To establish the efficacy of cell therapy in Wilson's disease, we used the Long-Evans Cinnamon (LEC) rat model with atp7b gene mutation and copper toxicosis. Several groups of LEC rats were established, including animals pretreated with retrorsine to exacerbate copper toxicosis and inhibit proliferation in native hepatocytes followed by partial hepatectomy to promote liver repopulation. Hepatocytes from normal, syngeneic LEA rats were transplanted intrasplenically. Animal survival, biliary copper excretion, and hepatic copper were determined. The magnitude of liver repopulation was demonstrated by measuring serum ceruloplasmin and hepatic atp7b mRNA. Long-term survival in LEC rats treated with retrorsine, partial hepatectomy, and cell transplantation was up to 90%, whereas fewer than 10% of animals pretreated with retrorsine, without cell therapy, survived, P < 0.001. Liver repopulation occurred gradually after cell transplantation, ranging from <25% at 6 weeks, 26 to 40% at 4 months, and 74 to 100% at 6 months or beyond. Liver repopulation restored biliary copper excretion capacity and lowered liver copper levels. Remarkably, liver histology was completely normal in LEC rats with extensive liver repopulation, compared with widespread megalocytosis, apoptosis, oval cell proliferation, and cholangiofibrosis in untreated animals. These data indicate that liver repopulation with functionally intact cells can reverse pathophysiological perturbations and cure Wilson's disease.

The inability of transplanted cells to proliferate in the normal liver hampers cell therapy. We considered that oxidative hepatic DNA damage would impair the survival of native cells and promote proliferation in transplanted cells. Dipeptidyl peptidase-deficient F344 rats were preconditioned with whole liver radiation and warm ischemia-reperfusion followed by intrasplenic transplantation of syngeneic F344 rat hepatocytes. The preconditioning was well tolerated, although serum aminotransferase levels rose transiently and hepatic injury was observed histologically, along with decreased catalase activity and 8-hydroxy adducts of guanine, indicating oxidative DNA damage. Transplanted cells did not proliferate in the liver over 3 months in control animals and animals preconditioned with ischemia-reperfusion alone. Animals treated with radiation alone showed some transplanted cell proliferation. In contrast, the liver of animals preconditioned with radiation plus ischemia-reperfusion was replaced virtually completely over 3 months. Transplanted cells integrated in the liver parenchyma and liver architecture were preserved normally. These findings offer a paradigm for repopulating the liver with transplanted cells. Progressive loss of cells experiencing oxidative DNA damage after radiation and ischemia-reperfusion injury could be of significance for epithelial renewal in additional organs.oxidative damage ͉ hepatocyte L iver repopulation with transplanted cells is of considerable interest for cell and gene therapy (1). Transplanted hepatocytes integrate in the liver parenchyma, function normally, and survive life-long (2-4). However, transplanted cells do not proliferate in the normal adult liver, whereas specific therapies require a significant transplanted cell mass. Proliferation in transplanted cells depends on whether native cells are at survival͞proliferation disadvantages, as suggested by animal studies using exogenous toxins or natural disease, e.g., fumaryl acetoacetate hydroxylase (FAH) mice (hereditary tyrosinemia type-1), Long-Evans Cinnamon (LEC) rats (Wilson's disease), P-glycoprotein-2 (Pgy-2) mutant mice (progressive familial intrahepatic cholestasis), etc. (5-12). Initial clinical studies in familial hypercholesterolemia (FH) or Crigler-Najjar syndrome substantiated these principles (13,14).Genotoxic liver injury is a potent stimulus for transplanted cell proliferation. Rats exposed to retrorsine, a pyrrolizidine alkaloid, or whole liver radiation (RT), which produce DNA adducts and oxidative injury, respectively, lead to extensive transplanted cell proliferation in conjunction with two-thirds partial hepatectomy (PH) (15, 16). Although PH induces hepatic DNA synthesis, its additional effects include oxidative DNA damage, senescence-type changes, including p21 expression, polyploidy, attenuated proliferation capacity, and hepatocyte apoptosis (17-19). Both retrorsine and RT increase PH-induced hepatic polyploidy and apoptosis (20,21). Moreover, the thyroid hormone T3, which regulates PH-induced polyplo...

To determine whether K ATP channels control liver growth, we used primary rat hepatocytes and several human cancer cell lines for assays. K ATP channel openers (minoxidil, cromakalim, and pinacidil) increased cellular DNA synthesis, whereas K ATP channel blockers (quinidine and glibenclamide) attenuated DNA synthesis. The channel inhibitor glibenclamide decreased the clonogenicity of HepG2 cells without inducing cytotoxicity or apoptosis. To demonstrate the specificity of drugs for K ؉ channels, whole-cell patch-clamp recordings were made. Hepatocytes revealed K ؉ currents with K ATP channel properties. These K ؉ currents were augmented by minoxidil and pinacidil and attenuated by glibenclamide as well as tetraethylammonium, in agreement with established responses of K ATP channels. Reverse transcription of total cellular RNA followed by polymerase chain reaction showed expression of K ATP channel-specific subunits in rat hepatocytes and human liver cell lines. Calcium fluxes were unperturbed in glibenclamide-treated HepG2 cells and primary rat hepatocytes following induction with ATP and hepatocyte growth factor, respectively, suggesting that the effect of K ATP channel activity upon hepatocyte proliferation was not simply due to indirect modulation of intracellular calcium. The regulation of mitogen-related hepatocyte proliferation by K ATP channels advances our insights into liver growth control. The findings have implications in mechanisms concerning liver development, regeneration, and oncogenesis.

Mechanisms directing position-specific liver gene regulation are incompletely understood. To establish whether this aspect of hepatic gene expression is an inveterate phenomenon, we used transplanted hepatocytes as reporters in dipeptidyl peptidase IV-deficient F344 rats. After integration in liver parenchyma, the position of transplanted cells was shifted from periportal to perivenous areas by targeted hepatic ablations with carbon tetrachloride. In controls, transplanted cells showed greater glucose-6-phosphatase and lesser glycogen content in periportal areas. This pattern was reversed when transplanted cells shifted from periportal to perivenous areas. Transplanted hepatocytes in perivenous areas exhibited inducible cytochrome P450 activity, which was deficient in periportal hepatocytes. Moreover, cytochrome P450 activity was rapidly extinguished in activated hepatocytes when these cells were transplanted into the nonpermissive liver of suckling rat pups. In cells isolated from the normal F344 rat liver, cytochrome P450 inducibility was originally greater in perivenous hepatocytes; however, periportal cells rapidly acquired this facility in culture conditions. These findings indicate that the liver microenvironment exerts supremacy over prior differentiation state of cells in directing position-specific gene expression. Therefore, persistence of specialized hepatocellular function will require interactions with regulatory signals and substrate availability, which bears upon further analysis of liver gene regulation, including in progenitor and/or stem cells.