WO2020019023A1

WO2020019023A1 - Composition and method

Info

Publication number: WO2020019023A1
Application number: PCT/AU2019/050768
Authority: WO
Inventors: Melissa H LITTLE; Lorna J. HALE
Original assignee: Murdoch Childrens Research Institute
Priority date: 2018-07-23
Filing date: 2019-07-23
Publication date: 2020-01-30

Abstract

The present disclosure relates to an isolated 3D organoid glomerulus, wherein the glomerulus is isolated from a stem cell-derived kidney organoid. These structures may be used in a variety of applications such as disease modelling, drug screening and toxicity screening.

Description

COMPOSITION AND METHOD

FIELD OF THE INVENTION

The present disclosure relates to isolated 3D organoid glomeruli. These structures may be used in a variety of applications such as disease modelling, drug screening and toxicity screening.

BACKGROUND OF THE INVENTION

A number of kidney diseases leading to proteinuria and/or haematuria, including congenital nephrotic syndrome and Alport syndrome, result from defects in the glomerular basement membrane (GBM), or functional and structural alterations to the podocytes of the glomeruli that lead to foot process effacement and loss of slit diaphragms.

Understanding the basis of human podocytopathies is hampered by the non proliferative nature and architecturally constrained morphology of primary podocytes. In particular, it is difficult to obtain primary podocytes in sufficient quantities for high throughput screening applications. In response, researchers turned to an in vitro model of the human podocyte, the temperature- sensitive SV40 conditionally immortalised podocyte cell line (Saleem, M.A., et al. (2002) J Am Soc Nephrol. 13:630-8). However, these conditionally immortalised podocytes are cultured as monolayers restricting their suitability as a morphologically accurate podocyte model. Hence, validation studies are commonly performed in animal models which can be costly and may not always replicate the human condition. For example, pre-clinical animal studies may not predict the toxicity of drug candidates in humans due to species differences.

New tools for studying kidney disease and treatments thereof are therefore required.

SUMMARY OF THE INVENTION

The present inventors have produced organoid-derived glomeruli “organoid glomeruli” (also referred to as an organoid glomerulus (in the singular) or“OrgGlom(s)”) which represent an accurate and reproducible three dimensional model of podocytes within the human glomeruli of the kidney. Importantly, comprehensive transcriptional and proteomic characterisation has revealed that podocytes within organoid glomeruli display a greater degree of maturation compared with isolated podocytes. The present inventors have also surprisingly identified that organoid glomeruli can be successfully isolated from kidney organoids and maintained in suspension culture. One reason this may be surprising is that previous attempts to culture glomeruli have resulted in glomeruli attachment to the cell culture vessel followed by podocyte outgrowth and destruction of the overarching glomeruli structure. Accordingly, in an example, the present disclosure encompasses a 3D organoid glomerulus isolated from a stem cell- derived kidney organoid. In an example, the glomerulus is PECAM1+ and/or PDGFR /+. In an example, the glomerulus is PECAM1+ and PDGFR /+. In an example, the glomerulus is suspended in culture. In an example, the glomerulus is suspended in culture for at least 3 days. In another example, the glomerulus is suspended in culture for at least 10 days. In another example, the diameter of the glomerulus is between 50 pm and 160 pm. In another example, the glomerulus comprises podocytes having apicobasal polarity as determined by immunohistochemistry for one or more or all of SYNAPTOPODIN, PODOCALYXIN, PODOCIN, NEPHRIN and NEPH1. In another example, the glomerulus incorporates additional cell types that are PECAM1+ (endothelial cells) and PDGFR /+ (mesangial cells). In another example, the glomerulus is positive for one or more of the following markers that provide evidence of a maturing glomerular basement membrane, including COL4A3, COL4A4, COL4A5 and LAMB2.

In another example, kidney organoids are derived from a culture expanded human stem cell population. In an example, the stem cells are human pluripotent stem cells, including human embryonic stem cells or human induced pluripotent stem (iPS) cells. In an example, the stem cells are human iPS cells. In an example, the human iPS cells are derived from fibroblasts or white blood cells. In another example, the human iPS cells are derived from a subject with a kidney disease. For example, the human iPS cells can be derived from a subject with a genetic kidney disease. In this example, the genetic kidney disease may 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 another example, the kidney organoid is derived from a culture expanded population of intermediate mesoderm (IM) cells. In an example, the IM cells are one or more of PAX2⁺, FHXl⁺, OSRl⁺.

In another example, the glomerulus is isolated from a kidney organoid which comprises cells expressing high levels of NPHS 1, PAX2, CDH1 and GATA3. In another example, the glomerulus is isolated from a kidney organoid which comprises NPHS 1+ podocytes, FTF+ proximal segments, ECAD+ distal segments, ECAD+/GATA3+ collecting duct or a combination thereof. In another example, the glomerulus is isolated from a kidney organoid which is produced by a method comprising culturing a population of stem cells in a cell culture medium for at least 7 days, dissociating the cells and, further culturing the cells in cell culture medium under conditions that facilitate organoid formation. In an example, the cells are cultured in cell culture medium comprising at least 4 mM CHIR for up to 5 days with the remaining days before dissociation involving the culturing of cells in cell culture medium comprising at least lOOng/ml FGF9. In an example, the cells may be dissociated at day 7 and further cultured for at least 14 days, wherein the culture media comprises FGF9 for the first 5 days and FGF9 is removed from the culture media thereafter (e.g. d7+5). In an example, 0.5mM to 5mM retinoic acid is added to the cell culture after 5 days of culture. In an example, the cell culture medium prior to dissociation can contain up to I.OmM CHIR together with at least 100 ng/ml of FGF9. In an example, cells are swirled after dissociation.

The present inventors have also identified that with increasing time in organoid culture, OrgGloms also begin to express markers for endothelial and mesangial cells and display laminin and collagen switching suggestive of maturation. As such, OrgGloms according to the present disclosure may also provide a tractable three-dimensional (3D) model of the human glomerulus. Accordingly, in an example, the glomerulus is isolated from a d7+l4 or later kidney organoid. In another example, the glomerulus is isolated from a d7+l4 to d7+ 25 kidney organoid. In another example, the glomerulus is isolated from a d7+l5 kidney organoid. In another example, the glomerulus is isolated from a d7+l8 kidney organoid. In another example, the glomerulus is isolated from a kidney organoid after d7+l8.

In an example, the suspension culture is performed in a low attachment culture plate.

Maintaining organoid glomeruli defined herein in suspension culture may be particularly advantageous for screening applications such as screening candidate compounds for therapeutic efficacy or nephrotoxicity. Accordingly, in another example, the present disclosure encompasses, a method of screening a candidate compound for nephrotoxicity, the method comprising contacting an organoid glomerulus isolated from a kidney organoid with a candidate compound and determining whether or not the candidate compound is nephrotoxic. In another example, the present disclosure encompasses, a method of screening a candidate compound for therapeutic efficacy, the method comprising contacting an organoid glomerulus isolated from a kidney organoid with a candidate compound and determining whether or not the candidate compound is therapeutically effective. In an example, the method comprises contacting an organoid glomerulus with a candidate compound and a nephrotoxin and determining whether or not the candidate compound is therapeutically effective. In an example, the candidate compound is a small molecule. In an example, the candidate compound is a drug. In an example, the candidate compound is a silencing RNA, delivered by means of a virus or transfection, and is designed to reduce expression of a specific gene. In an example, the candidate compound is serum, including serum isolated from a subject with kidney disease. In these examples, the screening method can further comprise selecting a candidate compound which is not nephrotoxic and/or is therapeutically effective.

In an example, the present disclosure also encompasses an assay when used for screening a candidate compound for nephrotoxicity and/or therapeutic efficacy, the assay comprising an organoid glomerulus isolated from a kidney organoid and a candidate compound. For example, the assay may comprise an organoid glomeruli disclosed herein.

In another example, the present disclosure encompasses a method for stratifying a group of subjects for a clinical trial of a therapeutic agent, the method comprising:

isolating an iPS cell population from a group of subjects;

generating a kidney organoid from each subject’s iPS cell population and isolating an organoid glomerulus;

contacting the isolated organoid glomerulus with a therapeutic agent and determining whether the therapeutic agent is therapeutically effective;

using the results of the determination to select subjects more likely to be responsive to the therapy. For example, the method may use a kidney organoid disclosed herein. For example, the method may use a 3D organoid glomerulus disclosed herein. In an example, the method comprises contacting the organoid glomeruli with a therapeutic agent and a nephrotoxic agent. In these examples, the agent may be a small molecule, polynucleotide, peptide, protein, antibody, antibody fragment, virus, bacteria, stem cell, serum including kidney disease patient derived serum or a combination of one or more thereof.

In another example, the present disclosure encompasses an organoid glomerulus disclosed herein when used for modelling glomerular development. In another example, the present disclosure encompasses an organoid glomerulus disclosed herein when used for modelling kidney disease. In these examples, the glomerulus may be isolated from a d7+l4 kidney organoid.

Numbered statements of the invention are as follows: 1. An isolated 3D organoid glomerulus, wherein the glomerulus is isolated from a stem cell-derived kidney organoid, and wherein the glomerulus is PECAM1+ and/or PDGFR /+.

2. The organoid glomerulus according to statement 1, wherein the glomerulus is positive for one or more of the following markers: COL4A3, COL4A4, COL4A5,

LAMB 2, LAMA5, LAMC1, KDR, MMP2, CXCL12, ITGA2, TEK, FN1, ANGPTL2, IGFBP3, SHIS A3, MME, IGFBP5, EMCN, UCHL1, TAGLN, CDH5, GJA5, SMAD7, CX3CL1, FGF2, GAT A3, DES, WT1, FOXD1, and C1QTNF12.

3. The organoid glomerulus according to statement 2, wherein the glomerulus expresses high levels of one or more of said markers.

4. The organoid glomerulus according to any one of statements 1 to 3, wherein the kidney organoid is derived from a culture expanded human stem cell population.

5. The organoid glomerulus according to statement 4, wherein the stem cells are human pluripotent stem cells, including human embryonic stem cells or human induced pluripotent stem (iPS) cells.

6. The organoid glomerulus of statement 4 or 5, wherein the human iPS cells are derived from a subject with a genetic kidney disease.

7. The organoid glomerulus of statement 6, wherein the genetic kidney disease is 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.

8. The organoid glomerulus according to any one of statements 1 to 7, wherein the kidney organoid is derived from a culture expanded population of stem cell- derived intermediate mesoderm (IM) cells.

9. The organoid glomerulus according to any one of statements 1 to 8, comprising at least one reporter gene associated with at least one gene of interest.

10. The organoid glomerulus according to any one of statements 1 to 9, wherein the organoid glomerulus is in suspension culture.

11. The organoid glomerulus according to any one of statements 1 to 10, wherein the organoid glomerulus is in suspension culture for at least 24 to 96 hours. 2. The organoid glomerulus according to any one of statements 1 to 11, wherein the kidney organoid has been cultured for at least 18 days.

3. A method of screening a candidate compound for nephrotoxicity or therapeutic efficacy, the method comprising contacting an isolated 3D organoid glomerulus isolated from a stem cell-derived kidney organoid with a candidate compound and determining whether or not the candidate compound is nephrotoxic or therapeutically effective.

4. A method of screening a candidate compound for nephrotoxicity and/or therapeutic efficacy, the method comprising contacting an organoid glomerulus according to any one of statements 1 to 12 with a candidate compound and determining whether or not the candidate compound is therapeutically effective.5. The method of statement 13 or 14, comprising contacting said organoid glomerulus with a candidate compound and a nephrotoxin and determining whether or not the candidate compound is therapeutically effective.

16. The method of any one of statements 13 to 15, wherein determining whether or not the candidate compound is nephrotoxic or therapeutically effective comprises measuring one or more of: diameter of the organoid glomerulus; expression of one or more genes associated with cell death; expression of one or more genes associated with cell viability; expression of one or more nephron-associated genes; expression of one or more genes associated with glomerular extracellular matrix; expression of one or more genes associated with podocyte, endothelial or mesangial cell types; and intensity of expression of a reporter gene associated with at least one gene of interest.

7. The method according to statement 16, wherein said one or more genes comprises one or more of: COL4A3, COL4A4, COL4A5, LAMB 2, LAMA5, LAMC1,

WT1, KDR, MMP2, CXCL12, ITGA2, TEK, FN1, ANGPTL2, IGFBP3, SHIS A3, MME, IGFBP5, EMCN, UCHF1, TAGEN, CDH5, GJA5, SMAD7, CX3CL1, FGF2, GATA3, DES, FOXD1, and C1QTNF12.

8. The method according to statement 16 or 17, comprising measuring the expression of one or more of: SYNAPTOPODIN, PODOC ALYXIN, PODOCIN, NEPHRIN,

NEPH1, PEC AM and PDGFR ?.

9. The method according to any one of statements 16 to 18, wherein: i) a measured reduction in one or more of: diameter of the organoid glomerulus; expression of one or more genes associated with cell viability; expression of one or more nephron-associated genes; expression of one or more genes associated with glomerular extracellular matrix; expression of one or more genes associated with podocyte, endothelial or mesangial cell types; and intensity of said reporter gene; and/or ii) a measured increase in expression of one or more genes associated with cell death; is indicative of nephrotoxicity of the candidate compound.

20. The method according to any one of statements 16 to 18, wherein: i) a measured increase or absence of a measured reduction in one or more of: diameter of the organoid glomerulus; expression of one or more genes associated with cell viability; expression of one or more nephron-associated genes; expression of one or more genes associated with glomerular extracellular matrix; expression of one or more genes associated with podocyte, endothelial or mesangial cell types; and intensity of said reporter gene; and/or ii) a measured reduction in expression of one or more genes associated with cell death; is indicative of therapeutic efficacy of the candidate compound.

21. The method according to any one of statements 16 to 20, wherein the candidate compound is a small molecule, polynucleotide, peptide, protein, antibody, antibody fragment, serum, virus, bacteria, stem cell or combination thereof.

22. The method according to any one of statements 16 to 21, wherein the candidate compound is a small molecule.

23. The method according to any one of statements 16 to 21, wherein the candidate compound is serum including serum isolated from a subject with kidney disease.

24. The method according to any one of statements 16 to 22, further comprising selecting a candidate compound which is not nephrotoxic and/or is therapeutically effective.

25. An assay when used for screening a candidate compound for nephrotoxicity and/or therapeutic efficacy, the assay comprising an organoid glomerulus isolated from a kidney organoid and a candidate compound.

26. The assay of statement 25, wherein the assay comprises organoid glomeruli according to any one of statements 1 to 12.

27. A method for stratifying a group of subjects for a clinical trial of a therapeutic agent, the method comprising:

- isolating an iPS cell population from a group of subjects; - generating a kidney organoid from each subjects iPS cell population and isolating an organoid glomerulus;

- contacting the isolated organoid glomerulus with a therapeutic agent and determining whether the therapeutic agent is therapeutically effective;

- using the results of the determination to select subjects more likely to be responsive to the therapy.

28. The method of statement 27, wherein the organoid glomerulus is defined by any one of statements 1 to 12.

29. The method of statement 27 or statement 28, which comprises contacting the organoid glomeruli with a therapeutic agent and a nephrotoxic agent.

30. The method according to any one of statements 27 to 29, wherein the agent is a small molecule, polynucleotide, peptide, protein, antibody, antibody fragment, virus, bacteria, stem cell, serum including kidney disease patient derived serum or a combination of one or more thereof.

31. The organoid glomerulus according to any one of statements 1 to 11 when used for modelling glomerular development.

32. The organoid glomerulus according to any one of statements 1 to 110 when used for modelling kidney disease.

33. The organoid glomerulus according to statement 31 or 32, wherein the glomerulus is isolated from a d7+l4 kidney organoid.

Any example herein shall be taken to apply mutatis mutandis to any other example unless specifically stated otherwise.

The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the disclosure, as described herein.

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

The disclosure is hereinafter described by way of the following non-limiting Examples and with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

Figure 1. Capillary loop stage organoid glomeruli can be isolated intact from human iPSC kidney organoids. A. Pure populations of whole organoid glomeruli can be isolated from kidney organoids by sieving. Scale bars 200pm (left) and lOOpm (right). B. Organoid glomeruli (OrgGloms) are formed in the appropriate size range, comparable to both human glomeruli at 32 weeks gestation, and adult human glomeruli. One way ANOVA / <0.0001 ; error bars = S.E.M.; significant difference assessed by Tukey’s multiple comparisons test; F-value=l76.9; DF=2; biological replicates n=30 adult glomeruli, n=30 neonatal glomeruli, n=l55 OrgGlom. C. (i) Organoid glomeruli developed within kidney organoids show comparable morphology to capillary loop stage human glomeruli, highlighted by in situ immunofluorescent staining of serial sections with the podocyte protein NEPHRIN, which shows infolding of the glomerular surface layer. Scale bar lOpm, central cross-section (left), surface (right) (ii) Quantitative PCR analysis of isolated glomerular fractions compared to whole organoid show a significant enrichment in NPHS1 gene expression in sieved organoid glomeruli. Error bar = S.E.M.; significant difference assessed by Student’s unpaired t-test p= 0.0002, t-value=l3, DF=4, n=3 biological replicates shown by symbols. D. Transmission electron microscopy of kidney organoid glomeruli show podocyte cell bodies connected by primary and secondary processes (inset), surrounded by an outer layer of parietal epithelial cells (arrowhead). Scale bar lOpm. E. Immuno staining of whole OrgGloms shows appropriate apicobasal cell polarity. A single cross-sectional plane is shown on the left, and a 3D reconstruction using all Z-stack images acquired (444 sections) on the right. Expression levels of single channels shown in greyscale to preserve maximum contrast, merged images shown in colour.

