In Vitro Cell.Dev.Biol.--Animal 34:247-258,March 1998 © 1998Society for In Vitro Biology 1071-2690/98 $05.00 + 0.00 MATURATION-DEPENDENT GENE EXPRESSION IN A CONDITIONALLY TRANSFORMED LIVER PROGENITOR CELL LINE A. S. FIORINO,1 A. M. DIEHL, H. Z. LIN, I. R. LEMISCHKA, ANDL. M. REID2 Medical Scientist Training Program (A. S. E) and Departments of Microbiology and Immunology (L M. R.) and Molecular Pharmacology (A. S. E, L. M. R.), Albert Einstein College of Medicine, Bronx, New York 10461; Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 (A. M. D., H. Z. L.): Department of Molecular Biology; Princeton University. Princeton, New Jersey 08544 (L R. L) (Received 23 September 1996; accepted 6 March 1997) SUMMARY We have isolated a conditionally transformed liver progenitor cell line with phenotypic similarities to both hepatoblasts (bipotent embryonic liver cells that give rise to hepatocytes and intrahepatic biliary epithelial cells) and liver epithelial cells (primitive hepatic cells isolated from adult livers capable of generating both hepatocytic and biliary lineages). Cell line L2039 was derived from El4 fetal mouse liver after transformation with temperature-sensitive SV-40 large T antigen. At 33 ° C, these ceils have an epithelial morphology" with a high nucleocytoplasmic ratio and express both hepatocytic and biliary genes, including albumin, c~-fetoprotein, glutamine synthetase, insulinlike growth factor II receptor, fibronectin and laminin, and eytokeratins 8 and 19, a set of markers characteristic for hepatoblasts. The presence of cytokeratin 14, vimentin, and several oval-cell antigens link cell line L2039 to nonparenchymal liver epithelial cell populations thought to contain progenitor cells. Serum-free, hormonally defined media conditions and extracellular matrix requirements were determined for growth and differentiation of this cell line. During culture on type IV collagen at 39 ° C, L2039 cells cease dividing and demonstrate hepatocytic differentiation with the assumption of a hepatocytelike morphology and glucocorticoid- dependent regulation of liver-specific genes, including albumin, ct-fetoprotein, phosphoenolpyruvate carboxykinase, and liver-enriched transcription factors. The number of albumin-positive cells increases during culture at 39 ° C, indicating that L2039 cells convert from a prehepatocytic to a hepatocytic phenotype. Under conditions specific for hepatocytic differen- tiation, C/EBPs were expressed and differentially regulated, with C/EBPI3 and C/EBP6 upregulated early and C/EBPct only slightly expressed after 7 d, indicating that C/EBPct may not be a crucial factor in commitment to the hepatocytic phenotype. Key words: hepatoblast; hepatocyte differentiation; C/EBP; SV-40 large T antigen. INTRODUCTION From the endodermal cords that arise from the hepatic diverticu- lum and invade the hepatic mesenchyme during embryonal liver de- velopment derive hepatoblasts (5,12,29,36,81), bipotent epithelial cells that give rise to both bepatoeytes and intrahepatic biliary epi- thelial cells (43,44,47,76-81,88). The rodent liver, which begins de- velopment around embryonal Day 9 (E9), by El2 consists mainly of a morphologically homogeneous population of hepatoblasts, without mature hepatocytes or intrahepatic biliary epithelial cells (48,76- 79). Hepatoblasts express genes characteristic of hepatocytes and biliary epithelial ceils, including ct-fetoprotein (AFP), albumin (76,78,79,81), glutamine synthetase (GS) (5), insulinlike growth fac- tor (IGF) II receptor (15), laminin (61), fibronectin (61), and cyto- keratin (CK) 7, 8, 18, and 19 (48,60,79). As development proceeds, hepatoblasts become committed to either lineage and develop into 1To whom correspondence should be addressed at Department of Derma- tology, University of Pennsylvania Medical Center, Rhoads 2. Philadelphia, Pennsylvania 19104. -"To whom requests for reprints should be addressed at Department of Phys- iology and Program in Molecular Biology and Biotechnology, UNC School of Medicine, CB 7038, Chapel Hill, North Carolina 27514. mature cells with the appropriate morphology and gene expression (36,58). Certain nonparenchymal liver epithelial cells (LECs) isolated from adult rodents are thought to be capable of acting as hepatic progenitor cells (46,59,82,85). After intrahepatic transplantation, some LECs can incorporate into hepatic plates and undergo hepatocytic differ- entiation (25). Oval cells, LEC-like nonparenchymal epithelial cells isolated from the livers of carcinogen-treated rodents (3,36,47,73,74,82), differentiate in vivo into both hepatocytes (31,32,33) and duetlike cells (83) and, when transplanted, form col- onies of hepatoeytelike cells (35) and biliary structures (45). In cul- ture, oval cells can be induced to a hepatoeytie phenotype, as indi- cated by expression of albumin and liver enzymes (45,67). After in vitro transformation and transplantation, both LECs and oval cells form tumors of hepatoeytic and biliary phenotypes (14,84,91). To- gether, these results indicate thai some hepatic nonparenchymal ep- ithelial cells can act as progenitors, giving rise to both the hepato- cytic and biliary lineages. Hepatoblasts share with LECs and oval cells phenotypie charac- teristics as well as the ability to give rise to both liver lineages. It is unknown if LECs are hepatoblasts or bepatoblast precursors that are 247