An organoid

Abstract

The invention relates to organoids, and particularly, although not exclusively, to kidney organoids. The invention extends to novel methods for producing kidney organoids, to their uses in in vitro and ex vivo assays, and in screening methods during the evaluation of pharmaceuticals, particularly, although not exclusively, during the preclinical phase, as well as to apparatus for use in such screening. In addition, the invention extends to the use of the kidney organoids to study kidney disease, and for modelling disease.

Description

[0001] An Organoid

[0002] The present invention relates to organoids, and particularly, although not exclusively, to kidney organoids. The invention extends to novel methods for producing kidney organoids, to their uses in in vitro and ex vivo assays, and in screening methods during the evaluation of pharmaceuticals, particularly, although not exclusively, during the preclinical phase, as well as to apparatus for use in such screening. In addition, the invention extends to the use of the kidney organoids to study kidney disease, and for modelling disease.

[0003] The development of new pharmaceuticals is an expensive, time-consuming, and inefficient process, with a 96% failure rate for candidate drugs making it to market. This is in part due to inadequate preclinical models, particularly in oncology, where animal models and cell line assays do not sufficiently assess drug efficacy, organ-specific toxicity, and dosing, thus contributing to a 90% candidate drug failure rate in clinical trials. These shortcomings, along with ethical concerns and high costs, hinder the translation of preclinical results to human outcomes. In particular, drug safety and efficacy issues are responsible for 50% of clinical trial failures.

[0004] Acute kidney injury (AKI) induced by chemotherapeutic drugs causes toxic effects on kidneys (nephrotoxicity), and its prevalence is increasing each year. Importantly, kidney toxicity accounts for up to 8% of preclinical safety closures and drawbacks of new drugs.

[0005] There is, therefore, a need for an improved platform for nephrotoxicity analysis of pharmaceuticals, particularly during the preclinical phase.

[0006] In order to address the above problems, the inventors investigated methods to produce kidney organoids under three-dimensional conditions. As a result of their extensive experimentation, the inventors have developed a new process which enables the production of kidney organoids in a highly reproducible and consistent manner.

[0007] The inventors found that contacting at least one hiPSC-AIM with at least one hiPSC-PIM results in the production of a kidney organoid.

[0008] It will be appreciated that the term "hiPSC-AIM" can mean at least one human induced pluripotent stem cell (hiPSC) which will differentiate into at least one anterior intermediate mesoderm (AIM) progenitor cell. It will also be appreciated that the term "hiPSC-PIM" can mean at least one human induced pluripotent stem cell (hiPSC) which will differentiate into at least one posterior intermediate mesoderm (PIM) progenitor cell. Accordingly, it will be appreciated that the AIM progenitor cells differentiate from human induced pluripotent stem cells (hiPSCs), and thus may be referred to as human induced pluripotent stem cells (hiPSCs) (i.e., hiPSC-AIM). Indeed, upon culture with various compounds, the human induced pluripotent stem cells (hiPSCs) (i.e., hiPSC-AIM) differentiate to form AIM progenitor cells.

[0009] Accordingly, it will also be appreciated that the PIM progenitor cells differentiate from human induced pluripotent stem cells (hiPSCs), and thus may be referred to as human induced pluripotent stem cells (hiPSCs) (i.e., hiPSC-PIM). Indeed, upon culture with various compounds, the human induced pluripotent stem cells (hiPSCs) (i.e., hiPSC-PIM) differentiate to form PIM progenitor cells.

[0010] Thus, according to a first aspect of the invention, there is provided a 3D-differentiated kidney organoid comprising at least one anterior intermediate mesoderm (AIM) progenitor cell and at least one posterior intermediate mesoderm (PIM) progenitor cell.

[0011] Advantageously, the kidney organoids produced by the inventors have been shown to replicate the structural and functional features of the in vivo kidney. These features include: (i) water regulation mechanisms, as discussed in Examples 5 and 9, exemplified by the expression of water regulation proteins (AQP-2) and the main sodium transporter of the nephron (Na / K / ATPase) in the distal tubules, (ii) the ability to uptake molecules, as described in Example 6, exemplified by the selective uptake of 488-labeled dextran by Lotus tetragonolobus lectin (LTL)-positive tubules, and (iii) response to injury, as discussed in Example 7, exemplified by the induction of cystic phenotypes in the organoids by forskolin stimulation. Each of these features of the organoid of the invention means that it can be used as an improved platform that more accurately resembles kidney function over existing technologies.

[0012] It will be appreciated that the term "3D-differentiated" can mean that the kidney organoid comprises three dimensions, e.g., height (up / down), width (left / right), and depth (forwards / backwards), and has been permitted to grow in, move in, and interact with surfaces in all three of these dimensions. It will also be appreciated that a "3D- differentiated" kidney organoid comprises a height, width, and / or depth of more than one cell, i.e., a plurality of cells that is more than one cell thick.

[0013] The three-dimensional kidney organoid format of the present invention supports continuous cell-cell contact, mimicking aspects of early embryogenesis more closely than two-dimensional systems. This format allows for self-organisation, more uniform exposure to morphogens throughout the aggregate, and vastly improved reproducibility.

[0014] In contrast, it will be appreciated that "2D-differentiated" can mean a culture that only comprises two dimensions, e.g., only two of height (up / down), width (left / right), and depth (forwards / backwards), and has only been permitted to grow in, move in, and interact with surfaces in two of these dimensions. It will also be appreciated that a 'iodifferentiated" culture comprises a monolayer of cells, i.e., a layer of cells that is one cell thick.

[0015] In one embodiment, therefore, the kidney organoid may comprise three dimensions. The three dimensions may comprise height, width, and depth, typically each being more than one cell thick.

[0016] In another embodiment, the kidney organoid may comprise a height, width, and / or depth of more than 0.1 pm, 0.5 pm, 1 pm, 5 pm, or 10 pm. In another embodiment, the kidney organoid may comprise a height, width, and / or depth of more than 100 pm, 250 pm, 500 pm, 750 pm, or 1000 pm. Typically, however, the kidney organoid comprises a height, width, and / or depth of more than 10 pm.

[0017] In another embodiment, the kidney organoid may comprise a height, width, and / or depth of at least 20 pm, at least 30 pm, at least 40 pm, at least 50 pm, at least 60 pm, at least 70 pm, at least 80 pm, at least 90 pm, or at least 100 pm.

[0018] In one embodiment, the kidney organoid may comprise at least five, at least 10, at least 20, at least 30, at least 40, or at least 50 cell layers.

[0019] Advantageously, the organoids of the invention do not comprise a matrix. In one embodiment, therefore, the kidney organoid does not comprise a matrix.

[0020] In an embodiment, therefore, the kidney organoid comprises a structural feature of a kidney. The structural feature of a kidney may be selected from a list of structural features, including : renal fascia, perirenal fat capsule, renal capsule, cortex, medulla, renal pelvis, hilum, nephrons, renal pyramids, renal columns, lobes of the kidney, ureter, calyces, renal arteries, renal veins, inferior vena cava, segmental arteries, interlobar arteries, arcuate arteries, cortical radiate arteries, cortical nephrons, juxtamedullary nephrons, renal corpuscle, renal tubule, glomerulus, Bowman's capsule, proximal convoluted tubule, loop of Henle, descending limbs, ascending limbs, distal convoluted tubule, afferent arteriole, efferent arteriole, peritubular capillary network, and / or vasa recta.

[0021] The kidney organoid may comprise at least one distal convoluted tubule, at least one proximal convoluted tubule, and / or at least one collecting duct. Typically, however, the kidney organoid comprises at least one distal tubule, at least one proximal tubule, and / or at least one collecting duct.

[0022] In another embodiment, the kidney organoid comprises a functional feature of a kidney. The functional feature of a kidney may be selected from a list of functional features, including : blood filtering, blood pressure regulation, acid-base regulation, toxin removal, red blood cell production, activation of vitamin D, water regulation, electrolyte regulation, and / or hormone production.

[0023] It will be appreciated that arginine vasopressin (AVP), alternatively referred to as antidiuretic hormone (ADH), governs the permeability of water in principal cells (PC) and initiates the transcription of aquaporin-2 (AQP2). The AVP-triggered integration of AQP2 into the apical membrane of principal cells stands as the principal mechanism through which renal regulation of water equilibrium occurs in mammals.

[0024] The inventors have found that when the kidney organoids of the invention are subjected to AVP exposure for a duration of 3 hours following a 24-hour washout period of AVP and aldosterone, the AQP2 protein migrates from its predominantly cytosolic localisation to the apical membrane, which may be quantified by confocal immunostaining of AQP2. It will be appreciated that the apical membrane can comprise the part of the plasma membrane of a polarised cell that forms the surface facing the lumen.

[0025] In one embodiment, therefore, the kidney organoid of the invention may comprise a water regulation mechanism. In another embodiment, the water regulation mechanism may comprise the AQP2 protein. The AQP2 protein may migrate to the apical membrane. The AQP2 protein may migrate to the apical membrane of an epithelial cell. The AQP2 protein may migrate to the apical membrane of a tubular epithelial cell. The AQP2 protein may migrate to the apical membrane following exposure to AVP. The AQP2 protein may migrate to the apical membrane following exposure to AVP subsequent to a washout period of AVP and aldosterone.

[0026] The kidney organoid of the invention may comprise the ability to selectively uptake a molecule, wherein the kidney organoid selectively uptakes at least one molecule with which it is incubated. It will be appreciated that the term "selectively uptakes" can mean uptake of a specific molecule that is independent of the uptake of one or more other molecule.

[0027] The kidney organoid of the invention may comprise the ability to selectively uptake water, electrolytes (i.e., sodium, chloride), glucose, amino acids, phosphate, lactate, citrate, urea, creatinine, uric acid, hormones, drugs, toxins, and / or dextran. The kidney organoid may comprise the ability to selectively uptake at least one molecule by at least one Lotus tetragonolobus lectin (LTL)-positive tubule, glomerulus, loop of Henle, distal convoluted tubule, and / or collecting duct. Typically, the kidney organoid comprises the ability to selectively uptake dextran by at least one LTL-positive tubule.

[0028] The kidney organoid of the invention may comprise a response to injury. The response to injury may comprise activation of a cellular stress response, activation of a cell death pathway, inflammation, and / or fibrosis. The cellular stress response may comprise production and / or accumulation of oxygen reactive species (ROS). The cell death pathway may comprise caspase activation.

[0029] The kidney organoid may comprise a cystic phenotype in response to cyclic AMP (cAMP) signalling. Typically, the kidney organoid comprises a cystic phenotype in response to contact with forskolin. It will be appreciated that cAMP-driven mechanisms are central to the pathogenesis of polycystic kidney disease (PKD), and that cAMP-regulated mechanisms are fundamental to cyst formation and disease progression. It will also be appreciated that the term "cystic phenotype" can mean the presence of cysts on and / or in the kidney organoids.

[0030] Advantageously, the kidney organoid of the invention is produced under fully three- dimensional conditions, thereby recreating the in vivo three-dimensional physiological settings in vitro. As such, the kidney organoid allows the gathering of more biologically relevant data concerning organ-specific cell functionality.

[0031] Moreover, as described herein, since the organoids may be derived from induced pluripotent stem cells (iPSCs), such as human induced pluripotent stem cells (hiPSCs), the inventors' novel platform could be used to model a disease. For example, the platform could be used in combination with tubular swelling assays to provide a new means of studying polycystic kidney disease, amongst other genetic diseases. The inventors envisage the use of disease-specific iPSCs obtained from cell banks to produce a kidney organoid that models a disease. For example, iPSCs from polycystic kidney disease patients could be used to produce a kidney organoid that models polycystic kidney disease. The inventors also envisage the use of reprogrammed iPSCs, such as hiPSCs, obtained from disease specific patients. In addition, the inventors also envisage the use of their novel platform for assessing drug-induced renal injury during preclinical trials.

[0032] In one embodiment, therefore, the at least one anterior intermediate mesoderm (AIM) progenitor cell and / or the at least one posterior intermediate mesoderm (PIM) progenitor cell may be derived from at least one animal cell, which may be a mammal cell. In another embodiment, the at least one anterior intermediate mesoderm (AIM) progenitor cell and / or the at least one posterior intermediate mesoderm (PIM) progenitor cell may be derived from at least one human, pig, or mouse cell. In a further embodiment, the at least one anterior intermediate mesoderm (AIM) progenitor cell and / or the at least one posterior intermediate mesoderm (PIM) progenitor cell may be derived from at least one stem cell, which may be a pluripotent stem cell, or an induced pluripotent stem cell (iPSC). Typically, however, the at least one anterior intermediate mesoderm (AIM) progenitor cell and / or at least one posterior intermediate mesoderm (PIM) progenitor cell is derived from at least one human induced pluripotent stem cell (hiPSC).

[0033] In another embodiment, the cell from which the at least one anterior intermediate mesoderm (AIM) progenitor cell and / or the at least one posterior intermediate mesoderm (PIM) progenitor cell is derived may be a disease-specific cell. In another embodiment, the cell from which the at least one anterior intermediate mesoderm (AIM) progenitor cell and / or at least one posterior intermediate mesoderm (PIM) progenitor cell is derived may be genetically modified. In another embodiment, the cell from which the at least one anterior intermediate mesoderm (AIM) progenitor cell and / or at least one posterior intermediate mesoderm (PIM) progenitor cell is derived may be genetically modified to model a disease genotype.

[0034] In comparison with existing 2D cell lines and hiPSC-derived kidney cells, the kidney organoid of the invention has greater maturity and superior morphology, which provides an improved platform system for the evaluation of the simultaneous responses of multiple cell types. As such, the use of kidney organoids enables the collection of more biologically relevant data over currently available platforms.

[0035] In an embodiment, therefore, the kidney organoid comprises one or more cell type. The one or more cell type of which the kidney is comprised may be selected from a list of cell types, including : tubular epithelial cells, parietal epithelial cells, tubule epithelial cells, mesangial cells, podocytes, glomerular endothelial cells, juxtaglomerular cells, macula densa cells, principal cells, intercalated cells, epithelial progenitor cells, pericytes, Loop of Henle cells, thin descending limb cells, thin ascending limb cells, thick ascending limb cells, collecting duct cells, fibroblasts, smooth muscle cells, and renal interstitial cells. Typically, however, the kidney organoid comprises endothelial cells, podocytes, and interstitial cells.

[0036] In another embodiment, the kidney organoid of the invention comprises a cell-cell interaction in order to mimic the formation of a nephron. The kidney organoid may comprise a cell-cell interaction between an epithelial cell of the nephric duct and the surrounding nephrogenic mesenchyme, resulting in mesenchymal-to-epithelial transitions to form a nephron.

