Compositions and Methods

Abstract

To provide a kidney organoid and a manufacturing method thereof which can be used in various applications such as disease modeling, drug screening, regenerative medicine, and 5 scaling-up production of a kidney cell.SOLUTION: Provided is a kidney organoid including less than 50 nephrons.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to renal organoids and methods for their production, which can be used in a variety of applications, including disease modeling, drug screening, regenerative medicine, and scale-up production of renal cells. [Background technology]

[0002] The kidneys play a major role in removing waste products and maintaining body fluid volume. The functional unit is known as the nephron. Human kidneys contain up to 2 million epithelial nephrons, which are responsible for blood filtration and all arise after birth. Nephron progenitor cells are absent from postnatal human kidneys. This absence of a progenitor cell population means that nephrons cannot self-renew, and subsequent injury, aging, and disease can lead to end-stage renal disease (ESDR). Limited therapeutic options for ESDR treatment typically place additional stress on already damaged kidneys. At the end of kidney disease, the only available, but expensive, treatment options are dialysis and / or kidney transplantation, both of which have significant disadvantages and impair the patient's quality of life.

[0003] Directing the differentiation of human pluripotent stem cells (hPSCs), including both human embryonic stem cells (hES) and human induced pluripotent stem cells (hiPS), toward distinct cellular endpoints has enabled the generation of organoid models of various human tissues, including the kidney. Previous organoid models, such as those discussed in Takasato et al. (2015) Nature, Vol. 526:564-568, are expensive and can produce organoids with complex three-dimensional structures, limiting their utility for imaging and screening applications. Furthermore, these organoids can suffer from proliferation limitations after 3 weeks of culture, limiting their ability to expand and mature. This makes current renal organoid production protocols suboptimal as a cell source for regenerative medicine or disease modeling. Therefore, there is a current need for renal organoids and methods for their production. Summary of the Invention

[0004] Surprisingly, the present inventors have identified renal organoids with simplified three-dimensional structures. Such organoids are advantageous because they are easier to image and culture long-term. Thus, in one example, the present disclosure encompasses renal organoids comprising fewer than 50 nephrons. In another example, the renal organoids comprise fewer than 25 nephrons. In another example, the renal organoids comprise fewer than 15 nephrons. In another example, the renal organoids comprise 5-12 nephrons. In one example, renal organoids are produced by culturing a population of intermediate mesoderm (IM) cells in a cell culture medium under conditions sufficient to promote the development of renal organoids. In this example, the IM cell culture medium may comprise approximately 180-220 ng / ml of FGF9.

[0005] In one example, kidney organoids were 0.5 x 10 6 ~1.5×10 6 In another example, kidney organoids are produced by swirling IM at 0.8 x 10 cells / ml. 6 ~1.2×10 6Produced by circulating IM of about 1000 cells / ml.In another example, said renal organoid comprises the cell that expresses high levels of nephron markers.In another example, said renal organoid comprises the cell that expresses any one or more of PAX2, LHX1, SIX1, OSR1, WNT11 and GATA3 at high levels.In another example, said renal organoid comprises the cell that expresses any one or more of PAX2, LHX1, SIX1, OSR1, WNT11 and GATA3 at high levels.In another example, said renal organoid comprises the cell that expresses any one or more of PAX2, SIX1, LHX1, OSR1, WNT11 and GATA3 at high levels.In another example, said renal organoid comprises the cell that expresses any one or more of PAX2, SIX1, LHX1, OSR1, WNT11, GATA3, PAX8, EYA1 and CITED1 at high levels, and / or expresses any one or more of PDGFRA, MEIS2, WT1 and / or C-RET at low levels. In another example, the renal organoids comprise cells that express high levels of PAX2, LHX1, SIX1, OSR1, WNT11, GATA3, PAX8, EYA1, and CITED1.

[0006] In another example, the renal organoid is derived from stem cells selected from the group consisting of H9, hES3, iPSC GAPTrap td-Tomato, CRL1502.C32, CLR1502.3, hES3 SOX17mCherry, or H9 GAPTrap Luc2.

[0007] In another example, said renal organoid comprises the cell that expresses low levels of interstitial markers.In another example, said renal organoid comprises the cell that expresses low levels of PDGFRA, MEIS2, WT1 and / or C-RET.In another example, said renal organoid comprises NPHS+podocytes, LTL+proximal tubule segments, ECAD+distal tubule segments, ECAD+ / GATA3+collecting ducts or combinations thereof.In another example, said renal organoid comprises 1x10 4 ~5×10 4 In another example, the renal organoids contain about 1.5 x 10 cells. 4 ~2.5×10 4In another example, the renal organoids contain approximately 100 cells. In another example, the renal organoids have a diameter of approximately 250 to 500 μm. In another example, the renal organoids survive for at least 18 days under rotation culture. In another example, the renal organoids survive for at least 3 weeks under rotation culture. In another example, the renal organoids survive for at least 4 weeks under rotation culture. In another example, the nephrons of the renal organoids include collecting ducts (GATA3+; ECAD+), early distal tubules (GATA3-; LTL-; ECAD+), early distal tubules (LTL+; ECAD-), and glomeruli (WT1+).

[0008] Surprisingly, the present inventors have also confirmed that the cell of the renal organoid disclosed herein continues to divide under rotation culture after 7 days (for example, 7+7 days).Without wishing to be bound by any particular theory, this may indicate that the organoid that uses rotation culture to produce is more suitable for therapeutic applications such as transplantation.

[0009] In another example, the present disclosure encompasses the composition comprising the renal organoids described herein.In another example, the present disclosure encompasses the composition comprising the renal organoids described herein or their digestion products.For example, the present disclosure encompasses the composition comprising the renal organoids described herein or their enzymatic digestion products.

[0010] Again, without wishing to be bound by any particular theory, it is believed that the low complexity of the renal organoids disclosed herein may make them more suitable for producing transplant compositions.For example, the compositions produced from renal organoids with low complexity may be more likely to differentiate into correct cell types, rather than forming teratomas or cartilage.In another example, the present disclosure encompasses compositions comprising the enzyme digestion of the renal organoids described herein.

[0011] In another example, the present disclosure encompasses a method for treating kidney disease, comprising administering a composition described herein to a subject in need of treatment. In one example, the composition comprises whole organoids. In another example, the composition comprises digested material of the organoids disclosed herein. For example, the kidney disease can be kidney failure. In one example, the composition is administered intravenously. In another example, the composition is administered by intrarenal artery injection, intrarenal parenchyma injection, implantation, or subcapsular kidney transplantation.

[0012] In another example, the present disclosure includes an in vitro method for producing renal organoids, comprising spinning a population of intermediate mesoderm (IM) cells in a cell culture medium containing FGF.

[0013] In one example, the method provides a cost-effective means for scale-up production of renal cell types in vitro. In one example, the IM cells are spun in a medium containing FGF, CHIR, and heparin. In one example, the IM cells are spun in culture for at least 5 days, including spun in a cell medium containing FGF, heparin, CHIR, and a ROCK inhibitor for the first 24 hours, followed by culturing the cells in a cell medium containing FGF, heparin, and CHIR for the following 4 days. In this example, a ROCK inhibitor is present in the cell medium for the first 24 hours and not present thereafter. In one example, the cell medium contains 100-300 ng / ml FGF9. In one example, the cell medium contains 180-220 ng / ml FGF9. In one example, the cell medium contains PVA and MC. In one example, the first 24 hours involve spinning the cells in cell culture medium containing FGF, 0.5-1.5 μg / ml heparin, 0.5-1.5 μM CHIR, and 9-11 μM ROCK inhibitor. In another example, the subsequent four days involve spinning the cells in cell culture medium containing FGF, 0.5-1.5 μg / ml heparin, and 0.5-1.5 μM CHIR. In this example, ROCK inhibitor is not present in the cell culture medium during the subsequent four days. In another example, the subsequent four days involve culturing the cells in cell culture medium containing FGF9, heparin, CHIR, MC, and PVA. In another example, the subsequent four days involve culturing the cells in cell culture medium containing FGF9, heparin, CHIR, 0.05-0.2% MC, and 0.05-0.2% PVA. In another example, for the remaining culture days after at least 5 days have passed, the cells are cultured in a cell culture medium containing 0.05 to 1.5% PVA and 0.05 to 1.5% MC. In this example, the cell culture medium may contain PVA and MC without containing any of FGF9, heparin, CHIR, and a ROCK inhibitor. In another example, the FGF-containing cell culture medium contains at least 100 ng / ml FGF9. In another example, the FGF-containing cell culture medium contains at least 150 ng / ml FGF9.In another example, the cell culture medium containing FGF contains 150 to 250 ng / ml of FGF9. In another example, the cell culture medium containing FGF contains 180 to 220 ng / ml of FGF9.

[0014] In some examples, the methods for producing renal organoids disclosed herein improve cell yields compared to those obtained from organoids produced without rotation, such as those described by Takasato et al. (2015). In one example, after 10 days of rotational culture, a 30-fold increase in cell yield can be observed from the starting number of IM cells added to the rotational culture. In another example, after 10 days of rotational culture, a 35-fold increase in cell yield can be observed from the starting number of IM cells added to the rotational culture. In another example, after 12 days of rotational culture, a 40-fold increase in cell yield can be observed from the starting number of IM cells added to the rotational culture. In another example, after 12 days of rotational culture, a 45-fold increase in cell yield can be observed from the starting number of IM cells added to the rotational culture. In another example, after 12 days of rotational culture, a 30-40-fold increase in cell yield can be observed from the starting number of IM cells added to the rotational culture. In another example, after 18 days of rotational culture, a 30-40 fold increase in cell yield can be observed from the starting number of IM cells added to the rotational culture.

[0015] In one example, the IM cells are spun at 30-90 rpm, hi another example, the IM cells are spun for 18-24 days.

[0016] In another example, the IM cells are produced by culturing a stem cell population for at least 7 days, the first 4-5 days comprising culturing the stem cells in a cell culture medium containing at least 6 μM of a Wnt / β-catenin agonist, and the remaining days comprising culturing the cells in a cell culture medium containing FGF and at least 0.5 μM of a Wnt / β-catenin agonist. In one example, the Wnt / β-catenin agonist is CHIR. In these examples, the FGF-containing cell culture medium may contain 100-300 ng / ml of FGF9. In these examples, the FGF-containing cell culture medium may contain 0.5-1.5 μM of CHIR. In these examples, the FGF-containing cell culture medium may further contain 0.5-1.5 μg / ml of heparin. In one example, the IM cells are dissociated with EDTA or trypsin or TrypLE™ Select and passed through a mesh screen before spinning. In one example, the stem cells are pluripotent stem cells, embryonic stem cells, or induced pluripotent stem (iPS) cells. In one example, the stem cells are selected from the group consisting of H9, hES3, iPSC, GAPTrap td-Tomato, CRL1502.C32, CLR1502.3, hES3 SOX17mCherry, or H9 GAPTrap Luc2.

[0017] The present inventors have confirmed that the method of the present disclosure requires a relatively small number of starting cells.This is advantageous because it allows the cost-effective scaling up of organoid and cell culture.Therefore, in another example, the method of the present disclosure can be used to produce 0.5 × 10 6 cells / ml~3×10 6 In another example, the method of the present disclosure includes spinning an IM cell population of 0.8 x 10 cells / ml. 6 cells / ml~1.2×10 6 This method comprises rotating IM cell population at about 1000 cells / ml.Therefore, in one example, the present disclosure encompasses the renal organoid disclosed herein, which is produced by rotating IM cell population.The example of timing and medium used for rotating IM cell population to produce renal organoid is disclosed herein.In one example, the organoid is 0.5x106 cells / ml~3×10 6 In another example, the organoids are prepared by spinning an IM cell population containing 2 x 10 cells / ml of IM cells. 6 In another example, the organoids are prepared by spinning an IM cell population containing less than 0.5 x 10 IM cells. 6 cells / ml~1.5×10 6 In another example, the organoids are prepared by spinning an IM cell population containing 0.8 x 10 cells / ml of IM cells. 6 cells / ml~1.2×10 6 It is prepared by swirling an IM cell population containing approximately 1000 cells / ml of IM cells.

[0018] In another example, the disclosure includes renal organoids produced by the methods described herein.

[0019] In another example, the present disclosure encompasses the method for screening candidate compound for nephrotoxicity, comprising contacting renal organoid as herein described with candidate compound, thereby determining whether this candidate compound is nephrotoxic.In one example, said candidate compound is small molecule.

[0020] In another example, the present disclosure encompasses the use of renal organoid, cell population or composition as described herein in producing kidney or renal cell or renal tissue.In another example, the present disclosure encompasses the use of renal organoid, cell population or composition as described herein in treating renal disease.In some examples, cell population refers to the cell obtained from the enzyme digestion of renal organoid.

[0021] Surprisingly, the inventors also confirmed that culturing stem cells in medium containing low concentrations of CHIR and activating Wnt / β-catenin signaling for a long period of time is beneficial in producing improved intermediate mesoderm.

[0022] In one example, the disclosure includes an in vitro method for producing intermediate mesoderm (IM) cells, comprising culturing a population of posterior primitive streak (PPS) cells for 2 to 5 days in a cell culture medium containing FGF and less than 4 μM of a Wnt / β-catenin agonist.

[0023] In another example, stem cells may be first cultured in CHIR for 7 days, with the stem cells being cultured in a medium containing a high concentration of CHIR for the first 4-5 days, followed by culture in a medium containing a low concentration of CHIR and FGF for the remaining days. Thus, in another example, the present disclosure encompasses an in vitro method for producing intermediate mesoderm (IM) cells, comprising culturing a stem cell population for at least 7 days, wherein the first 4-5 days comprise culturing the stem cells in a cell culture medium containing at least 6 μM of a Wnt / β-catenin agonist, and the remaining days comprise culturing the cells in a cell culture medium containing FGF and at least 0.5 μM of a Wnt / β-catenin agonist. In one example, the remaining days comprise culturing the cells in a cell culture medium containing 0.5-3 μM of a Wnt / β-catenin agonist and FGF. In another example, the remaining days comprise culturing the cells in a cell culture medium containing 0.8-1.2 μM of a Wnt / β-catenin agonist and FGF. In another example, the first four days include culturing the stem cells in a cell culture medium containing at least 6 μM of a Wnt / β-catenin agonist. In another example, the first four days include culturing the stem cells in a cell culture medium containing 7 μM of a Wnt / β-catenin agonist. In one example, the FGF-containing cell culture medium contains 100 to 300 ng / ml of FGF9. In one example, the FGF-containing cell culture medium contains 180 to 220 ng / ml of FGF9. In another example, the FGF9-containing cell culture medium further contains heparin. For example, the cell culture medium may contain at least 1.0 μg / ml of heparin. In one example, the stem cells are pluripotent stem cells, embryonic stem cells, or induced pluripotent stem (iPS) cells. In another example, the stem cell is selected from the group consisting of H9, hES3, iPSC, GAPTrap td-Tomato, CRL1502.C32, CLR1502.3, hES3 SOX17mCherry, or H9 GAPTrap Luc2.

[0024] In another example, the present disclosure encompasses a method for bioprinting a kidney, comprising preparing a bioink from the organoid or cell population described herein, and bioprinting a kidney. In some examples, the cell population refers to cells obtained from enzymatic digestion of renal organoids.

[0025] In another example, the present disclosure comprises the use of the renal organoid, composition or cell population described herein in producing kidney or renal cell or renal tissue.In another example, the present disclosure comprises the method for producing the nephron cell type for cell therapy, comprising using the method described herein or IM cell population to produce renal organoid.

[0026] Unless otherwise stated, any example in this specification shall be deemed to apply mutatis mutandis to any other example.

[0027] The present disclosure is not limited in scope by the specific examples described herein, which are intended for illustrative purposes only. Functionally equivalent products, compositions, and methods are also expressly encompassed within the scope of the present disclosure as described herein.

[0028] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, a reference to a step, composition, group of steps, or group of compositions shall be deemed to encompass one and more (i.e., one or more) of that step, composition, group of steps, or group of compositions.

[0029] The present disclosure will now be illustrated by the following non-limiting examples with reference to the accompanying drawings. [Brief explanation of the drawings]

[0030] [Figure 1-1]Generation of renal microorganoids in suspension culture. (A) Overview of the renal microorganoid differentiation protocol with images of differentiation performed using CRL1502.C32 cells. (B) Brightfield image (100 mm scale) showing uniform renal microorganoids at days 7 + 18. (C, D1, and D2) Immunofluorescence and confocal images (100 mm scale) of microorganoids showing the formation of nephron segments, including the development of vasculature, independent of microorganoid size and shape. (E) Bar graph showing the mean fold change in gene expression profiling of intermediate mesoderm by qPCR at days 7 + 0 with FGF9 ± 1 μM CHIR99021. Data represent the mean values. (F) Immunofluorescence (50 μm scale) of PAX2 with FGF9 ± 1 μM CHIR99021 treatment (days 7 + 11). [Figure 1-2] (G) Immunofluorescence and confocal images of nephron compartments within renal microorganoids: podocytes (NPHS1+ and MAFB+), proximal tubules (LTL+, CUBN+, LRP2+, and HNF4A+), distal tubules (ECAD), collecting ducts (ECAD+, GATA3+), and endothelial cells (SOX17+ and PECAM1+) (50 μm scale). [Figure 2]Renal microorganoids in suspension culture exhibit functional proximal tubules, and early Wnt signaling is critical for renal organoid development and maturation. (A) Confocal images (5 μm scale) of renal microorganoid tubules at days 7+18 showing FITC albumin uptake. (B) Confocal images (50 μm scale) of renal microorganoids generated using four different cell lines, including hES (H9 GAP-Trap Luc2, hES3 SOX17mCheny) and iPS (CRL1502.C32 and CRL1502.3), at days 7+18, using antibodies labeling different nephron segments. (C, D, and E) Brightfield (C, 100 μm scale) and immunofluorescence confocal images showing SOX17+ vasculature (D) and MEIS1 / 2 / 3+ stroma (E) (100 μm scale) of hES3 SOX17mCherry-derived microorganoids generated after exposure to initial 7 μM CHIR99021 treatment for 3, 4, 5, and 6 days. [Figure 3-1] Transcriptional confirmation of kidney differentiation within microorganoids. (A) t-SNE plot after Seurat clustering of single-cell RNA-seq from CRL1502-C32 microorganoids at days 7+18, showing 11 distinct clusters. [Figure 3-2] (B) Heatmap showing scaled gene expression of significant marker genes within the cluster. [Figure 3-3] (C) t-SNE plot showing the expression of significant marker genes for selected nephron cell types. Color intensity is scaled per gene, with blue indicating higher expression. [Figure 4-1]Renal microorganoids provide a better platform for efficient scale-up of hPSC-derived kidney cells. (A) Brightfield image of a standard renal organoid at days 7+11 (left, 500 µm scale), immunofluorescence and confocal images (tile scans) of a whole standard organoid (center, 200 µm scale) showing that nephron structures are spatially restricted to the organoid's edge, and a magnified image of a nephron within this organoid (right, 200 µm scale). (B) Brightfield image of a renal microorganoid and a magnified brightfield image of a single renal microorganoid, and a confocal image of a renal microorganoid at days 7+11 (200 µm scale). [Figure 4-2] (C) Changes in organoid size at different developmental stages. (D) Changes in total cell number from the starting cell number over time and the scalability of micro-organoids compared to standard organoids. (E-F) Immunofluorescence and 3D reconstruction using Bitplane Imaris of C32 micro-organoids at days D7+18 showed distinct, polarized, interconnected nephron segments, starting from the glomerulus (NPHS1), proximal tubule (LTL+), distal tubule (ECAD+), collecting duct (ECAD+, GATA3+), and interstitial cells (GATA3+). [Figure 5-1] Comparative single-cell transcriptional profiling of standard renal organoids and microorganoids shows equivalent nephrogenic patterns. (A) t-SNE plot (CRL1502.32) after integrated Seurat analysis of 10x scRNA-Seq data from renal microorganoids (Micro-org) and standard organoids (Stand-org) at days 7+18. (B) t-SNE plot showing the contribution of microorganoids and standard organoids to cell types in each cluster, color-coded by organoid type. [Figure 5-2](C) Bar graphs representing the proportion of the Micro-org or Stand-org datasets assigned to each transcriptional cluster and differentiation lineage type. (D) Split dot plots showing gene expression of kidney markers in each cluster between renal micro-organoids and standard organoids. (E) Violin and scatter plots showing the log-normalized per-cell counts of nephron-related genes (PAX2, SIX1, LHX1) and interstitium-related genes (PDGFRA, MEIS2) within Micro-orgs and Stand-orgs. (F) Immunofluorescence (50 μm scale) showing the expression of PAX2 and MEIS1 / 2 / 3 in renal Micro-orgs and Stand-orgs. [Figure 6] Comparison of nephron and stromal markers within micro-organoids and stand-organs. (A and B) t-SNE signature plots for nephron and stromal genes in scRNA-Seq data from standard and microorganoids. [Figure 7] All-trans retinoic acid helps improve glomerular maturation in renal microorganoids. Immunofluorescence analysis of C32 gyral organoids generated by gyral suspension culture. (A) Organoids generated without atRA supplementation. (B) Organoids generated with atRA from days 7+5 to 7+10 show improved glomerular podocyte maturation. (C) qPCR analysis of organoids generated with and without atRA at different time points (days 7+11 and 7+18). [Figure 8] Renal microorganoids provide a platform for drug toxicity screening. Adriamycin treatment (24 h) induces dose-dependent toxicity in renal microorganoids by increasing the expression of the apoptosis marker TUNEL (A-C). (D) Adriamycin treatment also reduces the expression of kidney-specific genes in microorganoids. DETAILED DESCRIPTION OF THE INVENTION

[0031] Unless otherwise defined, all technical and scientific terms used herein shall be assumed to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., molecular biology, cell culture, stem cell differentiation, cell therapy, genetic engineering, disease modeling, biochemistry, physiology, and clinical research).

