Cell transplantation causes loss of gap junctions and activates GGT expression permanently in host liver.

Cell transplantation into hepatic sinusoids, which is necessary for liver repopulation, could cause hepatic ischemia. To examine the effects of cell transplantation on host hepatocytes, we transplanted Fisher 344 rat hepatocytes into syngeneic dipeptidyl peptidase IV-deficient rats. Within 24 h of cell transplantation, areas of ischemic necrosis, along with transient disruption of gap junctions, appeared in the liver. Moreover, host hepatocytes expressed gamma-glutamyl transpeptidase (GGT) extensively, which was observed even 2 years after cell transplantation. GGT expression was not associated with alpha-fetoprotein activation, which is present in progenitor cells. Increased GGT expression was apparent after transplantation of nonparenchymal cells and latex beads but not after injection of saline, fragmented hepatocytes, hepatocyte growth factor, or turpentine. Some host hepatocytes exhibited apoptosis, as well as DNA synthesis, between 24 and 48 h after cell transplantation. Changes in gap junctions, GGT expression, DNA synthesis, and apoptosis after cell transplantation were prevented by vasodilators. The findings indicated the onset of ischemic liver injury after cell transplantation. These hepatic perturbations must be considered when transplanted cells are utilized as reporters for biological studies.

Transplanted reporter cells help in defining onset of hepatocyte proliferation during the life of F344 rats.

Transplanted hepatocytes integrate in the liver parenchyma and exhibit gene expression patterns that are similar to adjacent host hepatocytes. To determine the fate of genetically marked hepatocytes in the context of hepatocellular proliferation throughout the rodent life span, we transplanted Fischer 344 (F344) rat hepatocytes into syngeneic dipeptidyl peptidase IV-deficient rats. The proliferative activity in transplanted hepatocytes was studied in animals ranging in age from a few days to 2 yr. Transplanted hepatocytes proliferated during liver development between 1 and 6 wk of age, each dividing an estimated two to five times. DNA synthesis in occasional cells was demonstrated by localizing bromodeoxyuridine incorporation. There was no evidence for transplanted cell proliferation between 6 wk and 1 yr of age. Subsequently, transplanted cells proliferated again, with increased sizes of transplanted cell clusters at 18 and 24 mo of age. The proliferative activity of transplanted cells was greater in rats entering senescence compared with during postnatal liver development. In old rats, some liver lobules were composed entirely of transplanted cells. We conclude that hepatocyte proliferation in the livers of very young and old F344 rats is regulated in a temporally determined, biphasic manner. The findings will be relevant to mechanisms concerning liver development, senescence, and oncogenesis, as well as to cell and gene therapy.

Transplanted hepatocytes engraft, survive, and proliferate in the liver of rats with carbon tetrachloride-induced cirrhosis.

Repopulation of the cirrhotic liver with disease-resistant hepatocytes could offer novel therapies, as well as systems for biological studies. Establishing whether transplanted hepatocytes can engraft, survive, and proliferate in the cirrhotic liver is a critical demonstration. Dipeptidyl peptidase IV-deficient F344 rats were used to localize transplanted hepatocytes isolated from the liver of syngeneic normal F344 rats. Cirrhosis was induced by administration of carbon tetrachloride with phenobarbitone and these drugs were withdrawn prior to cell transplantation. Cirrhotic rats showed characteristic hepatic histology, as well as significant portosystemic shunting. When hepatocytes were transplanted via the spleen, cells were distributed immediately in periportal areas, fibrous septa, and regenerative nodules of the cirrhotic liver. Although some transplanted cells translocated into pulmonary capillaries, this was not deleterious. At 1 week, transplanted cells were fully integrated in the liver parenchyma, along with expression of glucose-6-phosphatase and glycogen as reporters of hepatic function. Transplanted cells proliferated in the liver of cirrhotic animals and survived indefinitely. At 1 year, transplanted hepatocytes formed large clusters containing several-fold more cells than normal control animals, which was in agreement with increased cell turnover in the cirrhotic rat liver. The findings indicate that the cirrhotic liver can be repopulated with functionally intact hepatocytes that are capable of proliferating. Liver repopulation using disease-resistant hepatocytes will be applicable in chronic conditions, such as viral hepatitis or Wilson's disease.

Rapid clearance of syngeneic transplanted hepatocytes following transduction with E-1-deleted adenovirus indicates early host immune responses and offers novel ways for studying viral vector, target cell and host interactions.

