 mOsm / kg. There are no particular restrictions on the duration of culture under each osmotic pressure, but for example, in the case of four stages, the culture period can be 10-12 days under isotonic pressure, about 3-4 days under the first hyperosmolarity, about 1-2 days under the second hyperosmolarity, about 1-2 days under the third hyperosmolarity, and about 2-3 days under the fourth hyperosmolarity. For example, in the case of five stages, the culture period can be 10-12 days under isotonic pressure, about 2-3 days under the first hyperosmolarity, about 1-2 days under the second hyperosmolarity, about 1-2 days under the third hyperosmolarity, about 1-2 days under the fourth hyperosmolarity, and about 2-3 days under the fifth hyperosmolarity. Furthermore, it is possible to divide the culture under any of the hyperosmolarity conditions into multiple stages, or to add additional culture under additional hyperosmolarity conditions.For example, an additional period of hyperosmolarity (e.g., approximately 320–340 mOsm / kg for about 1 day) can be added before the first period of hyperosmolarity culture, or a culture under hyperosmolarity at any stage can be changed to a two- or three-stage culture. For example, but not limited to this, a three-day culture at approximately 350–450 mOsm / kg can be changed to a culture at approximately 350–370 mOsm / kg for 1 day and then at approximately 380–450 mOsm / kg for 2 days. In this way, the osmotic pressure and culture period in multi-stage hyperosmolarity culture can be arbitrarily set based on the type of ureteral bud, the degree of maturation of the collecting duct, and other factors.

[0028] The culture medium used in the culture method of the present invention (sometimes referred to as "branched medium" in this specification) can be prepared by appropriately adding necessary factors to the basal medium used for culturing animal cells. Examples of basal media include media used in the present art, such as DMEM (Dulbeccoo's modified Eagle medium), DMEM / F12 medium, GMEM (Glasgow MEM) medium, 199 medium, EMEM medium, Ham's F12 medium, PRMI 1640 medium, IMDM (Iscob's modified Dulbecco medium), αMEM (Eagle's minimal essential medium α modified), Fischer's medium, and mixed media thereof. The basal medium may contain serum or may be serum-free.

[0029] The culture medium used in the present invention may, as needed, include, for example, serum (e.g., FBS), albumin, ITS (insulin, transferrin, selenium), KSR (Knockout Serum Replacement), N-2 supplement (Thermo Fisher Scientific), B-27® supplement and B-27® supplement minus vitamin A (Thermo Fisher Scientific), 2-mercaptoethanol, 1-thioglycerol, amino acids, L-glutamine, glutamine supplements (e.g., L-alanyl-L-glutamine, GlutaMAX), non-essential amino acids, ascorbic acid, pyruvate, TGF-β, retinoic acid, dexamethasone, BMP, IGF, vitamins, Wnt ligand, EGF, FGF (FGF-1, FGF-2, FGF-7, etc.), noggin, LDN193189, and R-spondin. 1. The culture medium may also contain at least one culture medium additive selected from GDNF, NGF, antibiotics including penicillin and streptomycin, growth factors, signaling pathway promoters and inhibitors, and one or more other substances commonly added to animal culture media. The culture medium used in the present invention is preferably a DMEM / F12 medium, which is a basal medium, to which culture medium additives have been added. However, it is not limited to this, but examples include the culture medium described in Non-Patent Document 2 and the culture medium described in the examples of this specification.

[0030] The culture method of the present invention is preferably performed using three-dimensional culture. Three-dimensional culture refers to culturing cells in a three-dimensional manner. There are two types: the Scaffold type, in which cells are cultured within a scaffold material, and the Scaffold-free type, in which cells are cultured in a suspended state as a mass (spheroid). The culture method of the present invention is preferably the Scaffold type. An extracellular matrix can be used as the three-dimensional support. The extracellular matrix is ​​not particularly limited as long as it is a substance that serves as a scaffold for cells in cell culture, but examples include Matrigel®, collagen, laminin, alginate hydrogel, and Vitrigel, with Matrigel being preferred.

[0031] When Matrigel is used as a three-dimensional scaffold material in culture, the concentration of Matrigel in the culture medium may be about 10% or more, for example, about 20% to 80%, about 30% to 70%, about 40% to 60%, or about 50%. The concentration of Matrigel may also be about 10% or more, preferably about 20% or more, more preferably about 30% or more, even more preferably about 40% or more, and even more preferably about 50% or more.

[0032] In the culture method of the present invention, the culture vessel used for three-dimensional culture is not particularly limited as long as the osmotic pressure of the culture medium can be gradually increased to any desired high osmotic pressure during the culture process (maturation process of the collecting tube), and is not particularly limited as long as it is a normal culture vessel for cell or tissue culture. Examples include dishes, petri dishes, tissue culture dishes, microplates, microwell plates, multiplates, multiwell plates, chamber slides, trays, flasks, cell inserts, etc. Examples of materials for the culture vessel include inorganic materials such as metal, glass, ceramic, and silicon, and organic materials represented by elastomers and plastics (e.g., polyester resin, polyethylene resin, polypropylene resin, ABS resin, nylon, acrylic resin, fluororesin, polycarbonate resin, polyurethane resin, methylpentene resin, phenolic resin, melamine resin, epoxy resin, and vinyl chloride resin), but is not limited thereto.