Figure 2. Isolated organoid-derived glomeruli exhibit superior podocyte identity and maturation. A. Principle component analysis of RNA sequencing (RNA-Seq) data was performed on 3 biological replicates of OrgGloms, OrgPods and ciPods in both differentiated and undifferentiated states and compared to previously published data (Jiang et al. (2016) Sci Rep, 6:35671). B. Venn diagram displaying the intersections of each comparison, upregulated genes are shown in red (displayed on top) and downregulated genes are shown in blue. This shows the greatest number statistically significant upregulated genes were identified in the OrgGloms versus differentiated ciPods. There was little to no overlap of differentially expressed genes in differentiated ciPods versus undifferentiated ciPods compared with all other pairwise comparisons. C. Heatmap showing the top 50 differentially expressed genes of each sample triplicate. A stark contrast in expression levels between the 3D OrgGloms and the 2D podocyte cultures is observed, particularly in genes associated with the podocyte. Log-normalised gene expression levels depicted. D. Gene Ontology (GO) enrichment analysis of the top 100 upregulated genes differentially expressed between OrgGloms and differentiated ciPods found enrichment of GO terms associated with developmental processes, and slit diaphragm components including genes associated with podocyte foot processes and those of the podocyte actin cytoskeleton. The top 10 most statistically significant GO term categories are shown. P value of 0.05 depicted as dotted line. E. Heatmap representation of key podocyte-associated genes showed low expression levels in both undifferentiated and differentiated ciPods, regardless of the laboratory in which they were cultured. By contrast, significantly elevated gene expression levels were displayed in the OrgGlom samples. Log-normalised gene expression levels depicted. F. Quantitative PCR analysis of key podocyte-associated genes validated the RNA-seq data showing strikingly enhanced gene expression in OrgGloms compared to 2D podocyte models. Two way ANOVA /;< 0.0001 ; error bars = S.E.M.; significant difference was assessed by Tukey’s multiple comparisons test; n=3 biological replicates shown by symbols; F-value(Interaction)=58.22, DF(Interaction)=2l; F-value(Gene)=56.82, DF(Gene)=7; F-value(Cell Type)=544.4, DF(Cell Type)=3. G. A glomerular expression score was determined for each sample by calculating the average log expression across the top 100 upregulated genes identified from a human kidney glomerulus-enriched gene expression dataset. OrgGlom samples were found to have the highest scores for this gene set, showing greatest congruence to human glomerular isolates.

Figure 3. Isolated organoid glomeruli synthesise mature components of the human glomerular basement membrane. A. Serial chemical fractionation of organoid glomeruli (OrgGlom) and organoid proximal tubules (OrgPT) to derive fractions enriched for cellular and extracellular matrix (ECM) components, where Cl and C2 are predominantly cellular proteins, C3 nuclear proteins, and C4 enriched for ECM proteins. Representative blot showing one of the three biological replicates for each cell type. B. Principal component analysis of mass spectrometry data from Cl and C4 fractions of both isolated glomeruli and tubules. C. Mapping of organoid proteomic data onto the human matrisome database and the identification of 60 ECM proteins*. D. The expression profile of matrix proteins detected in OrgGloms and OrgPTs in the ECM enriched fraction (C4) and cellular fraction (Cl). Gene ontology (GO) classification** was used to group those detected into core matrix proteins (i), or proteoglycans and matrix-associated proteins (ii). Error bars = SEM, n=3 biological replicates. E. Immuno staining of isolated OrgGloms shows basal expression of the GBM protein Laminin-a5. Expression levels of single channels shown in greyscale to preserve maximum contrast, merged image shown in colour. Scale bar lOpm. F. A comparison of matrix protein identification in human organoid glomeruli (OrgGlom) versus: human glomerular matrix (GM), immortalised human glomerular endothelial cells (ciGEnC), immortalised human podocytes (ciPod) and GEnC and podocyte co-culture. Red bar denotes presence of this protein in the sample. *Human matrisome resource: MatrisomeDB. **GO terms are based on the MatrisomeDB Gene Ontology divisions and categories.

Figure 4. Temporal analysis of organoid glomeruli shows evidence of maturing cell types. A. Differentiation of MAFB-BFP2 iPSC into kidney organoids was successful with blue fluorescent protein 2 (BFP2) expression observed in live organoids from d7+7 onwards. Scale bar lOOOpm. B. (i) FACS plot showing the BFP2-positive cell population isolated (ii) RT-PCR analysis of BFP2 positive and negative organoid cell fractions showed MAFB expression is only found in BFP2 positive cells. C. Brightfield and BFP2 fluorescent live imaging of organoid glomeruli isolated from d7+l8 MAFB- reporter organoids at the time of plating and after 24hrs culture. Strong BFP2 signal is observed within organoid glomeruli when in suspension (Ohr and 24hr arrow), but does not remain active when the organoid glomeruli are adhered for culture; nor is it expressed in the migrating podocyte population (inset). Scale bar 200pm. D. A principle component analysis of RNA sequencing (RNA-Seq) data was performed on 3 biological replicates for d7+l0, d7+l4 and d7+l9 BFP2-positive cell populations and compared to OrgPod samples. A clear separation between the 2D cultured organoid podocytes and MAFB BFP2-positive cells isolated from whole organoids was observed in dimension 1, in dimension 2 the variable of time was evident in the separation of the triplicates. E. Venn diagram displaying the intersections of each comparison, upregulated genes are shown in red and downregulated genes are shown in blue. This shows the greatest number of statistically significant upregulated genes were identified in the OrgGlom (d7+l9) vs immature podocyte (d7+l0) MAFB-BFP2 population. F. Heatmap showing the top 50 upregulated differentially expressed genes between d7+l9 and d7+l0, with many enriched genes found to transcribe proteins in the human glomerular ECM proteome. Fold change of log-normalised gene expression levels for each of the triplicate samples presented. G. Gene Ontology (GO) enrichment analysis of the top 100 upregulated genes differentially expressed d7+l9 and d7+l0 found enrichment of GO terms associated with extracellular matrix (ECM) maturation, collagen maturation and cell adhesion. The top 10 most statistically significant GO term categories are shown. P value of 0.05 depicted as a dotted line. H. Top 15 most-significantly upregulated genes with time, identified using a human kidney glomerulus -enriched gene expression dataset. Significant upregulation of genes associated with the maturation of additional glomerular cells, including endothelium and mesangial cells alongside specific GBM components and associated proteoglycans. Two way ANOVA p<0.000l; error bars = S.E.M.; significant difference between time points assessed by Tukey’s multiple comparisons test; n=3 biological replicates shown by symbols; F-value(Interaction)=27. l6, DF(Interaction)=28; F-value(Gene)=37.58, DF(Gene)=l4; F-value(Time)=495.7, DF(Time)=2.

Figure 5. Analysis of organoid-derived podocytes and glomeruli. A. MAFB-BFP2 iPSC reporter cell line were successfully directly differentiated into kidney organoids Immuno staining of fixed organoids showed appropriate nephron segmentation with lotus tetragonolobus lectin (FTF) marking proximal tubule and E-CADHERIN marking maturing proximal tubule, distal tubule and collecting duct. MAFB-BFP2 signal was found to overlay with NEPHRIN in the glomeruli confirming the specificity of the MAFB-tagged line. Scale bar 200pm. B. Live imaging of MAFB-BFP2 reporter expression over time in sieved OrgGloms shows robust gene expression when in suspension up to 96 hours post-isolation. C. Immuno staining of isolated OrgGloms shows low level expression of the endothelial marker PLATELET ENDOTHELIAL CELL ADHESION MOLECULE ( PECAM1 ) and mesangial marker PLATELET- DERIVED GROWTH FACTOR RECEPTOR BETA {PDGFRfi). Expression levels of single channels shown in greyscale to preserve maximum contrast, merged images shown in colour. Scale bars lOpm.

Figure 6. Organoid Glomeruli accurately model congenital nephropathy syndrome in vitro. A. Description of the NPHS1 variants identified in the patient diagnosed with congenital nephrotic syndrome (CNS). B-D. Immunostaining of OrgGloms isolated from control organoids and Congenital Nephrotic Syndrome (CNS) patient organoids show reduced NEPHRIN and PODOCIN protein levels in the organoids derived from patient-iPSC, representative images shown. E. Higher power immunofluorescent images show the co-localisation of NEPHRIN with NEPH1 and PODOCIN in control OrgGloms, this is lost in CNS OrgGloms. F. Semi-quantitative analysis of fluorescence intensities from independent OrgGlom biological replicates shows significant reduction in NEPHRIN and PODOCIN protein levels. One way ANOVA /; <0.0001 ; Error bars=S.E.M.; significant difference assessed by Sidak’s multiple comparisons test; F- value=29.6; DF=l. Biological replicates; Controls: NEPHRIN n=29, PODOCIN h=10, CD2AP h=10, NEPH11 n=8; CNS: NEPHRIN n=30, PODOCIN h=10, CD2AP n=8, NEPH1 n=9. Figure 7. Cultured organoid glomeruli can be utilised for toxicity screening. A. MAFB- BFP2 OrgGloms can be cultured in isolation in a 96 well format. Live imaging of organoid glomeruli at 48hr post-treatment with doxorubicin showed a dose-dependent decrease of BFP2 -reporter intensity. B. Fixed organoid glomeruli immunolabelled with Caspase-3 showed activation of this pro-apoptotic pathway following doxorubicin treatment. Scale bar lOOpm. C. Semi-quantitative analysis of BFP2 -reporter intensity (blue) alongside OrgGlom diameter (red) showed a dose-dependent reduction in both glomerular diameter and BFP2-reporter intensity. One way ANOVA /;< 0.0001 ; Error bars=S.E.M.; significant difference between sequential doses assessed by Tukey’s multiple comparisons test. BFP2 -reporter intensity: F-value=80.3; DF=5; biological replicates n=l6 per dose. OrgGlom Diameter: F-value=60.94; DF=5; biological replicates n=l6 per dose.

DETAILED DESCRIPTION OF THE INVENTION

Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., molecular biology, cell culture, stem cell differentiation, drug screening, disease modelling, biochemistry and physiology).

Unless otherwise indicated, the molecular and statistical techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et ah, Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present), Michos Odysse (editor) Kidney Development: Methods and Protocols (Springer), Robert Lanza (editor) Handbook of Stem Cells, Volume 1, Embryonic Stem Cells (Elsevier).

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

As used herein, the term“about”, unless stated to the contrary, refers to +/- 10%, more preferably +/- 5%, more preferably +/- 1%, of the designated value.

The term“and/or”, e.g.,“X and/or Y” shall be understood to mean either“X and Y” or“X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

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

The term“suspension culture” is used to describe the culture of organoid glomeruli disclosed herein. Previous attempts to culture glomeruli have generally relied on adherent cultures that facilitate outward proliferation of podocytes from glomeruli over time (see for e.g., Saleem, M.A., et al. (2002) J Am Soc Nephrol. 13:630-8). This outward proliferation of podocytes can destroy the glomeruli structure making it difficult to sustain glomeruli in culture long term. The present inventors have surprisingly found that they can maintain structural integrity of organoid glomeruli disclosed herein by culturing them in a suspension culture which inhibits adherence of glomeruli to cell culture vessels. Accordingly, terms such as“suspension culture” and“suspended in culture” are used to refer to culture of organoid glomeruli disclosed herein, wherein the organoid glomeruli are suspended in a liquid culture medium. For the avoidance of doubt, organoid glomeruli in suspension culture according to the present disclosure are not attached to a surface of cell culture vessels. Functionally, suspension culture does not allow outward proliferation of podocytes from the 3D organoid glomeruli structure. This can be assessed visually or via immunostaining for various podocyte markers. Various methods of suspension culture suitable for organoid glomeruli culture are known in the art. For example, suspension culture may comprise culture using low-adhesion plates such as those that are commercially available from suppliers such as Corning (e.g. ultra- low-attachment cell culture plate (Corning, 3473) and plates described in US9,790,465). Such plates may employ various coatings to prevent attachment of organoid glomeruli including, for example, polymeric coatings such as non-ionic hydrogels and agarose. However, the coating is not particularly limited so long as it can sustain organoid glomeruli in suspension culture and inhibit attachment. In another example organoid glomeruli can be agitated while suspended in culture. For example, organoid glomeruli can be agitated in cell culture vessels using for example, an orbital shaker. Various examples of suitable speeds for agitating organoid glomeruli in suspension cell culture are discussed below in the context of swirler culture.

The culture vessels used for suspension culture of organoid glomeruli are also not particularly limited so long as they inhibit attachment of organoid glomeruli in culture. Various plates (e.g. 96-well) and flasks (T25) are known in the art and commercially available. In another example, the culture vessel is a bioreactor such as a stirred tank or rotating bioreactor. Such devices are designed for providing a 3D homogenous culture environment, maintaining organoid glomeruli in suspension and for enabling monitoring and control of culture parameters (e.g. temperature, pH, dissolved oxygen, nutrients/metabolites).

In an example, organoid glomeruli are suspended in culture for at least 24 hours. In another example, organoid glomeruli are suspended in culture for at least 48 hours. In another example, organoid glomeruli are suspended in culture for at least 72 hours. For example, organoid glomeruli can be maintained in suspension culture for between 2 to 4 days. In another example, organoid glomeruli are suspended in culture for at least 5 days. In another example, organoid glomeruli are suspended in culture for at least 6 days. In another example, organoid glomeruli are suspended in culture for at least 7 days. In another example, organoid glomeruli are suspended in culture for at least 8 days. In another example, organoid glomeruli are suspended in culture for at least 9 days. In another example, organoid glomeruli are suspended in culture for 10 days or longer. In another example, organoid glomeruli are suspended in culture for 24 to 48 hours. In another example, organoid glomeruli are suspended in culture for 48 to 72 hours. In another example, organoid glomeruli are suspended in culture for 48 hours to 5 days. In another example, organoid glomeruli are suspended in culture for 3 to 5 days. In another example, organoid glomeruli are suspended in culture for 3 to 7 days. In another example, organoid glomeruli are suspended in culture for 3 to 10 days.

Suspension cell culture may also be used to produce kidney organoids disclosed herein. In this context, the term“suspension cell culture” is used to refer to cell culture in which single cells or small aggregates of cells multiply while suspended in liquid medium. For example, the single cells or small aggregates of cells multiply in suspension culture and form kidney organoids.

The term “media” or“medium” as used in reference to suspension culture includes the components of the environment surrounding the cells. It is envisaged that the medium contributes to and/or provides the conditions sufficient for organoid glomeruli and cells disclosed herein. In an example, the feeder cell lines may be used to release additional supplements into the media as required. For example, feeder cells may be required during culture of stem cells. An example of a feeder cell type is fibroblast. For example, mouse embryonic fibroblasts (MEF) can be used as a feeder cell line. An example of culture media and conditions suitable for growing stem cells is also provided in Example 1.

Medium may be solid, liquid, gaseous or a mixture of phases and materials. Medium can include liquid growth medium as well as liquid medium that do not sustain cell growth. Exemplary gaseous medium include the gaseous phase that cells are exposed to.

The culture medium used in the method of the present disclosure can be prepared by using a culture medium for culturing of cells and organoid glomeruli disclosed herein as a basal culture medium. The basal culture medium includes, for example, Eagles minimal essential (MEM) culture medium and is not particularly restricted providing it can be used for culturing of cells and organoid glomeruli disclosed herein. Further, the culture medium of the present disclosure can contain any components such as fatty acids or lipids, vitamins, growth factors, cytokines, antioxidants, buffering agents, inorganic salts and the like. The cell culture medium used in the present disclosure contains all essential amino acids and may also contain non-essential amino acids. In general, amino acids are classified into essential amino acids (Thr, Met, Val, Leu, Ile, Phe, Trp, Lys, His) and non-essential amino acids (Gly, Ala, Ser, Cys, Gln, Asn, Asp, Tyr, Arg, Pro). In an example, the culture medium comprises murine embryonic fibroblast (MEF) conditioned hES medium. In other examples, the basal culture medium includes for example APEL, APEL2 or mTESR-E6 or E6 chemically defined medium (StemCell Technologies). Basal culture media may also be supplemented with protein free hybridoma media (PFHM) (e.g. 3.5%). In an example, basal media is supplemented with a serum replacement. For example, basal culture media can be supplemented with knockout serum replacement (Thermo Fisher).

In an example, the basal media for maintaining organoid glomeruli in suspension culture is RPMI 1640 medium. In this example, basal medium may be supplemented with serum such as 10% fetal calf serum (FCS) or a suitable serum replacement. Exemplary media and conditions for maintaining organoid glomeruli in suspension culture are also described in Saleem et al. (2002) J Am Soc Nephrol 13:630-8.

