Compositions and methods

Abstract

The present disclosure relates to kidney organoids and methods for making the same. The organoids and methods can be used in a variety of applications, such as disease modeling, drug screening, regenerative medicine, and the expanded production of kidney cells.

Description

Field of the Invention

[0001] The present disclosure relates to kidney organoids and methods for making the same. The organoids and methods can be used in a variety of applications, such as disease modeling, drug screening, regenerative medicine, and expanding kidney cell production. Background of the Invention

[0003] The kidneys play an important role in removing waste products and maintaining body fluid volume. Functional working units are called nephrons. The human kidney contains up to 2 million epithelial nephrons responsible for blood filtration, all of which are produced after birth. Nephron progenitor cells are absent in postnatal human kidneys. The lack of this progenitor cell population does not ensure the ability of the nephron to self-renew, and therefore, subsequent damage, aging and disease may lead to end-stage renal disease (ESDR). The limited therapeutic options for treating ESDR often put more stress on the already damaged kidneys. The only available but expensive treatment options for late-stage kidney disease are dialysis and / or kidney transplantation, both of which have significant disadvantages and affect the patient's quality of life.

[0004] The directed differentiation of human pluripotent stem cells (hPSC) (including human embryonic stem cells (hES) and human induced pluripotent stem cells (hiPS)) to different cell endpoints has made it possible to generate organoid models of various human tissues (including kidneys).Previous organoid models (such as those discussed in Takasato et al. (2015) Nature, Vol.526:564-568) are expensive, and may produce organoids with complex three-dimensional structures, thereby limiting their use in imaging and screening applications.These organoids may also be restricted by diffusion after three weeks of cultivation, thereby limiting their ability to produce increased cell numbers and their ability to mature.This in turn makes the current scheme for kidney organoid generation become the suboptimal cell source for regenerative medicine or disease modeling.Therefore, new kidney organoids and their manufacturing methods are needed. Summary of the invention

[0005] The inventors surprisingly identified kidney organoids with simplified three-dimensional structures. Such organoids are advantageous because they are easier to image and culture for a long time. Therefore, in one example, the present disclosure encompasses kidney organoids comprising less than 50 nephrons. In another example, the kidney organoids comprise less than 25 nephrons. In another example, the kidney organoids comprise less than 15 nephrons. In another example, the kidney organoids comprise 5 to 12 nephrons. In one example, kidney organoids are produced by vortexing an intermediate mesoderm (IM) cell colony in a cell culture medium under conditions sufficient to promote the development of kidney organoids. In this example, the IM cell culture medium may comprise about 180 to 220 ng / ml of FGF9.

[0006] In one example, by vortexing 0.5 × 10 6 to 1.5×10 6 In another example, approximately 0.8 to 1.2 × 10 cells / ml were used to generate kidney organoids. 6 In another example, the kidney organoids include cells expressing high levels of nephron markers. In another example, the kidney organoids include cells expressing high levels of PAX2, LHX1, SIX1, OSR1, WNT11 and GATA3. In another example, the kidney organoids include cells expressing high levels of PAX2, LHX1, SIX1, OSR1, WNT11 and GATA3. In another example, the kidney organoids include cells expressing high levels of PAX2, LHX1, SIX1, OSR1, WNT11 and GATA3. In another example, the kidney organoids include cells expressing high levels of PAX2, SIX1, LHX1, OSR1, WNT11, GATA3, PAX8, EYA1 and CITED1 and / or low levels of PDGFRA, MEIS2, WT1 and / or C-RET. In another example, the kidney organoids comprise cells expressing high levels of PAX2, LHX1, SIX1, OSR1, WNT11, GATA3, PAX8, EYA1, and CITED1.

[0007] In another example, the kidney 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 GAPTrapLuc2.

[0008] In another example, the kidney organoid comprises cells expressing low levels of stromal markers. In another example, the kidney organoid comprises cells expressing low levels of PDGFRA, MEIS2, WT1, and / or C-RET. In another example, the kidney organoid comprises NPHS+ podocytes, LTL+ proximal segments, ECAD+ distal segments, ECAD+ / GATA3+ collecting ducts, or a combination thereof. In another example, the kidney organoid comprises approximately 1×10 4 Up to 5×10 4 In another example, a kidney organoid contains approximately 1.5×10 4 Up to 2.5×10 4Cells. In another example, the diameter of the kidney organoid is about 250 to 500 μm. In another example, the kidney organoid remains alive in vortex culture for at least eighteen days. In another example, the kidney organoid remains alive in vortex culture for at least three weeks. In another example, the kidney organoid remains alive in vortex culture for at least four weeks. In another example, the kidney organoid nephron includes a collecting duct (GATA3+; ECAD+), an early distal tubule (GATA3-; LTL-; ECAD+), an early proximal tubule (LTL+; ECAD-) and a glomerulus (WT1+).

[0009] The inventors also surprisingly identified that the cells of the kidney organoids disclosed herein continued to divide after 7 days in vortex culture (e.g., D7+7). Without wishing to be bound by any particular theory, this may indicate that organoids generated using vortex culture are more suitable for therapeutic applications, such as transplantation.

[0010] In another example, the present disclosure encompasses a composition comprising a kidney organoid as defined herein. In another example, the present disclosure encompasses a composition comprising a kidney organoid as defined herein or a digest thereof. For example, the present disclosure encompasses a composition comprising a kidney organoid as defined herein or an enzymatic digest thereof.

[0011] Again, without wishing to be bound by any particular theory, it is believed that the low complexity of the kidney organoids disclosed herein may make them more suitable for producing compositions for transplantation. For example, a composition produced from a low complexity kidney organoid may be more inclined to differentiate into the correct cell type rather than forming a teratoma or cartilage. In another example, the present disclosure encompasses a composition comprising an enzymatic digestion of a kidney organoid as defined herein.

[0012] In another example, the present disclosure encompasses a method for treating kidney disease, comprising administering a composition as defined herein to a subject in need thereof. In one example, the composition comprises a whole organoid. In another example, the composition comprises a digest of an organoid disclosed herein. For example, kidney disease can be renal failure. In one example, the composition is administered intravenously. In another example, the composition is administered by renal artery injection, renal parenchymal injection, implantation, or subcapsular transplantation.

[0013] In another example, the present disclosure encompasses an in vitro method of generating kidney organoids, the method comprising vortexing a population of intermediate mesoderm (IM) cells in a cell culture medium comprising FGF.

[0014] In one example, the method provides a cost-effective means for expanding the production of renal cell types in vitro. In one example, IM cells are vortexed in a culture medium containing FGF, CHIR and heparin. In one example, IM cells are vortexed in culture for at least 5 days, wherein the first 24 hours include vortexing the cells in a cell culture medium containing FGF, heparin, CHIR and ROCK inhibitor, and the next four days include culturing the cells in a cell culture medium containing FGF, heparin and CHIR. In this example, the ROCK inhibitor is present in the cell culture medium within the first 24 hours and is not present in the cell culture medium in the following days. In one example, the cell culture medium includes 100 to 300ng / ml FGF9. In one example, the cell culture medium includes 180 to 220ng / ml FGF9. In one example, the cell culture medium includes PVA and MC. In one example, the first 24 hours include vortexing the cells in a cell culture medium comprising FGF, 0.5 to 1.5 μg / ml heparin, 0.5 to 1.5 μM CHIR and 9 to 11 μM ROCK inhibitor. In another example, the following four days include vortexing the cells in a cell culture medium comprising FGF, 0.5 to 1.5 μg / ml heparin and 0.5 to 1.5 μM CHIR. In this example, there is no ROCK inhibitor in the following four days of cell culture medium. In another example, the following four days include culturing the cells in a cell culture medium comprising FGF9, heparin, CHIR, MC and PVA. In another example, the following four days include culturing the cells in a cell culture medium comprising FGF9, heparin, CHIR, 0.05 to 0.2% MC and 0.05 to 0.2% PVA. In another example, the cells are cultured in a cell culture medium comprising 0.05 to 1.5% PVA and 0.05 to 1.5% MC after at least five days for the remaining days of culture. In this example, the cell culture medium may comprise PVA and MC without FGF9, heparin, CHIR or ROCK inhibitor. In another example, the cell culture medium comprising FGF comprises at least 100 ng / ml FGF9. In another example, the cell culture medium comprising FGF comprises at least 150 ng / ml FGF9. In another example, the cell culture medium comprising FGF comprises 150 to 250 ng / ml FGF9. In another example, the cell culture medium comprising FGF comprises 180 to 220 ng / ml FGF9.

[0015] In some examples, the methods for producing kidney organoids disclosed herein result in improved cell yields compared to cell yields obtained from organoids produced without vortexing (e.g., those described in Takasato et al. (2015)). In one example, after 10 days of vortex culture, a 30-fold increase in cell yield can be observed from the starting number of IM cells added to the vortex culture. In another example, after 10 days of vortex culture, a 35-fold increase in cell yield can be observed from the starting number of IM cells added to the vortex culture. In another example, after 12 days of vortex culture, a 40-fold increase in cell yield can be observed from the starting number of IM cells added to the vortex culture. In another example, after 12 days of vortex culture, a 45-fold increase in cell yield can be observed from the starting number of IM cells added to the vortex culture. In another example, after 12 days of vortex culture, a 30 to 40-fold increase in cell yield can be observed from the starting number of IM cells added to the vortex culture. In another example, after 18 days of vortex culture, a 30- to 40-fold increase in cell yield was observed from the starting number of IM cells added to the vortex culture.

[0016] In one example, the IM cells are vortexed between 30 and 90 rpm. In another example, the IM cells are vortexed for 18 to 24 days.

[0017] In another example, IM cells are produced by culturing a stem cell population for at least 7 days, wherein the first 4 to 5 days comprise culturing the stem cells in a cell culture medium comprising at least 6 μM Wnt / β-catenin agonist, and the remaining culturing days comprise culturing the cells in a cell culture medium comprising FGF and at least 0.5 μM Wnt / β-catenin agonist. In one example, the Wnt / β-catenin agonist is CHIR. In these examples, the cell culture medium comprising FGF may comprise 100 to 300 ng / ml of FGF9. In these examples, the cell culture medium comprising FGF may comprise 0.5 to 1.5 μM CHIR. In these examples, the cell culture medium comprising FGF may further comprise 0.5 to 1.5 μg / ml heparin. In one example, the IM cells are cultured with EDTA or trypsin or TrypLE TM select dissociation and pass through a screen before vortexing. In one example, the stem cell is a pluripotent stem cell, an embryonic stem cell or an induced pluripotent stem (iPS) cell. In one example, the stem cell is selected from the group consisting of H9, hES3, iPSC GAPTrap td-Tomato, CRL1502.C32, CLR1502.3, hES3 SOXUmCherry or H9 GAPTrapLuc2.

[0018] The inventors have identified that the methods of the present disclosure require relatively low starting cell numbers. This is advantageous because it allows for cost-effective expansion of organoid and cell cultures. Thus, in another example, the methods of the present disclosure include 0.5×10 6 IM cells / ml to 3×10 6 In another example, the method of the present disclosure comprises vortexing a population of about 0.8×10 6 IM cells / ml to 1.2×10 6 IM cells / ml. Thus, in one example, the disclosure encompasses a kidney organoid disclosed herein, wherein the organoid is produced by vortexing a population of IM cells. Exemplary occurrence times and culture media for vortexing a population of IM cells to produce kidney organoids are disclosed herein. In one example, by vortexing a population of IM cells containing 0.5×10 6 IM cells / ml to 3×10 6 Organoids are generated by vortexing a population of IM cells containing less than 2×10 6 Organoids were generated by vortexing a population of IM cells containing 0.5 × 10 6 IM cells / ml to 1.5×10 6 Organoids were generated by vortexing a population of IM cells containing 0.8×10 6 IM cells / ml to 1.2×10 6 Organoids were generated by vortexing the IM cell population at 10 IM cells / ml.

[0019] In another example, the present disclosure encompasses kidney organoids produced by the methods defined herein.

[0020] In another example, the present disclosure encompasses a method of screening a candidate compound for nephrotoxicity, the method comprising contacting a kidney organoid as defined herein with a candidate compound to determine whether the candidate compound is nephrotoxic. In one example, the candidate compound is a small molecule.

[0021] In another example, the disclosure encompasses kidney organoids, cell colonies or compositions as defined herein when used to produce kidney or kidney cells or tissues. In another example, the disclosure encompasses kidney organoids, cell colonies or compositions as defined herein for treating kidney disease. In some examples, the cell colony refers to cells derived from enzymatic digestion of kidney organoids.

[0022] The present inventors have also surprisingly identified that prolonged cultivation of stem cells in a medium comprising low concentrations of CHIR and activated Wnt / β-catenin signaling is beneficial for the generation of modified intermediate mesoderm.

[0023] In one example, the present disclosure encompasses an in vitro method of generating intermediate mesoderm (IM) cells, the method comprising culturing a population of postprimate filament (PPS) cells in a cell culture medium comprising FGF and less than 4 μM of a Wnt / β-catenin agonist for 2 to 5 days.

[0024] In another example, stem cells may be initially cultured in CHIR for about 7 days, wherein the stem cells are cultured in a medium containing a high concentration of CHIR for the first 4 to 5 days, and then cultured in a medium containing a low concentration of CHIR and FGF for the remaining days. Thus, in another example, the disclosure encompasses an in vitro method for producing intermediate mesoderm (IM) cells, the method comprising culturing a stem cell population for at least 7 days, wherein the first 4 to 5 days include culturing the stem cells in a cell culture medium containing at least 6 μM of a Wnt / β-catenin agonist, and the remaining culture days include 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 culture days include culturing cells in a cell culture medium containing FGF, the cell culture medium containing 0.5 to 3 μM of a Wnt / β-catenin agonist. In another example, the remaining culture days include culturing cells in a cell culture medium containing FGF, the cell culture medium containing 0.8 to 1.2 μM of a Wnt / β-catenin agonist. In another example, the first 4 days include culturing the stem cells in a cell culture medium comprising at least 6 μM of a Wnt / β-catenin agonist. In another example, the first 4 days include culturing the stem cells in a cell culture medium comprising 7 μM of a Wnt / β-catenin agonist. In one example, the cell culture medium comprising FGF includes 100 to 300 ng / ml of FGF9. In one example, the cell culture medium comprising FGF includes 180 to 220 ng / ml of FGF9. In another example, the cell culture medium comprising FGF9 also includes heparin. For example, the cell culture medium may include 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, hES3SOX17mCherry, or H9 GAPTrap Luc2.

[0025] In another example, the present disclosure encompasses a method of bioprinting a kidney, the method comprising preparing a bio-ink from an organoid or cell population as defined herein and bioprinting a kidney. In some examples, the cell population refers to cells derived from enzymatic digestion of a kidney organoid.

[0026] In another example, the present disclosure encompasses organoids, compositions or cell populations defined herein when used to generate kidney or kidney cells or tissues. In another example, the present disclosure encompasses methods of generating nephron cell types for cell therapy, the method comprising generating kidney organoids using the methods or IM cell populations defined herein.

[0027] Unless expressly stated otherwise, any example herein should apply mutatis mutandis to any other example.

[0028] The scope of the present disclosure is not limited to the specific examples described herein, which are intended for illustrative purposes. As described herein, functionally equivalent products, compositions and methods are clearly within the scope of the present disclosure.

[0029] Throughout the specification, references to individual steps, compositions of matter, groups of steps or groups of compositions of matter shall be considered to cover both one and multiple (ie, one or more) those steps, compositions of matter, groups of steps or groups of compositions of matter, unless expressly stated otherwise or the context requires otherwise.

[0030] Hereinafter, the present disclosure is described by means of the following non-limiting examples and with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1. Generation of kidney organoids in suspension culture. (A) Overview of the kidney micro-organoid differentiation protocol by using images of differentiation performed with CRL1502.C32 cells. (B) Bright field images showing uniform kidney micro-organoids at day 7+18 (scale 100 mm). (C, D1 and D2) Immunofluorescence and confocal images of micro-organoids showing the formation of nephron segments independent of the size and shape of the micro-organoids, including the development of vascular structures (scale 100 mm). (E) Bar graph showing the average fold change of the intermediate mesoderm gene expression profile by qPCR for FGF9+ / -1 μM CHIR99021 at day 7+0. Data are expressed as mean values, (F) Immunofluorescence for PAX2 treated with FGF9+ / -1 μM CHIR99021 (scale 50 μm) (D7+11). (G) Immunofluorescence and confocal images of nephron compartments within kidney microorganoids; podocytes (PHS1+ and MAFB+), proximal tubules (LTL+, CUBN+, LRP2+, and HNF4A+), distal tubules (ECAD), collecting ducts (ECAD+GATA3+), and endothelial cells (SOX17+ and PECAM1+) (scale 50 μm).

[0032] Figure 2.Micro-kidney organoids in suspension culture show functional proximal tubules, and initial Wnt signaling is important for kidney organoid development and maturation. (A) Confocal image of micro-kidney organoid tubules at day 7+18, showing FITC albumin uptake (scale 5μm). (B) Confocal images of micro-kidney organoids generated using 4 different cell lines (including hES (H9 GAP-Trap Luc2, hES3SOX17mCheny) and iPS (CRL1502.C32 and CRL1502.3)) at day 7+18, with antibodies labeling different nephron segments (scale 50μm). (C, D, and E) hES3 SOX17mCherry-derived microorganoids generated after exposure to initial 7 μM CHIR99021 for different days for 3, 4, 5, and 6 days of treatment, showing bright field (C, scale 100 μm) and immunofluorescence confocal images showing SOX17+ vasculature (D) and MEIS1 / 2 / 3+ stroma (E) (scale 100 μm).

