Urine derived kidney organoids and methods of producing and using same

Abstract

A method of producing a urine derived kidney organoid (uORG) is provided. Also provided is an isolated uORG ex vivo produced from urine-derived human epithelial cells as well as methods of using same and secretome thereof in methods of regenerating renal function, drug design and a treatment selection.

Description

[0001] URINE DERIVED KIDNEY ORGANOIDS AND METHODS OF PRODUCING AND USING SAME

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of priority of US Provisional Patent Application No. 63 / 762,779 filed on February 25, 2025, the contents of which are incorporated herein by reference in their entirety.

[0004] FIELD AND BACKGROUND OF THE INVENTION

[0005] The present invention, in some embodiments thereof, relates to urine derived kidney organoids and methods of producing and using same.

[0006] Kidneys play a crucial role in regulating the body's fluid balance, filtering waste products, and maintaining electrolyte homeostasis. However, their function can be impaired by various factors, including diabetes, hypertension, and drug-induced toxicity. To better understand normal kidney physiology and the development of renal diseases, researchers require representative preclinical models. In recent years, several in vitro kidney models have been developed based on organoid technology, offering new possibilities for kidney research and personalized medicine.

[0007] Organoids are three-dimensional, multicellular structures that mimic the morphology and physiology of the tissue they originate from, whether normal or diseased. They can be generated from two main sources: pluripotent stem cells (PSCs) or adult stem cells (ASCs), each with distinct characteristics and applications in kidney research.

[0008] PSC-derived kidney organoids aim to replicate nephrogenesis, the process of kidney development. These organoids can be created from patient-derived committed cells through induced pluripotency (iPSCs). By exposing iPSCs to specific growth factor cocktails, researchers can induce differentiation into various nephron cell types, which comprise the kidney's functional unit. While these organoids form a mini-organ in a dish, their establishment is time-consuming, often taking several weeks. Additionally, iPSCs may exhibit genetic instability due to the reprogramming process, which can be a concern for certain applications. A notable limitation of PSC-derived organoids is their inability to fully mature into adult kidney cells. Instead, they display a transcriptome profile more akin to the fetal kidney at an early developmental stage, which may limit their applicability in modeling adult kidney diseases or drug responses.

[0009] In contrast, ASC-derived human kidney tubuloids replicate the renewal of adult kidney epithelium. These organoids primarily represent the proximal tubule, loop of Henle, distal tubules, and collecting duct, as confirmed by the expression of various transporter proteins characteristicof these segments. The tubuloid culture protocol enables the rapid expansion of patient-derived kidney tissue while maintaining a stable genome, which is crucial for accurate disease modeling and drug testing. Research applications for tubuloids include studying normal kidney physiology, investigating nephrotoxicity, conducting drug screening, and modeling various kidney diseases.

[0010] The ability to generate kidney organoids from urine has several advantages. First, it provides a non-invasive means of obtaining patient-specific cells, which is particularly beneficial for studying rare kidney diseases or when repeated sampling is necessary. Second, it allows for the creation of personalized models to test drug efficacy and toxicity, potentially leading to more tailored treatment approaches. Lastly, urine-derived tubuloids can be used to study the effects of genetic variations on kidney function, as they maintain the genetic profile of the patient.

[0011] Both PSC-derived organoids and ASC-derived tubuloids have their place in kidney research, with each offering unique insights into different aspects of kidney biology. PSC-derived organoids are particularly useful for studying developmental processes and congenital kidney diseases. PSC-organoids are not able to fully mature into adult kidney cells, revealing a transcriptome profile that resembles the fetal kidney at an early development stage, while ASC- derived tubuloids represent adult kidney epithelium (the proximal tubule, loop of Henle, distal tubules, and collecting duct) excel in modeling adult kidney function and disease states.

[0012] One potential limitation of patient-derived organoid cultures, including tubuloids, is the availability of fresh tissue. However, recent studies have shown that urine can serve as a source for kidney epithelial cells, offering a simpler, less invasive method to obtain patient material for useful spheroid cultures (WO2022 / 084998), however unlike organoids they are a much more primitive structure with no ability to proliferate or respond to drugs ex vivo.

[0013] Attempts to generate organoids from urine epithelial cells were either inefficient as described by Schutgens, F., Rookmaaker, M.B., Margaritis, T. et al. Tubuloids derived from human adult kidney and urine for personalized disease modeling. 2017 Nat Biotechnol 37, 303- 313; or resulted in a structure which is non-cystic as disclosed inGuo H, Deng N, Dou L, Ding H, Criswell T, Atala A, Furdui CM, Zhang Y. 3-D Human Renal Tubular Organoids Generated from Urine-Derived Stem Cells for Nephrotoxicity Screening. ACS Biomater Sci Eng. 2020 Dec 14;6(12):6701-6709; or Sun G, Ding B, Wan M, Chen L, Jackson J, Atala A. Formation and optimization of three-dimensional organoids generated from urine-derived stem cells for renal function in vitro. Stem Cell Res Ther. 2020 Jul 22;ll(l):309.In fact, a paper by Schutgens et al. (Tissue Engineering, Part C, Volume 27, Number 3, 2021) confirmed the lack of efficiency in production of urine-derived organoids and suggested that this can be increased by immediatelyexposing the cells to organoind formation (without further culturing prior to organoid derivation) and minimize the time in culture.

[0014] SUMMARY OF THE INVENTION

[0015] According to an aspect of some embodiments of the present invention there is provided a method of producing a urine derived kidney organoid, the method comprising:

[0016] (a) isolating urine derived epithelial cells (UD-EpCs) from urine of a human subject; (b) expanding the UD-EpCs in a 2 dimensional (2D) setting under conditions that allow obtaining expanded UD-EpCs;

[0017] (c) culturing the expanded UD-EpCs under conditions which allow formation of urine derived organoids (uORGs); and

[0018] (d) passaging the uORGs.

[0019] According to some embodiments of the invention, the expanding is until P0.

[0020] According to some embodiments of the invention, the conditions of step (b) comprise coated dishes which ensure substrate adherence.

[0021] According to some embodiments of the invention, the conditions of step (b) comprise presence of serum.

[0022] According to some embodiments of the invention, the conditions of step (b) comprise RE:MC or KSFM.

[0023] According to some embodiments of the invention, the conditions of step (b) comprise RE:MC.

[0024] According to some embodiments of the invention, the conditions of step (b) comprise presence of CD40 ligand and optionally at least one of PDGF-AB-10, EGF and FGF-basic.

[0025] According to some embodiments of the invention, the culturing the expanded UD-EpCs comprises formation of spheroids followed by subjecting the spheroids to the conditions which allow formation of urine derived organoids (uORGs).

[0026] According to some embodiments of the invention, the formation of spheroids is under low adherence conditions, followed by subjecting the spheroids to the conditions which allow formation of uORGs in the presence of extracellular matrix (ECM) that encapsulates the spheroids.

[0027] According to some embodiments of the invention, the culturing the expanded UD-EpCs does not allow formation of spheroids.

[0028] According to some embodiments of the invention, the conditions which allow formation of urine derived organoids (uORGs) comprise at least one of N-acetylcysteine (NAC) or an analogthereof, ROCK inhibitor, epidermal growth factor (EGF), fibroblast growth factor 10 (FGF-10), B27 and a Wnt activator.

[0029] According to some embodiments of the invention, the culturing the UD-EpCs is by encapsulating the UD-EpCs within an extracellular matrix (ECM).

[0030] According to some embodiments of the invention, the ECM comprises basement membrane extract (BME).

[0031] According to some embodiments of the invention, culturing the expanded UD-EpCs is in the absence of serum.

[0032] According to some embodiments of the invention, the passaging is for at least 10 passages. According to some embodiments of the invention, the method further comprises isolating cells from UD-EpCs or uORGs and / or analyzing the expanded UD-EpCs or uORGs or the cells thereof.

[0033] According to an aspect of some embodiments of the present invention there is provided an isolated urine derived kidney organoid (uORG) ex vivo produced from urine-derived human epithelial cells and comprising a cystic interior, wherein the organoid is proliferative and a polarized tubule structure prior to transplantation.

[0034] According to an aspect of some embodiments of the present invention there is provided an isolated urine derived kidney organoid (uORG) obtainable according to the method as described herein.

[0035] According to some embodiments of the invention, the uORG comprises cells expressing renal progenitor markers e.g., CD24, PROMI, CD44, VIM and / or PAX2 as determined by singlecell RNA sequencing.

[0036] According to some embodiments of the invention, the uORG expresses sternness markers e.g., inhibitor of DNA-binding (ID1, ID2, ID3 and ID4) as determined by single-cell RNA sequencing

[0037] According to some embodiments of the invention, the uORG expresses higher level of early epithelial markers and / or progenitor epithelial markers selected from the group consisting of NCAM1, ALHD1A2 and VCAM, as compared to a nephro spheroid as determined by single-cell RNA sequencing.

[0038] According to some embodiments of the invention, the uORG expresses higher level of cell adhesion markers selected from the group consisting of ICAMN1, CAV 1 and CDH2, as compared to a nephro spheroid as determined by single-cell RNA sequencingAccording to some embodiments of the invention, the uORG comprises a cell cluster expressing cell proliferation markers selected from the group consisting of MKI67, CDK1, TOP2A, CDC20 and CCNA2, as determined by single-cell RNA sequencing.