Organoid glomeruli

The present inventors surprisingly identified that organoid glomeruli can be isolated from kidney organoids and maintained in suspension culture for a sufficient duration to be suitable for use in various applications. Suspension culture according to the present disclosure creates an environment in which organoid glomeruli are permitted to grow as an intact three dimensional structure interacting with their surrounding media from all sides without any adherence to a surface. Thus, suspension culture may be generally more representative of the in-vivo environment when compared to 2- dimensional (2D) adherent culture systems. 2D cultures can also suffer from loss of tissue-specific architecture, mechanical and biochemical cues, and cell-to-cell and cell- to-matrix interactions thus making them relatively poor models for screening. Accordingly, in various examples, suspension culture according to the present disclosure is not subject to the same limitations of 2D culture. Suspension culture of organoid glomeruli may also be seen as advantageous as adherent culture generally results in the outward proliferation of podocytes which form a monolayer of morphologically compromised cells (Saleem, M.A., et al. (2002) J Am Soc Nephrol. 13:630-8).

Accordingly, in one example, the present disclosure encompasses an organoid glomerulus suspended in culture, wherein the glomerulus is isolated from a kidney organoid derived from pluripotent stem cells. The term“organoid glomerulus” (plural, “organoid glomeruli”) is used in the context of the present disclosure to refer to glomeruli that are isolated from kidney organoids disclosed herein. The organoid glomeruli have a 3-dimensional (3D) structure. As noted below, organoid glomeruli generally present as podocyte clusters in kidney organoids that are resistant to enzymatic dissociation using trypsin or other suitable trypsin-like protease such as the commercially available TrypLE (Gibco). Those of skill in the art will appreciate that organoid glomeruli isolated from kidney organoids are artificial products and, while they share a number of physiological and biochemical hallmarks of mammalian glomeruli, do not occur naturally. In an example, organoid glomeruli disclosed herein are not connected to intact vasculature. For example, organoid glomeruli disclosed herein have no or low numbers of endothelial cells. In an example, organoid glomeruli have no or low levels of CD31, CD34. For example, organoid glomeruli comprise less than 10% of cells that are positive for CD31 or CD34 as determined by immunohistochemistry. In an example, organoid glomeruli comprise less than 5% of cells that are positive for CD31 or CD34 as determined by immunohistochemistry. In another example, organoid glomeruli comprise less than 3% of cells that are positive for CD31 or CD34 as determined by immunohistochemistry. In another example, organoid glomeruli comprise less than 2% of cells that are positive for CD31 or CD34 as determined by immunohistochemistry. In another example, organoid glomeruli comprise less than 1% of cells that are positive for CD31 or CD34 as determined by immunohistochemistry. In another example, organoid glomeruli comprise less than 0.01% of cells that are positive for CD31 or CD34 as determined by immunohistochemistry. In another example, organoid glomeruli comprise less than 0.001% of cells that are positive for CD31 or CD34 as determined by immunohistochemistry.

In another example, the basement membrane of organoid glomeruli disclosed herein has an expression profile which differs from naturally occurring glomeruli. The inventors have surprisingly found that in the organoid glomeruli disclosed herein there is an increasingly more complex glomerular basement membrane (GBM) than that of other models made in vitro. The organoid glomerulus comprises elements of a mature basement membrane. The trimer of collagen IV deposited into the immature GBM is made up of three strands: two strands of COL4A2 and a single strand of COL4A1. When the GBM becomes suitably mature this is replaced by another trimer, this time composed of COL4A3, COL4A4 and COL4A5. Accordingly, in an example the organoid glomeruli are COL4A3+, COL4A4+ and COL4A5+. A second change which has been observed to occur is with respect to laminin isoforms, wherein the profile changes from a trimer composed of LAMA1 and LAMB 1, to a trimer made up of LAMA5, LAMB 2 and LAMC1. Accordingly, in another example, the organoid glomeruli are LAMA5+, LAMB2+ and LAMC1+. In a further example, organoid glomeruli can be COL4A3+, COL4A4+ and COL4A5+, wherein the expression level of COL4A4 and COL4A5 is higher than the expression level of COL4A3. In another example, organoid glomeruli can be LAMA5+, LAMB2+ and LAMC1+. In another example, organoid glomeruli can be LAMA5+, LAMB2+ and LAMC1+, wherein the expression level of LAMA5, LAMB2 and LAMC1 is higher than the expression level of LAMA1 and LAMB 1. In another example, organoid glomeruli can be COL4A3+, COL4A4+, COL4A5+, LAMA5+, LAMB2+ and LAMC1+, wherein the expression level of COL4A4 and COL4A5 is higher than the expression level of COL4A3 and the expression level of LAMA5, LAMB2 and LAMC1 is higher than the expression level of LAMA1 and LAMB 1.

Generally, the species identity of organoid glomeruli encompassed by the present disclosure, whether it is mammalian, such as mouse, human or otherwise is dictated by the cells used to generate the organoid from which it was isolated. In one example, the present disclosure encompasses mammalian glomeruli. In this example, mammalian pluripotent stem cells are used to generate the kidney organoid from which the mammalian glomeruli are isolated. The mammalian glomeruli may be representative of glomeruli from a companion animal such as a canine or feline, or a livestock animal such as an equine or a bovine. Thus, in these examples, stem cells from canines, felines etc. are used to generate the kidney organoid from which the glomeruli are isolated. In another example, the mammalian glomeruli are representative of glomeruli from a mouse or rat. In another example, the mammalian glomeruli are representative of glomeruli from higher order primates such as cynomolgus monkey or rhesus monkey. In another example, the mammalian glomeruli are representative of glomeruli from humans. Where pluripotent stem cells from a particular species are used to generate a kidney organoid, the resulting isolated glomerulus may be identified based on that species. For example, when using human stem cells to generate a kidney organoid, the resulting organoid glomerulus can be identified as a human organoid glomerulus. Thus, in an example, organoid glomeruli encompassed by the present disclosure include human organoid glomeruli isolated from kidney organoids derived from human stem cells. Various other examples of stem cells that are suitable for generating kidney organoids are discussed below.

Organoid glomeruli disclosed herein can be“isolated” from kidney organoids using various methods. In an example, kidney organoids are digested using a protease and organoid glomeruli are filtered from the resulting cell suspension. For example, organoid glomeruli can be filtered by passing the cell suspension through a mesh screen such as a sieve. A detailed overview of an exemplary method for isolating organoid glomeruli is discussed below in Example 4. In another example, kidney organoids are digested using a protease and organoid glomeruli are retrieved from the resulting cell suspension using a microscope and manual selection. Exemplary proteases include trypsin or other suitable trypsin-like enzymes such as the commercially available TrypLE (Gibco).

Accordingly, in an example, the present disclosure encompasses an organoid glomerulus suspended in culture, wherein the glomerulus is isolated from a kidney organoid and wherein the glomerulus is isolated by digesting the kidney organoid using a protease to form a cell suspension and filtering the cell suspension through a mesh screen. In this example, the protease can be trypsin. In an example, the mesh screen can be a sieve. In an example, the cell suspension is filtered using one or more sieves having pores of a diameter less than 50pm sieve. In an example, the cell suspension is filtered using a 40pm sieve followed by a 30pm sieve.

Organoid glomeruli of the present disclosure may be characterised by their diameter. In an example, organoid glomeruli of the present disclosure can have a diameter greater than 50 pm. For example, organoid glomeruli of the present disclosure can have a diameter of at least 60 pm, 80 pm, 100 pm, 120 pm, 140 pm, 160 pm, 180 pm. In other examples, organoid glomeruli can have a diameter of between 50 pm and 160 pm, 60 pm and 140 pm, 70 pm and 120 pm. In another example, organoid glomeruli can have a diameter of between 90 mih and 110 mih. In an example, organoid glomeruli can have a width consistent with the above exemplified diameters when determined using Feret’s Diameter. In other examples, organoid glomeruli can be characterised by the width across the widest point of their three-dimensional structure.

In another example, organoid glomeruli can be characterised based on expression of molecular markers. Marker expression can be characterised using various techniques such as immunohistochemistry or fluorescent activated cell sorting. Immunohistochemistry generally involves using a primary antibody specific for the marker of interest. Binding of the primary antibody to a marker can be visualised via various known methods. For example, a labelled secondary antibody that recognises the primary antibody can be used. In this example, the label could be an enzyme such as horse radish peroxidase, a radioactive isotope, a fluorescent reporter, an electro- chemiluminescent tag. Binding of the labelled secondary antibody to the primary antibody can be detected via cytological assessment or via an automated plate reader.

In a particular example, an organoid glomeruli or section or sample thereof is contacted with a specific primary antibody. The organoid glomeruli or section or sample thereof is then washed to remove any unbound primary antibody and then a secondary antibody specific for the primary antibody and linked to a peroxidase enzyme is applied to the sample. The organoid glomeruli or section or sample thereof is then washed to remove any unbound secondary antibody and 3,3'-Diaminobenzidine (DAB) is applied to the sample. The conversion of DAB into a coloured product is visualised by routine cytological assessment with the presence of a coloured product indicating that the marker is present in the sample. In an example, the level of coloured product may be quantified using Image J or various other software packages that are commercially available from suppliers such as Perkin Elmer and Leica.

In another example, a cell suspension is produced from an organoid glomeruli or section or sample thereof. Cells in suspension are contacted with a fluorescently labelled antibody that is specific for a particular maker. Cells positive for a particular marker are identified using techniques such as fluorescent activated cells sorting (FACS).

A cell that is referred to as being "positive" for a given marker may express either a low (lo or dim) or a high (bright, bri) level of that marker depending on the degree to which the marker is present on the cell surface, where the terms relate to intensity of fluorescence or other marker used in the sorting process of the cells. The distinction of lo (or dim or dull) and bri will be understood in the context of the marker used on a particular cell population being sorted. A cell that is referred to as being "negative" for a given marker is not necessarily completely absent from that cell. This term means that the marker is expressed at a relatively low or very low level by that cell or population, and that it generates a very low signal when detectably labelled or is undetectable above background levels, e.g., levels detected using an isotype control antibody.

In an example, markers of organoid glomeruli and organoids described herein can be detected using a fluorescent reporter gene. For example, expression of particular markers can be monitored to track development of organoid glomeruli, kidney organoids or cells comprising the same in real time. For example, stem cells can be genetically engineered to express one or more fluorescent or chemiluminescent reporter(s) under a given set of conditions. Reporters can be used to track cell identity, cell viability or cell function in real time. An example of a suitable reporter gene is exemplified below where a knock-in iPSC line is generated that harbours the mTagBFP2 fluorescent reporter gene inserted at the start codon of the endogenous MAFB locus (MAFB^mTagBFP2/+). MAFB is highly expressed in developing podocytes and therefore, expression of MAFB can be monitored to track development of podocytes in organoid glomeruli and kidney organoids in real time. Other examples of reporter cell lines suitable for use in the methods disclosed herein include fluorescent reporters inserted into promoters for genes expressed in podocytes, including WT1, NPHS 1 or NPHS2 or the promoters of genes expressed in endothelial cells, including PECAM, VE-CADHERIN, KDR or ANGPT1. In another example, cells used to generate organoid glomeruli are engineered to express one or more reporter genes in association with one or more genes which are expressed predominantly in more mature glomeruli. In one example, the organoid glomeruli prepared according to methods described herein and used in the screening methods and screening systems herein described are engineered to express a reporter in association with one or more of the following genes: KIRREL, CD2AP, SYNPO, PODXL, WT1, MAFB, LMX1B, TCF21, NPHS 1, and NPHS2. In another example, the organoid glomeruli prepared according to methods described herein and used in the screening methods and screening systems herein described are engineered to express a reporter in association with one or more of the following genes: KIRREL, CD2AP, SYNPO, PODXL, WT1, LMX1B, TCF21, NPHS 1, and NPHS2. In another example, the organoid glomeruli prepared according to methods described herein and used in the screening methods and screening systems herein described are engineered to express a reporter in association with SYNPO and/or NPHS 1.

As outlined above, reporters can be used to monitor cell viability. Accordingly, in one example, the organoid glomeruli prepared according to methods described herein and used in the screening methods and screening systems herein described are engineered to express a reporter in association with one or more apoptosis-related genes. In one example, the organoid glomeruli prepared according to methods described herein and used in the screening methods and screening systems herein described are engineered to express a reporter in association with one or more of the following genes: ASM1, BAD, BAK1, BAX, BCL2, BCL10, BclXL, BclXS, BIK, BINCARD, BIRC8, CARD 8, CASP1, CASP2, CASP3, CASP4, CASP5, CASP6, CASP7, CASP8, CASP9, CASP10, CASP12, CFLAR, CRADD, DIABLO, EMAP2 (AIMP1), FADD, FASL, GAX (MEOX2), HBXIP, HRD1 (SYVN1), LCN2, LTBR, MAPT, MFN2, MLKL, NAIP1, NAIP5, NFIL3, Noxa (PMAIP1), OPTN, p35, PCNA, PDCD4, PDCD8, PIDD, PTPN6, PUMA, RFC4, SARP2, SERPINB9, Survivin, TGFB 1, TGFB2, TNFAIP8, TNFRSF10A, TNFRSF10B, TNFRSF10C, TNFRSF10D, TNFRSF11B, TRADD, TRAIL (TNFSF10), XIAP, CST3, HAVCR1, CLU, TFF3, B2M, AMBP, and MIF. In another example, the organoid glomeruli prepared according to methods described herein and used in the screening methods and screening systems herein described are engineered to express a reporter in association with CASP3.

In an example, organoid glomeruli according to the present disclosure can express one or more or all of SYNAPTOPODIN, PODOCALYXIN, PODOCIN, NEPHRIN, NEPH1, PEC AM 1 and PDGFR ?. For example, organoid glomeruli can comprise podocytes having apicobasal polarity as determined by immunohistochemistry for PODOCALYXIN, PODOCIN, NEPHRIN and NEPHL For example, organoid glomeruli can be characterised by immunohistochemistry which shows PODOCALYXIN localising to the apical surface of cells with NEPHRIN and NEPH1 localising to the intracellular junction between adjacent podocytes. In another example, organoid glomeruli express SYNAPTOPODIN. For example, organoid glomeruli can express SYNAPTOPODIN at a higher level than 2D cultured podocytes. In an example, the podocytes are isolated from an organoid glomeruli described herein and cultured in 2D before SYNAPTOPODIN expression levels are compared with organoid glomeruli. In these examples, expression of SYNAPTOPODIN can be determined using immunofluorescence.

In an example, organoid glomeruli are PECAM1+ and PDGFR /+ indicating the presence of endothelial and mesangial cells. In another example, organoid glomeruli disclosed herein are positive for one or two or all of the following markers that provide evidence of a maturing glomerular basement membrane, including COL4A3, COL4A4, COL4A5 and LAMB2. In another example, organoid glomeruli disclosed herein are positive for COL4A3, COL4A4 and COL4A5. In another example, organoid glomeruli disclosed herein are positive for LAMA5, LAMB2 and LAMC1. Thus in an example, organoid glomeruli according to the present disclosure can be COL4A3+. In another example, organoid glomeruli according to the present disclosure can be COL4A5+. In another example, organoid glomeruli according to the present disclosure can be LAMB2+. For example, organoid glomeruli according to the present disclosure can be COL4A3, COL4A5 and LAMB2+. In another example, organoid glomeruli according to the present disclosure can be LAMA5+. For example, organoid glomeruli according to the present disclosure can be COL4A3+, COL4A5+, LAMB2+ and LAMB5+. For example, organoid glomeruli according to the present disclosure can be COL4A3+, COL4A5+, LAMB2+, LAMA5+ and LAMC1+.

In an example, the organoid glomeruli according to the present disclosure are positive for one or more of the following markers: COL4A3, COL4A4, COL4A5, LAMB 2, LAMA5, LAMC1, KDR, MMP2, CXCL12, ITGA2, TEK, FN1, ANGPTL2, IGFBP3, SHIS A3, MME, IGFBP5, EMCN, UCHL1, TAGLN, CDH5, GJA5, SMAD7, CX3CL1, FGF2, GAT A3, DES, WT1, FOXD1, and C1QTNF12. In another example, the organoid glomeruli according to the present disclosure express high levels of the foregoing markers. In the above examples, reference to high (or low) levels of expression are relative to expression levels determined in a control sample, wherein 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 an 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. In another example, the control sample represents kidney organoids cultured via the method described in Takasato et al. (2015) Nature, Vol. 526:564-568, or organoid glomeruli isolated from kidney organoids at a different stage of development such as one or more of those discussed below (e.g. d7+l0). In an example, the control sample is an organoid glomerulus isolated from a d7+l0 kidney organoid. In an example, the control sample is an organoid glomerulus isolated from a d7+l2 kidney organoid. In another example, the control sample is an organoid glomerulus isolated from isolated from a d7+l4 kidney organoid. In another example, the control sample is an organoid glomerulus isolated from kidney organoids at d7+l5. In another example, the control sample is an organoid glomerulus isolated from a d7+l7 kidney organoid. In an example, the organoid glomeruli express one or more of COL4A3, COL4A4, COL4A5, LAMB2, LAMA5, LAMC1, KDR, MMP2, CXCL12, ITGA2, TEK, FN1, ANGPTL2, IGFBP3, SHIS A3, MME, IGFBP5, EMCN, UCHL1, TAGLN, CDH5, GJA5, SMAD7, CX3CL1, FGF2, GAT A3, DES, WT1, FOXD1, and C1QTNF12 at a level which is at least 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 11 fold, 12 fold, 13 fold, 14 fold, 15 fold, 16 fold, 17 fold, 18 fold, 19 fold, 20 fold, 21 fold, 22 fold, 23 fold, or 24 fold higher than that observed for a control sample.

