Tissue regeneration

Abstract

Populations of cells for use in the treatment of kidney disease and methods of making the same.

Description

Tissue regeneration1 BACKGROUIND

[0001] Approximately 10% of the population worldwide is affected by chronic kidney disease (CKD), a number expected to rise due to increased prevalence of risk factors such as diabetes, hypertension, obesity, and an aging population. CKD is one of the areas where there is a great unmet need for innovative pharmacological therapies [1],

[0002] The anatomical complexity of the kidney makes the kidney a particularly challenging target for tissue regenerative therapy. One challenge is the production of off-target cells in the kidney organoid, and particularly the production of fibrotic regions and cartilage. The challenge is therefore to maximise production of on-target cell types associated with kidney nephrons (podocytes, proximal tubule, distal tubule, collecting duct and endothelial cells), whilst minimizing off-target populations.

[0003] Nephron progenitor cells (NPC) express the markers intermediate mesoderm SIX2, SALL1 , WT 1 , and PAX2 and are found in humans only during kidney organogenesis (i.e. not in the adult kidney).

[0004] The majority of studies focused on recapitulating kidney tissue development in vitro have focused on the generation of culture systems for use in the study of human kidney development and drug development. Morizane et al. 2015 describes NPC derived cellular model derived from posterior intermediate mesoderm (IM) cells [2], Borestrom et al. describes NPC derived cellular model derived from iPSC [1],

[0005] Tsujimoto et al. describes kidney organoids produced from separate lineages of NPCs and UBs [3], WO 2019 / 084612 A1 describes kidney organoids having simplified structures derived from a single population of stem cells, according to the methods described in Freedman et al. 2015 [4], WO 2016 / 094948 A1 describes a method for preparing organoids in a 3D matrix from a single lineage of differentiated iPSCs. The organoids comprise about 500,000 (500k) cells. WO 2023 / 033137 A1 describes a method for producing kidney organoids from a single lineage of intermediate mesoderm cells. WO 2020 / 019023 A1 describes a method for producing kidney organoids from a single lineage of intermediate mesoderm.2 SUMMARY

[0006] The present invention relates to products and methods for tissue regeneration in the kidney. The invention particularly relates to cells and cell populations for use in methods of tissue regeneration of diseased kidney tissue.

[0007] The present invention particularly relates to micro-assembloids and their use to treat kidney disease. The present inventors have surprisingly shown that micro-assembloids, comprising cells derived separately from 2 linages, mature into functional kidney tissue when transplanted into the kidney.3 BRIEF DESRIPTION OF THE DRAWINGSFigure 1 : Differentiation protocols

[0008] Summaries of UB (Figure 1A), NPC (Figure 1B) and ASB (Figure 1c) protocols.Figure 2: UB differentiationFigure 3: In vitro cultured assembloids of different sizes

[0009] After assembloid formation, assembloids starting from 10,000 cells or 120,000 cells were cultured in vitro and diameter was evaluated at regular intervals during culture.Figure 4: Kidney structures in assembloids of different sizes

[0010] After in vitro culture to day 23 of differentiation, assembloids staring from 10,000 cells or 120,000 cells were fixed, embedded and sectioned. H&E (hematoxylin and eosin) staining was ued to identify basic kidney related structuresFigure 5: RNAseq profile of assembloids of different sizes

[0011] Bulk RNA sequencing of assembloids starting from 10,000 cells or 120,000 cells after in vitro culture to day 23 of differentiation. Analysis of top differential expressed genes between 10k ASB and 120k ASBs and top enriched gene sets in 120k ASB.Figure 6: RNAseq profile of assembloids versus NPC kidney organoids

[0012] Cell type deconvolution comparing NPC and ASB 10kFigure 7: Glomerular and tubular structures in assembloids

[0013] H&E and 4-plex staining on representative assembloid, ASB10. Assembloids = 10k assembloids. D23.Figure 8: Expression of kidney related markers in 10k and 120k ASBs

[0014] Assembloids = 10k or 120k assembloids. Gene expression measured by bulk RNA-seq. Columns (genes) have been mean centred and scaled to unit variance (z-score).Figure 9: UB marker expression in assembloids versus NPC kidney organoids

[0015] The expression of UB markers were assessed at day 9 of differentiation in assembloids and NPC kidney organoids. RNA seq > 3 replicates, mean-centred scaled to unit variance. Assembloids = 10k assembloidsFigure 10: Collecting duct development in assembloids versus kidney organoids

[0016] Assembloids = 10k assembloids.Figure 11 : Expression of kidney related markers in ASBs and NPC kidney organoids

[0017] NPC kidney organoids and 10k Assembloids. Gene expression measured by bulk RNA-seq.Figure 12: H&E staining of engrafted NPC kidney organoids and assembloids

[0018] 2 weeks post graft. Assembloids = 10k assembloids.Figure 13: IHC staining of specific kidney tissue markers

[0019] 2 weeks post graft. Assembloids = 10k assembloids.Figure 14: Assembloid gene expression

[0020] Assembloid gene expression was assessed by qPCR. Assembloids = 10k assembloids.4 DETAILED DESCRIPTION

[0021] The disclosure relates to an isolated cell population, wherein the cell population expresses a) UB cell lineage associated genes (UB genes), and b) NPC lineage associated genes (NPC genes), wherein the cell population is in the form of a three-dimensional (3D) tissue capable of maturing to form one or more components of kidney organ architecture, e.g. in vivo or following implantation into a subject. The cell population may be a kidney organoid. The cell population may be produced by first separately differentiating UB cells and NPC, and then combining the UB cells and NPC in a single cell population, followed by further culturing in conditions suitable for kidney organoid formation (e.g. in 3D culture, in the presence of a GSK-3 inhibitor, Fibroblast growth factor (FGF) and / or a ROCK inhibitor for about 2 days).4.1 Assembloid cells & structural features

[0022] The 3D tissue may have a diameter of less than about 800 pm. The 3D tissue may have a diameter of about 200 to 450 pm. The 3D tissue may have a diameter of about 250 to 300 pm. The 3D tissue may have a diameter of about 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480 pm. The 3D tissue may have a diameter of about 0.25 to 0.3 mm. The 3D tissue may have a diameter of about 0.2, 0.21 , 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31 , 0.32, 0.33, 0.34, 0.34, 0.35, 0.36, 0.37, 0.38. 0.39, 0.4, 0.41 , 0.42, 0.43, 0.44, or 0.45 mm.

