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.

1 - 26 . (canceled)  
   
   
       27 . An isolated single-cell bipotent hepatic progenitor which 
 (a) expresses at least one intercellular adhesion molecule (ICAM) antigen;    (b) does not express major histocompatibility complex (MHC) class Ia antigen; and    (c) exhibits at least one of the following characteristics: (1) expression of at least one of CD44H, alpha-fetoprotein, albumin or CK19, or (2) dull expression of a nonclassical MHC class Ia antigen, or (3) higher side scatter (SSC) relative to non-parenchymal cells as measured in a flow cytometer;    in which the bipotent hepatic progenitor has a capacity to differentiate into a hepatocyte or a biliary cell when exposed to differentiation-inducing growth conditions.    
   
   
       28 . An isolated single-cell hepatic progenitor in which the hepatic progenitor: 
 (a) expresses at least one MHC class Ib antigen;    (b) exhibits a numerically higher sidescatter value determined by flow cytometry than the sidescatter value of nonparenchymal cells of the same species;    (c) expresses alpha-fetoprotein, albumin, CK19, or combinations thereof; and    (d) wherein the hepatic progenitor is capable of differentiating into a hepatocyte or a biliary cell when exposed to differentiation-inducing growth conditions.    
   
   
       29 . A composition consisting essentially of isolated single-cell bipotent hepatic progenitors which (a) express at least one intercellular adhesion molecule (ICAM) antigen; 
 (b) do not express major histocompatibility complex (MHC) class Ia antigen; and    (c) exhibit at least one of the following characteristics: (1) expression of at least one of CD44H, alpha-fetoprotein, albumin or CK19, or (2) dull expression of a nonclassical MHC class Ia antigen, or (3) higher side scatter (SSC) relative to non-parenchymal cells as measured in a flow cytometer;    in which the bipotent hepatic progenitors have a capacity to differentiate into hepatocytes or biliary cells when exposed to differentiation-inducing growth conditions.    
   
   
       30 . A composition consisting essentially of isolated single-cell hepatic progenitors in which the hepatic progenitors: 
 (a) express at least one MHC class Ib antigen;    (b) exhibit a numerically higher sidescatter value determined by flow cytometry than the sidescatter value of nonparenchymal cells of the same species;    (c) express alpha-fetoprotein, albumin, CK 19, or combinations thereof; and    (d) wherein the hepatic progenitors are capable of differentiating into a hepatocytes or biliary cells when exposed to differentiation-inducing growth conditions.    
   
   
       31 . A composition comprising a population of isolated single cells enriched in bipotent hepatic progenitors which (a) express at least one intercellular adhesion molecule (ICAM) antigen; 
 (b) do not express major histocompatibility complex (MHC) class Ia antigen; and    (c) exhibit at least one of the following characteristics: (1) expression of at least one of CD44H, alpha-fetoprotein, albumin or CK19, or (2) dull expression of a nonclassical MHC class Ia antigen, or (3) higher side scatter (SSC) relative to non-parenchymal cells as measured in a flow cytometer;    in which the bipotent hepatic progenitors have a capacity to differentiate into hepatocytes or biliary cells when exposed to differentiation-inducing growth conditions.    
   
   
       32 . The composition of  claim 31  in which the bipotent hepatic progenitors express at least one MHC class Ib antigen.  
   
   
       33 . The composition of  claim 32  in which the MHC class Ib antigen is weakly expressed in comparison to expression of ICAM as indicated by a dull positive response to immunostaining with fluorescent anti-MHC class 1b antibody in comparison to a positive response to immunostaining with anti-ICAM antibody.  
   
   
       34 . The composition of  claim 31  in which the ICAM antigen is ICAM-1.  
   
   
       35 . The composition of  claim 31  in which the hepatic progenitors have a sidescatter value determined by flow cytometry which is numerically less than the sidescatter value of mature parenchymal cells of the same species.  
   
   
       36 . The composition of  claim 31  in which the hepatic progenitors have a sidescatter in flow cytometry which is between the sidescatter of nonparenchymal cells of the same species and the sidescatter of mature parenchymal cells of the same species.  
   
   
       37 . The composition of  claim 31  in which the hepatic progenitors are capable of dividing and giving rise to progeny.  
   
   
       38 . The composition of  claim 37  in which the hepatic progenitors exhibit a capacity for clonal growth.  
   
   
       39 . The composition of  claim 38  in which the clonal growth requires extracellular matrix.  
   
   
       40 . The composition of  claim 39  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.  
   
   
       41 . A composition comprising a population of isolated single cells enriched in hepatic progenitors in which the hepatic progenitors: 
 (a) express at least one MHC class Ib antigen;    (b) exhibit a numerically higher sidescatter value determined by flow cytometry than the sidescatter value of nonparenchymal cells of the same species;    (c) express alpha-fetoprotein, albumin, CK 19, or combinations thereof; and    (d) wherein the hepatic progenitors are capable of differentiating into hepatocytes or biliary cells when exposed to differentiation-inducing growth conditions.    
   
   
       42 . The composition of  claim 41  in which the hepatic progenitors are derived from endoderm or bone marrow.  
   
   
       43 . The composition of  claim 42  in which the endoderm is selected from liver, pancreas, lung, gut, thyroid, gonad, or combinations thereof.  
   
   
       44 . The composition of  claim 42  in which the progenitors express ICAM antigen.  
   
   
       45 . The composition of  claim 44  in which the ICAM antigen is ICAM-1.  
   
   
       46 . The composition of  claim 42  in which the progenitors do not express MHC class Ia.  
   
   
       47 . The composition of  claim 42  in which the progenitors weakly express at least one MHC class Ib antigen in comparison to expression of ICAM as indicated by a dull positive response to immunostaining with fluorescent anti-MHC class 1b antibody in comparison to a positive response to immunostaining with anti-ICAM antibody.  
   
   
       48 . A cell culture comprising a population of single-cell bipotent hepatic progenitors that 
 (a) express at least one intercellular adhesion molecule (ICAM) antigen;    (b) do not express major histocompatibility complex (MHC) class Ia antigen; and    (c) exhibit at least one of the following characteristics: (1) expression of at least one of CD44H, alpha-fetoprotein, albumin or CK19, or (2) dull expression of a nonclassical MHC class Ia antigen, or (3) higher side scatter (SSC) relative to non-parenchymal cells as measured in a flow cytometer;    in which the bipotent hepatic progenitors have a capacity to differentiate into hepatocytes or biliary cells when exposed to differentiation-inducing growth conditions.    
   
   
       49 . The cell culture of  claim 48  further comprising extracellular matrix.  
   
   
       50 . The cell culture of  claim 49  in which the extracellular matrix comprises collagen, fibronectin, laminin, or combinations thereof.  
   
   
       51 . The cell culture of  claim 49  further comprising feeder cells.  
   
   
       52 . The cell culture of  claim 51  in which the feeder cells are fibroblast cells.  
   
   
       53 . The cell culture of  claim 51  in which the feeder cells are embryonic or fetal cells.  
   
   
       54 . The cell culture of  claim 49  further comprising a serum-free culture medium.