[0033] In the culture method of the present invention, it is preferable to use a cell insert as the culture vessel, from the viewpoint of changing the osmotic pressure of the culture medium to a hyperosmolar state during the culture process. As one embodiment of the culture method of the present invention, for example, cells are seeded in a three-dimensional culture medium (for example, a medium containing about 50% Matrigel) placed in a cell insert (Upper Chamber), while the hyperosmolar medium, which is to be changed stepwise, is added to the base chamber (Lower Chamber), and the cells are cultured sequentially in the hyperosmolar medium.

[0034] As a combination of a cell culture vessel consisting of a cell insert having a porous membrane on the bottom surface and a base chamber that can be used in the method of the present invention, commercially available products can be used, for example. Examples include cell culture inserts (cell inserts) and culture vessels (base chambers) for holding the inserts provided by Corning International, Thermo Scientific, Greiner Bio-One International, etc. Examples of cell inserts include Corning's Transwell® permeable support, Snapwell® insert, Netwell® insert, and Falcon cell culture insert, which can be suitably used in the present invention. Examples of base chambers include Corning's Falcon cell culture plate and Falcon multi-cell culture plate, which can be suitably used in the present invention.

[0035] In one embodiment, the culture method of the present invention may include a step of culturing in a hyperosmolar medium supplemented with forskolin (hereinafter sometimes referred to as "FSK-supplemented hyperosmolar medium"). The FSK-supplemented hyperosmolar medium can be any of one or more hyperosmolar media used in the culture step of the present invention, and forskolin may be contained in all of the hyperosmolar media used, or it may be contained in a part of the hyperosmolar media used, preferably in the later stages, in other words, in the hyperosmolar media used at the more mature stage. Although not limited to this, for example, if the method includes culturing in six stages of hyperosmolar media, it is preferable that the last four stages of hyperosmolar media be FSK-supplemented hyperosmolar media (hereinafter referred to as "the last four stages / all six stages" or "four stages / six stages"). The culture process using the FSK-supplemented hyperosmolar medium of the present invention can be, for example, the latter two stages / total five stages, three stages / five stages, four stages / five stages, the latter two stages / total six stages, three stages / six stages, four stages / six stages, five stages / six stages, the latter two stages / total seven stages, three stages / seven stages, four stages / seven stages, five stages / seven stages, or six stages / seven stages. Furthermore, forskolin can also be included in the isosmolar medium, and in such cases, it can be included only for a portion of the culture period in the isosmolar medium, preferably only during the later stages. The culture process of the present invention using the FSK-supplemented hyperosmolar medium is preferably used when using human iPS-derived ureteral bud organoids. The concentration of forskolin in the hyperosmolar medium is not particularly limited as long as it is sufficient to achieve maturation of the ureteral bud or collecting duct, but for example, it is about 1 μM to about 100 μM, preferably about 2 μM to about 50 μM, more preferably about 3 μM to about 30 μM, even more preferably about 5 μM to about 20 μM, and most preferably about 10 μM.

[0036] In one aspect, the culture method of the present invention preferably includes culturing in a hypertonic medium (hereinafter sometimes referred to as "hormone-added hypertonic medium") to which a hormone acting on the renal tubule is further added. Examples of the hormone acting on the renal tubule include vasopressin, desmopressin, and aldosterone, and preferably vasopressin and / or aldosterone. Vasopressin (VP) can include arginine vasopressin (AVP), lysine vasopressin (LVP), phenylalanine vasopressin, etc., and any of these can be used, but preferably AVP.

[0037] The hormone-added hypertonic medium can be used in any one or more of the hypertonic media used in the culture process of the present invention, can be used in all hypertonic media, or can be used in a part of the hypertonic media. Further, the hormone can also be contained in an isotonic medium, and in such a case, it can also be contained only during a part of the period of culturing in the isotonic medium, for example, only during the later period. The culture process of the present invention using the hormone-added hypertonic medium is preferably used when using human iPS-derived ureteric bud organoids.

[0038] The concentration of the hormone in the hypertonic medium is not particularly limited as long as it can sufficiently achieve the maturation of the ureteric bud or the collecting duct, for example, a concentration that can achieve sufficient expression of aquaporin 2 (AQP2). For example, it is about 1 nM to about 10 μM, preferably about 2 nM to about 1 μM, more preferably about 5 nM to about 500 nM, still more preferably about 10 nM to about 200 nM, and most preferably about 100 nM.

[0039] The culture temperature is about 30 to about 40 °C, for example, about 37 °C, and the culture is carried out in an atmosphere containing air, and the CO ２ concentration is, for example, about 2 to about 5%. ２

[0040] ​Collecting duct organoids prepared by the method of the present invention can also be used as disease models or pathological models. Therefore, one aspect of the present invention is a collecting duct organoid prepared by the method of the present invention, characterized by reproducing the in vitro pathological state of a disease. Diseases and pathological states that can be reproduced include, but are not limited to, congenital nephrogenic diabetes insipidus, ADPKD pathological models, and salt-sensitive hypertension.

[0041] Collecting tubules or collecting tubule organoids produced by the method of the present invention have at least one, preferably at least two, more preferably at least three, and even more preferably at least four of the following features (a) to (g): (a) Increased expression of any, preferably multiple, genes selected from the group consisting of Aqp2, Slc14a2, Avpr2, Akr1b3, Slc6a12, Slc5a3, Elf5, and Pax2. (b) Expression of aquaporin 2 and / or KRT8 can be confirmed on the apical side of the collecting tubule, and polarity exists in the constituent cells. (c) They react to vasopressin and have the property of aquaporin 2 moving to the apical side. (d) They are accompanied by lumen dilation and cytoplasmic enlargement of constituent cells. (f) They are urea resistant, for example, resistant to 450 mM urea. (g) They produce prostaglandins.

