Differentiation of pluripotent stem cells into nephrosome-like bodies

Abstract

A method for producing a nephrosome is provided, the nephrosome comprising nephrons, ureteric buds and vascular systems and / or progenitor cells of these. In one embodiment, the method includes contacting the mid-segment mesoderm cells with the following under conditions that promote the formation of vascularized nephrosomes: fibroblast growth factor 9 and / or fibroblast growth factor 20 and / or fibroblast growth factor 2, optionally selected from one or more substances of the group consisting of: bone morphogenetic protein 7; heparin; Wnt agonists; retinoic acid; and RA antagonists. The nephrosomes may have such end uses, such as for kidney repair and regeneration, bioprinting of kidneys or their functional components, renal cell arrays, and compounds for screening nephrotoxicity.

Description

[0001] The present application is a divisional application of an invention patent application with an application date of December 15, 2015, application number 201580076197.X, and invention name “Differentiation of pluripotent stem cells to form nephrosomes”. Technical Field

[0002] The present invention relates to kidney development. More specifically, the present invention relates to an in vitro method of generating a nephroid, comprising nephrons and / or ureteric buds and / or progenitors of these, which are at least partially vascularized and contain a renal interstitial compartment. Background Art

[0003] With the prevalence of end-stage renal disease rising by 8% per year worldwide, renal regeneration strategies are urgently needed. The kidney is a mesodermal organ that differentiates from the adjacent intermediate mesoderm (IM)-derived metanephric mesenchyme (MM) through the formation of the ureteric bud (UB) and the interaction between this bud and the adjacent intermediate mesoderm (IM)-derived metanephric mesenchyme (MM). The nephron is derived from a population of nephron progenitor cells that originate from the MM. Other progenitor cells in the IM or MM are thought to contribute to components of the renal matrix / interstitium and the renal vasculature, including glomerular capillaries. The IM itself originates from the posterior primitive streak. Although the developmental origins of the kidney are well understood, nephron formation in the human kidney is completed before birth. 5 . Therefore, there are no postnatal stem cells capable of replacing lost nephrons.

[0004] Human pluripotent stem cells have great potential for generating cell-based therapies for kidney disease. However, the realization of human pluripotent stem cells as a cell source for clinical use and as a treatment for diseases such as kidney disease has been hampered by a lack of understanding of how to make the cell types necessary to generate nephrons and other structures of the kidney. Summary of the invention

[0005] The inventor uses the growth factor used during normal embryogenesis to successfully guide the differentiation of human pluripotent stem cells through the posterior primitive streak and the middle segment mesoderm (IM) under completely chemically defined monolayer culture conditions. This differentiation scheme leads to the synchronous induction of ureteric bud (UB) and metanephric mesenchyme (MM), and the synchronous induction forms a self-organizing structure in vitro, including nephron formation and segmentation, to form distal tubules, proximal tubules and Bowman's capsule. Organoids also contain mesenchyme-derived renal matrix. The components in this hESC source show a wide range of in vitro renal potential, illustrating the potentiality of renal regeneration based on pluripotent stem cells. In addition, the inventor has guided the differentiation of the vascular system in the nephroid body, including the nephron and matrix in the ureteric bud (UB), metanephric mesenchyme (MM) and MM sources of differentiation. In a particular form, the present invention provides the generation of nephron progenitor cells and ureteric epithelial progenitor cells that form the aggregation of nephroid bodies in the culture period shortened. More specifically, the present invention provides a method for directing human pluripotent stem cells to form complex multicellular nephrosomes that contain fully segmented nephrons surrounded by endothelium and renal stroma and are transcriptionally similar to human fetal kidneys.

[0006] Therefore, one aspect of the present invention provides a method for producing renal unit progenitor cells and ureteric epithelial progenitor cells, the method comprising the step of contacting intermediate mesoderm (IM) cells with: fibroblast growth factor 9 (FGF9) and / or fibroblast growth factor 20 (FGF20) and / or fibroblast growth factor 2 (FGF2); and optionally one or more agents selected from the group consisting of: bone morphogenetic protein 7 (BMP7); heparin; Wnt agonists; retinoic acid (RA), analogs or agonists; and RA antagonists; thereby producing renal unit progenitor cells and ureteric epithelial progenitor cells from IM cells under conditions that induce or promote the aggregation of renal unit progenitor cells and ureteric epithelial progenitor cells into one or more nephron-like bodies, which are at least partially vascularized and / or contain vascular progenitor cells.

[0007] In some embodiments, at least partial vascularization and / or the presence of vascular progenitor cells is promoted by conditions that promote or direct the development of vascular endothelial or vascular progenitor cells from mesenchymal cells or tissues.

[0008] In one embodiment, vascularization of the nephron-like body is promoted by including one or more human pluripotent stem cells and / or endothelial progenitor cells differentiated therefrom.

[0009] In another embodiment, vascularization of nephroid somatic cells is promoted by using an appropriate oxygen tension that promotes angiogenesis.

[0010] In one embodiment, IM cells are derived from or differentiated from posterior primitive streak cells.

[0011] In one embodiment, the posterior primitive streak cells are derived from or differentiated from human pluripotent stem cells (hPSCs). Non-limiting examples of hPSCs include human embryonic stem cells (hESCs) and induced human pluripotent stem cells (iPSCs).

[0012] A related aspect of the invention provides a method for producing mesodermal cells, the method comprising the steps of contacting hPSCs with a Wnt agonist, thereby producing mesodermal cells. The mesodermal cells can be a mixed population of mesodermal cells such as definitive mesoderm and mid-segment mesoderm (IM) including rostral IM and / or caudal IM.

[0013] The method may further comprise a subsequent step of contacting the definitive mesoderm cells with fibroblast growth factor 9 (FGF9) and / or fibroblast growth factor 20 (FGF20) and / or fibroblast growth factor 2 (FGF2).

[0014] Suitably, a subsequent step of contacting the definitive mesoderm cells with fibroblast growth factor 9 (FGF9) and / or fibroblast growth factor 20 (FGF20) and / or fibroblast growth factor 2 (FGF2) facilitates the formation of the intermediate mesoderm (IM), which subsequently produces the differentiation of ureteric epithelial cells and nephron progenitors from the IM cells.

[0015] In another embodiment, the method also comprises that cells are dissociated and reaggregated into cell mass for the subsequent step of cultivating in the presence of FGF2, FGF9 and / or FGF20.Suitably, this step helps produce the class nephron that comprises nephron.Cultivation in the presence of FGF2, FGF9 and / or FGF20 can be carried out on the floating filter at air / culture medium interface.

[0016] In another embodiment, the method also includes adding a Wnt agonist before removing FGF2, FGF9 and / or FGF20, and cultivating in the absence of growth factors subsequently. This step further promotes the formation of nephrons in the kidney-like body. Suitably, the Wnt agonist is in a relatively high concentration in a relatively short time (such as 30-60 minutes).

[0017] Optionally, culturing cells in the presence of a Wnt agonist, fibroblast growth factor 9 (FGF9) and / or fibroblast growth factor 20 (FGF20) and / or fibroblast growth factor 2 (FGF2) in any of the above steps may also include one or more of the following: a retinoic acid (RA) antagonist, RA or RA agonist, bone morphogenetic protein 7 (BMP7); and / or retinoic acid.

[0018] Preferably, the nephron is at least partially vascularized.

[0019] Preferably, the nephroid body comprises segmented nephrons surrounded by endothelium, perivascular cells and renal interstitium.

[0020] In one embodiment, vascularization of the nephron-like body is promoted by including one or more human pluripotent stem cells and / or endothelial progenitor cells differentiated therefrom.

[0021] In another embodiment, vascularization of the nephroid is promoted by using an appropriate oxygen partial pressure that promotes vascularization.

[0022] In one embodiment, the method further comprises the step of identifying viable nephron progenitor cells and / or ureteric epithelial progenitor cells.

[0023] In certain embodiments, identification of viable nephron progenitor cells and / or ureteric epithelial progenitor cells comprises measuring or detecting the co-expression of multiple nucleic acids and / or proteins that are markers of viable nephron progenitor cells and / or ureteric epithelial progenitor cells.

[0024] In another aspect, the present invention provides isolated, enriched or purified nephron and / or ureteric epithelial progenitor cells and / or nephron-like bodies produced according to the methods of the preceding aspects.

[0025] Preferably, the nephroid body comprises segmented nephrons surrounded by endothelium and renal interstitium.

[0026] In yet another aspect, the present invention provides a method for producing a kidney or renal cells or tissue, the method comprising the steps of differentiating the kidney or renal cells or tissue from the renal unit progenitor cells and / or ureteric epithelial progenitor cells and / or nephrons of the aforementioned aspects, thereby producing a kidney or renal cells or tissue.

[0027] In some embodiments, nephron progenitor cells and / or ureteric epithelial progenitor cells and / or nephronoids can be used for recellularization of a whole organ decellularized kidney to produce a reconstructed or replacement kidney.

[0028] In other embodiments, nephron progenitor cells and / or ureteric epithelial progenitor cells and / or nephronoids can be used as a source for cell therapy of kidney diseases and conditions.

[0029] In certain aspects, nephron progenitor cells and / or ureteric epithelial progenitor cells and / or nephron-like bodies can be used as a source of cells or tissue for bioprinting or bioengineering a whole kidney, a source of kidney cells and / or tissue for kidney transplantation or treatment of chronic renal damage or disease.

[0030] One particular aspect provides a method for bioprinting a renal structure, the method comprising depositing a plurality of hPSCs or other progenitor cells disclosed herein to form a renal structure having one or more functional characteristics of a kidney or its components, or which is capable of displaying one or more functional characteristics of a kidney or its components.

[0031] Suitably, the renal structure is at least partially vascularised and / or comprises vascular progenitor cells.

[0032] Suitably, hPSCs or other progenitor cells are subjected to the methods disclosed herein for generating nephron progenitor cells and ureteric epithelial progenitor cells in a three-dimensional structure.

[0033] This particular aspect also provides a bioprinted kidney structure having one or more functional characteristics of the kidney or its components, or capable of displaying one or more functional characteristics of the kidney or its components, produced by the aforementioned method.

[0034] Another specific aspect provides a method for bioprinting a renal structure, the method comprising depositing a plurality of nephron progenitor cells and ureteric epithelial progenitor cells disclosed herein to form a renal structure having one or more functional characteristics of the kidney or its components, or being capable of displaying one or more functional characteristics of the kidney or its components.

[0035] Suitably, the bioprinted renal structure is at least partially vascularized and / or comprises vascular progenitor cells.

[0036] Suitably, nephron progenitor cells and ureteric epithelial progenitor cells have been generated from hPSCs by the methods disclosed herein.

[0037] This particular aspect also provides a bioprinted kidney structure having one or more functional characteristics of the kidney or its components, or being capable of displaying one or more functional characteristics of the kidney or its components, produced by the above-described method.

[0038] Another aspect of the invention provides arrays of nephron progenitor cells and ureteric progenitor cells having a planar geometry.

[0039] The array may include 2-15 or more stacked arrays.

[0040] The array may be stacked in a tessellation pattern.

[0041] A related aspect of the invention provides nephroids obtained by maturing or differentiating the array of the preceding aspects or cells therein.

[0042] Suitably, the nephroid is at least partially vascularized and / or comprises vascular progenitor cells.

[0043] On the other hand, the present invention provides a method for determining the renal toxicity of one or more compounds, the method comprising the step of contacting the one or more compounds with isolated or purified nephron progenitor cells and / or ureteric epithelial progenitor cells, bioprinted renal structures, arrays and / or nephrons of the aforementioned aspects, or renal cells or tissues differentiated therefrom or otherwise obtained, thereby determining whether the one or more compounds are nephrotoxic.

[0044] In one embodiment, this aspect provides for bioprinting of nephron progenitor cells and / or ureteric epithelial progenitor cells into nephrotoxicity-like bodies for nephrotoxicity screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Differentiation effects of CHIR culture for 3, 4, or 5 days followed by FGF9 alone (b) or FGF9 (b) in combination with RA (a) or RA antagonists (c).

[0046] Figure 2 . MEIS1 is present between the forming nephrons + Presence of matrix population. Red = MEIS1; blue = nuclei.

[0047] Figure 3 .CD31 + Presence of vascular progenitor cells. Red = NPHS1 (podocytes), blue = nuclei, green = CD31.

[0048] Figure 4 All segments of the normally developing nephron are interconnected, including the collecting ducts (GATA3 + PAX2 + ECAD + ), distal tubule (ECAD + GATA3 - LTL - ), proximal tubule (LTL + AQP1 + ) and glomerulus (WT1 + NPHS1 + SYNPO + ), indicating normal embryonic organogenesis. Pink = GATA3, green = ECAD, blue = LTL, red = WT1, collecting ducts (pink and green), distal tubules (green), proximal tubules (blue), glomeruli (red in nuclei).

[0049] Figure 5. Nephroid bodies cultured 11 days after cell aggregation. a = bright field image with a diameter of 3 to 5 cm. b = immunofluorescence staining image. Green = ECAD, red = NPHS1, blue = LTL, distal tubule (green), proximal tubule (blue), glomerulus (red).

[0050] Figure 6 . A 45-minute pulse of high CHIR (5 μM) was added immediately after reaggregation, followed by 5 days of culture in FGF9 with or without AGN (retinoic acid inhibitor), BMP7, low CHIR, or RA, and then 6 days of culture without these factors. a = 4-day pellet without CHIR; b = 4-day pellet with 45-minute CHIR pulse; c = 11-day pellet without CHIR; d = 11-day pellet with 45-minute CHIR pulse.