[0033] Figure 3. Transcriptional validation of renal differentiation within microorganoids. (A) t-SNE plot after Seurat clustering of single-cell RNA-seq of CRL1502-C32 microorganoids at day 7+18, showing 11 different clusters. (B) Heat map showing proportional gene expression of key marker genes within clusters. (C) t-SNE plot demonstrating expression of key marker genes for selected nephron cell types. Color intensity is scaled by gene, with blue indicating higher expression.

[0034] Figure 4. Kidney microorganoids provide a better platform for efficient hPSC-derived kidney cell expansion. (A) Brightfield image of a standard kidney organoid at day 7+11 (left, scale 500 μm), immunofluorescence and confocal images of the entire standard organoid (tile scan), which shows that the nephron structure is spatially restricted at the edge of the organoid (middle, scale 200 μm) and a magnified image of the nephron within the organoid (right, scale 200 μm). (B) Brightfield image of kidney microorganoid and magnified brightfield image of a single kidney microorganoid, confocal image of kidney microorganoid at day D7+11 (scale 200 μm). (C) Size changes of organoids at different developmental stages. (D) Changes in total cell number over time from the starting cell number and the scalability of microorganoids compared to standard organoids. (EF) Immunofluorescence and Bitplane-Imaris 3D reconstruction of C32 microorganoids at D7+18 showed clear nephron segments interconnected in a polarized manner, starting from the glomerulus (PHS1), proximal tubule (LTL+), distal tubule (ECAD+), collecting duct (ECAD+GATA3+), and interstitial cells (GATA3+).

[0035] Figure 5. Comparative single-cell transcriptional profiles of standard kidney organoids and micro-organoids show equivalent nephrogenic patterns. (A) t-SNE plots of 10x scRNA-Seq data from kidney micro-organoids (Micro-org) and standard organoids (Stand-org) from day 7+18 (CRL1502.32) after comprehensive Seurat analysis. (B) t-SNE plots representing the contribution of micro-organoids and standard organoids to cell types in each cluster, colored by organoid type (C) Bar graphs representing the proportion of each micro-organoid or standard organoid dataset assigned to each transcriptional cluster and differentiation lineage type. (D) Split dot plots showing gene expression of kidney markers between kidney micro-organoids and standard organoids in each cluster. (E) Violin and scatter plots showing log-normalized counts per cell for nephrons (PAX2, SIX1, LHX1) and matrix-related genes (PDGFRA, MEIS2) in micro-organoids and standard organoids. (F) Immunofluorescence showing the expression of PAX2 and MEIS1 / 2 / 3 between micro-organoids and standard organoids (scale 50 μm).

[0036] Figure 6 Comparison of nephron and stromal markers in microorganoids and standard organoids. (A and B) t-SNE feature plots of nephron and stromal genes in standard and microorganoid scRNA-Seq data.

[0037] Figure 7 All-trans retinoic acid contributes to improved glomerular maturation in renal microorganoids. Immunofluorescence analysis of C32 vortexer organoids generated in vortex suspension culture. (A) Organoids generated without supplementation under atRA. (B) Organoids generated with atRA from D7+5 to D7+10 show improved glomerular podocyte maturation. (C) qPCR analysis of organoids generated with and without atRA at different time points (D7+11 and D7+18).

[0038] Figure 8 Kidney microorganoids provide a platform for drug toxicity screening. Doxorubicin treatment (24 h) induced dose-dependent toxicity in kidney microorganoids (AC) by increasing the expression of the apoptotic marker TUNEL. (D) Doxorubicin treatment also reduced the expression of kidney-specific genes in microorganoids. DETAILED DESCRIPTION

[0039] Unless specifically defined otherwise, all technical and scientific terms used herein shall have the same meanings as commonly understood by one of ordinary skill in the art (e.g., molecular biology, cell culture, stem cell differentiation, cell therapy, gene modification, disease modeling, biochemistry, physiology, and clinical research).

[0040] Unless otherwise indicated, the molecular and statistical techniques used in this disclosure are standard procedures well known to those skilled in the art. This technique is described and explained throughout the literature, for example, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), TA Brown (ed.), Essential Molecular Biology: A Practical Approach, Vols. 1 and 2, IRL Press (1991), DM Glover and BD Hames (eds.), DNA Cloning: A Practical Approach, Vols. 1-4, IRL Press (1995 and 1996), and FM Ausubel et al. (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed Harlow and David Lane (eds.) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), JE Coligan et al. (eds.) Current Protocols in Immunology, John Wiley & Sons (includes all updates to date), Michos Odysse (editor) Kidney Development: Methods and Protocols (Springer), Robert Lanza (editor) Handbook of Stem Cells, Volume 1, Embryonic Stem Cells (Elsevier).

[0041] As used in this specification and the appended claims, the singular and the singular forms of the terms "a," "an," and "the," for example, optionally include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a kidney organoid" optionally includes one or more kidney organoids.

[0042] As used herein, unless indicated to the contrary, the term "about" means + / - 10%, more preferably + / - 5%, more preferably + / - 1% of the specified value.

[0043] The term "and / or", such as "X and / or Y", should be understood to mean "X and Y" or "X or Y", and should be construed to provide explicit support for both meanings or either meaning.

[0044] Throughout the specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of stated elements, integers or steps, or groups of elements, integers or steps, but not the exclusion of any other elements, integers or steps, or groups of elements, integers or steps.

[0045] Cell compositions according to the present disclosure can be administered to various 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 a cow. In another example, the subject is a human. Terms such as "subject," "patient," or "individual" are terms that can be used interchangeably in the present disclosure in context.

[0046] As used herein, the term "treatment" refers to a clinical intervention designed to change the natural course of the treated individual or cell during clinical pathology. Ideal therapeutic effects include reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. For example, if one or more symptoms associated with the disease are alleviated or eliminated, the individual is successfully "treated". In one example, the term "treatment" refers to therapeutic treatments and defensive or preventive measures for nephropathy, anemia, EPO deficiency, tubular transport defects, or glomerular filtration defects, wherein the purpose is to reverse, prevent, or slow down (alleviate) the target disorder. Patients in need of treatment include patients who have already suffered from nephropathy, anemia, EPO deficiency, tubular transport defects, or glomerular filtration defects, patients who tend to suffer from such disorders, or patients who need to prevent such disorders. In one example, treatment includes stabilization and / or improvement of renal function.

[0047] "Effective amount" refers to an amount that effectively achieves the desired treatment or preventive effect at least within a certain dose and a desired time period. The 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 for achieving the treatment of a kidney disease or condition as described above. The effective amount can vary according to the disease or condition to be treated and also according to other factors related to body weight, age, ethnic background, sex, health and / or physical condition and the mammal being treated. Typically, the effective amount will fall within a relatively wide range (e.g., "dosage" range) that can be determined by routine tests and experiments of a physician. The effective amount can be administered in a single dose or in a dosage repeated once or multiple times during the treatment period.

[0048] "Therapeutically effective amount" is at least the minimum concentration required to achieve a measurable improvement in a specific nephropathy (e.g., nephritis, renal cell carcinoma). Therapeutically effective amount herein can also vary according to factors such as the patient's disease state, age, sex and weight, and the ability of the cell composition to cause a desired response in an individual. Therapeutically effective amount is also such an amount, wherein the therapeutic beneficial effect exceeds any toxic or deleterious effect of the composition. In the case of renal cell carcinoma, the therapeutically effective amount can reduce the number of cancer cells; reduce the size of the primary tumor; inhibit (i.e., slow down to some extent and stop in some instances) cancer cells from infiltrating surrounding organs; inhibit (i.e., slow down to some extent and stop in some instances) tumor metastasis; inhibit or delay tumor growth or tumor progression to some extent; and / or alleviate one or more symptoms related to renal cell carcinoma to some extent. For renal cell carcinoma therapy, for example, in vivo efficacy can be measured by assessing survival time, disease progression time (TTP), response rate (RR), response time and / or quality of life.

[0049] "Intermediate mesoderm (IM)" cells refer to embryonic mesoderm cells that are derived from the definitive mesoderm, which in turn derives from the posterior protonema and ultimately develops into the urogenital system, including the ureters and kidneys, as well as other tissues, such as gonads. Non-limiting examples of markers that characterize or represent the intermediate mesoderm include PAX2, OSR1, and / or LHX1.

[0050] It should also be understood that the production of IM cells does not mean that the IM cells are a pure or homogeneous population of IM cells without the presence of other cell types (e.g., definitive mesoderm). Therefore, reference to "IM cells" or "IM cell populations" refers to cells or cell populations that contain IM cells. Suitably, according to the present invention, IM cells are produced by contacting posterior protonema cells with one or more agents that promote the differentiation of posterior protonema cells into IM cells, as will be described in more detail below. Preferably, IM cells are produced by contacting posterior protonema cells with one or more agents that promote the differentiation of posterior protonema cells into IM cells.

[0051] "Posterior protoline (PPS)" cells refer to cells that can be obtained from the cells at the posterior end of the protoline structure formed in the blastocyst during early mammalian embryonic development, or cells that correspond functionally and / or phenotypically thereto. The posterior protoline establishes bilateral symmetry, determines the site of gastrulation and initiates germ layer formation. Typically, the posterior protoline is a progenitor cell of the mesoderm (i.e., the presumed mesoderm), while the anterior protoline is a progenitor cell of the endoderm (i.e., the presumed endoderm). Non-limiting examples of markers that characterize or represent the posterior protoline include Brachyury (T). Non-limiting examples of markers that characterize or represent the anterior protoline are SOX17. MIXL1 can be expressed by the anterior and posterior protolines.

[0052] It should also be understood that the generation of posterior protoline cells does not mean that the posterior protoline cells are pure or homogeneous populations of posterior protoline cells, without the presence of other cell types. Therefore, reference to "posterior protoline cells" or "posterior protoline cell populations" refers to cells or cell populations that include posterior protoline cells. Posterior protoline cells are produced by contacting hPSC cells with one or more agents that promote the differentiation of hPSC cells into posterior protoline cells, which will be described in more detail below. For example, one or more agents may include bone morphogenetic protein 4 (BMP4), activin A and / or Wnt agonists, such as CHIR99021.

[0053] Kidney organoids

[0054] The present disclosure encompasses the generation 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 are derived from the definitive mesoderm, which in turn derives from the posterior protonema and ultimately develops into the urogenital system, including the ureters and kidneys, as well as other tissues, such as gonads. Non-limiting examples of markers that characterize or represent the intermediate mesoderm include PAX2, OSR1, and / or LHX1.

[0055] In one example, culture conditions are provided to allow these IM cells to "self-organize" and form kidney organoids. The term "kidney organoids" is used in the context of the present disclosure to refer to heterogeneous 3D aggregations of cells that summarize aspects of cell self-organization, structure, and signaling interactions present in native kidneys. Examples of kidney organoids are described in Takasato et al. (2015) Nature, Vol. 526: 564-568, WO 2014 / 197934, and WO 2016 / 094948. In the context of the present disclosure, the terms "kidney organoids" and "kidney organoids" can be used interchangeably.

[0056] The inventors have surprisingly identified kidney organoids with a simplified three-dimensional structure. Such organoids are advantageous because they are easier to image and culture for a long time. For example, there are 0.8 to 2 million nephrons in each kidney of a healthy adult, typically about 1 million. In contrast, the organoids encompassed by the present disclosure contain a much lower number of nephrons. Therefore, in one example, the present disclosure encompasses kidney organoids that contain structural features of a natural kidney with a reduced number of nephrons. In one example, the kidney organoids encompassed by the present disclosure may include one or more nephrons. In one example, the nephrons are segmented into distal and proximal tubules, early loops of Henle, and glomeruli. In another example, the organoids include segmented nephrons surrounded by endothelial cells, perivascular cells, and renal interstitium. In another example, the organoids of the present disclosure do not show the presence of a vasculature.

[0057] In other examples, organoids according to the present disclosure are at least partially vascularized. For example, an organoid can include a nephron containing podocytes that form foot processes and undergo vascularization.

[0058] In one example, the kidney organoid is characterized by % nephron, % matrix, and / or % vasculature. In this example, single-cell RNA sequencing can be used to characterize the kidney organoid. Examples of single-cell sequencing are provided below. In one example, the kidney organoid contains at least 20% mature nephrons. In another example, the kidney organoid contains at least 25% mature nephrons. In another example, the kidney organoid contains at least 30% mature nephrons. In another example, the kidney organoid contains at least 31% mature nephrons. In another example, the kidney organoid contains at least 32% mature nephrons. In these examples, the kidney organoid also contains at least 15% matrix. In another example, the kidney organoid also contains at least 20% matrix. In another example, the kidney organoid also contains at least 25% matrix. In another example, the kidney organoid does not contain any substantial vasculature. In another example, the kidney organoid does not contain vasculature.

[0059] In one example, a kidney organoid according to the present invention comprises less than 100 nephrons. In another example, a kidney organoid according to the present invention comprises less than 90, less than 80, less than 70, less than 60 nephrons. In another example, a kidney organoid according to the present invention comprises less than 50 nephrons. In another example, a kidney organoid according to the present disclosure comprises less than 40, less than 30, less than 20, less than 10 nephrons. In another example, a kidney organoid according to the present disclosure comprises less than 5 nephrons. In another example, a kidney organoid according to the present disclosure comprises less than 4, less than 3 nephrons.

[0060] In another example, the kidney organoids according to the present disclosure comprise 2 to 100 nephrons. In another example, the kidney organoids according to the present disclosure comprise 2 to 50 nephrons. In another example, the kidney organoids according to the present disclosure comprise 2 to 10 nephrons. In another example, the kidney organoids according to the present disclosure comprise 5 to 12 nephrons. In another example, the kidney organoids according to the present disclosure comprise 6 to 10 nephrons. In another example, the kidney organoids according to the present disclosure comprise 2 to 6 nephrons. In another example, the kidney organoids according to the present disclosure comprise 2 to 4 nephrons.

[0061] "Nephron" is the functional working unit of the kidney, which plays an important role in removing waste from the blood / plasma and maintaining body fluid volume. Those skilled in the art can use various methods to identify and count them in the organoids disclosed herein. For example, confocal microscopy and immunofluorescence markers (e.g., WT1+ glomeruli; NPHS+ podocytes, LTL+ECAD-proximal tubules, ECAD+ distal tubules, and ECAD+GATA3+ collecting ducts) can be used to visualize and count nephrons.

[0062] Generally, the species identity of the kidney organoids covered by the present disclosure, whether mammals, such as mice, humans or other, is determined by the cells used to produce kidney organoids. In one example, the present disclosure covers mammal kidney organoids. In this example, mammal pluripotent stem cells are used to produce kidney organoids. Mammalian kidney organoids can represent kidneys from companion animals (such as canines or felines) or livestock animals (such as horses or cattle). Therefore, in these examples, stem cells from canines, felines, etc. are used to produce kidney organoids. In another example, mammal kidney organoids represent kidneys from mice or rats. In another example, kidney organoids represent kidneys from higher primates such as cynomolgus monkeys or rhesus monkeys. In another example, mammal kidney organoids represent kidneys from humans. When pluripotent stem cells from a specific species are used to generate kidney organoids, the resulting kidney organoids can be identified according to the species. For example, when human stem cells are used to generate kidney organoids, the resulting kidney organoids can be identified as human kidney organoids. Therefore, in one example, the kidney organoids covered by the present disclosure include human kidney organoids derived from human stem cells. Various other examples of stem cells suitable for generating kidney organoids are discussed below.

[0063] In another example, kidney organoids can be characterized based on the expression of molecular markers. Marker expression can be characterized using a variety of techniques, such as immunohistochemistry or fluorescence activated cell sorting. Immunohistochemistry generally involves the use of a primary antibody that is specific to the target marker. The binding of a primary antibody to a marker can be visualized by various known methods. For example, a labeled secondary antibody that recognizes a primary antibody can be used. In this example, the label can be an enzyme, such as horseradish peroxidase, a radioisotope, a fluorescent reporter gene, an electrochemiluminescent label. The binding of the labeled secondary antibody to the primary antibody can be assessed by cytology or detected by an automatic plate reader.

[0064] In a specific example, kidney organoid or its slice or sample is contacted with a specific primary antibody.Then the kidney organoid or its slice or sample is washed to remove any unbound primary antibody, and then the secondary antibody that is specific to the primary antibody and connected with peroxidase is applied to the sample.Then the kidney organoid or its slice or sample is washed to remove any unbound secondary antibody, and 3,3'-diaminobenzidine (DAB) is applied to the sample.DAB can be converted into a colored product visualization by conventional cytological assessment, and the presence of the colored product indicates that the marker is present in the sample.In an example, the level of the colored product can be quantified using Image J or various other software packages that can be purchased from suppliers such as Perkin Elmer and Leica.

