Intrahepatic expansion and maturation of telomerase-immortalized human fetal hepatocytes

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