[0039] According to some embodiments of the invention, the uORG comprises a cell cluster expressing distal tubule related markers selected from the group consisting of MUC1, CDH1, and SLC8A1, as determined by single-cell RNA sequencing.

[0040] According to some embodiments of the invention, the uORG comprises a cell cluster expressing metabolic activity markers selected from the group consisting of EEF1A1, ACTG1 and ATP5F1A, as determined by single-cell RNA sequencing.

[0041] According to some embodiments of the invention, the urine-derived epithelial cells are from a healthy subject.

[0042] According to some embodiments of the invention, the urine-derived epithelial cells are from a subject diagnosed with a chronic kidney disease (CKD).

[0043] According to some embodiments of the invention, the CKD is selected from the group consisting of ciliopathy, Fanconi Bickel Syndrome, Pearson syndrome and Autosomal recessive polycystic kidney disease (ARPKD).

[0044] According to some embodiments of the invention, the urine-derived epithelial cells are from a subject under 18 years of age.

[0045] According to some embodiments of the invention, the urine-derived epithelial cells are from a subject being at least 18 years of age.

[0046] According to an aspect of some embodiments of the present invention there is provided a composition of matter comprising multiple uORGs from a single human subject, the multiple uORGs comprising all tubular segments including a distal tubule, a proximal tubule, a collecting duct and a loop of Henle yet being devoid of glomerulus.

[0047] According to an aspect of some embodiments of the present invention there is provided a bank comprising multiple compositions as described hereom from multiple subjects.

[0048] According to an aspect of some embodiments of the present invention there is provided a secretome of the uORG as described herein.

[0049] According to an aspect of some embodiments of the present invention there is provided a method of producing a secretome, the method comprising:

[0050] (a) providing a culture of the uORGs as described herein;

[0051] (b) collecting conditioned medium of the culture, the conditioned medium comprising the secretome.According to some embodiments of the invention, the method comprises isolating the secretome or a portion thereof from the conditioned medium following step (b).

[0052] According to an aspect of some embodiments of the present invention there is provided a method of regenerating renal function, the method comprising administering to a subject in need thereof the uORGs as described herein or secretome thereof, thereby regenerating renal function.

[0053] According to an aspect of some embodiments of the present invention there is provided a method of treating a chronic kidney disease (CKD), the method comprising administering to a subject in need thereof the uORGs as described herein or secretome thereof, thereby treating the CKD.

[0054] According to some embodiments of the invention, the uORGs or secretome thereof as described herein for use in treating a chronic kidney disease (CKD).

[0055] According to an aspect of some embodiments of the present invention there is provided a method of drug design, the method comprising determining an effect of a test drug on the uORGs as described herein or secretome thereof.

[0056] According to some embodiments of the invention, a method of selecting treatment to a subject diagnosed with a kidney disease, the method comprising:

[0057] (a) contacting a uORG produced from UD-EpCs of a subject diagnosed with the kidney disease; and

[0058] (b) determining an alleviation in a renal symptom evident in the uORG following the contacting, the alleviation being indicative of an efficacious therapy.

[0059] According to some embodiments of the invention, the test drug is Tolvaptan and / or Everolimus.

[0060] According to some embodiments of the invention, the effect is cyst size.

[0061] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0062] BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0063] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail,it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0064] In the drawings:

[0065] FIGs. 1A-E describe a protocol for establishment of urine-derive kidney organoids from healthy individuals and their morphological and molecular characterization;

[0066] FIGs. 2A-E show establishment of urine-derive kidney organoids from kidney disease patients;

[0067] FIGs. 3A-C show establishment of urine-derive kidney organoids from Pearson syndrome patients;

[0068] FIGs. 4A-F show organoids from an autosomal recessive polycystic kidney disease (ARPKD) patient as a model for pathology investigation and treatment development;

[0069] FIGs. 5 A- J show transcriptome analysis of uORGs.

[0070] DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

[0071] The present invention, in some embodiments thereof, relates to urine derived kidney organoids and methods of producing and using same.

[0072] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

[0073] Multiple attempts to establish kidney organoids from urine of human beings have failed, despite following a previously reported protocol by Schutgens et al. 2017 (supra). In this protocol freshly isolated kidney cells from human urine were seeded directly on an ECM matrix (basement membrane extract), however, no organoids were formed.

[0074] Whilst conceiving embodiments of the invention, the present inventors devised a novel approach for generating human urine-derived kidney organoids. This method involves expanding the urine isolated kidney cells in 2D conditions, and then transitioning them into 3D culture to form organoids which are also termed tubuloids or cystoids. In an alternative embodiment, the expanded urine isolated kidney cells are subjected to conditions, which induce nephrosphere formation and these in turn are subjected to 3D conditions that allow formation of organoids.

[0075] The organoids of some embodiments of the invention are proliferative and can be subjected to numerous passaging (e.g., above 25 passages). Urine-derived organoids were positively stained for distal tubule markers, SLC12A1 and EMA, and for proximal tubule markers, LTL and LRP2(Figure 1C). Positive staining for Ki67 is also evident suggesting that the cells have proliferative nature.

[0076] Ultrastructural analysis using electron microscopy further confirmed the polarity of urine derived organoids (uORG), with the basal membrane positioned on the outer side of the tubule, tight junctions between tubular cells, and brush borders at the luminal (apical) inner side (Figure IE). The same results were achieved for both adult and human derived kidney cells.

[0077] The organoids were found useful as a model for testing treatment with various drugs as shown in Figures 2A-E to 4A-F. Gene expression analysis revealed that the organoids are endowed with a unique gene expression which is different than that of nephrospheroids.

[0078] Thus, the novel method disclosed herein offers several advantages. The initial 2D expansion phase allows for greater control over cell proliferation and increases the overall yield of cells available for organoid formation. By expanding cells in 2D first, it is also possible to perform quality checks and potentially enrich for specific cell populations before initiating 3D culture (for instance to deplete urothelial epithelial cells). The subsequent transfer to 3D conditions promotes the self-organization of cells into more complex, tissue-like structures that better recapitulate kidney architecture and function. This approach may result in more mature and stable organoids compared to direct 3D culture methods.

[0079] Hence, the method of organoids formation of some embodiments of the invention, expands the toolkit available to researchers studying kidney biology and disease, offering a non-invasive way to generate patient-specific kidney models with potentially enhanced maturity and stability which can be used in therapy too.

[0080] Thus, according to an aspect of the invention there is provided a method of producing a urine derived kidney organoid, the method comprising:

[0081] (a) isolating urine derived epithelial cells (UD-EpCs) from urine of a human subject; (b) expanding said UD-EpCs in 2 dimensional (2D) settings under conditions that allow obtaining expanded UD-EpCs;

[0082] (c) culturing said expanded UD-EpCs under conditions which allow formation of urine derived organoids (uORGs); and

[0083] (d) passaging said uORGs.

[0084] As used herein “kidney organoid” refers to a 3 dimensional structure of kidney cells, having a cystic (hollow) interior. The structure exhibits polarity with the same orientation as in the nephron tubule. Cells within the organoid are capable of proliferation. It can be maintained in culture (ex-vivo) for at least 25 passages. The organoid comprises epithelial cells from various kidney segments (proximal, distal, loop). The organoid responds ex vivo to drugs which reduce cystexpansion and preserve kidney function. The term organoid, does not include a whole kidney or a fragment thereof, or a nephrosphere which is an amorphous aggregate structure of non-proliferative nature in culture.

[0085] The phrase “urine derived epithelial cells” abbreviated as UD-EpCs, refers to epithelial cells which are shed from the human kidney to the urine. The cells are Pax8+p63-, which identify kidney epithelial cells.

[0086] Notably, urine samples comprise, aside from epithelial cells, also squamous cells (mostly observed in samples derived from women, likely of urethral origin), leukocytes and semen, in accordance with previous reports. However, these cell types typically do not adhere to the plate surface and gradually disappear following a few medium aspirations.

[0087] Thus, in effect the passaging is performed to enrich UD-EpC and deplete squamous epithelial cells of vagina and / or bladder origin.

[0088] Therefore at P0 or Pl and certainly in later passages the culture is pure, i.e., more than 90 %, 95 % or 99 % cells are kidney epithelial cells.

[0089] According to some embodiments of the invention, in order to isolate the cells from urine, mid-stream urine samples are collected from male or female human subjects, at any age e.g., adult - 18 years or above, or child, below 18 years of age. The subject can be healthy or diagnosed with a kidney disease, such as chronic kidney disease (CKD), such as representing inherited or acquired glomerular and / or tubular and / or tubulo-interstitial disorders: glomerulonephritis, glomerulosclerosis, interstitial nephritis, any form of a ciliopathy, renal tubular acidosis (RTA), Pearson syndrome, Fanconi syndrome, proximal tubulopathy, renal tubule dysgenesis (RTD), Bickel-Fanconi, any form of congenital anomalies of the kidney and urinary tract (CAKUT).

[0090] According to a specific embodiment, the CKD is selected from the group consisting of ciliopathy, Fanconi Bickel Syndrome, Pearson syndrome and Autosomal recessive polycystic kidney disease (ARPKD), such as exemplified in the Examples section which follows.