[0032] Unless otherwise specified, the molecular and statistical methods used in this disclosure are standard procedures, well known to those skilled in the art. Such techniques are reported and described in the following references: J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984); J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989); T.A. Brown (ed.), Essential Molecular Biology: A Practical Approach, Vols. 1 and 2, IRL Press (1991); D.M.G.lover and B.D.Hames (eds.), DNA Cloning: A Practical Approach, Vols. 1-4, IRL Press (1995 and 1996); and F.M.Ausubel et al. (eds.), Current Protocols in Molecular Biology, Greene Publication Associates and Wiley-Interscience. Pub. Associates and Wiley-Interscience (1988, including all current updates), Ed Harlow and David Lane (eds.), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988), JE Coligan et al. (eds.), Current Protocols in Immunology, John Wiley & Sons (including all current updates), Michos Odysse (ed.), Kidney Development: Methods and Protocols (Springer), Robert Lanza (ed.), Handbook of Stem Cells, Vol. 1, Embryonic Stem Cells (Elsevier).

[0033] When used in this specification and appended claims, singular terms and singular forms " a ", " an " and " the " can optionally include plural referents unless otherwise clearly indicated by context.That is, for example, when referring to " renal organoid ", can optionally include one or more renal organoids.

[0034] As used herein, the term "about" refers to ±10%, more preferably ±5%, more preferably ±1% of the specified value, unless otherwise specified.

[0035] The term "and / or," e.g., "X and / or Y," shall be understood to mean either "X and Y" or "X or Y," and shall be considered to explicitly endorse both meanings or either meaning.

[0036] Throughout this specification the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated element, integer, or step, or group of elements, integers, or steps, but not the exclusion of other elements, integers, or steps, or groups of elements, integers, or steps.

[0037] The cell composition of the present disclosure can be administered to a variety of subjects. In one example, the subject is a mammal. The mammal can be a companion animal such as a dog or cat, or a livestock animal such as a horse or cow. In another example, the subject is a human. Terms such as "subject," "patient," or "individual" are terms that can be used interchangeably within the context of the present disclosure.

[0038] As used herein, the term "treatment" refers to a clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. Favorable therapeutic effects include a reduction in the rate of disease progression, an improvement or alleviation of disease symptoms, and remission or improved prognosis. For example, an individual is "treated" if one or more symptoms associated with a disease are alleviated or eliminated. In one example, the term "treatment" refers to both therapeutic and prophylactic or preventative treatment for kidney disease, anemia, erythropoietin deficiency, renal tubular transport failure, or glomerular filtration failure, with the aim of reversing, preventing, or slowing (alleviating) the targeted disorder. Those in need of treatment include those already suffering from kidney disease, anemia, erythropoietin deficiency, renal tubular transport failure, or glomerular filtration failure, those susceptible to such disorders, or those in whom such disorders are to be prevented. In one example, treatment involves stabilizing and / or improving kidney function.

[0039] An "effective amount" refers to at least an amount effective, at a dosage and for a period of time necessary, to achieve the desired therapeutic or prophylactic result. An effective amount can be provided in one or more administrations. In some examples of the present disclosure, the term "effective amount" is used to refer to the amount necessary to achieve treatment of a renal disorder or disorder as described herein below. The effective amount will vary depending on the disease or disorder being treated, and may also vary depending on the weight, age, ethnic background, sex, health and / or physical condition, and other factors related to the mammal being treated. Typically, an effective amount will fall within a relatively broad range (e.g., a "dosage" range) that can be determined through routine clinical trial and experimentation by a medical practitioner. An effective amount can be administered in a single dose, or in one or several repeated doses over the course of treatment.

[0040] A "therapeutically effective amount" refers to at least the minimum concentration required to achieve measurable improvement in a particular kidney disorder (e.g., nephritis, renal cell carcinoma). Herein, a therapeutically effective amount may also vary depending on factors such as the patient's condition, age, sex, and weight, as well as the ability of the cell composition to elicit a desired response in an individual. A therapeutically effective amount is also an amount in which any toxic or adverse effects of the composition are outweighed by the therapeutically beneficial effects. In the case of renal cell carcinoma, a therapeutically effective amount may reduce the number of cancer cells; reduce primary tumor size; inhibit (i.e., delay to some extent, and in some cases prevent) cancer cell invasion into peripheral organs; inhibit (i.e., delay to some extent, and in some cases prevent) tumor metastasis; inhibit or delay tumor growth or tumor progression to some extent; and / or alleviate to some extent one or more symptoms associated with renal cell carcinoma. In the treatment of renal cell carcinoma, in vivo efficacy may be measured, for example, by survival time, time to progression (TTP), response rate (RR), duration of response, and / or quality of life assessment.

[0041] "Intermediate mesoderm (IM)" cells refer to embryonic mesoderm cells that arise from definitive mesoderm and can derive from the posterior primitive streak and ultimately develop into the urogenital system, including the ureters and kidneys, as well as other tissues such as the gonads. Examples of markers characteristic or representative of intermediate mesoderm include, but are not limited to, PAX2, OSR1, and / or LHX1.

[0042] It should also be understood that the production of IM cells does not imply that the IM cells are a pure or homogeneous IM cell population free of other cell types (such as committed mesoderm). Therefore, references to "IM cells" or "IM cell population" refer to cells or cell populations containing IM cells. According to the present invention, IM cells are suitably produced by contacting posterior primitive streak cells with one or more agents that promote the differentiation of posterior primitive streak cells into IM cells, as described in more detail below. Preferably, IM cells are produced by contacting posterior primitive streak cells with one or more agents that promote the differentiation of posterior primitive streak cells into IM cells.

[0043] "Posterior primitive streak (PPS)" cells refer to cells derived from, or cells functionally and / or phenotypically corresponding to, the posterior end of the primitive streak structure formed in the blastula during early mammalian embryogenesis. The posterior primitive streak establishes bilateral symmetry, determines the site of gastrulation, and initiates germ layer formation. Typically, the posterior primitive streak is a precursor of mesoderm (i.e., presumptive mesoderm), and the anterior primitive streak is a precursor of endoderm (i.e., presumptive endoderm). Non-limiting examples of markers characteristic or representative of the posterior primitive streak include Brachyury (T). Non-limiting examples of markers characteristic or representative of the anterior primitive streak include SOX17. MIXL1 can be expressed by both the posterior and anterior primitive streak.

[0044] It should also be understood that the production of posterior primitive streak cells does not imply that the posterior primitive streak cells are a pure or homogeneous population of posterior primitive streak cells, free of other cell types. Thus, references to "posterior primitive streak cells" or "posterior primitive streak cell populations" refer to cells or cell populations that contain posterior primitive streak cells. Posterior primitive streak cells are produced by contacting hPSC cells with one or more agents that promote differentiation of hPSC cells into posterior primitive streak cells, as described in more detail below. For example, the one or more agents may include bone morphogenetic protein 4 (BMP4), activin A, and / or a Wnt agonist (e.g., CHIR99021).

[0045] Renal organoids The present disclosure encompasses the production of intermediate mesoderm (IM) cells. The term "intermediate mesoderm (IM)" is used in the context of the present disclosure to refer to embryonic mesoderm cells that arise from committed mesoderm and can derive from the posterior primitive streak and ultimately develop into the urogenital system, including the ureters and kidneys, as well as other tissues such as the gonads. Examples of markers characteristic or representative of intermediate mesoderm include, but are not limited to, PAX2, OSR1, and / or LHX1.

[0046] In one example, the culture conditions are provided to allow these IM cells to " self-organize " and form renal organoid.In the context of the present disclosure, the term " renal organoid " refers to the heterogeneous three-dimensional cell aggregate, which repeats the aspects of cell self-organization, construction and signaling interaction that exist in the original kidney.Takasato et al. (2015) Nature, Vol.526:564-568, International Publication No. 2014 / 197934 and International Publication No. 2016 / 094948 describe examples of renal organoid.In the context of the present disclosure, the terms " renal organoid " and " renal organoid " can be used interchangeably.

[0047] Surprisingly, the present inventors have identified renal organoids with simplified three-dimensional structures. Such organoids are advantageous because they are easier to image and cultivate for long periods. For example, a healthy adult has 800,000 to 2 million nephrons in each kidney, typically about 1 million. In contrast, the organoids encompassed by the present disclosure contain a much smaller number of nephrons. Thus, in one example, the present disclosure encompasses renal organoids with the structural characteristics of the original kidney, but with a reduced number of nephrons. In one example, the renal organoids encompassed by the present disclosure may contain one or more nephrons. In one example, the nephrons are segmented into distal tubules, proximal tubules, early Henle's loops, and glomeruli. In another example, the organoids comprise segmented nephrons surrounded by endothelial cells, perivascular cells, and renal interstitium. In another example, the organoids disclosed herein do not exhibit the presence of vasculature.

[0048] In other examples, the organoids of the present disclosure are at least partially vascularized, for example, the organoids may be composed of nephrons containing podocytes that develop foot processes and undergo vascularization.

[0049] In one example, renal organoids are characterized in terms of nephron percentage (%), interstitium percentage (%), and / or vasculature percentage (%). In this example, renal organoids can be characterized using single-cell RNA sequencing. An example of single-cell sequencing is described below. In one example, renal organoids comprise at least 20% mature nephrons. In another example, renal organoids comprise at least 25% mature nephrons. In another example, renal organoids comprise at least 30% mature nephrons. In another example, renal organoids comprise at least 31% mature nephrons. In another example, renal organoids comprise at least 32% mature nephrons. In these examples, the renal organoids also comprise at least 15% interstitium. In another example, the renal organoids also comprise at least 20% interstitium. In another example, the renal organoids also comprise at least 25% interstitium. In another example, the renal organoids are substantially free of vasculature. In another example, the renal organoids do not contain vasculature.

[0050] In one example, the renal organoid of the present disclosure comprises less than 100 nephrons.In another example, the renal organoid of the present disclosure comprises less than 90, less than 80, less than 70, or less than 60 nephrons.In another example, the renal organoid of the present disclosure comprises less than 50 nephrons.In another example, the renal organoid of the present disclosure comprises less than 40, less than 30, less than 20, or less than 10 nephrons.In another example, the renal organoid of the present disclosure comprises less than 5 nephrons.In another example, the renal organoid of the present disclosure comprises less than 4, or less than 3 nephrons.

[0051] In another example, the renal organoids of the present disclosure comprise between 2 and 100 nephrons. In another example, the renal organoids of the present disclosure comprise between 2 and 50 nephrons. In another example, the renal organoids of the present disclosure comprise between 2 and 10 nephrons. In another example, the renal organoids of the present disclosure comprise between 5 and 12 nephrons. In another example, the renal organoids of the present disclosure comprise between 6 and 10 nephrons. In another example, the renal organoids of the present disclosure comprise between 2 and 6 nephrons. In another example, the renal organoids of the present disclosure comprise between 2 and 4 nephrons.

[0052] " Nephron " is the functional operating unit of the kidney, which plays a major role in removing waste products from blood / plasma and maintaining body fluid volume.Those skilled in the art can use various methods to identify and count the nephron in organoid disclosed herein.For example, nephron can be visualized and counted using confocal microscopy and immunofluorescence labeling (for example, WT1+ glomerulus; NPHS+ podocyte, LTL+ ECAD- proximal tubule, ECAD+ distal tubule, and ECAD+ GATA3+ collecting duct).

[0053] Generally, the species identification of the renal organoids encompassed by the present disclosure is determined by the cells used to generate the renal organoids, regardless of whether they are mammals such as mice or humans.In one example, the present disclosure encompasses mammalian renal organoids.In this example, mammalian pluripotent stem cells are used to generate the renal organoids.The mammalian renal organoids may represent the kidneys of companion animals such as dogs or cats, or livestock animals such as horses or cows.That is, in these examples, stem cells derived from dogs, cats, etc. are used to generate the renal organoids.In another example, the mammalian renal organoids represent the kidneys of mice or rats.In another example, the renal organoids represent the kidneys of higher primates such as cynomolgus monkeys or rhesus monkeys.In another example, the mammalian renal organoids represent the kidneys of humans.If pluripotent stem cells from a specific species are used to generate renal organoids, the resulting renal organoids can be identified based on their species. For example, when human stem cell is used to generate renal organoid, the renal organoid obtained can be identified as human renal organoid.That is, in one example, the renal organoid included in the present disclosure includes the human renal organoid derived from human stem cell.Other various examples of stem cell suitable for generating renal organoid are described below.

[0054] In another example, renal organoids can be characterized based on the expression of molecular markers. Marker expression can be characterized using various techniques, such as immunohistochemistry or fluorescence-activated cell sorting. Immunohistochemistry typically involves the use of a primary antibody specific to the marker of interest. The binding of the primary antibody to the marker can be visualized by various known methods. For example, a labeled secondary antibody that recognizes the primary antibody can be used. In this example, the label can be an enzyme such as horseradish peroxidase, a radioisotope, a fluorescent reporter, or an electrochemiluminescent tag. The binding of the labeled secondary antibody to the primary antibody can be detected by cytological evaluation or via an automated plate reader.

[0055] In a particular example, renal organoid or its section or sample is contacted with specific primary antibody.Then, the renal organoid or its section or sample is washed to remove unbound primary antibody, and then the secondary antibody specific to the primary antibody is added to this sample, which is linked to peroxidase enzyme.Then, the renal organoid or its section or sample is washed to remove unbound secondary antibody, and 3,3'-diaminobenzidine (DAB) is added to this sample.Then, the conversion of DAB into colored product is visualized by conventional cytological evaluation, and the existence of colored product represents the presence of the marker in the sample.In one example, the level of colored product can be quantified using ImageJ or other various software packages commercially available from suppliers such as PerkinElmer and Leica.

[0056] In another example, cell suspension is prepared from representative kidney organoid, its group, or its segment or sample.The suspended cell is contacted with the fluorescent-labeled antibody specific to specific marker.The cell that is positive for specific marker is identified by such a method as fluorescence-activated cell sorting (FACS).

[0057] Cells that are said to be "positive" for a given marker may express that marker at low (lo or dim) or high (bright, bri) levels, depending on how present the marker is on the cell surface. Here, these terms refer to the intensity of the fluorescence or other marker used in the cell sorting process. The distinction between lo (or dim or dull) and bri is understood in relation to the marker used on the particular cell population being sorted. When a cell is said to be "negative" for a given marker, the cell does not necessarily have to be completely absent. This term means that the marker is expressed at a relatively low or very low level by the cell or population, so that detectably labeling produces very little signal, or is undetectable above background levels (e.g., levels detected using an isotype control antibody).

[0058] In one example, fluorescent reporter gene can be used to detect the marker of renal organoid described herein.For example, by monitoring the expression of certain marker, the development of renal organoid or the cells that comprise it can be tracked in real time.For example, stem cells can be genetically modified under a given set of conditions to express one or more fluorescent reporters or chemiluminescent reporters.By using reporter, cell identity information, cell survival rate or cell function can be tracked in real time.

[0059] An example of a suitable reporter gene is shown below, which has the mTagBFP2 fluorescent reporter gene inserted into the start codon of the endogenous MAFB locus (MAFB mTagBFP2 / + ) knock-in iPSC line is generated. MAFB is highly expressed in developing podocytes, so by monitoring MAFB expression, the development of podocytes in renal organoids can be tracked in real time. Other examples of reporter cell lines suitable for use in the methods disclosed herein include GATA3mCherry, RETtdTOMATO, or Six2Cre.

[0060] In another example, renal organoids comprise cells that express one or more nephron markers at high levels. In another example, renal organoids comprise cells that express one or more of PAX2, SIX1, LHX1, OSR1, WNT11, GATA3, PAX8, EYA1, and CITED1 at high levels. For example, renal organoids can express high levels of PAX2. In another example, renal organoids can express high levels of SIX1. In another example, renal organoids can express high levels of LHX1. In another example, renal organoids can express high levels of OSR1. In another example, renal organoids can express high levels of WNT11. In another example, renal organoids can express high levels of GATA3. In another example, renal organoids comprise cells that express high levels of PAX2, SIX1, LHX1, OSR1, WNT11, and GATA3. In another example, renal organoids comprise cells that express PAX2, SIX1, LHX1, OSR1, WNT11, GATA3, PAX8, EYA1 and CITED1 at high levels.In these examples, renal organoids can express reference markers such as one or more of PAX2, SIX1, LHX1, OSR1, WNT11, GATA3, PAX8, EYA1 and CITED1 at high levels compared with renal organoids with at least 100 nephrons.In another example, renal organoids can express reference markers such as one or more of PAX2, SIX1, LHX1, OSR1, WNT11, GATA3, PAX8, EYA1 and CITED1 at high levels compared with renal organoids with more than 50 nephrons.In another example, renal organoids can express reference markers such as one or more of PAX2, SIX1, LHX1, OSR1, WNT11, GATA3, PAX8, EYA1 and CITED1 at high levels compared with renal organoids with more than 50 nephrons. 5 In another example, the renal organoids may express at a higher level one or more reference markers, such as PAX2, SIX1, LHX1, OSR1, WNT11, and GATA3, compared to renal organoids containing at least 1 x 10 cells. 6Compared with the renal organoid that comprises cells, renal organoid can express one or more reference markers such as PAX2, SIX1, LHX1, OSR1, WNT11 and GATA3 at high levels.In another example, compared with the renal organoid that is produced without rotation, such as the renal organoid described in Takasato et al. (2015), renal organoid can express one or more reference markers such as PAX2, SIX1, LHX1, OSR1, WNT11 and GATA3 at high levels.

