Origin and fate of oval cells in dipin-induced hepatocarcinogenesis in the mouse.

We have studied the development and differentiation of oval cells in the Dipin model of hepatocarcinogenesis in the mouse and compared this process to generation of biliary epithelial cells by bile duct ligation using light and electron microscopy. The Dipin model of hepatocarcinogenesis consists of a single injection of an alkylating drug, Dipin (1,4-bis[N,N'-di(ethylene)-phosphamide]-piperazine), followed by partial hepatectomy. The Dipin treatment resulted in irreversible damage and gradual death of hepatocytes by necrosis and apoptosis. Earlier work provided evidence that regeneration of parenchyma occurred via oval cell proliferation and subsequent differentiation into hepatocytes that replaced the degenerating hepatocytes. Both autoradiographic and morphological data indicated that oval cells were derived from ductular cells of Hering canals. The first oval cells labeled with [3H]thymidine were similar in size and ultrastructure to ductular cells of Hering canals with whom intracellular connections existed. The proliferation of ductular cells of Hering canals gave rise to a new system of oval cell ducts that spread into the liver acinus. In the periportal areas, the transition of oval cells into hepatocytes was observed inside the ducts. Both growth patterns and ultrastructure of oval cells were different from the biliary epithelial cells in bile duct-ligated liver. Also, oval cells retained the property to interact with adjacent hepatocytes through desmosomes and intermediate junctions. Oval cell population was heterogeneous in terms of proliferating potential. A proportion of proliferating cells (38 to 45%) in the Hering canals and small oval cell ducts located in the periportal areas was similar throughout the period of oval cell development. The extent of proliferation of oval cells decreased from 62% at the stage of active migration into the acinus to 22% at maximum formation of oval cell ducts. These data suggest that in the mouse liver cells of the terminal biliary ductules harbor the hepatic stem cell compartment from which oval cells, capable of differentiating into hepatocytes, may be derived.     

Differentiation processes of oval cells into hepatocytes: proposals based on morphological and phenotypical traits in carcinogen-treated hamster liver

Hepatic stem cells participate in the recovery process of liver with severe injury or impaired hepatocyte regeneration. Oval cells (an oval-shaped liver cell population newly emerging from the portal or periportal zones following severe hepatic cellular damage) are believed to be the progeny of liver stem cells and precursor cells of both hepatocytes and bile duct cells. An attempt was made to define the differentiation processes of hepatic oval cells into mature hepatocytes in hamsters fed a choline-deficient diet and treated with diethylnitrosamine and 2-acetyl aminofluorene, on the basis of histopathological, electron microscopical, histochemical and immunohistochemical characterization of hepatic cell components. Two putative differentiation pathways of oval cells toward mature hepatocytes are proposed, namely (1) the differentiation of ductular-like oval cells via ductular/acinar-type hepatocytes, and (2) the differentiation of individual oval cells via small hepatocytes. Those proposals were strongly supported by consistent immunoreactivity of the cells for OV-6, an oval cell marker, and differential expression patterns for CK19 and PAS-positive cytoplasmic glycogen granules.     

The inhibitory effect of rapamycin on the oval cell response and development of preneoplastic foci in the rat

Oval cell activation occurs under conditions of severe liver injury when normal hepatocyte proliferation is blocked. Recent studies have shown that a subset of hepatocellular carcinomas expresses oval cell markers, suggesting that these cells are targets of hepatocarcinogens. However, the signaling pathways that control oval cell activation and proliferation are not well characterized. Based on the role of the nutrient signaling kinase complex, mTORC1, in liver development, we investigated the role of this pathway in oval cell activation. Oval cell proliferation was induced in male Fisher rats by a modification of the traditional choline deficient plus ethionine model (CDE) or by 2-acetylaminoflourene treatment followed by 2/3 partial hepatectomy with or without initiation by diethylnitrosamine. To assess the role of mTOR in the oval cell response and development of preneoplastic foci, the effect of the mTORC1 inhibitor, rapamycin, was studied in all models. Rapamycin induced a significant suppression of the oval cell response in both models, an effect that coincided with a decrease in oval cell proliferation. Rapamycin administration did not affect the abundance of neutrophils or natural killer cells in CDE-treated liver or the expression of key cytokines. Gene expression studies revealed the fetal hepatocyte marker MKP-4 to be expressed in oval cells. In an experimental model of hepatic carcinogenesis, rapamycin decreased the size of preneoplastic foci and the rate of cell proliferation within the foci. mTORC1 signaling plays a key role in the oval cell response and in the development of preneoplastic foci. This pathway may be a target for the chemoprevention of hepatocellular carcinoma.     