[0065] In another example, a cell suspension is generated from a representative kidney organoid, a population thereof, or a slice or sample thereof. The cells in the suspension are contacted with a fluorescently labeled antibody specific for a particular marker. Cells that are positive for a particular marker are identified using techniques such as fluorescence activated cell sorting (FACS).

[0066] Cells that are referred to as "positive" for a given marker may express low (lo or dim) or high (bright, bri) levels of the marker, depending on the extent of presence of the marker on the cell surface, where the term relates to fluorescence intensity or other markers used in the cell sorting process. The distinction between lo (or dim or fuzzy) and bri will be understood in the context of the markers used on the particular cell population being sorted. Cells that are referred to as "negative" for a given marker are not necessarily completely absent from the cell. The term means that the marker is expressed at a relatively low or very low level by the cell or population, and when the marker is detectable it will produce a very low signal or cannot be detected above background levels (e.g., levels detected using an isotype control antibody).

[0067] In one example, fluorescent reporter genes can be used to detect markers of kidney organoids described herein. For example, the expression of specific markers can be monitored to track the development of kidney organoids or cells contained therein in real time. For example, stem cells can be genetically engineered to express one or more fluorescent or chemiluminescent reporter genes under a given set of conditions. Reporter genes can be used to track cell identity, cell viability, or cell function in real time.

[0068] An example of a suitable reporter gene is illustrated below, in which a knock-in iPSC line was generated that had the mTagBFP2 fluorescent reporter gene (MAFB mTagBFP2 / + ). MAFB is highly expressed in developing podocytes, therefore, the expression of MAFB can be monitored to track the development of podocytes in kidney organoids in real time. Other examples of reporter cell lines suitable for the methods disclosed herein include GATA3mCherry, RETtdTOMATO or Six2Cre.

[0069] In another example, the kidney organoid comprises cells expressing high levels of one or more nephron markers. In another example, the kidney organoid comprises cells expressing high levels of one or more of PAX2, SIX1, LHX1, OSR1, WNT11, GATA3, PAX8, EYA1, and CITED1. For example, the kidney organoid may express high levels of PAX2. In another example, the kidney organoid may express high levels of SIX1. In another example, the kidney organoid may express high levels of LHX1. In another example, the kidney organoid may express high levels of OSR1. In another example, the kidney organoid may express high levels of WNT11. In another example, the kidney organoid may express high levels of GATA3. In another example, the kidney organoid comprises cells expressing high levels of PAX2, SIX1, LHX1, OSR1, WNT11, and GATA3. In another example, the kidney organoid comprises cells expressing high levels of PAX2, SIX1, LHX1, OSR1, WNT11, GATA3, PAX8, EYA1, and CITED1. In these examples, the kidney organoid can express high levels of a reference marker, such as one or more of PAX2, SIX1, LHX1, OSR1, WNT11, GATA3, PAX8, EYA1, and CITED1, relative to a kidney organoid having at least 100 nephrons. In another example, the kidney organoid can express high levels of a reference marker, such as one or more of PAX2, SIX1, LHX1, OSR1, WNT11, GATA3, PAX8, EYA1, and CITED1, relative to a kidney organoid having more than 50 nephrons. In another example, relative to a kidney organoid comprising at least 1×10 5 The kidney organoids can express high levels of a reference marker, such as one or more of PAX2, SIX1, LHX1, OSR1, WNT11, and GATA3. In another example, relative to a kidney organoid comprising at least 1×10 6 In another example, the kidney organoids can express high levels of reference markers, such as one or more of PAX2, SIX1, LHX1, OSR1, WNT11, and GATA3, relative to kidney organoids without vortexing, such as those described by Takasato et al. (2015).

[0070] In another example, the kidney organoid comprises cells expressing low levels of WT1. In another example, the kidney organoid comprises cells expressing low levels of C-RET. In another example, the kidney organoid comprises cells expressing low levels of FOXD1. In another example, the kidney organoid comprises cells expressing low levels of PDGFRA. In another example, the kidney organoid comprises cells expressing low levels of MEIS2. In another example, the kidney organoid comprises cells expressing low levels of WT1 and C-RET. In another example, the kidney organoid comprises cells expressing low levels of WT1, C-RET, and FOXD1. In these examples, the kidney organoid may express low levels of reference markers, such as one or more of WT1, C-RET, and FOXD1, relative to kidney organoids having at least 100 nephrons. In another example, the kidney organoid may express low levels of reference markers, such as one or more of WT1, C-RET, and FOXD1, relative to kidney organoids having more than 50 nephrons.

[0071] In another example, the kidney organoids comprise cells expressing high levels of PAX2, SIX1, LHX1, OSR1, WNT11, and GATA3 and low levels of WT1, C-RET, PDGFRA, MEIS2, and FOXD1. In another example, the kidney organoids comprise cells expressing high levels of PAX2, SIX1, LHX1, OSR1, WNT11, GATA3, PAX8, EYA1, and CITED1 and low levels of WT1, C-RET, PDGFRA, MEIS2, and FOXD1.

[0072] In the above examples, high and low expression levels are relative to kidney organoids cultured without vortexing, 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-fold lower. In another example, low expression is at least 1.5-fold lower. In another example, low expression is at least 2-fold lower.

[0073] Expression levels can be measured using techniques such as polymerase chain reaction including appropriate primers for the marker of interest. For example, total RNA can be extracted from organoids prior to reverse transcription and PCR and analysis.

[0074] In one example, the kidney organoid comprises a nephron comprising one or more of WT1+ glomeruli, NPHS+ podocytes, LTL+ECAD-proximal tubules, ECAD+ distal tubules, and ECAD+GATA3+ collecting ducts. In another example, the kidney organoid comprises a nephron comprising NPHS+ podocytes, LTL+ proximal segments, ECAD+ distal segments, and ECAD+GATA3+ collecting ducts. The kidney organoids comprising the above exemplary components can be identified in various ways. In one example, the kidney organoids can be fixed and mounted as a whole prior to visual evaluation using confocal microscopy and immunofluorescence labeling.

[0075] In one example, after being used for the screening method discussed below, kidney organoids can be characterized based on one or more of the above-mentioned reference markers. In another example, before selecting kidney organoids expressing suitable markers for the screening method discussed below, kidney organoids representing a wider population can be characterized based on one or more of the above-mentioned reference markers. For example, a population of kidney organoids can be generated using the methods disclosed herein. Before selecting kidney organoids for the screening method discussed below, the expression of one or more of the above-mentioned markers can be confirmed in kidney organoids from a population.

[0076] In another example, an organoid according to the present disclosure comprises 0.5×10 4 Up to 8×10 4 In another example, a kidney organoid comprises 0.8×10 4 Up to 7×10 4 In another example, an organoid according to the present disclosure comprises 1×10 4 Up to 5×10 4 In another example, an organoid according to the present disclosure comprises at least 1×10 4 In another example, an organoid according to the present disclosure comprises less than 3×10 4 In another example, an organoid according to the present disclosure comprises 2×10 4 Up to 2.5×10 4 cells.

[0077] In another example, the diameter of the organoid according to the present disclosure is less than 2,500 μm. In another example, the diameter of the organoid according to the present disclosure is less than 2,000 μm. In another example, the diameter of the organoid according to the present disclosure is less than 1,000 μm. In another example, the diameter of the organoid according to the present disclosure is less than 500 μm. In another example, the diameter of the organoid according to the present disclosure is less than 400 μm. In another example, the diameter of the organoid according to the present disclosure is less than 300 μm. In another example, the diameter of the organoid according to the present disclosure is 150 to 600 μm. In another example, the diameter of the organoid according to the present disclosure is 200 to 500 μm. For example, the diameter of the organoid according to the present disclosure may be 200 μm to 2,000 μm. In another example, the diameter of the organoid according to the present disclosure may be 200 μm to 1,000 μm. In another example, the diameter of the organoid according to the present disclosure may be between 200 μm and 400 μm. In another example, the diameter of the organoid according to the present disclosure may be between 200 μm and 300 μm. In another example, the diameter of the organoid according to the present disclosure may be between 250 μm and 300 μm.

[0078] One skilled in the art will appreciate that organoid size can be measured, for example, by light microscopy and accompanying software such as Image J. For example, one skilled in the art can identify organoids of the exemplary sizes described above by measuring the width of the widest point of its three-dimensional structure.

[0079] In another example, the organoid according to the present disclosure remains alive in culture for at least three weeks. In another example, the organoid according to the present disclosure remains alive in culture for at least four weeks. In another example, the organoid according to the present disclosure remains alive in culture for at least six weeks. In another example, the organoid according to the present disclosure remains alive in culture for at least three to four weeks. For example, the kidney organoid disclosed herein may only have diffusion restriction (i.e., the restriction that affects the transfer of nutrients to cells containing organoid structures) after three to six weeks of culture. In these examples, the reference time period is measured from D7+1. Therefore, in other words, the organoid according to the present disclosure remains alive in culture until at least D7+21, D7+28 or D7+42. In one example, the cell proliferation rate can be used as a measure of diffusion restriction. This is because cells generally do not continue to divide in the absence of sufficient nutrients. Therefore, cell proliferation in the organoid disclosed herein can be tracked over time to determine when diffusion restriction occurs. In one example, a reduction in cell proliferation rate over a period of 3 to 6 days indicates diffusion restriction. In another example, a plateau in cell proliferation over a period of 3 to 6 days indicates proliferation restriction.

[0080] The present disclosure encompasses kidney organoids that contain a small number of nephrons compared to human kidneys. One skilled in the art will appreciate that kidney organoids are artificial products, and while they share many physiological and biochemical characteristics of mammalian kidneys, they do not exist in nature. For example, the kidney organoids disclosed herein may not be connected to a complete vasculature and / or one or more of the following features:

[0081] - Have fewer than 50 nephrons;

[0082] - Includes approximately 0.5×10 4 Up to 8×10 4 cells;

[0083] - have a diameter of less than 100 μm (preferably about 250 to 350 μm);

[0084] - is an independent three-dimensional structure that is not part of a tissue or organ; and,

[0085] - Produced by a method involving in vitro differentiation of stem cells.

[0086] Cellular compositions and therapeutic methods

[0087] The organoids or cells of the present disclosure can be used to produce therapeutic compositions. In one example, the entire organoid disclosed herein can be provided as a therapeutic composition. In another example, the present disclosure encompasses a cell composition produced by the kidney organoid disclosed herein. For example, the cell composition is prepared by enzymatic digestion of the organoid according to the present disclosure. For example, the cell composition can be prepared by digesting the organoid defined herein using a protease such as trypsin, either alone or in combination with ethylenediaminetetraacetic acid (EDTA). In another example, the cell composition can be prepared by digesting the organoid defined herein with a collagenase such as collagenase I and / or collagenase II (e.g., commercially available liberase TM (Roche)). In one example, the enzymatic digest is partially purified or purified to deplete one or more cell types. For example, vascular and / or endothelial cells can be depleted. In another example, the enzymatic digest is partially purified or purified to enrich for one or more cell types. For example, the enzymatic digest can be partially purified or purified to provide an enriched composition of nephron progenitor cells and / or ureteric epithelial progenitor cells.

[0088] In another example, the compositions encompassed by the present disclosure include cells cultured using the methods disclosed herein. For example, the compositions may include modified IM cells expressing high levels of PAX2, LHX1, and OSR1 (cap mesenchyme) and Wnt11 and GATA3 (ureteric epithelium). As described in the above examples, IM cells may be cultured using the methods defined herein and partially purified or purified to enrich for one or more cell types, such as nephron progenitor cells and / or ureteric epithelial progenitor cells.

[0089] Thus, in another example, the present disclosure encompasses a cell composition comprising a population of nephron progenitor cells and / or ureteric epithelial progenitor cells purified from an organoid or cell population produced by a method defined herein.

[0090] In one example, the therapeutic composition disclosed herein comprises a pharmaceutically acceptable carrier and / or excipient. The terms "carrier" and "excipient" refer to a composition of matter conventionally used in the art to promote storage, administration and / or biological activity of the active compound (see, e.g., Remington's Pharmaceutical Sciences, 16th edition, MacPublishing Company (1980)). The carrier can also reduce any undesirable side effects of the active compound. Suitable carriers are, for example, stable, such as being unable to react with other components in the composition. In one example, at the dose and concentration used for treatment, the carrier does not produce significant local or systemic adverse reactions in the recipient.

[0091] Suitable carriers for the present disclosure include those conventionally used, for example, saline, aqueous dextrose, lactose, Ringer's solution, buffered solutions, hyaluronan and glycols are exemplary liquid carriers, particularly (when isotonic) for solutions. Suitable pharmaceutical carriers and excipients include starch, cellulose, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glyceryl monostearate, sodium chloride, glycerol, propylene glycol, water, ethanol, etc.

[0092] In another example, the carrier is a culture medium composition, such as a cell or whole organoid, in which it is grown or suspended. For example, such a culture medium composition does not cause any adverse reactions in a subject to which it is administered. In one example, the cell culture medium may include a basal medium disclosed herein. In one example, the basal medium may include PVA and MC. For example, the basal medium may include 0.05 to 0.5% PVA and 0.05 to 0.5% MC.

[0093] In one example, the carrier or excipient provides buffering activity to maintain the cells at a suitable pH to exert biological activity, for example, the carrier or excipient is phosphate buffered saline (PBS). PBS represents an attractive carrier or excipient because it has minimal interaction with cells and factors and allows rapid release of cells and factors, in which case the compositions of the present disclosure can be produced in liquid for direct administration to the bloodstream or into the kidney or areas surrounding or adjacent to the kidney, such as by injection. Therefore, in one example, the cell compositions or whole organoids disclosed herein are provided in phosphate buffered saline (PBS).

[0094] In another example, the cell composition or the whole organoid can be incorporated into or embedded in a scaffold that is compatible with the recipient and degrades into a product that is harmless to the recipient. These scaffolds provide support and protection for the cells to be transplanted into the recipient subject. Natural and / or synthetic biodegradable scaffolds are examples of such scaffolds. Various scaffolds can be successfully used in the practice of the present disclosure. Exemplary scaffolds include, but are not limited to, biodegradable scaffolds. Natural biodegradable scaffolds include collagen, fibronectin and laminin scaffolds. Suitable synthetic materials for cell transplant scaffolds should be able to support a wide range of cell growth and cell function. Such scaffolds can also be resorbable. Suitable scaffolds include polyglycolic acid scaffolds, for example, as described 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 acid. In another example, cells can be administered in a gel scaffold (e.g., Gelfoam from 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) component.

[0095] In one example, the composition comprises an effective amount or a therapeutically effective amount of cells or whole organoids. In another example, the cells or whole organoids are contained in a chamber that does not allow the cells or organoids to enter the subject's circulatory system, but allows factors secreted by the cells or organoids to enter the circulatory system. In this way, soluble factors can be administered to a subject by allowing cells or organoids to secrete factors into the subject's circulatory system. Such chambers can also be implanted in the subject's site to increase the local level of soluble factors, such as implanted in or near the kidney.

[0096] In one example, the compositions disclosed herein can be systemically administered, for example, by intravenous, intra-arterial or intraperitoneal administration. In one example, the compositions disclosed herein are administered intravenously. In another example, the compositions are administered intra-arterially. In another example, the compositions are administered by renal artery injection, renal parenchymal injection or subcapsular transplantation into a normal or diseased kidney. In another example, the compositions are implanted. For example, the entire organoid can be implanted near the kidney of a subject.

[0097] In one example, the cell composition according to the present disclosure can be cryopreserved. Cryopreservation of cells or whole organoids can be performed using slow cooling methods or "fast" freezing protocols known in the art. Preferably, the method of cryopreservation maintains similar phenotypes, cell surface markers, and growth rates of cryopreserved cells or whole organoids compared to unfrozen cells or whole organoids. The cryopreserved composition can include a cryopreservation solution. The pH of the cryopreservation solution is generally 6.5 to 8, preferably 7.4.

[0098] Examples of cryopreservation solutions include non-pyrogenic isotonic solutions, e.g., PlasmaLyte 100mLPlasmaLyte Contains 526 mg sodium chloride, USP (NaCl); 502 mg sodium gluconate (C6H 11 NaO7); 368 mg sodium acetate trihydrate, USP (C2H3NaO2·3H2O); 37 mg potassium chloride, USP (KC1); and 30 mg magnesium chloride, USP (MgCl2·6H2O). It does not contain an antimicrobial agent.

[0099] In one example, the present disclosure encompasses a method of cell therapy, which includes administering a composition disclosed herein to a subject in need thereof. For example, the present disclosure encompasses a method of treating nephropathy by administering a composition disclosed herein to a subject in need thereof. The term "nephropathy" is used in the context of the present disclosure to refer to an obstacle associated with any stage or degree of acute or chronic renal failure, which results in a loss of the ability of the kidney to perform blood filtration and remove excess fluid, electrolytes and waste from the blood. Examples of nephropathy include endocrine dysfunction, such as anemia (erythropoietin deficiency) and mineral imbalance (vitamin D deficiency). Nephropathy may also originate from the kidney or may be secondary to a variety of conditions, including (but not limited to) heart failure, hypertension, diabetes, autoimmune disease or liver disease or drug-induced toxicity. In one example, nephropathy may be a condition of chronic renal failure that develops after acute injury to the kidney. For example, damage to the kidney caused by ischemia and / or exposure to poisons may lead to acute renal failure; incomplete recovery after acute renal injury may lead to the development of chronic renal failure. Other examples of renal diseases include congenital nephrotic syndrome (CNS), including steroid-resistant nephrotic syndrome and Finnish nephropathy, focal segmental glomerulonephritis (FSGS), Alport syndrome, and PierSon syndrome.