[0051] Figure 7 .Selective induction of collecting duct or kidney mesenchymal lineages. a, Schematic illustration of the AP patterning mechanism of the IM during embryogenesis 13 . The timing of PSM cell migration determines the timing of exposure to FGF9 and RA, resulting in a fate choice between AI and PI. PSM, presomitic mesoderm; AI, anterior midsegment mesoderm; PI, posterior midsegment mesoderm; UE, ureteric epithelium; MM, metanephric mesenchyme. b, Schematic diagram of the three experimental timelines. c, Time course qPCR of the initial 7 days of differentiation with the above time settings. Experiments were performed using monolayer culture conditions. (Mean ± sd, n = 3 independent experiments) d, Immunofluorescence on day 7 of differentiation with AI marker GATA3 and PI marker HOXD11. Scale bar = 100 μm. Experimental replicates = 3e, Schematic illustration of RA signaling at the posterior primitive streak stage. The RA metabolizing enzyme CYP26 is expressed in the PSM region to shield PSM cells from RA signaling. f, Schematic diagram of the three experimental timelines. RA or AGN193109 (AGN) was added to FGF9 after CHIR99021, followed by growth factor withdrawal (no GF). Experiments were performed using monolayer culture conditions. g, Immunofluorescence at day 18 of differentiation following 3 days of CHIR99021 followed by ±RA / AGN. AGN inhibits AI specification of early migratory cells, leading to posteriorization. At day 18, GATA3 and HOXD11 marked UE and MM, respectively (left panel). GATA3 + PAX2 + ECAD + Cells represent UE, while GATA3 - PAX2 + Cell expression MM (ECAD - ) and its derivatives (ECAD +) (right panel). Experimental replicates = 3. Scale bar = 100 μm.

[0052] Figure 8 .Generation of nephron-like bodies equivalent to human fetal kidney in vitro. a, Schematic diagram of the hPSC differentiation protocol. b, Global bright field observation of self-organized nephron-like bodies across a time series. The success rate of organoid differentiation was 94.2% (138 organoids, 5 experiments). Scale bar = 1 mm. c, Immunofluorescence of plain scans of whole nephron-like bodies showed structural complexity. Scale bar = 1 mm d, High-power immunofluorescence microscopy showed that the nephron was divided into 4 compartments, including the collecting duct (CD, GATA3 + ECAD + ), distal tubule (DT, GATA3 - ECAD + LTL - ), proximal tubule (PT, ECAD - LTL + ) and glomerulus (G, WT1 + ). Scale bar = 100 μm. e, Confocal microscopy generated serial z-stack images from the bottom to the top of the day 11 nephron (Extended Data Videos 1 and 2). Schematic diagram illustrating the location of different structures within the organoid. e', e", and e"' are representative images taken through the organoid at the positions indicated in e. Each segment of the nephron is labeled (or colored in the schematic) as follows: collecting duct, GATA3 + ECAD + (green dots in yellow); distal tubule, ECAD + (yellow); proximal tubule, LTL + (red); glomerular NPHS1 (green circle). Scale bar = 100 μm. f, Visualization of the relative transcriptional identity of nephrosomes to 13 human fetal tissues (scores from 0 to 1, using the KeyGene algorithm 15 RNA-seq was performed on whole nephroids from 4 time points (days 0, 3, 11, 18 after clustering) x 3 individual organoids from 1 experiment / time point (see Supplementary Table 2). g, Dendrogram showing hierarchical clustering of human fetal organoids from the first and second trimesters with nephroids at days 0, 3, 11, and 18 based on the 85 key genes previously defined (Supplementary Table 3). 15 This clearly shows a close match between the 1st gestation fetal kidneys cultured from day 11 and day 18.

[0053] Fig. 9 .The nephroid contains differentiated nephrons, matrix, and vasculature, and matures over time.

[0054] a, Schematic diagram illustrating the developmental pathway of each cellular component from IM to kidney. CD, collecting duct; DT, distal tubule; LoH, loop of Henle; PT, proximal tubule; POD, podocyte; VASC, vasculature; STROM, renal interstitium. bj, Immunofluorescence of nephroids at day 11 or day 18. b, Collecting ducts marked by PAX2, GATA3, and ECAD. Scale bar = 50 μm. c, d, LTL at day 11 + ECAD - The early proximal tubule (open arrow). + ECAD + Mature proximal tubules appear at day 18 (white arrows). Scale bar = 100 μm. e, Proximal tubules express Cubilin (CUBN). Scale bar = 50 μm. f, Loops of Henle marked by UMOD and ECAD. Scale bar = 50 μm. g, Developing glomerulus with podocytes marked by WT1 and NPHS1. Scale bar = 50 μm. h, CD31 in the renal interstitium + Endothelial cells. Scale bar = 200 μm. i, Evidence of endothelial invasion into glomeruli at day 18 of culture. Scale bar = 50 μm. j, Renal interstitium marked by MEIS1. Scale bar = 100 μm. km, Transmission electron micrograph of nephroids. k, Putative distal tubules with relatively sparse short microvilli (m) and tight junctions (tj). l, Putative proximal tubules with lumen filled with extensively dense microvilli characteristic of a brush border (bb). m, Podocyte (p) with characteristic large nucleus and primary (pf) and secondary (sf) foot processes. Data represent at least 3 independent experiments.

[0055] Fig.10 .The functional maturity of the proximal tubules.

[0056] a, display LTL + Dextran uptake assay of tubule endocytosis capacity. Scale bar = 50 μm. b, Nephrosomes treated with 20 μM cisplatin resulted in LTL + ECAD + Proximal tubular cell apoptosis. Apoptotic cells were detected by staining with cleaved caspase 3 antibody (CASP3). Scale bar = 100 μm. c, Quantification of the number of apoptotic tubules showing mature proximal tubule-specific apoptosis by the nephrotoxic agent cisplatin. In response to 5uM and 20uM cisplatin, LTL + ECAD + Mature proximal tubules (PTs) undergo apoptosis in a dose-dependent manner. + ECAD - Immature PTs were unresponsive to cisplatin. P values ​​were calculated by independent t-test (mean ± SE, n = 5 independent experiments).

[0057] Fig.11 Regulation of nephrogenesis in nephrosomes. a, Stimulation of organoids with 5 μM CHIR99021 for 1 h immediately after aggregation promoted nephrogenesis (CHIR pulse), whereas only a limited number of nephrogenic events occurred in the absence of CHIR99021 (no pulse). Scale bar = 1 mm. b, After this CHIR99021 pulse, without the addition of FGF9, organoids did not induce nephrogenesis (-FGF9). Scale bar = 200 μm.

[0058] Fig.12 . Changes in gene expression during nephroid development. ac, Graphs showing changes in expression of selected marker genes at four time points (days 0, 3, 11, and 18) of nephroid cultures. The X-axis represents the detection counts of each gene in RNA sequencing analysis. Markers for nephron progenitors (cap mesenchyme) and collecting duct progenitors (ureter tip) peak at day 3 and then decline (a). Markers for early nephrons increase at day 3, while markers for mature nephron components (proximal and distal tubules and podocytes) begin after day 3. Graphs showing the expression region (blue) of each selected gene in the developing kidney (b).

[0059] Fig.13 Dendrogram showing hierarchical clustering of D0, D3, D11, D18 differentiation experiments and 21 human fetal organs from first and second trimesters (GSE66302) 15 The sample name consists of the individual ID followed by the organ name and gestational age. For example, "DJ1 kidney_9" represents a kidney from the 9th week of pregnancy from individual ID: DJ1. The D0 and D3 nephrosomes have gonads, consistent with the common origin of gonads and kidneys in the mid-segment mesoderm. The D11 and D18 nephrosomes show the strongest similarity to human kidneys in gestational age 1. The classification genes used for this analysis are detailed in Table 3.

[0060] Fig.14 Characterization of non-epithelial structures in nephroids. All images were taken from day 18 nephroids a, attached to KDR + Vascular PDGFRA + Pericytes. Scale bar = 50 μm. b, Some glomeruli contain PDGFRA that may represent early mesangial cells + cell 19 Scale bar = 50 μm. DETAILED DESCRIPTION

[0061] The present invention is based, at least in part, on the identification of specific in vitro culture conditions tailored to promote synchronous, simultaneous differentiation of nephron progenitors and ureteric epithelial progenitors from the mid-segment mesoderm (IM) to produce at least partially vascularized nephrosomes or other renal cells or tissue aggregates. More specifically, FGF9 plus heparin alone, or in combination with one or more agents including bone morphogenetic protein 7 (BMP7), retinoic acid (RA), RA antagonists; Wnt agonists and / or FGF20 plus heparin; and / or FGF2 plus heparin, can promote differentiation of mid-segment mesoderm into nephron progenitors and ureteric epithelial progenitors. In addition, the in vitro culture method provides a system for differentiating human embryonic stem cells through the posterior primitive streak, IM and metanephric mesenchyme stages to produce nephron progenitors and ureteric epithelial progenitors. Advantageously, the presence or absence of certain molecules such as RA, RA antagonists and / or Wnt agonists can be manipulated to preferentially promote the generation of nephron progenitors and ureteric epithelial progenitors, and vice versa. More specifically, the invention is also based on the discovery that human pluripotent stem cells can be directed to form a complex multicellular nephrosome that contains fully segmented nephrons surrounded by endothelium and renal stroma and is transcriptionally similar to human fetal kidney. Angiogenesis can be promoted by conditions that promote or direct the development of vascular endothelium from mesenchymal cells or tissues.

[0062] Nephron progenitor cells and ureteric epithelial progenitor cells are induced simultaneously, guiding each other to differentiate in vivo, and are able to develop into different tubular epithelial structures including the ureteric tree and nephron progenitor mesenchyme, during which the epithelial structure replaces the ureteric tip to maintain the nephron progenitor cells. It is therefore proposed that the hESC-derived ureteric epithelial and / or nephron progenitor cells produced according to the present invention can guide differentiation into renal cells from both the ureteric and mesenchymal compartments. In addition, the ability of these cells to "self-organize" into aggregated organoid structures can be further utilized to promote kidney repair, for example, with the aid of kidney bioengineering. As described above, nephron progenitor cells, nephrons derived therefrom, or "self-organized" nephrons may also be suitable for renal toxicity tests that were previously hindered by the inability to produce cells suitable for testing.

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described.

[0064] As used herein, the term "comprise" and variations of the term such as "comprising", "comprises" and "comprised" are not intended to exclude further additives, components, integers or steps, unless the context requires otherwise.

[0065] It will be understood that the indefinite articles "a" and "an" cannot be regarded as singular indefinite articles or do not include more than one or more than one single object to which the indefinite article refers. For example, "a" cell includes one cell, one or more cells and a plurality of cells.

[0066] For purposes of the present invention, "isolated" means material that has been removed from its natural state or otherwise artificially manipulated. An isolated material (e.g., a cell) may be substantially or essentially free of components that normally accompany it in its natural state, or may be manipulated to be in an artificial state with components that normally accompany it in its natural state.

[0067] "Enriched" or "purified" means having a higher incidence, representation or frequency in a particular state (eg, an enriched or purified state) compared to a previous state prior to enrichment or purification.

[0068] The terms "differentiate", "differentiating" and "differentiated" refer to the progression of cells from an earlier or initial stage of a developmental pathway to a later or more mature stage of a developmental pathway. It will be understood that in this context, "differentiated" does not mean or imply that the cells are fully differentiated and have lost pluripotency or the ability to progress further along the developmental pathway or along other developmental pathways. Differentiation may be accompanied by cell division.

[0069] A "progenitor cell" is a cell that is capable of differentiating along one or more developmental pathways, with or without self-renewal. Typically, a progenitor cell is unipotent or oligopotent and capable of at least limited self-renewal.

[0070] As will be well understood in the art, the stage or state of cell differentiation can be characterized by expressing and / or not expressing one or more markers. In this case, "marker" refers to a nucleic acid or protein encoded by the genome of a cell, cell population, pedigree, compartment, or subset, whose expression or expression pattern changes throughout the developmental process. Nucleic acid marker expression can be detected or measured by any technology known in the art, including nucleic acid sequence amplification (e.g., polymerase chain reaction) and nucleic acid hybridization (e.g., microarray, Northern hybridization, in situ hybridization), but is not limited thereto. Protein marker expression can be detected or measured by any technology known in the art, including flow cytometry, immunohistochemistry, immunoblotting, protein arrays, protein analysis (e.g., 2D gel electrophoresis), but is not limited thereto.

[0071] One aspect of the present invention provides a method for producing renal unit progenitor cells and ureteric epithelial progenitor cells, the method comprising the steps of contacting intermediate mesoderm (IM) cells with: BMP7; retinoic acid (RA); RA antagonist; Wnt agonist; fibroblast growth factor 9 (FGF9) and / or FGF20; and heparin; thereby generating renal unit progenitor cells and ureteric epithelial progenitor cells from IM cells.

[0072] "Retinoic acid" or "RA" referred to herein includes all forms of retinoic acid (e.g., including all trans-RA and 9-cis RA), analogs and / or retinoic acid receptor (RAR) agonists having similar biological activity to RA. Various RA analogs and RAR agonists (including non-selective and selective agonists to RARα, β or γ) are commercially available from, for example, R&D Systems and Tocris Bioscience.

[0073] Specifically contemplated "RA antagonists" include retinoic acid receptor (RAR) antagonists and any other molecules that inhibit, block or prevent RA signal transduction through RAR. Non-limiting examples of RAR antagonists include AGN193109, LE 135, ER50891, BMS 493, BMS 453 and MM 11253, but are not limited thereto. This definition does not exclude the possibility that RA antagonists also or alternatively mimic the possibility of blocking signal transduction via RAR by binding of another ligand.

[0074] As used herein, "Wnt agonists" are molecules that inhibit GSK3 (e.g., GSK3-β) in the context of a canonical Wnt signaling pathway, but preferably not in the context of other non-canonical Wnt signaling pathways. Non-limiting examples of Wnt agonists include CHIR99021, LiCl SB-216763, CAS 853220-52-7, and other Wnt agonists commercially available from companies such as Santa Cruz Biotechnology and R&D Systems. This definition should not be considered to absolutely exclude the possibility that a Wnt agonist mimics one or more other inhibitors of GSK3β activity.

[0075] It will also be understood that although FGF9 is preferred, fibroblast growth factors such as FGF2, FGF9 and FGF20 may be interchangeable. Heparin is typically included to promote or enhance the biological activity of fibroblast growth factors such as FGF2, FGF9 and / or FGF20.

[0076] The preferred concentrations of FGF9, BMP7, retinoic acid (RA); RA antagonists; Wnt agonists; FGF20 and heparin are described in more detail below. It will also be mentioned that the presence or absence of certain molecules such as RA agonists or analogs, RA antagonists and / or Wnt agonists can be controlled or manipulated to preferentially promote the generation of nephron progenitor cells and ureteric epithelial progenitor cells, and vice versa.

