To analyze mechanisms of liver repopulation, we transplanted normal hepatocytes into syngeneic rats deficient in dipeptidyl peptidase IV activity. When
To understand regulation of transplanted hepatocyte proliferation in the normal liver, we used genetically marked rat or mouse cells. Hosts were subjected to liver injury by carbon tetrachloride (CCl4), to liver regeneration by a two-thirds partial hepatectomy, and to hepatocellular DNA synthesis by infusion of hepatocyte growth factor for comparative analysis. Transplanted hepatocytes were documented to integrate in periportal areas of the liver. In response to CCl4 treatments after cell transplantation, the transplanted hepatocyte mass increased incrementally, with the kinetics and magnitude of DNA synthesis being similar to those of host hepatocytes. In contrast, when cells were transplanted 24 h after CCl4 administration, transplanted hepatocytes appeared to be injured and most cells were rapidly cleared. When hepatocyte growth factor was infused into the portal circulation either subsequent to or before cell transplantation and engraftment, transplanted cell mass did not increase, although DNA synthesis rates increased in cultured primary hepatocytes as well as in intact mouse and rat livers. These data suggested that procedures causing selective ablation of host hepatocytes will be most effective in inducing transplanted cell proliferation in the normal liver. The number of transplanted hepatocytes was not increased in the liver by hepatocyte growth factor administration. Repopulation of the liver with genetically marked hepatocytes can provide effective reporters for studying liver growth control in the intact animal.
For hepatic gene therapy or applications of hepatocyte transplantation in liver failure, survival and function of transplanted cells is critical. Insights into site-specific gene regulation will significantly facilitate development of appropriate strategies for transplanting hepatocytes. To assess the function of transplanted cells, we used a transgenic hepatitis B virus (HBV) hepatocyte system, which allowed analysis of cellular gene expression with HBV surface antigen (HBsAg) mRNA expression, as well as secretion of HBsAg into peripheral circulation. When congeneic HBV hepatocytes were transplanted into the liver (via spleen), serum HBsAg promptly appeared in circulation and persisted for the entire duration of the studies. In contrast, transplantation of hepatocytes into the peritoneal cavity or dorsal fat pad resulted in serum HBsAg levels that were either significantly lower or gradually rose after a lag period. HBsAg mRNA expression was several-fold greater in transplanted hepatocytes in liver or spleen versus in peritoneal cavity or dorsal fat pad. Despite persistence of transplanted hepatocytes in peritoneal cavity or dorsal fat pad, serum HBsAg was cleared by antibody to HBsAg (anti-HBs) but this was not observed after hepatocyte transplantation into spleen. As the function of transplanted hepatocytes is optimally regulated in the liver, hepatic reconstitution with cell transplantation will be most appropriate for gene therapy.
Hepatocytes transplanted into the host liver engraft promptly, retain normal function, and survive indefinitely. Although intrasplenic transplantation is effective in delivering hepatocytes to the liver, to define potentially limiting complications, we studied its safety in normal, cirrhotic, and partial portal vein-ligated rats. In normal rats, portal pressures increased severalfold after hepatocyte transplantation but returned to normal within 3 weeks. In contrast, in portal hypertensive rats with partial portal vein ligation or cirrhosis, portal pressures were either unchanged or increased less after hepatocyte transplantation. However, more transplanted cells migrated to the lungs along with a rise in right atrial pressures in portal hypertensive rats. Further quantitative studies using 111Indium-labeled hepatocytes showed that intrasplenic retention of transplanted hepatocytes was similar in all animal groups. Intrahepatic cell translocation was comparable in normal and cirrhotic rats, whereas fewer cells migrated to the liver in partial portal vein-ligated rats. The most remarkable difference, however, was significantly greater intrapulmonary translocation of hepatocytes in portal hypertensive rats, which was presumably related to portosystemic shunting. These results indicate that because intrasplenic hepatocyte transplantation induces only temporary portal hypertension in normal subjects, potential strategies to augment liver repopulation could include repeated cell transplantation. This should be useful for optimizing the results of ex vivo gene therapy, or other hepatocyte-based therapies. However, the hepatic and portal hemodynamic status requires careful evaluation in portal hypertensive or cirrhotic subjects if serious complications are to be avoided.
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