[0023] The cell population may contain less than about 120,000 cells. The cell population may contain 10,000 cells or less. The cell population may contain about 2,000 to 10,000 cells. The cell population may contain about 1 ,000 to 8,000 cells. The cell population may contain about 2,000, 2,500, 3,000, 3,5000, 4,000, 4,500, 5,000, 5,500, 6,000, 6.500, 7,000, 7,500, 8,000, 8,500, 9,000, 9,500, 10,000, 10,500, 1 1 ,000, 11 ,500, 12,000, 12,500, 13,000, 13,500, 14,500, 15,000, 15,500, 16,000 cells. Cells may be counted by methods known in the art. Cells may be counted directly using a hemacytometer or an automated cell counter. Cells may be counted indirectly e.g. using a cell viability assay such as MTT or MTS assays. Cell count may relate to live cells.4.2 Gene expression markers

[0024] UB genes may be UB lineage specific genes, i.e. genes expressed in UB cells and / or cells derived from UB cells but are not expressed in NPC or cells derived from NPC. NPC genes may be NPC lineage specific genes, i.e. genes that are expressed in NPC and / or cells derived from NPC but are not expressed in UB cells or cells derived from UB cells. UB genes may be genes associated with collecting duct tissue.

[0025] The cell population may express RET. The cell population may express PAX2. The cell population may express PAX8. The cell population may express CALB1. The cell population may express ETV5. The cell population may express Six2. The cell population may express WT1. The cell population may express HOXD1 1 . The cell population may express HOXC10. The cell population may express OSR1 . The cell population may express RET, PAX2, PAX8, CALB1 , and / or ETV5. Cell population may express Six2, WT 1 , HOXD11 , HOXC10 and / or OSR1 . The cell population may express HOXD1. The cell population may express RET, PAX2, PAX8, CALB1 , ETV5. The cell population may express Six2, WT1 , HOXD1 1 , HOXC10 and / or OSR1 .

[0026] The cell population may express one or more podocyte associated gene. The cell population may express PODXL. The cell population may express a proximal tubuli associated gene. The cell population may express PRP2.

[0027] The cell population may express one or more distal tub associated gene. The cell population may express BTN1. The cell population may express GATA3. The cell population may express BRN1 and GATA3. The cell population may express one or more collecting duct associated gene. The cell population may express CALB1. The cell population may express GAT A3 and CALB1. The cell population may express CALB1 and AQP2. The cell population may express one or more endothelial cell associated gene. The cell population may express CD31 (PECAM1). The cell population may express one or more proximal tubuli associated gene. The cell population may express AQP1 . The cell population may express one or more renal pelvis associated gene. The cell population may express SCNN1 B. The cell population may express UPK1A.

[0028] Gene expression may be defined relative to a control cell population not expressing the gene. Gene expression may be defined as at least 2-fold higher gene expression relative to a control cell population. UB gene expression may be defined relative to a control cell population consisting of NPC lineage cells. NPC gene expression may be defined relative to a control cell population consisting of UB cells or UB lineage cells. UB genes expression may be defined relative to gene expression of the same gene a control cell population consisting of NPC lineage cells or NPC. UB lineage cells are cells derived from UB cells. NPC lineage cells are cells derived from NPC.

[0029] Gene expression in the cell population may be measurable by polymerase chain reaction, RNA sequencing, immunohistochemistry and / or immunofluorescence.4.3 Cell types

[0030] The cell population may be self-organised. The cell population may comprise NPCs and / or NPC lineage cells. The cell population may comprise UB cells and / or UB lineage cells. UB lineage cells are cell derived from UB cells. NPC lineage cells are cells derived from NPC.

[0031] The cell population may comprise podocytes. The cell population may comprise tubular cells. The cell population may comprise epithelial cells. The cell population may be at an early stage of maturation. The cell population may not comprise a nephron or nephrons.

[0032] Kidney organ architecture is observable by immunohistochemistry or immunofluorescence.4.4 Function

[0033] The cell population of any preceding claim, wherein the cell population does not comprise a functional nephron. Once implanted into the kidney of a subject, the cell population may be capable of maturing into tissue comprising a functional nephron. Once implanted into the kidney of a subject, the cell population may be capable of maturing into tissue comprising glomeruli and / or collecting ducts.

[0034] The cell population of any preceding claim, wherein the cell population performs an [in vivo function associated with kidney tissue.4.5 Cell source

[0035] The cell population may be derived from cells comprising stem cells. The cell population may be derived from cells comprising embryonic stem cells. The cell population may be derived from cells comprising induced pluripotent stem cells (iPSCs). The cell population may be derived from cells comprising mesoderm cells. The cell population may be derived from cells comprising intermediate mesoderm cells. The cells may be derived from cells comprising intermediate mesoderm cells.

[0036] The cell population may be is derived from a cell or cells that have been expanded ex vivo. The cell population may be derived from cell or cells isolated from a subject. The cell population may be artificial. The cell population may not exist in nature. The cell population may comprise a cell or cells that have been genetically modified or gene edited. The cell population may be isolated from the body and may be comprised in a pharmaceutical composition.

[0037] The stem cells may be selected from the group consisting of H9, hES3, IPSC GAPTrap td- Tomato, CRM 502..C32, CI..R1502.3, hES3 SOXUmCherry or H9 GAPTrap Luc2.4.6 Culture time course

[0038] The cell population may have been cultured in vitro 3D conditions for less than 4 day. The cell population may have been cultured in vitro 3D conditions for about 2 days. The cell population may have been cultured in vitro in media comprising media comprising a GSK-3 inhibitor, Fibroblast growth factor (FGF) and / or a ROCK inhibitor, for about 2 days.4.7 Manufacturing / Process

[0039] The disclosure also relates to a method of producing a cell population according to the disclosure., the method comprising a) combining NPC cells and UB cells to form a mixed population of cells, and b) culturing the mixed population of cells in vitro in conditions suitable for the formation of a kidney organoid.