[0037] In another embodiment, the kidney organoid comprises at least one extracellular protein. The one or more extracellular protein may be selected from a list of extracellular proteins, including : Aggrecan, Agrin, Aspein, Asporin, Biglycan, BMP binding endothelial regulator, Bone sialoprotein, Brevican, Cartilage oligomeric matrix protein, CCDC80, Chondronectin, Cochlin, CRISPLD2, Cysteine rich secretory protein led domain containing 1, Decorin, Dentin sialophosphoprotein, Dermatopontin, DMBT1, DMP1, Elastin, Extracellular matrix protein 1, Extracellular matrix protein 2, Fibromodulin, Fibronectin, FRAS1, HAPLN1, Hemicentin 1, Hemicentin 2 Hyalin, Ladinin 1, Laminin, Matricellular protein, Matrilin, Matrilin-1, Matrilin-2, Matrilin-3, Matrix Gia protein, MEPE, MFAP4, MXRA5, Neurocan, Nidogen, Nidogen-1, Nidogen-2, Osteoglycin, Osteonectin, Osteopontin, Periostin, Perlecan, Pikachurin, Proteoglycan 4, R-spondin 1, R-spondin 2, R-spondin 3, R-spondin 4, SCO-spondin, SIBLING proteins, SNED1, SPARCL1, Spondin 1, Spondin 2, SRPX, SRPX2, TECTA, TECTB, Tenomodulin, TGFBI, Tsukushi, Versican, Vitronectin, and / or VWA2. Typically, however, the extracellular protein is Laminin.

[0038] The responses which the kidney organoid comprise may include a response to injury, indicated by increased inflammatory biomarkers, cell death biomarkers, changes in filtration rate, changes in electrolyte balance, serum creatinine, urea levels, tubular injury, interstitial fibrosis, changes in vascular cells, and / or oxidative stress.

[0039] The responses which the kidney organoid comprise may be measured by flow cytometry, confocal microscopy, transepithelial resistance measurement, qRT-PCR, ELISA, immunostaining, and / or fluorescence / absorbance readings. The kidney organoid of the invention may comprise simultaneous responses of more than one cell type. Typically, the kidney organoid comprises simultaneous responses of tubular epithelial cells, parietal epithelial cells, tubule epithelial cells, mesangial cells, podocytes, glomerular endothelial cells, juxtaglomerular cells, macula densa cells, principal cells, intercalated cells, epithelial progenitor cells, pericytes, Loop of Henle cells, thin descending limb cells, thin ascending limb cells, thick ascending limb cells, collecting duct cells, fibroblasts, smooth muscle cells, and / or renal interstitial cells.

[0040] It will be appreciated that the kidney organoid of the invention may simultaneously comprise any one of the above structural features, functional features, regulation mechanisms, or cell types and an AIM or a PIM progenitor cell (i.e., an early progenitor cell or progenitor-like cell). This can result from incomplete organoid maturation and spatial patterning that permits the formation of niches where less differentiated cells are maintained. Indeed, it is typical that existing methods of producing organoids result in organoids comprising heterogeneous cell populations, including cells expressing intermediate mesoderm markers (e.g., PAX2, LHX1, OSR.1), and cells co-expressing early mesodermal and renal progenitor markers. Thus, it will be appreciated that such heterogeneity is common in organoid models, and reflects both the plasticity of mesodermal progenitors and the inherent complexity of achieving complete and uniform in vitro differentiation.

[0041] In one embodiment, therefore, the kidney organoid of the invention may simultaneously comprise: i) a structural feature, a functional feature, a regulation mechanism, and / or one or more cell type, and ii) an AIM progenitor cell and / or a PIM progenitor cell.

[0042] The inventors envisage the kidney organoids of the invention as being vascularised.

[0043] In one embodiment, therefore, the kidney organoid of the invention may be vascularised.

[0044] As discussed in the Examples, the inventors have demonstrated that the assembly of AIM and PIM aggregates together with endothelial (Endo) spheroids results in the presence of endothelial cells, as suggested by the presence of the key endothelial marker CD31.

[0045] It will be appreciated that the presence of endothelial cells in the kidney organoids suggests vascularisation because endothelial cells are the key cell type that forms the inner lining of blood vessels. It will also be appreciated that the term "spheroid" (e.g., endo spheroid) can mean a three-dimensional cell culture, where cells self-assemble into sphere-like formations during cell proliferation, promoting cell-cell interactions. The term "spheroid" can also mean a cell model which provides cell migration, polarisation, differentiation, survival, and growth. The terms "spheroid" and "aggregate" may be used interchangeably herein.

[0046] In one embodiment, therefore, the kidney organoid may comprise a blood vessel. The kidney organoid may comprise a blood vessel that is an artery, an arteriole, a capillary, a venule, and / or a vein.

[0047] The inventors' novel and innovative methodology to produce kidney organoids under fully three-dimensional conditions relies on the initial and separate derivation of the posterior intermediate mesoderm (PIM) and anterior intermediate mesoderm (AIM) using size-controlled human induced pluripotent stem cells (hiPSC). It will be appreciated that the term "three-dimensional conditions" can mean that cells are permitted to grow, and move, left and right, up and down, forwards and backwards, and interact with surfaces in all three of these dimensions. Such conditions recreate the three-dimensional physiological settings of an in vivo kidney in vitro, thus allowing the kidney organoids to mimic kidney tissue and its microarchitecture.

[0048] During their extensive experimentation, the inventors discovered that the induction of specific AIM and PIM progenitor cells as size-controlled aggregates, and the contacting of these two populations, results in the production of a kidney organoid.

[0049] Thus, in a second aspect of the invention, there is provided a method of producing a kidney organoid, the method comprising contacting at least one anterior intermediate mesoderm (AIM) progenitor cell, or hiPSC-AIM, and at least one posterior intermediate mesoderm (PIM) progenitor cell, or hiPSC-PIM, to thereby produce a kidney organoid.

[0050] As discussed, it will be appreciated that the term "hiPSC-AIM" can mean at least one human induced pluripotent stem cell (hiPSC) which will differentiate into at least one anterior intermediate mesoderm (AIM) progenitor cell. It will also be appreciated that the term "hiPSC-PIM" can mean at least one human induced pluripotent stem cell (hiPSC) which will differentiate into at least one posterior intermediate mesoderm (PIM) progenitor cell. Accordingly, it will be appreciated that the AIM progenitor cells differentiate from human induced pluripotent stem cells (hiPSCs), and thus may be referred to as human induced pluripotent stem cells (hiPSCs) or hiPSC aggregates (i.e., hiPSC-AIM). Indeed, upon culture with various compounds, the human induced pluripotent stem cells (hiPSCs) or hiPSC aggregates (i.e., hiPSC-AIM) differentiate to form AIM progenitor cells / AIM progenitor cell aggregates.

[0051] Accordingly, it will also be appreciated that the PIM progenitor cells differentiate from human induced pluripotent stem cells (hiPSCs), and thus may be referred to as human induced pluripotent stem cells (hiPSCs) or hiPSC aggregates(i.e., hiPSC-PIM). Indeed, upon culture with various compounds, the human induced pluripotent stem cells (hiPSCs) or hiPSC aggregates (i.e., hiPSC-PIM) differentiate to form PIM progenitor cells / PIM progenitor cell aggregates.

[0052] Thus, in an aspect of the invention, there is provided a method of producing a kidney organoid, the method comprising contacting at least one hiPSC-AIM and at least one hiPSC-PIM, to thereby produce a kidney organoid.

[0053] It will be appreciated that upon contacting the at least one anterior intermediate mesoderm (AIM) progenitor cell, or hiPSC-AIM, and the at least one posterior intermediate mesoderm (PIM) progenitor cell, or hiPSC-PIM, the AIM progenitor cell, or hiPSC-AIM, and PIM progenitor cell, or hiPSC-PIM, assemble. In one embodiment, therefore, the method may comprise the assembly of the at least one anterior intermediate mesoderm (AIM) progenitor cell, or hiPSC-AIM, and the at least one posterior intermediate mesoderm (PIM) progenitor cell, or hiPSC-PIM.

[0054] It will also be appreciated that the presence of two distinct and organised progenitor populations, i.e., AIM and PIM progenitor cells, or hiPSC-AIM and hiPSC-PIM, aims to replicate the events that occur during in vivo embryonic development more accurately than existing models, as the presently disclosed methods result in improved cross-tissue cell interactions over existing models.

[0055] Advantageously, the methods producing the organoids of the invention do not require a matrix.

[0056] In one embodiment, therefore, the method of the second aspect does not comprise contacting at least one anterior intermediate mesoderm (AIM) progenitor cell, or hiPSC- AIM, and / or at least one posterior intermediate mesoderm (PIM) progenitor cell, or hiPSC-PIM, with a matrix.

[0057] In one embodiment, the at least one anterior intermediate mesoderm (AIM) progenitor cell, or hiPSC-AIM, and the at least one posterior intermediate mesoderm (PIM) progenitor cell, or hiPSC-PIM, may be contacted at a ratio of between 1:1 and 1:1,000, or between 1:1 and 1:100, or between 1:1 and 1:50, or between 1:1 and 1:25, or between 1:1 and 1:20, or between 1:1 and 1:10, or between 1:1 and 1:5, or between 1:1 and 1:4, or between 1:1 and 1:3, or between 1:1 and 1:2. Typically, however, the at least one anterior intermediate mesoderm (AIM) progenitor cell, or hiPSC-AIM, and the at least one posterior intermediate mesoderm (PIM) progenitor cell, or hiPSC-PIM, are contacted at a ratio of 1:10.

[0058] In another embodiment, the at least one posterior intermediate mesoderm (PIM) progenitor cell, or hiPSC-PIM, and the at least one anterior intermediate mesoderm (AIM) progenitor cell, or hiPSC-AIM, may be contacted at a ratio of between 1:1 and 1:1,000, or between 1:1 and 1:100, or between 1:1 and 1:50, or between 1:1 and 1:25, or between 1:1 and 1:20, or between 1:1 and 1:10, or between 1:1 and 1:5, or between 1:1 and 1:4, or between 1:1 and 1:3, or between 1:1 and 1:2. Typically, however, the at least one posterior intermediate mesoderm (PIM) progenitor cell, or hiPSC-PIM, and the at least one anterior intermediate mesoderm (AIM) progenitor cell, or hiPSC-AIM, are contacted at a ratio of 1:10.

[0059] In one embodiment, the at least one anterior intermediate mesoderm (AIM) progenitor cell, or hiPSC-AIM, and / or at least one posterior intermediate mesoderm (PIM) progenitor cell, or hiPSC-PIM, may be derived from at least one animal cell, which may be a mammal cell. In another embodiment, the kidney organoid may be derived from at least one human, pig, or mouse cell. In a further embodiment, the kidney organoid may be derived from at least one stem cell, which may be a pluripotent stem cell, or an induced pluripotent stem cell (iPSC). Typically, however, the at least one anterior intermediate mesoderm (AIM) progenitor cell, or hiPSC-AIM, and / or at least one posterior intermediate mesoderm (PIM) progenitor cell, or hiPSC-PIM, is derived from at least one human induced pluripotent stem cell (hiPSC).

[0060] In one embodiment, at least one at least one anterior intermediate mesoderm (AIM) progenitor cell, or hiPSC-AIM, and at least one posterior intermediate mesoderm (PIM) progenitor cell, or hiPSC-PIM, may be contacted to thereby produce at least one kidney organoid in a three-dimensional environment. It will be appreciated that the term "three-dimensional environment" can mean (as in respect of the first aspect) an environment where a cell, or cells, are permitted to grow and / or move in three dimensions, e.g., up / down, left / right, and forwards / backwards. In one embodiment, therefore, the at least one at least one anterior intermediate mesoderm (AIM) progenitor cell, or hiPSC-AIM, and the at least one posterior intermediate mesoderm (PIM) progenitor cell, or hiPSC-PIM, may be contacted and permitted to grow and / or move in three dimensions to thereby produce at least one kidney organoid in a three-dimensional environment. The three dimensions may be up / down, left / right, and forwards / backwards.

[0061] In one embodiment, the at least one anterior intermediate mesoderm (AIM) progenitor cell, or hiPSC-AIM, and the at least one posterior intermediate mesoderm (PIM) progenitor cell, or hiPSC-PIM, may be size-controlled.

[0062] In one embodiment, the at least one anterior intermediate mesoderm (AIM) progenitor cell, or hiPSC-AIM, and the at least one posterior intermediate mesoderm (PIM) progenitor cell, or hiPSC-AIM, may comprise aggregates.

[0063] It will be appreciated that an aggregate is formed by the clustering together and / or adhesion of initially separate cells.

[0064] Typically, the at least one anterior intermediate mesoderm (AIM) progenitor cell, or hiPSC-AIM, and the at least one posterior intermediate mesoderm (PIM) progenitor cell, or hiPSC-PIM, are size-controlled by initial aggregate cell number and / or aggregate diameter.

[0065] It will be appreciated that the term "initial aggregate" can mean an aggregate of at least one anterior intermediate mesoderm (AIM) progenitor cell, or hiPSC-AIM, and / or an aggregate of at least one posterior intermediate mesoderm (PIM) progenitor cell, or hiPSC-AIM, which are contacted according to the method of the second aspect.

[0066] It will also be appreciated that the term "initial aggregate" or "aggregate" can mean an aggregate of at least one human induced pluripotent stem cell (hiPSC) which will differentiate into mesoderm AIM progenitor cells (hiPSC-AIM) and / or an aggregate of at least one human induced pluripotent stem cell (hiPSC) which will differentiate into mesoderm PIM progenitor cells (hiPSC-PIM), which are contacted according to the method of the second aspect. The initial aggregate of anterior intermediate mesoderm (AIM) progenitor cells, or hiPSC- AIMs, may comprise between 2 and 100,000 cells per aggregate. The initial aggregate of anterior intermediate mesoderm (AIM) progenitor cells, or hiPSC-AIMs, may comprise between 100 and 10,000 cells per aggregate, between 300 and 5,000 cells per aggregate, between 500 and 3,000 cells per aggregate, between 700 and 1,500 cells per aggregate, between 800 and 1,200 cells per aggregate, between 900 and 1,100 cells per aggregate, or between 950 and 1,050 cells per aggregate. Typically, however, the initial aggregate of anterior intermediate mesoderm (AIM) progenitor cells, or hiPSC-AIMs, comprises about 1,000 cells per aggregate.

[0067] The initial aggregate of anterior intermediate mesoderm (AIM) progenitor cells, or hiPSC- AIMs, may comprise a diameter of between 1 and 1,000 pm. The initial aggregate of anterior intermediate mesoderm (AIM) progenitor cells, or hiPSC-AIMs, may comprise a diameter of between 40 and 600 pm, between 80 and 300 pm, between 120 and 250 pm, between 150 and 210 pm, between 160 and 200 pm, or between 170 and 190 pm. Typically, however, the initial aggregate of anterior intermediate mesoderm (AIM) progenitor cells, or hiPSC-AIMs, comprises a diameter of 178 ± 7 pm.