[0042] Preparation of Ureteric Buds or Ureteric Bud Organoids The ureteric buds used in the culture method of the present invention can be prepared by referring to previously reported methods. For example, ureteric buds can be isolated from embryos, particularly embryonic kidneys, and used in the method of the present invention. For example, they can be prepared according to the inventors' previously published findings (Non-Patent Literature 2). The ureteric bud organoids used in the culture method of the present invention can be prepared by referring to previously published methods. Ureteric bud organoids can be prepared from iPS cells by differentiating iPS cells, for example, by referring to the inventors' previously published findings (Non-Patent Literature 2).

[0043] Hereinafter, the present invention will be specifically described by way of examples. However, the present invention is not limited to the following examples. (Materials and Methods) All animal experiments were conducted with the approval of the Ethics Approval Committee of Kumamoto University (approval numbers: A2021-008, A2023-009, A2024-073) in accordance with the ethical guidelines of the facility.

[0044] Mouse Nfat5 flox / flox Mice were created according to the report by Katherine Drews-Elger et al. (PLos One, 4, e5245 (2009)). Hoxb7-Cre mice were purchased from the Jackson Laboratory. Nfat5 flox / flox By mating Nfat5 mice with Hoxb7-Cre mice, mice with specifically deleted Nfat5 in the ureteric bud (UB) / collecting duct (CD) (Nfat KO mice) were created.

[0045] Human iPSC The 201B7 and RN7iPSC lines were established from healthy female and male providers, respectively. Both were maintained on iMatrix-511 (Nippi) in StemFit AK03N medium (AJINOMOTO).

[0046] Immunohistochemical staining Immunohistological staining (hereinafter sometimes simply referred to as "immunostaining") was performed by fixing the specimens with 10% formalin and embedding them in paraffin. After deparaffinizing the paraffin-embedded sections, antigen retrieval was performed in citrate buffer. Immunostaining was performed according to a conventional method. A secondary antibody conjugated with an Alexa Fluor dye was used, and fluorescence images were captured with a confocal microscope. For NFAT5 staining, signals were amplified using an ImmPRESS HRP reagent kit (Vector Laboratories) and a tyramide signal amplification (TSA) kit (Thermo Fisher Scientific).

[0047] Measurement of PGE2 production by MS: Collecting tubes (mCDs) of cultured mice were collected on day 9, recovered using a Hand Style Simple Grinding Container (ASONE #2-1825-01), and stored at -80°C. The tissue was homogenized in 500 μL of MeOH:formic acid (100:0.2) containing an internal standard consisting of deuterium-labeled PGE2, using microchip sonication. The samples were solid-phase extracted using an Oasis HLB cartridge (5 mg; Waters, Milford, MA). Subsequently, PGE2 was quantified using a triple quadrupole mass spectrometer. PGE2 production in mCDs was calculated by subtracting the amount of PGE2 in the Matrigel from the amount of PGE2 in the cultured mCDs, since the cultured mCDs were embedded in Matrigel.

[0048] Single-cell RNA sequencing (scRNA-seq) analysis: ScRNA-seq data from mouse kidneys at E15.5 (embryonic day 15.5), P0 (postnatal day 0), and P7 (postnatal day 7) have already been reported (Naganuma, H. et al. Dev. Biol. 470, 62-73, 2021). ScRNA-seq data from nephron progenitor cells and stromal progenitor cells induced from embryonic stem cells (iNPs and iSPs) have also been reported (Tanigawa, S. et al. Nature communications 13, 1-15, 2022). Therefore, cultured mCD and human CD organoids were analyzed according to standard methods. Cultured mCD or human CD organoids were incubated in 0.25% trypsin / EDTA for 10 minutes. After washing the cells with a buffer containing DNase, the cells were filtered through a 40 μm pore cell strainer. A portion containing 5,000 dissociated cells (aliquote) was applied to a Chromium Controller (10x Genomics) from each sample. A cDNA library was then constructed using the Chromium Single Cell 3' Library & Gel Beads Kit v3 (10x Genomics), and subsequently sequenced using Illumina HiSeq X Ten.

[0049] RNA extraction and RT-qPCR analysis were performed according to standard procedures. Total RNA was isolated using the RNeasy Plus Micro Kit (Qiagen) and reverse transcribed using the Superscript VILO cDNA synthesis kit (Invitrogen). Quantitative PCR was performed using the Real-Time PCR System (Takara Bio) and the TB Green Fast qPCR Mix (Takara Bio). Relative mRNA expression levels were standardized by β-actin gene expression.

[0050] In the following examples, ureteral bud maturation culture was performed using mouse or human branched media. The osmotic pressure of the mouse and human branched media was measured using an osmometer (Fiske 210 Micro-Sample Osmometer) and was found to be approximately 320 mOsm / kg. For simplicity, we will assume 300 mOsm / kg and use this value in the following examples. Therefore, when it is stated in this specification that culture is performed under an osmotic pressure of 300 mOsm / kg, it means that the culture is performed in a normal branched media without osmotic pressure adjustment, and does not mean that the osmotic pressure of the medium is exactly 300 mOsm / kg.

[0051] (Example 1) The effect of hyperosmolarity on the maturation of mouse ureteral buds / collecting ducts and the formation of collecting ducts (CDs) was investigated as follows. E11.5 Ureteral buds (UBs) that give rise to collecting ducts (CDs) were isolated from the embryonic kidney of ICR mouse embryos (Jcl: ICR; SLC, Inc. Japan). The isolated UBs were cultured in hyperosmolarity medium with an osmotic pressure gradually increased from 300 mOsm / kg to 750 mOsm / kg, as shown in Figure 1. The osmotic pressure was adjusted by adding NaCl solution. UBs isolated from wild-type (WT) and Nfat KO mice were used.