[0077] As used herein, "nephron progenitor cells" are progenitor cells derived from the metanephric mesenchyme that can differentiate into all nephron segments (except collecting ducts) by an initial mesenchymal to epithelial transition, including nephron epithelial cells such as connecting segments, distal tubule curvature (DCT) cells, distal tubule straight (DST) cells, proximal tubule straight (PST) segments 1 and 2, PST cells, podocytes, glomerular endothelial cells, ascending loop of Henle and / or descending loop of Henle, but are not limited thereto. Nephron progenitor cells are also capable of self-renewal.

[0078] Non-limiting examples of markers specific to or representative of metanephric mesenchyme include WT1, SIX1, SIX2, SALL1, GDNF and / or HOXD11, but are not limited thereto. Non-limiting examples of markers specific to or representative of nephron progenitor cells include WT1, SIX1, SIX2, CITED1, PAX2, GDNF, SALL1, OSR1 and HOXD11, but are not limited thereto.

[0079] "Ureteric epithelial progenitor cell" means an epithelial progenitor cell derived from, obtainable from or originating from the mesonephric duct or its derivative, the ureteric bud, which can develop into renal tissue and / or structures such as the collecting duct.

[0080] Non-limiting examples of markers specific to or representative of ureteric epithelial progenitor cells include, but are not limited to, HOXB7, cRET, GATA3, CALB1, E-CADHERIN, and PAX2.

[0081] As described above, nephron progenitors and ureteric epithelial progenitors are differentiated from the intermediate mesoderm (IM) in the presence of FGF9 alone or in combination with one or more agents comprising BMP7, retinoic acid (RA), agonists or analogs, RA antagonists such as AGN193109 and / or FGF20, preferably heparin.

[0082] "Mid-mesoderm (IM)" cells refer to embryonic mesodermal cells that arise from the definitive mesoderm, which is derived from the posterior primitive streak and can ultimately develop into the urogenital system, including the ureters and kidneys, as well as other tissues such as gonads. Non-limiting examples of markers that are unique to or representative of the mid-mesoderm include PAX2, OSR1, and / or LHX1.

[0083] It will also be understood that the production of IM cells does not mean that the IM cells are a pure or homogenous population of IM cells without the presence of other cell types (e.g., definitive mesoderm). Thus, reference to "IM cells" or "IM cell populations" refers to cells or cell populations that contain IM cells.

[0084] Suitably, according to the present invention, IM cells are produced by contacting posterior primitive streak cells with one or more agents that promote differentiation of posterior primitive streak cells into IM cells, as will be described in more detail below.

[0085] Preferably, IM cells are produced by contacting posterior primitive streak cells with one or more agents that promote differentiation of posterior primitive streak cells into IM cells.

[0086] Typically, the one or more agents include fibroblast growth factor 9 (FGF9) and optionally a RA antagonist such as AGN 193109 and / or one or more other FGFs such as FGF2 and / or FGF20.

[0087] "Posterior primitive streak (PPS)" cells refer to cells that can be obtained from the cells at the rear end of the primitive streak structure formed in the blastocyst in the early stages of mammalian embryonic development, or cells that correspond functionally and / or phenotypically to the cells. The posterior primitive streak establishes bilateral symmetry, determines the position of gastrulation and initiates germ layer formation. Typically, the posterior primitive streak is a progenitor cell of the mesoderm (i.e., the putative mesoderm), and the anterior primitive streak is a progenitor cell of the endoderm (i.e., the putative endoderm). Non-limiting examples of markers that are peculiar to or represent the posterior primitive streak include Brachyury (T). Non-limiting examples of markers that are peculiar to or represent the anterior primitive streak are SOX17. MIXL1 can be expressed by the posterior primitive streak and the anterior primitive streak.

[0088] It will also be understood that the production of posterior primitive streak cells does not mean that the posterior primitive streak cells are a pure or homogenous population of posterior primitive streak cells without the presence of other cell types. Thus, reference to "posterior primitive streak cells" or "population of posterior primitive streak cells" means a cell or cell population comprising posterior primitive streak cells.

[0089] Suitably, according to the present invention, as will be described in more detail below, posterior primitive streak cells are generated by contacting hPSC cells with one or more agents that promote differentiation of hPSC cells into posterior primitive streak cells.

[0090] Typically, the one or more agents include bone morphogenetic protein 4 (BMP4), activin A, and / or a Wnt agonist such as CHIR99021.

[0091] The terms "human pluripotent stem cells" and "hPSC" refer to cells derived from, obtainable from, or originating from human tissue that exhibit pluripotency. The hPSC may be a human embryonic stem cell or a human induced pluripotent stem cell.

[0092] Human pluripotent stem cells can be derived from the inner cell mass or reprogrammed using Yamanaka factors from many fetal or adult somatic cell types. hPSCs may be generated using somatic cell nuclear transfer.

[0093] The terms "human embryonic stem cell", "hES cell" and "hESC" refer to cells derived from, obtainable from or derived from a human embryo or blastocyst, which are self-renewing and pluripotent or omnipotent, having the ability to generate all cell types present in a mature animal. Human embryonic stem cells (hESC) can be isolated from, for example, a human blastocyst obtained from a human preimplantation embryo in vivo, an in vitro fertilized embryo, or a single-cell human embryo expanded to the blastocyst stage.

[0094] The terms "induced pluripotent stem cells" and "iPSCs" refer to cells derived from, obtainable from, or derived from any type of human adult somatic cell that is reprogrammed to a pluripotent state by expression of exogenous genes, such as transcription factors, including a preferred combination of OCT4, SOX2, KLF4, and c-MYC. hiPSCs display a level of pluripotency equivalent to hESCs, but can be derived from patients undergoing autologous therapy (with or without simultaneous gene correction prior to differentiation and cell delivery).

[0095] More generally, the methods disclosed herein can be applied to any pluripotent stem cell derived from any patient, or subsequently modified using gene editing to generate a mutant model hPSC, or mutant hPSC corrected using gene editing.Gene editing can be performed by CRISPR, TALEN or ZF nuclease technology.

[0096] From the foregoing it will be appreciated that a preferred broad form of the invention provides a method comprising the following sequential steps:

[0097] (i) contacting hPSCs with one or more agents that promote differentiation of hPSCs into postprimitive streak cells;

[0098] (ii) contacting the posterior primitive streak cells with one or more agents that promote differentiation of the posterior primitive streak cells into mid-mesodermal cells; and

[0099] (iii) contacting the mid-mesoderm cells with FGF9 and optionally one or more of: BMP7; retinoic acid; a RA antagonist such as AGN193109; a Wnt agonist such as CHIR99021; FGF20; and heparin; thereby generating metanephric mesenchymal cells and ureteric epithelial progenitor cells from the mid-mesoderm cells.

[0100] These sequential steps are described below.

[0101] (i) Differentiation of hPSCs into posterior primitive streak

[0102] It can be understood from the above that hPSCs are contacted with BMP4, activin A and / or CHIR99021 in a suitable serum-free medium such as APEL differentiation medium (Ng et al., 2008, Nat. Protoc. 3:768), but not limited thereto, to produce posterior primitive streak cells that appropriately include posterior primitive streak cells. hPSCs can be hESCs or iPSCs.

[0103] Suitably, the concentration of BMP4 is about 5-40ng / mL, and the concentration of activin A is about 3-40ng / mL. In one embodiment, the concentration of BMP4 is about 20-35ng / mL, or more preferably about 30ng / mL. In one embodiment, the concentration of activin A is about 5-30ng / mL, or more preferably 10ng / mL. Suitably, the best relative activity ratio is in the range of 3:1 to 1:6 BMP4 to activin A. Preferably, the best relative activity ratio is in the range of 3:1 to 1:1 BMP4 to activin A.

[0104] In some embodiments, the concentration of a Wnt agonist such as CHIR99021 may be in the range of about 0.5 to 50 μM, preferably about 4-30 μM, more preferably about 5-20 μM or advantageously about 8 μM. In certain embodiments, CHIR99021 is present alone in the absence of BMP4 and Activin A.

[0105] The stem cell population can be cultured in a medium containing BMP4, Activin A, and / or a Wnt agonist such as CHIR99021 for 36-120 hours.

[0106] In some non-limiting embodiments, cells can be contacted with BMP4, Activin A and / or CHIR99021 for a longer period of time than required for hESCs. For example, cells such as iPSCs can be contacted with BMP4, Activin A and / or CHIR99021 for up to 96-120 hours.

[0107] The culture medium can be changed every 24-48 hours.

[0108] Although not wishing to be bound by theory, as disclosed herein, contacting hPSCs with BMP4, activin A and / or Wnt agonists such as CHIR99021 results in the formation of primitive streaks (PS) including the posterior primitive streak. This is the initial step in generating mesoderm and endoderm tissues. Typically, differentiation of hPSCs is toward a mixed cell population comprising cells expressing markers characteristic of the posterior primitive streak (i.e., putative mesoderm) and cells expressing markers characteristic of the anterior primitive streak (i.e., putative endoderm).

[0109] Non-limiting examples of markers characteristic of the posterior primitive streak (putative mesoderm) include Brachyury (T).

[0110] A non-limiting example of a marker characteristic of the preprimitive streak (putative endoderm) is SOX17.

[0111] (ii) Differentiation of posterior primitive streak cells into intermediate mesoderm (IM)

[0112] Suitably, the posterior primitive streak cells or a mixed primitive streak population comprising posterior primitive streak cells are contacted with one or more fibroblast growth factors (FGFs) comprising at least FGF9 and optionally FGF2 and / or FGF20 and / or a retinoic acid (RA) antagonist in a suitable medium in the absence of serum, such as APEL differentiation medium.

[0113] Typically, the retinoic acid signaling antagonist is a retinoic acid receptor (RAR) inhibitor or antagonist, such as AGN193109.

[0114] Suitably, the concentration of FGF2, FGF9 and / or FGF20 is about 100 to 400 ng / mL. In a preferred embodiment, the concentration of FGF2, FGF9 and / or FGF20 is about 150 to 300 ng / ml or advantageously about 200 ng / mL. In one embodiment, the concentration of the RA antagonist (e.g., AGN193109) is about 0.1-10 μM, more preferably 0.5-5 μM.

[0115] The cells are contacted with FGF9, alone or with FGF9 and FGF2 and / or FGF20 and / or RA antagonists (e.g., AGN193109), for at least about 96 hours but not more than about 190-200 hours. Preferably, the cells are contacted with FGF9 alone or with FGF9 and FGF2 and / or FGF20 and / or RA antagonists (e.g., AGN193109) for about 96 hours.

[0116] The culture medium can be changed every 40-48 hours.

[0117] In one embodiment, contacting the posterior primitive streak cells, which typically express markers characteristic of the posterior primitive streak (putative mesoderm) and anterior primitive streak (putative endoderm), with FGF9 alone or with FGF9 together with FGF2 and / or FGF20 results in differentiation of the cells into a population of cells expressing markers characteristic of the intermediate mesoderm (IM). Non-limiting examples of markers characteristic of the intermediate mesoderm include PAX2, LHX1, and OSR1.

[0118] (iii) Differentiation of the mid-segment mesoderm (IM) into nephron progenitor cells and ureteric epithelial progenitor cells

[0119] Suitably, after contacting the posterior primitive streak cells with FGF2, FGF9 and / or FGF20, the resulting IM cells are contacted with FGF9 alone or in combination with one or more of BMP7, RA, RA antagonists, FGF20, Wnt agonists and / or heparin in a suitable serum-free culture medium such as APEL differentiation medium.

[0120] Suitably, the FGF9 concentration is about 20 ng to 1 μg / mL. In a preferred embodiment, the concentration of FGF9 is about 50-500 ng / mL, more preferably about 100-300 ng / mL or advantageously about 200 ng / mL. Typically, heparin is included at a concentration of about 0.1-10 μg / mL, preferably about 0.3-5 μg / mL, 0.5-2 μg / mL or advantageously about 1 μg / mL.

[0121] In one embodiment, the concentration of FGF20 is about 20 ng to 1 μg / mL. In a preferred embodiment, the concentration of FGF20 is about 50-500 ng / mL, more preferably about 100-300 ng / mL or advantageously about 200 ng / mL.

[0122] In one embodiment, the FGF2 concentration is about 20 ng to 1 μg / mL. In a preferred embodiment, the FGF2 concentration is about 50-500 ng / mL, more preferably about 100-300 ng / mL or advantageously about 200 ng / mL.

[0123] It will be appreciated that FGF20 and FGF2 may replace or supplement FGF9 as these agents have similar biological activities.

[0124] In one embodiment, the concentration of BMP7 is about 25 to 75 ng / mL. In a preferred embodiment, the concentration of BMP7 is about 35-60 ng / mL, 45-55 ng / mL, or advantageously about 50 ng / mL.

[0125] In one embodiment, RA is at a concentration of about 10 pM to 1 μM. In a preferred embodiment, the concentration of RA is about 30 pM to 0.5 μM, more preferably about 50 pM to 0.2 μM or advantageously about 0.1 μM. Although not binding on the present invention, preliminary data indicate that higher concentrations of RA promote a relative increase in the proportion of ureteric epithelial progenitor cells, while lower concentrations of RA promote a relative decrease in the proportion of ureteric epithelial progenitor cells.

[0126] In one embodiment, the concentration of the RA antagonist, such as AGN193109, is about 50 pM to 10 μM. In a preferred embodiment, the concentration of AGN193109 is about 0.01 μM to 5 μM, more preferably about 0.1 μM to 5 μM or advantageously about 1 μM. Although not binding on the present invention, preliminary data indicate that higher concentrations of AGN193109 promote a relative increase in the proportion of metanephric mesenchymal cells.

[0127] In one embodiment, the Wnt agonist, such as CHIR99021, is present at a concentration of about 0.1 μM to 10 μM, preferably about 0.2 μM to 5 μM or more preferably about 1-2 μM.

[0128] Although not binding on the present invention, Wnt agonists promote a relative increase in the production of nephron progenitor cells from IM cells. Preferably, cells are contacted with FGF9 alone or with FGF9 together with one or more of BMP7, RA, Wnt agonists, RA antagonists and / or FGF20 and / or FGF2 plus heparin for at least 72 hours but not more than 360 hours. Preferably, cells are contacted for about 160-220 hours, or more preferably about 190-200 hours.

[0129] The culture medium can be changed every 48-72 hours.

