Oval cell proliferation was induced in twelve male Fischer rats by administration of 2-acetylaminofluorene (2-AAF) for 2 weeks and by performing partial hepatectomy one week after the beginning of 2-AAF administration. Albumin expression in liver was studied by using in situ hybridization of 3H-labeled rat albumin riboprobe. Radiolabeled thymidine was administered to a group of animals at day 6 after partial hepatectomy. The animals were killed at 0, 3, 7, 9, 11 and 13 days after partial hepatectomy. Both oval cells and basophilic hepatocytes showed a prominent expression of albumin, whereas albumin expression in acidophilic pre-existing hepatocytes was decreased. Oval cell nuclei were exclusively labeled one day after administration of [3H]thymidine. At day 9, 11 and 13 basophilic hepatocytes became labeled and the area occupied by these cells increased. This is the first demonstration of the transfer of radiolabeled thymidine from oval cells to newly formed hepatocytes in vivo. Thus the precursor-product relationship between oval cells and basophilic hepatocytes has been established.

The cellular distribution and temporal expression of transcripts from transforming growth factor-beta 1 (TGF-beta 1) and procollagen alpha 1(I), alpha 1(III), and alpha 1(IV) genes were studied in carbon tetrachloride (CCl4)-induced rat liver fibrosis by using in situ hybridization technique. During the fibrotic process, TGF-beta 1 and procollagen genes were similarly and predominantly expressed in Desmin-positive perisinusoidal cells (e.g., fat-storing cells and myofibroblasts) and fibroblasts and their expression continued to be higher than those observed in control rats. These transcripts were also observed in inflammatory cells mainly granulocytes and macrophage-like cells at the early stages of liver fibrosis. The production of extracellular matrix along small blood vessels and fibrous septa coincided with the expression of these genes. Expression of TGF-beta 1 and procollagen genes were not detected in hepatocytes throughout the experiment. No significant differences in cellular distribution or time course of gene expression among procollagen alpha 1(I), alpha 1(III), and alpha 1(IV) were observed. Desmin-positive perisinusoidal cells and fibroblasts appeared to play the principal role in synthesis of collagens in CCl4-induced hepatic fibrosis. The simultaneous expression of TGF-beta 1 and procollagen genes in mesenchymal cells, including Desmin-positive perisinusoidal cells, during hepatic fibrosis suggests the possibility that TGF-beta 1 may have an important role in the production of fibrosis.

Proliferation of a new population of epithelial cells with distinct structure, as well as cytokeratin and alpha-fetoprotein expression, was observed in nonneoplastic liver tissues from 14 cases (13 hepatitis B virus-positive) of human hepatocellular carcinoma. These cells were characterized by oval nuclei; scant, pale cytoplasm; small cell size; and cross-reaction with a monoclonal antibody against rat oval cells. These putative human oval cells were strongly positive for cytokeratin 19 and displayed considerable heterogeneity in alpha-fetoprotein and albumin expression. The oval cells were most prominent in actively regenerating nodules and in liver tissue surrounding the cancer. Oval cells and transitional types of cells appear to be the principal producers of alpha-fetoprotein in the regenerating liver. Cancer cells positive for cytokeratins 8, 18 and 19 were observed in half the hepatocellular carcinomas studied. The data suggest that a new cell population structurally similar to oval cells seen in early stages of chemical hepatocarcinogenesis in rats is consistently present in regenerating liver lesions associated with human hepatocellular carcinoma. Furthermore, it is possible that the proliferation of these oval-type cells may partly account for the elevation of serum alpha-fetoprotein frequently seen in precancerous stages of hepatitis B virus-associated human hepatocellular carcinoma.

Both the level of expression and cellular distribution of transcripts for transforming growth factor-alpha (TGF-alpha) were studied in adult rat liver after partial hepatectomy and during hepatic differentiation in fetal, neonatal, and adult livers by northern blot analysis and in situ hybridization. A marked increase in the expression of TGF-alpha was observed in neonatal livers and in adult livers after partial hepatectomy and during hepatic regeneration following modification of the Solt-Farber protocol. Quantitation of silver grains after in situ hybridization with a TGF-alpha riboprobe revealed a sixfold to eightfold increase in fetal and neonatal hepatocytes. Moreover, the expression of TGF-alpha in the liver 3 wk after birth was still fourfold higher than that of the adult quiescent liver. Both proliferating oval cells and basophilic foci of hepatocytes generated by modification of the Solt-Farber protocol were positive for TGF-alpha transcripts. The level of TGF-alpha transcripts was sixfold higher in the basophilic foci than in the surrounding liver. High concentrations of TGF-alpha transcripts were observed in the oval cells that lined pseudoducts and in the transitional cells proliferating within the ducts. The combination of in situ hybridization and immunocytochemistry using cell-specific antibodies revealed the presence of TGF-alpha transcripts in both oval cells and in perisinusoidal stellate cells. The observation that TGF-alpha transcripts were found both in primitive liver epithelial cells and perisinusoidal stellate cells suggests that this growth factor, in addition to its mitogenic action, may also have other important functions in the liver.

ported by the finding that the early population of oval cells, The existence of a facultative hepatic stem cell comwhich represent the progeny of the hepatic stem cell compartpartment in bile ductules that participates in the rement, are both phenotypically similar to bile ductular cells newal process of epithelial cell populations in the liver and indeed may at least at an early stage be anatomically is well documented. The present study was undertaken connected to the bile tree. 3 The differentiation of oval cells to determine whether the immature bile epithelium reinto hepatocytes has now been shown in several experimental sponds differently to growth stimulus induced by bile models in which normal liver regeneration was impaired bestasis to that seen in the adult animal. In addition, the cause of the inability of hepatocytes to enter the cell cycle. 9-11 possible involvement of the growth factor/receptor sys-The most common experimental model used to induce extems associated with early activation of hepatic stem tensive and prolonged proliferation of bile epithelial cells in cells in bile duct proliferation was also examined. Bile duct ligation was used to induce the proliferation of bile the adult rat is the ligation of the common bile duct. 12 The epithelial cells. The expression of full-length alpha-feto-results from these experiments have shown that proliferation protein (AFP) was used as an indicator for activation of of bile-like cells are derived from existing biliary epithelium the stem cell compartment. AFP was highly and selec-and retain its characteristics; that bile duct cells divide irretively expressed in small bile ducts 7 days after bile duct spective of size of the ducts in which they are located, and ligation in immature rats up to 5 weeks of age. Although retain a lumen that is continuous with the preexisting biliary no significant increase in the expression of stem cell fac-tree. 13 Furthermore, no indications of a functioning stem cell tor (SCF) c-kit, hepatocyte growth factor (HGF), and compartment for the proliferation of either biliary epithelium transforming growth factor-a (TGF-a) was observed 7 or other cell lineages have been observed in any particular days after bile duct ligation in adult rats, the expression duct size or within the periportal connective tissue. 13 These of all these growth factors was increased in bile duct data suggest that the bile epithelium, and in particular the ligated rats up to 5 weeks of age. These results suggest bile ductular epithelium responds in a dramatically different that the bile ductular epithelium in the young rats re-way to growth stimulation induced by the ligation of the comsponds to bile stasis in a fashion that is phenotypically mon bile duct to that observed in the experimental models in similar to that seen during early activation of hepatic which oval cell proliferation occurs. Because the development stem cells in adult liver. (HEPATOLOGY 1997;25:1115-1122.) and maturation of the biliary system extends well into the perinatal period...