Immortalization of human hepatocytes by temperature-sensitive SV40 large-T antigen

Dear Editor: Primary hepatocyte cultures tend to lose differentiated function in vitro, and in the case of human cells, are difficult to obtain for experimental studies. Simian Virus 40 (SV40) large-T antigen can bind p53 and prevent growth arrest and apoptosis of primary cells. Rat hepatocytes have been immortalized with whole SV40 (Georoff et al., 1984; Nakamura et al., 1995) or SV40 deoxyribonucleic acid (Livingstone et al., 1994) giving cell-lines with continued proliferation with retention of a variety of hepatoeyte functional markers. A temperature-sensitive (ts) T-antigen which binds p53 at the permissive temperature of 33.5 and not 39.5 ~ C has been used to transduce rat hepatocytes (Fox et al., 1995) producing cells with little differentiated function at the growth-permissive temperature (33.5 ~ C) but a marked functional increase at 39.5 ~ C, but this approach has not been reported with human hepatocytes. We established cell lines from human hepatoeytes by transduetion with the gene sequence for ts large-T antigen, and describe the characteristics of the initial lines and subsequent daughter clones. We used a replication-deficient retrovirus containing full-length large-T antigen gene sequence including tsA58 (temperature sensitivity) and U19 mutations plus the neor-seleetable marker gene (Stamps et al., 1994) to infect human hepatoeytes isolated from portions of normal liver necessarily removed during surgery for metastatic colonic cancer. Hepatoeytes isolated after eollagenase perfusion were separated at 50 x g, and plated at 2 x 10 a cells/cm 2 on a layer of irradiated (20 Gy) 3T3 mouse fibroblast feeder cells, in a-minimal essential medium containing nueleosides and nueleotides, glucose, 10% fetal calf serum, antibiotics, L-glutamine, 25 mg/ml bovine serum albumin/linoleic acid, 10 p.g/ml insulin, 10 6 M hydrocortisone, 10 ng/ml cholera toxin, 1.2 g/L nicotiuamide, and 10 ng/ml epidermal growth factor (EGF). Forty-eight hours after plating, the cultures were exposed for 24 h to retroviral supernatant of a helper-virus-free clone (PA/tsA58-U19/8) of an amphotropic packaging cell line (PA317) containing ts large-T antigen sequence in the presence of 8 Ixg/ml polybrene. From 7to 28-d cells were exposed to G418 selection. Colonies with a euboidal-epithelial appearance were then marked and all other cells removed by scraping, yielding three epithelial colonies from five independent sets of retrovirus-infeeted primary hepatoeyte cultures. The colonies were proliferatively quiescent for about 6 wk after selection. When proliferation resumed they were transferred from 37 to 33.5 ~ C. Cultures were passaged every 2 -3 wk, using medium as above except for EGF, for up to 11 passages. Cells in these cultures were morphologically heterogeneous, islands of tightly packed cuboidal cells being interspersed with scarcer elongated cells (Fig. 1). Growth was temperature-sensitive, and growth arrest occurred within 24-48 h of shift to 39.5 ~ C. Their doubling time at 33.5 ~ C was between 7 and 14 d. Despite the rapid growth arrest at 39.5 ~ C, nuclear Tantigen staining by indirect immunofluorescence persisted for over a wk, although much less intensely than in 33.5 ~ C cells. Albumin secretion by parent cells could be detected at low levels but there was little difference between cultures at 33.5 and 39.5 ~ C (0.352 + 0.027 and 0.376 -+ 0.065 p~g protein/106cells/24 h, respectively). However, secretion of cd-antiwypsin rose steadily over a 9-d period at 39.5 ~ C to 10 times the mean value at 33.5 ~ C (0.05 compared to 0.005 Ixg/106 cells/24 h). Cytochrome P450 activity measured in passage 7 ceils after 8 d at 39.5 ~ C, following treatment for 24 h with 3-methylcholanthrene or 1,2:5,6 dibenzanthracene as inducers, was 321.4 -+ 19.3 and 256.6 -432.4 pmol/h/mg cellular protein for the two inducers. No activity was seen at 33.5 ~ C. By passage 10, no P450 activity was detectable at either temperature. At passage 3 >90% of cells stained for cytokeratin 18, >90% also stained for vimentin, approximately 75% for cytokeratin 19, and 50% for cytokeratin 8, with the same pattern of cytoskeletal staining at the permissive and nonpermissive temperatures. To isolate clonal daughter lines, ceils of parent cultures of passage 4 -6 were plated at 50-200 cell/cm z on a feeder layer of irradiated (20 Gy) 3T3 fibroblasts. After 2 wk at 33.5 ~ C, colonies of 100-200 cells were visible. Discrete colonies of uniform morphology were used for subcloning. A 75-mm plastic ring over a clone was filled with 100 txl of 1 mg/ml trypsin in phosphate-buffered saline/0.02% ethylenediamine-tetraacetic acid, the contents were then aspirated and placed onto irradiated 3T3 feeder ceils. After further recloning, colonies were maintained in the usual growth medium. Thirteen clones were propagated. Two distinct morphological clonal types were observed at 33.5 ~ C (Fig. 2). Type I ceils grew as islands or sheets of cuboidal cells. Type II cells grew with a more dispersed cuboidal-elongated morphology. We examined in detail HTLC-17 with type 1 morphology, and HTLC-7, 11, 15, and 19 with type II morphology. HLTC-17 had a 7-10-d doubling time, and was passaged every 2 wk. The doubling times of the others were 14-20 d, and cells were passaged after 3 4 wk; in all lines, shift to 39.5 ~ C resulted in growth arrest and flattening and spreading of the cells within 24M~8 h (Fig. 3). HLTC-11 cells, passage 14 (after cloning) were stained for the ts T-antigen. Moderate or strong nuclear staining was observed in >95% of the cells fixed at 33.5 ~ C. After both 2 and 7 d at 39.5 ~ C, only 5-10% cells had moderate or strong staining and the rest were weakly stained or not stained at all. Different patterns of secretion of liver-specific proteins were observed. At passage 6 after cloning, fibrinogen secretion could be detected in all lines at a fairly constant rate (0.1-0.63 l,~g/106 cells/24 h) and was not affected by shifting the ceils to the nonpermissive temperature. In some lines (HLTC-7, HLTC-15, HLTC19), a l-ant i t rypsin was detected at 33.5 ~ C (mean values 0.11, 0.22,