Telomerase reconstitution immortalizes human fetal hepatocytes without disrupting their differentiation potential.
OBJECTIVE
The availability of in vitro expandable human hepatocytes would greatly advance liver-directed cell therapies. Therefore, we examined whether human fetal hepatocytes are amenable to telomerase-mediated immortalization without inducing a transformed phenotype and disrupting their differentiation potential. Telomerase is a ribonucleoprotein 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 telomeres trigger replicative senescence.


METHODS
The catalytic subunit, telomerase reverse transcriptase, was expressed in human fetal hepatocytes. Transduced cells were characterized for telomerase activity, telomere length, proliferative capacity, hepatocellular functions, oncogenicity, and their in vivo maturation potential.


RESULTS
The expression of human telomerase reverse transcriptase restored telomerase activity in human fetal hepatocytes. Telomerase-reconstituted cells were capable of preserving elongated telomeres, propagated in culture beyond replicative senescence for more than 300 cell doublings (to date), and maintained their liver-specific nature, as analyzed by a panel of hepatic growth factors, growth factor receptors, and transcription factors as well as albumin, glucose-6-phosphatase, glycogen synthesis, cytochrome P450 (CYP) expression profiles, and urea production. Moreover, the immortalized cells exhibited no oncogenicity, and no up-regulation of c-Myc was detected. The cells engrafted and survived in the liver of immunodeficient mice with hepatocellular gene expression.


CONCLUSIONS
Reconstitution of telomerase activity induces indefinite replication in human fetal hepatocytes and offers unique opportunities for examining basic biologic mechanisms and for considering development of stable cell lines for liver-directed therapies.
Wege
Henning
H
Transplant Research Institute, University of California, Davis Medical Center, Sacramento 95817, USA.
Le
Hai T
HT
Chui
Michael S
MS
Liu
Li
L
Wu
Jian
J
Giri
Ranjit
R
Malhi
Harmeet
H
Sappal
Baljit S
BS
Kumaran
Vinay
V
Gupta
Sanjeev
S
Zern
Mark A
MA
D000818:Animals; D002454:Cell Differentiation; D002461:Cell Line, Transformed / Q000502:physiology; D004268:DNA-Binding Proteins; D005260:Female; D005333:Fetus / Q000166:cytology*; D022781:Hepatocytes / Q000166:cytology / Q000201:enzymology / Q000502:physiology*; D006801:Humans; D051379:Mice; D009369:Neoplasms / Q000209:etiology; D011247:Pregnancy; D019098:Telomerase / Q000235:genetics / Q000378:metabolism*; D016615:Telomere / Q000648:ultrastructure; D014161:Transduction, Genetic
D004268:DNA-Binding Proteins; D019098:Telomerase