A novel strategy for in vivo expansion of transplanted hepatocytes using preparative hepatic irradiation and FasL-induced hepatocellular apoptosis (original) (raw)
Related papers
PLoS ONE, 2012
Background and Aims: Preparative hepatic irradiation (HIR), together with mitotic stimulation of hepatocytes, permits extensive hepatic repopulation by transplanted hepatocytes in rats and mice. However, whole liver HIR is associated with radiation-induced liver disease (RILD), which limits its potential therapeutic application. In clinical experience, restricting HIR to a fraction of the liver reduces the susceptibility to RILD. Here we test the hypothesis that repopulation of selected liver lobes by regional HIR should be sufficient to correct some inherited metabolic disorders.
International Journal of Radiation Oncology*Biology*Physics, 2007
Purpose/Objective(s): Hepatocyte transplantation is being considered as an alternative to orthotopic liver transplantation for inherited metabolic liver diseases. Transplantation of autologous hepatocytes following ex vivo transduction of the deficient gene is attractive because it avoids the need for lifelong immunosuppression associated with allogeneic cell transplantation. A major obstacle in ex vivo gene therapy has been primarily the loss of transgene expression after cell transplantation and the number of transduced cells that are necessary to overcome the deficiency. In this report we evaluated a preparative regimen of hepatic irradiation for ex vivo lentiviral gene therapy of autologous hepatocytes and studied the long-term gene expression and correction of serum hyperbilirubinemia in Gunn rats, which is a rodent model of type I Crigler-Najjar syndrome. Materials/Methods: The median lobe was resected from UDP-glucuronosyltransferase (UGT1A1)-deficient jaundiced Gunn rats. Freshly isolated Gunn hepatocytes, purified by two-step collagenase perfusion, were transduced ex vivo with a recombinant lentivirus vector, expressing the human UGT1A1 gene. The ex vivo gene transduced, autologous hepatocytes were then transplanted into donor Gunn rats by intrasplenic injection, following partial hepatectomy and preparative hepatic irradiation (HIR, 50 Gy). Animals were subsequently followed for serum bilirubin. Liver repopulation by transplanted hepatocytes was determined by UGT1A1 immunohistochemistry and immunoblots of liver homogenates. Liver histology was examined after H&E staining. Results: Serum levels of unconjugated bilirubin progressively decreased over time in the transplanted animals. At 8 weeks, serum hyperbilirubinemia decreased by 50-70% of the pretreatment levels (p \ 0.001). Immunoblot analyses of UGT1A1 demonstrated the expression of the enzyme in liver homogenates from transplanted rats. There was progressive repopulation of the irradiated liver lobes by UGT1A1-expressing autologous Gunn hepatocytes (40-65%). H&E staining failed to detect any pathological features of radiation injury. The correction of serum hyperbilirubinemia was maintained throughout the course of the present study (6 months). Conclusions: Ex vivo liver-directed UGT1A1 gene therapy was successful in ameliorating serum hyperbilirubinemia in Gunn rats. This study demonstrates successful isolation of autologous hepatocytes from a resected liver lobe, followed by transduction of the deficient transgene by lentiviral gene therapy. A preparative regimen of HIR augmented the engraftment and preferential proliferation of the transplanted gene-transduced hepatocytes.
Hepatology, 2002
The treatment of inherited metabolic liver diseases by hepatocyte transplantation (HT) would be greatly facilitated if the transplanted normal hepatocytes could be induced to proliferate preferentially over the host liver cells. We hypothesized that preparative hepatic irradiation (HIR) should inhibit host hepatocyte proliferation in response to partial hepatectomy (PH). Normal nonirradiated hepatocytes transplanted in this setting should have a selective growth advantage over the host liver cells and should progressively repopulate the liver. To test this hypothesis, we transplanted 5 million hepatocytes from normal Wistar-Roman High Avoidance (RHA) rats into the livers of congeneic bilirubin-uridine 5-diphosphoglucuronate glucuronosyltransferase (UGT1A1)-deficient jaundiced Gunn rats by intrasplenic injection after one of the following treatments: (1) 68% PH, (2) HIR (50 Gy), or (3) HIR ؉ PH. In rats receiving either PH or HIR alone before HT, serum bilirubin concentrations declined by 25% to 30% in 28 weeks. In contrast, serum bilirubin levels were normalized completely in rats receiving HIR ؉ PH before HT. Massive repopulation of the Gunn rat liver by the UGT1A1-positive Wistar-RHA hepatocytes was shown by UGT1A1 enzyme assay, immunoblot analysis, and immunohistochemical staining of the recipient liver. High-performance liquid chromatography analysis of the bile collected from Gunn rats 5 months after PH, HIR, and HT showed normalization of the pigment profile, with bilirubin diglucuronide and monoglucuronide as the predominant pigments. In conclusion, a preparative regimen of HIR ؉ PH results in massive repopulation of the liver with functionally normal transplanted hepatocytes, resulting in complete correction of a metabolic deficiency. Noninvasive strategies to replace PH for providing proliferative stimuli to the transplanted cells should make this regimen valuable in augmenting the effects of HT for the treatment of liver diseases. (HEPATOLOGY 2002; 36:354-362.)