[0052] Specifically, the isolated UBs were embedded in 150 μL of mouse branched medium containing 50% growth factor-reduced Matrigel in a 24-well Transwell insert, and the well insert was placed on 500 μL of medium without Matrigel (bottom medium). The mouse branched medium consisted of DMEM / F12 containing 10% FBS, 1% penicillin / streptomycin, 0.1 μM retinoic acid, 100 ng / mL human R-spongin 1, 2 ng / mL human GDNF, and 100 ng / mL mouse FGF1. From day 6 of culture, the osmotic pressure of the entire medium was gradually increased by adding 5 M NaCl solution to the base medium until the medium containing the cells reached the desired osmotic pressure (day 6: 450 mOsm / kg, day 7: 600 mOsm / kg, day 8: 750 mOsm / kg). Mature tissue was collected on day 9. Collecting ducts (mCDs) prepared from ureteral buds of wild-type mice tolerated culture at 750 mOsm / kg. However, collecting ducts prepared from ureteral buds of Nfat KO mice showed inhibited growth during culture at 750 mOsm / kg, resulting in a total culture period of 8 days and a final osmotic pressure of 600 mOsm / kg.

[0053] Figure 2 shows the results of culturing ureteral buds isolated from wild-type mice. Furthermore, the luminal diameter, cell height, cytoplasmic area, and ciliary length were measured using ImageJ software. Luminal diameter was measured using bright-field imaging, while cell height and cytoplasmic area were measured using hematoxylin and eosin-stained sections. Ciliary length was measured by observing sections stained with both AcTUB and ARL13B. The measurement results for luminal diameter, cell height, and cytoplasmic area are shown in Figure 3.

[0054] As can be seen from the figure, hyperosmolarity (750 mOsm / kg) stimulation caused lumen dilation, increasing cell height and cytoplasmic region. Furthermore, primary cilia, which protrude from the apical surface of renal epithelium as flow sensors, elongated in response to hyperosmolarity stimulation. In addition, immunohistochemistry revealed that hyperosmolarity stimulation upregulates cadherin 16 (CDH16), also known as a kidney-specific (Ksp-) cadherin expressed on the basolateral membrane of renal tubules, and mucin 1 (MUC1), a transmembrane glycoprotein that forms a mucus barrier at the apical end of CD. Furthermore, as shown in Figure 4, reverse transcription quantitative PCR (RT-qPCR) analysis revealed that Aqp2, Slc14a2, Avpr2, Akr1b3, Slc6a12, Slc5a3, Elf5, and Pax2, genes reported to be regulated by osmotic pressure, are upregulated in the collecting duct by hyperosmotic stimulation. Slc14a2 encodes urea transporter protein A (UT-A), which is expressed in the collecting duct. To protect against osmotic stress, cells accumulate organic osmoregulators such as sorbitol, betaine, and inositol. Akr1b3 encodes aldose reductase, a key enzyme in sorbitol production, while Slc6a12 and Slc5a3 encode betaine and inositol transporters, respectively. Elf5 and Pax2 are important transcription factors in kidney development and physiology.

[0055] UB cells were cultured by adding either a 2 M sucrose solution or an 8 M urea solution instead of a 5 M NaCl solution, thereby gradually increasing the osmotic pressure. The results of bright-field imaging are shown in Figure 5, the results of hematoxylin and eosin staining are shown in Figure 6, and the results of RT-qPCR analysis are shown in Figure 7. Morphological changes, including the formation of lumen dilation, were observed not only with salt (NaCl) but also with sucrose, whereas, in contrast, urea did not induce these changes. Furthermore, the expression of characteristic genes was observed not only with salt (NaCl) but also with sucrose, but not with urea. NaCl and sucrose are cell membrane impermeable molecules, while urea is a cell membrane permeable molecule. From these results, it was found that the changes in epithelial morphology and gene expression were caused not by NaCl itself or osmotic pressure stimulation, but by hypertonic stimulation. Furthermore, single-cell RNA sequencing (scRNA-seq) analysis of cultured mCDs suggested that the cultured mCDs were mainly composed of principal cells. In addition, by comparing the transcriptional profiles of mCD cells cultured under normal and hyperosmolar conditions, 3942 genes that are upregulated by hyperosmolarity were identified.

[0056] (Example 2) Effects at Different Osmotic Pressures Next, we confirmed the effects at different osmotic pressures (750 mOsm / kg and 450 mOsm / kg). In the same manner as in Example 1, mCDs were prepared from ureteral buds of wild-type mice, but one group was cultured at 450 mOsm / kg on day 6 and then maintained at the same osmotic pressure until day 9. When gene expression was evaluated by RT-qPCR, it was found that the culture condition of 750 mOsm / kg increased gene expression more than that of 450 mOsm / kg overall. Specifically, when the expression level of marker genes was set to 1 at 300 mOsm / kg, the expression levels at 450 mOsm / kg and 750 mOsm / kg were 3.4 times and 6.0 times (Slc14a2), 3.7 times and 7.0 times (Avpr2), 4.9 times and 39.0 times (Akr1b3), 22 times and 1330 times (Slc6a12), 5.7 times and 10.9 times (Slc5a3), 2.3 times and 3.0 times (Elf5), and 2.3 times and 3.8 times (Pax2).