[0091] According to a specific embodiment, isolating urine cells is by centrifugation. Thus, according to a specific embodiment, to obtain kidney cells from the urine, urine samples are centrifuged (e.g., 400 x g), washed with buffer e.g., PBS and centrifuged, such as at higher, same or lower gravity e.g., at 200xg, than the first round. Then the cell pellet is seeded on an adherent substrate.

[0092] As mentioned, passaging is also performed to achieve higher numbers of urine derived epithelial cells which is critical to the process of producing organoids.According to a specific embodiment “expanding” is by at least 10 fold increase in epithelial cells number compared to the amount adhered to the container following isolation from the urine sample.

[0093] According to a specific embodiment, the adherent conditions comprise gelatin coating, though other adherent natural or synthetic matrices may be used. Examples include, but are not limited to Poly-L-lysine (PLL) and Poly-D-lysine (PDL), Poly-ornithine (PLO), Dendritic polyglycerol amine (dPGA), SARSTEDT Cell+ plates and Coming® CellBIND® surface.

[0094] The phrase “adherent conditions” refers to conditions in which the cells attach to the surface of a container in which they are cultured such that a substantial portion of the cells cannot be removed from the surface of the container by mechanical manipulations that do not cause significant damage to the cells.

[0095] The conditions for culturing (expanding) urine epithelial cells further include serumcontaining medium.

[0096] According to a specific embodiment, RE:MC or KSFM media are used for establishing UD-EpC cultures.

[0097] RE:MC: (renal growth medium: Mesenchymal Cell proliferation at a 1:1 ratio) (e.g., REGM Renal Growth BulletKit: REB M™ Basal Medium with Additives: hEGF, Insulin, Hydrocortisone, FBS, Transferrin, Triiodothyronine, Epinephrine Lonza, Cat No. CC-3190) and mesenchymal cell media, MC (Dulbecco's Modified Eagle Medium (DMEM) High Glucose, 1 % L-glutamine solution, 1 % Penicillin-Streptomycin Solution, Human PDGF-AB-10 (10-25ng / ml), Animal Free Human EGF (10-50 ng / ml), Human FGF-basic (10-50 ng / ml) and 5-10 % FBS] supplemented with CD40L (2-10 ng / ml).

[0098] KSFM: PR (keratinocyte serum free medium: progenitor cell medium at a 1:1 ratio) KSFM (Keratinocyte-SFM Medium with Additives: Glutamine, Phenol Red, EGF and Bovine Pituitary Extract, ThermoFisher Cat No. 17005075), PR (DMEM-F12, hydrocortisone, cholera toxin, insulin, transferrin, Animal Free Human EGF and 10 % FBS)

[0099] According to a specific embodiment, the conditions for expansion in 2D culture comprise presence of CD40 ligand (CD40L) (2-10 ng / ml and optionally at least one of platelet-derived growth factor (PDGF)-AB-IO (10-25 ng / ml), epidermal growth factor (EGF) (10-50ng / ml)and basic fibroblast growth factor (FGF-basic. bFGF, FGF2) (10-50 ng / ml)).

[0100] According to a specific embodiment, the medium is supplemented with CD40L.

[0101] According to a specific embodiment, the 2D culture medium for urine derived cells comprise RE:MC: (renal growth medium: Mesenchymal Cell proliferation at a 1:1 ratio) (e.g., REGM Renal Growth BulletKit: REB M™ Basal Medium with Additives: hEGF, Insulin, Hydrocortisone, FBS, Transferrin, Triiodothyronine, Epinephrine Lonza, Cat No. CC-3190) and mesenchymal cellmedia, MC [Dulbecco's Modified Eagle Medium (DMEM) High Glucose, 1 % L-glutamine solution, 1 % Penicillin-Streptomycin solution, Human PDGF-AB-10 (10-25 ng / ml), Animal Free Human EGF (10-50ng / ml), Human FGF-basic (10-50 ng / ml) and 5-10% FBS] supplemented with CD40E (2-10 ng / ml).

[0102] As used herein, “serum” refers to naturally occurring serum or serum replacement. The use of serum replacement may be beneficial to avoid xeno-contamination.

[0103] According to a specific embodiment, the serum is fetal bovine serum (FBS), e.g., 5-10 %. Other sera and sera replacements include but are not limited to: human serum, human platelet lysate (hPE), human AB serum, human serum albumin, and human plasma. Animal-derived alternatives encompass bovine ocular fluid, fish serum, and serum from bovine adults. Plant-derived alternatives feature sericin protein and plant-based protein hydrolysates. Chemically defined alternatives consist of serum-free media with defined components, recombinant growth factors, and synthetic supplements such as lipids, vitamins, and trace elements.

[0104] When cells reach confluency (P0) e.g., 80-100 %, they are harvested such as by the use of a protease, e.g., trypsin and seeded to the next passage (Pl) or subjected to conditions which allow the formation of urine-derived organoids (uORGs).

[0105] According to a specific embodiment, expanding is until P0.

[0106] According to a specific embodiment, expanding is until no later than Pl.

[0107] According to a specific embodiment, expanding is until no later than P2.

[0108] According to a specific embodiment, expanding is until no later than P3.

[0109] According to a specific embodiment, expanding is until no later than P4.

[0110] According to some embodiments of the invention, higher passage number (e.g., Pl, P2, P3, P4) is used in the 2 step protocol as described below.

[0111] The formation of uORGs can be directly from the expanded UD-EpCs (also referred to herein as the “one step protocol”) or preceded by a step of nephro spheroid formation (also referred to herein as the “two step protocol”). According to a specific (and preferred) embodiment, the one step protocol is used.

[0112] Briefly, in the one step protocol, culturing is done without the formation of spheroids. As used herein “nephro spheroid” or “spheroid” refers to an aggregate of kidney epithelial cells (such as urine derived). The aggregate is about 50-200 pm in diameter (as opposed to an organoid where the size is 30-800 pm in diameter)

[0113] Thus, the passaged cells of the 2D culture are encapsulated in extracellular matrix (ECM) such as 2000-8000 cells / pl, e.g., 4000 cells / pl. The cells are then seeded in domes (e.g., 15 pl-100 pl, e.g., 50 pl) which are allowed to solidify.Examples of ECM which can be used include basement membrane extract (BME), VitroGel ORGANOID, Self-Assembling Peptide Hydrogels (PeptiGel Technologies), Gelatin Methacrylate (GelMA) Hydrogels (Advanced BioMatrix), Collagen Type I (Coming), and Sodium Alginate Hydrogel (Sigma- Aldrich). The vendors are just exemplary and should not be considered binding.

[0114] According to a specific embodiment, all ingredients used in the formation of uORGs throughout the method are xeno-free (devoid of animal contaminants).

[0115] According to specific embodiments, conditions which allow formation of urine derived organoids (uORGs) comprise at least one of N-acetylcysteine (NAC) or an analog thereof, ROCK inhibitor, epidermal growth factor (EGF), fibroblast growth factor 10 (FGF-10), B27, and a Wnt activator, e.g., R-spondin or R-spondin conditioned medium (others are listed below).

[0116] According to a specific embodiment, the ROCK inhibitor is Y-27632, though others can be used such as: fasudil, H-1152 and HA-1077 (a Fasudil analog).

[0117] Table 1 below provides some embodiments of such media. The functional annotation indicates that other ingredients can be used to mediate the designated function. Some substitutes are mentioned, e.g., in the case of ROCK inhibitor, each is category or ingredient is considered an individual embodiment.

[0118] Table 1

[0119]

[0120]

[0121] Culturing at this stage and passaging thereafter is being done in the absence of serum. In an alternative embodiment, the two-step protocol is used, where the cells are first subjected to formation of spheres that are then encapsulated to form uORGs under the conditions described above.

[0122] To form spheres, the expanded UD-EpCs are cultured under non-adherence or low-adherence conditions.

[0123] The phrase “non-adherent conditions” or “low adherent conditions” refers to conditions in which the cells do not attach to the surface of a container in which they are cultured such that a substantial portion of the cells can be removed from the surface of the container by mechanical manipulations that do not cause significant damage to the cells.

[0124] Contemplated culture media include, but are not limited to IMDM (Invitrogen) or DMEM (Invitrogen).

[0125] According to another embodiment, the culture medium, when generating the nephrospheres is devoid of serum.

[0126] According to a specific embodiment, the dish is coated with polyHEMA. Other reagents can be used to achieve such conditions as the use of commercial low-attachment flasks.The nephro spheroid is not hollow and does not demonstrate a cystic structure ex vivo, rather it is a cell aggregate which at this stage is non-proliferative but once encapsulated in ECM becomes proliferative when cultured in a uORG medium, as described above. The nephrospheroids can be subject to dissociation or left intact for organoids formation.

[0127] Thus, according to a specific embodiment, the formation of spheroids is under low adherence conditions, followed by subjecting said spheroids to said conditions which allow formation of uORGs in the presence of extracellular matrix (ECM) that encapsulates said spheroids.

