Establishment And Transplantation Of Reversibly Immortalized Human Fetal Hepatocytes For Prevention Of Acute Liver Failure In Mice

Background and objectivesAcut or chronic liver failure is an end-stage liver disease caused by various agents. Orthotopic allogeneic liver transplantation remains the only treatment option that significantly improves the prognosis of patients with terminal liver failure and cirrhosis. Transplantation of the liver, however, is a complex procedure that is associated with significant morbility and mortality. In addition, the supply of livers available for transplantation limits the number of procedures to a small fraction of those needed and imposes a need to allocate transplantable livers to some patients and deny livers to others. Liver cells transplantation and bioartificial liver have been used clinically to bridge patients to whole organ transplantation and as an alternative to whole organ transplantation, and are attractive alternative to orthotopic allogeneic liver transplantation. The lack of an adequate supply of human hepatocytes, however, severely limits the use of hepatocyte transplantation as an alternative strategy in the treatment of liver diseases. The ideal hepatocytes for clinical use should exhibit at least the following specific properties: (1) being human in origin, (2) of normal (nonmalignant) phenotype, (3) readily available, (4) rapidly and easily grown in cell culture to high density, (5) stably remaining in a well-differentiated state for days or weeks, and (6) capable of the full range of synthetic and detoxifying features of mature hepatocytes. In theory, human hepatocytes (include adult and fetal hepatocytes) are the most ideal. But the source of adult liver donor is very limited and only used in liver transplantation. Further more, adult hepatocytes have poor proliferation potential in vitro. So it is impossible to obtain plentiful adult hepatocytes. Fetal hepatocyte is also difficult to be used widely for ethical reasons. Using animal hepatocytes will take risk of immune reaction and transmission of zoogenic viruses. Clinical use of bioartificial livers (BAL) and hepatocytes transplantation relie heavily on the development of human liver cell lines. When cultured in vitro, primary human hepatocytes almost do not proliferate, and rapidly lose its hepatic function in one to two weeks. It is a difficult problem to subculture primary human hepatocytes. An attractive alternative to using primary human hepatocytes would be to utilize clonal hepatocyte cell lines that can grow economically in culture and exhibits the characteristics of differentiated hepatocytes. The aim of this study is to reversibly immortalize human fetal hepatocytes by using the retroviral vector pLNCTIGlox containing SV40T gene and investigate whether the immortalized cells can rescue mice with acute liver failure induced by 90% hepatectomy.Methods1. The pLNCTIGlox retrovirus particles were packaged by the PT67 amphotropic packaging cell line. The viral titer was determined by infecting NIH3T3 cells. And expression of SV40T from the retrovector was verified by immunocytochemical staining of SV40T in infected NIH3T3 cells.2. Human fetal hepatocytes were isolated from the liver of a legally aborted 20-week-old male fetus with two steps collagenase perfusion.3. Human fetal hepatocytes were infected with pLNCTIGlox retrovirus supernantant and succesfully transduced immortalized cells were selectively cultured using G418.4. Indirect immunochemical staining of albumin, cytokeratin 18, cytokeratin 19 and SV40T was carried out for the immortalized cells. Glycogen of the immortalized cells was also stained. Cell growth characteristics was analysed.5. Supernatant of culture were collected for assessment of albumin secretion and urea synthesis using radio-immnoassay and automatic biochemistry analyzer respectively.6. Reverse transcription polymerase chain reaction(RT-PCR) was performed to detect mRNA of genes related with hepatic differentiation in the immortalized cells.7. To evaluate the potential tumorigenicity of the immortalized hepatocytes, 4×10~6 cells in the tenth passage were inoculated in the left oxter of severe combined immunodeficiency (SCID) mouse. And 4×10~6 fetal hepatocytes were inoculated in the right oxter as control.8. SCID mice acute liver failure were surgically induced by 90% hepatectomy. Animals were divided into four groups: Group 1 (G1), 1×10~6 immortalized hepatocytes; Group 2 (G2), 1×10~6 primary human fetal hepatocytes; Group 3 (G3), 1×10~6 transformed HepG2 human liver cells; and Group 4 (G4), intrasplenic injection of 