[0040] The disclosure also relates to a method of producing a cell population, the method comprising a) combining NPC and UB cells to form a mixed population of cells, and b) culturing the mixed population of cells in vitro in conditions suitable for the formation of a kidney organoid, wherein the cell population is in the form of a three-dimensional (3D) tissue and comprises a component of kidney organ architecture. The cell population may be a kidney organoid. Step b) of the method may compriseculturing the mixed population of cells in media comprising a GSK-3 inhibitor (also referred to as a Wnt agonist), Fibroblast growth factor (FGF) and / or a ROCK inhibitor.

[0041] The GSK-3 inhibitor may be CHIR99021. The FGF may be FGF2, FDF9 and / or FGF20. The media may comprise 200 ng / ml FGF2. The ROCK inhibitor may be Y-27632. The media may also comprise heparin. The GSK-3 inhibitor may LiCI SB-216763, CAS 853220-52-7 and other Wnt agonists that are commercially available.

[0042] The method may comprise any or all of the steps outlined in Figure 1 . The mixed population of cells may be cultured in vitro for 3 days or less. The mixed population may be cultured for about 2 days. The mixed population may be cultured for about 48 hours. The mixed population may be cultured for a day. The mixed population may be cultured for about 24 hours. The method may comprise deriving the NPC, e.g. according to the steps outlined in Figure 1. The method may comprise deriving the UB cells, e.g. according to the steps outlined in Figure 1 . The method of deriving the UB may comprise culturing pluripotent stem cells in APS media for at least 24 hours. Either or both the UB and NPC may be derived from ESC and / or iPSC.

[0043] The UB cells used in the method may express UB specific or UB associated genes. The UB cells may express FOXA1 , KRT8, KRT18, CDH1 , FOXA1 , SPRED1 , WNT11 , RET, UPK1A, RBP1 , GFRA1 , LHX1 , KRT19 and / or AXIN2. The NPC may not express UB associated genes. The NPC may not express FOXA1 , KRT8, KRT18, CDH1 , FOXA1 , SPRED1 , WNT11 , RET, UPK1A, RBP1 , GFRA1 , LHX1 , KRT 19 and / or AXIN2. Expression of one or more UB gene by the UB cells may be at least 2- fold higher than the expression of the same UB gene by the NPC, wherein the one or more UB gene may comprise FOXA1 , KRT8, KRT18, WNT1 1 , RET, UPK1A, RBP1 , GFRA1 , LHX1 and / or KRT19. Gene expression may be defined relative to a control cell population not expressing the gene or relative to the expression of a house-keeping gene, e.g. GAPDH. Expression of the UB gene may be at least 2-fold higher than the expression of the gene in the control cell population, where the control cell population may be NPC. Gene expression may be measured by anyone method known in the art, e.g. by polymerase chain reaction, RNA sequencing, immunohistochemistry and / or immunofluorescence.

[0044] In the method, the NPC and UB cells may be combined in a ratio of about 1 .3, 1 .4 or 1 .5.4.8 Method of treating

[0045] The disclosure also relates to method of treating or preventing disease in a subject, the method comprising administering to a subject in need thereof an effective amount of the cell population disclosed herein. The method may use allogeneic or autologous cells. The disease may be end-stage kidney disease. The method may comprise implanting the cell population in the kidney of the subject. The subject may be a human. The subject may be an adult human with kidney disease. Following implantation, the cell population may mature to form functional kidney architecture and thereby treat the kidney disease.4.9 Product-by-process

[0046] The disclosure also relates to a cell population obtainable by any of the methods disclosed herein.4.10 Pharmaceutical composition

[0047] The disclosure also relates to a pharmaceutical composition comprising any of the populations of cells disclosed herein, and a pharmaceutically excipient. The disclosure further relates to methods of making such a pharmaceutical composition.4.11 EPC 2000

[0048] The disclosure also relates to a pharmaceutical composition for use in a method of treatment of a disease, wherein the method comprises administering the pharmaceutical composition to a subject, and wherein the pharmaceutical composition comprises any of the cell populations disclosed herein, of and a pharmaceutical excipient.5 DEFINITIONS5.1 Assembloid

[0049] An assembloid is an improved kidney organoid formed by combining at least two separately derived lineages of kidney progenitor cells, for example the combination of separately derived UB cell and NPC lineages. As demonstrated herein, combining cells of the UB and NPC lineages into a composite kidney assembloid can generate complex, organised structures that comprise the key components of a functional nephron unit once implanted.

[0050] Assembloids are generally spherical in shape. The diameter of an assembloid is the longest cross-sectional linear longest distance through the sphere.5.2 Gene expression markersTable 1 : Kidney associated gene expression markers5.3 Kidney organ architecture

[0051] The kidneys are bilateral organs placed retroperitoneally in the upper left and right abdominal quadrants and are part of the urinary system. Their shape resembles a bean, where we can describe the superior and inferior poles, as well as the major convexity pointed laterally, and the minor concavity pointed medially. The main function of the kidney is to eliminate excess bodily fluid, salts and by products of metabolism -this makes kidneys key in the regulation of acid-base balance, blood pressure, and many other homeostatic parameters. Each kidney is composed of millions of microscopic filtering units called nephrons. The structure of the nephron can be divided into two main parts: the renal corpuscle and the renal tubule. The renal corpuscle includes the glomerulus, a network of tiny blood vessels, and Bowman's capsule, a cup-like structure that surrounds the glomerulus. Blood enters the glomerulus and is filtered through its walls, which allow small molecules such as water, electrolytes, and waste products to pass through, while preventing larger molecules like blood cells and proteins from entering. The filtered fluid, called the glomerular filtrate, then enters the renal tubule, which is a long, twisted tube that is divided into several sections, each with its own specific function. The tubule reabsorbs some of the filtered fluid, including important substances like glucose and amino acids, back into the bloodstream while also secreting additional waste products into the filtrate. As the filtrate moves through the tubule, it becomes more concentrated as water and other substances are reabsorbed, and eventually exits the kidney as urine.