[0128] According to some embodiments of the invention, the medium used for nephrospheroids formation comprise SFM: DMEM / F-12 [HAM] 1:1, 1% L-glutamine solution, 1% Penicillin-Streptomycin Solution (*10,000 units / ml), 1-10% Non-essential Amino acids (NEAA), 1-3% Sodium Pyruvate, 0.02% Chemically defined (CD), Lipid mix, 2% B27 supplement (X50), Heparin sodium Fresenius 5000IU / lml, Animal Free Human EGF (10-50 ng / ml), Human FGF-basic (10-50 ng / ml), 0.06-0.1 mg / ml Glucose, 0.01-1 mg / ml Transferrin, 2.5-10 ug / ml Insulin, 0.1 ug / ml Putrescine, 3 nM Sodium Selenite, 2 nM Progesterone).

[0129] The formation of the nephrosphroid is for about 18-36 hours (e.g., 24 hours). Then the protocol proceeds to the next stage of uORG formation as described above (encapsulation in BME).

[0130] Regardless of the protocol, whether it is the one step protocol or the two step protocol, once uORGs are obtained they are passaged.

[0131] According to a specific embodiment, passaging is effected by mechanical disruption of the uORGs. The matrix is first depleted. For example, BME is dissolved in 4 °C (on ice) and then subjected to mechanical fractionation and split 2-20 (e.g., 2-10, 2-8). The passaging rate depends on the proliferation rate.

[0132] According to an embodiment of the invention, passaging is for at least 10, 20, 30, 50 or more passages e.g., up to 100 passages, e.g., 10-40 passages, 10-30 passages, 15-30 passages, 20-30 passages.

[0133] At any step of the protocol the cells can be analyzed for any specific parameter, such as morphology, functionality, proliferation rate and / or marker expression, using methods which are well known in the art and rely (not exclusively) on molecular biology, single cell RNA sequencing, immunology, protein expression and cell biology (e.g., microscopy).

[0134] Thus, according to a specific embodiment, the method comprises isolating cells from UD-EpCs or uORGs and / or analyzing said expanded UD-EpCs or uORGs or said cells thereof.

[0135] For example, UD-EpC and / or uORGs may be analyzed for markers of renal epithelial cells (e.g., tubular), morphology of renal cells (epithelial cells do not have a spindle shape for instance),confluence (e.g., 80-100 %); uORG may be analyzed for markers such as decribed herein, as well as cyst shape, cyst size, effect of drugs (e.g., forskolin expands the cyst, while mTOR inhibitors reduce cyst size), mutation analysis which is especially important for mitochondrial diseases (heteroplasmia) or mosaic (Pearson).

[0136] The present teachings also contemplate the uORGs, secretome or fraction thereof derived from the methods described herein.

[0137] Thus, according to an aspect of the invention, there is provided an isolated urine derived kidney organoid (uORG) ex vivo produced from urine-derived human epithelial cells, wherein the organoid is proliferative and comprises a polarized tubule structure prior to transplantation. It will be appreciated that a spheroid generated as described in WO2022 / 084998 is proliferative only following transplantation (in vivo).

[0138] According to an additional or an alternative aspect there is provided an isolated urine derived kidney organoid (uORG) obtainable according to the method as described herein.

[0139] uORGs described herein are endowed with a unique gene expression. Methods of determining gene expression are well known in the art. They can be determined at the level of RNA or protein. Global gene expression can be determined and alternatively or additionally they can be determined at the single cell level. Cell cluster data derived from single-cell RNA sequencing refers to the grouping of individual cells based on their gene expression profiles. These clusters represent distinct cell populations with similar molecular characteristics.

[0140] Tools for analyzing are well known in the art and include Seurat, Scanpy, Monocle, SingleR, Cell Ranger, CiteSeq-Count, and SC3. These tools are commonly used for analyzing single-cell RNA sequencing data, offering functionalities for clustering, trajectory analysis, celltype annotation, and data preprocessing.

[0141] In the context of the Seurat tool, which is commonly used for analyzing single-cell RNA sequencing data, clustering is typically performed by first normalizing and scaling the data, then identifying genes that contribute to the differences between cells. Seurat uses algorithms like Louvain clustering or k-means clustering to group cells into clusters, allowing researchers to identify cell types, subtypes, or states based on their gene expression patterns.

[0142] According to some embodiments of the invention, the uORG comprises cells expressing renal progenitor markers e.g., CD24, PROMI, CD44, VIM and / or PAX2 as determined by singlecell RNA sequencing.

[0143] According to some embodiments of the invention, the uORG comprises cells expressing sternness markers e.g., inhibitor of DNA-binding (ID1, ID2, ID3 and ID4) as determined by singlecell RNA sequencing.According to some embodiments of the invention, the uORG comprises cells expressing higher level of early epithelial markers and / or progenitor epithelial markers selected from the group consisting of NCAM1, ALHD1A2 and VCAM, as compared to a nephro spheroid as determined by single-cell RNA sequencing.

[0144] According to some embodiments of the invention, the uORG comprises cells expressing higher level of cell adhesion markers selected from the group consisting of ICAMN1, CAV1 and CDH2, as compared to a nephro spheroid as determined by single-cell RNA sequencing.

[0145] According to some embodiments of the invention, the uORG comprises a cell cluster expressing cell proliferation markers selected from the group consisting of MKI67, CDK1, TOP2A, CDC20 and CCNA2, as determined by single-cell RNA sequencing and for MKI67 optionally also by immunofluorescent staining.

[0146] According to some embodiments of the invention, the uORG comprises a cell cluster expressing distal tubule related markers selected from the group consisting of MUC1, CDH1, and SLC8A1, as determined by single-cell RNA sequencing, MUCl(EMA) and for CDHl(E-CAD) optionally also by immunofluorescent staining.

[0147] According to some embodiments of the invention, the uORG comprises a cell cluster expressing metabolic activity markers selected from the group consisting of EEF1A1, ACTG1 and ATP5F1A, as determined by single-cell RNA sequencing.

[0148] Strikingly, the present inventors identified that uORGs generated according to the present teachings are endowed by all the tubular segments.

[0149] Thus according to an aspect of the invention there is provided a composition of matter comprising multiple uORGs from a single human subject, said multiple uORGs comprising all tubular segments including a distal tubule, a proximal tubule, a collecting duct and a loop of Henle yet being devoid of glomerulus.

[0150] Such compositions can be collected and put in a bank in which is composition is indentified or indexed according to the donor.

[0151] Thus, according to an aspect there is provided a bank comprising multiple compositions (each being from a single subject) from multiple subjects.

[0152] Also contemplated herein is the secretome of the uORG described herein.

[0153] As used herein “a secretome” refers to the set of secreted metabolites, proteins expressed by the uORGs and secreted into the environment, cell-derived particles such as exosomes, nucleic acid molecules (e.g., RNA), carbohydrates, lipids, small molecules which are secreted to the medium in which the uORGs are cultured. It can also be referred to as a uORG conditioned medium. It may include, for example, cytokines, growth factors, extracellular matrix proteins andregulators, and shed receptors. The secretome can be measured by mass spectrometry as well as other analytic methods.

[0154] Also contemplated is a method of producing a secretome, the method comprising:

[0155] (a) providing a culture of the uORGs as described herein; and

[0156] (b) collecting conditioned medium of the culture, the conditioned medium comprising the secretome.

[0157] According to a specific embodiment, the method further comprises isolating the secretome or a portion thereof from the conditioned medium following step (b).

[0158] Isolation of the secretome or fractions thereof typically comprises filtering, centrifugation, affinity chromatography, gel filtration chromatography, liquid chromatography or precipitation and for example protein isolation methods such as SDS-PAGE, Western blotting, ELISA, or mass spectrometry, the skilled artisan would know which methods to apply. According to some embodiments, following expansion of organoids, secretome is collected every 36-72 homes (e.g., 48 hours), allowing for repeatable collection.

[0159] The ability of the organoids to form renal structures ex vivo and their response to renal drugs suggests that they can function in treatment of renal diseases.

[0160] Thus, according to an aspect of the invention there is provided a method of regenerating renal function, the method comprising administering to a subject in need thereof a therapeutically effective amount of the uORGs or secretome thereof, thereby regenerating renal function.

[0161] Thus, uORGs of the present invention can be used to treat any form of acute or chronic kidney disease, diabetic nephropathy, renal disease associated with hypertension, hypertensive acute tubular injury (ischemic, toxic), interstitial nephritis, congenital anomalies (Aplasia / dysplasia / obstructive uropathy / reflux nephropathy); hereditary conditions (Juvenile nephronophtisis, ARPCKD, Alport, Cystinosis, Primary Hyperoxaluria); Glomerulonephritides (Focal Segmental Glomerulosclerosis); Multisystem Diseases (SLE, HSP, HUS) or any form of genetic or inherited glomerular, tubular, tubulo-interstitial kidney disease.

[0162] According to a specific embodiment, the disease is a chronic kidney disease.

[0163] According to a specific embodiment, the CKD is selected from the group consisting of ciliopathy, Fanconi Bickel Syndrome, Pearson syndrome and Autosomal recessive polycystic kidney disease (ARPKD).

[0164] The present teachings contemplate administration of the intact uORGS though, partly dissociated uORG-cells can also be used. Each of these should be considered as a separate independent embodiment of the invention.The uORGs in any form or secretome may be administered per se or as part of a pharmaceutical composition where they are mixed with a suitable carrier or excipient.

[0165] As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0166] Herein the term "active ingredient" refers to the cells in any form or secretome accountable for the biological effect.

