Differentiation (1995) 59:35-42 zyxwvutsrq Differentiation Ontogeny, Neoplasia and DifferentiationTherapy zyx 0 Springer-Verlag 1995 Evidence for a terminal differentiation process in the rat liver Samuel H. SigaP4, Sanjeev Gupta2. zyxwvuts 4, David F. Gebhard Jr.3, Patricia Holst3,David Neufeld4, Lola M. ReidI4 Departments of I Molecular Pharmacology, 2 Medicine, Microbiology and Immunology, Marion Bessin Liver Research Center, Albert Einstein College of Medicine, I300 Morris Park Avenue, Bronx, NY 10461, USA Accepted in revised form: 18 April 1995 Abstract. In rapidly renewing epithelia, such as skin and gut, as well as hemopoietic cells and stromal fibroblasts, the process of progenitor cell maturation, terminal dif- ferentiation and senescence from cells of a fetal pheno- type is strikingly similar. To examine hepatocellular maturation, we studied embryonic, suckling and young adult rat liver cells with multiparametric fluorescence activated cell sorting (FACS), after exclusion of hemo- poietic, endothelial, Kupffer, and nonviable cells. With maturation, cell granularity and autofluorescence expo- nentially increased from fetal liver to suckling and adult liver as the proportion of S phase cells progressively de- clined from 33.8%+1.3% to 4.9%*2.8% and 1.1%&0.6% zyxwvu (P<0.05), respectively. In liver from fetal and suckling rats, all hepatocytes were mononuclear and contained diploid DNA whereas 21.2%+5.9% hepatocytes in adult liver were binucleated. Analysis of nuclear DNA content in adult hepatocytes demonstrated that 53.3%&3.9% of the nuclei were diploid, 43.6%+3.5% tetraploid and 0.5&0.6%octaploid. However, in the adult liver, small, mononuclear cells were also present with granularity and autofluorescence comparable to fetal hepatoblasts, as well as glucose-6-phosphatase activity, diploid DNA in 89.0%+2.1% of the nuclei, and with increased granu- larity in culture. Since general features of terminal cellu- lar differentiation and senescence include cessation of mitotic activity, polyploidy and accumulation of autoflu- orescent secondary lysosomes, our data suggest that liv- er cells too undergo a process of terminal differentiation. Introduction Hepatic development is an extended process that contin- ues through early postnatal life. In the rat, the liver is first evident on embryonic day 10 (E10) as the hepatic diverticulum invaginates into the septum transversum [31]. Through E14, most hepatoblasts are thought to be zyxwvu Correspondence to: S. Gupta bipotent with an ability to differentiate into hepatocytes as well as biliary cells, but by El5 most hepatoblasts are committed to the hepatocyte lineage [9, 311. During the remaining period of gestation and the first 4 postnatal weeks, hepatoblasts progressively acquire morphologic features, zyxw lineage-position-specific cytokeratins, plasma membrane proteins and functions of the differentiated phenotype as the adult hepatic cord structure and meta- bolic zonation evolve [9, 19, 311. Associated with this process of hepatocyte differentiation is a progressive de- cline in cellular replication. At birth, 16%-18% of hepa- tocytes synthesize DNA with a uniform distribution throughout the hepatic acinus [17, 261. However, as ear- ly as day 1, DNA synthesis rapidly decreases in hepatic zone 3 followed 3 days later by a decrease in zone 2 [17]. By the age of 10-12 days, only 6% of hepatocytes synthesize DNA, and 80% of these cells are located in hepatic zone 1. By 24 weeks of age, less than 0.2% of the hepatocytes synthesize DNA with an even greater decrease in mitotic activity as the cells become increas- ingly polyploid [17, 261. Overall, these findings suggest an inverse relationship between hepatic differentiation and cell proliferation. Furthermore, the in vitro prolifera- tive capacity of hepatocytes progressively declines with maturation. Fetal hepatoblasts can multiply 4- to 6-fold in vitro [14]. Under optimal hormonal stimulation, a large proportion of suckling hepatocytes undergo DNA synthesis and mitosis, whereas adult hepatocytes exhibit only limited DNA synthesis or mitosis [2]. The limited proliferative potential of adult hepa- tocytes in vitro has long been a paradox in view of the tremendous regenerative capacity of the liver. To account for this discrepancy, a role has been proposed for hepa- tocyte progenitor cells in the adult liver [l, 10, 11, 18, 30-32, 391. Though the vast majority of adult hepa- tocytes are capable of several cycles of cell division fol- lowing two-thirds partial hepatectomy, early experimen- tal studies suggested that this capacity is limited and a precursor cell population is ultimately responsible for hepatocyte renewal following liver injury [38]. This hy- pothesis was subsequently strongly supported by numer-