Recently, we described a new model for hepatocyte transplantation with nearly total replacement of the liver by exogenous hepatocytes (E. Laconi et al., Am. J. Pathol., 153: 319–329, 1998). The model is based on the mitoinhibitory effect of the pyrrolizidine alkaloid retrorsine on hepatocytes in the resident liver while transplanted hepatocytes proliferate. In this study, we exploit this novel approach to address the important and controversial issue of whether hepatocytes, when proliferating extensively, undergo dedifferentiation and give rise to foci of undifferentiated hepatocytes. Genetically marked hepatocytes (isolated from normal Dipeptidyl peptidase IV+ Fischer 344 rats) were delivered intraportally (2 × 106 cells) into the liver of retrorsine-treated Dipeptidyl peptidase IV- mutant Fischer 344 rats in conjunction with partial hepatectomy. Transplanted hepatocytes were detected histochemically or immunohistochemically, and cell proliferation was studied by in situ hybridization for histone-3 mRNA. Expression of α-fetoprotein (AFP) mRNA, a marker of hepatocyte dedifferentiation, was also revealed by in situ hybridization. One day after partial hepatectomy and hepatocyte transplantation, endogenous hepatocytes and oval cells expanding in the liver expressed histone-3 mRNA (cells had entered S phase); 2 days later, transplanted hepatocytes and nonparenchymal cells also expressed histone-3 mRNA. Although the majority of endogenous hepatocytes did not divide and became arrested as quiescent megalocytes, the exogenous hepatocytes, as well as newly formed small hepatocytes, most probably derived from liver progenitor cells, underwent extensive proliferation. After 7–14 days, the nonparenchymal cells stopped proliferating, but transplanted hepatocytes and small endogenous hepatocytes continued to proliferate for 1 month, forming foci of dividing parenchymal cells. Although many of the hepatocytes in clusters were in S phase (histone-3 mRNA positive), none expressed AFP mRNA. In contrast, high expression of AFP mRNA was observed in proliferating oval and transitional cells, forming duct-like structures of cytokeratin-19-positive cells. From these studies, we conclude that hepatocyte proliferation in the adult liver is not associated with dedifferentiation.
Supported by NIH Grants RO1 DK 17609, RO1 DK 50636, and P30 DK41296 (to D. A. S.). E. L. is the recipient of a fellowship from the Italian Foundation for Cancer Research.