[0130] Typically, as disclosed herein, mid-segment mesoderm cells are contacted with FGF9 alone or with FGF9 together with one or more of BMP7, RA, RA antagonists, Wnt agonists and / or FGF20 and / or FGF2 and preferably heparin to differentiate mid-segment mesoderm cells into cells of the metanephric mesenchyme and ureteric epithelial cell lineages. The metanephric mesenchyme lineage includes nephron progenitor cells that are optimally produced after culturing in FGF9 and heparin for about 72 hours. It is also proposed that the presence, absence and / or concentration of RA analogs or agonists and / or RA antagonists can be selected to manipulate the relative amount of ureteric epithelium produced by the method compared to the metanephric mesenchyme produced by the method. As previously described, RA promotes the formation of ureteric epithelium at the expense of metanephric mesenchyme, while RA antagonists such as AGN193109 promote the formation of metanephric mesenchyme at the expense of ureteric epithelium. Wnt agonists such as CHIR99021 may also promote the survival and / or formation of the metanephric mesenchyme at the expense of the ureteric epithelium.

[0131] Non-limiting examples of markers specific to or representative of cells of the metanephric mesenchymal lineage or cells thereof include WT1, SIX1, SIX2, SALL1, GDNF and / or HOXD11, but are not limited thereto.

[0132] Non-limiting examples of markers specific to or representative of nephron progenitor cells include WT1, SIX2, CITED1, PAX2, GDNF, SALL1, and HOXD11, but are not limited thereto.

[0133] Non-limiting examples of markers characteristic of or representative of cells of the ureteric epithelial lineage include, but are not limited to, HOXB7, GATA3, CALB1, E-CADHERIN, PAX2 and / or cRET.

[0134] Based on the co-expression of WT1, SIX2, CITED1, PAX2, GDNF, SALL1 and HOXD11, nephron progenitor cells can be maximally generated 11-15 days or advantageously 14 days (range of days 11 to 15) after initiation of this method from hPSC cell culture.

[0135] Based on the co-expression of HOXB7, cRET, E-CADHERIN and PAX2, ureteric epithelial progenitor cells can be maximally generated after at least 10 days or advantageously 14 days following this method starting from hPSC culture.

[0136] In a preferred form of the method, FGF9 is present in at least a portion of or completely throughout both steps (ii) and (iii) described herein. More preferably, a Wnt agonist such as CHIR99021 is present in at least a portion of step (i) described herein.

[0137] A particularly preferred method comprises the following steps in sequence:

[0138] (a) Human pluripotent stem (hPSC) cells were exposed to CHIR99021 to promote the differentiation of hPSC cells into postnatal

[0139] Strip cells;

[0140] (b) contacting the posterior primitive streak cells with FGF9 (alone or together with a RA antagonist such as AGN 193109) to promote differentiation of the posterior primitive streak cells into IM cells; and

[0141] (c) Treating IM cells with FGF9 and heparin (alone or together with RA antagonists such as AGN193109)

[0142] contact, thereby generating nephron progenitor cells and ureteric epithelial progenitor cells from IM cells.

[0143] According to this preferred form, renal differentiation from an initial population of hES cells can be promoted within a total culture period of about 18-20 days.

[0144] Rapid generation of nephrosomes from hPSCs

[0145] A related aspect of the invention provides a method for producing definitive mesoderm cells, the method comprising the steps of: contacting hPSC with a Wnt agonist for a longer period of time (optimally 3-5 days). Suitably, the method of this aspect produces a mesodermal cell population comprising one or more of definitive mesoderm cells and IM cells, the IM cells may include beak and tail IM. Generally, the longer the duration of culture with a Wnt agonist, the more tail IM appears and the less beak IM remains.

[0146] In one embodiment, the method further comprises the subsequent step of contacting the mesodermal cells with fibroblast growth factor 9 (FGF9) and / or fibroblast growth factor 20 (FGF20) and / or fibroblast growth factor 2 (FGF2). Suitably, this step facilitates differentiation of the caudal and rostral IMs. Suitably, the caudal and rostral IMs will in turn differentiate into nephron progenitor cells and ureteric epithelial cells, respectively. As previously described, the inclusion of RA or an analog or agonist may increase the relative production of ureteric epithelial progenitor cells.

[0147] In another embodiment, after contacting the middle mesoderm (IM) cells with fibroblast growth factor 9 (FGF9) and / or fibroblast growth factor 20 (FGF20) and / or fibroblast growth factor 2 (FGF2), the method also includes a subsequent step of dissociating and reaggregating the cells. This can be cultured on a floating filter at the air-medium interface. Suitably, this step helps to form a nephron-like body containing nephrons, matrix and vascular system.

[0148] In another embodiment, after forming the aggregates for culture (eg, on a floating filter), the method further comprises subsequently adding a Wnt agonist for 30-60 minutes. Suitably, this step facilitates the generation of aggregated nephrosomes with the largest nephrons.

[0149] It should be understood that the preferred purpose of this method is to produce aggregated, differentiated nephron progenitor cells and ureteric epithelial progenitor cells that form organoids or other at least partially organized renal structures. Preferably, the presence of all segments of the normally developing nephron can be present in the collecting duct (phenotype is GATA3 + ECAD + ), early distal tubules (phenotyped as GATA3 - LTL - ECAD + ), early proximal tubules (phenotype LTL + ECAD - ) and glomeruli (phenotype WT1 + ) organoids, suggesting normal embryonic organogenesis.

[0150] In one embodiment, aggregates of differentiated cells for culture as organoids can be formed in about 7 days of culture as described above.

[0151] The preferred concentration of the Wnt agonist (eg CHIR99021) is about 1-50 mM, preferably about 1-20 μM, 5-15 μM or advantageously about 8 μM. Preferably, the duration of contact with the Wnt agonist is about 4 days.

[0152] Suitably, the FGF9 concentration is about 20 ng to 1 μg / mL. In a preferred embodiment, the FGF9 concentration is about 50-500 ng / mL, more preferably about 100-300 ng / mL or advantageously about 200 ng / mL. Preferably, the duration of subsequent contact with FGF9 / FGF20 / FGF2 is about 3 days including heparin.

[0153] Preferably, a short pulse of a Wnt agonist such as CHIR99021 is added immediately after reaggregation after culture in FGF9 / FGF20 / FGF2 plus heparin. The preferred concentration of the Wnt agonist (e.g., CHIR99021) is about 1-15 μM, preferably about 2-10 μM or advantageously about 5 μM. The short pulse is typically 0.5 to 2 hours, for example about 45 minutes or 1 hour.

[0154] The formation of aggregated, at least partially organized structures such as nephrosomes can be assisted by maintaining or promoting physical contact between cultured cells. In this regard, the clumping of cells before adding a "short pulse" of a Wnt agonist (e.g., CHIR99021) can help organoid formation.

[0155] Optionally, the culture may further include: one or more of a retinoic acid (RA) antagonist, RA or RA agonist, bone morphogenetic protein 7 (BMP7) and / or heparin. The respective concentrations and effects of retinoic acid (RA) antagonists, RA agonists, bone morphogenetic protein 7 (BMP7); and / or heparin may be as described above.

[0156] Induce angiogenesis

[0157] Suitably, at least part of the vascularisation and / or presence of vascular progenitor cells in the nephroids or aggregates is promoted by conditions that promote or direct the development of vascular endothelial or vascular progenitor cells from mesenchymal cells or tissues.

[0158] Suitably, aggregates of differentiated cells and / or organoids produced according to the foregoing aspects can be cultured under conditions that promote at least partial vascularization, particularly angiogenesis of glomerular structures, or at least the generation of progenitor cells of vascular endothelial or other vascular cells or tissues. In some embodiments, angiogenesis is promoted by conditions that promote or direct the development of vascular endothelial cells from mesenchymal cells or tissues.

[0159] In one embodiment, the method can include co-culturing vascular endothelial progenitor cells (e.g., differentiated from human pluripotent stem cells) with IM cells as described above, or adding to a culture of at least partially differentiated nephron progenitor cells and ureteric epithelial progenitor cells, thereby generating vascular cells or tissue such as vascular endothelium.

[0160] In another embodiment, the method may include a reduced oxygen partial pressure during the culture period. Typically, 21% O2 is a common oxygen partial pressure present in a standard tissue culture incubator. The present invention contemplates 5 to 12% O2, which may be more equivalent to the oxygen partial pressure experienced in a developing embryo. This may increase the ability of the metanephric mesenchyme to produce VEGFA, thereby inducing Flk1 + Formation and migration of endothelial progenitor cells.

[0161] In view of the above, the protein agents mentioned, such as BMP4, BMP7, activin A, FGF2, FGF9 and FGF20, should be understood to include natural or recombinant or chemically synthesized proteins of any mammalian (including human, mouse and rat) origin, but are not limited thereto. In addition, these proteins may include chemical modifications known in the art, glycosylation, lipidation, labels such as biotin and additional amino acid sequences such as epitope tags or fusion partners. Typically, the above proteins can be commercially available and / or prepared as recombinant or chemically synthesized proteins by conventional laboratory or manufacturing methods.

[0162] In another aspect, the invention provides isolated or purified nephron progenitor cells, ureteric epithelial progenitor cells, and / or nephron-like bodies produced according to the methods disclosed herein.

[0163] The nephroid body preferably includes segmented nephrons surrounded by endothelium and renal interstitium.

[0164] In certain embodiments, the nephron is divided into four (4) or more components, including the collecting duct (phenotyped as GATA3 + ECAD + ), early distal tubules (phenotyped as GATA3 - LTL - ECAD + ), early proximal tubules (phenotype LTL + ECAD - ) and glomeruli (phenotype WT1 + ). Appropriately, a collecting duct tree forms at the bottom of the organoid, connecting to the distal and proximal tubules in the middle, with glomeruli at the top of the organoid.

[0165] It should be understood that nephron progenitor cells and / or ureteric epithelial progenitor cells can be obtained after an appropriate culture period as described above, and in some optional embodiments can be further enriched or purified based on the co-expression of surface markers. Cell enrichment or purification can be performed by any technique or method known in the art, including flow cytometry cell sorting (e.g., FACS), by magnetic immunobeads (e.g., Dynabeads TM ) for positive or negative cell selection, panning, density separation or complement-mediated lysis, but are not limited thereto.

[0166] Kidney regeneration and transplantation

[0167] Chronic kidney disease is a serious medical condition that affects 31 million Americans and 1.7 million Australians each year. Patients may lose 90% of their kidney function before developing symptoms, leading to kidney failure, dialysis or a kidney transplant. The medical costs of end-stage renal disease in the United States were estimated at $28 billion in 2010.

[0168] Therefore, one aspect of the present invention provides a method for producing kidney or renal cells or tissues, the method comprising the step of differentiating kidney or renal cells or tissues from isolated or purified nephron and / or ureteric epithelial progenitor cells to produce kidney or renal cells or tissues. In addition, this aspect of the present invention provides at least partial angiogenesis and / or produces vascular progenitor cells under conditions that promote or guide vascular endothelial or vascular progenitor cells to develop from mesenchymal cells or tissues.

[0169] The present invention provides a method for producing cells of ureteric epithelial and metanephric mesenchymal lineages or compartments. Preferably, these cells are induced simultaneously and guide each other's differentiation in vivo. These cells can develop into different tubular epithelial structures, including ureteric tree and nephron progenitor mesenchyme. Therefore, it is proposed that the ureteric epithelial and / or nephron progenitor cells derived from hPSC cells prepared according to the present invention can be guided to differentiate into nephrocytes from ureteric and metanephric mesenchymal compartments. Under appropriate conditions, nephron progenitor cells can be able to differentiate into any nephron segment (except collecting duct) including nephron epithelial cells, such as connecting segments, distal tubule curvature (DCT) cells, distal tubule straight (DST) cells, proximal tubule straight (PST) segments 1 and 2, PST cells, podocytes, glomerular endothelial cells, Henle's ascending loop and / or Henle's descending loop, but not limited thereto.

[0170] Furthermore, the ability of these cells to "self-organize" could therefore be exploited to promote kidney repair, for example through renal tissue or organ bioengineering.

[0171] It will be appreciated that one embodiment of the method of this aspect may include adoptively transferring or transplanting isolated or purified nephron and / or ureteric epithelial progenitor cells into a human, thereby generating kidney or renal cells or tissue.

[0172] According to this embodiment, differentiation of the isolated or purified nephron and / or ureteric epithelial progenitor cells into renal or kidney cells or tissue occurs in vivo.

[0173] Another embodiment of the method of this aspect can include at least partially differentiating the isolated or purified nephron and / or ureteric epithelial progenitor cells in vitro into kidney, or renal cells or tissue, or progenitor cells of these. Suitably, at least part of the in vitro differentiated cells kidney, or renal cells or tissue, or progenitor cells of these are adoptively transferred or transplanted into a human.

[0174] According to either or both embodiments, the kidney, kidney cells or tissue may promote or aid in the regeneration or repair of the kidney, its cells or tissue.

[0175] One embodiment provides organoids, the nephron progenitor cells and / or ureteric epithelial progenitor cells separated therefrom to produce the purposes of engineering or artificial kidney.For example, separated nephron progenitor cells and / or ureteric epithelial progenitor cells can be incorporated into a scaffold, such as decellularized human kidney or its extracellular matrix (ECM) components, polyester villi or biodegradable polymer scaffolds, so as to produce regenerated tubular structures.For example, such a method may include one or more of the following: (a) one or more differentiated cell types and / or intermediate progenitor cell types are separated from organoids; and (b) one or more differentiated cell types and / or intermediate progenitor cell types are delivered to decellularized kidney scaffolds.In some embodiments, the ECM from kidney scaffolds can be used as a matrix (e.g., produced from a single ECM or an ECM combined with a hydrogel), wherein one or more differentiated cell types and / or intermediate progenitor cell types are inoculated or bioprinted so that kidney scaffolds or matrix are recellularized.

[0176] Another embodiment may involve repairing a damaged or diseased kidney. For example, the method may include one or more of the following: (i) isolating one or more differentiated cell types and / or intermediate progenitor cell types from the organoid; (ii) delivering one or more differentiated cell types and / or intermediate progenitor cell types to the damaged or diseased kidney, thereby promoting repair and / or regeneration of the diseased or damaged kidney. Direct delivery to the damaged or diseased kidney by intraparenchymal injection or by vascular access.

