A symphony of techniques for liver cell therapy, only applicable to rats?
Authors: Michael Ott
Journal: Journal of Hepatology (2008)

The present issue of the Journal includes a study by Kawashita et al., that investigates the potential of immortalized hepatocytes to repopulate the liver of Gunn rats after preparative irradiation and partial hepatectomy [[1]Kawashita Y. Guha C. Moitra R. Wang X. Fox I.J. Roy-Chowdhury J. et al.Hepatic repopulation with stably transduced conditionally immortalized hepatocytes in the Gunn rat.J Hepatol. 2008; 49: 99-106Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar]. Primary hepatocytes from Gunn rats were transduced with a thermolabile mutant simian virus 40 T-antigen as well as the human UGTA1a1 gene. The transduced cells were transplanted into recipient Gunn rats via intrasplenic injection. After various time points, serum bilirubin concentrations, human UGT1A1 levels and other parameters were measured. Three treatment groups and one control group receiving the parental cell line without transduction with the human UGTA1a1 gene were followed over a time period of 140 days. The results demonstrate that the in vitro amplified and transduced hepatocytes engraft, function and proliferate in the recipient liver. Radiation therapy of the recipient liver in combination with partial hepatectomy was most effective in reducing serum bilirubin levels. All single components of this study are well known and have been published before. It was one of the many achievements of Jayanta and Namita Roy-Chowdhury’s laboratory to have thoroughly characterized the Gunn rat as a model for cell and gene therapy [2Chowdhury J.R. Kondapalli R. Chowdhury N.R. Gunn rat: a model for inherited deficiency of bilirubin glucuronidation.Adv Vet Sci Comp Med. 1993; 37: 149-173PubMed Google Scholar, 3Kim B.H. Takahashi M. Tada K. Bosma P.J. Roy-Chowdhury J. Roy-Chowdhury N. Cell and gene therapy for inherited deficiency of bilirubin glucuronidation.Proc Natl Acad Sci USA. 1999; 96: 10349-10354Crossref PubMed Scopus (190) Google Scholar]. Their laboratory was also among the first to create and characterize immortalized hepatocytes, to study ex vivo gene therapy for metabolic liver diseases and, in co-operation with other groups at the Albert Einstein College, to apply preparative hepatic irradiation in combination with hepatectomy to induce liver repopulation by transplanted cells. The present paper now orchestrates all the pieces into one therapeutic approach and shows that a bilirubin glucuronidation deficiency state can be corrected in rats. The authors provide evidence that immortalized hepatocytes can be expanded in culture and transplanted into a recipient liver. This is both, a surprising and a promising result, since primary adult hepatocytes trypsinized from collagen coated cell culture plates hardly engraft in a recipient liver after transplantation. Of course, favourable conditions were created for the cells. Preparative irradiation of the liver combined with partial hepatectomy has been shown before to provide a favourable environment for cell engraftment and repopulation and probably was the key for the generation of sufficient liver mass derived from UGT1A1 gene corrected Gunn rat hepatocytes [4Takahashi M. Deb N.J. Kawashita Y. Lee S.W. Furgueil J. Okuyama T. et al.A novel strategy for in vivo expansion of transplanted hepatocytes using preparative hepatic irradiation and FasL-induced hepatocellular apoptosis.Gene Ther. 2003; 10: 304-313Crossref PubMed Scopus (34) Google Scholar, 5Christiansen H. Koenig S. Krause P. Hermann R.M. Rave-Frank M. Proehl T. et al.External-beam radiotherapy as preparative regimen for hepatocyte transplantation after partial hepatectomy.Int J Radiat Oncol Biol Phys. 2006; 65: 509-516Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar]. Additional help for reducing the bilirubin levels after transplantation was provided by transducing the immortalized hepatocytes with a retrovirus expressing the UGT1A1 gene. Compared to freshly isolated “wild-type” hepatocytes, the transduced immortalized Gunn rat hepatocytes were previously shown to over-express the UGT1A1 gene by twofold [[6]Tada K. Roy-Chowdhury N. Prasad V. Kim B.H. Manchikalapudi P. Fox I.J. et al.Long-term amelioration of bilirubin glucuronidation defect in Gunn rats by transplanting genetically modified immortalized autologous hepatocytes.Cell Transplant. 1998; 7: 607-616Crossref PubMed Scopus (52) Google Scholar]. In contrast, endogenously produced glucuronidation activity is likely to be down-regulated with a gradual loss of the differentiated hepatic phenotype in immortalized and expanded “wild-type” hepatocytes. It would be interesting to know whether immortalized “wild-type” hepatocytes would be as efficient for the correction of hyperbilirubinemia as isolated primary adult hepatocytes or gene corrected immortalized Gunn rat-derived hepatocytes [[7]Guha C. Parashar B. Deb N.J. Garg M. Gorla G.R. Singh A. et al.Normal hepatocytes correct serum bilirubin after repopulation of Gunn rat liver subjected to irradiation/partial resection.Hepatology. 