[0167] Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.

[0168] Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

[0169] The cells in any form or secretome can be administered into a subject such as surgically or by infusion. For example, renal cells are injected in vivo into a kidney that is in the postischemic recovery phase. This can be tested easily in an animal model predictive of ischemic kidney damage, the renal pedicle of an anesthetized mouse is clamped for 30 minutes to induce kidney ischemia. Renal stem cells are then injected into the juxtamedullary region (approximately 2000 cells at a depth of 2-4 mm). Otherwise, the cell secretome is injected into the renal artery / tail vain. After 2 weeks of recovery, immunohistochemical analysis is used as described above to look for differentiated cells surface markers GP33O, Tamm-Horfall, Dolichos Biflorous, and the like. In addition, immunohistochemical analysis for collal and chemical staining (such as: Masson-Trichrome and Sirius red) are used to quantify the fibrotic tissue. Post-incorporation differentiation status can then be compared to pre-injection marker status.

[0170] According to a specific embodiment, intra-renal administration of the nephrospheresm is contemplated herein.

[0171] The uORGs in any form or secretome can be used to construct artificial kidney systems. Such a system can be based on a hollow fiber filtration system.

[0172] In one example of a filtration device, the uORGs or cells derived therefrom are grown on the interior of hollow fibers having relatively high hydraulic conductivity (i.e., ultrafiltration coefficient). The hollow fiber passes through a chamber that is provided with a filtrate outlet port.Arterial blood containing metabolic waste and other unwanted material is introduced into one end of the hollow fiber through an inlet port. Blood passed through the fiber and exits the other end of the fiber through an outlet port where it passed into the patient's vascular venous flow. As blood passes through the fiber, filtrate pass through the uORGs or cells derived therefrom lining the interior of the fiber and through the hollow fiber itself. This filtrate then passes out of the chamber containing the fiber through the filtrate outlet port. The device preferably includes many such hollow fibers each of which can be in its own chamber. Alternatively many, many hollow fibers (100-100,000 or even more) can be bundled together in a single chamber.

[0173] The uORGs or cells derived therefrom of the invention can be used to create a tubuleprocessing device. In such a device the uORGs or cells derived therefrom can be grown in a layer on the exterior of the semipermeable hollow fiber (i.e. a scaffold). The fiber is placed in a chamber that is provided with an inlet port and an outlet port. As ultrafiltrate from filtered blood flows through the chamber, reabsorbant passes through the cell layer and through the wall of the fiber into the lumen of the fiber from which it can be directed back into the patient's systemic circulation. Ultrafiltrate that is not reabsorbed passes through the outlet port of the chamber.

[0174] In the devices described above, it can be desirable to coat the fiber surface that will bear the cell layer with extracellular matrix components. For example, the fiber can be coated with materials such as collagen (e.g., Type I collagen or Type IV collagen), proteoglycan, fibronectin, and laminin or combinations thereof. It can be desirable to combine various cell types on the inner or outer surface of the fibers. For example, it can be desirable to include endothelial cells and pericyte, vascular smooth muscle cells or mesangial cells or fibroblasts or combinations thereof. It can also be useful to provide a feeder layer of cells, e.g., irradiated fibroblasts or other cells that can provide soluble factors and structural support to cells they are indirectly or directly in contact with.

[0175] The above-described filtration system and the above-described tubule processing system can be combined to create an artificial kidney. Such systems are described in U.S. Pat. No. 6,150,164, hereby incorporated by reference. A number of suitable materials for forming the hollow fiber are described in U.S. Pat. No. 6,150,164, hereby incorporated by reference.

[0176] The present invention provides a method of using the cell populations of the present invention to characterize cellular responses to biologic or pharmacologic agents involving producing the cells as described herein and culturing with one or more biologic or pharmacologic agents, identifying one or more cellular responses to the one or more biologic or pharmacologic agents, and comparing the one or more cellular responses of the cultures. Tissue culture techniques known to those of skill in the art allow mass culture of hundreds of thousands of cell samples fromdifferent individuals, providing an opportunity to perform rapid screening of compounds suspected to be, for example, teratogenic or mutagenic.

[0177] Also provided is a method of drug design comprising determining an effect of a test drug on the uORG or secretome thereof.

[0178] The effect can be any selected by the skilled artisan, such as cyst size or cyst shape.

[0179] These teachings can be further harnessed towards tailored therapy against a disease. Thus, according to an aspect of the invention, there is provided a method of selecting treatment for a subject diagnosed with a kidney disease, the method comprising:

[0180] (a) contacting a uORG produced from UD-EpCs of a subject diagnosed with the kidney disease; and

[0181] (b) determining an alleviation in a renal symptom evident in the uORG following the contacting, the alleviation being indicative of an efficacious therapy.

[0182] According to a specific embodiment, the uORG is autologous.

[0183] According to a specific embodiment, the uORG is allogeneic.

[0184] Examples of drugs used in renal therapy and CKD in particular include, but are not limited to, Vasopressin receptor antagonists, such as Tolvaptan; mTOR inhibitors like Everolimus utilized in kidney transplant management and to slow cyst growth in polycystic kidney disease. Angiotensin-converting enzyme (ACE) inhibitors, such as Enalapril and Lisinopril, and angiotensin receptor blockers (ARBs) like Losartan and Valsartan help manage blood pressure in chronic kidney disease. Diuretics, including Furosemide and Spironolactone, are used to manage fluid retention, while erythropoiesis- stimulating agents like Epoetin alfa and Darbepoetin alfa treat anemia. Phosphate binders, such as Sevelamer and Lanthanum carbonate, help manage phosphate levels in the blood. Sodium bicarbonate is used for metabolic acidosis, and calcimimetics like Cinacalcet are used for mineral bone disorder management. Immunosuppressants such as Prednisone and Mycophenolate mofetil are prescribed to prevent organ rejection in kidney transplant recipients. SGLT2 inhibitors like Empagliflozin and Canagliflozin are used to manage diabetes-related kidney damage.

[0185] As used herein the term “about” refers to ± 10 %.

[0186] The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

[0187] The term “consisting of’ means “including and limited to”.

[0188] The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, stepsand / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0189] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0190] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0191] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0192] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0193] As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

[0194] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in thecontext of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0195] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0196] EXAMPLES

[0197] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

[0198] Establishment of primary culture from Urine samples - uEpC (urine derived epithelial cells)

[0199] Mid-stream urine samples were collected from healthy individuals or CKD patients who provided their informed consent and following the approval of The Chaim Sheba Medical Center ethics committee. The urine samples were centrifuged at 400 x g, washed with PBS and centrifuged again at 200 x g. Then the cell pellet was seeded on two different gelatin-coated wells in a 24 well plate (0.1 %, Biological Industries, 01-944- IB), with RE:MC: (renal growth medium: Mesenchymal Cell proliferation at a 1:1 ratio) (e.g., REGM Renal Growth BulletKit: REBM™Basal Medium with additives: hEGF, Insulin, Hydrocortisone, FBS, Transferrin, Triiodothyronine, Epinephrine Lonza, Cat No. CC-3190) and mesenchymal cell media, MC [Dulbecco's Modified Eagle Medium (DMEM) High Glucose, 1 % L-glutamine solution, 1 % Penicillin-Streptomycin solution, Human PDGF-AB-10 (10-25ng / ml), Animal Free Human EGF (10-50ng / ml), Human FGF-basic (10-50 ng / ml) and 5-10% FBS] supplemented with CD40L (2-10ng / ml).

[0200] R-spondin conditioned

[0201] R-spondin conditioned medium was prepared using HEK293T cells stably transfected with an R-spondinl expression plasmid. Cells were maintained in Advanced DMEM / F12 (Thermo Fisher Scientific) supplemented with 10 % heat-inactivated fetal bovine serum (FBS; Gibco), 1 % GlutaMAX (Thermo Fisher Scientific), 1 % Penicillin-Streptomycin (Thermo Fisher Scientific), 1 % sodium pyruvate, and 300 pg / mL Zeocin (InvivoGen). For conditioning, cells at 70-80 % confluence were washed with PBS and cultured in Advanced DMEM / F12 with 1 % GlutaMAX and 1% Penicillin-Streptomycin and 1 % NEAA (without FBS or Zeocin) for 4-7 days. The conditioned medium was then collected, centrifuged at 500 x g for 5 minutes, and filtered through a 0.22 pm filter. Aliquots were stored at -80°C until use.

[0202] Urine-derived organoids formation:

[0203] One step protocol: Upon reaching 80-100 % confluence at P0, cells grown as 2D uEpC were trypsinized and seeded with Cultrex BME (basement membrane extract, R&D Systems) at 4000cells / pl, and plated on 24-well culture plates as 50-pl domes (concentration of 200,000 cells / 50 ul BME). After incubation at 37 °C to allow the BME to solidify, uORG- serum-free medium was added. The medium included R-spondin conditioned medium which was used at 10 % (v / v) concentration or recombinant protein Human R-Spondinl, peprotech (120-38-100ug; 250ng / ml) 1%NAC, lOng / ml FGF-10, 50ng / ml EGF, 5ug A83-01, 1.5% B27 (X50), 1 % L-glutamine solution, 1 % Penicillin- Streptomycin Solution, XI NEAA, 10 uM Y-27632. uORGs were mechanically disrupted with a bovine serum albumin (BSA)-treated 1,000-pl pipette tip and passaged at a 1:2 ratio every 2-3 weeks, depending on the proliferation rate.