[0068] The initial aggregate of posterior intermediate mesoderm (PIM) progenitor cells, or hiPSC-PIMs, may comprise between 2 and 100,000 cells per aggregate. The initial aggregate of posterior intermediate mesoderm (PIM) progenitor cells, or hiPSC-PIMs, may comprise between 100 and 20,000 cells per aggregate, between 500 and 8,000 cells per aggregate, between 1,500 and 4,000 cells per aggregate, between 1,700 and 2,500 cells per aggregate, between 1,800 and 2,200 cells per aggregate, between 1,900 and 2,100 cells per aggregate, or between 1,950 and 2,050 cells per aggregate. Typically, however, the initial aggregate of posterior intermediate mesoderm (PIM) progenitor cells, or hiPSC-PIMs, comprises about 2,000 cells per aggregate.

[0069] The initial aggregate of posterior intermediate mesoderm (PIM) progenitor cells, or hiPSC-PIMs, may comprise a diameter of between 1 and 1,000 pm. The initial aggregate of posterior intermediate mesoderm (PIM) progenitor cells, or hiPSC-PIMs, may comprise a diameter of between 50 and 800 pm, between 100 and 500 pm, between 150 and 300 pm, between 180 and 280 pm, between 200 and 260 pm, or between 220 and 240 pm. Typically, however, the initial aggregate of posterior intermediate mesoderm (PIM) progenitor cells, or hiPSC-PIMs, comprises a diameter of 233 ± 4 pm.

[0070] The initial anterior intermediate mesoderm (AIM) progenitor cell, or hiPSC-AIM, aggregate and the initial posterior intermediate mesoderm (PIM) progenitor cell, or hiPSC-PIM, aggregate may be the same size. The initial anterior intermediate mesoderm (AIM) progenitor cell, or hiPSC-AIM, aggregate and the initial posterior intermediate mesoderm (PIM) progenitor cell, or hiPSC-PIM, aggregate may be different sizes. Typically, however, the initial AIM progenitor cell, or hiPSC-AIM, aggregate is of a smaller size than the initial PIM progenitor cell, or hiPSC-PIM, aggregate.

[0071] In one embodiment, the initial AIM progenitor cell, or hiPSC-AIM, aggregate comprises a smaller diameter than the initial PIM progenitor cell, or hiPSC-PIM, aggregate.

[0072] The initial anterior intermediate mesoderm (AIM) progenitor cell, or hiPSC-AIM, aggregate and the initial posterior intermediate mesoderm (PIM) progenitor cell, or hiPSC-PIM, aggregate may be comprised of the same number of cells per aggregate. The initial anterior intermediate mesoderm (AIM) progenitor cell, or hiPSC-AIM, aggregate and the initial posterior intermediate mesoderm (PIM) progenitor cell, or hiPSC-PIM, aggregate may be comprised of a different number of cells per aggregate. Typically, however, the initial AIM progenitor cell, or hiPSC-AIM, aggregate comprises fewer cells than the initial PIM progenitor cell, or hiPSC-PIM, aggregate.

[0073] It will be appreciated that CHIR99021 (CHIR) is an aminopyrimidine derivative, and a potent glycogen synthase kinase (GSK) 3 inhibitor. CHIR is commonly used as a stem cell differentiation and reprogramming reagent. It will also be appreciated that Activin A is a pleiotropic cytokine and member of the TGF-0 family. Activin A is a critical regulator in human embryonic stem cells (hESCs) maintenance and differentiation.

[0074] The inventors unexpectedly discovered, following substantial experimentation, that typical concentrations of cell differentiation and reprogramming reagents used for the production of organoids were suboptimal for the methods of the present invention. As discussed in the Examples, the inventors found that contacting the hiPSC-AIM cells (hiPSCs which will differentiate into AIM progenitor cells) and / or the or hiPSC-PIM cells (hiPSCs which will differentiate into PIM progenitor cells) with about 11 pM CHIR was optimal to result in the desired kidney gene expression.

[0075] Accordingly, the method may comprise inducing the AIM progenitor, or hiPSC-AIM, cells by contacting the cells with CHIR, typically for at least 12 hours, at least 24 hours, at least 36 hours or at least 48 hours. In some embodiments, the AIM progenitor, or hiPSC- AIM, cells are contacted with at least lpM CHIR, at least 2pM CHIR, at least 3pM CHIR or at least 4pM CHIR. In some embodiments, the AIM progenitor, or hiPSC-AIM, cells are contacted with at least 5pM CHIR, at least 6pM CHIR, or at least 7pM CHIR. In some embodiments, the AIM progenitor, or hiPSC-AIM, cells are contacted with at least 8pM CHIR, at least 9pM CHIR, at least lOpM CHIR or at least llpM CHIR.

[0076] The method may comprise inducing the AIM progenitor, or hiPSC-AIM, cells by contacting the cells with Activin A, ideally for at least 12 hours, at least 24 hours, at least 36 hours or at least 48 hours. The method may comprise inducing the AIM progenitor, or hiPSC-AIM, cells by contacting the cells with Activin A, typically for at least 60 hours, or at least 72 hours. In some embodiments, the AIM progenitor, or hiPSC-AIM, cells are contacted with at least 1 ng / mL Activin A, at least 2 ng / mL Activin A, at least 3 ng / mL Activin A or at least 4 ng / mL Activin A. In some embodiments, the AIM progenitor, or hiPSC-AIM, cells are contacted with at least 5 ng / mL Activin A, at least 6 ng / mL Activin A, at least 7 ng / mL Activin A or at least 8 ng / mL Activin A. In some embodiments, the AIM progenitor, or hiPSC-AIM, cells are contacted with at least 9 ng / mL Activin A, or at least 10 ng / mL Activin A.

[0077] Typically, however, the method comprises the induction of the AIM progenitor, or hiPSC- AIM, cells using 2 days induction with 11 pM CHIR, and 3 days with 10 ng / mL Activin A.

[0078] In another embodiment, the method may comprise inducing the PIM progenitor, or hiPSC-PIM, cells by contacting the cells with CHIR, typically for at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, or at least 96 hours. In some embodiments, the PIM progenitor, or hiPSC-PIM, cells are contacted with at least lpM CHIR, at least 2pM CHIR, at least 3pM CHIR or at least 4pM CHIR. In some embodiments, the PIM progenitor, or hiPSC-PIM, cells are contacted with at least 5pM CHIR, at least 6pM CHIR, or at least 7pM CHIR. In some embodiments, the PIM progenitor, or hiPSC-PIM, cells are contacted with at least 8pM CHIR, at least 9pM CHIR, at least lOpM CHIR, or at least llpM CHIR.

[0079] The method may comprise inducing the PIM progenitor, or hiPSC-PIM, cells by contacting the cells with Activin A, ideally for at least 12 hours, at least 24 hours, at least 36 hours or at least 48 hours. The method may comprise inducing the PIM progenitor, or hiPSC- PIM, cells by contacting the cells with Activin A, typically for at least 60 hours, or at least 72 hours. In some embodiments, the PIM progenitor, or hiPSC-PIM, cells are contacted with at least 1 ng / mL Activin A, at least 2 ng / mL Activin A, at least 3 ng / mL Activin A or at least 4 ng / mL Activin A. In some embodiments, the PIM progenitor, or hiPSC-PIM, cells are contacted with at least 5 ng / mL Activin A, at least 6 ng / mL Activin A, at least 7 ng / mL Activin A or at least 8 ng / mL Activin A. In some embodiments, the PIM progenitor, or hiPSC-PIM, cells are contacted with at least 9 ng / mL Activin A, or at least 10 ng / mL Activin A.

[0080] The method may comprise inducing the PIM progenitor, or hiPSC-PIM, cells by contacting the cells with FGF-9, ideally for at least 12 hours, at least 24 hours, at least 36 hours or at least 48 hours. In some embodiments, the PIM progenitor, or hiPSC-PIM, cells are contacted with at least 1 ng / mL FGF-9, at least 2 ng / mL FGF-9, at least 3 ng / mL FGF-9, or at least 4 ng / mL FGF-9. In some embodiments, the PIM progenitor, or hiPSC-PIM, cells are contacted with at least 5 ng / mL FGF-9, at least 6 ng / mL FGF-9, at least 7 ng / mL FGF-9, or at least 8 ng / mL FGF-9. In some embodiments, the PIM progenitor, or hiPSC-PIM, cells are contacted with at least 9 ng / mL FGF-9, or at least 10 ng / mL FGF-9.

[0081] Typically, however, the method comprises the induction of the PIM progenitor, or hiPSC- PIM, cells using 4 days induction with 11 pM CHIR, 3 days with 10 ng / mL Activin A, and 2 days with 10 ng / mL FGF-9.

[0082] Advantageously, the at least one anterior intermediate mesoderm (AIM) progenitor cell or hiPSC-AIM and at least one posterior intermediate mesoderm (PIM) progenitor cell or hiPSC-PIM are contacted and cultured, and typically cultured with CHIR, Activin A, and FGF-9 for induction of the AIM and PIM progenitor cells, in an ultra-low attachment plate, thus eliminating the need for an adhesion substrate and / or a dissociation step, which are typically required in processes for the generation of organoids. Accordingly, the three-dimensional aggregate format facilitated by the methods of the present invention support continuous cell-cell contact, mimicking aspects of early embryogenesis more closely than two-dimensional systems. This format allows for self-organisation, more uniform exposure to morphogens throughout the aggregate, and vastly improved reproducibility.

[0083] It will also be appreciated that a dissociation step refers to the process of breaking apart larger tissue structures or cell aggregates into smaller pieces or single cells, where typical techniques of dissociation comprise mechanical trituration, mincing, filtering, and / or centrifugation. However, such dissociation techniques introduce additional manipulation and shifts cells into a different physical context, potentially affecting their developmental trajectory.

[0084] In one embodiment, therefore, the at least one anterior intermediate mesoderm (AIM) progenitor cell or hiPSC-AIM and at least one posterior intermediate mesoderm (PIM) progenitor cell or hiPSC-PIM are contacted and cultured in an ultra-low attachment plate. In one embodiment, the method of the second aspect does not comprise contacting the at least one anterior intermediate mesoderm (AIM) progenitor or hiPSC-AIM cell and / or the at least one posterior intermediate mesoderm (PIM) progenitor hiPSC-PIM cell with an adhesion substrate.

[0085] In one embodiment, the method of the second aspect does not comprise a dissociation step.

[0086] As discussed in the Examples, the inventors have demonstrated that the assembly of AIM and PIM, or hiPSC-AIM and hiPSC-PIM, aggregates together with endothelial (Endo), hiPSC-EC, spheroids / aggregates results in the presence of endothelial cells, as suggested by the presence of the key endothelial marker CD31, and thus, the kidney organoids may be vascularised.

[0087] As is the case for the hiPSC-AIMs and hiPSC-PIMs, it will be appreciated that the term "hiPSC-EC" can mean at least one human induced pluripotent stem cell (hiPSC) which will differentiate into at least one endothelial cell.

[0088] Thus, it will be appreciated that the endothelial spheroids / aggregates differentiate from human induced pluripotent stem cells (hiPSCs), and thus may be referred to as human induced pluripotent stem cell (hiPSC) aggregates. Upon culture with various compounds, the human induced pluripotent stem cell (hiPSC) aggregates differentiate to form endothelial spheroids.

[0089] Referring to Figure 16, there is shown schematics of PIM, AIM, and endothelial differentiation prior and after assembly. In parallel with AIM and PIM differentiation, human induced pluripotent stem cells (hiPSC) were seeded into plates to generate hiPSC-EC aggregates.

[0090] Referring to Figure 17, there is shown the assembly of AIM, PIM, and Endothelial (Endo) spheroids. Indeed, on day 6 of differentiation, endothelial spheroids were assembled with AIM and PIM spheroids (i.e., aggregates). Referring to Figure 17D, immunofluorescence staining (CD31) of day 30 kidney organoids cryosections indicates the presence of endothelial cells.

[0091] In one embodiment, therefore, the at least one anterior intermediate mesoderm (AIM) progenitor, or hiPSC-AIM, cell aggregate and the at least one posterior intermediate mesoderm (PIM) progenitor, or hiPSC-PIM, cell aggregate may be contacted with at least one human induced pluripotent stem cell-endothelial cell (hiPSC-EC) aggregate.

[0092] In one embodiment, the at least one human induced pluripotent stem cell-endothelial cell (hiPSC-EC) aggregate may comprise at least 10, at least 20, at least 30, at least 40, or at least 50 cells. In one embodiment, the at least one human induced pluripotent stem cell-endothelial cell (hiPSC-EC) aggregate may comprise at least 60, at least 70, at least 80, at least 90, or at least 100 cells. Typically, however, the at least one human induced pluripotent stem cell-endothelial cell (hiPSC-EC) aggregate comprises about 50 cells.

[0093] In one embodiment, the at least one human induced pluripotent stem cell-endothelial cell (hiPSC-EC) aggregate may be cultured in media (e.g., mTeSRPIus), typically wherein the media is supplemented with Y-27632. Typically, the media is supplemented with at least 5 pM, at least 6 pM, at least 7 pM, at least 8 pM, at least 9 pM, or at least 10 pM Y- 27632.

[0094] In one embodiment, the at least one human induced pluripotent stem cell-endothelial cell (hiPSC-EC) aggregate may be cultured at at least 30°C, or at least 35°C. Typically, the at least one human induced pluripotent stem cell-endothelial cell (hiPSC-EC) aggregate is cultured at about 37°C.

[0095] In one embodiment, the at least one human induced pluripotent stem cell-endothelial cell (hiPSC-EC) aggregate may be cultured in media for at least 5 hours, at least 10 hours, at least 15 hours, or at least 20 hours. Typically, the at least one human induced pluripotent stem cell-endothelial cell (hiPSC-EC) aggregate is cultured in media for about 24 hours.

[0096] Typically, the at least one human induced pluripotent stem cell-endothelial cell (hiPSC- EC) aggregate is cultured in media supplemented with 10 pM Y-27632 at 37°C for 24 hours.