[0061] In another example, renal organoids comprise cells that express low levels of WT1. In another example, renal organoids comprise cells that express low levels of C-RET. In another example, renal organoids comprise cells that express low levels of FOXD1. In another example, renal organoids comprise cells that express low levels of PDGFRA. In another example, renal organoids comprise cells that express low levels of MEIS2. In another example, renal organoids comprise cells that express low levels of WT1 and C-RET. In another example, renal organoids comprise cells that express low levels of WT1, C-RET, and FOXD1. In these examples, renal organoids may express at low levels reference markers such as one or more of WT1, C-RET, and FOXD1, compared to renal organoids with at least 100 nephrons. In another example, renal organoids may express lower levels of reference markers, such as one or more of WT1, C-RET, and FOXD1, compared to renal organoids having more than 50 nephrons.

[0062] In another example, renal organoid comprises the cell that expresses high level PAX2, SIX1, LHX1, OSR1, WNT11 and GATA3, and low level WT1, C-RET, PDGFRA, MEIS2 and FOXD1.In another example, renal organoid comprises the cell that expresses high level PAX2, SIX1, LHX1, OSR1, WNT11, GATA3, PAX8, EYA1 and CITED1, and low level WT1, C-RET, PDGFRA, MEIS2 and FOXD1.

[0063] In the above examples, high and low levels of expression are relative to renal organoids cultured without rotation, such as those described in Takasato et al. (2015) Nature, Vol. 526:564-568. In this example, high expression is at least 1-fold higher. In another example, high expression is at least 1.5-fold higher. In another example, high expression is at least 2-fold higher. In one example, low expression is at least 1 / 1-fold lower. In another example, low expression is at least 1 / 1.5-fold lower. In another example, low expression is at least 2-fold lower.

[0064] Expression level can be measured by using methods such as polymerase chain reaction, which includes the primer suitable for the target marker.For example, total RNA can be extracted from organoid, reverse transcribed, and subjected to PCR analysis.

[0065] In one example, renal organoid comprises nephrons comprising one or more of WT1+ glomeruli, NPHS+ podocytes, LTL+ ECAD- proximal tubules, ECAD+ distal tubules, and ECAD+ GATA3+ collecting ducts.In another example, renal organoid comprises nephrons comprising NPHS+ podocytes, LTL+ proximal tubule segments, ECAD+ distal tubule segments, and ECAD+ GATA3+ collecting ducts.The renal organoids comprising the above-exemplified components can be identified by various methods.In one example, renal organoids can be fixed and whole-mounted, and then visually evaluated using confocal microscopy and immunofluorescence labeling.

[0066] In one example, renal organoid can be characterized by one or more of the above-mentioned markers after being used in the screening method described below.In another example, the renal organoid that shows a broader group is characterized by one or more of the above-mentioned markers, and then the renal organoid that expresses the marker that is suitable for being used in the screening method described below can be selected.For example, renal organoid population can be produced by the method disclosed herein.After the renal organoid of said population is confirmed to express one or more of the above-mentioned markers, the renal organoid that is used in the screening method described below can be selected.

[0067] In another example, the organoids of the present disclosure are 0.5 x 10 4 ~8×10 4 In another example, a kidney organoid contains 0.8 x 10 cells. 4 ~7×10 4 In another example, an organoid may contain 1 x 10 cells. 4 ~5×10 4 In another example, the organoid comprises at least 1 x 10 cells. 4 In another example, an organoid may contain 3 x 10 cells. 4 In another example, an organoid may contain less than 2 x 10 cells. 4 ~2.5×10 4 Contains cells.

[0068] In another example, the organoids of the present disclosure have a diameter of less than 2,500 μm. In another example, the organoids of the present disclosure have a diameter of less than 2,000 μm. In another example, the organoids of the present disclosure have a diameter of less than 1,000 μm. In another example, the organoids of the present disclosure have a diameter of less than 500 μm. In another example, the organoids of the present disclosure have a diameter of less than 400 μm. In another example, the organoids of the present disclosure have a diameter of less than 300 μm. In another example, the organoids of the present disclosure have a diameter of 150 to 600 μm. In another example, the organoids of the present disclosure have a diameter of 200 to 500 μm. For example, the organoids of the present disclosure may have a diameter of 200 μm to 2,000 μm. In another example, the organoids of the present disclosure may have a diameter of 200 μm to 1,000 μm. In another example, the organoids of the present disclosure may have a diameter of 200 μm to 400 μm. In another example, the organoids of the present disclosure may have a diameter of 200 μm to 300 μm. In another example, the organoids of the present disclosure may have a diameter of 250 μm to 300 μm.

[0069] Those skilled in the art can understand that the size of organoid can be measured by, for example, optical microscopy and the accompanying software such as ImageJ.For example, those skilled in the art can identify the organoid of the above-exemplified size by measuring the width at the widest point of its three-dimensional structure.

[0070] In another example, the organoids of the present disclosure survive in culture for at least 3 weeks. In another example, the organoids of the present disclosure survive in culture for at least 4 weeks. In another example, the organoids of the present disclosure survive in culture for at least 6 weeks. In another example, the organoids of the present disclosure survive in culture for at least 3-4 weeks. For example, the renal organoids disclosed herein may develop diffusion limitations (i.e., limitations affecting the movement of nutrients to the cells that comprise the organoid structure) only after 3-6 weeks in culture. In these examples, the period is measured from day 7+1. Thus, in other words, the organoids of the present disclosure survive in culture for at least day 7+21, day 7+28, or day 7+42. In one example, cell proliferation rate can be used as a measure of diffusion limitations, since cells typically do not continue to divide in the absence of sufficient nutrients. Therefore, by tracking cell proliferation in the organoids disclosed herein over time, it is possible to determine when diffusion limitations occur. In one example, a decrease in cell growth rate over a period of 3-6 days indicates a diffusion limitation. In another example, a stagnant cell growth over a period of 3-6 days indicates a diffusion limitation.

[0071] The present disclosure encompasses renal organoids, which comprise fewer nephrons than human kidney.It is understood by those skilled in the art that renal organoids are artificial products, and share many physiological and biochemical characteristics of mammalian kidney, and do not occur naturally.For example, renal organoids disclosed herein may not be associated with intact vasculature and / or one or more of the following characteristics: having fewer than 50 nephrons; 0.5×10 4 ~8×10 4 Contains approximately 10 cells; have a diameter of less than 1000 μm (preferably about 250 to 350 μm); are independent three-dimensional structures that are not part of a tissue or organ; and It is produced by a method involving differentiation of stem cells in vitro.

[0072] Cellular Compositions and Therapies The organoids or cells of the present disclosure can be used to produce therapeutic compositions. In one example, the whole organoids disclosed herein can be provided as therapeutic compositions. In another example, the present disclosure encompasses cell compositions produced from the renal organoids disclosed herein. For example, the organoids of the present disclosure can be enzymatically digested to produce cell compositions. For example, the organoids described herein can be digested with a protease such as trypsin, alone or in combination with ethylenediaminetetraacetic acid (EDTA), to produce cell compositions. In another example, the organoids described herein can be digested with collagenase, such as collagenase I and / or collagenase II (e.g., commercially available Liberase™ (Roche)), to produce cell compositions. In one example, the enzymatic digest can be partially purified or purified to deplete one or more cell types. For example, vascular cells and / or endothelial cells can be depleted. In another example, the enzymatic digest can be partially purified or purified to enrich one or more cell types. For example, the enzymatic digest may be partially purified or purified to obtain an enriched composition of nephron progenitor cells and / or ureteral epithelial progenitor cells.

[0073] In another example, compositions encompassed by the present disclosure include cells cultured using the methods disclosed herein. For example, the composition may include improved IM cells that express high levels of PAX2, LHX1, and OSR1 (cap mesenchyme), and Wnt11 and GATA3 (ureteral epithelium). As in the above example, IM cells may be cultured and partially purified or purified using the methods described herein to enrich for one or more cell types, such as nephron progenitor cells and / or ureteral epithelial progenitor cells.

[0074] Thus, in another example, the present disclosure encompasses a cell composition comprising a population of nephron progenitor cells and / or ureteral epithelial progenitor cells purified from an organoid or cell population produced by the methods described herein.

[0075] In one example, the therapeutic compositions disclosed herein include a pharmaceutically acceptable carrier and / or excipient. The terms "carrier" and "excipient" refer to compositions conventionally used in the art to facilitate the storage, administration, and / or biological activity of an active compound (see, for example, Remington's Pharmaceutical Sciences, 16th Edition, Mac Publishing Company (1980)). A carrier may also reduce any undesirable side effects of an active compound. A suitable carrier is, for example, a stable carrier, e.g., a carrier that cannot react with other components in the composition. In one example, the carrier does not cause significant local or systemic adverse effects in the recipient at the dosages and concentrations used for therapy.

[0076] Suitable carriers for the present disclosure include conventionally used carriers, for example, saline, aqueous glucose, lactose, Ringer's solution, buffer, hyaluronan, and glycols are exemplary liquid carriers, especially for aqueous solutions (when isotonic). Suitable pharmaceutical carriers and excipients include starch, cellulose, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, glycerol, propylene glycol, water, ethanol, etc.

[0077] In another example, the carrier is a medium composition, for example, in which cells or whole organoids are grown or suspended. For example, such a medium composition does not cause any adverse effects to the subject to which it is administered. In one example, the cell culture medium may include the basal medium disclosed herein. In one example, the basal medium may contain PVA and MC. For example, the basal medium may contain 0.05-0.5% PVA and 0.05-0.5% MC.

[0078] In one example, carrier or excipient provides buffering activity, so that cells are maintained at appropriate pH, thereby exerting biological activity, for example, carrier or excipient is phosphate buffered saline (PBS).PBS is attractive as carrier or excipient, because it has minimal interaction with cells and factors and allows the cells and factors to be rapidly released, and in this case, the composition of the present disclosure can be prepared as a liquid for direct application into bloodstream, or to kidney, or to the area surrounding or adjacent to kidney, such as by injection.Therefore, in one example, the cell composition or whole organoid disclosed herein is provided in phosphate buffered saline (PBS).

[0079] In another example, cell compositions or whole organoids can be incorporated or embedded in scaffolds that are compatible with recipients and decompose into products that are not harmful to recipients. These scaffolds provide support and protection to the cells that are transplanted into recipient subjects. Natural and / or synthetic biodegradable scaffolds are examples of such scaffolds. Various scaffolds can be used in the implementation of the present disclosure. Exemplary scaffolds include, but are not limited to, biodegradable scaffolds. Natural biodegradable scaffolds include collagen scaffolds, fibronectin scaffolds, and laminin scaffolds. Synthetic materials suitable for cell transplantation scaffolds should also be able to support a wide range of cell growth and cell function. Such scaffolds can also be resorbable scaffolds. Suitable scaffolds include polyglycolic acid scaffolds, as described, for example, in Vacanti, et al. J. Ped. Surg. 23:3-9 1988; Cima, et al. Biotechnol. Bioeng. 38: 145 1991; Vacanti, et al. Plast. Reconstr. Surg. 88:753-9 1991; or synthetic polymers such as polyanhydrides, polyorthoesters, and polylactic acids. In another example, cells can be administered in a gel-like scaffold (e.g., Gelfoam manufactured by Upjohn Company). In another example, cells can be administered in a decellularized kidney scaffold. In one example, cells can be administered in a decellularized human kidney or its extracellular matrix (ECM) components.

[0080] In one example, the composition comprises effective amount or therapeutically effective amount of cells or whole organoid.In another example, cells or whole organoid are contained in a container, which does not allow cells or organoid to escape into the blood circulation of the subject, but allows the factors secreted by cells or organoid to enter into the blood circulation.In this way, soluble factors can be administered to the subject by making cells or organoid secrete factors into the blood circulation of the subject.Similarly, this container can be implanted at a certain site in the subject, for example, implanted in or near the kidney, to increase the local level of soluble factors.

[0081] In one example, the compositions disclosed herein can be administered systemically, for example, by intravenous administration, intraarterial administration or intraperitoneal administration.In one example, the compositions disclosed herein are administered intravenously.In another example, the compositions are administered intraarterially.In another example, the compositions are administered by intrarenal artery injection, intrarenal parenchyma injection or subrenal capsule transplantation to normal kidney or diseased kidney.In another example, the compositions are transplanted.For example, the whole organoid can be transplanted close to the kidney of the target.

[0082] In one example, the cell composition of the present disclosure can be cryopreserved. Cryopreservation of cells or whole organoids can be performed using slow cooling or "rapid" freezing protocols known in the art. Preferably, the cryopreservation method maintains the phenotype, cell surface markers, and growth rate of the cryopreserved cells or whole organoids similar to those of unfrozen cells or whole organoids. The cryopreservation composition can include a cryopreservation solution. The pH of the cryopreservation solution is typically 6.5-8, preferably 7.4.

[0083] Examples of cryopreservation fluids include non-pyrogenic isotonic solutions, such as PlasmaLyte A®. 100 mL of PlasmaLyte A® contains 526 mg of sodium chloride, USP (NaCl); 502 mg of sodium gluconate (C6H 11NaC7); 368 mg of sodium acetate trihydrate, USP (C2H3NaO2·3H2O); 37 mg of potassium chloride, USP (KC1); and 30 mg of magnesium chloride, USP (MgCl2·6H2O). PlasmaLyte A® does not contain antimicrobial agents.

[0084] In one example, the present disclosure encompasses cell therapy comprising administering a composition disclosed herein to a subject in need of treatment. For example, the present disclosure encompasses a method of treating kidney disease by administering a composition disclosed herein to a subject in need of treatment. The term "renal disease," in the context of the present disclosure, refers to disorders associated with any stage or degree of acute or chronic kidney failure that result in the loss of the kidney's ability to perform its functions of filtering and removing excess fluid, electrolytes, and waste products from the blood. Examples of kidney disease include endocrine dysfunction, such as anemia (erythropoietin deficiency) and mineral imbalance (vitamin D deficiency). The kidney disease may be kidney-derived or may be kidney disease secondary to various abnormalities, including, but not limited to, heart failure, hypertension, diabetes, autoimmune disease, or liver disease or drug-induced toxicity. In one example, the kidney disease may be a state of chronic kidney failure that occurs after acute injury to the kidney. For example, injury to the kidney due to ischemia and / or toxic exposure can cause acute renal failure; incomplete recovery after acute kidney injury can lead to the development of chronic renal failure. Other examples of kidney diseases include congenital nephrotic syndrome (CNS) (including steroid-resistant nephrotic syndrome and Finnish nephropathy), focal segmental glomerulonephritis (FSGS), Alport syndrome, and Pearson syndrome.

[0085] In one example, the present disclosure comprises the method for treating kidney disease by transplanting the whole organoid disclosed herein into the subject that needs treatment.In another example, the present disclosure comprises the method for treating kidney disease by administering the cell composition disclosed herein into the subject that needs treatment.

[0086] In other examples, the compositions or cells disclosed herein may be provided for the recellularization of a decellularized kidney scaffold. In another example, the present disclosure encompasses biomaterials or scaffolds comprising the compositions or cells disclosed herein.

[0087] stem cells Each embodiment of the present disclosure encompasses the culture of stem cells. The term "stem cell," in the context of this disclosure, refers to a subset of progenitor cells that, under certain circumstances, have the capacity or potential to differentiate into a more specialized or differentiated phenotype and, under certain circumstances, retain the ability to proliferate without substantial differentiation. In one example, the term stem cell typically refers to a naturally occurring mother cell whose progeny specialize, often by differentiating in various directions, e.g., by acquiring complete individuality, as occurs during the diversification of fetal cells and tissues. Cell differentiation is a complex process that typically occurs through many cell divisions. Differentiated cells can be derived from pluripotent cells, which themselves are derived from other multipotent cells. While each of these pluripotent cells can be considered a stem cell, the range of cell types each can give rise to can be quite diverse. Some differentiated cells have the capacity to give rise to cells with greater developmental potential. Such capacity can be natural or induced artificially by treatment with various factors. In many biological examples, stem cells are "pluripotent" because they can give rise to progeny of two or more distinct cell types, but this is not required for "stemness." Self-renewal is the other part of the classical definition of a stem cell. In principle, self-renewal can occur through either of two major mechanisms: stem cells can divide asymmetrically, with one daughter cell retaining the stemness state and the other daughter cell expressing some other specialized function and phenotype. Alternatively, a fraction of stem cells within a population can divide symmetrically into two stem cells, thereby maintaining some stemness in the population as a whole, while other cells in the population give rise only to differentiated progeny.

[0088] In one example, the stem cells are human stem cells. In one example, the stem cells are a culture-expanded human stem cell population. In one example, the stem cells can be culture-expanded in vitro or ex vivo. In one example, the culture-expanded stem cells have been passaged at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

[0089] In one example, the stem cells are pluripotent stem cells. In another example, the stem cells are human embryonic stem cells. Pluripotent stem cells typically express OCT4, NANOG, and SSEA1 when in the pluripotent state, and the expression of these markers typically disappears with differentiation. In another example, the stem cells are human embryonic stem cells. The term "human embryonic stem cell" and its abbreviations, such as "hES" and "hESC," refer to cells derived from, available from, or derived from a human embryo or blastocyst, which are self-renewing and pluripotent, or totipotent, capable of generating all cell types present in an adult animal. Human embryonic stem cells (hESCs) can be isolated, for example, from human blastocysts obtained from human in vivo preimplantation embryos, in vitro fertilized embryos, or one-cell human embryos expanded to the blastocyst stage.

[0090] In another example, the stem cells are induced pluripotent stem cells. For example, the stem cells can be human induced pluripotent stem cells. The term "induced pluripotent stem cells" and its abbreviation "iPSC" refer to cells inducible, obtainable, or derived from any type of human adult somatic cell that have been reprogrammed into a pluripotent state through the expression of exogenous genes, such as transcription factors, including a preferred combination of OCT4, SOX2, KLF4, and c-MYC. Human iPSCs exhibit a level of pluripotency comparable to hESCs, but can be obtained from patients for autologous therapy and subsequently differentiated and delivered, with or without parallel gene correction. Suitable methods for generating induced pluripotent stem cells are described, for example, in U.S. Patent No. 7,615,374 and U.S. Patent Application Publication No. 2014273211, Barberi et al. Plos medicine, Vol 2(6):0554-0559 (2005), and Vodyanik et al. Cell Stem cell, Vol 7:718-728 (2010). In one example, iPSCs are derived from fibroblasts. In another example, iPSCs are derived from blood. For example, iPSCs are derived from leukocytes. In another example, iPSCs are derived from fibroblasts. In another example, iPSCs are derived from leukocytes or fibroblasts.

[0091] In one example, the stem cell is H9 or hES3. Thus, in one example, the present disclosure encompasses the renal organoid disclosed herein that is derived from H9 stem cells. In another example, the present disclosure encompasses the renal organoid disclosed herein that is derived from hES3 stem cells (Kao et al., 2016; Ng et al., 2016; van den Berg et al., 2018). For example, the renal organoid can be derived from hES3-SOX17mCherry or H9 GAPTrapLuc2. Thus, in one example, the present disclosure encompasses the renal organoid that is derived from H9 stem cells or hES3 stem cells and comprises less than 50 nephrons. In another example, the renal organoid comprises less than 15 nephrons and is derived from H9 stem cells or hES3 stem cells. In these examples, the stem cells can express a reporter gene.

[0092] In another example, the stem cell is iPSC GAPTrap td-Tomato, CRL1502.C32 or CLR1502.3 (Briggs et al., 2013; Takasato et al., 2015).Therefore, in one example, the present disclosure encompasses the kidney organoid described herein that is derived from iPSC GAPTrap td-Tomato.

[0093] Therefore, in one example, the present disclosure encompasses the renal organoid that comprises less than 50 nephrons, and is derived from iPSC GAPTrap td-Tomato, CRL1502.C32 or CLR1502.3.In another example, the renal organoid comprises less than 15 nephrons and is derived from iPSC GAPTrap td-Tomato, CRL1502.C32 or CLR1502.3.Similarly, in these examples, the stem cell can express reporter gene.