Bile ductular damage induced by methylene dianiline inhibits oval cell activation.

Administration of 2-acetylaminofluorene (2-AAF) given before a two-thirds partial hepatectomy (PHx), results in suppression of hepatocyte proliferation and stimulation of oval cell proliferation. Our objective in this study was to examine the oval cell response and associated alpha-fetoprotein (AFP) gene expression by combining 2-AAF with selective hepatic damage caused by either carbon tetrachloride (CCl4) exposure or by PHx. We also examined oval cell response with the above two protocols (2-AAF/CCl4 and 2-AAF/PHx) as affected by previous bile ductular damage caused by 4,4'-methylene dianiline (4,4'-diaminodiphenylmethane, DAPM) exposure. DAPM is an aromatic diamine, known to cause bile ductular damage in both humans and animals. Using the protocols of 2-AAF/ CCl4 and 2-AAF/PHx, when DAPM was given 24 hours before the hepatic injury, no oval cell proliferation was seen (histological) and AFP expression was not detected by Northern blot analysis. These results provide direct evidence that oval cells are closely associated with the biliary epithelial cells and supports the theory that hepatic oval cells may originate from cells derived from either intraportal or periportal ductules.     

Cytosine deaminase versus thymidine kinase: a comparison of the antitumor activity

Abstract. The efficacy of single and combination suicide gene therapy was evaluated using a Herpes simplex virus thymidine kinase/ganciclovir system and Escherichia coli cytosine deaminase/5-fluorocytosine system on the rat prostate tumor cell line R3327 AT-1. The wild-type R3327 AT-1 cell line was transfected with a bifunctional fusion gene CDglyTK, which had the advantage that the resulting R3327 AT-1/CDglyTK cell line has the same amount of cytosine deaminase and thymidine kinase molecules. The percentage of viable R3327 AT-1/CDglyTK cells after 96 h incubation with 0.1 µg/ml ganciclovir or 10 µg/ml 5-fluorocytosine were 85% and 52% of controls, respectively. The cell viability when both suicide genes systems were activated was 43%. For in vivo analysis, Copenhagen rats were injected subcutaneously with R3327 AT-1 or R3327 AT-1/CDglyTK cells and treated with 30 mg/kg ganciclovir, 500 mg/kg 5-fluorocytosine, or both prodrugs together. A survival of 83% with the thymidine kinase/ganciclovir and 57% with the CD/5-FC could be observed. Only co-administration of thymidine kinase- and cytosine deaminase-specific prodrugs resulted in a 100% recurrence-free survival of the Copenhagen rats with a Dunning R3327 AT-1/CDglyTK prostate tumor and showed an additive cytotoxic effect. Calculation of the degree of activation and the potential of activation can be used to predict the success of a suicide gene therapy. In our case, the cytosine deaminase/5-fluorocytosine system had a low degree of activation (value 40), which is also found in the low response to 5- fluorocytosine in vivo (57% tumor free).     

Cytosine deaminase versus thymidine kinase: a comparison of the antitumor activity