[0097] In one embodiment, subsequent to culture with Y-27632, the at least one human induced pluripotent stem cell-endothelial cell (hiPSC-EC) aggregate may be cultured in mesoderm induction media. In one embodiment, the mesoderm induction media may be supplemented with CHIR-99021 and / or BMP4. Typically, the mesoderm induction media is supplemented with CHIR-99021 and BMP4. In one embodiment, the mesoderm induction media may be supplemented with at least 1 pM, at least 5 pM, or at least 10 pM CHIR-99021. Typically, the mesoderm induction media is supplemented with about 11 pM CHIR-99021. In one embodiment, the mesoderm induction media may be supplemented with at least 10 ng / mL, at least 15 ng / mL, or at least 20 ng / mL BMP4. Typically, the mesoderm induction media is supplemented with about 25 ng / mL BMP4.

[0098] In one embodiment, the at least one human induced pluripotent stem cell-endothelial cell (hiPSC-EC) aggregate may be cultured in mesoderm induction media for at least 24 hours, at least 36 hours, at least 48 hours, or at least 60 hours. Typically, the at least one human induced pluripotent stem cell-endothelial cell (hiPSC-EC) aggregate is cultured in mesoderm induction media for about 72 hours (3 days).

[0099] In one embodiment, subsequent to culture in mesoderm induction media, the at least one human induced pluripotent stem cell-endothelial cell (hiPSC-EC) aggregate may be cultured in endothelial cell (EC) induction media. In one embodiment, the EC induction media may be supplemented with VEGF and / or forskolin. Typically, the EC induction media is supplemented with VEGF and forskolin. In one embodiment, the EC induction media may be supplemented with at least 50 ng / mL, at least 80 ng / mL, or at least 90 ng / mL VEGF. Typically, the EC induction media is supplemented with about 100 ng / mL VEGF. In one embodiment, the EC induction media may be supplemented with at least 0.5 pM, at least 1 pM, or at least 1.5 pM forskolin. Typically, the EC induction media is supplemented with about 2 pM forskolin.

[0100] In one embodiment, the at least one human induced pluripotent stem cell-endothelial cell (hiPSC-EC) aggregate may be cultured in EC induction media for at least 24 hours, at least 36 hours, at least 48 hours, or at least 60 hours. Typically, the at least one human induced pluripotent stem cell-endothelial cell (hiPSC-EC) aggregate is cultured in EC induction media for about 72 hours (3 days).

[0101] In one embodiment, therefore, the at least one anterior intermediate mesoderm (AIM) progenitor, or hiPSC-AIM, cell aggregate and the at least one posterior intermediate mesoderm (PIM) progenitor, or hiPSC-PIM, cell aggregate may be contacted with at least one human induced pluripotent stem cell-endothelial cell (hiPSC-EC) aggregate, wherein the at least one human induced pluripotent stem cell-endothelial cell (hiPSC-EC) aggregate is a day 6 aggregate (i.e., on day 6 of differentiation).

[0102] In one embodiment, the at least one anterior intermediate mesoderm (AIM) progenitor, or hiPSC-AIM, cell aggregate, the at least one posterior intermediate mesoderm (PIM) progenitor, or hiPSC-PIM, cell aggregate, and the at least one human induced pluripotent stem cell-endothelial cell (hiPSC-EC) aggregate may be co-cultured may be co-cultured in Basal Medium supplemented with VEGF. In one embodiment, the Basal Medium may be supplemented with at least 20 ng / mL, at least 30 ng / mL, or at least 40 ng / mL VEGF. Typically, the Basal Medium is supplemented with about 50 ng / mL VEGF.

[0103] In one embodiment, the at least one anterior intermediate mesoderm (AIM) progenitor, or hiPSC-AIM, cell aggregate, the at least one posterior intermediate mesoderm (PIM) progenitor, or hiPSC-PIM, cell aggregate, and the at least one human induced pluripotent stem cell-endothelial cell (hiPSC-EC) aggregate may be co-cultured for at least 5 days, at least 10 days, at least 15 days, at least 20 days, or at least 25 days. Typically, the at least one anterior intermediate mesoderm (AIM) progenitor, or hiPSC-AIM, cell aggregate, the at least one posterior intermediate mesoderm (PIM) progenitor, or hiPSC- PIM, cell aggregate, and the at least one human induced pluripotent stem cell- endothelial cell (hiPSC-EC) aggregate are co-cultured for about 30 days.

[0104] In a third aspect of the invention, there is provided a kidney organoid obtained, or obtainable by, the method according to the second aspect.

[0105] The kidney organoid of the third aspect may be as defined as the organoid of the first aspect.

[0106] The inventors believe that the organoid of the first and third aspects invention may be used in a screen for new, or existing, therapeutic compounds in the drug discovery and evaluation process, particularly for nephrotoxicity analysis of drugs. For example, methods and assays using the organoid may be used for candidate compound screening (e.g., High Throughput Screening, selective library screens, and structure-based design etc.), for identifying hits that may offer effective treatment, as well as secondary assays (e.g., in vitro and ex vivo secondary assays), for identifying nephrotoxicity of candidate drugs, as part of the "hit to lead" development of a pharmaceutical.

[0107] Accordingly, in a fourth aspect of the invention, there is provided use of the kidney organoid according to the first or third aspect, in a drug evaluation screen.

[0108] Accordingly, in a fifth aspect of the invention, there is provided a method of carrying out a drug evaluation screen, the method comprising contacting the kidney organoid according to the first or third aspect with a test agent, and analysing the effects of the test agent on the kidney organoid. The inventors envisage the use of the kidney organoid of the first or third aspect in various stages of drug evaluation, but particularly during the pre-clinical phase. These stages of drug evaluation may include toxicity analysis, pharmacokinetics analysis, pharmacodynamics analysis, and functional analysis. Furthermore, the inventors envisage the use of the kidney organoid in determining a drug's mechanism of action, the adverse effects of a drug, and / or for assessing drug-induced renal injury during preclinical trials.

[0109] In one embodiment, therefore, the kidney organoid is used for drug evaluation. The kidney organoid may be used for drug toxicity analysis, pharmacokinetics analysis, pharmacodynamics analysis, and / or functional analysis. Typically, however, the kidney organoid is used for nephrotoxicity analysis.

[0110] The kidney organoid may be used during the drug research and discovery stage of drug development, during the preclinical stage of drug development, during the clinical stage of drug development, during the drug review stage of drug development, and / or during the post-market drug safety monitoring stage of drug development. The kidney organoid may be used during the drug research and discovery stage of drug development, during the preclinical stage of drug development, and / or during the clinical stage of drug development. Typically, however, the kidney organoid is used during the preclinical stage of drug development.

[0111] The kidney organoid may be used for assessing drug mechanism of action, adverse effects, and / or drug-induced renal injury.

[0112] The kidney organoid may be used in an in vitro and / or ex vivo assay. The kidney organoid may be used in a preclinical in vitro and / or ex vivo assay. The kidney organoid may be used in a preclinical efficacy in vitro and / or ex vivo assay. In some embodiments, the kidney organoid is used in a preclinical in vitro and / or ex vivo assay for assessing drug-induced renal injury. Typically, the kidney organoid is used in a preclinical in vitro and / or ex vivo assay for assessing drug-induced renal injury caused by nephrotoxic drugs.

[0113] Therefore, in a sixth aspect, there is provided a method of analysing the nephrotoxicity of a test agent, the method comprising the steps of:

[0114] (i) contacting, in vitro or ex vivo, the kidney organoid according to the first or third aspect with a test agent; and (ii) analysing the kidney organoid to detect any damage caused by the test agent, wherein the damage, as compared to a control kidney organoid in the absence of the test agent, is an indicator of the nephrotoxicity of the test agent.

[0115] The kidney organoid of the invention may be analysed by its function and / or structure. The kidney organoid of the invention may be analysed by its function and / or structure before and / or after contact with the test agent.

[0116] The analysis of organoid function and / or structure may comprise a physical integrity analysis, structural organisation analysis, and / or functional analysis.

[0117] The physical integrity analysis may comprise analysis of apoptosis, oxidative stress, genotoxic damage, cell membrane permeability, endoplasmic reticulum integrity, mitochondrial membrane potential, the percentage of apoptotic cells, caspase activity, reactive oxygen species (ROS) activity, whole-cell reactive oxygen detection, mitochondrial superoxide detection, quantification of phosphorylated histone Y-H2AX, and / or fluorescence intensity quantification.

[0118] The structural organisation analysis may comprise analysis of kidney organoid development (kinetics and / or structure), kidney cell function and / or organisation, kidney cell structure, kidney cell morphology and / or activity, mitochondria activity and / or structure, and / or progression of maturation.

[0119] The functional analysis may comprise analysis of a water regulation mechanism, the ability to uptake and / or accumulate a molecule, endocrine function and / or response to injury.

[0120] It will be appreciated that the term "damage" can mean pathophysiology, and / or pathological changes to the kidney organoid. For example, the damage may include changes in organoid appearance and / or function.

[0121] The kidney organoid may be analysed to detect damage by biomarker analysis. The biomarker which, when expressed at an elevated level as compared to baseline expression, indicate kidney organoid damage, may be selected from a list of biomarkers, including : KIM-1, NGAL, creatinine, and / or urea. Biomarker analysis may be performed using an ELISA assay, qRT-PCR, and / or confocal microscopy. Damage may be quantified by comparing a test organoid, e.g., an organoid contacted with a test agent, with at least one negative control, e.g., an organoid not contacted with a test agent, using statistical analysis to evaluate the significance of the results.

[0122] It will be appreciated that the term "damage" can mean pathophysiology, and / or pathological changes to the kidney organoid. For example, the damage may include changes in glomerular hemodynamics, tubular cell toxicity, inflammation, crystal nephropathy, rhabdomyolysis, thrombotic microangiopathy, metabolic acidosis, hydronephrosis, ischemic injury, proteinuria, and / or hypoalbuminemia.

[0123] In a seventh aspect, there is provided an apparatus for identifying the nephrotoxicity of a test agent, the apparatus comprising :

[0124] (i) a kidney organoid according to the first or third aspect; and

[0125] (ii) a vessel configured to permit contacting the kidney organoid and a test agent.

[0126] The vessel may be a test tube, a microtube, a well plate, a spot plate, a microplate, a T-Flask, a slide, and / or a slide cover. The microplate may be used for qRT-PCR. and / or ELISA, the slide may be used for immunostaining, the well plates and / or T- Flask may be used for cell culture.

[0127] It will be appreciated that the kidney organoid can be used to detect drug-induced nephrotoxicity in the apparatus and methods of the invention. Hence, if the kidney organoid comprises damage in response to the presence of the test agent or compound, then this would indicate that the test agent does not represent a useful candidate therapeutic compound. Conversely, if the kidney organoid does not comprise damage in the presence of the test compound, then this would indicate that the test compound does represent a useful candidate therapeutic compound, and could be taken forward in subsequent trials. Typically, the methods and apparatus of the invention involve identifying the nephrotoxicity of a therapeutic agent.

[0128] As discussed in the Examples, the inventors have demonstrated that disease can be induced in the kidney organoids of the invention.

[0129] As such, the inventors also believe that the kidney organoids of the invention can be used to study diseases, such as genetic diseases, of the kidney.

[0130] Therefore, in an eighth aspect, there is provided use of the kidney organoid according to the first or third aspect, to study a kidney disease. The inventors envisage the kidney organoids of the invention for use in disease modelling and / or for studying a kidney disease.

[0131] In one embodiment, the kidney organoid may be contacted with an agent to induce a disease state.

[0132] It will be appreciated that inducing a disease state in the kidney organoid of the invention can mean that, subsequent to contacting the kidney organoid with the agent, the kidney organoid displays a genotype and / or phenotype typical of a diseased kidney, including but not limited to alterations in gene expression, protein expression, cellular morphology, functional parameters (e.g., filtration or reabsorption capacity), and the composition or quantity of secreted molecules such as cytokines, metabolites, or extracellular vesicles.

[0133] In one embodiment, the agent which induces a disease state may be a nephrotoxic drug. In one embodiment, the nephrotoxic drug may be selected from a list of therapeutic classes consisting of: chemotherapeutic, analgesic, antibiotic, and immunosuppressant. In one embodiment, the nephrotoxic drug may be selected from a list of compounds consisting of: cisplatin, acetaminophen, ibuprofen, gentamicin, and thalidomide.

[0134] In one embodiment, the agent which induces a disease state may be contacted with the kidney organoid at a concentration of at least 1 nM, at least 10 nM, at least 100 nM, at least 250 nM, at least 500 nM, at least 750 nM, or at least 1000 nM.

[0135] In one embodiment, the agent which induces a disease state may be contacted with the kidney organoid at a concentration of between 1 pM and 1 M, between 5 pM and 0.1 M, between 10 pM and 50 mM, between 15 pM and 25 mM, between 20 pM and 20 mM, or between 25 pM and 15 mM. In one embodiment, the agent which induces a disease state may be contacted with the kidney organoid at a concentration of between 30 pM and 10 mM, between 35 pM and 8 mM, between 40 pM and 7 mM, between 45 pM and 6 mM, or between 50 pM and 5 mM.

[0136] In one embodiment, the agent which induces a disease state may be contacted with a day 1, day 5, day 10, or day 20 kidney organoid. In one embodiment, the agent which induces a disease state may be contacted with a day 30, day 40, day 50, or day 60 kidney organoid. Typically, however, the agent which induces a disease state is contacted with a day 30 kidney organoid. It will be appreciated that the term "day" refers to a day of the method of producing the kidney organoid of the invention, i.e., the method of the second aspect.

[0137] In one embodiment, the agent which induces a disease state may be contacted with a kidney organoid for at least 1, 5, 15, 30, or 45 minutes. In one embodiment, the agent which induces a disease state may be contacted with a kidney organoid for at least 1, 2, 5, 10, 20, or 24 hours. In one embodiment, the agent which induces a disease state may be contacted with a kidney organoid for at least 36, 40, 45, or 48 hours. Typically, however, the agent which induces a disease state may be contacted with a kidney organoid for about 48 hours.

[0138] In one embodiment, inducing a disease state in the kidney organoid may comprise decreasing the cell viability, metabolic activity, cellular respiration, membrane integrity, esterase activity of the cells of the kidney organoid, inducing alterations in gene or protein expression, inducing oxidative stress, inducing secretion of pro-inflammatory or fibrotic mediators, inducing functional impairments (such as reduced solute transport or albumin uptake), and / or inducing morphological changes characteristic of diseased kidney tissue, when compared to a control kidney organoid that is not contacted with an agent to induce a disease state.

[0139] In one embodiment, inducing a disease state in the kidney organoid may comprise increasing the expression of a biomarker of renal injury in the cells of the kidney organoid when compared to a control kidney organoid that is not contacted with an agent to induce a disease state. In one embodiment, the biomarker of renal injury may comprise a biomarker of tubular epithelial damage, such as Kidney Injury Molecule-1 (KIM-1), NGAL, FABP1, IL-6, MCP-1, TGF-pi, collagen types I and III, fibronectin, HO-1, caspase-3, albumin, podocin, nephrin, a proximal tubule-specific biomarker, or a biomarker of inflammation, fibrosis, oxidative stress, glomerular dysfunction, and / or cellular senescence.