[0177] Another embodiment of the present invention provides the use of renal cells or tissues differentiated from isolated nephron progenitor cells and / or ureteric epithelial progenitor cells in a device for assisting or promoting renal dialysis. For example, a bioartificial kidney can be prepared by inoculating renal cells or their progenitor cells into a reactor to produce a "renal assist device" used in parallel with dialysis.

[0178] Also contemplated is "bioprinting" of a kidney or other nephron-containing organ, organoid, or organ-like structure using renal cells or tissue differentiated or otherwise obtained from the isolated nephron progenitor cells and / or ureteric epithelial progenitor cells described herein.

[0179] Thus, a particular aspect of the present invention provides a method for bioprinting a kidney structure, the method comprising depositing a plurality of hPSCs or other progenitor cells disclosed herein to form a kidney structure having one or more functional characteristics of a kidney or its components, or which is capable of displaying one or more functional characteristics of a kidney or its components.

[0180] Suitably, hPSCs or other progenitor cells are subjected to the methods disclosed herein for generating nephron progenitor cells and ureteric epithelial progenitor cells in a three-dimensional structure.

[0181] This particular aspect also provides a bioprinted kidney structure having one or more functional characteristics of the kidney or its components, or capable of displaying one or more functional characteristics of the kidney or its components, prepared by the above method.

[0182] Another specific aspect of the present invention provides a method for bioprinting a renal structure, the method comprising depositing a plurality of nephron progenitor cells and ureteric epithelial progenitor cells disclosed herein to form a renal structure, the renal structure having one or more functional characteristics of the kidney or its components, or being capable of displaying one or more functional characteristics of the kidney or its components.

[0183] Suitably, nephron progenitor cells and ureteric epithelial progenitor cells have been generated from hPSCs by the methods disclosed herein. In some embodiments, the method produces a bioprinted kidney structure having or capable of displaying at least partial vascularization and / or vascular progenitor cells.

[0184] This particular aspect also provides a bioprinted kidney structure having one or more functional characteristics of the kidney or its components, or capable of displaying one or more functional characteristics of the kidney or its components, prepared by the above method. In some embodiments, a bioprinted kidney structure having or capable of displaying at least partial angiogenesis and / or vascular progenitor cells.

[0185] Suitably, the bioprinted kidney structure is a three-dimensional kidney structure.

[0186] It will also be understood that, as will be described in more detail below, three-dimensional structures can be constructed or formed from multiple bioprinted "layers" or "arrays."

[0187] The bioprinted kidney structural components can be or include any structural and / or functional components of the kidney, such as, but not limited to, glomeruli, juxtaglomerular organelles, interstitial tissue, collecting ducts, Bowman's capsule, proximal and / or distal tubular curvature, vascular systems such as arterioles, arteries, veins and / or capillaries.

[0188] Suitably, the bioprinted renal structure is at least partially vascularized and / or comprises vascular progenitor cells.

[0189] In some embodiments, the bioprinted kidney or kidney components can be implantable or otherwise adoptively transferred into a host.

[0190] As used herein, "bioprinting" includes and encompasses three-dimensional, precise cell deposition (e.g., cell solutions, cell-containing gels, cell suspensions, cell concentrates, multicellular aggregates, organoids, multicellular bodies, etc.) using methods compatible with automated or semi-automated computer-assisted three-dimensional prototyping equipment (e.g., bioprinters). In this regard, reference is made to U.S. patent applications US20120116568, US20130164339, and US20140012407, which are incorporated herein by reference, and provide non-limiting examples of potentially suitable bioprinting techniques.

[0191] For example, in some embodiments, at least one component of engineered, implantable renal tissue and / or organ can be bioprinted. In other embodiments, engineered, implantable tissue and / or organ are completely bioprinted. In other embodiments, a method of preparing a bioprinted construct using rapid prototyping techniques based on three-dimensional, automated, computer-assisted deposition of renal cells as disclosed herein (including cell solutions, cell suspensions, gels or pastes containing cells, cell concentrates, multicellular bodies (e.g., cylinders, spheres, ribbons, etc.)) and limiting materials to a biocompatible surface (e.g., composed of hydrogels and / or porous membranes) through a three-dimensional delivery device (e.g., a bioprinter) is used. As used herein, in some embodiments, the term "engineering" refers to renal tissue and / or organ, meaning that cells, cell solutions, cell suspensions, gels or pastes containing cells, cell concentrates, multicellular aggregates, and layers thereof are positioned to form a three-dimensional structure by a computer-assisted device (e.g., a bioprinter) according to a computer script. In other embodiments, a computer script is, for example, one or more computer programs, computer applications, or computer modules. In still other embodiments, three-dimensional tissue structures are formed by post-printing fusion of cells or multicellular bodies similar to the self-assembly phenomenon in early morphogenesis.

[0192] Although many methods can be used to arrange cells, multicellular aggregates and / or layers thereof on biocompatible surfaces to produce three-dimensional structures, it is advantageous to include manual placement and positioning by automated computer-assisted machines (e.g., biobeaters). Advantages of using this technology to deliver cells or multicellular bodies include rapid, accurate and reproducible placement of cells or multicellular bodies to produce constructs that exhibit a planned or predetermined orientation or pattern of cells, multicellular aggregates and / or layers thereof with various compositions. Advantages also include ensuring high cell density while minimizing cell damage. In some embodiments, the method of bioprinting is continuous and / or substantially continuous. A non-limiting example of a continuous bioprinting method is to dispense bio-ink from a bioprinter via a dispensing tip (e.g., a syringe, a capillary, etc.) connected to a bio-ink reservoir. In a further non-limiting embodiment, a continuous bioprinting method is to dispense bio-ink in a repetitive pattern of functional units. In various embodiments, the repeating functional unit has any suitable geometry, including, for example, a circle, a square, a rectangle, a triangle, a polygon and an irregular geometry. In other embodiments, the repeating pattern of the bioprinted functional unit comprises a layer or array, and a plurality of layers or arrays are bioprinted (e.g., stacked) adjacently to form an engineered tissue or organ. In various embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more layers or arrays are bioprinted (e.g., stacked) adjacently to form an engineered kidney tissue or organ.

[0193] In some embodiments, the functional units of bioprinting are repeated in a checkerboard pattern. A "checkerboard pattern" is a graphic plane that has no overlap and no gaps. Advantages of continuous and / or checkerboard bioprinting include, by way of non-limiting example, increased productivity of bioprinted tissues. Another non-limiting exemplary advantage is the elimination of the need to align the bioprinter with previously deposited bio-ink elements. Continuous bioprinting can also facilitate printing of larger tissues from large bio-ink reservoirs, optionally using a syringe mechanism.

[0194] In various embodiments, methods for continuous bioprinting include optimizing and / or balancing parameters such as print height, pump speed, robot speed, or combinations thereof, independently or relative to each other. In one example, the bioprinter head speed for deposition is 3 mm / s, the dispensing height of the first layer is 0.5 mm, and the dispensing height of each subsequent layer increases by 0.4 mm. In some embodiments, the dispensing height is approximately equal to the diameter of the bioprinter dispensing tip. Indefinite, appropriate and / or optimal dispensing distances do not cause material to flatten or adhere to the dispensing needle. In various embodiments, the bioprinter dispensing tip has an inner diameter of approximately 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 650, 600, 650, 700, 750, 800, 850, 900, 950, 1000 μM or more, including increments therein. In various embodiments, the bio-ink reservoir of the bioprinter has a volume of about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 cm 3 or more, including increments therein. In some embodiments, the pump speed is suitable and / or optimal when the residual pressure buildup in the system is low. In some embodiments, a favorable pump speed depends on the ratio between the cross-sectional area of ​​the reservoir and the dispensing needle, wherein a larger ratio requires a lower pump speed. In some embodiments, a suitable and / or optimal printing speed enables deposition of uniform lines without affecting the mechanical integrity of the material.

[0195] By way of example only, Organovo has collaborated with Invetech to develop an organ printer that uses a hydrogel scaffold to place human cells in a desired orientation to reconstruct human organs. Kidney cells or tissue differentiated from the isolated nephron progenitor cells and / or ureteric epithelial progenitor cells described herein or otherwise obtained can be used with an Organovo machine such as that described above to develop a "bioprinted" human nephron or kidney.

[0196] Another aspect of the invention provides arrays of nephron progenitor cells and ureteric progenitor cells having a planar geometry.

[0197] The array may comprise a plurality of stacked arrays, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 or more stacked arrays.

[0198] Arrays can be stacked in a checkerboard pattern.

[0199] A related aspect of the invention provides nephroids obtained by maturing the array of the preceding aspects.

[0200] Suitably, the nephroid is at least partially vascularized and / or comprises vascular progenitor cells.

[0201] It will also be appreciated that the directed differentiation of the isolated nephron progenitor cells and / or ureteric epithelial progenitor cells, organoids, and bioprinted renal structures described herein can be a potential source of purified, differentiated renal cell subtypes, organoids, and / or bioprinted renal structures for cell therapy.

[0202] For example, the nephron progenitor cells and / or ureteric epithelial progenitor cells separated as described herein can be used to produce kidney cells or tissues after gene correction in certain genetic renal diseases. For example, the repair of monogenic nephropathy such as Alport syndrome (COL4A3 mutation) and polycystic kidney disease (PKD1, PKD2, etc.) can be assisted or promoted by regenerating renal tissue from nephron progenitor cells and / or ureteric epithelial progenitor cells separated as described herein after gene correction.

[0203] In a specific embodiment, iPSC lines derived from, obtained from or originating from a patient with a genetic kidney disease can be used to repair a genetic mutation in vitro.Such cells can be used according to the methods of the invention and then administered to a patient for autologous cell therapy.

[0204] Nephrotoxicity screening

[0205] It will also be appreciated that the directed differentiation of the isolated nephron progenitor cells and / or ureteric epithelial progenitor cells described herein can provide a potential source of purified differentiated renal cells, bioprinted renal structures, nephrosomes, arrays, or renal tissue subtypes for renal toxicity screening.

[0206] The development of interventions aimed at preventing disease, including drugs and cell-based therapies, is hampered by the lack of availability of primary human kidney cells for in vitro drug testing.

[0207] Therefore, another aspect of the present invention provides a method for determining the renal toxicity of one or more compounds, the method comprising the step of contacting one or more compounds with nephron progenitor cells and / or ureteric epithelial progenitor cells described herein (as organoids or after isolation and purification, or renal cells or tissues differentiated therefrom or otherwise obtained therefrom), thereby determining whether the one or more compounds are nephrotoxic.

[0208] Preferably, the method is performed using organoids or renal cells or tissues derived from isolated or purified nephron progenitor cells.

[0209] Many useful drugs have nephrotoxic side effects, such as through direct tubular effects (e.g., aminoglycosides, cisplatin, radioactive control media, NSAIDs, ACE inhibitors), interstitial nephritis (e.g., beta-lactam antibiotics, lithium, CsA, anti-epileptic drugs such as phenytoin), or glomerulonephritis. Therefore, it is advantageous to test new or existing drugs using defined, specific renal cell and tissue types differentiated from or otherwise obtained from the isolated or purified nephron progenitor cells described herein. The above-mentioned "bioprinted" kidneys or bioprinted kidney-like bodies can also be used for nephrotoxicity screening.

[0210] Nephrotoxicity can be assessed or measured by any suitable assay for in vitro renal cell function, including decreased creatinine clearance or biomarker expression, such as by the Human Nephrotoxicity RT from Qiagen. 2 Profiler TM PCR Array or High Content Analysis (HCA) Multiplexed Nephrotoxicity Assay from Eurofins, but not limited thereto.

[0211] As described in more detail in the Examples, cisplatin is a nephrotoxic substance that induces caspase-mediated acute apoptosis of proximal tubular cells in the kidney. Cisplatin treatment of nephrosomes induces specific acute apoptosis of mature proximal tubular cells, while immature cells do not undergo apoptosis.

[0212] In order that the present invention may be readily understood and put into practical effect, reference is made to the following non-limiting examples.

[0213] Example

[0214] The research leading to the present invention has determined specific in vitro culture conditions, which are customized to promote the synchronization and simultaneous differentiation of nephron progenitor cells and ureteric epithelial progenitor cells from the middle mesoderm (IM). More specifically, FGF9 plus heparin alone, or in combination with one or more agents including bone morphogenetic protein 7 (BMP7), retinoic acid (RA), RA antagonists; Wnt agonists and / or FGF20 plus heparin, can promote the differentiation of the middle mesoderm into nephron progenitor cells and ureteric epithelial progenitor cells. In addition, the in vitro culture method provides a system for differentiating hPSCs through the posterior primitive streak, IM and metanephric mesenchyme stages to produce nephron progenitor cells and ureteric epithelial progenitor cells. The presence or absence of certain molecules such as RA, RA antagonists and / or Wnt agonists can be manipulated to preferentially promote the generation of nephron progenitor cells and ureteric epithelial progenitor cells, and vice versa. The posterior PS is a progenitor cell population for mesoderm such as IM, and is induced from hPSC using a Wnt agonist (e.g., CHIR99021). The IM differentiates into two key renal progenitor cell populations: the ureteric epithelium (UE), the progenitor of the collecting ducts, and the metanephric mesenchyme (MM), the progenitor of the nephrons. While the anterior IM gives rise to the UE, the posterior IM develops into the MM. Here, we present a method to simultaneously induce the anterior and posterior IM over carefully defined time periods using CHIR99021. This simultaneous induction leads to the successful generation of nephron-like bodies containing all expected renal components, including nephrons, mesenchyme, and endothelium. We also propose that the addition of inhibitors of RA signaling, such as the synthetic and potent pan-retinoic acid receptor (RAR) antagonist AGN 193109, will promote the formation of the metanephric mesenchyme.

[0215] Example 1

[0216] Materials and methods

[0217] Non-limiting examples of sources for reagents mentioned in these methods are provided in Table 1.

[0218] The culture medium was as follows:

[0219] Gelatin solution: 0.1% gelatin in PBS. The solution was then autoclaved.

[0220] FDMEM: 89% DMEM high glucose, 10% fetal bovine serum, 1% GlutaMAX supplement, 0.5% penicillin / streptomycin.