[0057] (Example 3) Synergistic effect of hypertonic stimulation and hormones We investigated the maturation of collecting ducts with hormones (AVP and aldosterone) that have been previously reported to increase several UB / CD markers (Non-Patent Literature 8 and Non-Patent Literature 6), and the maturation of collecting ducts with hypertonic stimulation. The maturation of collecting ducts with hypertonic stimulation was carried out in the same manner as in Example 1. In addition, AVP and aldosterone were added at a concentration of 100 nM each from day 6 to day 9 of culture. Gene expression of Aqp2, Slc14a2, Avpr2, Akr1b3, Slc6a12, Slc5a3, Elf5 and Pax2 was evaluated by RT-qPCR. In maturation under isotonic conditions, Slc14a2 increased 2.78 times with hormone addition, but other genes that increased with hypertonic stimulation did not increase. Aqp2 expression showed an increasing trend with hypertonic stimulation alone (750 mOsm / kg) or with hormone alone, but the difference was not statistically significant. On the other hand, when both were combined, Aqp2 expression increased synergistically (by approximately 4250 times). This indicates that hypertonic stimulation and hormones play different roles in collecting duct maturation, and that a synergistic effect can be obtained by combining them.

[0058] (Example 4) Examination of the physiological function of mCD One of the main functions of collecting tubules (CDs) is water reabsorption, which is regulated by an endocrine hormone called arginine vasopressin (AVP) or antidiuretic hormone. This hormone acts on the AVP receptor 2 (AVPR2) of CDs, inducing the translocation of aquaporin 2 (AQP2), a water channel, to the apical membrane (luminal membrane) of CDs, thereby increasing osmotic water reabsorption. To examine AVP reactivity, in the same manner as in Example 1, 100 nM AVP was added to the culture medium from Day 6 to Day 9 in a culture to mature UB into CDs. In addition, 450 mM urea was added to wild-type mouse collecting tubules on Day 9 to examine urea tolerance. As shown in Figure 8, when AVP was added during the maturation process, it was found that AQP2 accumulated at the apical end under high tonicity (750 mOsm / kg) in the presence of AVP, but did not accumulate under normal tonicity (300 mOsm / kg). This result demonstrates that mCD cultured using the culture method (maturation method) of the present invention acquires functional responsiveness to AVP.

[0059] Another characteristic of CDs is their ability to withstand extremely harsh conditions with very high urea concentrations in the medulla. To investigate this protective function, mCDs cultured under normal or hypertonic conditions were exposed to 450 mM urea on day 9 and cultured for one day. The results are shown in Figure 9. mCDs cultured under hypertonic conditions (750 mOsm / kg) maintained their structure, but mCDs cultured under normal tonicity (300 mOsm / kg) did not. This indicates that mCDs acquired urea tolerance upon hypertonic stimulation.

[0060] Furthermore, ureteral buds are known to produce prostaglandin E2 (PGE2), which is involved in sodium and water homeostasis in the body. The expression of Ptgsl and Ptges2, which encode cyclooxygenase-1 and PGE2 synthase 2, respectively, that are involved in PGE2 synthesis, was increased at high tonicity (750 mOsm / kg) compared to normal tonicity (300 mOsm / kg). In addition, when the amount of PGE2 produced was quantified by mass spectrometry, the amount of PGE2 produced increased more than 10 times in high-tonic mCD.

[0061] (Example 5) Study using ureteral buds isolated from Nfat KO mice. Activated T cell-5 nuclear factor (NFAT5), also known as osmotic-responsive enhancer-binding protein (TonEBP), is a unique transcription factor that converts extracellular hypertonic stress into downstream signaling pathways. In P7 kidney, it is expressed in the nuclei of epithelial and stromal cells in the medulla, but not in the cortex. When NFAT5 matured under hypertonic conditions, it accumulated in the nucleus, so to investigate its role in the maturation process, Nfat5 flox / flox By crossing mice with Hoxb7-Cre mice, Nfat5 was specifically deleted in UB / CDs, and E11.5 Nfat5 knockout (KO) UBs were isolated and cultured. Since the growth of Nfat5 KO mCDs was poor at high osmolality (750 mOsm / kg), the final osmolality of the culture medium was 600 mOsm / kg (8 days of culture). The results are shown in Figure 10. At 600 mOsm / kg, the proliferation of Nfat5 KO mCDs was impaired, accompanied by epithelial breakdown, which is consistent with previous reports that Nfat5 KO mice have impaired medullary development in the kidney.

[0062] A comparison of Nfat5-expressing cells from control mCDs with Nfat5-deficient cells from knockout mCDs revealed 3260 downregulated genes in Nfat5-deficient cells. The results showed that 35.3% (1390 genes) of the 3942 hypertonic-induced genes were regulated by NFAT5, suggesting that hypertonic-induced maturation is mediated by both NFAT5-dependent and independent pathways. Among hypertonic-inducing transcription factors, Elf5, Ehf, Pax2, Pax8, Gata3, and Hifla were regulated by NFAT5, while Gata2 was not. Furthermore, genes involved in ion and molecular transport and osmotic protection were primarily downstream of NFAT5, while genes involved in epithelial characteristics, signaling pathways, and metabolism were mediated by both pathways. In particular, 55.5% (379 genes) of the 683 tonicity-inducible maturation markers were regulated by NFAT5, indicating that NFAT5 regulates the majority of the gene circuit programs triggered by hypertonicity. These results indicate that NFAT5 is a major regulator of mCD maturation.

