Improved kidney organoid culture method by modulating pka

Abstract

The invention describes an improved method to culture a kidney-like structure such as a kidney organoid. In particular the method describes culturing cells during the differentiation process in the presence of a PKA inhibitor or a PKA activator. The invention further relates to kidney-like structures or organoids obtained by the method and uses thereof.

Description

[0001] Title: Improved kidney organoid culture method by modulating PKA.

[0002] Field of the invention

[0003] The invention describes a method for culturing kidney organoids using exposure to a PKA activator and / or a PKA inhibitor. The method results in improved kidney morphology in the resulting organoids, in particular the organoids have reduced cartilage induction and deterioration of nephron structures at later stages. The invention further relates to organoids obtained by the method and uses thereof.

[0004] Background of the invention

[0005] Chronic kidney disease (CKD) is characterized by a gradual loss of kidney function and is linked to a diminished glomerular filtration rate. Its prevalence varies from 3.3 to 17.3% and has risen in the last decades. Currently, kidney replacement is the most reliable therapeutic option for CKD. Worldwide, 9.7 million people need kidney replacement therapy, but due the shortage of donor organs and healthcare costs, only 2.6 million will receive it. The numbers of kidney transplants needed are expected to double within the next 10 years, increasing the pressure to find alternative solutions. Recent developments to generate organoids in vitro have opened the possibility for a regenerative medicine-based approach with the potential to provide a functional substitute to the failing kidney. Indeed, using the knowledge of developmental biology, human induced pluripotent stem cells (hiPSCs) are widely used to produce organoids mimicking various organ systems, including kidney organoids. Organoids present advantages compared to 2D-culture as they are isolated multicellular systems. This 3D-culture model is more physiologic and allows the study of interactions between different cell types. Indeed, kidney organoids can recapitulate renal structures as well as the cellular complexity of human kidney, and have demonstrated the potential to restore glomerular filtration upon transplantation. Moreover, it was shown that renin production is responsive to regulation by parathyroid hormone in iPSC-derived kidney organoids under cyclic AMP stimulation, reflecting their physiological functionality in the endocrine system.

[0006] This physiologic functionality in vitro demonstrates that kidney organoids might be a good model to study kidney diseases as well as a good therapeutic approach to treat kidney deficiency. Indeed, kidney diseases commonly involve interactions between different cell types, and the multicellular aspect of kidney organoids allows their use as in vitro kidney disease models. Actually, kidney organoids are already used to model diseases such as ciliopathies or polycystic kidney disease, and to study the mechanisms of renal pathologies as well as potential treatments. More than modelling diseases, the physiologic potential of kidney organoids makes them good candidates for transplant-based therapeutic approaches.

[0007] Despite their great therapeutic potential, kidney organoids also present several drawbacks. A major example of such drawbacks is that kidney organoids produced according to existing protocols are not stable. After a certain time point, cartilage starts forming and structural components such as tubules, collecting ducts and nephron structures in the organoid start to deteriorate. For both medical applications and drug screening purposes it is highly desirable to maintain the kidney organoid structure for a prolonged time or even to further mature the structure to a structure more closely resembling a kidney. Therefore improved methods are needed to fill this need. This need is met by the methods, products and uses described in the appended claims.

[0008] Brief description of the figures

[0009] Fig. 1 depicts an overview of a reference protocol (R) and two embodiments according to the invention where a PKA inhibitor (I) or a PKA activator (A) are used to differentiate providing a cell population comprising ureteric epithelium cells and metanephric mesenchyme cells to a kidney-like structure. The grey arrows in the reference protocol (R) indicate the 2D culturing phase and the 3D culturing phase of the protocol.

[0010] Fig. 2 depicts an overview of the same reference protocol (R) as used in Fig. 1 for comparison and a third embodiment of the invention where both a PKA inhibitor and a PKA activator (l+A) are used to differentiate providing a cell population comprising ureteric epithelium cells and metanephric mesenchyme cells to a kidney-like structure. Fig. 3 depicts immunohistochemistry staining of a kidney-like structure grown according to the reference protocol. The top left depicts the blue channel showing staining of Dapi (cell nuclei), the top right panel depicts the red channel showing staining with LTL (lotus tetragonolobus lectin), the bottom left panel depicts the green panel showing staining with a NPHS1 antibody. The bottom right panel depicts a merger of all three color channels. Fig. 4 depicts immunohistochemistry staining of a kidney-like structure grown according to the “I” protocol where only a PKA inhibitor is used during differentiation of the cell population comprising ureteric epithelium cells and metanephric mesenchyme cells. The top left depicts the blue channel showing staining of Dapi (cell nuclei), the top right panel depicts the red channel showing staining with an LTL (lotus tetragonolobus lectin), the bottom left panel depicts the green panel showing staining with a NPHS1 antibody. The bottom right panel depicts a merger of all three color channels.

[0011] Fig. 5 depicts immunohistochemistry staining of a kidney-like structure grown according to the “A” protocol where only a PKA activator is used during differentiation of the cell population comprising ureteric epithelium cells and metanephric mesenchyme cells. The top left depicts the blue channel showing staining of Dapi (cell nuclei), the top right panel depicts the red channel showing staining with an LTL (lotus tetragonolobus lectin), the bottom left panel depicts the green panel showing staining with a NPHS1 antibody. The bottom right panel depicts a merger of all three color channels.

[0012] Fig. 6 depicts immunohistochemistry staining of a kidney-like structure grown according to the “l+A” protocol where both a PKA inhibitor and a PKA activator are used during differentiation of the cell population comprising ureteric epithelium cells and metanephric mesenchyme cells. The top left depicts the blue channel showing staining of Dapi (cell nuclei), the top right panel depicts the red channel showing staining with an LTL (lotus tetragonolobus lectin), the bottom left panel depicts the green panel showing staining with a NPHS1 antibody. The bottom right panel depicts a merger of all three color channels. of the invention

[0013] In a first aspect the invention relates to a method for producing a kidney-like structure, the method comprising: providing a cell population comprising ureteric epithelium cells and metanephric mesenchyme cells; culturing the cell population under conditions that allow the formation of a three dimensional kidney-like structure; wherein the provided cell population is a population of cell aggregates showing early nephrogenesis, and wherein the cell population is cultured in the presence of a PKA inhibitor and / or a PKA activator. In a second aspect the invention relates to a kidney-like structure or kidney organoid obtained or obtainable by the method according to the first aspect of the invention.

[0014] In a third aspect the invention relates to a kidney-like structure or kidney organoid according to the second aspect of the invention for use in the treatment of a kidney disease, preferably wherein the kidney disease is chronic kidney disease.

[0015] In a fourth aspect the invention relates to the in vitro or ex vivo use of a kidney-like structure or kidney organoid according to claim 11 or 12 for one or more of:

[0016] - target identification for a drug;

[0017] - target validating for a drug;

[0018] - performing safety studies for a drug;

[0019] - performing pharmacodynamics studies for a drug;

[0020] - drug development;

[0021] - drug discovery;

[0022] - performing stratification studies for a drug;

[0023] - predicting an individual patient’s response to a drug;

[0024] - modelling a disease;

[0025] - modelling development; or

[0026] - studying kidney biology.

[0027] Definitions

[0028] A portion of this disclosure contains material that is subject to copyright protection (such as, but not limited to, diagrams, device photographs, or any other aspects of this submission for which copyright protection is or may be available in any jurisdiction.). The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent Office patent file or records, but otherwise reserves all copyright rights whatsoever.

[0029] Various terms relating to the methods, compositions, uses and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention pertains, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. “A,” “an,” and “the”: these singular form terms include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.

[0030] “About” and “approximately": these terms, when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1 %, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0031] As used herein, the term "at least" a particular value means that particular value or more. For example, "at least 2" is understood to be the same as "2 or more" i.e. , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, ... , etc. As used herein, the term "at most " a particular value means that particular value or less. For example, "at most 5 " is understood to be the same as "5 or less" i.e., 5, 4, 3, ... .-10, -11 , etc.

