Primitive and proximal hepatic stem cells

Abstract

Hepatic progenitors comprise two populations of human hepatic stem cells, primitive and proximal hepatic stem cells, and two populations of committed progenitors, one for biliary cells and one for hepatocytes. Human primitive hepatic stem cells are a very small fraction of the liver cell population and give rise to proximal hepatic stem cells constituting a much larger fraction of the liver. Human proximal hepatic stem cells give rise to biliary and hepatocyte committed progenitors. Primitive and proximal stem cells are the primary stem cells for the human liver. Human primitive hepatic stem cells may be isolated by immunoselection from human livers or culturing human liver cells under conditions which select for a human primitive hepatic stem cell. Proximal hepatic stem cells may be isolated by immunoselection, or by culturing human liver cells under conditions which include a developmental factor. Proximal hepatic stem cells may also be isolated by culturing colonies comprising a primitive hepatic stem cell under conditions which include a developmental factor. Resulting compositions may be used for treating liver disorders and for producing bioartificial organs.

1 - 12 . (canceled) 
     
     
         13 . A composition comprising an isolated proximal hepatic stem cell, wherein said isolated proximal hepatic stem cell expresses EpCAM and ICAM-1 and gives rise to a hepatocytic progenitor or biliary progenitor, wherein the isolated proximal hepatic stem cell is a single cell. 
     
     
         14 . The composition according to  claim 13 , wherein said isolated proximal hepatic stem cell expresses alpha-fetoprotein, albumin and cytokeratin 19. 
     
     
         15 . The composition according to  claim 14 , wherein said isolated proximal hepatic stem cell expresses hedgehog proteins, cytokeratin 7, CD44 and CD133. 
     
     
         16 . The composition according to  claim 15 , wherein said isolated proximal hepatic stem cell expresses P450-A7, SOX9 and telomerase protein in the nucleus and the cytoplasm. 
     
     
         17 . An isolated proximal hepatic stem cell, which expresses EpCAM and ICAM-1 and gives rise to a hepatocytic progenitor or biliary progenitor, wherein the isolated proximal hepatic stem cell is a single cell. 
     
     
         18 . The isolated proximal hepatic stem cell according to  claim 17 , wherein said isolated proximal hepatic stem cell co-expresses alpha-fetoprotein, albumin and cytokeratin 19. 
     
     
         19 . The isolated proximal hepatic stem cell according to  claim 18 , wherein said isolated proximal hepatic stem cell expresses hedgehog proteins, cytokeratin 7, CD44 and CD133. 
     
     
         20 . The isolated proximal hepatic stem cell according to  claim 19 , wherein said isolated proximal hepatic stem cell expresses P450-A7, SOX9 and telomerase protein in the nucleus and the cytoplasm. 
     
     
         21 . A method for producing a medicament for treating liver dysfunction or disease comprising admixing an effective amount of the proximal hepatic stem cell according to  claim 17  with a pharmaceutically acceptable carrier, excipient, or diluent. 
     
     
         22 . A method of isolating a proximal hepatic stem cell capable of giving rise to both hepatocytic progenitors or biliary progenitors, the method comprising:
 (a) providing a suspension of cells derived from liver tissue;   (b) culturing the cells under conditions including the use of serum-free medium supplemented with insulin, transferrin/Fe, a mixture of lipids comprising free fatty acids bound to albumin and a lipoprotein, trace elements and co-cultured with secondary cells that provide a developmental factor.   (c) selecting those cells that express EpCAM, ICAM-1, alpha-fetoprotein, albumin and cytokeratin 19.   
     
     
         23 . The method according to  claim 22 , wherein said secondary cells comprise feeder cells. 
     
     
         24 . The method according to  claim 23 , wherein said feeder cells are selected from the group consisting of STO feeder cells, endothelial cells, stromal cells, and embryonic liver stromal cells. 
     
     
         25 . The method according to  claim 24 , wherein said endothelial cells comprise endothelial precursor cells. 
     
     
         26 . The method according to  claim 24 , wherein said stromal cells comprise stromal precursor cells. 
     
     
         27 . The method according to  claim 22 , further comprising selecting those cells that express CD133. 
     
     
         28 . The method according to  claim 27 , further comprising selecting those cells that express hedgehog proteins, cytokeratin 7, and CD44. 
     
     
         29 . The method according to  claim 28 , further comprising selecting those cells that express P450-A7, SOX9 and telomerase protein in the nucleus and the cytoplasm. 
     
     
         30 . The method according to  claim 22 , wherein the liver tissue is obtained from fetus or a neonate. 
     
     
         31 . The method according to  claim 22 , wherein the liver tissue is obtained from a child or an adult. 
     
     
         32 . The method according to  claim 22 , wherein the proximal hepatic stem cell further comprises exogenous nucleic acid. 
     
     
         33 . A method of isolating a proximal hepatic stem cells from a liver, the method comprising:
 (a) providing a suspension of cells derived from liver tissue;   (b) selecting cells simultaneously expressing EpCAM, ICAM-1 and CD133 via positive immunoselection.