[0100] In one example, the present disclosure encompasses a method of treating kidney disease by implanting a whole organoid disclosed herein into a subject in need thereof. In another example, the present disclosure encompasses a method of treating kidney disease by administering a cell composition disclosed herein to a subject in need thereof.

[0101] In other examples, the compositions or cells disclosed herein can be provided for recellularization of decellularized renal scaffolds. In another example, the present disclosure encompasses a biomaterial or scaffold comprising the compositions or cells disclosed herein.

[0102] Stem Cells

[0103] Aspects of the present disclosure encompass the cultivation of stem cells. The term "stem cell" is used in the context of the present disclosure to refer to a subset of progenitor cells that have the ability or potential to differentiate into a more specialized or differentiated phenotype under a specific environment, and retain the ability to proliferate without substantial differentiation under certain circumstances. In an example, the term stem cell generally refers to a naturally occurring mother cell, whose progeny (offspring) are generally specialized in different directions by differentiation (e.g., by acquiring complete individual characteristics), as occurs in the gradual diversification of embryonic cells and tissues. Cell differentiation is a complex process that generally occurs through many cell divisions. Differentiated cells can be derived from pluripotent cells, which themselves are derived from pluripotent cells, etc. Although each of these pluripotent cells can be considered a stem cell, the range of cell types may each cause considerable changes. Some differentiated cells also have the ability to produce cells with greater developmental potential. Such an ability can be natural or artificially induced after being treated with various factors. In many biological examples, stem cells are also "pluripotent" because they can produce offspring of more than one different cell type, but this is not required for "stemness". Self-renewal is another classic part of the definition of stem cells. In theory, self-renewal can occur by either of two main mechanisms. Stem cells may divide asymmetrically, with one progeny retaining a stem state and the other progeny expressing some different other specific functions and phenotypes. Alternatively, some stem cells in a population may be symmetrically divided into two stems, thereby maintaining some stem cells in the population as a whole, while other cells in the population only produce differentiated offspring.

[0104] In one example, the stem cells are human stem cells. In one example, the stem cells are a population of culture-expanded human stem cells. In one example, the stem cells can be cultured and expanded in vitro or in vitro. In one example, the culture-expanded stem cells have been passaged at least once, twice, three times, four times, five times, six times, seven times, eight times, nine times, or ten times.

[0105] In one example, the stem cell is a pluripotent stem cell. In another example, the stem cell is a human embryonic stem cell. Typically, pluripotent stem cells show expression of OCT4, NANOG, and SSEA1 when in a pluripotent state, and the expression of these markers is typically lost with differentiation. In another example, the stem cell is a human embryonic stem cell ("hES" or "hESC").

[0106] In another example, stem cells are induced pluripotent stem cells. For example, stem cells can be induced pluripotent stem cells of people. The term "induced pluripotent stem cells" and its abbreviation "iPSC" refers to cells derived from, obtainable from or derived from any type of human adult somatic cells reprogrammed to a pluripotent state by expressing exogenous genes (e.g., transcription factors, including a preferred combination of OCT4, SOX2, KLF4 and c-MYC). The pluripotency level of human iPSC is comparable to that of hESC, but can be derived from patients for autologous therapy that is used for or without gene correction before differentiation and cell delivery. Suitable methods for producing induced pluripotent stem cells are described in, for example, US 7,615,374 and US2014273211, 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, iPSC is derived from fibroblasts. In another example, iPSCs are derived from blood. For example, iPSCs can be derived from leukocytes. In another example, iPSCs are derived from fibroblasts. In another example, iPSCs are derived from leukocytes or fibroblasts.

[0107] In one example, the stem cell is H9 or hES3. Therefore, in one example, the present disclosure encompasses kidney organoids disclosed herein, wherein the kidney organoids are derived from H9 stem cells. In another example, the present disclosure encompasses kidney organoids disclosed herein, wherein the kidney organoids are derived from hES3 stem cells (Kao et al., 2016; Ng et al., 2016; vanden Berg et al., 2018). For example, kidney organoids can be derived from hES3-SOX17mCherry or H9GAPTrapLuc2. Therefore, in one example, the present disclosure encompasses kidney organoids comprising less than 50 nephrons, wherein the kidney organoids are derived from H9 or hES3 stem cells. In another example, the kidney organoids comprise less than 15 nephrons and are derived from H9 or hES3 stem cells. In these examples, stem cells can express reporter genes.

[0108] In another example, the stem cell is iPSC GAPTrap td-Tomato, CRL1502.C32 or CLR1502.3 (Briggs et al., 2013; Takasato et al., 2015). Thus, in one example, the present disclosure encompasses kidney organoids as defined herein, wherein the kidney organoids are derived from iPSC GAPTrap td-Tomato.

[0109] Thus, in one example, the disclosure encompasses kidney organoids comprising less than 50 nephrons, wherein the kidney organoids are derived from iPSC GAPTrap td-Tomato, CRL1502.C32, or CLR1502.3. In another example, the kidney organoids comprise less than 15 nephrons and are derived from iPSC GAPTrap td-Tomato, CRL1502.C32, or CLR1502.3. Again, in these examples, the stem cells can express a reporter gene.

[0110] In one example, it may be desirable to produce kidney organoids representing a particular subject and / or disease. Various examples of this embodiment are described below. Related to this section are iPS cells that can be used to produce kidney organoids. In one example, human iPS cells are derived from human subjects suffering from hereditary nephropathy. In this example, blood samples can be separated from subjects suffering from hereditary nephropathy, and iPS cells can be induced from cells (e.g., leukocytes) in the blood sample. Subjects can suffer from one of a variety of exemplary hereditary nephropathy. Examples include congenital nephrotic syndrome (CNS), including steroid-resistant nephrotic syndrome and Finnish nephropathy, focal segmental glomerulonephritis (FSGS), Alport syndrome, and PierSon syndrome. Therefore, in one example, the present disclosure encompasses kidney organoids representing nephropathy 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 PierSon syndrome. Therefore, in one example, kidney organoids represent CNS. In another example, kidney organoids represent steroid-resistant nephrotic syndrome.

[0111] In one example, kidney organoids can be used to model developing kidneys and / or nephropathy. Therefore, in one example, the disclosure encompasses kidney organoids disclosed herein, wherein the kidney organoids are used to model kidney development. In another example, the disclosure encompasses kidney organoids disclosed herein, wherein the kidney organoids are used to model nephropathy. In one example, nephropathy is CNS or another of the above-mentioned diseases. In this example, iPS cells can be induced from subjects suffering from the above-mentioned nephropathy and kidney organoids can be produced therefrom to model the disease. In this example, gene editing (e.g., CRISPR / Cas9 gene editing) can be used to introduce mutations into genes of iPS cells of subject-derived sources that are associated with or potentially associated with nephropathy development. In other examples, gene editing is used to correct mutations in iPS cells of subject-derived sources. In one example, iPS cells (e.g., Forbes et al. (2018) Am J Hum Genet.102:816-831) of isogenic editing can be produced. Kidney development and disease can use different developmental stages such as those discussed below in one or more (such as d7+15) kidney organoids over time (such as 2,5,10 or more days) modeling.In these examples, organoid glomeruli can be grouped and cultured, and each group represents different developmental stages (such as d7+11, d7+15, d7+18, d7+20) and / or cultivates the time period limited (for example, 2,5 and 10 days in vortex culture).Can use such as visual assessment, immunohistochemistry, gene and protein expression analysis to assess kidney organoids to determine development or disease stage.In one example, during these studies, kidney organoids can also be made to contact with nephrotoxins, candidate compounds and / or therapeutic compounds, and nephrotoxicity and / or therapeutic efficacy can be measured.As mentioned above, the kidney organoids used in the above examples can be produced by iPS cells through genetic modification to express reporter genes.

[0112] Cell culture methods

[0113] The term "media" or "medium" for cell culture includes the composition of the environment surrounding the cells. It is envisioned that the culture medium facilitates and / or provides conditions sufficient for cell differentiation and organoid formation. The culture medium can be solid, liquid, gaseous, or a mixture of phases and materials. The culture medium can include liquid growth medium as well as liquid culture medium that does not sustain cell growth. Culture media also include gel-like culture media, such as agar, agarose, gelatin, and collagen matrices. Exemplary gaseous culture media include gas phases to which cells grown on culture dishes or other solid or semisolid supports are exposed. The term "culture medium" also refers to a substance used for cell culture, even if it has not yet come into contact with cells.

[0114] The culture medium used in the method of the present disclosure can be prepared by using a culture medium for culturing stem cells or IM cells as a basal culture medium. The basal culture medium includes, for example, Eagles minimum essential (MEM) medium, and is not particularly limited as long as it can be used to culture stem cells or IM cells. In addition, the culture medium of the present disclosure may contain any components, such as fatty acids or lipids, vitamins, growth factors, cytokines, antioxidants, buffers, inorganic salts, etc. The cell culture medium used in the present disclosure contains all essential amino acids and may also contain non-essential amino acids. Generally, amino acids are divided into essential amino acids (Thr, Met, Val, Leu, He, Phe, Trp, Lys, His) and non-essential amino acids (Gly, Ala, Ser, Cys, Gin, Asn, Asp, Tyr, Arg, Pro). In other examples, the basal culture medium includes, for example, APEL, mTESR-E6 or E8 chemically defined culture medium (StemCell Technologies). The basal culture medium may also be supplemented with protein-free hybridoma culture medium (PFHM) (e.g., 3.5%). In one example, the basal culture medium is supplemented with a serum substitute. For example, basal medium can be supplemented with knockout serum replacement (Thermo Fisher).

[0115] As will be appreciated by those skilled in the art, the culture medium disclosed herein will need to be replaced over time. It is considered within the skill of the skilled person to determine the appropriate time to replace the culture medium. For example, various commercially available colorimetric indicators are commonly used in cell culture media to indicate when the culture medium needs to be replaced. As a guide, for cell cultures in porous culture dishes, the culture medium can be replaced every 24 or 48 hours. For example, the culture medium can be replaced every two days.

[0116] Kidney organoids

[0117] The present disclosure encompasses a method for producing kidney organoids. In one example, kidney organoids are produced by vortexing a culture medium containing an intermediate mesoderm (IM) cell population. In one example, IM cells are vortexed in suspension culture. For the avoidance of doubt, "suspension culture" is used in the context of the present disclosure to refer to a cell culture in which a single cell or small cell aggregate proliferates while suspended in a stirred liquid culture medium. For example, a single cell or small cell aggregate proliferates in suspension culture and forms a kidney organoid.

[0118] In one example, the IM cell culture medium comprises FGF. In some examples, the FGF can 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 can include FGF from D7 to at least D7+10. In one example, the IM cell culture medium can include FGF from D7 to at least D7+15. Exemplary concentrations of FGF are provided below. For example, the cell culture medium comprises at least 50 ng / ml of FGF. In another example, the cell culture medium comprises at least 100 ng / ml of FGF. In another example, the cell culture medium comprises at least 150 ng / ml of FGF. In another example, the cell culture medium comprises at least 200 ng / ml of FGF. In another example, the cell culture medium comprises at least 300 ng / ml of FGF. In another example, the cell culture medium comprises at least 350 ng / ml of FGF. In another example, the cell culture medium comprises at least 400 ng / ml of FGF. In another example, the cell culture medium comprises at least 500 ng / ml of FGF. In another example, the cell culture medium comprises 50 ng / ml to 400 ng / ml of FGF. In another example, the cell culture medium comprises 50 ng / ml to 300 ng / ml of FGF. In another example, the cell culture medium comprises 50 ng / ml to 250 ng / ml of FGF. In another example, the cell culture medium comprises 100 ng / ml to 200 ng / ml of FGF. In another example, the cell culture medium comprises 180 ng / ml to 220 ng / ml of FGF. In another example, the cell culture medium comprises 190 ng / ml to 210 ng / ml of FGF.

[0119] In one example, the IM cell culture medium may include FGF9. In one example, the cell culture medium includes at least 50 ng / ml of FGF9. In another example, the cell culture medium includes at least 100 ng / ml of FGF9. In another example, the cell culture medium includes at least 150 ng / ml of FGF9. In another example, the cell culture medium includes at least 200 ng / ml of FGF9. In another example, the cell culture medium includes at least 300 ng / ml of FGF9. In another example, the cell culture medium includes at least 350 ng / ml of FGF9. In another example, the cell culture medium includes at least 400 ng / ml of FGF9. In another example, the cell culture medium includes at least 500 ng / ml of FGF9. In another example, the cell culture medium includes 50 ng / ml to 400 ng / ml of FGF9. In another example, the cell culture medium includes 50 ng / ml to 300 ng / ml of FGF9. In another example, the cell culture medium includes 50 ng / ml to 250 ng / ml of FGF9. In another example, the cell culture medium comprises 100 ng / ml to 200 ng / ml of FGF9. In another example, the cell culture medium comprises 180 ng / ml to 220 ng / ml of FGF9. In another example, the cell culture medium comprises 190 ng / ml to 210 ng / ml of FGF9.

[0120] In another example, the above reference level of FGF9 is 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.

[0121] In another example, the above reference level of FGF9 is 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 FGF 16. In another example, the cell culture medium contains 190 ng / ml to 210 ng / ml of FGF16.

[0122] In another example, the above reference level of FGF9 is replaced with FGF20. For example, the IM cell culture medium can include 50ng / ml to 400ng / ml of FGF20. In another example, the cell culture medium includes 50ng / ml to 300ng / ml of FGF20. In another example, the cell culture medium includes 50ng / ml to 250ng / ml of FGF20. In another example, the cell culture medium includes 100ng / ml to 200ng / ml of FGF20. In another example, the cell culture medium includes 180ng / ml to 220ng / ml of FGF20. In another example, the cell culture medium includes 190ng / ml to 210ng / ml of FGF20. In one example, FGF is removed from the culture medium after 5 days in vortex culture. In one example, FGF is removed from the culture medium after 6 days in vortex culture. In one example, FGF is removed from the culture medium after 4 to 6 days of vortex culture.

[0123] In one example, nephrogenesis is initiated in organoids by supplementing the culture medium with a Wnt / β-catenin agonist. The term "Wnt / β-catenin agonist" is used in the context of the present disclosure to refer to molecules that inhibit GSK3 (e.g., GSK3-P) in the case of a typical Wnt signaling pathway, but preferably not in the case of other atypical Wnt signaling pathways. In some instances, the Wnt / β-catenin agonist is a GSK3β inhibitor. Examples of Wntβ-catenin agonists include CHIR99021 (CHIR), LiCl, SB-216763, CAS 853220-52-7, and other Wnt / β-catenin agonists commercially available from sources such as Santa Cruz Biotechnology and R&D Systems.

[0124] Thus, in one example, the IM cell culture medium may contain the above-referenced 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 Wnt / β-catenin agonist. In another example, the cell culture medium may contain 1.3 μM or less of a Wnt / β-catenin agonist. In another example, the cell culture medium may contain 1.4 μM or less of a Wnt / β-catenin agonist. In another example, the cell culture medium may contain 1.5 μM or less of a Wnt / β-catenin agonist. It should be understood that the culture medium may contain any combination of these upper and lower limits of 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 a Wnt / β-catenin agonist. In another example, the cell culture medium may contain 0.8 μM to 1.2 μM of a Wnt / β-catenin agonist. In one example, the cell culture medium may contain at least 0.5 μM 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 should be understood that the culture medium may contain any combination of these upper and lower limits of 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 can contain 0.8 μM to 1.2 μM CHIR.

[0125] In another example, the IM cell culture medium can include a Rho kinase inhibitor (ROCKi), such as Y-27632 (StemCell Technologies). In one example, the cell culture medium can include at least 8 μM ROCKi. In another example, the cell culture medium can include about 10 μM ROCKi. In another example, the cell culture medium can include 12 μM or less ROCKi. In another example, the cell culture medium can include 8 μM to 12 μM ROCKi.

[0126] In the above example, the IM cell culture medium may contain one or more or all of FGF9, Wnt / β-catenin agonists such as CHIR and heparin, poly(vinyl alcohol) (PVA) and methylcellulose (MC). In another example, the cell culture medium may also contain ROCKi.

[0127] In one example, the IM cell culture medium comprises at least 0.5 μg / ml of heparin. In another example, the cell culture medium comprises about 1 μg / ml of heparin. In another example, the cell culture medium comprises 1.5 μg / ml or less of heparin. In another example, the cell culture medium comprises 2 μg / ml or less of heparin. In another example, the cell culture medium comprises 0.2 μg / ml to 2 μg / ml of heparin. In another example, the cell culture medium comprises 0.5 μg / ml to 1.5 μg / ml of heparin. In another example, the cell culture medium comprises 0.8 μg / ml to 1.2 μg / ml of heparin.

[0128] In one example, the IM cell culture medium comprises at least 0.05% PVA. In another example, the cell culture medium comprises about 0.1% PVA. In another example, the cell culture medium comprises 0.15% or less PVA. In another example, the cell culture medium comprises 0.1% to 0.15% PVA.