[0032] "Agonist": This term, as used herein refers to a compound or agent having the ability to initiate or enhance a biological function of a target protein or polypeptide, such as increasing the activity or expression of the target protein or polypeptide. Accordingly, the term "agonist" is defined in the context of the biological role of the target protein or polypeptide. While some agonists herein specifically interact with (e.g., bind to) the target, compounds and / or agents that initiate or enhance a biological activity of the target protein or polypeptide by interacting with other members of the signal transduction pathway of which the target polypeptide is a member are also specifically included within this definition.

[0033] "Antagonist" and "inhibitor": These terms are used interchangeably, and they refer to a compound or agent having the ability to reduce or inhibit a biological function of a target protein or polypeptide, such as by reducing or inhibiting the activity or expression of the target protein or polypeptide. Accordingly, the terms "antagonist" and "inhibitor" are defined in the context of the biological role of the target protein or polypeptide. An inhibitor need not completely abrogate the biological function of a target protein or polypeptide, and in some embodiments reduces the activity by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. While some antagonists herein specifically interact with (e.g., bind to) the target, compounds that inhibit a biological activity of the target protein or polypeptide by interacting with other members of the signal transduction pathway of which the target protein or polypeptide are also specifically included within this definition. Non-limiting examples of biological activity inhibited by an antagonist include those associated with the development, growth, or spread of a tumor, or an undesired immune response as manifested in autoimmune disease.

[0034] “And / or”: The term “and / or” refers to a situation wherein one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

[0035] “Conventional techniques” or “methods known to the skilled person”: These terms refer to a situation wherein the methods of carrying out the conventional techniques used in methods of the invention will be evident to the skilled worker. The practice of conventional techniques in molecular biology, biochemistry, cell culture, genomics, sequencing, medical treatment, pharmacology and related fields are well- known to those of skill in the art and are discussed, for example, in the following literature references: Human Embryonic Stem Cell: The Practical Handbook. Publisher: John Wiley & Sons, LTD, Editors (Sullivan, S., Cowan, C. A., Eggan, K.) Harvard University, Cambridge, MA, USA (2007); Human Stem Cell, a Laboratory Guide (2ndEdition) by Peterson, S., and Loring, J. F. (2012).

[0036] “Comprising”: this term is construed as being inclusive and open ended, and not exclusive. Specifically, the term and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components.

[0037] “Differentiating” and “differentiation”: these terms, in the context of living cells, relate to progression of a cell further down the developmental pathway. A “differentiated cell” is a cell that has progressed further down the developmental pathway than the cell it is being compared with; differentiation is the process of progression. Human pluripotent stem cells can differentiate into lineage-restricted progenitor cells (cells that, like a stem cell, have a tendency to differentiate into a specific type of cell, but are already more differentiated than a stem cell and are pushed to eventually differentiate into its end-stage cell; e.g. endoderm, mesoderm and ectoderm), which in turn can differentiate into further restricted cells (e.g., cardiomyocyte progenitors, neuronal cell progenitors), which can differentiate into terminally differentiated cells (e.g., cardiomyocytes or neurons). Differentiation is controlled by the interaction of a cell's genes with the physical and chemical conditions outside the cell, usually through signaling pathways involving proteins embedded in the cell surface. In the present invention, “differentiation” is the biological process whereby an unspecialized human pluripotent stem cell (population) acquires the features of a specialized cell such as a cardiomyocyte under controlled conditions in in vitro culture.

[0038] “Embryonic stem cells”: abbreviated as ‘ES cells’ or ESC (or if of human origin ‘hES cells’ or ‘hESCs’) refers to stem cells that are derived from the inner cell mass of a blastocyst. The skilled person understands how to obtain such embryonic stem cells, for example as described by Chung (Chung et al (2008) Stem Cell Lines, Vol 2(2): 113- 117), which employs a technique that does not cause the destruction of the donor embryo(s). Various ESC lines are listed in the NIH Human Embryonic Stem Cell Registry.

[0039] "Exemplary": this terms means "serving as an example, instance, or illustration," and should not be construed as excluding other configurations disclosed herein.

[0040] "In vivo": This term refers to an event that takes place in a subject's body

[0041] "In vitro": This term refers to an event that takes places outside of a subject's body. For example, an in vitro assay encompasses any assay conducted outside of a subject. In vitro assays encompass cell-based assays in which cells, alive or dead, are employed. In vitro assays also encompass a cell-free assay in which no intact cells are employed.

[0042] “Induced pluripotent stem cell” or “iPSC”: These terms refer to pluripotent stem cells that are derived from a cell that is not a pluripotent stem cell (i.e. , from a cell this is differentiated relative to a pluripotent stem cell). When used herein the term pluripotent stem cells may refer to either naive or primed pluripotent cells. Naive pluripotent cells represent the cellular state of the preimplantation blastocyst inner cell mass, while primed pluripotent cells are representative of the post-implantation epiblast cells. Induced pluripotent stem cell can be derived from multiple different cell types, including terminally differentiated cells. Induced pluripotent stem cells generally have an ES cell-like morphology, growing as flat colonies with large nucleo- cytoplasmic ratios, defined borders and prominent nuclei. In addition, induced pluripotent stem cells may express one or more key pluripotency markers known by one of ordinary skill in the art, including but not limited to Alkaline Phosphatase, SSEA3, SSEA4, Sox2, Oct3 / 4, Nanog, TRA160, TRA181 , TDGF 1 , Dnmt3b, FoxD3, GDF3, Cyp26a1 , TERT, and zfp42. Examples of methods of generating and characterizing induced pluripotent stem cells may be found in, for example, U.S. Patent Publication Nos. US20090047263, US20090068742, US20090191159, US20090227032, US20090246875, and US20090304646. To generate induced pluripotent stem cells, somatic cells may be provided with reprogramming factors (e.g. Oct4, SOX2. KLF4, MYC, Nanog, Lin28, etc.) known in the art to reprogram the somatic cells to become pluripotent stem cells (see, for example, Takahashi et. al, Cell. 2007 Nov. 30; 131 (5):861-72; Takahashi et. al, Nat Protoc. 2007; 2(12):3081-9; Yu et. al, Science. 2007 Dec. 21 :318(5858): 1917-20. Epub 2007 Nov. 20).

[0043] “Markers” or “lineage-specific markers”: these terms refer to a characteristic specifically associated with the phenotype of cells of a lineage and can be used to assess the differentiation of cells. The terms may refer to nucleic acid or polypeptide molecules that are differentially expressed in a cell of interest. The detectable level of the marker is sufficiently higher or lower in the cells of interest compared to other cells, such that the cell of interest can be identified and distinguished from other cells using any of a variety of methods known in the art.

[0044] “Media”: This term refers to an aqueous solution, including buffers, suitable for maintaining human or animal cells for a sufficient period. The term is used interchangeably with its singular form medium. For example, a media is suitable if it allows the treatment of cells for a period required to obtain the effect intended by the treatment. The term “media” also, and preferably, includes growth media that are suitable for the in vitro cell culture of human or animal cells. A “defined media” refers to a (growth) media suitable for the in vitro cell culture of human or animal cells and in which all of the chemical components are known. Such defined media does not or essentially not comprise any ill-defined source of nutrients and / or other ill-defined factors. Within the context of the current invention the defined media used may still contain defined amounts of products such as (purified) albumin, growth factors, and hormones, but is essential free of serum (i.e. less than 1 % w / w, preferably less than 0.5% w / w. even more preferably less than 0.1 % w / w, even more preferably less than 0.05% w / w of the medium ready for use, most preferably the medium is free of serum (i.e. 0% w / w serum; albeit it might contain defined amount of specified compounds like (recombinant) albumin. Although widely used, serum has many limitations. It contains high levels of numerous and unknown proteins and compounds which interfere dramatically with the small quantities of the desired proteins produced by the cells. The presence of serum may also affect in vitro testing results with the cells obtained since some compounds may bind up to 99% to serum proteins. Another limitation is the serum batch-to-batch inconsistencies, resulting in serious regulatory concern about various serum protein contaminations in the product.