[0094] In one example, it may be preferable to produce renal organoids that represent specific subjects and / or diseases.Various examples of this embodiment are described below.This section relates to the iPS cells that can be used to produce renal organoids.In one example, the human iPS cells are derived from a human subject with genetic renal disease.In this example, blood samples can be isolated from subjects with genetic renal disease, and iPS cells can be derived from cells (e.g., white blood cells) in the blood samples.The subject can have one of various exemplary genetic renal diseases.Examples include congenital nephrotic syndrome (CNS) (including steroid-resistant nephrotic syndrome and Finnish nephropathy), focal segmental glomerulonephritis (FSGS), Alport syndrome, and Pearson syndrome. Thus, in one example, the present disclosure encompasses the renal organoids that represent renal diseases selected from the group consisting of congenital nephrotic syndrome (CNS) (including steroid-resistant nephrotic syndrome and Finnish nephropathy), focal segmental glomerulonephritis (FSGS), Alport syndrome and Pearson syndrome.Therefore, in one example, said renal organoids represent CNS.In another example, said renal organoids represent steroid-resistant nephrotic syndrome.

[0095] In one example, renal organoids can be used to model developing kidneys and / or kidney diseases. Thus, in one example, the present disclosure encompasses the renal organoids disclosed herein that are used to model kidney development. In another example, the present disclosure encompasses the renal organoids disclosed herein that are used to model kidney disease. In one example, the kidney disease is CNS or another of the above diseases. In this example, the disease can be modeled by deriving iPS cells from a subject with the above kidney disease and then producing renal organoids. In this example, gene editing can be used to introduce mutations into genes in iPS cells derived from a subject that are important or may be important in the development of kidney disease (e.g., CRISPR / Cas9 gene editing). In another example, gene editing can be used to correct mutations in iPS cells derived from a subject. In one example, isogenic gene-edited iPS cells can be generated (e.g., Forbes et al. (2018) Am J Hum Genet. 102:816-831). Renal development and disease can be modeled over a long period of time (for example, 2 days, 5 days, 10 days, or longer) using renal organoids at various developmental stages, such as one or more of the following (for example, 7+15 days).In these examples, organoid glomeruli are cultured in groups, each group representing a different developmental stage (for example, 7+11 days, 7+15 days, 7+18 days, 7+20 days), and / or can be cultured for a certain period of time (for example, 2 days, 5 days, and 10 days of rotation culture).Renal organoids can be evaluated using visual evaluation, immunohistochemistry, gene and protein expression analysis, etc. to determine the stage of development or disease.In one example, renal organoids can also be contacted with nephrotoxic substances, candidate compounds, and / or therapeutic compounds during these tests, and nephrotoxicity and / or therapeutic effect can be measured.As mentioned above, the renal organoids used in the above examples can be generated from iPS cells that are genetically modified to express reporter genes.

[0096] Cell culture method The term "media" or "medium" when used in connection with cell culture includes components of the environment surrounding cells. It is assumed that the medium contributes to and / or realizes conditions sufficient for cell differentiation and organoid formation. The medium can be solid, liquid, gaseous, or a mixture of phases and substances. The medium can include liquid growth media as well as liquid media that do not sustain cell growth. The medium also includes gelatinous media such as agar, agarose, gelatin, and collagen matrix. Exemplary gaseous media include the gas phase to which cells growing on a Petri dish or other solid or semi-solid phase are exposed. The term "medium" refers to a substance intended for use in cell culture, even if it is not yet in contact with cells.

[0097] The medium used in the disclosed methods can be prepared using a medium used for culturing stem cells or IM cells as the basal medium. Examples of basal media include Eagle's Minimum Essential Medium (MEM), but are not limited to any medium that can be used for culturing stem cells or IM cells. Furthermore, the disclosed medium can contain any component, such as fatty acids or lipids, vitamins, growth factors, cytokines, antioxidants, buffers, and inorganic salts. The cell culture medium used in the disclosed methods contains all essential amino acids and may also contain non-essential amino acids. Amino acids are generally classified as essential (Thr, Met, Val, Leu, He, Phe, Trp, Lys, His) and non-essential (Gly, Ala, Ser, Cys, Gin, Asn, Asp, Tyr, Arg, Pro). In other examples, the basal medium includes, for example, chemically defined media such as APEL, mTESR-E6, or E8 (StemCell Technologies). The basal medium may also be supplemented with protein-free hybridoma medium (PFHM) (e.g., 3.5%). In one example, the basal medium is supplemented with serum replacement. For example, the basal medium may be supplemented with Knockout Serum Replacement (Thermo Fisher).

[0098] As will be understood by those skilled in the art, the culture medium disclosed herein needs to be changed over time. Identifying the appropriate timing for changing the culture medium is considered to be well within the skill of those skilled in the art. For example, there are various commercially available color indicators that are commonly used in cell culture media to indicate when the culture medium needs to be changed. As a guideline, when culturing cells in multi-well culture dishes, the culture medium can be changed every 24 or 48 hours. For example, the culture medium can be changed every 2 days.

[0099] Renal organoids The present disclosure encompasses the method for producing renal organoid.In one example, renal organoid is produced by rotating the medium containing intermediate mesoderm (IM) cell population.In one example, the IM cell is rotated under suspension culture.For avoidance of doubt, in the context of the present disclosure, " suspension culture" refers to the cell culture in which single cell or small cell clusters are grown in agitated liquid medium while floating.For example, the single cell or small cell clusters are grown under suspension culture to form renal organoid.

[0100] In one example, the IM cell culture medium contains FGF. In some examples, the FGF may be selected from the FGF9 superfamily, which includes FGF9, FGF16, and FGF20. In some examples, the FGF is FGF9. For example, the IM cell culture medium may contain FGF from day 7 to at least day 7+10. In one example, the IM cell culture medium may contain FGF from day 7 to at least day 7+15. Examples of FGF concentrations are described below. For example, the cell culture medium contains at least 50 ng / ml of FGF. In another example, the cell culture medium contains at least 100 ng / ml of FGF. In another example, the cell culture medium contains at least 150 ng / ml of FGF. In another example, the cell culture medium contains at least 200 ng / ml of FGF. In another example, the cell culture medium contains at least 300 ng / ml of FGF. In another example, the cell culture medium contains at least 350 ng / ml of FGF. In another example, the cell culture medium contains at least 400 ng / ml of FGF. In another example, the cell culture medium contains at least 500 ng / ml of FGF. In another example, the cell culture medium contains 50 ng / ml to 400 ng / ml of FGF. In another example, the cell culture medium contains 50 ng / ml to 300 ng / ml of FGF. In another example, the cell culture medium contains 50 ng / ml to 250 ng / ml of FGF. In another example, the cell culture medium contains 100 ng / ml to 200 ng / ml of FGF. In another example, the cell culture medium contains 180 ng / ml to 220 ng / ml of FGF. In another example, the cell culture medium contains 190 ng / ml to 210 ng / ml of FGF.

[0101] In one example, the IM cell culture medium may contain FGF9. In one example, the cell culture medium contains at least 50 ng / ml of FGF9. In another example, the cell culture medium contains at least 100 ng / ml of FGF9. In another example, the cell culture medium contains at least 150 ng / ml of FGF9. In another example, the cell culture medium contains at least 200 ng / ml of FGF9. In another example, the cell culture medium contains at least 300 ng / ml of FGF9. In another example, the cell culture medium contains at least 350 ng / ml of FGF9. In another example, the cell culture medium contains at least 400 ng / ml of FGF9. In another example, the cell culture medium contains at least 500 ng / ml of FGF9. In another example, the cell culture medium contains 50 ng / ml to 400 ng / ml of FGF9. In another example, the cell culture medium contains 50 ng / ml to 300 ng / ml of FGF9. In another example, the cell culture medium contains 50 ng / ml to 250 ng / ml of FGF9. In another example, the cell culture medium contains 100 ng / ml to 200 ng / ml of FGF9. In another example, the cell culture medium contains 180 ng / ml to 220 ng / ml of FGF9. In another example, the cell culture medium contains 190 ng / ml to 210 ng / ml of FGF9.

[0102] In another example, the above levels of FGF9 are replaced with FGF2. For example, the IM cell culture medium may contain 50 ng / ml to 400 ng / ml of FGF2. In another example, the cell culture medium contains 50 ng / ml to 300 ng / ml of FGF2. In another example, the cell culture medium contains 50 ng / ml to 250 ng / ml of FGF2. In another example, the cell culture medium contains 100 ng / ml to 200 ng / ml of FGF2. In another example, the cell culture medium contains 180 ng / ml to 220 ng / ml of FGF2. In another example, the cell culture medium contains 190 ng / ml to 210 ng / ml of FGF2.

[0103] In another example, the above levels of FGF9 are replaced with FGF16. For example, the IM cell culture medium may contain 50 ng / ml to 400 ng / ml of FGF16. In another example, the cell culture medium contains 50 ng / ml to 300 ng / ml of FGF16. In another example, the cell culture medium contains 50 ng / ml to 250 ng / ml of FGF16. In another example, the cell culture medium contains 100 ng / ml to 200 ng / ml of FGF16. In another example, the cell culture medium contains 180 ng / ml to 220 ng / ml of FGF16. In another example, the cell culture medium contains 190 ng / ml to 210 ng / ml of FGF16.

[0104] In another example, the above-mentioned levels of FGF9 are replaced with FGF20. For example, the IM cell culture medium may contain 50 ng / ml to 400 ng / ml of FGF20. In another example, the cell culture medium contains 50 ng / ml to 300 ng / ml of FGF20. In another example, the cell culture medium contains 50 ng / ml to 250 ng / ml of FGF20. In another example, the cell culture medium contains 100 ng / ml to 200 ng / ml of FGF20. In another example, the cell culture medium contains 180 ng / ml to 220 ng / ml of FGF20. In another example, the cell culture medium contains 190 ng / ml to 210 ng / ml of FGF20. In one example, FGF is removed from the medium after 5 days of rotational culture. In one example, FGF is removed from the medium after 6 days of rotational culture. In one example, FGF is removed from the medium after 4 to 6 days of rotational culture.

[0105] In one example, Wnt / β-catenin agonist is added to the culture medium to induce nephrogenesis in organoid.In the context of the present disclosure, the term " Wnt / β-catenin agonist " is used to refer to a molecule that preferably inhibits GSK3 (for example, GSK3-β) in the context of classical Wnt signaling pathway, but does not inhibit in the context of other non-classical Wnt signaling pathway.In some examples, the Wnt / β-catenin agonist is a GSK3β inhibitor.Examples of Wnt / β-catenin agonist include CHIR99021 (CHIR), LiCl, SB-216763, CAS853220-52-7 and other Wnt / β-catenin agonists commercially available from suppliers such as Santa Cruz Biotechnology and R&D Systems.

[0106] Thus, in one example, the IM cell culture medium may contain the above levels of FGF and Wnt / β-catenin agonist. For example, the IM cell culture medium may contain at least 0.5 μM of Wnt / β-catenin agonist. In another example, the cell culture medium may contain at least 0.6 μM of Wnt / β-catenin agonist. In another example, the cell culture medium may contain at least 0.7 μM of Wnt / β-catenin agonist. In another example, the cell culture medium may contain at least 0.8 μM of Wnt / β-catenin agonist. In another example, the cell culture medium may contain at least 0.9 μM of Wnt / β-catenin agonist. In another example, the cell culture medium may contain about 1 μM of Wnt / β-catenin agonist. In another example, the cell culture medium may contain 1.1 μM or less of Wnt / β-catenin agonist. In another example, the cell culture medium may contain 1.2 μM or less of the Wnt / β-catenin agonist. In another example, the cell culture medium may contain 1.3 μM or less of the Wnt / β-catenin agonist. In another example, the cell culture medium may contain 1.4 μM or less of the Wnt / β-catenin agonist. In another example, the cell culture medium may contain 1.5 μM or less of the Wnt / β-catenin agonist. It will be understood that the medium may include any combination of these upper and lower limits for the concentration of the Wnt / β-catenin agonist. In another example, the cell culture medium may contain 0.5 μM to 1.5 μM of the Wnt / β-catenin agonist. In another example, the cell culture medium may contain 0.8 μM to 1.2 μM of the Wnt / β-catenin agonist. In one example, the cell culture medium may contain at least 0.5 μM of CHIR. In another example, the cell culture medium may contain at least 0.6 μM CHIR. In another example, the cell culture medium may contain at least 0.7 μM CHIR. In another example, the cell culture medium may contain at least 0.8 μM CHIR. In another example, the cell culture medium may contain at least 0.9 μM CHIR. In another example, the cell culture medium may contain about 1 μM CHIR. In another example, the cell culture medium may contain 1.1 μM or less CHIR. In another example, the cell culture medium may contain 1.2 μM or less CHIR. In another example, the cell culture medium may contain 1.3 μM or less CHIR. In another example, the cell culture medium may contain 1.4 μM or less CHIR.In another example, the cell culture medium may contain 1.5 μM or less CHIR. It is understood that the medium may include any combination of these upper and lower limits for CHIR concentration. In another example, the cell culture medium may contain 0.5 μM to 1.5 μM CHIR. In another example, the cell culture medium may contain 0.8 μM to 1.2 μM CHIR.

[0107] In another example, the IM cell culture medium may contain a Rho kinase inhibitor (ROCKi), such as Y-27632 (Stem Cell Technologies). In one example, the cell culture medium may contain at least 8 μM of ROCKi. In another example, the cell culture medium may contain about 10 μM of ROCKi. In another example, the cell culture medium may contain 12 μM or less of ROCKi. In another example, the cell culture medium may contain 8 μM to 12 μM of ROCKi.

[0108] In the above example, the IM cell culture medium may contain FGF9, a Wnt / β-catenin agonist such as CHIR, and one or more or all of heparin, poly(vinyl alcohol) (PVA), and methylcellulose (MC). In another example, the cell culture medium may also contain ROCKi.

[0109] In one example, the IM cell culture medium may contain at least 0.5 μg / ml of heparin. In another example, the cell culture medium contains about 1 μg / ml of heparin. In another example, the cell culture medium contains 1.5 μg / ml or less of heparin. In another example, the cell culture medium contains 2 μg / ml or less of heparin. In another example, the cell culture medium contains 0.2 μg / ml to 2 μg / ml of heparin. In another example, the cell culture medium contains 0.5 μg / ml to 1.5 μg / ml of heparin. In another example, the cell culture medium contains 0.8 μg / ml to 1.2 μg / ml of heparin.

[0110] In one example, the IM cell culture medium contains at least 0.05% PVA. In another example, the cell culture medium contains about 0.1% PVA. In another example, the cell culture medium contains 0.15% or less PVA. In another example, the cell culture medium contains 0.1% to 0.15% PVA.

[0111] In one example, the IM cell culture medium contains at least 0.05% MC. In another example, the cell culture medium contains about 0.1% MC. In another example, the cell culture medium contains less than 0.15% MC. In another example, the cell culture medium contains 0.1% to 0.15% MC.

[0112] In the context of the present disclosure, the terms "swirl," "orbiting," and "orbiting" are used interchangeably and refer to the movement of cell culture medium in a circular, twisting, or spiral pattern. In one example, the cell culture medium is rotated by applying sufficient agitation to the cell culture in a circular motion. For example, the cell culture can be rotated using an orbital shaker. Other examples of suitable devices for rotating cell culture include a shaker platform, a shaker incubator, or a rotary flask. Appropriate rotational speed allows IM cells to aggregate and form organoids.

[0113] In one example, the IM cell culture is spun at at least 30 rpm. In another example, the cell culture is spun at at least 40 rpm. In another example, the cell culture is spun at at least 50 rpm. In another example, the cell culture is spun at at least 60 rpm. In another example, the cell culture is spun at at least 70 rpm. In another example, the cell culture is spun at at least 80 rpm. In another example, the cell culture is spun at 40-80 rpm. In another example, the cell culture is spun at 50-70 rpm. In another example, the cell culture is spun at 55-65 rpm. In another example, the cell culture is spun at 30-150 rpm. In another example, the cell culture is spun at 30-90 rpm.

[0114] In one example, the IM cells are cultured for at least 5 days. In another example, the IM cells are cultured for at least 7 days. In another example, the IM cells are cultured for at least 10 days. In another example, the IM cells are cultured for at least 12 days. In another example, the IM cells are cultured for at least 14 days. In another example, the IM cells are cultured for at least 20 days. In another example, the IM cells are cultured for up to 42 days. In another example, the IM cells are cultured for 5-20 days. In another example, the IM cells are cultured for 5-18 days. In another example, the IM cells are cultured for 7-14 days. For example, the IM cells are cultured for at least 10 days with rotation. For example, the IM cells can be rotated for at least 20 days. In another example, the IM cells can be rotated for at least 30 days.

[0115] In one example, the IM cells are cultured for 10-30 days. In another example, the IM cells are cultured for 15-30 days.

[0116] In one example, the IM cell population is dissociated and cultured in the above-described medium under rotation. In one example, the IM cells can be dissociated using EDTA. In another example, the IM cells can be dissociated using trypsin or TrypLE. In one example, the dissociated IM cells are further cultured after being passed through a mesh screen. In one example, the cells are cultured under rotation for at least 12 days after dissociation. In another example, the cells are cultured under rotation for at least 13 days after dissociation. In another example, the cells are cultured under rotation for at least 14 days after dissociation. In another example, the cells are cultured under rotation for at least 15 days after dissociation. In another example, the cells are cultured under rotation for at least 20 days after dissociation. In another example, the cells are cultured under rotation for at least 25 days after dissociation. In another example, the cells are cultured under rotation for at least 35 days after dissociation. In another example, the cells are cultured under rotation for 5 to 18 days after dissociation.

[0117] In other examples, the IM cells described herein can be cultured in various media containing various components. For example, cells can be subjected to staged culture, with each stage associated with a different medium. In one example, IM cells are cultured in two stages. In this example, the first stage medium contains the above levels of FGF (e.g., FGF9), CHIR, ROCKi, heparin, PVA, and MC, while the second stage medium contains the above levels of FGF (e.g., FGF9), CHIR, heparin, PVA, and MC. For example, the first stage medium can contain 200 ng / ml FGF9, 1 μM CHIR, 10 μM ROCKi, 1 μg / ml heparin, 0.1% PVA, and 0.1% MC, while the second stage medium can contain FGF9, 1 μM CHIR, 1 μg / ml heparin, 0.1% PVA, and 0.1% MC.

[0118] In another example, IM cells are cultured in a three-stage rotation. In this example, the first and second stage media are as defined above, and the third stage media contains the above-mentioned levels of PVA and MC. For example, the third stage media may contain 0.1% PVA and 0.1% MC.

[0119] In one example, IM cells are cultured in stage 1 medium for 1 day, followed by culture in stage 2 medium for 4 days. In one example, cells are cultured in stage 1 medium for 18-24 hours, followed by culture in stage 2 medium for 4 days. In these examples, cells may be further cultured in stage 3 medium for 7-20 days. In another example, cells may be further cultured in stage 3 medium for at least 15 days. In another example, cells may be further cultured in stage 3 medium for at least 30 days.

[0120] The present inventors have also confirmed that adding retinoic acid to IM cells after approximately 5-10 days of rotation culture improves glomerular maturation (improved glomerular podocyte maturation) of organoids. Therefore, in one example, retinoic acid is added to the cell culture medium after 5-10 days of rotation culture. In one example, all-trans retinoic acid (atRA) is added to the cell culture medium. In one example, at least 0.07 μM of retinoic acid is added to the cell culture medium. In another example, at least 0.1 μM of retinoic acid is added to the cell culture medium. In another example, at least 0.2 μM of retinoic acid is added to the cell culture medium. In another example, at least 0.5 μM of retinoic acid is added to the cell culture medium.

[0121] In another example, at least 1.5 μM of retinoic acid is added to the cell culture medium. In one example, at least 1.8 μM of retinoic acid is added to the cell culture medium. In one example, at least 2.0 μM of retinoic acid is added to the cell culture medium. In another example, at least 2.5 μM of retinoic acid is added to the cell culture medium. In another example, 1.5 μM to 3 μM of retinoic acid is added to the cell culture medium. In another example, 2.0 μM to 3 μM of retinoic acid is added to the cell culture medium.