[0052] Nephrons are the functional working units of kidney which play a major role in removal of waste products from blood / plasma 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 (see for example WO 2019 / 084612 A1).5.4 Population of cells

[0053] The invention also relates to a population or plurality of cells. The population of cells may be heterogenous. The population of cells may contain cells of different cell types, for example UB lineage cells and NPC lineage cells.5.5 Stem cells

[0054] For the avoidance of doubt, the human embryonic cells used in the disclosure can be derived from parthenogenetically activated human oocytes by methods known in the art [5], The invention does not encompass human embryos or relate to uses of human embryos.6 EXAMPLE 1 : Methods6.1 Differentiation protocols6.1.1 UB differentiation

[0055] UB lineage cells were produced from Human pluripotent stem cells (hPSCs) according to the protocol summarised in Figure 1. hPSCs were seeded at around 20,000 cells / cm2on iMatrix-511 coated plates. The day after the medium was changed to UB stage 1 medium containing CHIR99021 and ActivinA. Cells were kept in UB stage 1 medium for 20-24 hours. Thereafter the medium was changed to stage 2 medium containing FGF8, TTNPB, A83-01 and LDN193189. Cells were kept in UB stage 2 medium for 2 days. On day 4 of differentiation, cells were dissociated and reseeded at around 100,000cells / cm2 in UB stage 3 medium containing 10% FBS, FGF8, TTNPB, A83-01 , LDN193189 and Y27632. 24 hours later the medium was changed to UB stage 4 medium containing CHIR99021 , FGF8, TTNPB, LDN193189 and GDNF. On day 7 of differentiation UB cells were dissociated, counted and aggregates of around 10,000 cells formed. UB aggregates were cultured for 2-3 days in UB stage 5 medium containing 10% FBS, CHIR99021 , FGF8, TTNPB, LDN193189, GDNF and Y27632. On day 9 or 10 of differentiation UB aggregated were dissociated, cells counted and used for assembloid formation. The components of the relevant media are shown in Table 2. Basal medium: Gibco Essential 6 (E6).Table 2: UB media6.2 NPC differentiation

[0056] NPCs were produced from Human pluripotent stem cells (hPSCs) according to the protocol summarised in Figure 1. hPSCs were seeded at around 10,000 cells / cm2one day ahead of induction / differentiation. The day after the medium was changed to NPC stage 1 medium containing CHIR99021 . Cells were kept in stage 1 medium for 4 days (medium refreshed after 2 days). Thereafter the medium was changed to stage 2 medium containing FGF9. Cells were kept in NPC stage 2 mediumfor 4 days (medium refreshed after 2 days). On day 9 of differentiation NPC cells were dissociated, counted, and used for organoid or assembloid formation. Basal Medium: Stem cell technologies STEMdiff APEL 2. The components of the relevant media are shown in Table 3.Table 3: NPC media6.3 Assembloid protocol

[0057] For the production of assembloids, single cell suspensions of UB and NPC lineage cells were mixed at a 1 :3 ratio in ASB stage 1 medium containing CHIR99021 , FGF2 and Y27632 and plated on AggreWell plates for aggregate formation (Figure 1). The total number of cells was determined by the size and number of assembloids needed, according to the values provided in Table 5. After two days, assembloids were transferred to suspension culture in ASB stage 2 medium containing FGF2. Basal medium: DMEM / F12 + Glutamax (supplemented with 20 % Knock-out serum replacement (KORS), 0,5% 2-mercaptoethanol (0,5x), 1 % MEM-NEAA (1x), 1 % PenStrep (1x))Table 4: ASB mediaTable 5: Assembloids starting cell count6.4 RNA sequencing

[0058] RNA sequencing was performed using standard protocols. RNA from organoids were extracted using RNadvanced Tissue method (Beckman) on the i7 liquid handler (Beckman). The extracted RNA quantity and quality were measured using the Agilent Fragment analyser instrument and verified to have RQN score >7 to enable mRNA sequencing. 500ng RNA was used for library preparation on the Fluent liquid handler (Tecan) using KAPA mRNA hyperPrep kit (Roche) where samples undergo reverse transcription, double stranded cDNA generation and indexing to facilitate multiplexing. Afterthe quantity, quality and the fragment size of the prepared library was measured using Fragment Analyser (Agilent) the libraries were pooled and diluted to 1.65nM for sequencing on NovaSeq 6000 (Illumina) using the 100 cycle SP reagent kit (Illumina) where paired end sequencing was performed 2 x 51 cycles.6. Data processing and analysis

[0059] Fastq files from paired-end sequenced libraries, generated from stem cell derived UB- / NPC- / ASB-organoids, were collected and read quality for all libraries was accessed using FastQC (vO.11 .9) [6], Qualimap (v2.2.2d) [7] and samtools stats (v1.15) [8], Quality control (QC) metrics for Qualimap were based on a STAR (v2.7.10a) [9] alignment against the human genome (GRCh38, Ensembl v105). Next, QC metrics were summarized using MultiQC (v1 .12)

[0010] , All libraries were of good quality for further analysis. Sequencing adapters were trimmed from the libraries using NGmerge (v0.3)

[0011] , A human transcriptome index consisting of cDNA and ncRNA entries from Ensembl (v105) was generated and reads were mapped to the index using Salmon (v1 .7.0)

[0012] , The bioinformatics workflow was organized using Nextflow workflow management system (v20.10)

[0013] and Bioconda software management tool

[0014] , Differential gene expression were assessed with DESeq2 (v 1 .34.0 in R (v 4.1 .0)

[0015] , Estimated counts was used as input for DESeq2 with lowly expressed genes excluded (requiring at least 10 counts in all samples). For visualization, gene expression were plotted as variance stabilized transformation (vst)