[0063] These results indicate that exposure of mouse embryonic collecting ducts to hypertonic conditions induces morphological changes in the epithelium, upregulates maturation-related genes, and confers responsiveness to antidiuretic hormone, primarily dependent on the transcription factor NFAT5 (nuclear factor 5 of activated T cells).

[0064] (Example 6) Maturation of collecting duct (CD) organoids from ureteral bud (UB) organoids The present inventors have reported a method for inducing ureteral bud (UB) organoids from human iPS cells (Non-Patent Literature 2). Therefore, the collecting duct maturation protocol of the present invention was applied to UB organoids induced by the method of Taguchi et al. Specifically, UB organoids were induced from human iPS cells using the method of Taguchi et al. The UB organoids induced by the method of Taguchi et al. were immature and sensitive to hypertonicity, and therefore could not form mature collecting duct organoids. Therefore, collecting duct organoids were produced by increasing the tonicity more slowly and stepwise. Specifically, this was done as follows: 1% sodium pyruvate was added to the original differentiation medium of the UB organoids. On day 0, the UB organoids were embedded in 150 μL of human branched medium containing 50% growth factor-reduced Matrigel (upper medium) in a 24-well transwell insert. The well inserts were placed on 500 μL of medium (lower medium) without Matrigel. DMEM / F12 containing 10% FBS, 1% GlutaMAX (Gibco), 1% non-essential amino acids (Gibco), 1% pyruvate, 1% P / S, 0.1 μM RA, 100 ng / mL human R-spondin 1, 2 ng / mL human GDNF, 100 ng / mL human FGF1, 30 ng / mL human FGF7, and 10 nM LDN193189 was used as the differentiation medium. Culture was started at 300 mOsm / kg, and the osmotic pressure of the medium was gradually increased from day 12 by adding 5M NaCl (day 12: 330 mOsm / kg, day 13: 360 mOsm / kg, day 14: 400 mOsm / kg, day 16: 550 mOsm / kg, day 17: 700 mOsm / kg, day 18: 900 mOsm / kg). The medium was changed on days 6, 9, 12, 13, 14, 16, 17, and 18. Culture was carried out for 20 days. Furthermore, forskolin (FSK), an adenylyl cyclase activator, was added at a concentration of 10 μM from day 6 of culture. A schematic of the maturation protocol is shown in Figure 11. Also, the CDs after 20 days of culture are shown in Figure 12.

[0065] The addition of FSK allowed the CD organoids to withstand higher tonicity, maintain better morphology, and exhibit lumen dilation. Furthermore, while the culture period was two weeks without FSK, its addition extended it to nearly three weeks. Immunostaining confirmed lumen dilation and apical localization of KRT8 in CD organoids cultured at high tonicity (900 mOsm / kg) in the presence of FSK. In contrast, KRT8 was not expressed at the apical end at normal tonicity (300 mOsm / kg). This suggests that, even in the presence of FSK, CD organoids remain immature without high tonicity. Gene expression analysis also showed that several important markers, including transcription factors such as AQP2, SLC14A2, AVPR2, and ELF5, PAX2, and PAX8, were upregulated by high tonicity.

[0066] AVP and aldosterone, other important hormones in sodium homeostasis, have been previously reported to increase several UB / CD markers (Non-Patent Literature 8 and Non-Patent Literature 6). Next, CD organoids were cultured with these two hormones at normal tonicity (300 mOsm / kg) and compared with culture at hypertonicity (900 mOsm / kg). AVP and aldosterone were added at a concentration of 100 nM, respectively, from day 6. Figure 13 shows the results of RT-qPCR analysis of marker gene expression. CD organoids cultured at hypertonicity showed significantly higher levels of marker gene expression than those cultured with the hormones alone.

[0067] Furthermore, the cultured CD organoids were immunostained. The results are shown in Figure 14. CD organoids matured using the method of the present invention (FSK + hypertonicity: "high osmotic pressure" in the figure) showed localization of the AQP2 protein to the apical membrane, whereas organoids matured with FSK or aldosterone to normal tonicity ("normal osmotic pressure" or "hormone" in the figure, respectively) did not show localization.

[0068] In the method of the present invention, considering that AVPR2 expression increases approximately 50-fold at high tonicity, after maturation culture (after 20 days of culture), FSK was removed from the culture medium and desmopressin (1-deamino-8-D-arginine vasopressin: ddAVP), a synthetic analog of AVP, was added instead and cultured until day 25 to investigate the AVP responsiveness mediated by AVPR2. Natural AVP acts on both AVPR1A and AVPR2, but ddAVP is known to act selectively on AVPR2. Therefore, in order to evaluate the responsiveness mediated by AVPR2, the responsiveness to ddAVP was evaluated. At high tonicity, AQP2 moved to the apical side (localized to the luminal side) in response to ddAVP, whereas at normal tonicity, CD organoids matured with FSK or a hormone (aldosterone) did not respond to ddAVP (did not express AQP2). This indicates that human CD organoids acquired responsiveness to vasopressin and ddAVP by being cultured at hypertonic levels, and that culturing at hypertonic levels + FSK promotes the maturation of human CD organoids. Therefore, it was found that the maturation method using hypertonic stimulation of the present invention can be used to create a model that reproduces congenital nephrogenic diabetes insipidus in humans.