[0204] Two-step protocol: Upon reaching 80-100 % confluence at P0, cells grown as 2D uEpC were trypsinized and seeded on poly (2-hydroxyethylmethacrylate) (poly-HEMA; Sigma- Aldrich) pre-coated plates, in serum free media (SFM: DMEM / F-12 [HAM] 1:1, 1% L-glutamine solution, 1% Penicillin- Streptomycin Solution (*10,000 units / ml), 1-10% Non-essential Amino acids (NEAA), 1-3% Sodium Pyruvate, 0.02% Chemically defined (CD), Lipid mix, 2% B27 supplement (X50), Heparin sodium Fresenius 5000IU / lml, Animal Free Human EGF (10-50 ng / ml), Human FGF-basic (10-50 ng / ml), 0.06-0.1 mg / ml Glucose, 0.01-1 mg / ml Transferrin, 2.5-10 ug / ml Insulin, 0.1 ug / ml Putrescine, 3 nM Sodium Selenite, 2 nM Progesterone. 24 hours later the floating aggregates were collected and seeded with BME and the protocol continued as a one step protocol.

[0205] Single cell RNA sequencing

[0206] Preparation of single cell suspension from uSPH.’ uSPH culture medium was centrifuged and resuspended in 3 ml IX TrypLe express (#12605010, Gibco) Resuspend spheroid pellet in TrypLE Express (IX) - 2ml for a T175 flask and 1ml for a T75 flask. (Ref. 12605-010, Gibco). The tubes were incubated at 37°C for 10-minute cycles after which the uSPH are disturbed by pipetting U&D 15 times. After each cycle, a 10- l cell suspension is checked under a microscope to determine whether a single cell suspension was achieved. 1-2 cycles are usually enough to break down the uSPH to single cells without affecting cell viability.

[0207] Preparation of single cell suspension from uORG: The culture medium was aspirated and 1 ml of warm 1 mg / ml Dispase (# 07923, Stem cell Technologies) was added to a 24-well containing a BME droplet with uORGs. The droplets were then gently broken-down by pipetting up and down (U&D) using a treated 1,000-pl pipette tip. Untreated tips are treated by 5X U&D of 1% BSA-PBS solution to prevent uORGs fragments from sticking to the pipette tip. After 40 minutes at 37°C, the BME is dissolved and the uORGs were transferred to 15 ml tubes containing ice cold DMEMfl2, centrifuged and resuspended in 3 ml IX TrypLe express (#12605010, Gibco) supplemented with lOuM ROCKi. The tubes were incubated at 37 °C for 10-minute cycles afterwhich the uORGs are disturbed by pipetting U&D 15 times. After each cycle, a 10- pl cell suspension is checked under a microscope to determine whether a single cell suspension was achieved. 2-3 cycles are usually enough to break down the uORGs to single cells without affecting cell viability.

[0208] Single-cell RNA-seq library preparation and sequencing: single cells were (harvested, washed and) resuspended in phosphate buffered saline (PBS) supplemented with 0.5% bovine serum albumin (BSA) to achieve an optimal concentration of approximately 1000 cells / pL for loading onto the Next GEM Chip (lOx Genomics). Libraries were prepared using the lOx Genomics Chromium Controller in conjunction with the single-cell 3’ v3.1 kit, protocol revision E. The cDNA synthesis, barcoding and library preparation were carried out according to the manufacturer's instructions. Briefly, cDNA amplification was performed for 11 cycles and sample index PCR was performed for 12 cycles using Chromium i7 Sample Indices. The resulting libraries were quantified and analysed by Qubit and TapeStation and were sequenced on the NextSeq 500 platform (Illumina), following the manufacturer's protocol, and a NextSeq 500 / 550 High Output Kit v2.5 (75 cycles) kit (Illumina).

[0209] Pre-processing: Cell ranger (v3) pipeline was used to perform sample demultiplexing to produce FASTQ files for each sample. Sample were aligned to the GRCh38 reference human genome and cell barcodes were filtered so only barcodes connected to unique molecular identifiers and gene ids were used in a matrix file compatible with Seurat.

[0210] Quality control: The standard QC workflow as presented in Seurat vignettes from the Satija Lab were followed. In brief, low-quality cells, empty droplets with low gene counts, or doublets / multiplets with abnormally high gene counts were filtered out. Cells with high mitochondrial gene counts were also excluded. After filtration, 16,727 cells from uSPH and 17,957 cells from uORG remained. The data was then normalized, highly variable features were identified, scaling and linear dimension reduction was performed.

[0211] Clustering analysis: The Seurat’ sOick or tsp hete to text. (v5.0.0) FindNeighborsQ and FindClusters() were used to produce a UMAP containing the various single-cell clusters. The resolution was set to 0.5 and number of dimensions to 1:35. This produced 8 clusters of uSPH and 9 clusters of uORG. Differentially expressed genes were found for each cluster using FindAllMarkersQ, with parameters min.pct=0.25, logfc.threshold=0.25 and p_val_adj<0.05.

[0212] Projection analysis to FKO dataset: To compare the single-cell datasets of uSPH and uORG with fetal kidney organoid (FKO) data, we performed a projection analysis using Seurat’s MapQueryO function. This method projects the test data (uSPH and uORG) onto the referenceFKO dataset by identifying anchors between the reference and test datasets, enabling the alignment and comparison of shared cell populations.

[0213] Comparison Analysis between uSPH and uORG: To compare uSPH and uORG datasets, we performed dataset integration using the RunHarmonyO function on a merged Seurat object, allowing for batch effect correction and harmonization of the data. Differential expression analysis was then conducted using the FindAllMarkers() function with parameters only.pos = TRUE, min.pct = 0.25, and logfc. threshold = 0.5. Genes with an adjusted p-value (p_val_adj) < 0.05 were considered significant.

[0214] W hole-mount staining

[0215] uORG were cultured in imaging chambers (p-Slide 8 well, ibidi) and fixed for 15 min in 4% paraformaldehyde (PFA) solution. The PFA solution was then replaced with 0.3% Triton X-100 in PBS for 30 min at room temperature for permeabilization. Samples were then washed three times with PBST (0.1% Triton X-100 in PBS) for 5 min. Blocking solution (3% BSA in PBST) was added for 1 h at room temperature, and this was followed by an overnight (ON) incubation at 4°C in blocking solution supplemented with the primary antibodies. After three washes with PBST, secondary antibodies — Alexa488-conjugated anti-rabbit (1:500, A21202, Invitrogen), Alexa555-conjugated anti-mouse (1:500, A31570, Invitrogen), Alexa647-conjugated anti-goat (1:500, abl50135, Abeam), and Streptavidin-conjugated Alexa647 (1:500, S21374, Thermo Fisher) — and actin stain (1:1000, abl76759, Phalloidin-iFluor 647 Reagent, Abeam) were added in blocking solution and the samples incubated overnight at 4°C. The samples were then washed in PBST, and DAPI-PBST solution was added (1:250, #62248, Thermo Fisher) for 10 min. The samples were washed again and imaged with a confocal microscope ZEISS LSM700; photo processing was done using ZEN blue. Widefield z-stack images were taken with a Nikon CSU-W1 Yokogawa Spinning Disk Field Scanning Confocal System and processed using Fiji.

[0216] Example 1

[0217] Establishment and characterization of kidney organoids derived from urine of healthy individuals

[0218] In order to establish urine derived organoids, the present inventors employed a previously published protocol by Schutgens et al [Schutgens, F., Rookmaaker, M.B., Margaritis, T. et al. Tubuloids derived from human adult kidney and urine for personalized disease modeling. Nat Biotechnol 37, 303-313]. In this protocol, the urine was collected from healthy adult donors and immediately seeded on a matrix (after centrifugation and wash). Attempts to repeat the protocol described by Schutgens et al. have failed: specifically, following four attempts of using freshlyisolated urine derived cells, the cells failed to grow and organize into viable organoids (Figure 1A). It is believed that small cell numbers of urine derived cells were insufficient to robustly form organoids. Therefore, an additional step of expending the urine-derived cells prior to organoid formation was attempted. First, a urine sample was collected and cells were seeded (after centrifugation and wash) on gelatin coated plates to let the cells proliferate and expand in 2D (adherent) conditions with serum containing media (Figure IB). After a week in 2D culture the cells were harvested and seeded as follows: 1. One-step protocol: on matrix comprising basement membrane extract (BME) with serum free media, for organoids generation, or 2. Two-step protocol: first, on low adherent conditions (as spheroids- 3D) for one day in order to allow aggregates formation and then transferred onto matrix (Figure IB -left panel). Following this protocol, organoids were formed from both 2D and 3D expanded cells (Figure IB- right panel), and therefore it was decided to continue with the one-step protocol.

[0219] Next, the established kidney organoids derived from the urine were characterized by immunostaining for kidney epithelial markers. Urine-derived organoids were positively stained for distal tubule markers, SLC12A1 and EMA, and for proximal tubule markers, LTL and LRP2 (Figure 1C). Positive staining for Ki67 was evident suggesting that the cells have proliferative nature of organoid residing cells.