[0140] In one embodiment, inducing a disease state in the kidney organoid may comprise increasing the expression of a secreted biomarker of renal injury in the culture of the kidney organoid, typically the culture supernatant, when compared to a control kidney organoid that is not contacted with an agent to induce a disease state. In one embodiment, the biomarker of renal injury may comprise a biomarker of injury-induced protein release, such as Kidney Injury Molecule-1 (KIM-1), neutrophil gelatinase- associated lipocalin (NGAL), liver-type fatty acid binding protein (FABP1), monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), clusterin, p2-microglobulin, retinol-binding protein 4 (RBP4), and osteopontin (SPP1), or kidney function impairment, such as creatinine.

[0141] In one embodiment, a disease state in the kidney organoid may be detected and / or measured using an alamarBlue assay, LIVE / DEAD staining, immunostaining, fluorescence microscopy, and / or ELISA.

[0142] In one embodiment, the kidney organoid of the invention may be genetically modified. Typically, the kidney organoid may be genome edited. The kidney organoid may be genome edited by means of an AAV vector or a lentiviral vector.

[0143] In another embodiment, the kidney organoid of the invention may be genome edited for the construction of a disease model, typically a genetic disease model.

[0144] Accordingly, in a ninth aspect of the invention, there is provided a method of carrying out a kidney disease study, the method comprising contacting the kidney organoid according to the first or third aspect with a test agent, and analysing the effects of the kidney organoid in a kidney disease.

[0145] The kidney organoid of the invention may be used in an in vitro and / or ex vivo assay in the study of a kidney disease. The disease may be selected from a list of diseases, including : kidney disease, chronic kidney disease, acquired cystic kidney disease, amyloidosis, diabetes insipidus, ectopic kidney, glomerular disease, anti-GBM (Goodpasture's) disease, IgA nephropathy, IgA vasculitis, kidney infection (pyelonephritis), kidney stones, lupus nephritis, medullary sponge kidney, multicystic dysplastic kidney, nephrotic syndrome, polycystic kidney disease (PKD), renal artery stenosis, renal tubular acidosis, kidney cysts, Alport syndrome, cystinosis, Fabry disease, Gitelman syndrome, tuberous sclerosis complex, nephronophthisis, Hypophosphatemic ricketts, Bartter syndrome types 1-4, Gitelman syndrome, Hypomagnesemia, Liddle syndrome, Gordon syndrome (PHA type 2), Pseudohypoaldosteronism type 1, SeSAME syndrome, Diabetes insipidus, Lowe syndrome, and / or Hemolytic uremic syndrome.

[0146] In some embodiments, the kidney organoid is used in an in vitro and / or ex vivo assay in the study of a genetic disease. The genetic disease may be selected from a list of genetic diseases, including : polycystic kidney disease, Alport syndrome, cystinosis, Fabry disease, Gitelman syndrome, tuberous sclerosis complex, nephronophthisis, steroid resistant nephrotic syndrome, Denys-Drash syndrome, Frasier syndrome, Nail-Patella syndrome, Schimke immuno-osseous dystrophy, Renal hypodysplasia, Multicystic renal dysplasia, HDR syndrome, Kallman syndrome, Townes-Brocks syndrome, Medullary cystic kidney disease, Meckel-Gruber syndrome, Bardet-Biedl syndrome types 1-12, von-Hippel-Lindau disease, and / or Wilms-tumor-aniridia syndrome. Typically, the kidney organoid is used in an in vitro and / or ex vivo assay in the study of polycystic kidney disease.

[0147] Typically, the kidney organoid is used in an acute tubular necrosis (ATN) assay, an acute kidney injury (AKI) assay, a tubular swelling assay, a glomerular filtration assay, and / or a hormone production assay, to analyse the effects of the test agent on the kidney organoid.

[0148] The test agent may be a drug or pharmaceutical. The test agent is a candidate drug. Typically, however, the test agent is a candidate drug in the preclinical stage of drug evaluation.

[0149] The test agent may be selected from a list of therapeutic classes including : analgesic, antibiotic, anticancer, anticoagulant, antidepressant, antidiabetic, antiepileptic, antipsychotic, antispasmodic, antiviral, depressant, sedative, and stimulant.

[0150] Any of the methods described herein may be carried out in vitro or ex vivo. Any of the methods may comprise screening a test agent that shows a positive indication for the same activity in a cell-based system and / or in vivo in a non-human mammal.

[0151] All of the features described herein (including any accompanying claims, abstracts and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some features and / or steps are mutually exclusive.

[0152] For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which:-

[0153] Figure 1 (A) is a schematic of one embodiment of a method of kidney organoid production from human pluripotent stem cells (hPSCs). PIM : posterior intermediate mesoderm; AIM : anterior intermediate mesoderm; CHIR: CHIR99021; GDNF: glial- derived neurotrophic factor; RA: retinoic acid; EGF: epidermal growth factor; FGF-9: fibroblast growth factor-9; AVP: arginine-vasopressin. (B) is a schematic representation of a second embodiment of a method for kidney organoid production from hPSCs. AIM : anterior intermediate mesoderm; PIM : posterior intermediate mesoderm; ROCKi: 10 pM Y-27632; CHIR: 11 pM CHIR99021; Activin A: 10 ng / mL; FGF-9: 10 ng / mL; FHGRE: 200 ng / mL FGF-9 (F), 1 pg / mL heparin (H); 10 ng / mL GDNF (G), 100 nM retinoic acid (R), 10 nM EGF (E); AVP: 10 nM arginine-vasopressin; 10 nM aldosterone.

[0154] Figure 2 shows brightfield images of size-controlled human pluripotent stem cell (hPSC) aggregates at the start and at the end of AIM and PIM differentiation inside microwells. AIM: anterior intermediate mesoderm; PIM : posterior intermediate mesoderm. Scale bars: 250 pm.

[0155] Figure 3 shows gene expression of mesoderm, endoderm, and pluripotency markers at different timepoints for AIM and PIM specification for DF6, GEpi, and TCLab human induced pluripotent stem cell (hiPSC) lines. Error represents standard deviation (STDEV).

[0156] Figure 4 shows gene expression levels for posterior intermediate mesoderm (HOXD11) and anterior intermediate mesoderm (GATA3) markers at the end of AIM and PIM specification. Error represents standard error of the mean (SEM).

[0157] Figure 5 shows the production of a kidney assembloid from AIM and PIM progenitor cells. (A) Model of assembly of PIM and AIM aggregates. (B) Brightfield images of the assembloids through a differentiation period of 60 days. Scale bars: 100 pm.

[0158] Figure 6 is the immunological characterisation of structures within day 30 kidney organoids. (A) Staining for distal tubules (characterised by the expression of E-cadherin (ECAD)) and proximal tubules (characterised by the expression of Lotus tetragonolobus lectin (LTL)). (B) Staining for interstitium (FOXD11) and maturing glomeruli develop glomerular basement membrane (LAM). (C) Staining for collecting ducts (ECAD and GATA3) and distal tubules (ECAD). (D) Staining for podocytes (NPHS1, WT1). (A, B) : Scale bar: 50 pm. (C, D) : Scale bar: 100 pm.

[0159] Figure 7 is the immunological characterisation of structures within day 60 kidney organoids. (A) Staining for distal tubules (ECAD) and proximal tubules (LTL). (B) Maturing glomeruli develop glomerular basement membrane (LAM) in the middle of the podocytes (NPHS1), which cover the outside border of the organoid. (C) Staining for renal interstitium (FOXD1, WT1) and podocytes (FOXD1+ WT1 + ). (D) Presence of endothelial cells (CD31). (E) Distal tubules present water regulation proteins (AQP-2) and the main sodium transporter of the nephron (Na / K / ATPase). Scale bars: 50 pm. Figure 8 (A) shows brightfield and (B) fluorescent images of a kidney organoid after 24 hours of exposure to 488-labeled dextran, and indicates the successful uptake of this molecule.

[0160] Figure 9 shows kidney organoid response to forskolin stimulation. (A) Kidney organoids respond to forskolin stimulation and increase in size in different cell lines - DF6 (i), GEpi (ii), TCLab (iii). (B) Kidney organoids after 48 hours of exposure to forskolin - DF6 (i'), GEpi (ii'), TCLab (iii'). The seeding of AIM and PIM progenitor cells on transwells induces visible changes under control conditions (C) or under exposure to forskolin (D). n.d. : no data.

[0161] Figure 10 shows cell viability assessment following 48-hour drug exposure to evaluate acute kidney injury (AKI) potential. Percent cell survival relative to control was measured using the alamarBlue assay. Data represent mean ± SEM. * p<0.05; ** p<0.01.

[0162] Figure 11 shows representative fluorescence images of LIVE / DEAD staining after 48- hour drug treatment to assess acute kidney injury. Live cells are stained green (calcein- AM), and dead cells are stained red (ethidium homodimer-1). Images reflect differential cytotoxic responses across treatments.

[0163] Figure 12 shows immunofluorescence staining of Kidney Injury Molecule-1 (KIM-1, green) in treated kidney cells, indicating tubular epithelial injury. Nuclei are counterstained with DAPI (blue). Increased KIM-1 expression correlates with drug- induced cellular damage.

[0164] Figure 13 shows ELISA quantification of KIM-1 and creatinine levels in culture supernatants. KIM-1 secretion was measured in the DF6 cell line (n = l; mean ± SD); data for gentamicin treatment were not available. Creatinine levels were quantified for DF6 (n=3; mean ± SEM) and GEpi (n = l; mean ± SD) cell lines.

[0165] Figure 14 shows relative expression profiles of mesodermal and renal markers during renal differentiation of hiPSCs upon exposure to WNT signalling for 96 hours. Cells were exposed to 10 pM of CHIR. and 100 nM of Dorsomorphin or 11 pM of CHIR for 96 hours. Error bars represent SEM. (n=2). Figure 15 shows relative expression profiles of mesodermal and renal markers during renal differentiation of hiPSCs treated with CHIR99021 for 72 hours. Error bars represent SD. (n=l).

[0166] Figure 16 shows schematics of PIM, AIM, and endothelial differentiation prior and after assembly.

[0167] Figure 17 shows the assembly of AIM, PIM, and Endothelial (Endo) spheroids. One endothelial spheroid (upper panel) or multiple endothelial spheroids (bottom panel) were included in each organoid. (A) Schematics of assembly of AIM, PIM, and endothelial spheroids. (B) Brightfield images at assembly (t=Oh). (C) Brightfield images 5 days after assembly. (D) Immunofluorescence staining of day 30 kidney organoids cryosections. CD31 indicates endothelial cells. Scale bar: 50 pm.

[0168] Examples

[0169] The inventors have developed a novel method that enables the reproducible production of kidney organoids which replicate the structural and functional features of the kidney. Specifically, the kidney organoids have been shown by the inventors to be capable of selective uptake of molecules, to show responsiveness to injury, and to comprise water regulation mechanisms. In addition, the inventors have discovered that the kidney organoids of the invention may be vascularised. These organoids can, therefore, be used in assays for testing the nephrotoxicity of pharmaceuticals, or for assessing kidney disease.

[0170] Materials and Methods

[0171] Cell Seeding for Posterior Intermediate Mesoderm (PIM)

[0172] 1. Aspirate culture medium, and rinse once with PBS to remove debris and exhausted culture medium.

[0173] 2. After washing, add 1 mL / well Accutase and incubate at 37°C for 5 minutes.

[0174] 3. Add double the volume of DMEM / F12 or Washing medium to inactivate the enzyme.

[0175] 4. Transfer the content of the wells to a Falcon tube.

[0176] 5. Centrifuge at 1000 rpm for 3 minutes.

[0177] 6. Discard supernatant and resuspend the cells in mTeSR Plus supplemented with 10 pM ROCK inhibitor.

[0178] 7. Add 10 pL of cell suspension to 40-190 pL of Trypan Blue, and count cells using a hemocytometer.

[0179] 8. Calculate cell volume for the needed cell number:

[0180] 9. Rinse each well with 2 mL of DMEM / F12.

[0181] 10. Add 500 pL of mTeSR Plus supplemented with 10 pM ROCK inhibitor to each well and centrifuge at 3500 rpm for 3 minutes.

[0182] 11. Verify that there are no air bubbles on the microwells. If bubbles remain, centrifuge again.

[0183] 12. In each well, add the appropriate volume of cell suspension.

[0184] 13. Calculate the remaining volume of mTeSR Plus + 10 pM ROCK inhibitor to fulfil

[0185] 1.5 ml (1500 pL - 500 pL already on the well - cell suspension volume).

[0186] 14. Add the remaining volume to each well, doing 1-3 up-down movements with the pipette to ensure proper cell homogeneity.

[0187] 15. Centrifuge plate at 1000 rpm for 3 minutes.

[0188] 16. Incubate in an incubator with humidified atmosphere of 5% of CO2 in air at 37 °C.

[0189] 17. With the remaining cells, seed Matrigel-coated plated according to C.

[0190] 18. If any cell remains, centrifuge at 1000 rpm for 3 minutes.

[0191] 19. Remove supernatant.

[0192] 20. Resuspend pellet in 1 mL PBS, transfer to a 1.5 mL Eppendorf tube, and centrifuge at 1000 rpm for 3 minutes.

[0193] 21. Remove supernatant, and store the dry pellet at -80°C for RNA extraction (day 0 sample).

[0194] Differentiation into Posterior Intermediate Mesoderm

[0195] 1. 24 hours after seeding, verify the diameter of the aggregates by taking several photographs of the wells, and measuring their diameter using Fiji software.

[0196] 2. If the aggregates average 233 ± 4 pm, start the differentiation protocol.

[0197] 3. On day 0, replace medium for 1.5 mL / well Basal Medium supplemented with 11 pM CHIR99021.

[0198] 4. On day 2, replace medium for 1.5 mL / well Basal Medium supplemented with 11 pM CHIR99021.

[0199] 5. On day 4, replace medium for 2.0 mL / well Basal Medium supplemented with 10 ng / mL Activin A.

[0200] 6. On day 7, replace medium for 1.5 mL / well Basal Medium supplemented with 10 ng / mL FGF-9.

[0201] 7. On day 9, PIM aggregates are ready. Cell Seeding for Expansion

[0202] 1. Remove Matrigel from a previously prepared Matrigel-coated plate and replace it with 1.5 mL / well mTeSR Plus + 10 pM ROCK inhibitor.

[0203] 2. Seed cells at 10,000 cells / cm2.

[0204] 3. After 24 hours, replace the medium with mTeSR Plus.

[0205] 4. Change medium daily until day 4 after seeding.