Hepatic irradiation augments engraftment of donor cells following hepatocyte transplantation
Hepatology, 2009
Engraftment of donor hepatocytes is a critical step that determines the success of hepatocyte transplantation. Rapid and efficient integration of donor cells would enable prompt liver repopulation of these cells in response to selective proliferative stimuli offered by a preparative regimen. We have earlier demonstrated that hepatic irradiation (HIR) in combination with a variety of hepatotrophic growth signals, such as partial hepatectomy and hepatocyte growth factor, can be used as a preparative regimen for liver repopulation of transplanted hepatocytes. In this study, we investigated the effects of HIR on engraftment of transplanted dipeptidyl peptidase
Hepatology, 1995
Viral vectors and protein carriers utilizing asialoglycoprotein receptor (ASGR)-mediated endocytosis are being developed to transfer genes for the correction of bilirubin-UDP-glucuronosyltransferase (bilirubin-UGT) deficiency. Ex vivo evaluation of these gene transfer vectors would be facilitated by a cell system that lacks bilirubin-UGT, but expresses differentiated liver functions, including ASGR. We immortalized primary Gunn rat hepatocytes by transduction with a recombinant Moloney murine leukemia virus expressing a thermolabile mutant SV40 large T antigen (tsA58). At 33°C, the immortalized hepatocyte clones expressed SV40 large T antigen, synthesized DNA, and doubled in number every 2 to 3 days. At this temperature, differentiated hepatocyte markers, e.g., albumin, ASGR, and androsterone-UGT, were expressed at 5% to 10% of the levels found in primary hepatocytes maintained in culture for 24 hours. Glutathione-S-transferase Yp (GST-Yp), an oncofetal protein, was expressed in these cells at 33°C, but was undetectable in primary hepatocytes. In contrast, when the cells were cultured at 39°C or 37°C, the large T antigen was degraded, DNA synthesis and cell growth stopped, and morphologic characteristics of differentiated hepatocytes were observed. The expression of albumin, ASGR, and androsterone-UGT, and their corresponding mRNAs, increased to 25% to 40% of the level in primary hepatocytes, whereas GST-Yp expression decreased.
Human Gene Therapy, 2001
Hepatocyte transplantation might represent a potential therapeutic alternative to liver transplantation in the future 1,2 ; however, transplanted cells have a limited capacity to repopulate the liver, as they do not proliferate under normal conditions. Recently, studies in urokinase (uPA) transgenic mice 3-5 and in fumarylacetoacetate hydrolase (FAH)-deficient mice 6 have shown that the liver can be repopulated by genetically engineered hepatocytes harboring a selective advantage over resident hepatocytes 7 . We have reported that transgenic mice expressing human Bcl-2 in their hepatocytes are protected from Fas/CD95-mediated liver apoptosis 8 . We now show that Bcl-2 transplanted hepatocytes selectively repopulate the liver of mice treated with nonlethal doses of the anti-Fas antibody Jo2. FK 506 immunosuppressed mice were transplanted by splenic injection with Bcl-2 hepatocytes. The livers of female recipients were repopulated by male Bcl-2 transgenic hepatocytes, as much as 16%, after 8 to 12 administrations of Jo2. This only occurred after anti-Fas treatment, confirming that resistance to Fas-induced apoptosis constituted the selective advantage of these transplanted hepatocytes. Thus, we have demonstrated a method for increasing genetic reconstitution of the liver through selective repopulation with modified transgenic hepatocytes, which will allow optimization of cell and gene therapy in the liver.
Tissue Engineering Part A, 2016
Cell-based therapies represent a promising alternative to orthotopic liver transplantation. However, therapeutic effects are limited by low cell engraftment rates. We recently introduced a technique creating human hepatocyte spheroids for potential therapeutic application. The aim of this study was to evaluate whether these spheroids are suitable for engraftment in diseased liver tissues. Intrasplenic spheroid transplantation into immunodeficient uPA/SCID/beige mice was performed. Hepatocyte transduction ability prior to transplantation was tested by lentiviral labeling using red-greenblue (RGB) marking. Eight weeks after transplantation, animals were sacrificed and livers were analyzed by immunohistochemistry and immunofluorescence. To investigate human hepatocyte-specific gene expression profiles in mice, quantitative real-time-PCR was applied. Human albumin and alpha-1-antitrypsin concentrations in mouse serum were quantified to assess the levels of human chimerism. Precultured human hepatocytes reestablished their physiological liver tissue architecture and function upon transplantation in mice. Positive immunohistochemical labeling of the proliferating cell nuclear antigen revealed that human hepatocytes retained their in vivo proliferation capacity. Expression profiles of human genes analyzed in chimeric mouse livers resembled levels determined in native human tissue. Extensive vascularization of human cell clusters was detected by demonstration of von Willebrand factor activity. To model gene therapy approaches, lentiviral transduction was performed ex vivo and fluorescent microscopic imaging revealed maintenance of RGB marking in vivo. Altogether, this is the first report demonstrating that cultured and retroviral transduced human hepatocyte spheroids are able to engraft and maintain their regenerative potential in vivo. The works were performed at the University Medical Centers Bonn and Hamburg-Eppendorf.
PLOS One, 2010
Background: Severe Combined Immune Deficient (SCID)/Urokinase-type Plasminogen Activator (uPA) mice undergo liver failure and are useful hosts for the propagation of transplanted human hepatocytes (HH) which must compete with recipient-derived hepatocytes for replacement of the diseased liver parenchyma. While partial replacement by HH has proven useful for studies with Hepatitis C virus, complete replacement of SCID/uPA mouse liver by HH has never been achieved and limits the broader application of these mice for other areas of biomedical research. The herpes simplex virus type-1 thymidine kinase (HSVtk)/ganciclovir (GCV) system is a powerful tool for cell-specific ablation in transgenic animals. The aim of this study was to selectively eliminate murine-derived parenchymal liver cells from humanized SCID/uPA mouse liver in order to achieve mice with completely humanized liver parenchyma. Thus, we reproduced the HSVtk (vTK)/GCV system of hepatic failure in SCID/uPA mice.