[0069] ScRNA-seq analysis was performed on CD organoids cultured under normal and hypertonic conditions. The results showed that CD organoids were primarily composed of chief cells rather than interstitial cells. Furthermore, a comparison of CD populations cultured under hypertonic and normal conditions identified 7717 upregulated genes, including several markers. These genes covered a wide range of biological categories, including transcription factors, signal ligands, signal receptors, metabolic enzymes, channels and transporters, extracellular matrix and cell adhesion molecules, and osmotic protection. Comparing hypertonic-induced genes in humans with their corresponding mouse genes, duplicated genes (977 genes) accounted for 12.7% of human genes (7717 genes) and 24.8% of mouse genes (3942 genes). The relatively low duplication is thought to be due to species differences as well as the different developmental stages of the two experimental systems. Assuming that common genes are likely to be biologically important, we performed hyperexpression analysis on these 977 genes using the RNAseqChef program, based on GO terms and molecular signature databases. We found that genes involved in humoral regulation were enriched. This indicates that exposure to hypertonicity is a key developmental signal for human CD maturation. These results suggest that human CD organoids undergo transcriptional maturation in response to hypertonicity.

[0070] (Example 7) Disease Modeling Using Mature Human CD Organoids Disease modeling of mature human CD organoids was performed as follows. Using genome editing via CRISPR-Cas9, NFAT5 KO and control iPSC lines were created by targeting exon 4 upstream of the Rel-homology DNA binding domain as follows. Two NFAT5 KO human iPSC lines were created from the 201B7 line. A single guide RNA (sgRNA) was designed to target exon 4 upstream of the Rel-homology domain. After preparing a single-cell suspension of the 201B7 strain, the sgRNA and Cas9 protein were electroporated into the dissociated cells. The electroporated cells were cloned into single cells by dilution. The target region was sequenced, and NFAT5 KO strains were selected based on analysis using analytical tools. UB organoids were induced from these two lines, and then mature CD organoids were induced under hypertonic conditions in the same manner as in Example 6. The results are shown in Figure 15. As shown in Figure 15, the NFAT5 KO CD organoids could not withstand culture at 900 mOsm / kg, so in subsequent experiments, the osmotic pressure of the culture medium was increased to 700 mOsm / kg. Immunostaining and scRNA-seq analysis were performed on the KO organoids. NFAT5 protein was not detected in the 700 mOsm / kg KO organoids. In addition, AQP2 mRNA expression was reduced in the KO organoids, and AQP2 protein was not detected on the apical side of the epithelium.

[0071] Considering that serum osmolality is determined by water reabsorption in cyclophosphamide (CD), it can be hypothesized that NFAT5 mutants reduce AQP2 expression and CD water reabsorption capacity, potentially leading to increased serum osmolality. Furthermore, when SCNN1A and SCNN1G expression were measured in CD organoids cultured at 300 and 700 mOsm / kg, SCNN1A and SCNN1G, which encode subunits of the epithelial sodium channel (ENaC), were significantly increased only in KO organoids cultured under hypertonic conditions. These channels reabsorb sodium in CD under aldosterone regulation, thereby determining the amount of salt in the body and, consequently, blood pressure. Therefore, the upregulation of SCNN1A and SCNN1G in KO organoids suggests that NFAT5 mutants increase ENaC expression levels, likely leading to salt-sensitive hypertension in adult humans. Thus, NFAT5 KO CD organoids may be useful as a model for these pathological conditions.

[0072] Next, mature collecting tubule organoids were created from the two NFAT5 KO human iPSC lines using a maturation protocol combining hypertonic stimulation and hormones. The hormones (AVP and aldosterone) were added at a concentration of 100 nM each starting from day 6 of culture. Similarly, mature collecting tubule organoids induced from NFAT5 KO iPS cells were successfully produced using the maturation protocol combining hypertonic stimulation and hormones. Figure 16 shows the results of evaluating AQP2 expression by immunostaining. In the control group, AQP2 expression was confirmed on the luminal side (apical side) (the arrows in the figure show a portion of this), whereas in the KO organoids, a decrease in AQP2 expression was observed. Figure 17 shows the results of gene expression measurement by RT-qPCR. In the KO organoids, the expression level of SCNN1A / G was elevated. This indicates that the maturation method combining hypertonic stimulation and hormones can also create a model illustrating the pathological conditions caused by NFAT5 abnormalities in humans.

[0073] (Example 8) Preparation of a congenital nephrogenic diabetes insipidus (NDI) model organoid Congenital nephrogenic diabetes insipidus (NDI) patients suffer from polyuria due to impaired urine concentration. Most have mutations in AVPR2 and are reported to show impaired response to AVP. AVPR2 is located on the X chromosome, and this disease is inherited in an X-linked recessive form. In addition, in men with a missense mutation (g.882A>G) in AVPR2, a missense substitution of Q174R occurs in the transmembrane region of AVPR2 (AVPR2 Q174R ) and is reported to cause severe congenital NDI (Non-Patent Document 9). On the other hand, from the results of Example 6, the mature CD organoids prepared by the method of the present invention had high expression of AVPR2 mRNA and clearly responded to ddAVP. Therefore, two clones of human iPSC lines (AVPR2 Q174R iPSCs) having the AVPR2 c.521A>G substitution were prepared from the RN7 iPSC line. Specifically, an sgRNA targeting the region surrounding the 521st base of the coding region was designed, and the sgRNA and Cas9 protein were electroporated into a single-cell suspension of the RN7 line. The remaining procedures were carried out in the same manner as the preparation of NFAT5 KO. Using UBs induced from AVPR2 WT and two clones of AVPR2 Q174R iPSCs, the matured CD organoids prepared by the method of the present invention were exposed to ddAVP. Specifically, the UB induced from iPSCs was cultured by the method of the present invention shown in FIG. 11, and on the 20th day, FSK was removed and ddAVP was added, followed by culturing for 5 days. As a result of immunostaining analysis, the WT organoids showed accumulation of AQP2 on the apical side of the epithelium, but no accumulation was observed in the AVPR2 Q174R organoids. The phenotype in this organoid is highly likely to reproduce the pathophysiology in congenital NDI patients.