[0221] KSR medium: 77.8% DMEM / F-12, 20% Knockout serum replacement, 1% NEAA, 1% GlutaMAX, 0.5% penicillin / streptomycin, 0.2% 2-mercaptoethanol (55 mM). The medium was then filtered with Stericup-GP.

[0222] MEF-conditioned KSR medium: Add 40 mL of KSR medium to 10 million mouse embryonic fibroblasts in a T175 flask for one day. Collect the medium the next day and add another 40 mL of KSR medium. After repeating 6 times, collect and combine the medium and filter it through Stericup-GP.

[0223] • APEL: Refill one bottle of STEMdiff APEL (100 mL) with 0.5 mL of antibiotic-antimycotic.

[0224] For other reagents, methods, PCR primers, etc., see also International Publication WO 2014 / 197934 and Takasato, M. et al., 2014, . Nat. Cell Biol. 16, 118-26, which are incorporated herein by reference in their entirety.

[0225] Inoculation of feeder layer (8th day before last)

[0226] At 25cm 2 Coat the tissue culture flask with 3 mL of 0.1% gelatin solution.

[0227] A vial of frozen mitotically inactivated mouse embryonic fibroblasts (MEF) was thawed by heating the vial at 37°C until small ice pellets remained. 5 mL of warm FDMEM medium was added dropwise to the vial and gently mixed. Collected into a 15 mL tube and centrifuged at 1500 rpm for 3 minutes.

[0228] Remove the supernatant and resuspend MEFs in FDMEM. Plate at 12,000 cells / cm in FDMEM. 2 Inoculate in flasks and incubate overnight in a CO2 incubator at 37 °C.

[0229] Thawing hESC / iPSC (7th day before the end)

[0230] Thaw the frozen vials of iPSC / hESC by heating the vials at 37°C into the prepared 25 cm medium containing mitotically inactivated MEFs. 2 1. Add 5 mL of warm KSR medium (see Appendix A) dropwise into the vial and mix gently. Collect into a 15 mL tube and centrifuge at 1,500 rpm for 3 minutes.

[0231] Every 25cm 2 Prepare 5 mL of KSR medium in a tissue culture flask. Add 10 ng / mL bFGF to the KSR medium.

[0232] The supernatant was removed and iPSC / hESC was resuspended in KSR medium containing 10 ng / mL bFGF, seeded into flasks and incubated in a CO2 incubator at 37°C for 3 days.

[0233] The spent KSR medium was aspirated daily and supplemented with 5 mL of fresh KSR medium containing 10 ng / mL bFGF.

[0234] Dissociation of hESC / iPSC and Matrigel adaptation (3rd day before the end)

[0235] Matrigel coating:

[0236] Aliquot 3 mL of cold DMEM / F12 basal medium into 15 mL tubes.

[0237] Add 25uL of hESC-qualified Matrigel to DMEM / F12. Mix well and transfer to a 25cm 2 (*Handle Matrigel on ice in solidified form during heating.)

[0238] Keep the flask at room temperature for at least 30 min to allow the Matrigel to coat the surface.

[0239] Prepare 5 mL of MEF-conditioned KSR medium (see Appendix B) and add 10 ng / mL bFGF to the medium.

[0240] Dissociate cells:

[0241] (*For best results, cells should be approximately 80-90% confluent. If cells are not confluent, allow an additional day for incubation or perform a lower split ratio.)

[0242] Wash the confluent 25 cm 2 flask twice.

[0243] Add 2 mL of TrypLE Select to the cells and incubate at 37 °C for 3 minutes.

[0244] Pipette 5 mL of DMEM / F12 basal medium onto the cells, mix and ensure that the cells have detached from the plastic surface.

[0245] Collect the cell suspension at a 1:3 split ratio in a 15 mL tube and centrifuge at 1,500 rpm for 3 min.

[0246] Seeding cells onto Matrigel-coated flasks:

[0247] Remove the supernatant and add 5 mL of the prepared conditioned KSR medium to the cells. Mix gently.

[0248] Aspirate the DMEM / F12 containing Matrigel from the prepared tissue culture flask and seed the cells.

[0249] Incubate overnight in a CO2 incubator at 37 °C for two days.

[0250] The spent conditioned KSR medium was aspirated daily and supplemented with 5 mL of fresh conditioned KSR medium containing 10 ng / mL bFGF.

[0251] Seeding cells for differentiation (1st to last day)

[0252] Wash 25 cm with 3 mL of PBS 2 flask twice.

[0253] Add 2 mL of TrypLE Select to the cells and incubate at 37 °C for 3 minutes.

[0254] Pipette 5 mL of DMEM / F12 basal medium onto the cells, mix and ensure that the cells have detached from the plastic surface.

[0255] Collect the cell suspension into a 15 mL tube. Count.

[0256] Calculate the number of cells needed to achieve 4,000 cells per well (12,500 cells / cm 2 ).

[0257] Aliquot the desired number of cells into 15 mL tubes and centrifuge at 1,500 rpm for 3 minutes.

[0258] Resuspend cells in conditioned KSR containing 10 ng / mL bFGF, 50 uL per well. Seed cells into 96-well glass bottom plates and incubate at 37°C overnight.

[0259] Differentiation stage 1 (day 0)

[0260] For a newly opened bottle of APEL, add antibiotic-antimycotic at 1 / 100 (100x).

[0261] Prepare 8 μM CHIR in APEL.

[0262] Aspirate the conditional KSR from the 96-well plate.

[0263] 100 μl of APEL containing 8 μM CHIR was added to the cells.

[0264] Incubate at 37 °C for 4 days, refreshing the medium every 2 days.

[0265] Differentiation stage 2 (Day 4)

[0266] Prepare 200 ng / mL FGF9 + 1 μg / mL heparin in APEL.

[0267] Aspirate the depleted APEL containing CHIR from the 96-well plate.

[0268] 100 μL APEL containing 200 ng / mL FGF9 + 1 μg / mL heparin was added to the cells.

[0269] Incubate at 37°C for 2 days and on day 6 refresh the medium with fresh APEL containing 200 ng / mL FGF9 + 1 μg / mL heparin.

[0270] 3D organoid culture (Day 7)

[0271] C32 IPS cells were cultured in 96-well plates under different conditions.

[0272] Aspirate the medium and wash quickly with sterile PBS.

[0273] Aspirate PBS.

[0274] Add 50 μL of trypsin EDTA (0.25%) to each well.

[0275] Place in a 37 °C incubator for up to 3 min to allow cells to detach from the surface.

[0276] Observe under a microscope to ensure that all cells have detached from the glass (96-well) surface. If cells are still attached to the surface, gently aspirate the cells with trypsin and return to the incubator for an additional 2 minutes.

[0277] Neutralize trypsin with 100 μL DMEM + 10% FBS + 1% P / S.

[0278] The entire culture was aliquoted into 15 mL Falcon tubes.

[0279] The cells were centrifuged at 1500 rpm for 5 minutes.

[0280] Aspirate the medium until only the cell pellet remains.

[0281] Resuspend cells in 3 mL CHIR.

[0282] Take out 10 μL of the cell suspension and count the cells using a hemocytometer.

[0283] Each 3D pellet organoid will have approximately 5×10 5 cells and aliquot the required amount of cell suspension into 15 mL Falcon tubes.

[0284] The tubes were centrifuged at 2000 RPM for 2 minutes.

[0285] 1.2 mL APEL + 5 mL CHIR were aliquoted into 6-well Transwell polyester membrane cell culture plates.

[0286] Using a P1000 pipette, slowly pipette the precipitate out of the APEL-containing solution.

[0287] The pellet was removed by using a P1000 wide-bore pipette tip.

[0288] Carefully place the pellet onto the Transwell filter with minimal culture medium.

[0289] Place in cell culture incubator for 1 hour.

[0290] 1.2 mL APEL + 200 ng / mL FGF9 + 1 μg / mL heparin was aliquoted into Corning Costar 6-well plates.

[0291] The filter cultured in APEL+5μM CHIR for 1 hour was transferred to APEL+200ng / mL FGF9+1μg / mL heparin plate and cultured for 5 days. (The culture medium was changed every two days)

[0292] After 5 days, the filters were removed and placed into freshly prepared Corning Costar 6-well plates with 1.2 ml of fresh APEL.

[0293] Organoids were cultured for an additional 6 days in medium containing only APEL. (The medium was changed every two days)

[0294] 3D organoid immunofluorescence (Day 18)

[0295] After 6 days of organoid culture, pellets were fixed with 1% paraformaldehyde at 4 °C for 20 min.

[0296] Remove the paraformaldehyde and wash three times with PBS.

[0297] Once fixed and washed, organoids can be stored at 4 °C for up to one week prior to immunofluorescence staining.

[0298] Approximately 150 uL of blocking buffer (10% donkey serum / 0.3% Triton-x / PBS) was aliquoted into MatTek glass bottom culture dishes.

[0299] Carefully cut the organoids out of the filter and submerge the filter in blocking buffer.

[0300] Block organoids for 2-3 hours.

[0301] Prepare the primary antibody of choice in blocking buffer.

[0302] Most antibodies are listed at a dilution of 1:300.

[0303] Aspirate the Blocking Buffer from the MatTek dish and aliquot 150 μL of Blocking Buffer + primary antibody into the MatTek dish.

[0304] Organoids were incubated with primary antibodies overnight at 4°C.

[0305] Aspirate the primary antibody from the dish and wash six times with PBTX for 10 min each.

[0306] Prepare the secondary antibody of choice (1:400 diln) in PBTX (0.1% Triton-X / PBS).

[0307] Organoids were incubated in secondary antibodies for 4 h.

[0308] The secondary antibody was removed and the sections were incubated with DAPI (1:1000 dilution) in PBS for 1 h.

[0309] Wash three times with PBS, 10 min each time.

[0310] Prepare for imaging.

[0311] result

[0312] like Figure 1 As shown, in order to select the collection tube (ECAD + ,GATA3 + ,PAX2 + ), fewer days (2-3) of initial Wnt agonism and / or subsequent culture with FGF9 and activation of retinoic acid signaling are optimal. + HOXD11 + ECAD - ) and generate epithelial structures expressing early nephron markers), more days (3-5) of initial Wnt agonism and / or subsequent culture with FGF9 and inhibition of retinoic acid signaling are optimal.

[0313] We now have evidence that organoids formed through aggregation of human pluripotent stem cells using the methods disclosed herein display the following features indicative of normal kidney organogenesis. Figure 2 As shown, Meis1 + The presence of a matrix population has been shown to be present between forming nephrons. This population is known to be generated by the metanephric mesenchyme, is present between developing nephrons in the embryonic kidney, and has been shown to contribute to the formation of the perivascular space of the final organ. Figure 3 CD31 + Vascular progenitor cells. We see evidence of endothelium as assessed by CD31, however CD31 is a marker for mature endothelium and we do not yet see early endothelial progenitor cells that show active invasion of the glomerulus to form functional filtration units. Figure 4 and Figure 5 Shows the presence of all segments of the normally developing nephron, including the collecting duct (GATA3 + PAX2 + ECAD + ), distal tubule (ECAD + GATA3 - LTL - ), proximal tubule (LTL + AQP1 + ) and glomerulus (WT1 + NPHS1 + SYNPO + ), which are interconnected, suggesting normal embryonic organogenesis.

[0314] Variations in a number of parameters increased the overall size, complexity, maturity, and number of nephrons present within organoids formed from human pluripotent stem cells.

[0315] These include the formation of aggregates of differentiated cells for culture as organoids after 7 days of induction. Initially, these were shown to be able to form after 14 or 18 days of existing culture. We can now reliably obtain organoid formation after 7 days, which includes 4 days of culture in 8 μM CHIR followed by 3 days of culture in FGF9 with or without AGN (retinoic acid inhibitor), BMP7, low CHIR or RA, in a range of concentrations as described previously. Figure 6 As shown, aggregate formation was enhanced by adding a 45 min pulse of high CHIR (5 μM) immediately after reaggregation, followed by incubation in FGF9 with or without AGN (retinoic acid inhibitor), BMP7, low CHIR, or RA at concentrations as previously described.

[0316] The formation of vascularized glomeruli is essential for kidney function. Demonstration that this glomerular vasculature can even form in vitro would significantly increase the credibility of organoids as a kidney model.

[0317] One approach combines human pluripotent stem cells differentiated into kidneys with human pluripotent stem cells differentiated into endothelial progenitor cells, such as Orlova 31 plan.

[0318] Another approach is to reduce the oxygen tension during differentiation from 21% O2 (common in a standard tissue culture incubator) to 5 to 12% O2 (more similar to that experienced in a developing embryo). We anticipate that this will significantly improve the ability of the metanephric mesenchyme to produce VEGFA and induce the formation and migration of Flk1+ endothelial progenitor cells.

[0319] Example 2

[0320] Cell culture and differentiation

[0321] All experiments presented used the previously described wild-type human iPSC line CRL1502 (clone #C32), which was episomal reprogrammed using 28 Undifferentiated human iPSCs were maintained on mouse embryonic fibroblasts (MEFs) (Millipore) as a feeder layer using human ES cell (hES) medium as previously described. 1 . Identify cells and test for mycoplasma infection 28 Human iPSCs were plated on Matrigel-coated (Millipore) culture dishes and cultured in MEF-conditioned hES medium (MEF-CM) until 60-100% confluence was reached. Then, the cells were plated again at 5000 cells / cm 2 The cells were plated on Matreigel-coated culture dishes and cultured in MEF-CM. The next day, when the cells reached 40-50% confluence, the cells were treated with 8 μM CHIR99021 for 2-5 days in APEL basal medium (STEMCELL Technologies) supplemented with antibiotic-antimycotic (Life Technologies), followed by FGF9 (200 ng mL-1) and heparin (1 μg mL -1 ) for another 5-2 days, with the medium changed every other day. On day 7, cells were harvested and dissociated into single cells using trypsin or TrypLE select (Life Technologies). Cells (0.5 × 10 6 ) was centrifuged at ×400 g for 2 min to form a precipitate, which was then transferred to a Transwell 0.4 μm pore polyester membrane (#CLS3450 Corning). The precipitate was treated with 5 μM CHIR99021 in APEL for 1 h and then with FGF9 (200 ng mL -1 ) and heparin (1 μg mL -1 ) for 5 days and then cultured in APEL basal medium for another 6-13 days, with medium changes 3 times a week. The medium should not exceed the membrane. For differentiation in monolayer culture, cells induced with CHIR99021 were treated with FGF9 (200 ng mL-1 ) and heparin (1 μg mL -1 ) for 10 days, followed by APEL basal medium for another 6 days. In some experiments, RA (0.1 μM) or AGN193109 (5 μM) was added to the FGF9 medium. A step-by-step protocol describing the generation of nephrosomes is provided in Example 1.

