Expansion and maintenance of adult primary human hepatocytes in culture

Abstract

Methods for derivation, culture, and maturation of small hepatic progenitor cells are described.

1 - 6 . (canceled) 
     
     
         7 . A substantially pure, isolated population of CD44 +  small hepatocyte progenitor cells is obtained by culturing human hepatocytes in the presence of embryonic fibroblasts in a culture medium that comprises Y27632, A-083, and CHIR99021, epidermal growth factor, N2 and B27. 
     
     
         8 . A pharmaceutical composition comprising the cell population of  claim 7  and a pharmaceutically acceptable carrier. 
     
     
         9 . A method for treating a liver disease or injury in a patient, comprising administering to the patient a therapeutic dose of CD44 +  small hepatocyte progenitor cells of  claim 7 . 
     
     
         10 . The method of  claim 9 , wherein the disease or injury is selected from the group consisting of hepatitis liver infection, cirrhosis of the liver, alpha-1-antitrypsin deficiency, Wilson's disease, inherited liver disease, chronic liver disease and acute liver damage. 
     
     
         11 . A method for obtaining a population of mature human hepatocytes, the method comprising:
 culturing the population of small hepatocyte progenitor cells of  claim 7  in the presence of sinusoidal endothelial cells in a culture medium that supports survival of sinusoidal endothelial cells and under conditions suitable for generation of mature human hepatocytes, whereby a cell population comprising mature hepatocytes is obtained.   
     
     
         12 . The method of  claim 11 , wherein the sinusoidal endothelial cells are human liver sinusoidal endothelial cells. 
     
     
         13 . The method of  claim 11 , wherein the culture medium comprises N2 and B27. 
     
     
         14 . The method of  claim 13 , wherein the culture medium further comprises dexamethasone and epidermal growth factor. 
     
     
         15 . A method of obtaining a population of mature human hepatocytes, the method comprising:
 culturing the population of small hepatocyte progenitor cells of  claim 7  in a culture medium comprising a MAPK inhibitor, whereby a cell population comprising mature hepatocytes is obtained.   
     
     
         16 . The method of  claim 15 , wherein the population of small hepatocyte progenitor cells is cultured in the presence of mouse embryonic fibroblasts or human embryonic fibroblasts. 
     
     
         17 . The method of  claim 15 , wherein the population of small hepatocyte progenitor cells is cultured in the presence of human sinusoidal endothelial cells. 
     
     
         18 . The methods of  claim 15 , wherein the MAPK inhibitor is U0126. 
     
     
         19 . The method of  claim 15 , wherein the culture medium additionally comprises N2 and B27. 
     
     
         20 . The method of  claim 19 , wherein the culture medium additionally comprises dexamethasone and epidermal growth factor. 
     
     
         21 . The cell population of  claim 7 , wherein the CD44 +  small hepatocyte progenitor cells proliferate in culture for at least 3 passages. 
     
     
         22 . The cell population of  claim 7 , wherein after 3 passages, the small hepatocyte progenitor cells retain metabolic enzyme activity at a level at least 95% as high as that of the cultured human hepatocytes. 
     
     
         23 . The cell population of  claim 7 , wherein the SHPCs are genetically engineered and comprise a heterologous nucleic acid. 
     
     
         24 . A substantially pure, isolated population of genetically engineered mature hepatocytes derived from the cell population of  claim 23 . 
     
     
         25 . A cell culture medium comprising epidermal growth factor, Y27632, A083, CHIR99021, N2, and B27. 
     
     
         26 . The cell culture medium of  claim 25 , comprising between about 10 ng/ml and about 200 ng/ml epidermal growth factor, between about 0.5 μM and about 50 μM Y27632, between about 0.05 μM and about 10 μM A083, between about 0.1 μM and about 20 μM CHIR99021, 1X N2, and 1X B27. 
     
     
         27 . The cell culture medium of  claim 25 , additionally comprising dexamethasone, Oncostatin M, fetal bovine serum, insulin, nicotinamide, or combinations thereof. 
     
     
         28 . The cell culture medium of  claim 27  comprising 1 μM dexamethasone, 100 ng/ml epidermal growth factor, 10 μg/ml Oncostatin M, 20% fetal bovine serum, 2 μg/ml insulin, 100 nM nicotinamide, 10 μM Y27632, 0.5 μM A083, 3 μM CHIR99021, 1X N2 and 1X B27.