The efficacy of single and combination suicide gene therapy was evaluated using a Herpes simplex virus thymidine kinase/ganciclovir system and Escherichia coli cytosine deaminase/5-fluorocytosine system on the rat prostate tumor cell line R3327 AT-1. The wild-type R3327 AT-1 cell line was transfected with a bifunctional fusion gene CDglyTK, which had the advantage that the resulting R3327 AT-1/CDglyTK cell line has the same amount of cytosine deaminase and thymidine kinase molecules. The percentage of viable R3327 AT-1/CDglyTK cells after 96 h incubation with 0.1 micro g/ml ganciclovir or 10 micro g/ml 5-fluorocytosine were 85% and 52% of controls, respectively. The cell viability when both suicide genes systems were activated was 43%. For in vivo analysis, Copenhagen rats were injected subcutaneously with R3327 AT-1 or R3327 AT-1/CDglyTK cells and treated with 30 mg/kg ganciclovir, 500 mg/kg 5-fluorocytosine, or both prodrugs together. A survival of 83% with the thymidine kinase/ganciclovir and 57% with the CD/5-FC could be observed. Only co-administration of thymidine kinase- and cytosine deaminase-specific prodrugs resulted in a 100% recurrence-free survival of the Copenhagen rats with a Dunning R3327 AT-1/CDglyTK prostate tumor and showed an additive cytotoxic effect. Calculation of the degree of activation and the potential of activation can be used to predict the success of a suicide gene therapy. In our case, the cytosine deaminase/5-fluorocytosine system had a low degree of activation (value 40), which is also found in the low response to 5- fluorocytosine in vivo (57% tumor free).     

肝脏干细胞的研究进展

干细胞为机体进化过程中高度保守的细胞族，在机体细胞再生调控中具有特殊的作用。目前认为以肝内胆管系统源性为主的多潜能肝脏干细胞分化群，即可向胆管细胞分化，又可向肝细胞分化。近年来，在现代细胞生物学、分子生物学及分子遗传学研究推动下，有关肝脏干细胞定位、来源、发生、演变、功能及体外培养、鉴定技术等研究取得了较大进展，本文将近年来的主要进展作一综述。     

Oxidative stress and oval cell accumulation in mice and humans with alcoholic and nonalcoholic fatty liver disease

In animals, the combination of oxidative liver damage and inhibited hepatocyte proliferation increases the numbers of hepatic progenitors (oval cells). We studied different murine models of fatty liver disease and patients with nonalcoholic fatty liver disease or alcoholic liver disease to determine whether oval cells increase in fatty livers and to clarify the mechanisms for this response. To varying degrees, all mouse models exhibit excessive hepatic mitochondrial production of H(2)O(2), a known inducer of cell-cycle inhibitors. In mice with the greatest H(2)O(2) production, mature hepatocyte proliferation is inhibited most, and the greatest number of oval cells accumulates. These cells differentiate into intermediate hepatocyte-like cells after a regenerative challenge. Hepatic oval cells are also increased significantly in patients with nonalcoholic fatty liver disease and alcoholic liver disease. In humans, fibrosis stage and oval cell numbers, as well as the number of intermediate hepatocyte-like cells, are strongly correlated. However, cirrhosis is not required for oval cell accumulation in either species. Rather, as in mice, progenitor cell activation in human fatty liver diseases is associated with inhibited replication of mature hepatocytes. The activation of progenitor cells during fatty liver disease may increase the risk for hepatocellular cancer, similar to that observed in the Solt-Farber model of hepatocarcinogenesis in rats.     

Loss of keratin 19 favours the development of cholestatic liver disease through decreased ductular reaction

Keratins (K) are cytoprotective proteins and keratin mutations predispose to the development of multiple human diseases. K19 represents the most widely used marker of biliary and hepatic progenitor cells as well as a marker of ductular reaction that constitutes the basic regenerative response to chronic liver injury. In the present study, we investigated the role of K19 in biliary and hepatic progenitor cells and its importance for ductular reaction. K19 wild‐type (WT) and knockout (KO) mice were fed: (a) 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC); (b) cholic acid (CA); (c) a choline‐deficient, ethionine‐supplemented (CDE) diet; or (d) were subjected to common bile duct ligation (CBDL). The bile composition, liver damage, bile duct proliferation, oval cell content and biliary fibrosis were analysed. In untreated animals, loss of K19 led to redistribution of the K network in biliary epithelial cells (BECs) but to no obvious biliary phenotype. After DDC feeding, K19 KO mice exhibited (compared to WTs): (a) increased cholestasis; (b) less pronounced ductular reaction with reduced ductular proliferation and fewer oval cells; (c) impaired Notch 2 signalling in BECs; (d) lower biliary fibrosis score and biliary bicarbonate concentration. An attenuated oval cell proliferation in K19 KOs was also found after feeding with the CDE diet. K19 KOs subjected to CBDL displayed lower BEC proliferation, oval cell content and less prominent Notch 2 signal. K19 deficiency did not change the extent of CA‐ or CBDL‐induced liver injury and fibrosis. Our results demonstrate that K19 plays an important role in the ductular reaction and might be of importance in multiple chronic liver disorders that frequently display a ductular reaction. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Hepatic stem cells: a review