[0322] Immunohistochemistry

[0323] For monolayer cells, antibody staining was performed as previously described. 1For nephrosomes, organoids were fixed with 2% paraformaldehyde in PBS for 20 min at 4°C and then washed three times with PBS. Organoids were then blocked with 10% donkey serum, 0.3% Triton X / PBS for 2-3 h at room temperature and incubated with primary antibodies overnight at 4°C. After washing five times with 0.1% Triton X / PBS, secondary antibodies were incubated for 4 h at room temperature.The following antibodies and dilutions were used: rabbit anti-PAX2 (1:300, #71-6,000, Zymed Laboratories), goat anti-SIX1 (1:300, #sc-9709, Santa Cruz Biotechnology), rabbit anti-SIX2 (1:300, #11562-1-AP, Proteintech), mouse anti-ECAD (1:300, #610181, BD Biosciences), rabbit anti-WT1 (1:100, #sc-192, Santa Cruz Biotechnology), mouse anti-HOXD11 (1:300, #SAB1403944, Sigma-Aldrich), goat anti-GATA3 (1:300, #AF2605, R&D Biotechnology). Systems), rabbit anti-JAG1 (1:300, #ab7771, Abcam), goat anti-Cubilin (1:150, #sc-20607, Santa Cruz Biotechnology), sheep anti-NPHS1 (1:300, AF4269, R&D Systems), LTL-biotin conjugate (1:300, B-1325, Vector Laboratories), DBA-biotin conjugate (1:300, B-1035, Vector Laboratories), mouse anti-KRT8 (1:300, #TROMA, DSHB), mouse anti-CD31 (1:300, #555444, BD Pharmingen), rabbit anti-KDR (1:300, #2479, CellSignaling Technology), goat anti-SOX17 (1:300, #AF1924, R&D Systems), rabbit anti-NG2 (1:300, #AB5320, Merck Millipore), rabbit anti-SMA (1:300, #ab15267, Abcam), mouse anti-PDGFRA (1:200, #556001, BD Pharmingen), rabbit anti-laminin (1:300, #L9393, Sigma-Aldrich), rabbit anti-UMOD (1:300, #BT-590, Biomedical Technologies), mouse anti-MEIS1 (1:300, #ATM39795, activemotif), goat anti-FOXD1 (1:200, #sc-47585, Santa Cruz Biotechnology) and rabbit anti-cleaved CASP3 (1:300, #9661, Cell Signaling Technology).Images were taken using a Nikon Ti-U microscope or a Zeiss LSM 780 confocal microscope. All immunofluorescence analyses were successfully repeated more than three times and representative images are shown.

[0324] Electron microscopy

[0325] Organoids were processed for electron microscopy analysis using the following method. A solution of 5% glutaraldehyde in 2×PBS was added directly to an organoid culture dish of equal volume to the growth medium and placed under vacuum for 5 minutes. The organoid size was reduced by cutting into small pieces (~2×2mm) and irradiated again in 2.5% fresh fixative under vacuum for 6 minutes in a Pelco Biowave (Ted Pella In, Redding, CA) at 80W power. The samples were then washed 4×2 minutes in 0.1M cacodylate buffer. The samples were then immersed in a solution containing potassium ferricyanide (3%) and osmium tetroxide (2%) in 0.1M cacodylate buffer for 30 minutes at room temperature. After washing 6×3 minutes in distilled water, the tissue blocks were incubated at room temperature for 30 minutes in a filtered solution containing thiocarbamide (1%). After subsequent washing in distilled water (6×2 minutes), the samples were incubated in an aqueous solution of osmium tetroxide (2%) for 30 minutes, then in distilled water (6×2 minutes), and in a 1% aqueous solution of uranyl acetate at 4°C for 30 minutes. After further washing in distilled water (2×2 minutes), a freshly prepared filtered solution of 0.06% lead nitrate in aspartic acid (pH 5.5) warmed to 60°C was added to the culture dish and further incubated at 60°C for 20 minutes, followed by rinsing in distilled water at room temperature (6×3 minutes). In a Pelco Biowave, tissue blocks were dehydrated twice at 250 watts for 40 seconds in each of 30%, 50%, 70%, 90% and absolute ethanol solutions. Epon LX112 resin was used to embed tissues in Pelco Biowave at 25%, 50% and 75% resin: absolute ethanol immersion for 3 minutes under 250 watt vacuum and finished with 100% resin (2 times) followed by final embedding / blocking and curing at 60°C for 12 hours.

[0326] qRT-PCR analysis

[0327] Total RNA was extracted from cells using Purelink RNA Mini Kit (Life Technologies), and cDNA was synthesized from >100 ng total RNA using Super Script III reverse transcriptase (Life Technologies). qRT-PCR analysis was performed using GoTaq qPCR Master Mix (Promega) using a Roche LightCycler 96 real-time PCR machine. All absolute data were first normalized relative to GAPDH and then normalized relative to control samples (δ-δ-Ct method). Primer sequences for qRT-PCR are shown in Table 2.

[0328] Next-generation RNA sequencing and comparative analysis using KeyGenes

[0329] Sequencing was performed using the Illumina NextSeq500 (NextSeq Control Software v1.2 / Real Time Analysis v2.1) platform. The library pool was diluted and denatured according to the standard NextSeq500 protocol and sequenced using the NextSeq500 High Yield Kit (Catalog No. FC-404-1005) for 75 cycles to generate single-end 76 bp reads. STAR was used to sequence the library pool. 29 Reads were aligned to the reference human genome (hg19) and read counts for each gene in the UCSC annotation were generated using htseq-count in the HTSeq python package (http: / / www-huber.embl.de / users / anders / HTSeq / doc / index.html). The number of uniquely mapped reads ranged from 18810634 to 36706805 per sample. 30 Calculate the normalized read count.

[0330] KeyGenes have been used to generate identity scores for different first trimester human organs, including kidneys, for D0, D3, D11, and D18 nephrosomes (GSE66302). 15 Dendrogram showing hierarchical clustering of D0, D3, D11, D18 renal somatic cells and 21 human fetal organs from the first and second trimesters (GSE66302) ( Fig.13) is based on Pearson correlation analysis of the expression levels of 85 classifier genes determined by KeyGenes (www.keygenes.nl; Table 3). Classifier genes were calculated by KeyGenes using the top 500 most differentially expressed genes of human fetal data, without including extraembryonic tissues from this data set.

[0331] Functional analysis of the proximal tubule

[0332] For the dextran uptake assay, day 17 organoids were treated with 10 μg mL -1 10,000MW dextran Alexa488 conjugate (D-22910, Life Technologies) was cultured for 24 hours. The organoids were fixed and stained with LTL without permeabilization. For nephrotoxicity assays, organoids on day 17 were cultured with 0, 5, 20 or 100 μM cisplatin (Sigma-Aldrich) for 24 hours. In 2 or 3 representative areas of each experiment, the ratio of apoptotic proximal tubules to total proximal tubules was manually counted using ImageJ. A total of n=5 independent experiments. Images were taken using a Zeiss LSM 780 confocal microscope.

[0333] Results and Discussion

[0334] We have previously demonstrated in vitro that IM formation requires FGF9 or FGF2 1 Therefore, in vivo, we hypothesized that UE formation in early migratory PSM cells exposed to FGF9 and RA shortly after the primitive streak stage, whereas cells migrating later, and therefore exposed to longer Wnt signaling, should induce MM 13 ( Figure 7 a). To confirm this and following the data presented in Example 1, we varied the duration of initial Wnt signaling (CHIR99201) prior to the addition of FGF9 ( Figure 7 b) and monitored markers of AI and PI by qPCR. Shorter periods of CHIR99021 induced AI markers LHX1 and GATA3, whereas longer days of CHIR99021 increased PI markers HOXD11 and EYA1 at day 7. As expected, prolonged expression of PSM markers TBX6 and T after longer periods in CHIR99021 indicated a delay in FGF9-induced fate commitment ( Figure 7 c). Immunofluorescence analysis showed that longer (or shorter) duration of CHIR99021 induced less (more) AI but more (less) PI, respectively, as indicated by GATA3 and HOXD11, respectively, at day 7 of differentiation ( Figure 7d). These observations persisted after 18 days in culture, with a predominant UE induction after fewer days in CHIR99201 (GATA3 + PAX2 + ECAD + ), in CHIR66201, MM(PAX2 + ECAD - ) and its derivatives (PAX2 + ECAD + ) are preferentially induced. Furthermore, we investigated whether RA signaling also controls the AP fate pattern of IM using RA or the RAR antagonist AGN193109 together with FGF9 ( Figure 7 e, f). RA promotes UE induction, while AGN193109 inhibits UE but enhances the induction of the MM lineage ( Figure 7 g).

[0335] These results increase our understanding of embryogenesis and provide a method by which to regulate the relative induction of two IM-derived progenitor cell populations essential for kidney formation. We therefore modified an existing renal differentiation procedure to increase the proportion of MM formation, increase the 3D culture time, and actively trigger nephron formation. This optimized method was applied to hESCs or human iPSCs and consisted of an initial 4 days of CHIR99021, which resulted in the induction of UE and MM in monolayer culture, followed by 3 days of FGF9 before transfer to organoid culture ( Figure 8 a). The resulting aggregates were cultured for up to 20 days, during which time they spontaneously formed complex nephrosomes ( Figure 8 b) During normal kidney development, nephron formation from MM is initiated in response to Wnt9b secreted from UE. In mice, ectopic nephron formation can be triggered by the addition of canonical Wnt agonists 14 In fact, the maximum number of nephrons per organoid required a pulse of CHIR99021 for 1 h after pellet formation ( Figure 8 a and Fig.11 a). Furthermore, continued presence of FGF9 after the CHIR99021 pulse is essential for nephrogenesis, suggesting an additive role for FGF signaling after Wnt-mediated nephron induction ( Fig.11 b) Within each organoid, the nephron is properly divided into four components, including the collecting duct (GATA3 + ECAD + ), early distal tubule (GATA3 - LTL - ECAD + ), early proximal tubule (LTL + ECAD -) and glomerulus (WT1 + )( Figure 8 c, d). In addition, the nephroids showed a complex morphogenetic pattern, with a collecting duct tree formed at the base of the organoids, connected to the distal and proximal tubules in the middle, and a glomerulus at the top of each organoid ( Figure 8 e', e", e"'). This pattern mimics the organization of the tissue observed in vivo, where glomeruli emerge in the cortex and collecting ducts radiate through the organ from the middle. Here, the relative levels of collecting ducts to nephrons within individual organoids can be varied depending on the timing of the initial CHIR99201-to-FGF9 switch. We next performed RNA sequencing of whole nephrons at days (d) 0, 3, 11, and 18 following aggregation and 3D culture. Over this time period, we observed a transient loss of nephron progenitor gene expression, but an increase in markers for multiple nephron segments including podocytes, proximal and distal tubules ( Fig.12 Transcriptional profiling was performed and compared with human fetal transcriptome datasets from 21 human fetal organs / tissues from the first and / or second trimester of pregnancy using an unbiased approach. The analysis clustered nephrosomes ( Figure 8 f, g; Fig.13 At slightly earlier culture time points (d0, d3), organoids more closely matched fetal gonads (tissues derived from the IM that are closely related to the embryo).

[0336] In the kidney, epithelial cell types (nephrons and collecting ducts) are surrounded by the renal interstitium (stroma), in which a vascular network exists. In addition to forming the MM, the IM produces stromal and vascular progenitor cells ( Fig. 9 a) 16,17 We examined the nephroids to demonstrate additional cell types and functional maturation. Collecting ducts can be distinguished based on co-expression of PAX2, GATA3, and ECAD ( Fig. 9 b). At d11, the nephron epithelium shows the proximal (LTL + ECAD - ) and remote (LTL - ECAD + )element( Fig. 9 c). By d18, the proximal tubules mature to co-express ECAD and LTL, with Cubilin evident on the apical surface ( Fig. 9 d, e). Transmission electron microscopy (TEM) shows different epithelial subtypes; cells that are characteristic of collecting ducts / distal tubules and have few short microvilli surrounding an open lumen ( Fig. 9 k) and typical proximal tubular epithelial cells showing apical brush border with tight junctions ( Fig. 9 l). To d18, Henle loop (UMOD+ ) begins to form( Fig. 9 f) By d11, WT1 cells containing Bowman's capsules with centropod formation were observed + NPHS1 + Early glomeruli 18 Connected to the proximal tubule ( Fig. 9 g). Nephroids also develop CD31 with cavity formation + KDR + SOX17 + Endothelial network ( Fig. 9 h). TEM shows the presence of primary and secondary foot processes that are unique to podocytes ( Fig. 9 m) In the developing kidney, renal mesenchyme differentiates into pericytes and mesangial cells 19 As expected, as in human fetal kidney 20 As observed in the nephrotic cells, the nephrotic bodies contain KDR + Endothelium and PDGFRA + PDGFRA in early mesangial cell invasion of glomeruli + Perivascular cells ( Fig.14 a, b). Early avascular glomeruli contain a basement membrane, as shown by laminin staining and TEM, and show foot processes attached to the basement membrane. In some cases, glomeruli show evidence of endothelial invasion ( Fig. 9 i), a feature not seen in transplanted embryonic mouse kidneys 21 Finally, the nephron is expressed by MEIS1 + Renal interstitial cells 22 Surrounded by some of them are also FOXD1 + ( Fig. 9 j), indicating the presence of cortical (FOXD1 + MEIS1 + ) and medulla (FOXD1 - MEIS1 + ) matrix. Thus, all expected renal components form, configure, and begin to mature in these hPSC-derived nephrosomes. Consistent with these observations are transcriptional changes over time in culture, with a gradual decrease in nephrogenic mesenchyme and ureteric tip markers, followed by an upregulation of genes specific to podocytes, proximal tubules, distal tubules, and loops of Henle. 23,24 ( Fig.11 ).