[0016] , Plots were generated in R (v 4.1.0) using ggplot2 (v 3.4.0) and pheatmap (v 1 .0.12) and EnhancedVolcano (v 1 .16.0). Gene set enrichment analysis was performed in R (v 4.1 .0) using gprofiler2 (v 0.2.1) [17,18],6.6 Cell type deconvolution

[0060] Cell type deconvolution is a computational method for estimating cell type fractions in bulk transcriptomics based on gene expression patterns specific to the individual cell types. The tool Scaden was used to train a neural network based on labelled single¬cell data from human adult kidney (data from 'www.kpmp.org). That model was then applied to bulk RNA-seqsequencing data from the NPC / UB / ASB protocols.6.7 H&E and 4-plex staining

[0061] The paraffin-embedded assembloids were sectioned into 4 pm slices. H&E staining was performed on Leica ST5020 autostainer. All immunohistochemistry methods and protocols were set up on the automated Ventana Discovery Ultra autostainer (Roche, Basel, Switzerland). Immunohistochemistry for detection of desired epitopes (table 1) were carried out according to manufactures recommendation and all reagents except antibodies were Ventana products (Roche, Basel, Switzerland). Antigen retrieval was done in Ventana Cell Conditioner 1 for 40 min at 95°C. Primary antibody was added for 1 hr at 37° followed secondary anti-rabbit reagent or anti-mouse HRP, purple, teal, green, yellow chromogenic detection in single or up to four plex staining. The slides were digitized using a Aperio XT whole slide scanner and the images were viewed and exported on Aperio ImageScope Software (v12.3.3.5048).Table 6: IHC antibody list6.8 Organoid & assembloid engraftment

[0062] Assembloids and NPC kidney organoids were engrafted into a murine model, as summarized in Figure 1. The grafts were allowed to mature for 2 weeks.

[0063] Engraftment of assembloids and NPC kidney organoids were studied in a murine model. Organoids or assembloids were isolated from culture on day 11 (2 days post formation), rinsed in unsupplemented medium and keept on ice until transplantation. Male immunocompromised NSG mice (7-8 weeks) were anesthetized with isoflurane (induction dose 4-5% & maintenance dose 1.5-2%), shaved and disinfected with Descutan of the left side flank. The kidney was localized and 1-2 cm incision was made below the ribs through the dermis and into the peritoneal wall. The kidney was gently forced out through the peritoneal opening and rested stably on the surface of the skin while keeping it moisturized with sterile saline. Using a 27 gauge needle, a 0.2 cm small incision was made through the kidney capsule across the anterior surface of the kidney and a subcapsular pouch created using a smooth capillary glass rod. An aliquot of blood (8-10 ul) collected from a donor animal was added to the cell pellet to form a blood clot. The clot was collected into a 16G Tuohy needle and transferred to the subcapsular pouch. The anterior opening of the pouch was sealed and the kidney pushed back into the peritoneal cavity. Finally, the abdominal wall was closed with sutures and wound clips.6.9 qPCR

[0064] Quantitative PCR was performed using standard protocols. The primers and probes are provided in Table 6.Table 7: TaqMan qPCR primer and probes7 EXAMPLE 2: UB differentiation protocol

[0065] The protocol for differentiating hESC into a UB lineage of cells was optimised. A summary of the basic protocol for UB lineage differentiation is shown in Figure 1. It was surprisingly ascertained that an APS induction step of at least 24 hours was critical to ensure proper UB differentiation from ESC cells (Figure 2).8 EXAMPLE 3: Impact of cell count on assembloid structure and function

[0066] The structural and functional characteristics of assembloids formed from populations of cells having different cell numbers were compared. Assembloids were derived by combining 2 lineages of cells that had been differentiated from stem cells separately, an NPC lineage and a UB cell linage. The UB and NPC lineages were mixed in a ratio of 1 :3. The starting number of cells following combination of the two lineages was either about 10k or 120k (Figure 1 , Table 5). The mixed cell populations were allowed to mature. The assembloids derived from about 10k cells were smaller than the assembloids derived from about 120k cells (Figure 3, Table 7).Table 8: Assembloids cell count and size*Cell count accuracy ± 10% within the listed range

[0067] After the mixed cell population had been allowed to mature for 2 days (D11), assembloids derived from about 10k cells (“10k assembloids” or “micro-assembloids”) were about 250 to 450 pm in diameter and comprised or consisted of about 2,000 to 10,000 number of cells (Table 7, Figure 3).

[0068] After the assembloids had been allowed to mature for 14 days in vitro (Day 23, D23) the selforganisation and structural characteristics of the assembloids were assessed. The micro-assembloids (i.e. assembloids derived from a mixed population of about 1 ,000 to 10,000 cells) comprised collections of glomerular and renal tubular-like structures (Figure 4).. By contrast, assembloids derived from a greater number of cells (e.g. 120k cells, “large assembloids”), had a diameter of about 800,000 to 120,000 and comprised far fewer defined kidney structures (Table 7, Figure 4).

[0069] The transcription profile of the matured assembloids and micro-assembloids was assessed by RNAseq. Figure 5 shows the top differential expressed genes between large assembloids and micro- assembloids. Assembloids with a greater cell count and diameter surprisingly displayed an upregulatedhypoxia and ECM signal, e.g. increased expression of LOX, a gene associated with collagen formation (Figure 5A). The top enriched gene sets annotated to the ECM and collagen related pathways (Figure 5B). The larger assembloids thus had a more rigid structure, less nephron space, and a poorsecretome profile. The large assembloids (120 ASB) had poor self-organisation and decreased vascularisation, whilst the extensive presence of well differentiated glomerular and tubular structure was confirmed in the matured micro-assembloids (Figure 7).

[0070] Further analysis of the gene expression in the micro-assembloids (10k ASB) was performed, as compared to large assembloids (120k ASB). The expression of individual genes associated with UB cells, NPC or kidney structure and function are shown in Figure 8. These data clearly showed that micro-assembloids (10k ASB) have more complete kidney cell type coverage than large assembloids (120k ASB). Particularly, 10k ASBs had overall a more favourable expression profile of kidney related markers (Figure 8).