In an example, organoid glomeruli can be characterised based on one or more of the above referenced markers after use in a method of screening discussed below. In another example, an organoid glomerulus representative of a broader population can be characterised based on one or more of the above referenced markers before the remaining glomeruli from the population are used in a method of screening discussed below. For example, a population of glomeruli can be isolated from a kidney organoid using methods discussed above. Expression of one or more of the above markers can be confirmed in organoid glomeruli from the population before the remaining organoid glomeruli isolated from the kidney organoid are used in a method of screening discussed below.

Stem cells

Aspects of the present disclosure encompass cultures of stem cells. For example, organoid glomeruli disclosed herein can be produced from pluripotent stem cells. For example, a kidney organoid can be produced from pluripotent stem cells and organoid glomeruli can be isolated therefrom.

The term "stem cell" is used in the context of the present disclosure to refers to a subset of progenitors that have the capacity or potential, under particular circumstances, to differentiate to a more specialized or differentiated phenotype, and which retain the capacity, under certain circumstances, to proliferate without substantially differentiating. In one example, the term stem cell refers generally to a naturally occurring mother cell whose descendants (progeny) specialize, often in different directions, by differentiation, e.g., by acquiring completely individual characters, as occurs in progressive diversification of embryonic cells and tissues. Cellular differentiation is a complex process typically occurring through many cell divisions. A differentiated cell may derive from a multipotent cell which itself is derived from a multipotent cell, and so on. While each of these multipotent cells may be considered stem cells, the range of cell types each can give rise to may vary considerably. Some differentiated cells also have the capacity to give rise to cells of greater developmental potential. Such capacity may be natural or may be induced artificially upon treatment with various factors. In many biological instances, stem cells are also "multipotent" because they can produce progeny of more than one distinct cell type, but this is not required for "stem-ness." Self-renewal is the other classical part of the stem cell definition. In theory, self-renewal can occur by either of two major mechanisms. Stem cells may divide asymmetrically, with one daughter retaining the stem state and the other daughter expressing some distinct other specific function and phenotype. Alternatively, some of the stem cells in a population can divide symmetrically into two stems, thus maintaining some stem cells in the population as a whole, while other cells in the population give rise to differentiated progeny only.

In an example, the stem cells are human stem cells. In an example, the stem cells are a population of culture expanded human stem cells. In an example, stem cells can be culture expanded in-vitro or ex-vivo. In an example, culture expanded stem cells have been passaged at least once, twice, three, four, five, six, seven, eight, nine, 10 times.

In an example, the stem cells are human pluripotent stem cells. Generally, pluripotent stem cells show expression of OCT4, NANOG and SSEA1 when in a pluripotent state and expression of these markers is generally lost with differentiation. In another example, stem cells are human embryonic stem cells. The terms "human embryonic stem cell" and abbreviations thereof such as "hES” and "hESC” refer to cells derived, obtainable or originating from human embryos or blastocysts, which are self- renewing and pluri- or toti-potent, having the ability to yield all of the cell types present in a mature animal. Human embryonic stem cells (hESCs) can be isolated, for example, from human blastocysts obtained from human in vivo preimplantation embryos, in vitro fertilized embryos, or one-cell human embryos expanded to the blastocyst stage.

In another example, the stem cells are induced pluripotent stem cells. For example, the stem cells can be human induced pluripotent stem cells. The term "induced pluripotent stem cell" and abbreviation thereof "iPSC” refer to cells derivable, obtainable or originating from human adult somatic cells of any type reprogrammed to a pluripotent state through the expression of exogenous genes, such as transcription factors, including a preferred combination of OCT4, SOX2, KLF4 and c-MYC. Human iPSC show levels of pluripotency equivalent to hESC but can be derived from a patient for autologous therapy with or without concurrent gene correction prior to differentiation and cell delivery. Suitable processes for generation of induced pluripotent stem cells are described, for example, in US 7,615,374 and US 2014273211, Barberi et al; Plos medicine, Vol 2(6):0554-0559 (2005), and Vodyanik et al. Cell Stem cell, Vol 7:718- 728 (2010). In an example, iPSC are derived from fibroblasts. In another example, iPSC are derived from blood. For example, iPSC can be derived from white blood cells. In another example, iPSC are derived from fibroblasts. In another example, iPSC are derived from white blood cells or fibroblasts.

In an example, it may be desirable to produce an organoid glomeruli that is representative of a particular subject and/or disease. Various examples of this embodiment are described below. Relevant to this section is the iPS cells that may be used to produce the organoid glomeruli. In an example, the human iPS cells are derived from a human subject with a genetic kidney disease. In this example, a blood sample may be isolated from the subject with a genetic kidney disease and iPS cells may be induced from cells in the blood sample (e.g. white blood cells). The subject may have one of various exemplary genetic kidney diseases. Examples include congenital nephrotic syndrome (CNS) including steroid resistant nephrotic syndrome and Finnish nephropathy, focal segmental glomerulonephritis (FSGS), Alport syndrome and Pierson syndrome. Accordingly, in an example, the present disclosure encompasses organoid glomeruli that are representative of a kidney disease 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. Accordingly, in an example, the organoid glomeruli are representative of CNS. In another example, the organoid glomeruli are representative of steroid resistant nephrotic syndrome. In these examples, iPS cells can be used to produce a kidney organoid described herein and organoid glomeruli are isolated therefrom.

In an example, organoid glomeruli isolated from stem cell (e.g. iPS cell) derived kidney organoids can be used to model glomeruli in the developing kidney and/or in kidney disease.

Accordingly, in an example, the present disclosure encompasses an organoid glomerulus disclosed herein suspended in culture, wherein the glomerulus is used for modelling glomeruli development. In another example, the present disclosure encompasses an organoid glomerulus suspended in culture, wherein the glomerulus is isolated from a kidney organoid that is representative of kidney disease. In an example, the kidney disease is CNS or another of the above referenced diseases. In this example, disease can be modelled by inducing iPS cells from subjects with an above referenced kidney disease and culturing organoid glomeruli isolated from renal organoids produced therefrom. In this example, gene editing can be employed (e.g. CRISPR/Cas9 gene editing) to introduce mutations into genes of the subject derived iPS cells that are relevant or potentially relevant to kidney disease development. In other examples, gene editing is employed to correct mutations in the subject derived iPS cells. In an example, isogenic gene edited iPS cells can be generated (e.g. Forbes et al. (2018) Am J Hum Genet. 102:816-831). Organoid glomeruli development and disease can be modelled over time (e.g. 2, 5, 10 or more days) using organoid glomeruli isolated from kidney organoids at various developmental stages such as one or more of those discussed below (e.g. d7+l4 or 15). In these examples, organoid glomeruli may be cultured in groups with each group being representative of a different developmental stage (e.g. d7+l l, d7+l5, d7+l8, d7+20) and/or being cultured for a defined period of time (e.g. 2, 5, and 10 days). Organoid glomeruli can be assessed using for example, visual assessment, immunohistochemistry, gene and protein expression analysis to determine developmental or disease stage. In an example, organoid glomeruli can also be contacted with a nephrotoxin, candidate compound (including a therapeutic compound) during these studies and nephrotoxicity and/or therapeutic efficacy can be determined. As noted above, organoid glomeruli used in the above examples can be generated from iPS cells that have been genetically modified to express a reporter gene.

Kidney Organoid

Organoid glomeruli defined herein are isolated from kidney organoids and suspended in culture. The term“kidney organoid” is used in the context of the present disclosure to refer to a heterogeneous 3D agglomeration of cells that recapitulates aspects of cellular self-organization, architecture and signalling interactions present in the native kidney. Kidney organoids disclosed herein are derived from stem cells such as induced pluripotent stem cells or a stem cell derived intermediate such as intermediate mesoderm cells. Examples of kidney organoids are described in Takasato et al. (2015) Nature, Vol. 526:564-568, Takasato et al. (2016) Nat Protocols, 11: 1681-1692, Takasato et al. (2014) Nat. Cell Biol., 16: 118-127, WO 2014/197934 and WO 2016/094948.

In an example, the kidney organoid is derived from stem cells. Various examples of stem cells suitable for deriving kidney organoids described herein are discussed above. For example, the kidney organoid can be derived from a culture expanded population of human stem cells. Accordingly, in an example, the kidney organoid can be derived from human iPS cells.

In another example, the kidney organoid is derived from a culture expanded population of intermediate mesoderm (IM) cells. Non- limiting examples of markers characteristic or representative of intermediate mesoderm cells include PAX2, OSR1 and/or LHX1. Accordingly, in an example, the IM cells are one or more of PAX2+, LHX1+, OSR1+. For example, the IM cells can be PAX2+. In another example, the IM cells are FHX1+. In another example, the IM cells are OSR1+.

In another example, the kidney organoid is derived from a culture expanded population of IM cells that are characterised by the method used for culture expansion and/or production.

Accordingly, in an example, the method of producing IM cells comprises, culturing a population of stem cells for around 3 to 4 days in a cell culture medium comprising a Wnt/p-catcnin agonist followed culturing the cells for around 3 to 4 days in a cell culture medium comprising FGF such as FGF9. In this example, the cells can be cultured 7 days in total, after which the IM cells are dissociated. The term "Wnt/b- catenin agonist" is used in the context of the present disclosure to refer to a molecule that inhibits GSK3 (e.g GSIG-b) in the context of the canonical Wnt signalling pathway, but preferably not in the context of other non-canonical, Wnt signalling pathways. Examples of Wnt b-catenin agonists include CHIR99021 (CHIR), LiCl SB-216763, CAS 853220- 52-7 and other Wnt^-catenin agonists that are commercially available from sources such as Santa Cruz Biotechnology and R & D Systems.

In an example, cells are cultured in cell culture media comprising between 4 and 8 mM of a Wnt^-catenin agonist before they are cultured in cell culture media comprising FGF. In another example, cells are cultured in cell culture media comprising 4 mM of a Wnt^-catenin agonist before they are cultured in cell culture media comprising FGF. In another example, cells are cultured in cell culture media comprising 5 pM of a Wnt/b- catenin agonist before they are cultured in cell culture media comprising FGF. In another example, cells are cultured in cell culture media comprising 6 pM of a Wnt^-catenin agonist before they are cultured in cell culture media comprising FGF. In another example, cells are cultured in cell culture media comprising 7 pM of a Wnt^-catenin agonist before they are cultured in cell culture media comprising FGF. In another example, cells are cultured in cell culture media comprising 8 pM of a Wnt^-catenin agonist before they are cultured in cell culture media comprising FGF. In these examples the Wnt^-catenin agonist can be CHIR. For example, cells can be cultured in cell culture media comprising 8 pM of CHIR before they are cultured in cell culture media comprising FGF.

In an example, the IM cell culture medium comprises at least 50 ng/ml FGF9. In another example, the cell culture medium comprises at least 100 ng/ml FGF9. In another example, the cell culture medium comprises at least 150 ng/ml FGF9. In another example, the cell culture medium comprises at least 200 ng/ml FGF9. In another example, the cell culture medium comprises at least 300 ng/ml FGF9. In another example, the cell culture medium comprises at least 350 ng/ml FGF9. In another example, the cell culture medium comprises at least 400 ng/ml FGF9. In another example, the cell culture medium comprises at least 500 ng/ml FGF9. In another example, the cell culture medium comprises between 50 ng and 400 ng/ml FGF9. In another example, the cell culture medium comprises between 50 ng and 300 ng/ml FGF9. In another example, the cell culture medium comprises between 50 ng and 250 ng/ml FGF9. In another example, the cell culture medium comprises between 100 ng and 200 ng/ml FGF9. In another example, an above referenced level of FGF9 is substituted for FGF2. For example, the IM cell culture medium can comprise between 50 ng and 400 ng/ml FGF2. In another example, the cell culture medium comprises between 50 ng and 300 ng/ml FGF2. In another example, the cell culture medium comprises between 50 ng and 250 ng/ml FGF2. In another example, the cell culture medium comprises between 100 ng/ml and 200 ng/ml FGF2.

In another example, an above referenced level of FGF9 is substituted for FGF20. For example, the IM cell culture medium can comprise between 50 ng and 400 ng/ml FGF20. In another example, the cell culture medium comprises between 50 ng and 300 ng/ml FGF20. In another example, the cell culture medium comprises between 50 ng and 250 ng/ml FGF20. In another example, the cell culture medium comprises between 100 ng/ml and 200 ng/ml FGF20.

In an example, the IM cell culture medium which comprises FGF also comprises heparin. In an example, the cell culture medium comprises 0.5 pg/ml heparin. In another example, the cell culture medium comprises 1 pg/ml heparin. In another example, the cell culture medium comprises 1.5 pg/ml heparin. In another example, the cell culture medium comprises 2 pg/ml heparin. In another example, the cell culture medium comprises between 0.5 pg/ml and 2 pg/ml heparin. In another example, the cell culture medium comprises between 0.5 pg and 1.5 pg/ml heparin. In another example, the cell culture medium comprises between 0.8 pg/ml and 1.2 pg/ml heparin.

In another example, the method of producing IM cells comprises culturing a population of stem cells in a high concentration of Wnt/p-catenin agonist such as CHIR followed by culturing the cells in a low concentration of Wnt/p-catenin agonist and a FGF such as FGF9. In this example, a“high concentration” of CHIR is at least 4 pM and a“low concentration” of CHIR is less than 2 pM. In another example, a“high concentration” of CHIR is at least 6 pM and a“low concentration” of CHIR is less than 1 pM. In another example, a“high concentration” of CHIR is at least 7 pM and a“low concentration” of CHIR is 0.5 pM or less. In these examples, cells can be cultured in culture medium comprising high concentration CHIR for at least three to four days before being cultured in culture medium comprising low concentration CHIR and FGF for at least two to three days. In another example, cells can be cultured in culture medium comprising high concentration CHIR for four days before being cultured in culture medium comprising low concentration CHIR and FGF for three days. In another example, stem cells are cultured for at least seven days, wherein cells are cultured in culture medium comprising high concentration CHIR for the first four days before being cultured in culture medium comprising low concentration CHIR and FGF. In these examples, the culture medium comprising low concentration CHIR can further comprise FGF9. For example, the culture medium can comprise lOOng/ml FGF9. In another example, the culture medium can comprise 200ng/ml FGF9.

In an example, the IM cells are produced by culturing stem cells for 7 days, wherein days 3 to 5 involve culturing stem cells in cell culture medium comprising an above referenced high concentration of CHIR and the remaining days involve culturing cells in cell culture medium comprising an above referenced concentration of an FGF. For example, the IM cells can be produced by culturing stem cells for 7 days, wherein days 3 to 5 involve culturing stem cells in cell culture medium comprising at least 4 mM CHIR and the remaining days involve culturing cells in cell culture medium comprising at least lOOng/ml FGF9.

In another example, IM cells can be produced by culturing stem cells for 8 days. In another example, IM cells can be produced by culturing stem cells for 9 days. In another example, IM cells can be produced by culturing stem cells for 10 days. In each of these examples, days 3 to 5 can involve culturing stem cells in cell culture medium comprising at least 4 pM CHIR, wherein cells are cultured in cell culture medium comprising FGF9 for the remaining days. For example, days 3 to 5 can involve culturing stem cells in cell culture medium comprising between 4 pM and 8 pM CHIR, wherein cells are cultured in cell culture medium comprising FGF9 for the remaining days.

In other examples, IM cells used to produce kidney organoids can be cultured in culture mediums comprising different or additional components. Exemplary components and timing for their use in cell culture is discussed below.

In an example, the cell culture medium can comprise a Rho kinase inhibitor (ROCKi) such as Y-27632 (StemCell Technologies). In this example, stem cells are cultured in a cell culture medium comprising ROCKi for 24 hours before being cultured in a cell culture medium comprising at least 4 pM CHIR for around 3 to 4 days. In this example, cells can subsequently be cultured in a cell culture medium comprising FGF for a further 3 to 4 days. In an example, the cell culture medium can comprise 8 pM ROCKi. In another example, the cell culture medium can comprise 10 pM ROCKi. In another example, the cell culture medium can comprise 12 pM ROCKi. In another example, the cell culture medium can comprise between 8 pM and 12 pM ROCKi.

In an above example, after culturing with ROCKi for 24 hours and at least 4 pM CHIR for around 3 to 4 days, the cells can be cultured in a culture medium which comprises FGF9 and one or more or all of a Wnt/p-catcnin agonist such as CHIR at a low concentration (e.g. less than 3 pM), an above referenced concentration of Heparin, poly(vinyl alcohol) (PVA) and methyl cellulose (MC). In this example, the IM cell culture medium can comprise at least 0.05% PVA. In another example, the cell culture medium comprises 0.1% PVA. In another example, the cell culture medium comprises 0.15% PVA. In another example, the cell culture medium comprises between 0.1% and 0.15% PVA. In an example, the cell culture medium can comprise at least 0.05% MC. In another example, the cell culture medium comprises 0.1% MC. In another example, the cell culture medium comprises 0.15% MC. In another example, the cell culture medium comprises between 0.1% and 0.15% MC.

