Separation of mouse hepatocytes of distinct biological phenotypes based on their asialoglycoprotein receptor-mediated adhesion to an artificial ligand

The ability to isolate highly proliferative hepatocytes is important for gaining insight into the molecular mechanisms of liver regeneration as well as for the development of a bio-hybrid artificial liver, because it is difficult to proliferate hepatocytes in vitro. The aim of this study was to isolate highly proliferative hepatocytes in the adult liver. We tried to separate hepatocytes expressing low levels of asialoglycoprotein receptor (ASGP-R) based on differences of adhesion of hepatocytes for poly[N-p-vinylbenzyl-O-β-D-galactopyranosyl-(1→4)-D-gluconamide (PVLA). PVLA is a β-galactose-carrying styrene polymer. This polymer is regarded as an artificial ligand for ASGP-R. We were able to get hepatocytes that could not adhere to PVLA when we weakened the interaction between PVLA and hepatocytes by decreasing calcium ion in the incubation buffer. Those hepatocytes that could not adhere to PVLA and had low ASGP-R expression were approximately 5% to 15% of the total number of hepatocytes. It is of interest that the hepatocytes that could not adhere to PVLA, which had lower ASGP-R expression levels, had higher (more than two times) DNA synthesizing activity (i.e., were more proliferative) than the hepatocytes that could adhere to PVLA, which had higher ASGR-R expression levels. These results suggest that the hepatocytes with lower adhesion to PVLA due to their low ASGP-R expression may be potential candidates for progenitor-like hepatocytes due to their highly proliferative capacity. These findings indicate that isolation of highly proliferative hepatocytes using PVLA may provide a new research tool for a better understanding of the biology of hepatocytes and the mechanisms regulating their proliferation and differentiation in health and disease.