[0045] “Pluripotency": This term is generally understood by the skilled person and refers to an attribute of a (stem) cell that has the potential to differentiate into all cells constituting one or more tissues or organs, for example, any of the three germ layers: endoderm (e.g. interior stomach lining, gastrointestinal tract, the lungs), mesoderm (e.g. heart, muscle, bone, blood, urogenital tract), or ectoderm (e.g. epidermal tissues and nervous system).

[0046] “Pluripotent stem cell” or “PSC”: This is a stem cell capable of producing all cell types of the organism and can produce cells of the germ layers, e.g. endoderm, mesoderm, and ectoderm, of a mammal and encompasses at least pluripotent embryonic stem cells and induced pluripotent stem cells. Pluripotent stem cells can be obtained in different ways. Pluripotent embryonic stem cells may, for example, be obtained from the inner cell mass of an embryo. Induced pluripotent stem cells (iPSCs) may be derived from somatic cells. Pluripotent stem cells may also be in the form of an established cell line.

[0047] Times used for indicating organoid age: when used herein the age of cells or organoids is indicated in hours or days. When done so, the age refers to the time since initiating the first differentiation stage. For example, “3 day” or “day 3” refers to cells cultured for three days in the first differentiation stage (in a 2D culture environment) and “day 10” or “10 day” refers to organoids where the cumulative time culturing in the first and the second differentiation stage is 10 days (e.g. 7 days in the first differentiation stage and 3 days in the second differentiation stage). Alternatively the age can be annotated as “day A+B” or “A+B day”, here A indicates the number of days cultured in the first differentiation stage (in 2D culture) and B indicates the number of days cultured in the second differentiation stage. For example day 7+12 indicates organoids which are grown for 7 days in the first differentiation stage (2D culture) and 12 days in the second differentiation stage (3D culture environment), and thus are cultured for an accumulative of 19 days in the first and second differentiation stage together. When used herein, the term “after onset”, when for example used in the context of “from onset of the 3D culture” intends to indicate immediately after the start thereof (e.g. the 3D culture), but allows also to include shortly after onset of the intended event (e.g. 3D culture), such as 1 , 2, 3, 4, 6, 12, 24, 36 or 48 hours after initiating the referred event.

[0048] Detailed description of the invention

[0049] It is contemplated that any method, use or composition described herein can be implemented with respect to any other method, use or composition described herein. Embodiments discussed in the context of methods, use and / or compositions of the invention may be employed with respect to any other method, use or composition described herein. Thus, an embodiment pertaining to one method, use or composition may be applied to other methods, uses and compositions of the invention as well.

[0050] A much used protocol in the field of kidney organogenesis is developed in the Little lab and described in Takasato et al (Nat Protoc. 2016 Sep;11 (9):1681-92.). Briefly this protocol describes a two stage differentiation protocol where first pluripotent cells are differentiated to induce intermediate mesoderm. Thus stage is typically performed in a regular (2D) culture dish or flask, so using adherent cells. After about 2-5 days the culture is switched to an FGF9 containing medium and at day 7 switched to a 3D culture environment (like a transwell filter, second stage) to allow aggregate formation. At day 12 (day 7+5) all growth factors are withdrawn from the medium. This protocol is also schematically depicted as protocol “R” in Figures 1 and 2. This protocol, henceforward referenced to Takasato protocol, results in organoids that show a kidney-like morphology with structure resembling and expressing markers of nephrons and ducts. A major drawback of the protocol as that only few cells show specialized kidney-like morphology or marker expression, and typically the organoids start expressing cartilage markers and display morphological deterioration at day 7+18. There is thus a need for improved protocols to reducing cartilage formation and morphological deterioration.

[0051] The reason for the consistent observation of chondrocytes in kidney organoid culture and transplantation is not understood. Cartilage formation involves the condensation of mesenchyme tissue, which differentiates into chondrocytes and produces the extracellular matrix protein collagen 2 (COL2A1). The pathways leading to chondrocyte differentiation mainly involve the SOX protein family, particularly SOX9. In the study of Bantounas et al. (Stem Cell Reports 10, 766-779 (2018)), the authors used kidney progenitors to transplant mice and still observed the appearance of cartilage weeks after the graft, highlighting the presence of a potential dedifferentiation of renal structures. After injury or stress, adult kidney tissue is able to regenerate via dedifferentiation of tubes, notably through the EGFR pathway. This pathway also induces a transitory increase of SOX9 expression, particularly in proximal tubules, and can last for 2-3 days to induce healing of the injured tissue18. The transitory aspect of SOX9 expression is highly important as this nuclear factor is a key player in other processes including chondrogenesis. Indeed, during cartilage formation and maintenance, SOX9 secures the chondrocytic lineage, cell survival and regulate genes implicated in cartilage structure.

[0052] Dedifferentiation of renal structures is also linked to epithelial to mesenchymal transition (EMT). EMT is known to happen during the development of kidney organoids and is associated with expression of several markers as vimentin, alpha-smooth muscle actin (aSMA) and collagen 1a1 (COL1A1), see e.g. Rutier et al. (Adv Sci (Weinh) 9, e2200543 (2022)). During this process, epithelial cells lose their characteristics, as cell polarity and cell-cell adhesion, to a mesenchymal and ECM component — secreting cell phenotype and ability to differentiate in other cell types.

[0053] The inventors now surprisingly found that either inhibiting or activating Protein Kinase A (PKA) during the second stage of differentiation resulted in improved kidney-like morphology and stability of the organoids. Even more surprisingly, by consecutive treatment with a PKA inhibitor followed by a PKA activator the morphology and stability could be improved much further.

[0054] Therefore, in a first aspect the invention relates to a method for producing a kidneylike structure, the method comprising: providing a cell population comprising ureteric epithelium cells and metanephric mesenchyme cells; culturing the cell population under conditions that allow the formation of a three dimensional kidney-like structure; wherein the provided cell population is a population of cell aggregates showing early nephrogenesis, and wherein the cell population is cultured in the presence of a PKA inhibitor and / or a PKA activator. The invention describes an improvement of the second stage of the Takasato reference protocol, however it is understood that any other protocol could be used to provide cell aggregates comprising ureteric epithelium cells and metanephric mesenchyme cells.

[0055] Day 7 of the Takasato protocol represents the stage of intermediate mesoderm that is considered to include not only progenitors of ureteric epithelium and metanephric mesenchyme but also progenitors of renal interstitium and endothelium. A proper 3D environment allows this kidney progenitor mixture to undergo self-nephrogenesis to form a kidney organoid. The kidney progenitor aggregate includes the ureteric epithelium and the metanephric mesenchyme; hence, their reciprocal interaction spontaneously initiates nephrogenesis.

[0056] Therefore, in an embodiment the invention describes a method for producing a kidneylike structure, the method comprising providing a kidney progenitor aggregate; culturing the kidney progenitor aggregate under conditions that allow the formation of a three dimensional kidney-like structure; wherein the cell population is cultured in the presence of a PKA inhibitor and / or a PKA activator. In an embodiment the kidney progenitor aggregate includes the ureteric epithelium and the metanephric mesenchyme. In an embodiment the kidney progenitor includes progenitors of ureteric epithelium and metanephric mesenchyme preferably also progenitors of renal interstitium and endothelium.

[0057] When used herein, a kidney-like structure is a cell aggregate wherein at least part of the cells express kidney marker genes. Preferably at least part of the cells show kidney-like morphology such as but not limited to nephron-like structures and tubulelike structures. The kidney-like structure may be an organoid.

