The relationship between differentiation and proliferation in late gestation fetal rat hepatocytes

Hepatocyte proliferation and differentiation occur simultaneously during la te mammalian gestation, We hypothesized that regulation of hepatocyte growt h and differentiation would be coordinated in late gestation fetal hepatocy te cultures such that proliferation would be most active in a population of less well-differentiated cells. Cultured fetal hepatocytes (embryonic d 19 and 21; E19 and E21) were studied using double staining immunofluorescent microscopy. Differentiation was assessed as staining for cw-fetoprotein (AF P), three markers of enzymic differentiation (glucokinase [GK], phosphoenol pyruvate carboxykinase [PEPCK], and carbamoyl phosphate synthase [CPS]), an d a hepatocyte cell-cell adhesion molecule (C-CAM). Proliferation was asses sed using immunocytochemical detection of proliferating cell nuclear antige n (PCNA) or 5-bromo-2'-deoxy-uridine (BrdU) incorporation into DNA. Fetal h epatocyte cultures consisted of a heterogeneous population of cells, slight ly more than half of which were proliferative under defined, growth factor- free conditions. These cultures were heterogeneous for AFP expression. Ther e was no correlation between the expression of AFP and PCNA or AFP and S-ph ase entry (BrdU staining) during the first 48 h in culture. Similar results were obtained in staining for the enzymic differentiation markers and C-CA M. In addition, the differentiation status of cultured fetal hepatocytes wa s unrelated to a presumed indicator of mature growth regulation, mitogenic responsiveness to transforming growth factor alpha (TGF alpha), and hepatoc yte growth factor (HGF). Finally, absence of any correlation between prolif eration and differentiated phenotype was supported by in vivo studies using staining for PCNA, AFP, CPS, and PEPCK in liver sections. These results in dicate that the developmental program governing differentiation of late ges tation fetal rat hepatocytes is independent from mechanisms controlling pro liferation.