0.1 mL of DMEM. Cell transplantation was performed simultaneously during the procedure of 90% hepatectomy. The ammonia levels of 90% hepatectomized mice were measured in blood obtained from the tail vein of the mice, and all transplanted mice were followed 1 month for survival. At 2 weeks after transplantation the liver tissue was sampled. Serial tissue sections were cut and immunohistochemical staining was carried out to track the transplanted cells.9. The pQCXIP-NLS-iCre-ERT2 retrovirus particles were packaged by the Phoenix amphotropic packaging cell line, and the Cre-ER gene was transduced into the immortalized liver cells by infection with the pQCXIP-NLS-iCre-ERT2 retrovirus supernantant.10. Tamoxifen treatment induced the activety of Cre-ER recombinase. The immortalizing gene SV40T were excised in the immortalized liver cells. The expression and integration of SV40T gene were analyzed by immunocytochemical staining and polymerase chain reaction(PCR) respectively.Results1. The pLNCTIGlox viral titer was determined as 3×104CFU/ml. And expression of SV40T from the retrovector was positive with immunocytochemical staining in infected NIH3T3 cells.2. Hepatocyte yields were 1×10~9 cells, and the cells were about 95% viable by trypan blue dye exclusion.3. Five cell clones with epithelial phenotypes survived after selection with 500μg/mL G418. One resulting clone obtained after extensive screening is referred to as HepCL.4. The population doubling time of HepCL cells in logarithmic growth phase was calculated to be approximately 17 hours. HepCL cells were positive for albumin, cytokeratin 18, cytokeratin 19 and SV40T immunocytochemical staining. The former three stain localise in cytoplasm, and the latter in nuclei. Glycogen staining of HepCL cells was weakly positive.5. Quantitative assays of human albumin and urea demonstrated that the average synthetic efficacy of HepCL cells for albumin and urea was 3.6pg per cell per day and 0.22pmol per cell per day respectively.6. Approximately 4×106 HepCL cells were transplanted into the left oxter of SCID mice, and same amount of unmodified primary human fetal hepatocytes were transplanted into the right oxter of each mice as a control. The transplanted mice were observed for more than 16 months and no tumor was formed in any animal.7. RT-PCR assessment showed that HepCL cells express hepatocyte specific markers albumin, CK8, CK18, HGF receptor,α-1-antitrypsin and cytochrome P450 1B1, and the biliary-specific markers CK 7, CK19, biliary glycoprotein andγ-glutamyl transpeptidase. The cell cycle regulator p21 was also expressed. The POU domain transcription factor octamer-binding protein 4(Oct-4) was present in HepCL cells. But transforming growth factor beta receptor type II and Rex-1 was not expressed.8. Mice receiving HepCL cells (G1) and primary human fetal hepatocytes (G2) showed significantly lower blood ammonia levels after 90% hepatectomy compared with mice in G3 or G4. The mean survival time of mice in G1 and G2 were significantly longer than those in G3 and G4. Two weeks after 90% hepactectomy and cell transplantation, clusters of transplanted human hepatocytes could be observed in the liver of survived mice receiving HepCL cells or primary human fetal hepatocytes.9. Phoenix A cells stably packaging pQCXIP-NLS-iCre-ERT2 retrovirus particles were successfully obtained, and the viral supernantant was titrated as 5×10~4CFU/ml.10. A Cre-HepCL cell line harbored Cre-ER gene was successfully obtained by infection of HepCL with the pQCXIP-NLS-iCre-ERT2 retrovirus supernantant.11. After OHT treatment induced recombination, a few Cre-HepCL cells were still positive for SV40T immumocytochemical staining, and SV40T PCR were also weakly positive, suggesting not 100% of Cre-HepCL cells can reverse. Conclusions1. The immortalized liver cell line HepCL was successfully established by infection of primary human fetal hepatocytes with retroviral vector pLNCTIGlox.2. HepCL cells have similar characteristics as primary human fetal hepatocytes and show lack of tumorigenicity. The POU domain transcription factor octamer-binding protein 4(Oct-4) was present in HepCL cells. But transforming growth factor beta receptor type II and Rex-1 was not expressed.3. HepCL cells can provide hepatic support and improve survival for mice with acute liver failure. HepCL may be useful as a source of hepatic function in cell-based therapeutics of acute liver failure.4. Reversibly immortalized Cre-HepCL cells were obtained by infection of HepCL with the pQCXIP-NLS-iCre-ERT2 retrovirus supernantant. But reversion of Cre-HepCL cells can not reach 100%.