[0058] When used herein a kidney-like structure refers to a cell aggregate comprising different cell types representing different kidney structures. Preferably the kidney-like structure at least comprises tubule like structures or expresses tubule markers, and / or comprises nephron like structure or expresses nephron markers. Exemplary proximal tubule markers are LTL (lotus tetragonolobus lectin which preferentially binds to the brush borders in the proximal tubules), AQP1 , and CDH16, exemplary distal tubular markers are CDH1 , THP, and CDH16. Nephron markers include NPHS1 (the gene encoding nephrin), however other markers are known to the skilled person and for example described in Agarwal et al. (Renal Physiology, Volume 321 , Issue 6, December 2021

[0059] Pages F715-F739). When described herein, the kidney-like structures preferably express NPHS1 or can be stained with LTL (lotus tetragonolobus lectin), to demonstrate the presence of proximal tubule like structures and nephron like structures.

[0060] It is understood that the cell population comprising ureteric epithelium cells and metanephric mesenchyme cells can be obtained in multiple ways. For example, pluripotent cells can be cultured in the presence of a WNT activator such as CHIR99021 (which activates WNT by inhibiting the enzyme GSK-3) for about 4 days, followed by culturing the cells in the presence of FGF9 (optionally supplemented with heparin), pulse treating the cells with a WNT activator (such as CHIR99021) and harvesting the cells at around day 7 to transfer the cells to a 3D culture environment, as also described in Takasato et al supra.

[0061] The stepwise differentiation of pluripotent stem cells to kidney begins with the induction of the primitive streak which is the progenitor population for both endoderm and mesoderm. While the anterior primitive streak gives rise to the endoderm, the posterior primitive streak has potential to develop into the mesoderm, including the axial, paraxial, intermediate and lateral plate mesoderm. The intermediate mesoderm differentiates to the ureteric epithelium and the metanephric mesenchyme, which are two key kidney progenitor populations subsequently undergoing a reciprocal interaction to form the kidney (Takasato et al. Nat Cell Biol 16, 118-126 (2014)). When culturing kidney organoids it is attempted to recreate these developmental steps in vitro.

[0062] When used herein the term first differentiation stage refers to culturing stem cells in a 2D (monolayer) culture under conditions to allow differentiation of the stem cells to a cell culture comprising cells in the intermediate mesoderm stage. The first differentiation stage may comprise several distinct steps. Generally as a first step the stem cells are cultured in the presence of a GSK3 inhibitor or a WNT activator. Such compounds are known to the skilled person, an exemplary compound that may be used is for example CHIR99021 which is a GSK3 inhibitor. During this step induction of both the ureteric epithelium and the metanephric mesenchyme takes places in the monoculture. The ureteric epithelium is characterized by expression of the markers PAX2, GATA3 and CDH1. The metanephric mesenchyme is characterized by the expression of PAX2, Wnt4 and BMP7.

[0063] The Takasato method generates a cell population comprising ureteric epithelium cells and metanephric mesenchyme cells which generally undergoes self-nephrogenesis to form a kidney-like structure (like a kidney organoid). Other methods to obtain populations of cells comprising ureteric epithelium cells and metanephric mesenchyme cells are for example described in Morizane and Bonventre, Nat. Protoc. 2017 Jan; 12(1): 195-207, henceforth referred to as the Morizane protocol.

[0064] In a second step during the first differentiation stage, the metanephric mesenchyme is further differentiated to nephron progenitor cells, while further differentiating the ureteric epithelium. This step is performed by culturing the partially differentiated cells at least in the presence of FGF9 or an activator of FGFR1 and / or FGFR3. The nephron progenitor cells are characterized by the expression of the markers SIX2, HOXD11 , WT1 and PAX2. The ureteric epithelium is at this stage also characterized by expression of the markers PAX2, GATA3 and CDH1.

[0065] Both the Takasato and the Morizane protocols describe culturing cell aggregates in a 3D environment (second stage of the protocols) where briefly a WNT agonist is added and where the cell aggregates are treated with FGF9 for a predetermined amount of time (both protocols specify 5 days counting form the onset of the 3D cell culture). In the present invention it was found that modulating PKA activity in the cell aggregate has a positive effect on kidney-like structure formation, when compared to using a common protocol like the Takasato or the Morizane protocol.

[0066] In a first experiment the inventors added a PKA inhibitor and found that doing so induces tubule formation. The resulting kidney-like structure has more tubule like structures compared to kidney-like structures grown according to the reference (Takasato) protocol. Thus in an embodiment, the cell population is cultured in the presence of a PKA inhibitor to induce tubule formation in the cell population. The results of this experiment are shown in Figure 4.

[0067] In a second experiment the inventors added a PKA activator and found that doing so induces nephron formation. The resulting kidney-like structure has more nephron-like structures compared to kidney-like structures grown according to the reference (Takasato) protocol. Thus in an embodiment, the cell population is cultured in the presence of a PKA activator to induce nephron formation in the cell population. The results of this experiment are shown in Figure 5.

[0068] In a third experiment the inventors first added a PKA inhibitor and then added a PKA activator and found that doing so induces both tubule and nephron formation. The resulting kidney-like structure has more tubule and nephron like structures compared to kidney-like structures grown according to the reference (Takasato) protocol. Thus in an embodiment, the cell population is first cultured in the presence a PKA inhibitor to induce tubule formation in the cell population, followed by culturing the cell population with increased tubule formation in the presence of a PKA activator to induce nephron formation in the cell population. The results of this experiment are shown in Figure 6.

[0069] Protein kinase A (PKA) is a family of serine-threonine kinase whose activity is dependent on cellular levels of cyclic AMP (cAMP). PKA is also known as cAMP- dependent protein kinase and classified as EC 2.7.11.11. PKA has several functions in the cell, including regulation of glycogen, sugar, and lipid metabolism. When inactive, the PKA holoenzyme exists as a tetramer which consists of two regulatory subunits and two catalytic subunits. The catalytic subunit contains the active site, a series of canonical residues found in protein kinases that bind and hydrolyse ATP, and a domain to bind the regulatory subunit. The regulatory subunit has domains to bind to cyclic AMP, a domain that interacts with catalytic subunit, and an auto inhibitory domain. There are two major forms of regulatory subunit; Rl and RII. In humans, the following genes encode PKA subunits: catalytic subunit - PRKACA, PRKACB, PRKACG; regulatory subunit type I - PRKAR1A, PRKAR1 B; and regulatory subunit type II - PRKAR2A, PRKAR2B. Thus when used herein the term PKA refers to a protein complex comprising at least one preferably two PRKAC (e.g. PRKACA, PRKACB, or PRKACG or a combination thereof) catalytic subunits and at least one, preferably two regulatory subunits selected from PRKAR1 (PRKAR1 A or PRKAR1 B or a combination thereof) and PRKAR2 (e.g. PRKAR2A or PRKAR2B or a combination thereof).

[0070] The human versions of the different PKA subunits are encoded as follows: gene identifier

[0071] PRKACA ENSG00000072062

[0072] PRKACB ENSG00000142875 PRKACG ENSG00000165059

[0073] PRKAR1A ENSG00000108946

[0074] PRKAR1 B ENSG00000188191

[0075] PRKAR2A ENSG00000114302

[0076] PRKAR2B ENSG00000005249

[0077] PKA inhibitors are known to the skilled person. When used herein a PKA inhibitor refers to a compound or biological (such as but not limited to a peptide, protein, nucleotide or sugar oligomer or -polymer) capable of inhibiting PKA enzymatic activity. The inhibitor may work directly on the PKA enzyme complex or one of its subunits, or it may function upstream by modifying, inhibiting or activating a factor which downstream causes a reduction of PKA enzymatic activity. Non-limiting examples of PKA inhibitors include: PKI, KT5720, H89 dihydrochloride, staurosporine, fasudil hydrochloride, HA-1004, Aplithianines A, K-252a, PKItide, daphnetin, AT13148, HA- 100, CCG215022 and AT7867. More preferably the PKA inhibitor is selected from PKI, KT5720, or H89 dihydrochloride, most preferably PKI. When used herein PKI refers the muscle PKA inhibitor protein or a fragment thereof, such as but not limited to PKA inhibitor fragment 5-24, 14-22 (myristoylated), or 6-22:

[0078] 5-24 SEQ ID NO: 4 TTYADFIASGRTGRRNAIHD

[0079] 14-22 SEQ ID NO: 5 GRTGRRNAI

[0080] 6-22 SEQ ID NO: 6 TYADFIASGRTGRRNAI

[0081] The skilled person is able to determine the desired concentration to be used in the protocol. For example, PKI has a Kj of approximately 2.5 nM and the desired concentration to be used in the culture medium is between 1 and 25 pM, preferably between 2 and 20 pM more preferably between 2.5 and 10 pM, most preferably approximately 5 pM. For other inhibitors the desired concentration range can thus be deduced based on the respective Kj of the inhibitor. Thus in an embodiment, the PKA inhibitor is selected from PKI, KT5720, H89 dihydrochloride. Alternatively the PKA inhibitor is selected from the list provided above. Kj values for the different inhibitors are typically provided by the supplier. The PKA inhibitor may be provided to the cell culture starting from day 3, 4, 5, 6, or 7 of the onset of the 3D culture stage. Preferably the inhibitor is provided starting from day 4, 5, or 6 starting from the onset of the 3D culture stage. Ideally the inhibitor is provided for about a week, for example for 4, 5, 6, 7, 8, 9, or 10 days, preferably 5, 6, 7, 8, or 9 days, more preferably 6, 7, or 8 days, preferably consecutive days.

[0082] PKA activators are known to the skilled person. When used herein a PKA activator refers to a compound or biological (such as but not limited to a peptide, protein, nucleotide or sugar oligomer or -polymer) capable of activating PKA enzymatic activity. The activator may work directly on the PKA enzyme complex or one of its subunits, or it may function upstream by modifying, inhibiting or activating a factor which downstream causes an increase in PKA enzymatic activity. Examples of PKA activators are cAMP analogs, preferably the cAMP analogs are cAMP modified to be more cell permeable and / or resistant to hydrolysis. Non-limiting examples of PKA activators are CW008, DbcAMP (dibutyryl-cAMP), 8-Bromo-cAMP, 8-C PT-cAMP, Taxol, N6-Benzoyladenosine-3',5'-cyclic monophosphate or a salt thereof, preferably a sodium salt, Adenosine 3',5'-cyclic monophosphate, Belinostat, (S)-Adenosine, cyclic 3',5'-(hydrogenphosphorothioate) triethylammonium, Sp-Adenosine 3',5'-cyclic monophosphorothioate triethylammonium salt, Sp-5,6-DCI-cBiMPS, 8- Bromoadenosine 3',5'-cyclic Monophosphothioate Sp-lsomer sodium salt, Adenosine 3',5'-cyclic Monophosphorothioate, 8-Bromo-, Sp-lsomer, Sodium Salt, Sp-8-pCPT- cyclic GMPS Sodium, 8-Bromoadenosine 3',5'-cyclic monophosphate, N6- Monobutyryladenosine 3':5'-cyclic monophosphate sodium salt, 8-PIP-cAMP and Sp- cAMPS.

[0083] The skilled person is able to determine the desired concentration to be used in the protocol. For example, DbcAMP is typically used in cell culture in a concentration range of 50 pM to 1 mM, preferably 100 pM to 500 pM more preferably around 250 pM. For other activators the desired concentration range can thus be calculated by performing an assay to compare the concentration that achieves a comparable PKA activation with respect to DbcAMP in the above listed concentration ranges. In a preferred embodiment the PKA activator is selected from a cAMP analog and CW008, more preferably wherein the cAMP analog is selected from DbcAMP and 8-Bromo- cAMP. Alternatively the PKA activator is selected from the list provided above. Preferred concentration ranges may be provided by the supplier of the PKA activator. The PKA activator may be provided to the cell culture starting from day 3, 4, 5, 6, or 7 of the onset of the 3D culture stage. Preferably the activator is provided starting from day 4, 5, or 6 starting from the onset of the 3D culture stage. Ideally the activator is provided for about a week, for example for 4, 5, 6, 7, 8, 9, or 10 days, preferably 5, 6, 7, 8, or 9 days, more preferably 6, 7, or 8 days, preferably consecutive days.

[0084] In a particularly preferred embodiment the method includes providing both a PKA inhibitor and an activator. In such case it is preferred that the inhibitor and the activator are not provided simultaneously but in sequence. It is further preferred that first the PKA inhibitor is provided, followed by providing the PKA activator. In between the cells may be cultured for a few days without growth factors, although it is preferred that the activator treatment follows directly after the inhibitor treatment. When combining the activator and inhibitor treatment of the cell aggregates, it is preferred that the PKA inhibitor is provided to the cell culture starting from day 3, 4, 5, 6, or 7 of the onset of the 3D culture stage. Preferably the inhibitor is provided starting from day 4, 5, or 6 starting from the onset of the 3D culture stage. Ideally the inhibitor is provided for about a week, for example for 4, 5, 6, 7, 8, 9, or 10 days, preferably 5, 6, 7, 8, or 9 days, more preferably 6, 7, or 8 days, preferably consecutive days. The PKA inhibitor treatment is followed by the PKA activator treatment, by supplementing the medium with the PKA activator to the cell culture starting from day 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16 or 17 of the onset of the 3D culture stage. Preferably the activator is provided starting from day 9, 10, 11 , 12, 13, 14, or 15, preferably day 10, 11 , 12, 13, or 14, starting from the onset of the 3D culture stage. Ideally the inhibitor is provided for about a week, for example for 4, 5, 6, 7, 8, 9, or 10 days, preferably 5, 6, 7, 8, or 9 days, more preferably 6, 7, or 8 days, preferably consecutive days. In an embodiment the protocol is performed such that the PKA inhibitor and the PKA activator are not provided at the same time or simultaneously to the cell culture.

[0085] As described above the present invention provides an improved method to further differentiate a cell population comprising ureteric epithelium cells and metanephric mesenchyme cells to kidney-like structures, and as such corresponds to the second stage of the Takasato or the Morizane protocols.

[0086] When used herein, the cell population comprising ureteric epithelium cells and metanephric mesenchyme cells refers to a population comprising at least one cell aggregate. It is understood that when culturing the cell population the conditions may be chosen such that a single cell aggregate is present in the culture vessel or multiple cell aggregates are present. This may be achieved by selecting the number of cells that are included in the culture vessel and the size of the culture vessel. Typically the step of differentiating is performed in a 3D culture environment as defined below.

[0087] For the purpose of the invention the term 3D culture environment should be interpreted as any culture method that allows the formation of kidney-like structures from the cell population comprising ureteric epithelium cells and metanephric mesenchyme cells, provided the appropriate culture conditions are provided. Typically the culturing involves a transient period of providing FGF9 or an alternative factor that activates FGFR1 and / or FGFR3 as described herein below. For example the Takasato protocol describes a trans-well membrane system to culture cells on a liquid air interface, while the Morizane protocol describes the use of ultra-low attachment plates. Other culture methods that allow the formation of organoids are however known to the skilled person and should be construed as included under the term 3D culture environment. Nonlimiting examples are hydrogels, scaffolds or suspension culture. Therefore in an embodiment the cell population is cultured in a 3D culture environment, preferably wherein the 3D culture environment is selected from culturing on a trans-well membrane, culturing in a low attachment well, culturing in a hydrogel, culturing on an air-liquid interface, culturing on or in a scaffold, or a suspension culture.

