Three-dimensional differentiation of epiblast spheroids to kidney organoids models stage-specific epithelial physiology, morphogenesis, and disease

Abstract

Human pluripotent stem cells (hPSCs) have dual value as microphysiological laboratory models and regenerative therapeutics. hPSCs are epithelial cells, but the extent to which hPSCs and descendant epithelia can reconstitute lineage-specific functions remains poorly understood. Here the Inventors show that hPSCs in three-dimensional cultures and their differentiated descendants can functionally recapitulate tissue-specific epithelial morphogenesis, physiology, and disease.

1 . A method of screening a compound for an effect on tubular organoids, the method comprising:
 providing a quantity of tubular organoids;   adding one or more compounds to the tubular organoids; and   determining changes to phenotype or activity of the tubular organoids.   
     
     
         2 . The method of  claim 1 , wherein determining changes to phenotype or activity comprises detecting and/or measuring expression levels of one or more markers in the tubular organoids. 
     
     
         3 . The method of  claim 2 , wherein the one or more markers comprise kidney injury molecule (KIM-1). 
     
     
         4 . The method of  claim 3 , wherein an increase in KIM-1 mRNA or KIM-1 polypeptide expression correlates with a toxic effect of the compound. 
     
     
         5 . The method of  claim 1 , wherein the tubular organoids are kidney organoids. 
     
     
         6 . The method of  claim 1 , wherein the tubular organoids express one or more of podocalyxin (PODXL), zonula occluden (ZO-1), and lotus tetragonolobus lectin (LTL). 
     
     
         7 . The method of  claim 1 , wherein the kidney organoids are differentiated from human pluripotent stem cells (hPSCs). 
     
     
         8 . The method of  claim 1 , wherein determining the phenotype or activity of tubular organoids comprises an assay selected from the group consisting of:
 (a) a KIM-1 induction assay;   (b) a permeability assay;   (c) an immunofluorescence assay;   (d) an RT-PCR assay;   (e) transcriptome profiling; and   (f) microscopy.   
     
     
         9 . The method of  claim 1 , wherein the tubular organoids are genome-modified kidney organoids. 
     
     
         10 . The method of  claim 1 , wherein the tubular organoids are derived from a subject with a kidney disease. 
     
     
         11 . The method of  claim 9 , wherein the tubular organoids are modified by a CRISPR/Cas9 genome editing system. 
     
     
         12 . The method of  claim 10 , wherein the kidney disease is polycystic kidney disease. 
     
     
         13 . The method of  claim 1 , wherein the tubular organoids are generated by the method comprising:
 (i) providing a quantity of human pluripotent stem cells (hPSCs);   (ii) culturing the hPSCs in a first culture medium comprising a ROC kinase inhibitor for at least 24 hours and then culturing the hPSCs sandwiched between two layers of a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells to form epiblast spheroids; wherein the first medium does not comprise exogenous fibroblast growth factor 2 (FGF2), activin or bone morphogenetic protein; and   (iii) contacting the epiblast spheroids from step (ii) with a second culture medium comprising at least 12 μM CHIR99021 for at least 24 hours, wherein the second medium does not comprise exogenous fibroblast growth factor 2 (FGF2), activin or bone morphogenetic protein; and   (iv) culturing the epiblast spheroids from step (iii) with a third culture medium comprising B27 for at least 48 hours, wherein the third medium does not comprise exogenous fibroblast growth factor 2 (FGF2), activin or bone morphogenetic protein, thereby differentiating the epiblast spheroids into tubular organoids.   
     
     
         14 . A method of screening a compound for the treatment of polycystic kidney disease, the method comprising:
 providing a quantity of genome-modified kidney organoids;   adding one or more compounds to the kidney organoids; and   determining changes to phenotype or activity of the kidney organoids.