[0220] Next, the same one step protocol was effected on cells derived from healthy children to expand age range (can serve as a control for pediatric kidney diseases). Accordingly, a 2D culture was generated from a five-year old child (Figure ID) and then upon reaching confluence the cells were transferred onto a matrix to form organoids (Figure ID). The cells created massive number of organoids that expanded along consecutive passages (e.g., 25 passages which correspond to about 19 months in cultur). Ultrastructural analysis using electron microscopy further confirmed the polarity of urine derived organoids (uORG), with the basal membrane positioned on the outer side of the tubule, tight junctions between tubular cells, and brush borders at the luminal (apical) inner side (Figure IE).

[0221] Example 2

[0222] Establishment and characterization of kidney organids from derived from urine of CKD patients

[0223] Next, the protocol for uORG formation was implemented on patients with a variety of kidney tubular diseases. First, urine was collected from an 18-year-old patient with a ciliopathy, kidney disease manifested by the presence of kidney cysts that develop due to abnormal epithelial cell proliferation, growth, and polarity, downstream of dysregulated ciliary dependent signaling.Due to cystic-associated kidney injury and systemic inflammation, cases result in kidney failure requiring dialysis and transplantation. The one-step protocol was employed: first, culturing the cells in 2D conditions followed by seeding on BME (Figure 2A). The urine derived kidney cells from the ciliopathy patient were able to form kidney organoids and expand along passages (Figure 2B shows till passage 4).

[0224] Next, a urine sample was collected from a seven-year-old child diagnosed with Fanconi Bickel Syndrome. Fanconi-Bickel syndrome is a rare glycogen storage disease leading to severe proximal renal tubular dysfunction and impaired glucose and galactose metabolism. This type of patient can serve as an excellent control because of the development of a renal Fanconi syndrome not due to a mitochondrial defect. This is of significance, since, as mentioned, they can serve as a control for mitochondrial diseases. Second, due to their ability to generate organoids from CKD patients with various etiologies, including Fanconi-Bickel syndrome.

[0225] kidney cells derived from the patient’s urine (Figure 2C) had the capacity to form well established kidney organoids that were growing for about six months and expanded into many passages (p 14) without losing their morphology (Figure 2C) and expansion capacity (Figure 2D). Next, the organoids were characterized by immunostaining: the organoids were positively stained for the kidney epithelial markers E-cadherin, EpCAM and LRP2 (Figure 2E). Noteworthy is that one of the Fanconi-Bickel organoids exhibited kidney polarity as it positively stained for phalloidin in the apical membrane (purple), E-cadherin in the tight junctions (green) concomitantly with EpCAM (red) in the basolateral membrane (Figure 2E white arrow).

[0226] Finally, urine samples were collected from two Pearson patients. Pearson syndrome is a rare mitochondrial disease. It is a severe disorder that usually begins in infancy and affects various parts of the body including the kidney. Mitochondrial dysfunction can lead to a variety of renal manifestations. Examples of tubular manifestations are renal Fanconi Syndrome, which is often found in patients diagnosed Pearson syndrome. Urine was collected from a young adult (17y old-donor I) and from a child (9y old-Donor II) (Figure 3A). It was observed that urine derived kidney cells had the capacity to generate kidney organoids from both patients (Figure 3B). Urine derived kidney organoids generated from Pearson syndrome could expand and propagate along passages (Figure 3B). Two organoids underwent EM imaging, one from a patient with Pearson syndrome, showing polarized cells with microvilli in a surface opposing the basal membrane, along with tight junctions, presenting abnormal contours and cristae of the mitochondria (Figure 3C).Example 3

[0227] ARPKD Human Kidney Organoid - Model for Pathology Investigation and Treatment Development

[0228] Autosomal recessive polycystic kidney disease (ARPKD) is a severe genetic disorder characterized by the formation of cysts predominantly in the distal tubules and collecting ducts, leading to significant kidney enlargement and eventual renal failure. The development of effective treatments has been limited by the lack of accurate disease models that replicate the complex renal cystic phenotype observed in ARPKD patient.

[0229] The present inventors have successfully generated organoids from the urine and kidney tissue of ARPKD patients, accurately replicating the cystic morphology characteristic of the disease (Figure 4A). These organoids exhibit proper nephron tubule orientation (Figure 4B) and positive KI67 staining, indicating ongoing cellular proliferation (Figure 4B). The distal tubules are larger than the proximal ones (Figure 4C), consistent with ARPKD pathology. ARPKD is primarily caused by mutations in the PKHD1 gene, resulting in dysregulated cyclic adenosine monophosphate (cAMP) levels within cells. Elevated cAMP levels have been associated with cyst growth, affecting fluid accumulation and cell proliferation. Consequently, the present study aimed at identifying potential ARPKD treatments that can target both liquid accumulation and cellular proliferation. Elevated cAMP levels in the ARPKD organoids match the expected disease profile (Figure 4D).

[0230] Next, two FDA-approved drugs, Tolvaptan and Everolimus, were selected for evaluation. Tolvaptan, a selective arginine vasopressin V2-receptor (V2R) antagonist, reduces cAMP levels and cyst enlargement by inhibiting the action of arginine vasopressin (A VP). Tolvaptan is already approved for the treatment of Autosomal Dominant Polycystic Kidney Disease (ADPKD) and is currently being investigated in clinical trials with ARPKD patients. Everolimus, an mTOR pathway inhibitor, modulates various cellular processes, including cell growth and proliferation. By inhibiting mTOR, Everolimus may impede abnormal cell proliferation and cyst growth.

[0231] ARPKD uORGs were treated with Tolvaptan, Everolimus, or a combination of both drugs to assess their impact on cyst size. Quantitative analysis of cyst area was performed before and after treatment. Remarkably, treatment with Tolvaptan (10 pM) or Everolimus (1 pM) individually resulted in significant reductions in cyst size (Figure 4E). However, the combination therapy demonstrated the most substantial reduction in cyst size (Figure 4E). These findings suggest that the combination of Tolvaptan and Everolimus could offer a promising therapeutic strategy for ARPKD patients. Similar results were observed when treating kidney organoids derived from ARPKD patient kidney tissue, with a significant reduction in cyst size followingtreatment (Figure 4F). These findings further support the similarity between organoids originating from different kidney sources.

[0232] Example 4

[0233] Transcrip tomic Insights and Therapeutic Potential of uORG

[0234] The present inventors have previously demonstrated the generation of 3D kidney spheroids derived from expanded kidney cells obtained from urine (see e.g., WO2022 / 084998). Both spheroids and organoids, collectively termed kidney organospheroids (uKOS). RNA-seq analysis revealed that the uSPH exhibit epithelial re-differentiation, with the upregulation of genes associated with various renal tubular epithelial cell types, including proximal tubule, distal tubule, and loop of Henle. Additionally, an upregulation in Wnt signaling pathway was observed, accompanied by a reduction in profibrotic and inflammatory genes (as presented in WO2022 / 084998). Overall, the uSPH exhibited an anti-fibrotic, anti-inflammatory gene signature, suggesting a potential therapeutic application for uSPH. To characterieze uORG transcriptome the present inventors interrogated uKOS at the single cell level, specifically separately analyzing and comparing uSPH and uORG. S ingle-cell RNA sequencing (scRNA-seq) analysis of uSPH further revealed cells expressing renal maturation markers from the different nephron segments (PT, LOH, DT / CNT, CD), confirming the presence of kidney marker genes consistent with bulk analysis and more resemblance to adult kidney in the 3D kidney cultures (Figure 5A). Close examination of uPSH and uORG revealed to some extent varying enrichment patterns. Differential expression analysis allowed identification of markers of specific segments enriched in certain uSPH clusters, showing significant enrichment for LOH representation, with three LOH-like clusters: LOH1 (cluster 1; SPP1, CLU, CLDN10), LOH2 (cluster 3; SLC12A1, CLDN16, KCNJ1), and LOH3 (cluster 6; UMOD, PTPN4, LAMA1) (Figure 5B). Additionally, cluster 4 is enriched with TMEM129+cells, which are predominantly found in the LOH segment. Also identified were a proximal-like cluster (Cluster 0; SLC3A1, BICC1) and a parietal epithelial-like cluster (cluster 2; CDH6, AKAP12, MYH9) (Figure 5B). Interestingly, an OLFM3+cluster (cluster 7) was identified, which is a gene expressed during early podocyte development, and a SIX1+cluster (cluster 5). Additionally, a stress response cluster marked by NPC1, SIRT1, and SOD2 was also observed (cluster 8) (Figure 5B). While single-cell analysis of uORG revealed that, as found for uSPH, there is expression of renal tubule markers from different nephron segments (Figure 5C). Distinct cell populations within the organoids were discernible, including a small population of proliferating cells identified within cluster 7, marked by the expression of MKI67, CDK1, TOP2A, CDC20, and CCNA2 (Figure 5D). Cluster 8 represented a more distal-like population with markers such as MUC1, SLC8A1, KRT7, CDH1, and GATA3. Additionally, cluster 9 wasmetabolically active, characterized by the expression of proteins associated with high metabolic activity, including EIF4B, HSP90B1, HSPA8, MT-CO1, ATP5F1A, and PDIA6 (Figure 5D). It was found that similarly to uSPH, all identified clusters expressed markers associated with progenitor and early differentiation stages, such as CD24, PR0M1, CD44, VIM, and PAX2, indicating a predominant state of progenitor or developing epithelial cells (Figure 5C). Additionally, these clusters exhibited the expression of inhibitors of DNA-binding proteins (ID1-4), which are known for inhibiting differentiation and promoting proliferation in progenitor cells (Figure 5D). This widespread progenitor-like expression profile is consistent with findings from previous studies of organoids derived directly from human kidney tissue and CD24+kidney cells. This intriguing finding suggested performing projection analysis using human fetal kidney organoid (hFKO) single cell RNAseq data to show that both uSPH and uORG have populations corresponding to the developed tubular regions of hFKO (Figure 5E). Since both uSPH and uORG were both projected on developed epithelia of hFKO, their maturity levels were compared. Accordingly, differential expression analysis between the single-cell data of uSPH and uORG revealed that while there was a notable enrichment of proximal tubule populations in uORG compared to uSPH, LOH-related genes, such as SLC12A1, TFAP2B, and TMPRSS4, were more highly expressed in uSPH, consistent with our cluster annotation findings (Figure 5F). Moreover, uSPH appeared to be more mature than uORG, as suggested by the higher expression of progenitor / de-differentiation genes like NCAM1, ALDH1A2, and VCAM1 in uORG (Figure 5F), as well as gene ontology analysis showed an enrichment of kidney development processes in uORG compared to uSPH (Figure 5G and 5H). Additionally, uSPH demonstrated an enrichment of HLA class I genes, further confirming its more differentiated state relative to uORG (Figure 5G and 5H). Gene ontology analysis also identified pathways related to stress response in uSPH, indicating a potential protective capability (Figure 5G-5I).

