Differentiation of pluripotent stem cells to form renal organoids

Abstract

A method is provided for producing renal organoids comprising nephrons, ureteric bud and vasculature and/or progenitors of these. In one embodiment, the methods includes contacting intermediate mesoderm cells with: fibroblast growth factor 9 and/or fibroblast growth factor 20 and/or fibroblast growth factor 2 and optionally, one or more selected from the group consisting of: bone morphogenic protein 7; heparin; a Wnt agonist; retinoic acid; and an RA antagonist under conditions that promote formation of vascularized renal organoids. Another embodiment includes producing mesoderm cells by sequentially contacting pluripotent stem cells with a Wnt agonist and fibroblast growth factor 9 and/or fibroblast growth factor 20 and/or fibroblast growth factor 2, followed by a relatively short re-exposure to the Wnt agonist. The renal organoids may have end uses such as for kidney repair and re generation, bioprinting of kidneys or functional components thereof, renal cell arrays and screening compounds for nephrotoxicity.

1 . A method of producing nephron progenitor cells and ureteric epithelial progenitor cells comprising contacting intermediate mesoderm (IM) cells with: fibroblast growth factor  9  (FGF 9 ) and/or fibroblast growth factor  20  (FGF 20 ) and/or fibroblast growth factor  2  (FGF 2 ); and optionally, one or more selected from the group consisting of: bone morphogenic protein  7  (BMP 7 ); heparin; a Wnt agonist; retinoic acid (RA), analog or agonist; and an RA antagonist; to thereby produce nephron progenitor cells and ureteric epithelial progenitor cells under conditions that induce aggregation of nephron progenitor cells and ureteric epithelial progenitor cells into one or more renal organoids whereby the renal organoids are at least partly vascularized and/or comprise vascular progenitors. 
     
     
         2 . The method of  claim 1 , wherein vascularization is facilitated by conditions that promote or direct development of vascular endothelium or vascular progenitors from mesenchymal cells or tissues. 
     
     
         3 .- 4 . (canceled) 
     
     
         5 . The method of  claim 1 , wherein RA, analog or agonist increases the relative production of ureteric epithelial progenitor cells from the IM cells. 
     
     
         6 . (canceled) 
     
     
         7 . The method of  claim 1 , wherein the RA antagonist increases the relative production of nephron progenitor cells from the IM cells. 
     
     
         8 . (canceled) 
     
     
         8 . The method of  claim 1 , wherein the Wnt agonist increases the relative production of nephron progenitor progenitor cells from the IM cells. 
     
     
         9 .- 11 . (cancelled) 
     
     
         12 . The method of  claim 1 , wherein the nephron progenitor cells and ureteric epithelial progenitor cells are produced synchronously or simultaneously from the IM cells. 
     
     
         13 . The method of  claim 1 , which includes further comprising contacting posterior primitive streak cells with one or more agents that facilitate differentiation of the posterior primitive streak cells into said IM cells. 
     
     
         14 . The method of  claim 13 , further comprising contacting human pluripotent stem cells (hPSCs) with one or more agents that facilitate differentiation of the hPSCs into said posterior primitive streak cells. 
     
     
         15 . A method of producing mesoderm cells, comprising contacting hPSCs with a Wnt agonist to produce the mesodeini cells. 
     
     
         16 . The method of  claim 15 , wherein the mesodeini cells are a mixed population comprising one or more of definitive mesoderm and intermediate mesoderm. 
     
     
         17 .- 23 . (canceled) 
     
     
         24 . The method of  claim 15 , which further includes the production of further comprising producing vasculature and/or vascular progenitor cells. 
     
     
         25 .- 26 . (canceled) 
     
     
         27 . Isolated, enriched or purified nephron progenitor cells, ureteric epithelial progenitor cells and/or renal organoids produced according to the method of  claim 1 . 
     
     
         28 . The renal organoid of  claim 27 , which comprises segmented nephrons, endothelia and renal interstitium. 
     
     
         29 . (canceled) 
     
     
         30 . A method of producing a kidney, or kidney cells or tissues, comprising differentiating the isolated or purified nephron progenitor cells, ureteric epithelial progenitor cells and/or renal organoids of  claim 27 , to produce the kidney, or kidney cells or tissues. 
     
     
         31 . The Isolated, enriched, or purified nephron progenitor cells,, ureteric epithelial progenitor cells, and/or renal organoids produced according to the method of  claim 30 . 
     
     
         32 . A method of bioprinting a renal structure, comprising depositing a plurality of hPSCs or other progenitor cells to form a renal structure that is at least partly vascularized and/or comprises vascular progenitors and having one or more functional characteristics of a kidney or component thereof, or which is capable of developing one or more functional characteristics of a kidney or component thereof. 
     
     
         33 . The method of claim  3332 , wherein the hPSCs or other progenitor cells are prepared by contacting intermediate mesoderm (IM) cells with: fibroblast growth factor  9  (FGF 9 ) and/or fibroblast growth factor  20  (FGF 20 ) and/or fibroblast growth factor  2  (FGF 2 ); and optionally, one or more selected from the group consisting of: bone morphogenic protein  7  (BMP 7 ); heparin; a Wnt agonist; retinoic acid (RA), analog or agonist; and an RA antagonist, to thereby produce nephron progenitor cells, ureteric epithelial progenitor cells and vasculature in the bioprinted renal structure. 
     
     
         34 . A method of bioprinting a renal structure, comprising depositing a plurality of nephron progenitor cells and ureteric epithelial progenitor cells disclosed herein to form a renal structure that is at least partly vascularized and/or comprises vascular progenitors and having one or more functional characteristics of a kidney or component thereof, or which is capable of developing one or more functional characteristics of a kidney or component thereof. 
     
     
         35 . (canceled) 
     
     
         36 . A bioprinted renal structure which is at least partly vascularized having one or more functional characteristics of a kidney or component thereof, or which is capable of developing one or more functional characteristics of a kidney or component thereof, produced by the method of  claim 32 . 
     
     
         37 . A method of (i) bioprinting or bio-engineering whole kidneys and kidney tissue for kidney transplant or treating chronic kidney disease; (ii) recellularisation of whole organ decellularised kidney to thereby create a reconstituted or replacement kidney; or (iii) cellular therapy of a damaged kidney or one or more kidney diseases or conditions comprising bioprinting the isolated, enriched, or purified nephron progenitor cells, ureteric epithelial progenitor cells, and/or renal organoids of  claim 31 . 
     
     
         38 .- 42 . (canceled) 
     
     
         43 . A method of determining the nephrotoxicity of one or a plurality of compounds, comprising contacting the one or plurality of compounds with the isolated or purified nephron progenitor cells, ureteric epithelial progenitor cells, and/or renal organoids of any one of  claim 27 , or kidney cells or tissues differentiated or otherwise obtained therefrom, and determining whether or not the one or plurality of compounds is nephrotoxic. 
     
     
         44 . (canceled)