[0337] The utility of stem cell-derived nephrons for disease modeling or drug screening will depend on the functional maturation of the nephrons within these organoids. To test this, we focused on the proximal tubule, a nephron segment that plays an important role in the reabsorption of solutes, vitamins, hormones, and amino acids.+ The selective uptake of dextran-Alexa488 by tubules from the culture medium demonstrated the ability of cubilin to mediate proximal tubule-specific endocytosis ( Fig.10 a). The proximal tubules are involved in multidrug resistance (e.g. ABCB1, ABCG2) and anion and cation transporters (e.g. SLC22 gene family) 23,24 Cisplatin is one of the nephrotoxic agents that induces caspase-mediated acute apoptosis of proximal tubular cells in the kidney. 25,26 We treated nephroids with 0, 5, and 20 μM cisplatin for 24 h and then examined cleaved CASP3 antibody staining. Although control organoids sometimes showed apoptotic interstitial cells, both 5 μM and 20 μM cisplatin induced apoptotic proliferation of mature proximal tubule cells (LTLs). + ECAD + ) specific acute apoptosis, while immature cells (LTL + ECAD - ) No apoptosis occurred ( Fig.10 b, c).

[0338] In conclusion, this study demonstrates that by carefully balancing IM with A / P patterning by small molecules, human pluripotent stem cells can be directed to form composite multicellular nephrosomes that contain fully segmented nephrons surrounded by endothelium and renal stroma and are transcriptionally similar to human fetal kidney. This will therefore improve our understanding of human kidney development. Each nephrosome reached a substantial size of >500 nephrons / organoid, a number equivalent to the mouse kidney at 14.5 dpc. 27 Although there is room for further improvement regarding tubular functional maturation, glomerular vascularization, and a continuous collecting duct epithelium with a single outlet pathway for urine, the observed tissue complexity and degree of organoid functionalization support their use for screening drug toxicity, modeling genetic kidney diseases, or as a source of specific kidney cell types for cell-based therapies.

[0339] The fact that we can form organoids from separate cultures of differentiated hES cells opens the possibility of generating tissue-based nephrotoxicity screens, in vitro disease models, or developing transplantable organoids to supplement kidney function. It also demonstrates the feasibility of generating specific mature kidney cell types for later purification.

[0340] Specific uses for cells generated using this method may include:

[0341] Generate miniature nephrosomes or purified renal cells for nephrotoxicity screening;

[0342] Generation of miniature nephrosomes or purified kidney cells for disease modeling, either generally or for each patient; and / or

[0343] Generate miniature nephrosomes or purified renal cell types for drug screening for the treatment of renal diseases.

[0344] These can be performed in microbioreactors or after bioprinting into larger format screens. For disease modeling or drug screening, we might purify individual cell types and culture them in a way or format that provides useful information based on the specific disease. For example, we can isolate UB and grow them in matrigel culture to assess cyst formation (e.g. for diseases such as nephronopthisis) or isolate MM to generate podocytes (e.g. for diseases such as Finnish nephropathy or Alport syndrome).

[0345] Specific examples of cell therapy and organ replacement or repair may include:

[0346] Producing kidney cell types for cell therapy (acute kidney injury or chronic kidney injury);

[0347] Generating kidney cell types for whole-organ replacement bioengineering, which may require joining multiple smaller kidneys together to form a replacement “organ”; and / or

[0348] • Generation of renal cell types for recellularization of decellularized scaffolds.

[0349] Other applications include:

[0350] 1) Generate nephrosomes from human pluripotent stem cell lines that are engineered to report one or more fluorescent reporters as readouts for differentiation into specific renal cell types. This will allow the generation of organoids from a single starting cell line, will report the complexity of differentiation without the need for specific antibody visualization, and will facilitate combined FACS sorting to purify multiple specific renal cell types from one differentiation. Such sorted cells may be useful in cell therapy.

[0351] 2) Generate nephrosomes from human pluripotent stem cell lines engineered to report one or more fluorescence or luciferase-based reporters as a readout of cell injury (predicted response to nephrotoxicity, cytotoxicity, or induction of apoptosis / necrosis). This will allow the generation of organoids for drug or nephrotoxicity screening with a quantifiable response readout.

[0352] 3) Produce separate collecting ducts or separate metanephric mesenchyme to combine with other bioprinted structures. For example, produce separate nephron-forming mesenchyme for combination with collecting duct cells that are differentiated separately but bioprinted into the ureteric tree. The advantage here would be to create a specifically patterned collecting duct network around which nephrons can differentiate so that the final product can serve as a transplantable organ capable of directing urine to a single outlet.

[0353] Throughout the specification, the purpose is to describe the preferred embodiments of the present invention without limiting the present invention to any one embodiment or specific feature set. Therefore, it will be appreciated by those skilled in the art that various modifications and changes may be made in the specific embodiments shown, based on this disclosure, without departing from the scope of the present invention.

[0354] All computer programs, algorithms, patents and scientific literature cited herein are incorporated by reference.

[0355] Table 1. Sources of reagents

[0356]

[0357]

[0358] Table 2: Primer sequences used for qRTPCR

[0359]

[0360] Table 3

[0361]

[0362]

[0363]

[0364]

[0365] References

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[0382] 17.Sims-Lucas,S.et al.Endothelial Progenitors Exist within the Kidneyand Lung Mesenchyme.PLoS One 8,1–8(2013).

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Claims

1. A method for producing one or more nephrosomes, the method comprising: contacting human pluripotent stem cells (hPSCs) with a Wnt agonist to generate cells containing posterior primitive streak cells; contacting the cells comprising posterior primitive streak cells with fibroblast growth factor 9 (FGF9) alone or with a combination of FGF9 and one or more of heparin, an RA receptor antagonist, fibroblast growth factor 2 (FGF2) and fibroblast growth factor 20 (FGF20), thereby generating cells comprising intermediate mesoderm (IM) cells; forming the cells obtained in (b) into aggregates; and The cell aggregates formed in (c) are cultured with fibroblast growth factor 9 (FGF9) or with a combination of FGF9 and one or more of the group consisting of bone morphogenetic protein 7 (BMP7), heparin, Wnt agonist, retinoic acid (RA), RA receptor antagonist, fibroblast growth factor 2 (FGF2) and fibroblast growth factor 20 (FGF20); thereby producing nephron progenitor cells and ureteric epithelial progenitor cells under conditions that induce the nephron progenitor cells and ureteric epithelial progenitor cells to aggregate into one or more nephron-like bodies, which are at least partially vascularized and / or contain vascular progenitor cells. Wherein (c) after the cell aggregates are formed, the method further comprises adding a Wnt agonist and culturing for 0.5 to 2 hours.

2. A method for producing kidney or kidney cells or kidney tissue in vitro, the method comprising: contacting human pluripotent stem cells (hPSCs) with a Wnt agonist to generate cells containing posterior primitive streak cells; contacting the cells comprising posterior primitive streak cells with fibroblast growth factor 9 (FGF9) alone or with a combination of FGF9 and one or more of heparin, an RA receptor antagonist, fibroblast growth factor 2 (FGF2) and fibroblast growth factor 20 (FGF20), thereby generating cells comprising intermediate mesoderm (IM) cells; forming the cells obtained in (b) into aggregates; and The cell aggregates formed in (c) are cultured with fibroblast growth factor 9 (FGF9) or with a combination of FGF9 and one or more of the group consisting of bone morphogenetic protein 7 (BMP7), heparin, Wnt agonist, retinoic acid (RA), RA receptor antagonist, fibroblast growth factor 2 (FGF2) and fibroblast growth factor 20 (FGF20); thereby generating nephron progenitor cells and / or ureteric epithelial progenitor cells and further differentiating to generate kidneys or renal cells or renal tissues that are at least partially vascularized and / or contain vascular progenitor cells, Wherein (c) after the cell aggregates are formed, the method further comprises adding a Wnt agonist and culturing for 0.5 to 2 hours.

3. A method for bioprinting a kidney structure, the method comprising: contacting human pluripotent stem cells (hPSCs) with a Wnt agonist to generate cells containing posterior primitive streak cells; contacting the cells comprising posterior primitive streak cells with fibroblast growth factor 9 (FGF9) alone or with a combination of FGF9 and one or more of heparin, an RA receptor antagonist, fibroblast growth factor 2 (FGF2) and fibroblast growth factor 20 (FGF20), thereby generating cells comprising intermediate mesoderm (IM) cells; allowing the cells obtained in (b) to form aggregates; bioprinting (c) cell aggregates on a biocompatible surface; and The bioprinted cells of (d) are cultured with fibroblast growth factor 9 (FGF9) or with a combination of one or more of the group consisting of FGF9 and bone morphogenetic protein 7 (BMP7), heparin, Wnt agonist, retinoic acid (RA), RA receptor antagonist, fibroblast growth factor 2 (FGF2) and fibroblast growth factor 20 (FGF20); thereby generating nephron progenitor cells and / or ureteric epithelial progenitor cells and further differentiating to generate renal tissue that is at least partially vascularized and / or contains vascular progenitor cells, Wherein (c) after the cell aggregates are formed, the method further comprises adding a Wnt agonist and culturing for 0.5 to 2 hours.

4. A method for determining the nephrotoxicity of one or more compounds, the method comprising: contacting human pluripotent stem cells (hPSCs) with a Wnt agonist to generate cells containing posterior primitive streak cells; contacting the cells comprising posterior primitive streak cells with fibroblast growth factor 9 (FGF9) alone or with a combination of FGF9 and one or more of heparin, an RA receptor antagonist, fibroblast growth factor 2 (FGF2) and fibroblast growth factor 20 (FGF20), thereby generating cells comprising intermediate mesoderm (IM) cells; allowing the cells obtained in (b) to form aggregates; Culturing the cell aggregates formed in (c) with fibroblast growth factor 9 (FGF9) or with a combination of FGF9 and one or more of the group consisting of bone morphogenetic protein 7 (BMP7), heparin, Wnt agonist, retinoic acid (RA), RA receptor antagonist, fibroblast growth factor 2 (FGF2) and fibroblast growth factor 20 (FGF20); thereby producing nephron progenitor cells and ureteric epithelial progenitor cells under conditions that induce the nephron progenitor cells and ureteric epithelial progenitor cells to aggregate into one or more nephron-like bodies, which are at least partially vascularized and / or contain vascular progenitor cells; contacting one or more compounds with the nephrotoxic body of (d) to thereby determine whether the one or more compounds are nephrotoxic, Wherein (c) after the cell aggregates are formed, the method further comprises adding a Wnt agonist and culturing for 0.5 to 2 hours.

5. The method of any one of claims 1-4, wherein forming cell aggregates in (c) comprises culturing on a floating filter.

6. The method according to any one of claims 1 to 4, comprising culturing the cell aggregates of (c) under an oxygen partial pressure of 5% to 12%.

7. The method according to claim 2, wherein the cell aggregates formed in (c) are cultured in (d) for 20 days.

8. The method of claim 3, wherein the cell aggregates formed in (c) are bioprinted onto a biocompatible surface.

9. The method according to any one of claims 1 to 4, wherein the concentration of FGF9 and / or FGF20 and / or FGF2 is in the range of 20 ng to 1 μg / mL.

10. The method according to claim 9, wherein the concentration of FGF9 and / or FGF20 and / or FGF2 is in the range of 50-500 ng / mL.

11. The method according to claim 10, wherein the concentration of FGF9 and / or FGF20 and / or FGF2 is in the range of 100-300 ng / mL.

12. The method according to claim 11, wherein the concentration of FGF9 and / or FGF20 and / or FGF2 is 200 ng / mL.

13. The method according to any one of claims 1 to 4, wherein the concentration of BMP 7 is in the range of 25 to 75 ng / mL.

14. The method of claim 13, wherein the concentration of BMP 7 is in the range of 35 to 60 ng / mL.

15. The method of claim 14, wherein the concentration of BMP 7 is in the range of 45 to 55 ng / mL. The method according to claim 15 , wherein the concentration of BMP 7 is 50 ng / mL.

17. The method according to any one of claims 1 to 4, wherein the concentration of RA is in the range of 10 pM to 1 μM.

18. The method according to claim 17, wherein the concentration of RA is in the range of 30 pM to 0.5 μM. The method according to claim 18 , wherein the concentration of RA is in the range of 50 pM to 0.2 μM.

20. The method according to claim 19, wherein the concentration of RA is 0.1 μM.

21. The method according to any one of claims 1-4, wherein the concentration of the RA receptor antagonist is in the range of 0.50 pM to 10 μM.

22. The method of claim 21, wherein the concentration of the RA receptor antagonist is in the range of 0.01 μM to 5 μM.

23. The method of claim 22, wherein the concentration of the RA receptor antagonist is in the range of 0.1 μM to 5 μM.

24. The method of claim 23, wherein the concentration of the RA receptor antagonist is 1 μM.

25. The method of any one of claims 1-4, wherein the concentration of the Wnt agonist is in the range of 0.5 μM to 50 μM.

26. The method of claim 25, wherein the concentration of the Wnt agonist is in the range of 4 μM to 30 μM.

27. The method of claim 26, wherein the concentration of the Wnt agonist is in the range of 5-20 μM.

28. The method of any one of claims 1-4, wherein the concentration of the Wnt agonist is in the range of 0.1 μM to 10 μM.

29. The method of claim 28, wherein the concentration of the Wnt agonist is in the range of 0.2 μM to 5 μM.

30. The method of claim 29, wherein the concentration of the Wnt agonist is in the range of 1-2 μM.

31. The method of any one of claims 1-4, wherein heparin is present at a concentration of 0.1-10 μg / mL.

32. The method of any one of claims 1-4, wherein the concentration of the Wnt agonist is 8 μM.

33. The method of any one of claims 1-4, wherein the concentration of the Wnt agonist is 5 μM.

34. The method according to any one of claims 1-4, wherein after (c) forming cell aggregates, the method further comprises adding the Wnt agonist and culturing for 1 hour.

35. The method of any one of claims 1-4, wherein the Wnt agonist is CHIR99021.

36. The method of any one of claims 1-4, wherein the hPSC is a human embryonic stem cell or an induced human pluripotent stem cell.

37. The method of claim 1, wherein the nephroid comprises segmented nephrons, endothelium, and renal interstitium.

38. The method of claim 2, wherein the kidney, renal cell, or renal tissue comprises segmented nephrons, endothelium, and renal interstitium.

Images