[0235] In addition to their unique signature, uSPH demonstrated tubulogenic and therapeutic effects when injected into a CKD mouse model, notably reducing fibrosis in the injured kidney. This anti-fibrotic effect was further supported by in vitro tests using activated fibroblasts treated with secreted factors from uSPH (the secretome). To evaluate the therapeutic potential of uORG, the present inventors applied the same in vitro tests with uORG-derived secretome. Notably, the uORG secretome showed significant effects on activated fibroblasts, reducing fibrotic markers at both the RNA and protein levels. (Figure 5 J).

[0236] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to thoseskilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0237] It is the intent of the Applicant(s) that all publications, patents and patent applications referred to in this specification are to be incorporated in their entirety by reference into the specification, as if each individual publication, patent or patent application was specifically and individually noted when referenced that it is to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is / are hereby incorporated herein by reference in its / their entirety.

Claims

WHAT IS CLAIMED IS:

1. A method of producing a urine derived kidney organoid, the method comprising: (a) isolating urine derived epithelial cells (UD-EpCs) from urine of a human subject; (b) expanding said UD-EpCs in a 2 dimensional (2D) setting under conditions that allow obtaining expanded UD-EpCs;(c) culturing said expanded UD-EpCs under conditions which allow formation of urine derived organoids (uORGs); and(d) passaging said uORGs.

2. The method of claim 1, wherein said expanding is until PO.

3. The method of any one of claims 1-2, wherein said conditions of step (b) comprise coated dishes which ensure substrate adherence.

4. The method of any one of claims 1-2, wherein said conditions of step (b) comprise presence of serum.

5. The method of any one of claims 1-4, wherein said conditions of step (b) comprise RE:MC or KSFM.

6. The method of any one of claims 1-4, wherein said conditions of step (b) comprise RE:MC.

7. The method of any one of claims 1-6, wherein said conditions of step (b) comprise presence of CD40 ligand and optionally at least one of PDGF-AB-10, EGF and FGF-basic.

8. The method of any one of claims 1-7, wherein said culturing said expanded UD-EpCs comprises formation of spheroids followed by subjecting said spheroids to said conditions which allow formation of urine derived organoids (uORGs).

9. The method of claim 8, wherein said formation of spheroids is under low adherence conditions, followed by subjecting said spheroids to said conditions which allow formation of uORGs in the presence of extracellular matrix (ECM) that encapsulates said spheroids.

10. The method of any one of claims 1-7, wherein said culturing said expanded UD-EpCs does not allow formation of spheroids.

11. The method of any one of claims 1-10, wherein said conditions which allow formation of urine derived organoids (uORGs) comprise at least one of N-acetylcysteine (NAC) or an analog thereof, ROCK inhibitor, epidermal growth factor (EGF), fibroblast growth factor 10 (FGF-10), B27 and a Wnt activator.

12. The method of any one of claims 1-11, wherein said culturing said UD-EpCs is by encapsulating said UD-EpCs within an extracellular matrix (ECM).

13. The method of claim 9 or 12, wherein said ECM comprises basement membrane extract (BME).

14. The method of any one of claims 1-13, said culturing said expanded UD-EpCs is in the absence of serum.

15. The method of any one of claims 1-14, wherein said passaging is for at least 10 passages.

16. The method of any one of claims 1-15 further comprising isolating cells from UD-EpCs or uORGs and / or analyzing said expanded UD-EpCs or uORGs or said cells thereof.

17. An isolated urine derived kidney organoid (uORG) ex vivo produced from urine-derived human epithelial cells and comprising a cystic interior, wherein the organoid is proliferative and a polarized tubule structure prior to transplantation.

18. An isolated urine derived kidney organoid (uORG) obtainable according to the method of claim 1.

19. The uORG of any one of claims 17-18, comprising cells expressing renal progenitor markers e.g., CD24, PR0M1, CD44, VIM and / or PAX2 as determined by single-cell RNA sequencing.

20. The uORG of any one of claims 17-19, expressing sternness markers e.g., inhibitor of DNA-binding (ID1, ID2, ID3 and ID4) as determined by single-cell RNA sequencing.

21. The uORG of any one of claims 17-20, expressing higher level of early epithelial markers and / or progenitor epithelial markers selected from the group consisting of NCAM1, ALHD1A2 and VCAM, as compared to a nephro spheroid as determined by single-cell RNA sequencing.

22. The uORG of any one of claims 17-21, expressing higher level of cell adhesion markers selected from the group consisting of ICAMN1, CAV1 and CDH2, as compared to a nephro spheroid as determined by single-cell RNA sequencing.

23. The uORG of any one of claims 17-22 comprising a cell cluster expressing cell proliferation markers selected from the group consisting of MKI67, CDK1, TOP2A, CDC20 and CCNA2, as determined by single-cell RNA sequencing.

24. The uORG of any one of claims 17-23, comprising a cell cluster expressing distal tubule related markers selected from the group consisting of MUC1, CDH1, and SLC8A1, as determined by single-cell RNA sequencing.

25. The uORG of any one of claims 17-24, comprising a cell cluster expressing metabolic activity markers selected from the group consisting of EEF1A1, ACTG1 and ATP5F1A, as determined by single-cell RNA sequencing.

26. The method or uORG of any one of claims 1-25, wherein said urine-derived epithelial cells are from a healthy subject.

27. The method or uORG of any one of claims 1-25, wherein said urine-derived epithelial cells are from a subject diagnosed with a chronic kidney disease (CKD).

28. The method or uORG of claim 27, wherein said CKD is selected from the group consisting of ciliopathy, Fanconi Bickel Syndrome, Pearson syndrome and Autosomal recessive polycystic kidney disease (ARPKD).

29. The method or uORG of any one of claims 1-28, wherein said urine-derived epithelial cells are from a subject under 18 years of age.

30. The method or uORG of any one of claims 1-28, wherein said urine-derived epithelial cells are from a subject being at least 18 years of age.

31. A composition of matter comprising multiple uORGs from a single human subject, said multiple uORGs comprising all tubular segments including a distal tubule, a proximal tubule, a collecting duct and a loop of Henle yet being devoid of glomerulus.

32. A bank comprising multiple compositions of claim 31 from multiple subjects.

33. Secretome of the uORG of any one of claims 17-30.

34. A method of producing a secretome, the method comprising:(a) providing a culture of the uORGs of any one of claims 17-30;(b) collecting conditioned medium of said culture, said conditioned medium comprising said secretome.

35. The method of claim 34, further comprising isolating said secretome or a portion thereof from said conditioned medium following step (b).

36. A method of regenerating renal function, the method comprising administering to a subject in need thereof the uORGs of any one of claims 17-30 or secretome thereof, thereby regenerating renal function.

37. A method of treating a chronic kidney disease (CKD), the method comprising administering to a subject in need thereof the uORGs of any one of claims 17-30 or secretome thereof, thereby treating the CKD.

38. The uORGs of any one of claims 17-30 or secretome thereof for use in treating a chronic kidney disease (CKD).

39. A method of drug design, the method comprising determining an effect of a test drug on the uORGs of any one of claims 17-30 or secretome thereof.

40. A method of selecting treatment to a subject diagnosed with a kidney disease, the method comprising:(a) contacting a uORG produced from UD-EpCs of a subject diagnosed with the kidney disease; and(b) determining an alleviation in a renal symptom evident in said uORG following said contacting, said alleviation being indicative of an efficacious therapy.

41. The method of claim 39 or 40, wherein said test drug is Tolvaptan and / or Everolimus.

42. The method of any one of claims 39-41, wherein said effect is cyst size.

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