The existence of a liver stem cell population has only gained credence recently, following the results of animal experiments. These cells are thought to reside in the terminal bile ductules (canals of Hering). Hepatocyte division is responsible for liver regeneration after most causes of injury. However, stem cells may contribute to hepatocyte regeneration, or even take over this role if the liver injury is severe and associated with an impairment of hepatocyte proliferation as in cirrhosis or submassive/massive necrosis, due to drugs, toxins or viruses. "Oval" cells are the descendants of the stem cells and are found in the portal and periportal regions in experimental animals within days of the liver injury. These cells proliferate to form narrow ductules, which may stain positively for biliary cytokeratins CK 19, and radiate out into the damaged parenchyma. Both in vitro and in vivo animal studies now suggest that oval cells can differentiate into bile ductular cells or hepatocytes to allow repopulation of the injured liver. As the oval cells differentiate into hepatocytes they may show positive staining for pyruvate kinase isoenzyme L-PK, albumin and alpha-fetoprotein. There is also growing evidence that bone marrow stem cells may contribute to liver regeneration. The possible involvement of hepatic stem cells in the development of dysplastic nodules, hepatocellular carcinoma and cholangiocarcinoma has been suggested but remains highly controversial. Oval cell isolation and culture techniques, together with stem cell transplantation strategies, may in the future provide novel treatments for individuals with inherited and acquired hepatic disorders.     

Nuclear receptor CAR (NR1I3) is essential for DDC-induced liver injury and oval cell proliferation in mouse liver

The liver is endowed with the ability to regenerate hepatocytes in response to injury. When this regeneration ability is impaired during liver injury, oval cells, which are considered to be postnatal hepatic progenitors, proliferate and differentiate into hepatocytes. Here we have demonstrated that 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) activates the nuclear receptor constitutive active/androstane receptor (CAR), resulting in proliferation of oval cells in mouse liver. Activation of CAR by DDC was shown by hepatic nuclear CAR accumulation and cytochrome P450 (CYP)2B10 mRNA induction after feeding a 0.1% DDC-containing diet to Car(+/+) mice. After being fed the DDC diet, Car(+/+), but not Car(-/-) mice, developed severe liver injury and an A6 antibody-stained ductular reaction in an area around the portal tract. Oval cell proliferation was confirmed by laser capture microdissection and real-time PCR; mRNAs for the two oval cell markers epithelial cell adhesion molecule and TROP2 were specifically induced in the periportal region of DDC diet-fed Car(+/+), but not Car(-/-) mice. Although rates of both hepatocyte growth and death were initially enhanced only in DDC diet-fed Car(+/+) mice, growth was attenuated when oval cells proliferated, whereas death continued unabated. DDC-induced liver injury, which differs from other CAR activators such as phenobarbital, occurred in the periportal region where cells developed hypertrophy, accumulated porphyrin crystals and inflammation developed, all in association with the proliferation of oval cells. Thus, CAR provides an excellent experimental model for further investigations into its roles in liver regeneration, as well as the development of diseases such as hepatocellular carcinoma.     