In an example, the kidney organoids are derived by producing IM cells using an above referenced method, dissociating the IM cells and then further culturing the IM cells in a method of producing a kidney organoid discussed below. For example, IM cells can be produced using an above exemplified method, dissociated and then re aggregated to form kidney organoids. In examples, re-aggregation can be performed in culture on a floating filter. For example, IM cells can be produced using an above exemplified method, dissociated and then cultured for at least 14 days on Transwell™ filters. In this example, dissociated cells can be centrifuged to form a pellet. Pellets can then be transferred to a suitable medium for further culture such as Transwell™ filters. In an example, pellets are briefly contacted with a cell culture medium comprising CHIR before being cultured further. For example, pellets can be contacted with a cell culture medium comprising 4 to 6 mM CHIR for one to two hours before being cultured further. In another example, pellets can be contacted with a cell culture medium comprising 5 mM CHIR for one hour before being cultured further.

In another example, re-aggregation can be performed using swirler culture. For example, IM cells can be produced using an above exemplified method, dissociated and then cultured in swirler culture to produce organoids. The terms“swirler”,“swirled” and “swirl” are used interchangeably in the context of the present disclosure to refer to the movement of cell culture medium in a twisting or spiralling pattern. In one example, cell culture medium is swirled by applying sufficient agitation in a circular motion to a cell culture. For example, cell cultures can be swirled using an orbital shaker. In an example, the cell culture is swirled at least at 40 rpm. In another example, the cell culture is swirled at least at 50 rpm. In another example, the cell culture is swirled at least at 60 rpm. In another example, the cell culture is swirled at least at 70 rpm. In another example, the cell culture is swirled at least at 80 rpm. In another example, the cell culture is swirled at between 40 and 80 rpm. In another example, the cell culture is swirled at between 50 and 70 rpm. In another example, the cell culture is swirled at between 55 and 65 rpm.

In an example, cells can be dissociated using EDTA. In another example, cells can be dissociated using trypsin or TrypLE. In an example, dissociated cells are passed through a mesh screen before being cultured further. In an example, cells are cultured for at least 12 days after dissociation. In another example, cells are cultured for at least 13 days after dissociation. In another example, cells are cultured for at least 14 days after dissociation. In another example, cells are cultured for at least 15 days after dissociation. In another example, cells are cultured for at least 20 days after dissociation. In another example, cells are cultured for at least 25 days after dissociation. In another example, cells are cultured for at least 35 days after dissociation.

In an example, IM cells are dissociated after 7 days in culture (d7) and then re aggregated to produce kidney organoids. In an example, cells are re-aggregated by culturing in a cell culture medium comprising FGF. For example, cells are cultured in a cell culture medium comprising an above referenced level of FGF9, FGF2 or FGF20 after dissociation. In an example, cells are cultured in a cell culture medium comprising lOOng/ml FGF9 after dissociation. In another example, cells are cultured in a cell culture medium comprising 200ng/ml FGF9 after dissociation. In these examples, the cell culture medium can also comprise heparin. For example, the cell culture medium can comprise FGF9 and 1 pg/ml heparin after dissociation. In these examples, cells can be cultured in cell culture medium comprising FGF and heparin for 4 to 6 days after dissociation. In an example, cells can be cultured in cell culture medium comprising FGF and heparin for 5 days after dissociation.

In an example, FGF is removed from the cell culture medium 4 to 6 days after dissociation. In another example, FGF is removed from the cell culture medium 5 days after dissociation. In an example, no growth factors are provided in the culture medium 5 days after dissociation.

In an example, the cell culture medium used after dissociation can also comprise retinoic acid. In an example, all trans retinoic acid (atRA) is added to cell culture medium after dissociation. In an example, at least 0.07 mM retinoic acid is added to the cell culture medium. In an example, at least 0.1 pM retinoic acid is added to the cell culture medium. In an example, at least 0.2 pM retinoic acid is added to the cell culture medium. In an example, at least 0.5 pM retinoic acid is added to the cell culture medium.

In another example, at least 1.5 pM retinoic acid is added to the cell culture medium. In an example, at least 1.8 pM retinoic acid is added to the cell culture medium. In an example, at least 2.0 pM retinoic acid is added to the cell culture medium. In another example, at least 2.5 pM retinoic acid is added to the cell culture medium. In another example, between 1.5 pM and 3 pM retinoic acid is added to the cell culture medium. In another example, between 2.0 pM and 3 pM retinoic acid is added to the cell culture medium. In an example, retinoic acid is added to the cell culture medium 4 days after dissociation. In another example, retinoic acid is added to the cell culture medium 5 days after dissociation. In another example, retinoic acid is added to the cell culture medium 4 to 6 days after dissociation.

In an example, at least 5xl0⁵ IM cells are re-aggregated to from kidney organoids. In another example, at least 4xl0⁵ IM cells are re-aggregated to from kidney organoids.

In another example, at least 3xl0⁵ IM cells are re-aggregated to from kidney organoids.

In another example, at least lxlO⁵ IM cells are re-aggregated to from kidney organoids.

In another example, at least 5xl0⁴ IM cells are re-aggregated to from kidney organoids.

In another example, at least 4xl0⁴ IM cells are re-aggregated to from kidney organoids.

In another example, at least 3xl0⁴ IM cells are re-aggregated to from kidney organoids.

In another example, at least 2xl0⁴ IM cells are re-aggregated to from kidney organoids.

In another example, at least lxlO⁴ IM cells are re-aggregated to from kidney organoids.

In another example, between 5xl0³ and 5 xlO⁶ IM cells are re-aggregated to from kidney organoids. In another example, between 5xl0⁴ and 5 xlO⁶ IM cells are re-aggregated to from kidney organoids.

In an example, 1,000 to 10,000 IM cells are re-aggregated to from a kidney organoid. In another example, 4,000 to 500,000 IM cells are re-aggregated to from a kidney organoid.

Kidney organoids encompassed by the present disclosure can be described based on number of days in culture. The days in culture can be separated into two components including days for production of IM cells from stem cells (X) and days for formation of kidney organoid from IM cells (Y). In an example, the step distinguishing production of IM cells from stem cells and production of kidney organoid from IM cells is the dissociation of IM cells. One way of representing the days in culture for production of IM cells from stem cells and days for formation of kidney organoid from IM cells is day (d) X+Y (e.g. d7+l2 would describe 7 days of producing IM cells from stem cells followed by dissociation of IM cells and 12 days of organoid formation from IM cells (i.e. Y = number of days as an organoid).

In an example, organoid glomeruli are isolated from a d7+l2 kidney organoid. In another example, organoid glomeruli are isolated from a d7+l4 kidney organoid. In another example, organoid glomeruli are isolated from kidney organoids at d7+l5 or later. In another example, organoid glomeruli can be isolated from a d7+l7 kidney organoid. In another example, organoid glomeruli can be isolated from a d7+l8 kidney organoid. In another example, organoid glomeruli can be isolated from a d7+l9 kidney organoid. In another example, organoid glomeruli can be isolated from a d7+20 kidney organoid. In another example, organoid glomeruli can be isolated from a d7+2l kidney organoid. In another example, organoid glomeruli can be isolated from a d7+22 kidney organoid. In another example, organoid glomeruli can be isolated from a d7+23 kidney organoid. In another example, organoid glomeruli can be isolated from a d7+24 kidney organoid. In another example, organoid glomeruli can be isolated from a d7+25 kidney organoid.

In another example, organoid glomeruli are isolated from a d7+20 kidney organoid. In another example, organoid glomeruli are isolated from a d7+22 kidney organoid. In another example, organoid glomeruli are isolated from a d7+25 kidney organoid. In another example, organoid glomeruli are isolated from a d7+30 kidney organoid. In another example, organoid glomeruli are isolated from kidney organoids between d7+l3 and d7+30. In another example, organoid glomeruli are isolated from kidney organoids between d7+l4 and d7+30. In another example, organoid glomeruli are isolated from kidney organoids between d7+l5 and d7+30. In another example, organoid glomeruli are isolated from kidney organoids between d7+l5 and d7+25. In another example, organoid glomeruli are isolated from kidney organoids between d7+l5 and d7+20. In another example, organoid glomeruli are isolated from kidney organoids between d7+l5 and d7+l8. In another example, organoid glomeruli are isolated from kidney organoids between d7+l4 and d7+l9. In another example, organoid glomeruli are isolated from kidney organoids between d7+l8 and d7+30. In another example, organoid glomeruli are isolated from kidney organoids between d7+l9 and d7+30. In another example, organoid glomeruli are isolated from kidney organoids between d7+l8 and d7+25. In another example, organoid glomeruli are isolated from kidney organoids between d7+l9 and d7+25. In the above referenced examples IM cells may be cultured for 8, 9 or 10 days (i.e. d8+Y, d9+Y or dlO+Y).

Kidney organoids disclosed herein such as those produced using one or more of the above referenced methods can be characterised based on various structural features.

In an example, kidney organoids disclosed herein comprise 60 to 150 organoid glomeruli. In another example, kidney organoids disclosed herein comprise 80 to 120 organoid glomeruli. In another example, kidney organoids disclosed herein comprise 90 to 110 organoid glomeruli. In another example, kidney organoids disclosed herein comprise around 100 organoid glomeruli.

In other examples, such as when kidney organoids are produced using swirler culture, kidney organoids can comprise 4 to 10 organoid glomeruli. In these examples, it may be desirable to produce large numbers of kidney organoids before isolating organoid glomeruli. The numbers of organoid glomeruli that can be isolated from a single kidney organoid, and the numbers of kidney organoids that can be generated from a single differentiation make the organoid glomeruli particularly suitable for applications such as screening.

In an example, organoid glomeruli described herein are isolated from bio-printed kidney organoids. Terms such as“bioprinted” or“bioprinting” are used in the context of the present disclosure to refer to a process utilizing three-dimensional, precise deposition of cells (e.g., cell solutions, cell-containing gels, cell suspensions, cell concentrations, multicellular aggregates, multicellular bodies, bio-ink etc.) via methodology that is compatible with an automated, computer-aided, three-dimensional prototyping device (e.g. a bio-printer). Examples of methods suitable for bio-printing are disclosed in WO 2012/054195 and WO 2013/040087. In an example, bio-printing is performed using an organ printing machine which uses a hydrogel scaffold to place human cells in a desired orientation to produce kidney organoids disclosed herein (e.g. Organovo/Invetech) .

In an example, the kidney organoids described herein comprise architectural hallmarks of a native kidney with reduced numbers of nephrons. In an example, a kidney organoid encompassed by the present disclosure can comprise one or more nephrons. In an example, nephron(s) segment into distal and proximal tubules, early loops of Henle, and glomeruli. In another example, organoids comprise segmented nephrons surrounded by endothelial cells, perivascular cells and kidney interstitium. In another example, organoids of the present disclosure do not show the presence of vasculature.

In other examples, organoids according to the present disclosure are at least partially vascularised. For example, organoids can comprise nephrons containing podocytes elaborating foot processes and undergoing vascularisation.

In an example, kidney organoids are characterised in terms of % nephron, % stroma and/or % vasculature. In this example, kidney organoids can be characterised using single cell RNA sequencing. An example of single cell sequencing is provided below. In an example, kidney organoids comprise at least 20% mature nephron. In another example, kidney organoids comprise at least 25% mature nephron. In another example, kidney organoids comprise at least 30% mature nephron. In another example, kidney organoids comprise at least 31% mature nephron. In another example, kidney organoids comprise at least 32% mature nephron. In these examples, the kidney organoids also comprise at least 15% stroma. In another example, the kidney organoids also comprise at least 20% stroma. In another example, the kidney organoids also comprise at least 25% stroma. In another example, the kidney organoids do not comprise any substantial vasculature. In another example, the kidney organoids do not comprise vasculature.

In an example, kidney organoids according to the present disclosure comprise less than 100 nephrons. In another example, kidney organoids according to the present disclosure comprise less than 90, less than 80, less than 70, less than 60 nephrons. In another example, kidney organoids according to the present disclosure comprise less than 50 nephrons. In another example, kidney organoids according to the present disclosure comprise less than 40, less than 30, less than 20, less than 10 nephrons. In another example, kidney organoids according to the present disclosure comprise less than 5 nephrons. In another example, kidney organoids according to the present disclosure comprise less than 4, less than 3 nephrons.

In another example, kidney organoids according to the present disclosure comprise between 2 and 100 nephrons. In another example, kidney organoids according to the present disclosure comprise between 2 and 50 nephrons. In another example, kidney organoids according to the present disclosure comprise between 2 and 10 nephrons. In another example, kidney organoids according to the present disclosure comprise between 2 and 6 nephrons. In another example, kidney organoids according to the present disclosure comprise between 2 and 4 nephrons.

“Nephrons” are the functional working units of kidney which play a major role in removal of waste products and maintenance of body fluid volume. They can be identified and counted in organoids disclosed herein by those of skill in the art using various methods. For example, nephrons can be visualized and counted using confocal microscopy and immunofluorescence labelling (e.g. WT1+ glomerulus; NPHS+ podocytes, LTL+ECAD- proximal tubule, ECAD+ distal tubule and ECAD+GATA3+ collecting duct).

In another example, kidney organoids comprise cells expressing high levels of one or more of NPHS 1, PAX2, CDH1 and GATA3. For example, kidney organoids can express high levels of NPHS 1. In another example, kidney organoids can express high levels of PAX2. In another example, kidney organoids can express high levels of CDH1. In another example, kidney organoids can express high levels of GATA3.

In another example, kidney organoids comprise cells expressing low levels of WT1. In another example, kidney organoids comprise cells expressing low levels of C- RET. In another example, kidney organoids comprise cells expressing low levels of FOXD1. In another example, kidney organoids comprise cells expressing low levels of WT1 and C-RET. In another example, kidney organoids comprise cells expressing low levels of WT1, C-RET and FOXD1. In another example, kidney organoids comprise cells expressing high levels of NPHS 1, PAX2, CDH1 and GAT A3 and low levels of WT1, C-RET and FOXD1.

In the above examples, high and low levels of expression are relative to kidney organoids cultured via the method described in Takasato et al. (2015) Nature, Vol. 526:564-568, Takasato et al. (2016) Nat Protocols, 11: 1681-1692, or Takasato et al. (2014) Nat. Cell Biol., 16: 118-127. 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 an 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.

Expression levels can be measured using techniques such as polymerase chain reaction comprising appropriate primers for markers of interest. For example, total RNA can be extracted from cells before being reverse transcribed and subject to PCR and analysis.

In an example, kidney organoids comprise nephron(s) comprising one or more of WT1+ glomerulus, NPHS+ podocytes, LTL+ECAD- proximal tubule, ECAD+ distal tubule and ECAD+GATA3+ collecting duct. In another example, kidney organoids comprise nephron(s) comprising NPHS+ podocytes, LTL+ proximal segments, ECAD+ distal segments and ECAD+GATA3+ collecting duct. Kidney organoids comprising above exemplified components can be identified in various ways. In one example, kidney organoids can be fixed and whole mounted before being visually assessed using confocal microscopy and immunofluorescence labelling.

In an example, kidney organoids can be characterised based on one or more of the above referenced markers after glomeruli have been isolated. In another example, a kidney organoid(s) representative of a broader population can be characterised based on one or more of the above referenced markers before glomeruli are isolated from the remaining kidney organoids in the population. For example, a population of kidney organoids can be produced using methods discussed above. Expression of one or more of the above markers can be confirmed in a kidney organoid(s) from the population before organoid glomeruli are isolated from the remaining kidney organoids.

In an example, kidney organoids according to the present disclosure are representative of kidney disease. In an example, the kidney disease is a genetic kidney disease insofar as it is characterised by a genetic mutation(s).

Examples of kidney disease include congenital nephrotic syndrome (CNS) including steroid resistant nephrotic syndrome and Finnish nephropathy, focal segmental glomerulonephritis (FSGS), Alport syndrome and Pierson syndrome. Accordingly, in an example, the present disclosure encompasses organoid glomeruli that are representative of an above reference kidney disease. For example, organoid glomeruli encompassed by the present disclosure can be representative of a kidney disease 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. For example, the organoid glomeruli can be representative of congenital nephrotic syndrome (CNS).

Screening Methods

Organoid glomeruli encompassed by the present disclosure can be used in various screening applications. In an example, organoid glomeruli can be used to screen for toxicity. For example, organoid glomeruli can be used to screen for nephrotoxicity.

Accordingly, in an example, the present disclosure encompasses a method of screening a candidate compound for nephrotoxicity, the method comprising contacting an organoid glomerulus disclosed herein with a candidate compound and determining whether or not the candidate compound is nephrotoxic.

