Hepatoblasts and method of isolating same

Abstract

This invention relates to methods of isolating hepatoblasts utilizing panning techniques and fluorescence activated cell sorting. This invention further relates to isolated hepatoblasts and to a method of treating liver dysfunction as well as to methods of forming artificial livers.

We claim:  
     
         1 . A method of isolating hepatoblasts from embryonic or neonatal liver comprising: 
 (a) preparing a single cell suspension of embryonic or neonatal liver cells;    (b) panning said suspension utilizing antibodies specific for hemopoietic cells, including red blood cells, endothelial cells or other mesenchymal cells so as to remove hemopoietic cells, including red blood cells, endothelial cells and other mesenchymal cells from said suspension; and    (c) performing fluorescence activated cell sorting utilizing said antibodies so as to remove hemopoietic cells, including red blood cells, including red blood cells, endothelial cells and other mesenchymal cells from said suspension and performing multiparametric fluorescence activated cell sorting on said suspension utilizing at least one antibody to a hepatic cell marker, side scatter, forward scatter and/or autofluorescence such that the cells remaining in said suspension are isolated hepatob lasts.    
     
     
         2 . The method of  claim 1  wherein the antibody-specific for hemopoietic cells is a monoclonal antibody.  
     
     
         3 . The method of  claim 2  wherein said monoclonal antibody is OX-43 and/or OX-44.  
     
     
         4 . The method of  claim 1  wherein the antibody to a hepatic cell marker is monoclonal antibody 374.3.  
     
     
         5 . The method of  claim 1  wherein said hepatic cell marker is OC.3.  
     
     
         6 . The method of  claim 1  wherein said single cell suspension contains an agent capable of removing calcium from liver cell surface.  
     
     
         7 . The method of  claim 1  wherein said single cell suspension contains EGTA.  
     
     
         8 . The method of  claim 1  wherein said single cell suspension contains an enzyme capable of dissociating liver cells.  
     
     
         9 . The method of  claim 1  wherein said single cell suspension contains collagenase.  
     
     
         10 . The method of  claim 1  wherein said single cell suspension is chilled.  
     
     
         11 . The method of  claim 1  wherein said single cell suspension is at a temperature of between about 2 and 20° C.  
     
     
         12 . Hepatoblasts isolated by the method of  claim 1 .  
     
     
         13 . A method of isolating hepatoblasts from adult liver comprising: 
 (a) preparing a single cell suspension of adult. liver cells;    (b) panning said suspension utilizing antibodies specific for mature hepatocytes, mature bile duct cells, endothelial cells and mesenchymal cells so as to remove mature hepatocytes, mature bile duct cells, endothelial cells and mesenchymal cells from said suspension; and    (c) performing fluorescence activated cell sorting utilizing said antibodies so as to remove mature hepatocytes, mature bile duct cells, endothelial cells and mesenchymal cells from said suspension and performing multiparametric fluorescence activated cell sorting on said suspension utilizing antibody to a hepatic cell marker, side scatter, forward scatter and/or autofluorescence such that the cells remaining in said suspension are isolated hepatoblasts.    
     
     
         14 . The method of  claim 13  wherein the antibody to a hepatic cell marker is monoclonal antibody 374.3.  
     
     
         15 . The method of  claim 13  wherein the hepatic cell marker is OC.3.  
     
     
         16 . The method of  claim 13  wherein the single cell suspension contains an agent capable of removing calcium from the surface of liver cells.  
     
     
         17 . The method of  claim 13  wherein the single cell suspension contains EGTA.  
     
     
         18 . The method of  claim 13  wherein the single cell suspension contains an enzyme capable of dissociating adult liver cells.  
     
     
         19 . The method of  claim 13  wherein the single cell suspension contains collagenase.  
     
     
         20 . The method of  claim 13  where in the single cell suspension is chilled.  
     
     
         21 . The method of  claim 13  wherein the single cell suspension is at a temperature of between about 2 and 20° C.  
     
     
         22 . Hepatocytes isolated by the method of  claim 13 .  
     
     
         23 . A method of treating liver dysfunction comprising the administration of hepatoblasts.  
     
     
         24 . The method of  claim 23  wherein the administration comprises injecting said hepatoblasts into the liver via a vascular vessel.  
     
     
         25 . The method of  claim 23  wherein the administration of comprises injecting said hepatoblasts into an ectopic site.  
     
     
         26 . The method of  claim 23  wherein the administration comprises injecting said hepatoblasts into an ectopic site of the spleen.  
     
     
         27 . The method of  claim 23  wherein the hepatoblasts are isolated by the method of  claim 1 .  
     
     
         28 . The method of  claim 23  wherein the hepatoblasts are isolated by the method of  claim 13 .  
     
     
         29 . A method of forming an artificial liver comprising the utilization of hepatoblasts with a bioreactor.  
     
     
         30 . The method of  claim 29  wherein the hepatoblasts are isolated by the method of  claim 1 .  
     
     
         31 . The method of  claim 29  wherein the hepatoblasts are isolated by the method of  claim 13 .  
     
     
         32 . A method of forming an artificial liver comprising the utilization of hepatoblasts in a culture apparatus.  
     
     
         33 . The method of  claim 32  wherein the hepatoblasts are isolated by the method of  claim 1 .  
     
     
         34 . The method of  claim 32  wherein the hepatoblasts are isolated by the method of  claim 13.