[0122] In another example, the first and second stage media are as defined above, and the third stage media contains the above levels of PVA, MC, and atRA, e.g., 0.1% PVA, 0.1% MC, and 2.5 μM atRA.

[0123] The present inventors have confirmed that by rotating a small number of IM cells in a suitable medium, the development of kidney organoids with a simple three-dimensional structure can be directed.Therefore, in one example, the method of the present disclosure can be used to generate 5x10 5 In another example, the methods of the present disclosure include spinning an IM cell population comprising less than 4 x 10 IM cells. 5 In another example, the method of the disclosure includes spinning an IM cell population comprising less than 3 x 10 IM cells. 5In another example, the methods of the present disclosure include spinning an IM cell population comprising less than 1 x 10 IM cells. 5 In another example, the method of the disclosure includes spinning an IM cell population comprising less than 5 x 10 IM cells. 4 In another example, the method of the disclosure includes spinning an IM cell population comprising 4 x 10 IM cells. 4 In another example, the method of the disclosure includes spinning an IM cell population comprising 1 x 10 IM cells. 4 ~1×10 5 In another example, the method of the disclosure includes spinning an IM cell population comprising 2 x 10 IM cells. 4 ~1×10 5 In another example, the method of the disclosure includes spinning an IM cell population comprising 5×10 IM cells. 3 ~3×10 5 The method includes spinning an IM cell population comprising 10 IM cells.

[0124] In another example, the method of the present disclosure 5 cells / ml~5×10 6 In another example, the method of the present disclosure involves spinning 5×10 IM cells / ml. 5 ~4×10 6 In another example, the method of the present disclosure involves spinning 5×10 IM cells / ml. 5 ~3×10 6 In another example, the method of the present disclosure involves spinning 5 x 10 IM cells / ml. 6 In another example, the method of the present disclosure involves spinning an IM cell population of less than 4 x 10 cells / ml. 6 In another example, the method of the present disclosure involves spinning an IM cell population of less than 3 x 10 cells / ml. 6 In another example, the method of the present disclosure involves spinning an IM cell population of less than 2 x 10 cells / ml. 6 In another example, the method of the present disclosure involves spinning an IM cell population of less than 1 x 10 cells / ml. 6 In another example, the method of the present disclosure involves spinning an IM cell population of 5×10 cells / ml or less.5 ~2×10 6 In another example, the method of the present disclosure involves spinning 5×10 IM cells / ml. 5 ~1.5×10 6 This involves spinning IM cells at 1 x 10 cells / ml. In these examples, approximately 5,000-15,000 organoids can be produced. In another example, approximately 8,000-10,000 organoids can be produced. In these examples, the total cell number increases 30-40-fold over the culture period from the number of cells at the start of the spinning culture. In these examples, the total cell number increases 3-4-fold compared to the increase in total cell number using the protocol described by Takasato et al. (2015) Nature, Vol. 526:564-568. In this example, the total cell number is 1 x 10 5 ~5×10 6 cells / ml to 3 x 10 6 ~2×10 8 It increases to about cells / ml.

[0125] In one example, the IM cells are obtained by the methods disclosed herein.

[0126] In one example, the disclosure provides a method for producing renal organoids, the method comprising: a process for producing IM cells by culturing a stem cell population for 7 days, wherein the first 4 to 5 days involve culturing the stem cells in a high concentration of a Wnt / β-catenin agonist (such as CHIR), and the remaining days involve culturing the cells in a cell culture medium containing FGF9 and a low concentration of a Wnt / β-catenin agonist; dissociating the IM cells; A process for producing renal organoids by spinning IM cells in a cell culture medium containing FGF9 for at least 5 days, comprising culturing the cells in a cell culture medium containing FGF9, heparin, a low concentration of a Wnt / β-catenin agonist, and ROCKi for the first 24 hours, and culturing the cells in a cell culture medium containing FGF9, heparin, a low concentration of a Wnt / β-catenin agonist, PVA, and MC for the next 3 or 4 days; The present invention encompasses a method for producing renal organoids, comprising:

[0127] In the above example, the stem cells may be cultured for 7 days, with the first 4 days involving culturing the stem cells in a high concentration of a Wnt / β-catenin agonist (e.g., CHIR), and the remaining days involving culturing the cells in a cell culture medium containing FGF9 and a low concentration of a Wnt / β-catenin agonist. In one example, the Wnt / β-catenin agonist is CHIR. In one example, the high concentration of CHIR is about 3 μM to about 12 μM. In another example, the high concentration of CHIR is about 4 μM to about 10 μM, about 5 μM to about 9 μM, about 6 μM to about 8 μM, about 6.5 μM to about 8 μM, or about 6.5 μM to about 7 μM. In one example, the low concentration of CHIR is 1 μM. In one example, the IM cells are dissociated using trypsin. In another example, the cells are dissociated using EDTA. In one example, IM cells are spun in cell culture medium containing 200 ng / ml FGF9 for at least 4 days, including culturing the cells for the first 24 hours in cell culture medium containing 200 ng / ml FGF9, 1 μg / ml heparin, 1 μM CHIR, and 10 μM ROCKi, and for the next 3 or 4 days in cell culture medium containing 200 ng / ml FGF9, 1 μg / ml heparin, 1 μM, 0.1% PVA, and 0.1% MC.

[0128] In another example, the method further comprises spinning the cells in a cell culture medium containing PVA and MC, but not containing CHIR or FGF9. In one example, the cell culture medium contains 0.1% PVA and 0.1% MC. In one example, the cells are spun in the cell culture medium containing PVA and MC, but not containing CHIR or FGF9, for 5 days or more.

[0129] intermediate mesoderm Surprisingly, the present inventors also found that culturing stem cells in a medium containing a low concentration of CHIR and activating Wnt / β-catenin signaling for a longer period of time is beneficial in producing improved intermediate mesoderm. This in vitro culture method provides a system for differentiating stem cells through posterior primitive streak (PPS) cells and intermediate mesoderm (IM) cells to produce renal organoids.

[0130] Thus, in one example, the disclosure includes an in vitro method for producing intermediate mesoderm (IM) cells, comprising culturing a population of posterior primitive streak (PPS) cells in a cell culture medium containing FGF and less than 4 μM of a Wnt / β-catenin agonist for 2 to 5 days.

[0131] Suitable concentrations of FGF and Wnt / β-catenin agonist, as well as suitable culture periods, are as described below in connection with the method for producing intermediate mesoderm (IM) cells after the initial 3 or 4 day culture period.

[0132] Thus, in one example, the present disclosure encompasses a method for producing intermediate mesodermal (IM) cells, comprising culturing a stem cell population in a cell culture medium containing CHIR for about 7 days, with an FGF, such as FGF9, being added to the medium after the first 3 or 4 days of culture. In one example, 50 ng / ml to 400 ng / ml of FGF9 is added to the medium after the first 3 or 4 days of culture. In another example, 50 ng / ml to 300 ng / ml of FGF9 is added to the medium after the first 3 or 4 days of culture. In another example, 50 ng / ml to 250 ng / ml of FGF9 is added to the medium after the first 3 or 4 days of culture. In another example, 100 ng / ml to 200 ng / ml of FGF9 is added to the medium after the first 3 or 4 days of culture. In another example, 150 ng / ml to 250 ng / ml of FGF9 is added to the medium after the first 3 or 4 days of culture. In another example, after the first three or four days of culture, 175 ng / ml to 225 ng / ml of FGF9 is added to the medium. In another example, after the first three or four days of culture, 190 ng / ml to 210 ng / ml of FGF9 is added to the medium. In another example, after the first three or four days of culture, 195 ng / ml to 205 ng / ml of FGF9 is added to the medium. In another example, after the first three or four days of culture, about 200 ng / ml of FGF9 is added to the medium. In one example, heparin is also added to the medium after the first three or four days of culture. In one example, 0.5 μg / ml to 2 μg / ml of heparin is added to the medium after the first three or four days of culture. In another example, 0.5 μg / ml to 1.5 μg / ml of heparin is added to the medium after the first three or four days of culture. In another example, 0.8 μg / ml to 1.2 μg / ml of heparin is added to the medium after the first 3 or 4 days of culture. In another example, 1 μg / ml of heparin is added to the medium after the first 3 or 4 days of culture. In one example, the above levels of FGF and heparin are added to the medium after 4 days of culture. In one example, the stem cells are cultured in a cell culture medium containing CHIR for 7 days.

[0133] In particular, the inventors have confirmed that improved IM cells can be produced by culturing stem cells in a high concentration of a Wnt / β-catenin agonist (such as CHIR) and then culturing the stem cells in a low concentration of a Wnt / β-catenin agonist and FGF (such as FGF9). For example, the improved IM cells express high levels of PAX2, LHX1, and OSR1 (cap mesenchyme), as well as Wnt11 and GATA3 (ureteral epithelium). Thus, in one example, stem cells can be cultured in a medium containing a high concentration of a Wnt / β-catenin agonist (such as CHIR), and then cultured in a medium containing a low concentration of a Wnt / β-catenin agonist and FGF (such as FGF9, FGF16, FGF20, or FGF2).

[0134] In one example, the disclosure encompasses a method for producing intermediate mesoderm (IM) cells, comprising culturing a stem cell population in a cell medium containing a high concentration of CHIR, followed by culturing the stem cell population in a cell medium containing a low concentration of CHIR and an FGF (such as FGF9). In this example, a "high concentration" of CHIR is at least 5 μM, and a "low concentration" of CHIR is less than 3 μM. In another example, a "high concentration" of CHIR is at least 6 μM, and a "low concentration" of CHIR is less than 2 μM. In another example, a "high concentration" of CHIR is 7 μM, and a "low concentration" of CHIR is 1 μM or less.

[0135] Thus, in another embodiment, the disclosed method includes an in vitro method for producing intermediate mesoderm (IM) cells. In one example, the IM cell production method includes culturing a stem cell population for about 7 days, where the first 4-5 days involve culturing the stem cells in a high concentration of a Wnt / β-catenin agonist (e.g., CHIR), and the remaining days involve culturing the cells in a cell culture medium containing FGF9 and a low concentration of a Wnt / β-catenin agonist. In one embodiment, the IM cell production method includes, after the first 4-5 days of culturing in a high concentration of a Wnt / β-catenin agonist, culturing the stem cell population in a cell culture medium containing FGF9 and at least 0.5 μM of a Wnt / β-catenin agonist. In another example, after the first 4-5 days of culturing in a high concentration of a Wnt / β-catenin agonist, the cell culture medium can contain at least 0.6 μM of a Wnt / β-catenin agonist. In another example, after culturing in a high concentration of a Wnt / β-catenin agonist for the first 4-5 days, the cell culture medium may contain at least 0.7 μM of a Wnt / β-catenin agonist. In another example, after culturing in a high concentration of a Wnt / β-catenin agonist for the first 4-5 days, the cell culture medium may contain at least 0.8 μM of a Wnt / β-catenin agonist. In another example, after culturing in a high concentration of a Wnt / β-catenin agonist for the first 4-5 days, the cell culture medium may contain at least 0.9 μM of a Wnt / β-catenin agonist. In another example, after culturing in a high concentration of a Wnt / β-catenin agonist for the first 4-5 days, the cell culture medium may contain about 1 μM of a Wnt / β-catenin agonist. In another example, after culturing in a high concentration of a Wnt / β-catenin agonist for the first 4-5 days, the cell culture medium may contain 1.1 μM or less of a Wnt / β-catenin agonist. In another example, after the first 4-5 days of culture in a high concentration of a Wnt / β-catenin agonist, the cell culture medium may contain 1.2 μM or less of a Wnt / β-catenin agonist. In another example, after the first 4-5 days of culture in a high concentration of a Wnt / β-catenin agonist, the cell culture medium may contain 1.3 μM or less of a Wnt / β-catenin agonist. In another example, after the first 4-5 days of culture in a high concentration of a Wnt / β-catenin agonist, the cell culture medium may contain 1.4 μM or less of a Wnt / β-catenin agonist.In another example, after the first 4-5 days of culture in a high concentration of Wnt / β-catenin agonist, the cell culture medium may contain 1.5 μM or less of the Wnt / β-catenin agonist. It will be understood that after the first 4-5 days of culture in a high concentration of Wnt / β-catenin agonist, the medium may contain any combination of these upper and lower limits for the concentration of the Wnt / β-catenin agonist. In another example, after the first 4-5 days of culture in a high concentration of Wnt / β-catenin agonist, the cell culture medium may contain 0.5 μM to 1.5 μM of the Wnt / β-catenin agonist. In another example, after the first 4-5 days of culture in a high concentration of Wnt / β-catenin agonist, the cell culture medium may contain 0.8 μM to 1.2 μM of the Wnt / β-catenin agonist. Thus, in one example, after the first 4-5 days of culture in a high concentration of a Wnt / β-catenin agonist, the cell culture medium may contain less than 2 μM of a Wnt / β-catenin agonist, and in another example, after the first 4-5 days of culture in a high concentration of a Wnt / β-catenin agonist, the cell culture medium may contain less than 1.5 μM of a Wnt / β-catenin agonist.

[0136] In one example, the Wnt / β-catenin agonist is CHIR. Thus, in one example, after culturing in a high concentration of CHIR for the first 4 to 5 days, the cell culture medium may contain at least 0.5 μM CHIR. In another example, after culturing in a high concentration of CHIR for the first 4 to 5 days, the cell culture medium may contain at least 0.6 μM CHIR. In another example, after culturing in a high concentration of CHIR for the first 4 to 5 days, the cell culture medium may contain at least 0.7 μM CHIR. In another example, after culturing in a high concentration of CHIR for the first 4 to 5 days, the cell culture medium may contain at least 0.8 μM CHIR. In another example, after culturing in a high concentration of CHIR for the first 4 to 5 days, the cell culture medium may contain at least 0.9 μM CHIR. In another example, after culturing in a high concentration of CHIR for the first 4 to 5 days, the cell culture medium may contain about 1 μM CHIR. In another example, after culturing in a high concentration of CHIR for the first 4 to 5 days, the cell culture medium may contain 0.5 μM to 1.5 μM of CHIR. In another example, after culturing in a high concentration of CHIR for the first 4 to 5 days, the cell culture medium may contain 0.8 μM to 1.2 μM of CHIR. Thus, in one example, after culturing in a high concentration of CHIR for the first 4 to 5 days, the cell culture medium may contain less than 2 μM of CHIR. In another example, after culturing in a high concentration of CHIR for the first 4 to 5 days, the cell culture medium may contain less than 1.5 μM of CHIR.

[0137] In one example, after the first 4 to 5 days of culture in a high concentration of CHIR, the medium may contain a low concentration of CHIR and FGF9, as exemplified above. In one example, the cell culture medium contains at least 50 ng / ml of FGF9. In another example, the cell culture medium contains at least 100 ng / ml of FGF9. In another example, the cell culture medium contains at least 150 ng / ml of FGF9. In another example, the cell culture medium contains at least 200 ng / ml of FGF9. In another example, the cell culture medium contains at least 300 ng / ml of FGF9. In another example, the cell culture medium contains at least 350 ng / ml of FGF9. In another example, the cell culture medium contains at least 400 ng / ml of FGF9. In another example, the cell culture medium contains at least 500 ng / ml of FGF9. In another example, the cell culture medium contains 50 ng / ml to 400 ng / ml of FGF9. In another example, the cell culture medium contains 50 ng / ml to 300 ng / ml of FGF9. In another example, the cell culture medium contains 50 ng / ml to 250 ng / ml of FGF9. In another example, the cell culture medium contains 100 ng / ml to 200 ng / ml of FGF9. In another example, the cell culture medium contains 180 ng / ml to 220 ng / ml of FGF9. In another example, the cell culture medium contains 190 ng / ml to 210 ng / ml of FGF9.

[0138] In another example, the levels of FGF9 are replaced with FGF2.

[0139] For example, the cell culture medium may contain 50 ng / ml to 400 ng / ml of FGF2. In another example, the cell culture medium contains 50 ng / ml to 300 ng / ml of FGF2. In another example, the cell culture medium contains 50 ng / ml to 250 ng / ml of FGF2. In another example, the cell culture medium contains 100 ng / ml to 200 ng / ml of FGF2. In another example, the cell culture medium contains 180 ng / ml to 220 ng / ml of FGF2. In another example, the cell culture medium contains 190 ng / ml to 210 ng / ml of FGF2.

[0140] In another example, the above levels of FGF9 are replaced with FGF16. For example, the cell culture medium may contain 50 ng / ml to 400 ng / ml of FGF16. In another example, the cell culture medium contains 50 ng / ml to 300 ng / ml of FGF16. In another example, the cell culture medium contains 50 ng / ml to 250 ng / ml of FGF16. In another example, the cell culture medium contains 100 ng / ml to 200 ng / ml of FGF16. In another example, the cell culture medium contains 180 ng / ml to 220 ng / ml of FGF16. In another example, the cell culture medium contains 190 ng / ml to 210 ng / ml of FGF16.

[0141] In another example, the above levels of FGF9 are replaced with FGF20. For example, the cell culture medium may contain 50 ng / ml to 400 ng / ml of FGF20. In another example, the cell culture medium contains 50 ng / ml to 300 ng / ml of FGF20. In another example, the cell culture medium contains 50 ng / ml to 250 ng / ml of FGF20. In another example, the cell culture medium contains 100 ng / ml to 200 ng / ml of FGF20. In another example, the cell culture medium contains 180 ng / ml to 220 ng / ml of FGF20. In another example, the cell culture medium contains 190 ng / ml to 210 ng / ml of FGF20.

[0142] In one example, the medium containing the high concentration of CHIR does not contain FGF.

[0143] In another example, after the first 4 to 5 days of culture in a high concentration of CHIR, the cell culture medium may contain a low concentration of CHIR, as exemplified above, and FGF and heparin at the levels exemplified above. In one example, the cell culture medium containing the low concentrations of CHIR and FGF also contains heparin. In this example, the cell culture medium may contain 1 μg / ml of heparin. In another example, the cell culture medium may contain 1.5 μg / ml of heparin. In another example, the cell culture medium may contain 2 μg / ml of heparin. In another example, the cell culture medium may contain 0.5 μg / ml to 2 μg / ml of heparin. In another example, the cell culture medium may contain 0.5 μg / ml to 1.5 μg / ml of heparin. In another example, the cell culture medium may contain 0.8 μg / ml to 1.2 μg / ml of heparin.

[0144] In one example, the method of the present disclosure involves combining the above-described IM cell production method with the use of these IM cells in the renal organoid production method exemplified above.

[0145] In one example, stem cells can be cultured using the above-mentioned IM cell production method, then dissociated, and then cultured under the above-mentioned rotation culture to produce renal organoid.In this example, IM cells can be dissociated using EDTA, trypsin, or TrypLE.In another example, cells can be dissociated using EDTA, then passed through a mesh screen, and then cultured under the above-mentioned rotation culture to produce renal organoid.In another example, cells can be dissociated using trypsin, then centrifuged, and the obtained pellet is resuspended and placed under the above-mentioned rotation culture to produce renal organoid.

[0146] The renal organoid encompassed by the present disclosure can be described based on the number of days of culture.This number of days of culture can be divided into two components, including the number of days (X) for producing IM cells from stem cells and the number of days (Y) for forming renal organoid from IM cells.In one example, the step that distinguishes between producing IM cells from stem cells and producing renal organoid from IM cells is the dissociation step of IM cells.One way to express the number of days of culture for producing IM cells from stem cells and the number of days for forming renal organoid from IM cells is X+Y day (for example, 7+12 day describes that 7 days are spent producing IM cells from stem cells, then IM cells are dissociated, and IM cells are spent 12 days forming organoid (that is, Y=number of days as organoid)).