[0129] 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.

[0130] The terms "swirled," "swirl," and "swirling" are used interchangeably in the context of the present disclosure and refer to the movement of a cell culture medium in a circular, torsional, or spiral pattern. In one example, the cell culture medium is vortexed by sufficiently agitating the cell culture in a circular motion. For example, an orbital shaker can be used to vortex the cell culture. Other examples of suitable equipment for vortexing the cell culture include a shaking platform, a shaking incubator, or a spinner flask. The appropriate rpm for vortexing allows the IM cells to aggregate and form organoids.

[0131] In one example, the IM cell culture is vortexed at least at 30 rpm. In another example, the cell culture is vortexed at least at 40 rpm. In another example, the cell culture is vortexed at least at 50 rpm. In another example, the cell culture is vortexed at least at 60 rpm. In another example, the cell culture is vortexed at least at 70 rpm. In another example, the cell culture is vortexed at least at 80 rpm. In another example, the cell culture is vortexed at 40 to 80 rpm. In another example, the cell culture is vortexed at 50 to 70 rpm. In another example, the cell culture is vortexed at 55 to 65 rpm. In another example, the cell culture is vortexed at 30 to 150 rpm. In another example, the cell culture is vortexed at 30 to 90 rpm.

[0132] In one example, the IM cells are cultured for at least five days. In another example, the IM cells are cultured for at least seven days. In another example, the IM cells are cultured for at least ten 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 to 20 days. In another example, the IM cells are cultured for 5 to 18 days. In another example, the IM cells are cultured for 7 to 14 days. For example, the IM cells may be vortexed for at least ten days. For example, the IM cells may be vortexed for at least 20 days. In another example, the IM cells may be vortexed for at least 30 days. In one example, the IM cells are vortexed for 10 to 30 days. In another example, the IM cells are vortexed for 15 to 30 days.

[0133] In one example, the IM cell colony is dissociated and vortexed in the above-mentioned reference medium. 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 passed through a mesh screen before further culture. In one example, the cells are vortexed for at least 12 days after dissociation. In another example, the cells are vortexed for at least 13 days after dissociation. In another example, the cells are vortexed for at least 14 days after dissociation. In another example, the cells are vortexed for at least 15 days after dissociation. In another example, the cells are vortexed for at least 20 days after dissociation. In another example, the cells are vortexed for at least 25 days after dissociation. In another example, the cells are vortexed for at least 35 days after dissociation. In another example, the cells are vortexed for 5 to 18 days after dissociation.

[0134] In other examples, the IM cells defined herein can be cultured in a variety of culture media comprising different components. For example, cells can be cultured in stages, wherein each stage is associated with a different culture medium. In one example, the IM cells are vortex cultured in two stages. In this example, the stage 1 culture medium comprises the above reference levels of FGF, such as FGF9, CHIR, ROCKi, heparin, PVA and MC, and the stage 2 culture medium comprises the above reference levels of FGF, such as FGF9, CHIR, heparin, PVA and MC. For example, the stage 1 culture medium may comprise 200ng / ml FGF9, 1μM CHIR, 10μM ROCKi, 1μg / ml heparin, 0.1%PVA and 0.1%MC, and the stage 2 culture medium may comprise FGF9, 1μM CHIR, 1μg / ml heparin, 0.1%PVA and 0.1%MC.

[0135] In another example, IM cells are vortex cultured in three stages. In this example, stage 1 and stage 2 culture media are as defined above, and stage 3 culture media includes PVA and MC at the above reference levels. For example, stage 3 culture media can include 0.1% PVA and 0.1% MC.

[0136] In one example, the IM cells are cultured in stage 1 medium for one day and then cultured in stage 2 medium for four days. In one example, the cells are cultured in stage 1 medium for 18 to 24 hours and then cultured in stage 2 medium for four days. In these examples, the cells can be further cultured in stage 3 medium for 7 to 20 days. In another example, the cells can be further cultured in stage 3 medium for at least 15 days. In another example, the cells can be further cultured in stage 3 medium for at least 30 days.

[0137] The inventors also found that adding retinoic acid to IM cells after about 5 to 10 days of vortex culture improves glomerular maturation (improved glomerular podocyte maturation) in organoids. Therefore, in one example, after 5 to 10 days of vortex culture, retinoic acid is added to cell culture medium. In one example, all-trans retinoic acid (atRA) is added to cell culture medium. In one example, at least 0.07 μM retinoic acid is added to cell culture medium. In another example, at least 0.1 μM retinoic acid is added to cell culture medium. In another example, at least 0.2 μM retinoic acid is added to cell culture medium. In another example, at least 0.5 μM retinoic acid is added to cell culture medium.

[0138] In another example, at least 1.5 μM retinoic acid is added to the cell culture medium. In one example, at least 1.8 μM retinoic acid is added to the cell culture medium. In one example, at least 2.0 μM retinoic acid is added to the cell culture medium. In another example, at least 2.5 μM retinoic acid is added to the cell culture medium. In another example, 1.5 μM to 3 μM retinoic acid is added to the cell culture medium. In another example, 2.0 μM to 3 μM retinoic acid is added to the cell culture medium.

[0139] In another example, the stage 1 and stage 2 medium are as defined above, and the stage 3 medium comprises the above referenced levels of PVA, MC, and atRA. For example, the stage 3 medium may comprise 0.1% PVA and 0.1% MC. 2.5 μM atRA.

[0140] The inventors have discovered that vortexing a small number of IM cells in an appropriate culture medium can direct the development of kidney organoids with a simplified three-dimensional structure. Thus, in one example, the methods of the present disclosure encompass vortexing containing less than 5×10 5 In another example, the methods of the present disclosure encompass a vortex containing less than 4×10 5 In another example, the methods of the present disclosure encompass a vortex containing less than 3×10 5 In another example, the methods of the present disclosure encompass a vortex containing less than 1×10 5 In another example, the methods of the present disclosure encompass vortexing a population of IM cells containing 5×10 4 In another example, the methods of the present disclosure encompass a vortex containing 4×10 4 In another example, the methods of the present disclosure encompass vortexing a population of IM cells containing 1×10 4 Up to 1×10 5In another example, the methods of the present disclosure encompass a vortex containing 2×10 4 Up to 1×10 5 In another example, the methods of the present disclosure encompass vortexing a population of IM cells containing 5×10 3 Up to 3×10 5 A population of IM cells.

[0141] In another example, the method of the present disclosure encompasses a vortex of 1×10 5 IM cells / ml to 5×10 6 In another example, the method of the present disclosure encompasses vortexing 5×10 5 Up to 4×10 6 In another example, the method of the present disclosure encompasses vortexing 5×10 5 IM to 3×10 6 In other examples, the methods of the present disclosure encompass vortexing of less than 5×10 6 In another example, the method of the present disclosure encompasses a population of 1M cells / ml. 6 In another example, the method of the present disclosure encompasses a population of 1M cells / ml. 6 In another example, the method of the present disclosure encompasses a population of 1M cells / ml. 6 In another example, the method of the present disclosure encompasses vortexing 1×10 6 In another example, the method of the present disclosure encompasses vortexing 5×10 5 Up to 2×10 6 In another example, the method of the present disclosure encompasses vortexing 5×10 5 to 1.5×10 6 IM cells / ml. In these examples, about 5,000 to 15,000 organoids can be produced. In another example, about 8,000 to 10,000 organoids can be produced. In these examples, during the culture period, the total cell number increased by 30 to 40 times from the number of cells at the beginning of the vortex culture. In these examples, the total cell number increased by 3 to 4 times compared to the total cell number increase using the protocol described in Takasato et al. (2015) Nature, Vol. 526: 564-568. In this example, the total cell number increased from about 1×10 5 Up to 5×10 6 The cells / ml increased to about 3×10 6 Up to 2×10 8 cells / ml.

[0142] In one example, IM cells are obtained by the methods disclosed herein.

[0143] In one example, the present disclosure encompasses a method of generating a kidney organoid comprising:

[0144] - culturing the stem cell population for 7 days to generate IM cells, 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 comprising FGF9 and a low concentration of a Wnt / β-catenin agonist;

[0145] - Dissociation of IM cells;

[0146] - Kidney organoids were generated by vortexing IM cells in cell culture medium containing FGF9 for at least 5 days, wherein the first 24 hours involved culturing the cells in cell culture medium containing FGF9, heparin, a low concentration of a Wnt / β-catenin agonist, and ROCKi and the subsequent 3 or 4 days involved culturing the cells in cell culture medium containing FGF9, heparin, a low concentration of a Wnt / β-catenin agonist PVA, and MC.

[0147] In the above examples, stem cells can be cultured for 7 days, wherein the first 4 days involve culturing stem cells in a high concentration of a Wnt / β-catenin agonist such as CHIR, and the remaining days involve culturing cells in a cell culture comprising 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, IM cells are dissociated using trypsin. In another example, cells are dissociated using EDTA. In one example, IM cells are vortexed in a cell culture medium comprising 200 ng / ml FGF9 for at least 4 days, wherein the first 24 hours involve culturing the cells in a cell culture medium comprising 200 ng / ml FGF9, 1 μl / ml heparin, 1 μM CHIR and 10 μM ROCKi, and the subsequent 3 or 4 days involve culturing the cells in a cell culture medium comprising 200 ng / ml FGF9, 1 μg / ml heparin, 1 μM, 0.1% PVA and 0.1% MC.

[0148] In another example, the method further comprises vortexing the cells in a cell culture medium comprising PVA and MC that does not comprise CHIR or FGF9. In one example, the cell culture medium comprises 0.1% PVA and 0.1% MC. In one example, the cells are vortexed in a cell culture medium comprising PVA and MC that does not comprise CHIR or FGF9 for 5 days or longer.

[0149] Intermediate mesoderm

[0150] The inventors have also surprisingly found that culturing stem cells for a longer period of time in a medium containing low concentrations of CHIR and activated wnt / β-catenin signaling is beneficial for generating improved intermediate mesoderm. The in vitro culture method provides a system for differentiating stem cells through postprimordial line (PPS) cells and intermediate mesoderm (IM) cells to generate kidney organoids.

[0151] Thus, in one example, the disclosure encompasses an in vitro method for generating intermediate mesoderm (IM) cells, the method comprising culturing a population of postprimordial line (PPS) cells for 2 to 5 days in a cell culture medium comprising FGF and a Wnt / β-catenin agonist at less than 4 μM. Suitable concentrations of FGF and Wnt / β-catenin agonists and duration of culture are as described below for methods of generating intermediate mesoderm (IM) cells within a time period after the first 3 or 4 days of culture.

[0152] Therefore, in one example, the disclosure encompasses a method for producing an intermediate mesoderm (IM) cell, the method being included in a cell culture medium comprising CHIR and culturing a stem cell colony for about 7 days, wherein FGF such as FGF9 is added to the culture medium after the first 3 or 4 days of cultivation. In one example, after the first 3 or 4 days of cultivation, 50ng / ml to 400ng / ml of FGF9 is added to the culture medium. In another example, after the first 3 or 4 days of cultivation, 50ng / ml to 300ng / ml of FGF9 is added to the culture medium. In another example, after the first 3 or 4 days of cultivation, 50ng / ml to 250ng / ml of FGF9 is added to the culture medium. In another example, after the first 3 or 4 days of cultivation, 100ng / ml to 200ng / ml of FGF9 is added to the culture medium. In another example, after the first 3 or 4 days of cultivation, 150ng / ml to 250ng / ml of FGF9 is added to the culture medium. In another example, after the first 3 or 4 days of culture, 175ng / ml to 225ng / ml of FGF9 is added to the culture medium. In another example, after the first 3 or 4 days of culture, 190ng / ml to 210ng / ml of FGF9 is added to the culture medium. In another example, after the first 3 or 4 days of culture, 195ng / ml to 205ng / ml of FGF9 is added to the culture medium. In another example, after the first 3 or 4 days of culture, about 200ng / ml of FGF9 is added to the culture medium. In one example, after the first 3 or 4 days of culture, heparin is also added to the culture medium. In one example, after the first 3 or 4 days of culture, 0.5μg / ml to 2μg / ml of heparin is added to the culture medium. In another example, after the first 3 or 4 days of culture, 0.5μg / ml to 1.5μg / ml of heparin is added to the culture medium. In another example, after the first 3 or 4 days of culture, 0.8 μg / ml to 1.2 μg / ml of heparin is added to the culture medium. In another example, after the first 3 or 4 days of culture, 1 μg / ml of heparin is added to the culture medium. In one example, after 4 days of culture, the above reference levels of FGF and heparin are added to the culture medium. In one example, stem cells are cultured in a cell culture medium comprising CHIR for 7 days.

[0153] In particular, the inventors have found that 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 produces improved IM cells. For example, the improved IM cells express high levels of PAX2, LHX1, and OSR1 (cap mesenchyme) and Wnt11 and GATA3 (ureteric epithelium). Thus, in one example, stem cells can be cultured in a medium comprising a high concentration of a Wnt / β-catenin agonist such as CHIR and then cultured in a medium comprising a low concentration of a Wnt / β-catenin agonist and FGF such as FGF9, FGF16, FGF20, or FGF2.

[0154] In one example, the present disclosure encompasses a method for producing intermediate mesoderm (IM) cells, the method comprising: culturing a stem cell population in a cell culture medium comprising a high concentration of CHIR, and then culturing the stem cell population in a cell culture medium comprising a low concentration of CHIR and FGF such as FGF9. In this example, the "high concentration" of CHIR is at least 5 μM, while the "low concentration" of CHIR is less than 3 μM. In another example, the "high concentration" of CHIR is at least 6 μM, while the "low concentration" of CHIR is less than 2 μM. In another example, the "high concentration" of CHIR is 7 μM, while the "low concentration" of CHIR is 1 μM or less.

[0155] Therefore, on the other hand, the methods of the present disclosure encompass in vitro methods for producing intermediate mesoderm (IM) cells. In one example, the method for producing IM cells includes culturing a stem cell population for about 7 days, wherein the first 4 to 5 days involve culturing stem cells in a high concentration of a Wnt / β-catenin agonist such as CHIR, and the remaining days involve culturing cells in a cell culture medium comprising FGF9 and a low concentration of a Wnt / β-catenin agonist. On the one hand, the method for producing IM cells includes culturing a stem cell population in a cell culture medium comprising FGF9 and at least 0.5 μM Wnt / β-catenin agonist after culturing in a high concentration of a Wnt / β-catenin agonist for the first 4 to 5 days. In another example, after culturing in a high concentration of a Wnt / β-catenin agonist for the first 4 to 5 days, the cell culture medium may contain at least 0.6 μM of a Wnt / β-catenin agonist. In another example, the cell culture medium may contain at least 0.7 μM of the Wnt / β-catenin agonist after the first 4 to 5 days of culture at a high concentration of the Wnt / β-catenin agonist. In another example, the cell culture medium may contain at least 0.8 μM of the Wnt / β-catenin agonist after the first 4 to 5 days of culture at a high concentration of the Wnt / β-catenin agonist. In another example, the cell culture medium may contain at least 0.9 μM of the Wnt / β-catenin agonist after the first 4 to 5 days of culture at a high concentration of the Wnt / β-catenin agonist. In another example, the cell culture medium may contain about 1 μM of the Wnt / β-catenin agonist after the first 4 to 5 days of culture at a high concentration of the Wnt / β-catenin agonist. In another example, after the first 4 to 5 days of culturing in a high concentration of a Wnt / β-catenin agonist, the cell culture medium may contain 1.1 μM or less of a Wnt / β-catenin agonist. In another example, after the first 4 to 5 days of culturing 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 to 5 days of culturing 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 to 5 days of culturing 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 to 5 days of culture at a high concentration of the Wnt / β-catenin agonist, the cell culture medium may contain 1.5 μM or less of the Wnt / β-catenin agonist. It should be understood that after the first 4 to 5 days of culture at a high concentration of the Wnt / β-catenin agonist, the culture medium may contain any combination of these upper and lower limits of the Wnt / β-catenin agonist concentration.In another example, after the first 4 to 5 days of culturing in a high concentration of a Wnt / β-catenin agonist, the cell culture medium may contain 0.5 μM to 1.5 μM of a Wnt / β-catenin agonist. In another example, after the first 4 to 5 days of culturing in a high concentration of a Wnt / β-catenin agonist, the cell culture medium may contain 0.8 μM to 1.2 μM of a Wnt / β-catenin agonist. Thus, in one example, after the first 4 to 5 days of culturing in a high concentration of a Wnt / β-catenin agonist, the cell culture medium may contain 2 μM of a Wnt / β-catenin agonist. In another example, after the first 4 to 5 days of culturing 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.

