Aims: Telomerase reconstitution permits the unrestricted expansion of human fetal hepatocytes
in vitro. As expected, the enhanced proliferative capacity of telomerase-immortalized
hepatocytes is associated with an undifferentiated phenotype. In this study, we investigated
the kinetic of intrahepatic expansion and maturation of immortalized human cells in
a murine liver repopulation model. Methods: Scid/beige uPA mice (n=27) were intrasplenically transplanted with 2×106 immortalized human cells at population doubling 80 to 100. Engraftment and intrahepatic
expansion were monitored by human-specific PCR for beta-globin at 3 hours (n=4), 2.5
(n=5), 5 (n=4), 8 (n=10), and 12 weeks (n=5) after transplantation. To evaluate maturation,
we measured the expression of hepatocyte-specific genes employing human-specific RT-PCR
and relative quantification in comparison to control cells before engraftment (expression
level=1) with GADPH as housekeeping control (ΔΔCt-method). Results: Human cells were detected in the livers of all transplanted mice. As described previously,
about 10% of transplanted cells translocated to the liver 3 hours after transplantation.
Although the number of engrafted human cells did not increase significantly within
the first 8 weeks, a 4-fold expansion of engrafted human cells was observed at week
12, reaching repopulation rates of 3%. At week 2.5, we observed a 50- to 500-fold
increase in the expression of HNF4alpha. At week 5, the expression of HNF4alpha was
decreased to approximately baseline levels and was not consistently detectable at
week 8 and 12. Simultaneously, a 100- to 10,000-fold increase in the expression of
albumin, alpha1-antitrypsin, and transferrin was observed at week 2.5. Interestingly,
between week 5 and 12, a second significant increase in the expression of albumin
(median expression level at week 12 in comparison to control cells before engraftment
6.0×105, Mann-Whitney test in comparison to week 5 p=0.029) and transferrin (1.6×104, p=0.036) was observed. Discussion: Our data confirm the potential of immortalized hepatocytes to differentiate in the
liver environment. Intrahepatic maturation was associated with an early transient
upregulation of HNF4alpha and correlated to the expansion kinetic. We conclude that
telomerase-immortalized human fetal hepatocytes respond to signals governing proliferation
and differentiation in the liver parenchyma.
HNF4alpha - gene expression - hepatocyte transplantation - liver repopulation - telomerase