In an example an organoid glomerulus described herein is contacted with a candidate compound before being assessed for nephrotoxic side effects. 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 kidney cell function in vitro , including analysis of biomarker expression using commercially available tools including, for example, the Human Nephrotoxicity RT² Profiler™ PCR Array from Qiagen or the High Content Analysis (HCA) Multiplexed Nephrotoxicity Assay from Eurofins. In another example, nephrotoxicity is assessed by measuring acute apoptosis of glomerular cells in organoid glomeruli disclosed herein following contact with a candidate compound. In other examples, nephrotoxicity can be assessed using electron microscopy such as transmission EM or scanning EM. Other examples of criteria indicative of nephrotoxicity include loss of podocyte marker gene expression or protein expression and loss of foot processes (loss of effacement). In an example, determining whether or not the candidate compound is nephrotoxic comprises measuring one or more of: diameter of the organoid glomerulus; expression of one or more genes associated with cell death; expression of one or more genes associated with cell viability; expression of one or more nephron-associated genes; expression of one or more genes associated with glomerular extracellular matrix; expression of one or more genes associated with podocyte, endothelial or mesangial cell types; and intensity of expression of a reporter gene associated with at least one gene of interest. In an example, determining whether or not the candidate compound is nephrotoxic comprises measuring the expression of one or more genes selected from the group consisting of: KIRREL, CD2AP, SYNPO, PODXL, WT1, MAFB, LMX1B, TCF21, NPHS 1, and NPHS2. In an example, determining whether or not the candidate compound is nephrotoxic comprises measuring the expression of one or more genes selected from the group consisting of: COF4A3, COF4A4, COF4A5, FAMB2, FAMA5, FAMC1, KDR, MMP2, CXCF12, ITGA2, TEK, FN1, ANGPTF2, IGFBP3, SHIS A3, MME, IGFBP5, EMCN, UCHF1, TAGEN, CDH5, GJA5, SMAD7, CX3CL1, FGF2, GATA3, DES, FOXD1, and C1QTNF12. In an example, determining whether or not the candidate compound is nephrotoxic comprises measuring the expression of one or more or all of SYNAPTOPODIN, PODOCALYXIN, PODOCIN, NEPHRIN, NEPH1, PECAM and PDGFR ?. In an example, any one of the aforementioned genes is a so-called “gene of interest” associated with a reporter gene. In an example, a measured reduction in one or more of the foregoing markers following contacting the organoid glomerulus is indicative of nephrotoxicity of the candidate compound. In an example, following contacting the organoid glomerulus with the candidate compound, i) a measured reduction in one or more of: diameter of the organoid glomerulus; expression of one or more genes associated with cell viability; expression of one or more nephron-associated genes; expression of one or more genes associated with glomerular extracellular matrix; expression of one or more genes associated with podocyte, endothelial or mesangial cell types; and intensity of a reporter gene; and/or ii) a measured increase in expression of one or more genes associated with cell death; is indicative of nephrotoxicity of the candidate compound.

In another example, determining whether or not the candidate compound is nephrotoxic comprises measuring the expression of one or more apoptosis-related genes selected from the group consisting of: ASM1, BAD, BAK1, BAX, BCL2, BCL10, BclXL, BclXS, BIK, BINCARD, BIRC8, CARD 8, CASP1, CASP2, CASP3, CASP4, CASP5, CASP6, CASP7, CASP8, CASP9, CASP10, CASP12, CFLAR, CRADD, DIABLO, EMAP2 (AIMP1), FADD, FASL, GAX (MEOX2), HBXIP, HRD1 (SYVN1), LCN2, LTBR, MAPT, MFN2, MLKL, NAIP1, NAIP5, NFIL3, Noxa (PMAIP1), OPTN, p35, PCNA, PDCD4, PDCD8, PIDD, PTPN6, PUMA, RFC4, SARP2, SERPINB9, Survivin, TGFB 1, TGFB2, TNFAIP8, TNFRSF10A, TNFRSF10B, TNFRSF10C, TNFRSF10D, TNFRSF11B, TRADD, TRAIL (TNFSF10), and XIAP. In another example, determining whether or not the candidate compound is nephrotoxic comprises measuring the expression of CASP3. In another example, a measured increase in expression of one or more of the aforementioned apoptosis associated genes is indicative of nephrotoxicity of the candidate compound. In an example, any one of the aforementioned genes is a so-called“gene of interest” associated with a reporter gene.

In another example, determining whether or not the candidate compound is nephrotoxic comprises measuring the expression of the expression of any one or more of: CST3, HAVCR1, CLU, TFF3, B2M, AMBP, and MIF. In another example, a measured increase in the expression of any one or more of: CST3, HAVCR1, CLU, TFF3, B2M, AMBP, and MIF is indicative of nephrotoxicity. In an example, any one of the aforementioned genes is a so-called“gene of interest” associated with a reporter gene.

For studies involving contacting organoid glomeruli with a candidate compound, nephrotoxicity may be determined based on a comparison with an organoid glomerulus that is not contacted candidate compound, or on a comparison of a measurement of the same glomerulus before and after contacting the glomerulus with the candidate compound.

In another example, the present disclosure encompasses a method of screening a candidate compound for therapeutic efficacy in treating kidney disease, the method comprising contacting an organoid glomerulus disclosed herein with a candidate compound under conditions to determine whether or not the candidate compound is therapeutically effective. In this example, the method may comprise contacting an organoid glomerulus disclosed herein with a candidate compound in the presence of a nephrotoxic compound and determining whether or not the candidate compound is therapeutically effective.

In other examples of screening for therapeutic efficacy, the organoid glomeruli may be isolated from a kidney organoid that is representative of a kidney disease. For example, the organoid glomeruli may be isolated from a kidney organoid that is derived from cells obtained from a patient with kidney disease. For example, the kidney disease can be selected from the group consisting of congenital nephrotic syndrome (CNS) including steroid resistant nephrotic syndrome and Finnish nephropathy, focal segmental glomerulonephritis (FSGS), Alport syndrome and Pierson syndrome. In an example, the kidney disease is CNS.

The term“therapeutic efficacy” is used in the context of the present disclosure to refer to a response in which any toxic or detrimental effects of a candidate compound or composition comprising the same is outweighed by the therapeutically beneficial effects. Therapeutic efficacy can be determined based on improved kidney cell function; maintained kidney cell function; inhibition (i.e., slowing to some extent and, in some examples, stopping) decline in kidney cell function; inhibiting (i.e., slowing to some extent and, in some examples, stopping) kidney cell death. In an example, therapeutic efficacy is determined based on the presence of appropriate podocyte proteins and evidence that they are appropriately polarised. An example includes localisation of NPHS 1, NPHS2 and NEPH-l at the membrane of podocytes, wherein NPHS1, NPHS2 and NEPH-l is determined using immunohistochemistry.

For studies involving organoid glomeruli isolated from a kidney organoid that is representative of a kidney disease, nephrotoxicity and therapeutic efficacy can be determined relative to a pre-determined standard ascertained based on corresponding kidney cell function in a disease-free organoid glomerulus. In another example, improved kidney cell function may be determined based on a comparison of kidney cell function between an organoid glomerulus isolated from a kidney organoid representative of kidney disease and an organoid glomerulus isolated from a kidney organoid representative of healthy glomeruli. In an example, determining whether or not the candidate compound is therapeutically effective comprises measuring one or more of: diameter of the organoid glomerulus; expression of one or more genes associated with cell death; expression of one or more genes associated with cell viability; expression of one or more nephron-associated genes; expression of one or more genes associated with glomerular extracellular matrix; expression of one or more genes associated with podocyte, endothelial or mesangial cell types; and intensity of expression of a reporter gene associated with at least one gene of interest. In an example determining whether or not the candidate compound is therapeutically effective comprises measuring one or more genes selected from the group consisting of: KIRREL, CD2AP, SYNPO, PODXL, WT1, MAFB, LMX1B, TCF21, NPHS 1, and NPHS2. In an example determining whether or not the candidate compound is therapeutically effective comprises measuring one or more genes selected from the group consisting of: COL4A3, COL4A4, COL4A5 LAMB 2, LAMA5, LAMC1, KDR, MMP2, CXCL12, ITGA2, TEK, FN1, ANGPTL2, IGFBP3, SHIS A3, MME, IGFBP5, EMCN, UCHL1, TAGLN, CDH5, GJA5, SMAD7, CX3CL1, FGF2, GAT A3, DES, WT1, FOXD1, and C1QTNF12. In an example, the method comprises measuring the expression of one or more or all of SYNAPTOPODIN, PODOCALYXIN, PODOCIN, NEPHRIN, NEPH1, PECAM and PDGFR//. In another example, a measured increase, or the absence of a decrease in expression of one or more of the aforementioned genes is indicative of therapeutic efficacy of the candidate compound. In an example, i) a measured increase or absence of a measured reduction in one or more of: diameter of the organoid glomerulus; expression of one or more genes associated with cell viability; expression of one or more nephron-associated genes; expression of one or more genes associated with glomerular extracellular matrix; expression of one or more genes associated with podocyte, endothelial or mesangial cell types; and intensity of expression of a reporter gene associated with a gene of interest; and/or ii) a measured reduction in expression of one or more genes associated with cell death; is indicative of therapeutic efficacy of the candidate compound. In an example, any one of the aforementioned genes is a so-called“gene of interest” associated with a reporter gene.

In another example, determining whether or not the candidate compound is therapeutically effective comprises measuring the expression of one or more apoptosis- related genes selected from the group consisting of: ASM1, BAD, BAK1, BAX, BCL2, BCL10, BclXL, BclXS, BIK, BINCARD, BIRC8, CARD8, CASP1, CASP2, CASP3, CASP4, CASP5, CASP6, CASP7, CASP8, CASP9, CASP10, CASP12, CFLAR, CRADD, DIABLO, EMAP2 (AIMP1), FADD, FASL, GAX (MEOX2), HBXIP, HRD1 (SYVN1), LCN2, LTBR, MAPT, MFN2, MLKL, NAIP1, NAIP5, NFIL3, Noxa (PMAIP1), OPTN, p35, PCNA, PDCD4, PDCD8, PIDD, PTPN6, PUMA, RFC4, SARP2, SERPINB9, Survivin, TGFB 1, TGFB2, TNFAIP8, TNFRSF10A, TNFRSF10B, TNFRSF10C, TNFRSF10D, TNFRSF11B, TRADD, TRAIL (TNFSF10), and XIAP. In another example, determining whether or not the candidate compound is therapeutically effective comprises measuring the expression of CASP3. In another example, a measured reduction, or the absence of an increase in expression of one or more of the aforementioned apoptosis associated genes is indicative of therapeutic efficacy of the candidate compound. In an example, any one of the aforementioned genes is a so-called “gene of interest” associated with a reporter gene.

In another example, determining whether or not the candidate compound is therapeutically effective comprises measuring the expression of the expression of any one or more of: CST3, HAVCR1, CLU, TFF3, B2M, AMBP, and MIF. In another example, a measured decrease in the expression of any one or more of: CST3, HAVCR1, CLU, TFF3, B2M, AMBP, and MIF is indicative of therapeutic efficacy of the candidate compound. In an example, any one of the aforementioned genes is a so-called“gene of interest” associated with a reporter gene.

For studies involving contacting organoid glomeruli with a candidate compound, therapeutic efficacy may be determined based on a comparison with an organoid glomerulus that is not contacted candidate compound, or on a comparison of a measurement of the same glomerulus before and after contacting the glomerulus with the candidate compound.

For studies involving contacting organoid glomeruli with a nephrotoxic compound and a candidate compound, therapeutic efficacy of the candidate compound, such as represented by improved kidney cell function, may be determined based on a comparison with organoid glomeruli that is not contacted with the nephrotoxic compound and/or organoid glomeruli contacted with nephrotoxic compound alone.

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

It is considered that terms such as“contacting”,“exposing” or“applying” are terms that can, in context, be used interchangeably in the present disclosure. The term contacting, requires that the candidate compound(s) be brought into contact with a glomerulus disclosed herein. In an example, the compound can be dissolved in cell culture media if the compound is water soluble or water-immiscible. Otherwise, a suitable substrate may be soaked in the compound and placed over organoid glomeruli in culture. For the screening of volatile candidate compounds, organoid glomeruli disclosed herein can be exposed to air or other gas mixtures comprising the compound(s). Alternatively, organoid glomeruli can be exposed to a solution or suspension of the volatile compound in cell culture media. Again, if possible, volatile compounds can be dissolved or stabilised. Otherwise, a suitable substrate may be soaked in the compound and placed over organoid glomeruli in culture.

In performing the methods of the present disclosure a plurality of candidate compounds can be contacted with organoid glomeruli. 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, 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 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 organoid glomeruli. In an example, candidate compounds can be contacted with the same or separate organoid glomeruli. For example, specific combinations of candidate compounds can be screened.

In an example, candidate compounds are labelled prior to screening. In an example, the candidate compound can be a composition. For example, the candidate compound may be present in a formulation or comprise 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 with kidney disease. In an example, the serum isolated from a subject with CNS. For example, the serum can be isolated from a subject that has steroid resistant nephrotic syndrome. In another example, the serum is isolated from a subject that has had a kidney transplant. In another example, the serum is isolated from a subject with nephrotic syndrome that has presented post kidney transplant.

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

As noted above, organoid glomeruli can be isolated from kidney organoids after varying days in culture. These organoid glomeruli can be used in the screening applications disclosed herein. Thus, as one example, organoid glomeruli isolated from kidney organoids at d7+l5 or later can be used in screening. Thus, as one example, organoid glomeruli isolated from kidney organoids at d7+l8 or later can be used in screening. In another example, organoid glomeruli isolated from kidney organoids between d7+l8 and d7+30 can be used in screening. In another example, organoid glomeruli isolated from kidney organoids between d7+l9 and d7+30 can be used in screening. In another example, organoid glomeruli isolated from kidney organoids between d7+l8 and d7+25 can be used in screening. In another example, organoid glomeruli isolated from kidney organoids between d7+l9 and d7+25 can be used in screening. In another example, organoid glomeruli from immature kidney organoids can be used in screening. For example, organoid glomeruli isolated from kidney organoids between d7+l4 and d7+25 can be used in screening. Again, IM cells may be cultured for longer and thus d8+Y, d9+Y or dlO+Y organoids can be used in screening.

The organoid glomeruli employed in the screening methods, compositions and kits described herein are may be used several days after they have been isolated when maintained in suspension culture. In an example, organoid glomeruli used for screening methods are suspended in culture for at least 24 hours. In another example, organoid glomeruli used for screening methods are suspended in culture for at least 48 hours. In another example, organoid glomeruli used for screening methods are suspended in culture for at least 72 hours. In another example, organoid glomeruli used for screening methods are suspended in culture for at least 96 hours. For example, organoid glomeruli used for screening methods can be maintained in suspension culture for between 2 to 4 days. In another example, organoid glomeruli used for screening methods are suspended in culture for at least 5 days. In another example, organoid glomeruli used for screening methods are suspended in culture for at least 6 days. In another example, organoid glomeruli used for screening methods are suspended in culture for at least 7 days. In another example, organoid glomeruli used for screening methods are suspended in culture for at least 8 days. In another example, organoid glomeruli used for screening methods are suspended in culture for at least 9 days. In another example, organoid glomeruli used for screening methods are suspended in culture for 10 days or longer. In another example, organoid glomeruli used for screening methods are suspended in culture for 24 to 48 hours. In another example, organoid glomeruli used for screening methods are suspended in culture for 48 to 72 hours. In another example, organoid glomeruli used for screening methods are suspended in culture for 48 hours to 5 days. In another example, organoid glomeruli used for screening methods are suspended in culture for 3 to 5 days. In another example, organoid glomeruli used for screening methods are suspended in culture for 3 to 7 days. In another example, organoid glomeruli used for screening methods are suspended in culture for 3 to 10 days. According to the foregoing examples, the candidate compound(s) used in the methods of screening may be brought into contact with the organoid glomeruli for a defined period of time during suspension culture, including after at least 24h of suspension culture. In an example, the candidate compound is brought into contact with the organoid glomeruli after at least 24h of suspension culture.

In an example, the screening method comprises contacting candidate compound(s) with a library of organoid glomeruli. For example, candidate compounds can be screened using organoid glomeruli isolated from kidney organoids at different developmental stages. For example, organoid glomeruli from d7+l0, d7+l5, d7+l9 and d7+25 kidney organoids can be used. In another example, candidate compounds can be screened using organoid glomeruli isolated from kidney organoids representative of different kidney diseases.

As the skilled person would appreciate, there are a wide variety of different screening procedures which could be adapted to screen candidate compounds. For example, organoid glomeruli disclosed herein can be provided in a single or multiwell format and contacted with candidate compounds for a set period of time. In an example, organoid glomeruli are provided in a multi well plate. In an example, one organoid glomerulus is provided per well. In another example, two organoid glomeruli are provided per well. In another example, three organoid glomeruli are provided per well. In another example, four organoid glomeruli are provided per well. In another example, five organoid glomeruli are provided per well. In another example, 10 organoid glomeruli are provided per well. In another example, 20 organoid glomeruli are provided per well. In an example, the organoid glomeruli are provided in a 96 well plate.

High throughput screening methods are encompassed by the present disclosure. In this example, high throughput screening involves providing a library containing a large number of candidate compounds. Such libraries are then screened in one or more assays to identify those library members (e.g. particular chemical species or subclasses) that display a desired level of activity (e.g. therapeutic efficacy).

High throughput screening systems are commercially available and typically automate entire procedures, including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of a culture plate (e.g. 96 well formats) in detectors appropriate for the assay. These configurable systems provide rapid start up as well as a high degree of flexibility and customization. The manufacturers of such systems (e.g. Invitrogen, Thermo Fisher Scientific etc.) provide detailed protocols for use.