[0206] Cell Seeding for Anterior Intermediate Mesoderm (AIM)

[0207] 1. Aspirate culture medium, and rinse once with PBS to remove debris and exhausted culture medium.

[0208] 2. After washing, add Accutase and incubate at 37°C for 5 minutes.

[0209] 3. Add double the volume of DMEM / F12 or Washing medium to inactivate the enzyme.

[0210] 4. Transfer the content of the wells to a Falcon tube.

[0211] 5. Centrifuge at 1000 rpm for 3 minutes.

[0212] 6. Discard supernatant and resuspend the cells in mTeSR Plus supplemented with 10 pM ROCK inhibitor.

[0213] 7. Add 10 pL of cell suspension to 40-190 pL of Trypan Blue, and count cells using a hemocytometer.

[0214] 8. Calculate cell volume for the needed cell number:

[0215] 9. Rinse each well with 2 mL of DMEM / F12.

[0216] 10. Add 500 pL of mTeSR Plus supplemented with 10 pM ROCK inhibitor to each well and centrifuge at 3500 rpm for 3 minutes.

[0217] 11. Verify that there are no air bubbles on the microwells. If bubbles remain, centrifuge again.

[0218] 12. In each well, add the appropriate volume of cell suspension.

[0219] 13. Calculate the remaining volume of mTeSR Plus + 10 pM ROCK inhibitor to fulfill

[0220] 1.5 ml (1500 pL - 500 pL already on the well - cell suspension volume).

[0221] 14. Add the remaining volume to each well, doing 1-3 up-down movements with the pipette to ensure proper cell homogeneity.

[0222] 15. Centrifuge plate at 1000 rpm for 3 minutes.

[0223] 16. Incubate in an incubator with humidified atmosphere of 5% of CO2 in air at 37 °C.

[0224] Differentiation into Anterior Intermediate Mesoderm 1. 24 hours after seeding, verify the diameter of the aggregates by taking several photographs of the wells, and measuring their diameter using Fiji software.

[0225] 2. If the aggregates average 178 ± 7 pm, start the differentiation protocol.

[0226] 3. On day 0, replace medium for 1.5 mL / well Basal Medium supplemented with 11 pM CHIR99021.

[0227] 4. On day 2, replace medium for 2.0 mL / well Basal Medium supplemented with 10 ng / mL Activin A.

[0228] 5. On day 5, AIM aggregates are ready.

[0229] Production of Kidney Organoids on Transweiis

[0230] 1. Prepare 2 Falcon tubes named "AIM" and "PIM".

[0231] 2. Gently pipette up and down using a P1000 to remove aggregates from the microwells, and transfer to the respective Falcon tube.

[0232] 3. Using 1 mL Basal Medium per well, flush each well of the plate to completely remove the aggregates from the microwells, and transfer to the same Falcon tube.

[0233] 4. Allow aggregates to settle, and carefully remove the culture medium.

[0234] 5. Add 2 mL of PBS to each tube.

[0235] 6. Allow aggregates to settle, and carefully remove the culture medium.

[0236] 7. Add 3 mL of Accutase per tube, and incubate for 5-10 minutes at 37°C, pipetting at minute 5, 7, and 10, until the aggregates dissociate.

[0237] 8. Add 7 mL of Basal Medium per tube.

[0238] 9. Centrifuge at 1000 rpm for 3 minutes.

[0239] 10. Remove supernatant and add 2 mL of Basal Medium to resuspend the pellet.

[0240] 11. Add 10 pL of cell suspension to 40-190 pL of Trypan Blue and count cells using a hemocytometer.

[0241] 12. Calculate cell suspension volume for the needed cell numbers:

[0242] 13. Add the appropriate volume of AIM cell suspension to a 1.5 mL Eppendorf tube.

[0243] 14. Add the appropriate volume of PIM cell suspension to the previous tube.

[0244] Note: each Eppendorf tube will represent an organoid.

[0245] 15. Centrifuge the Eppendorfs at 200g for 3 minutes.

[0246] 16. Carefully remove the supernatant, leaving only a dry pellet.

[0247] 17. Using a P200, carefully aspirate the pellet and transfer it into a transwell membrane. Note: The pellet should remain as whole as possible.

[0248] 18. Add 1.3 mL (6-well plate) or 400 pL (24-well plate) of Basal Medium supplemented with 5 pM CHIR99021 to the bottom of the well.

[0249] 19. Incubate for 1 hour at 37°C in a humidified incubator.

[0250] 20. If any cell remains, centrifuge at 1000 rpm for 3 minutes.

[0251] 21. Remove supernatant.

[0252] 22. Resuspend pellet in 1 mL PBS, transfer to a 1.5 mL Eppendorf tube, and centrifuge at 1000 rpm for 3 minutes.

[0253] 23. Remove supernatant, and store the dry pellet at -80°C for RNA extraction (day 5 AIM and day 9 PIM sample).

[0254] 24. After 1 hour, carefully remove the culture medium and replace it with 150 pL of Basal Medium supplemented with 200 ng / mL FGF-9, 1 pg / mL Heparin, 10 ng / mL GDNF, 100 nM Retinoic Acid, and 10 nM EGF.

[0255] 25. Incubate at 37°C.

[0256] Production of Kidney Assembloids through Dissociation-Reaggregation (under optimisation)

[0257] 1. Prepare 2 Falcon tubes named "AIM" and "PIM".

[0258] 2. Gently pipette up and down using a P1000 to remove aggregates from the microwells and transfer to the respective Falcon tube.

[0259] 3. Using 1 mL Basal Medium per well, flush each well of the plate to completely remove the aggregates from the microwells, and transfer to the same Falcon tube.

[0260] 4. Allow aggregates to settle and carefully remove the culture medium.

[0261] 5. Add 2 mL of PBS to each tube.

[0262] 6. Allow aggregates to settle, and carefully remove the culture medium.

[0263] 7. Add 3 mL of Accutase per tube and incubate for 5-10 minutes at 37°C, pipetting at minute 5, 7 and 10, until the aggregates dissociate.

[0264] 8. Add 7 mL of Basal Medium per tube.

[0265] 9. Centrifuge at 1000 rpm for 3 minutes.

[0266] 10. Remove supernatant and add 2 mL of Basal Medium to resuspend the pellet.

[0267] 11. Add 10 pL of cell suspension to 40-190 pL of Trypan Blue, and count cells using a hemocytometer.

[0268] 12. Calculate cell suspension volume for the needed cell numbers: 13. Prepare an AggreWell 800 plate according to the protocol.

[0269] 14. Add the appropriate volume of AIM cell suspension to a well of an AggreWell 800 plate.

[0270] 15. Add the appropriate volume of PIM cell suspension to the same well.

[0271] 16. Fill the well up to 1.5 mL.

[0272] 17. Centrifuge at 1000 rpm for 3 minutes.

[0273] 18. Carefully remove the medium, and replace it with 1 mL of Basal Medium supplemented with 5 pM CHIR99021.

[0274] 19. Incubate for 1 hour at 37°C in a humidified incubator.

[0275] 20. After 1 hour, carefully remove the culture medium, and replace it with 150 pL of Basal Medium supplemented with 200 ng / mL FGF-9, 1 pg / mL Heparin, 10 ng / mL GDNF, 100 nM Retinoic Acid, and 10 nM EGF.

[0276] Production of Kidney Assembloids through Assembly

[0277] 1. Prepare 2 Falcon tubes named "AIM" and "PIM".

[0278] 2. Gently pipette up and down using a P1000 to remove aggregates from the microwells and transfer to the respective Falcon tube.

[0279] 3. Using 1 mL Basal Medium per well, flush each well of the plate to completely remove the aggregates from the microwells, and transfer to the same Falcon tube.

[0280] 4. Remove a sample of ~20 aggregates to a 1.5 mL Eppendorf for histology.

[0281] 5. Add 3 mL of Basal Medium to each Falcon tube.

[0282] 6. Using a P1000, carefully transfer individual AIM aggregates to U-shapped UltraLow Attachment 96-well plate wells (1 aggregate per well).

[0283] 7. Repeat step 6 for the PIM aggregates.

[0284] 8. Centrifuge the plate at 200 rpm for 30 seconds.

[0285] 9. Carefully remove the supernatant, and add 100 pL of Basal medium supplemented with 5 pM CHIR99021 to each well.

[0286] 10. Incubate for 1 hour at 37°C in a humidified incubator.

[0287] 11. After 1 hour, carefully remove the culture medium, and replace it with 150 pL of Basal Medium supplemented with 200 ng / mL FGF-9, 1 pg / mL Heparin, 10 ng / mL GDNF, 100 nM Retinoic Acid, and 10 nM EGF.

[0288] Kidney Organoid Maturation

[0289] 1. Maintain organoids derived in F, G, or H in Basal Medium supplemented with 200 ng / mL FGF-9, 1 pg / mL Heparin, 10 ng / mL GDNF, 100 nM Retinoic Acid, and 10 nM EGF for 5 days, replacing the medium every 2 days.

[0290] 2. At day 5 after organoid formation, replace medium for Basal Medium supplemented with 10 nM Aldosterone and 10 nM Arginine-Vasopressin (AVP). 3. Change media every other day until day 19 (counting from the day of AIM and PIM junction).

[0291] Table 1 - Materials used in methods

[0292] Table 2 - Basal Medium components

[0293] Example 1 - Development of differentiation protocol for kidney organoid production from human pluripotent stem cells (hPSCs)

[0294] With reference to Figure 1, the inventors have developed an innovative protocol for the production of kidney organoids from hPSCs. The strategy to produce kidney organoids under fully three-dimensional conditions relies on the initial and separate derivation of posterior intermediate mesoderm (PIM) and anterior intermediate mesoderm (AIM) using size-controlled human induced pluripotent stem cells (hiPSC). The inventors have surprisingly discovered that the induction of specific AIM and PIM progenitor cells as size-controlled aggregates results in the assembly of these two populations into a kidney organoid.

[0295] In order to produce the kidney organoids, hiPSC aggregates containing 2,000 cells each were produced using mTeSR Plus supplemented with 10 pM of ROCK inhibitor (Y-27632) for 24 hours. Subseguently, the medium was replaced with basal medium, composed of Advanced RPMI-1640 supplemented with lx Glutamax, and 0.5% (v / v) penicillin / streptomycin. For the first 4 days of PIM induction, the basal medium was also supplemented with 11 pM CHIR99021, with the medium being replaced by day 2. On day 4, CHIR99021 was withdrawn from the media, and cells were cultivated in basal medium supplemented with Activin A for a total of 3 days. On day 7, 10 ng / mL FGF-9 was supplemented in the basal medium until day 9. For AIM induction, the hiPSC aggregates were initially composed of 1,000 cells each. The protocol comprised an exposure 11 pM CHIR99021 for 2 days, followed by 3 days in the presence of Activin A.

[0296] Example 2 - Growth of AIM and PIM aggregates

[0297] Through extensive experimentation, the inventors discovered that aggregates of a deduced average diameter were reguired to initiate AIM and PIM specification, as indicated in Table 3. Three different cell lines were used to produce aggregates with 1,000 and 2,000 cells each, for AIM and PIM induction, respectively.

[0298] Table 3 - Average diameter of aggregates reguired to initiate the differentiation protocol for different cell lines. Error is represented as SEM. AIM: anterior intermediate mesoderm : PIM : posterior intermediate mesoderm

[0299] With reference to Figure 2, throughout the differentiation protocol, both AIM and PIM aggregates increased in size, with AIM aggregates having a bigger size compared to PIM aggregates.

[0300] Example 3 - Gene expression of mesoderm, endoderm, and pluripotency markers at different timepoints for AIM and PIM specification

[0301] Aggregates were collected at different timepoints, and gene expression was assessed by qRT-PCR. The inventors discovered that intermediate mesoderm genes, including OSR1 and PAX2, demonstrate a higher expression when compared to lateral mesoderm (KDR), paraxial mesoderm (PDGFR-a), endoderm (SOX17), and pluripotency (OCT4) genes, as shown in Figure 3. The kidney derives from the intermediate mesoderm, and thus, the inventors' assessment of gene expression in the aggregates verified that there was an upregulation of this population, i.e., intermediate mesoderm markers, and a downregulation of off-target populations, i.e., lateral mesoderm, paraxial mesoderm, endoderm, and pluripotency markers

[0302] At the end of AIM and PIM specification, gene expression of AIM (GATA3) and PIM (HOXD11) specific markers was analysed. With reference to Figure 4, the expression of HOXD11 was found to be higher in PIM aggregates, whereas GATA3 expression was enhanced in AIM aggregates. These results confirmed that the inventors had successfully derived the two progenitors separately.

[0303] Example 4 - Production of a kidney assembloid from AIM and PIM progenitor cells With reference to Figure 5 (A), after AIM and PIM induction, one aggregate from each lineage was collected and transferred to a 96-welL With reference to Figure 5 (B), after 24 hours of culture, both aggregates were already starting to assemble, and surprisingly, by day 19, some level of organisation was already observable.

[0304] Example 5 - Immunological characterisation of structures within day 30 and day 60 kidney organoids

[0305] The inventors identified that maturation of kidney organoids occurred throughout a period of up to 60 days. With reference to Figures 6 and 7, organoid sections were analysed at day 30 and day 60, respectively. To the surprise of the inventors, the presence of different kidney-specific structures was detected, including distal and proximal tubules, and collecting ducts.

[0306] Example 6 - Kidney organoids are capable of selective uptake of molecules

[0307] The success of employing stem-cell-derived kidney organoids for disease modelling or drug screening relies on how well the nephrons within these organoids mature functionally. Proximal tubules represent the section of the nephron that has an important role in solute, vitamin, hormone, and amino acids reabsorption.

[0308] To demonstrate the ability of the kidney organoids to perform such functions in vitro, kidney organoids were incubated with fluorescently labelled dextran. With reference to Figure 8, the inventors observed selective uptake of 488-labeled dextran by LTL-positive tubules after 24 hours of exposure, showing that the kidney organoids are capable of selective uptake of molecules.

[0309] Example 7 - Kidney organoids show responsiveness to forskolin stimulation

[0310] Induction of cystic phenotypes in kidney organoids can be attained by using forskolin, a stimulator of cAMP signalling. In order to evaluate this hypothesis, day 30 kidney organoids were exposed to 10 pM of forskolin for a period of 48 hours, and the alterations in organoid size were measured, as shown in Figure 9 (A) and (B).

[0311] Moreover, a model of dissociation-reaggregation of AIM and PIM progenitor cells on transwells yielded visible results in the induction of cysts upon exposure to forskolin, as shown in Figure 9 C and D. It has been demonstrated that incubation with forskolin leads to the accumulation of fluid inside proximal tubules, resulting in the increase in size of the kidney organoids, thus suggesting the induction of renin expression and release. The inventors predict that the above described process resulting in increased organoid size reverses with the depletion of forskolin from the culture medium.