2002; 36: 354-362Crossref PubMed Scopus (91) Google Scholar]. Will this strategy lead us to new horizons in the cell therapy of metabolic liver diseases? Yes and no. One of the major limitations for the development of liver cell therapy programs is the shortage of human hepatocytes. Many laboratories have focussed on stem cells as a renewable source of hepatocytes, but overall, extensive liver tissue formation has not been achieved with either adult or embryonic stem cells. It is one of the main messages of this report that immortalized hepatocytes can engraft, proliferate and correct a disease phenotype of a recipient liver. Safety of the cells remains a concern. Activation of the SV40 T-antigen is known to trigger molecular events in the target cell, which are not fully reversible by inactivation of the gene. Although tumours have not been detected in the animals, activation of alternative pathways inducing proliferation and new technologies such as flanking transferred genes with loxP targets for Cre recombinase mediated excision may be needed for future clinical application [[8]Kobayashi N, Fujiwara T, Westerman KA, Inoue Y, Sakaguchi M, Noguchi H, et al. Prevention of acute liver failure in rats with reversibly immortalized human hepatocytes. Science 2000;287:1258–1262.Google Scholar]. It is obvious from the published data that hepatocyte transplantation provides a therapeutic benefit in patients with hereditary metabolic liver disease, but cannot completely and stably correct a disease phenotype [9Nussler A. Konig S. Ott M. Sokal E. Christ B. Thasler W. et al.Present status and perspectives of cell-based therapies for liver diseases.J Hepatol. 2006; 45: 144-159Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 10Fisher R.A. Strom S.C. Human hepatocyte transplantation: worldwide results.Transplantation. 2006; 82: 441-449Crossref PubMed Scopus (373) Google Scholar]. Most patients, which are considered as targets for cell therapy do not have signs of a liver pathology and thus would not provide an intrinsic growth advantage for transplanted cells. Although well established in surgical departments, partial hepatectomy of the recipient liver as part of a cell therapy protocol will probably not be accepted for the often very young patients with metabolic liver disease. Mild transient ischemia/reperfusion damage of the liver may provide an alternative method in order to increase engraftment and proliferation of transplanted hepatocytes. In a conclusive study Malhi and co-workers have demonstrated that ∼90 min of ischemia and reperfusion in combination with preparative hepatic irradiation can result in massive repopulation by transplanted cells [[11]Malhi H. Gorla G.R. Irani A.N. Annamaneni P. Gupta S. Cell transplantation after oxidative hepatic preconditioning with radiation and ischemia-reperfusion leads to extensive liver repopulation.Proc Natl Acad Sci USA. 2002; 99: 13114-13119Crossref PubMed Scopus (84) Google Scholar]. The inherent risks of the procedure may be significantly reduced by restriction to single liver lobes. Cell transplantation combined with regional ischemia/reperfusion of less than half of the liver mass partially corrected hypercholesterolemia even in the absence of radiation theraphy [[12]Attaran M. Schneider A. Grote C. Zwiens C. Flemming P. Gratz K.F. et al.Regional and transient ischemia/reperfusion injury in the liver improves therapeutic efficacy of allogeneic intraportal hepatocyte transplantation in low-density lipoprotein receptor deficient Watanabe rabbits.J Hepatol. 2004; 41: 837-844Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar]. The radiation dosage required for inhibition of hepatocyte proliferation would not necessarily impair liver function, but would definitely cause DNA damage and carry the risk for tumour formation. Stephenne et al. have recently reviewed the application of radiation therapy in children with liver organ transplantation and did not find an increased risk for tumour formation or other pathologies in the follow-up [[13]Stephenne X. Najimi M. Janssen M. Reding R. de Ville de Goyet J. Sokal E.M. Liver allograft radiotherapy to treat rejection in children: efficacy in orthotopic liver transplantation and long-term safety.Liver Int. 2005; 25: 1108-1113Crossref PubMed Scopus (9) Google Scholar]. Would any of these procedures be too risky for patients with hereditary metabolic liver disease? Lessons may be learned from the hematologists. For years bone marrow transplantation has been a common practice for the treatment of congenital immune disorders and other defects affecting bone marrow-derived cells. Complete or partial myeloablation with chemotherapeutic drugs is often part of the protocol to facilitate the engraftment of donor bone marrow stem cells [[14]Notarangelo L.D. Forino C. Mazzolari E. Stem cell transplantation in primary immunodeficiencies.Curr Opin Allergy Clin Immunol. 2006; 6: 443-448Crossref PubMed Scopus (21) Google Scholar]. Advocacy for more aggressive cell therapy protocols would have to consider that liver organ transplantation is available for most of the children in the western world and provides long-term correction of the metabolic defect. Nevertheless, cell therapy concepts as an alternative or an additional approach to organ transplantation may offer potential benefits for selected patients with metabolic liver disease. It should be the objective of the international scientific community to develop the best protocols and techniques for the benefit of patients. The fascinating experiments presented by Kawashita et al. should help us to move closer to the routine application of cell therapies for liver diseases.