In an example, the above referenced methods further comprise selecting a compound which displays therapeutic efficacy. For example, compounds that, in the presence of a nephrotoxin and/or when contacted with organoid glomeruli isolated from kidney organoids representative of kidney disease, maintain kidney cell function; inhibit (i.e., slow to some extent and, in some examples, stop) decline in kidney cell function; inhibit (i.e., slow to some extent and, in some examples, stop) kidney cell death. In another example, the above referenced methods further comprise selecting a compound which reduces nephrotoxicity. For example, compounds that inhibit glomerulonephritis can be selected. In another example, compounds that improve kidney cell function may be selected. In these examples, kidney cell function may be determined based on biomarker expression using commercially available tools including, for example, the Human Nephrotoxicity RT² Profiler™ PCR Array from Qiagen or the High Content.

In another example according to any one of the foregoing screening methods, the method comprises a step of selecting organoid glomeruli to be contacted with a candidate compound and/or nephrotoxin in the screening method on the basis of expression of a gene of interest. In one example, the screening method comprises determining the level of expression of one or more of SYNAPTOPODIN, PODOCALYXIN, PODOCIN, NEPHRIN, NEPH1, PEC AM 1 and PDGFR ? and selecting glomeruli which are positive for expression of one or more of the same. In one example, the screening method comprises determining the level of expression of PEC AM 1 and/or PDGFR ? and selecting glomeruli which are PECAM1+ and/or PDGFR /+. In another example, the screening method comprises determining the level of expression of one or more of COL4A3, COL4A4, COL4A5, LAMB2, LAMA5, LAMC1, KDR, MMP2, CXCL12, ITGA2, TEK, FN1, ANGPTL2, IGFBP3, SHIS A3, MME, IGFBP5, EMCN, UCHF1, TAGEN, CDH5, GJA5, SMAD7, CX3CL1, FGF2, GAT A3, DES, WT1, FOXD1, and C1QTNF12 and selecting glomeruli which are positive for expression of one or more of the same. In another example, the screening method comprises determining the level of expression of one or more of COL4A3, COL4A4, COL4A5, LAMB2, LAMA5, LAMC1, KDR, MMP2, CXCL12, ITGA2, TEK, FN1, ANGPTL2, IGFBP3, SHIS A3, MME, IGFBP5, EMCN, UCHL1, TAGLN, CDH5, GJA5, SMAD7, CX3CL1, FGF2, GATA3, DES, WT1, FOXD1, and C1QTNF12 and selecting glomeruli which express high levels of one or more of the same. In one example, the screening method comprises determining the level of expression of KDR and selecting glomeruli which are KDR+. In another example, the screening method comprises determining the level of expression of KDR and selecting glomeruli which express high levels of KDR.

Personalised medicine and stratification

As outlined above, organoid glomeruli can be isolated from various kidney organoids representative of kidney disease in a subject. A candidate compound showing therapeutic efficacy in organoid glomeruli isolated from kidney organoids representative of kidney disease in a subject may be more likely to display therapeutic efficacy in the subject. Accordingly, in an example, organoid glomeruli isolated from these kidney organoids can be used to select agents that are more likely to affect treatment or prophylaxis of kidney disease in the subject.

In another example, organoid glomeruli can be isolated from kidney organoids representative of a kidney disease in multiple subjects. These organoid glomeruli can be used to select agents that are more likely to affect treatment or prophylaxis of kidney disease in multiple subjects or identify groups of subjects that are more likely to respond to treatment with a particular agent. Such methods may be useful for stratifying subjects in clinical trials of agents being tested for capacity to treat kidney disease. Grouping subject populations based on organoid glomeruli screening may eliminate or reduce variation in treatment outcome due to genetic factors, leading to a more accurate assessment of the efficacy of a potential drug. Accordingly, in an example, the present disclosure encompasses a method for stratifying a group of subjects for a clinical trial of a therapeutic agent, the method comprising obtaining an iPS cell population from a group of subjects generating a kidney organoid from each subjects iPS cell population and isolating organoid glomeruli, contacting the isolated organoid glomeruli with a therapeutic agent and determining whether the therapeutic agent is therapeutically effective using the results of the determination to select subjects more likely to be responsive to the therapy. In this example, the method may comprise contacting organoid glomeruli with a therapeutic agent and a nephrotoxin before determining whether the therapeutic agent is therapeutically effective. Examples of therapeutic agents include candidate compounds discussed above such as, for example, one or more small molecules, polynucleotides, peptides, proteins, antibodies, antibody fragments, viruses, bacteria, stem cells, serum including kidney disease patient derived serum. For the avoidance of doubt, serum can be isolated from a subject with a particular kidney disease and contacted with kidney organoids disclosed herein. Various examples of kidney disease are discussed herein and serum can be isolated from various subjects representative of these diseases. Methods of isolating serum from subjects are known in the art. In an example, serum is purified from a whole blood sample using centrifugation.

Compositions/Kits

In one example, the present disclosure relates to a kit or assay for use in screening applications. For example, the present disclosure encompasses a kit or assay for use in screening candidate compounds for nephrotoxicity and/or therapeutic efficacy. In an example, an organoid glomerulus described herein is provided in suspension culture, candidate compounds can then be contacted with organoid glomeruli and screened for nephrotoxicity and/or therapeutic efficacy. Accordingly, in an example, the present disclosure encompasses an assay when used for screening, the assay comprising an organoid disclosed herein in suspension culture. In an example, the assay is used for nephrotoxicity screening. In an example, the assay is used for therapeutic efficacy screening. In an example, organoid glomeruli are provided with culture media or other components for maintaining organoid glomeruli in culture. In an example, organoid glomeruli are provided with written instructions for performing the methods of the present disclosure. In an example, the assay comprises a glomerulus described herein. In other examples, the assay comprises more than one glomerulus. For example, the assay can comprise 10, 20, 30 or more organoid glomeruli. Glomeruli can be provided in a single or multi-well format such as a 96 well plate. In these examples, glomeruli are provided in suspension culture.

When using an above exemplified assay, glomeruli are contacted with a candidate compound. The effect of the candidate compound on the glomeruli can then be assessed. For example, cell survival and/or viability can be assessed after contacting organoid glomeruli with a candidate compound. Various candidate compounds and methods of assessing effects thereof on organoids disclosed herein are discussed above in relation to screening. EXAMPLES

EXAMPLE 1 - Culturing and maintenance of hPSC

Human ES cells (H9 cells) were grown on mouse embryonic fibroblast (MEF) feeders in a DMEM media supplemented with 10% KOSR (Life technologies) and bFGF to 80% confluency before splitting using TrypLE (Life technologies). Before differentiation, ES cells were adapted to matrigel (Coming) surface in the absence of MEF feeders in a MEF conditional media and bFGF.

Human iPS cells were grown as individual colonies on geltrex (Life technologies) coated plates in an E8 media (Life technologies). Passaging of the iPS cells was performed once with EDTA once the cells reached 60-70% confluency or every 3 days.

Dissociation of hPSC into single cells was achieved using TrypLE and cells were seeded on a matrigel coated plated at 15K cells/cm². Matrigel adapted hES cells were seeded using MEF conditional media. Human iPS cells were seeded as single cells in E8 media using revita cell (1 : 100 dilution) on matrigel coated plate overnight.

EXAMPLE 2 - Differentiation of iPSC to kidney organoid

In short, iPSC were differentiated for 7 days, then separated to single cells and re aggregated to form an organoid. Organoids were analysed at various time points following re-aggregation, described in these examples as d7+’Y’ number of days as an organoid, to a maximum of d7+l8. Further detail on the differentiation method follows.

Human iPS cells were plated on a Matrigel-coated (Millipore) culture dish and cultured in MEF-conditioned hES medium (MEF-CM). Cells were re-plated at 15,000 cells per cm² in MEF-CM. Next day, cells were treated with 8 mM CHIR99021 in APEL basal medium (STEMCELL Technologies) for 2-5 days, followed by FGF9 (200 ng/ml) and heparin (1 mg/ml) (medium changed every second day). At day 7, cells were collected and dissociated into single cells. Cells were spun down and then transferred onto a Transwell 0.4 pm pore polyester membrane (CLS3450 Corning). Pellets were treated with 5 mM CHIR99021 in APEL for 1 h, and then cultured with FGF9 (200 ng/ml) and heparin (1 mg/ml) for 5 days, followed by another 6-13 days in APEL basal medium (medium changed three times a week).

EXAMPLE 3 - Alternative method for differentiation of iPSC to kidney organoid hPSCs or hES were differentiated into intermediate mesoderm (IM) by exposing cells to high concentration of CHIR (7 pM) for the first 4 days in APEL2 (Stem cell technologies) media with 3.5% protein free hybridoma media (PFHM) (Thermo Fischer), APEL (Stem cell technologies) or E6 media (Stem cell technologies). The cultures were subjected to an additional 3 days (days 5 to 7 from seeding) of a low concentration of CHIR (1 mM) in addition to FGF9 and heparin.

On day 7, IM cells were dissociated by washing with 1 ml of EDTA solution, further incubation in 1 ml EDTA for 3 minutes at 37°C and EDTA solution was removed by aspiration without disturbing the IM cell layer. Stage 1 Media (Base media, FGF9 200 ng/ml, Heparin 1 pg/ml, 1 mM CHIR, 0.1% PVA, 0.1% MC) (2ml) was added along with 10 pM Rho kinase inhibitor (ROCKi, 1:1000 dilution, stem cell technologies) and cells were gently detached as a clump using Gilson pipette. The cell suspension was transferred to 6 cm² low adhesion dishes (Greiner bio) and passed through 40 pm cell strainers (BD biosciences).

For organoid formation, Stage 1 Media was toped up to 5 ml and the dishes were swirled in a Ratek orbital shaker at 60 rpm in a standard cell culture incubator at 37°C and 5% C0₂. Stage 1 Media was replaced with Stage 2 Media (base media, FGF9 200 ng/ml, Heparin 1 pg/ml, 1 mM CHIR, 0.1% PVA, 0.1% MC) after 24 hours in swirler culture. The cells were cultured in in Stage 2 Media for another 4 days. From day 7+5 onwards, all the organoids were refreshed with Stage 3 Media (base media, 0.1% PVA, 0.1% MC) every alternative day and cultured until day 7+18.

EXAMPLE 4 -Isolation of organoid glomeruli from kidney organoid

iPSC-derived kidney organoids with an initial starting cell number of 200,000 differentiated iPSC were dissociated together in groups. Organoids were dissociated by incubation with TrypLE select enzyme (Thermo Fisher) for 12 minutes at 37°C. Gentle mixing using a lml pipette was applied every 3 minutes to aid dissociation, resulting in a homogenous cell solution. Per group, a single 70pm cell strainer (Falcon) was placed onto a 50ml tube (Falcon) and the mesh hydrated with phosphate-buffered saline (PBS). The cell solution was added to the strainer in a stepwise manner using a lml pipette, allowing flow through of the solution by gravity. The plunger from a lml sterile syringe (Terumo) was used to gently push the remaining cell solution captured on the strainer through the mesh. The strainer was washed thoroughly using PBS then discarded, cell flow through was retained. A single 40pm cell strainer (Falcon) was placed onto a fresh 50ml tube (Falcon) before hydrating the mesh with PBS. Flow through cell mix was applied to the strainer allowing the single cells to flow through by gravity. Again, the sieve was washed extensively with PBS to remove any remaining single cells, cell flow through was retained. The largest organoid glomeruli were collected from the 40pm cell strainer by inverting the sieve inside a 10cm petri dish before washing from below using PBS. The above process was repeated using a 30pm cell strainer (Miltenybiotec) to collect smaller organoid glomeruli.

EXAMPLE 5 - Culture of sieved organoid glomeruli

For the culture of 3D organoid glomeruli in suspension, sieved organoid glomeruli were cultured in low attachment culture plates (Coming) in the deliberate absence of any compound allowing adhesion. Cells were supplemented with podocyte growth media (Saleem, M.A., et al. (2002) J Am Soc Nephrol. 13:630-8) and incubated in a humidified atmosphere at 37°C plus 5% C0₂. Media was changed every other day post-seeding.

EXAMPLE 6 - Capillary loop stage organoid glomeruli can be isolated intact from human iPSC kidney organoids.

The formation of podocyte clusters that were resistant to enzymatic dissociation within human iPSC-derived kidney organoids was observed. These arose between day 11 and day 18 of organoid development (d7+l l, d7+l8). These clusters were organoid glomeruli, and hence were isolated using the glomerular sieving technique described above at example 4. Enzymatic dissociation of organoids at day 11 post-aggregation (d7+l l) yielded a predominantly single cell population (data not shown), whilst enzymatic dissociation of more mature d7+l8 organoids yielded 3D aggregates of podocytes representing forming organoid glomeruli (OrgGloms). These structures could be isolated consistently in large numbers (Figure 1 A), yielding approximately 3000-4500 OrgGloms per differentiation (150 OrgGloms per organoid; 20-30 organoids per single well differentiation in a 6 well plate), and surprisingly bore a striking resemblance to whole glomeruli isolated from post-natal human tissue. Average OrgGlom diameter was between that of glomeruli from late trimester three human foetal kidney and adult human glomeruli (Figure 1B). OrgGloms at d7+l8 were comprised of an involuting podocyte layer surrounded by a Bowman’s capsule (Figure lCi) suggestive of the capillary loop stage of glomerular development, with enriched NPHS1 gene expression in sieved organoid glomeruli compared with whole organoids (Figure lCii). Transmission electron microscopy of OrgGloms in situ revealed podocyte cell bodies connected by primary and secondary processes and surrounded by an outer layer of parietal epithelial cells (Figure 1D). This likely reduced enzymatic dissociation, facilitating the isolation of OrgGloms by sieving. Immunofluorescence showed appropriate apicobasal polarity of the podocytes within the OrgGloms (Figure 1E). PODOCALYXIN was shown to localise to the apical surface of the cells, with co-localisation of NEPHRIN and NEPH1 proteins at the intracellular junction between adjacent podocytes, as would be expected in the slit diaphragm.

EXAMPLE 7 - Isolated organoid glomeruli exhibit superior podocyte identity and maturation

While OrgPods showed a podocyte identity similar to that previously reported for conditionally immortalised human podocytes (ciPods), both of these culture approaches lack the three dimensional context of a glomerulus in vivo. To address this, the quality of our 3D OrgGlom model compared to OrgPods and ciPods was investigated using RNA sequencing (RNA-seq). A principle component analysis (PCA) revealed that each cell types clustered tightly indicating highly consistent gene expression within biological replicates (Figure 2A). A differential expression analysis was performed comparing all sample groups (false discovery rate <5% and absolute log2-fold-change > 1). The greatest difference between culture models was seen when comparing 3D OrgGloms with differentiated ciPods (Figure 2B). An unbiased heatmap depicting expression levels of the top 50 differentially expressed genes showed distinct differences between all 2D cultures and 3D OrgGloms, with many of the significantly upregulated genes in OrgGloms associated with the podocyte (Figure 2C). GO terms associated with slit diaphragm components, kidney filtration cell differentiation and glomerular development were the most significantly enriched in OrgGloms compared with differentiated ciPods (Top 100 upregulated genes; Figure 2D).

A heatmap showing expression levels of key podocyte-associated genes demonstrated a clear distinction between the 3D organoid glomeruli and the 2D cell models (Figure 2E). This was validated using qPCR which also showed strikingly enhanced gene expression in OrgGloms compared to the 2D models (Figure 2F). Again, ciPods showed much lower podocyte gene expression levels in both undifferentiated and fully differentiated states. While OrgPods showed slightly improved expression levels in comparison to the immortalised line, they were also suboptimal compared to the OrgGloms. To confirm that the OrgGlom model was reproducible we examined OrgGloms isolated from three independent iPSC lines. Finally, to relate the expression data back to human kidney, we further defined a panel of 100 human glomerular-enriched genes by analysing publicly available adult glomerular gene expression data. Glomerular expression scores were determined for each sample, with OrgGloms found to be the most congruent with human glomeruli (Figure 2G). Whilst there have been previous reports of the generation of kidney organoids using iPSC (Kim et al. (2017) Stem cells, 35:2366- 2378), until recently no in depth transcriptomic analysis of the podocytes generated by these approaches had been completed. By comparing our RNA-seq data to that performed by Kim et al. (2017) Stem cells, 35:2366-2378, we were able to evaluate the quality of the OrgGlom model in the context of iPSC-derived podocytes. A PCA found the nephron-like structures derived by Kim et al. (2017) Stem cells, 35:2366-2378 separated clearly from both 2D podocytes and 3D OrgGloms, expressing lower levels of both podocyte-specific genes and podocyte-enriched genes identified from human glomerular isolates. In conclusion, OrgGloms have improved cellular identity to that of previously reported iPSC-derived podocyte approaches, with profiling of OrgGloms suggesting a clear benefit to suspension culture for the maintenance of podocyte identity in vitro.

EXAMPLE 8 - Isolated organoid glomeruli synthesise mature components of the human glomerular basement membrane.

Serial chemical fractionation of OrgGloms and OrgPTs was performed to derive fractions enriched for cellular and ECM components (Figure 3 A ). Fractions Cl and C2 were predominantly cellular proteins, C3 nuclear proteins and C4 enriched for ECM proteins. A PCA demonstrated good separation between cellular and ECM fractions. There was close overlap of Cl fractions from both cell types, while C4 fractions showed close congruence in principle component 1, with greater differences seen in principle component 2 (Figure 3B).

