Generation of hepatocytes from pluripotent stem cells

Abstract

Methods are provided for producing differentiated cells from stem cells, including producing hepatocytes. Compositions thereof are also provided, as are methods of treating a liver disorder.

1 . A method of producing a differentiated cell from a pluripotent stem cell, the method comprising maintaining the pluripotent stem cell in a medium comprising two or more of phenacetin, phytosphingosine HCl, and pyridoxal HCl, or a medium comprising conditioned medium from hepatoblasts, for a time sufficient to produce the differentiated cell. 
     
     
         2 . The method of  claim 1 , wherein the differentiated cell is a hepatocyte. 
     
     
         3 . The method of  claim 1 , wherein the differentiated cell exhibits a meso-endodermal phenotype of a fetal human hepatocyte. 
     
     
         4 . The method of  claim 1 , wherein the differentiated cell exhibits ureagenesis and/or albumin synthesis and/or vimentin expression. 
     
     
         5 . The method of  claim 1 , wherein the pluripotent stem cell is an inducible pluripotent stem cell or is an embryonic stem cell. 
     
     
         6 . The method of  claim 1 , wherein the hepatoblasts are immortalized human hepatoblasts. 
     
     
         7 . The method of  claim 1 , wherein the medium does not comprise serum. 
     
     
         8 . The method of  claim 1 , wherein the conditioned medium from hepatoblasts comprises medium conditioned by a culture of immortalized human hepatoblasts cultured in a medium comprising a basal medium. 
     
     
         9 . The method of  claim 1 , wherein the immortalized human hepatoblasts are immortalized human fetal hepatoblasts. 
     
     
         10 - 18 . (canceled) 
     
     
         19 . A composition comprising conditioned medium obtained from a culture of hepatoblasts cultured in a medium comprising a basal medium, or comprising a basal medium comprising two or more of phenacetin, phytosphingosine HCl, and pyridoxal HCl. 
     
     
         20 . The composition of  claim 19 , wherein (i) the medium comprising a basal medium from which the conditioned medium is obtained, or (ii) the basal medium further comprising two or more of phenacetin, phytosphingosine HC, and pyridoxal HCl, further comprises L-glutamine, non essential amino acids, and an antibiotic. 
     
     
         21 . The composition of  claim 19 , wherein basal medium is a Dulbecco's modified Eagle's medium (DMEM). 
     
     
         22 . The composition of  claim 20 , wherein the antibiotic is penicillin-streptomycin. 
     
     
         23 . The composition of  claim 20 , wherein the non-essential amino acids are glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline and L-serine. 
     
     
         24 . The composition of  claim 20 , wherein (i) the medium comprising a basal medium from which the conditioned medium is obtained, or (ii) the basal medium further comprising two or more of phenacetin, phytosphingosine HCl, and pyridoxal HCl,
 further comprises an artificial serum replacement.   
     
     
         25 . The composition of  claim 19 , wherein the hepatoblasts are immortalized. 
     
     
         26 . The composition of  claim 19 , wherein the hepatoblasts are human hepatoblasts. 
     
     
         27 . The composition of  claim 19 , wherein the hepatoblasts are fetal hepatoblasts. 
     
     
         28 - 35 . (canceled) 
     
     
         36 . A method of treating a liver disorder in a subject comprising administering to the subject an amount of the differentiated cells produced by the method of  claim 1 , or a conditioned media from a culture of such cells comprising a basal medium in an amount effective to treat a liver disorder.