[0088] It is understood that instead of providing a cell population comprising ureteric epithelium cells and metanephric mesenchyme cells, the method may also start from pluripotent cells or stem cells and involve a first stage wherein the pluripotent cells or stem cells are differentiated to a cell population comprising ureteric epithelium cells and metanephric mesenchyme cells. Thus in an embodiment, the method according to the invention is preceded by differentiating pluripotent cells or stem cells to a cell population comprising ureteric epithelium cells and metanephric mesenchyme cells. The skilled person is aware that different protocols are available to differentiate pluripotent cells or stem cells such as the Takasato protocol or the Morizane protocol. In an exemplary embodiment pluripotent cells are first cultured in the presence of a WNT activator, such as but not limited to CHIR99021. The cells may be cultured for example for 2, 3, 4, 5, or 6 days, preferably 3, 4, or 5 days, in the presence of the WNT activator. Exemplary concentrations when CHIR99021 is used are between 1 to 10 micromolar, for example 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 micromolar, where the concentration is preferably lower when using short treatment compared to when using pulse treatment, e.g. 5 micromolar for pulse treatment and 3 micromolar for short treatment. When a different WNT activator is used the concentration can be adapted accordingly. Typically during this step induction of both the ureteric epithelium and the metanephric mesenchyme takes place, which is indicated by cells expressing PAX2, GATA3 and CDH1 and cells expressing PAX2, Wnt4 and BMP7.

[0089] After culturing the cells in the presence of a WNT activator the WNT activator is withdrawn and the cells may be cultured in the presence of FGF9 or an activator of FGFR1 and / or FGFR3. This step may be performed for example for 2, 3, 4, or 5 days, preferably about 3 days. The FGF9 or an activator of FGFR1 and / or FGFR3 containing medium may be supplemented by heparin. Exemplary concentrations when using FGF9 in this step are between 40 and 1000 ng / pL, for example 40, 50, 60, 70, 80, 90, 100, 120, 150, 175, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, or 1000 ng / pL. When instead an activator of FGFR1 and / or FGFR3 is used the concentration can be adapted accordingly. If the medium is supplemented with heparin, the concentration used may for example be between 50 ng / mL and 10 pg / mL, e.g. 50, 60, 70, 80, 90, 100, 120, 150, 175, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 ng / mL.

[0090] In an embodiment the method of the invention is preceded or partially coincides with a step of differentiating the cell population comprising ureteric epithelium cells and metanephric mesenchyme cells under conditions that induce aggregate formation and early nephrogenesis in the cell population comprising ureteric epithelium cells and metanephric mesenchyme cells. In an embodiment the induction of aggregate formation and early nephrogenesis in the cell population comprising ureteric epithelium cells and metanephric mesenchyme cells is performed by culturing the cell population in the presence of an activator of FGFR1 and / or FGFR3, preferably wherein the activator of FGFR1 and / or FGFR3 is selected from FGF20, FGF2, or Smoothened agonist (SAG).

[0091] It is understood that a cell population comprising ureteric epithelium cells and metanephric mesenchyme cells as described herein is placed in a 3D culture environment and cultured under conditions that allow the formation of a three dimensional kidney-like structure. In principle the cell aggregates of cells comprising ureteric epithelium cells and metanephric mesenchyme cells will undergo self- organogenesis when cultured in a 3D environment as descried herein and when transiently provided with FGF9 (or an activator of FGFR1 and / or FGFR3). FGF9 or an activator of FGFR1 and / or FGFR3 is ideally provided from onset of the 3D culture stage. Preferably FGF9 or an activator of FGFR1 and / or FGFR3 is provided to the cells for 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16 days, preferably 4, 5, 6, 7, 8, 9, 10, 11 , 12, or 13 days. The Takasato protocol dictates providing FGF9 for about 5 days, however the inventors found that better results may be achieved when the period is prolonged to about 12 days, such as for example 9-15 days, preferably ID-

[0092] 14 days, more preferably 11-13 days, however for the effect of the present invention (modulating PKA activity) prolonged GF9 treatment is not essential.

[0093] Exemplary concentrations when using FGF9 in this step are between 40 and 1000 ng / pL, for example 40, 50, 60, 70, 80, 90, 100, 120, 150, 175, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, or 1000 ng / pL. When instead an activator of FGFR1 and / or FGFR3 is used the concentration can be adapted accordingly.

[0094] During the provision of FGF9 or an activator of FGFR1 and / or FGFR3 the medium may be supplemented with heparin, the concentration used may for example be between 50 ng / mL and 10 pg / mL, e.g. 50, 60, 70, 80, 90, 100, 120, 150, 175, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 ng / mL.

[0095] At the onset of the 3D culture stage, the cells may be pulse treated with a WNT activator, such as CHIR99021. The pulse treatment may for example be provided for

[0096] 15 minutes to 4 hours, preferably 30 minutes to 2 hours, more preferably about 1 hour, or for a short period such as 12 hours to 4 days, preferably 1 to 3 days, more preferably about 3 days. Exemplary concentrations when using CHIR99021 are between 1 to 10 micromolar, where the concentration is preferably lower when using short treatment compared to when using pulse treatment, e.g. 5 micromolar for pulse treatment and 3 micromolar for short treatment.

[0097] During the 3D culturing of the cell population comprising ureteric epithelium cells and metanephric mesenchyme cells, immature kidney-like structures are formed, which demonstrate renal structures and start to express renal factors such as ECAD and NPHS1. Thus in an embodiment the cells are cultured under conditions that allow the formation of immature kidney-like structures. Alternatively the comprises a step wherein the cells are cultured under conditions that allow induction of cells expressing ECAD and NPHS1 . During this step the medium is transiently supplemented with FGF9 or an activator of FGFR1 and / or FGFR3. During this step the medium is also transiently supplemented with a PKA activator and / or a PKA inhibitor according to the invention. Following this step the cells are cultured in growth factor free medium to allow maturation further differentiation of the kidney-like structures. This step is performed by culturing the immature kidney-like structures in the absence of any growth factors. The mature kidney-like structures are characterized by an increased expression of ECAD and NPHS1 compared to the immature kidney-like structures and further express SLC12A1. Thus in an embodiment the second differentiation stage comprises culturing the immature kidney-like structures under conditions that allow differentiation to mature kidney-like structures. Alternatively the step comprises culturing immature kidney-like structures under conditions that allow induction of the kidney-like structures to express ECAD, NPHS1 and SLC12A1. Thus in an embodiment the method of the invention is followed by a step comprising culturing the stem cells in the absence of growth factors. In an embodiment the step is performed for at least 5 days, preferably at least 6, 7, 8, 9, 10, 11 or at least 12 days.

[0098] When used herein the term FGF9 or an activator of FGFR1 and / or FGFR3 is intended to refer to FGF9, a functional homolog of FGF9 or an alternative of FGF9 that can be used to replace FGF9 in the presented method, wherein the alternative activates FGFR1 and / or FGFR3.

[0099] FGF9 is a member of the fibroblast growth factor (FGF) family. FGF family members possess broad mitogenic and cell survival activities, and are involved in a variety of biological processes, including embryonic development, cell growth, morphogenesis, tissue repair, tumor growth and invasion. FGF9 was isolated as a secreted factor that exhibits a growth-stimulating effect on cultured glial cells. The peptide sequence of FGF9 is represented by SEQ ID NO: 1 below. Thus in embodiment of the method of the invention the second differentiation step is characterized in that a protein having or comprising a peptide sequence as defined in SEQ ID NO 1 , is provided for 8 to 16 days after onset of the 3D culture. Preferably the protein is provided in the culture medium, thus allowing contact of the cells with the protein.

[0100] It is further understood that functional homologs of FGF9 (the protein defined by SEQ ID NO: 1) can be used in the context of the invention. Therefore in an embodiment of the method of the invention the second differentiation step is characterized in that a protein having or comprising a peptide sequence as defined in SEQ ID NO 1 or a functional homolog thereof, is provided for 8 to 16 days after onset of the 3D culture, wherein the functional homolog is a protein having or comprising a peptide sequence with 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, preferably 99% sequence homology with SEQ ID NO: 1. Preferably the functional homolog is a protein having or comprising a peptide sequence with 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, preferably 99% sequence homology with SEQ ID NO: 1 which able to activate FGFR1 and / or FGFR3.