[0074] (Example 9) Production of model organoids from iPS cells of NDI patients Based on a protocol approved by the Kumamoto University Ethics Committee, iPS cells were produced from the blood of healthy individuals with informed consent. The AVPR2 gene mutation reported in NDI patients was introduced into the iPS cells using the CRISPR / Cas9 system. Subsequently, mature collecting tubule organoids were produced from UBs induced from the produced iPSCs using a maturation protocol combining the method of the present invention and hormones, as shown in Figure 11. Hormones (AVP and aldosterone) were added at a concentration of 100 nM each from day 6 of culture. On day 20 of culture, FSK, AVP, and aldosterone were removed, and ddAVP was added. The cells were cultured for 5 days, and vasopressin reactivity was evaluated. The results of immunohistochemical analysis are shown in Figure 18. Mature collecting duct organoids induced from iPS cells of healthy individuals showed AQP2 expression on the luminal side (apical side) (the arrows in the figure indicate a portion of this expression), whereas mature collecting duct organoids induced from iPS cells with an AVPR2 mutation showed decreased AQP2 expression (green) and reduced responsiveness to vasopressin. This indicates that a model that reproduces congenital nephrogenic diabetes insipidus in humans can be constructed using a maturation method combining hypertonic stimulation and hormones.

[0075] (Example 10) Study of stepwise increase in osmotic pressure In the same manner as in Example 6, collecting duct organoids were matured using human ureteral bud organoids induced from human iPS cells. However, the stepwise increase in osmotic pressure was increased by 100 mOsm / kg per day starting from day 12 (Day 12: 330 mOsm / kg, Day 13: 400 mOsm / kg, Day 14: 500 mOsm / kg, Day 15: 60 mOsm / kg, Day 16: 700 mOsm / kg). The result was that when the osmotic pressure was increased by 100 mOsm / kg per day, the morphology of the organoids, i.e., the epithelial morphology, could no longer be maintained when it reached 700 mOsm / kg. Figure 19 shows the results of microscopic observation of the organoid morphology at the stage when it reached 700 mOsm / kg. From the figure, it can be seen that the morphology was not maintained. This indicates that it is desirable to gradually increase osmotic pressure in the initial stages of hypertonic stimulation.

[0076] The above detailed description merely illustrates the object and subject matter of the present invention and does not limit the scope of the appended claims. Various modifications and substitutions to the embodiments described without departing from the scope of the appended claims will be apparent to those skilled in the art from the teachings described herein.

[0077] Collecting tubes or collecting tube organoids produced by the method of the present invention are useful in the field of regenerative medicine, as well as in elucidating the pathogenesis of diseases and screening pharmaceuticals.

Claims

1. A method for producing a renal collecting tubule, comprising the step of culturing ureteral buds in an isotonic osmotic medium, and then culturing them in one or more media having an osmotic pressure exceeding isotonic pressure (hyperosmolar media), wherein at least one hyperosmolar media has an osmotic pressure of at least about 600 mOsm / kg or more.

2. The manufacturing method according to claim 1, wherein the step of culturing in a hyperosmotic medium comprises culturing in a hyperosmotic medium having an osmotic pressure of at least about 700 mOsm / kg or more.

3. The manufacturing method according to claim 1, wherein the step of culturing in a hyperosmolar medium includes the step of culturing in hyperosmolar mediums having at least two or more different osmotic pressures, and the culturing is carried out sequentially in media having progressively higher osmotic pressures.

4. The manufacturing method according to claim 1, wherein the step of culturing in a hyperosmolar medium is a step of culturing in a hyperosmolar medium having at least three or more different osmotic pressures.

5. The manufacturing method according to claim 1, wherein the osmotic pressure of the hyperosmolar medium is that of a medium prepared by adding a cell membrane-impermeable molecule to the medium.

6. The method for producing the product according to claim 1, wherein the hyperosmolar medium is a medium to which forskolin has been added.

7. The manufacturing method according to claim 1, wherein the hyperosmolar medium is a medium to which a hormone that acts on the renal tubules has been added.

8. The manufacturing method according to claim 1, wherein the culture step is carried out in a culture medium containing approximately 10% or more Matrigel.

9. The method for producing a ureteral bud according to any one of claims 1 to 8, wherein the ureteral bud is a ureteral bud induced from iPS cells.

10. The manufacturing method according to claim 9, wherein the renal collecting duct is a renal collecting duct organoid derived from iPS cells.

11. The method for producing iPS cells according to claim 9, wherein the iPS cells are human-derived iPS cells.

12. The method for producing human iPS cells according to claim 11, wherein the human iPS cells are human iPS cells in which the AVPR2Q174R mutation has been introduced into the aquaporin 2 receptor gene by genetic manipulation.

13. The method for producing human-derived iPS cells according to claim 11, wherein the human-derived iPS cells are human iPS cells in which the NFAT5 gene has been knocked out by genetic manipulation.

14. A manifold organoid manufactured by the manufacturing method described in any one of claims 1 to 13.

15. A collecting tubule organoid manufactured by the manufacturing method described in claim 10, which is used as a disease model.

16. A collecting tubule organoid manufactured by the manufacturing method of claim 13, which is used as a model of salt-sensitive hypertension.

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