[0312] Example 8 - Use of the kidney organoid in an assay to assess the nephrotoxicity of a pharmaceutical

[0313] The kidney organoids of the invention may be used to assess the nephrotoxicity of a drug. Said drug could include a drug undergoing clinical trial, or an existing drug that lacks relevant nephrotoxic data.

[0314] For example, the inventors, using the kidney organoids of the invention, would be able to assess the impact of drug exposure on normal organoid function and / or structure. Thus, the inventors would be informed of the nephrotoxicity of a drug, based on how exposure of said drug impacts normal in vivo kidney function and / or structure.

[0315] The inventors would select an appropriate range of test agent (drug) concentrations, and timings for drug exposure. For example, the inventors would incubate the kidney organoids at drug concentrations ranging from 0.00001 mM to 100 mM for a period of 0 to 96 hours. The experiment could be designed to assess the impact of acute drug exposure, i.e., up to 24 hours of exposure, by selecting timings for drug exposure of 3, 6, 12, and 24 hours, for example. Alternatively, the experiment could be designed to assess the impact of prolonged drug exposure, by selecting timings for drug exposure between 2 and 7 days, for example. The experiment could also be designed to assess chronic drug exposure, by selecting timings for drug exposure within a range of physiological concentrations from 7 to 30 days, for example.

[0316] The inventors would use, as controls, 1) organoids exposed to a nephrotoxic drug, such as cisplatin, gentamicin, or vancomycin, i.e., positive controls; 2) organoids exposed only to the vehicle in which the drug is diluted or dissolved, for example DMSO or H2O, i.e., negative controls; 3) untreated organoids, i.e., negative controls; and 4) organoids exposed to a non-nephrotoxic drug, such as glucose or saline solution, i.e., negative controls.

[0317] After drug exposure to the defined time, the inventors would wash the organoids with PBS, and culture the organoids in a drug-free culture medium for three hours. After that time, the inventors would proceed to organoid function and / or structure analysis. Parameters evaluated by the inventors would include cell survival and biomarker expression, using methods including, but not limited to, LIVE / DEAD assays, MTT, Alamar Blue, caspase activation, immunostaining, qRT-PCR, and ELISA. All parameters selected for analysis after drug exposure would also be acquired before drug exposure to allow for comparison.

[0318] Example 9 - Use of the kidney organoid in an assay to assess water regulation mechanisms

[0319] Water regulation within the kidney depends on the translocation of the AQP-2 protein from a primarily cytosolic localisation to the apical membrane of the principal cells of collecting ducts. Accordingly, the inventors, using the kidney organoids, measured the relative distance of the AQP-2 protein from the apical membrane using confocal microscopy in combination with AQP-2 and Na / K / ATPase immunostaining. The inventors then depleted the kidney organoid culture media of arginine vasopressin (AVP) and aldosterone for a 24-hour washout period, before incubating the organoids with AVP for a duration of 3 hours, and fixing the organoids using paraformaldehyde (PFA). The inventors then remeasured the relative distance of the protein from the apical membrane, and found that the AQP2 protein had migrated from its predominantly cytosolic localisation to the apical membrane, thus suggesting that the organoids comprise the ability to regulate water permeability, and therefore comprises a water regulation mechanism.

[0320] Example 10 - Inducing acute kidney injury in the kidney organoid

[0321] As discussed, the kidney organoids of the invention can be used to study diseases of the kidney, such as acute kidney injury (AKI).

[0322] To induce AKI in the kidney organoids, day 30 organoids were exposed to various different drugs for a period of 48 hours, as indicated in Table 4.

[0323] Table 4 - Drugs used for evaluation of acute kidney injury in human kidney organoids and respective concentration

[0324] Referring to Figure 10, cell viability and metabolic activity were assessed using the alamarBlue assay, which measures cellular respiration as an indicator of survival. Complementary to this, LIVE / DEAD staining was conducted to differentiate live cells from dead cells based on membrane integrity and esterase activity, as shown in Figure 11, providing a gualitative and guantitative measure of cytotoxicity.

[0325] Assessment of cell viability using the alamarBlue assay revealed a significant decrease in survival in samples treated with gentamicin, acetaminophen, and ibuprofen compared to control conditions, where organoids were not treated with vehicle. LIVE / DEAD staining further confirmed these findings, showing localised regions of cell death around the periphery of the organoids, with particularly pronounced cytotoxicity observed in the acetaminophen-treated sample. The inventors expect that the regions of cell death may be particularly localised in the area of the proximal tubules. Renal injury was further characterised by immunostaining for Kidney Injury Molecule-1 (KIM-1), a well-established biomarker of tubular epithelial damage. The expression levels and localization of KIM-1 in the cultured cells were analysed using fluorescence microscopy to assess the extent of cellular injury, as shown in Figure 12.

[0326] Referring to Figure 12, immunofluorescence analysis of KIM-1 expression revealed the highest levels of positive staining in samples treated with cisplatin, gentamicin, and acetaminophen, indicating significant tubular epithelial injury. To further characterise the affected cell populations, the inventors envisage performing additional staining using proximal tubule-specific markers.

[0327] To complement the cellular analyses, secreted biomarkers were quantified in the culture supernatant. Referring to Figure 13, enzyme-linked immunosorbent assays (ELISA) were performed to measure the concentrations of KIM-1 and creatinine, reflecting injury- induced protein release and kidney function impairment, respectively. These combined approaches allowed for a comprehensive assessment of drug-induced nephrotoxicity at both the cellular and molecular levels.

[0328] ELISA analysis showed elevated KIM-1 levels in samples treated with cisplatin, thalidomide, ibuprofen, and DMSO compared to control, where organoids were not .treated with vehicle. Creatinine measurements in the DF6 cell line indicated higher concentrations following ibuprofen and acetaminophen treatment relative to control. In the GEpi cell line, creatinine levels were also higher in samples treated with acetaminophen and DMSO.

[0329] Example 11 - Optimisation of CHIR99021 concentration and incubation duration

[0330] As discussed, the methods of producing the kidney organoid the invention may comprise inducing the AIM progenitor cells or the PIM progenitor cells by contacting the cells with CHIR99021, optionally along with another compound, such as Activin A and / or FGF-9.

[0331] CHIR99021 concentration optimisation has not been previously explored for kidney differentiation. However, CHIR99021 concentrations have been tested in other differentiation protocols, such as for cardiac mesoderm induction.

[0332] Referring to Figure 14, there is shown relative expression profiles of mesodermal and renal markers during renal differentiation of hiPSCs upon exposure to CHIR99021 for 96 hours. Cells were exposed to 10 pM of CHIR99021 and 100 nM of Dorsomorphin, or 11 pM of CHIR.99021 for 96 hours.

[0333] Up to day 9 of the differentiation protocol, both conditions (10 pM CHIR99021 + Dorsomorphin and 11 pM CHIR99021) showed increased expression of renal lineage markers. However, the 11 pM CHIR99021 condition consistently resulted in higher expression levels for most assessed genes, including PAX2, GATA3, WT1, and HOXD11. These markers are associated with intermediate mesoderm (IM), metanephric mesenchyme (MM), and nephron progenitor cells (NPCs), indicating enhanced lineage specification under higher CHIR99021 concentration.

[0334] These results suggest that 11 pM CHIR99021 promotes stronger and / or more advanced differentiation toward renal lineages by day 9, particularly for posterior IM and MM markers.

[0335] Referring to Figure 15, the inventors have also demonstrated that aggregates exposed to CHIR99021 for 72 hours, followed by 72 hours of Activin and 48 hours of FGF9 treatment (day 8), exhibited generally lower relative expression levels of key renal lineage genes compared to cells exposed to 96 hours of CHIR99021 (day 9). Nevertheless, even with 72 hours of CHIR99021 treatment, aggregates treated with 8 pM CHIR showed reduced expression of WT1, PAX2, and SIX2 relative to the other experimental groups, further emphasising the importance of the optimised concentrations (i.e., 11 pM CHIR) used in the methods disclosed herein.

[0336] Example 12 - Vascularisation of kidney organoids

[0337] As discussed, the inventors envisage the kidney organoids of the invention as being vascularised in some embodiments.

[0338] Vascularised organoid models are advantageous as they more accurately mimic the complex tissue architecture and microenvironment of native organs, including essential blood vessel networks. This improved physiological relevance enhances nutrient and oxygen delivery, waste removal, and cellular signalling, leading to more predictive and functional in vitro systems. In the context of the kidney organoids of the invention, vascularisation is especially critical for replicating glomerular filtration and tubular function, thereby advancing disease modelling, drug testing, and regenerative medicine applications. Indeed, the inventors have surprisingly developed a preliminary model approach to generate a vascularised kidney organoid.

[0339] Referring to Figure 16, there is shown schematics of PIM, AIM, and endothelial differentiation prior and after assembly. In parallel with AIM and PIM differentiation, human induced pluripotent stem cells (hiPSC) were seeded into AggreWell400 plates at a density of 3.5xl05cells per well to generate hiPSC aggregates containing 50 cells each.

[0340] Following cell seeding, cells were kept at 37°C in the incubator for 24 hours in mTeSRPIus supplemented with 10 pM Y-27632 for self-assembly of hiPSCs into 3D aggregates. After 1 day, the medium was replaced by mesoderm induction medium, thus starting the endothelial differentiation process (this day was defined as day 0 of differentiation). The mesoderm induction medium consisted of N2B27 medium supplemented with 11 pM CHIR-99021 and 25 ng / mL BMP4. After 3 days, the entire medium was changed to EC induction medium, composed of N2B27 medium supplemented with 100 ng / mL VEGF and 2 pM forskolin.

[0341] Referring to Figure 17, there is shown the assembly of AIM, PIM, and Endothelial (Endo) spheroids. Indeed, on day 6 of differentiation, endothelial spheroids were assembled with AIM and PIM spheroids (i.e., aggregates). Through maturation until day 30, the same medium previously described for organoid maturation was employed, further supplemented with 50 ng / mL VEGF. Referring to Figure 17D, immunofluorescence staining (CD31) of day 30 kidney organoids cryosections indicates the presence of endothelial cells.

[0342] Summary

[0343] The inventors have demonstrated a methodology that enables the consistent and reproducible production of kidney organoids which replicate the structural and functional features of the kidney. The inventors' novel and innovate methodology relies on the initial and separate derivation of the posterior intermediate mesoderm (PIM) and anterior intermediate mesoderm (AIM) using size-controlled human induced pluripotent stem cells (hiPSC). The inventors have discovered that the induction of specific AIM and PIM progenitor cells as size-controlled aggregates, specifically by diameter, and the contacting of these two populations, results in the production of kidney organoids that are capable of selective uptake of molecules, show responsiveness to injury, and comprise water regulation mechanisms. The inventors have also discovered that the kidney organoids of the invention may be vascularised, by assembling AIM and PIM aggregates together with an endothelial spheroid. As such, the present invention provides an improved platform that more accurately resembles kidney function over existing technologies. This novel platform could be used in combination with other assays to provide a new and improved means of studying genetic diseases, such as polycystic kidney disease. The inventors also envisage the use of their platform for assessing drug-induced renal injury during preclinical trials, thereby providing a more effective, robust, and animal-friendly alternative to the current preclinical tests. The establishment of this cutting-edge platform promises to revolutionise drug development, paving the way for safer and more efficacious treatments in the future.

[0344] References

[0345] Forbes, T. A., Howden, S. E., Lawlor, K., Phipson, B., Maksimovic, J., Hale, L., Wilson, S., Quinlan, C., Ho, G., Holman, K., Bennetts, B., Crawford, J., Trnka, P., Oshiack, A., Patel, C., Mallett, A., Simons, C., & Little, M. H. (2018). Patient-IPSC-Derived Kidney Organoids Show Functional Validation of a Ciliopathic Renal Phenotype and Reveal Underlying Pathogenetic Mechanisms. The American Journal of Human Genetics, 102(5), 816-831. https: / / doi.Org / 10.1016 / j.ajhg.2018.03.014

[0346] Lawlor, K. T., Vanslambrouck, J. M., Higgins, J. W., Chambon, A., Bishard, K., Arndt, D., Er, P. X., Wilson, S. B., Howden, S. E., Tan, K. S., Li, F., Hale, L. J., Shepherd, B., Pentoney, S., Presnell, S. C., Chen, A. E., 8i Little, M. H. (2021). Cellular extrusion bioprinting improves kidney organoid reproducibility and conformation. Nature Materials, 20(2), 260-271. https: / / doi.org / 10.1038 / s41563-020-00853-9

[0347] Low, J. H., Li, P., Chew, E. G. Y., Zhou, B., Suzuki, K., Zhang, T., Lian, M. M., Liu, M., Aizawa, E., Rodriguez Esteban, C., Yong, K. S. M., Chen, Q., Campistol, J. M., Fang, M., Khor, C. C., Foo, J. N., Izpisua Belmonte, J. C., 8i Xia, Y. (2019). Generation of Human PSC-Derived Kidney Organoids with Patterned Nephron Segments and a <em>De Novo< / em> Vascular Network. Cell Stem Cell, 25(3), 373-387. e9. https: / / doi.Org / 10.1016 / j.stem.2019.06.009

[0348] Mae, S.-I., Ryosaka, M., Sakamoto, S., Matsuse, K., Nozaki, A., Igami, M., Kabai, R., Watanabe, A., 8i Osafune, K. (2020). Expansion of Human iPSC-Derived Ureteric Bud Organoids with Repeated Branching Potential. Cell Reports, 32(4). https: / / doi.Org / 10.1016 / j.celrep.2020.107963

[0349] Mae, S.-I., Ryosaka, M., Toyoda, T., Matsuse, K., Oshima, Y., Tsujimoto, H., Okumura, S., Shibasaki, A., 8i Osafune, K. (2018). Generation of branching ureteric bud tissues from human pluripotent stem cells. Biochemical and Biophysical Research Communications, 495(1), 954-961. https: / / doi.Org / https: / / doi.org / 10.1016 / j.bbrc.2017.ll.105

[0350] Morizane, R., 8i Bonventre, J. V. (2017). Generation of nephron progenitor cells and kidney organoids from human pluripotent stem cells. Nature Protocols, 12(1), 195-207. https: / / doi.org / 10.1038 / nprot.2016.170

[0351] Morizane, R., Lam, A. Q., Freedman, B. S., Kishi, S., Valerius, M. T., 8i Bonventre, J. V. (2015). Nephron organoids derived from human pluripotent stem cells model kidney development and injury. Nature Biotechnology 2015 33: 11, 33(11), 1193-1200. https: / / doi.org / 10.1038 / nbt.3392