[0101] Fibroblast growth factor receptor 1 (FGFR1), also known as basic fibroblast growth factor receptor 1 , fms-related tyrosine kinase-2 I Pfeiffer syndrome, and CD331 , is a receptor tyrosine kinase whose ligands are specific members of the fibroblast growth factor family. Fibroblast growth factor receptor 3 is a protein that in humans is encoded by the FGFR3 gene. FGFR3 has also been designated as CD333 (cluster of differentiation 333). The gene, which is located on chromosome 4, location q16.3, is expressed in tissues such as the cartilage, brain, intestine, and kidneys. FGFR1 and FGFR3 are cell surface membrane receptors that possess tyrosine kinase activity. A full-length representative of these four receptors consists of an extracellular region composed of three immunoglobulin-like domains which bind their proper ligands, the fibroblast growth factors (FGFs), a single hydrophobic stretch which passes through the cell's surface membrane, and a cytoplasmic tyrosine kinase domain. When bonded to FGFs, these receptors form dimers with any one of the four other FGFRs and then cross-phosphorylate key tyrosine residues on their dimer partners. These newly phosphorylated sites bind cytosolic docking proteins such as FRS2, PRKCG and GRB2 which proceed to activate cell signalling pathways that lead to cellular differentiation, growth, proliferation, prolonged survival, migration, and other functions. The FGFR1 receptor is in humans encoded by the gene annotated as

[0102] ENSG00000077782 (Ensembl) or HGNC:3688.

[0103] The FGFR3 receptor is in humans encoded by the gene annotated as

[0104] ENSG00000068078 (Ensembl) or HGNC:3690.

[0105] It is thought that the main function of FGF9 in the kidney organoid differentiation protocol is the activation of FGFR1 and / or FGFR3, therefore the skilled person is aware that FGF9 may also be replaced by other factors capable of activating these receptors. Non limiting examples of activators of the FGFR1 and / or FGFR3 receptors are FGF20, FGF2, or Smoothened agonist (SAG).

[0106] FGF20 is a member of the fibroblast growth factor (FGF) family and capable of activating both FGFR1 and FGFR3. The peptide sequence of FGF20 is represented by SEQ ID NO: 2 below. Thus in embodiment of the method of the invention the second differentiation step is characterized in that a protein having or comprising a peptide sequence as defined in SEQ ID NO 2, is provided for 8 to 16 days after onset of the 3D culture. Preferably the protein is provided in the culture medium, thus allowing contact of the cells with the protein.

[0107] It is further understood that functional homologs of FGF20 (the protein defined by SEQ ID NO: 2) can be used in the context of the invention. Therefore an activator of FGFR1 and / or FGFR3 may be a protein having or comprising a peptide sequence as defined in SEQ ID NO 2 or a functional homolog thereof, wherein the functional homolog is a protein having or comprising a peptide sequence with 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, preferably 99% sequence homology with SEQ ID NO: 2. Preferably the functional homolog is a protein having or comprising a peptide sequence with 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, preferably 99% sequence homology with SEQ ID NO: 2 which able to activate FGFR1 and / or FGFR3. FGF2 is a member of the fibroblast growth factor (FGF) family and capable of activating both FGFR1. The peptide sequence of FGF2 is represented by SEQ ID NO: 3 below. Therefore an activator of FGFR1 and / or FGFR3 may be a protein having or comprising a peptide sequence as defined in SEQ ID NO 3 or a functional homolog thereof, wherein the functional homolog is a protein having or comprising a peptide sequence with 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, preferably 99% sequence homology with SEQ ID NO: 3. Preferably the functional homolog is a protein having or comprising a peptide sequence with 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, preferably 99% sequence homology with SEQ ID NO: 3 which able to activate FGFR1 and / or FGFR3.

[0108] Smoothened agonist (SAG) was one of the first small-molecule agonists developed for the protein Smoothened, a key part of the hedgehog signalling pathway, which is involved in brain development as well as having a number of other functions in the body. It is shown that SAG increases expression of FGF9 and thus indirectly stimulates FGFR1 and FGFR3 by increasing expression of FGF9. SAG is represented by formula 1 below and also known as 3-Chloro- / V-[trans-4-(methylamino)cyclohexyl]- / \ / -[[3-(4- pyridinyl)phenyl]methyl]benzo[b]thiophene-2-carboxamide with CAS registry number 912545-86-9

[0109] Formula 1 :

[0110] In a second aspect the invention relates to a kidney-like structure or kidney organoid obtained or obtainable by the methods as broadly described herein. As demonstrated by the appended Figures, treatment with a PKA inhibitor increases tubule formation (compared to the reference protocol) and treatment with a PKA activator increases nephron formation (compared to the reference protocol), and treatment with a PKA inhibitor followed by a PKA activator results in kidney-like structures with increased presence of tubules and nephrons. Therefore kidney-like structures, such as organoids, grown according to the method described herein can be distinguished from kidney-like structures grown according to the reference protocol due to the increased presence of tubule and / or nephron like structures. This difference can be demonstrated with tubule or nephron markers such as LTL (lotus tetragonolobus lectin, which binds to the brush borders of proximal tubules) and / or NPHS1. Other marker are known to the skilled person and described herein. In an embodiment the kidneylike structure or kidney organoid according has reduced cartilage or exhibits delayed morphological deterioration.

[0111] In a third aspect the invention relates to a kidney-like structure or kidney organoid according to the second aspect of the invention for use in the treatment of a kidney disease, preferably wherein the kidney disease is chronic kidney disease. It is understood that the kidney-like structures may form the basis for an artificial kidney to be used in the treatment of a subject with a kidney disease such as chronic kidney disease. Non limiting examples include growing the kidney-like like structure to organ size and implanting the kidney-like structures in a patient. Alternatively, one or more kidney-like structures may be assembled in a medical device to be used in dialysis. Such device may be used externally or implanted in the subject. Therefore in an embodiment the invention further relates to a kidney-like structure or kidney organoid according to the second aspect of the invention for use in a surgical method the method comprising implanting the kidney-like structure or kidney organoid. In an alternative embodiment the invention relates to a medical device comprising one or more kidney-like structures according to the second aspect of the invention. The medical device may be an implantable device or a dialysis device. Thus in an embodiment the invention further relates to a medical device comprising one or more kidney-like structures or organoids according to the second aspect of the invention for use in the treatment of a kidney disease in a subject, the use comprising implanting the medical device in the subject or using the device to perform dialysis on the subject. Alternatively, the invention relates top a method of treating a kidney disease in a subject in need thereof, the method comprising introducing a kidney-like structure or kidney organoid according to the second aspect of the invention in the subject, or using the . In an embodiment the kidney disease is chronic kidney disease.

[0112] In a fourth aspect the invention relates to an in vitro or ex vivo use of a kidney-like structure or kidney organoid according to the second aspect of the invention for one or more of:

[0113] - target identification for a drug;

[0114] - target validating for a drug;

[0115] - performing safety studies for a drug;

[0116] - performing pharmacodynamics studies for a drug;

[0117] - drug development;

[0118] - drug discovery;

[0119] - performing stratification studies for a drug;

[0120] - predicting an individual patient’s response to a drug;

[0121] - modelling a disease;

[0122] - modelling development; or

[0123] - studying kidney biology. Examples

[0124] Having now generally described the invention, the same will be more readily understood through reference to the following examples which is provided by way of illustration and is not intended to be limiting of the present invention.

[0125] Reference Example 1 - Reference protocol

[0126] Kidney organoids were generated at the air liquid interface using previously described protocol (Takasato, M. et al. Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis. Nature 526, 564-568 (2015)). Briefly, 105cells were seeded in a 6-well plate and cultured in Stemdiff APEL2 medium (STEM CELL Technologies), supplemented with 1% Protein-Free Hybridoma Medium II (Thermo Fisher Scientific) and 1% antibiotic-antimycotic, and treated with CHIR99021 (8pM GSK-3 inhibitor, R&D system) and FGF9 (fibroblast growth factor 9, 200ng / ml, R&D system) / heparin (1 pg / ml, Sigma-Aldrich) cocktail. After 7 days of differentiation (denoted day 7+0), cells were treated with CHIR99021 for 1 h and trypsinized. 500000 cells were aggregated by centrifugation at 425 RCF and cultured at the air-liquid interface on transwell tissue culture plates 1 pm pore polyester membrane inserts (CelIQart, Sabeu). Organoids are kept in complete Stemdiff APEL2 medium with a FGF9 / heparin cocktail for 5 additional days (denoted day 7+5), after which the cocktail was removed. Kidney organoids were them cultures until day 7+25 and 7+32 in complete Stemdiff APEL2 medium. The protocol is schematically depicted in Figure 1 , bottom part.