[0147] In one example, the renal organoid encompassed in the present disclosure is the renal organoid of 7+12 days.In another example, the renal organoid encompassed in the present disclosure is the renal organoid of 7+14 days.In another example, the renal organoid encompassed in the present disclosure is the renal organoid of 7+15 days or later.In another example, the renal organoid encompassed in the present disclosure is the renal organoid of 7+17 days.

[0148] In another example, the renal organoids encompassed by the present disclosure are renal organoids at day 7+20. In another example, the renal organoids encompassed by the present disclosure are renal organoids at day 7+22. In another example, the renal organoids encompassed by the present disclosure are renal organoids at day 7+25. In another example, the renal organoids encompassed by the present disclosure are renal organoids at day 7+30. In another example, the renal organoids encompassed by the present disclosure are renal organoids at day 7+13 to day 7+30. In another example, the renal organoids encompassed by the present disclosure are renal organoids at day 7+14 to day 7+30. In another example, the renal organoids encompassed by the present disclosure are renal organoids at day 7+15 to day 7+30. In another example, the renal organoids encompassed by the present disclosure are renal organoids at day 7+15 to day 7+25. In the above example, the IM cells may be cultured for 8, 9, or 10 days (ie, day 8+Y, day 9+Y, or day 10+Y).

[0149] In another example, the cells of the renal organoids disclosed herein proliferate after day 7+7. In another example, the cells of the renal organoids disclosed herein proliferate after day 7+10. In another example, the cells of the renal organoids disclosed herein proliferate after day 7+12. In another example, the cells of the renal organoids disclosed herein proliferate from day 7+5 to day 7+10. In another example, the cells of the renal organoids disclosed herein proliferate from day 7+5 to day 7+12. In these examples, cell proliferation can be detected by using the methods disclosed herein to generate an organoid population, isolating and dissociating organoids from the population at specific time points (e.g., day 7+5, day 7+7, day 7+10, etc.), and determining the cell number at each time point, for example, using a trypan blue dye exclusion test on an automated cell counter (e.g., Life Technologies).

[0150] screening The renal organoids included in the present disclosure can be used for various screening applications.In one example, renal organoids can be used for toxicity screening.For example, renal organoids can be used for nephrotoxicity screening.

[0151] Thus, in one example, the present disclosure encompasses a method for screening a candidate compound for nephrotoxicity, comprising contacting the renal organoid disclosed herein with the candidate compound and determining whether the candidate compound is nephrotoxic.

[0152] In one example, renal organoid as described herein is contacted with candidate compound, and then nephrotoxicity side effect is evaluated.Exemplary nephrotoxicity side effect includes direct effect on renal tubule, podocyte injury, interstitial nephritis and glomerulonephritis.Nephrotoxicity can also be evaluated or measured by any suitable in vitro renal cell function test, including biomarker expression analysis using commercially available tools, such as the Human Nephrotoxicity RT (Human Nephrotoxicity RT) from Qiagen. 2The renal toxicity assay method can be used for evaluating renal toxicity.The ...

[0153] In another example, the present disclosure encompasses the method for screening candidate compounds for therapeutic effects in treating kidney disease, by contacting the renal organoid disclosed herein with the candidate compound under conditions for determining whether the candidate compound is therapeutically effective.In this example, the method can include contacting the renal organoid disclosed herein with the candidate compound in the presence of nephrotoxic compounds, and determining whether the candidate compound is therapeutically effective.

[0154] Another example of screening therapeutic effect includes the evaluation of renal organoid that shows renal disease.For example, said renal disease can be selected from the group consisting of congenital nephrotic syndrome (CNS) (including steroid-resistant nephrotic syndrome and Finnish nephropathy), focal segmental glomerulonephritis (FSGS), Alport syndrome and Pearson syndrome.In one example, said renal disease is CNS.

[0155] The term "therapeutic effect," in the context of the present disclosure, refers to a response in which the therapeutically beneficial effects of a candidate compound or a composition comprising the same outweigh any toxic or adverse effects. Therapeutic effect can be determined based on the improvement of renal cell function; the maintenance of renal cell function; the inhibition (i.e., some delay, or in some cases prevention) of the decline of renal cell function; or the inhibition (i.e., some delay, or in some cases prevention) of renal cell death. In one example, the therapeutic effect is determined based on the presence of appropriate podocyte proteins and evidence that the podocyte proteins are properly polarized. One example is the localization of NPHS1, NPHS2, and NEPH-1 in the membrane of podocytes, where NPHS1, NPHS2, and NEPH-1 are determined by immunohistochemistry.

[0156] In the study using the renal organoid that represents kidney disease, nephrotoxicity and therapeutic effect can be compared with the predetermined standard that is determined based on the corresponding kidney cell function in disease-free kidney organoid.In another example, the improvement of kidney cell function can be determined based on the comparison of kidney cell function between the renal organoid that represents kidney disease and the renal organoid that represents healthy kidney.

[0157] In studies involving contacting renal organoids with a nephrotoxic compound and a candidate compound, improvements in renal cell function can be determined based on a comparison with renal organoids not contacted with the nephrotoxic compound and / or renal organoids contacted with the nephrotoxic compound alone.

[0158] The term "candidate compound" in the context of the present disclosure refers to a drug to be screened. Examples of candidate compounds include small molecules such as low-molecular-weight organic compounds (e.g., organic molecules having a molecular weight of about 50 to about 2,500 Da), peptides or mimetics thereof, ligands including peptide and non-peptide ligands, polypeptides, nucleic acid molecules such as aptamers, peptide nucleic acid molecules, and components, combinations, and derivatives thereof.

[0159] In the present disclosure, terms such as "contact," "exposure," or "application" are considered to be synonymous terms in context. The term "contact" requires that a candidate compound be brought into contact with the glomeruli disclosed herein. In one example, if the compound is water-soluble or water-immiscible, it can be dissolved in cell culture medium. Alternatively, a suitable substrate can be soaked in the compound and then seeded on renal organoids in culture. In screening for volatile candidate compounds, the renal organoids disclosed herein can be exposed to air or other gas mixtures containing the compound. Alternatively, the renal organoids can be exposed to a solution or suspension of the volatile compound in cell culture medium. Similarly, if possible, the volatile compound can be dissolved or stabilized. Alternatively, a suitable substrate can be soaked in the compound and then seeded on renal organoids in culture.

[0160] In the implementation of the method of the present disclosure, can be contacted with renal organoid with a plurality of candidate compounds.For example, at least 2 kinds, at least 3 kinds, at least 4 kinds, at least 5 kinds, at least 6 kinds, at least 7 kinds, at least 8 kinds, at least 9 kinds, at least 10 kinds, at least 11 kinds (least 11), at least 12 kinds, at least 13 kinds, at least 14 kinds, at least 15 kinds, at least 16 kinds, at least 17 kinds, at least 18 kinds, at least 19 kinds, at least 20 kinds, at least 25 kinds, at least 30 kinds, at least 35 kinds, at least 40 kinds, at least 45 kinds, at least 50 kinds, at least 55 kinds, at least 60 kinds, at least 70 kinds, at least 80 kinds, at least 90 kinds, at least 100 kinds, at least 200 kinds, at least 300 kinds, at least Can be contacted with renal organoid with at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 2,000, at least 3,000, at least 5,000, at least 10,000, at least 20,000, at least 40,000, at least 50,000, at least 100,000, at least 200,000 or more candidate compounds.In one example, can be contacted with the same or different renal organoid with candidate compounds.For example, can be screened for a specific combination of candidate compounds.

[0161] In one example, the candidate compound is labeled and then screened. In one example, the candidate compound can be a composition. For example, the candidate compound can be present in a formulation or can include a mixture of compounds or molecules. For example, the candidate compound can be serum. For example, the candidate compound can be serum isolated from a subject suffering from kidney disease. In one example, the serum is isolated from a subject suffering from CNS disease. For example, the serum can be isolated from a subject suffering from steroid-resistant nephrotic syndrome. In another example, the serum is isolated from a subject who has undergone a kidney transplant. In another example, the serum is isolated from a subject suffering from nephrotic syndrome that developed after a kidney transplant.

[0162] Exemplary nephrotoxic agents include aminoglycoside antibiotics, beta-lactam antibiotics, cisplatin, contrast media, NSAIDs, ACE inhibitors, lithium, CsA, and antiepileptic drugs such as phenytoin.

[0163] Renal organoids that have been cultured for various periods can be used for the screening purposes disclosed herein. That is, for example, renal organoids after 7+15 days can be used for screening. In another example, renal organoids from 7+18 days to 7+25 days can be used for screening. In another example, immature renal organoids can be used for screening. For example, those from 7+11 days to 7+18 days can be used for screening. In various examples, IM cells can be cultured for longer periods, that is, renal organoids from 8+Y days, 9+Y days, or 10+Y days can be used for screening.

[0164] In one example, said screening method comprises contacting candidate compound with the library of renal organoid.For example, can use renal organoid of different development stages to screen candidate compound.For example, can use renal organoid of 7+10 days, 7+15 days and 7+25 days.In another example, can use renal organoid of different renal diseases to screen candidate compound.

[0165] Those skilled in the art will understand that there are various screening procedures that can be configured to screen candidate compounds.For example, the renal organoid disclosed herein can be prepared in single-well format or multi-well format, and contacted with candidate compounds for a certain period of time.In one example, renal organoid is prepared in a multi-well plate.In one example, one renal organoid is prepared per well.In another example, two renal organoids are prepared per well.In another example, three renal organoids are prepared per well.In another example, four renal organoids are prepared per well.In another example, five renal organoids are prepared per well.In another example, ten renal organoids are prepared per well.In another example, more than 20 renal organoids are prepared per well.In one example, renal organoid is prepared in a 96-well plate.

[0166] High-throughput screening methods are encompassed by the present disclosure. In this example, high-throughput screening involves preparing a library containing a large number of candidate compounds. Such libraries are then screened in one or more assays to identify library members (e.g., specific chemical species or subclasses) that exhibit a desired level of activity (e.g., therapeutic effect).

[0167] High-throughput screening systems are commercially available and typically automate all steps, including pipetting all samples and reagents, dispensing liquids, timed incubations, and final reading of culture plates (e.g., 96-well format) with a detector appropriate for the assay. These configurable systems offer not only a high degree of flexibility and customization, but also fast startup. Manufacturers of such systems (e.g., Invitrogen, Thermo Fisher Scientific, etc.) provide detailed usage protocols.

[0168] In one example, the method further comprises selecting a compound that shows therapeutic effect.For example, in the presence of nephrotoxic substances and / or when contacted with renal organoids that represent renal disease, the compound maintains renal cell function; suppresses (i.e., delays to some extent, and in some cases prevents) the decline of renal cell function; suppresses (i.e., delays to some extent, and in some cases prevents) renal cell death.In another example, the method further comprises selecting a compound that reduces nephrotoxicity.For example, the compound that suppresses glomerulonephritis can be selected.In another example, the compound that improves renal cell function can be selected.In these examples, renal cell function can be measured by Qiagen Human Nephrotoxicity RT. 2 It can be measured based on biomarker expression using commercially available tools including Profiler™ PCR Array or High Content.

[0169] Personalized Medicine and Stratification The candidate compound that shows therapeutic effect in renal organoid that represents renal disease in object is also likely to show therapeutic effect in said object.Therefore, in one example, these renal organoids can be used to select the drug that is likely to affect the treatment or prevention of renal disease in object.

[0170] In another example, renal organoids can be generated that represent renal disease in multiple subjects. These renal organoids can be used to select drugs that are likely to affect the treatment or prevention of renal disease in multiple subjects, or to identify a group of subjects that are likely to respond to treatment with a specific drug. Such methods can be useful for stratifying subjects in clinical trials of drugs being tested for the therapeutic potential of renal disease. By classifying a subject population based on renal organoid screening, the variability in treatment outcome due to genetic factors can be eliminated or reduced, and the effectiveness of candidate drugs can be more accurately evaluated. Thus, in one example, the present disclosure encompasses a method for stratifying a subject group in a clinical trial of a therapeutic drug, the method comprising: obtaining a population of iPS cells from a group of subjects; generating a renal organoid or a renal organoid population from the iPS cell population of each subject; contacting the renal organoid with a therapeutic drug; determining whether the therapeutic drug has a therapeutic effect; and using the result of the determination to select subjects that are likely to respond to the therapeutic drug. In this example, the method may include contacting renal organoid with a therapeutic agent and a nephrotoxic substance, and then determining whether the therapeutic agent has a therapeutic effect.Examples of therapeutic agents include the above-mentioned candidate compounds, such as one or more small molecules, polynucleotides, peptides, proteins, antibodies, antibody fragments, viruses, bacteria, stem cells, serum, including serum from patients with renal disease.For the avoidance of doubt, serum can be isolated from subjects suffering from specific renal diseases and contacted with the renal organoids disclosed herein.Although various examples of renal diseases are described herein, serum can be isolated from various subjects representing these diseases.Methods for isolating serum from subjects are known in the art.In one example, serum is purified from whole blood sample using centrifugation.

[0171] Bioprinting In one example, the present disclosure encompasses "bioprinted" renal structures, such as kidneys or other nephron-containing organs, organoids, or organ-like structures, produced using the compositions or cells disclosed herein. Terms such as "bioprinted" or "bioprinting," in the context of the present disclosure, refer to methods that utilize precise three-dimensional cell deposition (e.g., cell solutions, cell-containing gels, cell suspensions, cell concentrates, multicellular aggregates, multicellular bodies, bioinks, etc.) by methods compatible with automated, computer-assisted, three-dimensional prototyping devices (e.g., bioprinters). Examples of suitable methods for bioprinting are disclosed in WO 2012 / 054195 and WO 2013 / 040087. In one example, bioprinting is performed using an organ printing device (e.g., Organovo (Invetech)) that uses a hydrogel scaffold to place human cells in a desired configuration to recreate a human organ.

[0172] In one example, the kidney structure is bioprinted from a bioink. The term "bioink" is used in the context of this disclosure to refer to a liquid, semi-solid, or solid formulation containing the compositions or cells described herein. In one example, the bioink comprises a cell solution, a cell aggregate, a cell-containing gel, a multicellular body, or a tissue. In another example, the bioink further comprises a support material.

[0173] The bioprinted renal structures encompassed by the present disclosure may have or develop one or more functional characteristics of a kidney or its components. For example, the bioprinted renal structure may include glomeruli, juxtaglomerular apparatus, interstitial tissue, collecting ducts, Bowman's capsule, proximal convoluted tubules, and / or distal convoluted tubules. In one example, the bioprinted renal structure is non-vascularized. In another example, the bioprinted renal structure includes vasculature, such as arterioles, arteries, veins, and / or capillaries.

[0174] In one example, the bioprinted kidney construct is implantable or adoptively transferable into a host.

[0175] Composition / Kit In one example, the present disclosure relates to a kit or assay used for screening purposes. For example, the present disclosure encompasses a kit or assay used to screen candidate compounds for nephrotoxicity and / or therapeutic effects. In one example, after the renal organoids described herein are prepared in culture, a candidate compound can be contacted with the renal organoids and screened for nephrotoxicity and / or therapeutic effects. Thus, in one example, the present disclosure encompasses an assay used for screening, comprising the renal organoids disclosed herein in culture. In one example, the assay is used for nephrotoxicity screening. In one example, the assay is used for therapeutic effect screening. In one example, the renal organoids are provided with a medium or other components for maintaining the renal organoids in culture. In one example, the renal organoids are provided with written instructions for carrying out the method of the present disclosure. In one example, the assay comprises the renal organoids described herein. In another example, the assay comprises two or more renal organoids. For example, the assay can comprise 10, 20, 30, or more renal organoids. Renal organoids are provided in single-well format or multi-well formats such as 96-well plates. [Example]

[0176] Example 1: Culture and maintenance of hPSCs Human ES cells (H9 cells) were grown on mouse embryonic fibroblast (MEF) feeders in DMEM medium supplemented with 10% KOSR (Life Technologies) and bFGF. Cells were cultured until 80% confluent and then dissociated using TrypLE (Life Technologies). After differentiation, ES cells were conditioned onto a Matrigel (Corning) surface in MEF-conditioned medium and bFGF in the absence of MEF feeders.

[0177] Human iPS cells were grown as individual colonies in E8 medium (Life Technologies) on Geltrex (Life Technologies)-coated plates. iPS cells were passaged once using EDTA when the cells reached 60–70% confluence or every 3 days.

[0178] hPSCs were dissociated into single cells using TrypLE and plated onto matrigel-coated plates at 15,000 cells / cm. 2 Cell numbers were determined using a hemocytometer. Matrigel-adapted hES cells were plated using MEF-conditioned medium. Human iPS cells were plated as single cells overnight on Matrigel-coated plates using RevitaCell (1:100 dilution) in E8 medium.

[0179] Example 2: Improved IM production hPSCs or hESCs were differentiated into intermediate mesoderm by exposing the cells to a high concentration of CHIR (7 μM) in APEL2 or TeSR-E6 medium (StemCell Technologies), APEL medium (StemCell Technologies), or E6 medium (StemCell Technologies) containing 3.5% protein-free hybridoma medium (PFHM) (Thermo Fisher Scientific) for the first 4 days. The medium was changed on day 2. Cultures were then exposed to a low concentration of CHIR (1 μM) as well as FGF9 and heparin for an additional 3 days (days 5–7 after seeding) (Figure 1A, Examples 1–3), which induces a mixture of intermediate mesoderm (IM) cells.

[0180] Gene expression profiling by qPCR on day 7+0 rotating suspension cultures showed that low-concentration, long-term CHIR exposure induced an increase in cap mesenchymal cells compared with short-term exposure (Figure IE, left panel). Expression of PAX2, LHX1, and OSR1 cells was more than two-fold higher than that of the short-term CHIR exposure group (Figure IE, left panel). Exposure of cells to low concentrations of CHIR also induced increases in the ureter epithelium; for example, expression of WNT11 and GATA3 was more than two-fold higher compared with the short-term CHIR exposure group (Figure IE, right panel). Long-term activation of Wnt / β-catenin signaling by the addition of low concentrations of CHIR was shown to be a method for producing improved intermediate mesoderm.

[0181] Example 3: Rotating suspension culture On day 7, IM cells prepared using the method of Example 2 were dissociated using 1 ml of EDTA solution and further incubated in 1 ml of EDTA at 37°C for 3 minutes, and the EDTA solution was aspirated off without disturbing the IM cell layer (Figure 1A). IM cells were also dissociated using 1.5 ml of TrypLE™ Select at 37°C for 3 minutes, and excess TrypLE™ was removed by centrifugation at 1500 RPM in a 15 ml Falcon tube.

[0182] Two ml of the first stage medium (basal medium, 200 ng / ml FGF9, 1 μg / ml heparin, 1 μM CHIR, 0.1% PVA, 0.1% MC) was added along with 10 μM Rho kinase inhibitor (ROCKi, 1:1000 dilution, 10 μM, StemCell Technologies). The cells were gently detached as cell clumps using a Gilson pipette. The cell suspension was then dispensed into a 6 cm well. 2 The cells were transferred to a low-attachment dish (Greiner Bio) and passed through a 40 μm cell strainer (BD Biosciences).

[0183] The culture dishes were replenished with 5 ml of stage 1 medium and rotated at 60 rpm in a Ratek orbital shaker in a standard cell culture incubator at 37°C and 5% CO2. Organoids measuring 20–40 μm in diameter spontaneously formed within 24 hours of placement on the orbital shaker. After 24 hours of rotational culture, the stage 1 medium was replaced with stage 2 medium (basal medium, 200 ng / ml FGF9, 1 μg / ml heparin, 1 μM CHIR, 0.1% PVA, 0.1% MC). Cells were cultured in stage 2 medium for an additional 4 days. From day 7+5, all organoids were cultured with stage 3 medium (basal medium, 0.1% PVA, 0.1% MC) every other day until day 7+18.

