Telomerase Reconstitution Immortalizes Human Fetal Hepatocytes Without Disrupting Their Differentiation Potential HENNING WEGE,* HAI T. LE,* MICHAEL S. CHUI,* LI LIU,* JIAN WU,* RANJIT GIRI, ‡ HARMEET MALHI, ‡ BALJIT S. SAPPAL, ‡ VINAY KUMARAN, ‡ SANJEEV GUPTA, ‡ and MARK A. ZERN* *Transplant Research Institute, University of California, Davis Medical Center, Sacramento, California; and ‡ Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York See editorial on page 568. Background & Aims: The availability of in vitro expand- able human hepatocytes would greatly advance liver- directed cell therapies. Therefore, we examined whether human fetal hepatocytes are amenable to telomerase-mediated immortalization without induc- ing a transformed phenotype and disrupting their dif- ferentiation potential. Telomerase is a ribonucleopro- tein that plays a pivotal role in maintaining telomere length and chromosome stability. Human somatic cells, including hepatocytes, exhibit no telomerase activity. Consequently, their telomeres progressively shorten with each cell cycle until critically short telo- meres trigger replicative senescence. Methods: The catalytic subunit, telomerase reverse transcriptase, was expressed in human fetal hepatocytes. Trans- duced cells were characterized for telomerase activ- ity, telomere length, proliferative capacity, hepatocel- lular functions, oncogenicity, and their in vivo maturation potential. Results: The expression of hu- mantelomerasereversetranscriptaserestoredtelom- erase activity in human fetal hepatocytes. Telomer- ase-reconstituted cells were capable of preserving elongated telomeres, propagated in culture beyond replicative senescence for more than 300 cell dou- blings (to date), and maintained their liver-specific nature, as analyzed by a panel of hepatic growth factors,growthfactorreceptors,andtranscriptionfac- tors as well as albumin, glucose-6-phosphatase, gly- cogen synthesis, cytochrome P450 (CYP) expression profiles, and urea production. Moreover, the immor- talized cells exhibited no oncogenicity, and no up- regulation of c-Myc was detected. The cells engrafted andsurvivedintheliverofimmunodeficientmicewith hepatocellular gene expression. Conclusions: Recon- stitution of telomerase activity induces indefinite rep- lication in human fetal hepatocytes and offers unique opportunities for examining basic biologic mecha- nisms and for considering development of stable cell lines for liver-directed therapies. L iver failure constitutes a major health problem. Liver- directed cell therapies, such as hepatocyte transplan- tation 1 and bioartificial liver support, 2,3 offer alternatives to orthotopic liver transplantation, which, as the only currently available effective treatment for liver failure, is restricted by the shortage of donor organs. Although use of primary human hepatocytes would be ideal for liver- directed cell therapies, the availability of these cells is limited, and expansion of human hepatocytes in culture is difficult. Using malignant cell lines, e.g., HepG2 or C3A cells, 2 or xenogenic (porcine) hepatocytes 3 for liver- directed cell therapies carries potential risks to recipi- ents, e.g., inoculation of tumor cells 4 or zoonotic dis- eases, notably transmission of porcine endogenous retrovirus. 5,6 This situation has prompted intensive re- search to generate human hepatocyte-derived cell lines and to explore various strategies for immortalizing and expanding the number of human hepatocytes. Telomeres consist of kilobases (kb) of reiterated hexa- nucleotides with the sequence 5'-TTAGGG-3' and con- stitute the specialized ends of eukaryotic chromosomes. 7 An adequate telomere length is required for chromosome stability because telomere looping is necessary to provide capping and protection of chromosome ends against deg- radation and ligation. 7 Normal somatic cells lose telo- meric DNA at a rate of 50 to 200 base pairs (bp) per population doubling (PD) because of replication-associ- ated attrition. 8 Eventually, this telomeric loss leads to critically short telomeres that are incapable of looping, and replicative senescence is activated with loss of cell Abbreviations used in this manuscript: bp,basepair;BrdU,5-bromo- 2-deoxyuridine; C/EBP, CCAAT/enhancer binding protein; CYP, cyto- chrome P450; FH, fetal hepatocytes; FH-hTERT, hTERT-transduced fetal hepatocytes; HGFR, hepatocyte growth factor receptor; HNF, hepatocyte nuclear factor; hTERT, human telomerase reverse tran- scriptase; kb, kilobase; PCR, polymerase chain reaction; PD, popula- tion doublings; RT-PCR, reverse transcription-polymerase chain reac- tion; TRF, terminal restriction fragment. © 2003 by the American Gastroenterological Association 0016-5085/03/$35.00 doi:10.1053/gast.2003.50064 GASTROENTEROLOGY 2003;124:432– 444