[0127] 4 days of CHIR99021 resulted in the induction of both the ureteric epithelium and the metanephric mesenchyme in monolayer culture and is followed by 3 days of FGF9 / heparin. Maximal nephron number per organoid required a pulse of CHIR99021 for one hour. The continued presence of FGF9 after this CHIR99021 pulse was essential for nephrogenesis.

[0128] Example 2 - materials and method use in the experiments

[0129] Culture of human induced pluripotent stem cells (hiPSCs)

[0130] Induced pluripotent stem cells were maintained in culture using E8 medium supplemented with 1% penicillin-streptomycin in truncated recombinant human vitronectin — coated 6-well plates. Pluripotency and karyotype were previously assessed in our lab. Medium was refreshed every day and cells were passaged twice a week. After passaging, cells were seeded in presence of Revitacell overnight for both maintenance or differentiation. iPSCs-derived kidney organoid generation and treatment

[0131] Kidney organoids were generated at the air liquid interface using previously described protocol (Takasato et al., supra). Briefly, 105cells were seeded in a 6-well plate and cultured in Stemdiff APEL2 medium (STEMCELL Technologies), supplemented with 1% Protein-Free Hybridoma Medium II (Thermo Fisher Scientific) and 1% antibiotic- antimycotic, and treated with CHIR99021 (8 pM GSK-3 inhibitor, R&D system) and FGF9 (fibroblast growth factor 9, 200ng / ml, R&D system) / heparin (1 pg / ml, Sigma- Aldrich) cocktail. After 7 days of differentiation (denoted day 7+0), cells were treated with CHIR99021 for 1 h and trypsinized. 500000 cells were aggregated by centrifugation at 425 RCF and cultured at the air-liquid interface on transwell tissue culture plates 1 pm pore polyester membrane inserts (CelIQart, Sabeu). Organoids are kept in complete Stemdiff APEL2 medium with a FGF9 / heparin cocktail for 5 additional days (denoted day 7+5), after which the cocktail was removed. Kidney organoids were them cultures until day 7+25 and 7+32 in complete Stemdiff APEL2 medium.

[0132] For treatment of the organoids, PKI or DbcAMP was added from day 7+5 to day 7+12, or PKI was added from day 7+5 to 7+12 and DbcAMP was added from day 7+12 to day 7+18. Following treatment with PKI and / or DbcAMP, kidney organoids were maintained in complete Stemdiff APEL2 medium without growth factors until day 7+25 or day 7+32.

[0133] Immunofluorescence

[0134] In order to assess renal structure development, cryosectioning and mounting on slides were performed as described above. Gelatin / sucrose embedding was removed by placing the slides in PBS for 15 min at 37°C. Slides are incubated for 20 min at RT with blocking solution consisting in PBS supplemented with 10% BSA (bovine serum albumin) and 0.2% Tween. Primary antibodies were diluted in PBS-BSA1 %-Tween 0.2% and incubated overnight at 4°C. Staining was performed with: Anti-Nephrin 1 (R&D system, AF4269) , and LTL (Brunschwig B-1325). After rinsing in PBS-Tween 0.2%, slides were incubated with secondary antibodies for 1 h at RT. Nuclei were stained using DAPI (1 / 1000) in PBS-Tween 0.2%. Slides were mounted using Dako fluorescent mounting medium. Imaging was performed on an automated inverted Nikon Ti-E microscope, equipped with a Lumencor Spectra light source, an Ador Zyla 5.5 sCMOS camera, and an MCL NANO Z200-N Tl z-stage. Objective used was CFI PLAN APO LBDA 10X 0.45 / 4 mm. Images were analyzed using NIS-Element software.

[0135] Results

[0136] The grown organoids were kept in culture until day 7+25 and stained using LTL (lotus tetragonolobus lectin; tubule marker), NPHS1 (nephron marker) and Dapi to stain nuclei. The resulting images are shown in Figures 3-6. Use of DbcAMP (PKA activator) alone increases the number of nephrons (and increased expression of NPHS1), while use of PKI (PKA inhibitor) alone increases tubule formation (as demonstrated with the LTL marker) and cartilage appearance. Combined use of PKI and DbcAMP induces a balance between tubules and nephrons with engulfment of the nephron in the tubules and no cartilage appearance.

Claims

CLAIMS1. A method for producing a kidney-like structure, the method comprising:- providing a cell population comprising ureteric epithelium cells and metanephric mesenchyme cells;- culturing the cell population under conditions that allow the formation of a three dimensional kidney-like structure; wherein the provided cell population is a population of cell aggregates showing early nephrogenesis, and wherein the cell population is cultured in the presence of a PKA inhibitor and / or a PKA activator.

2. The method according to claim 1 wherein the cell population is cultured in the presence of a PKA inhibitor to induce tubule formation in the cell population.

3. The method according to claim 1 or 2 wherein the cell population is cultured in the presence of a PKA activator to induce nephron formation in the cell population.

4. The method according to any one of the preceding claims wherein the cell population is first cultured in the presence a PKA inhibitor to induce tubule formation in the cell population, followed by culturing the cell population with increased tubule formation in the presence of a PKA activator to induce nephron formation in the cell population.

5. The method according to any one of the preceding claims wherein the PKA inhibitor is selected from PKI, KT5720, H89 dihydrochloride and / or wherein the PKA activator is selected from a cAMP analog and CW008, preferably wherein the cAMP analog is selected from DbcAMP and 8-Bromo-cAMP.

6. The method according to any one of the preceding claims wherein the cell population is cultured in a 3D culture environment, preferably wherein the 3D culture environment is selected from culturing on a trans-well membrane, culturing in a lowattachment well, culturing in a hydrogel, culturing on an air-liquid interface, culturing on or in a scaffold, or a suspension culture.

7. The method according to any one of the preceding claims wherein the method is preceded by differentiating stem cells to a cell population comprising ureteric epithelium cells and metanephric mesenchyme cells.

8. The method according to any one of the preceding claims wherein the method is preceded or partially coincides with a step of differentiating the cell population comprising ureteric epithelium cells and metanephric mesenchyme cells under conditions that induce aggregate formation and early nephrogenesis in the cell population comprising ureteric epithelium cells and metanephric mesenchyme cells.

9. The method according to claim 8 wherein the induction of aggregate formation and early nephrogenesis in the cell population comprising ureteric epithelium cells and metanephric mesenchyme cells is performed by culturing the cell population in the presence of an activator of FGFR1 and / or FGFR3, preferably wherein the activator of FGFR1 and / or FGFR3 is selected from FGF20, FGF2, or Smoothened agonist (SAG).

10. The method according to any one of the preceding claims wherein the kidneylike structure is a kidney organoid.

11. A kidney-like structure or kidney organoid obtained or obtainable by the method according to any one of claims 1 to 10.

12. A kidney-like structure or kidney organoid according to claim 11 having reduced cartilage or exhibiting delayed morphological deterioration.

13. Kidney-like structure or kidney organoid according to claim 11 or 12 for use in the treatment of a kidney disease, preferably wherein the kidney disease is chronic kidney disease.

14. The in vitro or ex vivo use of a kidney-like structure or kidney organoid according to claim 11 or 12 for one or more of:- target identification for a drug;- target validating for a drug; - performing safety studies for a drug;- performing pharmacodynamics studies for a drug;- drug development;- drug discovery;- performing stratification studies for a drug; - predicting an individual patient’s response to a drug;- modelling a disease;- modelling development; or- studying kidney biology.

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