[0156] In one example, the Wnt / β-catenin agonist is CHIR. Thus, in one example, after the first 4 to 5 days of culture in a high concentration of CHIR, the cell culture medium may contain at least 0.5 μM CHIR. In another example, after the first 4 to 5 days of culture in a high concentration of CHIR, the cell culture medium may contain at least 0.6 μM CHIR. In another example, after the first 4 to 5 days of culture in a high concentration of CHIR, the cell culture medium may contain at least 0.7 μM CHIR. In another example, after the first 4 to 5 days of culture in a high concentration of CHIR, the cell culture medium may contain at least 0.8 μM CHIR. In another example, after the first 4 to 5 days of culture in a high concentration of CHIR, the cell culture medium may contain at least 0.9 μM CHIR. In another example, after the first 4 to 5 days of culture in a high concentration of CHIR, the cell culture medium may contain about 1 μM CHIR. In another example, after the first 4 to 5 days of culturing in a high concentration of CHIR, the cell culture medium may contain 0.5 μM to 1.5 μM CHIR. In another example, after the first 4 to 5 days of culturing in a high concentration of CHIR, the cell culture medium may contain 0.8 μM to 1.2 μM CHIR. Thus, in one example, after the first 4 to 5 days of culturing in a high concentration of CHIR, the cell culture medium may contain less than 2 μM CHIR. In another example, after the first 4 to 5 days of culturing in a high concentration of CHIR, the cell culture medium may contain less than 1.5 μM CHIR.

[0157] In one example, after the first 4 to 5 days of cultivation in a high concentration of CHIR, the culture medium may include a low concentration of CHIR (such as those exemplified above). In one example, the cell culture medium includes at least 50 ng / ml of FGF9. In another example, the cell culture medium includes at least 100 ng / ml of FGF9. In another example, the cell culture medium includes at least 150 ng / ml of FGF9. In another example, the cell culture medium includes at least 200 ng / ml of FGF9. In another example, the cell culture medium includes at least 300 ng / ml of FGF9. In another example, the cell culture medium includes at least 350 ng / ml of FGF9. In another example, the cell culture medium includes at least 400 ng / ml of FGF9. In another example, the cell culture medium includes at least 500 ng / ml of FGF9. In another example, the cell culture medium includes 50 ng / ml to 400 ng / ml of FGF9. In another example, the cell culture medium includes 50 ng / ml to 300 ng / ml of FGF9. In another example, the cell culture medium comprises 50 ng / ml to 250 ng / ml of FGF9. In another example, the cell culture medium comprises 100 ng / ml to 200 ng / ml of FGF9. In another example, the cell culture medium comprises 180 ng / ml to 220 ng / ml of FGF9. In another example, the cell culture medium comprises 190 ng / ml to 210 ng / ml of FGF9.

[0158] In another example, the above reference level of FGF9 is replaced with FGF2. For example, the cell culture medium may include 50 ng / ml to 400 ng / ml of FGF2. In another example, the cell culture medium includes 50 ng / ml to 300 ng / ml of FGF2. In another example, the cell culture medium includes 50 ng / ml to 250 ng / ml of FGF2. In another example, the cell culture medium includes 100 ng / ml to 200 ng / ml of FGF2. In another example, the cell culture medium includes 180 ng / ml to 220 ng / ml of FGF2. In another example, the cell culture medium includes 190 ng / ml to 210 ng / ml of FGF2.

[0159] In another example, the above reference level of FGF9 is 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.

[0160] In another example, the above reference level of FGF9 is 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.

[0161] In one example, the medium comprising a high concentration of CHIR does not comprise FGF.

[0162] In another example, after the first 4 to 5 days of culture in a high concentration of CHIR, the cell culture medium may include a low concentration of CHIR (such as those exemplified above), FGF and heparin at the above exemplified levels. In one example, the cell culture medium comprising a low concentration of CHIR and FGF also comprises heparin. In this example, the cell culture medium may include 1 μg / ml of heparin. In another example, the cell culture medium may include 1.5 μg / ml of heparin. In another example, the cell culture medium may include 2 μg / ml of heparin. In another example, the cell culture medium may include 0.5 μg / ml to 2 μg / ml of heparin. In another example, the cell culture medium may include 0.5 μg / ml to 1.5 μg / ml of heparin. In another example, the cell culture medium may include 0.8 μg / ml to 1.2 μg / ml of heparin.

[0163] In one example, the methods of the present disclosure encompass combining the above-mentioned methods of generating IM cells with using these IM cells in the above-exemplified methods of generating kidney organoids.

[0164] In one example, stem cells can be cultured using the methods mentioned above to produce IM cells, and then dissociated and cultured in the vortex culture mentioned above to produce kidney organoids. In this example, the IM cells can be dissociated with EDTA, trypsin or TrypLE. In another example, the cells can be dissociated with EDTA before passing through a mesh and culturing in the vortex culture mentioned above to produce kidney organoids. In another example, the cells can be dissociated with trypsin before centrifugation and resuspending the resulting pellet in the vortex culture mentioned above to produce kidney organoids.

[0165] The kidney organoids covered by the present disclosure can be described based on the number of days in culture. The number of days in culture can be divided into two parts, including the number of days (X) for producing IM cells from stem cells and the number of days (Y) for forming kidney organoids from IM cells. In one example, the step of distinguishing the production of IM cells from stem cells from the production of kidney organoids from IM cells is the dissociation of IM cells. A method of representing the number of days in culture for producing IM cells from stem cells and the number of days for forming kidney organoids from IM cells is (d) X+Y days (e.g., d7+12 will describe 7 days of producing IM cells from stem cells, then dissociating the IM cells, and 12 days of forming organoids from IM cells (i.e., Y=days as organoids).

[0166] In one example, the kidney organoids encompassed by the present disclosure are d7+12 kidney organoids. In another example, the kidney organoids encompassed by the present disclosure are d7+14 kidney organoids. In another example, the kidney organoids encompassed by the present disclosure are d7+15 or later kidney organoids. In another example, the kidney organoids encompassed by the present disclosure are d7+17 kidney organoids.

[0167] In another example, the kidney organoids encompassed by the present disclosure are d7+20 kidney organoids. In another example, the kidney organoids encompassed by the present disclosure are d7+22 kidney organoids. In another example, the kidney organoids encompassed by the present disclosure are d7+25 kidney organoids. In another example, the kidney organoids encompassed by the present disclosure are d7+30 kidney organoids. In another example, the kidney organoids encompassed by the present disclosure are d7+13 to d7+30. In another example, the kidney organoids encompassed by the present disclosure are d7+14 to d7+30. In another example, the kidney organoids encompassed by the present disclosure are d7+15 to d7+30. In another example, the kidney organoids encompassed by the present disclosure are d7+15 to d7+25. In the above-mentioned examples, IM cells can be cultured for 8, 9 or 10 days (i.e., d8+Y, d9+Y or d10+Y).

[0168] In another example, the cells of kidney organoids disclosed herein proliferate after D7+7. In another example, the cells of kidney organoids disclosed herein proliferate after D7+10. In another example, the cells of kidney organoids disclosed herein proliferate after D7+12. In another example, the cells of kidney organoids disclosed herein proliferate between D7+5 and D7+10. In another example, the cells of kidney organoids disclosed herein proliferate between D7+5 and D7+12. In these examples, the colony of organoids can be prepared by using the methods disclosed herein, and at a specific time point (e.g., D7+5, D7+7, D7+10, etc.), organoids are separated and dissociated from the colony, and the number of cells is determined by using, for example, an automatic cell counter (e.g., Life Technologies) at each time point, the trypan blue dye exclusion test is used to measure the number of cells to detect cell proliferation.

[0169] filter

[0170] The kidney organoids encompassed by the present disclosure can be used in a variety of screening applications. In one example, the kidney organoids can be used to screen for toxicity. For example, the kidney organoids can be used to screen for renal toxicity.

[0171] Thus, in one example, the present disclosure encompasses a method of screening a candidate compound for nephrotoxicity, the method comprising contacting a kidney organoid disclosed herein with a candidate compound and determining whether the candidate compound is nephrotoxic.

[0172] In one example, before assessing nephrotoxic side effects, kidney organoids described herein are contacted with a candidate compound. Exemplary nephrotoxic side effects include direct tubular effects, podocyte injury, interstitial nephritis, and glomerulonephritis. Nephrotoxicity can also be assessed or measured by any appropriate test for in vitro renal cell function, including using commercially available tools (including, for example, Human Nephrotoxicity RT from Qiagen). 2 Profiler TM PCR Array or High Content Analysis (HCA) multiple nephrotoxicity assay from Eurofins) analyzes the expression of biomarkers. In another example, nephrotoxicity is assessed by measuring the acute apoptosis of glomerular cells in kidney organoids disclosed herein after contact with the candidate compound. In other examples, nephrotoxicity can be assessed using an electron microscope (e.g., transmission EM or scanning EM). Other examples of the standard for indicating nephrotoxicity include the loss of podocyte marker gene expression or protein expression and the loss (falling off) of foot processes.

[0173] In another example, the present disclosure encompasses a method of screening for a candidate compound having therapeutic efficacy in treating kidney disease, the method comprising contacting a kidney organoid disclosed herein with a candidate compound under conditions that determine whether the candidate compound is therapeutically effective. In this example, the method may include contacting a kidney organoid disclosed herein with a candidate compound in the presence of a nephrotoxic compound, and determining whether the candidate compound is therapeutically effective.

[0174] Other examples of screening for therapeutic efficacy include evaluating kidney organoids representing nephropathy. For example, the nephropathy 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 PierSon syndrome. In one example, the nephropathy is CNS.

[0175] The term "therapeutic efficacy" is used in the context of the present disclosure to refer to a reaction in which the therapeutic beneficial effects exceed any toxic or deleterious effects of the candidate compound or composition comprising it. The therapeutic efficacy can be determined based on improved renal cell function; maintaining renal cell function; inhibiting (i.e., slowing down to some extent and stopping in some instances) renal cell function decline; inhibiting (i.e., slowing down to some extent and stopping in some instances) renal cell death. In one example, the therapeutic efficacy is determined based on the presence of appropriate podocyte proteins and evidence that they are appropriately polarized. An example includes positioning NPHS1, NPHS2, and NEPH-1 on the membrane of the podocyte, wherein NPHS1, NPHS2, and NEPH-1 are determined using immunohistochemistry.

[0176] For studies involving kidney organoids representing kidney disease, renal toxicity and therapeutic efficacy can be determined relative to predetermined criteria determined based on corresponding renal cell function in disease-free kidney organoids. In another example, improved renal cell function can be determined based on a comparison of renal cell function between kidney organoids representing kidney disease and kidney organoids representing healthy kidneys.

[0177] For studies involving contacting renal organoids with a nephrotoxic compound and a candidate compound, improved renal cell function can be determined based on comparison to renal organoids not contacted with the nephrotoxic compound and / or renal organoids contacted with the nephrotoxic compound alone.

[0178] The term "candidate compound" is used in the context of the present disclosure to refer to an agent to be screened. Candidate compounds can include, for example, small molecules, such as small organic compounds (e.g., organic molecules with a molecular weight between about 50 and about 2,500 Da), peptides or their mimetics, ligands including peptides and non-peptide ligands, polypeptides, nucleic acid molecules such as aptamers, peptide nucleic acid molecules, and components, combinations and derivatives thereof.

[0179] It is considered that terms such as "contacting," "exposing," or "applying" are terms that can be used interchangeably in the present disclosure in context. The term contacting requires that the candidate compound is in contact with the glomerulus disclosed herein. In one example, if the compound is water-soluble or immiscible with water, the compound can be dissolved in the cell culture medium. Otherwise, a suitable substrate can be immersed in the compound and placed above the cultured kidney organoids. To screen volatile candidate compounds, the kidney organoids disclosed herein can be exposed to air or other gas mixtures containing the compound. Alternatively, the kidney organoids can be exposed to a solution or suspension of the volatile compound in the cell culture medium. Likewise, if possible, the volatile compound can be dissolved or stabilized. Otherwise, a suitable substrate can be immersed in the compound and placed above the cultured kidney organoids.

[0180] In performing the methods of the present disclosure, a plurality of candidate compounds can be contacted with a kidney organoid. For example, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 70, at least 80, at least 90, at least 100, at least 115, at least 120, at least 130, at least 140, at least 150, at least 161, at least 171, at least 182, at least 193, at least 204, at least 256, at least 257, at least 261, at least 262, at least 263, at least 264, at least 265, at least 271 100, at least 200, at least 300, 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 can be contacted with kidney organoids. In one example, candidate compounds can be contacted with the same or separate kidney organoids. For example, specific combinations of candidate compounds can be screened.

[0181] In one example, the candidate compound is labeled before screening. In one example, the candidate compound can be a composition. For example, the candidate compound can be present in a formulation or 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 nephropathy. In one example, serum is isolated from a subject suffering from CNS. For example, serum can be isolated from a subject suffering from steroid-resistant nephrotic syndrome. In another example, serum is isolated from a subject who has undergone a kidney transplant. In another example, serum is isolated from a subject presenting nephrotic syndrome after a kidney transplant.

[0182] Exemplary nephrotoxins include aminoglycoside antibiotics, beta-lactam antibiotics, cisplatin, radiocontrast media, NSAIDs, ACE inhibitors, lithium, CsA, and anti-epileptic drugs such as phenytoin.

[0183] Kidney organoids cultured for various lengths of time can be used for screening applications disclosed herein. Thus, as an example, kidney organoids from d7+15 or later can be used for screening. In another example, kidney organoids between d7+18 and d7+25 can be used for screening. In another example, immature kidney organoids can be used for screening. For example, d7+11 to d7+18 can be used for screening. In various examples, IM cells can be cultured for longer periods of time, so d8+Y, d9+Y, or d10+Y kidney organoids can be used for screening.

[0184] In one example, the screening method includes contacting the candidate compound with a kidney organoid library. For example, kidney organoids at different developmental stages can be used to screen candidate compounds. For example, D7+10, D7+15, and D7+25 kidney organoids can be used. In another example, kidney organoids representing different kidney diseases can be used to screen candidate compounds.

[0185] As will be appreciated by those skilled in the art, a variety of different screening procedures can be used to screen candidate compounds. For example, the kidney organoids disclosed herein can be provided in a single or multi-well form and contacted with a candidate compound for a set period of time. In one example, kidney organoids are provided in a multi-well plate. In one example, one kidney organoid is provided per well. In another example, two kidney organoids are provided per well. In another example, three kidney organoids are provided per well. In another example, four kidney organoids are provided per well. In another example, five kidney organoids are provided per well. In another example, 10 kidney organoids are provided per well. In another example, 20 or more kidney organoids are provided per well. In one example, kidney organoids are provided in a 96-well plate.

[0186] The present disclosure encompasses high throughput screening methods. In this example, high throughput screening involves providing a library comprising a large number of candidate compounds. Such libraries are then screened in one or more assays to identify those library members (e.g., specific chemical species or subclasses) that exhibit a desired level of activity (e.g., therapeutic efficacy).

[0187] High-throughput screening systems are commercially available and typically automate the entire procedure, including pipetting of all samples and reagents, liquid dispensing, timed incubations, and final reading of the culture plate (e.g., 96-well format) in a detector suitable for the assay. These configurable systems offer rapid startup as well as a high degree of flexibility and customization. Manufacturers of such systems (e.g., Invitrogen, Thermo Fisher Scientific, etc.) provide detailed protocols for use.

[0188] In one example, the above-mentioned method also includes selecting a compound showing therapeutic efficacy. For example, maintaining renal cell function in the presence of nephrotoxin and / or when contacting with a kidney organoid representing nephropathy; inhibiting (i.e., slowing down to some extent and stopping in some instances) renal cell function decline; inhibiting (i.e., slowing down to some extent and stopping in some instances) compounds of renal cell death. In another example, the above-mentioned method also includes selecting a compound that reduces nephrotoxicity. For example, a compound that inhibits glomerulonephritis can be selected. In another example, a compound that improves renal cell function can be selected. In these examples, commercially available tools (including, for example, HumanNephrotoxicity RT from Qiagen) can be used. 2 Profiler TM PCR Array or High Content) to determine renal cell function based on the expression of biomarkers.

[0189] Personalized medicine and stratification

[0190] Candidate compounds that show therapeutic efficacy in kidney organoids representing the subject's kidney disease are more likely to show therapeutic efficacy in the subject. Thus, in one example, these kidney organoids can be used to select agents that are more likely to affect the treatment or prevention of the subject's kidney disease.

[0191] In another example, kidney organoids representing nephropathy of multiple subjects can be prepared. These kidney organoids can be used to select the treatment or prevention of nephropathy that is more likely to affect multiple subjects or to identify the agent that is more likely to respond to a specific agent treatment. Such methods can be used to stratify subjects in clinical trials of agents that test the ability to treat nephropathy. Grouping the subject population based on kidney organoid screening can eliminate or reduce the differences in treatment results due to genetic factors, thereby more accurately evaluating the efficacy of potential drugs. Therefore, in one example, the present disclosure covers a method for stratifying a group of subjects for clinical trials of therapeutic agents, the method including obtaining an iPS cell population from a group of subjects, or generating kidney organoids or their populations from each subject iPS cell population, contacting the kidney organoids with a therapeutic agent, determining whether the therapeutic agent has a therapeutic effect, and using the results of the determination to select subjects who are more likely to respond to the treatment. In this example, the method may include contacting the kidney organoids with a therapeutic agent and a nephrotoxin before determining whether the therapeutic agent is effective in treatment. Examples of therapeutic agents include candidate compounds discussed above, for example, one or more small molecules, polynucleotides, peptides, proteins, antibodies, antibody fragments, viruses, bacteria, stem cells, serum including serum from patients with nephropathy. For the avoidance of doubt, serum can be isolated from a subject suffering from a specific nephropathy and contacted with the kidney organoid disclosed herein. Various examples of nephropathy are discussed herein, and serum can be isolated from various subjects representing these diseases. Methods for separating serum from a subject are known in the art. In one example, serum is purified from a whole blood sample using centrifugation.