[0071] In summary, kidney assembloids with a diameter of about 150 to 450 pm (comprising about 800 to 10,000 cells) after days of initial maturation (Day 11 , D11) go on to mature into assembloids having more favourable characteristics and more closely resemble kidney tissue then assembloids with a diameter of 800 pm or more (comprising about 800,000 cells or more) after 3 days of initial maturation, as demonstrated by histological and transcriptome analysis. Micro-assembloids therefore possess a more beneficial functional profile than large assembloids. Micro-assembloids are thus a candidate for therapeutic use in kidney disease.9 EXAMPLE 3: NPC kidney organoids versus assembloids9.1 Transcription profile

[0072] The transcription profile of large assembloids (“120k assembloids”), micro-assembloids and conventional NPC kidney organoids (containing approx. 1000 cells) were compared. Cell type deconvolution of bulk RNA-sequencing data demonstrated that after 14 days of maturation, the micro- assembloids showed a better representation of cell types covering all regions of the kidney compared to conventional NPC kidney organoids (i.e. organoids produced from a single population of cells) (Figure 6). 120k assembloids showed a higher fraction of VSMC / pericytes and fibroblasts compared to both micro-assembloids and NPC kidney organoids. These data further highlight that micro- assembloids surprisingly represent a better candidate for therapeutic use than large (“120k”) assembloids. Micro-assembloids further represent a better candidate for therapeutic use than conventional NPC kidney organoids.

[0073] Further analysis revealed that within the 120k assembloids and micro-assembloids, UB cells expressed markers of the developing ureteric compartment, contributing to collecting duct specification over time within the assembloids. Early UB markers were expressed in UB cells within the UB cell lineage before they were mixed with the NPC lineage (Day 9, D9) (Figure 9). Furthermore, UB markers were surprisingly not observed in the NPC lineage, even after 23 days isolated incubation without comixing with the UB lineage (Figure 9). Additionally, genes associated with collecting duct development(AQP2, CALB1 and GATA3) were enriched in micro-assembloids but not NPC kidney organoids after 14 days of maturation (Day 23, D23) (Figure 10). The combination of the UB cell lineage and NPC lineage was therefore required for collecting duct formation in the micro-assembloids. Without the UB lineage, collecting duct associated genes were not expressed in the NPC kidney organoids (Figure 10). Conventional NPC kidney organoids therefore do not possess the same functional capabilities as micro- assembloids.

[0074] Further analysis of the gene expression in the micro-assembloids (10k ASB) was performed, as compared to gene expression of NPC kidney organoids and large assembloids (120k ASB). The expression of individual genes associated with UB cells, NPC or kidney structure and function are shown in Figure 11 and Figure 8. These data clearly showed that ASBs (10k and 120k) have more complete kidney cell type coverage. 10k ASBs had overall a more favourable expression profile of kidney related markers.

[0075] The micro-assembloids were found to express markers of key renal cell types as well as key early UB makers (POU3F3, GATA3, CALB1) and NPC developmental kidney markers (PODXL, CDH1 , LRP2) following further differentiation (Figure 14). It is therefore expected that following implantation at after 2 days of maturation (Day 11 , D11), that the micro-assembloids would continue to mature and produce functional kidney tissue architecture such as a functional nephron.9.2 In vivo engraftment

[0076] The ability of the organoids and assembloids to develop following implantation was assessed. The micro-assembloids, assembloids or NPC kidney organoids were implanted into mice. The assembloids and micro-assembloids were allowed to mature for 2 days before implantation (i.e. from D9-D11).

[0077] Following implantation, the assembloids and organoids continued to develop and mature in vivo. The micro-assembloids showed superior in vivo maturation versus NPC kidney organoids at 2 weeks post-implantation. Particularly, the micro-assembloids displayed a greater proportion of mature renal structures and fewer non-renal structures (Figure 12). At 2 weeks following graft, the NPC kidney organoids had few structural elements and showed mostly early renal differentiation. A large proportion of the graft was composed of cartilage. By contrast, in the engrafted micro-assembloids, multiple elements showing advanced differentiation into various renal compartments was visible 2 weeks postimplantation, including ureteric bud derived distal tubular structures. Only a minority of the graft volume was composed of off-target cartilage (Figure 12).

[0078] Further analysis of the micro-assembloid graft by immunohistochemical (ICH) staining revealed several different kidney-like structures in the micro-assembloid grafts 2 weeks post-implantation, including collecting ducts, glomeruli, distal tubules and the growth of host capillaries (Figure 13). The staining also revealed integration of the host endothelium with the graft-derived glomerular structure. Notably, the NPC lineage expressed HDXC10, Six2 and WT1 at D9 (i.e. before mixing with the UB cell lineage cells for assembloid formation) (Figure 13). The UB lineage expressed ETV5, RET, CAT A3 and CALB1 at D9 (i.e. before co-mixing with the NPC lineage for assembloid formation) (Figure 13).REFERENCES

[0079] All publications mentioned in the specification are herein incorporated by reference.

[0080] [1] C. Borestrbm et al., Kidney Int 94, 1099 (2018).[2] R. Morizane et al., Nat Biotechnol 33, 1193 (2015).[3] H. Tsujimoto et al., Cell Reports 31 , 107476 (2020).[4] B. S. Freedman et al., Nat Commun 6, 8715 (2015).[5] Q. Mai et al., Cell Res 17, 1008 (2007).[6] (n.d.).[7] K. Okonechnikov, A. Conesa, and F. Garcia-Alcalde, Bioinformatics 32, 292 (2016).[8] H. Li et al., Bioinformatics 25, 2078 (2009).[9] A. Dobin etal., Bioinformatics 29, 15 (2013).

[0010] P. Ewels et al., Bioinformatics 32, 3047 (2016).

[0011] J. M. Gaspar, BMC Bioinformatics 19, 536 (2018).

[0012] R. Patro et al. , Nat Methods 14, 417 (2017).

[0013] P. Di Tommaso et al., Nat Biotechnol 35, 316 (2017).

[0014] B. Griming et al., Nat Methods 15, 475 (2018).

[0015] M. I. Love, W. Huber, and S. Anders, Genome Biology 15, 550 (2014).