Mass spectrometry-based proteomic analysis of the isolated extracellular matrix (ECM) and cell lysate of OrgGloms was performed and compared to organoid proximal tubular cells (OrgPT). Mapping of organoid proteomic data onto the human matrisome database (matrisomeproject.mit.edu) identified 60 enriched matrix proteins, 30 found within the predominantly ECM-rich fraction (C4), 20 found in the predominantly cellular protein fraction (Cl) and 10 matrix proteins common to both (Figure 3C, Table 1 & Table 2).

Enrichment of mature GBM components was evident in OrgGloms. In particular, the laminin chains a.5, b2 and gΐ were highly abundant, confirmed by immunostaining of laminin subunit alpha-5 (LAMA5) protein in OrgGloms (Figure 3E). Type IV collagen chains al and a2 were also abundant and indicative of basement membrane formation in OrgGloms. The mature type IV collagen a5 and a6 chains were also highly abundant, these are expressed in the Bowman’ s capsule as the a5a5a6 network, however a3 and a4 chains were at the limit of detection, suggesting that alternative cues are required for assembly of the a3a4a5 network (Figure 3Di, Table 1 & Table 2). In addition, a number of key ECM-associated glycoproteins and proteoglycans were produced in OrgGloms which are essential for kidney development. The matrix components Fraser extracellular matrix subunit 1 (FRAS1) and Fraser extracellular matrix subunit 2 (FREM2) known to have distinct roles in kidney development, were found to be significantly enriched in the OrgGlom C4 fraction. Also enriched in OrgGloms were hemicentin-2 (HMCN2), a key protein required for connecting adjacent basement membranes, and nidogen-l/2 (NID1, NID2) which bind collagen IV and laminin networks and are also subsequently important in membrane fusion. In addition agrin and heparan sulfate proteoglycan 2, two of the most abundant heparan sulfate proteoglycans in the GBM were found to be highly expressed in OrgGloms (Figure 3Di). To achieve a wider perspective, the OrgGlom matrix data was compared to proteomic data from human glomerular tissue alongside the matrices of the conditionally immortalised human podocytes (ciPod) and conditionally immortalised Glomerular endothelial cells (ciGEnC) (Figure 3F). OrgGloms show greater congruence to human glomerular tissue by comparison to monoculture-derived matrix (ciPod or ciGEnC) or coculture-derived matrix (ciPod-ciGEnC).

EXAMPLE 9 - Temporal analysis of organoid glomeruli shows evidence of maturing cell types.

The MAF bZIP transcription factor B gene ( MAFB ) is highly expressed in developing podocytes. In order to monitor podocyte development within an organoid context, we generated a knock-in iPSC line that harbours the mTagBFP2 fluorescent reporter gene inserted at the start codon of the endogenous MAFB locus (MAFB^mTagBFP2/+). Kidney organoids were generated from MAFB^mTagBFP2/+ iPSCs, allowing podocyte development to be imaged in real time (Figure 4A). Isolation of the mTagBFP2+ cells from dissociated organoids at day 10 post-aggregation (d7+l0) by fluorescence-activated cell sorting (FACS) (Figure 4Bi) followed by RT-PCR analysis confirmed MAFB expression was restricted to the mTagBFP2+ fraction and absent from mTagBFP2 non-expressing cells (Figure 4Bii). Co-localisation of mTagBFP2 and NEPHRIN was also observed in whole organoids analysed by immunostaining at d7+l8. Isolation of d7+l8 OrgGloms from live organoids by sieving yielded a pure mTagBFP2+ cell population (Figure 4C, Ohr). However 2D culture of OrgPods initially derived from mTagBFP2+ organoid glomeruli demonstrated rapid reduction of reporter gene expression within 24 hours (Figure 4C, 24hr). In contrast, when sieved mTagBFP+ OrgGloms were cultured in suspension, strong reporter gene expression could be maintained for up to 96 hours post-isolation (Figure 4C, arrow and Figure 5B). These data indicate that OrgGloms are better tools for screening applications than OrgPods, in particular because they may allow reporter gene expression to be assessed over a longer period of time.

Through differentiation of MAFB^mTagBFP2/+ iPSCs to kidney organoids, podocytes could be isolated at various developmental stages by virtue of the fluorescent reporter. RNA-seq analysis of mTagBFP2+ podocytes and organoid glomeruli resulted in a transcriptional profile of human podocyte development across time. Podocytes were examined early in glomerular development soon after MAFB expression is first observed (d7+l0), mid- way through organoid development (d7+l4) and late in organoid development (d7+l9). A PCA was performed to assess the overall similarity between samples across time, with each triplicate found to cluster together closely (Figure 4D). The d7+l4 mTagBFP2+ population showing greatest similarity to OrgPods as compared to OrgGloms. A differential expression analysis comparing all time points (false discovery rate <5% and absolute log2-fold-change >1) showed few genes that were significantly up- or downregulated when comparing early to mid (d7+l4 v d7+l0) or mid to late time points (d7+l9 v d7+l4). The greatest difference was seen when comparing OrgGloms (d7+l9) to immature podocytes (d7+l0) (Figure 4E). Heatmaps depicting expression levels of the top 50 upregulated genes across time show gradual increases in expression between d7+l0 and d7+l4, increasing dramatically by d7+l9, with many of these genes found to be members of the human glomerular ECM proteome (Figure 4F). GO terms derived from the top 100 upregulated genes in OrgGlom (d7+l9) versus immature podocytes (d7+l0) were associated with maturation of the ECM, cell adhesion and collagen trimer formation (Figure 4G). When comparing this data to a panel of genes found to be enriched in adult human glomeruli, significant temporal upregulation was observed in a number of non-podocyte glomerular genes (Figure 4H), suggesting the accumulation with time of endothelial and mesangial cell types within the organoid glomeruli, supported by immunofluorescence of OrgGloms for the endothelial marker PLATELET ENDOTHELIAL CELL ADHESION MOLECULE ( PECAM1 ) and mesangial marker PLATELET-DERIVED GROWTH FACTOR RECEPTOR BETA (PDGFRfi) (Figure 5C). In particular, KDR (VEGFR-2), which plays a critical and specific role in the maintenance and integrity of glomerular endothelial cells, showed a 24-fold upregulation between d7+l0 and d7+l9 (Figure 4H, Table 3). Endothelial cell markers (TEK, EMCN), mesangial markers ( IGFBP5 , TAGEN, MMP2), as well as critical GBM collagens ( COL4A3 , COL4A4 ) and genes essential for maintaining the kidney vasculature ( CXCL12 , ANGPTL2 ) were all significantly upregulated with time (Table 3). This suggests increasing cellular complexity and individual cellular maturity occurs within OrgGloms present in kidney organoids over time.

Table 3. All significantly upregulated glomerular-enriched genes: MAFB-BFP2+ cell copulation d7+ 19 v d7+!0.

EXAMPLE 10 - Summary of OrgGlom analysis

OrgGloms showed greater correlation to mature podocytes than that displayed in both the gold-standard conditionally immortalised human podocyte cell line (e.g. Saleem, M.A., et al. (2002) J Am Soc Nephrol. 13:630-8) or organoid-derived podocytes cultured in 2D. Indeed, within 24 hours of plating, component podocytes from within OrgGloms showed rapid downregulation of MAFB, further highlighting the advantages of culture in suspension.

In depth proteomic analysis of OrgGloms revealed a distinct and complex ECM. OrgGloms showed evidence of more mature GBM components with an abundance of laminin-521, suggesting that cellular crosstalk and the 3D conformation of OrgGloms promotes synthesis of this mature laminin network.

Furthermore, when examining maturation of OrgGloms across time, many of these glomerular ECM components became progressively enriched, whilst evidence of additional cells types, including both mesangial and endothelial populations, was apparent. This is an important observation, as cellular crosstalk has long been known to play a critical role in the glomerulus, with secreted growth factors and signalling ligands acting as cross-talk effector molecules, through autocrine effects on the same cell or paracrine effects on those nearby. The ECM plays a crucial role in this process and has been shown to be mature and complex in OrgGloms compared to podocytes in 2D culture. It is therefore possible that OrgGloms will allow examination of the mechanisms that induce collagen and laminin isoform switching and the role of additional components involved in GBM assembly, maintenance and repair. Accordingly, the data presented herein suggest that OrgGloms in suspension culture represent a superior approach for modelling podocyte biology compared with adherent culture where outward proliferation of podocytes results in destruction of the organoid glomeruli structure. EXAMPLE 11 - Organoid Glomeruli accurately model congenital nephropathy syndrome in vitro

Congenital nephrotic syndrome (CNS) is characterised by severe proteinuria evident in the first months of life. The majority of CNS cases result from mutations in genes encoding critical podocyte proteins, such as NPHS1 (encodes NEPHRIN) and NPHS2 (encodes PODOCIN). To investigate whether OrgGloms could accurately model such a human podocytopathy, an iPSC line was derived from sample isolated from a patient with CNS. The patient was a compound heterozygote with mutations in exon 10 (c. l235delG) and exon 27 (c.348l+4G>T) of the NPHS1 gene (Figure 6A). The variant in exon 10 has not been previously reported and was predicted to create a frameshift, resulting in the introduction of a premature stop codon with the mRNA produced likely targeted for nonsense mediated decay (NMD). The mutation in exon 27 has been previously reported, with the single nucleotide substitution located in the donor splice site of intron 27. Bioinformatic analysis predicted this variant to result in the skipping of exon 27. It was deemed highly likely that both variants would be pathogenic. The iPSC line derived from this patient was differentiated into kidney organoids alongside a wildtype iPSC line and OrgGloms isolated for immunostaining. Confocal Z-stack images were acquired through entire OrgGlom structures using matched parameters for image detection (Figure 6B-D). Notable differences in protein expression were observed, particularly for NEPHRIN and PODOCIN (Figure 6E). Semi-quantitative analysis of fluorescence intensity within individual organoid glomeruli (Figure 6F) revealed a significant reduction in NEPHRIN and PODOCIN proteins in the patient OrgGloms compared to control, whereas levels of CD2AP were comparable and NEPH1 slightly elevated. These data indicate that OrgGloms can be isolated from organoids that are representative of various kidney diseases and used for various applications such as screening and disease modelling.

EXAMPLE 12 - Cultured organoid glomeruli can be utilised for toxicity screening

The ability to reliably generate and isolate OrgGloms in large numbers from kidney organoids provides the opportunity for drug screening, both for toxicity and efficacy. As a proof of principle, individual MAFB-BFP2 OrgGloms were placed in a 96-well format and exposed to increasing concentrations of doxorubicin. MAFB-BFP2 intensity was assessed via live imaging at 48 hours post-treatment, revealing fragmentation of organoid glomeruli and loss of BFP2 signal with increasing dose (Figure 7A). Fixation of organoid glomeruli followed by immunostaining for the apoptosis marker caspase-3 showed an increase in activity at the lower doses of doxorubicin, before cell death prevailed resulting in the destruction of organoid glomeruli (Figure 7B). MAFB-BFP2 intensity showed a dose-dependent decrease with a comparable reduction in glomerular size following doxorubicin treatment (Figure 7C).

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the disclosure as shown in the specific embodiments without departing from the spirit or scope of the disclosure as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

1. An isolated 3D organoid glomerulus, wherein the glomerulus is isolated from a stem cell-derived kidney organoid, and wherein the glomerulus is PECAM1+ and/or PDGFR /+.

2. The organoid glomerulus according to claim 1, wherein the glomerulus is positive for one or more of the following markers: COL4A3, COL4A4, COL4A5, LAMB 2, LAMA5, LAMC1, KDR, MMP2, CXCL12, ITGA2, TEK, FN1, ANGPTL2, IGFBP3, SHIS A3, MME, IGFBP5, EMCN, UCHL1, TAGLN, CDH5, GJA5, SMAD7, CX3CL1, FGF2, GAT A3, DES, WT1, FOXD1, and C1QTNF12.

3. The organoid glomerulus according to claim 2, wherein the glomerulus expresses high levels of one or more of said markers.

4. The organoid glomerulus according to any one of claims 1 to 3, wherein the kidney organoid is derived from a culture expanded human stem cell population.

5. The organoid glomerulus according to claim 4, wherein the stem cells are human pluripotent stem cells, including human embryonic stem cells or human induced pluripotent stem (iPS) cells.

6. The organoid glomerulus of claim 4 or 5, wherein the human iPS cells are derived from a subject with a genetic kidney disease.

7. The organoid glomerulus of claim 6, wherein the genetic kidney disease is 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.

8. The organoid glomerulus according to any one of claims 1 to 7, wherein the kidney organoid is derived from a culture expanded population of stem cell-derived intermediate mesoderm (IM) cells.

9. The organoid glomerulus according to any one of claims 1 to 8, comprising at least one reporter gene associated with at least one gene of interest.

10. The organoid glomerulus according to any one of claims 1 to 9, wherein the organoid glomerulus is in suspension culture.

11. The organoid glomerulus according to any one of claims 1 to 10, wherein the organoid glomerulus is in suspension culture for at least 24 to 96 hours.

12. The organoid glomerulus according to any one of claims 1 to 11, wherein the kidney organoid has been cultured for at least 18 days.

13. A method of screening a candidate compound for nephrotoxicity or therapeutic efficacy, the method comprising contacting an isolated 3D organoid glomerulus isolated from a stem cell-derived kidney organoid with a candidate compound and determining whether or not the candidate compound is nephrotoxic or therapeutically effective.

14. A method of screening a candidate compound for nephrotoxicity and/or therapeutic efficacy, the method comprising contacting an organoid glomerulus according to any one of claims 1 to 12 with a candidate compound and determining whether or not the candidate compound is therapeutically effective.

15. The method of claim 13 or 14, comprising contacting said organoid glomerulus with a candidate compound and a nephrotoxin and determining whether or not the candidate compound is therapeutically effective.

16. The method of any one of claims 13 to 15, wherein determining whether or not the candidate compound is nephrotoxic or therapeutically effective comprises measuring one or more of: diameter of the organoid glomerulus; expression of one or more genes associated with cell death; expression of one or more genes associated with cell viability; expression of one or more nephron-associated genes; expression of one or more genes associated with glomerular extracellular matrix; expression of one or more genes associated with podocyte, endothelial or mesangial cell types; and intensity of expression of a reporter gene associated with at least one gene of interest.

17. The method according to claim 16, wherein said one or more genes comprises one or more of: COL4A3, COL4A4, COL4A5, LAMB 2, LAMA5, LAMC1, WT1, KDR, MMP2, CXCL12, ITGA2, TEK, FN1, ANGPTL2, IGFBP3, SHIS A3, MME, IGFBP5, EMCN, UCHF1, TAGEN, CDH5, GJA5, SMAD7, CX3CL1, FGF2, GAT A3, DES, FOXD1, and C1QTNF12.

18. The method according to claim 16 or 17, comprising measuring the expression of one or more of: SYNAPTOPODIN, PODOCALYXIN, PODOCIN, NEPHRIN, NEPH1, PEC AM and PDGFR ?.

19. The method according to any one of claims 16 to 18, wherein: i) a measured reduction in one or more of: diameter of the organoid glomerulus; expression of one or more genes associated with cell viability; expression of one or more nephron-associated genes; expression of one or more genes associated with glomerular extracellular matrix; expression of one or more genes associated with podocyte, endothelial or mesangial cell types; and intensity of said reporter gene; and/or ii) a measured increase in expression of one or more genes associated with cell death; is indicative of nephrotoxicity of the candidate compound.

20. The method according to any one of claims 16 to 18, wherein: i) a measured increase or absence of a measured reduction in one or more of: diameter of the organoid glomerulus; expression of one or more genes associated with cell viability; expression of one or more nephron-associated genes; expression of one or more genes associated with glomerular extracellular matrix; expression of one or more genes associated with podocyte, endothelial or mesangial cell types; and intensity of said reporter gene; and/or ii) a measured reduction in expression of one or more genes associated with cell death; is indicative of therapeutic efficacy of the candidate compound.

21. The method according to any one of claims 16 to 20, wherein the candidate compound is a small molecule, polynucleotide, peptide, protein, antibody, antibody fragment, serum, virus, bacteria, stem cell or combination thereof.

22. The method according to any one of claims 16 to 21, wherein the candidate compound is a small molecule.

23. The method according to any one of claims 16 to 21, wherein the candidate compound is serum including serum isolated from a subject with kidney disease.

24. The method according to any one of claims 16 to 22, further comprising selecting a candidate compound which is not nephrotoxic and/or is therapeutically effective.

26. The assay of claim 25, wherein the assay comprises organoid glomeruli according to any one of claims 1 to 12.

- isolating an iPS cell population from a group of subjects;

- generating a kidney organoid from each subjects iPS cell population and isolating an organoid glomerulus;

28. The method of claim 27, wherein the organoid glomerulus is defined by any one of claims 1 to 12.

29. The method of claim 27 or claim 28, which comprises contacting the organoid glomeruli with a therapeutic agent and a nephrotoxic agent.

30. The method according to any one of claims 27 to 29, wherein the agent is a small molecule, polynucleotide, peptide, protein, antibody, antibody fragment, virus, bacteria, stem cell, serum including kidney disease patient derived serum or a combination of one or more thereof.

31. The organoid glomerulus according to any one of claims 1 to 11 when used for modelling glomerular development.

32. The organoid glomerulus according to any one of claims 1 to 110 when used for modelling kidney disease.

33. The organoid glomerulus according to claim 31 or 32, wherein the glomerulus is isolated from a d7+l4 kidney organoid.