[0192] Bioprinting

[0193] 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 compositions or cells disclosed herein. Terms such as "bioprinted" or "bioprinting" used in the context of the present disclosure refer to a process of three-dimensional precise deposition of cells (e.g., cell solutions, cell-containing gels, cell suspensions, cell concentrates, multicellular aggregates, multicellular bodies, bio-inks, etc.) via methods compatible with automated computer-assisted three-dimensional prototyping equipment (e.g., bioprinters). Examples of methods suitable for bioprinting are disclosed in WO 2012 / 054195 and WO 2013 / 040087. In one example, bioprinting is performed using an organ printer that uses a hydrogel scaffold to place human cells in a desired direction to reconstruct a human organ (e.g., Organovo / Invetech).

[0194] In one example, the kidney structure is bioprinted from bio-ink. The term "bio-ink" is used in the context of the present disclosure to refer to a liquid, semi-solid or solid composition comprising a composition or cells as defined herein. In one example, the bio-ink includes a cell solution, a cell aggregate, a gel containing cells, a multicellular body or a tissue. In another example, the bio-ink also includes a support material.

[0195] The bioprinted renal structures encompassed by the present disclosure have one or more functional characteristics of the kidney or its components, or are capable of developing one or more functional characteristics of the kidney or its components. For example, the bioprinted renal structure may include a glomerulus, a juxtaglomerular organelle, interstitial tissue, a collecting duct, a Bowman's capsule, a proximal and / or distal convoluted tubule. In one example, the bioprinted renal structure is not vascularized. In another example, the bioprinted renal structure includes a vasculature, such as arterioles, arteries, veins, and / or capillaries.

[0196] In one example, the bioprinted kidney structure is implantable or otherwise adoptively transferred into a host.

[0197] Composition / Kit

[0198] In one example, the present disclosure relates to a kit or assay for screening applications. For example, the present disclosure encompasses a kit or assay for screening the nephrotoxicity and / or therapeutic efficacy of a candidate compound. In one example, a kidney organoid as described herein is provided in a culture, and then the candidate compound is contacted with the kidney organoid and its nephrotoxicity and / or therapeutic efficacy is screened. Therefore, in one example, the present disclosure encompasses an assay for screening, which includes kidney organoids disclosed herein in a culture. In one example, the assay is used for nephrotoxicity screening. In one example, the assay is used for therapeutic efficacy screening. In one example, kidney organoids are provided with culture medium or other components to maintain kidney organoids in culture. In one example, kidney organoids are provided with written instructions for performing the method disclosed herein. In one example, the assay includes kidney organoids as described herein. In other examples, the assay includes more than one kidney organoid. For example, the assay can include 10, 20, 30 or more kidney organoids. Kidney organoids can be provided in a single or multi-hole form, such as a 96-well plate.

[0199] Examples

[0200] Example 1 - Cultivation and maintenance of hPSCs

[0201] Human ES cells (H9 cells) were grown on mouse embryonic fibroblast (MEF) feeder cells in DMEM medium supplemented with 10% KOSR (Life technologies) and bFGF. Cells were cultured to 80% confluence before splitting using TrypLE (Life technologies). Prior to differentiation, ES cells were conditioned to a Matrigel (Corning) surface in MEF conditioned medium and bFGF in the absence of MEF feeder cells.

[0202] Human iPS cells were grown as single colonies on Geltrex (Life Technologies) coated plates in E8 medium (Life Technologies). iPS cells were passaged once with EDTA once the cells reached 60-70% confluence or every 3 days.

[0203] hPSCs were dissociated into single cells using TrypLE and the cells were plated at 15,000 cells / cm 2 Seed on matrigel-coated plates. Count the cells using a hemocytometer. Seed Matrigel-adapted hES cells using MEF-conditioned medium. Human iPS cells were seeded as single cells in E8 medium using revita cells (1:100 dilution) on matrigel-coated plates overnight.

[0204] Example 2 - Generation of Improved IM

[0205] hPSCs or hES were differentiated into intermediate mesoderm by exposing the cells to high concentrations of CHIR (7 μM) in APEL2 or TeSR-E6 medium (Stem cell technologies) containing 3.5% protein-free hybridoma medium (PFHM) (Thermo Fischer), APEL (Stem cell technologies) or E6 medium (Stem cell technologies) for the first 4 days. The medium was renewed on day 2. In addition to FGF9 and heparin, the culture was subjected to low concentrations of CHIR (1 μM) (Figure 1A, Examples 1-3) for another 3 days (5-7 days after inoculation). This resulted in the induction of a mixture of intermediate mesoderm (IM) cells.

[0206] Gene expression profiling of vortexed suspension cultures at D7+0 by qPCR showed that the addition of low concentrations and longer duration exposures of CHIR resulted in an increase in cap mesenchymal cells compared to shorter exposure times (Figure 1E left panel). Expression of PAX2, LHX1, and OSR1 cells was more than 2-fold higher compared to the short CHIR exposure group (Figure IE left panel). Exposing cells to low concentrations of CHIR also resulted in an increase in ureteric epithelium, such as a more than 2-fold increase in expression of Wnt11 and GATA3 compared to the short CHIR exposure group (Figure 1E right panel). Adding low concentrations of CHIR to activate Wnt / β-catenin signaling for longer durations demonstrates a method to generate modified intermediate mesoderm.

[0207] Example 3 - Vortex suspension culture

[0208] On day 7, IM cells generated using the method in Example 2 were dissociated using 1 ml of EDTA solution, further incubated in 1 ml of EDTA at 37°C for 3 minutes, and the EDTA solution was removed by aspiration without disturbing the IM cell layer (Figure 1A). IM cells can also be dissociated using 1.5 ml of TrypLE TM Select was dissociated at 37°C for 3 minutes and excess TrypLETM was removed by centrifugation at 1500 RPM in a 15 ml falcon tube.

[0209] Add stage 1 medium (basal medium, FGF9 200ng / ml, heparin 1μg / ml, 1μM CHIR, 0.1% PVA, 0.1% MC) (2ml) and 10μM Rho kinase inhibitor (ROCKi, 1:1000 dilution, stem cell technologies) Use a Gilson pipette to gently dissociate the cells into clumps. Transfer the cell suspension to a 6cm 2 The cells were plated in low-adhesion culture dishes (Greiner bio) and passed through a 40 μm cell strainer (BD biosciences).

[0210] The stage 1 culture medium is loaded to 5ml, and the culture dish is vortexed at 60rpm in a Ratek orbital shaker at 37°C and 5% CO2 in a standard cell culture incubator. After the culture dish is placed on an orbital shaker for 24 hours, organoids with a diameter of 20 to 40 μm are spontaneously formed. After 24 hours of vortex culture, the stage 1 culture medium is replaced with stage 2 culture medium (basal medium, FGF9 200ng / ml, heparin 1μg / ml, 1μMCHIR, 0.1%PVA, 0.1%MC). The cells are cultured in stage 2 culture medium for another 4 days. Starting from the 7th + 5th day, all organoids are updated every few days with stage 3 culture medium (basal medium, 0.1%PVA, 0.1%MC) and cultured to the 7th + 18th day.

[0211] After 18 days post-aggregation (days 7+18), each kidney organoid displayed tubular epithelial structures, as confirmed by the presence of 6–10 nephrons by bright field, periodic acid Schiff (PAS) staining, and confocal microscopy analysis ( Figures 1 and Figure 2 ). These nephrons show evidence of early patterning and segmentation. Glomerular formation is evident from positive staining for NPHS1 and MAFB. The proximal nephron segments are EpCAM + , and stained positively for LTL, CUBN, LRP2, and HNF4A (Figure 1G). + The segments were able to internalize FITC-albumin within 24 hours of addition to the culture medium, indicating a functional albumin uptake pathway. The distal nephron segments were stained with ECAD and EpCAM, while the presumptive collecting ducts were stained with ECAD + / GATA3 + When renal organoids were generated using a SOX17mCherry reporter cell line (Ng et al., 2016), it was also noted that endothelial cells (PECAM1 + / SOX17 + ) exists (Figure ID; Figure 2 C, D, and E). As an indication of the transferability of this protocol between human pluripotent stem cell lines, data are provided on the successful generation of kidney organoids from 4 different cell lines, including hESC reporter cell lines (H9GAPTrapLuc2, 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 consistently to the protocol and had similar patterns for kidney organoids ( Figure 2 B).

[0212] Example 4 - Effects of duration, concentration, and timing of canonical Wnt signaling on kidney organoid models

[0213] To optimize differentiation in the kidney organoid protocol, hPSC monolayers were stimulated with a fixed concentration of CHIR99021 (7 μM) for different durations (3, 4, 5, and 6 days) before continuing culture in the presence of low CHIR and FGF9 + heparin until day 7 ( Figure 2 C, 2D, and 2E). After 18 days, the renal structure of the resulting kidney organoids was assessed using confocal microscopy ( Figure 2 C, 2D and 2E). Canonical Wnt activation for only 3 days failed to produce kidney morphology ( Figure 2 B, left). In contrast, the epithelial structures that were present showed an indeterminate epithelium with large cystic cavities and no evidence of nephron formation. Initial induction with 7 μM CHIR99021 for 4 or 5 days generated kidney organoids containing stereotypical nephrons, including surrounding SOX17 indicative of endothelial and mesenchymal stromal cells, respectively. + and MEIS1 / 2 / 3 + The existence of a group Figure 2 C, 2D, and 2E, middle panels). However, Wnt activation for 6 days, despite generating larger kidney organoids with greater NPHS1 staining, contained expanded MEIS1 + stromal population, with significant damage to epithelial structures ( Figure 2 C, 2D and 2E, right panels). An initial 7 μM CHIR99021 induction for 4 days was confirmed to be optimal and was used for further studies.

[0214] Example 5 - Dissociation of Kidney Organoids

[0215] Kidney organoids represent heterogeneous epithelial structures of approximately 250-300 μm in diameter. The use of harsh enzymes may damage cell surface markers, resulting in loss of cell identity for later use. Mild dissociation is performed with cold active protease (LiberaseTM, Roche) to produce maximum viable single cells. Transfer the kidney organoids to a 15 ml falcon tube with a 5 ml serological pipette and allow to settle. Use vacuum to remove the culture medium supernatant, and then wash the organoid pellet three times with 0.1 M PBS. The organoids are then treated with 500 μl of 1 μg / ml Liberase TM Solution was treated and incubated at 4°C for 20 min with trituration every 5 min. After 20 min, kidney organoids were dissociated into single cells and washed twice with DMEM medium containing 10% FCS to inactivate Liberase TM The final cell pellet was suspended in DMEM medium containing 10% FCS.

[0216] Example 6 - Vortexed intermediate mesoderm cells generate micro-kidney organoids in suspension culture

[0217] Existing methods for producing kidney organoids are labor-intensive, expensive and produce low-quality organoids. The inventors have produced economical, simple and fast methods to produce kidney organoids in suspension culture. Compared with previous methods, due to the minimum dissociation and low-speed vortex of the monolayer before culturing in low-adhesion culture plates, cell aggregates are formed in the intermediate mesoderm (IM) stage (the 7th day) of differentiation. This results in the formation of 8,000-10,000 kidney organoids, which are much smaller than the organoids produced using the previous method. After 18 days of suspension culture, each kidney organoid contains about 6-10 nephrons, with evidence of early patterns and segmentation, including the formation of proximal and distal epithelium and podocytes containing glomeruli. Importantly, single-cell transcriptional profiles have revealed the equivalence between these smaller organoids and the standard organoids produced using the previous method in terms of cell diversity and maturity. Compared to previous methods (e.g., those based on Takasato et al. (2015) Nature, Vol. 526: 564-568), directed differentiation using this approach resulted in a 30-40-fold expansion of cells over the course of 21 days in culture, representing a 3- to 4-fold increase in the yield of organoid-derived kidney cells per million and a 4-fold reduction in cost.

[0218] The disclosed method illustrated in Figure 1A and described in Examples 1-3 involves adding 0.1% polyvinyl alcohol (PVA) and 0.1% methylcellulose (MC) to the culture medium of the vortex culture to enhance the cohesion of the intermediate mesoderm (IM) cells, thereby spontaneously aggregating into 3D spherical organoids. After 24 hours of vortex culture, the organoids formed an outer layer of adhesion protein basement membrane.

[0219] C32 organoids cultured for another 12 to 18 days were collected, mounted and stained for NPHS, LTL, ECAD and GATA3 for confocal microscopy analysis. To assess the presence of vasculature, organoids were stained with mouse anti-human CD31 (1:300, BDbiosciences) and for mature proximal tubules, goat anti-human CUBN was used. Immunofluorescence staining analysis of organoids at D7+12 showed that the major nephron segments were nephrin-positive glomeruli, LTL-positive proximal tubules, ECAD-positive distal tubules, and different expression patterns of ECAD and GATA3 double-positive collecting duct cells (including GATA3-positive stromal cells) (Figures 1G and 4E and F), which were comparable to the transwell organoid system (Figure 4A). Vortex micro-kidney organoids exhibited a simple morphology compared to transwell organoids in terms of the number of nephrons (5 to 10 nephrons per organoid) and the presence of non-renal cell types.

[0220] In summary, the results demonstrate a vortex suspension method that produces organized organoids and has the ability to generate complex multicellular kidney organoids with all nephron segments. The method has been successfully tested with human ES and iPS cell lines to produce organoids. Different basal media conditions such as APEL, APEL2, and E6 have been shown to be suitable for the disclosed method. Therefore, the method may be useful for the expansion of kidney cell cultures for personalized medicine, drug screening, and regenerative cell therapy.

[0221] Example 7 - Micro-kidney organoids in suspension culture suitable for expansion of iPS-derived kidney cells

[0222] To evaluate the expansion of suspension cultures, C32 iPS cells were differentiated to generate IMs as described in Examples 1 and 2. The growth of organoids was monitored by measuring the size and total number of cells in the culture starting at D7+0 (Figures 4C and 4D).

[0223] Brightfield images of C32 organoids generated using vortex suspension culture showed an increase in size and maintenance of epithelial architecture (Figures 4A and 4B). Organoid size was measured in brightfield images of up to 10 randomly sampled organoids using NIS-Elements microscopy software (Nikon). Up to 10 organoids were randomly sampled at different growth rate intervals and the diameter of the organoids was measured. Sizes were reported to range from low to high. A consistent increase in organoid size was observed over time, and the diameters of the detected organoids ranged from 30 μm to 300 μm (Figure 4C). TM Total cell number was assessed after select dissociation and counted manually using a hemocytometer. A 40-fold increase in cell number was observed between D7+12 and D7+18 compared to the seeding density at D7+0 (Figure 4D).

[0224] Example 8 - Micro-kidney organoids in suspension culture show organized nephron segments and clear tubular lumen

[0225] Classical transwell organoids exhibit complex morphology, limiting the ability to study the 3D structure of individual nephrons. The vortex micro-kidney organoids generated by the methods described here are simpler and contain a smaller number of nephrons.

[0226] Organoids have clear 3D morphology, allowing the study of nephrons in 3D space. C32-derived kidney organoids were generated using the methods described in Examples 1-3 and immunostained with antibodies against NPHS1, LTL, ECAD, and GATA3 to visualize nephron segments in high-throughput confocal microscopy with Z resolution matching the pinhole.

[0227] Kidney organoids were collected in 15 ml Flacon tubes and washed with PBS (twice) to remove excess medium and fixed in freshly prepared 2% PFA for 20 minutes at 4°C. Excess PFA was then removed by washing the organoids 3 times with PBS containing 0.3% TritonX100 (PBST) 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 hour and then incubated with primary antibodies diluted in blocking buffer. The evaluation of the differentiation capacity of kidney organoids was confirmed by staining the main nephron segments, and the primary antibodies used were: sheep anti-human NPHS1 (1:300 R&D Systems), biotin anti-human LTL (1:300 Vectorlaboratories), mouse anti-human ECAD (1:300 Life technologies) and rabbit anti-human GATA3 (1:300 Cellsignalling technologies) mouse anti-human CD31 (1:300, BD biosciences) and goat anti-human CUBN (1:300 Santa Cruz) and LRP2 (1:300 Sapphire Bioscience). Organoids were incubated in primary antibodies at 4°C overnight, then washed 5 times in PBST and then incubated with species-matched secondary antibodies with fluorescent labels. After staining, organoids were dehydrated using different concentrations of methanol and then cleared using BABB (benzyl alcohol and benzyl benzoate, ratio 1:2) as previously described by DodtHU et al. (Dodt et al., 2007). Clarified organoids were mounted on MatTek glass-bottom dishes and confocal microscopy was performed using an inverted Zeiss LSM 780 microscope. Images were analyzed using ZEN software (Zeiss).

[0228] Images were analyzed using Imaris software to reconstruct 3D renderings of the acquired confocal images (Figures 4E and 4F). The 3D images showed clear nephron segments connected to each other in a polarized manner, starting with the glomerulus (NPHS1), proximal tubule (LTL+), distal tubule (ECAD+), collecting duct (ECAD+GATA3+), and interstitial cells (GATA3+) (Figure 5E). The use of the snipping tool allowed visualization of the lumen formed in the tubular cells (Figure 5F). The results in Figures 5E and 5F demonstrate that the vortexer method described herein can be used to allow the study of the morphology of developing kidney organoids in 3D space.