[0352] Taguchi, A., Kaku, Y., Ohmori, T., Sharmin, S., Ogawa, M., Sasaki, H., 8i Nishinakamura, R. (2014). Redefining the in vivo origin of metanephric nephron progenitors enables generation of complex kidney structures from pluripotent stem cells. Cell Stem Cell, 14(1), 53-67. https: / / doi.Org / 10.1016 / j.stem.2013.ll.010

[0353] Taguchi, A., 8i Nishinakamura, R. (2017). Higher-Order Kidney Organogenesis from Pluripotent Stem Cells. Cell Stem Cell, 21(6), 730-746. e6. https: / / doi.Org / 10.1016 / j.stem.2017.10.011 Takasato, M., Er, P. X., Becroft, M., Vanslambrouck, J. M., Stanley, E. G., Elefanty, A. G., & Little, M. H. (2013). Directing human embryonic stem cell differentiation towards a renal lineage generates a selforganizing kidney. Nature Cell Biology 2013 16: 1, 16(1), 118-126. https: / / doi.org / 10.1038 / ncb2894

[0354] Takasato, M., Er, P. X., Chiu, H. S., Maier, B., Baillie, G. J., Ferguson, C., Parton, R. G., Wolvetang, E. J., Roost, M. S., Chuva de Sousa Lopes, S. M., 8i Little, M. H. (2015). Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis. Nature, 526(7574), 564-568. https: / / doi.org / 10.1038 / naturel5695

[0355] Tsujimoto, H., Kasahara, T., Sueta, S., Araoka, T., Sakamoto, S., Okada, C., Mae, S., Nakajima, T., Okamoto, N., Taura, D., Nasu, M., Shimizu, T., Ryosaka, M., Li, Z., Sone, M., Ikeya, M., Watanabe, A., 8i Osafune, K. (2020). A Modular Differentiation System Maps Multiple Human Kidney Lineages from Pluripotent Stem Cells. Cell Reports, 31(1). https: / / doi.Org / 10.1016 / j.celrep.2020.03.040

[0356] Uchimura, K., Wu, H., Yoshimura, Y., 8i Humphreys, B. D. (2020). Human Pluripotent Stem Cell-Derived Kidney Organoids with Improved Collecting Duct Maturation and Injury Modeling. Cell Reports, 33(11), 108514. https: / / doi.Org / 10.1016 / j.celrep.2020.108514

[0357] Zeng, Z., Huang, B., Parvez, R. K., Li, Y., Chen, J., Vonk, A. C., Thornton, M. E., Patel, T., Rutledge, E. A., Kim, A. D., Yu, J., Grubbs, B. H., McMahon, J. A., Pastor-Soler, N. M., Hallows, K. R., McMahon, A. P., 8i Li, Z. (2021). Generation of patterned kidney organoids that recapitulate the adult kidney collecting duct system from expandable ureteric bud progenitors. Nature Communications, 12(1), 3641. https: / / doi.org / 10.1038 / s41467-021-23911-5

Claims

Claims1. A 3D-differentiated kidney organoid comprising at least one anterior intermediate mesoderm (AIM) progenitor cell and at least one posterior intermediate mesoderm (PIM) progenitor cell.

2. The kidney organoid according to claim 1, wherein the kidney organoid comprises three dimensions comprising height, width, and depth, each being more than one cell thick.

3. The kidney organoid according to either claim 1 or claim 2, wherein the kidney organoid comprises at least one distal convoluted tubule, at least one proximal convoluted tubule, and / or at least one collecting duct.

4. The kidney organoid according to any preceding claim, wherein the kidney organoid comprises a functional feature of a kidney, optionally wherein the functional feature of a kidney is selected from a list of functional features, including : blood filtering, blood pressure regulation, acid-base regulation, toxin removal, red blood cell production, activation of vitamin D, water regulation, electrolyte regulation, and / or hormone production.

5. The kidney organoid according to any preceding claim, wherein the kidney organoid comprises a water regulation mechanism, the ability to selectively uptake a molecule, and / or a response to injury.

6. The kidney organoid according to any preceding claim, wherein the at least one anterior intermediate mesoderm (AIM) progenitor cell and / or the at least one posterior intermediate mesoderm (PIM) progenitor cell are derived from at least one animal cell, optionally wherein the animal cell is a mammal cell.

7. The kidney organoid according to any preceding claim, wherein the at least one anterior intermediate mesoderm (AIM) progenitor cell and / or the at least one posterior intermediate mesoderm (PIM) progenitor cell are derived from at least one stem cell, optionally wherein the stem cell is a pluripotent stem cell, or an induced pluripotent stem cell (iPSC).

8. The kidney organoid according to any preceding claim, wherein the cell from which the at least one anterior intermediate mesoderm (AIM) progenitor cell and / or the atleast one posterior intermediate mesoderm (PIM) progenitor cell is derived is a diseasespecific cell and / or genetically modified.

9. The kidney organoid according to any preceding claim, wherein the kidney organoid comprises one or more cell type selected from a list of cell types, including : tubular epithelial cells, parietal epithelial cells, tubule epithelial cells, mesangial cells, podocytes, glomerular endothelial cells, juxtaglomerular cells, macula densa cells, principal cells, intercalated cells, epithelial progenitor cells, pericytes, Loop of Henle cells, thin descending limb cells, thin ascending limb cells, thick ascending limb cells, collecting duct cells, fibroblasts, smooth muscle cells, and renal interstitial cells.

10. A method of producing a kidney organoid, the method comprising contacting at least one anterior intermediate mesoderm (AIM) progenitor cell, or hiPSC-AIM, and at least one posterior intermediate mesoderm (PIM) progenitor cell, or hiPSC-PIM.

11. The method according to claim 10, wherein the method comprises the assembly of the at least one anterior intermediate mesoderm (AIM) progenitor cell, or hiPSC-AIM, and the at least one posterior intermediate mesoderm (PIM) progenitor cell, or hiPSC- PIM.

12. The method according to either claim 10 or claim 11, wherein:(i) the at least one anterior intermediate mesoderm (AIM) progenitor cell, or hiPSC-AIM, and the at least one posterior intermediate mesoderm (PIM) progenitor cell, or hiPSC-PIM, are contacted at a ratio of between 1 : 1 and 1: 100; or(ii) the at least one posterior intermediate mesoderm (PIM) progenitor cell, or hiPSC-PIM, and the at least one anterior intermediate mesoderm (AIM) progenitor cell, or hiPSC-AIM, are contacted at a ratio of between 1 : 1 and 1 : 100.

13. The method according to any one of claims 10 to 12, wherein the at least one at least one anterior intermediate mesoderm (AIM) progenitor cell, or hiPSC-AIM, and the at least one posterior intermediate mesoderm (PIM) progenitor cell, or hiPSC-PIM, are contacted to thereby produce at least one kidney organoid in a three-dimensional environment.

14. The method according to any one of claims 10 to 13, wherein the at least one at least one anterior intermediate mesoderm (AIM) progenitor cell, or hiPSC-AIM, and the at least one posterior intermediate mesoderm (PIM) progenitor cell, or hiPSC-PIM, are contacted and permitted to grow and / or move in three dimensions to thereby produce atleast one kidney organoid in a three-dimensional environment, optionally wherein the three dimensions are up / down, left / right, and forwards / backwards.

15. The method according to any one of claims 10 to 14, wherein the at least one anterior intermediate mesoderm (AIM) progenitor cell, or hiPSC-AIM, and the at least one posterior intermediate mesoderm (PIM) progenitor cell, or hiPSC-PIM, comprise aggregates.

16. The method according to claim 15, wherein the at least one anterior intermediate mesoderm (AIM) progenitor cell, or hiPSC-AIM, and the at least one posterior intermediate mesoderm (PIM) progenitor cell, or hiPSC-PIM, are size-controlled.

17. The method according to claim 16, wherein the at least one anterior intermediate mesoderm (AIM) progenitor cell, or hiPSC-AIM, and the at least one posterior intermediate mesoderm (PIM) progenitor cell, or hiPSC-PIM, are size-controlled by initial aggregate cell number and / or aggregate diameter.

18. The method according to either claim 15 or 17, wherein:(i) the initial aggregate of anterior intermediate mesoderm (AIM) progenitor cells, or hiPSC-AIMs, comprises a diameter of between 1 and 1,000 pm; and / or(ii) the initial aggregate of posterior intermediate mesoderm (PIM) progenitor cells, or hiPSC-PIMs, comprises a diameter of between 1 and 1,000 pm.

19. The method according to any one of claims 15, 17, or 18, wherein the initial AIM progenitor cell, or hiPSC-AIM, aggregate comprises a smaller diameter than the initial PIM progenitor cell, or hiPSC-PIM, aggregate.

20. The method according to any one of claims 10 to 19, wherein the method comprises inducing the AIM progenitor cells, or hiPSC-AIMs, by contacting the cells with CHIR and / or Activin A.

21. The method according to claim 20, wherein the method comprises the induction of the AIM progenitor cells, or hiPSC-AIMs, using 2 days induction with 11 pM CHIR and 3 days with 10 ng / mL Activin A.

22. The method according to any one of claims 10 to 19, wherein the method comprises inducing the PIM progenitor cells, or hiPSC-PIMs, by contacting the cells with CHIR, Activin A, and / or FGF-9.

23. The method according to claim 22, wherein the method comprises the induction of the PIM progenitor cells, or hiPSC-PIMs, using 4 days induction with 11 pM CHIR, 3 days with 10 ng / mL Activin A, and 2 days with 10 ng / mL FGF-9.

24. A kidney organoid obtained, or obtainable by, the method according to any one of claims 10-23.

25. Use of the kidney organoid according to any one of claims 1 to 9 or claim 24, in a drug evaluation screen.

26. A method of carrying out a drug evaluation screen, the method comprising contacting the kidney organoid according to any one of claims 1 to 9 or claim 24, and analysing the effects of the test agent on the kidney organoid.

27. Use of the kidney organoid according to claim 25, wherein the kidney organoid is used for drug evaluation, drug toxicity analysis, pharmacokinetics analysis, pharmacodynamics analysis, functional analysis, and / or nephrotoxicity analysis.

28. Use of the kidney organoid according to either claim 25 or claim 27, wherein the kidney organoid is used during the drug research and discovery stage of drug development, during the preclinical stage of drug development, during the clinical stage of drug development, during the drug review stage of drug development, and / or during the post-market drug safety monitoring stage of drug development.

29. Use of the kidney organoid according to any one of claims 25, 27 or 28, wherein the kidney organoid is used for assessing drug mechanism of action, adverse effects, and / or drug-induced renal injury.

30. Use of the kidney organoid according to any one of claims 25, or 27-29, wherein the kidney organoid is used in an in vitro and / or ex vivo assay.

31. A method of analysing the nephrotoxicity of a test agent, the method comprising the steps of:(i) contacting, in vitro or ex vivo, the kidney organoid according to any one of claims 1 to 9 or claim 24 with a test agent; and(ii) analysing the kidney organoid to detect any damage caused by the test agent, wherein the damage, as compared to a control kidney organoid in the absence of the test agent, is an indicator of the nephrotoxicity of the test agent.

32. The method according to claim 31, wherein the kidney organoid is analysed by its function and / or structure, optionally wherein the analysis of organoid function and / or structure comprises a physical integrity analysis, structural organisation analysis, and / or functional analysis.

33. An apparatus for identifying the nephrotoxicity of a test agent, the apparatus comprising :(i) a kidney organoid according to any one of claims 1 to 9 or claim 24; and(ii) a vessel configured to permit contacting the kidney organoid and a test agent.

34. The apparatus according to claim 33, wherein the vessel is a test tube, a microtube, a well plate, a spot plate, a microplate, a T-Flask, a slide, and / or a slide cover.

35. Use of the kidney organoid according to any one of claims 1 to 9 or claim 24, to study a kidney disease.

36. Use of the kidney organoid according to claim 35, wherein: i) the kidney organoid is contacted with an agent to induce a disease state, optionally wherein the agent comprises a nephrotoxic drug; and / or ii) the kidney organoid is genetically modified and / or genome edited, optionally wherein the kidney organoid is genome edited for the construction of a disease model.

37. A method of carrying out a kidney disease study, the method comprising contacting the kidney organoid according to any one of claims 1 to 9 or claim 24 with a test agent, and analysing the effects of the kidney organoid in a kidney disease.

38. The method according to claim 37, wherein the kidney organoid is used in an in vitro and / or ex vivo assay in the study of a kidney disease, optionally wherein the disease is selected from a list of diseases, including : kidney disease, chronic kidney disease, acquired cystic kidney disease, amyloidosis, diabetes insipidus, ectopic kidney, glomerular disease, anti-GBM (Goodpasture's) disease, IgA nephropathy, IgA vasculitis, kidney infection (pyelonephritis), kidney stones, lupus nephritis, medullary spongekidney, multicystic dysplastic kidney, nephrotic syndrome, polycystic kidney disease (PKD), renal artery stenosis, renal tubular acidosis, kidney cysts, Alport syndrome, cystinosis, Fabry disease, Gitelman syndrome, tuberous sclerosis complex, nephronophthisis, Hypophosphatemic ricketts, Ba rtter syndrome types 1-4, Gitelman syndrome, Hypomagnesemia, Liddle syndrome, Gordon syndrome (PHA type 2), Pseudohypoaldosteronism type 1, SeSAME syndrome, Diabetes insipidus, Lowe syndrome, and / or Hemolytic uremic syndrome.

39. The method according to either claim 37 or claim 38, wherein the kidney organoid is used in an in vitro and / or ex vivo assay in the study of a genetic disease, optionally wherein the genetic disease is selected from a list of genetic diseases, including : polycystic kidney disease, Alport syndrome, cystinosis, Fabry disease, Gitelman syndrome, tuberous sclerosis complex, nephronophthisis, steroid resistant nephrotic syndrome, Denys-Drash syndrome, Frasier syndrome, Nail-Patella syndrome, Schimke immuno-osseous dystrophy, Renal hypodysplasia, Multicystic renal dysplasia, HDR syndrome, Kallman syndrome, Townes-Brocks syndrome, Medullary cystic kidney disease, Meckel-Gruber syndrome, Bardet-Biedl syndrome types 1-12, von-Hippel- Lindau disease, and / or Wilms-tumor-aniridia syndrome.

40. The method according to any one of claims 25, 31-34, or 37-39, wherein the test agent is a drug or pharmaceutical, optionally wherein the test agent is selected from a list of therapeutic classes including : analgesic, antibiotic, anticancer, anticoagulant, antidepressant, antidiabetic, antiepileptic, antipsychotic, antispasmodic, antiviral, depressant, sedative, and stimulant.

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