To distinguish between transduced cell clearance and transgene regulation following adenoviral gene transfer, we infected F344 rat hepatocytes with an E-1-deleted adenovirus (Ad beta gal) and studied cell survival in the liver of dipeptidyl peptidase IV-deficient (DPPIV-) F344 rats. Transplanted cells were localized with histochemical staining for DPPIV and transgene expression localized with staining for beta-galactosidase (lacZ). The transgene was expressed in 90-100% hepatocytes without impairment in cell viability in vitro, although transplanted cells were cleared mostly within 1 day by infiltrates containing activated macrophages, CD4+ or CD8+ lymphocytes, and phagocytes. When Ad beta gal-transduced hepatocytes were transplanted repeatedly at 14-day intervals, transplanted cells were cleared rapidly each time. LacZ expression following Ad beta gal administration to intact animals was associated with apoptosis and unscheduled DNA synthesis in the liver. To determine whether adenoviral antigen expression activated consequential MHC-restricted liver injury, we transplanted Ad beta gal-hepatocytes followed subsequently by transplantation of nontransduced hepatocytes. Transplanted cells expressing Ad beta gal were rapidly cleared as before, whereas nontransduced hepatocytes engrafted with progressive liver repopulation. The findings indicated that adenovirally transduced cells are cleared early in the host liver. Use of ex vivo strategies will facilitate analysis of modified adenoviral vectors in the context of immunoregulatory, cellular and viral mechanisms.

Differentiation-specific regulation of transgene expression in a diploid epithelial cell line derived from the normal F344 rat liver.

To establish the differentiation potential of progenitor cells, non-parenchymal epithelial cells from the F344 rat liver (FNRL cells) were studied. These cells reacted with the OV-6 antibody marker of oval cells, but were negative for hepatocyte markers (albumin, transferrin, glycogen, glucose-6-phosphatase, H4 antigen), biliary markers (gamma glutamyl transpeptidase, cytokeratin-19), and alpha-fetoprotein, although exposure to sodium butyrate induced nascent albumin and alpha-fetoprotein mRNA transcription. When stably transduced, FNRL cells expressed a retroviral promotor-driven lacZ reporter in vitro, similar to transgene expression in hepatocyte-derived HepG2 cells. However, lacZ expression in FNRL cells was rapidly extinguished in intact animals, whereas the reporter remained active in HepG2 cells. Transplanted FNRL cells showed copious glucose-6-phosphatase expression; however, the cell differentiation programme remained incomplete, despite two-thirds partial hepatectomy, D-galactosamine treatment or bile duct ligation. Interestingly, lacZ expression resumed in cultures of FNRL cells explanted from recipients. Moreover, lacZ expression was down-regulated by gamma-interferon in FNRL cells, without affecting lacZ activity in HepG2 cells. The data indicate that although subpopulations of oval cells may not fully differentiate into mature hepatocytes, these cells might serve critical functions, such as glucose utilization, and help survival after liver injury. Also, introduced genes may be regulated in progenitor cells at multiple levels, including by interactions between regulatory sequences, differentiation-specific cellular factors, and extracellular signals; in vivo studies are thus especially important for analysing gene regulation in progenitor cells.

Partial hepatectomy-induced polyploidy attenuates hepatocyte replication and activates cell aging events.

In understanding mechanisms of liver repopulation with transplanted hepatocytes, we studied the consequences of hepatic polyploidization in the two-thirds partial hepatectomy model of liver regeneration. Liver repopulation studies using genetically marked rodent hepatocytes showed that the number of previously transplanted hepatocytes did not increase in the liver with subsequential partial hepatectomy. In contrast, recipients undergoing partial hepatectomy before cells were transplanted showed proliferation in transplanted hepatocytes, with kinetics of DNA synthesis differing in transplanted and host hepatocytes. Also, partial hepatectomy caused multiple changes in the rat liver, including accumulation of polyploid hepatocytes along with prolonged depletion of diploid hepatocytes, as well as increased senescence-associated beta-galactosidase and p21 expression. Remnant hepatocytes in the partially hepatectomized liver showed increased autofluorescence and cytoplasmic complexity on flow cytometry, which are associated with lipofuscin accumulation during cell aging, and underwent apoptosis more frequently. Moreover, hepatocytes from the partially hepatectomized liver showed attenuated proliferative capacity in cell culture. These findings were compatible with decreased proliferative potential of hepatocytes experiencing partial hepatectomy compared with hepatocytes from the unperturbed liver. Attenuation of proliferative capacity and other changes in hepatocytes experiencing partial hepatectomy offer novel perspectives concerning liver regeneration in the context of cell ploidy.

Position-specific gene expression in the liver lobule is directed by the microenvironment and not by the previous cell differentiation state.

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.