Fate of extrahepatic human stem and precursor cells after transplantation into mouse livers.

UNLABELLED: In recent years, a large number of groups studied the fate of human stem cells in livers of immunodeficient animals. However, the interpretation of the results is quite controversial. We transplanted 4 different types of human extrahepatic precursor cells (derived from cord blood, monocytes, bone marrow, and pancreas) into livers of nonobese diabetic/severe combined immunodeficiency mice. Human hepatocytes were used as positive controls. Tracking of the transplanted human cells could be achieved by in situ hybridization with alu probes. Cells with alu-positive nuclei stained positive for human albumin and glycogen. Both markers were negative before transplantation. However, cells with alu-positive nuclei did not show a hepatocyte-like morphology and did not express cytochrome P450 3A4, and this suggests that these cells represent a mixed cell type possibly resulting from partial transdifferentiation. Using antibodies specific for human albumin, we also observed a second human albumin-positive cell type that could be clearly distinguished from the previously described cells by its hepatocyte-like morphology. Surprisingly, these cells had a mouse and not a human nucleus which is explained by transdifferentiation of human cells. Although it has not yet been formally proven, we suggest horizontal gene transfer as a likely mechanism, especially because we observed small fragments of human nuclei in mouse cells that originated from deteriorating transplanted cells. Qualitatively similar results were obtained with all 4 human precursor cell types through different routes of administration with and without the induction of liver damage. CONCLUSION: We observed evidence not for transdifferentiation but instead for a complex situation including partial differentiation and possibly horizontal gene transfer.

Molecular and functional characterization of drug-metabolizing enzymes and transporter expression in the novel spontaneously immortalized human hepatocyte line HC-04.

In toxicological research, immortalized human hepatocytes provide a useful alternative to primary hepatocytes because interindividual variability in the expression of drug-metabolizing enzymes and drug transporters can largely be eliminated. However, it is essential that the cell line retain the original phenotype. The purpose of this study was to characterize a novel spontaneously immortalized human hepatocyte cell line, HC-04, with respect to the transcript and functional protein expression profile for the major drug-metabolizing enzymes and transmembrane transporters. HC-04 cells retained hepatocyte-specific function including albumin production and ornithine transcarbamoylase and glucose-6-phosphatase activity. Most of the major CYP forms were expressed at basal levels and responsive to inducing agents. In particular, CYP3A4 was expressed abundantly, and HC-04 cells were able to metabolize the CYP3A4 probe, midazolam, at a rate similar to primary human hepatocytes. Furthermore, the major human sulfotransferase and UDP-glucuronosyltransferase forms, as well as members of the ABC and SLC transporter superfamilies, nuclear receptors, and hepatic transcription factors were also expressed. HC-04 cells readily responded to standard hepatotoxicants that are dependent on CYP-mediated bioactivation, while another, tumor-derived cell line remained refractory to the drug challenge. Collectively, HC-04 cells provide a reliable, stable, and reproducible model for biomechanistic studies in drug toxicology.

Leflunomide or A77 1726 protect from acetaminophen-induced cell injury through inhibition of JNK-mediated mitochondrial permeability transition in immortalized human hepatocytes.

Leflunomide, a disease-modifying anti-rheumatic drug, protects against T-cell-mediated liver injury by poorly understood mechanisms. The active metabolite of leflunomide, A77 1726 (teriflunomide) has been shown to inhibit stress-activated protein kinases (JNK pathway), which are key regulators of mitochondria-mediated cell death. Therefore, we hypothesized that leflunomide may protect from drugs that induce the mitochondrial permeability transition (mPT) by blocking the JNK signaling pathway. To this end, we exposed cultured immortalized human hepatocytes (HC-04) to the standard protoxicant drug acetaminophen (APAP), which induces CsA-sensitive mPT-mediated cell death. We determined the effects of leflunomide on the extent of APAP-induced hepatocyte injury and the upstream JNK-mediated mitochondrial signaling pathways. We found that leflunomide or A77 1726 concentration-dependently protected hepatocytes from APAP (1 mM)-induced mitochondrial permeabilization and lethal cell injury. This was not due to proximal inhibition of CYP-catalyzed APAP bioactivation to its thiol-reactive metabolite. Instead, we demonstrate that leflunomide (20 microM) inhibited the APAP-induced early (3 h) activation (phosphorylation) of JNK1/2, thus inhibiting phosphorylation of the anti-apoptotic protein Bcl-2 and preventing P-Bcl-2-mediated induction of the mPT. This greatly attenuated mitochondrial cytochrome c release, which we used as a marker for mitochondrial permeabilization. The specific JNK2 inhibitor SP600125 similarly protected from APAP-induced cell death. In conclusion, these findings are consistent with our hypothesis that leflunomide protects from protoxicant-induced hepatocyte injury by inhibiting JNK signaling and preventing mPT induction.