Sequential analysis of hepatic carcinogenesis. Regeneration of liver after carbon tetrachloride-induced liver necrosis when hepatocyte proliferation is inhibited by 2-acetylaminofluorene

The effect of inhibition of hepatocyte proliferation by dietary 2-acetylaminofluorene (2-AAF) on the restoration of liver in rats after a necrogenic dose of carbon tetrachloride has been studied. The liver weights remained low during the entire feeding period of the 2-AAF, and virtually no hepatocyte proliferation was seen, as determined autoradiographically after thymidine incorporation and by the absence of mitotic figures. Oval cell proliferation was extensive. Morphometric analysis showed (a) equal and maximum liver cell necrosis by 24 hours in both the experimental and control groups, (b) similar kinetics of removal of dead liver cells, and (c) similar values for the mean liver cell area. The distance between the portal triad and terminal hepatic vein in animals on the dietary 2-AAF was considerably reduced. Massive hepatocyte proliferation began after termination of the 2-AAF diet, and the liver returned to normal appearance within 14 days. The oval cells disappeared during this period of liver cell restoration. A new hypothesis for oval cell proliferation based on differential inhibition of hepatocyte proliferation resulting in unbalanced growth of ductular cells is presented.     

In-vitro immune response of splenic lymphocytes to portal serum agents from rats undergoing hepatic regeneration or hepatic carcinogenesis.

To clarify the physiologic response of splenic lymphocytes to liver damage and the role of this response in regeneration versus malignant transformation, we cultured rat spleen lymphocytes with portal sera from rats subjected either to partial (70%) hepatectomy or to long-term oral administration of the hepatic carcinogen 3'-methyl-4-dimethylaminoazobenzene. Sera taken within 24h after partial hepatectomy contained a previously described signal protein which serves as a marker of liver damage. The MW 5,000-10,000 serum fraction also contained a factor that promoted cell growth, DNA synthesis, glucose utilization, and the production of anti-sheep erythrocyte plaque-forming cells in cultures of rat splenic lymphocytes. In contrast, the sera of rats subjected to liver damage by the carcinogen had no more effect on the cultured lymphocytes than sera from sham-operated or untreated controls. The signal protein was present initially in portal sera from carcinogen-treated rats, but decreased as hepatitis gave way to cirrhosis. Subsequent malignant transformation was marked by the appearance of serum alpha-fetoprotein. Our results suggest that activation of splenic lymphocytes by serum factor(s) is involved in hepatic regeneration and that this process is deranged in carcinogenesis.     

Fate-mapping evidence that hepatic stellate cells are epithelial progenitors in adult mouse livers

Liver injury activates quiescent hepatic stellate cells (Q-HSC) to proliferative myofibroblasts. Accumulation of myofibroblastic hepatic stellate cells (MF-HSC) sometimes causes cirrhosis and liver failure. However, MF-HSC also promote liver regeneration by producing growth factors for oval cells, bipotent progenitors of hepatocytes and cholangiocytes. Genes that are expressed by primary hepatic stellate cell (HSC) isolates overlap those expressed by oval cells, and hepatocytic and ductular cells emerge when HSC are cultured under certain conditions. We evaluated the hypothesis that HSC are a type of oval cell and, thus, capable of generating hepatocytes to regenerate injured livers. Because Q-HSC express glial fibrillary acidic protein (GFAP), we crossed mice in which GFAP promoter elements regulated Cre-recombinase with ROSA-loxP-stop-loxP-green fluorescent protein (GFP) mice to generate GFAP-Cre/GFP double-transgenic mice. These mice were fed methionine choline-deficient, ethionine-supplemented diets to activate and expand HSC and oval cell populations. GFP(+) progeny of GFAP-expressing precursors were characterized by immunohistochemistry. Basal expression of mesenchymal markers was negligible in GFAP(+)Q-HSC. When activated by liver injury or culture, HSC downregulated expression of GFAP but remained GFP(+); they became highly proliferative and began to coexpress markers of mesenchyme and oval cells. These transitional cells disappeared as GFP-expressing hepatocytes emerged, began to express albumin, and eventually repopulated large areas of the hepatic parenchyma. Ductular cells also expressed GFAP and GFP, but their proliferative activity did not increase in this model. These findings suggest that HSC are a type of oval cell that transitions through a mesenchymal phase before differentiating into hepatocytes during liver regeneration. Disclosure of potential conflicts of interest is found at the end of this article.     