[0184] After 18 days of aggregation (days 7+18), brightfield periodic acid-Schiff (PAS) staining confirmed that each renal organoid exhibited tubular epithelial structures, and confocal microscopy confirmed the presence of 6–10 nephrons (Figures 1 and 2). These nephrons showed evidence of early patterning and segmentation. Positive staining for NPHS1 and MAFB revealed the formation of glomeruli. Proximal nephron segments were stained with EpCAM. + The cells stained positive for LTL, CUBN, LRP2, and HNF4A (Fig. 1G). +The segments were able to endocytose FITC-albumin within 24 hours of adding the medium, indicating a functional albumin uptake pathway. Distal nephron segments stained for ECAD and EpCAM, and the presumptive collecting ducts stained for ECAD. + / GATA3 + When generating renal organoids using the SOX17mCherry reporter cell line (Ng et al., 2016), endothelial cells (PECAM1 + / SOX17 + We also noted the presence of a phenotype similar to that of the hPSCs (Figure ID; Figure 2C, Figure 2D, and Figure 2E). Demonstrating the interchangeability of the protocol across human pluripotent stem cell lines, we have reported data demonstrating successful generation of renal organoids from four different cell lines, including hESC reporter lines (H9 GAPTrapLuc2, hES3-SOX17mCherry) (Kao et al., 2016; Ng et al., 2016; van den Berg et al., 2018) and human iPSCs (CRL1502.C32, CRL1502.3) (Briggs et al., 2013; Takasato et al., 2015). All hPSC lines responded uniformly to the protocol and patterned similarly into renal organoids (Figure 2B).

[0185] Example 4: Effects of duration, concentration, and timing of canonical Wnt signaling on renal organoid patterning To optimize differentiation within the renal organoid protocol, hPSC monolayers were stimulated with a fixed concentration of CHIR99021 (7 μM) for various periods (3, 4, 5, and 6 days) and then cultured in the presence of low concentrations of CHIR, FGF9, and heparin until day 7 (Figure 2C, Figure 2D, and Figure 2E). After 18 days, the renal structure of the resulting renal organoids was assessed using confocal microscopy (Figure 2C, Figure 2D, and Figure 2E). Canonical Wnt activation for only 3 days failed to produce kidney morphology (Figure 2B, left panel). Instead, the epithelial structures present showed an ill-defined epithelium with large cystic lumens and no evidence of nephron formation. Initial induction with 7 μM CHIR99021 for 4 or 5 days resulted in the formation of surrounding SOX17 endothelial and interstitial stromal cells, respectively. + Group and MEISl / 2 / 3 + Renal organoids containing patterned nephrons were generated, including the presence of clusters of MEIS1-expressing NPHS1-positive nephrons (Figure 2C, 2D, and 2E, center panels). However, 6 days of Wnt activation produced larger renal organoids with greater NPHS1 staining, but with clearly impaired epithelial architecture and expanded MEIS1-expressing nephrons (Figure 2D). + The stromal population was included (Figures 2C, 2D, and 2E, right panels). An initial induction of 4 days with 7 μM CHIR99021 was found to be optimal and was used in further studies.

[0186] Example 5: Dissociation of Renal Organoids Renal organoids are heterogeneous epithelial structures approximately 250–300 μm in diameter. Harsh enzymes can destroy cell surface markers and result in loss of cell identity for subsequent use. Gentle dissociation using cold-activated protease (Liberase™, Roche) yields the greatest number of single viable cells. Renal organoids were transferred to a 15 ml Falcon tube using a 5 ml serological pipette and allowed to settle. After aspirating the medium supernatant, the organoid pellet was washed three times with 0.1 M PBS. Next, the organoids were treated with 500 μl of 1 μg / ml Liberase™ solution and incubated at 4°C for 20 minutes, triturating every 5 minutes. After 20 minutes, the renal organoids were completely dissociated into single cells. The Liberase™ was inactivated by washing twice with DMEM containing 10% FCS, and the final cell pellet was suspended in DMEM containing 10% FCS.

[0187] Example 6: Rotation of intermediate mesodermal cells generates renal microorganoids in suspension culture Existing methods for generating renal organoids are labor-intensive, expensive, and produce low-quality organoids. We have developed an economical, simple, and rapid method for generating renal organoids in suspension culture. Unlike previous methods, minimal dissociation and slow rotation of the monolayer followed by culture on low-adhesion culture plates results in the formation of cell aggregates at the intermediate mesoderm (IM) differentiation stage (day 7). This results in the formation of 8,000–10,000 renal organoids, far fewer than those generated using previous methods. After 18 days of suspension culture, each renal organoid contains approximately 6–10 nephrons, with evidence of early patterning and segmentation, including the formation of a proximal epithelium containing podocytes, a distal epithelium, and glomeruli.

[0188] Importantly, single-cell transcriptional profiling revealed that these miniature organoids were equivalent to standard organoids produced using conventional methods in terms of cell diversity and maturity. This directed differentiation approach resulted in a 30-40-fold increase in cell expansion over 21 days of culture, which is 1 × 10 compared to conventional approaches, such as those based on Takasato et al. (2015) Nature, Vol. 526:564-568. 6 This resulted in a 3-4x improvement in yield and a 4x reduction in cost per organoid-derived kidney cell.

[0189] The method of the present disclosure, illustrated in Figure 1A and described in Examples 1-3, involves adding 0.1% polyvinyl alcohol (PVA) and 0.1% methylcellulose (MC) to the rotation culture medium to enhance the cohesion of intermediate mesoderm (IM) cells, causing them to spontaneously aggregate into three-dimensional spherical organoids. After 24 hours of rotation culture, the organoids formed an outer laminin basement membrane.

[0190] C32 organoids cultured for an additional 12–18 days were harvested, whole-mounted, and stained for NPHS, LTL, ECAD, and GATA3 for confocal microscopy analysis. To assess the presence of vasculature, the organoids were stained with mouse anti-human CD31 (1:300, BD Biosciences) and goat anti-human CUBN for mature proximal tubules. Immunofluorescence analysis of organoids at days 7–12 revealed major nephron segmentation, characterized by nephrin-positive glomeruli, LTL-positive proximal tubules, ECAD-positive distal tubules, and ECAD / GATA3-double-positive collecting duct cells, including GATA3-positive interstitial cells (Figures 1G, 4E, and 4F), comparable to the results observed in the Transwell organoid system (Figure 4A). With regard to nephron number (5–10 nephrons in each organoid) and the presence of non-renal cell types, gyratory kidney micro-organoids exhibit a simpler morphology compared to Transwell organoids.

[0191] Taken together, these results demonstrate that the suspension spinning method can form organized organoids capable of generating complex multicellular renal organoids with all nephron segments. This method was tested using not only human iPS cell lines but also human ES cell lines, and organoids were successfully formed. Various basal medium conditions, such as APEL, APEL2, and E6, were shown to be suitable for the disclosed method. Therefore, this method may be useful for scaling up renal cell culture for personalized medicine, drug screening, and regenerative cell therapy.

[0192] Example 7: Renal microorganoids in suspension culture suitable for scale-up of iPS-derived kidney cells To evaluate suspension culture for scale-up, C32 iPS cells were differentiated to generate IMs as described in Examples 1 and 2. Organoid growth was monitored by measuring the size and total cell number in culture from day 7+0 (Figures 4C and 4D).

[0193] Brightfield images of C32 organoids generated using rotating suspension culture showed an increase in size and maintenance of epithelial structure (Figure 4A and Figure 4B). Organoid size was measured using brightfield images of up to 10 randomly selected organoids in NIS-Elements microscopy software (Nikon). Random samples of up to 10 organoids were taken at various growth rate intervals, and organoid diameters were measured. Sizes were reported as ranging from small to large. A consistent increase in organoid size was observed over time, with organoids ranging from 30 µm to 300 µm in diameter (Figure 4C). Total cell number was assessed after dissociation using TrypLE™ Select and manually counted using a hemocytometer. A 40-fold increase in cell number was observed from days 7+12 to 7+18 compared to the seeding density on day 7+0 (Figure 4D).

[0194] Example 8: Renal microorganoids in suspension culture exhibit organized nephron segments and distinct tubule lumens While conventional Transwell organoids exhibit complex morphology, which limits the study of the 3D structure of individual nephrons, the gyratory kidney micro-organoids produced by the methods described herein are much simpler and contain fewer nephrons.

[0195] These organoids exhibit distinct 3D morphology, enabling the study of nephrons in 3D space. C32-derived renal organoids were generated using the methods described in Examples 1-3 and immunostained with antibodies against NPHS1, LTL, ECAD, and GATA3 to visualize individual nephron segments using high-throughput confocal microscopy with a Z-resolution matched to a pinhole microscope.

[0196] Renal organoids were collected in 15 ml flacon tubes, washed twice with PBS to remove excess medium, and fixed in freshly prepared 2% PFA for 20 min at 4 °C. Next, organoids were washed three times with 0.3% TritonX100 in PBS (PBST) to remove excess PFA and stored in PBST at 4 °C until staining. Fixed organoids were blocked with 10% donkey serum in PBST (blocking buffer) for at least 1 h and then incubated with primary antibodies diluted in blocking buffer. The differentiation potential of renal organoids was assessed by staining of major nephron segments with the following primary antibodies: sheep anti-human NPHS1 (1:300, R&D Systems), biotin anti-human LTL (1:300, Vector Laboratories), mouse anti-human ECAD (1:300, Life Technologies), rabbit anti-human GATA3 (1:300, Cell Signaling Technologies), mouse anti-human CD31 (1:300, BD Biosciences), goat anti-human CUBN (1:300, Santa Cruz), and LRP2 (1:300, Sapphire Bioscience). Organoids were incubated overnight in primary antibodies at 4°C, washed five times with PBST, and then incubated with species-matched, fluorescently labeled secondary antibodies. After staining, organoids were dehydrated using various concentrations of methanol and then cleared using BABB (benzyl alcohol and benzyl benzoate, 1:2 ratio) as previously reported by Dodt HU et al. (2007). Cleared organoids were mounted on MatTek glass-bottom dishes and subjected to confocal microscopy using an inverted Zeiss LSM 780 microscope. Images were analyzed using ZEN software (Zeiss).

[0197] Images were analyzed using Imaris software, and 3D renderings of the acquired confocal images were reconstructed (Figure 4E and Figure 4F). The 3D images showed distinct, interconnected nephron segments, starting from the glomerulus (NPHS1), proximal tubule (LTL+), distal tubule (ECAD+), collecting duct (ECAD+GATA3+), and interstitial cells (GATA3+) (Figure 5E). Using a snipping tool, the lumen formed within the tubular cells was visualized (Figure 5F). The results in Figure 5E and Figure 5F demonstrate that the rotation method described herein is useful for enabling morphological studies of developing renal organoids in 3D space.

[0198] Example 9: Single-cell RNA sequencing analysis reveals promising renal phenotypes To further characterize the gyratory renal organoids, we performed single-cell RNA sequencing of C32-derived renal microorganoids at days 7+18. Approximately 40–50 renal microorganoids and one standard whole organoid were cultured using the same hPSC line (CRL1502.C32 in APEL medium) for up to days 7+18. The organoids were harvested and washed three times with PBS to remove excess medium. The organoids were treated with 400 μl of 1 μg / ml Liberase™ (Roche) solution at 4°C for 20 minutes, stirring with a 1 ml pipette every 5 minutes. Within 20 minutes, the organoids dissociated into single cells. Liberase™ was inactivated by adding 2 ml of cell culture media. The cells were centrifuged at 1300–1500 rpm for 3–5 minutes to form a pellet. The supernatant was removed, and the pellet was resuspended in fresh DMEM-F12 medium, passed through a 20 μm cell strainer to remove clumps, and stored on ice until analysis. Viability and cell count were analyzed by FACS and trypan blue exclusion using an automated cell counter (Life Technologies). Cells were stored on ice until analysis. Cells were thoroughly mixed using a large-bore 1 ml pipette tip, and approximately 4,000 viable cells were used for RNA-seq analysis. The Chromium Single Cell 3' Solution, developed with TenX Genomics technology, was used. Sample preparation was performed according to the TenX Genomics single cell protocol (details are provided in the Chromium Single Cell 3' Reagent Kits v2 User Guide, accessible online). The single-cell suspension, gel beads, and partitioning oil were loaded into the appropriate wells of a 10x Chromium Chip. The 10x Chromium Chip was then secured in a 10x Gasket, and the complete assembly was loaded into a 10x Chromium Controller. This automatically generates a suspension of single cells coated with oil droplets containing their unique UMIs, which can then be employed in conventional RT-PCR to amplify transcripts.

[0199] Cells were barcoded using nanoliter-scale Gel Bead-In-EMulsions (GEMS) and UMIs to individually index each cell's transcriptome. Magnetic beads were used to remove residual reagents and primers after barcoding. Full-length barcoded cDNAs were PCR-amplified to generate sufficient quantities for library construction. These libraries were simultaneously sequenced for UMIs and cDNA fragments in two separate reads. Library analysis was performed using Cell Ranger™, which enables the study of expression data at single-cell resolution. The Cell Ranger pipeline (vl.3.1) was used for sample demultiplexing, barcode processing, and single-cell gene counting (Zheng et al., 2017). Samples were demultiplexed to generate a pair of FASTQ files for each sample. Reads containing sequence information were aligned to the GRCh38 reference genome. Cell barcodes were filtered to remove empty droplets, and PCR duplicates were removed by selecting unique combinations of cell barcode, UMI, and gene ID. The final result was a gene expression matrix, which was used for further analysis, thus allowing the study of expression data at single-cell resolution. Further analysis to demonstrate cell clustering, cell type classification, and differential gene expression was performed using the Seurat R package (version 2.3.1).

[0200] The gene expression matrix generated in Cell Ranger was imported into Seurat (Satija et al., 2015) for quality control and further analysis. All cells underwent initial filtering to remove genes expressed in fewer than three cells and cells expressing fewer than 200 genes. Further filtering removed a single cell with more than 15% mitochondrial transcripts. Using the cyclone function in Sscran (Lun et al., 2016; Scialdone et al., 2015), each cell was assigned a score related to its likelihood of being in G1, S, or G2M phase, and the cell cycle phase was assigned based on this scoring.

[0201] Expression data were normalized and scaled, and variability associated with the number of UMIs, mitochondrial expression rate, ribosomal expression rate, and G2M score was regressed using the ScaleData function in Seurat. Cells were clustered using the first 15 principal components and a resolution value of 1.2 using a shared nearest neighbor modularity maximization-based clustering algorithm implemented in Seurat. A marker gene list was created using the FindAllMarkers function in Seurat to find differentially expressed genes between clusters with a logarithmic fold change of 0.25 or greater.

[0202] For the combined analysis of standard and renal organoid datasets, a gene-cell matrix was generated in Cell Ranger as described above. All cells were initially filtered for genes expressed in fewer than three cells and for cells with fewer than 200 genes. Each dataset was normalized and scaled, and regressions were performed in Scran on the number of UMIs, percentage of mitochondrial expression, percentage of ribosomal expression, and S, G1, and G2M scores. Clustering was based on aligned composite components calculated in Seurat using the RunCCA and AlignSubspace functions (Butler et al., 2018). For the combined dataset, clustering was performed at a resolution of 0.6 (Butler et al., 2018).

[0203] We analyzed single-cell RNA gene expression profiling of organoids produced by the method described herein and the conventional Transwell method. Based on the genes present in the cells, the number of UMIs was plotted as a tSE plot and automated clustering was performed (Figure 6A-6B). GO enrichment analysis of all clusters revealed 22.3% nephrons, 37.5% total interstitium, and 9.8% vascular tissue (Figure 6A). In contrast, in the gyratory renal microorganoids, 32.5% mature nephrons (excluding cap-like mesenchyme and nephron precursors) and 25.9% interstitium were present, whereas in the gyratory C32 organoids, no vascular tissue was present (Figure 6B). Thus, the gyratory renal microorganoids exhibited better markers of kidney development compared to Transwell-cultured organoids. Renal microorganoids also showed enhanced nephron organization compared to Transwell-cultured organoids (Figure 4).

[0204] Example 10: Transcriptional validation of kidney differentiation within renal organoids Characterization of cell types present within renal organoids was performed using single-cell RNA sequencing (scRNA-seq). Pools of 20–30 renal organoids were dissociated into single viable cells using the cold-activated protease Liberase™. As a result, 89.4% single cells were generated, of which 88.5% were viable (data not shown). Cell Ranger (Tenx Genomes) was used to generate a matrix of UMIs per cell, which was imported for further analysis using the Seurat R package (version 2.3.1) (Satija et al., 2015). Filtered data revealed 1,673 cells with a median number of expressed genes per cell of 3,759. Clustering using the Seurat R package generated seven distinct cell clusters at a resolution of 0.6 (Figure 3A and Figure 3B, Table 1). Differential expression studies were performed to identify markers for each cluster, and gene ontology and functional enrichment analyses for the top significantly upregulated genes within each cluster were performed using the PANTHER gene ontology suite ( Mi et al., 2013 ; Table 1 ).

[0205] [Table 1]

[0206] As evident using immunofluorescence of whole-mount organoids, endothelium (a subset of cluster 1) and podocytes (cluster 6, 18 cells) were represented by very few individual cells in the scRNA-seq data. Cluster 3 (293 cells) and cluster 5 (122 cells) showed expression of genes consistent with kidney nephron epithelium; cluster 0 showed expression of kidney vesicle / S-shaped body genes (early nephrons), while epithelial cell cluster 5 showed expression of distal tubule / collecting duct markers such as GFRA1 (Figure 3C). Cluster 2 showed expression of nephron precursor markers SIX1, SIX2, and CITED1, as well as the stromal marker PAX3, previously associated with myogenic Wilms tumor (Hueber et al., 2009). Cells in cluster 2 also expressed markers of myogenic fate, such as MYF5 and MYF6, but not PAX7, MYOD1, or TBX6. Cluster 0 (430 cells), representing the largest cluster, exhibited characteristics of more committed nephron progenitors, with expression of early renal vesicle markers PAX2, PAX8, LHX1, and JAG1, as well as human P markers LYPD1 and DAPL1 (Lindstrom et al., 2018). Cluster 1 (337 cells) exhibited mesenchymal features, such as PDGFRB and MEIS2. Cluster 4 expressed the early nephron marker cadherin CDH6, but also displayed neural transcriptional signatures, suggesting the presence of a neural off-target population, as previously reported in renal organoids (Wu et al., 2017). This analysis strongly supported the cell type identity observed within renal organoids at the immunofluorescence level.

[0207] Example 11: Comparison of Standard Organoids and Renal Organoids. Single-cell transcriptional profiling demonstrates equivalence in nephrogenesis patterning. To directly compare the cellular components within renal organoids with other renal organoid methods, scRNA-seq data from renal organoids was combined with data from 1,421 renal organoid cells generated using the same iPSC cell line (CRL1502.C32) and the standard renal organoid protocol of Takasato et al., 2016. The two datasets were combined using an alignment algorithm implemented in Seurat (Butler et al., 2018) that uses correlated component analysis followed by dynamic time warping. Clustering revealed the following: committed nephron precursors (cluster 0), nephron epithelium (cluster 6), podocytes (cluster 7), interstitium (clusters 1 and 3), endothelial cells (cluster 5), and PAX3. +ve Eight transcriptional clusters were identified in the combined dataset, representing nephron cells (cluster 2), neural off-target populations (cluster 3), and neural off-target populations (cluster 4) (Figure 5A and Figure 5B). While the proportion of cells belonging to each cluster varied, all clusters were represented in both datasets (Figure 5B-Figure 5D). Direct comparison of key markers for each cluster indicates that while there were clear differences in the proportion contributing to each cluster between protocols (Figure 5B and Figure 5C), there was strong transcriptional agreement between cells identified in any given cluster between both protocols (Figure 5D). The neural off-target populations identified in renal organoids were also evident in standard organoids. Overall, the renal organoid dataset contained a higher proportion of nephron cells and a lower proportion of interstitial cells than the standard organoid dataset (Figure 5E). An increase in PAX2-expressing nephron cells and a decrease in MEIS1 / 2 / 3-expressing interstitial cells in renal organoids compared to standard organoids was confirmed using immunofluorescence analysis of whole-mount organoids (Figure 5F and Figure 6).

