Methods of Inducing Metabolic Maturation of Human Pluripotent Stem Cells-Derived Hepatocytes

Abstract

Provided are methods of increasing metabolic maturation of an immature hepatocyte, by contacting an immature hepatocyte which expresses alpha-fetoprotein (AFP) and albumin with an effective amount of a fatty acid or a small molecule selected from the group consisting of: an amphipathic carboxylic acid Thiazolidinedione (TZD), WY-14643 (Pirinixic Acid), GW409544, GW6471, Leukotriene B4, GW 7647, Perfluorooctanesulfonic Acid, Perfluorooctanoic Acid, CP-775146, CP-865520, UNII-999KY5ZIGB, and Gemfibrozil. Also provided are isolated hepatocytes and uses thereof.

1 - 27 . (canceled) 
     
     
         28 . An isolated cell population of metabolically mature hepatocytes wherein one or more hepatocyte cells exhibit Cytochrome P450 3A4 (CYP3A4) activity which oxidizes at least 1 pmol of 7-benzyloxy-4-trifluoromethylcoumarin (BFC) per minute per milligram of cellular protein and an alpha-fetoprotein (AFP) activity of at least 60 μg/day/mg cellular protein as assayed by ELISA when cultured in the presence of a culture medium which comprises Insulin-Transferrin-Selenium (ITS), Glutamax, Dexamethasone, hepatocyte growth factor (HGF), Oleic acid and 9CLA. 
     
     
         29 . (canceled) 
     
     
         30 . An isolated population of cells as claimed in  claim 28 , wherein at least 50% of the cells exhibit said activities. 
     
     
         31 - 32 . (canceled) 
     
     
         33 . The isolated hepatocyte population of  claim 28 , wherein said hepatocyte population is characterized by nuclear expression of pregnane X receptor (PXR). 
     
     
         34 - 37 . (canceled) 
     
     
         38 . The isolated hepatocyte population of  claim 28 , obtained from an immature hepatocyte which produces at least 1 μg to 25 μg Albumin/ml/mg cellular protein and cannot differentiate into bile duct cells. 
     
     
         39 . The isolated hepatocyte population of  claim 28 , wherein said hepatocyte is distinguishable from an adult hepatocyte by at least the expression of AFP. 
     
     
         40 . The hepatocyte population of  claim 28 , wherein the hepatocyte contains an increased mitochondrial mass per cell as compared to the mitochondrial mass in a control immature hepatocyte. 
     
     
         41 . The metabolically mature hepatocyte population of  claim 38 , wherein said immature hepatocytes exhibit an alpha-fetoprotein (AFP) + /Albumin + /CYP347 + /SOX2 − /OCT4 −  expression signature. 
     
     
         42 . The metabolically mature hepatocyte population of  claim 28 , wherein the mature hepatocytes exhibit an albumin + /CY3A4 + /E-cadherin + /OCT4 − /SOX2 − /A1AT + /HNF4α +  expression signature. 
     
     
         43 . An isolated cell population of metabolically mature hepatocytes generated from immature hepatocytes contacted with one or more of a PXR agonist, a fatty acid or a small molecule selected from the group consisting of: an amphipathic carboxylic acid, Thiazolidinedione (TZD), WY-14643 (Pirinixic Acid), GW409544, GW6471, Leukotriene B4, GW 7647, Perfluorooctanesulfonic Acid, Perfluorooctanoic Acid, CP-775146, CP-865520, UNII-999KY5ZIGB, and Gemfibrozil, said contacting increasing the metabolic maturation of the immature hepatocyte, said agonist causing increased mitochondrial mass and mitochondrial proliferation rate per cell when compared to untreated immature hepatocytes. 
     
     
         44 . The isolated metabolically mature hepatocytes of  claim 43 , where the PXR agonist is a small molecule, a bile acid and a steroid, contact with said agonist increasing expression of CYP3A4 and CYP2C9 by at least two-fold. 
     
     
         45 . The metabolically mature hepatocyte population of  claim 43 , wherein the mature hepatocytes exhibit an albumin + /CY3A4 + /E-cadherin + /OCT4 − /SOX2 − /A1AT + /HNF4α +  expression signature. 
     
     
         46 . The metabolically mature hepatocyte population of  claim 44 , wherein the mature hepatocytes exhibit an albumin + /CY3A4 + /E-cadherin + /OCT4 − /SOX2 − /A1AT + /HNF4α +  expression signature.