Methods of isolating bipotent hepatic progenitor cells

Abstract

A method of obtaining a mixture of cells enriched in hepatic progenitors is developed which comprises methods yielding suspensions of a mixture of cell types, and selecting those cells that are classical MHC class I antigen(s) negative and ICAM-1 antigen positive. The weak or dull expression of nonclassical MHC class I antigen(s) can be used for further enrichment of hepatic progenitors. Furthermore, the progenitors can be selected to have a level of side scatter, a measure of granularity or cytoplasmic droplets, that is higher than that in non-parenchymal cells, such as hemopoietic cells, and lower than that in mature parenchymal cells, such as hepatocytes. Furthermore, the progeny of the isolated progenitors can express alpha-fetoprotein and/or albumin and/or CK19. The hepatic progenitors, so isolated, can grow clonally, that is an entire population of progeny can be derived from one cell. The clones of progenitors have a growth pattern in culture of piled-up aggregates or clusters. These methods of isolating the hepatic progenitors are applicable to any vertebrates including human. The hepatic progenitor cell population is expected to be useful for cell therapies, for bioartificial livers, for gene therapies, for vaccine development, and for myriad toxicological, pharmacological, and pharmaceutical programs and investigations.

We claim:  
     
         1 . A composition comprising bipotent hepatic progenitors which express At least one intercellular adhesion molecule (ICAM) antigen and do not express major histocompability complex (MHC) class Ia antigen, in which the bipoint hepatic progenitors have a capacity to differentiate.  
     
     
         2 . The composition of  claim 1  in which the hepatic progenitors express at least one MHC class  1   b  antigen.  
     
     
         3 . The composition of  claim 2  in which the MHC class  1   b  antigen is weakly expressed.  
     
     
         4 . The composition of claim I in which the ICAM antigen is ICAM-1.  
     
     
         5 . The composition of  claim 1  in which the hepatic progenitors have a sidescatter in flow cytometry which is less than the sidescatter of mature parenchymal cells.  
     
     
         6 . The composition of  claim 1  in which the hepatic progenitors have a sidescatter in flow cytometry which is between the sidescatter of non-parenchymal cells and the sidescatter of mature parenchymal cells.  
     
     
         7 . The composition of  claim 1  in which the hepatic progenitors are capable of dividing and giving rise to progeny.  
     
     
         8 . The composition of  claim 7  in which the hepatic progenitors exhibit a capacity for clonal growth.  
     
     
         9 . The composition of  claim 8  in which the clonal growth requires extracellular matrix.  
     
     
         10 . The composition of  claim 7  in which the progeny grow in piled-up clusters.  
     
     
         11 . The composition of  claim 7  in which the progeny express alpha-fetoprotein, albumin, CK19, or combinations thereof.  
     
     
         12 . The composition of  claim 7  in which the progeny are hepatocytes or biliary cells.  
     
     
         13 . The composition of  claim 12  in which the hepatocytes or biliary cells additionally express a cell adhesion molecule that can be used for selection or identification of a particular subpopulation.  
     
     
         14 . A composition comprising hepatic progenitors, their progeny, or a combination thereof in which the hepatic progenitors and their progeny: 
 (a) weakly express at least one MHC class Ib antigen,    (b) exhibit a higher side scatter in flow cytometry than non-parenchymal cells, and    (c) express alpha-fetoprotein, albumin, CK19, or combinations thereof.    
     
     
         15 . The composition of  claim 14  in which the hepatic progenitors, their progeny, or a combination thereof are derived from endoderm or bone marrow.  
     
     
         16 . The composition of  claim 15  in which the endoderm is selected from liver, pancreas, lung, gut, thyroid, gonad, or combinations thereof.  
     
     
         17 . The composition of  claim 15  in which the progenitors express ICAM antigen.  
     
     
         18 . The composition of  claim 17  in which the ICAM antigen is ICAM-1.  
     
     
         19 . The composition of  claim 15  in which the progenitors do not express MHC class  1   a .  
     
     
         20 . The composition of  claim 15  in which the progenitors express at least one MHC class  1   b  antigen.  
     
     
         21 . A method of obtaining a mixture of vertebrate cells enriched in hepatic progenitors comprising: 
 (a) obtaining a cell suspension comprising vertebrate liver cells and    (b) removing from the cell suspension those cells that express at least one MHC class Ia antigen to provide a mixture of cells enriched in hepatic progenitors.    
     
     
         22 . A method of obtaining a mixture of vertebrate cells enriched in progenitors comprising: 
 (a) obtaining a cell suspension of vertebrate cells and    (b) sequentially in either order, or substantially simultaneously, removing from the cell suspension those cells that express at least one MHC class Ia antigen and isolating from the cell suspension those cells that are positive for an ICAM antigen, to provide a mixture of cells enriched in progenitors.    
     
     
         23 . A method for identification of progenitor cells, comprising: 
 (a) providing a cell suspension suspected of including progenitor cells; and    (b) identifying cells which express ICAM antigen and do not express MHC class Ia antigen.    
     
     
         24 . A method of obtaining a mixture of vertebrate cells enriched in hepatic progenitors comprising: 
 (a) providing a vertebrate embryonic stem cell,    (b) expanding the embryonic stem cell to give embryonic stem cell progeny, and    (c) isolating those embryonic stem cell progeny which express at lease one ICAM antigen and do not express MHC class  1   a  antigen.    
     
     
         25 . A method of treating a liver disorder or dysfunction with liver progenitors in a subject in need thereof comprising: administering to the subject an effective amount of cells enriched in human liver progenitors, their progeny, or a combination thereof, in a pharmaceutically acceptable carrier, in which the human liver progenitors express an ICAM antigen and do not express MHC class  1   a  antigen.  
     
     
         26 . A method of treating a genetic disorder in an individual in need thereof comprising administering to an individual in need thereof an effective amount of a bipotent hepatic progenitor harboring a gene which corrects a genetic disorder.