Oval cell response in 2-acetylaminofluorene/partial hepatectomy rat is attenuated by short interfering RNA targeted to stromal cell-derived factor-1

Stromal cell-derived factor-1 (SDF-1) is known to play an essential role in the regulation of stem/progenitor cell trafficking. During hepatic stem, or oval, cell activation, SDF-1 has been reported to be up-regulated within the liver, implying a possible role in oval cell-aided liver regeneration. In the present study, SDF-1 expression was knocked down in the liver of 2-acetylaminofluorene/partial hepatectomy-treated rats using short interfering RNA delivered by recombinant adenovirus. The oval cell response was compromised in these animals, as evidenced by a decreased number of OV6-positive oval cells. In addition, knockdown of SDF-1 expression caused a dramatic decrease in alpha-fetoprotein expression, implying impaired oval cell activation in these animals. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling assay showed no significant apoptosis related to SDF-1 suppression. Instead, as revealed by Ki67 immunohistochemistry, the suppression of SDF-1 resulted in decrease of hepatic cell proliferation, implying the repair process had been inhibited in these animals. These results indicate that SDF-1 is an essential molecule needed in oval cell activation.     

Modulation of the Gene Network Connected to Interferon-γ in Liver Regeneration from Oval Cells

Suppression subtractive hybridization was used to clone genes associated with proliferation of oval cells in rat liver regenerating after a 70% partial hepatectomy combined with the feeding of 2-acetylaminofluorene. A subset of the identified genes comprised interferon-gamma receptor alpha subunit (IFN-gammaRalpha), gp91phox, interleukin-1beta (IL-1beta), lymphocyte function-associated molecule-1alpha (LFA-1), eukaryotic initiation factor-2-associated 67-kd protein (eIF-2-associated 67-kd protein), and alpha-fetoprotein, which constitute part of the cellular program modulated by IFN-gamma. Therefore, expression analysis performed by Northern blotting and immunohistochemistry were extended to include IFN-gamma, the IFN-gamma receptor beta subunit (IFN-gammaRbeta), three secondary response genes induced by interaction of IFN-gamma with IFN-gamma receptor complexes, ie, IL-1beta-converting enzyme (ICE), intercellular adhesion molecule-1 (ICAM-1), and urokinase-type plasminogen activator receptor (uPAR), and a cytokine inducing IFN-gamma expression, ie, interleukin-18 (IL-18). The Northern blot analysis showed that all examined genes were modulated when progenitor-like oval cells were activated and recruited for liver regeneration. Immunohistochemistry localized the subunits of the IFN-gamma receptor complex, IFN-gammaRalpha and IFN-gammaRbeta, the secondary response genes uPAR and ICAM-1, the IFN-gamma-inducing factor IL-18, and ICE to the ductular structures of oval cells. In contrast, during liver regeneration after a 70% partial hepatectomy, only modulation of IL-1beta and ICE was observed. Our results, therefore, indicate that IFN-gamma-mediated events may be particularly important when cells in the bile ductules must respond to liver damage by production of ductular oval cells.     

Transforming growth factor-beta1 protein, proliferation and apoptosis of oval cells in acetylaminofluorene-induced rat liver regeneration

Administering of 2-acetylaminofluorene (2-AAF) before a two-thirds partial hepatectomy (PHx) results in suppression of hepatocyte proliferation and stimulation of oval cell proliferation. The objectives of this study was to examine the oval cell behaviour and associated transforming growth factor-beta1 (TGF-beta1) protein expression by combining 2-AAF with selective hepatic damage caused by PHx. We also studied the temporal relationship between TGF-beta1 expression, and proliferation and apoptosis of oval cells. Oval cells emerged from the portal areas and became more numerous with time fanning out into the periportal and midzonal hepatic parenchyma. Both smooth muscle actin (SMA) and TGF-beta1 immunostain revealed that TGF-beta1-positive cells were SMA-positive hepatic stellate cells (HSCs). Coinciding with the proliferation of oval cells, an increase expression of TGF-beta1 produced by SMA-positive HSCs was observed, thereafter apoptosis of oval cells reached its peak. This result implicated that TGF-beta1 produced by HSCs is intimately associated with proliferation and apoptosis of oval cells, and plays a role in the cessation of oval cell activation and remodeling of liver parenchyma in 2-AAF induced liver regeneration.