[0208] Example 12: Renal organoids provide a better platform for efficient scale-up of hPSC-derived kidney cells Standard renal organoids cultured on Transwell™ filters may face diffusion limitations after 3 weeks of culture due to the size of the generated organoid tissue (Figure 4A). Immunofluorescent staining of nephron segments after this period suggested that the nephron structure was spatially restricted to the organoid's edge. In contrast, renal organoids contain renal tubules throughout their structure (Figure 4B). Furthermore, large numbers of renal organoids can be simultaneously formed using an orbital shaker, avoiding the laborious manual steps associated with standard organoid protocols. As a result, approximately 8,000–10,000 uniformly sized renal organoids can be generated in 5–10 minutes, compared to approximately 30 organoids generated in 60 minutes with standard organoid protocols. Renal organoids exhibit a much smaller final size (250–300 µm) compared to standard organoids (3,000–5,000 µm) (Figure 4C). As demonstrated by immunofluorescence, nephrons formed within standard organoids are located at the periphery of the tissue compared to renal organoids (Figure 4A). However, these structures are much larger than those in renal organoids. To directly compare the efficiency and cost of each approach, standard and renal organoids generated using iPSC and hESC reporter lines, respectively, were dissociated into single-cell suspensions at multiple time points throughout the differentiation protocol, beginning with day 7, for quantification of total cell number (Figure 4D). While standard organoids showed no significant change in total cell number per organoid after day 7+7, renal organoids continued to increase in total cell number until day 7+12. Overall, cell number increased 8-10-fold under standard organoid conditions, but increased 30-40-fold in renal organoids. This represents a 3-4-fold improvement in cell yield using this modified protocol.

[0209] Example 13: Addition of all-trans retinoic acid improves maturation of glomerular podocytes To determine whether the addition of all-trans retinoic acid (atRA) promotes glomerular maturation in organoids produced using the convoluted culture method described in Examples 1-3, atRA was added to the culture medium from day 7+0 onward. All-trans retinoic acid (2.5 μM) was added to C32-derived convoluted organoids from day 7+5 to day 7+10 (Figure 5). After day 7+18, immunofluorescence analysis showed that the addition of atRA improved the podocyte phenotype compared to the control group (Figures 7A and 7B). This result was confirmed by mRNA expression and qPCR analysis of samples from day 7+11 and day 7+18, which showed that the addition of 2.5 μM atRA increased the expression of glomerular markers such as NPHS1 and proximal tubule markers such as CUBN compared to the control (Figure 7C).

[0210] Example 14: Drug Toxicity Testing in Renal Microorganoids To evaluate the suitability of the suspension culture method described herein for in vitro renal drug toxicity testing, a drug toxicity study was performed. Organoids generated by this method were harvested on days 7+18 and randomly assigned to treatment groups in 250 μl of medium in 24-well low-attachment plates. Organoids were stimulated with various concentrations of the cytotoxic drug adriamycin (0, 2.5, and 5 μg / ml) for 24 hours. After stimulation, the medium was removed, and organoids were fixed in 2% PFA for immunofluorescence analysis of apoptosis using TUNEL staining. Some organoids were lysed for RNA analysis (Figures 8A-8D).

[0211] Example 15: Summary of improvements in kidney organoid production Renal organoids obtained from the above exemplary method demonstrate reliable formation of renal nephron epithelial, interstitial, and endothelial cell components, comparable at the single-cell transcriptional level to those present in previously reported renal organoid protocols (Takasato et al., 2016; Takasato et al., 2015). However, by modifying culture conditions, relative cell yields were improved 3-4-fold, resulting in a 4-fold reduction in generation cost per million kidney cells. The robustness of the exemplified method is evident from the successful replication of renal organoid generation using two different hESC (H9 and hES3) and three different iPSC lines (iPSC GAPTrap td-Tomato, CRL1502.C32, and CLR1502.3), including the hES3 SOX17mCherry, H9 GAPTrap Luc2, and iPSC GAPTrap td-Tomato fluorescent reporter lines.

[0212] Those skilled in the art will appreciate that the present disclosure as set forth in the specific embodiments may be subject to numerous variations and / or modifications without departing from the spirit of the disclosure as broadly described, and therefore, the present embodiments are to be considered in all respects as illustrative and not restrictive.

[0213] This application claims priority to Australian Patent Application Publication No. 2017904424, filed October 31, 2017, which is incorporated herein by reference.

[0214] All publications discussed and / or referenced herein are incorporated herein by reference in their entirety.

[0215] Any discussion of documents, acts, materials, devices, articles and the like which has been included in the present specification is solely for the purpose of providing a context for the present invention and is not to be construed as an admission that any or all of such matters form part of the prior art or were general general knowledge in the art relevant to the present invention as existing prior to the priority date of each claim of this application.

[0216] References Barberi et al., (2005) Plos Medicine 2(6): 0554-0559. Briggs et al., (2013) Stem Cells 31: 467-78. Butler et al., (2018) Nat Biotechnol 36: 411-420. Cima et al., (1991) Biotechnol. Bioeng. 38: 145 1991. Dodt et al., (2007) Nat Methods 4: 331-6. Forbes et al., (2018) Am J Hum Genet. 102:816-831 Hueber et al., (2009) Pathol. 12: 347-54. Kao et al., (2016) Stem Cell Reports 7: 518-526. Lindstrom et al., (2018) J Am Soc Nephrol. 29: 806-824. Lun et al., (2016) FlOOORes 5: 2122. Mi et al., (2013) Nat Protoc 8: 1551-1556. Ng et al., (2016) Nat Biotechnol 34: 1168-1179. Satija et al., (2015) Nat Biotechnol, 33: 495-502. Scialdone et al., (2015) Methods 85: 54-61. Takasato et al., (2015) Nature 526: 564-8. Vacanti, et al., (1988) J. Ped. Surg. 23 :3-9. Vacanti, et al., (1991) Plast. Reconstr. Surg. 88:753-9. van den Berg et al., (2018) Stem Cell Reports 10: 751-765. Vodyanik et al., (2010) Cell Stem cell 7:718-728. Wu et al., (2017). Zheng et al., (2017) Nat Commun 8: 14049. Some aspects of the invention are described below. 1. Renal organoids containing fewer than 50 nephrons. 2. The renal organoid of item 1, comprising fewer than 25 nephrons. 3. The renal organoid of item 1, comprising fewer than 15 nephrons. 4. Renal organoids described in section 1, containing 5 to 12 nephrons. 5. The renal organoid of any one of items 1 to 4, comprising cells that express any one or more of PAX2, SIX1, LHX1, OSR1, WNT11, GATA3, PAX8, EYA1, and CITED1 at high levels, and / or any one or more of PDGFRA, MEIS2, WT1, and / or C-RET at low levels. 6. The renal organoid according to any one of items 1 to 4, comprising cells expressing high levels of PAX2, SIX1, LHX1, OSR1, WNT11, and GATA3. 7. The renal organoid according to any one of items 1 to 6, comprising NPHS+ podocytes, LTL+ proximal tubule segments, ECAD+ distal tubule segments, ECAD+ / GATA3+ collecting ducts, or a combination thereof. 8. The renal organoid according to any of items 1 to 7, wherein the renal organoid is derived from a stem cell selected from the group consisting of H9, hES3, iPSC GAPTrap td-Tomato, CRL1502.C32, CLR1502.3, hES3 SOX17mCherry, or H9 GAPTrap Luc2. 9.1×10 4 ~5×10 4 9. The renal organoid according to any one of items 1 to 8, comprising approximately cells. 10.1.5×10 4 ~2.5×10 4 9. The renal organoid according to any one of items 1 to 8, comprising approximately cells. 11. The renal organoid according to any one of items 1 to 10, having a diameter of about 250 to 500 μm. 12. The renal organoid according to any one of items 1 to 11, which survives for at least 3 weeks under rotational culture. 13. The renal organoid according to any one of items 1 to 11, which survives in culture for at least 4 weeks. 14. The renal organoid according to any of items 1 to 13, wherein the nephron comprises a collecting duct (GATA3+; ECAD+), an early distal tubule (GATA3-; LTL-; ECAD+), an early proximal tubule (LTL+; ECAD-), and a glomerulus (WT1+). 15. The renal organoid according to any of items 1 to 14, which is produced by spinning a population of intermediate mesoderm (IM) cells in a cell culture medium under conditions sufficient to promote the development of renal organoids. 16. The renal organoid of item 15, wherein the cell culture medium contains 200 ng / ml of FGF9. 17.0.5×10 6 ~1.5×10 6 17. The renal organoid of item 15 or item 16, produced by swirling IM at 100 cells / ml. 18.0.8×106 ~1.2×10 6 17. The renal organoid of item 15 or item 16, which is produced by swirling IM at 100 cells / ml. 19. A therapeutic composition comprising the renal organoid or an enzymatic digest thereof according to any one of items 1 to 18. 20. A method for treating kidney disease, comprising administering the composition according to item 19 to a subject in need of treatment. 21. An in vitro method for producing renal organoids, comprising spinning a population of intermediate mesoderm (IM) cells in a cell culture medium containing FGF. 22. The method of claim 21, wherein the IM cells are spun in culture for at least 5 days, the first 24 hours comprising spinning the cells in a cell medium containing FGF, heparin, CHIR, and a ROCK inhibitor, and the subsequent 4 days comprising culturing the cells in a cell medium containing FGF, heparin, and CHIR. 23. The method according to item 22, wherein the remaining number of days of culture comprises culturing the cells in a cell culture medium containing PVA and MC. 24. The method according to item 22, wherein the cell culture medium contains 100 to 300 ng / ml of FGF9. 25. The method of any of items 21 to 24, wherein the first 24 hours comprises spinning the cells in a cell culture medium containing FGF, 0.5 to 1.5 μg / ml heparin, 0.5 to 1.5 μM CHIR, and 9 to 11 μM ROCK inhibitor. 26. The method of any of items 21 to 25, wherein the subsequent 4 days comprise spinning the cells in a cell culture medium containing FGF, 0.5 to 1.5 μg / ml heparin, and 0.5 to 1.5 μM CHIR. 27. The method according to any of items 21 to 26, wherein the subsequent 4 days comprise culturing the cells in a cell culture medium containing FGF9, heparin, CHIR, MVA, and PVC. 28. The method according to item 27, wherein the cell culture medium containing FGF contains 0.05 to 0.2% MVA and 0.05 to 0.2% PVC. 29. The method according to any one of items 21 to 28, wherein the remaining number of days of culture comprises culturing the cells in a cell culture medium containing 0.05 to 0.2% PVA and 0.05 to 0.2% MC, but not FGF, heparin CHIR, or a ROCK inhibitor. 30. The method according to any one of items 21 to 29, wherein the IM cells are spun at 30 to 90 rpm. 31. The method according to any of items 21 to 30, wherein the IM cells are rotated for 18 to 24 days. 32. The method of any of items 21 to 31, wherein the IM cells are produced by culturing a stem cell population for at least 7 days, the first 4 to 5 days comprising culturing the stem cells in a cell culture medium containing at least 6 μM of a Wnt / β-catenin agonist, and the remaining days of culture comprising culturing the cells in a cell culture medium containing FGF and at least 0.5 μM of a Wnt / β-catenin agonist. 33. The method of item 32, wherein the Wnt / β-catenin agonist is CHIR and the FGF is FGF9. 34. The method according to item 32 or 33, wherein the FGF-containing cell culture medium contains 100 to 300 ng / ml of FGF9. 35. The method according to any one of items 33 to 34, wherein the FGF-containing cell culture medium contains 0.5 to 1.5 μM CHIR. 36. The method according to any one of items 32 to 35, wherein the cell culture medium containing FGF further contains 0.5 to 1.5 μg / ml of heparin. 37. The method of any of items 21 to 36, wherein the IM cells are dissociated with EDTA or trypsin and passed through a mesh screen before spinning. 38. The method according to any one of items 32 to 37, wherein the stem cells are pluripotent stem cells, embryonic stem cells, or induced pluripotent stem (iPS) cells. 39.0.5×10 6 cells / ml~1.5×10 6 39. The method of any of items 21 to 38, comprising spinning an IM cell population comprising 100 cells / ml of IM. 40. A renal organoid produced by the method described in any one of items 21 to 39. 41. A method for screening a candidate compound for nephrotoxicity, comprising contacting the renal organoid of any one of items 1 to 18 or item 40 with the candidate compound to determine whether the candidate compound is nephrotoxic. 42. The method of item 41, wherein the candidate compound is a small molecule. 43. Use of the renal organoid of any one of items 1 to 18 or item 40, or the composition of item 19, in producing kidneys, or renal cells or renal tissue. 44. An in vitro method for producing intermediate mesoderm (IM) cells, comprising culturing a population of posterior primitive streak (PPS) cells for 2 to 5 days in cell culture medium containing FGF and less than 4 μM of a Wnt / β-catenin agonist. 45. An in vitro method for producing intermediate mesodermal (IM) cells, comprising culturing a stem cell population for at least 7 days, wherein the first 4-5 days comprise culturing the stem cells in a cell culture medium containing at least 6 μM of a Wnt / β-catenin agonist, and the remaining days comprise culturing the cells in a cell culture medium containing FGF9 and at least 0.5 μM of a Wnt / β-catenin agonist. 46. ​​The method according to item 45, wherein the FGF-containing medium contains 0.5 to 3 μM of a Wnt / β-catenin agonist. 47. The method according to item 45, wherein the medium contains 0.8 to 1.2 μM of a Wnt / β-catenin agonist. 48. The method according to any of items 45 to 47, wherein the first 4 days comprise culturing the stem cells in a cell culture medium containing at least 6 μM of a Wnt / β-catenin agonist. 49. The method according to any one of items 45 to 47, wherein the first 4 days comprise culturing the stem cells in a cell culture medium containing 7 μM of a Wnt / β-catenin agonist. 50. The method according to any one of items 44 to 49, wherein the FGF-containing cell culture medium contains 100 to 300 ng / ml of FGF9. 51. The method according to any one of items 44 to 50, wherein the cell culture medium containing FGF further contains heparin. 52. The method according to item 51, wherein the cell culture medium contains 0.5 to 2 μg / ml of heparin. 53. The method according to any one of items 45 to 52, wherein the stem cells are pluripotent stem cells, embryonic stem cells, or induced pluripotent stem (iPS) cells. 54. The method of item 20, wherein the composition is administered intravenously. 55. The method of item 20, wherein the composition is administered by intrarenal artery injection, intrarenal parenchymal injection, implantation, or subcapsular transplantation. 56. A method for bioprinting a kidney, comprising: Preparing a bioink from the organoid according to any one of items 1 to 18 or item 40; Bioprinting a kidney, A method comprising: 57. Use of the organoid according to any one of items 1 to 18 or item 40, or the composition according to item 19, in the manufacture of kidneys, or kidney cells or kidney tissue. 58. A method for generating nephron cell types for cell therapy, comprising producing renal organoids using the method described in any one of items 21 to 39, or producing IM cells using the method described in items 44 to 53.

Claims

1. 1. An in vitro method for producing renal organoids, comprising the steps of culturing stem cells in a monolayer to produce intermediate mesoderm (IM) cells, dissociating the IM cells, and spinning the IM cell population in a cell culture medium containing FGF, wherein the IM cells are spinned in a suspension culture, and the renal organoids comprise NPHS1+ podocytes, LTL+ proximal tubule segments, ECAD+ distal tubule segments, and ECAD+ / GATA3+ collecting ducts, and the IM cells are spinned for at least 5 days, the first 24 hours comprising spinning the cells in a cell culture medium containing FGF, heparin, CHIR, and a ROCK inhibitor, and the subsequent 4 days comprising spinning the cells in a cell culture medium containing FGF, heparin, and CHIR.

2. 2. The in vitro method of claim 1, wherein the cell culture medium comprises poly(vinyl alcohol) (PVA) and methylcellulose (MC).

3. 3. The in vitro method according to claim 1, wherein the cell culture medium contains 100 to 300 ng / ml of FGF9.

4. 4. The in vitro method of any one of claims 1 to 3, wherein the first 24 hours comprises spinning the cells in a cell culture medium containing 0.5 to 1.5 μg / ml heparin, 0.5 to 1.5 μM CHIR, and 9 to 11 μM ROCK inhibitor.

5. 5. The in vitro method of any one of claims 1-4, wherein the subsequent four days comprise spinning the cells in cell culture medium containing 0.5-1.5 μg / ml heparin and 0.5-1.5 μM CHIR.

6. The in vitro method of any one of claims 1 to 5, wherein the IM cells are spun at 30 to 90 rpm.

7. The in vitro method of any one of claims 1 to 6, wherein the IM cells are rotated for 18 to 24 days.

8. 8. The in vitro method of claim 1, wherein the IM cells are produced by culturing a population of stem cells for at least 7 days, the first 4 to 5 days comprising culturing the stem cells in a cell culture medium containing at least 6 μM of a Wnt / β-catenin agonist, and the remaining days of culture comprising culturing the cells in a cell culture medium containing FGF and at least 0.5 μM of a Wnt / β-catenin agonist.

9. 9. The in vitro method of claim 8, wherein the Wnt / β-catenin agonist is CHIR and the FGF is FGF9.

10. The in vitro method according to claim 8 or 9, wherein the cell culture medium containing FGF further contains 0.5 to 1.5 μg / ml of heparin.

11. 11. The in vitro method of any one of claims 1 to 10, wherein the IM cells are dissociated with EDTA or trypsin and passed through a mesh screen before spinning.

12. The in vitro method according to any one of claims 8 to 11, wherein the stem cells are pluripotent stem cells, embryonic stem cells, or induced pluripotent stem (iPS) cells.

13. 0.5 x 10 6 cells / ml ~ 1.5 x 10 6 13. The in vitro method of any one of claims 1 to 12, comprising spinning an IM cell population comprising cells / ml of IM.

14. 14. A renal organoid comprising fewer than 50 nephrons produced by the in vitro method of any one of claims 1 to 13, wherein the renal organoid has a diameter of less than 500 μm and comprises NPHS1+ podocytes, LTL+ proximal tubule segments, ECAD+ distal tubule segments, and ECAD+ / GATA3+ collecting ducts.

15. The renal organoid of claim 14, comprising cells that express any one or more of PAX2, SIX1, LHX1, OSR1, WNT11, GATA3, PAX8, EYA1 and CITED1 at high levels, wherein said high level of expression is at least 1-fold higher than that of renal organoids cultured without rotation.

16. The renal organoid of claim 14, comprising cells that express any one or more of PDGFRA, MEIS2, WT1, and / or C-RET at low levels, wherein the low levels of expression are at least 1-fold lower compared to renal organoids cultured without rotation.

17. The renal organoid of any one of claims 14 to 16, comprising at least 25% mature nephrons and at least 15% interstitium.

18. 1 x 10 4 ~5 x 10 4 The renal organoid of any one of claims 14 to 17, comprising cells.

19. The renal organoid of any one of claims 14 to 18, having a diameter of 250 to 500 μm.

20. The renal organoid of any one of claims 14 to 19, which survives for at least 3 weeks under rotational culture.

21. The renal organoid of any one of claims 14 to 20, wherein the nephron comprises collecting duct (GATA3+; ECAD+), early distal tubule (GATA3-; LTL-; ECAD+), early proximal tubule (LTL+; ECAD-), and glomerulus (WT1+).

22. A therapeutic composition comprising the renal organoid of any one of claims 14 to 21.

23. A composition comprising the renal organoid of any one of claims 14 to 21 for use in treating renal disease.

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