[0016] S. Anders and W. Huber, Genome Biology 11 , R106 (2010).

[0017] L. Kolberg et al., F1000Res 9, ELIXIR (2020).

[0018] U. Raudvere et al., Nucleic Acids Res 47, W191 (2019).

Claims

CLAIMS1 . An isolated cell population, wherein the cell population expresses: a) UB cell lineage associated genes (UB genes), and b) NPC lineage associated genes (NPC genes), wherein the cell population is in the form of a three-dimensional (3D) tissue capable of maturing to form one or more component of kidney organ architecture.

2. The isolated cell population of claim 1 , wherein the cell population is a kidney organoid.

3. The cell population of any preceding claim, wherein the 3D tissue has a diameter of less than about 800 pm4. The cell population of any preceding claim, wherein the 3D tissue has a diameter of about 200 to 450 pm.

5. The cell population of any preceding claim, wherein the 3D tissue was a diameter of about 250 to 300 pm.

6. The cell population of any preceding claim, wherein the cell population contains less than about 120,000 cells.

7. The cell population of any preceding claim, wherein the cell population contains 10,000 cells or less.

8. The cell population of any preceding claim, wherein the cell population contains about 2,000 to 10,000 cells.

9. The cell population of any preceding claim, wherein the cell population contains about 1 ,000 to 8,000 cells.

10. The cell population of any preceding claim, wherein the UB genes are UB lineage specific genes.

11. The cell population of any preceding claim, wherein the NPC genes are NPC lineage specific genes.

12. The cell population of any preceding claim, wherein the UB genes comprise RET, PAX2, PAX8, CALB1 , and / or ETV5.

13. The cell population of any preceding claim, wherein NPC genes comprise Six2, WT1 , HOXD11 , HOXC10 and / or OSR1.

14. The cell population of any preceding claim, wherein the UB genes comprise RET and the NPC genes comprise HOXD1.

15. The cell population of any preceding claim, wherein the cell population expresses one or more podocyte associated gene, optionally wherein the podocyte associated gene is PODXL.

16. The cell population of any preceding claim, wherein the cell population expresses one or more proximal tubuli associated gene, optionally wherein the proximal tub associated gene is PRP2.

17. The cell population of any preceding claim, wherein the cell population expresses one or more distal tub associated gene, optionally wherein the distal but associated gene is BRN1 and / or GATA3.

18. The cell population of any preceding claim, wherein the cell population expresses one or more collecting duct associated gene, optionally wherein the collecting duct associated gene is CALB1 and / or GATA3.

19. The cell population of any preceding claim, wherein the cell population expresses one or more collecting duct associated gene, optionally wherein the collecting duct associated gene is CALB1 and / or AQP2.

20. The cell population of any preceding claim, wherein the cell population expresses one or more endothelial cell associated gene, optionally wherein the endothelial cell associated gene is CD31 (PECAM1).21 . The cell population of any preceding claim, wherein the cell population expresses one or more proximal tubuli associated gene, optionally wherein the proximal tubuli associated gene is AQP1.

22. The cell population of any preceding claim, wherein the cell population expresses one or more renal pelvis associated gene, optionally wherein the renal pelvis associated gene is SCNN1 B and / or UPK1A.

23. The cell population of any preceding claim, wherein gene expression is defined relative to a control cell population not expressing the gene.

24. The cell population of claim 23, wherein gene expression is defined as at least 2-fold higher expression relative to a control cell population.

25. The cell population of any of claims 1 to 22, wherein UB genes expression is defined relative to a control cell population consisting of NPC or NPC lineage cells, wherein gene expression is defined as at least 2-fold higher expression relative to the control cell population.

26. The cell population of any of claims 1 to 22 or claim 25, wherein NPC genes expression is defined relative to a control cell population consisting of UB cells or UB lineage cells, wherein gene expression is defined as at least 2-fold higher expression relative to the control cell population.

27. The cell population of any preceding claim, wherein gene expression in the cell population is measurable by polymerase chain reaction, RNA sequencing, immunohistochemistry and / or immunofluorescence.

28. The cell population of any preceding claim, wherein the cell population is self-organised.

29. The cell population of any preceding claim, wherein the cell population comprises NPCs.

30. The cell population of any preceding claim, wherein the cell population comprises UB cells.31 . The cell population of any preceding claim, wherein the cell population comprises podocytes.

32. The cell population of any preceding claim, wherein the cell population comprises tubular cells.

33. The cell population of any preceding claim, wherein the cell population comprises epithelial cells.

34. The cell population of any preceding claim, wherein the cell population does not comprise mature kidney tissue architecture35. The population of any preceding claim, wherein the cell population does not comprise a nephron or nephrons.

36. The cell population any preceding claim, wherein kidney architecture may be visualised by immunohistochemistry or immunofluorescence.

37. The cell population of any preceding claim, wherein the cell population does not comprise a functional nephron.

38. The cell population of any preceding claim, wherein once implanted into the kidney of a subject, the cell population is capable of maturing into tissue comprising a functional nephron.

39. The cell population of any preceding claim, wherein once implanted into the kidney of a subject, the cell population is capable of maturing into tissue comprising glomeruli and / or collecting ducts.

40. The cell population of any preceding claim, wherein the cell population is derived from cells comprising stem cells.41 . The cell population of any preceding claim, wherein the cell population is derived from cells comprising embryonic stem cells.

42. The cell population of any preceding claim, wherein the cell population is derived from cells comprising induced pluripotent stem cells.

43. The cell population of any preceding claim, wherein the cell population is derived from cells comprising mesoderm cells.

44. The cell population of any preceding claim, wherein the cell population is derived from cells comprising intermediate mesoderm cells.

45. The cell population of any preceding claim, wherein the cell population is derived from a cell or cells that have been expanded ex vivo.

46. The cell population of any preceding claim, wherein the cell population is derived from a cell or cells isolated from a subject.

47. The cell population of any preceding claim, wherein the cell population is artificial or wherein the cell population does not exist in nature.

48. The cell population of any preceding claim, wherein the cell population comprises a cell that has been genetically modified or gene edited.

