Maturation of hepatocyte-like cells derived from human pluripotent stem cells

Abstract

The present invention relates to directed differentiation and maturation of hepatocyte-like cells. In particular, the present invention relates to exposure of hepatocyte-like cells to an activator of a retinoic acid responsive receptor, such as retinoic acid (RA), optionally in combination with an inhibitor of GSK-3 (Glycogen synthase kinase 3) or activator of Wnt signalling and/or with the overlay of the cells with one or more components characteristic of the mammalian extracellular matrix (matrix overlay). The present invention also relates to exposure of hepatocyte-like cells to an activator of a retinoic acid responsive receptor, such as retinoic acid (RA), optionally in combination with an inhibitor of a cycline dependent kinase (CDK) and/or with the overlay of the cells with one or more components characteristic of the mammalian extracellular matrix (matrix overlay). The hepatocyte-like cells obtained in accordance with the present invention show a phenotype which is more similar to that of primary hepatocytes than previously shown.

1 - 88 . (canceled) 
     
     
         89 . A composition comprising at least one activator of a retinoic acid responsive receptor and at least one selected from GSK3 inhibitor, activator of Wnt signalling and CDK inhibitor. 
     
     
         90 . A composition according to  claim 89 , comprising at least one activator of a retinoic acid responsive receptor and at least one GSK-3 inhibitor, and optionally at least one CDK inhibitor; or comprising at least one activator of a retinoic acid responsive receptor and at least one activator of Wnt signalling, and optionally at least one CDK inhibitor; or comprising at least one activator of a retinoic acid responsive receptor and at least one CDK inhibitor, and optionally at least one GSK3 inhibitor or at least one activator of Wnt signalling. 
     
     
         91 . The composition according to  claim 89 , comprising at least one activator of a retinoic acid responsive receptor and at least one GSK-3 inhibitor, and optionally at least one CDK inhibitor. 
     
     
         92 . The composition according to  claim 91 , wherein the activator of a retinoic acid responsive receptor is a retinoic acid. 
     
     
         93 . The composition according to  claim 91 , wherein the activator of a retinoic acid responsive receptor is a retinoic acid isomer, a retinoic acid analogue, or a retinoid. 
     
     
         94 . The composition according to  claim 91 , wherein the activator of a retinoic acid responsive receptor is 9-cis-retinoic acid, 13-cis-retinoic acid or a combination thereof. 
     
     
         95 . The composition according to  claim 91 , wherein the activator of a retinoic acid responsive receptor is 9-cis-retinoic acid. 
     
     
         96 . The composition according to  claim 91 , wherein the GSK3-inhibitor is selected from the group consisting of Kenpaullone, 1-Aza-Kenpaullone, Alsterpaullone, Aminopyrimidine CHIR99021 and Indirubin-3′-monoxime. 
     
     
         97 . The composition according to  claim 91 , wherein the GSK3-inhibitor is selected from the group consisting of Kenpaullone, 1-Aza-Kenpaullone, and Alsterpaullone. 
     
     
         98 . The composition according to  claim 91 , wherein the GSK3-inhibitor is Kenpaullone. 
     
     
         99 . The composition according to  claim 91 , wherein the at least one activator of a retinoic acid responsive receptor is selected from the group consisting of 9-cis-retinoic acid, 13-cis-retinoic acid or a combination thereof, and the at least one GSK-3 inhibitor is selected from the group consisting of Kenpaullone, 1-Aza-Kenpaullone and Alsterpaullone. 
     
     
         100 . The composition according to  claim 91 , comprising 9-cis-retinoic acid and Kenpaullone. 
     
     
         101 . The composition according to  claim 89 , wherein the activator of Wnt signalling is a Wnt protein. 
     
     
         102 . The composition according to  claim 89 , wherein the at least one CDK inhibitor is selected from the group consisting of Kenpaullone, 1-Aza-Kenpaullone, Alsterpaullone, Indirubin-3′-monoxime, SNS-032(BMS-387032), AT-7519 and AZD5438. 
     
     
         103 . A kit comprising at least one activator of a retinoic acid responsive receptor and at least one selected from GSK3 inhibitor, activator of Wnt signalling, and CDK inhibitor. 
     
     
         104 . The kit according to  claim 103 , comprising at least one activator of a retinoic acid responsive receptor, at least one GSK3 inhibitor, and optionally at least one extracellular matrix (ECM) component or ECM component mixture; or comprising at least one activator of a retinoic acid responsive receptor, at least one activator of Wnt signalling, and optionally at least one extracellular matrix (ECM) component or ECM component mixture; or comprising at least one activator of a retinoic acid responsive receptor, at least one CDK inhibitor, and optionally at least one extracellular matrix (ECM) component or ECM component mixture. 
     
     
         105 . The kit according to  claim 103 , comprising at least one activator of a retinoic acid responsive receptor, at least one GSK3 inhibitor, and optionally at least one extracellular matrix (ECM) component or ECM component mixture. 
     
     
         106 . The kit according to  claim 103 , wherein the kit comprises at least one extracellular matrix (ECM) component or ECM component mixture. 
     
     
         107 . The kit according to  claim 106 , wherein the at least one ECM component is selected from the group consisting of collagen, fibronectin, elastin, chondroitin sulfate proteoglycan, dermatan sulfate proteoglycan, heparin proteoglycan, heparan sulfate proteoglycan, such as glypicans, syndecans or perlecans, glycosaminoglycans, nidogen/entactin, laminins, biglycan, tenascin, and hyaluronans. 
     
     
         108 . The kit according to  claim 106 , wherein the ECM component mixture comprises collagen and fibronectin.