[0229] Example 9 - Single-cell RNA sequencing analysis reveals promising kidney phenotypes

[0230] To further extensively characterize the vortexer kidney organoids, single-cell RNA sequencing analysis was performed on C32-derived micro-kidney organoids at D7+18. Approximately 40-50 micro-kidney organoids and 1 full standard organoid were cultured to day 7+18 using the same hPSC line (CRL1502.C32 in APEL medium). Organoids were collected and washed 3 times with PBS to remove excess medium. The cells were incubated with 400 μl of 1 μg / ml Liberase at 4°C by stirring every 5 minutes using a 1 ml pipette. TM Treat organoids with Liberase (Roche) solution for 20 minutes. Organoids dissociate into single cells within 20 minutes. Add cell culture medium (2 ml) to the inactive Liberase TM In. The cells were centrifuged at 1300-1500rpm for 3-5 minutes to form a precipitate. The supernatant was removed, and the precipitate was resuspended in fresh DMEM F12 culture medium, and passed through a 20 μm cell strainer to remove clumps, and stored on ice until analysis. Viability and cell number were analyzed by FACS and trypan blue dye exclusion test in an automatic cell counter (life technologies). The cells were stored on ice until analysis. The cells were fully mixed using a wide-bore 1ml pipette tip, and approximately 4000 live cells were used for RNA sequencing analysis. The chromium single cell 3' solution developed by 10X genomics technology was used. Sample preparation was performed according to the 10x Genomics single cell protocol (further information can be obtained in the chromium single cell 3' reagent kit v2 user guide available online). Single cell suspension, gel beads, and distribution oil were loaded into the appropriate wells of the 10x chromium chip. The chip will be fixed with a 10x gasket, and the complete assembly will be loaded into the 10x chromium controller. This will automatically generate a suspension of single cells coated in oil droplets containing a unique UMI for each cell. This suspension will be used for conventional RT-PCR to amplify transcripts.

[0231] Cells were barcoded using nanoliter Gel Bead-In-EMulsions (GEMs) and UMIs to index the transcriptome of each cell separately. After barcoding, magnetic beads were used to remove residual reagents and primers. The transcriptome was amplified by PCR using full-length barcoded cDNA to generate sufficient quality for library construction. These libraries were sequenced simultaneously with 2 different reads for UMIs and cDNA fragments. TMLibrary analysis was performed to enable the study of expression data at single-cell resolution. The Cell Ranger pipeline (version 1.3.1) was used to perform sample demultiplexing, barcode processing, and single-cell gene counting (Zheng et al., 2017). The samples were demultiplexed to produce a pair of FASTQ files for each sample. The 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 barcodes, UMIs, and gene IDs. The final result was a gene expression matrix for further analysis, which enabled the study of expression data at single-cell resolution. Further analysis was performed using the Seurat R package (version 2.3.1) to represent cell aggregation, cell type classification, and differential gene expression.

[0232] The gene expression matrix generated in Cell Ranger was imported into Seurat (Satija et al., 2015) for quality control and further analysis. All cells passed an initial filter to remove genes expressed in less than 3 cells and cells expressing less than 200 genes. Further filtering removed 1 cell with a mitochondrial transcript greater than 15%. The swirl function in Sscran (Lun et al., 2016; Scialdone et al., 2015) was used to assign a score associated with the likelihood of each cell being in G1, S, or G2M phase, and the cell cycle phase was assigned based on this score.

[0233] Expression data were normalized and scaled, with variability related to the number of UMIs, percent mitochondrial expression, percent ribosomal expression, and G2M score regressed out using the Seurat ScaleData function. Cells were clustered using the shared nearest neighbor modularity optimization based clustering algorithm implemented in Seurat using the first 15 principal components and a resolution value of 1.2. Marker gene lists were generated using the Seurat FindAllMarkers function to find genes differentially expressed between clusters with a log fold change above 0.25.

[0234] For combined analysis of standard and kidney organoid datasets, gene-cell matrices were generated in Cell Ranger as described above. All cells passed an initial filter to filter out genes expressed in fewer than 3 cells and cells expressing fewer than 200 genes. Each dataset was normalized and scaled, and regression analyses were performed on the number of UMIs, percent mitochondrial expression, percent ribosomal expression, and S, Gl, and G2M scores generated in Scran. Clustering was based on alignment composition 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).

[0235] Single-cell RNA gene expression profiles of organoids generated by the methods described here and by the classical transwell method were analyzed. UMI counts were plotted as tSNE plots and automatically clustered according to the genes present within the cell ( Figure 6 A-6B). GO enrichment analysis of all clusters showed 22.3% for nephron, 37.5% for total matrix, and 9.8% for vasculature ( Figure 6 A), while the Vortex micro-kidney organoids showed 32.5% mature nephrons (excluding cap mesenchyme and nephron progenitor cells) and 25.9% stroma, however, the Vortex C32 organoids did not show the presence of vasculature ( Figure 6 B). As a result, vortexed micro-kidney organoids displayed improved markers of kidney development compared to trans-well organoids. Micro-kidney organoids also displayed enhanced nephron composition compared to trans-well cultured organoids (Figure 4).

[0236] Example 10 - Transcriptional Validation of Renal Differentiation in Kidney Organoids

[0237] Single-cell RNA sequencing (scRNA-seq) was used to characterize the cell types present in kidney organoids. TM20-30 kidney organoid pools were dissociated into live single cells. This resulted in 89.4% single cells, of which 88.5% were alive (data not shown). Cell Ranger (10×Genomics) was used to generate a UMI count matrix for each cell, which was imported using the Seurat R software package (version 2.3.1) for further analysis (Satija et al., 2015). The filtered data represented 1673 cells, with a median of 3759 expressed genes per cell. Clustering was performed using the Seurat R software package, generating 7 different cell clusters at a resolution of 0.6 (Figures 3A and 3B, Table 1). Differential expression tests were performed to identify markers for each cluster, and the PANTHER gene ontology suite was used to perform gene ontology and functional enrichment analysis on the most significantly upregulated genes in each cluster (Mi et al., 2013; Table 1).

[0238] Table 1: Gene ontology terms used for different clusters in kidney micro-organoids.

[0239]

[0240]

[0241] Although clearly visible using immunofluorescence of whole-mounted organoids, in the scRNA-seq data, endothelium (a subset of cluster 1) and podocytes (cluster 6, 18 cells) are represented by only a few single cells. Clusters 3 (293 cells) and clusters 5 (122 cells) show gene expression consistent with nephron epithelium, with cluster 0 showing expression of renal alveolar / S-shaped body genes (early nephrons), while epithelial cluster 5 also shows expression of distal tubule / collecting duct markers such as GFRA1 (Figure 3C). Cluster 2 shows expression of nephron progenitor cell markers SIX1, SIX2, and CITED1, as well as the matrix marker PAX3 previously associated with myogenic Wilms tumor (Hueber et al., 2009). Cells in cluster 2 also express markers of myogenic fate, such as MYF5 and MYF6, but not PAX7, MYOD1, and TBX6. Cluster 0 (430 cells), representing the largest cluster, showed a more committed nephron progenitor cell signature, expressing early renal vesicle markers PAX2, PAX8, LHX1, and JAG1 as well as human NP markers LYPD1 and DAPL1 (Lindstrom et al., 2018). Cluster 1 (337 cells) showed stromal signatures, such as PDGFRB and MEIS2. Cluster 4 showed a neural transcriptional signature while expressing the early nephron marker cadherin CDH6, suggesting the presence of a neural off-target population, as previously reported in kidney organoids (Wu et al., 2017). This analysis strongly supported the identification of the cell types observed within kidney organoids at the immunofluorescence level.

[0242] Example 11 - Comparative single-cell transcriptional profiling of standard and kidney organoids demonstrates equivalence of nephrogenic patterns.

[0243] To directly compare the cellular components in kidney organoids with another kidney organoid approach, kidney organoid scRNA-seq data were combined with data from 1421 kidney organoid cells generated using the same iPSC cell line (CRL1502.C32) and the standard kidney 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 related component analysis followed by dynamic time warping. Clustering identified eight transcriptional clusters within the combined dataset, representing committed nephron progenitors (cluster 0), nephron epithelium (cluster 6), podocytes (cluster 7), stroma (clusters 1 and 3), endothelial cells (cluster 5), PAX3 +ve cells (cluster 2) and neural off-target populations (cluster 4) (Figures 5A and 5B). All clusters are shown in both datasets, but the proportion of cells belonging to each cluster varies (Figures 5B-5D). Direct comparison of key markers for each cluster showed that although there were clear differences in the proportion of contribution to each cluster between the protocols (Figures 5B and 5C), there was strong transcriptional consistency between the cells identified in any given cluster between the two protocols (Figure 5D). The neural off-target populations identified in kidney organoids were also evident in standard organoids. Overall, the kidney organoid dataset contained a higher proportion of nephron cells and a lower proportion of stromal cells compared to the standard organoid dataset (Figure 5E). Immunofluorescence analysis of whole-mounted organoids confirmed an increase in nephron cells expressing PAX2 and a decrease in stromal cells expressing MEIS1 / 2 / 3 in kidney organoids compared to standard organoids (Figures 5F and Figure 6 ).

[0244] Example 12 - Kidney organoids provide a better platform for efficient hPSC-derived kidney cell expansion

[0245] Due to the size of the generated organoid tissue, TMStandard kidney organoids cultured on filters may face diffusion restrictions after 3 weeks of culture (Fig. 4A). Immunofluorescence staining of nephron segments thereafter showed that the nephron structure was spatially restricted at the edge of the organoid. In contrast, kidney organoids contain tubules throughout the structure (Fig. 4B). Orbital oscillators can also be used to simultaneously form kidney organoids in large quantities, thereby avoiding the cumbersome manual manipulation process involved in the standard organoid protocol. As a result, approximately 8000 to 10000 kidney organoids of uniform size may be generated in 5-10 minutes, while the standard organoid protocol generates approximately 30 organoids in 60 minutes. Compared with standard organoids (3000-5000 μm), kidney organoids exhibit a much smaller final size (250-300 μm) (Fig. 4C). As shown by immunofluorescence, compared with kidney organoids, the nephrons formed in standard organoids are present in the edges around the periphery of the tissue (Fig. 4A). However, these structures are much larger than kidney organoids. To directly compare the efficiency and cost of each method, standard organoids and kidney organoids generated using iPSC and hESC reporter cell lines were dissociated into single cell suspensions at multiple time points of the differentiation protocol starting from day 7 to quantify the total cell number (Figure 4D). After day 7+7, standard organoids did not show significant changes in the total cell count per organoid, while the total cell count of kidney organoids continued to increase until day 7+12. Overall, cell counts increased 8-10 times under standard organoid conditions, and 30-40 times in the case of kidney organoids. This represents a 3-4-fold increase in cell yield using this modified protocol.

[0246] Example 13 - Addition of all-trans retinoic acid improves maturation of glomerular podocytes

[0247] To determine whether the addition of all-trans retinoic acid (atRA) would promote glomerular maturation of organoids generated by the vortex culture method described in Examples 1-3, atRA was supplemented in the culture medium after D7+0. All-trans retinoic acid (2.5 μM) was added to C32-derived vortex organoids from D7+5 to D7+10 (Figure 5). After D7+18, immunofluorescence analysis showed that the addition of atRA improved the podocyte phenotype ( Figure 7 A and 7B). The results were confirmed by mRNA expression, and qPCR analysis of D7+11 and D7+18 samples showed that the addition of 2.5 μM atRA increased the expression of glomerular markers such as MPHS1 and proximal tubule markers such as CUBN compared to the control ( Figure 7 C).

[0248] Example 14 - Drug toxicity testing in micro kidney organoids

[0249] In order to evaluate the suitability of in vitro testing of kidney drug toxicity using the suspension culture method described herein, drug toxicity studies were performed. Organoids produced by this method were collected on days 7+18 and randomly divided into treatment groups in 24-well low-attachment plates in 250 μl of culture medium. Organoids were stimulated with different concentrations of the cytotoxic drug doxorubicin (0, 2.5 and 5 μg / ml) for 24 hours. After stimulation, the culture medium was removed and the organoids were fixed with 2% PFA for immunofluorescence staining analysis of apoptosis using TUNEL staining, and some organoids were lysed for RNA analysis ( Figure 8 A-8D).

[0250] Example 15 - Summary of Improvements in Kidney Organoid Generation

[0251] Kidney organoids generated by the above example methods showed reliable formation of renal nephron epithelial, stromal, and endothelial cell components equivalent to those present in previously described kidney organoid protocols at the single-cell transcriptional level (Takasato et al., 2016; Takasato et al., 2015). However, changes in culture conditions resulted in a 3-4-fold increase in relative cell yield, while the cost per million kidney cells produced was reduced by 4-fold. The robustness of the example method was demonstrated by the ability to successfully recapitulate kidney organoid generation using 2 different hESCs (H9 and hES3) and 3 different iPSC lines (iPSC GAPTrap td-Tomato, CRL1502.C32, and CLR1502.3), including hES3 SOX17mCherry, H9 GAPTrap Luc2, and iPSC GAPTrap td-Tomato fluorescent reporter cell lines.

[0252] It will be appreciated by those skilled in the art that many changes and / or modifications may be made to the present disclosure as shown in the specific embodiments without departing from the spirit or scope of the present disclosure as broadly described. Therefore, the present embodiments are to be considered in all aspects as illustrative and not restrictive.

[0253] This application claims priority to AU 2017904424 filed on October 31, 2017, the disclosure of which is incorporated herein by reference.

[0254] All publications discussed and / or cited herein are incorporated in their entirety.

[0255] Any discussion of documents, acts, materials, devices, articles or the like that has been included in this specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

[0256] References

[0257] Barberi et al. (2005) Plos Medicine 2(6):0554-0559.

[0258] Briggs et al. (2013) Stem Cells 31:467-78.

[0259] Butler et al. (2018) Nat Biotechnol 36:411-420.

[0260] Cima et al. (1991) Biotechnol. Bioeng. 38:145 1991.

[0261] Dodt et al. (2007) Nat Methods 4:331-6.

[0262] Forbes et al. (2018) Am J Hum Genet. 102:816-831.

[0263] Hueber et al. (2009) Pathol. 12:347-54.

[0264] Kao et al. (2016) Stem Cell Reports 7:518-526.

[0265] Lindstrom et al. (2018) J Am Soc Nephrol. 29:806-824.

[0266] Lun et al., (2016) Fl000Res 5:2122.

[0267] Mi et al. (2013) Nat Protoc 8:1551-1556.

[0268] Ng et al. (2016) Nat Biotechnol 34:1168-1179.

[0269] Satija et al., (2015) Nat Biotechnol, 33: 495-502.

[0270] Scialdone et al. (2015) Methods 85:54-61.

[0271] Takasato et al. (2015) Nature 526:564-8.

[0272] Vacanti et al., (1988) J. Ped. Surg. 23:3-9.

[0273] Vacanti, et al., (1991) Plast. Reconstr. Surg. 88:753-9.

[0274] van den Berg et al. (2018) Stem Cell Reports 10:751-765.

[0275] Vodyanik et al. (2010) Cell Stem Cell 7:718-728.

[0276] Wu et al., (2017).

[0277] Zheng et al., (2017) Nat Commun 8:14049.

Claims

1. A kidney organoid comprising less than 50 nephrons, wherein the kidney organoid has a diameter of less than 500 μm and comprises NPHS1+ podocytes, LTL+ proximal segments, ECAD+ distal segments, and ECAD+GATA3+ collecting ducts.

2. The kidney organoid according to claim 1, wherein The kidney organoids comprise cells expressing high levels of any one or more of PAX2, SIX1, LHX1, OSR1, WNT11, GATA3, PAX8, EYA1 and / or CITED1 relative to kidney organoids cultured without vortexing.

3. The kidney organoid according to claim 2, wherein: The kidney organoids comprise cells expressing low levels of any one or more of PDGFRA, MEIS2, WT1 and / or C-RET relative to kidney organoids cultured without vortexing.

4. The kidney organoid of claim 3, wherein the kidney organoid comprises at least 25% mature nephrons and at least 15% stroma.

5. The kidney organoid of claim 4, wherein the kidney organoid comprises 1×10 4 Up to 5×10 4 cells. The kidney organoid of claim 5 , wherein the kidney organoid has a diameter of 250 to 500 μm.

7. The kidney organoid of claim 6, wherein the kidney organoid remains viable in vortex culture for at least three weeks.

8. The kidney organoid of claim 7, wherein the organoid nephron comprises GATA3+ECAD+ collecting ducts, GATA3-LTL-ECAD+ early distal tubules, LTL+ECAD- early proximal tubules, and WT1+ glomeruli.

9. Use of the kidney organoid according to any one of claims 1 to 8 or a cell composition of the kidney organoid prepared by dissociating the kidney organoid with a single protease, a combination of a protease and ethylenediaminetetraacetic acid, or collagenase in the preparation of a composition for treating kidney disease.

Images