49. The cell population of any preceding claim, wherein the cell population is isolated from the body and is comprised in a pharmaceutical composition.

50. The cell population of any preceding claim, wherein the cell population has been cultured in vitro 3D conditions for less than 4 day.

51. The cell population of any preceding claim, wherein cell population has been cultured in vitro 3D conditions for about 2 days.

52. The cell population of any preceding claim, wherein the cell population has been cultured in vitro in media comprising media comprising a GSK-3 inhibitor, Fibroblast growth factor (FGF) and / or a ROCK inhibitor for about 2 days.

53. A method of producing a cell population of any of claims 1 to 52, the method comprising: a) combining NPC cells and UB cells to form a mixed population of cells, and b) culturing the mixed population of cells in vitro in conditions suitable forthe formation of a kidney organoid.

54. A method of producing a cell population, the method comprising: a) combining NPC and UB cells to form a mixed population of cells, b) culturing the mixed population of cells in vitro in conditions suitable forthe formation of a kidney organoid, wherein the cell population is in the form of a three-dimensional (3D) tissue capable of maturing to form one or more components of kidney organ architecture.

55. The method of any of claims 53 to 54, wherein the cell population is a kidney organoid.

56. The method of any of claims 53 to 55, wherein step b) comprises culturing the mixed population of cells in media comprising a GSK-3 inhibitor, Fibroblast growth factor (FGF) and / or a ROCK inhibitor.

57. The method of claim 56, wherein the GSK-3 inhibitor comprises CHIR99021.

58. The method of claim 56 or 57, wherein the FGF comprises FGF2.

59. The method of any of claims 56 to 58, wherein the ROCK inhibitor comprises Y-27632.

60. The method of any of claims 56 or 59, wherein the media comprises 200 ng / ml FGF2.

61. The method of any of claims 53 to 60, wherein the mixed population of cells is cultured in vitro for 3 days or less.

62. The method of any of claims 53 to 61 , wherein the mixed population of cells is cultured in vitro for about 2 days.

63. The method of any of claims 53 to 62, comprising deriving the NPC.

64. The method of any of claims 53 to 63, comprising deriving the UB cells.

65. The method of claim 64, wherein deriving the UB cells comprises culturing pluripotent stem cells in APS media for at least 24 hours.

66. The method of any of claims 53 to 65, wherein the NPC and / or UB cells are derived from cells comprising or consisting of ESC and / or iPSC.

67. The method of any of claims 53 to 66, wherein the UB cells express UB genes, wherein the UB genes comprise FOXA1 , KRT8, KRT18, CDH1 , FOXA1 , SPRED1 , WNT11 , RET, UPK1A, RBP1 , GFRA1 , LHX1 , KRT19 and / or AXIN2.

68. The method of any of claims 53 to 67, wherein the NPC do not express UB associated genes, wherein the UB genes comprise FOXA1 , KRT8, KRT18, CDH1 , FOXA1 , SPRED1 , WNT11 , RET, UPK1A, RBP1 , GFRA1 , LHX1 , KRT19 and / or AXIN2.

69. The method of any of claims 53 to 68, wherein the expression of one or more UB gene by the UB cells is at least 2-fold higher than the expression of the same UB gene by the NPC, wherein the one or more UB gene may comprise FOXA1 , KRT8, KRT18, WNT11 , RET, UPK1A, RBP1 , GFRA1 , LHX1 and / or KRT19.

70. The method of any of claims 67 to 69, wherein UB gene expression is defined relative to a control cell population not expressing the gene.71 . The cell population of any of claims 67 to 70, wherein gene expression is defined as at least 2- fold higher expression relative to a control cell population.

72. The cell population of any of claims 67 to 71 , wherein UB genes expression is defined relative to a control cell population consisting of NPC.

73. The cell population of any preceding claim, wherein gene expression in the cell population is measurable by polymerase chain reaction, RNA sequencing, immunohistochemistry and / or immunofluorescence.

74. The method of any of claims 54 to 73, wherein NPC and UB cells are combined in a ratio of about 1 :3.

75. A method of treating or prevent a disease in a subject, the method comprising administering to a subject in need thereof an effective amount of the cell population of any of claims 1 to 52.

76. The method of claim 75, wherein the cell population is derived from cells comprising an allogenic cell or cells consisting of allogenic cells.

77. The method of claim 75, wherein the cell population is derived from cells comprising an autologous cell or cells consisting of autologous cells.

78. The method of any of claims 75 to 77, wherein the disease is kidney disease, optionally endstage kidney disease.

79. A method of treating or prevent a disease in a subject, the method comprising: a) producing in vitro a cell population according to the method of any of claims 53 to 74, b) and implanting the cell population into the kidney of the subject.

80. A cell population obtainable by the method of any of claims 53 to 74.

81. A cell population obtainable by a method, the method comprising: a) combining NPC and UB cells to form a mixed population of cells; and b) culturing the mixed population of cells in vitro in conditions suitable forthe formation of a kidney organoid, wherein the cell population is in the form of a three-dimensional (3D) tissue capable of maturing to form one or more components of kidney organ architecture.

82. A pharmaceutical composition comprises the population of cells of any of claims 1 to 52, and a pharmaceutically excipient.

83. A pharmaceutical composition comprising a pharmaceutical excipient and a cell population comprising: a) UB cell lineage associated genes (UB genes), and b) NPC lineage associated genes (NPC genes), wherein the cell population is in the form of a three-dimensional (3D) tissue capable of maturing to form one or more components of kidney organ architecture.

84. A method of making a pharmaceutical composition, the method comprising combining the cell population of any of claims 1 to 52 with a pharmaceutical excipient.

85. A pharmaceutical composition for use in a method of treatment of a disease, wherein the method comprises administering the pharmaceutical composition to a subject, and wherein thepharmaceutical composition comprises the cell population of any of claims 1 to 52 and a pharmaceutical excipient.

86. The pharmaceutical composition for the use of claim 85, wherein the disease comprises kidney disease and the method comprises implanting the cell population into the kidney of the subject.

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